CA2192972A1 - Rapamycin amidino carbamates and pharmaceutical compositions containing them - Google Patents

Abstract

A compound of the structure (I) wherein R and R1 are each, independently, hydrogen, or (a); R2 and R3 are each, independently, hydrogen, alkyl, alkenyl, alkynyl, -CO2R5, -COR5, -CN, -NO2, -SO2R5, -SO3R5, -OR5, -SR5, or Ar; R4 is hydrogen, alkyl, alkenyl, alkynyl, -CF3, -NR5R6, -CO2R5, -COR5, CONR5R6, -NO2, halogen, -OR5, -SR5, -CN, -SO2R5, -SO3R5, -SO2NR5R6, or Ar; R5 and R6 are each, independently, hydrogen, alkyl, alkenyl, alkynyl, or Ar; Ar is phenyl, naphthyl, or hetaryl, wherein the foregoing may be optionally substituted;
with the proviso that R and R1 are both not hydrogen, or a pharmaceutically acceptable salt thereof which is useful as an immunosuppressive, anti-inflammatory, antifungal, antiproliferative, and antitumor agent.

Description

w 09sl34s6s 2 1 9 2 9 7 2 PCTAU595/07412 RAPAMYCIN AMIOINO CARBAMATES ANO PHARMACEUTICAL COMPOSITIONS CONTAINING THEM

BACKGROUND OF THE INVENTION
This invention relates to amidino . ' of rapamycin and a method for using them for inducing ~ and in the treatment of i . ' rejection, graft vs. host disease, ~ diseases, diseases of ;,. nA~ll m ~ adult T-cell leukemiallymphoma, solid tumors, fungal infections, and ~ vli~ a~iv~;
vascular disorders.
Rapamycin is a .II_.,.v~ ,lic triene antibiotic produced by S~ v~ly-,.,s hy~.v~cvy; ~ which was found to have antifungal activity, ~ h.ul~ly against ~An~ nhirAn~ both in vitro and in vivo tc Vezina et al., J. Antibiot. 28, 721 (1975); S.N. Sehgal 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 ~ ;.. 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 ~ allergic . , ' ' , ~ model, a model for multiple sclerosis; in the adjuvant arth~itis model, a model for .; .. ~ 1 arthritis; and effectively inhibited the formation of IgE-like antibodies.
The ., ~ ., effects of rapamycin have been disclosed in FASEB 3, 3411 (1989). Cyclosporin A and FK-506, other ~ ,lv~ ,l;c molecules, also have been shown to be effective as ;~ ~~P~ , agents, therefore useful in preventing transplant rejection tFASEB 3, 3411 (1989); FASEB 3, 5256 (1989); R.
Y. Calne et al., Lancet 1183 (1978); and U.S. Patent 5,100,899].
Rapamycin has also been shown to be useful in preventing or treating systernic lupus Clyi' [U.S. Patent 5,078,999], pulmonary i~n~ - ;.. [U5~ Patent 5,080,899], insulin dependent diabetes mellitus [Fifth Int. Conf. Inflamm. Res. Assoc.
121 (Abstract), (1990)], smooth muscle cell proliferation and intimal thickeningfollowing vascular injury [Morris, R. J. Heart Lung Transplant 11 (pt. 2): 197 (1992)], adult T-cell leukemia~lymphoma [European Patent Application 525,960 Al], and ocular ~ j rj-.. ~li.. [EuropeanPatentApplication532,862Al].
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 aminoacyl prodrugs of rapamycin (U.S. Paten wo 9sl34s6s , r~l,u~. /412 , -4,650,803). Recently, the numbering convenion for rapamycin has been changed;
therefore according to Chemical Abstracts 1..,.. ~rl-n"G, the esters described above would be at the 31- and 42- positions. U.S. Patents 5,118,678 and 5,302,584 discloses c.ui of rapamycin that are useful as l r ~ Dai~." anti-5 ~ '' y, antifungal, r~ lif~ ivc, and antitumor agents.

DESCRIPTlON OF TE~ I~VENTION
This invention provides d~ r~ of rapamycin which are useful as ."I.. ".. ~ ai~" y, antifungal, r~ JliÇtlr~livG, and antitumor 10 agents having the structure ~OR

"J~ ' OMe ~X, o I~ORI

H ~~ MeO~ ~
~ OMe ~\>

o R4 wherein R and Rl are each, '~ n hydrogen, or --C N C~N~ R

R2 and R3 are each, ' 1 ' 'y, hydrogen, aLkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, -CO2R5, -CoR5, -CN, -NO2, -SO2RS, -SO3R5, -oR5, -SR5, or Ar;
R4 is hydrogen, aLlcyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, -CF3, -NR5R6, -Co2R5, -CoR5, CoNR5R6, -NO2, halogen, -oR5, -SR5, -CN, -SO2RS, -So3R5, -So2NR5R6~ or Ar;
R5 and R6 are each, ;~L 1, L lly, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, or Ar, W0 95134565 2 1 ~ 2 9 7 2 ~ ....5 . ~4l2 ~\

Ar is phenyl, naphthyl, or hetaryl, wherein the foregoing may be optionally mono-, di-, or tri . ~ - 1 with a group selected from alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, arylaL~yl of 7-10 carbon atoms, allcoxy of 1-6 carbon atoms, cyano, halo, hydroxy, nitro, carbaLkoxy of 2-7 carbon atoms, i ~ . ' yl, .r~ ~ y~ amino, dialkylarnino of 1-6 carbon atoms per alkyl group, dialkyl~,l;..oalkyl of 3-12 carbon atoms, LylLuAy~Lkyl of 1-6 carbon atoms, aLwAy~yl of 2-12 carbon atoms, alkylthio of 1-6 carbon atoms, -SO3H, and -CO~H;
with the proviso that R and Rl are both not hydrogen, or a ~ ", r~ lly acceptable 10 salt thereof.

