CA2051782A1 - Aminoesters of rapamycin - Google Patents

Abstract

ABSTRACT
A compound of the structure wherein R1 and R2 are each, independently, hydrogen or with the proviso that R1 and R2 are not both hydrogen;
R3 is hydrogen, alkyl, aralkyl, -(CH2)qCO2R6, -(CH2)r,NR7CO2R8, carbamylalkyl, aminoalkyl, hydroxyalkyl, guanylalkyl, mercaptoalkyl, alkylthioalkyl, indolylmethyl, hydroxyphenylmethyl, 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;
R4 and R7 are each, independently, hydrogen, alkyl, or aralkyl;
R5, R6, and R8 are each, independently, alkyl, aralkyl, fluorenylmethyl, 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;
m is 0-4;
n is 0-4;

AHP-9675/9675-1-Nl p is 1-2;
q is 0-4;
r is 0-4;

wherein R3, R4, m, and n are independent in each of the subunits when p = 2;
or a pharmaceutically acceptable salt thereof, 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.

Description

5~"

AMINOE~ l'cS OF RAP~Y~IN

BACKGROUND OF THE INVENTION
This invention relates to novel esters of rapamycin and a method 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 Streptomvces hvgroscopicus, which was found to have antifungal activity, particularly againstCandida albicans, both ~ vitro and in yivo [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.
[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 i.nmunosuppressive effects of rapamycin have been disclosed in FASEB 3, 3411 (1989), rapamycin has been shown to be effective in inhibiting transplant rejection (U.S. 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 irnmunosuppressive 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 an~ 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 a'oove would be at the 31- and 42- positions.

AHP-9675/9Ç75-1-N1 - 2 - ~5~

DESCRIPI~ON OF THE INVENTION
This invention providcs derivatives of rapamycin which are useful ai immunosuppressive, anti-inflammatory, and anti-fungal agents having the structure ~OMe ~oR2 ~o MeO' ~0 OMe , " ~~

S wherein Rl and R2 are each, independently, hydrogen or o --[C(CH2)mCH(CH2)nN]pCo2R5 with the proviso that Rl and R2 are not both hydrogen;
R3 is hydrogen, alkyl of 1-6 carbon atoms, aralkyl of 7-10 carbon atoms, -(CH2)qCO2R6~ -(CH2);NR7Co2R8~ 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, imidazoylmethyl 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;
R4 and R7 are each, independently, hydrogen, alkyl of 1-6 carbon atoms, or aralkyl of 7-10 car'oon atoms;

5~L 7~ ~ rJ
~ 3 ~
R5, R6, and R8 are each, independently, alkyl of 1-6 carbon atoms, aralkyl of 7-10 carbon atoms, fluorenylmethyl, or phenyl which is optionally mono-, di-, or ~i-substituted with a substituent selected from alkyl of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms, hydroxy, cyano, halo, nitro, carbaL~coxy of 2-7 carbon S atoms, trifluoromethyl, arnino, or a carboxylic acid;
misO-4;
n is O - 4;
pis 1 - 2;
qisO-4;
risO-4;

wherein R3, R4, m, and n are independent in each of the [C(CH2)mCH(CH2)nN]
subunits when p = 2;
or a pharmaceutically acceptable salt thereof. R3 R4 Of the compounds, preferred members are those in which m = O, n = O, and p = 1; m = O, n = O, and p = 2; n = O, and R3 is -(CH2)qC02R6; m = O, n = O, and R3 is ~(CH2)rNR7Co2R8; and m = O, n = O, and R3 is hydrogen.

The pharmaceutically acceptable salts may be formed from inorganic cations such as sodium, potassium, and the like, and organic acids such as acetic, lactic, citrict tartaric, succinic, maleic, malonic, gluconic, and the like, when R3 contains a basic 20 amino group.
The compounds of this invention can be prepared by acylating rapamycin with an acylating agent having the general structure 1l X- [c(cH2)mcH(cH2)nNlpco2Rs 1 3 l 4 where X is OH in the presence of a coupling reagent, such as dicyclohexyl-25 carbodiimide. The compounds of this invention also can be prepared using a mixed anhydride of the above described carboxylic acid as the acyla~ing species.
Alternatively, the acylating species can be an acid halide, where X 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.

