Rapamycin acetals as immunosuppressant and antifungal agents

Derivatives of Rapamycin where the hydroxy group at position 31 and/or 42 are reacted with acetal forming reagents have been shown to have immunosuppressant and antifungal properties. These derivatives are represented by the formula: ##STR1## wherein R.sup.1 and R.sup.2 independently are hydrogen, --CH.sub.2 YX, --C(CH.sub.3).sub.2 YX, --CH.sub.2 (CH.sub.3)YX, or L; PA1 Y is O or S; X is --CH.sub.3, --(CH.sub.2).sub.n CH.sub.3, --CH.sub.2 C.sub.6 H.sub.6, --(CH.sub.2).sub.2 OCH.sub.3, --CH.sub.2 Cl.sub.3 or --CH.sub.2 CH.sub.2 Si(CH.sub.3).sub.3 and L is selected from tetrahydrofuran-2-yl, tetrahydrothiophen-2-yl, tetrahydrothiopyran-2-yl, tetrahydropyran-2-yl, 4-methoxytetrahydropyran-2-yl, 4-methoxytetrahydrothiopyran-2-yl, or 4-methoxytetrahydrothiopyran-S, S-dioxide-2-yl with a proviso that R.sup.1 and R.sup.2 cannot simultaneously be hydrogen and n is 1-5.

BACKGROUND OF THE INVENTION 
This invention relates to acetals of rapamycin, their immunosuppressant 
properties and their antifungal activity. This invention further relates 
to pharmaceutical compositions containing rapamycin acetals. 
Rapamycin is a macrolide antibiotic produced by Streptomyces hydroscopicus 
having profound antifungal activity, especially against Candida albicans, 
and low toxicity [U.S. Pat. Nos. 3,929,992 and 3,993,749; Journal of 
Antibiotics 28(10), 721-726, 727-732 (1975) and journal of Antibiotics 
31(6), 539-545 (1978)]. 
Rapamycin was reported to inhibit the immune response in Can. J. Physiol. 
Pharmacol. 55, 48-51 (1977). U.S. Pat. No. 4,316,885 discloses monoacyl 
and diacyl derivatives of rapamycin, especially the acetylated derivatives 
which are useful antifungal antibiotics. A. U.S. Pat. application Ser. No. 
957,626 filed Nov. 3, 1978 which ultimately resulted in U.S. Pat. No. 
4,885,171 discloses a method for treating carcinogenic tumors in mammals 
with rapamycin. U.S. Pat. No. 4,401,653 discloses a method of using 
rapamycin and picibanil in combination for the treatment of transplantable 
carcinogenic tumors. U.S. Pat. No. 4,650,803 discloses 
aminoalkylcarboxylic acid esters of rapamycin at positions 31 and/or 42 
which are water soluble prodrug forms of rapamycin. 
SUMMARY OF THE INVENTION 
The present invention is concerned with acetals of rapamycin of Formula I 
or pharmaceutically acceptable salts thereof, which possess 
immunosuppressive and/or antifungal and/or antitumor and/or 
antiinflammatory activity in vivo and/or inhibit thymocyte proliferation 
in vitro and are therefore useful in the treatment of transplantation 
rejection, autoimmune diseases (i.e., lupus, rheumatoid arthritis, 
diabetes mellitus, multiple sclerosis), Candida albicans injections, and 
diseases of inflammation. 
##STR2## 
Under Formula I, R.sup.1 and R.sup.2 are independently hydrogen, 
--CH.sub.2 YX, --C(CH.sub.3).sub.2 YX, --CH(CH.sub.3)YX, or L; 
where 
Y is O or S; 
X is --CH.sub.3, --(CH.sub.2).sub.n CH.sub.3, --CH.sub.2 Ar, 
--(CH.sub.2).sub.2 OCH.sub.3 --CH.sub.2 CCl.sub.3, --CH(CH.sub.3).sub.2 or 
--CH.sub.2 CH.sub.2 SiMe.sub.3 ; 
L is tetrahydrofuran-2-yl, tetrahydrothiophen-2-yl, 
tetrahydrothiopyran-2-yl, tetrahydropyran-2-yl, 
4-methoxytetrahydropyran-2-yl, 4-methoxytetrahydrothiopyran-2-yl, or 
4-methoxytetrahydrothiopyran-2-yl S,S-dioxide; and where 
n=1-5, with proviso that R.sup.1 and R.sup.2 cannot both be hydrogen 
simultaneously.

