United States Patent m
`Skotnicki et al.
`
`[54] PHOSPHORYLCARBAMATES OF
`RAPAMYCEN AND OXIME DERIVATIVES
`THEREOF
`
`[75]
`
`Inventors: Jerauld S. Skotnicki, Allentown;
`Andri L. Smith, Princeton, both of
`N J.
`
`[73] Assignee: American Home Products
`Corporation, Madison, N.J.
`
`[21] Appl. No.: 134,428
`
`[22] Filed:
`
`Oct. 8, 1993
`
`[51] Int. Cl.*
`[52] U.S. Q
`[58] Field of Search
`
`A61K 31/395; C07D 491/06
`540/456
`540/456; 514/291
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`3,929,992 12/1975 Sehgal et al
`3,993,749 11/1976 Sehgal etal
`4,316,885 2/1982 Rakhit
`4,375,464 3/1993 Sehgal et al
`4,401,653 8/1983 Eng
`4,650,803 3/1987 Stella et al
`4,885,171 12/1989 Surendraetal
`5,023,262 6/1991 Caufield et al
`• 5,023,263 6/1991 Von Burg
`5,023,264 6/1991 Caufield et al
`5,078,999 1/1992 Warner et al
`5,080,899 1/1992 Sturm et al
`5,091,389 2/1992 Ondeyka et al
`5,100,883 3/1992 Schiehser
`5,100,899 3/1992 Calne
`5,102,876 4/1992 Caufield
`5.118.677 6/1992 Caufield
`5.118.678 6/1992 Kao et al
`5,120,842 6/1992 Failli et al
`5,130,307 7/1992 Failli et al
`5,138,051 8/1992 Hughes et al
`5,151,413 9/1992 Caufield et al
`5,169,851 12/1992 Hughes et al
`5,177,203 1/1993 Failli et al
`5,194,447 3/1993 Kao
`5,221,670 6/1993 Caufield
`5,233,036 8/1993 Hughes
`
`424/122
`424/122
`424/122
`424/122
`424/114
`514/291
`424/122
`514/291
`514/291
`514/291
`424/122
`424/122
`514/291
`514/183
`514/291
`514/183
`514/183
`514/183
`514/452
`514/321
`540/456
`514/63
`514/291
`540/456
`514/542
`514/183
`540/455
`
`US005391730A
`[ii] Patent Number:
`[45] Date of Patent:
`
`5,391,730
`Feb. 21,1995
`
`FOREIGN PATENT DOCUMENTS
`507555A1 7/1992 European Pat. Off.
`
`514/291
`
`OTHER PUBLICATIONS
`Kao et al.. Commonly owned U.S. patent application
`Ser. No. 08/054,655 filed: Apr. 23, 1993.
`Venzina, C. J. Antibiot. 28:721 (1975).
`Sehgal, S. N., J. Antibiot. 28:727 (1975).
`Baker, H. J., Antibiot. 31:539 (1978).
`Martel, R. R., Can. J. Phyisol. Pharmacol. 55:48 (1977).
`Staruch, M. J., FASEB 3:3411 (1989).
`Dumont, F. J., FASEB 3:5256 (1989).
`Calne, R. Y., Lancet 1183 (1978).
`Morris, R. E., Med. Sci. Res. 17:877 (1989).
`Baeder, W. L., Fifth Int. Conf. Inflamm. Res. Assoc.
`121 (Abstract) (1990).
`Meiser, B. M., J. Heart Lung Transplant. 11 (pt. 2): 197
`(1992).
`Stepkowski, S. M. Transplantation Proc. 23:507 (1991).
`
`Primary Examiner—Robert T. Bond
`Attorney, Agent, or Firm—Arnold S. Milowsky
`
`ABSTRACT
`[57]
`A compound of the structure
`
`(Abstract continued on next page.)
`
`West-Ward Pharm.
