.
`United States Patent
`Skotnicki et al.
`
`115
`
`US005362718A
`[11] Patent Number:
`[45] Date of Patent:
`
`5,362,718
`Noy. 8, 1994
`
`[54] RAPAMYCIN 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.
`
`5,260,300 11/1993 HU cccssssssssssssssveesessesessssnseesee 540/456
`5,262,423 11/1993 Kao cossssssssssssessvessssesesssesee 514/291
`5,286,730 2/1994 Canfield et al.ese 514/291
`
`2/1994 Caufield et al.
`cee 514/291
`5,286,731
`5,302,584 4/1994 Kao et ab. cececsccscsssesen 514/291
`FOREIGN PATENT DOCUMENTS
`50755SA1
`7/1992 European Pat. Off. ov... 540/456
`OTHER PUBLICATIONS
`
`(21] Appl. No.: 229,261
`
`Apr. 18, 1994
`[22] Filed:
`oonereeserene ONDdetComb1/04
`[51] nt. C1?
`[52] UsS. Che ceessssccessscsenseceessnseseveses 514/63; 514/291,
`[58] Field of Search................ 540/456, 452; 514/291,
`514/63
`
`2
`
`.
`
`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).
`Calne, R. Y., Lancet 1183 (1978).
`Morris, R. E., Med. Sci. Res. 17:877 (1989).
`Baeder, W. L., Fifth Int. Conf. Inflamm. Res. Assoc.
`References Cited
`121 (Abstract) (1990).
`U.S. PATENT DOCUMENTS
`Meiser, B. M., J. Heart Lung Transplant, 11 (pt. 2):197
`(1992).
`3993749 11/1976 Snel Cb al srr~ Daign_Stepkowski, S. M., Transplantation Proc. 23:507 (1991).
`vee 424/122
`4,316,885 2/1982 Rakhit.......
`
`
`4,375,464 3/1983 Sehgal etal. .
`w. 424/122
`8/1983 Eng .....
`-. 424/124
`4,401,653
`
`...
`«. 540/456
`4,650,803
`3/1987 Stella et al.
`4,885,171 12/1989 Surendraet al.
`.
`» 424/122
`
`5,023,262 6/1991 Caufield et al. ..
`wa 340/456
`
`6/1991 Von Burg.........
`5,023,263
`... 540/456
`
`5,023,264 6/1991 Caulfield etal. ....
`.. 514/291
`5,078,999
`1/1992 Warneretal. ...
`we 424/122
`5,080,899
`1/1992 Sturm etal. ......
`. 424/122
`
`a 514/291
`5,091,389 2/1992 Ondeyka etal. .
`3/1992 Schiehser.........
`5,100,883
`.- 514/183
`
`
`. 514/291
`5,100,899 3/1992 Calne........
`
`5,102,876 4/1992 Caufield....
`« 514/18.3
`5,118,677 6/1992 Caufield ....
`«+» 314/183
`
`6/1992 Kao etal......
`see 514/183
`5,118,678
`
`5,120,842 6/1992 Failli et al.
`...
`wese 340/452
`5,130,307 7/1992 Failli et al.
`.......
`w 514/291
`
`5,138,051
`8/1992 Hughesetal. ...
`we» 340/456
`5,151,413
`9/1992 Caufield etal. ......
`we 540/456
`
`5,169,851 12/1992 Hughes etal. .......
`w. 514/291
`1/1993 Failli et al. ow...
`.. 340/456
`5,177,203
`
`we. 540/456
`3/1993 Kao...
`5,194,447
`
`wee 314/183
`5,221,670
`6/1993 Caufield
`8/1993 Hughes occccccccssssessessesssseens 540/456
`5,233,036
`
`5
`
`[56]
`
`Primary Examiner—Robert T. Bond
`Attorney, Agent, or Firm—Arnold 8. Milowsky
`
`ABSTRACT
`[57]
`A compoundofthe structure
`.
`(Abstract continued on next page.)
`
`West-Ward Pharm.
`Exhibit 1046
`Page 001
`
`West-Ward Pharm.
