CA2051781A1 - Carboxylic acid esters of rapamycin - Google Patents

Abstract

ABSTRACT
A compound of the structure wherein R1, R2, and R3 are each, independently, hydrogen or with the proviso that R1, R2, and R3 are not all hydrogen;
R4 is -(CH2)mX(CH2)nCO2R5 or ;
R5 and R6 are each, independently, alkyl, aralkyl, or phenyl which is optionallymono-, di-, or tri-substituted with a substituent selected from alkyl, alkoxy, hydroxy, cyano, halo, nitro, carbalkoxy, trifluoromethyl, amino, or a carboxylic acid;
X is , O, or S;
R7 and R8 are each, independently, hydrogen or alkyl;
Y is CH or N;
m is 0-4;
n is 0-4;
with the proviso that m and n are not both O when X is O or S;
or a pharmaceutically acceptable salt thereof, which is by virtue of its immunosuppressive activity is useful in treating transplantation rejection, host vs. graft disease, autoimmune diseases, and diseases of inflammation, and by virtue of itsantifungal activity is useful in treating fungal infections.

Description

A~ 9580 CARBQ~YLTC AClD ESTEI~S OF R~lP~lYCIN

BACKGROUND OF THE INVENTION
This invention relates to novel estcrs of raparnycin and a method for using themin 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 hvgrosc~cus, which was found to have antifungal activity, particularly against 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.
[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 rhewmatoid arthritis; and effectively inhibited the fornnation of IgE-like antibodies.
The immwnosuppressive effects of rapamycin have been disclosed in FASEl~ 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 immunosuppressive agents, therefore useful in preventing transplant rejection [FASEB
3, 3411 (1989); FASEB 3, 5256 (1989); and R. Y. Calne et al., Lancet 1183 (l978).
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 the 31- and 42- positions.

- 2- 2~5~7~ :
DESCRIPTION OF THE INVENTION
This invention provides derivatives of rapamycin which are useful as immunosuppressive, anti-inflammatory, and antifungal agents having the st~ucture ~ORI

' OMe ~0 ~oR2 R3 ~ MeO~O
~0 OMe o wherein R1, R~, and R3 are each, independently, hydrogen or - CR4 with the proviso that Rl, R2, and R3 are not all hydrogen;
R4 i8 -(CH2)mX(CH2) nCO2Rs or --~--C02R6 0 RS and R6 are each, independently, alkyl of 1-6 carbon atoms, aralkyl of 7-10 carbon atoms, or phenyl which is optionally mono-, di-, or tri-substituted with a substituent selected from alkyl of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms, hydroxy, cyano, halo, nitro, carbalkoxy of 2-7 carbon atoms, trifluoromethyl, amino, or a carboxylic acid;

Xis -¢-, O, orS;

R7 and R8 are each, independently, hydrogen or alkyl of 1-6 carbon atoms;
Y is CH or N;
20 misO-4;
nisO-4;
with the proviso that m and n are not both 0 when X is O or S;
or a pharmaceutically acceptable salt thereof.

' 3 2~5~
Of the compounds, preferred members are those in which R4 is -(CH2)mX (C~I2) nCO2Rs The pharmaceutically acceptable salts may be formed from inorganic cations such as sodium, potassium, and the like; mono-, di-, and triaLlcyl amines of 1-6 carbon S atoms, per aLkyl group and mono-, di-, and trihydroxyalkyl amines of 1-6 carbon atoms per alkyl group. Preferred 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 structure Il 4 XCR
where X is OH, in the presence of a coupling reagent, such as dicyclohexyl-carbodimide. The compounds of this invention also can be prepared using an anhydride of the above described carboxylic acid as the acylating species.
Alternatively, the acylating species can be an acid halide, where X can be Cl, Br, or I.
15 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.

Immunosuppressive activity was evaluated in an in vitro standard pharmacological test proçedure to measure Iymphocyte proliferation (LAF) and in two 20 in vivo standard pharmacological test procedures. The first in vivo procedure was a popliteal Iymph node (PLN) test procedure which measured the effect of compounds of this invention on a mixed Iymphocyte reaction and the second in yivo procedure evaluated the survival time of a pinch skin graft.

