CA2372639C - 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

DEUTERATED CYCLOSFORINE ANALOGS AND THEIR USE AS IMMUIYOMODULATING
AGENTS
This application is divided from Canadian Patent Application 2, 298,572, tiled October 8,1998.
Cyclosporin derivatives of the present invention are disclosed which possess enhanced efficacy and reduced toxicity over naun~ally occurring and other presently known cyclosporins and cyclosporine derivatives.
The cyclosporin derivatives of the present invention are produced by chemical and isotopic substiwtion of the cycIosporine A (CsA) molecule by:
I. Chemical substitution and optionally deuterium substitution of amino acid I; and

2. Deuterium substitution at key sites of metabolism of the cyclosporine A
molecule such as amino acids 1, 4, 9.
The most acri~ie derivatives of the invention were those possessing both chemical and deuterium substitution.
The cyclosporins are a family o~ neutral, hydrophobic cyclic undecapepddes, containing a novel nine-carbon amino acid (MeBmt) at position 1 of the ring that exhinit potent immunosuppressive, antiparasitic, fungicidat, and chronic anti-iafIammatory;properties. The naturally occurring members of this icy of structurally related compounds are produced by various fungi impcrfecti.
CycIosporines A and C; are the major coniponeats. Cyclosporine A, which is discussed further below, is a particularly important member of the cycIosporin family of compounds. Twenty four minor metabolites, also oligopeptides, have been identified:
Lawen et aI,_J. Antcbiotics 42,1283 (I989); Traber et sI, Helv. Chim. Acta 70, 13 (1987); Von VJartburg and 20. Traber Prop. Med: Chem., 25,1 (I988).
Isolation of cyclosporines A and C, as well as the structure of A were reported by A. Raegger d sL, Heiv. Chim. Acts 59, I075(19,76); M. Dreyfuss et al., J. Appl. MierobioL 3,125 (197f~ Crystal and molecular structures of the iodo derivative of A have been reported by T. J. Petcher ei al., Helv. Chim. Acts 59, 1480 ZS (1976). The strucuue of C was reported by R Traber et al., tbid. 60;1247 (I977j. Production of A and C has been reported by E. Harri ct aL, U.S. Pat. No. 4, I 17, I I8 (1978 to Saadoz~
Isolation, characta~ization and aatifungal activity of H, D, E, as well as the structures of A through D nave been reported by R Traber of al., Helv. Chief. Acts 60,1568(1977). Isolation and siructurts ofE, F, G, H, I:
cidem, ibid. 65, 1655 (1982).
Preparation of (2-Deutcro-3-fluoro-D-Alaj=-CsA is disclosed by Paxchett d a1 in GB 2,206, I99A which was 30 published on Dec. 29, 1988.
Cyclosporin was discovered to be immunosuppressive when it was observed to suppress ant:'body production in mice during the screening of fungal extracu. Specifically, its suppressive effects appear to be retazed to the inhibition of T-cell receptor-tnodiated activation events. It accomplishes this by iniarupting calcium dependent signal transduction during T-cell activation by inactivating eahaodulin and eyelophdin, a peptidly propyi isomerase. It also inhibits IymphoIcine production by T:
helper cells in vitro and arrests the development of mature CD8 and CD4 cells in the thymus. Other in vitro properties include inhibition of IL~2 producing T-lymphocytes and cytotoxic T lymphocytes, inhibition of IL 2 released by activated T-cells, inhibition of resting T-Iyrnphocytes in response to alloantigen and exogenous lymphokine, inhibition of IL-I
production, and inIn'bition of m'rtogen activation of IL-2 producing T-lymphoCytes..Further evidence indicates that the above effects involve the T-lymphocytes at the activation and mauuatio~ stages.
Stimulation of TCR (T cell receptor) by foreign antigen on a major histocampat~t~ity (MHC}
molecule on the surface of the T cell results in the aemration of a TCR signal transmission pathway (exact method of transmission unlatown) through the cytoplasm causing the signal results in the activation of nuclear ID transcription factors, i.e. nuclear factors of activated T-cells (I~IF-AT}
which regulate transcription of T-cell activation genes. These genes include that of lyraphokine imerleulin 2 (IL-2~
Translation of the message is followed by secretion of.IL-2. T-cell activation also involves the appearance of the Iyrapholcine receptor IL 2R
on the cell srface. After iL. 2 binds to IL-2It, a lymphokine receptor (LKR}
signal transmission pathway is activated. Tlie immunosuppressive drug, rapamycim, inht'bits this pathway.
15 CsA is a potent inhibitor of TCR-mediated signal transduction pathway. It inhibits binding of NF AT
to the IL 2 enhances, and thus inhibits transcriptions! activation. CsA binds to cyclophilin, which binds to calcineurin, which is a key enzyme in the T-cell signal transductioa cascade:
Cycloplu'lin is found in prokaryotic and eukarotic organisms and is ubiduitous and abundant.
. Cyclophilin is a single poiypeptide chain vrith 165 amino acid residues. It has a molecular mass of I7.8 kD. A
20 roughly spherical molecule with a radius of I 7 angstroms, cyclophilin has a eight-stranded antiparallel beta barrel. Inside the barrel, the tightly pgcked core contains mostly hydrophobic side chains: CsA has numerous hydrophobic side chains which allow it to fit into the cyclophiIin beta barrel. Cyclophillin catalyzes the interconversion of the cis and traps-rotamets of the peGIFdyI-prolyl amide bond of peptide and protein substrates. Cyclophilin is identical in structure with peptidyl prolyl cis-traps isomerase and bears structural 25 resemblance to the superfamily of proteins that transports Iigands such as retinoi-binding protein (RBP}. These proteins carry the ligand in the barrel core. But cyclophiIin actually carries the ligand binding site on the outside of the barnl. The tetrapepdde ligand binds in a long deep groove on the proteia surface between one face of the beta barrel and the Thrl ld-GIy130 loop.
Further properties have also been reported in studies of the biological activity of CsA: 3. F. Borel et 30 al., Agents tlctions 6, 468 (1976). Pharmacology: Eidem. immunology 32, l01? (i977}; R Y. CaIne, Clin.
F.xp. Immunol. 35,1 (I979). human studio: R. Y. Calve et al., Lancet 2,1323(1978); R L: Powles et aL, bid.
1327; R. L. Powles ei aL, ibid 1, 327 (I980). In vitro activity (porcine T-ceiLs): D. J. White et at., Transplantation 2?, 55 (1979}. Effects on human lymphoid and myeloid cells: M.
Y. Gordon, J. W. Singer, Afature 279, 433(1979}. Clinical study of CsA in graft-versus-host disease: P.
J. Tutschka et aL,'Hlood 61, 35 3i8(i983).
Mechanism of Cvclosoorine A Action Cyclosporine A-CycIophilin A coneplex CsA, as discussed above, binds to the cyciophdin beta barrel. Thirteen CyP A
residues define the CsA binding site. These residues are Arg 55, Phe b4, Met 6I, Gln 63, Gly 72, AIa I01, Asn 102, AIa 143, GIa i i i, Phe 1 I3, Trp 121, Leu 122, Isis 128. The largest side-chaixt movements are I .3 A for Arg 55 and np to 0.7 A for Phe 60, Gln 63, and Trp I2I. There are four direct hydrogen bonds between the CyP A aad CsA.
Residues 4, 5, '6, ?, 8 of CsA protrude out into the solvem and are thought to be involved in binding the effecior protein, salcineurin (Pflugl, G., KalIen, J., Schirmer,.T., Jansonius, J.H., Zurini, M.G:M., F~ .
Walkinshaw, M.Dc (1993)Namre 36I, 91-94.) Function of CsA-CyP A complex..
The C$A-CyP A complex inhibits the phosphatase activity of the heterodimeric protein serine!
threonine phosplzatase or cakineurin (I:iu, J., Farmer, J.D., Lane, W.S., Friedmaatt, J:, Weissrnan, L, &
Schreiber, S.L. (1991) Celt 66, 807-IS:; Swatuon, S.K., Born, T., Zydowsky, C.D., Cho, H., Chang, Fi.Y., &
Walsh, C.T. (I992) Proc. Nail. Acad. Sci:USA 89, 368&90): CyP A binds CsA with an amity of ca.10 nM.
The complex is then presented to calcineurin (Liu, J., Farmer, J.D., Lane, W.S., Friedmate, J., Weissman, L, &
Scltreiber, S.L. (199.I) Cell 66, 807-15.).
13 Calcineurin dephosphoryhues the transcription factor NEAT found in the cytoplasm of T-cells.
Dephosphorylation allows NFAT to translocate to the mucleus, combine with jun/
fos genes and activate the transcription of the IL-2 gene responsible for cell cycle progression, leading to immune response. CsA-CyP A
complex inhibits the phosphatase activity of calcinettrist and ulrimately immunosuppression (Rtzkorn,.F. A., Chang, Z, Stalz, L.A., 8cWaL~I~ C.T. (1994) Sioeheznistry 33, 2380-2388.).
Neither CsA or CyI? A alone are 20. ~ . important immunologically. Only their complex is important (Liu, J., Farmer, J.D., Lane, W.S., Friedrnan, J., Weissman, L, & Schceiber, S.L. (I99I) Cell 6&, 807-15).
Metabolism Qf Cy elospurine:
Cyclasporine is tnetaboliied in liver, small imestine and kidney to more than 30 metatioIites. The 25_ structure of I3 metabolites and 2 phase II metabolites have been identified amdat least.23 further metabolises have beet isolated by FiPLC arid their structures characterized by mass spearotnetry. The, reactions involved in phase I metabolism of cyclosporine are &ydmxyIation, demethylation a$ well as oxidation and cyclisation at . amino acid 1. Several clinical studies and reports showed an association between blood concentrations of cyclosporine metabolites and neuro- or. nephrotoxicity. In vitro experiments indicate that metabolites are 30 considerably less immsmosupi'eg5ive and more toxic than CsA.
As exemplified by the ever expanding list of indications for which CsA has bees found useful, the cyctosporin fam4y of cvzepounds stud ut~y in the prevention of rejection or organ and bone marrow transpIants;~and in the treatment of psoriasis, and a number of autoiaunune disorders such as type 1 diabetes mellitus, multiple sclerosis, autoimmune,meitis, and~rheumatoid aathritis.
Additional indications are discussed 35 infra, As is generally accepted by-those of slaZl in the art, inhibition of secretion of interleukin-2 (IL-2) and other Iympholtines from Lymphocytes, is a useful Indicator of intrinsic immudosuppressive actirilry of a cyelosporin analoc For a recent review of cyclosporia uses and meebartisms of action see Wenger et a1 Cyclosporine: Chemistry, Structure-Activity Relationships and Mode of Acdoa, Progress in Clinical CA 02372639 2002-03-07 ' Biochemistry and Medicine; wol. 2,'176 (198.

