GB2207678A - Novel immunosuppressive fluorinated cyclosporin analogs - Google Patents

Abstract

New cyclosporin analogs containing fluorinated amino acid residues at the various positions around the ring have been synthesized end found to exhibit unexpectedly superior activity over cyclosporin A as immunosuppressive agents.

Description

TITLE OF THE INVENTION NOVEL IMMUNOSUPPRESSIVE FLUORINATED CYCLOSPORIN ANALOGS BACKGROUND OF THE INVENTION Immunoregulatory abnormalities have been shown to exist in a wide variety of "autoimmune" and chronic 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 Crohns disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, psoriasis, ichthyosis, and Graves ophthalmopathy. Although the underlying pathogenesis of each of these conditions may be quite different, they have in common the appearance of a variety of autoantibodies and selfreactive lymphocytes. Such self-reactivity may be due, in part, to a loss of the homeostatic controls under which the normal immune system operates.

Similarly, following a bone-marrow or an organ transplantation, the host lymphocytes recognize the foreign tissue antigens and begin to produce antibodies which lead to graft rejection.

One end result of an auto immune or a rejection process is tissue destruction caused by inflammatory cells and the mediators they release.

Ant-iinflammatory agents such as NSAID's and corticosteroids act principally by blocking the effect or secretion of these mediators but do nothing to modify the immunologic basis of the disease. On the other hand, cytotoxic agents such as cyclophosphamide, act in such a nonspecific fashion that both the normal and autoimmune responses are shut off. Indeed, patients treated with such nonspecific immunosuppressive agents are as likely to succumb from infection as they are from their autoimmune disease.

The cyclosporins are a family of immunosuppressive compounds isolated from fermentation broths of various fungal species, including Tolypocladium inflatum and Cylindrocarpon lucidum.

The generic structure of the class of cyclosporins has been established as a cyclic peptide of formula I which contains 11 amino acids.

For example, cyclosporin A of formula II contains several N-methylated amino acids and one novel amino acid, "MeBMT", designated as the 1- "C-9 amino acid". This novel amino acid is located in position 1 and has been found to be important for the biological activity of cyclosporin. We now find that fluorinating the amino acid at positions 2, 4, 5, 6, 7, 9, 10 and 11 around the ring, provides potent immunosuppressive compounds and, unexpectedly, in some cases with biological activity greater than that measured for cyclosporin A. Biological activity is measured in terms of binding affinity for cyclophilin, the cytosolic receptor for cyclosporin tR. Handschumacher et al., Science, 226 (1984) 544], inhibition of interleukin-2 production, and inhibition of T-cell proliferation.

Structure of Cvclosorin Aa

10 11 1 2 3 MeL?u - MeVal - MeBmt - Abu - Sar 9 Metes j (II) D-Ala - Ala ~ MeLeu ~ Val ~ MeLeu 8 7 6 5 4 Bmt = (4R)-4-[(E)-2-butenyl3-4-methyl-L- threonine Me = Methyl Abu = a-Aminobutyric acid Val = Valine Ala = Alanine MeLeu = N-Methyl-leucine MeVal = N-Methyl-valine Sar = Sarcosine a. Unless specified, the amino acid is of the L-configuration at the C-2 position.

Generally a cyclosporin such as cyclosporin A is not cytotoxic nor myelotoxic. It does not inhibit migration of monocytes nor does it inhibit granulocytes and macrophage action. Its action is specific and leaves most established immune responses intact. However, it is nephrotoxic and is known to cause the following undesirable side effects: (1) abnormal liver function; (2) hirsutism; (3) gum hypertrophy; (4) tremor; (5) neurotoxicity; (6) hyperaesthesia; and (7) gastrointestinal discomfort.

Accordingly, an object of the present invention is to provide new cyclosporin analogs which will (1) restore the balance of the help-andsuppression mechanism of the immune system by acting at an earlier point than the anti-inflammatory agents and (2) induce specific long-term transplantation tolerance through a suppressor cell circuit without increasing the body's susceptibility to infection.

Another object of the present invention is to provide pharmaceutical compositions for administering to a patient in need of the treatment one or more of the active immunosuppressive agents of the present invention.

Still a further object of this invention is to provide a method of controlling graft rejection, autoimmune and chronic inflammatory diseases by administering a sufficient amount of one or more of the novel immunosuppressive agents in a mammalian species in need of such treatment.

Finally, it is the object of this invention to provide processes for the preparation of the active compounds of the present invention.

DETAILED DESCRIPTION OF THE INVENTION A. Scope of the Invention This invention relates to cyclosporins of formula I

wherein R1 is MeBMT; or dihydro MeBMT; R2 is an amino acid residue selected from the group consisting of the residues of Nva (norvaline), Thr, Ser, and Val, or a fluorinated analog thereof, wherein a fluorinated analog represents the amino acid residue in which one or more of the various C-H bonds in the side chain are replaced with C-F bonds; for example, fluorinated analog of Abu represents

wherein X1-X5 independently is H or F with the proviso that at least one of X1, X2, X3, X4, and X5 is F.

