NZ239852A - Esters of rapamycin and pharmaceutical and fungicidal composition thereof - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £39852 239 85^ Priority., .... : J?X?< Coiii^ioio opewification Filed: CQIP^.?IU;... C??.^!<#><■. .{%>!&■!>?)p?-;. .QPJMtfi ^9, Public:; ;;on Dm,: ....? 6 .W. 1993....

P.O. Journal, No: . "i ■&***• «» $ ft ?f & *<r^sK\ &§W s WI Patents Form No. 5 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION AMINOESTERS OF RAPAMYCIN WE, AMERICAN HOME PRODUCTS CORPORATION, a corporation under the state of Delaware, U.S.A. of 685 Third Avenue, New York, NY 10017, U.S.A. hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: (followed by page la) N.Z. PATENT OFFICE 19 SEP 1991 RECE4V~D # 239853 AHP-9580/9675-1-N1 -la- AMINO ESTERS OF RAPAMYCIN BACKGROUND OF THE INVENTION This invention relates to novel esters of rapamycin and a method for using them 5 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 Streptomvces hvgroscopicus. which was found to have antifungal activity, particularly against 10 Candida albicans, both in vitro and in vivo [C. 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 (U.S. Patent 4,885,171) or in combination with picibanil (U.S. Patent 4,401,653) has been shown to have antitumor activity. R. Martel et al. 15 [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, 20 3411 (1989), rapamycin has been shown to be effective in inhibiting transplant rejection (U.S. Patent No 5100899. 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)]. 25 Mono- and diacylated derivatives of rapamycin (esterified at the 28 and 43 positions) have been shown to be useful as antifungal agents (U.S. Patent 4,316,885) and used to make water soluble prodrugs of rapamycin (U.S. 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 30 the 31- and 42- positions. /yCr Q-s ! ^ !.H ' 2 t JAN1993' r e 1 vj 239852 AHP-9580/9675-1 -N1 DESCRIPTION OF THE INVENTION This invention provides derivatives of rapamycin which are useful as immunosuppressive, anti-inflammatory, and antifungal agents having the structure (I) wherein R1, R2 and R3 are each, independently, hydrogen, O II • [C(CH2)mCH(CH2)nN]pC02R7 I I R5 R6 O ■c"^y9~c°2r12; o —C - (CH2)tX(CH2)uC02R 11 or R5 is hydrogen, alkyl of 1-6 carbon atoms, aralkyl of 7-10 carbon atoms, -(CH2)qC02R8, -(CH2)rNR9C02R10, carbamylalkyl of 2-3 carbon atoms, aminoalkyl of 1-4 carbon atoms, hydroxyalkyl of 1-4 carbon atoms, guanylalkyl of 2-4 carbon atoms, mercaptoalkyl of 1-4 carbon atoms, alkylthioalkyl of 2-6 carbon atoms, indolylmethyl, hydroxyphenylmethyl, imidazolylmethyl 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, aiui carboxy; ' 4, R6 and R^ are each, independently, hydrogen, alkyl of 1-6 carbon ato^s or aralkyl «< of 7-10 carbon atoms; 1 (^2 1 JAN1993 239852 AHP-9580/9675- 1-N1 R7, R8, and R10 are each, independently, alkyl of 1-6 carbon atoms, aralkyl of 7-10 carbon atoms, fluorenylmethyl, 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 5 atoms, trifluoromethyl, amino and carboxy; R11 and R12 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, 10 trifluoromethyl, amino and carboxy; R*3 X is -C-, O orS; R14 R13 and R14 are each, independently, hydrogen or alkyl of 1-6 carbon atoms; 15 YisCHorN; m is 0 - 4; n is 0 - 4; p is 1 - 2; q is 0 - 4; r is 0 - 4; tis0-4 and u is 0 - 4; O II wherein R5, R6, m and n are independent in each of the [C(CH2)mCH(CH2)nN] I I R5 r6 subunits when p = 2; or a pharmaceutical^ acceptable salt thereof, with the proviso that R1, R2 and R3 are 25 not all hydrogen, further provided that R1, R2 and R3 are not all O II — [C(CH2)mCH(CH2)nN]pC02R7 , and still further provided that t and u are not I I R5 R6 both 0 when X is O or S. 239852 — Zj.— AHP-9 580/967 5-1-Nl o II Of the compounds when R4 is — [C(CH2)mCH(CH2)nN]pC02R , I I R5 R6 preferred members are those in which m = 0, n = 0 and p = 1; m = 0, n = 0 and 5 p = 2; n = 0 and R5 is -(CH2)qC02R8; m = 0, n = 0. and RS is -(CH2)rNR9C02R10; and m = 0, n = 0 and R5 is hydrogen. Preferred compounds also include those O 4. 11 11 members in which R is —C-(CH2)tX(CH2)uC02R The pharmaceutically acceptable salts may be formed from inorganic bases such as those containing sodium or potassium cations; mono-9di-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 O 0 II 7 'I 17 Z—[CCCH - ) CH(CH_) N] CO_R , Z-C-(CH~) X(CH_) CO^RiX z mi 2 n i p 2 2 t 2u2 6t> % or where Z is OH; in the presence of a coupling reagent, such as dicyclohexyl-carbodiimide. The compounds of this invention also can be prepared using an anhydride or mixed anhydride of the above described carboxylic acid as the acylating species. Alternatively, the acylating species can be an acid halide, where X can be CI, 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 A having two or three different R groups then seguen^^rjj.j. acylation may be performed using appropriate acy^qping agents as defined above, if necessary isolating Jhfie 1 ta t JAN1993 h ■ •£e' 23! AHP-9580/9675-1-N1 -5- desired product by appropriate purification 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 required.

Immunosuppressive activity was evaluated in an i_n vitro standard 5 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 15 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 Jvmphoproliferation of 1 |iM. 3H-control thymus cells - H3-rapamvcin-treated thymus cells 3H-control thymus cells - H3-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 25 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 30 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 i.p., 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. 35 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: 2 39 AHP-9580/9675-1-N1 3H-PLN cells control C3H mouse - 3H-PLN cells rapamvcin-treated C3H mouse ^H-PLN cells control C3H mouse - 3H-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 P.B., 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 10 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 ± S.D.) of the drug treatment group is compared with 15 the control group.

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

Compound TABLE LAF* (ratio) 1 PLN* (ratio) Skin Graft (davs + SE Example 1 1.8 0.61 12.0 ±_1.6 Example 2 0.33 0.62 11.5 ±0.6 Example 3 0.20 + 9.0 ± 0.9 Example 4 4.9 0.18 12.3 ± 0.5 Example 5 0.006 + 8.8 ± 0.9 Example 6 .4 0.33 11.5 ±3.5 Example 7 3% at l[iM** + 7.7 ±1.5 Example 8 0.03 0.41 + Example 9 0.96 1.34 .3 ±0.8 Example 10 2.0 0.96++ 12.7 ± 1.2 Example 11 0.004 + .5 ± 1.3 Example 12 19.8 -2.87 12.0 ±2.0 Example 13 22% at ljiM** + 7.0 ±0.6 Example 14 0.37 + 8.2 ± 1.2 Example 15 0.9 0.69 .7 ± 1.2 AHP-9580/9 2 39 B B t 675-1-N1 TABLE 1 (Continued) LAF* PLN* Skin Graft Compound (ratio) ("ratio) (davs + SD) Example 16 3.27 1.04## 12.7 ± 0.9 Example 17 0.56 1.68### .2 ± 1.7 Example 18 0.02 1.11## 8.0 ± 1.7 Example 19 0.01 0.48 8.0 ± 0.9 Example 20 0.97 0.70 9.3 ± 1.6 Example 21 0.22 -1.93 12.0 ± 1.7 Example 22 0.22 0.41 .2 ± 1.2 Example 23 0.18 0.39 .8 ±0.8 Example 24 0.00 0.09 7.8 ±_\.l Rapamycin 1.0 1.0 12.0 ±1.7 * Calculation of ratios was described supra.

