AU733685B2 - Intermediate in production of small molecule inhibitors of rotamase enzyme activity - Google Patents

Description

P/00/011 Regulation 3.2

AUSTRALIA

PATENTS ACT 1990 COMPLETE

SPECIFICATION

o eo FOR A STANDARD PATENT

ORIGINAL

of Applicant: Actual Inventor(s): Address for Service: TO BE COMPLETED BY APPLICANT GUILFORD PHARMACEUTICALS,

INC.

Gregory S. Hamilton; Joseph P. Steiner CALLINAN LAWRIE, 711 High Street, Kew, 3101, Victoria, Australia SMALL MOLECULE INHIBITORS OF ROTAMASE ENZYME

ACTIVITY

Invention Title: The following statement is a full description of this invention, including the best method of performing it known to me:- 15/0 6 9 9 ,1p10625.cs,l INTERMEDIATE IN PRODUCTION OF SMALL MOLECULE INHIBITORS

OF

ROTAMASE ENZYME ACTIVITY BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to production of neurotrophic compounds having an affinity for FKBP-type immunophilins, and their use as inhibitors of the enzyme activity associated with immunophilin proteins, and particularly inhibitors of peptidyl-prolyl isomerase or rotamase enzyme activity.

2. Description of the Prior Art The term immunophilin refers to a number of proteins that serve as receptors for the principal immunosuppressant drugs, cyclosporin A (CsA), FK506, and rapamycin. Known classes of immunophilins are cyclophilins, and FK506 binding proteins, such as FKBP. Cyclosporin A binds to cyclophilin while FK506 and rapamycin bind to FKBP. These immunophilin-drug complexes interface with a variety of intracellular signal transduction systems, especially in the immune system and the nervous system.

Immunophilins are known to have peptidyl-prolyl isomerase (PPlase) or S rotamase enzyme activity. It has been determined that rotamase activity has a role in the catalyzation of the interconversion of the cis and trans isomer of immunophilin proteins.

Immunophilins were originally discovered and studied in immune tissue. It 25 was initially postulated by those skilled in the art that inhibition of the immunophilins rotamase activity leads to the inhibition of T-cell proliferation, thereby causing the immunosuppressive action exhibited by immunosuppressive drugs such as cyclosporin A, FK506, and rapamycin. Further study has shown that the inhibition of rotamase activity, in and of itself, is not sufficient for 30 immunosuppressant activity. Schreiber et al., Science, 1990 vol. 250 pp. 556-559.

It has been shown that the immunophilin-drug complexes interact with ternary protein targets as their mode of action. Schreiber et al., Cell, 1991, vol. 66, pp.

807-815. In the case of FKBP-FK506 and FKBP-CsA, the drug-immunophilin omplexes bind to the enzyme calcineurin, inhibitory T-cell receptor signalling 2 7/03/01.mc10625.speci, 1 leading to T-cell proliferation. Similarly, the complex of rapamycin and FKBP interacts with the RAFT1/FRAP protein and inhibits signalling from the IL-2 receptor.

Immunophilins have been found to be present at high concentrations in the central nervous system. Immunophilins are enriched 10-50 times more in the central nervous system than in the immune system. Within neural tissues, immunophilins appear to influence neuronal process extension, nitric oxide synthesis, and neurotransmitter release.

It has been found that picomolar concentrations of an immunosuppressant such as FK506 and rapamycin stimulate neurite out growth in PC12 cells and sensory nervous, namely dorsal root ganglion cells (DRGs). Lyons et al., Proc. of Natl. Acad. Sci., 1994 vol. 91, pp. 3191-3195. In whole animal experiments, FK506 has been shown to stimulate nerve regeneration following facial nerve injury and results in functional recovery in animals with sciatic nerve lesions.

Surprisingly, it has been found that drugs with a high affinity for FKBP are potent rotamase inhibitors causing a neurotrophic effect. Lyons et al. These findings suggest the use of immunosuppressants in treating various peripheral neuropathies and enhancing neuronal regrowth in the central nervous system (CNS). Studies have demonstrated that neurodegenerative disorders such as i" :20 Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS) may occur due to the loss, or decreased availability, of a neurotrophic substance :specific for a particular population of neurons affected in the disorder.

Several neurotrophic factors effecting specific neuronal populations in the central nervous system have been identified. For example, it has been 25 hypothesized that Alzheimer's disease results from a decrease or loss of nerve growth factor (NGF). It has thus been proposed to treat Alzheimer's patients with exogenous nerve growth factor or other neurotrophic proteins such as brain derived nerve factor (BDNF), glial derived nerve factor, ciliary neurotrophic factor, and neurotropin-3 to increase the survival of degenerating neuronal populations.

Clinical application of these proteins in various neurological disease states is hampered by difficulties in the delivery and bioavailability of large proteins to nervous system targets. By contrast, immunosuppressant drugs with neurotrophic are relatively small and display excellent bioavailability and specificity.

7.However, when administered chronically, immunosuppressants exhibit a number 27/03/01,mc10625.speci.2 3 of potentially serious side effects including nephrotoxicity, such as impairment of glomerular filtration and irreversible interstitial fibrosis (Kopp et al., 1991, J. Am.

Soc. Nephrol. 1:162); neurological deficits, such as involuntary tremors, or nonspecific cerebral angina such as non-localized headaches (De Groen et al., 1987, N. Engl. J. Med. 317:861); and vascular hypertension with complications resulting therefrom (Kahan et al., 1989 N. Engl. J. Med. 321: 1725).

In order to prevent the side effects associated with use of the immunosuppressant compounds, the present invention provides an intermediate for producing non-immunosuppressive compounds containing small molecule FKBP rotamase inhibitors for promoting neuronal growth and regeneration in various neuropathological situations where neuronal repair can be facilitated including peripheral nerve damage by physical injury or disease state such as diabetes, physical damage to the central nervous system (spinal cord and brain) brain damage associated with stroke, and for the treatment of neurological disorders relating to neurodegeneration, including Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis.

SUMMARY OF THE INVENTION S* This application is a divisional application of Australian application No.