R3 is Sar or D-MeAla; R41 R61 R91 and R10 are MeLeu or a fluorinated analog thereof; R5 is Val or a fluorinated analog thereof; R7 is Ala or a fluorinated analog thereof; R8 is D-Ala; and R11 is MeVal or a fluorinated analog thereof.

With the proviso that at least one of R2 to R11 is a fluorinated analog of an amino acid.

Preferably, this invention relates to a cyclosporin analog of formula I wherein R1 is MeBMt or dihydro MeBmt; R2 is (a) a fluorinated analog of L-alanine; (b) a fluorinated analog of L-2-aminobutyric acid; (c) a fluorinated analog of L-norvaline; (d) a fluorinated analog of L-valine; or (e) L-2-aminobutyric acid, L-norvaline, or L-threonine; R , R and R6 to R10 are as previously defined; R5 is valine or a fluorinated analog of valine; and Rll is N-methyl-valine or a fluorinated analog of N-methyl-valine.

2 the proviso that at least one of R 11 With the proviso that at least one of R2 to R is a fluorinated analog of an amino acid.

In a even more preferred embodiment of this invention: R1 is MeBmt or dihydro MeBmt; is is (a) 4,4-difluoro-Abu; (b) 4-f luoro-Abu; (c) 5-fluoro-Nva; (d) 4-fluoro-Nva; (e) 4-fluoro-Val; (g) Abu; (h) Nva; or (i) Thr; R , R4 and R6 to R10 are as previously defined; is is Val or 4-fluoro-Val; and R11 is Me Val or 4-fluoro-MeVal.

With the proviso that at least one of R2 to R11 is a fluorinated analog of an amino acid.

B. Preparation of the compounds within the scope of the present invention The cyclosporins of this invention are prepared via cyclization of appropriate linear undecapeptides following well-established procedures, which were slightly modified for better results. The procedure most used is published by R.M. Wenger et al. in Helv. Chim. Acta, 67, 502(1984). Scheme I below illustrates the application of this procedure to the cyclosporins of this invention. Starting materials for the process are either known or available commercially.

Scheme I

According to the scheme, 4,4-difluoro-Laminobutyric acid (1) is N-protected as its BOC derivative, then coupled with sarcosine benzyl ester, in the presence of bis-(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-C1) as the peptide coupling reagent, to afford after hydrogenolysis the O-deprotected dipeptide 2. Dipeptide 2 is then coupled with the known H-MeLeu-Val-MeLeu-Ala-O-Benzyl tetrapeptide (R.N. Wenger, Helv. Chim.Acta. 67 (1984) 502] with BOP-C1 as the coupling reagent to afford, after N-deprotection with trifluoroacetic acid, the hexapeptide 5. Heptapeptide 4 is then formed by first coupling with N,Q-isopropylidene-MeBMT in the presence of N-hydroxybenzotriazole and 1, 3-dicyclohexylcarbodiimide, and subsequent removal of the isopropylidene acetal group by treatment with an acid, for example, HC1 in methanol. The protected linear undecapeptide S is prepared by condensation of 4 with the known Boc-D-Ala-MeLeu-MeLeu-MeValOH tetrapeptide tR.M. Wenger, Helv. Chim. Acta. 66 (1983) 2672) in the presence of benzotriazol-l-yloxytris (dimethylamino)phosphonium hexafluorophosphate (BOP reagent).Q-deprotection is achieved by treatment with aqueous NaOH in ethanol and N-deprotection by treatment with trifluoroacetic acid. The linear unprotected undecapeptide i is then cyclized at high dilution in the presence of a peptide coupling reagent, such as l-propanephosphonic acid cyclic anhydride in the presence of 4-dimethylaminopyridine, to afford (4,4-difluoro-Abu2)-cyclosporin A (7).

The dihydro MeBMT derivatives of the fluorinated cyclosporing analogs are prepared by hydrogenation in an alcohol, such as methanol or ethanol, in the presence of a catalyst, such as, 10% palladium-on-charcoal.

Table I below lists the representative compounds prepared by following essentially the same procedures described in Scheme I, but employing the appropriate tetrapeptide ss or e, substituted with a fluorinated amino acid.a Table I. - Reoresentative Comoounds

11 1 2 MeLeu - R - R1. R2 Sar Meieu MeLeu D-Ala Ala -MeLeu-R5 / Compound R R2 R5 R11 FAB-MSb (M + 1) (1) MeBMT 4,4-difluoro-Abu Val MeVal 1238 (2) dihydroMerMt 4,4-difluoro-Abu Val MeVal 1240 (3) MeBMT 4-fluoro-Abu Val MeVal 1220 (4) MeBMT 4-fluoro-Nva Val MeVal 1234 (5) MeBMT 5-fluoro-Nva Val MeVal 1234 (6) MeBMT Abu 4-fluoroval MeVal 1220 (7) dihydroMeBMT Abu 4-fluoroVal MeVal 1222 (8) ZeBT Abu Val 4-fluoro- 1220 ZkVal a The fluorinated amino acids employed in synthesis of the cyclosporins described in this invention are novel. For their synthesis. the general method of photofluorination was employed. (J. Kollonitsch, Isr. J. Chem., 17. 51-59, 1978, and reference therein.) For example, photofluorination of L-2- aminobutyric acid gives 4,4-difluoro-L-2-aminobutyric acid. Photo fluorination of L-valine gives 4-fluoro-3(S)-L-valine and 4-fluoro-3(R) L-valine. Photofluorination of L-norvaline gives 4-fluoro-4(S)-L-norval ine 4-fluoro-4(R)-L-norval ine and 5-fluoro-L-norval ine.

