MXPA00002068A - Deuterated cyclosporine analogs and their use as immunomodulating agents - Google Patents

Abstract

Cyclosporine derivatives are disclosed which possess enhanced efficacy and reduced toxicity over naturally occurring and other presently known cyclosporins and cyclosporine derivatives. The cyclosporine derivatives of the present invention are produced by chemical and isotopic substitution of the cyclosporine A (CsA) molecule by:(1) chemical substitution and optionally deuterium substitution of amino acid 1, and (2) deuterium substitution at key sites of metabolism of the cyclosporine A molecule such as amino acids 1, 4, 9. The most active derivatives of the invention were those possessing both chemical and deuterium substitution. Also disclosed are methods of producing the cyclosporine derivatives and method of producing immunosuppression with reduced toxicity with the disclosed cyclosporine derivatives.

Description

DETERMINED CYCLOSPORINE ANALOGS AND THEIR USE AS IMMUNOMODULATOR AGENTS DESCRIPTION OF THE INVENTION This application is a continuation in part of the United States provisional application serial number 60 / 061,360 filed on October 8, 1997, which is entrusted to and incorporated into its whole. Cyclosporin derivatives of the present invention having improved efficacy and reduced toxicity occurring naturally and other currently known cyclosporins and cyclosporin derivatives are disclosed. The cyclosporin derivatives of the present invention are produced by chemical and isotopic substitution of the cyclosporin A molecule (CsA) by: 1. Chemical substitution and optionally deuterium substitution of amino acid 1; and 2. Replacing deuterium as key sites of metabolism of the cyclosporin A molecule such as amino acids 1, 4, 9. The most active derivatives of the invention are those that possess both chemical and deuterium substitutions. Cyclosporins are a family of neutral, hydrophobic, cyclic, undecapeptides containing a novel nine carbon amino acid (MeBmt) at the 1-position of the ring that exhibits anti-inflammatory properties of immunosuppressive, antiparasitic, fungal and chronic potency. The elements that naturally occur from this family of structurally related compounds are produced by various fungal imperfections. Cyclosporins A and C are the main components. Cyclosporin A, which is discussed further below, is a particularly important member of the cyclosporin family of compounds. Twenty-four minor metabolites, also oligopeptides, have been identified: La et al, J. Antibiotics 42, 1283 (1989); Traber et al, Helv. Chim. Acta 70, 13 (1987); Von artburg and Traber Prog. Med. Chem. 25, 1 (1988). Isolation of the A / C cycle / sporinas, as well as the structure of A were reported by A. Ruegger et al., Helv. Chim. Acta 59, 1075 (1976); M. Dreyfuss et al., J. Appl. Microbiol. 3, 125 (1976). The crystal and molecular structures of the iodine derivative of A have been reported by T. J. Petcher et al., Helv. Chim. Acta 59, 1480 (1976). The structure of C was reported by R. Traber et al., Ibid. 60, 1247 (1977). The production of A and C has been reported by E. Harri et al., US Patent No. 4,117,118 (1978 to Sandoz). The isolation, characterization and antifungal activity of B, D, E, as well as the structures from A to D have been reported by R. Traber et al., Helv. Chim. Acta 60, 1568 (1977). The isolation and structures of E, F, G, H, I: eidem, ibid. 65, 1655 (1982). The preparation of [2-Deutero-3-fluoro-D-Ala] 8-CsA is described by Patchett et al in GB 2,206,199A which was published on December 29, 1988. Cyclosporine was discovered to be immunosuppressive when it was observed to suppress antibody production in mice during screening of fungal extracts. Specifically, these suppressive effects appear to be related to the inhibition of activation cases mediated by the T-cell receptor. This is carried out to interrupt the calcium-dependent signal transduction during activation of the T cell by inactivating calmodulin and cyclophilin, a peptidyl propyl isomerase. It also inhibits the production of lymphokine by T-helper cells in vitro and stops the development of mature CD8 and CD4 cells in the thymus. Other in vitro properties include the inhibition of IL-2 that produces T lymphocytes and cytotoxic T lymphocytes, inhibition of IL-2 released from activated T cells, inhibition of resting T lymphocytes in response to alloantigene and exogenous lymphokine, inhibition of production of IL-1, and inhibition of mitogen activation of IL-2 that produces T lymphocytes. Further evidence indicates that the above effects involve T lymphocytes at the activation and maturation stages. The stimulation of TCR (T cell receptor) by the foreign antigen in a major histocompatibility molecule (MHC) on the surface of the T cell results in the activation of a TCR signal transmission path (exact unknown transmission method) to through the cytoplasm causing signal results in the activation of nuclear transcription factors, that is, the nuclear factors of activated T cells (NF-AT) that regulate the transcription of the T cell activation genes. These genes include those of the lymphokine interleukin-2 (IL-2). The translation of the message is followed by the secretion of IL-2. T-cell activation also involves the appearance of the lymphokine receptor IL-2R on the cell surface. After IL-2 binds to IL-2R, a lymphocyte receptor (LKR) transmission signal path is activated. The immunosuppressant drug, rapamycin, inhibits this trajectory. CsA is a potent inhibitor of the TCR-mediated transduction signal path. This inhibits the binding of NF-AT to IL-2 enhancer, and thus inhibits transcriptional activation. CsA binds to cyclophilin, which binds to calcineurin, which is a key enzyme in the transduction signal cascade of the T cell. Cyclophilin is found in prokaryotic and eukaryotic organisms and is ubiquitous and abundant. Cyclophilin is a single peptide chain with residues of 165 amino acids. This has a molecular mass of 17.8 kD. An approximately spherical molecule with a radius of 17 angstroms, cyclophilin has an antiparallel beta barrel of eight threads. Instead of the barrel, the tightly packed center contains more hydrophobic side chains. CsA has numerous hydrophobic side chains that allow adjustment in the beta-barrel of cyclophilin. Cyclophilin catalyses the interconversion of the cis and trans rotamers of the peGIFdyl-prolylamide linkage of the peptide and protein substrates. Cyclophilin is identical in structure with cis-trans peptidyl propyl isomerase and supports structural similarity to the superfamily of proteins that carry ligands such as bound retinol protein (RBP). These proteins carry the ligand in the barrel core. But cyclophilin currently carries the linked ligand site at the outlet of the barrel. The tetrapeptide ligand binds in a long deep groove on the protein surface between one side of the beta barrel and the Thrll6-Glyl30 linkage. Additional properties have also been reported in studies of biological activity of CsA: J.F. Borel et al., Agents Actions 6, 468 (1976). Pharmacology: Eidem. Immunology 32, 1017 (1977); R. Y. Calne, Clin. Exp. Immunol. 35, 1 (1979). Human studies: R. Y. Calne et al., Lancet 2, 1323 (1978); R.L. Poles et al., Ibid. 1327; R. L. Powles et al., Ibid, 1, 327 (1980). In vitro activity (porcine T-cells): D. J. White et al., Transplantation 27, 55 (1979). Effects on human lymphoid and myeloid cells: M. Y. Gordon, J.W. Singer, Nature 279, 433 (1979). Clinical study of CsA in graft-versus-host disease: P .J. Tutschka et al., Blood 61, 318 (1983). Mechanism of Action of Cyclosporin A Cyclosporin Complex A-Cyclophilin A CsA, as discussed above, binds to the barrel of beta cyclophilin. Thirty CyP A residues define the CsA binding site. These residues are Arg 55, Phe 60, Met 61, Gln 63, Gly 72, Ala 101, Asn 102, Ala 103, Gln 111, Phe 113, Trp 121, Leu 122, His 126. The longest side chain movements are 1.3 A for Arg 55 and above 0.7 A for Phe 60, Gln 63, and Trp 121. There are four direct hydrogen bonds between CyP A and CsA. The CsA residues 4, 5, 6, 7, 8 protrude out of the solvent and are thought to be involved in the binding of the determinant protein, calcineurin (Pflugl, G., Kallen J., Schirmer, T., Jansonius, JN , Zurini, MGM, &Walkinshaw, MD (1993) Nature 361, 91-94). Function of the CsA-Cyp complex A. The CsA-CyP A complex inhibits the phosphatase activity of the heterodimeric phosphatase protein serine / threonine calcineurin (Liu, J., Farmer, JD, Lane, WS, Friedman, J., Weissman, I., &Schreiber, SL (1991) Cell 66, 807-15. S anson, SK, Born, T., Zydowsky, C. D., Cho. H., Chang. H. Y. & Walsh, C. T. (1992) Proc.

Nati Acad. Sci. USA 89, 3686-90). CyP A binds CsA with an affinity of approximately 10 nM. The complex is then presented to calcineurin (Liu, J., Farmer, J. D., Lane, W.