`Exhibit 1046
`Page 001
`
`

`

`5,362,718
`
`Page 2
`
`
`
`wherein R! and R2are each, independently, hydrogen
`or —CO(CR3R4)i(CR5R9)gCR7RERS;
`R3 and R4are each, independently, hydrogen,alkyl,
`alkenyl, alkynyl, trifluoromethyl, or —F;
`R5 and R§ are each, independently, hydrogen, alkyl,
`alkenyl, alkynyl, —(CR3R4)OR!0, —CF3, —F, or
`—CO2RH, or Ri and R§ may be taken together to
`form X or a cycloalkyl ring that
`is optionally
`mono-, di-, or tri-substituted with —(CR3R4)OR!®,
`
`R? is hydrogen, alkyl, alkenyl, alkynyl, —(CR3R*+
`)/OR!9, —CF3, —F, or —CO2R}!;
`R8 and R°9are each, independently, hydrogen,alkyl,
`alkenyl, alkynyl, —(CR3R4)/OR!°, —CF3, —F, or
`—CO?2R!!, or R8 and R? may be taken together to
`form X or a cycloalkyl ring that
`is optionally
`mono-, di-, or
`tri-substituted with —(CR3R*+
`OR10;
`R19 is hydrogen, alkyl, alkenyl, alkynyl, tri-(alkyl)si-
`lyl,
`tri-(alkylsilylethyl,
`triphenylmethyl, benzyl,
`alkoxymethyl,tri-(alky])silylethoxymethy]l, chloro-
`ethyl, or tetrahydropyranyl;
`R!! is hydrogen, alkyl, alkenyl, alkynyl, or phenylai-
`kyl;
`X is 5-(2,2-dialkyl)[1,3]dioxanyl, 5-(2,2-dicycloalk-
`yDf[1,3]dioxanyl,
`4-(2,2-dialky)f1,3]dioxanyl,
`4-
`(2,2-dicycloalkyl)[1,3]dioxanyl, 4-(2,2dialkyl)[1,3-
`Jdioxalanyl, or 4-(2,2-dicycloalky]l[1,3]dioxalany);
`b=0-6;
`d=0-6; and
`f=0-6
`
`with the proviso that R! and R2 are both not hydrogen
`and further provided that either R! or R? contains at
`least one —(CR3R4)/OR!9, X, or —(CR3R4)/OR!9 sub-
`stituted cycloalkyl group, or a pharmaceutically accept-
`able salt thereof which is useful as an immunosuppres-
`sive, antiinflammatory, antifungal, antiproliferative, and
`antitumor agent.
`
`24 Claims, No Drawings
`
`West-Ward Pharm.
`Exhibit 1046
`Page 002
`
`West-Ward Pharm.
`Exhibit 1046
`Page 002
`
`

`

`1
`
`5,362,718
`
`RAPAMYCIN HYDROXYESTERS
`
`BACKGROUND OF THE INVENTION
`
`This invention relates to hydroxyesters of rapamycin
`and a method for using them for inducing immunosup-
`pression, and in the treatment of transplantation rejec-
`tion, graft vs. host disease, autoimmune diseases, dis-
`eases of inflammation, adult T-cell
`leukemia/lym-
`phoma,solid tumors, fungal infections, and hyperprolif-
`erative vascular disorders.
`Rapamycin is a macrocyclic triene antibiotic pro-
`duced by Streptomyces hygroscopicus, which was found
`to have antifungal activity, particularly against Candida
`albicans, both in vitro and in vivo [C. Vezina etal., 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-
`bination with picibanil (U.S. Pat. No. 4,401,653) has
`been shownto have antitumoractivity. 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 FASEB3, 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-
`tion [FASEB3, 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 shownto 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),
`(1990)], smooth muscle cell proliferation and intimal
`thickening following vascular injury [Morris, R. J.
`Heart Lung Transplant 11 (pt. 2): 197 (1992)j, adult
`T-cell leukemia/lymphoma [European Patent Applica-
`tion 525,960 Al], and ocular inflammation [European
`Patent Application 532,862 Al].
`Mono- and diacylated derivatives of rapamycin (es-
`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 aminoacy! 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.