The comitogen-induced thymocyte proliferation procedure (LAF~ was used as an m ro measure of the immunosuppressive effects of representative compounds.
Briefly, cells from the thymus of normal BALB/c mice are cultured for 72 hou~rs with PHA and IL 1 and pulsed with tritiated thymidine during the last six hours. Cells are cultured with and without various concentrations of rapamycin, cyclosporin A, or test compound. Cells are harvested and incorporated; radioactivity is determined.
Inhibition of Iymphoproliferation is assessed in percent change in counts per minute from non-drug treated controls. The results are expressed by the following ratio:
3H-control thymus çells - H3-rapamycin-treated thYmus çells 3H-çontrol thymus cells - H3-test compound-treated cells - 4 - Z~L'71 A mixed Iymphocyte reaction (MLR) occurs when lymphoid cells from genetically distinct animals are combined in tissue culture. Each stimulates the other to undergo blast transformation which results in increased DNA synthesis that can be qu~ntified 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 colTelates 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 Iymph nodes are removed and dissolved, and radioactivity counted. The corresponding left RLN 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 q3% suppression. Results are expressed by the ~ollowing ratio:
3H-PLN cells control G3H mouse - 3H-PLN cells rapamycin-treated C3H mouse 3H-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 aut~ograft is used as control in the same region. The recipients are tr~ated 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 the control group.
The following table summarizes the results of representative compounds of this invention in these three standard test procedures.

2~ 7~

LAF* PLN* Skin Graft Compound (ratio) (ratio~~davs + SD
S Example 1 0.37 + 8.2 + 1.2 Example2 0.9 0.69 10.7 + 1.2 Example 3 3.27 1.04~+12.7 + 0.9 Example 4 0.56 1.68++~10.2 + 1.7 Example S 0.02 1 . 1 1++8.0 +1 .7 Example 6 0.01 0.48 8.0 + 0.9 Example7 0.97 0.70 9.3 + 1.6 Example 8 0.22 -1.93 12.0 + 1.7 Exampleg 0.22 0.41 10.2+ 1.2 Example 100.18 0.39 10.8 + 0.8 Example 110.00 0.09 7.8 + 1.7 Rapamycin 1.0 1.0 12.0 + 1.7 * Calculation of ratios was described ~.
+ Notevaluated ++ 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 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 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 8 may cause T cell proliferation in the PLN test procedure, it is believed a negative ratio in this test procedure coupled with an increased survival time observed in the skin graft test procedure indicates a proliferation of TSuppressor cells, which are implicated in suppressing the immune response. (see, I. Roitt et al. Immunology,C. V. Moseby Co. 1989, p 12.8-12.11).

2~!5~7 Antifungal activity of the compounds of this invention was measured against 5 strains of Candida albicans using a plate test procedure for measurement of inhibition.
The fol1Owing represents the typical procedure used. Compound to be tested was placed on sterile dried 114" plate disks, and allowed to dry. Agar plates were seeded S with fungi and allowed to solidify. The impregnated disks were placed on the seeded Agar surface and incubated for the time required for the particular culture. Results are expressed in MIC ( ~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 that the parent10 compound, rapamycin.
Table 2*
Strain of Candida albicans Cornl~oundATCC 10231ATCC 38246ATCC 38247ATCC 38248 3669 Example 1> 0.4 >0.4 >0.4 >0.4 >0.4 Exarnple 2> 0.4 0.4 > 0.4 0.4 0.4 Exarnple3 0.2 0.1 0.4 0.1 0.1 Exarnple 4> 0.4 0.2 > 0.4 0.2 0.4 Exarnple S0.4 > 0.4 > 0.4 >0.4 >0.4 Example 6 0.4 > 0.4 0.4 >0.4 >0.4 Example7 0.1 0.4 0 1 0.1 0.2 Example 8 0.4 > 0.4 0-4 >0-4 >0-4 Example 9 0.2 > 0.4 0.2 0.4 >0.4 Example 100.1 > 0.4 0.2 0.4 >0.4 Example 11> 0.4 > 0.4 >0.4 >0.4 >0.4 Rapamycin0.003 0.025 0.003 0.006 0.025 expressed as MIC (~,lg/ml) Based on the results of these standard pharmacological test procedures, the 30 compounds are useful in the treatment of transplantation rejection such as, heart, kidney,1iver, bone marrow, and skin transplants; autoi ~mmune diseases such as, lupus, rheumatoid arthritis, diabetes mellitus, myasthenia gravis, and multiple sclerosis;
diseases of inflammation such as, psoriasis, dermatitis, eczema, seborrhea, and inflammatory bowel disease; and fungal infections.

