AU653175B2 - Carboxylic acid esters of rapamycin - Google Patents

Description

OPI DATE 15/04/92 AOJP DATE 28/05/92 APPLN. TD 86599 91 PCT NUMBER PCT/ IS91/0n624 INTERNA I fREATY(PCT) (51) International Patent Classification 5 (11) International Publication Number: WO 92/05179 C07D 498/18, C07K 5/06 A61K 31/395, 37/02 A (43) International Publication Date: 2 April 1992 (02.04.92) (C07D 498/18, 311/00, 273/00) (21) International Application Number: PCT/US91/06824 (74) Agents: ALICE, Ronald, W. et al.; American Home Products Corporation, 685 Third Avenue, New York, NY (22) International Filing Date: 19 September 1991 (19.09.91) 10017 (US).

Priority data: (81) Designated States: AT (European patent), AU, BE (Euro- 584,833 19 September 1990 (19.09.90) US pean patent), CH (European patent), DE (European pa- 589,878 28 September 1990 (28.09.90) US tent), DK (European patent), ES (European patent), FI, 657,294 19 February 1991 (19.02.91) US FR (European patent), GB (European patent), GR (European patent), HU, IT (European patent), JP, KR, LU (European patent), NL (European patent), SE (Euro- (71) Applicant: AMERICAN HOME PRODUCTS CORPOR- pean patent), SU' ATION [US/US]; 685 Third Avenue, New York, NY 10017 (US).

Published (72)Inventors: CAUFIELD, Craig, Eugene 30-08 Raven's With international search report.

Crest Drive, Plainsboro, NJ 08536 FAILLI, Amedeo, Arturo 14 Landing Lane, Princeton Junction, NJ 08550 STEFFAN, Robert, John 263 Wheat- 6 sheaf Lane, Langhorne, PA 19047

M

(54) Title: CARBOXYLIC ACID ESTERS OF RAPAMYCIN 0

II

R

4 is f-IC(CH 2 )mCH(CH 2 R1 5

R

6 (a) 0

II

(CH,),X(CH,'lC0 2

R'

R1 3 Xis 0.

R1 4 0 -C CO,R2 0

II

[C(CH)mCH(CH)nN1 I I

R

5 R6 (57) Abstract A compound of structure wherein RI, R 2 and R 3 are each, independently, hydrogen, or R 4

R

4 is or R is hydrogen, alkyl, aralkyl, -(CH 2 )qCO 2 R8, -(CH 2 )rNR9CO 2

R

io carbamylalkyl, aminoalkyl, hydroxyalkyl, guanylalkyl, mercaptoalkyl, alkylthioalkyl, indolylmethyl, hydroxypehnylmethyl, imidazoylmethyl or phenyl which is optionally mono-, di-, or tri-substituted with a substituent selected from alkyl, alkoxy, hydroxy, cyano, halo, nitro, carbalkoxy, trifluoromethyl, amino, or a carboxylic acid; R 6 and R 9 are each, independently, hydrogen, alkyl, or aralkyl; R 7

R

8 and R 10 are each, independently, alkyl, aralkyl, fluorenylmethyl, or phenyl which is optionally mono-, di-, or tri-substituted; RI and R 12 are each, independently, alkyl, aralkyl, or phenyl which is optionally mono-, di-, or tri-substituted; X is O, or S; R 13 and R 1 4 are each, independently, hydrogen or alkyl; Y is CH or N; m is 0-4; n is 0-4; p is 1-2; q is 0-4; r is 0-4; t is 0-4; u is 0-4; wherein Rs, R 6 m, and n are independent in each of(e) subunits when p=2; or a pharmaceutically acceptable salt thereof, with the proviso that RI, R 2 and R 3 are not all hydrogen, further provided that R 1

R

2 and R 3 are not all and still further provided that t and u are not both 0 when X is O or S, which by virtue of its immuno-suppressive activity is useful in treating transplantation rejection, host vs. graft disease, autoimmune diseases, and diseases of inflammation, and by virtue of its antifungal activity is useful in treating fungal infections.

See back of page WO 92/05179 PCT/US91/06824 -1- CARBOXYLIC ACID ESTERS OF RAPAMYCIN BACKGROUND OF THE INVENTION This invention relates to novel esters of rapamycin and a iethod for using them in the treatment of transplantation rejection, host vs. graft disease, autoimmune diseases, diseases of inflammation, and fungal infections.

Rapamycin is a macrocyclic triene antibiotic produced by Streptomyces hygroscopicus, which was found to have antifungal activity, particularly against Candida albicans, both in vitro and in vivo Vezina et al., J. Antibiot. 28, 721 (1975); S.N. Seghal et al., J. Antibiot. 28, 727 (1975); H. A. Baker et al., J. Antibiot.

31, 539 (1978); U.S. Patent 3,929,992; and U.S. Patent 3,993,749].

Rapamycin alone Patent 4,885,171) or in combination with picibanil Patent 4,401,653) has been shown to have antitumor activity. R. Martel et al.

[Can. J. Physiol. Pharmacol. 55, 48 (1977)] disclosed that rapamycin is effective in the experimental allergic encephalomyelitis model, a model for multiple sclerosis; in the adjuvant arthritis model, a model for rheumatoid arthritis; and effectively inhibited the formation of IgE-like antibodies.

The immunosuppressive effects of rapamycin have been disclosed in FASEB 3, 3411 (1989), rapamycin has been shown to be effective in inhibiting transplant rejection Patent Application Ser. No. 362,544 filed June 6, 1989). Cyclosporin A and FK-506, other macrocyclic molecules, also have been shown to be effective as immunosuppressive agents, therefore useful in preventing transplant rejection [FASEB 3, 3411 (1989); FASEB 3, 5256 (1989); and R. Y. Calne et al., Lancet 1183 (1978)].

Mono- and diacylated derivatives of rapamycin (esterified at the 28 and 43 positions) have been shown to be useful as antifungal agents Patent 4,316,885) and used to make water soluble prodrugs of rapamycin Patent 4,650,803).

Recently, the numbering convention for rapamycin has been changed; therefore according to Chemical Abstracts nomenclature, the esters described above would be at the 31- and 42- positions.

WO 92/05179 WO 92/05179PCT/1.S91 /06824 -2- DESCRIPTION OF THE INVENTION This invention provides derivatives of rapamycin which are useful as immunosuppressive, anti-inflarnmatory, and antifungal agents having the structure wherein R1, R 2 and R 3 are each, independently, hydrogen, orR4 0 11 R4 is C(CH 2 )mjCH(CH 2 )nN~pCO 2

R'

I I

R

5

R

0 1I

(CH

2 )tX(CH 2 )uCO 2 R'l ,or 0 -CL -t AC0 2

R

1 2

R

5 is hydrogen, alkyl of 1-6 carbon atoms, aralkyl of 7-10 carbon atoms, -(CH2)qCO2R 8 -(CH2)rNR 9 CO2Rl 0 carbamylalkyl of 2-3 carbon atoms, aminoalkyl of 1-4 carbon atoms, hydroxyaikyl of 1-4 carbon atoms, guanylalkyl of 2-4 carbon atoms, mercaptoalkyl of 1-4 carbon atoms, alkylthioalcyl of 2-6 carbon atoms, indolylmethyl, hydroxyphenylmethyl imidazolylinethyl or phenyl which is optionally mono-, di-, or tni-substituted with a substituent selected from alkyl of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms, hydroxy, cyano, halo, nitro, carbalkoxy of 2-7 carbon atoms, trifluoromethyl, amino, or a carboxylic acid;

R

6 and R 9 are each, independently, hydrogen, alkyl of 1-6 carbon atoms, or aralkyl of 7-10 carbon atoms; WO 92/05179 PCT/US91/06824 -3-

R

7

R

8 and R 10 are each, independently, alkyl of 1-6 carbon atoms, aralkyl of 7-10 carbon atoms, fluorenylmethyl, or phenyl which is optionally mono-, di-, or trisubstituted with a substituent selected from alkyl of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms, hydroxy, cyano, halo, nitro, carbalkoxy of 2-7 carbon atoms, trifluoromethyl, amino, or a carboxylic acid;

R

11 and R 12 are each, independently, alkyl of 1-6 carbon atoms, aralkyl of 7-10 carbon atoms, or phenyl which is optionally mono-, di-, or tri-substituted with a substituent selected from alkyl of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms, hydroxy, cyano, halo, nitro, carbalkoxy of 2-7 carbon atoms, trifluoromethyl, amino, or a carboxylic acid;

R

13 Xis O, orS;

R

1 4

R

13 and R 14 are each, independently, hydrogen or alkyl of 1-6 carbon atoms; Y is CH or N; m is 0 4; n is 0 4; pis 1-2; qis 0 4; r is 0 4; t is 0 4; u is 0 4; 0 wherein R 5

R

6 m, and n are independent in each of the [C(CH 2 )mCH(CH 2 )nN] I I

R

5 R6 subunits when p 2; or a pharmaceutically acceptable salt thereof, with the proviso that R 1

R

2 and R 3 are not all hydrogen, further provided that R 1

R

2 and R 3 are not all

O

II

[C(CH

2 )mCH(CH 2 )nN]pCO 2

R

7 and still further provided that t and u are not I I

R

5

R

6 both 0 when X is O or S.

WO 92/05179 PCT/US91/06824 -4- 0 Of the compounds when R 4 is [C(CH 2 )mCH(CH 2 )nN]pCO 2

R

7 I I

R

5

R

6 preferred members are those in which m 0, n 0, and p 1; m 0, n 0, and p 2; n 0, and R 5 is -(CH2)qCO2R 8 m 0, n 0, and R 5 is -(CH2)rNR 9

CO

2 R1 0 and m 0, n 0, and R 5 is hydrogen. Preferred compounds also include those 0

II

members in which R 4 is -C-(CH 2

)X(CH

2

)CO

2 R" The pharmaceutically acceptable salts may be formed from inorganic ations such as sodium, potassium, and the like; mono-, di-, and trialkyl amines of 1-6 carbon atoms, per alkyl group and mono-, di-, and trihydroxyalkyl amines of 1-6 carbon atoms per alkyl group; and organic acids such as acetic, lactic, citric, tartaric, succinic, maleic, malonic, gluconic, and the like. Preferred basic salts are formed from sodium cations and tris(hydroxymethyl)methylamine.