b FAST ATOM BOMBARDMENT MASS SPECTROSCOPY C. Utility of the compounds within the scope of the invention This invention also relates to a method of treatment for patients suffering from immunorgulatory abnormalities involving the administration of a compound of formula I as the active constituent.

For the treatment of these conditions and diseases caused by immunoirregularity a compound of formula I may be administered orally, topically, parenterally, by inhalation spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.

The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparation. Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients may 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) granulating and disintegrating 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 intestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by the techniques described in the U.S. Patents 4,256,108; 4,160,452; and 4,265,874 to form osmotic therapeutic tablets for 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 oil medium, for example peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions normally 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, methyl cellulose, hydroxypropylmethyl cellulose, sodium alginate, polyvinyl pyrrolidone, gum tragacanth and gum acacia; (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, heptadeca ethyleneoxycetanol, (d) a condensation product of ethylene oxide with å partial ester derived from a fatty acid and a hexitol such as polyoxyethylene sorbitol monooleate, or (e) a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride, for example polyoxy ethylene sorbitan monooleate.

The aqueous suspensions may also contain one or more preservatives, for example, ethyl or n-propyl p-hydroxybenzoate; one or more colouring agents; one or more flavoring agents; and one or more sweetening agents such as sucrose or saccharin.

Oily suspension may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The-oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents may be. added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

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 already 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 oily'phase may be a vegetable oil such as olive oil or arachis oils, or a mineral oil such as liquid paraffin or a mixture thereof.

Suitable emulsifying agents may be (1) naturallyoccurring 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 or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents.

The pharmaceutical compositions may be in 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 1,3-butanediol. Among the acceptable vehicles and solvents 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 addition1 fatty acids such as oleic acid find use in the preparation of injectibles.

A compound of formula I 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 immunoregulants are employed.

Dosage levels of the order from about 25 mg to about 2.5 gms per patient per day are useful in the treatment of the above-indicated conditions.

The amount of active ingredient that may be 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 5-250 mg of an active 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 from about 25 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.

D. Biological evidence in support of utility of the compounds within the scope of the invention It has been found that the compounds of formula I have immunosuppressive activities and are thereby useful in the treatment of various "autoimmune" and chronic inflammatory diseases. They may also be useful in the prevention of graft rejection or rejection of "donor" organs in transplantation operations.The following table illustrates and supports the utility of the compounds of the present invention: IMMUNOSUPPRESSIVE ACTIVITIES OF FLUORINATED CYCLOSPORIN ANALOGS

MeLeu - R11 - R1 - Abu - Sar MeLeu MeLeu D-Ala - Ala - MeLeu - R5 T-Cell Cyclophilin Proliferation a0 g11 R5 Binding ' Inhibition ' MeBMT MeVal 4-fluoro-Val 232 144 dihydroMeBMT MeVal 4-fluoro-Val 239 163 MeBMT 4-fl uoro-MeVal Val 69 47 a This assay is described in detail by R. Handschumacher et al., Science, 226 (1984) 544.

b The data are expressed as % CsA's activity (CsA(cyclosporin A)=100).

c T-Cell Proliferation Assay: T-cell proliferation was measured in-mouse T-cell cultures stimulated with ionomycin plus phorbol myristate acetate (PMA). Spleen cell suspensions from C57B1/6 mice were prepared and separated on nylon wool columns. The recovered T-cells were suspended at 106 cells /ml in complete culture medium with addition of ionomycin (250 ng/ml) and PMA (10 ng/ml). The cell suspension was immediately distributed in 96 well-flat bottom microculture plates at 100 pl/well. Control medium or various concentrations of test compound were added in triplicate wells at 10 l/well. The plates were incubated at 370C in a humidified atmosphere of 5% CO,-95 air for 44 hours.At 44 hours of culture, the plates received 20 ml/well of a solution of (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MMT) in PBS (10 mg/ml). To dissolve the purple crystals of MTT formazan produced by metabolically active cells, 100 ml of a 10% SDS-0.01 N hydrochloric acid solution was added to each well. The culture plates were incubated at 37"C in a 5% CO2 incubator. The plates were read at 570-600 nm in a multiwell scanning spectrophotometer.