The r~ ~ly acceptable salts are those derived from such inorganic cations such as sodium, potassium, and the like; organic bases such as: mono-, di-, and trialkyl amines of 1-6 carbon atoms, per alkyl group and mono-, di-, and 15 Il;L y~LUAyalkyl amines of 1-6 carbon atoms per aLcyl group, and the like; and organic and inorganic acids as: acetic, Iactic, citric, tartaric, succinic, maleic, malonic, gluconic, Ly~Lu~ Llv~ Lubl~,ll"c, pl~cp'~ , nitric, sulfuric, ' '~ and similarly known acceptable acids.

The terms alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, and alkynyl of 2-7 carbon atoms, include both straight chain as well as branched carbon chains.
When any of the generic terms (i.e., R5) are contained more than once in a givencompound, each may be the sarne or different. Examples of alkyl as a group or part of a group, e.g. arylalkyl, alkoxy or alkanoyl (alhyl~,~l yl) are straight or branched chains of 1-6 carbon atoms, preferably 1-4 carbon atoms, e.g. methyl, ethyl, propyl, isopropyl and n-buyl. Examples of arylalkyl are phenalkyl groups such as benzyl and phenethyl, eg 2-phenethyl, Hetaryl is defined as an ' or partially saturated L~,t~"~y~.lic radical of 5-12 atoms having 1 ring or 2 fused rings. r'referred L~,t~,lu~ yclic radicals include 1 ~ }.~ .,u~,y.,li~, radicals such as furanyl, thiophenyl, pyrrolyl, pyrazolyl, imidazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2-dithiolyl, 1,3-dithiolyl, 1,2,3-oxa-thiolyl, isoxazolyl, oxazolyl, thiazolyl, isothiazolyl, 1,2,3-oxadiazolyl, 1,2,5-oxa-diazolyl, 1,3,4-oAadia~vlyl, 1,2,3,4-oxatriazolyl, 1,2,3,5-uA~Llia~ulyl~ 1,2,3-dioxa-zolyl, 1,2,4-dioxazolyl, 1,3,2-dioxazolyl, 1,3,4-dioxazolyl, 1,2~5-uAh~ ulyl~ 1,3-oxathiolyl, 1,2-pyranyl, I,~pyranyl, pyridinyl, I'Y ' yl, I,J,ill i.li.,yl, pyrazinyl, 1,3,5-triazinyl, 1,2,4-triazinyl, 1,2,3-triazinyl, 1,2,4-oxazinyl, 1,3,2-oxazinyl, 1,2,6-WO 95/34565 PCT/I~S95/07412 21 ~2~72 ,~

oxazinyl, 1,4-oxazinyl, isoxazinyl, 1,2,5-u~.alllia~,hlyl, 1,4-oxazinyl, o-isoxazinyl, p-isoxazinyl, 1,2,5-UAalll;a~,illyl, 1,2,6-vAdlll;a~illyl, 1,3,5,2-uAa~liaaillyl, azepinyl, oxepinyl, thiepinyl, 1,2,4-diazepinyl, l r ~1 ;'UI.P.~ r~ yl~
indolyl, indolenyl, 2 ~ lyl~ 1,5-pyrindinyl, pyrano[3,4-b]pyrrolyl, benzpyra-S zolyl, b .~ YI, b,.~ YI, anthranilyl, 1~2-b~ ,vpylallyl, quinolinyl, l, cinnolinyl, ~. ' yl, ~ JhLll.y~idh~ pyrido[3,4-b]pyridinyl. pyrid~
[4,3-b]pyridinyl, pyrido[2,3-b]pyridinyl, 1,3,2-b ~ . ~ ..yl, 1l4,2-b~,llzuAa~ill~l, 2,3, I --IJ . , Y I 1 3,1,4--1, ~ y 1, 1,2--L,. ,; ~. ~, , yl, 1,4--b~n 7i CCIY ~ 7i n yl, carbazolyl, purinyl, and parlially saturated h~,t~,~ucy~,lic radicals selected from the list 10 above. A11 of the preferred h~ u~yclic radicals contain at least one double bond.
When the h.,~c~u~,y~,lil, radical is partially saturated, one or more of the olefins in the ~ ( ' ring sysum is saturated; the partially saturated h~ ucy~lic radical still contains at least one double bond. It is more preferred that hetaryl is pyridinyl.
Of the c , ' of this invention preferred members are those in which Rl is 15 hydrogen.

('I , ' of this invention having the amidino carbamate moiety at the 42- or 31,42-positions can be prepared by conversion of lapanlyl,ill to the - 4- u~h~,.lylcallJun~c (as illustrated in Example 1), followed by reaction with an alJ~ r 'y r.. -- n~ AT; . .T amidine. Mixtures of 42- and 31,42-carbamates can be separated by ' , - O . ' y.
The 31-amidino carbamates of this invention can be prepared by protecting the 42-alcohol of rapamycin with a protecting group, such as with a tert-butyl dh~ ybiiyl group, followed by ~,alballlylàLiu~ of the 31-position by the procedures described 25 above. The ~ pulaliUII of rapamycin 42-silyl ethers is described in U.S. Patent B1 5,120,842, which is hereby iUCUI~ ' by reference. Removal of the proucting group provides the 31-esterified c~lmprll~A~Tc In the case of the urt-butyl ~" ' yl~ilJI
protecting group, d~,p~utc~,liu.. can be ~ pT; I d under mildly acidic conditions, such as acetic acid / water / THF. The ~ ,.utc r l i.... procedure is described in Exarnple 15 of U.S. Patent S,118,678, which is hereby pl - ' by reference.
Having the 31-position c~I)àlll~' - ' and the 42-position .l p vt~ 1, the 42-position can be ~,A b~ using a different amidine agent than was reacted with the31-carbonau, to give - --r ~ having different amidino carbamates at the 31- and 42- positions. Alternatively, the 42-Calbd~lly~ - r - ~, prepared as described above, can be converted to the 31-~,1i q2-carbamate and reacted with a differentamidine to provide compounds having different carbamates at the 31- and 42-positions.

