AU756253B2 - Inhibitors of interleukin-1beta converting enzyme - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT *go* Name of Applicant: *o Actual Inventors: go 0 0* .o 0 Address for Service: 9 .0o.

0 so Vertex Pharmaceuticals Incorporated Mark J. Batchelor, David Bebbington, Guy W. Bemis, Wolf Herman Fridman, Roger J. Gillespie, Julian M. C. Golec, Yong Gu, David J. Lauffer, David J. Livingston, Saroop S. Matharu, Michael D.

Mullican, Mark A. Murcko, Robert Murdoch, Philip L. Nyce, Andrea L.

C. Robidoux, Michael Su, M. Woods Wannamaker, Keith P. Wilson and Robert E. Zelle CULLEN CO., Patent Trade Mark Attorneys, 239 George Street, Brisbane, Qld. 4000, Australia.

Invention Title: Inhibitors of Converting Enzyme Interleukin-lp The following statement is a full description of this invention, including the best method of performing it known to us: VPI96-01 CIP2 DIV INHIBITORS OF INTERLEUKIN-1B CONVERTING ENZYME TECHNICAL FIELD OF THE INVENTION The present invention relates to novel classes of compounds which are inhibitors of interleukin-lp converting enzyme This invention also relates to pharmaceutical compositions comprising these compounds. The compounds and pharmaceutical compositions of this invention are 10 particularly well suited for inhibiting ICE activity and consequently, may be advantageously used as agents against interleukin-1- apoptosis-, interferon gamma inducing factor- ("IGIF") and interferon-ymediated diseases, including inflammatory diseases, autoimmune diseases, destructive bone, proliferative disorders, infectious diseases and degenerative diseases. This invention also relates to methods for inhibiting ICE activity, and decreasing IGIF production and IFN-y production and methods for treating interleukin-1-, apoptosis-, IGIF- and IFN-ymediated diseases using the compounds and compositions of this invention. This invention also relates to *methods of preparing N-acylamino compounds.

BACKGROUND OF THE INVENTION 25 Interleukin 1 is a major proinflammatory and immunoregulatory protein that stimulates fibroblast differentiation and proliferation, the production of prostaglandins, collagenase and phospholipase by synovial cells and chondrocytes, basophil and eosinophil degranulation and neutrophil activation. Oppenheim, J.H. et al, Immunolovy Today, 7, pp. 45-56 (1986). As such, it is involved in the pathogenesis of chronic and acute inflammatory and autoimmune diseases. For example, in rheumatoid arthritis, IL-1 is both a mediator of 2 inflammatory symptoms and of the destruction of the cartilage proteoglycan in afflicted joints. Wood, D.D.

et al., Arthritis Rheum. 26, 975, (1983); Pettipher, E.J. et al., Proc. Natl. Acad. Sci. UNITED STATES OF AMERICA 71, 295 (1986); Arend, W.P. and Dayer, J.M., Arthritis Rheum. 38, 151 (1995). IL-1 is also a highly potent bone resorption agent. Jandiski, J. Oral Path 17, 145 (1988); Dewhirst, F.E. et al., J. Immunol.

8, 2562 1985). It is alternately referred to as "osteoclast activating factor" in destructive bone diseases such as osteoarthritis and multiple myeloma.

Bataille, R. et al., Int. J. Clin. Lab. Res. 21(4), 283 (1992). In certain proliferative disorders, such as acute myelogenous leukemia and multiple myeloma, IL-1 can promote tumor cell growth and adhesion. Bani, J. Natl. Cancer Inst. 83, 123 (1991); Vidal- Vanaclocha, Cancer Res. 54, 2667 (1994). In these disorders, IL-1 also stimulates production of other cytokines such as IL-6, which can modulate tumor development (Tartour et al., Cancer Res. 54, 6243 (1994). IL-1 is predominantly produced by peripheral blood monocytes as part of the inflammatory response and exists in two distinct agonist forms, IL-la and IL- Mosely, B.S. et al., Proc. Nat. Acad. Sci., 84, pp. 4572-4576 (1987); Lonnemann, G. et al., Eur.J.

Immunol., 19, pp. 1531-1536 (1989).

is synthesized as a biologically inactive precursor, pIL-10. pIL-10 lacks a conventional leader sequence and is not processed by a signal peptidase. March, Nature, 315, pp. 641-647 (1985). Instead, pIL-10 is cleaved by interleukin-lp converting enzyme between Asp- 3 116 and Ala-ll7 to produce the biologically active C-terminal fragment found in human serum and synovial fluid. Sleath, et al., J. Biol. Chem., 265, pp. 14526-14528 (1992); A.D. Howard et al., J.

Immunol., 147, pp. 2964-2969 (1991). ICE is a cysteine protease localized primarily in monocytes. It converts precursor IL-10 to the mature form. Black, R.A.

:et al., FEBS Lett., 247, pp. 386-390 (1989); Kostura, et al., Proc. Natl. Acad. Sci. UNITED STATES OF 10 AMERICA, 86, pp. 5227-5231 (1989). Processing by ICE is also necessary for the transport of mature through the cell membrane.

eoeICE, or its homologs, also appears to be involved in the regulation of programmed cell death or apoptosis. Yuan, J. et al., Cell, 75, pp. 641-652 Miura, M. et al., _Cell, 75, pp. 653-660 (1993); ooo• Nett-Fiordalisi, M.A. et al, J. Cell Biochem., 17B, p. 117 (1993). In particular, ICE or ICE homologs are o •thought to be associated with the regulation of apoptosis in neurodegenerative diseases, such as Alzheimer's and Parkinson's disease. Marx, J. and M.

Baringa, Science, 259, pp. 760-762 (1993); Gagliardini, V. et al., Science, 263, pp. 826-828 (1994).

Therapeutic applications for inhibition of apoptosis may include treatment of Alzheimer's disease, Parkinson's disease, stroke, myocardial infarction, spinal atrophy, and aging.

ICE has been demonstrated to mediate apoptosis (programmed cell death) in certain tissue types. Steller, Science, 267, p. 1445 (1995); Whyte, M. and Evan, Nature, 376, p. 17 (1995); Martin, S.J. and Green, Cell, 82, p. 349 (1995); Alnemri, et al., J. Biol. Chem., 270, p. 4312 4 (1995); Yuan, J. Curr. Opin. Cell Biol., 7, p. 211 (1995). A transgenic mouse with a disruption of the ICE gene is deficient in Fas-mediated apoptosis (Kuida, K. et al., Science 267, 2000 (1995)). This activity of ICE is distinct from its role as the processing enzyme for pro-ILlp. It is conceivable that in certain tissue types, inhibition of ICE may not affect secretion of mature IL-10, but may inhibit apoptosis.

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50 00 9 @0 60 Enzymatically active ICE has been previously described as a heterodimer composed of two subunits, and plO (20kDa and 10kDa molecular weight, respectively). These subunits are derived from a proenzyme (p45) by way of a p30 form, through an activation mechanism that is autocatalytic.

Thornberry, N.A. et al., Nature, 356, pp. 768-774 (1992). The ICE proenzyme has been divided into several functional domains: a prodomain (p14), a p22/20 subunit, a polypeptide linker and a pl0 subunit.

Thornberrv et al., supra; Casano et al., Genomics, pp. 474-481 (1994).

Full length p45 has been characterized by its cDNA and amino acid sequences. PCT patent applications WO 91/15577 and WO 94/00154. The p20 and plO cDNA and amino acid sequences are also known. Thornberry et al., supra. Murine and rat ICE have also been sequenced and cloned. They have high amino acid and nucleic acid sequence homology to human ICE. Miller, D.K. et al., Ann. N.Y. Acad. Sci., 696, pp. 133-148 (1993); Molineaux, S.M. et al., Proc. Nat. Acad. Sci., 90, pp. 1809-1813 (1993). The three-dimensional structure of ICE has been determined at atomic resolution by X-ray crystallography. Wilson, et al., Nature, 370, pp. 270-275 (1994). The active 5 enzyme exists as a tetramer of two p20 and two pl0 subunits.

Additionally, there exist human homologs of ICE with sequence similarities in the active site regions of the enzymes. Such homologs include TX (or ICErel-II or ICH-2) (Faucheu, et al., EMBO 14, p.

1914 (1995); Kamens et al., J. Biol. Chem., 270, p.

15250 (1995); Nicholson et al., J. Biol. Chem., 270 15870 (1995)), TY (or ICErel-iiI) (Nicholson et al., J.

10 Biol. Chem., 270, p. 15870 (1995); ICH-1 (or Nedd-2) (Wang, L. et al., Cell, 78, p. 739 (1994)), MCH-2, (Fernandes-Alnemri, T. et al., Cancer Res., 55, p. 2737 (1995), CPP32 (or YAMA or apopain) (Fernandes-Alnemri, T. et al., J. Biol. Chem., 269, p. 30761 (1994); Nicholson, D.W. et al., Nature, 376, p. 37 (1995)), and CMH-1 (or MCH-3) (Lippke, et al., J. Biol. Chem., 0 (1996); Fernandes-Alnemri, T. et al., Cancer Res., (1995)). Each of these ICE homologs, as well as ICE itself, is capable of inducing apoptosis when overexpressed in transfected cell lines. Inhibition of one or more of these homologs with the peptidyl ICE inhibitor Tyr-Val-Ala-Asp-chloromethylketone results in inhibition of apoptosis in primary cells or cell lines.

Lazebnik et al., Nature, 371, p. 346 (1994). The compounds described herein are also capable of inhibiting one or more homologs of ICE (see Example Therefore, these compounds may be used to inhibit apoptosis in tissue types that contain ICE homologs, but which do not contain active ICE or produce mature Interferon-gamma inducing factor (IGIF) is an approximately 18-kDa polypeptide that stimulates T-cell production of interferon-gamma (IFN-y). IGIF is 6 produced by activated Kupffer cells and macrophages in vivo and is exported out of such cells upon endotoxin stimulation. Thus, a compound that decreases IGIF production would be useful as an inhibitor of such Tcell stimulation which in turn would reduce the levels of IFN-y production by those cells.

IFN-y is a cytokine with immunomodulatory effects on a variety of immune cells. In particular, IFN-y is involved in macrophage activation and Thl cell 0@ 10 selection Belardelli, APMIS, 103, p. 161 (1995)).

IFN-y exerts its effects in part by modulating the expression of genes through the STAT and IRF pathways Schindler and J.E. Darnell, Ann. Rev. Biochem., 64, p. 621 (1995); T. Taniguchi, J. Cancer Res. Clin.

Oncol., 121, p. 516 (1995)).

Mice lacking IFN-y or its receptor have multiple defects in immune cell function and are resistant to endotoxic shock Huang et al., Science, 259, p. 1742 (1993); D. Dalton et al., Science, 259, p. 1739 (1993); B. D. Car et al., J. Exp. Med., 179, p. 1437 (1994)). Along with IL-12, IGIF appears to be a potent inducer of IFN-y production by T cells (H.

Okamura et al., Infection and Immunity, 63, p. 3966 (1995); H. Okamura et al., Nature, 378, p. 88 (1995); S. Ushio et al., J. Immunol., 156, p. 4274 (1996)).

IFN-Y has been shown to contribute to the pathology associated with a variety of inflammatory, infectious and autoimmune disorders and diseases.

Thus, compounds capable of decreasing IFN-y production would be useful to ameliorate the effects of IFN-Y related pathologies.

The biological regulation of IGIF and thus IFN-y has not been elucidated. It is known that IGIF 7 is synthesized as a precursor protein, called "pro- IGIF". It has been unclear, however, how pro-IGIF is cleaved and whether its processing has biological importance.

Accordingly, compositions and methods capable of regulating the conversion of pro-IGIF to IGIF would be useful for decreasing IGIF and IFN-y production in vivo, and thus for ameliorating the detrimental effects of these proteins which contribute to human disorders and diseases.

However, ICE and other members of the ICE/CED-3 family have not previously been linked to the conversion of pro-IGIF to IGIF or to IFN-y production in vivo.

ICE inhibitors represent a class of compounds useful for the control of inflammation or apoptosis or both. Peptide and peptidyl inhibitors of ICE have been described. PCT patent applications WO 91/15577; WO 93/05071; WO 93/09135; WO 93/14777 and WO 93/16710; and 0* European patent application 0 547 699. Such peptidyl inhibitors of ICE has been observed to block the production of mature IL-10 in a mouse model of inflammation (vide infra) and to suppress growth of leukemia cells in vitro (Estrov et al., Blood 84, 380a (1994)). However, due to their peptidic nature, such inhibitors are typically characterized by undesirable pharmacologic properties, such as poor cellular penetration and cellular activity, poor oral absorption, poor stability and rapid metabolism.

Plattner, J.J. and D.W. Norbeck, in Drug Discovery Technologies, C.R. Clark and W.H. Moos, Eds. (Ellis Horwood, Chichester, England, 1990), pp. 92-126. This has hampered their development into effective drugs.

8 Non-peptidyl compounds have also been reported to inhibit ICE in vitro. PCT patent application WO 95/26958; US Patents 5,552,400; Dolle et al., J. Med. Chem., 39, pp. 2438-2440 (1996); However, it is not clear whether these compounds have the appropriate pharmacological profile to be therapeutically useful.

Additionally, current methods for the preparation of such compounds are not advantageous.

10 These methods use tributyltin hydride, a toxic, moisture sensitive reagent. Thus, these methods are inconvenient to carry out, pose a health risk and create toxic-waste disposal problems. Furthermore, it is difficult to purify compounds prepared by these methods.

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Accordingly, the need exists for compounds that can effectively inhibit the action of ICE in vivo, for use as agents for preventing and treating chronic and acute forms of IL-1-mediated diseases, apoptosis-, IGIF-, or IFN-y-mediated diseases, as well as inflammatory, autoimmune, destructive bone, proliferative, infectious, or degenerative diseases.

The need also exists for methods of preparing such compounds.

SUMMARY OF THE INVENTION The present invention provides novel classes of compounds, and pharmaceutically acceptable derivatives thereof, that are useful as inhibitors of ICE. These compounds can be used alone or in combination with other therapeutic or prophylactic agents, such as antibiotics, immunomodulators or other anti-inflammatory agents, for the treatment or prophylaxis of diseases mediated by IL-1, apoptosis, 9 IGIF or IFN-y. According to a preferred embodiment, the compounds of this invention are capable of binding to the active site of ICE and inhibiting the activity of that enzyme. Additionally, they have improved cellular potency, improved pharmacokinetics, and/or improved oral bioavailability compared to peptidyl ICE inhibitors.

S C 6O 0 *0 0 6 It is a principal object of this invention to provide novel classes of compounds which are inhibitors of ICE represented by formula: 0 (m OR 1 3

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wherein the various substituents are described herein.

It is a further object of this invention to provide a process of preparing N-acylamino compounds by coupling a carboxylic acid with an alloc-protected amine.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1A ICE cleaves pro-IGIF in vivo. Cell lysates from Cos cells transfected with the various indicated expression plasmids or controls were analyzed for the presence of IGIF by separating proteins by SDS-PAGE and immunoblotting with anti-IGIF antisera (lane 1, mock transfected cells; lane 2, pro-IGIF alone; lanes 3-12, pro-IGIF in combination with ICE, ICE-C285S, CPP32, CPP32-C163S, CMH-1, CMH-1-C186S, Tx, Tx-C258S, respectively). Mobilities of pro-IGIF and the 18-kDa mature IGIF are indicated on the right. Molecular weight markers in kDa are shown on the left (Example 23).

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0@0e 0* *0 Fig. 1B ICE cleaves pro-IGIF at the authentic processing site in vitro as shown by Coomassie blue staining of proteolytic reaction products separated by SDS-PAGE (Example 23). The proteases and inhibitors used were: lane 1, buffer control; lane 2, 0.1 nM ICE; lane 3, i nM ICE; lanes 4 and 5, 1 nM ICE with 10 nM Cbz-Val-Ala-Asp-[(2,6-dichlorobenzoyl)oxy]methyl ketone and 100 nM Ac-Tyr-Val-Ala-Asp-aldehyde, respectively; lanes 6 and 7, 15 nM CPP32 with and without 400 nM 0 Ac-Asp-Glu-Val-Asp-aldehyde W. Nicholson et al., Nature, 376, p. 37 (1995)), respectively; lane 8, 100 nM CMH-1; lane 9, 10 units/ml granzyme B; and M, molecular weight markers in kDa.

Fig. 1C ICE cleavage converts inactive pro-IGIF to 5 active IGIF which induces IFN-y production in Thl helper cells. Uncleaved (Pro-IGIF), ICE-cleaved (Pro- IGIF/ICE), CPP32-cleaved (Pro-IGIF/CPP32), and recombinant mature IGIF (rIGIF) were incubated with A.E7 Thl cells at 12 ng/ml (open bar) and 120 ng/ml '0 (hatched bar) for eighteen hours and the levels of IFNy released into the culture medium assayed by ELISA (Example 23). A.E7 cells cultured with buffer, ICE alone (ICE) or CPP32 alone (CPP32) were assayed similarly for negative controls. The numbers represent the average of three determinations.

Fig. 2A Mature IGIF (18-kDa) is produced by Cos cells co-transfected with pro-IGIF and ICE-expressing plasmids. Cell lysates (left) and conditioned medium (right) from Cos cells transfected with a pro-IGIF ;0 expression plasmid in the absence or presence of an expression plasmid encoding wild type (ICE) or inactive mutant (ICE-C285S) ICE. Transfected cells were metabolically labeled with 3 "S-methionine, proteins from

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55 11 cell lysates and conditioned medium immunoprecipitated with anti-IGIF antisera and separated by SDS-PAGE (Example 24). Mobilities of pro-IGIF and the 18-kDa mature IGIF are indicated on the right. Molecular weight markers in kDa are shown on the left.

Fig. 2B IFN-y inducing activity is detected in Cos cells co-transfected with pro-IGIF and ICE-expressing plasmids. Cell lysates (hatched bar) and conditioned medium (open bar) from Cos cells transfected with a 10 pro-IGIF expression plasmid in the absence (Pro-IGIF) or presence (Pro-IGIF/ICE) of an expression plasmid encoding wild type (ICE) were assayed for IFN-y levels (ng/ml) by ELISA. Cos cells transfected with buffer (Mock) or an ICE-expressing plasmid alone (ICE) served as negative controls (Example 24).

Fig. 3A Kupffer cells from mice lacking ICE are defective in the export of IGIF. Kupffer cells from eeO wild type mice (ICE or ICE-deficient mice e00* homozygous for an ICE mutation were isolated and primed with LPS for three hours. The levels of immunoreactive IGIF polypeptides in the conditioned media (ng/ml) of wild type cells were measured by ELISA (Example 25). N.D. (not detectable) indicates that the IGIF concentration was less than 0.1 ng/ml.

Fig. 3B Kupffer cells from mice lacking ICE are defective in the export of mature IGIF. Kupffer cells from wild type mice (ICE or ICE deficient mice homozygous for an ICE mutation (ICE were isolated and primed with LPS for three hours. Primed cells were metabolically labeled with 35 S-methionine, proteins from cell lysates and conditioned medium immunoprecipitated with anti-IGIF antisera and separated by SDS-PAGE (Example 25). Mobilities of pro-IGIF and the 18-kDa 12 mature IGIF are indicated on the right. Molecular mass markers in kDa are shown on the left.

Fig. 3C Serum from ICE-deficient mice contains reduced levels of IGIF. Serum samples from wild type mice (ICE or ICE deficient mice homozygous for an ICE mutation (ICE were assayed for IGIF levels (ng/ml) by ELISA (Example Fig. 3D Serum from ICE-deficient mice contains reduced levels of IFN-y. Serum samples from wild type 1 0 mice (ICE or ICE deficient mice homozygous for an

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ICE mutation (ICE were assayed for IFN-y levels (ng/ml) by ELISA (Example S' Fig. 4 Serum IFN-y levels are significantly reduced in ICE-deficient mice after an acute challenge with LPS (Example 26). Serum samples from wild type mice (filled squares) or ICE-deficient mice (filled circles) were assayed for IFN-y levels (ng/ml) by ELISA as a function of time (hours) after LPS challenge.

Temperatures of the animals during the time course in degrees Celcius is shown for wild type mice (open squares) or ICE-deficient mice (open circles).

Fig. 5 The ICE inhibitor, AcYVAD-aldehyde (AcYVAD- CHO), inhibits LPS-stimulated IL-13 and IFN-y synthesis by human peripheral blood mononuclear cells (PBMC).

Percent inhibition as a function of inhibitor concentration (uM) is shown for IL-18 (open squares) and IFN-y (open diamonds) synthesis.

Fig. 6 Compound 214e inhibits IL-1p production in LPS-challenged mice. Serum samples from CD1 mice were assayed for IL-1 levels (pg/ml) by ELISA after LPS challenge. Compound 214e was administered by intraperitoneal (IP) injection one hour after LPS challenge. Blood was collected seven hours after LPS 13 challenge (see Example 7).

Fig. 7 Compound 217e inhibits IL-1S production in LPS-challenged mice. Serum samples from CD1 mice were assayed for IL-1P levels (pg/ml) by ELISA after LPS challenge. Compound 217e was administered by intraperitoneal (IP) injection one hour after LPS challenge. Blood was collected seven hours after LPS challenge (see Example 7) Fig. 8 Compound 214e, but not compound 217e, 10 inhibits IL-13 production in LPS-challenged mice when 0 administered by oral gavage. This assay measures oral S. absorption under similar conditions as those described for Figs. 6 and 7. These results indicates that 214e is potentially orally active as an ICE inhibitor (see Example 7).

Fig. 9 Compound 214e and analogs of 214e also inhibit IL-1P production after IP administration.

@see These results were obtained in the assay described for Figs. 6 and 7 and Example 7.

Fig. 10 Compound 214e, and analogs of 214e, also inhibit IL-13 production after oral (PO) administration. These results were obtained in the 9 0 assay described for Figs. 6 and 7 and Example 7.

Figs. 11A/B Compounds 302 and 304a show detectable blood levels when administered orally (50mg/kg, in carboxymethylcellulose) to mice. Blood samples were collected at 1 and 7 hours after dosing.

Compounds 302 and 304a are prodrugs of 214e and are metabolized to 214e in vivo. Compound 214e shows no blood levels above 0.10 pg/ml when administered orally (Example 8).

Fig. 12 Compound 412f blocks the progression of type II collagen-induced arthritis in male DBA/1J mice 14 @0 @0 0 00 S 0 0 *s S 0

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0 @5 (Wooley, Methods in Enzvmoloqy, 162, pp. 361-373 (1988) and Geiger, Clinical and Experimental Rheumatology, 11, pp. 515-522 (1993)). Compound 412f was administered twice a day (10, 25 and approximately 7h apart, by oral gavage. Inflammation was measured on the Arthritis Severity Score on a 1 to 4 scale of increasing severity. The scores of the two front paws were added to give the final score (see Example 21).

10 Fig. 13 Compound 412d blocks the progression of type II collagen-induced arthritis in male DBA/1J mice. The results were obtained as described for Fig. 12 and in Example 21.

Fig. 14 Compound 696a blocks the progression of type 15 II collagen-induced arthritis in male DBA/1J mice. The results were obtained as described for Fig. 12 and in Example 21.

ABBREVIATIONS AND DEFINITIONS 0 *c 6

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0@ S 0 0 Designation Ala Arg Asn Asp Cys Gln Glu Gly His Ile Leu Lys Met Abbreviations Reagent or Fragment alanine arginine asparagine aspartic acid cysteine glutamine glutamic acid glycine histidine isoleucine leucine lysine methionine 15 S@ 0 0 0@ 0

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0 0@@ 000 Phe phenylalanine Pro proline Ser serine Thr threonine Trp tryptophan Tyr tyrosine Val valine Ac20 acetic anhydride n-Bu normal-butyl DMF dimethylformamide DIEA N,N-diisopropylethylamine EDC 1-(3-Dimethylaminopropyl)-3ethylcarbodiimide hydrochloride Et 2 0 diethyl ether EtOAc ethyl acetate Fmoc 9-fluorenylmethyoxycarbonyl HBTU O-benzotriazol-l-yl-N,N,N' N' tetramethyluronium hexafluorophosphate HOBT 1-hydroxybenzotriazole hydrate MeOH methanol TFA trifluoroacetic acid Alloc allyloxycarbonyl Definitions The following terms are employed herein: The term "interferon gamma inducing factor" or "IGIF" refers to a factor which is capable of stimulating the endogenous production of IFN-y.

The term "ICE inhibitor" refers to a compound which is capable of inhibiting the ICE enzyme. ICE inhibition may be determined using the methods described and incorporated by reference herein. The skilled practitioner realizes that an in vivo ICE 00

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C0 S. 0 00 0@ 16inhibitor is not necessarily an in vitro ICE inhibitor.

For example, a prodrug form of a compound typically demonstrates little or no activity in in vitro assays.

Such prodrug forms may be altered by metabolic or other biochemical processes in the patient to provide an in vivo ICE inhibitor.

The term "cytokine" refers to a molecule which mediates interactions between cells.

Se The term "condition" refers to any disease, 10 disorder or effect that produces deleterious biological eeee consequences in a subject.

"The term "subject" refers to an animal, or to one or more cells derived from an animal. Preferably, the animal is a mammal, most preferably a human. Cells 15 may be in any form, including but not limited to cells retained in tissue, cell clusters, immortalized cells, 0@SO transfected or transformed cells, and cells derived from an animal that have been physically or phenotypically altered.

The term "active site" refers to any or all of the following sites in ICE: the substrate binding site, the site where an inhibitor binds and the site where the cleavage of substrate occurs.

The term "heterocycle" or "heterocyclic" refers to a stable mono- or polycyclic compound which may optionally contain one or two double bonds or may optionally contain one or more aromatic rings. Each heterocycle consists of carbon atoms and from one to four heteroatoms independently selected from a group including nitrogen, oxygen, and sulfur. As used herein, the terms "nitrogen heteroatoms" and "Sulphur heteroatoms" include any oxidized form of nitrogen or sulfur and the quaternized form of any basic nitrogen.

17 Heterocycles defined above include, for example, pyrimidinyl, tetrahydroquinolyl, tetrahydroisoquinonlinyl, purinyl, pyrimidyl, indolinyl, benzimidazolyl, imidazolyl, imidazolinoyl, imidazolidinyl, quinolyl, isoquinolyl, indolyl, pyridyl, pyrrolyl, pyrrolinyl, pyrazolyl, pyrazinyl, quinoxolyl, piperidinyl, morpholinyl, thiamorpholinyl, furyl, thienyl, triazolyl, thiazolyl, 3-carbolinyl, tetrazolyl, thiazolidinyl, benzofuranoyl, 10 thiamorpholinyl sulfone, benzoxazolyl, oxopiperidinyl, oxopyrroldinyl, oxoazepinyl, azepinyl, isoxazolyl, tetrahydropyranyl, tetrahydrofuranyl, thiadiazolyl, benzodioxolyl, benzothienyl, tetrahydrothiophenyl and sulfolanyl. Further heterocycles are described in A.R.

15 Katritzky and C.W. Rees, eds., Comprehensive Heterocyclic Chemistry: The Structure, Reactions, 0 Synthesis and Use of Heterocyclic Compounds, Vol. 1-8, Pergamon Press, NY (1984).

The term "cycloalkyl" refers to a mono- or polycyclic group which contains 3 to 15 carbons and may optionally contain one or two double bonds. Examples include cyclohexyl, adamantyl and norbornyl.

The term "aryl" refers to a mono- or polycyclic group which contains 6, 10, 12, or 14 carbons in which at least one ring is aromatic.

Examples include phenyl, naphthyl, and tetrahydronaphthalene.

The term "heteroaromatic" refers to a monoor polycyclic group which contains 1 to 15 carbon atoms and from 1 to 4 heteroatoms, each of which is selected independently from a group including sulphur, nitrogen and oxygen, and which additionally contains from 1 to 3 five or six membered rings, at least one of which is 18 aromatic.

The term "alpha-amino acid" (c-amino acid) refers to both the naturally occurring amino acids and other "non-protein" a-amino acids commonly utilized by those in the peptide chemistry arts when preparing synthetic analogues of naturally occurring peptides, including D and L forms. The naturally occurring amino acids are glycine, alanine, valine, leucine, iso- .ee. 10 leucine, serine, methionine, threonine, phenylalanine, 10 tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, ycarboxyglutamic acid, arginine, ornithine and lysine.

Examples of "non-protein" alpha-amino acids include hydroxylysine, homoserine, homotyrosine, homo- 15 phenylalanine, citrulline, kynurenine, 4-aminophenylalanine, 3- (2-naphthyl) -alanine, 3- (1-naphthyl)alanine, methionine sulfone, t-butyl-alanine, 0O55 ;t-butylglycine, 4-hydroxyphenyiglycine, aminoalanine, phenylglycine, vinylalanine, propargyl-glycine, 1,2,4-triazolo-3-alanine, 4,4,4-trifluoro-threonine, OSthyronine, 6-hydroxytryptophan, 3-hydroxykynurenine, 3-aminotyrosine, trifuoromethylalanine, 2-thienylalanine, (4-pyridyl) ethyl)cysteine, 3,4-dimethoxy-phenylalanine, 3-(2-thiazolyl)alanine, ibotenic acid, 1-amino-l-cyclopentanecarboxylic acid, l-amino-1-cyclohexanecarboxylic acid, quisqualic acid, 3-trifluoromethylphenylalanine, 4-trifluoro-methylphenylalanine, cyclohexylalanine, cyclo-hexylglycine, thiohistidine, 3-methoxytyrosine, elastatinal, norleucine, norvaline, alloisoleucine, homoarginine, thioproline, dehydroproline, hydroxyproline, isonipectotic acid, homoproline, cyclohexylglycine, c-amino-n-butyric acid, cyclohexylalanine, 19 aminophenylbutyric acid, phenylalanines substituted at the ortho, meta, or para position of the phenyl moiety with one or two of the following: a (C 1

-C

4 alkyl, a

(C

1

-C

4 alkoxy, halogen or nitro groups or substituted with a methylenedioxy group; 3-2- and 3-thienylalanine, 3-2- and 3-furanylalanine, 3- and 4-pyridylalanine, 3-(benzothienyl-2- and 3-yl)alanine, and 2-naphthyl)alanine, O-alkylated derivatives of serine, threonine or tyrosine, S-alkylated cysteine, S-alkylated homocysteine, O-sulfate, O-phosphate and Ocarboxylate esters of tyrosine, 3-sulfo-tyrosine, 3carboxy-tyrosine, 3-phospho-tyrosine, 4-methane sulfonic acid ester of tyrosine, 4-methane phosphonic acid ester of tyrosine, 3,5-diiodotyrosine, 3-nitro- 15 tyrosine, e-alkyl lysine, and delta-alkyl ornithine.

Any of these a-amino acids may be substituted with a sees methyl group at the alpha position, a halogen at any aromatic residue on the a-amino side chain, or an appropriate protective group at the O, N, or S atoms of the side chain residues. Appropriate protective groups are disclosed in "Protective Groups In Organic Synthesis," T.W. Greene and P.G.M. Wuts, J. Wiley Sons, NY, NY, 1991.

The term "substitute" refers to the replacement of a hydrogen atom in a compound with a substituent group. In the present invention, those hydrogen atoms which form a part of a hydrogen bonding moiety which is capable of forming a hydrogen bond with the carbonyl oxygen of Arg-341 of ICE or the carbonyl oxygen of Ser-339 of ICE are excluded from substitution. These excluded hydrogen atoms include those which comprise an -NH- group which is alpha to a -CO- group and are depicted as -NH- rather than an X 20 group or some other designation in the following diagrams: through through The term "straight chain" refers to a contiguous unbranching string of covalently bound atoms. The straight chain may be substituted, but these substituents are not a part of the straight *o chain.

The term "Ki" refers to a numerical measure of the effectiveness of a compound in inhibiting the 10 activity of a target enzyme such as ICE. Lower values ,o of K i reflect higher effectiveness. The K i value is a o derived by fitting experimentally determined rate data to standard enzyme kinetic equations (see I. H. Segel, Enzyme Kinetics, Wiley-Interscience, 1975).

15 The term "patient" as used in this application refers to any mammal, especially humans.

The term "pharmaceutically effective amount" refers to an amount effective in treating or ameliorating an IL-1-, apoptosis-, IGIF- or IFN-ymediated disease in a patient. The term "prophylactically effective amount" refers to an amount effective in preventing or substantially lessening IL-1-, apoptosis-, IGIF or IFN-y mediated diseases in a patient.

The term "pharmaceutically acceptable carrier or adjuvant" refers to a non-toxic carrier or adjuvant that may be administered to a patient, together with a compound of this invention, and which does not destroy the pharmacological activity thereof.

The term "pharmaceutically acceptable derivative" means any pharmaceutically acceptable salt, ester, or salt of such ester, of a compound of this invention or any other compound which, upon 21 0* 0S eoeoee

S

S

S..

administration to a recipient, is capable of providing (directly or indirectly) a compound of this invention or an anti-ICE active metabolite or residue thereof.

Pharmaceutically acceptable salts of the compounds of this invention include, for example, those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acids include hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic and benzenesulfonic acids. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, 15 may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts. Salts derived from appropriate bases include alkali metal sodium), alkaline earth metal magnesium), ammonium and N-(C 1 4 alkyl)4+ salts.

00*0 0 0 0000 0 0 0 0eSS 0e 0 0

OS

00 S 0* 0 This invention also envisions the "quaternization" of any basic nitrogen-containing groups of the compounds disclosed herein. The basic nitrogen can be quaternized with any agents known to those of ordinary skill in the art including, for example, lower alkyl halides, such as methyl, ethyl, propyl and butyl chloride, bromides and iodides; dialkyl sulfates including dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; and aralkyl halides including benzyl and phenethyl bromides. Water or oil-soluble or 22 dispersible products may be obtained by such quaternization.

The ICE inhibitors of this invention may contain one or more "asymmetric" carbon atoms and thus may occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual S* diastereomers. All such isomeric forms of these compounds are expressly included in the present invention. Each stereogenic carbon may be of the R or 10 S configuration. Although specific compounds and scaffolds exemplified in this application may be s" depicted in a particular stereochemical configuration, compounds and scaffolds having either the opposite stereochemistry at any given chiral center or mixtures 15 thereof are also envisioned.

The ICE inhibitors of this invention may comprise ring structures which may optionally be substituted at carbon, nitrogen or other atoms by various substituents. Such ring structures may be singly or multiply substituted. Preferably, the ring structures contain between 0 and 3 substituents. When multiply substituted, each substituent may be picked independently of any other substituent as long as the combination of substituents results in the formation of a stable compound.

Combinations of substituents and variables envisioned by this invention are only those that result in the formation of stable compounds. The term "stable", as used herein, refers to compounds which possess stability sufficient to allow manufacture and administration to a mammal by methods known in the art.

Typically, such compounds are stable at a temperature of 40 0 C or less, in the absence of moisture or other 23 chemically reactive conditions, for at least a week.

Substituents may be represented in various forms. These various forms are known to the skilled practitioner and may be used interchangeably. For example, a methyl substituent on a phenyl ring may be represented in any of the following forms: 4u* c 00 0O a 0@ 0 0000 6 0#0*

H,

H C F13 Me

ZPI

Various forms of substituents such as methyl are used herein interchangeably.

DETAILED DESCRIPTION OF THE INVENTION In order that the invention herein described may be more fully understood, the following detailed description is set forth.

The ICE inhibitors of one embodiment C of this invention are those of formula (II): 0 (m OR 13 RT- R4

(II)

00 q 0 CS 0 a wherein: m is 1 or 2;

R

1 is: (el0)

R

3 is -CN, -C -CH 2

-T

1

-R

1 1

-C(O)-CH

2

-F,

-C=N-O-R

9 or -CO-Ar 2

R

5 is -C(O)-R 1 0

-C(O)O-R

9

-C(O)-N(R

1 0

(R

1 0 2

-R

9 -C(0)-CH 2

-O-R

9 C(0)-R 1 0

-R

9 or 24 -C(0)C(0)-OR 1 0

X

5 is -CH-

Y

2 is H 2 or O; each T 1 is independently or S* each R 9 is independently -Ar 3 or a -C1-6 straight or branched alkyl group optionally substituted with 10 Ar 3 wherein the -C1-6 alkyl group is optionally 00. unsaturated; each R 10 is independently -Ar 3 a C3-6 cycloalkyl group, or a -C 1 6 straight or branched alkyl group optionally substituted with Ar 3 wherein the -C 1 -6 alkyl group is optionally unsaturated; each R 11 is independently -Ar 4

-(CH

2 )1- 3 -Ar 4

-H,

or -C(O)-Ar 4

R

1 3 is H, Ar 3 or a C1-6 straight or branched alkyl group optionally substituted with Ar 3

-CONH

2

-OR

5 -OH, -OR 9 or -C0 2

H;

-OR

1 3 is optionally -N(H)-OH; each R 21 is independently -H or a -C 1 -6 straight or branched alkyl group; Ar 2 is independently selected from the following group, in which any ring may optionally be singly or multiply substituted by -Q 1 (hh) and 25 (ii)

Y

N D S. 0 5e 0@ 0S S

OS

0 @6 6 6 6000

S.

60

S

S

0 0O S 0O 0 0@ S. 0 0O *0 wherein each Y is independently 0 or S; each Ar 3 is a cyclic group independently selected from the set consisting of an aryl group which contains 6, 10, 12, or 14 carbon atoms and between 1 and 3 rings and an aromatic heterocycle group containing between and 15 ring atoms and between 1 and 3 rings, said heterocyclic group containing at least one heteroatom 10 group selected from SO 2 and -NH-,

-N(R

5 and said heterocycle group optionally containing one or more double bonds, said heterocycle group optionally comprising one or more aromatic rings, and said cyclic group optionally being singly or 15 multiply substituted by -Q

I

each Ar 4 is a cyclic group independently selected from the set consisting of an aryl group which contains 6, 10, 12, or 14 carbon atoms and between 1 and 3 rings, and a heterocycle group containing between 5 and 20 15 ring atoms and between 1 and 3 rings, said heterocyclic group containing at least one heteroatom group selected from SO 2

-NH-,

-N(R

5 and -N(R 9 said heterocycle group optionally containing one or more double bonds, said heterocycle group optionally comprising one or more aromatic rings, and said cyclic group optionally being singly or multiply substituted by -Q 1 each Q 1 is independently -NH 2 -C02H, -Cl, -Br,

-NO

2 -CN, -OH, -perfluoro C1- 3 alkyl, R 5

-OR

5

-NHR

5

OR

9

-NHR

9

R

9

-C(O)-R

1 0 or 26

CH

2 provided that when -Ar 3 is substituted with a Q1 group which comprises one or more additional -Ar 3 with another -Ar 3 Preferred compounds of this embodiment 10 include, but are not limited to compounds 220b, 223b, 223e, 226e, 227e, 308a, 308b, and 429.

In more preferred compounds of embodiment C,

SR

3 is CO-Ar 2 Alternatively, R 3 is -C(O)-CH 2

-T

1

-R

1 1 and

R

11 is -(CH 2 )1- 3 -Ar 4 Alternatively, R 3 is -C(O)-CH 2

T

1

-R

11

T

1 is 0, and R 11 is -C(O)-Ar 4 Alternatively,

R

3 is Alternatively, R 3 is -CO-CH 2

-T

1

-R

1 1 and

R

11 is -Ar 4 o* More preferably, in these more preferred compounds, R 5 is -C(O)-R 1 0 -C(0)O-R 9 or -C(O)-NH-R 1 0 20 Alternatively, in these more preferred compounds, R 5 is -S(0) 2

-R

9 -S(0) 2

-NH-R

1 0

-R

1 0

-R

9 or C(0)-OR 1 0 Most preferably, in these more preferred compounds: m is 1;

T

1 is O or S;

R

13 is H or a -C1-4 straight or branched alkyl group optionally substituted with -Ar 3 -OH, -OR 9 or

-CO

2 H, wherein the R 9 is a -C1.4 branched or straight alkyl group, wherein Ar 3 is morpholinyl or phenyl, wherein the phenyl is optionally substituted with Q 1

R

21 is -H or -CH 3 Ar 2 is (hh); Y is 0; and 27 Ar 3 is phenyl, naphthyl, thienyl, quinolinyl, isoquinolinyl, pyrazolyl, thiazolyl, isoxazolyl, benzotriazolyl, benzimidazolyl, thienothienyl, imidazolyl, thiadiazolyl, benzo[b]thiophenyl, pyridyl benzofuranyl, or indolyl; Ar 4 is phenyl, tetrazolyl, pyridinyl, oxazolyl, naphthyl, pyrimidinyl, or thienyl; each Q 1 is independently -NH 2 -Cl, -Br, -OH,

-R

9

-NH-R

5 wherein R 5 is -C(O)-R 10 or -S(0) 2

-R

9

-OR

0 wherein R 5 is -C(O)-R 10

-OR

9

-NHR

9 or 0 0O S

SO

SO

0O S S 0000 0O 05 5*

CH

2

S

0 0650 065O 06 5* 0 wherein each R 9 and R10 are independently a -C1-6 straight or branched alkyl group optionally substituted with Ar 3 wherein Ar 3 is phenyl; provided that when -Ar 3 is substituted with a Q1 group which comprises one or more additional -Ar 3 groups, said additional -Ar 3 groups are not substituted 0B 0 0O 5O 0

S.

S.

with another -Ar 3 Preferred but are not limited 246, 257, 280, 281, 404, 405, 406, 407, 416, 417, 418, 419, 431, 432, 433, 434, 442, 443, 444, 445, 453, 454, 455, 456, 464, 465, 466, 467, 475, 476, 477, 478, 483, 484, 485, 486, 495, 496, 497, 498, compounds of embodiment C include, to 214c, 214e, 217c, 217d, 217e, 282, 283, 284, 285, 286, 287, 302, 408, 409, 410, 411, 412, 413, 415, 420, 422, 423, 424, 425, 426, 430, 435, 436, 437, 438, 439, 440, 441, 446, 447, 448, 449, 450, 451, 452, 457, 458, 459, 460, 461, 462, 463, 468, 469, 470, 471, 472, 473, 474, 479, 480, 481, 481s, 482, 482s, 487, 488, 489, 490, 491, 493, 494, 499, 421, 427, and 428.

28 The ICE inhibitors of another embodiment E of this invention are those of formula (II): 0 m R

R

1 -N R 3 (I 0* 0 00 ae 0 0* 6O 0 000000 0 00 0 000* 00 0 000 0 006000

U

Oge.

0 *000 0000

OG

0 00 0 0 00 6* 0 00 00 5 wherein: each R 5 is -C (0)-R 1 0 -CO(0)O-R 9 -C (R 1 0

(R

1 0 -S 2

-R

9 -S 2 -NH-Rl 0 -c (0)-CH 2

-O-R

9 -C (0)0 (0)-Rl 1 0

-R

9 -C0(0)C0) -OR 1 0 or -C C(0)-N (R 9

(R

1 0

R

1 5 is -OH, -OAr 3 or a -001-6 straight 10 or branched alkyl group optionally substituted with -Ar 3

-CONH

2

-OR

5 -OH, -OR 9 or -C0 2

H;

each Q, is independently -NH 2 -C0 2 H, -01, -Br,

-NO

2 -ON, -OH, -perfluoro C1-3 alkyl, R 5

-OR

5

-NHR

5

OR

9

-N(R

9 (Rjo), R 9

-C(O)-R

1 0 or 15 0

OH

2 0 provided that when -Ar 3 is substituted with a Q group which comprises one or more additional -Ar 3 groups, said additional -Ar 3 groups are not substituted with another -Ar 3 and the other substituents are as described above in embodiment In more preferred compounds of embodiment E,

R

3 is 00-Ar 2 Alternatively, R 3 is -O(O)-0H 2

-T

1 -Rll and

R

1 1 is -(0H 2 1 3 -Ar 4 Alternatively, R 3 is -0(O)-OH 2

T

1 -Rjj, T, is 0, and R 1 1 is -0(O)-Ar 4 Alternatively,

R

3 is Alternatively, R 3 is -OO-0H 2 -Tj-Rjj and

R

11 is -Ar 4 More preferably, in these more preferred compounds, R 5 is -O(O)-R 10

-C(O)O-R

9 or -C(O)-NH-Rl 0 29 Alternatively, in these more preferred compounds, R 5 is -S(0) 2

-R

9 -S(0) 2

-NH-R

1 0 1 0

-R

9 or C(0)-OR 1 0 Most preferably, in these more preferred compounds: m is 1;

T

1 is O or S (except as defined above);

R

15 is -OH or -OC_14 straight or branched alkyl group optionally substituted with -Ar 3 -OH, -OR 9 or 10 -CO 2 H, wherein the R 9 is a -C 1 4 branched or straight alkyl group, wherein Ar 3 is morpholinyl or phenyl, wherein the phenyl is optionally substituted with Q 1

R

21 is -H or -CH 3 Ar 2 is (hh); Y is 0; and each Ar 3 cyclic group is independently phenyl, naphthyl, thienyl, quinolinyl, isoquinolinyl, pyrazolyl, thiazolyl, isoxazolyl, benzotriazolyl, benzimidazolyl, thienothienyl, imidazolyl, thiadiazolyl, benzo[b]thiophenyl, pyridyl, benzofuranyl, or indolyl, and said cyclic group optionally being singly or multiply substituted by -Q

I

each Ar 4 cyclic group is independently phenyl, tetrazolyl, pyridinyl, oxazolyl, naphthyl, pyrimidinyl, or thienyl, said cyclic group being singly or multiply substituted by -Q

I

each Q 1 is independently -NH 2 -Cl, -Br, -OH,

-R

9

-NH-R

5 wherein R 5 is -C(O)-R 1 0 or -S(0) 2

-R

9

-OR

wherein R 5 is -C(O)-R 1 0

-OR

9

-N(R

9

(R

1 0 or

CH

2

O

30 wherein each R 9 and R 10 are independently a -Cg6 straight or branched alkyl group optionally substituted with Ar 3 wherein Ar 3 is phenyl; provided that when -Ar 3 is substituted with a Q1 group which comprises one or more additional -Ar 3 groups, said additional -Ar 3 groups are not substituted with another -Ar 3 The ICE inhibitors of another embodiment H of this invention are those of formula wherein: 10 R 21 is -CH 3 Ar 2 is independently selected from the following group, in which any ring may optionally be singly or multiply substituted by -Q 1 or phenyl, optionally substituted by -Q 1 15 o 20 *o 20 Y(hh) and (ii)

Y

N

wherein each Y is independently O or S; and the other substituents are as defined above in embodiment E.

Compounds of another form of embodiment I (form 1) are those of formula wherein: each R 5 is -C(O)C(O)-OR 10 Ar 2 is as defined above in embodiment H; and the other substituents are as defined above in embodiment E. Alternatively, compounds of this form of embodiment I (form 2) are those wherein R 21 is -CH 3 Compounds of another form of embodiment J (form 1) are those of formula wherein: Ar 2 is as defined above in embodiment H; and 31 the other substituents are as described above in embodiment E; provided that when: m is 1;

R

15 is -OH;

R

21 is and Y~2 is 0 and R 3 is then R 5 cannot be: -C(O)-Rj 0 wherein R 10 is -Ar 3 and the Ar 3 cyclic group is phenyl, unsubstituted by -Q 1 4- :10 (carboxymethoxy)phenyl,. 2-fluorophenyl, 2-pyridyl, N- (4-methylpiperazino)methylphenyl, or -C(O)-0R 9 wherein R 9 is -CH -Ar 3 and the Ar 3 cyclic group is phenyl, unsubstituted by -Qj; and when Y'2 is 0, R 3 is 2

-T

1 -Rll, T, is 0, and R 1 is 1 is Ar 4 wherein the Ar 4 cyclic group is S....'chlorophenyl)-3-trifluoromethyl)pyrazolyl), then R cannot be: -C(O)-Rj 0 wherein R 10 is -Ar 3 and the Ar 3 cyclic group is 4-(dimethylaminomethyl)phenyl, phenyl, 4- 00 20 (carboxymethylthio)phenyl, 4- (carboxyethylthio)phenyl, 4- (carboxyethyl)phenyl, 4- (carboxypropyl)phenyl, 2fluorophenyl, 2-pyridyl, N-(4methylpiperazino) methyiphenyl, or -C(O)-0R 9 wherein R 9 is -CH 2 -Ar 3 and the Ar 3 cyclic group is phenyl; and when R 1 is Ar 4 wherein the Ar 4 cyclic group is 5- (l-phenyl-3-trifluoromethyl)pyrazolyl), then R cannot be: -C(0)-0R 9 wherein R 9 is -CH 2 -Ar 3 and the Ar 3 cyclic group is phenyl; and when R 1 is Ar 4 wherein the Ar 4 cyclic group 32 is 5- (1-(2-pyricyl)-3-trifluoromethyl)pyrazolyl), then

R

5 cannot be:

-C(O)-R

10 wherein R 10 is -Ar 3 and the Ar 3 cyclic group is 4-(dimethylaminomethyl)phenyl, or -C(0)-0R 9 wherein R 9 is -CH 2 -Ar 3 and the Ar 3 cyclic group is phenyl, unsubstituted by -Qj; and when es go @0 0* 0* 0 *ee.os 0

S

10

OS

0 000 0 15 0000 0 *000 0 0 0S 0

S.

S S 0* Y~2 is 0, R 3 is -C(O)-CH 2

-T

1 -Rll, T, is 0, and R 1 is -C (0)-Ar 4 wherein the Ar 4 cyclic group is dichiorophenyl, then R 5 cannot be: -C(0)-R 10 wherein R 10 is -Ar 3 and the Ar 3 cyclic group is 4- (dimethylaminomethyl)phenyl, 4- (Nmorpholinomethyl)phenyl, 4- (Nrethylpiperazino)methyl)phenyl, 4- (2methyl) imidazolylmethyl)phenyl, 5-benzimidazolyl, benztriazolyl, N-carboethoxy-5-benztriazolyl, Ncarboethoxy-5-benzimidazolyl, or -C(0)-0OR 9 wherein R 9 is -CH 2 -Ar 3 and the Ar 3 cyclic group is phenyl, unsubstituted by -Qj; and when Y2 is H 2

R

3 is -C(0)-CH 2

-T

1 -Rll, T, is 0, and RI, 0 so -C(O)-Ar 4 wherein the Ar 4 cyclic group is dichiorophenyl, then R 5 cannot be: -C(O)-0R 9 wherein R 9 is -CH 2 -Ar 3 and the Ar 3 cyclic group is phenyl; Compounds of another form of embodiment J (form 2) are those wherein R 21 is -CH 3 Compounds of another form of embodiment J (form 3) are those wherein

R

5 is -C(O)-C(0)-0R 10 Compounds of another form of embodiment J (form 4) are those wherein R 5 is C(0)-0R 10 and R 2 1 is -CH 3 Preferred compounds of embodiments H, I, and 33 J employ formula wherein R 3 is -CO-Ar 2 More preferably, R 3 is -CO-Ar 2 and Y is O.

Preferred compounds of embodiments H, I, and J employ formula wherein R 3 is -C(O)-CH 2

-T

1

-R

1 1 and

R

11 is -(CH 2 )1_ 3 -Ar 4 More preferably, when R 3 is

-C(O)-CH

2

-T

1

-R

1 1 and R 11 is -(CH 2 )1- 3 -Ar 4

T

1 is O.

Preferred compounds of embodiments H, I, and S" J employ formula wherein R 3 is -C(O)-CH 2

-T

1

-R

1 1

T

1 is 0, and R 11 is -C(O)-Ar 4 10 Preferred compounds of embodiments H, I, and J employ formula wherein R 3 is Preferred compounds of embodiments H, I, and J employ formula wherein R 3 is -CO-CH 2

-T

1

-R

1 1 and

R

1 1 is -Ar 4 More preferably, when R 3 is -CO-CH 2

-T

1

-R

11 and R 11 is -Ar 4

T

1 is O or S.

More preferred compounds of embodiments H and J (forms 1 and 2) are those wherein R 5 is -C(O)-R 10 -C(0)O-R 9 or -C(O)-NH-R 1 0 Alternatively, R 5 is -S(0) 2

-R

9 -S(0) 2

-NH-R

1 0 1 0

-R

9

C(O)-OR

1 0 or 9

(R

1 0 Most preferably, R 5 is 1 0 Alternatively, R 5 is -C(O)-C(O)-ORI 0 More preferred compounds of embodiments H, I (form and J (forms 2 and 4) are those wherein: m is 1; Y2 is O;

R

15 is -OH or -OC 1 _4 straight or branched alkyl group optionally substituted with Ar 3 -OH, -OR 9

-CO

2

H,

wherein the R 9 is a C1- 4 branched or straight chain alkyl group; wherein Ar 3 is morpholinyl or phenyl, wherein the phenyl is optionally substituted with Q 1 Ar 2 is (hh); Y is 0, and 34 @0 0 6 6 0 6O 0000 0 0000

S

@00 0000 0 0 @0 4@ 1 1 each Ar 3 cyclic group is independently phenyl, naphthyl, thienyl, quinolinyl, isoquinolinyl, pyrazolyl, thiazolyl, isoxazolyl, benzotriazolyl, benzimidazolyl, thienothienyl, imidazolyl, thiadiazolyl, benzo[b]thiophenyl, pyridyl, benzofuranyl, or indolyl, and said cyclic group optionally being singly or multiply substituted by -Q 1 each Ar 4 cyclic group is independently phenyl, tetrazolyl, pyridyl, oxazolyl, naphthyl, pyrimidinyl, 0 or thienyl, and said cyclic group optionally being singly or multiply substituted by -Q 1 each Q 1 is independently -NH 2 -Cl, -Br, -OH,

-R

9

-NH-R

5 wherein R 5 is -C(O)-R 10 or -S(0) 2

-R

9

-OR

wherein R 5 is -C(O)-R 10

-OR

9

(R

1 0 and 5 0

CH

2 0 wherein each R 9 and R 10 are independently a -C1-6 straight or branched alkyl group optionally substituted with Ar 3 wherein the Ar 3 cyclic group is phenyl, and said cyclic group optionally being singly or multiply substituted by -Q

I

provided that when -Ar 3 is substituted with a Q1 group which comprises one or more additional -Ar 3 groups, said additional -Ar 3 groups are not substituted with another -Ar 3 More preferred compounds of embodiments I (form and J (form 3) are those wherein: m is 1;

R

21 is -H or -CH 3

R

51 is a C1-6 straight or branched alkyl group optionally substituted with Ar 3 wherein the Ar 3 cyclic group is phenyl, said cyclic group optionally being 35 multiply or singly substituted by -Q 1 each Ar 3 cyclic group is independently phenyl, naphthyl, thienyl, quinolinyl, isoquinolinyl, pyrazolyl, thiazolyl, isoxazolyl, benzotriazolyl, benzimidazolyl, thienothienyl, imidazolyl, thiadiazolyl, benzo[b]thiophenyl, pyridyl, 0 benzofuranyl, or indolyl, and said cyclic group Soptionally being singly or multiply substituted by -Q 1 each Q 1 is independently

-NH

9 -Cl, -Br, -OH, 10 -R 9

-NH-R

5 wherein R 5 is -C(O)-R 10 or -S(0) 2

-R

9

-OR

wherein R 5 is -C(O)-R 10

-OR

9

-N(R

9 (R10), or 0

O

CH

2 0 *oo *wherein each R 9 and R 10 are independently a -C1-6 straight or branched alkyl group optionally substituted 20 with Ar 3 wherein the Ar 3 cyclic group is phenyl, and o said cyclic group optionally being singly or multiply substituted by -Q 1 provided that when -Ar 3 is substituted with a -Q1 group which comprises one or more additional -Ar 3 25 groups, said additional -Ar 3 groups are not substituted with another -Ar 3 Preferably, in these more preferred compounds the Ar 3 cyclic group is phenyl, naphthyl, thienyl, quinolinyl, isoquinolinyl, pyrazolyl, thiazolyl, isoxazolyl, benzotriazolyl, benzimidazolyl, thienothienyl, imidazolyl, thiadiazolyl, benzo[b]thiophenyl, benzofuranyl, or indolyl, and said cyclic group optionally being singly or multiply substituted by -Q 1 Preferred compounds of embodiments H, and J 36

S

S

*5

S

S.