The compounds of this invention can be prepared by acylating rapamycin with an acylating agent having the general structures 0 0 II o I I Z-[C(CH2)mCH(CH2)nN]pCO2R 7 Z-C-(CH2)tX(CH2)uC

O

2R 1 1 or

R

5

R

6 0 Z-C- CO- 2

R

12 tYJ where Z is OH in the presence of a coupling reagent, such as dicyclohexylcarbodiimide. The compounds of this invention also can be prepared using an anhydride or a mixed anhydride of the above described carboxylic acid as the acylating species. Alternatively, the acylating species can be an acid halide, where Z can be Cl, Br, or I. The acylating groups used to prepare the compounds of this invention are commercially available or can be prepared by methods that are disclosed in the literature.

Where it is desired to prepare acyl derivatives having two or three different R 4 groups then sequential acylation may be performed using appropriate acylating agents as defined above, if necessary isolating the desired product by appropriate purification WO 92/05179 PCT/US91/06824 techniques. In general the 42-position is acylated first and such a monoacylated product may be isolated prior to the second acylation and so forth. Appropriate protecting groups may be used to block any position where acylation is not requ: Immunosuppressive activity was evaluated in an in vitro standard pharmacological test procedure to measure lymphocyte proliferation (LAF) and in two in vivo standard pharmacological test procedures. The first in vivo procedure was a popliteal lymph node (PLN) test procedure which measured the effect of compounds of this invention on a mixed lymphocyte reaction and the second in vivo procedure evaluated the survival time of a pinch skin graft.

The comitogen-induced thymocyte proliferation procedure (LAF) was used as an in vitro measure of the immunosuppressive effects of representative compounds.

Briefly, cells from the thymus of normal BALB/c mice are cultured for 72 hours with PHA and IL-1 and pulsed with tritiated thymidine during the last six hours. Cells are cultured with and without various concentrations of rapamycin, cyclosporin A, or test compound. Cells are harvested and incorporated; radioactivity is determined.

Inhibition of lymphoproliferation is assessed in percent change in counts per minute from non-drug treated controls. The results are expressed by the following ratio, or as the percent inhibition of lymphoproliferation of 1 gM.

3 H-control thvmus cells H 3 -rapamycin-treated thvmus cells 3 H-control thymus cells H 3 -test compound-treated cells A mixed lymphocyte reaction (MLR) occurs when lymphoid cells from genetically distinct animals are combined in tissue culture. Each stimulates the other to undergo blast transformation which results in increased DNA synthesis that can be quantified by the incorporation of tritiated thymidine. Since stimulating a MLR is a function of disparity at Major Histocompatibility antigens, an in vivo popliteal lymph node (PLN) test procedure closely correlates to host vs. graft disease. Briefly, irradiated spleen cells from BALB/c donors are injected into the right hind foot pad of recipient C3H mice. The drug is given daily, p.o. from Day 0 to Day 4. On Day 3 and Day 4, tritiated thymidine is given b.i.d. On Day 5, the hind popliteal lymph nodes are removed and dissolved, and radioactivity counted. The corresponding left PLN serves as the control for the PLN from the injected hind foot. Percent suppression is calculated using the non-drug treated animals as allogenic control.

Rapamycin at a dose of 6 mg/kg, p.o. gave 86% suppression, whereas cyclosporin A at the same dose gave 43% suppression. Results are expressed by the following ratio: WO 92/05179 PCT/US91/06824 -6- 3 H-PLN cells control C3H mouse 3 H-PLN cells rapamycin-treated C3H mouse 3 H-PLN cells control C3H mouse 3 H-PLN cells test compound-treated C3H mouse The second in vivo test procedure is designed to determine the survival time of pinch skin graft from male DBA/2 donors transplanted to male BALB/c recipients. The method is adapted from Billingham R.E. and Medawar J. Exp. Biol. 28:385- 402, (1951). Briefly, a pinch skin graft from the donor is grafted on the dorsum of the recipient as a homograft, and an autograft is used as control in the same region. The recipients are treated with either varying concentrations of cyclosporin A as test control or the test compound, intraperitoneally. Untreated recipients serve as rejection control.

The graft is monitored daily and observations are recorded until the graft becomes dry and forms a blackened scab. This is considered as the rejection day. The mean graft survival time (number of days of the drug treatment group is compared with the control group.

The following table summarizes the results of representative compounds of this invention in these three standard test procedures.

TABLE 1 Compound Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Example 8 Example 9 Example 10 Example 11 Example 12 Example 13 Example 14 Example 15

LAF*

(ratio) 1.8 0.33 0.20 4.9 0.006 5.4 3% at lltM** 0.03 0.96 2.0 0.004 19.8 22% at lM* 0.37 0.9

PLN*

L(rfiQ) 0.61 0.62 0.18 0.33 0.41 1.34 0.96++ -2.87 0.69 Skin Graft (days SD) 12.0 1.6 11.5 0.6 9.0 0.9 12.3 8.8 0.9 11.5 7.7 10.3 0.8 12.7 1.2 10.5 1.3 12.0 7.0 0.6 8.2+ 1.2 10.7 1.2 WO 92/05179 PCr/US91/06824 -7- TABLE 1 (Continued) Compound Example 16 Example 17 Example 18 Example 19 Example 20 Example 21 Example 22 Example 23 Example 24 Rapamycin

LAF*

(ratio) 3.27 0.56 0.02 0.01 0.97 0.22 0.22 0.18 0.00 1.0

PLN*

(ratio) 1.04## 1.68### 1.11## 0.48 0.70 -1.93 0.41 0.39 0.09 1.0 Skin Graft (days SD) 12.7 0.9 10.2 1.7 8.0+ 1.7 8.0 0.9 9.3 1.6 12.0 1.7 10.2 1.2 10.8 0.8 7.8 1.7 12.0 1.7 Calculation of ratios was described supra.

Result expressed as percent inhibition of lymphoproliferation at 1 [IM.

Not evaluated Results obtained using cremophore/ethanol as a vechicle for administration.

Ratios of 0.33 and 1.07 were also obtained using carboxymethyl cellulose as a vehicle for administration.

Results obtained using cremophore/ethanol as a vechicle for administration.

Ratios of 0.20 and 1.08 also were obtained using carboxymethyl cellulose as a vehicle for administration.

A ratio of 0.42 also was obtained for this compound.

The results of these standard pharmacological test procedures demonstrate immun- .uppressive activity both in vitro and in vivo for the compounds of this invention. Positive ratios in the LAF and PLN test procedures indicate suppression of T cell proliferation. As a transplanted pinch skin grafts are typically rejected within 6-7 days without the use of an immunosuppressive agent, the increased survival time of the skin graft when treated with the compounds of this invention further demonstrates their utility as immunosuppressive agents. While it appears that the compound disclosed by Examples 12 and 21 may cause T cell proliferation in the PLN test procedure, it is believed a negative ratio in this test procedure coupled with an increased survival time observed in the skin graft test procedure indicates a proliferation of Tsuppressor cells, WO 92/05179 PCT/US91/06824 0 which are implicated in suppressing the immune response. (see, I. Roitt et al.

Immunology, C.V.Moseby Co. 1989, p 12.8-12.11).

Antifungal activity of the compounds of this invention was measured against strains of Candida albicans using a plate test procedure for measurement of inhibition.

The following represents the typical procedure used. Compound to be tested was placed on sterile dried 1/4" plate disks, and allowed to dry. Agar plates were seeded with fungi and allowed to solidify. The impregnated disks were placed on the seeded Agar surface and incubated for the time required for the particular culture. Results are expressed in MIC gg/ml) to inhibit growth. The results of this test procedure showed that the compounds of this invention have antifungal activity; however, it was surprising that the compounds of this invention were less active than the parent compound, rapam ycin.

Table 2* Strain of Candida albicans Compound ATCC 10231 ATCC 38246 Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Example 8 Example 9 Example 10 Example 11 Example 12 Example 13 Example 14 Example 15 Example 16 Example 17 Example 18 Example 19 0.4 0.1 0.4 0.1 0.4 0.1 0.4 0.4 0.2 0.4 0.2 0.4 0.4 0.4 0.2 0.4 0.4 0.4 0.2 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.1 0.2 0.4 ATCC 38247 0.4 0.2 0.4 0.1 0.4 0.2 0.4 0.4 0.2 0.4 0.1 0.4 0.4 0.4 0.4 0.4 0.4 ATCC 38248 >0.4 0.2 >0.4 0.1 >0.4 0.4 >0.4 >0.4 0.4 >0.4 0.2 >0.4 >0.4 0.4 0.1 0.2 >0.4 3669 >0.4 0.1 0.4 0.2 >0.4 >0.4 >0.4 >0.4 0.4 >0.4 0.4 >0.4 >0.4 0.4 0.1 0.4 >0.4 0.4 0.4 >0.4 >0.4 WO 92/05179 PCT/US91/06824 -9- Table 2* (Continued) Strain of Candida albicans Compound ATCC 10231 ATCC 38246 ATCC 38247 ATCC 38248 366 Example 20 0.1 0.4 0.1 0.1 0.2 Example 21 0.4 0.4 0.4 >0.4 >0.4 Example 22 0.2 0.4 0.2 0.4 >0.4 Example 23 0.1 0.4 0.2 0.4 >0.4 Exaxple 24 0.4 0.4 >0.4 >0.4 >0.4 Rapaiycin 0.003 0.025 0.003 0.006 0.025 expressed as MIC pg/ml) not evaluated Based on the results of these standard pharmacological test procedures, the compounds are useful in the treatment of transplantation rejection such as, heart, kidney, liver, bone marrow, and skin transplants; autoimmune diseases such as, lupus, rheumatoid arthritis, diabetes mellitus, myasthenia gravis, and multiple sclerosis; and diseases of inflammation such as, psoriasis, dermatitis, eczema, seborrnea, inflammatory bowel di'sese; and fungal infections.

The compounds may be administered neat or with a pharmaceutical carrier to a mammal in need thereof. The pharmaceutical carrier may be solid or liquid.

A solid carrier can include one or more substances which may aiso act as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet-disintegrating agents; it can also be an encapsulating material. In powders, the carrier is a finely divided solid which is in admixture with the finely divided active ingredient. In tablets, the active ingredient is mixed with a carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain up to 99% of the active ingredient. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.

