Proline derivatives

The invention relates to a process for the preparation of therapeutically useful proline derivatives of formula (I)

<img file="FI66596C_D0001.tif" />

(I) wherein R 1 is hydroxy or lower alkoxy, R 1 and R 2 independently represent hydrogen, lower alkyl or phenyl-lower alkyl and R 1 may further be phenyl, R 1 is hydrogen, hydroxy or lower alkyl, R 1₂ is hydrogen, lower alkyl, phenyl-lower alkyl, triphenylmethyl or R 1 CO-, wherein R 1 is lower alkyl, phenyl or phenyl-lower alkyl and n is 0, 1 or 2.

The compounds of the invention are generally prepared by acylation of a compound of formula (II)

<img file="FI66596C_D0002.tif" />

COR (II) with an acid of formula

R₂ - S

R,

I r, (CH) -CH-COOH (III) or a chemical equivalent thereof.

Thus, the final product can be prepared not only by direct acylation with an acid of formula III, but also with an intermediate such as

(a) όίΛ haloalkanoic acid of formula ΐ⁴

X- (CH)

- CH - COOH (purple) wherein X is bromine, chlorine or iodine, or

6 5 9 6

b) tosyloxyalkanoic acid, wherein X in formula IIIa is tosyloxy (CH₃..

• SOgO-) or

c) a substituted acrylic acid of formula ^RU ^R1 Ah = A - COOH (Illb)

The compound obtained in this acylation is then subjected to a substitution or addition reaction with a thiol or thioic acid anion of the formula

Rg - SH, i.e. Rg S

The acylation can also be performed with a thiolactone of formula

R,

Wnΐ¹

-CH (IV)

-c wherein n is 1 or 2, or with mercaptoalkanoic acid of formula

R „R,

Y - S - (CH)_of- CH COR (IIId) wherein Y is Rg, or in addition if a compound of formula I wherein Rg is hydrogen is desired, then Y can also be a protecting group such as

a)

b)

<img file="FI66596C_D0003.tif" />

c)

CHgCONHCHg, Rn

R,

d)

ROC-CH- (CH) -S n or other sulfur protecting group. The deprotection can be carried out in a manner known per se, such as treatment with hot trifluoroacetic acid, cold trifluoromethanesulfonic acid, mercuric acetate, sodium in liquid ammonia, zinc and hydrochloric acid or the like. These methods are described in: Methoden der Organischen Chemie (Houben-Weyl),

Butter. XV, Part I, from page 736 (1974).

When an acid of formula III is used as the acylating agent, the acylation may be performed in the presence of a coupling agent such as dicyclohexyl carbodiimide or the like, or the acid may be activated to form its mixed anhydride, symmetrical anhydride, acid chloride or acid ester, or using Woodward reagent K, N-ethoxy ethoxy-1,2-dihydroxyquinoline or the like, (Methoden der Organischen Chemie (Houben-Weyl), Vol. XV, Part II, from page 1 (1974)).

Compounds of formula II include, for example, proline, hydroxyproline, 4-methylproline and their lower alkyl esters. The acylation of such compounds is described in detail below.

According to a preferred embodiment, in the preparation of compounds of formula I, especially when R₂ is R 1 -CO-, coupling an acid or ester of formula II with a haloalkanoic acid of formula

X - (CH) - CH - COOH (Ula) n

wherein X is halogen, preferably chlorine or bromine. This can be accomplished using any known method in which acid IIIa is activated prior to reaction with acid II, comprising the formation of a mixed anhydride, symmetrical anhydride, acid chloride or active ester, or Woodward reagent K, EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2 -dihydroxyquinoline) or a similar use.

The product obtained in this reaction is a compound of formula?

<img file="FI66596C_D0004.tif" />

(V)

The compound of formula V is reacted with a thioic acid anion of formula

R₂-SH, (i.e. RgS) to give a compound of formula

<img file="FI66596C_D0005.tif" />

(I)

When R_o on R_rCO, this compound can then be converted to? 3

<img file="FI66596C_D0006.tif" />

R₄ R_x

HS - (CH) - CH - CO - N n

COR (Ia) by ammonolysis. When Rg is a protecting group, then the compound of formula Ia can be obtained by deprotection as described above. When R is an ester group (R is lower alkoxy) the ester group can be removed, e.g. when R is tert-butoxy or tert-amyloxy, by treating the ester of formula I or Ia with trifluoroacetic acid and anisole to give the corresponding free acid. When other alkoxy groups are present, the corresponding acid is obtained by alkaline hydrolysis.

According to a variant of this method, acrylic acid having the formula! ^of T -L

CH = C-C00H (IIIb)

This acrylic acid is first converted to an acid halide and then reacted with a compound of formula II to give a compound of formula

CH = C

C/O

<img file="FI66596C_D0007.tif" />

COR and this intermediate are subjected to an addition reaction with a thiol or thioic acid as described above.

Tosyloxyalkanoic acid of formula

CH

<img file="FI66596C_D0008.tif" />

—SO, O- (CH) h

CH

COOH n

can also be used as an acylating agent of an acid of formula II and the resulting acylated product is then subjected to a substitution reaction, e.g. as described above.

Alternatively, an acrylic acid of formula IIIb may be reacted, e.g., with a thioic acid anion I 1 S to give a compound of formula

I IC

R₂-S-CH-CH-C00H which is converted to its acid halide, e.g. with thionyl chloride, is then coupled to the compound of formula II and the same reaction sequence as above is followed.

The acid or ester of formula II may also be acylated with a protected form of Co 'mercaptoalkanoic acid of formula * 1

R_q-S- (CH) -ch-cooh ο Π (VI) wherein Rg is a protecting group. These protecting groups may be as described above.

After acylation, the product can be deprotected in a manner known per se.

The acylating agent may also be a thiolactone, e.g. β-propiothiolactone or β-methyl-β-propiothiolactone.

Detailed information on the methods for preparing the compounds of the invention can be found in the following description and working examples.

According to a particularly preferred embodiment, the acid or ester of formula II is acylated with a haloalkanoyl halide of formula

I ^of I

X- (CH) -CH-COX n wherein each X is independently halogen, preferably chlorine or bromine, is hydrogen, lower alkyl or phenyl-lower alkyl and n is 0, 1 or 2. This reaction is carried out in an alkaline medium, e.g. dilute in an alkali metal hydroxide solution,

Ί

665 9 6 in an early metal bicarbonate or alkali metal carbonate solution at a reduced temperature, e.g. at about 0-15 ° C. The reaction product is also subjected to a substitution reaction using the anion of thiol or thioic acid described above also in an alkaline medium, preferably an alkali metal carbonate solution, and then treated in a conventional manner. The compound obtained in the reaction in which the substituent Rg in formula I is R1-CO is converted into a compound in which Rg is hydrogen by ammonolysis, e.g. by means of alcoholic ammonia or concentrated ammonium hydroxide solution or by alkaline hydrolysis using e.g. a metal hydroxide solution. When an acid of formula II is used as a starting material, the final product is obtained as a free carboxylic acid which can then be converted to an ester, e.g. by esterification with a diazoalkane such as diazomethane, 1-alkyl-3β-tolyltriazene such as 1n-butyl-3-p-tolyltriazene or the like.

According to another embodiment, the ester of formula II, preferably t-butyl ester, is treated in an anhydrous medium such as dichloromethane, tetrahydrofuran or dioxane with thioalkanoic acid of formula

R₀-S- (CH₀) - CH - COOH

2 n coupling of dicyclohexylcarbodiimide, N, N'-carbonylbisimidazole, ethoxyacetylene, diphenylphosphoryl azide or the like in the temperature range of about 0-10 ° C. The ester group (R) can then be removed, e.g., by treatment with trifluoroacetic acid and anisole at about room temperature.

When an ester of formula II (e.g. R is lower alkoxy, variously t-butoxy) is acylated with a thiolactone, e.g. β-propiothiolactone or β-methyl- (3-propiothiolactone), the reaction may be performed in an anhydrous solvent such as tetrahydrofuran, dioxane or methylene chloride. between about 0 ° C and room temperature. The ester group can be removed with anisole and trifluoroacetic acid as described above.

The compounds of formula I have one or more asymmetric carbon atoms. When R 1, R 2 or R 2 represent other than hydrogen, the carbon to which they are attached is asymmetric.

These carbon atoms are marked with an asterisk in formula I. The compounds are thus in stereoisomeric forms or as racemic mixtures thereof. All of these forms are within the scope of the invention. The racemate or enantiomer may be used as starting materials in the methods described above. When a racemic starting material is used in the synthesis described above, the stereoisomers contained in the compound can be separated by known chromatographic or fractional crystallization methods. In general, the L-isomer relative to the amino acid carbon comprises the preferred isomeric form. _ Also the D-isomer of the acyl side chain

-carbon (carbon with R) is preferred.

The compounds of the invention form basic salts with various inorganic and organic bases and are also within the scope of 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 inorganic bases, e.g. dicyclohexylamine salt, benzazine, N-methyl-D-glucamine, hydrabamine salts, salts with amino acids such as arginine and lysine. Non-toxic, physiologically acceptable salts are preferred, although other salts are also useful, e.g., in isolating or purifying the product as described in connection with the dicyclohexylamine salt in the working examples.

Salts are formed in a manner known per se by reacting the free acid form of the product with an equivalent of a base to give the desired cation, in a solvent or medium in which the salt is soluble, or in water and then removing the water by freeze-drying. When neutralizing a 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 can give the free acid and, if desired, form a second salt.