The absorbance (specific OD) of experimental wells was corrected for that of wells with unstimulated cells or no cells. The percent inhibition of proliferation was calculated according to the formula: Specific OD experimental % Inhib. = 100 - ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ x 100 Specific OD control medium The following examples illustrate the processes for making an active compound to be used in the present invention.

EXAMPLE 1 Cyclo(-((2S,3R,4R, 6E)-3-hydroxy-4-methyl-2-methyl- amino-6-octenoyl)-L-2-aminobutyryl-sarcosyl-N-methyl-L- Leucyl-4-fluoro-valyl-N-methyl-L-leucyl-alanyl-D- alanyl-N-methyl-L-Leucyl-N-methyl-L-leucyl-N-methyl- L-valvll Step 1: Boc-4-fluoro-3-(R.S)-L-valine To a suspension of 990 mg (0.0073 mole) of (R,S)-4-fluoro-(L)-valine in 10 ml of dioxane and 10 ml of water was added 1.12 g (0.0111 mole) of triethylamine. 2.12 g (0.0086 mole) of BOC-ON was added and stirred at room temperature for 16 hours.

The reaction was diluted with 40 ml of water and 60 ml of ether. The aqueous layer was separated and made acid with 2.5 N hydrochloric acid, then extracted with 3 x 40 ml of ethyl acetate. The organic layers were dried over magnesium sulfate and evaporated leaving the crude product as a thick oil. This was reacted in step 2 without further purification. The crude yield is 94% of theory. Fast atom bombardment mass spectroscopy (FAB m.s.) showed a molecular ion peak at 236.

Step 2: BOC-4-fluoro-3 (R,S)-li-Valyl-N-Methyl-lieucyl- L-Alanine benzyl ester A solution of 1.6 g (0.0069 mole) of Boc-4fluoro-3(R,S)-L-valine and 2.11 g (0.0069 mole) of N-methyl-L-leucyl-L-alanine benzyl ester [R.M.

Wenger, Helv. Chim. Acta 67 (1984)502) in 70 ml of dry methylene chloride was stirred and cooled at -100C. After 15 minutes, 1.69 g. (0.0167 mole) of dry triethylamine was added. The reaction was stirred for 5 minutes and 1.93 g (0.0076 mole) of N,N-bis (2- oxo-3-oxazolidinyl)phosphorodiamidic chloride was added. The reaction mixture was stirred at -100C for 16 hours, then evaporated to a small volume. The residue was taken up between 150 ml of ether and 50 ml of 10% potassium bisulfate. The ether layer was separated, washed with 3 x 35 ml of saturated sodium bicarbonate, dried over magnesium sulfate, and evaporated. The residue was chromatographed on silica gel with 1:1 ether-huxane as the eluent. The title compound was obtained as a mixture of diastereoisomers and weighed 1.79 g.THe yield was 50% of theory and FAB m.s. showed a molecular ion peaks at 524.

Stew 3: 4-fluoro-3(R,S)-L-Valyl-N-Methyl-L-Leucyl-L- alanine benzvl ester A solution of 1.75 g (0.0033 mole) of Boc-4fluoro-3(R,S)-L-valyl-N-methyl-L-leucyl-li-alanine benzyl ester in 17 ml of dry methylene chloride was stirred and cooled at -10 C as 17 ml of trifluoroacetic acid, which was previously cooled at the same temperature, was added. The reaction was stirred and kept at -5 C for 16 hours, then poured carefully with stirring into a mixture of 21 g of sodium bicarbonate, ice, and 100 ml of methylene chloride. After 5 minutes the organic layer was separated and washed with 3 x 30 ml of saturated sodium bicarbonate solution. The methylene chloride layer was dried over magnesium sulfate, and evaporated in vacuo to give 1.38 g of a stiff oil as the product. The yield was 99% of theory and the FAB m.s. showed a molecular ion peak at 424.

Step 4: Boc-N-Methyl-L-leucyl-4-fluoro-3 (R, 5)-li-Valyl- N-Methvl-L-Leucol-L-Alanine benzYl ester A solution of 1.46 g (0.0059 mole) of Boc-N methyl-li-leucine in 90 ml of dry methylene chloride was stirred and cooled at OOC while one-half of 1.68 g. (0.0130 mole) of diisopropylethylamine was added, followed by 1.65 g. (0.0065 mole) of N,N-bis(2-oxo-3-oxazolidinyl)phosphorodiamidic chloride. The reaction was stirred at OOC for 4 hours. The remaining diisopropylethylamine was added and a solution of 4-fluoro-3(R,S)-L-valyl-N-methyl L-leucyl-L-alanine benzyl ester in 15 ml of dry methylene chloride. This was stirred and kept at 0 C for 20 hours.The reaction mixture was diluted with 50 ml of methylene chloride, extracted with 35 ml of 5% potassium bisulfate solution, and washed with 2 x 30 ml of saturated sodium bicarbonate solution. The organic layer was dried over magnesium sulfate and chromatographed on silica gel using 2:1 ether-hexane as the eluent. The faster moving diastereoisomer was obtained as the major component and weighed 700 mg.

