United States Patent [191
`Patchett et a1.
`
`HlllllllllllllllIlllllllllllllllllllIlllllllllllllllllllllllllllllllllllll
`5,122,511
`Jun. 16, 1992
`
`USOO5122511A
`Patent Number:
`Date of Patent:
`
`[11]
`[45]
`
`[54]
`
`[75]
`
`[73]
`[21]
`[22]
`[51]
`
`[52]
`
`[53]
`[56]
`
`IMMUNOSUPPRESSIVE CYCLOSPORIN
`ANALOGS WITH MODIFIED AMINO ACIDS
`AT POSITION-8
`Inventors: Arthur A. Patchett, West?eld; David
`Taub, Metuchen; Robert T.
`Goegelman, Linden, all of NJ.
`Assignee: Merck & Co., Inc., Rahway, NJ.
`Appl. No.: 485,920
`Filed:
`Feb. 27, 1990
`
`Int. Cl.5 ........................ .. C07K 5/12; C07K 7/64;
`A6lK 37/00
`US. Cl. .................................... .. 514/11; 530/317;
`530/321
`Field of Search ................. .. 514/11; 530/317, 321
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`298,516 1/1990 Durette et a1. .
`4.102.877 7/1978 Nutt .
`4,108,985 8/1978 Riiegger et a1. .................. .. 530/321
`4.117.118 9/1978 Harri et a1. .
`4.210,581 7/1980 Riiegger et a1. .................. .. 530/321
`4.220.641 9/1980 Traber et a]. .
`4188,43] 9/1981 Traber et a1. .
`4,289,851 9/1981 Traber et a1. .
`4,384,996 5/1983 Bollinger et a1. .
`4.396.542 8/1983 Wenger .
`4,639,434 1/1987 Wenger et a1. ..................... .. 514/11
`4,681,754 7/1987 Siegl .
`4.703.033 10/1987 Seebach .............................. .. 514/11
`4.764.503 8/1988 Wenger
`.. 514/11
`4.798,823 l/l989 Witzel ................................. .. 514/11
`4.914,188 4/1990 Durette et al. .
`
`FOREIGN PATENT DOCUMENTS
`
`0056782 7/1982 European Par. Off. .
`
`0194972 9/1986
`0296122 12/1988
`002206119 12/1988
`2206119 12/1988
`
`European Pat. Off. .
`European Pat. Off. .
`Fed. Rep. of Germany .
`United Kingdom .
`
`OTHER PUBLICATIONS
`Traber et al., Chemical Abstracts, 1988, BA 88(5):
`48607.
`H. Kobe] and R. Traber, Directed Biosynthesis of Cy
`closporins, European J. Appln. Microbiol Biotechnol,
`14, 237-240 (1982).
`J. Kollonitsch, Isr. J. Chem, 17, 53-59, 1978.
`R. Wenger, Cyclosporine vol. I. pp. 14-25 (1983).
`R. Wenger, Total Synthesis-Change in Molecular
`Structure--Biological Effect: Cyclosporin as Example,
`Sandorama, 1984/ 111, pp. 4-11.
`R. M. Wenger, Synthesis of Cyclosporine and Ana
`logues: Structural Requirements for Immunosuppres
`sive Activity, Angewandte Chemic 24:2, 77-138 (Feb.
`1985).
`P. L. Durette et al., A Study of the Correlation Be
`tween Cyclophilin Binding and In Vitro 1mmunosup—
`pressive Activity of Cyclosporine A and Analogues,
`Transplantation Proceedings, vol. X, No. 2. Suppl. 2
`(Apr.), 1988; pp. 51-57.
`Primary Examiner—Lester L. Lee
`Assistant Examiner-A. M. Davenport
`Attorney. Agent, or Firm—Curtis C. Panzer; Hesna J.
`Pfeiffer
`ABSTRACT
`[57]
`New immunosuppressive cyclosporin analogs are dis
`closed consisting of [dehydro-Ala]8 cyclosporins and
`derived therefrom cyclosporins having a sulfur contain
`ing amino acid at position-8.
`
`11 Claims, No Drawings
`
`NOVARTIS EXHIBIT 2009
`Par v Novartis, IPR 2016-00084
`Page 1 of 9
`
`

`
`1
`
`IMMUNOSUPPRESSIVE CYCLOSPORIN
`ANALOGS WITH MODIFIED AMINO ACIDS AT
`POSITION-8
`
`5,122,511
`2
`of Action, Progress in Clinical Biochemistry and Medi
`cine, vol. 2, 176 (1986).
`Cyclosporin A is a cyclic peptide which contains
`several N-methyl amino acids and, at position-8, con
`tains a D-alanine.
`
`5
`
`Structure of Cyclosporin A”
`
`3
`2
`1
`ll
`10
`MeLeu-MeVal-MeBmt-Abu-Sar
`l
`9 MeLeu
`
`D-Ala-Ala-MeLcu-Val-MeLeu
`8
`7
`6
`5
`4
`
`(l)
`
`Abu = L-aAminobutyric acid
`Ala = L-Alanine
`MeBmt = N-Methyl-(4R)-4h[(E)-2-butenyl]-4-methyl-L-threonine
`Leu = L-Leucine
`MeLeu = N-Methyl-L~leucine
`MeVal = N-Methyl-L-valine
`Nva = L-Norvaline
`Sar = Sarcosine
`Thr = L-Threonine
`Val = L-Valine
`‘
`<7Unless otherwise speci?ed. each of the amino
`acids of the disclosed cyclosporin is of the
`L~con?guration.