20 61602/96 (hereinafter "the parent application"), the specification of which is herein incorporated by reference.

The present invention relates to an intermediate useful in the production of novel neurotrophic compounds having an affinity for FKBP-type immunophilins.

Once bound to this protein the neurotrophic compounds are potent inhibitors of the S 25 enzyme activity associated with immunophilin proteins and particularly rotamase enzyme activity, thereby stimulating neuronal regeneration and outgrowth. A key feature of the compounds of the parent invention is that they do not exert any significant immunosuppressive activity in addition to their neurotrophic activity.

More specifically the present invention provides the compound dioxo-3,3-dimethylpentyl)-2-pyrrolidine carboxylic acid. This compound may be used as an intermediate in the production of the compounds of the parent application.

T R A preferred embodiment of the parent application is a neurotrophic ound of the formula: c ound of the formula: 27/03/01,mc10625.speci,3

Y-Z

0

X

where Ri is selected from the group consisting of a Ci-C9 straight or branched chain alkyl or alkenyl group optionally substituted with C3-Cs cycloalkyl, C3 or Cs cycloalkyl, Cs-C7 cycloalkenyl, Arl, where said alkyl, alkenyl, cycloalkyl or cycloalkenyl groups may be optionally substituted with C 1

-C

4 alkyl, C1-C4 alkenyl, or hydroxy, where Ar is selected from the group consisting of 1-napthyl, 2-napthyl, 2-indolyl, 3-indolyl, 2-furyl, 3-furyl, 2thiazolyl, 2-thienyl, 3-thienyl, 4-pyridyl, and phenyl, having one to three substituents which are independently selected from the group consisting of hydrogen, halo, hydroxyl, nitro, trifluoromethyl, Ci-C6 straight or branched alkyl or alkenyl, C1-C4 alkoxy or C1-C4 alkenyloxy, phenoxy, benzyloxy, and amino; X is selected from the group consisting of oxygen, sulfur, methylene 15 (CH 2 or H 2 S.Y is selected from the group consisting of oxygen or NR 2 where R 2 is hydrogen or CI-C6 alkyl; and Z is H or is selected from the group consisting of C2-06 straight or branched chain alkyl or alkenyl, wherein the alkyl chain is substituted in one or more positions with Arl as defined above, C3-C8 cycloalkyl, cycloalkyl connected by a C1-C6 straight or unbranched alkyl or alkenyl chain, and Ar 2 where Ar 2 is selected from the group consisting of 2-indolyl, 3-indolyl, 2-furyl, 3-furyl, 2- thiazolyl, 2-thienyl, 3-thienyl, or 4-pyridyl, and phenyl, having one to three substituents which are independently selected from the group consisting of hydrogen, halo, hydroxyl, nitro, trifluoromethyl, C0-C6 straight or branched alkyl or alkenyl, Ci-C4 alkoxy or C1-C4 alkenyloxy, phenoxy, benzyloxy, and amino; Z may also be the fragment: 27/03/01,mc10625.speci,4 0 -CH X 2

-R

4 3 where

R

3 is selected from the group consisting of straight or branched alkyl C 1 Ca optionally substituted with C 3

-C

8 cycloalkyl, or Arl as defined above, and unsubstituted Ar l

X

2 is 0 or NRs, where R 5 is selected from the group consisting of hydrogen, C 1

-C

6 straight or branched alkyl and alkenyl;

R

4 is selected from the group consisting of phenyl, benzyl, CI-C straight or branched alkyl or alkenyl, and C 1

-C

5 straight or branched alkyl or alkenyl substituted with phenyl; or pharmaceutically acceptable salts or hydrates thereof.

Another preferred embodiment of the parent application is a neurotrophic compound of the formula:

N

0 where RI is a Ci-C 9 straight or branched chain alkyl or alkenyl group optionally substituted with C 3

-C

8 cycloalkyl, C 3 or C 5 cycloalkyl, C 5

-C

7 9 cycloalkenyl, or Arl, where said alkyl, alkenyl, cycloalkyl or cycloalkenyl groups may be optionally substituted with C 1

-C

4 alkyl, .:20 C0-C 4 alkenyl, or hydroxy, and where Arl is selected from the group consisting of 1-napthyl, 2-napthyl, 2-indolyl, 3-indolyl, 2-furyl, 3-furyl, 2-thiazolyl, 2-thienyl, 3-thienyl, or 4-pyridyl, or phenyl, having one to three substituents which are independently selected from the group consisting of hydrogen, halo, hydroxyl, nitro, trifluoromethyl,

CI-C

6 straight or branched alkyl or alkenyl, C 1

-C

4 alkoxy or C 1

-C

4 alkenyloxy, phenoxy, benzyloxy, and amino; f Z is H or is a C 2

-C

6 straight or branched chain alkyl or alkenyl, wherein the alkyl chain is substituted in one or more positions with Ar as 27/03/01,mc10625.speci.5 defined above, C 3

-C

8 cycloalkyl, cycloalkyl- connected by a-C 1

-C

6 straight or unbranched alkyl or alkenyl chain, or Ar 2 where Ar 2 is selected from the group consisting of 2-indolyl, 3-indolyl, 2-furyl, 3furyl, 2- thiazolyl, 2-thienyl, 3-thienyl, or 4-pyridyl, or phenyl, having one to three substituents which are independently selected from the group consisting of hydrogen, halo, hydroxyl, nitro, trifl uoromnethyl, Cl-C 6 straight or branched alkyl or alkenyl, Cl-C 4 alkoxy or CI-C 4 alkenyloxy, phenoxy, benzyloxy, and amino; or pharmaceutically acceptable salts or hydrates thereof.