S., Friedman, J., Weissman, I., & Schreiber, S. L. (1991) Cell 66, 807-15.). The dephosphorylates of calcineurin of the transcription factor NFAT are found in the cytoplasm of T cells. Dephosphorylation allows NFAT to translocate to the nucleus, which combines with the jun / fos genes and activate the transcription of the IL-2 gene responsible for the progression of cell cycle, directing the immune response. The CsA-CyP complex inhibits calcineurin phosphatase activity and final immunosuppression. (Etzkorn, F.A., Chang.Z., Stolz, L.

A., & Walsh, C. T. (1994) Biochemistry 33, 2380-2388.). Neither CsA or CyP A alone are immunologically important. Only its complex is important (Liu, J., Farmer, J. D., Lane, W. S., Friedman, J., Weissman, I., &Schreiber, S. L. (1991) Cell 66, 807-15). Cyclosporin metabolism: Cyclosporine is metabolized in the liver, small intestine and kidney to more than 30 metabolites. The structure of 13 metabolites and 2 metabolites of phase II has been identified and at least 23 additional metabolites have been isolated by HPLC and their structures characterized by mass spectrometry. The reactions involved in the phase I metabolism of cyclosporin are hydroxylation, demethylation as well as oxidation and cyclization to amino acid 1. Several clinical studies and reports show an association between blood concentrations of cyclosporin metabolites and neuro or nephrotoxicity. In vitro experiments indicate that the metabolites are considerably less immunosuppressive and more toxic than CsA. As exemplified by the increasingly expanded list of indications for which CsA have been found useful, the cyclosporin family of the compounds find utility in the prevention of rejection or organ and bone marrow transplants; and in the treatment of psoriasis, and a number of autoimmune disorders such as diabetes mellitus type 1, multiple sclerosis, autoimmune uveitis, and rheumatoid arthritis. Additional indicators are discussed below. As is generally accepted by those of skill in the art, inhibition of secretion of interleukin-2 (IL-2) and other lymphokine lymphokines is a useful indicator for the intrinsic immunosuppressive activity of a cyclosporin analogue. For a recent review of the uses and mechanisms of action of cyclosporin see Wenger et al Cyclosporine: Chemistry, Structure-Activity Relationships and Mode of Action, Progress in Clinical Biochemistry and Medicine, vol. 2, 176 (1986). Cyclosporin A is a cyclic peptide that contains several N-methyl amino acids and, in position 8, contains a D-alanine. The structure of Ciclosporin Aa is given as follows: H3CN "Q * M-aU * MaL.au 10 MaVa. 11 J MaBmt l Wing 7 MaLau ß V «I 5 MaL.au 4 a Unless otherwise specified, each of the amino acids of the cyclosporin described is of the L configuration. As is the practice in the field, a particular cyclosporin analog can be named using an abbreviated notation that identifies how the analog Thus, cyclosporin C, which differs from cyclosporine A by threonine in position 2, can be identified as [Thr] 2-cyclosporin or [Thr] 2-CsA. Similarly, cyclosporin B is [Ala] 2-CsA; cyclosporin D is [Val] 2-CsA; cyclosporin E is [Val] 11-CsA; cyclosporin F is [3-DeoxyMebt] 1-CsA; cyclosporin G is [NVa] 2-CsA; and cyclosporin H is [D-MeVal] n-CsA. Serine D and Threonine T have been introduced into position 8 of cyclosporin A by resulting biosynthesis in active compounds. See R. Traber et al. J. Antibiotics 42, 591 (1989). D-chloroalnin has also been introduced in position 8 of Cyclosporin A by biosynthesis. See A. La et al. J. Antibiotics 52, 1283 (1989). Indications for Cyclosporine Therapy Immunoregulatory abnormalities have been shown to exist in a wide variety of chronic autoimmune and inflammatory diseases, including systemic lupus erythematosis, chronic rheumatoid arthritis, type 1 diabetes mellitus, inflammatory bowel disease, biliary cirrhosis, uveitis, multiple sclerosis and other disorders such as Crohn's disease, ulcerative colitis, pemphigoid bolus, sarcoidosis, psoriasis, ichthyosis, and Graves' ophthalmopathy. Although the underlying pathogenesis of each of these conditions may be very different, they may have in common the appearance of a variety of autoantibodies and self-reactive lymphocytes. Such auto-reactivity may be due, in part, to the loss of the homeostatic controls under which the normal immune system operates. Similarly, following the bone marrow or an organ transplant, the host lymphocytes recognize the antigens of foreign tissue and are to produce antibodies that lead to graft rejection. A final result of an autoimmune process or a rejection is tissue destruction caused by inflammatory cells and mediators released. Anti-inflammatory agents, such as NSAIDs (Non-Spheroidal Anti-Inflammatory Drugs), and corticosteroids act mainly by blocking the effect, or secretion, of these mediators, but none to modify the immunological basis of the disease. On the other side, cytotoxic agents such as cyclophosphamide, act in such a way that the normal and autoimmune responses are not interrupted. Instead, patients treated with such non-specific immunosuppressive agents are thus likely to succumb to infection as they are to their autoimmune disease. Generally, a cyclosporin, such as cyclosporin A, is neither cytotoxic nor myelotoxic. This does not inhibit the migration of monocytes nor inhibits the action of granulocytes and macrophages. This action is specific and leaves more established immune responses intact. However, this is nephrotoxic and is known to cause the following undesirable side effects: (1) abnormal liver function; (2) hirsutism; (3) rubber hypertrophy; (4) shudder; (5) neurotoxicity; (6) hyperaesthesia; and (7) gastrointestinal discomfort. A number of cyclosporins and analogs have been described in the patent literature: U.S. Patent No. 4,108,985, issued to Ruegger, et al. on August 22, 1978 entitled "Dihydrocyclosporin C", describes dihydrocyclosporin C, which can be produced by hydrogenation of cyclosporin C. US Patent No. 4,117,118 issued to Harri, et al., on September 26, 1978, entitled "Organic Compounds ", describes cyclosporins A and B, and the production thereof by fermentation. U.S. Patent No. 4,210,581, issued to Ruegger, et al., On July 1, 1980, entitled "Organic Compounds", describes cyclosporin C and dihydrocyclosporicin C which can be produced by hydrogenation of cyclosporin C. US Patent No. 4,220,641, issued to Traber, et al., on September 2, 1980, entitled "Organic Compounds", describes cyclosporin D, dihydrocyclosporin D, and isocyclosporin D. US Patent No. 4,288,431 issued to Traber, et al., on September 8, 1981 entitled "Ciclosporin Derivatives, Their Production and Pharmaceutical Compositions Containing Them ", describes cyclosporin G, dihydrocyclosporin G, and isocyclosporin G. US Patent No. 4,289,851, issued to Traber, et al., On September 15, 1981, entitled" Process for Producing Cyclosporin Derivatives ", describes cyclosporin D , Dihydrocyclosporin D, and Isocyclosporin D, and a process for producing same, US Patent No. 4,384,996, issued to Bollinger, et al., on May 24, 1983, entitled "Novel Cyclosporins", describes cyclosporins having a β-vinylene-a-amino acid residue at position 2 and / or a β-hydroxy-α-amino acid residue at position 8. Cyclosporins described include either MeBmt or dihydro-MeBmt at position 1. US Patent No. 4,396,542, issued to Wenger on August 2, 1983, entitled "Method for the Total Synthesis of Cyclosporins, Novel Cyclosporins and Novel Intermediates and Methods for theis Production", describes the synthesis of cyclosporins, where the residue in position 1 is either MeBmt, dihydro-MeBmt, and protected intermediates. U.S. Patent No. 4,639,434, issued to Wenger, et al., On January 27, 1987, entitled "Novel Cyclosporins", describes cyclosporins with substituted residues at positions 1, 2, 5 and 8. US Patent No. 4,681,754 , issued to Siegel, on July 21, 1987, entitled "Counteracting Cyclosporin Organ Toxicity", describes methods for the use of cyclosporin comprising co-dergocrine. U.S. Patent No. 4,703,033, issued to Seebach on October 27, 1987, entitled "Novel Cyclosporins", describes cyclosporins with substituted residues at positions 1, 2 and 3. Substitutions at position 3 include halogen. H. Kobel and R. Traber, Directed Biosynthesis of Cyclosporins, European J. Appln. Microbiol Biotechnol., 14, 237B240 (1982), describes the biosynthesis of cyclosporins A, B, C, D and G by fermentation. Additional cyclosporin analogs are described in US Patent No. 4,789,823, issued to Witzel, entitled "New Cyclosporin Analogs with Modified" amino acids C-9, which describe analogs of cyclosporin with sulfur-containing amino acids at the 1-position.

