Title of Invention	"SYNTHESIS OF CYCLOSPORIN ANALOGS"
Abstract	A method of preparing an isomeric mixture of cyclosporin A analogs modified at the 1-amino acid residue, wherein the synthetic pathway comprises the steps of (a) heating an acetyl-n-halocyclosporin A with a first compound selected from the group consisting of triaryl phosphine, trialkylphosphine, arylalkylphosphine, and triarylarsine to produce an intermediate; (b) preparing a mixture of (E) and (Z)-isomers of acetyl-1,3-diene by stirring the intermediate with a second compound selected from the group consisting of acetaldehyde, formaldehyde, deuterated formaldehyde, 2-chlorobenzaldehyde, and benzaldehyde; and (c) preparing a mixture of (E) and (Z)-isomers of IS ATX247 by treating the mixture of (E) and (Z)-isomers of acetyl-1,3-diene with a base.

Title of Invention

"SYNTHESIS OF CYCLOSPORIN ANALOGS"

Abstract

A method of preparing an isomeric mixture of cyclosporin A analogs modified at the 1-amino acid residue, wherein the synthetic pathway comprises the steps of (a) heating an acetyl-n-halocyclosporin A with a first compound selected from the group consisting of triaryl phosphine, trialkylphosphine, arylalkylphosphine, and triarylarsine to produce an intermediate; (b) preparing a mixture of (E) and (Z)-isomers of acetyl-1,3-diene by stirring the intermediate with a second compound selected from the group consisting of acetaldehyde, formaldehyde, deuterated formaldehyde, 2-chlorobenzaldehyde, and benzaldehyde; and (c) preparing a mixture of (E) and (Z)-isomers of IS ATX247 by treating the mixture of (E) and (Z)-isomers of acetyl-1,3-diene with a base.

