IE47971B1 - Halogen substituted mercaptoacylamino acid - Google Patents

Abstract

Compounds which have the general formula (I> wherein R is hydrogen, lower alkanoyl or m is 1 or 2; n is 0 or 1; R1 is hydrogen or lower alkyl; R2 and R'2 each is hydrogen or halogen; R3 is hydrogen, lower alkyl or CF3, and also halogen when n is 1; R4 is hydrogen, lower alkyl or trifluoromethyl; R6 is hydrogen, and also halogen when m is 1; at least one of R2, R'2, R3, R4 and R6 being halogen or CF3 as represented by the symbols and only R2 and R'2 can both be halogen at the same time, and basic salts thereof may be used as hypotensive agents.

Description

This invention provides compounds which have the general formula (I) * wherein R is hydrogen, lower alkanoyl or Ro R ’ 3 \r/ 2 h2c (CH), CO— N(JWm - CH-COOR m is 1 or 2; n is 0 or 1; R^ is hydrogen or lower alkyl; R2 and R'2 each is hydrogen or halogen; R^ is hydrogen, lower alkyl or CF^, and also halogen when n is 1; R4 is hydrogen, lower alkyl or trifluoromethyl; Rg is hydrogen, and also halogen when m is 1; with the provisos that at least one of R2, R'2, R3, and Rg is halooun or CF3; only R2 and R'2 can both be halogen at the same time; and, 'vhen R3 is trifluoromethyl, at least one of R2 and R'2 is halogen. When is hydrogen, these rarpounds may be in the form of salts formed with a base. Ihe asterisk indicates an asymmetric carbon atom. - 3 The invention thus concerns halogenated derivatives of mercaptoacyl proline and mercaptoacyl pipecolic acids having the formula I above.

With respect to the prolines (when m is 1) two preferential groups of compounds within formula I are those having the following formulas (II) wherein R is hydrogen, lower alkanoyl or R1 is hydrogen or lower alkyl; R^ and R'2 each is hydrogen or fluorine; R^ and R^ each is hydrogen or trifluoromethyl, one being hydrogen and the other trifluoromethyl; and n is 0 or 1 ; with the proviso that, when is trifluoromethyl, at least one of R2 and R'2 is halogen; (III) R— S -(CH)-— CH — CO- N-lCOOR1 - 4 wherein R and R^ have the same meaning as defined above for formula II; R2 and R'2 each is hydrogen or halogen; R3 is hydrogen, halogen or lower alkyl, R3 being t halogen when both R2 and R 2 are hydrogen, R3 being other r * 3 than halogen when R2 or R 2 is halogen; R^ is hydrogen; and n is 0 or 1; and salts of said compounds of formula II and III, respectively.

Thus in the case of formula II, when n is 1 10 either R^ or R4 is trifluoromethyl and the other is hydrogen. That is to say there is one trifluoromethyl oroup in the acyl side chain of the molecule. It is on the carbon a to the carbonyl group (R3 = CFj) when n is 0. It is on either the carbon a to the carbonyl group (R3 = CF3, R^ = H) or on the carbo β t.o the carbonyl group (R3 = H, R^ = CFj) when n is 1, the ether of the pair of symbols (Rj, R^) is then hydrogen. When is trifluoromethyl, R2 and R'2each is hydrogen or halogen, but when R3 is trifluoromethyl at least one of R2 and R'2 is halogen.

In the case of formula III, preferably one or both of R2 and R'2 are halogen and R3 and R^ each is hydrogen or lower alkyl, or both R2 and R'2 are hydrogen, R3 is halogen, preferably chlorine or bromine, and is hydrogen.

Preferred particularly are those compounds of formula 1 wherein R is hydrogen or lower alkanoyl, 79 7i especially hydrogen or acetyl; R1 is hydrogen or lower alkyl, especially hydrogen; R2 and R'2 each is hydrogen or halogen, especially hydrogen or fluorine; and n is 0 or 1, especially 1.

One or two halogens can be present on the pyrrolidine ring. A single halogen can be on either the carbon in the 3-position or the carbon in the 4-position. Two halogens can be present in the 4position and preferably they are the same. Fluorine is preferred on this ring, especially one or two fluorine atoms on the carbon in the 4-position.

In the case of the pipecolic acids (m is 2), those compounds of formula 1 are preferred wherein R is hydrogen or lower alkanoyl, especially hydrogen or acetyl; R^ is hydrogen or lower alkyl, especially hydrogen; R2 and R‘2 each is hydrogen or halogen especially hydrogen or fluorine; one of Rj and R^ is CF3 and the other is hydrogen; n is 0 or 1, especially 1. When R4 is CF3, both R2 and R'2 are preferably hydrogen.

The L-configuration for the prolines or pipecolic acids is especially preferred.

The lower alkyl groups represented by any of the variables are straight and branched chain hydrocarbon radicals from methyl to heptyl, for example, 4797 1 methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl and the like. The C^-C^ members, especially and C2 members, are preferred The lower alkanoyl groups are those having the 5 acyl radicals of the lower (C2-C?) fatty acids, for example, acetyl, propionyl, butyryl and the like. Similarly, those lower alkanoyl groups having up to four carbons, and especially acetyl, are preferred.

The halogens are the four common halogens, chlorine, bromine and fluorine being preferred, especially fluorine.

The products of formula I can be produced by various methods of synthesis.

In general, these compounds can be synthesized 15 by coupling the acid of the formula (IV) R4 R3 I I R-S — (CH) -- CH-COOH to the amino acid of the formula HN--CH -COOR^ by any method which can be used to form amide bonds. See, for example, Methoden der Organischen Chemie (Houben-Weyl) part I, p. 376 et seq., part III, ρ. 1 et seq. (1974). ‘17 9 71 The acids of formula XV, when n is 1 can be obtained by the addition of a thioacid R-SH to a suitably substituted acrylic acid. As a temporary protection of the mercapto group in compounds of formula IV, R can be a p-methoxy-benzyl group. This group is then removed with trifluoroacetic acid and mercuric acetate. The acids of formula IV, when n is 0, are obtained by a displacement reaction using a thioacid R-SH and a 2-halo acid.

According to one method, preferred when n is 0, an acid of formula V is coupled with a haloalkanoic acid of the formula (VI) X— (CH)--CH-COOH wherein X is halogen, preferably chlorine or bromine, by one of the known procedures in which the acid VI. is activated, prior to reaction with the acid V, involving formation of a mixed anhydride, symmetrical anhydride, acid chloride, active ester, or use of Woodward reagent K, EEDQ (N-ethoxy-carbonyl-2-ethoxy-l,2-dihydroquinoline) or the like.

The product of this reaction is a compound of the formula (VIX) R' h2c X-(CH), CH--CO—NCOOR, 2 4797 1 This product is subjected to a displacement reaction with the anion of a thioacid of the formula (VIII) R?-CO-SH wherein is of the formula (IX) lower alkyl yielding a product R' R? — CO —S — R. R, H0C (CHR,)m I III {CH) -jj— CH—CO - N-L COORj which can then be converted to the product (X) HS- (CH) —CH— CO—Nn ^'m COOR, by conventional alkaline hydrolysis or anmonolysis. When is an ester group (i.e., R^ is lower alkyl, obtained when an ester of the starting acid V is used),the ester group can be removed by conventional techniques. For example, when is tert-butoxy or tert-amyloxy, treatment of the ester of formula IX or X with trifluoroacetic acid and anisole will give the corresponding free acid. When other alkoxy groups are present alkaline hydrolysis will yield the corresponding acid.

When an acid of formula V is used as starting material, or the final product is obtained as the free carboxylic acid, this acid can be converted to its ester, for example, by esterification with a diazoalkane, such as diazomethane, l-alkyl-3-p-tolyl-triazene, such as 1-nbutyl-3-p-tolyltriazene or the like.

According to another variation, an ester, preferably the methyl or t-butyl ester, of formula V, in an anhydrous medium such as dichloromethane, tetrahydrofuran, dioxane or the like, is treated with an acylthioalkanoic acid of the formula (XI) R.

Ry- CO- S — (CH) — CH — COOH in the presence of dicyclohexylcarbodiimide, N,Ν'-carbonylbisimidazole, ethoxyacetylene, diphenylphosphoryl azide or similar coupling agents at a temperature in the range of about to 10°C. The ester group can then be removed for example, by treatment with trifluoroacetic acid and anisole at about room temperature to yield the free acid (R^ = H).

A variation, preferred when n is 1, R^ is CF3 and R3 is H, is to react a thioacid of formula VIII with an acrylic acid derivative of the formula (XII) H.

R. 2.

CH - CH- C- N CHR,) o m *— COOR-l instead of with the compound of formula VII, and then continue as described above. The compounds of formula XII are obtained from 3-trifluoromethylacrylic acid and an ester of formula V by the method described in Example 14 below. 4-7971 - 10 10 Compounds of formula I wherein R is R2 r<2 V *4 |3 H2^ 'Wm -s (CH) — CH -- CO — N-«—COOR^ are produced by direct oxidation of a compound of formula I in which R is hydrogen, e.g., with iodine, to obtain the symmetrical bis compound.

Halogenated oanpounds of formula V which are used as starting materials can be produced by methods known in the art, e.g.. Biochemistry £, 2509 (1965), Aust. J. Chem. 20, 1493 (1967), J. Amer. Chem. Soc, 86, 4709 (1964), J. Med Chem. , 1176 (1977).