** Result expressed as percent inhibition of lymphoproliferation at 1 jiM. + 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 immunosuppressive activity both in vitro and in vivo for the compounds of this 30 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 35 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 Tsuppress0r cells, AHP-9580/! 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 5 5 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 10 expressed in MIC (M-g/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, rapamycin.

Table 2* Strain of Candida albicans B B 1 Compound ATCC 10231 ATCC 38246 ATCC 38247 ATCC 38248 3669 Example 1 >0.4 >0.4 >0.4 >0.4 >0.4 Example 2 0.1 0.2 0.2 0.2 0.1 Example 3 0.4 >0.4 > 0.4 >0.4 0.4 Example 4 0.1 0.4 0.1 0.1 0.2 Example 5 >0.4 >0.4 >0.4 >0.4 >0.4 ; Example 6 0.1 >0.4 0.2 0.4 >0.4 Example 7 + + + + + Example 8 >0.4 >0.4 . >0.4 >0.4 >0.4 Example 9 0.4 >0.4 0.4 >0.4 >0.4 Example 10 0.2 >0.4 0.2 0.4 0.4 Example 11 >0.4 >0.4 >0.4 >0.4 >0.4 Example 12 0.2 >0.4 0.1 0.2 0.4 Example 13 >0.4 > 0.4 >0.4 >0.4 >0.4 Example 14 >0.4 >0.4 >0.4 >0.4 >0.4 Example 15 >0.4 0.4 >0.4 0.4 0.4 Example 16 0.2 0.1 0.4 0.1 0.1 Example 17 >0.4 0.2 >0.4 0.2 0.4 Example 18 0.4 >0.4 >0.4 >0.4 >0.4 Example 19 0.4 >0.4 0.4 >0.4 >0.4 2 39 AHP-9580/9675-l-Nl Table 2* (Continued) Strain of Candida albicans Compound ATCC 10231 ATCC 38246 ATCC 38247 ATCC 38248 3669 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 Example 24 >0.4 >0.4 >0.4 >0.4 >0.4 Rapamycin 0.003 0.025 0.003 0.006 0.025 * expressed as MIC (|ig/ml) + not evaluated Based on the results of these standard pharmacological test procedures, the 15 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, seborrhea, inflammatory bowel disease; 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 also act as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet-disintegrating agents; it can also be an encapsulating 25 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, 30 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 35 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 AHP-9580/967< 2.3.9 B 5 can contain other 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 5 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 (e.g. 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 10 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 15 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 20 packaged compositions, for example, packeted powders, vials, ampoules, prefilled 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 solution, cream, or lotion by formulation with pharmaceutically acceptable vehicles containing 0.1-5 percent, preferably 2%, of active compound which may be administered to a fungally affected area.

Accordingly this invention also provides a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier- The following examples illustrate the preparation of representative compounds of this invention. 2 3 9 B 5 2 AHP-9580/9675-1 -N1 Example 1 Rapamvcin-42-ester with N-IYl.l -dimethvlethoxvkarbonvll-glvcvlglvcine a ) Under anhydrous conditions, a solution of rapamycin (3 g, 3.28 mmole) and N-[(l,l-dimethylethoxy)carbonyl]-glycylglycine (3.04 g, 13.1 mmole) in 40 mL of anhydrous dichloromethane was treated with dicyclohexylcarbodiimide (1.35 g, 6.56 mmole) 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 10 dichloromethane. The combined filtrates were absorbed directly onto silica gel Merck 60 by adding the gel and evaporation to dryness. Flash chromatography of the preabsorbed material (using a gradient elution with ethylacetate-toluene from 2:1 to 1:0 v/v) afforded 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 15 monoester is a 8.3:1 mixture of two conformers.

*H NMR (CDC13, 400 MHz): 5 1.46 (m, 9H, COOBu1), 1.654 (s, 3H, CH3C=C), 1.751 (s, 3H, CH3C=C), 3.14 (s, 3H, CH3O), 3.33 (s, 3H, CH3O), 3.36 (s, 3H, CH30), 4.18 (d, 1H, C//OH), 4.75 (m, 1H, 42-CHO ), 4.79 (s, 1H, OH); High Res. MS (neg. ion FAB) Calcd for C60H93N3O17: 1127.6504, measured mass 1127.6474.

Anal. Calcd for C60H93N3O17 • 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 from rapamycin and the appropriate terminally-N-substituted amino acid by employing the method used to prepare the title compound in Example 1. b )Rapamycin-42-ester with N-[(fluorenylmethoxy)carbonyl]-alanylserine c) Rapamycin-42-ester with N-[(fluorenylmethoxy)carbonyl]-glycylglycine d) Rapamycin-42-ester with N-[(ethoxy)carbonyl]-arginylmethionine e) Rapamycin-42-ester with N-[(4'-chlorophenoxy)carbonyl]-histidylarginine f) Rapamycin-42-ester with N-[(phenoxy)carbonyl]-tryptophanylleucine g) Rapamycin-42-ester with N-[(phenylmethoxy)carbonyl)]-N-methylglycyl-N-ethylalanine AHP-9580/96^-lJlQ 0 h)Rapamycin-42-ester with N-[(phenylmethoxy)carbonyl]-N-methyl-P-alanylphenylalanine ^ Rapamycin-42-ester with N-[( 1,1 -dimethylethoxy)carbonyl]-cysteinylglycine Example 2 Rapamvcin-31.42-die.ster with N-1Y1 .l-dimethylethoxvlcarbonvll-glvcylglycine a) 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. HPLC analysis showed that the diester is a 8.1:1 mixture of conformers. !h NMR (CDCI3, 400 MHz): 8 1.452 (m, 18H, COOBu1), 1.6612 (s, 3H, CH3C=C), 1.7815 (s, 3H, CH3C=C), 3.14 (s, 3H, OCH3), 3.34 (s, 3H, OCH3), 15 3.35 (s, 3H, OCH3), 4.52 (s, IH, OH), 4.79 (m, IH, 42-CH0 ); High Res. MS (neg. ion FAB): Calcd for C69H107N5O21 1341.7458, measured mass: 1341.7463.

Anal. Calcd for C69H107N5O21 • 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. b) Rapamycin-31,42-diester with N-[(fluorenylmethoxy)carbonyl]-alanylserine c) Rapamycin-31,42-diester with N-[(fluorenylmethoxy)carbonyl]-glycylglycine d) Rapamycin-31,42-diester with N-[(ethoxy)carbonyl]-arginylmethionine e) Rapamycin-31,42-diester with N-[(4'-chlorophenoxy)carbonyl]-histidylarginine f) Rapamycin-31,42-diester with N-[(phenoxy)carbonyl]-tryptophanylleucine g) Rapamycin-31,42-diester with N-[(phenylmethoxy)carbonyl)]-N-methylglycyl-N-ethyl-alanine h) Rapamycin-31,42-diester with N-[(phenylmethoxy)carbonyl]-N-methyl-(3-alanylphenyl- alanine i) Rapamycin-31,42-diester with N-[(l,l-dimethylethoxy)carbonyl]-cysteinylglycine 239 852 AHP-9580/9675-1-N1 Example 3 Rapamvcin-31.42-diester with N-IY1.1 -dimethylethoxv)carbonvll-N-methvlglvcine a) 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 dichloromethane 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 10 dichloromethane. The combined filtrates were evaporated to dryness to give an amorphous amber solid (3 g). The crude product was purified by flash chromatography ( on silica Merck 60, elution with hexane-ethylacetate 1:1, v/v) 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 15 NMR peaks suggests the presence of amide rotamers.