This was reacted further in step 5. The yield was 33% of theory and FAB m.s. shows a molecular ion peak at 651.

Step 5: N-methyl-L-leucyl-4-fluoro-L-valyl-N-methyl- L-leucyl-L-alanine benzyl ester To a stirred solution of 650 mg (0.001 mole) of Boc-N-methyl-L-leucyl-4-fluoro-li-valyl-N-methyl-li leucyl-l-alanine benzyl ester in 10 ml of dry methylene chloride and cooled at 100C was added 6.5 ml of trifluoroacetic acid, which was previously cooled at -150C. The reaction was stirred for 16 hours, then carefully poured into a mixture of 7.7 g. of sodium bicarbonate, ice, and 75 ml of methylene chloride with stirring. The organic layer was extracted with 3 x 20 ml of saturated sodium bicarbonate solution, then dried over magnesium sulfate. The solvent was removed in vacuo leaving the product as a stiff oil, which weighed 540 mg.The yield is 989% of theory and the FAB m.s. showed a molecular ion peak at 551.

Step 6: Boc-L-2-aminobutyryl-Sa rcosyl-N-methyl-L- Leucyl-4-Fluoro-L-valyl-N-methyl-L-leucyl-L alanine benzyl ester A solution of 274 mg (0.00010 mole) of Boc-L2-amino-butyryl-sarcosine [R.M. Wenger, Helv. Chim.

Acta, 67 (1984) 502) and 500 mg (0.009 mole) of N-methyl-L-leucyl-4-fluoro-valyl-N-methyl-L-leucylalanine benzyl ester in 40 ml of dry methylene chloride was stirred and cooled at -50C. 245 mg of dry triethylamine was added. After 10 minutes 280 mg (0.0011 mole) of N,N-bis(3-oxo-3-oxazolidinyl) phosphordiamidic chloride was added and the reaction was stirred and cooled at -100C for 16 hours. The reaction mixture was concentrated to a small volume and placed on a column of silica gel. The column was first eluted with ether, then with 5 ethyl acetate in ether giving 613 mg of the product as a thick oil. The yield is 84% of theory and FAB m.s. showed a molecular ion peak at 807.

Step 7: L-2-aminobutyryl-sarcosyl-N-methyl-li-leucyl- 4-fluoto-L-valyl-N-methyl-l-leucyl-l-ala benzyl ester A solution of 550 mg (0.0007 mole) of Boc-L2-aminobutyryl-sarcosyl-N-methyl-L-leucyl-4-fluoro-Lvalyl-N-methyl-L-leucyl-L-alanine benzyl ester in 5.5 ml of dry methylene chloride was stirred and cooled at -100C, as 5.5 ml of trifluoroacetic acid cooled at -150C was added. The reaction was stirred and cooled at -50C for 16 hours, then poured carefully into a mixture of 6 g. of sodium bicarbonate, ice, and 100 ml of methylene chloride. After stirring for 10 minutes the organic layer was separated and washed with 3 x 25 ml of saturated sodium bicarbonate solution. The organic layer was dried over magnesium sulfate and evaporated in vacuo giving 470 mg. of product as a glass. The yield is 95% of theory, and FAB showed m.s. a molecular ion peaks at 707.

Step 8: ((4S,5R,1'R,3'E)-2,2,3-trimethyl-5-(1'-methyl- 3'-pentenyl)-4-oxazolidinecarbonyl)-L-2-amino- butyryl-sarcosyl-N-methyl-li-leucyl-4-fluoro-L- valyl-N-methyl-l-leucyl-l-alanine benzyl ester To a mixture of (4S,5R,1'R,3'E)-2,2,3-tri methyl-5-(1 ' -methyl-3 '-pentenyl)-4-oxazolidine- carboxylic acid (prepared by heating at reflux temperature a solution of (2S,3R,4R,6E)-3-hydroxy4-methyl-2-methylamino-6-octenoic acid prepared by the process set forth in R.M. Wenper. Helv. Chim.

Acta 66 (1983) 2308] (129 mg., 0.64 mmol.) in dry acetone (60 ml.) for 48 hours and subsequent evaporation under diminished pressure) in .3 ml.

acetone were added, successively with stirring under a nitrogen atmosphere, dry tetrahydrofuran (6.5 ml.), N-methylmorpholine (81 ml., 0.74 mmol.), N-hydroxybenzotriazole (175 mg., 1.30 mmol.), and a solution of L-2-aminobutyryl-sarcosyl-N-methyl-L-leucyl-4- fluoro-L-valyl-N-methyyl-li-leucyl-L-alanine benzyl ester (465 mg., 0.658 mmol.) in dry tetrahydrofuran (4 ml.). The reaction mixture was cooled in an ice bath and 1,3-dicyclohetylcarbodiimide (139 mg., 0.67 mmol.) added. The mixture was allowed to attain room temperature, and stirring was continued for an additional 24 hours.The mixture was then diluted with dichloromethane (30 ml.) and washed with saturated sodium hydrogencarbonate solution (20 ml.) The aqueous layer was extracted with dichloromethane (20 ml.), and the combined organic extracts were dried (sodium sulfate) and evaporated. The residue was triturated with diethyl ether, filtered, and evaporated. The resulting crude material was applied to a column of silica gel (Merck #7734, packed as a slurry in 28 methanol in dichloromethane). Elution was effected with 2% methanol in dichloromethane.