AHP-9675/9675-1-Nl 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 ~ivo procedure was a popliteal Iymph node (PLN) test procedure which measured the effect of compounds of S this invention on a mixed Iymphocyte reaction and the second in ~Q 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 10 PHA and lL-l 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 Iymphoproliferation is assessed in percent change in counts per minute from non-drug treated controls. The results are expressed by the following ratio, or as 15 the percent inhibition of Iymphoproliferation of 1 ',lM.
3H-control thvmus cells - H3-rapamvcin-treated thvmus cells 3H-control thymus cells - H3-test compound-treated cells A mixed Iymphocyte reaction (MLR) occurs when Iymphoid 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 quanti~led by the incorporation of tritiated thymidine. Since stimulating a MLR is a function of disparity at Major Histocompatibility antigens, an in vivo popliteal Iymph 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 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.
Rapamycin at a dose of 6 mg/kg, p.o. gave 86% suppression, whereas cyclosporin Aat the same dose gave 43% suppression. Results are expressed by the following ratio:
3H-PLN cells control C3H mouse - 3H-PLN cells rapamvcin-treated C3H mouse 3H-PLN cells control C3H mouse - 3H-PLN cells test compound-treated C3H mouse ., .

AHP-9675/9675- 1 -Nl ~5:~l'7~
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 5 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 10 survival time (number of days + S.D.) 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.

LAF* PLN* Skin Graft Compound (ratiol fratio)__ (davs + Sl)!
Examplel 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 5.4 0.33 11.5 + 3.5 Example 7 3% at l ~,lM** + 7.7 + 1.5 Example 8 0.03 0.41 +
Example9 0.96 1.34 10.3 + 0.8 Example 10 2.0 0.96++ 12.7 + 1.2 Example 11 0.004 + 10.5 + 1.3 Example 12 19.8 -2.87 12.0 + 2.0 Example 13 22% at l~M** + 7.0 + 0.6 Rapamycin 1.0 1.0 12.0 + 1.7 * Calculation of ratios was described supra.
Result expressed as percent inhibition of Iymphoproliferation at 1 ~M.
+ 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.

AHP-9675/9675-1-Nl 2~5~

The results of these standard pharmacological test procedures demonstrate immunosuppressive 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 ~7 5 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 Example 12 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 10 the skin graft test procedure indicates a proliferation of TSuppressor cells, which are implicated in suppressing the immune response. (see, I. Roitt et al. Immunology,C.V.Moseby Co. 1989, p 12.8-12.11).

Andfungal activity of the compounds of this invention was measured against 5 15 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 st~rile 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 20 expressed in MIC ( ,ug/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 parentcompound, rapamycin ~5~ 7~ ~?

Table 2*
Strain of Candida albicans CompoundATCC 10231ATCC 38246ATCC 38247ATCC 38248 3669 Examplel>0.4 >0.4 >0.4 >0.4 >0.4 S Example20.1 0.2 0.2 0.2 0.1 Example 30.4 > 0.4 > 0-4 >0.4 0-4 Exarnple40.1 0.4 0.1 0.1 0.2 Example 5> 0.4 > 0.4 > 0.4 >0.4 >0.4 Example 60.1 > 0.4 0.2 0.4 >0.4 Example 7 + + + + +
Example 8> 0.4 > 0.4 > 0-4 ~0-4 >0-4 Example 90.4 > 0.4 0.4 >0.4 >0.4 Example 100.2 > 0.4 0.2 0.4 0.4 Example 11> 0.4 > 0.4 > 0.4 >0.4 >0.4 lS Example 120.2 > 0.4 0.1 0.2 0.4 Example 13> 0.4 > 0.4 > 0.4 >0.4 >0.4 Rapamycin0.003 0.025 0.003 0.006 0.025 * expressed as MIC (~g/ml) 20 + not evaluated Based on the results of these standard pharmacological test procedures, the compounds are useful in the treatrnent 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 25 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 rnarnmal 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 30 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 AHP-9675/9675-1-Nl 2~S~7 the shape and size ~esired. 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 5 and ion exch~nge resins.
Liquid carriers are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressuriæd 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 10 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 containing additives as above, e.g. cellulose derivatives, preferably sodium 15 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 in sterile liquid form compositions for parenteral administration. The liquid carrier for 20 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 25 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, prefilled30 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.

