(12) United States Patent
`Naicker et al.
`
`USOO661.3739B1
`(10) Patent No.:
`US 6,613,739 B1
`(45) Date of Patent:
`Sep. 2, 2003
`
`(54) DEUTERATED CYCLOSPORINE ANALOGS
`AND THEIR USE AS
`IMMUNOMODULATING AGENTS
`(75) Inventors: Selvaraj Naicker, Edmonton (CA);
`Randall W. Yatscoff, Edmonton (CA);
`Robert T. Foster, Edmonton (CA)
`
`(73) Assignee: Isotechnika, Inc., Edmonton (CA)
`(*) Notice:
`Subject to any disclaimer, the term of this
`tent is extended
`diusted under 35
`ps g 5), O Gy JuSled under
`a --
`(21) Appl. No.: 09/634,945
`(22) Filed:
`Aug. 7, 2000
`Related U.S. Application Data
`(63) Continuation of application No. 09/184,109, filed on Nov. 2,
`1998.
`(51) Int. Cl." ................................................ A61K 38/00
`(52) U.S. Cl. ......................... 514/11; 530/321, 530/350
`530/363; 530/806; 530/807
`(58) Field of Search ............................ 514/11; 530/321,
`530/350, 363, 806, 807
`References Cited
`U.S. PATENT DOCUMENTS
`4,108,985 A 8/1978 Riegger et al. .............. 514/11
`4,117,118 A 9/1978 Harri et al.
`4,201,771 A 5/1980 Onishi et al.
`4,210,581 A 7/1980 Riegger et al.
`4,220,641 A 9/1980 Traber et al.
`4,288.431 A 9/1981 Traber et al.
`4,384,996 A 5/1983 Bollinger et al.
`4,396,542 A 8/1983 Wenger ...................... 530/321
`4,404,194 A 9/1983 Arala-Chaves
`4,554,351 A 11/1985 Wenger ...................... 544/177
`4,639.434 A 1/1987 Wenger et al................. 544/11
`4,681,754. A
`7/1987 Siegl
`
`(56)
`
`EP
`EP
`EP
`WO
`
`4,703,033. A 10/1987 Seebach
`4,727,018 A 2/1988 Eichner et al.
`(List continued on next page.)
`FOREIGN PATENT DOCUMENTS
`O 283 8O1
`9/1988
`O 373 260
`6/1990
`O 557 544
`1/1994
`WO952.6325
`10/1995
`OTHER PUBLICATIONS
`Curran, D., et al. “Intramolecular Hydrogen Transfer Reac
`tions of o-(Bromophenyl)dialkylsilyl Ethers. Preparation of
`Rapamycin-d” Tetrahedron Letters. 33(17):2295-2298
`(1992).
`1.
`Eberle, M. K., et al. “Modifications of the MeBmt Side
`Chain of Cyclosporin A.” Bioorganic & Medicinal Chem
`istry Letters. 5(15):1725–1728 (1995).
`(List continued on next page.)
`Primary Examiner Brenda Brumback
`ASSistant Examiner Anish Gupta
`(74). Attorney, Agent, or Firm-Burns Doane Swecker &
`Mathis LLP
`ABSTRACT
`(57)
`Cyclosporine derivatives are disclosed which possess
`enhanced efficacy and reduced toxicity over naturally occur
`ring and other presently known cyclosporins and cyclospo
`rine derivatives. The cycloSporine derivatives of the present
`invention are produced by chemical and isotopic Substitution
`of the cyclosporine A (CSA) molecule by: (1) Chemical
`Substitution and optionally deuterium Substitution of amino
`acid 1; and (2) deuterium Substitution at key sites of metabo
`lism of the cycloSporine A molecule Such as amino acids 1,
`4,9. Also disclosed are methods of producing the cycloSpo
`rine derivatives and method of producing immunosuppres
`Sion with reduced toxicity with the disclosed cycloSporine
`derivatives.
`
`16 Claims, 4 Drawing Sheets
`
`Apotex Ex. 1013
`
`Apotex v. Auspex
`IPR2021-01507
`
`

`

`US 6,613,739 B1
`Page 2
`
`U.S. PATENT DOCUMENTS
`
`
`
`5,643,870 A 7/1997 Boelsterli et al. ............. 514/11
`5,709,797 A
`1/1998 Bocchiola et al.
`5,741,512 A 4/1998 Hauer et al. ................ 424/450
`5,741,775 A
`4/1998 Balkovec et al.
`5,747,330 A 5/1998 Casareto et al.
`5,767,069 A 6/1998 Ko et al. ...................... 514/11
`
`OTHER PUBLICATIONS
`
`of
`Preparation
`“The
`al.
`et
`O.,
`HenSens,
`Cyclosporin A By
`2-Deutero-3-Fluoro-D-ALA
`Directed Biosynthesis.” J. Antibiotics. 45(1): 133-135
`(1992).
