Europaisches Patentamt
`
`e Veroffentlichungsnummer:
`e Publication number:
`Office europeen des brevets e Numero de publication:
`
`European Patent Office
`
`0 763 039
`
`Internationale Anmeldung veroffentlicht durch die
`
`Weltorganisation fiir geistiges Eigentum unter der Nummer:
`
`WO 95/28406
`
`(art.158 des EPU).
`
`International application published by the World
`
`Intellectual Property Organisation under number:
`
`WO 95/28406
`
`(art.158 of the EPC).
`
`Demande internationale publiee par l' Organisation
`
`Mondiale de la Propriete sous le numero:
`
`WO 95/28406
`
`(art.158 de la CBE).
`
`

`

`P B 5818 - Patentlaan 2
`2280 HV RIJSWIJk (ZH)
`Z'
`(070) 3 40 20 40
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`
`Europaisches
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`
`European
`Patent Office
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`in Den Haag
`PriJfungs(cid:173)
`abtftlhlOQ
`
`Branch at
`The Hague
`Examining
`d!YISIOC
`
`Wileman, David Francis, Dr.
`c/o Wyeth Laboratories
`Huntercombe Lane South
`Taplow
`Maidenhead
`Berkshire SL6 OPH
`GRANDE BRETAGNE
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`r
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`7
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`_J
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`I D,t,m/0,t,
`
`05/02/97
`
`Office europeen
`des brevets
`Departement a
`La Haye
`D1V1sIon
`d'examen
`COPY
`
`ZeIch en/Ref/Ref
`PCT/US950460
`AHP-93171
`Anmelder/Appl1canVDemandeur//Paten1lnhaber/Propnetor/T1tulaire
`AMERICAN HOME PRODUCTS CORPORATION
`
`Anmeldung Nr /AppllcatIon No /Oemande n' //Patent Nr /Patent No /Brevet n'
`95915671. 2-2101
`
`I 0763039
`
`NOTIFICATION OF EUROPEAN PUBLICATION NUMBER AND INFORMATION
`ON THE APPLICATION OF ARTICLE 67(3) EPC
`
`-
`
`The provisional protection under Article 67(1) and (2) EPC in the
`individual Contracting States becomes effective only when the
`conditions referred to in Article 67(3) EPC have been fulfilled
`(for further details, see information brochure of the European
`Patent Office "National Law relating to the EPC" and additional
`information in the Official Journal of the European Patent Office).
`
`A request has been made for extension of the patent to
`LT SI .
`See Official Journal 1-2/1994 for further information on
`provisional protection in LT SI .
`
`Pursuant to Article 158(1) EPC the publication under Article 21 PCT
`of an international application for which the European Patent Office
`is a designated Office t~kes the place of the publication of a
`European patent application.
`
`The bibliographic data of the above-mentioned Euro-PCT application
`will be published on 19.03.97 in Section I.1 of the European Patent
`Bulletin.
`The European publication number is 0763039.
`
`In all future communications to the European Patent Office, please
`quote the application number plus Directorate number.
`
`RECEIVING SECTION
`
`EPO Form 1219
`
`(02.94)
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`31/01/97
`
`

