BG60368B2 - Derivatives of aminoacids of antihypertension activity - Google Patents

Abstract

The invention includes methods for preparing carboxyalkyldipeptide derivatives and related compounds that are used as angiotensin-converting enzyme inhibitors and as antihypertensive and other pharmaceutical preparations containing these carboxyalkyldipeptide compounds in combination with another compound with antihypertensive and/or diuretic action. 33 claims

Description

U.S. Patent Nos. 4,129,571 and 4,154,960 describe substituted acyl derivatives of amino acids which are useful as inhibitors of angiotensin converting enzyme. In particular, these compounds are mercapto-substituted acylamino acids and their derivatives, including the clinically effective antihypertensive compound captopril O-3-mercapto-2-methylpropanoyl-1-proline.

The compounds of the prior art are not dipeptide derivatives, as are the compounds of the invention. Furthermore, the compounds of the prior art contain a sulfhydryl substituent or a derivative thereof, while the compounds of the invention do not contain such a substituent. The dipeptide compounds of the invention are unusual dipeptides, the N-terminal of which is linked to a carboxymethylene group, which is preferably further substituted with a methyl group. In addition, the carboxyl group(s) can also be converted to derivatives, such as esters, amides, salts. In fact, the compounds of the invention are hybrids formed by linking α-amino acids in dipeptides via a nitrogen atom which is common to these two-part structures. Such a structural arrangement is unusual in the field of synthetic and natural peptides and is characteristic of the mercaptoacyl type groups in the prior art described above.

U.S. Patent No. 4,052,511 discloses N-carboxyalkanoylamino acids which are useful as inhibitors of angiotensin converting enzyme. Since the compounds of the invention are dipeptide derivatives, they can be considered in a formal sense to be related to some of the compounds described in U.S. Patent No. 4,052,511. When one of the methylene groups is replaced by an amino group, compounds of the invention are obtained with very high potency. For example, the preferred compounds of the invention can be administered in low doses, e.g. 2.5 mg per patient per day, compared to the lowest dose of 1 mg/kg per day for the preferred compounds of U.S. Patent No. 4,052,511, which corresponds to 60 mg/day per patient, with an average patient weight of about 68 kg.

Description of the invention

The invention relates to carboxyalkyl dipeptides and their derivatives, which are useful as converting enzyme inhibitors and as antihypertensive agents. The compounds according to the invention can be represented by the following formula

About r1 r3 r4 r5

II I I I I II

R-C-C-NH-CH-C—N-C-C-R6

L· III ,

R2 OR ⁷ j in which R and R ⁶ are the same or different and are hydroxy, lower alkoxy, lower alkenoxy, lower dialkylamino lower alkoxy (dimethylaminoethoxy), acylamino lower alkoxy (acetylaminoethoxy), acyloxy lower alkoxy (pivaloyloxymethyl), aryloxy, such as phenoxy, aryllower alkoxy, such as benzyloxy, substituted aryloxy or substituted aryllower alkoxy, the substituent being methyl, halogen or methoxy, amino, lower alkylamino, lower dialkylamino, hydroxyamino, aryllower alkylamino such as benzylamino;

R* is hydrogen, alkyl of 1 to 20 carbon atoms, including side and cyclic and unsaturated (such as allyl) alkyl groups, substituted lower alkyl, the substituent may be halogen, hydroxy, lower alkoxy, aryloxy such as phenoxy, amino, di-lower alkylamino, acylamino such as acetamino and benzamido, arylamino, guanidino, imidazolyl, indolyl, mercapto, lower alkylthio, arylthio such as phenylthio, carboxy or carboxamido, carbonyl alkoxy, aryl such as phenyl or naphthyl, substituted aryl such as phenyl, the substituent being lower alkyl, lower alkoxy or halogen, aryl lower alkyl, aryl lower alkenyl, heteroaryl lower alkyl or heteroaryl lower alkenyl such as benzyl, styryl or indolyl ether, substituted aryl-lower alkyl, substituted aryl-lower alkenyl, substituted heteroaryl-lower alkyl, or substituted heteroaryl-lower alkenyl, the substituent(s) being/are halogen, dihalogen, lower alkyl, hydroxy, lower alkoxy, amino, aminomethyl, acylamino (acetylamino or benzoylamino) di-lower alkylamino, lower alkylamino, carboxyl, halogenated lower alkyl, cyano or sulfonamido, aryl-lower alkyl or heteroaryl-lower alkyl substituted in the alkyl portion with amino or acylamino (acetylamino or benzoylamino);

R ² and R ⁷ are the same or different and are hydrogen or lower alkyl;

R ³ is hydrogen, lower alkylphenyl lower alkyl, aminophenylmethyl lower alkyl, hydroxyphenyl lower alkyl, acylamino lower alkyl, (such as benzoylamino lower alkyl, acetylamino lower alkyl), amino lower alkyl, dimethylamino lower alkyl, halogenated lower alkyl, indolyl lower alkyl, mercapto lower alkyl, lower alkylthio lower alkyl;

R ⁴ is hydrogen or lower alkyl;

R ⁵ is hydrogen or lower alkyl, phenyl, phenyl lower alkyl, hydroxyphenyl lower alkyl, hydroxy lower alkyl, amino lower alkyl, guanidino lower alkyl, imidazolyl lower alkyl, indolyl lower alkyl, mercapto lower alkyl or lower alkylthio lower alkyl;

R ⁴ and R ³ may be linked together to form an alkylene bridge of 2 to 4 carbon atoms, an alkylene bridge of 2 to 3 carbon atoms and one sulfur atom, an alkylene bridge of 3 to 4 carbon atoms containing one double bond or an alkylene bridge, as above substituted with hydroxy, lower alkoxy, lower alkyl or lower dialkyl; and their pharmaceutically acceptable salts.

Lower alkyl or lower alkenyl groups, unless otherwise noted, described in any of the embodiments of the invention contain hydrocarbon radicals of one to six carbon atoms, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, n-butyl, pentyl, isopentyl, hexyl or vinyl, allyl, butenyl, etc. Arylalkyl groups described in any of the above embodiments have from one to four carbon atoms in their alkyl portion and include, for example, benzyl, p-methoxybenzyl and the like. Halogen means chlorine, bromine, iodine or fluorine. Aryl in any of the embodiments, unless otherwise noted, represents phenyl or naphthyl. Heteroaryl groups, when present, include, for example, pyridyl, thienyl, furyl, indolyl, benzthienyl, imidazolyl and thiazolyl.

The substituted lower alkyl moieties R, R and R are clarified by groups such as

N' I

HO-CH _? -, HS-CH ₂ -, H ₂ N-(CH2) ₄ -, CH ₃ -S-(CH2) ₂ -, H ₂ N-(CH2) ₃ etc.

When R ⁴ and R ⁵ are linked through the carbon and nitrogen atoms to which they are attached, they form a 4- to 6-membered ring which may contain one sulfur atom or one double bond. Preferred rings have the following formula

in which Y is CH ₂ , S or CHOCH ₃ .

Preferred are those compounds of formula I in which

R and R ⁶ can each and independently be hydroxy, lower alkoxy, lower alkenoxy, aryllower alkoxy, amino, lower dialkylamino lower alkoxy, acylamino lower alkoxy, acyloxy lower alkoxy, the substituent being methyl, halogen or methoxy;

R ² and R ⁷ are hydrogen;

R ³ is lower alkyl, amino lower alkyl, imidazoyl lower alkyl, halogen lower alkyl;

R ⁴ and R ⁵ are joined to form the preferred indicated rings, in which Y is CH ₂ , S or CH-OCH ₃ ;

R ¹ is as defined above.

Even more preferred are those compounds of formula I in which R ¹ is additionally alkyl having from 1 to 8 carbon atoms; substituted lower alkyl where the alkyl group has 1 to 5 carbon atoms and the substituent is amino, arylthio, aryloxy or arylamino, arylalkyl or heteroarylalkyl where the alkyl moiety has 1 to 3 carbon atoms, such as phenethyl or indolylethyl or substituted arylloweralkyl (phenyl lower alkyl or naphthyl lower alkyl) and substituted heteroarylloweralkyl where the alkyl groups have 1 to 3 carbon atoms and where the substituent(s) is/are halogen, dihalogen, amino, aminoalkyl, hydroxy, lower alkoxy or lower alkyl. Most preferred are compounds of formula I in which R and R ⁶ are hydroxy, lower alkoxy, arylalkyloxy;

R ² and R ⁷ are hydrogen;

R ^J is methyl or amino lower alkyl;

R ⁴ and R ⁵ are linked through the carbon and nitrogen atoms to form pyrroline, 4-thiapyrroline or 4-methoxypyrroline;

R ¹ is alkyl of 1 to 8 carbon atoms, substituted lower alkyl, the alkyl group having 1 to 5 carbon atoms and the substituent is amino, arylthio or aryloxy, arylalkyl or heteroarylalkyl, the alkyl group having 1 to 3 carbon atoms, such as phenethyl, or indolylethyl or substituted arylalkyl (phenyl lower alkyl or naphthyl lower alkyl) and substituted heteroalkyl, the alkyl group of the substituent(s) being/are halogen, dihalogen, amino, aminoalkyl, hydroxy, lower alkoxy or lower alkyl.

Said preferred compounds also include their pharmaceutically acceptable salts, in particular compounds such as:

N-(1(3)-carboxy-3-phenylpropyl)-1-alanyl-1-proline;

N-(1(8)-ethoxycarbonyl-3-phenylpropyl)-1-alanyl-1-proline and its salt with mapeic acid;

N-(1(c)-ethoxycarbonyl-4-methylpentyl)-1-alanyl-1-proline; N-(1-carboxy-5-aminopentyl)-1-alanyl-1-proline;

N-α-(1(3)-carboxy-3-phenylpropyl)-1-lysyl-L-proline;

N-α-(1(3)-ethoxycarbonyl-3-phenylpropyl)-E-lysyl-proline;

N-α-[1 (3)-carboxy-3-(3-indolyl)propyl]-1-lysyl-L-proline:

N-α-[1 (3)-carboxy-3-(4-chlorophenyl)propyl]<-lysyl-L-proline; N-α-[1 (3)-carboxy-2-phenylthiomethyl]<-lysyl-L-proline;

N-α-[1(3)-carboxy-3-(4-chlorophenyl)propyl]<-lysyl<-4-methoxyproline;

N-α-[1 (3)-carboxy-5-aminopentyl]<-lysyl-L-proline:

Ethyl N-(1(3)-ethoxycarbonyl-3-phenylpropyl)-E-alanyl<prolinate hydrochloride;

etc.

Products with formula (I)

R1R3

II II

R—C—C—NH—CH—C l _o II

R20 and the preferred subgroups can be obtained by one or more of the methods described below.

As shown in the examples, reactive groups not involved in the condensation, such as amino, carboxy, mercapto, etc., can be protected by methods standard in peptide chemistry before the coupling reactions take place and then deprotected to provide the desired compounds.

Method 1. Reaction Scheme 1 (R ² = H)

R—C—C=O + H ₂ N-CH-CNC—C—R6 NaBHsCN »

R7

II HI

A keto acid (or ester, amide or hydroxamic acid) II is condensed with the dipeptide of formula III in aqueous solution, optimally near the neutral point, or in a suitable organic solvent (e.g. CH ₃ CN) in the presence of sodium cyanoborohydride to give I (R ² = H). Optionally, the intermediate Schiff base, enamine or aminol can be catalytically reduced to give product I, for example with hydrogen in the presence of 10% palladium on carbon or Raney nickel. The ratio of diastereoisomeric products formed can be varied by the choice of catalyst.

If R and ^R are carboxy protecting groups such as alkoxy or benzyloxy or the like, they can be converted by known methods, such as hydrolysis or hydrogenation, into a compound of formula I in which R or ^R are hydroxy groups. This is valid for all subsequent methods carried out under the conditions described.

Optionally, the compound of formula II can be condensed with amino acid IV.

R ³ NaBH ₃ CN О R ¹ R ³

H ₂ NCH-COOH < II ---------► R—C-CHNHCH—COOH

IV V under the same conditions to obtain amino acid V. When coupled by known methods with the amino acid derivative VI, a compound of formula I is obtained.

Known methods include protection of reactive groups during the coupling reaction, for example, by N-formyl, N-tert-butyloxycarbonyl and N-carbobenzyloxy groups, followed by their removal to give a compound of formula I. In addition, the substituent R may include removable ester groups, such as benzyl, ethyl or tert-butyl. Condensing agents in this synthesis are those commonly used in peptide chemistry such as dicyclohexylcarbodiimide (DCC) or diphenylphosphoryl azide (DPPA), or a compound of formula V may be activated through the mediation of active esters, such as those derived from 1-hydroxybenzothiazole.

R ⁴ R ⁵ DCC

V * HN—C—CO—R ⁶ ------* I

R ⁷ (VI)

DCC = dicyclohexylcarbodiimide

DPPA = diphenylphosphorylazide

Reaction scheme 2

VII

VIII

Amino acid (or ester, amido or hydroxamic acid) VII is condensed with ketone VIII under the conditions described for reaction scheme 1 to give a compound of formula I.

In a given case, the synthesis can be carried out in steps by condensing a compound of formula VII with a keto acid of formula IX.

R ³ I VI * O=c—COOH — R ¹ R ³ II —► RC—C—NH—CH—COOH R2 IX X

to obtain amino acid X. By known methods, as set forth above in reaction scheme 1, X can be condensed with the amino acid derivative of formula VI to obtain a compound of formula I.

R< R5

I I

X * HN-C-CO—R6

^R7

VI

R1 R3 r4 r5 o

II I I I I II R—C—C—NH—CH—C—N—C—C—RS ι ο II 17

R2 Or R7

In a case where R ¹ has an α-amino substituent, the carbonyl and amino groups may be suitably protected as a β-lactam group.

Method 2. Reaction Pathway 1

R3 O R ⁴ R ⁵

I II I I

_H2I4 -CH-CNC-CO-R6 * ^2I

R7

111

R ¹

I

X-C-COR

I

R2

XI

O R ¹ II I

^R3

I

R ⁴ R ⁵ O

I I II

R—C—C—NH—CH—C—N—C—C

I, II I,

R2 OR ⁷

^R6

The dipeptide of formula III is alkylated with the appropriate α-haloacid (ester or amide) or α-sulfonyloxyacid (ester or amide) of formula XI under alkaline conditions in water or an organic solvent.

X is chlorine, bromine, iodine or an alkylsulfonyloxy or arylsulfonyloxy group.

In a given case, the synthesis can be carried out in stages in the following order:

R3

I h ₂ n-ch—soon

R ¹

I

X-C-COR

I

R2

IV

XI

R1 ^R3

I I

RCO—C—NH—CH-COOH

I

R2

X ^R ⁴ R ⁵

HN—C—COR ⁶

I.

VI

OR ¹ r3 R4 r5 o

II I I I I II

R—C—C—NH—CH—C—N—C—C—R ⁶

I o II I -, r2 o R ⁷

X is chlorine, bromine, iodine, alkylsulfonyloxy or arylsulfonyloxy.

The amino acid of formula IV is alkylated with a halogen-containing acid (ester or amide) of formula XI under alkaline conditions to give compounds of formula X. The latter is condensed by standard methods as outlined in reaction path 1 with the amino acid (ester or amide) of formula VI to give a compound of formula I.

Reductive cleavage of a benzyl ester of formula I in which ^R6 is benzyloxy and R6 is alkoxy gives compounds of formula I in which R6 is alkoxy and ^R6 is hydroxy, and when ^R6 is alkoxy and R6 is benzyloxy, compounds of formula I in which R6 is hydroxy and ^R6 is alkoxy are given.

Reaction Scheme 2 R1 I R3 ^{0 R4 R5} R—C-C—NH _{? +} I n I I _ II I ² X-CH-CNC-COR ⁶ 0 R2 ^R7

VI

XI

OR ¹ R3 r4 r5 0

II IIII II _c

RCC-NH-CH-CNCC-R6 l _o II 1 ₇

R2 ^OR7 in which X is a chlorine, bromine, iodine, alkylsulfonyloxy or arylsulfonyloxy group.

The amino acid or derivative thereof of formula VII is alkylated with the appropriately substituted α-haloacetyl or α-sulfonyloxyacetyl amino acid of formula XII, preferably with R ⁶ other than hydroxyl, under alkaline conditions in water or another solvent to give the compounds of formula I.

In a given case, the synthesis can be carried out in steps by condensing the amino acid ester of formula VII with a substituted α-halogenated acetic acid or α-sulfonyloxyacetic acid (XIII) to give the intermediate of formula X. By known methods described in reaction path 1, X can be coupled with an amino acid of formula VI or a derivative thereof to give a compound of formula I. In the described methods, the arylalkyloxy moiety in R or in R ⁶ or in both substituents can be used to

obtain an ester or diester. R-C- R1 I -C-NH, ^R3 || I I 0 R2 h-sn-soon VII XHI

^{R1 R3}

I I

RCO-C-NH—CH—COOH

R2

R ³ R ⁴ R ⁵ Q

I I I fl NH—CH—C—N—C—C—R6 II I,

Or r7

In some cases, the protecting groups can be removed by known methods.

The starting materials required for the processes described above are known from the literature or can be prepared by known methods from known starting compounds.

In the compounds of general formula I, the carbon atoms which are connected to R ¹ , R ³ and R ⁵ may be asymmetric. The compounds therefore exist in diastereoisomeric forms or in mixtures thereof. The described syntheses may use racemates, enantiomers or diastereomers as starting compounds. When diastereoisomeric products are obtained in the syntheses, the latter may be separated by known methods, such as chromatography and fractional crystallization.

The amino acid portion of the structures is usually preferred

R ¹

II I R—C—C—NH—

I R2

^R3

I —NH—CH—CO and

R ⁴ R ⁵ O

I I I —N—C—C—

I R7 in formula I may be in the L or S configuration, but the diastereomers containing the D-amino acid have activity dependent on their structures and have advantages with respect to metabolic stability in vivo and therefore can be used in a mixture or as pure diastereomeric compounds.

The compounds of the invention form salts with various inorganic or organic acids and bases, which are also within the scope of the invention. Such salts include ammonium salts, alkali metal salts, such as sodium and potassium salts, alkaline earth metal salts, such as calcium and magnesium salts, salts with organic bases, e.g. dicyclohexylammonium salts, N-methyl-O-glucamine, salts with amino acids, such as arginine, lysine, etc. Salts with organic and inorganic acids, e.g. hydrochloric, hydrobromic, sulfuric, phosphoric, methanesulfonic, toluenesulfonic, maleic, fumaric, camphorsulfonic acids, can also be prepared. Non-toxic physiologically acceptable salts are preferred, although other salts are also useful, e.g. in the isolation or purification of the product.

Salts can be formed by known methods, such as reacting the free acid or free base form of the product with one or more equivalents of the appropriate base or acid in a solvent such as water, which is then removed in vacuo or by freeze-drying, or by exchanging the cations of the existing salt with another cation on a suitable ion exchange resin.

The compounds of the invention inhibit the enzyme, angiotensin converting enzyme, and thus block the conversion of the decapeptide angiotensin I to angiotensin II. Angiotensin II is a potent blood pressure-increasing substance. Therefore, a decrease in blood pressure can be obtained as a result of inhibition of its biosynthesis, especially in animals and humans whose high blood pressure is associated with angiotensin II. The converting enzyme breaks down the vasodepressor substance bradykinin. Therefore, inhibitors of the angiotensin converting enzyme may also lower blood pressure by enhancing the effects of bradykinin. Although the relative importance of these and other possible mechanisms remains to be established, angiotensin converting enzyme inhibitors are effective agents with antihypertensive activity in various animal models and are used clinically, for example in many patients with renovascular, malignant and congenital hypertension. For example, D.W. Cushman et al., Biochemistry 16, 5484 (1977).

The evaluation of the converting enzyme inhibitors is carried out with in vitro enzyme inhibition assays. For example, the method described in Y. Piquilloud, A. Reinharz, M. Roth, Biochem. Biophys. Acta, 206, 136 (1970) is used in which the hydrolysis of carbobenzyloxyphenylalanylhistidinelyl leucine is measured. In vivo evaluations can be carried out, for example, with normotensive rats challenged with angiotensin I according to the method of J. R. Weeks and J. A. Jones, Proc. Soc. Exp. Biol. Med. 104, 646 (1960), and in high-renin rat models as described in S. Koletsky et al., Proc. Soc. Exp. Biol. Med., 125, 96 (1967).

The compounds of the invention are therefore useful in the treatment of high blood pressure. They are also valuable in the treatment of acute and chronic congestive heart failure, in the treatment of secondary hyperaldosteronism, primary and secondary pulmonary hypertension, renal failure and renal vascular hypertension, and in the treatment of vascular disorders such as migraine or Raynaud's disease. The use of the compounds of the invention for these and similar disorders is obvious to those skilled in the art.

In the treatment of high blood pressure and the described clinical conditions, the compounds of the invention can be used in compositions such as tablets, capsules or elixirs for oral administration, suppositories for rectal insertion, sterile solutions or suspensions for parenteral or intramuscular administration, etc. The compounds of the invention can be administered to animals or humans in need of treatment in doses that provide maximum pharmaceutical effectiveness. Although the dosage will depend on the individual needs of the patient depending on the disease, the weight of the patient and special diets that must be imposed by other medications taken at the same time and other factors known to those skilled in the art, the dosage range is usually about 1 to 1000 mg per patient per day, which can be administered in a single dose or multiple doses. Preferably the dosage is from 2.5 to 250 mg per patient per day, with the most preferred dose being 2.5 to 75 mg per patient per day.

The compounds of the invention may also be administered in combination with other antihypertensives and/or diuretics, for example the compounds of the invention may be administered in combination with such compounds as amiloride, atenolol, bendroflumethiazide, chlorothalidone, clonidine, cryptene aminoacetates and cryptene aminotannates, deserpidine, diazoxide, guanethidine sulfate, hydrolysine hydrochloride, hydrochlorothiazide, hydroflumethiazide, metolazone, metoprolol tartrate, methyclothiazide, methyldopa, methyldopa hydrochloride, minoxidil, pargyline hydrochloride, polythiazide, prazosin, racinamine, reserpine, sodium nitroprusside, spironolactone, timolol, trichlormethiazide, trimethophan, camsylate, benzthiazide, quinethazone, ticrinaphan, triamterene, acetazolamide, aminophylline, cyclothiazide, ethacrynic acid, furosemide, meretoxylin procaine, sodium ethacrynate and others, as well as mixtures and combinations thereof.

Typically, individual daily doses for these combinations can vary from one-fifth of the recommended minimum clinical dose to the maximum doses when given separately.

To illustrate these combinations, one of the antihypertensive compounds according to the invention, effective clinically, at doses of 2.5-250 mg per day can be effectively combined at doses of 0.5-250 mg per day with the following compounds at the indicated daily dose: hydrochlorothiazide (15-200 mg), chlorothiazide (125-2000 mg), ethacrynic acid (15-200 mg), amiloride (5-20 mg), furosemide (5-80 mg), propanolol (20-480 mg), timolol (5-60 mg) and methyldopa (65-2000 mg). The triple drug combination of hydrochlorothiazide (15-200 mg) plus amiloride (5-20 mg) plus an angiotensin converting enzyme inhibitor of the invention (3-200 mg) or hydrochlorothiazide (15-200 mg) plus timolol (5-60 mg) plus angiotensin converting enzyme inhibitor of the invention (0.5-200 mg) are effective compositions for controlling blood pressure in hypertensive patients. Of course, the limits of these doses can be adjusted to a unit basis when it is necessary to determine a separate daily dose, which varies depending on the disease, the weight of the patient, special diet and other factors.

Typically, these combinations can be formulated into pharmaceutical compositions, which we will discuss below.

About 1 to 100 mg of a compound or mixture of compounds of general formula I or a physiologically acceptable salt is mixed with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavoring agent, etc. in the form of a unit dose. The amount of active substance in these compositions is such that a suitable dose within the indicated range is obtained.