*H NMR (CDCI3, 400 MHz): 5 1.411, 1.438, 1.448 and 1.474 (m, 18 H, COOBut), 2.91 (m, 6H, NCH3), 3.14 (s, 3H, CH30), 3.34 (s, 3H, CH3O), 3.37 (s, 3H, CH3O), 4.73 (broad, IH, 42-CHO), 4.82 (2s, IH, OH); High Res. MS (neg. ion FAB): Calcd. for C67H105N3O19 1255.7342, measured mass 1255.7289. 20 Anal. Calcd for C67H105N3O19: C, 64.04; H, 8.42; N, 3.34 Found: C, 64.14; H, 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 25 prepare the title compound in Example 3. b) Rapamycin-31,42-diester with N-[(ethoxy)carbonyl]-tyrosine c) Rapamycin-31,42-diester with N-[(fluorenylmethoxy)carbonyl]-phenylalanine d) Rapamycin-31,42-diester with N-[(3',4',5'-trihydroxyphenoxy)carbonyl]-isoleucine 30 e) Rapamycin-31,42-diester with N-[(l,l-dimethylethoxy)carbonyl)-glutamine f) Rapamycin-31,42-diester with N-[(phenoxy)carbonyl]-N-methylalanine g) Rapamycin-31,42-diester with N-[(propyloxy)carbonyl]-4-aminobutryic acid h) Rapamycin-31,42-diester with N-[(phenylmethoxy)carbonyl]-7-aminoheptanoic acid i) Rapamycin-31,42-diester with N-[(fluorenylmethoxy)carbonyl]-serine 2L39 852 AHP-9580! Example 4 Rapam vcin-42-ester with N-\( 1.1 -dimethvlethoxv)carbonvll -N-methvl glycine a) Under anhydrous conditions, an ice cold solution of rapamycin (0.95 g, 1.02 mmole) and Na-Boc sarcosine (0.21 g, 1.1 mmole) in 20 mL of anhydrous dichloromethane was treated with dicyclohexylcarbodiimide 0.21 g, 1 mmole) followed by 4-dimethylaminopyridine (0.12 g, 1 mmole). After stirring for 4 hours at ambient temperature, the precipitated solid was collected and washed with dichloromethane. The 10 combined filtrates were concentrated in vacuo to give an amorphous amber solid. Flash chromatography of the crude product (on silica Merck 60, elution with hexane-ethylacetate 1:1 v/v to remove the diester of Example 3, followed by chloroform-ethylacetate-methanol 75:25:1 v/v) provided partially purified title compound (0.38 g, 35%). Pure product was obtained by preparative HPLC (Waters Prep 500, silica gel, 15 chloroform-ethylacetate-methanol 75:25:1 v/v, flow rate 250 mL/min). 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. lH NMR (CDCI3, 400 MHz): 8 1.42-1.46 (ds, 9H, COOBu1), 2.91 (ds, 3H, NCH3), 1.644 (s, 3H, CH3C=C), 1.738 (s, 3H, CH3C=C), 3.12 (s, 3H, CH3O), 20 3.32 (s, 3H, CH3O), 3.35 (s. 3H, CH3O), 4.18 (d, IH, CHOH), 4.71 (broad, IH, 42-CHO), 4.78 (broad s, IH, OH); High Res. MS (neg. ion FAB): Calcd for C59H92N2O16 1084.6446, measured mass 1084.6503.

Anal. Calcd for C59H92N2O16: C, 65.29; H, 8.54; N, 2.58 Found: C, 65.25; H, 8.52; N, 2.42 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 4. b) Rapamycin-42-ester with N-[(ethoxy)carbonyl]-tyrosine c) Rapamycin-42-ester with N-[(fluorenylmethoxy)carbonyl]-phenylalanine d) Rapamycin-42-ester with N-[(3,,4',5'-trihydroxyphenoxy)carbonyl]-isoleucine e) Rapamycin-42-ester with N-[(l,l-dimethylethoxy)carbonyl)-glutamine f) Rapamycin-42-ester with N-[(phenoxy)carbonyl]-N-methylalanine g) Rapamycin-42-ester with N-[(propyloxy)carbonyl]-4-aminobutryie acid h) Rapamycin-42-ester with N-[(phenylmethoxy)carbonyl]-7-aminoheptanoic acid 239855 AHP-9580/9675-1 -N1 i) Rapamycin-31,42-diester with N-[(fluorenyImethoxy)carbonyl]serine Example 5 Rapamvcin-31.42-diester with 5-( 1.1 -dimethylethoxvV2-fIY 1.1 -dimethvlethoxv)-carbonvllaminol-5-oxopentanoic acid a) Under anhydrous conditions, an ice cold solution of rapamycin (4 g, 4.37 mmole) and L-glutamic acid Na-Boc-y-tert-butylester (4.9 g, 16.1 mmole) in 40 mL 10 of dry dichloromethane 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 15 (on silica Merck 60, gradient elution with hexane-ethylacetate from 2:1 to 1:1, v/v) to yield 4.52 g (69.6%) 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.

*H NMR (CDCI3, 400 MHz): 5 1.42 (m, 36 H, COOBu1), 1.646 (s, 3H, CH3C=C), 1.701 (s, 3H, CH3C=C), 3.13 (s, 3H, CH30), 3.34 (s, 3H, CH30), 20 3.36 (s, 3H, CH3O), 4.735 (m, 2H, 0H+42-CH-0); High Res. MS (neg. ion FAB): calc. for C79Hj25N3023 1483.8715, measured mass 1483.8714.

Anal. Calcd for C79Hi25N3023: 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 rapamycin and the appropriately terminally-N-substituted amino diacid monoester by employing the method used to prepare the title compound in Example 5. b) Rapamycin-31,42-diester with 6-(phenylmethoxy)-2-[[fluorenylmethoxy)carbonyl]-amino]-6-oxohexanoic acid c) Rapamycin-31,42-diester with 6-(4'-methylphenoxy)-3-[[(phenylmethoxy)carbonyl]-amino-6-oxohexanoic acid d) Rapamycin-31,42-diester with 6-(ethoxy)-4-[[(phenoxy)carbonyl]amino]-6-oxo-35 hexanoic acid 239 ArtP-9580/9675-l-Nl e)Rapamycin-31,42-diester with 6-(methoxy)-5-[[(ethoxy)carbonyl]amino]-6-oxo-hexanoic acid f)Rapamycin-31,42-diester with 4-(phenoxy)-2-[N-[(l,l-dimethylethoxy)carbonyl]-N-methylamino]-4-oxobutanoic acid g) Rapamycin-31,42-diester with 4-(phenylmethoxy)-3-[N-[(methoxy)carbonyl]-N-methylamino]-4-oxobutanoic acid Example 6 Rapamvcin-42-ester with 5-( 1.1 -dimethylethoxvV2-IT( 1.1 -dimethvlethoxv)-carbonyllaminol-5-oxopentanoic acid a) The title compound (1.14 g, 20.6%) was separated from the 31,42-diester as 15 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.

*H NMR (CDCI3, 400 MHz): 5 1.425 (m, 18H, COOBu1), 1.643 (s, 3H, CH3C=C), 1.737 (s, 3H, CH3C=C), 3.13 (s, 3H, CH30), 3.32 (s, 3H, CH30), 3.36 (s, 3H, CH30), 4.17 (d, IH, C//OH), 4.71 (M, IH, 42-CHO), 4.785 (s, IH, 20 OH); High Resolution MS ( neg. ion FAB): Calc. for C65H102N2O18 1198.7127, measured mass 1198.7077.