`

`PCT
`WORLD INJ'ELLECI1JAL PROPERTY ORGANIZATION
`International Bureau
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`WO 95/28406
`(51) International Patent Classification 6 :
`C07D 498/18, A61K 31/435 // (C07D
`498/18, 311:00, 273:00, 221:00)
`
`( 43) International Publication Date:
`
`26 October 1995 (26.10.95)
`
`(11) International Publication Number:
`
`Al
`
`(21) International Application Number:
`
`PCT/US95/04603
`
`(22) International Filing Date:
`
`14 April 1995 ( 14.04.95)
`
`(30) Priority Data:
`08/229,261
`
`18 April 1994 (18.04.94)
`
`us
`
`(81) Designated States: AM, AU, BB, BG, BR, BY, CA, CN, CZ,
`EE, FI, GE, HU, IS, JP, KG, KP, KR, KZ, LK, LR, LT,
`LV, MD, MG, MN, MX, NO, NZ, PL, RO, RU, SG, SI,
`SK, TJ, TM, TT, UA, UG, UZ, VN, European patent (AT,
`BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL,
`PT, SE), OAPI patent (BF, BJ, CF, CG, Cl, CM, GA, GN,
`ML, MR, NE, SN, TD, TG), ARIPO patent (KE, MW, SD,
`SZ, UG).
`
`(71) Applicant: AMERICAN HOME PRODUCTS CORPORA-
`TION [US/US]; Five Giralda Farms, Madison, NJ 07940- Published
`With international search report.
`0874 (US).
`
`(72) Inventors: SKOTNICKI, Jerauld, Stanley; 4 Tyler Court,
`Allentown, NJ 08501 (US). LEONE, Christina, Louise;
`99 Bayard Lane, Princeton, NJ 08540 (US). SCHIEHSER,
`Guy, Alan; 658 Bayberry Lane, Yardley, PA 19067 (US).
`
`(74) Agents: ALICE, Ronald, W.; American Home Products Corpo(cid:173)
`ration, Five Giralda Farms, Madison, NJ 07940-0874 (US)
`et al.
`
`(54) Title: RAPAMYCIN HYDROXYESTERS, PROCESS FOR THEIR PREPARATION AND PHARMACEUTICAL COMPOSIDONS
`CONTAINING THEM
`
`(57) Abstract
`
`structure
`compound of
`A
`(I) wherein R 1 and R2 are each,
`or
`independently,
`hydrogen
`-CO(CR3R4)b(CR5R6)dCR7R8R9;
`R3 and R4 are each, independently,
`hydrogen, alkyl, alkenyle, alkynyl,
`trifluoromethyl, or -F; R5 and R6 are
`each, independently, hydrogen, alkyl,
`-(CR3R4)tOR 10,
`alkenyl,
`alkynyl,
`or R5
`-CO2R11 ,
`-CF3,
`-F, or
`and R6 may be
`taken
`together
`to form X or a cycloalkyl ring
`that
`is optionally mono-, di-, or
`tri-substituted with -(CR3R4)tOR IO;
`R7
`is hydrogen,
`alkyl,
`alkenyl,
`alkynyl, -(CR3R4)tOR10,
`-CF3,
`-F,
`or -CO2R11 ; R8 and R9 are each,
`independently,
`hydrogen,
`alkyl,
`-(CR3R 4)tOR10,
`alkenyl,
`alkynyl,
`or R8
`-CO2R11 ,
`-CF3,
`-F, or
`and R9 may be
`taken
`together
`to
`form X or a cycloalkyl ring
`that
`is optionally mono-, di-, or
`tri-substituted with -(CR3R4)tOR 10; R 10 is hydrogen, alkyl, alkenyl, alkynyl, tri-(alkyl)silyl, tri-(alkyl)silylethyl, triphenylmethyl, benzyl,
`alkoxymethyl, tri-(alkyl)silylethoxymethyl, chloroethyl, or tetrahydropyranyl; R11 is hydrogen, alkyl, alkenyl, alkynyl, or phenylalkyl;
`X is 5-(2,2-dialkyl)[ l ,3]dioxanyl, 5-(2-spiro-cycloalkyl)[ l ,3]dioxanyl, 4-(2,2-dialkyl)[ l ,3]dioxanyl, 4-(2-spiro-cycloalkyl)[ l ,3]dioxanyl,
`4-(2,2-dialkyl)[l,3]-dioxalanyl, or 4-(2-spiro-cycloalkyl)[l,3]dioxalanyl; b - 0-6; d - 0-6; and f - 0-6 with the proviso that RI and R2
`are both not hydrogen and further provided that either R 1 or R2 contains at least one -(CR3R4)tOR10, X, or -(CR3R4)tOR10 substituted
`cycloalkyl group, or a pharmaceutically acceptable salt thereof which is useful as an immunosuppressive, antiinflammatory, antifungal,
`antiproliferative, and antitumor agent.
`
`(I)
`
`...
`'
`
`