`
`A generic structure, useful for describing cyclosprin
`A and analogs thereof is
`
`25
`
`BACKGROUND OF THE INVENTION
`The cyclosporins are a family of, neutral, hydropho
`bic cyclic undecapeptides, containing a novel nine-car
`bon amino acid (MeBmt) at position 1 of the ring that
`exhibit potent immunosuppressive, antiparasitic, fungi
`cidal, and chronic anti-in?ammatory properties. The
`naturally occuring members of this family of structur
`ally related compounds are produced by various fungi
`imperfecti. Cyclosporins A and C, are the major com
`ponents. Cyclosporin A, which is discussed further
`below, is a particularly important member of the cyclos
`porin family of compounds. Twenty four minor metab
`olites, also oligopeptides, have been identi?ed: Lawen
`et al, J. Antibiotics 42, 1283 (1989); Traber et al, Helv.
`Chim. Acta 70, 13 (1987); Von Wartburg and Traber
`Prog. Med. Chem., 25, l (1988).
`Isolation of cyclosporins A and C, as well as the
`structure of A were reported by A. Ruegger et al.,
`Helv. Chim. Acta 59, 1075(1976); M. Dreyfuss et al., J.
`Appl. Microbiol. 3, 125 (1976). Crystal and molecular
`structures of the iodo derivative of A have been re
`ported by T. J. Petcher et al., Helv. Chim. Acta 59, 1480
`(1976). The Structure of C was reported by R. Traber et
`al., ibid. 60, 1247 (1977). Production of A and C has
`been reported by E. Harri et al., US. Pat. No. 4,117,118
`(1978 to Sandoz). Isolation, characterization and anti
`fungal activity of B, D, E, as well as the structures of A
`through D have been reported by R. Traber et al., Helv.
`Chim. Acta 60, 1568( 1977). Isolation and structures of
`E, F, G, H, I: eidem, ibid. 65, 1655 (1982). Preparation
`of [2-Deutero-3-?uoro-D-Ala]8-CsA is disclosed by
`Patchett et al in GB 2,206,199A which was published on
`Dec. 29, 1988.
`Further properties have also been reported in studies
`of the biological activity of A: J. F. Borel et al., Agents
`Actions 6, 468 (1976). Pharmacology: eidem, Immunol
`ogy 32, 1017 (1977); R. Y. Calne, Clin. Exp. Immunol.
`35, l (1979). Human studies: R. Y. Calne et al., Lancet
`2, 1323(1978); R. L. Powles et al., ibid. 1327; R. L.
`Powles et al., ibid 1, 327 (1980). In vitro activity (por
`cine T-cells): D. J. White et al., Transplantation 27, 55
`(1979). Effects on human lymphoid and myeloid cells:
`M. Y. Gordon, J. W. Singer, Nature 279, 433(1979).
`Clinical study of A in graft-versus-host disease: P. J.
`Tutschka et al., Blood 61, 318(1983).
`As exempli?ed by the ever expanding list of indica
`tions for which Cyclosporin A has been found useful,
`the cyclosporin family of compounds ?nd utility in the
`prevention of rejection or organ and bone marrow
`transplants; and in the treatment of psoriasis, and a num
`ber of autoimmune disorders such as type 1 diabetes
`mellitus, multiple sclerosis, autoimmune uveitis, and
`rheumatoid arthritis. Additional indications are dis
`cussed infra.
`As is generally accepted by those of skill in the art,
`inhibition of secretion of interleukin~2 (IL-2) and other
`lymphokines from lymphocytes, is a useful indicator of
`intrinsic immunosuppressive activity of a cyclosporin
`analog. For a recent review of cyclosporin uses and
`mechanisms of action see Wenger et a1 Cyclosporine:
`Chemistry, Structure-Activity Relationships and Mode
`
`wherein the superscript number de?nes the position of
`the amino acid. Because of our speci?c interest in the
`amino acid at position 8, we will hereinafter replace
`“R3” with “Y”, thereby emphasizing that amino acid.
`As is the practice in the ?eld, a particular cyclosporin
`analog may be named using a shorthand notation identi
`fying how the analog differs from cyclosporin A. Thus,
`cyclosporin C which differs from cyclosporin A by the
`threonine at position-2 may be identi?ed as [ThrP
`cyclosporin or [ThrP-CsA. Similarly, cyclosporin B is
`[AlaP-CsA; cyclosporin D is [ValP-CsA; cyclosporin E
`is [Val]1l-CsA; cyclosporin F is [3-DesoxyMeBmt]1
`CsA; cyclosporin G is [NVaP-CsA; and cyclosporin H
`is [D-MeValPI-CsA.