Cyclosporin A is a cyclic peptide which contains several N-methyl amino acids and, at position-8, contains a D-alanine. The structtn~e of CycIosporin A' is given below:
IH3C~ yH
C .-~eLea 10 MsVat-'t1 ~ MeBMt 1 C~ lC~ C~ /C~ H~~~'t2 C..,, CH CH C CH ~
CHz CH2 C~, ~ H~ ~' j H ~~; H2 CH=-CO -N --CH -CO-N-CH-CO-N-CFt-CO-N--~tt pbu 2 CH3 i ~~ CHI Cti9 H CD
H C-~H CH3-N

3 I ~ I H3 j~ ~eay 3 l~ c.ata tt .
CO--CH-! -CO-CH-N-CO--CH-N--CO- ;H-N-CO
C~ H ~~ / \ H
. j H tt3C CH3 CN
ti3C Cti~. H3C ~~
Ata T Met.eu 6 Yal 5 NteLeu 4 'Unless otherv~ise specified, each of the amino acids of the disclosed ayclosporin is. of the L-canfignration.
As is the practice in the field; a particular cyclasporin analog may be named using a shorthand notation identifying bow the analog differs from cyclosporin A. Thus, cyclosporia C which differs frotn cyciosporin A by the dtreonine at position-2 may be identified as [1'hr]~-cycIosporin or [Thr]a-CsA. ~imiia~iy, 25 cyclosporin H is [Ala~-CsA; cyclosporin D i's [Yal]=-CsA; cycIosporin E is [Yan"-CsA; cyclosporin F is [3-DesoxyMeHmtj'-CsA; cyclosporirt G is [NVaj~-CsA; and cycIosporin8 is [D-MeVal]'~-CsA.
D-Serine and D-Threoniae have been inirodaced into the 8-position of cyclosporin A by biosynthesis t~esu3ting in active compounds. See R Traber et al. J. Antibtotics 42, 59I
(f989~ D-Chloroalanitu has a~ .
been introduced irno position-8 of Cyclos~rin A by biosynthesis. See A. Lawen et'aI J. :antibiotics 52, 1283 30 (1989).
Fadications for Cyclosnorine Z'heranv Immunoregulatory abnormalities have been sown to exist in a wide variety of autoiatmune and chronic inflamr<ratory diseases, including systemic lupus erythematosis, chronic rheumatoid arthritis, type 1 35 diabetes mellitus, 'm#lammatory bowel disease, bt'liary cirrhosis, uveitis, multiple sclerosis and other disorders such as Crohn's disease, ulcerative colitis; bulious pemphigoid, sarcoidosis, psoriasis, ichthyosis, and Graves aphtbaImopathy. Although the. underlying pathogenesis of each of these condiiioas may be cite different; they have iu common the appearance, of a variety of autoantibodies and self reactive lymphocytes ;5neh self reacdviry shay be due, lit part, ~to a loss of the homeostatic controls under which the normal izmnune system OpeLateS.
Similarly, following a bone marrow or aworgan transplantation, the host lymphocytes recognize the foreign tissue antigens and begin to produce antibodies which lead-to graft rejection.
One end result of as autoimmune or a rejection process is tissue destruction caused by inflaznmatory S cells and the mediators they release. Asiti i~Iammatory agents, such as NSATCYs (Non-Steroidal Ami-inflammatory Dnigs), and corticosteroids ac; principally by blocking the effect of'~ or secretion oi; these mediators, but do nothing to modify the itmnunologic basis of the disease. On the other hand, cytotoxic agents, such as cyclophosphamide, act in such a nonspecific fashion that both the normal and antoimmune responses are shut ofd Indeed, patients treated with such nonspecific itnmunosuppressive agents are as fkeiy to succumb to infection as they are to their autoimmune disease. .
Generally, a cyclosporin, such as cyclosporine A, is not cytotnxic nor myelomxic. It does not inhibit migration of monocytes nor doe's it inhibit granulacyres and macrophage action. Its action is specific and leaves most established immune responses intact, However, it is nephrotaxic and is known to cause the following undesirable side effects:
(I ) abnormal liver function;
(2) hirsutism;
(3) gum hypertrophy;

(4) tremor;

(5) neurotoxicity;.
(~ hyperaesthesia; and (7} gastrointestinal discomfort.
A number of cyclosporines and analogs have been described in the patent literature:
U.S: Pat. No. 4,108,985 issued to Ruegger, ei al, on Aug. 22,1978 entitled, "Dihydracyclosporin C", discloses d~ydrocyclosporin C, which can be produced by hydrogenation of cycIosporia C.
U.S. Pat. No. 4,117,118 issued to Harri, et al. on Sep. 26, T978 entitled, "Organic Compounds", discloses cyciosporins A and B, and the production thereof by fermentation.
U.S. PaL..No. 4,210,581 issued to Rue~er, et al. on TuL 1,1980 entitled, "Organic Compounds", discloses cyclasporin C and d~3ydracyclosporin C which can be produced by hydrogenation of cyclosporin C:
U.S. Pat. No. 4,220,641, issued to Tiab~; et al. on Sep. 2,1980 etttiiled, "Organic Compounds", discloses cycFosporin D, dihydrocyclosporin D, and isocyclosporin~D.
U.S. Pat. No. 4,288,431 ~ issued to Traber, et aL on Sep. 8,1981 entitled, "Cyclosporin Derivatives, Their Production and Pharmaceutical Cotupositiarrs Containing Them", discloses cyciosporirt G, d~ydrocylaspoiin G, and isocyciasporin G.
U.S, Pat. No. 4,289,851, issued to T'raber, et al. oa Seg. 15, I981 entitled, "Process for Producing Cyclosporin Derivatives", discloses c. yc~osporin D, d~ydrocyclospor'ra D, and isoGyclosporia D, and a process for producing same.
U.S, Pat. No. 4,384,996, issued to Bolliager, et al. on May 24,1983 entitled "Alovel Cyclosporims", discloses cyclasporins baying a ~i-vinylene-a-amino acid residue at the 2-position andlor a -~-hydroxy x-amino acid residue at the 8~position. The cycIosporins disclosed included either MeBmt or dihydro-MeBmt at the I position.
U.S. Pat. No. 4,396,542, 'issued to Wenger on Auo. 2,1983 entitled, "Method for the Total Symhesis of Cyclosporins, Novel CycIosporins and Novel Intermediates and Methods for their Production", discloses the synthesis of cyclosporins, wherein the residue at the 1 position is either MeBmt; d'hydro-MeBtnt, and protected intermediates.
IJ.S. Pat. No. 4,639,434, issued to Wenger, et al on Ian. 27, I98~, entitled'~Vovel Cyciospotins~, discloses cyclosporins with substitumd residues at positions I,, 2, 5 and 8.
U.S. Pat. No. 4,681,754, issued to Siegel on Jul. 21,1987 entitled, "Counteracting CycIosporin Grgan Toxicity", discloses methods of use of cyclosporin comprising ca-dergotxnae.
U.S. Pat: No. .4,703,033 issued to Seebach on OcK. 2'~, 198'7 entitled, "l~Iovet Cyclosporins", discloses cyclosporins witfi substituted residues at positions 1, 2 and ~3. The substitutions at position-3 include halogen.
H. Kobel and R Traber, Directed Biosynthesis of Cyclosporiru, European J.
Apple: MicrobioI
Biotechnol., l4, 237B240 (1982), discloses the biosynthesis of cycIosporins A, _B, C, D & G by fermentation.
IS . Additional cyclosparin analogs are disclosed in U.S. Pat: No.
4,798,823,.issued to Witzel, entitled, New Cyclosporin Analogs with Modified "C-9 amino acids", ~ubich discloses cyclosporin analogs with sulfur-containing amino acids at position-1.
SUMMARY OP THE INVENTION
The present invention concerns chemically substituted and deuterated analogs of cyclosporine A and related cyclosporines.
An object of the present invention is to provide new eyclosporine analogs vsrhieh have enhanced e~cacy and aliened pharmaeolcinetic and pharmacodynamic parameters. Another object, of the present invention is to provide a cyclosporine analog for the care of immunoregulatory disordersand diseases, ZS including the prevention, control and trea:merit thereof. An additional atiject of the present invention is to provide phatmaceuticai compositions far administering to a pollen in_ need of the treatment one or more of the active immuno~ive agents afthe present invemion. StdI a further object of this invention Is to provide a method of controlling graft rejection, autoimmune and clsranic immflaxnmatory diseases by administering a stz~cient amourn of one or more of the novel immunosuppressive agents in a mammalian species in need of such treatment. Finally, it is the object of this invention to provide processes for the preparation of the active compounds of the presegt invention.
Substitution and deuteration of the cyclosporine molecule results in altered physicochemical and pharmacokinetic properties which enhance its usefulness in the treatment of transplantation xejectioa, host vs.
graft disease, graft vs. host disease, apIastic anemia, focal and segmental glomerulosclerosis, myasthenia 3 S grouts, psoriatic arthritis, relapsing poiychandritis and ulcerative colitis.
Embodiments of the invention include CsA derivatives wherein one or more hydrogen atoms in the I , 3 and 9 amino acid positions are substituted with a deuterium atom and wherein the cyclosporine A derivatives arc optionally chemically substituted at the amino acid 9 position. A further specific embodiment of the invention is the CsA derivative repxeserned by formula 1:

CHR
v CH
H3C ~CH3 CH2 CH H3C ~CH3 R,\ CH-CH3 CH3 -N-CH-CO-N-CH-CO-N-CH-CO-N-Cti CH3 CH3 CH3 H CO tn C, CH3 ~ Z
-N-CO-CH-N--CO-CH-N-GO- i H-N'-CO

~CH H3C ~3 ,CH

where R is (i) a deuterium or (ii) a saturated or unsaturated straight or branched aliphatic chain of from I to 16 carbon atoms and containing one or more deuterium atoms or an ester, ketone or alcohol of the carbon chain and optionally containing one or more substituents selected from halogen, nitro, amino, amindo, aromatic, and heterocyclic, or (iii) R is an aromatic or heterocyclic group optionally containing a deuterium atom or (iv) R is a methyl group; and X, Y, and Z are hydrogen or deuterium provided that at least one of X, Y, or Z is deuterium; and R' is an OH or an ester or is an O and together with a carbon adjacent to a double bond on amino acid 1 form a heterocyclic ring such as 5-membered rings where the heteroatom is oxygen.
Preferably, R is an unsaturated straight or branched aliphatic carbon chain of from 2 to 3 carbons, X, Y and Z are hydrogen or deuterium, and R' is an -0H or acetoxy.
Other specific embodiments of the present invention include the CsA derivative of formula I
where R is a saturated or unsaturated carbon chain of from 2 to 3 carbons containing one or more deuterium atoms.

The invention also specifically contemplates the compounds, and mixtures thereof, represented by the structures A and B:
R
w HO,~ f MeLeu-MeVah ~N Abu-Sar O
MeLeu-D-Ata-Ala-MeLeu-Val-MeLeu (A) R
HO.,r MeLeu-MeVal->~ ~--Abu-Sar O
MeLeu-O AIa-Ala-MeLeu-Val-MeLeu CB) wherein R is selected from the group consisting of : (i)-CH=CH-CH3, (ii)-CH=CH2, and (iii)-CD=CD2.
Further specific embodiments include those of formulas Sg and Se below:
(5e) MeLeu -MeVat '-~r MeLeu-MeVat MeLeu -D-Ala -Ala -MeLeu -Vat-MeLeu MeLeu-D-,41a-DESCRIPTION OF THE FIGURES
Figure I is the structure of cyclosparine A showing a site of deuteration at the amino acid 3 position.
l figure 2 is the structure of cyelospotzne A showing a site of deuteration at the amino acid 9 position.
Figure 3 is scheme I of the syathesis~ of the cyclosporine derivatives.
S Figure 4 is scheane II of the synthesis ofthe cyclosporine derivatives.
Figure 5~ is a graph of the results of the calcineurin assay of Example 9:
Figctre d is a graph of the results of a mixed lysnpliocyte reaction assay ofF.xampIe 10.
DETAILED DESCRIPTION OF THE INVENTION
I4 Substitution of deuterium for ordinary hydrogen and deuterated substrates for protio metabolites can produce profound changes in biosystems. Isatopicaliy altered drugs have shown widely divergent pharmacological effects. Pettersen et aL, found increased anti-cancer effect with deuterated 5,6-benzylidene-d1-L-ascorbic acid (Zilascorb} [Anticancer Res. I2, 33 (1992?].
Substitution of deuterium in methyl groups of ayclosporine wi'Il result in a slower rate~of a~idaaon of I S the C-D bond relative to the rate of oxidation of a non-deuterium substituted C-H bond. Tlie isotopic effect acts to reduce formation of demethylated metabefrtes and thereby alters the pharmacokinetic parameters.of the drub. Lower rates of oxidation, metabolism and clearance result in greater and mare sustained biological activity. Deuteration is targeted at various sites of the cycIosporin molecule to increase the potency of drug, reduce toxicity of the drug, reduce the clearance of the pharmacologically active moiety and improve the 20 stability of the molecule.
Isotonic Substitution: .
Stable isotopes (eg., deuteriura; t'C, tsN, is ~) are nonradioactive isotopes which cantaln one 25 additions! neutron than the normally abundant isotope of the respective atom. Deuterated corupounds have been used in pharmaceutical research to investigate the in vivo metabolic fate of the compounds by evaluation of the mechanism of action and W etabolic pathway of the non deuterated parent compound (Blake et al. J.
Pharm. Sci. 64, 3, 36T-391,I97S7: Such axetabalic studies are ~ in the design of safe, eil~ive therapeutic drugs, either because the in vipo active cornpaund administered~to the p~atiert or because the 30 metabolites produced from the parent compound prove to be toxic or carcinogenic (Foster et al., Advances in Drug Research Vol. I4, pp. 2 36, Academic press, London,1985~
Incorporation of a heavy atom p~rticulariy substitution of deuterium for hydrogen, can give rise to an isotope effect that could alter the pharmacokinetics of the drug. This effect is nsualty insignificant if the IabeI is placed at a metabolically inert position of the molecule.
35 Stable isotope labeling of a drug can alter its physico-chemical pzoperties such as. pKa and lipid solubility. These changes may influence the fate of the drug at different steps along its passage through the body. Absorption, distr~benion, metabolism or excretion can be changed:
Absorption and distn'bettion are processes that depend primary on the moleGaIar size and the IipophiIicity of the substance. These effects and alterations can affect the pbarmacodynamic response of the drug molecule if the isotopic substitution affects a region involved in a ligand-receptflr interaction.
I~
Drug metabolism can give rise to large isotopic effect if the breaking of a chemical bond to a deuterium atom is the rate limiting step in the process. Wlu'le some of the physical properties of a stable isotope-labeled molecnIe are different from those of the unlabeled one, the chenuca~ and hiaIogicai properties are the same, with one important exception: because of the increased mass of the heavy isotope, any ~bopd involving the heavy~isotope and another atom wi'II be stronger than the same bond between the light isotope and that atom. In any reaction is which he breaking of this bond is the rate limiting step, the reacti~ will proceed slower for the molecule v~rith the heavy isotope due to "Iinetic isotope effect" A reaction involving breaking a C D bond can he up to 700 per cent slower than a similar reaction involving breaking a C H bond.
If the C D bond is. not involved in arty of the steps leading to the metabolite , there may not be any effect to alter the behavior of the drug. If a deuterium is placed .at a site involved in the metabolism of a drug , an isotope effect will tie observed only if breaking of the G-D band is the rate limiting step There is evidence to suggest that whenever cleavage of an aliphatic C-H bond occurs, usually by oxidation catalyzed by a raixed function oxidase, replacement of the hydrogen by deuterium will lead to observable isotope effect. It is also I S raigortant to understand that the incorporation of deuterium at the site of metabolism slows its raft to the point where another metabolite produced by attack at a carbon atom not substituted by deuterium becomes the nor pathway a process called "metabolic switching". It is also observed that one of the most important metabolic pathways of compounds containing aromatic systems is hydroxylation leading to a phenolic group in the 3 or 4 position to carbon substituents. Ahhough this pathway involves cleavage of the C-H bond, it is often not accompanied by an isotope effect, because the cleavage of this bond mostly not involved in the rate limiting step. The substitution of hydrogen by deuterium at the stereo center wilt induce a greater effect oa the activity of the drug.
Synthesls of Cyclos~SOrIne Derivatives.~
ZS The staring material for the preparation of the compounds of this invention is cyclosporine A: The process for preparing the compounds of the present invention are illustrated as shown in scheme I in figure 3:
It~wilI be readily apparent to one of ordinary skill in the art reviewing the syrnhetic;oute depicted helow that other compounds with formula h can be synthesized by suvstiwtion of appropriate reactants and agents in the synthesis shown below.
The first step in the process for making deuterated cyclosporin analogs is the pre~ration of the key inte;mecilate 3 and 6 . This can be achieved by the oaddation of the double bond in the amino acid 1.
Treatixnent of cyclosporh~ with acetic anhydride and excess of dimethylaminopyridine provided the hydroxyl ~ ~~YI cyclosporia. 2 .Although cleavage of the double bond could then be accomplished by treatment of 2 with ozone; or KMnO,J NaIO,~, it was found out that Os04INaI0, was the reagent of choice 3f for the transformation to the aIdehyde product 3. The reaction was generally found to be cleaner, producing the required material gad to proceed in higher yield. The drawback to this reaction is that OsOa is expensive gad highly toatic, so that its use is limited : But the results can be accomplished morn economically by the use of HzO, with Os04 present in catalytic amounts. t butyl hydroxide in alkaline solution and N-methylmorgholine-N-oxide can be substituted for HZOI in this pmcess. The aIdehyde compound 3_ was fiu~h~

