MERCAPTO-AMIDE DERIVATIVES AS INHIBITORS OF THE NEUTRAL ENDOPEPTIDASE.

TECHNICAL FIELD

This invention relates to new mercapto-amide derivatives and pharmaceutically acceptable salts thereof which are useful as a medicament.

BACKGROUND ART

Some mercapto-amide derivatives have been known as inhibitor enkephalinase which is an enkephalin-degrading enzyme, for example, in EP Patent Application Publication Nos. 0 110 484, 0 115 997, 0 159 254, 0 280 627 and 0 419 327.

DISCLOSURE OF INVENTION

This invention relates to new mercapto-amide _. derivatives and pharmaceutically acceptable salts thereof. More particularly, it relates to new mercapto-amide derivatives and pharmaceutically acceptable salts thereof which possess an inhibitory activity against the neutral endopeptidase (hereinafter NEP), e.g. neutral endopeptidase EC 3. 4. 24. 11, to processes for the preparation thereof, to a pharmaceutical composition comprising the same and to a method for the treatment and/or prevention of various cardiovascular disorders such as hypertension, heart failure, angina pectoris or the like, renal insufficiency, cyclic edema, hyperaldosteronism, hypercalciuria and the like in human beings or animals. Additionally, the object compound is expected to be useful as therapeutical and/or preventive agents for glaucoma, asthma, inflammation, pain, epilepsy, dementia, obesity and gastrointestinal disorders (especially diarrhoea and irritable bowel syndrome); the

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modulation of gastric acid secretion and the treatment of hyperreninaemia.

One object of this invention is to provide new and useful mercapto-amide derivatives which possess an inhibitory activity against NEP.

Another object of this invention is to provide processes for the preparation of said mercapto-amide derivatives and salts thereof.

A further object of this invention is to provide a pharmaceutical composition comprising, as an active ingredient, said mercapto-amide derivatives and pharmaceutically acceptable salts thereof.

Still further object of this invention is to provide a therapeutical method for the treatment and/or prevention of aforesaid diseases in human beings or animals, using said mercapto-amide derivatives and pharmaceutically acceptable salts thereof.

It is well known that NEP is involved in the breakdown of several peptide hormones, including atrial natriuretic peptides (hereinafter, ANP) which has potent vasodilatory, diuretic and natriuritic activities, and enkephalin which is a endogenous morphine-like peptide. Thus, NEP inhibitors can potentiate the biological effects of ANP and enkephalin. Therefore, the compounds inhibiting NEP are useful for the treatment and/or prevention of various cardiovascular disorders such as hypertension, heart failure, angina pectoris or the like, renal insufficiency, cyclic edema, hyperaldosteronism, hypercalαiuria, and the other diseases mentioned above.

The object mercapto-amide derivatives of this invention are new and can be represented by the following general formula [I] :

wherein R is hydrogen or a mercapto-protective group,

2 R is lower alkyl or aryl which may be substituted with lower alkylenedioxy, 3 R is tetrazolyl, thiazolyl or thiadiazolyl, each of which may be substituted with substituent(s) selected from the group consisting of acyl and acyl(lower)alkyl,

A is lower alkylene,

X is lower alkylene or S, and

Y is a single bond or lower alkylene, provided that when R is tetrazolyl or thiazolyl, then Y is lower alkylene, and pharmaceutically acceptable salts thereof.

The object compound [I] or its salt can be prepared by the following processes.

Process 1

i

R -S-A- CONH-Y-R3

[II] [III] [I] or its reactive or its salt or its salt derivative at the carboxy group or a salt thereof

_ A -

fication

Έ 1 3 Deesteri Γ -S-A -Y- a

Lie] lid] or its salt or its salt

Process 4

' R'

X X

1 I -R^3 Amidation T-S-A-CHCONH-Y R 1-S-A-C'HCONH-Y- 3 c

[Id] [Ie] or its reactive or its salt derivative at the carboxy group or a salt thereof

Process 5

_R 2 Introduction of the _R 2

I mercapto-protective |

X X

5 I group _ _> j

H-S-A-CHCONH-Y-R ³ R 3 ¹ .-S-A-CHCONH-Y-R ³

[Ib] [la] or its salt or its salt

¹⁰ wherei.n of which is substituted with substituent(s) selected from the group consisting of - _r - esterified carboxy and esterified carboxy(lower)alkyl,

R, 3 i.s tetrazolyl, thiazolyl or thiadiazolyl, each of which is substituted with substituent(s) selected from the group consisting of

2 _Q carboxy and carboxy(lower)alkyl,

R is tetrazolyl, thiazolyl or thiadiazolyl, each of which is substituted with substituent(s) selected from the group consisting of N-containing heterocycliccarbonyl,

25 N-containing heterocycliccarbonyl(lower)- alkyl, a group of the formula : -CO-Z-OR , wherein Z is amino acid(s) residue, and R is hydrogen or a carboxy protective group, lower alkyl substituted with a group of the formula : -C 4, 4

30 O-Z-OR wherein Z and R are each as defined above, carba oyl and carbamoyl(lower)alkyl, carbamoyl of which may be substituted with substituent(s) selected from the group consisting of lower - _r - alkyl, cyclo(lower)alkyl, aryl,

ar(lower)alkyl, lower alkoxy(lower)alkyl and a heterocyclic group, and R 1, R2, R3, A, X and Y are each as defined above.

In the above and subsequent descriptions of the present specification, suitable examples of the various definitions to be included within the scope of the invention are explained in detail in the following.

The term "lower" is intended to mean a group having 1 to 6 carbon atom(s), unless otherwise provided.

The lower moiety in the term "cyclo(lower)alkyl" is intended to mean a group having 3 to 6 carbon atoms. Suitable lower alkyl moiety in the terms "acyl(lower)alkyl" "esterified carboxy(lower)alkyl", "carboxy(lower)alkyl", "carbamoyl(lower) lkyl", ¹¹ ar(lower)alkyl", "lower alkoxy(lower}alkyl" and "N-containing heterocycliccarbonyKlower)alkyl" may be a straight or branched one such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl or the like, in which preferable one is methyl, ethyl or isopropyl.

Suitable "cyclo(lower)alkyl" may be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. Suitable "aryl" may be phenyl, naphthyl, phenyl substituted with lower alkyl [e.g. tolyl, mesityl, cumenyl, xylyl, diethylphenyl, diisopropylphenyl, di-tert-butylphenyl, etc.] and the like, in which preferable one is- phenyl or tolyl. Suitable "ar(lower)alkyl" may be benzyl, phenethyl, diphenylmethyl, triphenylmethyl, naphthylmethyl, and the like, in which preferable one is benzyl.

Suitable lower alkoxy moiety in the term "lower alkoxy(lower)alkyl" may be methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy and the like,

in which preferable one is methoxy.

Suitable "lower alkylene" may be a straight or branched one such as methylene, ethylene, trimethylene, propylene, tetramethylene, pentamethylene, hexa ethylene, ethylethylene, or the like, in which preferable one is methylene.

Suitable "lower alkylenedioxy" may be a straight or branched one such as methylenedioxy, ethylenedioxy, trimethylenedioxy, dimethylmethylenedioxy, propylenedioxy, or the like, in which preferable one is methylenedioxy. Suitable "mercapto-protective group" may be lower alkyl [e.g. tert-butyl, etc.], lower alkoxy(lower)alkyl [e.g. methoxymethyl, isobutoxymethyl, etc.], substituted or unsubstituted ar(lower)alkyl [e.g. benzyl, methoxybenzyl, nitrobenzyl, diphenylmethyl, bis(methoxyphenyl)methyl, triphenylmethyl, etc.], substituted or unsubstituted aryl [e.g. phenyl, dinitrophenyl, etc.], acyl such as lower alkanoyl [e.g. formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, etc.], lower alkoxycarbonyl [e.g. methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl, pentyloxycarbonyl, hexyloxycarbonyl, etc.], aroyl [e.g. benzoyl, etc.], substituted or unsubstituted ar(lower)alkoxycarbonyl [e.g. benzyloxycarbonyl, methoxybenzyloxycarbonyl, etc.], a group of the formula: , wherein R ² , R ³ , A, X and Y are each as defined above, and the like, in which preferable one is lower alkanoyl or aroyl and the most preferable one is acetyl or benzoyl.

Suitable "acyl" and acyl moiety in the term

"acyl(lower)alkyl" may include carboxy; esterified

4 carboxy; a group of the formula : -CO-Z-OR , wherein Z 4 and R are each as defined above; carbamoyl optionally

substituted with substituent(s) selected from the group consisting of lower alkyl, cyclo(lower)alkyl, aryl, ar(lower)alkyl, lower alkoxγ(lower)alkyl and a heterocyclic group ; lower alkanoyl; aroyl; a heterocycliccarbonyl; lower alkylsulfonyl; and the like, in which preferable one is carboxy, esterified carboxy, a group of the formula : -CO-Z-OR 4, wherein Z and R4 are each as defined above or carbamoyl optionally substituted with substituent(s) selected from the group consisting of lower alkyl, ar(lower)alkyl and lower alkoxγ(lower)alkyl .

The esterified carboxy may be substituted or unsubstituted lower alkoxycarbonyl [e.g. methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, tert-butoxycarbonyl, hexyloxycarbonyl, 2-iodoethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, etc.], substituted or unsubstituted aryloxycarbonyl [e.g. phenoxycarbonyl, 4-nitrophenoxycarbonyl,

2-naphthyloxycarbonyl, etc.], substituted or unsubstituted ar(lower)alkoxycarbonyl [e.g. benzyloxycarbonyl, phenethyloxycarbonyl, benzhydryloxycarbonyl,

4-nitrobenzyloxycarbonyl, etc.] and the like, in which preferable one is lower alkoxycarbonyl or ar(lower) lkoxycarbonyl.

The lower alkanoyl may be formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl and the like.