Products of formula I -have one or more asymmetric centers, the basic being indicated by an asterisk in formula I. The compounds accordingly exist in stereoisomeric forms or in racemic mixtures thereof. All of these are within the scope of the invention. The above described synthesis can utilize the racemate or one of the enantiomers as starting material. When the racemic starting material is used in the synthetic procedure, the stereoisomers obtained in the product can be separated by conventional chromatographic or fractional crystallization methods. In general, the L-isomer with respect to the basic asymmetric carbon constitutes the preferred isomeric form.

The compounds of this invention form salts with various inorganic and organic bases which are also within the scope of the invention. Such - 11 salts include ammonium salts, alkali metal salts like sodium and potassium salts (which'are preferred), alkaline earth metal salts such as the calcium and magnesium salts, salts with organic bases, e.g., dicyclohexylamine salts, benzathine, N-methy1-D-glucamine, hydrabamine salts, salts with amino acids such as arginine, lysine and the like.

The non-toxic, physiologically acceptable salts are preferred, although other salts are also useful, e.g., in isolating or purifying the product.

The salts are formed in conventional manner by reacting the free acid form of the product with one or more equivalents of the appropriate base providing the desired salt ion, in a solvent or medium in which the salt is insoluble, or in water and removing the water by freeze drying. By neutralizing the salt with an insoluble -acid such as a cation exchange resin in the hydrogen form (e.g., polystyrene sulfonic such as Dowex (Trade Mark) 50 ) or with an aqueous acid and extraction with an organic solvent, e.g., ethyl acetate, dichioromethane or the like, the free acid form can be obtained, and, if desired, another salt formed.

Additional experimental details are found in the examples which are preferred embodiments and also serve as models for the preparation of other compounds.

The compounds of this invention are used as hypotensive agents. They inhibit the conversion of the decapeptide angiotensin I to angiotensin XI and therefore are useful in reducing of relieving 4797 1 - 12 angiotensin related hypertension. The action of the enzyme renin on angiotensinogen, a'pseudoglobulin in blood plasma, produces angiotensin I. Angiotensin I is converted by angiotensin converting enzyme (ACE) to angiotensin II. The latter is an active pressor substance which has been implicated as the causative agent in various forms of hypertension in various mammalian species, e.g., rats and dogs. The compounds of this invention intervene in the angiotensinogen -* (renin) ·+ angiotensin I + (ACE) + angiotensin II sequence by inhibiting angiotensin converting enzyme and reducing or eliminating the formation of the pressor substance angiotensin II. Thus by the administration of a composition containing one or a combination of compounds of formula I or a physiologically acceptable salt thereof, angiotensin dependent hypertension in the species of mammal suffering thereform is alleviated. A single dose, or prefera20 bly two to four divided daily doses, provided on a basis of about 0.1 to 100 mg. per kilogram per day, preferably about 1 to 50 mg. per kilogram per day.is appropriate to reduce blood pressure as indicated in the animal model experiments described by S.L. Engel, T.R. Schaeffer, Μ. H.Waugh and B. Rubin, Proc. Soc. Exp. Biol. Med. 143 (1973). The substance is preferably administered orally, but parenteral routes such as subcutaneously, intramuscularly, intravenously or intraperitoneally can also be employed.

The invention thus extends to a pharmaceutical composition comprising a compound of the invention and a pharmaceutical carrier. The a impounds of this invention can be utilized to achieve the reduction of blood oressure by - 13 formulating in compositions such as tablets, capsules or elixirs for oral administration or in sterile solutions or suspensions for parenteral administration. About 10 to 500 mg. of a compound or mixture of compounds of formula I or physiologically acceptable salt is compounded with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavor, etc., in a unit dosage form as called for by accepted pharmaceutical practice. The amount of active substance in these compositions or preparations is such that a suitable dosage in the range indicated is obtained.

Illustrative of the adjuvants which may be incorporated in tablets, capsules and the like are the following: a binder such as gum tragacanth, aoacia, corn starch or gelatin; an excipient such as dicalcium phosphate or microcrystalline cellulose; a disintegrating agent such as corn starch, potato starch, alginic acid and the like, a lubricant such as magnesium stearate; a sweetening agent such as sucrose, lactose or saccharin; a flavoring agent such as peppermint, oil of Wintergreen or cherry. When the dosage unit form is a capsule, it may contain in addition to materials of the above type a liquid carrier such as a fatty oil. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propyl parabens as preservatives, a dye and a flavoring such as cherry or orange flavor. 4797 1 - 14 Sterile compositions for injection can be formulated according to conventional pharmaceutical practice bydissolving or suspending the active substance in a vehicle such as water for injection, a naturally occurring vegetable oil like sesame oil, coconut oil, peanut oil, cottonseed oil, etc.

The following examples are illustrative of the invention and constitute especially preferred embodiments.

All temperatures are in degrees Celsius.

Preparation 1 3-Acetylthio-2-trifluoromethylpropanoic acid A mixture of thiolacetic acid (50 g.) and 2(trifluoromethyl) acrylic acid [M.W. Buxton, et al.

J. Chem. Soc., 366 (1954)] (66 g.) is heated on the steam bath for one hour and then stored at room temperature for eighteen hours. The reaction mixture is distilled in vacuo to give 3-acetylthio-2-trifluoromethylpropanoic acid.

Preparation 2 2-Bromo-3,3,3-trifluoropropanoic acid 3,3,3-Trifluoroalanine (88 g.) is dissolved in a mixture of potassium bromide (250 g.) and 2.5 N sulfuric acid (1.240 ml.). The solution is chilled to 0° with an ice-salt bath and sodium nitrite (65.5g). is added in small portions over a one hour period with vigorous stirring. the reaction mixture is stirred in the cooling bath for another hour and then extracted with ether. The organic layer is washed with water, dried over magnesium sulfate and concentrated to dryness in vacuo to yield 2-bromo-3,3,330 trifluoropropanoic acid. - 15 Preparation 3 2-Bromo-3,3,3-trifluoropropanoic acid chloride A solution of 2-bromo-3,3,3-trifluoropropanoic acid (5 g.) in thionyl chloride (5 ml.) is refluxed in the steam bath for two hours. The excess thionyl chloride is removed in vacuo, and the residue distilled under reduced pressure to yield 2-bromo-3,3,3-trifluoropropanoic acid chloride.

Example 1 1- (2-Acetylthio-3,3,3-trifluoropropanoyl)-L-proline To a solution of L-proline (5.75 g.) in IN sodium hydroxide (50 ml.), chilled in an ice-water bath, 2- bromo-3,3,3-trifluoropropanoic acid chloride (12 g.) is added and the mixture is vigorously stirred at room temperature for three hours. A solution of thiolacetic acid (4 ml.) and potassium carbonate (4.8 g.) in water (50 ml.) is added and the mixture is stirred at room temperature for sixteen hours. After extraction with ethyl acetate, the aqueous layer is acidified with concentrated hydrochloric acid and extracted again with ethyl acetate. This last organic phase is dried over magnesium sulfate and concentrated to dryness in vacuo.

The residue is chromatographed on a silica gel column with a mixture of benzene-acetic acid (7:2) to yield 1-(2acetylthio-3,3,3-trifluoropropanoyl-)-L-proline.

Example 2 1-(2-Mercapto-3,3,3-tri£luoropropanoyl)-L-proline 1-(2-Acetylthio-3,3,3-trifluoropropanoyl)-L-proline (4 g.) is dissolved in a mxiture of water (8 ml.) and concentrated ammonia (8 ml.) under a blanket of nitrogen. - 16 After thirty minutes at room temperature, the reaction mixture is acidified and extracted with ethyl acetate.

The organic phase is dried over magnesium sulfate and concentrated to dryness in vacuo to yield 1 -(2-mercapto5 3,3,3,-trifluoropropanoyl)-L-proline Example 3 1- (3-Acetylthio-2-trifluoromethylpropanoyl)-4,4difluoro-L-proline To a solution of 4,4-difluoro-2-proline (7.5 g.) in N sodium hydroxide (50 ml.) chilled in an ice-water bath, 3-acetylthio-2-trifluoromethylpropanoic acid chloride (prepared from 3-acetylthio-2-trifluoromethylpropanoic acid and thionyl chloride by the procedure of Example 6,) (12 g.) is added and the mixture is vigorously stirred at room temperature for two hours. After acidification with concentrated hydrochloric acid, the aqueous mixture is extracted with ethyl acetate. The organic phase is dried over magnesium sulfate and concentrated to dryness to yield 1-(3-acetylthio-2-trifluoromethyIpropanoyl) 4,4-difluoro-L-proline.

Example 4 1-(3-Mercapto-2-trifluoromethylpropanoyl)-4,4-difluoro-Lproline By substituting 1-(3-acetylthio-2-trifluoromethylpropanoyl)-4,4-difluoro-L-proline for the 1 -(3-acetylthio-2trifluoromethylpropanoyl)-L-proline in the procedure of Example 4, 1 -(3-mercapto-2-trifluoro-47971 - 17 methylpropanoyl)-4,4-difluoro-L-proline is obtained. Example 5 1,1'-(Dithiobis-(2-trifluoromethyl-3-propanoyl)]bis-4,4-difluoro-L-proline By substituting 1-(3-mercapto-2-trifluoromethylpropanoyl)-4,4-difluoro-L-proline for the l-(3-mercapto-2-trifluoromethylpropanoy1) - Lproline in the procedure of Example 9, 1,1[dithiobis-(2-trifluoromethyl-3-propanoyl)]-bis10 4,4-difluoro-L-proline is obtained.

