NO790568L - PROCEDURE FOR THE PREPARATION OF HALOGEN-SUBSTITUTED MARKAPTOACYLAMINO ACIDS - Google Patents

Description

"Procedure for the production of halogen-substituted mercaptoacyl amino acids"

This invention relates to the preparation of new halogenated compounds with the general formula

(IN)

where R is hydrogen, lower alkanoyl or

m is 1 or 2; n is 0 or 1

R 1 is hydrogen or , lower alkyl;

R 2 and R 2 are each hydrogen or halogen

R^ is hydrogen, lower alkyl or CF^ and also halogen when n is 1; R 1 is hydrogen, lower alkyl or trifluoromethyl;

Rg is hydrogen and also halogen when m is 1;

wherein at least one of R„, R', R0, R. and R, is halogen or CF_ as represented by the symbols, and only R.sub.2 and R.sub.2 can both be halogen at the same time.

The star denotes an asymmetric carbon atom.

The invention thus generally relates to halogenated derivatives of mercaptoacylproline and mercaptoacylpipecolic acid of formula I.

Regarding the prolines (where m is 1), two preferred groups of compounds of formula I are those having the following formulas:

(II)

where R is hydrogen, lower alkanoyl or

R 1 is hydrogen or lower alkyl;

R 2 and R 1 are each hydrogen or fluorine;

R 1 and R 2 are each hydrogen or trifluoromethyl, one being hydrogen and the other trifluoromethyl; and

n is 0 or 1;

(III)

where R and R^ have the same meaning as stated above for formula II;

R 2 and R 1 - are each hydrogen or halogen;

R^ is hydrogen, halogen or lower alkyl, R^ being halogen when both R 2 and R 1 are hydrogen, and R^ being different from halogen when R 2 or R 1 - is halogen;

R 4 is hydrogen; and

n is 0 or 1;

and basic salts of said compounds of formulas II and III, respectively.

Thus when n is 0 and and both are hydrogen i

formula II, R 1 is trifluoromethyl. When n is 1, either R 1 or R 2 is trifluoromethyl and the other is hydrogen. This means that there is one trifluoromethyl group on the acy1 side chain in the molecule. It is on the carbon atom in the a position of the carbonyl group (R^ = CF^) when n is 0. It is on either the carbon atom in the a position of the carbonyl group

(R^= CF^, R4 = H) or on the carbon atom in the p-position to the carbonyl group (R^ = H>R^= CF^) when n is 1, the other of the two symbols (R^, R^ ) then' is hydrogen. When either R₁ or R₁ is trifluoromethyl, R₁ and R₁ are each hydrogen or halogen.

In the case of formula III, preferably one of or both R 2 and R 1 are halogen, and R 2 and R 3 are each hydrogen or lower alkyl, or both R 2 and R 1 . is hydrogen, R^ is halogen, preferably chlorine or bromine, dg R^ is hydrogen.

Particularly preferred are those compounds of formula I where

R is hydrogen or lower alkanoyl, especially hydrogen or acetyl; is hydrogen or lower alkyl, especially hydrogen; R 2 and R 1 are each hydrogen or halogen, especially hydrogen or fluorine;

R₂ and R₂ are each hydrogen, trifluoromethyl or lower alkyl, one of R₂ or R₂ being trifluoromethyl and the other hydrogen when R₂, R₂ and R₂ are all hydrogen; and n is 0 or 1, especially 1.

,One or two halogen atoms may be present on the pyrrolidine ring. A single halogen atom can be on either the carbon atom in the 3-position or on the carbon atom in the 4-position.

Two halogen atoms may be present in the 4-position, and preferably they are equal. Fluorine is preferred on this ring, especially one or two fluorine atoms on the carbon atom in the 4-position.

In the case of pipecolic acids (m is 2), those compounds of formula I where R is hydrogen or lower alkanoyl, especially hydrogen or acetyl; R 1 is hydrogen or lower alkyl, especially hydrogen; R 2 and R 1 are each hydrogen or halogen, especially hydrogen or fluorine; one of R^ and R^ is CF^ and the other is hydrogen; n is 0 or 1, especially 1. L and R'2 can independently be hydrogen or halogen, especially fluorine. When R 1 or R 4 is CF 2 , both R 2 and R 2 are preferably hydrogen. The L-configuration for the prolines or pipecholic acids is particularly preferred.

The lower alkyl groups denoted by any of the variables include linear and branched hydrocarbon radicals from methyl to heptyl, e.g. methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl and the like. The C^-C^ groups, especially C, and the C2~ groups are preferred.

The lower alkanoyl groups are those which have acyl radicals of the lower {C^-C^) fatty acids, e.g. acetyl, propionyl, butyryl and the like. Correspondingly, the lower alkanoyl groups which have up to 4 carbon atoms, especially acetyl, are preferred.

The halogens are the four common halogens, preferably

chlorine, bromine or fluorine, especially fluorine.

The compounds of formula I are prepared according to

the invention by several synthesis methods.

In general, these compounds can be synthesized by the acid with the formula

is connected to the amino acid with the formula:

by any method that can be used to form amide bonds. See e.g. "The Method of Organic Chemistry"

(Houben-Weyl), Part I, page 376 et seq., Part III, page 1 et seq. (1974) ..

The acids of formula IV can, when n is 1, be prepared by adding a thioacid R-SH to a suitably substituted acrylic acid.

As a temporary protection of the mercapto group in the compounds of formula IV, R may be a p-methoxy-benzyl group. This group is then removed with trifluoroacetic acid and mercury(II) acetate. The acids of formula IV, when n is 0, are obtained by a displacement reaction using a thioacid R-SH and a 2-haloacid.

According to a preferred method when n is 0, an acid of formula V is coupled to a haloalkanoic acid of the formula

where X is halogen, preferably chlorine or bromine, by one of the known methods where the acid VI is activated before reaction with the acid V, which includes the formation of a mixed anhydride, symmetrical anhydride, acid chloride, active ester, or the use of Woodward's reagent K , EEDQ (N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline) or the like.

The product obtained by this reaction is a compound with the formula:

This product is subjected to a displacement reaction with the anion of a thioacid of the formula

(VIII)

R7- CO SH

where R_ is lower alkyl, whereby a product with the formula is obtained

which can then be converted into the product:

by ordinary alkaline hydrolysis or ammonolysis. When R 1 is an ester group (ie R 1 is lower alkyl, obtained when an ester of the starting acid V is used), the ester group can be removed in the usual way. When e.g. R^ 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

present, alkaline hydrolysis will yield the corresponding acid.

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

such as 1-n-butyl-3-p-tolyltriazene or the like.

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

in the presence of dicyclohexylcarbodiimide, N,N1-carbonylbisimidazole, ethoxyacetylene, diphenylphosphorylazide or similar coupling agents at a temperature in the range from approx. 0 to 10°C. The ester group can then be removed, e.g. by treatment with trifluoroacetic acid and anisole at about room temperature to give the free acid

(R-L = H) .

A variation, which is preferred when n is 1, R₂ is CF₂ and R₂ is H, is to react a thioacid of formula VIII with an acrylic acid derivative of the formula instead of with the compound of formula VII, and then proceed 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.

Compounds of formula I where R is

is produced by direct oxidation of a compound of formula I where R is hydrogen, e.g. with iodine, to form the symmetrical bis compound.

Halogenated compounds of formula V, which are used as starting materials, can be prepared by methods known per se, e.g. as stated in Biochemistry 4, 2509 (1965), Aust. J. Chem.

20, 1493 (1967), J. Amer. Chem. Soc. 86_, 4709 (1964), J. Med. Chem. 20, 1176 (1977).

The products of formula I have one or more asymmetric centers, and the solid is that which is denoted by an asterisk in formula I. The compounds thus exist in stereoisomeric forms or in racemic mixtures thereof. All of these can be produced according to the invention. The syntheses described above can be carried out with the racemate or one of the enantiomers as starting material. When the racemic starting material is used in the synthesis, the stereoisomers obtained in the product can be separated by usual chromatographic or fractional crystallization methods. In general, the L-isomer with respect to the fixed asymmetric carbon atom is the preferred isomeric form.