5

S

0055 0

S

50 5 0 (forms 1 and 1) are those wherein:

R

3 is -CH2-TI-R1;

T

1 is 0; and

R

11 is -C(O)-Ar 4 wherein the Ar 4 cyclic group is tetrazolyl, pyridyl, oxazolyl, pyrimidinyl, or thienyl, and said cyclic group optionally being singly or multiply substituted by -Q 1 Preferred compounds of embodiments H, I, and J employ formula wherein R 3 is -CO-CH 2

-T

1

-R

11 and

R

11 is -Ar 4 wherein the Ar 4 cyclic group is pyridyl, and said cyclic group optionally being singly or multiply substituted by -Q 1 Preferred compounds of embodiment J (form 1) are those wherein:

R

3 is and

R

5 is -C(0)-R 10 wherein:

R

1 0 is Ar 3 wherein the Ar 3 cyclic group is phenyl optionally being singly or multiply substituted by:

-F,

20 -Cl,

-N(H)-R

5 wherein -R 5 is -H or -C(O)-R 1 0 wherein

R

1 0 is a -C 1 -6 straight or branched alkyl group optionally substituted with Ar 3 wherein Ar 3 is phenyl,

-N(R

9

)(R

10 wherein R 9 and R 1 0 are independently a -C1- 4 straight or branched alkyl group, or

-O-R

5 wherein R 5 is H or a -C 1 4 straight or branched alkyl group.

More preferably, Ar 3 is phenyl being optionally singly or multiply substituted at the 3- or 5-position by -Cl or at the 4-position by -NH-R 5

-N(R

9

(R

1 0 or -O-R 5 37 *0

S

S

1 *505ee 0 Other more preferred compounds of embodiment J (form 1) are those wherein R 3 is and R 5 is

-C(O)-R

10 wherein R 10 is Ar 3 and the Ar 3 cyclic group is indolyl, benzimidazolyl, thienyl, or benzo[b]thiophenyl, and said cyclic group optionally being singly or multiply substituted by -Q 1 Other more preferred compounds of embodiment J (form 1) are those wherein R 3 is and R 5 is

-C(O)-R

10 wherein R 10 is Ar 3 and the Ar 3 cyclic group 0 is quinolyl or isoquinolyl, and said cyclic group optionally being singly or multiply substituted by -QI.

Other more preferred compounds of embodiment J (form 1) are those wherein R 3 is and R 5 is

-C(O)-R

10 wherein R 10 is Ar 3 and the Ar 3 cyclic group 5 is phenyl, substituted by 0

CH

2 5 A preferred compound of embodiment J includes, but is not limited to 2002.

@5 0 0o 38 The ICE inhibitors of another embodiment L of this invention are those of formula 0 m R

R

1 -N R 3

H

wherein: 5 m is 1;

S**

R

1 is: (elO-B) 21 Rs-NO 0* 0 0 H O 0 each T 1 is independently or 10 R21 is -H or -CH3; Ar 2 is: (hh) wherein Y is O; each Ar 3 is independently phenyl, naphthyl, thienyl, quinolinyl, isoquinolinyl, pyrazolyl, thiazolyl, isoxazolyl, benzotriazolyl, benzimidazolyl, thienothienyl, imidazolyl, thiadiazolyl, benzo[b]thiophenyl, pyridyl benzofuranyl, or indolyl, and said cyclic group optionally being singly or multiply substituted by -Q 1 each Ar 4 is independently phenyl, tetrazolyl, pyridinyl, oxazolyl, naphthyl, pyrimidinyl, or thienyl, and said cyclic group optionally being singly or multiply substituted by -Q 1 39 each Q 1 is independently -NH 2 -Cl, -Br, -OH,

-R

9

-NH-R

5 wherein R 5 is -C(O)-R 1 0 or -S(0) 2

-R

9

-OR

wherein R 5 is -C(O)-R 1 0

-OR

9

-NHR

9 or

O

CH

2 0*

S

S

5 0 005 0S@S*0 0 0 0OSS 5*.

S

S. S 0 provided that when -Ar 3 is substituted with a Q1 group which comprises one or more additional -Ar 3 groups, said additional -Ar 3 groups are not substituted with another -Ar 3 and the other substituents are as defined above in embodiment E; provided that when: m is 1;

R

15 is -OH;

R

21 is and Y2 is O and R 3 is then R 5 cannot be: 20 -C(O)-R 10 wherein R 10 is -Ar 3 and the Ar 3 cyclic group is phenyl, unsubstituted by -Q 1 4- (carboxymethoxy)phenyl, 2-fluorophenyl, 2-pyridyl, N- (4-methylpiperazino)methylphenyl, or

-C(O)-OR

9 wherein R 9 is -CH 2 -Ar 3 and the Ar 3 cyclic group is phenyl, unsubstituted by -Q 1 and when Y2 is O, R 3 is -C(0)-CH 2

-T

1

-R

1 1

T

1 is 0, and R 11 is Ar 4 wherein the Ar 4 cyclic group is chlorophenyl)-3-trifluoromethyl)pyrazolyl), then R cannot be:

-H;

-C(O)-R

10 wherein R 10 is -Ar 3 and the Ar 3 cyclic group is 4-(dimethylaminomethyl)phenyl, phenyl, 4- (carboxymethylthio)phenyl,4-(carboxyethylthio)phenyl, 4-(carboxyethyl)phenyl, 4-(carboxypropyl)phenyl, 2- O0 40 fluorophenyl, 2-pyridyl, N-(4methylpiperazino) methyiphenyl, or -C(O)-0R 9 wherein R 9 is isobutyl or -CH 2 -Ar 3 and the Ar 3 cyclic group is phenyl; and when R 1 is Ar 4 wherein the Ar 4 cyclic group is 5- (1-phenyl-3-trifluoromethyl)pyrazolyl or 5-Cl- (4chloro-2-pyridinyl) -3-trifluoromethyl)pyrazolyl, then

R

5 cannot be: -C(O)-0R 9 wherein R 9 is -CH 2 -Ar 3 and the Ar 3 cyclic group is phenyl; *and when R 1 is Ar 4 wherein the Ar 4 cyclic group is 5-Cl- (2-pyridyl)-3-trifluoromethyl)pyrazolyl), then

R

5 cannot be:

-C(O)-R

10 wherein R 10 is -Ar 3 and the Ar 3 cyclic group is 4-(dimethylaminomethyl)phenyl, or -C(O)-0R 9 wherein R 9 is -CH 2 -Ar 3 and the Ar 3 cyclic group is phenyl, unsubstituted by -Qj; and when Y~2 is 0, R 3 is -C -CH 2

-T

1 -Rll, T, is 0, and R 1 is -C(O)-Ar 4 wherein the Ar 4 cyclic group is dichlorophenyl, then R 5 cannot be:

-C(O)-R

10 wherein R 10 is -Ar 3 and the Ar 3 cyclic group is 4- (dimethylaminomethyl) phenyl, 4- (Na. morpholinomethyl)phenyl, 4- (Nmethylpiperazino)methyl)phenyl, 4- (2methyl) imidazolylmethyl)phenyl, 5-benzimidazolyl, benztriazolyl, N-carboethoxy-5-benztriazolyl, Nor -C(O)-0R 9 wherein R 9 is -CH 2 -Ar 3 and the Ar 3 cyclic group is phenyl, unsubstituted by and when

Y

2 is H 2

R

3 is -C (0)-CH 2 -Tl-Rll, T 1 is 0, and R 1 is -C(O)-Ar 4 wherein the Ar 4 cyclic group is dichlorophenyl, then R 5 cannot be: -C(O)-0R 9 wherein R 9 is -CH 2 -Ar 3 and the Ar 3 41 .9 0 0* *6 00 *r 6 0@ 6 8 0 0 e g.

0060 00 S 06 6 *0 00 0 @0

S.

cyclic group is phenyl.

Preferably, R 3 is -C(O)-Ar 2 Alternatively,

R

3 is -C(O)CH 2

-T

1

_R

11 Alternatively, R 3 is Preferably, in any of the above compounds of embodiment L, R 3 is and R 5 is -C(O)-R 10 or 10 and the other substituents are as defined above.

More preferably R 10 is -Ar 3 More preferably in these more preferred compounds: R 5 is -C(O)-R 10 and R 10 is Ar 3 wherein the Ar 3 cyclic group is phenyl optionally being singly or multiply substituted by: -R 9 (wherein R 9 is a C_-4 straight or branched alkyl group), -Cl, -N(H)-R (wherein -R 5 is -H or -C(O)-R 10 wherein R 10 is a -C1_ 6 straight or branched alkyl group optionally substituted with Ar 3 wherein Ar 3 is phenyl), -N(R 9

)(R

10 (wherein

R

9 and R 10 are independently a -C1- 4 straight or branched alkyl group), or -O-R 5 (wherein R 5 is H or a

-C

1 4 straight or branched alkyl group).

Most preferably, Ar 3 is phenyl being singly or multiply substituted at the 3- or 5-position by -Cl or at the 4-position by -NH-R 5

-N(R

9

)(R

10 or -O-R 5 Other preferred compounds of this most preferred embodiment include, but are not limited to 214k and 214m.

Alternatively, Ar 3 is phenyl being singly or multiply substituted at the 3- or 5-position by -R 9 wherein R 9 is a C1- 4 straight or branched alkyl group; and at the 4-position by -O-R 5 Another preferred compound of this most preferred embodiment includes, but is not limited to 214w.

Alternatively, in this more preferred 42 embodiment: R 5 is -C(O)-R 10 wherein R 10 is Ar 3 and the Ar 3 cyclic group is selected from the group consisting of is indolyl, benzimidazolyl, thienyl, quinolyl, isoquinolyl and benzo[b]thiophenyl, and said cyclic group optionally being singly or multiply substituted by -Q 1 Most preferably, the Ar 3 cyclic group is isoquinolyl, and said cyclic group optionally being singly or multiply substituted by -Q 1 10 Another preferred compound of this most S. preferred embodiment includes, but is not limited to eq..

412.

Alternatively, in this more preferred embodiment R 5 is -C(O)-R 10 wherein R 10 is -Ar 3 and the Ar 3 cyclic group is phenyl, substituted by 0

CH

2 20 0 A preferred compound of this more preferred embodiment includes, but is not limited to 415.

Other compounds of embodiment L include, but are not limited to 214f, 214g, 214h, 214i, 214j, 2141, 246b, 280b, 280c, 280d, 283b, 283c, 283d, 284, 285, 286, 308c, 308d, 500, 501, 505b, 505c, 505d, 505e, 505f, 510a, 510b, 510c, 510d, 511c, 2100i, and 2100j.

The most preferred compounds of embodiment L are those wherein the Ar 3 cyclic group is isoquinolyl.

Compounds of this invention are described in co-pending United States Application Serial Nos.

08/575,641 and 08/598,332 the disclosures of which are herein incorporated by reference.

The compounds of this invention have a 43 molecular weight of less than or equal to about 700 Daltons, and more preferably between about 400 and 600 Daltons. These preferred compounds may be readily absorbed by the bloodstream of patients upon oral administration. This oral availability makes such compounds excellent agents for orally-administered treatment and prevention regimens against IL-1-, apoptosis-, IGIF- or IFN-y mediated diseases.

*9 S @0 *0 9 £0

S

5S 00 £900 It should be understood that the compounds of this invention may exist in various equilibrium forms, depending on conditions including choice of solvent, pH, and others known to the practitioner skilled in the art. All such forms of these compounds are expressly included in the present invention. In particular, many of the compounds of this invention, especially those which contain aldehyde or ketone groups in R 3 and carboxylic acid groups in T, may take hemi-ketal (or hemi-acetal) or hydrated forms. For example, compounds of embodiment C may take the forms depicted below: EQ1 0 L 0 b 0

(CJ

2

O

^OH

R--N--X

1

O/H

I d OH Ri-N-X H (CHH )g

OH

Hydrated Form 0

(CJ

2 )M

O

\OH

(CH2)g-C Rl3

A

R

1

-N-X

0

(CJ

2 )m 1 0

(CH

2

_R-

1H Hemi-ketal or Hemi-acetal Form Depending on the choice of solvent and other conditions known to the practitioner skilled in the art, compounds of this invention may also take acyloxy ketal, acyloxy acetal, ketal or acetal form: 44 0 0

(CJ

2 )m-d (CJ2)MA

OH

It, i.R) I (CH2)9g-_ C R3HCg 0

(CJ

2 )m- N-X Or Ketal or Acetal Form Acyloxy Ketal or Acyloxy Acetal Form 0* S 9. 0 g 0 .9 9 S S In addition, it should be understood that the equilibrium forms of the compounds of this invention may include tautomeric forms. All such forms of these compounds are expressly included in the present invention.

It should be understood that the compounds of this invention may be modified by appropriate S• functionalities to enhance selective biological properties. Such modifications are known in the art and include those which increase biological penetration into a given biological system blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow .00 administration by injection, alter metabolism and alter 15 rate of excretion. In addition, the compounds may be altered to pro-drug form such that the desired compound is created in the body of the patient as the result of the action of metabolic or other biochemical processes on the pro-drug. Such pro-drug forms typically demonstrate little or no activity in in vitro assays.

Some examples of pro-drug forms include ketal, acetal, oxime, imine, and hydrazone forms of compounds which contain ketone or aldehyde groups, especially where they occur in the R 3 group of the compounds of this invention. Other examples of pro-drug forms include 45 the hemi-ketal, hemi-acetal, acyloxy ketal, acyloxy acetal, ketal, and acetal forms that are described in EQ1 and EQ2.

ICE and TX Cleave and Thereby Activate Pro-IGIF The ICE protease was identified previously by virtue of its ability to process inactive pro-IL-1l to :mature active IL-1B, a pro-inflammatory molecule, in vitro and in vivo Here we show that ICE and its close homologue TX (Caspase-4, C. Faucheu et al., EMBO, 10 14, p. 1914 (1995)) can proteolytically cleave inactive pro-IGIF. This processing step is required to convert pro-IGIF to its active mature form, IGIF. Cleavage of pro-IGIF by ICE, and presumably by TX, also facilitates the export of IGIF out of cells.

We first used transient co-expression of plasmids transfected into Cos cells to determine whether any known members of the ICE/CED-3 protease family can process pro-IGIF to IGIF in cultured cells *e S' *(Example 23) (Fig. 1A).

Fig. 1A demonstrates that ICE cleaves pro-IGIF in Cos cells co-transfected with plasmids that express pro-IGIF in the presence of active ICE. Cos cells were transfected with an expression plasmid for pro-IGIF alone (lane 2) or in combination with the indicated expression plasmids encoding wild type or inactive mutants of ICE/CED-3 family of proteases (lanes 3-12). Cell lysates were prepared and analyzed for the presence of IGIF protein by immunoblotting with an anti-IGIF antiserum. Lane 1 contained lysates from mock transfected cells.

Co-expression of pro-IGIF with ICE or TX resulted in the cleavage of pro-IGIF into a polypeptide similar in size to the naturally-occurring 18-kDa 46 mature IGIF. This processing event is blocked by single point mutations that alter the catalytic cysteine residues and thus inactivate ICE and TX Gu et al., EMBO, 14, p. 1923 (1995)).

Co-expression with CPP32 (Caspase-3), a protease involved in programmed cell death (T.

Fernandes-Alnemri et al., J. Biol. Chem., 269, p. 30761 (1994); D. W. Nicholson et al., Nature, 376, p. 37 (1995)), resulted in the cleavage of pro-IGIF into a 10 smaller polypeptide, while co-expression with CMH-1 (Caspase-7), a close homolog of CPP32 A. Lippke et al., J. Biol. Chem., 271, p. 1825 (1996)), failed to cleave pro-IGIF to any significant extent. Thus, ICE and TX appear to be capable of cleaving pro-IGIF into a polypeptide similar in size to the naturally-occurring 18-kDa IGIF.

We next examined the ability of these cysteine proteases to cleave pro-IGIF in vitro using a purified, recombinant (His) 6 -tagged pro-IGIF as a substrate (Example 23).

Fig. 1B demonstrates that pro-IGIF is cleaved in vitro by ICE. Purified recombinant (His),-tagged pro-IGIF (2 .Lg) was incubated with the indicated cysteine protease in the presence or absence of ICE or CPP32 inhibitors as described in Example 23. The cleavage products were analyzed by SDS-PAGE and Coomassie Blue staining.

ICE cleaved the 24 kDa pro-IGIF into two polypeptides of approximately 18-kDa and 6-kDa.

N-terminal amino acid sequencing of the ICE cleavage products indicated that the 18-kDa polypeptide contains the same N-terminal amino acid residues (Asn-Phe-Gly-Arg-Leu) as the naturally occurring IGIF.

47 This shows that ICE cleaves pro-IGIF at the authentic processing site (Asp35-Asn36) Okamura et al., Infection and Immunity, 63, p. 3966 (1995); H. Okamura et al., Nature, 378, p. 88 (1995)). N-terminal amino acid sequencing of the CPP32 cleavage products indicated that CPP32 cleaved pro-IGIF at Asp69-Ile70.

The cleavage by ICE of pro-IGIF is highly S* specific with a catalytic efficiency (kcat/KM) of 1.4 x 107 -1 -1 M 1 s (KM= 0.6 0.1 M; kcat 8.6 0.3 and 10 is inhibited by specific ICE inhibitors (Ac-Tyr-Val-Ala-Asp-aldehyde) and Cbz-Val-Ala-Asp-

S.

[(2,6-dichlorobenzoyl)oxy]methylketone,

(N.A.

Thornberry et al., Nature, 356, p. 768 (1992); R. E.

Dolle et al., J. Med. Chem., 37, p. 563 (1994)).

Fig. 1C demonstrates that ICE cleavage in vitro activates pro-IGIF. Uncleaved pro-IGIF, ICE- or CPP32-cleaved products of pro-IGIF, or recombinant mature IGIF (rIGIF) were each added to A.E7 cell cultures to a final concentration of 12 ng/ml or 120 ng/ml (see, Example 23). Eighteen hours later, IFN-y in the cultural medium was quantified by ELISA. While the uncleaved pro-IGIF had no detectable IFN-y inducing activity, ICE-cleaved pro-IGIF was active in inducing IFN-y production in Thl cells.

Like ICE, the ICE homolog TX also cleaved pro-IGIF into similarly sized polypeptides. However, its catalytic efficiency was about two orders of magnitude lower than that shown for ICE.

Consistent with the observations from the Cos cell experiments above, CPP32 cleaved pro-IGIF at a different site (Asp69-Ile70) and the resulting polypeptides had little IFN-y inducing activity (Fig.

48 1C). CMH-1 and granzyme B each failed to cleave pro-IGIF to any significant extent.

Together, these results demonstrate that, both in Cos cells and in vitro, ICE and TX are capable of processing the inactive pro-IGIF precursor at the authentic maturation site to generate a biologically active IGIF molecule.

Processing of Pro-IGIF by ICE Facilitates Its Export 0S 0 00 0 *0 0 0 S0*

S

S

0e*S 0500

S

S@ S 0* 0 5* S S 0* IGIF is produced by activated Kupffer cells 10 and macrophages in vivo and is exported out of the cells upon stimulation by endotoxin Okamura et al., Infection and Immunity, 63, p. 3966 (1995); H. Okamura et al., Nature, 378, p. 88 (1995). We used the Cos cell co-expression system (Example 23) to examine whether the intracellular cleavage of pro-IGIF by ICE would facilitate the export of mature IGIF from the cell. Such is the case for pro-IL-l when it is cleaved by ICE into active IL-18 Thornberry et al., Nature, 356, p. 768 (1992)).

In Fig. 2A, Cos cells transfected with an expression plasmid for pro-IGIF alone (lanes 2 and 6) or in combination with an expression plasmid encoding wild type (lanes 3 and 7) or inactive mutant ICE (lanes 4 and 8) were metabolically labeled with 35 S-methionine (see, Example 24). Cell lysates (left) and conditioned medium (right) were immunoprecipitated with an anti-IGIF antiserum. The immunoprecipitated proteins were analyzed by SDS-PAGE and fluorography (Fig. 2A).

An 18-kDa polypeptide corresponding in size to mature IGIF was detected in the conditioned medium of Cos cells co-expressing pro-IGIF and ICE, while Cos cells co-expressing pro-IGIF and an inactive ICE mutant (ICE-C285S), or pro-IGIF alone exported only very 49 low levels of pro-IGIF and no detectable mature IGIF.

We estimate that about 10% of the mature IGIF was exported from co-transfected cells, while greater than 99% of pro-IGIF was retained within the cells.

We also measured the presence of IFN-y inducing activity in cell lysates and in the conditioned medium of the above transfected cells (see, Example 24). IFN-y inducing activity was detected in both cell lysates and the conditioned medium of Cos cells co-expressing pro-IGIF and ICE, but not in cells expressing either pro-IGIF or ICE alone (Fig. 2B).

These results indicate that ICE cleavage of pro-IGIF facilitates the export of mature, active IGIF from cells.

Pro-IGIF is a Physiological Substrate of ICE In Vivo To study the role of ICE in the proteolytic activation and export of IGIF under physiological conditions, we examined the processing of pro-IGIF and export of mature IGIF from lipopolysaccharide (LPS)-activated Kupffer cells harvested from Propiobacterium acnes-elicited wild type and ICE deficient mice (Example As shown in Fig. 3A, Kupffer cells from ICE-/- mice are defective in the export of IGIF.

Kupffer cell lysates of wild type and ICE-/- mice contained similar amounts of IGIF as determined by ELISA. IGIF, however, could be detected only in the conditioned medium of wild type but not of the ICE-/cells. Thus, ICE-deficient mice synthesize pro-IGIF, but fail to export it as extracellular pro-or mature IGIF.

To determine whether ICE-deficient mice process intracellular pro-IGIF but fail to export 50 0 0 OS 0 0 0@

S.

S

0**S

OS

SSS

0 00e0

SO

S.

S.

0O 0 0S *0 0 0* IGIF, Kupffer cells from wild type and ICE-/- mice were metabolically labeled with 35 S-methionine and IGIF immunoprecipitation experiments were performed on cell lysates and conditioned media as described in Example 25. These experiments demonstrated that unprocessed pro-IGIF was present in both wild type and ICE-/- Kupffer cells. However, the 18-kDa mature IGIF was present only in the conditioned medium of wild type and not ICE-/- Kupffer cells (Fig. 3B). This shows that 10 active ICE is required in cells for the export of processed IGIF out of the cell.

In addition, conditioned medium from wild type but not from ICE-/- Kupffer cells contained IFN-y inducing activity that was not attributed to the action of IL-12 because it was insensitive to a neutralizing anti-IL-12 antibody. The absence of IGIF in the conditioned medium of ICE-/- Kupffer cells is consistent with the finding in Cos cells that the processing of pro-IGIF by ICE is required for the export of active IGIF.

Figs. 3C and 3D show that, in vivo, ICE-/mice have reduced serum levels of IGIF and IFN-y, respectively. Wild type and ICE-/- mice (n=3) primed with heat-inactivated P. acnes were challenged with LPS (Example 26), and the levels of IGIF (Fig. 3C) and IFN-y (Fig. 3D) in the sera of challenged mice were measured by ELISA three hours after LPS challenge (Example The sera of ICE-/- mice stimulated by P. acnes and LPS contained reduced levels of IGIF (Fig. 3C) and no detectable IFN-y inducing activity in the presence of an anti-IL-12 antibody. The reduced 51 serum levels of IGIF likely accounts for the significantly lower levels of IFN-y in the sera of ICE-/- mice (Fig. 3D), because we have observed no significant difference in the production of IL-12 in ICE-/- mice under these conditions. Consistent with this interpretation is the finding that non-adherent splenocytes from wild type and ICE-/- mice produced similar amounts of IFN-y when stimulated with recombinant active IGIF in vitro. Thus the impaired production of IFN-y is not due to any apparent defect in the T cells of the ICE-/- mice.

S" Taken together, these results establish a critical role for ICE in processing the IGIF precursor and in the export of active IGIF both in vitro and in 15 vivo.

To examine in more detail the relationship between serum levels of IFN-y and ICE activity in vivo, a time course after challenge of wild type and ICE-deficient mice with LPS was performed (Example 26) (Fig. 4).

Fig. 4 shows a time course increase of serum S"IFN-y in wild type mice, with sustained levels of 17 ng/ml occurring from 9-18 hrs after LPS challenge.

As predicted by the experiments discussed above, serum IFN-y levels were significantly lower in ICE-/- mice, with a maximum of 2 ng/ml achieved over the same time period, which is approximately 15% of the level observed in wild type mice (Fig. 4).

Animals were also observed for clinical signs of sepsis and body temperature was measured at 4-hour intervals in wild type and ICE-/- mice challenged with mg/kg or 100 mg/kg LPS (ICE-/-only). Results in 52 Fig. 4 show that wild type mice experienced a significant decrease in body temperature (from 36'C to 26'C) within 12 hours of LPS challenge. Signs of clinical sepsis were evident and all animals expired within 24-28 hours.

In contrast, ICE-/- mice challenged with 30 mg/kg LPS experienced only a 3'-4'C decrease in body temperature with minimal signs of distress and with no observed lethality. ICE-/- mice challenged with 10 100 mg/kg LPS experienced clinical symptoms, a decrease in body temperature, and mortality similar to wild type mice at the 30 mg/kg LPS dose.

The ICE Inhibitor Ac-YVAD-CHO is an Equipotent Inhibitor of IL-1R and IFN-V Productinn

S

S

*000 00 S@ 6 0e 0 00 *S 0 Since the processing and secretion of biologically active IGIF is mediated by ICE, we compared the activity of a reversible ICE inhibitor (Ac-YVAD-CHO) on IL-1P and IFN-y production in a peripheral blood mononuclear cell (PBMC) assay (Examples 27) Results in Fig. 5 show a similar potency for the ability of the Ac-YVAD-CHO ICE inhibitor to decrease IL-1 and IFN-y production in human PBMCs, with an IC 50 of 2.5 p.M for each. Similar results were obtained in studies with wild type mouse splenocytes.

These findings provide additional evidence that pro-IGIF is a physiological substrate for ICE and suggest that ICE inhibitors will be useful tools for controlling physiological levels of IGIF and IFN-y.

In summary, we have found that ICE controls IGIF and IFN-y levels in vivo and in vitro and that ICE inhibitors can decrease levels of IGIF and IFN-y in human cells. These results have been described in co- 53 0 00 0 4 15 0@*060 fee* 0* 0 0ee 15 6 pending United States Application Serial No.

08/712,878, the disclosure of which is herein incorporated by reference.

Compositions and Methods The pharmaceutical compositions and methods of this invention will be useful for controlling IL-1, IGIF and IFN-y levels in vivo. The methods and compositions of this invention will thus be useful for treating or reducing the advancement, severity of effects of IL-1, IGIF- and IFN-y-mediated conditions.

The compounds of this invention are effective ligands for ICE. Accordingly, these compounds are capable of targeting and inhibiting events in IL-1-, apoptosis-, IGIF-, and IFN-y-mediated diseases, and, thus, the ultimate activity of that protein in inflammatory diseases, autoimmune diseases, destructive bone, proliferative disorders, infectious diseases, and degenerative diseases. For example, the compounds of this invention inhibit the conversion of precursor IL- 00 0O 0 00 p1 to mature IL-10 by inhibiting ICE. Because ICE is essential for the production of mature IL-1, inhibition of that enzyme effectively blocks initiation of IL-1mediated physiological effects and symptoms, such as inflammation, by inhibiting the production of mature IL-1. Thus, by inhibiting IL-10 precursor activity, the compounds of this invention effectively function as IL-1 inhibitors.

Similarly, compounds of this invention inhibit the conversion of precursor IGIF to mature IGIF. Thus, by inhibiting IGIF production, the compounds of this invention effectively function as inhibitors of IFN-y production.

54 Accordingly, one embodiment of this invention provides a method for decreasing IGIF production in a subject comprising the step of administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an ICE inhibitor and a pharmaceutically acceptable carrier.

:Another embodiment of this invention provides a method for decreasing IFN-y production in a subject

S

S comprising the step of administering to the subject a 10 pharmaceutical composition comprising a therapeutically 0effective amount of an ICE inhibitor and a •pharmaceutically acceptable carrier.

In another embodiment, the methods of this invention comprise the step of administering to a subject a pharmaceutical composition comprising an inhibitor of an ICE-related protease that is capable of cleaving pro-IGIF to active IGIF, and a pharmaceutically acceptable carrier. One such ICE-related protease is TX, as described above. This invention thus provides methods and pharmaceutical compositions for controlling IGIF and IFN-y levels by O administering a TX inhibitor.

Other ICE-related proteases capable of processing pro-IGIF into an active IGIF form may also be found. Thus it is envisioned that inhibitors of those enzymes may be identified by those of skill in the art and will also fall within the scope of this invention.

The compounds of this invention may be employed in a conventional manner for the treatment of diseases which are mediated by IL-1, apoptosis, IGIF or IFN-y. Such methods of treatment, their dosage levels and requirements may be selected by those of ordinary 55 skill in the art from available methods and techniques.

For example, a compound of this invention may be combined with a pharmaceutically acceptable adjuvant for administration to a patient suffering from an IL-1-, apoptosis-, IGIF- or IFN-y-mediated disease in a pharmaceutically acceptable manner and in an amount effective to lessen the severity of that disease.

0, Alternatively, the compounds of this invention may be used in compositions and methods for 10 treating or protecting individuals against IL-1-, apoptosis-, IGIF- or IFN-y-mediated diseases over extended periods of time. The compounds may be employed in such compositions either alone or together with other compounds of this invention in a manner consistent with the conventional utilization of ICE 0 o. inhibitors in pharmaceutical compositions. For example, a compound of this invention may be combined with pharmaceutically acceptable adjuvants e conventionally employed in vaccines and administered in prophylactically effective amounts to protect individuals over an extended period of time against IL-1-, apoptosis-, IGIF- or IFN-y- mediated diseases.

The compounds of this invention may also be co-administered with other ICE inhibitors to increase the effect of therapy or prophylaxis against various IL-1-, apoptosis, IGIF- or IFN-y-mediated diseases.

In addition, the compounds of this invention may be used in combination either conventional antiinflammatory agents or with matrix metalloprotease inhibitors, lipoxygenase inhibitors and antagonists of cytokines other than The compounds of this invention can also be 56 administered in combination with immunomodulators bropirimine, anti-human alpha interferon antibody, IL-2, GM-CSF, methionine enkephalin, interferon alpha, diethyldithiocarbamate, tumor necrosis factor, naltrexone and rEPO) or with prostaglandins, to prevent or combat IL-1-mediated disease symptoms such as inflammation.

When the compounds of this invention are administered in combination therapies with other agents, they may be administered sequentially or concurrently to the patient. Alternatively, pharmaceutical or prophylactic compositions according to this invention comprise a combination of an ICE inhibitor of this invention and another therapeutic or prophylactic agent.

SPharmaceutical compositions of this invention 5 comprise any of the compounds of the present invention, and pharmaceutically acceptable salts thereof, with any pharmaceutically acceptable carrier, adjuvant or vehicle. Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as da-tocopherol polyethyleneglycol 1000 succinate, or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, 57 colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Cyclodextrins such as 3- and y-cyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2-and 3-hydroxypropyl-B-cyclodextrines, or other solubiliezed derivatives may also be advantageeously used to enhanve delivery of compounds of this invention.

The pharmaceutical compositions of this invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. We prefer oral administration. The pharmaceutical compositions of this invention may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compounds or its delivery form. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, 58 S.

S

51

*SSOSS

t

OS

S

500 Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterallyacceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending 0 medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides.

Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant such as Ph. Helv or a similar alcohol.

e

I

0555 0

S.

55

S

55 55 0 S S

S.

The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspensions and solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.

59- The pharmaceutical compositions of this invention may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa 10 butter, beeswax and polyethylene glycols. OO0 Topical administration of the pharmaceutical compositions of this invention is especially useful when the desired treatment involves areas or organs readily accessible by topical application. For application topically to the skin, the pharmaceutical composition should be formulated with a suitable ooeoo ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and S. water. Alternatively, the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of this invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topically-administered transdermal 60 patches are also included in this invention.

The pharmaceutical compositions of this invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.

Dosage levels of between about 0.01 and about 100 mg/kg body weight per day, preferably between about 1 and 50 mg/kg body weight per day of the active ingredient compound are useful in the prevention and treatment of IL-1-, apoptosis, IGIF and IFN-y-mediated diseases, including inflammatory diseases, autoimmune diseases, destructive bone disorders, proliferative disorders, infectious diseases, degenerative diseases, necrotic diseases, osteoarthritis, acute pancreatitis, chronic pancreatitis, asthma, adult respiratory distress syndrome, glomeralonephritis, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, chronic thyroiditis, Graves' disease, autoimmune gastritis, insulin-dependent diabetes mellitus (Type autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia, chronic active hepatitis, myasthenia gravis, inflammatory bowel disease, Crohn's disease, psoriasis, graft vs. host disease, osteoporosis, multiple myeloma-related bone disorder, acute myelogenous leukemia, chronic myelogenous leukemia, metastatic melanoma, Kaposi's sarcoma, multiple myeloma sepsis, septic shock, Shigellosis, Alzheimer's disease, Parkinson's disease, 61 cerebral ischemia, myocardial ischemia, spinal muscular atrophy, multiple sclerosis, AIDS-related encephalitis, HIV-related encephalitis, aging, alopecia, and neurological damage due to stroke. Typically, the pharmaceutical compositions of this invention will be administered from about 1 to 5 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient that may be 1 0 combined with the carrier materials to produce a single 0 dosage form will vary depending upon the host treated and the particular mode of administration. A typical *preparation will contain from about 5% to about active compound Preferably, such preparations contain from about 20% to about 80% active compound.

Upon improvement of a patient's condition, a maintenance dose of a compound, composition or e g.

6 combination of this invention may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved *'"."condition is retained when the symptoms have been alleviated to the desired level, treatment should cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence or disease symptoms.

As the skilled artisan will appreciate, lower or higher doses than those recited above may be required. Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of 62 excretion, drug combination, the severity and course of the disease, and the patient's disposition to the disease and the judgment of the treating physician.

The IL-1 mediated diseases which may be treated or prevented by the compounds of this invention include, but are not limited to, inflammatory diseases, autoimmune diseases, destructive bone disorders, proliferative disorders, infectious diseases, and degenerative diseases. The apoptosis-mediated diseases which may be treated or prevented by the compounds of this invention include degenerative diseases.

Inflammatory diseases which may be treated or prevented include, but are not limited to osteoarthritis, acute pancreatitis, chronic pancreatitis, asthma, and adult respiratory distress syndrome. Preferably the inflammatory disease is osteoarthritis or acute pancreatitis.

Autoimmune diseases which may be treated or prevented include, but are not limited to, glomeralonephritis, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, chronic thyroiditis, Graves' disease, autoimmune gastritis, insulindependent diabetes mellitus (Type autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia, chronic active hepatitis, myasthenia gravis, multiple sclerosis, inflammatory bowel disease, Crohn's disease, psoriasis, and graft vs. host disease.

Preferably the autoimmune disease is rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, or psoriasis.

Destructive bone disorders which may be treated or prevented include, but are not limited to, osteoporosis and multiple myeloma-related bone 63 disorder.

Proliferative diseases which may be treated or prevented include, but are not limited to, acute myelogenous leukemia, chronic myelogenous leukemia, metastatic melanoma, Kaposi's sarcoma, and multiple myeloma.

Infectious diseases which may be treated or prevented include, but are not limited to, sepsis, septic shock, and Shigellosis.

The IL-l-mediated degenerative or necrotic S diseases which may be treated or prevented by the compounds of this invention include, but are not limited to, Alzheimer's disease, Parkinson's disease, cerebral ischemia, and myocardial ischemia.

Preferably, the degenerative disease is Alzheimer's disease.

The apoptosis-mediated degenerative diseases which may be treated or prevented by the compounds of this invention include, but are not limited to, Alzheimer's disease, Parkinson's disease, cerebral ischemia, myocardial ischemia, spinal muscular atrophy, multiple sclerosis, AIDS-related encephalitis, HIVrelated encephalitis, aging, alopecia, and neurological damage due to stroke.

The methods of this invention may be used for treating, or reducing the advancement, severity or effects of an IGIF-or IFN-y-mediated inflammatory, autoimmune, infectious, proliferative, destructive bone, necrotic, and degenerative conditions, including diseases, disorders or effects, wherein the conditions are characterized by increased levels of IGIF or IFN-y production.

Examples of such inflammatory conditions 64 include, but are not limited to, osteoarthritis, acute pancreatitis, chronic pancreatitis, asthma, rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, ulcerative collitis, cerebral ischemia, myocardial ischemia and adult respiratory distress syndrome.

Preferably, the inflammatory condition is rheumatoid arthritis, ulcerative collitis, Crohn's disease, hepatitis and adult respiratory distress syndrome.

S. S 6O 0@ @0 0 0@@O 0@ 0e 00090e

S

*SS*

0SO0 0000 00 @9 0 0* 0* 0@ S 5O 00 10 Examples of such infectious conditions include, but are not limited to, infectious hepatitis, sepsis, septic shock and Shigellosis.

Examples of such autoimmune conditions include, but are not limited to, glomerulonephritis, systemic lupus erythematosus, scleroderma, chronic thyroiditis, Graves' disease, autoimmune gastritis, insulin-dependent diabetes mellitus (Type juvenile diabetes, autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia, myasthenia gravis, multiple sclerosis, psoriasis, lichenplanus, graft vs.

host disease, acute dermatomyositis, eczema, primary cirrhosis, hepatitis, uveitis, Behcet's disease, acute dermatomyositis, atopic skin disease, pure red cell aplasia, aplastic anemia, amyotrophic lateral sclerosis and nephrotic syndrome.

Preferably the autoimmune condition is glomerulonephritis, insulin-dependent diabetes mellitus (Type juvenile diabetes, psoriasis, graft vs. host disease, including transplant rejection, and hepatitis.

Examples of such destructive bone disorders include, but are not limited to, osteoporosis and multiple myeloma-related bone disorder.

Examples of such proliferative conditions 65 include, but are not limited to, acute myelogenous leukemia, chronic myelogenous leukemia, metastatic melanoma, Kaposi's sarcoma, and multiple myeloma.

Examples of such neurodegenerative conditions include, but are not limited to, Alzheimer's disease, Parkinson's disease and Huntington's disease.

Although this invention focuses on the use of the compounds disclosed herein for preventing and treating IL-1, apoptosis, IGIF- and IFN-y-mediated 10 diseases, the compounds of this invention can also be used as inhibitory agents for other cysteine proteases.

The compounds of this invention are also useful as commercial reagents which effectively bind to ICE or other cysteine proteases. As commercial reagents, the compounds of this invention, and their derivatives, may be used to block proteolysis of a target peptide in biochemical or cellular assays for eeO ICE and ICE homologs or may be derivatized to bind to a stable resin as a tethered substrate for affinity chromatography applications. These and other uses which characterize commercial cysteine protease inhibitors will be evident to those of ordinary skill in the art.

Process of Preparing N-Acylamino Compounds The ICE inhibitors of this invention may be synthesized using conventional techniques.

Advantageously, these compounds are conveniently synthesized from readily available starting materials.

The compounds of this invention are among the most readily synthesized ICE inhibitors known.

Previously described ICE inhibitors often contain four or more chiral centers and numerous peptide linkages.

The relative ease with which the compounds of this 66 invention can be synthesized represents an advantage in the large scale production of these compounds.

For example, compounds of this invention may be prepared using the processes described herein. As can be appreciated by the skilled practitioner, these processes are not the only means by which the compounds described and claimed in this application may be synthesized. Further methods will be evident to those of ordinary skill in the art. Additionally, the 1 0 various synthetic steps described herein may be performed in an alternate sequence or order to give the desired compounds.

This invention also provides a preferred method for preparing the compounds of this invention.

Accordingly, in another embodiment M is provided a process for preparing an N-acylamino compound comprising the steps of: a) mixing a carboxylic acid with an Nalloc-protected amino in the presence of an inert solvent, triphenylphoshine, a nucleophilic scavenger, and tetrakis-triphenyl phosphine palladium(0) at ambient temperature under an inert atmosphere; and b) adding to the step a) mixture, HOBT and EDC; and optionally comprising the further step of: c) hydrolyzing the step b) mixture in the presence of a solution comprising an acid and wherein the step b) mixture is optionally concentrated, prior to hydrolyzing.

Preferably, the inert solvent is CH 2 C12, DMF, or a mixture of CH 2 C1 2 and DMF.

Preferably, the nucleophilic scavenger is dimedone, morpholine, trimethylsilyl dimethylamine, or dimethyl barbituric acid. More preferably, the 67 nucleophilic scavenger is trimethylsilyl dimethylamine or dimethyl barbituric acid.

Preferably, the solution comprises trifluoroacetic acid in about 1-90% by weight. More preferably, the solution comprises trifluoroacetic acid in about 20-50% by weight.

Alternatively, the solution comprises hydrochloric acid in about 0.1-30% by weight. More preferably, the solution comprises hydrochloric acid in about 5-15% by weight.

More preferably, in the above process, the inert solvent is CH 2 C12, DMF, or a mixture of CH 2 Cl 2 and DMF and the nucleophilic scavenger is dimedone, morpholine, trimethylsilyl dimethylamine, or dimethyl barbituric acid.

Most preferably, in the above process the a inert solvent is CH 2 C1 2 DMF, or a mixture of CH 2 Cl 2 and DMF and the nucleophilic scavenger is trimethylsilyl dimethylamine or dimethyl barbituric acid.

Preferably, the N-acyclamino compound is represented by formula (VIII):

R

1

-N-R

2

H

wherein: R1 is as defined above in embodiment C; R2 is: 0

H

wherein R 51 is R 9

-C(O)-R

9 or 9 and

R

9 is as defined herein; 68 o

OH

H

,or 0* 0 0*

OS

wherein m is as defined herein.

Preferably, the N-alloc-protected amine is: 0 Alloc-N ORs, wherein R 51 is as defined above.

006004 10 0 0

S*

00 0 In order that this invention be more fully understood, the following examples are set forth.

These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any way.

69 0S 00 0 *5 00 00 0 6 0

S.

S. C

C

SS

Example 1 Inhibition of ICE We obtained inhibition constants (K i and IC 50 values for compounds of this invention using the three methods described below: 1. Enzyme assay with UV-visible substrate This assay is run using an Succinyl-Tyr-Val- Ala-Asp-pNitroanilide substrate. Synthesis of analogous substrates is described by L. A. Reiter (Int.

10 J. Peptide Protein Res. 43, 87-96 (1994)). The assay mixture contains: 65 il buffer (10mM Tris, 1 mM DTT, 0.1% CHAPS @pH 8.1) pl ICE (50 nM final concentration to give a rate of ~imOD/min) 5 p DMSO/Inhibitor mixture 20 ul 400pM Substrate (80 pM final concentration) 100pl total reaction volume The visible ICE assay is run in a 96-well microtiter plate. Buffer, ICE and DMSO (if inhibitor 20 is present) are added to the wells in the order listed.

The components are left to incubate at room temperature for 15 minutes starting at the time that all components are present in all wells. The microtiter plate reader is set to incubate at 37 oC. After the 15 minute incubation, substrate is added directly to the wells and the reaction is monitored by following the release of the chromophore (pNA) at 405 603 nm at 37 OC for minutes. A linear fit of the data is performed and the rate is calculated in mOD/min. DMSO is only present during experiments involving inhibitors, buffer is used to make up the volume to 100 pl in the other experiments.

2. Enzyme Assay with Fluorescent Substrate This assay is run essentially according to Thornberry et al. (Nature 356: 768-774 (1992)), using substrate 17 referenced in that article. The substrate 70 is: Acetyl-Tyr-Val-Ala-Asp-amino-4-methylcoumarin (AMC). The following components are mixed: pl buffer(l0mM Tris,lmM DTT, 0.1% CHAPS @pH8.1) 1l ICE (2 10 nM final concentration) 5 pi DMSO/inhibitor solution ul 150 uM Substrate (30 pM final) 100pl total reaction volume The assay is run in a 96 well microtiter plate. Buffer and ICE are added to the wells. The 10 components are left to incubate at 37 OC for 15 minutes in a temperature-controlled wellplate. After the minute incubation, the reaction is started by adding substrate directly to the wells and the reaction is monitored @37 oC for 30 minutes by following the release of the AMC fluorophore using an excitation wavelength for 380 nm and an emission wavelength of 460 nm. A linear fit of the data for each well is performed and a rate is determined in fluorescence units per second.

For determination of enzyme inhibition constants (K i or the mode of inhibition (competitive, uncompetitive or noncompetitive), the rate data determined in the enzyme assays at varying inhibitor concentrations are computer-fit to standard enzyme 25 kinetic equations (see I. H. Segel, Enzyme Kinetics, Wiley-Interscience, 1975).

The determination of second order rate constants for irreversible inhibitors was performed by fitting the fluorescence vs time data to the progress equations of Morrison. Morrison, Mol. Cell.

Biophys., 2, pp. 347-368 (1985). Thornberry et al.

have published a description of these methods for measurement of rate constants of irreversible inhibitors of ICE. Thornberry, et al.

Biochemistry, 33, pp. 3923-3940 (1994). For compounds 71 0* p 9 0 @00n 0 0S00 @0 0

*C:

S S r 0* *0 *0 0* where no prior complex formation can be observed kinetically, the second order rate constants (kinact) are derived directly from the slope of the linear plots of kobs vs. For compounds where prior complex formation to the enzyme can be detected, the hyperbolic plots of kobs vs. are fit to the equation for saturation kinetics to first generate K i and The second order rate constant kinact is then given by k'/Ki.

3. PBMC Cell assay Assay with a Mixed Population of Human Peripheral Blood Mononuclear Cells (PBMC) or Enriched Adherent Mononuclear Cells Processing of pre-IL-lp by ICE can be measured in cell culture using a variety of cell sources. Human PBMC obtained from healthy donors provides a mixed population of lymphocyte subtypes and mononuclear cells that produce a spectrum of interleukins and cytokines in response to many classes 20 of physiological stimulators. Adherent mononuclear cells from PBMC provides an enriched source of normal monocytes for selective studies of cytokine production by activated cells.

Experimental Procedure: 25 An initial dilution series of test compound in DMSO or ethanol is prepared, with a subsequent dilution into RPMI-10% FBS media (containing 2 mM L-glutamine, 10 mM HEPES, 50 U and 50 ug/ml pen/strep) respectively to yield drugs at 4x the final test concentration containing 0.4% DMSO or 0.4% ethanol.

The final concentration of DMSO is 0.1% for all drug dilutions. A concentration titration which brackets the apparent K i for a test compound determined in an ICE inhibition assay is generally used for the primary compound screen.

72 a a.

S 0 0 Generally 5-6 compound dilutions are tested and the cellular component of the assay is performed in duplicate, with duplicate ELISA determinations on each cell culture supernatant.

PBMC Isolation and IL-1 Assay: Buffy coat cells isolated from one pint human blood (yielding 40-45 ml final volume plasma plus cells) are diluted with media to 80 ml and LeukoPREP separation tubes (Becton Dickinson) are each overlaid with 10 ml of cell suspension. After 15 min centrifugation at 1500-1800 xg, the plasma/media layer is aspirated and then the mononuclear cell layer is collected with a Pasteur pipette and transferred to a ml conical centrifuge tube (Corning). Media is 15 added to bring the volume to 15 ml, gently mix the cells by inversion and centrifuge at 300 xg for 15 min.

Resuspend the PBMC pellet in a small volume of media, count cells and adjust to 6 x 106 cells/ml.

For the cellular assay, 1.0 ml of the cell suspension is added to each well of a 24-well flat bottom tissue culture plate (Corning), 0.5 ml test compound dilution and 0.5 ml LPS solution (Sigma #L-3012; 20 ng/ml solution prepared in complete RPMI media; final LPS concentration 5 ng/ml). The 0.5 ml additions of test compound and LPS are usually sufficient to mix the contents of the wells. Three control mixtures are run per experiment, with either LPS alone, solvent vehicle control, and/or additional media to adjust the final culture volume to 2.0 ml.

The cell cultures are incubated for 16-18 hr at 37 °C in the presence of 5% CO 2 At the end of the incubation period, cells are harvested and transferred to 15 ml conical centrifuge tubes. After centrifugation for 10 min at 73 200 xg, supernatants are harvested and transferred to ml Eppendorf tubes. It may be noted that the cell pellet may be utilized for a biochemical evaluation of pre-IL-l1 and/or mature IL-10 content in cytosol extracts by western blotting or ELISA with pre-IL-lp specific antisera.

Isolation of Adherent Mononuclear cells: S. PBMC are isolated and prepared as described above. Media (1.0 ml) is first added to wells followed by 0.5 ml of the PBMC suspension. After a one hour incubation, plates are gently shaken and nonadherent cells aspirated from each well. Wells are then gently washed three times with 1.0 ml of media and final resuspended in 1.0 ml media. The enrichment for adherent cells generally yields 2.5-3.0 x 10 5 cells per well. The addition of test compounds, LPS, cell incubation conditions and processing of supernatants proceeds as described above.

ELISA:

S 20 We have used Quantikine kits (R&D Systems) for measurement of mature IL-10. Assays are performed 9. according to the manufacturer's directions. Mature IL-10 levels of about 1-3 ng/ml in both PBMC and adherent mononuclear cell positive controls are observed. ELISA assays are performed on 1:5, 1:10 and 1:20 dilutions of supernatants from LPSpositivecontrols to select the optimal dilution for supernatants in the test panel.

The inhibitory potency of the compounds can be represented by an IC 50 value, which is the concentration of inhibitor at which 50% of mature is detected in the supernatant as compared to the positive controls.

74 The skilled practitioner realizes that values obtained in cell assays, such as those described herein, can depend on multiple factors, such as cell type, cell source, growth conditions and the like.

Example 2 Pharmacokinetic Studies in the Mouse Peptidyl ICE inhibitors are cleared rapidly with clearance rates greater than 100 p/min/kg.

Compounds with lower clearance rates have improved 10 pharmacokinetic properties relative to peptidyl ICE inhibitors.

We obtained the rate of clearance in the mouse (p/min/kg) for several compounds of this invention using the method described below: Sample Preparation and Dosing Compounds were dissolved in sterile TRIS solution (0.02M or 0.05M) at a concentration of 2.5mg/ml. Where necessary to ensure a complete solution, the sample was first dissolved in a minimum 20 of dimethylacetamide (maximum of 5% of total solution volume) then diluted with the TRIS solution.

The drug solution was administered to CD-1 mice (Charles River Laboratories 26-31g) via the tailvein at a dose volume of 10ml/kg giving a drug dose of Mice were dosed in groups of 5 for each timepoint (generally from 2 minutes to 2 hours) then at the appropriate time the animals were anaesthetised with halothane and the blood collected into individual heparinized tubes by jugular severance. The blood samples were cooled to 0 OC then the plasma separated and stored at -20 OC until assayed.

75 6 0 6* *6

S

0 *6*e

S

Bioassay Drug concentration in the plasma samples were determined by HPLC analysis with UV or MS (ESP) detection. Reverse phase chromatography was employed using a variety of bonded phases from C1 to C18 with eluents composed of aqueous buffer/acetonitrile mixtures run under isocratic conditions.

Quantitation was by external standard methods with calibration curves constructed by spiking plasma with drug solutions to give concentrations in the range of 0.5 to Prior to analysis the plasma samples were deproteinated by the addition of acetonitrile, methanol, trichloroacetic acid or perchloric acid followed by centrifugation at 10,000g for 10 minutes.

Sample volumes of 20pl to 50pl were injected for analysis.

Compound 214e Dosing and sampling 20 The drug was dissolved in sterile 0.02M Tris to give a 2.5mg/ml solution which was administered to 11 groups of 5 male CD-1 mice via the tail vein at a dose of 25mg/kg. At each of the following timepoints: 2, 5, 10, 15, 20, 30, 45, 60, 90 and 120 minutes a group of animals was anaesthetised and the blood collected into heparinized tubes. After separation the plasma was stored at -20 OC until assayed.

Assay Aliquots of plasma (150pl) were treated with 5% perchloric acid (5pl) then mixed by vortexing and allowed to stand for 90 minutes prior to centrifugation. The resulting supernatant was separated and 20pl was injected for HPLC analysis.

90@6 9 S* 6

S

.3 0* 6 9 6 06 76 HPLC Conditions Column 100 x 4.6mm Kromasil KR 100 5C4 Mobile Phase 0.lm Tris pH7.5 86% Acetonitrile 14% Flowrate Iml/min Detection UV at 210nm Retention Time 3.4 mins The results of the analysis indicated a decrease in the mean plasma level of the drug from 10 70pg/ml at 2 minutes to 2pg/ml at 90 and 120 minutes.

Compound 217e Dosing and sampling The drug was dissolved in sterile 0.02M Tris to give a 2.5mg/ml solution which was administered to 11 groups of 5 male CD-1 mice via the tail vein at a dose of 25mg/kg. At each of the following timepoints: 2, 5, 10, 15, 20, 30, 45, 60, 90 and 120 minutes a group of animals was anaesthetised and the blood collected into heparinized tubes. After separation the 20 plasma was stored at -20 oC until assayed.

Assay Aliquots of plasma (100ul) were diluted with acetonitrile (100il) then mixed by vortexing for seconds before centrifugation for 10 minutes. The resulting supernatant was separated and 20l was injected for HPLC analysis.

HPLC Conditions Column 150 x 4.6mm Zorbax SBC8 Mobile Phase 0.05M Phosphate 72% buffer ph7.1 Acetonitrile 28% Flowrate 1.4ml/min Detection UV at 210nm Retention Time 3.0 and 3.6 mins (diasteromers) 77 The results of the analysis indicated a decrease in mean plasma concentrations from at 2 minutes to 0.2pg/ml at 60-120 minutes.

Example 3 Peptidyl ICE inhibitors are cleared rapidly with clearance rates greater than 80 ml/min/kg.

Compounds with lower clearance rates have improved pharmacokinetic properties relative to peptidyl ICE inhibitors.

10 We obtained the rate of clearance in the rat (ml/min/kg) for several compounds of this invention using the method described below: In vivo Rat Clearance Assay Cannulations of the jugular and carotid vessels of rats under anesthesia were performed one day prior to the pharmacokinetic study. M.J. Free, R.A.

Jaffee; 'Cannulation techniques for the collection blood and other bodily fluids'; in: Animal Models; p. 480-495; N.J. Alexander, Ed.; Academic Press; 20 (1978). Drug (10mg/mL) was administered via the jugular vein in a vehicle usually consisting of: propylene glycol/saline, containing 100mM sodium bicarbonate in a 1:1 ratio. Animals were dosed with 10-20 mg drug/kg and blood samples were drawn at 0, 2, 5, 7, 10, 15, 20, 30, 60, and 90 minutes from an indwelling carotid catheter. The blood was centrifuged to plasma and stored at -20 OC until analysis.

Pharmacokinetic analysis of data was performed by nonlinear regression using standard software such as RStrip (MicroMath Software, UT) and/or Pcnonlin (SCI Software, NC) to obtain clearance values.

78 Analytical: Rat plasma was extracted with an equal volume of acetonitrile (containing 0.1% TFA). Samples were then centrifuged at approximately 1,000 x g and the supernatant analyzed by gradient HPLC. A typical assay procedure is described below.

200 pL of plasma was precipitated with 200 pL of 0.1% trifluoroacetic acid (TFA) in acetonitrile and 10 pL of a 50% aqueous zinc chloride solution, vortexed 10 then centrifuged at -1000 x g and the supernatant collected and analyzed by HPLC.

HPLC procedure: Column: Zorbax SB-CN (4.6 x 150 mm) S***particle size) Column temperature: 50 °C Flow rate: 1.0 mL/min Injection volume: 75 pL.

Mobile phase: A=0.1% TFA in water and B=100% acetonitrile S 20 Gradient employed: 100% A to 30% A in 15.5 min 0% A at 16 min 100% A at 19.2 min Wavelength: 214 nm A standard curve was run at 20, 10, 5, 2 and 25 1 pg/mL concentrations.

Example 4 Whole Blood Assay for IL-11 Production We obtained IC 50 values for several compounds of this invention using the method described below: Purpose: The whole blood assay is a simple method for measuring the production of IL-lb (or other cytokines) and the activity of potential inhibitors. The complexity of this assay system, with its full complement of lymphoid and inflammatory cell types, spectrum of plasma proteins and red blood cells is an ideal in vitro representation of human in vivo physiologic conditions.

79 Materials: Pyrogen-free syringes 30 cc) Pyrogen-free sterile vacuum tubes containing lyophilized Na 2 EDTA (4.5 mg/10 ml tube) Human whole blood sample 30-50 cc) ml eppendorf tubes Test compound stock solutions 25mM in DMSO or other solvent) Endotoxin-free sodium chloride solution and HBSS 10 Lipopolysaccharide (Sigma; Cat.# L-3012) stock solution at Img/ml in HBSS IL-l1 ELISA Kit (R D Systems; Cat TNFa ELISA Kit (R D Systems; Cat Water bath or incubator Whole Blood Assay Experimental Procedure: Set incubator or water bath at 30 OC.