Preparation 4 I-(4,4,4-Trifluoro-2-butenoyl)-L-proline Boric anhydride (7.0 g., 0.1 mole)(prepared by fusing boric acid in a platinum crucible and IS crushing under nitrogen)is combined with ethyl 3-hydroxy-4,4,4~trifluorobutanoate (32.2 g., 0.173 mole) in a 50 ml. flask equipped with a Dean-Stark trap and the mixture is heated at 180° with a salt bath until all of the anhydride dissolves (6 hours). The heat is increased to 350° during which time 23 ml. of distillate accumulates in the trap. The distillate is returned to the reaction flask and the heating step is repeated. This process is repeated 4 times to assure complete dehydration of the hydroxy ester.

The distillate is dissolved in petroleum ether, dried over phosphorous pentoxide and distilled, yielding 10 g. of 4,4,4-trifluoro-2-butenoic acid ethyl ester (b.p. 115-120°) and 650 mg. of 4,4,4-trifluoro-2-butenoic acid (b.p. 150°, 53-55° recrystallization from pentane). 7 9 7 1 - 18 10 The ester is combined with 10% aqueous sodium hydroxide (24 ml.) and stirred at 25° for 6 hours.

The mixture is diluted with water and extracted with methylene chloride to remove unchanged material.

The aqueous layer is adjusted to pH 3 with concentrated hydrochloric acid and this mixture is extracted with methylene chloride ( 3 x 50 ml.). The organic layers are combined, dried over sodium sulfate, concentrated and the residue distilled giving crystalline 4,4,4-trifluoro-2-butenoic acid (b.p. 145-153°) Recrystallized from pentane, the acid melts at 54-55°, yield 4.6 g.

A mixture of the 4,4,4-trifluoro-2-butenoic acid (4.91 g., 35 mmole), hydroxybenzotriazole (4.73 g., 35 mmole), L-proline-t-butyl ester (6.00 g., 35 mmole) and dicyclohexylcarbodiimide (7.22 g., 35 mmole) in methylene chloride (200 ml.) is stirred under nitrogen overnight at room temperature. The mixture is filtered, the filtrate washed with 5% sodium bisulfate (2 x 50 ml.) and saturated sodium bicarbonate (2 x 50 ml.), dried over sodium sulfate and concentrated to yield an oil. This is dissolved in ether and the solution is chilled and filtered free of precipitate. The filtrate is concentrated, yielding a solid ( m.p. 95-100°, 8.7 g.) which shows a single spot by TLC (silica gel EM 50/50, EtOAc/CH2Cl2, Rf = .85).

A mixture of the above obtained l-(4,4,4-trifluoro-2-butenoyl)-L-proline t-butyl ester (4.0 g., 13.6 mmole) is mixed with trifluoroacetic acid (60 ml.) and anisole (13 ml.) and stirred under nitrogen for one hour. The solvents are removed - 19 under vacuum and the residue, dissolved in ether (10 ml.), is poured into pentane (500 ml.). This precipitation technique is repeated and the o residue allowed to stand at 0 for 72 hours during which time crystallization occurs. The 1-(4,4,4trifluoro-2-butenoyl)-L-proline is recrystallized from ethyl acetate-hexane; yield 2.48 g., m.p. 119-120°.

Example 6 1-(3-Mercapto-4,4,4-trifluorobutanoyl)-L-proline Thiolacetic acid (1.5 ml.) is combined with 1-(4,4,4-trifluoro-2-butenoyl)-L-proline (720 mg., mmole) under argon and the mixture stirred at room temperature overnight. The excess thiolacetic acid is removed under vacuum and the residual 1-(3-acetylthio-4,4,4-trifluorobutanoyl) L-proline is mixed with aqueous ammonia (15 ml. cone. NH^ + 15 ml. water) and stirred for 2 hours at room temperature. The mixture is then diluted with ice and acidified with concentrated hydrochloric acid. The acid mixture is extracted with methylene chloride (3 x 50 ml.), the extracts dried over sodium sulfate and concentrated to yield an oil. This is purified by dissolving in water (double distilled), treating the solution with carbon and filtering through a millipore filter (0.4 m followed by 0.08 m). Lyophilization of this solution gives 700 mg. of 1-(3-mercapto-4,4,4trifluorobutanoyl)-L-proline as a colorless glass.

Rf (benzene:acetic acid 7:1) 0.24 - 20 Preparation 5 3-Acetylthio-4 ,4,4-trifluorobutanoic acid chloride By substituting 4,4,4-trifluoro~2-butenoic acid for the 2-trifluoromethyl acrylic acid in the procedure of Example 1, 3-acetylthio-4,4,4-trifluorobutanoic acid is obtained, then chlorinating with thionyl chloride as in Example 6, 3-acetylthio-4,4,4trifluorobutanoic acid chloride is obtained.

Example 7 1(3-Mercapto-4,4,4-trifluorobutanoyl)-4,4difluoro-L-proline By substituting 1-(3-acetylthio-4,4,4-trifluorobutanoic acid chloride for the 3-acetylthio-2trifluoromethylpropanoic acid chloride in the procedure of Example 3 and then submitting the product to the procedure of Example 4, of British Patent Specification Mo. 2014987A, 1-(3-mercapto-4,4,4-trifluorobutanoyl)-4,4-difluoro-L-proline is obtained.

Example 8 1,1'-[Dithiobis-(4,4,4-trifluoro-3-butanoyl)]-bis-Lproline By substituting 1-(3-mercapto-4,4,4-trifluorobutanoyl)-L-proline for the 1-(3-mercapto-2-trifluoromethylpropanoyl)-L-proline in the procedure of Example 11, 1,1[dithiobis-(4,4,4-trifluoro-3butanoyl)1-bis-L-proline is obtained.

Preparation 6 cis-4-Pluoro-L-proline, hydrobromide a) N-Carbobenzyloxy-4-hydroxy-L-proline, methyl ester 30 N-Carbobenzyloxy-4-hydroxy-L-proline [12.4 g. (0.047 mole)] is esterified with diazomethane in dioxane-ether as described in JACS, 79, 191 (1957). - 21 To avoid freezing of the dioxane the addition of Ό the diazomethane solution is begun at 10 and completed at 0-2°. The yield of nearly colorless viscous oil is 14.6 g. (100%). b) N-Carbobenzyloxy~4-tosyloxy-L-proline, methyl ester A stirred solution of 14.5 g. (0.052 mole) of N-carbobenzyloxy-4-hydroxy-L-proline, methyl ester in 30 ml. of pyridine is treated dropwise at -5° to -8 with a solution of 11 g. (0.058 mole) of tosyl chloride in 15 ml. of pyridine. The pale yellow solution is stored in the cold for 3 days, then added with stirring to 300 ml. of ice-cold 2 N hydrochloric acid. The precipitated gum is extracted with 200 ml. of chloroform. The aqueous phase is extracted with additional chloroform (3 x 100 ml.). The organic·layers are combined, dried (MgSO^), and the solvent evaporated to give a pale yellow viscous oil. The oil is dissolved in 100 ml. of methanol and diluted to 400 ml. with water to precipitate the product as an oil which gradually crystallizes on seeding, rubbing, and cooling: yield 17.4 g. (77%); m.p. 62-65°.

Following crystallization from 85 ml. of isopropanol, the colorless solid N-carbobenzyloxy-4-tosyloxyL-proline, methyl ester weighs 15.9 g. (70%); m.p. 67-69°; -30° ( c = 1; methanol), c) cis-N-Carbobenzyloxy-4-fluoro-L-proline, methyl ester A stirred suspension of 19.1 g. (0.044 mole) of N-carbobenzyloxy-4-tosyloxy-L-proline, methyl ester in 100 ml. of redistilled diethylene glycol o is treated at 42 (under argon) with 19.1 g. - 22 (0.33 mole) of anhydrous potassium fluoride and the resulting solution is heated at 81-*84° for 20 hours. After cooling, the light yellow solution is worked up to give 18.6 g. (100%) of cis-N5 carbobenzyloxy-4-fluoro-L-proline, methyl ester as a light yellow oil. d) cis-N-Carbobenzyloxy -4-fluoro-L-proline The cis-N-carbobenzyloxy-4-fluoro-L-proline, methyl ester (18.4 g., approximately 0.044 mole) jQ is dissolved in 140 ml. of methanol, treated dropwise at -1° to 4° with 33 ml. (0.066 mole) of 2 N sodium hydroxide, then kept at 0° for one hour, and at room temperature overnight. After removing about 1/2 of the solvent on a rotary evaporator, the solution is diluted with 300 ml. of water, washed with ether (wash discarded), acidified while cooling with 12.5 ml. of 1:1 hydrochloric acid to pH 2, and extracted with ethyl acetate (4 x 150 ml.) The extracts are combined, washed with 100 ml. of saturated sodium chloride solution, dried (MgSO^) and the solvent evaporated to give 13.8 g. of a pale yellow viscous oil. The latter is dissolved in 60 ml. of ethanol, treated with 5.1 g. of cyclohexylamine in 10 ml. of ethanol and diluted to -900 ml. with ether. On seeding and rubbing, crystalline cis-N-carbobenzyloxy-4-fluoro-L-proline, cyclohexylamine salt separates: weight after cooling overnight, 11.0 g., m.p. 180-183° (s. 175°).