2~5~7~

In addition, the sompounds of this invention may be employed as a solution, cream, or lotion by formulation with pharmaceutically acceptable vehicles containing 0.1-0.5 percent, preferably 2%, of active compound which may 'De administered to a fungally affected area.
s The ~ollowing examples illus~ate the preparation of representalive compounds of this invention.

10 E~ample 1 Rap~mycin-42-ester with N-r(l~l-dimethvlethoxv!carbony~ lvcvl~lvcine Under anhydrous conditions, a solution of rapamycin (3 g, 3.28 mmole) and N-[(1,1-dimethylethoxy)carbonyl]-glycylglycine (3.04 g, 13.1 mmole) in 40 mL of anhydrous dichloromethane was treated with dicyclohexylcar'oodiimide (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 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 monoester is a 8.3:1 mixture of two conformers.
lH NMR (CDC13, 400 MHz): o 1.46 (m, 9H, COOBut), 1.654 (s, 3H, CH3C=C), 1.751 (s, 3H, CH3C=C), 3.14 (s, 3H, CH30), 3.33 (s, 3H, CH30~, 3.36 (s, 3H, CH30), 4.18 (d, lH, CHOH), 4.75 (m, lH, 42-CHO ), 4.79 (s, lH, OH); High Res. MS (neg. ion FAB) Calcd for C60Hg3N3017: 1127.6504, measured mass 1127.6474.
Anal. Calcd for C6nHg3N3017 0.75 PhCH3: C, 65.45; H, 8.33; N, 3.51 Found: C, 65,23; H, 8.32; N, 3.86 2 ~S~ d 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.
Rapamycin-42-ester with N-[(fluorenylmethoxy)carbonyl]-alanylserine S Rapamycin-42-ester with N-[(fluorenylmethoxy)carbonyl]-glycylglycine Rapamycin-42-ester with N-[(ethoxy)carbonyl]-arginylmethionine Rapamycin-42-ester with N-~(4'-chlorophenoxy)carbonyl]-histidylarginine Rapamycin-42-ester with N-[(phenoxy)carbonyl]-tryptophanylleucine Rapamycin-42-ester with N-[(phenylmethoxy)carbonyl)]-N-methylglycyl-N-ethyl-10 alanine Rapamycin-42-ester with N-[(phenylmethoxy)carbonyl]-N-methyl-,l~-alanylphenyl-alanine Rapamycin-42-ester with N-[(l,l-dimethylethoxy)carbonyl]-cysteinylglycine Example 2 Rapamvcin-31.42-diester with N-f(l .l-dimethylethoxy!carbonvll-glvcylglYcine 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.
lH NMR (CDC13, 400 MHz): ~ 1.452 (m, 18H, COOBut), 1.6612 (s, 3H, CH3C=C), 1.7815 (s, 3H, CH3C=C), 3.14 (s, 3H, OCH3), 3.34 (s, 3H, OCH3), 3.35 (s, 3H, OCH3), 4.52 (s, lH, OH), 4.79 (m, lH, 42-CHO ); High Res. MS (neg.
ion FAB): Calcd for C6gH107Nso2l 1341.7458, measured mass: 1341.7463.
Anal. Calcd for C6gH107Nso2l 0.75 PhCH3: C, 63.17; H, 8.06; N, 4.96 Found: C, 62.83; H, 8.09; N, 5.00 .:

S~
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 Exarnple 2.

S Rapamycin-31,42-diest~r with N-[(fluorenylmethoxy)carbonyl]-alanylserine Rapamycin-31,42-diester with N-l(fluorenylmethoxy)carbonyl]-glycylglycine Rapamycin-31,42-diester with N-[(ethoxy)carbonyl]-arginylrnethionine Rapamycin-31,42-diester with N-[(4'-chlorophenoxy)carbonyl]-histidylarginine Rapamycin-31,42-diester with N-[(phenoxy)carbonyl]-tryptophanylleucine 10 Rapamycin-31,42-diesterwith N-[(phenylmethoxy)carbonyl)]-N-methylglycyl-N-ethyl-alanine Rapamycin-31,42-diester with N-[(phenylmethoxy)carbonyl]-N-methyl-~-alanylphenyl- alanine Rapamycin-31,42-diester with N-[(1,1-dimethylethoxy)carbonyU-cysteinylglycine Example 3 l~amvcin-31~42-dieste~with N-r(l.l-dimethvlethoxv~carbonvll-N-methvl~lvcine 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 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 analysisshowed that the diester is a 19.8:1 mixture of two conformers. The multiplicity of the NMR peaks suggests the presence of amide rotamers.
1H NMR (CDC13, 400 MHz): ~ 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, CH30), 3 37 (s, 3H, CH30), 4 73 (broad, lH, 42-CHO), 4 82 (2s, lH, OH); High Res. MS (neg. ion ~AB): Calcd. for C67H10sN3ol9 1255.7342, measured mass 1255.7289.
Anal. Calcd for C67H105N3019: C, 64.04; H, 8.42; N, 3.34 Found: C, 64.14; H, 8.74; N, 3.63 - 12 - 2~S~l'7~3~