Liquid carriers are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid carrier WO 92/05179 PCT/US91/06824 can contain rther suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid carriers for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils fractionated coconut oil and arachis oil). For parenteral administration, the carrier can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are useful in sterile liquid form compositions for parenteral administration. The liquid carrier for pressurized compositions can be halogenated hydrocarbon or other pharmaceutically acceptable propellent.

Liquid pharmaceutical compositions which are sterile solutions or suspensions can be utilized by, for example, intramuscular, intraperitoneal or subcutaneous injection. Sterile solutions can also be administered intravenously. The compound can also be administered orally either in liquid or solid composition form.

Preferably, the pharmaceutical composition is in unit dosage form, e.g. as tablets or capsules. In such form, the composition is sub-divided in unit dose containing appropriate quantities of the active ingredient; the unit dosage forms can be packaged compositions, for example, packeted powders, vials, ampoules, prefi. A syringes or sachets containing liquids. The unit dosage form can be, for example, a capsule or tablet itself, or it can be the appropriate number of any such compositions in package form. The dosage to be used in the treatment must be subjectively determined by the attending physician.

In addition, the compounds of this invention may be employed as a soluton, cream, or lotion by formulation with pharmaceutically acceptable vehicles containing 0.1- 5 percent, preferably of active compound which may be administered to a fungally affected area.

The following examples illustrate the preparation of representative compounds of this invention.

WO 92105179 WO 9205179PCI'/US91 /06824 Exa2niple 1 Rapamvcin-42-ester with N-r1 .1 -dimethylethoxv)carbonvUl-glycvlglvcine Under anhydrous conditions, a solution of rapamycin (3 g, 3.28 mmole) Lind N-[(1,1-dimethylethoxy)carbonyl]-glycylglycine (3.04 g, 13.1 mrnole) in 40 mL of anhydrous dichioromethare was treated with dicyclohexylcarbodiimide (1.35 g, 6.56 mmoie) followed by 4-dimethylaminopyridine (0.8 g, 6.56 mmole). After stirring at ambient temperature for 48 hours, the precipitated solid was collected and washed with dichioromethane. The combined filtrates were absorbed directly onto silica gel Merck by adding the gel and evaporation to dryness. Flash chromatography of the preabsorbedA material (using a. gradient elution with ethylacetate-toluene from 2:1 to vlv) nfforded 1.05 g (28.3 of the title compound isolated as a three quarter toluene solvate, along with the 31,42-diester of Example 2. HPLC analysis showed that the monoester is a 8.3:1 mixture of two conformers.

I H NMR (CDCl 3 400 MHz): 8 1.46 (in, 9H, COOBut), 1.654 3H,

CH

3 1.751 3H, 013C=0), 3.14 3H1, CH 3 3.33 3H, 01130), 3.36 3H, 01130), 4.18 1H1, CHOH), 4.75 (in, 1H, 42-CHIO), 4.79 1H1, OH); High R1,es. MS (neg. ion FAB) Calcd for C 6 0

)H

93

N

3 01 7 1127.6504, measured mass 1127.6474.

Anal. Calcd for C6 0 H93N30 1 7 0.75 PhCH3: C, 65.45; H, 8.33; N, 3.51 Found: C, 65,23; H, 8.32; N, 3.86 The following representative compounds can be prepared ftrm rapamycin and the appropriate terminally-N-substituted amino acid by employing the method used to prepare the title compound in Example 1.

Rapaxnycin-42-ester with N-[(fluorenylmethoxy)carbonyliI-alanylserine Rapamycin-42-ester with N-[(fluorenylmethoxy)carbonyl]-glycylglycine Rapainycin-42-ester with N-[(ethoxy)carbonyl]-arginylmnethionine Rapaxnycin-42-ester with N-[(4'-chlorophenoxy)carbonylI-histidylarginine Rapamycin-42-ester with N-I(phenoxy)carbonyl]-tryptophanylleucine Rapamycin-42-ester with N-[(phenylmethoxy)carbonyl)]-N-methylglycyl-Nethylalanine WO 92/05179 WO 9205179PCTIUS9I /06824 -12- Rapaniycin-42-ester with N-[(phenylmethoxy)carbonyll-N-methyl-3alanylphenylalar-* .,e Rapaznycin-42-ester with ,1-dimethylethoxy)carbonyl]-cysteinylAglycine Example 2 Rapaniycin-3 1 42-diester with 1.1 -dimethylethoxv)carbonyll-glycylglvcine The title compound (1.85 g, 42%) was separated from the 42-monoester as described in Example 1 and isolated as a three quarter toluene solvate. I-PLC analysis showed that the diester is a 8. 1:1 mixture of conformers.

1 H NMR (CDCL3, 400 MHz): 5 1.452 (in, 18H, COOBut), 1.6612 3H, CH3C=C), 1.7815 3H, CH3C=C), 3.14 3H, 3.34 3H, OCH3), 3.35 3H, OCH3), 4.52 1H, OH), 4.79 (in, 1H, 42-CHO High Res. MIS (neg.

ion FAB): Calcd for C69H107IN5021 1341.7458, measured mass: 1341.7463.

Anal. Calcd for C69H107N5021 0.75 PhCH3: C, 63.17; H, 8.06; N, 4.96 Found: C, 62.83; H, 8.09; N, 5.00 The following representative compounds can be prepared from rapamycin and the appropriate terminally-N-substituted amino acid by employing the method used to prepare the title compound in Example 2.

Rapamycin-3 1,42-diester with N-[(fluorenylmethoxy)carbonyl]-alanylserine Rapamycin-3 1,42-diester with N-[(fluorenylmethoxy)carbonyl]-glycylglycine Rapamycin-31 ,42-diester with N-[(ethoxy)carbonyl3-arginylmethionine Rapamycin-3 1,42-diester with N-[(4'-chlorophenoxy)carbonyl]-histidylarginine Raparnycin-3 1,42-diester with N-I(phenoxy)carbonyl]-tryptophanylleucine Rapainycin-3 1,42-diester with N-[(phenylmethoxy)carbonyl)]-N-methylglycyl-Nethyl-alanine Rapamycin-3 1,42-diester with N-[(phenylmethoxy)carbony]-N-methyl-palanylpheriyl- alanine Rapamycin-3 1,42-diester with I -dimethylethoxy)carbonyl] -cysteinylglycine WO 92/05179 WO 92/51 79PCT/!US9 1/06824 13 Example 3 Rapamvcin-3 1 42-diester with 1.1-dimethvlethoxy)carbonvll-N-methyl glvcn Under anhydrous conditions, an ice cold solution of rapamycin (2 g, 2.18 mmole) and Na-Boc sarcosine (1.65 g, 8.75 mmole) in 20 ml of anhydrous dichioromethane was treated with dicyclohexylcarbodiimide (1.8 g, 8.7 mmole) followed by 4-dimethylaminopyridine (1 g, 8.7 mmole). After stirring overnight at ambient temperature, the precipitated solid was collected and washed with dichloromethane. The combined filtrates were evaporated to dryness to give an amorphous amber solid (3 The crude product was purified by flash chromatography on silica Merck 60, elution with hexane-ethylacetate 1: 1, vlv) to provide the title compound (0.75 g, 27.4%) along with the 42-monoester of Example 4. HPLC analysis showed that the diester is a 19.8:1 mixture of two conformers. The multiplicity of the NMR peaks suggests the presence of amide rotamers.

1 H NMR (CDCl3, 400 MHz): 5 1.411, 1.438, 1.448 and 1.474 (in, 18 H, COOBut), 2.91 (in, 6H1, NCH3), 3.14 3H, CH3O), 3.34 3H, CH3O), 3.37 (s, 3H, CH3O), 4.73 (broad, 1H1, 42-CHO), 4.82 (2s, 1H1, OH); High Res. MS (neg. ion FAB): Calcd. for C67H1105N3019 1255.7342, measured mass 1255.7289.

Anal. Calcd for C67H1105N3019: C, 64.04- H, 8.42; N, 3.34 Found: C, 64.14; H1, 8.74; N, 3.63 The following representative compounds can be prepared from rapamycin and the appropriate terminally-N-substituted amino acid by employing the method used to prepare the title compound in Example 3.

Rapaxnycin-3 1,42-diester with N-[(ethoxy)carbonyl]-tyrosine Rapamycin-3 1,42-diester with N-[(fluorenylmethoxy)carbonyl]-phe.-ylalanine Rapamycin-3 1,42-diester with N-[(3',4',5'-trihydroxyphenoxy)carbonyl]-isoleucine Rapamycin-3 1,42-diester with ,1-dimethylethoxy)carbonyl)-glutaniine Rapamycin-3 1,42-diester with N-[(phenoxy)carbonyl]-N-methylalanine Rapainycin-3 1,42-diester with N-[(propyloxy)carbonyl-4-aiinob&* i acid Rapamycin-3 1,42-diester with N-[(phenylmethoxy)carbonyl]-7-aminoheptanoic acid Rapamycint-3 1,42-diester with N-[(fluorenylmethoxy)carbonyl]-serine WO 92/05179 WO 92/51 79PCI'/US91/06 i24 -14- Example 4 Rapamycin-42-ester with 1 1-dimethvlethoxv)carbonyll-N-methyl glycine Under anhydrous conditions, an ice cold solution of rapamycin (0.95 g, 1.02 mmole) and N(a-Boc sarcosine (0.21 g, 1.1 mmole) in 20 niL of anhydrous dichioromethane was treated with dicyclohexylcarbodlimide 0.21 g, 1 mmole) followed by 4-dimethylamninopyridine (0.12 g, 1 mmole). After stirring for 4 hours at ambient temperature, the precipitated solid was collected and washed with dichioromethane. The combined filtrates were concentrated in vacuo to give an amorphous amber solid. Flash chromatography of the crude product (on silica Merck 60, elution with hexaneethylacetate 1:1 v/v to remove the diester of Example 3, followed by chloroformethylacetate-methanol 75:25:1 v/v) provided partially purified title compound (0.38 g, Pure product was obtained by preparative HPLC (Waters Prep 500, silica gel, chioroformn-ethylacetate-methanol 75:25:1 v/v, flow rate 250 mL/rnin). HPLC analysis showed that the ester is a 6.6:1 mixture of two conformers. The multiplicity of NMR peaks suggests the presence of amide rotamers.