The compounds of the invention inhibit the conversion of decapeptide angiotensin I to angiotensin II and are therefore useful in alleviating or relieving angiotensin-induced hypertension. The action of the enzyme renin on angiotensinogen, a pseudoglobulin in the plasma, produces angiotensin I. Angiotensin I is converted to angiotensin II by angiotensin-producing enzyme (ACE). The latter is an active antihypertensive agent that has been found to cause various forms of hypertension in various mammals, e.g., rats and dogs. The compounds of the invention act on a series of transformations angiotensin (renin) angiotensin 1- $ · angiotensin II by blocking the action of an angiotensin converting enzyme and reducing or eliminating the formation of pressure-inducing angiotensin II. Thus, administration of a composition comprising one or more compounds of formula I or a physiologically acceptable salt thereof will improve angiotensin-dependent hypertension in mammals. A single dose or preferably 2-4 divided daily doses in an amount of about 0.1-100 mg per kg per day, preferably about 1-50 mg per kg per day is suitable for lowering blood pressure, as demonstrated by animal experiments described in: SL Engel, TR Schaeffer, MH Waugh and B. Rubin, Proc. Soc.

Exp. Biol. Med. 143, 483 (1973). The agent is preferably administered orally, but parenteral administration, such as intravenous, intramuscular, subcutaneous or intraperitoneal administration, may also be used.

The compounds of the invention may be used to lower blood pressure by formulating compositions such as tablets, capsules or liquids for oral administration or sterile solutions or suspensions for parenteral administration. About 10 to 500 mg of a compound of formula I or a mixture of compounds or a physiologically acceptable salt thereof is combined with a physiologically acceptable medium, carrier, additive, binder, preservative, stabilizer, flavoring agent, etc. in unit dosage form according to accepted pharmaceutical practice. The amount of active ingredient in these compositions or preparations is such that a suitable amount will be obtained within the range indicated.

Exemplary excipients that may be used with tablets, capsules, and the like include: a binder such as tragacanth, acacia, corn starch, or gelatin; an additive such as dicalcium phosphate; a disintegrant such as corn starch, potato starch, alginic acid and the like; a lubricating agent such as magnesium stearate; a sweetening agent such as sucrose, lactose or saccharin; a flavoring agent such as peppermint, wintergreen oil, 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 substances may be present as coatings or for other modification of the physical form of the unit dose. For example, 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 paraben as a preservative, a coloring agent and a sweetening agent such as cherry or pomegranate essence.

Suitable formulations for injection are obtained in accordance with standard pharmaceutical practice by dissolving or suspending the active ingredient in a vehicle such as water for injection, natural vegetable oils such as sesame oil, peanut oil, coconut oil or coconut oil, cottonseed oil, etc. or the like. Buffers, preservatives, antioxidants and the like may be added as needed.

The following examples illustrate the method according to the invention and particularly preferred embodiments. All temperatures are in degrees Celsius.

Example 1

1- (2-benzpylthioacetyl) -L-proline

L-Proline (5.75 g) is dissolved in N sodium hydroxide (50 ml) and the solution is cooled in an ice-water bath. Sodium hydroxide 2N (26 ml) and chloroacetyl chloride (5.65 g) are added and the mixture is stirred vigorously at room temperature for 3 hours. A suspension of thiobenzoic acid (7.5 g) and potassium carbonate (4.8 g) in water (50 ml) is added. After stirring for 18 hours at room temperature, the reaction mixture is acidified and extracted with ethyl acetate. The ethyl acetate layer is washed with water, dried over magnesium sulfate and concentrated to dryness in vacuo. The residue (14.6 g) is dissolved in ethyl acetate (150 ml) and dicyclohexylamine (11 ml) is added. The crystals are filtered and recrystallized from ethyl acetate to give 5.7 g, m.p. 151-152 ° C.

To convert the salt to the acid, the crystals are dissolved in a mixture of 5% potassium bisulfate (100 ml) and ethyl acetate (300 ml). The organic phase is washed once with water, dried over magnesium sulphate and concentrated to dryness in vacuo, yield 3.45 g.

Example 2

1- (2-mercaptoacetyl) -L-proline

1- (2-Benzoylthioacetyl) -L-proline (3.4 g) is dissolved in a mixture of water (10.5 ml) and concentrated ammonia (6.4 ml). After 1 hour, the reaction mixture is diluted with water and filtered. The filtrate is extracted with ethyl acetate and acidified with concentrated hydrochloric acid, saturated with sodium chloride and extracted twice with ethyl acetate. The ethyl acetate extracts are washed with saturated sodium chloride and concentrated to dryness, yield 1.5 g. The product 1- (2-mercaptoacetyl) -L-proline is crystallized from ethyl acetate (m.p. 133-135 °).

Example 3

1- (2-benzothiopropanoyl) -L-proline

L-Proline (5.75 g) is dissolved in aqueous N sodium hydroxide (50 ml) and the solution is cooled in an ice bath with stirring. 2N Sodium hydroxide (25 ml) and 2-bromopropionyl chloride (8.57 g) are added in this order, and the mixture is removed from the ice bath and stirred at room temperature for 1 hour. A mixture of thiobenzoic acid (7.5 g) and potassium carbonate (4.8 g) in water (50 ml) is added and the mixture is stirred overnight at room temperature. After acidification with concentrated hydrochloric acid, the aqueous solution is extracted with ethyl acetate and the organic phase is washed with water, dried and concentrated to dryness. The residue (14.7 g) is chromatographed on a column of 440 g of silica gel with a benzene / acetic acid mixture (7: 1). The fractions containing the desired substance are collected, concentrated to dryness and the residue is precipitated twice with ether / hexane and converted into the dicyclohexylamine salt from ether / hexane to give 9.4 g of product. Sp. 148-156 °. The dicyclohexylamine salt is converted back to the acid according to Example 1, yield

5.7 g.

Example 4 _

1- (2-Mercaptopropanoyl) -L-proline 1- (2-Benzoylthiopropanoyl) -L-proline (5.7 g) is dissolved in a mixture of water (12 ml) and concentrated ammonium hydroxide (9 ml) with stirring. After 1 hour, the mixture is diluted with water (10 mL) and filtered. The filtrate is extracted twice with ethyl acetate, concentrated to 1/3 volume, acidified with concentrated hydrochloric acid and extracted with ethyl acetate. The organic phase is washed with sat

6659 6 sodium chloride, dried and concentrated to dryness in vacuo. The residue 1- (2-mercaptopropanoyl) -L-proline is crystallized from ethyl acetate / hexane, yield 3 g, m.p. (105) 116-120 ° C.

Example 5

1- (3-benzoylthiopropanoyl) -L-proline

L-proline (5.75 g) is dissolved in 1N sodium hydroxide (50 ml) and the solution^-cooled in an ice bath. 3-Bromopropionyl chloride (8.5 g) and 2N sodium hydroxide (27 ml) are added and the mixture is stirred for 10 minutes in an ice bath and for 3 hours at room temperature. A suspension of thiobenzoic acid (7.5 g) and potassium carbonate (4.5 g) in water (50 ml) is added and the mixture is stirred for 18 hours at room temperature. After acidification with concentrated hydrochloric acid, the aqueous phase is extracted twice with ethyl acetate. The organic layers are dried over magnesium sulfate and concentrated to dryness in vacuo to give 7.1 g of 1- (3-benzoylthiopropanoyl) -L-proline, m.p. 101-102 ° (ethyl acetate / hexane).

Example 8

L-proline tert-butyl ester

L-proline (230 g) is dissolved in a mixture of water (1 L) and 5N sodium hydroxide (400 mL). The solution is cooled in an ice bath while 5N sodium hydroxide and (460 ml) and benzyloxycarbonyl chloride (340 ml) are added with vigorous stirring in five equal portions over 1/2 hour. After stirring for one hour at room temperature, the mixture is extracted twice with ether and acidified with concentrated hydrochloric acid. The precipitate is filtered off and dried. Yield 442 g, m.p. 78-80 °.

The benzoyloxycarbonyl-L-proline thus obtained (180 g) is dissolved in a mixture of dichloromethane (300 ml), liquid isobutylene (800 ml) and concentrated sulfuric acid (7.2 ml). The solution is shaken in a pressure flask for 72 hours. The pressure is removed, the isobutylene is allowed to evaporate and the solution is washed with 5% sodium carbonate, water, dried over magnesium sulphate and concentrated to dryness in vacuo to give 205 g of benzyloxycarbonyl-L-proline tert-butyl ester.

Benzyloxycarbonyl-L-proline tert-butyl ester (205 g) is dissolved in absolute ethanol (1.2 L) and hydrogenated under normal pressure using 10% Pd on carbon (10 g) until only traces of carbon dioxide in the dehydrogenation gas are detected (24 hours). The catalyst is filtered off and the filtrate is concentrated in vacuo at 30 mm Hg. The residue is distilled in vacuo to give L-pro13 line tert-butyl ester,_1πυη 50-51°.

Example 7

1- (3-acetylthiopropanoyl) -L-proline tert-butyl ester

L-Proline tert-butyl ester (5.13 g) is dissolved in dichloromethane (40 ml) and the solution is cooled in an ice-water bath. A solution of dicyclohexylcarbodiimide (6.18 g) in dichloromethane (20 ml) is added immediately followed by 3-acetylthiopropionic acid (4.45 g). After stirring for 15 minutes in an ice-water bath and 16 hours at room temperature, the precipitate is filtered off and the filtrate is concentrated to dryness in vacuo. The residue is dissolved in ethyl acetate and washed until neutral. The organic layer is dried over magnesium sulfate and concentrated to dryness in vacuo to give 9.8 g of 1- (3-acetylthiopropanoyl) -L-proline tert-butyl ester.