Particularly preferred neurotrophic N-glyoxyl prolyl ester compounds according to the parent application are selected from the group consisting of: 5-dimethoxyphenyl -propyl (2S)-1 3-dimethyl-1 ,2-dioxopentyl pyrrol idinecarboxyl ate, 5-di methoxyph enyl1)- 1 -pro E)-enyl (2S)-1 3-dimethyl-1 ,2dioxopentyl)-2-pyrrolidine- carboxylate, 5-trimethoxyphenyl)-1 -ethyl (2S)-1 -(3,3-dimethyl-1 ,2-dioxopentyl pyrrolidinecarboxylate, 3-(3-Pyridyl)-1 -propyl (2S)-1 3-dimethyl-1 ,2-dioxopentyl)-2- .::20pyrrol idinecarboxyl ate, 00,203-(2-Pyridyl)-1 -propyl (2S)-1 3-dimethyl-1 ,2-dioxopentyl)-2pyrrolidinecarboxylate, 3-(4-Pyridyl)-1 -propyl (2S)-1 3-dimethyl-1 ,2-dioxopentyl)-2pyrrolidinecarboxylate, 3-phenyl-1 -propyl (2S)-1 -(2-tert-butyl-1 ,2-dioxoethyl pyrrolidinecarboxylate, 3-phenyl-1 -propyl (2S)-1 -(2-cyclo hexyl ethyl-1, ,2-d ioxoethyl1)-2pyrrolidinecarboxylate, 3-(3-pyridyl -propyl (2S)-1 -(2-cyclohexylethyl-1 ,2-dioxoethyl)-2- *:pyrrol idi necarboxylate, 303-(3-pyridyl)-1 -propyl (2S)-1 -(2-tert-butyl-1 ,2-dioxoethyl)-2pyrrolidi necarboxylate, 3, 3-diphenyl-1 -propyl (2S)-1 3-dimethyl-1 ,2-dioxopentyl)-2- \sTF pyrrolidinecarboxylate, 27/03/01,mcI0625.speci,6 3-(3-pyridyl)-1 -propyl (2S -(2-cyclohexyl-1 ,2-dioxoethyl pyrrolidinecarboxylate, 3-(3-Pyridyl)-l -propyl (2S)-N-([2-thienyl] glyoxyl)pyrrolidinecarboxylate, 3, 3-Diphenyl-1 -propyl (2S)-l 3-dimethyl-1 ,2-dioxobutyl pyrrol idi necarboxylate, 3, 3-Diphenyl-1 -propyl (2S)-l -cycl ohexyl glIyoxyl-2-pyrrol id ineca rboxy late, and 3, 3-Diphenyl-1 -propyl (2S)-1 -(2-thi enyl )glIyoxyl -2-pyrrolIid i neca rboxylIate.

Preferred compounds of the parent invention include R, groups which are not stereochemically bulky in relation to the known shape and size of the hydrophobic core of the FKBP active site. Thus, very large and/or highly substituted R, groups would bind with less affinity to the FKBP active site.

Preferred compounds of the parent invention include: 3-phenyl-l -propyl (2S)-l 3-di methyl-i ,2-dioxopentyl pyrrolidinecarboxylate, 3-phenyl-1 -prop-2-( E)-enyl (2S)-1 3-dimethyl-1 ,2-dioxopentyl pyrrol idinecarboxyl ate, 5-trimethoxyphenyl)-1 -propyl (2S)-1 3-dimethyl-1 ,2-dioxopentyl)-2- 5-trimethoxyphenyl)-1 -prop-2-(E)-enyl (2S)-1 -(3,3-dimethyl-1 ,2d ioxopentyl)-2-pyrrol idine carboxylate, 3-(4,5-methylenedioxyphenyl)-1 -propyl (2S)-1 -(3,3-dimethyl-1 ,2d i doxopentyl )-2-pyrrol id ineca rboxylIate, 3-(4,5-m ethylIe ned ioxyp he nyl)- 1 -pro p-2 -(E)-enyl (2S)-1 -(3,3-dimethyl-1 ,2d ioxopentyl )-2-pyrrol idinecarboxyl ate, 3-cyclohexyl-1 -propyl (2S)-1 3-dimethyl-1 ,2-dioxopentyl)-2pyrrolidinecarboxylate, 3-cyclohexyl-1 -prop-2-( E)-enyl (2S)-1 3-dimethyl-1 ,2-dioxopentyl pyrrolidinecarboxylate, (1 R)-1 ,3-diphenyl-1 -propyl (2S)-1 -(3,3-dimethyl-1 ,2-dioxopentyl)-2pyrrolidinecarboxylate, 3-phenyl-1 -propyl (2S 2-d ioxo-2-[2-fu ranyl ])ethyl -2pyrrolidinecarboxylate, 27/03/01 ,mc10625.speci,7 3-phenyl-1 -propyl (2S)-l ,2-dioxo-2-[2-thienyl])ethyl-2pyrrolidinecarboxylate, 3-phenyl-l -propyl (2S)-1 2-d ioxo-2-[2-thi azol yl ])ethyl -2pyrrolidinecarboxylate, 3-phenyl-l -propyl (2S)-1 2 -d ioxo-2 -phenyl)ethyl -2-pyrroIid ineca rboxyl ate, 5-dimethoxyphenyl -propyl (2S)-l 3-dimethyl-l, 2-dioxopentyl pyrrolidinecarboxylate, 5-dimethoxyphenyl -prop-2-(E)-enyl (2S)-1 3-dimethyl-l ,2dioxopentyl)-2-pyrrolidine- carboxylate, 5-trimethoxyphenyl)-l -ethyl (2S 3-dimethyl-l ,2-dioxopentyl)-2pyrrolidinecarboxylate, 3-(3-Pyridyl)-l -propyl (2S)-I 3-d im ethyl-1, 2-d ioxope ntyl)-2pyrrolidinecarboxylate, 3-(2-Pyridyi)-l -propyl (2S)-1 3-dimethyl-I ,2-dioxopentyl)-2pyrrolidinecarboxylate, 3-(4-Pyridyl)-I -propyl (2S)-l 3-dimethyl-I ,2-dioxopentyl)-2pyrrolidinecarboxylate, 3-phenyl-I -propyl (2S)-l -(2-cyclohexyl-I 2-dioxoethyl 20 pyrrolidinecarboxylate, 203-phenyl-1 -propyl (2S)-l -(2-tert-butyl-I ,2-dioxoethyl)-2pyrrolidinecarboxylate, :.3-phenyl-1 -propyl (2S)-l -cycloh exylIethyl-I 1,2-d ioxoethyl)-2pyrrol idi necarboxylate, 3-(3-pyridyl -propyl (2S)-I -(2-cyclohexyl ethyl-1, ,2-d ioxoethyl)-2pyrrolidinecarboxylate, 3-(3-pyridyl -propyl (2S)-I -(2-tert-butyl-I ,2-dioxoethyi)-2pyrrol id inecarboxylate, 3-diphenyi-l -propyl (2S 3-di methyl-I ,2-dioxopentyl)-2- 4: pyrrol id inecarboxylate, 303-(3-pyridyl -propyl (2S)-l -(2-cyclohexyl-1 ,2-dioxoethyl pyrrolidinecarboxylate, 3-(3-Pyridyl)-l -propyl (2S)-N-([2-thienyl] glyoxyl )pyrrolidinecarboxylate, 3,3-Diphenyl-1 -propyl (2S)-1 -(3,3-dimethyl-1 ,2-dioxobutyl)-2- <.rroidinecarboxylate, 27/03/01, mcl 0625.speci, 8 3, 3-Diphenyl-l -propyl (2S)-l -cycl ohexylIglIyoxyl-2-pyrrol id ineca rboxy late, 3, 3-Diphenyl-l -propyl (2S)-1 -(2-th ienyl )glIyoxyl-2-pyrroIid ineca rboxylIate.

