BG60339B2 - L-PROLINE DERIVATIVES AS ANTI-HYPERTENSIVE AGENTS AND PHARMACEUTICAL COMPOSITIONS - Google Patents

Abstract

The new compounds are used in medicine as antihypertensive agents. A group of new carboxyalkyl dipeptides and their derivatives were obtained, pharmaceutical compositions and methods for the treatment of high blood pressure were created. 82 claims

Description

Substituted acyl derivatives of amino acids are known which are useful as inhibitors of angiotensin-converting enzyme (1) and (2). In particular, these compounds are mercapto-substituted acyl amino acids and their derivatives include the clinically effective antihypertensive compound captopril or O-3-mercapto-2-methylpropanoyl-1-proline.

The compounds known from the prior art are not dipeptide derivatives, as are the compounds according to the invention. The compounds known from the prior art contain as an essential substituent a sulfhydryl or a derivative thereof, while those according to the present invention do not contain one. In addition, the dipeptide compounds according to the invention are dipeptides whose N-termini bear a carboxymethyl group, which is preferably further substituted by a methyl group. The carboxyl group(s) can also be converted into an ester, an amide or a salt. In fact, the compounds according to the invention are hybrids formed by the coupling of ./-amino acids with dipeptides by means of a nitrogen which is common to these two partial structures. Such a structural arrangement is rare in the field of synthetic and natural peptides and is not described in the mercaptoacyl type of functions in the two prior art patents mentioned.

β-Carboxyalkanoyl amino acids are known to be useful as inhibitors of anti-pyotensin converting enzyme (3). Since the compounds of the invention are dipeptide derivatives, in a formal sense, they can be considered related to some of the compounds described in the patent reference (3). When one of the methylene groups is replaced by an amino function, the compounds of the invention exhibit high activity. For example, the preferred compounds of the invention can be administered in very low doses of 2.5 mg per day per patient, as opposed to the lowest doses of about 1 mg per kilogram of body weight per day of the most preferred compounds described in the reference (3 ₎ , which corresponds to about 60 mg per day per patient, based on an average patient weight of about 150 pounds.

Essence of the invention

The invention in its broadest aspect relates to carboxylic dipeptides and their derivatives, which are useful as inhibitors of the converting enzyme and as antihypertensive agents. The compounds according to the invention can be included in the following formula

0 II ?1 *h 1 ^{J R5} 0 AND R - C - with - 1 NHCH · - C II - N -C - 1 C - Rg *2 0 *7

in which

R and Rg are the same or different and represent hydroxy, lower alkoxy, lower alkenyloxy, dinitroalkylamino lower alkoxy (dimethylaminoethoxy), acylamino lower alkoxy (acetylaminoethoxy), acyloxy lower alkoxy (pivaloyloxymethoxy), aryloxy, such as phenoxy, arylnitroalkoxy, such as benzyloxy, substituted aryloxy or substituted arylnitroalkoxy, wherein the substituent is methyl, halogen or methoxy, amino, lower alkylamino, dinitroalkylamino, hydroxyamino, arylnitroalkylamino, such as benzylamine;

R.J represents hydrogen, alkyl containing from 1 to 20 carbon atoms, which may have a branched or cyclic chain and includes unsaturated (such as allyl) alkyl groups, substituted lower alkyl, wherein the substituent may be halogen, hydroxy, lower alkoxy, aryloxy, e.g. phenoxy, amino, dinitrialkylamino, acylamino, e.g. acetamido and benzamido, arylamino, guanidino, imidazolyl, indolyl, mercapto, lower alkylthio, arylthio, e.g. phenylthio, carboxy, or carboxamido, carboxynitrialkoxy, aryl, such as phenyl or naphthyl, substituted aryl such as phenyl, wherein the substituent may be lower alkyl, lower alkoxy or halogen, arylnitrialkyl, arylnitrialkenyl, heteroarylnitrialkyl or heteroarylnitrialkenyl, such as benzyl, styryl or indolylethyl, substituted aryl-lower alkyl, substituted aryl-lower alkenyl, substituted heteroaryl-lower alkyl or substituted heteroaryl-lower alkenyl, wherein the substituent(s) may be halogen, dihalogen, lower alkyl, hydroxy, lower alkoxy, amino, aminomethyl, acylamino, acetylamino or benzoylamino, dinitrile alkyl amino, lower alkyl amino, carboxy, haloalkyl, cyano or sulfonamino group, aryl-lower alkyl, heteroaryl-lower alkyl, the alkyl moiety being substituted with amino or acylamino (acetylamino or benzoylamino);

R 1 and R 2 are the same or different and represent hydrogen or lower alkyl;

K ₃ represents hydrogen, lower alkyl, phenyl lower alkyl, aminomethylphenyl lower alkyl, hydroxy lower alkyl, acylamino lower alkyl, such as benzoylamine lower alkyl, acetylamino lower alkyl, acetylamino lower alkyl, amino lower alkyl, dimethylamine lower alkyl, haloalkyl, guanidino lower alkyl, imidazolyl lower alkyl, indolyl lower alkyl, mercapto lower alkyl, lower alkylthio lower alkyl;

represents hydrogen or lower alkyl;

R1 represents hydrogen, lower alkyl, phenyl, phenyl lower alkyl, hydroxyphenyl lower alkyl, hydroxylower alkyl, amino lower alkyl, guanidino lower alkyl, imidazolyl lower alkyl, indolyl lower alkyl, mercapto lower alkyl, lower alkylthio lower alkyl;

^R 4 and R ₅ ^may be linked together to form an alkylene bridge containing 2 to 4 carbon atoms, an alkylene bridge containing 2 to 3 carbon atoms and one sulfur atom, an alkylene bridge containing 3 or 4 carbon atoms containing a double bond or an alkylene bridge substituted as above with hydroxy, lower alkoxy, lower alkyl or di-lower alkyl; and their pharmaceutically acceptable salts.

A lower alkyl or lower alkenyl group, unless otherwise specified, is any of the group consisting of straight or branched chain hydrocarbon radicals containing 1 to 6 carbon atoms, for example methyl, ethyl, propyl, isopropyl, tert-butyl, pentyl, isopentyl, hexyl or vinyl, allyl, butenyl, etc. Aralkyl groups are defined by any of the above containing 1 to 4 carbon atoms in the alkyl portion and include, for example, benzyl, p-methoxy benzyl, etc. Halogen means chlorine, bromine, iodine or fluorine. The term aryl, where it appears unless otherwise specified, means phenyl or naphthyl. Heteroaryl groups include, for example, pyridyl, thienyl, indolyl, benzthienyl, furyl, imidazoyl and thiazolyl.

R1, _R3 and _R5 substituted lower alkyl moieties are illustrated by the following groups of the formulas

H0-CH ₂ -,H5-CH ₂ - ^H ,H ₂ N-/CH ₂ / ₄ -, *CH ₃ -5-/CH ₂ / ₂ -,H ₂ M-/CH ₂ / ₃ -,

NH

H ₂ NC -NH- /CH ₂ / ₃ - and others.

When _R4 and R6 are connected through a carbon atom and a nitrogen atom to which they are attached, they form a four to six membered ring which may contain a sulfur atom or a double bond. Preferred rings have the following formulae in which Y represents a methylene group, a sulfur atom or _CHOCH3 . Preferred are those compounds of the general formula (I) in which R and R6 can independently represent hydroxy, lower alkoxy, lower alkenyloxy, arylnialkoxy, amino, dinialkylamino lower alkoxy, acylamino lower alkoxy, ocyloxy lower alkonyi, the substituent being methyl, halo or n-methoxy;

R ₂ and R 3 represent hydrogen;

_1-3 represents lower alkyl, amino lower alkyl, imidazoyl lower alkyl, halo lower alkyl;

& are linked to form rings preferably as indicated above, in which Y represents _CH2 , sulfur or

CH-OCH ₃ ; 1C has the meanings indicated above.

Even more preferred are compounds of the general formula (I) in which R<| represents alkyl containing from one to eight carbon atoms, substituted lower alkyl, wherein the alkyl group contains one to five carbon atoms and the substituent is amino, alkylthio, arylthio, aryloxy or arylamino, arylalkyl or heteroarylalkyl, the alkyl moiety having one to three carbon atoms, such as phenylethyl or indolylethyl or substituted arylloweralkyl (phenyl lower alkyl or phenyl lower alkyl) and substituted heteroarylloweralkyl, the alkyl group having one to three carbon atoms and the substituent(s) are halogen, two halogen atoms, amino, aminoalkyl, hydroxy, lower alkoxy or lower alkyl. Most preferred compounds of the general formula (I) are those in which

R and Rg represent hydroxy, lower alkoxy, aralkyloxy;

R ₂ and R ₇ represent hydrogen;

R ₃ represents methyl or amino lower alkyl;

R| and Κς are linked through a carbon and nitrogen atom to form proline, 4-thiaproline or 4-methoxyproline;

R^ represents alkyl containing from 1 to 8 carbon atoms, substituted lower alkyl, the alkyl group containing one to five carbon atoms and the substituent is amino, arylthio or aryloxy, arylalkyl or heteroarylalkyl, the alkyl moiety containing one to three carbon atoms, such as phenethyl or indolylethyl or substituted aryl-alkyl (phenyl/lower alkyl or naphthyl lower alkyl) and substituted heteroarylalkyl, the alnyl substituent having one to three carbon atoms and being substituted with one or two substituents such as halogen, dihalogen, amino, aminoalkyl, hydroxy, lower alkoxy or lower alkyl.

Preferred, particularly preferred and most preferred compounds also include their pharmaceutically acceptable salts and are exemplified by the following compounds: N-(1/5)-carboxy-3-phenylpropyl)-L-alanyl-L-proline; N-(1/6)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline and their salts;

N-(1/5)-ethoxycarbonyl-4-methylpentyl(-b)-alanyl-L-proline; b 1 -[1-carboxy-5-aminopentyl b -alanyl-L-proline;

-[1(5)-carboxy-3-phenylpropyl]-L-lysyl-L-proline;

N-Z, (1/5)-ethoxycarboxyl-3-phenylpropyl)-L-proline; N-/ (1/5)-carboxy-3-(3-indolyl)propyl)-L-lysyl-L-proline; N-Z (1/5)-carboxy-3-(4-chlorophenyl)-propyl)-L-lysyl-L-proline;

N-Z (1/6)-carboxy-2-phenylthioethyl)-L-lysyl-L-proline; N-/ -(1/S)-carboxy-3-(4-chlorophenyl)-propyl)-L-lysyl-14)-methoxypropyl;

N-Z (1/5 /-carboxy-5-aminopentyl)-L-lysyl-L-proline: Ethyl N-(1/5 /-ethoxycarbonyl)-3-phenylpropyl)-L-alanyl-L-prolinate hydrochloride and others.

The compounds of formula (I) and the preferred subgroups may be prepared by one or more methods selected from those described below.

0 th ^{R1 R3} 0 II % ^R5 0 p R - C - C -NHC - 1 With - • N - C - 1 C - R _{6 r2} ^7

As shown in the examples, reactive groups not included in the conjugations, such as amino, carboxy, mercapto, etc., can be pre-protected by some of the standard methods used in peptide chemistry before the addition reactions and then released to obtain the desired products.

Method I. Way I /R ₂ means H/

OR, R_ 0 R _£ Re 0

I ¹ I ³ 11 I * I ⁵ ll

R - C - C=0 ♦ H ₂ NC -CNCC-Rg NaBHgCHH /I/ ^R 7 /II/ /III/

A keto acid (or ester, amide or hydroxamic acid derivative) (II) is condensed with a dipeptide (III) in aqueous solution, optimally under near-neutral conditions, or in a suitable organic solvent (e.g. CH ₃ CK) in the presence of sodium cyanoborohydride to give (I) where R ₂ is H. Optionally, the intermediate Schiff base, enamine or aminol can be catalytically reduced to a compound of formula (I) e.g. with hydrogen in the presence of 10% palladium on carbon or Raney nickel. The ratio of diastereomeric compounds produced can be varied by the choice of catalyst.

If R and _R are protected carboxy groups, such as alkoxy or benzyloxy or the like, they can be converted by known methods such as hydrolysis or hydrogenation to a product of formula (I) in which I and/or R are hydroxy. This also applies to all subsequent methods in which a similar situation exists.

In a given case, a compound of formula (II) can condense with an amino acid of formula (II) % 0 R«R ³ - μ 1 ¹ 1

H ₂ NCH-C00H * II PaVN ₃ SC R - C -CHNNCHCOOH /I//Y/ under the same conditions to obtain amino acid Ϊ. Subsequent addition by known methods to an amino acid derivative of formula ΪΙ leads to the preparation of a compound of formula /I/.

Known methods provide reactive groups with protection during coupling reactions, for example with N-formyl, N-tert-butoxycarbonyl and N-carbobenzyloxy groups, followed by their removal to give product I. Furthermore, the substituent R may include ester groups which can be removed, such as benzyl, ethyl or tert-butyl. The coupling agents in this synthetic route are those commonly used in peptide chemistry, such as dicyclohexylcarbodiimide (DCC) or diphenylphosphoryl oside (DPPA), or a compound of formula Y may be activated via intermediate active esters similar to those derived from 1-hydroxybenzotriazole.

/VAT/ /I/ *7 /¥1/ /VAT/ = /DPPA/=

Dicyclohexylcarbodiimide Diphenylphosphoryl azide

Way /¥P/ /¥W/

An amino acid (or ester, amide or hydroxamic acid) ¥11 reacts with a ketone ¥111 under the conditions described above.

Method I for preparing a compound of formula I.

In a given case, the synthesis can be carried out in stages by condensation of a compound of formula ¥11 with a keto acid of formula IX

R, 0 R.R

I II I

YII * 0=C - COOH. R-C-C-NH-CH-COOH

--------------->I ^R 2 /IX//X/ to obtain the amino acid of formula X. By known methods, as indicated above in Experiment /method/ 1, a compound of formula X can be added to the derivative of an amino acid of formula ¥1 to obtain a compound of formula I.

HN /P/ >

n -Ο/I/ case when it carries a Jj -amino substituent, the carbonyl and amino groups can be protected in the form of a β-Lactam function.

Model

2. Way I ^R 1 *1 ^R 3 ^{0 R} 4 ^R S

I ’ ) il < I

-C-COR —* ROCCNHCH-CNC-CORg ^R ?

I

_R2

I

R ₂ /I/ /XI/ iodine or ylyl sulfonyloxy and aryl sulfo /III/

X represents chlorine, bromine, nyloxy. The dipeptide of formula III is alkylated with a suitable X haloacid (in the form of an ester or amide) or with a <Z-sulfonyloxyacid (ester or amide) of formula XI under alkaline conditions in water or in an organic solvent.

In a given case, the synthesis can be carried out in stages. ^R 3 /

H ₂ K1CH-S00H /I/ /P/

I ¹ | ³ I ⁵ Ri R, 0 Ri Rc I ¹ i ³ // I ⁴ P RC0-C-NH-CH-C0H+HN- 1 -C-COR _g ——>RCO-C-NH-CH-C -NC-COR _g is ₂ *7 ^R2

/X/ /VI/ /I/

X represents CH2O, iodine, alkylsulfonyloxy or arylsulfonyloxy.

The amino acid of formula IV is alkylated with a c-halo acid (ester or amide) or with a (/_ _C u _L f _ONYLOXY acid (ester or amide) of formula XI under alkaline conditions to give a compound of formula X. The latter is condensed by known methods, as indicated above in Method I, with the amino acid (ester or amide) of formula I to give a compound of formula I.

Reductive cleavage of the benzyl group from the corresponding ester of formula I, in which R _g represents benzyloxy and R g represents alkoxy, leads to the preparation of compounds of formula I, in which P represents alkoxy and R _g represents hydroxy, and in which R _g represents alkoxy and R is benzyloxy, leads to the preparation of compounds of formula I, in which R represents hydroxy and R _g represents alkoxy.

Way 2 ?1 Ϊ Г5 J1 fs Ϊ

RCC-NH,*XCH-CNC-COR _e ----4RCC-NH-CH-C I ² I ⁶ 11' o r ₂ r ₇ o r ₂

ΪΙΙΙ

XII

X represents chlorine, bromine, iodine, sulfonyloxy or aryl sulfonyloxy.

The amino acid or its derivative with formula ΪΙΙ is

- 13 alkylates with an appropriately substituted 1-haloacetyl or 7-sulfonyloxyacetyl amino acid of formula XII (preferably with Rg other than hydroxy) under alkaline conditions in water or another solvent to give compounds of formula I.

In a given case, the synthesis can be carried out in stages by condensing the amino acid ester of formula ΪΙΙ with a substituted

*3 1 ^J I ^{1 R} 3 1 ³ RCO - with - NH ₂ + X-CH-COOH —A RCO -c - i NH -CH - COOH _{r2 r2} III XIII X *4 ^R5 *1 _R3 0 r5 ₀ 1 I 1 ί ii X + HN- •CH-COR ₆ _—} RC-C- II I NH- -c-c - N-C-C-fb- or ₂ *7 ΪΙ I

£ - chalcoacetic acid or £ -sulfonyloxy acetic acid of formula XIII to obtain the intermediate of formula X. By known methods, described in Method I, a compound of formula X can be reacted with an amino acid of formula ΪΙ or a derivative thereof to obtain a compound of formula I. In the methods described above, an aralkyloxy moiety can be used for one or both of the substituents R and Rg to obtain an ester or diester.

As described above, the protecting groups can be removed by known methods.

and

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

In the compounds of general formula I, the carbon atoms to which Rp, R^ and R^ are attached may be asymmetric. The compounds therefore exist in diastereoisomeric forms or as mixtures thereof. The syntheses described above may employ racemates, enantiomers or diastereoisomers as starting materials. When diastereoisomeric compounds are obtained as a result of the syntheses carried out, the latter may be separated by conventional chromatographic methods or by known fractional crystallization methods.

Although amino acid structures, for example

Ri a ' ¹

R - C - C - NH-

I» of formula 1 are generally preferred in the L - and β -configurations, the diastereoisomers containing D -amino acids have an activity that depends on their structures and have an advantage with respect to metabolic stability ϊπ Ϊ1Υ0 and therefore can be used in a mixture or as pure diastereoisomeric compounds.

The compounds of the invention form salts with various inorganic and organic bases, which are also included 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 or magnesium salts, organic base salts, such as dicyclohexylamine salts, N-methyl-])-glucamine, amino acid salts, such as arginine, lysine, and the like. Salts with inorganic or organic acids, such as hydrochloric, hydrobromic, sulfuric, phosphoric, methanesulfonic, toluenesulfonic, maleic, fumaric, camphorsulfonic, can also be prepared. Non-toxic pharmaceutically acceptable salts are preferred, although other salts may also be useful, for example in the isolation or purification of the products.

The salts can be prepared by known methods by reacting the free base of the product with one or two of a suitable base or acid in a solvent or medium in which the resulting salt is insoluble, or in a solvent such as water, which is then removed by distillation in vacuo or by freezing or by cation exchange of the existing salt with a cation on a suitable cation exchange resin.

The compounds of the invention inhibit the angiotensin converting enzyme and thus block the conversion of the decapentide angiotensin of formula I to angiotensin of formula II, which is a potent stimulatory (blood pressure-increasing) substance. Thus, the lowering of blood pressure may result from the inhibition of its biosynthesis, particularly in animals and humans whose elevated blood pressure is associated with the formation of angiotensin of formula II. Furthermore, the converting enzyme degrades the vasodepressant substance bradykinin. Therefore, inhibitors of angiotensin converting enzyme may also lower blood pressure by potentiating bradykinin. Although the relative importance of these and other possible mechanisms remains to be established, angiotensin converting enzyme inhibitors are effective antihypertensive agents in a variety of animal models and are also useful in clinical settings, for example in many patients with renal vascular, malignant and essential hypertension (4).

The evaluation of the inhibitors of the converting enzyme is carried out by an in vitro enzyme inhibition assay. For example, the method of Y. Piquilloud, A. Reinharz and M. Roth /5/, in which the hydrolysis of carbobenzyloxyphenylalanylhistidyleucine is measured. In vivo evaluation can be carried out, for example, with normotensive rats immunized with angiotensin I according to the method of J. R. Weeks and J. A. Jones /6/ or in rats with increased blood pressure of renal origin, such as those of O. Koletsky /7/.

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

For the treatment of high blood pressure and the clinical conditions mentioned above, the compounds according to the invention can be administered in the form of tablets, capsules or elixirs for oral use, suppositories for rectal use,

- 17 administration, sterile solutions or suspensions for parenteral or intramuscular administration, etc. The compounds of the invention can be administered to patients (animals or humans) in need of such treatment in doses that provide maximum pharmaceutical efficacy. Although the doses will vary depending on the nature and severity of the disease, the weight of the patient, the special diet, concomitant diseases, etc. Factors that the person skilled in the art should take into account, the dose is usually from 1 to 1000 mg per patient per day and can be given once or in several doses. Preferably the dose is from about 2.5 to about 250 mg per patient / day, and even more preferably the dose is from about 2.5 to about 75 mg per patient per day. The compounds of the invention can also be administered in combination with other antihypertensive agents and/or diuretics. For example, the compounds of the invention may be administered in combination with compounds such as amiloride, atenolol, bendroflumethiazide, chlorothalidone, chlorothiazide, chlonidine, cryptenamine acetates and cryptenamine tannates, deserpidine, diazoxide, guanethidine sulfate, hydralazine hydrochloride, hydrochlorothiazide, hydroflumethiazide, metalazone, metoprolol tartrate, methylclothiazide, methyldopa, methyldonate hydrochloride, minoxidil, pergyline hydrochloride, prazosin, propranolol, rauvolfia serpentina, rescinamine, reserpine, sodium nitroprusside, spironolactone, timolol, trichlormethiazide, trimethophan, camsylate, benzthiazide, kinetazone, triamterene, acetazolamide, aminophylline, cyclothiazide, ethacrynic acid, furosomide, meretoxyline procaine, sodium ethacrynate, and the like. as well as mixtures or combinations thereof.

Typically, individual daily doses for these combinations may range from about one-fifth of the minimum recommended clinical dose to the maximum recommended dose for cases where

- 18 are given separately.

To illustrate these combinations, one of the antihypertensive agents according to the invention, clinically effective in doses of 2.5 - 250 mg/day, can be effectively combined in an amount of 0.5-250 mg/day with the following compounds in the indicated daily doses - hydrochlorothiazide /15-200 mg/, chlorothiazide /125-2000 mg/, ethocrynic acid /15-200 mg/, amiloride /5-20 mg/, furosemide /5-80 mg/, propranolol /20-480 mg/, timolol /5-60 mg/ and methyldopa /65-2000 mg/. Triple drug combinations of hydrochlorothiazide (15-200 mg) and amiloride (5-20 mg) and an angiotensin converting enzyme inhibitor of the invention (3-200 mg) or hydrochlorothiazide (15-200 mg) and timolol (5-60 mg) and angiotensin converting enzyme inhibitor of the invention (0.5250 mg) are effective combinations for controlling blood pressure in patients with elevated blood pressure. These dosage ranges can be adjusted on an individual basis to obtain divided daily doses and, as noted above, the dosage depends on the nature and severity of the disease, the weight of the patient, special diets and other factors.