`
`DESCRIPTION OF THE INVENTION
`
`25
`
`30
`
`45
`
`This invention provides derivatives of rapamycin
`which are useful as immunosuppressive, antiinflamma-
`tory, antifungal, antiproliferative, and antitumor agents
`having the structure
`
`65
`
`
`
`wherein R! and R2are each, independently, hydrogen
`or —CO(CR3R*),(CR5R5)gCR7RERS:
`R3and Rare 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 Rare each, independently, hydrogen,alkyl of
`1-6 carbon atoms, alkenyl of 2-7 carbon atoms,
`alkynyl of 2-7 carbon atoms, —(CR3R4)OR!0,
`—CF3, —F, or —COR', or R5 and R® may be
`taken together to form X or a cycloalkyl ring of
`3-8 carbon atomsthat is optionally mono-, di-, or
`tri-substituted with —(CR3R4)OR!0,
`R7is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of
`2-7 carbon atoms, alkynyl of 2-7 carbon atoms,
`—(CR3R4OR!0, —CF3, —F, or —CO2R!;
`R8and Rare each, independently, hydrogen,alkyl of
`1-6 carbon atoms, alkenyl of 2-7 carbon atoms,
`alkynyl of 2-7 carbon atoms, —(CR3R4)OR,
`—CF3, —F, or —CO2R!, 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)OR10;
`R10is 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
`tetrahydropyrany];
`R!lis hydrogen, alkyl of 1~6 carbon atoms,alkenyl of
`2-7 carbon atoms, alkynyl of 2-7 carbon atoms, or
`phenylalky! of 7-10 carbon atoms;
`X is 5-(2,2-di-(alkyl of 1-6 carbon atoms)){1,3]dioxa-
`nyl,
`5-(2,2-di-(cycloalkyl
`of
`3-8
`carbon
`atoms))[1,3]dioxanyl, 4-(2,2-di-(alkyl of 1-6 carbon
`atoms))[1,3]dioxanyl, 4-(2,2-di-(cycloalkyl of 3-8
`carbon atoms))[1,3]dioxanyl, 4-(2,2-di-(alkyl of 1-6
`carbon atoms))[1,3]dioxalanyl, or 4-(2,2-di-(cy-
`cloalkyl of 3-8 carbon atoms))[1,3]dioxalany];
`b=0-6;
`d=0~6; and
`f=0-6
`with the proviso that R! and R2 are both not hydrogen
`and further provided that either R! or R? contains at
`least one —(CR3R*)OR!0, X, or—(CR3R4)/OR" sub-
`stituted cycloalkyl of 3-8 carbon atoms group, or a
`pharmaceutically acceptable salt thereof.
`
`West-Ward Pharm.
`Exhibit 1046
`Page 003
`
`West-Ward Pharm.
`Exhibit 1046
`Page 003
`
`

`

`5,362,718
`
`3
`The pharmaceutically acceptable salts are those de-
`rived from such inorganic cations such as sodium, po-
`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
`1-6 carbon atomsper 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 compoundsof this invention can contain more
`than one —(CR3R*)/OR!9 group, R3, R4, f, and R!0can
`be the sameor different. Similarly, when other generic
`substituent descriptions are repeated in the samestruc-
`ture, they can be the sameordifferent.
`For a compound in which R! contains R® and R9
`taken together to form X, where X is 5-(2,2-di-(alkyl of
`1-6 carbon atoms)){1,3]dioxanyl, the alkyl group of X
`contains 1 carbon atom, and d=0, R! would have the
`following structure.
`
`—CO(CR3R4)5
`R
`
`o
`
`o
`
`K
`
`CH3
`
`CHB
`
`Similarly, for a compound in which R! contains R8
`and R° taken together to form X, where X is 4-(2,2-di-
`(cycloalkyl of 3-8 carbon atoms))[1,3]dioxanyl,
`the
`cycloalkyl group of X contains 6 carbon atom, and
`d=0, R! would havethe following structure.
`
`corn—oO
`
`R
`
`oO
`
`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!9 is not hydrogen,alkyl, alkenyl, or alkynyl,
`it is intended that R!0 is a group that can serve as an
`alcohol protecting group. Thus, these groups are inter-
`mediates of free hydroxylated compounds, as well as
`being biologically active in their own right. R!° covers
`tri-(alkyl of 1-6 carbon atoms)silyl,
`tri-(alkyl of 1-6
`carbon atoms)silylethyl,
`triphenylmethyl, benzyl, al-
`koxymethy! of 2-7 carbon atoms,tri-(alkyl of 1-6 car-
`bon atoms)silylethoxymethyl, chloroethyl, and tetrahy-
`dropyranyl groups. Other alcohol protecting groups are
`knownbyoneskilled in the an and are also considered
`pan of this invention.