The following representative compounds can be prepared from rapamycin and the appropriately terminally-N-substituted amino diacid monoester by employing the 30 method used to prepare the title compound in Example 6. b) Rapamycin-42-ester with 6-(phenylmethoxy)-2-[[fluorenylmethoxy)carbonyl]-amino]-6-oxohexanoic acid c) Rapamycin-42-ester with 6-(4'-methylphenoxy)-3-[[(phenylmethoxy)carbonyl]-amino-35 6-oxohexanoic acid Anal. Calcd for C55H102N2O18 • CH3COOEt • 0.25 H2O: C, 64.13, H, 8.60; N, 2.17 Found: C, 64.18; H, 8.52: N, 2.01 AHP-9580/9675-1 2'3 9 8 5 d ^Rapamycin-42-iester with 6-(ethoxy)-4-[[(phenoxy)carbonyl]amino]-6-oxo- hexanoic acid e) Rapamycin-42-ester with 6-(methoxy)-5-[[(ethoxy)carbonyl]amino]-6-oxo- hexanoic acid f) Rapamycin-42-ester with 4-(phenoxy)-2-[N-[(l,l-dimethylethoxy)carbonyl]-N-methylamino]-4-oxobutanoic acid g) Rapamycin-42-ester with 4-(phenylmethoxy)-3-[N-[(methoxy)carbonyl]-N-methylamino]-4-oxobutanoic acid Example 7 Rapamvcin-31.42-diester with 2-\\( 1.1 -dimethylethoxy)carbonyllaminol-4-oxo-4-(phenvlmethoxv') butanoic acid Under anhydrous conditions, 295mg (1.21mmol) of 2,4,6 trichlorobenzoyl chloride was added to a solution of 391mg(1.21mmol) of Na-Boc-L-aspartic acid-P-benzyl ester and 170(j.L (1.21mmol) of Et3N in 1 mL of THF at room temperature. After stirring for 30 minutes, 500 rug (0.55mmol) of rapamycin and 295 mg ( 2.42 mmol) of dimethylaminopyridine was added and the reaction was left to stir overnight. 20 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.

*H NMR (CDCI3, 400 MHz) 5 7.347 (s, 10 H, Ar), 6.223, 5.126 (s, 4 H, CH2Ph), 25 4.698 (m, 1 H, CH-C02), 4.587 (m, 2 H, NH), 3.353 (s, 3 H, CH3O), 3.337 (s, 3 H, CH3O), 3.301 (s, 3 H, CH3O), 2.775 (m, 4 H, CH2C02); IR (KBr) 3420 (OH), 2935 (CH), 2920 (CH), 1730 (C=0), 1650, 1500, 1455, 1370, 1170 cm*1; MS (neg. ion FAB) 1523 (M"), 1433, 297, 248, 205, 148, 44, 25 (100).

Anal. Calcd for C83Hi i7N3023-7H20 C, 60.40; H, 7.09; N, 2.54 30 Found: C, 60.54; H, 7.28; N, 2.56 239 851 AHP-9580/9675-1 -N1 Example 8 Rapamycin-31.42-diester with 3-rr(l.l-dimethvlethoxv')carbonvllaminol-4-oxo-4-(phenvlmethoxv) 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 Na-Boc-L-aspartic acid-a-benzyl ester and 303 |iL (2.18 mmol) of Et3N 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 HC1 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 (20-40 % ethyl acetate/hexane as eluant) to give 532 mg (33 %) of the title compound which was isolated as the hydrate.

!H NMR (CDC13, 400 MHz) 6 7.362 (s, 10 H, Ar), 5.193 (s, 4 H, CH2Ph), 4.596 (m, 1 H, CH-C02), 4.586 (m, 2 H, NH), 3.336 (s, 3 H, CH3O), 3.306 (s, 3 H, CH3O), 3.145 (s, 3 H, CH3O); IR (KBr) 3410 (OH), 2950 (CH), 2920 (CH), 1735 (C=0), 1710 (C=0), 1640, 1490, 1445, 1350, 1150 cm -1; MS (neg. ion FAB) 1524 (M-), 1434, 297, 248, 232, 214, 205, 167, 148, 42 (100), 26.

Anal. Calcd for C83H117N3023 • 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-[T(l.l-dimethylethoxy)carbonynaminol-4-oxo-4-(phenvlmethoxv) 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. 239 8 5 * AHP-9580/9675-1-N1 19- lH NMR (CDC13, 400 MHz) 5 7.356 (s, 5 H, Ar), 5.185 (s, 2 H, CH2Ph), 4.635 (m, 1 H, CH-C02), 4.582 (m, 1 H, NH), 3.330 (s, 6 H, CH3O), 3.135 (s, 3 H, CH3O); IR (KBr) 3410 (OH), 2950 (CH), 2920 (CH), 1735 (C=0), 1710 (C=0), 1640, 1490, 1445, 1350, 1150 cm -1; MS (neg. ion FAB) 1218 (M*), 1127, 590, 168, 5 42, 25, 17 (100).

Anal. Calcd for Q57H98N2O18 • 1.5 H20: C, 63.64; H, 8.21; N, 2.22 Found: C, 63.64; H, 7.51; N, 2.13 Example 10 Rapamvcin-42-ester with 5-(l. 1 -dimethvloxy)-4-fIY1.1 -dimethylethoxv)carbonyl1 -aminol-5-oxopentanoic acid Under anhydrous conditions, an ice cold solution of rapamycin (4 g, 4.37 15 mmole) and L-glutamic acid Na-Boc-a-tert-butylester (4.9 g, 16.1 mmole) in 40 mL of anhydrous dichloromethane was treated with dicyclohexylcarbodiimide (1.8 g, 8.7 mmole) followed by 4-dimethylamino pyridine (1 g, 8.7 mmole). After stirring overnight at ambient temperature, the precipitated solid was collected and washed with dichloromethane. The combined filtrates were concentrated in vacuo to give 9 g of an 20 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, v/v) to provide 1.35 g (25.7%) 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.

NMR (CDCI3, 400 MHz): 5 1.43 (s, 9H, COOBu1) and 1.46 (s, 9H, COOBu1), 1.65 (s, 3H, CH3C=C), 1.75 (s, 3H, CH3C=C), 3.14 (s, 3H, CH30), 3.34 (s, 3H, CH30), 3.38 (s, 3H, CH30), 4.18 (d, IH, CH-OH), 4.65 (m, IH, 42- CHO), 4.80 (s, IH, OH); High Res. MS (neg. ion FAB): Calc. for C65Hi02N2Ol8: 1198.7126, measured mass 1198.7135.

Anal. Calcd for C65Hi02N2Oi8: C, 65.09; H, 8.57; N, 2.34 Found C, 65.04; H, 8.33; N, 2.64 239 852 AHP-9580/9675-1 -N1 Example 11 Rapamvcin-31.42-diester with 5-(l .1 -dimethvlethoxvW-nY 1. 1-dimethylethoxv)-carbonvll- amino!-5-oxopentanoic acid The title compound was prepared (0.83 g, 12.8%) along with the 42-monoester as described in Example 10. HPLC analysis showed that the diester is a 7.7:1 mixture of two conformers. !h NMR (CDCI3, 400 MHz): 5 1.43 (s, 18H, COOBu1), 1.46 (s, 18H, COOBu*), 1.659 (s, 3H, CH3C=C), 1.759 (s, 3H, CH3C=C), 3.14 (s, 3H, CH30), 3.34 (s, 3H, CH30), 3.38 (s, 3H, CH30), 4.66 (m, IH, 42-CHO), 4.72 (s, IH, OH); High Res. MS (neg. ion FAB): Calcd for C79H125N3O23: 1483.8704, 15 measured mass 1483.8636.