Typically, these combinations can be formulated into pharmaceutical forms as described hereinafter.

About 1 to 100 mg of a compound or mixture of compounds of formula I or their pharmaceutically acceptable salts are mixed with a physiologically acceptable carrier, excipient, binder, preservative, stabilizer, flavoring agent, etc. in a unit dosage form, as is known in conventional pharmaceutical practice. The amount of active ingredient in these compositions or preparations is such as to provide a suitable dosage within the dosage ranges indicated above.

Examples of excipients which may be included in tablets, capsules, etc. are the following substances: a binder, such as tragacanth, corn starch, gelatin; an excipient, such as microcrystalline cellulose; a dispersing agent, such as corn starch, pregelatinized starch, alginic acid, etc.; a lubricant, such as magnesium stearate; a sweetening agent, such as sucrose, lactose or saccharin; and a flavoring agent, such as peppermint, oil of wintergreen or cherry. When the dosage form is formulated in a capsule, it may contain, in addition to the materials listed, a liquid carrier, such as animal brain. Many other materials may also be present as coatings or otherwise incorporated into the final dosage form. For example, tablets may be coated with shellac and/or sugar. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propyl parabens as preservatives, a dye, and cherry or orange essence for flavoring.

Sterile injectable compositions may be prepared by methods known in the art of pharmacy by dissolving or suspending the active ingredient in a vehicle such as water for injection, essential vegetable oils such as sesame oil, coconut oil, peanut oil, cottonseed oil, etc., or synthetic fatty vehicles such as ethyl oleate or the like. Buffers, preservatives, antioxidants and the like may be included as necessary.

The following examples illustrate the invention and represent particularly preferred embodiments. The preferred diastereoisomers are isolated by column chromatography or by fractional crystallization.

Example 1. N-(1-carboxy-2-phenylethyl)-L-alanyl-L-proline.

A mixture of phenylprogross niselin (753 mg) and L-alanyl-L-proline (171 mg) in a mixture of water and methanol with a pH adjusted to 6.8 was 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; H-(1-carboxy-2-phenylethyl)-L-alanyl-L-proline. A portion of this product was purified by gel filtration (L H-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 doublet at 1.5 ppm.

Example 2. L1-(1-carboxyethyl)-L-alanyl-L-proline.

A solution of L-alanyl-L-proline (372 mg) and pyruvic acid (881 mg) in water adjusted to pH 7 was treated with 377 mg of sodium borohydride at room temperature until the reaction was complete. The product was adsorbed onto a strong ion exchange resin in the acidic form and then eluted with a 2X solution of pyridine in water. Freeze drying gave 472 mg of K-(1-carboxyethyl)-L-alanyl-L-proline. The NMR spectrum and mass spectrogram were consistent with the structure. The NMR spectrum showed multiplets centered at 4.5; 3.7 and 22 ppm and a pair of doublets at 1.6 ppm.

- 21 Example 3. N-(carboxy-2-cyclohexylethyl)-L-alanyl-L-proline.

3-Cyclohexyl-2-oxopropionic acid (cyclohexylpyruvic acid) (0.98 mg) and L-alanyl-L-proline (0.22 g) were treated with sodium cyanoborohydride (0.22 g) as described above. A light colorless precipitate of solid M-(1-carboxy-2-cyclohexylethyl)-L-alanyl-L-proline, 0.31 g, was obtained. After purification by chromatography, the mass spectrum showed peaks at 340 (molecular ion), 322, 277, 249, and 226. The NMR spectrum showed complex absorption in the range of 4.8 to 3.6 and peaks at 2.2, 1.7, and 1.2 ppm.

Example 4. b 1 -[1-Carboxy-5-methylhexyl b -alanyl-L proline.

6-Methyl-2-oxohentanoic acid (0.90 g) and L-alanyl-L-proline (0.21 g) were treated with sodium cyanoborohydride (0.21 g) as described above. A white fluffy substance, L1-(1-carboxy-5-methylhexyl)-L-alanyl-L-proline, 0.24 g, was obtained. After purification by chromatography, the mass spectrum showed a peak at 472 (disilyl derivative). The NMR spectrum showed absorption centered at 4.5; 3.65; 2.0, 1.6, 1.3, and 0.85 ppm.

Example 5. N-(1-carboxy-3-methylbutyl)-L-alanyl-L·proline.

4-Methyl-2-oxopentanoic acid /1.29 g/ and L-alanyl-L-proline /0.32 g/ were treated with sodium cyanoborohydride /0.32 g/ as described above. A fluffy white solid, L1-/Ι,-carboxy-3-methylbutyl/-L· -alanyl-L-proline /0.40 g/, was obtained. A portion of this was purified by chromatography. The mass spectrum showed a peak at 429 /molecular ion of disilyl

- 22 derivative minus methyl, 444-15/. The NMR spectrum shows resonances centered at 4.4, 3.6, 2.1, 1.6 and at 0.95 ppm.

Example b. 1H-[1-Carboncypropyl]-L-alanyl-L·-proline.

2-Oxobutyric acid (1.02 g) and L-alanyl-L-proline (0.37 g) were treated with sodium cyanoborohydride (0.38 g) as described above. The crude L-L-carboxypropyl-1-alanyl-L-proline was obtained in a yield of (0.42 g). A portion of it was purified by chromatography for spectral analysis. The mass spectrum showed distinct peaks at 254/M-18/ and 210/M-62/. The NMR spectrum showed a complex absorption of 4.5 to 3.4 multiplet centered at 2.0 and methyl resonance centered at 1.55 and 0.95 ppm.

Example 7. N -[1-Carboxy-2-methylpropyl] - b -alanyl-L proline.

A mixture of 3-methyl-2-oxobutyric acid, as the sodium salt (1.46 g), and L-alanyl-L-proline (0.40 g) were treated with sodium cyanoborohydride (0.41 g) as described above. The crude N-(L-carboxy-2-methylpropyl)-L-alanyl-L-proline was obtained by elution from an ion exchange resin (0.45 g). The product melted at 131-142°C. The NMR spectrum showed a complex absorption in the region from 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)-alanyl-L-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-dinarboxyproline)-L-alanyl-L-proline (0.47 g) was obtained with mp 140-160°C. The mass spectrum of the silylated material showed an ion at 517 m/s, equivalent to the molecular ion for the trisilyl derivative minus the methyl.

- 23 /532-15/. The NMR spectrum confirms the structure. The methyl resonances are centered at 1.4 ppm.

Example 9. N-(1,4-dicarboxybutyl)-b-alanyl-L-proline.

2-Oxoadipic acid (1.74 g) and L alanyl-L -proline (0.41 g) were treated with sodium cyanoborohydride (0.42 g) as described above. Crude P-(1,4-dicarboxybutyl)-L -alanyl-L -proline (0.35 g) was obtained with mp 106-132°C. The highest peak in the mass spectrum was 312, corresponding 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. L1-[L-carboxy-3-methylbutyl]-L-alanyl-L-isoleucine.

A solution of L-alanyl-L-isoleucine (150 mg) and 4-methyl-2-oxopentanoic acid, as the sodium salt (564 mg) in water adjusted to pH 7 was treated with 140 mg of sodium cyanoborohydride at room temperature for several days. The reaction was quenched with a strongly acidic ion exchange resin, added to a column of the same resin and eluted with 2X aqueous pyridine. Freeze drying gave 200 mg (84.9X) of a white powder, N-(Icarboxy-3-methylbutyl)-L-alanyl-L-isoleucine. The mass spectrum showed peaks at 460 for the disilylated product and at 445 for the disilyl molecular ion minus methyl (460-15). The NMR spectrum shows a broad doublet centered at 0.95 ppm, a complex absorption in the range from 1.2 to 1.8 ppm, and a broad weak singlet at 3.7 ppm.

Example 11. N -(1-Naroxy-3-methylbutyl) N -alanyl-L-phenylalanine.

and

- 24 Solution of L -alonyl-L· -phenylalanine /150 mg/ and 4-methyl-

2-Oxypentanoic acid, sodium salt (483 mg) in water adjusted to pH 7 was treated with 120 mg sodium cyanoborohydride at room temperature for several days. The reaction was quenched with Dowex 50 (H+) added to the same resin belt and eluted with 2% aqueous pyridine. Freeze drying gave 197 mg (88.7%) of a white powder, N-(1-carboxy-3-methylbutyl)-L-alanyl-L-phenylalanyl. The mass spectrum showed peaks at 551 for the trisilyl derivative minus methyl (566-15), 479 for the disilyl derivative minus methyl (494-15), and 449 for the trisilyl derivative minus -COOTMS (566-117). The NMR spectrum shows broad doublets at 0.95 and 1.5 ppm, a complex absorption in the range of 2.8-3.4 ppm (weak) and a singlet at 7.1 ppm. The integration is consistent with the structure, giving the correct ratio of aromatic to aliphatic protons.

Example 12. N-Carboxymethyl-L-alinyl-L·-proline.

In a small flask equipped with a pH electrode, combine

1.5 g L-alanyl-L-proline and 1.2 ml 4M sodium hydroxide solution. Add 0.53 g chloroacetic acid in 1.2 ml 2M sodium hydroxide solution. Adjust the pH to 8-9, heat to 85°C and maintain the pH of the solution in the range 8-9 for 15 minutes by adding sodium hydroxide solution if necessary. Add another 0.53 g chloroacetic acid and sodium hydroxide solution if necessary over a period of 15 minutes. Add a third portion of 0.53 g chloroacetic acid and maintain the pH in the range 8-9 for 15 minutes, hold for a further 15 minutes at 85°C and cool.

The reaction mixture was passed through a column of Dowex 50 (I*), washed with water and eluted with 2X aqueous pyridine. The combined functions which showed a positive ninhydrin reaction were concentrated to a small volume in vacuo and freeze-dried.

The resulting material was dissolved in a few milliliters of water and loaded onto a column of Dowex 50 (Na+). It was eluted with 0.5M citric acid, the pH adjusted to 3.3 with sodium hydroxide. The unreacted product appeared first (ninhydrin test), well separated from the unreacted alanylproline. The product fraction was concentrated in vacuo to about 300 g.

This solution was loaded onto a column of Dowex 50 (H+G). Washed with water, then eluted with a 2% solution of pyridine in water. The product fraction was concentrated in vacuo to a small volume and freeze-dried. 417 mg of L-carboxymethyl-L-alanyl-L-proline were obtained.

The NMR spectrum /deuterated water, methanol internal standard/ 1.58 ppm/ 0.1=6/ with a small peak at 1.53 /d,I=6/ /total 3H/, 1.77-2.68 /broad m, 4H/, 3.63 /3/ over 3.28-3.92 /m//total 4H/, 4.05-4.72 /broad m, 2H/ overlapped with the water peak 4.68.

Example 13. M-(1-carboxyethyl)-L-alanyl-L-proline Dissolve 45 g of benzyl pyruvate and 4.5 g of L-alanine in a mixture of water (115 ml) and 250 ml of p-dioxane. Adjust the pH to 5.5 with sodium hydroxide. Add 9.4 g of sodium cyanoborohydride and stir at room temperature for 6 days. Adjust the pH to 1 with concentrated hydrochloric acid.

The solution was loaded onto a column of Dowex 50 (H+) prepared in 50X-dioxane-water. Washed with 50% dioxane in water then with water alone. Elute with 2X pyridine solution in

- 26 water, the product fractions are combined and concentrated in vacuo to dryness. The solid residue is triturated with water, filtered and washed with water. Dry to give 6.8 g of l-/1r-carbobenzoxyethyl/-l-alanine as a mixture of diastereoisomers. A second crop of 1.0 g of solid can be collected from the mother liquor.

Dissolve 208 mg of the product obtained above and 217 mg of benzyl ester of L-proline hydrochloride in dry dimethylformamide. Cool to 0°C. Add 0.193 ml of diphenylphosphoryl azide dissolved in dimethylformamide (DMF). Then add dropwise over 10 min. a solution of 0.24 ml of triethylamine in DMF, maintaining the temperature at 0°C. Stir for 3 hours at 0°C and overnight at room temperature.

The mixture was diluted with ethyl acetate, washed with water and 5X sodium bicarbonate solution. Concentrated in vacuo and chromatographed on a silica gel preparative thin layer chromatography plate, developed with ethyl acetate. A broad band developed at b = 0.5-0.6, eluted with ethyl acetate and the solvent was distilled off to give 212 mg of the mixture of diastereoisomers of N-(1-carbobenzoxyethyl)-L-alanyl-L-proline benzyl ester.

135 mg of the above mixture was dissolved in methanol and water. 50 mg of 10% ladium/carbon was added and hydrogenated at 40 psi hydrogen pressure at room temperature. Filtered, concentrated in vacuo and freeze-dried to give 95 mg of the mixture of diastereoisomers of L1-(1-carboxyethyl)-L-alanyl-L-proline. The NMR spectrum was comparable to that obtained in Example 2 and the mass spectrum of the silylated derivative showed the same fragments.

Example 14. N-(1-Carboxyethyl)-L-alanyl-L-proline.

Dissolve 0.75 g of L-[1-carbobenzoxyethyl]-L-alanine in pyridine and add 7.5 ml of a 1 M solution of triethylamine in pyridine. Cool and add 1.09 g of L-proline as benzyl ester hydrochloride and 0.678 g of dicyclohexylcarbodiimide. Store at 0°C for 20 hours. Filter, then concentrate in vacuo. Dissolve the residue in ethyl acetate and wash this solution with saturated potassium carbonate solution, then with saline. The organic phase is dried, concentrated in vacuo, then the residue obtained is chromatographed on silica gel and ethyl acetate-hexane is used to isolate the diastereoisomeric mixture of L-[1-carbobenzoxyethyl]-L-alanine-L-proline.

Hydrogenation in the usual manner with 10% palladium/carbon in aqueous ethanol and after isolation and freeze-drying work-up afforded N-(1-carboxyethyl)-L-alanyl-L-proline as a white solid.

NMR spectrum in deuterated water - 1.65 ppm/d,6H/, 1.9-

2.6 /M,4H/, 3.5 - 4.2 /M,3H/, 4.3-4-8 /M,2H/.

Example 15. L1-(1-Carbomethoxyethyl)-L-alanyl-L-proline. A solution of 1.4 g of methyl L-alaninate hydrochloride and 3.1 g of p-bromopropionic acid in a mixture of dioxane and water was neutralized to pH 9 with sodium hydroxide. The mixture was heated to a temperature of 70°C and maintained for 30 min., and the pH of the medium was maintained in the range of 8 to 9, if necessary by adding sodium chloride. After cooling, the mixture was loaded onto a Dowex 50 (H+) I column with ion exchange resin, washed with water, and eluted with a 2X aqueous solution of pyridine.

- 28 Combine the product fractions and freeze-dry. This crude product is purified by chromatography on an ion exchange resin loaded in Dowex 50 (Na+) in 0.5 M sodium citrate buffer solution at pH 3.3. The product fractions are collected and concentrated in vacuo to a small volume, then rechromatographed on Dowex 50 (H+). The product fraction is freeze-dried to give pure K-[b-carbomethoxyethyl]-alanine.

The intermediate was reacted with butyl ester of L-proline using diphenylphosphoryl azide as described in Example 13. The tert-butyl ester group was removed by dissolving in trifluoroacetic acid at room temperature for 3 hours, tetrafluoroacetic acid was distilled off and the mixture was purified on a Dowex 50 (H+) column and after elution with 2% aqueous pyridine as described, N-(1-napromethoxyethyl)-L-alanyl-L-proline was obtained as a mixture of diastereoisomers.

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

A solution of niruvoyl-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 standing at room temperature for 3 days, the product was adsorbed onto a strongly acidic ion exchange resin and eluted with 2% aqueous pyridine to give 80 mg of product. The NMR spectrum showed a methoxy group at 3.95 δ, a thiomethyl group at 2.2 δ and CH-CH- at 1.55 and 1.7 δ'. The mass spectrometry program of the silylated

W

- 29 material shows the expected molecular ion at 404 m/e.

Example 17. 1\1-[1\5\-carboxy-3-methylthiopropyl]-alanyl\-proline.

A solution of N-(1/6)-methoxycarbonyl-(-3-methylthiopropyl)-DL-alanyl-L·-proline (27.5 mg; 0.384 mM) in 2 mL of water was treated under nitrogen with 7.82 mL of 0.100 I sodium chloride (0.782 mM) and stirred for two and a half hours at room temperature. The product was absorbed from the reaction mixture onto 30 mL of Dowex 50 (H+) and eluted with 4% aqueous pyridine to give 73.5 mg of product, which was further purified on a Ij H-20 column to give 55.7 mg of product. The NMR spectrum in deuterated water showed thiomethyl at 2.1; CH-CH ₃ at 1.5 and 1.6 P and no methyl ester. The mass spectrogram of the silylated material shows the expected molecular ion at 462 m/e.

Example 18. N-(N-methoxycarbonyl-2-(3-indolyl)-ethyl)alanyl-L-proline.

The procedure is as described in Example 16, whereby the methyl ester of tryptophon is condensed with pivaloyl-L-proline in the presence of sodium cyanoborohydride to give N-(1'-methoxycarbonyl-2-(3-indolyl)-ethyl)-alanyl-1'-proline.

The NMR spectrum in deuterated chloroform shows aromatic protons at 6.9 to 7.7; protons adjacent to the aromatic nuclei and adjacent to the nitrogen 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 material shows an ion at 516 m/e consistent with a disilylated material that has lost a methyl group.

Example 19. N-(1(5)-carboxy-2-(3-indolyl)-ethyl)-O-olanyl-L-proline.

- 30 The procedure was as described in Example 17, hydrolyzing the product to give the expected diacid. The D2O-c^PYR NMR spectrum showed five aromatic protons at 6.8 to 7.7; 7 protons adjacent to the aromatic nucleus and adjacent to the nitrogen at 2.8 to 7.4 and 7 aliphatic protons at 1.0 to 2.2 in accordance with the expected structure. The mass spectrogram of the silylated material showed a peak at 431 m/e, interpreted as a protonated minisilylated ion that had lost a methyl group.

Example 20. N-(1-Carboxy-3-phenylpropyl)-b-alanyl-L-thiazolidine narcarboxylic acid.

Tert-Boc-alanine /1.8 g/ and L-thiadolidine-4-carboxylic acid in the form of benzyl ester, hydrochloride /2.6 g/ in methylene chloride are mixed. At a temperature of 0-5°C it is treated with triethylamine /1.4 ml/, then dicyclohexylcarbodiimide /DCC/ /2.3 g/ in methylene chloride, then it is left to stand overnight. After filtering and washing the filter with water and sodium bicarbonate solution, the solvent is distilled and the product is chromatographed with silica gel 6-60 /E.Merck/ in ethyl acetate-hexane. The solvent of the collected fractions containing the product is distilled in vacuo. The benzyl ester is hydrolyzed in acetonitrile - water at pH 13.5 /sodium hydroxide/ for one hour at room temperature. The medium was neutralized to pH 8 with hydrochloric acid, washed with ether, the aqueous layer was concentrated in vacuo and then freeze-dried. The tert-butoxycarbonyl protecting group was removed in a 4 M solution of hydrogen chloride in ethyl acetate. The product was precipitated with ether, filtered and dried to give L-alanyl-L-thiazolidine-4-carboxylic acid. 0.385 g of this product was condensed with 1.88 g of 2-oxo-4-phenylbutyrate.

- 31 acid in water using 0.354 grams of sodium cyanoborohydride according to the method described in Example 24 to obtain 0.53 g of the mixture of diastereoisomers of N-L-carboxy-3-phenylpropyl)-3-alanyl-L-4-thiazolidine carboxylate. The NMR spectrum in deuterated water and deuterated sodium hydroxide contains a doublet at 1.2 ppm (3H), a singlet at 7.1 (5H), a broad absorption in the range from 1.6 to 2.0 (2H), a broad absorption in the range from 2.2 to 4.1 and a broad water peak at 4.6 ppm. The mass spectrum of the silylated product shows the molecular ion of the disilylated derivative at m/e=556.

Example 21. N-[b]carboxy-3-phenylpropyl[b]-alanyl-L-pipecolic acid.

By replacing the thiazolidine carboxylic ester of Example 20 with L-pipecolic acid, methyl ester, hydrochloride (1.8 g) the title compound can be obtained by applying the method described in Example 20.

The NMR spectrum in deuterated methanol shows a broad multiplet at 1.3 to 1.9 ppm (9H8), a singlet at 7.22 (5H), and a series of multiplets in the region of 2.0-4.8 ppm. The mass spectrum of the silylated material shows a peak at m/e=580 for the disilylated molecular ion.

Example 22. N -(1-carboxy-3-phenylpropyl) - b -alanyl-L N -methyl-alanine.

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

Example 23. N-(1-Narboxy-1-methylethyl)-L-alanyl-L-proline.

- 32 The title compound can be obtained by mixing 7.7 g of 2-bromoisobutyric acid, benzyl ester, 2.4 g of L -alanyl-L -proline, tert. butyl ester, 7.0 g of silver oxide in 40 ml of benzene. After refluxing for 24 hours, an additional 7.7 g of the bromoester and 7.0 g of silver oxide are added and refluxing is continued for another 24 hours. Cooling, filtration, distillation of the solvent and the diester of the product are isolated by conventional chromatographic methods. After cleavage of the tert. butyl ester group in trifluoroacetic acid and of the benzyl group by catalytic hydrogenation by conventional methods, the desired free acid is obtained.

Example 24. N -[1-Carboxy-3-phenylpropyl] - b -alanyl-L proline.

A mixture of 4-phenyl-2-oxobutyric acid (1.49 g) and L alanyl-L -proline (0.31 g) in water adjusted to pH 7.5 with sodium hydroxide was treated with sodium cyanoborohydride (0.32 g) overnight. The product was absorbed onto a strongly acidic ion exchange resin and eluted with a 2% solution of pyridine in water to give 0.36 g of the crude diastereomeric product, 1H-[1H-carboxy-3-phenylpropyl]-alanyl-L-proline. A portion of this was purified by gel filtration (L H-20) for spectrographic analysis. The NMR spectrum in dimethyl sulfoxide shows an aromatic hydrogen at 7.20, a broad singlet at 4.30, a broad multiplet in the range 3.0 to 3.9, 2.67 and 1.94, and a doublet at 1.23 and at 1.15. The mass spectrum shows a molecular ion at 492 m/e for the ditrimethylsilylated materials.

Example 25. N-[1-carboxy-3-phenylpropyl]-L-alanyl-L-proline.