The aroyl may be benzoyl, naphthoyl, benzoyl substituted with lower alkyl [e.g. toluoyl, xyloyl, etc.] and the like. Suitable "heterocyclic group" and heterocyclic moiety in the term "heterocycliccarbonyl" may include saturated or unsaturated, monocyclic or polycyclic one containing at least one hetero atom such as nitrogen atom, oxygen atom or sulfur atom. The preferred examples of thus defined "heterocyclic

group" may be unsaturated, 3 to 8-membered, more preferably 5 or 6-membered heteromonocyclic group containing 1 to 4-nitrogen atom(s), for example, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyridyl N-oxide, dihydropyridyl, tetrahydropyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazinyl, triazolyl, tetrazinyl, tetrazolyl, etc. ; saturated, 3 to 8-membered, more preferably 5 or

6-membered heteromonocyclic group containing 1 to 4 nitrogen atom(s), for example, pyrrolidinyl, imidazolidinyl, piperidino, piperazinyl, etc.; unsaturated, condensed heterocyclic group containing

1 to 5 nitrogen atom(s), for example, indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl, etc.; unsaturated, 3 to 8-membered heteromonocyclic group containing 1 to 2 oxygen atom(s) and 1 to 3 nitrogen atom(s) for example, oxazolyl, isoxazolyl, oxadiazolyl, etc. ; saturated, 3 to 8-membered heteromonocyclic group containing 1 to 2 oxygen atom(s) and 1 to 3 nitrogen atom(s), for example, morpholino, sydnonyl, etc.; unsaturated, condensed heterocyclic group containing

1 to 2 oxygen atom(s) and 1 to 3 nitrogen atom(s), for example, benzoxazolyl, benzoxadiazolyl, etc.; unsaturated, 3 to 8-membered heteromonocyclic group containing 1 to 2 sulfur atom(s) and 1 to 3 nitrogen atom(s), for example, thiazolyl, isothiazolyl, thiadiazolyl, etc.; unsaturated, 3 to 8-membered heteromonocyclic group containing 1 to 2 sulfur atom(s), for example, thienyl, etc. ; unsaturated condensed heterocyclic group containing 1 to 2 sulfur atom(s) and 1 to 3 nitrogen atom(s), for example, benzothiazolyl, benzothiadiazolyl, etc. ;

unsaturated, 3 to 8-membered heteromonocyclic group containing an oxygen atom, for example, furyl, etc.; unsaturated, condensed heterocyclic group containing 1 to 2 sulfur atom(s), for example, benzothienyl, etc.; unsaturated, condensed heterocyclic group containing 1 to 2 oxygen atom(s), for example, benzofuranyl, etc.; or the like.

Suitable "amino acid(s) residue" means a bivalent residue derived from amino acid(s), and such amino acid may be neutral amino acid such as glycine, D- or

L-alanine, β-alanine, D- or L-valine, D- or L-leucine, D- or L-isoleucine, D- or L-serine, D- or L-threonine, D- or L-cysteine, D- or L-methionine, D- or L-phenylalanine, D- or L-tryptophan, D- or L-tγrosine, D- or L-proline, D- or L-4-hγdroxyproline, D- or L-pyroglutamic acid, acidic amino acid such as D- or L-glutamic acid, D- or L-aspartic acid, D- or L-β-aspartic acid, D- or L-glutamine, D- or L-asparagine, and basic amino acid such as D- or L-lγsine, D- or L-arginine, D- or L-histidine, D- or L-ornithine, and combination of two of such amino acid, in which preferable one is phenylalanine.

Suitable "carboxy protective group" may include a conventional protective group, which is used in the field of amino acid and peptide chemistry, that may be lower alkyl as mentioned above, aryl (e.g. phenyl, tolyl, naphthyl, etc.), ar(lower)alkyl (e.g. benzyl, phenethyl, etc.), and the like, in which preferable one is tert-butyl.

Suitable "N-containing heterocycliccarbonyl" and N-containing heterocyclic moiety in the term "N-containing heterocycliccarbonyl(lower)alkyl" may be pyrrolidinylcarbonyl, imidazolidinylcarbonyl, piperidinocarbonyl, piperazinylcarbony1, N-methylpiperazinylcarbonyl or the like. Preferable compound [I] is one which has hydrogen or

lower alkanoyl for R ; phenyl optionally substituted with

2 methylenedioxy for R ; tetrazolyl substituted with carboxy(lower)alkyl or esterified carboxy(lower)alkyl, thiazolyl substituted with carboxy, esterified carboxy, carboxy(lower)alkyl or esterified carboxy(lower)alkyl, or thiadiazolyl substituted with carboxy or esterified

3 carboxy for R ; methylene for A; methylene for X and a single bond for Y; or hydrogen, lower alkanoyl or aroyl for R 1; phenyl for R2; tetrazolyl substituted with carbamoyl(lower)alkyl or lower alkylcarbamoyKlower)alkyl 3 for R ; methylene for A; methylene for X and a single bond for Y.

More preferable compound [I] is one which has hydrogen for R 1, phenyl for R2, carboxymethyltetrazolyl, carboxymethylthiazolyl, carboxythiazolyl or

3 carboxythiadiazolyl for R , methylene for A, methylene for

X and a single bond for Y; or acetyl for R , phenyl for

R 2, methylcarbamoylmethyltet*razolyl or

3 dimethylcarbamoylmethyltetrazolyl for R , methylene for A, methylene for X and a single bond for Y.

Suitable pharmaceutically acceptable salts of the object compound [I] are conventional non-toxic salts and include a metal salt such as an alkali metal salt [e.g. sodium salt, potassium salt, etc.] and an alkaline earth metal salt [e.g. calcium salt, magnesium salt, etc.], an ammonium salt, an organic base addition salt [e.g. trimethylamine salt, triethyla ine salt, etc.] or the like.

The processes for preparing the object compound [I] are explained in detail in the following.

Process 1

The compound [I] or its salt can be prepared by reacting a compound [II] or its reactive derivative at the -carboxy group or a salt thereof with a compound [III] or

its salt.

Suitable salts of the compound [II] and its reactive derivative at the carboxy group can be referred to the same salt as exemplified for the compound [I]. Suitable salt of the compound [III] may be an acid addition salt such as an inorganic acid addition salt [e.g. hydrochloride, hydrobromide, sulfate, phosphate, etc.], an organic addition salt [e.g. formate, acetate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, toluenesulfoante, etc.] or the like.

Suitable reactive derivative at the carboxy group of the compound [II] may include an acid halide, an acid anhydride, an activated amide, an activated ester, and the like. Suitable examples of the reactive derivatives may be an acid chloride; an acid azide; a mixed acid anhydride with an acid such as substituted phosphoric acid [e.g. dialkylphosphoric acid, phenylphosphoric acid, diphenylphosphoric acid, dibenzylphosphoric acid, halogenated phosphoric acid, etc.], dialkylphosphorous acid, sulfurous acid, thiosulfuric acid, sulfuric acid, sulfonic acid [e.g. methanesulfonic acid, etc.], aliphatic carboxylic acid [e.g. acetic acid, propionic acid, butyric acid, isobutyric acid, pivalic acid, pentanoic acid, isopentanoic acid, 2-ethylbutyric acid, trichloroacetic acid; etc.] or aromatic carboxylic acid [e.g. benzoic acid, etc.]; a symmetrical acid anhydride; an activated amide with imidazole, 4-substituted imidazole, dimethylpyrazol , triazole or tetrazole; or an activated ester [e.g. cyanomethyl ester, methoxymethyl ester, dimethyliminomethyl [(CH ₃ ) ₂ N=CH-] ester, vinyl ester, propargyl ester, p-nitrophenyl ester, 2,4-dinitrophenyl ester, trichlorophenyl ester, pentachlorophenyl ester, mesylphenyl ester, phenylazophenyl ester, phenyl thioester, p-nitrophenyl thioester, p-cresyl thioester, carboxymethyl thioester, pyranyl ester, pyridyl ester.

piperidyl ester, 8-quinolyl thioester, etc.], or an ester with an N-hydroxy compound [e.g. N,N-dimethylhydroxylamine, l-hγdroxy-2-(1H)-pyridone, N-hydroxysuccinimide, N-hydroxyphthalimide, 1-hydroxy-lH-benzotriazole, etc.], and the like. These reactive derivatives can optionally be selected from them according to the kind of the compound [II] to be used.

The reaction is usually carried out in a conventional solvent such as water, alcohol [e.g. methanol, ethanol, etc.], acetone, dioxane, acetonitrile, chloroform, methylene chloride, ethylene chloride, tetrahydrofuran, ethyl acetate, N,N-dimethγlformamide, pyridine or any other organic solvent which does not adversely influence the reaction. These conventional solvent may also be used in a mixture with water.

In this reaction, when the compound [II] is used in a free acid form or its salt form, the reaction is preferably carried out in the presence of a conventional condensing agent such as N,N'-dicyclohexylcarbodiimide; N-cγclohexyl-N'-morpholinoethylcarbodiimide;

N-cγclohexγl-N'-(4-diethγlaminocyclohexyl)carbodiimide ; N,N*-diethylcarbodiimide, N,N'-diisopropylcarbodiimide; N-ethγl-N'-(3-dimethylaminopropγl)carbodiimide; N,N'-carbonγlbis-(2-methylimidazole) ; pentamethγleneketene-N-cyclohexylimine; diphenγlketene-N-cyclohexylimine; ethoxyacetylene; 1-alkoxy-l-chloroethγlene; trialkyl phosphite; ethyl polyphosphate; isopropyl polyphosphate; phosphorus oxychloride (phosphoryl chloride); phosphorus trichloride; diphenyl phosphorylazide; diphenylphosphinic chloride; thionyl chloride; oxalyl chloride; lower alkyl haloformate [e.g. ethyl chloroformate, isopropyl chloroformate, etc.]; triphenylphosphine; 2-ethyl-7-hydroxγbenzisoxazolium salt; 2-ethyl-5-(m-sulfophenγl)isoxazolium hydroxide intramolecular salt; l-(p-chlorobenzenesulfonyloxy)-6-

chloro-lH-benzotriazole; so-called Vilsmeier reagent prepared by the reaction of N,N-dimethylformamide with thionyl chloride, phosgene, trichloromethyl chloroformate, phosphorus oxychloride, etc.; or the like. The reaction may also be carried out in the presence of an inorganic or organic base such as an alkali metal bicarbonate, tri(lower)alkylamine, pyridine, N-(lower)alkylmorpholine, ,N-di(lower)alkylbenzylamine, or the like. The reaction temperature is not critical, and the reaction is usually carried out under cooling to heating.