The compounds produced according to the invention form basic salts with various inorganic and organic bases which can also be produced according to the invention. Such salts include ammonium salts, alkali metal salts such as sodium and potassium salts (which are preferred), alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases, e.g. dicyclohexylamine salts, benzathine, N-methyl-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. when isolating or cleaning the product.

The salts are formed in the usual way by reacting the free acid form of the product with one or more equivalents of the appropriate base which gives the desired salt ion, in a solvent or medium in which the salt is insoluble, or in water, after which the water is removed by freeze-drying. By neutralizing the salt with an insoluble acid such as a cation exchange resin in hydrogen form, (e.g. polystyrene sulfonic acid resin such as "Dowex" 50) or with an aqueous acid and extraction with an organic solvent, e.g. ethyl acetate, dichloromethane or the like,

the free acid form can be obtained and possibly another salt formed.

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

The new compounds are useful as hypotensive agents. They inhibit the conversion of the decapeptide angiotensin I to angiotensin II and are therefore useful in reducing or alleviating angiotensin-induced hypertension. The action of the enzyme renin on angiotensinogen, a pseudoglobulin in blood plasma, leads to the formation of angiotensin I. Angiotensin I is converted by angiotensin-converting enzyme (ACE) to angiotensin II.

The latter is an active precursor that is believed to be the component

which cause different forms of hypertension in different mammals, e.g. rats and dogs. The new compounds intervene in the angiotensinogen ->-(renin)-»• angiotensin I (ACE) ->-angiotensin II course by inhibiting the angiotensin-converting enzyme and reducing or eliminating the formation of the precursor angiotensin II. Thus, by administering a preparation containing one or more of the compounds of formula I or physiologically acceptable salts thereof, angiotensin-dependent hypertension is alleviated in mammals having this disorder. A single dose,

or preferably two to four daily doses, given on a basis

of approx. 0.1 to 100 mg/kg per day, preferably approx. 1 to 50 mg/kg per day, is appropriate for reducing blood pressure as shown by the animal model experiments described by S. L. Engel, T. R. Schaeffer,

M. H. Waugh and B. Rubin, Proe. Soc. Exp. Biol. With. 143, 483

(1973).'The compound is preferably administered orally, but parenteral administration can also be used such as subcutaneous, intramuscular, intravenous or intraperitoneal administration.

The new compounds can be used to achieve reduction

of blood pressure, by preparation in preparations 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 salts thereof are mixed with a physiologically acceptable carrier, excipient, binder, preservative, stabilizer, flavoring agent, etc. in a unit dosage form suitable according to accepted pharmaceutical practice. The amount of active substance in these preparations is such that a suitable dosage in the specified range is achieved.

Illustrative examples of the excipients that can be incorporated into tablets, capsules and the like are the following: a binder such as tragacanth gum, acacia gum, corn starch or gelatin; an auxiliary such as dicalcium phosphate or microcrystalline cellulose, a disintegrant such as corn starch, potato starch, alginic acid and the like, a lubricant such as magnesium stearate, a sweetener such as sucrose, lactose or saccharin; a flavoring such as peppermint, wintergreen oil or cherry. When the unit dosage form is a capsule, it may, in addition to materials of the above type, contain 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 unit dose. E.g. tablets can be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetener, methyl and propyl parabens as a preservative, a coloring agent and a flavoring agent such as cherry or orange flavor.

Sterile preparations for injection may be prepared according to ordinary pharmaceutical practice by dissolving or suspending the active substance in a carrier such as water for injection, a naturally occurring vegetable oil such as sesame oil, coconut oil, peanut oil, cottonseed oil, etc.

The following examples shall serve to further illustrate the invention and constitute particularly preferred embodiments. All temperatures are in °C.

Example 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 a steam bath for 1 hour and then stored at room temperature for 18 hours. The reaction mixture is distilled in vacuo to give 3-acetylthio-2-trifluoromethylpropanoic acid.

Example 2

1-( 3- acetyl- thio- 2- trifluoromethylpropanoyl)- L- proline- tert-butyl ester

L-proline tert-butyl ester (5.1 g) is dissolved in dichloromethane (40 mg), and the solution is stirred and cooled in an ice bath. Dicyclohexylcarbodiimide (6.9 g) dissolved in dichloromethane (15 ml) is added, immediately followed by a solution of 3-acetylthio-2-trifluoromethylpropanoic acid (6.5 g) in dichloromethane (5 ml). After 15 minutes of stirring in an ice bath and 16 hours at room temperature

the formed precipitate is filtered off, and the filtrate is concentrated to dryness in vacuo. The residue is dissolved in ethyl acetate and washed neutrally. The organic phase is dried over magnesium sulfate and concentrated to dryness in vacuo to give 1-(3-acetylthio-2-trifluoromethylpropanoyl)-L-proline tert-butyl ester.

Example 3

1-(3-acetylthio-2-trifluoromethylpropanoyl)-L-proline 1-(3-acetylthio-2-trifluoromethylpropanoyl)-L-proline tert-butyl ester (8 g) is dissolved in a mixture of anisole (55 ml) and trifluoroacetic acid (110ml). After 1 hour of storage at room temperature, the solvent is removed in vacuo, and the residue is precipitated several times from ether-hexane to give 1-(3-acetylthio-2-trifluoromethyl-propanoyl)-L-proline.

Example 4

1-(3-mercapto-2-trifluoromethylpropanoyl)-L-proline

1-(3-acetylthio-2-trifluoromethylpropanoyl)-L-proline (4 g) is dissolved in a mixture of water (8 ml) and concentrated ammonia (8 ml) under a blanket of nitrogen. After 25 minutes of stirring

at room temperature, the reaction mixture is cooled, acidified and extracted with ethyl acetate. The organic layer is concentrated to dryness in vacuo to give 1-(3-mercapto-2-trifluoromethyl-pxopanoyl)-L-proline.

Example 5

2- bromo- 3, 3, 3- trifluoropropanoic acid

3,3,3-trifluoroalanine (.88 g) is dissolved in a mixture of potassium bromide (250 g) and 2.5N sulfuric acid (1,240 ml). The solution is cooled to 0° with an ice-salt bath and sodium nitrite (65.5 g) is added in small portions over a period of 1 hour with vigorous stirring. The reaction mixture is stirred in the cooling bath for a further 1 hour and then extracted with ether. The organic layer is washed with water, dried over magnesium sulfate and concentrated to dryness in vacuo to give 2-bromo-3,3,3-trifluoropropanoic acid.

Example 6

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 a steam bath for 2 hours. Excess thionyl chloride is removed in vacuo, and the residue is distilled under reduced pressure to give 2-bromo-3,3,3-trifluoropropanoic acid chloride.

Example 7

1-( 2- acetylthio- 3, 3, 3- trifluoropropanoy1)- L- proline

To a solution of L-proline (5.75 g) in 1N sodium hydroxide (50 ml), cooled in an ice-water bath, is added 2-bromo-3,3,3-trifluoropropanoic acid chloride (12 g) and the mixture is stirred vigorously at room temperature for 3 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 16 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 give 1-(2-acetylthio-3,3,3-trifluoro-propanoyl)-L-proline.

Example 8

1-( 2- mercapto-3, 3, 3- trifluoropropanoyl)- L- proline

1-(2-acetylthio-3,3,3-trifluoropropanoyl)-L-proline (4 g) is dissolved in a mixture of water (8 ml) and concentrated ammonia (8 ml) under a blanket of nitrogen. After 30 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 give 1-(2-mercapto-3,3,3-trifluoropropanoyl)-L-proline.

Example 9

1, 1'-[dithiobis-(2-trifluoromethyl-3-propanoyl)]-bis-L-proline

1-(3-mercapto-2-trifluoromethylpropanoyl)-L-proline (1 g) is dissolved in water adjusted to pH 7 with N sodium hydroxide. An ethanolic solution of iodine is added dropwise while the pH value is kept between 6 and 7 by careful addition of N sodium hydroxide. When a permanent yellow color is obtained, the addition of iodine is stopped,

and the color is removed with sodium thiosulphate. The reaction mixture is acidified and extracted with ethyl acetate. The organic layer is dried over magnesium sulfate and concentrated to dryness to give 1,1'-[dithiobis-(2-trifluoromethyl-3-propanoyl)]-bis-L-proline.