, . .
: . :
' ' 7 ~35~ ~7 The compounds may be administered neat or with a pharmaeeutieal earrier to a mammal in need thereof. The pharmaeeutieal earrier may be solid or liquid.
A solid earrier ean include one or more substances which may also act as flavoring agents, lubrieants, 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 eompaeted in the shape and size desired. The powders and tablets preferably contain up to 99~o of the aetive ingredient. Suitable solid carriers include, for example, calcium phosphat~e, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.
Liquid earriers are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized eompositions. The aetive ingredient can be dissolved or suspended in a pharmaeeutieally aeeeptable liquid carrier such as water, an organic solvent, a mixture of both or pharrnaceutically aeeeptable oils or fats. The liquid earrier ean eontain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, eolors, viseosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid earriers for oral and parenteral administration inelude water (partially eontaining additives as above, e.g. eellulose derivatives, preferably sodium earboxymethyl eellulose solution), aleohols (ineluding monohydrie aleohols and polyhydrie aleohols, e.g. glyeols) and their derivatives, and oils (e.g. fraetionated eoeonut oil and araehis 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 eompositions for parenteral administration. The liquid earrier for pressurized compositions can be halogenated hydroearbon or other pharmaeeutieally aeeeptable propellent.
Liquid pharmaeeutieal eompositions whieh are sterile solutions or suspensions ean be utilized by, for example, intramuscular, intraperitoneal or subcutaneous injeetion. Sterile solutions can also be administered intravenously. The compound ean also be administered orally either in liquid or solid composition form.
Preferably, the pharmaeeutieal eomposition is in unit dosage form, e.g. as tablets or eapsules. In sueh form, the eomposition is sub-divided in unit dose eontaining appropriate quantities of the active ingredient; the unit dosage forrns can be 8 2~5~
packaged compositions, for example, packeted powders, vials, ampoules, prefilledsyringes 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 S by the attending physician.

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

10 Example 1.

Rapan~vcin- 14!3142-tris(monobenzvlsuccinate!

To a solution of 5.0 g (5.47 mmol) of rapamycin, 3.41 g (16.41 mmol) of monobenzylsuccinate, and 3.15 g (16.41 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in 20 mL of dry dichloromethane was added 200 mgof 4-dimethylaminopyridine. The solution was stirred at room temperature for 3 days.
The reaction mixture was poured into 2 N HCl and extracted three times with ethyl 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. Flash chromatography on a 60 mm x 150 mm sil;ca 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-(monobenzylsuccinate).
IH NMR (CDCl3, 400 MHz) ~ 7.353 (bs, 15 H, arom), 5.168 (d~ J = 2.0 Hz, 1 H, CH-O2C), 5.148 (m, 6 H, CH2Pn), 4.672 (m, 1 H, CO2CH-CHOMe), 3.355 (s, 3 H, CH30-), 3.337 (s, 3 H, CH30-), 3.327 (s, 3 H, CH30-), 2.697 ( m, 12 H, O2CCH2CH2CO2CH2Ph), 1.745 (s, 3 H, CH3C=C), 1.655 (s, 3 H, CH3C=C);
IR (KBr) 3450 (OH), 2950 (CH), 1745 (C=O), 1650, 1460, 1385, 1360, 1160, 1105, 995 cm~l.
AnalysisCalcdforCg4HlogNO2l 3H20 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 1.