`Exhibit 1047
`Page 001
`
`

`

`5,391,730
`Page 2
`
`wherein R1 and R2 are each, independently, hydro(cid:173)
`gen, or
`
`O OR3
`O
`II
`. 11/
`-C—NR5—P
`
`;
`OR4
`
`R3 and R4 are each, mdependently, hydrogen, Ar, or
`— ( C R ^ V ^ C R S R ^ i Z, or R3 « ^ « 4m ay be taken
`together to form a 5-7 membered ring;
`R5 is hydrogen, alkyl, alkenyl, alkynyl, alkoxyalkyl,
`arylalkyl, cycloalkyl, or Ar;
`R6, R7, R8, and R9, are each, independently, hydro(cid:173)
`gen, alkyl, alkenyl, alkynyl, hydroxyalkyl, alkoxy(cid:173)
`alkyl, alkylthioalkyl, alkylaminoalkyl, dialkylami(cid:173)
`noalkyl, arylalkyl, cycloalkyl, —OR10, —SR10,
`halogen, —CN, —NO2, —CF3, —COR10,
`—CO2R10, — CONHRl0, —SO2R10, —SO3R10,
`—OSO3RI0, — NRIOR", —NHCORi0, —NH-
`CO2R10, —NHSO2R10, —NHSO3R10 or Ar;
`
`X is 0 or NOR12;
`Y is —O— —CH2—, —NR13—, —S—, —S(0)—,
`- S ( 0 ) 2 -, or - C ( 0 ) -;
`R10, R11, R12, and R13 are each, independently, hy(cid:173)
`drogen, alkyl, or arylalkyl;
`Z is hydrogen, alkyl of 1-6 carbon atoms, or Ar;
`Ar is aryl which may be optionally mono-, di-, or
`tri-substituted;
`a = l -6 and;
`b=0-6;
`or a pharmaceutically acceptable salt thereof, with
`the proviso that R1 and R2 are not both hydrogen;
`and further provided that when a is greater than 1,
`each of the (CR6R7) subunits may be the same or
`different and when b is greater than 1, each of the
`(CR8R9) may be the same or different which is
`useful as an immunosuppressive, antiinflammatory,
`antifungal, antiproliferative, and antitumor agent.
`
`9 Claims, No Drawings
`
`West-Ward Pharm.
`Exhibit 1047
`Page 002
`
`

`

`5,391,730
`
`PHOSPHORYLCARBAMATES OF RAPAMYCIN
`AND OXIME DERIVATIVES THEREOF
`
`OR1
`
`10
`
`15
`
`20
`
`25
`
`BACKGROUND OF THE INVENTION
`This mvention relates to phosphorylcarbamates of
`rapamycin and oxime derivatives thereof and a method
`for using them for inducing immunosuppression, and in
`the treatment of transplantation rejection, host vs. graft
`disease, autoimmune diseases, diseases of inflammation,
`solid tumors, fungal infections, and hyperproliferative
`vascular disorders.
`Rapamycin is a macrocyclic triene antibiotic pro(cid:173)
`duced 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. Baker et al., J. Antibiot. 31,539
`(1978); U.S. Pat. No. 3,929,992; and U.S. Pat. No.
`3,993,749].
`Rapamycin alone (U.S. Pat. No. 4,885,171) or in com(cid:173)
`bination with picibanil (U.S. Pat. No. 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 en(cid:173)
`cephalomyelitis 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 rejec(cid:173)
`tion [FASEB 3, 3411 (1989); FASEB 3, 5256 (1989); R.
`Y. Calne et al., Lancet 1183 (1978); and U.S. Pat. No.
`5,100,899].
`Rapamycin has also been shown to be useful in pre(cid:173)
`venting or treating systemic lupus erythematosus [U.S.
`Pat. No. 5,078,999], pulmonary inflammation [U.S. Pat.
`No. 5,080,899], insulin dependent diabetes mellitus
`[Fifth Int. Conf. Inflamm. Res. Assoc. 21 (Abstract),
`(1990)], and smooth muscle cell proliferation and inti-
`mal thickening following vascular injury [Morris, R. J.
`Heart Lung Transplant 11 (pt. 2): 197 (1992)].
`Mono- and diacylated derivatives of rapamycin (es(cid:173)
`terified at the 28 and 43 positions) have been shown to
`be useful as antifungal agents (U.S. Pat. No. 4,316,885)
`and used to make water soluble prodrugs of rapamycin
`(U.S. Pat. No. 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-posi-
`tions. U.S. Pat. No. 5,118,678 discloses carbamates of 55
`rapamycin that are useful as immunosuppressive, anti(cid:173)
`inflammatory, antifungal, and antitumor agents. U.S.
`Pat. No. 5,194,447 discloses sulfonyl carbamates useful
`as immunosuppressive, anti-inflammatory, antifungal,
`and antitumor agents. U.S. Pat. No. 5,023,264 discloses 60
`oximes of rapamycin useful as immunosuppressive, anti(cid:173)
`inflammatory, and antifungal agents.