Example 10

1-( 3- acetylthio- 2- trifluoromethylpropanoyl)- L- proline sodium salt

A suspension of 1-(3-acetylthio-2-trifluoromethylpropanoyl)-L-proline (1 g) in water (10 ml) is adjusted to pH 8

by addition of N sodium hydroxide. The resulting solution is freeze-dried to give 1-(3-acetylthio-2-trifluoromethylpropanoyl)-L-proline sodium salt.

Example 11

1-( 3- acetylthio- 2- trifluoromethylpropanoyl)- 4, 4- difluoro- L- proline

To a solution of 4,4-difluoro-2-proline (7.5 g) i

N sodium hydroxide (50 ml) cooled in an ice-water bath, 3-acetylthio-2-trifluoromethylpropanoic acid chloride (prepared from 3-acetylthio-2-trifluoromethylpropanoic acid and thionyl chloride by the method according to example 6) (12 g) is added, and the mixture is stirred vigorously at room temperature for 2 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 give 1-(3-acetylthio-2-trifluoromethylpropanoyl)-4,4-difluoro-L-proline.

Example 12

1-( 3- mercapto- 2- trifluoromethylpropanoyl)- 4, 4- difluoro- L- proline

By using 1-(3-acetylthio-2-trifluoromethylpropanoyl)-4,4-difluoro-L-proline instead of 1-(3-acetylthio-2-trifluoromethylpropanoyl)-L-proline in the method according to example 4,

1-(3-mercapto-2-trifluoromethylpropanoyl)-4,4-difluoro-L-proline is obtained.

Example 13

1,1'-[dithiobis-(2-trifluoromethyl-3-propanoyl)]-bis-4,4-difluoro-L-proline

By using 1-(3-mercapto-2-trifluoromethylpropanoyl)-4,4-difluoro-L-proline instead of 1-(3-mercapto-2-trifluoromethyl-propanoyl)-L-proline in the method according to example 9, one obtains 1,1-[dithiobis-(2-trifluoromethyl-3-propanoyl)]-bis-4,4^difluoro-L-proline.

Example 14

1-(4,4,4-trifluoro-2-butenyl)-L-proline

Boric anhydride (7.0 g, 0.1 mol) (prepared by melting boric acid in a platinum crucible and crushing it under nitrogen) is mixed with ethyl 3-hydroxy-4,4,4-trifluorobutanoate (32.2 g, 0.173 mol ) in a 50 mL flask fitted with a Dean-Stark trap,

and the mixture is heated at 180° with a salt bath until all the anhydride is dissolved (6 hours). The temperature is raised to 350°

and during this time 23 ml of distillate is collected in the trap. The distillate is returned to the reaction flask, and the heating step is repeated. This process is repeated 4 times to ensure complete dehydration of the hydroxyester. The distillate is dissolved in petroleum ether, dried over phosphorus pentoxide and distilled to give 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).

The ester is mixed 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 collected, dried over sodium sulfate, concentrated, and the residue distilled to give crystalline 4,4,4-trifluoro-^2-butenoic acid (b.p. 145-153°). After recrystallization from pentane, the acid melts at 54-55°, yield 4.6 g.

A mixture of 4,4,4-trifluoro-2-butenoic acid (4.91 g,

35 mmol), hydroxybenzotriazole (4.73 g, 35 mmol), L-proline-t-butyl ester (6.00 g, 35 mmol) and dicyclohexylcarbodiimide (7.22 g, 35 mmol) in methylene chloride (200 mL) stirred under nitrogen overnight at room temperature. The mixture is filtered, and the filtrate is washed with 5% sodium bisulfate (2 x 50 ml) and saturated sodium bicarbonate (2 x 50 ml), dried over sodium sulfate and concentrated to give an oil. This is dissolved in ether, and the solution is cooled and filtered free of precipitate. The filtrate is concentrated to give a solid (m.p. 95-100°, 8.7 g) which shows a single spot by TLC (silica gel EM 50/50, Et0Ac/CH2Cl2, Rf = 0.85).

A mixture of the 1-(4,4,4-trifluoro-2-butenoyl)-L-proline-t-butyl ester obtained above (4.0 g, 13.6 mmol) is mixed with trifluoroacetic acid (60 ml) and anisole ( 13 ml) and stirred under nitrogen for 1 hour. The solvents are removed under vacuum and the residue, dissolved in ether (10 ml) is poured into pentane (500 ml).

This precipitation technique is repeated, and the residue is allowed to stand at 0° i

72 hours, during which time crystallization takes place.

1-(4,4,4-trifluoro-2-butenoyl)-L-proline is recrystallized from ethyl acetate-hexane; yield 2.48 g, m.p. 119-120°.

Example 15

1-( 3- mercapto-4, 4, 4- trifluorobutanoy1)- L- proline

Thiolacetic acid (1.5 mL) is mixed with 1-(4,4,4-trifluoro-2-butenoyl)-L-proline (720 mg, 3 mmol) under argon, and the mixture is stirred at room temperature overnight. Excess thiolacetic acid is removed under vacuum, and remaining 1-(3-acetylthio-4,4,4-trifluorobutanoyl)-L-proline is mixed with aqueous ammonia

(15 ml concentrated 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 are dried over sodium sulfate and concentrated to give an oil. This is cleaned by dissolution

in water (double-distilled), the solution is treated with charcoal and

filtered through a "Millipore" filter (0.4 pm followed by 0.08 ym). Lyophilization of this solution gives 700 mg of 1-(3-mercapto-4,4,4-trifluorobutanoyl)-L-proline as a harmless glass.

Rf (benzene:acetic acid 7:1) 0.24.

Example 16

3- acetylthio- 4, 4, 4- trifluorobutanoic acid chloride

By using 4,4,4-trifluoro-2-butenoic acid instead of 2-trifluoromethyl-acrylic acid in the method according to example 1, 3-acetylthio-4,4,4-trifluorobutanoic acid is obtained, and this is then chlorinated with thionyl chloride as in example 6 for to give 3-acetylthio-4,4,4-trifluorobutanoic acid chloride.

Example 17

1-(3-mercapto-4,4,4-trifluorobutanoyl)-4,4-difluoro-L-proline

By using 1-(3-acetylthio-4,4,4-trifluorobutanoic acid chloride instead of 3-acetylthio-2-trifluoromethylpropanoic acid chloride in the method according to example 11 and then subjecting the product to the method according to example 4, one obtains 1-(3-mercapto-4, 4,4-trifluorobutanoyl)-4,4-difluoro-L-proline.

Example 18

1,1'-[dithiobis-(4,4,4-trifluoro-3-butanoyl)]-bis-L-proline

By using 1-(3-mercapto-4,4,4-trifluorobutanoyl)-L-proline instead of 1-(3-mercapto-2-trifluoromethylpropanoyl)-L-proline in the method according to example 11, one obtains 1,1' -[dithiobis-(4,4,4-trifluoro-3-butanoyl)]-bis-L-proline.

Example 19

cis- 4-fluoro-L-proline hydrobromide

a) N-carbobenzyloxy-4-hydroxy-L-proline methyl ester N-carbobenzyloxy-4-hydroxy-L-proline [12.4 g (0.047 mol)]

is esterified with diazomethane in dioxane ether as described in JACS, 79, 191 (1957). To avoid the dioxane freezing, the addition of the diazomethane solution is started at 10° and completed at 0-2°. The yield of almost 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 mol) of N-carbo-benzyloxy-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 chilled for 3 days and is then added, with stirring, to 300 ml of ice-cold 2N 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 4 ), 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°. After crystallization from 85 ml of isopropanol, the colorless solid N-carbobenzyloxy-4-tosyloxy-L-proline methyl ester weighs 15.9 g (70%); sm.p. 67-69°, [α]^ -30° (c = 1, methanol).

c) cis-N-carbobenzyloxy-4-fluoro-L-proline-methyl ester

A stirred suspension of 19.1 g (0.044 mol) of N-carbo-benzyloxy-4-tosyloxy-L-proline-methyl ester in 100 ml of redistilled diethylene glycol is treated at 42° (under argon) with 19.1 g (0. 33 moles) of anhydrous potassium fluoride, and the resulting solution is heated at 81-84° for 20 hours. After cooling, the pale yellow solution is worked up to give 18.6 g (100%) of cis-N-carbobenzyloxy-4-fluoro-L-proline methyl ester as a pale yellow oil.

d) cis-N-carbobenzyloxy-4-fluoro-L-proline Cis-N-carbobenzyloxy-4-fluoro-L-proline methyl ester

(18.4 g, ca. 0.044 mol) is dissolved in 140 ml of methanol, treated dropwise at -1° to 4° with 33 ml (0.066 mol) of 2N sodium hydroxide, then kept at 0° for 1 hour and at room temperature overnight. After removing approx. half of the solvent on a rotary evaporator, dilute the solution with 300 ml of water,

washed with ether (washing liquids are 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 saturated sodium chloride solution, dried (MgSO 4 ), 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. During inoculation and rubbing, crystalline cis-N-carbobenzyloxy-4-fluoro-L-proline-cyclohexylamine salt is secreted: weight after cooling overnight 11.0 g, m.p. 180-183° (p. 175°). After crystallization from 70 ml of ethanol, the colorless solid weighs 7.6 g, m.p. 185-187°, [cc]j^ -40° (c = 1, methanol).