2~53 '~

Rapamycin- 14,31,42-tris (monom.-thylsuccinate) Rapamycin-14,31,42-tris (monophenyl-3',3'-dimethylglutarate~
Rapamycin- 14,31,42-tris (mono t-butyl-3'-methylglutarate) Rapamycin-14,31,42-tris (monobenzylthiodiglycolate) Rapamycin- 14,31,42-tris (monohexyldiglycolate) Rapamycin-14,31,42-tris (monopropylphthalate) Rapamycin-14,31,42-tris (monoethyl-2',6'-pyridinedicarboxylate) Example 2.
Ra~amy~in-31.42-bis(monobenzvlsuccinatel Fraction # 2, obtained from the procedure employed in Example 1, gave 1.25 g (17.7 %) of pure rapamycin-31,42-bis(monobenzylsuccinate) upon concen-tration.
IH NMR (CDCl3, 400 MHz) ~ 7.351 (bs, 10 H, arom), 5.168 (d, J = 2.0 Hz, 1 H, CH-O2C), 5.125 (m, 4 H, CH2Ph), 4.680 (m, 1 H, CO2C~-CHOMe), 3.356 (s, 3 H, CH30-), 3.329 (s, 3 H, CH30-), 3.146 (s, 3 H, CH30-), 2.639 ( m, 8 H, 02CC~2CH2C02CH2Ph), 1.748 (s, 3 H, CH3C=C), 1.654 (s, 3 H, CH3C=C);
IR (KBr) 3450 (OH), 2940 (CH), 1740 (C=O), 1650, 1455, 1380, 1355, 1160, 1105, 995 cm-l; 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 C73HggNOIg 1293.68108, found 1293.6811.
AnalysisCalcdforC73H99NOl9 H2o 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 2.

Rapamycin-31,42-bis (monomethylsuccinate) Rapamycin-31,42-bis (monophenyl-3',3'-dimethylglutarate) Rapamycin-31,42-bis (mono t-butyl-3'-methylglutarate) Rapamycin-31,42-bis (monobenzylthiodiglycolate) Rapamycin-31,42-bis (monohexyldiglycolate) Rapamycin-31,42-bis (monopropylphthalate) Rapamycin-31,42-bis (monoethyl-2',6'-pyridinedicarboxylate) Example 3.

Ra,~bi~Y~ccinate s Fraction # 3, obtained from the procedure employed in Example 1, gave 930 mg (15.4 %) of pure rapamycin-42-monobenzylsuccinate upon concentration.
IH NMR (CDC13, 400 MHz) o 7.355 (bs, 5 H, arom), 5.141 (m, 2 H, CH2Ph), 4.680 (m, 1 H, CO2CH-CHOMe), 3.364 (s, 3 H, CH30-), 3.333 (s, 3 H, CH30-), 3.141 (s, 3 H, CH30-), 2.698 ( m, 4 H, 02CCH2CH2CO2CH2Ph), 1.751 (s, 3 H, CH3C=C), 1.655 (s, 3 H, CH3C=C); IR (KBr) 3450 (OH), 2940 (CH), 1740 (C=O), 1645, 1455, 1380, 1165, 1105, 990cm-~; MS (neg. ionFAB) 1103 (M-), 1045, 1012, 624, 590, 167, 99 (100); High Res. MS (neg. ion FAB) Calcd for C62HggNO16 1103.6181, found 1103.6048.
AnalysisCalcdforC62HggNOl6-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 3.

Rapamycin-42-(monomethylsuccinate) Rapamycin-42-monophenyl-3',3'-dimethylglutarate) Rapamycin-42-(mono t-butyl-3'-methylglutarate) Rapamycin-42-(monobenzylthiodiglycolate) Rapamycin-42-(monohexyldiglycolate) Rapamycin-42-(monopropylphthalate) Rapamycin-42-(monoethyl-2',6'-pyridinedicarboxylate) .
:
, 035~a7 ~