`
`30
`
`35
`
`40
`
`45
`
`50
`
`DESCRIPTION OF THE INVENTION
`This mvention provides derivatives of rapamycin 65
`which are useful as immunosuppressive, antiinflamma(cid:173)
`tory, antifungal, antiproliferative, and antitumor agents
`having the stmcture
`
`wherein R1 and R2 are each, independently, hydro(cid:173)
`gen, or
`
`O OR3
`O
`. 11/
`II
`—C—NR5—P
`
`;
`OR4
`
`R3 and R4 are each, mdependently, hydrogen, Ar, or
`—(CRSR'OaY^RSR^Z, or R ^ - R4m ay be taken
`together to form a 5-7 membered ring;
`R5 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of
`2-7 carbon atoms, alkoxyalkyl of 2-7 carbon
`atoms, arylalkyl of 7-10 carbon
`atoms, cycloalkyl of 3-8 carbon atoms, or Ar;
`R6, R7, R8, and R9, are each, independently, hydro(cid:173)
`gen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 car(cid:173)
`bon atoms, alkynyl of 2-7 carbon atoms, hydroxy(cid:173)
`alkyl of 1-6 carbon atoms, alkoxyalkyl of 2-12
`carbon atoms, alkylthioalkyl of 2-12 carbon atoms,
`alkylaminoalkyl of 2-12 carbon atoms, dialkylami(cid:173)
`noalkyl of 3-12 carbon atoms, arylalkyl of 7-10
`carbon atoms, cycloalkyl of 3-8 carbon atoms,
`—OR10 —SR10 halogen, —CN, —NO2 , — CF3,
`—COR10 —CO2R10, —CONHR10 —SO2R10
`—SO3R10 —OSO3R10, —NR^R11, —NHCOR10,
`—NHCO2R10, —NHSO2R10 — NHSO3R10, or
`Ar;
`X is O or NOR12;
`is —O—,—CH2—,—NR13—, — S—,—S-
`Y
`( 0 ) - , - S ( 0 ) 2 -, or - C ( 0 ) -;
`R10, R11, R12, and R13 are each, independently, hy(cid:173)
`drogen, alkyl of 1-6 carbon atoms, or arylalkyl of
`7-10 carbon atoms;
`Z is hydrogen, alkyl of 1-6 carbon atoms, or Ar;
`Ar is aryl which may be optionally mono-, di-, or
`tri-substituted with a group selected from alkyl of
`1-6 carbon atoms, arylalkyl of 7-10 carbon atoms,
`alkoxy of 1-6 carbon atoms, cyano, halo, hydroxy,
`nitro, carbalkoxy of 2-7 carbon atoms, trifluoro(cid:173)
`methyl, amino, dialkylamino of 1-6 carbon atoms
`per alkyl group, dialkylaminoalkyl of 3-12 carbon
`atoms, hydroxyalkyl of 1-6 carbon atoms, alkoxy(cid:173)
`alkyl of 2-12 carbon atoms, alkylthio of 1-6 carbon
`atoms, —SO3H, —PO3H, and —CO2H;
`a= 1-6 and;
`b=0-6;
`
`West-Ward Pharm.
`Exhibit 1047
`Page 003
`
`

`

`5,391,730
`
`_jf
`,
`,
`O2N—<
`RAP—OH
`
`/
`
`>
`o
`\_
`, -OCCI
`)—c
`
`4
`3
`protecting group, deprotection can be accomplished
`or a pharmaceutically acceptable salt thereof, with
`the proviso that R1 and R2 are not both hydrogen;
`under mildly acidic conditions. The protection and
`deprotection of the 42-hydroxyl group of rapamycin
`and further provided that when a is greater than 1,
`each ofthe (CR«R7) subunits may be the same or
`Was disclosed in U.S. Pat. No. 5,120,842 , which is
`different and when b is greater than 1, each ofthe 5 hereby incorporated by reference.
`(CR8R9) may be the same or different.
`Having the 31-position carbamylated and the 42-posi-
`It is prefened that the aryl moiety of the Ar group or
`tion deprotected, the 42-position can be reacted with a
`of the arylalkyl group is a phenyl, naphthyl, pyridyl,
`different
`isocyanate than was reacted with the 31-
`qumolyl, isoquinolyl, quinoxalyl, thienyl, thionaphthyl,
`alcohol, to give compounds having different carba-
`furyl, benzofuryl benzodioxyl, benzoxazolyl, ben- 10 m a teS at the 31- and 42-positions. Aitematively, the
`zoisoxazolyl indolyl, thiazolyl, isoxazolyl, pyrimidinyl,
`42-carbamylated compounds, prepared as described
`pyrazmy, benzopyranyl benz[b]thiophenolyl, ben-
`i soC y a n a te to
`a b o v e) c an be
`r e a c t ed w i th a d i f f e r e nt
`zumdazolyl benzthiazolyl, benzodioxolyl, pipendyl,
`i de Compounds having different carbamates at the
`morpholinyl, piperazinyl, tetrahydrofuranyl, or pyr-
`31 . ^d 42-positions
`rolidinyl group which may be optionally mono- di- or 15
`F or t he Comp0 u n ds 0f t his in v e nt i0„ in which R5 is
`£ " £T
`^ , 1 ^?
