United States Patent 119J
`Failli et at.
`
`[54] RAPA\IYCJN 42·Sl:LFONATES AND
`42·CI':CARBOALKOXYJSULFAMATES
`USEFUL AS IMMUNOSUPPRESSIVE
`AGENTS
`
`(75)
`
`Inventors: Amedeo A. Failli, Princeton
`Junction. NJ.; Wenling Kao, Paoli;
`Robert J. Steffan, Langhorne, both
`of Pa.; Robert L. Vogel, Stratford,
`N.J.
`
`[73] Assignee; American HQme Products
`Corporation, New York, N.Y.
`
`[21] Appl. No,, 917.555
`
`[22] Filed'
`
`Jul. 21, 1992
`
`[62]
`
`[51]
`[52]
`[58]
`
`[56]
`
`Related U.S. Application Data
`Division of Ser. No. 846,637, Mar. 5. 1992, Pat. No.
`5,177,203
`
`Int. a.~
`lJ.S. Cl.
`Field of Search .
`
`............................. A61K 31/395
`...... 514/291; 540/456
`. 514/291
`
`References Cited
`t:.S. PATENT DOCI:MENTS
`5.1'77,20.3 1/1993 Fa:lli et aJ
`
`''""'"" 540/456
`
`Primar)' Examiner-Roben T. Bond
`Attorney. AgeJ:t, or Firm-Walter Patton
`
`111111111111111111111111111111111111111111111111111111111111111111111111111
`US005260299A
`[11] Patent Number:
`[45] Date of Patent:
`
`5,260,299
`Nov. 9, 1993
`
`[57]
`ABSTRACT
`A derivative of rapamycin of general formula (I)
`
`(I)
`
`where Rl is alkyl, alkcnyl, or alkynyl containing 1 to 6
`carbon atoms; or an aromatic moiety selected from the
`group cons1sting of phenyl and naphthyl or a heterocy(cid:173)
`clic moiety selected from the group consisting of thio(cid:173)
`phenyl and quinolinyl or NHC02R2 wherein R2 is
`lower alkyl containing 1 to 6 carbon atoms or a pharma(cid:173)
`ceutically acceptable salt thereof, which by virtue of its
`immunosuppressive activity is useful in treating trans(cid:173)
`plantation rejection, hosT versus graft disease, autoim(cid:173)
`mune diseases, and diseases of inflammatiun.
`
`1 Oaim, 1'\n Drawings
`
`N PC02231755
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`RAPAMYCIN 42-SULFONATES AND
`42-(]1;-CARBOALKOXYJSULFAMATES USEFUL AS
`IMMt:NOSUPPRESSIVE AGEJI."TS
`
`This is a division of application Ser. No. 07/846,637
`filed Mar. 5, 1992, no"' U.S. Pat. No. 5,177,203.
`
`(I)
`
`BACKGROUND OF THE INVENTION
`This invention relates to rapamycin 42-sulfonates and 10
`42-(N-carboalkoxy)sulfamates and a method for using
`them in the treatment of transplantation rejection, host
`versus graft disease, autoimmune diseases, diseases of
`inflammation, and fungal infections.
`Rapamycin is a macrocydic triene antibiotic pro- 15
`duced by Streptomyces hygroscopicus, which was found
`to have antifungal activity, particularly against Candida
`o.lbicans, both in vitro and in vivo [C. Vezina et al., J.
`Antibiat. 28, 721 (1975); S. N. Sehgal ct al., J. Antibiot.
`28, 727 (1975}; H. A. Baker et at., J. Antihiot. 31, 539 20
`(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. 25
`[Can. J. Physiol. Pharmacal. 55,48 (1976)] disclosed thar
`rapamycin is effective in the experimental allergic en(cid:173)
`cephalom~elitis model, a model for mulliple sclerosis: in
`the adjuvant arthritis model, a model for rheumatoid
`arthritis; and effectively inhibited the formation of IgE- 30
`like antibodies.
