CA2046105A1 - Rapamycin oximes - Google Patents

Abstract

ABSTRACT
A derivative of rapamycin in which the 27-position has the structure wherein R1 is hydrogen, alkyl, or -CH2Ar;

A1 is , , , or ;

wherein R2, R3, and R4 are each, independently, hydrogen, alkyl, aralkyl, alkoxy, hydroxy, cyano, halo, nitro, carbalkoxy, trifluoromethyl, amino, or a carboxylic acid;
X is N, O, or S;
or a pharmaceutically acceptable salt thereof, which is by virtue of its immunosuppresive activity is useful in treating transplantation rejection host vs. graft disease, autoimmune diseases, and diseases of inflammation.

Description

AHP-()G65 ~ ~J /~ J'~

BACKGROUND OF THE INVENTION
This invention relates to novel oxirne der~vatives of rapamycin and a meth~ for using them in the treatment of transplantation rejection, host vs. graft disease, 5 autoimmune diseases, and diseases of inflammation.
Rapamycin is a macrocyclic triene antibiotic produced by StreDtomyces hy~oscopicus, which was found to have antifungal activity, particularly against andida 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 3,922,992; 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. Martei et al.
[Can. J. Physiol. Pharmacol. 55, 48 (1976) disclosed that rapamycin is effective in the expenmental allergic encephalomyelitis model, a model for multiple sclerosis; in the adjuvant arthritis model, a model for rheumatoid arthritis; and effectively inhibited the formation of I(~E-like antibodies.
The immwnosuppressive effects of rapamycin have been disclosed in FAS~B 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, 198~). Cyclosporin A and ~K 5()6, other macrocyclic molecules, also have been shown to bc eff~ctive as itnmlmosllppressive aL~ents, therefore useful in preventing transplant rejection LI~ASEB
3, 3411 (1989); l~AS~B 3, 5256 (1989); and R. Y. Calne et al., Lancet 1183 (1978).
Mono- and diacylated derivatives of rapamycin (esterified at the 28 and 43 positions) have been shown to be useful as antifungal agents (U.S. Patent 4,316,885) and used to make water solub}e prodrugs of rapamycin (U.S. Patent 4,650,803).
Recently, ~he numbering convention for rapamycin has been changed; therefore according to Chemical Abstracts nomenclature, the esters described aboYe would be at the 31- and 4~- positions.

AHP~()665 - 2- ~ ~3 .~ f~3 ~J
DESCRIPTION O~7 TH:L~ INVENTION
This invention provides oxime derivatives of rapamycin which are usefi11 as immunosuppressive and anti-inflammatory agents possessing the general structure of rapamycin shown in Fig 1, where the 27 position has been transformed into an oxime 5 having the structure N
RlO
wherein Rl is hydrogen, aLkyl of 1-6 carbon atoms, or -CH2Ar;

Ar is ~ R4 ~ONJ ~X--R3 ~X~R3 wherein R2, R3, and R4 are each, independently, hydrogen, aLkyl of 1-6 carbon 10atoms, aralkyl of 7-10 carbon atoms, alkoxy of 1-6 carbon atoms, hydroxy, cyano, halo, nitro, carbalkoxy of 2-7 carbon atoms, trifluoromethyl, amino, or a carboxylic acid;
XisN,O,orS;
or a pharmacelltically acceptable salt thereof.
15Of the compounds, preferred members are those in which Rl is hydrogen, alkyl of l 6ca~bonatoms,or ~ Iz~
R