DETAILED DESCRIPTION OF THE INVENTION 
The Formula I compounds are prepared by reacting rapamycin with acetal 
forming reagents using standard literature procedures. The acetal forming 
reagents are those employed in protecting secondary hydroxyl groups and 
are described in Theodora Green, Protective Groups in Organic Synthesis 
(John Wiley and Sons, Inc., 1981), pp 16-26. These reactions consist of 
reacting rapamycin with various halomethylethers or thioethers in the 
presence of a base, for example, sodium hydride, triethylamine, or ethyl 
diisopropylamine; or reacting rapamycin with a vinyl ether under acidic 
conditions to obtain the acetals of the instant invention as shown in the 
reaction schemes 1 and 2 below. 
##STR3## 
The various acetal-forming agents include chloromethlylmethyl ether, 
chromomethylmethyl sulfide, benzyl chloromethyl ether, t-butyl 
chloromethyl ether, 2-methoxyethoxymethyl chloride, 
2,2,2-trichloroethoxymethyl chloride, bis(2-chloroethoxy)methyl chloride, 
2-(trimethylsilyl)ethoxymethyl chloride, 2,3-dihydropyran, 
2,3-dihydrothiopyran, 4-methoxy-2,3-dihydropyran, 
4-methoxy-2,3-dihydrothiopyran-S, S-dioxide, tetrahydrofuran (and thionyl 
chloride), 2,3-dihydrofuran, ethyl vinyl ether, 2-methoxy-1-propene and 
isopropyl vinyl ether. 
The following experimental procedures are included for illustrative 
purposes. 
EXAMPLE 1 
31-[O-(1-Methoxy-1-methyl)ethyl]rapamycin 
To a solution of 5.0 g (5.47 mmol) of rapamycin in 5 mL of dry 
dichloromethane is added 1.05 mL (10.96 mmol) of 2-methoxy-1-propene 
followed by a catalytic amount of pyridinium p-toluenesulfonate and 
allowed to stir at room temperature for 4 h. The reaction was worked up by 
pouring into water and allowing to stir for 20 min at which time, the 
organic layer was separated, washed with brine, dried over sodium sulfate, 
filtered, and concentrated in vacuo to give a pale yellow foamy solid. The 
residue was purified via preparative HPLC chromatography (Rainin HPXL 
pumps, 2 diameter Dynamax silica column, 60% ethyl acetate/hexane, 35 
mL/min), collecting 1.43 g (27%) of 
31-[O-(1-methoxy-1-methyl)ethyl]rapamycin as a later eluting peak. The 
spectral data follow: .sup.1 H NMR (CDCl.sub.3, 400 MHz) .delta.4.82 (s, 1 
H, anomeric OH), 3.41 (s, 3 H, --OCH.sub.3), 3.28 (s, 3 H, -- OCH.sub.3), 
3.15 (s, 3 H,--OCH.sub.3), 3.10 (s, 3 H, --C(CH.sub.3).sub.2 CH.sub.3), 
1.81 (s, 3 H, CH.sub.3 C.dbd.C--), 1.67 (s, 3 H, CH.sub.3 C.dbd.C--); IR 
(KBr) 3430 (OH), 2920, 2860, 1725 (C.dbd.O), 1645 (C.dbd.O), 1450, 1375, 
1190, 1090, 985 cm.sup.-1 ; MS (neg.ion FAB) 985 (M-, 100), 167. 
Analysis: Calc'd for C.sub.55 H.sub.87 NO.sub.14.4 H.sub.2 O: C 62.41; H 
9.05; N 1.32; Found: C 62.15; H 8.39; N 1.35. 
EXAMPLE 2 
42-[O-(1-Methoxy-1-methy]rapamycin 
Using the above procedure, 120 mg (2.3%) of another compound, 
42-[O-(1-methoxy-1-methyl)ethyl]rapamycin was isolated as a less polar 
fraction. The spectral data follow: .sup.1 H NMR (CDCl.sub.3, 400 MHz) 
.delta.4.82 (s, 1 H, anomeric OH), 3.34 (s, 3 H, --OCH.sub.3), 3.28 (s, 3 
H, --OCH.sub.3), 3.14 (s, 3 H, -C(CH.sub.3).sub.2 OCH.sub.3), 3.10 (s, 3 
H, -OCH.sub.3), 1.81 (s, 3 H, CH.sub.3 C.dbd.C--), 1.65 (s, 3 H, CH.sub.3 
C.dbd.C--); IR (KBr) 3440 (OH), 2930, 2860, 1720 (C.dbd.O), 1645 
(C.dbd.O), 1450, 1375, 1190, 1090, 985 cm.sup.-1 ; MS (neg. ion FAB) 985 
(M-, 100 ), 590, 446 (100), 175, 167. 
Analysis: Calc'd for C.sub.55 H.sub.87 NO.sub.14.1H.sub.2 O; C 65.77; H 
8.93; N 1.40; Found: C 65.37; H 8.44; N 1.08. 