The present invention relates chemically to substituted and deuterated analogs of cyclosporin A and related cyclosporins. An object of the present invention is to provide novel cyclosporin analogues that have improved efficacy and altered pharmacokinetic and pharmacodynamic parameters. Another object of the present invention is to provide a cyclosporin analogue for the care of disorders and immunoregulatory diseases, including the prevention, control and treatment thereof. A further object of the present invention is to provide pharmaceutical compositions for administering to a patient in need of treatment one or more of the active immunosuppressive agents of the present invention. A still further object of this invention is to provide a method for controlling graft rejection, autoimmune and chronic inflammatory diseases by administering a sufficient amount of one or more of the immunosuppressive agents in a mammalian species in need of such treatment. Finally, it is an object of this invention to provide processes for the preparation of the active compounds of the present invention. The replacement and deuteration of the cyclosporin molecule results in altered physicochemical and pharmacokinetic properties that improve its usefulness in the treatment of transplant rejection, host against graft disease, graft against host disease, aplastic anemia, focal and segmental glomerulosclerosis, myasthenia gravis, arthritis psoriatic, recurrent polychondritis and ulcerative colitis. Modalities of the invention include derivatives of CsA wherein one or more hydrogen atoms at the positions of 1, 3 and 9 amino acids are substituted with a deuterium atom and wherein the cyclosporin A derivatives are optionally chemically substituted at position 9 of the amino acid. A further specific embodiment of the invention is the derivative CsA represented by formula I: wherein R is (i) a deuterium or (ii) a linear or branched, saturated or unsaturated aliphatic chain of 2 to 16 carbon atoms and containing one or more deuterium atoms or an ester, ketone or carbon chain alcohol and optionally contains one or more substituents selected from halogen, nitro, amino, amido, aromatic and heterocyclic, or (iii) R is an aromatic or heterocyclic group optionally containing a deuterium atom, or (iv) R is a methyl group, and X, Y and Z are hydrogen or a deuterium that provides at least one of X, Y or Z is deuterium and R 'is an OH or an ester or is a 0 and together with a carbon adjacent to a double bond or an amino acid 1 it forms a heterocyclic ring such as 5-membered rings wherein the heteroatom is oxygen. Other specific embodiments of the present invention include the CsA derivative of the formula I wherein R is a saturated or unsaturated carbon chain of 2 to 3 carbons containing one or more deuteriums. Additional specific modalities include those of the following formulas 5g and 5e: BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is the structure of cyclosporin A showing a deuteration site at position 3 of the amino acid. Figure 2 is the structure of cyclosporin A showing a deuteration site at position 9 of the amino acid. Figure 3 is scheme I of the synthesis of cyclosporin derivatives. Figure 4 is scheme II of the synthesis of cyclosporin derivatives. Figure 5 is a graph of the results of the calcineurin test of Example 9. Figure 6 is a graph of the results of a mixed lymphocyte reaction test of Example 10. The substitution of deuterium for ordinary hydrogen and deuterated substrate for protium Metabolites can produce profound changes in biosystems. Isotopically altered drugs have shown widely divergent pharmacological effects. Pettersen et al. Found increases in anti-cancer effect with deuterated 5,6-benzylidene-dl-L-ascorbic acid (Zilascorb) [Anticancer Res. 12, 33 (1992)]. The substitution of deuterium to cyclosporin methyl groups will result in a lower oxidation ratio of the C-D bond relative to the oxidation ratio of a non-deuterium substituted C-H bond. The isotopic effect acts to reduce the formation of demethylated metabolites and therefore alters the pharmacokinetic parameters of the drug. The lower oxidation, metabolism and purification relationships result in a greater and more sustained biological activity. The deuteration is directed to several sites of the cyclosporin molecule to increase the potency of the drug, reduces drug toxicity, reduces the clearance of the pharmacologically active portion and improves the stability of the molecule. Isotopic substitution: Stable isotopes (eg, deuterium, 13C, 15N, 180), are non-radioactive isotopes that contain an additional neutron than the normally abundant isotope of the respective atom. The deuterated compounds have been used in pharmaceutical investigations to investigate the in vivo metabolic fate of the compounds by evaluating the mechanism of action and metabolic pathway of the deuterated parent compound (Blake et al., J. Pharm. Sci. 64, 3, 367-391 , 1975). Such metabolic studies are important in the design of safety, effective therapeutic drugs, either due to the active compound in vivo administered to the patient or due to the metabolites produced from the paternal compound provided to be toxic or carcinogenic (Foster et al., Advances in Drug Research Vol. 14, pp. 2-36, Academic press, London, 1985). The incorporation of a heavy atom, particularly the substitution of deuterium for hydrogen, can give rise to an isotopic effect that could alter the pharmacokinetic effects of the drug. This effect is usually insignificant if the tag is placed at a metabolically inert position of the molecule. Stable isotope radioactivation of a drug can alter its physicochemical properties such as pKa and lipid solubility. These changes can influence the fate of the drug at different stages throughout its passage through the body. The absorption, distribution, metabolism or excretion can be changed. Absorption and distribution are processes that depend mainly on the molecular size and lipophilicity of the substance. These effects and alterations can affect the pharmacodynamic response of the drug molecule if the isotopic substitution affects a region involved in a ligand-receptor interaction. The metabolism of the drug can elevate to a large isotopic effect if the breaking of a chemical bond to a deuterium atom is the limiting relationship stage in the process. While some of the physical properties of an isotope-labeled stable molecule are different from those of non-labeled ones, the chemical and biological properties are the same, with one important exception: due to the increased mass of the heavy isotope, any linkage implies the heavy isotope and another atom will be stronger than the limiting ratio stage, the reaction will be processed lower for the heavy isotope molecule due to the "kinetic isotope effect". A reaction involving the breakdown of the C-D bond can be up to 700 percent lower than a similar reaction involving the breaking of a C-H- bond. If the C-D bond is not involved in any of the outgoing stages to the metabolites, these may have no effect on altering the behavior of the drug. If a deuterium is placed in a site involved in the metabolism of a drug, an isotope effect will be observed only if the breakdown of the C-D bond is the limiting relationship stage. This is evidence to suggest that whenever the cleavage of an aliphatic C-H bond occurs, usually by oxidation catalyzed by a mixed oxidase-function, replaced by hydrogen by deuterium will lead to the observable isotope effect. It is also important to understand that the incorporation of deuterium to the site of light metabolism is the relationship to the point where another metabolite produced by attack on a carbon atom not replaced by deuterium becomes the main trajectory of a process called "metabolic disruption".