1 H NMR (CDCI3, 400 MHz): 8 1.42-1.46 (ds, 9H, CC)OBut), 2.91 (ds, 3H, NCH3), 1.644 3H, CH3C=C), 1.738 3H, CH3C=C), 3.12 3H, 3.32 3H, CH3O), 3.35 3H, CH3O), 4.18 1H, CHOB), 4.71 (broad, 1H, 42-CHO), 4.78 (broad s, 1H, OH); High Res. MS (neg. ion FAB): Calcd for C59H92N2O16 1084.6446, measured mass 1084.6503.

Anal. Calcd for C59H92N20 16: C, 65.29; 8.54; N, 2.58 Found: C, 65.25; H, 8.52; N, 2.42 The following representative compounds can be prepared from raparnycin and the appropriate terminally-N-substituted amino acid by employing the method used to prepare the title compound in Example 4.

Raparnycin-42-ester with N-[(ethoxy)carbonyl]-qtrosine Raparnycin-42-ester with N-[(fluorenylmethoxy)carbonyl]-phenylalanine Rapamycin-42-ester with N-Ij(3,4',5'-trihydroxyphenoxy)carbonyl]-isoleucine Rapaniycin-42-ester with 1,1-dimethylethoxy)carbonyl)-giutamine Rapamycin-42-ester with N-[(phenoxy)carbonyl]-N-methylalanine Rapamycin-42-ester with N-[(propyloxy)carbonyl]-4-aminobutryic acid Rapamycin-42-ester with N-[(phenylmethoxy)carbonyl]-7-aminoheptanoic acid WO 92/05179 WO 9205179PCTr/US9I /06824 Rapamycin-3 1,42-diester with N-[(fluorenylmethoxy)carbonyl~serine Example Rapamycin-31I 42-diester with 5-0 11 -dimethvlethoxy)-2-rr(l 1I -dimethylethoxy)acid Under anhydrous conditions, an ice cold solution of rapamycin (4 g, 4.37 wmole) and L-glutarnic acid Na-Boc-y~-tert-butylester (4.9 g, 16.1 mmole) in 40 niL of dry dichioromethane was treated with dicyclohexylcarbodiimide (1.8 g, 8.7 mmole) followed by 4-dimethylaminopyridine (1 g, 8.7 mmole). After stirring overnight at room temperature, the precipitated solid was collected and washed with dichloromethane. The combined filtrates were concentrated in vacuo to provide 11 g of an amorphous amber solid. The crude product was purified by flash chromatography (on silica Merck 60, gradient elution with hexane-ethylacetate from 2: 1 to 1: 1, v/v) to yield 4.52 g of the title compound along with the 42-monoester of Example 6.

HPLC analysis showed that the diester consists of a 6.6:1 mixture of two conformers.

1 H NMR (CDCI 3 400 MHz): 8 1.42 (in, 36 H, COOBut), 1.646 3W,

CH

3 1.701 3H, CH 3 3.13 3H, CH 3 3.34 3H, CH 3 0), 3.36 3H, CH3O), 4.735 (in, 2H, OH+42-CH-O); igh Res. MS (neg. ion FAB): calc. for C 7 9

H

1 25

N

3 0 2 3 1483.87 15, measured mass 1483.87 14.

Anal. Calcd for C 7 9 H1 1 2 5

N

3 0 2 3 C, 63.90; H, 8.49; N, 2.83 Found: C, 63.63; H, 8.41; N, 2.44 The following representative compounds can be prepared from rapaniycin and the appropriately terminally-N-substituted amino diacid monoester by employing the method used to prepare the title compound in Example Rapamycin-3 1,42-diester with 6-(phenylmethoxy)-2-[[fluorenylmethoxy)carbonyl]amino]-6-oxohexanoic acid Rapamycin-3 1,42-diester with 6-(4'-methylphenoxy)-3-[[(phenylmethoxy)carbonyl]arnino-6-oxohexanoic acid Rapamycin-3 1,42-diester with 6-(ethoxy)-4-[[(phenoxy)carbonyl] ariino]-6-oxohexanoic acid WO 92/05179 WO 9205179PC1'/US91/06824 -16- Rapamycin-3 1,42-diester with 6-(methoxy)-5- [[(ethoxy)carbonyllamino]-6-oxohexanoic acid Rapamnycin-3 1,42-diester with 4-(phenoxy)-2-[N-[(1 ,1-dimethylethoxy)carbonyl]-Nmethylaxnino]-4-oxobutanoic acid Rapamycin-3 1,42-diester with 4-(phenylmethoxy)-3-[N-[(methoxy)carbonyl]-Nmethylaniino]-4-oxobutanoic acid Example 6 Rapamycin-42-ester with 11 -diniethylethoxy)-2-[r(l 1I -dimethylethoxv)acid The title compound (1.14 g, 20.6%) was separated from the 31,42-diester as described in Example 5 and isolated as the quarter hydrate/mono-ethyl acetate solvate.

HPLC analysis showed that the monoester is a 11.5: 1 mixture of two conformers.

1 H NMR (CDCl 3 400 MHz): 8 1.425 (in, 18H, COOBut), 1.643 3H,

CH

3 1.737 3H, CH 3 3.13 3111, CH 3 3.32 3H, CH 3

O),

3.36 3H, CH 3 4.17 1H, CHOM), 4.71 1H, 42-CHO), 4.785 1H, OH); High Resolution MS neg. ion FAB): Calc. for C 6 5

H

1 0 2

N

2 0 1 8 1198.7127, measured mass 1198.7077.

Anal. Calcd for C 6 5

H

10 2

N

2 0 18

-CH

3 COOEt 0.25 H 2 0: C, 64.13, H, 8.60; N, 2.17 Found: C, 64.18; H, 8.52: N, 2.01 The following-representative compounds can -td from rapainycin and the appropriately terminally-N-substituted amino diacid timonoester by employing the method used to prepare the title compound in Example 6.

Raparnycin-42-ester with 6-(phenylmethoxy)-2-[[fluorenylmethoxy)-carbonyl-amino]- 6-oxohexanoic acid Rapamycin-42-ester with 6-(4'-methylphenoxy)-3-[[(phenylmethoxy)carbonyll-arnino- 6-oxohexanoic acid WO 92/05179 WO 9205179PCT/US9I /06824 -17- Rapaxnycin-42-iester with 6-(ethoxy)-4-[[(phenoxy)carbonyl] amino] -6-oxo- hexanoic acid Rapaniycin-42-ester with 6- (methoxy)-5-[[(ethoxy)carbonyl] amino]- 6-oxo- hexanoic acid Rapamycin-42-ester with 4-(phenoxy)-2-[N-[(1, 1-dimethylethoxy)carbonyl]-Nmethylainino]-4-oxobutanoic acid Rapamycin-42-ester with 4-(phenylmethoxy)-3-[N-[(methoxy)carbonyl]-Nmethylamino]-4-oxobutanoic acid Example 7 Rapamycin-3 1 42-diester with 2-rr(1.1 -dirnethvlethoxv)carbonvllaminol-4-oxo-4- (phenvimethoxy) butanoic acid Under anhydrous conditions, 295mg (1.2lmmol) of 2,4,6 trichlorobenzoyl chloride was added to a solution of 391mg(1.2lmmol) of Na-Boc-L-aspartic acid-pbenzyl ester and 170.tL (1.2 immol) of Et 3 N in 1 rnL of TIHF at room temperature.

After stirring for 30 minutes, 500 mg (0.S5mmol) of rapamycin and 295 mg 2.42 mmol) of dimethylaminopyridine was added and the reaction was left to stir overnight.

The reaction mixture was then filtered and the filtrate concentrated in vacuo. Pure product (200 mg, 25%) was obtained by preparative HPLC (5 cm column, 40 ethyl acetate-hexane). The product was isolated as the heptahydrate.

1 H NMR (CDCI3, 400 MHz) 8 7.347 10 H, Ar), 6.223, 5.126 4 H, CH2Ph), 4.698 (in, 1 H, OH-GO 2 4.587 (in, 2 H, NH), 3.353 3 H, CH 3 3.337 3 H, CH 3 3.301 3 H, CH 3 2.775 (in, 4 H, 0H 2 002); IR 3420 (OH), 2935 2920 1730 1650, 1500, 1455, 1370, 1170 cnv 1 MS (neg.

ion FAB) 1523 1433, 297, 248, 205, 148, 44, 25 (100).

Anal. Calcd for C 83

HI

17

N

3

O

23 -7H 2 O 0, 60.40; H, 7.09; N, 2.54 Found: C, 60.54; H, 7.28; N, 2.56 WO 92/05179 PCT/US91/06824 -18- Example 8 Rapamvcin-31,42-diester with 3-[1(1,1-dimethvlethoxv)carbonvllaminoj-4-oxo-4- (phenylmethoxy) butanoic acid Under anhydrous conditions, 532 mg (2.18 mmol) of 2,4,6 trichlorobenzoyl chloride in 1 mL THF was added to a solution of 704 mg (2.18 mmol) of Nt-Boc-Laspartic acid-ca-benzyl ester and 303 pL (2.18 mmol) of Et 3 N in 5 mL of THF at room temperature. After stirring for 20 minutes, the reaction mixture was filtered over sintered glass, and the precipitate was washed with THF. The filtrate was concentrated in vacuo to give a thick oil. The oil was dissolved in 5 mL of benzene and 1.00 g (1.09 mmol) of rapamycin and 532 mg (4.36 mmol) of dimethylaminopyridine in 1 mL of benzene was added dropwise. The reaction was stirred for 2 hr, poured into ethyl acetate, and washed consecutively with 0.5 N HCI and brine. The solution was dried over sodium sulfate, decanted, concentrated in vacuo to give a white foamy solid, which was purified via flash chromatography on a 60 mm x 100 mm silica column ethyl acetate/hexane as eluant) to give 532 mg (33 of the title compound which was isolated as the hydrate.