Aliquot 0.25ml of blood into 1.5 ml eppendorf tubes.

Note: be sure to invert the whole blood sample tubes after every two aliquots. Differences in replicates 20 may result if the cells sediment and are not uniformly suspended. Use of a positive displacement pipette will also minimize differences between replicate aliquots.

Prepare drug dilutions in sterile pyrogenfree saline by serial dilution. A dilution series which brackets the apparent K i for a test compound determined in an ICE inhibition assay is generally used for the primary compound screen. For extremely hydrophobic compounds, we have prepared compound dilutions in fresh plasma obtained from the same blood donor or in PBS-containing 5% DMSO to enhance solubility.

Add 25 pl test compound dilution or vehicle control and gently mix the sample. Then add 5.0 pl LPS solution (250 ng/ml stocked prepared fresh: 5.0 ng/ml 80 final concentration LPS), and mix again. Incubate the tubes at 30 °C in a water bath for 16-18 hr with occasional mixing. Alternatively, the tubes can be placed in a rotator set at 4 rpm for the same incubation period. This assay should be set up in duplicate or triplicate with the following controls: negative control- no LPS; positive control- no test inhibitor; vehicle control- the highest concentration of DMSO or compound solvent used in the experiment.

10 Additional saline is added to all control tubes to normalize volumes for both control and experimental whole blood test samples After the incubation period, whole blood samples are centrifuged for 10 minutes at 2000 rpm in the microfuge, plasma is transferred to a fresh microfuge tube and centrifuged at 1000 x g to pellet residual platelets if necessary. Plasma samples may be stored frozen at -70 °C prior to assay for cytokine levels by ELISA.

20 ELISA: We have used R D Systems (614 McKinley Place N.E. Minneapolis, MN 55413) Quantikine kits for measurement of IL-10 and TNF-a. The assays are performed according to the manufacturer's directions.

We have observed IL-10 levels of 1-5 ng/ml in positive controls among a range of individuals. A 1:200 dilution of plasma for all samples has been sufficient in our experiments for ELISA results to fall on the linear range of the ELISA standard curves. It may be necessary to optimize standard dilutions if you observe differences in the whole blood assay. Nerad, J.L. et al., J. Leukocvte Biol., 52, pp. 687-692 (1992) 81 Example Inhibition of ICE homoloqs 1. Isolation of ICE homologs Expression of TX in insect cells using a baculovirus expression system. We have subcloned Tx cDNA (Faucheu et al., supra 1995) into a modified pVL1393 transfer vector, co-transfected the resultant plasmid (pVL1393/TX) into insect cells with viral DNA and identified the recombinant baculovirus. After the 10 generation of high titer recombinant virus stock, the medium was examined for TX activity using the visible ICE assay. Typically, infection of Spodoptera frugiperda (Sf9) insect cells at an MOI of 5 with recombinant virus stock resulted in a maximum expression after 48 hours of 4.7pg/ml. ICE was used as a standard in the assay.

Amino terminal T7 tagged versions of ICE or .TX were also expressed. Designed originally to assist the identification and purification of the recombinant S 20 proteins, the various constructs have also allowed examination of different levels of expression and of C the relative levels of apoptosis experienced by the different homologs. Apoptosis in the infected Sf9 cells (examined using a Trypan Blue exclusion assay) was increased in the lines expressing ICE or TX relative to cells infected with the viral DNA alone.

Expression and purification of N-terminally (His) 6 tagged CPP32 in E. coli. A cDNA encoding a CPP32 (Fernandes-Alnemri et al, supra 1994) polypeptide starting at Ser (29) was PCR amplified with primers that add in frame XhoI sites to both the 5' and 3' ends of the cDNA and the resulting XhoI fragment ligated into a Xho I-cut pET-15b expression vector to create an in frame fusion with (his) 6 tag at the n-terminus of 82 the fusion protein. The predicted recombinant protein starts with the amino acid sequence of MGSSHHHHHHSSGLVPRGSHMLE, where LVPRGS represents a thrombin cleavage site, followed by CPP32 starting at Ser E. coli BL21(DE3) carrying the plasmid were grown to log phase at 30 oC and were then induced with 0.8 mM IPTG. Cells were harvested two hours after IPTG addition. Lysates were prepared and soluble proteins were purified by Ni-agarose chromatography. All of the 10 expressed CPP32 protein was in the processed form. Nterminal sequencing analysis indicated that the processing occurred at the authentic site between Asp (175) and Ser (176). Approximately 50 pg of CPP32 protein from 200 ml culture. As determined by active site titration, the purified proteins were fully active. The protease preparation were also very active in vitro in cleaving PARP as well as the synthetic DEVD-AMC substrate (Nicholson et al, supra 1995).

2. Inhibition of ICE homologs 20 The selectivity of a panel of reversible inhibitors for ICE homologs is depicted in Table 1. ICE enzyme assays were performed according to Wilson et al (supra 1994) using a YVAD-AMC substrate (Thornberry et al, supra 1992). Assay of TX activity was performed using the ICE substrate under identical conditions to ICE. Assay of CPP32 was performed using a DEVD-AMC substrate (Nicholson et al., supra 1995). In general, there is low selectivity between ICE and TX for a wide range of scaffolds. None of the synthetic ICE compounds tested are effective inhibitors of CPP32. Assay of the reversible compounds at the highest concentration (1 pM) revealed no inhibition.

83 Table 1 Compound K i ICE (nM) K i TX (nM) K i CPP32 (nM) 214e 7.5 7.0 1.1 1000 135a 90 55 9 >1000 125b 60 57 13 1000 137 40 40 7 1000 Second-order rate constants for inactivation of ICE and ICE homologs with selected irreversible inhibitors are presented below (Table The 10 irreversible compounds studied are broad spectrum inhibitors of ICE and its homologs. Some selectivity, however, is observed with the irreversible compounds a comparing inhibition of ICE and CPP32.

Table 2 Compound kinact kinact (TX) kinact (ICE) (CPP32) M-1 M- 1

S-

1

M-

1 s- 1 138 120,000 150,000 550,000 S* 217d 475,000 250,000 150,000 Example 6 Inhibition of apoptosis 20 Fas-Induced Apoptosis in U937 cells. Compounds were S"evaluated for their ability to block anti-Fas-induced apopotosis. In a preliminary experiment using RT-PCR, we detected mRNA encoding ICE, TX, ICH-1, CPP32 and CMH-1 in unstimulated U937 cells. We used this cell line for apoptosis studies. U937 cells were seeded in culture at 1 x 105 cells/ml and grown to -5 x 106 cells/ml. For apoptosis experiments, 2 x 10 cells were plated in 24-well tissue culture plates in 1 ml RPMI-1640-10% FBS and stimulated with 100 ng/ml anti- Fas antigen antibody (Medical and Biological Laboratories, Ltd.). After a 24 hr incubation at 84 37 the percentage of apoptotic cells was determined by FACS analysis using ApoTag reagents.

All compounds were tested initially at 20 pM and titrations were performed with active compounds to determine IC50 values. Inhibition of apoptosis 75% at 20 pM) was observed for 136 and 138. An of 0.8 pM was determined for 217e compared to no inhibition of anti-Fas-induced apoptosis by 214e at S=0 •pM.

10 Example 7 In vivo acute assay for efficacy as @000 oanti-inflammatory agent LPS-Induced IL-1I Production.

Efficacy of 214e and 217e was evaluated in CD1 mice (n=6 per condition) challenged with LPS mg/kg IP). The test compounds were prepared in olive *o oil:DMSO:ethanol (90:5:5) and administered by IP injection one hour after LPS. Blood was collected seven hours after LPS challenge. Serum IL-10 levels were measure by ELISA. Results in Fig. 6 show a dose dependent inhibition of IL-10 secretion by 214e, with an ED 50 of approximately 15 mg/kg. Similar results were obtained in a second experiment. A significant inhibition of IL-10 secretion was also observed in 217e treated mice (Fig. However, a clear dose response was not apparent.

Compounds 214e and 217e (50 mg/kg) were also administered by oral gavage to assess absorption.

Results in Fig. 8 show that 214e, but not 217e when administered orally inhibited IL-10 secretion, suggesting potential for oral efficacy of ICE inhibitors as anti-inflammatory agents.

85 C. C C a a.

*i C

C.

Oe 0

B

0.

Ca C 0*

S

a..

0 CaSge*

S

CC

a a Ce..

Ca..

*5 a. 0 The efficacy of analogs of 214e were also evaluated in LPS challenged mice after IP administration (Fig. 9) and PO administration (Fig. Table 3 Inhibition of IL-P3 production by analogs of 214e in LPs-chellenged mice after P0 and IP administration (50 mg/kg).

Table 3 P0% IP% Compound Inhibition Inhibition 0 214e 75 78 416 52 39 434 80 74 438 13 442 10 0 5 2002 78 Table 4 Comparison of 214e Prodrugs for Efficacy in LPS Challenged Mice: Time Course Inhibition of IL-13 Production Time of Compound Administration (relative to time of LPS challenge, PO, 50 mg/kg D Compound -2 hr -1 hr 0 hr hr 214e 39* 43* 44* 48* 11* 47* 304a 30 33 68 37 2100e 49 54 94 66 2100a 8 71 67 58 213e 0 48 41 89 302 0 27 21 26 2100c 0 0 85 2100d 42 35 52 26 2100b 0 0 47 26 a C Ce S. a C C Ce 86 2001 -63 62 -57 -54 64* 62* 58* Values obtained in subsequent assays Example 8 Measurement of blood levels of prodruqs of 214e.

Mice were administered a p.o. dose of compounds 302 and 304a (50 mg/kg) prepared in 0.5 carboxymethylcellulose. Blood samples were collected at 1 and 7 hours after dosing. Serum was extracted by o precipitation with an equal volume of acetonitrile 10 containing 2 formic acid followed by centrifugation.

,The supernatant was analyzed by liquid chromatographymass spectrometry (ESI-MS) with a detection level of 0.03 to 3 pg/ml. Compounds 302 and 304a showed detectable blood levels when administered orally, 214e itself shows no blood levels above 0.10 pg/mL when Sadministered orally. Compounds 302 and 304a are prodrugs of 214e and are metabolized to 214e in vivo (see Fig. 11).

Example 9 We obtained the following data (see Tables and 6) for compounds of this invention using the methods described in Examples 1-8. The structures of the compounds of Example 9 are shown in Example 10-12.

Table Cell Whole Clearance UV- PBMC human learance Rat, i.v.

Compound Visible avg. blood Mouse, ml/min/kg Ki (nM) IC50 IC50 l/min/ (nM) (nM) ml/min/kg 47b 27 1800 <600 338 47a 19 2600 5100 79 32 135a 90 2800 5000 >100 135b 320 1600 1700 125b 60 800 4500 137 40 1700 14000 139 350 2000 87 *0 0 0@ 0* 0

SO

@0

S

000000 0

S@

0 S 0000 0@ 0

SSS

0@@005

S

0 @000 0 0 0@ S Cell Whole Clearance Rlaance UV- PBMC human Mouse, mi/mm/kg.

Compound Visible avg. blood iv lmnk Ki (nM) 1C50 1C50 ml/min/kg I(nM) (nM) 213e 130 900 600 400* 214c 1200 5000 214e 7.5 1600 1300 23 12 217c 1700 7000 5 217e 175 2000 220b 600 2125 223b 99 5000 >100 223e 1.6 3000 >20000 89 226e 15 1100 1800 109 10 227e 7 234 550 230e 325 300 67 232e 1100 4500 22 2 6 235e 510 4750 36 238e 500 4250 15 246 12 950 10000 31 257 13 11000 6 600 281 50 600 2 302 4500 >20000 >20000 304a 200 1,400 2400 14 000* 20 307a 55 14500 16000 307b 165 14000 404 2.9 1650 1100 64 24 180 0* 405 6.5 1700 2100 406 4 1650 2300 407 0.4 540 1700 408 0.5 1100 1000 41 23 409 3.7 2500_____ 410 17 2000 2800 32 411 0.9 540 1900 412 1.3 580 700 660* 1000* 413 750 6200 415 2.5 990 450 26 18 1000* 3500n*

OS

S S @0 50 0 S 55 50 88 0 0@ 0@ 0@ 0*

S.

0

S

0@ 0 @00@ 0*

S@S

@OSOOO

OS..

0S5* 0000 S 0O S Cell Whole Clearance Rlaance UV- PBMC human Mouse, Rati/kg Compound Visible avg. blood iV lmnk Ki IC50 1C50 ml/min/kg (rim) (nM) 416 12 1200 3400 47 417 8 2000 6000 33 22 418 2.2 1050 7800 13 5.9 2200* 1800* 419 280 >8000 '420 1200 8000 >80 00 421 200 4300 4 600* 422 50 2200 1200 423 10 2100 1500 18 00* 424 45 2500 4000 425 0.8 650 650 700* 426 90 4500 2 500* 427 180 4500 36 428 280 429 7000 430 60 >8000 431 8 >8000 8000 432 1.6 560 2000 433 2.9 1000 1100 434 4.9 1600 1800 1200* 1300* 435 8 4400 436 7.5 2700 437 12 1800 5000 438 28 1000 700 22 2 90 0* 439 3.7 2800 3200 34 00* 440 2.3 5000 2000 441 1 2500 4500 442 3.2 900 2000 54 443 3.6 2800 1500 444 15 3500 3500 445 135 4000 5* 0 00 @5 0 0O 0 55 89 0S S S S *5 0S

S*

SOS...

9 5O

S

S.

0 @00 00 0 U Cell Whole C anceClearance Copond Vbl PBMC human Mouse, Rat/mi/kg CopudVsbe avg. blood lmnk Ki (nM) IC50 1C50 i*v.

446 62 3000 447 5.8 2500 1500 448 130 4000 449 12 1500 3200 00* 450 5 800 2200 18 12 17 00* 451 4 1800 1500 900 0* 452 4.5 600 650 27.3 800* 1600* 453 0.65 1300 1900 1600* 454 45 2500 455 1.2 400 2800 54 2 600* 456 4.5 600 600 12.7 1400* 457 6.2 2000 3500 458 20 2900 459 5 1800 460 115 400 2400 461 47 462 463 14 2400 28 00* 464 2.5 1000 >1000 2 50 465 3 1000 800 466 0.8 1400 600 467 11 1900 468 4.5 850 2500 470 5 500 360 63 500* 471 1 750 400 17 472 140 473 1 1000 400 450* 474 5 0 S Se 50 5 S S S S 55 90 C. 0

C

0@ C C 0S

S

oem...

0 0O 0 S

S.

C

.me 0 0 @0CC

C

.me.

*0@O S C em S Cell Whole Clearance Rlarance UV- PBMC human Mouse, Ratmi/k Compound Visible avg. blood iv lmnk K i (n1M) IC50 IC50 ml/mV*k (rim) (nM) m/m/k 475 5.5 690 400 31 21 650* 350* 476 7 1600 2500 477 478 380 479 15 900 700 24 00* 480 25 2300 481 1.2 390 600 34 930* 500* 482 <0.2 340 380 260* 483 1.7 900 700 484 2 1550 5000 14 00* 485 2 900 900 486 2.3 480 500 37 570* 487 2.4 650 500 950* 400* 488 1.5 940 750 489 6 2250 15000 17 00* 490 4.3 980 700 1000* 1900* 491 5 2500 493 25 1200 800 850* 494 15 1350 7000 0* 495 43 496 16 1550 6000 1600* 497 3.5 740 350 700* 498 1.5 560 500 400* 499 3.5 1200 9000 800* 2100e 250 800 600 2100a 100 1100 850

S.

S S em *5 0 me em 91 0* 0 0 S @0 0@ 0* 00 0 *00000 0 00 0 S 0000

S.

S

*0@ Cell Whole Clearance Rlaaice UV- PBMC human Mouse, Ral/mi/kg Compound Visible avg. blood iv lmnk K i (riM) IC50 IC50 1/in/V (nM) (rim) mlmnk 2002 4 810 70 32 860* 1400* 2100d >1000 00 >20000 >20000 2100c 7400 >20000 >20000 2100b 8000 >20000 >20000 5 2001 135 1800 3500 Table 6 1Fluorescent Cell1 WholeClanc Assay PBMC human Clenc Clearance Compound kiat avg. blood Rat, i.v.

1 1 C50 C50ml/min/kg ml/min/kg M s (n14) (riM) 136 5.4xl10 5 870 2800 93 138 1.2x10 5 900 2900 116 10 217d 4.7xl10 5 340 4000 280 4x10 5 650 >1000 187 283 1X10 5 <200 450 104 284 3.5x10 5 470 550 77 .100 285 4.0 5 810 1000 130 *Values obtained upon reassay.

Examiple Compound 139 was synthesized by a method similar to the method used to synthesize 47a.

0 0@S@S0

S

OSS0 @000 S S 0S S 0 0 0 00 139 CH 3 Compounds 136 and 138 were synthesized by a method similar to the method used to synthesize 57b.

92 0 0N0 HN O H 0 CI H 0 ;and *0 S 0@

OS

0@ S @5 S0 *5@005 0 0O 0 *550 @0 0 *0S Compounds 135a, 135b, and 137 were 5 synthesized by a method similar to the method used to synthesize 69a.

a 505005 0 *050 0 0550 0O 0@ S 0 1 1 55 0 0 @5 05 5 00 ;and Compounds 830e, 832e, 835e, 838e, 846, 857, 865, 907a, 907b, 1015-1045, and 1070-1091 were synthesized by methods similar to those used to synthesize compound 264 and the corresponding compounds in Examples 10 and 11.

93 Compounds 47a, 47b, 108a, 108b, 125b, 213e, 214c, 217c, 217d, 217e, 220b, 223b, 223e, 226e, 227e, 230e, 232e, 235e, 238e, 246, 257, 280-287, 302, 304a, 307a, and 307b were synthesized as described below.

X X o o H O CO-t-Bu H O CO 2

H

5 44 X X S o o 0 0S r^^N^N o

N

r C" 0 2

H

H o0 H N0

CHO

H OBn H 46 47 X X H 2 (44a). To a solution of (1S,9S)t-butyl 9-amino-6,10dioxo-octahydro-6H-pyridazino [1,2-a][1,2]diazepine-1carboxylate (690mg; 2.32mmol; GB 2128984) in dioxane (16ml) and water (4ml) at 0 OC was added solid sodium bicarbonate (292mg; 3.48mmol) followed by dropwise 15 addition of 3-phenylpropionyl chloride (470mg; 2.78mmol). The mixture was stirred at room temperature for 2h then more sodium bicarbonate (200mg; 2.38mmol) and 3-phenylpropionyl chloride (100mg; 0.6mmol) were added. The mixture was stirred for a further 2h at room temperature, diluted with ethyl acetate washed with saturated sodium bicarbonate (2 x then dried (MgSO 4 and concentrated. The residue was purified by flash chromatography (0-50% ethyl acetate/chloroform) and finally crystallized by

-L

94 trituration with ether to afford 860mg of a white solid: mp. 137-138 [a1D 23_95.10 (c 0.549, CH 2 C1 2 IR (KBr) 3327, 1736, 1677, 1664, 1536, 1422, 1156; 1NMR (ODC1 3 (5 7.24 (5H, in), 6.50 (1H, d, 5.24 (1H, in), 4.90 (1H, mn), 4.60 (1H, mn), 3.44 (1H, mn), 2.93 (2H, mn), 2.84 (1H, mn), 2.64 (1H, mn), 2.54 (2H, in), 2.26 (2H, mn), 1.70 (4H, in), 1.70 (9H, MS(FAB, in/z): 430 (M 374, 242, 105, 91.

(44b) was prepared from (1S,9S) t-butyl 9-aminosee,: 10 octahydro-10-oxo-6H-pyridazino[1,2-a] [1,2]diazepine-1- Osseo:carboxylate (Attwood et al., J. Chemn. Soc. Perkin 1, .00. pp. 1011-19 (1986)) as for 44a, to afford 810mg (81%) of a colorless oil: [Ct]D 2 33.50 (c 0.545, CH 2 C1 2

IR

(film) 3334, 2935, 1737, 1728, 1659, 1642; 1H NNR (ODC1 3 5 7.24 (5H, mn), 6.75 (1H, d, 5.27 (1H, in), 4.92 (1H, in), 3.39 (1H, in), 3.03 (4H, in), 2.55 (3H, Osseoin), 2.33 (1H, mn), 2.17 (1H, in), 1.80 (5H, mn), 1.47 (9H, 0 *0*000 1.39 (1H, in). MS (FAB, mn/z) 4 16 (M 1) 3 '00:94'211, 143, 97.

0* 0 20 (45a). To a solution of (1S,9S) t-butyl 6,10-dioxooctahydro-9- (3-phenylpropionylanino) -6Hpyridazino[1,2-a] [l,2]diazepine-1-carboxylate (44a) (800mg; 1.863mmio1) in dry dichloroinethane (5mi) at 0 OC was added trifluoroacetic acid (5m1) The solution was stirred at room temperature for 3h then concentrated.

Dry ether (l1inl) was added to the residue then removed under vacuum. This process was repeated three times to afford a crystalline solid. The solid was triturated with ether and filtered to afford 590mg of a white crystalline solid: mp. 196-197.5 [cx] 0 23 -129.50 (c 0.2, CH 3 OH); IR (KBr) 3237, 1729, 1688, 1660, 1633, 1574, 1432, 1285, 1205; 1H NNR (CD 3 OD) 6 8.28 (1H, d, 7.22 (5H, mn), 5.32 (1H, dd, J=5-9, 4.75 (1H, in), 4.51 (1H, in), 3.50 (1H, in), 3.01 95 (1H, mn), 2.91 (2H, in), 2.55 (2H, mn), 2.29 (3H, in), 1.95 (2H, mn), 1.71 (2H, mn). Anal. Calcd for C 1 9

H

2 3

N

3 0 5

C,

61.12; H, 6.21; N, 11.25. Found: C, 60.80; H, 6.28; N, 10.97. MS(FAB, m/z) 374 242, 105, 91.

(45b) was prepared from (1S,9S) t-butyl octahydro-lOoxo-9- (3-phenylpropionylamino) -6Hpyridazino[1,2-a] (1,2]diazepine-1-carboxylate (44b) by the method described for compound 45a to afford 657mg OS of 45b as a crystalline solid: mp. 198-202 0

C;

23D 86.20 (c 0.5, CH 3 OH) ;IR (KBr) 3294, 2939, 1729, 1645, 1620, 1574, 1453, 1214; 1 H NMR (CD 3 OD) (5 7.92 (1H, d, 7.20 (5H, in), 5.29 (1H, mn), 4.90 (1H, inm), 3.47 (1H, in), 3.08'(2H, in), 2.90 (2H, mn), 2.55 (3H, mn), 2.36 (1H, mn), 1.81 (5H, in), 1.43 (2H, mn). MS(FAB, m/z) 360 (M 211,143,91.

(46a). To a solution of (1S,9S) 6,10-dioxo-octahydro- 9- (3-phenyl-propionylamino) -6H-pyridazino[1,2-a] obes* [1,2]diazepine-1-carboxylic acid. (45a) (662mg; 1.773mniol) in dry dichioroinethane (9m1) and dry dimethyl forinamide (3in1) at room temperature was added bis (triphenylphosphine)palladiun chloride (30mg) and (3S, 2R,S) -3-allyloxycarbonylamino-2-benzyloxy-5oxotetrahydrofuran (Chapman, Bioorq. Med. Chem. Lett., 2, pp. 613-18 (1992)) (568mg; 1.95inmol) followed by dropwise addition of tri-n-butyltin hydride (1.19g; 4.O9mmiol) 1-Hydroxy-benzotriazole (479mg; 3.S46ramol) was added to the mixture and the mixture was cooled to 0 'C before addition of 1-(3-dimethylaminopropyl)-3ethylcarbodiimide hydrochloride (408mg; 2.l28mmiol).

The mixture was stirred at room temperature for 3.25h then diluted with ethyl acetate (S5inl), washed twice with dilute hydrochloric acid (20in1), twice with saturated sodium bicarbonate (20m1), once with brine then dried (MgSO 4 and concentrated. The resulting oil 96 was purified by flash chromatography (0-100% ethyl acetate/chloroform) to afford 810mg of 46a as a mixture of anomers: mp. 92-94 0 C; IR (KBr) 3311, 1791, 1659, 1651, 1536; 1H NMR(CDCl 3 (5 7.49, 6.56 (1H, 2d, J=6.7, 7.29 (10H, in), 6.37, 6.18 (1H, 2d, 5.56, 5.34 (1H, d, s, 5.08-4.47 3.18-2.80 2.62-2.28 2.04-1.53 MS (FAB, m/ z) 5 63 (M 328, 149, 91.

(46b) was prepared from 45b by the method described for 46a to yield 790mg of a glass: m.p. 58-60'C; IR (KBr) 3316, 2940, 1793, 1678, 1641, 1523, 1453, 1120; 1 H NNR (CDCl 3 5 7.28 (iON, in), 6.52, 6.42 (1H, 2d, **oeJ=7.2, 5.53, 5.44 (1H, d, s, 5.35 (1H, in), 4.6-4.9, 4.34 (4H, in), 3.1-2.8 (6H, in), 2.6-2.1 1.95-1.05 MS(FAB, 549 (M 400, 310, 279, 91.

(47a). A mixture of [3S, 2R,S, (1S,9S)] N-(2benzyloxy-5-oxotetrahydrofuran-3-yl) octahydro-9- (3-phenylpropionylamino) -6Hpyridazino[1,2-a] [1,2]diazepine-1-carboxanide (46a) (205mg; 0.364inmo1), 10% palladium on carbon (200mg) and methanol (20m1) was stirred under hydrogen at atmospheric pressure for 5h. The mixture was filtered then concentrated to yield 154mg of a glass: mp.

116-118'C; [Ct]D 23 _1400 (c 0.1, CH 3 OH); IR (KBr) 3323 1783, 1731, 1658, 1539, 1455, 1425; 1H NMR

(CD

3 OD) 5 7.21 (5H, in), 5.17 (1H, in), 4.73 (1H, mn), 4.50 (2H, in), 4.23 (1H, in), 3.38 (1H, in), 3.06 (1H, in), 2-91 (2H, in), 2.73-2.18 (6H, in) and 2.01-1.59 (5H, mn).

Anal. Calcd for C 2 3

H

2 7

N

4 0 7

H

2 0: C, 56.32; H, 6.16; N, 11.42. Found: C, 56.29; H, 6.11; N, 11.25. MS(FAB, in/z) 473 (M 1) 176, 149, 105, 91.

(47b) was prepared from 46b by the method described for 47a. The residue was purified by flash chromatography 97 (0-10% methanol/chloroform) to afford 65mg of a glass; m.p. 87-90 0 C; [a]D 2 3 -167.00 (c 0.1, methanol); IR (KBr) 3329, 2936, 1786, 1727, 1637; 1H NMR (CD 3 0D) 7.23 (5H, 5.29 (1H, 4.83 (1H, 4.59 (1H, d, 4.29 (1H, 3.3-3.0 (3H, 2.91 (2H, m), 2.70-2.34 (5H, 2.19 (2H, 1.75 (4H, 1.36 (2H, Anal. Calcd for C 2 3

H

3 0

N

4 0 6 0.5H 2 0: C, 59.09; H, 6.68; N, 11.98. Found: C, 58.97; H, 6.68; N, 11.73.

MS(FAB, m/z) 459 310, 149, 105, 91.

A solution of 5-(2,6-Dichlorophenyl)oxazole described in Tet. Lett. 23, p. 2369 (1972)) in tetrahydrofuran (65mL) was cooled to -78 oC under a nitrogen atmosphere. To this solution was added n- 15 butyl lithium (1.5M solution in hexanes, 13.3mmol) and stirred at -78 oC for 30min. Magnesium bromide etherate (3.6g, 13.9mmol) was added and the solution was allowed to warm to -45 oC for 15min. The reaction was cooled to -78 oC and aldehyde 58 (3.26g, 12.7mmol; Graybill et al., Int. J. Protein Res., 44, pp. 173-182 (1993)) in tetrahydrofuran (65mL) was added dropwise. The reaction was stirred for 25min., then allowed to warm to -40 °C and stirred for 3h, and then at room temperature for lh. The reaction was quenched with 5% NaHCO 3 (12mL) and stirred for 3h. The tetrahydrofuran was removed in vacuo and the resulting residue was extracted with dichloromethane. The organic layer was washed with saturated sodium chloride solution and dried over magnesium sulfate, filtered, 98 and concentrated to yield 6.14g of the title compound.

Purification gave 4.79g of 99: 1H NNR (CDCl 3 )6 1.45(s, 9H), 2.7-2.5(m, 2H), 2.8(dd, 1H), 4.2, 4.4(2 x d, 1H), 4.7-4.5(m, 3H), 5.35-5.1Cm, 2H), 5.6, 5.7(2 x d, 1H), 6.0-5.8(m, 1H), 7.2(d, 1H), 7.3(m, 1H), 7.4(m, 2H).

C 2tBu C02-tBu ~H H be122 123 S C02-tBu cC C H H

N.H

124 125 N. 0 0 a R =0o b 0 (123) Potassium fluoride (273mg, 4.70rnmol) and then 2-chiorophenylmethyl thiol (373mg, 2.35inmol) were added to a stirred solution of (3S) t-butyl N- Callyloxycarbonyl) -3-amino-5-bromo-4-oxo-pentanoate (122; 749mg, 2.l4mmol; WO 93 16710) in dimethylformamide (20m1) The mixture was stirred for quenched with water (50m1) and extracted with ethyl acetate (2 x 50m1) The combined organic extracts were washed with water (4 x 5Oml) then brine (50m1) They were dried (MgSO 4 and concentrated to afford an oil which was purified by flash chromatography (10-35% ethyl acetate/hexane) to afford 832 mg of a colourless solid: mp. 45-6 [a]D 2 0 -19.01 Cc 1.0, CH 2 Cl 2 IR (film) 3340, 2980, 2935, 99 0@ S 0 0* es S 0 @0

S

See..0

S

00 0 @000

S.

0 000 0 000050

S

*505 0 @550 0 0 00 0 *5 0 0 00 *5 So 05 1725, 1712, 1511, 1503, 1474, 1446, 1421, 1393, 1368, 1281, 1244, 1157, 1052, 1040, 995, 764, 739; 1H NMR (ODCd 3 5 7.36 (2H, in), 7.21 (2H, mn), 5.91 (2H, in), 5.27 (2H, in), 4.76 (1H, in), 4.59 (2H, 3.78 (2H, 3.36 (2H, in), 2.91 (1H, dd), 2.74 (1H, dcl), 1.43 (9H. s).

Anal. Calcd for C 2 0

H

2 6 ClNO 5 S: C, 56.13; H, 6.12; N, 3.27; S, 7.49. Found: C, 56.08; H, 6.11; N, 3.26; S, 7.54. MS 43b/28 1, 374/2 (100).

(124a) 6-Benzyl-1,2-dihydro-2-oxo-3-(3- 10 phenylpropionylamino)-pyridyl acetic acid (52b; 300mg, 0.76rnmol) in THF (7m1) was stirred with 1hydroxybenzotriazole (205mg, 1.S2mmiol) and l-(3dimethylaminopropy-3-ethylcarbodiimide hydrochloride).

After 3 min, water (12 drops) was added and the mixture stirred 10mmn then treated with t-butyl (3S) N- (allyloxycarbonyl) -3-amino-5- (2chlorophenylmethylthio)-4-oxopentanoate (123) (325mg, 0.76mmol), bis (triphenyiphosphine) palladium II chloride (20mg) and tributyltin hydride (0.6m1, 2.28mnol) The mixture was stirred for 5h at room temperature, poured into ethyl acetate and washed with aqueous 1M HCl aqueous sodium bicarbonate, brine, dried (MgSO 4 and concentrated. The residue was triturated with pentane and the supernatant discarded.

Chromatography (silica gel, 50% ethyl acetate/hexane) afforded a colourless foam (439mg, 81%) [a3D 21_18.30 (c 0.5, CH 2 Cl 2 IR (KBr) 3356, 3311, 1722, 1689, 1646, 1599, 1567, 1513, 1367, 1154; 1H NMR (CDC 3 56 8. 39 (1 H, 8.23 (1H, 7.24 (14H-, in), 6.16 (1H, 4.95 (1H, mn), 4.63 (2H, in), 4.02 (2H, 3.74 (2H, 3.27 (2H, 2.85 (6H, in), 1.40 (9H, Anal. Calcd for

C

39

H

42 C1N 3 0 6 S: C, 65.39; H, 5.91; N, 5.87. Found: C, 65.51; H, 5.99; N,5.77.

100 (124b) was prepared by a similar method as 124a from the thioether 123 and 3S(1S,9S)-3-(6,10-dioxo- 1,2,3,4,7,8,9,10-octahydro)-9-(3-phenylpropionylamino)- 6H-pyridazino[l,2-a][1,2]diazepine-1-carboxylic acid (45a) to afford 452mg of colourless foam: mp 7 [a]D 2 2 -94.00 (c 0.12, CH 2 C1 2 IR (KBr) 3288, 2934, 1741, 1722, 1686, 1666, 1644, 1523, 1433, 1260, 1225, 1146, 757; 1H NMR (CDC13) 6 7.35 (3H, 7.20 S (7H, 6.46 (1H, 5.21 (1H, 4.97 (2H, 4.56 (1H, 3.75 (2H, 3.25 (3H, 2.93 (5H, 2.71 (1H, dd), 2.55 (2H, 2.30 (1H, 1.92 (3H, m), 1.66 (2H, 1.42 (9H, Anal. Calcd for 0

C

35

H

4 3 C1N 4 0 7 S. 0.25H 2 0: C, 59.73; H, 6.23; Cl, 5.04; N, 7.96; S, 4.56. Found: C, 59.73; H, 6.19; Cl, 5.10; N, 7.79; S, 4.58. MS (-FAB) 697 100).

(125a). t-Butyl-3(2(6-benzyl-l,2-dihydro-2-oxo-3- (3phenylpropionylamino)-1-pyridyl)acetyl-amino-5-(2chlorophenylmethylthio)-4-oxopentanoate (124a) (400mg, 0.56mmol) in dichloromethane (3ml) at 0 oC was treated with trifluoroacetic acid (3ml) and stirred at 0 °C for lh and room temperature for 0.5h. The solution was sconcentrated then redissolved in dichloromethane and reconcentrated. This procedure was repeated three times. The residue was stirred in ether for lhr and filtered to yield a colourless solid (364mg, mp.

165-7 [a]D 2 2 -27.70 (c 0.2, CH 2 C1 2 IR (KBr) 3289, 1712, 1682, 1657, 1645, 1593, 1562, 1527, 1497, 1416, 1203, 1182; 1H NMR (CDC13) d 8.47 (1H, 8.21 (1H, 7.70 (1H, 7.22 (14H, 6.24 (1H, 5.03 (1H, 4.65 (2H, 4.06 (2H, 3.69 (2H, 3.23 (2H, 2.88 (6H, m).

(125b) was prepared by a similar method as 125a from the t-butyl ester 124b to afford 362mg of 101 colourless powder: mp 76-80 [a]D 21 -134 (c 0.10, MeOH); IR (KBr) 3309, 2935, 1725, 1658, 1528, 1445, 1417, 1277, 1219, 1175; 1H NMR (D 6 -DMSO) 6 8.80 (1H, d), 8.19 (1H, 7.31 (9H, 5.09 (1H, 4.74 (1H, m), 4.63 (1H, 4.35 (1H, 3.76 (2H, 3.28 (3H, m), 2.80 (5H, 2.52 (4H, 2.16 (2H, 1.90 (3H, m).

Anal. Calcd for C 31

H

35 C1 2

N

4 0 7 S. 0.25H 2 0: C, 57.49; H, 5.53; N, 8.65; S, 4.95. Found: C, 57.35; H, 5.43; N, 8.45; S, 4.88. MS (-FAB) 641 100).

CO

2 -t-Bu C0 2 -t-Bu Allocr-N" OH Alloc-. 0 H

H

81 201 2-Chlorophenylmethyliodide. A mixture of 2chlorophenylmethylbromide (4g, 19.47mmol) and Nal (14g, 97.33mmol) in acetone (40ml) was stirred under reflux for 1 hour. The reaction mixture was cooled, filtered and concentrated in vacuo. The residue was triturated with hexane and filtered. The solution was concentrated in vacuo, and the resulting oil purified by flash chromatography (silica, hexane) to afford the 1 title compound (4.67g, 63%) as an oil: 1H NMR (CDCI 3 6 7.34 (4H, 4.54 (2H, s).

(201). (3S) t-Butyl hydroxy-4-oxopentanoate (81, Chapman, et al., Bioorg. Med. Chem. Lett., 2, pp. 613-618 (1992) 0.144g, and 2-chlorophenylmethyliodide (0.569g, in CH 2 C12 (4ml) were stirred vigorously with silver oxide (0.231g, Immol) and heated at 38 °C for hours. The reaction mixture was cooled, filtered and the filtrate evaporated. The residue was purified by flash chromatography (silica, 0-20% ethylacetate in hexane) to afford the product as a colourless oil 102 (0.138g, [a]D 2 4 +3.90 (c 1.3, CH 2 C1 2 1 H NMR (CDC1 3 5 7.37 (4H, 5.88 (2H, 5.26 (2H, m), 4.69 (2H, 4.57 (3H, 4.50 (1H, 4.35 (1H, d), 3.03 (1H, dd), 2.76 (1H, dd), 1.42 (9H, s).

CO

2 t-Bu H Alloo-- H" AT- H OH I Sa a a 202 203 204 5 (203). A solution of 2,4-dichloro-6-nitrophenol (202, oo0. 40g containing 20% moisture) in EtOAc (500ml) was dried using MgSO 4 filtered and the filter cake washed with a little EtOAc. Platinum on carbon sulphided 2g) was added and the mixture hydrogenated until uptake of 10 H 2 ceased. Triethyl orthoformate (160ml) and p-toluene o 0:000 sulphonic acid (160mg) were added and the mixture 0 0 0 Soooo° refluxed for 4h. After cooling and removal of spent 0000 catalyst by filtration the solution was washed with Oo 0 sat. NaHCO 3 solution, water and brine, dried with MgSO 4 and evaporated to dryness. Trituration with hexane S0oo gave a solid which was collected by filtration, washed 0 0 o0 with hexane and dried to give the title compound P 00 (25.5g, 88%) as a crystalline solid: mp 98-99 oC; IR (KBr) 3119, 1610, 1590, 1510, 1452, 1393, 1296, 1067, 850; 1H NMR (CDC1 3 6 8.16 (1H, 7.69 (1H, d, J 7.42 (1H, d, J Anal. Calcd for C 7

H

3 Cl 2

NO:

C, 44.72; H, 1.61; N, 7.45; Cl, 37.70. Found: C, 44.84; H, 1.69; N, 7.31; Cl, 37.71.

(204). Magnesium bromide was prepared by reaction of Mg (7.45g, 0.30mole) in THF (516ml) with 12 (50mg) and 1,2-dibromoethane (26.3ml, 5 7 .3g, 0.30mole) at reflux for 2h and then cooling to -40 oC. To the above was added rapidly via cannula a solution of 2-lithio-5,7- 104 t-BuO 2 C. CO 2 H t-BuO2C, CO 2 H t-BuO 2 C O

H

NH

2 Alloc-NH Alloc-NH S 205a 206a R 205b 206b H

H

t-BuO2CN.NH2 t-BuO 2 C

O

Alloc-NH O Alloc-NH 208a 207a 208b 207b

*S

(205a). To a mixture of THF (200ml) and water (100ml) containing NaHCO 3 (16.6g, 0.2mol) was added glutaric acid t-butyl ester (10g, 49.2mmol) and then dropwise over 20 minutes allyl chloroformate (6.8ml, 64mmol).

The mixture was stirred for 2 hours, extracted with EtOAc, washed with a sat. hydrogenocarbonate solution, water and a sat. salt solution, dried and evaporated to an oil 205a (9.5g, [a]D 2 -6 (c 1.5, MeOH) 1 1 H NMR (D 6 -DMSO) 5 6.10 (1H, 5.96-5.88 (1H, m), 5.31-5.12 (2H, 4.45 (2H, 3.90-3.84 (1H, t), 2.18 (2H, 1.85-1.76 (2H, 1.36 (9H, s).

(205b) was prepared by an analogous method to 205a to afford a colourless oil (6.27g, []D 2 0 +16 0 (c 0.095, MeOH); IR (KBr) 3678, 3332, 3088, 2980, 2937, 1724, 1530, 1453, 1393, 1370, 1331, 1255, 1155, 1056, 995, 935, 845, 778, 757, 636, 583; H NMR (CDCl 3 6 9.24 (1H, broad 5.94-5.79 (1H, 5.58 (1H, d), 5.33-5.17 (2H, 4.55 (2H, 4.38-4.31 (1H, m), 2.41-1.95 (4H, 1.42 (9H, Anal. Calcd for 105

C

1 3

H

2 1NO 6 C, 54.35; H, 7.37; N, 4.88. Found: C, 54.4; H, 7.5; N, 4.8.

(206a). To a solution of 205a (3.6g, 12.5mmol) in THF (100ml) at 0 °C was added N-methyl morpholine 13mmol) followed by isobutyl chloroformate, (1.lml, 13mmol). After 15 minutes, this mixture was added to a suspension of NaBH 4 (0.95g, 25mmol) in THF (100ml) and MeOH (25ml) at -78 After 2 hours at -70 oC, the mixture was quenched with acetic acid, diluted with 10 EtOAc, washed with a sat. hydrogenocarbonate solution 3 times, water and a sat. solution of salt, dried and evaporated. Flash chromatography MeOH in CH 2 C1 2 afforded 206a as a colourless oil (2.4g, -100 (c 3.88, CH 2 C1 2 H NMR (CDCl 3 6 5.84 (1H, m), 5.34-5.17 (3H, 4.56-4.53 (2H, 3.68-3.59 (2H, 2.98 (1H, 2.40-2.30 (2H, 1.84-1.78 (2H, m), 1.43 (9H, Anal. Calcd for C 1 3

H

2 3

NO

5 C, 57.13; H, 8.48; N, 5.12. Found: C, 57.1; H, 8.6; N, (206b) was prepared by an analogous method to 206a which afforded the title compound as a light yellow oil (3.42g, [1 D20 +14 (c 0.166, MeOH); IR (KBr) 3341, 3083, 2976, 2936, 2880, 1724, 1533, 1454, 1419, 00 1369, 1332, 1251, 1156, 1062, 997, 933, 846, 777, 647; H NMR (CDC1 3 6 5.98-5.81 (1H, 5.35-5.10 (3H, m), 4.55 (2H, 3.70-3.56 (3H, 2.50-2.47 (1H, broad 2.37-2.30 (2H, 1.89-1.74 (2H, 1.44 (9H, s); Anal. Calcd for C 1 3

H

2 3

NO

5 C, 57.13; H, 8.48; N, 5.12.

Found: C, 56.9; H, 8.6; N, 5.6 (207a). To a solution of DMSO (1.51g, 19.3mmol) in

CH

2 Cl 2 (25ml) at -70 °C was added oxalyl chloride (1.34g, 19.3mmol). After 10 minutes at -70 OC, a solution of (206a) (2.4g, 8.8mmol) in CH 2 C1 2 (10ml) was added dropwise and the mixture stirred for 15 minutes at -70 oC. Diisopropylethylamine (3.4g, 26.3mmol) was 106 added and the mixture stirred at -25 °C for 15 minutes then diluting with EtOAc (50ml) washed with a solution of sodium hydrogen sulfate 2M, concentrated to give an oil which was used immediately without purification: 1H NMR (CDC1 3 5 9.5 (1H, 6.0-5.5 (2H, 5.5-5.1 (2H, 4.5 (2H, 4.2 (1H, 2.4-2.10 (2H, m), 2.05 (2H, 1.36 (9H, s).

(207b) was prepared by an analogous method to 207a which afforded an oil (2.95g, 96%) which was used 20 without further purification in the next step: [a] 20 +21° (c 0.942, MeOH); H NMR (CDC1 3 5 9.58 (1H, s), 6.05-5.80 (1H, 5.57 (1H, broad 5.35-5.18 (2H, 4.56 (2H, 4.34-4.24 (1H, 2.38-2.16 (3H, m), 1.96-1.73 (1H, 1.43 (9H, s).

(208a). To a solution of 207a (2.39g, 8.8mmol), in MeOH (20ml) was added sodium acetate (0.72g, 8.8mmol) and semicarbazide (0.98g, 8.8mmol) stirred overnight, concentrated and diluted with CH 2 C12 (100ml), washed with water, dried and concentrated. Flash chromatography MeOH in CH 2 C1 2 afforded 208a (2.10g, 73%) as an oil: []D20 -21 (c 2.550, CH 2 Cl 2 1 H NMR (CDC1 3 5 9.98 (1H, 7.27 (1H, 5.8 (1H, 5.5 (1H, 5.35-5.19 (2H, 4.58 (2H, 4.14 (1H, 2.37 (2H, 2.09 (1H, 2.0-1.75 (2H, m); Anal. Calcd for C 14

H

24

N

4 0 5 C, 51.21; H, 7.37; N, 17.06. Found: C, 50.2; H, 7.3; N, 16.1 (208b) was prepared by an analogous method to 208a which afforded a glassy oil (2.37g, []D20 (c 0.26, CHC1 3 IR (KBr) 3476, 3360, 2979, 2923, 1700, 1586, 1527, 1427, 1394, 1369, 1338, 1253, 1156, 1060, 997, 929, 846, 775; 1H NMR (CDC1 3 5 9.87 (1H, 7.09 (1H, 6.05-5.75 (3H, 5.58 (1H, 5.32-5.16 (2H, 4.54 (2H, 4.35 (1H, 2.32-2.26 (2H, m), 2.15-1.55 (2H, 1.41 (9H, Anal. Calcd for 107

C

14

H

24

N

4 0 5 C, 51.21; H, 7.37; N, 17.06. Found: C, 51.0; H, 7.5; N, 16.7.

0 0 Ri -N Ri-T H H 00 OHU *211 R1 MeSO 2 212 R1 MeSO 2 c) R 1 MeCO R1= MeCO 1 5 R PhCH 2 000 R1 PhCH 2 0C0 @01 1 e) R~ PhCO R PhCO R1 Fmoc (if) R 1 Fmoc (211b). A solution of t-butyl 9-amino-6, lO-dioxo- 1,2,3,4,7,8,9, 10-octahydro-6Hpyridazino[l,2-a] [l,2]diazepine-1-carboxylate

(GB

2,128,984; 831mg, 2.79mmol) and diisopropylethylamine (1.22m1, 6.99mmol, 2.5 equiv) in CH 2 Cl 2 (l0mi) under dry nitrogen was treated with methanesuiphonyl chloride (237ii1, 3.O7mmol 1.1 equiv) The mixture was stirred for lh, diluted with EtOAc (75m1) and washed with saturated NaHCO 3 (50m1) and saturated aqueous sodium chloride (30m1), dried (MgSO 4 and concentrated. Flash chromatography (10-35% EtOAc in CH 2 Cl 2 afforded 211b (806mg, 77%) as a colourless solid: mp 68-70 00; lot]D -_109 Cc 1.09, CH 2 Cl 2 IR (KBr) 3270, 2980, 2939, 1735, 1677, 1458, 1447, 1418, 1396, 1370, 1328, 1272, 1252, 1232, 1222, 1156, 1131, 991; 1H NMR (CDC1 3 6.15 (1H, 5.31 (1H, in), 4.65-4.11 (2H, in), 3.47 (1H, m) 2.99 O3H, 2.89 (1H, in), 2.72-2.51 (2H, in), 2.34 (1H, in), 2.26 (1H, mn), 2.05-1.62 (4H, in), 1.47 (9H, Anal. Calcd for C 15

H

23

N

3 0 6 S: C, 47.97;-H, 6.71; N, 11.19; S, 8.54. Found: C, 48.28; H, 6.68; N, 108 10.86; S, 8.28. MS FAB) 376 (M 1, 320 (100).

(211c). Acetic anhydride (307mg, 3.Olmmol) was added to a stirred mixture of t-butyl 9-amino-6,10-dioxo- 1,2,3,4,7,8,9,10-octahydro-6Hpyridazino[1,2-a] [1,2]diazepine-1-carboxylate (GB 2,128,984; 813.7mg, 2.74mmol), go diisopropylethylamine (884mg, 6.B4mmol) and CH 2 Cl 2 (20m1). The mixture was kept for 1h then diluted with 10 EtOAc, washed with NaHCO 3 solution then brine, dried (MgSO 4 and concentrated to yield a colourless oil.

product was purified by flash chromatography 8% MeOH/CH 2

C

2 to afford 211c (804mg, 71%) of 2C123 colourless powder: mp 162-3 Ka1D -_109 (c 1.03,

CH

2 C1 2 IR(KBr) 3358, 2974, 1733, 1693, 1668, 1528, 1462, 1431, 1406, 1371, 1278, 1271, 1250, 1233, 1217, **1154, 1124; 6 1H NMR (CDC1 3 d 6.32 (1H, 5.29-5.25 eg(1H, in), 4.98-4.85 (1H, in), 4.68-4.58 (1H, in), 3.55- 3.39 (1H, in), 2.91-2.66 (2H, in), 2.39-2.18 (2H, in), 2.03 (3H, 1.88-1.64 (4H, in), 1.47 (9H, Anal.

Calcd for C 16

H

25

N

3 0 5 C, 56.62; H, 7.43; N, 12.38.

Found: C, 56.62; H, 7.43; N,12.36; MS FAB) 340 (M+ 1, 40%) 284 (100).

(211d). Benzyl chloroformate (1.07g) was added dropwise to a stirred ice cold mixture of the (1S,9S) t-butyl 9-amino-6, 10-dioxo-l,2,3, 4,7,8,9,10-octahydro- 6H-pyridazino[1,2-a] [1,2]diazepine-1-carboxylate (GB 2,128,984; 1.55g, 5.2lmmol), NaHCO 3 (0.66g, 7.B2inmol), dioxan (32m1) and water (8m1) The mixture was kept at 5 'C for 15mmn then for 2h at room temperature. The mixture was diluted with EtOAc (50m1), washed twice with sat. NaHCO 3 solution, dried (MgSO 4 and concentrated. The oily residue was purified by flash chromatography to afford 211d (1.98g, 88%) of a 109 24 colourless oil: [a]D2 -56.4 (c 1.0, CH 2 Cl 2 IR(thin film) 3325, 2979, 2946, 1728, 1677, 1528, 1456, 1422, 1370, 1340, 1272, 1245, 1156, 1122, 1056, 916, 734, 699; 1H NMR (CDC1 3 6 7.29 (5H, 5.81-5.72 (1H, m), 5.26-5.20 (1H, 5.05 (2H, 4.69-4.51 (2H, m), 3.48-3.36 (1H, 2.81-2.51 (2H, 2.34-2.19 (2H, 1.90-1.54 (4H, 1.41 (9H, Anal. Calcd for

C

2 2

H

2 9

N

3 0 6 oH 2 0: C, 58.79; H, 6.92; N, 9.35. Found: C, 59.10; H, 6.57; N, 9.25; MS (ES 454 (M +Na, 87%), S* 10 432 (M 100).

o(211e). A solution of benzoyl chloride (1.61g, 11.47mmol) in CH 2 Cl 2 (15ml) was added dropwise to a stirred ice cold mixture of (1S,9S) t-butyl 9-amino- 6,10-dioxo-1,2,3,4,7,8,9,10-octahydro-6Hpyridazino[1,2-a][1,2]diazepine-l-carboxylate (GB 2,128,984; 3.1g, 10.43mmol), dry CH 2 Cl 2 (20ml) and diisopropylethylamine (4.54ml, 26.06mmol). The mixture was kept cold for lh then left at room temperature for The mixture was diluted with CH 2 C12, washed twice with brine, dried (MgSO 4 and concentrated. The residue was purified by flash chromatography methanol in CH 2 C12) to afford 211e (4.0g, 96%) of a colourless glass: mp 74-76 OC; [a]D 30 -75.0° (c 0.12,

CH

2 C1 2 IR (KBr) 3350, 2979, 2938, 1736, 1677, 1662, 1536, 1422, 1276, 1250, 1155; 1H NMR (CDC1 3 5 8.72 (2H, 7.53-7.40 (3H, 7.07 (1H, d, J 5.30 (1H, dd, J 3.0, 5.12 (1H, 4.66 (1H, m), 3.51 (1H, 2.90 (2H, 2.38 (1H, dd, J 13.2, 6.8), 2.25 (1H, 1.9 (2H, 1.70 (1H, Anal. Calcd for C 21

H

27

N

3 0 5 0.5H 2 0: C, 61.45; H, 6.88; N, 10.24.

Found C, 61.69; H, 6.71; N, 10.18.

(211f) was prepared in a similar manner to 211e, except 9 -fluorenylmethylchloroformate was used instead of benzoylchloride to give a white glassy solid 211f 110 (2.14g, mp 190-192 0C; [a]D 2 5 -81.50 (c 0.1,

CH

2 C1 2 IR (KBr) 3335, 2977, 1731, 1678, 1450, 1421, 1246, 1156, 742; IH NMR (CDC1 3 6 7.60 (2H, 7.57 (2H, 7.50-7.26 (4H, 5.60 (1H, d, J 5.28 (1H, 4.67 (2H, 4.38 (2H, 4.23 (1H, m), 3.59-3.41 (1H, 2.92-2.65 (2H, 2.41-2.21 (2H, 1.95-1.58 (4H, 1.47 (9H, MS(ES, m/z) 520 (M 1, 179 (100%).

(212b) was synthesized by the same method as compound V 10 212e (635mg, 85%) as a colourless powder: mp 209- 12 [a]D -132 (c 0.12, MeOH); IR (KBr) 3308, 2940, 1717, 1707, 1699, 1619, 1469, 1456, 1442, 1417, 1391, •1348, 1339, 1330, 1310, 1271, 1247, 1222, 1175, 1152, 1133, 993, 976; H NMR (CD 3 OD) 6 5.35 (1H, 4.58-4.48 (1H, 4.46-4.36 (1H, 3.60-3.42 (1H, 3.01- 2.87 (1H, 2.95 (3H, 2.55-2.39 (1H, 2.32- 604 2.20 (2H, 2.09-1.89 (2H, 1.78-1.62 (2H, m); Anal. Calcd for CIIH 17

N

3 0 6 S: C, 41.37; H, 5.37; N, 13.16; S, 10.04. Found: C, 41.59; H, 5.32; N, 12.75; *6 0 S, 9.76; MS(ES Accurate Mass calculated for CllHl 8

N

3 0 6 S (MH 320.0916. Found: 320.0943.

go(212c) was prepared from 211e the same method as *o compound 212e as a white glassy solid (595mg, mp 24 >250 []D 24 -153 (c 0.10, MeOH); IR (KBr) 3280, 2942, 1742, 1697, 1675, 1650, 1616, 1548, 1470, 1443, 1281, 1249, 1202, 1187, 1171; 1H NMR (CD 3 OD) 6 5.35- 5.31 (1H, 4.81-4.71 (1H, 4.61-4.46 (1H, m), 3.59-3.44 (2H, 3.11-2.94 (1H, 2.58-2.39 (1H, 2.36-2.19 (2H, 2.11-1.83 (3H, 1.99 (3H, s), 1.78-1.56 (2H, m) Anal. Calcd for C 12

H

17

N

3 0 5

C,

50.88; H, 6.05; N, 14.83. Found: C, 50.82; H, 6.02; N, 14.58; MS (ES 282 100%): Accurate Mass calculated for C 12

H

18

N

3 0 5 284.1246. Found: 284.1258.

ll (212d) was prepared from 211d by the same method as compound 212e as colourless crystals (170mg, mp 60-100 OC; [a]D 2 2 -103 (c 0.10, MeOH); IR (KBr) 3341, 2947, 1728, 1675, 1531, 1456, 1422, 1339, 1272, 1248, 1221, 1174, 1122, 1056, 982, 699; 1H NMR (CDC13) 6 7.35 5.65 (1H, 5.48-5.40 (1H, 5.10 (2H, s), 4.76-4.57 (2H, 3.49-3.30 (2H, 2.92-2.59 (2H, 2.40-2.27 (2H, 1.97-1.67 (4H, MS (ES 374 (M 1, 100%). Accurate mass calculated for C 18

H

22

N

3 0 6 (MH 376.1509. Found: 376.1483. Accurate mass calculated for C 18

H

21

N

3 06Na (MNa+) 398.1328. Found: 398.1315.