`D-Serine and D-Threonine have been introduced into
`the 8-position of cyclosporin A by biosynthesis result
`ing in active compounds. See R. Traber et al. J. A ntibior
`ics 42, 591 (1989). D-Chloroalanine has also been intro
`duced into position-8 of Cyclosporin A by biosynthesis.
`See A. Lawen et al J. Antibiotics 52, 1283 (1989).
`The present invention concerns new analogs of cy
`closporin A and related cyclosporins for the care of
`immunoregulatory disorders and diseases, including the
`prevention, control and treatment thereof.
`
`45
`
`50
`
`55
`
`SUMMARY OF THE INVENTION
`This invention relates to [dehydro-Ala]8 cyclosporins
`and their preparation and conversion to novel cyclospo
`rin analogs useful as alternatives to cyclosporin A.
`More speci?cally, the invention relates to [dehydro
`Ala]8 cyclosporins and derived therefrom cyclosporin
`analogs having a sulfur containing amino acid at posi
`tion-8.
`
`65
`
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`

`
`5,122,511
`
`3
`DETAILED DESCRIPTION OF THE
`INVENTION
`This invention relates to cyclosporin analogs of the
`formula
`
`5
`
`4
`R4 may be, but is not limited to MeLeu or MeVal;
`R5 may be, but is not limited to Val or Nva;
`R6 may be. but is not limited to, MeLeu or MeVal;
`R7 may be. but is not limited to Ala. Abu. or L-phenyl
`alanyl;
`R9 may be, but is not limited to MeLeu or MeVal;
`Rlomay be, but is not limited to MeLeu, or MeVal; and
`R11 may be, but is not limited to MeVal, D-MeVal or
`MeNva.
`One embodiment within the scope of the invention, is
`the cyclosporin analogs selected from the group con
`sisting of:
`(a) [3-DesoxyMeBmt1l[Y]3—CsA;
`
`(e) [Nva]2[Y]8-CsA and dihydro and iso [Nva]2[Y]8
`
`One class of compounds within the embodiment is the
`compounds wherein, R is CH3(OCH3CH3),, —~S(O)m
`wherein m is O or] and n is 1,2,3 or 4 or R,,S(O)m; m is
`O is l; and Ra is selected from the group consisting of
`l) H unless m is l,
`2) C14, alkyl, such as methyl, ethyl, isopropyl or tert
`butyl;
`.
`3)substituted C14, alkyl wherein the substitutent is se
`lected from the group consisting of,
`(a)
`
`wherein R}, is C14, alkyl or hydrogen,
`(b) CH, acylamino-,
`(c) —NR1,R( wherein RC is C14, alkyl or hydrogen;
`(d) -hydroxy; and
`(e) CM acyloxy-.
`A second embodiment within the scope of the inven
`tion, is the compounds of formula II
`
`3
`2
`1
`ll
`10
`MeLeu--MeVal-MeBmt—Abu-Sar
`|
`9 MeLeu
`|
`Y- Ala — MeLeu — Val -— MeLeu
`8
`7
`6
`5
`4
`
`II
`
`One class of compounds within this embodiment is
`the compounds wherein, R is CH3(OCH2CH2)n—-S
`(0),,I wherein m is 0 or 1 and n is 1,2,3 or 4 or R,,S(O)m;
`wherein m is O or 1; and Ra is selected from the group
`consisting of
`a
`l) H provided that m is 0,
`2) C14, alkyl, such as methyl, ethyl, isopropyl or tert
`butyl;
`3) substituted CM alkyl wherein the substitutent is se
`lected from the group consisting of,
`(a)
`
`wherein R1, is C14, alkyl or hydrogen,
`(b) C14, acylamino-,
`
`wherein the amino acid moiety at position-8 is Y, and Y
`is [dehydro-Ala], namely
`
`wherein
`R is CH3(O—CH3——CI-Ig),,—-S(O)m—, wherein m is O
`or 1 and n is 1,2,3, or 4: or
`RaS(O),,,—, wherein R, is selected from the group con
`sisting of
`l) H, provided that m is O;
`2) C14, alkyl, such as methyl, ethyl, isopropyl or tert
`butyl;
`3) substituted C14, alkyl wherein the substitutent is
`selected from the group consisting of,
`(a)
`
`wherein Rb is C14, alkyl or hydrogen,
`(1)) —NRbRC wherein R( is CH, alkyl or hydrogen;
`(c) C14, acylamino-;
`(d) -hydroxy; and
`(e) Cm acyloxys
`4) benzyl or phenyl;
`5) substituted benzyl or phenyl wherein substitutents
`are selected from the group consisting of C1.4alkyl,
`hydroxyl, C14 alkyloxy, and halo,
`
`20
`
`25
`
`35
`
`55
`
`wherein
`R1 may be, but is not limited to MeBmt, 3-desox
`yMeBmt or dihydroMeBmt;
`R2 may be, but is not limited to Abu, Ala, Nva, SeR,
`Thr or Val;
`R3 may be, but is not limited to, Sar or N-methyl-D-ala
`nyl;
`
`65
`
`NOVARTIS EXHIBIT 2009
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`

`
`5
`(c) —NR1,Rr wherein R,- is C14, alkyl or hydrogen;
`(d) -hydroxy; and
`(e) C14, acyloxy-.