Full Text	SYNTHESIS OF CYCLOSPORIN ANALOGS CROSS-REFERENCE TO RELATED APFLICATIQNS [0001] This application claims the benefit of U.S. Application Serial Nos. 60/346,201 filed October 19,2001 and 60/370,596 filed April 5, 2002. The entire disclosure of each of these applications is incorporated herein by reference in its entirety. FIELD OF THE INVENTION [0002] The invention is directed to isomeric mixtures of cyclosporin analogues"that are related to cyclosporine A. It is contemplated that the mixtures possess enhanced efficacy and/or reduced toxicity over the individual isomers and over naturally occurring and other presently known cyclosporines and cyclosporine derivatives. In addition, the present invention relates to synthetic pathways for producing isomers of cyclosporin A analogs, where such pathways vary in the degree of stereoselectivity depending on the specific reaction conditions. References [0003] The following references are related hereto or referred to herein by patent or application number or in parenthesis by author and year at the relevant portions of this specification: [0004] Bennett, W.M., "The nephrotoxicity of new and old immunosuppressive drugs," Renal Failure, Vol. 20, pp. 687-90 (1998). [0005] J.-F. Biellmann, J.-B. Ducep in "Allylic and benzylic carbanions substituted by heteroatoms," Organic Reactions, Vol. 27 (Wiley, New York, 1982), p. 9. [0006] H.J. Carlsen et al in "A Greatly Improved Procedure for Ruthenium Tetroxide Catalyzed Oxidations of Organic Compounds,"^ Org. Chem., Vol. 46, No. 19, pp 3736-3738 (1981). [0007] T. Chang, L.Z. Benet, M.F. Hebert, "The effect of water-soluble vitamin E on cyclosporine pharmacokinetics in healthy volunteers," Clin. Pharmacol. Ther., Vol. 59, pp. 297-303 (1996). [0008] E.J. Corey, M.C. Desai in Tetrahedron Letters, Vol. 26, No. 47, pp. 5747-8, (1985). [0009] M.K. Eberle, F, Nuninger, "Synthesis of the main metabolite (OL-17) of cyclosporin A,"/. Org. Chem., Vol. 57,pp. 2689-2691 (1992). [0010] E. Ehlinger, P. Magnus in "Silicon in synthesis. 10. The (trimethylsilyl)allyl anion: A P-acyl anion equivalent for the conversion of aldehydes and ketones into y-lactones," J. Am. Chem. Soc, Vol. 102, No. 15, pp. 5004-5011 (1980). [0011] D.S. Fruman, C.B. Klee, B.E. Bierer, S.J. Burakoff, "Calcineurin phosphatase activity in T lymphocytes is inhibited by FK506 and cyclosporin A," Proc. Natl. Acad. Sci. USA, Vol. 89, pp. 3686-90 (1992), [0012] A. Granelli-Piperno, L. Andrus, R.M. Steinman, "Lymphokine and nonlymphokine mRNA levels in stimulated human cells: kinetics, mitogen requirements, and effects of cyclosporin A," J. Exp. Med., Vol. 163, p. 922 (1986). [0013] J.R. Hanson, 'The Protection of Alcohols," Protecting Groups in Organic Synthesis, Ch. 2, pp. 24-25 (Sheffield Academic Press, Sheffield, England, 1999). [0014] M.F. Hebert, IP. Roberts, T. Prueksaritanont, L.Z. Benet, "Bioavailability of cyclosporin,with concomitant rifampin administration is markedly less than predicted by hepatic enzyme induction," Gin. Pharmacol. Ther., Vol. 52, pp. 453-7 (1992). [0015] R.W. Hoffinann, Angewandte Chemie International Edition, Vol. 555 (1982). [0016] R.W. Hoffinann, H.-J Zei, "Stereoselective synthesis of alcohols. 8. Diastereoselective synthesis of P-methylhomoallyl alcohols via crotylboronates," J. Org. Chem., Vol. 46, pp. 1309-1314 (1981). [0017] P.F. Hurdlik and D. Peterson in "Stereospecific Olefin-Forming Elimination Reactions of p-Hydroxysilanes," J. Am. Chem. Soc, Vol. 97, No. 6, pp. 1464-1468 (1975). [0018] Y. Dceda, J. Ukai, N. Bceda, H.Yamamoto, "Stereoselective synthesis of (Z)- and (E)-l,3-alkadienes from aldehydes using organotitanium and lithium reagents," Tetrahedron, Vol. 43, No. 4, pp. 723-730 (1987). [0019] Kobel et al., Europ. J. Applied Microbiology and Biotechnology, Vol. 14, pp. 237-240 (1982). [0020] J. McMurry, Organic Chemistry, 5th Ed. (Brooks/Cole, Pacific Grove, 2000), pp. 780-783. [0021] M.T. Reetz in Organotitanium Reagents in Organic Synthesis (Springer- Verlag, Berlin, 1986), pp. VE, 148-149, and 164-165. [0022] Rich et al., J. Med. Chem., Vol. 29, p. 978 (1986). [0023] W.R. Roush, "Allylorganometallics," Comprehensive Organic Synthesis, Pergamon Press, Vol. 2, pp. 1-53. [0024] S.L. Schreiber, G.R. Crabtree, "The mechanism of action of cyclosporin A and FK506," Immunol. Today, Vol. 13, pp. 136-42 (1992). [0025] I. Sketris, R. Yatscoff, P. Keown, D.M. Canafax, M.R. First, D.W. Holt, TJ. Schroeder, M. Wright, "Optimizing the use of cyclosporine in renal transplantation," Clin. Biochem., Vol. 28, pp. 195-211 (1995). [0026] M.B. Smith and J. March, March's Advanced Organic Chemistry (Wiley, New York, 2001), pp. 144-147. [0027] A. Streitwieser, C. H. Heathcock, Introduction to Organic Chemistry, 2nd ed. (Macmillan, New York, 1981), pp, 845-846. [0028] J.A. Thliveris, R.W. Yatscoff, M.P. Lukowski, K.R. Copeland, J.R. Jeffery, G.F. Murphy, "Chronic ciclosporin nephrotoxicity: A rabbit model," Nephron. Vol. 57, pp. 470-6 (1991). [0029] J.A. Thliveris, R.W. Yatscoff, MJ. Mihatsch, "Chronic cyclosporine-induced- nephrotoxicity: A rabbit model," Transplantation, Vol. 57, pp. 774-6 (1994). [0030] S. E. Thomas in Organic Synthesis: The Roles of Boron and Silicon (Oxford University Press, New York, 1991), pp. 84-87. [0031] Traber et al., Helv. Chim. Acta, Vol. 60, pp. 1247-1255 (1977). [0032] Traber et al., Helv. Chim. Acta, Vol. 65, pp. 1655-1667 (1982). [0033] D.S. Tsai, D.S. Matteson, "A stereocontrolled synthesis of (Z) and (E) terminal dienes from pinacol (E)-l-trimethylsilyl-l-propene-3-boronate," Tetrahedron Letters, Vol. 22, No. 29, p. 2751-2752 (1981). [0034] H.A. Valantine, J.S. Schroeder, "Recent advances in cardiac transplantation" [editorial; comment], N. Engl. J. Med., Vol. 333, No. 10, pp. 660-1 (1995). [0035] von Wartburg et al., Progress in Allergy, Vol. 38, pp. 28-45 (1986). [0036] Wenger, Transpl. Proc, Vol. 15, Suppl. 1, p. 2230 (1983). [0037] Wenger, Angew. Chenu Int. Ed, Vol. 24, p. 77 (1985), [0038] Wenger, Progress in the Chemistry of Organic Natural Products, Vol. 50, p. 123 (1986). [0039] Y. Yamamoto, N. Asao, Chemical Reviews, p. 2307 (1993). [0040] Dan Yang, et al., "A C2 Symmetric Chiral Ketone for Catalytic Asymmetric Epoxidation of Unfunctionalized Olefins," J. Am. Chem. Soc, Vol. 118, pp. 491-492 (1996). [0041] Dan Yang, et al., "Novel Cyclic Ketones for Catalytic Oxidation Reactions," J. Org. Chem., Vol. 63, pp. 9888-9894 (1998) [0042] U.S. Pat. No. 4,108,985. [0043] U.S. Pat. No. 4,160,452. [0044] U.S. Pat. No. 4,210,581. [0045] U.S. Pat. No. 4,220,641. [0046] U.S. Pat. No. 4,256,108. [0047] U.S. Pat. No. 4,265,874. [0048] U.S. Pat. No. 4,288,431. [0049] U.S. Pat. No. 4,384,996. [0050] U.S. Pat. No. 4,396,542. [0051] U.S. Pat. No. 4,554,351. [0052] U.S. Pat. No. 4,771,122. [0053] U.S. Pat. No. 5,284,826. [0054] U.S. Pat. No. 5,525,590. [0055] European Patent Publication No. 0 034 567. [0056] European Patent Publication No. 0 056 782. [0057] International Patent Publication No. WO 86/02080. [0058] International Patent Publication No. WO 99/15120. [0059] The disclosure of each of the above-referenced patents, patent applications and publications is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION [0060] Cyclosporine derivatives compose a class of cyclic polypeptides, consisting of eleven amino acids, that are produced as secondary metabolites by the fungus species Tolypocladium inflatum Gams. They have been observed to reversibly inhibit immunocompetent lymphocytes, particularly T-lymphocytes, in the Go or G\ phase of the cell cycle. Cyclosporine derivatives have also been observed to reversibly inhibit the production and release of lymphokines (Granelli-Pipemo et al, 1986). Although a number of cyclosporine derivatives are known, cyclosporine A is the most widely used. The suppressive effects of cyclosporine A are related to the inhibition of T-cell mediated activation events. This suppression is accomplished by the binding of cyclosporine to the ubiquitous intracellular protein, cyclophilin. This complex, in turn, inhibits the calcium- and calmodulin-dependent serine-threonine phosphatase activity of the enzyme calcineurm. Inhibition of calcineurin prevents the activation of transcription factors such as NFATp/c and NF-KB, which are necessary for the induction of the cytoldne genes (IL-2, IFN-y, IL-4, and GM-CSF) during T-cell activation. Cyclosporine also inhibits lymphokine production by T-helper cells in vitro and arrests the development of mature CD8 and CD4 cells in the thymus (Granelli-Piperno et al, 1986). Other in vitro properties of cyclosporine include the 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 exogenous lymphokine, inhibition of IL-1 production, and inhibition of mitogen activation of IL-2 producing T-lymphocytes (Granelli-Piperno et al, 1986). [0061] Cyclosporine is a potent immunosuppressive agent that has been demonstrated to suppress humoral immunity and cell-mediated immune reactions such as allograft rejection, delayed hypersensitivity, experimental allergic encephalomyelitis, Freund's adjuvant arthritis and graft vs. host disease. It is used for the prophylaxis of organ rejection subsequent to organ transplantation; for treatment of rheumatoid arthritis; for the treatment of psoriasis; and for the treatment of other autoimmune diseases, including type I diabetes, Crohn's disease, lupus, and the like. [0062] Since the original discovery of cyclosporin, a wide variety of naturally occurring cyclosporins have been isolated and identified and many further non-natural cyclosporins have been prepared by total- or semi-synthetic means or by the application of modified culture techniques. The class comprised by the cyclosporins is thus now substantial and includes, for example, the naturally occurring cyclosporins A through Z.[c.f. Traber et al. (1977); Traber et al. (1982); Kobel et al. (1982); and von Wartburg et al. (1986)], as well as various non-natural cyclosporin derivatives and artificial or synthetic cyclosporins including the dihydro- and iso-cyclosporins; derivatized cyclosporins (e.g., in which the 3'-O-atom of the -MeBmt- residue is acylated or a further substituent is introduced at the a-carbon atom of the sarcosyl residue at the 3-position); cyclosporins in which the -MeBmt-residue is present in isomeric form (e.g., inwhichthe.configurationL across positions 6' and 7 of the -MeBmt-residue is cis rather than trans); and cyclosporins wherein variant ainino acids are incorporated at specific positions within the peptide sequence employing, e.g., the total synthetic method for the production of cyclosporins developed by R. Wenger-see e.g. Traber et al. (1977), Traber et al. (1982) and Kobel et al. (1982); U.S. Pat. Nos. 4,108,985, 4,210,581,4,220,641,4,288,431,4,554,351 and 4,396,542; European Patent Publications Nos. 0 034 567 and 0 056 782; International Patent Publication No. WO 86/02080; Wenger (1983); Wenger (1985); and Wenger (1986). Cyclosporin A analogues containing modified amino acids in the 1 -position are reported by Rich et al. (1986). Immunosuppressive, anti-inflammatory, and anti-parasitic cyclosporin A analogues are described in U.S. Pat. Nos. 4,384,996; 4,771,122; 5,284,826; and 5,525,590, all assigned to Sandoz. Additional cyclosporin analogues are disclosed in WO 99/18120, assigned to Isotechnika. The terms Ciclosporin, ciclosporin, cyclosporine, and Cyclosporine are interchangeable and refer to cyclosporin. [0063] There are numerous adverse effects associated with cyclosporine A therapy, including nephrotoxicity, hepatotoxicity, cataractogenesis, hirsutism, parathesis, and gingival hyperplasia to name a few (Sketris et al., 1995). Of these, nephrotoxicity is one of the more serious, dose-related adverse effects resulting from cyclosporine A administration. Immediate-release cyclosporine A drug products (e.g., Neoral® and Sandimmune®) can cause nephrotoxicities and other toxic side effects due to their rapid release and the absorption of high blood concentrations of the drug. It is postulated that the peak concentrations of the drug are associated with the side effects (Bennett, 1998). The exact mechanism by which cyclosporine A causes renal injury is not known; however, it is proposed that an increase in the levels of vasoconstrictive substances in the kidney leads to the vasoconstriction of the afferent glomerular arterioles. This can result in renal ischemia, a decrease in glomerular filtration rate and, over the long term, interstitial fibrosis. When the dose is reduced or another irnrnunosuppressive agent is substituted, renal function improves (Valantine and Schroeder, 1995). [0064] Accordingly, there is a need for immunosuppressive agents which are effective and have reduced toxicity. [0065] Cyclosporin analogs containing modified amino acids in the 1-position are disclosed in WO 99/18120, which is assigned to the assignee of the present application, and incorporated herein in its entirety. Also assigned to the present assignee is U.S. Provisional Patent Application No. 60/346,201, in which applicants disclosed a particularly preferred cyclosporin A analog referred to as "ISATX247." This analog is structurally identical to cyclosporin A except for modification at the 1-amino acid residue. Applicants discovered that certain mixtures of cis and trans isomers of ISArx247 exhibited a combination of enhanced potency, and/or reduced toxicity over the naturally occurring and presently known cyclosporins. Certain alkylated, arylated, and deuterated derivatives of ISArx247 were also disclosed. [0066] Typically, the disclosed mixtures in U.S. Provisional Patent Application No. 60/346,201 range from about 10 to 90 percent by weight of the trans-isomer and about 90 to 10 percent by weight of the os-isomer, in another embodiment, the mixture contains about 15 to 85 percent by weight of the trans-isomer and about 85 to 15 percent of the cis-isomer; in another embodiment, the mixture contains about 25 to 75 percent by weight of the trans-isomer and about 75 to 25 percent by weight of the cir-isomer; in another embodiment, the mixture contains about 35 to 65 percent by weight of the trans-isomer and about 65 to 35 percent by weight of the cw-isomer; in another embodiment, the mixture contains about 45 to 55 percent by weight of the trans-isomer and about 55 to 45 percent of the cis-isomer. In another embodiment, the isomeric mixture is an ISArx247 mixture which comprises about 45 to 50 percent by weight of the trans-isomer and about 50 to 55 percent by weight of the cis-isomer. These percentages by weight are based on the total weight of the composition. In other words, a mixture might contain 65 percent by weight of the (E)-isomer and 35 percent by weight of the (Z)-isomer, or vice versa. In an alternate nomenclature, the m-isomer may also be described as a (Z)-isomer, and the trans-isomer could also be called an (E)-isomer. [0067] Accordingly, there is a need in the art for methods of preparation of cyclosporin analogs, including isomers of ISATX247. Synthetic pathways are needed that produce enriched compositions of the individual isomers, as well mixtures of the isomers having a desired ratio of the two isomers. Methods of preparation of derivatives of ISAi%247 are needed as well. SUMMARY OF THE INVENTION [0068] Cyclosporine and its analogs are members of a class of cyclic polypeptides having potent immunosuppressant activity. Despite the advantages these drugs offer with respect to their immunosuppressive, anti-inflammatory, and anti-parasitic activities, there are numerous adverse effects associated with