Example 8

1- (3-acetylthiopropanoyl) -L-proline 1- (3-acetylthiopropanoyl) -L-proline tert-butyl ester (4.7 g) is dissolved in a mixture of anisole (34 ml) and trifluoroacetic acid (68 ml), and the mixture is kept at room temperature for 1 hour. The solvents are removed in vacuo and the residue is precipitated from ether / hexane several times. The residue (3.5 g) is dissolved in acetonitrile (25 ml) and dicyclohexylamine (2.8 ml) is added. The crystalline salt is filtered and recrystallized from isopropanol. Yield 3.8 g, m.p. 176-177 °. The salt is reconverted to 1- (3-acetylthiopropanoyl) -L-proline as described in Example 1, yield 1.25 g, m.p. 89-90 ° (ethyl acetate / hexane).

.Example 9

1- (3-mercaptopropanoyl) -L-proline tert-butyl ester

To a solution of L-proline tert-butyl ester (3.42 g) in anhydrous tetrahydrofuran (10 ml) cooled in an ice bath is added propiothiolactone (1.76 g). After 5 minutes in an ice bath and 3 hours at room temperature, the reaction mixture is diluted with ethyl acetate and washed with 5% potassium bisulfate and water. The organic layer is dried over magnesium sulfate and concentrated to dryness in vacuo. The residue 1- (3-mercaptopropanoyl) -L-proline tert-butyl ester is crystallized from ether / hexane, yield 3.7 g, m.p. 57-58 °.

Example 10

1- (3-mercaptopropanoyl) -L-proline

Method A

1- (3-Benzoylthiopropanoyl) -L-proline (4.9 g) is dissolved in a mixture of water (8 ml) and concentrated ammonium hydroxide (5.6 ml), and the solution is kept under stirring under an argon atmosphere for 1 hour. The reaction mixture is diluted with water, filtered and the filtrate is extracted with ethyl acetate. The aqueous phase is acidified with concentrated hydrochloric acid, saturated with sodium chloride and extracted with ethyl acetate. The organic layers are washed with saturated sodium chloride, dried over magnesium sulfate and concentrated to dryness in vacuo. The residue 1- (3-mercaptopropanoyl) -L-proline is crystallized from ethyl acetate / hexane, yield 2.5 g, m.p. 68-70 °.

Method B 1- (3-Acetylthiopropanoyl) -L-proline (0.8 g) is dissolved in 5.5 N methanolic ammonia (5 ml) and the solution is kept under argon at room temperature. After 2 hours, the solvent is removed in vacuo, the residue is dissolved in water and taken up in an ion exchange column which is H⁺stage / t) owex 50 (analytical quality)? and eluted with water. The fractions giving a thiol-positive reaction are collected and concentrated to dryness, yield 0.6 g This product is crystallized from ethyl acetate / hexane according to Method A to give 1- (3-mercaptopropanoyl) -E-proline.

Method C 1- (3-Mercaptopropanoyl) -L-proline t-butyl ester (2.3 g) is dissolved in a mixture of anisole (20 ml) and trifluoroacetic acid (45 ml). After 1 hour at room temperature under argon, the reaction mixture is concentrated to dryness in vacuo and the residue is precipitated from ethyl acetate / hexane several times. The residue (1.9 g) is dissolved in ethyl acetate (30 ml) and dicyclohexylamine (85 ml) is added. The crystalline salt is filtered and recrystallized from isopropanol, yield 2 g, m.p. 187-188 °.

The salt is converted to the acid according to Example 1, yield 1.3 g. The product is crystallized from ethyl acetate / hexane according to Method A.

Salts

Sodium 1- (3-mercaptopropanoyl) -L-proline (500 mg) is dissolved in a mixture of water (2.5 ml) and N sodium hydroxide (2.5 ml). The solution is lyophilized to give the sodium salt.

Magnesium 1- (3-mercaptopropanoyl) -L-proline (500 mg), magnesium oxide (49.5 mg) and water (10 ml) are stirred gently with heating until a complete solution is formed. The solvent is then removed by freeze-drying to give the magnesium salt

Potassium 1- (3-mercaptopropanoyl) -2-proline (500 mg) is dissolved in a mixture of calcium hydroxide (91 mg) and water (10 ml), and the solution is lyophilized to give the calcium salt.

Potassium

1- (3-Mercaptopropanoyl) -L-proline (500 mg) is dissolved in a mixture of potassium bicarbonate (246 mg) and water (10 ml) and lyophilized to give the potassium salt.

N-methyl-D-glucamine 1- (3-mercaptopropanoyl) -L-proline (500 mg) and N-methylD-glucamine (480 mg) are dissolved in water (10 ml) and lyophilized to give the N-methyl-D-glucamine salt. .

Example 11

1- (3-mercaptopropanoyl) -L-hydroxyiproline

Using L-hydroxyproline in the process of Example 3 instead of L-proline and then treating the product with Method A of Example 10 gives 1- (3-benzoylthiopropanoyl) -L-hydroxyproline and 1- (3-mercaptopropanoyl) -L-hydroxyproline, respectively. 193.5 195 ° C as dicyclohexylanine salt).

6596

Example JL2_

1- (3-mercaptopropanoyl) -D-proline

Using D-proline in the process of Example 5 instead of L-proline and then treating the product with the method of Example 10 to give 1- (3-benzoylthiopropanoyl) -D-proline 1- (3-mercaptopropanoyl) -D-proline, m.p. 6870 °.

.Example 13

A. .3-Acetylthio-2-methylpropanoic acid

A mixture of thioacetic acid (50 g) and methacrylic acid (40.7 g) is heated on a steam bath for 1 hour and then kept at room temperature for 18 hours. After MNR spectroscopy shows that the methacrylic acid has completely reacted, the reaction mixture is distilled in vacuo and the desired 3-acetylthio-2-methylpropanoic acid is separated off in a fraction having a b.p. 128.5-131 ° (2.6 mm Hg), yield 64 g.

Β. 1- (3-Acetylthio-2-methylpropanoyl) -L-proline tert-butyl ester

L-Proline tert-butyl ester (5.1 g) is dissolved in dichloromethane (40 ml) and the solution is stirred and cooled in an ice bath. Dicyclohexylcarbodiimide (6.2 g) dissolved in dichloromethane (15 ml) is added and then a solution of 3-acetylthio-2-methylpropanoic acid (4.9 g) in dichloromethane (5 ml) is added immediately. After stirring for 15 minutes in an ice bath and 16 hours at room temperature, the precipitate is filtered off and the filtrate is concentrated to dryness in vacuo. The residue is dissolved in ethyl acetate and washed until neutral. The organic phase is dried over magnesium sulfate and concentrated to dryness in vacuo. The residue 1- (3-acetylthio-2-methylpropanoyl) -L-proline tert-butyl ester is purified by column chromatography (silica gel / chloroform), yield 7.9 g as an oil

Example iL

1- (3-acetylthio-2-methylpropanoyl) -L-proline

Method A

1- (3-Acetylthio-β-methylpropanoyl) L-proline tert-butyl ester (7.8 g) from Example 13R is dissolved in a mixture of anisole (55 mL) and trifluoroacetic acid (110 mL). After one hour at room temperature, the solvent is removed in vacuo and the residue is precipitated several times from ether / hexane. The residue (6.8 g) is dissolved in acetonitrile (40 ml) and dicyclohexylamine (4.5 ml) is added. The crystalline salt is boiled with fresh acetonitrile (100 ml), cooled to room temperature and filtered, yield 3.8 g, m.p. (165) 187-188 °. This material is recrystallized from isopropanol M + -67 ° (C 1.4, EtOH). The crystalline dicyclohexylamine salt is suspended in a mixture of 5% potassium disulfate and ethyl acetate. The organic phase is washed with water and concentrated to dryness. The residue is crystallized from ethyl acetate / hexane to give 1- (3-acetylthio-2-D-methylpropanoyl-L-proline, mp 83-8 ° C?_{162 (Cj 1j7j EtOH}).

Method B

3-Acetylthio-2-methylpropanoic acid (8.1 g) and thionyl chloride (7 g) are stirred and the suspension is stirred for 16 hours at room temperature. The reaction mixture is concentrated to dryness and distilled in vacuo (b.p. 80 °). This 3-acetylthio-2-methylpropanoic acid chloride (5.4 g) and 2N sodium hydroxide (15 ml) are added to a solution of L-proline (3.45 g) in 1N sodium hydroxide (30 ml) cooled in an ice-water bath. After stirring for 3 hours at room temperature, the mixture is extracted with ether, the aqueous phase is acidified and extracted with ethyl acetate. The organic phase is dried over magnesium sulfate and concentrated to dryness to give 1- (3-acetylthio-2-DL-methylpropanoyl-L-proline.

Method C

Methacryloyl chloride (4.16 g) is added to a solution of L-proline (3.45 g) in a mixture of water (100 ml) and sodium bicarbonate (12 g) cooled in an ice-water bath while stirring vigorously. After the addition is complete, the mixture is stirred at room temperature for 2 hours and then extracted with ether. The aqueous phase is acidified with N-hydrochloric acid and extracted with ethyl acetate. The organic phase is concentrated to dryness in vacuo, the residue is taken up in thiolacetic acid (3.5 g), a few crystals of azobisisobutyronitrile are added and the mixture is heated on a steam bath for 2 hours. The reaction solution is dissolved in benzene / acetic acid (75:25) and applied to a silica gel column. Elution with the same solvent mixture gives 1- (3-acetylthio-2-DL-methylpropanoyl) -L-proline.

Example 15

1- (3-mercapto-2-D-methylpropanoyl) -L-proline

1- (3-Mercapto-2-methylpropanoyl) -L-proline is obtained by treating the compound of Example 14 as follows:

The thioester (0.85 g) is dissolved in 5.5 N methanolic ammonia and the solution is kept at room temperature for 2 hours.