`fn
`f
`T
`^l
`%
`hydrogenorisasubstituentotherthanhydrogen,carba-
`carbonatoms, arylalkyl of 7-10 carbon atoms, alkoxy of
`_ *
`A A AU
`-,,
`u c
`A A AU
`,1
`3
`1
`J
`,
`,-
`.
`-A ""•""J'™
`mates can be formed at the 42 - and at the 31 - and
`, 1 u A
`1-6 carbon atoms, cyano, halo, hydroxy, nitro, carbalk-
`AI
`-A-
`u
`r A
`™, nf 1 n ~o..i 1 .•« I A -a
`AU 1
`•
`J -I
`42-positions by first convenmg rapamycin to a carbon-
`oxy of 2-7 carbon atoms, tnfluoromethyl, amino, dial-
`„ . „.
`„„„,.•
`•
`-.u
`-A UI
`UI
`C
`kylamino of 1-6 carbon atoms per alkyl group, dialkyl- 20 a t e
`by T
`g
`rfp a ,Py C 1" ^th % SUltfle. ?1
`hl0rO,f?.r-
`t
`aminoalkyl of 3-12 carbon a t o L, hydroxyalkyl of 1-6
`m a t e;. ^ ch ^P-mtrophenyl chloroformate, followed by
`ff *10" of J6 f o n a te with an appropnately substi-
`carbon atoms, alkoxyalkyl of 2-12 carbon atoms, alkyl-
`thio of 1-6 carbon atoms, - S O 3 H, - P O 3 H, and
`^
`Phosphoramidate amon, as shown in the scheme
`—CO2H. It is more prefened that the aryl moiety is a
`e o w'
`phenyl group that may be optionally substituted as de- 25
`scribed above.
`When R3 and R4 are defmed as being taken together
`to form a 5-7 membered ring, it is prefened that R3R4
`are _ ( C H 2 ) 2-
`- ( C H 2 ) 3- or _ ( C H 2 ) 4 -.
`W h en X is N O R1 3, t he 27-oxime can exist in both the 30
`E and the Z forms; this disclosure covers b o th of these
`forms.
`The pharmaceutically acceptable salts are those de-
`rived from such inorganic cations such as sodium, po-
`tassium, and the like; organic bases such as: mono-, di-, 35
`and trialkyl amines of 1-6 carbon atoms, per alkyl group
`and mono-, di-, and trihydroxyalkyl amines of 1-6 car(cid:173)
`bon atoms per alkyl group, and the like; and organic and
`inorganic acids as: acetic, lactic, citric, tartaric, suc(cid:173)
`cinic, maleic, malonic, gluconic, hydrochloric, hydro- 40
`bromic, phosphoric, nitric, sulfuric, methanesulfonic,
`and similarly known acceptable acids.
`Ofthese compounds, prefened members are those in
`which R3 and R4 are alkyl of 1-6 carbon atoms; those in
`which R5 is hydrogen; those in which R2 and R5 are 45 B Ap _n
`hydrogen; and those in which R2 and R5 are hydrogen
`and R3 and R4 are alkyl of 1-6 carbon atoms.
`The compounds of this invention in which R5 is hy(cid:173)
`The phosphoramidate anion can be generated by
`drogen, that are carbamylated at the 42-position or at
`both the 31- and 42-positions can be prepared by react- 50 treating the appropriate phosphoramidate with a strong
`ing rapamycin with an isocyanate having the general
`base, such as sodium hydride or lithium diisopropyla-
`structure
`mide, at low temperatures, typically —78° C. The 31-
`carbamylated compounds of this invention can be pre-
`pared using this route by first protecting the 42-position
`55 as described above, followed by conversion of the 31-
`hydroxyl group to a carbonate and subsequent treat-
`ment with a phosphoramidate anion.
`For the compounds of this invention in which X is
`NOR12, the oximation of the 27-ketone of rapamycin
`60 can be accomplished following the carbamylation by
`treatment of the rapamycin phosphonylcarbamate with
`an appropriately substituted hydroxylamine, as dis-
`closed in U.S. Pat. No. 5,023,264 , which is hereby
`incorporated by reference.
`The isocyanates, phosphoramidates, and hydroxyl-
`amines used to prepare the compounds of the invention
`am commercially available or can be prepared by meth-
`ods that are disclosed in the literature.
`
`either in the presence of a base, such as pyridine, or in
`the absence of a base.