lI
trued with various deuterated alkyl or aryl triphenyl phosphonium derivattves(wittig reagents) and hydrolysis by alkaline solution provided the final derivatives ( 5_ a-~: We also developed a general procedure to obtain various compounds as shown in Scheme ll in figure 4. .
In ibis approach , the aldehyde derivative 3_ was treated with the Witdg reagent prepared by using .
standard procedure. The restthant product on mt'Id acid hydrolysis provided the key irdermediate sldehyde product G_ This was further treated with second deu~rated aikyl or aryl tripheirylpbosphflnium halide reagents and on mild acid hydrolysis yielded the. required products. This method provides control over the extension of the diene system. By using this approch; olefinic double bonds can be introduced step by step.
A third approach to prepare the deuterated compounds Sa-b- is by heating non deuterated cyclosporiQ
analogs descn'bed earlier, in a deueraied Solvent such as deuterated water, deuterated acetic acid in ibe presence of acid or Base catalyst.
Preferred cyclosporins of the prGSent invention are those which both contain s deuterium and a chemical substitution on amino acid 1 such as those of formula II: .
X-Abu-Sar-MeLeu-Vat =MeLeu-Ata-(D)Ata-MeLeu-Meteu -Me 1 2 a 4 b B 7 8 9 40 11 .
Where X is R~'CHZ
HC~ ~CH1CH, CH a -H-CH-Ca-And R~ -CHO, -CDO, -CH=CD-CDa : C~CD-CD3 ,-CH~H-CHAD-CD3 ; CD--CH-CD~D.CD3 , -CH~H-CH=CD=,-CD=CH-CD=CDr,-CHI,-CH=CHiand-CII~CCI3i.
EXAMPLES:
Example 1.
To a stirred solution of cycIasporme 1(1.018, 4.84mmoI) in acetic anhydride (20mL) at room temperature was added DMAP (150mg, I23mrnol, LSeq) After stirring overnight, the reaction mixture was partitioned between EtOAc (~Om1) and water (25m1). The separated EtOAc layer was then washed with water (SOmL} and brine (SOmh), dried (MgS04) and the solvent removed irr vacuo to give the crude product as a glassy solid.
Purification by flash chromatography through a short column of silica (2%
MeOFIIDCM} and Iyophitlisation -from benzene yielded 2_ (1.0~4g, 0.84mmol, quart.} as a fluffy, colourless solid; ta~ -305.? (c. 03; CI3CI3), vo,~, (CFIC13 cast)iciri i 3328m, 2963m, I746m,1627s, I528m; I472m, 1233m; SH
(600MHz, C6D~ 8.73 (1H, d, J= 9SHz, NHS, 8.30 (IH, d, J=7.OHz, NI-~1, ?.92 (IH, d,J= 75Hz; NCI,), 7 49 (IH, d, J=7.SHz, NI-~, . 6.05 (IH, d, J~ 11 SHz), 5.$8 (1H, dd, J= 3S,1l.SHz), 5.82 (1H, d, J=
II.SHz), 5:65 (1H; dd, J=4:0, t2.OHz), 5.60 (1H, dd, J= 3.5, l2SHz), 5.63-5.57 (1H, m], 5SI-5.45 (IH, m);
5.37 (1>3, dd, J= 5.5, 8SHz), S.OS-5.01 (2H, complex 499 (1H, d, J= I l.OHz}, 4.7fi (IH, p; J~ 7.OHz), 4.58 (1H; pJ= 7.OHz), 4.02 (IF3, ~d, J=13.5p1z), 3.47 (3H, s), 3.30 (3H, s), 3.I? (3H, s), 3.11 (3H, s), 2.98 (3H, s), 2.68-2.62 (1H, m),2.63 (3H, s); 2.51-239 (2H, complex), 234-2.25 (8H; coatpIex), 2.03 (3H; s},19?-L85 (2H,'coanple~c),1.$3 (3H, dd, J
=1.0, 6.SHz},1.82 1.77 (ZH, complex),1.68-L6I (3H, complex), L55 (3H,. d, J=
7,OHz),.1.55-1.51.(1H, m), 1.44-L3$ (1H, m), 132-1.20 {SH, complex),1.29 {3H, d; J= 7.OHz), 121 (3H, d, J= 6SHz),1.I7 (3H, d, J=
I O 6SHz),1:14 (3H, d, J= 6SHz), 1:08 (3H, d,-J= 6.5Hz),1.04 {3Hf, d, J=

6.OHz),1.03 (3H, d, J=:7.Oliz), I .00 (3H, d, J=?.OHz), 0.93 (3H, d; J= 6:OHz), 0.92 (3H, d; J= 6:SHz), 0.88-0.84 (9H; complex), 0.76 (3H, d, J= 6SHz); 0.5? (3H, d,J= 6SHz); ~ {'75MHz, C6D~
I73.b,173.2,172.8,172.6,1713, ~I7l.l,.T70.71, 170.67,170.4, I70.2,.I69.8, Ifi7:9 LCD), I29.0,126:2 (C-C,~, 73.1 (C_OAc], 58.1, 57.i, 56.0, 55.0, 54.6, 34:2, 50.3, 499, 48.6, 48.1, 47.8, 44.5, 40.8, 3.9.1, 35.7, 33.6, 32.9, 32.1;
3 IS, 31.2, 30.0, 29.7, 29.5, 293, I5 24:9, 24.6, 24.4, 24.0, 23.6, 23.4, 233, 21,7, 21.1, 21.0, 20.6,20.3,19.5,18.5,18.0,17.7,17.5, 1?.4, I4.9; .
9.7; rrr~ (Eleczmspray) ExamnTe 2 To a solution of compound 2 (289tag, 0?3~no>) in a 1:1 mixture of dloxar~e arid water (5mL) was added 20 firstly sodium metaperiodate (IOOmg, 0.47mmo1, 2eq) and secondly a solution of osmium tetraoxide (3mL;
0.5g OsO., iu 250tnL of solvent). 'Iwo-phase work up, purif canon by flash cohimri ciuomatography (40%
acetone in petroleum ether) and lyophilisation from benzene gave compound 3_.
(22fimg; O.I8mmol, 80%) as a fluffy, colourless solid; ~a~ 260.0 (c. 0.1, CHC13); v",~ (CHC13 cast)Iciri ' 3325m, 2962m; I748w,1724w;
1677rn,1626s, IZ'8ra, 755; 8x (300MHz, C6I?~ $.63 (iH, d, J= 9SHz, N~, 8.16 (IH, d, J=?.OHz, N~,I), 25 795 (1H; d, J= 7SHz, NHS, 7.48~(IH, d; J= 9.pliz, NHS, 5.93 (I H, d, J=