`

`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
`applications under the PCT.
`
`AT
`AU
`BB
`BE
`BF
`BG
`BJ
`BR
`BY
`CA
`CF
`CG
`CH
`CI
`CM
`CN
`cs
`CZ
`DE
`DK
`ES
`FI
`FR
`GA
`
`Austria
`Australia
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`COte d'Ivoire
`Cameroon
`China
`Czechoslovakia
`Czech Republic
`Germany
`Denmark
`Spain
`Finland
`France
`Gabon
`
`GB
`GE
`GN
`GR
`HU
`IE
`iT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LI
`LK
`LU
`LV
`MC
`MD
`MG
`ML
`MN
`
`United Kingdom
`Georgia
`Guinea
`Greece
`Hungary
`Ireland
`Italy
`Japan
`Kenya
`Kyrgystan
`Democratic People's Republic
`of Korea
`Republic of Korea
`Kazakhstan
`Liechtenstein
`Sri Lanka
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`Mali
`Mongolia
`
`MR
`MW
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SI
`SK
`SN
`TD
`TG
`TJ
`TT
`UA
`us
`uz
`VN
`
`Mauritania
`Malawi
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Slovenia
`Slovakia
`Senegal
`Chad
`Togo
`Tajikistan
`Trinidad and Tobago
`Ukraine
`United States of America
`Uzbekistan
`Viet Nam
`
`,.
`
`

`

`WO95/28406
`
`PCT/US95/04603
`
`RAPAMYCIN HYDROXYESTERS, PROCESS FOR THEIR PREPARATION AND PHARMACEUTICAL
`COMPOSITIONS CONTAINING THEM
`
`- 1 -
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`BACKGROUND OF THE INVENTION
`This invention relates to hydroxyesters of rapamycin and a method for using
`them for inducing immunosuppression, and in the treatment of transplantation rejection,
`graft vs. host disease, autoimmune diseases, diseases of inflammation, adult T-cell
`leukemia/lymphoma, solid tumors, fungal infections, and hyperproliferative vascular
`disorders.
`
`Rapamycin is a macrocyclic triene antibiotic produced by Streptomyces
`hygroscopicus, 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. Sehgal et al., J. Antibiot. 28, 727 (1975); H. A. Balcer et al., J. Antibiot.
`31, 539 (1978); U.S. Patent 3,929,992; and U.S. Patent 3,993,749].
`Rapamycin alone (U.S. Patent 4,885,171) or in combination with picibanil
`(U.S. Patent 4,401,653) has been shown to have antitumor activity. R. Martel et al.
`[Can. J. Physiol. Pharmacol. 55, 48 (1977)] disclosed that rapamycin is effective in
`the experimental allergic encephalomyelitis model, a model for multiple sclerosis; in the
`adjuvant arthritis model, a model for rheumatoid arthritis; and effectively inhibited the
`formation of IgE-like antibodies.
`The immunosuppressive effects of rapamycin have been disclosed in FASEB 3,
`3411 (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); 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 systemic
`lupus erythematosus [U.S. Patent 5,078,999], pulmonary inflammation [U.S. Patent
`5,080,899], insulin dependent diabetes mellitus [Fifth Int. Conf. Inflamm. Res. Assoc.
`121 (Abstract), (1990)], smooth muscle cell proliferation and intimal thickening
`following 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
`inflammation [European Patent Application 532,862 Al].
`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. Patent
`
`

`

`WO95/28406
`
`PCT/US95/04603
`
`- 2 -
`
`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.
`
`5 DESCRIPTION OF THE INVENTION
`This invention provides derivatives of rapamycin which are useful as
`immunosuppressive, antiinflammatory, antifungal, antiproliferative, and antitumor
`agents having the structure
`
`(I)
`
`10
`
`15
`
`20
`
`25
`
`hydrogen or
`
`independently,
`
`R 1 and R2
`each,
`are
`wherein
`-CO(CR3R4)b(CR5R6)ctCR7R8R9;
`R3 and R4 are each, independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of
`2-7 carbon atoms, alkynyl of 2-7 carbon atoms, trifluoromethyl, or -F;
`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, -(CR3R4)tOR 10, -CF3, -F, or
`-CQiR 11, or R 5 and R6 may be taken together to form X or a cycloalkyl ring of
`3-8 carbon atoms that is optionally mono-, di-, or tri-substituted with
`-(CR3R4)t0RlO;
`R7 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-
`7 carbon atoms, -(CR3R4)tOR10, -CF3, -F, or -CO2R11 ;
`R8 and R9 are each, independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of
`2-7 carbon atoms, alkynyl of2-7 carbon atoms, -(CR3R4)tOR10, -CF3, -F, or
`-CQiRll, or R8 and R9 may be taken together to form X or a cycloalkyl ring of
`
`•
`
`