The following are examples of excipients that may be included in tablets, capsules, etc. A binder, such as tragacanth, gum arabic, starch or gelatin, an inert substance, such as microcrystalline cellulose; a disintegrating agent, such as starch, gelatinized starch, alginic acid, etc.; a lubricant, such as magnesium stearate; a sweetener, such as sucrose, lactose or saccharin; a flavoring agent, such as mint, wintergreen, cherry oil. When the unit dose is in capsule form, it may contain, in addition to the materials listed, a liquid carrier, such as an oil of vegetable or animal origin. Various other materials may be present as coatings or as modifiers of the physical form of the unit dose. The tablets may be coated with lacquer, sugar or both. A syrup or elixir may contain the active compound, sucrose, as a sweetener, methyl and propyl parabens as a preservative, a dye, and a cherry or orange flavoring.

Formulations for sterile injectable compositions may be prepared according to accepted pharmaceutical practice by dissolving or suspending the active substance in a solvent such as water for injection, a natural vegetable oil such as sesame oil, coconut oil, peanut oil, cottonseed oil, etc. or a synthetic fatty vehicle such as ethyl oleate, etc. Buffers, preservatives, antioxidants, etc. may be included as required.

The following examples illustrate the invention and particularly preferred embodiments. The preferred diastereomers of these examples are isolated by column chromatography or fractional crystallization.

Example 1. (1)Carboxy-2-(phenylethyl)-1-aphanyl-1-proline.

A mixture of phenylpyruvic acid (753 mg) and L-alanyl-L-proline (171 mg) in methanol-water was adjusted to pH 6.8 and treated with sodium cyanoborohydride (173 mg) at room temperature until the reaction was complete. The product was adsorbed onto a strong cation exchange resin and eluted with 2% pyridine in water to give 294 mg of the crude diastereomeric product, L-(1-carboxy-2-phenylethyl)-1-alanyl-L-proline. A portion was purified by gel filtration (LH-20) for spectrographic analysis. The NMR spectrum showed a broad singlet at 7.2, a complex absorption from 3.0 to 4.6, a multiplet at 2.1, and a pair of doublets at 1.5 ppm (parts per million).

Example 2. N-(1-Carboxyethyl)-1-alanyl-1-proline.

A solution of L-alanyl-L-proline (372 mg) and pyruvic acid (881 mg) in water was adjusted to pH 7 and treated with 337 mg of sodium cyanoborohydride at room temperature until the reaction was complete. The product was absorbed onto a strong acid ion exchange resin and then eluted with 2% pyridine in water. Freeze drying gave 472 mg of N-(1-carboxyethyl)-1_-alanyl-1_-proline. NMR and mass spectrometry were consistent with the structure. The NMR spectrum showed multiplets centered at 4.5, 3.7, and 2.2 ppm and a pair of doublets at 1.6 ppm.

Example 3. N-(1-Carboxy-2-cyclohexylethyl)-1-alanyl-1-proline.

3-Cyclohexyl-2-oxopropionic acid (cyclohexylpyruvic acid) (0.98 g) and 1-alanyl-1-proline (0.22 g) were treated with sodium cyanoborohydride (0.22 g) as described above. A faintly colored solid, N-(1-carboxy-2-cyclohexylethyl)-1-alanyl-1-proline, 0.31 g, was obtained. After purification by chromatography, the mass spectrum showed peaks at 340 (molecular ion), 322, 227, 249, and 226. The NMR spectrum showed a complex absorption in the range 4.8 to 3.6, as well as peaks (maxima) at 2.2, 1.7, and 1.2 ppm.

Example 4. N-(1-Carboxy-5-methylhexyl)-1-alanyl-bproline.

6-Methyl-2-oxoheptanoic acid (90 g) and L-alanyl•L-proline (0.21 g) were treated with sodium cyanoborohydride (0.21 g) as described above. A white powder of 1\1-(1-carboxy-5-methylhexyl)-1_alanyl-1_-proline (0.24 g) was obtained. After purification by chromatography, the mass spectrum showed a maximum at 472 (disilyl derivative). The NMR spectrum showed absorption centered at 4.5, 3.65, 2.0, 1.6, and 0.85 ppm.

Example 5. N-(1-Carboxy-3-methylbutyl)-1-alanyl-1-proline.

4-Methyl-2-oxopentanoic acid (4-methyl-2-levulinic acid) (1.29 g) and 1-alanyl-1-proline (0.32 g) were treated with sodium cyanoborohydride (0.32 g) as described above. A white loose mass of N-(1-carboxy-3-methylbutyl)-1-alanyl-1-proline (0.40 g) was obtained. A portion was purified by chromatography. The mass spectrum showed a maximum at 429 (molecular ion of the disilyl derivative minus methyl, 444-15). The NMR spectrum showed resonances centered at 4.4, 3.6, 2.1, 1.6, and 0.95 ppm.

Example 6. N-(1-Carboxypropyl)-1-alanyl-1-proline.

2-Oxobutyric acid (ketobutyric acid) (1.02 g) and 1-alanyl-1-proline (0.37 g) were treated with sodium cyanoborohydride (0.38 g) as described above. Crude N-(1-carboxypropyl)-1-alanyl-1-proline (0.42 g) was obtained. A portion was chromatographed for spectral analysis. The mass spectrum showed prominent peaks at 254 (M-18) and 210 (M-62). The NMR spectrum revealed a complex absorption of 4.5 to 3.4 multiplet centered at 2.0, and methyl resonances centered at 1.55 and 0.95 ppm.

Example 7 N-(1-Carboxy-2-methylpropyl)-1-alanyl-1-proline.

A mixture of 3-methyl-2-ketobutyric acid sodium salt (1.46 g) and 1-alanyl-1-proline (0.40 g) was treated with sodium cyanoborohydride (0.41 g) as described above. Elution from an ion exchange resin gave crude N-(1-carboxy-2-methylpropyl)-1-alanyl-1-proline (0.45 g). The product melted at 131-142°. The NMR spectrum showed a complex absorption in the region 4.6 to 3.3, a broad multiplet centered at 2.2, and doublets at 1.65 and 1.1 ppm.

Example 8. N-(1,3-dicarboxypropyl)-1-alanyl-1-proline.

2-Oxoglutaric acid (1.46 g) and L-alanyl-L-proline (0.37 g) were treated with sodium cyanoborohydride (0.38 g) as described above. Crude N-(1,3-dicarboxypropyl)-1-alanyl-1-proline (0.47 g) was obtained, mp 140-160° C. The mass spectrum of the silylated material showed a single ion at 517 m/e, equivalent to the molecular ion for the trisilylated derivative minus methyl (532-15). NMR was consistent with the structure. The methyl resonances were centered at 1.4 ppm.

Example 9. (1,4-Dicarboxybutyl)-1-alanyl-1-proline.

2-Oxoadipic acid (1.74 g) and 1_-alanyl-1_proline (0.41 g) were treated with sodium cyanoborohydride (0.42 g) as described above. Crude N-(1,4-dicarboxybutyl)-1_-alanyl-1_-proline (0.35 g) was obtained, mp 106-132°. The highest peak in the mass spectrum was at 312 and corresponded to the molecular ion minus water. The methyl resonances in the NMR spectrum showed a pair of doublets centered at 1.55 ppm.

Example 10. N-(1-carboxy-3-methylbutyl)-alanyl-isoleucine.

A solution of 1-alanyl-1-isoleucine (150 mg) and 4-methyl-2-oxopentanoic acid sodium salt (564 mg) in water was adjusted to pH 7 and treated with 140 mg of sodium cyanoborohydride at room temperature for several days. The reaction was quenched with a strong acid ion exchange resin, loaded onto a column of the same resin, and eluted with 2% pyridine in water. After freeze-drying, 200 mg (84.9%) of a white powder of N-(1-carboxy-3-methylbutyl)-1-alanyl-isoleucine were obtained. The mass spectrum showed peaks at 460 for the disilylated derivative and at 445 for the disilyl molecular ion minus methyl (460-15). The NMR spectrum showed a broad doublet centered at 0 95 ppm, a complex absorption at 3.7 ppm.

Example 11. (1-Carboxy-2-methylbutyl)-1-alanyl-1-phenylalanine.

A solution of b-alanyl-b-phenylalanine (150 mg) and sodium salt of 4methyl-2-oxopentanoic acid (483 mg) in water was adjusted to pH 7 and treated with 120 mg of sodium cyanoborohydride at room temperature for several days. The reaction was quenched with Dautex 50 (H'), the reaction mixture was passed through a column of the same resin and eluted with 2% pyridine in water. After freeze-drying, 197 mg (88.7%) of a white powder of N-(1-carboxy-2methylbutyl)-1-alanyl-1-phenylalanine were obtained. The mass spectrum shows a maximum at 55 for the trisilyl derivative minus methyl (566-15), 479 for the disilyl derivative minus methyl (494-15> and 449 for the trisilyl ¹ d'-yu minms -COO~MS ^: ~.zz 11 ⁷ ) The NMR spectrum shows broad

Ш · Г · - · · Л "· * '*· ' ,Р - η X ,-4 £ . , . . — O s y . . . Кg » I Ch/ · С X* » ' · W /w Х-> · . Aw .,·.. .W. »ν· .Xi 4 -4 w. —4 v4</ h/Ц» . Hm> χΛ \ □ 4-4 . C1 4_. .3 h orgp /.с ⁷ .. ί ν!ntegration is in accordance with ρτιηι/ζτ4/ηατθ m pzod tpiiptp €ΈΟΤΜθι_πθΜΜθ nr ЗРОМаТНН КАМ 3ΠΗφ3Τ ^| '!ΐ

In a small flask fitted with a pH electrode, combine 1.05 g of L· alanyl-B-pyrrolyl-! and 1.2 ml of 4 M NaOH. Add 0.53 g of chloroacetic acid in 1.2 ml of 2 M NaOH. Adjust the pH to c - 9. Heat to 35°. Maintain the pH at 8 - 9 for 15 min by adding sodium hydroxide as necessary. Add another 0.53 g of urorhodetic acid and sodium hydroxide as necessary over 15 min. Charge a third portion of 0.53 g of chloroacetic acid, maintain the pH at 8 - 9 for 15 min. then another 15 min at 85° and cool. The reaction mixture is passed through a column of Dautex 50 (H~), rinsed with water and eluted with 2% pyridine in water. The collected fractions, showing a positive ninhydrin reaction, were concentrated under a small eight-bar vacuum and dried over ⁴ l of air.

4J Cl »w -W . Ca. 4- » '. C» 4 ·—» 4 t 4-* zg se raz ¹ var? ex 50 (Na ).

A few milliliters of water and eluted with 0.5 M citric acid and washed with sodium hydroxide. The required

The product appeared first (ninhydrin test. well separated from the unreacted alanylproline. The product fraction was concentrated in vacuo to a weight of about 300 mg.

i ν V» > |- 3·- W . й. FW V- 4- - -· •у - 4-4 -2 ·' 4z / . 4W, 'A Cl , Cl J . w *\x/ \ 1 I / . I IpC .Ilf iOCl h 3 vol then the eluate is prepared with 2 % ηιί'Ρι/ΙΠΐΊΗ IN VSDα. FmsJiiI ^; s of the product are concentrated in vacuo and subjected to freeze-drying. A yield of 417 mg of M-1-carboxymethyl-1_-alanyl-'Prsl: .-i.

BAD ORIGINAL spectrum ' ¹ j 0 MeS ^{, n} internal st?np?.o ^tg 1.58 oom <d J=6* ίθ Γn I — Ο f'-'F'' Ρ Л 4' 1 . О ¢0 ί,,,,,ηΛ., m JU' O CO Pl Ο Ο Qp /o-Р

68.

Example 13. (1-carboxyethyl)-alanyl-L-proline.

Dissolve 45 g of benzylpyruvic acid and 4.5 g of L-alanine in p-dioxane. ^and adjust to 5.5 with g of sodium cyanoborohydride, then the temperature is maintained for 6 days with sodium hydroxide and the mixture is stirred with a standard ''PCC'/KTO pH is adjusted to > with '<сн ^;: e ^and ' ^g О' ^, '^' ^?и ' ^:1 k'/cp'·'' ^,и! a

This solution was loaded onto a column of Dauseco 50 (H ⁴ '). ^prepared in om -<s Dimethylsulfonate water, washed with om to dioxane - water, then with water. The product was eluted with 2% pyridine in water, all fractions were combined and concentrated to dryness in vacuo. This was followed by trituration with water, filtration and washing with water. The mixture of N-1-cassobenzoyl-c-alapril was obtained.

A diastase of 10 g can be obtained from the solids containing OE in the mother liquor.

ύΤ6Ό

L-proline

Π'-ίΜογρ pr^rMaM·'! ^p |Sl..'f'. Lupo^pya ro pp

ML

o.aztvsoen in

HS p f. ι; p· . 6 hours at 0°. then _ -C’ trmrepatipo

HΟΙΟΙ οιειου Αδικαρόνητα. κ chromatographed on a pre-clarification plate for thin-layer chromatography with silica gel, developed with ethyl acetate. The broad band at rf = 0.5-0.6 is removed, eluting with ethyl acetate!

To obtain 212 mg of the mixture, the solvent was distilled off to obtain 1 ml.

from

BAD OP!G L ec'rp -a \-i-carbobenzoxyethyl-L0 ... v ... ../1 c. I OO it. I t> . ί .0 j ί у Η ν H Ι τι ί ί 0-4 VX ί 3 CM cv ... i*. . ci π G j . i . roda 50 mg of 10% palladium on charcoal as a catalyst are added and the mixture is heated at a pressure of h _? 276 κΝ/μ and room temperature.

Ж r-ι »1 г» f3 - % - 1^;. <l ? /Λ! \Ζ of sucking through

About Art.t. ks Ci ύ ·. O C* ΓΊ sf kats Ct-

\ 1 - ο τ Κ-Ι ! k> I

Γ> Τ

I

r.-i ji ·-aci ο Ον ρχ ισ ί i io;-L-alanyl- L-proline. The NMR spectrum is comparable to that of 2 and the mass ^spectrum of the silylated derivative shows

Dissolve 0.75 g of N-d-carbobenzyloxyethyl)-L-alanyl in pyridine and add 7.5 ml of 1 M triethylamine in pyridine, cool and add 1.09 g of L-pyridyl benzyl ester hydrochloride and 0.678 g of dicyclohexylcarbodiimide. Store at 0° for 20 h, filter and concentrate the reaction mixture in vacuo. The residue is dissolved in ethyl acetate; the solution is washed with saturated potassium carbonate, and then

The organic phase was dried, concentrated in vacuo, and the residue was chromatographed on silica gel with ethyl acetate/hexane to isolate the diastereoisomeric mixture of N-(1-

uh ' n ^A u !_-! 100 P:

, and after processing and sublimation

Su^e-n se .3. u^ava ^-10-kaos<o<sietiptsC-s1ann.)1- L-προ.'τ.η as oyalo tvoatsa ve^eztzs. o spgutur > St. 1.65 ρρ^ι 'Dog·;. 1 in - s.o oi, 4pd

V · W V- · . ^: - heard Ο- I— I ! .0,..030 %. Ν-·.!-carsomethoxyethyl;-L-alanyl-L-prslin.

A mixture of a solution of 1.4 g of methyl L-alaninate hydrochloride and 3.1 g of abromopyrgonic acid in dioxane was neutralized to pH 9 with sodium hydroxide, heated to 70° and this temperature was maintained for 30 min., the pH being maintained at 8 to 9 by adding the necessary

BAD ORIGINAL co-ipcTRo warpiee hydroxide After cooling, the mixture was placed in a column with ion exchange resin Dautex 50 (H'') _s washed with water and eluted with 2% pyridine in a 200 ml flask. The collected product fractions were freeze-dried. The crude product was purified ^by chromatography on an ion exchange column of Dautex 50 (Na*) in 0.5 M sodium citrate buffer with pH 3.3. The collected product fractions were concentrated to a small volume in vacuo and the chromatography was repeated with Dautex 50 (Na'). The product fractions were freeze-dried, yielding pure N-(1-carbomethoxyethyl)-alanine.

The intermediate was coupled with L-proline-tert-butyl ester using diphenylphosphoryl azide as described in Example 13, then the tert-butyl ester was removed by dissolving in trifluoroacetic acid at room temperature for 3 hours, TFA was distilled off and the reaction mixture was purified on a column with Dautex 50 (H) - 2% pyridine as described, and N-(1-carbomethoxyethyl)-1-alanyl-L-proline was obtained as a mixture of diastereoisomers.

Example 16. N-(1-Methoxycarbonyl-3-methylthiopropyl)-alanyl-L-proline.

A solution of pyruvoyl-L-proline (185 mg), L-methionine, methyl ester (600 mg) and sodium cyanoborohydride (200 mg) in 20 ml of methanol was neutralized with dilute methanolic sodium hydroxide solution. After three days at room temperature, the product was absorbed onto a strongly acidic ion exchange resin and eluted with 2% pyridine in water to give 80 mg of product. The NMR spectrum showed OCH< at 3.35, 3-CH ₃ at 2.2 δ and CH-CH ₃ at 1.55 and 1.7 δ. The mass spectrogram of the silylated product showed the expected molecular ion at 404 m/e.

Example 17. M-(1(5)-carboxy-3-methylthiopropyl)-alanyl-LVAP ORIGINAL

A solution of N-(1(c)-methoxycarbonyl-3-methylthiopropyl)-O-alanyl·L-proline (127.5 mg; 0.384 mM) in 2 mL of water was treated under nitrogen with 7.82 mL of 0.180 N sodium hydroxide (0.782 mM) and stirred for 2.5 hours at room temperature. The product was absorbed from the reaction mixture onto 30 mL of Dautex 50 (H*) and eluted with 4% pyridine in water to give 73.5 mg, which was further purified via LH-20 column to give 55.7 mg of product. NMR spectrum in D ₂ O showed -S-CH ₃ at 2.1; -CH ₃ at 1.5 and 1.6 δ and the absence of methyl ester. The mass spectrogram of the silylated product shows the expected molecular ion at 462 ppm.

Example 18 [[1-Methoxycarbonyl-2-(3-indolyl)-ethyl]-alanyl-L-proline.

Working as described in Example 16, tryptophan methyl ester was condensed with pyruvyl-L-proline in the presence of sodium cyanoborohydride to give N-[1-methoxycarbonyl-2-(3-indolyl)ethyl]-alanyl-L-proline. The NMR spectrum in CDCl ₃ showed aromatic protons at 6.9 to 7.7, protons adjacent to the aromatic nucleus and adjacent to the nitrogen atom at 2.8 to 3.9: aliphatic methylene protons at 1.4 to 2.7 and alanine methyl at 1.0 to 1.4. The mass spectrogram of the silylated product showed a single ion at 516 m/e and corresponded to the loss of ^i'· ^{1 r>} n ^, .'G'P 0^· -j '3?

Example 19,- N-[1(c)-Carboxy-2-(3-indolyl)-ethyl]-O2-alanyl-1-proline.

It is worked up as described in Example 17. The product is hydrolyzed to give the expected diacid. The NMR spectrum in D>Od ₅ Pyr shows 5 aromatic protons at 6.8 to 7.7: protons adjacent to the aromatic nucleus and adjacent to the nitrogen atom at 2.8 to 7.4 and 7 aliphatic protons at 1.0 to 2.2 δ consistent with the expected structure. The mass spectrogram of the silylated material shows a peak at 431 m/e, interpreted as a deprotonated monosilylated ion that has lost one CH ₃ group.

IMPR OHIG1NAL

I ipnwep 20 N-(1-Carboxy-3-phenylpropyl)-1-alanyl-C-4-thiazolidinecarboxylic acid.

-! O

< --. γ n ·

Aj 1 Ο kz *

of the benzylic ester of

L-thiasglycidine-D-carboxylic acid (2.6 g) was reacted in ^methanol . It was treated at 0-5°C with triethylamine (1.4 ml). Then with dicyclohexylcarbodiimide (DCC) (2.3 g) in methylene chloride and the mixture was left to stand overnight. After filtration and washing of the filtrate with water and sodium bicarbonate solution, the solvent was distilled off and the product was chromatographed on silica G60 (E.Merck) in ethyl acetate-hexane. The solvent was distilled off from the combined product fractions in vacuo and the petrol ester was hydrolyzed in acetonitrile-water at pH 13.5 (sodium base) for 1 hour at room temperature. After neutralization to pH 8 with hydrochloric acid, it was washed with ether, the aqueous layer was concentrated in vacuo and freeze-dried. The tert-butyloxycarbonyl protecting group was removed in 4 M hydrogen chloride in ethyl acetate, the product was precipitated with ether, filtered and dried to give 1-alanyl-1-thiazolidine-4-carboxylic acid. 0.385 g of

acid in water using 0.354 g of sodium cyanohydride by the method described in the example to obtain 0.53 g of a mixture of diastereoisomers of N-(1-carboxy-3-phenylpropyl)-1-alanyl-1-4-thiazolidinecarboxylic acid. The NMR spectrum (D ₂ O - NaOD) contains a split doublet at 1.2 ppm (3H), a singlet at 7.1 (5H), a broad absorption in the region 1.6 to 2.0 (2H),

range 2.2 to 4.1 and large water

I » Ύ ₍ , η · 4 k-ν k-h .. ;SchG\^k*.. y .1 1 _g Mk» ίζ fJvH rivtl i“Ci Example 21 m. The mass spectrum of the silylated material shows DC , > 1« i k . 1 * *f*^* ¹ * GT*H h>chuO. N-(1-carboxy-3-phenylpyridyl)<-alanyl-L-

pipecolic acid.

By replacing the thiazolidine carboxylate ester of the example with the hydrochloride of the methyl ester of L-pipecolic acid (1.8 ^g) the title of the example is obtained by the method P: \ ·_^ Q GrIROr ·?·-Π •'ll . .. I y , . h. < i. \_G .

The NR*P spectrum of tCDODi shows a broad multiplet at 1.3 - 1.9 ppm (?n>. a singlet at 7.22 (5H) and a series of multiplets in the range 2.0 - 4.8 ppm. The mass spectrum of the silylated material shows a maximum at m/e = 580 for the disilylated molecular ion.

Example 22. N-(1-jarboxy-3-phenylpropyl)-1-alanyl-1-methylalanine

By replacing the thiazolidine carboxylic ester of Example 20 with L-N-methylalanine methyl ester hydrochloride (1.5 g) the title compound of the example can be obtained by the method described in Example 20.

Example 23. N-(1-carboxy-1-methylethyl)-1-alanyl-1-proline.

The above compound can be obtained by combining 7.7 g of 2-bromoisobutyric acid benzyl ester, 2.4 g of blanyl-bproline tert-butyl ester and 7.0 g of silver oxide in 40 ml of benzene. After refluxing for 24 h, an additional 7.7 g of benzyl ester and 7.0 g of silver oxide are added and refluxing is continued for another 24 h. After cooling, filtering and distilling off the solvent, the diester of the product is isolated by conventional chromatographic methods. The required free acid is obtained by removing ^both the tert-butyl ester group in trifluoroacetic acid and the benzene group by catalytic hydrogenolysis in the established manner.

Example 24. {N-(carboxy-1-phenylpropyl)-alanyl}-proline.

A mixture of 4-phenyl 2-soxobutyric acid (1.49 g) and 1-alanyl-1-proline (0.31 g) in water was adjusted to pH 7.5 with sodium hydroxide and treated with sodium cyanoborohydride (0.32 g) overnight. The product was absorbed onto a strongly acidic ion exchange resin and eluted with 2% pyridine in water to give 0.36 g of the crude diastereomeric product, N'(1-carboxy-1-phenylpropyl)-1-alanyl-1-proline. One portion was

BAD OFUG'NAL j purified by gel filtration (LH-20) for spectrographic analysis. The NMR spectrum in dimethylsulfoxide (DMSO) shows aromatic hydrogen 7 20, a broad singlet at 3.40. broad multiplets at 3.0 to 3.9. 2.67 and 1.94 .·; one doublet at 1 23 and 1 15. The mass spectrum shows a molecular ion at 492 m/e for the ditrimethylsilylated groups.

Example 25. N'-(1-Crbsxy-3-feccylgrsgg^-L-panyl-L-proline).