Example 4.
Rapamvcin-31 ~2-bishemigl~rate 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 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 ~IPLC on a Clg column eluting starting with 60 % acetonitrile/water. Collected, after, concentration, 586 mg (24 %) of rapamycin-31,42-bishemiglutarate.
IH NMR (CDC13, 400 MHz) ~ 5.398 (m, 1 H, -CO2CHCHOMe), 4.683 (m, 1 H, -CO2CHCHOMe), 3.364 (s, 3 H, CH30-), 3.362 ~s, 3 H, CH30-), 3.106 (s, 3 H, CH30-), 2.407 (m, 8 H, -O2CCH2CH2CH2CO2H), 1.960 (m, 4 H, -O2CCH2CH2CH2CO2H). 1.770 (s, 3 H, CH3C=C), 1.653 (s, 3 H, CH3C=C);
13C NMR (CDC13, MHz) 211.45 (C=O), 206.84 (C=O), 200.44 (C=O), 177.83 (C=O), 177.04 (C=O), 172.43 (C=O), 171.20 (C=O), 165.27 (C=O), 159.08 (C=O);
IR (KBr) 3430 (OH), 2940 (CH), 2880 (CH), 1745 (C=O), 1685, 1625, 1580, 1450, 1385, 1330, 1200, 1140, 1100, 990 cm~l; 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 C61HgoOIgN (M-H) 1140.6107, Found 1140.6106.
AnalysisCalcdforC6lH9lOlsN 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 the appropriate anhydride by employing the method used to prepare the title compound in Example 4.
Rapamycin-31,42-bishemi-3'-methylglutarate Rapamycin-31,42-bishemi-3',3'-dimethylglutarate Rapamycin-31,42-bishemi-3'-oxoglutarate Rapamycin-31,42-bishemi-3'-thioglutarate Rapamycin-31,42-bishemi-phthalate Rapamycin-31,42-bishemi-2',3'-pyridine dicarboxylate - 12- ~ ~S~L7 Example 5.

Rapamycin-31,42-hemiglutarate bissodium salt Purified bis-31,42-hemiglutarate of rapamycin (740 mg, 649 umol), prepared as described in Example 4, was dissolved in 5 mL of 95 ~o ethanol and 107 mg (1.27 mmol) of sodium bicarbonate was added. Water (I mL) was added to completely dissolve the salt. Once dissolved, the light yellow solution was 10 concentrated in vacuo to give a foamy yellow solid. The foam was dried in a drying pistol for 24 h, refluxing over acetone at reduced pressure to give 520 mg of the bissodium salt.
lH NMR (d6-DMSO, 400 MH2) ~ 5.235 (m, 1 H, -CHO2C), 4.498 (m, 1 H, MeOCHCHO2C-), 3.287 (s, 6 H, 2 CH30-), 3.236 (s, 3 H, CH30-), 2.245 (m, 8 H, 02CCH2CH2CH2C02-), 1.712 (s, 3 H, CH3C=C), 1.593 (s, 3 H, CH3C=C);
IR (KBr) 3420 (OH), 2920 (CH), 1725 (C=O), 1675, 1620, 1560, 1450, 1400, 1375, 1230, 1195, 1130, 1090, 980 cm~l; 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 C61HggOlgNNa (M-Na) 1162.5926, Found 1162.5899.
AnalysisCalcdforC61HggOlgNNa2-H2O C 60.85; H 7.56; N 1.16 Found C 60.67; H 7.36; N 1.58 Example 6.
Rap~mycin-31.42-bishemigllltara~ç bistromethamine salt Purified bis-31,42 hemiglutarate of rapamycin (950 mg,833 umol), prepared as described in Example 4, was dissolved in 5 mL of 95 % ethanol and 197 mg 30 (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 concentrated in vacuQ 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.

' ' ~ ' ',, . , ' - 13- 2~.5~'7~
IH NMR (d6-DMSO, 400 MHz) o 5.253 (m, 1 H, -CHO2C), 4.523 (m, 1 H, MeOCHCHO2C-), 3.347 (s, 6 H, 2 CH30-), 3.276 (s, 3 H, CH30-), 2.289 (m, 8 H, 02CCH2CH2CH2C02-), 1.681 (s, 3 H, CH3C=C), 1.595 (s, 3 H, CH3C=C);
IR (KBr) 3400 (OH), 2920 (CH), 1730 (C=O), 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 C~lHgoOlgN (M-H, free acid) 1140.6107, Found 1140.6069.
Analysis Calcd for C6gH103o2sN3 - 2 H2O C 58.77; H 7.58; N 2.98 Found C 58.47; H 7.94; N 3.58 Example 7.