Process 2

The compound [lb] or its salt can be prepared by subjecting a compound [la] or its salt to elimination reaction of the mercapto-protective group.

Suitable salts of the compounds [la] and [lb] may be the same as those exemplified for the compound [I].

The reaction is carried out in accordance with a conventional method such as hydrolysis or the like. The hydrolysis is preferably carried out in the presence of a base or an acid including Lewis acid.

Suitable base may include an inorganic base and an organic base such as an alkali metal [e.g. sodium, potassium, etc.], an alkaline earth metal [e.g. magnesium, calcium, etc.], the hydroxide or carbonate or bicarbonate, thereof, ammonia, cysteamine, trialkylamine [e.g. trimethylamine, triethylamine, etc. ], picoline, 1,5-diazabicyclo[4.3.0]non-5-ene, 1,4-diazabicγclo[2.2.2]- octane, l,8-diazabicyclo[5.4.0]undec-7-ene, or the like. Suitable acid may include an organic acid [e.g. formic acid, acetic acid, propionic acid, trichloroacetic acid, trifluoroacetic acid, etc.] and an inorganic acid [e.g. hydrochloric acid, hydrobromic acid, sulfuric acid, etc.]. The reaction is usually carried out in a solvent such

as water, an alcohol [e.g. methanol, ethanol, etc.], methylene chloride, tetrahydrofuran, a mixture thereof or any other solvent which does not adversely influence the reaction. A liquid base or acid can be also used as the solvent. The reaction temperature is not critical and the reaction is usually carried out under cooling to warming.

In this reaction, in case that the compound [la] having tetrazolyl, thiazolyl or thiadiazolyl, each of which is substituted with substituent(s) selected from the group consisting of esterified carboxy and esterified

3 carboxy(lower)alkyl for R is used as a starting compound, the compound [lb] having tetrazolyl, thiazolyl or thiadiazolyl, each of which is substituted with substituent(s) selected from the group consisting of

3 carboxy and carboxy(lower)alkyl for R may be obtained according to reaction conditions. This case is also included within the scope of the present reaction.

Process 3 The compound [Id] or its salt can be prepared by subjecting a compound [Ic] or its salt to deesterification reaction.

Suitable salts of the compounds [Ic] and [Id] may be the same as those exemplified for the compound [I]. This reaction can be carried out in substantially the same manner as Process 2, and therefore the reaction mode and the reaction condition [e.g. solvent, reaction temperature, etc.] of this reaction are to be referred to those as explained in Process 2. In this reaction, in case that the compound [Ic] ι having acyl for R is used as a starting compound, the compound [Id] having hydrogen for R may be obtained according to reaction conditions. This case is also included within the scope of the present reaction.

Process 4

The compound [IeJ or its salt can be prepared by reacting a compound [Id] or its reactive derivative at the carboxy group or a salt thereof with an amine. Suitable salts of the compounds [Ie] and [Id] and its reactive derivative at the carboxy group may be the same as those exemplified for the compound [I].

Suitable "amine" may be ammonia, lower alkylamine, cyclo(lower)alkylamine, arylamine, ar(lower)alkylamine, lower alkoxy(lower)alkylamin , amine substituted with a heterocyclic group, an amino acid, an amino acid ester,

N-containing heterocyclic compound and the like.

The lower alkylamine may be mono or di(lower)alkylamine such as methylamine, ethylamine, propylamine, isopropylamine, butylamine, isobutylamine, pentylamine, hexylamine, dimethylamine, diethylamine, dipropylamine, dibutylamine, di-isopropylamine, dipentylamine, dihexylamine or the like, in which preferable one is methylamine or dimethylamine. The arylamine may be aniline, naphthylamine and the like. The cγclo(lower)alkylamine may be cyclopropylamine, cyclobutylamine, cyclopentylamine, cyclohexylamine and the like, in which preferable one is cyclopropylamine.

The amino acid may be glycine, alanine, β-alanine, phenylalanine, isoleucine, tyrosine and the like, in which preferable one is phenylalanine.

The amino acid ester may be lower alkyl ester of above-mentioned amino acid and the like, in which preferable one is phenylalanine tert-butyl ester. The ar(lower)alkylamine may be benzylamine, phenylethylamine, phenylpropylamine and the like, in which preferable one is benzylamine.

The lower alkoxy(lower)alkylamine may be methoxvmethylamine, methoxyethylamine, ethoxymethylamine, ethoxyethylamine and the like, in which preferable one is

methoxyethylamine.

The amine substituted with a heterocyclic group may be one substituted with a heterocyclic group as afore-mentioned such as aminothiazole, aminothiadiazole, aminotriazole, aminotetrazole or the like.

The N-containing heterocyclic compound may be saturated 5 or 6-membered N-, or N- and S-, or N- and O- containing heterocyclic compound such as pyrrolidine, imidazolidine, piperidine, piperazine, N-(lowerJalkylpiperazine [e.g. N-methylpiperazine,

N-ethylpiperazine, etc.], morpholine, thiomorpholine or the like.

This reaction can be carried out in substantially the same manner as Process 1, and therefore the reaction mode and the reaction condition [e.g. solvent, reaction temperature, etc.] of this reaction are to be referred to those as explained in Process 1.

In this reaction, in case that the compound [Id] having acyl for R is used as a starting compound, the compound [Ie] having hydrogen for R may be obtained according to reaction conditions. This case is also included within the scope of the present reaction.

Process 5 The compound [la] or its salt can be prepared by subjecting a compound [lb] or its salt to introduction reaction of the mercapto-protective group.

Suitable salts of the compounds [la] and [lb] may be the same as those exemplified for the compound [I]. Suitable introducing agent of the mercapto-protective group used in this reaction may be alkylating agent, which is capable of introducing the alkyl group as afore-mentioned such as isobutylene, lower alkoxy(lower)alkyl halide [e.g. methoxymethyl chloride, isobutoxymethyl chloride, etc.], substituted or

unsubstituted ar(lower)alkyl halide [e.g. benzyl chloride, methoxybenzyl chloride, nitrobenzyl chloride, etc.] or the like, acylating agent, which is capable of introducing the acyl group as afore-mentioned, such as carboxylic acid, carbonic acid, sulfonic acid, carbamic acid and their reactive derivative, for example, an acid halide, an acid anhydride, an activated amide, an activated ester, isocyanate, and the like. Preferable example of such reactive derivative may include acid chloride, acid bromide, a mixed acid anhydride with an acid such as substituted phosphoric acid (e.g. dialkylphosphoric acid, phenylphosphoric acid, diphenylphosphoric acid, dibenzylphosphoric acid, halogenated phosphoric acid, etc. ) , dialkylphosphorous acid, sulfurous acid, thiosulfuric acid, sulfuric acid, alkyl carbonate (e.g. methyl carbonate, ethyl carbonate, propyl carbonate, etc.), aliphatic carboxylic acid (e.g. pivalic acid, pentanoic acid, isopentanoic acid, 2-ethylbutyric acid, trichloroacetic acid, trifluoroacetic acid, etc.), aromatic carboxylic acid (e.g. benzoic acid, etc.), a symmetrical acid anhydride, an activated acid amide with a heterocyclic compound containing imino function such as imidazole, 4-substituted imidazole, dimethylpyrazole, triazole and tetrazole, an activated ester (e.g. p-nitrophenyl ester, 2,4-dinitrophenyl ester, trichlorophenyl ester, pentachlorophenyl ester, mesylphenyl ester, phenylazophenyl ester, phenyl thioester, p-nitrophenyl thioester, p-cresyl thioester, carboxymethyl thioester, pyridyl ester, piperidinyl ester, 8-guinolyl thioester, or an ester with a N-hydroxy compound such as N,N-dimethylhydroxylamine, l-hydroxy-2-(1H)-pyridone, N-hydroxysuccinimide, N-hydroxyphthalimide, 1-hγdroxybenzotriazole, l-hydroxγ-6-chlorobenzotriazole, etc.), isocyanate, and the like.

This reaction is preferably conducted in the presence of an organic or inorganic base such as alkali metal (e.g. lithium, sodium, potassium, etc.), alkaline earth metal (e.g. calcium, etc.), alkali metal hydride (e.g. sodium hydride, etc.), alkaline earth metal hydride (e.g. calcium hydride, etc.), alkali metal hydroxide (e.g. sodium hydroxide, potassium hydroxide, etc.), alkali metal carbonate (e.g. sodium carbonate, potassium carbonate, etc.), alkali metal hydrogen carbonate (e.g. sodium hydrogen carbonate, potassium hydrogen carbonate, etc.), alkali metal alkoxide (e.g. sodium methoxide, sodium ethoxide, potassium tert-butoxide, etc.), alkali metal alkanoic acid (e.g. sodium acetate, etc.), trialkylamine (e.g. triethylamine, etc. ) , pyridine compound (e.g. pyridine, lutidine, picoline, 4-N,N-dimethylaminopyridine, etc. ) , quinoline, and the like.

In case that the acylating agent is used in a free form or its salt in this reaction, the reaction.is preferably conducted in the presence of a conventional condensing agent such as a carbodiimide compound [e.g. N,N'-dicγclohexγlcarbodiimide, N-cyclohexyl-N'-(4- diethylaminocyclohexyl)carbodiimide,

N,N'-diethylcarbodiimide, N,N'-diisopropylcarbodiimide, N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide, etc. ] , a ketenimine compound (e.g. N,N'-carbonylbis(2- methylimidazole) , pentamethyleneketene-N-cyclohexγlimine, diphenylketene-N-cyclohexylimine, etc. ) ; an olefinic or acetylenic ether compound (e.g. ethoxyacetylene, β-cyclovinylethyl ether), a sulfonic acid ester of N-hydroxybenzotriazole derivative [e.g. l-(4-chlorobenzenesulfonyloxy)-6-chloro-lH-benzotriazole, etc. ] or the like.

The reaction is usually conducted in a conventional solvent which does not adversely influence the reaction such as dioxane, chloroform, dichlormethane,

tetrahydrofuran, pyridine, benzene, N,N-dimethylformamide, etc. , and further in case that the base or the introducing agent of the mercapto-protective group is in liquid, it can be used as a solvent. The reaction temperature is not critical and the reaction can be carried out under cooling to heating.