The cyclohexylamine salt is suspended in 75 ml of ethyl acetate, stirred and treated with 45 ml of hydrochloric acid. The teams are separated,

the aqueous phase is extracted with further ethyl acetate (2 x 75 ml), then the combined organic layers are dried (MgSO 4 ),

and the solvent is evaporated. The remaining free acid, cis-N-carbobenzyloxy-4-fluoro-L-proline, crystallizes after drying at 0.2 mm and 45°, yield 5.7 g (49%), m.p. 116-118°.

e) cis-4-fluoro-L-proline hydrobromide

Cis-N-carbobenzyloxy-4-fluoro-L-proline (5.5 g, 0.021 mol)

treated with 28 ml of hydrogen bromide in acetic acid (30-32%), carefully covered and stirred for 1 hour. Ether (300 ml) is added to the yellow mixture, and when the crystalline product has settled, the ethereal liquid is decanted, and the material is washed with 300 ml of fresh ether by decantation. The product is finally heated in a steam bath with 70 ml of methyl ethyl ketone, cooled for 2 hours, washed with cold methyl ethyl ketone and with ether and dried in vacuum. The yield of almost colorless solid, cis-4-fluoro-L-proline hydrobromide is 3.8 g (86%), m.p. 189-191° (dec.), [α]^<6>-19° (c = 1, methanol).

Some of the crude hydrobromide salt is converted to the free acid by passing it through a column of "Dowex" 1-X8 ion exchange resin.

Example 20

cis-1-[D-3-(acetylthio)-2-methylpropanoyl]-4-fluoro-L-proline cis-4-fluoro-L-proline hydrobromide (4.5 g, 0.021 mol)

and 4.2 g (0.023 mol) 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 (about 20 minutes). The mixture is worked up after a further 1 hour by washing with ethyl acetate (2 x 50 ml), covering with a layer of 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 collected, dried and evaporated. The solid residue from the ethyl acetate evaporation

rubbed under ether, and the evaporation repeated; weight of colorless product, 5.4 g (93%), m.p. 146-148° (p. 133°), [a]^<6>-132°

(c = 1, methanol). The dicyclohexylamine salt is prepared by adding dicyclohexylamine to cis-1-[D-3-(acetylthio)-2-methylpropanoyl-4-fluoro-L-proline in 70 ml of ethyl acetate. 8.1 g of salt, which

crystallizes, is obtained, m.p. 202-204° (p. 187°), [a]^6 -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. or [a]D-

The dicyclohexylamine salt (16.9 g) is converted back to

the free acid by partitioning between 10% potassium bisulphate and ethyl acetate (60 ml 10% KHSO^, 4 x 50 ml ethyl acetate extractions). The organic layers are combined and evaporated to dryness to give 4.1 g (71%) of colorless, free acid, m.p. 154-156° (p. 140°) [a]^<6>-142°

(c = 1, methanol).

Example 21

cis- 4- fluoro- 1-( D- 3- mercapto- 2- methylpropanoyl)- L- proline

cis-1-[D-3-(acetylthio)-2-methylpropanoyl]-4-fluoro-L-proline (3.9 g, 0.014 mol) 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 glassy product which slowly crystallizes when dried at 0.2 mm and 50°. The material is rubbed out with 20 ml of ethyl acetate (with gentle heating

under argon), diluted with 25 ml of hexane, rubbed and cooled overnight (under argon). After filtration under argon, washing with hexane and drying in vacuum, the colorless solid cis-4-fluoro-1-(D-3-mercapto-2-methylpropanoyl)-L-proline weighs 2.8 g (85%), sm. p. 135-137° (p. 129°) [α]^<6>-116° (c = 1, methanol).

Example 22

1- [ 3-( acetylthio)- 2- chloropropanoyl]- L- proline

(Isomer A)

L-proline (1.44 g) and sodium carbonate (667 mg) dissolve

in 17 ml of water and stirred in an ice bath. To this, sodium carbonate (2 g) is added to 8.5 mg of water, immediately followed by 3-acetylthio-2-chloropropanoic acid chloride (2.5 g). 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 cooled, acidified with concentrated hydrochloric acid, saturated with sodium chloride, extracted into ethyl acetate, dried over magnesium sulfate and concentrated to dryness in vacuo to give 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 give 2 g of product which crystallizes from water to give 450 mg of 1-[ 3-(acetylthio.)-2-chloropropanoyl]-L-proline, sm.p. 111-113°. [alD-170° (c = 1, ethanol).

Example 2 3

1-[ 3-(acetylthio)-2-chloropropanoyl]-L-proline

(Isomer B)

The aqueous mother liquors from example 22 are lyophilized and chromatographed on silica gel with benzene/acetic acid 7:1. The fractions containing the UV-absorbing material and shown to be homogeneous by TLC are collected, concentrated to dryness and crystallized from water, yield 800 mg, m.p. 90-109°

[ct]D 25 -4 o(c = 2.1, ethanol). The mother liquors are concentrated to dryness by freeze-drying, and the remaining 1-[3-(acetylthio)-2-chloro-propanoyl-L-proline (isomer B) is crystallized from ether-hexane, yield 380 mg, m.p. 108-110°, [α]^<5>+ 17.6° (c = 1.25, ethanol).

Example 2 4

1- [ 3-( acetylthio)- 2- bromopropanoyl]- L- proline

By using 3-acetylthio-2-bromopropanoic acid chloride instead of 3-acetylthio-2-chloropropanoic acid chloride in the method according to example 22, 1-[3-(acetylthio)-2-bromopropanoyl]-L-proline is obtained, m.p. 109-110°, [α]D- 162° (c - 1.39, ethanol).

Example 25

cis- 4- chloro- l-( D- 3- mercapto- 2- methylpropanoyl- L- proline

By using cis-4-chloro-L-proline [Aust. J. Chem. 20, 1493 (1967)] instead of cis-4-fluoro-L-proline hydrobromide in the method according to example 20 and then subjecting the product to the method according to example 21, one obtains cis-1-[D-3-(acetylthio)-2- methylpropanoyl-4-chloroproline and cis-4-chloro-1-(D-3-mercapto-2-methylpropanoyl)-L-proline.

Example 26

trans- 4- bromo- l-( D- 3- mercapto- 2- methylpropanoyl)- L- proline

By using trans-4-bromo-L-proline [Aust. J. Chem. 20, 1493 (1967)] instead of cis-4-fluoro-L-proline hydrobromide in the method according to Example 20, and then subjecting the product to the method according to Example 21, one obtains trans-1-

[D-3-(acetylthio)-2-methylpropanoyl]-4-bromo-L-proline and trans-4-bromo-1-(D-3-mercapto-2-methylpropanoyl)-L-proline.

Example 2 7

cis- 4- iodo- l-( D- 3- mercapto- 2- methylpropanoyl- L- proiin

By using cis-4-iodo-L-proline instead of cis-4-fluoro-L-proline hydrobromide in the method according to Example 20, and then subjecting the product to the method according to Example 21, one obtains cis-1-[D-3- (acetylthio)-2-methylpropanoyl-4-iodo-L-proline and cis-4-iodo-1-[D-3-mercapto-2-methylpropanoyl]-L-proline.