cyclosporine A therapy that include nephrotoxicity and hepatotoxicity. Accordingly, there is a need for new immunosuppressive agents that are as pharmacologically active as the naturally occurring compound cyclosporin A, but without the associated toxic side effects. [0069] Embodiments of the present invention provide certain mixtures of cis and trans-isomsrs of cyclosporin A analogs, which are pharmaceutically useful. A preferred analog is referred to as ISAJX247. Mixtures of ISATX247 isomers exhibit a combination of enhanced potency and reduced toxicity over the naturally occurring and presently known cyclosporins. [0070] The present invention is based in part on the discovery that certain isomeric mixtures of analogues of cyclosporine provide superior immunosuppressive effects without the adverse effects associated with cyclosporine A. In particular, we have unexpectedly found that isomeric mixtures (i.e., mixtures of both cis- and trans- isomers) ranging from about 10:90 to about 90:10 (trans- to cis-) of cyclosporine analogues modified at the 1-amino acid residue provide superior efficacy and safety. Examples of such analogues are disclosed in WO 99/18120, and include deuterated and non-deuterated compounds. In particular, mixtures in the range of about 45:55 to about 50:50 (trans- to cis-) and in the range of about 50% to about 55% trans- and about 45% to about 50% cis- are found to be particularly efficacious. Moreover, it has been demonstrated that these isomer mixtures exhibit a combination of superior potency and reduced toxicity over naturally occurring and other presently known cyclosporines and cyclosporine derivatives. [0071] A particularly preferred analogue (referred to herein as "ISATX247") is structurally similar to cyclosporine A except for a modified functional group .on the periphery of the molecule, at the 1-amino acid residue. The structure of this particular isomeric analogue mixture compared to the structure of cyclosporine A is shown in FIGS. 1A, IB, 2A, 2B. [0072] The isomeric mixtures can be used, among other things, for immunosuppression, and the care of various immune disorders, diseases and conditions, including the prevention, control, alleviation and treatment thereof. . [0073] According to embodiments of the present invention, ISATX247 isomers (and derivatives thereof) are synthesized by stereoselective pathways that may vary in their degree of selectivity. Stereoselective pathways produce compositions that are enriched in either of the (E) and (Z)-isomers, and these compositions may be combined such that the resulting mixture has a desired ratio of the two isomers. Alternatively, the reactions conditions of a stereoselective pathway may be tailored to produce the desired ratio directly in a prepared mixture. The percentage of one isomer or another in a mixture can be verified using nuclear magnetic resonance spectroscopy (NMR) or other techniques well known in the art. [0074] Each of the pathways typically proceeds with the application of a protecting group to a sensitive alcohol functional group. In one embodiment the alcohol is protected as an acetate; in other embodiments the protecting groups are benzoate esters or silyl ethers. Although acetate protecting groups are common in the art, it is important to emphasize that in many of the exemplary embodiments described herein certain undesirable side-reactions involving an acetate protecting group may be avoided through the use of protecting groups such as benzoate esters or silyl ethers. [0075] The protected compound may then serve as a precursor for a variety of stereoselective synthetic pathways including some that utilize phosphorus-containing reagents as participants in a Wittig reaction, and inorganic elements as members of organometallic reagents. The latter type may proceed through six-membered ring transition states where steric hindrance dictates the configurational outcome. Many organometallic reagents are available, including those that feature inorganic elements such as boron, silicon, titanium, lithium, and sulfur. Individual isomers may be prepared from single or multiple precursors. [0076] The ratio of the (E) to (Z)-isomers in any mixture, whether produced stereoselectively or non-stereoselectively, may take on a broad range of values. For example, the mixture may comprise from about 10 to 90 percent of the (E)-isomer to about 90 to 10 percent of the (Z)-isomer. In other embodiments, the mixture may contain from about 15 to 85 percent by weight of the (E)-isomer and about 85 to 15 percent of the (Z)-isomer; in another embodiment, the mixture contains about 25 to 75 percent by weight of the (E)-isomer and about 75 to 25 percent by weight of the (Z)-isomer; in another embodiment, the mixture contains about 35 to 65 percent by weight of the (E)-isomer and about 65 to 35 percent by weight of the (Z)-isomer; in another embodiment, the mixture contains about 45 to 55 percent by weight of the (E)-isomer and about 55 to 45 percent of the (Z)-isomer. In another embodiment, the isomeric mixture is an ISATX247 mixture which comprises about 45 to 50 percent by weight of the (E)-isomer and about 50 to 55 percent by weight of the (Z)-isomer. These percentages by weight are based on the total weight of the composition, and it will be understood that the sum of the weight percent of the (E)-isomer and the (Z)-isomer is 100 weight percent. In other words, a mixture might contain 65 percent by weight of the (E)-isomer and 35 percent by weight of the (Z)-isomer, or vice versa. [0077] Accordingly, in one aspect, the invention provides methods of preparing an isomeric mixture of cyclosporin A analogs modified at the 1-amino acid residue, wherein the synthetic pathway comprises the steps of: heating an acetyl-T\|-halocyclosporin A with triakylphosphine, triarylphosphine (e.g. triphenylphosphine), arylalkylphosphine, and triarylarsine to produce an intermediate phosphonium halide of acetyl cyclosporin A; preparing a mixture of (E) and (Z)-isomers of acetyl-l,3-diene by stirring the intermediate phosphonium halide of acetyl cyclosporin A with formaldehyde, optionally in the presence of a lithium halide; and preparing a mixture of (E) and (Z)-isomers of ISATX247 by treating the mixture of (E) and (Z)-isomers of acetyl- 1,3-diene with a base. [0078] In another aspect, the invention is directed to methods of preparing an isomeric mixture of cyclosporin A analogs modified at the 1-amino acid residue, wherein the synthetic pathway comprises the steps of: converting an intermediate, e.g., protected trimethylsilyl (TMS) cyclosporin A aldehyde or acetyl cyclosporin A aldehyde to a mixture of (E) and (Z)-isomers of acetyl-1,3-diene by reacting the intermediate with a phosphorus ylide via a Wittig reaction, optionally in the presence of a lithium halide; and preparing a mixture of (E) and (Z)-isomers of LSATX247 by treating the mixture of (E) and (Z)-isomers of acetyl-1,3-diene with a base in the case of acetyl-protecting group or, e.g., an acid in case of a TMS-protecting group. [0079] In a further aspect, the invention is directed to methods of producing an E- isomer enriched mixture of cyclosporin A analogs modified at the 1-amino acid residue, wherein the stereoselective synthesis of the E-isomer enriched material comprises the steps of: reacting an acetyl cyclosporin A aldehyde with a reagent selected from the group consisting of trialkylsilylallyl boronate ester and E-y-(trialkylsilylallyI) diaikylborane to form a p-trialkylsilyl alcohol; treating the P-trialkylsilyl alcohol with acid to form acetyl-(E)-l,3-diene; and treating the acetyl-(E)-1,3-diene with base to form the (E)-isomer of ISATX247. [0080] In yet a further aspect, the invention is directed to methods of producing a Z- isomer enriched mixture of cyclosporin A analogs modified at the 1-amino acid residue, wherein the stereoselective synthesis of the Z-isomer enriched material comprises the steps of: reacting an acetyl cyclosporin A aldehyde with a reagent selected from the group consisting of trialkylsilylallyl boronate ester and (E-^-trialkylsilylallyl) diaikylborane to form a P-trialkylsilyl alcohol; treating the p-trialkylsilyl alcohol with base to form acetyl-(Z)-1,3-diene; and treating the acetyl-(Z)-l,3-diene with base to form the (Z)-isomer of ISATX247. [0081] In a still further aspect, the invention is directed to methods of producing an E- isomer enriched mixture of cyclosporin A analogs modified at the 1-amino acid residue, wherein the stereoselective synthesis of the E-isomer enriched material comprises the steps of: reacting an acetyl cyclosporin A aldehyde with a lithiated allyldiphenylphosphine oxide to form acetyl-(E)-l,3-diene; and treating the acetyl-(E)-l,3-diene with base to form the (E)-isomer of ISArx247. [0082] In yet a further still aspect, the invention provides method of producing a Z- isomer enriched mixture of cyclosporin A analogs modified at the 1-amina acid residue, wherein the stereoselective synthesis of the Z-isomer enriched material comprises the steps of: reacting an acetyl cyclosporin A aldehyde with [3-(diphenylphosphino)allyl] titanium to form a titanium-containing intermediate; allowing the titanium-containing intermediate to proceed to an erythro-a-adduct; converting the erythro-cc-adduct to an p-oxidophosphonium salt by treatment of iodomethane; converting the p-oxidophosphonium salt to an acetyl-(Z)-1,3-diene; and treating the acetyl-(Z)-l,3-diene with base to form the (Z)-isomer of ISATX247. [0083] In still a further aspect, the invention provides mixtures (E) and (Z)-isomers prepared by a process comprising the steps of: protecting the P-alcohol of cyclosporin A to form acetyl cyclosporin A; brominating the r)-carbon of the side chain of the 1-amino acid residue of acetyl cyclosproin A to produce a first intermediate acetyl-rj-bromocyclosporin A; heating the first intermediate acetyl-T\|-bromocyclosporin A with a reagent selected from the group consisting of triphenyl phosphine and trialkyl phosphine to produce a second intermediate selected from the group consisting of triphenyl- and trialkyl phosphonium bromides of acetyl cyclosporin A; preparing a mixture of (E) and (Z)-isomers of acetyl-1,3-diene by stirring the ylide generated from the triphenyl- or trialkyl salt (second intermediate triphenylphosphonium bromide) of acetyl cyclosporin A with formaldehyde; and preparing the mixture of (E) and (Z)-isomers of ISATX247 by treating the mixture of (E) and (Z)-isomers of acetyl- 1,3-diene with a base. [0084] The invention is also directed to compositions of matter, including triphenyl- and trialkyl phosphonium bromides of acetyl cyclosporin A, the product prepared by a process comprising the steps of: protecting the f3-alcohol of cyclosporin A; brominating the •n-carbon of the side chain of the 1-amino acid residue of cyclosporin A to produce a first intermediate acetyl-T\|-bromocyclosporin A; and heating the first intermediate acetyl-rj-bromocyclosporin A with a reagent selected from the group consisting of triphenylphosphine and trialkylphosphine to produce a bromide of acetyl cyclosporin A selected from the group consisting of the triphenyl- and trialkylphosphonium bromides of acetyl cyclosporin A. Also provided are compositions comprising a triphenyl or trialkyl phosphonium bromide derivative of acetyl cyclosporin A and compositions comprising a P-trimethylsilyl alcohol derivative of cyclosporin A. [0085] In an additional aspect, the invention provides methods for the selective preparation of cyclosporin A aldehyde comprising the steps of: protecting the P-alcohol of cyclosporin A by forming acetyl cyclosporin A or trimethylsilyl (IMS) cyclosporin A; and oxidizing the acetyl cyclosporin A or TMS cyclosporin A with ozone as the oxidizing agent used with a reducing agent. [0086] In another added aspect, the invention is directed to methods of preparing an isomeric mixture of cyclosporin A analogs modified at the 1-amino acid residue, wherein the synthetic pathway comprises the steps of: converting an intermediate acetyl cyclosporin A aldehyde to a mixture of (E) and (Z)-isomers of acetyl-1,3-diene by reacting the intermediate with a phosphorus ylide prepared from a tributylallylphosphonium halogenide via a WIttig reaction, optionally in the presence of a lithium halide; and preparing a mixture of (E) and (Z)-isomers of ISATX247 by treating the mixture of (E) and (Z)-isomers of acetyl-l,3-diene with a base. [0087] In an additional aspect, the invention provides methods for the stereoselective synthesis of the E-isomer of ISATX247 comprising the steps of: reacting a trimethylsilyl (TMS) cyclosporin A aldehyde with trialkylsilylallyl borane to form a p-trialkylsilyl alcohol; treating the p-trialkylsilyl alcohol to form directly the E-isomer of ISATX247. [0088] Another aspect of the invention is directed to methods for the stereoselective synthesis of the Z-isomer of ISATX247 comprising the steps of: reacting a trimethylsilyl (TMS) cyclosporin A aldehyde with trialkylsilylallyl borane to form a P-trialkylsilyl alcohol; treating the p-trialkylsilyl alcohol with base to form TMS-(Z)-l,3-diene; and deprotecting the TMS-(Z)-l,3-diene to form the Z-isomer of ISArx247. [0089] The invention is also directed to methods of preparing isomeric mixtures of cyclosporin A analogs modified at the 1-amino acid residue, the method comprising a synthetic pathway that prepares an (E)-isomer and a (Z)-isomer of ISATX247 such that the (E)-isomer and the (Z)-isomer are present in the mixture in a predetermined ratio, wherein the synthetic pathway comprises the steps of: protecting the p alcohol of cyclosporin A; oxidizing the protected cyclosporin A to produce a second intermediate protected cyclosporin A aldehyde; converting the second intermediate protected cyclosporin A aldehyde to a mixture of E- and Z- isomers of protected 1,3 diene by reacting the second intermediate with a phosphorus ylide via a Wittig reaction, optionally in the presence of a lithium halide; and preparing a mixture of E- and Z- isomers by deprotecting the protected 1,3 diene. [0090] Other methods of preparing such mixtures also provided by the invention include methods of preparing an isomeric mixture of cyclosporin A analogs modified at the 1-amino acid residue, the method comprising a synthetic pathway that prepares an (E)-isomer and a (Z)-isomer of ISArx247 such that the (E)-isomer and the (Z)-isomer are present in the mixture in a predetermined ratio, wherein the ratio of isomers in the mixture ranges from about 45 to 55 percent by weight of the (E)-isomer to about 55 to 45 percent by weight of the (Z)-isomer, based on the total weight of the mixture. [0091] The invention also provides methods of preparing a predetermined isomeric mixture of cyclosporin A analogs modified at the 1-amino acid residue, the method comprising: preparing a first material enriched in an (E)-isomer of ISATX247; preparing a second material enriched in a (Z)-isomer of ISATX247; and mixing the first and second materials in a ratio designed to give the desired isomeric composition. BRIEF DESCRIPTION OF THE DRAWINGS [0092] FIG. 1A shows the structure of cyclosporin A, illustrating the 11 amino acid residues that comprise the cyclic peptide ring of the molecule, as well as the structure of the side chain of the 1-amino acid residue; [0093] FIG. IB is another illustration of the structure of cyclosporin A with particular emphasis on the definition of the term "CsA" as it is used in the present description; [0094] FIG. 2A shows the structure of the E-isomer (or trans-isomer) of