`

`WO95/28406
`
`PCT/US95/04603
`
`- 3 -
`
`5
`
`3-8 carbon atoms that is optionally mono-, di-, or tri-substituted with
`-(CR3R4)f0R10;
`R 10 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-
`7 carbon atoms, tri-(alkyl of 1-6 carbon atoms)silyl, tri-(alkyl of 1-6 carbon
`atoms)silylethyl, triphenylmethyl, benzyl, alkoxymethyl of 2-7 carbon atoms,
`tri-(alkyl of 1-6 carbon atoms)silylethoxymethyl, chloroethyl, or
`tetrahydropyranyl;
`R11 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-
`7 carbon atoms, orphenylalk:yl of7-10 carbon atoms;
`10 Xis 5-(2,2-di-(alkyl of 1-6 carbon atoms))[l,3]dioxanyl, 5-(2-spiro(cycloalkyl of 3-8
`carbon atoms))[l,3]dioxanyl, 4-(2,2-di-(alkyl of 1-6 carbon atoms))[l,3](cid:173)
`dioxanyl, 4-(2-spiro(cycloalkyl of 3-8 carbon atoms))[l,3]dioxanyl, 4-(2,2-di(cid:173)
`(alkyl of 1-6 carbon atoms))[l,3]dioxalanyl, or 4-(2-spiro(cycloalkyl of 3-8
`carbon atoms))[l,3]dioxalanyl;
`b = 0-6;
`d = 0-6; and
`f=0-6
`with the proviso that R 1 and R2 are both not hydrogen and further provided that either
`RlorR2containsatleastone -(CR3R4)f0Rl0, X, or -(CR3R4)f0Rl0 substituted
`cycloalk:yl of 3-8 carbon atoms group, or a pharmaceutically acceptable salt thereof.
`
`15
`
`20
`
`The pharmaceutically acceptable salts are those derived from such inorganic
`cations such as sodium, potassium, and the like; and organic bases such as: mono-, di-,
`and trialkyl amines of 1-6 carbon atoms, per alkyl group and mono-, di-, and
`trihydroxyalkyl amines of 1-6 carbon atoms per alkyl group, and the like.
`
`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.
`As the compounds of this invention can contain more than one -(CR3R4)f0R 10 group,
`R3, R4, f, and RIO can be the same or different. Similarly, when other generic
`substituent descriptions are repeated in the same structure, they can be the same or
`different.
`For a compound in which R1 contains R8 and R9 taken together to form X,
`where Xis 5-(2,2-di-(alkyl of 1-6 carbon atoms))[l,3]dioxanyl, the alkyl group of X
`contains 1 carbon atom, and d = 0, R 1 would have the following structure.
`
`-
`
`25
`
`30
`
`35
`
`•
`
`