`Illustrating this class are:
`D-[3-methylthio-Ala]3-CsA;
`D-[3-carbomethoxymethylthio-Ala]3-CsA;
`D-[3(2-hydroxyethylthio)Ala]3-CsA; [dehydro-AlaP
`CSA;
`D-[S-benzylthio-Ala]S-CsA;
`D-[3-phenylthio-AlaFeCsA;
`D-[3-methylthio-Ala]3-CsA sulfoxide;
`D-[3-(2-hydroxyethylthio)Ala]8-CsA sulfoxide; and
`D-[methoxyethoxy)ethoxyethylthio-Ala]B-CsA.
`Compounds of the present invention are conveniently
`prepared using the procedures described generally
`below and more explicitly in the Example Section
`thereafter.
`Now turning to Scheme 1, in one embodiment the
`cyclosporin analogs of this invention are conveniently
`prepared via conversion of [X-D-AlaF-CsA to [A
`Ala]3-CsA.
`‘
`
`10
`
`,... 5
`
`Scheme 1
`
`R SH
`[A-AlaP-CsA L9 [RaS-Ala]8-CsA
`
`25
`
`5,122,511
`6
`lithium or potassium methoxide or hydride, of which
`sodium methoxide is preferred.
`The second solvent includes. but is not limited to
`C1_galkano1s corresponding to the selected second base,
`such as methanol and ethers (as de?ned above) such as
`l,2-dimethoxyethane, or tetrahydrofuran, of which
`methanol and tetrahydrofuran are preferred. ()ne exam
`ple of corresponding second solvent and base is metha
`nol and sodium methoxide.
`The sulfur nucleophile includes, but is not limited to
`RSH wherein R is CH3(O—CH2——CH2)n-—S(O)m, and
`RuSH wherein Ra is given its broadest de?nition pro
`vided above.
`T he reaction can be conveniently conducted in a
`temperature range of 0° to 50° C., of which 15° to 30° C.
`is preferred. The reaction is allowed to proceed to com
`pletion in l to 36 hours, of which 15 to 18 hours is
`preferred.
`In Scheme 1 the A-Ala moiety of [A-Ala]8-CsA
`serves as a Michael acceptor to various nucleophiles.
`As an alternative to the above, [A-Ala]-CsA can be
`produced from [D-Ser]8-CsA. In this procedure [D
`Ser]3-CsA is treated with a slight excess of methanesul
`fonyl chloride or toluenesulfonyl chloride in methylene
`chloride in the presence of 4-dimethylaminopyridine to
`yield, after chromatography, [methane or toluene sub
`stituted sulfonyloxy-D-SerP-CsA. These compounds
`can be treated with excess LDA in THF at low temper
`atures to yield [A-AlaP-CsA. Similarly [D-Chloro
`Ala]8-CsA can be treated with excess LDA in THF at
`low temperatures to yield [A-AlaP-CSA.
`Compounds [D-RS-AlaP-CsA (wherein R is
`CH3(O—CH3--CH2)n-S(O)m) and [D-RaS-AlaP-CsA
`may also be produced by an alternate route from [D
`Cys]3-CsA (which is the same as [D-HS-AlaPCsA) by
`reaction with RX and RaX (wherein R0 is not phenyl or
`substituted phenyl and X is chlorine, bromine or a sul
`fonyloxy aryl or alkyl group such as mesyloxy or tosy
`loxy) as indicated in Scheme 2 which also shows pro
`duction of [D-HS-AlaF-CsA by reaction of [A-Ala]3
`C'sA with the sodium salt of thiolacetic acid followed
`by hydrolysis.
`As appreciated by those of skill in the art, the remain
`ing compounds within the scope of the invention can be
`produced in an analogous manner.
`‘The SRa groups in the disclosed compounds can be
`oxidized to the corresponding sulfoxides. A convenient
`route to sulfoxides is by periodate oxidation as de
`scribed below.
`
`wherein X is fluoro, chloro, methanesulfonyloxy,
`toluenesulfonyloxy and Ra is the broadest de?nition of
`Ra provided above.
`According to Scheme 1 [2-deutero-3-?uoro-D-Ala]8
`CsA (abbreviated as [F-D-AlaP-CsA), in an aprotic
`solvent, is reacted with an aprotic base to yield [AAla]3
`CsA.
`[F-D-AlaF-CsA possesses 17 active hydrogens (l2
`aCH, 4-NH AND l-OH). Accordingly, a large excess
`of aprotic base is required to generate the polyanion.
`The molar ratio of aprotic base to [F-D-AlaP-CsA may
`range from 17 to 35 of which 20-25 is preferred. Suit
`able aprotic bases include, but are not limited to, mono
`or diCMalkylamido derivatives such as lithium diethyl
`amide, lithium diisopropylamide, sodium bis (trimethyl
`silyl)amide, lithium bis (trimethylsilyl)amide of which
`lithium diisopropylamide is preferred. Suitable aprotic
`solvents include, but are not limited to, diC1_4alkoxy
`C14alkane derivatives such as 1,2-dimethoxyethane;
`ethers such as diethyl ether di-n-butyl and diisopentyl
`ethers, cyclic ethers such as tetrahydropyran, dihydro
`pyran, tetrahydrofurfuryl methyl ether, furan, tetrahy
`drofuran and Z-ethoxytetrahydrofuran, and mono or
`diC1_4alkyl carbonyl amines such as dimethylformam
`ide. Tetrahydrofuran is preferred.