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.
s Rapamycin-31,42-diester with N- [(ethoxy)carbonyl]-tyrosine Rapamycin-31,42-diester with N-~(fluorenylmethoxy)carbonyl]-phenylalanine Rapamycin-31,42-diester with N- [(3',4' ,S'-trihydroxyphenoxy)carbonyl] -isoleucine Rapamycin-31,42-diester with N- [(1,1 -dimethylethoxy)carbonyl)-glutamine 10 Rapamycin-31,42-diester with N- [(phenoxy)carbonyl] -N-methylalanine Rapamycin-31,42-diester with N-[(propyloxy)carbonyl]-4-aminobutryic acid Rapamycin-31,42-diester with N-[(phenylmethoxy)carbonyl]-7-aminoheptanoic acid Rapamycin-31,42-diester with N-[(fluorenylmethoxy)carbonyl]-serine 15 Exampl~ 4 RaDanly~ilL-4~-e~t~with N-~ l-dim~hy]ethoxv!carbonyl]-N-methyl~ly~ç

Under anhydrous conditions, an ice cold solution of rapamycin (0.95 g, 1.02 20 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 combined filtrates were concentrated in vacuo to give an amorphous amber solid. Flash 25 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, chloroform-ethylacetate-methanol 75:25: 1 v/v, flow rate 250 mL/min). HPLC analysis 30 showed that the ester is a 6.6:1 mixture of two confolTners. The multiplicity of NMR
peaks suggests the presence of amide rotamers.
lH NMR (CDCl3, 400 MH~ 1.42-1.46 (ds, 9H, COOBut), 2.91 (ds, 3H, NCH3), 1.644 (s, 3H, CH3C=C), 1.738 (s, 3H, CH3C=C), 3.12 (s, 3H, CH30), 3.32 (s, 3H, CH30), 3.35 (s. 3H, CH30), 4.18 (d, lH, CHOH), 4.71 (broad, lH, 35 42-CHO), 4.78 (broad s, lH, OH); High Res. MS (neg. ion FAB): Calcd for Cs9H92N2O16 1084.6446, measured mass 1084.6503.

2~S~

Anal. Calcd for CsgHg2N2016: 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 5 the appropriate terminally-N-substituted amino acid by employing the method used to prepare the title compound in Example 4.

Rapamycin-42-ester with N-[(ethoxy)carbonyl]-tyrosine Rapamycin-42-ester with N-~(fluorenylmethoxy)carbonyl]-phenylalanine Rapamycin-42-ester with N-[(3',4',5'-trihydroxyphenoxy)carbonyl]-isoleucine Rapamycin-42-ester with N-[(l,l-dimethylethoxy)carbonyl)-glutamine 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 Rapamycin-31,42-diester with N-[(fluorenylmethoxy)carbonyl]serine Example S

Rap~amycin-31 ~42-diester with S-( l . I -dimethvlethoxv!-2-rrf 1 ~ I -dimethvlethoxyl-calrl2Qny~ oxopentanoic acid Under anhydrous conditions, an ice cold solution of rapamycin (4 g, 4.37 mmole) and L-glutamic acid Na~Boc-~tert-butylester (4.9 g, 16.1 mmole) in 40 mL
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(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 con.sists of a 6.6: 1 mixture of two conformers.
lH NMR (CDC13, 400 MHz): ~ 1.42 (m, 36 H, COOBut), 1.646 (s, 3H, CH3C=C), 1.701 (s, 3H, CH3C=C), 3.13 (s, 3H, CH30), 3.34 (s, 3H, CH30), 3.36 (s, 3H, CH30), 4.735 (m, 2H, OH~42-CH-O); High Res. MS (neg. ion FAB):
calc. for qgH12sN3023 1483.8715, measured mass 1483.8714.