Following crystallization from 70 ml. of ethanol, the colorless solid weighs 7.6 g., m.p. 185-187°, [a]33 -40° (c = 1; methanol). - 23 The cyclohexylamine salt is suspended in 75 ml. of ethyl acetate, stirred, and treated with 45 ml. of hydrochloric acid. The layers are separated, the aqueous phase is extracted with additional ethyl acetate (2 x 75 ml.), then the combined organic layers are dried (MgSO^), and the solvent evaporated. The residual free acid, cis-N-carbobenzyloxy-4fluoro-L-proline crystallizes when finally dried at 0.2 mm. and 45°; yield 5.7 g. (49%); m.p. 116-118°. e) cis-4-Fluoro-L-proline, hydrobromide The cis-N-carbobenzyloxy-4-fluoro-L-proline (5.5 g., 0.021 mole) is treated with 28 ml. of hydrogen bromide in acetic acid (30-32%), stoppered loosely, and stirred for one hour. Ether (300 ml.) is added to the yellow mixture and when the crystalline product has settled the ethereal liquor is decanted and the material washed with 300 ml. of fresh ether by decantation. The product is I finally heated in the steam bath with 70 ml. of methyl ethyl ketone, cooled for two hours, washed with cold methyl ethyl ketone and with ether, and dried in vacuo. The yield of nearly colorless solid, cis-4-fluoro-L-proline, hydrobromide is 3.8 g, (86%), m.p. 189-191° (dec.), [a]36 -19° (c = 1, methanol).

A portion of the crude hydrobromide salt is converted to the free acid by passing through a column of Dowex 1-X8 ion exchange resin. - 24 Example 9 cis-1-|D— 3—(Acetylthio)-2-nielhylpropan»yl1-4fluoro-L-prolinc cis-4-Fluoro-L-proline, hydrobromide (4.5 g., 0.021 mole) and 4.2 g. (0.023 mole) of D-3-acetylthio-2-methylpropanoic acid chloride are reacted in 50 ml. of water in the presence of sodium carbonate to stabilize the pH at 8.0 - 8.2 during the acylation (approximately 20 minutes). The mixture is worked up after an additional hour by washing with ethyl acetate (2 x 50 ml.),layering over with ethyl acetate, acidifying with hydrochloric acid to pH 2, saturating with sodium chloride and then separating the layers. The aqueous phase is extracted with additional ethyl acetate and the organic layers are combined, dried and evaporated. The solid residue from the ethyl acetate evaporation is rubbed under ether and the evaporation repeated; weight of colorless product, 5.4 g. (93%), m.p. 146-148° (s,133°) 2o [a]^ 1^2° (c = 1; methanol). The dicyclohexylamine salt is prepared by adding dicyclohexylamine to the cis-1-[D-3-(acetylthio)-2-methylpropanoyl-4-fluoro-Lproline in 70 ml. of ethyl acetate. 8.1 gm. of salt, which crystallizes out, are obtained, m.p. 202-204° (s. 187°); [a]^ "72° (c = 1; methanol).

Crystallization from 90 ml. of isopropanol gives 7.0 g., m.p. 205-207° (s, 190°). [a]^6 -74°.

A sample recrystallized from ethanol shows no further change in m.p. of [α]ρ26.

The dicyclohexylamine salt (16.9 g.) is converted back to the free acid by distribution between 10% potassium bisulfate and ethyl acetate (60 ml. - 25 10% KIISO^; 4 x 50 ml. ethyl acetate extractions).

The organic layers are combined and evaporated to dryness to obtain 4.1 g. (71%) of colorless free acid, m.p. 154-156° (s. 140°) fct]Z6 -142° (c = 1; methanol).

Example 10 cis-4-Fluoro-l-(D-3-mercapto-2-methylpropanoy1)-Lproline cis-1-[D-3-(Acetylthio)-2-methylpropanoyl]-4fluoro-L-proline (3.9 g., 0.014 mole) is hydrolyzed in 22 ml. of water containing 9 ml. of concentrated ammonium hydroxide. The reaction mixture is acidified with hydrochloric acid and extracted with ethyl acetate. The organic layer is concentrated to dryness to give 3.3 g. of glass-like product which slowly crystallizes when dried at 0.2 mm. and 50°. The material is triturated with·20 ml. of ethyl acetate (with slight warming under argon), diluted with 25 ml. of hhxane, rubbed, and cooled overnight (under argon). Following filtration under argon, washing with hexane, and drying in vacuo, the colorless solid cis-4-fluoro-1-(D-3-mercapto-2-methylpropanoyl)-Lproline weighs 2.8 g. (85%), m.p. 135-137° (s. 129°), [<χ]θ6 -116° (c = 1; methanol).

Example 11 1-[3-(Acetylthio)-2-chloropropanoyl]-L-proline (Isomer A) L-Proline (1.44 g.) and sodium carbonate (667 mg.) are dissolved in 17 ml. of water and stirred in an ice bath. To this sodium carbonate (2 g.) in 8.5 mg. of water is added, followed immediately by 3-acetylthio-2-chloropropanoic acid chloride (2.5 g.). 79 71 - 26 The ice bath is removed. After 30 minutes a precipitate forms which is solubilized with the addition of 17 ml. of water. After a total of 1.5 hours, the reaction mixture is extracted twice with ethyl acetate.

The aqueous layer is chilled, acidified with concentrated hydrochloric acid, saturated with sodium chloride, extracted into ethyl acetate, dried over magnesium sulfate and concentrated to dryness in vacuo to obtain the product as a crude oil, yield 3.3 g.

The oil is applied to a 100 g. silica gel column and eluted with benzene/acetic acid 7:1 to yield 2 g. of product which is crystallized from water to yield 450 mg. of 1-[3-(acetylthio)-2-chloropropanoyl]-L-proline, m.p. 111-113 . [α]β -170 (c = 1; ethanol) Example 12 1-[3-(Acetylthio)-2-chloropropanoyl]-L-proline (Isomer B) The aqueous mother liquors from Example 22 are 20 lyophilized and chromatographed on silica gel with benzene/acetic acid 7:1. The fractions containing the UV absorbing material and shown to be homogensous by TLC are pooled, concentrated to dryness and crystallized from water, yield 800 mg., m.p. 90-109° -4° (c = 2.1; ethanol). The mother liquors are concentrated to dryness by freeze-drying and the residual 1-[3-(acetylthio)-2-chloropropanoyl-Lproline, (Isomer B) is crystallized from ethero 25 o hexane; yield 380 mg., m.p. 108-110 , [α]θ +17.6 (c = 1.25; ethanol). - 27 Example 13 1- [3- (Acetylthio)-2-byoniopropanoyl]-L-proline By substituting 3-acetylthio-2-bromopropanoic acid chloride for the 3-acetylthio-2-chloropropanoic acid chloride in the procedure of Example 11 1-[3-(acetylthio)-2-bromopropanoyl]-L-proline is obtained, m.p. 109-110°, [a)D25 - 162° (c = 1.39; ethanol).

Example 14 cis-4-Chloro-l-(P-3-mercapto-2-methylpropanoyb-Lproline By substituting cis-4-chloro-L-proline [Aust.

J. Chem. 20, 1493 (1967)] for the cis-4-fluoro-Lproline, hydrobromide in the procedure of Example 20 and then submitting the product to the procedure of Example 10 cis-l-[D-3-(acetylthio)-2-methylpropanoyl-4-chloroproline and cis-4-chloro-l-(D-3mercapto-2-methylpropanoyl)-L-proline are obtained.

Example 15 trans-4-Bromo-l-(D-3-mercapto-2-methylpropanoyl)-Lproline By substituting trans-4-bromo-L-proline [Aust.

J. Chem. 20, 1493 (1967)] for the cis-4-fluoro-Lproline hydrobromide in the procedure of Example 9 and then submitting the product to the procedure of Example 10 trans-l-[D-3-(acetylthio)-2-methylpropanoylb 1-bromo-L-proline and trans-4-bromo-l- (D3-mercapto-2-methylpropanoyl)-L-proline are obtained.

Example 16 cis-4-Iodo-1-(D-3-mercapto-2-methylpropanoy 3b L-proline By substituting cis-4-iodo-L-proline for the cis-4-fluoro-L-proline hydrobromide in the procedure 4797 1 - 28 of Example 9, and then submitting the product to the procedure of Example 21, cis-1-[D-3-{acetylthio)2-methylpropanoyl-4-iodo-L-proline, and cis 4-iodo1-[D-3-mercapto-2-methylpropanoyl]-L-proline are obtained.

Example 17 crs-4-Fluoro-l-(3-mercapto-2-trifluoromethylpropanoyl)L-proline By substituting 3-acetylthio-2-trifluoromethyl10 propanoic acid chloride for the 3-acetylthio-2-methylpropanoic acid chloride in the procedure of Example q and then submitting the product to the procedure of Example 10, cis-1-[3-(acetylthio)-2-trifluoromethylpropanoyl]-4-fluoro-L-proline and cis-4-fluoro-l15 (3-mercapto-2-trifluoromethylpropanoyl)-L-proline, respectively, are obtained.

Preparation 6 cis-3-Fluoro-DL-proline, hydrobromide By substituting N-carbobenzyloxy-3-hydroxy-DL20 proline [J. Am. Chem. Soc. 85, 2824 (1963)] for the N-carbobenzyloxy-4-hydroxy-L-proline in the procedure of Preparation 6, cis-3-fluoro-DL-proline hydrobromide is obtained.