- 33 Grind and sieve XAD-2 polyester resin (Rohm and

Haas Co.), The 200-400 mesh fraction was separated and 440 ml was loaded onto a chromatography column. Equilibrated with 0.1 M ammonium hydroxide and a 95 to 5 v/v water-methanol mixture. The column was loaded with 350 mg of N-[1-carboxy-3-phenylpropyl]L-alanyl-L-proline, prepared as described in Example 24, dissolved in 10 ml of the same solvent. The first isomer left the column in the eluate volume range of 375-400 ml. The second isomer in the eluate range of 440-480 ml with an intermediate fraction containing a mixture of isomers. After freeze-drying the fraction containing the first isomer, 130 mg of a white solid was obtained. After recrystallization from 1 ml of water adjusted to pH 3, 94 mg of white needle-like crystals were obtained, mp 148-151°C. This is the most active isomer and has the $,$, 3 configuration, as determined by X-ray analysis ΛΖ/ _α = -67.0° /0.1 M HCl/ after drying in vacuo over diphosphorus pentaoxide. The NMR spectrum in dimethylsulfoxide showed a single doublet for the methyl protons at 1.22 ppm. Freeze-drying of the fraction containing the second isomer gave 122 mg of a white solid. Recrystallization of 103 mg of this from 2.5 ml of water adjusted to pH 3 gave 64 mg of light white crystals, mp 148-151°C. 140-145°C d.,/c//d = -101.6° (0.1 M HCl) after drying. NMR spectrum in dimethylsulfoxide shows the methyl doublet at 1.17 ppm.

Example 26. [1-(5-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline.

Dissolve ethyl-2-oxy-4-phenylbutyrate (1.03 g) and L-alanyl-L-proline (0.19 g) in a 1 to 1 ethanol/water mixture. Stir the resulting solution for 2 hours at room temperature.

- 34 A solution of sodium cyanoborohydride (0.19 g) in ethanol-water was added dropwise. When the reaction was complete, the product was absorbed onto a strongly acidic ion exchange resin and eluted with 2% aqueous pyridine. The francium-rich product was freeze-dried to give 0.25 g of crude N-[2-(2,3-ethoxycarbonyl-3-phenylpropyl)-[beta]-alanyl-L-proline. The mass spectrum showed a molecular ion at 448 m/e for the monosilylated material. Chromatography provided the desired isomer.

Example 27. [1-[1-aminocarbonyl-3-phenylpropyl]-L-alanylL·-proline.

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

Example 28. N-[1H-Carboxy-3-phenylpropyl]-L-alinyl-L-tryptophan.

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

Example 29. N-(1-carboxy-3-phenylpropyl)-L-alanyl-L-4-hydroxy-proline.

By the method described in Example 24, 2-oxo-4-phenylbutyric acid and L-alanyl-4-hydroxyproline are condensed in the presence of cyanoborohydride to give the L1-(1-carboxy)-

3-Phenyl-prolyl/-b-alanyl-L-4-hydroxylroline.

The NMR spectrum in deuterated methanol shows a doublet centered at 1.53 ppm (3H), a singlet at 7.13 (5H), a series of multiplets in the range from 2.0 to 4.7 ppm. The mass spectrum of the silylated product shows a molecular ion of the trisilyliron product at m/e=580.

Example 30. ΓΊ-/Ι-κορ6οκ with η-3-phenylpropyl/L· -serinyl-L proline.

By the method described in Example 24, 2-oxo-4-phenylbutyric acid and L-serinyl-L-proline were condensed in the presence of sodium cyanoborohydride to give L1-(1-carboxy-3-phenylpropyl)-L-serinyl-L-proline.

The mass spectrum shows a molecular ion at 580 m/s for the trisilylated species. The NMR spectrum in deuterated water confirms the structure.

Example 31. N-[1-carboxy-3-phenylpropyl]-L-phenylalanyl-L-proline.

By the method described in Example 24, 2-oxo-4-phenylbutyric acid and L-phenylalanyl-L-proline were condensed in the presence of sodium cyanoborohydride to give N1-[1-carboxy-3-phenylpropyl-L-proline.

The mass spectrum shows a single ion at 406 m/e for the molecular ion minus water /424-18/. The NMR spectrum in deuterated water is consistent with the structure.

Example 32. L1-(1-carboxy-3-phenylpropyl)-L-cysteinyl-L proline.

By the method described in Example 24, 2-oxo-4-phenylbutyric acid can be condensed with L-5-benzylcysteinyl-L-proline in the presence of sodium cyanoborohydride. The product can then be treated with sodium in liquid ammonia to give L1-(1-carboxy-3-phenylpropyl)-L-cysteinyl-L-proline.

Example 33. N-(1-carboxy-3-phenylpropyl)-L-histidinyl-36L-leucine.

By the method described in Example 24, one can condense

2-oxo-4-phenylbutyric acid with L-histidinyl-L· -leucine in the presence of sodium cyanoborohydride to obtain

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

Example 34. N-(1-carboxy-3-phenylpropyl)-b-phenylalanyl-L-arginine.

By the method described in Example 24, one can condense

2-oxo-4-phenylbutyric acid with L-phenylalanyl-L-arginine in the presence of sodium cyanoborohydride to obtain N-(1-carboxy-3-phenylpropyl)-L-phenylalanyl-L-arginine.

Example 35. N-[1<-carboxy-3-phenylpropyl]-b-phenylalanyl-L-tryptophan.

By the method described in Example 24, one can condense

2-oxo-4-phenylbutyric acid with L· -phenylalanyl-L-tryptophan in the presence of sodium cyanoborohydride to obtain N-(1-carboxy-3-phenylpropyl)-L-phenylalanyl-L-tryptophan.

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

By the method described in Example 24, 4-(3-indolyl)-2-oxo-butyric acid and L-alanyl-L-proline were condensed in the presence of sodium cyanoborohydride to give N-(Icarboxy-3-(3-indolyl)propyl)-alanyl-L-proline. The mass spectrum of the trisilylated material showed a molecular ion at 603 m/e and an M♦-15 peak at 588 m/e.

Example 37. L1-(1-carboxy-3-phenylpropyl)-olanyl-3,4-dihydroproline. A mixture of 3,4-dihydroproline (2.3 g), tert. Boc-L-alanine, N-hydroxysuccinimide ester (7.2 g) and sodium carbonate (2.5 g) was stirred in a dioxane-water mixture at

- 37 temperature 0°C overnight. The pH of the medium was neutralized to 8 with hydrochloric acid. It was concentrated to a small volume in vacuo and freeze-dried. The tert. Boc-protecting group was cleaved with trifluoroacetic acid in the usual manner and the product was chromatographed eluting with 2N aqueous pyridine using Dowex 50 (H+) by the method described in Example 2. The dipeptide was isolated by freeze-drying. The resulting product was reacted with 2-keto-4-phenylbutyric acid by the method 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 species.

The diastereomeric product (140 mg) obtained above was separated into components by chromatography on XAD-2 resin as described in Example 25. The major component (70 mg) which left the column first was with Ζα (c=1.3 methanol). The mass spectrum of each component showed a molecular ion at 490 m/s for the ditrimethylsilylated species.

Example 38. N-(1-Carboxy-3-phenylpropyl)-1-galanyl-2-methyl-thiozolidin-4-carboxylic acid.

This compound can be prepared by the method described in Example 37, replacing (2.3 g) 3,4-dihydroproline with 2.9 g

2-methyl-thiazolidine-4-carboxylic acid.

Example 39. b1-[1-Carboxy-3-phenylpropyl]-1'-alanyl-L-methylalanine.

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

Example 40. Dry-filled capsules containing 50 mg

- 38 active ingredient.

Each capsule (size 1) of 200 mg contains the following components, in mg: 1M-(1-carboxy-2-phenylethyl)-L-alanyl-L· proline 50; lactose 149; Magnesium stearate 1.

Grind the N-[1,2-carboxy-2-phenylethyl]-L-alanyl-L-proline to a particle size of 60 mesh and then sieve the lactose and magnesium stearate over the powdered active ingredient. The ingredients are mixed for 10 minutes and then filled into a dry gelatin capsule size of 1 mesh.

Example 41. T[-[1'-ethoxycarbonyl-3-phenylpropyl]-b-alanyl L-proln.

A solution of L-alanyl-L-proline (7.7 g) and ethyl-2-oxo-4-phenylbutyrate (42.6 g) in 140 ml of ethanol was stirred with 64 g of powdered molecular sieve at room temperature for half an hour. Then, a solution of sodium cyanoborohydride (2.6 g) in 40 ml of ethanol was added slowly over 6 hours. After filtering the sieve, the reaction mixture was concentrated in vacuo to a small volume. The resulting 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 fortified 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 diastereomers. High-performance liquid chromatography (HPLC) showed that the main product was the desired -/1-(3)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline.

The NMR spectrum shows aromatic absorption at 7.1 δ and diastereomeric methyls as a multiplet centered at 1.3 δ.

The mass spectrometer shows a molecular ion at 376 m/e and a strong

- 39 peak at 358 m/e /M ♦ -HgO/.

Example 42. N-(1-(5)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L·-proline, maleate salt.

A solution of N-(1-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline, a mixture of isomers, (13.8 g) in 69 ml of acetonitrile was treated with 4.25 g of maleic acid in 69 ml of acetonitrile. After stirring for 1 hour at room temperature, the solid was filtered, washed with acetonitrile and dried to give 8.4 g of the maleate salt, mp 141-145°C. High performance liquid chromatography (HPLC) showed the product to be 96% pure. The crude maleate salt was recrystallized from acetonitrile to give 7.1 g of N-(1/5)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline, maleate, mp 148-150°C, 99% pure (HPLC).

Example 43.

A. N-[1-ethoxycarbonyl-3-phenylpropyl]-L-alanyl-L-proline.

A mixture of 0.814 g L-alanyl-L-proline, 0.206 g ethyl 2-oxo-

4-Phenylbutyrate and 1.6 g molecular sieve in 10 ml ethanol were hydrogenated at room temperature at 40 psi with 0.1 g of 10% palladium/carbon as catalyst. After stopping the flow of hydrogen, the crude product was obtained by filtration and concentration, then absorbed onto an ion exchange resin (Dowex 50, H+) and eluted with a 2% solution of pyridine in water to give 0.224 g of N-(1'-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline. HPLC showed that the isomer ratio was 55 to 45.

B. N-(1-ethoxycarbonyl-3-phenylpropyl)-E-alanyl-L proline, maleic acid salt.

- 40 A mixture of 3 g L-alanyl-L-proline, 5 g ethyl-2-oxo-4phenyl-butanoate, 13 g 3A molecular sieve and 3.6 g Raney nickel in 85 ml ethanol was hydrogenated at 25°C and 40 psi of hydrogen until hydrogen uptake ceased. The solids were filtered, washed with 80 ml ethanol and the filtrates were combined. HPLC analysis showed that the diastereoisomers ratio in the mixture was 87 to 13 in favor of the desired product. The ethanol was removed in vacuo to give an oil which was dissolved in 60 ml water and 20 ml ethyl acetate. The pH of the resulting biphasic mixture was adjusted to 1 with stirring.

8.6 with 50% sodium hydroxide. The layers were separated and the aqueous phase was extracted with twice 20 ml of ethyl acetate. The pH of the aqueous phase was adjusted to 4.25 with hydrochloric acid, 12 g of sodium chloride dissolved in water were added and the product was extracted with five times 12 ml of ethyl acetate. The extracts were combined and dried over sodium sulfate. The crude product δ1-(1/.57-ethoxycarbonyl-3-phenylpropyl)-1>-alanyl-L-proline was preacetylated as its salt with maleic acid by adding 1.86 g of the acid. After stirring for 4 hours the salt was filtered, washed with ethyl acetate and after drying 5.2 g of pure product were obtained, mp 150-151°C.

Example 44. L1-(1-benzyloxycarbonyl-3-phenylpropyl)-L-alanyl-L·-proline.

A solution of L-alanyl-L-proline (167 g) and benzyl 2-exo-4-phenylbutyrate (1.20 g) in 5 ml of ethanol was stirred at room temperature with 3 g of powdered molecular sieve, type 4A. Sodium cyanoborohydride (75 mg) was then added portionwise over 3 hours. The product was purified by absorption.

- 41 on a strong cation exchange resin and eluted with a 2X solution of pyridine in water. After passing through a gel filtration column (B H-20), 220 mg of N-N-benzyloxycarbonyl-3-phenylpropyl-L-alanyl-L-proline were obtained as a mixture of isomers. Thin layer chromatography on silica gel eluting with 1 ethyl acetate, 1 n-butanol, 1 water, 1 acetic acid showed one major spot at R _F 0.71. The isomers were separated using reverse phase HPLC to give N-N-N-benzyloxycarbonyl-3-phenylpropyl-L-alanyl-L-proline.

In an analogous manner, L-acetylaminoethyl-2-oxo-4-phenylbutyrate and L-alanyl-L-proline are reduced with sodium cyanoborohydride to give N-(1-(2-acetylamino)ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline.

Similarly, dimethylaminoethyl 2-oxo-4-phenylbutyrate and L-alanyl-L-proline give N-(1-(2-dimethylamino)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline.

In a similar manner, benzyl 2-oxo-5-methylhexanoate and L-alanyl-L-proline gave N-(1-benzyloxocarbonyl-4-methylpentyl)-L-alanyl-L-proline.

The mass spectrum of the silylated material shows peaks at 510 (M ⁺ , very weak), 495 (M ⁺ -15), 464, 420, 419, 385 and 296 m/s (major peak).

The NMR spectrum is consistent with the structure.

Example 45. [1-Butoxycarbonyl-3-phenylpropyl]-L-alanyl-L-proline.

A solution of 3-benzyloxycarbonyl-L-alanyl-L-proline butyl ester (452 mg) in 5 ml of benzene was hydrogenated over 150 mg of ηθ-ene palladium/carbon until the nitrogen protecting group was removed. After filtration and removal of the solvent, the L-alanyl-L-proline 3° butyl ester was dissolved in 8 ml of tetrahydrofuran.

- 42 and treated with 1.41 g of butyl 2-oxo-4-phenylbutyrate and 3 g of powdered molecular sieve. Sodium cyanoborohydride (150 mg) was added portionwise over several hours, after which the mixture was stirred at room temperature overnight. After filtration and concentration in vacuo, the residue obtained was treated with 25 ml of trifluoroacetic acid at room temperature for 2 hours. After removal of the acid, the product was purified by absorption on an ion exchange resin and by gel filtration (L H-20). After concentration and drying of the fractions containing the product, 182 mg of L1-(1-butoxycarbonyl-3-phenylpropyl)-L -alanyl-L -proline were obtained in the form of a mixture of isomers. Thin layer chromatography (silica gel, 1 ethyl acetate, 1 butanol, 1 water, 1 acetic acid) showed two spots, R _F 0.67 and 0.72. The mass spectrum showed peaks at 548 (disilylated molecular ion) and at 476 (monosilylated molecular ion). The isomers were separated using reverse phase high performance liquid chromatography to give t-(1/6)-butoxycarbonyl-3-(phenylpropyl)-alanyl-L-proline.

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

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

Example 47. L1-(1-carboxy-3-phenylpropyl)-L-alanyltrans-4-methoxy-L-proline.

Methyl trans-4-methoxy-L-prolinate hydrochloride is prepared from L-hydroxyproline by the method of E.A.C.A.M. et al. 1.Biol.Chem. 208,573 /1954/, by esterification with methanolic hydrochloric acid in the known manner. This is followed by reaction with Boc-L-alanine in methylene chloride with dicyclohexylcarbodiimide in the manner described above, purification of the intermediate Boc-L-alanyl-L-methoxy Pzo-OMe by chromatography on silica gel eluting with a mixture of ethyl acetate and hexane 1/1. Hydrolysis of the ester with sodium hydroxide in acetonitrile-water, adjustment of pH to 7.5, freeze-drying, removal of the amine protecting group in 4M hydrochloric acid in ethyl acetate in the known manner. Condensation of 0.54 g of this L-alanyl-L-TpaHC-4-MeTOKCH-L-proline with 2.0 g of 2-oxo-4-phenylbutyric acid in 6 mm of water using 0.43 g of sodium cyanoborohydride by the method described in Example 24. Isolation of the product described in the same example to obtain 0.92 g of a mixture of diastereoisomers of N-(L-carboxy-3-phenylpropyl)-L-alanyl-trans-4-L-proline.

The NMR spectrum in deuterated water shows a doublet centered at 1.58 ppm/3H/, singlets at 3.37 /3H/ and 7.35 ppm /5H/, a nomplex 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 m/e=360 /M-18/ and at 256 /M-122/.

Example 48. [1-(1-benzyloxycarbonyl-3-phenylpropyl)-L-alanyl-1-trans-4-methoxy-L-proline.

By adding L-alanyl-L-4-methoxy-cyproline,

- 44 obtained in Example 47, with benzyl-2-oxo-4-phenylbutyrate in ethanol using sodium cyanoborohydride according to the method described in Example 44, can be obtained a mixture of diastereoisomers of L1-(1'-benzyloxycarbonyl-3-phenylpropyl)-L-alanyl-trans-4-methoxy-L-proline. The isomers can be separated using reverse phase HPLC to obtain N-(I/S)benzyloxycarbonyl-3-phenylpropyl)-L-alanyl-trans-4-methoxy-L-proline.

Example 49. N -[1H-Benzylaminocarbonyl-3-phenylpropyl]-L-alanyl-L-proyline.

The benzylamide of 2-oxo-4-phenyl-butyric acid was prepared by dissolving 3.0 g of the acid, 2.4 ml of benzylamine and 4.7 ml of diphenylphosphoryl azide in 60 ml of cold dimethylformamide and adding 2.6 ml of triethylamine in dimethylformamide dropwise, the temperature being maintained at about -10°C for two and a half hours. After standing at room temperature overnight, the dimethyl formamide 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 in a ratio of 1 to 4. From the fractions containing the product, the solvent was evaporated to give 2.2 g of crystalline n-benzyl-2-oxo-4-phenylbutyramide. 1.26 g of this product was reacted with 0.19 g of L-alanyl-L-proline using 0.125 g of sodium cyanoborohydride in ethanol according to the method described in Example 44. The crude product was purified by gel filtration (L H-20) to give a mixture of diastereoisomers of N-(1-benzyl-aminocarbonyl)-3-phenylpropyl)-L-alanyl-L-proline. The NMR spectrum in deuterated chloroform showed a doublet at 1.1 ppm (3H), a close pair of singlets at 7.3 (10H).

- 45 and complex absorption in the region of 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 shows prominent peaks at m/e ~ 509 (monosilylated derivative) and at 581 (disilylated derivative).

Example 50. N-[N]-Carboxy-3-phenylpropyl-L-L-alanyl-L-N-methylphenylalanine.

By replacing the thiazolidine carboxylic ester from example

20, with M-methyl-L-phenylalanine, methyl ester, is obtained

L-Alanine-N-methyl-L-phenylalanine. By condensing 0.85 g of this product with 0.32 g of 2-oxo-4-phenylbutyric acid using 0.64 g of sodium cyanoborohydride in water as described above, acidifying the mixture to pH 1.5 and extracting with ester, after distillation of the ether, dissolving the residue in 70% methanol in water and chromatographing on Dowex 50 (H+) ⁸ the same solvent and eluting with a 3X solution of pyridine in the same solvent mixture, collecting the fractions containing the product, concentrating, freeze-drying. After purification on L H-20 in methanol, 0.54 g of the mixture of isomers of N-(1-carboxy-3-phenylpropyl)-L-alanyl-L-N-methylphenylalanine is obtained. The NMR spectrum (deuterated water, deuterated sodium hydroxide) shows a doublet centered at 1.18 ppm/3H, two overlapping singlets at 7.3 (10H), a singlet at 2.95 (3H), and a broad multiplet absorption in the ranges 1.4-2.1 and 2.3-4.4. The mass spectrum of the silylated material shows a molecular ion of the disilylated material at m/e = 556.

Example 51. L1-(1-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline amide.

L-alanyl-L-proline amide is obtained by addition

- 46 no tert.-Boc-L-alanine with L-proline amide by known methods using dicyclohexylcarbodiimide in a mixture of 4 to 1 methylene and chloroform to dimethylformamide. Purification of the intermediate tert. Boc-L-alanyl-L-proline amide by chromatography in L H-20 in methanol, then removal of the tert. Boc protecting group in a 4 M solution of hydrochloric acid in ethyl acetate. Coupling of 0.5 g of the tert. L-alanyl-L-proline amide hydrochloride in 10 ml of absolute ethanol, neutralized with one equivalent of triethylamine, with 2.4 g of ethyl-2-oxo-4-phenylbutyrate using molecular sieves and 0.30 g of sodium cyanoborohydride in the manner described in Example 41. In this example, the product was determined in the chloroform extract at pH 8.5, concentrated in vacuo, the residue dissolved in 50X ethanol/water, chromatographed on Dowex 50 (H+) filled with 50X ethanol/water and eluted with 2% pyridine in the same solvent. The fractions containing the product were pooled and further purified by chromatography on L H-20 in methanol. The solvent was distilled in vacuo to give 0.40 g of N-(1-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline amide as a mixture of diastereoisomers. The NMR spectrum in deuterated chloroform shows a triplet overlapping a doublet at 1.1-1.5 ppm (6H), a series of five multiplets in the region of 1.5-4.7 ppm (15H) and a singlet at 7.17 ppm (5H). The mass spectrum of the silyliron product shows prominent peaks at m/e=477 (monosilylated production) and at 519 (disilylated production).

Example 52. N-(1'-carboxy-3-phenylpropyl)-L-alanyl-L-proline amide.

L-alanyl-L-proline amide, obtained in Example 51, was reacted with 2-oxo-4-phenylbutyric acid using sodium cyanoborohydride in a 50% mixture of ethanol and water according to the method described in Example 2. After elution from an ion exchange resin, concentration in vacuo to a small volume, pouring in water and freeze-drying, 1H-[1-carboxy-3-phenylpropyl]L-alanyl-L-proline amide was obtained as a mixture of diastereoisomers.

The NMR spectrum shows an aromatic absorption at 7.35 λ and a pair of doublets for the diastereomeric methyls at 1.55 (P.

Example 53. N-[1-[6]]hydroxyaminocarbonyl-(3-phenylpropyl)-L-alanyl-L-proline.

To a cold solution of 0.19 g of L-[1-ethoxycarbonyl-3-phenylpropyl]-L-alanyl-L-proline, maleate salt, obtained in Example 42, in 1 ml of ethanol, was added 0.85 g of potassium hydroxide in 0.57 ml of ethanol. 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. The mixture was kept in an ice bath for two hours, then at room temperature overnight. The supernatant was decanted, diluted with 10 ml of water, the pH was adjusted to 2.5 with hydrochloric acid and washed with chloroform. After neutralization, freeze-drying of the aqueous layer and purification by chromatography on XAD-2 resin with a gradient of 0.1 M ammonium hydroxydeethanol, L1-[1-X$]-hydroxyaminocarbonyl-3-phenylpropyl]-L-alanyl-L-proline was obtained. The mass spectrum of the silylated product showed an ion at m/e=579 for the trisilylation reaction and the NMR spectrum was consistent with the structure.

Example 54. N-(1-Carboxy-3-methylbutyl)-(1)-alanyl-L· tryptophan.

Sodium salt solution of 4-methyl-3-oxo-pentanoene

- 48 acid (414 mg) and L-alanyl-L-tryptophan (150 mg) in water adjusted to pH 7 with sodium hydroxide were treated with sodium cyanoborohydride (103 mg) at room temperature for several days. The product was adsorbed onto a strongly acidic ion exchange resin and eluted with a 2% solution of pyridine in water. The product-rich fractions were pooled and freeze-dried to give 189 mg of a slightly white solid. The mass spectrum showed a molecular ion at 389 m/s and peaks at 187 m/s and 158 m/s for the following fragments:

sn _? sn

I 1

H00C-CH-IIH-CH

The NMR spectrum in deuterated water confirmed the structure.