It is also observed that one of the most important trajectories of the compounds containing aromatic systems is the hydroxylation leaving a phenolic group in the 3 or 4 position to the carbon substituents. Although this trajectory implies unfolding of the C-H bond, it is often not accompanied by an isotope effect, due to the splitting of this link, which is not involved in the limiting relationship stage. The substitution of hydrogen by deuterium to the stereo center will induce a greater effect on the activity of the drug. Synthesis of Cyclosporin Derivatives: The starting material for the preparation of the compounds of this invention is cyclosporin A. The process for preparing the compounds of the present invention are illustrated as shown in Scheme I in Figure 3. It will be readily apparent for a person of ordinary skill in the art to review the synthetic route represented below that other compounds with formula I can be synthesized by substitution of appropriate reagents and agents in the synthesis shown below. The first step in the process to perform deuterated cyclosporin analogues is the preparation of key intermediates 3 and 6. This can be carried out by the oxidation of the double bond in amino acid 1. The treatment of cyclosporine with acetic anhydride and excess of dimethylaminopyridine provides the protected cyclosporine of hydroxylacetyl 2. Although the double bond splitting could be effected by treatment of 2 with ozone , or KMn04 / NaI04, it was found out that Os04 / NaI04 was the reagent of choice for the transformation to the aldehyde product 3. The reaction was generally found to be clean, producing the required material and proceeding in high yield. The setback to this reaction is that 0s04 is expensive and highly toxic, so its use is limited. But the results can be achieved more economically by the use of H20, with Os0 present in catalytic amounts, t-butyl hydroxide in alkaline solution and N-methylmorpholin-N-oxide can be replaced by H202 in this process. The aldehyde compound 3 ^ was further treated with various deuterated alkyl or aryltriphenylphosphonium derivatives (deliberate reagents), and hydrolysis by alkaline solution provided the final derivatives (5 a -h). A general procedure was also developed to obtain various compounds as shown in Scheme II in Figure 4. In this regard, the aldehyde derivative 3 was treated with deliberate reagent prepared using a standard procedure. The resulting product in moderate acid hydrolysis provides the key intermediate aldehyde product 6. This was further treated with second deuterated alkyl halide or alkyltriphenylphosphonium reagents and a moderate hydrolysis of acid produced the required products. This method provides control over the extent of the diene system. By using this approach, olefinic double bonds can be introduced step by step. A third aspect for preparing the deuterated 5a-h compounds is by heating the non-deuterated cyclosporin analogs described above, in a deuterated solvent such as deuterated water, deuterated acetic acid in the presence of an acid or base catalyst. Preferred cyclosporins of the present invention are those containing a deuterium and chemical substitution at an amino acid 1 such as that of formula II: Where X is "H ^ CH3 I - N-CH-CO- , EXAMPLES Example 1. To a stirred solution of cyclosporin 1 (1.01 g, 0.84 mmol) in acetic anhydride (20 ml) at room temperature is added DMPA (150 mg, 1.23 mmol, 1.5 eq). After stirring overnight, the reaction mixture is partitioned between EtOAc (50 ml) and water (25 ml). The separated EtOAc layer was then washed with water (50 ml) and brine (50 ml), dried (MgSO) and the solvent removed in vacuo to give the crude product as a glassy solid. Purification by flash chromatography through a short column of silica (2% MeOH / DCM) and lyophilization of benzene yielded 2 (1044 g, 0.84 mmol, quant.) As a clear, colorless solid.; [OÍ] D25 -305.7 (c.0.3, CHCI3); vmax (CHCl3 cast) / cm = 1 3328m, 2963m, 1746m 1627s, 1528m, 1472m, 1233m; dH (600MHz, C6D6 8.73 (1H, d, J = 9.5Hz, NH), 8.30 (1H, d, J = 7.0Hz, NH), 7.92 (1H, d, J = 7.5Hz NH), 7.49 (1H, d, J = 7.5Hz NH), 6.05 (1H, d, J = 11.5Hz), 5.88 (1H, dd, J = 3.5, 11.5Hz), 5.82 (1H, d, J = 11.5Hz), 5.65 (1H , = dd, J = 4.0, 12.0Hz), 5.60 (1H, dd J = 3.5, 12.5Hz), 5.63-5.57 (1H, m), 5.51-5.45 (1Hm), 5.37 (1H, dd, J = 5 5, 8.5Hz), 5.05-5.01 (2H, complex), 4.99 (1H, d, J = 11.0Hz), 4.76 (1H, p, J = 7.0Hz), 4.58 (1H, p, J = 7.0Hz ), 4.02 (1H, d, J = 13.5Hz), 3.47 (3H, s), 3.30 (3H, s), 3.17 (3H, s), 3.11 (3H, s), 2.98 (3H, s), 2.68 -2.62 (1H, m), 2.63 (3H, s), 2.51-2.39 (2H, complex), 2.34-2.25 (8H, complex), 2.03 (3H, s), 1.97-1.85 (2H, complex), 1.83 (3H, dd, J = 1.0, 6.5Hz), 1.82-1.77 (2H, complex), 1.68-1.61 (3H, complex), 1.55 (3H, d, J = 7.0Hz), 1. 55-1.51 (1H, m), 1.44-1.38 (1H, m), 1.32-1.20 (5H, complex), 1.29 (3H, d, J = 7.0Hz, 1.21 (3H, d, J = 6.5Hz), 1. 17 (3H, d, J = 6.5Hz), 1.14 (3H, d, J = 6.5Hz), 1.08 (3H, d, J = 6.5Hz), 1.04 (3H, d, J = 6.0Hz), 1.03 (3H, d, J = 7.0Hz), 1. 00 (3H, d, J = 7.0Hz), 0.93 (3H,, J = 6.0Hz), 0.92 (3H, d, J = 6.5Hz), 0.88-0.84 (9H, complex), 0.76 (3H, d, J = 6.5Hz), 0.57 (3H, d, J = 6.5Hz, dc (75MHz, C6D6) 173.6, 173.2, 172.8, 172. 6, 171 3, 171.1, 170.71, 170.67, 170.4, 170.2, 169.8, 167. 9 (C = 0), 129.0, 126.2 (C = C), 73.1 (COAc), 58.1, 57.1, 56. 0, 55.0, 54.6, 54.2, 50.3, 49.9, 48.6, 48.1, 47.8, 44.5, 40. 8, 39.1, 35.7, 33.6, 32.9, 32.1, 31.5, 31.2, 30.0, 29.7, 29.5, 293, 24.9, 24.6, 24.4, 24.0, 23.6, 23.4, 23.3, 21.7, 21. 1, 21.0, 20.6, 20.3, 19.5, 18.5, 18.0, 17.7, 17.5, 17.4, 14. 9, 9.7; m / z (Electroaspersion) Example 2 To a solution of compound 2 ^ (289 mg, 0.23 mmole) in a 1: 1 mixture of dioxane and water (5 ml) was first added sodium metaperiodate (100 mg, 0.47 mmoles, 2 eq.) And secondly an osmium tetraoxide (5 ml; 0.5 g Os04 in 250 ml of the solvent). Two phases were treated, purification by flash column chromatography (40% acetone in petroleum ether) and lyophilization of benzene gave compound 3_ (226 mg, 0.18 mmole, 80%) as a colorless solid emollient [] D25 -260.0 (0.1, CHC13); vmax (CHC13 cast) / cm "1 3325m, 2962m, 1748w, 1724w, 1677m, 1626s, 1228m, 755m, dH (300MHz, C5D6) 8.63 (1H, d, J = 9.5Hz, NH), 8.16 (1H, d , J = 7.0Hz, NH), 7.95 (1H, d, J = 7.5Hz, NH), 7.48 (1H, d, J = 9.0Hz, NH), 5.93 (1H, d, J = 7.5Hz), 5.84 (1H, dd, J = 4.0, 11.5Hz), 5.70 (1H, d, J = 11.5Hz) 5.56-5.54 (1H, m), 5.32 (1H, dd, J = 5.5, 8.0Hz), 5.07-4.88 (3H, complex), 4.72 (1H, p, J = 7.0Hz), 4.49 (1H, p, J = 7.0Hz), 3.98 (1H, d, J = 14.0Hz), 3.42 (3H, s, CH3N) , 327 (3H, s, CH3N), 3.12 (3H, s, CH3N, 3.07 (3H, s, CH3N), 2.91 (3H, s, CH3N), 2.79 (3H, s, CH3N), 2.59 (3H, s, CH3N), 2.42-2.08 (10H, complex) 1.94 (3H, s, CH3C02), 1.47 (3H , d, J = 7.0Hz), 1.24 (3H, 7.0Hz), 1.14-1.09 (9H, complex), 1.04 (3H, d, J = 6.5Hz), 1.01 (3H, d, J = 7.0Hz), 0.96 (3H, d, J = 6.5Hz), 0.92 (3H d, J = 6.5Hz), 0.91 (3H, d, J = 6.5Hz), 0.89 (3H, d, J = 6.0Hz), 0.83 (6H) , d, J = 6.5Hz), 0.74 (3H d, J = 6.5Hz), 0.59 (3H, d, J = 6.5Hz); dc (75MHz, C6D6) 202.5 (CHO), 174.4, 174.0, 173.7, 172.8, 171.6, 171.5, 171.2, 171.1, 170.6, 170.2, 170.2, 168.1, 73.0, 58.7, 57.6, 56.7, 55.5, 55.0, 54.5, 49.4 , 48.9, 48.5, 48.1, 45.0, 44.6, 41. 3, 39.8, 38.8, 37.7, 36.2, 32.5, 32.0, 31.6, 30.9, 30.3, 30.0, 29.8, 29.6, 25.6, 25.3, 25.0, 24.8, 24.5, 24.0, 23.8, 23. 4, 22.0, 21.7, 21.2, 20.5, 20.0, 19.8, 18.8, 18.5, 18.2, 17.4, 15.2, 10.0; m / z (Electroaspersion) 1232.8 (MH +, 100%).