Example 18 cis-3-Fluoro-l-(D-3-mercapto-2-methylpropanoyU-DL-proline By substituting cis-3-fluoro-DL-proline hydrobromide for the cis-4-fluoro-DL-proline in the procedure of Example 9 and then submitting the product to the procedure of Example 10, cis-1-[D-3-(acetylthio)30 2-methylpropanoyl]-3-fluoro-DL-proline and cis-3-fluoro1-(D-3-mercapto-2-methylpropanoyl)-DL-proline are obtained. - 29 Example 19 cis-3-Chloro-l-(D-3-mercapto-2-mcthylpropanoylL-proline By substituting cis-3-chloro-L-proline [obtained from 3-hydroxyproline by the procedure described in Aust. J. Chem. 20, 1493 (1967)) for the cis-4fluoro-L-proline hydrobromide in the procedure of Example 9 and then submitting the product to the procedure of Example 10, cis-l-[D-3-(acetylthio)-2methylpropanoyl-3-chloro-L-proline, and cis-3-chloro1- (D-3-mercapto-2-methylpropanoyl)-L-proline are obtained.

Example 20 4,4-Dichloro-l-(D-3-mercapto-2-methylpropanoyl)L-proline By substituting 4,4-dichloro-L-proline [prepared from 4-keto-L-proline diketopiperazine and phosphorus pentachloride by the procedure described in J. Med. Chem. 20, 1176 (1977)) for the cis-4-fluoroL-proline hydrobromide in the procedure of Example 20 and then submitting the product to the procedure of Example 1-[D-3-(acetylthio)-2-methylpropanoyl]-4, 4-dichloro-L-proline and 4,4-dichloro-l-(D-3-mercapto2- methylpropanoyl,-L-proline are obtained.

Example 21 1,1'-[Dithiobis-(2-D-methylpropanoyl)]-bis-[(cis4-fluoro)-L-proline) By substituting cis-4-fluoro-1-(D-3-mercapto2-methyIpropanoyl)-L-proline for the 1-(3-mercapto2-trifluoromethylpropanoyl)-L-proline in the procedure of Example 9, of British Patent Specification No. 2014967a, 1,1'- [dithiobis-(2-D-methyl-propanovl)bis-[(cis-4-flucro)-L-proline] is obtained. 9 7 1 - 30 Example 22 4.4- Difluoro-1-(3-mercaptobutanoyl) -L-proline By substituting 3-acetylthiobutanoic acid chloride for the 3-acetylthio-2-trifluoromethy 1" propanoic acid chloride in the procedure of Example 3 and then submitting the product to the procedure of Example 4 , 1-(3-acetylthio-butanoy1)-4,4-difluoro-L-proline and 4.4- difluoro-1-(3-mercaptobutanoyl)-L-proline are obtained Preparation 7 3- (4-Methoxybenzyl)thio-2-trifluoromethylpropanoic acid A neat mixture of 1-trifluoromethylacrylic acid (3.9 g.) and 4-methoxybenzylthiol (4.3 g.) is stirred at 100-110° for one hour. The mixture is allowed to cool to room temperature and the solid is recrystallized from cyclohexane, m.p. 72-74°.

Preparation 8 3-Acetylthio-2-chloropropanoic acid chloride By substituting 2-chloroacrylic acid for the 20 2-trifluoromethylacrylic acid in the procedure of Preparation 1 and then allowing the product to react with thionyl chloride, 3-acetyithio-2-chloropropanoic acid and 3-acetylthio-2-chloropropanoic acid chloride are obtained. - 31 Preparation 9 D-3-Acetylthio-2-roethylpropanoic acid chloride A suspension of 1-(D-3~mercapto-2-methylpropanoyl)-L-proline, (150 g. 690 mmoles), in 1274 ml. of water and 426 ml. of concentrated hydrochloric acid (5.526 moles) is refluxed under nitrogen with stirring for 8 hours. The resulting solution is kept at room temperature overnight and then extracted with 400 ml. of chloroform (10 x). The combined chloroform extracts are dried over magnesium sulfate under nitrogen and then evaporated. To the residue, 81.2 g. is added acetic anhydride, (176 ml., 1.809 mole), and pyridine, 180 ml., and the mixture is kept at room temperature for 20 hours. The mixture is then evaporated and the oily residue is dissolved in 1000 ml. of ethyl acetate and the solution is washed in sequence with 200 ml, % hydrochloric acid-saturated sodium chloride (washing pH 2), 200 ml. of saturated sodium chloride solution (2 times, second washing pH 7) and then stripped of the solvent. To the clear oily residue, [96.9 g., 86.5%, had [α]θ = 61.8° (CHCl^lJis added freshly distilled thionyl chloride, (83 ml., 1.173 mole) and the resulting solution is stirred at room temperature with evolution of gas for 18 hours. The excess thionyl chloride is evaporated under vacuum and a 50° bath and the residue is distilled at reduced pressure to obtain 56.9 g. of D-3-acetylthio-2-methylpropanoic acid chloride, b.p. 40-4 ° (0.17 -0.2 mm Hg.) [ J25 -42.5° (c 2; methanol). - 32 Preparation 10 3- Acetylthio~2-bromopropanoic acid chloride By substituting 2-bromoacrylic acid for the 2-trifluoromethylacrylic acid in the procedure of Example 1 and then allowing the product to react with thionyl chloride, 3-acetylthio-2-bromopropanoic acid and 3-acetylthio-2-bromopropanoic acid chloride are obtained.

Example 23 Trans-1-[D-3-(acetylthio)-2-methyl-l-oxopropyl]-4fluoro-L-proline A) Trans-l-carbobenzyloxy-4-fluoro-L-proline A solution of 6.2 grams of trans-1-carbobenzyloxy4- fluoro-L-proline, methyl ester in 50 ml of methanol 15 is treated dropwise at 0-5°, with 11.5 ml of 2N sodium hydroxide solution, and after 1 hour at 0°, allowed to warm to room temperature overnight. The reaction mixture is concentrated under reduced pressure to about one-half its original volume and is then diluted with 100 ml of water. The aqueous reaction mixture is extracted with ether and the ether extracts discarded. The aqueous solution is acidified, with cooling, with dilute hydrochloric acid to pH 2 and then extracted with ethyl acetate (4 x 50 ml). The combined ethyl acetate extracts are dried over anhydrous magnesium sulfate, and then concentrated under reduced pressure to yield the desired product. It is purified by conversion to the cyclohexylamine salt, mp. 194196°, [a] 25 -44° (c=l% in methanol). 7 9 71 - 33 The free acid is obtained by suspension of cyclohexylamine salt in 25 ml of ethyl acetate with 22 ml of N hydrochloric acid and extracting the aqueous layer with 4 x 35 ml of ethyl acetate. The combined ethyl acetate extracts are dried over anhydrous magnesium sulfate and the solvent concentrated under reduced pressure to yield the desired trans -l-carbobenzyloxy-4-fluoro-L proline.

B) Trans-4-fluoro-L-proline, hydrobromide A mixture of 3 grams of trans-l-carbobenzyloxy-4fluoro-L-proline and 15 ml of hydrogen bromide in acetic acid (30-32%) is stirred for 1 hour, and then 150 ml of anhydrous ether is added. The solvent is decanted from the precipitate that is then triturated with fresh ether and finally with methyl ethyl ketone. The trans-4-fluoro-L-proline hydrobromide melts at 162-164° (dec), [nj^ -30° (c=l% in methanol).

C) Trans-1-[D-3-(acetylthlo)-2-methyl-l-oxopropyl]4-fluoro-L-proline To a stirred solution of 1.9 grams of trans 4fluoro-L-proline hydrobromide in 25 ml of cold water there is added 1 gram of sodium carbonate to adjust pH to 3.2. Then with continued cooling (5°) and stirring there is added dropwise 1.8 grams of D-3acetylthio-2-methylpropionyl chloride in 2.5 ml of ether, while maintaining the pH at about 8.2-8.3 by the dropwise addition of a 25% aqueous sodium carbonate solution. The stirring and cooling is continued for one hour after the addition is completed. The reaction mixture is extracted with ethyl acetate (2 x 25 ml) and the extracts discarded. To the aqueous layer is added 50 ml of ethyl acetate and with stirring and 9 7 1 - 34 cooling, concentrated hydrochloric acid is added dropwise to a pH of 2.0. The aqueous layer is saturated with sodium chloride and the ethyl acetate layer separated. The aqueous layer is extracted with additional ethyl acetate (3 x 25 ml), the combined ethyl acetate extracts dried over anhydrous magnesium sulfate and concentrated under reduced pressure to yield the desired product. The dicyclohexylamine salt is prepared by dissolving the product in 25 ml of ethyl acetate and adding a solution of 1.8 gram of dicyclohexylamine in 35 ml of ethyl acetate. The precipitated salt is filtered and recrystallized from isopropanol to yield the dicyclohexylamine salt of trans l-[D-3(acetylthio)-2-methyl-l-oxopropyl]-4-fluoro-L-proline; mp. 209-211°, [a]^5 -85° (c=l% methanol).

The free acid is recovered by dissolving the dicyclohexylamine salt in 5% aqueous potassium acid sulfate and extraction with ethyl acetate. The ethyl acetate solution is dried over anhydrous magnesium sulfate and concentrated under reduced pressure to yield the desired trans 1-[D-3-(acetylthio)-2-methyll-oxopropyl] -4-fluoro-L-proline.

Example 24 trans-4-fluoro-l-(D-3~mercapto-2-methyl-l-oxopropyl)25 L-proline Argon is passed tnrough a cold solution of 4.2 ml of concentrated ammonium hydroxide in 16 ml of water.