The solvent is removed in vacuo and the residue is dissolved in water, fed to an ion exchange column which is H⁺(Dowex 50, analytical grade) and eluted with water. The fractions which give a positive thiol reaction are collected and freeze-dried. The residue is crystallized from ethyl acetate / hexane, yield 0.3 g. 1- (3-mercapto-2-D-methylpropanoyl-L-proline melts at 103-104 °, [7]

-131 (C, 2, EtOH). „. ...,_c 'Example 16

A · 3-Acetylthio-2-benzylpropanoic acid

Using 2-benzylacrylic acid in the process of Example 13A instead of methacrylic acid gives 3-acetylthio-2-benzylpropanoic acid.

B. N- (3-Acetylthio-2-benzylpropanoyl) -L-proline tert-butyl ester

Using 3-acetylthio-2-benzylpropanoic acid in the process of Example 7 instead of 3-acetylthiopropanoic acid, 1- (3-acetylthio-2-benzylpropanoyl) -L-proline tert-butyl ester is obtained.

C. 1- (3-Acetylthio-2-benzylpropanoyl) -L-proline The product of B above is used in place of 1- (3-acetylthio-2-methylpropanoyl-L-proline) -tert-butyl ester in Method A of Example 14 to give 1- (3-acetylthio-2-benzylpropanoyl) -L-proline.

D. 1- (3-Mercapto-2-benzylpropanoyl) -L-proline

1- (3-Acetylthio-2-benzylpropanoyl) -L-proline is treated with methanolic ammonia according to Example 15 to give 1- (3-mercapto-2-benzylpropanoyl) -L-proline as an oil, R_f = 0.47 (silica gel, benzene / acetic acid 75:25).

Example 17

1- (3-mercapto-2-methylpropanoyl) -L-hydroxyproline

Using L-hydroxy-proline tert-butyl ester in the process of Example 13, treating the resulting product according to Method A of Example 14 and then further treating according to Example 16 affords 1- (3-acetyl-thio-2-methylpropanoyl) -L-hydroxyproline, respectively. -tert-butyl ester, tert-butyl ether-1- (3-acetylthio-2-methylpropanoyl) -L-hydroxyproline and 1- (3-mercapto-2-methylpropanoyl) -L-hydroxyproline, the latter as an amorphous substance L _D -61.90 (c = 2, methanol).

.Example 18

1- (4-benzoylthiobutanoyl) -L-proline

To a solution of L-proline (2.88 g) in 1N sodium hydroxide (25 ml) cooled in an ice bath is added 2N sodium hydroxide (12.5 ml) and 4-chlorobutyryl chloride (3.5 g). The reaction mixture is stirred at room temperature for 3.5 hours and a suspension of thiobenzoic acid (3.75 g) and potassium carbonate (2.4 g) in water (25 ml) is added. After stirring overnight at room temperature, the reaction mixture is acidified with concentrated hydrochloric acid and extracted with ethyl acetate. The organic layer is dried over magnesium sulfate and concentrated to dryness in vacuo. The residue is chromatographed on a silica gel column, eluting with benzene / acetic acid (7: 1). The fractions containing the desired substance are collected and concentrated to dryness, yield 1.35 g. A small amount of this material is dissolved in ethyl acetate and dicyclohexylamine is added to pH 8-10 (on wet pH paper). The dicyclohexylamine salt crystallizes immediately, m.p. 159-161 °.

Example 19.

1- (4-Mercaptobutanoyl) -L-proline 1- (4-Benzylthiobutanoyl) -L-proline (1.08 g) is dissolved in a mixture of water (4 ml) and concentrated ammonia (2.7 ml). After stirring for 1 hour at room temperature, the mixture is diluted with water, filtered, extracted with ethyl acetate and the aqueous phase is concentrated in vacuo. The ammonium salt of 1- (4-mercaptobutanoyl) -L-proline is purified by ion exchange chromatography using a diethylaminoethyl-Sephadex (cross-linked with dextran) column and eluting with ammonium bicarbonate, yield 0.7 g. The ammonium salt is dissolved in water (2 ml) and applied to a column of Dowex 50 sulfonic acid resin (analytical grade) in hydrogen form, and the free acid is eluted with water. Fractions containing the desired substance (sulfhydryl reagent and carboxyl reagent positive) are collected and lyophilized to give 1- (4-mercaptobutanoyl) -Lproline. The dicyclohexylammonium salt is obtained according to Example 18, m.p. 157-158 °.

2Q

Example 20

A. 1- (3-Acetylthiobutanoyl) -L-proline tert-butyl ester

Dicyclohexylcarbodiimide (6.2 g) and 3-acetylthiobutyric acid (4.86 g) are added to a solution of L-proline tert-butyl ester (5.1 g) in dichloromethane (60 ml) with stirring in an ice bath. After 15 minutes, the ice bath is removed and the mixture is stirred at room temperature for 16 hours. The precipitate is filtered off, the filtrate is concentrated to dryness and the residue is chromatographed on a silica gel column eluting with chloroform to give 1- (3-acetylthiobutanoyl) -L-proline tert-butyl ester, yield 5.2 g.

Β. 1- (3-acetylthiobutanoyl) -L-proline

1-C3-Acetylthiobutanoyl) -L-proline tert-butyl ester (5.2 g) according to A above is dissolved in a mixture of trifluoroacetic acid (60 ml) and anisole (30 ml) and the solution is kept at room temperature for 1 hour. The solvents are removed in vacuo and 1- (3-acetylthiobutanoyl) -L-proline is reprecipitated from ether / hexane several times, yield 4 g. The dicyclohexylamide salt is prepared according to Example 44, m.p. 175-176 °.

Example 21

1- (3-mercaptobutanoyl) -L-proline

1- (3-Acetylthiobutanoyl) -L-proline (0.86 g) from Example 20B is dissolved in 5.5 N methanolic ammonia (20 mL) and the reaction mixture is kept at room temperature for 2 hours. The solvent is removed in vacuo and the residue is chromatographed using an ion exchange column (Dowex 50) and water as eluent. Fractions containing the desired 1- (3-mercaptobutanoyl) -L-proline are collected and lyophilized, yield 0.6 g. The dicyclohexylamine salt is prepared according to Example 19, m.p. 183-184 ° C.

Example 22

1- / 3 - / (ethoxy) thiocarbonyl / thio / propanoyl / -L-proline

Aqueous 2N sodium hydroxide (25 mL) and 3-bromopropion21

6596 nyl chloride (8.5 g) is added to a solution of L-proline (5.75 g) in N-sodium hydroxide (50 ml) while cooling and stirring in an ice bath. After 5 minutes, the ice bath is removed and stirring is continued at room temperature. After 3 hours, ethyl 5-xanthogenic acid-potassium salt (9.6 g) is added and the mixture is stirred overnight at room temperature. The solution is acidified with concentrated hydrochloric acid and extracted with ethyl acetate. The organic layer is concentrated to dryness and the residue is chromatographed on a silica gel column eluting with benzene / acetic acid (7: 1) to give 1- [3 - [(ethoxy) thiocarbonyl] thio? Propanoyl] -L-proline, m.p. 94-95 °.

Example 23

3 - [(methylamino) thiocarbonyl 4? tiq? propion ihappo

Methyl isothiocyanate (4 g) is added to a solution of 3-mercaptopropionic acid (5.3 g) in a mixture of pyridine (250 ml) and 0.5N sodium hydroxide (100 ml). The solution is kept at 40 ° for 2 hours and concentrated to dryness in vacuo. The residue is dissolved in water (100 ml), acidified with concentrated hydrochloric acid and extracted with ether. The organic phase is concentrated to dryness to give 3- (N- (methylamino) thiocarbonyl). propion ihappo, m.p. 86-87 °.

Example 24 1-Za-Z (methylamino) thiocarbonyl-thio-t'-propanoyl-T'-L-proline-tert. -butyl ester

To a solution of L-proline tert-butyl ester (1.71 g) and hydroxybenzotriazole (1.35 g) in dichloromethane (10 ml) cooled and stirred in an ice bath are added dicyclohexylcarbodiimide (2.06 g) and 3-methylaminothiocarbonylthiopropionic acid thionic acid (1.79 g). After 14 minutes, the bath is removed and stirring is continued overnight. The precipitate is filtered off and the filtrate is diluted with ethyl acetate and washed until neutral. The organic phase is concentrated to dryness to give 1- [3- [Y (methylamino) thiocarbonyl?^_thiopropanoyl-L-proline tert-butyl ester, m.p. 129-130 °.

Example 25 .1- ^ 3- [? (Methylamino) thiocarbonyl4? thio? Pr opanoyl 1 R-proline 1- (methylaminothiocarbonylthiopropanoyl) L-proline tert-butyl ester (0.98 g) is dissolved in a mixture of anisole (3.6 ml) and trifluoroacetic acid (7.5 ml). after standing at room temperature, the mixture is concentrated to dryness in vacuo and the residue is precipitated three times from ether / hexane. The material obtained is chromatographed on a silica gel column eluting with a benzene / acetic acid mixture (75:25) to give 1- (N - (N (methylamino) thiocarbonyl) -propionoyl-L-proline, R1. = 0.4 ^ silica gel-benzene: acetic acid (75:25)? '. The dicyclohexylammonium salt melts at 127-12 ° C.

Example 26

1- (3-methylthiopropanoyl) -L-proline

Methyl 3-methylthiopropionate (51 g) is saponified with% sodium hydroxide solution (150 ml, 30 minutes at 100 °). The cooled solution is extracted with ether and acidified to give the crude acid thus obtained and converted into the acid chloride with thionyl chloride.