7.SHz), 5.84 {I H, dd, J= 4:0, I LSHz), 5.70 (lI#, d, J=1 I:SHz), 5.56-5.54 (IH, m), 532 (1H, dd, J= 5.5, S.OHz), 5.07-4.88 (3H, complex), 4.T2 (IH, p, J=?.OHz), 4.49 (1H, p, J=7:UHz), 3.98 (IH, d; J=14.0Hz), 3.42 (3H, s, Cue, 3.27 {3H, s, CH I~, 3.12 {3H; s, CSI I~, 3.07 (3H; s, CH~1~, Z.91 (3H, s; CI"~NN), 2.79 (3H;~ s, CSI 1V)2.59 (3H, s, CHIN}, 2.42 208 (IOH, complex),1.94 (3H, s, CF~,,~I CO~,1:47 (3H, d,J=?:OHz), L24 {3H, 7.OHz),1.14-L09 (9H, 30 . Iex),1.04 (3H, d, J= 6.SHz}, LOI (3H, d, J= 7.Oliz), 0.96 (3H, d, J=
6.SHz), 0.92 (3H, d, J= 6.SHz), 4.91 (3H, d, J= fi.SHz), 0.89 (3H, d, J= fi.OHz); 0.83 (6H, d, J= 6.5Hz), 0.74 (3H, d, J= 6SHz}, 0.59 (3H, d, J= 6.SHz); 8c (TSMFIz, C6D~ 202.5 (C_HO),1?4.4,174.0; I73.7,1?2.8,17I.6, I?I.S, I?I2, I7L1,170.6;
1702, 170.2,168.1, 73.0, 58.7, 57.6; 56.7, 55.5, 55:0, 545, 49.4, 4$:9, 48.5, 48.1, 45.0, 44.6, 413, 39.8, 38.8, 3?.7; 362, 32.5, 32.0, 31.6, 30.9, 30.3, 30.0; 29.8, 29:6, 25.6, 25.3, 25.0, 24.8, 245, 24.0, 23.8, 23.4, 22.0, 35- 21.7, 2I Z, 205, 20.0,19.8,18.8,18.5,182,17.4,15:2,10.0; m/3 {Electrospray) 1232.8 (MH''', 100°i6).

Exaenule 3 Method A: To a solution of compound 3_( 3 l5mg, 026nunoI) in THF (5mL) at 0°C iwas added a solution of the deutero-phosphorus ylid (2,67mmol, ~lOec~, Prepared from drethyhriphenylghosphonium~iodide. After w~k-up, purification by Sash column chromatography (30% to 60% acetone in FE) and HPLC (60% to 65% MeCN
in water) , then Iyoplu'lisation from benzene yielded compound 4 (153mg, 0.12mmo1, 47%) as a fluffy, colourless solid, Method B: To a stirred salution of compound 3 (287mg, 0.23thmoI) in THF (5mL) under Ar at 78°C was carefully added a soIt>tion of phosphanu yIid (formed by the addition of sodium hexamethyldis'ly3arrtide (1.OM; 2.25tnL, 2.2Smtnol,--I Oeq) to a.suspension of d~-ethyltripheaylphosphoaiurn iodide (480mg, ~ 1.l3mmol; ~5eq) in THF (lOmL) under Ar at room temperature). After stirring for 2hr with gradual warming to room temperatt>re; the reaction mixture was cooled to 0°C and was quenched by the addition of I O°!o AeOHfTHF (IOtnL).,The reaction mixture was concentrated in varuo and partitioned between water (20tnL) and EtOAc (20mL). The aqueous layer was further extracted with EtOAc (20nL) and the combined organic extracts were then washed with IN HCl (20mL) and water (24m1,), dried (MgSOd) and the solvent removed in I S vacuo to give the crude product. Purification by flash column chromatogaphy (40% acetone in petroleum ether) and lyophilisation from benzene yielded compound 4d (84mg, 67~rmol, 29%) as a fluffy, colourless solid; It'X~ -283.0 (c. 0.1, CHCl3x v;"~ (CHC13 cast)lari f 3320th, 301 Om, 2959x, 2924s, 287Im, 2853an, I743nt,1626s, 756x; &H.(b00MHz, CsD6) 8.78 (1H, d, J= 9.SHz), 8.33 (II~i, d, J= 7.UHz), 7.99 (IH, d, J=
7.SHz), 7.59 (IH, d, J= 9.OHz), 6.09 (IH, d, J=1 LSHz~ 5.92 (IH, dd, J= 4.0, I
I .OHz), 5.86 (1H; d; J=
1 LSHz), 5.72-5.64 (ZH, complex), 5.b2 (IH, dd, J= 3.5, I2.5Hz~ 5.40 (IH, dd, J= 5.5; 8.SHz), 5.10-5.02 (3H, complex), 4.80 (tli, q, J= 7.0Hz), 4.60 (IH, q, J= 7.OHz), 4.05 (1H, d, J=14.OHx), 3 S I (3H, s~~ 331 (3H, s), 3.20 (3H, s~ 3.13 (3H, s~ 3.01 (3H, s), 2.87 (3H, s), 2.64 (3H, s), 2.45 (IH, dt, J= 4.0, L2.3Hz~ 236-2.20 (I OH, cornpIex), 2.06 (3H, s), L93-1.79 (3H, complex); c~ (84MHz, C6H~ , Sc (125MHz,'CsD~ 17.4.5,173.7, 173.6,. i73.I,17,1:7,171.4, 170.9;170.7,170.6, 170.3;170.0,168.4,1302 (CSC), 123.8 (C'~, ?3:8 (Mel3rat 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, 342, 33.9, 32.6, 32.3, 32.0, 31.4, 309, 30.8, 30:2, 30.1, 30.0, 299, 29.8, 29.6, 28.5, 25.6, 25.3, 25.0, 249, 24.8, 24.1, 23.9, 23.8, ?3.6, 23.1, 22.1, 21.7, 2I:4, 20.7, 20.0,19.9, I9.&, 18.9,1$.7, .
18.6,18.3,17.4,153,143,102; mlz (Elec~cospray) 1270 ([M+Naj'',100%j,1286 ([M+Kj ; 20~
ExamEle 4 To a stirred sohttion of 4d (84ing, 67pmol) in MeOH (5m1.) and water (2.5mL) at room te~nper~re was added potassium carbonate (99mg; 0.72mmol, ~IOeq?: After stirring overnight, the MeOH was removed fn vacuo and the aqueous residue was partitioned between EiOAc (I OmL) and Solo citric acid solution (IOmL).
The EtOAc layer was thetl washed with water (I OmL) and brine (IOmL), dried (MgSO,j and the solvent removed in varuo to give the crude product. I3PLC purification (60% to 65%
MeCN in water) and Iyophilisation from benzene yielded compound 5d (59mg, 49pmo1, ?0%) as a fluffy, colourless solid; ~Gx~
262.0 (c. O.OS, CHC13); v",~ (CHCI3 cast~ari' 3318m, 3008m; 29GOs, 2872ia, I627s,1519m, I470m, I4I I m, I295m, I095m, 754m; ~ (b00MHz, Cue) 8~.2? (1H, d, J= 9.Slllz~ 796 (IH, d, J=
7.SHx), 7.63 (1H, d, J=
B.OHz~ 7.45 (IH, d, J= 9.0Hz),.5.87 (IH, dd, J= 3.5, ILOHz}, 5.74 (IH, d, J=
7.SHz~ 5.73-5.69 (ll~, m), 5.66-5.64 (1H, br.d,.J= ll.OHz), 5.79 {IH, dd, J=4:0; Il.SHz), 3.39 (IH, dd, J= S.S,10.5Hz), 533 (IH, dd, J= 3.5, 8.5Hz), 524.(1H, d, J=1 i.OHz~ 5:12 (1H, dt, J= 7.5, I0.0Hz), 4.88-4.79 (3H, complex), 4.22 (I H, dd, J= 5.5, 7.SHz~ 4.00 (1H, d, I3.SHz~ 3.72 (3H, s), 322 (3H, s~ 3.06 (3H;
s), 2.97 {3H, s), 2:92 (3H, s~
2.85 (3H; s), 2.67-2.60 (IH, m), 2S8 (3H, s~ 2.56-2S0 (IH, br m~ 2.33-2.23 {4H, complcac~ 220-2.07 (4H, camplex),1.80-1.74 (3H, complex); L67 (3H, d, J= 7.OHz~ 1.56-L50 (2H, complex),1.46-i 23 (9H, cornplex~ 1.i7~L13 (16H, complex), i.06 {3H, d, J= 6.SHz),1.02 (3H, d, J=7.0I~Tz~, 0:98 (3H, d; J=
6.SHz), 0.96 (3H, d, Ja ?.OHz~ 092-0.89 (9H eomplex~ 0.86 (3H, t, J= 7.SHz), 0.83 (3Ii, d, J= 6.OHz~
0.64 (3H, d, J= 6.SHz~ ~ (84MHz, C6H~ 1.64 (CDR); &c (75MHz, e6Fi~ 174.2,174.
i; 174.0, I73.7,.17i.8, 171.4,171.2,170:5,170.4, I?0.3,169.>1,130.2, I24.I, (99.2,) 74.3, (67.1,) 663, 66.1, 6I .0, 59.5, 58.3, 57.8, 55:7,.55.5, 55,4, 49.4, 49.0, 48.4, 453; 414, 39.6, 39.0, 37.8; 365, 36.I, 35.8, 33.?, 31.6, 30.8, 30.4, 30.i, 29:9, 29.3, 29.4, 23.5, 25.2, 25.0, 24.9,.24.5, 24.2, 23.8, 23.?, 23.6, 22.0, 2L4; 20:0,18.8,18.5, 17.8, I6.0, I O.I; m!a (EIectrospray) 1206 ((M-i-H]', 30%),.1228 ([M+Naj'',100), 1244 (HIV!+KI+, 25).