Ra~amvcin-42-hemi-3'-oxoglutarate 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 by1.32 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
HCI, and extracted three times with dichloromethane. The combined organic extracts were washed wi~h brine, dried over anhydrous sodium sulfate, decanted, and concentrated vacu~ to give a yellow foam. The crude product was purlfied via reverse phase HPLC on a Clg column eluting starting with 60 % acetonitrile/water.
After concentration, 870 mg ( 26 %) of rapamycin-42-hemi-3'-oxoglutarate and 500 mg (13 %) of rapamycin-31,42-bishemi-3'oxoglutarate were isolated.
lH NMR (CDC13, 400 MHz) o 4.768 (m, 1 H, CO2CH-CHOMe), 4.250 (m, 4 H, O2CCH2OCH2CO2), 3.356 (s, 3 H, CH30-), 3.331 (s, 3 H, CH30-), 3.139 (s, 3 H, CH30-), 1.759 (s, 3 H, CH3C=C), 1.653 (s, 3 H, CH3C=C);
IR (KBr) 3420 (OH), 2920 (CH), 2875 (CH), 1740 (C=O), 1720 (C=O), 1640, 1625, 1445, 1370, 1320, 1200, 1135, 1095, 980 cm-l; MS (neg. ion FAB) 1028 (M - H), 327, 167 (100), 148, 133, 115; High Res. MS (neg. ion FAB) Calcd for CssH82ol7N (M - H) 1028.5597, Found 1028.5599.
Analysis Calcd for CssHg3O17N 3 H2O C 60.97; H 8.22; N 1.29 Found C 61.33; H 7.74; N 1.69 14 2~5~7~
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 7.

S Rapamycin-42-hemi-3'-methylglutarate Rapamycin-42-hemi-3',3'-dimethylglutarate Rapamycin-42-hemi-3'-thioglutarate Rapamycin-42-hemi-phthalate Rapamycin-42-hemi-2',3'-pyridine dicarboxylate Example 8.

Ra~amycip-~f 1.42-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 by2.17 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 HCI, and extracted three times with dichloromethane. The combined organic extracts were washed with brine, dried over anhydrous sodium sulfate, decanted, and concentrated ~n Yacuo to give a yellow foam. The crude product was purified via reverse phase HPLC on a Clg column eluting starting with 60 % acetonitrile/water. After concentration, 1.75 g ( 28 %) of rapamycin-31,42-bishemi-3'-oxoglutarate was isolated.
1H NMR (CDC13, 400 MHz) o 4.785 (m, 1 H, CO2CHCHOMe), 4.260 (m, 8 H, O2CCH2OCH2CO2), 3.360 (s, 3 H, CH30-), 3.343 (s, 3 H, CH30-), 3.143 (s, 3 H, CH30-), 1.775 (s, 3 H, CH3C=C), 1.656 (s, 3 H, CH3C=C);
13C NMR (CDC13, MHz) 211.12 (C=O), 207.73 (C=O), 193.11 (C=O), 171.90 (C=O), 171.59 (C=O), 170.15 (C=O), 169.35 (C=O), 168.83 (C=O), 166.63 (C=O);
IR (KBr) 3420 (OH), 2920 (CH), 2850 (CH), 1740 (C=O), 1645~ 1625, 1440, 1370, 1190, 11300, 980 cm~l; 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 CsgH86O21N (M - H) 1144.5701, Found 1144.5702.
AnalysisCalcdforCsgHg7O21N C 61.82; H 7.65; N 1.22 Found C 61.59; H 7.36; N 1.84 - 15 - 21~53 Example 9.