`Of the compoundsofthis invention preferred mem-
`bers are those in which R? is hydrogen; those in which
`R2 is hydrogen, b=0, and d=0; those in which R?is
`hydrogen, b=0, d=0, and R8 and R9are each, indepen-
`dently hydrogen, alkyl, or —(CR3R*4OR!, or are
`taken together to form X.
`Compoundsofthis invention having the ester group
`—CO(CR3R4),CR5R®)ACR7RER9), at the 42- or 31,42-
`positions can be prepared by acylation of rapamycin
`using protected hydroxy and polyhydroxyacids, alkoxy
`or polyalkoxy carboxylic acids that have been acti-
`vated, followed by removal of the alcohol protecting
`
`4
`groups, if so desired. Several procedures for carboxyl-
`ate activation are known in the art, but the preferred
`methods utilize carbodiimides, mixed anhydrides, or
`acid chlorides. For example, an appropriately substi-
`tuted carboxylic acid can be activated as a mixed anhy-
`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. Alternatively, the acylation
`reaction can be accomplished with 1-(3-dimethylamino-
`propyl)-3-ethylcarbodiimide hydrochloride and dime-
`thylaminopyridine. Mixtures of 42- and 31,42-esters can
`be separated by chromatography.
`The 31-ester-42-hydroxy compounds of this inven-
`tion can be prepared by protecting the 42-alcohol of
`rapamycin with a protecting group, such as witha tert-
`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. B1 5,120,842, which is hereby incorpo-
`rated by reference. Removal of the protecting group
`provides the 31-esterified compounds.In the case of the
`tert-butyl dimethylsily] protecting group, deprotection
`can be accomplished under mildly acidic conditions,
`such as acetic acid/water/THF. The deprotection pro-
`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 compoundshaving different esters at
`the 31- and 42- positions. Alternatively, the 42-esterified
`compounds, prepared as described above, can be re-
`acted with a different acylating agent to provide com-
`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-
`mycin derivatives in which the double bondsin the 1-,
`3-, and/or 5-positions have been reduced [U.S. Pat. No.
`5,023,262]; 29-desmethylrapamycin [U.S. Pat. No.
`5,093,339, 32-desmethylrapamycin under C.A. nomen-
`clature]; 7,29-bisdesmethylrapamycin [U.S. Pat. No.
`5,093,338, 7,32-desmethylrapamycin under C.A. no-
`menclature]; and 15-hydroxyrapamycin [U.S. Pat. No.
`5,102,876]. The disclosures in the above cited U.S. Pa-
`tents are hereby incorporated by reference.
`Immunosuppressive activity for representative com-
`pounds 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.
`Thepinch skin graft test procedure measures the immu-
`nosuppressive activity of the compoundtested as well
`as the ability of the compoundtested to inhibit ortreat
`transplant rejection. The adjuvant arthritis standard
`pharmacological test procedure, which measures the
`ability of the compoundtested to inhibit immune medi-
`ated inflammation. The adjuvant arthritis test proce-
`dure is a standard pharmacological test procedure for
`rheumatoid arthritis. The procedures for these standard
`pharmacological test procedures are provided below.
`The comitogen-induced thymocyteproliferation pro-
`cedure (LAF) was used as an in vitro measure of the
`immunosuppressive effects of
`representative com-
`
`20
`
`25
`
`30
`
`35
`
`45
`
`60
`
`65
`
`West-Ward Pharm.
`Exhibit 1046
`Page 004
`
`West-Ward Pharm.
`Exhibit 1046
`Page 004
`
`

`

`5,362,718
`
`5
`pounds. Briefly, cells from the thymus of normal
`BALB/c miceare 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.Celis are harvested and incorporated radio-
`activity is determined. 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-
`pose of comparison. An ICso was obtained for each test
`compound as well as for rapamycin. When evaluated as
`a comparator for the representative compoundsofthis
`invention, rapamycin had an ICs9 ranging from 0.6-1.5
`nM.Theresults obtained are provided as an ICso and as
`the percent inhibition of T-cell proliferation at 0.1 pM.
`Theresults obtained for the representative compounds
`of this invention were also expressed as a ratio com-
`pared with rapamycin. A positive ratio indicates immu-
`nosuppressive activity. A ratio of greater than 1 indi-
`cates that the test compound inhibited thymocyte pro-
`liferation to a greater extent than rapamycin. Calcula-
`tion of the ratio is shown below.
`
`ICso of Rapamycin
`ICso of Test Compound
`
`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 concentra-
`tions of test compounds intraperitoneally or orally.