_ _ _ _ _ _ . ... . _ . _ .. . .. .. . .. .. . . _ . ... . ..

W0 95134565 - 2 1 9 2 C~ 7 2 I ~l".~, " ,4l2 ~' Accordingly this invention provides a process for preparing the rapamycin cn ~ron~ of this invention having formula I as defined above which comprises:
reacting a carbonate derivative of rapamycin selected from a 31-O-(aLcyl-, aralkyl- or aryl-u,.y.,~ul,ul.yl)-42-OH protected rapamycin; 31-ORI-42-O-(aUcyl-, aralkyl- or aryl-5 O~y~.albu..yl)la~ ,;ll or 31,42-bis-[O-(alkyl-, aralkyl- or aryl-o~.yualuu~lyl)]
rapamycin where Rl is as defined above, with a compound of formula II

\NHR2' ~) wherein R4 is as defined above and R2 and R3 are each ,--~ l - ly as defined abovc for R2 to give a cullc r " g compound of formula I or protected derivative10 thereof and if required removing any protecting groups present.

It is most preferred that the carbonate derivative of rapamycin used as startingmaterial in the process above has a 4-~ u~lL.,l~u~.yc~ul/u.l.yl group. However tbe c....,l.u~ of formula I can also be prepared by reacting rapamycin to form otherLS suitably substituted carbonates at the 42-, 31,42-bis-, or 31-positions, followed by treatment with a compound of formula II, as described above. Examples of suitable carbonates that can be formed at the 42-, 31,42-bis-, or 31-postiions include, but are not limited to, alku,.ycall,ull~l and optionally substituted I ' yu~ubull.yl derivatives of rapamycin. It is preferred that the substituent on the phenyl ring (when ' ')be an electron w;l~L~wLl~; group. The carbonates of rapamycin can be formed by reacting rapamycin or an ~ , protected or substituted rapamycin with an alkyl-, araLcyl- or optionally substituted aryl- ~,LIulurullll~ltc or carbonate. Preferably the alkyl group has I to 6 carbon atoms, eg I to 4 carbon atoms such as methyl.
Preferably the aryl group has 6 to 10 carbon atoms, eg phenyl and preferably the2~; araUcyl group has 7 to 12 carbon atoms.

This invention also covers analogous carbamates of other ld~,a~ ,,h.s such as, but not limited to, 29-d~ ,dlu/~yl~ ;ll, [U.S. Patent 4,375,464, 32-demethoxy-rapamycin under C.A. ' ]: rapamycin derivatives in which the double bonds in the 1-, 3-, and/or 5-positions have been reduced [U.S. Patent 5,023,262];
29-d~ L~ ,Ll [U.S. Patent 5,093,339, 32-dc;,n~ ,alll.~,;ll under C.A.

... .... ....... .. ...... ... .. .. _ _ _ = ... , , , , _ wo ss/34s6s 21 9 2 9 7 2 PCTIUS95/07412 c]; 7~29-b;~dl,i~ yL_~)allly~,;ll [U.S. Patent 5,093,338, 7,32-desmethyl-rapamycin under C.A. n~ ' c]; and 15-L~Lu~.yl_~JAllly~ l [U.S. Patent 5,102,876]. The disclosures in the above cited U.S. Patents are hereby ~Uldt~by reference.
s T r .~ , aCtivity for I qJl C ' ' ~/C S I of this invention was evaluated in an in vitro standard p~ gi( Al test procedure to measure the inhibition of Iymphocyte proliferation (LAF) and in two in. vivo standard pl,-.",~ ~lr~ test procedures. The pinch skin graft test procedure measures the 1O ' - ~ r ~ iVC activity of the compound tested as well as the ability of the compound testcd to inhibit or treat transplant rejection. The adjuvant arthritis standard gi~Al test proccdure, which measures the ability of the compound tested to inhibit immune mediated ;"r~ ;u" The adjuvant arthritis test procedure is a standard ~ g; ~.1 oest procedure for li ~ ' arthritis. The procedures for 15 these standard l,l -- . ., -- . .l~.~;, Al test procedures are provided below.

The ~UIIL~ induced thymocyte ~ulir~ iu.. proccdure (LAF) was uscd as aniDLy~measureofthe - Ir c~;veeffectsof lcl)lc~cll~a~ r~
Briefly, cells from the thymus of normal BALB/c mice are culturcd for 72 hours with 20 PHA and IL-1 and pulsed with tritiated thymidine during the last six hours. Cells are cultured with and without various ~ ~ of rapamycin, CyclU~lJulill A, or test compound. Cells are harvcstcd and i"cu,~ ' l~iUa~,~iVi~y is dPt~rrninPA Inhibition of Iy~ ",lirrlAl;.. - is assessed as percent change in counts per minute from non-drug treated controls. For each compound evaluated, rapamycin was also evaluated for 25 the purpose of c-,~ - An ICso was obtained for each oest compound as well as for rapamycin. When evaluatcd as a cUlll~Jal_lUl for the ~c~c v~ compounds of this invention, rapamycin had an ICso ranging from 0.4 - 1.0 nM. The results obtained are provided as an ICso and as the percent inhibition of T-cell l" . .l; r... A I i~ ~" at 0.1 ~LM.
The results obtained for the lc~lc~ a~ivc c~ of this invention were also 30 expressed as a ratio compared with rapamycin. A positive ratio indicates , activity. A ratio of greater than 1 indicates that the test compound inhibited thymocyte IJlvlLrclatiull to a greater extent than rapamycin. Calculation of the ratio is shown below.
ICso of Rapamycin ICso of Test Compound WO 95134565 2 1 9 2 ~ 7 2 PCT/US9~/07412 .