(212e). TFA (20ml) was added to an ice cold stirred solution of the t-butyl ester 211e (4.15g, 10.34mmol) in dry CH 2 Cl 2 (20ml). The mixture was kept cold for 1.5h then left for 2.5h at rt, concentrated. TFA was •removed by repeated concentrations of CH 2 Cl 2 \ether and ether solutions of the residue. Finally trituration of the residue with ether afforded 212e 3.05g of a white glassy solid: mp 118-126 []D24 -70.50 (c 0.1, CH 2 C1 2 IR (KBr) 3361, 2943, 1737, 1659, 1537, 1426, 1220, 1174; H NMR (CDC13) 6 7.80 (2H, 7.54- 7.33 (4H, 8.83 (brs), 5.44 (1H, 5.26-5.13 (1H, 4.66 (1H, 3.59-3.41 (1H, 2.97, 2.76 (2H, 2m), 2.36 (2H, 1.98 (2H, 1.75 (2H, MS(ES m/z) 344 (M 1, 100%).

(212f) was prepared from 211f in 96% yield by the same method as for 212e: mp 120-126 [a]D 2 5 -72.50 (c 0.1, CH 2 C1 2 IR (KBr) 3406, 2950, 1725, 1670, 1526, 1449, 1421, 1272, 1248, 1223, 1175, 761, 741; 1 H NMR (CDC1 3 6 7.76 (2H, 7.62-7.26 (4H, 6.07, 5.76 (2H, brs, d, d, J 5.46, 5.36 (1H, 2m), 4.79-4.54 (2H, 4.77 (2H, 4.21 (1H, 3.41 (1H, 2.89 (1H, 2.69 (1H, 2.35 (2H, m), 112 1.98, 1.73 (4H, 2m). MS(ES-, m/z) 462 1, 240 (100%).

0 0 RI- 0 o Rl- OH H4 CF-c H OBn H .(213) R~ MeCO (214) R MeCO e) R =PhCO R PhCO (213c) was synthesized from 212c by the same method as compound 213e to afford a mixture of diastereomers (193mg, 36%) as colourless crystals: IR (KBr) 3272, 1799, 1701, 1682, 1650, 155, 1424, 1412, 1278, 1258, 1221, 1122, 937; 1H NMR (ODC1 3 5 7.41-7.28 (5H, in), 6.52 (0.5H, 6.38 (0.5H, 6.22 (0.5H, 5.57 5.36 (0.5H, s) 5.10-5.05 (1H, in), 5.00-4.45 in), 3.19-2.84 (3H, in), 2.72-2.56 (1H, in), 2.51- 2.25 (2H, in), 2.02 (3H, 1.98-1.70 C3H, mn), 1.66- 1.56 C3H, in); Anal. Calcd for C 2 3

H

2 8

N

4 0 7 C, 58.47; H, 5.97; N, 11.86. Found: C, 58.37; H, 6.09; N, 11.47.

MS (ES 471 100%) Accurate mass calculated for C 23

H

29

N

4 0 (NH 473.2036. Found: 473.2012.

go Accurate mass calculated for C 2 3

H

2 8

N

4

O

7 Na (Mna) 495.1856. Found: 495.1853.

(213e). Tributyltin hydride (2.2m1, 8.l8mmol) was added dropwise to a solution of acid 212e C1.95g, 5.6mmol), (3S, 2RS) 3-allyloxycarbonylamino-2- (Chapman, Bioora. Med.

Chein. Lett., 2, pp. 615-618 (1992); 1.80g, 6.l6mnol) and (Ph 3

P)

2 PdCl 2 (50mg) in dry CH 2 Cl 2 (36in1), with stirring, under dry nitrogen. After 5 min 1hydroxybenzotriazole (1.51g, 11.2mmiol 6.72mniol) was added followed after cooling (ice/H 2 0) by 113 ethyldimethylaminopropyl carbodiimide hydrochloride (1.29g, 6.72mmol). After 5 mins the cooling bath was removed and the mixture was kept at room temperature for 4h, diluted with EtOAc, washed with 1M HC1, brine, sat. aq. NaHCO 3 and brine, dried (MgS0 4 and concentrated. Flash chromatography (silica gel, 0-90% EtOAc in CH 2 C12) gave the product as a white solid (2.34g, IR (KBr) 3499, 1792, 1658, 1536, 1421, 1279, 1257, 1123, 977, 699; 1H NMR (CDCl 3 5 7.81 (2H, 10 7.54-7.34 (8H, 7.1, 6.97, 6.89, 6.48 (2H, m, d, J 7.7, d, J 7.5, d, J 5.57, 5.28 (1H, d, J= S..S 5.2, 5.23-5.07 (2H, 4.93-4.42, 3.22-2.70, 2.51- 2.26, 2.08-1.69, 1.22 (15H, 5m). Anal. Calcd for

C

28

H

30

N

4 0 7 0.5H 2 0: C, 61.87; H, 5.75; N, 10.32. Found C, 62.02; H, 5.65; N, 10.25.

(214c) was synthesized from 213c by a method similar to the method used to synthesize 214e from 213e to provide colourless crystals (140mg, mp 90-180 oC; [a]D 2 2 -114 (c 0.10, MeOH); IR (KBr) 3334, 3070, 2946, 1787, 20 1658, 1543, 1422, 1277, 1258; H NMR (d6-DMSO) 5 8.66 (1H, 8.18 (1H, 6.76 (1H, 5.08 (1H, 4.68 (1H, 4.30 (1H, 2.92-2.70 (2H, 2.27-2.06 (3H, 1.95-1.72 (4H, 1.85 (3H, 1.58 (2H, m); MS(ES 381 100%); Accurate mass calculated for

C

1 6

H

2 3

N

4 0 7 (MH 383.1567. Found: 383.1548.

(214e). A mixture of 213e (2.29g, 4.28mmol), palladium on carbon (1.8g) and MeOH (160ml) was stirred under H 2 at atmospheric pressure for 6.3h.

After filtering and concentrating the hydrogenation was repeated with fresh catalyst (1.8g) for 5h. After filtering and concentrating the residue was triturated with diethyl ether, filtered and washed well with ether to give 214e as a white solid (1.67g, mp 143- 147 [aa]D 2 3 -1250 (c 0.2, CH 3 0H). IR (KBr) 3391, 114 1657, 1651, 1538, 1421, 1280, 1258; 1H NMR (CD 3 0D) 6 7.90 (2H, 7.63-7.46 (3H, 5.25 (1H, 5.08- 4.85 (1H, 4.68-4.53 (2H, 4.33-4.24 (1H, m), 3.62-3.44, 3.22-3.11, 2.75-2.21, 2.15-1.92, 1.73-1.66 (11H, 5m). Anal. Calcd for C 21

H

24

N

4 0 7

H

2 0: C, 54.54; H, 5.67; N, 12.11. Found C, 54.48; H, 5.63; N, 11.92.

0

O

OS 0 S *CO-rt-Bu SoRC-N0 2 Bu H a 00 H H OH C O 0 H O- 0 S***see (215) (216) 0 0 8000 O O Cl H

O

C

2 H a ci

R

1 MeCO (217) H O C4 0

R

1 PhCH 2

OCO

1" R 1 PhCO (215c) was synthesized from 214c by the same method as compound 215e, to afford a mixture of diastereomers as a white glassy solid (398mg, IR (KBr) 3338, 2977, 1738, 1658, 1562, 1541, 1433, 1368, 1277, 1150; 1H NMR (CDC1 3 5 7.36-7.32 (3H, 6.91 (1H, 6.30 (1H, 5.15-5.09 (1H, m) 5.01-4.88 (1H, 4.61-4.44 (2H, 4.37-4.08 (3H, 3.32-3.18 (1H, 3.04- 2.89 (1H, 2.82-2.51 (4H, 2.39-2.29 (1H, m), 2.08-1.64 (4H, m) 2.02 (3H, Anal. Calcd for

C

28

H

34

N

4 Cl 2 0 9 C, 52.26; H, 5.64; N, 8.71. Found: C, 52.44; H, 5.87; N, 8.16. MS (ES 645/3/1 26%), 115 0 o *o 0 o *000 *0 0 0 189 134 (100). Accurate mass calculated for

C

28

H

37

N

4 C1 2 0 9 643.1938. Found: 643.1924.

Accurate mass calculated for C 28

H

36

N

4 C120 9 Na (MNa 665.1757. Found: 665.1756.

(215d) was synthesized from 214d by the same method as compound 215e to afford a mixture of diastereomers (657mg, 70%) as a glassy white solid: IR (KBr) 3420, 3361, 2975, 2931, 1716, 1658, 1529, 1434, 1367, 1348, 1250, 1157, 1083, 1055; 1H NMR (CDC13) 6 7.32 (8H, m), 10 7.14 (1H, 5.81 (1H, 5.15 (1H, 5.07 (2H, s), 4.74-4.65 (1H, 4.58-4.22 (4H, 4.15-4.06 (1H, 3.72 (1H, 3.32-3.21 (1H, 3.04-2.94 (1H, m), 2.69-2.52 (3H, 2.33-2.27 (1H, 1.95-1.59 (4H, 1.28 (9H, Anal. Calcd for C 34

H

40

N

4 C1 2 0 10

H

2 0: C, 54.70; H, 5.54; N, 7.50. Found: C, 54.98; H, 5.59; N, 7.24. MS (ES 737/5/3 193/1/89 (100). Accurate mass calculated for C 34

H

41

N

4 C1 2 0 10 (MH 735.2120. Found: 735.2181.

(215e). Tributyltin hydride (4.6ml; 11.4mmol) was added dropwise to a stirred mixture of (3S,4RS) t-Butyl (N-allyloxycarbonyl)-3-amino-5-(2,6dichlorobenzoyloxy)-4-hydroxypentanoate (prepared by a method similar to the method described in Revesz et al., Tetrahedron. Lett., 35, pp. 9693-9696 (1994)) (2.64g; 5.7mmol), (Ph 3

P)

2 PdCl 2 (50mg), CH 2 C1 2 (100ml) and DMF (20ml) at room temperature. The mixture was stirred for a further 10min was then 1hydroxybenzotriazole (1.54g, 11.4mmol)was added. The mixture was cooled to 0 °C then ethyldimethylaminopropyl carbodiimide hydrochloride (1.31g; 6.84mmol) added. The mixture was kept at this temperature for 15min then at room temperature for 17h.

The mixture was diluted with EtOAc (300ml), washed with 1M HC1 (2xl00ml), sat. aq. NaHCO 3 (3xl00ml) and brine 116 (2xl00ml), dried (MgSO 4 and concentrated. The residue was purified by flash chromatography (MeOH/CH 2 Cl 2 to afford 3.24g of 215e as a glassy solid: mp 106-110 IR (KBr) 3354, 1737, 1659, 1531, 1433, 1276, 1150; 1H NMR (CDC 3 5 7.80 (2H, dd, J 7.9 and 7.75-7.26 (6H, 7.14-6.76 (2H, m), 5.30-5.02 (2H, 4.63-4.11 (5H, 3.44-3.26 (2H, 3.10-2.30 (5H, 2.10-1.60 (5H, 1.44 (9H, s); Anal. Calcd for C 33

H

38 Cl 2

N

4 0 9 0.75H 2 0: C, 55.12; H, ee 10 5.54; N, 7.79; Cl, 9.86. Found: C, 55.04; H, 5.34; N, *se: 7.80; Cl, 10.24. MS (ES 709/7/5 (M 378 (59), :324 322 (100).

(216c) was synthesized from 215c by the same method as compound 216e as a glassy white solid (300mg, mp 23 80-125 []D 2 3 -89.1 (c 1.08, CH 2 C1 2 IR (KBr) 3356, 2979, 2935, 1740, 1659, 1532, 1434, 1369, 1276, 1260, 1151; H NMR (CDC1 3 5 7.39-7.32 (3H, 7.13 (1H, d), 6.34 (1H, 5.22-5.17 (1H, 5.11 (1H, 5.04 (1H, 4.99-4.88 (2H, 4.64-4.52 (1H, 3.29- 20 3.11 (1H, 3.05-2.67 (4H, 2.39-2.29 (1H, m), 2.02 (3H, 1.98-1.75 (4H, 1.46 (9H, Anal.

Calcd for C 28

H

34

N

4 C1 2 0 9 C, 52.42; H, 5.34; N, 8.73.

Found: C, 52.53; H, 5.70; N, 7.85. MS (ES-) 643/41/39 100%). Accurate mass calculated for

C

2 8

H

3 5

N

4 Cl 2 0 9 (MH 641.1781. Found: 641.1735.

Accurate mass calculated for C 2 8

H

3 4

N

4 Cl20 9 Na (Mna+) 663.1601. Found: 663.1542.

(216d) was synthesized from 215d by the same method as compound 216e to afford 216d as a white glassy solid (688mg, mp 90-170 []D 2 5 -83.4 (c 1.01,

CH

2 C1 2 IR (KBr) 3338, 2933, 1736, 1670, 1525, 1433, 1417, 1368, 1258, 1151, 1056, 1031; 1H NMR (CDC1 3 7.33 (8H, 7.18 (1H, 5.65 (1H, 5.19 (1H, m), 5.09 (2H, 4.98-4.86 (1H, 4.82-4.49 (2H, d), 117 3. 30-3. 07 (1H, mn), 3. 05-2 .59 (4H, mn), 2. 42-2 .27 (1H, in), 2. 18-1. 59 (5H, mn), 1. 42 (9H, s) MS 737/5/3 13%) 185 (100).

(216e). Dess-Martin reagent (3.82g; 9.Ommxol) was added to a stirred solution of the alcohol 215e (3.17g; in CH 2 Cl 2 (lOini) The mixture was sirred for lh, diluted with EtOAc (300m1), then washed with a 1:1 mixture of sat. Na 2

S

2

O

3 and sat. NaH-C0 3 (lO0rni) followed by brine (100m1) The mixture was dried (MgSO 4 then 10 concentrated. The residue was purified by flash chromatography to afford 2.2g of 216e as a 0 *0 colourless solid: mp 102-107 0 C; Ea1D -_82.5 (c 0.1,

CH

2 Cl 2 IR (KBr) 3374, 2937, 1739, 1661, 1525, 1433, 1275, 1260, 1152; 1 NMR (ODC1 3 5 7.85-7.78 (2H, mn), 7.57-7.32 (6H, mn), 7.09 (1H, d, J 7.01 (1H, d, J 5.25-5.16 (1H, mn), 5.16-5.05 (1H, in), 5.15 (1H, 5.03 (1H, 4.99-4.90 (1H, mn), 4.68-4.54 (1H, mn), 0e0S 3.31-3.17 (1H, in), 3.17-2.72 (4H, mn), 2.45-2.35 (1H, in), 2.30-1.66 (5H, mn), 1.44 (9H, Anal. Calcd for 20 C 3 3 H 6 1N0. 0.5H 2 0 C,,55.62; H, 5.23; N, 7.86; Cl, 9.95. Found: C, 55.79; H, 5.15; N, 7.80; Cl 9.81. MS (ES 729/7/5 (M Na) 707/5/3 (M 1) 163 (100%).

S

(217c) was synthesized from 216c by the same method as :.690 .0 compound 217e as a glassy white solid (166mg, 66%) mp 85-175 0 C; [ca]D -5_156 (c 0.13, NeOH); IR (KBr) 3373, 2929, 1742, 1659, 1562, 1533, 1433, 1412, 1274, 1266, 1223, 1197, 1145, 1138; 1H NNR (CD 3 OD) 5 7.38 O3H, S), 5.14-5.03 (1H, in), 4.49-4.32 (2H, in), 3.50-3.27 (1H, in), 3.11-2.92 (1H, in), 2.84-2.62 (2H, mn), 2.46-2.11 (2H, in), 2.05-1.46 (5H, mn), 1.92 (3H, Anal. Calcd for C 2 4

H

2 6

N

4 C1 2 0 9

.H

2 0: C, 47.77; H, 4.68; N, 9.29.

Found: C, 47.75; N, 4.59; N, 9.07. MS (ES 627/5/3 611/9/7 (N+Na, 87), 589/7/5 71),- 118 266 (100); Accurate mass calculated for C 24

H

27

N

4 C1 2 0 9 (MH 585.1155. Found: 585.1134.

(217d) was synthesized from 216d by the same method as compound 217e to afford 217d as a white glassy solid (310mg, mp 85-110 OC; [a]D 2 4 -85.9 (c 0.13, MeOH); IR (KBr) 3351, 2945, 1738, 1669, 1524, 1433, 1258, 1147, 1057; 1H NMR (CD 3 0D) 5 7.56 (4H, 7.45 5.32 (2H, 5.20 (2H, 4.76-4.48 (3H, m), 3.65-3.38 (3H, 3.27-3.09 (2H, 3.03-2.89 (2H, 2.65-2.24 (3H, 2.19-1.62 (5H, MS (ES 679/7/5 100%); Accurate mass calculated for

SC

30

H

31

N

4 Cl 2 0 10 (MH 677.1417. Found: 677.1430.

(217e) TFA (25ml) was added dropwise to an ice cold stirred solution of the ester 216e (2.11g, The mixture was stirred at 0 oC for 20min then at room temperature for lh. The mixture was evaporated to dryness then coevaporated with ether three times.

e Addition of dry ether (50 ml) and filtration afforded ee 1.9g of 217e as a colourless solid: mp 126- 20 130 OC; [C]D 3 -122.0 (c 0.1, MeOH); IR (KBr) 3322, 1740, 1658, 1651, 1532, 1433, 1277, 1150; 1H NMR (D 6 DMSO) 5 8.87 (1H, d, J 8.61 (1H, d, J 7.8), 7.92-7.86 (2H, 7.65-7.43 (6H, 5.25-5.12 (3H, 4.94-4.60 (2H, 4.44-4.22 (1H, 3.43-3.10 (1H, 3.00-2.52 (3H, 2.45-2.10 (3H, 2.10- 1.75 (2H, 1.75-1.50 (2H, Anal. Calcd for

C

29

H

28 C1 2

N

4 0 9 1H 2 0: C, 52.34; H, 4.54; N, 8.42; Cl, 10.66. Found: C, 52.02; H, 4.36; N, 8.12; Cl, 10.36.

MS (ES 649/7/5 (M 411 (100%).

119 0 0

R

1 -N C0 2 -t-Bu CI I 1 N C 2 tuC

CCI

218b R, MeSO 2 219b

C

0 NC0 2 H C1

R

1

-N

H 00

H

220b C1 S .5'(218b) was prepared from the acid 212b and 99 in an analogous way to compound 215e to afford a mixture of diastereomers (865mg, 80%) as a colourless solid: IR (KBr) 3298, 2974, 1723, 1659, 1544, 1518, 1430, 1394, 1370, 1328, 1273, 1256, 1156, 1134; 1H MR (CDC1 3 7.45-7.28 (4H, in), 7.26-7.15 (2H, in), 5.26-5.10 (2H, in), 4.80-4.67 (1H, in), 4.59-4.42 (2H, in), 3.32-3.17 (1H, in), 2.96 (3H, 2xs), 2.93-2.79 (1H, in), 2.71-2.53 (4H, in), 2.38-2.28 (1H, in), 2.07-1.81 (4H, in); Anal.

Calcd for C 28

H

35

N

5 C1 2 0 9 S. 0.5 H 2 0: C, 48.21; H, 5.20; 10.03. Found: C,48.35; H, 5.26; N, 9.48. MS (ES 714/2/0 (M Na, 692/90/88 (M 1, 51), 636/4/2 246 (100) Accurate mass calculated for

C

2 8

H

3 6

N

5 C1 2 0 9 S 688.1611. Found: 688.1615.

(219b) was prepared from 218b in an analogous way to compound 216e as an off-white powder (675mg, 81%) mp 100-200 [ojID -_84.9 (c 1.01, CH 2 Cl 2 IR (KBr) 3336, 2978, 2936, 1719, 1674, 1510, 1433, 1421, 1369, 1329, 1274, 1257, 1155, 991, 789; 1H MR (CDCl 3 7.47-7.38 (4H, in), 7.24 (1H, 5.61-5.53 (1H, mn), 5.48 (1H, 5.38-5.30 (1H, in), 4.67-4.45 (2H, mn), 120 3.4 8 18 (2H, in), 3.0 4 90 (2H, in), 2. 97 (3H, s) 2.69-2.54 (1H, in), 2.42-2.32 (1H, mn), 2.22-2.15 (1H, mn), 2.07-1.93 (3H, in), 1.71-1.65 (2H, in), 1.38 (9H, s); Anal. Calcd for C 28

H

33

N

3 01 2 0 9 S: C, 48.98; H, 4.84; N, 10.20; S, 4.67. Found: C, 48.73; H, 4.95; N, 9.65; S, 4.54. MS (ES 692/90/88 CM+ 1, 100%), 636/4/2 (71) Accurate mass calculated for C 28

H

34

N

5 C1 2 0 9

S

686.1454. Found: 686.1474.

*(220b) was prepared from 219b in an analogous way to 10 compound 217e as a pale cream powder (396mg, 87%) mp 100-200 00; [a1D 27- 129 Cc 0.12, MeOR); IR (KBr) 3310, 3153, 1713, 1667, 1557, 1510, 1432, 1421, 1329, 1273, *0:006 1258, 1221, 1193, 1153, 1134, 992, 789; 1H NNR Cd6 DMSO) 6 7.88 (1H, 7.81-7.60 (4H, in), 5.49-5.28 (1H, in), 5.24-5.14 (1H, in), 4.46-4.22 (2H, in), 3.30-3.03 (2H, in), 2.97-2.76 (3H, in), 2.96 (3H, 2.46-2.24 (1H, in), 2.16-2.05 (1H, in), 2.03-1.78 C3H, in), 1.68- 1.46 (2H, mn); MS 632/30/28 CM 1, 149/7/5 (100). Accurate mass calculated for C 24

H

26

N

5 C1 2 0 9

S

20 (NH 630.0828. Found: 630.0852.

*0 0 q 0 121 *Bu

P

221b R 1 MeSO 2 C1 222b R, MeSO 2

CI

221e R, PhCO 222e R, =,PhCO 00 0

II

H0 H 0 N! 223 CO meho as. Hhtue o opud25 oafr 0~ 5.2 (1Hb was preare from the (3 aci obiand, (3S,4s),t 10%,62 M_ )C17 tbutyl N- (allyloxycarbonyl) -3-amino-4-hydroxy-4- (5,7dichlorobenzoxazol-2-yl)butanoate (204) by an analogous as that used for compound 215e to afford a mixture of diastereoiners (60mg, 87%) as a glass: IR 122 (film) 3328, 1729, 1660, 1534, 1454, 1422, 1399, 1276, 1254, 1155; 1H NMR (CDC13) 5 7.80 (2H, 7.60-7.35 7.05 (2H, 5.13 (3H, 4.74 (1H, 4.51 (1H, 3.25 (1H, 3.1-2.6 (5H, 2.33 (1H, m), 2.1-1.5 (5H, 1.43 and 1.41 (9H combined, 2 x s).

MS ES Da/e 688 (M 1) Cl 3 5 55%, 690 (M C137 328 100%.

(222b) was prepared from 221b by an analogous method as that used for compound 216e to afford a colourless 26 1* 0 glass (371mg, [1]D -81.0 (c 0.1, CH 2 C1 2

IR

(KBr) 3324, 2979, 2936, 1726, 1664, 1394, 1370, 1328, 1155, 991; H NMR (CDCl 3 5 7.78 (1H, 7.57 (2H, m), 5.87 (1H, 5.69 (1H, 5.47 (1H, 4.55 (2H, m), 3.24 (2H, 3.0 (5H, m 2.59 (1H, 2.39 (1H, 2.2 1.7 (4H, 1.65 (1H, 1.40 (9H, s).

(222e) was prepared from 221e by an analogous method as •that used for compound 216e to afford a colourless 0 glass (480mg, 84%) -86.40 (c 0.1 CH 2 Cl 2

IR

(KBr) 3337, 2978, 2938, 1728, 1657, 1534, 1456, 1422, 20 1395, 1370, 1277, 1250, 1154; H NMR (CDC13) 5 7.80 (3H, 7.50 (4H, 7.20 (1H, 7.02 (1H, 5.60 (1H, 5.28 (1H, 5.15 (1H, 4.11 (1H, 3.34 (2H, 2.96 (3H, 2.40 (1H, 2.20 (1H, 1.92 (2H, 1.67 (2H, 1.38 (9H, MS ES Da/e 684 (M 1) Cl 3 5 47%, 686 (M 1) C13 7 32%.

(223b) was prepared from 222b by an analogous method as that used for compound 217e to afford an off-white solid (257mg, [a]D 25 -105.70 (c 0.1, CH 2 Cl 2

IR

(KBr) 3321, 1723, 1663, 1407, 1325, 1151, 992; H NMR

(D

6 -DMSO) 5 8.96 (1H, 8.18 (1H, 7.96 (1H, d), 5.50 (1H, 5.15 (1H, 4.30 (2H, 3.06 (2H, m), 2.87 (5H, m 2.29 (1H, 1.99 (4H, 1.56 (2H, m).

123 (223e) was prepared from 222e by an analogous method as that used for compound 217e to afford a pale cream solid (311mg, mp 167-180 0 C; EcUID 23_88.6' (c 0.1 CH 2 Cl9); IR (KBr) 3331, 1724, 1658, 1534, 1458, 1421, 1279, 1256, 991; 1H NNR (CDCl 3 5 7.77 (4H, in), 7.4 (5H, in), 5.57 (1Hi, bs), 5.33 (1H, bs), 5.47 (1H, 4.56 (1H, bd), 3.60 (2H, in), 3.20 (3H, in), 2.76 (1H, in), 2.36 (1H, dd), 2.0 (3H, in), 1.66 (1H, in). MS :ES Da/e 628 (M Cl 35 630 (M C137 2.3%, 584 100%.

@0 0000 COL2-C- 2

H

t.a C0 2 HtB a H 0 H 0aC

H

0

X.H

224e R, =PhCO, X =S 226e R 1 PhCO, 0.se225e

R,

1 =PhCO, X=O0 227e R, =PhCO, X=O0 (224e) l-Hydroxybenzotriazole (0.23g, l.7lnunol) and ethyl diinethylaminopropyl carbodiimide hydrochloride was added to a stirred solution of the acid 212e (0.295g, 0.853mmo1) in THF (5m1) After 5mmn water C.5m1) was added followed, after a further 7mmn, by the addition of a solution of (3S) t-butyl-3- (2-chloro-phenyl)methylthio-4oxopentanoate (123, 0.478g, 1.O2mnol) and (PPh 3 2 PdCl 2 in THE (2m1) Tributyltin hydride (0.65in1, 2.33mmol) was added dropwise during 20mmn. The mixture was kept for 4.5h then diluted with EtOAc, washed with 1M HCl, brine, sat. aq. NaHCO 3 and then brine again.

The mixture was dried (MgSO 4 and concentrated. The residue was triturated several times with hexane, which was decanted and discarded, then purified by flash 124 chromatography (10-100% EtOAc in CH 2 Cl 2 to afford 0.2g of a white glassy solid: mp 70-72 [ac]D 26 -82.5° (c 0.02, CH 2 C1 2 IR (KBr) 3404, 1726, 1660, 1534, 1524, 1422, 1277, 1254, 1154; 1H NMR (CDCl 3 6 7.83-7.78 (2H, 7.7, 7.75-7.32, 7.26-7.20 (7H, 3m), 7.12 (1H, d, J 7.01 (1H, d, J 5.23-5.08 (2H, 5.03-4.94 (1H, 4.62 (1H, dt, J 14.5), 3.78 (2H, 3.38-3.29 (1H, 3.26 (2H, 3.06- 1 2.82 (4H, 2.71 (1H, dd, J 17.2, 2.39 (1H, 1. 0 dd, J 13.2, 2.15-1.83, 1.73-1.63 (5H, 1.45 o° (9H, Anal. Calcd for C 3 3

H

3 9 C1N 4 0 7 S: C, 59.05; H, S o 5.86; N, 8.35. Found: C, 59.00; H, 5.80; N, 7.92.

(225e) was prepared from acid 212e and (3S) t-butyl N- *e (allyloxycarbonyl)-3-amino-5-(2-chlorophenylmethyloxy)- 4-oxopentanoate (201) using a method similar to that used for compound 224e, to afford 40mg of a glassy solid: H NMR (CDC 3 6 7.83-7.73 (2H, m), 7.67-7.10 (9H, 5.23-5.09 (2H, 4.59 (1H, m), 4.45-4.22 (2H, 3.7-3.19, 3.08-2.72, 2.71-2.47, 20 2.05-1.85, 1.72-1.61, 1.45-1.26 (20H, 6m).

(226e) was prepared from 224e by an analogous method as that used for compound 217e which afforded 0.22g (81%) Oe of an off-white solid: mp 95-100 [Ca]D 2 3 -95.6° 00 (c 0.2, CH 2 C1 2 IR (KBr) 3393, 1720, 1658, 1529, 1422, 1279; H NMR (D 6 -DMSO) 6 8.80 (1H, d, J 7.89 (2H, 7.7 (1H, d, J 7.56-7.28 (7H, m), 5.10 (1H, 4.87-4.73 (2H, 4.39 (1H, 3.77 (2H, 3.44, 3.35 (2H, +H 2 0, 2m), 2.97-2.56, 2.2, 1.92, 1.61 (11H, 4m). Anal. Calcd for C 2 9

H

3 1 ClN 4 0 7

S

0.5H 2 0: C, 55.02; H, 5.10; N, 8.85. Found: C, 55.00; H, 5.09; N, 8.71.

(227e) was prepared from 225e by an analogous method as that used for compound 217e. The product was further purified by flash chromatography MeOH/CH 2 Cl 2 to 125 afford 19mg of a glassy solid: 1H NMR (CDC1 3 6 7.79 (2H, 7.66-7.18 (9H, 5.30-5.10 (2H, m), 4.85 (1H, 4.65 (2H, 4.53 (1H, 4.28 (2H, m), 3.28, 3.01, 2.72, 2.33, 1.94, 1.60 (11H, 6m). MS (ES-, m/z) 597 (M 1, 100%).

S. C

C.

0

C

0

OSS

228e X 229e X 230e X

C

S

5500

CC..

S

C. S

CO

2 -t-Bu N F F

OH

CO-t-Bu N F 0

CO

2

H

0 (228e). l-Hydroxybenzotriazole (0.23g, 1.68mmol) followed by ethyldimethylaminopropyl carbodiimide hydrochloride (0.21g, 1.09mmol) were added to a stirred solution of the acid 212e (0.29g, 0.84mmol) in CH 2 C12 (3ml) at rt. The mixture was kept for 10min then a solution of (3RS,4RS) t-butyl 3-amino-5-fluoro-4hydroxypentanoate (Revesz, L. et al. Tetrahedron Lett., 52, pp. 9693-9696 (1994); 0.29g, 1.40mmol) in CH 2 Cl 2 (3ml) was added followed by 4-dimethylaminopyridine The solution was stirred for 17h, diluted with EtOAc, washed with 1M HC1, brine, sat. aq. NaHCO 3 and 126 brine again, dried (MgSO 4 and concentrated. The residue was purified by flash chromatography (50-100% EtOAc/CH 2 Cl 2 and 5% MeOH/EtOAc) to afford 0.25g (56%) of a white glassy solid: IR (KBr) 3343, 1726, 1658, 1536, 1426, 1279, 1257, 1157; 1H NMR (CDCI 3 5 7.84- 7.79 (2H, 7.57-7.40 (3H, 7.05-6.92, 6.73 (2H, 2m), 5.17-5.04 (2H, 4.56, 4.35-4.21, 4.04 (5H, 3m), 3.36, 3.09-2.34, 2.00 (11H, 3m), 1.46 (9H, Anal.

Calcd for C 26

H

35

FN

4 0 7 0.5H 2 0: C, 57.45; H, 6.65; N, 10 10.31. Found: C, 57.64; H, 6.56; N, 10.15.

(229e) was prepared from 228c by an analogous method to that used for compound 216e. After purification by flash chromatography (30-50% EtOAc/CH 2 Cl 2 the product 0 was obtained as a white glassy solid (0.194g, IR (KBr) 3376, 1728, 1659, 1529, 1424, 1279, 1256, 1156.

(230e) was prepared from 229e by an analogous method to that used for compound 217e to afford 230e as a white glassy solid mp 105-125 oC; [ID 2 3 -91.40 (c 0.72, CH 3 0H). IR (KBr) 3336, 1789, 1737, 1659, 20 1535, 1426, 1279, 1258, 1186; 1H NMR (CD 3 OD) 5 7.71- 7.68 (2H, 7.37-7.23 (3H, 5.02, 4.88-4.63, 4.37- (6H, 3m), 3.30, 2.97, 2.68-2.60, 2.37-1.54 (11H, 4m). MS(ES-, m/z) 475 (M 1, 100%).

*00 0: 0 0 o a Ph H %O C02Me CO2H H 0 NCN H 00 NCN H

H

(231e) (232e) (231e). N-Fluorenylmethyloxy-carbonyl-3-amino-3cyanopropionic acid methyl ester (EP0547699A1, 385mg, l.lmmol) was treated with 17ml of diethylamine. After stirring at room temperature the solution was 127 concentrated. The residue was chromatographed on silica gel methanol in CH2C1 2 and gave the free amine as a pale yellow oil. To a solution of this oil and hydroxybenzotriazole (297mg, 2.19mmol) in DMF (5ml), was added at 0 °C ethyldimethylaminopropyl carbodiimide (232mg, 1.21mmol, 1.1 equiv) followed by (1S,9S) 9-(benzoylamino)-[6,10-dioxo-1,2,3,4,7, 8,9,10octahydro-6H-pyridazino[1,2-a][1,2]diazepine-lcarboxylic acid (212e). After stirring at 0 oC for e° 10 min and then at room temperature overnight, the mixture was diluted with CH 2 Cl 2 (50ml) and the resulting Ssolution washed successively with 1M HC1 (2 x

H

2 0 (30ml), 10% NaHCO 3 (2 x 30ml) and sat. aq.

NaCl,dried (MgSO 4 and concentrated. Purification by flash chromatography on silica gel methanol in

CH

2 C1 2 afforded the compound 231e (404mg, 83%) as a solid: [a]D 20 -1210 (c 0.14, CH 2 C12); H NMR (CDC13) 7.40-7.83 (5H, 7.38 (1H, 6.96 (1H, 5.27- 5.07 (2H, 4.66-4.50 (1H, 3.79 (3H, 3.23- 20 2.73 (6H, 2.47-2.33 (1H, 2.15-1.82 (4H, m); Anal. Calcd for C 2 2

H

2 5

N

5 0 6 C, 58.0; H, 5.53; N, 15.38.

Found: C, 57.6; H, 5.6; N, 15.0.

(232e). A solution of methyl ester 231e (400mg, 0.88mmol) in methanol (30ml) and water (30ml) was cooled at 0 oC and treated with diisopropylethylamine.

The solution was stirred at 0 °C for 10min and then at room temperature overnight. The heterogeneous mixture was concentrated and the solid obtained was chromatographed on silica gel methanol/l% formic acid in CH 2 C1 2 affording the free acid 232e (170mg, 44%) as a white solid: mp 155 °C (dec); [a]D 2 0 -1170 (c 0.1, MeOH); IR (KBr) 3343, 3061, 2955, 1733, 1656, 1577, 1533, 1490, 1421, 1342, 1279, 1256, 1222, 1185, 708; H NMR (D4-MeOH) 5 7.88-7.28 (5H, 5.20-5.03 128 (1H, mn), 4 .98-4 .84 (2H, in), 4 .75-4. 53 (1H, mn), 4. 51- 4. 34 (1H, in), 3. 45-3. 22 (1H, mn), 3 .14-2. 94 (1H, m) 3. 14-2 .94 (1H, mn), 2 .88-2. 61 (2H, mn), 2. 53-1 .50 (8H, mn); Anal. Calcd for C 2 1

H

2 3

N

5 0 6 1.5H 2 0: C,53.84; H, 5.59; N, 14.95; 0, 25.61. Found: 0, 54.3; H, 5.4; N, 14.3.

0* 0* 0O

S

o e 0

SS

0 000000 0 0S 0 0 0000 00 0 000 H H C0 2 -t-Bu 233e R= C0 2 -t-Bu 236e R C0 2 -t-BU 234e R= C0 2 -t-BU 237e RI 0 000000 0 0000

S

0000 0006 0 0 @0 0 0 0N Ph ),NR H 0 N CHO

S.

@0 0 00 @0 0 00

OS

C0 2

H

235eR C0 2

H

238eR (233e). A solution of (1S,9S) 6,10-dioxo- 1,2,3,4,7,8,9, 10-octahydro-9- (benzoylamino) -6Hpyridazino[1,2-a] [1,2]diazepine-1-carboxylic acid 129 (212e) (345mg, 1.0mmol), (208a) (361mg, 1.lmmol, 1.1 equiv) and (Ph 3

P)

2 PdCl 2 (20mg) in CH 2 C1 2 (5ml), was treated dropwise with n-Bu 3 SnH (0.621ml, 2.3mmol, 2.1 equiv). The resulting orange brown solution was stirred at 25 °C for 10min and then 1hydroxybenzotriazole (297mg, 2.2mmol, 2 equiv) was added. The mixture was cooled to 0 oC and ethyldimethylaminopropyl carbodiimide (253mg, 1.3mmol, 1.2 equiv) added. After stirring at 0 oC for 10min and 10 then at room temperature overnight, the mixture was diluted with EtOAc (50ml) and the resulting solution washed successively with 1M HC1 (3 x 25ml), 10% NaHCO 3 (3 x 25ml) and sat. aq. NaCl, dried (MgSO 4 and concentrated. Flash chromatography on silica gel (2- 10% methanol in CH 2 C1 2 afforded compound 233e (280mg, 49%) as a tan solid: [Q]D2 -95 (c 0.09, MeOH); IR (KBr) 3477, 3333, 2968, 2932, 1633, 1580, 1535, 1423, 1378, 1335, 1259, 1156, 1085, 709; H NMR (CDC13) 6 9.32 (1H, 7.83-7.39 (6H, 7.11-7.09 (1H, m), 20 6.30-5.30 (2H, brs), 5.17-5.05 (2H, 4.62-4.38 (2H, 3.30-3.15 (1H, 3.13-2.65 (2H, 2.46-2.19 (3H, 2.15-1.54 (8H, 1.42 (9H, s) (236e) was prepared by an analogous method to that used for 233e using (4R) t-butyl N-allyloxycarbonyl-4-amino- 5-oxo-pentanoate semicarbazone (208b, 435mg, 1.33mmol).

The product was obtained as a foam (542mg, 71%): -990 (c 0.19, CHC1 3 IR (KBr) 3473, 3331, 3065, 2932, 2872, 1660, 1580, 1533, 1488, 1423, 1370, 1337, 1278, 1254, 1223, 1155, 1080, 1024, 983, 925, 877, 846, 801, 770, 705; 1H NMR (CDC13) 6 9.42 (1H, 7.81 (2H, 7.51-7.40 (4H, 7.06 (1H, 6.50-5.50 (2H, broad 5.25-5.00 (2H, 4.60-4.45 (2H, 3.15- 2.85 (2H, 2.75-2.35 (1H, 2.30-1.23 (11H, m), 1.42 (9H, 130 SO 0 @0 e 6 0000 0 4 0000

S

0660 (234e). A solution of semicarbazone 233e (390mg, 0.68mmol) in methanol (10ml) was cooled at 0 °C and then treated with a 38% aq. solution of formaldehyde (2ml) and 1M HC1 (2ml). The reaction mixture was then stirred overnight at room temperature. The solution was concentrated to remove the methanol. The aq.

solution was extracted with EtOAc (30ml). The organic solution was successively washed with 10% NaHCO 3 and sat. aq. NaCl (30ml), dried (MgSO 4 and concentrated. Purification by flash chromatography on silica gel methanol in CH 2 Cl 2 afforded 234e (179mg, 51%) as a white foam: [a]D20 -1010 (c 0.064, MeOH); IR (KBr)3346, 2976, 2934, 1730, 1657, 1535, 1456, 1425, 1278, 1255, 1156, 708; 1H NMR (CDC13) 15 9.56 (1H, 7.88-7.38 (5H, 7.01 and 6.92 (2H, 2d), 5.27-5.08 (2H, 4.69-4.46 (1H, 3.50-3.27 (2H, 3.15-2.73 (2H, 2.46-1.83 (10H, 1.45 (9H, s).

(237e) was prepared from 236e by an analogous method to 20 234e to afford a white foam (390mg, [aD20 -1130 (c 0.242, CHC1 3 IR (KBr) 3352, 3065, 2974, 1729, 1657, 1536, 1489, 1454, 1423, 1369, 1338, 1278, 1255, 1223, 1156, 1078, 1026, 981, 846, 709.

(235e). A solution of t-butyl ester 234e (179mg, 0.35mmol) in dry CH 2 C12 (3ml) was cooled to 0 °C and treated with trifluoroacetic acid (2ml). The resulting solution was stirred at 0 °C for 30min and then at room temperature for 2h. The solution was concentrated, the residue taken up in dry CH 2 C1 2 (5ml) and the mixture again concentrated. This process was repeated once again with more CH 2 C1 2 (5ml). The residue obtained was crystallized in diethyl ether. The precipitate was collected and purified on silica gel column methanol in CH 2 C1 2 which afforded compound 235e as a 131 .9 6 *9

SO

9. B o 9

S.

9 9000e0 6 6 *e 9 9

S.

0S* 0 *09066

B

0e4* a .*e white solid (111mg, mp 142 0 C (dec) tc]D 20 -85.5 (c 0.062, MeOH); IR 3409, 3075, 2952, 1651, 1541, 1424, 1280, 1198, 1136, 717; 1HNMR (D 6 -DMSO) 9.40 (1H, 8.62 (2H, in), 7.96-7.38 (5H, in), 5.19- 5.02 in), 4.98-4.79 (1H, in), 4.48-4.19 (1H, mn), 3.51-3.11 (2H, mn), 3.04-2.90 (2H, mn), 2.38-1.46 Mn).

(238e) was prepared from 237e by an analogous route to 235e which afforded a beige foam (190mg, 20 10 -78 (c 0.145, MeOH); IR .(KBr 3400, 3070, 2955, 2925, 2855, 1653, 1576, 1541, 1490, 1445, 1427, 1342, 1280, 1258, 1205, 1189, 1137, 1075, 1023, 983, 930, 878, 843, 801, 777, 722; H NMR (D 6 -DMSO) 5 9.40 (1H, 8.72- 8.60 (2H, mn), 7.89 (2H, 7.56-7.44 (3H, mn), 5.17 (1H, in), 4.90-4.83 (1H, in), 4.46-4.36 mn), 4.20- 4.15 (1H, mn), 3.40-3.30 (1H, mn), 2.98-2.90 (2H, mn), 2.50-1.60 (10H, in).

0 0 (243) 0S S B

S.

*9 0 66 (244) -0

PI

H H OBn (246) (245) (243) was prepared from (1S,9S) t-butyl 9-aminooctahydro-10-oxo-6H-pyridazino[l,2-a] [1,2]diazepine-1carboxylate (Attwood, et al. J. Chem. Soc., Perkin 1, pp. 1011-19 (1986)), by the method described for 211e, to afford 2.03g of a colourless foam: [a1D 2 132 -15.9" (c 0.5, CH 2 C1 2 IR (KBr) 3400, 2976, 2937, 1740, 1644, 1537, 1448, 1425, 1367, 1154; 1H NMR (CDC1 3 5 7.88-7.82 (2H, 7.60-7.38 (4H, 5.48 (1H, 4.98 (1H, 3.45 (1H, 3.22-2.96 (2H, m), 2.64 (1H, 2.43-2.27 (2H, 1.95 (2H, 1.82- 1.36 (4H, 1.50 (9H, Anal. Calcd for C 2 1

H

2 9

N

3 0 4 0.25H 2 0: C, 64.35; H, 7.59; N, 10.72. Found: C, 64.57; H, 7.43; N, 10.62. MS (ES m/z) 388 (100%, M 1).

10 (244) was prepared from (1S,9S) t-butyl 9-benzoylamino- Seoctahydro-10-oxo-6H-pyridazino- [1,2]diazepine-l-carboxylate (243), by the method described for 212e, to afford 1.52g of a •25 white powder: mp. 166-169 °C (dec); [a]D 2 -56.40 (c 0.5, CH 3 0H); IR (KBr) 3361, 2963, 2851, 1737, 1663, 1620, 1534, 1195, 1179; 1H NMR (D 6 -DMSO) 5 12.93 (1H, brs), 8.44 (1H, d, J 7.93 (2H, 7.54 (3H, ^.oo 5.46 (1H, 4.87 (1H, 3.12 (2H, 2.64 (1H, 2.64 (1H, 2.27 (1H, 1.98-1.68 (7H, 1.40 20 (1H, Anal. Calcd for C 17

H

21

N

3 0 4 0.25H 2 0: C, 60.79; H, 6.45; N, 12.51. Found: C, 61.07; H, 6.35; N, 12.55. MS m/z) 332 M+ 211 (100).

(245) was prepared from (1S,9S) 9-benzoylaminooctahydro-10-oxo-6H-pyridazino[1,2-a][1,2]-diazepine-1carboxylic acid (244), by the method described for 213e, to afford 601mg of a colourless foam: IR (KBr) 3401, 2945, 1794, 1685, 1638, 1521, 1451, 1120; H NMR (CDC1 3 5 7.87-7.77 (2H, 7.57-7.14 (10H, m), 5.59-5.47 (2H, 4.97-4.32 (4H, 3.27-1.35 (14H, Anal. Calcd for C 2 8

H

3 2

N

4 0 6 0.5H 2 0: C, 63.50; H, 6.28; N, 10.58. Found: C, 63.48; H, 6.14; N, 10.52.

MS (ES m/z) 521 (100%, M+ 1).

(246) was prepared from [3S, 2RS (1S,9S)]N-(2benzyloxy-5-oxotetrahydrofuran-3-yl)-9-benzoylamino- 133 octahydro-10-oxo-6H-pyridazino[1,2-a][1,2]diazepine-1carboxamide (245), by the method described for 214e, to afford 396mg of a white powder: mp. 110-115 OC; [a]D 2 6 -126.30 (c 0.2, CH 3 0H); IR (KBr) 3345, 2943, 1787, 1730, 1635, 1578, 1528, 1488, 1450, 1429; H NMR

(CD

3 0D) 6 7.88 (2H, 7.48 (3H, 5.55 (1H, m), 4.91 (1H, 4.56 (1H, 4.29 (1H, 3,41-3.05 (3H, 2.76-2.41 (3H, 2.28-2.01 (3H, 1.86- 1.65 (4H, 1.36 (1H, Anal. Calcd for C 2 1

H

2 6

N

4 06- 1.25H 2 0: C, 55.68; H, 6.34; N, 12.37. Found: C, 55.68; H, 6.14; N, 12.16. MS (ES m/z) 429 (100%, M 1).

S.

95

S

S

S.

*09550

S

S

90 5 C0 2

CH

3 (248) c *c S

O

5* 0

SS

S

(247)

CO

2 H CH O COH HC*IH2Nw.,,,,. 0 ONO v 0

OCH

3 0 0OC (249) (250) (247). n-Butyllithium (1.6M in hexane) (22.3ml, 35.7mmol) was added dropwise over 20min to a solution of (2R)-(-)-2,5-dihydro-3,6-dimethoxy-2-(1- 134 methylethyl)pyrazine (5.8ml, 6.0g, 32.4mmol) in THF (250ml) cooled to -75 °C at a rate such that the temperature was maintained below -72 The reaction mixture was stirred for lh at -75 °C and a solution of 2,6-di-t-butyl-4-methoxyphenyl-2-butenoate (Suzuck et al. Liebiqs Ann. Chem. pp. 51-61 (1992)) (9.9g, 32.5mmol) in THF (60ml) was added over 30 minutes maintaining the temperature below -72 °C during the addition. The reaction mixture was kept at -75 °C for 10 1.5h and a solution of glacial acetic acid (6ml) in THF (25ml) was added at -75 °C and the solution warmed to room temperature. The solution was poured onto

NH

4 Cl (300ml) and extracted with diethyl ether (3 x 250ml). The combined organic phases were washed with brine (2 x 200ml), dried over Na 2

SO

4 and evaporated to dryness under reduced pressure. The residual oil was purified by flash chromatography on silica gel heptane in CH 2 Cl 2 which afforded the title compound as *eO- a light yellow oil (13.5g, [1]D -640 (c 0.22, 20 MeOH); IR (KBr) 2962, 2873, 2840, 1757, 1697, 1593, 1460, 1433, 1366, 1306, 1269, 1236, 1187, 1157, 1126, 1063, 1038, 1011, 970, 924, 892, 867, 846, 831, 797, 773, 754; 1H NMR (CDC13) 5 6.85 (2H, 4.21 (1H, t, J 3.98 (1H, t, J 3.79 (3H, 3.71 25 (3H, 3.69 (3H, 3.15 (1H, dd, J 17.8, 7.9), 2.86-2.81 (1H, 2.58 (1H, dd, J 17.8, 2.28- 2.19 (1H, 1.33 (18H, 1.02 (3H, d, J 6.8), 0.70 (6H, dd, J 13, 6.8).

(248). A solution of (247) (22.4g, 45.8mmol) in acetonitrile (300ml) and 0.25N HC1 (366ml, 2 equiv) was stirred at room temperature under nitrogen atmosphere for 4 days. The acetonitrile was evaporated under reduced pressure and diethylether (250ml) was added to the aq. phase. The pH of the aq. phase was adjusted to 135 pH8-9 with concentrated ammonia solution and the phases separated. The aq. phase was extracted with diethylether (2 x 250ml). The combined organic phases were dried over Na 2

SO

4 and evaporated to dryness under reduced pressure. The residual oil was purified by flash chromatography on silica gel methanol in

CH

2 Cl 2 which afforded the required product as a light yellow oil (8.2g, [W]D 20 +20 (c 0.26, MeOH); IR(KBr) 3394, 3332, 3000, 2962, 2915, 2877, 2838, 1738, S* 10 1697, 1593, 1453, 1430, 1419, 1398, 1367, 1304, 1273, 1251, 1221, 1203, 1183, 1126, 1063, 1025, 996, 932, 891, 866, 847, 800, 772, 745; 1H NMR (CDC13) 5 6.85 (2H, 3.79 (3H, 3.74 (3H, 3.72-3.69 (1H, m), 3.05-2.85 (1H, 2.67-2.50 (2H, 1.32 (18H, s), 0.93. (3H, d, J Anal. Calcd for C 2 2

H

3 5

NO

5

C,

67.15; H, 8.96; N, 3.56. Found: C, 67.20; H, 9.20; N, 3.70.

(249). A solution of (2S,3S)-5-[2,6-di-t-butyl-4methoxyphenyl]3-methylglutamate (248) (8.0g, 20.3mmol) 20 in 5N HCI (200ml) was heated at reflux for 2h. The reaction mixture was evaporated to dryness under reduced pressure. The residue was dissolved in cyclohexane (x4) and evaporated to dryness (x4) which afforded a white solid (7.9g, mp 230 oC; [a]D 25 +220 (c 0.27, MeOH); IR (KBr) 3423, 2964, 1755, 1593, 1514, 1456, 1421, 1371, 1303, 1259, 1201, 1179, 1138, 1106, 1060, 966, 926, 861, 790, 710; 1H NMR (MeOD) 6 6.76 (2H, 4.02 (1H, d, J 3.67 (3H, s), 3.05-2.85 (1H, 2.80-2.55 (2H, 1.22 (18H, s), 1.09 (3H, d, J 13C NMR (MeOD) 5 174.5, 171.4, 158.6, 145.2, 143.1, 113.2, 58.3, 56.3, 39.8, 36.9, 32.5, 16.6; Anal. Calcd for C 2 1

H

3 4

CINO

5 C, 60.64; H, 8.24; N, 3.37. Found: C, 60.80; H, 8.40; N, 3.40.

136 (250) Diisopropylethylamine (4.1ml, 3.04g, 23.5mmol, 1.25 equiv) and phthalic anhydride (3.5g, 23.6mmol, 1.25 equiv) were added to a solution of (2S,3S)-5-[2,6di-t-butyl-4-methoxyphenyl]3-methylglutamate (249) (7.8g, 18.6mmol) in toluene (300ml). and the resulting mixture was heated at reflux for 3 hours. After cooling to room temperature, the reaction mixture was evaporated to dryness and the resulting oil purified by flash chromatography on silica gel methanol in 10 CH 2 C1 2 which afforded the required product as a white 20 foam (8.35g, [C]D -200 (c 1.04, MeOH); IR (KBr) 3480, 2968, 2880, 1753, 1721, 1594, 1462, 1422, 1388, 1303, 1263, 1216, 1183, 1148, 1062, 1003, 933, 899, 755, 723; 1 H NMR (CDCl 3 5 7.92-7.87 (2H, m), 7.78-7.73 (2H, 6.84 (2H, 4.95 (1H, 3.78 (3H, 3.30-3.05 (2H, 2.85-2.65 (1H, 1.30 (18H, 1.13 (3H, d).

137 0 C0 2 H t-Bu N 0 CO2-t-Bu t-Bu 0 t-Bu OMe (250) (251) :C0 2 H CBz 0* H' 0 COZ-t-Bu eNN N

OH

0 00 0S*(253)1 (252) 0 0 *00 0N

N

(255)1 0 0 N NO 4 H ON CHO H 0 N (257 )H(256) H OBn (251). A solution of the amino acid (250) (1.2g, 2.35mmol) in dry diethylether (l0mi) was treated with phosphorus pentachioride (0.52g, 2.5mmol) at room temperature for 2h. The mixture was concentrated and.

treated several times with toluene and again evaporated to dryness. The resulting acid chloride was dissolved 138 in dry THF (5ml) and CH 2 C12 (5ml) and cooled to 0 0C.

t-Butyl-l-(benzyloxycarbonyl)-hexahydro-3-pyridazinecarboxylate (0.753g, 2.35mmol, 1 equiv) and Nethylmorpholine (3ml) were added to the solution. The reaction mixture was stirred for 30min at 0 °C and then overnight at room temperature. The mixture was evaporated and the resulting residue taken up with

CH

2 Cl 2 (30ml). The solution was washed with 1M HC1, water, 10% NaHCO 3 dried (MgSO 4 and evaporated. The S. 10 resulting white foam was purified on silica gel (0-2% methanol in CH2C1 2 which afforded the required compound 251 as a pale yellow glassy solid (740mg, [a]D 2 -22 (c 0.42, MeOH); IR (KBr) 3441, 2966, 1725, 1693, 1386, 1255, 1221, 1186, 1154, 1123, 1063, 724;1 H NMR (CDC13) 5 7.94-7.89 (4H, 7.56-7.28 6.84 (2H, 2s), 5.29-5.20 (2H, AB), 4.91-4.81 (1H, 4.05-3.88 (1H, 3.78 (3H, 3.75-3.80 (1H, m), 3.28-2.95 (2H, 2.23-1.51 (6H, 1.45 (9H, s), 1.31 (9H, 1.28 (9H, 1.27 (3H, d).

(254). A solution of the protected acid (251) (715mg, 0.893mmol) in acetonitrile was treated with Cerium (IV) ammonium nitrate (1.8g, 3.3mmol, 3.7 equiv) in water (3ml) for 4h at room temperature. Mannitol (600mg, 3.3mmol, 3.7 equiv) was added and the mixture was 25 stirred for lh. Diethylether (50ml) and water were added to the mixture. After decantation, the aq.

phase was extracted with diethylether (4 x 50ml). The combined organic phase was washed with water, dried (MgSO 4 and concentrated. Chromatography on silica gel (10% methanol in CH 2 Cl 2 afforded 5-(1benzyloxycarbonyl-3-t-butoxycarbonylhexahydropyridazin-2-yl)carbonyl-3-methyl-4phthalimidopentanoic acid (252) (360mg, [a]D 20 -49.2 c 0.118, MeOH). This product was used without 139 further purification (360mg, 0.609mmol), and was hydrogenated in methanol (30ml) using 10% Pd/carbon (36mg) for 3h. The reaction mixture was filtered and the resulting solution concentrated to afford the amine (253) as a foam (270mg, 96%) [a]D 2 0 -56.1 (c 0.18 MeOH). The amine (253) was dissolved in dry THF and phosphorous pentachloride (305mg, 1.47mmol, equiv) was added. The mixture was then cooled to -5 °C and N-ethylmorpholine was added under nitrogen. The *o 10 reaction mixture was stirred overnight at room temperature. The mixture was concentrated and the residue taken up with CH 2 C1 2 (20ml), cold H 2 0 (20ml), 1M HC1 (20ml). After decantation, the aq. phase was reextracted with CH 2 Cl 2 (2 x 20ml). The combined organic phase was washed with 10% NaHCO 3 and water, dried (MgSO 4 and concentrated. The resulting oil was purified on silica gel methanol in CH 2 Cl 2 S• affording the bicyclic compound (254) as a solid 20 [a]D -77 (c 0.208, MeOH); IR (KBr) 3471, 3434, 2975, 2928, 1767, 1723, 1443, 1389, 1284, 1243, 1151, 1 1112, 720; H NMR (CDC1 3 6 7.94-7.69 (4H, 5.34- 5.27 (1H, 4.89-4.66 (2H, 3.94-3.64 (2H, m), 3.02-2.84 (1H, 2.34-2.19 (2H, 1.94-1.61 (3H, 1.47 (9H, 1.14 (3H, Anal. Calcd for

C

23

H

27

N

3 0 6 C, 62.57; H, 6.17; N, 9.52. Found: C, 62.60; H, 6.40; N, 9.10.