`Rapamycin was used as a test control. Untreated recipi-
`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
`consideredas the rejection day. The mean graft survival
`time (number of days+S.D.) of the drug treatment
`group was compared with the control group. Thefol-
`lowing table showsthe results that were obtained. Re-
`sults are expressed as the mean survival time in days.
`Untreated (control) pinch skin grafts are usually re-
`jected within 6-7 days. Compounds weretested using a
`dose of 4 mg/kg.
`The adjuvantarthritis standard pharmacological test
`procedure measures the ability of test compounds to
`prevent immune mediated inflammation and inhibit or
`treat rheumatoid arthritis. The following briefly de-
`scribes the test procedure used. A group of rats (male
`inbread Wistar Lewis rats) are pre-treated with the
`compoundto 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 sched-
`ule from day 0-14 for a total of 7 doses. Both hind paws
`are measured on days 16, 23, and 30. Thedifferencein
`paw volume (mL) from day 16 to day 0 is determined
`and a percent change from controlis obtained. Theleft
`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 non-
`
`6
`specific inflammation. Compoundsweretested 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% changeversus control, indicating that rapa-
`mycin treated rats had between 70-90% less immune
`induced inflammation than controlrats.
`Theresults obtained in these standard pharmacologi-
`cal test procedures are provided following the proce-
`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 compoundsof this
`invention. The results obtained in the LAF test proce-
`dure indicates
`suppression of T-cell proliferation,
`thereby demonstrating the immunosuppressive activity
`of the compoundsofthis invention. Further demonstra-
`tion of the utility of the compoundsof this invention as
`immunosuppressive agents was shown by the results
`obtainedin the skin graft and adjuvantarthritis standard
`pharmacological test procedures. Additionally, the re-
`sults obtained in the skin graft test procedure further
`demonstrates the ability of the compoundsofthis inven-
`tion to treat or inhibit transplantation rejection. The
`results obtained in the adjuvant arthritis standard phar-
`macological
`test procedure further demonstrate the
`ability of the compoundsof this invention to treat or
`inhibit rheumatoid arthritis.
`Based on theresults of these standard pharmacologi-
`cal test procedures, the compounds are useful in the
`treatmentor inhibition of transplantation rejection such
`as kidney, heart, liver, lung, bone marrow, pancreas
`(islet cells), cornea, small bowel, and skin allografts, and
`heart valve xenografts; in the treatment or inhibition of
`autoimmune diseases such as lupus, rheumatoid arthri-
`tis, diabetes mellitus, myasthenia gravis, and multiple
`sclerosis; and diseases of inflammation suchas 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-
`pounds of this invention also are considered to have
`antitumor, antifungal activities, and antiproliferative
`activities. The compoundsof this invention therefore
`also useful in treating solid tumors, adult T-cell leuke-
`mia/lymphoma, fungal infections, and hyperprolifera-
`tive vascular diseases such as restenosis and atheroscler-
`osis. When used for restenosis, it is preferred that the
`compoundsof this invention are used to treat restenosis
`that occurs following an angioplasty procedure. When
`used for this purpose, the compoundsofthis invention
`can be administered prior to the procedure, during the
`procedure, subsequent to the procedure, or any combi-
`nation of the above.
`When administered for the treatment or inhibition of
`the above disease states, the compoundsof this inven-
`tion can be administered to a mammalorally, parenter-
`ally, intranasally, intrabronchially, transdermally, topi-
`cally, intravaginally, or rectally.
`It is contemplated that when the compoundsofthis
`invention are used as an immunosuppressive or antiin-
`flammatory agent, they can be administered in conjunc-
`tion with one or more other immunoregulatory agents.
`
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`West-Ward Pharm.
`Exhibit 1046
`Page 005
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`West-Ward Pharm.
`Exhibit 1046
`Page 005
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`

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`5,362,718
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`7
`Such other immunoregulatory agents include, but are
`not limited to azathioprine, corticosteroids, such as
`prednisone and methylprednisolone, cyclophospha-
`mide, rapamycin, cyclosporin A, FK-506, OKT-3, and
`ATG. By combining the compoundsof this invention
`with such other drugs or agents for inducing immuno-
`suppression or treating inflammatory conditions, the
`lesser amounts of each of the agents are required to
`achieve the desired effect. The basis for such combina-
`tion therapy was established by Stepkowski whose re-
`sults showedthatthe use of a combination of rapamycin
`and cyclosporin A at subtherapeutic dosessignificantly
`prolonged heart allograft survival time. [Transplanta-
`tion Proc. 23: 507 (1991)}.