R.,~l~ v.,: ~ . ' of this invention were also evaluated in an in vivo test procedure designed to determine the survival time of pinch skin graft from male BALB/c donors , ' ' to male C3H(H-2K) recipients. The method is adapted from Rillingh~rn R.E. and Medawar P.B., J. Exp. Biol. 28:385-402, (1951). Briefly, 5 a pinch skin graft from the donor was grafted on the dorsum of the recipient as a allograft, and an isograft was used as control in the same region. The recipients were treatedwitheithervarying ~ oftest. . '; n A~ lly ororally.
Rapamycin was used as a test control. Untreated recipients serve as rejection control.
The graft was monitored daily and U~ iUlls were recorded until the graft became 10 dry and formed a blackened scab. This was considered as the rejection day. The mean graft survival time (number of days + S.D.) of the drug treatment group was compared with the control group. The following table shows the results that were obtained.
Results are expressed as the mean survival time in days. Untreated (control) pinch skin grafts are usually rejected within 6-7 days. ~'~ . ' were tested using a dose of 4 L5 mg/kg.

The adjuvant arthritis standard ~ " ~ ,;. Al test procedure measures the ability of test r~lnro~ fic to prevent immune mediated i~nA~ ) and inhibit or treat ~ ' arthritis. The following briefly describes the test procedure usc-d. A group20 of rats (male inbread Wistar Lewis rats) are pre-treated with the compound to be tested (1 h prior to antigen) and then injected with Freud's Complete Adjuvant (FCA) in the right hind paw to induce arthritis. The rats are then orally dosed on a Monday, Wednesday, Friday schedule from day 0-14 for a total of 7 doses. Both hind paws are measured on days 16, 23, and 30. The difference in paw volume (rnL) from day 16 to 25 day 0 is ~ ~ and a percent change from control is obtained. The left hind paw (uninjected paw) ;"n- ,... -~;..,. is caused by T-cell mediated i..nA. . -~;..., and is recorded in the above table (% change from control). The right hind paw r~ .
on the other hand~ is caused by no~ ;rlc il lnA~ were tested at a dose of 5 mg/kg. The results are expressed as the percent change in the uninjected paw 30 at day 16 versus control; the more negative the percent change, the more potent the cf~mro~ Rapamycin provided -90% change versus control, indicating that rapamycin treated rats had 90% less immune induced; .n- ....~ than control rats.
The results obtained in these standard ph~rm~ gi~Al test procedures are 35 provided following the procedure for making the specific . . ~ that were tested.

wossl34!i6s 21 q2972 r~l~u_,_. ~412 The results of these standard ph~ test procedures .L .. n .
~r '~ activity both inY~ and in vivo for the . , ~ of this invention. The results obtained in the LAF test procedure indicates ! .1: ' of T-cell ~ thereby I g the ~ .r CDD;~ activity of the 5 ~ r ~ of this invention. Further fl~ of the utility of the f r ~ of this invention as .r ODD;V.~ agents was shown by the results obtained in the skin graft and adjuvant arthritis standard ph,~ , fAI test ~)lv~,dul~,D.
Additionally, the results obtained in the skin graft test procedure further d-the ability of the compounds of this invention to treat or inhibit i , ' rejection.10 The results obtained in the adjuvant arthritis standatd ~ f~gi.,l test procedure further r' the ability of the c ....l.u ,.k of this invention to treat or inhibit rheumatoid arthritis.
Based on the results of these standard r~ g;~ Al test procedures, the c. ~ v ~- -1~ are useful in the treatment or inhibition of ~ p ~ ;- ~'~ rejection such as 15 kidney, heart, liver, lung, bone marrow, pancreas (islet cells), cornea, small hcowe and skin allografts, and heart valve xenografts; in the treatment or inhibition of gTaft vs.
host disease; in the treatment or inhibition of _ ~ diseases such as lupus, ,h....,.AI..;.l arthritis, diabetes mellitus, IllyrDLL~ ;a gravis, and multiple sclerosis; and diseases of '' such as psoriasis"lfrmAriti~ eczema, seborrhea, 20 in~lAmmAtr~ry bowel disease, pulmonary ;..n- ...~.AI;r~.. (including asthma, chronic obD~Iu~ ., pulmonary disease, e...~l.y~ .a, acute respiratory distress syndrome,bronchitis, and the like), and eye uveitis.
Because of the activity profile obtaincd, the ~ of this invention also are considered to have antitumor, antifungal activities, and ~ lir~ ivG activities.
25 The c~ -r ' of this invention therefore also useful in treating solid turnors, adult T-cell leukemially , I fungal infections, and Ly~ ulircl~livG vascular discases such as restenosis and ~ -,lc.~ . When uscd for restenosis, it is preferrcd thatthe .~-- I-.n 1~ of this invention are used to treat restenosis that occurs following an . .gi~ y procedure. When used for this purpose, the .,, , ' of this invention 30 can be a.l.l i..i~t".Gd prior to the procedure, during the procedure, subscquent to the procedure, or any .. 1.;. -~ of the above.
When A~ ;' ''' '' .I for the treatment or inhibition of the above disease states, the f.. l.~,.. l~ of this invention can be a~-,h.i~.~,.Gd to a mammal orally, parenterally, intranasally, ~,.I.,I.;i,lly, i ' 'Iy, topically, ;..I.~.v..g;..,lly, orrcctally.