Example 5 To a vigorously stirred tnixmre of compound 3 (49mg; 39.8Iemol) ands deuterated d~-alIyiiripheuylphosphonium ltromide (3I lmg, 8l2prnot, ~20e~ in benzene (3mL.) at room tempera'dzre was added IN NaOH {3mL~ Stirring was continued at room temperature for 5days, ailer which time the 2 layers were separated, the benzene Layer was washed with water (SmL), dried (MgS~~ a~
the soIveut removed in vacuo to give the crude pradud. Purifu~ion by HP1.C (20% to 60% MeCN is water) and Iyophdisation from benzene yielded compound 4g (23mg,183pno1, 47%) as a flufi~, colourless solid;
~a~ Z64:2.(c. 0.24, CHCl3); v""~ (CHC13 cast}lari' 3322m, 2959m, ,1?44m, I626s, 1231 m, 754m; 8H
(300MI<3z, C6D~} complex due to I :I ratio of geometrical isomers 8.73 (d, J= 9.5Hz, NHS, 8.T2 (d, J=
9.SHz, N~1, 829 {d, J= 6SHz, N~), 8.26 {d, J= b.SHz, N_H}, ?.92 (d, J=?.Sllz, NCI , ?.86 (d, J= 7:SHz, NH ;
T:53 (d, J= 9.OHz, NI-~I , ?.49 (d, J= 9.0Hz, NH,,, 7.10-6.70 (complex); 6:33 (br t, J= Z I.OHz), 6.18 (d, J
=10.5Hz~ 6.12 (d, J = i 0,5Idz), 6.05 (d, J= I I.OHz~ 6.03 (d,.J=1 LOHz~-5.90-5.53 (complex 537 (dd, J= 6.0,-

8.OHz~ 5.20 (d, J=
12.OHz), 5.14 (d, J=12.OHz~ 5.07-497 (complex), 4.80-4.70 (complex), 4.57 (p;
J= 7.OHz), 4.02 (d, J=
I4.OHz), 4.01 (d, J= I4.OHz~ 3.47 (s), 3:46 (s~ 328 {s), 3.26 (s), 3.16 (s), 3.15 (s~ 3.09 (s), 2.97 (s), 2.96 (s), 2.84 (s), 2.62 (s~ 2.48 2.23 (complex), 2:05 (s), 2:03 (x),1.95-I .59 (comglex),.1;54 (d, J= 7_.OHz), I .53-0.80 (coanFlex), 0.?7 (d, J= 6.5Hz~ 0.58 (d, J= 6.SHz}, ,0.5? (d, J= 6.3Hz); &c (?SMHz, CsDs) 174.5,174.0, I73.9,173.6,173.5,173.I,17L7, IZL6, I7L4,170.9,170.8, 170.6, 170.6,170,3, I69.8,:I69.?, 168.4, 13?9, 133.9,133.5,132:8,1323, 131.6; 130.1, I I6.9; I 15.0, 73.6,.58.6, 57.6, 57.0, 56.8, 55.7; 55.5, 55,0, 549, 54.7, 54.5, 49.4, 48.9, 48.5, 48.2, 48..I, 44.9, 41.5, 39:9, 39.0, 38.9; 37.8, 37.6, 36.6, 363, 34.1, 33.7, 32.?, 32;1, 32.0, 31.5, 30.9, 30.7, 30.0, 29.8, 29.6, 25.6, 25.5, 253, 25.2, 25.0, 24.9, 24.1, 239, 23.7, 23.6, 22.1, 21,7, 2L6, 21.4, 21.3, 20.7, 20.0,19.9,189,18.6,18.3,17.6, 15.3,102; rrrla (Electrospray) 1258.8 (MHO, 100%).

Example 6 To a vigorously stirred mixhu~e of compound 3 (56mg, .45.Slunot) and deuterated d~-cmtyitriphenylghosphonium bromide (360mg, 907iunol, ZOeq) in benzene (3mL) at room temperature was added IN NaOH (3mL). Stirring was continued at roam temperature for 5days;
after which time the 2 layers S were sepmated, the benzene layer was washed with water (3mi.), dried (MgS04) and the solvent removed in vacuo to give tile. exude product. Purification by IdPLC (20'/o to 60°~
MeCN in water) and lyophilisatioa from benzene yielded compouad 4e (33mg, IB.Ipmol, 40%) as a fluffy, colourless solid; ~Cx~s -236.0 (c. 0~, CHCI3); v,",x (CHCI3 cast~cni ~ 3324m, 2959m, 287Im, 1T45w, 1626s,123Im; &H
(300MHz, CsD~ complex due to presence of 4 isomers 8.76 (d, J= 6.OHz), 8.?3 (d, J= 6.OHz); 8.29 (d, J= 7.OHz), ?:93 (d, J= 7.SI3z~
10 7.88 (d, J= 7.SHz}, 7.53 (d, J= 9.SHz~ 7:62-731 {I H, complex), 7.16-6.88 (2H, complex 6.59-.6:39 (complex), 6.28 (t, J=1 I.OHz~ 6.15 {d; J=-IO.SHz), 6.09 (d, J=10:5IIz), 6,05 (d, J=~ 1 LSHz), 6.03 (d, J=
I I.SHz), 5.90-5.82~(complex), S.b8:5.3S (complex), 5.08-4.97 {complex), 4.81-4.?2 (ca~rrpiex), 4.63-4.53.
(complex), 4.03 (d, J= l4.Olviz), 3.47 (s), 3.46 (s), 3.28 (s), 3.26 (s}, 3.17 (s), 3:I5 (s), 3.09 (s~ 2.98 (s~ 2.97 (s), 2.83 (s), 2.63 {s), 2.62 (s), ? 7I 2.Sb (complex), 2.47-2.23 (campiex), 2.05 (s), 2.04 (s); 2.03 (s); 2.02 (s}, 15 L98-0.82 (complex), 0.77 {d, J= 6.SHz), fl.58 (d, J= 6.SIiz), 0.58 (d, J=
b.SHz); ml: (Electrospray) I273,8 (' 1~)~
Examnte'I
To a stirred solution of compound 4g (20mg, I5.9pmot) in methanol {5mL) and water (1mL) at room temperature was added potassium carbonate {30mg, 217pmol). After stirring overnight, the reaction mixture was partitioned lieZween EtOAe (I OmL) and S% aqueous citric acid {1 OmL). The aqueous layer was further - extracted with EtOAc (SmL}, the combined or~nic layers were tl~ washed with 5% citric acid (1 OmL) and brine (IOmL), dried {MgSO,} and the solvem iemoved in vacuo to give the crude producK. Purification by HPLC (85~o MeCN) and tyophilisation from benzene yielded compound Sg (I Omg, 8.2~tmol, 52%) as a fluffy, colourless solid; ~lz~ -2852 {c. 0.Z9, CHCI~; v~ {CHCI; cast~ari ~ .3500-3200bc, 33 I9m, 2958m,, 2927m, I b26s,1520m, I4b8m, 754m; &n (300MHz, C6T),~ complex due to the presence of 2 isomers 8:25 (d, J=
I O.OHz, ~i~, 8.I3 (d, J=10.0Hz, I~B~, ?93 (d, J= 7.0Hz, NHS, 7.84 {d, J=
7.OHz, NI~, 7.67 (d, J= 8.0Hz, Ice, 7.61 (d, J= 8.OHz, N~i , 7.55 (d, J= 8.SHz, N~, x.54 (d, J= B.SHz, NCI , 6.84 (t,.J= IO.SHz), 6.79 (t, ..J=1Q.SHz~ 6.58 (t, J=10.SHz), 6.52 (t, J= IO.SHz~ 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= I4.UHz), 3.93 {d, J=14.OHa), 3.72 (s), 3.68 {s~ 3.I9 (s), 3.I7 (s~ 3.05 ~(s~ 3.03 (s}; 2.94 (s}, 2.93 (s}, 2.89 (s~
2.8b (s), 2.82 {s), 2.81 (s), 2.72 2.53 (complex 2.55 {s), 2_54 {s); 2.49-236 (compl~~ 2.32 2.03 (eomplelc), I.BI-0:81 {complex), 0.65 (d, J=
6.5Hz)), m!z (Electmspray) 1216.8 (AdH*, 1009~0~ 607.9 ([M+2HJ~', _15'""~