Particularly preferred neurotrophic N-glyoxyl prolyl ester compounds of the parent invention are selected from the group consisting of: 3-(2,5-dimethoxyphenyl)-1 -propyl (2S)-l -(3,3-dimethyl-1 ,2-dioxopentyl)-2pyrrolidinecarboxylate, 5-dimethoxyphenyl)-I -prop-2-( E)-enyl (2S)-I 3-di methyl-I ,2dioxopentyl)-2-pyrrolidine- carboxylate, 5-trimethoxyphenyl)-1 -ethyl (2S)-1 3-dimethyl-1 ,2-dioxopentyl pyrrolidinecarboxylate, 3-(3-Pyridyl)-I -propyl (2S)-I 3-dimethyl-I ,2-dioxopentyl)-2pyrrol idinecarboxyl ate, 3-(2-Pyridyl)-1 -propyl (2S)-1 3-dimethyl-I ,2-dioxopentyl)-2pyrrol idi necarboxyl ate, 3-(4-Pyridyl)-1 -propyl (2S)-I 3-dimethyl-1 ,2-dioxopentyl)-2pyrrolidinecarboxylate, 3-phenyl-1 -propyl (2S)-I -(2-tert-butyl-I ,2-dioxoethyl)-2pyrrolidinecarboxylate, 3-phenyl-I -propyl (2S)-I -(2-cycloh exyl ethyl- 1 2-dioxoethyl)-2pyrrolidinecarboxylate, 3-(3-pyridyl -propyl (2S)-1 -(2-cyclohexylethyl-1 ,2-dioxoethyl)-2pyrrolidinecarboxylate, 3-(3-pyridyl)-1 -propyl (2S)-1 -(2-tert-butyl-1 ,2-dioxoethyl)-2pyrrolidinecarboxylate, 25 3,3-diphenyl-1 -propyl (2S)-I -(3,3-dimethyl-1 ,2-dioxopentyl)-2gobs pyrrolidinecarboxylate, 3-(3-pyridyl -propyl (2S)-1 -(2-cyclohexyl-1 ,2-dioxoethyl)-2-

S

pyrrolidinecarboxylate, 3-(3-Pyridyl)-1 -propyl (2S)-N-([2-thienyl] glyoxyl)pyrrolidinecarboxylate, 3,3-Diphenyl-1-propyl im ethyl 2-d ioxobutyl)-2pyrrol idinecarboxyl ate, 3,3-Diphenyl-I -propyl (2 S)-1 -cycl ohexyl glIyoxyl-2-pyrrol id inecarboxyl ate, and 3, 3-Di phenyl-1 -propyl (2S)-1 -(2-th ienyl )glIyoxyl -2-pyrroIid inecarboxylIate.

27/03/01, ,MCI0625.speci, 9 The following examples are illustrative of preferred embodiments of the invention and are not to be construed as limiting the invention thereto. All polymer molecular weights are mean average molecular weights. All percentages are based on the percent by weight of the final delivery system or formulation prepared unless otherwise indicated and all totals equal 100% by weight.

EXAMPLES

The compounds of the parent application and that of the present invention may be prepared by a variety of synthetic sequences that utilize established chemical transformations. The general pathway to the compounds is described in Scheme 1. N-glyoxylproline derivatives may be prepared by reacting L-proline methyl ester with methyl oxalyl chloride as shown in Scheme I. The resulting oxamates may be reacted with a variety of carbon nucleophiles to obtain intermediates compounds. These intermediates are then reacted with a variety of alcohols, amides, or protected amino acid residues to obtain the propyl esters and amides of the parent invention.

o* Scheme I o 0 RUor RMgX

OCH,

NH COOCH, OCH3 0 0 U H

Y-Y-

2 EXAMPLE 1 SSynthesis of 3-phenyl-1 -propyl (2S)-1 Coupmethyl-ing1, 2-dioxopentyl)-2- S'-rrolidinecarboxylate (Example 1).

R

EXAMPLE 1 Synthesis of 3-phenyl-1-propy (2S)-1 -(3,3-dimethyl-l ,2-dioxopentyl)-2- ^rrolidinecarboxylate (Example 1).

27/03/01,mc10625.speci,10 Synthesis of methyl (2S)-1-(1,2-dioxo-2-methoxyethyl)-2pyrrolidinecarboxylate.