1 H NMR (CDC1 3 400 MHz) 8 7.362 10 H, Ar), 5.193 4 H, CH 2 Ph), 4.596 1 H, CH-CO2), 4.586 2 H, NH), 3.336 3 H, CH 3 3.306 3 H, CH30), 3.145 3 H, CH 3 IR (KBr) 3410 2950 2920 (CH), 1735 1710 1640, 1490, 1445, 1350, 1150 cm 1 MS (neg. ion FAB) 1524 1434, 297, 248, 232, 214, 205, 167, 148, 42 (100), 26.

Anal. Calcd for C 83

H

1 17

N

3 0 23 H20: C, 65.38; H, 7.73; N, 2.76 Found: C, 64.85; H, 7.67; N, 2.56 Example 9 Rapamvcin-42-ester with 3-F (1,1-dimethylethoxy)carbonyllaminol-4-oxo-4- (phenvlmethoxy) butanoic acid The title compound (374 mg, 23%) was prepared by the method described in the previous Example and separated from the compound described in the previous Example by flash chromatography (20-40% ethyl acetate/hexane as the eluant) and isolated as the sesquihydrate.

WO 92105179 WO 9205179PCT/US91 t06824 -19- 1 H NMR (CDCl3, 400 MHz) 8 7.356 5 H, Ar), 5.185 2 H, CH 2 Ph), 4.635 (mn, 1 H, CH-CO2), 4.582 (mn, 1 H, NH), 3.330 6 H, CH 3 3.135 3 H, CH 3 IR (KBr) 3410 2950 2920 1735 1710 1640, 1490, 1445, 1350, 1150 cm. MIS (neg. ion FAB) 1218 1127, 590, 168, 42, 25, 17 (100).

Anal. Calcd for C 67

H

98

N

2

O

18 1.5 H20: C, 63.64; H, 8.21; N, 2.22 Found: C, 63.64; H, 7.5 1; N, 2.13 Example Rapamvcin-42-ester with Ii -dimethyiox)-4-r( 1,1 -dime-thylethoxv)carbonvlI acid Under anhydrous conditions, an ice cold solution of rapamycin (4 g, 4.37 inmole) and L-glutamic acid NaBc(-etbtlse (4.9 g, 16.1 inmole) in 40 mL of anhydrous dichioromethane was treated with dicyclohexylcarbodiimide (1.8 g, 8.7 inmole) followed by 4-dimethylamino pyridine (1 g, 8.7 inmole). After stirring overnight at ambient temperature, the precipitated solid was collected and washed with dichioromethane. The combined filtrates were concentrated in vacuo to give 9 g of an amorphous amber solid. The crude product was purified by flash chromatography (on silica Merck 60, gradient elution with hexane-ethylacetate from 2:1 to 3:2, vlv) to provide 1.35 g of the title compound along with the 31,42-diester of Example 11. HPLC analysis showed that the monoester is a 7.5 :1 mixture of two conformers.

1 H NMR (CDCl 3 400 MHz): 8 1.43 9H, COOBut) and 1.46 9H, COOBut), 1.65 311, CH3C=C), 1.75 3H, CH 3 3.14 3H, CH 3

O),

3.34 3H, CH 3 3.38 3H, CH 3 4.18 1I-, CH-OH), 4.65 (in, 1H, 42- CHO), 4.80 1H, OH); High Res. MS (neg. ion FAB): Caic. for C 6 5

H

10 2 N2018: 1198.7126, measured mass 1198.7135.

Anal. Calcd for C 6 5

H

1 0 2

N

2 0 1 8 C, 65.09; H, 8.57; N, 2.34 Found C, 65.04; H, 8.33; N, 2.64 WO 92/05179 PCT/US91/06824 Example 11 Rapamycin-31,42-diester with 11 -dimethvlethoxy)-4-T[( 1,1-dimethylethoxy)carbonyll- aminol-5-oxopentanoic acid The title compound was prepared (0.83 g, 12.8%) along with the 42monoester as described in Example 10. HPLC analysis showed that the diester is a 7.7:1 mixture of two conformers.

1 H NMR (CDC1 3 400 MHz): 8 1.43 18H, COOBut), 1.46 18H, COOBut), 1.659 3H, CH 3 1.759 3H, CH 3 3.14 3H, 3.34 3H, CH 3 3.38 3H, CH30), 4.66 1H, 42-CHO), 4.72 1H, OH); High Res. MS (neg. ion FAB): Calcd for C 7 9

H

12 5

N

3 0 2 3 1483.8704, measured mass 1483.8636.

Anal. Calcd for C79H125N3023: C, 63.90; H, 8.49; N, 2.83 Found: C, 63.68; H, 8.60; N, 3.20 Example 12 Rapamvcin-42-ester with N. N-bisf1. -dimethylethoxy)carbonvll-L-lysine Under anhydrous conditions, a solution of rapamycin (3 g, 3.28 mmole) and

N

a NE-bis-Boc-L-lysine (4.5 g, 13 mmole) in 40 mL of anhydrous dichloromethane was treated with dicyclohexylcarbodiimide (1.35 g, 6.56 mmole) followed by 4dimethylaminopyridine (0.8 g, 6.56 m mole). After stirring overnight at ambient temperature, the precipitated solid was collected and washed with dichloromethane. The combined filtrates were concentrated in vacuo to give an amorphous amber solid. Flash chromatography of the crude product (on silica Merck 60, elution with hexaneethylacetate 1:1 v/v) gave partially purified title compound. Pure product (0.8 g, 19.6%) was obtained by preparative HPLC (Waters Prep 500, silica gel, hexane- Sethylacetate 3:2 v/v, flow rate 250 mL/min). HPLC analysis showed that the monoester is a 9:1 mixture of two conformers.

WO 92/05179 WO 9205179PCT/US91 /06824 -21- IH NMR (CDC1 3 400 MHz): 8 1.438 (in, 9H, COOBut), 1.455 9H, COOBut) 1.652 3H, CH 3 1.752 3H, CH 3 3.14 3H, CH 3

O),

3.33(s, 3H, CH 3 3.37 3H, CH 3 4.18 1H, CH~hj, 4.72 (in, 1H, 42- CHO), 4.79 1H, OH); High Res. MS (neg. ion FAB): Calcd for C 6 7

H

10 7

N

3 0 18 1241.7549, measured mass 1241.7604.

Anal. Calcd for C 6 7

H

1 07

N

3

O

1 8 C, 64.76; H, 8.68; N, 3.38 Found: C, 64.58; H, 9.01; N, 3.10 Example 13 Rapamvcin-31 .42-diester withi N N-bisLF( 1-dimethvlethoxvlcarbonyll-L-lvsine Under a nitrogen atmosphere, a solution of NONF bis-Boc-L-lysine (1.038 g, 3 inmole) and triethylamine (0.42 mL, 3 mmole) in 10 mL of anhydrous THFf was treated in one portion with 2,4,6-trichlorobenzoyl chloride (0.73 g, 3 mmole). After stirring for 20 minutes at ambient temperature, the precipitated solid was collected and the filtrate was concentrated in vacuo The resulting mixed anhydride was dissolved in mL of benzene and added to a stirred solution of rapamycin (1 g, 1.09 mino3e) containing 4-diinethylaimno pyridine (0.59 g, 4.8 minole) in 10 mL of benzene. After stirring at ambiefnt temperature overnight, the precipitated solid was collected and the filtrate was evaporated to dryiness (yellow foamn). The crude product was purified by flash chromatography on silica Merck 60, elution with hexane-ethylacetate 1: 1) to provide title compound (1.15 g, HPLC analysis shows that the diester is a 9:1 mixture of two conformers.

IH NMR (CDCl3, 400 MHz): 5 1.426 (in, 9H, COOBut), 1.438 9H, COOBut), 1.443 9H, COOBut), 1.446 9H, COOBut), 3.141 3H, CH3O), 3.36 3H, CH-, 3.378 3H, CH3O), 4.68-4.76 (mn, 2H, OH and 42-CHO); High res. MS (neg. FAB): Caicd. for C83H135N5023 1569.9526, measured mass 1569.9537.

Anal. Calcd. for C83H135N5023: C, 63.46; H, 8.66; N, 4.46 Found: C, 63.06; H, 8.84; N, 4.09 WO 92/05179 WO 9205179PCT/US91 /06824 22 Example 14.

Rapamycin- 14,31 .42-tris(monobenzvlsuccinate) To a solution of 5.0 g (5.47 mmol) of rapamycin, 3.41 g (16.41 Mmol) of monobenzylsuccinate, and 3.15 g (16.41 mmol) of 1-(3-dimethylaminopropyl)-3ethylcarbodlimide hydrochloride in 20 mL of dry dichioromethane was added 200 mg of 4-dimethylaminopyridine. The solution was stirred at room temperature for 3 days.

Thui reaction mixture was poured into 2 N HC1 and extracted three times with ethyl acetate. The org, .lic layers werrc combined, washed with brine, dried over anhydrous sodium sulfate, decanted, and concentrated in vacuo to give a light yellow foam. Flash chromatography on a 60 mm x 150 mm silica gel column eluting with 20 ethyl acetate/hexane to 75 ethyl acetate/hexane gave three fractions. Fraction upon concentration, gave 330 mg (4.1 of pure rapamycin-14,31,42-tris- (monobenzylsuccinate).

1 NMR (CDCl 3 400 MHz) 8 7.353 (bs, 15 H, arom), 5.168 J 2.0 Hz, 1 H, CH-0 2 5.148 (in, 6 H, CH 2 Ph), 4.672 (in, I H, CO2CH-CHOMe), 3.355 3 H, CH 3 3.337 3 H, CH 3 3.327 3 H, CH3O-), 2.697 (i,12 H,

O

2 CCH2CH 2

CO

2

CH

2 Ph), 1.745 3 H, CH 3 1.655 3 H, CH 3

C=C);

IR (KBr) 3450 2950 1745 1650, 1460, 1385, 1360, 1160, 1105, 995 cm- 1 Analysis Calcd for C 84 1 Q9N0 21 -3 H20 C 66.27; H 7.56; N 0.92 Found C 65.96; H 7.24; N 1.00 The following representative compounds can be prepared from rapamycin and the appropriate half acid-ester by employing the method used to prepare the title compound in Example 14.