(255). A solution of the bicyclic compound (254) 0.16mmol) in ethanol was treated with hydrazine hydrate (0.02ml, 4mmol, 2.5 equiv). After 5h stirring at room temperature, the mixture was concentrated and the resulting residue taken up in toluene and reevaporated. The residue was treated with 2M acetic acid (2ml) for 16h. The resulting precipitate was filtered and washed with 2M acetic acid (10ml). The 140 filtrate was basified with solid NaHCO 3 and then extracted with EtOAc. The organic solution was washed with water, dried (MgSO 4 and concentrated.

Purification by flash chromatography on silica gel (2% methanol in CH 2 C12) afforded the free amine as a foam 100%). The amine (50mg, 0.16mmol) was dissolved in dioxane (1ml) and water (0.25ml) and treated with NaHCO 3 (0.034g, 0.04mmol) followed by benzoylchloride (0.047ml, 0.40mmol, 2.8 equiv). The mixture was 10 stirred overnight at room temperature, then diluted with EtOAc (15ml). The organic solution was washed with 10% NaHCO 3 and sat. aq. NaCl, dried (MgSO 4 and concentrated. Purification by flash chromatography on silica gel methanol in CH 2 C12) afforded the benzamide 255 as a foam (67mg, 100%): 1H NMR (CDCl 3 6 7.89-7.39 (5H, 6.79 (1H, 5.32-5.20 (1H, m), 4.98-4.82 (1H, 4.75-4.64 (1H, 3.84-3.65 (1H, 3.09-2.89 (1H, 2.45-2.18 (2H, 2.00-1.61 (4H, 1.48 (9H, 1.28 (3H, d).

(257). A solution of t-butyl ester 255 (67mg, e 0.16mmol) in CH 2 Cl 2 (1ml) was treated at 0 °C with trifluoroacetic acid (1ml). The resulting solution was stirred at 0. C for 15min and then at room temperature for lh. The solution was concentrated, the residue 25 taken up in dry CH 2 C12 (2 x 2ml) and the mixture again concentrated The residue was crystallized from diethylether. Filtration of the precipitate afforded the free acid of 255 as a grey solid (40mg, A solution of acid (40mg, 0.llmmol), N-allyloxycarbonyl- 4-amino-5-benzyloxy-2-oxotetrahydrofuran (Chapman, Bioorq. Med. Chem. Lett., 2, pp. 615-18 (1992); 39mg, 0.13mmol, 1.2equiv) and (Ph 3

P)

2 PdCl 2 (3mg) in a mixture of dry CH 2 C1 2 (1ml) and dry DMF (0.2ml) was treated dropwise with n-Bu 3 SnH (0.089ml, 0.33mmol, 3 equiv).

141 w 9* 0 6O 0 0

S

S

*5 S 0

S

@5 The resulting solution was stirred at 25 OC for and then 1-hydroxybenzotriazole (36mg, 0.266mmol, 2.4 equiv) was added. The mixture was cooled to 0 oC and ethyldimethylaminopropyl carbodiimide (31mg, 0.16mmol, l.5equiv) was added. After stirring at 0 OC for and then at room temperature overnight, the mixture was diluted with EtOAc (20ml) and the resulting solution washed successively with 1M HC1 (2 x 5ml), 10% NaHCO 3 (2 x 5ml) and sat. aq. NaCl (5ml), dried (MgSO 4 and 10 concentrated. Flash chromatography on silica gel (2% methanol in CH 2 Cl 2 afforded a mixture of diastereoisomers (256) as a grey solid (50mg, 82%).

This product (256) was used without further purification (50mg, 0.091mmol) and was hydrogenated in methanol (5ml) using 10% Pd/carbon (30mg) for 24h. The reaction mixture was filtered and the resulting solution concentrated. Flash chromatography on silica gel (2-20% methanol in CH 2 Cl 2 afforded compound 257 (9mg, 21%) as a white solid: 1H NMR (D4-MeOH) 5 7.88- 20 7.29 (5H, 5.18-4.99 (1H, 4.59-4.35 (3H, m), 4.26-4.11 (1H, 3.65-3.41 (2H, 3.18-2.91 (1H, 2.62-1.47 (8H, 1.29-1.00 (3H, 2d) (mixture of acetal and hemiacetal). MS (ES 457.

Compounds 280-283 were prepared from 212b by a method similar to the method used to prepare 226e.

Compounds 284-287 were prepared by a method similar to the method used to prepare 217e.

142 0

OH

0 N R

H

280-287 compound R5 R

N

0S 280 *281 BF 282

SS

283 UN @0 a 284 Ib,3C,.ck a 285 9 0 0 H3C 286 H 3 COX _0 I 287 H3C 143

C

2 -t-Bu C0 2 -t-Bu AIIocN 4 H H 2 N-OR rlo- 306 0 N 0 0 N1 q N H 00 O0 N 0 N 0 C N2~ sea. 212e OHNC2tB 0 00 0 S@0 seN 11CO2H 06000H 308 H 308 N OR

CI

a R= (306a) was prepared by a similar procedure as 208a except that 2,6-dichiorophenylmethoxyamine (prepared by se. a similar method as 306b) was used instead of semicarbazide to give 870mg (quant.) as a clear oil.

(306b) was prepared by a similar procedure as 208a except that 2-(phenyl)ethoxyamine (US 5 346 911) was used instead of semicarbazide to give 395mg (quant.) as a clear oil.

(307a) was prepared by a procedure similar to 233e except 306a was used instead of 207a to give 23 mg(23%) of 307a as a white solid.

144 (307b) was prepared by a procedure similar to 2 33e except 306b was used instead of 207a to give 43 mg(48%) of 307b as a white solid.

(308a) was prepared by from 307a a procedure similar to the preparation of 235e from 234e to give 15.2 mg (74%) as white solid: 1 NMR(CD 3 OD) 5 1.3(s), 2.3(dd), 2.4- 3.4-3.5(m), 7.2- 10 7.8(dd), 8.4(dd), (308b) was prepared by from 307b a procedure similar to the preparation of 235e from 234e to give 25.2 mg (68%) as white solid: 1 NNR(CD 3 OD) 6 1.6-1.7(m), 2.6-2.7(dd), 4.25(m), 7.35(dd), 7.8(dd), 8.3(dd).

@0 0 .4 eq

S

0O 0S

S

*5600e 4 0 4.

0 0055 0* 0 See 0 000000 e *005

S

0*SS *.eS Se 05 e 0* S 0 0g S. C 0* 0e 145 O 0 N CO-t-Bu J H 0> H 0H H NN

H

H.

(212e) (301) NH O NH 2 o 0 O I O N CO7t-Bu C0 2 -t-Bu I OO N H H OR H O H OR (302) (303a) R=CH 3 0 o N 0 0 H

OR

5 (304a) R=CH 3 (302).

Step A: 301 was prepared by procedure similar to 605a (Step except 212e was used instead of 603a to give 540 mg to give a white solid.

Step B: 302. A solution of 301 (50.7 mg; 0.091 mmol) in 2.8 ml of MeOH/HOAc/37% aq. formaldehyde was stirred at rt for 5.5 h. and the reaction was concentrated to 0.7 ml in vacuo. The residue was dissolved in 3 ml of CH 3 CN and concentrated to 0.7 ml dissolved in toluene and concentrated to 0.7 ml in vacuo and concentrated to dryness.

Chromatography (flash, SiO 2 5% isopropanol/CH 2 Cl 2 gave 302 (45.5 mg, 78%) as a white solid: 1H NMR(DMSO-d 6 146 1.0-1.15(m, 2H), 1.4(s, 9H), 1.65(m, 2H), 1.9-2.1(m, 2H), 2.15-2.4(m, 3H), 2.55(m, 1H), 2.7-3.0Cm, 2H), 4.3- 4.6(m, 2H), 4.9(m, 1H), 5.2(m, 1H), 7.4-7.6(m, 2H), 7.8-8.0Cm, 2H), 8.6Cm, 1H), 8.8(m,1H), 1H).

(304a).

Step A: A solution of 302 (90 mg; 0.18 nunol) in 10 ml of MeOH was treated with trimethylorthoformate Cimi) and p-toluene sulfonic acid hydrate (5 mg; 0.026 mmol) and the reaction was stirred for 20 h. The reaction 10 was treated with 3 ml of aq. sat. NaH-C0 3 and concentrated in vacuo. The residue was taken up in EtOAc and washed with dilute aq. NaHCO 3 dried over 0 S IMgSO 4 and concentrated in vacuo to give 80 mg of 303a.

VD*. Step B: 303a was dissolved in 2 ml of TEA and stirred at rt for 15 min. The reaction was dissolved in CH 2 Cl 2 and concentrated in vacuo (3x) Chromatography (flash, *SiO 2 1% to 3% MeOH/CH 2 Cl 2 gave 43 mg of 304a as a white solid: ~H NNR(CDCl 3 45 1.55-l.8(m, 2H), 1.9- *0*6 2.15(m, 4H), 2.25-2.5(m, 2H), 2.7-3.3(m, 4H), 3.45, 3.6(s, s, 3H), 4.4, 4.75(2m, 1H), 4.6(m, 1H), 4.95, 5.4(t,d, 1H), 5.1-5.2(m, 1H), 6.45, 7.05(2d, 1H), 6.95(m, 1H), 7.45(m, 2H), 7.5Cm, 1H), 7.85Cm, 2H).

Exam-Ple 11 .Compounds 214e, 404-413, 415-445, 446-468, 470-491, and 493-499 were synthesized as described in Example 11 and Table 7.

147 -Bu

H

2 N Rs Step A 400 401 sor** 0* 0 *qees see* r oft Step B 402 Step C H

H

0 00 a 0e SS S S0 a a.

403 Step D ,0 2

H

H

.N-s~2 214e, 404-499 148 Step A. Synthesis of 401. TentaGel S® NH 2 resin (0.16 mmol/g, 10.0 g) was placed in a sintered glass funnel and washed with DMF (3 x 50 mL), 10% DIEA in DMF (2 x 50 mL) and finally with DMF (4 x 50 mL).

Sufficient DMF was added to the resin to obtain a slurry followed by 400 (1.42 g, 2.4 mmol, prepared from (3S)-3-(fluorenylmethyloxycarbonyl)-4-oxobutryic acid t-butyl ester according to A.M. Murphy et. al. J. Am.

Chem. Soc., 114, 3156-3157 (1992)), 1- 10 hydroxybenzotriazole hydrate (HOBT.H 2 0; 0.367 g, 2.4 mmol), O-benzotriazol-l-yl-N,N,N,N'-tetramethyluronium hexafluorophosphate (HBTU; 0.91 g, 2.4 mmol), and DIEA •o (0.55 mL, 3.2 mmol). The reaction mixture was agitated overnight at rt using a wrist arm shaker. The resin was isolated on a sintered glass funnel by suction filtration and washed with DMF (3 x 50 mL). Unreacted amine groups were then capped by reacting the resin with 20% Ac20/DMF (2 x 25 mL) directly in the funnel (10 min/wash). The resin was washed with DMF (3 20 x 50 mL) and CH 2 Cl 2 (3 x 50.mL) prior to drying overnight in vacuo to yield 401 (1.1.0 g, quantitative yield).

Step B. Synthesis of 402. Resin 401 (6.0 g, 0.16 mmol/g, 0.96 mmol) was swelled in a sintered glass funnel by washing with DMF (3 x 25 mL). The Fmoc protecting group was then cleaved with 25% (v/v) piperidine/DMF (25 mL) for 10 min (intermittent stirring) and then for 20 min with fresh piperidine reagent (25 ml). The resin was then washed with DMF (3 x 25 ml), followed by N-methypyrrolidone (2 x 25 mL).

After transferring the resin to a 100 mL flask, Nmethypyrrolidone was added to obtain a slurry followed by 212f (0.725 g, 1.57 mmol), HOBT.H 2 0 (0.25 g, 1.6 mmol), HBTU (0.61 g, 1.6 mmol) and DIEA (0.84 mL, 4.8 149 mmol). The reaction mixture was agitated overnight at rt using a wrist arm shaker. The resin work-up and capping with 20% Ac 2 0 in DMF were performed as described for 401 to yield 402 (6.21 g, quantitative yield).

Step C. Synthesis of 403. This compound was prepared from resin 402 (0.24 g, 0.038 mmol)-using an Advanced ChemTech 396 Multiple Peptide synthesizer.

The automated cycles consisted of a resin wash with DMF 10 (3 x 1 mL), deprotection with 25% piperidine in DMF (1 mL) for 3 min followed by fresh reagent (1 mL) Sfor 10 min to yield resin 403. The resin was washed with DMF (3 x 1 mL) and N-methypyrrolidone (3 x 1 mL).

Step D. Method 1. (409). Resin 403 was acylated with a solution of 0.4M thiophene-3-carboxylic acid and 0.4M HOBT in N-methypyrrolidone (1 mL), a solution of 0.4M HBTU in N-methylpyrrolidone (0.5 mL) and a solution of 1.6M DIEA in N-methypyrrolidone (0.35 mL) and the reaction was shaken for 2 hr at rt. The acylation step was repeated. Finally, the resin was washed with DMF (3 x 1 mL), CH 2 Cl 2 (3 x 1 mL) and dried in vacuo. The aldehyde was cleaved from the resin and globally deprotected by treatment with 95% TFA/5% H 2 0 1.5 mL) for 30 min at rt. After washing the resin with cleavage reagent (1 mL), the combined filtrates were added to cold 1:1 Et 2 0:pentane (12 Ml) and the resulting precipitate was isolated by centrifugation and decantation. The resulting pellet was dissolved in 10% CH 3 CN/90% H 2 0/0.1% TFA (15 mL) and lyophilized to obtain crude 409 as a white powder. The compound was purified by semi-prep RP-HPLC with a Rainin Microsorb m C18 column (5 p, 21.4 x 250 mm) eluting with a linear CH 3 CN gradient containing 0.1% TFA over 45 min at 12 mL/min.

150 Fractions containing the desired product were pooled and lyophilized to provide 409 (10.8 mg, 63%).

Step D. Method 1A. Synthesis of 418. Following a similar procedure as method 1, resin 403 was acylated with 4-(l-fluorenylmethoxycarbonylamino)benzoic acid and repeated. The Fmoc group was removed as described in Step C and the free amine was acetylated with Ac20 in DMF (1 mL) and 1.6M DIEA in Nmethylpyrrolidone (0.35 mL) for 2 hr at rt. The 10 acetylation step was repeated. Cleavage of the e aldehyde from the resin gave 418 (3.2 mg).

C

Step D. Method 1B. Synthesis of 447. Following Soa similar procedure as method 1A, resin 403 was acylated with 0.4M 4-(1fluorenylmethoxycarbonylamino)benzoic acid. The acylation step was repeated once. The Fmoc group was removed as before and the free amine was reacted with 1M methanesulfonyl chloride in CH 2 Cl 2 (0.5 mL) and 1M pyridine in CH 2 Cl 2 (0.60 mL) for 4 hr at rt. Cleavage 20 of the aldehyde from the resin gave 447 (10.0 mg) Step D. Method 2. Synthesis of 214e. Following a similar procedure as method 1, resin 403 was acylated with 0.5M benzoyl chloride in N-methypyrrolidone (1 mL) "and 1.6M DIEA in N-methypyrrolidone (0.35 mL) for 2 hr at rt. The acylation step was repeated. Cleavage of the aldehyde from the resin gave 214e (5.1 mg, Step D. Method 3. Synthesis of 427. Following a similar procedure as method 1, resin 403 was reacted with 1.OM benzenesulfonyl chloride in CH 2 C12 (0.5 mL) and 1M pyridine in CH 2 Cl 2 (0.60 mL) for 4 hr at rt.

The reaction was repeated. Cleavage of the aldehyde from the resin gave 427 (7.2 mg, 151 Step D. Method 4. Synthesis of 420. Following a similar procedure as method 1, resin 403 was reacted with 0.5M methylisocyanate in N-methypyrrolidone (1 mL) and 1.6M DIEA in N-methypyrrolidone (0.35 mL) for 2 hr at rt. The reaction was repeated. Cleavage of the aldehyde from the resin gave 420 (8.3 mg, Step D. Method 5. Synthesis of 445. Following a similar procedure at method 1, resin 403 was acylated with 0.27M imidazole-2-carboxylic acid (1 mL) in 2:1 S. 10 DMF:H 2 0 (with 1 eq. DIEA) and 1M 1-(3dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in 2:1 N-methypyrrolidone/H 2 0 (0.35 mL) for 3 hr at rt. Cleavage of the aldehyde from the resin gave 445 (9.5 mg).

15 Analytical HPLC methods: Waters DeltaPak C18, 300A (5p, 3.9 x 150 mm).

Linear CH 3 CN gradient 45%) containing 0.1% TFA over 14 min at 1 mL/min.

Waters DeltaPak C18, 300A (5p, 3.9 x 150 mm).

Linear CH 3 CN gradient 25%) containing 0.1% TFA over 14 min at 1 mL/min.

Waters DeltaPak C18, 300A (5p, 3.9 x 150 mm).

Isocratic elution with 0.1% TFA/water at 1 mL/min.

Waters DeltaPak C18, 300A (5p, 3.9 x 150 mm).

Linear CH 3 CN gradient 30%) containing 0.1% TFA over 14 min at 1 mL/min.

Waters DeltaPak C18, 300A (5p, 3.9 x 150 mm).

Linear CH 3 CN gradient 35%) containing 0.1% TFA over 14 min at 1 mL/min.

0 C C C C 0

C

0 0

C

0 C C C C C 0 S C CS S C 0 S C S S C. S 050 000 OS S C C S 0 05 S S C 0 55 0 C S 0 0 C 0 S S 0 C 0 0 S .55 CCC SOC Table 7 Cmpd. Structure jMF MW HPLC RT MS Syn.

214e 0 0 Q1g <H C21H24N407 444.45 6.67 (2) 98% 445 4. 4 4 404 0 0 'S0 0 N 0

OH

C22H2 6N407 458.48 6.66 (2) 97 459 I I 4- 4 405 C22H2 6N408 474.47 8.2 (1) 98% 475 0 a

S

0

S

0 0* 5 0@ 0@ S S0 0 0 0 0@ 00 0 000 000 @5 0 0 0 OS 5 0 00 5 0 5 0 0 S 6 0 0 SOS @00 560 HPLC RT MS Syn.

Cmpd. Structure MF MWMethod 406 407

OH

N 0 0 H0 0 H C21H23C1N407 478.89 6.33 98% 4 I- 4 C25H26N407 479 494.51 9.90 98% 495 408 (1) C25H26N407 494.51 495 2 98% 0 @0 0 S

S

0* 0 S 000 0 0 0 0 0 0 00 0@ 0 S0 S 0 0* S 0* S @00 SO 5 0 0 0 @0 0 0 5 5 0 @5 S 9 S S S 0 0 0 5@ 0 0 5 0 005 0~~S 005 Cmpd. Structure IMF MW I-PCR MSy.

min Method 409 410 0 H 0

NH

0 0 ~N4H 0 0

)H

C27H28N407 520.55 11.14 (1) 98% 4.87 (1) C19H22N407S 450.47 4511 98% 10.7 (1) C24H25N507 495.50 4961 98% 521 411 S 9 S 9 0 9 9*9 S

S

0 0 5 Se S 9 0 9O S 0 0S S 55 0 @09 @5 0 0 C S@ C 6 0 S S@ C S S S 5 0 0 5 5* 0 S S 5 @55 099 OSO Cmpd. Structure MF MW HPCR S Sn i _M+H Method 412 0 00 0.

0 C2 4H25N507 495.50 8.57 98% 496 413 C18H24N407 408.41 409 1 98% 415 0 0 C22H2 4N409 488.46 7.58 (1) 98% 489

S

S S 0 0@ S 4 0 S 0 S S SO 0S 0e S @0 S S S SO 0 @0 S @50 000 50 0 0 0 S 60 5 0 0 0 0 00 0 0 0 SO S 4 5 S S S S 0 0 550 04. 550 Cmpd. Structure MF MW IPCR s Sn min J(M+H) Method 416 C2 1H23C1N407 478.89 9.66 (1) 98% 479 t I 417 C24H30N4010 534 .53 8.12 (1) 535 535 418 C23H27N508 501 .50 5.93 (1) 98% 502 0 00 0 0 0 *6 00 006 0 0 S 0 0 0 60 @0 0e S 0O 0 0 6 0 0* 0 6* 0 606 SSO OS 0 0 0 S 00 0 0 6 0 50 S 5 0 00 0 0 6 0* 0 0 0 0 000 0*0 500 Cmpd. Structure MF MW HPL T Sn mi Method 419 Cl 6H22N408 398.38 6.84 (2) 98% 399 I 4* I I 420 421 0 0 00 0 ?4H 0 0 i Cl 6H23N507 397.39 5.25 (2) 98% 7.13 (2) C16H24N408S 432.46 433 3 98% 398 0 0000 S S 0e 0@

S

S

0 0 S OS S 00 0 S S 5 0* S 55 0 000 050 00 0 5 0 0 00 S 5 0 0 0 *S 0 5 0 50 0 0 0 S 0 0 0 0 S 000 000 000 IIH PLC RT MS Syn.

Cmpd. Structure IMF I MWI 1 Method 422 0 0 N H C2 1H2 8N607 476.49 6.89 98% 477 4. 4. 4 4. 4.

423 C2 0H2 5N507S 479.52 5.62 (1) 98% 480 424 6.28 (1) C19H23N508 449.42 4501 450 425 0 HD'0

I

C25H2 6N408 510.51 8.25 (1) 98% 511 0 0* S 0 a C C 00 000 0 S 0 *6 em 0s @0 0 0 0 0 SO 0 04 0 000 a* So 0: 00C0 *0 0 :0 See50 00 HPLC RT MS Syn.

Cmnpd.l Structure MF MWI min Method 426 C21H30N407 450.50 (1) 98% 1 4 4 4 4 427 0 N 0 0 0

I

C2 0H2 4N408BS 480.50 7.87 (1) 98% 428 5.13 (1) C16H25N508S 447.47 448 3 98%

C

en C 0 4 Sm

C.

mm.

S

4 0 CO CC S C S~ C C C C CC 0 C. S SOS COS C C S S S* C 5 C C S CS 0 S I 55 S 0 C em a a C PCC SI'e Cm.

Cmpd. Structure MF MW HPL T Myn min Method 429 C14H20N406 340.34 3.19 98% 430 00 N N 0OH C23H27N508 501.50 502 1A H 0H 98% 0 HC21H25N507 459.46 4601 431 432 H0

N')

C21H23N707 485.46 5.59 (1) 98% 486 1 I L a a a o a 0*

U

U SS 0 S0 S S *a S a a aa Sd S 055 *50 OS S 5 1 U' a a U 55 a a a a a S a S S a a so. 050 HPLC RT MS Syn.

Cmpd. Structure MF MW i mmn Method 433 434 0 2Q40 OH ooH 0 0 H Cl H 0 0

OO

N 0H

OH

H

N H 0O

-I

C24H27N507 497.51 11.07 (1) 97% C22H24N607 498 484.47 4.43 (1) 98% 485 435 5.10 (1) C24H25N507 495.50 496 1 98% 8.20 (4) C24H25N507 495.50 496 1 98% 436 0 S 0

S

~0 0 000 S 0 0 S S 5 00 S 0 S 0@ S S 0 S S S @0 555 550 Se 5 0 6 0 55 0 0 S 0 0 .5 S S 0 0 0 0 5 9 055 000 050 Cmpd. Structure MF MW IPL IT M y 1min JMethod 437 N 0

N-O

C2 5H2 7N508 525.52 12.78 98% 526 438 439 0 0 4H 0 4.85 (1) IIC24H25N507 495.50 496 1 98% H C24H25N507 495.50 496 1 98% 9.96 )HC25H27N507 509.52 510 1 98% 440 0 H O~ S 0 S 0 0 S S SS* 0 S S S @0 5 0 S S @5 S 0 0 5* S 05 0 055 *00 @5 5 0 0 S 55 S S S S CS S S S 0 0 S S S S S S S S @55 555 SOS Cmpd.j Structure MF MW HPLC RT MS Syn.

min j Method 441 442 0 N H 0 0,0 0 N 0

H

C27H31N507 537 .58 6.15 (1) 98% 10.10 (1) C21H22N40752 506.56 5071 98% 13.12 (1) C27H28N408 536.55 5371 98% 538 443 0 0 0 0 0 0 0* 0 0

S

0 0 0 00 0 0S S OS 0 0 0 ~0 0 0 *00 0S 00 0 0 #00 se Cmpd Structure MF MW MethodMS yn .1min. Method 444 C2lH22C12N407 513.34 9.96 98% 510 445 0 0 0 5.72 (1) OH C18H22N607 434.41 9%435 ~NH' 8 0 5.00 (1) N A HC17H20N607S 452.45 9%453 98 N/O H D H_ 446 S S 0 5

S

S S S 0 S 0 5 0 00 S 0

SO

S*

05 5 0 5 0 @5 SS S. S 0 S 5 0 05 S 0 S 505 555 555 Cmpd. Structure MF MW HPLC RT MS Syn.

min Method 447 448 0 0 N 0

OH

O 0 0 0

N

Ne H 0 0 H

OC:CH

0 O

H

-I

C22H27N509S 537.55 6.32 (1) 98% I 4 4 C24H29N508 538 515.53 6.36 (1) 98% 516 449 13.86 (1) C25H26N408 510.51 511 1 98% 0 0 0 S S S *0O S S S S 0 0S S S S S@ S S 5 5* 55 5 @50 Se 5 0 SS 0 5 0 S S. S S S S S S S S 0@ 5 5 S S SOS 555 555 Cmpd.j Structure j MF MW mmCRT M Sn min___ Method 450 0 H 0 0 C2 3H2 7N508 501 .50 6.10 (1) 98% 502 451 0~ O 0 8.02 (1) N HC22H26N408 98%.4 475 2 110 0 0NOHC22H26N408 474.47 475 2 452 0

S

0 *0O S 0 5 S 0 0 S

SO

*0 S S S S 55 S @50 555 05 5 0 S S *5 S S 55 5 S S0 0 5 @0 S S 0 905 555 505 I HPLC RT MS tSyn.

Structure MF MW I S min Method H 0 C23H24N407S 500.53 11.11 (1) 98% 501 0 N0 O OH N H H 0o N 0 CI 0 6.24 (2) C20H23N507 445.44 446 2 98% 9.45 (1) C21H23C1N407 478.89 479 2 98% 5.58 (M+Na) C21H24N408 460.45 1 98% 483 H NO)

CH

*.O

S S S 0 S

S

S S 6*e 0@ SO 0 Se S 0@ S S 0 S S S .5 5 555 S*S 00 0 5 S 5 05 5 5 5 5 S. 5 5 5 0 5 0 5 55 0 0 5 500 555 506 Cmpd Structure MF MW HPCR ISn I min Method 457 0 N.0

HO

0

-I

C28H28N4010 580.56 10.42 (1) 98% (M+Na) 603 458 8.65 (1) C21H22F2N407 480.43 9% 481.1 1 10.11 (1) C21H22C1FN407 496.88 498.3 1 98% 459 0 S 0 0 0* 9 0 0 0 0 4 0 *e 4 S S S 0S S S S S S S S *SS SS* 55 6 0 5 OS S 0 0 0 50 0 S S 0@ 5 4 S S S S S 550 *0e **S Cmpd. Structure JMF MW HLO RT MS Syn.

460 N~4 H

O

H0 C22H2 6N409S 522.54 6.16 (1) 9 8% 523.6

H

3

C._

1 461 0 FO0

N"

FY gH C21H23FN407 462.44 7.41 (1) 98% 463.3 462 7.71 (1) C21H23FN407 462.44 463.3 1 98%

S

0 00 0 0 0 *S S 00 00 0 *0 0 S 0 5* @5 5 *00 555 @0 0 S S 6 OS 50 0 9 0 0 0 S 0 0 @00 509 HPLC RT MS Syn.

Cmpd. Structure I MF I MW mi (+MS etJ 463 464 0

HO

F

H

HO

0 H 0 0 Cle N Y X H C, NI:r OH 0

H

C21H23FN407 462.44 7.64 (1) 98% 11.59 (1) H C21H22C12N407 513.34 414.5 1 98% 9.65 (1) C22H25C1N407 492.92 493.9 1 98% 464 465 *00 S 0 00 500 0 S 0 0 0 @0 00 0e S @0 0 5 0 0 00 S @0 0 050 000 *0 0 0 0 0 @0 0 0 0 0 00 0 0 00 0 0 S @0 0 0 5 0 000 *00 500 Imd tutr FM H PLC RT MS Syn.

I min Method 466

H

3 N 0

CI

C22H25C1N407 492.92 9.63 (1) 98% 493.9 I I I I 467 0 F NCH O 4HOH C23H2 4N408 484.47 9.73 (1) 98% 485.8 4. 1 1 468 C26H26F'3N507S 1 609.59 14.84 (1) 98% 609.7 0 0

S

0 0 0 S SO 0 S S 0 00 5 5 0 S S 0O 0 *50 SOto 0:so 0 00 Cmpd. Structure MF MW IP IT M Sn min J(M+H) JMethod 470 N H 0 H 3 C' CH 3 C2 3H2 9N507 487.52 4.57 (1) 98% 489.5 4. 4. 4 4 471 0 H0C NO0 3CN 0 H 3 0 0 0- 0 C23H29N507 487.52 5.74 (1) 98% 488.2 I I I I 472 C22H25N507 471.47 4.00 (1) 98% 474 0*OO

S

0

S

0e@ S

S

5 00 0O SO 0 0@ 0 0 0 0@ 0S S SOS 0B 0 B 5 @0 *0 0S 0 OS 0 0 S S 0 S 000 @00 500 HPC RT MS Syn.

Cmpd. Structure MF MW min Method 473 0H N N) (CH a O~Q4

H

3 CH

H

Ye 0H 0O 0 0~J N

OH

H

C23H26N409 502.49 7.65 (1) 98% 503.6 474 7.16 (1) C23H26N408 486.49 488.1 98% 9.77 (1) C23H25N507 483.49 485.1 97% 475 0 *000 0 0

SO

0O 0 0 0 0 0 0 00 0e 0e 0 0O 0 0 0 0 SO 0 .0 0 050 00* 0 00 CmpdC Stucur MF MWyn.

HPL. RTucur MS Syn min Method 476 477 H3C 0 N 0 0H 0 C22H26N408 474.47 5.25 (1) 98% 475.8 0 N 02H359 595 N O 4.76 (1) I H 0 2H359 595 561.8 1 HO 0 9 0,0 0 0 N 05.25 (1) N HC21H25N5095 523.53 9% 524.3 1 0 H 478

A

S A a

S

S

0 S SO 6O 0O A 0* 5 0 S A A AS S 055 @55 05 A 0 0 S 00 0 0 5 0 S 05 0 A S 50 0 0 S 0* 0 0 0 0 @50 055 SOS Cmpd. Structure jMF MW PCR S Sn 479 0 HO,, N -l

N--

C22H2 6N408 474.47 5.35 (1) 98% 475.8 480

H

3

CO,

)H C25H30N609 558.55 5.11 593 1 7.10 (1) C21H24C1N507 493.9 495.1 1 98% 481 0 0 0 0 U U 0

S

0 S 0 00 0 0B *0 0 0 0 0 *O S. 0 SOS *50 00 0 0 0 5 0 05 0 0 0 0 50 4 0 0 *S 0 0 U C 5 550 IHPLC RT MS ISyn.

Cmpd. Structure MF MW )+I mi jM+H) jMethod 482 C2 1H23C12N507 528.4 9.05 (1) 98 529. 8 1 4 9 t 483

HH

0 H 0 010

H

3

CHN

C2 8H2 9N508 563. 57 10.01. (1) 98% 565. 6 1,2 -I 4 9 4 484 C25H31N508 529.55 7.88 (1) 98% 531 1,2 0 *0O3 0 a a a 300 0 0 0 0 00 0 0* 0 0* 0 0 0 a eG a 0. 0 000 036 6 0 0 0 00 a a a a a 0 a. a a 06 0 0 a *ee *0a *a.

IIHPLC RT MS Syn.

Cmpd. Structure IMF I MW m i (MH IMto 485 C24H2 9N508 515.53 7.00 (1) 98% 517.6 1,2 4- 4 4 4.

486 0 QNA~NO N 0)IN~

HO

0 C29H31N508 577. 60 10.43 (1) 98% 579.4 1,2 487 9.30 (1) C26H33N508 543.58 545.7 1,2 98% S S

S

S S S S SOS S S S S S 0 55 a OS S 5 0 5 5 5 S. S 500 55 4 5 S 0 05 5 S S S 50 0 5 0 5 0 0 5 00 S S S 5 550 505 505 HPLC RT MS S yn.

Structure MF MW I min IMethod 0

HH

0 0-1N^N 0 H H OH 0 0 0

H

C25H31N508 529.55 8.13 (1) 98% 531 .1 1,2 5.89 (1) -jC23H28N608 516.52 517.8 1,4 98% 7.27 (M+Na) C23H27N509 517.50 1,2 98% 540.8

S

S S 0 0 0 0* S S

S

S 0 0 0 0 00 50 50 S 00 S S S 0 5 0 5 55 5 500 005 50 5 5 0 S 5 00 5 5 0 0 0 50 S 0 0 05 0 0 5 5* 5 0 0 555 @50 SOS HPLC RT MS ISyn.

Cmpd Structure MF 1 MWII min (M+H Method 491 0 0N 0

H

0 i N 0NJNOH 0 F. 0

-I

02 8H2 8N409 564 .56 12.9 (1) 98% 565.3 493 8.31 (1) C22H25F'N408 492.46 493.9 1 98% 9.34 (1) C23H26N407 470.49 471.2 2 98% 494 0 0 0 0 0 S 0 S 00 S 0 0 0 S0 S S 0 0 0 0 00 0 000 00 0 0 0 0 @0 0 0 S S 0 00 S 0 0 @0 S S S S 0 0 S 0 00-0 555 Cmpd.J Structure JMF MW IHPLC RT MS Syn.

j min (M+H Method 495 0

HOH

0H 0 0 0 NC 0

CI

C22H26N407 458.48 7.24 98% 459.9 496 9.47 (1) C22H26N408 474.47 475.7 2 98% 9.58 (1) C22H25C1N408 508.92 509.5 1 98% 497 0 0 0 S 0 S S S 5 5

S

5 0 S S S SC S S 0 6 0@ S 0 0 0 0 6 5 Se 0 000 000 00 0 S S @6 0 50 S 0 0 0 0 0 S S S @00 0 0 0

S

0

S

S~0 SOS Cmpd. Structure IMF IMW PL IT M Sn min 1 Method 498 0 HOIII" HOH

CIH

C2lH23C1N408 494.89 7.18 98% 495.1 4. 4 4 4.

499 0H 0 C2 8H30N408 550.57 13.27 (1) 98% 552 182 Example 12 The preparation of compounds 2001, 2002, 2100a-e, and 2201 is described below.

0 0

O

N N O OtBu 0

O

0 OH 0* S

S

S

S. S 5

WOO

1999 2000 0000

S

0 N t N, COOH

H

2002 N N 0

N

H

0 N H OBn 2001 6S 0 *5 C. 0 5e *5 (2000). To a solution of t-butyl 9-amino-6,10-dioxo- 5 1,2,3,4,7,8,9,10-octahydro-6H-pyridazino[ 1,2-a][l,2]diazepine-l-carboxylate (GB 2,128,984; 340 mg, 1.15 mmol) in CH 2 Cl 2 was added benzoylformic acid (260 mg, 1.7 mmol), HOBT (230 mg, 1.7 mmol) and EDC (340 mg, 1.7 mmol). The resulting mixture was stirred at ambient temperature for 16 hours, poured into 1N HC1 and extracted with CH 2 Cl 2 The organic extracts were further washed with saturated NaHCO 3 dried over MgSO 4 and concentrated to afford 1999 as a pale yellow solid.

The solid was dissolved in CH 2 C1 2 (25 ml) and TFA ml) and stirred overnight and concentrated in vacuo to give 560 mg of 2000 as an oil.

183 (2001) was synthesized from 2000 by methods similar to compound 213e to afford 410 mg of 2001 as a white solid: 1H NMR (CDC1 3 mixture of diastereomers) 5 8.25 (1H, 8.23 (1H, 7.78 (1H, dd), 7.65 (1H, bm), 7.50 (2H, 7.40-7.25 (4H, 6.55 (1H, 5.57 (1H, 5.10 (1H, 5.05-4.95 (2H, 4.90, (1H, 4.80 (1H, 4.72 (1H, bm), 4.65 (1H, 4.55 (1H, 4.45 (1H, 3.25 (1H, 3.15 (1H, 3.00 S(2H, bm), 2,90 (1H, dd), (1H 2.70 (1H, 2.47 (1 dd), 2.45 (1H, 2.35 (1H, 2.00-1.75 (4H, 1.60 (1H, bm).

(2002). Compound 2001 (58.6 mg, 0.10 mmol) was treated with 15 ml of TFA/MeCN/water and stirred at room temperature for 6.5 h. The reaction was extracted 15 with ether. The aqueous layer was concentrated with S. azeotropic removal of the water using MeCN. The product was suspended in CH 2 Cl 2 concentrated in vacuo and precipitated with ether to give 46.8 mg of 2002 as a white solid: 1H NMR (CD 3 0D) 6 9.05 (0.25H, 8.15 (1H, 7.68 (1H, 7.64 (0.25H, 7.55 (3H, 7.35 (0.5H, 5.22 (1H, 4.90 (1H, m), 4.58 (1H, dd), 4.50 (1H, 4.28 (1H, bm), 3.45 (1H, 3.10 (1H, bt), 2.68 (1H, ddd), 2.60-2.45 (2H, m), 2.30 (1H, dd), 2.15-2.05 (2H, 1.90 (2H, bm), 1.68 (1H, bm).

184 0 0N ON N~iII r 214e H 2- 0ON 2100

H

*0 0 *2100a R= 02100c OMe *OiPr OMe O Et COiPr 2100b R 1 f-,OEt 2100d R4 oiPr QEt OiPr (2100a). A solution of 214e (101 mg, 0.23 nunol) in isopropanol (10 ml) was stirred at room temperature with a catalytic amount of p-toluenesulfonic acid mg). After 75 minutes, the reaction mixture was poured 5 into saturated NaHCO 3 and extracted with CH 2 Cl 2 The combined extracts were dried over Na 2

SO

4 and concentrated. Flash chromatography (SiO 2

CH

2 Cl 2 to a EtOAc) afforded 56 mg of 2100a as a white solid: .H NMR (CDCl 3 mixture of diastereomers) 67.9-7.8 (211,m), 7.6-7.5 (lH, in), 7.5-7.4 (211, in), 7.1 6.9 (0.5H1, 6.4 5.6 (0.5H, 5.3 5.2-5.1 (1H, in), 4.95 (0.SH, in), 4.75-4.5 mn), 4.35 (0.5H1, 4.1 (0.5H, in), 3.98 (0.5H1, in), 3.3-2.75 (4H, in), 2.5-2.4 2.25 (1H, mn), 2.1-1.9 (311,m) 1.75-1.55 (2H,m).

(2100b). A solution of 214e (16 mg, 0.036 mmol) in ethanol (2 ml) was stirred at room temperature with a catalytic a mount of p-toluenesulfonic acid (2 mg).

After 5 days, the reaction mixture was poured into 185 saturated NaHCO 3 and extracted with CH 2 C12. The combined extracts were dried over Na 2

SO

4 and concentrated. Flash chromatography (Si02, CH 2 Cl 2 :EtOAc 95:5 v/v) afforded 16 mg of 2100b as a white solid: 1H NMR (CDCl 3 d 7.85-7.74 7.55-7.38 7.04-6.95 6.61-6.48 5.15-5.08 4.63-4.53 4.52-4.45 4.42-4.35 4.15-4.05 3.74-3.60 3.57-3.42 3.39-3.28 3.03-2.93 2.92-2.82 10 2.65-2.52 2.42-2.25 2.20-1.88 1.76-1.50 1.35-1.10 (9H,m).

(2100c). A solution of 214e (165 mg, 0.37 mmol) in methanol (5 ml) was stirred at room temperature with a catalytic amount of p-toluenesulfonic acid (17.5 mg).

After 4 days, the reaction mixture was diluted with EtOAc and washed with 10% NaHCO 3 (3x) and brine. The combined extracts were dried over Na 2

SO

4 and concentrated. Flash chromatography (Si0 2 EtOAc) afforded 127 mg of 2100c as a white solid: H NMR (CDC1 3 6 7.82 (2H, 7.55-7.50 (1H, 7.47-7.43 (2H, 7.02 (1H, 6.53 (1H, 5.20-5.10 (1H, m), 4.56-4.50 (1H, 4.45-4.50 (1H each, two 3.69 (3H, 3.41 (3H, 3.43 (3H, 3.35-3.25 (1H, m), 3.06-2.98 (1H, 2.94-2.83 (1H, 2.65-2.53 (2H, 2.35-2.32 (1H, 2.15-2.07 2.00-1.89 (3H, 1.75-1.56 (2H, m).

(2100d). A solution of 214e (53 mg, 0.12 mmol) in isopropanol (5 ml) was stirred at 50 oC with a catalytic amount of p-toluenesulfonic acid (5 mg).

After 3 days the reaction mixture was poured into saturated NaHCO 3 and extracted with CH 2 Cl2. The combined extracts were dried over Na 2

SO

4 and concentrated. Flash chromatography (Si0 2

CH

2 C12:EtOAc 186 (4:1 to 1:1 afforded 49 mng of 2100d as a white solid: 1 H NR (CDCl 3 5 7. 85 (2H, d) 7. 50-7. 43 (1H, in), 7.41-7.35 (2H, in), 7.02 (1K, 6.47 (1H, d), 5.13-5.07 (1H, mn) 5.00-4.9 (1H, mn), 4.61-4.55 (2H, mn), 4.37-4.30(1K, mn), 3.80-3.70 (1H, mn), 3.90-3.80 (1H, mn), 3.42-3.35 (1H, in), 3.03-2.93 (1H, mn), 2.91-2.81 (1H, in), 2.62-2.50 (2K, in), 2.38-2.33 (1H, mn), 2.12-2.06 (1H,in), 1.97-1.81 (3K, mn), 1.70-1.60 (2H, in), 1.28-1.05 (18H,m).

w* 0

S

NK

H 0 H OEt 2100e (2100e) was synthesized from 302 via inethods used to *synthesize 304a to afford 2100e, except ethanol and triethylorthofornate were used instead of methanol and triinethylorthoformate. Chromatography (SiO 2 **ethanol/CK 2 Cl 2 afforded 92 mg of a white solid: 15 K NM'R (CDCl 3 mixture of diastereoners) 6 7.90-7.80 (2H, in), 7.60-7.50 (1K, in), 7.50-7.40 (2H, in), 7.30 7.00 (0.5K, 6.50 (0.5K, 5.50 5.20-5.10 (1.5K, mn), 4.95 (0.5K, in), 4.75-4.65 in), 4.65-4.50 (1K, in), 4.38 (0.05K, 4.00- 3.90 (0.5K, in), 3.85-3.75 (0.5K, in), 3.75-3.65 mn), 3.65-3.55 (0.5K, in), 3.30-2.70 (4K, in), 2.50-2.35 (2K, mn), 2.30 (1H, 2.15-1.90 (3K, in), 1.80-1.60 (2K, mn), 1 .25-1.20 (3K, two t).

187

C

0 0 5 0 e g

C.

C.

0 5 0*Oe Example 13 We obtained the following data for selected compounds of this invention using the methods described herein (Table 8, see Example 7; Tables 9 and 10, see Examples The structures and preparations of compounds of this invention are described in Examples 23-24.

Table 8 Comparison of Prodrugs for Efficacy in LPS Challenged Mice: Inhibition of IL-10 Production.

The percent inhibition of IL-10 production after treatment with a compound of the invention is shown as a function of time after LPS challenge indicates that no value was obtained at that relative time).

Time of Compound Administration (relative to time of LPS challenge, PO, 50 mg/kg) Compound -2h -lh Oh +1h 213f 8 213h 9 53 213i 62 20 213k 0 68 2131 80 213m 26 42 213o 4 8 2 1 3 p 21 29 25 213q 17 91 213r 59 37 213x 0 78 213y 29 50 214e 39 70 43 44 48 11 47 214k 12 31 2141 0 54 214m 0 17 214w 11 91 404 56 6 00SS 'be 0 0 0 0* 412 0 0 11 37- 188 *6 0 0* 0 0 00 00 s Compound 1 -2h -lh Oh +lh 418 64.

-52 434 0 -63 450 0 -35 452 28 -89 456 56 41 69 470 0 36 471 0 34 4 75 0 15 481 27 0 486 19 15 487 17 20 550f 0 50 550h 55 73 550i 23 550k 36 34 5501 9 38 550m 45 52 550n 19 65 550o 19 64 50p 30 60 2001 64 62 58 2001a 10 16 2002 5 87- 2100h 34 32- 2100i 19 74 2 100j 48 41 0 33 2100k 30 50 32 72 21001 52 28 2100m 40 42 2100n 21 9 64 73 2100o 31 44 68 64 189 00-.

Table 9 Data for selected compounds of this invention obtained using the methods described in Examples 1-4.

LUV- Cell PBMC WoeCarneClearance Compound Visible avg. IC50 human Mouse Rat, i.v.

Ki (nM (nM) IC50(nM) ml/min/kgmlink 213f 3000 213g 2200 213h 1500 213i 1100 213j 213k 2000 2131 2000 213m 2500 213o 5000 3300 213p <300 213q <300 213r <300 213v 0.5 1,100 1100 41 23 213x 4500 2500 213y 930 214j 4.2 2500 6000 214k 0.2 500 580 22 2141 6 1900 1100 12 214m 1.5 530 2200 33.4 214w 0.6 620 370 246b 30000 >30000 87 280b 13000 280c 10000 86 280d 25000 283b 1750 41 283c 4000 283d >8000 10000 308c 3000 308d 3000 500 25 1800 1800 501 2.5 1800 1600 505c 1500 505d >20000 505f 550 510a 65 200 267 510d 2300 >20000 190 *0 0 0O *0 @6 00 00 0 ~00e00 0 0@ 3 0 e.g.

B.

0 00.

rnr T Twm/-Whole Clearance UV- Cell PBMC ole Clearance Clearance Compound Visible avg. IC50 human Mouse, Rat, i.v.

blood i.v.

Ki (nM) (nM) IC50(nM) ml/min/kg ml/min/kg 511c 730 >20000 78 550f 1100____ 550h 550i 1400 550k 3000 5501 750__ 550m 2000_____ 550n 550o 450 3000 550p 550q 2001a 3000 2100f 2100g 2100h 2100i 2100j 30000 12000 2100k 520 4000 600 21001 750 2200 2100m 2100n 670 770 4000_____ 2100o 670 1150 1500_____ 0 0 a.

0

S

00 0S We obtained the following data for selected compounds of this invention (Table 10) using the methods described herein (see Examples The structures and preparations of compounds of this invention are described in Examples 23-24.

Table Fluorescent Whole Fluoescnt WoleClearance Clearance Assay Cell PBMC human Clearance Clearance Cmpd. kinact avg. IC50 blood Mouse, Rat fl C0 i.v. i .v.

i(n M I C 5 0 -1 s-1 (n ml/min/kg ml/min/kg m s rm 286 370000 300 1600 119 505b 190000 1500 2100 161 196 505e 420000 9000 191 Example 14 In vivo acute assay for efficacy as anti-inflammatory agent Results in the Table 11 show that 412f and 412d inhibit IL-10 production in LPS-challenged mice after oral adminstration using ethanol/PEG/water, 0-cyclodextrin, labrosol/water or cremophor/water as vehicles. The compound was dosed at time of LPS

D**

challenge. The protocol is described in Example 7.

10 Table 11 Inhibition of IL-10 production in LPSchallenged mice.

Compound 10 mg/kg 25 mg/kg 50 mg/kg dose dose dose 412f 17% 25% 32% 412e 5% 17% 61% Example Mouse Carrageenan Peritoneal Inflammation Inflammation was induced in mice with an intraperitoneal (IP) injection of 10 mg carrageenan in ml of saline (Griswold et al., Inflammation, 13, 20 pp. 727-739 (1989)). Drugs are administered by oral gavage in ethanol/PEG/water, P-cyclodextrin, labrosol/ water or cremophor/water vehicle. The mice are S" sacrificed at 4 hours post carrageenan administration, 0 .e then injected IP with 2 ml of saline containing heparin. After gentle massage of the peritoneum, a small incision is made, the contents collected and volume recorded. Samples are kept on ice until centrifuged (130 x g, 8 mins at 4 to remove cellular material, and the resultant supernatant stored at -20 OC. IL-10 levels in the peritoneal fluid are determined by ELISA.

Results in the Table 12 show prodrug 412f inhibits IL-10 production in carrageenan-challenged mice after oral adminstration of drug. Compound 214e 192 .0 0 0 0 0 0 0* 0 0 0 10 did not inhibit IL-10 production when dosed orally at mg/kg.

Table 12 Inhibition of IL-10 production by 412f and 412d in carrageenan-challenged mice.

Dose Compound 412f Compound 412d (mg/kg) 1 30% 0 54% 32% 25 49% 31% 50 73% 36% 100 75% 53% Example 16 Type II Collagen-induced Arthritis Type II collagen-induced arthritis was established in male DBA/1J mice at described Wooley and Geiger (Wooley, Methods in Enzvmology, 162, pp. 361-373 (1988) and Geiger, Clinical and Experimental Rheumatology, 11, pp. 515-522 (1993)).

Chick sternum Type II collagen (4 mg/kg in 10 mM acetic acid) was emulsified with an equal volume of Freund's complete adjuvant (FCA) by repeated passages (400) between two 10 ml glass syringes with a gauge 16 double-hub needle. Mice were immunized by intradermal injection (50 ul; 100ul CII per mouse) of collagen emulsion 21 days later at the contra-lateral side of the tail base. Drugs were administered twice a day 25 and 50 mg/kg) by oral gavage approximately 7 h apart. Vehicles used included ethanol/PEG/water, 0-cyclodextrin, labrosol/water or cremophor/water.

Drug treatments were initiated within 2 h of the CII booster immunization. Inflammation was scored on a 1 to 4 scale of increasing severity on the two front paws and the scores are added to give the final score.

Results in the Figs. 12 and 13 show prodrugs 412f and 412d inhibit inflammation in collagen-induced arthritits in mice after oral adminstration. Compound 193 214e did not inhibit inflammation when dosed (50 mg/kg) once a day by oral gavage.

Example 17 In vivo bioavailabilitv determination The drugs (10-100 mg/kg) were dosed orally to rats (10 mL/kg) in ethanol/PEG/water, 0-cyclodextrin, labrosol/water or cremophor/water. Blood samples were drawn from the carotid artery at 0.25, 0.50, 1, 1.5, 2, 3, 4, 6, and 8 hours after dosing, centrifuged to 10 plasma and stored at -70 C until analysis. Aldehyde concentrations were determined using an enzymatic assay. Pharmacokinetic analysis of data was performed by non-linear regression using RStrip (MicroMath Software, UT). Drug availability values were determined as follows: (AUC of drug after oral prodrug dosing/AUC of drug after i.v. dosing of drug)x(dose i.v./dose x100%.

Results in Table 13 show that prodrugs 412f 1 and 412d give significant blood levels of drug and have good drug availability when dosed orally. Blood levels of 214e were not detected when it was dosed orally.

Table 13 Oral Bioavailability of 412f, 412d, and 214e in Rat.

25 Compound Dose Cmax Drug (mg/kg) (pg/ml) Availability 412f 25 2.4 32 412d 25 2.6 214e 45 0.2 0.9% Example 18 ICE cleaves and activates pro-IGIF ICE and ICE homolog expression plasmids A 0.6 kb cDNA encoding full length murine pro-IGIF Okamura et al., Nature, 378, p. 88 (1995) was ligated into the mammalian expression vector pCDLSRo Takebe et al., Mol. Cell Biol., 8, p. 466 (1988)).

194 Generally, plasmids (3 pg) encoding active ICE (above), or the three ICE-related enzymes TX, CPP32, and CMH-1 in the pCDLSRa expression vector Faucheu et al., EMBO, 14, p. 1914 (1995); Y. Gu et al., EMBO, 14, p. 1923 (1995); J. A. Lippke et al., J. Biol. Chem., 271, p. 1825 (1996)), were transfected into subconfluent monolayers of Cos cells in dishes using the DEAE-dextran method Gu et al., EMBO 14, p. 1923 (1995)). Twenty-four hours later, 10 cells were lysed and the lysates subjected to SDS-PAGE :and immunoblotting using an antiserum specific for IGIF Okamura et al., Nature, 378, p. 88 (1995).

Polymerase chain reaction was used to introduce Nde I sites at the 5' and 3' ends of the 15 murine pro-IGIF cDNA using the following primers: GGAATTCCATATGGCTGCCATGTCAGAAGAC (forward) and GGTTAACCATATGCTAACTTTGATGTAAGTTAGTGAG (reverse). The resulting NdeI fragment was ligated into E. coli expression vector pET-15B(Novagen) at the NdeI site to create a plasmid that directs the synthesis of a polypeptide of 213 amino acids consisting of a 21residue peptide (MGSSHHHHHHSSGLVPRGSHM, where LVPRGS represents a thrombin cleavage site) fused in-frame to the N-terminus of pro-IGIF at Ala2, as confirmed by DNA 25 sequencing of the plasmid and by N-terminal sequencing of the expressed proteins. E. coli strain BL21(DE3) carrying the plasmid was induced with 0.8 mM isopropyl- 1-thio-P-D-galactopyranoside for 1.5 hours at 370C, harvested, and lysed by microfluidization (Microfluidic, Watertown, MA) in Buffer A (20 mM sodium phosphate, pH 7.0, 300 mM NaC1, 2 mM dithiothreitol, glycerol, 1 mM phenylmethylsulfonyl fluoride, and pg/ml leupeptin). Lysates were cleared by centrifugation at 100,000 x g for 30 min. (His)6- 195 tagged pro-IGIF protein was then purified from the supernatant by Ni-NTA-agarose (Qiagen) chromatography under conditions recommended by the manufacturer.

In Vitro pro-IGIF Cleavage Reactions In vitro cleavage reactions (30 pl) contained 2 pg of purified pro-IGIF and various concentrations of the purified proteases in a buffer containing 20 mM Hepes, pH 7.2, 0.1% Triton X-100, 2 mM DTT, 1 mM PMSF and 2.5 pg/ml leupeptin and were incubated for 1 hour 10 at 37'C. Conditions for cleavage by granzyme B were as described previously Gu et al., J. Biol. Chem., 9* 271, p. 10816 (1996)). Cleavage products were analyzed by SDS-PAGE on 16% gels and Coomassie Blue staining, and were subjected to N-terminal amino acid sequencing 15 using an ABI automated peptide sequencer under conditions recommended by the manufacturer.

Kinetic Parameters of IGIF Cleavage by ICE The kinetic parameters (kcat/KM, KM, and kcat) for IGIF cleavage by ICE were determined as follows.

S-methionine-labeled pro-IGIF (3000 cpm, prepared by in vitro transcription and translation using, the TNT T7-coupled reticulocyte lysate system (Promega) and pro-IGIF cDNA in a pSP73 vector as template) were incubated in reaction mixtures of 60 pl containing 0.1 25 to 1 nM recombinant ICE and 190 nM to 12 pM of unlabeled pro-IGIF for 8-10 min at 37'C. Cleavage product concentrations were determined by SDS-PAGE and Phospholmager analyses. The kinetic parameters were calculated by nonlinear regression fitting of the rate vs. concentration data to the Michaelis-Menten equation using the program Enzfitter (Biosoft).

IFN-y Induction Assays A.E7 Thl cells Quill and R. H. Schwartz, J. Immunol., 138, p. 3704 (1987)) (1.3 x 105 cells in 196 0.15 ml Click's medium supplemented with 10% FBS, 50 IuM 2-mercaptoethanol and 50 units/ml IL-2) in 96-well plates were treated with IGIF for 18-20 hours and the culture supernatant were assayed for IFN-y by ELISA (Endogen, Cambridge, MA).