EXAMPLE 3 
31-[O-[2-(trimethylsilyl)ethoxylmethyl]rapamycin 
To a stirred solution of 914 mg (1 mmol) of rapamycin in 5 ml of methylene 
chloride under an atmosphere of nitrogen was added 333 mg (1.5 mmol, 26.5 
.mu.l) of 2-(trimethylsilyl)ethoxymethyl chloride followed by dropwise 
addition of 258 mg (2.0 mmol, 348 .mu.l) of ethyl diisopropylamine. After 
30 minutes, the analysis (silica gel, eluted with ether) indicated the 
reaction was complete. 
The reaction mixture was diluted with ethyl acetate and 0.1 N aqueous 
hydrochloric acid. The mixture was extracted twice with ethyl acetate and 
the combined extract washed successively with 0.1 N hydrochloric acid 
solution and aqueous sodium bicarbonate. The ethyl acetate solution was 
dried (magnesium sulfate) and concentrated to obtain the title compound, 
mp 80.degree.-84.degree. C. Tlc showed 1 spot. 
Analysis: Calc'd for C.sub.57 H.sub.93 NO.sub.14 S; C, 65.99; H, 8.97; N, 
1.24; Found: C, 65.49; H, 8.98; N, 1.34. 
Immunosuppressive activity was evaluated in an in vitro standard 
pharmacological test procedure to measure lymphocyte proliferation (LAF) 
and in an in vivo procedure in which the survival time of a pinch skin 
graft was evaluated. 
The comitogen-induced thymocyte proliferation procedure (LAF) was used as 
an in vitro measure of the immunosuppressive effects of representative 
compounds. Briefly, cells from the thymus of normal BALB/c mice are 
cultured for 72 hours with PHA and IL-1 and pulsed with tritiated 
thymidine during the last six hours. Cells are cultured with and without 
various concentrations of rapamycin, cyclosporin A, or test compound. 
Cells are harvested and incorporated; radioactivity is determined. 
Inhibition of lymphoproliferation is assessed in percent change in counts 
per minute from non-drug treated controls. The results are expressed by 
the following ratio, or as the percent inhibition of lymphoproliferation 
of 1 .mu.M. 
##EQU1## 
The in vivo test procedure is designed to determine the survival time of 
pinch skin grafts 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 ski graft from the 
donor is grafted on the dorsum of the recipient as a homograft, and an 
autograft is used as control in the same region. The recipients are 
treated with either varying concentrations of cyclosporin A as test 
control or the test compound, intraperitoneally. Untreated recipients 
serve as rejection control. The graft is monitored daily and observations 
are recorded until the graft becomes dry and forms a blackened scab. This 
is considered as the rejection day. The mean graft survival time (number 
of days .+-.S.D.) of the drug treatment group is compared with the control 
group. 
The following table summarizes the result obtained with the compounds of 
Examples 1 and 2 in the three standard test procedures. 
______________________________________ 
Compound LAF.sup.1 
Skin Graft.sup.2 
______________________________________ 
Rapamycin 3.3-5.8 12.5 
Example 1 3.2 10.7 
Example 2 8.3 -- 
Example 3 17.2 -- 
______________________________________ 
.sup.1 IC.sub.50 (nM) 
.sup.2 Mean survival days 
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 following represents the typical procedure used. 
Compound to be tested was placed on sterile dried 1/4" plate disks, and 
allowed to dry. Agar plates were seeded with fungi and allowed to 
solidify. The impregnated disks were placed on the seeded Agar surface and 
incubated for the time required for the particular culture. Results are 
expressed in MIC (.mu.g/ml) to inhibit growth. The results of this test 
procedure showed that the compounds of this invention have antifungal 
activity. 
______________________________________ 
Anti-Candida Activity (.mu.g/mL)* 
ATCC ATCC ATCC ATCC 
Compound 10231 38246 38247 38248 3669 
______________________________________ 
Rapamycin 
0.03 0.25 0.03 0.006 0.25 
Example 1 
0.025 0.4 0.025 0.10 0.4 
Example 2 
0.025 0.4 0.025 0.1 0.2 
______________________________________ 
*Minimal Inhibitory Concentration (MIC) 
Based on the results of these standard pharmacological test procedures, the 
compounds are useful in the treatment of transplantation rejection such 
as, heart, kidney, liver, bone marrow, and skin transplants; automimmune 
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 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 ingredients. Suitable solid 
carriers include, for example, calcium phosphate, magnesium stearate, 
talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl 
cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine, low 
melting waxes and ion exchange resins. 
Liquid carriers are used in preparing solutions, suspensions, emulsions, 
syrups, elixirs and pressurized compositions. The active ingredient can be 
dissolved or suspended in a pharmaceutically acceptable liquid carrier 
such as water, an organic solvent, a mixture of both or pharmaceutically 
acceptable oils or fats. The liquid carrier 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 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 
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 
administrated intravenously. The compound can also be administered orally 
either in liquid or solid composition form.