Example 3 Method A: To a solution of compound 3_ (315 mg, 0.26 mmol) in THF (5 ml) at 0 ° C was added a solution of the iodide deutero-phosphorus (2.67 mmol, approximately 10 eq.), Prepared from iodide of d5-ethyltriphenylphosphonium. After treatment, purification by flash column chromatography (30% to 60% acetone in PE) and HPLC (60% to 65% MeCN in water), then lyophilization of benzene yielded compound 4 (153 mg, 0.12 mmole, 47%) as a colorless solid. Method B: To a stirred solution of compound 3 ^ (287 mg, 0.23 mmol) in THF (5 ml) under Ar at -78 ° C was carefully added a solution of phosphorus ylide (formed by the addition of sodium hexamethyldisilamide ( 1.0 M, 2.25 ml, 2.25 mmole, -10 eq.) To a suspension of d5-ethyltriphenylphosphonium iodide (480 mg, 1.13 mmoles, ~ 5 eq.), In THF (10 L) under Ar at room temperature). After stirring for 2 hours with gradual warming to room temperature, the reaction mixture was cooled to 0 ° C and then divided by the addition of 10% AcOH / THF (10 mL). The reaction mixture was concentrated in vacuo and partitioned between water (20 mL) and EtOAc (20 mL). The aqueous layer was then extracted with EtOAc (20 ml) and the combined organic extracts were then washed with 1 N HCl (20 ml) and water (20 ml), dried (MgSO 4) and the solvent removed in vacuo to give the product without purify. Purification by flash column chromatography (40% acetone in petroleum ether) and benzene lyophilization yielded the compound 4d (84 mg, 67 μmol, 29%) as a colorless solid; [a] D25 -283.0 (c.1.1, CHC13); vmax (CHC13 cast) / cm "1 3320m 3010m 2959s, 2924s, 2871m 2853m, 1743m, 1626s, 756s; dH (600MHz, C6D6) 8.78 (1H, d, J = 9.5Hz), 8.33 (1H, d, J = 7.0Hz), 7. 99 (1H, d, J = 7.5Hz), 7.59 (1H, d, J = 9.0Hz), 6.09 (1H, d, J = 11.5Hz), 5.92 (1H, dd, J = 4.0, 11.0Hz), 5.86 (1H, d, J = 11.5Hz), 5.72-5.64 (2H, complex), 5.62 (1H, dd, J = 3.5 , 12. 5Hz), 5.40 (1H, dd, J = 5.5, 8.5Hz), 5.10-5.02 (3H, complex), 4.80 (1H, q, J = 7.0Hz), 4.60 (1H, q, J = 7.0Hz), 4. 05 (1H, d, J = 14.0Hz), 3.51 (3H, s), 3.31 (3H, s), 3 20 (3H, s), 3.13 (3H, s), 3.01 (3H, s), 2.87 ( 3H, s), 2.64 (3H, s), 2.45 (1H, dt, J = 4.0, 12.5Hz), 2.36-2.20 (10H, complex), 2. 06 (3H, s), 1.93-1.79 (3H, complex); dD (84MHz, C6H6) dc (125MHz, C6D6) 174.5, 173.7, 173.6, 173.1, 171.7, 171.4, 170.9, 170.7, 170.6, 170.3, 170.0, 168.4, 130.2 (C = C), 123.8 (C = C), 73.8 (MeBmt C-3), 58.7, 58.1, 57.6, 57.1, 55.5, 55.0, 54.5, 49.4, 49.0, 48.6, 48.2, 45.0, 41.4, 39.9, 39.0, 37.8, 34.2, 33.9, 32.6, 32.3, 32.0, 31.4, 30.9, 30.8, 30.2, 30.1, 30.0, 29.9, 29.8, 29.6, 28 5, 25.6, 25.3.25, 24.9, 24.8, 24.1, 23.9, 23.8, 23.6, 23.1, 22.1, 21.7, 21.4, 20.7, 20.0 , 19.9, 19.8, 18.9, 18.7, 18.6, 18.3, 17.4, 153, 14.3, 10.2; m / z (Electroaspersion) 1270 ([M + Na] +, 100%), 1286 ([M + K] +, 20). Example 4 To a stirred solution of 4_d (84 mg, 67 μmol) in MeOH (5 mL) and water (2.5 mL) at room temperature was added potassium carbonate (99 mg, 0.72 mmol, ~ 10 eq.). After stirring overnight, the MeOH was removed in vacuo and the aqueous residue was partitioned between EtOAc (10 mL) and 5% citric acid solution (10 mL). The EtOAc layer was then washed with water (10 mL) and brine (10 mL), dried (MgSO4) and the solvent removed in vacuo to give the product without purification. Purification of HPLC (60% to 65% MeCN in water) and lyophilization of benzene yielded compound 5d (59 mg, 49 μmol, 70%) as a colorless solid: [a] D25 -262.0 (c 0.05, CHC13); vmax (CHC13 cast) / cm "1 3318m, 3008m, 2960s, 2872m, 1627s, 1519m, 1470m, 1411m, 1295m, 1095m, 754m, dH (600MHz, C6H6) 8.27 (1H, d, J = 9.5Hz), 7.96 (1H, d, J = 7.5Hz), 7.63 (1H, d, J = 8.0Hz), 7.45 (1H, d, J = 9.0Hz), 5.87 (1H, dd, J = 3.5, 11.0Hz), 5.74 (1H, d; J = 7.5Hz), 5.73-5.69 (1H, m), 5.66-5.64 (1H, broad d, J = 11.0Hz), 5.79 (1H, dd, J = 4.0, 11.5Hz), 3.39 (1H, dd, J = 5.5, 10.5Hz), 5.33 (1H, dd, J = 5.5, 8.5Hz), 5.24 (1H, d, < J = 11.0Hz), 5.12 (1H, dt J = 7.5, 10.0Hz), 4.88-4.79 (3H, complex), 4.22 (1H, dd, J = 5.5, 7.5Hz), 4.00 (1H, d, 13.5Hz), 3.72 (3H, s), 3.22 (3H, s) , 3.06 (3H, s), 2.97 (3H, s), 2.92 (3H, s), 2.85 (3H, s), 2.67-2.60 (1H, m), 2.58 (3H, s), 2.56-2.50 (1H , m broad), 2.33-2.23 (4H, complex), 220-2.07 (4H, complex), 1.80-1.74 (3H, complex), 1.67 (3H, d, J = 7.0Hz), 156-1.50 (2H, complex), 1.46-1 23 (9H, complex), 1.17-1.13 (16H, complex), 1.06 (3H, d, J = 6.5Hz), 1.02 (3H, d, J = 7.0Hz), 0.98 (3H, d, J = 6.5Hz), 0.96 (3H, d, J = 7.0Hz), 0.92-0.89 (9H complex), 0.86 (3H, t, J = 7.5Hz), 0.83 (3H, d, J = 6.0Hz), 0.64 (3H, d, J = 6.5Hz); dD (84MHz, C6 H6) 1.64 (CD3); dc (75MHz, C6H6) 174.2, 174.1, 174.0, 173.7, 171.8, 171.4, 171.2, 170.5, 170.4, 170.3, 169.8, 130.2, 124.1, (99.2,) 74.3, (67.1,) 66.3, 66.1, 61.0, 59.5, 58.3, 57.8, 55. 7, 55.5, 55.4, 49.4, 49.0, 48.4, 45.3, 41.4, 39.6, 39.0, 37. 8, 36.5, 36.1, 35.8, 33.7, 31.6, 30.8, 30.4, 30.1, 29.9, 29. 5, 29.4, 25.5, 25.2, 25.0, 24.9, 24.5, 24.2, 23.8, 23.7, 23. 6, 22.0, 21.4, 20.0, 18.8, 18.5, 17.8, 16.0, 10.1; m / z (Electroaspersion) 1206 ([M + H] +, 30%), 1228 ([M + Na] +, 100), 1244 ([M + K] +, 25). Example 5 To a vigorously stirred mixture of compound 3_ (49 mg, 39.8 μmol) and deuterated d3-allyltriphenylphosphonium bromide (311 mg, 812 μmol, ~ 20 eq.) In benzene (3 ml) at room temperature were added NaOH IN ( 3 mL). The stirring was continued at room temperature for 5 days, after which time the 2 layers were separated, the benzene layer was washed with water (5 mL), dried (MgSO4) and the solvent removed in vacuo to give the crude product. Purification by HPLC (20% to 60% MeCN in water) and lyophilization of benzene yielded compound 4_cf (23 mg, 18.3 μmol, 47%) as an emollient, colorless solid: [] D25 -264.2 (c. , CHC13); vmax (CHC13 cast) / cm "1 3322m, 2959m, 1744m, 1626s, 1231m, 754m, dH (300MHz, C6D6) complex due to 1: 1 ratio of geometrical isomers 8.73 (d, J = 9.5Hz, NH), 8.72 (d, J = 9.5Hz, NH), 8.29 (d, J = 6.5Hz, NH), 8.26 (d, J = 6.5Hz, NH), 7.92 (d, J = 7.5Hz, NH), 7.86 (d) , J = 7.5Hz, NH), 7.53 (d, J = 9.0Hz, NH), 7.49 (d, J = 9.0Hz, NH 7.10-6.70 (complex), 6.33 (broad t, J = 11.0Hz), 6.18 (d, J = 10.5Hz), 6.12 (d, J = 10.5Hz), 6.05 (d, J = 11.0Hz), 6.03 (d, J = 11.0Hz), 5.90-5.53 (complex), 537 (dd, J = 6.0, 8.0Hz), 5.20 (d, J = 12.0Hz), 5.14 (d, J = 12.0Hz), 5.07-4.97 (complex), 4.80-4.70 (complex), 4.57 (p, J = 7.0Hz ), 4.02 (d, J = 14.0Hz), 4.01 (d, J = 14.0Hz), 3.47 (s), 3.46 (s), 3.28 (s), 3.26 (s), 3.16 (s), 3.15 (s) ), 3.9 (s), 2.97 (s), 2.96 (s), 2.84 (s), 2.62 (s), 248-2.23 (complex), 2.05 (s), 2.03 (s), 1.95-1.59 (complex) , 1.54 (d, J = 7.0Hz), 1.53-0.80 (complex), 0.77 (d, J = 6.5Hz), 0.58 (d, J = 6.5Hz), 0.57 (d, J = 6.5Hz), dc ( 75MHz, C6D6) 174.5, 174.0, 173.9, 173.6, 173.5, 173.1, 17 1.7, 171.6, 171.4, 170.9, 170.5, 170.6, 170.6, 170.3, 169.8, 169.7, 168.4, 137.9, 133.9, 133.5, 132.8, 132.3, 131.6, 130.1, 116.9, 115.0, 73.6, 58.6, 57.6, 57.0, 56.8, 55.7, 55.5, 55.0, 54.9, 54.7, 54.5, 49.4, 48.9, 48.5, 48.2, 48.1, 44.9, 41.5, 39.9, 39.0, 38.9, 37.8, 37.6, 36.6, 36.3, 34.1, 33.7, 32.7, 32.1, 32.0, 31.5 , 30 9, 30.7, 30.0, 29. 