To this solution there is added, with stirring in an atmosphere of argon, 1.8 grams of trans-1-[D-3-(acetyl30 thio)-2-methyl-l-oxopropyl]4-fluoro-L-proline. The reaction mixture is stirred for an additional two hours and is then extracted with ethyl acetate which is - 35 discarded. The aqueous layer is stirred, 30 ml of ethyl acetate added, and the aqueous layer acidified with concentrated hydrochloric acid. The aqueous layer is saturated with sodium chloride and the ethyl acetate layer separated. The aqueous layer is extracted with ethyl acetate (3 x 30 ml). The combined ethyl acetate extracts are dried over anhydrous magnesium sulfate and concentrated under reduced pressure to yield the desired trans-4-fluoro-1-[D3-mercapto-2-methyl-l-oxopropyl)-L-proline; [ <*] -112°(c=l% in methanol).

Example 25 1-[D-3-(acetylthio)-2-methyl-l-oxopropyl]-4,4-difluoroL-proline A) l-carbobenzyloxy-4,4-difluoro-L-proline, methyl ester To a cooled stirred solution of 3.3 grams of 1carbobenzyloxy-4-keto-L-proline, methyl ester in 80 ml of methylene dichloride there is added dropwise 3.3 ml diethylaminosulfurtrifluorids. The reaction mixture is allowed to remain overnight at room temperature. About 100 grams of crushed ice is added with stirring and the reaction mixture stirred for 45 minutes. The organic layer is separated and the aqueous layer extracted with methylene chloride (2 x 40 ml). The combined extracts are dried over anhydrous magnesium sulfate and then concentrated under reduced pressure to yield the desired 1-carbobenzyloxy-4,4-difluoro-L-proline, methyl ester. 4797 1 - 36 B) l-carbobenzyloxy-4,4-difluoro-L-proline A solution of 5.6 grams of l-carbobenzyloxy-4,4difluoro-L-proline, methyl ester in 50 ml of methanol is treated dropwise with 11.5 ml of 2 N sodium hydrox5 ide solution at 0-5°. The reaction mixture is left at 0° for 1 hour and is then allowed to warm to room temperature overnight. The reaction mixture is concentrated under reduced pressure to about one-half its original volume and is then diluted with 100 ml of water. The aqueous reaction mixture is extracted with ether and the ether extracts discarded. The aqueous solution is acidified with cooling with dilute hydrochloric acid to pH 2 and is then extracted with ethyl acetate (3 x 50 ml). The ethyl acetate ex15 tracts are combined and washed with saturated sodium chloride solution, dried over anhydrous magnesium sulfate and concentrated to yield the desired product, l-carbobenzyloxy-4,4-difluoro-L-proline.

It is purified by conversion to the cyclohexylamine salt. mp. 180-185°!o]^6 = -24° (c=l% in ethanol).

The free acid is obtained by treating an aqueous solution of the cyclohexylamine salt with hydrochloric acid and extracting the mixture with ethyl acetate (4 x 30 ml). The ethyl acetate extracts are dried over anhydrous magnesium sulfate and concentrated under reduced pressure to yield the desired 1-carbobenzyloxy-4,4-difluoro-L-proline.

C) 4,4-Difluoro-L-proline, hydrobromide A mixture of 2.4 grams of l-carbobenzyloxy-4,430 difluoro-L-proline and 12 ml of hydrogen bromide in acetic acid (30-32%) is stirred for 30 minutes at room - 37 temperature and then 300 ml of anhydrous ether is added. The mixture is cooled and the precipitated solid is filtered and dried under reduced pressure.

The desired 4,4-difluoro-L-proline, hydrobromide melts at 163-165° (dec); [ OJ = -14°(C=1% in methanol).

D) 1-IP-3-(acetylthio)-2-methyl-l-oxopropyl]-4,4difluoro-L-proline To a stirred solution of 2.7 grams of 4,4-difluoroL-proline, hydrobromide in 30 ml of water, cooled to 5°, there is added solid carbonate to adjust the pH to 8,4. Then with continued cooling and stirring there is added dropwise 2.4 grams of D-3-acetylthio2-methylpropionyl chloride in 3 ml of anhydrous ether, while maintaining the pH of the solution at 8.1-8.3 by the addition of a 25% aqueous sodium carbonate solution: The stirring and cooling is continued for one hour after the addition is completed. The reaction mixture is extracted with ethyl acetate (2 x 25 ml) and the extracts discarded. To the aqueous layer is added 50 ml of ethyl acetate and, with stirring and cooling, there is added dropwise concentrated hydrochloric acid to a pH of 2.0. The aqueous layer is saturated with sodium chloride and the ethyl acetate layer separated. The aqueous layer is extracted with ethyl acetate (3 x 25 ml) and the combined ethyl acetate extracts dried over anhydrous magnesium sulfate and concentrated under reduced pressure to yield the desired product. The dicyclohexylamine salt is prepared by dissolving the product in 40 ml of ethyl acetate and adding a solution of 2.3 grams of dicyclohexylamine in 5 ml of ethyl acetate. The pre47971 - 38 cipitated salt is filtered and recrystallized from ethanol. The dicyclohexylamine salt of l-[D-3(acetylthio)-2-methyl-l-oxopropyl]4,4-difluoro-Lproline melts at 225-227°; [ct]15 * * * * 20 * * 23 * 25 * * * * 30 = -70° (c=0.5% in methanol).

The free acid is recovered by dissolving the dicyclohexylamine salt in 5% aqueous potassium acid sulfate and extraction with ethyl acetate. The ethyl acetate solution is dried over anhydrous magnesium sulfate and concentrated under reduced pressure to yield the desired 1-[D-3-(acetylthio)-2methyl-l-oxopropyl]-4,4-difluoro-L-proline.

Example 26 4,4-Di fluoro-1-(D-3-mercapto-2-methyl-l-oxopropyl)15 L-proline Argon is passed through a cold solution of 4.6 ml of concentrated ammonium hydroxide in 11 ml of water.

To this solution there is added, with stirring in an atmosphere of argon 2.1 grams of 1-[D-3-(acetylthio)-220 methyl-l-oxopropyl]-4,4-difluoro-L-proline. The reaction mixture is stirred an additional two hours and is then extracted with ethyl acetate, which is discarded The aqueous layer is stirred, 30 ml of ethyl acetate is added and the aqueous layer acidified with concentrated hydrochloric acid. The aqueous layer is saturated with sodium chloride and the ethyl acetate layer separated. The aqueous layer is extracted with ethyl acetate (3 x 25 ml). The combined ethyl acetate extracts are dried over anhydrous magnesium sulfate and concentrated under reduced pressure to yield the desired 4,4-difluoro-1-(D-3-mercapto-2-methyl-loxopropyl)-L-proline. 7971 - 39 Example 27 1- [D-3-(acetylthio)-2-methyl-l-oxopropyl·]-5-fluoro2- pipecolic acid A) l-Carbobenzyloxy-5-hydroxy-L-pipecolic acid, methyl ester A solution of 5 grams of l-carbobenzyloxy-5hydroxy-L-pipecolic acid in 60 ml of dioxane is treated with an ethereal solution of diazomethane portionwise, until the yellow color persists. The temperature is maintained at about 5° during the addition. The excess diazomethane is destroyed with glacial acetic acid and the resulting solution dried over anhydrous magnesium sulfate. The solution is concentrated under reduced pressure to yield the desired l-carbobenzyloxy-5-hydroxy-L-pipecolic acid, methyl ester as a pale yellow viscous oil.

B) l-Carbobenzyloxy-5-tosyloxy-I.-pipecolic acid, methyl ester To a stirred solution of 5.7 grams of 1-carbo20 benzyloxy-5-hydroxy-L-pipecolic acid, methyl ester in 12 ml of pyridine, there is added dropwise at 5° to 8° a solution of 4 grams of tosyl chloride in 6 ml of pyridine. The reaction mixture is kept at 5° for 72 hours and is then treated, with cooling, with 200 ml of ice-cold 2-N hydrochloric acid. The precipitate is dissolved in chloroform and the aqueous solution extracted with additional chloroform (3 x 75 ml). The combined chloroform solutions are dried over anhydrous magnesium sulfate and concentrated under reduced pressure to yield the desired l-carbobenzyloxy-5-tosyloxy-Lpipecolic acid, methyl ester, melting at 74-76°, after crystallization from isopropanol, Ια] θθ = -5°, c=l% - 40 26 ο in methanol, i«lD = -11 , c=l% in chloroform.

C) l-Carbobenzyloxy-5-fluoro-L-pipecolic acid, methyl ester A stirred suspension of 5.2 grams of 1-carbo5 benzyloxy-5-tosyloxy-L-pipecolic acid, methyl ester in ml of diethylene glycol is treated with 5.2 grams of anhydrous potassium fluoride and the mixture heated at 80°, with stirring for 14 hours. The cooled solution is diluted with 50 ml of water and extracted with ethyl acetate (3 x 100 ml). The ethyl acetate extracts are combined, washed with a saturated sodium chloride solution and dried over anhydrous magnesium sulfate. The solvent is removed under reduced pressure to yield the desired l-carbobenzyloxy-5-fluoro-L15 pipecolic acid, methyl ester as a yellow oil.