`

`WO95/28406
`
`PCT/US95/04603
`
`- 4 -
`
`- CO(CR3R4)b__rO)<CH3
`
`~O CH3
`R7
`
`Similarly, for a compound in which R1 contains R8 and R9 taken together to form X,
`where Xis 4-(2-spiro(cycloalkyl of 3-8 carbon atoms))[l,3]dioxanyl, the cycloalkyl
`group of X contains 6 carbon atoms, and d = 0, R 1 would have the following structure.
`
`5
`
`10
`
`15
`
`20
`
`25
`
`For compounds containing X, preferred compounds include those in which the alkyl
`group of X, if present, is methyl and the cycloalkyl group of X, if present, is
`cyclohexyl.
`
`When R 10 is not hydrogen, alkyl, alkenyl, or alkynyl, it is intended that R 10 is
`a group that can serve as an alcohol protecting group. Thus, these groups are
`intermediates of free hydroxy lated compounds, as well as being biologically active in
`their own right. RIO covers tri-(alkyl of 1-6 carbon atoms)silyl, tri-(alkyl of 1-6 carbon
`atoms)silylethyl, triphenylmethyl, benzyl, alkoxymethyl of 2-7 carbon atoms, tri-(alkyl
`of 1-6 carbon atoms)silylethoxymethyl, chloroethyl, and tetrahydropyranyl groups.
`Other alcohol protecting groups are known by one skilled in the art and are also
`considered part of this invention.
`
`Of the compounds of this invention preferred members are those in which R2 is
`hydrogen; those in which R2 is hydrogen, b = 0, and d = 0; those in which R2 is
`.hydrogen, b = 0, d = 0, and R8 and R9 are each, independently hydrogen, alkyl, or
`-(CR3R4)tOR 10, or are taken together to form X.
`
`ester group
`the
`invention having
`this
`Compounds of
`-CO(CR3R4)b(CR5R6)ctCR7R8R9 at the 42- or 31,42-positions can be prepared by
`acylation of rapamycin using protected hydroxy and polyhydroxy acids, alkoxy or
`polyalkoxy carboxylic acids that have been activated, followed by removal of the
`
`

`

`WO 95/28406
`
`PCT/US95/04603
`
`- 5 -
`
`alcohol protecting groups, if so desired. Several procedures for carboxylate activation
`are known in the art, but the preferred methods utilize carbodiimides, mixed
`anhydrides, or acid chlorides. For example, an appropriately substituted carboxylic
`acid can be activated as a mixed anhydride, with an acylating group such as 2,4,6-
`trichlorobenzoyl chloride. Treatment of rapamycin with the mixed anhydride under
`mildly basic condition provides the desired compounds. Alternatively, the acylation
`reaction can be accomplished with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
`hydrochloride and dimethylaminopyridine. Mixtures of 42- and 31,42-esters can be
`separated by chromatography.
`
`Accordingly, the invention also provides a process for preparing the rapamycin
`compounds of this invention. In particular this invention provides a process for
`preparing hydroxy esters of rapamycin including those of formula I as defined above
`which comprises:
`a)
`acylating rapamycin or a functional derivative or analogue thereof with an
`acylating agent;
`or
`sequentially acylating rapamycin or a functional derivative or analogue thereof
`b)
`with two acylating agents;
`said acylatiung agent(s) being selected from acids of formula
`
`or a reactive derivative thereof wherein R3-R9, band dare as defined above providing
`that free hydroxy groups are protected, if desired protecting the 42-position of
`rapamycin or functional derivative with an appropriate protecting group and after the
`reaction removing any protecting groups present as required.
`
`The reaction may be carried out in the presence of a coupling reagent, such as a
`suitably substituted carbodiimide coupling reagent. The above-mentioned compounds
`of this invention can also be prepared by acylation using reactive derivatives of the acid
`of formula II such as an anhydride, a mixed anhydride, or a acid halide such as the
`chloride.
`
`The 31-ester-42-hydroxy compounds of this invention can be prepared by
`protecting the 42-alcohol of rapamycin with a protecting group, such as with a tert(cid:173)
`butyl dimethylsilyl group, followed by esterification of the 31-position by the
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`