Rapamycin- 14,3 1,42-tris (monomethylsuccinate) Rapaniycin-14,3 1,42-t-is (inonophenyl-3',3'-dimethylglutarate) Rapamycin-14,3 1 42-iris (mono t-butyl,-3'-methylglutarate) Rapamycin-14,3 1,42-iris (monobenzylthiodiglycolate) Rapamycin-14,3 1,42-iris (monohexyldiglycolate) Rap amycin-14,3 1,42-t-is (monopropylphthalate) Rapaxnycin-14,3 1,42-tris (monoethyl-2',6'-pyridinedicarboxylate) WO 92/05179 WO 9205179PCT/US91 /06824 23 Example Rap~amycin-3 1.42-bi s(monobenzylsuccin ate) Fraction 2, obtained from the procedure employed in Exani. 2 24, gave g (17.7 of pure rapamycin-31,42-bis(monobcnzylsuccinate) upon concentration.

IH NMR (CDC13, 400 MHz) 8 7.35 1 (bs, 10 H, arom), 5.168 J 2.0 Hz, 1 H, CH-02C), 5.125 (in, 41H, CH 2 Ph), 4,680 (mn, 1 H, CO2CH-CHOMe), 3.356 3 H, CH3O-), 3.329 3 H, CH3O-), 3.146 3 H, CH 3 2.639 (i,8 A1

O

2

CCH

2

CH

2

CO

2

CH

2 Ph), 1,748 3 H, CH3C=C), 1.654 3 H, CH 3

C=C);

IR (KBr) 3450 2940 1740 1650, 1455, 1380, 1355, 1160, 1105, 995 cnn1; MIS (neg. ion FA13) 1294 1202, 1103, '012, 590, 511, 475, 297, 207, 167, 148, 99 (100); High Res. MS (neg. ion FAB) Calcd for C 7 3

H

99 N0 1 9 1293.68108, found 1293.6811.

Analysis Calcd for C 7 3H9 9 N0 1 9 -H20 C 66.82; H 7730; N 1,07 Found C 67.17; H 7.67; N 1.23 The following representative compounds can be prepared from rapamycin and the appropriate half acid-ester by employing the method used to prepare the title compound in Example Rapaxnycin-3 1,42-bis (inonomethylsuccinate) Rapainycin-3 1 A2-bis (monophenyl-3',3'-dimethylgiutarate) Rapaxnycin-3 1,4'2-bis (mono t-butyl-3'-methylglutarate) Rapaniycin-3 1,42-bis (monobenzylthiodiglycolate) Rapamycin-3 1,42-bis (inonohexyldiglycolate) Rapainycin-3 1,42-bis (inonopropylphthalate) Rapainycin-3 1,42-bis (inonoethyl -2',6'-pyridinedicarboxylate) Example 16.

Rapainycin-42-(monobenzvlsuccinate) Fraction 3, obtained from the procedure employed in Example 14, gave 930 mng (15.4 of pure rapamycin-42-monobenzylsuccinate upon concentration.

WO 92/05179 WO 9205179PCT/US91 /06824 -24 IH NMR (CDCI 3 400 MHz) 5 7.355 (bs, 5 H, arom), 5.141 (mn, 2 H,

CH

2 Ph), 4.680 (mn, 1 H, CO2CH-CHOMe), 3.364 3 H, CH3O-), 3.333 3 H,

C'H

3 3.141 3 H, CH 3 2.698 (in, 4 H, O2CCH 2

CH

2

CO

2

CH

2 Ph), 1.751 3 H, CH 3 1.655 3 H, CH 3 IR (KBr) 3450 2940 1740 1645, 1455, 1380, 1165, 1105, 990 cm- 1 MS (neg. ion FAB) 1103 1045, 1012, 624, 590, 167, 99 (100); Hi-gh Res. MS (neg. ion FAB) Calcd for C62H89N0 1 6 1103.6181, found 1103.6048.

Analysis Calcd for C6 2

H

8 9N0 16 -H20 C 66.36; H 8.02; N 1.24 Found C 66.02; H 7.69; N 1.26 The following representative compounds can be prepared from rpamycin and the appropriate half acid-ester by employing the method used to prepare the title compound in Example 16.

Rapamycin-42-(inonomethylsuccinate) Rapainycin-42-monophenyl-3',3'-diinethylglutarate) Rapamycin-42-(mono t-butyl-3'-methylglutarate) Rapamycin-42-(monobenzylthiodiglycolate) Rapamycin-42-(monohexyldiglycolate) Raparnycin-42-(inonopropylphthalate) Rapamycin-42-(monoethyl-2',6'-pyridinedicarboxylate) Example 17.

Rapamvcin-3 1 42-bishemi glutarate To a solution of 2.0 g (2.2 inmol) of rapamnycin in 10 mL of dry dichloromethane was added 1.24 g (10.9 minol) of glutaric anhydride followed by 881 uL (861 mng, 10.9 minol) of pyridine. To this was added 200 mg of 4-diinethylaminopyridine and the reaction mixture was allowed to reflux for 8 h. The solution was cooled to room temperature, poured into 2 N HCI, and extracted three times with dichloroinethane. The combined organic extracts were washed with brine, dried over anhydrous sodium sulfate, decanted, and concentrated in vacuo to give a yellow foam. The crude product was purified via reverse phase HPLC on a C 1 8 column eluting starting with 60 acetonitrile/water. Collected, after, concentration, 586 mg (24 of rapaniycin-31,42-bishemiglutarate.

WO 92/05179 WO 9205179PCr/US9 1/06824 IH NMR (CDCl 3 400 MHz) 8 5.398 (in, 1 H, -CO2CHCHOMe), 4.683 (mn, 1 H, -CO2CHCHOMe), 3.364 3 H, CH 3 3.362~ 3 H, CH 3 3.106 3 H, CH3gO-), 2.407 (in, 8 H, -02CCH2CH 2

CHJ

2

CO

2 1.960 (in, 4 H, -02CCH2CH 2 CH2CO2H), 1.770 3 H, C'H 3 1.653 3 H, C'H 3

C=C);

13 C NMR (CDCl 3 MHz) 211.45 206.84 200.44 177.83 177.04 172.43 171.20 165.27 159.08 IR (KBr) 3430 2940 2880 1745 1685, 1625, 1580, 1450, 1385, 1330, 1200, 1140, 1100, 990 cm- 1 MS (neg. ion FAB) 1140 1122, 1026, 990, 946, 913, 590, 475, 435, 321, 167, 148, 131 (100), 113; High Res.

MS (neg. ion FAB) Caled for C6lH9(0I 9 N 1140.6107, Found 1140.6106.

Analysis Calcd for C 61

H

9 1 0 19 N H20 C 63.15; H 8.02; N 1.20 Found C 63.35; H 7.88; N 1.40 The following representative compounds can be prepared from rapamycin and the appropriate anhydride by employing the method used to prepare the title compound in Example 17.

Rapamycin-3 1,42-bishemi-3'-methylglutarate Rapamycin-3 1,42-bishenii-3',3'-dimethylglutarate Rapamycin-3 1,42-bishemi-3'-oxoglutarate Rapamycin-3 1,42-bisherri-3'-thioglutarate Rapamycin-3 1,42-bishemi-phthalate Rapainycin-3 1,42-bishemi-2',3'-pyridine dicarboxylate.

Example 18.

Rapamvcin-31 .42-hemniglutarate bissodium salt Purified bis-31,42-hemiglutarate of rapamycin (740 mng, 649 umol), prepared as described in Example 17, was dissolved in 5 mL of 95 ethanol and 107 mng (1.27 minol) of sodium bicarbonate was added. Water (1 m.L) was added to completely dissolve the salt. Once dissolved, the light yellow solution was concentrated in vacuo to give a foamy yellow solid. The foam was dried in a drying pistol for 24 h, refluxing over acetone at reduced pressure to give 520 mng of the bissodium. salt.

WO 92/05179 WO 9205179PCr/US91 /06824 -26- 1H NMR (d 6 -DMk.SO, 400 MHz) 8 5.235 (in, I H, -CHO 2 4.498 (in, 1 H, McOCHCH0 2 3.287 6 H, 2 Cft 3 3.236 3 H, CH 3 2.245 (in, 8 H,

O

2

CCH

2 CH2CH 2

CO

2 1.712 3 H, CH 3 1.593 3 H, CH 3

C=C);

IR (KBr) 3420 2920 1725 1675, 1620, 1560, 1450, 1400, 1375, 1230, 1195, 1130, 1090, 980 cm- 1 MS (neg. ion FAB) 1112 free acid), 994, 589, 475, 297, 167, 148, 117, 99 (100); High Res. MS (neg. ion FAB) Calcd for

C

6 1H890l9NNa (M-Na) 1162.5926, Found 1162.5899.

Analysis Calcd for C61H89Ol9NNa 2 1{20 C 60.85; H 7.56; N 1.16 Found C 60.67; H 7.36; N 1.58 Example 19.

Rap~amycin-31 .42-bishemiglutarate bistromethainine salt, Purified bis-31,42 hemiglutarate of rapamycin (950 mng, 833 uinol), prepared as described in Example 17, was dissolved in 5 mL of 95 ethanol and 197 mg (1.63 inrol) of tris(hydroxyinethyl)inethylamine was added. Water (1 nfL) was added to completely dissolve the amine. Once dissolved, the yellow solution was concentrated in vacuo to give a foamy yellow solid. The vy hygroscopic foam was dried in a drying pistol for 24 h, refluxing over acetone at reduced pressure to give 900 mng (78 of the bistroinethamine salt.

1 H NMR (d6-DMSO, 400 MHz) 5 5.253 (mn, 1 H, -CHO 2 4.523 (in, 1 H, MeOCHCHO 2 3.347 6 H, 2 CH 3 3.276 3 H, CH 3 2.289 (in, 8 H,

O

2

CCH

2

CH

2

CH

2 CO2), 1.681 3 H, CH 3 1.595 3 H, CH 3

C=C);

IR (KBr) 3400 2920 1730 1620, 1555, 1450, 1400, 1370, 1185, 1060, 980 cm- 1 MS (neg. ion FAB) 1140 free acid), 1028, 167, 148, 131 (100), 113; High Res. MS (neg. ion FAB) Calcd for C 61 H900 1 9N free acid) 1140.6107, Found 1140.6069.