The pharmaceutically acceptable salts may be formed from inorganic cations such as so~ium, potassium, and the like Although compounds of this invention can be prepared by conventional 20 methods that are disclosed in the literature, because functional groups are contained in a large macrocyclic ring, functional group reactivity cannot be readily predicted [R.B.
Woodward et al., J Am. Chem. Soc. 103, 3215 (1981)]. Rapamycin has carbonyl groups at the 14- and 27- and 33- positions Based on x-ray crystallographic analysis, the 33-position was predicted to be the most reactive center; unexpectedly however, 25 oxime formation occurred exclusively at the 27- position A~ t9665 2 ~ .3 The compounds of this invention exist as geometric isomers (~ and Z~ or mixtures thereof due to tlle con~iguration of the -N-R growp, All sueh isomeric forms and mixtures thereof are within the scope of this invention, Where a cornpound is isolated in the form of one of the geometric isomers it may be converted at least in part 5 to the other by treating under mildly aeidic conditions, e,g" using hydrochlorie acid in ether or chloroform, Such a geometric isomer is therefore an intermediate to the other geometric isomer, The compounds of the invention can be prepared by the following route from rapamycin.

S5~ RION
o NaOAc 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 ~1rst In ViVQ procedure was a popliteal lymph node (PLN) test procedure which measured the effeet o~ eompounds of 15 this invention on a mixed lymphocyte reaetion ancl the seeond in vivo procedure evaluated the survival ~ime of a pinch skin graft.
The comitogen-induced thymocyte prol;feration procedure (LAP) was used as an in y~Q measure of the immunosuppressive eft'eets of repres~ntative compounds,Briefly, cells from the thymus of nvrrnal BAL13/c mice alre cultured Çor 72 hollrs with 20 PHA and IL~1 and pulx~d with tritiated thymidine during the last si~ hours, Cclls are eultured with and witllout various eoncentrationx of rapamycin, cyclosporin A, or test compound. Cclls are harvested iand incorporated; radioactivity is determined.
Inhibition of lymphoproliferation is assessed in percent change in counts per minwte from non-drug treated controls, The results are expressed by the following ratio:
3H-control thymus cells - H3-rapamvcin-treated thymus cells 3H-control thymas cells - H3-test compound-treated cells A mixed lymphocyte reaction (MLR) occurs when lymphoid cells from genetically distinct animals are combined in tissue culture, Each stimulates the other to undergo blast transformation which results in increased DNA synthesis that can be quantified by the incorporation of tritiated thymidine, Since stimulating a MLR is a ~P~)6~5 function of dispality at Major Histocompatibility antigens, an in ~Y~ popli~eal lymph node (PLN) test procedure c]osely correlates to host vs. graft di~ease. l~riefly, 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 5 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 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 A10 at 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 The second in vivo test procedure is designed to determine the survival time of pinch skin graf~ from male DBA/2 donors ~ransplanted to male BALB/c recipients. The method is adapted from Billingham R.E. and Medawar P.B., J. Exp. Biol. 28:385-(402), 1951. Briefly~ a pinch skin graft from the donor is grafted on the dorsum of the recipient as a homograft, and an autograft is used as control in the same region. The recipients are treated with either varying concentrations of cyclosporin A as test control or the test compound, intraperitoneally. Untreated recipients serve as rejection control.
20 The graft is monitored daily and observations cue recorded until the graft becomes dry and forms a blackened scab. 'I'his iS considered as the rejection day. The mean graft survival time (number of days ~ S.D.) of the druL~ treatment group is compared with the control group.