A solution of L-proline methyl ester hydrochloride (3.08 g; 18.60 mmol) in dry methylene chloride was cooled to 0°C and treated with triethylamine (3.92 g; 38.74 mmol; 2.1 eq). After stirring the formed slurry under a nitrogen atmosphere for 15 min, a solution of methyl oxalyl chloride (3.20 g; 26.12 mmol) in methylene chloride (45 mL) was added dropwise. The resulting mixture was stirred at 0°C for hr. After filtering to remove solids, the organic phase was washed with water, dried over MgSO 4 and concentrated. The crude residue was purified on a silica gel column, eluting with 50% ethyl acetate in hexane, to obtain 3.52 g of the product as a reddish oil. Mixture of cis-trans amide rotamers; data for trans rotamer given. 'H NMR (CDCI 3 d 1.93 (dm, 2H); 2.17 2H); 3.62 2H); 3.71 3H); 3.79, 3.84 s, 3H total); 4.86 (dd, 1H, J 8.4, 3.3).

Synthesis of methyl (2S)-1-(1,2-dioxo-3,3-dimethylpentyl)-2pyrrolidinecarboxylate.

A solution of methyl (2S)-1-(1,2-dioxo-2-methoxyethyl)-2pyrrolidinecarboxylate (2.35 g; 10.90 mmol) in 30 mL of tetrahydrofuran (THF) S was cooled to -780C and treated with 14.2 mL of a 1.0 M solution of 1,1dimethylpropylmagnesium chloride in THF. After stirring the resulting homogeneous mixture at -780C for three hours, the mixture was poured into saturated ammonium chloride (100 mL) and extracted into ethyl acetate. The S* organic phase was washed with water, dried, and concentrated, and the crude S material obtained upon removal of the solvent was purified on a silica gel column, eluting with 25% ethyl acetate in hexane, to obtain 2.10 g of the oxamate as 25 a colorless oil. 1 H NMR (CDCI 3 d 0.88 3H); 1.22, 1.26 3H each); 1.75 (dm, 2H); 1.87-2.10 3H); 2.23 1H); 3.54 2H); 3.76 3H); 4.52 (dm, 1H, J 8.4, 3.4).

Synthesis of (2S)-1-(1,2-dioxo-3,3-dimethylpentyl)-2-pyrrolidinecarboxylic acid.

A mixture of methyl 2 2 -dioxo-3,3-dimethylpentyl)-2pyrrolidinecarboxylate (2.10 g; 8.23 mmol), 1 N LiOH (15 mL), and methanol mL) was stirred at 0°C for 30 min and at room temperature overnight. The mixture was acidified to pH 1 with 1 N HCI, diluted with water, and extracted into 100 mL of S4VT/iNmethylene chloride. The organic extract was washed with brine and concentrated 27/03/01,mc10625.speci, 1 12 to deliver 1.73 g of snow-white -solid which did not require further purification. 1 H NMR (CDCI 3 d 0.87 3H); 1.

2 2 1.25 3H each); 1.77 (din, 2H); 2.02 (in, 2H); 2.17 (in, 1 2.25 (in, 1IH); 3.53 (dd, 2H, J 10.4, 4.55 (dd, 1 H, J 8.6, 4. 1).

Synthesis of 3-phenyl-1 -propyl (2S)-1 3-dimethyl-1 ,2-dioxopentyl)-2pyrrolidinecarboxylate (Example A mixture of (2S)-i ,2-dioxo-3,3dimethylpentyl)-2-pyrrolidine-carboxylic acid (600 ing; 2.49 iniol), 3-phenyl-1 propanol (508 mg; 3.73 iniol), dicyclohexylcarbodijinide (822 mg; 3.98 minol), cainphorsuiphonic acid (190 mg; 0.8 iniol) and 4-diinethylaininopyridine (100 mg; 0.8 iniol) in methylene chloride (20 mL) was stirred overnight under a nitrogen atmosphere. The reaction mixture was filtered through Celite to remove solids and concentrated in vacuo, and the crude material was purified on a flash column ethyl acetate in hexane) to obtain 720 ing of Example 1 as a colorless oil. 1 H NMR (GOC1h): d 0.84 3H); 1.19 3H); 1.23 3H); 1.70 (din, 2H); 1.98 (in, 5H); 2.22 (in, 1IH); 2.64 (in, 2H); 3.47 (in, 2H); 4.14 (in, 2H); 4.51 1IH); 7.16 (in, 3H); 7.26 (in, 2H).

The method of Example 1 was utilized to prepare the following illustrative examples: Example 2: 3-phenyl-1 -prop-2-(E)-enyl (2S)-1 3-diinethyl-1, 2- 20 dioxopentyl)-2-pyrrolidinecarboxylate, 80%, 1 H NMR (360 Mhz, COC13): d 0.86 (t, 3 3H); 1. 21 3 1. 25 3 1. 54-2. 10 (in, 5 2.10-2.37 (in, 1 3.52-3.55 (in, 2H); 4.56 (dd, 1 H, J 3.8, 4.78-4.83 (in, 2H); 6.27 (in, 1 6.67 (dd, 1 H, J= 15.9); 7.13-7.50 (in, 25 Example 3: 3-(3,4,5-trimethoxyphenyl)-1 -propyl (2S)-I -(3,3-diinethyl-1 ,2dioxopentyl)-2-pyrrol idine-. carboxylate, 61 1 H NMR (CDO13): d 0.84 3H); 1. 3H); 1.24 3H); 1. 71 (din, 2H); 1. 98 (in, 5H); 2.24 (in, 1IH); 2.63 (in, 2H); 3.51 2H); 3.79 3H); 3.83 3H); 4.14 (in, 2H); 4.52 (in, 1 6.36 2H).

Example 4: 3 -(3,4,5-trimethoxyphenyl)-1 -prop-2-(E)-enyl (2S)-I diinethyl-1,2-dioxopentyl)-2pyrrolidine carboxylate, 66%, 1 H NMR (COCl 3 d 0.85 3H); 1.22 3H), 1.25 3H); 1.50-2.11 (in, 5H); 2.11-2.40 (in, 1 3.55 (in, 2H); 3.85 3H); 3.88 6H); 4.56 (dd, 1 4.81 (in, 2H); 6.22 (in, 1 6.58 (d, 1 H, J 16); 6.63 2H).