`The reaction may be conveniently conducted in a
`temperature range of - 100° to — 10° C., of which — 70°
`to —30" C. is preferred. The reaction is allowed to
`proceed to completion in l to 24 hours, of which a 4 to
`5 hour reaction time is preferred.
`The [A-AlaP-CsA product can be isolated by stan
`dard chromatography, HPCL or TLC on silica gel
`plates as is known in the art.
`[A-AlaP-CsA is then converted into the thio com
`pound [R”S-Ala]-CsA by reaction in a second solvent
`with a sulfur nucleophile in the presence of a second
`base.
`The second base includes, but is not limited to, the
`alkali metal C1-6alkoxides and hydrides, such as sodium,
`
`45
`
`Scheme 2
`
`55
`
`[D-Hs-AuPcsA
`
`65
`
`As shown in Scheme 2 treatment of [A-Ala]3CsA
`_with 5-10 equivalents of CH3COS-—Na (generated in
`situ from equivalent quantities of CH3COSH and
`CH3ONa) in CH3OH for 15-18 hr at 20-25“ C. pro
`
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`

`
`Scheme 3
`
`Scheme 4
`
`NalO4
`
`5,122,511
`8
`7
`duces [D-CH3COS-Ala]8-CsA which is partly deacetyl
`animal body weight. conveniently given in divided
`ated. Deacetylation to [D-HS-alaPCsA is completed by
`doses 2 to 4 times a day or in sustained release form. For
`reaction with CH3ONa (l-5 equivalents) in a C1-galk
`the larger mammals, the total daily dosage is in the
`anol such as methanol for 3-18 hr at 20-25" C. Reaction
`range from about 50 to about 5000 mg. and dosage
`of [D-I‘IS-AlaF-CSA with R‘X (5-10 equivalents) in the
`forms suitable for oral administration comprise from
`presence of CHgONa (l-2 equivalents) in an C|.galk
`about 15 mg to about 500 mg (e.g. 25-300 mg) of the
`anol such as CH3OH for 15-18 hr at 20-25" C. yields [D
`compounds admixed with a solid or liquid pharmaceuti
`RaS-AlaPCsA. In this procedure Ra is not phenyl or
`substituted phenyl.
`cal carrier or diluent.
`For example a compound accessible by this route is
`The present invention also provides a pharmaceutical
`[D-3-thia-Lys]8-CsA, ([D-HgNCHgCHgS-Ala]S-CsA).
`composition comprising a compound of formula II in
`This compound is useful in preparing af?nity chroma
`association with a pharmaceutical carrier or diluent.
`tography columns for cyclosporin receptor isolation
`Such compositions may be in the form of, for exam
`and to prepare cyclosporin antibodies.) [D-3-Thia
`ple, a solution, a tablet or a capsule and in ointments
`LysPCsA may be prepared as indicated in Scheme 3.
`especially for the treatment of psoriasis.
`The cyclosporins of formula II may be administered
`by any conventional route, in particular in accordance
`with means currently practiced in relation to adminis
`tration of cyclosporine, in particular via intravenous
`infusion, e.g. in the case or organ transplant, pre- and
`immediately post-‘transplant, as well as during episodes
`of gastrointestinal disturbance which might otherwise
`impair absorption, or orally, e.g. in the form of an oral
`solution.
`Biological activity can be measured in terms of bind
`ing af?nity for cyclophilin, the cytosolic receptor for
`cyclosporin (R. Handschumacher et al., Science. 226
`(1984) 544), inhibition of interleukin-2 production, and
`inhibition of T-cell proliferation. Table 2 illustrates the
`pharmacological activity of representative compounds
`of the present invention.
`T-cell proliferation was measured in mouse T-cell
`cultures stimulated with ionomycin plus phorbol myris
`tate acetate (PMA). Spleen cell suspensions from
`C57Bl/6 mice were prepared and separated on nylon
`wool columns. The recovered T-cells were suspended
`at 106 cells/ml in complete culture medium with addi
`tion of ionomycin (250 ng/ml) and PMA (10 ng/ml).
`The cell suspension was immediately distributed in 96
`well-flat bottom microculture plates at 100 til/well.
`Control medium or various concentrations of test com
`pound were added in triplicate wells at 10 ul/well. The
`plates‘ were incubated at 37‘ C. in a humidi?ed atmo
`sphere of 5% Cog-95% air for 44 hours. At 44 hours of
`culture, the plates received 20 ul/well of a solution of
`(3-(4,5-dimethylthiazol-2-yl)-2,S-diphenyltetrazolium
`bromide (MMT) in PBS (10 mg/ml). To dissolve the
`purple crystals of MTT formazan produced by metabol
`ically active cells, 100 pl of_a 10% SDS-0.0l N hydro
`chloric acid solution was added to each well. The cul
`ture plates were incubated at 37' C. in a 5% CO2 incuba
`tor. The plates were read at 570-600 nm in a multiwel]
`scanning spectrophotometer. The absorbance (speci?c
`OD) of experimental wells was corrected for that of
`wells with unstimulated cells or no cells. The percent
`inhibition of proliferation was calculated according to
`the formula:
`
`20
`
`25
`
`30
`
`As shown in Scheme 4, the [RaS-Ala]8 cyclosporins
`are converted into the corresponding sulfoxides by
`treatment with sodium periodate in aqueous alcohol
`with methanol-water in the ratio 3:1 as the preferred
`solvent. The time may range from 3 to 36 hours with
`15-18 hours preferred. The preferred temperature
`range is 20—25° C.