The followin~ tuble summurizes the results of representutive compolln(ls of this25 inventioll in theso tllree stundurd test proccdllres.
TABL,E 1 LAF* PLN* SkinGraft 5~nd (ratio) (ratio)(davs + SD) Example 1 0.25 -1.4010.0 + 1.6 Example 2 0.87 1.399.3 + 1.4 Example3 0.007 + 9.5 + 1.4 Rapamycin 1.0 1.012.0 +1,7 * Calculation of ratios was described ~, + Not evaluated A~-lP-t)~5 The results of these standard pharmacological test procedures demonstrate immunosuppressive activity both in vitro and in y~Q for the compounds of this invention. Positive ratios in the LA~ and Pl,N test procedures indicate suppression of T cell proliferation. Transplanted pinch skin grafts are typically rejected within 6-7 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 1 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 10 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) Antifungal activity of the compounds of this invention was measured against 5 strains of Candida a!bicans using a plate test procedure for measurement of inhibition.
15 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 ( ~g/ml) to inhibit growth. Surprisingly, the oxime derivatives of 20 this invention have substantially less antifungal activity than the parent compound, rapamycin.
Table 2~
Strain of Candida albicans ~Qme~ ~l rrc~~ ~ç~ ~T55~. ~)~
Llxample 1 0,05 0.2 0.05 0.1 0.2 ~x~lmple 2 ~0,4 ~0,4 ~0,4 >0.4 ~0.4 E~xample 3 0.2 0.05 0.2 0.05 0.2 Rapamycin 0.003 0.025 0.003 0.006 0.025 * expressed as MIC (,ug/ml) Based on the results of these standard pharmacological test procedures, the compounds are useful in the treatment of transplantation rejection such as, heart, kidney, liver, bone marrow, and skin transplants; autoimmune diseases such as, lupus, rheumatoid arthritis, diabetes mellitus, myasthenia gravis, and multiple sclerosis; and AHP-9~65 P. ~3 ~;, diseases of inflammation such as, psoriasis, dermatitis, eczema, seborrhea, and inflammatory bowel disease.
The compounds may be administered neat or with a pharmaceutical carrier to a mamrnal 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-disinteg,rating agents; it can also be an encapsulating material. In powders, the carrier is a finely divided solid which is in admixture with the filnely 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 9g% of the active ingredient. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.
Liquid carriers are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active ingredient can be d.issolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both or pharmaceutically acceptable vils or fats. The liquid carrier can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thiclcening agents, colors, viscosity reglllators, stabilizers or osmo-regulators. Suitable examples of liquid carriers for oral and parenteral administration include water (partLllly containing additives as above, e,g. celllllose derivut;ves, preferably so~lium curboxymethyl cellulose solution), alcohols (includillg monohy~lric ulcohols undpolyhydric alcohols, eg, glycols) and their derivatives, and vils (e.g. fractionated coconut oil and arachis oil), ~or parenteral administration, the carrier can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liqwid carriers are useful in sterile liquid ~orm compositions for parenteral administration. The liquid carrier for pressurized compositions can be halogenated hydrocarbon or other pharmaceutically acceptable propellent.
Liquid pharmaceutical compositions which are s~erile solu~ions or suspensions can be utilized by, for example, intramuscular, intraperitoneal or subcutaneous injection. Sterile solutions can also be administered intravenously. The compound can also be administered orally either in liquid or solid composition form.