W095134565 21 ~?2q~ 1~ PCrnTS95~07412 _ 9_ It is: l l ' that when the . , ' of this invention are used as an ; " " " " ~ : v~, or: ~ y agent, they can be r ' ' ~ ' in .
with one or more other illlU. IIUI~ y agents. Such other illllllUllUI~,, ' ' y sgents include, but are not limited to n ~ q ;--~, cultiuu~l~,.u;d~, such as prednisone S and Ill~lI.yl~ y~ Il.t.h ,~t~l ",' lr., rspamycin, ~ ,lU7~ulhl A, FK-506, OKT-3, and ATG. By combining the, ~,,,,1,,, l~ of this invention with such otherdrugs or agents for inducing . ~ ivll or treating ;. ,n --, . . ~- ., y conditions, the lesser smounts of each of the agents are required to achieve the desired effect. The basis for such c~ ll l.U;~ therspy was . 7~ by Stepkowski whose results 10 showedthattheuseofa~ h -~,...,ofrapamycinand1~,1u~t~uli..Aat ' ' doses cit ~ ly prolonged heart sllograft survival time. [T.,...~ ;.... Proc. 23:
507 (1991)].

The cv..l~uu,lds of this invention can be ro.,."~ d neat or with a 15 ~ -l carrier to a mammal in need thereof. The ~ carrier msy be solid or liquid. When formulated orally, it has been found that 0.01% Tween 80 in PHOSAL PG-50 (1 ' . ' 'i, ', with 1,2-propylene glycol, A.
& Cie. GmbH) provides sn acceptable oral ' A solid carrier can include one or more substsnces which may also act as 20 flavoring agents, lubricants, sr~h~hili7rr~ ~.,~I....l;,~g agents, fillers, glidants, Cull~ iull aids, binders or tsblet~ agents; it csn also be an ~ .. ~y- l ~ g msterial In powders, the carrier is a finely divided solid which is in admixture with the finely divided sctive ingredient In tablets, the active ingredient is mixed with a carrier having the necessary ~ properties in suitable l~-ul~u~Iiul~7 and compacted in the shape and si_e desired. The powders and tablets preferably contain up to 999'o of tbe active ingredient. Suitable solid carriers include, for example, calcium phosphate, mrgn~cil-m stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, sodium u uhu~yl~ llyl cellulose, polyYi"yll,y~lulidine~ low melting waxes and ion exchange resins.
Liquid carriers are used in preparing solutions, ~ , emulsions, syrups, elixirs and 1~ ' c- . q.. .- :~ c The active ingredient can be dissolved or suspended in a ~ u, lly acceptable liquid carrier such as water, an organic - solvent, a mixture of both or i ' - ~Iy acceptable oils or fats. The liquid carrier can contain other suitable ~h~ additives such as ~ ~ 1i7~rc,; ~ ~
3~ buffers, ~ d~ ,S, sweeteners, flavoring agents" ~ll;.~E agents, thickening agents, colors, viscosity regulators, stabili_ers or osmo-regulators. Suitable examples of liquid carriers for oral and parenteral A~ ,rl;l include water (partiallycontaining additives as above, e.g. cellulose d~.liY~ , preferably sodium c.~ubu~ ,tll,Yl cellulose solution), alcohols (including ' y~Lic alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, lethicins, and oils (e.g.
5 ' . t; ' coconut oil and arachis oil). For parenteral r ' the carrier can also bc an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are useful in sterile liquid form ~ .... ~l ..,- ~ ;n ~; for parenteral - ' The liquid carrier for p~ d ~ o~ can be l.Alnt,. -~ d LyLu1 - l)un or other ly acceptable propellant.
Liquid I ' ~ " which are sterile solutions or ~
can be utilized by, for example, i~.UA.I...;. ..lrr, h.LI~ ( 1 or ~h~ f~) injecion. Sterile solutions can also be ' ~d hlh~ u .,ly. The compound can also be d~ ~i orally either in liquid or solid c. ~ form.
The - - . ' of this invention may be A.l. . ,;, .; ~ . cd rectally in the form of a 15 cu..~ iu~dl I, ' y. ForA.l,..:. 1,A l;nn byintranasalorimrrAhn~nr~ inhalationor rrl - l ;.... the C,UII~IJU_ ' of this invention may be formulated into an aqueous or partially aqueous solution, which can then be utilized in the form of an aerosol. The compounds of this invention may also be A.l...~ .. cJ u,...~ 1. ~-- lly through the use of a u.- 1, .,.. 1 patch containing the active compound and a carrier that is inert to the 20 active compound, is non toxic to the skin, and allows delivery of the agent for systemic absorption into the blood stream via the skin. The carrier may take any number of forms such as creams and ointments, pastes, gels, and occlusive devices. The creams and ointments may be viscous liquid or semisolid emulsions of either the n;l _.~.. or water-in-oil type. Pastes comprised of absorptive powders dispersed in petroleum or hydrophilic petroleum containing the active ingredient may also be suitable. A variety of occlusive devices may be used to release the active ingredient into the blood stream such as a ~ membrane covering a reservoir containing the active ingredient with or without a carrier, or a matrix containing the active ingredient. Other occlusive devices are known in the literature.
In addition, the c .,,~l~u ,.1~ of this invention may be employed as a solution,cream, or lotion by r..., .. 1 ~ ;"" with ~ r ~ , acceptable vehicles containing 0.1 - 5 percent, preferably 2%, of active compound which may be ad..~ tc.cd to afungally affected area.
The dosage IC 1 Clll.~ vary with the particular c ~ - employed, the 35 route of rA.l",;,,i~l".l;r~n the severity of the symptoms presented and the particular subject being treated. Based on the results obtained in the standard 11 - ~l ~rn~