In this reaction, in case that the compound [lb] having tetrazolyl, thiazolyl or thiadiazolyl, each of which is substituted with substituent(s) selected from the

3 group consisting of carboxy and carboxy(lower)alkyl for R is used as a starting compound, the compound [la] having tetrazolyl, thiazolyl or thiadiazolyl, each of which is substituted with substituent(s) selected from the group consisting of esterified carboxy and esterified

3 carboxy(lower)alkyl for R may be obtained according to reaction conditions. This case is also included within the scope of the present reaction.

The compounds obtained by the above processes can be isolated and purified by a conventional method such as pulverization, recrystallization, column chromatography, reprecipitation, or the like.

It is to be noted that the compound [I] and the other compounds may include one or more stereoisomers due to asymmetric carbon atoms, and all of such isomers and mixture thereof are included within the scope of this invention.

The object compound [I] and pharmaceutically acceptable salts thereof which possess an inhibitory activity against NEP, and are useful for the treatment and/or prevention of various cardiovascular disorders such as hypertension, heart failure, angina pectoris or the like, renal insufficiency, cyclic edema, hyperaldosteronism, hypercalciuria and the like in human beings or animals. Additionally, the object compound [I] is expected to be useful as therapeutical and/or

preventive agents for glaucoma, asthma, inflammation, pain, epilepsy, dementia, obesity and gastrointestinal disorders (especially diarrhoea and irritable bowel syndrome); the modulation of gastric acid secretion and the treatment of hyperreninaemia.

In order to illustrate the usefulness of the object compound [I], the pharmacological data of the compound [I] are shown in the following.

Test :

Neutral endopeptidase (NEP) inhibitory activity

Method :

Purified NEP was used, which was prepared from male Sprague-Dawley rat kidney by the method of J.L. Sonnenberg et al. described in Peptide, Vol. 9, page 173-180 (1988).

NEP inhibitory activity was determined as follows.

The incubation mixture (total volume of 262 μl) contained 0.1M Tris buffer (pH 7.4), 0.1 mg/ml α-hANP (α-human ANP) , test compound (dissolved in 2 μl

N,N-dimethγlformamide) and NEP (45-50 U/ml). The reaction mixture was incubated for 15 min. at 37°C and was terminated with the addition of 50 μl 10% acetic acid.

Fifty microliters of the reaction mixture was injected into a HPLC and measured the hydrolysis of α-hANP by the reverse phase HPLC using C _lg column (YMC, ODS-A 200S).

A 15 min. linear gradient elution from 0.05% trifluoroacetic acid : ^' 60% CH ₃ CN (70:30) to 0.05% trifluoroacetic acid : 60% CH^CN (54:46) was used. NEP inhibitory activity was defined as the inhibition of hydrolysis of α-hANP.

2 -

Results

10

15

For therapeutic purpose, the compound [I] of the

20 present invention can be used in a form of pharmaceutical preparation containing one of said compounds, as an active ingredient, in admixture with a pharmaceutically acceptable carrier such as an organic or inorganic solid, semi-solid or liquid excipient suitable for oral,

25 parenteral or external (topical) administration. The pharmaceutical preparations may be capsules, tablets, dragees, granules, suppositories, solution, lotion, suspension, emulsion, ointment, gel, or the like. If desired, there may be include in these preparations,

30 auxiliary substances, stabilizing agents, wetting or emulsifying agents, buffers and other commonly used additives.

While the dosage of the compound [I] will vary depending upon the age and condition of the patient, an

35 average single dose of about 0.1 mg, 1 mg, 10 mg, 50 mg,

100 mg, 250 mg, 500 mg and 1000 mg of the compound [I] may be effective for treating the above-mentioned diseases. In general, amounts between 0.1 g/body and about 1,000 mg/body may be administered per day.

The following Preparations and Examples are given for the purpose of illustrating this invention.

Preparation 1 A mixture of lH-5-aminotetrazole (7.24 g) , tert-butyl 3-bromopropionate (15.07 g) and powdered potassium carbonate (11.76 g) in acetone (100 ml) was stirred for 8.5 hours under refluxing, and filtered. After evaporation of the solvent, the residue was purified by silica gel column chromatography using a mixture of

^"" chloroform and methanol (50:1 to 20:1) as an eluent to afford ^" tert-butyl 2-(5-amino-2H-tetrazol-2-yl)propionate (1.56 g).

IR (Film) : 3340, 3240, 1725 cm ^-1 NMR (DMSO-d ₆ , δ) : 1.35 (9H, s), 2.86 (2H, t,

J=6.3Hz), 4.56 (2H, t, J=6.3Hz), 5.99 (2H, br s) MASS (m/z) : 214

Preparation 2 The following compound was obtained according to a similar manner to that of Preparation 1.

Ethyl 2-(5-amino-2H-tetrazol-2-yl)propionate mp : 72-74°C IR (Nujol) : 3400, 3320, 1740 cm ^"1

NMR (DMSO-d ₆ , δ) : 1.17 (3H, t, J=7.1Hz), 1.72 (3H, d, J=7.2Hz), 4.15 (2H, q, J=7.1Hz), 5.68 (1H, q, J=7.2Hz), 6.12 (2H, br s) MASS (m/z) : 185

Preparation 3

To a mixture of thiophenol (11.02 g) and sodium hydroxide (4.00 g) in ethanol (60 ml) was added dropwise a solution of tert-butyl 2-bromopropionate (20.91 g) in ethanol (21 ml) at 0°C for 10 minutes. The reaction mixture was stirred overnight at ambient temperature and evaporated in vacuo. The residue was partitioned between water and diethyl ether. The organic layer was washed successively with saturated sodium bicarbonate solution, water and brine, dried over anhydrous magnesium sulfate and evaporated in vacuo. The residue was purified by silica gel column chromatography using a mixture of ethyl acetate and n-hexane (1:100 to 1:20) as an eluent to afford tert-butyl 2-phenylthiopropionate (23.00 g) . IR (Film) : 1720 cm ^"1

NMR (CDC1 ₃ , δ) i 1.35 (9H, s), 1.44 (3H, d,

J=7.1Hz), 3.73 (1H, q, J=7.1Hz), 7.2-7.6 (5H, m) MASS (m/z) : 238

Preparation 4

To a solution of tert-butyl 2-phenylthiopropionate (7.15 g) in diethyl ether (100 ml) was added dropwise a solution of m-chloroperbenzoic acid (80%, 6.47 g) in diethyl ether (30 ml) at 0°C for 20 minutes. The reaction mixture was stirred at 0°C for 10 minutes, evaporated in vacuo and diluted with dichloromethane. The solution was washed successively with sodium thiosulfonate solution, saturated sodium bicarbonate solution and brine, dried over anhydrous magnesium sulfate and evaporated in vacuo. The residue was purified by silica gel column chromatography using a mixture of ethyl acetate and n-hexane (1:9) as an eluent to afford tert-butyl 2-phenylsulfinγlpropionate (8.13 g) . IR (Film) : 1720 cm ^"1 NMR (CDCK, δ) : 1.28 and 1.47 (all 3H, each d.

J=7.1Hz), 1.37 and 1.41 (all 9H, each s), 3.42 and 3.71 (all 1H, each q, J=7.1Hz), 7.45-7.7 (5H, m) MASS (m/z) : 254

Preparation 5

A mixture of tert-butyl 2-phenylsulfinylpropionate (4.34 g), acetic anhydride (2.42 ml) and methanesulfonic acid (0.12 ml) in dibromomethane (107 ml) was stirred for 6 hours under reflux and evaporated in vacuo. The residue was partitioned with water and diethyl ester. The organic layer was washed with brine, dried over anhydrous magnesium sulfate and evaporated in vacuo. To the residue was added thioacetic acid (2.43 ml). The reaction mixture was stirred for 2 hours at 60°C and evaporated in vacuo twice with toluene for removal of excess thioacetic acid. The residue was purified by silica gel column chromatography using a mixture of ethyl acetate and n-hexane (1:20) as an eluent to afford tert-butyl 3-acetylthio-2-phenylthiopropionate (2.72 g) . IR (Film) : 1720, 1685 cm ^"1

NMR (CDC1 ₃ , δ) : 1.40 (9H, s), 2.32 ( 3H, s), 3.14 (1H, dd, J=9.9Hz, 13.6Hz), 3.35 (1H, dd, J=5.5Hz, 13.6Hz), 3.68 (1H, dd, J=5.5Hz, 9.9Hz) , 7.25-7.6 (5H, m)

MASS (m/z) : 312, 256, 238, 236

Preparation 6

To a solution of tert-butyl 3-acetylthio-2- phenylthiopropionate (2.19 g) in dichloromethane (22 ml) was added trifluoroacetic acid (5.4 ml) at 0°C. The reaction mixture was stirred at 0°C for 30 minutes and at ambient temperature for 2 hours and evaporated in vacuo. The residue was purified by silica gel column chromatography using a mixture of methanol and chloroform

(1:50) as an eluent to afford 3-acetylthio-2- phenylthiopropionic acid (1.13 g) . IR (Film) : 2630, 1700 cm ^"1

NMR (CDC1 ₃ , δ) : 2.35 (3H, s) , 3.14 (1H, dd, J=9.8Hz, 13.7Hz), 3.37 (1H, dd, J=5.7Hz, 13.7Hz), 3.76 (1H, dd, J=5.7Hz, 9.8Hz), 7.25-7.6 (5H, m), 9.90 (1H, br s) MASS (m/z) : 256, 238, 180

Example 1

To a suspension of methyl 5-amino-2H-tetrazol-2- ylacetate (0.94 g) and pyridine (0.58 ml) in dichloromethane (17 ml) was added dropwise a solution of 2-acetylthiomethyl-3-phenylpropionyl chloride (1.69 g) in dichloromethane (2.8 ml) under ice-water cooling. The reaction mixture was stirred at 3.5 to 4.0°C for 1.5 hours and then concentrated under reduced pressure. The residue was partitioned between ethyl acetate and 5% hydrochloric acid, and the organic layer was washed successively with water, aqueous sodium bicarbonate and brine, and dried over anhydrous magnesium sulfate. The solvent was evaporated in vacuo and the residue was purified by column chromatography on silica gel using a mixture of chloroform and methanol (100:1) as an eluent to give an oily product of methyl 5-(2-acetylthiomethγl-3-phenγlpropionamido)-2H- tetrazol-2-ylacetate (1.98 g) .