`Hughes, P. et al. “The Isolation, Synthesis and Character
`99
`ization of An Isomeric Form of Rapamycin.” Tetrahedron
`L
`3333): 4739-4742 (1992
`etters. 33(33):
`(1992).
`Park, S.B., et al. “A Semi-Synthetic Approach to Olefinic
`Analogs of Amino Acid One (MeBMT) in Cyclosporin A.”
`Tetrahedron Letters, 30, 32, 4215-4218 (1989).
`Patchett, A., et al. “Analogs of Cyclosporin A Modified at
`the D-ALA Position.” J. Antibiotics. 45(1):94-102 (1992).
`
`4,764,503 A 8/1988 Wenger - - - - - - - - - - - - - - - - - - - - - - - 514/11
`4,765,980 A 8/1988 DePrince et al.
`4,771,122 A
`9/1988 Seebach
`4,798.823 A
`1/1989 Witzel ......................... 514/11
`4,839,342 A 6/1989 Kaswan
`4.885,276 A 12/1989 Witzel ......................... 514/11
`4.914,188 A 4f1990 Dumont et al. ............. 530/317
`4.963,362 A 10/1990 Rahman et al.
`4,996,193 A 2/1991 Hewitt et al.
`4997,648 A 3/1991 Galpin et al.
`5,013,719 A
`5/1991 Bowlin
`5,051.402 A 9/1991 Kurihara et al.
`5,079,341 A
`1/1992 Galpin et al. ............... 530/321
`5,116,816. A 5/1992 Dreyfuss et al. .............. 514/11
`5,122,511. A 6/1992 Patchett et al. ......
`... 514/11
`5,214,130 A
`5/1993 Patchett et al. ............... s3033
`5,227.467 A 7/1993 Durette ....................... 530/321
`5,236,899 A 8/1993 Durette ........................ 514/11
`5,239,057 A
`8/1993 Wang et al. ................ 530/321
`5,256,547 A 10/1993 Rudat et al.
`Seebach, D., et al. “Modification of CycloSporin A (CS)');
`5,284.826 A 2/1994 Eberle - - - - - - - - - - - - - - - - - - - - - - - - - 514/11
`5,318.901 A 6/1994 Patchett et al.
`Generation of an Enolate at the Sarcosine Residue and
`ith El
`hiles.
`liti
`himi
`5,342.625 A 8/1994 Hauer et al. ................ 424/455
`5,350,574. A
`9/1994 Erlanger et al. ............... so Reactions with Electrophiles." Helvitica Chimica Acta. 76
`5,382,655 A
`1/1995 Szanya et al.
`(1993).
`5,389,382 A 2/1995 List et al.
`Yohannes, D., et al. “Degredation of Rapamycin: Retrieval
`5,401,649 A 3/1995 Davalian et al.
`of Major Intact Subunits.” Tetrahedron Letters. 33(49):
`5,405,785 A 4/1995 Erlanger et al. ............ 436/531
`7469–7472 (1992).
`5,409,816. A 4/1995 Lundell et al.
`von Wartberg, A., R. Traber. “Chemistry of the Natural
`5,411,952 A 5/1995 Kaswan
`Cyclosporin Metabolites.” Prog. Allergy. 38 28–45 (1986).
`5,427,960 A 6/1995 Wang et al. ................ 436/536
`Kobel H
`d R. Trab
`“Directed Bi
`thesi
`f
`5,489,668 A 2/1996 Morrison et al.
`obel, H. and K. I raper
`releasiosyntness, o
`5,525,590 A * 6/1996 Bollinger et al. ............. s14/11
`Cyclosporins." European Journal of Applied Microbiology
`5,540,931 A 7/1996 Hewitt et al.
`and Biotechnology. (14) 237-240 (1982);
`5,616,595 A 4/1997 Chu et al.
`von Traber, René, et al. “The Structure of Cyclosporin C.”
`5,624.902 A 4/1997 Blondelle et al.
`Helvetica Chimica Acta 60(4) 1247–1255 (1977).
`5,639,724 A 6/1997 Cavanak
`5,639,852 A 6/1997 Rich et al. .................. 530/317
`* cited by examiner
`
`Apotex Ex. 1013
`
`

`

`U.S. Patent
`
`Sep. 2, 2003
`
`Sheet 1 of 4
`
`US 6,613,739 B1
`
`Meeu 9
`
`MeLeu10
`
`Meeu 11
`
`HC H Y
`
`MeBnt 1
`
`-C
`H N
`CH2
`HC CH
`H3C, CH
`CH
`ch CH
`HC CH
`3 'cA 3.
`e? 3
`3
`u13
`HO
`3
`3.