Anal. Calcd for C79Hl25N3023: 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£-bislY 1.1 -dimethvlethoxv)carbonyn-L-lysine Under anhydrous conditions, a solution of rapamycin (3 g, 3.28 mmole) and Na, 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 4-dimethylaminopyridine (0.8 g, 6.56 m mole). After stirring overnight at ambient temperature, the precipitated solid was collected and washed with dichloromethane. The 30 combined filtrates were concentrated in vacuo to give an amorphous amber solid. Flash chromatography of the crude product (on silica Merck 60, elution with hexane-ethylacetate 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-ethylacetate 3:2 v/v, flow rate 250 mL/min). HPLC analysis showed that the monoester 35 is a 9:1 mixture of two conformers.

AHP-9580/967 *H NMR (CDCI3, 400 MHz): 5 1.438 (m, 9H, COOBut), 1.455 (s, 9H, COOBu1), 1.652 (s, 3H, CH3C=C), 1.752 (s, 3H, CH3C=C), 3.14 (s, 3H, CH3O), 3.33(s, 3H, CH3O), 3.37 (s, 3H, CH3O), 4.18 (d, IH, CHOH), 4.72 (m, IH, 42-CHO), 4.79 (s, IH, OH); High Res. MS (neg. ion FAB): Calcd for C67H107N3Oi8: 1241.7549, measured mass 1241.7604.

Anal. Calcd for C67H107N3O18 : 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 with N&. NS-bislYLl-dimethylethoxykarbonyll-L-lysine Under a nitrogen atmosphere, a solution of Na,Ne bis-Boc-L-lysine (1.038 g, 15 3 mmole) and triethylamine (0.42 mL, 3 mmmole) in 10 mL of anhydrous THF 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 5 mL of benzene and added to a stirred solution of rapamycin (1 g, 1.09 mmole) 20 containing 4-dimethylamino pyridine (0.59 g, 4.8 mmole) in 10 mL of benzene. After stirring at ambient temperature overnight, the precipitated solid was collected and the filtrate was evaporated to dryness (yellow foam). 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, 67%). HPLC analysis shows that the diester is a 9:1 25 mixture of two conformers. lH NMR (CDCI3, 400 MHz): 5 1.426 (m, 9H, COOBuO, 1.438 (s, 9H, COOBut), 1.443 (s, 9H, COOBut), 1.446 (s, 9H, COOBut), 3.141 (s, 3H, CH3O), 3.36 (s, 3H, CH3O), 3.378 (s, 3H, CH3O), 4.68-4.76 (m, 2H, OH and 42-CHO); High res. MS (neg. ion FAB): Calcd. for C83H135N5O23 1569.9526, measured mass 1569.9537. 30 Anal. Calcd. for C83H135N5O23: C, 63.46; H, 8.66; N, 4.46 Found: C, 63.06; H, 8.84; N, 4.09 239 8K AHP-9580/9675-1 -N1 Example 14.

Rapamvcin-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 l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in 20 mL of dry dichloromethane was added 200 mg of 4-dimethylaminopyridine. The solution was stirred at room temperature for 3 days. The reaction mixture was poured into 2 N HC1 and extracted three times with ethyl 10 acetate. The organic layers were combined, washed with brine, dried over anhydrous sodium sulfate, decanted, and concentrated in vacuo to give a light yellow foam. Rash 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 #1, upon concentration, gave 330 mg (4.1 %) of pure rapamycin-14,31,42-tris-15 (monobenzylsuccinate).

NMR (CDC13, 400 MHz) 6 7.353 (bs, 15 H, arom), 5.168 (d, J = 2.0 Hz, 1 H, CH-02C), 5.148 (m, 6 H, CH2Ph), 4.672 (m, 1 H, C02C//-CH0Me), 3.355 (s, 3 H, CH3O-), 3.337 (s, 3 H, CH3O-), 3.327 (s, 3 H, CH3O-), 2.697 ( m, 12 H, 02CC//2C//2C02CH2Ph), 1.745 (s, 3 H, CH3C=C), 1.655 (s, 3 H, CH3C=C); 20 IR (KBr) 3450 (OH), 2950 (CH), 1745 (C=0), 1650, 1460, 1385, 1360, 1160, 1105, 995 cm*1.

Analysis Calcd for C84Hi09NO2i • 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. b) Rapamycin-14,31,42-tris (monomethylsuccinate) 30 c ^ Rapamycin-14,31,42-tris (monophenyl-3',3'-dimethylglutarate) d) Rapamycin-14,31,42-tris (mono t-butyl-3'-methylglutarate) e) Rapamycin-14,31,42-tris (monobenzylthiodiglycolate) f) Rapamycin-14,31,42-tris (monohexyldiglycolate) 9) Rapamycin-14,31,42-tris (monopropylphthalate) Rapamycin-14,31,42-tris (monoethyl-2',6'-pyridinedicarboxylate) 239 AHP-9580/9675-1 -N1 Example 15.

Rapamvcin-31.42-bisfmonobenzvlsuccinate') a) Fraction # 2, obtained from the procedure employed in Example 14, gave 1.25 g (17.7 %) of pure rapamycin-31,42-bis(monobenzylsuccinate) upon concentration.

NMR (CDCI3, 400 MHz) 5 7.351 (bs, 10 H, arom), 5.168 (d, J = 2.0 Hz, 1 H, CH-02C), 5.125 (m, 4 H, CH2Ph), 4.680 (m, 1 H, C02C7/-CH0Me), 3.356 10 (s, 3 H, CH3O-), 3.329 (s, 3 H, CH3O-), 3.146 (s, 3 H, CH3O-), 2.639 ( m, 8 H, 02CCH2C//2C02CH2Ph), 1.748 (s, 3 H, CH3C=C), 1.654 (s, 3 H, CH3C=C); IR (KBr) 3450 (OH), 2940 (CH), 1740 (C=0), 1650, 1455, 1380, 1355, 1160, 1105, 995 cm*1; MS (neg. ion FAB) 1294 (M-), 1202, 1103, 1012, 590, 511, 475, 297, 207, 167, 148, 99 (100); High Res. MS (neg. ion FAB) Calcd for C73H99NO19 15 1293.68108, found 1293.6811.

Analysis Calcd for C73H99NO19 • H20 C 66.82; H 7.70; 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 15. b) Rapamycin-31,42-bis (monomethylsuccinate) 25 c) Rapamycin-31,42-bis (monophenyl-3',3'-dimethylglutarate) d) Rapamycin-31,42-bis (mono t-butyl-3'-methylglutarate) e) Rapamycin-31,42-bis (monobenzylthiodiglycolate) f) Rapamycin-31,42-bis (monohexyldiglycolate) g) Rapamycin-31,42-bis (monopropylphthalate) h) Rapamycin-31,42-bis (monoethyl-2',6'-pyridinedicarboxylate) Example 16.

Rapamycin-42-("monobenzylsuccinate) a) Fraction # 3, obtained from the procedure employed in Example 14, gave 930 mg (15.4 %) of pure rapamycin-42-monobenzylsuccinate upon concentration.

AHP-9580/9675-fa 239 !H NMR (CDC13, 400 MHz) 5 7.355 (bs, 5 H, arom), 5.141 (ra, 2 H, C//2Ph), 4.680 (m, 1 H, C02C//-CH0Me), 3.364 (s, 3 H, CH3O-), 3.333 (s, 3 H, CH3O-), 3.141 (s, 3 H, CH3O-), 2.698 ( m, 4 H, O2CC//2C//2CO2CH2PI1), 1.751 (s, 3 H, CH3C=C), 1.655 (s, 3 H, C//jC=C); IR (KBr) 3450 (OH), 2940 (CH), 1740 (C=0), 1645, 1455, 1380, 1165, 1105, 990 cm*1; MS (neg. ion FAB) 1103 (M-), 1045, 1012, 624, 590, 167, 99 (100); High Res. MS (neg. ion FAB) Calcd for C62H89NO16 1103.6181, found 1103.6048.