Analysis Calcd for C 69 HI0 3 02 5 N3 -2 H 2 0 C 58.77; H 7.5 8; N 2.9 8 Found C 5 8.47; H 7.94; N 3.58 WO 92/05179 WO 9205179PCT/US91/06824 -27 Example Rgpamycin-42-heri-3'-oxoo1utarate To a solution of 3.0 g (3.3 mmol) of rapamycin in 20 mL of dry dichioromethane was added 1.90 Ij (16.4 mmol) of diglycolic anhydride followed by 1.32 mL (1.29 g, 16.4 mmol) c pyridine. To this was added 200 mg of 4-dimethylaminopyridine and the reection mixture was allowed to stir at room temperature for 2 days. The solution was cooled to room temperature, poured into 2 N HC1, and extracted three times with dichloromethane. The combined organic extracts were washed with brine, dried over anhydrous sodc'um sulfate, decanted, and concentrated in vacuo to give a yellow foam. The crude product was purified via reverse phase HPLC on a C 1 8 column eluting starting with 60 acetonitrile/water.

After concentration, 870 mg 26 of rapamycin-42-hemi-3'-oxoglutarate and 500 mg (13 of rapamycin-31,42-bishemi-3'oxoglutarate were isolated.

IH NMR (CDCl3, 400 MHz) 8 4.768 (in, 1 H, CO 2 CH-CHOMe), 4.250 (in, 4 H, O2CCH 2

OCH

2

CO

2 3.356 3 H, CH 3 3.331 3 H, CH 3 3.139 3 H, CH 3 1.759 3 H, CH 3 1.653 3 H, CH 3

C=C);

IR (KBr) 3420 2920 2875 1740 1720 1640, 1625, 1445, 1370, 1320, 1200, 1135, 1095, 980 cm- 1 MS (neg. ion FAB) 1028 (M 327, 167 (100), 148, 133, 115; High Res. MS (neg. ion FAB) Calcd for 5

H

82 0 17 N (M H) 1028.5597, Found 1028.5599.

Analysis Calcd for C 55

H

83 0 17 N 3 H20 C 60.97; H 8.22; N 1.29 Found -C 61.33; H 7.74; N 1.69 The following representative compounds can be prepared from rapamycin and the appropriate half acid-ester by employing the method used to prepare the title compound in Example Rapamycin-42-hemi-3'-methylglumarte Rapamnycin-42-hemi-3',3'-dimethylglutarate Rapamycin-42-hemi-3'-thioglutarate Rapamycin-42-hemi-phthalate Rapamycin-42-hemi-2',3'-pyridine dicarboxylate WO 92/05179 PCT/US91/06824 -28- Example 21.

Rapamycin-31,.42-bishemi-3'-oxoglutarate To a solution of 5.0 g (5.47 mmol) of rapamycin in 20 mL of dry dichloromethane was added 3.17 g (27.3 mmol) of diglycolic anhydride followed by 2.17 mL (2.12 g, 27.3 mmol) of pyridine. To this was added 400 mg of 4-dimethylaminopyridine and the reaction mixture was allowed to stir at reflux for 24 h.

The solution was cooled to room temperature, poured into 2 N HC1, and extracted three times with dichloromethane. The combined organic extracts were washed with brine, dried over anhydrous sodium sulfate, decanted, and concentrated in vacuo to give a yellow foam. The crude product was purified via reverse phase HPLC on a

C

1 8 column eluting starting with 60 acetonitrile/water. After concentration, 1.75 g (28 of rapamycin-31,42-bishemi-3'-oxoglutarate was isolated.

1 H NMR (CDC13, 400 MHz) 8 4.785 1 H, CO2CHCHOMe), 4.260 8 H, O 2

CCH

2 0CH 2

CO

2 3.360 3 H, CH30-), 3.343 3 H, CH 3 3.143 3 H, CH30-), 1.775 3 H, CH3C=C), 1.656 3 H, CH 3

C=C);

13 C NMR (CDCl 3 MHz) 211.12 207.73 193.11 171.90 171.59 170.15 169.35 168.83 166.63 IR (KBr) 3420 2920 2850 1740 1645, 1625, 1440, 1370, 1190, 11300, 980 cm- 1 MS (neg. ion FAB) 1140 1122, 1026, 990, 946, 913, 590, 475, 435, 321, 167, 148, 131 (100), 113; High Res. MS (neg. ion FAB) Calcd for C59H860 2 1 N (M H) 1144.5701, Found 1144.5702.

Analysis Calcd for C59H870 2 1N C 61.82; H 7.65; N 1.22 Found C 61.59; H 7.36; N 1.84 Example 22.

Rapamycin-31,42-bishemi-3'-oxoglutarate disodium salt Purified bis-31,42 hemi-3'-oxoglutarate of rapamycin (720 mg, 629 umol) prepared by the procedure employed in Example 21, was dissolved in 10 mL of 95 ethanol and 106 mg (1.26 mrm!l) of sodium bicarbonate was added. Water (1 mL) was added to completely dissolve the salt. Once dissolved, the light yellow solution was concentrated in vacuo to give a foamy yellow solid. The foam was dried in a drying WO 92/05179 WO 9205179PCT/US91 /06824 29 pistol for 48 h, refluxing over dichioromethane at reduced pressure to give 435 mg (58 of the disoclium salt.

1 H NMR (d 6 -DMSO, 400 MHz) 8 4.975 (in, 1 H, -CH02CQ, 4.593 (in, 1 H, MeOCHCHO2C-), 4.135 2 H, O0 2

CCH

2 OCH2CO2R), 3.617 (s,2 H, O0 2

CCH

2 OCH2CO 2 3.299 6 H, 2 CH 3 3.232 3 H, CH3O-), 1.614 3 H, CH 3 1.553 3 H, CH 3 IR (KBr) 3420 2920 (CH), 1735 1615, 1445, 1395, 1380, 1320, 1220, 1130, 1090, 980 cm- 1 MS (neg. ion FAB) 1188 1166 1144, 1051, 1028, 590, 459, 167, 155 (100), 148, 133, 115.

Analysis Calcd for C 59 H8 5

O

2 INNa2 2H 2 0 C 57.79; H 7.26; N 1. 14 Found C 57.94; H 7.11; N 1.26 Example 23.

Rap~amvcin-31I 42-bishemi-3'-oxoglutarate bistromethamine salt Purified bis-31,42 hemi-3'-oxoglutarate of rapamycin (1.01 g, 882 uinol), prepared by the procedure employed in Example 21, was dissolved in 10 mL of 95 ethanol and 213 mng (1.76 minol) of tris(hydroxymethyl)> methylamnine was added.

Water (1 mL) was added to completely dissolve the amnine. Once dissolved, the yellow solution was concentrated in vacuo to give a foamy yellow solid. The very hygroscopic foam was dried in a drying pistol for 48 h, refluxing over dichloromethane at reduced pressure to give 805 mg (66 of the bistrometh amine salt.

IH NMR (d 6 -DMSO, 400 MHz) 8 4.955 (in, 1 H, -CH0 2 CQ, 4.600 (in, 1 H, MeOCHCHO2C-), 4.149 2 H, O0 2

CCH

2 OCH2CO2R), 3.770 2 H,

-O

2

CCH

2 OCH2CO2R), 3.407 6 H, 2 CH 3 3.257 3 H, CH3O-), 1.806 3 H, CH 3 1.614 3 H, CH 3 IR (K-Br) 3400 2920 (CH), 1730 1620, 1550, 1450, 1395, 1370. 1200, 1060, 985 cm-1; MS (neg. ion FAB) 1144 free acid), J.028, 167, 148, 133 (100), 115.

Analysis Calcd for Ce 5 7

H

1 0 9 O02 7 N3 -H20 C 57.22; H 7.90; N 2.98 Found C 57.26; H 7.90; N 3.15 WO 92/05179 PCT/US91/06824 Example 24.

Rapamvcin-31,42-bishemisuccinate.

To solution of 2.0 g (2.2 mmol) of rapamycin in 10 mL of dry dichloromethane was added 1.19 g (10.9 mmol) of succinic anhydride followed by 881 uL (861 mg, 10.9 mmol) of pyridine. To this was added 200 mg of 4-dimethylaminopyridine and the reaction mixture refluxed for 24 h. The solution was cooled to room temperature, poured into 2 N HC1, and extracted three times with dichloromethane.

The combined organic extracts were washed with brine, dried over anhydrous sodium sulfate, decanted, and concentrated in vacuo to give a yellow foam. The crude product was purified via reverse phase HPLC on a C 18 column gradient eluting starting wit;.

acetonitrile/water to 60 acetonitrile/water. Collected, after, concentration, 770 mg (31 of rapamycin-31,42-bishemisuccinate.

The purified bis-31,42 hemisuccinate of rapamycin (770 mg, 686 umol) was dissolved in 10 mL of 95 ethanol and 166 mg (1.37 mmol) of tris(hydroxymethyl)methylamine was added. Water (1 mL) was added to completely dissolve the amine.

Once dissolved, the yellow solution was concentrated in vacuo to give a foamy yellow solid. The very hygroscopic foam was dried in a drying pistol for 24 h, refluxing over acetone at reduced pressure to give 890 mg (95 of the bistromethamine salt. The bistromethane salt was evaluated in the standard pharmacological test procedures.

1H NMR (d 6 -DMSO, 400 MHz) 5.231 1 H, -CHO2C), 4.554 1 H, MeOCHCHO 2 3.426 6 H, 2 CH30-), 3.249 3 H, CH30-), 2.431 8 H, O2CCH2CM2CO2-), 1.700 3 H, CH 3 1.554 3 H, CH 3 13 C NMR (d6-DMSO,) 211.28 205.23 199.59 174.86 173.62 171.72 171.50 166.56 166.53 IR (KBr) 3420 2940 1735 1630, 1580, 1460, 1400, 1380, 1170, 1070, 990 cm-1; MS (neg. ion FAB) 1112 free acid), 994, 589, 475, 297, 167, 148, 117, 99 (100).