Example 55. 1N-(1-carboxy-3-methylbutyl)-L-histidyl-L-leucine.

By the method described in Example 54, 4-methyl-2-oxopyranoic acid and L-histidyl-L-leucine are condensed in the presence of sodium cyanoborohydride to give N-/I-carbohydrate-

3-methylbutyl-L-histidyl-L·-leucine. In this case, the product was eluted from the ion exchange resin with 10X ammonia solution. The mass spectrum showed a molecular ion at 408 m/e for the disilylated species minus 18. The structure was confirmed by NMR spectroscopy.

Example 56. N - N -carboxy-3-methylbutyl- b -phenylalanyl L -arginine.

By the method described in Example 54, 4-methyl-2-oxopentanoic acid and L-phenylalanyl-L-arginine are condensed

- 49 in the presence of sodium cyanoborohydride to give N-(1<-carboxy-3-methylbutyric)-L-phenylalanyl-L-arginine. The product was eluted from the ion exchange resin with 10% ammonia solution. The NMR spectrum of deuterated water showed a singlet at 7.2 (5H), a broad triplet concentrated at 3.0 (6H), a broad multiplet at 1.3 (6H) and a broad doublet at 0.9 (6H).

Example 57. L1-(1-carboxy-3-phenylpropyl)-L-lysyl-L-proline.

By the method described in Example 54, 2-oxo-4-phenylbutyric acid and tert. Boc-L-lysyl-L-proline were condensed in the presence of sodium cyanoborohydride. Essentially all of the tert. Boc protecting groups were cleaved off, while the product was adsorbed onto the strongly acidic ion exchange resin. The crude L1-(1-carboxy-3-phenylpropyl)-L-lysyl-L-proline was eluted from the resin with 10% ammonia solution, freeze-dried, and purified by gel filtration (L H-20). The minor peak for tert. Boc protons in the NMR spectrum disappeared when the product was treated with ethyl acetate with 4 N hydrogen chloride gas. The NMR spectrum of the resulting hydrochloride salt of the product was consistent with the structure. The mass spectrum showed a molecular ion at 693 m/e for the trisilylated species. Chromatography on XAP-2 resin using 3.5% acetonitrile in 0.1 M ammonium hydroxide afforded D1-X-(1/5)-carboxy-3-phenylpropyl)-L-lysyl-L-proline.

B. L-[(1,5)-narc-boxy-3-phenylpropyl]-L-lysyl-L-proline.

By the method described in Example 54, 2-oxo-4-phenylbutyric acid and N-tert.Boc-L-lysyl-L-proline were condensed in the presence of sodium cyanoborohydride. The product was absorbed onto a strongly acidic ion exchange resin and eluted with 2X-ene.

- 50 solution of pyridine in water. The product-rich fractions were distilled to dryness and treated with 4 N hydrogen chloride in ethyl acetate to remove the tert. Boc-protecting group. The resulting hydrochloride salt was converted to the free base by absorption onto a strongly acidic ion exchange resin and eluted with a 2X solution of pyridine in water. The product-rich fractions were freeze-dried to give N·^(1-carboxy-3-phenylpropyl)-L-lysyl-L-proline as a white powder. The NMR spectrum was consistent with the structure. The mass spectrum showed a molecular ion at 549 for the disilylated species. Chromatography yielded the desired isomer.

Example 58. L1-(1-carboxy-3-phenylpropyl)-L-3-fluoroalanyl-L-proline.

To a solution of D-3-fluoroalanine (420 mg) in 4 ml of acetone-water (1:1) was added triethylamine (590 mg) and 2-tert-butoxycarbonyloximino-2-phenylacetonitrile (1.060 g). The mixture was stirred for two and a half hours. A cold 5X aqueous solution of potassium bicarbonate was added and the mixture was extracted with ethyl acetate. The aqueous phase was acidified with 1 N hydrochloric acid and extracted with ethyl acetate. The latter 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 a mp of 91-93°C.

While stirring, to a solution of the product obtained above (800 mg) and proline benzyl ester (1.5 g) in methylene chloride (ml) at a temperature of 0°C, dicyclohexylcarbodiimide (845 mg) in methylene chloride (6 ml) was added and the mixture was maintained at a temperature of 0°C for 2 hours and at a temperature of

- 51 20°C for 18 hours. The mixture is filtered, the precipitate is washed with methylene chloride, the filtrate and washings are combined and extracted with cold 1 N hydrochloric acid solution, with cold 5X aqueous potassium bicarbonate solution, with saturated aqueous sodium chloride solution, dried over sodium sulfate and concentrated to dryness. Dry column chromatography is carried out on silica gel H, eluting with 6% acetone in chloroform to obtain the purified protected dipeptide.

The tert-Boc group was removed by treatment with 4 N hydrogen chloride solution in ethyl acetate (8 ml) at 0°C for one hour. Ether (about 20 ml) was added and the precipitated

1-3-Fluoralanyl-L-proline benzyl ester, hydrochloride (450 mg) was collected by filtration (mp 158-161°C). Hydrogenation in 6 ml water and 2 ml ethanol over 60 mg of 10H-ene palladium/carbon at one atmosphere pressure and 20°C for 90 min, followed by filtration and concentration to dryness gave L-3-fluoroalanyl-L-proline hydrochloride (330 mg). The mass spectrum showed a molecular ion at 348 m/e for the ditrimethylsilylated species.

To a mixture of 4-phenyl-2-oxomaleic acid (375 mg) and L-fluorolanyl-L-proline hydrochloride (100 mg) in 3 ml of water (pH adjusted to 7 with sodium hydrochloride) was added sodium cyanoborohydride (80 mg). The mixture was stirred for 20 hours and then worked up as described in Example 24. The mass spectrum of the product purified by L H-20 showed a molecular ion at 510 m/e for the ditrimethylsilylated species. Thin layer chromatography on silica gel showed a single spot at c = 0.7 using a 1:1:1:1 ethyl acetate/acetic acid/n-butanol/water system.

- 52 The diastereoisomers are separated by chromatography on XAD-2 resin according to the method described in example 25.

L1-(1'-ethoxycarbonyl-3-phenylpropyl)-L-3-fluoroaniline was prepared by the method described in Example 26.

Example 59. L1-(1-ethoxycarbonyl-3-phenylpropyl)-L-alanylL-3,4-dehydroproline.

By the method described in Example 26, L-alanyl-L-3,4-dehydroproline obtained in Example 37 was converted into 11-(1)-ethoxycarbonyl-

3-phenylpropyl/-L-alanyl-L-3,4-dehydroproline in the form of a two-component diastereomeric mixture. Thin-layer chromatography on silica gel showed two spots cR^ 0.79 /additionally small/ in two developments in the system of n-butanol/water/acetic acid in the ratio 4:1:1. The mass spectrum showed a molecular ion at 518 m/e for the ditrimethyl silylated species.

Example 60. 1N-(1/57-methoxycarbonyl-2-(1H-imidazol-4-yl)ethyl)-O-galanyl-L-proline.

By the method described in Example 16, L-histidine methyl ester was condensed with pyrroyl-L-proline in the presence of sodium cyanoborohydride to give N-(1-methoxycarbonyl-2-(1H-imidazol-4-yl)ethyl)-1-alanyl-L-proline. The NMR spectrum in deuterated water showed imidazole protons at δ 8.6 and δ 7.3. The protons adjacent to the imidazole and the methyl ester protons appeared at δ 3.7, and the olanyl methyl at 1.1 to 1.38.

Example 61. N-[1-(S)-carboxy-2-(1H-imidazol-4-yl)ethyl]-L-alanyl-L-proline.

By a method similar to that described in Example 17, the product obtained in Example 60 was characterized to give the expected diacid. The NMR spectrum in deuterated water showed the imidazole protons at 7.2 and 8.5; and the alanine methyl at

1.25 ί.

Example 62. N-(1(R)-ethoxycarbonyl-5-aminopentyl(-1),L-alanyl-L-proline.

A solution of e-benzyloxycarbonyl-L-lysine ethyl ester hydrochloride (2.94 g) in water (10 ml) was made alkaline with 15 ml of saturated potassium bicarbonate solution and extracted with methylene chloride. The extract was dried over magnesium sulfate and concentrated to dryness. The residue of e-benzyloxycarbonyl-L-lysine ethyl ester was dissolved in tetrahydrofuran (THF) (20 ml) and pyruvoylproline (555 mg) and powdered molecular sieve No. 4A (1.0 g) were added. The mixture was stirred at room temperature for 4 hours. A solution of sodium cyanoborohydride (630 mg) in 1 ml of methanol was added over 2 hours and the mixture was stirred overnight. It was then filtered, concentrated to dryness, and the residue partitioned between water (10 mL) and methylene chloride (15 mL). The aqueous phase was absorbed onto a strongly acidic ion exchange resin and eluted with a 4X solution of pyridine in water to give 470 mg of N-(I)(5)-(ethoxycarbonyl-5-benzyloxycarbonylaminopentyl)-B,L-alanyl-L-proline. The protecting group was removed by hydrogenation in ethanol-water (1 to 1) over 10% palladium on charcoal at 40 psi/pounds per square inch-absolute pressure. The mixture was filtered and the filtrate evaporated to dryness. The residue was dissolved in methanol and chromatographed on a L H-20 column to give the desired N-(1/$‘/-ethoxycarbonyl-5-aminopentyl)-L,L-alanyl-L-proline. The mass spectrum of the silylated material showed a ppeak at 559 m/e for the trisilylated derivative and a peak at 487 m/e for the disilylated derivative. The NMR spectrum in deuterated water confirmed the structure.

- 54 Example 63. M-(1/5/>carboxy-5-aminopentyl)-b-alanyl-L proline.

The M-[1/5/-ethoxycarbonyl-5-benzyloxycarbonyl-aminolentyl]-])-L-alanyl-L-proline obtained in Example 62 was treated with 0.1 M sodium hydroxide solution at room temperature overnight. After absorption of the product onto a strongly acidic ion exchange resin, the product was eluted with a 4% solution of pyridine in water to give the N-[1/5/-carboxy-

5-benzyloxycarbonylaminopentyl/-L ,L -alanyl-L -proline, which shows a single spot with R 0.4 on thin layer chromatography (developing system butanol-water-pyridine-acetic acid in the ratio 10:4:3:1). By a method similar to that described in Example 62, the protecting group is removed by hydrogenation to give 1H-/1///-carboxy-5-ominipentyl/-5,L -alanyl-L -proline. The mass spectrum of the trimethylsilylated product is consistent with the structure and has a mass peak at 531 m/e. Chromatography yields the desired isomer.

Example 64. 1,1-[b-carboxy-6-aminohexyl]-b-alanyl-L-proline.

Benzyl 2-oxo-7-phthalimidoheptanoate, obtained after alkylation of benzyl 1,3-dithiane-2-carboxylate with 5-phthalimidopentyl bromide and after oxidative conversion to the ketone with N-bromosuccinimide, was condensed with L-alanyl-L-proline in the presence of excess sodium cyanohydride. The condensation product N-(1-benzyloxycarbonyl-6-phthalimidohexyl)-L-alanyl-L-proline (390 mg) in 25 ml of a 50% mixture of ethanol and water was hydrogenated at 40 psi (abs. pressure in pounds per square inch) over 10% palladium/carbon. Removal of the solvent and catalyst gave 1H-(1-carboxy-6-phthalimidohexyl)-55 L-alanyl-L-proline (320 mg) which had the expected spectral and chromatographic characteristics. A portion of the above intermediate (152 mg) in 2 ml of ethanol was refluxed with hydrazine (32 mg) for 1.5 hours. The phthalhydrazide was removed by filtration; the ethanol was distilled in vacuo and the residue was adsorbed onto a strongly acidic ion exchange resin. Elution with 2% aqueous pyridine and freeze-drying gave the desired N-(1-carboxy-

6-aminohexyl-L-alanyl-L-proline (58 mg). The spectral data are consistent with the structure. The mass spectrum shows a peak at 311 for the molecular ion minus water (329-18).

Example 65. N-[1H-benzyloxycarbonyl-6-aminohexyl]-L-alanyl-L-proline.

When the hydrolysis of L1-[L-benzyloxycarbonyl-6-phthalimidohexyl]-L-alanyl-L-proline is carried out in the manner described in Example 64, a mixture is obtained from which N-[L-benzyloxycarbonyl-6-aminohexyl]-L-alanyl-L-proline can be isolated. Thin layer chromatography on silica gel, eluting with a mixture of 50 butanol, 41 acetic acid and 30 water, gives a spot with R _F 0.32.

Example 66. N-(1-carboxy-2-phenoxyethyl)-L-alinyl-L·proline.

A suspension of phenoxypyruvic acid (1.8 g), prepared by condensation of ethyl phenoxyacetate with diethyl oxalate, followed by cathodic acid hydrolysis and decarboxylation, and L-alanyl-L-proline (0.37 g*) in 10 ml of water, pH adjusted to 7 with dilute sodium hydroxide, was treated with sodium cyanoborohydride (0.18 g) and stirred at room temperature for five days. On the second and third days, additional ketoacid (0.9 g) and sodium cyanoborohydride (0.18 g) were added.

- 56 The product was adsorbed onto a strongly acidic ion exchange resin, eluted with a 2% solution of pyridine in water and freeze-dried to give 0.5 g of 1H-(1-carboxy-2-phenoxyethyl)-L-alanyl-L-proline. The NMR spectrum was consistent with the structure. The mass spectrum showed a peak at 479 for the silyl molecular ion minus methyl (494-15).

Example 67. N-[1-ethoxycarbonyl-2-phenoxyethyl]-L-alanyl-L-proline.

By reacting ethyl phenoxypyruvate, obtained by acid-catalyzed esterification of the corresponding acid, and L-alanyl-L-proline with sodium cyanoborohydride in ethanol solution and isolating the product as described in Example 66, L1-(1-ethoxycarbonyl-2-phenoxyethyl)-L-alanyl-L-proline can be obtained.

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

A mixture of phenylthiopyrogrape niselin (1.96 g), obtained by condensation of ethyl phenylthioacetate with diethyl oxalate and then by acidic hydrolysis and decarboxylation, and L-alanylL-proline (0.37 g) in 10 ml of water, with the pH adjusted to 7.0 with dilute sodium hydroxide solution, was treated with sodium cyanoborohydride (0.18 g) in 2 ml of water. After stirring overnight at room temperature, the product was absorbed onto a strongly acidic ion exchange resin and eluted with a 2% solution of pyridine in water to give 0.36 g of 1\[-/1-narboxy-

2-phenylthioethyl/^-alanyl-L-proline. NMR and mass spectra confirm the desired structure. Mass peak at 348 indicates the molecular ion /366/ minus water /18/.

- 57 Example 69. N-(1'-ethoxycarbonyl-2-phenylthioethyl)-L-alanylL-proline.

By reacting ethyl phenylthiopyruvate, obtained from the acid by esterification, and L-alanyl-L-proline with sodium cyanoborohydride in ethanol solution as described in Example 68 and isolating the product as described there, N-[1-ethoxycarbonyl-2-phenylthioethyl]-L-alanyl-L-proline can be obtained.

Example 70. 1N-(1-ethoxycarbonyl-3-p-chlorophenylpropyl)-alanyl-L-proline.

A solution of ethyl 4-p-chlorophenyl-2-oxobutyrate, obtained from the acid by esterification with ethanol in carbon tetrachloride at reflux, and L-alanyl-L-proline in ethanol is treated with excess sodium cyanoborohydride at room temperature and stirred until the reaction is complete. The ethanol is removed in vacuo and the product is absorbed onto a strongly acidic ion exchange resin. By eluting with a 2X solution of pyridine in water, N-(1-methoxycarbonyl-3-p-chlorophenylpropyl)-L-alanyl-L-proline can be obtained.

Example 71. [1-[1-Ethoxycarbonyl-2-(3-imidyl)ethyl]-alanyl-L-proline.

In the manner described in Example 26, indole-3-pyruvic acid ethyl ester was condensed with L-alanyl-L-proline in a 1:1 mixture of ethanol and water using tarium cyanoborohydride. Isolation on Dowex 50 in the manner described gave a mixture of isomers of N-(1-ethoxycarbonyl-2/3-indolyl (ethyl)-L-alanyl-L-proline. The NMR spectrum showed absorption at 7.0 λ (5H), 4.2 λ (5H), 3.4 λ (2H), 2.9 λ (6H), 1.9 λ (6H) and 1.2 λ (6H).

- 58 Example 72. 1N-[1-ethoxycarbonyl-2-p-aminomethylphenylethyl]-L-alanyl-L-proline.

Ethyl 2-oxo-3-p-cyanophenylprolanoate, obtained by reaction of p-cyanobenzyl bromide with ethyl 1,3-dithiane-2-carboxylate and subsequent oxidative hydrolysis by the method described by Eliel and Hartmann, J. Org, Chem., 37, 505 (1972), is condensed with L-alanyl-L-proline and the product is purified by the method described in Example 1. Hydrogenation of the resulting mixture of isomers of N-(1-ethoxycarbonyl-2-p-cyanophenylethyl)~ L-alanyl-L-proline in an ethanol solution containing hydrogen chloride and palladium on carbon as a catalyst. Distillation of the solvent and excess hydrogen chloride in vacuo, pouring in ethanol and concentrating to dryness to obtain the hydrochlorides of the mixtures of diastereoisomers of the desired compound.

Example 73. L1-[1'-Carboxy-2-p-aminomethylphenylethyl]-L-alanyl-L-proline.

A sample of N-(1-ethoxycarbonyl-2-p-cyanophenylethyl)-alanyl-L-proline, prepared above in Example 72, was treated with one equivalent of sodium hydroxide in a mixture of methanol and water as solvent at room temperature overnight. Distillation of the solvents in vacuo gave the sodium salts of the mixture of isomers of N-(L-carboxy-2-cyanophenylethyl)-L-alanyl-L-proline. This mixture was hydrogenated in ethanolic hydrochloric acid solution and worked up as described in Example 72 to give the hydrochlorides of the mixture of diastereoisomers of the desired product.

Example 74. 1N-(1-ethoxycarbonyl-2(5)-amino-3-phenylpropyl)-D,1-galanyl-L-proline.

To a mixture of N-phthaloyl-L-2-amino-3-phenylpropionaldehyde

- 59 (Petf.rson et al., J. Am. Chem. Soc., 79, 1398 (1957), /2.18 g/ and potassium metabisulfide /0.87 g/ in a mixture of water and methanol in a ratio of 1:1 sodium cyanide /0.55 g/ is added with vigorous stirring. Stirring is continued for 90 min, after which the mixture is diluted with ethyl acetate and filtered. The organic layer is washed with water and dried over magnesium sulfate. The solvent is removed in vacuo to obtain N-phthaloyl-

3-amino-4-phenyl-2-hydroxybutyronitrile, thin layer chromatography with ethyl acetate/hexane in a ratio of 1:1 gives a spot with RF 0.5.

A solution of this product is prepared in absolute ethanol, which is saturated with ammonia and left for 6 days at room temperature. The solvent is removed, the residue is taken up in dioxane/concentrated hydrochloric acid in a ratio of 1:1, heated to a temperature of 70°C and kept at this temperature for 20 hours. Evaporation of the solvent to dryness, suspension of the residue with hot water, filtration and purification on a strongly acidic ion exchange resin in the usual way to obtain /2R,S ; 35 /-2-amino-4-phenyl-3-phthaloylaminobutanoic acid. Dissolution of the acid in anhydrous ethanol, passage of anhydrous hydrogen chloride until saturation and maintaining the mixture at room temperature for 16 hours. Removal of the solvent in vacuo to obtain the hydrochloride of the ethyl ester of the amino acid. Condensation of this ethyl 2-amino-4-phenyl-3-phthalylamino-butanoate with pyruvoyl-L-proline sodium cyanoborohydride, as described in Example 16, to give l-[1-ethoxycarbonyl-3-phenyl-

2-Phthaloylaminopropyl-L-alanyl-L-proline as a mixture of isomers. Refluxing this material in ethanol with one mole of hydrazine for one and a half hours, cooling, filtering the phthalhydrazide, and isolating the desired product from the resulting mixture by chromatographic methods gave N-(1-carbethoxy-2(5)-amino-3-phenylpropyl)-L-alanyl-L-proline. The NMR spectrum at 300 MHz has peaks at 7.3 <δ (5H), 3.2 and 4.4 <δ (3H), 2.75 <δ (2H), 2.0 <δ (4H), and 1.18 <δ (M).

Example 75. N-(1-carboxy-2(5)-amino-3-phenylpropyl)-L,L-alanyl-L-proline.

The 2-amino-4-phenyl-3-(5)-3-phthaloylaminobutanoic acid obtained in Example 74 was condensed with pyruvoyl L-proline using sodium cyanoborohydride as described in Example 16 to give L-[1,1'carboxy-3-phenyl-2-phthaloylamino-1,1',L alanyl-L-proline as a mixture of isomers. This material was refluxed in ethanol with one equivalent of hydrazine for 1.5 hours, cooled, the phthaloyl hydrazide was filtered, and the desired product was isolated by chromatographic methods to give the title compound. Thin layer chromatography on silica gel eluting with ethyl acetate to 5: pyridine to 1: acetic acid to 3: water gave a clear spot with an R _F of 0.15.

Example 76. N-(1-Carboxy-2(5)-benzoylamino-3-phenylpropyl)·D,L-alanyl-L-proline.

A solution of P-phthaloyl-3-amino-4-phenyl-2-hydroxy butyronitrile, obtained in Example 74, in ethanol saturated with anhydrous ammonia was allowed to stand for 3 days at room temperature. The solvent was removed in vacuo and the residue was refluxed for 6 hours in concentrated hydrochloric acid. Evaporated to dryness, the residue was purified on a column of Dowex 50 (H+) ion exchange resin, eluting with

- 61 successively with water /methanol/ 10:1, water/pyridine/ 50:1 and finally with 0.5 M ammonium hydroxide solution. From this last eluent, after concentration to dryness, the desired 2,3-diamino-4-phenyl propionic acid is isolated. A solution of the copper complex of this amino acid and benzoylate at the amino group in the tertiary position is prepared in situ with benzoyl chloride under alkaline conditions and by the method described by R· Hoeske et al., J, Am. Senm. Soc. ,> 73, 5883 (1956). Cleavage of the copper complex with hydrogen sulfide and treatment of the mixture in the described manner to obtain 2amino-3-/$/-benzoylamino-4-phenyl butyric acid. Condensation of this intermediate with pyruvoyl 2-proline using sodium cyanoborohydride in the manner described in Example 16 afforded the desired [1-(-carboxy-2-(5-benzoylamino-3-phenylpropyl)-B _L _ _alanyl - L -proline in the form of a mixture of isomers which can be separated by chromatographic methods if appropriate. Mass spectral analysis showed a peak at 596 m/e for the disilylated molecular ion minus IS. The NMR spectrum showed absorption for two phenyls at 7.0-7.5 $ and for diastereomeric methyls at II cP .

Example 77. N-(1-Ethoxycarbonyl-2-(S-benzoylamino-3-phenyl-propyl)-],L-alanyl-L-proline.