`\ |
`V /
`CH:
`ch
`ch
`CH,
`ch,
`p-co-ch-co-oh-oo--on-co-p Abu 2
`Hic-
`H to
`CH3
`CH3
`CH
`CO
`HC-CH
`CH,
`N
`co-- co-ch-n-co-h- co-h- to Megly 3
`CH H
`CH2
`CH H
`CH2
`CH3
`Hic? CH
`3
`3
`
`D-Ala 8
`
`HC-N
`CD2
`
`CH
`HC CH3
`
`CR
`H3C
`CH3
`
`Ala 7
`
`Meeu 6
`
`Wa 5
`
`Meeu 4
`
`Fig. 1
`
`Apotex Ex. 1013
`
`

`

`U.S. Patent
`
`Sep. 2, 2003
`
`Sheet 2 of 4
`
`US 6,613,739 B1
`
`MeLeu 9
`
`Meeu 10
`
`MeLeu 11
`
`MeBmt 1
`
`HC H Y
`C 1 N
`H YCH
`CH
`HQ pH-CH,
`CH:
`GH
`ch–co-p Abu 2
`H to
`HC-N
`CH2
`
`H3C CH3
`D
`V /
`H3Cs-CH
`H. CH
`h
`3
`ch
`CH:
`H:
`p-co-ch-co-ch-co
`Hic-
`CH3
`CH3
`CO
`HC-CH
`CH
`HN
`D-Ala 8 co-ch-N-co-h-n-co-ch-y-co-ch- to Megly 3
`CH H
`CH2
`CE H
`CH2
`CH3
`H.C
`CH
`c:
`'
`CF
`'
`H3C CH3
`H3C CH3
`
`CH3
`
`Ala 7
`
`Meleu 6
`
`Wa 5
`
`MeLeu 4
`
`Fig. 2
`
`Apotex Ex. 1013
`
`

`

`U.S. Patent
`
`Sep. 2, 2003
`
`Sheet 3 of 4
`
`US 6,613,739 B1
`
`HO,
`
`AcQ,
`
`C——Abu-Sar
`MeLeu-MeVal-N)
`Ac,0, Py
`Pou
`oO —
`
`MeLeu-MeVal—N
`
`C——Abu- Sar
`
`MeLeu-D-Ala-Ala-MeLeu-Val—MeLeu
`
`MeLeu-D-Ala—Ala-MeLeu-Val—MeLeu
`
`
`
`4 2|1) Os04
`
`R
`
`|
`
`AcQ,
`
`2) NalOq
`
`AcO,
`
`CHO
`
`|
`
`MeLeu-MeVal-N” C—Abu-Sar
`'
`6
`MeLeu-D-Ala-Ala-MeLeu-Val~MeLeu
`
`1) RCH=PPh3P*T
`2) KyCO3
`
`& —Abu-Sar
`MeLeu-MeVal-N
`od
`i
`MeLeu-D-Ala-Ala-MeLeu-Val-MeLeu
`
`4
`
`3
`
`R
`
`|
`
`HO,
`
`MeLeu-MeVal-N C—Abu-Sar
`
`R= -H (Sa)
`~CH=CH,(5b)
`-CH=CH-CH3 (5c)
`
`-CD3 (5d)
`
`(5e)
`
`-CH=CH-CD,
`-D (5f)
`-CH=CDz (5g)
`
`-CD=CDz(5h)
`
`MelLeu-D-Ala-Ala-MeLeu-Val-MeLeu
`
`5
`
`Fig. 3
`
`Apotex Ex. 1013
`
`Apotex Ex. 1013
`
`

`

`U.S. Patent
`
`Sep. 2, 2003
`
`Sheet 4 of 4
`
`US 6,613,739 B1
`
`HO
`
`MeLeu-MeVal NY C-Abu-Sar
`O
`Meleu-D-Ala Ala-Melleu-Val-MeLeu
`
`1.
`
`ACO,
`
`Ac2O, Py
`
`MeLeu-MeVal-NY C-Abu-Sar
`b
`
`MeLeu-D-Ala-Ala-Melleu-Val-Melleu
`2
`
`1) OsO4
`2) NalO4
`
`CHO
`
`CHO
`
`ACO,
`
`ACO,
`
`Meleu-MeValN C-Abu-Sar
`s
`MeLeu-D-Ala-Ala-Meleu-Val-MeLeu
`6
`
`1) (EtO)2CHCH=PPh3Br
`q-m-m-m-
`2) acid hydrolysis
`
`Meleu-MeValNY C-Abu-Sar
`5
`Meleu-D-Ala-Ala-MeleuVal-MeLeu
`3
`
`se -D (5g)
`-CD (5e)
`
`1) RCD=PPh3PBr
`2) K2CO3
`
`D.
`
`R
`
`HO,
`Meley-MeVal N' G-Abu-Sar
`O
`Meleu-D-Ala-Ala-Meleu-Val-Meeu
`5
`
`Fig. 4
`
`Apotex Ex. 1013
`
`

`

`US 6,613,739 B1
`
`1
`DEUTERATED CYCLOSPORINE ANALOGS
`AND THEIR USE AS
`IMMUNOMODULATINGAGENTS
`
`2
`Stimulation of TCR (T cell receptor) by foreign antigen
`on a major histocompatibility (MHC) molecule on the
`surface of the T cell results in the activation of a TCR signal
`transmission pathway (exact method of transmission
`unknown) through the cytoplasm causing the signal results
`in the activation of nuclear transcription factors, i.e. nuclear
`factors of activated T-cells (NF-AT) which regulate tran
`Scription of T-cell activation genes. These genes include that
`of lymphokine interleukin-2 (IL-2). Translation of the mes
`sage is followed by secretion of IL-2. T-cell activation also
`involves the appearance of the lymphokine receptor IL-2R
`on the cell surface. After IL-2 binds to IL-2R, a lymphokine
`receptor (LKR) signal transmission pathway is activated.