Analysis Calcd for C62H89NO16 • 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 rapamycin and the appropriate half acid-ester by employing the method used to prepare the title compound in Example 16. k>) Rapamycin-42-(monomethylsuccinate) c) Rapamycin-42-monophenyl-3',3'-dimethylglutarate) d) Rapamycin-42-(mono t-butyl-3'-methylglutarate) e) Rapamycin-42-(monobenzylthiodiglycolate) f) Rapamycin-42-(monohexyldiglycolate) g) Rapamycin-42-(monopropylphthalate) h) Rapamycin-42-(monoethyl-2',6'-pyridinedicarboxylate) Example 17.

Rapamvcin-31.42-bishemi glutarate a) To a solution of 2.0 g (2.2 mmol) of rapamycin in 10 mL of dry dichloromethane was added 1.24 g (10.9 mmol) of glutaric 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 was allowed to reflux for 8 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 Ci8 column eluting starting with 60 % acetonitrile/water. Collected, after, concentration, 586 mg (24 %) of rapamycin-31,42-bishemiglutarate. 239 AHP-9580/9675-1-N1 - lH NMR (CDC13i 400 MHz) 5 5.398 (m, 1 H, -C02C//CH0Me), 4.683 (m, 1 H, -C02C//CH0Me), 3.364 (s, 3 H, CH3O-), 3.362 (s, 3 H, CH3O-), 3.106 (s, 3 H, CH3O-), 2.407 (m, 8 H, -O2CCH2CH2CH2CO2H), 1.960 (m, 4 H, -O2CCH2C//2CH2CO2H), 1.770 (s, 3 H, CH3C=C), 1.653 (s, 3 H, C7/jC=C); 5 !3C NMR (CDCI3, MHz) 211.45 (C=0), 206.84 (C=0), 200.44 (C=0), 177.83 (C=0), 177.04 (C=0), 172.43 (C=0), 171.20 (C=0), 165.27 (C=0), 159.08 (C=0); IR (KBr) 3430 (OH), 2940 (CH), 2880 (CH), 1745 (C=0), 1685, 1625, 1580, 1450, 1385, 1330, 1200, 1140, 1100, 990 cm'1; MS (neg. ion FAB) 1140 (M-H), 1122, 1026, 990, 946, 913, 590, 475, 435, 321, 167, 148, 131 (100), 113; High Res. 10 MS (neg. ion FAB) Calcd for C61H90O19N (M-H) 1140.6107, Found 1140.6106.

Analysis Calcd for C61H91O19N H2O 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 15 the appropriate anhydride by employing the method used to prepare the title compound in Example 17. b) Rapamycin-3 l,42-bishemi-3'-methylglutarate c) Rapamycin-3 l,42-bishemi-3',3'-dimethylglutarate 20 d) Rapamycin-3 l,42-bishemi-3'-oxoglutarate e) Rapamycin-3 l,42-bishemi-3'-thioglutarate f) Rapamycin-31,42-bishemi-phthalate g) Rapamycin-31,42-bishemi-2' ,3'-pyridine dicarboxylate Example 18.

Rapamycin-31.42-hemiglutarate bissodium salt Purified bis-31,42-hemiglutarate of rapamycin (740 mg, 649 umol), prepared as described in Example 17, was dissolved in 5 mL of 95 % ethanol and 107 mg (1.27 mmol) 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 35 pistol for 24 h, refluxing over acetone at reduced pressure to give 520 mg of the bissodium salt.

AHP-9580/9675-1- 239 85 2 75-1-N1 V U *H NMR (d6-DMSO, 400 MHz) 5 5.235 (m, 1 H, -CHQ2C), 4.498 (m, 1 H, MeOCHCT/O2C-), 3.287 (s, 6 H, 2 C//3O-), 3.236 (s, 3 H, C//3O-), 2.245 (m, 8 H, O2CC//2CH2C//2CO2-), 1-712 (s, 3 H, CH3C=C), 1.593 (s, 3 H, CH3C=C); IR (KBr) 3420 (OH), 2920 (CH), 1725 (C=0), 1675, 1620, 1560, 1450, 1400, 1375, 1230, 1195, 1130, 1090, 980 cm*1; MS (neg. ion FAB) 1112 (M-l, free acid), 994, 589, 475, 297, 167, 148, 117, 99 (100); High Res. MS (neg. ion FAB) Calcd for C6iH890i9NNa (M-Na) 1162.5926, Found 1162.5899.

Analysis Calcd for C6iH890i9NNa2 • H20 C 60.85; H 7.56; N 1.16 Found C 60.67; H 7.36; N 1.58 Example 19.

Rapamvcin-31.42-bishemiglutarate bistromethamine salt Purified bis-31,42 hemiglutarate of rapamycin (950 mg, 833 umol), prepared as described in Example 17, was dissolved in 5 mL of 95 % ethanol and 197 mg (1.63 mmol) of tris(hydroxymethyl)methylamine was added. Water (1 mL) was added to completely dissolve the amine. Once dissolved, the yellow solution was 20 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 900 mg (78 %) of the bistromethamine salt.

*H NMR (d6-DMSO, 400 MHz) 6 5.253 (m, 1 H, -CH02C), 4.523 (m, 1 H, Me0CHC//02C-), 3.347 (s, 6 H, 2 C//3O-), 3.276 (s, 3 H, C//3O-), 2.289 (m, 8 H, O2CCH2CH2CH2CO2-), 1.681 (s, 3 H, CH3C=C), 1.595 (s, 3 H, CH3C=C); IR (KBr) 3400 (OH), 2920 (CH), 1730 (C=0), 1620, 1555, 1450, 1400, 1370, 1185, 1060, 980 cm"1; MS (neg. ion FAB) 1140 (M-H, free acid), 1028, 167, 148, 131 (100), 113; High Res. MS (neg. ion FAB) Calcd for C61H90O19N (M-H, free acid) 1140.6107, Found 1140.6069.

Analysis Calcd for C69H103O25N3 • 2 H20 C 58.77; H 7.58; N 2.98 Found C 58.47; H 7.94; N 3.58 t 3 9 8 5"2 AHP-9580/9675-l-Nl Example 20.

Rapamvcin-42-hemi-3'-oxoglutarate a) To a solution of 3.0 g (3.3 mmol) of rapamycin in 20 mL of dry dichloromethane was added 1.90 g (16.4 mmol) of diglycolic anhydride followed by 1.32 mL (1.29 g, 16.4 mmol) of pyridine. To this was added 200 mg of 4-dimethylaminopyridine and the reaction mixture was allowed to stir at room temperature for 2 days. The solution was cooled to room temperature, poured into 2 N 10 HCI, 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 Cis column eluting starting with 60 % acetonitrile/water. After concentration, 870 mg ( 26 %) of rapamycin-42-hemi-3'-oxoglutarate and 15 500 mg (13 %) of rapamycin-3l,42-bishemi-3'oxoglutarate were isolated.

*H NMR (CDC13, 400 MHz) 5 4.768 (m, 1 H, C02C//-CH0Me), 4.250 (m, 4 H, O2CCH2OCH2CO2), 3.356 (s, 3 H, CH3O-), 3.331 (s, 3 H, CH3O-), 3.139 (s, 3 H, CH3O-), 1.759 (s, 3 H, CH3C=C), 1.653 (s, 3 H, CH3C=C)\ IR (KBr) 3420 (OH), 2920 (CH), 2875 (CH), 1740 (C=0), 1720 (C=0), 1640, 1625, 20 1445, 1370, 1320, 1200, 1135, 1095, 980 cm*1; MS (neg. ion FAB) 1028 (M - H), 327, 167 (100), 148, 133, 115; High Res. MS (neg. ion FAB) Calcd for C55H82O17N (M - H) 1028.5597, Found 1028.5599.

