United States Patent [19]
`Skotnicki et al.
`
`[54] RAPAMYCENJ HYDROXYESTERS
`
`[75]
`
`Inventors: Jerauld S. Skotnicki, Allentown;
`Christina L. Leone, Princeton, both
`of N.J.; Guy A. Schiehser, Yardley,
`Pa.
`
`[73] Assignee: American Home Products
`Corporation, Madison, N J.
`
`[21] Appl. No.: 229,261
`
`[22] FUed:
`
`Apr. 18,1994
`
`[51] Int. a .5
`
`[52] U.S. a
`
`[58] Field of Search
`
`A61K 31/695; A61K 31/395;
`C07D 498/16; C07D 7/04
`514/63; 514/291;
`540/452; 540/456
`540/456, 452; 514/291,
`514/63
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`Sehgal et al
`Sehgal et al
`Rakhit
`Sehgal et al
`Eng
`Stella et al
`Surendra et al
`Caufield et al
`Von Burg
`Caulfield etal
`Warner et al
`Sturm etal
`Ondeyka et al
`Schiehser
`Caine
`Caufield
`Caufield
`Kao etal
`Failli et al
`Failli et al
`Hughes et al
`Caufield et al
`Hughes etal
`Failli et al
`Kao
`Caufield
`Hughes
`
`12/1975
`3,929,992
`11/1976
`3,993,749
`2/1982
`4,316,885
`4,375,464
`3/1983
`8/1983
`4,401,653
`3/1987
`4,650,803
`4,885,171
`12/1989
`6/1991
`5,023,262
`6/1991
`5,023,263
`6/1991
`5,023,264
`1/1992
`5,078,999
`1/1992
`5,080,899
`2/1992
`5,091,389
`3/1992
`5,100,883
`3/1992
`5,100,899
`4/1992
`5,102,876
`6/1992
`5,118,677
`6/1992
`5,118,678
`6/1992
`5,120,842
`5,130,307
`7/1992
`8/1992
`5,138,051
`9/1992
`5,151,413
`12/1992
`5,169,851
`1/1993
`5,177,203
`3/1993
`5,194,447
`6/1993
`5,221,670
`8/1993
`5,233,036
`
`424/122
`424/122
`424/122
`424/122
`424/124
`540/456
`424/122
`540/456
`540/456
`514/291
`424/122
`424/122
`514/291
`514/183
`514/291
`514/18.3
`514/183
`514/183
`540/452
`514/291
`540/456
`540/456
`514/291
`540/456
`540/456
`514/183
`540/456
`
`iiiwiiiiiiiiiiiiiiiiiiiiiiniiiiiiiiiiiiiiiiiiiiiiii
`US005362718A
`[ii] Patent Number:
`5,362,718
`[45] Date of Patent:
`Nov. 8, 1994
`
`5,260,300 11/1993 Hu
`5,262,423 11/1993 Kao
`5.286.730 2/1994 Caufield et al
`5.286.731 2/1994 Caufield et al
`5,302,584 4/1994 Kao et al
`
`540/456
`514/291
`514/291
`514/291
`514/291
`
`FOREIGN PATENT DOCUMENTS
`507555A1 7/1992 European Pat. Off.
`
`540/456
`
`OTHER PUBLICATIONS
`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. Physiol. Pharmacol. 55:48 (1977).
`Staruch, M. J., FASEB 3:3411 (1989).
`Dumont, F. J., FASEB 3:5256 (1989).
`Caine, 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.)
`
`Par Pharm., Inc.