`The immunosuppressive drug, rapamycin, inhibits this path
`way.
`CSA is a potent inhibitor of TCR-mediated signal trans
`duction pathway. It inhibits binding of NF-AT to the IL-2
`enhancer, and thus inhibits transcriptional activation. CSA
`binds to cyclophilin, which binds to calcineurin, which is a
`key enzyme in the T-cell Signal transduction cascade.
`Cyclophilin is found in prokaryotic and eukarotic organ
`isms and is ubiquitous and abundant. Cyclophilin is a Single
`polypeptide chain with 165 amino acid residues. It has a
`molecular mass of 17.8 kD). A roughly spherical molecule
`with a radius of 17 angstroms, cyclophilin has a eight
`stranded antiparallel beta barrel. Inside the barrel, the tightly
`packed core contains mostly hydrophobic side chains. CSA
`has numerous hydrophobic side chains which allow it to fit
`into the cyclophilin beta barrel. Cyclophillin catalyzes the
`interconversion of the cis and trans-rotamers of the
`peGIFdyl-prolyl amide bond of peptide and protein Sub
`Strates. Cyclophilin is identical in Structure with peptidyl
`prolyl cis-trans isomerase and bears structural resemblance
`to the Superfamily of proteins that transports ligands Such as
`retinol-binding protein (RBP). These proteins carry the
`ligand in the barrel core. But cyclophilin actually carries the
`ligand binding site on the outside of the barrel. The tet
`rapeptide ligand binds in a long deep groove on the protein
`Surface between one face of the beta barrel and the Thr116
`Gly130 loop.
`Further properties have also been reported in Studies of
`the biological activity of CSA: J. F. Borel et al., Agents
`Actions 6,468 (1976). Pharmacology: Eidem. Immunology
`32, 1017 (1977); R. Y. Calne, Clin. Exp. Immunol. 35, 1
`(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 (porcine 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 CSA in
`graft-versus-host disease: P. J. Tutschka et al., Blood 61,
`318(1983).
`Mechanism of CycloSnorine A Action
`CycloSporine A-Cyclophilin A Complex
`CSA, as discussed above, binds to the cyclophilin beta
`barrel. Thirteen CyPA residues define the CSA binding site.
`These residues are Arg55, Phe 60, Met 61, Gin 63, Gly 72,
`Ala 101, ASn 102, Ala 103, Gln111, Phe 113, Trp 121, Leu
`122, His 126. The largest side-chain movements are 1.3 A
`for Arg55 and up 0.7 A for Phe 60, Gln 63, and Trp 121.
`There are four direct hydrogen bonds between the CyPA and
`CSA. Residues 4, 5, 6, 7, 8 of CSA protrude out into the
`solvent and are thought to be involved in binding the effector
`protein, calcineurin (Pflugl, G., Kallen, J., Schirmer, T.,
`Jansonius, J. N., Zurini, M. G. M., & Walkinshaw, M. D.
`(1993) Nature 361,91–94)
`
`5
`
`15
`
`This application is a continuation of application Ser. No.
`09/184,109, filed Nov. 2, 1998.
`INTRODUCTION AND BACKGROUND
`CycloSporin derivatives of the present invention are dis
`closed which possess enhanced efficacy and reduced toxicity
`over naturally occurring and other presently known
`cyclosporins and cycloSporine derivatives. The cycloSporin
`derivatives of the present invention are produced by chemi
`cal and isotopic Substitution of the cyclosporine A (CSA)
`molecule by:
`1. Chemical Substitution and optionally deuterium Sub
`Stitution of amino acid 1; and
`2. Deuterium Substitution at key sites of metabolism of the
`cyclosporine A molecule Such as amino acids 1, 4, 9.
`The cycloSporins are a family of, neutral, hydrophobic
`cyclic undecapeptides, containing a novel nine-carbon
`amino acid (MeBmt) at position 1 of the ring that exhibit
`potent immunosuppressive, antiparasitic, fungicidal, and
`chronic anti-inflammatory properties. The naturally occur
`ring members of this family of Structurally related com
`25
`pounds are produced by various fungi imperfecti. CycloSpo
`rines A and C, are the major components. CycloSporine A,
`which is discussed further below, is a particularly important
`member of the cyclosporin family of compounds. Twenty
`four minor metabolites, also oligopeptides, have been iden
`tified: 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, 1 (1988).