`In view of their immunosuppressive activity, end
`product cyclosporins eg of formula II, are useful for
`the prophylaxis and treatment of diseases and condi
`tions requiring a reduction of the immune response.
`45
`Thus they may be used to suppress the proliferation of
`lymphocytes and immunocytes, e.g. in treatment of
`autoimmune diseases or in preventing the rejection of
`transplants e.g. skin, lung, heart, heart-lung, bone-mar
`row, kidney, spleen and corneal transplants.
`Speci?c auto-immune diseases for which the cyclos
`pon'ns of formula II are useful include all of those for
`which treatment with cyclosporine has been proposed
`or used, for example, aplastic anaemia, pure red cell
`anaemia, idopathic thrombocytopaenia, systemic lupus
`erythematodes, polychondritis, sclerodoma, Wegener
`granulomatosis, chronic active hepatitis, myasthenia
`gravis, psoriasis, Steven-Johnson syndrome, idiopathic
`sprue, Crohn’s diseases, Graves opthalmopathy, sarcoi
`dosis, multiple sclerosis, primary biliary cirrhosis, pri
`mary juvenile diabetes, uveitis posterior, interstital lung
`?brosis and psoriatic arthritis as well as insulin-depend
`ent diabetes mellitus, nephrotic, syndrome and AIDS.
`For all these uses the dosage will, of course, vary
`depending on the compound employed, mode of admin
`istration and treatment desired. However, in general,
`satisfactory results are obtained when administered at a
`daily dosage of from about 1 mg to about 200 mg per kg
`
`50
`
`60
`
`. _
`7‘ lnhlb' — ‘00
`
`_ Spgci?c OD expgrimental
`Speci?c OD control medium X 100
`
`NOVARTIS EXHIBIT 2009
`Par v Novartis, IPR 2016-00084
`Page 5 of 9
`
`

`
`9
`TABLE 2
`IMMUNOSUPPRESSIVE ACTIVITIES OF
`CYCLOSPORIN ANALOGS
`
`5,122,511
`
`10
`-continued
`Culture: Tolvpocladium in?arum MFSOBO. NRRL-8044
`g/L
`
`seed Medium B
`l|vleLeu—MeVal—MeBmt-Abu—Sar
`70.0
`Malt Ext.
`M eLe“
`50-0
`Glucose
`Y——-Ala—MeLeu-—Val—MeLeu W
`Glucose
`40.0
`Caeinpeptone
`l0.0
`MgSO4.7H;O
`0.5
`KH2PO4
`2.0
`NaNO;
`3.0
`KC]
`05
`Feso4.7H2o
`0.01
`
`5
`
`T-Cell
`Proliferation
`lnhibitionb
`
`10
`
`'
`
`Y
`
`_
`[CH3S Ala]
`
`Cyclophilin
`Bindinga-b
`
`76.
`
`n
`[CH3O—C—CH2'-S-Ala]
`
`3.
`
`24.
`:L
`3()_
`
`15
`
`.
`
`.
`
`.
`
`A lyophtle tube was aseptlcally opened and grown In
`seed medium B (20 ml in a 250 ml 3-baffle Erlenmeyer
`flask) for 4 days on a rotary shaker (220 rpm) at 27° C.
`This seed was then used to inoculate slants (medium
`20 A) for future studies. The slants were incubated at 27°
`C. for 14 days after which time they were stored at 4° C.
`until used
`The spores were washed from an entire slant with 5
`ml of medium C and used to inoculate a preculture flask
`(50 ml medium C in a 250 ml Erlenmeyer flask). This
`preculture was incubated for 5 days at 27° C.
`Five ml of the preculture was used to inoculate the
`production medium (50 ml of medium C and 5 mg/ml of
`2-deutero'3-fluoro-D-alanine in a 250 ml Erlenmeyer
`?ask). The ?lter sterilized Z-deutero-3-fluoro-D'alanine
`was added (5 mg/ml, ?nal concentration) post-steriliza
`tion and prior to inoculation. Forty-four flasks contain
`ing a total of 2.2 liters of production medium were
`incubated 14 to 21 days with agitation (220 rpm) at 27°
`C. Following incubation, the fermentation broths were
`extracted by procedures described below in item C.
`
`[HOCHZCH2_S_M31
`
`23'
`85:
`54,
`30.
`approx.
`[Dehydro-Ala]
`"This assay is desscribed in detail by R. Handschumacher er al.. Science. 220 (I984)
`544.
`I’l'he data are expressed as ‘7: CsA activity (CsA(cyclosporin A) = 100).