Example 2 8

cis- 4- fluoro- 1-( 3- mercapto- 2- trifluoromethylpropanoyl)- L- proline

By using 3-acetylthio-2-trifluoromethylpropanoic acid chloride instead of 3-acetylthio-2-methylpropanoic acid chloride at . the method according to example 20 and then subjecting the product to the method according to example 21, one obtains cis-1-[3-(acetylthio)-2-trifluoromethylpropanoyl]-4-fluoro-L-proline and cis-4-fluoro-1-(3- mercapto-2-trifluoromethylpropanoyl)-L-proline.

Example 29

cis- 3- fluoro- DL- proline- hydrobromide

By using N-carbobenzyl.oxy-3-hydroxy-DL-proline

[J. Am. Chem. Soc. 8_5 , 2824 (1963)] instead of N-carbobenzyloxy-4-hydroxy-L-proline in the method according to example 19, cis-3-fluoro-DL-proline hydrobromide is obtained.

Example 30

cis- 3- fluoro- 1-( D- 3- mercapto- 2- methylpropanoy1- DL- proline

By using cis-3-fluoro-DL-proline hydrobromide instead of cis-4-fluoro-DL-proline in the method according to Example 20 and then subjecting the product to the method according to Example 21, one obtains cis-1-[D-3-( acetylthio)-2-methylpropanoyl]-3-fluoro-DL-proline and cis-3-fluoro-1-(D-3-mercapto-2-methylpropanoyl)-DL-proline.

Example 31

cis- 3- chloro- l-( D- 3- mercapto- 2- methylpropanoyl- L- proline

By using cis-3-chloro-L-proline [obtained from 3-hydroxy-proline by the method described in Aust. J. Chem. 20, 149 3

(1967)] instead of cis-4-fluoro-L-proline hydrobromide in the method according to Example 20 and then subjecting the product to the method according to Example 21, one obtains cis-1-[D-3-(acetylthio)-2-methylpropanoyl-3 -chloro-L-proline and c,is-3-chloro-1-(D-3-mercapto-2-methylpropanoyl)-L-proline.

Example 32

4, 4- dichloro- l-( D- 3- mercapto- 2- methylpropanoyl)- L- proline

By using 4,4-dichloro-L-proline [prepared from 4-keto-L-proline-diketopiperazine and phosphorus pentachloride by the method described in J. Med. Chem. 20, 1176 (1977)] instead of cis-4-fluoro-L-proline hydrobromide in the method according to Example 20, and then subjecting the product to the method according to Example 21, one obtains 1-[D-3-(acetylthio)-2-methylpropanoyl ]-4,4-dichloro-L-proline and 4,4-dichloro-1-(D-3-mercapto-2-methylpropanoyl)-L-proline.

Example 33

1, 1'-[ Dithiobis-( 2- D- methylpropanoyl)]- bis-[( cis- 4- fluoro)- L- proline]

By using cis-4-fluoro-1-(D-3-mercapto-2-methylpropanoyl)-L-proline instead of 1-(3-mercapto-2-trifluoromethylpropanoyl)-L-proline in the method according to example 9, one obtains 1,1'-[dithiobis-(2-D-methylpropanoyl)]-bis-[(cis-4-fluoro)-L-proline].

Example 34

4, 4-difluoro-1-(3-mercaptobutanoyl)-L-proline

By using 3-acetylthiobutanoic acid chloride instead of 3-acetylthio-2-trifluoromethylpropanoic acid chloride in the method according to example 11 and then subjecting the product to the method according to example 12, one obtains 1-(3-acetylthiobutanoyl)-4,4-difluoro-L-proline and 4, 4-difluoro-1-(3-mercaptobutanoyl)-L-proline.

Example 35

1-(3-propanoylthio-2-trifluoromethylpropanoyl)-L-proline

By using thiopropanoic acid instead of thioacetic acid

by the method according to example 1, and then submit

the product the methods according to examples 2 and 3, 3-propanoylthio-2-trifluoromethylpropanoyl, 1-(3-propanoylthio-2-trifluoromethylpropanoyl)-L-proline tert-butyl ester and 1-(3-propanoylthio-2-trifluoromethylpropanoyl) are obtained respectively -L-proline.

Example 36

3-(4-Methoxybenzyl)-thio-2-trifluoromethylpropanoic acid

A uniform mixture of 1-trifluoromethylacrylic acid (3.9 g)

and 4-methoxybenzylthiol (4.3 g) is stirred at 100-110° for 1 hour. The mixture is allowed to cool to room temperature, and the solid is recrystallized from cyclohexane, m.p. 72-74°.

Example 37

1-[ 3-( 4- methoxybenzyl) thio- 2- trifluoromethylpropanoyl)- L- proline tert- butyl ester

A solution of 3-(4-methoxybenzyl)thio-2-trifluoromethylpropanoic acid (6.5 g) and proline tert-butyl ester (3.76 g) in dichloromethane (500 ml) is stirred at 0° and treated with dicyclohexylcarbodiimide (4, 53 g). After 30 minutes at 0° and overnight at room temperature, the mixture is filtered, and the filtrate is washed neutrally. The organic layer is dried and concentrated to dryness in vacuo. TLC [silica gel, methylene chloride/ethyl acetate (95:5)] shows two main spots, Rf 0.46 and 0.51, corresponding to the two diastereoisomers.

Example 38

1-(3-mercapto-2-trifluoromethylpropanoyl)-L-proline

A solution of 1-[3-(4-methoxybenzyl)thio-2-trifluoromethylpropanoyl)-L-proline tert-butyl ester (4.47 g) obtained in Example 37 and anisole (10 ml) is cooled to 0°, and trifluoroacetic acid (100 ml)' are added, followed by mercuric (II) acetate (3.18 g). The bath is removed, and the mixture is stirred at room temperature for 1 hour. The mixture is concentrated to dryness in vacuo, and the residue is triturated with ether-hexane. The insoluble material is suspended in water, and hydrogen sulphide is bubbled through for 10 minutes. The precipitate is removed by filtration, and the filtrate is freeze-dried

to give the product, 1-(3-mercapto-2-trifluoromethylpropanoyl)-L-proline as an amorphous solid. Rf 0.29-0.31 (silica gel - benzene:

acetic acid 7:1).

Example 39

3- acetylthio- 2- chloropropanoic acid chloride

By using 2-chloroacrylic acid instead of 2-trifluoromethylacrylic acid in the method according to example 1 and then allowing the product to react with thionyl chloride, 3-acetylthio-2-chloropropanoic acid and 3-acetylthio-2-chloropropanoic acid chloride are obtained.

Example 40

D- 3- acetylthio- 2- methylpropanoic acid chloride

A suspension of 1-(D-3-mercapto-2-methylpropanoyl)-L-proline (150 g, 690 mmol) in 1274 mL of water and 426 mL of concentrated hydrochloric acid (5.526 mol) is refluxed under nitrogen with stirring for 8 h. The resulting solution is kept at room temperature overnight and then extracted with 400 ml of chloroform (10x). The combined chloroform extracts are dried over. magnesium sulfate under nitrogen and then evaporated. To the residue, 81.2 g, are added acetic anhydride (176 ml/1.809 mol) and pyridine, 180 ml, and the mixture is kept at room temperature for 20 hours. The mixture is then evaporated and the oily residue dissolved

in 1000 ml of ethyl acetate, and the solution is washed successively with 200 ml of 5% hydrochloric acid-saturated sodium chloride (wash liquids pH 2),

200 ml of saturated sodium chloride solution (2 times, other washing liquid, pH 7), and then the solvent is evaporated. To the clear, oily residue, [96.9 g, 86.5%, la]^ 5 = -61.8° (CHCl 3 )] is added freshly distilled thionyl chloride (83 ml, 1.173 mol) and the resulting solution is stirred at room temperature with gas development for 18 hours. Excess thionyl chloride is evaporated under vacuum with 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) , [α]p<5->42.5° (c 2, methanol) .

Example 41

3- acetylthio- 2- bromopropanoic acid chloride

By using 2-bromoacrylic acid instead of 2-trifluoromethylacrylic acid in the method according to 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 4 2

Trans- 1-[ D- 3-( acetylthio)- 2- methyl- l- oxopropyl]- 4- fluoro- L- proline

A) Trans-1- carbobenzylox. y- 4- fluoro- L- proline

A solution of 6.2 g of trans-1-carbobenzyloxy-4-fluoro-L-proline methyl ester in 50 ml of methanol is treated dropwise at 0-5° with 11.5 ml of 2N sodium hydroxide solution, and after 1 hour at 0° it obtains is heated to room temperature overnight. The reaction mixture is concentrated under reduced pressure to approx. half of its original volume and then diluted with 100 ml of water. The aqueous reaction mixture is extracted with ether, and the ether extracts are discarded. The aqueous solution is acidified under 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 give the desired product.