the cyclosporin A analog called ISATX247; [0095] FIG. 2B shows the structure of the Z-isomer (or cw-isomer) of the cyclosporin A analog ISATX247; [0096] FIG. 3 shows an overview of exemplary synthetic pathways that may be used to prepare the cyclosporin analogs of the present invention, where stereoselective pathways are grouped according to reactive conditions; [0097] FIG. 4 illustrates a synthetic pathway that produces a mixture of (E) and (Z)- isomers of ISATX247 from a bromine precursor; [0098] FIG. 5 illustrates another synthetic pathway that produces a mixture of (E) and (Z)-isomers of ISATX247 from an aldehyde precursor, [0099] FIG. 6 illustrates an exemplary stereoselective reaction scheme that may be used to prepare compositions enriched in either the (E) or (Z)-isomers of ISATX247, wherein either isomer may be prepared from the same precursor alcohol; [00100] FIG. 7 illustrates an alternative reaction scheme for the stereoselective synthesis of a composition enriched in the (Z)-isomer of ISATX247; [00101] FIG. 8 illustrates an alternative reaction scheme for the stereoselective synthesis of a composition enriched in the (E)-isomer of ISATX247; [00102] FIGS. 9A-C illustrate exemplary synthetic pathways for producing a mixture of the (E) and (Z)-isomers of ISATX247, the conditions of each reaction having been tailored to produce a particular exemplary ratio of the two isomers; [00103] FIG. 10 illustrates exemplary stereoselective pathways for producing a mixture of the (E) and (Z)-isomers of ISATX247, where compositions enriched in one of the two isomers are first prepared, and then mixed accordingly in predetermined proportions to achieve the desired ratio; [00104] FIG. 11 provides the results of an assay which shows that the inhibition of calcineurin phosphatase activity by ISATX247 (45-50% of E-isomer and 50-55% of Z-isomer) was up to a 3-fold more potent (as determined by IC50) as compared to cyclosporine A. [00105] FIG. 12 sets forth the structure and isomeric composition of some deuterated and non-deuterated analogue isomeric mixtures. [00106] FIG. 13 provides the results of an assay which shows that the inhibition of calcineurin phosphatase activity by various deuterated and non-deuterated analogue isomeric mixtures was at least as potent (as determined by IC50) as compared to cyclosporine A. DETAILED DESCRIPTION OF THE INVENTION Synthesis [00107] Cyclosporin and its analogs are members of a class of cyclic polypeptides having potent immunosuppressive activity. Despite the advantages these drugs offer with respect to their immunosuppressive, anti-inflammatory, and anti-parasitic activities, there are numerous adverse effects associated with cyclosporine A therapy mat include nephrotoxicity and hepatotoxicity. Accordingly, there is a need for new immunosuppressive agents mat are as pharmacologically active as the naturally occurring compound cyclosporin A, but without the associated toxic side effects. [00108] Applicants have previously disclosed a cyclosporin A analog referred to as '1SATX247." This analog is structurally similar to cyclosporin A except for modification at the 1-amino acid residue. Applicants discovered that certain mixtures of cis and trans-isomers of ISArx247 exhibited a combination of enhanced potency, and reduced toxicity, over the naturally occurring and presently known cyclosporins. [00109] According to embodiments of the present invention, ISArx247 isomers (and derivatives thereof) are synthesized by stereoselective pathways that may vary in their degree of stereoselectivity. Stereoselective pathways produce compositions that are enriched in either of the (E) and (Z)-isomers, and these compositions may be combined such that the resulting mixture has a desired ratio of the two isomers. Alternatively, the reaction conditions of a stereoselective pathway may be tailored to produce the desired ratio directly in a prepared mixture. [00110] The chemical name of one immunosuppresive cyclosporin analog of the present invention, called ISArx247, is chemically described by the name cyclo{{E^)-(2S,3R,4R)-3-hydroxy-4-memyl-2-(memylammo)-6,8-nonadienoyl}-L-2-aminobutyryl-N-methyl-glycyl-N-methyl-L-Leucyl-L-valyl-N-methyl-L-leucyl-L-alanyl-D-alanyl-N-methyl-L-leucyl-N-methyl-L-leucyl-N-methyl-L-valyl}. Its empirical formula is C63H111N11O12, and it has a molecular weight of about 1214.85. The term "ISArx247" is a trade designation given to this pharmacologically active compound. [00111] The structure of IS ATX247 has been verified primarily through nuclear magnetic resonance (NMR) spectroscopy. Both the !H and 13C spectra were assigned using a series of one and two dimensional NMR experiments, and by comparison to the known NMR assignments for cyclosporin A. The absolute assignment of the (E) and (Z)-isomers of ISATX247 was confirmed by Nuclear Overhauser Effect (NOE) experiments. Additional supporting evidence was provided by mass spectral analysis, which confirmed the molecular weight, and by the infrared spectrum, which was found to be very similar to cyclosporin A. The latter result was expected, given the similarity between the two compounds. [00112] The structure of cyclosporin A is illustrated in FIG. 1 A. The structure includes identification of the 11 amino acid residues that comprise the cyclic peptide ring of the molecule. These 11 amino acid residues are labeled with numbers increasing in a clockwise direction, starting with the amino acid shown at the top center of the ring (and identified with reference label "1-amino acid"). The first amino acid is enclosed in a dashed box for clarity. The side chain of the 1-amino acid residue has been drawn out chemically since it is at this general location that the synthetic reactions described herein take place. Conventionally, the carbon adjacent to the carbonyl group of an amino acid is labeled as the a-carbon, with progressive letters in the Greek alphabet used to label adjacent carbons in a direction down the chain, away from the peptide ring. In the case of cyclosporin A, as shown in FIG. 1 A, the p-carbon of the side chain is bonded to a hydroxyl group, and there is a trans-oriented double bond between the e and ^-carbons of the side chain. [00113] Another schematic of the cyclosporin A structure is drawn in FIG. IB, where a different portion of the molecule has been enclosed in a dashed box. This figure defines the nomenclature to be used in the present description, where the term "CsA" refers to the portion of the cyclosporin A enclosed in the box. The present nomenclature provides a shorthand means of displaying the region where the synthetic reactions described herein will take place (i.e., the side chain of the 1-amino acid residue, which has been drawn outside the dashed box in FIG. IB), without having to re-draw the remainder of the molecule each time a, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , We Claim: 1. A method of preparing an isomeric mixture of cyclosporin A analogs modified at the 1-amino acid residue, wherein the synthetic pathway comprises the steps of: a) heating an acetyl-n-halocyclosporin A with a first compound selected from the group consisting of triaryl phosphine, trialkylphosphine, arylalkylphosphine, and triarylarsine to produce an intermediate; b) preparing a mixture of (E) and (Z)-isomers of acetyl-1,3-diene by stirring the intermediate with a second compound selected from the group consisting of acetaldehyde, formaldehyde, deuterated formaldehyde, 2-chlorobenzaldehyde, and benzaldehyde; and c) preparing a mixture of (E) and (Z)-isomers of ISATX247 by treating the mixture of (E) and (Z)-isomers of acetyl-l,3-diene with a base. 2. The method as claimed in claim 1, wherein the acetyl-r\|-halocyclosporin A is acetyl-n-bromocyclosporin A. 3. The method as claimed in claim 1, further including the step of halogenating the n-carbon of the side chain of the 1-amino acid residue of cyclosporin A using a bromination reaction carried out by refluxing acetyl cyclosporin A with N-bromosuccinimide and azo-bis-isobutyronitrile. 4. The method as claimed in claim 1, wherein the first compound is triphenylphosphine and the intermediate is triphenylphosphonium halide of acetyl cyclosporin A. 5. The method as claimed in claim 1 or 4, wherein the second compound is formaldehyde. 6. A method of preparing an isomeric mixture of cyclosporin A analogs modified at the 1-amino acid residue, wherein the synthetic pathway comprises the steps of: a) converting an acetyl cyclosporin A aldehyde to a mixture of (E) and (Z)-isomers of acetyl-1,3-diene by reacting the acetyl cyclosporin A aldehyde with a phosphorus ylide via a Wittig reaction, optionally in the presence of a lithium halide; and b) preparing a mixture of (E) and (Z)-isomers of ISATX247 by treating the mixture of (E) and (Z)-isomers of acetyl-1,3-diene with a base. 7. The method as claimed in claim 6, in which the phosphorus ylide used in the Wittig reaction is selected from the group consisting of triphenylphosphine, triarylphosphine, . trialkylphosphine, and arylalkylphosphine. 8. A method of preparing an isomeric mixture of cyclosporin A analogs modified at the 1-amino acid residue, wherein the synthetic pathway comprises the steps of: a) protecting the ß-alcohol of cyclosporin A by forming a first intermediate acetyl cyclosporin A; b) oxidizing the acetyl cyclosporin A to produce a second intermediate acetyl cyclosporin A aldehyde; c) converting an intermediate acetyl cyclosporin A aldehyde to a mixture of .(E) and (Z)-isomers of acetyl-1,3-diene by reacting the intermediate with a phosphorus ylide via a Wittig reaction, optionally in the presence of a lithium halide; and d) preparing a mixture of (E) and (Z)-isomers of ISATX247 by treating the mixture of (E) and (Z)-isomers of acetyl-1,3-diene with a base. 9. The method as claimed in claim 8, wherein the oxidizing step is carried out with an oxidizing agent selected from the group consisting of ozone, potassium permanganate, ruthenium tetroxide, osmium tetroxide, polymer-supported osmium tetroxide, and ruthenium chloride. 10. The method as claimed in claim 9, wherein the ruthenium tetroxide and ruthenium chloride oxidizing agents are used with a co-oxidant selected from the group consisting of periodate and hypochlorite. 11. The method as claimed in claim 9, wherein the ruthenium tetroxide and ruthenium chloride oxidizing agents are used with acetonitrile. 12. The method as claimed in any of claims 1, 6 and 8, wherein the base that is used to treat the acetyl-l,3-diene is selected from the group consisting of sodium hydroxide, sodium carbonate, potassium carbonate, sodium alkoxide, and potassium alkoxide. 13. The method as claimed in claim 1, wherein the intermediate is triphenyl- or trialkyl phosphonium bromide of acetyl cyclosporin A, and wherein the step that stirs the intermediate with formaldehyde is done in the presence of a lithium halide. 14. A method of preparing an isomeric mixture of cyclosporin A analogs modified at the 1-amino acid residue, wherein the synthetic pathway comprises the steps of: a) converting an intermediate acetyl cyclosporin A aldehyde to a mixture of (E) and (Z)-isomers of acetyl-1,3 -diene by reacting the intermediate with a phosphorus ylide prepared from a tributylallylphosphonium halide or triphenylphosphonium halide via a Wittig reaction, optionally in the presence of a lithium halide; and b) preparing a mixture of (E) and (Z)-isomers of ISATX247 by treating the mixture of (E) and (Z)-isomers of acetyl-1,3-diene with a base. 15. The method as claimed in claim 14, wherein the phosphonium halide is a phosphonium bromide. 16. The method as claimed in claim 15, wherein the Wittig reaction is carried out in a solvent selected from the group consisting of tetrahydrofuran and toluene, and wherein the solvent is used in the presence of a compound selected from the group comprised of butyllithium, sodium lower alkoxide, potassium lower alkoxide, and carbonate at a temperature between about -80°C and 110°C. 17. The method as claimed in claim 16, wherein the potassium lower alkoxide is potassium-tert-butoxide. 18. The method of claim 17, wherein the solvent is tetrahydrofuran used in the presence of potassium-tert-butoxide at a temperature between about -70°C and -100°C. 19. A method of preparing an isomeric mixture of cyclosporin A analogs modified at the 1-amino acid residue, the method comprising a synthetic pathway that prepares an (E)-isomer and a (Z)-isomer of ISATX247 such that the (E)-isomer and the (Z)-isomer are present in the mixture in a predetermined ratio, wherein the synthetic pathway comprises the steps of: a) protecting the ß alcohol of the 1 amino acid of cyclosporin A; b) oxidizing the protected cyclosporin A to produce a protected cyclosporin A aldehyde; c) converting the protected cyclosporin A aldehyde to a mixture of E- and Z- isomers of protected 1,3 diene by reacting the protected cyclosporin A aldehyde with a phosporus glide via a Wittig reaction, optionally in the presence of a lithium halide; and d) preparing a mixture of E- and Z- isomers by deprotecting the protected 1, 3 diene. 20. The method as claimed in claim 19, wherein the ß alcohol of the 1 amino acid of cyclosporin A is protected by reacting cyclosporin A with a reagent to give a protected cyclosporin A selected from the group consisting of acetate esters, benzoate esters, substituted benzoate esters, ethers, and silyl ethers. 21. A method of preparing an isomeric mixture of cyclosporin A analogs modified at the 1-amino acid residue, the method comprising a synthetic pathway that prepares an (E)-isomer and a (Z)-isomer of ISATX247 such that the (E)-isomer and the (Z)-isomer are present in the mixture in a predetermined ratio, wherein the ratio of isomers in the mixture ranges from about 45 to 55 percent by weight of the (E)-isomer to about 55 to 45 percent by weight of the (Z)-isomer, based on the total weight of the mixture. 22. A method of preparing a predetermined isomeric mixture of cyclosporin A analogs modified at the 1-amino acid residue, the method comprising: a) preparing a first material enriched in an (E)-isomer of ISATX247; b) preparing a second material enriched in a (Z)-isomer of ISATx247; and c) mixing the first and second materials in a ratio designed to give the desired isomeric composition. 23. A method of preparing an isomeric mixture of cyclosporin A analogs modified at the 1-amino acid residue, wherein the synthetic pathway comprises the steps of: a) converting an intermediate TMS cyclosporin A aldehyde to a mixture of (E) and (Z)-isomers of TMS-l,3-diene by reacting the intermediate with a phosphorus glide prepared from a tributylallylphosphonium halide or triphenylphosphonium halide via a Wittig reaction, optionally in the presence of a lithium halide; and b) preparing a mixture of (E) and (Z)-isomers of ISATX247 by deprotecting the mixture of (E) and (Z)-isomers of TMS-l,3-diene with an acid. 24. The method as claimed in claim 23, wherein the phosphonium halide is a phosphonium bromide. 25. The method as claimed in claim 23, wherein step a) is carried out in a solvent which comprises tetrahydrofuran and/or toluene used in the presence of a sodium or potassium lower alkoxide, or a carbonate, at a temperature between about -80°C and 110°C. 26. The method as claimed in claim 25, wherein the sodium or potassium lower alkoxide is potassium-tert-butoxide. 27. The method as claimed in claim 23, wherein the acid is selected from the group consisting of hydrochloric acid, acetic acid, citric acid, a Lewis acid, and HF-based reagents.