`

`WO95/28406
`
`PCT/US95/04603
`
`- 6 -
`
`5
`
`procedures described above. The preparation of rapamycin 42-silyl ethers is described
`in U.S. Patent Bl 5,120,842, which is hereby incorporated by reference. Removal of
`the protecting group provides the 31-esterified compounds. In the case of the tert-butyl
`dimethylsilyl protecting group, deprotection can be accomplished under mildly acidic
`conditions, such as acetic acid / water / THF. The deprotection procedure is described
`in Example 15 of U.S. Patent 5,118,678, which is hereby incorporated by reference.
`Having the 31-position esterified and the 42-position deprotected, the 42-
`position can be esterified using a different acylating agent than was reacted with the 31-
`alcohol, to give compounds having different esters at the 31- and 42- positions.
`10 Alternatively, the 42-esterified compounds, prepared as described above, can be reacted
`with a different acylating agent to provide compounds having different esters at the 31-
`and 42-positions.
`
`..
`
`15
`
`20
`
`This invention also covers analogous hydroxy esters of other rapamycins such
`as, but not limited to, 29-demethoxyrapamycin, [U.S. Patent 4,375,464, 32-
`demethoxyrapamycin under C.A. nomenclature]; 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-desmethylrapamycin [U.S. Patent 5,093,339, 32-desmethylrapamycin
`under C.A. nomenclature]; 7,29-bisdesmethylrapamycin [U.S. Patent 5,093,338,
`7,32-desmethylrapamycin under C.A. nomenclature]; and 15-hydroxyrapamycin [U.S.
`Patent 5,102,876]. The disclosures in the above cited U.S. Patents are hereby
`incorporated by reference.
`
`25
`
`Immunosuppressive activity for representative compounds of this invention was
`evaluated in an in vitro standard pharmacological test procedure to measure the
`inhibition of lymphocyte proliferation (LAF) and in two in vivo standard
`pharmacological test procedures. The pinch skin graft test procedure measures the
`immunosuppressive activity of the compound tested as well as the ability of the
`compound tested to inhibit or treat transplant rejection. The adjuvant arthritis standard
`30 · pharmacological test procedure, which measures the ability of the compound tested to
`inhibit immune mediated inflammation. The adjuvant arthritis test procedure is a
`standard pharmacological test procedure for rheumatoid arthritis. The procedures for
`these standard pharmacological test procedures are provided below.
`
`35
`
`The comitogen-induced thymocyte proliferation procedure (LAF) was used as
`an in vitro measure of the immunosuppressive effects of representative compounds.
`
`