W0 9S/3456S 2 ~ ~ ~ 9 7 ~ 4l2 test procedures, projected daily dosages of active compound would be 0.1 llg/kg - 100 mg/kg, preferably between 0.001 - 25 mgtkg, and more preferably between 0.01 - 5mgtkg. Treatment will generally be initiated witb small dosages less than the optimum dose of the compound. Thereafter the dosage is increased until the optimum effect 5 under the: is reached; precise dosages for oral, parenteral, nasal, or L ~ ' 1. ' will be ~ ' by the ~ ' ~ physician based on experience with the individual subject treated. Preferably, tbe r~ I
c~mp-~;m n is in unit dosage form, e.g. as tablets or capsules. In such form, the ;. ). . is sub-divided in unit dose containing ~ ul ~ quantities of the active 10 ingredient; the unit dosage forrns can be packaged o ....~ , for exarnple, packetcd powders, vials, ampoules, prefilled syringes or sachets containing liquids. The unit dosage form can be, for exarnple, a capsule or tablet itself, or it can be the appropriate number of any such ,~ v~ in package form.
A~ , this invention provides 1~ , t;- _I C~ comprising 15 cO r- ~ of formula I or a ~' r~lly acceptable salt thereof and a ";- .lly acceptable carrier.
The following examples illustrate the IJIClJdl~liUII and biological activities of lr~ ve r ofthisinvention l~xample 1 4'7-0-(4-l~itro-~ ,..v,~vclubv~ ~.vcin To a solution of 5.15 g (5.633 mmol) of rapamycin in 40 ml of met,'ylene chloride cooled to -78-C with dry ice / acetone bath, was added 0.7 ml dry pyridine and 1.70 g (8.450 mmol) of p-.. illo~h~,.. yl~ l.l.. r,.. ". ~ dissolved in 10 ml methylene chloride. The reacion mixture was allowed to warm to ambient and stirred overnight under nitrogen. The reaction rnixture was . ' in vacuo and partitioned between ether and water. The organic phase was washed with 0.1 N HCI (3x), then with a saturated sodium chloride solution (2x), dried over m~,,r sulfate, filtered and ' under vacuum to give a pale yellow solid. r ~ by flash column cl~l~ O , ' y (elution wihh 4û% then 50% ethyl /' ) gave 5.41 g (88 %) of the tihe compound as a pale yellow solid.
IH NMR (DMSO) o 8.3 and 7.5 (d and d, aromatic-H, 4H), 4.5 (m, 42C-H, lH).
MS (-) FAB mlz: 1078 (M-), 590 (Southern Fragment).

WO 95/34565 2 1 9 2 q 7 2 PC'rlUS95/07412 Exarnple 2 ~ycin 42-este} with ~imino-yhenyl-m-.thyl)carbamic acid To a solution of 1.0024 g (0.9287 mmol) of 42-0-(4-Nitro-r' y~.~ubu~yl)~
rapamycin in 5 ml of DMF was added 0.2231 g (1.8574 mmol) of IJ' - .;.l;~ The Sreaction mixture was allowed to stir under N2 for 2 hours at ambient I~c, then was diluted with ethyl acetate and washed with portions of H2O and brine. The organic phase was dried over ~ sulfate, filtered and: ' under vacuum to yield crude product. P~l.ir.~ ul by flash column .,h~ OIrpl y (elution with 60% then 80% EtOAc/hexanes) gave 0.1032 g (10%) of the title compound as a pale yellow solid.
IH NMR (DMSO) o 9.05 (m, N-H, 2H), 7.98-7.48 (m, aromatic-H, SH), 4.48 (m, 42C-H, lH).
MS (-) FAB m/z: 1059 (M-), 590 (Southern Fragment), 467 (Northern Fragment).
Results obtained in standard I .1 ", , .1. ,g; I test IJlU~,CdU.
LAF ICso: 2.05 nM
15 LAFratio: 0.47 Skin graft survival: 9.8 + 0.8 Percent change in adjuvant arthritis versus control: -91 %
Example 3 20 ~p~ycin 42-ester with (imin~pyridin-2-yl)methyl)carbamic acid To 0.585 g (3.7 mmol) of 2-~Iflllilw~yli~ lc hydrochloride was added one equivalent of 0.1M sodium LyLu~ide/ ' after which the solvent was removed invacuo. To the solution of the free base in 20 ml of DMF was added 4.0 g (3.7 mmol) of 42-0-(4-Nitro-I' y.,~bu..yl)rapamycin. The reaction mixture was allowed to stir 25 under nitrogen for 6 hours at ambient ~ u.c, then was diluted with ethyl acetate and washed with portions of H2O and brine. The organic phase was dried over 1.. .~, . -:- ..
sulfate, filtered and ' under vacuum to yield crude product. P ~ ~ by flash column .,hl. O, ' y (elution with 1:1 ethyl acetate: hexanes, then 100% ethyl acetate) gave 0.47 g (12 qo) of the title compound as an off-white solid.
IH NMR (DMSO) o 9.05 (m, N-H, 2H), 8.68-8.72 (m, Ar-H, lH), 8.28-8.24 (m, Ar-H, lH), 8.0-7.94 (m, Ar-H, IH), 7.66-7.62 (m, Ar-H, lH), 4.4 (m, 42C-H, IH).
MS (-) FAB m/z: 1060 (M-), 590 (Southern Fragment), 468 (Northern Fragment).
Results obtained in standard pl ~ .g;~ -I test ~,.. ~
LAF ICso: 0.60 and 0.55 nM
LAF ratio: 1.50 and 0.78 Skin graft survival: 11.7 _ 1.0