Example 19 Processing of pro-IGIF by ICE In Cos Cells Cos cells were transfected with various expression plasmid combinations as described in Example S. 10 18. Transfected Cos cells (3.5 x 105 cells in a dish) were labeled for 7 hours with 1 ml of methioninefree DMEM containing 2.5% normal DMEM, 1% dialyzed 35 S. fetal bovine serum and 300 pCi/ml S-methionine S- Express Protein Labeling-Mix, New England Nuclear).

15 Cell lysates (prepared in 20 mM Hepes, pH 7.2, 150 mM NaCl, 0.1% Triton X-100, 5 mM N-ethylmaleimide, 1 mM PMSF, 2.5 pg/ml leupeptine) or conditioned medium were immunoprecipitated with an antiIGIF antibody that recognizes both the precursor and the mature forms of IGIF Okamura et al., Nature, 378, p. 88 (1995)).

Immunoprecipitated proteins were analyzed by SDS-PAGE (polyacrylamide gel electrophoresis) and fluorography (Fig. 2A).

We also measured the presence of IFN-y 25 inducing activity in the cell lysates and the conditioned media of transfected cells (Fig. 2B).

Transfected Cos cells (3.5 x 105 cells in a 35-mm dish) were grown in 1 ml medium for 18 hours. Media was harvested and used at 1:10 final dilution in the IFN-y induction assay (Example 18). Cos cell pellets from the same transfection were lysed in 100 pl of 20 mM Hepes, pH 7.0, by freeze-thawing 3 times. Lysates were cleared by centrifugation as described above and were used at a 1:10 dilution in the assay.

197 Example IGIF is a physiological substrate of ICE Wild type and ICE-/- mice were primed with heat-inactivated P. acnes, and Kupffer cells were isolated from these mice 7 days after priming and were then challenged with 1 ug/ml LPS for 3 hours. The amounts of IGIF in the conditioned media were measured by ELISA.

Wild type or ICE-deficient mice were injected 10 intraperitoneally with heat-killed p. acnes as described Okamura et al., Infection and Immunity, 63, p. 3966 (1995)). Kupffer cells were prepared seven 0 days later according to Tsutsui et al. Tsutsui et al., Hepato-Gastroenterol., 39, p. 553 (1992)) except a 15 nycodenz gradient was used instead of metrizamide. For each experiment, Kupffer cells from 2-3 animals were pooled and cultured in RPMI 1640 supplemented with fetal calf serum and 1 pg/ml LPS. Cell lysates and conditioned medium were prepared 3 hours later.

Kupffer cells from wild type and ICE-/- mice 35 were metabolically labeled with S-methionine as for Cos cells (described above in Example 19) except that methionine-free RPMI 1640 was used in place of DMEM.

IGIF immunoprecipitation experiments were performed on 25 cell lysates and conditioned media and immunoprecipitates were analyzed by SDS-PAGE and fluorography as described in Example 18. See Fig. 3.

Example 21 Induction of IFN-y Production In Vivo LPS mixed with 0.5% carboxymethyl cellulose in PBS, pH 7.4, was administered to mice by intraperitoneal injection (30 mg/kg LPS) in a dose volume of 10 ml/kg. Blood was collected every 3 h for 24 h from groups of three ICE-deficient or wild type 198 mice. Serum IFN-y levels were determined by ELISA (Endogen).

Example 22 IGIF and IFN-y Inhibition Assays Inhibition of IGIF processing by ICE inhibitors was measured in ICE inhibition assays as described herein (see Example 1 and Table 14).

Human PBMC Assays Human buffy coat cells were obtained from 10 blood donors and peripheral blood mononuclear cells (PBMC) were isolated by centrifugation in LeukoPrep tubes (Becton-Dickinson, Lincoln Park, NJ). PBMC were 6 added (3 x 10 /well) to 24 well Corning tissue culture %.o plates and after 1 hr incubation at 37 0 C, non-adherent cells were removed by gently washing. Adherent mononuclear cells were stimulated with LPS (1 pg/ml) with or without ICE inhibitor in 2 ml RPMI-1640-10% FBS. After 16-18 hr incubation at 37 0 C, IGIF and IFN-y

S

were quantitated in culture supernatants by ELISA.

For example, we obtained the following data for compound 412 of this invention using the methods described herein. The structure of compound 412 is shown below.

S.

0. Table 14 compound UV-Visible Cell PBMC

K

i (nM) avg. IC50 (nM) 412 1.3 580 Example 23 Compounds of this invention may be prepared via various methods. The following illustrates a preferred method: 199 0 0 0 rR5 Nl N A°oo-

'-N

H OOH H O1

H

A-I A C-I H OR 5 1 To a solution of A (1.1 equivalent) in CH 2 C12 (or DMF, or CH 2 Cl 2 :DMF is added triphenylphosphine (0-0.5 equivalent), a nucleophilic scavenger (2-50 equivalents) and tetrakis-triphenylphosphine 5 palladium(0) (0.05-0.1 equivalent) at ambient temperature under inert atmosphere (nitrogen or argon).

After 10 minutes, the above reaction mixture is optionally concentrated, then a solution of acid A-I in

CH

2 Cl 2 (or DMF, or CH 2 Cl 2 :DMF is added followed by addition of HOBT (1.1 equivalent) and EDC (1.1 equivalent). The resulting reaction mixture is allowed to stir at ambient temperature 1 hour-48 hours to provide coupled product C-I.

Various nucleophilic scavengers may be used in the above process. Merzouk and Guibe, Tetrahedron Letters, 33, pp. 477-480 (1992); Guibe and Balavoine, Journal of Organic Chemistry, 52, pp. 4984-4993 a (1987)). Preferred nucleophilic scavengers that may be used include: dimedone, morpholine, trimethylsilyl dimethylamine and dimethyl barbituric acid. More preferred nuclophilic scavengers are trimethylsilyl dimethylamine (2-5 equivalents) and dimethyl barbituric (5-50 equivalents). When the nucleophilic scavenger is trimethylsilyl dimethylamine, the above reaction mixture must be concentrated prior to addition of A-I.

Other compounds of this invention may be prepared by hydrolyzing compounds represented by C-I to 200 compounds represented by H-I as described in the following scheme: O 0 N 0 N N 0 0 'N R5 N H O 'N

OH

O' 'N H H C-I H OR 5 1 O0 H H-1 H O The hydrolysis may be carried out under various conditions, provided that the conditions include an 5 acid and H 2 0. Acids that may be used include ee p-toluensulfonic, methanesulfonic acid, sulfuric, Sperchloric, trifluoroacetic, and hydrochloric. For example, trifluoroacetic acid (1-90% by weight) or hydrochloric acid (0.1-30% by weight) in CH 3

CN/H

2 0 (1-90% H 2 0 by weight) at between 0-50 °C may be used.

Example 24 Compounds 213f, 213g, 213h, 213i, 213j, 213k, 2131, 213m, 214f, 214g, 214h, 214i, 214j, 214k, 2141, 214m, 550f, 550g, 550h, 550i, 550j, 550k, 5501 and 550m 15 were prepared as follows.

0e 201 0 0 R4N§ N?0 a- R4N N? oH H OR 1 HO0 213f-m, R =Bn 214f-m 550f-m, R 1 Et 0 0 :f R 4 l j R 4

CI

0 r* g9 R 4 Ik R 4

=C

*HO l

R

4 I R 4 0

H

2 N-

JN

1 0 H iR 4 m R4= m0 *sees CH30C 3

O

(213f) was synthesized from 212f by the methods used to prepare 213e from 212e to afford 504 mng of 213f as a yellow solid, 1H NMR CCD 3 OD) 6 1.10 Cbr. mn, 0.25H), 1.30 (br. mn, 2H), 1.50Cbr. mn, 1H), 1.65(br. mn, 1.8O(br. mn, 0.25H), 1.9O(br. mn, 0.25H), 1.95(br. mn, 0.5H), 2.05(br. mn, 0.25H), 2.15Cm, 1H), 2.3(m, 1H), mn, 1H), 2.6(dd, 1H), 2.8(m, 1H), 3.1(br. s, 3H), 3.15(br. mn, 1H), 3.32(br. s, 3H), 3.5Cm, 1H), mn, 1H), 4.62(d, 0.25H), 4.72(m, 3H), 4.95(m, 1H), 5.1(br. t, 0.25H), 5.15(br. t, 0.75H), 5.7(d, 1H), 6.75(d, 2H), 7.35(br. s, 5H), 7.75(d, 2H).

(213g) was synthesized from 212g by the methods used to prepare 213e from 212e to afford 400 mng of 213g, 1H NNR

(CD

3 OD) 5 1.5Cbr. m, 1H), 1.65Cbr. mn, 2H), 1.70Cbr. mi, 0.25H), 1.90Cbr. mn, 1H), 1.95Cbr. mn, 1H), 2.O5Cbr. mn, 202 0.25H), 2.10(m, 1H), 2.3(m, 1H), 2.5(m, 2H), 2.59(d, 1H), 2.6(d, 1H), 2.78(d, 1H), 2.8(d, 1H), 2.93(br. s, 4H), 3.05(br. m, 1H), 3.15(br. m, 0.25H), 3.3(br. s, 3H), 3.5(m, 2H), 4.5(br. m, 2H), 4.65(d, 1H), 4.7(br.

m, 2H), 4.95(br. m, 1H), 5.15(br. t, 0.25H), 5.2(br. t, 0.75H), 5.2(d, 1H), 6.95(d, 1H), 7.15(d, 1H), 7.25(br.

s, 1H), 7.3(br. t, 2H), 7.45(br. s, 6H).

(213h) was synthesized from 212h by the methods used to prepare 213e from 212e to afford 296 mg of 213h, 1H NMR (ODC1 3 5 1.55-1. 68 1H) 1. 7 05 3H) 2 3-2. 2H), 2.65-2.8(m, 1H), 2 8 5-2.93(m, 1H), 2 .95-3.25(m, 3H), 4.44-4.65(m, 2H), 4.68-4.82(m, 1H), 4.9-4.95(d, 1H), 5.05-5.18(m, 2H), 5.28(s, 0.5H), 5.55-5.58(d, 6.52-6.58(d, 0.5H), 6.

7 -6.76(m, 2H), 6.82- 6.85(d, 0.5H), 7 5H), 7 .52-7.58(m, 1H), 7.75(s, 0.5H), 7.8(s, (213i) was synthesized from 212i by the methods used to **prepare 213e from 212e to afford 1.1 g of 213i, 1H NNR

S....(ODC

3 5 1.

5 5-2.05(m, 6H), 2 .26-2.5(m, 2H), 2.68- 2.82(m, iH), 2 .85-2.92(m, 1H), 2 .95-3.25(m, 2H), C3.82(s, 1.5H), 3.85(s, 1.5H), 4.4-4.65(m, 2H), 4.7- 4.78(m, iH), 4 .88-4.95(m, 1H), 5.05-5.23(m, 1H), 5.28(s, 0.5H), 5.55-5.58(d, 0.5H), 6 .6-6.65(m, 1H), 6 .8-6.84(m, 1H), 6.9-6.95(m, 3H), 7 .3-7.45(m, 4H), 7 7 8-7.85(m, 2H).

(213j) was synthesized from 212j by the methods used to prepare 213e from 212e to afford 367 mg of 213j, 1H NMR (ODC1 3 6 1.55-2.05(m, 12H), 2.25(d, iH), 2.35(m, 1H), 2.48Cm, 2H), 2.75(m, 2H), 2.9(m, 2.95-3.25(m, 4.45(t, 1H), 4.5-4.6(m, 4H), 4.7(m, 1H), 4.75(d, 1H), 4.88(m, 1H), 5.05(m, 2H), 5.15(q, 1H), 5.3(s, 1H), 5.58(d, 1H)l 6.5(d, 1H), 6.9(d, 1H), 7.05(d, 1H), 7.25- 7.35(m, 5H), 7.6(s, 2H), 7 2H).

203 (213k) was synthesized from 212k by the methods used to prepare 213e from 212e to afford 593 mg of 213k, 1HNMR

(CD

3 OD) 6 1.5(m, 1H), 1.6-1.7(m, 2H), 1.75-1.95(m,4H), 2.15(m, 2H), 2.3(m, 1H), 2.6(m, 1H), 2.7(m, 1H), 3.05(m, 2H), 3.15(m, 1H), 3.5(m, 2H), 4.45(m, 2H), 4.65(d, 1H), 4.7(m, 1H), 4.95(m, 1H), 5.15(m, 1H), 5.4(s, 1H), 5.7(d, 1H), 7.3(m, 5H), 7.85(s, 2H).

(2131) was synthesized from 2121 by the methods used to prepare 213e from 212e to afford 133 mg of 2131, 1H NMR (ODC1 3 5 1.55-1.7(m, 1H), 1.75-2.05(m, 3H), 2.25(s, 1.5H), 2.27(s, 1.5H), 2.3-2.48(m, 2H), 2.7-2.83(m, 1H), 2.85-2.94(dd, 1H), 2.95-3.25(m, 2H), 4.42-4.65(m, 2H), 4.8 4 8 (m H ,4 88 4 9 1H 50..1 5.32(s, 0.5H), 5.55-5.6(d, 0.5H), 6.48-6.55(d, 1H), 6.88-6.92(d, 1H), 7.0-7.04(d, 0.5H), 7.15-7.2(d, 7.3-7.4(m, 4H), 7.64-7.78(m, 2H), 7.88-7.94(m, 1H), 8.45-8.56(m, 1H).

(213m) was synthesized from 212m by the methods used to prepare 213e from 212e to afford 991 mg of 213m, 1H MR (CDC1 3 8 1.5-2.15(m, 5H), 2.2-2.55(m, 3H), 2.6-3.3(m, 4H), 3.95(2s, 3H), 4.45-4.7(m, 2H), 4.7-4.85(m, 1H), 4.8504.95(m, 1H), 5.05-5.25(m, 1H), 5.3Cs, 5.6(d, 0.5H), 6.55(d, 0.5H), 6.85(d, 0.5H), 7.25-7.6(m, 5.5H), 7.75(s, 1H), 7.85(s, 1H).

(550ff) was synthesized from 212ff by the methods used to prepare 213e from 212e to afford 420 mg of 550ff as an off white solid, 1H NMR (CDC1 3 6 1.2-1.25 (br. t, 3H) 1.35(m, 1H), 1.55Cbr. m, 1H), 1.88-2.02(br. m, 4H), 2.3(d, 1H), 2.35(m, 1H), 2.45(m, 1H), 2.55-2.75(m, 3H), 3.0(s, 6H), 3.25(m, 1H), 3.55(m, 1H), 3.65(m, 1H), 3.75(m, 1H), 3.9(m, 1H), 4.3(t, 1H), 4.55(m, 2H), 4.68(br. m, 1H), 3.9(m, 1H), 4.3(t, 1H), 4.55(m, 2H), 204 4 .68 (br. m, 1H) 4 .95 (br. m, 1H) 5. 1(br. m, 2H), 5.45 1H) 6.5 2H) 7.7 2H) (550h) was synthesized from 212h by the methods used to prepare 213e from 212e to afford 195 mg of 550h as a white solid, 1H NNR (DMSO-d 6 5 1.1-1.18 (2t, 3H) 1. 6- 1.7(m, 2H), 1.88-2.05(m, 2H), 2.1-2.35(m, 3H), 2.48- 2.56(m, 1H), 2.75-2.8(m, 0.75H), 2.88-3.08(m, 1.25H), 3.25-3.4(m, 1H), 3.55-3.8(m, 2H), 4.35-4.45(m, 1H), 4.55-4.62(m, 1H), 4.8-4.88(m, 1H), 4.98-5.03(m, 0.25H), 10 5.1-5.13(m, 0.75H), 5.33(s, 0.25H), 5.58-5.6(d, 0.75H), 0 5.9-6.O(br. s, 2H), 6.8-6.85(d, 1Hl), 7.58-7.62(d, 1H-), 64900:7.82(s, 1H), 8.22-8.28(d, 1H), 8.48-8.52(d, 0.75H), 8.72-8.76(d, 0.25H).

O*SO~

:0 (550i) was synthesized from 212i by the methods used to prepare 213e from 212e to afford 135 mg of 550i, 1H NMR (CDCl 3 5 1.18-1.28(2t, 3H), 1.6-1.75(m, 1.5H), 1.9- 2.1Cm, 3.5H), 2.22-2.3(d, 0.5H), 2.38-2.47(m, 2.7-2.8(m, 0.5H), 2.8-2.93(m, 1H), 2.94-3.15(m, 0000003.15-3.28(m, 1H), 3 .55-3.62(q, 0.5H), 3.62-3.73(q, Se0:4 20 0.5H), 3.

7 8-3..88(q, 0.5H), 3.88(s, 3H), 3.9-3.95(q, 4.33-4.4(m, 0.5H), 4.5-4.55(m, 1H), 4.68-4.76(m, 4.9-4.95(m, 0.5H), 5.1-5.2(m, 1.5H), 5.18(s, 5.48-5.52(d, 0.5H), 6.48-6.55(d, 0.5H), 6.85- 6.9(m, 1H), 6.9-6.95(m, 2H), 7.34-7.38(d, 0.5H), 7.78- 7.85(m, 2H).

(550k) wa s synthesized from 212k by the methods used to prepare 213e from 212e to afford 174 mg of 550k as a white solid, 1H NMR (DMSO-d 6 8 1.15(2t, 3H), 1.6- 1.75(m, 2H), 1.9-2.05(m, 2H), 2.1-2.4(m, 5H), 2.55(m, 1H), 2.7-2.8(m, 0.5H), 2.85-3.0Cm, 1H), 3.1Cm, 0.5H), 3.55-3.7(m, 1H), 3.7-3.8(m, 1H), 4.2(t, 4.35-4.45(m, 0.5H), 4.55-4.65(m, 0.5H), 4.8- 4 0.5H), 5.05(t, 0.5H), 5.15(t, 0.5H), 5.35(s, 205 0.SH) S. 6(d, 0. 5H) 7. 95 2H) 8.5C(d, 0.5SH) 8. 65 (d, 1H), 8.75(d, 0.5H), 10.9(br. s, 1H).

(5501) was synthesized from 2121 by the methods used to prepare 213e from 212e to afford 151 mg of 5501, 1H NMR (CDCl 3 5 1.2-1.28(2t, 3H), 1.6-1.72(m, 1.5H), 1.88- 2.15(m, 3.5H), 2.22-2.28(m, 0.5H), 2.28(s, 3H), 2.38- 2.48Cm, 1.5H), 2.66-2.92(m, 1.5H), 2.95-3.14(m, 3.2-3.34(m, 1H), 3.56-3.63Cq, 0.5H), 3.63-3.72(q, 3.8-3.85(q, O.5H), 3.9-3.95(q, 0.5H), 4.32- 4.38(m, 0.5H), 4.5-4.62(m, 1H), 4.68-4.75(m, 4.88-4.92(m, 0.5H), 5.08-5.2(m, 1.5H), 5.18(s, 5.46-5.5Cd, 0.5H), 6.5-6.55(d, 0.5H), 6.98-7.05(m, 1H), 7.42-7.48(d, 0.5H), 7.63-7.78(m, 2.5H), 7.9-7.94(d, 0 0 0 8.44-8.52(m, 1H).

(550m) was synthesized from 212m by the methods used to prepare 213e from 212e to afford 301 mg of 550m as a white solid, 1H NM'R (CDC1 3 5 1.2-1. 35 (2t, 3H) 0 0 1.8Cm, 2H), 1.9-2.15(5H), 2.25(d, 0.5H), 2.4-2.5(m, *got so*# 2H), 2.65-2.8(m, 0.5H), 0.5H), 3.0-3.2(m, 05:5 20 1H), 3.2-3.35(m, 0.5H), 3.55-3.65(m, 0.5H), 3.65- C 3.75(m, 0.5H), 3.8-3.9(m, 0.5H), 3.9-4.0Cm, 0.5H), 4.4- 4.45Cm, 0.5H), 4.55-4.65(m, 0.5H), 4.7-4.8(m, *0 4.85-4.95(m, 0.5H), 5.05-5.2(m, 0.SH), 5.2(s, 0.5H), 6.5(d, 0.5H), 6.9(d, 0.5H), 6.95(d, 0.5H), 7.35(d, 0.5H), 7.75(s, 1H), 7.85(s, 1H).

(214j) wa s synthesized from 213j by the method used to prepare 2002 from 2001 to afford 62 mg of 214j as a white solid, 1H NMR (CD 3 QD) 5 0. 9 Ct, 1H) 1. 3 Cbr. s, 1H), 1.7 Cbr. m, 1H), 1.9Cbr. m, 1H), 2.1(br. s, 1H), 2.25(q, 1H), 2.35Cm, 1H), 2.48(m, 2H), 2.65Ct, 1H), 3.lS(br. t, 1H), 3.5Cbr. m, 1H), 4.3Cbr. s, 1H), 4.55Cm, 2H), 4.95Ct, 1H), 5.25(br. s, 1H), 7.6Cbr. s, 1H), 7.85Cbr. s, 1H).

206 (214k) was synthesized from 213k by the method used to prepare 2002 from 2001 to afford 80 mg of 214k as a white solid, 1H NMR (CD 3 QD) 5 1. 6-1.7C(m, 1H) 1. 8-2.0 (m, 2H), 2.0-2.1Cm, 2H), 2.15-2.25(m, 1H), 2.3-2.4Cm, 1H), 2.4-2.55(m, 2H), 2.6-2.75(m,1H), 3.05-3.2(m, 1H), 3.4- 3.6(m, 2H), 4.2-4.3(m, 1H), 4.45-4.6(m, 1H), 4.8-5.0Cm, 1H), 5.1-5.2(Cm, 1H), 7.85(s, 2H).

(2141) was synthesized from 2131 by the method used to prepare 2002 from 2001 to afford 91 mg of 2141 as a 10 white solid, 1H NMR (DMSO-d 6 5 1. 65 (br. m, 6H) 1. 9(br.

0. ILm, 6H), 2.15(s, 3H), 2.3(m, 3H), 2.6-2.85Cm, 3H), 2.9Cm, 2H), 3.0Cm, 1H), 4.15(br. q, 1H), 4.4(m, 3H), 1H), 5.15(m, 1H), 5.45(s, 1H), 7.8(d, 2H), 1H), 8.05(s, 1H), 8.65(m, 2H), 9.65(s, 1H).

(214m) was synthesized from 213m by the method used to prepare 2002 from 2001 to afford 105 mg of 214m as a white solid, 1H NMR (CD 3 OD) 5 1.6-1.75(m, 1H), 1.85- 1.95(m, 1H), 2.0-2.1(m, 2H), 2.15-2.25(m, 1H), 2.3- Door 2.4(m, 1H), 2.45-2.55(m, 2H), 2.65-2.75(m, 1H), 3.4- 600:96 20 3.55Cm, 2H), 3.95(s, 3H), 4.2-4.3(m, 1H), 4.45-4.6(m, 1H), 4.9-5.0(m, 1H), 5.15-5.2(m, 1H), 7.9(s, 2H).

Compounds 308c and 308d were prepared as 6. follows.

0 N* 0 21 2e

CH

3 Oj rI 308c,dH

,R

c R 1 Me d R 1 308c) was synthesized from 212e via the methds used' to prepare 308b from 212e to afford 266 mg of 308c 1 H NMP. CCDC1 3 6 1.6-1.7(m, 1H), 1.88-1.98(m, 3H), 2.02- 207 2.15(m, 1H), 2.3-2.4(m, 1H), 2.65-2.95(m, 3H), 3.04- 3.09(m, 1H), 3.12-3.25(m, 1H), 3.84(s, 3H), 3.86(s, 3H), 4.5-4.58(m, 1H), 4.88-4.95(m, 1H), 5.1-5.25(m, 2H), 6.86-6.9(d, 2H), 7.15-7.25(m, 2H), 7.36-7.4(m, 1H), 7.75-7.8(d, 2H).

(308d) was synthesized from 212e via the methods used to prepare 308b from 212e to afford 270 mg of 308d, 1H NMR (CDC1 3 6 1.55-1.65(m, 1H), 1.8-2.1(m, 4H), 2.3- 2.4(m, 1H), 2.65-2.88(m, 3H), 2.9-3.3(m, 3H), e 10 4.58(m, 1H), 4.88-4.95(m, 1H), 5.05(s, 2H), 5.1-5.2(m, 1H), 6.82-6.95(m, 2H), 7.02-7.15(m, 2H), 7.28(m, 7.45(m, 1H), 7.72(d, 2H) 0• Compounds 2100f, 2100g, 2100h, 2100i and "2100j were prepared as described below.

Oie 0 0 U AIIocN c IcN 4OH H i H to@ 2101a *ice* *4 1 AllocN AllocN H _H 2101b 0 0 N I

N

CF H 0OCO-'- Hq". R1 0 OH O00H

H

212e 2100f, g f, RI= O g, RI= 0^ 0^ 208 (2101a) was synthesized from allyloxycarbonylamino-o3te-rt-butyl aspartate by the methods employed by Chapman CBioorg. Med. Chem. Lett., 2, pp.615-618 (1992)) to prepare (3S, 2RS) 3 oxotetrahydrofuran using 4-chlorobenzyl alcohol instead of benzyl alcohol to afford 1.84 g of 2101a as a :crystalline solid.

(2100f) was synthesized from 212e by the methods used 0 0 to prepare 213e from 212e using 2101a to afford 380 mg O.:Oo* 10 of 2100f, 1H NMR (CDC1 3 5 1. 8-2.0C(m, 10iON), 2.310 lIN), 00* 2.31-2.5(m, 3H), 2.7-2.9(m, 3H), 3.05(m, 2H), 3.1- 3.2Cm, 4H), 4.45(q, 1H), 4.5-4.6(m, 3H), 4.7(d, 2H), 4.85(d, 1H), 4.9(t, 1H), 5.2(t, 1H), 5.15Cm, 2H), 5.25(s, 1H), 5.55(d, 1N), 6.5(d, 1H), 6.9(d, 1H), 6.95(d, 1H), 7.25(m, 3H), 7.35(t, 2H), 7.45Cm, 2H), 7. 55 (1H) 7.8C(m, 3H).

(2101b) was synthesized from (3S,2RS) 3-allyloxyvia the method used to prepare 2100d from 214e using H 2 S0 4 0, a 20 instead of pTSA to afford 2101b.

00 .0 (2100g) was synthesized from 212e by the methods used to prepare 213e from 212e using 2101b to afford 31 mg of 2100g, 1H NMR (CDCl 3 5 1. 19 Cd) 1. 94 Cbr s) 2. 00- 2.12 2.24 2,42 2.71-2.83 Cm), 3.02 Cdd), 3.12-3.27 (overlapping in), 3.93 4.32-4.37 4.52-4.63 Cm), 4.90-4.95 Cm), 5.12-5.20 Cm), 5.28 Cs), 6.93 Cd), 7.10 7.41-7.50 7.51-7.58 7,84 209 214e soNZN_ 2100h H QAc (2100h). A souio f 214e (287 mg,0.6 mmol)1 in pyridine (5 mL) was treated with Ac 2 0 (0.4 mL, 3.62 mmol) After 6 hours, the reaction mixture was poured into 5% NaHSO 4 and extracted 3 times with EtOAc. The combined organics were washed with brine, dried over Na 2

SO

4 and concentrated in vacuo. Chromatography (SiO 2 EtOAc) afforded 119 mg of 2100h, 1HNNR (CDC1 3 mixture of four diastereoisomers) 8 1.80-2.05(m), 2.12(s), 2.19(s), 2.22(d), 2.67-2.75(m), 2.80-2.95(m), 3.00-3.20(m), 3.21-3.33(m), 3.50-3.95(four discrete multiplets), 4.19(m), 4.55(m), 4.57-4.65(m), 4.69(m), 4.85-4.95(m), 5.04(m), 5.10(s), 5.10-5.22(m), 6.46(d), 6.03(s), 6.50(d), 6.58(d), 6.75(d), 6.95-7.05(m), 7.22(m), 7.30(m), 7.71(d), 7.75-7.83(m).

2100b N

NO

2100i H (2100i). To a solution of 2100b (1.5 g, 2.7 nimol) in

CH

3 CN (10 mL) was added 1N HCl at ambient temperature.

After 6 hours solid NaHCO 3 was added and the product extracted with EtOAc, dried over MgSO 4 and concentrated in vacuo. Chromatography (SiO 2 30-100% CH 2 Cl 2 in EtOAc) afforded 123 mg of 2100i, 1HNMR (CDCl 3 8 1.25(t, 3H), 1.6-1.8(m, 1H), 1.9-2.2(m, 5H), 2.4-2.5(m, 210 1H) 2. 75-2.9 2H) 3. 0-3. 1(m, 2H) 3.2-3. 25 1H) 4.05-4.2(m, 1H), 4.5-4.7(m, 1H), 5.1-5.25(m, 1H), 7.2(m, 2H), 7.4-7.45(m, 2H), 7.5(t, 1H), 7.8(t, 2H), 9. 5(s, 1H).

0@ N 21 00i 0 21100i b~ 20j to prpr610 rm24 o fod37m Componds100 j Hn 50 aeecbdiTal 5(1.j waT snhes iznse prrom by0 viathd methoiuse to prepaethd used fro prepe ooafods 47 (se, 1xmle1)

S

S S S S S 0 55

S

S S 0 5 S 55 5 00 0 S. S 5 0 SO S @5 505 5e5 0. 0 5 S S 0 00 0 S 0 SS S S S S 5 0 5 5 S S S S S 5 555 555 055 Table HPLC RT min

MS

Compound Structure MF MW (method) ___Purity Cl 0 011 .448

(A)

500 0 Y0 C22H24C1N508 521.92 0.991 523.1 N

H

H 3

C

0H ,00

H

3

C

212 The compounds described below (213m, 213n, 213o, 213p, 213q, 213r, 213s, 213t, 213u, 213v, 213w, 213x, and 214w), were prepared by methods similar to the methods used to prepare compounds 213b-f.

5 Compounds 419, 415, 450, 456, 475, 404, 486, 487, 417, 408 and 418 may also be prepared as described below.

S. 0 o S S 0S S. S 0S

S.

0 500000 0* 0@ S

S.

0 See 0 H 0 H 0 0 N0 R'-N

OH

H 0

H

H 0 0

OS@SSO

0 0 0000 0050 S S S. 0 *5 0 5 *5 *S 0 00 @5 213m-x 214w, 404, 408, 415, 417, 418, 419, 450, 456, 475, 486, 487 compoundR 213m, 419 MeOC(O)- 213n, 415 0 213o, 450 0 HN 'rMe 0 213p, 4560 0OQ 213 213q, 475 0 C /N H e so 0 S 0 a 000 0 *0*6 600 1* 213r, 404 Me 0 4.

213s, 486

H

213t, 487

H

00 OS 0 0 0 213u, 417 0 M eC MeO OM e 213v, 408 213w, 214w 0 Me'A

HO';

M e 214

S*

S@

OS

0 S S

OS..

*0

S

S.*

0 5

S

*O 213x, 418 o

H

3 C ',N

H

(213n) was isolated as a mixture of diastereomers 10 (syn:anti isomer ratio 6:4) (1.43g, 82%) as a white solid: mp. 206-10"C; IR (KBr) 3288, 1787, 1680, 1657, 1651, 1619, 1548, 1440, 1256, 1135; H NMR (D 6 -DMSO) 8.75 (0.4H, 8.55 (0.6H, 8.45 and 8.43 (1H, 2 x 7.50 (1H, 7.42 (1H, 7.40-7.27 (5H, 7.01 (1H, 6.11 (2H, 5.67 (0.6H, 5.43 (0.4H, s), 5.10-5.00 (1H, 4.90-4.59 (3.5H, 4.45-4.25 3.47-3.20 (1H, 3.20-2.70 (2H, 2.65- 2.35 (1H, 2.35-2.00 (3H, 2.00-1.75 (2H, m), 1.65-1.40 (2H, Anal. Calcd for C 2 9

H

3 0

N

4 0 9

C,

60.20; H, 5.23; N, 9.68. Found: C, 60.08; H, 5.32; N, 9.50. MS (ES 580 (M 2, 579 (M 1, 100), 404 367 236 107 (213o) anti-isomer as a white foamy solid (0.73g, 69%): 21 3 mp. 135-40 [a]D -37.30 (c 0.1, CH 2 C1 2 IR (KBr) 3452, 3310, 1790, 1664, 1659, 1650, 1549, 1425, 1258, 1121; H NMR (D 6 -DMSO) 6 10.11 (1H, 8.77 (1H, d), 8.57 (1H, 8.01 (1H, 7.76 (1H, 7.55 (1H, d), 7.45-7.25 (6H, 5.43 (1H, 5.08-5.00 (1H, m), 4.95-4.73 (1H, 4.76 and 4.68 (2H, dd), 3.40-3.20 (1H, 3.09 (1H, dd), 3.02-2.75 (1H, 2.45-2.06 (4H, 2.06 (3H, 2.00-1.75 (2H, 1.70-1.40 (2H, Anal. Calcd for C 3 0

H

3 3

N

5 0 8 '0.75H 2 0: C, 59.54; H, 5.75; N, 11.57. Found: C, 59.40; H, 5.62; N, 11.50.

MS (ES 593 2, 592 (M 1, 100), 574 215 487 475 385 373 318 296 266 221 (22).

(213p) was isolated as a foam (1.2g, [1]D -1150 (c 0.20, CH 2 C1 2 IR (KBr) 3368, 2946, 1794, 1654, 5 1609, 1540, 1505, 1421, 1277, 1175, 1119, 980; 1H NMR

(D

6 -DMSO) 6 10.1 (1H, 8.80 (0.5H, d, J 8.60 see**: (0.5H, d, J 8.40-8.36 (1H, 2d), 7.82 (2H, d, J 7.41 (5H, bs), 6.86 (2H, d, J 5.72 d, J 5.49 (0.5H, bs), 5.13-5.07 (1H, 4.95- 10 4.65 (2.5H, 4.49-4.38 (2.5H, 3.49-3.30 (2H, m), 3.21, 2.79 (2H, 2.40-1.41 (7H, MS (ES 551.

(213q) was isolated as a white glassy solid mp.

23 145-149 0 C; [OID -56.00 (c 0.05, CH 2 C1 2 IR (KBr) 3399-3319, 1791, 1657, 1543, 1420, 1253, 1119; H NMR

(CDCI

3 69.54 (1H, 7.65 (1H, d, J 7.51 (1H, 000 d, J 7.44-7.25 (7H, 7.18-7.06 (3H, m), 5.30-5.20 (1H, 5.27 (1H, 4.84 (1H, 4.79 (1H, d, J 11.4), 4.56 (1H, d, J 11.3), 4.47 (2H, 3.28 (1H, 3.10-2.97 (2H, 2.71 (1H, m), 2.47-2.37 (1H, 2.26 (1H, d, J 17.9), 2.09 (1H, 1.83, 1.70, 1.51 (4H, 3m).

(213r) was isolated as a mixture of diastereomers (syn:anti isomer ratio 55:45) as a white foamy solid (1.46g, mp. 106-10"C; IR (KBr) 3306, 2947, 1791, 1659, 1650, 1535, 1421, 1256, 1122; H NMR (D 6 -DMSO)6 8.76 (0.45H, 8.56 (0.55H, 8.49 and 8.47 (1H, 2 x 7.41-7.19 (9H, 5.67 (0.55H, 5.43 (0.45H, 5.11-5.02 (1H, 4.86-4.55 (3.5H, 4.45-4.25 3.40-3.20 (1H, 3.20-2.70 (2H, 2.65- 2.40 (1H, 2.34 (3H, 2.30-1.70 (5H, 1.65- 1.40 (2H, Anal. Calcd for C 2 9

H

3 2

N

4 0 7 C, 62.66; H, 5.95; N, 10.08. Found: C, 62.91; H, 6.00; N, 9.70. MS 216 (ES 550 2, 549 1, 100), 374 280 279 118 (213s) was isolated as the anti-isomer as a white foamy 21 solid (0.64g, mp. 137-41 0 C; [a]D -48.20 (c 5 0.05, CH 3 OH); IR (KBr) 3477, 3314, 1791, 1659, 1599, 1529, 1499, 1406, 1256, 1122; 1H NMR (D 6 -DMSO) 6 10.45 (1H, 8.76 (1H, 8.50 (1H, 7.86 (2H, 7.69 (2H, 7.41-7.20 (10H, 5.43 (1H, 5.08-4.98 (1H, 4.90-4.73 (1H, 4.76 and 4.68 (2H, dd), 10 3.67 (2H, 3.40-3.20 (1H, 3.09 (1H, dd), 3.02- 2.75 (IH, 2.39 (1H, dd), 2.30-2.00 (3H, 2.00- 1.75 (2H, 1.70-1.40 (2H, Anal. Calcd for

C

36

H

37

N

5 0 8 0.5H 2 0: C, 63.90; H, 5.66; N, 10.35. Found: C, 63.68; H, 5.67; N, 10.24. MS (ES 669 (M 2, 668 1, 100), 640 435 425 (23), 403 328 302, 274 197 138 (17).

(213t) was isolated as a white foamy solid (0.63g, 21 0 o so"80%): mp. 159-64 0 C; [a]D -37.0 (c 0.05, CH 3 OH); IR 20 (KBr) 3463, 3321, 1790, 1680, 1658, 1650, 1644, 1595, 1525, 1501, 1408, 1251, 1113, 933; 1H NMR (D 6 -DMSO) 8 10.13 (1H, 8.76 (1H, 8.48 (1H, 7.85 (2H, 7.68 (2H, 7.40-7.25 (5H, 5.43 (1H, s), 5.08-4.95 (1H, 4.92-4.73 (1H, 4.76 and 4.68 (2H, dd), 3.40-3.20 (1H, 3.09 (iH, dd), 3.02-2.75 (1H, 2.39 (1H, dd), 2.35-2.00 (6H, 2.00-1.75 (2H, 1.70-1.40 (2H, 0.93 (6H, Anal. Calcd for. C 33

H

3 9

N

5 0 8 0.5H 2 0: C, 61.67; H, 6.27; N, 10.90.

Found: C, 61.49; H, 6.24; N, 10.86. MS (ES 635 2, 634 1, 100), 484 427 274 268 204 117 (13).

217 (213u) was isolated as a white solid mp. 120- 132'C; IR (KBr) 3361-3334, 1792, 1659, 1585, 1536, 1499, 1457, 1416, 1340, 1236, 1126, 989; 1H NMR (ODC1 3 8 7.39-7.29 (6H, in), 7.12 (1H, 7.03 (1H, 6.92, "too 9 5 6.83, 6.48 (approx 3H, 3d, J 8.1, 7.5, 5.57 (d, .0 J 5.27 (1H, 5.23-5.06, 4.91-4.71, 4.64- 4 .43, (6H, 3m) 3. 92, 3. 91, 3 .89, 3. 88 (9H, 4s) 3 .32- 4*oo* 4 2.70, 2.52-2.08, 1.91, 1.63 (1H, 4m).

(213v) was isolated as a white solid mp. 121s' ob 10 7*C; IR (KBr). 3534-3331, 1791, 1659, 1528, 1420, 1256, 1122; 1H NM'R (ODC1 3 8 8.34-8.29 (1H, in), 7.98-7.87 (2H, mn), 7.68-7.45 (4H, in), 7.34-7.24 (5H, in), 7.04 J= 6.78 J 6.66 J 6.48 (2H, d, J 7.5)5.56 J 5.15 (1H, 5.30-5.14, 5.0, 4.89 J 11.2), 4.71-4.41 3.18-2.80, *00:002.50-2.27, 2.08-1.60 (11H, 3m).

(213w) was isolated as a mixture of diastereoisoiners (65/35) as a white solid (0.9g, ip. 110-115*C 0 fog. (decomp.); IR (KBr) 3409, 2945, 1792, 1658, 1606, 1534, 20 1486, 1420, 1330, 1276, 1209, 1122, 980, 960; 1H NMR (ODC1 3 5 7. 66 35H, d, J 6. 9) 7. 46-7.20 (7H, in), 6.93 (0.35H, d, J 6.85 (0.65H, d, J 7.6), 6.73 (0.65H, d, J 5.96 (0.35H, bs), 5.85 (0.65H, bs), 5.56 (0.65H, d, J 5.28 (0.35H, bs), 5.20-4.98 (2H, in), 4.96-4.40 (4H, in), 3.28-2.55 (3H, in), 2.53-2..32 (1K, in), 2.23 (6H, 2s), 2.03-1.40 (7K, mn). MS 577, (ES 579.

(213x) was isolated as a colourless poweder (691mg, mp. 150-70 [a1D 22_l0.10 (c 0.10, Me 2 C0); IR (KBr) 3313, 1791, 1679, 1654, 1597, 1528, 1501, 1457, 1407, 1371, 1315, 1255, 1184, 1122, 933; 1H NMR (d6- DMSO) 858. 75 (1K, d) 8.47 (1H, 7. 84 (2H, d) 7.6 6 218 (2H, 7.35 (5H, 5.43 (1H, 5.06-5.00 (1H, m), 4.90-4.64 (3H, 4.46-4.26 (2H, 3.16-2.86 (2H, 2.45-2.05 (5H, 2.07 (3H, 2.00-1.84 (2H, m), 1.68-1.56 (2H, Anal. Calcd for C 3 0

H

3 3

N

5 0 8 oH 2 0: C, 5 59.11; H, 5.79; N, 11.49. Found: C, 59.38; H, 5.66; N, 0@ S g* 11.31; M.S. (ES 614 592 (415) was prepared by a similar method as compound 214e to afford a white solid (297mg, mp. 158-62 °C; 24 'S [L]D -109.5° (c 0.1, CH 3 OH); IR (KBr) 3700-2500 (br), 10 1783,1659, 1650, 1538, 1486, 1439, 1257, 1037; H NMR

(CD

3 OD) 6 7.48 (1H, dd), 7.35 (1H, 6.88 (1H, d), 6.03 (2H, 5.25-5.15 (1H, 5.02-4.90 (1H, m), 4.63-4.45 (2H, 4.30-4.20 (1H, 3.57-3.30 (1H, 3.20-3.05 (1H, 2.75-2.10 (5H, 2.10-1.60 *0°"15 (4H, MS (ES 488 487 (M 1, 100), 443 387 315 150 127 113 Accurate mass calculated for C 2 2

H

2 5

N

4 0 9 489.1621.

Found 489.1648.

00 (450) was prepared by a similar method as compound 214e 20 to afford a white foamy solid (378mg, mp. 175-9 22 7 [a]D -91.70 (c 0.1, CH 3 OH); IR (KBr) 3700-2500 3319, 1659, 1590, 1553, 1427, 1260; 1H NMR

(CD

3 OD) 6 8.01 (1H, 7.74 (1H, dd), 7.58 (1H, d), 7.45-7.35 (1H, 5.25-5.15 (1H, 5.05-4.90 (1H, 4.60-4.45 (2H, 4.30-4.20 (1H, 3.55-3.30 (1H, 3.20-3.00 (1H, 2.75-2.20 (5H, 2.14 (3H, 2.20-1.60 Anal. Calcd for

C

2 3

H

2 7

N

5 0 8 *1.5H 2 0: C, 52.27; H, 5.72; N, 13.25. Found: C, 52.31; H, 5.86; N, 12.85. MS (ES 501 26%), 500 (M 1, 100), 328 149 113 (456) was prepared by a similar method as compound 214e to afford a white solid (0.73g, mp. >260 °C; 219 20 [a]D -66° (c 0.34, MeOH); IR (KBr) 3401, 2946, 1651, 1609, 1584, 1506, 1426, 1277, 1257, 1177; H NMR (D 6 DMSO) 610.2 (1H, very bs), 9.17 (1H, bs), 8.65 (1H, s), 8.37 (1H, d, J 7.81 (2H, d, J 6.87 (2H, d, J 5.24 (1H, 4.92-4.86 (1H, 4.41-4.32 (2H, 3.68-3.21 (3H, 3.12-2.79 (1H, 2.50- 1.42 (7H, MS (ES 459.

(475) was prepared by a similar method to that 0@ described for compound 214e to afford a white solid 8@ 10 mp. 150 °C (softens) 190-210 [a]D 23 -97.5° (c 0.1, CH 3 OH); IR (KBr) 3319, 1658, 1650, 1549, 1421, 1256; H NMR (CD 3 OD) 67.61 (1H, d, J 7.43 (1H, ooo d, 7.21 (2H, 7.05 (1H, 5.21 (1H, m), 5.07-4.77 (1H, 4.54 (2H, 4.23 (1H, 3.46 (1H, 3.14 (1H, 2.66-1.71 (9H, MS (ES Sm/z), 482 1, 100%).

was prepared by a similar method as compound 214e to afford a white solid (0.79g, mp. 156-9 °C; S. 25 [aD -119.7° (c 0.1, CH 3 OH); IR (KBr) 3700-2500 (br), 3387, 3309, 2956, 1785, 1659, 1650, 1535, 1422, 1278; H. H NMR (CD 3 OD) 6 7.46-7.15 (4H, 5.25-5.15 (1H, m), 5.02-4.90 (1H, 4.58-4.45 (2H, 4.30-4.20 (1H, 3.55-3.30 (1H, 3.20-3.05 (1H, 2.80-2.20 (4H, 2.41 (3H, 2.20-1.60 (5H, MS (ES 458 457 (M 1, 100), 413 339 285 134 127 Accurate mass calculated for

C

2 2

H

2 7

N

4 0 7 (MH 459.1880. Found 459.1854.

(486) was prepared by a similar method as compound 214e to afford a white solid (325mg, mp. 165-9 °C; 22 [a]D -69.1° (c 0.1, CH 3 OH); IR (KBr) 3700-2500 (br), 3318, 1658, 1599, 1530, 1505, 1407, 1258; H NMR 220

(CD

3 OD) 6 7.85 (2H, 7.69 (2H, 7.38-7.20(5H, m), 5.25-5.15 (1H, 5.05-4.90 (1H, 4.57-4.45 (2H, 4.30-4.20 (1H, 3.70 (2H, 3.55-3.30 (1H, m), 3.20-3.00 (1H, 2.75-1.60 (9H, Anal. Calcd for

C

29

H

31

N

5 0 8 *1.5H 2 0: C, 57.61; H, 5.67; N, 11.58. Found: C, 57.81; H, 5.74; N, 11.47. MS (ES 577 33%), 576 (M 1, 100), 502 (487) was prepared by a similar method as compound 214e to afford a white foamy solid (335mg, mp. 176-80 22 10 2 ]D -88.0° (cO.1, CH 3 OH); IR (KBr) 3700-2500 3321, 2960, 1781, 1660, 1597, 1529, 1407, 1258, 1187; H NMR (CD 3 OD) 6 7.86 (2H, 7.69 (2H, 5.25- 5.15 (1H, 5.05-4.90 (1H, 4.60-4.45 (2H, m), 4.30-4.20 (1H, 3.57-3.30 (1H, 3.20-3.00 (1H, 2.75-1.60 (12H, 1.00 (6H, Anal. Calcd for

C

2 6

H

3 3

N

5 0 8

*H

2 0: C, 55.61; H, 6.28; N, 12.45. Found: C, 56.00; H, 6.37; N, 12.15. MS (ES 543 542 (M 1, 100), 498 468 (417) was prepared by a similar method to that described for compound 214e to afford a white solid .(0.63g, mp. 145-155 °C (approx., not sharp); 27 [o]D -114.6° (c 0.11, CH 3 OH); IR (KBr) 3327, 1658, 1586, 1548, 1501, 1416, 1341, 1238, 1126; H NMR

(CD

3 OD) 67.22 (2H, 5.21 (1H, 5.00 (1H, 4.56, 4.49 (2H, 2m), 4.25 (1H, 3.88 (6H, 3.80 (3H, 3.55-3.43 (1H, 3.12 (1H, 2.71-1.70 (9H, m).

Anal. Calcd for C 2 4

H

3 0

N

4 0 1 0 *2H 2 0: C, 50.52; H, 6.01; N, 9.82. Found: C, 50.49; H, 6.05; N, 9.68. MS (ES m/z) 533 (M 1, 100%).

(408) was prepared by a similar method to that described for compound 214e to afford a white solid mp. 157-165 C (not sharp); [a27 140.5 (c mp. 157-165 0 C (not sharp) ]D -140.5 (c 221 0.1, CH 3 OH); IR (KBr) 3325, 1658, 1531, 1420, 1278, 1257; 1H NMR (CD 3 OD) 8 8.33-8.28 (1H, in), 8.01-7.78 (2H, in), 7.71 (1H, d, J 7.59-7.52 (3H, mn), 5.27 (1H, in), 5.12-5.03 (1H, in), 4.55 (2H, in), 4.25 (1H, mn), 3.64-3.43 (1H, mn), 3.24-3.12 (1H, mn), 2.80-1.67 (9H, m) Anal. Calcd for C 2 5

H

2 6

N

4 0 7 *2H 2 0: C, 56.60; H, 5.70; N, 10.56. Found: C, 56.70; H, 5.80; N, 10.33. MS in/z) 493 1, 100%).

(214w) was prepared by a similar method as compound 214e to afford 210mg of a white solid: mp. >260 5: C; [a]D 20 930 (c 0.20, MeOH); IR (KBr) 3401, 2948, 1651, 1604, 1559, 1486, 1421, 1325, 1276, 1210; 1H NNR

(D

6 -DMSO) 6 9. 39 (1H, bs) 8.29 (1H, d, J 5. 9) 7. (2H, 6.64 (1H, d, J 5.79 (1H, 5.25-5.21 (1H, in), 1.90-1.82 (1H, in), 4.41-3.69 (2H, in), 3.47see**:3.20 (3H, in), 2.97-2.91 (1H, in), 2 .23 (6H, 2.25- 1.60 (7H, in).

e* 0 0 *0 0:550q R= Et HO 2 13y R= Bn (550q) was synthesized via methods used to prepare 213e to afford 550q.

(213y) was synthesized via methods used to prepare 213e to afford 2 1 3 y.

222 412 a4 a 0 b C c d 0J~ e f *5 0*

S.

00 C 0 0 5O 0@*OOS

S

0e C S

SOS.

S.

S

5O5 0

S

S

55..

S S 5@ 0

OS

S 0 0* @5 0 S S *5 (412a) was synthesized via methods used to prepare 550q using 513a-1 to afford 412a.

(412b) was synthesized via methods used to prepare 550q using 513a-2 to afford 412b.

5 (412c) was synthesized via methods used to prepare 550q using 513b-1 to afford 412c.

(412d) was synthesized via methods used to prepare 550q using 513b-2 to afford 412d: 1 H NMR (CDC1 3 6 9.5 (1H, 8.9 (1H, 8.5 (1H, 7.9-7.8 (2H, in), 7.8-7.65 10 in), 6.55 (1H, 5.55 (1H, 5.25-5.1 (2H, in), 4.75-4.65 (1H, mn), 4.65-4.6 (1H, in), 4.4-4.3 (1H, mn), 3.25-3.15 (1H, in), 3.15-3.05 (1H, in), 2.95-2.8 (2H, in), 2.55-2.4 (21H, mn), 2.15-1.5 (14H, in).

(412e) was synthesized via methods used to prepare 550q using 513f-1 to afford 412e.

(412f) was synthesized via methods used to prepare 550q using 513f-2 to afford 412ff.

Compounds 410 and 412 were prepared via methods used to prepare 605 from 604.

223 S. 0@

S.

.5 0*

SS

OSeOSO

S

0S 0 00 00S 5

OOSS

0* S. S

OS

S S 0@ 0S

SS

502y, 502z 410, 412 compoundR1 0 502y, 410/I

SD

502z, 412

N

0 5 (410) was purified by flash chromatography (5-25% methanol in dichloroinethane) to give 296mg of a colourless solid: inp. 90-200'C; IR (KBr) 3338, 3096,.

2950, 1787, 1726, 1657, 1546, 1420, 1279, 1258, 1125, 1092, 984, 933; 1H NMR (CD 3 OD) 6 8. 41 (1H, d) 8.13 (1H, 7.54-7.41 (3H, mn), 7.20 (1H, 5.19-5.11 (1H, mn), 4.54-4.30 (1H, in), 3.27 (1H, in), 3.18-3.03 (lH, in), 2.81-2.64 (2H, in), 2.56-1.59 (7H1, mn). Anal. Calcd for

C

19

H

22

N

4 0 7 S*2.5H 2 0: C, 46.05; H, 5.49; N, 11.31. Found: C, 46.36; H, 5.25; N, 11.10. MS (ES 449 (M 1, 113 (100). Accurate mass calculated for

C

1 9

H

2 3

N

4 0 7 S 451.1287. Found: 451.1295.

(412) was prepared by a similar method to that described for compound 605 to afford a white glassy 224 23 5 solid mp. 138-141 [a]D -105.50 (c

CH

2 C1 2 IR (KBr) 3375, 1787, 1659, 1515, 1421, 1278, 1256; H NMR (CDC1 3 6 9.32 (1H, 8.79 (1H, 8.47 (1H, 7.86-7.64 (4H, 5.31, 5.18, 4.59, 4.37 (4 or 5H, 3.55-2.76, 2.49-2.39, 2.05, 1.65 (11H, 4m).

Anal. Calcd for C 24

H

25

N

5 0 7 -1.5H 2 0: C, 55.17; H, 5.40; N, 13.40. Found: C, 54.87; H, 5.22; N, 13.15. MS (ES m/z) 494 (M 1, 100%) 5 02y) was synthesized via methods used to prepare 604 10 from 603 to afford a pale cream powder: mp. 120-180 °C; 0: :[aiD 2 3 -109° (c 0.18, CH 2 C1 2 IR (KBr) 3478, 3327, 0:04 1 1670, 1582, 1543, 1421, 1279, 1257, 1155; H NMR @00 (CDC1 3

CD

3 OD) 68.04 (1H, 7.49 (1H, 7.38 (1H, 7.17 (1H, 5.17-5.01 (2H, 4.86 (1H, m), 4.61-4.50 (1H, 3.45-3.29 (2H, 3.21-3.03 (1H, 2.79-2.54 (3H, 2.43-2.33 (1H, 2.11-1.66 (5H, 1.44 (9H, Anal. Calcd for C 2 4

H

3 3

N

7 07S-H 2 0: C, 49.56; H, 6.07; N, 16.86; S, 5.51. Found: C, 49.51; H, 5.93; N, 16.31; S, 5.17. MS (ES 586 564 (M 1, 1.59). Accurate mass calculated for

C

2 4

H

3 4

N

7 0 7 S 564.2240. Found: 564.2267.

(502z) was prepared by a similar method to that described for compound 604 to afford a pale yellow solid mp. 142-145 [a]D 2 4 -136.5° (c 0.06,

CH

2 C1 2 H NMR (CDC1 3 6 9.51-9.46 (1H, 9.11 (1H, 8.83 (1H, d, J 8.53 (1H, d, J 7.89- 7.83 (2H, 7.77-7.65 (2H, 7.55 (1H, d, J 7.2), 7.18 (1H, d, J 5.26-5.12 (2H, 4.87 (1H, m), 4.59 (1H, 3.25-3.12 (2H, 2.95-2.76 (2H, m), 2.59-2.38, 2.18-1.94, 1.70 (5H, 3m), 1.44 (9H, s).

225 *0 0 0@ @0 00 0 o co 0@ 0 000000 0 00 0 0 Oe 0 00S 0 000000 0 0S0* 0 @000 0*00 0* 00 S 0S 0 0 00 S. S 0* 00 226 compoundR4 0 214w-6 CH3;J.

0 214w-7

CH

3

L

Ho 412g 4 12h *0 S 0O *0 C o 00 0@ 0 0

S@

S

C.

S

OSe 6e e005 (415a), (415b), (415c), (214w-1), (214w-2), (214w-3), (214w-4), (214w-5), (214w-6), (214w-7), (412g) and (412h) were synthesized via methods used to prepare 550q.

OC

C 0 50 5* 0 0*

SC

N

<O aH 0

CHOH

N 1 HO O 0N H CH1 3 H0 214w 415 (415) was synthesized by the method used to prepare 2002 from 2001 to afford 415.

(214w) was synthesized by the method used to prepare 2002 from 2001 to afford 214w.