`The immunosuppressive effects of rapamycin have
`been disclosed in FASEB 3, 3411 (1989). Rapamycin
`therefore is also useful in preventing transplant rejec(cid:173)
`tion [FASEB 3, 3411 (1989); FASEB 3, 5256 (1989); and 35
`R. Y. Caine et al., Lancet 1183 (1978)].
`Mono· and diacylated deriva-tives of rapamycin 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 (lJ.S. Pat. No. 4,650,803). Recently, the 40
`numbering convention
`for
`rapamycin has been
`changed; therefore according to Chemical Abstracts
`nomenclature, the esters described above would be at
`the 3I~and 42-posirions.
`
`DESCRIPTION OF THE IKVENTIO:K'
`This invention relates to rapamycin 42-sulfonates and
`42-(N~carboalkoxy)sulfamates of general formula (I)
`
`where R I is alkyl, alkenyl, or alkynyl containing 1 to 6
`carbon atom~; or an aromatic moiety selected frum the
`group consisting of phenyl and naphthyl or a heterocy~
`clic moiety selected from the group consisting of thio(cid:173)
`phenyl and quinolinyl or NHC02R2 wherein R2 is
`lower alkyl containing 1 to 6 carbon atoms or a pharma(cid:173)
`ceutically acceptable salt thereof.
`The rapamycin 42-sulfonate~ of this invention can be
`prepared by the standard literarure procedure as out(cid:173)
`lined below.
`
`0
`0
`II
`II
`R-OH + CJ-S-Rl --7R-O-S-Rl
`II
`II
`0
`0
`
`The sulfonate formation between alcohol and sufonyl
`halide ha5 been described [Jerry ~arch, Advanced
`Organic Chemistry, 3rd edition, published in 1985, page
`444]. The specific reaction condition employed in this
`invention was developed by S. Rakhit of Ayersr Labo(cid:173)
`ratories and reported in U.S. Pat. No. 4,316,855 (Feb.
`23, 1982).
`The 42-(N-carboalkm;y)sulfamates of the pn:::sent
`invention can also be prepared by reaction ofrapamycin
`with an alkyl(carboxysulfamoyl)triethylammonium hy~
`45 droxide inner salt (Burgess Salts; see G. M. Atkins Jr.
`and E. M. Burgess, J. Am. Chern. Soc., 90,4744, 1968;
`E. M. Burgess, H. R. Penton Jr. and E. A. Taylor, J.
`Org. Chern. 38, 26, 1978).
`
`r.apamycin
`
`0
`11-
`+
`RloCNS02N(C2H5}~ )
`benz~n~. r.t
`
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`4
`
`wherein R 2 is as defined above.
`The pharmaceutically acceptable salts may be formed
`from inorganic cations such as sodium, potassium, and
`the like.
`The following examples illustrate the preparation of
`representative compounds of this in\lention.
`
`EXAMPLE I
`Rapamycin 42·ester with
`5·(dimethylamino )·1·naphthaienesulfonic acid
`A solution of 200 mg (0.22 mmol) of rapamycin in 2
`mL of pyridine was treated at oo C. under anhydr~us
`condltions with 840 mg (3.1 mmol) of dansyl chlonde
`and stirred at roam temperature for 24 hours. The reac(cid:173)
`tion mixture was diluted at 0° C. with 30 mL of 2N HCI
`and extracted with ethyl acetate. The ethyl acetate
`extract was washed with brine, dried with M!>S04 and
`evaporated. The residue was chromatographed on silica
`gel. Elution with 25% ethyl acetate in benzene afforded
`150 mg of the title product as a light yellow powder,
`m.p. 101"-104° C.
`IRo 3430 (OH). 1740 (sh), 1720 (both C~O), 1650
`(amide C=O), 1450, 1355, 1170 (sulfonate), 1100, 985,
`960cm-1.