IR (CHC1 ₃ ) : 3420, 3240, 1750, 1720, 1680, 1530 cm ^'1 NMR (CDC1 ₃ , δ) : 2.30 (3H, s) , 2.8-3.2 (5H, m) , 3.81 (3H, s), 5.40 (2H, s) , 7.1-7.3 (5H, m) , 9.66 (1H, br s)

MASS (m/z) : 377, 334, 302, 288

Example 2

The following compounds were obtained according to a similar manner to that of Example 1.

1) Methyl 5-( 2-acetylthiomethyl-3-phenylpropionamido)- lH-tetrazol-1-ylacetate p : 100-101°C

IR (Nujol) : 3180, 1750, 1710, 1690 cm ^"1 NMR (CDC1 ₃ , δ) : 2.28 (3H, s), 2.9-3.4 (5H, m) , 3.80

(3H, s), 5.24 (2H, s), 7.1-7.3 (5H, m) , 11.0 (1H, br s), MASS (m/z) : 377, 335, 302, 288

2) Ethyl 2-(2-acetylthiomethyl-3-phenylpropionamido)- thiazol-4-ylacetate

IR (Film) : 3250, 1720, 1680, 1650 cm ^"1 NMR (CDC1 ₃ , δ) : 1.24 (3H, t, J=7.lHz), 2.30 (3H, s), 2.7-3.2.(5H, m), 4.15 (2H, quartet, J=7.1Hz), 6.79 (1H, s), 7.0-7.3 (5H, m) , 9.4

(1H, br s) MASS (m/z) : 406, 363, 330, 317

3) Methyl 2-(2-acetylthiomethyl-3-phenγlpropionamido)- thiazol-4-ylcarboxylate mp : 140-141°C

IR (Nujol) : 3260, 3220, 1715, 1680, 1540 cm ^"1 NMR (CDC1 ₃ , δ) : 2.28 (3H, s), 2.8-3.3 (5H, m) , 3.88 (3H, s), 7.0-7.3 (5H, m) , 7.86 (1H, s), 10.23 (1H, br s)

MASS (m/z) : 378, 335, 289

4) Methyl 5-[2-acetylthiomethyl-3-( 3,4-methylene- dioxyphenγl)propionamido]-2H-tetrazol-2-ylacetate NMR (CDC1 ₃ , δ) : 2.30 (3H, s), 2.7-3.2 (5H, m) , 3.81

(3H, s), 5.41 (2H, s) , 5.88 (2H, s), 6.65 (2H, s), 6.71 (1H, s), 9.74 (1H, br s)

Example 3 To a solution of methyl 5-( 2-acetylthiomethyl-3-

phenylpropionamido)-2H-tetrazol-2-ylacetate (1.01 g) in methanol (6.4 ml) was added IN aqueous sodium hydroxide solution (6.42 ml) and the resulting solution was stirred for 30 minutes under ice-cooling. The reaction mixture was neutralized with IN hydrochloric acid (6.4 ml) and extracted with ethyl acetate. The extract was washed with brine, dried over anhydrous magnesium sulfate, and concentrated in vacuo. The residue was purified by column chromatography on silica gel using a mixture of toluene, ethyl acetate, and acetic acid (20:2:1) and recrystallized from diisopropyl alcohol to give crystals of 5-(2- mercaptomethyl-3-phenylpropionamido)-2H-tetrazol-2- ylacetic acid (0.63 g) . mp : 173-175°C (dec.) IR (Nujol) : 3200, 1760, 1690, 1680, 1640 cm ^"1

NMR (DMSO-d ₆ , δ) : 2.35 (1H, t, J=8.1Hz), 2.5-3.1 (5H, m), 5.57 (2H, s), 7.1-7.4 (5H, m) , 11.22 (1H, s) MASS (m/z) : 321, 288, 274

Example 4

The following compounds were obtained according to a similar manner to that of Example 3.

1) 5-{2-Mercaptomethyl-3-phenylpropionamido)-lH- tetrazol-1-ylacetic acid

IR (CHC1 ₃ ) : 3220, 1725, 1570 cm ^"1

NMR (CDC1 ₃ , d) : 1.35 (1H, t, J=8.1Hz), 2.4-3.0 (5H, m), 5.37 (2H, s), 6.8-7.3 (5H, m) , 9.22 (1H, br s), 10.69 (1H, br s)

2) 2-(2-Mercaptomethyl-3-phenylpropionamido)thiazol-4- ylacetic acid p : 149-150°C (dec.) IR (Nujol) : 3180, 2560, 1685, 1560 cm ^-1

NMR (DMSO-d _fi , δ) : 2.33 (1H, t, J=8.1Hz), 2.5-3.1 (5H, m), 3.58 (2H, s), 6.93 (1H, s), 7.1-7.4 (5H, m) , 12.27 (2H, br s) MASS (m/z) : 336, 303, 289

3) 2-(2-Mercaptomethyl-3-phenylpropionamido)thiazol-4- ylcarboxylic acid mp : 202-204°C

IR (Nujol) : 3170, 3140, 1680, 1550 cm ^"1 NMR (DMSO-dg, δ) : 2.40 (1H, t, J=8.1Hz), 2.6-3.1

(5H, m), 7.1-7.3 (5H, m) , 7.96 (1H, s), 12.56 (1H, s) , 12.84 (1H, br s) MASS (m/z) : 322, 289, 275

4) 5-[2-Acetylthiomethyl-3-(3,4-methylenedioxyphenyl)- propionamido]-2H-tetrazol-2-ylacetic acid mp : 159-161°C

IR (Nujol) : 3220, 1760, 1700, 1570, 1540 cm ^"1 NMR (DMSO-dg, δ) : 2.33 (1H, t, J=8.1Hz), 2.5-3.1 (5H, m), 5.60 (2H, s), 5.96 (2H, s), 6.66 (1H, dd, J=7.9Hz, 1.5Hz), 6.80 (1H, d, J=7.9Hz), 6.82 (1H, d, J=1.5Hz), 11.21 (1H, s) MASS (m/z) : 365, 321, 290

Example 5

To a solution of ethyl 5-amino-l,2,4-thiadiazol-3- ylcarboxylate (0.50 g) in a mixture of pyridine (0.49 ml) and dichloromethane (5.0 ml) was added dropwise a solution of 2-acetylthiomethyl-3-phenylpropionyl chloride (0.77 g) in dichloromethane (7.7 ml) at 0°C for 5 minutes. The reaction mixture was stirred at the same temperature for 1 hour, diluted with ethyl acetate and washed successively with 5% aqueous hydrochloric acid, water, saturated sodium bicarbonate solution and brine. The extract was dried over anhydrous magnesium sulfate and evaporated in vacuo.

The residue was purified by silica gel column chromatography using a mixture of ethyl acetate and n-hexane (1:3) as an eluent to afford a colorless oil of ethyl 5-(2-acetylthiomethyl-3-phenylpropionamido)-1,2,4- thiadiazol-3-ylcarboxylate (0.77 g) .

IR (Film) : 3200, 1720, 1680, 1530 cm ^"1 NMR (CDC1 ₃ , δ) : 1.44 (3H, t, J=7.1Hz), 2.28 (3H, s), 2.9-3.4 (5H, m), 4.46 (2H, q, J=7.1Hz), 7.0-7.2 (5H, m), 10.95 (1H, br s) MASS (m/z) : 393, 363, 317, 304

Example 6

The following compounds were obtained according to a similar manner to that of Example 5.

1) Ethyl 5-[2-acetylthiomethyl-3-(3,4-methylenedioxy- phenyl)propionamido]-1,2,4-thiadiazol-3-ylcarboxylate IR (Film) : 3500 (br) , 3200 (br) , 1700 (br) NMR (CDC1 ₃ , δ) : 1.45 (3H, t, J=7.1Hz), 2.29 (3H, s), 2.8-3.3 (5H, m) , 4.48 (2H, q, J=7.1Hz), 5.88

(2H, s), 6.53 (1H, dd, J=1.5Hz, 8.0Hz), 6.55-6.65 (2H, m), 10.94 (1H, br s) MASS (m/z) : 437 (M ⁺ )

2) Benzyl 5-(2-acetylthiomethyl-3-phenylpropionamido)- 2H-tetrazol-2-ylacetate

IR (Film) : 3225, 1750-1660 (br), 1520 cm ^'1 NMR (DMSO-dg, δ) : 2.30 (3H, s), 2.7-2.85 (1H, m), 2.9-3.1 (4H, m), 5.19 (2H, s), 5.84 (2H, s), 7.15-7.35 (5H, m) , 7.39 (5H, br s), 11.28 (1H, br s) MASS (m/z) : 453, 411

Example 7 To a solution of ethyl 5-(2-acetylthiomethyl-3-

phenylpropionamido)-1,2,4-thiadiazol-3-ylcarboxylate ( 0.67 g) in methanol (6.8 ml) was added IN aqueous sodium hydroxide solution (6.8 ml) at 0°C at nitrogen atmosphere. The reaction mixture was stirred at ambient temperature for 1 hour and evaporated. The residue was partitioned ^' with water and diethyl ether. The aqueous layer was neutralized with IN aqueous hydrochloric acid (6.8 ml) to afford white precipitate. The precipitate was recrystallized from 33% aqueous ethanol to afford a white crystal of 5-( 2-mercaptomethyl-3-phenylpropionamido) - l,2,4-thiadiazol-3-ylcarboxylic acid (0.28 g) . mp : 188-189°C (dec.)

IR (Nujol) : 3150, 2800-2400, 1710, 1680, 1535 cm ^"1 NMR (DMSO-dg, δ) : 2.45-2.6 (1H, m) , 2.65-2.85 (2H, m), 2.9-3.05 (2H, m) , 3.1-3.3 (1H, m) , 7.05-7.35

(5H, m), 13.38 (1H, br s), 13.67 (1H, br s) MASS (m/z) : 323, 277

Example 8 The following compounds were obtained according to a similar manner to that of Example 7.

1) 5-[2-Mercaptomethyl-3-( 3, 4-methylenedioxyphenyl)- propionamido]-1,2,4-thiadiazol-3-ylcarboxγlic acid p : 180-182°C (dec.)