Polystyrene resin XAD-2 (Rohm & iaas Co.) was ground and sieved. The 200-400 mesh fraction was determined and 440 ml was loaded onto a chromatography column which was calibrated with 0.1 M _NH4OH in 95:5 (v/v) water-methanol. 350 mg of N-(1-carboxy-3-phenylpropyl)-1-alanyl-1-proline, prepared and purified according to Example 24, dissolved in 10 ml of the same solvent was loaded onto the column. Elution was then carried out with this solvent. The first isomer was eluted from the column in the 375-400 ml eluate range, the second isomer in the 440-480 ml range, the intermediate fractions containing a mixture of the isomers. The fraction containing the first isomer was freeze-dried to give 130 mg of a white solid. After recrystallization from 1 ml of water with pH to 3 gives 94 mg of white needle crystals with mp 148 - 151°, which represent the more active isomer with S,S,S configuration, determined by X-ray analysis. ' ⁱ⁷ L· ⁼ * 6 ⁷ 4·-' go 1 M HO) after drying in vacuum over P.O ₅ . NMR (DMSO) shows a single doublet for methyl protons at 1.22 f-· : jj . μ# Ki-ί, vi Л i Ci, lz C Cl дцсХ □ 1 kz i ; Л >< Co іνί θ , Cz ζζ 4z y P CO 3 CM DciH θ And gives 122 mg of a white solid. After recrystallization of 103 mg from 2.5 ml of water with pH to 3, 64 mg of white feathery crystals are obtained, mp 140 - 145° (with decomposition). [α] _D = - 101.6°, (0.1 M HCl) after drying. NMR (DMSO) shows the methyl doublet at 1.17 ppm.

Example 26. N-(1'C'-ethoxycarbonyl-3-phenylpropyl)-alanyl-L-proline.

Ethyl 2-oxo-4-phenylbutyrate (1.03g) and 1-alanyl-E-pyrrolidin (0.19g) were dissolved in a solvent containing 1:1 ethanol-water. A solution of sodium cyanoborohydride (0.19g) in ethanol-water was added dropwise at room temperature.

BAD ORIGINAL temperature for two hours. When the reaction was complete, the product was absorbed onto a strongly acidic ion exchange resin and eluted with 2% π..:ρ.in water. The product-rich fractions were freeze-dried to give 0.25 g of crude . N-O-ethoxycarbonyl-3phenylpropyl)-B-alanyl-B-proline. The mass spectrum showed a molecular ion at 448 m/e for the monosilylated groups. The required isomer was obtained by chromatography.

Example 27. N-(1-Aminocarbonyl-3-phenylpropyl)-B-alanyl-Bproline.

Working as described in Example 26, 2-oxo-4-phenylbutyramide and B-alanyl-B-proline can be condensed in the presence of sodium cyanoborohydride to obtain N-(1-aminocarbonyl-3-phenylpropyl)-B-alanyl-B-proline.

Example 28. N-(1-Carboxy-3-phenylpropyl)-B-alanyl-B-tryptophan.

Working as described in Example 24, 2-oxo-4-phenylbutyric acid and β-alanyl-β-tryptophan can be condensed in the presence of sodium cyanoborohydride to give N(1-carboxy-3-phenylpropyl)-β-alanyl-β-tryptophan.

Example

2S. N-(1-Carboxy-3-phenylpropanyl)-B-alanyl-B-4-hydroxyproline.

P G in presence

B-alanyl-B-4-hydroxyproline is condensed on sodium cyanoborohydride to give N-(1kg carboxy-3-phenylpropyl)-B-alanyl-B-4-hydroxyproline. The NMR spectrum in deuteromethanol shows a doublet centered at 1.53 ppm (3H), a singlet at 7.13 (5H) and a series of multiplets in the range 2.0 to 4.7 ppm.

The mass spectrum of the silyllanyl material shows a molecular ion of the tosilylated product at m/β = 580.

Example 30. N-(1-carboxy-3-phenylpropyl)-B-serinyl-B-proline.

The procedure is as described in Example 24, with 2-oxo-4-phenylbutyric acid and B-serinyl-B-proline being condensed in

The

BAD ORIGINAL sodium cyanoborohydride reaction. to obtain N-(1-carboxyO · . -1 -4П fx f I γι' * 'X .... г-l I ^-1 »·> <% г-ι . « . .

h/ v- •Η'-*··'·'·; - — D” ^: H ^; '

MSCC. .иКТУЦз Ζ0Г- <y.'..ίp6H ИСН ПрН OCv ιТ.. 0 ЗД trisilylated products NMR spectrum in D ₂ O is consistent with the structure.

Example 31. N1-Carboxy-3-phenylpropyl)-bphenylalanyl-b d.rolnn.

Working as described in Example 24, 2-oxo-4-phenylbutyric acid and 1-phenylalanyl-1-proline are condensed in the presence of sodium cyanoborohydride to give N-(1-carboxy-3-phenylpropyl)-1-phenylalanyl-1-proline.

The mass spectrum shows an ion at 406 m/e for the molecular ion minus water (424 - 18). The NMR spectrum in D _? O is consistent with the structure.

Example 32. N-(1-Carboxy-3-phenylpropyl)-1-cysteinyl-1-proline.

Working as described in Example 24, 2-oxo-4-phenylbutyric acid and L-β-benzylcysteinyl-L-proline are condensed in the presence of sodium cyanoborohydride. The product can then be treated with sodium in liquid ammonia to give (1-(1-carboxy-3-phenylpropyl)-β-cysteinyl-L-proline).

Example 33. N-(1-carboxy-3-phenylpropyl)-L-histidinyl-L-leucine.

Working as described in Example 24, 2-oxo-4-phenylbutyric acid and 1-histipinyl-1-leucine are condensed in the presence of sodium cyanoborohydride to give N-(1-carboxy)-4-phenylbutyric acid.

Ή'-'· ^: 4·

XI

Example 34. 1m-(1-Carboxy-3-phenylpropyl)-1-phenylalanyl-1-arginine.

The procedure is as described in Example 24, with 2-oxo-4-phenylbutyric acid and l-phenylalanyl-l-arginine being condensed in

.1 presence of sodium cyanoborohydride. to obtain 1N-(1-carboxy-3-phenylpropyl)-1_-phenylalanyl-1-arginine

Psimes 35. ^•/-Carbocop-3-Phenylpro-plUc-Phe.nilala.-l-b trip I oran.

Working as described in Example 24, 2-oxo-4-phenylbutyric acid and B-phenylalanyl-B-tryptophan are condensed in the presence of sodium cyanoborohydride to give N-(1-carboxy-3-phenylpropyl)-B-phenylalanyl-B-tryptophan.

Example 36. N-(1-Carboxy-3-(3-indolyl)propyl)-1-alanyl-1-proline.

Working as described in Example 24, 2-oxo-4-phenylbutyric acid and 1-phenylalanyl-1-arginine were condensed in the presence of sodium cyanoborohydride to give N-(1-carboxy-3-(3-indolyl)propyl)-1-alanyl-1-proline. The mass spectrum of the trisilylated material showed a molecular ion at 603 m/e and an M ⁺ - 15 maximum at 588 m/e.

Example 37. (1-Carboxy-3-phenylpropyl)-1-alanyl-3,4-dehydroproline.

A mixture of 3,4-dehydroproline (2.3 g), tert-Boc-1-alanine-N-hydrosuccinimide ester (7.2 g) and sodium carbonate (2.5 g) in a dioxane-water mixture was stirred at 0° overnight. The mixture

8W

•Z ’ o U T im · G\ O

Г\ V/ I -¼ w t < >

DdKyVM heard C 4 p νό Cu р с1оЯ p o.

The tert-Boc protecting group was removed with trifluoroacetic acid in the usual manner and chromatographed on Dautex-50 (H') _eluting with 2% pyridine in water as described in Example 2. The dipeptide was isolated by freeze drying. This product was condensed with 2-keto-4-phenylbutyric acid as described in Example 24 to give the product as a mixture of diastereoisomers. The mass spectrum showed a molecular ion at 490 m/e for the disilylated product.

BAD ORIGINAL

The diastereomeric mixture obtained above (140 mg) was separated into its components by chromatography on XAD-2 resin as described in Example 25. The major component (70 mg) was the first to leave the column = - 143° (1.3 methanol). The mass spectrum of each component

1'Г η^ι/υπ «poi »'··/> LJ nr>t< 4 OH rv-\ /'ο οο < Χ-ζ ' U, -X . . -ζ. _ . J. . ₍ VX/: > ΗνΠ ' tti ~ ¹ · , Ch.· kU<D d itr i m ethyl e yl il i r a n products.

| |Example 36. |'\-(1-Carboxy-3-phenylpropyl)-1-alanyl-2-methylthiazolidine-4-carboxylic acid.

This compound can be prepared as described in Example 37 by replacing 2.3 g of 3,4-dehydroproline with 2.9 g of 2-methylthiazolidine-4-carboxylic acid.

Example 39. N-(1-Carboxy-3-phenylpropyl)-1-alanyl-2-methylalanine.

This compound can be prepared as described in Example 37 by replacing 2.3 g of 3,4-dehydroproline with 1.8 g of 2-methylalanine.

Capsules filled with dry matter ^and containing 50 mg of active ingredient per capsule and auxiliary components in mg:

for capsule N-(1-carboxy-2-phenylethyl)-C-alanine-1_- 50 mg Π ΓΊ 1 · — » ^{1 ;} '' j;a ktosis 149 mg ^? 'acetic acid stearate 1 mg "2g-su ^g h (size No. 1) 200 ug

Grind N-(1-carboxy-2-phenylethyl)-1-alanyl-1-proline to a No. 60 powder and then sieve the lactose and magnesium stearate over the top powder through a No. 60 mesh sieve. The combined ingredients are mixed for 10 minutes and then filled into No. 1 dry gelatin capsules.

Example 41. N-(1-ethoxycarbonyl-3-phenylpropyl)-1-alanyl-1-BAD ORIGINAL proline

A mixture of /a-alanyl-C-proline (7.7 g) and ethyl-2-oxo-4,.,μύ; iL.o g, ь I4C ml of ethanol was stirred with 64 g of powdered molecular sieves at room temperature for half an hour. Then a solution of ^ι ·?'ρ·.·?ζ cyanoborohydride (2 6 g) in 40 ml of ^ethanol was slowly added over 6 h. ^After filtering off the d * ι ί Ся 1 d р С d Г\ >ц ч I ч. 1Ί Г ,ч< I d I vl Ud чя С >Λ Η ЦС ΗΤ^μ d 1 'Ομ DdΚγ JM MV M Cj ί dK С b 0 М . The residue was partitioned between chloroform and water. The pH was adjusted to 8 5 and the chloroform layer was separated and discarded. The aqueous layer was acidified to pH 2.7 and the product was extracted with chloroform. The chloroform extract was dried over sodium sulfate and concentrated in vacuo to give 10.4 g of mixed piastereomers. High pressure liquid chromatography (HPLC) showed that the major product was the desired 1N-(1-[N]-ethoxycarbonyl-3-phenylpropyl)-L-anan-L-propan-N.

NMR spectrum shows aromatic absorption at 7.1 δ and

diastereomeric methyls, as a multiplet centered at 1.3 δ.

The mass spectrum shows a molecular ion at 376 m/e and a strong maximum at 358 (M* - H _? O).

Pgm ί» <19 ..ch-chya Ch_. 1-(1-[8]-ethoxycarbonyl-3-phenylpropyl)-E-alanyl-E-

ί .rslin maleate.

A solution of N-(1-ethoxycarbonyl-3-phenylpropyl)-E-alanyl-E- ^{proline maleate (} 38 ^g ) ⁱⁿ 69 ml of acetone was stirred with 4.25 g of •.'e.-eo'-OBd acid in 69 ml of acetonitrile. After stirring for 1 h at room temperature, the solid was filtered, washed with acetonitrile and air-dried to give 8.4 g of maleate with mp 141-145° by HPLC with a purity of about 96%. The crude maleate was recrystallized from acetonitrile to give 7.1 g of N-(1/B/-ethoxycarbonyl-3-phenylpropyl)<-alanyl-E-proline maleate with mp 148-150° by HrLC with a purity of about 99%.

BAD ORIGINAL

A β-(1-ethoxycar6omyl-3-phenylpropyl)-alanyl-spoolin

So st C.3H g 1_-alanyl-1.-proline. 0.206 g ethyl 2-oxo-4-phenylbutyrate and 1.6 g molecular sieves in 10 ml ethanol were hydrogenated at room temperature under a pressure of 276 kM/m ^? with 0.1 g catalyst 10% palladium on carbon. After the absorption of hydrogen was complete, the crude product obtained by filtration and concentration was absorbed onto an ion exchange resin (Dautex 50, H ') and eluted with 2% pyridine in water to give 0.224 g LB(1-ethoxycarbonyl-3phenylpropyl)-1_-alanyl-1_-proline. HPLC showed a ratio of isomers of 55 : 45.

B. N-(1-(3)-ethoxycarbonyl-3-phenylpropyl)-alanyl-proline maleate.

A mixture of 3 g of L-alanyl-E-proline, 5 g of ethyl 2-oxo-4-phenylbutanoate and 13 g of 3A molecular sieves and 3.6 g of Raney nickel in 85 ml of ethanol was hydrogenated at 25° C. and a hydrogen pressure of 276 kM/m' until hydrogen uptake ceased. The solid phase was filtered, washed with 80 ml of ethanol and the filtrates were combined. Analysis by high pressure liquid chromatography (HPLC) showed a diastereoisomeric ratio of 87:13 in favor of the desired product. The ethanol was removed ⁱⁿ vacuo to give an oily product which was ~ 5r-~ ? r? < π '''H . r, > 4 - p . .o-ι-^ιτ- nU ι_Ό Ο LZQP up-rn

J— - . — — . 11 s J I uz r-S'** i ' — · . i r . 4, I » IV · v. X. Wt. i . 11 ν* biphasic mixture was adjusted to 8.6 with 50% sodium hydroxide. The layers were separated and the aqueous phase was extracted with another 2 x 20 ml of ethyl acetate. The aqueous phase was adjusted to pH 4.25 with hydrochloric acid, 12 g of sodium chloride was dissolved in the water and the product was extracted with 5 x 12 ml of ethyl acetate. The extracts were combined and dried over sodium sulfate. The desired product (1-(8)-ethoxycarbonyl-3-phenylpropyl)-k-alanyl-zoline crystallized as its maleic salt by addition of 1.86 g of maleic acid. After stirring for

The BAD OI 1H salt was filtered, washed with ethyl acetate and dried to give 5.2 g of pure product with mp 150-151°C.

Example 44 N-(1-benzyloxycarbonyl-3-phenylpropyl)-N-alanyl-L-proline.

A solution of L-alanyl-L-proline (167 mg) and benzyl-2-oxo-4-phenylbutyrate (1.20 g) in 5 ml of ethanol was stirred at room temperature with 3 g of powdered molecular sieves, type 4A. Sodium cyanoborohydride (75 mg) was then added portionwise over three hours. The product was purified by absorption onto a strong cation exchange resin and eluted with 2% pyridine in water. After passing through a gel filtration column (LH-20) 220 mg of N-(1-benzyloxycarbonyl-3-phenylpropyl)-1_-alanyl-L-proline was obtained as a mixture of isomers. After thin layer chromatography on silica gel, elution with 1 ethyl acetate, 1 n-butanol, 1 water, 1 HOAc (acetic acid) showed one large main spot with Rf 0=71. The isomers were separated using reverse phase HPLC to give N-(1(8)-benzyloxycarbonyl-3-phenylpropyl)-alanyl-L-proline.

Similarly, reduction of N-acetylaminoethyl-2-oxo-4-phenylbutyrate and L-alanyl-L-proline with sodium cyanoborohydride yields N-[1-(2-acetylamino)ethoxycarbonyl-3-phenylpropyl]-alanyl-4-proline.

In the same way, from dimethylaminoethyl 2-oxo-4-phenylbutyrate and L-alanyl-L-proline, N-[1-(2-dimethylamino)ethoxycarbonyl-3-phenylpropyl]<-alanyl<-proline is obtained.

In the same way, 1N-(1-benzyloxycarbonyl-4-methylpentyl)-alanyl-L-proline is obtained from benzyl 2-oxo-4-phenylbutyrate and L-alanyl-L-proline.

The mass spectrum of the silylated material shows maxima at 510 (M~-15), 464, 420, 419, 385 and 296 m/e (main maximum).

The NMR spectrum is consistent with the structure.

Example 45. N-(1-Butoxycarbonyl-3-phenylpropyl)-E-alanylL-proline.

A solution of N-benzyloxycarbonyl- ^4- methylpentyl)-alanyl-L-proline 3° butyl ester (452 mg) in 5 ml of benzene was hydrogenated over 150 mg of 10% palladium on charcoal to remove the nitrogen protecting group. After filtration and evaporation of the solvent, the L-alanyl-L-proline 3° butyl ester was dissolved in 8 ml of tetrahydrofuran and treated with 1.41 g of butyl 2-oxo-4-phenylbutyrate and 3 g of powdered molecular sieves. Sodium cyanoborohydride (150) was added portionwise over several hours and the mixture was stirred overnight at room temperature. After filtration and concentration in vacuo, the residue was treated with 25 ml of trifluoroacetic acid for 2 h at room temperature. After removal of the acid, the product was purified by absorption on an ion exchange resin and by gel filtration (LH-2O). After concentration and drying of the product fractions, 182 mg of 1N-(1-butoxycarbonyl-3-phenylpropyl)-alanyl-L-proline were obtained as a mixture of isomers. After thin layer chromatography (silica gel, 1 ethyl acetate, 1 butanol, 1 water, 1 acetic acid), two spots with Rf 0.67 and 0.72 were shown. The mass spectrum showed maxima at 548 (disilylated molecular ion) and ⁴⁷⁶ (monosilylated molecular ion). The isomers were separated by reverse phase HPLC, yielding N-(1-(S)butoxycarbonyl-3-phenylpropyl)-1-alanyl-L-proline.

Example 46. N-(1-Ethoxycarbonyl-3-phenylpropyl)-1-alanyl-L-proline, ethyl ester.

A solution containing 0.63 g of N-(1-carboxy-3-phenylpropyl)-1-alanyl-L-proline in 9.7 ml of ethanol was saturated with HCl gas at 0°. After standing overnight at room temperature, the HCl and ethanol were removed in vacuo to give a light yellow oil which was purified by gel filtration (LH-20 column). The ethyl ester of N-(1-ethoxycarbonyl-3-phenylpropyl)-1-alanyl-L-proline was obtained from 0.39 g, one spot on thin layer chromatography. The NMR spectrum showed two ethyl groups per aromatic ring. The mass spectrum showed a molecular ion at 404 m/e.

Example 46. 1N-(1-carboxy-3-phenylpropyl)-E-alanyl-trans-4-methoxy-L-proline.

Methyl trans-4-methoxy-proline hydrochloride was prepared by the method of E. Adams et al., J.Biol.Chem. 208, 573 (1054) by esterification with methanolic hydrogen chloride in the standard manner. Boc-L-alanine in methylene chloride was coupled with dicyclohexylcarbodiimide as described above, and the intermediate Boc-L-Ala-L-methoxy Pro-OMe was purified by chromatography on silica gel eluting with 1:1 ethyl acetate:hexane. The ester was hydrolyzed with sodium hydroxide in acetonitrile-water, adjusted to pH 7.5, dried by sublimation, and the amine was liberated in 4M hydrogen chloride in ethyl acetate in the usual manner. 0.54 g of this L-alanyltrans-D-methoxy-L-proline was condensed with 2.0 g of 2-oxo-4-phenylbutyric acid in 6 ml of water using 0.43 g of sodium cyanoborohydride as described in Example 24. After isolation according to Example 24, 0.92 g of a mixture of diastereoisomers of . N-(N-[1-carboxy-3-phenylpropyl)-E-alanyl-trans-4-methoxy-L-proline was obtained. The NMR spectrum in _D2O showed a split doublet centered at 1.58 ppm (3H), singlets at 3.37 (3H) and 7.35 ppm (5H), a complex absorption in the region 1.9-3.5 and a broad multiplet at 4.0-4.6 ppm. The mass spectrum shows prominent peaks at 360 (M-18) and 256 (M-122).

Example 48 N-(1-Benzyloxycarbonyl-3-phenylpropyl)-alanyltrans-4-methoxy-L-proline.

By coupling L-alanyl-4-methoxyproline, prepared according to Example 47, with 2-oxo-4-phenylbutyrate in ethanol in the presence of sodium cyanoborohydride according to the method described in Example 44, a mixture of diastereoisomers of N-(1-benzyloxycarbonyl-3-phenylpropyl)<-alanyl-trans-4-methoxy-L-proline can be obtained. The isomers can then be separated by reverse phase HPLC to obtain N-(1/b/-benzyloxycarbonyl-3-phenylpropyl)<-alanyl-trans-4-methoxy-L-proline.

Example 49. N-(1-Benzylaminocarbonyl-3-phenylpropyl)-1-alanyl-L-proline.

The benzylamide of 2-oxo-4-phenylbutyric acid was prepared by dissolving 3.0 g of this acid, 2.4 ml of benzylamine and 4.7 ml of diphenylphosphorylazide in 60 ml of cold dimethylformamide and adding dropwise 2.6 ml of triethylamine in dimethylformamide (DMF) while maintaining the temperature at about -10° C. for 2.5 h and overnight at room temperature. The dimethylformamide was distilled off in vacuo and the residue was partitioned between water and ethyl acetate. The organic layer was chromatographed on silica gel, eluting with ethyl acetate:hexane 1:4. The solvent was evaporated from the product fractions to give 2.2 g of crystalline N-benzyl-2-oxo-4-phenylbutyramide. 1.26 g of this was coupled with 0.19 g of L-Ala-L-Pro. using 0.125 g of sodium cyanoborohydride in ethanol as described in Example 44. The crude product was purified by gel filtration (LH2O) to give a mixture of diastereoisomers of N-(1-benzylaminocarbonyl-3-phenylpropyl)-k-alanyl-L-proline. The NMR spectrum ( _COCl3 ) showed one doublet at 1.1 ppm (H). a pair of singlets at 7.3 (10H) and complex absorption at 1.6 - 2.3 (6H), 2.3 - 2.9 (2H), 2.9 - 3.8 (4H) and 4.0 - 4.6 (3H). The mass spectrum of the silylated material showed distinct peaks at m/e = 509 (monosilyl derivative) and 581 (disilyl derivative).

Example 50. N-(1-Carboxy-3-phenylpropyl)-1-alanyl-N-methylphenylalanine.

By replacing the thiazolidine carboxylic ester of Example 20 with 1N-methyl-L-phenylalanine methyl ester, L-alanyl-1N-methyl-1-phenylalanine was obtained. 0.85 g of this was condensed with 3.02 g of 2-oxo-4-phenylbutyric acid using 0.64 g of sodium cyanoborohydride in water as described. The mixture was acidified to pH 1.5 and extracted into ether. The ether was distilled off and the residue was dissolved in 70% methanol-water and chromatographed on a Doutex 50 (H') column prepared with this solvent. Elution was performed with a 3% pyridine solution in the same solvent. The product fractions were combined, concentrated, dried by sublimation and purified on LH-20 in methanol to give 0.54 g of a mixture of isomers of. N-(1-carboxy-3-phenylpropyl)-1-alanyl-N-methyl-phenylalanine. The NMR spectrum (D ₂ O, NaOD) shows one doublet centered at 1.18 ppm (3H), two overlapping singlets at 7.3 (10H), a singlet at 2.95 (3H), and a broad multiple absorption in the ranges 1.4 to 2.1 and 2.3 to 4.4. The mass spectrum of the silylated product shows the molecular ion of the desilylated product at m/e = 556.

Example 51. N-(1-Ethoxycarbonyl-3-phenylpropyl)-1-alanyl-1-prolinamide.