227 0 0 N 0 H 0 N---q e

H

0 *0

S

66 0* 00f

S

0 00e00 0 0 000 S 0 0 *0 S. 0 0 5O 0e 2100k-o compound R 2100k Pro 21001 -0 5 2100m O-Io 2100n 21000 o (2100k) was prepared by a similar method as compound 213e to afford a mixture of diastereoisomers (75/25) as a white solid (258mg, mp. 101 [a]D -96° (c 0.2, CH 2 C1 2 IR (KBr) 3328, 2935, 2978, 1732, 1669, 1603, 1483, 1450, 1414, 1237, 1155, 1082, 989, 755; H NMR (CDC1 3 67.84-7.80 (2H, 7.54-7.17 (8H, 7.06- 6.99 (1H, 6.25 (1H, d, J 7.9H), 5.41 (0.75H, d, J 5.4H), 5.31 (0.25H, bs), 5.23-5.09 (1H, 4.93-4.87 (1H, 4.68-4.51 (2H, 4.40-4.33 (0.25H, 4.24- 4.14 (0.75H, 3.95-3.70 (1H, 3.30-3.13 (1H, m), 228 3.14-2.78 (5H, 2.47-2.21 (2H, 2.05-1.50 Anal. Calcd for C 29

H

32

N

4 0 7 *0.5H 2 0: C, 62.47; H, 5.97; N, 10.05. Found: C, 62.17; H, 5.83; N, 9.97. MS (ES 549.

(21001) was prepared by a similar method as 213e, (74%) as a colourless solid: mp. 172-80 [aID 23 -91.5, (C 0.1, CH 2 C1 2 IR (KBr) 3290, 1792, 1677, 1657, 1642, 1544, 1425, 1280, 1259, 1124, 977; 1H NMR (CDC 3 67.80 a (2H, 7.46 (3.5H, 7.00 (1H, d, J 6.48 (0.5H, d, J 5.55 (0.5H, d, J 5.19 (2H, a s 4.93 (0.5H, 4.62 (1.5H, 4.34 (1H, m), tQe 4.18 (0.5H, 3.28-2.70 (4H, 2.49-2.29 (2H, m), 205-1.48 (15H, m).

(2100m) was prepared by a similar method as 213e, (76%) as a colourless solid: mp. -140"C, remelts 187-9 'C; 23 a [a]D -96.90 (c 0.11, CH 2 C1 2 IR (KBr) 3507, 3308, a 3251, 1772, 1660, 1641, 1566, 1545, 1457, 1424, 1346, 1326, 1302, 1275, 1258, 1136, 1085, 1018, 981; 1H NMR (CDCl 3 6 7.78 (2H, 7.53 (3H, 7.19 (4H, 6.91 (1H, d, J 6.27 (1H, d, J 5.66 (1H, d, J 5.10 (1H, 4.96 (1H, 4.75 (2H, 4.52 (iH, 3.08 (3H, 3.03-2.71 (5H, 2.48-2.31 (2H, 1.90-1.40 (4H, 1.22 (1H, m) (2100n) was prepared by a similar method to that described for compound 213e to afford a white glassy solid mp. 112-5 [ct]D -62.0* (c 0.1,

CH

2 C1 2 IR (KBr) 3305, 1789, 1677, 1665, 1535, 1422, 1279, 1256, 1119, 942, 700; IH NMR (CDCI 3 67.84 (2H, 7.58-7.27 (9H, 6.99 (1H, d, J 5.23 (1H, 5.23-5.11 (1H, 4.89 (1H, 4.76 (1H, d, J 11.3), 4.55 (1H, d, J 11.4), 4.58-4.43 (2H, 3.30- 2.96, 2.81-2.69, 2.46-2.37, 2.16-1.66 (10H, 4m), 2.27 229 (1H, d, J 17.8). Anal. Calcd for C 28

H

30

N

4 0 7 -0.5H 2 0: C, 61.87; H, 5.75; N, 10.32. Found: C, 61.88; H, 5.70; N, 10.33. MS (ES m/z) 535 (M 1, 100%).

(2100o) (containing about 7% of was prepared by a similar method to that described for compound 213e to afford a white glassy solid mp. 115-7 [a]D 23 -121.8° (c 0.11, CH 2 C12); IR (KBr) 3326, 1792, 1659, 0. 1535, 1421, 1278, 1257, 1124, 978; H NMR (CDCl 3 6 7.82 (2H, 7.58-7.24 (8H, 6.90 (1H, d, J 6.49 (1H, d, J 5.57 (1H, d, J 5.11 (2H, m), 4.91 (1H, d, J 11.4), 4.57 (1H, d, J 11.1), 4.81- S. 4.68 (1H, 4.65-4.54 (1H, 3.18-2.71 2.52-2.30, 2.05-1.62 (11H, 3m). Anal. Calcd for C 2 8

H

3 0

N

4 0 7 -0.5H 2 0: C, 61.87; H, 5.75; N, 10.32. Found: C, 61.70; H, 5.71; N, 10.15. MS (ES m/z) 535 (M 1, 557 (100%) 0000 *N 0 H

N

00 Me

NJ

0 550n (550n) was prepared by a similar method as compound 213e to afford a mixture of diastereoisomers (65/35) as a tan powder (390mg, mp. 139-145 [a]D -104° (c 0.2, MeOH); IR (KBr) 3318, 2405, 2369, 1792, 1660, 1591, 1549, 1484, 1422, 1257, 1117; 1H NNR (D 6 -DMSO) 6 230 1 (1H, s) 8. 80 65H, d, J 6) 8. 58 35H, d, J 6. 6) 8. 59 (1H1, d, J 7. 8.06 (1H, bs) 7. 83- 7.79. (1H, mn), 7.61-7.57 (1H, mn), 7.47-7.39 (1H, in), 61 35H, d, J 5. 0) 5. 37 65H, bs) 5.17-5.14 35H, mn), 5. 08-5. 06 65H, in), 4. 92-4. 86 (1H, m) 4.67-4.61 (0.35H, mn), 4.47-4.41 (0.65H, mn), 4.28-4.11 (1H, 2m), 3.80-3.59 (2H, in), 3.23-2.75 (3H, in), 2.61- 1.48 (7H, mn), 2.10 (3H, 1.25 and 1.17 (3H, 2t, J= MS 528.

S0 @05000 0 0@0 NH

H

H a, 0 10 5500 (550o) was synthesized by a similar method as compound 213e to afford a colourless solid (1.071g, inp.

155-70 [a1D 22_75.8* (c 0.26, CH 2 C1 2 IR (KBr) 05 3314, 2941, 1791, 1658, 1545, 1420, 1341, 1312, 1252, 1181, 1118, 939, 749; 1H NMR CCDCl 3 569.45 (0.5H, s) 9.34 (0.5H, 7.68-7.62 (1H, in), 7.49-7.39 (2H, mn), 7.33-7.26 (1H, in), 7.18-7.03 (3H, in), 5.49 (0.5H, d), 5.30 (0.5 H, 5.26-5.13 (1H, mn), 4.90-4.83 mn), 4.76-4.49 (iH, in), 4.42-4.35 (0.5H, in), 3.97-3.74 (1H, in), 3.72-3.53 (1H, mn), 3.35-2.64 (4H, in), 2.50- 2.37 (1H, mn), 2.20-1.82 (5H, mn), 1.69-1.50 (2H, in), 1.30-1.19 (3H, mn).

231

S

0@ S

SS

5.65..

S

S@

0 5500

S.

S

S

S

000* @005 0 0@ 0 0

NH

H N HO H O/ 5 0 p (550p) was prepared by a similar method as compound 213e to afford a mixture of diastereoisomers as a white foam (820mg, [a]D 2 4 -750 (c 0.16, CH 2 C12); IR 5 (KBr) 3401, 2937, 1791, 1657, 1609, 1539, 1505, 1423, 1277, 1177, 1118; 1H NMR (CDC1 3 68.07-8.05 (1H, m), 7.67 (2H, d, J 7.38-7.29 (2H, 6.80 (2H, d, J 5.49 (0.5H, d, J 5.23 (0.5H, bs), 5.24-5.20 (1H, 5.12-5.08 (1H, 4.68-4.29 (2H, 10 3.92-3.45 (3H, 3.32-2.30 (2H, 2.80-1.56 (11H, 1.21 (3H, t, J 7.OH).

O

Nj 0O

N

OtBuO MeSO-N u H 0O N O R H OH @0 6 55 0O S S S

SS

O

N 0 N2 OtBu O MeSO-H H O N O rorR H O 504a-e 503a-e 286, 505b-e 232

S

S S @0 00 C

S.

S*

S

005505 0 0* S S e.g.

C.

0 006 @00050 0

S

SOS.

compound

R

503a- 504a 286 5 503b Me 504b 505b Ph CN OPh 503c 504c 10 505C 503dOh 504d 50 503e

S

504e\/ SO~e Me (503a) was prepared from 212b and (3S,4R) t-butyl (Nallyloxycarbonyl) -3-amino-4-hydroxy-5- (1naphthoyloxy)pentanoate by the method described for (213e) to afford 533mg of an off-white foam: [aID 22_81.40 (c 0.5, CH 2 C129; IR(KBr) 3342, 2976, 1719, 1664, 1328, 1278, 1246, 1153, 1137. 1HNMR (CDC1 3 6 8.86 (1H, d, J 8.21 (1H, dd, J 7.3), 8.03 (1H, d, J 7.88 (1H, d, J 7.66-7.45 (3H, mn), 7.23 (1H, d, J 5.96 (1H, d, J 5.30 (1H, mn), 4.59-4.33 (5H, mn), 4.24 (1H, in), 3.96 (1H, brd), 3.29 (1H, mn), 2.95 (1H, mn), 2.93 (3H, s), 2.69-2.50 (3H, mn), 2.36 (1H, in), 1.96 (4H, mn), 1.62 .5.5 S S 00 0 0 5 0S @0 0

S.

OS

233 (1H, 1.41 (9H, Anal. Calcd for

C

31

HN

4 0 10 S'0.25H 2 0 C, 55.97; H, 6.14; N, 8.42.

Found: C, 55.90; H, 6.11; N, 8.23. M.S. (ES 683 (M+Na, 100%), 661 605 (78).

(504a) was synthesized from 503a via method used to prepare 216e from 215e to afford 446mg of a colourless foam: [a]D 2 1 -111.60 (c 0.5, CH 2 C1 2

IR

S""(KBr) 3319, 2978, 2936, 1723, 1670, 1413, 1370, 1329, 1278, 1246, 1153. 1H NMR (CDC1 3 68.87 (1H, d, J S 10 8.29 (1H, d, J 8.06 (1H, d, J 8.3), 7.90 (1H, d, J 7.66-7.48 (3H, 7.37 (1H, d, J 5.61 (1H, d, J 5.31 (1H, 5.22 (1H, AB, J 16.9), 5.09 (1H, AB, J 16.92), 4.99 (1H, 4.65-4.43 (2H, 3.28 (1H, 2.96 (3H, 2.86 15 (2H, 2.59 (1H, m) 2.38 (1H, dd, J 6.8, 13.2), 600000 6 2.21-1.70 (6H, 1.45 (9H, Anal. Calcd for

C

3 1

H

3 8

N

4 0 1 0 So0.25H 2 0. C, 56.14; H, 5.85; N, 8.45.

Found: C, 56.11; H, 5.83; N, 8.29. M.S. (ES 657 (M- 1, 100%).

(286) was prepared from 504a by the method described for 217 to afford 356mg of a white powder: mp 23 120-123 0C; [a]D -1210 (c 0.194, CH 2 C1 2 IR (KBr) 3314, 2937, 1722, 1663, 1412, 1328, 1278, 1245, 1195, 1132. H NMR (d6-DMSO) 612.63 (1H, brs), 8.94 (1H, d, J 8.78 (1H, d, J 8.26 (2H, 8.11 (1H, d, J 7.77-7.62 (4H, 5.28 (2H, 5.21 (1H, 4.82 (1H, 4.44-4.29 (2H, 3.31 (1H, 2.98 (3H, 2.98-2.86 (2H, 2.72 (1H, dd, J 7.3, 16.9), 2.40 (1H, 2.24-1.84 (4H, 1.69 (2H, m).

Anal. Calcd for C 2 7

H

3 0

N

4

O

1 0

S.H

2 0 C, 52.25; H, 5.20; N, 9.03. Found: C, 52.11; H, 4.97; N, 8.89. M.S. (ES 601 100%) 234 (503b) was synthesized by a similar method as compound 213e, to afford an off-white powder (671mg, mp.

90-120"C; IR (KBr) 3345, 2977, 1727, 1664, 1532, 1450, 1423, 1369, 1323, 1310, 1276, 1257, 1154, 1101, 990, 766; 1H NMR (CDCl 3 67.61-7.55 (2H, 7.51-7.42 (3H, 6.86 (1H, 5.69 (1H, 5.21 (1H, 4.64-4.38 (2H, 4.15-4.05 (3H, 3.84 (1H, 3.31-3.14 0 0: (2H, 2.97-2.87 (1H, 2.94 (3H, 2.76 (3H, s), 2.64-2.48 (3H, 2.39-2.29 (1H, 2.04-1.61 10 Anal. Calcd for C 3 1

H

4 1

N

5 0 1 1

S-H

2 0: C, 52.46; H, 0.00 6.11; N, 9.87; S, 4.52. Found: C, 52.34; H, 5.92; N, 9.56; S, 4.44. MS (ES 714 692 (M 1, 84), 0 000 636 (100).

(504b) was synthesized by a similar method as compound 216b to afford a colourless powder (601mg, mp.

75-115 [I]D 23 1040 (c 0.26, CH 2 C1 2 IR (KBr) 3324, 0000 2977, 2935, 1730, 1670, 1525, 1452, 1422, 1369, 1317, 1276, 1256, 1222, 1155, 1107, 990, 766; 1H NMR (CDC1 3 6 00 0 7.68-7.61 (2H, 7.47-7.38 (3H, 7.32-7.24 (1H, 5.56 (1H, 5.36-5.24 (1H, 5.04 (1H, 4.88 (1H, 4.86-4.77 (1H, 4.64-4.39 (2H, 3.32- 3.17 (IH, 2.97-2.85 (1H, 2.93 (3H, 2.76 2.80-2.71 (IH, 2.65-2.49 (1H, 2.41- 2.30 (1H, 2.12-1.61 (6H, 1.42 (9H, Anal.

Calcd for C 31

H

39

N

5 0 11

SOH

2 0: C, 52.61; H, 5.84; N, 9.90; S, 4.53. Found: C, 52.94; H, 5.69; N, 9.72; S, 4.51.

MS (ES 712 707 (100), 690 (M 1, 41), 634 (505b) was synthesized by a similar method as compound 217 to afford a colourless powder (499mg, mp. 145 0 C; [a]D 22 -1370 (c 0.12, MeOH); IR (KBr) 3323, 2936, 1732, 1665, 1529, 1452, 1421, 1312, 1275, 1256, 235 1221, 1183, 1153, 1135, 1101, 990; 1H NMR (CD 3 OD) 67.67- 7.56 (2H, 7.49-7.38 (4H, 5.23-5.12 (1H, m), 5.02 (1H, 4.79-4.73 (1H, 4.52-4.34 (3H, m), 3.48-3.25 (2H, 3.03-2.85 (2H, 2.94 (3H, s), 2.74 (3H, 2.79-2.66 (1H, 2.52-2.38 (1H, m), 2.29-2.14 (1H, 2.04-1.70 (4H, Anal. Calcd for

C

27

H

31

N

5 0 11

S*H

2 0: C, 49.77; H, 5.18; N, 10.75; S, 4.92.

Found: C, 49.83; H, 5.01; N, 10.27; S, 4.84. MS (ES 746 632 (M 1, 100), 386 Accurate mass calculated for C 27

H

32

N

5 0 11 S 634.1819. Found: 634.1807.

(503c) was synthesized by a similar method as compound 213e to afford a colourless solid (446mg, IR (KBr) 3345, 2976, 2935, 1727, 1664, 1603, 1535, 1483, 15 1451, 1416, 1395, 1369, 1328, 1297, 1277, 1237, 1155, 1

H

1135, 1076, 990, 755; H NMR (CDCI 3 67.98-7.89 (1H, m), 7.55-7.45 (IH, 7.39-7.18 (3H, 7.14-7.07 (1H, Se:. in), 7.00-6.90 (3H, 6.75 (1H, 5.57-5.50 (1H, m), 5.21-5.09 (1H, 4.64-4.42 (2H, 4.36-4.12 (3H, 3.95-3.87 (1H, 3.39-3.18 (1H, 3.00-2.82 4. (1H, 2.95 (3H, 2.69-2.48 (3H, 2.42-2.28 (1H, 2.07-1.62 (6H, 1.42 (9H, Anal. Calcd for C 3 3

H

42

N

4 0 1 1

SH

2 0: C, 54.99; H, 6.15; N, 7.77; S, 4.45. Found: C, 54.95; H, 5.95; N, 7.34; S, 4.20. MS (ES 725 720 703 1, 34), 433 (100), 403 (89).

(504c) was synthesized by a similar method as compound 216e to afford a colourless powder: mp. 85-100 °C; [C]D -91.30 (c 0.52, CH 2 C1 2 IR (KBr) 3328, 2978, 2935, 1732, 1669, 1603, 1524, 1483, 1450, 1396, 1369, 1296, 1276, 1237, 1155, 1132, 1082, 989, 755; 1 H NMR

(CDCI

3 6 8.03-7.98 (1H, 7.52-7.44 (1H, 7.37-7.07 236 7.01-6.92 (3H, 5.52 (IH, 5.28-5.20 (1H, 5.06-4.84 (3H, 4.64-4.39 (2H, 3.32- 3.14 (1H, 2.99-2.88 (1H, 2.94 (3H, 2.65- 2.45 (2H, 2.39-2.29 (1H, 2.12-1.58 (6H, m), 1.40 (9H, Anal. Calcd for C 33

H

40

N

4 0 11 S: C, 56.56; H, 5.75; N, 8.00; S, 4.58. Found: C, 56.37; H, 5.84; N, 7.69; S, 4.37. MS (ES 723 718 (100), 701

S

(M 1, 23), 645 (59).

(505c) was synthesized by a similar method as compound 217 to afford a colourless foam (252mg, mp. 125 0C; [2]D 3 1330 (c 0.11, MeOH); IR (KBr) 3314, 2938, 1792, 1734, 1663, 1604, 1535, 1483, 1448, 1415, 1250, 1132, 756; IH NMR (D 6 -DMSO) 6 8.81-8.76 (1H, m), 7.92 (1H, 7.68-7.54 (2H, 7.41-7.25 (3H, m), 15 7.16-6.91 (4H, 5.13-4.98 (2H, 4.72-4.63 (1H, 6@oeeo S 4.37-4.21 (2H, 2.92 (3H, 2.90-2.60 (3H, m), 2.35-2.26 (1H, 2.17-2.05 (2H, 1.99-1.80 (2H, im), 1.61-1.50 (iH, m).Anal. Calcd for

C

2 9

H

3 2

N

4 0 1 1 SO0.5H 2 0: C, 53.29; H, 5.09; N, 8.57; S, 4.90. Found: C, 53.57; H, 5.18; N, 8.32; S, 4.75. MS (ES 643 (M 1, 100%) (503d) was synthesized by a similar method as compound 213e to afford a colourless solid (563mg, IR (KBr) 3349, 2978, 2935, 1724, 1664, 1583, 1536, 1489, 1443, 1370, 1327, 1271, 1226, 1189, 1155, 1073, 990, 755; 1H NMR (CDCI 3 67.77 (1H, 7.67 (iH, 7.45- 7.10 (6H, 7.00 (2H, 5.93-5.80 (1H, 5.36- 5.30 (1H, 4.63-4.24 (5H, 4.15-4.09 (1H, m), 3.37-3.22 (1H, 2.98-2.74 (1H, 2.94 (3H, s), 2.70-2.47 (3H, 2.40-2.30 (1H, 2.15-1.60 1.42 (9H, Anal. Calcd for C 33

H

42

N

4 0 11

SH

2 0: C, 54.99; H, 6.15; N, 7.77; S, 4.45. Found: C, 54.60; H, 237 5.88; N, 7.49; S, 4.50. MS (ES 725 720 (91), 703 (M 1, 74), 647 629 (100), 433 (78).

(504d) was synthesized by a similar method as compound 216e to afford a colourless powder (466mg, mp.

22 75-100 0 C; [a]D -99.30 (c 0.60, CH 2 C1 2 IR (KBr) 3335, 2978, 2937, 1728, 1669, 1584, 1525, 1487, 1444, 1416, 1369, 1328, 1272, 1227, 1188, 1155, 989, 754; 1

H

NMR (CDCI 3 )6 7.82-7.77 (1H, 7.66-7.65 (1H, m), o• 0 7.46-7.32 (4H, 7.26-7.10 (2H, 7.04-6.98 (2H, 5.68 (1H, 5.37-5.31 (1H, 5.11 (1H, d), 5.02-4.88 (2H, 4.66-4.42 (2H, 3.35-3.17 (1H, 2.98-2.89 (1H, 2.96 (3H, 2.84-2.78 (1H, m), 2.72-2.47 (1H, 2.42-2.32 (1H, 2.14-1.58 (6H, 1.43 (9H. Anal. Calcd for C 33

H

40

N

4 0 11 S: C, 15 56.56; H, 5.75; N, 8.00. Found: C, 56.36; H, 5.82; N, oo.oo.

7.71. MS (ES 723 718 701 1, 36), 645 (100).

(505d) was synthesized by a similar method as compound 217 to afford a colourless foam (353mg, mp. 115 0C; [a]D 2 3 -1380 (c 0.11, MeOH); IR (KBr) 3327, *0O 2937, 1728, 1666, 1584, 1529, 1487, 1443, 1413, 1328, 1 1273, 1227, 1189, 1155, 1134, 989, 754; H NMR (D 6 DMSO) 6 8.82 (1H, 7.76-7.72 (1H, 7.61-7.53 (2H, 7.48-7.32 (4H, 7.24-7.17 (1H, 7.11-7.06 (2H, 5.14-5.06 (3H, 4.73-4.64 (1H, 4.38- 4.24 (2H, 2.92 (3H, 2.89-2.61 (3H, 2.38- 2.27 (1H, 2.19-2.06 (2H, 2.02-1.79 (3H, m), 1.63-1.52 (1H, Anal. Calcd for C 2 9

H

3 2

N

4 0 1 1 S-0.5H 2 0: C, 53.29; H, 5.09; N, 8.57; S, 4.90. Found: C, 53.24; H, 5.14; N, 8.34; S, 4.86. MS (ES 643 (M 1, 100%), 385 (62).

238

S.

0

S

o

*OS

0

O-

0 OSoo 0 0 0 0 00@0

O

*0 0 0 0e* @0 (503e) was prepared by a similar method to that described for compound 213e, to afford an off white o ~25 0 solid mp. 100-103 [IX]D -84.00 (c 0.05,

CH

2 C1 2 IR (KBr) 3459-3359, 1722, 1664, 1514, 1368, 1328, 1278, 1247, 1155; H NMR (CDC 3 67.52 (1H, m), 7.06-6.99 (2H, 5.69 (1H, d, J 5.23 (1H, m), 4.61-4.16 (6H, 3.36-3.19 (1H, 2.96 (3H, s), 2.67-2.49, 2.42-2.32, 2.06-1.89, 1.69 (10H, 4m), 1.43 (9H, s).

10 (504e) was prepared by a similar method to that described for compound 216e, to afford a white solid mp. 91-98 [a]D 25 -112.5°C (c 0.06, CH 2 C1 2 IR (KBr) 3453-3364, 1727, 1668, 1513, 1420, 1368, 1245, 1155; H NMR (CDC1 3 67.54 (1H, d, J 7.18 (1H, 15 d, J 7.18), 7.05 (1H, d, J 5.42 (1H, d, J 5.25 (1H, 5.02 (2H, 4.96-4.87 (1H, m), 4.65-4.42 (2H, 3.34-3.17 (1H, 2.97-2.93 (1H, 2.97 (3H, 2.87-2.78, 2.73-2.50, 2.38-2.32, 2.13-1.88, 1.69-1.60 (9H, 5m), 1.44 (9H, s).

20 (505e). A solution of 217 (0.33g, 0.51mmol) in dry dichloromethane (3ml) was cooled (ice/water) with protection from moisture. Trifluoroacetic acid (2ml) was added with stirring. The solution was kept at room temperature for 2h after removal of the cooling bath, then concentrated in vacuo. The residue was evaporated three times from dichloromethane, triturated with diethyl ether and filtered. The solid was purified by flash chromatography (silica gel, 0-6% methanol in dichloromethane) to give the product as a white glassy 22.5 solid (0.296g, mp 110-122 [a]D -163.50 (c 0.1, CH 3 OH); IR (KBr) 3514-3337, 1726, 1664, 1513, 1420, 1245, 1152, 1134, 990; H NMR (CD 3 OD) 67.79 (1H, -239 di, J 5.2) 7. 12 (1H, d, J 5.20 (1H, in), 5. 02- 4.72 (21H, mn, masked by H 2 4.59-4.32 (3H, in), 3.48- 3.29, 3.08-2.75, 2.50-2.41, 2.31-2.22, 2.08-1.89, 1.72- 1. 63 (1 1H, 6m) 2. 95 (3H, s) 0e S 0e 00 0S S 5@

SO

t6*0e0

S

@6 a 0e 0 0ss 0 see...

0 seed a *0*O see.

0 S S. S *5 5 6

C.

S. C Cd Cd 506a-c,g compound R 1 506a PhC(O)- 507a 506b MeS(O) 2 7b 506c MeOC 507c 506g

CH

3 C 507g 507a-c,g (506a). A solution of 212e (321mg, 0.929inmol) and (3S) t-butyl 3-amino-5-diazo-4-oxopentanoate (198mg, 0.929mmo1) in dichloromethane (3m1) was cooled to 0* and N,N-diisopropylethylamine (O.16m1, l.S6mmol) and (1H-benzotriazol-l-yl) 3-tetramethyl-uronium tetrafluoroborate (328mg, 1.O2mmol) were added. The solution was stirred overnight at room temperature, diluted with ethyl acetate and washed with 1M NaI{S0 4 aqueous NaHCO 3 brine, dried over magnesium sulphate and evaporated. Chromatography on silica gel eluting with ethyl acetate gave 506a (425mg, 85%) as a 240 23 colourless foam: [a]D -124.90 (C 0.2, CH 2 C1 2

IR

(KBr) 3332, 2111, 1728, 1658, 1532, 1421, 1392, 1367, 1279, 1256, 1155; 1H NMR (CDCI 3 67.82 (2H, 7.49 (3H, 7.28 (iH, d, J 7.05 (1H, d, J 7.3), 5.06 (IH, 5.18 (2H, 4.78 (1H, 4.62 (1H, m), 3.29 (IH, 3.08-2.79 (3H, 2.58 (1H, dd, J :16.8, 2.20-1.85 (4H, 1.70 (1H, 1.45 (9H, MS (ES 539.58 (M 1, 97.9%) 529.59 (100).

(506b) was prepared by a similar method as compound 506a. 74% as yellow orange solid: mp. 75 0 C (decomp.); [aiD -92.0' (c 0.036, CH 2 C1 2 IR (KBr) 3438, 2904, '"2113, 1728, 1669, 1523, 1368, 1328, 1155; H NMR (CDC1 3 67.48 (IH, d, J 8.1) 5.83-5.68 (1H, 5.55-5.50 (1H, 5.43-5.14 (1H, 4.83-4.45 (3H, 3.40-3.19 (1H, 2.98 (3H, 2.92-2.30 (4H, m), 2.24-1.70 (6H, 1.43 (9H, s).

(506c) was prepared by a similar method as compound go a 506a to afford a pale yellow foam (405mg, []D -1440 (c 0.2, CH 2 C1 2 IR (KBr) 3339, 2978, 2958, 2112, 20 1728, 1674, 1530, 1459, 1415, 1367, 1274, 1252, 1154, o" 1063; IH NMR (CDC1 3 )6 7.23 (1H, d, J 5.51-5.31 (2H, 5.21-5.16 (1H, 4.77-4.55 (3H, 3.68 (3H, 3.35-3.18 (1H, 3.04-2.51 (4H, 2.40- 2.30 (iH, 2.09-1.66 (5H, 1.45 MS (ES 493.

(506g) was prepared by a similar method as compound 506a. 81%: [a]D 2 8 -146.7o (c 0.4, CH 2 C1 2 IR (KBr) 3438, 2904, 2113, 1728, 1669, 1523, 1368, 1328, 1155; IH NMR (CDCI 3 67.32 (1H, 6.43 (1H, 5.50 (1H, 5.22 (1H, 4.94 (1H, 4.77 (1H, 4.60 (1H, 3.24 (1H, 3.03-2.52 (4H, 2.36 (IH, m), 241 O* *P *Q

*O

2.10-1.64 (5H, m) 2.02 (3H, 1.45 (9H, s) Anal.

Calcd for C21H20N607: C, 52.69; H, 6.32; N, 17.05.

Found: C, 52.51; H, 6.27; N, 17.36. MS (ES 477(M 1, 100%) (507a). 506a (3.0g, 5.55mmol) in dry dichloromethane was cooled to 0" and 30% hydrobromic acid in acetic acid (l.lml, 5.55mmol) was added dropwise over 4min. The mixture was stirred at 0' for 9min and quenched with aqueous sodium bicarbonate. The product was extracted into ethyl acetate, washed with aqueous sodium bicarbonate, brine, dried (MgS04) and evaporated to give 2.97g of a colourless foam: [a]D -82.3° (c 0.23, CH2C12); IR (KBr) 3333, 1726, 1659, 1530, 1458, 1447, 1422, 1395, 1368, 1279, 1256, 1222, 1155, 15 728; 1H NMR (CDCI3) §7.81 (2H, m) 7.50 (3H, m) 7.11 (1H, d, J 7.01 (1H, d, J 5.20 (2H, m), 5.00 (1H, 4.06 (2H, 3.28 (1H, 3.20-2.70 (4H, 2.42 (1H, 2.10-1.85 (4H, 1.72 (1H, m), 1.44 (9H, Anal. Calcd for C26H33N407Br.0.7H20: C, 51.53; H, 5.72 N, 9.24. Found: C, 51.55; H, 5.52; N, 9.09. MS (ES 595, 593 1).

(507b) was prepared by a similar method as compound 507a. as an orange foam: -135° (c 0.053, CH2C12); IR (KBr) 3429, 2944, 2935, 1723, 1670, 1458, 1408, 1327, 1225, 1154, 991; 1H NMR (CDCI3) 67.38 (1H, d, J 5.69 (1H, d, J 5,43-5.34 (1H, m), 5.07-4.97 (1H, 4.70-4.42 (2H, 4.12 (2H, s), 3.35-3.17 (1H, 3.10-2.69 (4H, 2.98 (3H, s), 2.43-2.33 (1H, 2.15-1.65 (5H, 1.43 (9H, s).

Anal. Calcd for C2oH31BrN408S: C, 42.33; H, 5.51; N, 9.87. Found: C, 42.69; H, 5.52; N, 9.97.

242 (507c) was prepared by a similar method as compound 507a to afford a pale yellow foam (320mg, [aID 2 0 -107' (c 0.2, CH 2 C1 2 IR (KBr) 3401, 2956, 1726, 1670, 1528, 1452, 1415, 1395, 1368, 1276, 1251, 1155, 1064; IH NMR (CDCl 3 )6 7.07 (1H, d, J 5.47 (IH, d, J 5.21-5.16 (1H, 5.03-4.94 (1H, 4.75-4.56 (2H, 4.06 (2H, 3.69 (3H, 3.31-3.13 (1H, m), 3.03-2.92 (2H, 2.81-2.58 (2H, 2.41-2.31 (1H, 2.10-1.66 (5H, 1.44 (9H, s).

S• 10 (507g) was prepared by a similar method as compound 507a to afford a pale yellow foam [a]D 22 -109.60 (c 0.1, CH 2 C1 2 IR (KBr) 3324, 1727, 1659, 1535, 1458, 1444, 1423, 1369, 1279, 1256, 1223, 1155; IH NMR (CDCI 3 )6 7.12 (1H, d, J 6.33 (IH, d, J 5.19 (1H, 4.97 (2H, 4.58 (1H, 4.06 .0000. (2H, 3.20 (1H, 3.05-2.69 (4H, 2.35 (1H, m), 2.14-1.68 (5H, 2.03 (3H, 1.44 (9H, Anal.

Calcd for C 21

H

31 BrN 4 07.0.3H 2 0: C, 46.99; 5.93; N, 10.44. Found: C, 46.97; H, 5.90; N, 10.35.

0 0 N 0 N 0 *0 0~ NOtBuO 0 OH 0 MeOw f MeO 00 N O R H 0 H 0 508a, b 284, 285 compound R C1 508a 284 cI 243 Me 508b Me (508a). To a solution of 506c (547mg, Immol) in DMF (4ml) was added potassium fluoride (145mg, 2.5mmol, equiv). After 10min stirring at room temperature, 2,6dichlorobenzoic acid (229mg, 1.2mmol, 1.2 equiv) was added. After 3h reaction at room temperature, ethyl acetate (30ml) was added. The solution was washed with

S.:

e a saturated solution of sodium bicarbonate 10 brine, dried over MgSO 4 and concentrated in vacuo to see afford 590mg of a pale yellow foam: [a]D 22 -850 (c 0.20, CH 2 C1 2 IR (KBr) 3400, 2956, 1737, 1675, 1528, 1434, 1414, 1368, 1344, 1272, 1197, 1152, 1061; SIH NMR (CDC1 3 67.36-7.33 (3H, 7.04 (1H, d, J 5.46 (1H, d, J 5.19-5.16 (1H, 5.08 o. (2H, AB), 4.97 4.55 (1H, 4.69-4.55 (2H, 3.68 (3H, 3.30-3.10 (1H, 3.01-2.50 (4H, 2.40- 2.33 (1H, 2.15-1.60 (5H, 1.44 (9H, Anal.

Calcd for C 28

H

34

C

2

N

4 0 10 C, 51.15; H, 5.21; N, 8.52.

20 Found: C, 51.35; H, 5.32; N, 8.56.

(284) was synthesized from 508a via method used to prepare 505 from 504 which afforded 330mg of a white solid: mp. 115 °C (decomp.); [a]D20 -1070 (c 0.2,

CH

2 C1 2 IR (KBr) 3340, 2954, 1738, 1664, 1530, 1434, 1272, 1198, 1148, 1060; H NMR (D 6 -DMSO) 6 8.91 (1H, d, J 7.2H), 7.67-7.63 (3H, 7.54 (1H, d, J 5.24 (2H, 5.20-5.15 (1H, 4.79-4.70 (1H, m), 4.46-4.37 (2H, 3.58 (3H, 3.33-3.20 (1H, m), 2.94-2.55 (4H, 2.30-1.60 (6H, Anal. Calcd for 244

C

24

H

26

C

1 2

N

4 0 10 oH 2 0: C, 46.54; H, 4.56; N, 9.05. Found: C, 46.36; H, 4.14; N, 8.88.

(508b) was synthesized by a similar method as compound 508a to afford a pale yellow foam (460mg, [c]D 22 -115° (c 0.20, CH 2 C1 2 IR (KBr) 3413, 2960, 1729, 1675, 1528, 1514, 1461, 1421, 1368, 1265, 1116, 1096; H NMR (CDC1 3 6 7.27-7.03 (4H, 5.48 (1H, d, J

C*

5.20-5.14 (1H, 5.04 (2H, AB), 4.93-4.86 (1H, 4.80-4.56 (2H, 3.77 (3H, 3.32-3.15 (1H, m),

S

0 00 10 3.00-2.56 (4H, 2.37 (6H, 2.19-1.77 (5H, m), 1.45 (9H, 2.41-2.25 (1H, MS (ES 617.

6 (285) was synthesized by a similar method as compound 284 to afford a white solid (303mg, mp. 110"C 20 (decomp.); [a]D -1280 (c 0.10, CH 2 C1 2 IR (KBr) 3339, 15 2958, 1731, 1666, 1529, 1420, 1266, 1248, 1115, 1070; o. NMR (D 6 -DMSO) 5 8.90 (1H, d, J 7.54 (1H, d, J 7.36-7.28 (1H, 7.17-7.14 (2H, 5.19-5.15 .o (3H, 4.84-4.74 (1H, 4.45-4.37 (2H, 3.59 (3H, 3.45-3.25 (1H, 2.95-2.64 (4H, 2.35 20 (6H, 2.30-1.60 (6H, Anal. Calcd for

.C

26

H

32

N

4 0 10

*H

2 0: C, 53.98; H, 5.92; N, 9.68. Found: C, "o 53.50; H, 5.52; N, 9.49. MS (ES 559.

509a-d 510a, 280, 283, 510d 245 0 0@0 0 0 compound R 9a/

I

510 Sa 9b 280

N-

~N

509C 283 0 9dN 510 OdI (510a). A solution of 506a (2.27g, 4.2Inmol) in dry dichioromethane (50mi) was treated with 30% hydrobromic acid in acetic acid (1.84m1, 9.2minol, 2.2equiv) at 0*C, under nitrogen. After 10mmn stirring at 0*C the reaction was complete and a white solid crystallised in the medium. The solid was filtered and washed with ethylacetate and diethylether to afford 2.20g (100%) of [3S(lS,9S)] 5-bromo-3- (9-benzoylamino-6, lO-dioxo- 1,2,3,4,7,8,9, 10-octahydro-6Hpyridazino[1,2-a] [1,2]diazepine-1-carboxamido)-4oxopentanoic acid which was used without further purification: 1H NMR (D 6 DMS0) 6 8. 87 (1 H, d, J 8.63 (1H, d, J 7.91-7.87 (2H, in), 7.60-7.44 (3H, in), 6.92 (1H, bs), 5.14 -5.09 (1H, in), 4.92-4.65 (2H, in), 4.43 (2H, AB), 4.41-4.35 (1H, in), 3.33-3.22 in), 2.98-2.90 (1H, in), 2.89-2.57 (2H, in), 2.35- 2.15 (3H, in), 1.99-1.91 (2H, in), 1.75-1.60 (2H, in). A 246 solution of the bromoketone (535mg, Immol) in dry DMF was treated with potassium fluoride (150mg, 2.5 equiv), under nitrogen. After stirring at room temperature, 2-mercaptothiazole (140mg, 1.2mmol, 1.2equiv) was added. After overnight reaction ethylacetate (150ml) was added and the organic solution was washed with brine, dried over magnesium sulphate and reduced in vacuo. The residue was crystallised in diethyl ether, filtered and purified on 10 silica gel using a gradient of MeOH to in dichloromethane. Evaporation afforded 344mg of a white solid: mp. 90-95 OC (decomp.); [a]D 2 0 -820 (c 0.2, CH 2 C12); IR (KBr) 3328, 2941, 1745, 1659, 1535, 1422, 1276, 1255, 1223, 1072; 1H NMR (D 6 -DMSO) 6 8.92 S 15 (1H, d, J 8.68 (1H, d, J 7.98-7.90 (2H, 7.75-7.67 (1H, 7.64-7.50 (4H, 5.22-5.18 (1H, 4.95-4.74 (2H, 4.58-4.38 (3H, 3.52- 3.19 (1H, 3.05-2.65 (4H, 2.40-1.50 (6H, m).

Anal. Calcd for C 2 5

H

2 7

N

5 0 4 S2-H 2 0: C, 50.75; H, 4.94 N, 11.84. Found: C, 51.34; H, 4.70; N, 11.58. MS (ES 572.

(509b). 507a (100mg, 0.17mmol) in dry dimethylformamide (1.5ml) was treated with 1-phenyl-lH- (33mg, 0.187mmol) and potassium fluoride (15mg, 0.34mmol). The mixture was stirred at room temperature for 2h, diluted with ethyl acetate, washed with aqueous sodium bicarbonate brine, dried (MgSO 4 and evaporated. The product was purified by flash chromatography on silica gel eluting with ethyl acetate to give 103mg as a colourless foam: [a]D 2 3 -92.20 (c 0.1, CH 2 C1 2 IR (KBr) 3334, 1726, 1660, 1528, 1501, 1417, 1394, 1368, 1279, 1253, 1155; 247 H NMR (CDCI 3 67.82 (2H, 7.60-7.40 (8H, 7.39 (1H, d, J 7.05 (1H, d, J 5.26 (1H, m), 5.15 (1H, 4.99 (1H, 4.60 (2H, 4.30 (1H, d, J 17.2H), 3.32 (1H, 3.10-2.75 (4H, 2.40 (1H, 2.24 (1H, 1.90 (3H, 1.75 (1H, 1.44 (9H, MS (ES 691.47 (M 1).

(280) was synthesized via method used to prepare 505 S* from 504. 509b (98mg, 0.142mmol) in dichloromethane (1ml) was cooled to 0' and trifluoroacetic acid (!ml) 10 was added. The mixture was stirred at 0" for 15min and at room temperature for 30min before evaporation under reduced pressure. The residue was triturated with dry toluene and evaporated. Chromatography on silica gel eluting with 10% methanol in dichloromethane gave a 15 colourless glass which was crystallised from dichloromethane/diethyl ether to give 62mg of 22 colourless solid: mp. 145 0C (decomp.);[X]D -80.9° (c 0.1, CH 2 C1 2 IR (KBr) 3400, 1727, 1658, 1530, 1501, 1460, 1445, 1416, 1280, 1254; H NMR (CDCl 3 68.00 (1H, 7.79 (2H, d, J 7.58-7.30 (9H, 5.25 (2H, 4.94 (1H, 4.53 (2H, 4.35 (1H, 3.35 (1H, 3.01 (3H, 2.73 (1H, 2.38 (1H, 1.98 (4H, 1.64 (1H, m) Anal. Calcd for !C 2 9

H

30

N

8 0 7 S*0.2TFA: C, 53.71; H, 4.63 N, 17.04. Found: C, 53.97; H, 4.92; N, 16.77. MS (ES 633.55 (M 1).

(509c) was prepared by a similar method as compound 22 1 509b to afford a colourless glass [a]D -77.10 (c 0.25, CH 2 C1 2 IR (film) 3311, 1724, 1658, 1603, 1578, 1536, 1488, 1458, 1426, 1368, 1340, 1279, 1256, 1231, 1155, 707; 1 H NMR (CDCl 3 6 8.29 (2H, 7.84 (2H, 7.48 (4H, 7.22 (3H, 5.20 (2H, 4.90 (2H, 4.58 (1H, 3.29 (1H, 3.20-2.70 (4H, m), 248 2. 38 (2H, mn), 1. 96 (4H, in), 1. 68 (1H, in), 1. 42 s).

MS 608.54 (M 1).

(283) was prepared by a similar method as compound 280 to afford a colourless foam mp. -125 00; [IOD'1 9 -84.10 (c 0.1, 20% MeOH/CH 2 Cl 2 IR (KBr) 3401, 1736, 1663, 1538, 1489, 1459, 1425, 1281, 1258, 1200, 1134; 1 H NMR (CDO/Cl) 5 8.38 (2H, in), 7.84-7.40 (8H, in), 5.16 (4H, in), 4.80 (iH, mn), .4.56 (1H, in), 3.50 (1H, mn), 3.12 (2H, in), 2.82 (2H, mn), 2.37 (1H, mn), 2.10-1.65 10 (5H, mn). Anal. Calcd for C 2 7

H

2 9

N

5 0 8 *0.4H 2 0: C, 51.77; H, 4.61; N, 10.41. Found: C, 52.19; H, 4.93; N, 9.99.

000 (509d) was synthesized by a similar method as compound 509b to afford a colourless solid (49.6mg, 1 H NMR (CDC1 3 6 8.02 (iH, 7.95-7.86 (1H, in), 7.84-7.76 15 (2H, in), 7.62-7.35 (4H, in), 7.22-7.07 (1H, in), 6.43 (iH, 5.26-5.08 (2H, mn), 5.03-4.72 (3H, in), 4.66- 4.50 (1H, in), 3.43-3.19 (1H, mn), 3.15-2.97 (1H, in), 2.86-2.72 (3H, in), 2.48-2.31 (1H, mn), 2.18-1.60 (6H, in), 1.43 (9H, s).

(510d) was synthesized by a similar method as compound 0 280 to afford a colourless solid (25.7mg, 57%) inp.

0 140-80*C; IR (KBr) 3391, 2945, 1733, 1664, 1530, 1422, 1363, 1277, 1259, 1204; 1H NMR (CD 3 OD) 6 8.23 (1H, s), 7.94 (1H, 7.87 (2H, 7.54-7.42 (3H, in), 6.48 (1H, 5.22-5.15 (1H, in), 4.57-4.46 (iN, in), 3.62- 3.41 (1H, in), 3.22-3.13 (1H, in), 3.02-2.81 (2H, mn), 2.70-1.80 (6H, m) Anal. Calcd for C 26

N

28

N

6 0 8 *1.5N 2 0: C, 54.30; H, 5.35; N, 14.61. Found: C, 54.14; H, 5.35; N, 13.04. MS (ES 551 (M 1, 100%). Accurate mass calculated for C 2 6

H

2 9

N

6 0 8 553.2047. Found: 553.2080.

249 Hb01 505f, 280b, 283b 504ff-h S@ S

S.

0*

S

0 0eSeeO 0 S0 SO S *0

S

000

S

S

S

S. compound R 0 504ff

NT~I~

505ff

N

0N 504h 283b (505ff) was prepared by a similar method as compound 508a using 507b and 3-chloro-2-hydroxy-4Hpyridolll,2-a~pyrimidin-4-one and directly followed by the hydrolysis of 504ff with trifluoroacetic to afford a tan powder (65mg, [a]D 20 l28* (c 0.10, MeOH); IR (KBr) 3414, 2928, 1667, 1527, 2459, 1407, 1328, 1274, 1153, 1134; 1H NNR (MeOD) 8 9.35 (1H, d, J 6.6H), 8.34 (1H, t, J 7.2H), 7.99-7.95 (1H, in), 7.76-7.69 (1H, mn), 5.85-5.45 (3H, in), 5.30-5.21 (1H, in), 4.93-4.66 (2H, mn), 3.81-3.65 (1H, in), 3.66 (3H, in), 3.45-2.52 (4H, in), 2.52-1.71 (6H, in). D.J. Hlasta et al., J.

Med. Chemn. 1995, 38, 4687-4692.

0* S S

S.

5@ 0 5 5

OS

250 (504g) was prepared by a similar method as compound 23.7 509b, as a colourless foam: [a]D -112.7' (c 0.2, CH 2 C1 2 IR (KBr) 3312, 1726, 1668, 1501, 1413, 1395, 1369, 1328, 1276, 1254, 1155; 1H NMR (CDC1 3 67.59 (5H, 7.48 (1H, d, J 5.68 (1H, d, J 5.37 (1H, 4.95 (1H, 4.62-4.31 (4H, 3.36 (1H, 2.98 (3H, 2.88 (4H, 2.66 (1H, 2.42 (2H, 1.98 (1H, 1.75 (IH, 1.43 (9H,s).

(280b) was prepared by a similar method as compound 10 280, (100%) as a colourless foam: mp. 120-5 0 C; [c]D -112.40 (c 0.1, CH 2 C1 2 IR (KBr) 3328, 1730, 1664, 1

H

1529, 1501, 1410, 1328, 1277, 1219, 1153, 1134, 991; H NMR (CDC1 3 68.07 (1H, d, J 7.58 (5H, 6.41 (1I, d, J 5.32 (iH, 5.04 (1H, 4.70 (1H, d, J 17.5), 4.60 (3H, 3.50-2.9 (3H, 2.98 (3H, 2.45 (2H, 2.06 (4H, 1.68 (1H, m).

(504h) was prepared by a similar method as compound goo: 02 509b as a colourless foam: [(ID 23 -101.00 (c 0.2, CH 2 C1 2 IR (KBr) 3330, 1727, 1669, 1425, 1396, 1369, 1328, 1276, 1256, 1231, 1155, 1137, 991; 1H NMR (CDCl 3 68.28 (2H, br d, J 7.71 (1H, d, J 7.22 (2H, 6.03 (1H, d, J 5.36 (1H, 4.95 (2H, 4.52 (2H, 3.29 (1H, 3.07 (3H, 3.23-2.75 (3H, 2.66-2.35 (2H, 2.30-1.60 (5H, 1.42 (9H, s).

(283b) was prepared by a similar method as compound 280, (100%) as a colourless foam: mp. 120-5 0 C [a]D 2 -85.20 (c 0.1, 10% CH 3

OH/CH

2 Cl 2 IR (KBr) 3337, 1738, 1667, 1560, 1457, 1424, 1326, 1317, 1278, 1258, 1200, 1189, 1150, 1133, 991; IH NMR (CDC1 3

/CD

3 OD) 68.35 (2H, 7.54 (2H, 5.32 (2H, 4.83 (2H, 4.45 (2H, 251 mn), 3. 43-2.77 (4H, in), 2. 97 (311, s) 2. 42 (211, n), 2.05-1.72 (5H, in).

0 0 MeO JLN OtBu 0 M-k 0O H R H R H 0 H 0 508c-e 511c, 280c, 283c 00 6*60@ 0 0 0 00 0 00 0O compound R 508C 280c

I

0'

N

508e 283c N (508c) was prepared by a similar method as compound 509b to afford 544mg of a pale yellow foam: [aID 20_860 (C 0.19, CH 2 Cl 2 IR (KBr) 3426, 2947, 1725, 1669, 1551, 1418, 1383, 1253, 1155, 1064; 1H NMR (CDCl 3 5 8.49 (2H, d, J 7.13 (1H1, d, J 7.9), 7.03-6.98 (1H, in), 5.47 (1H, d, J 5.23-5.19 (1H, in), 5.09-5.01 (1H1, in), 4.84-4.51 (2H, in), 4.04 (2H, AR), 3.69 (3H, 3.38-3.19 (1H1, in), 3.06-2.64 (4H, in), 2.40-1.76 (611, in), 1.43 (9H, Anal. Calcd 252 *oo& 04 0.

0 0 00*00* 4 00

*SSS

000 0 0 6 0

OS

00* *5 for C 2 5

H

3 4

N

6 0 8 S: C, 51.89; H, 5.92; N, 14.52. Found: C, 51.49; H, 6.04; N, 13.87. MS (ES 579.

(511c) was prepared by a similar method as compound 280 to afford 370mg of a white powder: mp. 105 °C 22 (dec); [a]D -94° (c 0.20, CH 2 C1 2 IR (KBr) 3316, 3057, 2957, 1724, 1664, 1252, 1416, 1384, 1254, 1189, 1063; H NMR (D 6 -DMSO) 68.85 (1H, d, J 8.62 (2H, d, J 7.53 (1H, d, J 7.28-7.23 (1H, 5.21-5.17 (1H, 4.87-4.79 (1H, 4.47-4.35 10 (2H, 4.23 (2H, AB), 3.58 (3H, 3.30-3.21 (1H, 2.95-2.50 (4H, 2.35-1.60 (6H, Anal. Calcd for C 21

H

26

N

6 0 8

S-H

2 0: C, 46.66; H, 5.22; N, 15.55.

Found: C, 46.66; H, 5.13; N, 15.07. MS (ES 523, (ES 521.

15 (508d) was synthesized by a similar method as compound 509b to afford a colourless solid (269mg, mp. 23 110 [a]D 2 3 -108 (c 0.60 CH 2 C1 2 IR (KBr) 3315, 2977, 1727, 1688, 1527, 1501, 1458, 1418, 1368, 1279, 1250, 1155, 1064; H NMR (CDCl 3 67.70 (1H, 7.63- 20 7.53 (5H, 5.84 (1H, 5.34-5.27 (1H, 5.05- 4.92 (1H, 4.78-4.54 (3H, 4.38 (1H, 3.66 (3H, 3.37-3.19 (1H, 3.07-2.94 (1H, 2.91- 2.82 (2H, 2.71-2.56 (1H, 2.40-2.30 (1H, m), 2.19-2.13 (1H, 2.08-1.68 (4H, 1.42 (9H, MS (ES 667 645 1, 100), 589 (62).

(280c) was synthesized by a similar method as compound 280 to afford a pale cream solid (203mg, mp. 105- 22 130 [M]D -235° (c 0.11 MeOH); IR (KBr) 3342, 2951, 1727, 1667, 1529, 1501, 1459, 1416, 1276, 1252, 1225, 1192, 1062; 1H NMR (D 6 -DMSO) 6 8.89 (1H, 7.69 7.50 (1H, 5.18-5.11 (1H, 4.79-4.69 (1H, 4.57 (2H, 4.42-4.32 (1H, 3.54 (3H, s), 253 2.92-2.63 (3H, 2.21-1.82 (5H, 1.65-1.57 (1H, MS (ES 587 (M 1, 100%).

(508e) was synthesized by a similar method as compound 509b to afford a pale orange solid (199mg, mp.

23 80-120 0 C; [a]D _89' (c 0.51 CH 2 C1 2 IR (KBr) 3333, 2978, 1726, 1669, 1578, 1536, 1478, 1426, 1368, 1277, 1253, 1232, 1155, 1064; IH NMR (CDCI 3 58.41-8.18 (2H, 7.81 (1H, 7.26-7.20 (2H, 5.91 (1H, d), Ce 5.24-5.16 (IH, 5.07-4.86 (3H, 4.81-4.51 (2H, 10 3.67 (3H, 3.34-3.16 (1H, 3.10-2.81 (3H, m), 0:o 2.72-2.54 (1H, 2.41-2.31 (1H, 2.07-1.62 1.47 (9H MS (ES 562 (M 1, 100%), 506 (38).

(283c) was synthesized by a similar method as compound 15 280 to afford an off-white powder (167mg, mp. 6 22 *6 E,.105 [a]D -106* (c 0.11 MeOH); IR (KBr) 3325, 3070, 2956, 1669, 1544, 1423, 1256, 1199, 1133, 1062; H NMR (D 6 -DMSO) 5 8.95 (1H, 8.45-8.20 (2H, m), 7.53-7.45 (3H, 5.19-5.08 (3H, 4.70-4.62 (1H, 4.41-4.30 (2H, 3.53 (3H, 2.92-2.68 (3H, m), a 0 S "2.22-2.06 (2H, 1.95-1.82 (2H, 1.63-1.53 (1H., MS (ES 506 (M 1, 100%).

0 0 Y (,OtBu go Me OH Me eW l 512a, 512b 280d, 283d 254 compound R

Q_

280d S 4,N I 512b 283d

OS

00 *0 a a.

SO

a a *9 0 0 *000 0e 0e 0*0 a *0ae06 a a a 0000 a aa 0* a a.

Oa a. a 00 5 (512a) was prepared by a similar method as compound 509b, to afford as a colourless foam: II2 -129.60 (c 0.1, CH 2 Cl 2 IR (KBr) 3323, 1726, 1664, 1531, 1501, 1444, 1415, 1394, 1369, 1279, 1254, 1156; 10 1 H NMR (CDCl 3 567.59 (5H, s) 7.37 (1H, d, J 7. 9) 6.38 (1H, d, J 5.27 (1H, in), 4.98 (2H, in), 4.58 (2H, d in), 4.28 (1H, d, J 17.2), 3.28 (1H, in), 3.10-2.65 (4H, in), 2.31 (2H, mn), 2.03 (3H, 2.10- 1.72 (4H, in), 1.48 (9H, s).

(280d) was prepared by a similar method as compound 280, to afford as a colourless foam: [c]D 2 2 9330 (c 0.1, CH 2 Cl 2 IR CKBr) 3316, 1728, 1659, 1531, 1501, 1415, 1341, 1278, 1253, 1222, 1185; 1H NIMR (CDCl 3 58 -05 (1H, d, J 7.57 (5H, br s) 5.30 (1H, mn), 5.01 (2H, in), 4.70-4.10 (4H, in), 3.40-2.85 (4H, mn), 2.62 (1H, mn), 2.33 (1H, mn), 2.27-1.65 (5H, mn), 2.01 (3H, s).

(512b) was prepared by a similar method as compound 509b, to afford as a colourless foam: IR (KBr) 3333, 1727,.1661, 1542, 1427, 1369, 1279, 1257, 1232, 1156; 1H NMR (CDCl 3 6 8. 30 (2H, in), 7.20 (3H, in), 6. (1H, d, J 5.17 (1H, in), 4.91 (3H, in), 4.55 (1H, 255 in3.27 (1H, mn), 3.14-2.70 (4H, mn), 2.41 (1H, mn), 2.04 (3H, 2.10-1.65 (6H, in), 1.44 (9H, s).

(283d) was prepared by a similar method as compound 280. (100%) as a colourless foam: [aID 22_106.0* (c 0.2, 10% CH 3

OH/CH

2 Cl 2 IR (KBr) 3312, 1735, 1664, 1549, 1426, 1279, 1258, 1200, 1135; 1H NMR (CDCl 3 568.27 (2H, in), 7.46 (2H, in), 5.09 (1H, in), 4.79 (3H, in), 4.47 (1H, mi), 3.40 (1H, in), 3.30-2.70 (3H, in), 2.54 (1H, in), 2.30 in), 1.98 (3H, 2.05-1.65 (4H, in).