Example 5: 3 4 ,5-methylenedioxyphenyl)lpropyl (2S)-1-(3,3-dimethyl- 2 -dioxopentyl)-2-pyrrolidine- carboxylate,82%, 'H NMR (360 MHz, CDC1 3 d 27/03101,mc10625.speci, 12 13 0.86 1.22 3H); 1.25 3H); 1.60-2.10 (in, 5H); 3.36-3.79 (in, 2H); 4.53 (dd, 1 H, J 3.8, 4.61-4.89 (in, 2H); 5.96 2H); 6.10 (mn, 1 6.57 (dd, 1IH, J 15.8); 6.75 1 H, J 6.83 (dd, 1 H, J 1.3, 6.93 1 H).

Example 6: 5-methylenedioxyphenyl)-1 -prop-2-( E)-enyl (2S dimethyl-1,2-dioxopentyl)-2-pyrrolidinecarboxylate,82%, 1 H NMR (360 MHz,

CDCI

3 d 0.86 3H); 1.22 3H); 1.25 3H); 1.60-2.10 (in, 5H); 2.10-2.39 (in, 1 3.36-3.79 (mn, 2H); 4.53 (dd, 1 H, J 3.8, 4.61-4.89 (in, 2H); 5.96 2H); 6.10 (in, 1 6.57 (dd, 1 H, J 6.2, 15.8); 6.75 1IH, J 8. 6.83 (dd, 1 H, J= 1. 3, 8. 6.93 1 H).

Example 8: 3-cyclohexyl-1 -prop-2-(E)-enyl (2S)-1 -(3,3-dimethyl-1 ,2d ioxopentyl)-2-pyrrol id in ecarboxylIate, 92%, 'H NMR (360 MHz, CDCI 3 d 0.86 (t, 3H); 1. 13-1.40 (in 2 singlets, 9H total); 1.50-1.87 (in, 8H); 1.87-2.44 (in, 6H); 3.34-3.82 (in, 2H); 4.40-4.76 (in, 3H); 5.35-5.60 (in, 1 5.60-5.82 (dd, 1 H, J= 6.5,16).

Example 9: (1 R)-1 ,3-Diphenyl-1 -propyl (2S)-1 -(3,3-dimethyl-1 ,2d ioxopentyl)-2-pyrrol id inecarboxylIate, 90%, 'H NMR (360 MHz, CDCI 3 d 0.85 (t, 3H); 1.20 3H); 1.23 3H); 1.49-2.39 (in, 7H); 2.46-2.86 (mn, 2H); 3.25-3.80 (in, 2 4.42-4.82 (in, 1IH); 5.82 (td, 1 H, J 1. 8, 7.05-7.21 (mn, 3 7.21 -7.46 (in, 7H).

Example 10: 3-phenyl-1 -propyl (2S)-1 ,2-dioxo-2-[2-furanyl])ethyl-2pyrrolidinecarboxylate, 99%, 1 H NMR (300 MHz, CDCI 3 d 1.66-2.41 (in, 6H); 2.72 2H, J 3.75 (in, 2H); 4.21 (in, 2H); 4.61 (in, 1IH); 6.58 (in, 1 7.16-7.29 (in, 5H); 7.73 (in, 2H).

Example 11: 3-phenyl-1 -propyl (2S)-1 ,2-dioxo-2-[2-thienyl])ethyl-2pyrrolidinecarboxylate, 81 1 H NMR (300 MHz, CDCI 3 d 1.88-2.41 (in, 6H); 2.72 (din, 2H); 3.72 (in, 2H); 4.05 (in, 1 4.22 (in, 1 4.64 (in, 1 7.13-7.29 (in, 6H); 7.75 (din, 1 8.05 (in, 1 H).

Example 13: 3-phenyl-1 -propyl 1 2-di oxo-2-phenyl) ethyl -2pyrrolidinecarboxylate, 99%, 1 H NMR (300 MHz, GOC1h): d 1.97-2.32 (in, 6H); 2.74 2H, J 3.57 (in, 2H); 4.24 (in, 2H); 4.67 (in, 1 6.95-7.28 (in, 5H); 7.51 7.64 (in, 3H); 8.03-8.09 (in, 2H).

Example 14: 3-(2,5-dimethoxyphenyl)-1 -propyl (2S)-1 -(3,3-dimethyl-1 ,2dioxopentyl)-2-pyrrolidine- carboxylate, 99%, 1 H NMR (300 MHz, CDCI 3 d 0.87 (t, 1.22 3H); 1.26 3H); 1.69 (in, 2H); 1.96 (in, 5H); 2.24 (in, 1 2.68 (in, 27/03101, mcl 0625.speci, 13 14 2H); 3.55 (in, 2H); 3.75 3H); 3.77 3H); 4.17 (in, 2H); 4.53 1 6.72 (in, 3H).

Example 15: 3-(2,5-dimethoxyphenyl)-1 -prop-2-(E)-enyl (2S)-1 dimethyl-1,2-dioxopentyl)-2-pyrrolidine- carboxylate, 99%, 1 H NMR (300 MHz,

CDCI

3 d 0.87 3H); 1.22 3H); 1.26 3H); 1.67 (in, 2H); 1.78 (mn, 1 2.07 (in, 2H); 2.26 (in, 1 3.52 (mn, 2H); 3.78 3H); 3.80 3H); 4.54 (in, 1 4.81 (in, 2H); 6.29 (dt, 1 H, J 15.9); 6.98 1 H).

Example 16:- 2-(3,4,5-triinethoxyphenyl)-1 -ethyl (2S)-1 -(3,3-dimethyl-1 ,2dioxopentyl)-2-pyrrolidine- carboxylate, 97%, 1 H NMR (300 MHz, CDCI 3 d 0.84 3H); 1.15 3H); 1.24 3H); 1.71 (din, 2H); 1.98 (in, 5H); 2.24 (mn, 1H); 2.63 (in, 2H); 3.51 2H); 3.79 3H); 3.83 3H); 4.14 (mn, 2H); 4.52 (in, 1IH); 6.36 (s, 2H).