IR (Nujol) : 3150, 2800-2400, 1715, 1680, 1530 cm ^"1 NMR (DMSO-dg, δ) : 2.45-2.6 (1H, m) , 2.65-2.9 (4H, m), 3.0-3.15 (1H, m) , 5.96 (2H, s), 6.61 (1H, dd, J=1.5Hz, 8.0Hz), 6.7-6.85 (2H, m) , 13.37 (1H, br s), 13.68 (1H, br s)

MASS (m/z) : 323

2) 5-[ (S)-2-Mercaptomethyl-3-phenylpropionamido]-2H- tetrazol-2-ylacetic acid mp : 172-174°C

IR (Nujol) : 3230, 2710, 2600, 2520, 1740, 1690,

1550 cm ^"1 NMR (DMSO-dg, δ) : 2.34 (1H, t, J=8.1Hz), 2.4-3.2 (5H, m), 5.60 (1H, s), 7.1-7.4 (5H, m) , 11.23 (1H, s), 13.5 (1H, br s)

3) 5-[(R)-2-Mercaptomethyl-3-phenylpropionamido]-2H- tetrazol-2-γlacetic acid

IR (Nujol) : 3200, 1730, 1680, 1535 cm ^"1 NMR (DMSO-dg, δ) : 2.34 (1H, t, J=8.1Hz), 2.5-3.1

(5H, m), 5.60 (2H, s), 7.1-7.4 (5H, m) , 11.22 (1H, s), 13.7 (1H, br) MASS (m/z) : 321, 288, 274

4) 2-{5-[(S)-Mercaptomethy1-3-phenylpropionamido]-2H- tetrazol-2-yl}propionic acid IR (Film) : 3400, 3000-2700, 1710, 1690, 1570,

1545 cm ^"1 NMR (DMSO-dg, δ) : 1.78 (3H, d, J=7.3Hz), 2.35 (1H, br t), 2.55-3.05 (5H, m), 5.78 (1H, q, J=7.3Hz),

7.1-7.35 (5H, m), 11.22 (1H, br s) MASS (m/z) : 335

5) 5-(2-Mercaptomethyl-4-methγlpentanoylamino)-2H- tetrazol-2-ylacetic acid mp : 155-157°C

IR (Nujol) : 3265, 3225, 2715, 2600, 2530, 1735,

1685, 1545 cm ^"1 NMR (DMSO-dg, δ) : 0.86 (3H, d, J=6.1Hz), 0.91 (3H, d, J=6.1Hz), 1.2-1.4 (1H, m) , 1.4-1.7 (2H, m) ,

2.28 (1H, br t) , 2.6-2.9 (3H, m) , 5.61 (2H, s) , 11.24 (1H, br s), 13.70 (1H, br s)

6) 5-[(S)-2-Mercaptomethyl-3-(2-methylphenyl)- propionamido]-2H-tetrazol-2-ylacetic acid

mp : 180-182°C

IR (Nujol) : 3220, 1730, 1680, 1575, 1550 cm ^"1

NMR (DMSO-dg, δ) : 2.31 (3H, s), 2.39 (1H, t,

J=8.0Hz), 2.5-3.1 (5H, m) , 5.59 (2H, s), 7.0-7.2 (4H, m), 11.17 (1H, s), 13.7 (1H, br s)

MASS (m/z) : 335, 302, 288 [α] ^25-6 : 87.8° (C=0.5, MeOH)

Example 9 1) To a solution of (S)-2-acetylthiomethyl-3-phenyl- propionic acid (1 g) in dry dichloromethane (10 ml) was added thionyl chloride (0.92 ml) and N,N-dimethylformamide (1 drop). The reaction mixture was stirred for 2 hours at ambient temperature and evaporated to give (S)-2- acetylthiomethyl-3-phenylpropionyl chloride (1.10 g) .'

2) -To a solution of tert-butyl 5-amino-2H-tetrazol-2- ylacetate (1.13 g) and pyridine (0.55 ml) in dry dichloromethane (15 ml) was added dropwise (S)-2-acetylthiomethyl-3-phenylpropionyl chloride (1.46 g) in dry dichloromethane (5 ml) at 5°C. The reaction mixture was stirred for 30 minutes at 5°C and for 1 hour at ambient temperature. Evaporation of the solvent in vacuo gave a residue, to which was added ethyl acetate. The solution was washed successively with IN aqueous hydrochloric acid and brine, dried over anhydrous magnesium sulfate, and evaporated in vacuo. The residue was purified by silica gel column chromatography using a mixture of methanol and chloroform (1:100) as an eluent to afford tert-butyl 5-[ (S)-2-acetylthiomethyl-3- phenylpropionamido]-2H-tetrazol-2-ylacetate (1.75 g) .

IR (Film) : 3225-3275 (br), 1750 (br), 1685 (br),

1530-1560 cm ^"1 NMR (CDC1 ₃ , δ) : 1.50 (9H, s), 2.33 (3H, s), 2.92-3.20 (5H, m) , 5.27 (2H, s) , 7.14-7.26 (5H,

m) , 8. 81 ( 1H, br s ) MASS (m/z ) : 419

Example 10 The following compounds were obtained according to a similar manner to that of Example 9.

1) Benzyl 5-[ (S)-2-acetγlthiomethyl-3-phenyl¬ propionamido]-2H-tetrazol-2-ylacetate mp : 96-100°C

IR (Nujol) : 3270, 1745, 1688, 1545, 1415, 1343 cm ^"1 NMR (CDC1 ₃ , δ) : 2.32 (3H, s), 2.92-3.20 (5H, m) , 5.23 (2H, s), 5.41 (2H, s) , 7.15-7.26 (5H, m) , 7.33-7.37 (5H, m) , 8.78 (1H, br s) MASS (m/z) : 453

[α] ^16*4 : -25.3° (C=0.49, CH ₃ OH)

2) 5-[(S)-2-Acetylthiomethyl-3-phenylpropionamido]-2H- tetrazol-2-ylacetate IR (Nujol) : 3220, 1750, 1690, 1540 cm ^"1

NMR (CDC1 ₃ , δ) : 2.35 (3H, s), 2.8-3.4 (5H, m) , 3.81 (3H, s), 5.38 (2H, s), 7.1-7.4 (5H, m) , 8.51 (1H, br s) MASS (m/z) : 377

3) Methyl 5-[(R)-2-acetylthiomethyl-3-phenyl¬ propionamido]-2H-tetrazol-2-γlacetate

IR (CHC1 ₃ ) : 3230, 1760, 1695, 1550 cm ^"1 NMR (CDC1 ₃ , δ) : 2.31 (3H, s), 2.9-3.3 (5H, m) , 3.8 (3H, s), 5.39 (2H, s), 7.1-7.3 (5H, m) , 9.31

(1H, br s) MASS (m/z) : 378, 288

4) tert-Butyl 3-{5-[(S)-2-acetγlthiomethγl-3-phenγl- propionamido]-2H-tetrazol-2-yl>propionate

p : 83-86°C

IR (Nujol) : 3220, 1710, 1690, 1565, 1535 cm ^"1

NMR (CDC1 ₃ , δ) : 1.44 (9H, s), 2.30 (3H, s), 3.02

(2H, t, J=7.2Hz), 2.85-3.2 (5H, m) , 4.82 (2H, t, J=7.2Hz), 7.1-7.25 (5H, m) , 9.39 (1H, br s)

MASS (m/z) : 434

5) tert-Butyl 5-(3-acetylthio-2-phenylthiopropionamido)- 2H-tetrazol-2-ylacetate mp : 130-133°C

IR (Nujol) : 3280, 3240, 3090, 1745, 1690, 1680,

1565, 1530 cm ^"1 NMR (CDC1 ₃ , δ) : 1.48 (9H, s), 2.33 (3H, s), 3.35 (1H, dd, J=8.8Hz, 13.8Hz), 3.45 (1H, dd, J=5.8Hz, 13.8HZ), 3.9-4.1 (1H, m) , 5.28 (2H, s),

7.25-7.35 (3H, m) , 7.45-7.55 (2H, m) , 9.59 (1H, br s) MASS (m/z) : 438

6) Ethyl 2-{5-[ (S)-2-acetylthiomethyl-3-phenyl¬ propionamido]-2H-tetrazol-2-yl}propionate mp : 109-113°C

IR (Nujol) : 3220, 3050, 1740, 1715, 1685, 1560, 1530 cm ^"1 NMR (CDC1 ₃ , δ) : 1.25 (3H, t, J=7.1Hz), 2.01 (3H, d,

J=7.4Hz), 2.30 (3H, br s), 2.85-3.2 (5H, m) , 4.23 (2H, q, J=7.1Hz), 5.57 (1H, q, J=7.4Hz), 7.05-7.25 (5H, m) , 9.67 (1H, br s) MASS (m/z) : 405

7) Methyl 5-(2-acetγlthiomethyl-4-methylpentanoγlamino)- 2H-tetrazol-2-ylacetate

IR (Film) : 3230, 1750, 1685, 1535 cm ^"1 NMR (CDC1 ₃ , δ) : 0.94 (6H, d, J=6.1Hz), 1.4-1.55 (1H, m), 1.6-1.9 (2H, m) , 2.30 (3H, s), 2.8-3.0

(1H, m), 3.08 (1H, dd, J=8.4Hz, 13.5Hz), 3.20 (1H, dd, J=5.6Hz, 13.5Hz), 3.82 (3H, s), 5.44 (2H, s), 10.36 (1H, br s) MASS (m/z) : 328, 300

8) Methyl 5-[(S)~2-acetylthiomethγl-3-(2-methylphenγl)- propionamido]-2H-tetrazol-2-ylacetate IR (Nujol) : 3230, 1750, 1680, 1540 cm ^"1 NMR (CDC1 ₃ , δ) : 2.32 (6H, s) , 2.8-3.4 (5H, m), 3.81 (3H, s), 5.38 (2H, s), 7.0-7.2 (4H, m) , 8.93

(1H, br s) MASS (m/z) : 392, 391, 316, 302

Example 11 To a solution of tert-butyl 5-[(S)-2-acetyl- thiomethyl-3-phenylpropionamido]-2H-tetrazol-2-ylacetate (1.74 g) in dichloromethane (18 ml) was added trifluoroacetic acid (3.2 ml) at 0°C. The reaction mixture was stirred at 0°C for 30 minutes and at ambient temperature overnight. Removal of the solvent in vacuo gave a residue which was purified by silica gel column chromatography using a mixture of methanol and chloroform (1:50) as an eluent and then triturated with diethyl ether to afford 5-[(S)-2-acetylthiomethyl-3-phenylpropionamido]- 2H-tetrazol-2-ylacetic acid (0.45 g). mp : 126-130°C

IR (Nujol) : 3225, 1760, 1685-1695, 1565, 1540 cm ^"1 NMR (CDC1 ₃ , δ) : 2.30 (3H, s), 2.76-2.89 (1H, m),

2.95-3.03 (4H, m) , 5-60 (2H, s), 7.20-7.33 (5H, m)

MASS (m/z) : 363

[α] ^6-7 : -25.5° (C=0.51, OL _j OH)

Example 12 The following compounds were obtained according to a

similar manner to that of Example 11.