By treating 2-amino-3-benzoylamino-4-phenyl butyric acid, obtained in Example 76, with a saturated solution of hydrogen chloride in absolute ethanol for 4 h and subsequent distillation of the solvent in vacuo, ethyl

2-amino-3-benzoylamino-4-phenyl butyrate hydrochloride. By condensing this intermediate with pyruvoyl-L-proline using sodium cyanoborohydride in the manner described in Example 16 and isolating the product in the manner described therein, it is possible to

- 62 gave the title compound.

Example 78. N-(2-amino-1-carboxy-4-methylpentyl)-1,L-alanyl-L-proline.

A solution of 0.731 g of trans-3-amino-4-(2-methylpropyl)-2-azetidinone, prepared by adding chlorosulfonyl isocyanate to 4-methyl-1-pentene; the resulting J-lactam was protected as a tert. butyldimethylsilyl derivative and then treated with lithium diisopropylamide, followed by tosyl azide and chlorotrimethylsilane; after acidification and chromatography on silica gel, trans-3-azido-4-(2-methylpropyl)-2-azetidinone is obtained, which is hydrogenated with 10% palladium/carbon in ethanol to the corresponding amino derivative and 4.58 g of benzylpyruvate in 20 ml of absolute ethanol, containing 10 g of powdered molecular sieve 4A, is treated dropwise with a solution of sodium cyanoborohydride /0.65 g/ in 8 ml of absolute ethanol with stirring until the reaction is complete. The reaction mixture is filtered and the filtrate is concentrated. The residue is dissolved in 50 ml of water and acidified with 1 N hydrochloric acid solution to pH3. The mixture is basified with 10% sodium carbonate solution, the aqueous solution is saturated with sodium chloride and extracted with ethyl acetate /5x40 ml/. The combined organic layers were dried over sodium sulfate and concentrated to an oil (4.94 g). Chromatography on silica gel (ethyl acetate) gave 1.1 g of product. NMR and mass spectra were consistent with the structure of P-(trans-4-(2-methylpropyl)-2-oxo-3-azetidinyl)-1,L-alanine benzyl ester. Debenzylation was accomplished by hydrogenation with palladium on carbon in a 2:1 ethanol/water mixture. A cold 0°C solution of the acid (428 mg)

- 63 and L-proline tert.butyl ester (377 mg) in 5 ml of dimethylformamide was treated with a solution of diphenylphosphoryl azide (605 mg) in 5 ml of dimethylformamide and then with a solution of triethylamine (223 mg) in 5 ml of dimethylformamide for 20 minutes. After three hours, the ice bath was removed and the reaction mixture was stirred at room temperature overnight. Ethyl acetate (100 ml) was added and the resulting solution was washed with water (2x40 ml), 5X sodium carbonate solution (3 x 30 ml) and water (1 x 50 ml) before drying with sodium sulfate. Concentration gave an oil (0.78 g) whose NMR and mass spectra were consistent with the structure of 1H-/trans-4-/

2-methylpropyl/-2-oxo-3-azetidinyl/-0,1/-alanyl-L·-proline butyl ester. The crude product was dissolved in 25 ml of trifluoroacetic acid at 0°C. The reaction mixture was stirred at 0°C for 20 min and then at room temperature for two and a half hours. The reaction mixture was concentrated to dryness and the residue was treated with 1N sodium hydroxide solution (30 ml) for four and a half hours at room temperature. The mixture was slowly added to a strongly acidic ion exchange ^resin and the product was extracted with 2% pyridine/water solution. Drying by evaporation

S' freezing leads to the formation ^{of r} a r M-/2-amino-1-carboxy-

4-methylpentyl-P,L-al-yl-proline, consisting of four

V diastereomers (S,S,S,o, R,$; R,R, R, 5 ; R, R, 5 » S'; /which can be separated by chromatography. NMR and mass spectra are consistent with the structure. The NMR spectrum shows multiplets centered at 4.5, 3.85, 2.3, 1.79 and at 1.16 ppm. The mass spectrogram shows a peak at 458 m/e /molecular ion of disilylated material minus 15/.

BAD ORIGINAL $

- 64 Example 79. Ν-/2-ομμηο-Ι· -ethoxycarbonyl-4-methylpentyl/P ,L -alanyl-L· -proline.

An intermediate product obtained in Example 78, I-/trans-

4-(2-Methylpropyl)-2-oxo-3-azetidinyl)-B,L-alanine (125 mg) can be condensed with L-proline benzyl ester hydrochloride (167 mg) in the presence of diphenylphosphoryl azide (191 mg) and triethylamine (140 mg) in dimethylformamide solution to give 217 mg of N-(trans-4-(2-methylpropyl)-2-oxo-3-azetidinyl)-B,L-alanyl-L-probenzyl ester. The benzyl protecting group can be removed by hydrogenation and the β-lactam can be opened with anhydrous nitrous ethoxide in ethanol to give L1-(2-amino-1-ethoxycarbonyl-4-methyl-pentyl)-D,L-alanyl-L-proline.

Example 80. N-(2-benzamido-1'-Carboxy-4-methylpentyl)-P,L-alanyl-L-proline.

A solution of N-(2-amino-1'-carboxy-4-methylpentyl)-P,L alanyl-L -proline) prepared as described in Example 78 in aqueous alkaline medium was treated with benzoyl chloride to give N-(2-benzamido-1-carboxy-4-methylpentyl)-P,L alanyl-L -proline. The mass spectrometer of the disilylated material had peaks at 562 (M ⁺ -15), 460, 389 (major peak), 364, 242 and 174 m/e.

Example 81. L1-(2-benzamido-1-ethoxycarbonyl-4-methylpentyl)-P,L-alanyl-L-proline.

When treating a solution of N-[2-amino-1· -ethoxycarbonyl]

4-Methylpentyl (-P,L-alanyl-L-proline), prepared as described in Example 79, in an organic solvent with benzoyl chloride can be obtained N-(2-benzamido-1-ethoxycarbonyl-4-methyl-65pentyl) (-P,L-alanyl-L-proline).

Example 82. N- 1Z -[1-ethoxycarbonyl-3-phenylpropyl]-L-alginyl-L-proline.

By the method described in Example 37, N-<Z-tert. Boc-N-N-nitro-B-arginine N-hydroxy succinimide ester was condensed with L-proline in a mixture of dioxane and water, the N-<Z-tert. Boc protecting group was removed with trifluoroacetic acid and the dipeptide was isolated as described. Coupling of the resulting product with ethyl 2-oxo-4-phenylbutyrate and isolation as described in Example 26 resulted in the material with the N-nitrogen of the arginine still protected with a nitro group. The protecting group was removed by catalytic hydrogenation in ethanol-acetic acid over palladium/charcoal as a catalyst at room temperature and 40 pounds of hydrogen pressure. By filtering the catalyst and distilling the solvents in vacuo, a mixture of isomers of N-(1-ethoxycarbonyl)-3-phenylpropyl)-L-arginyl-L-proline is obtained.

Example 83. N-Z, -(1-carboxy-3-phenylpropyl)-B,L-homolysyl-L-proline.

The condensation of N-^-benzyloxycarbonyl-N-,Z,-3°butoxycarbonyl-D ,L -homolysin/, obtained from hemolysin via a copper complex with L-proline- ³ ° butyl ester, can be carried out using definitylphosphoryl azide. The 3° butyl groups can then be removed with trifluoroacetic acid and the product UJ-benzyloxycarbonyl-D ,Xrhomolysin-L-proline can be reacted with 2-oxo-4-phenylbutyric acid and sodium cyanoborohydride. The condensation product can be debenzylated by catalytic hydrogenation to give N-<£-/!· -carboxy-3-phenylpropyl/-P ,L -homolysin-L -proline.

- 66 Example 84. N-Z -/I'ethoc_sicarbonyl-3-phenylpropyl/-B ,

L -homolysyl-L· -proline.

The intermediate described in Example 83, N- (1) -benzyloxycarbonyl-D,L-homolysyl-L-proline, can be reacted with ethyl 2-oxo-4-phenylbutyrate and sodium cyanoborohydride. The condensation product can then be hydrogenated over palladium/carbon to give N- (1) -(1)-ethonyoxycarbonyl-

3-phenylpropyl/-P,L-homolysyl-L-proline.

Example 85. N-5 H -(T-Carboxy-3-phenylpropyl)-H-aminoP,L-alanyl-L-proline.

Under alkaline conditions, B,B-^, β-diaminopropionic acid is reacted with an excess of benzyloxycarbonyl chloride to give, upon acidification, D,L-A/-bis/benzyloxycarbonylamino/-propionic acid/ mp 123.5-124°C. To a chloroform solution containing this product, phosphorus pentachloride is added to give, upon development, D,L-

4- (benzyloxycarbonylaminomethyl)-oxazolidine-2,5-dione. A solution of L-proline tert. butyl ester in methylene chloride was added to the N-carboxyanhydride in tetrahydrofuran at -60°C. After standing overnight under freezing conditions, the mixture was evaporated to dryness. The crude product was obtained. Trifluoroacetic acid, which effectively cleaved the tert. butyl ester group for two hours at room temperature, resulted in a mixture of L-proline and p-benzyloxycarbonylamino-L,L-alanyl-L-proline. Gel chromatographic filtration on a L H-20 column yielded the pure dipeptide.

Condensed 2-oxo-4-phenylbutyric niselin and benzyloxyS1 UP

BAD ORIGINAL D ^ka P ^boni/, amino°-1) ,L-alanyl-L-proline in the presence of sodium ^{1 11} ' ..... cyanoborohydride. After removal of the protecting group from the resulting product, D1-(1-carboxy-3-phenyl-propyl) ^-amino-D ,L-alanyl- L>-proline is obtained. The NMR spectrum in deuterated . ------„„ nplnlti pptpni poi 7.4 cP. methine protons

- 67 at 4.2 o and multiples at 3.7, 3.2, 2.9 and 2.1

Example 86. Ν-Ζ-/Ι-ethoxycarbonyl-3-phenylpropyl/-l amino-D,L-alanyl-L-proline.

A solution of β-benzyloxycarbonylamino-β,L-alanyl-L-proline, prepared as described in Example 85, and ethyl 2-oxo-4-phenylbutyrate are condensed in ethanol with sodium cyanoborohydride. The protecting group can be removed from the product by catalytic hydrogenation to give N-[1-methoxycarbonyl-3-phenylpropyl]-β-amino-β,L-alanyl-L-proline.

Example 87. N-[1/5]-ethoxycarbonyl-3-phenylpropyl-0,L-p-aminomethylphenylalanyl-L-proline.

Ethyl 2-oxo-3-p-cyanophenylpropionate prepared in Example

72, was hydrolyzed by stirring with 5% sodium hydroxide solution at room temperature overnight. The reaction mixture was washed with ether, the aqueous layer was acidified to pH 2 with concentrated hydrochloric acid, the product was extracted into a mixture of ether and ethyl acetate, and the solvent was removed to give 2-oxo-3-p-cyanophenylpropionic acid. After reductive addition of the acid with ethyl ester of L-homophenylalanine in the presence of sodium cyanoborohydride and subsequent purification as described in Example 13, the mixture of diastereoisomers of N-[1-[5]carboethoxy-3-phenyl-propyl]-P,L-p-cyanophenylalanyl-L-proline was obtained as the benzyl ester. After hydrogenation of this intermediate in ethanol containing hydrogen chloride on palladium/carbon, as described in Example 72, and working up the mixture as indicated in the same example, the desired product was obtained as a mixture of diastereoisomers.

- 68 Example 88. N-Z -(1/5/carboxy-3-phenylpropyl)-P,L-p-amino-methylphenylalanyl-L-proline.

N-(1-(5)-ethoxycarbonyl-3-phenylpropyl)-P,L-p-cyanophenylalanyl-L-proline benzyl ester, obtained in Example 87, was hydrolyzed by treatment with two equivalents of sodium hydroxide in a mixture of methanol and water at room temperature overnight. The solvent was removed from the reaction mixture by distillation in vacuo and the residue was hydrogenated in ethanolic hydrochloric acid by the method described in Example 72. After further treatment of the mixture by the method described in the same example, a mixture of diastereoisomers of the desired product was obtained.

Example 89. -[1-Ethoxycarbonyl-3-phenylpropyl]-M-acetyl-L-lysyl-L-proline.

According to the method described in Example 26, ethyl 2-oxo-4-phenylbutyrate was added to N-[beta]-acetyl-L-lysyl-L-proline in a solvent mixture of ethanol and water in the presence of sodium cyanoborohydride. After isolation with Dowex 50 as described and freeze-drying of the fractions rich in the desired product, the mixture of isomers of N-(Tethoxycarbonyl-3-phenylpropyl-]-11-[beta]-acetyl-L-lysyl-L-proline was obtained. The mass spectrum of the trisilylated material had peaks at 663 (M*), 648 (M + - 15) and at 546 m/e.

Example 90. N-Ζ -/b-ethoxycarbonyl/-b-histidyl-L-proline.

As described in Example 26, ethyl 2-oxo-4-phenylbutyrate was reacted with L-histidyl-L-proline in the presence of sodium cyanoborohydride. After purification in the manner described, the mixture of diastereoisomers of M-X-(1-ethoxycarbonyl)-L-histidyl-L-proline was obtained.

- 69 Example 91. N-(-)-[ethoxycarbonyl-3-(naphthylpropyl)-L-lysyl-L-proline.

Ethyl N-[alpha]-naphthyl-2-oxobutyrate, prepared by alkylation of ethyl 1,3-dithiane-2-carboxylate with 2-[alpha]-naphthyl bromide and subsequent ketone conversion with N-bromosuccinimide in aqueous acetone, is condensed with [beta]-3[beta]-butoxycarbonyl-L-lysyl-L-proline in ethanol in the presence of sodium cyanoborohydride and molecular sieves. The product is then absorbed onto a strongly acidic ion exchange resin and eluted with a 2% solution of pyridine in water. The tert-Boc protecting group can be removed by treatment with 4.0 N hydrochloric acid in ethyl acetate to give N-[alpha]-ethoxycarbonyl-3-[alpha]-naphthylpropyl-L-lysyl-L-proline.

B. D'1 _-! /,-[1'-carboxy-3-(naphthylpropyl)-L-lysyl-L-proline.

4-Ζ-Naphthyl-2-oxobutyric acid, obtained from the ester by hydrolysis, is suspended in water and the pH of the medium is adjusted to 7 with dilute sodium hydroxide solution, then freeze-dried. The residue is treated with -3O-butoxycarbonyl-L-lysyl-L-proline as described in Part A. to obtain L1-/-/L-carboxy-3/-naphthylpropyl/^-lysyl-L-proline.

Example 92. N-(-[1/5]carboxy-3-p-chlorophenylpropyl]-lysyl-L-proline.

A solution of Ha£-3°-6yTOKCHKap6oHnn-L -lysyl-L -proline /0.36 g/ and 4-p-chlorophenyl-2-oxobutyric acid /1.1 g/ in 5 ml of water after adjusting the pH of the medium to 7 using dilute sodium hydroxide, was treated with 0.07 g of cyanoborohydride in 1 ml of water for several hours. After stirring the mixture at room temperature overnight, the product

- 70 was absorbed on a strongly acidic ion exchange resin and eluted with a 2% solution of pyridine in water to give 0.058 g of product. The NMR spectrum showed that the tert-Boc protecting group was not completely cleaved. The product was treated with 4.5 N hydrogen chloride in ethyl acetate, followed by isolation using an ion exchange resin to give 0.048 g of the _< /-/1rcarboxy-

3-p-chlorophenylpropyl-L-lysyl-L-proline. NMR and mass spectra are consistent with the structure. Peak at 584 corresponds to the silylated molecular ion. Chromatography yields the desired isomer.

Example 93. N-,/-[1-ethoxycarbonyl-3-p-chlorophenyl]-L-lysyl-L-proline.

Upon condensation of ethyl 4-p-chlorophenyl-2-oxobutyrate and t-3°-butoxycarbonyl-L-lysyl-L-proline in ethanol solution in the presence of excess sodium cyanoborohydride and isolation of the product as described in Example 92, N-[(1-methoxycarbonyl-3-p-chlorophenylprolyl]-L-lysyl-L-proline is obtained.

Example 94. N-[(-/I-carboxy-3-(3,4-dichlorophenyl)-propyl)-L-lysyl-L-proline.

A solution of 4-(3,4-dichlorophenyl)-2-oxobutyric acid, obtained from dichlorodihydrocinnamate ester by condensation with ethyl oxalate and subsequent acidic hydrolysis and partial decarboxylation, was treated in water according to the method described in Example 92 to give N-(3,4-dichlorophenyl)propyl-L-lysyl-L-proline. The NMR spectrum in deuterated water showed an aromatic hydrogen at 7.30 δ, a broad singlet at 4.35 δ, and broad multiplets at 3.53, 3.00, 2.68, 1.98, and 1.65 δ. The mass spectrum showed a peak at 455 m/e corresponding to a loss of !

- 71 of water from the corresponding structure. Large M+2 /457 m/e/ isotope indicates the presence of 2 CI.

Example 95. N-[(-[1/6]-carboxy-3-(3-indolyl)propyl]-L-lysyl-L-proline.

2-Oxo-4-(3-indolyl)-butyric acid was prepared from homotryptophan by the method described by Weygand et al., Ann. 658,128 (1962). This acid was condensed with e-tert-Boc-L-lysyl-L-proline in the presence of sodium cyanoborohydride as described in Example 54 to give a crude mixture of diastereoisomers of N-[1'-Narbox-3-[3-indolyl]prolyl-L-[1-tert-Boc-L-lysyl-L-proline. The mass spectrum of the trisilylated material had peaks at 732 (M*), 717 (M*-15), 615, 660, 645 m/e. The protecting group on the lysine side chain was cleaved by treatment with 4 N hydrogen chloride in ethyl acetate and purification on a strongly acidic ion exchange resin as described in Example 54 to give the desired product. The desired isomer can be obtained by chromatography.

Example 96. N~t/-/6-aMMHO-Ir naroxyethyl/-b-lysyl-Lproline.

Benzyl 2-oxo-7-phthalimidoheptanoate and 6-3°-butoxycarbonyl-L-lysyl-L-proline are condensed with excess sodium cyanoborohydride in ethanolic solution to give N-Ζ-(1-benzyloxycarbonyl-6-phthalimidohexide)-Λ-ε-3 ⁰ -butoxycarbonyl-L-lysyl-L-proline. The benzyl group can then be removed by hydrogenation over palladium/carbon, the tert-Boc protecting group can be cleaved with 4.5 N hydrogen chloride in ethyl acetate, and the phthalimide group can be cleaved by treatment with hydrazine to give N- _0- (6-amino-L-carboxyhexyl)-L-lysyl-L-proline.

Example 97. N _-O- [6-amino-L-benzyloxycarbonylhexyl]-72 L-lysyl-L-proline.

By treating N-(b-benzyloxycarbonyl-6-phthalimidohexyl)-N-(t-butoxycarbonyl-L-lysyl-L-proline prepared by the method described in Example 96 with 4.0 N hydrogen chloride in ethyl acetate and then with one equivalent of hydrazine at reflux in 8 ethanol, a mixture can be obtained from which N-(b-amino-N-(benzyloxycarbonylhexyl)-b-lysyl-L-proline can be isolated.

Example 98. N-(5-amino-1-(5)-carboxypentyl)-lysyl-L-proline.

Benzyl 2-oxo-6-phthalimidohexanoate was treated as described in Example 96 to obtain N-[beta]-amino-

1-Carboxypentyl-L-lysyl-L-proline. After chromatography, the desired isomer was isolated. The mass spectrum showed a molecular ion for the disilylated species at 516 m/e, an M*-15 ion at 501 m/e, and a peak at 399 m/e corresponding to the loss of the silylated carboxyl.

Example 99. _D1-0 (-/5-amino-1'-benzyloxycarbonylpentyl/L-lysyl-L-proline.

Benzyl 2-oxo-6-phthalimidohexanoate can be treated as described in Example 96, except that the debenzylation with hydrogen on palladium is omitted. The desired _N -[(5-amino-1'benzyloxycarbonylpentyl)-L-lysyl-L-proline] can be isolated from the product mixture.

Example 100. A. N-(-[1R-carboxy-2-phenoxyethyl]-L-lysyl-L-proline.

Phenoxypyruvic acid /0.9 g/ was dissolved in water, the pH was adjusted to 7 with dilute sodium hydroxide solution and the solution was freeze-dried. The residue

- 73 The residue was dissolved in 10 ml of ethanol and treated with ε-3°butoxycarbonyl-L-lysyl-L-proline (0.36 g) and powdered molecular sieve 'η ^? 4A (3.0 g). Sodium cyanoborohydride (0.18 g in 3.5 ml of ethanol) was added portionwise to the mixture 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 elution with a 2X solution of pyridine in water, followed by freeze-drying to give 0.25 g of the unprotected product, N-(1 'Carboxy-2-phenoxyethyl)-L-lysyl-L-proline. The NMR and mass spectra of the product were consistent with its structure.

B. M- _c (1'-ethoxycarbonyl-2-phenoxyethyl)-b lysyl-L-proline.

Ethyl phenoxypyruvate was treated with N-3°-butoxycarbonyl-L-lysyl-L-proline as described in Example 100 A to give N-3°-[1-ethoxycarbonyl-2-phenoxyethyl]-L-lysyl-L-proline. The NMR spectrum showed a phenyl absorption centered at 7.3° and broad peaks at 4.6, 4.4, 4.2, 3.7, 3.2, and 1.9° (?).

Example 101m A. 1H- _with (-/1/5/-carbO1{N-2-phenylthioethyl)-L-lysyl-L-proline.

Phenylthiopyruvic acid was treated with ε-3°butoxycarbonyl-L-lysyl-L-proline as described in Example 100 to give N- _with ε-(1'-carboxy-2-phenylthioethyl)-Β-lysyl-L-proline. The mass spectrum showed a silylated molecular ion at 567 m/e. After chromatography, the desired isomer was obtained.

B. _L1-0 (-/1-ethoxycarbonyl-2-phenylthioethyl/^-lysyl-L proline.

Ethyl phenylthiopyruvate is treated analogously with 4-3°-74 butoxycarbonyl-L-lysyl-L-proline in the manner described in Example 100A to obtain [1-[1-ethoxycarbonyl-2-phenylthioethyl]-L-lysyl-L-proline.

Example 102. R- _with (-/1-carboxy-2-[5-amino-3-phenylpropyl],L-lysyl-L·-proline.

Ethyl 2-amino-4-phenyl-3-phthalimidobutanoate can be 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 by the method described in Example 13. The resulting intermediate can then be reacted with L-proline benzyl ester hydrochloride using diphenylphosphoryl azide, as described in the same Example, to give a mixture of isomers of N-(-[1r-ethoxycarbonyl-2/5]-phthalimido-3-phenylpropyl]-L-[beta]-phthaloyl-D,L-lysyl-L· proline benzyl ester, purified by column chromatography. The product can then be treated with two equivalents of sodium hydroxide in ethanol-water for four hours at room temperature, neutralized to pH 4 with concentrated hydrochloric acid, the ethanol distilled off in vacuo, the product extracted with ethyl acetate and the solvent removed in vacuo. The resulting residue is then refluxed in ethanol containing 2 equivalents of hydridine for one and a half hours and isolated as described in Example 74 to give the desired compound.