Example 17: 3-(3-Pyridyl)-1 -propyl (2S)-1 3-dimethyl-1,2-dioxopentyl)-2pyrrolidinecarboxylate, 80%, 1 H NMR (ODC1 3 300 MHz): d 0.85 3H); 1.23, 1.26 3H each); 1.63-1.89 (in, 2H); 1.90-2.30 (in, 4H); 2.30-2.50 (in, 1IH); 2.72 2H); 3.53 (in, 2H); 4.19 (mn, 2H); 4.53 (mn, 1IH); 7.22 (in, 1 7.53 (dd, 1IH); 8.45.

Example 18: 3-(2-Pyridyl)-1 -propyl (2S)-1 3-dimethyl-1 2-dioxopentyl)-2pyrrolidinecarboxylate, 88%, 1 H NMR (CDC1 3 300 MHz): d 0.84 3H); 1.22, 1.27 3H each); 1.68-2.32 (in, 8H); 2.88 2H, J 3.52 (in, 2H); 4.20 (in, 2H); 2o 4.51 (nm1H);709-7.19(m, 2H); 759 1 8.53 1H, J pyrrolidinecarboxylate, 91 1 H NMR (ODC1 3 300 MHz) d 6.92-6.80 (in, 4H); 6.28 (in, 1 5.25 1IH, J 4.12 (in, 1 4.08 3H); 3.79 3H); 3.30 (in, 2H); 2.33 (in, 1 1.85-1.22 (in, 7H); 1.25 3H); 1.23 3H); 0.89 3H, J= 25 Example 20:- 3-phenyl-1 -propyl (2S -(2-cyclohexyl-1 ,2-dioxoethyl)-2pyrrolidinecarboxylate, 91 1 H NMR (CDC1 3 300 MHz): d 1.09-1.33 (in, 1.62-2.33 (in, 12H); 2.69 2H, J 3.15 (din, 1 3.68 (in, 2H); 4.16 (in, 2H); 4.53, 4.84 1 H total); 7.19 (in, 3H); 7.29 (in, 2H).

Example 21: 3-phenyl-1 -propyl (2S)-1 -(2-tert-butyl-1 ,2-dioxoethyl)-2pyrrolidinecarboxylate, 92%, 1 H NMR (CDC 3 300 MHz): d 1.29 9H); 1.94-2.03 (in, 5H); 2.21 (in, 1IH); 2.69 (in, 2H); 3.50-3.52 (mn, 2H); 4.16 (in, 2H); 4.53 (mn, 1 H); (in, 3H); 7.30 (in, 2H).

27/03101,mcl0625.speci, 14 Example 22: 3-phenyl-1 -propyl (2S)-1 -(2-cyclohexyl- ethyl 1,2-d ioxoethyl)- 2-pyrrolidinecarboxylate, 97%, 'H NMR (CDC1 3 300 MHz): d 0.88(in, 2H); 1.16 (in, 4H); 1.43-1.51 (in, 2H); 1.67 (in, 5H); 1.94-2.01 (in, 6H); 2.66-2.87 (in, 4H); 3.62-3.77 (in, 2H); 4.15 (in, 2H); 4.86 (in, 1IH); 7.17-7.32 (in, Example 23:. 3-(3-pyridyl)-1 -propyl (2S)-1 -(2-cyclo- hexylethyl-1 2dioxoethyl)-2-pyrrolidinecarboxylate, 70%, 1 H NMR (ODC1 3 300 MHz): d 0.87 (in, 2H); 1.16 (in, 4H); 1.49 (in, 2H); 1.68 (in, 4H); 1.95-2.32 (in, 7H); 2.71 (in, 2H); 2.85 (in, 2H); 3.63-3.78 (in, 2H); 4.19 (in, 2H); 5. 30 (in, 1IH); 7.23 (in, 1 7.53 (in, 1 8.46 (in, 2H).

Exam pie 24: 3-(3-pyridyl -propyl (2S)-1 -(2-tert-butyl-1 ,2-dioxoethyl)-2pyrrolidinecarboxylate, 83%, 1 H NMR (CDCI 3 300 MHz): d 1.29 9H); 1.95-2.04 (in, 5H); 2.31 (in, 1 2.72 2H, J 3.52 (in, 2H); 4.18 (in, 2H); 4.52 (in, 1 7.19-7.25 (in, 1IH); 7.53 (in, 1 8.46 (in, 2H).

Example 25: 3, 3-diphenyl-1 -propyl (2S)-1 3-dimethyl-1 ,2-dioxopentyl)-2pyrrolid inecarboxyl ate, 99%, 'H NMR (CDC1 3 300 MHz): d 0.85 3H); 1.21, 1.26 3H each); 1.68-2.04 (in, 5H); 2.31 (in, 1 2.40 (in, 2H); 3.51 (in, 2H); 4.08 (in, 3H); 4.52 (in, 1IH); 7.18-7.31 (in, 1 OH).

Example 26: 3-(3-pyridyl)-1 -propyl (2S)-1 -(2-cyclo- hexyl-1 ,2-dioxoethyl)-2pyrrolidinecarboxylate, 88%, 1 H NMR (CDC1 3 300 MHz): d 1.24-1.28 (in, 1.88-2.35 (in, 11 2.72 2H, J 3.00-3.33 (din, 1IH); 3.69 (in, 2H); 4.19 (in, 2H); 4.55 (in, 1 7.20-7.24 (in, 1 7.53 (in, 1 8.47 (in, 2H).

Exam pie 27: 3-(3-Pyridyl)-1 -propyl (2S )-N-([2-thienyl] glyoxyl)pyrrolidinecarboxylate, 49%, 1 H NMR (CDCI 3 300 MHz): d 1.81 -2.39 (in, 6H); 2.72 (din, 2H); 3.73 (in, 2H); 4.21 (in, 2H); 4.95 (in, 1 7.19 (in, 2H); 7.61 (in, 1 7.80 1 8.04 1 8.46 (in, 2H).

Example 28: 3,3-Diphenyl-1 -propyl (2S)-1 -(3,3-diinethyl-1 ,2-dioxobutyl)-2pyrrolidinecarboxylate, 99%, 1 H NMR (ODC1 3 300 MHz): d 1.27 9H); 1.96 (in, 2H); 2.44 (in, 4H); 3.49 (in, 1 3.64 (in, 1 4.08 (in, 4H); 4.53 (dd, 1 7.24 (in 1OH).