A solution of L-proline (11.5 g) in N sodium hydroxide (100 ml) is cooled in an ice bath and 3-methylthiopropanoic acid chloride (6.9 g) is added dropwise with vigorous stirring over 10 minutes. After 1 hour, the reaction mixture is acidified and extracted with ethyl ether to give 1- (3-methylthiopropanoyl) -L-proline. The dicyclohexylammonium salt is prepared by adding dicyclohexylamine to a solution of the free acid in ethyl acetate, m.p. 169-171 °.

Example

1- (methylthioacetyl) -L-proline

Using methyl methylthioacetate instead of methyl 3-methylthiopropionate in Method A of Example 26 gives 1- (methylthioacetyl) -L-proline, m.p. 123-124 °.

Example 28

1- (benzylthioacetyl) -L-proline

Using benzylthioacetyl chloride instead of 3-methylthiopropanoyl chloride in Method A of Example 26, 1- (Benzylthioacetyl) -L-proline is obtained, m.p. 86-88 °.

Example 29

1,1 * - / dithiobis (3-propanoyl N-bis-L-proline

3-Mercaptopropanoyl-L-proline (0.95 g) is dissolved in water (20 ml) and the pH is adjusted to 6.5 using 1N sodium hydroxide. An ethanolic solution of iodine is added dropwise while maintaining the pH at 6.5 by careful addition of 1N sodium hydroxide. When a permanent yellow color is obtained, the addition of iodine is stopped and the color is removed with a small amount of sodium thiosulfate. The reaction mixture is acidified with concentrated hydrochloric acid and extracted with ethyl acetate. The organic phase is washed with water, dried and concentrated to dryness to give 1,1 '- / dithiobis (3-propanoyl-bis-L-proline. The dicyclohexylammonium salt is prepared by adding dicyclohexylamine to a solution of the free acid in acetonitrile, mp 179-180 °.

6596

Example 30

1,1'-Zdithiobis (2-D-methyl-3-propanoyl N-bis-L-proline

Using 3-mercapto-2-D-methylpropanoyl-L-proline in the process of Example 29 instead of 3-mercaptopropanoyl-L-proline gives 1,1'-Zdithiobis (2-D-methyl-3-propanoyl bis-L-proline. 236-237 °.

Example 31

A. 3-Acetylthio-2-phenylpropanoic acid

Using 2-phenylacrylic acid in the process of Example 13 instead of methacrylic acid, 3-acetylthio-2-phenylpropanoic acid is obtained.

B. 1- (3-Acetyl-thio-2-phenylpropanoyl) -L-proline tert-butyl ester

Using 3-acetylthio-2-phenylpropanoic acid as an example

In process B, instead of 3-acetylthio-2-phenylpropanoic acid, 1- (3-acetylthio-2-phenylpropanoyl) -L-proline-2,5-tert-butyl ester is obtained, which is converted to the free acid 1 + 23 (c = 1, ethanol ).

Example 32

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

Using 1- (3-acetylthio-2-phenylpropanoyl) -L-proline tert-butyl ester in the process of Example 14A instead of 1- (3-acetylthio-2-methylpropanoyl-L-proline tert-butyl ester) and subjecting the product to ammonolysis according to Example 15 1- (3-acetylthio-2-phenylpropanoyl) -L-proline and 1- (3-mercapto-2-phenylpropanoyl) -L-proline are obtained.

+ 2.5 ° (c = 1, ethanol respectively + 2.5 ° (c = 1, ethanol) The final product mp 49-52 ° C.

Example 33

3-Z- (4-Methoxy-phenyl) -methyl-thio-7-methyl-propionic acid p-methoxy-.alpha.-toluene-thiol (15.4 g, 0.1 mol) is added to a solution of methacrylic acid (8.6 g, , 1 mole) in 50 ml of 2N sodium hydroxide. The mixture is heated on a steam bath for 3 hours and then refluxed for 2 hours and cooled. The mixture is extracted with ether, the aqueous layer is acidified with concentrated HCl and extracted with dichloromethane. The acidic extracts are washed with brine, dried (MgSO 4) and evaporated in vacuo.

The semi-solid obtained is taken up in 50 ml of dichloromethane, diluted with 50 ml of hexane and cooled. 3 - [(4-methoxyphenyl) methylthio] -2-methylpropanoic acid is recovered as a white crystalline solid, m.p. 74-82 ° (5.5 g).

Example 34

1- [3- (4-Methoxyphenyl) methylthio] -2-methylpropanoyl] -proline tert-butyl ester

3 - [(4-methoxyphenyl) methylthio] -2-methylpropanoic acid (3.6 g, 0.015 mol), L-proline tert-butyl ester (2.6 g,

0.015 mol) and dicyclohexylcarbodiimide (3.1 g, 0.015 mol) are dissolved in 50 ml of dichloromethane and stirred for 30 minutes at 0 °. The cooling bath is removed and the mixture is stirred overnight (16 hours). The resulting suspension is filtered and the filtrate is washed with 5% potassium bisulfate, saturated sodium bicarbonate and brine, then dried (MgSO 4) and evaporated in vacuo. The resulting clear oil is applied to a 250 ml silica gel column and chromatographed using 20% ​​ethyl acetate / hexane as eluent. The main fraction (Rf = 0.70, silica gel, ethyl acetate) is evaporated to give 5.5 g (93%) of 1- [3- (4-methoxyphenyl) methyl] -2-methylpropanoyl-L-proline tert-butyl ester. as a clear oil. R f = 0.70 (silica gel, ethyl acetate); R f = 0.60 (silica gel, ether).

Example 35

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

The ester of Example 34 (1.2 g, 0.003 mol), anisole (5 mL) and trifluoromethanesulfonic acid (0.5 mL) are dissolved in 20 mL of trifluoroacetic acid under a nitrogen atmosphere and the resulting red solution is allowed to stand for 1 hour at room temperature. The solution is evaporated in vacuo to a red residue which is taken up in ethyl acetate and washed with water, brine, then dried (MgSO 4) and evaporated. The residue is stirred several times with hexane and the remaining hexane is evaporated; the oily residue is 0.4 g. A portion of this material (180 mg) is subjected to preparative thin layer chromatography using 2 suction silica gel plates and benzene / acetic acid 75:25 as eluent. The main nitroprusside-positive band (R Chromatography using benzene / acetic acid 75:25 (Rf = 0.40) and chloroform / methanol / acetic acid 50:40:10 (Rf = 0.62).

.Example 3 6

1- (3-mercapto-2-D-methylpropanoyl) -L-proline

Under an argon atmosphere, 1- [3- (acetylthio) -2-D-methylpropanoyl] -L-proline (10.0 g) is slurried in 150 ml of water at 10 °. To this mixture is added 5N sodium hydroxide and the pH of the solution is maintained at 13 for 1.5 hours. After consumption of sodium hydroxide is complete, the solution is acidified to pH 2.0 with concentrated sulfuric acid.

The aqueous solution is then extracted three times with methylene chloride (3 x 150 mL) and the combined methylene chloride fractions are concentrated to an oil. The concentrate is taken up in ethyl acetate, filtered and the filtrate is diluted with hexane (30 mL). After more than 1/2 hour, more hexane is added and the mixture is cooled to 10 ° for 1 hour.

The crystals are filtered and washed with hexane (2 x 25 mL) and dried to constant weight to give 6.26 g of 1- (3-mercapto-2-dimethylpropanoyl) -L-proline as white crystals, m.p. 100-102 °.

Example 37

A. 1- [3-Tosyloxy-2-methylpropanoyl] -L-proline

Using 3-tosyloxy-2-methylpropanoic acid chloride instead of 3-acetylthio-2-methylpropanoic acid chloride in the procedure of Example 14B,^&lt;3-tosyloxy-2-methylpropanoyl-L-proline.

B. 1- [5-Acetylthio-2-D-methylpropanoyl] -L-proline 1- [3-tosyloxy-2-methylpropanoyl] -7-L-proline (3.5 g) is added to a solution of thiolacetic acid (1.14 g). ) and triethylamine (3.5 mL) in ethyl acetate (20 mL). The solution is kept at 50 ° for 3 hours, cooled, diluted with ethyl acetate (100 ml) and washed with dilute hydrochloric acid. The organic layer is dried and concentrated to dryness in vacuo. The residue is dissolved in acetonitrile and dicyclohexylamine is added. The crystalline precipitate is recrystallized from isopropanol to give 1- [3-acetylthio-2-D-methylpropanoyl] -L-proline dicyclohexylamine salt, m.p. 187-188 °, / o /?²³ -67 ° (c 1.4, EtOH). This salt is converted to the free acid, m.p. 83-85 ° (melting point 104-105 ° of the isomorphic form is obtained if the crystallization solution is inoculated with a high melting agent).

Example 38

1- (3-mercaptopropanoyl) -L-proline t-butyl ester

To a stirred solution of 1.71 g (10 mmol) of proline t-butyl ester and 1.35 g (10 mmol) of 1-hydroxybenzotriazole hydrate in 20 ml of N, N-dimethylformamide at 0-5 ° are added 2, 06 g (10 mmol) of N, N'-dicyclohexylcarbodiimide. The mixture is stirred for 10 minutes and then 1.06 g (10 mmol) of 3-mercaptopropanoic acid in 2 ml of N, N-dimethylformamide are added. The mixture is stirred at 0-5 ° for 1 hour and at room temperature overnight.

The precipitated N, N'-dicyclohexylurea is filtered off and the filtrate is concentrated in vacuo. The residue is taken up in ethyl acetate, washed with well-saturated sodium bicarbonate solution, dried and concentrated in vacuo to give 2.5 g of an oil.

the oil is taken up in ethyl acetate / hexane 1: 1 and applied to a silica gel column (100 g). Elution with ethyl acetate / hexane 1: 1 gives 1.40 g (54%) of 1- (3-mercaptopropanoyl) -L-proline t-butyl ester as an oil which crystallizes on standing. Recrystallization from ether / hexane gives 0.9 g of a colorless crystalline substance, m.p. 55-60 °, which is identical to the compound of Example 9.