It is purified by conversion to the cyclohexylamine salt, m.p. 194-196°, [α]° -44° (c = 1% in methanol).

The free acid is obtained by suspending the 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 is concentrated under reduced pressure to give the desired trans-1-carbobenzyloxy-4-fluoro-L-proline.

B) Trans-4-fluoro-L-proline hydrobromide

A mixture of 3 g of trans-1-carbobenzyloxy-4-fluoro-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 are added. The solvent is decanted from the precipitate, which is then triturated with fresh ether and finally with methyl ethyl ketone. The trans-4-fluoro-L-proline hydrobromide melts at 162-164° (dec.), [cx]^ -30°

(c = 1% in methanol).

C) Trans- 1-[ D- 3-( acetylthio)- 2- methyl- l- oxopropyl]- 4- fluoro-L- proline

To a stirred solution of 1.9 g of trans-4-fluoro-L-proline hydrobromide in 25 ml of cold water is added 1 g of sodium carbonate to adjust the pH to 8.2. During continued cooling (5°) and stirring, 1.8 g of D-3-acetylthio-2-methylpropionyl chloride in 2.5 ml of ether are then added dropwise while the pH is kept at approx. 8.2-8.3 by dropwise addition of a 25% aqueous sodium carbonate solution. Stirring and cooling are continued for 1 hour after the addition is complete. The reaction mixture is extracted with ethyl acetate (2 x 25 ml), and the extracts are discarded. To. 50 ml of ethyl acetate are added to the aqueous layer, and with stirring and 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 is separated.

The aqueous layer is extracted with additional ethyl acetate (3 x 25 mL), and the combined ethyl acetate extracts are dried over anhydrous magnesium sulfate and concentrated under reduced pressure to give 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 g of dicyclohexylamine in 35 ml of ethyl acetate. The precipitated salt is filtered and recrystallized from isopropanol to give the dicyclohexylamine salt of trans-1-[D-3-(acetylthio)-2-methyl-1-oxopropyl]-4-fluoro-L-proline, m.p. 209-211°, [α]^<5>-85° (c = 1% methanol).

The free acid is recovered by dissolving the dicyclohexylamine salt

. in 5% aqueous potassium hydrogen sulphate and extract with ethyl acetate. The ethyl acetate solution is dried over anhydrous magnesium sulfate

and concentrated under reduced pressure to give the desired trans-1-[D-3-(acetylthio)-2-methyl-1-oxopropyl]-4-fluoro-L-proline.

Example 4 3

trans- 4- fluoro- 1-( D- 3- mercapto- 2- methyl- l- oxopropyl)- L- proline

Argon is passed through a cold solution of 4.2 ml of concentrated ammonium hydroxide in 16 ml of water. To this solution is added, with stirring in an atmosphere of argon, 1.8 g of trans-1-[D-3-(acetylthio)-2-methyl-1-oxopropyl]-4-fluoro-L-proline. The reaction mixture is stirred for a further 2 hours and then extracted with ethyl acetate which is discarded. The aqueous layer is stirred,

30 ml of ethyl acetate are added, and the aqueous layer is acidified with

concentrated hydrochloric acid. The aqueous layer is saturated with sodium chloride, and the ethyl acetate layer is 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 give the desired trans-4-fluoro-1-[D-3-mercapto-2-methyl-1-oxopropyl)-L-proline, [ot]D 26 -112 o (c = 1% in methanol).

Example 4 4

1-[ D- 3-( acetylthio)- 2- methyl- l- oxopropyl]- 4, 4- difluoro- L- proline A) 1- carbobenzyloxy- 4, 4- difluoro- L- proline- methyl ester

To a cooled, stirred solution of 3.3 g of 1-carbobenzyloxy-4-keto-L-proline methyl ester in 80 ml of methylene dichloride, 3.3 ml of diethylaminosulphur trifluoride is added dropwise. The reaction mixture is allowed to stand overnight at room temperature. About. 100 g crushed ice for

is stirred, and the reaction mixture is stirred for 45 minutes. The organic layer is separated, and the aqueous layer is extracted with methylene chloride (2 x 40 ml). The combined extracts are dried over anhydrous magnesium sulfate and then concentrated under reduced pressure to give the desired 1-carbobenzyloxy-4,4-difluoro-L-proline methyl ester.

B) l-carbobenzyloxy-4,4-difluoro-L-proline

A solution of 5.6 g of 1-carbobenzyloxy-4,4-difluoro-L-proline methyl ester in 50 ml of methanol is treated dropwise with

11.5 ml of 2N sodium hydroxide solution at 0-5°. The reaction mixture is allowed to stand at 0° for 1 hour and is then warmed to room temperature overnight. The reaction mixture is concentrated under reduced pressure to approx. half of its original volume and then diluted with 100 ml of water. The aqueous reaction mixture is extracted with ether, and the ether extracts are discarded. The aqueous solution is acidified by cooling with dilute hydrochloric acid to pH 2

and then extracted with ethyl acetate (3 x 50 ml). The ethyl acetate extracts are combined and washed with saturated sodium chloride solution, dried over anhydrous magnesium sulfate and concentrated to give the desired product, 1-carbobenzyloxy-4,4-difluoro-L-proline.

It is purified by conversion to the cyclohexylamine salt, m.p. 180-185°, [a]^<6>= -24° (c.= 1% 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 give the desired 1-carbobenzyloxy-4,4-difluoro-L-proline.

C) 4, 4-difluoro-L-proline hydrobromide

A mixture of 2.4 g of 1-carbobenzyloxy-4,4-difluoro-L-proline and 12 ml of hydrogen bromide in acetic acid (30-32%) is stirred

for 30 minutes at room temperature, and then add 300 ml

anhydrous ether. 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 16 3-165° (dec.), [ a]* 5 -14° (c = 1% in methanol).

D) 1-[ D- 3-( acetylthio)- 2- methyl- l- oxopropyl]- 4, 4- difluoro-L- proline

To a stirred solution of 2.7 g of 4,4-difluoro-L-proline hydrobromide in 30 ml of water, cooled to 5°, solid carbonate is added to adjust the pH to 8.4. With continued cooling and stirring, 2.4 g of D-3-acetylthio-2-methylpropionyl chloride in 3 ml of anhydrous ether are then added dropwise, while the solution's pH value is kept at 8.1-8.3 by adding a 25% aqueous sodium carbonate solution. Stirring and cooling are continued for 1 hour after the addition is complete. The reaction mixture is extracted with ethyl acetate (2 x 25 ml), and the extracts are discarded. 50 ml of ethyl acetate is added to the aqueous layer, and with stirring and 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 is separated. The aqueous layer is extracted with ethyl acetate (3 x 25 mL), and the combined ethyl acetate extracts are dried over anhydrous magnesium sulfate and concentrated under reduced pressure to give 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 g of dicyclohexylamine in 5 ml of ethyl acetate. The precipitated salt is filtered and recrystallized from ethanol. The dicyclohexylamine salt of 1-[D-3-(acetylthio)-2-methyl-l-oxopropyl]-4,4-difluoro-L-proline melts at 225-227°, [a]^<5>= -70° ( c = 0.5% in methanol).

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

Example 45

4, 4- difluoro- 1-( D- 3- mercapto- 2- methyl- l- oxopropyl)- 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 is added, with stirring in an atmosphere of argon, 2.1 g of 1-[D-3-(acetylthio)-2-methyl-1-oxopropyl]-4,4-difluoro-L-proline. The reaction mixture is stirred for a further 2 hours and then extracted with ethyl acetate which is discarded. The aqueous layer is stirred, 30 ml of ethyl acetate is added, and the aqueous layer is acidified with concentrated hydrochloric acid. The aqueous layer is saturated with sodium chloride, and the ethyl acetate layer is 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 give the desired 4,4-difluoro-1-(D-3-mercapto-2-methyl-1-oxopropyl)-L-prqline.