`Exhibit 1046
`Page 001
`
`

`
`5,362,718
`
`Page 2
`
`R7 is hydrogen, alkyl, alkenyl, alkynyl, — ( C R ^4-
`)fORw, —CFi, —F, or — COjR11;
`R8 and R9 are each, independently, hydrogen, alkyl,
`alkenyl, alkynyl, —(CR3R4)pR10, —CF3, —F, or
`—CO2R11, or R8 and R9 may be taken together to
`form X or a cycloalkyl ring that is optionally
`tri-substituted with —(CR3R4-
`mono-, di-, or
`)PR10;
`R10 is hydrogen, alkyl, alkenyl, alkynyl, tri-(alkyl)si-
`lyl, tri-(alkyl)silylethyl, triphenylmethyl, benzyl,
`alkoxymethyl, tri-(alkyl)silylethoxymethyl, chloro(cid:173)
`ethyl, or tetrahydropyranyl;
`R11 is hydrogen, alkyl, alkenyl, alkynyl, or phenylal(cid:173)
`kyl;
`X is 5-(2,2-dialkyl)[l,3]dioxanyl, 5-(2,2-dicycloalk-
`yl)[l,3]dioxanyl, 4-(2,2-dialkyl)[l,3]dioxanyl, 4-
`(2,2-dicycloalkyl)[l,3]dioxanyl, 4-(2,2dialkyl)[l,3-
`Jdioxalanyl, or 4-(2,2-dicycloalkyl)[l,3]dioxalanyl;
`b = 0 - 6;
`d = 0 - 6; and
`f=0-6
`with the proviso that R1 and R2 are both not hydrogen
`and further provided that either R1 or R2 contains at
`least one —(CR3R4)/3Rio, X, or —(CR3R4)pR10 sub(cid:173)
`stituted cycloalkyl group, or a pharmaceutically accept(cid:173)
`able salt thereof which is useful as an immunosuppres(cid:173)
`sive, antiinflammatory, antifungal, antiproliferative, and
`antitumor agent.
`
`24 Claims, No Drawings
`
`wherein R1 and R2are each, independently, hydrogen
`or —CO(CR3R4)ft(CR5R6)(iCR7R8R9;
`R3 and R4 are each, independently, hydrogen, alkyl,
`alkenyl, alkynyl, trifluoromethyl, or —F;
`R5 and R6 are each, independently, hydrogen, alkyl,
`alkenyl, alkynyl, — (CRSR^/DRio, —CF3, —F, or
`—CO2R11, or R5 and R6 may be taken together to
`form X or a cycloalkyl ring that is optionally
`mono-, di-, or tri-substituted with —(CR3R4)/)R10;
`
`Par Pharm., Inc.
`Exhibit 1046
`Page 002
`
`

`
`5,362,718
`
`RAPAMYCIN HYDROXYESTERS
`
`BACKGROUND OF THE INVENTION
`
`5
`
`20
`
`This mvention relates to hydroxyesters of rapamycin
`and a method for using them for inducing immunosup(cid:173)
`pression, and in the treatment of transplantation rejec(cid:173)
`tion, graft vs. host disease, autoimmune diseases, dis(cid:173)
`leukemia/lym- 10
`eases of inflammation, adult T-cell
`phoma, sohd tumors, fungal infections, and hyperprolif-
`erative vascular disorders.
`Rapamycin is a macrocyclic triene antibiotic pro(cid:173)
`duced by Streptomyces hygroscopicus, which was found 25
`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. Nos. 3,929,992; and 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 25
`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 macrocycUc molecules, also have
`been shown to be effective as immunosuppressive 35
`agents, therefore useful in preventing transplant rejec(cid:173)
`tion [FASEB 3, 3411 (1989); FASEB 3, 5256 (1989); R.
`Y. Caine et al.. Lancet 1183 (1978); and U.S. Pat. No.
`5,100,899].
`Rapamycin has also been shown to be useful in pre-
`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. 121 (Abstract), 45
`(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 Applica(cid:173)
`tion 525,960 Al], and ocular inflammation [European
`Patent Application 532,862 Al].
`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) 55
`and used to make water soluble aminoacyl 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- positions.
`
`60
`
`DESCRIPTION OF THE INVENTION
`This invention provides derivatives of rapamycin g5
`which are useful as immunosuppressive, antiinflamma(cid:173)
`tory, antifungal, antiproliferative, and antitumor agents
`having the structure
`
`wherein R1 and R2 are each, independently, hydrogen
`or —CO(CR3R4)i(CR5R6)(iCR7R8R9.