Example 3 9.

1- (3-mercaptopropanoyl) -L-proline

To a solution of 75 mg (0.27 mmol) of 1- (3- [Tetylamino) carbonyl] thi] propanoyl, β-L-proline in 1 ml of concentrated ammonium hydroxide and 1 ml of water is allowed to stand at room temperature for 18 hours under argon. in the atmosphere. The solution is diluted with a small amount of water and extracted with ether. The aqueous layer is acidified with cold concentrated hydrochloric acid and extracted with ethyl acetate. The combined extracts are dried and concentrated in vacuo to give a compound identical to the product of Example 10. Thin layer chromatography (silica gel, benzene: acetic acid 7: 3) Rf = 0.4.

Example 40

Methacryloyl-L-proline i

L-proline (23.0 g, 0.2 mol) is dissolved in 100 ml of water and stirred in an ice bath. Methacryloyl chloride (19.6 mL, 0.2 mol) in 25 mL of methyl isobutyl ketone is added dropwise over 3 hours. At the same time, sodium hydroxide solution (2N) is added and the pH is maintained at 7.0. The addition of base is continued for 4 hours after the addition of acid chloride is complete. The reaction mixture is adjusted to pH 5 with concentrated HCl and extracted with ethyl acetate. The aqueous layer is acidified to pH 2.5 and extracted well with ethyl acetate. The acidic extracts are washed with brine and dried (MgSO 4). The ethyl acetate solution is treated with dicyclohexylamine (40 mL) and cooled overnight. The resulting white precipitate was filtered and dried to give 29 g (39%) of a white solid, m.p. 202-210 °. The material is crystallized from 1.5 liters of acetonitrile / isopropanol 3: 1 to give 19.7 g of methacryloyl-L-proline dicyclohexylamine salt as fine white needles, m.p. 202210 °.

The salt is dissolved in water / ethyl acetate and the mixture is acidified with concentrated HCl. The resulting suspension is filtered to remove a fine white precipitate which is washed well with ethyl acetate.

The filtrate is saturated with sodium chloride and extracted well with ethyl acetate. The extracts are washed with brine, dried (MgSO 4) and evaporated to a clear oil which solidifies. Crystallization from ethyl acetate / hexane gives 7.5 g (83%) of methacryloyl-L-proline as a white crystalline solid, m.p. 89-93 °. An analytical sample is obtained by recrystallization, m.p. 95-98 °.

Example 41

1- (3-acetylthio-2-D-methylpropanoyl) -L-proline

Methacryloyl-L-proline (183 mg, 0.001 mol) is dissolved in thiolacetic acid (0.5 ml) and allowed to stand at room temperature for 16 hours. The solution is evaporated in vacuo to a yellow residue. Preparative: thin layer chromatography (silica gel, dichloromethane / methanol / acetic acid 90: 5: 5) can isolate the main oil (240 mg) as a major fraction. thin layer chromatography (dichloromethane / methanol / acetic acid 90: 5: 5) shows that this material is 1- (3-acetylthio-2-DL-methyl28 propanoyl-L-proline corresponding to the product of Example 14.

Rf = 0.35, (benzene / acetic acid 75:25) Rf = 0.38.

The oil is dissolved in 3 ml of acetonitrile, treated with dicyclohexylamine until the solution is basic and cooled. A white crystalline solid (106 mg) was collected, m.p. 175181 °. Crystallization from isopropanol gives 1- (3-acetylthio-2-dimethylpropanoyl) -L-proline dicyclohexylamine salt, m.p. 187188 °, which is identical to the product of Example 14.

Example 42 1- (3-Mercapto-2-methylpropanoyl) -L-proline

Zinc dust (10.0 g) is added to a slurry of 1 - [(dithiobis- (2-methyl-3-propanoyl) 7bis-L-proline (5.0 g)

100 1 ml of sulfuric acid, and the mixture is stirred at 18 ° for 4 hours under a nitrogen atmosphere. The solution is filtered, the zinc is washed with water (20 ml) and the combined filtrates are extracted with methylene chloride (3 x 75 ml). The methylene chloride washings are extracted with water (25 mL) and then the organic solution is concentrated to an oil. This oil is taken up in ethyl acetate (20 ml) and filtered. Hexane (15 mL) is added to the filtrate and the mixture is stirred for 15 minutes. Additional hexane (30 mL) is then added and the solution is cooled to 5 ° for 1 hour. The mixture is then filtered and the product is washed with hexane (2 x 10 mL) and dried to give 4.17 g of 1- (3-mercapto-2-methylpropanoyl) -L-proline as white crystals. TLC, Rf = 0.60 (solvent system: benzene / acetic acid 75:25).

Example 43

A. 3-Benzylthio-2-methylpropanoic acid_

Using o &lt;-toluene thiol in the process of Example 33 instead of p-methoxy toluene thiol gives 3-benzylthio-2-methylpropanoic acid.

Β. 1- [3- (benzylthio) -2-methylpropanovyl] -L-proline tert. butyl ester

Using 3-benzylthio-2-methylpropanoic acid in the process of Example 34 instead of 3 - [(4-methoxyphenyl) methylthio] -2-methylpropanoic acid, 1- [3- (benzylthio) -2-methylpropanoyl] -L-proline tert-butyl ester is obtained.

C. 1- [3- (benzylthio) -2-methylpropanoyl] -L-proline

1- [3- (Benzylthio) -2-methylpropanoyl] -L-proline tert-butyl ester (7.8 g) is dissolved in a mixture of anisole (55 ml) and trifluoroacetic acid (110 ml). After 1 hour at room temperature, the solvent is removed in vacuo and the residue is dissolved in ether, washed several times with saturated sodium chloride, dried over magnesium sulphate and evaporated to dryness in vacuo to give 1- [3- (benzylthio) -2-methylpropanoyl] -L-proline. . Rf = 0.5 (silica gel, benzene / acetic acid 3: 1), Rf = 0.5 (silica gel, methyl ethyl ketone / acetic acid / pyridine / water 14: 1: 2: 1).

Example 44

1- (3-Mercapto-2-methylpropanoyl) -L-proline 1- [3- (Benzylthio) -2-methylpropanoyl] -L-proline (0.1 g) is suspended in boiling liquid ammonia (10 ml) and small pieces are added. sodium while stirring until a permanent blue color. The color is removed by adding a few crystals of ammonium sulfate and the ammonia is allowed to evaporate under a stream of nitrogen. The residue is dissolved in a mixture of dilute hydrochloric acid and ethyl acetate. The organic layer is dried and concentrated to dryness in vacuo to give 1- (3-mercapto-2-methylpropanoyl) -L-proline. Rf: 0.5 (silica gel; benzene / acetic acid 3: 1); Rf 0.5 (silica gel methyl ethyl ketone / acetic acid / pyridine / water 14: 1: 2: 1), which is identical to the compound of Example 15.

Example 45

3-Triphenylmethylthio-2-methylpropanoic acid

A solution of 3-mercapto-2-methylpropanoic acid (1.2 g) and trityl chloride (2.9 g) in methylene chloride (50 ml) is kept at room temperature for 2 hours. The mixture is heated on a steam bath for 20 minutes and then evaporated to dryness in vacuo and the residue is dissolved in saturated sodium bicarbonate and the solution is washed with ethyl acetate. The aqueous phase is acidified to pH 3 and extracted with ethyl acetate. The organic layer is dried and concentrated to dryness to give 3-triphenylethylthio-2-methylpropanoic acid. Rf 0.8 (silica gel, benzene / acetic acid 3: 1).

Example 46

A. 1- [3- (Triphenylmethyl) -2-methylpropanoyl] -L-proline tert-butyl ester

Using 3-triphenylmethylthio-2-methylpropanoic acid in the process of Example 34 instead of 3 - [(4-methoxyphenyl) methylthio] -2-methylpropanoic acid, 1- [3- (triphenylmethylthio) -2-methylpropanoyl] -L-proline tert-butyl ester is obtained. .

B. 1- [3- (triphenylmethylthio) -2-methylpropanoyl] -L-proline

3-Triphenylmethylthio-2-methylpropanoic acid (1.8 g) and N, N'-carbonyldiimidazole (0.8 g) are dissolved in tetrahydrofuran (10 ml) with stirring at room temperature. After 20 minutes, the solution is added to a mixture of L-proline (0.6 g) and N-methylmorpholine (1 g) in dimethylacetamide (20 ml). The resulting mixture is stirred overnight at room temperature, concentrated to dryness and the residue is dissolved in a mixture of ethyl acetate and 10% potassium bisulfate. The organic layer is separated and dried and concentrated to dryness in vacuo to give 1- [3- (triphenylmethylthio) -2-methylpropanoyl] -L-proline. Rf: 0.4 (silica gel, benzene / acetic acid 3: 1); Rf 1.0 (silica gel, methyl ethyl ketone / acetic acid / pyridine / water 14: 1: 2: 1) (2.4 g) and freshly distilled 2,3-dihydro-4H-pyran (1.9 g) in benzene (60 g). ml), boron trifluoride etherate (2.8 g) is added. After 2 hours, potassium carbonate (4 g) is added, the mixture is stirred and filtered. The filtrate is concentrated to dryness with 3- (tetrahydropyran-2-ylthio) -2-methylpropanoic acid. to obtain.