Fractions containing pure product were combined and evaporated to afford the protected N,O-isopropylidene MeBmt-Abu-Sar-MeLeu-4-Fluoro-Val-MeLeu-AlaObzl heptapeptide as a thick syrup; yield 381 mg. (64t). Its 200 MHz NMR spectrum in chloroform-d was in accord with the desired structure.

Step ((2S,3R,4R, 6E)-3-hydroxy-4-methyl-2-methyl- amino-6-octenoyl) -L-2-aminobutyryl-sarcosyl-N- methyl-L-leucyl-4-fluoro-L-valyl-N-methyl-L- leucvl-L-alanine benzyl ester To a solution of N,O-isopropylidene-MeBmt Abu-Sar-MeLeu-4-Fluoro-Val-MeLeu-AlaOBzl (381 mg., 0.41 mmol.) in methanol (6 ml.) was added with stirring 1E hydrochloric acid (0.44 ml.). The reaction mixture was stirred for 18 hours at room temperature, then neutralized with solid sodium hydrogencarbonate (250 mg.). The mixture was filtered, the filter washed with methanol, and the combined filtrate and washings evaporated. The crude material was taken up in dichloromethane and filtered.

The resulting syrup was applied to a column of silica gel (Merck #7734, packed as a slurry in 4% methanol in dichloromethane). Elution was effected with 48 methanol in dichloromethane. Fractions containing slower-moving product were combined and evaporated to afford the partially deprotected H-MeBmt-Abu-Sar MeLeu-4-Fluoro-Val-MeLeu-AlaOBzl heptapeptide as a thick syrup: yield 257 mg. (70.58).

Step 10: Boc-D-Alanyl-N-methyl-L-leucyl-N-methyl-L- leucyl-N-methyl-;-valyl-((2S,3R,4R,6E)-3- hydroxy-4-methyl-2-methylamino-6-octenoyl) - L-2-aminobutyryl-sarcosyl-N-methyl-L-leucyl-4- fluoro-l-valyl-N-methyl-l-leucyl-l-alani benzvl ester To a solution of Boc-D-Ala-MeLeu-MeLeu MeValOH Prepared by the process set forth in R.M.

Wenger, Helv.Chim. Acta. 66 (1983) 2672J (161 mg., 0.289 mmol.) and H-MeBmt-Abu-Sar-MeLeu-4-Fluoro-Val- MeLeu-AlaOBzl (257 mg., 0.289 mmol.) in dry dichloromethane (10 ml.) were added N-methylmorpholine (38 nl, 0.346 mmol.) and benzotriazol-l-yloxytris (dimethylamino)phosphonium hexafluorophosphate (191 mg., 0.432 mmol). The reaction mixture was stirred 4 days at room temperature under a nitrogen atmosphere.

It was then diluted with dichloromethane (100 ml.), washed with water (50 ml.), dried (sodium sulfate) and evaporated. The crude product was chromatographed on a column of silica gel (Merck #7734, packed as a slurry in 4% methanol in dichloromethane). Elution was effected with 4% methanol in dichloromethane.

Fractions containing pure product were combined and evaporated to afford the desired Boc-D-Ala-MeLeu MeLeu-MeVal-MeBmt-Abu-Sar-MeLeu-4-Fluoro-Val-MeLeu AlaOBzl undecapeptide; yield 258 mg. (62.5%).

Step 11: Boc-D-Alanyl-N-methyl-L-leucyl-N-methyl-L leucyl-N-methyl-L-valyl-((2S,3R,4R,6E)-3- hydroxy-4-methyl-2-methylamino-6-octenoyl)-L- 2-aminobutyryl-sarcosyl-N-methyl-L-leucyl-9- fluoro-L-valvl-N-methvl-L-leucvl-li-alanine To a solution of Boc-D-Ala-MeLeu-MeLeu-MeVal MeBmt-Abu-Sar-MeLeu-4-Fluoro-Val-MeLeu-AlaOBzl (258 mg., 0.181 mmol.) in ethanol (7 ml.) cooled to ice temperature was added 0.2 N aqueous sodium hydroxide (0.9 ml.). The reaction mixture was kept at 50C for 24 hours, brought to pH .5 with several drops of glacial acetic acid and then evaporated under diminished pressure.The residue was taken up in dichloromethane (25 ml.) and washed with water (12 ml.). The aqueous layer was extracted with dichloromethane, and the combined organic extracts were dried (sodium sulfate) and evaporated. The resulting material was applied to a column of silica gel (Merck #7734, packed as a slurry in 4% methanol in dichloromethane). Elution initially with 4% methanol in dichloromethane gave unreacted starting material and benzyl alcohol; subsequent elution with 15% methanol in dichloromethane afforded the partially protected Boc-D-Ala-MeLeu-MeLeu-NeVal-MeBmt-Abu-Sar-MeLeu-4- Fluoro-Val-MeLeu-AlaOH undecapeptide; yield 159 mg.