8, 29.6, 25.6, 25.5, 25.3, 25.2, 25.0, 24.9, 24.1, 23.9, 23.7, 23.6, 22.1, 21.7, 21.6, 21.4, 21.3, 20.7, 20.0, 19.9, 18. 9, 18.6, 18.3, 17.6, 15.3, 10.2; m / z (Electroaspersion) 1258.8 (MH +, 100%). Example 6 To a vigorously stirred mixture of compound 3 (56 mg, 45.5 pmol) and deuterated d-crotyltriphenylphosphonium bromide (360 mg, 907 pmol, -20 eq.) In benzene (3 mL) at room temperature was added NaOH IN (3 mL). Stirring was continued at room temperature for 5 days, after which time the 2 layers were separated, the benzene layer was washed with water (5 mL), dried (MgSO4) and the solvent removed in vacuo to give the product without purification. . Purification by HPLC (20% to 60% MeCN in water) and lyophilization of benzene yielded compound 4_e (23 mg, 18.1 μmol, 40%) as a colorless emollient solid; [] D25 -236.0 (c 0.25, CHC13); vmax (CHC13 cast) / cm "1 3324m, 2959m, 2871m, 1745w, 1626s, 1231m, dH (300MHz, C6D6) complex due to presence of 4 isomers 8.76 (d, J = 6.0Hz), 8.73 (d, J = 6.0Hz), 8.29 (d, J = 7.0Hz), 7.93 (d, J = 7.5Hz), 7.88 (d, J = 7.5Hz), 7.53 (d, J = 9.5Hz), 7.62-7.31 (1H, complex), 7.16-6.88 (2H, complex), 6.59-6.39 (complex), 6.28 (t, J = 11.0Hz), 6.15 (d, J = 10.5Hz), 6.09 (d, J = 10.5Hz), 6.05 (d, J = 11.5Hz), 6.03 (d, J = 11.5Hz), 5.90-5.82 (complex), 5.68-535 (complex), 5.08-4.97 (complex), 4.81-4.72 (complex), 4.63-4.53 (complex), 4.03 (d, < J = 14.0Hz), 3.47 (s), 3.46 (s), 3.28 (s), 3.26 (s), 3.17 (s), 3.15 (s), 3.09 (s) , 2.98 (s), 2.97 (s), 2.83 (s), 2.63 (s), 2.62 (s), 2.71-2.56 (complex), 2.47-2.23 (complex), 2.05 (s), 2.04 (s), 2.03 (s), 2.02 (s), 1.98-0.82 (complex), 0.77 (d, J = 6.5Hz), 0.58 (d, J = 6.5Hz), 0.58 (d, J = 6.5Hz), m / z (Electrospray) 1273.8 (MH +, 100%) Example 7 To a stirred solution of the compound 4_g_ (20 mg, . 9 μmol) in methanol (5 mL) and water (1 mL) at room temperature was added potassium carbonate (30 mg, 217 μmol). After stirring overnight, the reaction mixture was partitioned between EtOAc (10 mL) and 5% aqueous citric acid (10 mL). The aqueous layer was further extracted with EtOAc (5 mL), the combined organic layers were then washed with 5% citric acid (10 mL) and brine (10 mL), dried (MgSO4) and the solvent removed in vacuo to give the Unpurified product. Purification by HPLC (65% MeCN) and lyophilization of benzene afforded compound 5g (10 mg, 8.2 μmol, 52%) as a colorless solid; [] D25 -285.2 (c 0.29, CHC13); vmax (CHC13 castj / cm "1 3500-3200ample, 3319m, 2958m, 2927m, 1626s, 1520m, 1468m, 754m; dH (300MHz, ßOß) complex due to the presence of 2 isomers 8.25 (d, J = 10.0Hz, NH ), 8.13 (d, J = 10.0Hz, NH), 7.93 (d, J = 7.0Hz, NH), 7.84 (d, J = 7.0Hz, NH), 7.67 (d, J = 8.0Hz, NH), 7.61 (d, J = 8.0Hz, NH), 7.55 (d, J = 8.5Hz, NH), 7.54 (d, J = 8.5Hz, NH), 6.84 (t, J = 10.5Hz), 6.79 (t, J = 10.5Hz), 6.58 (t, J = 10.5Hz), 6.52 (t, J = 10.5Hz), 6.30-6.14 (complex), 5.88-5.78 (complex), 5.75-5.66 (complex), 5.44-4.74 (complex), 4.22-4.15 (complex), 3.95 (d, J = 14.0Hz), 3.93 (d, J = 14.0Hz), 3.72 (s), 3.68 (s), 3.19 (s), 3.17 (s) , 3.05 (s), 3.03 (s), 2.94 (s), 2.93 (s), 2.89 (s), 2.86 (s), 2.82 (s), 2.81 (s), 2.72-2.53 (complex), 2.55 (s) s), 2.54 (s), 2.49-2.36 (complex), 2.32-2.03 (complex), 1.81-0.81 (complex), 0.65 (d, J = 6.5Hz)), m / z (Electroaspersion) 1216.8 (MH +, 100%), 607.9 ([M + 2H] 2+, 1_5) Example 8 To a stirred solution of the compound ^ e (18 mg, 14. 2 μmol) in methanol (5 mL) and water (1 mL) at room temperature was added potassium carbonate (35 mg, 254 μmol). After stirring overnight, the reaction mixture was partitioned between EtOAc (10 mL) and 5% aqueous citric acid (10 mL). The aqueous layer was further extracted with EtOAc (5 mL), the combined organic layers were then washed with 5% citric acid (10 mL) and brine (10 mL), dried (MgSO4) and the solvent removed in vacuo to give the Unpurified product. Purification by HPLC (65% MeCN) and lyophilization of benzene afforded compound 5_e (10 mg, 8.1 μmol, 57%) as a colorless solid; [.a] D25 -285.5 (c 0.11, CHC13); dH (300MHz C6D6) complex due to presence of 4 isomers 8 31 (d, J = 9.5Hz), 8.28 (d, J = 9.5Hz), 8.16 (d, J = 9.5Hz), 8.14 (d, J = 9.5 Hz), 7. 96 (d, J = 7.5Hz), 7.95 (d, J = 7.5Hz), 7.86 (d, J = 7.5Hz), 7.85 (d, J = 7.5Hz), 7.63 (d, J = 7.5Hz), 7.59 (d, J = 7.5Hz), 7.50-7.44 (complex), 6.60-6.49 (complex), 6.32-6.11 (complex), 5.88-5.83 (complex), 5.76-5.71 (complex), 5.64-5.22 (complex ), 5.17-5.08 (complex), 4.91-4.77 (complex), 4.26-4.18 (complex), 3.99 (d, J = 14.0Hz), 3.97 (d, J = 14.0Hz), 3.74 (s), 3.73 ( s), 3.71 (s), 3.69 (s), 3.22 (s), 3.21 (s), 3.20 (s), 3.19 (s), 3.07 (s), 3.06 (s), 3.05 (s), 2. 97 (s), 2.96 (s), 2.95 (s), 2.92 (s), 2.91 (s), 2.89 (s), 2.84 (s), 2.83 (s), 2.69-2.07 (complex), 2.58 (s) ), 2.57 (s), 1. 84-0.81 (complex), 0.64 (d, J = 6.5 Hz); m / z (Electroaspersion) 1269.8 ([M + K] +, 5%), 1253.8 ([M + Na] +, 30), 1231.8 (MH +). Example 9 The immunosuppressive activity was tested for the deuterated cyclosporin analogues as described in the following. Compound 5e and compound 5g are more potent than paternal cyclosporin. The calcineurin activity was analyzed using a modification of the method previously described by Fruman et al. (A Proc Nati Acad Sci USA, 1992).