1) N-{5-[ (S)-2-Acetylthiomethyl-3-phenylpropionamido]- 2H-tetrazol-2-ylacetyl}phenylalanine mp : 122-126°C (dec.)

IR (Nujol) : 3290, 3220, 2800-2500, 1675, 1560 cm ^"1 NMR (DMSO-dg, δ) : 2.29 (3H, s), 2.7-3.2 (8H, m) , 4.3-4.45 (1H, m), 5.33 (1H, d, J=17Hz), 5.41 (1H, d, J=17Hz), 7.1-7.3 (10H, ) , 8.62 (1H, d, J=7.9Hz), 11.24 (1H, br s)

MASS (m/z) : 434 [α] ²⁸ : 14.26° (C=1.15, CH ₃ OH)

2) 3-{5-[ (S) -2-Acetylthiomethyl-3-phenylpropionamido]- 2H-tetrazol-2-yl}propionic acid

IR (Film) : 3400, 1685, 1570 cm ^"1

NMR (DMSO-dg, δ) : 2.30 (3H, s), 2.7-2.85 (1H, m) , 2.9-3.1 (6H, m) , 4.76 (2H, t, J=6.5Hz), 7.15-7.35 (5H, m) , 11.19 (1H, br s) MASS (m/z) : 377

3) 5-(3-Acetylthio-2-phenylthiopropionamido) -2H- tetrazol-2-ylacetic acid IR (KBr) : 3000-2700, 1684, 1585 cm ^"1 NMR (DMSO-dg, δ) : 2.34 (3H, s), 3.13 (1H, dd,

J=10.5Hz, 13.4Hz), 3.31 (1H, dd, J=4.9Hz, 13.4Hz), 4.05-4.2 (1H, m) , 5.31 (2H, s), 7.3-7.5 (5H, m), 11.55 (1H, br s) MASS (m/z) : 306

Example 13

To a suspension of benzyl 5-[ (S)-2-acetylthiomethyl- 3-phenylpropionamido]-2H-tetrazol-2-ylacetate (0.5 g) in methanol (2.5 ml) was added dropwise 25% aqueous methylamine (0.68 g) at ambient temperature under nitrogen

atmosphere. The mixture was stirred for 20 minutes and then poured into water (30 ml). The formed precipitate was collected by filtration and purified by column chromatography on silica gel (10 g) using a mixture of chloroform and methanol (20tl) as an eluent. The

' fractions containing the desired product were collected and concentrated under reduced pressure and the residue was suspended in diethyl ether, and collected by filtration to give 5-[(S)-2-mercaptomethyl-3-phenyl~ propionamido]-2H-tetrazol-2-yl-N-methylacetamide (0.12 g) . p : 195-197°C

IR (Nujol) : 3360, 3220, 1680, 1580, 1550 cm ^"1 NMR (DMSO-dg, δ) : 2.34 (1H, t, J=8.3Hz), 2.64-2.98 (8H, m), 5.33 (2H, s), 7.18-7.32 (5H, m), 8.36 (1H, ), 11.2 (1H, s)

MASS (m/z) : 304, 301, 287 [α] ^22*4 : 69.6° (C=0.49, CH ₃ OH)

Example 14 To a suspension of benzyl 5-[(S)-2-acetylthio¬ methyl-3-phenylpropionamido]-2H-tetrazol-2-ylacetate (2.27 g) in methanol (9.9 ml) was added 28% aqueous ammonia solution under nitrogen atmosphere. The mixture was stirred for 20 minutes at ambient temperature ^• and then poured into water (50 ml) containing potassium bisulfate (8.17 g). The formed precipitate was collected by filtration, washed with water, and dried under reduced pressure to give 5-[(S)-2-mercaptomethyl-3- phenylpropionamido]-2H-tetrazol-2-γlacetamide (1.60 g) . p : 174-176°C

IR (Nujol) : 3390, 3260, 3220, 1675, 1608, 1550 cm ^"1 NMR (DMSO-dg, δ) : 2.34 (1H, t, J=8.0Hz), 2.5-3.1 (5H, m), 5.32 (2H, s), 7.1-7.4 (5H, m) , 7.52 (1H, br s), 7.85 (1H, br s), 11.20 (1H, br s) MASS (m/z) : 320, 287, 273

[α] ²² ' ⁵ : 73.2 (C=0.50, CH ₃ OH)

Example 15

To a suspension of benzyl 5-[ (S)-2-acetγlthiomethyl- 3-phenγlpropionamido]-2H-tetrazol-2-ylacetate (1.93 g) in ethanol (7 ml) was added dropwise 28% aqueous ammonia solution (2.87 ml) under a nitrogen atmosphere. The resulting mixture was stirred for 20 minutes at ambient temperature, poured into 5% aqueous sodium bisulfate solution, and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous magnesium sulfate, filtered, and evaporated in vacuo. The residue was purified by column chromatography on silica gel using a mixture of ethyl acetate and methanol (10:1) as an eluent to afford 5-[(S)-2-mercaptomethyl-3- phenγlpropionamido]-2H-tetrazol-2-ylacetamide (1.04 g) . mp : 172-174°C

IR (Nujol) : 3370, 3250, 3200, 1670/ 1605, 1540 cm ^"1 NMR (DMSO-dg, δ) : 2.33 (1H, t, J=8.1Hz) , 2.5-3.1 (5H, m), 5.33 (2H, s), 7.1-7.4 (5H, m) , 7.85

(1H, br s), 8.31 (1H, br s), 11.21 (1H, s) MASS (m/z) : 320

Example 16 The following compounds were obtained according to a similar manner to that of Example 15.

1) 5-[ (S)-2-Mercaptomethyl-3-phenylpropionamido]-2H- tetrazol-2-yl-N,N-dimethylacetamide IR (Nujol) : 1660 cm ^"1

NMR (CDC1 ₃ , δ) : 1.65 (1H, t, J=8.5Hz) , 2.5-2.8 (1H, m), 2.8-3.1 (4H, m) , 3.00 (3H, t) , 3.10 (3H, s), 5.47 (2H, s), 7.0-7.4 (5H, m) , 9.34 (1H, br s) MASS (m/z) : 348, 301

2) 5-[(S)-2-Mercaptomethyl-3-phenylpropionamido]-2H- tetrazol-2-yl-N-(2-methoxyethγl)acetamide mp : 150-152°C

IR (Nujol) : 3300, 3200, 1675, 1575, 1545 cm ^"1 NMR (DMSO-dg, δ) : 2.34 (1H, t, J=7.9Hz), 2.5-3.2

(5H, m), 3.2-3.5 (7H, m) , 5.36 (2H, s), 7.1-7.4 (5H, m), 8.56 (1H, br t, J=5Hz), 11.21 (1H, br s) MASS (m/z) : 378

3) 5-[(S)-2-Mercaptomethγl-3-phenylpropionamido]-2H- tetrazol-2-yl-N-benzylacetamide mp : 129-132°C

IR (Nujol) : 3270, 1675, 1660, 1540 cm ^"1 NMR (CDC1 ₃ , δ) : 1.53 (1H, t, J=8.5Hz), 2.4-2.7 (3H, m), 2.7-3.1 (4H, m) , 4.42 (1H, d, J=5.6Hz), 5.41 (2H, s), 7.0-7.4 (10H, m) , 9.67 (1H, br s) MASS (m/z) : 410, 363

Example 17 _

To a suspension of 5-[(S)-2-acetylthiomethyl-3- phenylpropionamido]-2H-tetrazol-2-ylacetic acid (0.45 g) , methylamine hydrochloride (0.084 g) , and 1-hydroxγbenzotriazole (0.17 g) in dichloromethane (4.5 ml) was added l-(3-dimethγlaminopropyl)-3-ethyl- carbodiimide (0.21 g) under ice-water cooling. The mixture was stirred overnight at ambient temperature and then evaporated in vacuo. The residue was diluted with chloroform, washed successively with 5% hydrochloric acid and brine, dried over anhydrous magnesium sulfate, and filtered. The filtrate was evaporated in vacuo to give crude powder (0.41 g) , which was recrystallized from a mixture of ethanol and diethyl ether to give 5-[(S)-2-acetγlthiomethyl-3-phenylpropionamido]-2H- tetrazol-2-yl-N-methylacetamide (0.18 g) .

p : 168-170°C

IR (Nujol) : 3340, 3180, 1670, 1565, 1535 cm ^"1

NMR (CDC1 ₃ , δ) : 2.34 (3H, s), 2.83 (3H, d,

J=4.8Hz), 2.9-3.3 (5H, m) , 5.30 (2H, s), 6.27 (1H, br), 7.1-7.3 (5H, m) , 8.76 (1H, br s)

MASS (m/z) : 376, 287

Example 18

The following compounds were obtained according to a similar manner to that of Example 17.