Full Text

SYNTHESIS OF CYCLOSPORIN ANALOGS
CROSS-REFERENCE TO RELATED APFLICATIQNS
[0001] This application claims the benefit of U.S. Application Serial Nos. 60/346,201
filed October 19,2001 and 60/370,596 filed April 5, 2002. The entire disclosure of each of these applications is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The invention is directed to isomeric mixtures of cyclosporin analogues"that
are related to cyclosporine A. It is contemplated that the mixtures possess enhanced efficacy and/or reduced toxicity over the individual isomers and over naturally occurring and other presently known cyclosporines and cyclosporine derivatives. In addition, the present invention relates to synthetic pathways for producing isomers of cyclosporin A analogs, where such pathways vary in the degree of stereoselectivity depending on the specific reaction conditions.
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BACKGROUND OF THE INVENTION
[0060] Cyclosporine derivatives compose a class of cyclic polypeptides, consisting of
eleven amino acids, that are produced as secondary metabolites by the fungus species Tolypocladium inflatum Gams. They have been observed to reversibly inhibit immunocompetent lymphocytes, particularly T-lymphocytes, in the Go or G\ phase of the cell cycle. Cyclosporine derivatives have also been observed to reversibly inhibit the production and release of lymphokines (Granelli-Pipemo et al, 1986). Although a number of cyclosporine derivatives are known, cyclosporine A is the most widely used. The suppressive effects of cyclosporine A are related to the inhibition of T-cell mediated activation events. This suppression is accomplished by the binding of cyclosporine to the ubiquitous intracellular protein, cyclophilin. This complex, in turn, inhibits the calcium- and calmodulin-dependent serine-threonine phosphatase activity of the enzyme calcineurm. Inhibition of calcineurin prevents the activation of transcription factors such as NFATp/c and NF-KB, which are necessary for the induction of the cytoldne genes (IL-2, IFN-y, IL-4, and GM-CSF) during T-cell activation. Cyclosporine also inhibits lymphokine production by T-helper cells in vitro and arrests the development of mature CD8 and CD4 cells in the thymus (Granelli-Piperno et al, 1986). Other in vitro properties of cyclosporine include the 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 exogenous lymphokine, inhibition of IL-1 production, and inhibition of mitogen activation of IL-2 producing T-lymphocytes (Granelli-Piperno et al, 1986).
[0061] Cyclosporine is a potent immunosuppressive agent that has been demonstrated
to suppress humoral immunity and cell-mediated immune reactions such as allograft rejection, delayed hypersensitivity, experimental allergic encephalomyelitis, Freund's adjuvant arthritis and graft vs. host disease. It is used for the prophylaxis of organ rejection subsequent to organ transplantation; for treatment of rheumatoid arthritis; for the treatment of psoriasis; and for the treatment of other autoimmune diseases, including type I diabetes, Crohn's disease, lupus, and the like.
[0062] Since the original discovery of cyclosporin, a wide variety of naturally
occurring cyclosporins have been isolated and identified and many further non-natural

cyclosporins have been prepared by total- or semi-synthetic means or by the application of modified culture techniques. The class comprised by the cyclosporins is thus now substantial and includes, for example, the naturally occurring cyclosporins A through Z.[c.f. Traber et al. (1977); Traber et al. (1982); Kobel et al. (1982); and von Wartburg et al. (1986)], as well as various non-natural cyclosporin derivatives and artificial or synthetic cyclosporins including the dihydro- and iso-cyclosporins; derivatized cyclosporins (e.g., in which the 3'-O-atom of the -MeBmt- residue is acylated or a further substituent is introduced at the a-carbon atom of the sarcosyl residue at the 3-position); cyclosporins in which the -MeBmt-residue is present in isomeric form (e.g., inwhichthe.configurationL across positions 6' and 7 of the -MeBmt-residue is cis rather than trans); and cyclosporins wherein variant ainino acids are incorporated at specific positions within the peptide sequence employing, e.g., the total synthetic method for the production of cyclosporins developed by R. Wenger-see e.g. Traber et al. (1977), Traber et al. (1982) and Kobel et al. (1982); U.S. Pat. Nos. 4,108,985, 4,210,581,4,220,641,4,288,431,4,554,351 and 4,396,542; European Patent Publications Nos. 0 034 567 and 0 056 782; International Patent Publication No. WO 86/02080; Wenger (1983); Wenger (1985); and Wenger (1986). Cyclosporin A analogues containing modified amino acids in the 1 -position are reported by Rich et al. (1986). Immunosuppressive, anti-inflammatory, and anti-parasitic cyclosporin A analogues are described in U.S. Pat. Nos. 4,384,996; 4,771,122; 5,284,826; and 5,525,590, all assigned to Sandoz. Additional cyclosporin analogues are disclosed in WO 99/18120, assigned to Isotechnika. The terms Ciclosporin, ciclosporin, cyclosporine, and Cyclosporine are interchangeable and refer to cyclosporin.
[0063] There are numerous adverse effects associated with cyclosporine A therapy,
including nephrotoxicity, hepatotoxicity, cataractogenesis, hirsutism, parathesis, and gingival hyperplasia to name a few (Sketris et al., 1995). Of these, nephrotoxicity is one of the more serious, dose-related adverse effects resulting from cyclosporine A administration. Immediate-release cyclosporine A drug products (e.g., Neoral® and Sandimmune®) can cause nephrotoxicities and other toxic side effects due to their rapid release and the absorption of high blood concentrations of the drug. It is postulated that the peak concentrations of the drug are associated with the side effects (Bennett, 1998). The exact mechanism by which cyclosporine A causes renal injury is not known; however, it is proposed that an increase in the levels of vasoconstrictive substances in the kidney leads to the vasoconstriction of the

afferent glomerular arterioles. This can result in renal ischemia, a decrease in glomerular filtration rate and, over the long term, interstitial fibrosis. When the dose is reduced or another irnrnunosuppressive agent is substituted, renal function improves (Valantine and Schroeder, 1995).
[0064] Accordingly, there is a need for immunosuppressive agents which are
effective and have reduced toxicity.
[0065] Cyclosporin analogs containing modified amino acids in the 1-position are
disclosed in WO 99/18120, which is assigned to the assignee of the present application, and incorporated herein in its entirety. Also assigned to the present assignee is U.S. Provisional Patent Application No. 60/346,201, in which applicants disclosed a particularly preferred cyclosporin A analog referred to as "ISATX247." This analog is structurally identical to cyclosporin A except for modification at the 1-amino acid residue. Applicants discovered that certain mixtures of cis and trans isomers of ISArx247 exhibited a combination of enhanced potency, and/or reduced toxicity over the naturally occurring and presently known cyclosporins. Certain alkylated, arylated, and deuterated derivatives of ISArx247 were also disclosed.
[0066] Typically, the disclosed mixtures in U.S. Provisional Patent Application No.
60/346,201 range from about 10 to 90 percent by weight of the trans-isomer and about 90 to 10 percent by weight of the os-isomer, in another embodiment, the mixture contains about 15 to 85 percent by weight of the trans-isomer and about 85 to 15 percent of the cis-isomer; in another embodiment, the mixture contains about 25 to 75 percent by weight of the trans-isomer and about 75 to 25 percent by weight of the cir-isomer; in another embodiment, the mixture contains about 35 to 65 percent by weight of the trans-isomer and about 65 to 35 percent by weight of the cw-isomer; in another embodiment, the mixture contains about 45 to 55 percent by weight of the trans-isomer and about 55 to 45 percent of the cis-isomer. In another embodiment, the isomeric mixture is an ISArx247 mixture which comprises about 45 to 50 percent by weight of the trans-isomer and about 50 to 55 percent by weight of the cis-isomer. These percentages by weight are based on the total weight of the composition. In other words, a mixture might contain 65 percent by weight of the (E)-isomer and 35 percent