L-alanyl-L-prolinamide was prepared by coupling tert,Boc-L-alanine with L-prolinamide by known methods using dicyclohexylcarbodiimide in a 4:1 methylene chloride:dimethylformamide ratio. Purify the intermediate tert-Boc-L-Ala-LPrO-N·-K by chromatography on LH-20 in methanol, then remove the tert-Boc protecting group in 4 M HCl in ethyl acetate. Combine 0.5 g of the product L-Ala-L-Pro-NH ₂ .HCl in 10 ml of absolute ethanol, neutralized with one equivalent of triethylamine with 2.4 g of ethyl 2-oxy-4-phenyl-butyrate using molecular sieves and 0.30 g of sodium cyanoborohydride as described in Example 41. In this example, the product is found in the chloroform extract at pH 8.5; concentrated in vacuo, dissolved in 50% ethanol-water and chromatographed on Dautex 50 (H ^g ) prepared in 50% ethanol-water, eluting with 2% pyridine in this solvent, the product fractions were pooled and further purified by chromatography on LH-20 in methanol. The solvent was distilled off in vacuo to give 0.40 g of N-(1BAD O. ' ethoxycarbonyl-3-phenylpropyl)-1_-alanyl-1_-prolinamide in the form of diastereoisomers. The NMR spectrum (CDCl3) showed one triplet, an overlapping doublet at 1.1 - 1.5 ppm (6H), a series of five multiplets in the region 1.5 - 4.7 ppm (15H) and a singlet at 7.17 ppm (5H). The mass spectrum of the silylated material showed distinct peaks at m/e = 477 (monosilyl derivative) and 519 (disilyl derivative).

Example 52. N-(1-Carboxy-3-phenylpropyl)-1-alanyl-1-prolinamide.

1-Alanyl-L-proline amide, prepared as described in Example 51, was coupled with 2-oxo-4-phenylbutyric acid using cyanoborohydride in 50% ethanol-water according to the method described in Example 2. After elution from the ion exchange resin, it was concentrated in vacuo to a small volume, washed with water and freeze-dried to give N-(1-carboxy-3-phenylpropyl)-alanyl-L-proline amide as a mixture of diastereoisomers.

The NMR spectrum shows an aromatic absorption at 7.35 δ and a pair of doublets for diastereomeric methyls at 1.55 δ.

Example 53. N-(1-[3]-hydroxyaminocarbonyl-3-phenylpropyl)-[-aphanyl-b-proline.

To a cold solution of 1N-(1-ethoxycarbonyl-3-phenylpropanyl)-1-alanyl-[_-proline-maleate (0.19 g), prepared as described in Example 42, in 1 ml of ethanol. 0.85 g of potassium hydroxide in 0.57 ml of ethanol was added. Then, a suspension of 0.07 g of hydroxylamine hydrochloride in 0.9 ml of ethanol containing 0.060 g of potassium hydroxide was added dropwise. It was kept in an ice bath, and then overnight at room temperature. The upper layer was decanted, diluted with 10 ml of water, adjusted to pH 2.5 with hydrochloric acid and washed with chloroform. Neutralize and freeze-dry the aqueous layer and purify by chromatography on XAD-2 resin in a 0.1 M ammonium hydroxide/methanol gradient to give N-(1-[S]hydroxyaminocarbonyl-3-phenylpropyl)-1-alanyl-L-proline. The mass spectrum of the silylated material showed a single ion at m/e = 579 for the trisilylated derivative, and the NMR was consistent with the structure.

Example 54. N-(1-carboxy-3-methylbutyl)-alanyl-B-tryptophan

A solution of the sodium salt of 4-methyl-2-oxopentanoic acid (414 mg) and B-alanyl-B-tryptophan (150 mg) in water was adjusted to pH 7 with sodium hydroxide and treated with sodium cyanoborohydride (103 mg) at room temperature for several days. The product was absorbed onto a strongly acidic ion exchange resin and eluted with 2% pyridine in water. The product-rich fractions were freeze-dried to give 189 mg of a white powder. The mass spectrum showed a molecular ion at 389 m/e and maxima at 187 m/e and 157 m/e for the fragments shown separately:

sun-sun ₃

I

CH ²

but ₂ s—sn—nh—sn

The NMR spectrum in D _? O is consistent with the structure.

Example 55. N-(1-Carboxy-3-methylbyl)-B-histidyl-B-leucine.

The procedure was as described in Example 54, 4-methyl-2-oxopentanoic acid and β-histidyl-β-leucine were condensed in the presence of sodium cyanoborohydride to give N-(1-carboxy-3-methylbutyl)-β-histidyl-β-leucine. In this example, the product was eluted from the ion exchange resin with 10% ammonia. The mass spectrum was consistent with the structure.

Example 56. N-(1-Carboxy-3-methylbugyl)-B-phenylalanyl-Barginine.

The procedure is as described in Example 54, with 4-methyl-2-oxopentanoic acid and B-phenylalanyl-B-arginine being condensed in

BAD ORIGINAL presence of sodium cyanoborohydride to give 1N-(1-carboxy-3-methylbutyl)-1_-phenylalanyl-1_-arginine. The product was eluted from the iodophor resin with 10% ammonia. The NMR spectrum in D _? 0 showed one sp-llet at ⁷ 2 (5H), a broad triplet centered at 3.0 (6H), a broad multiplet at 1.3 (6H), and a broad doublet at 0.9 (6H).

Example 57.

A. N-(1-carboxy-3-phenylpropyl)-1-lysyl-L-proline.

Working as described in Example 54, 2-oxo-4-phenylbutyric acid and tert-Boc-lysyl-l-proline were condensed in the presence of sodium cyanoborohydride. Essentially all of the tert,Boc protecting groups were removed when the product was adsorbed onto a strongly acidic ion exchange resin. The crude N-(1-carboxy-3-phenylpropyl)-lysyl-l-proline was eluted from the resin with 10% ammonia, dried by sublimation, and purified by gel filtration chromatography (LH-20). A faint peak for the tert-Boc protons in the NMR spectrum disappeared when the product was treated with 4 N ethyl acetate in hydrogen chloride gas. The NMR spectrum of the resulting hydrochloric acid salt of the product was consistent with the structure. The mass spectrum showed a molecular ion at 693 m/e for the tetrasilylated products. Chromatography on XAD-2 resin using 3.5% acetonitrile in 0.1 molar ammonium hydroxide gave N-(1-carboxy-3-phenylpropyl)-1-lysyl-1-proline.

B. 1N-α-(1N-carboxy-3-phenylpropyl)-1-lysyl-1-proline.

The procedure is as described in Example 54, 2-oxo-4-phenylbutyric acid and N-tert-Boc-lysyl-E-proline are condensed in the presence of sodium cyanoborohydride. The product is absorbed onto a strongly acidic ion exchange resin and eluted with 2% pyridine in water. The product-rich fractions are distilled off and treated with 4 N HCl in ethyl acetate to remove the tert-Boc protecting groups. The hydrochloride salt obtained is converted to the free base by absorption onto a strongly acidic ion exchange resin and elution with 2

BAD OFliGiNAL % pyridine in water. After freeze-drying the product-rich fractions, N-α-ί ((b)-carboxy-3-phenylpropyl)-1-lysyl-ζ-proline was obtained as a white loose mass. The NMR spectrum was consistent with the structure. The mass spectrum showed a molecular ion at 549 for the disilylated products. Chromatography gave the required isomer.

Example 58 N-(1-Carboxy-3-phenylpropyl)-1-3-fluoroalanyl-1-proline.

To a solution of L-3-fluoroalanine (420 mg) in 4 ml acetone - water (1:1) was added triethylamine (589 mg) and 2-tert-butoxycarbonyloximino-2-phenylacetonitrile (1.060). The mixture was stirred for 2.5 hours, cold aqueous potassium bicarbonate solution (5 %) was added and the mixture was extracted with ethyl acetate. The aqueous phase was acidified with cold 1N hydrochloric acid and extracted with ethyl acetate. The extract was washed with saturated aqueous sodium chloride solution, dried over sodium sulfate and concentrated to dryness to give L-tert-Boc-3-fluoroalanine (800 mg) with mp 91 - 93 ⁰ .

To a stirred solution of the resulting compound (800 mg) and proline benzyl ester (1.5 g) in methylene chloride (8 ml) at 0° was added dicyclohexylcarbodiimide (845 mg) in methylene chloride (6 ml) and the mixture was stirred at 0° for 2 hours and at 20° for 18 hours. The mixture was filtered, the precipitate was washed with methylene chloride and the combined filtrates and washings were extracted with cold 1 N hydrochloric acid, cold 5% aqueous potassium bicarbonate solution and saturated aqueous sodium chloride solution, dried over sodium sulfate and concentrated to dryness. After column chromatography on silica gel and elution with 6% acetone in chloroform, the pure protected dipeptide was obtained.

The tert-Boc group was removed by treatment with 4 N hydrogen chloride in ethyl acetate (8 ml) at 0° for 1 hour. About 20 ml of ether was added and the hydrochloride of the benzene ester of 3-fluoroalanyl-3-proline (450 mg) precipitated, mp 158-161° C., which separated

BAD ORIGINAL by filtration After hydrogenation in 6 ml of water and 2 ml of ethanol over 60 mg of 10% palladium on carbon at a pressure ^{of 1} atmosphere and 20° C in

dry, 330 mg of 1_-3-Fluorolanyl-1_- phenylpropanone hydrochloride are obtained

The mass spectrum shows a molecular ion at 348 m/s for the trimethylsilyl product.

To a mixture of 4-phenyl-2-oxobutyric acid (375 mg) and L-3fluoroalanyl-L-proline hydrochloride (100 mg) in 3 ml of water (pH adjusted to 7 with sodium hydroxide) was added sodium cyanoborohydride (80 g; The mixture was stirred for 20 hours and worked up as described in Example 24. The mass spectrum of the LH-20 purified product showed a molecular ion at 510 m/e for the ditrimethylsilyl washed materials. Thin layer chromatography on silica gel showed a single spot Rf = 0.7 - system 1:1:1:1 ethyl acetate : acetic acid : n-butanol :

water.

The diastereoisomers were separated on XAD-2 resin as described in Example 25.

N-(1-ethoxycarbonyl-3-phenylpropyl)-1-3-fluoroalanyl-L-proline was prepared as described in Example 26.

Example 53 N-(1-Ethoxycarboxyl-3-phenylpropyl)-1-alanyl-1-3,4-dehydroproline.

'-•aldynyl-O-3 4-dehydroproline, prepared according to Example 37, was converted according to the procedure described in Example 26 to '--(1-ethoxycarbonyl,3-phenylpropyl)-alanyl-1_-3 _: 4-dehydroproline as a two-component diastereomeric mixture, thin layer chromatography - silica gel plate Rf 0 82 (major component) and Rf 0.79 (minor), two developments in the system 4.1:1 - butanol : water : acetic acid. The mass spectrum showed a molecular ion at 518 m/e for the ditrimethylsilylated materials.

Example 60 N-[1(S)-Methoxycarbonyl-2-(1H-imidazol-4-yl)-ethyl]-O-alanyl-L-proline.

BAD ORIGINAL

_......J

The procedure is as described in Example 16, with the methyl

N - [' - m e t o k s i carbonyl i l 2na*z/. gv

(1H-imidazol-4-yl,1-ethyl]-C-alapayl-E-proline.

The NMR spectrum in D ₂ O shows imidazole protons at 8.6 and 7.3;

melts adjacent to the imindase group and protons of the methyl ester at 3.7 and alanyl methyl group: 1.1 to 1.33.

Example 61. N-[1(S)-Kapo-2-(1H-imidazol-4-yl)-ethyl]DL-alanyl-E-proline.

Working as described in Example 17, the product of Example 60 was hydrolyzed to give the expected dibasic acid. NMR in D ₂ O showed imidazole protons at 7.2 and 8.5; alanine methyl

at 1.25 δ. Example 62. N-[1 (c)-ethoxycarbsnyl-5-aminopentyl]- DL-alanyl-E-proline.

A solution of c-benzyloxycarbonyl-L-lysine ethyl ester, hydrochloride (2.94 g) in water (10 ml) was basified with 15 ml of saturated aqueous potassium bicarbonate solution and extracted with methylene chloride. The extract was dried over magnesium sulfate and concentrated to dryness.

The residue, ethyl ester of ε-benzyloxycarbonyl-E-lysine, dissolves

powdered molecular sieves No. 4A (1.0 g). The mixture was stirred at room temperature for 4 hours. After 2 hours, sodium cyanoborohydride (630 mg) in 1 ml of hydrochloric acid was added and allowed to stand overnight. Stirring was continued, then filtered, concentrated to dryness and the residue was partitioned between water (·0 ml) and methylene chloride (15 ml). The aqueous phase was absorbed onto a strongly acidic ion exchange resin and eluted with 4% pyridine in water to give 470 mg of P-[1(c)-ethoxycarbonyl-5-benzyloxycarbonylaminopentyl]-O1_-alanyl-1_-proline. The protecting group was removed by hydrogenation in ethanol - Boda 1:1 over 10% palladium

on charcoal at 276 kN/m". The mixture was filtered and the filtrate was evaporated to dryness. The residue in methanol was chromatographed on a MH-20 column to obtain the desired N-[1(3)-ethoxycarbonyl-5-aminopentyl]□ ω-alanyl-ω-proline. The mass spectrum of the silylated material showed a peak at 059 for the irisin derivative and a peak at the derivative. NMR in D?O confirmed the structure.

Example 63. N-[1(3)-Carboxy-5-aminopentyl]-1-alanyl-1-proline.

N-[1 (S)-Ethoxycarbonyl-5-benzyloxycarbonyl-aminopentyl]-B-alanyl-B-proline. prepared as described in Example 62, was treated with 0.1 M sodium hydroxide at room temperature overnight. The product was absorbed onto a strongly acidic ion exchange resin and eluted with 4% pyridine in water to give N-[1 (S)-carboxy-5-benzyloxycarbonylaminopentyl]- B-alanyl-B-proline. single spot by thin layer chromatography (Rf 0.4 - butanol : water : pyridine : acetic acid 10:4:3:1). As described in Example 62, the protecting group was removed by hydrogenation to give. N-[1 (S)carboxy-5-aminopentyl]- B-alanyl-B-proline. The mass spectrum of the trisilylated product was consistent with the structure, showing

Example 64. N-[1-Carboxy-6-aminohexyl]-alanyl-L-proline.

Benzyl 2-oxo-7-phthalimidoheptanoate. obtained by alkylation of benzyl 1 3-dithane-2-carboxylate with 5-phthalimidopentyl bromide and subsequent oxidative conversion to the ketone with N-bromosuccinimide, was condensed with B-alanyl-B-proline in the presence of excess NaBH ₃ CN. The condensation product 1\1-[1-benzyloxycarbonyl-6-phthalimidohexyl]- c-alanyl-L· proline (390 mg) was hydrogenated in 25 ml of 50% aqueous ethanol at 276 kN/m ² over 10% palladium on carbon. After removal of the solvent and the catalyst, N-8

[1-Carboxy-6-phthalimidohexyl]-1-alanyl-1-proline (320 mg) with the expected spectral and chromatographic properties. One hour of the resulting intermediate (152 mg) in 2 ml of ethanol was refluxed with hydrazine (32 mg) for .5 hours, the phthalhydrazine was removed by filtration, the ethanol was removed in vacuo and the residue was adsorbed on a strongly acidic ion exchange resin. After elution with 2% aqueous pyridine and freeze-drying, the required N-(6-carboxy-6-aminohexyl)-1-alanyl-1-proline (58 mg) was obtained. The mass spectrum showed a maximum at 311 for the molecular ion minus water (329 - 18).

Example 65. N-[beta]-benzyloxycarbonyl-b-aminohexyl]-L-alanyl-L-proline.

By carrying out hydrazinolysis as described in Example 64 on N-[1-benzyloxycarbonyl-6-phthalimidohexyl]-1-alanyl-1-proline a mixture is obtained from which N-[1-benzyloxycarbonyl-6-phthalimidohexyl]-L-alanyl-L-proline can be isolated. After thin layer chromatography on silica gel and elution with 50 butanol : 11 acetic acid : 30 water a spot is obtained, Rf 0.32.

Example 66. N-[1-Carboxy-2-phenoxyethyl]-L-alanyl-L-proline.

A suspension of phenoxypyruvic acid (1.8 g) prepared by condensation of ethylphenoxyacetate with diethyl oxalate, followed by acid-catalyzed hydrolysis and decarboxylation with L-aquatic sodium base. The mixture was treated with NabH ₃ CN (0.18 g) and left at

^With constant stirring for 5 days, additional keto acid 10.9% and sodium cyanoborohydride (0.18 g) were added. The product was absorbed onto a strongly acidic ion exchange resin and eluted with 2% pyridine in water to give, after freeze-drying, 0.5 g of N-[1-carboxy-2-phenoxyethyl]-L-alanyl-L-proline. The NMR spectrum was consistent with

,? _?τ3 l/zse-e/.ter shows a maximum at 479 for z /·/;. 's·' mp-,·? m-ztol ;494 15).

h/'.'cp ο· . _L ι-ε'Zle»iKapucr.i'l-2-phenCs.'ethyl]-L-alag-yl-lps-olin

By reacting ethylphenoxypyruvate, obtained from the acid by acid-catalyzed esterification, and L-alanyl-L-proline with NaSH/CN in an ethanolic solution and isolating the product as described in Example bb, N-[1-ethoxycarbonyl-2-phenoxyethyl]-1- ^alanyl -L-proline can be obtained.

Example 68. N [1-Carboxy-2-phenylthioethyl]-L-alanyl-L-proline.

A mixture of phenylthiopyruvic acid (1.96 g), prepared by condensation of ethylphenylthioacetate with diethyloxylate, followed by acid-catalyzed hydrolysis and decarboxylation, and L-alanyl-L-proline (0.37 g) in 10 mL of water was adjusted to pH 7.0 with dilute sodium hydroxide and treated with NaBH ₃ CN (0.18 g) in 2 mL of water. After stirring overnight at room temperature, the product was adsorbed ^onto a strongly acidic ion exchange resin and eluted with 2% pyridine in water to give 0.36 g of N-[1-carboxy-2-phenylthioethyl]-L-alanyl-L-proline. NMR and mass spectra indicated the desired structure. A single peak at 348 indicated the molecular ion »-V ·» proline.

d .· > ! l· I vzai:, wasp

8. The product can be prepared as described to obtain N-[1-ethoxycarbonyl-2-phenylthioethyl]z* in Example

L-alanyl70. N-[1-ethoxycarbonyl-3-p-chlorophenylpropyl]alanyl-L-proline.

L50 A solution of ethyl 4-p-chlorophenyl-2-oxobutyric acid by esterification with ethanol in CCl and _ al:

obtained from rt ’ r-l ΓΊ I | I I | m Ci 3 7Ί I m Ι L/C Gbp Kci I i jj » . room ”*?mparsgture. until the reaction is complete, then ethanol can be removed under vacuum 'and the product absorbed on a highly ' absorbent ion exchange resin. By elution with 2 % pyridine so-to MO.r.e to obtain N-fl-ethoxy/jarssg-nl-3 p-chlorofzplshzopil]· ,Α

TOOTH

L71. GD[1-ethoxycarbonyl-2-(3-indsyl)ethyl]-L-alanyl

L-proline.

is as in Example 26. as the ethyl ester of indole-3-acid is condensed with L-alanyl-L-proline in a 1:1 solution of ethanol:water the spectrum shows pyrogenation by means of sodium cyanoborohydride. Isolation on Dautex 50, as described, gives a mixture of isomers of 1M-[1-ethoxycarbonyl-2-(3-indolyl)ethyl]-L-alanyl-L-proline. NMR absorption at 7 0 δ *5H). 4 2δ (5H). 3.4 δ (2H), 1.9 δ (6H) and 1.2 δ (6H).

Example 72.

N-[1-ethoxycarbonyl-2-p-aminomethylphenylethyl]-L-alanyl-L-proline.

- 4-phenylpropanoate, obtained by p-o-diphenylpropanoate, is reacted with ethyl 1-diphenylpropanoate and subsequent oxidative hydrolysis as described by Elie: and ^u anmann. J. Org. Chem., 37, 505 (1972)) with L-alanyl-L-proline and the product is purified by the method described in Example V. The resulting 1-aminopropanoate isomer of 1-(1-ethoxycarbonyl-2-resent-ethyl)-L-alanyl-L-proline is hydrogenated in ethanol solution containing hydrogen chloride and palladium on carbon catalyst. The solvent and excess HCl are distilled off in vacuo, washed with ethanol and concentrated to dryness to obtain the hydrochlorides of the mixture of diastereoisomers of the desired compound.

η

Example 73 N-[1-Carboxy-2-p-aminomethylphenylethyl]-L- alanyl-proline A solution of N-[1-ethoxycarbonyl-3-p-cyclophenylethyl]-alanyl-l-proline obtained in Example 72 was treated with one equivalent of ^sodium hydroxide in a mixture of methanol and water as solvent at room temperature for one hour. The solvents were removed in vacuo to give the sodium salts of a mixture of isomers of N-[1-carboxy-2cyanophenylethyl]-L- alanyl-L- proline. This mixture was hydrogenated in ethanolic hydrogen chloride and serabogie as described in Example 72 to give the hydrochlorides of a mixture of isomers of the desired compound.

Example 74 N-[1-Ethoxycarbonyl-2(S)-amino-3-phenylpropyl]-O.L-alanyl-L-proline.

To a mixture of N-phthaloyl-1-2-amino-3-phenylpropionaldehyde (Peterson et al., J.Am.Chem.Soc.. 79. 1389 (1957)), (2.18 g) and potassium metabisulfite (0.87 g) in water:methanol 1:1 was added sodium cyanide (0.55 g) with vigorous stirring. Stir for 90 min, dilute with ethyl acetate and filter. Wash the organic layer with water and dry over magnesium sulfate. Remove the solvent in vacuo to give N-phthaloyl-3-amyl-3-phenyl-4-hydroxy-4-pyronitrile.

of these products (in o-szvsdsp ι 1 Q L

The mixture is left for two days at room temperature. The solvent is removed with concentrated hydrochloric acid, the residue is dissolved in dioxane (1:1). It is heated to 7°C and maintained at this temperature.

·“ V ” ~ /’•y·'·. η POitJ r O O t S O

4Z_? I — \- ·—· w Д"*! '-Z J ’ X/ , I Х-* XX ’ Vi » I С· 4- X. I X > —X

s_z /and 1 and k'/.». L cl C xy I C Θ i D C D aV 4-,.- xz . . - C - .X.X . . X- i 14-C Civi s. . ,. Ch 4-,v; .,0 love!· whose mother. to obtain Sh'p.e>:38)*2-amino-4-phenyl-o-

Phthalylamino butanoic acid. Dissolve the acid in anhydrous

ethanol, pass anhydrous hydrogen chloride until saturated and

leaves 16 parts at room temperature. The solvent is removed

ο. Щ ORIGINAL j

in vacuo to obtain the ethanolic ^< » ^ester of ethyl 2-amino-.·:-o-όβπ;:;:-3-O-ethylamine butanoate with Γ.?·ρ _ζ 5O-.'t-χ-pr-?gi- by sodium cyanoborohydride as described in Example 16. to obtain N-[1-ethoxycarbonyl-3-phenyl-2-phthaloylaminopropyl]-0, L-alanyl-L-proline in the form of an isomer and D product with ethanol with a reflux condenser and with one equivalent of hydrazine 1.5 h. cooled and the phthalhydrazide isothermally cooled and ^the desired ^product is isolated from the resulting mixture ^and by chromatographic methods to obtain N-[1-ethoxycarbonyl-2(c)amino-3-phenylpropyl]-0. L-alanyl-L-proline. The 300 MHz NMR spectrum has peaks at 7.3 δ (5H), 3.2 and 4.4 δ (3H), 2.75 δ (2H), 2.0 δ (4H) and 1.1 δ (6H).

Example 75. N-[1-Carboxy-2-(b)-amino-3-phenylpropyl]-O.L-phenyl-L-proline.

Condensate 2-amino-4-phenyl-3-(b)-3-phthaloylamino butanoic acid, prepared in Example 74, with pyruvoyl-1-proline via sodium cyanoborohydride as described in Example 16 to give N-[1-carboxy-3-phenyl-2-phthaloylamino]-O _; L-alanyl-1-proline as a mixture of isomers. Reflux this product with one drop of ethanol for ^1.5 h, cool, filter off, and isolate the ^desired product by chromatography to give the title product. After silica gel chromatography and ethyl acetate elution, the title product was obtained. In water, a spot with Rf C. 1o is obtained.

Example 76. Ν-Γ1 -Carboxy-2-(3)-benzoylamino-3-phenylpropyl]D,L-alanyl-L-proline.