`R3and R4are each, independently, hydrogen, alkyl of
`1-6 carbon atoms, alkenyl of 2-7 carbon atoms,
`alkynyl of 2-7 carbon atoms, trifluoromethyl, or
`- F;
`R5and R6are each, independently, hydrogen, alkyl of
`1-6 carbon atoms, alkenyl of 2-7 carbon atoms,
`alkynyl of 2-7 carbon atoms, —(CR3R4)/OR10,
`—CF3, —F, or —COaR11, or R5 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)/OR10;
`R7 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of
`2-7 carbon atoms, alkynyl of 2-7 carbon atoms,
`—(CR3R4)pRio, —CF3, —F, or —COaR11;
`R8 and R9 are each, independently, hydrogen, alkyl of
`1-6 carbon atoms, alkenyl of 2-7 carbon atoms,
`alkynyl of 2-7 carbon atoms, —(CR3R4)/)R10,
`—CF3, —F, or —CO2R11, or R8 and R9 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)/)R10;
`R10 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, or
`phenylalkyl of 7-10 carbon atoms;
`X is 5-(2,2-di-(alkyl of 1-6 carbon atoms))[l,3]dioxa-
`nyl,
`5-(2,2-di-(cycloalkyl
`of
`3-8
`carbon
`atoms))[l,3]dioxanyl, 4-(2,2-di-(alkyl of 1-6 carbon
`atoms))[l,3]dioxanyl, 4-(2,2-di-(cycloalkyl of 3-8
`carbon atoms))[l,3]dioxanyl, 4-(2,2-di-(alkyl of 1-6
`carbon atoms))[l,3]dioxalanyl, or 4-(2,2-di-(cy-
`cloalkyl of 3-8 carbon atoms))[l,3]dioxalanyl;
`b=0-6;
`d=0-6; and
`f=0-6
`with the proviso that R1 and R2 are both not hydrogen
`and further provided that either R! or R2 contains at
`least one —(CR3R4)pR1 0, X, or—(CR3R4)pR10 sub(cid:173)
`stituted cycloalkyl of 3-8 carbon atoms group, or a
`pharmaceutically acceptable salt thereof.
`
`Par Pharm., Inc.
`Exhibit 1046
`Page 003
`
`

`
`5,362,718
`
`The pharmaceutically acceptable salts are those de(cid:173)
`rived from such inorganic cations such as sodium, po(cid:173)
`tassium, 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 5
`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 io
`than one —(CR3R4)pR10 group, R3, R4, f, and R10 can
`be the same or different. Similarly, when other generic
`substituent descriptions are repeated in the same struc(cid:173)
`ture, they can be the same or different.
`For a compound in which R1 contains R8 and R9 15
`taken together to form X, where X is 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, R1 would have the
`followmg structure.
`
`-CO(CR3RVV
`
`R /\ -O
`
`CH3
`y^
`CH3
`
`Similarly, for a compound in which R1 contains R8
`and R9 taken together to form X, where X is 4-(2,2-di-
`(cycloalkyl of 3-8 carbon atoms))[l,3]dioxanyl, the
`cycloalkyl group of X contains 6 carbon atom, and
`d = 0, R1 would have the following structure.
`
`-CO(CR3R4)i-
`R
`
`20
`
`25
`
`30
`
`35
`
`40
`
`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 R10 is not hydrogen, alkyl, alkenyl, or alkynyl, 45
`it is intended that R10 is a group that can serve as an
`alcohol protecting group. Thus, these groups are inter(cid:173)
`mediates of free hydroxylated compounds, as well as
`being biologically active in their own right. R10 covers
`tri-(alkyl of 1-6 carbon atoms)silyl, tri-(alkyl of 1-6 50
`carbon atoms)silylethyl, triphenylmethyl, benzyl, al(cid:173)
`koxymethyl of 2-7 carbon atoms, tri-(alkyl of 1-6 car(cid:173)
`bon atoms)silylethoxymethyl, chloroethyl, and tetrahy(cid:173)
`dropyranyl groups. Other alcohol protecting groups are
`known by one skilled in the an and are also considered 55
`pan of this invention.
`Of the compounds of this invention preferred mem(cid:173)
`bers 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, indepen- 60
`dently hydrogen, alkyl, or —(CR3R4)/OR10, or are
`taken together to form X.