`'H NMR (CDC!;, 400 MHz) 88.58 (d, IH, HI), 8.32
`(d, IH, H3), 8.25 (m, !H, Hz), 7.53 (m, 2H, Hs and H,),
`7.19 (d, !H, H4), 3.31, 3.13, 2.72 (all s, JH, -0-CH,),
`
`CH3
`
`2.,9(~. OH, - f\' ) ppm
`
`/
`
`CH;
`
`4
`
`20
`
`EXAMPLE 2
`Rapamycin 42-ester with 4-methylbenzenesulfonic acid
`A solution of 6.0 g (31.6 mmol) p-toluenesulfonyl_
`chloride in 25 mL pyridine was added to a solution of
`25 10.0 g (10.9 mmol) rapamycin at oo C. and the resulting
`solution was stirred at 20° C. for 22 hours. Cold 2N HCl
`(240 mL) was added and the product was extracted into
`ethyl acetate, washed with brine, dried over MgS04
`and ~vaporated to a yellow solid. Chromatography on
`30 sllica gel .eluted with 20% ethyl acetate in methylene
`chloride afforded 5.3 g product as a white solid, m.p.
`108'-116" c.
`IR(KBr)o 3410, 2910, 1710, 1640, 1440, 1160 and 655
`cm- 1.
`NMR (CDCh, 400 MHz)o 37.80 (d, 2H, aromatic),
`732 (d, 2H, aromatic), 3.33 (s, 3H), 3.14 (s, 3H), 3.13 (s,
`JH), 2.44 (s, JH).
`MS (neg. ion FAB): 1067 (M-), 590, 171, 155.
`
`35
`
`40
`
`45
`
`50
`
`EXAMPLEJ
`Rapamycin 42-ester with 2~thiophenesu1fonic acid
`A solution ofO.lS g (0.2 mmol) rapamycin and 0.13 g
`(O.il mmol) 2-thiophenesulfonyl chloride in 2 mL pyri(cid:173)
`dine was heated at 55° C. for 4 hours, then cooled to 10"
`C. and treated with 40 mL tN HCI. The product was
`extracted into ethyl acetate, washed with brine, dried
`over MgS04 and stripped of solvent. Chromatography
`on si1ica gel eluted with 20% ethyl acetate in methylene
`chloride afforded 40 mg title compound as a white solid,
`m.p. 114'-119' C.
`lR (KBr)o 3420, 2915, 1712, 1644, 1440, 1365, 1170
`and 660 cm-1.
`NMR (CDCb, 400 MHz)o 87.67 (IH, aromatic), 7.62
`55 (lH, aromatic), 7.07 (lH, aromatic), 3.29 (s, 3H, OCH3),
`3.14 (s, JH, OCH3), 3.09 (s, JH, OCHJ),
`MS (neg. ion FAB)o 1059 (M-), 912, 590, !63.
`
`60
`
`EXAMPLE4
`Rapamycin 42-ester with
`4·([4-(dimethylamino)phenyl]aza}benzenesulfonic acid
`Dabsyl chloride (0.83 g, 2.57 mmol) was added to a
`solution of 0.54 g (0.59 mmol) rapamycin in 30 mL dry
`pyridine and the solution heated at 65°-70° C. for 24
`65 hours. Upon cooling, the reaction mixture was parti(cid:173)
`tioned between 200 mL 2N HCI and 50 mL ethyl ace(cid:173)
`tate. The product was dried over MgS04, stripped of
`solvent and chromatographed on silica gel eluted with
`
`MS (neg. ion FAB) 1146 (M-}, 912,590,250.
`
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`30% ethyl acetate in methylene chloride, to afford the
`title compound as a bright red solid, m.p. 118"-133"
`IR (KB')' 3430, 2930, 1720, 1600, 1360, 1142, 683 and
`602 cm- 1•
`NMR (CDCl3, 400 MHz): SS.OO (2H, aromatic), 7.93
`(4H, aromatic), 6.76 (2H, aromatic). 3.33 (s, 3H, OCH3),
`3.!35 (s, JH, OCHJ), 3.126 (s, 3H, OCHJ).
`MS (pas. FAB): 1223 (MNa+), 1169,1137,918,306.