Whole blood lysates were evaluated for their ability to dephosphorylate a peptide substrate of 32P-labeled 19 amino acids in the presence of okadaic acid, a phosphatase inhibitor type 1 and 2. Background phosphatase activity 2C (resistance activity of CsA and okadaic acid) was determined and subtracted from each sample, with the assay performed in the presence and absence of aggregate excess of CsA. The remaining phosphatase activity was taken as calcineurin activity. The results of the calcineurin assay are presented in Figure 5. The results are expressed as means ± the standard error of the medium. The results are plotted as concentration of the CsA derivative in ug / L against the percentage of inhibition of calcineurin. The structures of the compounds analyzed include: (Compound 2) 16 (Compound 3) (Compound 6) Example 10 A mixed lymphocyte reaction assay (MLR) was performed with cyclosporin and compounds 5e and 5g. The results are presented in Figure 6 and are plotted as the means of four experiments showing concentration of cyclosporin or derivatives against percent inhibition. The MLR assay is useful for identifying CsA derivatives with biological (immunosuppressive) activity and for quantifying this activity relative to the immunosuppressive activity of the paternal CsA molecule. An example of a lymphocyte proliferation assay procedure useful for this purpose is as follows: 1. Collect blood from two individuals (20 ml each) and isolate the lymphocytes using Ficoll-Paque (Pharmacia Biotech). 2. Count lymphocytes diluted 1:10 in 2% acetic acid (v / v). 3. Prepare 10 ml of each of the lymphocyte populations (A + B) at lxlO6 cells / ml in DMEM / 20% FCS (v / v). 4. Fix up to 96 wells of sterile tissue culture plate, flat bottom (Sarstedt, cat # 83.1835). For each well add: 5. 100 μl of aliquots per well of lymphocyte A population 6. 100 μl of aliquots per well of B lymphocyte population 7. 20 μl of aliquots per drug well (CSA and derivatives of CSA) at 0 , 2.5, 5, 10, 25, 50 and 100 μg / L in triplicate in DMEM without supplements. 8. To measure the effect of the drug on proliferation, incubate the plate for 5 days at 37 ° C in 5% C02 atmosphere. 9. On day 6, prepare 3.2 ml of 1:50 dilution of Methyl-3H-thymidine (Amersham Life Science, cat # TRK 120) in DMEM without supplements. Add 30 μl per well and incubate for 18 hours at 37 ° C in 5% CO atmosphere. 10. On day 7 the cells are harvested on GF / A vitreous microfiber filters (Whatman cat # 1820024) using a Cell Harvester (Skatron, cat # 11019). The cells washed 3x with 1.0 ml of sterile distilled water. Note: All procedures are performed using sterile techniques in a flow aspirator. 11. Place filters in scintillation bottles and add 1.5 ml of SciniSafe Plus 50% scintillation fluid (Fisher, cat # SX-25-5). 12. Measure the amount of radioactivity incorporated in the lymphocytes using a beta counter (Micromedic System Inc., TAURUS Automatic Liquid Scintillation Counter) for 1.0 minutes. 13. Calculate averages and standard deviations for each drug and express results such as:% of Inhibition - [1- CPM Average of test drug] X 100 CPM Average of drug zero Proliferation% = 100-% Inhibition The MLR assay can be used to select antibodies of the invention that bind biologically active CSA metabolites and to paternal CSA molecule. The antibodies could also be selected for reactivity to the biologically inactive metabolites. From the results of the calcineurin assays and the lymphocyte reaction assay, it was found that cyclosporins that have been chemically substituted and deuted to the position of amino acid 1 may possess significant immunosuppressive activity. In the case of derivatives 5e and 5g, immunosuppressive activity that is significantly greater than CsA was obtained. Example 11 Other cyclosporin derivatives of the invention that have been prepared include the following: STRUCTURE CODE # M M MeLeu- MeVal lf || Abu-Sar DBl-bl-31 MeLeu- D-Ala- Ala- MeLeu- Val- eLeu MeLeu-D-Ala-Ala-eLeu-al-eLeu MeLeu-D-Ala-Ala-MeLeu-Val-M? Leu Me-Leu-D-Ala-Ala-eLeu-Val-MeLeu MeLßu- D-Ala- Ala- MeLeu- Val- MeLeu - MeLeu- D-Ala- Ala-MeLe? - Val- MeLeu MeLeu- Ala- Ala- eLeu- Val- eLeu Formulation of Drug Composition and Immunosuppression Provocation The determination of the physicochemical, pharmacodynamic, toxicological and pharmacokinetic properties of the described cyclosporin derivatives can be made using standard chemical and biological assays and through the use of molding techniques that are known in the art. chemical and pharmacological / toxicological techniques. The therapeutic utility and dose regimen can be extrapolated from the results of such techniques and through the use of appropriate pharmacokinetic and / or pharmacodynamic models. The compounds of this invention can be administered clean or with a pharmaceutical carrier to a warm-blooded animal in need thereof. The pharmaceutical carrier can be solid or liquid. This invention also relates to a method for treating patients suffering from immunoregulatory abnormalities involving the administration of the described cyclosporins as the active constituent. For the treatment of these conditions and diseases caused by immunoregularity, a deuterated cyclosporin can be administered orally, topically, parenterally, by inhalation, spray or rectally in dosage unit formulations containing pharmaceutically acceptable non-toxic, conventional carriers, adjuvants and vehicles. The term parenteral, as used herein, includes subcutaneous, intravenous, intramuscular injections, injection techniques or intrasternal infusion. The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, lozenges, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules or syrups or elixirs. The compositions intended for oral use may be prepared according to any method known in the art for the manufacture of the pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents, and conservative agents to provide pharmaceutically pleasing and tasteful preparation. Tablets containing the active ingredient in admixture with pharmaceutically acceptable non-toxic excipients can also be manufactured by known methods. The excipients used may be, for example, (1) inert diluents such as calcium carbonate, lactose, calcium phosphate or sodium phosphate; (2) granulation and disintegration agents such as corn starch, or alginic acid; (3) binding agents such as starch, gelatin or acacia, and (4) lubricating agents such as magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a long period. For example, a time-delayed material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by techniques described in U.S. Patent Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotic therapeutic tablets by controlled release. In some cases, formulations for oral use may be in the form of hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin. They may also be in the form of soft gelatin capsules wherein the active ingredient is mixed with water or an oily medium, for example, peanut oil, liquid paraffin, or olive oil. Aqueous suspensions usually contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients may be (1) suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum and acacia gum; (2) dispersing or wetting agents which may be (a) a naturally occurring phosphatide such as lecithin, (b) a condensation product of an alkylene oxide with a fatty acid, for example, polyoxyethylene stearate, (c) ) a condensation product of ethylene oxide with a long-chain aliphatic alcohol, for example, heptadecaethyleneoxycetanol, (d) a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol such as a monooleate of polyoxyethylene sorbitol, or (e) a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride, for example, polyoxyethylene sorbitan monooolate. The aqueous suspensions may also contain one or more preservatives, for example, ethyl p-hydroxybenzoate or n-propyl; one or more coloring agents; one or more flavoring agents; and one or more sweetening agents such as sucrose, aspartame or saccharin. The oily suspension can be formulated by suspending the active ingredient in a vegetable oil, for example, arachis oil, olive oil, sesame oil or coconut oil, or a mineral oil such as liquid paraffin. Oily suspensions may contain a thickening agent, for example, beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents can be added to provide a pleasant oral preparation. These compositions can be preserved by the addition of an antioxidant such as ascorbic acid. The dispersible powders and granules are suitable for the preparation of an aqueous suspension. They provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing or wetting agents, and suspending agents are exemplified by those mentioned above. Additional excipients, for example, those sweetening, flavoring and coloring agents described above may also be present.