by weight of the (Z)-isomer, or vice versa. In an alternate nomenclature, the m-isomer may also be described as a (Z)-isomer, and the trans-isomer could also be called an (E)-isomer.
[0067] Accordingly, there is a need in the art for methods of preparation of
cyclosporin analogs, including isomers of ISATX247. Synthetic pathways are needed that produce enriched compositions of the individual isomers, as well mixtures of the isomers having a desired ratio of the two isomers. Methods of preparation of derivatives of ISAi%247 are needed as well.
SUMMARY OF THE INVENTION
[0068] Cyclosporine and its analogs are members of a class of cyclic polypeptides
having potent immunosuppressant activity. Despite the advantages these drugs offer with respect to their immunosuppressive, anti-inflammatory, and anti-parasitic activities, there are numerous adverse effects associated with cyclosporine A therapy that include nephrotoxicity and hepatotoxicity. Accordingly, there is a need for new immunosuppressive agents that are as pharmacologically active as the naturally occurring compound cyclosporin A, but without the associated toxic side effects.
[0069] Embodiments of the present invention provide certain mixtures of cis and
trans-isomsrs of cyclosporin A analogs, which are pharmaceutically useful. A preferred analog is referred to as ISAJX247. Mixtures of ISATX247 isomers exhibit a combination of enhanced potency and reduced toxicity over the naturally occurring and presently known cyclosporins.
[0070] The present invention is based in part on the discovery that certain isomeric
mixtures of analogues of cyclosporine provide superior immunosuppressive effects without the adverse effects associated with cyclosporine A. In particular, we have unexpectedly found that isomeric mixtures (i.e., mixtures of both cis- and trans- isomers) ranging from about 10:90 to about 90:10 (trans- to cis-) of cyclosporine analogues modified at the 1-amino acid residue provide superior efficacy and safety. Examples of such analogues are disclosed in WO 99/18120, and include deuterated and non-deuterated compounds. In particular,

mixtures in the range of about 45:55 to about 50:50 (trans- to cis-) and in the range of about 50% to about 55% trans- and about 45% to about 50% cis- are found to be particularly efficacious. Moreover, it has been demonstrated that these isomer mixtures exhibit a combination of superior potency and reduced toxicity over naturally occurring and other presently known cyclosporines and cyclosporine derivatives.
[0071] A particularly preferred analogue (referred to herein as "ISATX247") is
structurally similar to cyclosporine A except for a modified functional group .on the periphery of the molecule, at the 1-amino acid residue. The structure of this particular isomeric analogue mixture compared to the structure of cyclosporine A is shown in FIGS. 1A, IB, 2A, 2B.
[0072] The isomeric mixtures can be used, among other things, for
immunosuppression, and the care of various immune disorders, diseases and conditions, including the prevention, control, alleviation and treatment thereof. .
[0073] According to embodiments of the present invention, ISATX247 isomers (and
derivatives thereof) are synthesized by stereoselective pathways that may vary in their degree of selectivity. Stereoselective pathways produce compositions that are enriched in either of the (E) and (Z)-isomers, and these compositions may be combined such that the resulting mixture has a desired ratio of the two isomers. Alternatively, the reactions conditions of a stereoselective pathway may be tailored to produce the desired ratio directly in a prepared mixture. The percentage of one isomer or another in a mixture can be verified using nuclear magnetic resonance spectroscopy (NMR) or other techniques well known in the art.
[0074] Each of the pathways typically proceeds with the application of a protecting
group to a sensitive alcohol functional group. In one embodiment the alcohol is protected as an acetate; in other embodiments the protecting groups are benzoate esters or silyl ethers. Although acetate protecting groups are common in the art, it is important to emphasize that in many of the exemplary embodiments described herein certain undesirable side-reactions involving an acetate protecting group may be avoided through the use of protecting groups such as benzoate esters or silyl ethers.

[0075] The protected compound may then serve as a precursor for a variety of
stereoselective synthetic pathways including some that utilize phosphorus-containing reagents as participants in a Wittig reaction, and inorganic elements as members of organometallic reagents. The latter type may proceed through six-membered ring transition states where steric hindrance dictates the configurational outcome. Many organometallic reagents are available, including those that feature inorganic elements such as boron, silicon, titanium, lithium, and sulfur. Individual isomers may be prepared from single or multiple precursors.
[0076] The ratio of the (E) to (Z)-isomers in any mixture, whether produced
stereoselectively or non-stereoselectively, may take on a broad range of values. For example, the mixture may comprise from about 10 to 90 percent of the (E)-isomer to about 90 to 10 percent of the (Z)-isomer. In other embodiments, the mixture may contain from about 15 to 85 percent by weight of the (E)-isomer and about 85 to 15 percent of the (Z)-isomer; in another embodiment, the mixture contains about 25 to 75 percent by weight of the (E)-isomer and about 75 to 25 percent by weight of the (Z)-isomer; in another embodiment, the mixture contains about 35 to 65 percent by weight of the (E)-isomer and about 65 to 35 percent by weight of the (Z)-isomer; in another embodiment, the mixture contains about 45 to 55 percent by weight of the (E)-isomer and about 55 to 45 percent of the (Z)-isomer. In another embodiment, the isomeric mixture is an ISATX247 mixture which comprises about 45 to 50 percent by weight of the (E)-isomer and about 50 to 55 percent by weight of the (Z)-isomer. These percentages by weight are based on the total weight of the composition, and it will be understood that the sum of the weight percent of the (E)-isomer and the (Z)-isomer is 100 weight percent. In other words, a mixture might contain 65 percent by weight of the (E)-isomer and 35 percent by weight of the (Z)-isomer, or vice versa.
[0077] Accordingly, in one aspect, the invention provides methods of preparing an
isomeric mixture of cyclosporin A analogs modified at the 1-amino acid residue, wherein the synthetic pathway comprises the steps of: heating an acetyl-T|-halocyclosporin A with triakylphosphine, triarylphosphine (e.g. triphenylphosphine), arylalkylphosphine, and triarylarsine to produce an intermediate phosphonium halide of acetyl cyclosporin A;

preparing a mixture of (E) and (Z)-isomers of acetyl-l,3-diene by stirring the intermediate phosphonium halide of acetyl cyclosporin A with formaldehyde, optionally in the presence of a lithium halide; and preparing a mixture of (E) and (Z)-isomers of ISATX247 by treating the mixture of (E) and (Z)-isomers of acetyl- 1,3-diene with a base.
[0078] In another aspect, the invention is directed to methods of preparing an
isomeric mixture of cyclosporin A analogs modified at the 1-amino acid residue, wherein the synthetic pathway comprises the steps of: converting an intermediate, e.g., protected trimethylsilyl (TMS) cyclosporin A aldehyde or acetyl cyclosporin A aldehyde to a mixture of (E) and (Z)-isomers of acetyl-1,3-diene by reacting the intermediate with a phosphorus ylide via a Wittig reaction, optionally in the presence of a lithium halide; and preparing a mixture of (E) and (Z)-isomers of LSATX247 by treating the mixture of (E) and (Z)-isomers of acetyl-1,3-diene with a base in the case of acetyl-protecting group or, e.g., an acid in case of a TMS-protecting group.
[0079] In a further aspect, the invention is directed to methods of producing an E-
isomer enriched mixture of cyclosporin A analogs modified at the 1-amino acid residue, wherein the stereoselective synthesis of the E-isomer enriched material comprises the steps of: reacting an acetyl cyclosporin A aldehyde with a reagent selected from the group consisting of trialkylsilylallyl boronate ester and E-y-(trialkylsilylallyI) diaikylborane to form a p-trialkylsilyl alcohol; treating the P-trialkylsilyl alcohol with acid to form acetyl-(E)-l,3-diene; and treating the acetyl-(E)-1,3-diene with base to form the (E)-isomer of ISATX247.
[0080] In yet a further aspect, the invention is directed to methods of producing a Z-
isomer enriched mixture of cyclosporin A analogs modified at the 1-amino acid residue, wherein the stereoselective synthesis of the Z-isomer enriched material comprises the steps of: reacting an acetyl cyclosporin A aldehyde with a reagent selected from the group consisting of trialkylsilylallyl boronate ester and (E-^-trialkylsilylallyl) diaikylborane to form a P-trialkylsilyl alcohol; treating the p-trialkylsilyl alcohol with base to form acetyl-(Z)-1,3-diene; and treating the acetyl-(Z)-l,3-diene with base to form the (Z)-isomer of ISATX247.

[0081] In a still further aspect, the invention is directed to methods of producing an E-
isomer enriched mixture of cyclosporin A analogs modified at the 1-amino acid residue, wherein the stereoselective synthesis of the E-isomer enriched material comprises the steps of: reacting an acetyl cyclosporin A aldehyde with a lithiated allyldiphenylphosphine oxide to form acetyl-(E)-l,3-diene; and treating the acetyl-(E)-l,3-diene with base to form the (E)-isomer of ISArx247.
[0082] In yet a further still aspect, the invention provides method of producing a Z-
isomer enriched mixture of cyclosporin A analogs modified at the 1-amina acid residue, wherein the stereoselective synthesis of the Z-isomer enriched material comprises the steps of: reacting an acetyl cyclosporin A aldehyde with [3-(diphenylphosphino)allyl] titanium to form a titanium-containing intermediate; allowing the titanium-containing intermediate to proceed to an erythro-a-adduct; converting the erythro-cc-adduct to an p-oxidophosphonium salt by treatment of iodomethane; converting the p-oxidophosphonium salt to an acetyl-(Z)-1,3-diene; and treating the acetyl-(Z)-l,3-diene with base to form the (Z)-isomer of ISATX247.
[0083] In still a further aspect, the invention provides mixtures (E) and (Z)-isomers
prepared by a process comprising the steps of: protecting the P-alcohol of cyclosporin A to form acetyl cyclosporin A; brominating the r)-carbon of the side chain of the 1-amino acid residue of acetyl cyclosproin A to produce a first intermediate acetyl-rj-bromocyclosporin A; heating the first intermediate acetyl-T|-bromocyclosporin A with a reagent selected from the group consisting of triphenyl phosphine and trialkyl phosphine to produce a second intermediate selected from the group consisting of triphenyl- and trialkyl phosphonium bromides of acetyl cyclosporin A; preparing a mixture of (E) and (Z)-isomers of acetyl-1,3-diene by stirring the ylide generated from the triphenyl- or trialkyl salt (second intermediate triphenylphosphonium bromide) of acetyl cyclosporin A with formaldehyde; and preparing the mixture of (E) and (Z)-isomers of ISATX247 by treating the mixture of (E) and (Z)-isomers of acetyl- 1,3-diene with a base.
[0084] The invention is also directed to compositions of matter, including triphenyl-
and trialkyl phosphonium bromides of acetyl cyclosporin A, the product prepared by a

process comprising the steps of: protecting the f3-alcohol of cyclosporin A; brominating the •n-carbon of the side chain of the 1-amino acid residue of cyclosporin A to produce a first intermediate acetyl-T|-bromocyclosporin A; and heating the first intermediate acetyl-rj-bromocyclosporin A with a reagent selected from the group consisting of triphenylphosphine and trialkylphosphine to produce a bromide of acetyl cyclosporin A selected from the group consisting of the triphenyl- and trialkylphosphonium bromides of acetyl cyclosporin A. Also provided are compositions comprising a triphenyl or trialkyl phosphonium bromide derivative of acetyl cyclosporin A and compositions comprising a P-trimethylsilyl alcohol derivative of cyclosporin A.
[0085] In an additional aspect, the invention provides methods for the selective
preparation of cyclosporin A aldehyde comprising the steps of: protecting the P-alcohol of cyclosporin A by forming acetyl cyclosporin A or trimethylsilyl (IMS) cyclosporin A; and oxidizing the acetyl cyclosporin A or TMS cyclosporin A with ozone as the oxidizing agent used with a reducing agent.
[0086] In another added aspect, the invention is directed to methods of preparing an
isomeric mixture of cyclosporin A analogs modified at the 1-amino acid residue, wherein the synthetic pathway comprises the steps of: converting an intermediate acetyl cyclosporin A aldehyde to a mixture of (E) and (Z)-isomers of acetyl-1,3-diene by reacting the intermediate with a phosphorus ylide prepared from a tributylallylphosphonium halogenide via a WIttig reaction, optionally in the presence of a lithium halide; and preparing a mixture of (E) and (Z)-isomers of ISATX247 by treating the mixture of (E) and (Z)-isomers of acetyl-l,3-diene with a base.
[0087] In an additional aspect, the invention provides methods for the stereoselective
synthesis of the E-isomer of ISATX247 comprising the steps of: reacting a trimethylsilyl (TMS) cyclosporin A aldehyde with trialkylsilylallyl borane to form a p-trialkylsilyl alcohol; treating the p-trialkylsilyl alcohol to form directly the E-isomer of ISATX247.
[0088] Another aspect of the invention is directed to methods for the stereoselective
synthesis of the Z-isomer of ISATX247 comprising the steps of: reacting a trimethylsilyl