A solution of N-phthaloyl-3-amines-4-phenyl-2-hydroxybutyronitrile, obtained in Example 74, in ethanol saturated with anhydrous ammonia was left for 3 days at room temperature. The solvent was removed in vacuo and

BAD

ORIGINAL 1 is --ε s=va the residue with reflux for 6 h is concentrated salt knsesh’-·? H—apssa is dried and the residue is purified with Dautex-5 . .. . bold, eluted sequentially with water - methanol

...-ipn _< 4,i.··. > »ι nalraya with a solution of 1.5 M ammonium hindoo-js Isolate the desired 2 3-diamino-4-phenylpropionic acid from the last eluate by concentration to dryness. Prepare a solution of the copper complex of this amino acid and benzoylate the 3-amino group in situ with benzoyl chloride under alkaline conditions, according to the method described by R Aoeske et al! J. Am C.hem Soo.,78. 5883 (1956). The copper compound is destroyed with sulfur dioxide and worked up as described in the above literature source to obtain 2-amino-3-(B)benzoylamino-4-phenylbutyric acid. The resulting intermediate is condensed with pyruvoyl-2-proline via sodium cyanoborohydride as described in Example 16 to give the desired 1N-[1-carboxy-2(c)-benzoylamino-3-phenylpropyl]-D,L-alanyl-L-proline as a mixture of isomers which can optionally be separated by chromatographic methods. Mass spectral analysis shows a maximum at 596 nm for the disilylated molecular ion minus 15. The NMR spectrum shows absorption for two phenyls at 7.0 - 7.5 δ and diastesomes for methyls at 1.1 δ.

Example 77. N-[1-Ethoxycarbonyl-2-(5)-sensoylamino-3-enylpropyl]-D L -alanyl- L -proline.

When 2-amino-3-benzolamino-4-phenylbutyrate is treated with hydrogen chloride in

in vacuo to give ethyl 2-amino-3-benzoylamino-4-phenylbutyrate ^. Treatment of this intermediate with pyruvic acid by cyanohydrin isolation, as described in Example 16, affords the ^title compound.

Example 78. [[2-Amino-1-carboxy-4-methylpentyl]-D.L-alanyl-L-proline.

BAD ORIGINAL st trans-3-amino-4-(2-methylpropyl)-2-acetate ^t :'l:'o ^,: obtained by addition of chlorosulfonylisocyanate to 4·.·.rι ι. with <. j, .'-aba j>-r.ar/gom. which is protected as a trig.suy<щ _; :mepi1sinlin derivative and then treated with lithium diisopropylamide, followed by tosylazide and chlorotrimethylsilane After acid treatment and chromatography on silica gel, grams-3-azido-D-^-methylpropyl;-e-azetidinone is obtained. which is hydrogenated (H0 % g-q With ethanol) to the amino derivative and to 4.58 g benzylpyruvate in 20 ml absolute ethanol. containing Юг powdered molecular sieves 4A. A solution of sodium cyanoborohydride (0.65 g) in 8 ml of absolute ethanol was added dropwise. Stirring was continued until the reaction was complete. The reaction mixture was filtered and the filtrate was concentrated. The residue was dissolved in 50 ml of water and acidified with 1 N HCl to pH 3. The mixture was then adjusted to pH 9.5 with 10% sodium carbonate solution.

The solution was saturated with sodium chloride and extracted with ethyl ^acetate (5 x 40 mL). The combined organic layers were dried over sodium sulfate and concentrated to give 4.94 g of an oil. Chromatography on silica gel (ethyl acetate) gave 1.11 g of purified product. The NMR and mass spectra were consistent with the structure of the benzyl ester of K ^! -trans-4-(2-methylpropyl)-2-oxo-3-dipyridyl ^- nitrogen.

..... _{h 4} ,-r-. , »-s ς. ~ Q' dtr ц Г* п ' f* ⁴ t; γ* Λ u растесс c temperature 0° of the acid (428 mg) and L-proline-tert-butene: ^_ eo ί??7 mA in 5 ml of dimethyl<Ьformamide are treated with a solution of :d (605 mg) in 5 ml of dimethylformamide and after thesis with in 5 ml of dimethylformamide) at the hour the ice bath is removed and the stirrer is turned on overnight at t/pa acetate (100 ml) and the resulting

10, t. - 0/ il. k> /O solution' of triethylamine (223 g.roganization for 20 min. After three mg nn solution was washed with sodium carbonate (13 x 30 ml) and water (1! Λ x 50 ml) solution of sodium chloride and dried over sodium sulfate. On concentration, an oily liquid was obtained, 0 78 g,

whose ^NMR and mass spectrum are consistent with the structure N-trans-[2-methylpropan-2-ol]-3-azetidinyl-D L-alanyl-L-proline tert <>

The reaction mixture was stirred at room temperature for 25 minutes at room temperature. The reaction mixture was concentrated to dryness and the residue was treated with 1 N sodium hydroxide (30 ml) for 4.5 hours at room temperature. The alkaline mixture was slowly added to a strongly acidic ion exchange resin and the product was extracted with 2% pyridine in water. After freeze-drying, 0.30 g of M-[2-amino-1-carboxy-4-methylpentyl]-D. L-alanyl-L-proline was obtained. It consists of four diastereoisomers (S,S,S,S; S.S.FLS: R,R,R,S; R,R.S,S). which can be separated by chromatography. NMR and mass spectrometry showed consistency with the structure. The NMR spectrum shows multiplets centered at 4.5, 3.85, 2.3, 1.79, and 1.16 ppm. The mass spectrogram has a maximum at 458 (disilylated molecular ion minus 15/

Impurity 79. N-12-amino-1-ethoxycarboxyl-4-methylphenyl]-D L-alanyl-L-proline.

The intermediate of Example 78 N-trans-4-(2-methylpropyl-oxo-c-azetidinyl-D.l-alanine (125 mg) can be condensed with L-proline, benzene ester hydrochloride (167 mg) in the presence of diphenylphosphoryl azide (191 mg) and triethylamine (140 mg) in dimethylformamide to give 217 mg of N-/trans-4-(2-methylpropyl)-2-oxo-3-azetidinyl-D.l-alanyl-E-proline, benzene ester. The benzene protecting group can be removed by hydrogenolysis and the p-lactam can be opened with anhydrous sodium ethynate in ethanol to give N-12-amine-1-ethoxycarbonyl--;-mb

L-alanyl-L-proline

Example 80. N-[2-benzamido-1-carboxy-4-methylpentyl]aphanyl-L-proline.

M-[2-amino-1-carboxy-4-methylpentyl]-D,L-alanyl obtained according to Example 78, dissolved in base is

village

D.

D, LL-proline, treated with

- .f— — /* ps •ector of disn. ^p : ^l lnra ^and nya •X С

SNA

N-2-benzamido-1*ethoxycarbonyl-4-methylpentyl]-D.

L-alanyl-L-proline.

of a solution of \'-[2-amino-1-ethoxycarbonyl-4-sr-^ene solvent with benzoyl chloride, can be obtained •ds-1-ethoxycarbonyl-methylpentyl-O,E-alanyl-L proline.

Example 82. N-α-[1-ethoxycarbonyl-3-phenylpropyl]-LL-proline.

Arginyl is worked up as described in Example 37 by condensing N-α-tert-Soc-N-fo-HHTpo-L-apruHHH, N-hydroxy succinimide ester, with L-propanol in dioxane water and the N-α-tert-Boc protecting group is removed with acid. The dipeptide is isolated as follows:

Tri·ol described Ethyl 2-oxo-4-phenylbutyrate is coupled to the resulting product as described in Example 25 and isolated as described to obtain

SEMI-DUPLEX WITH <Ό-α30 ^: IN δρΓΗΗΗπδ BC IS PROTECTED WITH PI ι ROGUPZTA.

This protection is removed by catalytic hydrogenation over 'C/.na ki Cs;3 ι BCDH i cj iHTe etoko; jarse g in vacuo to obtain a mixture of isomers of N-α-[1-3-phenpl-ospil]- L- aop.'^and;'lPOOLNN |Gl-1Mer 83 N-(ί-ϊ1-ΚΑρ6θΚθΜ-3-φσ.Η1·!Γ.ρθ“;··Π]

L λ-Jr.'

The conjugation of n*(o-oepylO|\»>n-caroonylp1 ^_ and,“3 ωιυκνΉcarbonyl-0 _: ζ-homolysine. obtained from homolysine via the copper complex, with L-proline 3° butyl ester can be carried out with diphenylphosphorylazide. Then the 3° is removed with trifluoric acid and the product can react groups can be η OWGlNAL ··<*

5Т _ _i -j ---. K0'- ^, D- ^, ЧЗаЦ1!С'НН ^, . ^, 1Ja ^t poodmkt

:.'·<r>_1; - _IG ip ₂ through ca^alntchmo hydrogenmrane. so that to obtain·'

PL

The intermediate described in Example 83. N-N-benzyloxycarbonyl-D0-homolysyl-L-proline can be reacted with ethyl 2-oxo-4-phenylbutyrate and NaBH4CN. The condensation product can then be hydrogenated over palladium on carbon to give N-N-[hexoxycarbonyl-3-phenylpropanyl]D0, L-homolysyl-L-proline.

Example 85. Ν-α-[1-carboxy-3-phenylpropyl]-p-amino-O. Lapanil-L ^-p roline

DL-a β-diaminopropionic acid is reacted under alkaline conditions with an excess of benzyloxycarbonyl chloride to give blind acidification D _; La p-di(benzyloxycarbonylamino)-propionic acid with mp. 123.5 - 124° C. phosphorus pentachloride is added to a chloroform solution containing the di-Cbz product to give after workup 0.1_-4-(benzyloxycarbonylaminomethyl)oxazolidine-2.5 dione A solution of tert.-butyl ester of L-proline in methylene chloride is added to

tetrahydrofuran at -60° C. After co'>oanaya«ape n.-.ja no·^ n? orizer the mixture is distilled to dryness which gives the crude product. Trifluoroacetic acid effectively cleaves tert.-butyl ester in two hours at a low temperature. ~ i-g.se zhj-ja collected mixture of L prslin and N-benzyloxycarbonyl!'lamino- O.b-alanine-y-proline. By gel ^and o ane 'column LW-20) pure d| peptide is obtained. Condensed^

2-oxo-h-phenanthylbutyric acid and benzyloxycarbonylamino-D.L alanine-α-proline in the presence of sodium cyanoborohydride. After removal of the protecting group from the resulting product, the

p amino-D,L-alanyl-L-proline. NMR in

ABOUT ORIGINAL

.. J

D 0 -shows aromatic protons at 7.4 δ methine protons at 4.2 and Μν.-.Π'Π.πθ-Γ.! ггл· 3 7 3 2 2 ^ϋ and 2 10 δ

.. ' ~ ~ ~ · г ‘ - А ι_ι ι ι π г·· г~. D» η ι ι ·' I. P, . is. and m ui - Oh

........ - - · ' · - y -·.<· f- J f .

L- cuidr-H:- L- Proline

A solution of [3-benzyloxycarbonylamino-D L-alanyl-L-proline, prepared as described in Example 85, and ethyl 2-oxy-4-phenylbutyrate [••.;can be condensed in ethanol solution with NaS'--yC' The bridging group can be removed from the product by catalytic hydrogenation to give N-a-fl-ethoxycarbonyl-3phenylpropyl]-[5-amino-C. L-alanyl-L-proline.

Example 87 [1-Ethoxycarbonyl-3-phenylpropyl]-O,L-alanylL-pren; η.

Ethyl 2-oxo-3-p-cyanophenylpropionate, ^obtained in Example 72 ^, was hydrolyzed by stirring in 5% sodium hydroxide at room ^{temperature for 1 h} . The reaction mixture was taken up in ether, the aqueous layer was acidified to pH 2 with conc. hydrochloric acid, the product was extracted with a ^mixture of ether and ethyl acetate, and the solvent was removed to give 2-oxo-3-cyanophenylpropionic acid. This acid was reductively coupled with the ethyl ester ^of L-homophenylalanine in the presence of d; ts: LR (T' Li, 37 Ή (_/ Λ.» ^: ί*''*4 · f\Cl Λ. · U cl·; — -· ~ * .'·->, C . i'l L,.. .

riOcMkiCTBanc gives a mixture of diastereoisomers of N-: *-(£>.·ethoxycarb'nyl-d-phenyllss ^_ . _; d!-and L- p-cyanophenylalanine. The product is condensed with benzene L-polyol hydrochloride in dimethylformamide using the reagent diphenylphosphorylazide, as described in Example 13. to give a mixture of diastereoisomers of the benzene ester of N-. =--5.;-carbethoxy-3-phenylpropyl]-O. L- p-cyanophenylalanine. This intermediate is hydrogenated in ethanol to give a mixture of isomers. ϊ ' m and ca. si i, 1Od! op palladium on the outside and is grown as described in Example 72 to give the desired product as a mixture of isomers.

|BAD ORIGINAL

Polymer 88 M-<x-[1-1'8''-Carboxy-3-phenylpropanyl]-O. L-p-aminomethylphenylapanyl-L-groln.

- SALT POL\/chen IN Γ’ΟΗΜΟΟΟ

ΖΝ “a ^t rne° hydroxide in a mixture of . Ci . / t- · .ъ-t-kenpe overnight.

The solvent was removed from the reaction mixture in vacuo, the residue was hydrolyzed in ethanolic hydrogen chloride solution and worked up as described in Example 72 to give a mixture of diastereomers of the desired compound.

Example 89. N-α-[1-ethoxycarbonyl-3-phenylpropyl]-N-acetyl-L-lysyl-L-proline.

As described in Example 25, ethyl 2-oxo-4-phenylbutyrate was coupled with N-b-acetyl-L-lysyl-c-proline in an ethanol-water solution in the presence of sodium cyanoborohydride. The product-rich fractions were isolated on Dautex-50 as described and freeze-dried to give a mixture of isomers of N-[1-ethoxycarbonyl-3-phenylpropyl]-N ^| -r> ^acet !D1-l-lysyl-L-proline. The mass fraction had a maximum at 653 (M648 η v-h , ., , LL . _{ _ ' 7 1--« / ! t M 'L|^· j- |.1 · . A D p, . ' G .-: Λ G? · « ' i_- , -pOj !IN.

As t 3 described in ΊΡΟίΜβρ 26. ethyl ^-sxo-4- is bound phenylbutyrate ο L-histidyl- L- proline in the presence of sodium 1 ; ' ? gn* L> Π vjn • I i !Λ ,Och ' ¹ ' ii» · ι t Ch/ ' D . Ch». U' Λ H/ C Λ L ' l o r C Λ the instructions to get oeoizmeseri of Ν-α-ί ι -ethox ;;Carbonyl-3-0enyl.-.ropyg!-b

*ISTIDIL- L· POOLIN.

Example 91 A. N-α-[1-ethoxycarbonyl-3-α-naphthylpropyl]-1-lysyl-L-proline

Ethyl 4-1-naphthyl-2-oxybutyrate, obtained by alkylation of ethyl 13-diglycine-2-carboxylate with 2-a-naphthylethyl bromide and subsequent

BAD ORIGINAL

And —-μ.

Absorbed water, strongly acidic ion exchange resin and eluted with 2% pyridine in it is increased by treatment

-UH A-rt ТГ1Г-|Г\Г\Г91 1 πΊ . I .. PMOMN. I _

d. ι\-<.χ-ι ι-carboxy-3-a-naphthylpropylj-L-lysnl-L-proline.

A suspension of 4-α- ^and aphthyl-2-oxomaspe ^: -'2 acidic ¹ -'?, from es.e^a by hydrolysis, in water was adjusted to pH 7 with dilute sodium hydroxide and dried by sublimation. The residue was treated with ε-3 ^ϋ - 6-butoxycarbonyl-E-lysyl-1_-proline. as described in Example 91

A, to obtain \-α-[1-carboxy-3-α-naphthylpropyl]-1-lysylLΠ00πιχ ,'L

L-proline.

{ G\

·. V/ chloro-2-oxobutyric acid (1.1 g) in 5 ml of water was adjusted to pH / with dilute sodium hydroxide and treated with 0.07 g of NaSH ₃ CN in 1.ml of water for several ^hours . After dissolution, the solution was allowed to stand /· in water to give 0.08 g of product. NMR showed that the tert-iox protecting group was not j-ι η p m- g 0 0

- .Ki Г-i ο <— — ·0 — — — < о —> — ; ••'G I 09.C· L ίJ С. ** m_

m.<

1.

IL- L- PSOLIΗ ► * ΓΛ

I'VI is not with the structure. At 584 a molecular ion was found. ^through chromatography it is

PSL.u hso<z l\81 ίαπι ί Isag.sr

.. ; _<t; ORIGINAL. — — I*·

Ί^ΗΗΜί.Ι·'.') .n.ynf>nA^unn].i ko ^, .'kaossn:; ^, l-_-L; ¹ zil- ^! _-~rol ^, .!n in an ethanol solution with an excess of isyl-l-poslin.

Solution

Ν-α-[1-carbox. 1-3-(3,4-Dichlorophenyl)-L-lysyl-L-prelin.

of 4-(3 _: 4-dichlorophenyl)-2-oxobutyric acid obtained from dichlorodihydrocinnamate by condensation with ethyl oxalate and subsequent acid-catalyzed hydrolysis and partial decarboxylation in water, was treated as described in Example 92 to give N-α-[1-carboxy-3-(3,4-dichlorophenyl)-propyl]-1-lysylL-proline. The NM spectrum in 0 ₂ 0 showed an aromatic hydrogen at 7.30 ω, a broad singlet at 4.3 δ and broad multiplets at 3.53, 3.00, 2.68, 1.98 and

60 L. The mass spectrum shows a peak at 455 m/s which corresponds to the loss of the desired structure, and a large M - 2 (457 m/s) isotopic contribution confirms the presence of 2 Cl.

N-α-[1(5)-carboxy-3-(3-indolyl)propyl]-L-lysyl-L-proline.

— 0 _ p f· —

V' cho.iotryptophan by the method described by Weygand ef al..

App 658.

(1962) Tert-Boc-1-lysyl-1-proline was condensed in sodium cyanoborohydride as described in Example 4 to give a crude mixture of diastereoisomers of |\-<x-[1-carboxy-3-(3-dihydrogenpyranyl)-1-proline]-1-methyl-1-methyl-1-proline. The mass spectrum of the tetrasilylated product had a maximum at 732 (MH), 717 (MH-15), 615, 660 and 645 m/e ^. The lysine side chain was deprotected by treatment with 4 N-hydrogen chloride in ethyl ^acetate and purified with a strongly acidic ion exchange resin as described in _this example to give

/ *\ ν Γ. Ξ Η from· '^;:'od'/kt noez chrematograssination is obtained

.'zomeo.

’ ’ ' - ο •-•s.rS ^: _KO.-!lslS»:Lg_- LYSIL- !_ GroLIN.

Benzyl 2-oxo- ⁷ -phthalimidoheptanoate and ε-3 ^υ - butoxycarbonyl-L· .-proline are oxidized with excess NaBH ₃ CN in ethanolic solution to give Na-fl-benzyloxycarbonyl-6. The benzyl group can then be removed by hydrogenation over palladium, the tert.-Boc group is removed with 45 N HCl in ethyl acetate, and the phthalimido group is removed by treatment with hydrazine to give

N-[6-amino-1-carboxyhexyl]-1-lysyl-L-proline.

Example 97. N-α-[6-amino-1-benzyloxycarbonylhexyl]-1-lysyl-L-proline.

Treatment of N-α-(1-benzyloxycarbonyl-6-phthalimidohexyl)-N-8-3 ^with '-butoxycarbonyl-1-lysyl-1-proline, prepared according to Example 96, with 4.0 N HCl in ethyl acetate and then with one equivalent of hydrazine in ethanol at reflux gave a mixture from which N-α-[alpha]-amino-N-benzyloxycarbonylhexyl '-lysyl-L-proline could be isolated.

lysylLprslin.

Benzyl 2-oxo-6-phthalimidohexanoate was treated as described in Example 96 to give the desired product, 516 m/s, M'-15 ion at 510 m/s, and a single peak at 399 m/s, attributable to loss of ^silylated carboxyl.

Example 99 o-AmiPd-1-Oenznlox. sorry·-·. m.le-ti.=/ί-L- proline.

J 'I

R αι_ιθ| 1Д

2-oxo-6-othalimidohexanoate

as described in Example 96. except that the dezτ-.ί! '.’wound with hydrogen on palladium From the mixture of products

G 1 Q*~' dd Μ-,γ-Γ 1 .Κ^οόοκοίЧС<'^ ^t ' ^{l n} '~I gch.chomp. , _ proline.

Phenoxypyrusic acid (0.9 g) was dissolved in water, the pH was adjusted to 7 with dilute sodium hydroxide and the solution was freeze-dried. The residue was dissolved in 10 ml of ethanol and triturated with .1-8-3°-butoxycarbonyl-1.-lysyl-1_-proline (0.36 g) and powdered molecular sieves No. 4A (30 g). Sodium cyanoborohydride (0.13 g in 3.5 ml of ethanol) was added portionwise and the reaction mixture was stirred at room temperature until the reaction was complete. The product was isolated by absorption onto a strongly acidic ion exchange resin and eluted with 2% pyridine in water, followed by freeze-drying to give 0.25 g of product free of protecting groups Ν-α-[1-carboxy-2-phenoxyethyl]-2-lysyl-L-proline. The NMR and mass spectra were consistent with the structure

5. N-α-[1-ethoxycarbonyl-2-phenoxyethyl]-L-lysyl-L-proline.

^Ethyl phenyloxypyruvate was treated with E-3°-butoxycarbonyl-1-yl according to Example 100 A and N-α-[1-pyrrolidone] was obtained. The spectrum showed phenyl absorption.

centered at 7.3 δ, and broad prolines.

was treated with t-3- ^tert -butoxycarbonyl-L-lysyl-L-proline as described in Example 100 to give N-α-[1-carboxy-2-phenylthioethyl]-1-lysyl-L-proline. The mass spectrum showed a silylated molecular ion at 567 m/e. Chromatography gave the desired isomer.

ii .i I i •'i , r <μι ·, , , , . , . I; d .— ; i 7 f | i _ r . Γ , .: 1 Π _ ; ¹ W> · <-/ k Γ\ Va sz 4z I ' » ' ? I γ vl .»'>· .4/ v . I 7 ' : L_ , Λ . k > I L .

r* ·; >·_ ”·'·''-·.'= :-3441, se _ТО βν _; ,..- _ν - ρ- .^£.и _Ч гр- icyeer .- :·-3-сутокси•'С.обонпл L-ri'Ciin-L-nponnH according to ^d simer 100 (А ^; to obtain Nа-[1-ethoxycarbonyl-2-phenylthioethyl]-Е- lysyl-L-proline.

Example 102. N-α-[1-Carboxy-2'c)-phenylpropyl]-O1-lysyl-Lπροπ',Ή.

N-Amino-4-phenyl-O-(phthalimido) _-1- butanoate was condensed with 2-oxo-6-phthalimidohexanoic acid (prepared by alkylation of benzyl 1,3-dithiane-2-carboxylate with phthalimidobutyl bromide, followed by oxidation and hydrolysis) in the presence of sodium cyanoborohydride, as described in Example 13. The resulting intermediate was coupled with the hydrochloride of the benzyl ester of L-proline via diphenylphosphoryl azide, as described in Example 13, to give a mixture of isomers of the benzyl ester of N-α-[1-ethoxycarbonyl-2(S) phthalimido-3-phenylpropyl]-1H-o-phthalo[n-O,[_-lysyl-L-proline, purified by column chromatography. It was then treated with two equivalents of sodium hydroxide in solution. ethanol-water for 4 hours at room temperature. neutralized to pH ⁴ with concentrated hydrochloric acid, the ethanol distilled off in vacuo. The product was heated in a reflux condenser in ethanol containing 2 equivalents of hydrazine for 1.5 hours and isolated as described in

! Example 1i3.

Ν-(/-ί i -carboxy-2'ci-oenzoylamiΗθ-ό-ψeNi1lΓΊρ/ΛΓίΜημΡ i . ! - ^Γ Ό0'ΊΗΗ

Example /7, is condensed with 2-oxo-o-phthalimido-hexanoic acid in the presence of sodium cyanoborohydride by the method described in Example 13

The resulting N-α-[1-carbethoxy-2(3)-benzoylamino-3-phenylpropyl]-1,2-c-phthaloyl-0,1-lysyl can then be coupled with the hydrochloride of

BAD ORIGINAL sec-'lPhosphorylazide as

GC.*4 lysyl-1_-proline purification by chromatography Poi with two equivalents of sodium hydroxide in ethanol solution for one hour at room temperature.