Rapamycin-31~42-bishemi-3'-oxo~lutarate disodium salt S Purified bis-31,42 hemi-3'-oxoglutarate of rapamycin t720 mg, 629 umol),prepared by the procedure employed in Example 8, was dissolved in 10 mL of 95 %
ethanol and 106 mg (1.26 mmol) of sodium bicarbonate was added. Water (1 mL) wasadded to completely dissolve the salt. Once dissolved, the light yellow solution was concentrated in vacuo to give a foamy yellow solid. The foam was dried in a drying pistol for 48 h, refluxing over dichloromethane at reduced pressure to give 435 mg (58 %) of the disodium salt.
lH NMR ~d6-DMSO, 400 MHz) o 4.975 (m, 1 H, -CHO2C), 4.593 (m, 1 H, MeOCHC~O2C-), 4.135 (s, 2 H, -O2CCH2OCH2CO2R), 3.617 (s,2 H, -O2CCH2OCH2CO2R), 3.299 (s, 6 H, 2 CH30-), 3.232 (s, 3 H, CH3C)-), 1.614 (s, 3 H, CH3C=C), 1.553 (s, 3 H, CH3C=C); IR (KBr~ 3420 (OH), 2920 (CH), 1735 (C=O), 1615, 1445, 1395, 1380, 1320, 1220, 1130, 1090, 980 cm~l;
MS (neg. ion FAB) 1188 (M-l), 1166 (M-Na), 1144, 1051, 1028, 590, 459, 167, 155 (100~, 148, 133, 115.
AnalysisCalcdforCsgHgso2lNNa2-2H2o C 57.79; H 7.26; N 1.14 Found C 57.94; H 7.11; N 1.26 Example 10.

25 Rapa,mvcin~ 42-~Q $hemi-~'-QxQ~lutarate bistromethamine salt Purified bis-31,42 hemi-3'-oxoglutarate of rapamycin (1.01 g, 882 umol), prepared by the procedure employed in Example 8, was dissolved in 10 mL of 95 %
ethanol and 213 mg (1.76 mmol) of tris(hydroxymethyl)- methylamine was added.
30 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 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.

- 16- 2~S~L7~.
lH NMR (d~s-DMSO, 400 MHz) ~ 4.955 (m, 1 H, -CHO2C), 4.600 (m, 1 H, MeOCHCHO2C-), 4.149 (s, 2 H, -O2CCH2OCH2CO2R), 3.770 (s, 2 H, -02CCH20CH2C02R), 3.407 (s, 6 H, 2 CH30-), 3.257 (s, 3 H, CH30-), 1.806 (s, 3 H, CH3C=C), 1.614 (s, 3 H, CH3C=C); IR (KBr) 3400 (OH), 2920 (CH), 1730 (C=O), 1620, 1550, 1450, 1395, 1370, 1200, 1060, 985 cm~l;
MS (neg. ion FAB) 1144 (M-H, free acid), 1028, 167, 148, 133 (100), 115.
AnalysisCalcdforC67HlogO27N3- H20 C 57.22; H 7.90; N 2.98 Found C 57.26; H 7.90; N 3.15 Example 11.

~?amycin-31.42-bishemisuccinate.

To a solution of 2.0 g (2.2 mmol) of rapamycin in 10 n~ 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-dimethylamino-pyridine and the reaction mixture refluxed for 24 h. The solution was cooled to room temperature, poured into 2 N HCl, 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 purif1ed via reverse phase HPLC on a Clg 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 solid. The verv 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.

:

2~ 7~L

IH NMR (d6-DMSO, 400 MHz) 5.231 (m, 1 H, -CHO2C), 4.554 (m, 1 H, MeOCHCHO2C-), 3.426 (s, 6 H, 2 CH30-), 3.249 (s, 3 H, CH30-), 2.431 (m, 8 H, 02CCH2CH2C02-), 1.700 (s, 3 H, CH3C=C), 1.554 (s, 3 H, CH3C=C); 13C NMR
(d6-DMSO, ) 211.28 (C=O), 205.23 (C=O), 199.59 (C=O), 174.86 (C=O), 173.62 (C=O), 171.72 (C=O), 171.50 (C=O), 166.56 (C=O), 166.53 (C=O); IR (KBr) 3420 (OH), 2940 (CH), 1735 (C=O), 1630, 1580, 1460, 1400, 1380, 1170, 1070, 990 cm-l; MS (neg. ion FAB~ 1112 (M-l, free acid), 994, 589, 475, 297, 167, 148,117, 99 (100).
AnalysisCalcdforC67HIogO2sN3-2H2O C 57.80; H 8.12; N 3.01 Found C 57.91; H 8.21; N 2.37, : , .. ; . " . ~ , --- ,