D) l-Carbobenzyloxy-5-fluoro-L-pipecolic acid To a cooled (0°) solution of 4.6 grams of 1carbobenzyloxy-5-fluoro-L-pipecolic acid, methyl ester in 32 ml of methanol there is added 7.3 ml of 2N sodium hydroxide solution. The reaction mixture is allowed to remain at 0-5° for one hour and at room temperature overnight. The solution was concentrated to about one-half its volume under reduced pressure and diluted with 20 ml of water. The solution is extracted with ether, which is discarded. The aqueous solution is cooled, acidified with concentrated hydrochloric acid and extracted with ethyl acetate (3 x 50 ml). The combined ethyl acetate extracts are dried over anhydrous magnesium sulfate and concentrated under reduced pressure to yield the desired l-carbobenzyloxy-5-fluoroL-pipecolic acid. It is purified by conversion to the 17971 - 41 cyclohexylamine salt, which melts at 158-161° la]36 = -11° (c=l% in methanol.

The free acid is obtained by treating an aqueous solution of the salt with hydrochloric acid, extracting the mixture with ethyl acetate (4 x 25 ml) and concentrating the dried extracts under reduced pressure.

E) 5-Fluoro-L-pipecolic acid hydrobromide A mixture of 2.2 grams of l-carbobenzyloxy-5fluoro-L-pipecolic acid and 12 ml of hydrogen bromide in acetic acid (30-32%) is stirred for 30 minutes at room temperature and then 300 ml of anhydrous ether is added. The cooled mixture is filtered and the precipitated solid dried under reduced pressure to yield 5-fluoro-L-pipecolic acid hydrobromide.

F) 1-[D-3-(acetylthro)-2-methyl-l-oxopropyl]-5-fluoroL-pipecolic acid To a suspension of 5.1 grams of 5-fluoro-L-pipecolic acid hydrobromide in 100 ml of dimethylacetonide there is added 10 grams of N methylmorpholine. To this mixture there is then added, slowly, with vigorous stirring 5.4 grams of D-3-acetyl-2-methylpropionyl chloride and the reaction mixture heated at 90° for three hours. The cooled reaction mixture is filtered and concentrated under reduced pressure. The residue is treated with dilute hydrochloric acid and is extracted with ethyl acetate (3 x 150 ml). The ethyl acetate extracts are dried and then concentrated under reduced pressure to yield l-[D-3-(acetylthio)-2-methyl1-oxopropyl]-5-fluoro-L-pipecolic acid.

The acid is purified by conversion to the dicyclohexylamine salt followed by crystallization of the salt from acetonitrile. >47971 - 42 Example 28 -Fluoro-l-(D-3-mercapto-2-methyl-l-oxopropyl)-Lpipecolic acid Nitrogen is bubbled through a solution (5°) of 5 11 ml of concentrated ammonium hydroxide in 25 ml of water for 30 minutes. To this solution there is added 1.6 grams of 1-[D-3-(acetylthio)-2-methyl-l-oxopropyl]5-fluoro-L-pipecolic acid and the mixture stirred for 15 minutes at 5° and then 4 hours at room temperature.

The solution is then cooled, acidified with concentrated hydrochloric acid and extracted with ethyl acetate (3 x 50 ml). The ethyl acetate extracts are dried over anhydrous magnesium sulfate and concentrated under reduced pressure to yield 5-fluoro-l-(D-3-mercapto-215 methyl-l-oxopropyl)-L-pipecolic acid.

Example 29 1-(2-Mercapto-3,3,3-trifluoropropanoyl)-L-pipecolic acid By substituting L-pipecolic acid for the L-proline in the procedure of Example 1, and then submitting the product to the procedure of Example 2, 1-(2-mercapto3,3,3-trifluoropropanoyl)-L-pipecolic acid is obtained. 479 71 - 43 Example 30 1-(3-Mercapto-2-trifluoromethylpropanoyl)-5,5-difluoroDL-pipecolic acid By substituting 5,5-difluoro-DL-pipecolic 5 acid [obtained from 5-keto-DL-pipecolic acid by the procedure described in J. Med. Chem. 20, 1176 (1977)] for the 4,4-difluoro-L-proline in the procedure of Example 3 and submitting the product to the procedure of Example 4 , 1-(3-mercapto-2-trifluoro10 methylpropanoyl)-5,5-difluoro-DL-pipecolic acid is obtained.

Example 31 1,1'-[Dithiobis-[2-trifluoromethyl-3-propanoyl)]bis5,5-difluoro-DL-pipecolic acid 15 By substituting l-(3-mercapto-2-trifluoromethylpropanoyl)-5,5-difluoro-DL-pipecolic acid for the 1-(3-mercapto-2-trifluoromethylpropanoyl)-Lproline in the procedure of Example 9, of British Patent Specification No, 2014987A, 1,1'-[dithio-bis-(2-trifluoromethyl 3-propanoyl)]bis-5,5-difluoro-DL-pipecolic acid is obtained.

Example 32 1-(3-Mercapto-4,4,4-trifluorobutanoy])-L-pipecolic acid By substituting L-pipecolic acid tert.-butyl 25 ester for the L-proline tert.-butyl ester in the procedure of Preparation 4, and then submitting the product to the procedure of Example 6, l-(3-mercapto-4,4,4-trifluorobutanoyl)-L-pipecolic acid is obtained. 4797 1 - 44 Example 33 1-(3-Mercapto-2-trifluoromethylpropanoyl)-5,5-dichloro DL-pipecolic acid By substituting 5,5-dichloro-DL-pipecolic acid 5 [prepared from 5-keto-DL-pipecolic acid and phosphorus pentachloride by a procedure similar to that described in J. Med. Chem. 20, 1176 (1977)] for the 4.4- difluoro-L-proline in the procedure of Example 3 and then submitting the product to the procedure of Example 4, 1-(3-mercapto-2-trifluoromethylpropanoyl)5.5- dichloro-DL-pipecolic acid is obtained.

Example 34 1-(3-Mercapto-2-methylpropanoyl)-5,5-difluoro-DLpipecolic acid By substituting 3-acetylthio-2-methylpropanoic acid chloride for the 3-acetylthio-2-trifluoromethylpropanoic acid chloride in the procedure of Example 3 and then submitting the product to the procedure of Example 4, 1-(3-mercapto-2-methylpropanoyl)-5,520 difluoro-DL-pipecolic acid is obtained.

Example 35 1-(3-Mercapto-2-methylpropanoyl)-5-fluoro-DL-pipecolic acid By substituting 3-acetylthio-2-methylpropanoic acid chloride for the 3-acetylthio-2-trifluoromethylpropanoic acid chloride and 5-fluoro-DL-pipecolic acid [prepared from 5-hydroxypipecolic acid by a procedure similar to that descrbied in Biochemistry, 4_, 2507 (1965)] for the 4,4-dif luoro-L-proline in the procedure of Example 3, and then submitting the product to the procedure of Example 4, l-(3-acetylthio-2-methylpropanoyl)-5-fluoro-DL-pipecolic acid - 45 and 1-(3-mercapto-2-methylpropanoyl)-5-fluoro-DLpipecolic acid are obtained.

Example 36 1-(3-Mercaptopropanoyl)-5-bromo-DL-pj.pecolic acid By substituting 3-acetylthiopropanoyl chloride for the 3-acetylthio-2-trifluoromethylpropanoic acid chloride and 5-bromo-DL-pipecolic acid [prepared from 5-hydroxypipecolic acid by a procedure similar to that described in Aust. J. Chem., 20, 1493 (1967)] for 4,4-difluoro-L-proline in the procedure of Example 3 and submitting the product to the procedure of Example 4, 1-(3-acetylthiopropanoyl)-5-bromo-DLpipecolic acid and 1-(3-mercaptopropanoyl)-5-bromoDL-pipecolic acid are obtained. 4797 1 - 46 Example 37 1-(3-Propanoylthio-2-trifluoromethylpropanoy1)-55 fluoro-DL-pipecolic acid By substituting thiopropanoic acid for the thiolacetic acid in the procedure of Preparation 1and then submitting the product to the*procedure of Example 15, 1-(3-propanoylthio-2-trifluoromethylpro10 panoyl)-5-f1uoro-DL-pipecolic acid is obtained.

Example 38 1-(3-Mercapto-2-methylpropanoyl)-5-fluoro-DLpipecolic acid sodium salt An aqueous solution of 1-(3-mercapto-2-methyl15 propanoyl)-5-fluoro-DL-pipecolic acid is mixed with an equimolar amount of aqueous N-sodium hydroxide and the solution is freeze dried.

Example 39 1-(3-Mercaptopropanoyl)-5,5-dichloro-DL-pipecolic acid By substituting 3-acetylthiopropanoic acid chloride for the 3-acetylthio-2-trifluoromethylpropanoic acid chloride in the procedure of Example 33 1-(3-mercaptopropanoyl)-5,5-dichloro-DL-pipecolic acid is obtained.

The racemic forms of the final products in each of the foregoing examples are produced by utilizing the DL-form of the starting amino acid instead of the L-form.' Similarly, the D-form of the final products in each of the foregoing examples is produced by utilizing the D-form of the starting amino acid instead of the L-form. - 47 Example 40 1000 tablets each containing 100 mg. of 1—(Π— 3-mercaptopropanoyl)-ci s-4-fluoro-b-proline, are produced from the following ingredients: 1-(3-mercaptopropanoyl)-cis-4 f1uoro-L-proline 100 g· Corn starch 50 g· Gelatin 7.5 g. Avicel(Trade Mark for a microcrvstalline 25 g- Magnesium stearate cellulose) 2.5 g.