Examvle 8 To a stirred solution of compound 4e {l8mg14.2~no1) iti methanol (SmL) and water (1mL) at room te~xtpeiature was added potassium carbo~te (35mg, 254ptnoi). After stirring ovetuight, the reaction mixture was partitioned between F..tOAc (lOmL) and S°1o aqueous citric acid (l OmL). The aqueous layer was f~nthar extracted with EtUAc (SmL~ the combined organic layers were then washed with 5°Yo citric acid {lOmL) and .
brine (1 OmL~ dried (Mg50~ and the solvent removed i» vacrre to give the cmde product Purification by HP1.C (65°/. MeG'1~ and lsrophilisatioa from be»zene yielded end Se (IOmg, 8.I pnnol, 57%) as a fh~'y, coiouriess solid; ~Cr~ 2853.(c. 0.1 I, CHCIs); 8x (3t)OMIiz, C°D~
complex due to presence of4 isomem.
831 (d, J = 9.SHz~ 8:28 (d, J= 9.,SIia), 8.16 (d, J ~ 9S1~Iz~ 8.14 (d, J =

9:SIix~ 7:96 {d, J= 7SHz), 795 (d, IO .T= 7.SHz~ 7.86 (d, J= 9.SHz), 7.85 (d, J= l.SHz), 7.63 (d, J=7.SHx), 7.59 (d, J=7.SHx}x.50-7.44 (complex), 6.60-6.49 (complex 632-6.I 1 (complex}, 5.88-5:83 (complex), 5.76-5:?I (complex), 5.64-5,22 (complex), S.I7-5.08 (complex 49I-4.77 (complex), 4.26-4.18 (complex), 3.99 (d, J=14.OHz), 397 (d, J=
I4.OHx), 3.74 (s~ 3.T3 (s), 3.71 (s~ 3.69 (s~ 3.22 (s), 3.21 (s~ 320 (s~ 3.i9 (s~ 3.07 (s), 3.06 (s), 3.05 (s~
2.97 (s}, 2.96 (s~ 2.95 (s), 2.92 (s), 2:9I (s}; 2.89 (s), 2.84 (s), 2.83 (s), 2.69-zo~ (complex), 3.58 {s); 2.57 (s), I.84-0:81 (complex), 0.64 (d, J= 6.SHz); mla (Electrospray) 1269.8 ([M+Kj~', 5%);1253.8 ([M+Na]+, 30), 1231.8 (MFi') Examyle 9 The immunosupressive activity was tested for deuterated cycIosporin analogs as descn'bed below.
24 ~ Compound Se and compound Sg are more potent than the parent cyclosporin.
Calcineurin nativity was assayed using a modification of the method;previously described by Fruman et al. (Proc Natl Acad Sci USA, 89,3686-3690, 1992). Whole blood lysates were evaluated for their ability to dephosphorylate a szp_IabeIled 19 amino acid peptide substrate in the presence of okadaic acid, a phosphatase type I and 2 inhibitor. Background phosphatase 2C activity (CsA and okadaic acid resistant activity) was determined ZS and subtracted from each sample, with the assay performed in the presence and absence of excess added 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 t the standard error of the mean. The results are plotted as CsA derivative concentration in ug/L versus percentage of caIcineurin inhabitation.
The structrues of the compounds assayed include:
MeLeu--WIeVat Abw-Sar AIieLeu-D-Ala-AIa--Meleu-=VaI-Mefeu (t~~~d 2) Ha~~
MeLeu--MeVat i ~-- Abu-,Sar O.
MeLeu--D-Ala--A!a-MeLeu-Vat-MeLeu (~pc"md 3j Hb MeLeu-.fiAeVat---=~ Abu-far .I ° i MeLeu--D Ala~- Ala-~-MeLeer-VaHNeLeu (Compound 6) S ExamQie 10 A mixed Lymphocyte ceacrion (MLR) assay was performed with cyclosporine and compounds Se and Sg. The rest are presented in figure 6 and are plotted as the means of four experhnents showing concentration of cyclospmine or derivative versus percent inhbition.
The MLR assay is useful for ide~ify~g CsA derivxrsves with biolo~Cai ('mmmosuppressive) activity 1 ~ and to qtietuifyr this activity relative to the immunosuppressive acrivit3r of the. parent CsA molecule.
An example of a Lymphocyte proliferation assay procedure useful for this p~pose is as .follows:
Ttt .
1. Collect blood from two individuals (20m~s each) and isolate lymghocytes using ~coll-Paque (Pharma~ia Biotech).
2. ~ Count lymphocytes at 1:I0 di7uv'on is 2 ~b acetic acid (vlv):
IS 3. Prepare lOmIs of each lymphocyte pogulati~s (A +B) at 1xI06 nl is DMEM
l20 % FCS (vlv~
4. Set up a 96 well ster0e tissue true plate, $~ bott~t (Sarstedt, cwt #
83.1835. To each well add:
5. Aliquot I OOp.I per well lymphocyte population A
6. Aliquot I QOltt per well lymphocyte population B
7. Aliquot 20pi per well of drag (CSA and CSA derivatives) at 0, 2.5, ~, I 0, 25, SO and 100ug/L in triplicate in 2t1 DMEM with no supplements.
8. To measure the effect ofdrug on proliferation,,iacubate the plate for 5 days at 3T° C in 5 % COi atmoe.
9. On day 5, prepare 3.2nals of 1:50 cf lotion of Methyl-'Ii-Ihymi~ (Aarersbam Life Science, c~i # TRK 1Z0) in DNIEM with no supplements. Add 30111 per well and incubate for I8 hours at 37° C in 5 % COi aunosphere, ZS 10: On day-7 celis~arc.harvested onto glass microfiber filters GFIA
(Whattnaa, cat # 1820024) using a CeII-TM
Hxcvestor (Ska~a, cat # 11019). RTash cells 3x with 1.0-ral sterile dist;'lled water.
Note All pros are done using ttrile fadmiques-in a biolbgical flow hood.
TM
i I_ Pla<x fdtas in Scint~ion vials and add 1.5m1s of SaraSafe Plus 50 %
scinaIatiOn fluid (F'>sher, cat # SX 25- -Ig I2. Measure the amount of radioactivity incorporated in the lymphocytes using a beta counter (Micromedic System Inc., TAURUS Automatic Liquid Scintiiation Counter) for L0 minute.
13. Calculate averages and standard deviations for each dmg and express results as:
~Yo lrdubition ~ [1~ Ave CRM of test drug-] x I00 Ave CPM ofzero drug °lo Proliferation ~ 100 ~ % Inhr'bition The MLR assay caa be trtiIized to select anh'bodies of the invention which bind biologically active CSA metabolites and the parent CSA molecule. Arnt'bodies could also be selected for reacrivitY to biologically inactive metabolites.
From the results of the caieineurin assay and the mixed lyraphocyte reaction assay, it was found that cyciosporines that have been chemically substittuect and deuterated at the amino acid 1 position can possess significant imrnunosc~piessant activity. la the case of the derivatives Se and Sg, isnmunost~pressant activity that is significantly greater than GSA was obtained.
ExamQte 11 Other cyciosporine derivatives of tire invention which have been prepared include the following:
STRUCTURE
MeLe~i-MeVal Abu--Sar MeLeu-~D-Ala--Afa-MeLeu-Va!-Mel.eti ~'r/
MeLeu-lllleVal ! Abu-Sar MeLetH--D-Aia-Vila-u--Va(~-~AAeiets MeLeu-AAeVat---- i Abu-Sar O
MeLeu-t)-A~~--Aia--AAeLeu-Vat-MeLeu a~~
MeLect-MeVal i Abet-Sar MeL~-~-Ai~~,ta-Meteu-Vat--MeLeu HZ~i HO,j MeLeu-fiAsVat --- ~ Abu--Sat MeLsu--D~la-A1a-MeLeu-Vai-MeLeu MeLeu-MeVai Abu-Sar MeLeu-D~tla-Afa-MeLeu-Vat-MeLeu AcQ~/
MeLeu-MeVaf I ~ Abu-Sar MeLeu-D=Ata-~41a-MeLeu-Vat-MeLeu >u--Sar MeLeu-D Ata~4la-MeLeu-Vat-MeLeu MeLeu-MeVaE ~r--Sar MeLeu-D-Ala-Ala-Meixu--Va!-MeLeu MeLeu-MeVai -lit Abu-Sar Q
MeLeu-t)-Ata-Ala-MeLeu-Vai-Mel.eu MeLeu-MeVai ~u-Sar MeLeu-~-~-Ata Ata-MeLeu=Val-.-MeLeu HC~r~
MeLeu-MeVat j ~ ~---- Abu--Sar Melee-D-Ala--Vila-MeLeu-Vat-MeLeu bu=Sar MeLeu-fl-Ala- Ala-Melee-Vaf-MeLeu ar MeLeu-D Ata-Ata-MeLeu-Val-MeLeu ACCl~/
Melee-MeVa! i ~ ~-- Ab~i-~Sar MeLeu-D Ala--Ala~-MeLeu--Val-NIeLeu ~ar MeLeu-D-Ala Ata-MeLeu-Val-MeLeu MeLeu~-~D-Ata-Ala-Mele~,~-Vat -MeLeu HOi~
MeLeu--MeVal ~' ~"" Abu--Sar MeLeu--~.-Aia- AtaE--MeLeu-Vat-MeLeu MeLeu--MeVai Abu-Sar Met.eu--D-Ata-Vila--AAeLeu--Vat--IUleLeu HOy~
MeLeu-MeVa! ~ ~--- Abu--Sar MeLeu-D-Ala-Ala-MeLeu--Vat--MeLeu MeLeu-MeVa1 Atsu--Sar MeLeu--D-Ata--~41a-MeLeu-Va!-MeLeu MeLeu-MeVat i Abu--Sar Mete::---D~lta-AIa--MeLeu-V~--MeLeu DraE Composition Farmulation and Elicitationlof Immunosunressfion - Detezmination of the physicochemical, pharmacodynamic, toxicological and phar~cokinetic properties Of the cyclosporine derivatives disclosed can be made using standard chemical and biological assays and through the use of mathematical modeling Lechniqnes which are W own in the chemical and pharmacologicalhoxicologieal arts. The therapeutic utility and dosing regimen can be extrapolated front the results of such techniques and through the use of appropriate pharmacokinetic andlor pharmacodynamic I0 models.
The compounds of this invention may be administe;ed neat or with a pharmaceutical carrier to a warm blooded anir~l in need thereof. The pharmaceutical carrier may be solid or liquid.
This invention also relates to a method oftreatment for patients suffering from immunoregiilaiory abnoiwalities involving the administration of the disclosed cyclosporines as the active constituent I S . For the treatm~t of these conditions atxl diseases by imraunoitregularity, a deuterated ryclosporin map ~e ad~aistered orally, topically, parenteralty, by inhalation spray or reetaIly in dosage unit formulations containing ~nventional non-toxic pharmaceutically acceptably carriers, adjttvants and vehicles.
The term parentezal, as used herein, includes sinus injections, intravenous, iatramuscnlar, intrasternal .
injection ar infusion techniques.
20 The pharmaceutical compositions cog the active ingredient may be in a form suitable for oral use, for example, as tablets, tt, Ioyges, aqueous or oily suspensions, disln~le powders or ~annles, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be ~.
according to auy method known to the art for the mant~acaue of pharmaceutical compositions and such compositions may contain. one or more_agems selected from the group, consisting of sweetening agents, 23 ~ flavoring agerns, coloring agems and preserving agents 'm order to provide pharmaceutically elegant and palatable preparation.. Tablets containing the active ingredient in admixture wish non-toxic pharmaceutically acceptable excipients may also be manufactured by known methods. The excipiei~ts used may be for example, (1 ) inert diluents such as calcitmt carbonate, lcctose, calcium phosphate or sodium phosphate; (2) granulating and disintegrating agents such as com starch, cr alginic acid; {3) binding agents such as star, gelatin or 30 ~ acacia, and (4) lubricating agents such as anagnesium stearate, stearic acid or talc. The tablets rnay be uncoated or they maybe coated by known techniques to delay disintegration arid absorption in the, gastrointestinal tract and thereby provide a sustained action over a longerperiod. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. Tliey may also be coated by the techniques described in the U.S. Pat. Nos. 4,256,108; 4;160,452; and 4,26S,874 to form osmotic therapeutic tablets for 5 controlled release.
In some cases, formulations for oral use may ix in the form of hard gelatin capsules ~ the active ingredient is'mixed with an inert solid dilnenx, 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 oil medium, for-example pearnti orl, liquid paraffin, or olive off.