`Isolation of cycloSporines 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 reported 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). Produc
`tion of A and C has been reported by E. Harriet al., U.S. Pat.
`No. 4,117,118 (1978 to Sandoz). Isolation, characterization
`and antifungal 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-fluoro-D-Ala-CSA is disclosed by Patchett et
`al in GB 2,206, 199A which was published on Dec. 29, 1988.
`CycloSporin was discovered to be immunosuppressive
`when it was observed to Suppress antibody production in
`mice during the Screening of fungal extracts. Specifically, its
`Suppressive effects appear to be related to the inhibition of
`T-cell receptor-mediated activation events. It accomplishes
`this by interrupting calcium dependent signal transduction
`during T-cell activation by inactivating calmodulin and
`cyclophilin, a peptidly propyl isomerase. It also inhibits
`lymphokine production by T-helper cells in vitro and arrests
`the development of mature CD8 and CD4 cells in the
`thymus. Other in vitro properties include inhibition of IL-2
`producing T-lymphocytes and cytotoxic T-lymphocytes,
`inhibition of IL-2 released by activated T-cells, inhibition of
`resting T-lymphocytes in response to alloantigen and eXog
`enous lymphokine, inhibition of IL-1 production, and inhi
`bition of mitogen activation of IL-2 producing
`T-lymphocytes. Further evidence indicates that the above
`effects involve the T-lymphocytes at the activation and
`maturation Stages.
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`Apotex Ex. 1013
`
`

`

`3
`Function of CSA-CyP A Complex.
`The CSA-CyPA complex inhibits the phosphatase activity
`of the heterodimeric protein Serine/threonine phosphatase or
`calcineurin (Liu, J., Fariner, J. D., Lane, W. S., Friedman, J.,
`Weissman, I., & Schreiber, S. L. (1991) Cell 66,807–15.;
`Swanson, S. K., Bom, T., Zydowsky, C. D., Cho, H., Chang,
`H. Y., & Walsh, C.T. (1992) Proc. Natl. Acad. Sci. USA89,
`3686–90). CyPA binds CSA with an affinity of ca. 10 nM.
`The complex is then presented to calcineurin (Liu, J.,
`Farmer, J. D., Lane, W. S., Friedman, J., Weissman, I., &
`Schreiber, S. L. (1991) Cell 66,807–15.).
`Calcineurin dephosphorylates the transcription factor
`NFAT found in the cytoplasm of T-cells. Dephosphorylation
`allows NFAT to translocate to the nucleus, combine with
`jun/foS genes and activate the transcription of the IL-2 gene
`responsible for cell cycle progression, leading to immune
`response. CSA-CyP A complex inhibits the phosphatase
`activity of calcineurin and ultimately immunosuppression
`
`US 6,613,739 B1
`
`4
`type I diabetes mellitus, multiple Sclerosis, autoimmune
`uveitis, and rheumatoid arthritis. Additional indications are
`discussed 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 ana
`log. For a recent review of cycloSporin uses and mechanisms
`of action See Wenger et al CycloSporine: Chemistry,
`Structure-Activity Relationships and Mode of Action,
`Progress in Clinical Biochemistry and Medicine, vol. 2, 176
`(1986).
`CycloSporin A is a cyclic peptide which contains Several
`N-methyl amino acids and, at position-8, contains a
`D-alanine. The structure of Cyclosporin A' is given below:
`
`15
`
`MeLeu10
`
`MeLeO 9
`CH
`CH
`1 CH3 CH
`Not
`n
`CH
`CH2
`
`-CH3
`CH
`
`MeVal11
`
`HCN, -H
`
`CH
`
`CH3 HO
`
`MeBmt 1
`1Sch
`2
`th
`h
`y y YOH, fl.
`1.
`ti-co--ch-co--al-co--ch-co--" Abu 2
`CH
`CH
`H l
`cu-
`CH
`fo
`lic-h
`D-Ala 8
`
`H-N
`
`MeGly 3
`CH
`CH
`CO-CH-N-CO-CH-N-CO-CH-N-CO-CH-N-CO
`
`al CH2
`
`CH. H
`
`CH2
`
`CH
`1.
`YCH
`HC
`MeLeu 6
`
`Ala 7
`
`CH H
`/n
`HC
`CH
`
`Va15
`
`CH2
`
`CH
`1.
`HC YCH,
`MeLeu 4
`
`(Etzkom, F. A., Chang, Z., Stolz, L. A., & Walsh, C. T.
`(1994) Biochemistry 33, 2380-2388.). Neither CSA or CyP
`A alone are important immunologically. Only their complex
`is important (Liu, J., Farmer, J.D., Lane, W. S., Friedman, J.,
`Weissman, I., & Schreiber, S. L. (1991) Cell 66,807–15).