`Compounds of this invention having the ester group
`—CO(CR3R4)iCR5R6)d(CR7R8R9)eat the 42- or 31,42-
`positions can be prepared by acylation of rapamycin 65
`using protected hydroxy and polyhydroxy acids, alkoxy
`or polyalkoxy carboxylic acids that have been acti(cid:173)
`vated, followed by removal of the alcohol protectmg
`
`groups, if so desired. Several procedures for carboxyl(cid:173)
`ate activation are known in the art, but the preferred
`methods utilize carbodiimides, mixed anhydrides, or
`acid chlorides. For example, an appropriately substi(cid:173)
`tuted carboxylic acid can be activated as a mixed anhy(cid:173)
`dride, with an acylating group such as 2,4,6-trichloro-
`benzoyl chloride. Treatment of rapamycin with the
`mixed anhydride under mildly basic condition provides
`the desired compounds. Altematively, the acylation
`reaction can be accomplished with l-(3-dimethylamino-
`propyl)-3-ethylcarbodiiniide hydrochloride and dime(cid:173)
`thylaminopyridine. Mixtures of 42- and 31,42-esters can
`be separated by chromatography.
`The 31-ester-42-hydroxy compounds of this inven(cid:173)
`tion 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 procedures described above. The
`preparation of rapamycin 42-silyl ethers is described in
`U.S. Pat. No. Bl 5,120,842, which is hereby incorpo(cid:173)
`rated by reference. Removal of the protecting group
`provides the 31-esterified compounds. In the case ofthe
`tert-butyl dimethylsilyl protecting group, deprotection
`can be accompUshed under mildly acidic conditions,
`such as acetic acid/water/THF. The deprotection pro(cid:173)
`cedure is described in Example 15 of U.S. Pat. No.
`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. Altematively, the 42-esterified
`compounds, prepared as described above, can be re(cid:173)
`acted with a different acylating agent to provide com(cid:173)
`pounds having different esters at the 31-and 42-posi-
`tions.
`This invention also covers analogous hydroxy esters
`of other rapamycins such as, but not limited to, 29-
`demethoxyrapamycin, [U.S. Pat. No. 4,375,464, 32-
`demethoxyrapamycin under C A. nomenclature]; rapa(cid:173)
`mycin derivatives in which the double bonds in the 1-,
`3-, and/or 5-positions have been reduced [U.S. Pat. No.
`5,023,262]; 29-desmethylrapamycm
`[U.S. Pat. No.
`5,093,339, 32-desmethylrapamycin under C A. nomen(cid:173)
`clature]; 7,29-bisdesmethylrapamycin [U.S. Pat. No.
`5,093,338, 7,32-desmethylrapamycm under C A. no(cid:173)
`menclature]; and 15-hydroxyrapamycin [U.S. Pat. No.
`5,102,876]. The disclosures in the above cited U.S. Pa(cid:173)
`tents are hereby incorporated by reference.
`Immunosuppressive activity for representative com(cid:173)
`pounds of this invention was evaluated in an in vitro
`standard pharmacological test procedure to measure
`the inhibition of lymphocyte proliferation (LAP) and in
`two in vivo standard pharmacological test procedures.
`The pinch skin graft test procedure measures the immu(cid:173)
`nosuppressive activity of the compound tested as well
`as the ability of the compound tested to inhibit or treat
`transplant rejection. The adjuvant arthritis standard
`pharmacological test procedure, which measures the
`ability of the compound tested to inhibit immune medi(cid:173)
`ated inflammation. The adjuvant arthritis test proce(cid:173)
`dure is a standard pharmacological test procedure for
`rheumatoid arthritis. The procedures for these standard
`pharmacological test procedures are provided below.
`The comitogen-induced thymocyte proliferation pro(cid:173)
`cedure (LAF) was used as an in vitro measure of the
`immunosuppressive effects of representative com-
`
`Par Pharm., Inc.
`Exhibit 1046
`Page 004
`
`

`
`5,362,718
`
`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 thymidine during the last
`six hours. Cells are cultured with and without various
`concentrations of rapamycin, cyclosporin A, or test 5
`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- io
`pose of comparison. An IC50 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 IC50 ranging from 0.6-1.5
`nM. The results obtained are provided as an IC50 and as 15
`the percent inhibition of T-cell proliferation at 0.1 /iM.
`The results obtained for the representative compounds
`of this invention 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- 20
`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.
`
`IC50 of Rapamycin
`IC50 of Test Compoimd
`
`25
`
`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 30
`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 35
`isograft was used as control in the same region. The
`recipients were treated with either varying concentra(cid:173)
`tions of test compounds intraperitoneaUy or oraUy.