7~

Anal. Calcd for C7gH12sN3023: 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 S the appropriately terminally-N-substituted amino diacid monoester by employing the method used to prepare the title compound in Example S.

Rapamycin-31,42-diester with 6-(phenylmethoxy)-2-[~fluorenylmethoxy)carbonyl]-amino]-~oxohexanoic acid Rapamycin-31,42-diester with 6-(4'-methylphenoxy)-3-[[(phenylmethoxy)carbonyl]-amino-6-oxohexanoic acid Rapamycin-31,42-diester with 6-(ethoxy)-4-[[(phenoxy)carbonyl]amino]-6-oxo-hexanoic acid Rapamycin-31,42-diester with 6-(methoxy)-S-[[(ethoxy)carbonyl]amino]-6-ox~
hexanoic acid Rapamycin-31,42-diester with 4-(phenoxy)-2-[N-[(1,1-dimethylethoxy)carbonyl]-N-methylamino]-40xobutanoic acid Rapamycin-31,42-diester with 4-(phenylmethoxy)-3-[N-[(methoxy)carbonyl]-N-methylamino]-4-oxobutanoic acid Example 6 Ra~a~y~in-42-ester with 5-(1~1-dimethvlethoxvl-2-~r(l~l-dimethvlethoxv)-carbony~ ~oxo~entanoic acid The title compound (1.14 g, 20.6%) was separated from the 31,42-diester as described in Example S and isolated as the quarter hydrate/mono-ethyl acetate solvate.
HPLC analysis showed that the monoester is a l l.S: 1 mixture of two conformers.1H NMR (CDC13, 400 MHz): o 1.425 (m, 18H, COOBut), 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, CHOH), 4.71 (M, lH, 42-CHO), 4.785 (s, lH, OH); High Resolution MS ( neg. ion FAB): C~lc. for C65H102N218 1198-7127 measured mass 1198.7077.

AE IP-9675/9675-1-Nl - 15- ~ L7~?, Anal. Calcd for C6sH102N2olg CH3COOEt 0-25 H2O:
C, 64.13, H, 8.60; N, 2.17 Found: C, 64.18; H, 8.52: N, 2.01 S The following representative compounds can be prepared from ~apamycin and the appropriately terminally-N-substituted amino diacid monoester by employing the rnethod used to prepare the tide compound in Example 6.

Rapamycin-42-ester with 6-(phenylmethoxy)-2-[[fluorenylmethoxy)carbonyl]-amino]-~oxohexanoic acid Rapamycin-42-ester with ~(4'-methylphenoxy)-3-[[(phenylmethoxy)carbonyl]-amin~
~oxohexanoic acid Rapamycin-42-iester with ~(ethoxy)-4-[[(phenoxy)carbonyl]amino]-~oxo- hexanoic acid l S Rapamycin-42-ester with 6-(methoxy)-S-[[(ethoxy)carbonyl]amino~-6-oxo- hexanoic acid Rapamycin-42-ester with 4-(phenoxy)-2-[N-[(1,1-dimethylethoxy)carbonyl]-N-methylamino]-~oxobutanoic acid Rapamycin-42-ester with 4-(phenylmethoxy)-3-[N-[(methoxy)carbonyl]-N-methyl-amino]-4-oxobutanoic acid Example 7 Rapamvcin-31.42-diester with 2-~r( l . l -dimethvlethoxv~carbonvllaminol-4-oxo-4-(Dhenylme~hg~ 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-~-benzyl ester and 17011L (1.21mmol) of Et3N in 1 mL of THF at room temperature.
After stirring for 30 minutes, 500 mg (0.SSmmol) 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 (S cm column, 40 % ethyl acetate-hexane). The product was isolated as the heptahydrate.