Example 46

1-[ D- 3-( acetylthio)- 2- methyl- l- oxopropyl]- 5- fluoro- 2- pipecolic acid A) l- carbobenzyloxy- 5- hydroxy- L- pipecolic acid methyl ester

A solution of 5 g of 1-carbobenzyloxy-5-hydroxy-L-pipecolic acid in 60 ml of dioxane is treated with an ethereal solution of diazomethane in portions, until the yellow color remains. The temperature is kept at approx. 5° during the addition. Excess diazomethane is decomposed with glacial acetic acid, and the resulting solution is dried over anhydrous magnesium sulfate. The solution is concentrated under reduced pressure to give the desired 1-carbobenzyloxy-5-hydroxy-L-pipecolic acid methyl ester as a pale yellow viscous oil.

B) 1-carbobenzyloxy-5-tosyloxy-L-pipecolic acid methyl ester

To a stirred solution of 5.7 g of 1-carbobenzyloxy-5-hydroxy-L-pipecolic acid methyl ester in 12 ml of pyridine is added dropwise at 5 to 8° a solution of 4 g 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 2N hydrochloric acid. The precipitate is dissolved in chloroform, and the aqueous solution is extracted with further chloroform (3 x 75 ml). The combined chloroform solutions are dried over anhydrous magnesium sulfate and concentrated under reduced pressure to give the desired l-carbobenzyloxy-5-tosyloxy-L-pipecolic acid methyl ester, which melts at 74-76° after crystallization from isopropanol, [ct]^ = -5°, c = 1% in methanol, [a]D 2 6 = -11 o, C = 1% in chloroform.

C) l-carbobenzyloxy-5-fluoro-L-pipecolic acid methyl ester

A stirred suspension of 5.2 g of 1-carbobenzyloxy-5-tosyloxy-L-pipecolic acid methyl ester in 50 ml of diethylene glycol is treated with 5.2 g of anhydrous potassium fluoride, and the mixture is heated at 80° C. 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 give the desired 1-carbobenzyloxy-5-flupr-L-pipecolic acid methyl ester as a yellow oil.

D) 1-carbobenzyloxy-5-fluoro-L-pipecolic acid

To a cooled (0°) solution of 4.6 g of 1-carbobenzyloxy-5-fluoro-L-pipecolic acid methyl ester in 32 ml of methanol is added 7.3 ml

2N sodium hydroxide solution. The reaction mixture is allowed to stand

. at 0-5° for 1 hour, and at room temperature overnight. The solution is concentrated to approx. half of its original 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 give the desired 1-carbobenzyloxy-5-fluoro-L-pipecolic acid. It is purified by conversion to the cyclohexylamine salt which melts at 158-161°, [a]D = -11° (c = 1% 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 g of 1-carbobenzyloxy-5-fluoro-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 is dried under reduced pressure to give 5-fluoro-L-pipecolic acid hydrobromide.

F) 1-[ D- 3-( acetylthio)- 2- methyl- l- oxopropyl]- 5- fluoro-L-pipecolic acid

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

The acid is purified by conversion to the dicyclohexylamine salt followed by crystallization of the salt from acetonitrile.

Example 4 7

5- f fluoro- 1- ( D- 3- mercapto- 2- methyl- l- oxopro. py 1) - L- pipecolic acid

Nitrogen is bubbled through a solution (5°) of 11 ml of concentrated ammonium hydroxide in 25 ml of water for 30 minutes. 1.6 g of 1-[D-3-(acetylthio)-2-methyl-1-oxopropyl]-5-fluoro-L-pipecolic acid is added to this solution, and the mixture is stirred for 15 minutes at 5° and then for 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 give 5-fluoro-1-(D-3-mercapto-2-methyl-1-oxopropyl)-L-pipecolic acid.

Example 4 8

1-(3-mercapto-2-trifluoromethylpropanoyl)-L-pipecolic acid

By using L-pipecolic acid tert-butyl ester instead of L-proline tert-butyl ester in the method according to example 2,

and subjecting the product to the methods according to examples 3 and 4, 1-(3-mercapto-2-trifluoromethylpropanoyl-1-L-pipecolic acid is obtained).

Example 49

1-( 2- mercapto- 3, 3, 3- trifluoropropanoyl)- L- pipecolic acid

By using L-pipecolic acid instead of L-proline in the method according to example 7, and then subjecting the product to the method according to example 8, 1-(2-mercapto-3,3,3-trifluoropropanoyl)-L-pipecolic acid is obtained.

Example 50

1-(3-mercapto-2-trifluoromethylpropanoyl)-5,5-difluoro-DL-pipecolic acid Using 5,5-difluoro-DL-pipecolic acid [obtained from 5-keto-DL-pipecolic acid by the method described in J. Med . Chem. 20, 1176 (1977)] instead of 4,4-difluoro-L-proline by the method according to Example 11 and subjecting the product to the method

according to example 12, 1-(3-mercapto-2-trifluoromethylpropanoyl)-5,5-difluoro-DL-pipecolic acid is obtained.

Example 51

1, 1'-[ dithiobis-(2- trifluoromethyl- 3- propanoyl)]- bis- 5, 5- difluoro-DL-pipecolic acid

By using 1-(3-mercapto-2-trifluoromethylpropanoyl)-5,5-difluoro-DL-pipecolic acid instead of 1-(3-mercapto-2-trifluoromethylpropanoyl)-L-proline in the method according to Example 9,

1,1'-[dithiobis-(2-trifluoromethyl-3-propanoyl)]-bis-5,5-difluoro-DL-pipecolic acid is obtained.

Example 5 2

1-(3-mercapto-4,4,4-trifluorobutanoyl)-L-pipecolic acid

By using L-pipecolic acid tert-butyl ester instead of L-proline tert-butyl ester in the method according to example 14,

and then subjecting the product to the method according to example 15, 1-(3-mercapto-4,4,4-trifluorobutanoyl)-L-pipecolic acid is obtained.

Example 5 3

1-( 3- mercapto- 2- trifluoromethylpropanoyl)- 5, 5- dichloro- DL-pipecolic acid

By using 5,5-dichloro-DL-pipecolic acid [prepared from 5-keto-DL-pipecolic acid and phosphorus pentachloride by a method similar to that described in J. Med. Chem. 20, 1176 (1977)]

instead of 4,4-difluoro-L-proline in the method according to example 11 and then subjecting the product to the method according to example 12, 1-(3-mercapto-2-trifluoromethylpropanoyl)-5,5-dichloro-DL-pipecolic acid is obtained.

Example 5 4

1-( 3- mercapto- 2- methylpropanoyl)- 5, 5- difluoro- DL-pipecolic acid

By using 3-acetylthio-2-methylpropanoic acid chloride instead of 3-acetylthio-2-trifluoromethylpropanoic acid chloride in the method according to example 11, and then subjecting the product to the method according to example 12, one obtains 1-(3-mercapto-2-methylpropanoyl)-5,5 -difluoro-DL-pipecolic acid.

Example 55

1-( 3- mercapto- 2- methylpropanoyl)- 5- fluoro- DL- pipecolic acid

By using 3-acetylthio-2-methylpropanoic acid chloride instead of 3-acetylthio-2-trifluoromethylpropanoic acid chloride and 5-fluoro-DL-pipecolic acid .[prepared from 5-hydroxypipecolic acid by a method similar to that described in Biochemistry, 4, 2507 (1965) ] instead of 4,4-difluoro-L-proline by the method according to example 11, and then subjecting the product to the method according to example 12, one obtains 1-(3-acetylthio-2-methylpropanoyl)-5-fluoro-DL-pipecolic acid and 1- (3-Mercapto-2-methylpropanoyl)-5-fluoro-DL-pipecolic acid.

Example 56

1-(3-mercaptopropanoyl)-5-bromo-DL-pipecolic acid

By using 3-acetyltippropanoyl chloride instead of 3-acetylthio-2-trifluoromethylpropanoic acid chloride and 5-bromo-DL-pipecolic acid [prepared from 5-hydroxypipecolic acid by a method similar to that described in Aust. J. Chem. 2_0, 149 3 (196 7)] instead of 4,4-difluoro-L-proline in the method according to example 11, and subjecting the product to the method according to example 12, one obtains 1-(3-acetylthiopropanoyl)-5-bromo-DL-pipecolic acid and 1-(3-mercaptopropanoyl)-5-bromo-DL-pipecolic acid.