`
`10
`
`EXAMPLE 5
`Rapamycin 42-ester with 1-naphthalene sulfonic acid
`l-Naphthalenesulfonyl chloride (0.48 g. 0.2.1 rnmol)
`was added to a solution of (0.54 g, 0.59 mmol) rapamy(cid:173)
`cin in J l miL pyridine and the resulting solution was 15
`stirred at 20~ C. for 44 hours. Cold 2~ HCl (75 mL) was
`added and the product was extracted into ethyl acetate,
`washed with brine, dried over MgS04 and evaporated
`to a tan solid. Chromatography on slica gel eluted with
`20% ethyl acetate in methylene chloride yielded 30 mg 20
`product as a white solid, m.p. 110'-131° C.
`IR (KBr): 3440. 292.S, 1720, 1645,1450,1175 and 765
`em-'.
`NMR (CDCI3, 400 MHz)' 88.65 (!H), 8.26 (!H), 8.10
`(2H), 7.93 (!H), 7.70 (!H), 7.62-7.53 (complex, 2H), 25
`3.32 (s. JH, OCHJ), 3.13 (s, 3H, OCHJ), 2.64 (s, 3H,
`OCHJ).
`MS (neg. FAB), 1103 (M-), 912, 590.
`
`6
`MS (neg. ion FAB, m/z): 991 (M)-, 590,399.
`for Cs2HsrN01sS+0.5 H20+0.5
`Anal.
`cal'd
`C4H802: C, 62.05; H, 8.29; ~. 1.34. Found: C, 61.63; H,
`8.34: N. 1.49
`
`EXAMPLE 8
`Rapamycin 42-(2,2,2-trifluoroethane sulfonate),
`dihydrate
`Under an atmosphere of nitrogen, a solution of rapa-
`mycin (0.46 g, 0.5 mmol) and triethylamine (0.15 mL,
`l.l mmol) in 2 mL of dry CH2Cb was treated in one
`portion with 2,2,2-trifluoroethane sulfonyl chloride
`(0.06 mL, 0.55 mmol). The solution was stirred over·
`night at ambient temperature. The solvent was evapo(cid:173)
`rated in vacuo to give a yellow foam. The crude prod·
`uct mixture was purified by MPLC (silica Lichrosorb
`60, Merck 440*37 rnm, ethyl acetate-hexane 1:2, flow(cid:173)
`rate 20 mL/min) to give the tide compound.
`NMR (400 MHz, CDCh)' 8!.6l (s, 3H, CH,C=C),
`1.749+ 1.752 (1s, JH, CH,C=C), 3.14 (s, 3H, OCHJ),
`3.34 (s, 3H, OCH 3), 3.37 (s, JH, OCH 3).
`MS (neg. ion FAB, rn/z), 1059 (M)-, 590,560,427,
`163.
`Anal. cal'd for C!2Hs1N015S+2 H20: C, 58.02; H,
`7.72; N, 1.28. Found: C, 57.94; H, 7.96; N, 1.22.
`
`EXAMPI,.E 9
`42.0-[[ (Methox ycarbon yl) ]amino ]sulfony l]rapam ycin
`Under anhydrous conditions, a solution ofrapamycin
`(0.5 g, 0.55 mmol) and methyl(carboxysulfamoyl)trie(cid:173)
`thylammonium inner salt (0.25 g, 1.2 rnmol, prepared as
`described by Burgess et al., J_ Org. Chern. 38, 26, 1978)
`in 5 mL of benzene was stirred at ambient temperature
`overnight. The reaction mixture was then diluted with
`EtOAc (50 mL) and the solution was washed with
`water and brine and dried (Na2S04). Removal of the
`solvent in vacuo yielded an off-white solid which was
`further purified by MPLC (silica Merck 60 Lichroprep.
`440*37 mm, ethyl acetate-hexane 2:1-methanol) to
`provide the title product as a yellow solid {0.247 g,
`43%).
`(s, 3H,
`(CDC!;, 400 MHz), 81.65
`lH NMR
`CH3C=C). 1.78 (s, 3H, CH,C=C), 3.13 (rn, 3H,
`CH 30), 3.39 (m, 6H, CHJO), 3.71 (s, 3H, CO,CH,).
`:\-IS (neg. ion FAB, rn/z): lOSO (M-H)-.