2~5~7~3~

IH NMR (CDC13, 400 MHz) ~ 7.347 (s, 10 H, Ar), 6.223, 5.126 (s, 4 H, CH2Ph), 4.698 (m, 1 H, CH-CO2), 4.587 (m, 2 H, NH), 3.353 (s, 3 H, CH30), 3.337 (s, 3 H, CH30), 3.301 (s, 3 H, CH30), 2.775 (m, 4 H, CH2CO2); IR ~KBr) 3420 (OH), 2935 (CH), 2920 (CH), 1730 (C=O), 1650, 1500, 1455, 1370, 1170 cm-l; MS (neg.
S ion FAB) 1523 (M-), 1433, 297, 248, 205, 148, 44, 25 (100).
Anal. Calcd for Cg3HI l7N3O23-7H2O C, 60.40; H, 7.09; N, 2.54 Found: C, 60.54; H, 7.28; N, 2.56 Example 8 Rapamycin-~42-diester with 3-r~(l.l-dimethvlethoxy!carbc,nvllaminol-4-ox~4-(~henvlmethoxv! butanoic acid Under anhydrous conditions, 532 mg (2.18 mmol) of 2,4,6 trichloro'~enzoyl chloride in 1 mL THF was added to a solution of 704 mg (2.18 mrnol) of Na-Boc-L-aspartic acid-a-benzyl ester and 303 ',IL (2.18 mmol) of Et3N in S mL of THF at room temperature. After stirring for 20 minutes, the reaction mixture was ~lltered over sintered glass, and the precipitate was washed with THF. The filtrate was concentrated in vacuo to give a thick oil. l'he oil was dissolved in S rnL of 'Denzene 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 ethylacetate, 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 (20-40 % ethyl acetate/hexane as eluant) to give 532 mg (33 %) of the title compound which was isolated as the hydrate.
lH NMR (CDC13, 400 MHz) ~ 7.362 (s, 10 H, Ar), 5.193 (s, 4 H, CH2Ph), 4.596 (m, 1 H, CH-CO2), 4.586 (m, 2 H, NH), 3.336 (s, 3 H, CH30), 3.306 (s, 3 H, CH30), 3.145 (s, 3 H, CH30); IR (KBr) 3410 (OH), 2950 (CH), 2920 (CH), 1735 (C=O), 1710 (C=O), 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 C83H1l7N3O23 - H2O: C, 65.38; H, 7.73; N, 2.76 Found: C, 64.85; H, 7.67; N, 2.56 2~315 Example 9 Rapamycin-42-ester with 3-rr(l.l-dimethvlethoxv!carbonvllaminol-4-oxo-4-(phenylmethoxv~ butanoic acid s 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.
lH NMR (CDC13, 400 MHz~ o 7.356 (s, 5 H, Ar), 5.185 (s, 2 H, CH2Ph), 4.635 (m, 1 H, CH-CO2), 4.582 (m, 1 H, NH), 3.330 (s, 6 H, CH30), 3.135 (s, 3 H, CH30); IR (KBr) 3410 (OH), 2950 (CH), 2920 (GH), 1735 (C=O), 1710 (C=O), 1640, 1490, 1445, 1350, 1150 cm -1; MS (neg. ion FAB) 1218 (M-), 1127, 590, 168,42, 25, 17 (1~0).
Anal. Calcd for C67Hg8N2O18 - 1.5 H2O: C, 63.64; H, 8.21; N, 2.22 Found: C~ 63.64; H, 7.51; N, 2.13 Example 10 Ra~amvcin-42-ester with 5-(l.l-dimethyloxv~-4-lr(1.1-dimethvlethoxv!carbonvll aminol-5-oxopentanoic acid IJnder anhydrous conditions, an ice cold solution of rapamycin (4 g, 4.37 mmole) and L-glutamic acid Na-Boc-a-tert-butylester (4.9 g, 16.1 mmole) in 40 mLof 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 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: I mixture of two conformers.

, ~5 iL~;3 1H NMR (CDC13, 400 MHz): ~ 1.43 (s, 9H, COOBut) and 1.46 (s, 9H, COOBut), 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, lH, CH-OH), 4.65 (m, lH, 42-CHO), 4.80 (s, lH, OH);
S High Res. MS (neg. ion FAB): Calc. for C6sH102N2olg: 1198.7126, measured mass 1198.7135.
Anal. Calcd for C6sH102N2ol8: C~ 65-09; H~ 8-57; N~ 2-34 Found C, 65.04; H, 8.33; N, 2.64 Example 11 Rapamvcin-31.42-diester with 5-(1.1-dimethvlethoxv)-4-rL(1.1-dimethvlethoxv)-carbonvll- amino3-5-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 a7.7:1 mixture of two conformers.

lH NMR (CDC13, 400 MHz): ~ 1.43 (s, 18H, COOBut), 1.46 (s, 18H, COOBut), 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 C7gH12sN3O23: 1483.8704, 25 measured mass 1483.8636.
Anal. Calcd for C7gH125N3O23: C, 63.90; H, 8.49; N, 2.83 Found: C, 63.68; H, 8.60; N, 3.20 ~S~,~B,~.