f~
~ 7 -Preferably, tlle pharmaceutical composition is in unit dosage form, e,g. as tablets or capsules. In such form7 the composition is sub-divided in unit dose containing appropriate quantities of the active ingredient; the uni~ dosage forms can be packaged compositions, for example, packeted powders, vials, ampoules, prefilledsyringes or sachets containing liquids. The unit dosage form can be~ for example, a capsule or tablee 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.
The following examples illustrate the preparation of representative compounds of this invention.

}~xample 1 Rapamvcin-27-oxime To a solution of 50 mg (54.7 ~umol) of rapamycin in 1 mL of methanol was added at room temperature, 12 mg (143 ~lmol) of anhydrous sodium acetate and 10 mg (143 ~lmol) of hydroxylamine hydrochloride. After 2 h stirring at room temperature, the reaction was complete by TLC. The reaction was diluted with water and extracted three times with ethyl acetate. The combined organics we,re washed with brine and dried over anhydrous sodium sulfate, decanted, and concentrated in vacuo to give a white, foamy solid.1H NMR indicated a mixture of E and Z isomers. The solid was dissolved in hot ethyl acetate and upon cooling, white crystals ~ormed. ~acuum filtration gave 23 mg (45 % ) of isomerically pure mono-oxime, which was biologically inactive (results not presented).
IH NMR (CDCl3, ~00 MI-lz) ~ 8.015 (s, 1 I-l, NOH), 4.820 (s, 1 ll, CON), 3.414 (~, 3 H, (;~13C)-)~ 3.308 (s, 3 ll, Cfl3O~), 3.145 (s, 3 ~-1, C~13O-), 1.667 (~;, 3 H, CH3C=C), 1.652 (s, 3 H, CH3C-C); 13C NMK (CDC13, 100 MHz ) 214.()9 (C=O), 192.03 (C=O), 169.38 (C=O), 16'7.2,1 (C=O), 158.74 (C=NOH); IR (KBr) 3450 (OH), 2960 (CH), 2890 (CH), 1760 (C-O), 1740 (C=O), 1732 (C=O), 1660 (C=O, C=NOH), 1465, 1390, 1205, 1100, 1005 cm~l; MS (neg. ion FAB) 928 (M-), 896, 590, 546, 167 (100); High Res. MS (neg. ion FAB) Calc. for C51H80N213 928.5660, Found 928.5677.
AnalysisCalcdforCslH~30N2O13-2H20 : C 63.42; H 8.21; N 2.74 Found : C 63.47; H 8.11; N 2.60 AHP~1~6G5 Th~ filtrate obtained following the ~ryst~llization degcribed above was concentrated to give a white foamy solid l~e solid was dissolved in hot ethyl acetate and hexane was added until slightly cloudy. Upon cooling, white crystals -formedVacuum fi1ltration gave 13 mg ( 25 %) of pure mono oxime which was a geometric S isomer of the compound isolated in the previous example This isomer was tested and found to be active in the assays described supra lH NMR (CDCl3, 400 MHz) ~ 7 620 (s, 1 H, NOH), 5 028 (s, 1 H, COH), 3.40g (s, 3 H, CH30-), 3.306 (s, 3 H, CH30-), 3 172 (s, 3 H, CH30-), 1 807 (s, 3 H, CH3C=C), 1 660 (s, 3 H, C~3C=C); 13C MMR (CDCl3, 75 MHz ) 211.83 (C-O), 191.43 (C=O), 168.73 (C=O), 167.11 (C=O), 159.94 (C=NOH); IR (KBr) 3450 (OH), 2960 (CH), 292Q (CH), 1775 (C=O), 1745 (C=O), 1680, 1650 (C=O, C=NOH), 1475, 1400, 1220, 1120, 1015 cm-l; MS (neg. ion FAB) 928 (M-~, 167 (100). High Res. MS (neg ion FAB) Calc. for Cs1H80N2ol3 928.5660, Found 928.5660.
AnalysisCalcdforCslH80N2OI3 : C 65.93; H 8.68; N 3.01 Found : C 66.19; H 8.93; N 2.88 Example 2 RaDamycin-~benzYI-27 -oxime To a solution of 50 mg t54.7 ~lmol) of r~lpamycin in 1 mL of methanol was added 12 mg (143 ,umol) of anhyclrous sodium a~etate and 23 mg~ (143 llmol) vt benzyloxyamine hydrochloride at room temper~ture. After 72 h ~tirrin~ t roon temperature, the reaction wa~ dilllted with water and extracted three times with ethyl acetate. Th~ combined or~unics were washell with brine, dried over anhydrous sodium sulfate, decantcd, ;In(l concentrated }~5aQ to give a colorless oil. IEI NMR incUcated a mixture of 1~ and æ isomers. The oil was dissolved in hot diisopropyl ether/hexane and upon cooling, white crystals formed. Vacuum filtration gave 25 mg ( 45 % ) of pure mono-oxime.
IH NMR (CDC13, 400 MHz) 5.031 (s, 2 H, CH2Ph), 3.365 (s, 3 H, CH3O-), 3.273 (S3 3 H, CH30-), 3.118 (s, 3 H, CH30-), 1.661 (s, 3 H, CH3C=C), 1.544 (s, 3 H, CH3C=C); 13C NMR (CDC13, 100 MHz ) 211.65 (C=O), 191.76 (C=O), 168.78 (C=O), 166.94 (C=O), 158.46 (C=NOH); IR (KBr) 3450 (OH), 2940 (CH), 2890 (CH), 1750 (C=O), 1730 (C=O), 1650, 1630 (C=O, C-NOR), 1460, 1380, 1195, ~$ ~