`The 3 1-carbamylated compounds ofthis invention in
`which R5 is hydrogen can be prepared by protecting the
`42-alcohol of rapamycin with a protecting group, such
`as with a tert-butyl dimethylsilyl group, followed by
`carbamylation of the 31-position with an isocyanate 65
`with the general structure shown above. Removal of
`the protecting group provides the 31-carbamylated
`compounds. In the case of the tert-butyl dimethylsilyl
`
`^= = =/
`
`^
`
`/,
`o
`RAP—oco—^
`\
`
`\
`5—NO2
`/
`
`00
`!!,_M1,5_J_r,p3
`\
`OR4
`
`0
`3
`_ _ _ l l_
`O—c—N P ^ OR
`OR4
`
`West-Ward Pharm.
`Exhibit 1047
`Page 004
`
`

`

`5,391,730
`
`a comparator for the representative compounds of this
`invention, rapamycin had an IC50 ranging from 0.4-5.1
`nM. The results obtained are provided as an IC50 and as
`the percent inhibition of T-ceU proliferation at 0.1 fiM.
`The results obtained for the representative compounds
`of this mvention were also expressed as a ratio com(cid:173)
`pared with rapamycin. A positive ratio indicates immu(cid:173)
`nosuppressive activity. A ratio of greater than 1 indi(cid:173)
`cates that the test compound inhibited thymocyte pro(cid:173)
`liferation to a greater extent than rapamycin. Calcula(cid:173)
`tion of the ratio is shown below.
`
`10
`
`This invention also covers analogous carbamates of
`other rapamycins such as, but not limited to, 29-deme-
`thoxyrapamycin, [U.S. Pat. No. 4,375,464, 32-deme-
`thoxyrapamycin under C A. nomenclature]; rapamycin
`derivatives in which the double bonds in the 1 -, 3 -,
`and/or 5-positions have been reduced [U.S. Pat. No.
`5,023,262]; 42-oxorapamycin [U.S. Pat. No. 5,023,262];
`29-desmethylrapamycm [U.S. Pat. No. 5,093,339, 32-
`desmethylrapamycin under C A. nomenclature]; 7,29-
`bisdesmethylrapamycin [U.S. Pat. No. 5,093,338, 7,32-
`desmethylrapamycin under C A. nomenclature]; and
`15-hydroxy- and 15,27-bishydroxy-rapamycin [U.S.
`Pat. No. 5,102,876]. The disclosures in the above cited
`U.S. Patents are hereby incorporated by reference.
`This invention additionally covers derivatives of 15
`rapamycin in which one of the 31 - or 42-hydroxyl
`groups has been converted to a phosphorylcarbamate,
`as described above, and the other of the 31 - or 42-
`hydroxyl groups has been esterified with a moiety that
`is not a phosphonylcarbamate. Such other esters include 20
`acyl derivatives of rapamycin as described in U.S. Pat.
`No. 4,316,885, which is hereby incorporated by refer(cid:173)
`ence; fluorinated esters of rapamycin as described in
`U.S. Pat. No. 5,100,883, which is hereby mcorporated
`by reference; amide esters of rapamycin as described in
`U.S. Pat. No. 5,118,677^ which is hereby incorporated
`by reference; carbamates of rapamycin as described in
`U.S. Pat. No. 5,118,678, which is hereby mcorporated
`by reference; aminoesters of rapamycin as described in
`U.S. Pat. No. 5,130,337, which is hereby incorporated
`by reference; ethers and acetals of rapamycin as de(cid:173)
`scribed in U.S. Pat. No. 5,151,413, which is hereby
`incorporated by reference; aminoacyl esters of rapamy(cid:173)
`cin as described in U.S. Pat. No. 4,650,803, which is
`hereby incorporated by reference; sulfonates and sulfa-
`mates of rapamycin as described in U.S. Pat. No.
`5,117,203; silyl ethers of rapamycin as described in U.S.
`Pat. No. 5,120,842, which is hereby incorporated by
`reference; and sulfonylcarbamates of rapamycin as de(cid:173)
`scribed in U.S. Pat. No. 5,194,447, which is hereby
`incorporated by reference. Similarly, this invention
`covers compounds in which one hydroxyl of rapamycin
`has been converted to a phosphonyl carbamate and the
`other hydroxyl is an inorganic ester of the hydroxyl
`group. These esters mclude phosphate, nitrate, sulfinate, 45
`sulfonate esters, and the like, and organic esters ofthese
`morganic acids.
`Immunosuppressive activity for representative com(cid:173)
`pounds of this invention was evaluated in an in vitro
`standard pharmacological test procedure to measure
`lymphocyte proUferation (LAF) and in an in vivo stan(cid:173)
`dard pharmacological test procedure which evaluated
`the survival time of a pinch skin graft.