Example 103. N -[1-Carboxy-2-(5)-benzoylamino-3-phenylpropyl] -P ,b -lysyl-L· -proline.

Ethyl 2-amino-3-benzoylamino-4-phenylbutanoate, obtained in Example 77/, was condensed with 2-oxo-6-phthalimidohexanoene

- 75 koselin in the presence of sodium cyanoborohydride by the method described in Example 13. The resulting L1-(-/-/1-Carbethoxy-2(5)-benzoylamino-3-phenylpropyl)-N1-ε-phthaloyl-D,L-lysine can then be coupled to L-proline benzyl ester hydrochloride using diphenylphosphoryl azide in the manner described in the example to obtain a mixture of isomers of N-,/-/1-carbethoxy-2(5)-benzoylamino-3-phenylpropyl)-N-ε-phthaloyl-D,L-lysyl-proline benzyl ester, purified by chromatography. After treating this product with two equivalents of sodium hydroxide in an ethanol-water mixture for four hours at room temperature and working up the mixture as described in Example 102, 1M- _with (-/12-carboxy-2-(5)-benzoylamino-3-phenylpropyl)-D1-4-phthaloyl-D,L-lysyl-L-proline is obtained. By refluxing this product for one and a half hours in ethanol in the presence of one equivalent of hydrazine and isolating the product as described in Example 74, the desired compound can be obtained as a mixture of isomers.

Example 104. N- _< (-/2-amino-1-carboxy-4-methylphenyl)-P,L-lysyl-L-proline.

Trans-3-amino-4-(2-methylpropyl)-2-azetidinone, obtained in Example 78, can be reductively added to benzyl in ethanol.

2-oxo-6-phthalimidohexanoate using sodium cyanoborohydride and molecular sieves. The product N-(-)-(4-(2-methylpropyl)-2-oxo-3-azetidinyl)-N-£-phthaloyl-D ,L-lysine benzyl ester can then be debenzylated by hydrogenation over palladium, the resulting free acid and proline benzyl ester can be reacted in the presence of diphenylphosphoryl azide. The resulting product can then be debenzylated as described above to give N-(-)-(4-(2-methylpropyl)-2-oxo-3-azetidinyl)-N-£-phthaloyl-D ,L-lysine-L-proline. The phthaloyl group can be removed in ethanolic solution with one molar equivalent of hydrazine at room temperature to give L1 _-α -(-[4-(2-methylpropyl)-2-oxo-3-azetidinyl]-β,L-lysyl-L-proline. By hydrolysis with dilute sodium hydroxide solution upon dissolution of the β-lactam ring, R-α-(-[2-amino-β-carboxy-4-methylpentyl]-β,L-lysyl-L-proline can be obtained.

Example 105. N-[2-(2-methylpropyl)-2-oxo-3-azetidinyl]-N-[4-(3-butoxycarbonyl)-O,L-lysine benzyl ester can be prepared from 3-trans-4-(2-methylpropyl) ^-2 -azetidinone (Example 78) and benzyl 3-butoxycarbonylamino-2-oxohexanoate. The benzyl group can be cleaved by hydrogenation and the resulting product coupled with L-proline benzyl ester, the resulting product N- _< /- /4- /2-methylpropyl /-2-oxo-3-azetidinyl /- N- -3 °-butoxycarbonyl- P , L -lysyl-L -proline benzyl ester can then be debenzylated by hydrogenation and the L lactam hydrolyzed with dilute base to give N - /- /2-amino-1· -carboxy-4-methylpentyl /- N- -3 °-butoxycarbonyl- D , L -lysyl /- L -proline. After benzoylation with benzoyl chloride in an organic solvent and cleavage of the tert. Boc protecting group with trifluoroacetic acid can be obtained N-o(-/2-benzoamido-L-carboxy-4-menthylpentyl/-1),L-lysylL-proline.

Example 106. L1-(-[1-(5)-carboxy-3-p-chlorophenylpropyl]-L-lysyl-trans-4-methone-L-proline.

Methyl-trans-4-methoxy-L-prolinate hydrochloride is coupled to N-^-Tepq.Boc-N-t-Cbz-L-lysine using

- 77 dicyclohexylcarbodiimide and triethylamine in methylene chloride as described in Example 20. The resulting product was purified by chromatography, the ester was hydrolyzed, and the tert-Boc-protecting group was removed as described in the said Example. This t-Cbz-L-lysyl-L-4-methoxyproline is then reductively coupled to 2-oxo-4-p-chlorophenylbutyric acid, derived from p-chlorohydrocinnamic acid, ethyl ester, by alkali-catalyzed condensation with diethyl oxalate, followed by decarboxylation with anhydrous hydrogen chloride in acetic acid in the presence of sodium cyanohydride and further treatment of the mixture as described in Example 24 to obtain a mixture of isomers of N- _< /-/L-carboxy-3-p-chlorophenylpropyl/-N-Cbs-L-lysyl-trans-4-methoxy-L-proline. The benzyloxycarbonyl protecting group is removed by catalytic hydrogenation over palladium on carbon as a catalyst in a known manner.

After chromatography of the resulting mixture, the desired isomer was isolated. The mass spectrum had peaks at 584 (M ⁺ ♦1), 568 (m ⁺ -15), 466, 397, 366 and 352 (major peak) for the disilylated material.

Example 107. N-(-[1-carboxy-3-p-chlorophenylpropyl]-L-lysyl-L-4-thiazolidine carboxylic acid.

N-(Z-Tepq.Boc-bI-e-Cl^ -L-lysine can be coupled to I-thiazolidine-4-carboxylic acid, benzyl ester, hydrochloride, the resulting product purified by chromatography, the ester hydrolyzed and the tert.Boc-protecting group removed as described in Example 20. By reductive coupling of the resulting product with 2-oxo-4-p-chlorophenylbutyric acid as described in the example, a mixture of isomers of N-(-/J-carboxy-3-chlorophenylpropyl/- 78 N-£-Cb2 -L-lysyl-L· -4-thiazolidine carboxylic acid can be obtained.

By removing the benzyloxycarbonyl protecting group by treating with hydrogen bromide in acetic acid at room temperature in a manner standard for peptide chemistry, distilling off the solvent in vacuo, pouring water on the residue, and freeze-drying, the desired product can be obtained.

Example 108. N-N-N-carboxy-3-p-chlorophenylpropyl N-lysyl-D,L-trans-5-methylthiazolidine-4-carboxylic acid.

3.7 from trans-5-methylthiozolidin-4-carboxylic acid ethyl ester, hydrochloride, prepared from /, -bromocrotonic acid and thioacetamide, acidic hydrolysis of the resulting product to L methylcysteine and subsequent reaction with formaldehyde analogous to the method /used by Ch, F. Nutt et al., Abstractsj of the btn American Peptide Symposium, Washington, d.C.

(1979), 1+16, P.95). reacted with 7.4 r N-/-tert.Boc-Cl-L-lysine, using 2.8 ml triethylamine and

4.5 g of dicyclohexylcarbodiimide in methylene chloride as described in Example 20. By reductive addition of the resulting intermediate to 2-oxo-4-p-chlorophenylbutyric acid using sodium cyanoborohydride and then removal of the benzyloxycarbonyl protecting group as described in Example 107, H-(-)-(1· -carboxy-3-p-chlorophenylpropyl)-1/ -lysyl-D,L-trans-5-methylthiazolidine-

4-carboxylic acid in the form of a mixture of isomers.

Example 109. N-(-[1-carboxy-3-p-chlorophenylpropyl]-L-lysyl-L·-3,4-dehydroproline.

Condensation of L-3,4-dehydroproline, ethyl ester, hydrochloride with L-/-tert-Boc-L-E-Cl-11-lysine, removal of tert-Boc protecting group with 4 M hydrogen chloride in ethyl acetate,

- 79 reductive addition of the intermediate with 2-oxo-4-chlorophenylbutyric acid. After removal of the protecting group with hydrogen bromide in acetic acid and further treatment of the reaction mixture as described in Example 107, a mixture of the isomers of N-[beta]-[beta]-carboxy-3-[beta]-chlorophenylpropyl]-[beta]-lysyl-L-3,4-dehydrolyroline is obtained.

Example 110. 1'1-[1-carboxy-4-methylphenyl]-L-alanyl-L-proline.

A solution of 5-methyl-2-oxohexanoic acid (1.44 g) and L-alanyl-L-proline (0.37 g) in five ml of water with pH adjusted to 7 was treated with sodium cyanoborohydride (0.31 g). After stirring at room temperature for five days, the reaction product was absorbed onto a strongly acidic ion exchange resin and eluted with a 2% solution of pyridine in water to give 0.6 g of a freeze-dried solid. A portion (0.2 g) was purified by chromatography on L H-20 to give 0.18 g of D1-(1-carboxy-4-methylphenyl)-L-alanyl-L-proline. The NMR and mass spectra were consistent with the expected structure. The diastereomers could be isolated by chromatography. In the mass spectrum, peaks of the disilylated material are observed at 458 (M ⁺ ), 443 (M ⁺ -15), 341, 301 and 244 m/e (main peak).

Example III - N -[1/b] -Ethoxycarbonyl-4-methylphenyl N -b alanyl-L -proline.

Ethyl 5-methyl-2-oxohexanoate /3.44 g/ and L-alanyl-L-proline /0.74 g/ were stirred in 15 ml of ethanol with 6 g of powdered molecular sieve 4A. A solution of sodium cyanoborohydride /0.23 g/ was added dropwise over several days.

- 80 in ethanol. The ethanol was then removed in vacuo, the product was absorbed onto a strongly acidic ion exchange resin and eluted with a 2% solution of pyridine in water to give 1.08 g of 1\1-/1~ethonincarbonyl-4-methylphenyl/-L-alanyl-L-proline. A portion of the product was purified on a L H-20 column (chromatographically) for spectral analysis. The NMR spectrum was consistent with the structure. The mass spectrum showed a peak at 414 (silylated molecular ion - 15). After chromatography of the product, the desired isomer was obtained.

Example 112. N-[L-Narbon-3-p-phenoxyphenylpropyl]-L-alanyl-L-proline.

A mixture of 2-oxo-4-p-phenoxyphenylbutyric acid obtained by reacting p-phenoxyphenyl Grignard reagent with ethylene oxide, converting the resulting alcohol to the bromide, and condensing the product with ethyl 1,3-dithiane-2-carboxylate. After oxidative cleavage of the dithiane, followed by alkaline hydrolysis, the keto acid is obtained, and L-alanyl-L-proline in water adjusted to pH 7 with dilute base is treated with excess sodium cyanoborohydride. The product N-(Ircarboxy-3-p-phenoxyphenylpropyl)-L-alanyl-L-proline is isolated by chromatography. The mass spectrum of the disilylated material contains peaks at 584 (M*), 569 (M ⁺ -15), 468, and 370 m/e (main peak).

Example 113. N-(1-ethoxycarbonyl-3-p-phenoxyphenylpropyl)-L-alanyl-L-proline.

Ethyl 2-oxo-4-phenoxyphenylbutyric acid, prepared as described in Example 112, with only the final alkaline hydrolysis omitted, was condensed with L-alanyl-L-proline in the presence of sodium cyanoborohydride to give

- 8] N-(1-ethoxycarbonyl)-3-p-phenoxyphenylpropyl)-L-alanyl-L·proline.

Example 114. b1-[b-carboxy-3-phenylpropyl]-b-alanyl-L D ,L -3,3-dimethyl-proline.

3,3-Dimethyl-L,L-proline was prepared from 3-methyl-2-butenal by the method of Cox, J. Chem. Soc., 1964, 5024 ^and converted to the methyl ester hydrochloride with methanolic acid. The resulting product was coupled to tert-Boc-alanine, then the intermediate was condensed with 2-oxo-4-phenylbutyric acid by the method described in Example 47 to give a mixture of isomers of the desired product.

Example 115.

A. L-[1-carboxy-3-phenylpropyl]-5-benzyl-cysteinyl L-proline.

The condensation of 1-N-tert.Boc-5-benzylcysteine with L-proline tert. butyl ester in the presence of dicyclohexyl carbodiimide in the usual manner gives I -/N-tert.Boc-benzylcysteinyl/-L-proline-tert.Boc-butyl ester. This blocked dipeptide is then treated with 4N hydrogen chloride in ethyl acetate at 0°C to give L -/E benzylcysteinyl/-E -proline. Treatment of this dipeptide with 2-oxo-4-phenylbutyric acid in the presence of sodium cyanoborohydride gives N-(1-carboxy-3-phenylpropyl)-L (5-benzylcysteinyl/-L-proline as a mixture of isomers.

B. L-[1-carboxy-3-phenylpropyl]-L-cysteinyl-L-proline. L-[1-carboxy-3-phenylpropyl]-L-5-benzylcysteinyl-L-proline prepared above in Part A was treated with sodium in liquid ammonia to give the desired compound.

- 82 Example 116. N-(-[1-carboxy-3-phenylpropyl]-1/-ornithyl-L proline.

N-S-Tepq.Boc-L-ornithyl-L-proline and 2-oxo-4-phenylbutyric acid were condensed in the presence of sodium cyanoborohydride as described in Example 54. The protecting group was removed from the resulting product using ethyl acetate with 4 N hydrogen chloride gas. The crude diastereomeric hydrochloride was absorbed onto a strongly acidic ion exchange resin and eluted with 2X aqueous pyridine. The mass spectrum showed a molecular ion at 355 m/e for the product minus 36. The NMR spectrum was consistent with the structure of the product.

Example 117. L1- _with (-[1/5]-ethoxycarbonyl-3-phenylpropyl]-L-lysyl-L-proline.

Ethyl 2-oxo-4-phenylbutyrate (2.58 g) and N-tert-Boc-L-lysyl-L-proline (859 mg) were dissolved in absolute ethanol (5 mL) and ground 4A molecular sieves (2.0 g) were added to the solution. At the end of the reaction, the sieve was removed by filtration. After evaporation of the filtrate, the residue was dissolved in water, extracted with ester and absorbed onto a strongly acidic ion exchange resin. After elution with a 2% solution of pyridine in water, 639 mg of crude ethoxycarbonyl-3-phenylpropyl-L-tert-Boc-L-proline were obtained. The protecting group was removed with ethyl acetate, which was 4 N in gaseous hydrogen chloride. The resulting hydrochloride was adsorbed onto a strongly acidic ion exchange resin and eluted with 2 L of aqueous pyridine to give 270 mg of product. The mass spectrum showed a molecular ion at 678 m/e for the disilylarion species plus one. The NMR spectrum was consistent with the structure.

- 83 Chromatography yields the desired isomer.

Example 118. N-7-(1-carboxy-3-phenylpropyl)-N-[beta]-N-dimethyl-L-lysyl-L-proline.

B1-/-tert.Boc-B1-E,-Cb2-E-lysyl-L-proline benzyl ester was reductively methylated in formaldehyde/10% palladium/carbon at 40 psi hydrogen pressure. The /-tert.Boc protecting group was removed with ethyl acetate which was 4N in gaseous hydrogen chloride. By the method described in Example 54, 2-oxo-4-phenylbutyric acid and N-^-N-E-dimethyl-L-lysyl-L-proline were condensed in the presence of sodium cyanoborohydride. The mass spectrum showed a molecular ion at 415 for the product, minus 18. The NMR spectrum was consistent with the structure.

Example 119. N- _x (-[1/5]-carboxy-3-phenylpropyl]-L-lysyl-L-proline.

N-(-)-(1-carboxy-3-phenylpropyl)-L-lysyl-L-proline, a mixture of diastereomers, obtained by the method of Example 57B, was purified by gel filtration on a chromatographic column L H-20. An XAD-2 column, prepared as described in Example 25, was equilibrated at 53 ^° C with 0.1 M ammonium hydroxide containing 4% acetonitrile. The isomeric mixture (250 mg) was dissolved in 10 ml of the above solvent and added to the column. When the column was eluted with the same solvent, the first isomer appeared at an eluate volume in the range of 320-360 ml. The second isomer appeared in the range of 450-540 ml of eluate. The intermediate fractions contained a mixture of isomers. When the fractions containing the first isomer were freeze-dried, 72 mg of a fluffy white solid was obtained, which represented the more active isomer and had an SSS configuration analogous to the more active isomer of D1-./-/1.-carboxy-3-phenylpropyl/-L-alanyl-L-proline, which was shown by X-ray analysis to have an SSS configuration. On thin layer chromatography on silica gel in a 1:1:1:1:1 mixture of ethyl acetate/n-butanol/water/acetic acid, the solid gave a single spot having an Rf of 0.43.300 MHz. The NMR spectrum showed a triplet for the methine proton γ to the phenyl substituent at 3.40 ppm. When the fractions containing the second isomer were freeze-dried, 72 mg of a white fluffy solid were obtained, which substance on thin layer chromatography on silica gel gave a single spot with Rf of 0.39.300' MHz. The NMR spectrum showed the triplet of the methine proton γ relative to the phenyl substituent at 3.61 ppm.

Example 120. N-[1-carboxy-3-phenylpropyl]-L-[beta]-acetyl-L-lysyl-L-proline.

According to the method described in Example 54, 2-oxo-4-phenylbutyric acid and N-E-acetyl-L-lysyl-L-proline were condensed in the presence of sodium cyanoborohydride to give N-,N-[L-carboxy-3-phenylpropyl]-N-E-acetyl-L-lysyl-L-proline. The NMR spectrum of the product obtained was consistent with its structure. The mass spectrum showed a molecular ion at 663 for the trisilylated species.

Example 121. N-[1-carboxy-3-phenylpropyl]-[4-arginyl]-proline.

The required dipeptide is obtained by condensation of N-_/-Tepa.Boc-N-u/-HHTpo-L-arginine and L-proline benzyl ester hydrochloride by the carbodiimide method. The protecting -tert. Boc group is removed by a known method with 4 N hydrogen chloride gas in ethyl acetate and the resulting N-VU-hhtpo-L * arginyl-L-proline benzyl ester is condensed with 2-oxo-4-phenylbutyric acid, as described in Example 24.

The reaction produced N-(1-carboxy-3-phenylpropyl)-N-O-nitro-L-arginyl-L-proline benzyl ester in relatively low yield (25-33%). This compound (159 mg) was dissolved in a mixture (2.5 ml) of acetic acid (water)/methanol (84%, 8X, 8$) and hydrogenated at ⁴⁰ psi of hydrogen at room temperature in the presence of 130 mg of 10% palladium/carbon to simultaneously remove the N-nitro and benzyl ester protecting groups. The catalyst was filtered and the filtrate evaporated to dryness (94 mg), the water-soluble portion of the resulting residue being freeze-dried to give a fluffy white solid (90 mg). This solid is the acetate salt of the desired product and the free base is obtained by absorption onto a strongly acidic ion exchange resin, washing with water and then eluting with a 2% solution of pyridine in water. Freeze drying of the product-rich fractions affords 60 mg of N-[N'-carboxy-3-phenylprolyl]-arginyl-L-proline. The NMR spectrum is consistent with the structure, the mass spectrum showing a molecular ion at 793 for the pentasilylated species.

Example 122. N-(1'Carboxy-3-phenylpropyl)-L-histidylL-proline.

By the method described in Example 54, 2-oxo-4-phenylbutyric acid and L-histidyl-L-proline are condensed in the presence of sodium cyanoborohydride to give N-/L-carboxy-

3-phenylpropyl/-L-histidyl-L-proline. The product was purified by gel filtration in a chromatography column (L H-20) in methanol. Its NMR spectrum was consistent with the structure. The mass spectrometry

- 86 shows a molecular ion at 657 for the disilylated species.

Example 123. N-(-)-[L-carboxy-2-(3-indolyl)ethyl]-L-lysyl-L-proline.

By the method described in Example 54, indole-3-pyruvic acid was condensed with N-£-Tepq.Boc-L-lysyl-L-proline in the presence of sodium cyanoborohydride. The protecting-^.-tert.Boc group was cleaved from the product with 4 N hydrogen chloride in ethyl acetate. The resulting hydrochloride was absorbed onto Dowex 50 /H ⁺ / and eluted with a 2% solution of pyridine in water. Freeze-drying of the product-rich fractions gave the free base as a light brown fluffy substance. The NMR spectrum of this product was consistent with its structure. The mass spectrum showed a molecular ion at 718 for the trisilylated species.

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

Dissolve 2-oxo-4-methyl-ethylpentanoate (2.75 g) in 150 ml of ethanol containing 16 g of powdered 4A molecular sieve. Hydrogenate at 40 psi absolute pressure of hydrogen at room temperature over 1 g of 10% palladium/carbon. After 1 mole of hydrogen has been taken up, the catalyst is filtered, the filter cake being carefully washed with ethanol. The solvent is evaporated to give 5.87 g of an oil. The oil is suspended in water, the pH of the medium is adjusted to 8.5, and extracted with ethyl acetate (3 x 60 ml) to remove neutral materials. The pH of the medium is adjusted to 7, saturated with sodium chloride, and extracted with ethyl acetate (3 x 100 ml). The product solution is dried over anhydrous magnesium sulfate. The ethyl acetate was evaporated to give 4.38 g of crude N-(-/L-ethoxycarbonyl-4-methylpentyl)-L-tert-Boc-L-lysyl-L-proline. The tert-Boc protecting group was removed in the usual manner with 4 N hydrogen chloride in ethyl acetate. The resulting hydrochloride was converted to the free base with a strongly acidic ion exchange resin, which was eluted with a 2% solution of pyridine in water. Freeze-drying of the product-rich fractions yielded 2.1 g of a hygroscopic friable substance. The NMR spectrum of this product was consistent with the structure of N-(-/L-carbethoxy-4-methylphenyl)-L-lysyl-L-proline. The mass spectrum gave a peak at 472 for the monosilylated molecular ion plus one.

Example 125. L1-(-[1-carboxy-4-methylpentyl]-L-lysyl-L-proline.

Hydrolyze N-/-/1-ethoxycarbonyl-4-methylpentyl/-L-lysyl-L-proline to the corresponding carboxylic acid under stirring in aqueous sodium hydroxide solution (2.5 equivalents) at room temperature for several days. The reaction mixture is acidified to pH 5, absorbed onto a strongly acidic ion exchange resin and eluted with a 2% solution of pyridine in water. The product-rich fractions are freeze-dried to give N-/L-carboxy-

4-Methylpentyl-L-lysyl-L-proline as a white fluffy solid. The NMR spectrum is consistent with its structure. The mass spectrum shows a molecular ion at 516 for the disilylated species.

Example 126. N -/ - / I -Carboxy-3-phenylpropyl / -b -leucyl b -tryntophan.

By the method described in Example 54, 2-oxo-4-phenylbutyric acid

- 88 acid and L-leucyl-L-tryptophan were condensed in the presence of sodium cyanoborohydride. The product was freeze-dried from a dioxane/water mixture until it was only slightly water-soluble. The NMR spectrum was consistent with the structure. The mass spectrum showed a molecular ion at 695 for the trisilylated species.

Example 127.