Analysis Calcd for C 67

H

109 0 25

N

3 2 H20 C 57.80; H 8.12; N 3.01 Found C 57.91; H 8.21; N 2.37

Claims (11)

1. 2. A compound of claim 1 where R 4 is 0 11 kC(CH 2 )mCH(CH 2 )nN~pCO 2 R 7 I I R 5 R 6 m 0, n 0, and p 1 or a pharmaceutically acceptable salt thereof. i. A compound of claim 1 where R 4 is 0 11 [C(CH 2 )mnCH(CH 2 )nN~pCO 2 R' I I R 5 R mn 0, n 0, and p 2 or a pharmaceutically acceptable salt thereof.
2. 4. A compound of claim 1 where R 4 is 0 11 kC(CH 2 )mnCH(CH 2 )nNjpCO 2 R' I I n 0, and R 5 is -(CH2)qCO2R 8 or a pharmaceutically acceptable salt thereof. A compound of claim 1 where R 4 is mn 0, n 0, and R 5 is -(CH2)rNR 9 CO2RI 0 thereof. 0 II kC(CH 2 )MCJJ(CH 2 )nN~pCO 2 R 7 I I R 5 R or a pharmaceutically acceptable salt
3. 6. A compound of claim I where R 4 is [C(CH 2 )nCHl(CH 2 )nNIpCO 2 R 7 m 0, n 0, and R 5 is hydrogen or a pharmaceutically acceptable salt thereof. -34-
4. 7. c compound of Claim 1 where R4i -C-(CH 2 )t (CH 2 u CO 2 F or- a pharmaceutically acceptable salt thereof, a. A compound according to Claim I. which is one of the following: rapamvcin-42-ester with N-C (1,1-dimet hvlethoxy)- carbony3 3gycylqlyvcine; rapamycin-32.,42-diester with di-methylethoxy)carbonKL 3giycylglycine; rapamycin-31,42-diester with N-4(1,l- dimethylethox)carbony.-N-rnethyl"'glycine; rapamycin-42-ester with Nfll dimet-hylethoxy)carbonyl -met-hylglycine raoamycin-31,42-diester with 5-(1,1- d ime thy 1e thoxy) 2 dime thy 1 e tbQy) c a r*ony Yi amino]-S-oxopentanoi.c acid; rapamycin-42-coster with 5-(1Tl-dirnethylethoxy)- 2 1 -dime thy 1.e thox) c a,2born .1 amn ao-5oxope n tan o ic acid; rapamycin-31,42-*diester with 2-CC[Cll- d ime thy Ie thoxy)a rbo.y I am ino 1- 4-ox 4 -(phe n lme thoxy) butanoic acid;' rapamycin-3J.,42-diester with 3-CC(1,1- d ime thyl eth oxy) carbo ny1. arm n o1- 4 -ox o- 4 phe ny lme thoxy) butanoic acid; raDamvcin-42-ester with 3-['C(3,1-di-nethvliethoxy)- carbonyllarminol-4-oxo-4-(-phenylmet-hoxy)butario,'c; raparnvcin-42-ester with 5-(1,l-dirnethyloxy)- 4-Lt(l,1-dimethylet hoxy)carbonyl acid; IAJ~j rapamrycin-31,42-diester with 5-(1,1- 42 ~direthyet'oy)-4-[t(1,1-dimethylethoxy~carbonvi>- aminol-5-oxopentanoic acid; sUBSTI*TUTE SHEET 'C H'A-W I ACIIIA 2 H L. i (I U2$2t 1:11 ,gw +M i M 2: 101, H 1',7)
5. 8-9080' 9 67 5-1-NIZ rapamyc i 42-e5t-.er with N- -bis[C 1 ,1- raparvcir.-32.,42-diester w.ith bis-(l,l- dimee-hvlethoxv)c.arbonyl l-L-lysi4ne; rapanivcin-14,31,42-lris(monobenzyisucci-nat-e); rapamycin-31,42-bis(monolbenzylsuccinate); rapamv/cin-42-(mor.obenzylsucciLn=te); raparnycin-31,42--bisheTmiglUtarate; rapamyc in.- 42-heni- 3 '-oxoglutarate; rapamyci-i3, 42-bishemi--3'-oxogluta-r ate and rapamrycin-3'1 ,42-bishemisucc.in;at-e; or a pharmaceutically acceptable salt thereof.
6. 9. A compound accordin~g to Claim I which is one of the following: raLpamycin-31,42-bishemi-3'-oxo.}utl-arate disodium rapan, c in-31, 42-bi shamni -3'-oxoc .u tarate bistromethamine salt; rapoamycin-3l,42-bishemi-succnate bistromethane salt; raparnyci-n-32,42-heaiglutarate bissodium salt and rapanycin-32,42-bishemiglu-tarate bistromethamine salt, A method of treating transplantation rejection, host vs. graft disease, autoirrnmu-ne diseases, and diseases of inflamamation in a mammal by administering an immunosuppressive amount off a compound as claimed in Claim 1.
7. 11. A method of treating furngal infections which f-samiitrn an antifungal amount of a compound as claimed in Claim 1.
8. 12. A pharmaceutical, composition which a coimrpound as claimed In Claim I. and a pharmaceuticai~y acceptable carrier. SUBSTITUTE SHEET
9. 13. A process for preparing a compound of formula I as claimed in Claim 1 which includes acylating rapamycin with an acylating agent or sequentially acylating rapamycin with one or more acylating agents, said acylating agent(s) being selected from acids of formula; O O Z-tC(CH CH(CH N] CO 2 R 7 Z-C-(CH 2 )tX(CH 2 CO2R 1 0 mH 2 n p 2u 2 6 R R and 0 C Z-CJ CO2R 1 2 wherein Z is OH and the other variables are as defined in Claim 1, or reactive derivatives thereof, if desired protecting any of 42, 31 and 14 positions of rapamycin with an appropriate protecting group and removing said group as required, and further if desired isolating the product as a pharmaceutically acceptable salt.
10. 14. A compound according to Claim 1 substantially as hereinbefore described with reference to any one of the S 25 examples.
11. 15. A process according to Claim 13 substantially as hereinbefore described with reference to any one of the examples. Dated: 12 July, 1994 Phillips Ormonde Fitzpatrick Attorneys for: AMERICAN HOME PRODUCTS CORPORATION 9 CU J SI 83461 39 36 I. LLAaIILAiulu, SUBJLLI IvlAI ILK li several classification symbols apply, indicate allj 6 According to International Patent Classification (IPC) or to both National Classification and IPC C 07 D 498/18 C 07 K 5/06 A 61 K 31/395 A 61 K 37/02 07 D 498/18 C 07 D 311:00 C 07 D 273:00 II. FIELDS SEARCHED Minimum Documentation Searched Classification System Classification Symbol., C 07 D 498/00 C 07 H 19/00 C 07 K 5/00 A 61 K 31/00 A 61 K 37/00 Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included in the Fields Searched s II. DOCUMENTS CONSIDERED TO BE RELEVANT 9 Category O Citation of Document, 11 with indication, where appropriate, of the relevant passages 12 Relevant to Claim No. 13 A EP,A,0046661 (AYERST McKENNA AND 1,33-36 HARRISON LTD) 3 March 1982, see the whole document, US, A, 4316885 (cited in the application) A US,A,4650803 (VALENTINO J.S. AND 1,33-36 KENNEDY 17 March 1987, see the whole document (cited in the application) Special categories of cited documents :10 T later document published after the international filing dete or irioritv date and not in conflict with the application but document defining the general state of the art which is not cited to Indertand the principle or theor underlying tbo considered \i be of particular relevance invention earier ocument but published on or after the international "X document of particular relevance; the claimed invention filing date cannot be ccnsiderrA novel or canner be considered to document which may throw doubts on priority claim(s) or involve an inventivt step which is cited to establish the publication date of another document of particular relevance; tie claimed invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the document referrinp to an oral disclosure, use, exhibition or document is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled document published prior to the international filing date but l the art. later than the priority date claimed document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search 13-12-1991 International Searching Authority EUROPEAN PATENT OFFICE Form PCTISA/210 lecond Iheet) I(Jnuary 19851 Date of Mailing of this International Search Report International A, ,cation No. PCT/ US91 106824 FURTHER INFORMATION CONTINUED FROM TlHE SECOND SHEET V. [M OBSERVATION WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHABLE 1 This International search report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons: 1. a Claim numbers because they relate to subject matter not required to be searched by this Authonty, namely. Although claims 33-34 are directed to a method of treatment of the human/animal body, the search has been carried out and based on the alleged effects of the compounds 2. Claim numbers because they relate to parts of the International application that do not comply with the prescribed requirements to such an extent that no meaningful International search can be camed out. specifically 3. I Claim numbers the second and third sentences of PCT Rule 6.4(a). because they are dependent claims and are not drafted in accordance with OBSERVATIONS WHERE UNITY OF INVENTION IS LACKING 2 This International Searching Authority found multiple inventions in this International application as follows: 1. O As all required additional search fees were timely paid by the applicant, this International search report covers all searchable claims of the International application 2. As only some of the required additional search fees were timely paid by the applicant, this international search report covers only those claims of the International application for which fees were paid, specifically claims: 3. No required additional search fees were timely paid by the applicant. Consequently, this international search report is restricted to the invention first mentioned in the claims; it is covered by claim numbers: 4. As all searchable claims could be searched without effort justifying an additional fee, the International Searching Authorty did not invite payment of any additional fee. Remark on Protest SThe additional search fees were accompanied by applicant's protest. O No protest accompanied the payment of additional search fees. I Form PCT/ISA/210 (supplemental sheet P9412B 05/91 ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO. US 9106824 SA 52089 This annex lists the patent family members relating to the patent documents cited in the above-mentioned international search report. The members are as contained in the European Patent Office EDP file on 13/01/92 The European Patent Office is in no way liable for these particulars which are merely given for the purpose of information. Patent document Publication Patent family Publication cited in search report date member(s) date EP-A- 0046661 03-03-82 US-A- 4316885 23-02-82 AT-T- E7920 15-06-84 CA-A- 1159054 20-12-83 JP-A- 57118586 23-07-82 US-A- 4650803 17-03-87 AU-B- 583439 27-04-89 AU-A- 6608086 11-06-87 CA-A- 1273920 11-09-90 EP-A- 0227355 .1-07-87 EP-A- 0429436 29-05-91 GB-A,B 2183647 10-06-87 JP-A- 62215592 22-09-87 c M For more details about this annex see Official Journal of the European Patent Office, No. 12/82