(65.8%).

SteD 12: D-Alanyl-N-methyl-L-leucyl-N-methyl-L-Leucyl N-methyl-L-valyl-((2S,3R,4R,6E)-3-hydroxy-4- methyl-2-methylamino-6-octenoyl)-L-2-amino- butyryl-sarcosyl-N-methyl-li-leucyl-4-fluoro-L- valvl-N-methvl-l-leucvl-lalanine Boc-D-Ala-MeLeu-MeLeu-MeVal-MeBmt-Abu-Sar- MeLeu-4-Fluoro-Val-MeLeu-AlaOH (159 mg., 0.12 mmol.) was cooled to -150C and treated with precooled trifluoroacetic acid (3 ml.) for 90 minutes at -15 C.

The reaction mixture was then evaporated under diminished pressure (bath temperature of OOC) and coevaporated several times with dichloromethane. The crude material was taken up in dichloromethane (25 ml.) and washed with saturated sodium hydrogencarbonate solution (12 ml.). The organic layer was dried (sodium sulfate) and evaporated. The product was triturated with diethyl ether and the resulting amorphous solid was filtered, washed with ether and the resulting amorphous solid was filtered, washed with ether and dried in vacuo; yield 119 mg. (81%).

QteP 13: cyclo-((2S,3R,4R,6E)-3-hydroxy-4-methyl-2- methylamino-6-octenoyl)-l-2-aminobutyryl sarcosyl-N-methyl-li-leucyl-4-fluoro-valyl-N- methyl-L-leucyl-alanyl-D-alanyl-N-methyl-L- leucyl-N-methyl-L-leucyl-N-methyl-L-valyl - To a solution of H-D-Ala-MeLeu-MeLeu-MeVal MeBmt-Abu-Sar-MeLeu-4-Fluoro-Val-MeLeu-AlaOH (119 mg., 0.096 mmol.) in dichloromethane (400 ml.) were added 4-dimethylaminopyridine (60 mg., 0.491 mmol.) followed by l-propanephosphonic acid cyclic anhydride (50 wt.% solution in dichloromethane) (0.3 ml.). The reaction mixture was stirred at room temperature for 24 hours, concentrated to .25 ml. and washed with saturated sodium hydrogencarbonate solution. The organic layer was dried (sodium sulfate) and evaporated. The crude material was applied to a column of silica gel (Merck #7734, packed as a slurry in 2:1 hexane-acetone). Elution was effected with 2:1 hexane-acetone. Fractions containing pure product were combined and evaporated to give 4-fluoro-Val5 cyclosporin A as a white amorphous solid; yield 57 mg.

(48.7). Its 400 MHz NMR spectrum in chloroform-d-was in accord with the desired structure. FAB m.s.

showed a molecular ion at m/z 1220.

Claims (11)

WHAT IS CLAIMED IS:

1. - A compound of formula

wherein R1 is MeBMT; or dihydro MeBMT; R2 is an amino acid residue selected from the group consisting of the residues of Nva (norvaline), Thr, Ser, and Val, or a fluorinated analog thereof, wherein a fluorinated analog represents the amino acid residue in which one or more of the various C-H bonds in the side chain are replaced with C-F bonds; R is Sar or D-NeAla; R4, R6, R9, and R10 are MeLeu or a fluorinated analog thereof; R5 is Val or a fluorinated analog thereof; 7 R is Ala or a fluorinated analog thereof; R8 is D-Ala; and Rll is MeVal or a fluorinated analog thereof, 11 With the proviso that at least one of R2 to R is a fluorinated analog of an amino acid.

2. The compound of Claim 1 wherein R1 is MeBMt or dihydroMeBMt; R2 is (a) a fluorinated analog of L-alanine; (b) a fluorinated analog of L-2-aminobutyric acid; (cj a fluorinated analog of L-norvaline; (d) a fluorinated analog of L-valine; or (e) L-2-aminobutyric acid, L-norvaline, or L-threonine; R, R4 and R6 to R10 are as previously defined; R5 is Valine or a fluorinated analog of valine; and Rll is N-methyl-valine or a fluorinated analog of N-methyl-va line.

3. The compound of Claim 2 wherein R 1is MeBmt or dihydro-MeBmt; R2 is (a) 4,4-difluoro-Abu; (b) 4-fluoro-Abu; (c) 5-fluoro-Nva; (d) 4-fluoro-Nva; (e) 4-fluoro-Val; (f) Abu; (g) Nva; or (h) Thr; R3, R and R6 to R10 are as previously defined; R5 is Val or 4-fluoro-Val; and Rll is Me Val or 4-fluoro-MeVal.