`Metabolism of Cyclosporine:
`CycloSporine is metabolized in liver, Small intestine and
`kidney to more than 30 metabolites. The structure of 13
`metabolites and 2 phase 11 metabolites have been identified
`and at least 23 further metabolites have been isolated by
`HPLC and their structures characterized by mass spectrom
`etry. The reactions involved in phase I metabolism of
`cyclosporine are hydroxylation, demethylation as well as
`oxidation and cyclisation at amino acid 1. Several clinical
`Studies and reports showed an association between blood
`concentrations of cyclosporine metabolites and neuro- or
`nephrotoxicity. In Vitro experiments indicate that metabo
`lites are considerably less immunoSupressive and more toxic
`than CSA.
`AS exemplified by the ever expanding list of indications
`for which CSA has been found useful, the cyclosporin family
`of compounds find utility in the prevention of rejection or
`organ and bone marrow transplants, and in the treatment of
`pSoriasis, and a number of autoimmune disorderS Such as
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`“Unless otherwise specified, each of the amino acids of the
`disclosed cycloSporin is of the L-configuration.
`AS is the practice in the field, a particular cyclosporin
`analog may be named using a shorthand notation identifying
`how the analog differs from cycloSporin A. Thus,
`cyclosporin C which differs from cyclosporin A by the
`threonine at position-2 may be identified as Thr
`cyclosporin or Thr-CSA. Similarly, cyclosporin B is
`Ala-CSA; cyclosporin D is Val-CSA; cyclosporin E is
`Vall'-CSA; cyclosporin F is 3-DesoxyMeBmt-CSA;
`cyclosporin G is NVal-CSA; and cyclosporin H is
`D-MeVal-CSA.
`D-Serine and D-Threonine have been introduced into the
`8-position of cycloSporin A by biosynthesis resulting in
`active compounds. See R. Traber et al. J. Antibiotics 42,591
`(1989). D-Chloroalanine has also been introduced into
`position-8 of Cyclosporin Aby biosynthesis. See A. Lawen
`et al J. Antibiotics 52, 1283 (1989).
`Indications for Cyclosporine Therapy
`Immunoregulatory abnormalities have been shown to
`exist in a wide variety of autoimmune and chronic inflam
`matory diseases, including Systemic lupus erythematosis,
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`US 6,613,739 B1
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`S
`chronic rheumatoid arthritis, type 1 diabetes mellitus,
`inflammatory bowel disease, biliary cirrhosis, uveitis, mul
`tiple Sclerosis and other disorderS Such as Crohn's disease,
`ulcerative colitis, bullous pemphigoid, Sarcoidosis,
`pSoriasis, ichthyosis, and Graves ophthalmopathy. Although
`the underlying pathogenesis of each of these conditions may
`be quite different, they have in common the appearance of
`a variety of autoantibodies and Self-reactive lymphocytes.
`Such Self-reactivity may be due, in part, to a loSS of the
`homeostatic controls under which the normal immune SyS
`tem operates.
`Similarly, following a bone marrow or an organ
`transplantation, the host lymphocytes recognize the foreign
`tissue antigens and begin to produce antibodies which lead
`to graft rejection.
`One end result of an autoimmune or a rejection process is
`tissue destruction caused by inflammatory cells and the
`mediators they release. Anti-inflammatory agents, Such as
`NSAID's (Non-Steroidal Anti-inflammatory Drugs), and
`corticosteroids act principally by blocking the effect of, or
`Secretion of, these mediators, but do nothing to modify the
`immunologic basis of the disease. On the other hand,
`cytotoxic agents, Such as cyclophosphamide, act in Such a
`nonspecific fashion that both the normal and autoimmune
`responses are shut off. Indeed, patients treated with Such
`nonspecific immunosuppressive agents are as likely to Suc
`cumb to infection as they are to their autoimmune disease.
`Generally, a cycloSporin, Such as cycloSporine A, is not
`cytotoxic nor myelotoxic. It does not inhibit migration of
`monocytes nor does it inhibit granulocytes and macrophage
`action. Its action is specific and leaves most established
`immune responses intact. However, it is nephrotoxic and is
`known to cause the following undesirable Side effects:
`(1) abnormal liver function;
`(2) hirsutism;
`(3) gum hypertrophy;
`(4) tremor;
`(5) neurotoxicity;
`(6) hyperaesthesia; and
`(7) gastrointestinal discomfort.
`A number of cycloSporines and analogs have been
`described in the patent literature:
`U.S. Pat. No. 4,108,985 issued to Ruegger, et al. on Aug.
`22, 1978 entitled, “Dihydrocyclosporin C, discloses dihy
`drocycloSporin C, which can be produced by hydrogenation
`of cycloSporin C.
`U.S. Pat. No. 4,117,118 issued to Harri, et al. on Sep. 26,
`1978 entitled, “Organic Compounds”, discloses cyclospor
`ins A and B, and the production thereof by fermentation.
`U.S. Pat. No. 4,210,581 issued to Ruegger, et al. on Jul.
`1, 1980 entitled, “Organic Compounds”, discloses
`cyclosporin C and dihydrocycloSporin C which can be
`produced by hydrogenation of cyclosporin C.