The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oil phase may be a vegetable oil such as olive oil or arachis oils, or a mineral oil such as a liquid paraffin or a mixture thereof. Suitable emulsifying agents may be (1) naturally occurring gums such as gum acacia and gum tragacanth, (2) naturally occurring phosphatides such as soybean and lecithin, (3) esters or partial esters derived from fatty acids and hexitol anhydrides, for example, sorbitan monooleate, (4) condensation products of partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents. Syrups and elixirs can be formulated with sweetening agents, for example, glycerol, propylene glycol, sorbitol, aspartame and sucrose. Such formulations may also contain a demulcent, a preservative, and flavoring and coloring agents. The pharmaceutical compositions may be in the form of sterile injectable aqueous or oleogenous suspension. This suspension can be formulated according to known methods using those wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the vehicles and acceptable solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspension medium. For this purpose any fixed soft oil can be employed including synthetic mono or diglycerides. In addition, the fatty acids such as oleic acid found used in the preparation of injectable solutions. The cyclosporins described can also be administered in the form of suppositories by rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures, but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycols. For topical use, creams, ointments, jellies, solutions or suspensions, etc., which contain the described cyclosporins are employed. Levels of doses in the range of about 0.05 mg to about 50 mg per kilogram of body weight per day are useful in treating the conditions indicated above (from about 2.5 mg to about 2.5 grams per patient per day). The amount of the active ingredient that can be combined with carrier materials to produce a single dose form will vary depending on the host treated and the particular mode of administration. For example, a formulation intended for oral administration of humans may contain from 2.5 mg to 2.5 grams of active agent compound with an appropriate and convenient amount of carrier material which may vary from about 5 to about 95 percent of the total composition. Dosage unit forms will generally contain between about 5 mg to about 500 mg of the active ingredient. It will be understood, however, that the specific dose level for any particular patient will depend on a variety of factors including the activity of the specific compound employed, age, body weight, general health, sex, diet, time of administration, route of administration , speed of excretion, combination of the drug and the severity of the therapy suffering a particular disease. All references listed herein are incorporated by reference. In the case of conflict, the text of the application is controlled. Modifications and changes of the described compounds and methods will be apparent to those skilled in the art. Such modifications and changes are intended to be encompassed by this description and the appended claims thereto.

Claims (26)

1. CLAIMS 1. A cyclosporin A derivative characterized in that one or more hydrogen atoms at the amino acid position selected from the group consisting of 1.3 and 9 or combinations thereof are substituted with a deuterium atom and wherein the cyclosporin A derivative is optionally chemically substituted at the 9-position of the amino acid or pharmaceutically acceptable salts thereof.
2. 2. The cyclosporin A derivative represented by the formula: (I) characterized in that: R is: (i) deuterium; (ii) a linear or branched, saturated or unsaturated aliphatic carbon chain of 1 to 16 carbon atoms and optionally contains one or more deuterium atoms or an ester, ketone or a carbon chain alcohol and optionally contains one or more selected substituents halogen, nitro, amino, amido, aromatic, and heterocyclic; (iii) an aromatic or heterocyclic group containing one or more deuterium atoms, or (iv) a methyl group; and X, Y and Z are hydrogen or deuterium with the proviso that if R is methyl or -CH2OH then at least one of X, Y or Z is deuterium; and wherein R 'is an OH or acetate or other ester or is an O and together with a carbon adjacent to a double bond at amino acid 1 forms a heterocyclic ring or a pharmaceutically acceptable salt thereof.
3. 3. The cyclosporin A derivative represented by the formula (II): (H) characterized in that: R is: (i) deuterium; (ii) a linear or branched, saturated or unsaturated aliphatic carbon chain of 1 to 16 carbon atoms, optionally containing one or more deuterium atoms and optionally containing one or more substituents selected from halogen, nitro, amino, amido, aromatic and heterocyclic; or (ii) an aromatic or heterocyclic group containing one or more deuterium atoms; X, Y and Z are hydrogen or deuterium; and R 'is an OH or acetate or other ester or is, an O and together with a carbon adjacent to a double bond or amino acid 1 forms a heterocyclic ring or a pharmaceutically acceptable salt thereof with the proviso that if R does not contain a Deuterium atom then at least one of X, Y and Z is deuterium. .
4. The cyclosporin A derivative according to claim 3, characterized in that: R is: (i) deuterium; or (ii) an aliphatic, linear or branched, saturated or unsaturated carbon chain of 1 to 16 carbon atoms, optionally containing one or more deuterium atoms and optionally containing one or more substituents selected from halogen, nitro, amino or amido; X, Y and Z are hydrogen or deuterium; and R 'is an OH or acetate or a pharmaceutically acceptable salt thereof with the proviso that if R does not contain a deuterium atom then at least one of X, Y and Z is deuterium.
5. 5. The cyclosporin derivative A according to claim 4, characterized in that R is -D, CD3, -CH = CD-CD3, -CD = CHCD3, -CD = CD-CD3, -CD = CH-CD = CD- CD3, -CH = CH-CH = CD-CD3, -CH = CH-CH = CD2, -CD = CH-CD = CD2, -CH = CD2 or -CD = CD2; X, Y and Z are hydrogen; and R 'is OH.
6. 6. The cyclosporin A derivative according to claim 5, characterized in that it has the formula (III): (III) or a pharmaceutically acceptable salt thereof.
7. 7. The cyclosporin A derivative according to claim 5, characterized in that it has the formula (IV): (IV) or a pharmaceutically acceptable salt thereof.
8. 8. The cyclosporin derivative A represented by the formula (V): (V) characterized in that: R is hydrogen, an aliphatic, linear or branched, unsaturated carbon chain of 2 to 16 carbon atoms, optionally containing one or more substituents selected from nitro, amino and amido; and R 'is an OH or is an O and together with a carbon adjacent to a double bond an amino acid 1 forms a heterocyclic ring or a pharmaceutically acceptable salt thereof.
9. 9. The cyclosporin A derivative according to claim 8, characterized in that it has the formula (VI): or a pharmaceutically acceptable salt thereof.
10. 10. The cyclosporin A derivative according to claim 8, characterized in that it has the formula (VII): (VII) or a pharmaceutically acceptable salt thereof.
11. 11. The pharmaceutical composition characterized in that it comprises the cyclosporin A derivative according to claim 2 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
12. The pharmaceutical composition characterized in that it comprises the cyclosporin A derivative according to any one of claims 3 to 7 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
13. The pharmaceutical composition characterized in that it comprises the cyclosporin A derivative according to any of claims 8 to 10 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
14. The method for use in immunosuppression provided in a subject characterized in that it comprises administering to the subject a cyclosporin A derivative according to claim 2 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
15. 15. The method for use in immunosuppression provided in a subject characterized in that it comprises administering to the subject a cyclosporin A derivative according to any of claims 3 to 7 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
16. 16. The method for use in immunosuppression provided in a subject characterized in that it comprises administering to the subject a cyclosporin A derivative according to any of claims 8 to 10 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
17. 17. The method for use in preventing or ameliorating an autoimmune disease in a subject characterized in that it comprises administering to the subject a cyclosporin A derivative according to claim 2 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
18. 18. The method for use to prevent or lessen an autoimmune disease in a subject characterized in that it comprises administering to the subject a cyclosporin A derivative according to any of claims 3 to 7 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
19. The method to be used to prevent or lessen an autoimmune disease in a subject characterized in that it comprises administering to the subject a cyclosporin A derivative according to claims 8 to 10, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
20. 20. A cyclosporin A derivative selected from the group consisting of: or a pharmaceutically acceptable salt thereof.
21. 21. The pharmaceutical composition characterized in that it comprises the cyclosporin derivative according to claim 20 to 10 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
22. 22. The method for use in immunosuppression provided in a subject characterized in that it comprises administering to the subject a cyclosporin A derivative according to claim 20, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
23. 23. The method for use in preventing or ameliorating an autoimmune disease in a subject characterized in that it comprises administering to the subject a cyclosporin A derivative according to claim 20 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
24. 24. A method for preparing a cyclosporin A derivative of Formula (I): (I) characterized in that R is H, D, -CD3, -CH = CH-CD3, -CH = CD2, -CD = CD2, -CH = CH2 or -CH = CHCH3, X, Y, and Z are hydrogen, and R 'is acetate which comprises condensing an aldehyde of the formula (2): (2) with a deliberate reagent of the formula (3): RCH = PPh3P + I "where R is as defined above, in the presence of sodium hydroxide
25. 25. A method for preparing a cyclosporin A derivative of the Formula (I): (I) characterized in that R is H, D, -CD3, -CH = CH-CD3, -CH = CD2, -CD = CD2, -CH = CH2 or -CH = CHCH3, X, Y, and Z are hydrogen , and R 'is -OH which comprises treating a compound of Formula (I) wherein R' is acetate with potassium carbonate.
26. 26. A method for preparing a compound of Formula (I): (I) characterized in that R is D or -CD3 and R 'is -OH which comprises condensing an aldehyde of the formula (4): (4) with a deliberate reagent of the formula: RCD = PPh3P + I "where R is as defined above, followed by treatment with potassium carbonate.