00 0 00 H N H H 0 1025b26 rpre rm 1,R 9Bnoyaio 0, ,9 1 -c a y ro 1 -x -H pyiaio12-]12daepn--abxyi cdb th ehddecie or25t ffr 1m cooresfaS-: itreo iseeioes: 1119b) wa prpae fromD13 77 ,97.en-.oyla 1Hino- 4.8 (1H, 4.ocadr-45 (15,-o,4.o-6H-m, .3 m) 2.yr 0zi111,2-a azepin- fcroyi cdb (K2r)3392, 3052, 294, 12.72, 162 152, 150,9 1454,: 256 C, 63.10; H, 6.16; N, 10.21. MS (ES 521 (M 1, 100%).

(246b) was prepared from 245b by the method described for 246 to afford 104mg of a white powder: mp.

24 115-119 [a]D -19.8° (c 0.2 MeOH); IR (KBr) 3293, 2944, 1786, 1639, 1578, 1537, 1489, 1450, 1329, 1162, 1124; H NMR (CD 3 OD) 67.85 (2H, d, J 7.49 (3H, 5.49 (1H, 4.55 (1H, 4.30 (2H, 3.40 (1H, 3.19-2.89 (3H, 2.63 (2H, 2.16-1.81 (5H, m), 0@SeS* 10 1.60 (3H, Anal. Calcd for C 21

H

26

N

4 0 6

.H

2 0: C, 56.24; H, 6.29; N, 12.49. Found: C, 56.54; H, 6.05; N, 12.29.

MS (ES 429 (M 1, 100%) Compounds 513a-j were prepared as described below.

o 0 0 1 0 N o

H

15 513a-f O0 S S

OS

2

SS

compound R 513a° 513a -1 513a-2 513b 513b-1 257 0* 0 00 00 S0

S

@0 S S 513b-2 0 513c 513d 513e 513ff 513f-1 513f-2 0 0 OtBu 0 H H 0

S

6000 @500

S

00 0 0@

S

05 0* S 00 513g 0 0 513h 258 'OBu 0 51 3i 0O 0 S@ H *513j (53a wa.rprdb. iilrmto scmon 513/e o ffod amitur 0 OiaterOmr Me7mg 500)a a i :IR(5)331 96,19,103 73 1531 1329 127 16, 10,160 77 37 01 *M (C C 3 8S.67 8 (H 9 3 (H S 1 5.34 m) 5.28- .18 m 4 5 6 (H m) (513b) was prepared byas similtoarfetord as comp)ond 51det fodamixture of diastereoisomers asacla67l 0mg,13 50)5 as anoil: IR (KBr) 331, 296, 170, 173,173 1215312, 132, 1257 1120, 1060 97 937 01; (CDCl 3 57.362-7.180 (5H, in), 5.993-5.836 (2H, in), 5.41- 5.34 (2H, in), 5.258 (2H, .59- 4.56.4 (2H,.in, 4.32-.3964.25 (0.5in)),2-.6-2(3H.

1in, 2.49- 32.3 in).5-.0(8,m nl 259 Calcd for C 1 3

H

1 9

NO

5 C, 57.98; H, 7.11; N, 5.20. Found: C, 56.62; H, 7.22; N, 4.95. MS (ES 270.

(513c) was synthesized by a similar method as compound 513d/e to afford a single isomer as a pale yellow oil: [aD 2 4 -63.10 (c 0.2, CH 2 C1 2 IR (film) 3338, 2948, 1791, 1723, 1529, 1421, 1330, 1253, 1122, 984, 929, 746; H NMR (CDCl 3 67.20 (4H, 5.87 (1H, m), 5.61 (1H, d, J 5.33-5.10 (2H, 4.70 (1H, m), 4.56 (3H, 3.33-3.19 (2H, 3.10-2.94 (2H, m), 10 2.81 (1H, dd, J 8.3, 17.3), 2.43 (1H, dd, J 10.5, 17.3).

(513d) and (513d/e) were prepared [via method described by Chapman Biorg. Med. Chem. Lett., 2, pp. 615-618 (1992)]. Following work-up by extraction with ethylacetate and washing with NaHCO 3 the product was dried (MgSO 4 filtered and evaporated to yield an oil which contained product and benzyl alcohol. Hexane (200ml) (200ml hexane for every 56g of AllocAsp(CO 2 tBu)CH 2 0H used) was added and the mixture stirred and cooled overnight. This afforded an oily solid. The liquors were decanted and retained for chromatography. The oily residue was dissolved in ethyl acetate and evaporated to afford an oil which was crystallised from 10% ethyl acetate in hexane (~500ml).

The solid was filtered to afford 513d (12.2g, mp.

108-110 [a]D 2 4 +75.720 (c 0.25, CH 2 Cl 2

IR

(KBr)3361, 1778, 1720, 1517, 1262, 1236, 1222, 1135, 1121, 944, 930, 760; 1H NMR (CDCl 3 67.38 (5H, 5.90 (1H, 5.50 (1H, 5.37 (0.5H, 5.26 (2.5H, m), 4.87 (1H, ABq), 4.63 (3H, 4.31 (1H, 3.07 (1H, dd), 2.46 (1H, dd). Anal. Calcd for C 1 5

H

1 7 NO5: C, 61.85; H, 5.88; N, 4.81. Found: C, 61.85; H, 5.89; N, 4.80.

260 The liquors were combined and evaporated to yield an oil (~200g) containing benzyl alcohol.

Hexane/ethyl acetate 100ml) was added and the product purified by chromatography eluting with ethyl acetate in hexane to remove the excess benzyl alcohol, and then dichloromethane/hexane (1:1 containing 10% ethyl acetate). This afforded 513e 2 containing some 513d (20.5g, mp. 45-48 OC; [a]D 24 S* -71.260 (c 0.25, CH 2 C1 2 IR (KBr) 3332, 1804, 1691, 1536, 1279, 1252, 1125,976. 1H NMR (CDC 3 67.38 5.91 (1H, 5.54 (1H, d, J 5.38 (3H, m); 4.90 (1H, ABq); 4.60 (4H, 2.86 (1H, dd); 2.52 (1H, dd). Anal. Calcd for C 15

H

17 N0 5 *0.1H 2 0 C, 61.47; H, 5.91; N, 4.78. Found: C, 61.42; H, 5.88; N, 4.81.

(513f) was synthesized by a similar method as 513d/e to afford a colourless oil (152mg, IR (film) 3334, 2983, 2941, 1783, 1727, 1713, 1547, 1529, 1422, 1378, 1 1331, 1313, 1164, 1122, 1060, 938; H NMR (CDCl 3 66.09- 5.82 (2H, 5.50-5.18 (3H, 4.64-4.54 (2H, m), 4.27-4.16 (1H, 3.95-3.78 (1H, 3.73-3.56 (1H, 3.05-2.77 (1H, 2.56-2.37 (1H, 1.35-1.17 (4H, Anal. Calcd for C 10

H

1 5 N0 5 C, 52.40; H, 6.60; N, 6.11. Found: C, 52.16; H, 6.62; N, 5.99. MS (ES 229 (M 1, 100%).

(513g). 4-Dimethylamino-pyridine (76.0mg, 622mmol) was added to a solution of 2-phenoxybenzoyl chloride (579mg, 2.49mmol) and 517 (600mg, 2.07mmol) in pyridine The mixture was stirred at room temperature for 18h before adding brine (25ml) and extracting with ethyl acetate (30ml, 20ml). The combined organic extracts were washed with 1M hydrochloric acid (3 x saturated aqueous sodium hydrogen carbonate (2 x 261 and brine (25ml), dried (MgSO 4 and concentrated.

The pale orange oil was purified by flash column chromatography (1-10% acetone in dichloromethane) to afford 447mg of colourless oil: IR (film) 3375, 2980, 1721, 1712, 1602, 1579, 1514, 1484, 1451, 1368, 1294, 1250, 1234, 1161, 1137, 1081, 754; 1H NMR (CDCl 3 67.98-7.93 (1H, 7.50-7.41 (1H, 7.35-7.25 (2H, 7.22-7.03 (3H, 6.95 (3H, 5.95-5.76 (1H, m), 5.57 (1H, 5.30-5.13 (2H, 4.51 (2H, 4.25 10 (2H, 4.18-4.04 (1H, 3.88 (1H, 3.50 (1H, m), 2.51 (2H, 1.41 (9H, MS (ES 508 503 486 (M 1, 45), 468 412 (100). Accurate mass calculated for C 2 6

H

3 2 N0 8 (MH 486.2128. Found: 486.2158.

(513h) was prepared from (3S,4R) t-butyl (Nallyloxycarbonyl)-3-amino-4,5-dihydroxypentanoate by the method described for 513g to afford 562mg of a colourless oil: IR(film) 3418, 2980, 1722, 1711, 1512, 1368, 1278, 1245, 1198, 1157, 1139; 1H NMR (CDC1 3 68.90 (1H, d, J 8.21 (1H, dd, J 1.2, 8.04 (1H, d, J 7.89 (1H, dd, J 7.67-7.46 (3H, 5.88 (1H, 5.49 (1H, d, J 5.35-5.18 (2H, 4.57-4.46 (4H, 4.19 (2H, 2.67 (2H, 1.40 (9H, Anal. Calcd for

C

2 4

H

2 9 N0 7 C, 65.00; H, 6.59; N, 3.16. Found: C, 64.74; H, 6.56; N, 3.09. M.S. (ES 466 (M+Na, 100%), 444 39), 388 (44).

(513i) was synthesized by a similar method as compound 513g to afford a colourless oil (569mg, IR (film) 3400, 1723, 1712, 1584, 1528, 1489, 1443, 1367, 1276, 1232, 1190, 1161, 1098, 1074, 995, 755; 1 H NMR (CDC1 3 6 8.65-8.59 (1H, 7.84-7.66 (2H, 7.45-711 (5H, m), 262 7.05-6.97 (2H, 6.00-5.78 (1H, 5.54-5.14 (2H, 4.62-4.52 (2H, 4.42-4.32 (2H, 4.08-4.22 (2H, 2.78-2.47 (2H, 1.44 (9H, MS (ES 508 486 (M 1, 33. Accurate mass calculated for

C

2 6

H

3 2

NO

8 (MH 486.2128. Found: 486.2121.

(513j) was synthesized by a similar method as compound 513g to afford a pale orange oil (905mg, IR C (film) 3418, 3383, 2980, 1722, 1711, 1601, 1517, 1450, *1424, 1368, 1308, 1252, 1154, 1100, 994, 767, 698; 1 10 NMR (CDC1 3 67.62-7.55 (2H, 7.51-7.42 (3H, 5.98- 5.76 (1H, 5.33-5.18 (2H, 4.53 (2H, 4.18 (2H, 3.91 (1H, 3.80 (1H, 2.76 (3H, 2.50 (2H, 1.43 (9H, Anal. Calcd for

C

2 4

H

3 0

N

2 0 8 *0.5H 2 0: C, 59.62; H, 6.46; N, 5.79. Found: C, 59.46; H, 6.24; N, 5.72. MS (ES 497 475 1, 15), 419 (48).

o. 0 0 0 'OtBu 0 OtBu O" N' f OH

OO

H OH H 514 515 (514) was prepared by the method described in H.

Matsunaga, et al. Tetrahedron Letters 24, pp. 3009-3012 Cm 0 0 0 Ot~u 0 r^OtBu OH 5 0 N H OHH 517 516 (514) was prepared by the method described in H.

Matsunaga, et al. Tetrahedron Letters 24, pp. 3009-3012 (1983) as a pure diastereomer as an oil: [a]D 23 -36.9° (c 0.5, dichloromethane); IR (film) 2982, 2934, 263 1726, 1455, 1369, 1257, 1214, 1157, 1068; H NMR (CDCl 3 67.31 (5H, 4.10 (1H, q, J 4.05-3.75 (4H, 3.10 (1H, q, J 2.40 (2H, 1.42 (9H, 1.40 (3H, 1.34 (3H, s).

(516), 514 (3.02g, 9.00mmol) and 10% palladium on carbon (300mg) in ethanol (30ml) were stirred under hydrogen for 2h. The suspension was filtered through celite and a 0.45mm membrane and the filtrate Sconcentrated to give a colourless oil 515 (2.106g, which was used without purification. The oil (1.93g, 7.88mmol) was dissolved in water (10ml) and 1,4-dioxan and sodium hydrogen carbonate added (695mg, 8.27mmol).

The mixture was cooled to O'C and allyl chloroformate (1.04g, 919ml, 8.66mmol) added dropwise. After 3h the mixture was extracted with ether (2 x 50ml). The combined ether extracts were washed with water (2 x and brine (25ml), dried (MgSO 4 and concentrated to give a colourless oil. Flash column chromatography (10-35% ethylacetate in hexane) afforded a colourless solid (2.69g, mp. 64-5 [a]D 23 -210 (c 1.00,

CH

2 C1 2 IR (KBr) 3329, 1735, 1702; H NMR (CDCl 3 6 6.00-5.82 (1H, 5.36-5.14 (2H, 542 (1H, 4.56 (1H, 4.40-4.08 (2H, 4.03 (1H, m) 3.70 (1H, m), 2.52 (2H, 1.44 (12H, 2 x 1.33 (3H, Anal.

Calcd for C 16

H

27 NO6: C, 58.34; H, 8.26; N, 4.25. Found: C, 58.12; H, 8.16; N, 4.19; MS (+FAB) 320 41%), 274 216 (100).

(517). A solution 516 (2.44g, 7.41mmol) in 80% aqueous acetic acid (25ml) was stirred at room temperature for 24h then concentrated and azeotroped with toluene (2 x The residue was treated with brine (25ml) and extracted with ethylacetate (2 x 25ml). The organic 264 fractions were dried (MgSO 4 and concentrated to afford a colourless oil. Flash chromatography (20-80% ethyl acetate in dichioroinethane) gave a colourless solid (1.99g, 90%) inp. 74-5 [(XJD 25_l.30 (c

CH

2 Cl 2 IR (KBr) 1723, 1691; 1H NMR (ODC1 3 6 6. 02-5. 78 (2H, in), 5.35-5.16 (2H, mn), 4.55 (2H, 4.16-4.04 (2H, mn), 2.76 (2H, 3.56 (2H, in), 2.56 (2H, in), 1.43 (9H, Anal. Calcd for C 1 3

H

2 3 N0 6 C, 53.97; H, 8.01; N, 4.84. Found 53.79; H, 7.88; N, 4.81; MS(+FAB) 10 290 234 (100) Example Compounds 605a-j, 605m-q, 605s, 605t, and 605v were synthesized as described below.

S

0O

S.

5O 0 S S 0* a 5.55..

S

5* S S @000 So 555

S

so a so

S

.55.

0 0 0 @5 S 05 S S 55 S. 0 00 00

NO

2

R

2 Step A H Ste BOC-N- OH R 2 Se H 0

NH

2

R

2 BOC-N 4

HR

HO0

H-

BOCN>

NR

O OR 600 Step C-. Step D Compound no. R 600a/103 H CH 3 600b IH CH 2 Ph 600c ICH 3

CH

2 Ph 265 (600a/103).

Step A. (2S)-2-tert-Butoxycarbonylamino-3-(2nitrophenyl-amino)-propionic acid. (2S)-2-tert- Butoxycarbonylamino-3-aminopropionic acid (10 g, 49 mmol), 2-fluoronitrobenzene (5.7 ml, 54 mmol), and NaHCO 3 (8.25 g, 98 mmol) was taken into 130 ml of DMF and heated at 80 'C for 18 h. The reaction was evaporated in vacuo to give a viscous orange residue that was dissolved in 300 ml of H 2 0 and extracted with 10 Et 2 0 (3 x 150 ml). The aq. solution was acidified to pH 5 with 10% NaHSO 4 and extracted with EtOAc (3 x 250 ml). The combined extracts were dried over anhydrous Na 2

SO

4 filtered, and evaporated to give 12.64 g (83%) of the title compound as an orange amorphous solid: 1H NMR (CD 3 0D) 5 8.15-8.10 7.54-7.48 7.13- 7.08 (1H, 6.73-6.65 (1H, 4.45-4.35 (1H, m), 3.9-3.8 (1H, dd), 3.65-3.55 (1H, dd), 1.45 (9H, s).

Step B. (2S)-2-tert-Butoxycarbonylamino-3-(2aminophenyl-amino)-propionic acid. A mixture of (2S)- 2-tert-Butoxycarbonylamino-3-(2nitrophenylamino)propionic acid (12.65 g, 40.5 mmol) and 0.5 g of 10% Pd/C in 100 ml of MeOH under hydrogen at 1 atmosphere was stirred for 4 h. The solution was filtered through Celite 545 and the filtrate evaporated in vacuo to afford the 11.95 g of the title compound in quantitative yield as a dark brown solid that was used without purification: 1H NMR (CD 3 0D) 6 6.75-6.70 6.65-6.58 (1H, 4.35-4.3 1H, 3.6-3.38 (2H, 1.45 (9H, s).

Step C. (3S)- 2 -Oxo- 3 -tert-Butoxycarbonylamino-1,3,4,5tetrahydro-1H-1,5-benzodiazepine. 1-(3- Dimethylaminopropyl)- 3 -ethylcarbodiimide hydrochloride (8.54 g, 44.5 mmol) was added to a cooled (0 'C) 266 solution of (2S)-2-tert-butoxycarbonylamino-3-(2aminophenylamino)propionic acid (11.95 g, 40.5 mmol) in 100 ml of DMF and stirred for 18 h. The reaction was poured into 700 ml of EtOAc and washed four times with 100 ml of H 2 0. The organic layer was dried over anhydrous Na 2

SO

4 filtered, and evaporated to give a brown solid that was purified by flash chromatography eluting with 3:7 EtOAc/hexane to give 8 g of the 1 title compound: H NMR (CDCl 3 5 7.78 (1H, 7.02- 6.95 (1H, 6.88-6.82 (1H, 6.82-6.78 (1H, m), 6.75-6.70 (1H, 5.8-5.7 (1H, 4.55-4.45 (1H, m), 3.95 (1H, 3.9-3.82 (1H, 3.48-3.40 1.45 S (9H,s).

Step D. (600a/103). A 1.0 M solution of lithium bis(trimethylsilyl)amide (3.4 ml, 3.4 mmol) in THF was added dropwise to a -78 'C solution of (3S)-2-oxo-3tert-butoxycarbonylamino-2,3,4,5-tetrahydro-lH-l,5benzodiazepine (0.94 g, 3.38 mmol) in 20 ml of anhydrous THF and stirred for .30 min. Methyl bromoacetate (o.44 ml, 4 mmol) was added dropwise to the reaction mixture then warmed to RT. The reaction was diluted with 100 ml of EtOAc and washed with 0.3N

KHSO

4 (50 ml), H 2 0 (2 x 50 ml), and brine. The combined organics were dried over anhydrous Na 2

SO

4 filtered, and evaporated to afforded a gum that was purified by flash chromatography eluting with 3:7 EtOAc/Hex. to give 0.98 g of the title compound as a white solid. 1 H NMR (CDCl 3 5 7.15-7.07 (2H, m), 6.98-6.94 (1H, 6.88-6.84 (1H, 5.62-5.55 (1H, 4.71-4.65 (1H, 4.65-4.6 (1H, 4.33-4.27 (1H, 3.96-3.90 (1H, 3.78 (3H, 3.44-3.37 (1H, m), 1.4 (9H, s).

267 (600b). Prepared by a similar method described for the preparation of 600a/103 (Step except benzyl bromoacetate was used instead of methyl bromoacetate to give 600b in quantitative yield.

(600c).

Step A. (2S)-2-tert-Butoxycarbonylamino-3-(2-nitroacid. Prepared by a method similar as described for 600a/103 (Step A), except 2-fluoro-4,6-dimethyl-nitrobenzene was used 10 instead of 2-fluoronitrobenzene to give the desired compound in 93% yield.

Step B. (2S)-2-tert-Butoxycarbonylamino-3-(2-aminoacid. (2S)-2-tert- Butoxycarbonylamino-3-(2-nitro-3,5-dimethylphenylamino)propionic acid was converted to the title compound in quantitive yield as described in the prepartation of 600a/103 (Step B).

Step C. 2-Oxo-(3S)-3-tert-butoxycarbonylamino-2,3,4,5tetrahydro-7,9-dimethyl-1H-l,5-benzodiazepine. A 0 'C solution of (2S)-2-tert-butoxycarbonylamino-3-(2-aminoacid (763 mg, 2.36 mmol) and N-methylmorpholine (483 mg, 4.78 mmol) in S" ml of anhydrous THF was treated dropwise with isobutylchloroformate (352 mg, 2.5 mmol). The reaction was stirred for 2 h at 0 at RT for lh and poured over EtOAc. The mixture was washed with aq. 5% NaHSO 4 sat. aq. NaHCO 3 and sat. aq. NaCI, dried over NaSO 4 and concentrated in vacuo. Chromatography (flash, SiO 2 10% to 25% to 50 EtOAc/CH 2 Cl 2 gave 490 mg (68%) of the desired product.

Step D. (600c). (2S)-2-tert-Butoxycarbonylamino-3-(2acid was 268 converted to 600c, 75% by a similar method for the preparation of 600b.

R2 R2 H0 1) Step A. HC1 R 3 N 2) Step B. PhCOH N N R 2 N R 2 SBoc-N 3) Step C. R 3 X R-N H 0 0 H 0 0 602 600 R2

R

R

3 StepD N N R 2 R4-NNO H OH 603 (602a).

Step A. Anhydrous HC1 was bubbled into a solution of 5 (3S)-2-oxo-3-tert-butoxycarbonylamino-2,3,4,5tetrahydro-1H-1,5-benzodiazepine-l-acetic acid methyl ester (600a/103, 4.0 g, 11.4 mmol) in 20 ml of CH 2 Cl 2 for 20 min then stirred for 1 h at RT. The reaction was evaporated to give (3S)-2-oxo-3-amino-2,3,4,5tetrahydro-lH-1,5-benzodiazepine-l-acetic acid methyl ester hydrochloride as a white solid.

Step B. The white solid was dissolved in 70 ml of DMF and benzoic acid (1.5 g, 12.3 mmol) was added. The reaction was cooled in a ice/H 2 0 bath and treated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.4 g, 12.5 mmol), 1hydroxybenzotriazole (1.7 g, 12.6 mmol) and diisopropylethylamine (3.0g, 23.2 mmol). The reaction was stirred for 18 h at RT under nitrogen atmosphere 269 and poured onto H 2 0. The aq. mixture was extracted with EtOAc The combined organic layers were washed with aq. 0.5 N NaHSO 4

H

2 0, sat. aq. NaHCO 3

H

2 0 and sat. aq. NaCI, dried over MgSO 4 and concentrated in vacuo. Chromatography (flash, SiO 2 10% to EtOAc/CH 2 C12) gave 3.4 g of (3S)-2-oxo-3- (benzoylamino)-2,3,4,5-tetrahydro-lH-l,5benzodiazepine-l-acetic acid methyl ester as a white solid.

eo S 10 Step C. Method A. (602a). .A solution of (3S)-2-oxo- 3- (benzoylamino)-2,3,4,5-tetrahydro-lH-l,5benzodiazepine-l-acetic acid methyl ester (200 mg, 0.57 mmol) in CH 2 C1 2 (10 ml) was treated with triethylamine (119 mg, 1.13 mmol) and 3-phenylpropionyl chloride (114 mg, 0.68 mmol). The reaction was stirred at RT for min and diluted with CH 2 Cl 2 The solution was washed with aq. 10% HC1 sat. aq. NaHCO 3 and sat. aq. NaC1, dried over Na 2

SO

4 and concentrated in vacuo to give 240 mg of 602a as a white foam.

Step C. Method B. (602g). A 0 'C solution of (3S)-2oxo-3-(benzoylamino)-2,3,4,5-tetrahydro-lH-1,5- Se benzodiazepine-1-acetic acid benzyl ester (600b) (465 S" mg, 1.10 mmol) in CH 2 C1 2 (5 ml) was treated with acetoacetic acid in 1 ml of CH 2 Cl 2 followed by slow addition of 1-(3-dimethylaminopropyl)-3ethylcarbodiimide hydrochloride (431 mg, 2.2 mmol) in 2 ml of CH 2 Cl 2 under N 2 atmosphere. After 15 min the reaction was poured onto EtOAc, washed with aq. 5 NaHSO 4 dried over Na 2

SO

4 and concentrated in vacuo.

Chromatography (flash, SiO 2 0% to 10% to MeOH/CH 2 Cl 2 gave 580 mg of (3S)-2-oxo-3- (benzoylamino)-5-acetoacetyl-2,3,4,5-tetrahydro-lH-1,5- 270 benzodiazepine-1-acetic acid benzyl ester as a white solid.

Step C. Method C. (602j). A vigorously-stirred, 0 "C solution of (3S)-2-oxo-3-(benzoylamino)-2,3,4,5tetrahydro-1H-1,5-benzodiazepine-l-acetic acid benzyl ester (600b) (461 mg, 1.07 mmol) in THF (5 ml) and sat.

aq. NaHCO 3 (2.5 ml) was treated with a THF solution (0.35 ml) of methyl chloroformate (151 mg, 1.6 mmol) and the reaction was stirred for 45 min at RT. The reaction was poured onto CH 2 Cl 2 and washed with H 2 0, o*oo dried over Na 2

SO

4 and concentrated in vacuo.

Chromatography (flash, SiO2, 0% to 10% MeOH/CH 2 Cl 2 gave 525 mg of 602j as a white solid.

Step C. Method D. (602p). A solution of 600a/103 15 (400 mg, l.lmmol) and benzylisocyanate (166 mg, 1.2mmol) in 10 ml of CH 2 Cl 2 and 10 ml of DMF and heated at 80 'C for 3 days. The reaction was cooled to RT S.poured onto H 2 0 and extracted with EtOAc The combined organic layers were washed with H 2 0 (4x) and sat. aq. NaCI, dried over MgSO 4 and concentrated in vacuo. Chromatography (flash, SiO 2 50% to EtOAc/hexane) gave 440 mg of 602p as a white solid.

Step C. Method E. (602v). A solution of (3S) 2-oxo- 3-amino-5-(3-phenylpropionyl)-2,3,4,5-tetrahydro-lHacid methyl ester hydrochloride (560 mg, 1.34 mmol), benzaldehyde (146 mg, 1.34 mmol) and sodium acetate (220 mg, 2.68 mmol) in methanol (20 ml) was treated with 4A sieves (2 g) and NaCNBH 3 (168 mg, 2.68 mmol). The reaction was stirred for 2.5 h, acidified with 10% aq. HC1 to pH 2 and washed with Et 2 0 (2x75 ml). The organic layers 271 were concentrated in vacuo to give an oil.

Chromatography (flash, SiC 2 0 to 35% EtOAc/CH 2 Cl 2 gave 250 mg of 602v as a clear oil.

StpD Method A. (603a). (3S)-2-Oxo-3-benzoylamino- 5- (3-phenylpropionyl)-2, 3,4, 5-tetrahydro-lH-l, diazepine-1-acetic acid methyl ester (602a; 1.25 g, 2.57 mmol) was dissolved in 11 ml of THE, MeCH and H 2 0 0: and treated with LiOH.H 2 0 (42 mg, 0.62 mmol) stirred at RT for 64 h. The reaction was concentrated *e 10 in vacuo, diluted with H 2 0 and acidified with aq. 1N HCl to give 230 mg of 603a as a white solid.

StpD Method B. (603d). A mixture of (3S)-2-oxo-3- (benzoylamino) -5-acetyl-2, 3,4, 5-tetrahydro-lH-1, benzodiazepine-1-acetic acid benzyl ester (602d; 510 mg, 1.08 mniol) and 5% Pd/C (250 mg) in MeOR (10 ml) *stirred under H 2 (1 atm) for 0.5h. The reaction was filtered and concentrated in vacuo 410 mg of 603d as a white solid.

The compounds of Table 16 were prepared as 20 described in Table 17, using the methods of Example Table 16 Compound R 2

R

3

R

4

R

no.

602b H PhCH 2 C(O) PhC(O) CH 2 Ph 602c H PhC(O) PhC(O) CH 2 Ph 602d H CH 3 C(O) PhC CH 2 Ph 602e H CH 3

OCH

2 C PhC(O) CH 2 Ph 602f H (CH 3 2

CHCH

2 C(O) PhC(0) CH 2 Ph 602g H CH 3

C(O)CH

2 C(O) PhC(0) CH 2 Ph 1602h IH ICH 3 0C (0)C IPhC(0) ICH 2 Ph 272 Compound R2 R3 R4 no.

602i H CH3C(O)C(O) PhC(O) CH2Ph 602j H CH30C(O) PhC(O) CH2Ph 602k H CH3C(O) Boc CH2Ph 6021 CH3 CH3 C(O) Boc CH2Ph 602m H CH3S(02) PhC(O) CH3 602p H PhCH,)NHC(O) PhC(O) CH 3 602q H PhC(O) CH2Ph

OWIIOW

602r H PhCH2CH2C(O) PhCH2CH2C(O) CH2Ph 602s H 4-pyridylCH2C(O) PhC(O) CH2Ph Table 17 No. Starting R Step C Step

D

material 3X method/ method/ yield) yield) 603b 600b PhCH2C(O)Cl A (98) B (89) 603c 600b PhC(o)Cl A (quant.) B (quant.) 603d 600b CH3C(O)Cl A (quant.) B (quant.) 603eJ60-Ob CH30CH2C(O)Cl A (59) B (quant.) 603f 600b (CH3)2CHCH2C(O)Cl A (88) B 603g 600b CH3C(O)CH2CO2H B (quant.) B (quant.) 603h,600b CH30C C Cl A (96) B (quant.) 603iJ600b CH3C(O)CO2H B (87) B (94) 603jI600b CH30C(O)Cl C (quant.) B (quant.) 603k 600b CH3C(O)Cl A, Step C not run only I(quant.) is 273 9 0O 0S e p 00 0 *PP*00 *9 0 0'~06 00 0 000 Starting Step C Step D No. maeilR 3 X method/ method/ yield) yield) 6031 600c CH 3 C (0)Cl A, Step C. not run only ~(quant.) 603m 600a/103 CH 3

SO

3 Cl, NEt 3 A (76) A (92) instead of pyridine and THF instead of CH 2 Cl 2 603p, 600a/103 PhCH 2 C=N=O D (80) A (86) 603q 600b C (83) B (71) 0 oCoc 603r 600a/103 PhCH 2

CH

2 C(O)Cl A 603s 600b 4-pyridylCH 2 00 2 H B (90) B (98)

J

"0-es.

090 6 me..

C C 0* 0 H N R 2 B ac-N H 0 OIOR 5 600 1) Step C.

2) Step A.

3) Step C.

R

3 X R 3 R3X R4X H 0 01

OR

602 0.' 0 Oe 00 0 @0 00 Step D

R,

4 N N

R

H 0 0

-OH

603 The compounds of Table 18 were prepared as described in Table 19 using the methods of Example 274 0*

S

*5 0S S

S.

S.

S

555555 0 0@ S S

OOSS

S

SoS 00 S 0 *0 0 Table 18 Compound no. R2R 3 R4R 602n H ICH 3 C Naphthylene-2-C(0) CH 2 Ph 602o OH 3

ICH

3 C(0) PhC(0) CH 2 Ph 602t H 3-CH 3 PhCH 2 C(0) PhC(0) CH 2 Ph 602u H CH 3 C Fmoc CH 2 Ph 602v IH jPhCH 2

CH

2 00 jPhCH 2

CH

3 j Table 19 1) Step C. 3) Step C Step D Starting No. maeil R 3 X method R 4 X method method yield) yield) yield) 603n 602k CH 3 C(O)C1 naphthylene B(quant.) A (quant.) 2-0(0)01 (70) 603o 6021 CH 3 C(O)C1 PhC(0)Cl B(quant.) (quant.) A (73) 603t 602k 3- PhC(0)C1 B

CH

3 PhCH 2 C(0)C1 A (93) (quant.) 603u 602k CH 3 C(0)C1 Fmoc-C1 C (98) A (quant.) C (82) 603v 600a/103 PhCH 2

CH

2 C(0)C1 PhCHO A A JE(40)_ 275

R

2 N R 2

R

4

-N

H 0 O)1^OH 603 Step A 0

N

604 H

H

S

0O

OS

S

S S 0 eecee

S

OS

S S

S.

S

Step B

R

4

-N

6 *5*5

S.

0S9S S S 50 0

S.

0

S.

S

S.

The compound of Table 20 was prepared by the method described below.

Table compound no. IR 2

R

3 R4 605a IH IPhCH 2

CH

2 C(O) PhC(O) (605a).

Stp (3S) (l-Fluorenylmethyloxycarbonylamino)-4oxobutyric acid tert-butyl ester semicarbazone (210 mg, 0.45 mol, Prepared in a similar manner to the benzyloxycarbonyl analog in Graybill et al., Int. J.

Protein Res. 44, pp. 173-82 (1994).) was dissolved in ml of DMF and 2 ml of diethylamine and stirred for 2 h. The reaction was concentrated in vacuo to give 3

S)-

3 -amino-4-oxobutyric acid tert-butyl ester 276 semicarbazone. The 0 'C solution of the above residue and 603a (200 mg, 0.42mmol) in 5 ml of DMF and 5 ml of

CH

2 Cl 2 was treated with l-hydroxybenzotriazole (57 mg, 0.42mmol) and 1-(3-dimethylaminopropyl)-3ethylcarbodiimide hydrochloride (98 mg, 0.51 mmol).

The reaction was stirred at RT for 18 h, poured onto EtOAc (75 ml) and washed with aq. 0.3 N KHSO 4 sat. aq.

NaHCO 3 and sat. aq. NaCl, dried over NaSO 4 and e 0 concentrated in vacuo. Chromatography (flash, SiO2, 0% 10 to 4% MeOH/0.1% NH 4 0H/CH 2 Cl 2 to give 240 mg of 604a.

Step B. 604a was stirred with 10 ml of 33% TFA/H 2 0 for 4 h and concentrated in vacuo. The residue was dissolved in 7 ml of MeOH/acetic acid/37% aq.

15 formaldehyde and stirred for 18 h.

Chromatography (Reverse Phase C18, 4.4mm ID x 25 cm, to 70% CH 3 CN/0.1% TFA/H 2 0) gave 32 mg of 605a 1 as a white solid: H NMR (CD 3 0D, existing as diastereomers of the hemiacetal) 5 7.85-7.78 (2H, d), 7.5-7.32 (6H, 7.32-7.28 (1H, 7.18-6.98 (5H, m), 4.92-4.85 (2H, 4.5-4.32 (2H, 4.31-4.20 (2H, m), 3.7-3.6 (1H, 2.90-2.75 (2H, 2.65-2.5 (1H, m), 2.48-2.25 (3H, m).

Step D. (600c). (2S)-2-tert-Butoxycarbonylamino-3-(2amino-3,5-dimethylphenyl-amino)-propionic acid was converted to 600c, 75% by a similar method for the preparation of 600b.

277 600b

ON'

N H 0

N

696a-e H

R

a a 696a R 1 %0Bn 696c R1= 9 696 R1*B69 dR g o 696e 1 (696a) was synthesized from 600b via methods used to *:see prepare 690a from 600b to afford 696a. HNMR (CDCl 3 )6 0.95(t, 2H), 1.25(t, 1H), 1.4(m, 2H), 1.55(m, 1H), *2.55(m, 1H), 2.85(m, 1H), 2.95(dd, 1H), 3.15(m, 1H), 3.55(m, 1H), 3.9(m, 2H), 4.35(t, 1H), 4.4-4.55(m, 2H), 4.75(m, 1H), 4.8-5.05(m, 2H), 5.45(s, 1H), 5.55(d, 1H), *6.85(d, 1H), 7.15(d, 1H), 7.2-7.5(m, 5H), 7.6-7.8(m, 3H), 8.45(d, 1H), 9.05(d, 1H), 9.35(d, 1H).

The data of the examples above demonstrate that compounds according to this invention display sinhibitory activity towards IL-1I8 Converting Enzyme.

Insofar as the compounds of this invention are able to inhibit ICE in vitro and furthermore, may be delivered orally to mammals, they are of evident clinical utility for the treatment of IL-i-, apoptosis-, IGIF-, and IFN-y mediated diseases. These tests are predictive of the compounds ability to inhibit ICE in vivo.

278 While we have described a number of embodiments of this invention, it is apparent that our basic constructions may be altered to provide other embodiments which utilize the products and processes of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the appended claims, rather than by the specific embodiments which have been presented by way of example.

S

0

S

Claims (6)

1. A compound represented. by the formula: R 1 S OS 4 0 wherein: m is 1 or 2; R, is: (elO-B) S 0 0S*O *5S* 0 *0 0 *5 0 0* 5S 0 4 .5 10 R 3 is -CN, -C (0)-CH 2 -T 1 -k 1 1 -C(O)-CH 2 -F, -C=N-O-R 9 or -CO-Ar 2 each R 5 is -C(O)-R 1 0 -C(0)O-R 9 -N (R 0 (RIO) -S(0) 2 -R 9 -S(0) 2 -NH-R 1 0 -C(0 -CH 2 R 9 -R 1 0 -R9, -li -C(O)C(O)-0Rj 1 0 or -C(0)C(0)-N(R 9 (R 1 0 15 Y2 is H 2 or 0; each T 1 is or each R 9 is -Ar 3 or a -CI- 6 straight or branched alkyl group optionally substituted with -Ar 3 wherein the -C 1 -6 alkyl group is optionally unsaturated; each RIO is -Ar 3 a -C 3 -6 cycloalkyl group, or a -C 1 straight or branched alkyl group optionally substituted with -Ar 3 wherein the -C 1 alkyl group is optionally unsaturated; each R 1 1 is -Ar 4 -(CK 2 )1- 3 -Ar 4 or -C(O)-Ar 4 R 1 5 is -OH, -OA-r 3 or -OC-6, wherein C 1 6 is a straight or branched alkyl group optionally 280 substituted with -Ar 3 -CONH 2 -OR 5 -OH, -OR 9 or -C0 2 H; each R 21 is -H or a -C1_ 6 straight or branched alkyl group; Ar 2 is independently selected from the following group, in which any ring may optionally be singly or multiply substituted by -Q 1 or phenyl, optionally substituted by Q 1 0@ 0 00 0* 0@ 0 S. 0 0 (hh) and (ii) Y N D wherein each Y is independently 0 or S; each Ar 3 is a cyclic group independently selected from the set consisting of an aryl group which contains 15 6, 10, 12, or 14 carbon atoms and between 1 and 3 rings and an aromatic heterocycle group containing between and 15 ring atoms and between 1 and 3 rings, said heterocyclic group containing at least one heteroatom group selected from S0 2 and -NH-, 20 -N(R 5 and -N(R 9 said heterocycle group optionally containing one or more double bonds, said heterocycle group optionally comprising one or more aromatic rings, and said cyclic group optionally being singly or multiply substituted by -Q 1 each Ar 4 is a cyclic group independently selected from the set consisting of an aryl group which contains 6, 10, 12, or 14 carbon atoms and between 1 and 3 rings, and a heterocycle group containing between 5 and ring atoms and between 1 and 3 rings, said heterocyclic group containing at least one heteroatom group selected from S02, -NH-, 281 -N(R 5 and -N(R 9 said heterocycle group optionally containing one or more double bonds, said heterocycle group optionally comprising one or more aromatic rings, and said cyclic group optionally being singly or multiply substituted by -Q I each Q 1 is independently -NH 2 -C02H, -Cl, -F, -Br, -NO 2 -CN, -OH, -perfluoro C1.3 alkyl, R,, -OR 5 -NHR 5 -OR 9 -N(R 9 )(R 1 0 -R 9 -C(O)-R 1 0 or 0 0 CH 2 0 S 0s S S. S 0000 S 6*000: 0*S 0 0 0@*e S. 0 0.*0. 0 9 S. *5 0 provided that when -Ar 3 is substituted with a Q1 group which comprises one or more additional -Ar 3 groups, said additional -Ar 3 groups are not substituted with another -Ar 3 provided that when: m is 1; 20 R 15 is -OH; R 2 1 is -H; Y2 is 0; and R 3 is then R 5 is not: -S(0)2-CH3; -C(0)-OCH 2 phenyl; or -C(O)-R 1 0 wherein R 1 0 is: -CH 2 CH 2 phenyl, phenyl unsubstituted by -Q 1

4-(carboxymethoxy)phenyl, 2-fluorophenyl, 2-pyridyl, or N-(4- methylpiperazino)methylphenyl; and provided that when: m is 1; R 15 is -OH; R 21 is 282 Y~2 is H 2 and R 3 is then R 5 is not -C(O)-CH 2 CH 2 phenyl; 0O 0 0@ *0 0* 0 0* 000000 S 0@ a 0S S S.. and provided that when: m is 1; R 15 is -OH or -OC(CH 3 3 R 21 is -H; Y 2 is 0; and R 3 is -CO-Ar 2 wherein*Ar 2 is Cf H 3 CO or o OCH 3 O") 0 l ;then R 5 is not -C(O)-CH 2 CH 2 phenyl; S 0

5.5. S 005@ *SSS S S 5* 0 @0 S. 9 *5 5* 0 00 0S and provided that when: m is 1; 15 R 15 is -OH or -OC(CH 3 3 R 21 is -H; Y~2 is 0; and R 3 is -C(O)-CH 2 -S-CH 2 -2-chlorophenyl; then R 5 is not -C(O)-CH 2 CH 2 phenyl; and when R 3 is -CH 2 -O-C 6-dichiorophenyl; then R 5 is not: -S()2-H3,or -C (0)-O-CH 2 phenyl, -C(O)-Rl 0 wherein R 10 is: -CH 2 CH 2 phenyl, 4- (dimethylaminomethyl)phenyl, 4- (N- morpholinomethyl)phenyl, 4- (N- methylpiperazino)methyl)phenyl, 4- (2- methyl) imidazolylmethyl)phenyl, 5-benzimidazolyl, benztriazolyl, N-carboethoxy-5-benztriazolyl, or N- carboethoxy-5-benzimidazolyl; and when 283 S@ 0 0* S. 0* S 10 S 0 SO S S S eeeS 0e 00 60S R 3 is -C (0)-CH 2 (1-(4-chiorophenyl) -3- trifluoromethyl)pyrazolyl); then R 5 is not: -H, -C(O)-R 10 wherein R 10 is: 4- (dimethylaminomethyl) phenyl, phenyl, 4- (carboxymethylthio)phenyl, 4- (carboxyethylthio) phenyl, 4- (carboxyethyl) phenyl, or 4- (carboxypropyl) phenyl, or -C(O)-0R 9 wherein R 9 is isobutyl or C2 phenyl; and when R 3 is -C (0)-CH 2 -O-R 1 1 wherein R 1 is 5- (1-phenyl- 3-trifluoromethyl)pyrazolyl or 5- (1-(4-chloro-2- pyridinyl) 3 -trifluoromethyl)pyrazolyl; then R 5 is not -C(O)-O-CH 2 phenyl; and when R 3 is -C(O)-CH 2 -O-5-(l-(2-pyridyl)-3- trifluoromethyl)pyrazolyl), then R 5 is not: (dimethylaminomethyl)phenyl or -0- CH 2 phenyl; and provided that when: M is 1; R 1 5 is -OH or -OC (CH 3 3 R 21 is -H; Y2 is H 2 and R 3 is -C(O)-CH 2

6-dichiorophenyl, then R is not -C(O)-O-CH 2 phenyl. 2. The compound according to claim 1, selected from the group consisting of: S *050** 0 0000 0 0000 S S 05 0 5* 00 00 06 S @0 S 05 284 0 N 2002 NCO HO 0 N CHO H S 0O 0 S. S S @0 5 OS* 214f 214g S *000 *00S S *5 0 0 N& HH 0 0 4OH H N 0 OY O H H CH 3 O H 0 a 0 H NO H 214h 0 S *0 S. S S S S* 214i 214j 285 2141 0 0H OH 0 0 H H o0 Hb 246b 0 .OH H 0ec H 0 C. @00 280b OH MeSO 2 'HO 00 0000 Sek 0e 0 280c 01 OH N- :MeA N 0 H *0 280d No' H 28 3b 11 i 286 283c 0 Meo OH MeOh 0 H 283d H0 0I OH N 00 I 0H 0H 000000 0 0 H 0 N H 0 0 N 0 0 0 302 10 OH 0 H H N I 0 0 0 0 308d OO 308e 0 308d 0 I 287 cI 500 0 4 Z0? OH 0 1 0O 060 0 .:501 0 OH 'N O H HH 00 0 00 505 Nb MeS 2 N 'I O o Me H 00 N 0 .000*H 0 0_N 0@ 0 6 50c Ki~> OH 0 0 0 MeSO 2 -N' H 0 0 0. H 0 H 0 505d 1 288 00 S 04. 00 OS 0 Sf S 555050 S 50 a 00 555 505ff 510a 0* 500 0 510Ob a *e 5 *0 50 S 5 *0 510C pup N L~4d 0d HZ03 0 OHN NH A l 0e 0 H C N N 0O 0 H N S O aZSe as. HO N @OC 00E 0 H. 0S H 00 O H S III..II 0 H 0 05 H 0 O 0H T 0 HO r S N PT 68Z 290 2100i H 0 0 OHCH H 00 3. The compound according to claim 1, selected from the group consisting of: 00 N C" owe 220b Ob H N0 O H 0 Q N. CI 0 a 0 0 0 223b &HOH4-N H260 22e 0 0 Nc~ 291 O 227e N HO NO *307a N OH H 0 H O Cl 307b o and H H4 O 0 429 N" 0 2N. OH H O 5 4. The compound according to claim 1, selected from the group consisting of: 214c N H 3 C N OH H H H 0 0 217c r K n 0 a 292 217e 246 N H O~ 0H fN 0 e N 0 0 H 0 257 0NO OH 00 280 n OS 0 0 H N-N? H 0 OH H OS@0 0 281 0 OH 5F CI H 0 H 4 0 282 4 NII OHQ H 283 .I o Y "O 293 284 W, 284 H 3 CO, rfw OH 0 CI HH 0 Wr 0H too 285 H O H 9H H 3 uNI H 0 0 H H OH 3 C 286 0 .H 3 CONH H 0 S. H 0 H 050 287 0o 0 H 3 C N OH 0 C H @099 0 404 /O 0 -H 3 C H 0 H O S.. 0 0 405 N 0 j OH SH 0 0 406 O 4OH 0H 0 0 407 N 0 00W 0 H N- O 294 0 408 NZ N 0 o S* 409 N OH 0H 00 o 410 <QoL 409 OH

9. :OH HH 0, H 0 0 411 0 *o OH eesH N Ho 5 413N0 OH H 0 0* 0 o oO 0 4170 418 o OH 4 h OHo 0 295 0 419 N oONO ~N4H 0 0 420 0 H H 0 *422N 0 OO i aa (I 0 0 423 N N O @0 0 423 Na 1 0 H 0& 6 H 0 0 0000 42 N"0 00 H 425 0 0e0 426 N 0 N O H 0 0 0 HO H 0N0 296 0 431 N NOH H 0 432 NN 0 0 1~F HN N O NN 0 N I H 0 00

33. N N 0 OS jW~~ O~ 0 434 N 0 0~1 S...O 435 0 N OH N OH 0e 0 436 Nc: 0~ NjA H 0 437 297 0 438 00 SO oo H N 000 6 4 o *0O 00 440 N h O H 0 H NH 2 H 0 0 441 N 0 0 N r 0 H 0000 0 5 442 N 0 O 0OH 0 0 I\ SH 00 0 444 OHOIIJ 298 H H 0 Hg 44 NHs Ni H o 00 N 0 447ANl 0 00 0 0*00 448 Oo N H HN 0 O 0 wo N 0H 0N N 0H OH H o 0 450 N NH 0 0 299 0 N O0 451 N S4OH H N Ho 0 *.452 N 0 H O *0 H OH 453 N 0 OH71 O OH 0 454 N 0 0 CN* 0 0 455 N 0 H H 0 O CN I0 456 N 0 HO OH 0 457 0 N o OH 300 458 H 0 459 0O~~ 0 N H S.. H 0C H 0 H 0 460 I-' HG N H ?N 4 H 0 H 0 HN 3 AO 0 H 0H FI OH H 0 463N 0 0 k OH Fe N 04 H 00 N 0O 464 OHH cp'N OoN 0 H3465"" 465 N ()H 0 H 0 301 0 H 3 O 466H0 H 0 H CI N 0 467 N 0 r O 0* 0 N o 0*0 468 N F y OH 0 4690 see#00* 0 N cl- A N H H 3 C 0 00 0 4700 h H0 600 N HC.N*CH 0 471 OH H 3 Cd1OKN O~N~~H H 3 C H 0 0 472 N 0 OH N0-N H 302 0 473 0 0 CHO 3 0 H H0 474N 0 N CH H H OS 00 H O @475N 0 OO 6: H 0H 0 0 476 HHC 0 rq^i 0 0 H 00 S HI 477 0 0 HO H 0HO 0 0 0 478 N 0 H 2 N. -H O 0 H 0 479 0 N H 0 303 0 480 H 3 CNO 0 HN NO H H H H 3 CO 0 481 0 *OH H H4 es 00 N 0 H 00 0 482s 6O 0 H0' 000H 482s N 0 S 0 H l N 005*l O H Cl H2~ON4 0 0 483 0 OH$O OH Ne 60 0 H0 0 0 0 0 N 0 4840 H~ H H H 3C H 0i T6 06 f *0 b 0 00 S. S. OS 0 68 8 8 D *0 S S 0~0e 0 *00S 0 S *00 0 S. S0 OS4S 04 9* 0 U 50050S 0 .D I 0 0 *4 6 9 S C. 0 H L8 HO N Y 0P0 0 9987 H 0 O0E 305 0 0 O ,495 OrN If F H 0 0 494 0N 0 ~~cI OOH 00 H 49 O 0 N 0 a 0 G 495 0 S I/ H0 0 0~0 0 0*0* 5 497 0 N 0 *C 0 0 H 3 C. 0 ^N 0 **:498 CjyAN^ OH 0 acod o cli HlH o HO 0 *1 0 0 499 0 0 I eNY fOH HH The compound according to claim 1, selected from the group consisting of: 306 O 421 O O N N OH H O O H 0 427 N O and o OH 0 0 0 o 0 0 N O 428 H3C? 0 N H 3 CN IOH N-I-N HC H H H 6. The compound according to claim 1, wherein: m is 1; T 1 is O or S; R 2 1 is -H or -CH 3 Ar 2 is (hh); Y is O; each Ar 3 cyclic group is independently phenyl, naphthyl, thienyl, quinolinyl, isoquinolinyl, pyrazolyl, thiazolyl, isoxazolyl, benzotriazolyl, benzimidazolyl, thienothienyl, imidazolyl, thiadiazolyl, benzo[b]thiophenyl, pyridyl, benzofuranyl, or indolyl and said cyclic group being singly or multiply substituted by -Q 1 each Ar 4 cyclic group is phenyl, tetrazolyl, pyridinyl, oxazolyl, naphthyl, pyrimidinyl, or thienyl and said cyclic group being singly or multiply substituted by -Q 1 307 each Q 1 is -NH 2 -Cl, -Br, -OH, -R 9 -NH-R wherein R 5 is -C(O)-R 1 0 or -S(O) 2 -R 9 -OR 5 wherein R is -C(O)-R 1 0 -OR 9 -NHR 9 or 0 CH 2 10 0 0S 0O 0 S 15 wherein each R 9 and R 10 are independently a -CI-6 straight or branched alkyl group optionally substituted with -Ar 3 wherein Ar 3 is phenyl; provided that when -Ar 3 is substituted with a Q1 group which comprises one or more additional -Ar 3 groups, said additional -Ar 3 groups are not substituted with another -Ar 3 0 S 0@0@ S 00 S 7. The compound according to wherein R 3 is -C(O)-Ar 2 8. The compound according to wherein R 3 is -C(O)CH 2 -T 1 -R 11 9. The compound according to wherein R 3 is claim 6, claim 6, claim 6, 0* 0 0@ 5. 0* The compound according to any one of claims 1 or 6-9, wherein R 5 is -C(O)-R 10 or -C(0)C(0)-R 10 11. The compound according to claim wherein R 10 is Ar 3 12. The compound according to claim 11, wherein R 5 is -C(O)-R 10 and R 10 is Ar 3 wherein the Ar 3 308 S 10 S 0@ 5 S@O cyclic group is phenyl optionally being singly or multiply substituted by: -R 9 wherein R 9 is a C 1 4 straight or branched alkyl group; -F, -Cl, -N(H)-R 5 wherein -R 5 is -H or -C(0)-R 10 wherein R 10 is a -CI_ 6 straight or branched alkyl group optionally substituted with -Ar 3 wherein Ar 3 is phenyl, -N(R 9 )(R 10 wherein R 9 and R 10 are independently a -C1- 4 straight or branched alkyl group, or -0-R 5 wherein R 5 is H or a -C 1 -4 straight or branched alkyl group. 13. The compound according to claim 12, wherein Ar 3 is phenyl being singly or multiply substituted at the 3- or 5-position by -Cl or at the 4- position by -NH-R 5 -N(R 9 (R 10 or -O-R 5 S S 5 S* S 000 0000 14. The compound according to claim 13, selected from the group consisting of: S 65 5 5e 0 55 214k and 214m 309 The compound according to claim 12, wherein Ar 3 is phenyl being singly or multiply substituted at the 3- or 5-position by -R 9 wherein R 9 is a C 1 4 straight or branched alkyl group; and at the 4 -position by -O-R 5 16. The compound according to claim wherein the compound is: go O 214w 0 N O O •Me JJ OH H O N H HO I N H Me S 17. The compound according to claim 11, wherein R 5 is -C(O)-R 10 wherein R 10 is Ar 3 and the Ar 3 S.cyclic group is indolyl, benzimidazolyl, thienyl, quinolyl, isoquinolyl or benzo[b]thiophenyl, and said cyclic group optionally being singly or multiply substituted by -Q 1 18. The compound according to claim 17, wherein the Ar 3 cyclic group is isoquinolyl, and said cyclic group optionally being singly or multiply substituted by -Q 1 19. The compound according to claim 18, wherein the compound is: 0 412 N 0 H O H H 6 310 The compound according to claim 11, wherein R 5 is -C(O)-R 10 wherein R 10 is Ar 3 and the Ar 3 cyclic group is phenyl, substituted by 0 CH 2 0@ 10 S 0 O 0 5 OS 21. The compound according to claim wherein the compound is: 415 0 00 0 O:CAN 0H S OeSS @000 S S. S S. 5 0@ S. S @5 *5 22. A pharmaceutical composition comprising a compound according to any one of claims 1-21 and a pharmaceutically acceptable carrier. 15 23. A method for treating or preventing a disease selected from osteoarthritis, acute pancreatitis, chronic panceatitis, asthma, adult respiratory distress syndrome, infectious hepatitis, glomeralonephritis, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, chronic thyroiditis, Grave's disease, autoimmune gastritis, insulin- dependent diabetes mellitus (Type autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia, chronic active hepatitis, myasthenia gravis, inflammatory bowel disease, Crohn's disease, ulcerative collitis, multiple sclerosis, psoriasis, lichenplanus, graft vs host disease, acute dermatomyositis, eczema, primary cirrhosis, uveitis, Behcet's disease, acute aplasia, aplastic anemia, 311 amyotrophic lateral sclerosis, nephrotic syndrome, osteoporosis, multiple myeloma-related bone disorder, acute myelogenous leukemia, chronic myelogenous leukemia, metastatic melanoma, Kaposi's sarcoma, multiple myeloma, sepsis, septic shock, Shigellosis, Alzheimer's disease, Parkinson's disease, cerebral ischemia, and myocardial ischemia in a patient comprising the step of administering to the patient a pharmaceutical composition according to claim 22. e* 10 24. The method according to claim 23, wherein the disease is osteoarthritis, acute pancreatitis, rheumatoid arthritis, inflammatory bowel disease, ulcerative collitis, Crohn's disease, hepatitis, adult respiratory distress syndrome, glomerulonephritis, insulin-dependent diabetes mellitus (Type juvenile diabetes, psoriasis, graft vs. host disease, hepatitis, or Alzheimer's disease. 25. A method for treating or preventing a disease selected from Alzheimer's disease, Parkinson's 20 disease, cerebral ischemia, myocardial ischemia, spinal muscular atrophy, multiple sclerosis, AIDS-related encephalitis, HIV-related encephalitis, aging, alopecia, and neurological damage due to stroke in a patient comprising the step of administering to a patient a pharmaceutical composition according to claim 22. 26. The method according to claim wherein the disease is Alzheimer's disease. DATED this 28 th Day of September 2001 Vertex Pharmaceuticals Incorporated By their Patent Attorneys CULLEN CO.