`
`25
`The following examples illustrate the preparation of
`the invention compounds of formula II and as such are
`not to be considered as limiting the invention set forth in
`the claims appended thereto.
`Preparation of the cyclosporin analogs of the instant
`invention are depicted in Schemes 1-4 above.
`Preparation of [2-deutero-3-?uoro D-Ala]8-CsA is
`disclosed in GB 2,206,119A ?led by Patchett et al in
`Jun. 20, 1988. Preparation [2-deutero-3-fluoro-D-ala]8
`CsA is also disclosed in Example 1.
`Preparation of ?uorinated amino acids such as 3 fluo
`ro-alanine, is well known to those skilled in the art. See,
`for example, Kollonitsch, J. Israel J. of Chemistry Vol.
`17 pp 53-59 (1978) and Durette et al., Transplantation
`Proceedings Vol 20 No. 2 suppl 2 pp 51-77 (April
`1988).
`As described above, each of the cyclosporin analogs
`within the scope of the invention is prepared from a
`preferred cyclosporin analog starting material possess
`ing [2-deutero-3-fluoro-D-Ala]8 or [3-chloro-D-Ala]8 or
`[D-Ser]3. These substituted cyclosporin analogs can
`also be made by total synthesis as taught by Wenger in
`US. Pat. No. 4,396,542 issued Aug. 2, 1983 as ampli?ed
`in US. Pat. No. 4,798,823 issued Jan. 17, 1989, which
`patents are hereby incorporated by reference. In these
`total syntheses the D-Ala component is replaced by
`2-deutero-3-?uoro-D-Ala, 3-chloro-D-Ala or D-Ser to
`generate the corresponding 8-substituted cyclosporin.
`The remaining starting materials for the process are
`available commercially, and/or their method of prepa
`ration known.
`
`35
`
`EXAMPLE 2
`Preparation of [3-?uoro-D-alanine]8-CsA
`Following essentially the same procedures as de
`scribed in Example 1 except that the preculture was
`used to inoculate a production medium of a total vo
`lumn of 400 ml containing 5 mg/ml of 3-?uoro-D-ala
`nine instead of 2-deutero-3-?uoro-D-alanine, there was
`obtained the fermentation broth which was extracted
`by the procedures described below in item C.
`
`A. Extraction Methodology
`a. The cells were removed from the broth by centrifu
`' gation.
`. The clari?ed broth was extracted 3 times each with
`25 ml portions of methylene chloride.
`c. The cells were extracted 3 times each with 25 ml
`portions of acetone.
`. The methylene chloride and acetone extracts were
`pooled and taken to dryness under vacuum.
`e. The residue was solubilized with methanol, dried
`with anhydrous Na2SO4, ?ltered and taken to dryness
`under vacuum.
`f. The samples were submitted for HPLC analysis to
`determine and isolate the cyclosporin derivatives.
`
`EXAMPLE 1
`Preparation of [2-deutero-3-fluoro-D-alanine]8
`cyclosporin A
`
`Culture: Tolypoclaclium inflatum MF5080. NRRL-8044
`g/L
`
`Media: Slant Medium A
`Malt Ext.
`Yeast Ext.
`Agar
`
`20.0
`4.0
`20.0
`
`65
`
`B. HPLC Analysis of [F-D-Ala]8 CsA
`Crude extracts were assayed by HPLC chromatogra
`phy using the following chromatographic system.
`Solvent: 80/20 vzv acetonitrilezwater
`Flow rate: 0.6 mL/min
`
`NOVARTIS EXHIBIT 2009
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`Page 6 of 9
`
`

`
`11
`Column: DuPont Zorbax ODS 4.6 mm><25 cm main
`tained at 60° C.
`Detector: LDC Spectromonitor III, 210 nm 0.05 AUFS
`Integrator: Spectra-Physics SP4100 Computing Inte
`grator
`The extraction residue from one 400 ml fermentation
`was taken up in 1 m1 of methylene chloride and the
`solution chromatographed on a 40 ml column of Phar
`macia LH-20 previously equilibriated with methanol.
`The chromatography was carried out with methanol at
`a ?ow rate of 2 ml/min., collecting one ten ml fraction
`followed by 30X 1 ml fractions. Fractions 16 through 27
`were selected and combined, based on I-IPLC analysis.
`The combined fractions were concentrated to dryness
`and the residue labeled F.
`Sample F was taken up in 250 m] of methanol and
`subjected to preparative HPLC chromatography on a
`DuPont Zorbax ODS column 0.9 X25 cm maintained at
`60° C., Chromatography was carried out with a solvent
`system of 80:20 vzv acetonitrilezwater at a flow rate of 2
`ml/min. The effuent stream was monitored at 220 nm
`using an LDC Spectromonitor II equipped with a 1 mm
`path length cell and a setting 1.28 AUFS. The ultra-vio
`let signal was monitored using a Spectra-Physics
`SP4100 computing integrator and eleven fractions were
`collected based on the ultra-violet trace. Fraction 7
`contained 3.25 mg of 8-[3-?uoro-D-alanine]g-CsA with
`an ultra-violet purity of >99% at 210 nm by HPLC
`analysis. Fraction 7 was concentrated to dryness under
`30
`high vacuum to yield 3.3 mg of [3-?uoro~D-alanine]8
`CsA.