`The comitogen-induced thymocyte proliferation pro(cid:173)
`cedure (LAF) was used as an in vitro measure of the
`immunosuppressive effects of representative com(cid:173)
`pounds. Briefly, cells from
`the thymus of normal
`BALB/c mice are cultured for 72 hours with PHA and
`IL-1 and pulsed with tritiated thymidme 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 radio(cid:173)
`activity is detennined. Inhibition of lymphoprolifera-
`tion is assessed as percent change in counts per minute
`from nondrug treated controls. For each compound
`evaluated, rapamycin was also evaluated for the pur(cid:173)
`pose of comparison. An IC50 was obtained for each test
`compound as well as for rapamycin. When evaluated as
`
`50
`
`55
`
`60
`
`65
`
`25
`
`30
`
`35
`
`40
`
`/C50 of Rapamycin
`/C50 of Test Compound
`
`Representative compounds of this mvention 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 concentra(cid:173)
`tions of test compounds intraperitoneally or orally.
`Rapamycin was used as a test control. Untreated recipi(cid:173)
`ents 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 drag treatment
`group was compared with the control group. The fol(cid:173)
`lowing table shows the results that were obtained. Re(cid:173)
`sults are expressed as the mean survival time in days.
`Untreated (control) pinch skin grafts are usually re(cid:173)
`jected within 6-7 days. The results shown in Table 1 are
`based on a dose of 4 mgAg of test compound. A sur(cid:173)
`vival time of 12.0± 1.7 days was obtained for rapamycin
`at 4 mgAg.
`
`The followmg table summarizes the results of repre(cid:173)
`sentative compounds of this invention in these two
`standard test procedures.
`TABLE 1
`EVALUATION OF IMMUNOSUPPRESSIVE ACTIVITY*
`L AF
`Skin Graft
`(ratio)
`(days ± SD)
`0.04
`7.2 ± 0.4
`10.2 ± 0.4
`0.44
`10.0 ± 0.6
`
`Compound
`Example 1
`Example 2
`
`ICsoCnM)
`90.9
`10.0
`
`% Inhib.+
`72
`95
`
`99.0
`Example 3
`0.05
`41.9
`Example 4
`0.01
`•Calculation of the ratio was described supra.
`+Percent mhibition of T-cell proliferation at 0.1 jiM.
`
`51
`82
`
`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 L AF test proce(cid:173)
`dure
`indicates suppression of T-cell proliferation,
`thereby demonstrating the immunosuppressive activity
`of the compounds of this invention. The results ob(cid:173)
`tained for representative compounds ofthis invention in
`preventing skin graft rejection further demonstrates
`their utihty as immunosuppressive agents.
`Based on the results of these standard pharmacologi(cid:173)
`cal test procedures, the compounds are useful in the
`
`West-Ward Pharm.
`Exhibit 1047
`Page 005
`
`

`

`5,391,730
`
`8
`7
`cellulose solution), alcohols (including monohydric
`treatment or prevention of transplantation rejection
`alcohols and polyhydric alcohols, e.g. glycols) and their
`such as kidney, heart, liver, lung, bone marrow, pan-
`derivatives, and oils (e.g. fractionated coconut oil and
`creas (islet cells), cornea, small bowel, and skin alio-
`arachis oil). For parenteral administration, the carrier
`grafts, and heart valve xenografts; in the treatment of
`autoimmune diseases such as lupus, rheumatoid arthri- 5 can also be an oily ester such as ethyl oleate and isopro-
`tis, diabetes mellitus, myasthenia gravis, and multiple
`pyl myristate. Sterile liquid carriers are useful in sterile
`sclerosis; and diseases of inflammation such as psoriasis,
`hquid form compositions for parenteral administration,
`dermatitis, eczema, seborrhea, inflammatory bowel
`The liquid carrier for pressurized compositions can be
`disease, and eye uveitis.
`halogenated hydrocarbon or other pharmaceutically
`Based on the activity profile obtained, the com- 10 acceptable propellant.
`pounds of this invention also are considered to have
`Liquid pharmaceutical compositions which are sterile
`antitumor, antifungal activities, and antiproliferative
`solutions or suspensions can be utilized by, for example,
`activities. The compounds of this invention therefore
`intramuscular, intraperitoneal or subcutaneous injec-
`also useful in treating solid tumors, fungal infections,
`tion. Sterile solutions can also be administered intrave-
`and hyperprohferative vascular diseases such as reste- 15 nously. The compound can also be administered orally
`nosis and atherosclerosis.
`either in liquid or solid composition form.