Analysis Calcd for C55H83O17N • 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 20. b) Rapamycin-42-hemi-3'-methylglutarate c) Rapamycin-42-hemi-3',3'-dimethylglutarate d) Rapamycin-42-hemi-3'-thioglutarate e) Rapamycin-42-hemi-phthalate f) Rapamycin-42-hemi-2',3'-pyridine dicarboxylate £V9 V5^ AHP-9580/9675-l-Nl 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 jo give a yellow foam. The crude product was purified via reverse phase HPLC on a Ci8 column eluting starting with 60 % acetonitrile/water. After concentration, 1.75 g (28 %) of rapamycin-3l,42-bishemi-3'-oxoglutarate was isolated.

!H NMR (CDC13, 400 MHz) 5 4.785 (m, 1 H, C02C//CH0Me), 4.260 (m, 8 H, O2CCH2OCH2CO2), 3.360 (s, 3 H, CH3O-), 3.343 (s, 3 H, CH3O-), 3.143 (s, 3 H, CH3O-), 1.775 (s, 3 H, CH3C=C), 1.656 (s, 3 H, CH3C=C); !3C NMR (CDCI3, MHz) 211.12 (C=0), 207.73 (C=0), 193.11 (C=0), 171.90 (C=0), 171.59 (OO), 170.15 (C=0), 169.35 (C=0), 168.83 (C=0), 166.63 (C=0); IR (KBr) 3420 (OH), 2920 (CH), 2850 (CH), 1740 (C=0), 1645, 1625, 1440, 1370, 1190, 11300, 980 cm"1; MS (neg. ion FAB) 1140 (M-H), 1122, 1026, 990, 946, 913, 590, 475, 435, 321, 167, 148, 131 (100), 113; High Res. MS (neg. ion FAB) Calcd for C59H86O21N (M - H) 1144.5701, Found 1144.5702.

Analysis Calcd for C59H87O21N 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 mmol) 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 AHP-9580/9675^N? ^ pistol for 48 h, refluxing over dichloromethane at reduced pressure to give 435 mg (58 %) of the disodium salt.

*H NMR (d^-DMSO, 400 MHz) 8 4.975 (m, 1 H, -CH02C), 4.593 (m, 1 H, Me0CHC//02C-), 4.135 (s, 2 H, -O2CCH2OC//2CO2R), 3.617 (s,2 H, 5 -O2CC//2OCH2CO2R), 3.299 (s, 6 H, 2 C//3O-), 3.232 (s, 3 H, C//3O-), 1.614 (s, 3 H, CH3C=C), 1.553 (s, 3 H, CH3C=C); IR (KBr) 3420 (OH), 2920 (CH), 1735 (C=0), 1615, 1445, 1395, 1380, 1320, 1220, 1130, 1090, 980 cm"1; MS (neg. ion FAB) 1188 (M-l), 1166 (M-Na), 1144, 1051, 1028, 590, 459, 167, 155 (100), 148, 133, 115.

Analysis Calcd for C59H8502iNNa2 • 2H2O C 57.79; H 7.26; N 1.14 Found C 57.94; H 7.11; N 1.26 Example 23.

Rapamvcin-31.42-bishemi-3'-oxoglutarate bistromethamine salt Purified bis-31,42 hemi-3'-oxoglutarate of rapamycin (1.01 g, 882 umol), prepared by the procedure employed in Example 2 l,was dissolved in 10 mL of 95 % 20 ethanol and 213 mg (1.76 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 48 h, refluxing over dichloromethane at reduced pressure to give 805 mg (66 %) of the bistromethamine salt.

*H NMR (d6-DMSO, 400 MHz) 8 4.955 (m, 1 H, -CH02C), 4.600 (m, 1 H, Me0CHC//02C-), 4.149 (s, 2 H, -02CCU20CH2C02R), 3.770 (s, 2 H, -O2CC//2OCH2CO2R), 3.407 (s, 6 H, 2 C//3O-), 3.257 (s, 3 H, C//3O-), 1.806 30 (s, 3 H, CH3C=C), 1.614 (s, 3 H, CH3C=C); IR (KBr) 3400 (OH), 2920 (CH), 1730 (C=0), 1620, 1550, 1450, 1395, 1370, 1200, 1060, 985 cm"1; MS (neg. ion FAB) 1144 (M-H, free acid), 1028, 167, 148, 133 (100), 115.

Analysis Calcd for C67H109O27N3 • H20 C 57.22; H 7.90; N 2.98 Found C 57.26; H 7.90; N 3.15 239 AHP-9580/9675-1 -N1 Example 24.

Rapamycin-31.42-bishemisuccinate.

To a solution of 2.0 g (2.2 mmol) of rapamycin in 10 mL of dry dichloro methane 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. 10 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 Cis column gradient eluting starting with 20 % 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 20 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.

!H NMR (d6-DMSO, 400 MHz) 5.231 (m, 1 H, -CH02C), 4.554 (m, 1 H, 25 Me0CHC7/02C-), 3.426 (s, 6 H, 2 C//30-), 3.249 (s, 3 H, CH3O-), 2.431 (m, 8 H, 02CCH2CH2C02-), 1.700 (s, 3 H, CH3C=C), 1.554 (s, 3 H, CH3C=C); ^C NMR (d6-DMSO, ) 211.28 (C=0), 205.23 (C=0), 199.59 (C=0), 174.86 (C=0), 173.62 (C=0), 171.72 (C=0), 171.50 (C=0), 166.56 (C=0), 166.53 (C=0); IR (KBr) 3420 (OH), 2940 (CH), 1735 (C=0), 1630, 1580, 1460, 1400, 1380, 1170, 1070, 30 990 cm"1; MS (neg. ion FAB) 1112 (M-l, free acid), 994, 589, 475, 297, 167, 148, 117, 99 (100).

Analysis Calcd for C67Hio9025N3 • 2 H20 C 57.80; H 8.12; N 3.01 Found C 57.91; H 8.21; N 2.37 239852 AHP-9580/9675-1-N1

Claims (38)

WHAT WE CLAIM IS:

1. A compound of the structure wherein R^R2 and R"^ are each, independently, hydrogen, O O — [C(CH2)mCH(CH2)nN]pC02R7 , -C-(CH2)tX(CH2)uC02Rn or I I R5 R6 0 -c-fY9-c°2Rl2; R5 is hydrogen, alkyl of 1-6 carbon atoms, aralkyl of 7-10 carbon atoms, -(CH2)qC02R8, -(CJi2)rNR9C02R10, carbamylalkyl of 2-3 carbon atoms, aminoalkyl of 1-4 carbon atoms, hydroxyalkyl of 1-4 carbon atoms, guanylalkyl of 2-4 carbon atoms, mercaptoalkyl of 1-4 carbon atoms, alkylthioalkyl of 2-6 carbon atoms, indolylmethyl, hydroxyphenylmethyl, imidazolylmethyl 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, carbalkox^^i-ll tar^n atoms, trifluoromethyl, amino, and carboxy; i*2 IJAN19931 £ \ 23 9 8 5 2 AHP-9580/9675- 1-N 1 32 R6 and R9 are each, independently, hydrogen, alkyl of 1-6 carbon atoms or aralkyl of 7-10 carbon atoms; R7, R8, and R10 are each, independently, alkyl of 1-6 carbon atoms, aralkyl of 7-10 carbon atoms, fluorenylmethyl, 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 and carboxy; R11 and R12 are each, independendy, 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 and carboxy; R13 X is -C-, O orS; R14 R13 and R14 are each, independently, hydrogen or alkyl of 1-6 carbon atoms; 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; and u is 0 - 4; O wherein R , R , m and n are independent in each of the [C(CH2)mCH(CH2)nN] R5 R6 subunits when p = 2; or a pharmaceutically acceptable salt thereof, with the proviso that R1, R2 and R3 are not all hydrogen, further provided that R1, R2 and R3 are not all T R -I AHP-9580/9675-1-N1 -33- O - [ C C CH_) CH(CH0) N] CO-.R7 , and still further that 2 m 2 n p 2 RJ I6 t and u are not both 0 when X is 0 or S. 12 3

2. A compound of Claim 1 where R , R or R is -CC(CH-) CHCCH_) N] CO~R7 , 2 m 2 n P 2 5 .6 K R wherein m=0,"n=0 and p, R5, R^ and R7 are as defined in Claim 1 or a pharmaceutically acceptable salt thereof.