`
`5,122,511
`12
`less polar band is obtained 19 mg of recovered [2
`deutero-3-?uoro-D-Ala]8CsA.
`EXAMPLE 4
`D-[3-Methylthio-Ala]g-CsA
`To a stirred solution of [dehydro-Ala]8 CsA (45 mg;
`0.037 mMol) in methanol (1.0 ml) is added sodium me
`thylmercaptide (60 mg) in methanol (1.5 ml). The mix
`ture is kept 18 hours at 20‘ C. It is then added to 20 ml
`of saturated aqueous sodium chloride containing 0.3 g
`sodium bisulfate and the mixture is extracted with ethyl
`acetate (4X15 ml). The organic extract is washed with
`saturated aqueous sodium chloride (2X15 ml), dried
`over sodium sulfate and concentrated to dryness under
`sodium sulfate and concentrated to dryness under vac
`uum. The residue (38 mg) is puri?ed by HPLC (column
`:Dupont Zorbax ODS 0.94X25 cm; solvent system
`:acetonitrile:water=70:30; 2.65 ml/min. at 60" C.) to
`give 12 mg (26%) of D-[3-methylthio-Ala]8 CsA (esti
`mated amount present 16 mg (34%); R, 20.5 min
`(CsA=l7.4 min) FAB-MS: M++1=1248 -consistent
`with molecular formula Cal-1113151110125.
`13C NMR Chemical Shifts (CDC13, 75 MHz): 9.8,
`15.8, 16.6, 17.0, 17.9, 18.4, 18.8, 19.9, 20.3, 21.2, 21.7,
`22.2, 23.3, 23.5, 23.7, 23.81, 23.84, 24.4, 24.6, 24.89,
`24.94, 25.4, 29.2, 29.85, 29.93, 30.0, 31.1, 31.3, 31.5, 33.7,
`35.4, 35.8, 36.0, 37.1, 37.3, 39.5(2><), 40.6, 48.2, 48.7,
`48.8(2><), 50.3, 55.1, 55.36, 55.44, 57.5, 58.1, 58.8, 74.5,
`126.3, 129.7, 170.05, 170.07, 170.2, 170.3, 171.1, 171.5,
`171.7, 171.9, 173.4, 173.61 and 173.66 ppm. The carbon
`. count of 63 is consistent with the molecular formula.
`
`25
`
`35
`
`45
`
`EXAMPLE 3
`[Dehydro-Ala] 8-CsA
`To 2.0 ml of tetrahydrofuran stirred at -—78° under
`nitrogen is added 0.6 ml of 1.5M (0.9 mmol) lithium
`diisopropylamide in cyclohexane. To this solution is
`added 50 mg (0.042 mmol) of [2-deutero-3-tluoro D
`A]a]3 cyclosporin A in 1.0 ml of tetrahydrofuran. The
`mixture is stirred at —78° for 30 minutes and the tem
`perature is slowly raised to -—30° over 4 hours. The
`mixture is cooled to —78° and quenched by adding 0.15
`ml of acetic acid in 0.9 ml of water. It is then added to
`20 ml of saturated aqueous sodium chloride containing
`0.2 g of sodium bisulfate and extracted with ethyl ace
`tate (3X20 ml). The latter extract is washed with satu
`rated aqueous sodium chloride (2><2O ml), dried over
`sodium sulfate and taken to dryness under vacuum. The
`residue (47 mg) is puri?ed by preparative TLC (three
`500 v 20X 20 cm silica gel plates; system-chloroformze
`thanol=96:4: two developments) to give two major
`bands. From the more polar band is obtained [dehydro
`Ala]8-CsA (17 mg) as a colorless solid; 34% direct yield;
`55
`53% conversion yield. HPLC-Dupont Zorbax ODS
`column; 80:20=C1~13CN:H2O/60°; R,=14 minutes.
`FAB-MS=M+ +1=1200-consistent with molecular
`formula C62H109N11O12.
`l3C NMR Chemical Shifts (CDC13, 100 MHz): 9.9,
`15.8, 16.6, 17.9, 18.6, 18.9, 19.5, 20.2, 21.2, 21.8, 22.0,
`23.1, 23.4, 23.7(2X), 23.9, 24.6, 24.7, 24.9, 25.0, 25.2,
`29.0, 30.1, 30.4, 31.1, 31.2, 31.3, 32.5, 33.9, 35.6, 35.9,
`36.0, 37.1, 39.2, 39.3, 40.8, 48.9, 49.2, 49.3, 50.2, 54.9,
`55.2, 55.5, 57.5, 57.8, 58.3, 74.7, 108.3, 126.3, 129.5,
`134.8, 167.6, 170.0 170.1, 170.3, 170.5, 170.8, 171.1,
`171.9, 173.4, 173.50 and 173.53 ppm. The carbon count
`of 62 is consistent with the molecular formula. From the
`
`60
`
`65
`
`EXAMPLE 5
`D-[3-carbomethoxymethylthio-Ala]8CsA