`It is contemplated that when the compounds of this
`The compounds of this invention may be adminis-
`invention are used as an immunosuppressive or antiin-
`tered rectally in the form of a conventional suppository,
`flammatory agent, they can be administered in conjunc-
`For administration by intranasal or intrabronchial inha-
`tion with one or more other immunoregulatory agents. 20 lation or insufflation, the compounds of this invention
`Such other immunoregulatory agents include, but are may be formulated into an aqueous or partially aqueous
`not limited to azathioprine, corticosteroids, such as
`solution, which can then be utilized in the form of an
`prednisone and methylprednisolone, cyclophospha-
`aerosol. The compounds of this invention may also be
`mide, rapamycin, cyclosporin A, FK-506, OKT-3, and
`administered transdermaUy through the use of a trans-
`ATG. By combining the compounds of this invention 25 dermal patch containing the active compound and a
`with such other drags or agents for inducing immuno-
`carrier that is inert to the active compoimd, is non toxic
`suppression or treating inflammatory conditions, the
`to the skin, and allows delivery of the agent for systemic
`lesser amounts of each of the agents are required to
`absorption into the blood stream via the skin. The car-
`achieve the desired effect. The basis for such combina-
`rier may take any number of forms such as creams and
`tion therapy was established by Stepkowski whose re- 30 ointments, pastes, gels, and occlusive devices. The
`suits showed that the use ofa combination of rapamycin
`creams and ointments may be viscous liquid or semi-
`and cyclosporin A at subtherapeutic doses significantly
`solid emulsions of either the oil-in-water or water-in-oil
`prolonged heart allograft survival time. [Transplanta-
`type. Pastes comprised of absorptive powders dispersed
`tion Proc. 23: 507 (1991)].
`in petroleum or hydrophilic petroleum containing the
`The compounds of this invention can be formulated 35 active ingredient may also be suitable. A variety of
`neat or with a pharmaceutical carder to a mammal in
`occlusive devices may be used to release the active
`need thereof. The pharmaceutical carder may be solid
`ingredient into the blood stream such as a semipermia-
`or liquid.
`ble membrane covering a reservoir containing the ac-
`A solid carrier can include one or more substances
`tive ingredient with or without a carrier, or a matrix
`which may also act as flavoring agents, lubricants, solu- 40 containing the active mgredient. Other occlusive de-
`bilizers, suspending agents, fillers, ghdants, compression
`vices are known in the literature,
`aids, binders or tablet-disintegrating agents; it can also
`In addition, the compounds of this invention may be
`be an encapsulating material. In powders, the carder is
`employed as a solution, cream, or lotion by formulation
`a finely divided solid which is in admixture with the
`with pharmaceutically acceptable vehicles containing
`finely divided active ingredient. In tablets, the active 45 0.1-5 percent, preferably 2%, of active compound
`ingredient is mixed with a carrier having the necessary
`which may be administered to a frugally affected area,
`compression properties in suitable proportions and
`The dosage requirements vary with the particular
`compacted in the shape and size desired. The powders
`compositions employed, the route of administration, the
`and tablets preferably contain up to 99% of the active
`severity of the symptoms presented and the particular
`ingredient. Suitable sohd carriers include, for example, 50 subject being treated. Based on the results obtained in
`calcium phosphate, magnesium stearate, talc, sugars,
`the standard pharmacological test procedures, pro-
`lactose, dextrin, starch, gelatin, cellulose, methyl cellu-
`jected daily dosages of active compound would be 0.1
`lose, sodium carboxymethyl cellulose, polyvinylpynol-
`jugAg-lOO mgAg, preferably between 0.001-25
`idone, low melting waxes and ion exchange resins.
`mgAg, and more preferably between 0.01-5 mgAg.
`Liquid carriers are used in preparing solutions, sus- 55 Treatment will generally be initiated with small dosages
`pensions, emulsions, syrups, elixirs and pressurized
`less than the optimum dose of the compound. Thereaf-
`compositions. The active mgredient can be dissolved or
`ter the dosage is increased until the optimum effect
`suspended in a pharmaceutically acceptable liquid car-
`under the circumstances is reached; precise dosages for
`rier such as water, an organic solvent, a mixture of both
`oral, parenteral, nasal, or intrabronchial administration
`or pharmaceutically acceptable oils or fats. The liquid 60 will be determined by the administering physician based
`carrier can contain other suitable pharmaceutical addi-
`on experience with the individual subject treated. Pref-
`tives such as solubilizers, emulsifiers, buffers, preserva-
`erably, the pharmaceutical composition is in unit dosage
`tives, sweeteners, flavoring agents, suspending agents,
`form, e.g. as tablets or capsules. In such form, the com-
`thickening agents, colors, viscosity regulators, stabiliz-
`position is sub-divided in unit dose containing appropri-
`ers or osmo-regulators. Suitable examples of liquid car- 65 ate quantities of the active ingredient; the unit dosage
`riers for oral and parenteral administration include
`forms can be packaged compositions, for example,
`water (partially containing additives as above, e.g. cel-
`packeted powders, vials, ampoules, prefilled syringes or
`lulose derivatives, preferably sodium carboxymethyl
`sachets containing liquids. The unit dosage form can be,
`
`West-Ward Pharm.