Claims (18)

1. A compound of the structure wherein R1, R2, and R3 are each, independently, hydrogen or with the proviso that R1, R2, and R3 are not all hydrogen;
R4 is -(CH2)mX(CH2)nCO2R5 or ;
R5 and R6 are each, independently, alkyl of 1-6 carbon atoms, aralkyl of 7-10 carbon atoms, or phenyl which is optionally mono-, di-, or tri-substituted with a substituent selected from alkyl of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms, hydroxy, cyano, halo, nitro, carbalkoxy of 2-7 carbon atoms, trifluoromethyl, amino, or a carboxylic acid;
X is , O, or S;
R7 and R8 are each, independently, hydrogen or alkyl of 1-6 carbon atoms;
Y is CH or N;
m is 0-4;
n is 0-4;
with the proviso that m and n are not both O when X is O or S;
or a pharmaceutically acceptable salt thereof.

2. A compound of claim 1 where R4 is -(CH2)mX(CH2)nCO2R5 or a pharmaceutically acceptable salt thereof.

3. A compound of claim 1 where R4 is or a pharma-pharmaceutically acceptable salt thereof.

4. A compound of claim 1 which is rapamycin-14,31,42-tris(monobenzyl-succinate) or a pharmaceutically acceptable salt thereof.

5. A compound of claim 1 which is rapamycin-31,42-bis(monobenzylsuccinate) or a pharmaceutically acceptable salt thereof.

6. A compound of claim 1 which is rapamycin-42-(monobenzylsuccinate) or a pharmaceutically acceptable salt thereof.

7. A compound of claim 1 which is rapamycin-31,42-bishemiglutarate or a pharmaceutically acceptable salt thereof.

8. A compound of claim 1 which is rapamycin-31,42-hemiglutarate bissodium salt.

9. A compound of claim 1 which is rapamycin-31,42-bishemiglutarate bistromethamine salt.

10. A compound of claim 1 which is rapamycin-42-hemi-3'-oxoglutarate or a pharmaceutically acceptable salt thereof.

11. A compound of claim 1 which is rapamycin-31,42-bishemi-3'-oxoglutarate or a pharmaceutically acceptable salt thereof.

12. A compound of claim 1 which is rapamycin-31,42-bishemi-3'-oxoglutarate disodium salt.

13. A compound of claim 1 which is rapamycin-31,42-bishemi-3'-oxoglutarate bistromethamine salt.

14. A compound of claim 1 which is rapamycin-31,42-bishemisuccinate or a pharmaceutically acceptable salt thereof.

15. A compound of claim 1 which is rapamycin-31,42-bishemisuccinate bistromethane salt.

16. A method of treating transplantation rejection, host vs. graft disease, auto-immune diseases, and diseases of inflammation in a mammal by administering an effective amount of a compound having the structure wherein R1, R2, and R3 are each, independently, hydrogen or with the proviso that R1, R2, and R3 are not all hydrogen;
R4 is -(CH2)mX(CH2)nCO2R5 or ;
R5 and R6 are each, independently, alkyl of 1-6 carbon atoms, aralkyl of 7-10 carbon atoms, or phenyl which is optionally mono-, di-, or tri-substituted with a substituent selected from alkyl of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms, hydroxy, cyano, halo, nitro, carbalkoxy of 2-7 carbon atoms, trifluoromethyl, amino, or a carboxylic acid;

X is , O, or S;
R7 and R8 are each, independently, hydrogen or alkyl of 1-6 carbon atoms;
Y is CH or N;
m is 0-4;
n is 0-4;
with the proviso that m and n are not both O when X is O or S;
or a pharmaceutically acceptable salt thereof.

17. A pharmaceutical composition comprising a compound of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

18. A composition as claimed in claim 17, in unit dosage form.