Example 1~

Rapamycin-4~-çster with N~-bis~ l-dimethylethoxv!carbonyll-L-lvsine Under anhydrous conditions, a solution of rapamycin (3 g, 3.28 mmole) and Na, N-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 pr~cipitated 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 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 mLlmin). HPLC analysis showed that the monoester is a 9:1 mixture of two conformers.
lH NMR (CDC13, 400 MHz): o 1.438 (m, 9H, ~OOBut), 1.455 (s, 9H, COOBut), 1.652 (s, 3H, CH3C=C), 1.752 (s, 3H, CH3C=C), 3.14 (s, 3H, CH30), 3.33(s, 3~I, CH30), 3.37 (s, 3H, CH30), 4.18 (d, lH, CHOH), 4.72 (m, lH, 42-CHO), 4.79 (s, lH, OH); High Res. MS (neg. ion FAB): Calcd for C67H107N3Olg:
201241.7549, measured mass 1241.7604.
Anal. Calcd for C67H107N3ol8: C~ 64-76; H~ 8-68; N~ 3-38 Found: C, 64.58; H, 9.01; N, 3.10 Example 13 Ravamycin-31.42-diester with N--Nbisr(1.1-dimethylethoxy)carbonvll-L-lvsine Under a nitrogen atmosphere, a solution of Na,N~ bis-Boc-L-lysine (1.038 g, 3 mmole) and triethylarnine (0.42 mL, 3 mmmole) in 10 mL of anhydrous THF was 30 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 benzeme and added to a stirred solwtion of rapamycin (1 g, 1.09 mmole) containing 4-dimethylamino pyridine (0.59 g,4.8 mmole) in 10 mL of benzene. After 35 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 211~5~78 ~d -flæh chromatography ( on silica Merck 60, elution with hexane-ethylacetate 1:1) to provide title compound (l.lS g, 67%). HPLC analysis shows that the diester,is a 9:1 n~xture of two conformers.
~H NMR (CDC13, 400 MHz): ~ 1.426 (m, 9H, COOBut), 1.438 (s, 9H, COOBut), 1.443 (s, 9H, COOBut), 1.446 (s, 9H, COOBut), 3.141 (s, 3H, CH30), 3.36 (s, 3H, CH30), 3.378 (s, 3H, CH30), 4.68-4.76 (m, 2H, OH and 42-CHO); High res. MS
(neg. ion FAB): Calcd. for Cg3H13sNsO23 1569.9526, measured mass 1569.9537.
Anal. Calcd. for Cg3H13sNsO23: C, 63.46; H, 8.66; N, 4.46 Found: C, 63.06; H, 8.84; N, 4.09 .

.

Claims (22)

1. A compound of the structure wherein R1 and R2 are each, independently, hydrogen or with the proviso that R1 and R2 are not both hydrogen;
R3 is hydrogen, alkyl of 1-6 carbon atoms, aralkyl of 7-10 carbon atoms, -(CH2)qCO2R6, -(CH2)rNR7CO2R8, 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, imidazoylmethyl 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;
R4 and R7 are each, independently, hydrogen, alkyl of 1-6 carbon atoms, or aralkyl of 7-10 carbon atoms;

AHP-9675/9675-1-Nl R5, R6, and R8 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, or a carboxylic acid;
m is 0-4;
n is 0-4;
p is 1-2;
q is 0-4;
r is 0-4;

wherein R3, R4, m, and n are independent in each of the subunits when p = 2;
or a pharmaceutically acceptable salt thereof.

2. A compound of claim 1 where m = 0, n = 0, and p = 1 or a pharmaceutically acceptable salt thereof.

3. A compound of claim 1 where m = 0, n = 0, and p = 2 or a pharmaceutically acceptable salt thereof.

4. A compound of claim 1 where n = 0, and R3 is -(CH2)qCO2R6 or a pharmaceutically acceptable salt thereof.

5. A compound of claim 1 where m = 0, n = 0, and R3 is -(CH2)rNR7CO2R8 or a pharmaceutically acceptable salt thereof.

6. A compound of claim 1 where m = 0, n = 0, and R3 is hydrogen or a pharmaceutically acceptable salt thereof.

7. A compound of claim 1 which is rapamycin-42-ester with N-[(1,1-dimethyleth-oxy)carbonyl]-glycylglycine or a pharmaceutically acceptable salt thereof.