`U.S. Pat. No. 4,220,641, issued to Traber, et al. on Sep. 2,
`1980 entitled, “Organic Compounds”, discloses cyclosporin
`D, dihydrocycloSporin D, and isocycloSporin D.
`U.S. Pat. No. 4,288,431 issued to Traber, et al. on Sep. 8,
`1981 entitled, “Cyclosporin Derivatives, Their Production
`and Pharmaceutical Compositions Containing Them', dis
`closes cycloSporin G, dihydrocylosporin G, and isocy
`closporin G.
`U.S. Pat. No. 4,289,851, issued to Traber, et al. on Sep.
`15, 1981 entitled, “Process for Producing Cyclosporin
`Derivatives', discloses cyclosporin D, dihydrocycloSporin
`D, and isocycloSporin D, and a process for producing Same.
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`6
`U.S. Pat. No. 4,384,996, issued to Bollinger, et al. on May
`24, 1983 entitled “Novel Cyclosporins”, discloses
`cyclosporins having a B-Vinylene-C-amino acid residue at
`the 2-position and/or a B-hydroxy-C.-amino acid residue at
`the 8-position. The cyclosporins disclosed included either
`MeBmt or dihydro-Me Bmt at the 1-position.
`U.S. Pat. No. 4,396,542, issued to Wenger on Aug. 2,
`1983 entitled, “Method for the Total Synthesis of
`Cyclosporins, Novel Cyclosporins and Novel Intermediates
`and Methods for their Production”, discloses the synthesis of
`cyclosporins, wherein the residue at the 1-position is either
`MeBmt, dihydro-Me Bmt, and protected intermediates.
`U.S. Pat. No. 4,639,434, issued to Wenger, et all on Jan.
`27, 1987, entitled “Novel Cyclosporins”, discloses
`cyclosporins with Substituted residues at positions 1, 2, 5
`and 8.
`U.S. Pat. No. 4,681,754, issued to Siegel on Jul. 21, 1987
`entitled, "Counteracting CycloSporin Organ Toxicity', dis
`closes methods of use of cycloSporin comprising
`co-dergocrine.
`U.S. Pat. No. 4,703,033 issued to Seebach on Oct. 27,
`1987 entitled, “Novel Cyclosporins”, discloses cyclosporins
`with substituted residues at positions 1, 2 and 3. The
`Substitutions at position-3 include halogen.
`H. Kobel and R. Traber, Directed Biosynthesis of
`Cyclosporins, European J. Applin. Microbiol Biotechnol.,
`14, 237B240 (1982), discloses the biosynthesis of
`cyclosporins A, B, C, D & G by fermentation.
`Additional cycloSporin analogs are disclosed in U.S. Pat.
`No. 4,798,823, issued to Witzel, entitled, New Cyclosporin
`Analogs with Modified “C-9 amino acids”, which discloses
`cyclosporin analogs with Sulfur-containing amino acids at
`position-1.
`
`SUMMARY OF THE INVENTION
`
`The present invention concerns chemically Substituted
`and deuterated analogs of cycloSporine A and related
`cyclosporines.
`An object of the present invention is to provide new
`cyclosporine analogs which have enhanced efficacy and
`altered pharmacokinetic and pharmacodynamic parameters.
`Another object of the present invention is to provide a
`cyclosporine analog for the care of immunoregulatory dis
`orders and diseases, including the prevention, control and
`treatment thereof. An additional object of the present inven
`tion is to provide pharmaceutical compositions for admin
`istering to a patient in need of the treatment one or more of
`the active immunosuppressive agents of the present inven
`tion. Still a further object of this invention is to provide a
`method of controlling graft rejection, autoimmune and
`chronic inflammatory diseases by administering a Sufficient
`amount of one or more of the novel immunosuppressive
`agents in a mammalian Species in need of Such treatment.
`Finally, it is the object of this invention to provide processes
`for the preparation of the active compounds of the present
`invention.
`Substitution and deuteration of the cycloSporine molecule
`results in altered physicochemical and pharmacokinetic
`properties which enhance its usefulneSS in the treatment of
`transplantation rejection, host VS. graft disease, graft VS. host
`disease, aplastic anemia, focal and Segmental
`glomeruloScierosis, myasthenia gravis, pSoriatic arthritis,
`relapsing polychondritis and ulcerative colitis.
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`US 6,613,739 B1
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`7
`Embodiments of the invention include CSA derivatives
`wherein one or more hydrogen atoms in the 1, 3 and 9 amino
`acid positions can be Substituted with a deuterium atom and
`wherein the cycloSporine Aderivatives are optionally chemi
`cally substituted at the amino acid 9 position. A further
`specific embodiment of the invention is the CSA derivative
`represented by formula I:
`
`(I)
`
`R-H
`
`Y-sa
`
`X
`
`CH 3
`SH:
`H
`les-cl. CHs.
`CH-CH3 R'S -CH
`SH:
`(H.
`SH
`CH (H.