102 gives N-α-1-carboxy-2(8)-benzoylamino-3-phenylpyrrolidinyl]-\-e-phthalic-0 ₁ b lysyl-L-proline. By heating the latter under reflux in ethanol for 1.5 hours in the presence of one equivalent of hydrazine and isolating according to Example 74, the desired compound can be obtained as a mixture of isomers.

Example 104 1N1-(x-[2-Amino1-carboxyl-1-4-methylpentyl]-O,b lysylL-proline.

An ethanolic solution of trans-3-amino-4-(2-methylpropyl)-2-azetidinone, prepared according to Example 78, can be reductively coupled with benzyl 2-oxo-6-phthalimidohexanoate using sodium cyanoborohydride and molecular sieves. The product benzyl ester of N-α-[4-(2-methylpropyl)-2-oxo-3-azetidinyl]-N-ε-phthaloyl-0,1_-lysine can then be debenzylated by hydrogenation over palladium.

The free acid and the gasoline ester of polyol are bound to the λποπ τ,'.οο n-ργ''ΖΠ- ^τ ~ ^{Ω π} οβρι9νπ;<πα

WJ 1 VU ЧК _ ^: -4 . ¹ UV /Ц Jf ' I <4 i X-/ Ch«x _m U P- ~ I I ) ;

\Ca1\ IV described above to obtain N-α-U!2-methylprop1 ^: l)-2-oxo-3-

The Otaloil group is

N-α-4[2-methylpropynyl]-2-oxo-3-anoic acid

G.- ⁴

BAD ORIGINAL ⁿ wp 105 Ν·α· [2benzamido, ./syl- L- proline.

- k a p b ο / sh ’ - 4 - m e t i l p e ch t i l ] - D. L -

2-Oxy-2-azetidinyl-3-butoxycarbonyl-DL-lysine can be prepared from trans-3-amino-3-methylpropyl-2-azetidinyldione (impurity 78) and benzyl 3- ^L -butoxycarbonylamino-2-oxohexanoate. The benzene group is removed by hydrogenation and the product is coupled to the benzene ester of L-proline. The product benzene ester of N-α-4-[2-methylpropyl]-2-oxo-3azetidinyl- ^3- butoxycarbonyl-DL-lysine is then debenzylated with hydrogen and the β-lactam is hydrolyzed with dilute base to give N-α-4-[2-amino-1-carboxy-4-methylpentyl]- N-ε3°' butoxycarbonyl-DL- lysyl-E-proline. After benzoylation with benzoyl chloride in an organic solvent, the tert-Boc protecting group is removed with trifluoroacetic acid to give N-α-[2-benzamido-1-carboxy-4-methylpentyl]-O,E-lysyl-L-proline.

Example 106. N-α-[1(3)-carboxy-3-p-chlorophenylpropyl]-lysyltrans-4-methoxy-E-proline.

The hydrochloride of non-methyl-trans-4-methoxy-prolinate is coupled with N-α-tert-Bcc-N-c-Cbz-L-lysine using dicyclohexylcarbodiimide and triethylamine in methyl ethyl ketone.

as described in the example, purified by chromatography. The ester was hydrolyzed and the tert-Boc protecting group was removed, as described in the same example. 1H-8-Cb2-E-lysyl-E-4-methoxyproline was reductively coupled with

2-oxo-4-o-chlorophenylbutyric acid (obtained from the ethyl ester of p-chlorocinnamic acid by alkali-catalyzed condensation with diethyl oxalate followed by decarboxylation in anhydrous hydrogen chloride in acetic acid) in the presence of sodium cyanohydride and subsequent treatment according to Example 24 to obtain a mixture of isomers of N-α-11-carboxy-3-p-chlorophenylpropyl]- N-εCbz-L-lysyl-trans-4-methoxy-L-proline. The benzyloxyamine is removed.

..__

The carsomal product is prepared by catalytic hydrogenation over a palladium-on-charcoal catalyst in the usual manner. The desired product is obtained by distillation. The mass spectrometry has maxima at 584° C., 588° C., 158° C., 466° C., and 352° C. (major maximum) for the disilylated product.

P*i-[-carboxy-3-p-chlorophenylpropyl]-l-lysyl-4-pyrazotidine carboxylic acid.

aesthetics

N-se^ret.-Boc- N-o-Cbz-L-ruCHH with the hydrochloride of 1-thiazolidine-4-carboxylic acid, the product is purified chromatographically, the ester is hydrolyzed and the tert.-Boc protecting group is removed by the methods described in Example 20. By reductive coupling with 2-oxo-4-chlorophenylbutyric acid, as described in the same example, a mixture of isomers of N-α-ΓΙ-carboxy-3-p-chlorophenylpropyl)N-e-Cbz-L-lysyl - L-thiazolidine-carboxylic acid can be obtained. By removing the benzyloxycarbonyl protecting group by treatment with hydrogen bromide in acetic acid at room temperature in the standard peptide chemistry manner, distillation of the solvent and finally - freeze-drying,

S'oS

--1,.

mes 108 \'-[1-carboxy-3-p-hydroxyphenylpropyl)-L' lysyl'*'hypophopoid of ethyl ester *a trans-5-methyl-thiazolidine-4 thioacetamide,. acid reaction with formaldehyde, hydrolysis to β-methylcysteine and subsequent following the method used by R.F. Nutt et al.

Abstracts of the 6th Am tert.-Bcc-N-c-Cbz-L-OHSHH were coupled using 2.8 ml of triethylamine and 4.5 g of dicyclohexylcarbodiimide in methylene chloride as described in Example 20 by reductive coupling of this intermediate

Vr · c 2-o.xo-4-p-\lorphenylbutyric acid, using cyanoborohydride and protecting the benzyloxycarbonyl p-<··.υρςϊΗ.ι/;Γ ₁ ροπι·ιπ.·<- lysyl- and _-trans-5-methylthiazolidine1 carboxylic acid in the form ^Li a mixture of isomers.

Example 109. N-α-[1-carboxy-3-p-chlorophenylpropyl]-1-lysylL-3,4-dehydroproline

The hydrochloride of the ethyl ester of L-3,4-dehydroproline is condensed with N-α-tert-Boc-N-8-Cbz-L-nn3HH, the tert,Boc group is removed with 4 M HCl in ethyl acetate, then the intermediate is reductively coupled with 2-oxo-4-p-chlorophenylbutyric acid, the protecting group is removed with hydrogen bromide in acetic acid and further processed according to Example 107 to obtain a mixture of isomers of N-α-[1-carboxy-3-p-chlorophenylpropyl)-1-lysyl-L-3,4 dehydroproline.

Example 110. N-[1-carboxy-4-methylpentyl]-alanyl-L-proline.

A solution of 5-methyl-1-oxohexanoic acid (1.44 g) and L alanyl-L-proline (0.37 g) in 5 ml of water was adjusted to pH 7 and treated at room temperature for 10 days. The reaction product was

S'

O O/ η 1 l AM Π I L LJ

P- ν ’ b > jJ ν »m '· m a to obtain 0.5 g of product after freeze-drying

A portion (0.2 g) was purified by chromatography on a LH 20 column to give 18 g of N-1-carboxy-4-methylpentyl-c-alanyl-L-proline. The NMR μ?ββ~?κτ.;~μ· were in agreement with ^{the t} ?e ^t s ^t ?'' ¹ e with the niastereoisomers

'.*o ^g a'· be isolated by chromatography. In the mass spectrum of the disilylated product, maxima are observed at 443 (M~-15), 458 (MΊ. 341.301 and 244 m/e (main maximum).

^Example 111 N-[1(B)-ethoxycarbonyl-4-methylpentyl]-c-alanylL-proline.

BAD ORIGINAL :-:-1.7 >mixl-2-oxo ^y hexanoate (3 44 g' n L- alanyl- L-proline (0.74 :} c? sasrkvat e 15 ml ethanol with 6 g powdered molecular sieves 4A. E . - pa -'iKuj ik-j hour sodium cyanide was added dropwise.!dr1-!di id io ~< =. esh-s. ^- h,-then the ethanol was removed in vacuo the product was absorbed on a strongly acidic ion exchange resin.· eluted with 2% pyridine in water to give 1 08 g N-[1ethoxycarbonyl-4-methylpentyl)-E- alanyl- L-proline. A portion was purified by LH-20 chromatography for spectral analysis. NMR was consistent with the structure. The mass spectrum showed a maximum at 114 (silylated molecular sieve ion-15). The desired measurement is obtained by chromatography.

Example 112. (14)-((1-carboxy-3-p-phenoxypropyl)-1-alanyl-L-proline.

A mixture of 2-oxo-4-p-phenoxyphenylbutyric acid obtained by the reaction of p-phenoxyphenyl Grignard reagent with ethylene oxide to give an alcohol which is converted to the bromide and condensed with ethyl 1,3-dithiane-2-carboxylate by oxidative cleavage of the dithiane.

followed by alkaline hydrolysis. The resulting ketoacid and L-alanyl-L-proline were treated with excess sodium cyanoborohydride at pH 7 adjusted with dilute base. The product N-(1-carboxy-3-p-phenoxypropyl';-(_-alanyl-L- ^proline was extracted ^with disilylation (M ), obs (M -loj, 468 i.o/0 rii/e

N'-(1-ethoxycarbonyl) is a compound with maxima at

Psimer

584

113.

product contains (basic maximum).

-3-p-phenoxyphenylpropyl)2-oxo-4-c-phenoxyphenylbutyric acid, as

Oh! 1-!

Example 112. except that, -3 can be omitted from the above-mentioned alkaloids and L-alanyl-L-proline can be ^condensed in the presence of sodium cyanohydride to give N-(10-peroxypropyl-3-p-phenoxyphenylpropyl)-L-alanyl-L-proline.

Example 114. N-(1-Carboxy-3-phenylpropyl)<-alanyl-D, λ

3,3-dimethyl-proline.

BAD ORIGINAL

3-Dimethyl-P.L-pyrrole was obtained from 3-methyl-2-butenal by the method of Cox _: J. Chem. Soo. 1964 5024. and converted to the hydrochloride of Na Mg·.K, l;.·? c'-Tup - _ ·/. _ . ·. ~c; _ D DVDZss; 36 ш сv.-alani.p s> sc this was condensed with c-oxo-4-phenylbutane *..'one as described in Example 47 to give a mixture of isomers -e desired product.

Example 115. A. N-[1-Carboxy-3-phenylpropyl)-1--(3C-synylcysteinyl)-k- · .roline

Condensation of L-N-tert-Boc-b-benzylcysteine with L-proline tert-butyl ester in the presence of dicyclohexyl carbodiimide in the usual manner gives the blocked dipeptide L-(N-Tper-Boc-S-benzylcysteinyl)-L-proline-butyl ester. The latter is treated with 4 N HCl in ethyl acetate at 0° to give P(3-benzylcysteinyl)-k-proline. Treatment of this dipeptide with 2-oxo-4-phenylbutyric acid in the presence of sodium cyanoborohydride gives N-(1-oxo-3-phenylpropyl}-o-1-b-benzylcysteinyl)-k-proline as a mixture of isomers.

B. · N-(1-carboxy-3-phenylpropyl)-k-cysteinyl L-proline.

N-Carboxy-3-phenylpyridyl-n-(5-benzylcysteinyl)-n-proline obtained in Part A. was treated with sodium in liquid ammonia to give the desired compound.

Example 116. N-α-[1-Carboxy-3-phenylpropyl]-k-ornithyl-L-proline.

M-6-tert-Boc-c-ornithyl<-proline and 2-oxo-4-phenylbutyric acid were condensed in the presence of sodium cyanohydride in the presence of impurity 5-m. The protecting group was removed from the product in ethyl acetate. The crude was adsorbed onto a 3% aqueous solution using 4% hydrogen chloride gas, the resulting hydrochloride salt was acidified with an exchange resin and was eluted with pyridine. The mass spectrum showed a molecular ion at 355 m/e for the product - 36. The NMR spectrum was consistent with the structure.

BAD ORIGINAL

L_.

Χ-ε- ^τ Ω?τ - BOS-L-lysyl-'_· •Ι.-'ΖΛ ,7 . Γ 1 ;-U . Γϊ-Γ» u/- ' t.· ” · Γ· _.4ι Qu ! Γ Π Γ>Γ>ζλ π ι 1 π ·· - Г '' · ''* I > - · - '·- '· -~f- - - — - Τ - ''*-.' · lasyl-L-proline

Z- .-.Z-h :. -i, ·. ί- t ' a * ' } -

to which ¹ has been added.'

a.-\ ν on is cool !с «..· >,> -.after the reaction is complete, the sieves are removed by filtration. After evaporation, the residue of the filtrate is dissolved in water, extracted with ether and absorbed on a strongly acidic ion exchange resin. Elution with 2% pyridine in water gives 639 mg of crude protected product M-α-[1(B)-ethoxycarbonyl-3-

The protecting group was removed with 4 N hydrogen chloride gas in ethyl acetate. The resulting HCl salt was absorbed onto a strongly acidic ion exchange resin and eluted with 2% pyridine to give 270 mg of product. The mass spectrum showed a molecular ion at 678 lm/e for the disilylated products plus 1. NMR was consistent with the structure. Chromatography gave the desired monomer.

Example 1'3. N-α-[1-Carboxy-3-phenylpropyl]-N-c-Ν-ε-dimethyl-Elysyl-L-proline.

ester is aladin on charcoal, 27c *1 CT· •fcl . · hi — Γ* ⁴ * II . JO ' J 1 /: / . _t . .

>- ci p £ D j h ✓: U; i .λ

The protecting group was cleaved with 4 N hydrogen chloride gas in ethyl acetate. As described in ^Example 54, 2-oxo-4-phenylbutyric acid and N-ε-N-ε-dimethyl-L-lysyl-L-proline hydrochloride were condensed in the presence of sodium cyanoborohydride.

2 spectrum is a combination

proline.

L-α-[1-carboxy-3-phenylpropyl]-1-lysyl-L-proline, a mixture of diastereomers prepared according to Example 57 B, was purified by

BAD ORIGINAL

Ο _; -ι ^d *'··“-<'·· D· 42 D .'JCiTQuO P HAP = 2 columns prepared. a<t^ is s-mica but ^p poimeo is calibrated at 53° C with 0 1 M NN ₄ OH. * - - . _l ,- _ - — .? k ·- mo ^: '1“· i^gvarya in ;C ml from the same time: author and is added to the column. When

The knees are eluted with this solvent, the first isomer appears in the range of 320-360 ml eluate. The second isomer appears in the range of -540 ml eluate. The me.ldln.ne fractions contain a mixture of isomers. When the fractions containing the first isomer are freeze-dried, 72 mg of a loose white mass is obtained. This is the more active isomer ^m with the SSS configuration by analogy with the more active isomer of M-a-[1-carboxy-3-phenylpropyl]-1_-alanyl-L-proline, which was found by X-ray analysis to have the SSS configuration. Thin layer chromatography on silica gel in 1:1:1:1 ethyl acetate/n-butanol/water/acetic acid gives a single spot with an Rf value of 0.43. The 300 MHz NMR spectrum shows a triplet for the methine proton γ to the phenyl substituent at 3.40 ppm. Upon sublimation, a solid is obtained. On thin-layer chromatography of this substance, a spot with an Rf value of 0.39 is obtained. The 300 MHz NMR spectrum shows a single triplet for the methine proton γ to the phenyl substituent at 3.40 ppm.

61 st.

the methine proton γ to the phenyl substituent at ι is lysyl-depleted.

as described in the example

54. as 2-oxo-4-acid and

M-c-acetyl-L-lysyl-L- rath in the presence of sodium cyanoborohydride. ha

The NMR carboxy-3-phenylpropyl]-.K1-c-acetyl-L-lysyl spectrum shows a molecular ion at 663 for the trisilylated products.

Example 121. N-α-[1-Carboxy-3-phenylpropyl N-arginyl-L-proline.

BAD ORIGINAL

The m.shlmiyat d.:Γ·?-;τ.'.α is prepared by DCC condensation of the hydrochloride salt of the benzyl ester of L-proline and of N-ot-tert ^P o ^g - .'\-<o-ni'ro- L-arginnch N-α-tert -Eos-protecting group is —, .- _o Qb ^{! 1} uRJ4'!~ uoi.hiu -> .j NJHCl 5 ^Q T'**7° ^{! 1 s} rz ^t p POL^CH ⁰¹ - ¹ ','Я benzyl ester of N-go-nitro- k-arginyl-P-proline is condensed with 2oxn-4-phenylbutyric acid, as described in Example 54

The reaction gives a very low yield (25 - 33 %) of M-α-[1-carboxy-

3-phenylpropyl]- N-ω-nitro- -L- arginyl- L- proline. This compound (159 mg) was dissolved in a solution (2.5 ml) of acetic acid/water/methanol (84%, 8%. 8%) and hydrogenated at 276 kN/m ² at room temperature, over 130 mg of palladium on charcoal to simultaneously remove the ω-nitro and benzyl ester protecting groups. The catalyst was filtered off and the filtrate was evaporated to 94 mg, the water-soluble portion being dried by sublimation to a loose white mass (90 mg). This solid is the acetate of the desired product and is converted to the free base by absorption on a strongly acidic ion exchange resin, washed with water, eluted with 2% pyridine in water, and freeze-dried product-rich fractions yield 60 mg of N-α-[1-carboxy-3-phenylpropyl]-k-arginyl-L-proline. The structure is consistent with the structure. The mass spectrum shows a molecular ion of π 793 for the pentasilyl-propionic products.

Example 122. N-[1-carboxy-3-phenylpropyl]-1-histidyl L-proline.

The procedure is as described in Example 54. 2-octo-4-phenylbutyric acid and n-histidyl-n-proline are condensed in the presence of sodium cyanoborohydride to give N-[1-carboxy-3-phenylpropyl]histidyl] n-prospin. The product is purified by gel filtration in methanol (LH-20). The NMR spectrum is consistent with the structure. The mass spectrum shows a molecular ion at 657 for the disilylated products.

BAD UrtiGiNAL

u...----—

I.

LсHL.О C ^- G ι u < Сl ”7 D ι v*r. Ach 4 1 </ ΐ'ίΠ^ύ,.'ν'ί-κύ^ r· or. ? г- c·. ъ - ' ci - ι uj Ο κ'Ό and ^L -> Π Oui ι '»' l ~ — 4.. ...χ,^...· .4 h/ 4 ^Γ 'ν> _Μ νΠ 4» ^ν4 ~

^.

L-proline in the presence of sodium cyanoborohydride. The protecting group t-tert-Boc is removed from the product with 4N HCl in ethyl acetate. The resulting hydrochloride

on Datex 50 (HM) and eluted with 2% pyridine in water. After freeze-drying of the product-rich fractions, the free base salt was obtained as a light brown loose solid. The NMR spectrum was consistent with the structure. The mass spectrum showed a molecular ion at 718 for the tetrasilylated

Example 124. N-α-[1-ethoxycarbonyl-4-methylpentyl]-1-lysyl L-proline.

Dissolve 2-oxo-4-methyl-ethylpentoate (2.75 g) and N-ε-βτ.-βοο-Λ_lysyl-L-proline (2.75 g; in 150 ml of ethanol containing 16 g of powdered molecular sieves 4A. Hydrogenate at 276 kN/m ² at room temperature, over ' g '0% palladium on charcoal. After the absorption of 1 mole of hydrogen, filter through a filter mat and wash the catalyst on the filtered precipitate carefully with eia~cn. The solvent is evaporated

G trνM ^η '' ^τ

Price 4 K/ JI 4J *.· ¹ 44 14- Π 4 ¹ spend

mp) to remove neutral products. Adjust the pH of the aqueous layer to ⁷ , saturate with sodium chloride and extract —- - - --G, . ~ <0 y -1 G.N D P ST O <4 nL 'J? p r·. p m-ITP -*>·'.··« -JS- ¹ ,- - - - ..^CС.a. L .4.4, ! <4^.'_; .4 · · _{ .· K ! C. _ 4. sШ anhydrous magnesium sulfate. Evaporate the ethyl acetate to obtain /138 g of crude M-xh-[1-ethoxycarbonyl-4-methylpentyl]-1\1-c-tert.-Eco -Lrg-.zll L- proline. Remove the tert.-Boc protecting group in the usual way-,:.- with 4 HC! in ethyl acetate. The resulting hydrochloride was converted to the free base with a strongly acidic ion exchange resin, eluting with 2% pyridine in water. The product-rich fractions were subjected to g

BAD ORIGINAL k

ν у ο» «>-0.k«ii^g1ps drying. to learn hygroscopic ^properties The MRI spectrometer is ! I GL| .11 4бГ1 1 L^ar<nr.v.'' _l ,|..t.»*urunriGuTUr:. . .Πΐ'ΟιΛΓΊ I - ΓυΛΤΠΜφ

N-[1-[1-ethoxy-4-methylpentyl]-L-lysyl L-proline was converted to the corresponding carbolic acid by heating in a 5% sodium hydroxide solution at room temperature. The reaction mixture was acidified ^{to pH} 5 ^, absorbed onto a strongly acidic ion exchange resin, and eluted with 2% pyridine in water. The product-rich fractions were dried by sublimation to give N-[1-carboxy-4-methylpentyl]-L-lysyl L-proline as a white, fluffy solid. The NMR spectrum showed a molecular ion at 516 for the disilylated products.

Example 126. M-α-[1-Carboxy-3-phenylpropyl]-*1_-leucyl-LTrII and Οψαπ.

The procedure is as described in Example 54, where 2-oxo-4-phenylbutyric acid and c-leucyl-c-triltophane are condensed in the presence of sodium borohydride. The product is subjected to salting-out from a mixture of dioxane and water, since it is poorly water-soluble. The NMR spectrum is consistent with the structure.

M Q p O i u P O <-] i'

M0Ch Pr*/

695 for trisilylated

Tablets.

and drink it <p .op/ o nvr>vO λ i . ' ί m r* <z r Kry |_|m Π - ^l LPROPYL

1-alanyl-1-prol.:-: (25 mg), pregelatinized starch (under US patent (82 mg), microcrystalline cellulose (82 mg) and magnesium stearate N-, 1 (S'i-ethoxycarbonyl-3mg) can be formulated 3 - phenylpyrazole /- L-alanyl-L-pyrazole with the composition of pregelatinized starch.

ί 'x ί L>

Phenipprs ^d ChF'-b-li311L-1_-proline (20 k I · 4 f О X *“ —' f Λ < · < / ГП »~Ч ΓΛ | ' — и'.,ό . r.ci gg/ of

BAD ORIGINAL microcrystalline cellulose and magnesium stearate, as described above.

B. Combination tablets.

A combination tablet with a diuretic, e.g. hydrochlorothiazide, usually contains N-(1(c)-ethoxycarbonyl-3-phenylpropyl)-1-alanyl-L-proline (7.5 mg), hydrochlorothiazide (50 mg), pregelatinized starch (82 mg) and magnesium stearate (1 mg). Tablets with e.g. N-(1(c)-ethoxycarbonyl-3-phenylprolyl)-1-lysyl-1-proline (5 mg) and hydrochlorothiazide (50 mg) are prepared by substituting the former for N-(1(c)-ethoxycarbonyl-3-phenylpropyl) in the above-described composition.

C. Injections.

An injectable composition contains N-(3)-ethoxycarbonyl-3-phenylpropyl)-1-alanyl-L-proline (5.42 mg), dibasic anhydrous sodium phosphate (11.4 mg), benzyl alcohol (0.01 ml) and water for injection (1.0 ml). Similarly, this composition can be prepared using N-(1(c)-ethoxycarbonyl-3-phenylpropyl)-1-lysyl-1-proline in place of N-(8)-ethoxycarbonyl-3-phenylpropyl)-1-alanyl-L-proline. The injectable composition can also contain a pharmaceutically effective amount of another compound with antihypertensive and/or diuretic activity, for example, hydrochlorothiazide, as described in Subsection B of this Example.