8. A compound of claim 1 which is rapamycin-31,42-diester with N-[(1,1-di-methyl- ethoxy)carbonyl]-glycylglycine or a pharmaceutically acceptable salt thereof.

AHP-9675/9675-1-Nl

9. A compound of claim 1 which is rapamycin-31,42-diester with N-[(1,1-di-methylethoxy)carbonyl]-N-methylglycine or a pharmaceutically acceptable salt thereof.

10. A compound of claim 1 which is rapamycin-42-ester with N-[(1,1-di-methylethoxy)carbonyl]-N-methylglycine or a pharmaceutically acceptable salt thereof.

11. A compound of claim 1 which is rapamycin-31,42-diester with 5-(1,1-dimethylethoxy)-2-[[(1,1-dimethylethoxy)carbonyl]amino]-5-oxopentanoic acid or apharmaceutically acceptable salt thereof.

12. A compound of claim 1 which is rapamycin-42-ester with 5-(1,1-dimethylethoxy)-2-[[(1,1-dimethylethoxy)carbonyl]amino]-5-oxopentanoic acid or apharmaceutically acceptable salt thereof.

13. A compound of claim 1 which is rapamycin-31,42-diester with 2-[[(1,1-dimethylethoxy)carbonyl]amino]-4-oxo-4-(phenylmethoxy) butanoic acid or a pharma-ceutdcally acceptable salt thereof.

14. A compound of claim 1 which is rapamycin-31,42-diester with 3-[[(1,1-dimethylethoxy)carbonyl]amino]-4-oxo-4-(phenylmethoxy) butanoic acid or apharmaceutically acceptable salt thereof.

15. A compound of claim 1 which is rapamycin-42-ester with 3-[[(1,1-dimethylethoxy)carbonyl]amino]-4-oxo-4-(phenylmethoxy) butanoic acid or apharmaceutically acceptable salt thereof.

16. A compound of claim 1 which is rapamycin-42-ester with 5-(1,1-dimethyl-oxy)-4-[[(1,1-dimethylethoxy)carbonyl]amino]-5-oxopentanoic acid or a pharma-ceutically acceptable salt thereof.

17. A compound of claim 1 which is rapamycin-31,42-diester with 5-(1,1-dimethylethoxy)-4-[[(1,1-dimethylethoxy)carbonyl]amino]-5-oxopentanoic acid or apharmaceutically acceptable salt thereof.

18. A compound of claim 1 which is rapamycin-42-ester with N.alpha., N.epsilon.-bis[(1,1-dimethylethoxy)carbonyl]-L-lysine or a pharmaceutically acceptable salt thereof.

AHP-9675/9675-1-Nl

19. A compound of claim 1 which is rapamycin-31,42-diester with N.alpha., N.epsilon.
bis[(1,1 -dimethylethoxy)carbonyl]-L-lysine or a pharmaceutically acceptable salt thereof.

20. A method of treating transplantation rejection, host vs. graft disease, autoimmune diseases, and diseases of inflammation in a mammal by administering an effective amount of a compound having the structure wherein R1 and R2 are each, independently, hydrogen or with the proviso that R1 and R2 are not both hydrogen;
R3 is hydrogen, alkyl of 1-6 carbon atoms, aralkyl of 7-10 carbon atoms, -(CH2)qCO2R6, -(CH2)rNR7CO2R8, 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, imidazoylmethyl 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;
R4 and R7 are each, independently, hydrogen, alkyl of 1-6 carbon atoms, or aralkyl of 7-10 carbon atoms;
R5, R6, and R8 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, or a carboxylic acid;
m is 0-4;
n is 0-4;
p is 1-2;
q is 0-4;
r is 0-4;

wherein R3, R4, m, and n are independent in each of the subunits when p = 2;
or a pharmaceudcally acceptable salt thereof.

21. A pharmaceutical composition comprising a compound of claim 1 or a pharmaceutically acceptable salt thereof.

22. A composition as claimed in claim 21 in unit dosage form.