Claims (11)

What is claimed is:

1. A compound of the structure wherein R and R1 are each, independently, hydrogen, or ;

R2 and R3 are each,independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, -CO2R5, -COR5, -CN, -NO2, -SO2R5, -SO3R5, -OR5, -SR5, or Ar;
R4 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, -CF3, -NR5R6, -CO2R5, -COR5, CONR5R6, -NO2, halogen, -OR5, -SR5, -CN, -SO2R5, -SO3R5, -SO2NR5R6, or Ar;
R5 and R6 are each,independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, or Ar;
Ar is phenyl, naphthyl, or hetaryl, wherein the foregoing may be optionally mono-, di-, or tri-substituted with a group selected from alkyl of 1-6 carbon atoms, alkenylof 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, arylalkyl of 7-10 carbon atoms, alkoxy of 1-6 carbon atoms, cyano, halo, hydroxy, nitro, carbalkoxy of 2-7 carbon atoms, trifluoromethyl,trifluoromethoxy, amino, dialkylamino of 1-6 carbon atoms per alkyl group, dialkylaminoalkyl of 3-12 carbon atoms, hydroxyalkyl of 1-6 carbon atoms, alkoxyalkyl of 2-12 carbon atoms, alkylthio of 1-6 carbon atoms, -SO3H, and -CO2H;
with the proviso that R and R1 are both not hydrogen. or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1 wherein R1 is hydrogen or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 1 which is rapamycin 42-ester with (imino-phenyl-methyl)carbamic acid or a pharmaceutically acceptable salt thereof.

4. The compound according to claim 1 which is rapamycin 42-ester with (imino-pyridin-2-yl)methyl)carbamic acid or a pharmaceutically acceptable salt thereof.

5. A method of treating transplantation rejection or graft vs. host disease in amammal in need thereof, which comprises administering to said mammal an antirejection effective amount of a compound of the structure wherein R and R1 are each, independently, hydrogen, or ;

R2 and R3 are each,independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, -CO2R5, -COR5, -CN, -NO2, -SO2R5, -SO3R5, -OR5, -SR5, or Ar;
R4 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, -CF3, -NR5R6, -CO2R5, -COR5, CONR5R6, -NO2, halogen, -OR5, -SR5, -CN, -SO2R5, -SO3R5, -SO2NR5R6, or Ar;
R5 and R6 are each,independently,hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, or Ar, Ar is phenyl, naphthyl, or hetaryl, wherein the foregoing may be optionally mono-, di-, or tri-substituted with a group selected from alkyl of 1-6 carbon atoms, alkenylof 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, arylalkyl of 7-10 carbon atoms, alkoxy of 1-6 carbon atoms, cyano, halo, hydroxy, nitro, carbalkoxy of 2-7 carbon atoms, trifluromethyl,trifluoromethoxy, amino, dialkylamino of 1-6 carbon atoms per alkyl group, dialkylaminoalkyl of 3-12 carbon atoms, hydroxyalkyl of 1-6 carbon atoms, alkoxyalkyl of 2-12 carbon atoms, alkylthio of 1-6 carbon atoms, -SO3H, and -CO2H;
with the proviso that R and R1 are both not hydrogen or a pharmaceutically acceptable salt thereof.

6. A method of treating a fungal infection in a mammal in need thereof, which comprises administering to said mammal an antifungal effective amount of a compound of the structure wherein R and R1 arc each,independently, hydrogen, or ;

R2 and R3 are each, independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, -CO2R5, -COR5, -CN, -NO2, -SO2R5, -SO3R5, -OR5, -SR5, or Ar;
R4 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, -CF3, -NR5R6, -CO2R5, -COR5, CONR5R6,-NO2, halogen, -OR5, -SR5, -CN, -SO3R5, -SO2R5, -SO2NR5R6, or Ar;
R5 and R6 are each, independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, or Ar, Ar is phenyl, naphthyl, or hetaryl, wherein the foregoing may be optionally mono-, di-, or tri-substituted with a group selected from alkyl of 1-6 carbon atoms, alkenylof 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, atylalkyl of 7-10 carbon atoms, alkoxy of 1-6 carbon atoms, cyano, halo, hydroxy, nitro, carbalkoxy of 2-7 carbon atoms,trifluromethyl,trifluoromethoxy, amino, dialkylamino of 1-6 carbon atoms per alkyl group, dialkylaminoalkyl of 3-12 carbon atoms, hydroxyalkyl of 1-6 carbon atoms, alkoxyalkyl of 2-12 carbon atoms, alkylthio of 1-6 carbon atoms, -SO3H, and -CO2H;
with the proviso that R and R1 are both not hydrogen, or a pharmaceutically acceptable salt thereof.

7. A method of treating rheumatoid arthritis in a mammal in need thereof, which comprises administering to said mammal an antiarthritis effective amount of a compound of the structure wherein R and R1 are each, independently, hydrogen, or ;

R2 and R3 are each, independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, -CO2R5, -COR5, -CN, -NO2, -SO2R5, -SO3R5, -OR5, -SR5, or Ar;
R4 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, -CF3, -NR5R6, -CO2R5, -COR5, CONR5R6, -NO2, halogen, -OR5, -SR5, -CN, -SO2R5, -SO3R5, -SO2NR5R6, or Ar;
R5 and R6 are each, independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, or Ar;
Ar is phenyl, naphthyl, or hetaryl, wherein the foregoing may be optionally mono-, di-, or tri-substituted with a group selected from alkyl of 1-6 carbon atoms, alkenylof 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, arylalkyl of 7-10 carbon atoms, alkoxy of 1-6 carbon atoms, cyano, halo, hydroxy, nitro, carbalkoxy of 2-7 carbon atoms,trifluoromethyl,trifluoromethoxy amino, dialkylamino of 1-6 carbon atoms per alkyl group, dialkylaminoalkyl of 3-12 carbon atoms, hydroxyalkyl of 1-6 carbon atoms, alkoxyalkyl of 2-12 carbon atoms, alkylthio of 1-6 carbon atoms, -SO3H, and -CO2H;
with the proviso that R and R1 are both not hydrogen, or a pharmaceutically acceptable salt thereof.