A. Tablets. A typical tablet contains N-(1/5/-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline (25 mg), pregelatinized starch (SP) (82 mg), microcrystalline cellulose (82 mg) and magnesium stearate (1 mg). Similarly, for example, N-(1/5/-narboxy-3-phenylpropyl)-L-lysyl-L-proline (20 mg) may be tabletted in place of N-(1-($/-ethoxycarbonyl3-phenylpropyl)-L-alanyl-L-proline) with the composition of pregelatinized starch, microcrystalline cellulose and magnesium stearate described above.

B. Combination tablets. A combination tablet with a diuretic such as hydrochlorothiazide usually contains L1-(1/6'-ethoxycarbonyl-3-phenyl-propyl)-1-galanyl-L-proline (7.5 mg), hydrochlorothiazide (50 mg), pregelatinized starch//5P (82 mg), microcrystalline cellulose (82 mg) and magnesium stearate (1 mg). Tablets, for example, with N-(1/5')-carboxy-3-phenylpropyl/L-lysyl-L-proline (5 mg) and hydrochlorothiazide (50 mg) are prepared by substituting the active ingredient in place of L1-(1/5')-ethoxycarbonyl-3-phenylpropyl) in the composition described above.

B. Injections. A typical injection form contains N-(1/5)-carboxy-3-phenylpropyl)-L-alanyl-L-proline (5.42 mg), anhydrous sodium phosphate (11.4 mg), benzyl alcohol (0.01 ml) and water for injection (1.0 ml). Similarly, this formulation may be prepared using, for example, N-(1/5)-carboxy-3-phenylpropyl)-L-lysyl-L-proline in place of N-(1/5)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline. Such an injection form may also contain a pharmaceutically effective amount of another antihypertensive or diuretic agent, for example, hydrochlorothiazide, as described in Example B above.

D. Suppositories. Typical suppositories for rectal administration contain N-(1/5)-ethoxycarbonyl-3-phenylprolyl)-L-alanyl-L·-proline (1-25 mg), butylated hydroxyanisole (0.08-1.0 mg), disodium calcium ethylenediaminetetraacetate (0.25-0.5 mg), and polyethylene glycol (775-1600 mg). Other suppository forms can be prepared, for example, by replacing the above-mentioned active substance with N-(1-(5)-carboxy-3-phenylpropyl)-L-lysyl-L-proline and by replacing, for example, the disodium calcium ethylenediaminetetraacetate with butylated hydroxytoluene /0.04-0.08 mg/ and the polyethylene glycol with hydrogenated vegetable oil /675-1400 mg/, such as Supposir L, Vecobee P5, Vecobee

I.', Vitepsols and the like. Further, these suppository forms may include other antihypertensive or diuretic agents in a pharmaceutically effective amount, as described, for example, in parts B and C of the same example.

Example 128. N-(1-ethoxycarbonyl-3-(3-indolylpropyl)-L-alanyl-L-proline.

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/charcoal in the presence of 3.2 g of powdered molecular sieve (34A) at an absolute hydrogen pressure of 40 pounds per square inch. After completion of the reaction,

- 90 The suspension was filtered and the filtrate was concentrated to dryness. The distilled oil was dissolved in 5 ml of methanol and chromatographed on a Sephadex L H-20 column to give 0.67 g of crude product as an orange oil. A portion of the crude product /0.3 g/ was purified by dissolving in water and extracting with ether. The product was extracted from the aqueous phase by diluting with an equal volume of saturated sodium chloride solution and extracting with ethyl acetate to give 80 mg of N-/I-ethoxycarbonyl-

3-(3-indolyl)propyl-L-alanyl-L-proline in the form of a yellow oil. The mass spectrum shows peaks at 559 for the disilylated product and at 632 for the trisilylated product.

Example 129. Benzyl N-(1(6)-carboxy-3-phenylpropyl)-1/alanyl-L·-prolinate.

Thionyl chloride (13.1 ml) was added to 150 ml of benzyl alcohol while maintaining the temperature below 0°C (exothermic reaction). 15 g of N-(1/4)-carboxy-3-phenylpropyl)-L-alanyl-L· proline was added portionwise to the cold solution. The cooling bath was removed and the reaction mixture was stirred at room temperature overnight. After heating to 45°C in vacuo to remove dissolved gases, the reaction mixture was diluted with 500 ml of ether and washed with 10 x 100 ml of water. The solid that appeared in the organic layer was filtered and dried to give 4.9 g of crude monobenzyl ester.

The combined aqueous extracts were neutralized with sodium bicarbonate, yielding a second crop of 1.4 g of product. The product from both crops was pooled and a portion (1.0 g) was recrystallized from ethanol-water to give 0.95 g of pure monobenzyl ester, mp 120-125°C. The spectrum

- 91 shows that the benzyl ester is attached to the proline ring. Example 130. Ethyl N-(1-(6)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-prolinate hydrochloride.

A solution of 3.0 g of N-(1/5)-ethoxycarbonyl-(3-phenylpropyl)-L-alanyl-L-proline in 50 ml of absolute ethanol was saturated with hydrogen chloride gas at 0°C. After standing at 0°C overnight, the hydrogen chloride and ethanol were removed in vacuo to give 3.1 g of an oil which, on freeze-drying, gave a white solid. Thin-layer chromatography and high-performance liquid chromatography (HPLC) indicated that the product consisted of only one component. The NMR spectrum indicated two ethyl groups for the aromatic nucleus. The mass spectrum showed a molecular ion at 404 m/e.

Example 131. N1-(1-ethoxycarbonyl-3-(2-chlorophenyl)propyl)-L-alanyl-L-proline.

By the method described in Example 26, ethyl-2-oxo-4-(2-chlorophenyl) butyrate and L-alanyl-L-proline were condensed in the presence of sodium cyanoborohydride to give N-(butoxycarbonyl-3-(2-chlorophenyl)propyl)-L-alanyl-L-proline. The mass spectrum showed a peak at 411 (M +1) and a strong peak at 392 (M - 18).

Example 132. L1-(1-ethoxycarbonyl-3-(4-fluorophenyl)propyl)-L-alanyl-L-proline.

By the method 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)-L-alanyl-L-proline. The mass spectrum showed a molecular ion at 394 and strong peaks at 376 (M - 18, water) and 375 (M - 19, fluorine). The NMR spectrum was consistent with the structure.

- 92 Elemental analysis shows that the product is in the form of a sesquihydrate.

Example 133. N-(N-ethoxycarbonyl-3-(2-naphthyl)propyl)-L-alanyl-L-proline.

by the method described in Example 26, ethyl 2-oxo-4-(2-naphthyl) butyrate and L-alanyl-L-proline were condensed in the presence of sodium cyanoborohydride to give M-(1-methoxycarbonyl-(3-(2-naphthyl)-propyl)-L-alanyl-L-proline. The mass spectrum showed a molecular ion at 426 and a peak at 408 (M-18, water). The NMR spectrum was consistent with the proposed structure. Elemental analysis showed that the product was in the form of a hemihydrate.

Example 134. N-Z-[1'-carboxy-3-(2-chlorophenyl)propyl]-lysyl-L-proline.

By the method described in Example 26, ethyl 2-oxo-4-(2-chlorophenyl)butyrate and N-tert-Boc-L-lysyl-L-proline were condensed in the presence of sodium cyanoborohydride to give N-Z-(1-ethoxycarbonyl-3-(2-chlorophenylpropyl)-N-E-tert-Boc-L-lysyl-L-proline.

The tert-Voc protecting group is removed by treatment with

N hydrogen chloride gas in ethyl acetate and then the ethyl ester group was salified with dilute sodium hydroxide solution to give N-/-/I-carboxy-3-/2-chlorophenyl/propyl/L-lysyl-L-proline after absorption on a strongly acidic ion exchange resin and elution therefrom. Elemental analysis showed solvation with one and a half molecules of water. The NMR spectrum was consistent with the proposed structure of the compound. The mass spectrum of the silylated sample showed a peak at 568, which corresponded to a loss of mass 15 /methyl/ from the disilylated species.

- 93 Example 135. N-7-/L-carboxy-3-/4-fluorophenyl/propyl/-

L-lysyl-L-proline.

By the method described in Example 134, ethyl 2-oxo-4-(4-fluorophenyl)butyrate and N-,-tert-Boc-L-lysyl-L-proline were condensed in the presence of sodium cyanoborohydride. The resulting product was deblocked as described in Example 4 to give N-,-/1-carboxy-3-(4-fluorophenyl)propyl)-L-lysylL-proline. Elemental analysis showed that the product was in the form of the sesquihydrate. The NMR spectrum confirmed the proposed structure. The mass spectrum of a silylated sample showed a molecular ion at 711 for the tetrasilylated species.

Example 136. 1N- _3- (L-carboxy-3-(2-naphthyl)propyl)-L-lysyl-L-proline.

By the method described in Example 134, replacing ethyl 2-oxo-

4-(2-Chlorophenyl)butyrate with ethyl 2-oxo-4-(2-naphthyl)butyrate gave H- _3- (1-carboxy-3-(2-naphthyl)propyl)-lysyl-L proline. The NMR spectrum was consistent with the proposed structure, the mass spectrum showing a molecular ion at 599 for the disilylated species.

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

A mixture of phosphorus tribromide (18.95 g) and pyridine (3.76 cm ³ ) in 10 ml of benzene was cooled to -5°C and treated with a solution of 2-(2-thianyl)-ethanol (25 g) in 10 ml of benzene containing 1.25 cm ³ of pyridine at such a rate that the reaction temperature never exceeded 0°C. The mixture was then allowed to warm to room temperature with stirring overnight. The reaction mixture was then concentrated on a water pump at 30-40°C to remove the solvent and other volatiles, and the residue was subjected to

- 94 of Claisen distillation under vacuum, provided by a water pump, until 21 g of crude product was obtained (b.p. 115 - 119°C).

This product was then fractionally distilled at 0.3 mm mercury to give 19 g of pure 2-(2-thianyl)ethyl bromide (b.p. 50-51°C; purity 99.9%, determined by gas chromatography). This bromide was dissolved in dry tetrahydrofuran (40 ml) and added dropwise, with stirring, to a mixture of magnesium turnings (2.4 g) and 15 ml of dry tetrahydrofuran under a nitrogen atmosphere. The mixture was then stirred and refluxed for one hour, cooled to room temperature and added dropwise, under a nitrogen atmosphere and with stirring, to a mixture of diethyl oxalate (17.5 g) in 25 ml of dry tetrahydrofuran at a bath temperature of -15°C. After completion of the reaction, the mixture was stirred at -10°C for 15 min, 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 containing in wt% by gas chromatography: solvent 26; diethyl oxalate 10.3 and ethyl 2-oxo-(2-thienyl)butyrate 63.4. The NMR spectrum in deuterated chloroform was consistent with this mixture, indicating the presence of two specific types of ethyl esters and the mass spectrum showed a peak at m/e=212 for the molecular ion of a keto-ester together with a fragmentation pattern consistent with the structure.

Example 138. N-[L-ethoxycarbonyl-3-(2-thienyl)propyl]-L-alanyl-L-proline.

Add 4 g of molecular sieve type 4A (powdered) to

- 95 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 15 minutes at room temperature followed by slow addition over three and a half hours of a solution of sodium cyanoborohydride (0.785 g) in 15 ml of anhydrous ethyl alcohol and stirred overnight at room temperature. The reaction mixture was made slightly acidic with glacial acetic acid, filtered and the filtrate was concentrated in vacuo to give an orange oil.

After extraction of the chloroform solution of this oil with 10% potassium bicarbonate solution, followed by re-extraction of the acidified (to pH 3) bicarbonate extracts with chloroform and after drying and evaporation of the solvent, 400 mg of the desired substituted dipeptide were obtained. The NMR spectrum in deuterated chloroform showed the thienyl proton as a multiplet at 7.0 ppm, two active protons at 6.2 ppm as a broad singlet, six methyl protons as a broad multiplet at 1.22-1.43 ppm and a series of multiplets in the region of 1.8-4.4 ppm.

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

6.95 g of molecular sieves of type 4A (powder) were added to a mixture of L·-Ι-ε-Boc-lysyl-L-proline (2.98 g) and ethyl-2-oxo-4-(2-thienyl)-butyrate (6.4 g) in 40 ml of anhydrous ethyl alcohol. The mixture was stirred at room temperature for 45 min, followed by the slow addition (over four and a half hours) of a solution of sodium cyanoborohydride (1.36 g) in 25 ml of anhydrous ethyl alcohol, stirring continued overnight at room temperature, which reaction mixture was made slightly acidic with the aid of ice

- 96 acetic acid, filtered and concentrated in vacuo to give an orange foam (16.4 g). Trituration of this crude product with three portions of ether and decantation of the ether solution removed a significant amount of the excess ketoester present. The ether-insoluble oil was stirred in 150 ml of 4 N hydrogen chloride/ethyl acetate at 0°C for one hour and poured into 1 L of cold ether, filtered and concentrated in vacuo to give 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 at room temperature overnight. After careful acidification with 0.5 N hydrochloric acid, concentration in vacuo, and subsequent purification by column elution with 150 ml Dowex 50W-X2(H+) with water containing 2% pyridine, the substituted dipeptide was obtained as the dicarboxylic acid. The NMR spectrum in deuterated water D CI was consistent with the structure.

Example 140. N-(I-Carboxy-3-phenylpropyl)-b-Olanyl-Haminoisobutyric Acid.

A mixture of 3.9 g of methyl 7-amino-isobutyrate hydrochloride, 8.7 ml of triethylamine and 11.9 g of Boc-L-alanine in 135 ml of methylene dichloride 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 48 hours. The mixture was filtered and the filtrate was concentrated, the residue was partitioned between water and ethyl acetate. The organic phase was washed with 10% citric acid, water, 5% sodium bicarbonate solution and water. It was dried over magnesium sulfate and concentrated to give the crude dipeptide. The crude product was purified * 97 on silica gel by eluting with a 7:3 hexane-ethyl acetate mixture. 1.2 g of this dipeptide was dissolved in 30 ml of 4N hydrogen chloride/ethyl acetate and kept at room temperature for one hour. The mixture was concentrated and freeze-dried from water to give L-alanyl-7-aminoisobutyric acid, methyl ester, hydrochloride.

A solution of 688 mg of this amino acid as an ester of 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/carbon as a catalyst and at 40 psi absolute pressure of hydrogen. It was filtered and the filtrate was concentrated. 4 ml of a 7:3 hexane/ethyl acetate mixture was added, filtered and the filtrate was chromatographed on silica gel to give methyl 1-/-ethoxycarbonyl-

3-phenylpropyl[b]-alanyl-β-amino-isobutyrate.

Elemental analysis: Calculated for C 20 ^N 30^2^5 ^: C, 63.30;

H, 8.24, N,7.38.

Found: C ₍ 63.58; H ₍ 8.13 and 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 at room temperature overnight. The resulting solution was loaded onto a Dowex 50 (H+) column and eluted with a 5% solution of pyridine in water. After concentration of the appropriate fractions, N-(1-carboxy-3-phenylpropyl)-L-alanyl-c-amino-mixed acid was obtained.

Elemental analysis. Calcd. for C-2H27M2O52H2O2 C, 54.82; H, 7.58; N, 7.52. Found: C, 54.94; H, 7.50 and N, 7.48.

The NMR spectrum is consistent with the structure of the compound.

- 98 Example 141. N-[1-Carboxy-3-phenylprolyl]-L-alanyl-L]-methylphenylalanine.

The compound was prepared by the method described in Example 140, starting from L-/-methylphenylalanine methyl ester hydrochloride in place of -aminoisobutyric acid, methyl ester, hydrochloride.

Example 142. N-[1-carboxy-3-[4-[1-benzylimidazolyl]propyl-L-alanine-L-proline.

An excess of 4-(4-/J-benzylimidazolyl//-2-unoleic acid-, obtained by dibenzylation of 3-(4-imidazolyl/) propionic acid and subsequent condensation with diethyl oxalate, followed by acidic hydrolysis and decarboxylation, was condensed with L-alanyl-L-proline in water (pH 6.7) in the presence of excess sodium cyanoborohydride. After three 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 L H-20 chromatography column using methanol. The data from the and hP NMR spectra and from the IR (infrared) spectrum were consistent with the structure. A satisfactory fuel analysis was obtained for the monohydrate. The mass spectrum after silylation shows peaks at 572 (M ⁺ ), 557 (M ⁺ -15) and at 455 (M + - CO 2 , Me _{3 )} (m/e).

Example 143. N-p-Carboxy-3-(4-imidazolyl/propyl/L-alanyl-L-propion).

N-(1-carboxyl-3-(4-)-[L·-benzylimidazolyl]propyl]-L-alanyl-L-proline, prepared as described in Example 142, was hydrogenated at 40 bar.

- 99 2 inches / no hydrogen on an equivalent weight of 5% palladium/carbon mixture of acetic acid / water in a ratio of 3 parts 1 part te-ps-patu?s ^G ' ^/3 C '2 pgsd.yanig of 3 days. Cf?,.· separation of the precipitate through 'filtripsche of the .'filtrato soup product

Very sour

resin, followed by elution with 2% aqueous pyridine and freeze-drying. Further purification was carried out on a Secodex L H-20 column using methanol. The proton HF spectrum and thin-layer chromatography of the product showed that the neutral group had been removed to give the title compound, the spectrum showing a peak for / ♦ - 10/ at m/e 320.

Example 144. N-(1-carboxyl-3-[4-benzylimidazolyl]-L-proline and N-(1-carboxyl-3-[4-yl]-1-benzylimidazolyl)-L-proline

PROPYL/-L-lysyl-L-PROLINE.

The two title compounds were prepared in the same manner as N-(1-Narboxy-3-(4-(1-benzylimidazolyl)-propyl-alanyl-L-proline (Example 142) and N-(L-carboxy3-N-imidazolyl)-propyl-alanyl-L-proline by replacing L-alanyl-L-proline with Et-4-oc-L-lysyl-L-o/irH and removing the tert. C group with trifluoroacetic acid at 0 ^and C for 3 h after condensation.

Example 145. N-[1. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 60. 61. 62. 63. 64. 65. 66. 67.

The two title compounds were prepared in the same manner as N-(1-carboxy-5-(4-[1-benzylimidazolyl]-propyl)-L-proline (Example 142) and

BAD ORIGINAL

100 N-(1-carboxy-3-(4-imidazolyl)propyn-L·-alanyl-L-proline/ example 143/ using 4-(4--(1--benzylimidazolyl)-2-ketobutyric acid ethyl ester and carrying out the condensation in ethanol/ in the presence of molecular sieves 4A/ instead of water.

LITERATURE

1. US 4129571

2. US 4154960

3. US 4052511

4. Cushman D.W. et al., Biochemistry 16, 5484, 1977

5. PLQUILLOUD Y., A. REINHARZ AND M. ROTH, BlOCHEM BlOPHYS.

Acta 206, 136, 1970

6. Weeks JR. and J.A. Jones, Proc. Soc. Exp. Biol. Med., 104, 646, 1960

7. Koletsky S. et al., Proc. Soc. Exp. Biol. Med., 125, 96, 1967

Claims (82)