`The comitogen-induced thymocyte proliferation pro·
`cedure (LAF) was used as an in vitro measure of the
`50 ~~~n:~sWJ;~Y~v~en:rr~~~~ 0t~/e&~:~sta~~e no=i
`BALB/c mice were cultured for 72 hours with PHA
`and IL-l and pulsed with tritiated thymidine during the
`last six hours. Cells are cultured with and without vari-
`55 ous concentrations of rapamycin, cyclosporin A, or test
`compound. Cells are harvested and incorporated; radio(cid:173)
`activity is determined. Inhibition of lymphoprolifera(cid:173)
`tion is assessed in precent change in counts per minute
`from non-drug treated controls. The results are ex-
`60 pressed by the following ratio:
`
`30
`
`EXAMPLE 6
`Rapamycin 42-ester with 8-quinolinesulfonic acid
`A solution of (0.30 g, 0.33 mrnol) rapamycin and (0.29
`g. l.2B mmol) 8-quinolinesulfony! chloride in 5 mL
`pyridine wa~ ~tirred at 20Q C. for 24 houn,. The reaction
`mixture was partit10ned between 2N HCI (10 mL) and 35
`ethyl ace::ate.
`The organic layer was washed with brine, dried 0'-'er
`MgS04, stripped of solvent and chromatographed on
`silica gel elmed v.-"ith 30% ethyl acetate in methylene
`chloride. affording I 30 mg of title compound as a white 40
`solid, mp 120"-165" C.
`IR (KBr): 3430,2925. 1715, 1640, 1170,985 and 785
`cm-1.
`NMR (CDCIJ, 400 MHz), 89.18 (!H), 8.49 (!H), 8.25
`(!H). 8.09 (!H), 7.65 (!Hi, 7.55 (!H), 3.32 (s, JH,
`OCH;.). 3. I3 (s, 3H, OCH3), 2.60 (s, 3H. OCH3).
`MS (neg. FAR): 1104 (M-), 912, 5QO, 208.
`
`45
`
`EXAMPLE 7
`Rapamycin 42·methanesulfonate, hemiethylacetate,
`hemihydrate
`Under an atmosphere ofnitragen, an ice cold solution
`of rapamycin (0.46 g, 0.5 mmol) and triethylamine (0.14
`mL, 1.0 mmol) in 5 mL of dry CH2Cb was treated
`dropwise with methanesulfonyl chloride (0.943 mL,
`0.55 mmol). The ice bath was removed and the solution
`stirred at ambient temperature for one hour. The reac(cid:173)
`tion mixture was diluted with CH2Ch and washed sue·
`cessively with H20 and brine. After drying (Na2S04),
`the solvent was removed in vacuo to give a yellow
`foam. Purification by flash chromatography (silica
`Merck 60. erhyl acetate-hexane 1:1) afforded the title
`compound (0.37 g, 75o/t: white solid).
`NMR (400 MHz, CDCl3): 01.65 (3H. CH3C=C), 65
`1.74+ 1.75 (2s, 3H, CHJC=C), 3.06 (>, 3H, CH,SO,),
`3.13 (s, JH, OCH;), 3.34 (s, JH, OCH;J. 3.4003 (s, 3H,
`OCH,).
`
`3H-control thvmus c-~lls
`3H.comrol thymus celis H3-test c:ompound·trea~cd cells
`
`A mixed lymphocyte reaction (MLR) occurs when
`lymphoid cells from genetically distinct animals are
`combined in tissue culture. Each stimulates the other to
`undergo blast transformation which results in increased
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`lH-PLN cells
`control C3H mouse
`3H·PLN ~ell;
`cGnttcl C3H mouse
`
`3H-PLN cell~
`rapamvdn-lreat~d C3H mouse
`_'IH-PLS cell~
`tes\ compound-treated C3H mouse
`
`4
`
`1.2J (Lp.)
`
`0.92 (i.p.)
`0.08 (i.p.)
`0.36 (i.p.)
`0.83 (i.p.)
`0.93 (i.p.)