`The compoundsof 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 PHOSAL PG-50 (phospho-
`lipid concentrate with 1,2-propylene glycol, A. Natter-
`mann & Cie. GmbH) provides an acceptable oral formu-
`lation.
`A solid carrier can include one or more substances
`which mayalsoactas flavoring agents, lubricants, solu-
`bilizers, suspending agents,fillers, glidants, compression
`aids, binders or tablet-disintegrating agents; it can also
`be an encapsulating material. In powders, the carrieris
`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, methy] cellu-
`lose, sodium carboxymethyl cellulose, polyvinylpyr-
`rolidine, low melting waxes and ion exchangeresins.
`Liquid carriers are used in preparing solutions, sus-
`pensions, emulsions, syrups, elixirs and pressurized
`compositions. The active ingredient can be dissolved or
`suspended in a pharmaceutically acceptable liquid car-
`rier such as water, an organic solvent, a mixture of both
`or pharmaceutically acceptable oils or fats. The liquid
`carrier can contain other suitable pharmaceutical addi-
`tives such as solubilizers, emulsifiers, buffers, preserva-
`tives, sweeteners, flavoring agents, suspending agents,
`thickening agents, colors, viscosity regulators, stabiliz-
`ers or osmo-regulators. Suitable examplesofliquid car-
`riers for oral and parenteral administration include
`water (partially containing additives as above,e.g. cel-
`lulose 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 isopro-
`py! myristate. Sterile liquid carders are useful in sterile
`liquid form compositions for parenteral administration.
`Theliquid carrier for pressurized compositions can be
`halogenated hydrocarbon or other pharmaceutically
`acceptable propellant.
`Liquid pharmaceutical compositions whicharesterile
`solutions or suspensionscan beutilized by, for example,
`intramuscular,
`intraperitoneal or subcutaneous injec-
`tion. Sterile solutions can also be administered intrave-
`nously. The compound can also be administered orally
`either in liquid or solid composition form.
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`8
`The compounds of this invention may be adminis-
`tered rectally in the form of a conventional suppository.
`For administration by intranasal or intrabronchial inha-
`lation or insufflation, the compoundsofthis invention
`may be formulated into an aqueous or partially aqueous
`solution, which can then be utilized in the form of an
`aerosol. The compoundsofthis invention may also be
`administered transdermally through the use of a trans-
`dermal patch containing the active compound and a
`carrier that is inert to the active compound,is non toxic
`to the skin, and allowsdelivery of the agent for systemic
`absorption into the blood stream via the skin. The car-
`rier may take any numberof forms such as creams and
`ointments, pastes, gels, and occlusive devices. The
`creams and ointments may be viscous liquid or semi-
`solid 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 semipermia-
`ble membrane covering a reservoir containing the ac-
`tive ingredient with or without a carrier, or a matrix
`containing the active ingredient. Other occlusive de-
`vices are knownin the literature.
`In addition, the compoundsofthis invention may be
`employedas 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
`severity of the symptoms presented and the particular
`subject being treated. Based on the results obtained in
`the standard pharmacological
`test procedures, pro-
`jected daily dosages of active compound would be 0.1
`pg/kg-100 mg/kg,
`preferably between 0.001-25
`mg/kg, and more preferably between 0.01-5 mg/kg.
`Treatment will generally be initiated with small dosages
`less than the optimum dose of the compound. Thereaf-
`ter the dosage is increased until the optimum effect
`underthe circumstances is reached; precise dosages for
`oral, parenteral, nasal, or intrabronchial administration
`will be determined by the administering physician based
`on experience with the individual subject treated. Pref-
`erably, the pharmaceutical compositionis in unit dosage
`form, e.g. as tablets or capsules. In such form, the com-
`position is sub-divided in unit dose containing appropri-
`ate quantities of the active ingredient; the unit dosage
`forms can be packaged compositions, for example.,
`packeted powders, vials, ampoules, prefilled syringes or
`sachets containing liquids. The unit dosage form can be,
`for example, a capsule or tablet itself, or it can be the
`appropriate number of any such compositions in pack-
`age form.