D. Suppositories

A typical suppository composition for rectal administration may contain N-(8)-ethoxycarbonyl-3-phenylpropyl)-1-alanyl-B-proline (1-25 mg), butylated hydroxyanisole (0.08-1.0 mg), disodium calcium edetate (ethylenediaminetetraacetic acid salt) (0.25-0.50 mg) and polyethylene glycol (775-1600 mg). Another suppository composition may be prepared by replacing, for example, N-(1-(c)-ethoxycarbonyl-3-phenylpropyl)-B-alanyl-B-proline with N-(1-(c)-ethoxycarbonyl-3-phenylpropyl)-1-lysyl-1-proline and by replacing, for example, disodium calcium edetate with butylated hydroxytoluene (0.04-0.08 mg) and polyethylene glycol. with hydrogenated vegetable oil (675 - 1400 mg) as

BAD ORIGINAL ϋιϋΐυΐ). .it ί ν;ecobee PS. Wtcobee ivl Wttepsols etc. Suppository compositions may also contain other compounds with antihypertensive activity.

Kami with 01 РигО Ь POINTS Ь AND WITH HP ι .ROOM

11rimer 128 N-[1-Egoxycarone;1-3(3-indolylpropyl)]-b alanyl-L-psoline

A solution of L-alanyl-L-proline (0.35 g) and ethyl 4-(3-indolyl)-2-oxobutyrate (1.0 g) in 30 ml of ethanol was hydrogenated with 10% palladium on carbon in the presence of 3.2 g of powdered molecular sieves (4A) at a hydrogen pressure of 276 kN/m''. After completion of the reaction, it was filtered and the filtrate was concentrated to dryness.

The residue was washed with methanol and chromatographed on a Sephadex LH-20 column to give 0.67 g of crude product as an orange oil. A portion of the crude product was purified by dissolving in water and extracting with ether. The product was extracted by diluting the aqueous phase with an equal volume of saturated sodium chloride solution and extracting with ethyl acetate to give 80 mg of N-[1-ethoxycarbonyl-3-(3-indolylpropyl)]-1-alanyl-L-proline as a yellow oil. The mass spectrum showed a maximum at 553 for the disilylated product and at 631 for the trisilylated product.

Benzyl N-1-(8-carboxy-3-phenylpropyl]-1alanyl-L-prolinate. .

Thiosinyl chloride (13.1 ml) was added to 150 ml of benzyl alcohol, keeping the temperature below 0° (exothermic reaction). N-[1(c)-carboxy-3-phenylpropyl]L-alanyl-L-preline (15 g) was added dropwise to the cold ^solution . The cooling bath was removed and the mixture was stirred at room temperature overnight. After heating to 45° under vacuum to remove dissolved gases, the reaction mixture was diluted with 500 ml of ether and washed with 10 x 100 ml of water. A solid phase appeared in the organic layer, which was filtered and dried to give 4.9 g of crude menobenzyl ester.

oh UHIGINAL

The combined aqueous extracts, neutralized with sodium bicarbonate, gave a second crop weighing 1.4 g. The two crops were combined and one portion (1.0 g) was recrystallized from ethanol-water to give 0.95 g of pure monobenzyl ester, mp 120-125°. Mass spectral analysis showed that the benzyl ester was linked to the proline ring.

Example 130. Ethyl N-[1(8)-epoxycarbonyl-3-phenylpropyl]-alanyl-L-proline hydrochloride.

A solution of 3.0 g of N-[1(8)-ethoxycarbonyl-3-phenylpropyl)-1-alanyl-L-proline in 50 ml of absolute ethanol was saturated with hydrogen chloride gas at 0°. After standing overnight at 0°, the HCl and ethanol were removed in vacuo to give 3.1 g of an oily liquid which, after freeze-drying, gave a white solid. Thin layer chromatography and HPLC indicated only one component. The NMR spectrum showed two ethyl groups per aromatic ring. The mass spectrum showed a molecular ion at 404 ppm.

Example 131. N-(1-Ethoxycarbonyl[3-(2-Chlorophenyl)propyl]4-alanylN-chloroline.

Working as described in Example 26, 2-oxo-3-(2chlorophenyl)butyrate and L-alanyl-L-proline were condensed in the presence of sodium cyanoborohydride to give N-(1-ethoxycarbonyl-3-(2chlorophenyl)propyl]-1-alanyl-L-proline. The mass spectrum showed a single peak at 411, (M ⁺ + 1) and a strong peak at 392, (M-18).

Example 132. N-Egoxycarbonyl[(4-fluorophenyl)propyl]N-alanyl-N-proline.

As described in Example 26, ethyl-2-oxo-4-(4-fluorophenyl)butyrate and L-alanyl-L-proline were condensed in the presence of sodium cyanoborohydride to give N-[1-ethoxycarbonyl-3-(4-fluorophenyl)propyl]-1-alanyl-L-proline. The mass spectrum showed a molecular ion at 394 and strong peaks at 376 (M-18, H ₂ O) and 375 (M19, F). The NMR spectrum was consistent with the proposed structure. Elengar analysis indicated a hydrate.

Example 133. N-[1-Ethoxycarbonyl-3-(2-naphthyl)propyl]-Balanyl-B-proline.

As described in Example 26, ethyl-2-oxo-4-(2-naphthyl)-butyrate and B-alanyl-B-proline were condensed in the presence of sodium cyanoborohydride to give N-[1-ethoxycarbonyl-3-(2-naphthyl)propyl]-B-alanyl-B-proline. The mass spectrum showed a molecular ion at 426 and a maximum at 408 (M-18, H ₂ O). The NMR spectrum was consistent with the proposed structure. Elemental analysis showed one hemihydrag.

Example 134. α-[1-Carboxy-3-(2-chlorophenyl)propyl]-Blisyl-B-proline.

As described in Example 26, ethyl-2-oxo-4-(2-chlorophenyl)butyrate and N-ε-tert-Boc-B-lysyl-B-proline were condensed in the presence of sodium cyanoborohydride to give N-α-[1-carboxy-3-(2chlorophenyl)propyl]-N-ε-tert-Boc-B-lysyl-B-proline. The tert-Boc protecting group was removed by treatment with 4N HCl in ethyl acetate, and the ethyl ester group was then saponified with dilute sodium hydroxide solution to give N-α-[1-carboxy-3-(2chlorophenyl)propyl]-B-lysyl-B-proline after absorption on a strongly acidic ion exchange resin and subsequent elution. Elemental analysis indicated solvation with 1.5 mol of water. The NMR spectrum was consistent with the proposed structure. The mass spectrum of one silylated sample shows a peak at 568, which corresponds to a mass loss of 15 (methyl) from the disilylated products.

Example 135. N-α-[1-carboxy-3-(4-fluorophenyl)propyl]-Blisyl-B-proline.

As described in Example 134, ethyl-2-oxo-4-(4-fluorophenyl)butyrate and N-ε-tert-Boc-B-lysyl-B-proline were condensed in the presence of sodium cyanoborohydride. The resulting product was deprotected as described in Example 4 to give N-α-[1-carboxy-3-(4-fluorophenyl)propyl]-B-lysyl-B-proline. Elemental

Γ· · analysis shows sesquihydrate. NMR spectrum is consistent with the proposed structure. Mass spectrum of the silylated sample shows molecular ion at 711 for the tetrasilylated products.

Example 136. N-α-[1-Carboxy-3-(2-naphthyl)propyl]-1-lysyl-L-proline.

As described in Example 134, replacing ethyl 2-oxo-4-(2-chlorophenyl)butyrate with ethyl 2-oxo-4-(2-naphthyl)butyrate gave Na-[1-carboxy-3-(2-naphthyl)propyl]-1-lysyl-L-proline. The NMR spectrum was consistent with the proposed structure. The mass spectrum showed a molecular ion at 599 for the disilylated products.

Example 137. Ethyl 2-oxo-4(2-thienyl)butyrate.

A mixture of diphosphorus tribromide (18.95 g) and pyridine (3.76 ml) in 10 ml of benzene was cooled to -5° C and treated with a solution of 2-(2-thienyl)ethanol (25 g) in 10 ml of benzene containing 1.25 ml of pyridine at such a rate that the reaction temperature did not exceed 0° C. The mixture was then allowed to reach room temperature and stirred overnight. The reaction mixture was then concentrated on a water pump at 30-40° to remove the solvent and other volatiles, and the residue was subjected to Claisen distillation under water pump vacuum to give 21 g of crude bromide (b.p. 115-119° C). This product was fractionally distilled at 0.3 mm to give 19 g of pure 2-(2-thienyl)-ethyl bromide (b.p. 50-51° C; 99.9%). This bromide was dissolved in 40 ml of dry tetrahydrofuran and added dropwise with stirring to a mixture of magnesium turnings (2.4 g) and 15 ml of dry tetrahydrofuran under nitrogen. The mixture was then stirred and refluxed for 1 hour, cooled to room temperature and added dropwise under nitrogen to a mixture of diethyl oxalate (17.5 g) in 25 ml of dry tetrahydrofuran in an ice bath at -15° C. After completion of the reaction, the reaction mixture was stirred at -10° C. for 15 minutes, then allowed to warm to 10° C. and treated with 60 mL of saturated ammonium chloride solution. The aqueous layer was extracted twice with toluene; the combined organic layers were dried over magnesium sulfate and concentrated in vacuo to give 16.7 g of a mixture which was determined by gas chromatography to contain, by weight, 26% solvent, 10.5% diethyl oxalate, and 63.4% ethyl oxo-(2thienyl)butyrate. The NMR spectrum was consistent with this mixture, indicating the presence of two specific types of ethyl esters, and the mass spectrum exhibited a maximum at m/e = 212 for the molecular ion of a ketoester.

Example 138. N-[1-Ethoxycarbonyl-3-(2-thienyl)propyl]-L-alanylL-proline.

Type 4A molecular sieves (powder) were added to a mixture of L-alanyl-L-proline (0.93 g) and ethyl 2-oxo-4-(2-thienyl)-butyrate (3.18 g) in 25 ml of anhydrous ethyl alcohol. The mixture was stirred for 45 min at room temperature, followed by the slow addition (3.5 h) of a solution of sodium cyanoborohydride (0.785 g) in 15 ml of anhydrous ethyl alcohol and stirring was continued overnight at room temperature. The reaction mixture was made very slightly acidic with glacial acetic acid, filtered and the filtrate was concentrated in vacuo to an orange oil. Extraction of a chloroform solution of this oil with 10% potassium bicarbonate, followed by re-extraction of the acidified (pH 3) bicarbonate extracts with chloroform, drying and evaporation of the solvent, gave 400 mg of the desired disubstituted dipeptide. The NMR spectrum (CDCl3) showed the thienyl proton as a multiplet at 7.0 ppm, two active protons in a broad singlet at 6.2 ppm, six methyl protons as a broad multiplet 1.22 - 1.43 ppm, and a series of ill-defined multiplets in the range 1.8 - 4.4 ppm.

Example 139. N[1-Carboxy-3-(2-thienyl)propyl]4-lysyl-L-proline.

Powdered molecular sieves of type 4A (6.95 g) were added to a mixture of N-ε-β-Boc-L-lysyl-L-proline (2.96 g) and ethyl 2-oxo-4(2-thienyl)-butyrate (6.4 g) in 40 ml of anhydrous ethyl alcohol. The mixture was stirred for 45 min at room temperature, then a solution of sodium cyanoborohydride (1.36 g) in 25 ml of anhydrous ethyl alcohol was added slowly (4.5 h) and stirred overnight at room temperature. The reaction mixture was then acidified very slightly with glacial acetic acid, filtered and concentrated in vacuo to give an orange foam (16.4 g). After trituration of this crude foam with three portions of ether and decantation of the ether solution, a significant amount of the excess ketoether present was removed. The ether-insoluble oil was poured into 1.1 ml of cold ether, filtered and concentrated in vacuo to an orange oil. A solution of this deblocked product in 250 ml of ethyl alcohol was treated with 5 g of sodium hydroxide in 35 ml of water and stirred overnight at room temperature. After careful acidification with 0.5 N HCl, concentration in vacuo and subsequent purification by elution on a column of 150 ml of Dautex 50 W-X2(H*) with water containing 2% pyridine, the substituted dipeptide was obtained as the dicarboxylic acid. The NMR spectrum (D ₂ O/DCI) was consistent with the structure.

Example 140. N-[1-carboxy-3-phenylpropyl]-4-alanyl-α-aminoisobutyric acid.

A mixture of 8.9 g of methyl-aaminoisobutyrate hydrochloride, 8.7 ml of triethylamine and 11.9 g of Boc-L-alanine in 135 ml of methylene chloride was prepared. A solution of 14.2 g of dicyclohexylcarbodiimide in 20 ml of methylene chloride was added and the mixture was stirred at room temperature for 46 hours, filtered and the filtrate was concentrated, then the residue was separated, treating the portions with water and ethyl acetate respectively. The organic phase was washed with 10% citric acid, water, 5% sodium bicarbonate and water. It was dried over magnesium sulfate and concentrated to give the crude dipeptide. The crude product was purified on silica gel, eluting with hexane-ethyl acetate (7:3).

Dissolve 1.2 g of this dipeptide in 30 mL of 4N HCl/ethyl acetate and store for one hour at room temperature. Concentrate the mixture and freeze-dry the residue to obtain L-alanyl-α-aminoisobutyric acid methyl ester hydrochloride.

A solution of 688 mg of this aminoester, 949 mg of ethyl 2-oxo-4-phenylbutyrate and 328 mg of sodium acetate in 10 ml of ethanol was prepared. This mixture was hydrogenated over 0.5 g of 10% palladium on carbon at 276 kN/m ² . The filtrate was filtered and concentrated. 4 ml of 7:3 hexane:ethyl acetate was added, filtered and the filtrate was chromatographed on silica gel to give methyl N-ethoxycarbonyl-3-phenylpropyl]L-alanyl-α-aminoisobutyrate.

Elemental analysis: Calculated for (C 112 N 115 ): C - 63.30; H - 8.24; N - 7.38; found: C - 63.58; H - 8.13; N - 7.27;

The NMR spectrum is consistent with the structure

460 mg of this diester was stirred with 3 ml of 2 N sodium hydroxide solution overnight at room temperature. The resulting solution was applied to a Doutex 50 (H*) column and eluted with 5% pyridine in water. The appropriate fractions were concentrated to give N-(1-carboxy-3-phenylpropyl)-alanyl-@-aminobutyric acid. Elemental analysis. Calcd. for (C ₁₇ H ₂ 7N ₂ O 6 .2H ₂ O): C - 54.82; H - 7.58; Found: C 54.94; H - 7.51; N - 7.48.

The NMR spectrum is consistent with the structure.

Example 141. b1[1-Carboxy-3-phenylpropyl]-1-alanyl-L··methylphenylalanine.

The compound was prepared according to Example 140, starting from 1-α-methylphenylalanine methyl ester hydrochloride instead of γ-aminoisobutyric acid methyl ester hydrochloride.

Example 142. N-{-1-carboxy-3-[4-(1-benzylimidazolyl)]propyl}-1-alanyl-L-proline.

An excess of 4-(4-(1-benzylidazolyl)]-2-ketobutyric acid (obtained by dibenzylation of 3-(4-imidazolyl)propionic acid, by condensation with diethyl oxalate, followed by acid-catalyzed hydrolysis and decarboxylation) was condensed with L-alanyl-L-proline in water (pH 6.7) in the presence of excess sodium cyanoborohydride. After standing for 3 days at room temperature, the product was absorbed onto a strongly acidic ion exchange resin. After elution with 2% aqueous pyridine and freeze-drying, the crude product was obtained, which was further purified on a Sephadex LH-20 column using methanol. C ¹³ and H ¹ NMR and IR spectral data were consistent with the structure. Organic elemental analysis data were available for the monohydrate. The mass spectrum (after silylation) showed peaks at 572 (M*), 577 (M*-15), and 455 (M ⁺ - CQSiMea) m/e.

Example 143. N-[1-carboxy-3-(4-imidazolyl)propyl]-4-alanyl-L-proline.

N-1-Carboxy-3-4-(1-benzimidazolyl)propyl-1_-alanyl-L-proline, prepared as described in Example 142, was subjected to hydrogenolysis at 276 kN/μ ² over an equal weight of 5% palladium on carbon in a 3:1 acetic acid/water mixture at 60° for three days. After removal of the catalyst by filtration and concentration of the filtrate, the crude product was purified by absorption on a strongly acidic ion exchange resin, followed by elution with 2% aqueous pyridine and freeze-drying. Further purification was carried out on a Sephadex LH-20 column using methanol. H ¹ NMR and thin layer chromatography (silica gel) showed that the benzene group had been removed to give the title product. The mass spectrum shows a maximum for (M ⁺ -18) at m/e = 320.

Example 144. {N-1-carboxy-3-[4-(1-benzylimidazolyl)]propyl} ^, -1-lysyl-L-proline and

N[1-carboxy-3-(4-imidazolyl)propyl]-1-lysyl-L-proline.

The two title compounds of the example were prepared in the same way as N-[1-carboxy-3-[4-(1-benzylimidazolyl)]propyl-85 alanyl-L-proline (Example 142) and N-[1-carboxy-3-(4-imidazolyl)propyl]L-alanyl-L-proline were prepared, whereby L-alanyl-L-proline was replaced by Et-Boc-L-lysyl-1-proline and the tert-Boc group was removed with trifluoroacetic acid at 0° for three hours after condensation.

Example 145. N-1-ethoxycarbonyl-3-[4-(1-benzylimidazolyl)]propyl-N-alanyl-1-proline and

N[1-ethoxycarbonyl-3-(4-imidazolyl)propyl]N-alanyl-L-proline.

The two title compounds of the example were prepared in the same manner as N-1-carboxy-3-[4-(1-benzylimidazolyl)]propylL-alanyl-L-proline (Example 142) and N-[1-carboxy-3-(4-imidazolyl)propyl]L-alanyl-L-proline (Example 143), using 4-[4(1-benzylimidazolyl)]-2-ketobutyric acid ethyl ester and the condensation was carried out in the presence of sieve 4A, in ethyl alcohol instead of water.

Claims (10)