The 1-(D-3-mercaptopropanoyl)-cis-4-fluoroL-proline and corn starch are admixed with an aqueous solution of the gelatin. The mixture is dried and ground to a fine powder. The Avicel and then the magnesium stearate are admixed with the granulation. This is then compressed in a tablet press to form 1000 tablets each containing 100 mg. of active ingredient.

Example 41 1000 tablets each containing 200 mg. of 1[D-3-(acetylthio)-2-methylpropanoyl]-cis-4-fluoroL-proline are produced from the following ingredients: 1-[D-3-(acetylthio)-2-methylpro- panoyl]-cis-4-fluoro-L-proline 200 g· Lactose 100 g· Avicel 150 g· Corn starch 50 g. Magnesium stearate 5 g.

The 1-(D-3-(acetylthio)-2-methylpropanoyl]-cis4-fluoro-L-proline, lactose and Avicel are admixed, then blended with the corn starch. Magnesium stearate is added. The dry mixture is compressed in a tablet press to form 1000 505 mg. tablets each containing 200 mg. of active ingredient. The tablets are •27071 - 48 coated with a solution of Methocel (Trade Mark) E 15 (methyl cellulose) including as a color a lake containing yellow #6.

Example 42 Two piece #1 gelatin capsules each containing 250 mg. of 1-(3-mercapto-4,4,4-trifluorobutanoyl)-Lproline are filled with a mixture of the following ingredients : 1-(3-mercapto-4,4,4-trifluorobutanoyl-L-proline Magnesium stearate USP lactose 250 7 193 mg. mg. mg. 15 Example 43 An injectable solution is produced as ; follows cis-1- [D-3- (mercapto)-2-methylpropanoyl)]-cis-4-fluoro-Lproline 500 g. 20 Methyl paraben 5 g· Propyl paraben 1 g- Sodium chloride 25 g- Water for injection qs. 5 1.

The active substance, preservatives and sodium chloride are dissolved in 3 liters of water for injection and then the volume is brought up to 5 liters. The solution is filtered through a sterile filter and aseptically filled into pre30 sterilized vials which are then closed with presterilized rubber closures. Each vial contains 5 ml. of solution in a concentration of 100 mg. - 49 of active ingredient per ml. of solution for injection.

The products of each example directed to a compound similarly formulated as in Examples 40 to 43.

Claims (28)

CLAIMS:

1. (I) A compound of the qeneral formula R' R 4 R 3 H 2 C (CHR 6 ) m R— S — (CH)— CH-CO— N-CH-COOR, n * -L wherein R is hydrogen, lower alkanoyl or R_ R' h 2 c < CHR 6>m — CH-COOR, 0 or 1; lower alkvl: S — (CH)—-CH—CO m is 1 or 2; n is R^ is hydrogen or R 2 and R‘ 2 each is hydrogen or halogen; is hydrogen, lower alkyl or CF^, and also halogen when n is 1; is hydrogen, lower alkyl or trifluoromethyl; R, is hydrogen, and also halogen when m is 1; with the ® I provisos that at least one. of R 2 , R 2 , R 3 , and R g is halogen or CF 3 only R 2 and R' 2 can be both halogen at the same time; and, when R 3 is trifluoromethyl, at least one of R 2 and R) is halogen; or, when is hydrogen such a ccrapound. ir. the form of a salt formed with a base.

2. A conpound according to claim 1 of the formula ‘179 71 - 51 wherein R is hydrogen, lower alkanoyl or R 4 R 3 R 2 R 2 -S-(CH)-— CH-CO— N-!-COORj^ R^ is hydrogen or lower alkyl; R 2 , R' 2 and R g each is hydrogen or halogen; R 3 is hydrogen, lower alkyl, halogen or tri f1uoromethyl; R 4 is hydrogen, lower alkyl or trifluoromethyl; and n is 0 or 1; with the provisos that at least one of I ' Ra.Rj/RjfRs and R g is halogen or CF 3 , only R 2 and R 2 can both be halogen and when R 3 is halogen n must be 1; and wherein R 3 is trifluoromethyl, at least one of R 2 and R' 2 is halogen; or, when is hydrogen, such a compound in the form of a salt formed with a base.

3. A compound according to claim 1 of the formula R— S CO— N----COOR. η 1 wherein R is hydrogen, lower alkanoyl or (CH)CH— C0- COOR. 47S71 R1 is hydrogen or lover alkyl; R 2 and R' 2 each is hydrogen or fluorine? R^ and R^ each is hydrogen or trifluoromethyl, one being hydrogen and the other trifluoromethyl; and n is 0 or 1; with the proviso that, when R 3 is trifluoromethyl, at least one of R 2 and R' 2 is halogen? or, when R 1 is hydrogen, such a compound in the form of a salt formed with a base.

4. A compound of the formula wherein R and have the same meaning as in Claim 2; R 2 and R' 2 each is hydrogen or fluorine; R^ is hydrogen, halogen or lower alkyl; R^ being halogen when both R 2 and R'z are hydrogen and R^ being other than halogen when R 2 or R' 2 is halogen; R 4 is hydrogen; and n is 0 or 1; or, when R, is hydrogen, such a compound in the form of a salt formed with a base.

5. . A compound according to claim 1 or 2 wherein R is hydrogen or lower alkanoyl; R 1 is hydrogen or lower alkyl; R 2 and R' 2 is hydrogen or halogen; R^ is hydrogen or lower alkyl; R 4 is hydrogen or lower alkyl; and n is 0 or 1.

6. A compound according to claim 1 or 2 wherein n is 0 or 1? R is hydrogen or lower alkanoyl; P^ is hydrogen or lower alkyl; R 2 and R' 2 each is hydrogen - 53 or fluorine; Rj is hydrogen or lower alkyl and R 4 is hydrogen or lower alkyl.

7. A compound aocording to claims 1 or 2 wherein R is hydrogen and R 2 and R' 2 each is fluorine.

8. A compound according to claims 1 or 2 wherein R and R^ each is hydrogen, R 2 and R' 2 each is fluorine, and R 3 and R^ each is hydrogen or lower alkyl.

9. A compound according to claim 4 wherein R 2 is hydrogen or fluorine; Rg is fluorine; R is hydrogen and Rj, R 3 and R^ each is hydrogen or lower alkyl.

10. A compound according to claim 4 wherein R 2 is hydrogen, R g is fluorine, R is hydrogen and Rj, Rj and R^ each is hydrogen or lower alkyl.

11. A compound according to claims9 or 10 wherein Rj and each is hydrogen and R 3 is methyl.

12. A compound according to claim 1 of the formula „ D , \ X 2 c. h 2 c (CH 2>m R- S— (CH)” CH— CO- NCH-COOR, Π j wherein R is hydrogen, lower alkanoyl or R, R' 2 x / 2 c -Si 4 (CH); h 2 c (CH CH — CO 2 m I CH-COOR, m is 2; n is 0 or 1; 17 9 7 1 - 54 10 R^ is hydrogen or lower alkyl; R^ and R' 2 each is hydrogen or halogen; R^ and R 4 each is hydrogen, lower alkyl or trifluoromethyl, not more than one being trifluoromethyl, and at least one of R 2 , R' 2 < R 3 or R^ is halogen or CF 3 ; with the proviso that, when R 3 is trifluoromethyl, at least one of R 2 and R' 2 is halogen; or, when' is hydrogen, such a compound in the form of a salt formed with a base.

13. A compound according to claim 12 wherein R, R 2 , R' 2 and R^ all are hydrogen and R 3 is trifluoromethyl.

14. A compound according to claim 12 wherein R 2 is halogen and R' 2 is hydrogen.

15. A compound according to claim 14 wherein the halogen is fluorine.

16. A compound according to claim 12 wherein R is hydrogen and R 2 and R' 2 each is halogen.

17. A compound according to claim 16 wherein the halogen is fluorine.

18. A compound according to claim 12 wherein R, R^ is hydrogen; R 2 is hydrogen or fluorine; R' 2 is fluorine; R 3 is methyl; R^ is hydrogen and n is 1.

19. A process for preparing a compound according to claim 1 which comprises coupling the acid of the formula *4 p R— S-(CH)-CH-COOH n to the amino acid of the formula H,C (CHR,) 2 ί 6 m HN CH--COOR^ - 55 wherein R is hydrogen or lower alkanoyl or a pmethoxy benzyl protecting group which is removed afterwards; and the other groups a/e as defined in Claim .1 to form a product wherein R is hydrogen or lower alkanoyl, and oxidizing said product wherein R is hydrogen, by treatment with iodine, to form the product wherein R is -S-(CH)-CH-CO- N-COOR. n l

20. A process according to claim 19 wherein the product wherein R is lower alkanoyl is hydrolzyed to form the product wherein R is hydrogen.

21. A process according to claim 20 wherein the product wherein R is lower alkanoyl is subjected to ammonolysis.

22. A compound according to claim 1, when prepared by a process according to any one of Claims 19 to 21.

23. A compound according to claim 1, as named in any of the Examples 1-39.

24. An optical isomer of a compound according to any one of Claims 1 to 18.

25. A compound according to any one of Claims 1 to 18 and 22 to 24 for use in the treatment of hypotension.

26. A pharmaceutical composition comprising a compound according to any one of Claims 1 to 18 and 22 to 24 and a pharmaceutical carrier. - 56

27. A composition according to Claim 26 in the form of a tablet, capsule, elixir or sterile injectable preparation.

28. A composition according to Claim 26 or 27 5 including an excipient, binder, preservative, lubricant, sweetener, stabiliser or flavour.