lL090, 990 cm-~; MS (neg. ion FAB) 1018 (M-), 590, 546, 167 (1~); High E~es. MS
(neg. ion F~B) Calc for Cs~H86N2OI3 1()18 6130, ~und 1018 6157 AnalysisCalcdforC58H86N2Ol3 H2o : C 67.18; H 8.4g; M 270 Found : C 67.17; H 861; N 2.56 Example 3 R~amycin-O-methvl-27-oxime To a solution of 750 mg ( 820 llmol) of rapamycin in 15 mL of methanol was added 180 mg (2.15 mmol) of anhydrous sodium acetate and 180 mg (2.15 mmol) of methoxyarnine hydrochloride at room temperature. After st~rring overnight at room temperature, the reaction mixture was diluted with water and extracted three times with ethyl acetate. The combined organics were washed with brine, dried over anhydrous sodium sulfate, decanted and concen~ated in va~ to give a viscous oil. llI NMR
indicated a mixture of E and Z isomers. The solid was dissolved in hot diisopropyl ether/hexane and upon cooling, white cryslals formed. Vacuum ~lltration gave 370 mg ( 48 % ) of pure mono-oxime.
lH NMR (CDC13, 400 MHz) ~ 4.960 (bs, 1 H, COH), 3.794 (s, 3 H, CH3ON=C), 3.395 (s, 3 H, CH30-), 3.288 ~s, 3 H, C~3O-), 3.121 (s, 3 H, CH3O-), 1.645 (s, 3H, CH3C=C), 1.587 (s, 3 H, CH3C=C); 13C NMR (CDC13, 100 MHz ) 211.70 (C-O), 192.11 ~C-O), 168.75 tc=), 166.84 (C=O), 15B.08 (C=NOMe); IR (KBr) 3450 (OH), 2940 (CH), 2890 (CH), 1750 (C=O), 1725 (C-O), 1655, 1635 (C=O, C=NOCH3), 1455, 1380, 1195, 1090, 1050, 990 cm~l; MS (neg. iotl ~AB) (~42 (M-), 590, 546, 167 (100): Eligh Res. MS (neL~, ion Ç~AB) Calc. for C521f8~N
942.5818, Fvund 942.5863.
AnalysisCalcdforC5~l82N~ 3 ~f2 : C 65.()0; ~1 8.75; N 2.91 ~7ouncl : C 65.20; ~1 ~.83; N 2.50

Claims (9)

1. A derivative of rapamycin in which the 27-position has the structure wherein R1 is hydrogen, alkyl of 1-6 carbon atoms, or -CH2Ar;

Ar is , , , or ;

wherein R2, R3, and R4 are each, independently, hydrogen, alkyl of 1-6 carbon atoms, aralkyl of 7-10 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 N, O, or S;
or a pharmaceutically acceptable salt thereof.

2. A compound of claim 1 wherein R1 is alkyl of 1-6 carbon atoms or a pharmaceutically acceptable salt thereof.

3. A compound of claim 1 wherein R1 is or a pharma-ceutically acceptable salt thereof.

4. A compound of claim 1 which is rapamycin-27-oxime or a pharmaceutically acceptable salt thersof.

5. A compound of claim 1 which is rapamycin-O-benzyl-27-oxime or a pharmaceutically acceptable salt thereof.

6. A compound of claim 1 which is rapamycin-O-methyl-27-oxime or a pharmaceutically acceptable salt thereof.

7. 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 which is a derivative of rapamycin in which the 27-position has the structure wherein R1 is hydrogen, alkyl of 1-6 carbon atoms, or -CH2Ar;

Ar is , , , or ;

wherein R2, R3, and R4 are each, independently, hydrogen, alkyl of 1-6 carbon atoms, aralkyl of 7-10 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 N, O, or S;
or a pharmaceutically acceptable salt thereof.

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

9. A composition as claimed in claim 8, in unit dosage form