1. A pharmaceutical composition for the treatment of high blood pressure, comprising a pharmaceutically acceptable carrier and a pharmaceutically effective amount of an amino acid derivative of the formula in which R and R ⁸ are the same or different and represent hydroxy, lower alkoxy, lower alkenoxy, lower dialkylamino lower alkoxy, acylamino lower alkoxy, acyloxy lower alkoxy, aryloxy, aryl lower alkoxy, substituted aryloxy or substituted aryl lower alkoxy, the substituent being methyl, halogen or methoxy, amino, lower alkylamino, lower dialkylamino, aryl lower alkylamino or hydroxyamino, R ¹ is hydrogen, alkyl of 1 to 20 carbon atoms, including pendant and cyclic and unsaturated alkyl groups; substituted lower alkyl, the substituent may be halogen, hydroxy, lower alkoxy, aryloxy, amino, lower alkylamino, lower dialkylamino, acylamino, arylamino, guanidino, imidazolyl, indolyl, mercapto, lower alkylthio, arylthio, carboxy or carboxamido, carbonyl alkoxy, phenyl, substituted phenyl, the substituent may be lower alkyl, lower alkoxy or halogen, aryl lower alkyl or heteroaryl lower alkyl, aryl lower alkenyl or heteroaryl lower alkenyl, substituted aryl lower alkyl, substituted heteroaryl lower alkyl, substituted aryl lower alkenyl or substituted heteroaryl lower alkenyl, the substituent may be halogen or dihalogen, lower alkyl, hydroxy, lower alkoxy, amino, aminomethyl, tn acylamino, lower dialkylamino, lower alkylamino, carboxyl, halogenated lower alkyl, cyano or sulfonamido, aryl lower alkyl or heteroaryl lower alkyl substituted in the alkyl portion with amino or benzoylamino; R ² and R ⁷ are hydrogen or lower alkyl; R ³ is hydrogen, lower alkyl, phenyl lower alkyl, aminomethyl phenyl lower alkyl, hydroxyphenyl lower alkyl, hydroxy lower alkyl, acetylamino lower alkyl, acylamino lower alkyl, amino lower alkyl, dimethylamino lower alkyl, halogenated lower alkyl, guanidino lower alkyl, imidazolyl lower alkyl, indolyl lower alkyl, mercapto lower alkyl, lower alkylthio lower alkyl; R ⁴ is hydrogen or lower alkyl; R ⁵ is hydrogen or lower alkyl, phenyl, phenyl lower alkyl, hydroxy lower alkyl, amino lower alkyl, guanidino lower alkyl, imidazolyl lower alkyl, indolyl lower alkyl, mercapto lower alkyl or lower alkylthio lower alkyl; R ⁴ and R ⁵ may be linked together to form an alkylene bridge of 2 to 4 carbon atoms, an alkylene bridge of 2 to 3 carbon atoms and one sulfur atom, an alkylene bridge ^of 3 to 4 carbon atoms containing one double bond or an alkylene bridge as defined above substituted with hydroxy, lower alkoxy, lower alkyl, or pharmaceutically acceptable salts thereof and an antihypertensive and/or diuretic compound selected from the group consisting of amiloride, atenolol, bendroflumethiazide, chlorothalidone, chlorothiazide, clonidine hydrochloride, cryptenamine acetates and cryptenamine tannates, deserpidine, diazoxide, guanethidine sulfate, hydralazine hydrochloride, hydrochlorothiazide, hydroflumethiazide, metolazone, metoprolol tartrate, methyclothiazide, methyldopa, methyldopa hydrochloride, minoxide, pargyline hydrochloride, polythiazide, prazosin hydrochloride, propanolol, rauwolfia serpentina, retsinamine, reserpine, sodium nitroprusside, spironolactone, timolol, trichlormethiazide, trimethophan camsylate, benzthiazide, quinethazone, ticrinaphan, triamterene, acetazolamide, aminophylline, cyclothiazide, ethacrynic acid, furosemide, meretoxyline procaine, sodium ethacrynate and mixtures and combinations thereof. 2. A pharmaceutical composition for the treatment of high blood pressure, comprising a pharmaceutically acceptable carrier and a pharmaceutical compound in an effective amount of an amino acid of the formula b wherein R and R ⁶ can be separately and independently hydroxy, lower alkoxy, arylalkyloxy, R ² and R ⁷ are hydrogen, R ³ is methyl, amino lower alkyl, R ⁴ and R ⁵ are linked through a carbon and nitrogen atom to form proline, 4-thiaproline or 4-methoxyproline and R ¹ is alkyl having from 1 to 8 carbon atoms; substituted lower alkyl, where the alkyl group has 1 to 5 carbon atoms and the substituent is amino, arylthio or aryloxy, arylalkyl or heteroarylalkyl, where the alkyl moiety has 1 to 3 carbon atoms, substituted arylalkyl or heteroarylalkyl, where the alkyl group has 1 to 3 carbon atoms and the substituent(s) is halogen? dihalogenated amino, aminoalkyl, hydroxy, lower alkoxy or lower alkyl, its pharmaceutically acceptable salts and an antihypertensive and/or diuretic compound selected from the group consisting of amiloride, atenolol, bendroflumethiazide, chlorothalidone, chlorothiazide, clonidine hydrochloride, cryptenamine acetates and cryptenamine tannates, deserpidine, diazoxide, guanethidine sulfate, hydralazine hydrochloride, hydrochlorothiazide, hydroflumethiazide, metolazone, metoprolol tartrate, methyclothiazide, methyldopa, methyldopa hydrochloride, minoxide, pargyline hydrochloride, polythiazide, prazosin hydrochloride, propanolol, rauwolfia serpentina, rescinamine, reserpine, sodium nitroprusside, spironolactone, timolol, trichlormethiazide, trimethophan camsylate, benzthiazide, quinethazone, ticrinaphan, triamterene, acetazolamide, aminophylline, cyclothiazide, ethacrynic acid, furosemide, meretoxyline procaine, sodium ethacrynate and mixtures and combinations thereof. 3. A composition according to claim 2, wherein the amino acid compound is selected from the group consisting of: N-(1-carboxy-3-phenylpropyl)-1-alanyl-1-proline; N-(1-ethoxycarbonyl-3-phenylpropyl)-1.-alanyl-1.-proline N-(1-ethoxycarbonyl-4-methylpentyl)-1-alanyl-1-proline; N-(1-carboxy-5-aminopentyl)-1-alanyl-1-proline; N-α-(1-carboxy-3-phenylpropyl)-lysyl-L-proline; N-α-(1-ethoxycarbonyl-3-phenylpropyl)-1-lysyl-L-proline; N-α-[1-carboxy-3-(3-indolyl)propyl]-lysyl-L-proline; N-α-[1-carboxy-3-(4-chlorophenyl)propyl]-lysyl-L-proline; N-α-[1-carboxy-2-phenylthioethyl]-1-lysyl-L-proline; N-α-[1-carboxy-3-(4-chlorophenyl)propyl]-E-lysyl-trans-4-methoxy-E-proline; N-α-[1-carboxy-5-aminopentyl]N-lysyl-L-proline; N-α-[1-carboxy-3-phenylpropyl]-E-ornithyl-L-proline; Ethyl N-(1-ethoxycarbonyl-3-phenylpropyl)-alanyl-E-prolinate hydrochloride; N-[1-(ethoxycarbonyl)-3-(4-imidazolyl)propyl]-alanine<proline; N-(1(c)-carboxy-3-phenylpropyl)-E-alanyl-E-proline; N-(1(c)-ethoxycarbonyl-3-phenylpropyl)-E-alanyl-E-proline; N-(1(S)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-E-proline, maleic salt; N-α-(1(8)-carboxy-3-phenylpropyl)-4-lysyl-L-proline; Ethyl N-(1 (b)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L· prolinate hydrochloride; and N-α-(1(c)-ethoxycarbonyl-8-phenylpropyl)-E-lysyl-L-proline; 4. A pharmaceutical composition for treating high blood pressure, comprising a pharmaceutically effective amount of an amino acid compound according to claim 3, a pharmaceutically effective amount of hydrochlorothiazide and a pharmaceutically acceptable carrier. 5. A pharmaceutical composition for the treatment of high blood pressure, comprising a pharmaceutically effective amount of an amino acid compound according to claim 3, a pharmaceutically effective amount of hydrochlorothiazide and timolol, and a pharmaceutically acceptable carrier. 6. A pharmaceutical composition for treating high blood pressure, comprising a pharmaceutically effective amount of an amino acid compound according to claim 3, a pharmaceutically effective amount of timolol and a pharmaceutically acceptable carrier. 7. A pharmaceutical composition for treating high blood pressure, comprising a pharmaceutically effective amount of an amino acid compound according to claim 3, a pharmaceutically effective amount of hydrochlorothiazide and amiloride, and a pharmaceutically acceptable carrier. 8. A pharmaceutical composition for treating high blood pressure, comprising a pharmaceutically effective amount of an amino acid compound according to claim 3, a pharmaceutically effective amount of furosemide and a pharmaceutically acceptable carrier. 9. A pharmaceutical composition for the treatment of high blood pressure, comprising a pharmaceutically effective amount of N-(1(S)ethoxycarbonyl-3-phenylpropyl)-1-alanyl-1-proline, a pharmaceutically effective amount of hydrochlorothiazide and a pharmaceutically acceptable carrier. 10. A pharmaceutical composition for the treatment of high blood pressure, comprising a pharmaceutically effective amount of N-α-(1(S)carboxy-3-phenylpropyl)-1-lysyl-L-proline, a pharmaceutically effective amount of hydrochlorothiazide and a pharmaceutically acceptable carrier. 11. A pharmaceutical composition for the treatment of high blood pressure, comprising a pharmaceutically effective amount of N-(1(S)ethoxycarbonyl-3-phenylpropyl)-L-alanyl-1-proline, a pharmaceutically effective amount of hydrochlorothiazide and timolol, and a pharmaceutically acceptable carrier. 12. A pharmaceutical composition for the treatment of high blood pressure, comprising a pharmaceutically effective amount of N-α-(1(S)carboxy-3-phenylpropyl)-1-lysyl-L-proline, a pharmaceutically effective amount of hydrochlorothiazide and timolol, and a pharmaceutically acceptable carrier. 13. A pharmaceutical composition for the treatment of high blood pressure, comprising a pharmaceutically effective amount of N-(1(S)ethoxycarbonyl-3-phenylpropyl)-E-alanyl-k-proline, a pharmaceutically effective amount of timolol and a pharmaceutically acceptable carrier. 14. A pharmaceutical composition for the treatment of high blood pressure, comprising a pharmaceutically effective amount of N-α-(1(S)carboxy-3-phenylpropyl)-1-lysyl-L-proline, a pharmaceutically effective amount of timolol and a pharmaceutically acceptable carrier. 15. A pharmaceutical composition for the treatment of high blood pressure, comprising a pharmaceutically effective amount of N-(1(S)ethoxycarbonyl-3-phenylpropyl)-1-alanyl-E-proline, a pharmaceutically effective amount of hydrochlorothiazide and amiloride, and a pharmaceutically acceptable carrier. 16. A pharmaceutical composition for the treatment of high blood pressure, comprising a pharmaceutically effective amount of N-α-(1(S)carboxy-3-phenylpropyl)-1-lysyl-L-proline, a pharmaceutically effective amount of hydrochlorothiazide and amiloride, and a pharmaceutically acceptable carrier. 17. A pharmaceutical composition for the treatment of high blood pressure, comprising a pharmaceutically effective amount of N-(1(S)ethoxycarbonyl-3-phenylpropyl)-1-alanyl-1-proline, a pharmaceutically effective amount of furosemide and a pharmaceutically acceptable carrier. 18. A pharmaceutical composition for the treatment of high blood pressure, comprising a pharmaceutically effective amount of N-α-(1(S)carboxy-3-phenylpropyl)-lysyl-L-proline, a pharmaceutically effective amount of furosemide and a pharmaceutically acceptable carrier. 19. A method for preparing a compound of the formula wherein R and R ⁶ are the same or different and represent hydroxy, lower alkoxy, lower alkenoxy, lower dialkylamino lower alkoxy, acylamino lower alkoxy, acyloxy lower alkoxy, aryloxy, aryl lower alkoxy, substituted aryloxy or substituted aryl lower alkoxy, the substituent being methyl, halogen or methoxy, amino, lower alkylamino, lower dialkylamino, aryl lower alkylamino or hydroxyamino, R ² and R ⁷ are hydrogen or lower alkyl; R ³ is hydrogen, lower alkyl, phenyl lower alkyl, aminomethyl phenyl lower alkyl, hydroxyphenyl lower alkyl, hydroxy lower alkyl, acetylamino lower alkyl, acylamino lower alkyl, amino lower alkyl, dimethylamino lower alkyl, halogenated lower alkyl, guanidino lower alkyl, imidazolyl lower alkyl, indolyl lower alkyl, mercapto lower alkyl, lower alkylthio lower alkyl; R ¹ is hydrogen, alkyl of 1 to 20 carbon atoms, including pendant and cyclic and unsaturated alkyl groups; substituted lower alkyl, the substituent may be halogen, hydroxy, lower alkoxy, aryloxy, amino, lower alkylamino, lower dialkylamino, acylamino, arylamino, guanidino, imidazolyl, indolyl, mercapto, lower alkylthio, arylthio, carboxy or carboxamido, carbonyl alkoxy, phenyl, substituted phenyl, the substituent may be lower alkyl, lower alkoxy or halogen, aryl lower alkyl or heteroaryl lower alkyl, aryl lower alkenyl or heteroaryl lower alkenyl, substituted aryl lower alkyl, substituted heteroaryl lower alkyl, substituted aryl lower alkenyl or substituted heteroaryl lower alkenyl, the substituent may be halogen or dihalogen, lower alkyl, hydroxy, lower alkoxy, amino, aminomethyl, acylamino, lower dialkylamino, lower alkylamino, carboxyl, halogenated lower alkyl, cyano or sulfonamido, aryl lower alkyl or heteroaryl lower alkyl substituted in the alkyl moiety with amino or acylamino; R ⁴ is hydrogen or lower alkyl; R ⁵ is hydrogen or lower alkyl, phenyl, phenyl lower alkyl, hydroxy lower alkyl, amino lower alkyl, guanidino lower alkyl, imidazolyl lower alkyl, indolyl lower alkyl, mercapto lower alkyl or lower alkylthio lower alkyl; R ⁴ and R ⁵ . are . linked together to form an alkylene bridge of 2 to 4 carbon atoms, an alkylene bridge of 2 to 3 carbon atoms and one sulfur atom, an alkylene bridge of 3 to 4 carbon atoms containing one double bond or an alkylene bridge as defined above substituted with hydroxy, lower alkoxy or lower alkyl, and pharmaceutically acceptable salts thereof, wherein a ketone of formula wherein R ¹ includes suitable protection of any reactive group is reacted with a dipeptide or protected dipeptide of formula r· ·· PO in which R ³ and R ⁶ include suitable protection of each reactive group and the intermediates formed are reduced in the reaction in the presence of a reducing agent, followed by removal of the protecting groups, if necessary, to obtain the desired product, and if desired, a salt thereof is obtained by conventional methods and, if desired, a biologically active isomer is isolated by chromatography or fractional crystallization. 20. A method for preparing a compound according to claim 1 comprising reacting a ketone of formula PO19. wherein R is a reactive group, formula is not hydroxy, and R is a suitable protection of any amino acid or protected amino acid with R? HjN—UN R ³ includes suitable protection of any reactive group, in the presence of a reducing agent, to obtain an intermediate of formula in which and after the amino acid formula n-soon this coupling of the intermediate or with a protected amino acid compound derivative (VI) LhX^ooh and condensing a compound of formula (X) with an amino acid derivative of formula R ⁴ R5 (VI) wherein R ⁶ is not hydroxy and R ⁵ includes appropriate protection of any reactive group, followed by removal of the protecting groups, if necessary, to obtain a compound according to claim 18, wherein R and R ⁶ are not hydroxy and, if desired, R and/or R* are converted to hydroxyl groups by hydrolyzing or hydrogenating the corresponding precursor and further, if desired, preparing a salt thereof by conventional methods and further, if desired, isolating a biologically more active isomer by chromatography or fractional crystallization. 22. A method for preparing compounds according to claim 19, comprising reacting a dipeptide of formula reactive (>0 in which R ³ and R ⁵ include a suitable protection of each group with a compound of formula R2 (I) wherein ^R6 is not hydroxy and ^R5 includes suitable protection of any reactive group to provide a compound according to claim 19 wherein R and ^R6 are not hydroxy, followed by removal of the protecting groups, if desired, conversion of R and/or ^R6 to hydroxy by hydrolyzing or hydrogenating the corresponding intermediate, and, if desired, salt formation by conventional means and, if desired, isolation of the biologically more active isomer by chromatography or fractional crystallization. 21. A method for preparing a compound of formula according to claim 19, which comprises reacting an amine of formula reactive with an R ¹ group. in which R ³ and includes with ketone with appropriate protection of each formula O=-C~c—N-^-CO—R ⁸ (VIII) R ⁵ include appropriate protection of any reactive group, in the presence of a reducing agent, followed by removal of the protecting groups if necessary to obtain the desired product or, if desired, stepwise reaction by condensation of (VII), where R is not hydroxy with a keto acid of formula wherein R ³ includes suitable protection of any reactive group, in the presence of a reducing agent, to give a compound of formula wherein R ¹ includes suitable protection of any reactive group and X is a chlorine, bromine, iodine or sulfonyloxy derivative, followed by removal of the protecting groups if necessary to give the desired product, or, optionally, reacting (XI), wherein R is not OH, with an amino acid derivative of formula nD^OOH wherein R ³ includes suitable protection of any reactive group, to form an intermediate of formula and then the intermediate reacts with an amino acid derivative of the formula -CO-R ⁸ in which R ⁶ is not hydroxy, and R ⁵ includes suitable protection of any reactive group, followed by removal of the protecting group, if necessary, to obtain a compound according to claim 19, and. optionally, to obtain a salt thereof by conventional methods and, optionally, to isolate the biologically more active isomer by chromatography or fractional crystallization. 23. A method for preparing compounds according to claim 19, comprising reacting the derivative of formula IN WHICH reactive amino acid includes appropriate protection of each group with an α-substituted acyl derivative of wherein X is chlorine, bromine, iodine or a sulfonyloxy derivative, wherein preferably R® is not hydroxy and R ³ and R ⁵ include appropriate protection of any reactive group, followed by removal of the protecting group if necessary. to obtain the desired product or, optionally, the amino acid ester of formula (VII), wherein R is not hydroxy, is reacted with an α-substituted acid of formula R? X-^H-COOH in which R ³ includes suitable protection of any reactive group to give an intermediate ester of formula and reaction of an amino acid with the formula intermediate with an ester of wherein R ⁶ is not hydroxy, and R ⁵ includes appropriate protection of any reactive group, followed by removal of the protecting group, if necessary, to obtain a compound according to claim 19, and, if desired, converting R and/or R® to hydroxy by hydrolyzing or hydrogenating the corresponding precursor and further, if desired, preparing a salt thereof by conventional methods and, if desired, isolating the biologically more active isomer by chromatography or fractional crystallization. 24. A method according to claim 19 for preparing a compound of formula R and R® can be separately and independently hydroxy, alkoxy, arylalkyloxy, R ¹ is alkyl having from 1 to 8 carbon atoms; substituted lower alkyl, where the alkyl group has 1 to 5 carbon atoms and the substituent is amino, arylthio or aryloxy, arylalkyl or heteroarylalkyl, where the alkyl moiety has 1 to 3 carbon atoms, substituted arylalkyl or heteroarylalkyl, where the alkyl group has 1 to 3 carbon atoms, the substituent(s) being halogen, dihalogen, amino, aminoalkyl, hydroxy, lower alkoxy or lower alkyl, R ² and R ⁷ are hydrogen, R ³ is methyl, amino lower alkyl, R ⁴ and R ⁵ are linked through a carbon and nitrogen atom to form proline, 4-thiaproline or 4methoxyproline. 25.. A method according to claim 19 for preparing a compound of formula 100 involving the reaction of a ketone with the formula with a dipeptide with the formula in the presence of a reducing agent to obtain the desired product, and isolation of the biologically more active diastereoisomer by chromatography and fractional crystallization. 26. A process according to claim 25, wherein the N-[1(S)ethoxycarbonyl-3-phenylpropyl]-alanyl-l-proline maleate is obtained by fractional crystallization. 27. A method according to claim 19 for preparing a compound of formula NHj nrnr n which involves the reaction of a ketone with the formula 101 O CHpHiD no-c-ό-ο with a protected dipeptide of formula in which t-Boc is the tert-butyloxycarbonyl protecting group, in the presence of a reducing agent to obtain the protected form of the desired product, followed by reaction with a suitable acidic reagent to obtain the desired product, followed by isolation of the biologically more active diastereomer by chromatography or fractional crystallization. 28. A method according to claim 19 for preparing a compound of formula which involves the reaction of a ketone with the formula <feHs C2H5O-s-sn=-o 102 with a protected dipeptide of formula NhftBoc (SNL in which t-Boc is the tert-butyloxycarbonyl protecting group, in the presence of a reducing agent to obtain the protected form of the desired product, followed by reaction with a suitable acidic reagent to obtain the desired product, followed by isolation of the biologically more active diastereomer by chromatography or fractional crystallization. 29. A method according to claim 19 for preparing a compound of formula comprising reacting a ketone of formula with a protected dipeptide of formula 103 M-NVos in which t-Boc is the tert-butyloxycarbonyl protecting group, in the presence of a reducing agent to obtain the protected form of the desired product, followed by reaction with a suitable acidic reagent to obtain the desired product, and isolation of the biologically more active diastereomer by chromatography or fractional crystallization. 30. A method according to claim 19 for preparing the following compounds: N-(1-carboxy-3-phenylpropyl)-1-alanyl-bproline; 1N-(1-ethoxycarbonyl-3-phenylpropyl)-E-alanyl-L-proline N-(1-ethoxycarbonyl-4-methylpentyl)-1_-alanyl-1_-proline; N-(1-carboxy-5-aminopentyl)-1_-alanyl-L-proline; N-α-(1-carboxy-34enylpropyl)N-lysyl-L-proline; N-α-(1-ethoxycarbonyl-3-phenylpropyl)-E-lysyl-L-proline; N-α-[1 -carboxy-3-(3-indolyl)propyl]4_-lysyl-L-proline; N-α-[1 -carboxy-3-(4-chlorophenyl)propyl]<-lysyl-L-proline; N-α-[1-carboxy-2-phenylthioethyl]-lysyl-L-proline; N-α-[1-carboxy-3-(4-chlorophenyl)propyl]-lysyl-trans-4-methoxy-L-proline; N-α-[1-carboxy-5-aminopentyl]-1-lysyl-L-proline; N-α-[1-carboxy-3-phenylpropyl]-ornigyl-L-proline; Ethyl N-(1-ethoxycarbonyl-3-phenylpropyl)-alanyl-L-prolinate hydrochloride; N-[1-(ethoxycarbonyl)-3-(4-imidazolyl)propyl]-E-alanine-Eproline; N-[1-carboxy-3-(4-imidazolyl)propyl]-lysyl-L-proline; 104 N-(1(S)-carboxy-3-phenylpropyl)-alanyl-E-proline; N-(1(b)-ethoxycarbonyl-3-phenylpropyl)-1-alanyl-1-proline; N-(1(8)-ethoxycarbonyl-3-phenylpropyl)-1-alanyl-E-proline, maleic salt; N-α-(1(8)-carboxy-3-phenylpropyl)-lysyl-L-proline and Ethyl N-(1(8)-ethoxycarbonyl-3-phenylpropyl)-alanyl-L-proline hydrochloride; and N-α-(1(8)-ethoxycarbonyl-3-phenylpropyl)-1-lysyl-L-proline; 31. A pharmaceutical composition for the treatment of high blood pressure, comprising a pharmaceutically acceptable carrier and a pharmaceutically effective amount of an amino acid compound of the formula in which R and R* are the same or different and represent hydroxy, lower alkoxy, lower alkenoxy, lower dialkylamino lower alkoxy, acylamino lower alkoxy, acyloxy lower alkoxy, aryloxy, aryl lower alkoxy, substituted aryloxy or substituted aryl lower alkoxy, the substituent being methyl, halogen or methoxy, amino, lower alkylamino, lower dialkylamino, aryl lower alkylamino or hydroxyamino, R ¹ is hydrogen, alkyl of 1 to 20 carbon atoms, including pendant and cyclic and unsaturated alkyl groups; substituted lower alkyl, the substituent being halogen, hydroxy, lower alkoxy, aryloxy, amino, lower alkylamino, lower dialkylamino, acylamino, arylamino, guanidino, imidazolyl, indolyl, mercapto, lower alkylthio, arylthio, carboxy or carboxamido, carbonyl alkoxy, phenyl, substituted phenyl, the substituent being lower alkyl, lower alkoxy or halogen, aryl lower alkyl or heteroaryl lower alkyl, aryl lower alkenyl or heteroaryl lower alkenyl, substituted aryl lower alkyl, 105 substituted heteroaryl lower alkyl, substituted aryl lower alkenyl or substituted heteroaryl lower alkenyl, the substituent being halogen or dihalogen, lower alkyl, hydroxy, lower alkoxy, amino, aminomethyl, acylamino, lower dialkylamino, lower alkylamino, carboxyl, halogenated lower alkyl, cyano or sulfonamido, aryl lower alkyl or heteroaryl lower alkyl, substituted in the alkyl moiety with amino or benzoylamino; R ² and R ⁷ are hydrogen or lower alkyl; R ³ is hydrogen, lower alkyl, phenyl lower alkyl, aminomethyl phenyl lower alkyl, hydroxyphenyl lower alkyl, hydroxy lower alkyl, acetylamino lower alkyl, acylamino lower alkyl, amino lower alkyl, dimethylamino lower alkyl, halogenated lower alkyl, guanidino lower alkyl, imidazolyl lower alkyl, indolyl lower alkyl, mercapto lower alkyl, lower alkylthio lower alkyl; R ⁴ is hydrogen or lower alkyl; R ⁵ is hydrogen or lower alkyl, phenyl, phenyl lower alkyl, hydroxy lower alkyl, amino lower alkyl, guanidino lower alkyl, imidazolyl lower alkyl, indolyl lower alkyl, mercapto lower alkyl or lower alkylthio lower alkyl; R ⁴ and R ⁵ may be linked together to form an alkylene bridge of 2 to 4 carbon atoms, an alkylene bridge of 2 to 3 carbon atoms and one sulfur atom, an alkylene ^bridge of 3 to 4 carbon atoms containing one double bond or an alkylene bridge as defined above substituted with hydroxy, lower alkoxy, lower alkyl, or pharmaceutically acceptable salts thereof and an antihypertensive and/or diuretic compound and mixtures or combinations thereof. 106 32. A pharmaceutical composition for the treatment of high blood pressure, comprising a pharmaceutically acceptable carrier and a pharmaceutically effective amount of an amino acid compound of the formula wherein R and R ⁶ can separately and independently be hydroxy, lower alkoxy, arylalkyloxy, R ² and R ⁷ are hydrogen, R ⁵ is methyl, amino lower alkyl, R 1 and R ⁵ are linked through carbon and nitrogen atoms to form proline, 4-thiaproline or 4-methoxy-proline and R ¹ is alkyl having from 1 to 8 carbon atoms, substituted lower alkyl, such that the alkyl group has from 1 to 5 carbon atoms and the substituent is amino, arylthio or aryloxy, arylalkyl or heteroarylalkyl, such as THE ALKYL MOTION HAS FROM 1 TO 3 CARBON ATOMS, SUBSTITUTED ARYLAKYL OR HETEROALKYL, WHERE THE ALKYL GROUPS HAVE FROM 1 TO 3 CARBON ATOMS AND THE SUBSTITUENT(S) ARE HALOGEN, DIHALOGEN, AMINO, AMINOALKYL, hydroxy, LOWER alkoxy OR LOWER ALKYL OR PHARMACEUTICALLY ACCEPTABLE SALTS THEREOF AND AN ANTIHYPERTENSIVE AND/OR DIURETIC COMPOUND AND MIXTURES OR COMBINATIONS THEREOF. 33. A composition according to claim 32, wherein the amino acid compound is selected from the following compounds: N-(1-carboxy-3-phenylpropyl)-1-alanyl-1-proline; N-(1-ethoxycarbonyl-3-phenylpropyl)-1-alanyl-L-proline N-(1-ethoxycarbonyl-4-methylpentyl)-L-alanyl-L-proline; 10? N-(1-carboxy-5-aminopentyl)-k-alanyl-b-proline; N-α-(1-carboxy-3-phenylpropyl)-lysyl-L-proline; N-α-(1-ethoxycarbonyl-3-phenylpropyl)-E-lysyl-k-proline; N-α-[1-carboxy-3-(3-indolyl)propyl]-1-lysyl-L-proline; N-α-[1-carboxy-3-(4-chlorophenyl)propyl]-1-lysyl-L-proline; N-α-[1-carboxy-2-phenylmethyl]-L-lysyl-L-proline; N-α-[1-carboxy-3-(4-chlorophenyl)propyl]-lysyl-trans-4methoxy-L-proline; N-α-[1-carboxy-5-aminopontyl]-κ-lysyl-L-proline; N-α-[1-carboxy-3-phenylpropyl]4-ornithyl-L-proline; Ethyl N-(1-ethoxycarbonyl-3-phenylpropyl)-1-alanyl-k-prolinate hydrochloride; N-[1-(ethoxycarbonyl)-3-(4-imidazolyl)propyl]-k-alanine-Eproline; N-[1-carboxy-3-(4-imidazolyl)propyl]-k-lysyl-L-proline; L-(1(8)-carboxy-3-phenylpropyl)-l-alanyl-k-proline; N-(1(c)-ethoxycarbonyl-3-phenylpropyl)-k-alanyl-k-proline; N-(1(8)-ethoxycarbonyl-3-phenylpropyl)-E-alanyl-k-proline, maleic salt; N-α-(1(8)-carboxy-3-phenylpropyl)-k-lysyl-L-proline and Ethyl N-(1(b)-ethoxycarbonyl-3-phenylpropyl)-k-alanyl-k-proline hydrochloride; and N-α-(1(8)-6-hydroxycarbonyl-3-phenylpropyl)-L-lysyl-L-proline.