4. A pharmaceutical composition for the prevention, control or treatment of immunoregulatory disorders or diseases comprising a pharmaceutical carrier and therapeutically effective amount of a compound of formula I

wherein R1 is MeBMT; or dihydro MeBMT; R2 is an amino acid residue selected from the group consisting of the residues of Nva (norvaline), Thr, Ser, and Val, or a fluorinated analog thereof, wherein a fluorinated analog represents the amino acid residue in which one or more of the various C-H bonds in the side chain are replaced with C-F bonds; R is Sar or D-MeAla; R4, R6, R9, and R10 are MeLeu or a fluorinated analog thereof, R5 is Val or a fluorinated analog thereof; R7 is Ala or a fluorinated analog thereof; R8 is D-A1a;; and R11 is MeVal or a fluorinated analog thereof, With the proviso that at least one of R to R is a fluorinated analog of an amino acid.

5. The pharmaceutical composition of Claim 4 wherein R1 is MeBMt or dihydroMeBMt; R2 is (a) a fluorinated analog of L-alanine; (b) a fluorinated analog of L-2-aminobutyric acid; (c) a fluorinated analog of L-norvaline; (d) a fluorinated analog of L-valine; or (e) L-2-aminobutyric acid, L-norvaline, or li-threonine; R , R and R6 to R10 are as previously defined; R5 is Valine or a fluorinated analog of valine; and R11 is N-methyl-valine or a fluorinated analog of N-methyl-valine.

6. The composition of Claim 4 wherein R1 is MeBmt or dihydro-MeBmt; R2 is (a) 4,4-difluoro-Abu; (b) 4-fluoro-Abu; (c) 5-fluoro-Nva; (d) 4-fluoro-Nva; (e) 4-fluoro-Val; (f) Abu; (g) Nva; or (h) Thr; R , R and R6 to R10 are as previously defined; R5 is Val or 4-fluoro-Val; and Rll is Me Val or 4-fluoro-MeVal.

7. A method for the prevention, control or treatment of immunoregulatory disorders or diseases comprising the administration to a mammalian species in need of such treatment an effective amount of a compound of formula I

wherein R1 is MeBMT; or dihydro MeBMT; R2 is an amino acid residue selected from the group consisting of the residues of Nva (norvaline), Thr, Ser, and Val, or a fluorinated analog thereof, wherein a fluorinated analog represents the amino acid residue in which one or more of the various C-H bonds in the side chain are replaced with C-F bonds; R3 is Sar or D-MeAla; R4, R6, R9, and R10 are MeLeu or a fluorinated analog thereof; R5 is Val or a fluorinated analog thereof; R7 is Ala or a fluorinated analog thereof; R8 is D-Ala; and Rll is MeVal or a fluorinated analog thereof, With the proviso that at least one of R to R is a fluorinated analog of an amino acid.

8. The method of Claim 7 wherein R1 is MeBMt or dihydroMeBMt; R2 is (a) a fluorinated analog of L-alanine; (b) a fluorinated analog of L-2-amino-butyric acid; (c) a fluorinated analog of L-norvaline; (d) a fluorinated analog of L-valine; or (e) L-2-aminobutyric acid, L-norvaline, or L-threonine; R , R and R6 to R10 are as previously defined; R5 is Valine or a fluorinated analog of valine; and R11 is N-methyl-valine or a fluorinated analog of N-methyl-va line.

9. The method of Claim 7 wherein R1 is MeBmt or dihydro-MeBmt; R2 is (a) 4,4-difluoro-Abu; (b) 4-fluoro-Abu; (c) 5-fluoro-Nva; (d) 4-fluoro-Nva; (e) 4-fluoro-Val; (f) Abu; (g) Nva; or (h) Thr; R4 R and R6 to R10 are as previously defined; R5 is Val or 4-fluoro-Val; and Rll is Me Val or 4-fluoro-MeVal.

10. A process for the preparation of a compound of formula (I) according to Claim 1.

wherein R1 is MeBMT; or dihydro MeBMT; R2 is an amino acid residue selected from the group consisting of the residues of Nva (norvaline), Thr, Ser, and Val, or a fluorinated analog thereof, wherein a fluorinated analog represents the amino acid residue in which one or more of the various C-H bonds in the side chain are replaced with C-F bonds; R3 is Sar or D-MeAla; R4, R6, R9, and R10 are MeLeu or a fluorinated analog thereof; R5 is Val or a fluorinated analog thereof; R7 is Ala or a fluorinated analog thereof; R8 is D-Ala; and Rll is MeVal or a fluorinated analog thereof.

comprising treating a compound of formula H-D-Ala-MeLeu-MeLeu-MeVal-MeBMT

with a cyclization reagent.

11. A fluorinated amino acid selected from a group consisting of (a) 4,4-difluoro-L-aminobutyric acid; (b) 4-fluoro-L-valine; (c) 4-fluoro-L-methyl-valine; (d) 4-fluoro-L-norvaline; (e) 5-fluoro-L-norvaline; (f) 4-fluoro-L-aminobutyric acid.