Example 47 1-Z3-Mercapto-2-methylpropanoyl-7-L-proline

1-Z3- (Triphenylmethylthio) -2-methylpropanoyl-N-proline tert-butyl ester (5 g) is dissolved in a mixture of anisole (55 ml) and trifluoroacetic acid (110 ml). After 1 hour at room temperature, the solvent is removed in vacuo and the residue is applied to a silica gel column equilibrated with benzene / acetic acid (75:25) and eluted with the same solvent. Fractions corresponding to component Rf 0.40 (TLC on silica gel with the same system) are collected and concentrated to dryness to give 1- (3-mercapto-2-methylpropanoyl) -7-L-proline. Rf 0.62 (silica gel, chloroform / methanol / acetic acid / water 50:40:10 identical to the compound of Example 15).

Example 48

A. 3- (Tetrahydropyran-2-ylthio) -2-methylpropanoic acid

To a solution of 3-mercapto-2-methylpropanoic acid (2.4 g) and freshly distilled 2,3-dihydro-4H-pyran (1.9 g) in benzene (60 ml) is added boron trifluoride etherate (2.8 g). After 2 hours, potassium carbonate (4 g) is added, the mixture is stirred and filtered. The filtrate is concentrated to dryness to give 3- (tetrahydropyran-2-ylthio) -2-methylpropanoic acid i.

B. 1- [3- (Tetrahydropyran-2-ylthio) -2-methylpropanoyl? L-proline

Using 3- (tetrahydropyran-2-ylthio) -2-methylpropanoic acid in the process of Example 46B instead of 3-triphenylmethylthio-2-methylpropanoic acid, 1- [3- (tetrahydropyran-2-ylthio) -2-methylpropanoyl] -L-proline is obtained. R f: 0.8 (silica gel, benzene / acetic acid 3: 1, R f: 0.7 (silica gel, methyl ethyl ketone / acetic acid / pyridine / water, 14: 1: 2: 1).

Example 49

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

A solution of 1- [3- (tetrahydropyran-2-ylthio) -2-methylpropanoyl) -β-L-proline (1 g) in a mixture of methanol (25 ml) and concentrated hydrochloric acid (25 ml) is kept at room temperature for 30 minutes. The solvents are removed in vacuo to give 1- (3-mercapto-2-methylpropanoyl) -L-proline. Rf: 0.35 (silica gel, benzene / acetic acid, 3: 1), Rf. 0.5 (silica gel, methyl ethyl ketone / acetic acid / pyridine / water 14: 1: 2: 1), identical to the product of Example 15.

Example 50

A. 3-Acetamidomethylthio-2-methylpropanoic acid

3-Mercapto-2-methylpropanoic acid (2.4 g) and N-hydroxymethylacetamide (1.8 g) are dissolved in trifluoroacetic acid and the solution is kept at room temperature for 1 hour. The trifluoroacetic acid is removed in vacuo and the residue is dried in vacuo with potassium hydroxide to give 3-acetamidomethylthio-2-methylpropanoic acid.

B. 1- [3- (Acetamidomethylthio) -2-methylpropanoyl-7-lproline

Using 3-acetamidomethylthio-2-methylpropanoic acid in the process of Example 48B instead of 3- (tetrahydropyran-2-ylthio) -2-methylpropanoic acid, 1- [3- (acetamidomethylthio) -2-methylpropanoyl) -L-proline is obtained. Rf 0.2 (silica gel, benzene / acetic acid 3: 1), Rf 0.3 (silica gel, methyl ethyl ketone / acetic acid / pyridine / water 14: 1: 2: 1).

Example 51

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

1- [3- (Acetamidomethylthio) -2-methylpropanoyl] -L-proline (1.4 g) and mercuric acetate (1.93 g) are dissolved in a mixture of acetic acid (25 ml) and water (25 ml). After stirring for 1 hour on a steam bath, hydrogen sulfide is bubbled through until no more mercury sulfide precipitate is observed. The mixture is filtered, the precipitate is washed with ethanol and the filtrate is concentrated to dryness in vacuo to give 1- (3-mercapto-2-methylpropanoyl) -L-proline. R 2; 0.35 (silica gel, benzene / acetic acid 3: 1),

Rf: 0.5 (silica gel, methyl ethyl ketone / acetic acid / pyridine / water 14: 1: 2: 1), identical to the compound of Example 15.

Example 52

1- (3-Mercapto-2-methylpropanoyl) -L-proline tert-butyl ester

To a cold (5 °) solution of 1.2 g (10 mmol) of 3-mer-capto-2-methylpropanoic acid and 1.7 g (10 mmol) of L-proline tert. -butyl ester 25 in dichloromethane, 2.26 g of dicyclohexylcarbodiimide in 5 ml of dichloromethane are added portionwise. After 2 hours at room temperature, 5 drops of acetic acid are added, the mixture is filtered and the filtrate is evaporated to an oily eastern residue. This residue is taken up in 20 ml of petroleum ether / ethyl acetate (3: 1) and applied to a 150 ml silica gel column prepared with petroleum ether. The fraction eluted with petroleum ether / ethyl acetate (1: 1) contains the product

1- (3-Mercapto-2-methylpropanoyl) -L-proline tert-butyl ester. This fraction (0.6 g) is dried over P₂O &lt;in vacuo for 12 hours.

R 0.6 (silica gel, benzene / acetic acid 3: 1),

R_f 0.8 (silica gel, methyl ethyl ketone / acetic acid / pyridine / water 14: 1: 2: 1).

Example 53

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

Using 1- (3-mercapto-2-methylpropanoyl) -L-proline tert. -butyl ester In the process of Example 10C, 1- (3-mercapto-2-methylpropanoyl) -L-proline is obtained instead of 1- (3-mercaptopropanoyl) -L-proline tert-butyl ester.

Rf 0.35 (silica gel, benzene / acetic acid 3: 1), 0.5 (silica gel, methyl ethyl ketone / acetic acid / pyridine / water 14: 1: 2: 1), identical to the compound of Example 15.

Example 54

A. 1- (3-Mercapto-2-D-methyl-1-oxopropyl) -4-cis-methylL-proline

To an ice-cooled solution of 4-cis-methyl-L-proline hydrochloride (2.87 g, 17.4 mmol) and sodium hydrochloride (3.2 N, 10.8 mL) is added a solution of 3-acetyl-2-methylpropionyl chloride (2 , 88 g, 16 mmol) and ether (3 mL). The mixture is stirred vigorously for 35 min. maintaining the pH at 8.5 with sodium carbonate (4 N, 12 mL). Stir for an additional 1 h at room temperature. The mixture is extracted with ether and the aqueous solution is acidified with concentrated HCl (4.5 mL). The acidified solution is extracted thoroughly with ethyl acetate, the ethyl acetate is removed in vacuo and the residue is chromatographed on silica gel (E. Merck, 175 g) at 1.4 kp / cm using a mixture of benzene and acetic acid (10: 1) as eluent. The compound is purified by formation of a dicyclohexylamine salt in ethyl acetate. The pure desired product is obtained as an oil by regeneration of the 5 N amine salt of dicyclohexyl. Yield 1.8 g or 42%. £ &lt;* · J = -171.5 (c = 1.9,

CHCl 3), R f = 0.54 (silica gel, benzene: acetic acid 7: 3). An analytical sample of the salt is obtained by recrystallization from acetonitrile. = -69.3 ° (c = 1.76 CHCl 3₃) sp. 182-184 ° C

Analysis: C 1-4 H 2 NO 2 S -dicyclohexylamine:

Calculated C, 63.40, H, 9.31, N, 6.16, S, 7.05

Found: C, 63.30, H, 9.37, N, 6.19, S, 7.03

Β. 1- (3-mercapto-2-D-methyl-1-oxopropyl) -4-cismethyl-L-proline

To a solution of Compound A (1.67 g, 6.1 mmol) and methanol (10 mL) is added aqueous NaOH (1N, 15.25 mL) and the mixture is allowed to stand under argon for 15 min. I drive. The solution is centrifuged to separate a small amount of suspended solid and the methanol is removed from the supernatant in vacuo.

The remaining aqueous solution is acidified with HCl (3 N, 5 mL) and extracted thoroughly with ethyl acetate. The ethyl acetate is removed in vacuo and the residue is chromatographed on silica gel 2 (E. Merck, 70 g) at 1.4 kp / cm using a mixture of benzene and acetic acid (7: 3) as eluent. The product from the column is dissolved in chloroform, the chloroform is removed in vacuo to give the desired product as a viscous oil. Yield 950 mg (67%),

Λ / ??⁵ . _139j7^o (c = 1.26, CHCl 3). R_f = 0.54 (silica gel, benzene: acetic acid 7: 3). The dicyclohexylamine salt of the compound --- 25 ° is obtained by using ethyl acetate as a solvent. J = -62.6 (c = 1.07, CHCl 3), m.p. (175 °) 177-179 ° C.

Analysis: C 1 H 2 NO 2 S dicyclohexylamine

Calculated C, 64.04, H, 9.77, N, 6.79,

C, 63.67, H, 9.98, N, 6.74,

S, 7.77 S, 7.74

Found

HS

99,4

Example 55

1- (3-mercapto-2-D-methyl-1-oxopropyl) -4-cis-heptylL-proline

A. 1- [3- (Acetylthio) -2-D-methyl-1-oxopropyl] -4-cis-heptyl-L-proline

Stirred suspension of 3.0 g (0.013 mol) of cis-4heptyl-L-proline suspended in a solution containing 1 g of Na₂CO 2 and 50 ml of water, cooled to 10 ° C and the suspension is treated portionwise with a solution of 2.6 g (0.014 mol) of D'-3-acetylthio-2-methylpropionyl chloride and 5 ml of ether for 10 min. I drive. During this time, an additional 2.5 g of Na is added to the mixture₂C0₃(batchwise) to maintain the pH at 8. A clear solution is obtained. Remove the ice bath, allow the solution to stir for 90 min. at room temperature, the solution is cooled, 50 ml of EtOAc are added and the mixture is acidified with 10 ml of 6N hydrochloric acid.