`Exhibit 1047
`Page 006
`
`

`

`5,391,730
`
`Rapamycin 42-ester with
`(diethoxyphosphoryl)carbamic acid
`(E)-27-0-(methyl)oxime
`To a solution of rapamycin 42-ester with (diethoxy-
`phosphoryl) carbamic acid (860 mg; 0.79 mmol) in 6 ml
`methanol was added methoxylamine hydrochloride (66
`
`10
`9
`mg; 0.79 mmol) and sodium aceta (65 mg; 0.79 mmol).
`for example, a capsule or tablet itself, or it can be the
`The pale yellow reaction mixture was stirred at ambi-
`appropriate number of any such compositions in pack-
`dent temperature overnight and was monitored by thin
`age form.
`ia y er chromatography. The following morning distilled
`The followmg examples illustrate the preparation of
`5 w at er and ethyl acetate were added to the reaction
`representative compounds ofthis invention.
`mixture. The aqueous layer was extracted with ethyl
`EXAMPLE 1
`acetate five times. The combined organic layers were
`-AU
`«
`• ni
`A*
`A-
`A
`J -J
`Rapamycin 31,42-diester with
`A-
`IC A CIA
`A
`A
`A A A-
`dned over sodium sulfate, filtered, and concentrated m
`-A
`,,- AU
`U
`u
`I\
`u
`•
`. ,,
`,.
`, . ,—
`.-
`^
`(diethoxyphosphoryl)carbamic acid
`vacuo to yield a white solid. Punfication was accom-
`To a solution of rapamycin (1.0 g; 1.1 mmol) in 5 ml 10 plished by high performance hquid chromatography
`anhydrous methylene chloride was added diethylphos-
`(acetonitrile/water, 55% to 65% gradient; reversed
`phinyl isocyanate (0.72 g; 4.9 mmol) via syringe. The
`h
`! Coiumn), producing the pure +E"isomer
`h a se
`reaction mixture was allowed to stir under a mtrogen
`o x i me ^
`of ^
`a w h i te s o l id
`( 1 20
`2 0%
`i e ld
`atmosphere at 0 C. for one hour and then concentrated
`a c eto n itrile/water, 70:30, RP-8).
`R f =0 27 .
`'
`under reduced pressure. The crude product was pun- 15
`'
`fied by flash c^omatography (ethyl acetate, followed
`»
`(KBr) 3400, 2930, 1740, 620, 1450, 1370, 1325,
`by 4% methanol in ethyl acetate, silica), affording the
`1290-1240, 1200, 1165, 1 00-1010, 990, 885 c m - l.
`N^ C400 M H z; D M S O> l H: 6 9-23 (d' 1 H' Pr o t on on
`tille compound as a white solid (0.76 g/55% yield).
`IR (KBr) 3400, 2910, 1740, 1645, 1460, 1020 c m - l.
`nitrogen of carbamate at C-42) 5.27 (d, IH, H-29 result-
`NMR (400 MHz; DMSO) 1H: 89.4-9.18 (2-m, 2H, pro- 20 ing from E-methoxime at C-27) 4.46 (m, IH, H-42 re-
`tons on nitrogen of carbamate at C-42 and C-31) 5.25
`suiting from carbamate at C-42) 4.00 (m, 4H, methylene
`(m, lH,H-28 resulting from carbamate at C-28) 4.37 (m,
`protons of diethoxyphosphoryl at C-42) 3.68 (s, 3H,
`IH, H-42 resulting from carbamate at C-42) 4.08-3.96
`methoxime methyl protons at C-27) 3.14 (d, IH, H-28
`(m, 8H, methylene protons of diethoxyphosphoryl at
`resulting from E-methoxime wrt C-27) 1.23 (t, 6H,
`C-42 and C-31 ) 1.25-1.15 (m, 12H, methyl protons of 25 methyl protons of diethoxyphosphoryl at C-42) C: S158
`diethoxyphosphoryl at C-42 and C-31) MS (neg. ion
`(C-27) 153 (carbamate carbonyl carbon at C-42) 63
`FAB) m/z: 1271 ( M - ), 912, 894, 590, 331.
`(methylene carbon of diethoxyphosphoryl at C-42) 61
`EXAMPLE 2
`(methoxime methyl carbon at C-27) MS (neg. ion FAB)
`m/z: 1121
`( M - ), 942, 590, 297. Analysis
`for
`30 C57H92N3O17P. Calculated: C, 61.00;