`Rapamycin was used as a test control. Untreated recipi(cid:173)
`ents serve as rejection control. The graft was monitored 40
`daUy 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 dmg treatment
`group was compared with the control group. The fol- 45
`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. Compounds were tested using a
`dose of 4 mgAg.
`The adjuvant arthritis standard pharmacological test
`procedure measures the abUity of test compounds to
`prevent immune mediated inflammation and inhibit or
`treat rheumatoid arthritis. The following briefly de(cid:173)
`scribes the test procedure used. A group of rats (male 55
`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
`oraUy dosed on a Monday, Wednesday, Friday sched- 60
`ule 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 65
`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 non(cid:173)
`
`50
`
`specific inflammation. Compounds were tested at a dose
`of 5 mgAg. 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 rapa(cid:173)
`mycin treated rats had between 70-90% less immune
`induced inflammation than control rats.
`The results obtained in these standard pharmacologi(cid:173)
`cal test procedures are provided following the proce(cid:173)
`dure for making the specific compounds that were
`tested.
`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 obtamed in the LAF test proce(cid:173)
`dure
`indicates suppression of T-cell proliferation,
`thereby demonstrating the immunosuppressive activity
`ofthe compounds ofthis invention. Further demonstra(cid:173)
`tion 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 re(cid:173)
`sults obtained in the skin graft test procedure further
`demonstrates the ability ofthe compounds ofthis inven(cid:173)
`tion to treat or mhibit transplantation rejection. The
`results obtained in the adjuvant arthritis standard phar(cid:173)
`macological 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 pharmacologi(cid:173)
`cal 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), comea, small bowel, and skin allografts, and
`heart valve xenografts; in the treatment or inhibition of
`autoimmune diseases such as lupus, rheumatoid arthri(cid:173)
`tis, 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 com(cid:173)
`pounds 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 leuke(cid:173)
`mia/lymphoma, fungal infections, and hyperprolifera-
`tive vascular diseases such as restenosis and atheroscler(cid:173)
`osis. 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 combi(cid:173)
`nation of the above.
`When administered for the treatment or inhibition of
`the above disease states, the compounds of this inven(cid:173)
`tion can be administered to a mammal orally, parenter(cid:173)
`ally, intranasally, intrabronchially, transdermally, topi(cid:173)
`cally, intravaginally, or rectally.
`It is contemplated that when the compounds of this
`invention are used as an immunosuppressive or antiin(cid:173)
`flammatory agent, they can be administered in conjunc(cid:173)
`tion with one or more other immunoregulatory agents.
`
`Par Pharm., Inc.
`Exhibit 1046
`Page 005
`
`

`
`5,362,718
`
`8
`7
`The compounds of this invention may be adminis-
`Such other immunoregulatory agents include, but are
`tered rectally in the form of a conventional suppository,
`not limited to azathioprine, corticosteroids, such as
`For administration by intranasal or intrabronchial inha-
`prednisone and methylprednisolone, cyclophospha-
`lation or insufflation, the compounds of this invention
`mide, rapamycin, cyclosporin A, FK-506, OKT-3, and
`ATG. By combining the compounds of this invention 5 may be formulated into an aqueous or partiaUy aqueous
`with such other drugs or agents for inducing immuno-
`solution, wiiich can then be utUized in the form of an
`suppression or treating inflammatory conditions, the
`aerosol. The compounds of this invention may also be
`lesser amounts of each of the agents are required to
`administered transdermaUy through the use of a trans-
`achieve the desired effect. The basis for such combina-
`dermal patch containing the active compound and a
`tion therapy was established by Stepkowski whose re- 10 carrier that is inert to the active compound, is non toxic
`suits showed that the use of a combmation of rapamycin
`to the skin, and allows delivery ofthe agent for systemic
`and cyclosporin A at subtherapeutic doses significantly
`absorption into the blood stream via the skin. The car-
`prolonged heart allograft survival time. [Transplanta-
`rier may take any number of forms such as creams and
`tion Proc. 23: 507 (1991)].