(TMS) cyclosporin A aldehyde with trialkylsilylallyl borane to form a P-trialkylsilyl alcohol; treating the p-trialkylsilyl alcohol with base to form TMS-(Z)-l,3-diene; and deprotecting the TMS-(Z)-l,3-diene to form the Z-isomer of ISArx247.
[0089] The invention is also directed to methods of preparing isomeric mixtures of
cyclosporin A analogs modified at the 1-amino acid residue, the method comprising a synthetic pathway that prepares an (E)-isomer and a (Z)-isomer of ISATX247 such that the (E)-isomer and the (Z)-isomer are present in the mixture in a predetermined ratio, wherein the synthetic pathway comprises the steps of: protecting the p alcohol of cyclosporin A; oxidizing the protected cyclosporin A to produce a second intermediate protected cyclosporin A aldehyde; converting the second intermediate protected cyclosporin A aldehyde to a mixture of E- and Z- isomers of protected 1,3 diene by reacting the second intermediate with a phosphorus ylide via a Wittig reaction, optionally in the presence of a lithium halide; and preparing a mixture of E- and Z- isomers by deprotecting the protected 1,3 diene.
[0090] Other methods of preparing such mixtures also provided by the invention
include methods of preparing an isomeric mixture of cyclosporin A analogs modified at the 1-amino acid residue, the method comprising a synthetic pathway that prepares an (E)-isomer and a (Z)-isomer of ISArx247 such that the (E)-isomer and the (Z)-isomer are present in the mixture in a predetermined ratio, wherein the ratio of isomers in the mixture ranges from about 45 to 55 percent by weight of the (E)-isomer to about 55 to 45 percent by weight of the (Z)-isomer, based on the total weight of the mixture.
[0091] The invention also provides methods of preparing a predetermined isomeric
mixture of cyclosporin A analogs modified at the 1-amino acid residue, the method comprising: preparing a first material enriched in an (E)-isomer of ISATX247; preparing a second material enriched in a (Z)-isomer of ISATX247; and mixing the first and second materials in a ratio designed to give the desired isomeric composition.

BRIEF DESCRIPTION OF THE DRAWINGS
[0092] FIG. 1A shows the structure of cyclosporin A, illustrating the 11 amino acid
residues that comprise the cyclic peptide ring of the molecule, as well as the structure of the side chain of the 1-amino acid residue;
[0093] FIG. IB is another illustration of the structure of cyclosporin A with particular
emphasis on the definition of the term "CsA" as it is used in the present description;
[0094] FIG. 2A shows the structure of the E-isomer (or trans-isomer) of the
cyclosporin A analog called ISATX247;
[0095] FIG. 2B shows the structure of the Z-isomer (or cw-isomer) of the cyclosporin
A analog ISATX247;
[0096] FIG. 3 shows an overview of exemplary synthetic pathways that may be used
to prepare the cyclosporin analogs of the present invention, where stereoselective pathways are grouped according to reactive conditions;
[0097] FIG. 4 illustrates a synthetic pathway that produces a mixture of (E) and (Z)-
isomers of ISATX247 from a bromine precursor;
[0098] FIG. 5 illustrates another synthetic pathway that produces a mixture of (E) and
(Z)-isomers of ISATX247 from an aldehyde precursor,
[0099] FIG. 6 illustrates an exemplary stereoselective reaction scheme that may be
used to prepare compositions enriched in either the (E) or (Z)-isomers of ISATX247, wherein either isomer may be prepared from the same precursor alcohol;

[00100] FIG. 7 illustrates an alternative reaction scheme for the stereoselective
synthesis of a composition enriched in the (Z)-isomer of ISATX247;
[00101] FIG. 8 illustrates an alternative reaction scheme for the stereoselective
synthesis of a composition enriched in the (E)-isomer of ISATX247;
[00102] FIGS. 9A-C illustrate exemplary synthetic pathways for producing a mixture
of the (E) and (Z)-isomers of ISATX247, the conditions of each reaction having been tailored to produce a particular exemplary ratio of the two isomers;
[00103] FIG. 10 illustrates exemplary stereoselective pathways for producing a
mixture of the (E) and (Z)-isomers of ISATX247, where compositions enriched in one of the two isomers are first prepared, and then mixed accordingly in predetermined proportions to achieve the desired ratio;
[00104] FIG. 11 provides the results of an assay which shows that the inhibition of
calcineurin phosphatase activity by ISATX247 (45-50% of E-isomer and 50-55% of Z-isomer) was up to a 3-fold more potent (as determined by IC50) as compared to cyclosporine A.
[00105] FIG. 12 sets forth the structure and isomeric composition of some deuterated
and non-deuterated analogue isomeric mixtures.
[00106] FIG. 13 provides the results of an assay which shows that the inhibition of
calcineurin phosphatase activity by various deuterated and non-deuterated analogue isomeric mixtures was at least as potent (as determined by IC50) as compared to cyclosporine A.

DETAILED DESCRIPTION OF THE INVENTION
Synthesis
[00107] Cyclosporin and its analogs are members of a class of cyclic polypeptides
having potent immunosuppressive activity. Despite the advantages these drugs offer with respect to their immunosuppressive, anti-inflammatory, and anti-parasitic activities, there are numerous adverse effects associated with cyclosporine A therapy mat include nephrotoxicity and hepatotoxicity. Accordingly, there is a need for new immunosuppressive agents mat are as pharmacologically active as the naturally occurring compound cyclosporin A, but without the associated toxic side effects.
[00108] Applicants have previously disclosed a cyclosporin A analog referred to as
'1SATX247." This analog is structurally similar to cyclosporin A except for modification at the 1-amino acid residue. Applicants discovered that certain mixtures of cis and trans-isomers of ISArx247 exhibited a combination of enhanced potency, and reduced toxicity, over the naturally occurring and presently known cyclosporins.
[00109] According to embodiments of the present invention, ISArx247 isomers (and
derivatives thereof) are synthesized by stereoselective pathways that may vary in their degree of stereoselectivity. Stereoselective pathways produce compositions that are enriched in either of the (E) and (Z)-isomers, and these compositions may be combined such that the resulting mixture has a desired ratio of the two isomers. Alternatively, the reaction conditions of a stereoselective pathway may be tailored to produce the desired ratio directly in a prepared mixture.
[00110] The chemical name of one immunosuppresive cyclosporin analog of the present invention, called ISArx247, is chemically described by the name cyclo{{E^)-(2S,3R,4R)-3-hydroxy-4-memyl-2-(memylammo)-6,8-nonadienoyl}-L-2-aminobutyryl-N-methyl-glycyl-N-methyl-L-Leucyl-L-valyl-N-methyl-L-leucyl-L-alanyl-D-alanyl-N-methyl-L-leucyl-N-methyl-L-leucyl-N-methyl-L-valyl}. Its empirical formula is C63H111N11O12, and

it has a molecular weight of about 1214.85. The term "ISArx247" is a trade designation given to this pharmacologically active compound.
[00111] The structure of IS ATX247 has been verified primarily through nuclear
magnetic resonance (NMR) spectroscopy. Both the !H and 13C spectra were assigned using a series of one and two dimensional NMR experiments, and by comparison to the known NMR assignments for cyclosporin A. The absolute assignment of the (E) and (Z)-isomers of ISATX247 was confirmed by Nuclear Overhauser Effect (NOE) experiments. Additional supporting evidence was provided by mass spectral analysis, which confirmed the molecular weight, and by the infrared spectrum, which was found to be very similar to cyclosporin A. The latter result was expected, given the similarity between the two compounds.
[00112] The structure of cyclosporin A is illustrated in FIG. 1 A. The structure
includes identification of the 11 amino acid residues that comprise the cyclic peptide ring of the molecule. These 11 amino acid residues are labeled with numbers increasing in a clockwise direction, starting with the amino acid shown at the top center of the ring (and identified with reference label "1-amino acid"). The first amino acid is enclosed in a dashed box for clarity. The side chain of the 1-amino acid residue has been drawn out chemically since it is at this general location that the synthetic reactions described herein take place. Conventionally, the carbon adjacent to the carbonyl group of an amino acid is labeled as the a-carbon, with progressive letters in the Greek alphabet used to label adjacent carbons in a direction down the chain, away from the peptide ring. In the case of cyclosporin A, as shown in FIG. 1 A, the p-carbon of the side chain is bonded to a hydroxyl group, and there is a trans-oriented double bond between the e and ^-carbons of the side chain.
[00113] Another schematic of the cyclosporin A structure is drawn in FIG. IB, where a
different portion of the molecule has been enclosed in a dashed box. This figure defines the nomenclature to be used in the present description, where the term "CsA" refers to the portion of the cyclosporin A enclosed in the box. The present nomenclature provides a shorthand means of displaying the region where the synthetic reactions described herein will take place (i.e., the side chain of the 1-amino acid residue, which has been drawn outside the dashed box in FIG. IB), without having to re-draw the remainder of the molecule each time a, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,

We Claim:
1. A method of preparing an isomeric mixture of cyclosporin A analogs modified at the
1-amino acid residue, wherein the synthetic pathway comprises the steps of:
a) heating an acetyl-n-halocyclosporin A with a first compound selected from the group consisting of triaryl phosphine, trialkylphosphine, arylalkylphosphine, and triarylarsine to produce an intermediate;
b) preparing a mixture of (E) and (Z)-isomers of acetyl-1,3-diene by stirring the intermediate with a second compound selected from the group consisting of acetaldehyde, formaldehyde, deuterated formaldehyde, 2-chlorobenzaldehyde, and benzaldehyde; and
c) preparing a mixture of (E) and (Z)-isomers of ISATX247 by treating the mixture of (E) and (Z)-isomers of acetyl-l,3-diene with a base.