`SH
`SH-CO-)-CH-CO-)-CH-CO-)-CH-CO-N-H
`CH-N
`CH
`CH
`CH
`H lo
`CO
`CH-N
`HC-CH
`Y
`1
`SH,
`H-N
`Hinz
`Co-H-N-CO-H-N-CO-H-N-CO-H-N-Co
`SH:
`CH. H
`ACHH
`SH:
`CH
`HC CH
`CH
`H3C
`NCH,
`H3C
`YoH,
`
`where R is (i) a deuterium or (ii) a Saturated or unsaturated
`straight or branched aliphatic chain of from 2 to 16 carbon
`atoms and optionally containing one or more deuterium
`atoms or an ester, ketone or alcohol of the carbon chain and
`optionally containing one or more Substituents Selected from
`halogen, nitro, amino, amido, aromatic, and heterocyclic, or
`(iii) R is an aromatic or heterocyclic group optionally
`containing a deuterium atom, or (iv) R is a methyl group and
`X, Y, and Z are hydrogen or deuterium provided that at least
`one of X, Y or Z is deuterium and R' is an OH or an ester
`or is an O and together with a carbon adjacent to a double
`bond on amino acid 1 form a heterocyclic ring Such as
`5-membered rings where the heteroatom is oxygen. Other
`Specific embodiments of the present invention include the
`CSA derivative of formula I where R is a Saturated or
`unsaturated carbon chain of from 2 to 3 carbons containing
`one or more deuterium. Further specific embodiments
`include those of formulas 5g and 5e below:
`
`
`
`CD
`
`(5g)
`
`MeLeu-MeVal-N
`
`Abu-Sar
`
`MeLeu-D-Ala-Ala-MeLeu-Val-MeLeu
`
`
`
`(5e)
`
`5
`
`MeLeu-MeVal-N
`
`MeLeu-D-Ala-Ala-MeLeu-Val-MeLeu
`
`DESCRIPTION OF THE FIGURES
`FIG. 1 is the Structure of cyclosporine A showing a site of
`deuteration at the amino acid 3 position.
`FIG. 2 is the Structure of cyclosporine A showing a site of
`deuteration at the amino acid 9 position.
`FIG. 3 is scheme I of the synthesis of the cyclosporine
`derivatives.
`FIG. 4 is scheme II of the synthesis of the cyclosporine
`derivatives.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`Substitution of deuterium for ordinary hydrogen and
`deuterated Substrates for protio metabolites can produce
`profound changes in biosystems. Isotopically altered drugs
`have shown widely divergent pharmacological effects. Pet
`tersen et al., found increased anti-cancer effect with deuter
`ated 5,6-benzylidene-dl-L-ascorbic acid (Zilascorb)
`Anticancer Res. 12, 33 (1992)).
`Substitution of deuterium in methyl groups of cycloSpo
`rine will result in a slower rate of oxidation of the C-D
`bond relative to the rate of oxidation of a non-deuterium
`substituted C-H bond. The isotopic effect acts to reduce
`formation of demethylated metabolites and thereby alters the
`pharmacokinetic parameters of the drug. Lower rates of
`oxidation, metabolism and clearance result in greater and
`more Sustained biological activity. Deuteration is targeted at
`various Sites of the cycloSporin molecule to increase the
`potency of drug, reduce toxicity of the drug, reduce the
`clearance of the pharmacologically active moiety and
`improve the stability of the molecule.
`Isotopic Substitution:
`Stable isotopes (e.g., deuterium, C, N, 'O) are non
`radioactive isotopes which contain one additional neutron
`than the normally abundant isotope of the respective atom.
`Deuterated compounds have been used in pharmaceutical
`research to investigate the in Vivo metabolic fate of the
`compounds by evaluation of the mechanism of action and
`metabolic pathway of the non deuterated parent compound.
`(Blake et al. J. Pharm. Sci. 64, 3, 367-391,1975). Such
`metabolic Studies are important in the design of Safe, effec
`tive therapeutic drugs, either because the in Vivo active
`compound administered to the patient or because the
`metaboliteS produced from the parent compound prove to be
`toxic or carcinogenic (Foster et al., Advances in Drug
`Research Vol. 14, pp. 2-36, Academic press, London, 1985).
`Incorporation of a heavy atom particularly Substitution of
`deuterium for hydrogen, can give rise to an isotope effect
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`9
`that could alter the pharmacokinetics of the drug. This effect
`is usually insignificant if the label is placed at a metaboli
`cally inert position of the molecule.
`Stable isotope labeling of a drug can alter its physico
`chemical properties Such as pKa and lipid Solubility. These
`changes may influence the fate of the drug at different Steps
`along its passage through the body. Absorption, distribution,
`metabolism or excretion can be changed. Absorption and
`distribution are processes that depend primarily on the
`molecular size and the lipophilicity of the Substance. These
`effects and alterations can affect the pharmacodynamic
`response of the drug molecule if the isotopic Substitution
`affects a region involved in a ligand-receptor interaction.
`Drug metabolism can give rise to large isotopic effect if
`the breaking of a chemi