`ointments, pastes, gels, and occlusive devices. The
`The compounds of this invention can be formulated 15 creams and ointments may be viscous liquid or semi-
`neat or with a pharmaceutical carrier to a mammal in
`SoUd emulsions of either the oU-m-water or water-in-oU
`need thereof. The pharmaceutical carrier may be soUd
`type. Pastes comprised of absorptive powders dispersed
`or liquid. When formulated orally, it has been found
`in petroleum or hydrophUic petroleum containing the
`that 0.01% Tween 80 in PHOSAL PG-50 (phospho-
`active ingredient may also be suitable. A variety of
`Upid concentrate with 1,2-propylene glycol, A. Natter- 20 occlusive devices may be used to release the active
`mann & Cie. GmbH) provides an acceptable oral formu-
`ingredient mto the blood stream such as a semipermia-
`lation.
`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-
`containing the active mgredient. Other occlusive de-
`bUizers, suspending agents, fillers, glidants, compression 25 vjc es ^g known jn the Uterature.
`aids, bmders or tablet-disintegrating agents; it can also
`j^ addition, the compounds of this mvention may be
`be an encapsulating material. In powders, the carrier is
`employed as a solution, cream, or lotion by formulation
`a finely divided solid which is in admixture with the
`wth pharmaceuticaUy acceptable vehicles contaming
`finely divided active ingredient. In tablets, the active
`percent, preferably 2%, of active compound
`0 ^
`ingredient is mixed with a carrier having the necessary 30 w h i ch m ay be administered to a fungaUy affected area,
`compression properties in suitable proportions
`,and
`Tile 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
`mgredient. Suitable solid camers mclude, for example,
`s u bje ct b e i ng treated. Based on the results obtamed in
`calcium phosphate, magnesium stearate, talc, sugars, 35 t he
`pharmacological test procedures, pro-
`stmdald
`lactose, dextnn, starch, gelatm, ceUulose, methyl ceUu-
`je c t ed d a i ly d o s a geS of active compound would be 0.1
`lose, sodium carboxymethyl cellulose, polyvmylpyr-
`. g / k g - i oo mgAg, preferably between 0.001-25
`rolidine, low meltmg waxes and ion exchange resms.
`m g / k g; ^
`m o re preferably between 0.01-5 mgAg.
`Liquid camers are used m preparmg solutions, sus-
`T reatment wUl generaUy be initiated with small dosages
`pensions, emukions, symps, elixirs and pressunzed 40 ie ss than the optimum dose of the compound. Thereaf-
`^^
`compositions. The active mgredient can be dissolved or
`^
`t he optimum effect
`is ^
`t er
`t he d
`suspended m a pharmaceutically acceptable hquid car-
`t he c i r c u m s t a n c es is r e a c hed. precise dosages for
`^^
`ner such as water an orgamc solvent, a mixture of both
`^
`t e r a l) n a s a l> or intrabronchial administration
`or pharmaceutically acceptable ods or fats. The liqmd
`^
`be determined by the administering physician based
`earner can contam other suitable pharmaceutical addi- 45 on e
`individual subject treated. Pref-
`e r i e n ce ^
`the
`tives such as solubUizers, emulsifiers, buffers, preserva-
`e r a bl ^
`h a r m a c e ut ic ai composition is in unit dosage
`tives, sweeteners, flavonng agents, suspendmg agents,
`f
`^
`^ ^^
`or
`^ s u ch f
`^
`c o m.
`thickening agents, colors, viscosity regulators, stabUiz-
`dose containing appropri-
`^ ^
`snb.divided
`^
`ers or osmo-regulators. Smtab e examples of liquid car-
`m es of the active ingredient; the unit dosage
`^
`ners for oral and parenteral admimstration mclude 50 -
`,
`,
`,
`c
`_ i
`-s
`.
`u
`i
`forms can be packaged compositions, for example.,
`, _t. ,,
`t . •
`AA-S
`. ,
`,
`• ,
`n
`r.u A
`•
`»
`water (partiaUy contammg additives as above, e.g. cel-
`,
`, ,
`tu i
`packeted powders, vials, ampoules, prefilled syrmges or
`A • J
`c ui
`A-
`u
`lulose denvatives, preferably sodium carboxymethyl
`i- • j - r nl
`- IJ
`u 4
`4 • •
`C
`U
`u A •
`sachets contaimng liquids. The umt dosage form can be,
`,, .
`.
`.
`\
`1 u 1 i- 1 A-
`cellulose solution), alcohols (mcludmg monohydnc
`.