2. The method as claimed in claim 1, wherein the acetyl-r|-halocyclosporin A is acetyl-n-bromocyclosporin A.
3. The method as claimed in claim 1, further including the step of halogenating the n-carbon of the side chain of the 1-amino acid residue of cyclosporin A using a bromination reaction carried out by refluxing acetyl cyclosporin A with N-bromosuccinimide and azo-bis-isobutyronitrile.
4. The method as claimed in claim 1, wherein the first compound is triphenylphosphine and the intermediate is triphenylphosphonium halide of acetyl cyclosporin A.
5. The method as claimed in claim 1 or 4, wherein the second compound is formaldehyde.
6. A method of preparing an isomeric mixture of cyclosporin A analogs modified at the 1-amino acid residue, wherein the synthetic pathway comprises the steps of:
a) converting an acetyl cyclosporin A aldehyde to a mixture of (E) and (Z)-isomers of acetyl-1,3-diene by reacting the acetyl cyclosporin A aldehyde with a phosphorus ylide via a Wittig reaction, optionally in the presence of a lithium halide; and

b) preparing a mixture of (E) and (Z)-isomers of ISATX247 by treating the mixture of (E) and (Z)-isomers of acetyl-1,3-diene with a base.
7. The method as claimed in claim 6, in which the phosphorus ylide used in the Wittig reaction is selected from the group consisting of triphenylphosphine, triarylphosphine, . trialkylphosphine, and arylalkylphosphine.
8. A method of preparing an isomeric mixture of cyclosporin A analogs modified at the 1-amino acid residue, wherein the synthetic pathway comprises the steps of:
a) protecting the ß-alcohol of cyclosporin A by forming a first intermediate acetyl
cyclosporin A;
b) oxidizing the acetyl cyclosporin A to produce a second intermediate acetyl
cyclosporin A aldehyde;
c) converting an intermediate acetyl cyclosporin A aldehyde to a mixture of .(E) and (Z)-isomers of acetyl-1,3-diene by reacting the intermediate with a phosphorus ylide via a Wittig reaction, optionally in the presence of a lithium halide; and
d) preparing a mixture of (E) and (Z)-isomers of ISATX247 by treating the mixture of (E) and (Z)-isomers of acetyl-1,3-diene with a base.

9. The method as claimed in claim 8, wherein the oxidizing step is carried out with an oxidizing agent selected from the group consisting of ozone, potassium permanganate, ruthenium tetroxide, osmium tetroxide, polymer-supported osmium tetroxide, and ruthenium chloride.
10. The method as claimed in claim 9, wherein the ruthenium tetroxide and ruthenium chloride oxidizing agents are used with a co-oxidant selected from the group consisting of periodate and hypochlorite.
11. The method as claimed in claim 9, wherein the ruthenium tetroxide and ruthenium chloride oxidizing agents are used with acetonitrile.
12. The method as claimed in any of claims 1, 6 and 8, wherein the base that is used to treat the acetyl-l,3-diene is selected from the group consisting of sodium hydroxide, sodium carbonate, potassium carbonate, sodium alkoxide, and potassium alkoxide.

13. The method as claimed in claim 1, wherein the intermediate is triphenyl- or trialkyl phosphonium bromide of acetyl cyclosporin A, and wherein the step that stirs the intermediate with formaldehyde is done in the presence of a lithium halide.
14. A method of preparing an isomeric mixture of cyclosporin A analogs modified at the 1-amino acid residue, wherein the synthetic pathway comprises the steps of:

a) converting an intermediate acetyl cyclosporin A aldehyde to a mixture of (E) and (Z)-isomers of acetyl-1,3 -diene by reacting the intermediate with a phosphorus ylide prepared from a tributylallylphosphonium halide or triphenylphosphonium halide via a Wittig reaction, optionally in the presence of a lithium halide; and
b) preparing a mixture of (E) and (Z)-isomers of ISATX247 by treating the mixture of (E) and (Z)-isomers of acetyl-1,3-diene with a base.

15. The method as claimed in claim 14, wherein the phosphonium halide is a phosphonium bromide.
16. The method as claimed in claim 15, wherein the Wittig reaction is carried out in a solvent selected from the group consisting of tetrahydrofuran and toluene, and wherein the solvent is used in the presence of a compound selected from the group comprised of butyllithium, sodium lower alkoxide, potassium lower alkoxide, and carbonate at a temperature between about -80°C and 110°C.
17. The method as claimed in claim 16, wherein the potassium lower alkoxide is potassium-tert-butoxide.
18. The method of claim 17, wherein the solvent is tetrahydrofuran used in the presence of potassium-tert-butoxide at a temperature between about -70°C and -100°C.
19. A method of preparing an isomeric mixture of cyclosporin A analogs modified at the 1-amino acid residue, the method comprising a synthetic pathway that prepares an (E)-isomer and a (Z)-isomer of ISATX247 such that the (E)-isomer and the (Z)-isomer are present in the mixture in a predetermined ratio, wherein the synthetic pathway comprises the steps of:

a) protecting the ß alcohol of the 1 amino acid of cyclosporin A;
b) oxidizing the protected cyclosporin A to produce a protected cyclosporin A aldehyde;
c) converting the protected cyclosporin A aldehyde to a mixture of E- and Z- isomers of protected 1,3 diene by reacting the protected cyclosporin A aldehyde with a phosporus glide via a Wittig reaction, optionally in the presence of a lithium halide; and
d) preparing a mixture of E- and Z- isomers by deprotecting the protected 1, 3 diene.

20. The method as claimed in claim 19, wherein the ß alcohol of the 1 amino acid of cyclosporin A is protected by reacting cyclosporin A with a reagent to give a protected cyclosporin A selected from the group consisting of acetate esters, benzoate esters, substituted benzoate esters, ethers, and silyl ethers.
21. A method of preparing an isomeric mixture of cyclosporin A analogs modified at the 1-amino acid residue, the method comprising a synthetic pathway that prepares an (E)-isomer and a (Z)-isomer of ISATX247 such that the (E)-isomer and the (Z)-isomer are present in the mixture in a predetermined ratio, wherein the ratio of isomers in the mixture ranges from about 45 to 55 percent by weight of the (E)-isomer to about 55 to 45 percent by weight of the (Z)-isomer, based on the total weight of the mixture.
22. A method of preparing a predetermined isomeric mixture of cyclosporin A analogs modified at the 1-amino acid residue, the method comprising:

a) preparing a first material enriched in an (E)-isomer of ISATX247;
b) preparing a second material enriched in a (Z)-isomer of ISATx247; and
c) mixing the first and second materials in a ratio designed to give the desired
isomeric composition.
23. A method of preparing an isomeric mixture of cyclosporin A analogs modified at the
1-amino acid residue, wherein the synthetic pathway comprises the steps of:
a) converting an intermediate TMS cyclosporin A aldehyde to a mixture of (E) and (Z)-isomers of TMS-l,3-diene by reacting the intermediate with a phosphorus glide prepared from a tributylallylphosphonium halide or triphenylphosphonium halide via a Wittig reaction, optionally in the presence of a lithium halide; and

b) preparing a mixture of (E) and (Z)-isomers of ISATX247 by deprotecting the mixture of (E) and (Z)-isomers of TMS-l,3-diene with an acid.
24. The method as claimed in claim 23, wherein the phosphonium halide is a phosphonium bromide.
25. The method as claimed in claim 23, wherein step a) is carried out in a solvent which comprises tetrahydrofuran and/or toluene used in the presence of a sodium or potassium lower alkoxide, or a carbonate, at a temperature between about -80°C and 110°C.
26. The method as claimed in claim 25, wherein the sodium or potassium lower alkoxide is potassium-tert-butoxide.
27. The method as claimed in claim 23, wherein the acid is selected from the group consisting of hydrochloric acid, acetic acid, citric acid, a Lewis acid, and HF-based reagents.

Documents:

693-delnp-2004-abstract.pdf

693-DELNP-2004-Assignment-(30-09-2011).pdf

693-delnp-2004-assignment.pdf

693-delnp-2004-claims.pdf

693-DELNP-2004-Correspondence Others-(04-07-2011).pdf

693-DELNP-2004-Correspondence Others-(18-07-2011).pdf

693-DELNP-2004-Correspondence Others-(30-09-2011).pdf

693-delnp-2004-Correspondence-Others-(27-09-2012).pdf

693-delnp-2004-correspondence-others.pdf

693-delnp-2004-description (complete).pdf

693-delnp-2004-drawings.pdf

693-DELNP-2004-Form-1-(30-09-2011).pdf

693-delnp-2004-form-1.pdf

693-DELNP-2004-Form-2-(30-09-2011).pdf

693-delnp-2004-form-2.pdf

693-delnp-2004-form-26.pdf

693-DELNP-2004-Form-3-(04-07-2011).pdf

693-delnp-2004-form-3.pdf

693-delnp-2004-form-5.pdf

693-delnp-2004-form-6.pdf

693-DELNP-2004-GPA-(30-09-2011).pdf

693-delnp-2004-pct-101.pdf

693-delnp-2004-pct-105.pdf

693-delnp-2004-pct-210.pdf

693-delnp-2004-pct-220.pdf

693-delnp-2004-pct-304.pdf

693-delnp-2004-pct-401.pdf

693-delnp-2004-pct-402.pdf

693-delnp-2004-pct-408.pdf

693-delnp-2004-pct-409.pdf

693-delnp-2004-pct-416.pdf

693-DELNP-2004-Petition-137-(04-07-2011).pdf

693-DELNP-2007-Abstract-(09-06-2011).pdf

693-DELNP-2007-Claims-(09-06-2011).pdf

693-DELNP-2007-Correspondence Others-(09-06-2011).pdf

693-DELNP-2007-Drawings-(09-06-2011).pdf

693-DELNP-2007-GPA-(09-06-2011).pdf

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Patent Number

256418

Indian Patent Application Number

693/DELNP/2004

PG Journal Number

24/2013

Publication Date

14-Jun-2013

Grant Date

13-Jun-2013

Date of Filing

18-Mar-2004

Name of Patentee

ISOTECHNIKA PHARMA INC

Applicant Address

5120-75TH STREET, EDMONTON, ALBERTA T6E 6W2, CANADA

Inventors:

#	Inventor's Name	Inventor's Address
1	SELVARAJ NAICKER	3304-117TH STREET, EDMONTON, ALBERTA T6J 3J4, CANADA
2	MARK ABEL	11530-9A AVENUE, EDMONTON, ALBERTA T6J 7B2, CANADA
3	HANS-JURGEN MAIR	FRIEDRICH HECKER STR.3, D-79539 LORRACH, GERMANY
4	RANDALL W. YATSCOFF	1596 HECTOR ROAD, EDMONTON, ALBERTA T6R 2Z5, CANADA
5	ROBERT T. FOSTER	34 WESTBROOK DRIVE, EDMONTION, ALBERTA T6J 2C9, CANADA
6	JEAN-MICHEL ADAM	IM REINCHERHOF 187, CH-4153 REINACH BL, SWITZERLAND
7	BRUNO LOHRI	SCHELTENSTRASSE 5, CH-4153 REINACH BL, SWITZERLAND
8	SEETHARAMAN JAYARAMAN	11812-13 AVENUE, EDMONTON, ALBERTA T6J 7E9, CANADA

PCT International Classification Number

C07K 7/00

PCT International Application Number

PCT/CA02/01559

PCT International Filing date

2002-10-17

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	60/346,201	2001-10-19	U.S.A.
2	60/370,596	2002-04-05	U.S.A.