`
`9.5 ± 0.3
`10.7 ::!: 2.1
`9.83 ± 0.98
`JO_Q :!. ].4
`10.33 ± 0.24
`
`7
`DNA synthesis that can be quantified by the incorpora(cid:173)
`tion of tritiated thymidine. Since stimulating a MLR is a
`function of disparity at Major Histocompatibility anti(cid:173)
`gens, an in vivo popliteal lymph node (PLN) test proce(cid:173)
`dure closely correlates to host vs. graft disease. ·Briefly,
`irradiated spleen cells from BALB/c donors are in(cid:173)
`jected into the right hind foot pad of recipient C]H
`mice. The drug is given daily, p.o. from Day 0 to Day
`4. On Day 3 and Day 4, tritiated thymidine is given i.p.,
`b.i.d. On Day 5, the hind popliteal lymph nodes are
`removed and dissolved, and radioactivity counted. The
`corresponding left PLN serves as the control for the
`PLN from the injected hind foot. Percent suppression is
`calculated using the non-drug treated animals as allo(cid:173)
`genic control. Rapamycin at a dose of 6 mg/kg, p.o.
`gave 86% suppression, whereas cyclosporin A at the
`same dose gave 43% suppression. Results are expressed
`by the following ratio:
`
`8
`of the present invention further demonstrate their utility
`as immunosuppressive agents.
`Based on the results of these standard pharmacologi(cid:173)
`cal test procedures, the compounds of this invention are
`5 useful in the prevention and treatment of transplant
`rejection such as heart, kidney, liver, bone marrow, and
`skin transplants; graft versus host disease; autoimmune
`and proliferative diseases such as, systemic lupus ery(cid:173)
`thematosus, rheumatoid arthritis, type I diabetes, multi-
`10 pie sclerosis, glomerular nephritis, Hashimoto's thy(cid:173)
`roiditis, myastenia gravis, uveitis and psoriasis; diseases
`of inflammation such as dermatitis, eczema,. seborrhea
`and inflammatory bowel disease; and fungal infections.
`The compounds may be administered neat or with a
`15 pharmaceutical- carrier to a mammal in need thereof.
`The pharmaceutical carrier may be solid or liquid.
`A solid carrier can include one or more substances
`which may also act as flavoring agents, lubricants, solu(cid:173)
`bilizers, suspending agents, fillers, glidants, compression
`20 aids, binders or tablet-disintegrating agents; it can also
`be an encapsulating material. In powders, the carrier is
`a finely divided solid which is in admixture with the
`finely divided active ingredient. In tablets, the active
`de~~i~~~~~ ~~n~i~~ ~~e p~7~~~~~e s~ind~~~f~e~o~ 25 ~~~~~~es~~oi~ ~;;ge~t!~ 3i~a:~~:~:vi;~~~ert~~~:ss:~~
`compacted in the shape and size desired. The powders
`male DBA/2 dcmors transplanted to male BALB/c
`and tablets preferably contain up to 99% of the active
`recipients. The method is adapted from Billingham R.
`ingredient. Suitable solid carriers include, for example,
`E. and Medawar P. B., J. Exp. Biol. 28:385-402 (1951).
`Briefly, a pinch skin graft from the donor is grafted on 30 calcium phosphate, magnesium stearate, talc, sugars,
`lactose, dextrin, starch, gelatin, cellulose, methyl cellu-
`thC dorsum of the recipient as a homograft, and an
`lose, sodium carboxymethyl cellulose, polyvinylpyr-
`autograft is used a~ control in the same region. The
`rolidine, low melting waxes and ion exchange resins.
`recipients are treated with either varying concentra-
`Liquid carriers are used in preparing solutions, sus-
`tions of cyclosporin A as test control or the test com-
`pound, intraperitoneally. Untreated recipients serve as 35 pensions, emulsions, syrups, elixirs and pressurized
`compositions. The active ingredient can be dissolved or
`rejection control. The graft is monitored daily and ob-
`suspended in a pharmaceutically acceptable liquid car-
`servations are recorded until the graft becomes dry and
`rier such as water, an organic solvent, a mixmre of both
`forms a blackened scab. This is considered as the rejec-
`or pharmaceutically acceptable oils or fats. The liquid
`tion day. The mean graft survival time (number of
`days±S.D.) of the drug treatment group 1s compared 40 carrier can contain other suitable phannaceutical addi-
`tives such as solubilizen., emulsifiers, buffers, preserva-
`with the control group.