Example 57

1-( 3- acetylthio- 2- trifluoromethylpropanoyl)- DL- pipecoI acid methyl ester

By using DL-pipecolic acid methyl ester instead of L-proline tert-butyl ester in the method according to example 2, 1-(3-acetylthio-2-trifluoromethylpropanoyl)-DL-pipecolic acid methyl ester is obtained.

Example 5 8

1-( 3- mercapto- 2- trifluoromethylpropanoyl- DL-pipecolic acid- methyl ester

By using DL-pipecolic acid methyl ester instead of L-proline tert-butyl ester and 3-mercapto-2-trifluoromethylpropanoyl instead of 3-acetylthio-2-methylpropanoic acid in the method according to example 2, 1-(3-mercapto-2-trifluoromethylpropanoyl) is obtained )-DL-pipecolic acid methyl ester.

Example 59

1-(3-propanoyl'io-2-trifluoromethylpropanoyl)-5-fluoro-DL-pipecolic acid

By using thiopropanoic acid instead of thioacetic acid in the method according to example 1 and then subjecting the product to the method according to example 26, 1-(3-propanoylthio-2-trifluoromethylpropanoyl)-5-fluoro-DL-pipecolic acid is obtained.

Example 60

1-( 3- mercapto- 2- methylpropanoy1)- 5- fluoro- DL- pipecolic acid sodium salt

An aqueous solution of 1-(3-mercapto-2-methyl-propanoyl)-5-fluoro-DL-pipecolic acid is mixed with an equimolar amount of aqueous N sodium hydroxide, and the solution is freeze-stirred.

Example 61

1-( 3- mercaptopropanoyl)- 5, 5- dichloro- DL-pipecolic acid

By using 3-acetylthiopropanoic acid chloride instead of 3-acetylthio-2-trifluoromethylpropanoic acid chloride in the method according to example 53, 1-(3-mercaptopropanoyl)-5,5-dichloro-DL-pipecolic acid is obtained.

The racemic forms of the final products in each of the preceding examples are obtained by using the DL form of the starting amino acid instead of the L form.

Correspondingly, the D form of the final products is formed in each of the previous examples by using the D form of the starting amino acid instead of the L form.

Claims (22)

1. Process for the preparation of compounds of the general formula (I) where R is hydrogen, lower alkanoyl or m is 1 or 2; n is 0 or 1; R 1 is hydrogen or lower alkyl; and R' 2 is each hydrogen or halogen; R 2 is hydrogen, lower alkyl or CF 2 , and also halogen when n is 1; R 1 is hydrogen, lower alkyl or trifluoromethyl; Rg is hydrogen, and also halogen when m is 1; wherein at least one of R^ , R'2 , R^ , R^ and R^ is halogen or CF^ as represented by the symbols, and only R2 and R'2 can both be halogen at the same time, and basic salts thereof, characterized in that an acid with the formula is linked to an amino acid of the formula:- where R is hydrogen or lower alkanoyl or a p-methoxybenzy1 protecting group which is subsequently removed; and the other groups are as indicated above, to form a product where R is hydrogen or lower alkanoyl, and optionally said product where R is hydrogen is oxidized, by treatment with iodine, to form a product. where R is 2. Method as stated in claim 1, characterized in that the product where R is lower alkanoyl is hydrolysed to form one product where R is hydrogen. 3. Method as stated in claim 2, characterized in that a product where R is lower alkanoyl is subjected to ammonolysis. 4. Process for the preparation of compounds with the formula stated in claim 1, and basic salts thereof, characterized in that an acid with the formula is coupled to a haloalkane acid with the formula where X is halogen and the other symbols are as defined above, to form a connection with the formula which is further reacted with a thioacid with the formula where R-, is lower alkyl, to form a product where R is lower alkanoyl, and optionally this product is subjected to hydrolysis to form a product where R is hydrogen, and optionally this product is oxidized by treatment with iodine to form a product where R is 5. Process for the preparation of compounds with the formula specified in claim 1, and basic salts thereof, characterized in that a compound with the formula where the symbols are as indicated above, is reacted with a compound of the formula R^ -CO-SH where R? is lower alkyl, to form a product where R is lower alkanoyl, and optionally this product is hydrolyzed to form a product where R is hydrogen, and optionally this product where R is hydrogen is treated with iodine to form a product where R is 6. Method as stated in claims 1 to 5, characterized in that the product has the formula where R is hydrogen lower alkanoyl or R 1 is hydrogen or lower alkyl; , R<1>2°9 Rg is each hydrogen or halogen; R 1 is hydrogen, lower alkyl, halogen or trifluoromethyl; R 1 is hydrogen, lower alkyl or trifluoromethyl; with the proviso that at least one of R2 , R^ , R^ , R^ and Rg is a halogen substituent represented by the symbols, only. R2 and R^ may both be halogen, and when R^ is halogen, n must be 1; and basic salts thereof, in 7. Process as stated in claims 1 to 5, characterized in that the product has the formula where R is hydrogen, lower alkanoyl or R1 is hydrogen or lower alkyl; R 2 and R 1 are each hydrogen or fluorine; R 3 and R 3 are each hydrogen or trifluoromethyl, one being hydrogen and the other trifluoromethyl, and basic salts thereof. 8. Method as stated in claims 1 to 5, characterized in that the product has the formula where R is hydrogen, lower alkanoyl or R 1 is hydrogen or lower alkyl; R^ and Rj. is each hydrogen or fluorine; R 1 is hydrogen, halogen or lower alkyl; wherein R 1 is halogen when both R 1 and R 1 are hydrogen, and R 1 is different from halogen when R 2 or R 1 is halogen; and R^ is hydrogen, and basic salts thereof. 9. Process as stated in claims 1 to 5, characterized in that R is hydrogen or lower alkanoyl; R 1 is hydrogen or lower alkyl; and R' 2 is hydrogen or halogen; R 1 is hydrogen or lower alkyl; and R 1 is hydrogen or lower alkyl. 10. Method as stated in claims 1 to 5, characterized in that R is hydrogen or lower alkanoyl; R 1 is hydrogen or lower alkyl; and R' 2 is each hydrogen or fluorine; R 1 is hydrogen or lower alkyl and R 2 is hydrogen or lower alkyl. 11. Method as stated in claims 1 to 5, characterized in that R is hydrogen and R 2 and R' 2 are each fluorine. 12. Process as stated in claims 1 to 5, characterized in that R and R^ are each hydrogen, R 2 and R < 1> 2 are each fluorine and R 1 and R 2 are each hydrogen or lower alkyl. 13. Method as stated in claim 8, characterized in that is hydrogen or fluorine, Rj. is fluorine, R is hydrogen and R 1 , R 2 and R 3 are each hydrogen or lower alkyl. 14. Process as set forth in claim 8, characterized in that R^ is hydrogen, R^- is fluorine, R is hydrogen and R^, R^ and R^ are each hydrogen or lower alkyl. 15. Method as stated in claims 13 and 14, characterized in that R, and each is hydrogen and R^ is methyl. 16. Process as stated in claims 1 to 5, characterized in that the product has the formula where R is hydrogen, lower alkanoyl or m is 2, 1*2 is hydrogen or lower alkyl;; 1*2 and R'2 are each hydrogen or halogen; R3 and R3 are each hydrogen, lower alkyl or trifluoromethyl, no more than one of which is trifluoromethyl, and at least one of R^, ^'2' R3 °^ ^4 is a nal°cphene substituent represented by the symbol; and basic salts thereof. 17. Process as set forth in claim 16, characterized in that R, R^, and are all hydrogen, and R^ is trifluoromethyl. 18. Method as specified in claim 16, characterized in that. R2 is halogen and R'2 is hydrogen. 19. Method as stated in claim 18, characterized in that the halogen is fluorine. 20. Method as stated in claim 16, characterized in that R is hydrogen and R 2 and R' 2 are each halogen. 21. Method as stated in claim 20, characterized in that the halogen is fluorine. 22. Method as stated in claim 16, characterized in that R and R^ are hydrogen, R2 is hydrogen or fluorine, R'2 is fluorine, R^ is methyl, R^ is hydrogen and n is 1.