`The foliowing examples illustrate the preparation and
`biological activities of representative compoundsofthis
`invention.
`
`EXAMPLE1
`
`Rapamycin 42-ester with
`(tetrahydropyran-2-yloxy)acetic acid
`3.51
`2,4,6-Trichlorobenzoyl
`chloride
`(0.55 mL,
`mmol) was added via syringe to a solution of the gly-
`colic acid THP-ether (0.562 g, 3.51 mmol) and triethyl-
`amine (0.49 mL, 3.51 mmol) in 10 mL THF at 0° C.
`under nitrogen. The mixture was stirred for 4 h at room
`
`West-Ward Pharm.
`Exhibit 1046
`Page 006
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`West-Ward Pharm.
`Exhibit 1046
`Page 006
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`

`

`5,362,718
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`10
`(m, 1H), 3.60 (m, 2H), 3.42 (m, 1H), 1.13 (s, 3H), 1.11 (,
`3H). 3C NMR (100.6 MHz, d-6 DMSO) 8 175.0, 98.0,
`73.8, 60.7, 42.6, 30.0, 24.9, 22.0, 21.6, 18.7.
`Results obtained in standard pharmacological test
`procedures:
`LAF ICso: 7.10 nM
`LAFratio: 0.34
`
`9
`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
`DMAP(0.429 g, 3.51 mmol) were added and the mix-
`ture was stirred overnight at room temperature. The
`mixture was diluted with EtOAc, washed with cold 1N
`HCI (aq), saturated NaHCO3(aq) and brine, dried over
`MgSO, filtered and concentrated to an oily yellow
`solid. Flash chromatography (2X with 65% EtOAc-
`hexane) afforded the title compound(1.114 g, 33% ) as
`a white solid.
`(—)FAB-MSm/z 1055.5 (M—), 590.3 (southern frag-
`ment), 463.2 (northern fragment). 'H NMR (400 MHz,
`d-6 DMSO)6 4.60 (m, 1 H, C(42)H), 4.66 (m, 1H), 4.14
`(s, 2H), 3.73 (m, 1H), 3.42 (m, 1H). 13C NMR (100.6
`MHz, d-6 DMSO) 6 169.2, 97.4, 63.5, 61.2, 29.7, 24.8.
`18.8.
`
`EXAMPLE2
`
`Rapamycin 42-ester with hydroxyacetic acid
`p-Toluenesulfonic acid (10 mg) was addedto a solu-
`tion of the product of Example 1 (306 mg,0.29 mmol) in
`10 mL CH30OHat 0° C. The solution was stirred 2 h at
`room temperature, then quenched with saturated NaH-
`CO3solution. The aqueous phase was extracted 3X with
`EtOAc and the combined organic phases were washed
`with brine, dried over MgSOq, filtered and concen-
`trated to a white solid. Purification by flash chromatog-
`raphy (2X with EtOAc) afforded the title compound
`(145 mg, 51%) as a white solid.
`(—) FAB-MS m/z 971.3 (M—), 590 (southern frag-
`ment), 379.1 (northern fragment). 1H NMR (400 MHz,
`d-6 DMSO) 6 4.60 (m, 1H, C(42)H), 3.98 (s, 2H). 8C
`NMR(100.6 MHz, d-6 DMSO) 6 172.1, 59.7.
`Results obtained in standard pharmacological test
`procedures:
`LAF ICso: 1.80 nM
`LAFratio: 0.83
`Percent change in adjuvant arthritis versus control:
`—88%
`
`EXAMPLE3
`
`Rapamycin 42-ester with
`2.2-dimethyl-3-(tetrahydropyran-2-yloxy)propionic
`acid
`
`_ Toasolution of the 2,2-dimethy]-3-hydroxypropionic
`acid THP-ether (0.319 g, 1.58 mmol) and triethylamine
`(0.22 mL, 1.58 mmol) in 5 mL dry THF at 0° C. under
`nitrogen was added 2,4,6-trichlorobenzoy! chloride
`(0.25 mL, 1.58 mmol) dropwise via syringe. The mix-
`ture was stirred 4.5 h at room temperature. The white
`precipitate was removed by vacuum filtration and the
`filtrate was concentrated with a stream of nitrogen and
`a warm water bath. The residue was dissolved in 5 mL
`benzene,
`then rapamycin (1.31 g, 1.43 mmol) and
`DMAP (0.193 g, 1.58 mmol) were added.