`tives, sweeteners, flavoring agents. suspending agents,
`BJOLOGICAL DATA
`thickening agents, colors, viscosity regulators, stabiliz-
`ers or osmo-regulators. Suitable examples of liquid car-
`The following table summarizes the results of repre-
`sentative l:Ompounds of thi~ invention in these three 45 riers for oral and parenteral administration include
`water (partially containing additives as above, e.g. cel-
`standard test procedures.
`lulose derivatives, preferably sodium carboxymethyl
`T ABLE 1
`cellulose solution), alcohols (including monohydric
`LAF A5s:;olG)!ica!P:~vi~y
`alcohols and poly hydric alcohols, e.g. glycols) and their
`Skin Graft Model
`50 derivatives, and oils (e.g. fractionated coconut oil and
`(day~+ SDl
`(RIA ralio)
`(RIA ratio)
`Example
`-==::""'--:::::-;;:;:;::::::c._c::::.:.:..::::::"-..--'7,:-'-;';:"---
`arachis oil). For parenteral administration, the carrier
`B.O ± 0.9
`0.26
`can also be an oily ester such as ethyl oleate and isopro-
`8.7 ± 1.2
`0.21
`pyl myristate. Sterile liquid carriers are useful in sterile
`0.23
`9.3 ± o.B
`o.m
`liquid form compositions for parenteral administration.
`0.19
`55 The liquid carrier for pressurized compositions can be
`1.32
`halogenated hydrocarbon or other pharmaceutically
`1.70
`acceptable propellent.
`O.BS
`Liquid pharmaceutical compositions which are sterile
`001
`solutions or suspensions can be utilized by, for example,
`intramuscular intraperitoneal or subcutaneous injection.
`Sterile solutions can also be administered intravenously.
`The compound can also be administered orally either in
`liquid or solid compositions form.
`Preferably, the pharmaceutical composition is in unit
`65 dosage form, e.g., as tablets or capsules. In such fcrm,
`the composition is sub-divided in unit. dose containing
`appropriate quantities of the active ingredient; the unit
`dosage forms can be packaged compositions, for exam-
`
`The results of these standard pharmacological test 60
`procedures demonstrate high immunosuppressive activ-
`ity both in vitro and in vivo for the compounds of the
`present invention. A positive ratio in the LAF and PLN
`test procedures indicates suppression ofT -cell prolifera(cid:173)
`tion. A!\ transplanted pinch skin grafts are typically
`rejected within 6-7 days without the use of an immune·
`suppressive agent, the substantial increase in survival
`time of the skin grant when treated with the compounds
`
`N PC02231759
`
`NOVARTIS EXHIBIT 2122
`Par v Novartis, IPR 2016-00084
`Page 5 of 6
`
`

`
`9
`
`5,260,299
`
`10
`
`ple, packeted powders, vials, ampoules, prefilled syrin-
`
`ges 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
`
`package form. The dosage to be used in the treatment
`
`must be subjectively determined by the attending physi-
`
`cian.
`
`What is claimed is:
`
`10
`
`15
`
`"'
`
`1. A method of treating transplantation rejection, 20
`
`host versus graft disease, autoimmune di!>east:s, and
`
`diseases of inflammation in a mammal by administering
`
`an effective amount of a compound of formula (I}
`
`where R 1 is alkyl, alkenyl, or a.lkynyl containing 1 to 6
`carbon atoms; or an aromatic moiety selected from the
`group consisting of phenyl and naphthyl or a heterocy-
`clic moiety selected from the group consisting of thio·
`phenyl and quinolinyl or NHC02R2 wherein R2 is
`lower alkyl containing 1 to 6 carbon atoms or a phanna-
`2S ceutically acceptable salt thereof.
`• • • • *
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`N PC02231760
`
`NOVARTIS EXHIBIT 2122
`Par v Novartis, IPR 2016-00084
`Page 6 of 6