`,
`t ui t -t ic
`-t
`u 4U
`r
`1 u A - 1 u 1
`1
`1 \
`for example, a capsule or tablet itself, or it can be the
`.
`,
`.
`,
`A tu •
`alcohols and polyhydnc alcohols, e.g. glycols) and their
`. \
`•
`1
`u
`c
`u
`-s
`.
`.
`..
`A Cc appropnate number of any such compositions m pack-
`A
`-i /
`c
`s
`tA
`4-1
`denvatives, and oUs (e.g. fractionated coconut ou and 55 ^^ . r
`r
`*
`J
`arachis oU). For parenteral administration, the carrier
`®
`. "
`,
`.„
`,
`.
`,,
`.
`,.
`,
`A-
`«
`The foUowmg examples illustrate the preparation and
`su 1 1 4
`r
`.r
`l i.
`r
`can also be an oily ester such as ethyl oleate and isopro-
`, .,
`.,
`. ?.
`.
`-i,.
`
`pyl myristate. Sterile liquid carders are useful in sterile
`todogical
`activities of representative compounds ofthis
`mven • n-
`liquid form compositions for parenteral administration.
`EXAMPLE 1
`The liquid carrier for pressurized compositions can be 60
`.,
`. „
`_
`halogenated hydrocarbon or other pharmaceutically
`Rapamycm 42-ester with
`,
`acceptable propellant.
`(tetrahydropyran-2-yloXy)acetic ac.d
`Liquid pharmaceutical compositions which are sterile
`2,4,6-Trichlorobenzoyl chloride
`(0.55 mL, 3.51
`solutions or suspensions can be utUized by, for example,
`intramuscular, intraperitoneal or subcutaneous injec- 65 mmol) was added via syringe to a solution of the gly-
`tion. Sterile solutions can also be administered intrave-
`colic acid THP-ether (0.562 g, 3.51 mmol) and triethyl-
`nously. The compound can also be administered orally
`amine (0.49 mL, 3.51 mmol) in 10 mL THF at 0 ° C
`either in liquid or solid composition form.
`under nitrogen. The mixture was stined for 4 h at room
`
`^^
`
`,
`
`Par Pharm., Inc.
`Exhibit 1046
`Page 006
`
`

`
`5,362,718
`
`temperature, and a white precipitate formed. The white
`precipitate was removed by vacuum filtration and the
`filtrate was concentrated with a stream of nitrogen and
`warm water bath. The residue was dissolved in 10 mL
`benzene, then rapamycin (2.92 g, 3.19 mmol) and 5
`DMAP (0.429 g, 3.51 mmol) were added and the mix(cid:173)
`ture was stined ovemight at room temperature. The
`mixture was dUuted with EtOAc, washed with cold IN
`HCI (aq), saturated NaHCOs (aq) and brine, dried over
`MgSOt, filtered and concentrated to an oUy yellow JQ
`soUd. Flash chromatography (2X with 65% EtOAc-
`hexane) afforded the title compound (1.114 g, 33% ) as
`a white solid.
`(-)FAB-MS m/z 1055.5 (M-), 590.3 (southern frag(cid:173)
`ment), 463.2 (northern fragment). iH NMR (400 MHz, 15
`d-6 DMSO) 8 4.60 (m, 1 H, C(42)H), 4.66 (m, IH), 4.14
`(s, 2H), 3.73 (m, IH), 3.42 (m, IH). "C NMR (100.6
`MHz, d-6 DMSO) 8 169.2, 97.4, 63.5, 61.2, 29.7, 24.8.
`18.8.
`
`20
`
`EXAMPLE 2
`Rapamycin 42-ester with hydroxyacetic acid
`p-Toluenesulfonic acid (10 mg) was added to a solu(cid:173)
`tion ofthe product of Example 1 (306 mg, 0.29 mmol) in
`10 mL CH3OH at 0 ° C. The solution was stined 2 h at 25
`room temperature, then quenched with saturated NaH(cid:173)
`COs solution. The aqueous phase was extracted 3X with
`EtOAc and the combined organic phases were washed
`with brine, dried over MgS04, filtered and concen(cid:173)
`trated to a white solid. Purification by flash chromatog- 30
`raphy (2X