8. A method of treating restenosis in a mammal in need thereof, which comprises administering to said mammal an antiproliferative effective amount of a compound of the structure wherein R and R1 are each, independently, hydrogen, or ;

R2 and R3 are each,independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, -CO2R5, -COR5, -CN, -NO2, -SO2R5, -SO3R5, -OR5, -SR5, or Ar;
R4 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, -CF3, -NR5R6, -CO2R5, -COR5, CONR5R6, -NO2, halogen, -OR5, -SR5, -CN, -SO2R5, -SO3R5, -SO2NR5R6, or Ar;
R5 and R6 are each, independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, or Ar;
Ar is phenyl, naphthyl, or hetaryl, wherein the foregoing may be optionally mono-, di-, or tri-substituted with a group selected from alkyl of 1-6 carbon atoms, alkenylof 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, arylalkyl of 7-10 carbon atoms, alkoxy of 1-6 carbon atoms, cyano, halo, hydroxy, nitro, carbalkoxy of 2-7 carbon atoms, trifluoromethoxy, amino, dialkylamino of 1-6 carbon atoms per alkyl group, dialkylaminoalkyl of 3-12 carbon atoms, hydroxyalkyl of 1-6 carbon atoms, alkoxyalkyl of 2-12 carbon atoms, alkylthio of 1-6 carbon atoms, -SO3H, and -CO2H;
with the proviso that R and R1 are both not hydrogen, or a pharmaceutically acceptable salt thereof.

9. A method of treating pulmonary inflammation in a mammal in need thereof, which comprises administering to said mammal an antiflammatory effective amount of a compound of the structure wherein R and R1 are each, independently, hydrogen, or ;
R2 and R3 are each, independently hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, -CO2R5, -COR5, -CN, -NO2, -SO2R5, -SO3R5, -OR5, -SR5, or Ar;
R4 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, -CF3, -NR5R6, -CO2R5, -COR5, CONR5R6, -NO2, halogen, -OR5, -SR5, -CN, -SO2R5, -SO3R5, -SO2NR5R6, or Ar;
R5 and R6 are each, independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, or Ar;
Ar is phenyl, naphthyl, or hetaryl, wherein the foregoing may be optionally mono-, di-, or tri-substituted with a group selected from alkyl of 1-6 carbon atoms, alkenylof 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, arylalkyl of 7-10 carbon atoms, alkoxy of 1-6 carbon atoms, cyano, halo, hydroxy, nitro, carbalkoxy of 2-7 carbon atoms, trifluoromethyl, trifluoromethoxy, amino, dialkylamino of 1-6 carbon atoms per alkyl group, dialkylaminoalkyl of 3-12 carbon atoms, hydroxyalkyl of 1-6 carbon atoms, alkoxyalkyl of 2-12 carbon atoms, alkylthio of 1-6 carbon atoms, -SO3H, and -CO2H;
with the proviso that R and R1 are both not hydrogen, or a pharmaceutically acceptable salt thereof.

10. A pharmaceutical composition which comprises a compound of the structure wherein R and R1 are each, independently, hydrogen, or ;

R2 and R3 are each, independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, -CO2R5, -COR5, -CN, -NO2, -SO2R5, -SO3R5, -SO3R5, -OR5, -SR5, or Ar;
R4 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, -CF3,-NR5R6, -CO2R5, -COR5, CONR5R6,-NO2, halogen, -OR5, -SR5, -CN, -SO2R5, -SO2NR5R6, or Ar;
R5 and R6 are each, independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, or Ar;
Ar is phenyl, naphthyl, or hetaryl, wherein the foregoing may be optionally mono-, di-, or tri-substituted with a group selected from alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, arylalkyl of 7-10 carbon atoms, alkoxy of 1-6 carbon atoms, cyano, halo, hydroxy, nitro, carbalkoxy of 2-7 carbon atoms, trifluoromethyl, trifluoromethoxy, amino, dialkylamino of 1-6 carbon atoms per alkyl group, dialkylaminoalkyl of 3-12 carbon atoms, hydroxyalkyl of 1-6 carbon atoms, alkoxyalkyl of 2-12 carbon atoms, alkylthio of 1-6 carbon atoms, -SO3H. and -CO2H;
with the proviso that R and R1 are both not hydrogen, or a pharmaceutically acceptable salt thereof, and a pharmaceutical carrier.

11. A process for preparing the rapamycin compounds of this invention having formula I as defined above which comprises:
(a) reacting a carbonate derivative of rapamycin selected from a 31-O-(alkyl-, aralkyl- or aryl-oxycarbonyl)-42-OH protected rapamycin;
31-OR1-42-O-(alkyl-, aralkyl- or aryl-oxycarbonyl)rapamycin or 31,42-bis-[O-(alkyl-, aralkyl- or aryl-oxycarbonyl)]rapamycin where R1 is as defined above, (b) with a compound of formula II

wherein R4 is as defined above and R2' and R3' are each independently as defined above for R2 to give a corresponding compound of formula I
or protected derivative thereof, and if required (c) removing any protecting groups present.