3. A compound of Claim 1 or Claim 2 where n = 0, and is -CCF^) CC^R^ wherein q and R^ are as defined in Claim 1 or a pharmaceutically acceptable salt thereof.

4. A compound of Claim 2 where R^ is 9 l n 9 in - (CH) NR CO-.R wherein r, R and R are as 2 r 2 ' defined in Claim 1 or a pharmaceutically acceptable salt thereof. 5 r\kNr

5. A compound of Claim 2 where R is hydrogen or\a pharmaceutically acceptable salt thereof. , 17 FEB1993

6. A compound of Claim 1 where R^, R^ or R^ is •*> o -C-CCH^^XCCl^) CO-R^wherein t, X, u and are as defined in Claim 1 or a pharmaceutically acceptable salt thereof.

7. Rapamycin-42-ester with N-[(1,1-dimethy1ethoxy )-carbonylIglycylglycine or a pharmaceutically acceptable salt thereof. m AHP-9 5 80/9675-1-N1 •34- 239 8? 2

8. Rapamycin-31,42-diester with N-[(l,l-dimethylethoxy)carbony1]-glycylglycine or a pharmaceutically acceptable salt thereof.

9. Rapamycin-31,42-diester with N-[(l,l-dimethylethoxy)carbonyl]-N-methylglycine or a pharmaceutically acceptable salt thereof.

10. Rapamycin-42-ester with N-[(l,l-dimethylethoxy)carbonyl]-N-methylglycine or a pharmaceutically acceptable salt thereof.

11. Rapamycin-31,42-diester with 5-(l,l-dimethylethoxy)—2 — [[CI,1-dimethylethoxy)carbony1]-amino]-5-oxopentanoic acid or a pharmaceutically acceptable salt thereof.

12. Rapamycin-42-ester with 5-(1,1-dimethylethoxy)- 2-[[C1,1-dimethylethoxy)carbonyl]amino]-5-oxopentanoic acid or a pharmaceutically acceptable salt thereof.

13. Rapamycin-31,42-diester with 2—[[Cl,l— dimethylethoxy)carbony1]amino]-4-oxo-4-Cphenylmethoxy) -butanoic acid or a pharmaceutically acceptable salt thereof.

14. Rapamycin-31,42-diester with 3-[[(l,l- dimethylethoxy)carbony1]amino]-4-oxo-4-(phenylmethoxy) • butanoic acid or a pharmaceutically acceptable salt thereof.

15. Rapamycin-42-ester with 3-[[C1,1-dimethylethoxy)carbony1]amino]-4-oxo-4-Cphenylmethoxy)butanoic acid or a pharmaceutically acceptable salt thereof. 239852 AHP-9580/9675-1-N1 -35-

16. Rapamycin-4 2-ester with 5-C1, 1-dimethyl.oxy )-4 -[[(1,1-dimethylethoxy)carbonyl]amino]-5-oxopentanoic acid or a pharmaceutically acceptable salt thereof.

17. Rapamycin-31,42-diester with 5-(l,l-dimethylethoxy)-4 -[[C1,1-dimethylethoxy)carbony1]-amino]-5-oxopentanoic acid or a pharmaceutically acceptable salt thereof.

18. Rapamycin-42-ester with Na, N-bis[(l,l-dimethylethoxy )carbonyl. ]-L-l ysine or a pharmaceutically acceptable salt thereof.

19. Rapamycin-31,42-diester with Na, N^-bis[(l,l-dimethylethoxy)carbony1]-L-lysine or a pharmaceutically acceptable salt thereof.

20. Rapamycin-14,31,42-tris(monobenzylsuccinate) or a pharmaceutically acceptable salt thereof.

21. Rapamycin-31,42-bis(monobenzylsuccinate) or a pharmaceutically acceptable salt thereof.

22. Rapamycin-42-(monobenzy!succinate) or a pharmaceutically acceptable salt thereof.

23. Rapamycin-31,42-bishemiglutarate or a pharmaceutically acceptable salt thereof.

24. Rapamycin-32,42-hemiglutarate bissodium salt.

25. Rapamycin-31,42-bishemiglutarate bistromethamine salt. .

26. Rapamycin-42-hemi-3'-oxoglutarate or pharmaceutically acceptable salt thereof. 2 \V * - AHP-9580/9675-1-Nl -36-

27. Rapamycin-31, 42-bishemi-3'-oxoglutarate or a pharmaceutically acceptable salt thereof.

28. Rapamycin-31, 42-bishemi-3'-oxoglutarate disodium salt.

2 9. Rapamyc in-31 , 42-bishemi-3'-oxoglutarate bistromethamine salt.

30. Rapamycin-31,42-bishemisuccinate or a pharmaceutically acceptable salt thereof.

31. Rapamycin-31, 42-bishemisuccinate bistromethamine salt.

32. A compound as claimed in any one of Claims 1 to 23, 26, 27 and 30 when in the form of a salt prepared with an inorganic base; a mono-, di- or tri-alkyl. amine of 1 to 6 carbon atoms per alkyl group; a mono-, di- or tri-hydroxyalkyl amine of 1 to 6 carbon atoms per alkyl group or an acid selected from acetic, lactic, citric, tartaric, succinic, maleic, malonic and gluconic.

33. A process for preparing a compound of formula I as claimed in Claim 1 which comprises (a) acylating rapamycin with an acylating agent or (b) sequentially acylating rapamycin with one or more acylating agents, said acylating agent(s) being selected from acids of formula: O O Z-tC(CH2 )mCHCCH2)nN]pC02R7 , Z-C-(CH2 ) fcX C CH2 ) r11 and ,5 R 6 Z- O C02R 12 j/% 'e~\ L % \£i \ JANW93 y * If AHP-9 5 80/9 675-1-N1 239852 -37- 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.

34. A process as claimed in Claim 33 in which the acylation is carried out using a coupling agent.

35. A process as claimed in Claim 33 in which the reactive derivative is the anhydride, mixed anhydride derived from 2,4,6-trichlorobenzoy1 chloride or the acid chloride, bromide or iodide.

36. A process for preparing a compound according to Claim 1 or a pharmaceutically acceptable salt thereof substantially as hereinbefore described and illustrated in any one of Examples 1 a) to i); 2a) to i); 3 a) to i); 4 a) to i); 5 a) to g);- 6 a) to g); 7 to 13; 14 a) to h); 15 a) to h); 16 a) to h); 17 a) to g); 18, 19, 20 a) to f) and 21 to 24.

37. A compound of formula I whenever prepared by a process according to any one of Claims 33 to 36.

38. A pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof as claimed in any one of Claims 1 to 32 and a pharmaceutically acceptable carrier. AMERIC HOME PRODUCTSs=6©R£QRATION