Compound with formula II with NH ci II in which are the same or different and represent hydroxy, lower alkoxy, , dinitrialkylamino lower alkoxy, acylamino lower alkoxy, acyloxy lower alkoxy aryloxy, arylnitrialkyloxy, substituted aryloxy or substituted arylnitrialkyloxy, the substituent being methyl, halo, or methoxy amino, lower alkylamino, dinitrialkylamino, arylnitrialkylamino, R-1 represents hydrogen, alkyl containing 1 to 20 or hydroxyamino; carbon atoms, including branched, cyclic and unsaturated alkyl groups; substituted lower alkyl, wherein the substituent is halo, hydroxy, lower alkoxy, aryloxy, amino, lower alkylamino, dinitrilealkylamino, acyl, phenyl, imidazolyl, indolyl, mercapto, lower alkylthio, arylthio, carboxy, carboxamido, carbonyl alkoxy, phenyl, substituted phenyl, wherein the substituent is lower alkyl, lower alkoxy or halo; arylloweralkyl or heteroarylloweralkyl, arylloweralkenyl or heteroarylloweralkenyl, substituted arylloweralkyl, substituted heteroarylloweralkyl, substituted arylloweralkenyl or substituted heteroarylloweralkenyl, the substituent being halo or dihalo, lower alkyl, hydroxy, lower alkoxy, amino, aminomethyl, acylamino, diloweralkylamino, lower alkylamino, carboxyl, haloloweralkyl, cyano or sulfonamido; arylloweralkyl or heteroarylloweralkyl substituted in the alkyl portion with amino or acylamino groups; R2 and R3 represent hydrogen or alkyl; means 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, halo lower alkyl, guanidino lower alkyl, imidazolyl lower alkyl, indolyl lower alkyl, mercapto lower alkyl and lower alkylthio lower alkyl; R1 represents hydrogen or lower alkyl; R1 represents hydrogen, 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; and R5 may be linked together to form an alkylene group of 2 to 4 carbon atoms, an alkylene bridge of 2 or 3 carbon atoms and one sulfur atom, an alkylene bridge of 3 or 4 carbon atoms containing a double bond, or an alkylene bridge of the type set forth above substituted with hydroxy lower alkoxy, lower alkyl or di-lower alkyl and their pharmaceutically acceptable salts, wherein said aryl is a member of the group consisting of phenyl or naphthyl, and said heteroaryl is a member of the group consisting of pyridyl, thienyl, furyl, indolyl, benzthienyl, imidazolyl or thiazrylyl. 2. A compound according to claim 1, in which the carbon atom bearing the substituent is in the 5 configuration and the other absolute configurations belong to the 6-amino acids. 3. Compound with formula 0 I/ , ^R 1 *h ( ^J *5 0 H I? - with - C -NH I -CH -with II -N - - C - 1 C - Rg ^R 2 0 ^? 7 in which R and Rg may independently of one another represent hydroxy, lower alkyl lower alkenyloxy, aryl lower alkyloxy, amino, di-lower alkylamino lower alkoxy, acylamino lower alkoxy or acyloxy lower alkoxy; R<1 represents hydrogen, alkyl containing 1 to 20 carbon atoms, including branching, cyclic and unsaturated alkyl groups; substituted lower alkyl, wherein the substituent is halo, hydroxy, lower alkoxy, aryloxy, amino, lower alkylamino, dinitrile alkylamino, acylamino, arylamino, guanidino, imidazoyl, indolyl, mercapto, lower alkylthio, arylthio, carboxy, carboxamido or carbonyl alkoxy, phenyl, substituted phenyl, wherein the substituent is lower alkyl, lower alkoxy or halo; arylloweralkyl or heteroarylloweralkyl, arylloweralkenyl, or heteroarylloweralkenyl, substituted arylloweralkyl, substituted heteroarylloweralkenyl, substituted arylloweralkenyl or substituted heteroarylloweralkenyl, wherein the substituent is halo, dihalo, loweralkyl, hydroxy, loweralkoxy, amino, aminomethyl, acylamino, dinitrilealkylamino, loweralkylamino, carboxyl, haloalkyl cyano or sulfonamido, arylloweralkyl or heteroarylloweralkenyl, substituted in the alkyl part with amino or acylamino groups; R 1 and R ₇ represent hydrogen; R ₃ represents lower alkyl amino lower alkyl imidazolyl lower alkyl, halo lower alkyl; 5 are linked together to carbon R ₄ and R containing from 2 to 4 form an alkylene atom or an alkylene bridge, BRIDGE containing 2 or 3 carbon atoms, and one sulfur atom or an alkylene bridge containing from 3 to 4 carbon atoms, containing a double bond _and/ or an alkylene bridge of the above type, substituted with hydroxy, lower alkoxy or lower alkyl, or pharmaceutically acceptable salts thereof, wherein said aryl is selected from the group consisting of phenyl or naphthyl, and said heteroaryl is selected from the group consisting of pyridyl, thienyl, furyl, indolyl, benzthienyl, imidazoyl, or thiazolyl. 4. A compound according to claim 3, wherein the carbon - 4 atom, which carries the substituent R-^e in 6 configuration and the other absolute configurations belong to L-amino acids. 5. A compound according to claim 3, wherein R1 and R2 are joined together and the carbon and nitrogen atoms to which they are attached form a ring of the formula ΟΰΰΖζ in which Y represents CH^, a sulfur atom or CH-OCH ₃ and Rp Rg and R? have the meanings specified in claim 3. 6. Compounds according to claim 5, in which the carbon atom bearing the denominator has the S configuration and the other absolute configurations belong to the L-amino acids. 7. Compound with formula 0 % ^R 4 is ₅ o 1' I 1 1 II R - С -C -NH-CH - - With -N II -C-C -Rg 0 ^R 7 in which R and Rg may independently of one another represent hydroxy, lower alkoxy, lower allenyloxy, aryllower alkyloxy, amino, dilower alkylamino lower alkoxy, acylamino lower alkoxy or acyloxy lower alkoxy; 1-1 means alkyl containing 1 to 8 carbon atoms, substituted lower alkyl, the alkyl group having 1 to 5 carbon atoms and the substituent is amino, arylthio, aryloxy, arylamino, arylalkyl or heteroarylalkyl, the alkyl moiety having 1 to 3 carbon atoms, substituted arylalkyl or heteroarylalkyl, the alkyl moiety having 1 to 3 carbon atoms and the substituent(s) is halo, dihalo, amino, aminoalkyl, hydroxy, lower alkoxy or lower alkyl; ^and ^7 represent hydrogen; R ₃ represents lower alkyl or amino lower alkyl; R^ and Rg are bonded together to both the carbon and nitrogen atoms to which they are attached, forming a ring of formula wherein Y represents CH ₂ , a sulfur atom or CH-OCH ₃ or pharmaceutically acceptable salts thereof, wherein said aryl is selected from the group consisting of phenyl or naphthyl, and said heteroaryl is selected from the group consisting of pyridyl, thienyl, furyl, indole, benzyl, IMIDO30YL, or THIO30YL. 8. Compounds according to claim 7, in which the carbon atom bearing the substituent R1 is in the S configuration and the other absolute configurations belong to the L-amino acids. 9. Compound with formula ^{0 R} 1 ^R 3 ^R 4 ^R 5 i· I II * R -CC-NH-CH-CNCCR ₆ ' II R ₂ 0R in which R and Rg independently of each other represent hydroxy, lower alkoxy, arylalkyloxy; R? and Ry represent hydrogen; _R3 represents methyl, aminolower alkyl; R1 and Rg are bonded together and with the carbon and nitrogen atoms to which they are attached, form proline, 4-thiaproline or 4-methoxyproline and R^ represents alkyl containing from 1 to 8 carbon atoms, substituted lower alkyl, the alkyl group having 1 to 5 carbon atoms and the substituent is amino, arylthio, aryloxy, arylalkyl or heteroarylalkyl, the alkyl moiety having 1 to 3 carbon atoms, substituted arylalkyl or heteroarylalkyl, the alkyl group having 1 to 3 carbon atoms and the substituent(s) are halo, dihalo, amino, aminoalkyl, hydroxy, lower alkoxy or lower alkyl and pharmaceutically acceptable salts thereof, wherein said aryl is selected from the group consisting of phenyl or naphthyl, and said heteroaryl is selected from the group consisting of pyridyl, thienyl, furyl, indolyl, benzthienyl, imidazoyl or thiazolyl. 10. Compounds according to claim 9, in which the carbon atom carrying the substituent is in the 5 configuration and the other absolute configurations belong to the L-amino acids. 11. A compound according to claim 9, which is N-(1-carboxy-3-phenylpropyl)-13-alanyl-L-proline. 12. A compound according to claim 9, which is N-[i-ethoxycarbonyl-3-phenylpropyl]-L-alanyl-L-proline. 13. A compound according to claim 9, which is T<-[1-ethoxycarbonyl-4-methylpentyl]-L-alanyl-L-proline. 14. A compound according to claim 9, which is N-[1-carboxy-5-aminopentyl]-L-alanyl-L-proline. 15. A compound according to claim 9, which is N-Y -[1-carboxy-3-phenylprolyl]-L-lysyl-L-proline. 16. A compound according to claim 9, which is N-[1-ethoxycarbonyl-3-phenylpropyl]-L-lysyl-L-proline. 17. A compound according to claim 9, which is N-(1-carboxy-3-(3-indolyl)-propyl)-L-lysyl-L-proline. 18. A compound according to claim 9, which is L1-γ-[1-carboxy-3-(4-chlorophenyl)propyl]-L-lysyl-L-proline. 19. A compound according to claim 9, which is 1N-E-(1-carboxy-2-phenylthioethyl)-L-lysyl-L-proline. 20. A compound according to claim 9, which is N-[(1-carboxy-3-(4-chlorophenyl)propyl)-lysyl-trans-4-methoxy-L-proline. 21. A compound according to claim 9, which is N-(-[1-carboxy-5-aminopentyl]-1-lysyl-L-proline. 22. A compound according to claim 9, which is N-(1-carboxy-3-phenylpropyl)-L-ornithyl-L-proline. 23. A compound according to claim 9, which is ethyl N-(1-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-prolinate hydrochloride. 24. A compound according to claim 9, which is N-(1-(ethoxycarbonyl)-3-(4-imidazolyl)propyl)-L-alanyl-L-proline. 25. A compound according to claim 9, which is L1-(1-(carboxy-3-(4-imidazolyl)propyl)-L-lysyl-1'-proline. 26. The compound N-[1-(6)-carboxy-3-phenylpropyl]-B-alanyl-L-proline. 27. The compound N-(1-(5)-ethoxycarbonyl-3-phenyl-propyl)-L-alanyl-L-proline. 28. The compound N-(1/5)-ethoxycarbonyl-3-phenyl-propyl)-L-alanyl-L-proline maleate. 29. The compound NV-[I/S]-carboxy-3-phenyl-propyl]-L-lysyl-L-proline. 30. The compound ethyl N-(1/5)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-prolinate hydrochloride. 31. The compound N-2-[1/5]-ethoxycarbonyl-3-phenylpropyl]L-lysyl-L-proline. 32. A pharmaceutical composition for the treatment of high blood pressure, comprising a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a compound of formula |I ϊ I I P RCC-NH-CH-CNCC-Rr > II I ⁶ ξ ₂ 0 r ₇ in which R and Rg may be the same or different and represent hydroxy lower alkoxy, lower alkenyloxy, dinitrialkylamino lower alkoxy, acylamino lower alkoxy, acyloxy lower alkoxy, aryloxy, aryllower alkyloxy, substituted aryloxy or substituted aryllower alkoxy, the substituent being methyl, halo or methoxy, amino, lower alkyldintrialkylamino. amino, aryl-lower alkylamino, hydroxyamino; R.J represents hydrogen, alkyl containing 1 to 20 carbon atoms, including branched, cyclic or unsaturated alkyl groups; substituted lower alkyl, such as . the substituent is halo, hydroxy, lower alkoxy, aryloxy, amino, lower alkylamino, dinitrilealkylamino, acylamino, arylamino, guanidino, imidazolyl, indolyl, mercapto, lower alkylthio, arylthio, carboxy, carboxamido, carbonylalkoxy, phenyl, substituted phenyl, wherein the substituent is lower alkyl, lower alkoxy or halo, arylnitrilealkyl or heteroarylnitrilealkyl, arylnitrilealkenyl or heteroarylnitrilealkenyl, substituted arylnitrilealkyl, substituted heteroarylnitrilealkyl, substituted arylnitrilealkenyl or substituted heteroarylnitrilealkenyl, wherein the substituent is halo or dihalo, lower alkyl, hydroxy, lower alkoxy, amino, aminomethyl, acylamino, dinitrilealkylamino, lower alkylamino, carboxyl, halonitrilealkyl, cyano and so on sulfonamido; aryl-lower alkyl or heteroaryl-lower alkyl substituted in the alkyl moiety with amino or acylamino; and R ₇ represents hydrogen or lower alkyl; R ₃ represents hydrogen, lower alkyl, phenyllower alkyl, aminomethyl phenyllower alkyl, hydroxyphenyllower alkyl, hydroxylower alkyl, acetylaminolower alkyl, acylaminolower alkyl, aminolower alkyl, dimethylaminolower alkyl, guanidinolower alkyl, halolower alkyl, imidazalyllower alkyl, indolyllower alkyl, mercantolower alkyl, lower alkylthiolower alkyl; represents hydrogen or lower alkyl; 1-5 represents hydrogen, 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; and are linked together and with the carbon and nitrogen atoms to which they are attached, form an alkylene bridge containing 2 to 4 carbon atoms, an alkylene bridge containing 2 or 3 carbon atoms and one sulfur atom, an alkylene bridge containing 3 to 4 carbon atoms containing a double bond or an alkylene bridge of the above type substituted with hydroxy, lower alkoxy, or lower alkylene and pharmaceutically acceptable salts thereof, wherein said aryl is selected from the group consisting of phenyl or naphthyl and said heteroaryl is selected from the group consisting of pyridyl, thienyl, furyl, indolyl, benzthienyl, imidazoyl, or thiazolyl. 33. A pharmaceutical composition for the treatment of high blood pressure, which comprises a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a compound of formula 0 *1 R ₃ 1^5 II I I 11 RCC-NH-CH-C'NCC-Rzi li I and ^b 1^2 θ in which P and Rg independently represent hydroxy, lower alkoxy, lower alkenyloxy, arylalkyloxy, amino, dialkylamino lower alkoxy, acylamino lower alkoxy or acyloxy lower alkoxy; means alkyl containing 1 to 8 carbon atoms, substituted lower alkyl, the alkyl group having 1 to five carbon atoms and the substituent is amino, arylthio, aryloxy, or arylamino, arylalkyl or heteroarylalkyl, the alkyl moiety having 1 to 3 carbon atoms, substituted arylalkyl or heteroarylalnyl, the alkyl moiety having 1 to 3 carbon atoms and the substituent(s) are halo, dihalo, amino, aminoalkyl, hydroxy, lower alkoxy or lower alkyl; R ₂ and R y represent hydrogen; _1-3 represents lower alkyl or amino lower alkyl; and are bonded together with the carbon and nitrogen atoms to which they are attached and form a ring with the formula wherein Y represents CH ₂ , a sulfur atom or CH-OCH ₃ or pharmaceutically acceptable salts thereof, wherein said aryl is selected from the group consisting of phenyl or naphthyl and said heteroaryl is selected from the group consisting of pyridyl, thienyl, indolyl benzthienyl, imidazoyl or thiazolyl. 34. A pharmaceutical composition for the treatment of high blood pressure, comprising a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a compound of the general formula 0 R-> R ~ Rd Rc k ι ι ³ ) ⁴ Р RCC-NH-CH-CNCCR, ¹ II I I. ^{6 R} 2 0 P _? 0 in which R and R independently of each other denote hydroxy, lower alkoxy, arylalkyloxy; p _and denote hydrogen; R ₃ represents methyl, aminoloweralkyl; R^ and Rg are bonded together with the carbon and nitrogen atoms to which they are attached^ and form proline, 4-thiaproline, 4-methoxyproline and R-I represents alkyl containing 1 to 8 carbon atoms, substituted lower alkyl, wherein the alkyl group has one to five carbon atoms and the substituent is amino, arylthio, aryloxy; arylalkyl or heteroarylalkyl, wherein the alkyl moiety contains 1 to 3 carbon atoms, substituted arylalkyl or heteroarylalkyl, wherein the alkyl moiety contains 1 to 3 carbon atoms and the substituent(s) is halo, dihalo, amino, aminoalkyl, hydroxy, lower alkoxy or lower alkyl, and pharmaceutically acceptable salts thereof, wherein said aryl is selected from the group consisting of phenyl or naphthyl, and said heteroaryl is selected from the group consisting of pyridyl, thienyl, furyl, indolyl, benzthienyl, imidazoyl or thiazolyl. 35. A composition according to claim 34, wherein said compound is [1-carboxy-3-phenylpropyl]-L-alanyl-L-proline. 36. A composition according to claim 34, wherein said compound is N-(1-ethoxycarbonyl-3-phenylpropyl)-N-alanyl-L·proline. 37. A composition according to claim 34, wherein said compound is 1N-[(1-ethoxycarbonyl)-4-methylpentyl-L-alanyl-L-proline. 38. A composition according to claim 34, wherein said compound is N-(1-carboxy-5-aminopentyl)-L-olanyl-L-proline. 39. A composition according to claim 34, wherein said compound is _N -[(1-carboxy-3-phenylpropyl)-L-lysyl-L·-proline. 40. A composition according to claim 34, wherein said compound is N-[1-ethoxycarbonyl-3-phenylpropyl]-L-lysyl-L-proline. 41. A composition according to claim 34, wherein said compound is 1M- _with (-/1-carboxy-3-(3-indolyl)-propyl)-L-lysyl- L-proline. 42. A composition according to claim 34, wherein said compound is _N -[(1-carboxy-3-4-chlorophenyl)propyl]-L·-lysyl-L proline. 43. A composition according to claim 34, wherein said compound is L1-/-/1-carboxy-2-phenylthioethyl/-L-lysyl-L·-proline. 44. A composition according to claim 34, wherein said compound is N-[(1-carboxy-3-(4-chlorophenyl)propyl)-L-lysyltrans-4-methoxy-L-proline. 45. A composition according to claim 34, wherein said compound is M- _is (-[1-carboxy-5-ominopentyl]-L-lysyl-L-proline. 46. A composition according to claim 34, wherein said compound is N _- [(1-carboxy-3-phenylpropyl)-[(1-amino)-3-amino]-[(1-amino)-3-phenylpropyl]-[(1-amino]-amino]-proline. 47. A composition according to claim 34, wherein said compound is ethyl N-(1-ethoxynorbonyl)-3-phenylpropyl)-L-alonyl-L-prolinate hydrochloride. 48. A composition according to claim 34, wherein said compound is _N -[(1-carboxy-3-phenylpropyl)-L-ornithyl-L·proline. 49. A composition according to claim 34, wherein said compound is ethyl L1-(1-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-prolinate hydrochloride. 50. A composition according to claim 34, wherein said compound is N-(1-ethoxycarbonyl-3-(4-imidazolyl)propyl)-L-alanyl-L-proline. 51. A composition according to claim 34, wherein said compound is N-(1-carboxy-3-(4-imidazolyl)propyl)-L-lysyl- L· -proline. 52. A composition according to claim 34, wherein said compound is N-[1(5)-carboxy-3-phenylpropyl]-L-olonyl-L-proline. 53. A composition according to claim 34, wherein said compound is N-(1/5)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl- L· -proline. 54. A composition according to claim 34, wherein said compound is N-(1(5)-ethoxycaronyl-3-phenylpropyl)-L-alanyl- L· -proline maleate. 55. A composition according to claim 34, wherein said compound is N-(-[1/5]-carboxy-3-phenylpropyl]-L-lysyl-L-proline. 56. A composition according to claim 34, wherein said compound is ethyl N-(5-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-prolinate hydrochloride. 57. A composition according to claim 34, wherein said compound is N-(-[1/5]-ethoxycarbonyl-3-phenylpropyl]-L-lysyl-L-proline. 58. A method for treating high blood pressure, which comprises administering to the patient a pharmaceutically effective amount of a compound of formula 0 and *5 0 II RC- -C-NH 1 -sn - c - II - N · - C - 1 C -R ₆ *2 0 ^R 7 in noiato R and Rg are the same or different and represent hydroxy, lower alkoxy, lower alkenyloxy, dinitroalkylamino lower alkoxy, acylamino lower alkoxy, acyloxy lower alkoxy, aryloxy, aryllower alkyloxy, substituted aryloxy or substituted aryllower alkyloxy, wherein the substituent is methyl, halo or methoxy; amino, lower alkylamino, dinitroalkylamino, aryllower alkylamino or hydroxyamino; means hydrogen, alkyl containing 1 to 20 carbon atoms, including branched, cyclic or unsaturated alkyl; substituted lower alkyl, wherein the substituent is halo, hydroxy, lower alkoxy aryloxy, amino, lower alkylamino, dialkylamino, acylamino, arylamino, guanidino, imidazolyl, indolyl, mercapto, lower alkylthio, arylthio, carboxy, carboxamido, carbonylalkyloxy, phenyl, substituted phenyl, wherein the substituent is lower alkyl, lower alkoxy or halo; arylloweralkyl or heteroarylloweralkyl, arylloweralkenyl, or heteroarylloweralkyl, substituted arylloweralkyl, substituted heteroarylloweralkyl, substituted arylloweralkenyl or substituted heteroarylloweralkenyl, wherein the substituent is halo or dihalo, lower alkyl, hydroxy, lower alkoxy, amino, aminomethyl, acylamino, diloweralnylamino, lower alkylomino, carboxyl, haloloweralkyl, cyano or sulfonamido; arylloweralkyl or heteroarylloweralkyl substituted in the alkyl portion with amino or acylamino; R2 and Ry represent hydrogen or lower alkyl; R ₃ represents hydrogen, lower alkyl, phenyl lower alkyl, aminomethyl phenyl lower alkyl, hydroxyphenyl lower alkyl, hydroxylower alkyl, acetylamino lower alkyl, acylamino lower alkyl, amino lower alkyl, dimethylamino lower alkyl, guanidino lower alkyl, halo lower alkyl, imidazolyl lower alkyl, indolyl lower alkyl, mercapto lower alkyl and lower alkylthio lower alkyl; R1 represents hydrogen or lower alkyl; ^5 represents hydrogen, 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, lower alkylthio lower alkyl; ^R 4 and R ₅ may be linked together to form an alkylene bridge containing from 2 to 4 carbon atoms, an alkylene bridge containing from 2 to 3 carbon atoms and one sulfur atom, an alkylene bridge containing from 3 to 4 carbon atoms and a double bond, or an alkylene bridge of the above type substituted with hydroxy, lower alkoxy or lower alkynyl, and pharmaceutically acceptable salts thereof, wherein said aryl is selected from the group consisting of phenyl or naphthyl, and said heteroaryl is selected from the group consisting of pyridyl, thienyl, furyl, indolyl, benz thienyl, imidazolyl or thiazolyl. 59. A method of treating high blood pressure, which comprises administering to the patient a pharmaceutically effective amount of a compound of formula 0 tl у ь R - C - - c - 1 NHCH - c - 1» - N - - C - 1 C - R< 11 ⁶ У 0 ^R 7 0 in which R and Rg independently of each other represent hydroxy, lower alkoxy, arylalkyloxy, R.] means alkyl containing 1 to 8 carbon atoms, substituted lower alkyl, wherein the alkyl group has from one to five carbon atoms and the substituent is amino, arylthio or aryloxy, arylalkyl or heteroarylalkyl, the alkyl portion containing from 1 to 3 carbon atoms, substituted arylalkyl or heteroarylalkyl wherein the alkyl portion contains 1 to 3 carbon atoms and the substituent(s) are halo, dihalo, amino, aminoalkyl, hydroxy, lower alkoxy, lower alkyl; 1?2 and Ry represent hydrogen; R ₃ represents methyl, aminoloweralkyl; and may be linked together and with the carbon and nitrogen atoms to which they are attached to form proline, 4-thialroline, or 4-methoxyproline, or pharmaceutically acceptable salts thereof, wherein said aryl is selected from the group consisting of phenyl or naphthyl, and said heteroaryl is a member selected from the group consisting of pyridyl, thienyl, furyl, indolyl, benzthienyl, imidazoyl, or thiazolyl. 60. The method of claim 59, wherein said compound is N-(1-carboxy-3-phenylpropyl)-L-alanyl-L-proline. 61. The method of claim 59, wherein said compound is L1-(1-ethoxycarbonyl-3-phenylpropyl)-11-alanyl-L-proline. 62. The method of claim 59, wherein said compound is N-(1-ethoxycarbonyl-4-methylpentyl)- N -alanyl-N -proline. 63. The method of claim 59, wherein said compound is N-[1-carboxy-5-aminopentyl]-1,1-alanyl-b proline. 64. The method of claim 59, wherein said compound is N-(1-carboxy-3-phenylpropyl)-L-lysyl-L·-proline. 65. The method of claim 59, wherein said compound is N-(N-ethoxycarbonyl-3-phenylpropyl)-N-lysylN-proline. 66. The method of claim 59, wherein said compound is _N- [1-carboxy-3-(3-indolyl)-propyl]-L-lysyl-L-proline. 67. The method of claim 59, wherein said compound is _N- [1-carboxy-3-(4-chlorophenyl)propyl]-L-lysyl- b-proline. 68. The method of claim 59, wherein said compound is N- _O -(-[1-carboxy-2-phenylthioethyl]-L-lysyl- L-proline. 69. The method of claim 59, wherein said compound is 1N-(-[1-carboxy-3-(4-chlorophenyl)propyl]-L-lysyltrans-4-methoxy-L-proline. 70. The method of claim 59, wherein said compound is N-(-[1-carboxy-5-aminopentyl]-1-lysyl-L-proline. 71. The method of claim 59, wherein said compound is L1-E-[1-carboxy-3-phenylpropyl]-L1-ornithyl-L-proline. 72. The method of claim 59, wherein said compound is ethyl N-ethoxycarbonyl-3-phenylpropyl-L-alanyl L-prolinate hydrochloride. 73. The method of claim 59, wherein said compound is N-[1-carboxy-3-phenylpropyl]-L-ornithyl-L-proline. 74. The method of claim 59, wherein said compound is ethyl N-(1-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-prolinate hydrochloride. 75. The method of claim 59, wherein said compound is 1N-(1-(ethoxycarbonyl)-3-(4-imidazolyl)propyl) L· -alanyl-L· -proline. 76. The method of claim 59, wherein said compound is L1-(1-carboxy-3-(4-imidazolyl)propyl)-L-lysylL·-proline. 77. The method of claim 59, wherein said compound is N-(1-(5)-carboxy-3-phenylpropyl)-L-alanyl-L-proline. 78. The method of claim 59, wherein said compound is N-(1-(6)-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline. 79. The method of claim 59, wherein said compound is N-(1/5)-ethoxycarbonyl-3-phenylpropyl)-alanyl-L·-proline maleate. 80. The method of claim 59, wherein said compound is N-(1-(5)-carboxy-3-phenylpropyl)-L-lysyl-L·proline. 81. The method of claim 59, wherein said compound is ethyl N-[1(5)-ethoxycarbonyl-3-phenyl-propyl]-L-alanyl-L·-pyrolinate hydrochloride. 82. The method of claim 59, wherein said compound is N-(-[1/5]-ethonylcarbonyl-3-phenylpropyl]-L-lysyl-L·-proline.