Example 29: 3, 3-Diphenyl-1 -propyl (2 S)-1 -cyclohexyl glyoxyl-2pyrrolidinecarboxylate, 91 1 H NMR (ODC1 3 300 MHz): d 1.32 (in, 6H); 1.54- 2.41 (in, 1 OH); 3.20 (din, 1 3.69 (in, 2H); 4.12 (in, 4H); 4.52 1 7.28 (in,

H).

27/03/O1,mcl0625.speci, Example 30: 3,3-Diphenyl-1-propyl (2S)-1-(2-thienyl) glyoxyl-2pyrrolidinecarboxylate, 75%, 1 H NMR (CDCI 3 300 MHz): d 2.04 3H); 2.26 (m, 2H); 2.48 1H); 3.70 2H); 3.82-4.18 3H total); 4.64 1H); 7.25 (m, 11H); 7.76 (dd, 1H); 8.03 1H).

The requisite substituted alcohols may be prepared by a number of methods known to those skilled in the art of organic synthesis. As described in Scheme II, alkyl or aryl aldehydes may be homologated to phenyl propanols by reaction with methyl (triphenylphosphoranylidene)acetate to provide a variety of trans-cinnamates; these latter may be reduced to the saturated alcohols by reaction with excess lithium aluminum hydride, or sequentially by reduction of the double bond by catalytic hydrogenation and reduction of the saturated ester by appropriate reducing agents. Alternatively, the trans-cinnamates may be reduced to (E)-allylic alcohols by the use of diisobutylaluminum hydride.

Lithium aluminum Ph 3

P=CHCOOCH

3 hydride R-CHO R 'OH

THF

DiH 2 .m Lithium aluminum i Diisbutyaluminum Pd/C hydrideor hydride hdrideor Diisobutylaluminum hydride aR' OC H3

R

Scheme II Longer chain alcohols may be prepared by homologation of benzylic and higher aldehydes. Alternatively, these aldehydes may be prepared by conversion of the corresponding phenylacetic and higher acids, and phenethyl and higher 20 alcohols.

General procedure for the synthesis of acrylic esters, exemplified for methyl (3,3,5-trimethoxy)-trans-cinnamate: A solution of 3,4,5-trimethoxybenzaldehyde (5.0 g; 25.48 mmol) and methyl (triphenyl- phosphoranylidene)acetate (10.0 g; 29.91 mmol) in tetrahydrofuran (250 mL) was refluxed overnight. After cooling, the reaction mixture was diluted with 200 mL of ethyl acetate and washed with 2 x 200 mL of water, dried, and T i- concentrated in vacuo. The crude residue was chromatographed on a silica gel 27/03/01,mc10625.speci, 16 17 column, eluting with 25% ethyl acetate in hexane, to obtain 5.63 g of the cinnamate as a white crystalline solid, 1 H NMR (300 Mhz; CDCl3): d 3.78 3H); 3.85 6H); 6.32 1H, J 16); 6.72 2H); 7.59 1H, J 16).

General procedure for the synthesis of saturated alcohols from acrylic esters. Exemplified for (3,4,5-trimethoxy) phenylpropanol.

A solution of methyl (3,3,5-trimethoxy)-trans-cinnamate (1.81 g; 7.17 mmol) in tetrahydrofuran (30 mL) was added in a dropwise manner to a solution of lithium aluminum hydride (14 mmol) in THF (35 mL), with stirring and under an argon atmosphere. After the addition was complete, the mixture was heated to 750C for 4 hours. After cooling, it was quenched by the careful addition of 15 mL of 2N NaOH followed by 50 mL of water. The resulting mixture was filtered through Celite to remove solids, and the filter cake was washed with ethyl acetate. The combined organic fractions were washed with water, dried, concentrated in vacuo, and purified on a silica gel column, eluting with ethyl acetate to obtain 0.86 g of the alcohol as a clear oil, 1 H NMR (300 Mhz; CDCl3): d 1.23 (br, 1H); 1.87 (m, 2H); 2.61 2H, J 3.66 2H); 3.80 3H); 3.83 6H); 6.40 2H).

General procedure for the synthesis of trans-allylic alcohols from acrylic esters. Exemplified for (3,4,5-trimethoxy)phenylprop-2-(E)-enol.

A solution of methyl (3,3,5-trimethoxy)-trans-cinnamate (1.35 g; 5.35 mmol) 20 in toluene (25 mL) was cooled to -10°C and treated with a solution of diisobutylaluminum hydride in toluene (11.25 mL of a 1.0 M solution; 11.25 mmol).

S* The reaction mixture was stirred for 3 hrs at 00C and then quenched with 3 mL of methanol followed by 1 N HCI until the pH was 1. The reaction mixture was S extracted into ethyl acetate and the organic phase was washed with water, dried and concentrated. Purification on a silica gel column eluting with 25% ethyl acetate in hexane furnished 0.96 g of a thick oil, 'H NMR (360 Mhz; CDCI 3 d 3.85 3H); 3.87 6H); 4.32 2H, J 6.29 (dt, 1H, J 15.8, 6.54 1H, J 15.8); 6.61 2H).

The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention and all such modification are intended to be included within the scope of the following claims.

27/03/01,mc10625.speci, 17 The present application is a divisional application of Australian application No. 61062/96. The specification of which as published prior to acceptance is incorporated herein by reference.

@0 C 0000 0000 0 0000 00 0 0 000 0 00 0 0 0 0 000.0.

0 0 00 00 00 0 0 0 0 0@ 0e 00 0 00 00 27/03/01, ,mc0625.speci. 18

Claims (2)

1. A com 'pound which is (2S)-1 ,2-dioxo-3,3-dimethylpentyl)-2- pyrrolidine carboxylic acid.

2. A compound according to claim 1 substantially as hereinbefore described with reference to any one of the Examples. DATED this 27 t day of March, 2000 GUILFORD PHARMACEUTICALS, INC. By their Patent Attorneys: CALLINAN LAWRIE 27/03/01, mc1 0625.speci, 19