The phases are separated and the aqueous phase is extracted with 25 ml of EtOAc. The organic phases are combined, dried over MgSO 4, filtered and the solvent is evaporated off to give 5.5 g of an almost colorless viscous residue. This is dissolved in 25 ml of EtOAc and the solution is treated with a solution of 2.7 g of dicyclohexylamine and 5 ml of EtOAc. The resulting solution is seeded, whereupon the dicyclohexylamine salt crystallizes rapidly. After allowing to cool overnight, 6.0 g (79%) of an almost colorless solid are obtained, m.p. 157-159 ° (s. 151 °). After recrystallization from 80 ml ΟΗθΟΝ, 5.4 g (71%) of an almost colorless solid are obtained, m.p. 161-163 °, = -64.5 ° (1% in CHCl 3).

Analysis ^Ci8^B31^NG4^S*^C12^B23^OF:

Calculated C, 66.87, H, 10.10, N, 5.20, S, 5.95 Found C, 66.68, H, 10.06, N, 5.20, S, 5.98

The resulting dicyclohexylamine salt (5.4 g) is converted to the free acid by suspending in 60 mL of EtOAc and treating the suspension portionwise with 60 mL of 10% KHSO 4 solution. The layers are separated and the aqueous phase is extracted with 30 ml of EtOAc (2 times). The organic phases are combined, dried over MgSO 4, filtered and the solvent is evaporated off, yielding 3.6 g (74%) of an almost colorless syrup, -105 ° (1% in EtOH), Rf 0.26 toluene - 15% acetic acid on silica gel).

B. 1- (3-Mercapto-2-D-methyl-1-oxopropyl) -4-cisheptyl-L-proline

Compound A (3.3 g) is treated under argon with a cold solution of 10 ml of concentrated ammonia and 25 ml of water. When the mixture is stirred for several minutes, it turns into a solution. The solution is cooled and extracted with 20 mL of EtOAc (2 times). The aqueous phase is stirred, 20 ml of EtOAc are poured on it, it is acidified with 6N hydrochloric acid and the layers are separated. The aqueous phase is extracted with EtOAc (2 times). The organic phases are combined, dried over MgSO 4, filtered and the solvent is separated off to give 2.87 g (98%) of an almost colorless syrup, -74 ° (1% in EtOH), Rf 0.22 (85% toluene - 15% acetic acid on silica gel, 0.72 (95: 5: 5 CH 2 Cl 2 - MeOH - AcOH on silica gel).

Analysis: ^ci6^B29^WELL3^S'⁰'²⁵ HgO:

Calculated C, 60.05, H, 9.45, N, 4.38, S, 10.02, SH, 10 Found C, 60.44, H, 9.36, N, 4.48, S, 10 , 12, SH, 10

The 1-adamantanamine salt of this compound melts at 177-179 ° C (from EtOAc).

Example 56 1- [2- (Mercaptomethyl) -1-oxo-9-phenylnonyl] -L-proline

A. 1- [2- (acetylthiomethyl) -1-oxo-9-phenylinonyl) L-proline

5.0 g of 3- (acetylthio) - (2-phenyl-n-heptyl) -propionic acid and 2.38 g of hydroxybenzotriazole dissolved in 23 ml of tetrahydrofuran are stirred under cooling in an ice bath. To this mixture is added 3.2 g of dicyclohexylcarbodiimide and then L .... 3 proline t-butyl esters in 100 cm tetrahydrofuran. The reaction mixture is allowed to stand overnight at room temperature. The urea by-product is evaporated off, the filtrate is evaporated to dryness in vacuo, the residue is taken up in ethyl acetate and washed to neutral pH with 10% KHSO 4 and saturated NaHCO 3, the ethyl acetate is removed and the residue is chromatographed on silica gel the compound as a t-butyl ester which is converted to the free acid by treatment with 11.5 cm of trifluoroacetic acid, to give 4.2 g of the title compound.

B. 1-772- (Mercaptomethyl) -1-oxo-9-phenylnonyl] -7-lproline

Compound A is taken up in 15 cm of cold methanol under argon.

. 3

To this is added 15 cm of cold concentrated ammonium hydroxide. 30 min. after which time the reaction mixture is acidified with concentrated HCl and extracted with ethyl acetate to give 4 g of a clear oil (m.p. 104-110 ° C as the adamantylamine salt prepared from ether-hexane).

Example 57

1-Z-2- (mercaptomethyl) -1-oxononyl-7-L-proline

A. 1-Z-2- (acetylthiomethyl) -1-oxononyl-7-proline

2.46 g of 3-acetylthio-2-n-heptylpropionic acid and 1.53 g of hydroxybenzotriazole are taken up in 15 ml of tetrahydrofuran in an ice bath with stirring. To this is added 2.06 g of dicyclohexylcarbodiimide and then 1.71 g of L-proline t-butyl ester dissolved in 5 ml of tetrahydrofuran. The ice bath is removed for 15 min. and the reaction mixture is allowed to stir overnight at room temperature. The urea by-product is removed by filtration and the filtrate is evaporated to dryness and taken up in ethyl acetate and washed neutral with 10% KHSO 4 and saturated NaHCO 3 Cl, then the ethyl acetate is removed to give 3.9 g of product as t-butyl ester. The ester product is taken up in 11.3 ml of anisole and 20 ml of trifluoroacetic acid and allowed to stand for 1 h and evaporated to dryness in vacuo to give an oil which is chromatographed on a silica gel column using a mixture of benzene and ethyl acetate (7: 1) as eluent.

1.8 g of the title compound.

B. 1- (2-Mercaptomethyl) -1-oxononyl] -L-proline

1.7 g of compound A are taken up in 8 ml of cold methanol under argon and this is treated with 8 ml of cold concentrated ammonia.

3Q min. after cooling, the reaction mixture is acidified with concentrated HCl and extracted with ethyl acetate to give, after evaporation to dryness, 1.5 g of product as an oil (m.p. 159-162 ° as the adamantylamine salt).

Example 58 1- (3-Mercapto-1-oxodecyl) -L-proline

A. 3-Z- (acetyl lithio) -1-oxodecyl-7-L-proline t-butyl ester

6.75 g (0.05 mol) of hydroxybenztriazole, HOBT

10.3 g (0.05 mol) of dicyclohexylcarbodiimide, DCC

8.6 g (0.05 mol) of L-proline tributyl ester

12.3 g (0.05 mol) of (j +) - 3- (acetylthio) decanoic acid. .

500 cm of ethyl acetate HOBT is dissolved in ethyl acetate at 50 ° and cooled to room temperature. Add decanoic acid and then DCC. After a few minutes, crystalline matter begins to crystallize. The reaction mixture is stirred at room temperature for 2 1/2 h, L-proline t-butyl ester is added and stirred at room temperature overnight. The solid is filtered to give 9.6 g of dicyclourea. The ethyl acetate filtrate is washed 2-x 200 cm³: 5% NaHCO 3, 1 x 200 cm³: saturated NaCl, x 200 cm³: 11a 5% 1 x 200 cm³: saturated NaCl, and dried over MgSO 4. The solvent is evaporated to give 17.8 g (89%) of product.

Analysis: C_nnH NO S

37 i +

Calculated C, 63.12, H, 9.33, N, 3.51

Found C, 64.01, H, 9.66, N, 3.94

Β. 1- [3- (acetyl) -1-oxyecyl] -L-proline

17.3 g (0.043 mol) of (+) - 3- (acetylthio) -1-oxodecyl-L3 proline t-butyl ester from position A 135 cm trifluoroacetic acid 6 cm³ anisolia

The solution of components is stirred under nitrogen at room temperature for about 4 h. Excess trifluoroacetic acid is removed at 40 ° and 3 mmHg to give a viscous residue (25.6 g, theoretical weight 14.8 g). The residue is dissolved in 150 cm of ether and a solution of 10 g of 3-dicyclohexylamine and 50 cm of ether is added. A white, crystalline precipitate forms immediately and the temperature rises. Cool and filter off the solid to give 15 g of the dicyclohexylamine salt of trifluoroacetic acid (m.p. 225-230 °). The filtrate is partitioned between 10% KHSO 4 and water. The organic layer is washed with saturated NaCl and dried over MgSO 4. After removal of the solvent, 15 g of a dark viscous residue are obtained. Analysis:^ci7^B29^N04^S:

Calculated C, 59.44, H, 8.51, N, 4.08, S, 9.33 Found C, 59.64, H, 8.60, N, 3.81, S, 9.56

C. 1- (3-Mercapto-1-oxodecyl) -L-proline g (0.043 mol) of (1+) -1- (3-acetylthio) -1-oxodecyl-β-proline from B

160 cm³ water cm³ concentrated NH 2 OH

The mixture of components is stirred under nitrogen at room temperature for about 3 h. The alkali-insoluble matter is extracted with ethyl acetate. The pale yellow aqueous layer is covered with ethyl acetate and strongly acidified with 20% HCl. The layers are separated and the aqueous layer is extracted3 ...

with a further 200 cm of ethyl acetate. The combined organic layers are washed with saturated NaCl and dried over MgSO 4.

The solvent is removed to give 8.7 g (67%) of a residue which gives a strong SH test. The residue is dissolved in 200 cm of benzene and lyophilized to give 8.4 g (62%) of the title compound as a viscous liquid.

Analysis: C- ^? NO3 S. 0.5. B₂0:

calculated C, 58.02, H, 9.09, S, 10.33, N, 4.51, SH, 100% Found C, 57.77, H, 9.37, S, 10.24, M, 4.47, SH, 98.9%