`

`WO95/28406
`
`PCT/0S95/04603
`
`- 7 -
`
`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 as percent change in counts per minute from non(cid:173)
`drug treated controls. For each compound evaluated, rapamycin was also evaluated for
`the purpose of comparison. An ICso was obtained for each test compound as well as
`for rapamycin. When evaluated as a comparator for the representative compounds of
`this invention, rapamycin had an ICso ranging from 0.6 -1.5 nM. The results obtained
`are provided as an ICso and as the percent inhibition of T-cell proliferation at 0.1 µM.
`The results obtained for the representative compounds of this invention were also
`expressed as a ratio compared with rapamycin. A positive ratio indicates
`immunosuppressive activity. A ratio of greater than 1 indicates that the test compound
`inhibited thymocyte proliferation to a greater extent than rapamycin. Calculation of the
`ratio is shown below.
`
`ICso of Rapamycin
`IC50 of Test Compound
`
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`Representative compounds 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 transplanted to male C3H(H-2K) 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 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
`treated with either varying concentrations of test compounds intraperitoneally or orally.
`Rapamycin was used as a test control. Untreated recipients serve as rejection control.
`The graft was monitored daily and observations were recorded until the graft became
`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. Compounds were tested using a dose of 4
`35 mg/kg.
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`The adjuvant arthritis standard pharmacological test procedure measures the
`ability of test compounds to prevent immune mediated inflammation and inhibit or treat
`rheumatoid arthritis. The following briefly describes the test procedure used. A group
`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 (mL) from day 16 to
`day 0 is determined and a percent change from control is obtained. The left hind paw
`(uninjected paw) inflammation is caused by T-cell mediated inflammation and is
`recorded in the above table (% change from control). The right hind paw inflammation,
`on the other hand, is caused by nonspecific inflammation. Compounds were tested at a
`dose of 5 mg/kg. The results are expressed as the percent change in the uninjected paw
`at day 16 versus control; the more negative the percent change, the more potent the
`compound. Rapamycin provided between -70% and -90% change versus control,
`indicating that rapamycin treated rats had between 70-90% less immune induced
`inflammation than control rats.
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`The results obtained in these standard pharmacological test procedures are
`provided following the procedure for making the specific compounds that were tested.
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`The results of these standard pharmacological test procedures demonstrate
`immunosuppressive activity both in vitro and in vivo for the compounds of this
`invention. The results obtained in the LAF test procedure indicates suppression of
`T-cell proliferation, thereby demonstrating the immunosuppressive activity of the
`compounds of this invention. Further demonstration of the utility of the compounds of
`this invention as immunosuppressive agents was shown by the results obtained in the
`skin graft and adjuvant arthritis standard pharmacological test procedures.
`Additionally, the results obtained in the skin graft test procedure further demonstrates
`the ability of the compounds of this invention to treat or inhibit transplantation rejection.
`The results obtained in the adjuvant arthritis standard pharmacological test procedure
`further demonstrate the ability of the compounds of this invention to treat or inhibit
`rheumatoid arthritis.
`Based on the results of these standard pharmacological test procedures, the
`compounds are useful in the treatment or inhibition of transplantation rejection such as
`kidney, heart, liver, lung, bone marrow, pancreas (islet cells), cornea, small bowel,
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`and skin allografts, and heart valve xenografts; in the treatment or inhibition of
`autoimmune 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, pulmonary inflammation
`(including asthma, chronic obstructive pulmonary disease, emphysema, acute
`respiratory distress syndrome, bronchitis, and the like), and eye uveitis.
`Because of the activity profile obtained, the compounds of this invention also
`are considered to have antitumor, antifungal activities, and antiproliferative activities.
`The compounds of this invention therefore also useful in treating solid tumors, adult T-
`cell leukemia/lymphoma, fungal infections, and hyperproliferative vascular diseases
`such as restenosis and atherosclerosis. When used for restenosis, it is preferred that
`the compounds of this invention are used to treat restenosis that occurs following an
`angioplasty procedure. When used for this purpose, the compounds of this invention
`can be administered prior to the procedure, during the procedure, subsequent to the
`procedure, or any combination of the above.
`When administered for the treatment or inhibition of the above disease states,
`the compounds of this invention can be administered to a mammal orally, parenterally,
`intranasally, intrabronchially, transdermally, topically, intravaginally, or rectally.
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`It is contemplated that when the compounds of this invention are used as an
`immunosuppressive or antiinflammatory agent, they can be administered in conjunction
`with one or more other immunoregulatory agents. Such other immunoregulatory
`agents include, but are not limited to azathioprine, corticosteroids, such as prednisone
`and methylprednisolone, cyclophosphamide, rapamycin, cyclosporin A, FK-506,
`25 OKT-3, and ATG. By combining the compounds of this invention with such other
`drugs or agents for inducing immunosuppression or treating inflammatory conditions,
`the lesser amounts of each of the agents are required to achieve the desired effect. The
`basis for such combination therapy was established by Stepkowski whose results
`showed that the use of a combination of rapamycin and cyclosporin A at subtherapeutic
`doses significantly prolonged heart allograft survival time. [Transplantation Proc. 23:
`507 (1991)].
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`The compounds of this invention can be formulated neat or with a
`pharmaceutical carrier to a mammal in need thereof. The pharmaceutical carrier may be
`solid or liquid. When formulated orally, it has been found that 0.01 % Tween 80 in
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`PHOSAL PG-50 (phospholipid concentrate with 1,2-propylene glycol, A. Nattermann
`& Cie. GmbH) provides an acceptable oral formulation.
`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 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 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 propellant.
`Liquid pharmaceutical compositions which are sterile solutions or suspensions
`can be utilized by, for example, intramuscular, intraperitoneal or subcutaneous
`injection. Sterile solutions can also be administered intravenously. The compoynd can
`also be administered orally either in liquid or solid composition form.
`The compounds of this invention may be administered rectally in the form of a
`conventional suppository. For administration by intranasal or intrabronchial inhalation
`or insufflation, the compounds of this invention may be formulated into an aqueous or
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`partially aqueous solution, which can then be utilized in the form of an aerosol. The
`compounds of this invention may also be administered transdermally through the use of
`a transdermal patch containing the active compound and a carrier that is inert to the
`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 oil-in-water 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 semipermiable 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 compounds of this invention may be employed as a solution,
`cream, or lotion by formulation with pharmaceutically acceptable vehicles containing
`0.1 - 5 percent, preferably 2%, of active compound which may be administered to a
`fungally affected area.
`The dosage requirements vary with the particular compositions employed, the
`route of administration, the