10 ~ Aqueous suspensions normally contain the active materials, in admixture with excipients suitable foi the manufacture of aqueous suspensions. Such excipients may he (1 ) suspending agents such as sodium carbaxymetnylcellulose, methylcellulose, hY~~YPreFY~e~yIceilulose, sodium alginate, polyviaylpytrrolidoue, gum tragacanth and gum acacia;
(2) dispersing or wetting agerts which may be ~15 (a) a naturally occurring phosphatide such as lecithin;
(b) a condensation product of an alkylene oxide with a fatty acid, for example, polyoxyethyIene steatate, (c) ~a condensation product of ethylene oxide with a long chain aliphatic alcohol, for example, heptadecaethyleneoxycetanol, 20 (d) a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol such as polyoxyethyIene sorbitoi monooleate, or (e) a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitoI anhydride, for example poIyoxyethylene sorbitan monooleate.
The aqueous suspensions may also contain one or more preservatives, for exaasple, ethyl or a-propyl 25 p-hydroxybenzoate; one or more coloring agents; one or more flavoring agents; and one or more sweetening agents such as sucrose, aspartame or saccharin.
Oily suspension tnay be formulated by suspending the active ingredient in a vegetable oil, for exaanple arachis oil, olive oil; sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. :The o~7y suspensions may contain a thickensng agent, for example beavvax, hard paraffin or cetyl alcohol..Sweetening agents and 30' :flavoring agents may be added to provide a palatable oral preparation.
These ~compositiorta may he preserved by the addition of an antioxidant such as ascorbic acid.
Dispezsible powders and granules are suitable for the preparation of an aqueous suspension. They provide the active ingredient in admixteue with a dispersing or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing or wetting alts and suspending agents are exemplified by those 3S already mentioned above. Additional excipients, for example, those sweetening, flavoring and coloring agents descn'bed above may also be present.
The pharri~aceuiical compositions of the invention may also be in the form of oil-in-water emulsions.
The oily phase may be a vegetable oil sack as olive oil or arachis oils, ar a mineral oil such as liquid gara~n or a mixture thereof Suitable emulsifying agents may be (I) naturally occurring Gums such as gum acacia and gum tragacanth, (2} naturally-occurring phosphatides such as soy bean and lecithin, (3) esters or partial esters derived from fatty acids and hexitol anhydrides, for example, sorbitan monooleate, (4) condensation products of said partial esters with ethylene oxide, for example, polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.
Syrups and elixirs may be formulated with sweetening agents, for example, glycerol, propylene glycol, sorbitol, aspartame or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents.
The pharmaceutical compositions may be iw the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in I,3-butanediol. Among the acceptable vehicles and solvents I5 that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
In addition; sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono=
or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
The disclosed cyclosporins may also be administered in the form of suppositories for 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., containing the disclosed cyclosporins are employed.
Dosage levels of the order from about 0.05 mg to about 50 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (from about 2.5 mg to about 2.5 gms. per patient per day}.

The amount of active ingredient that maybe combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a formulation intended for the oral administration of humans may contain from 2.5 mg to 2.5 gm of active agent compounded 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 from about 5 mg to about 500 mg of active ingredient.
It will be understood, however, that the specific.dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed,: the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
In the case of conflict between the references listed herein and this application, the text of the application is controlling. Modifications and changes of the disclosed compounds and methods will be apparent to one skilled in the art. Such modifications and changes are intended to be encompassed by this disclosure and the claims appended hereto.

Claims (7)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A composition that comprises a mixture of compounds represented by structures A and B:

wherein R is selected from the group consisting of : (i)-CH=CH-CH3, (ii)-CH=CH2, and (iii)-CD=CD2.

2. The composition of claim 1, wherein R is -CH=CH-CH3.

3. The composition of claim 1, wherein R is -CH=CH2.

4. The composition of claim 1, wherein R is -CD=CD2.

5. A pharmaceutical composition comprising a mixture of compounds as defined in any one of claims 1 to 4, and a pharmaceutically-acceptable excipient.

6. Use of an effective amount of a composition as defined in any one of claims 1 to 4, for producing immunosuppression.

7. Use of an effective amount of a composition as defined in any one of claims 1 to 4, in the manufacture of a medicament for producing immunosuppression.