1) 5-[ (S)-2-Acetylthiomethyl-3-phenylpropionamido]-2H- tetrazol-2-yl-N,N-dimethylacetamide p : 145-149°C (dec.) IR (Nujol) : 3220, 1680, 1655, 1530 cm ^"1

NMR (CDC1 ₃ , δ) : 2.31 (3H, s) , 2.99 (3H, s), 3.09

(3H, s), 2.83-3.2 (5H, m) , 5.46 (2H, s), 7.1-7.3 (5H, m), 9.32 (1H, br s) MASS (m/z) : 391 [α] ²³ : -33.30° (C=1.06, CHC1 ₃ )

2) 5-[ (S)-2-Acetylthiomethyl-3-phenylpropionamido]-2H- tetrazol-2-yl-N-( 2-methoxyethyl)acetamide mp : 103-105°C IR (Nujol) : 3290, 3190, 1700, 1670, 1570, 1540 cm ^"1 NMR (CDC1 ₃ , δ) : 2.32 (3H, s), 2.9-3.3 (5H, m) , 3.31 (3H, s), 3.4-3.5 (4H, m) , 5.32 (2H, s), 6.73 (1H, br s), 7.1-7.4 (5H, m) , 9.20 (1H, br s) MASS (m/z) : 420, 331

3) N-{5-[ (S)-2-Acetylthiomethyl-3-phenylpropionamido]- 2H-tetrazol-2-ylacetyl}phenylalanine tert-butyl ester IR (Film) : 3320, 1730, 1680, 1535 cm ^"1

NMR (CDC1 ₃ , δ) : 1.39 (9H, s), 2.31 (3H, s), 2.9-3.25 (7H, m) , 4.65-4.8 (1H, m) , 5.32 (2H,

s), 6.82 (1H, d, J=7.5Hz), 7.05-7.3 (10H, m), 9.19 (1H, br s) MASS (m/z) : 567 [α] ²³ : 11.91° (C=0.99, CHCl ₃ )

Example 19

To a suspension of 5-[(S)-2-acetylthiomethyl-3- phenγlpropionamido]-2H-tetrazol-2-ylacetic acid (545 mg) , benzylamine (177 mg) , and 1-hγdroxybenzotriazole (203 mg) in dichloromethane (10 ml) was added l-(3-dimethyl- aminopropyl)-3-ethylcarbodiimide hydrochloride (316 mg) under ice-cooling. The resulting mixture was stirred for 2 hours at the same temperature and concentrated in vacuo. The residue was partitioned between ethyl acetate and 5% hydrochloric acid. The organic layer was washed successively with saturated aqueous sodium bisulfate solution and brine, dried over anhydrous magnesium sulfate, and concentrated in vacuo. The residue was purified by column chromatography on silica gel using a mixture of chloroform and methanol (15:1) as an eluent to afford 5-[(S)-2-acetylthiomethyl-3-phenylpropionamido]-2H- tetrazol-2-yl-N-benzylacetamide (502 mg) . mp : 162-163°C

IR (Nujol) Ϊ 3280, 1685, 1645, 1545 cm ^"1 NMR (DMSO-dg, δ) : 2.30 (3H, s), 2.7-3.2 (5H, m) ,

4.34 (2H, d, J=5.8Hz), 5.44 (2H, s), 7.1-7.4 (10H, m), 8.96 (1H, t, J=5.8Hz), 11.25 (1H, s) MASS (m/z) : 452

22

[ct] -19.8° (C=0.5, DMF)

Example 20

A suspension of 5-[(S)-2-acetγlthiomethyl-3-phenγl- propionamido]-2H-tetrazol-2-ylacetic acid (3.63 g) in 9.4% aqueous ammonia (20 ml) was stirred for 20 minutes at ambient temperature under nitrogen atmosphere to give

clear solution. The solution was poured into water (88.5 ml) containing potassium bisulfate (17.7 g) and extracted with ethyl acetate. The extract was washed with water, dried over anhydrous magnesium sulfate, and evaporated in vacuo. The residue was suspended in ethyl acetate and filtered to give 5-[ (S)-2-mercaptomethyl-3- phenylpropionamido]-2H-tetrazol-2-ylacetic acid (2.66 g) . mp : 172-174°C

3230, 2710, 2600, 2520, 1740, 1690, 1550 cm ^"1 δ) : 2.34 (IH, t, J=8.1Hz) , 2.4-3.2 5.60 (IH, s), 7.1-7.4 (5H, m) , 11.23 13.5 (IH, br s) (C=0.5, CH ₃ OH)

To a suspension of 3-[5-(S)-2-acetylthiomethyl-3- phenylpropionamido]-2H-tetrazol-2-yl}propionic acid (0.57 g) in water (1 ml) was added 28% aqueous ammonia solution (1 ml) at ambient temperature under a nitrogen atmosphere. The reaction mixture was stirred for 10 minutes at ambient temperature, acidified with 10% aqueous potassium bisulfate (30 ml) and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate and evaporated in vacuo. The residue was purified by silica gel column chromatography using a mixture of chloroform and methanol (30:1) to afford 3-{5-[ (S)-2-mercaptomethyl-3-phenylpropionamido]-2H- tetrazol-2-yl]propionic acid (0.32 g) . IR (Film) : 3400, 3000-2500, 1700, 1560 cm ^"1

NMR (DMSO-dg, δ) : 2.34 (IH, t, J=8.1Hz) , 2.55-3.1 (7H, m) , 4.76 (2H, t, J=6.5Hz), 7.1-7.35 (5H, m) , 11.15 (IH, br s) MASS (m/z) : 335

Example 22

The following compound was obtained according to a similar manner to that of Example 21.

5-(3-Mercapto-2-phenylthiopropionamido)-2H-tetrazol- 2-ylacetic acid mp : 196-199°C (dec.)

IR (Nujol) : 3175, 2560, 1760, 1685, 1540 cm ^"1 NMR (DMSO-dg, δ) : 2.65-3.0 (2H, m) , 3.95-4.25 (IH, m), 5.63 (2H, s), 7.25-7.4 (6H, m) , 11.61 (IH, br s), 13.75 (IH, br s) MASS (m/z) : 306

Example 23 To a suspension of 5-[(S)-2-mercaptomethγl-3-phenyl- propionamido]-2H-tetrazol-2-ylacetamide (0.83 g) and pyridine (0.32 ml) in dichloromethane (15 ml) was added dropwise benzoyl chloride (0.22 ml) under a nitrogen atmosphere at 0~5°C. The resulting mixture was stirred at 0~5°C for an hour, evaporated, and partitioned between ethyl acetate and water. The organic layer was washed successively with 5% hydrochloric acid, saturated aqueous sodium bicarbonate solution and brine, dried over magnesium sulfate, and concentrated in vacuo. The residue was purified by column chromatography on silica gel using a mixture of chloroform and methanol (10:1) as an eluent. The fractions containing the object compound were collected, concentrated in vacuo, and recrystallized from ethanol to afford 5-[(S)-2-benzoylthiomethyl-3-phenyl- propionamido]-2H-tetrazol-2-ylacetamide (0.84 g) . mp : 180-182°C

IR (Nujol) : 3410, 3260, 1675, 1650, 1550 cm ^"1 NMR (DMSO-dg, δ) : 2.7-3.4 (5H, m) , 5.32 (2H, s),

7.1-7.4 (5H, m), 7.4-8.0 (7H, m) , 11.27 (IH, s) MASS (m/z) : 424

[α] ^28* ° : -66.0° (C=0.5, DMF)

Example 24

The following compound was obtained according to a similar manner to that of Example 23.

5-[ (S)-2-Acetylthiomethyl-3-phenylpropionamido]-2H- tetrazol-2-ylacetamide mp : 166-167°C IR (Nujol) : 3375, 3270, 3210, 1675, 1610, 1545 cm ^"1

NMR (DMSO-dg, δ) : 2.30 (3H, s), 2.6-3.1 (5H, m) ,

5.33 (2H, s), 7.1-7.4 (5H, m) , 7.52 (IH, br s), 7.84 (IH, br s) MASS (m/z) : 363

Example 25

To a solution of 5-aminomethyl-lH-tetrazole hydrochloride (1.36 g) in dry pyridine (15 ml) was added dropwise a solution of 2-acetylthiomethyl-3- phenylpropionyl chloride (2.57 g) in dry dichloromethane (5 ml) at 0°C for 5 minutes. The reaction mixture was stirred for 4.5 hours at ambient temperature, poured into cold 10% aqueous hydrochloric acid, and extracted with ethyl acetate to give insoluble products. This was triturated with ethanol to give 5-[ (2-acetylthiomethyl-3- phenylpropionγlaminomethyl]-lH-tetrazole (0.53 g) . mp : 175-176°C (dec.)

IR (Nujol) : 3275, 1690, 1650, 1540 cm ^"1 NMR (DMSO-dg, δ) : 2.27 (3H, s), 2.6-3.0 (5H, m) , 4.49 (2H, d, J=5.7Hz), 7.05-7.3 (5H, m) , 8.74

(IH, br t) MASS (m/z) : 319

Example 26 To a suspension of 5-[ (2-acetylthiomethyl-3-

phenylpropionyl)aminomethγl]-lH-tetrazole (0.18 g) in methanol (3.6 ml) was added cysteamine (0.05 g) at 0°C. The reaction mixture was stirred for 6 hours, stand overnight at ambient temperature, and evaporated in vacuo. The residue was acidified with 10% aqueous hydrochloric acid and the insoluble product was triturated with water to give 5-[(2-mercaptomethyl-3-phenylpropionyl)amino- methyl]-IH-tetrazole (0.07 g) . mp : 157-160°C (dec.) IR (Nujol) : 3280, 2800-2500, 1690, 1655, 1545 cm ^"1

NMR (DMSO-dg, d) : 2.15-2.35 (IH, m) , 2.4-3.0 (5H, m), 4.52 (2H, d, J=5.7Hz), 5.95 (2H, s), 6.57 (IH, d, J=9.5Hz), 6.65-6.8 (2H, m) , 8.69 (IH, br t) MASS (m/z) : 321

Example 27

A solution of 5-[(S)-2-mercaptomethyl-3-phenyl¬ propionamido]-2H-tetrazol-2-ylacetic acid (0.82 g) in dimethyl sulfoxide (45 ml) was stirred overnight at 70°C, cooled to ambient temperature, and thereto was added IN aqueous sodium hydroxide (6.1 ml) . The resulting mixture was washed with ethyl acetate, and the aqueous layer was acidified by IN hydrochloric acid, extracted with ethyl acetate, dried over anhydrous magnesium sulfate, and concentrated in vacuo. The residue was triturated with chloroform to afford bis[(S)-(2-carboxγmethyl-2H- tetrazol-5-ylcarbamoyl)-3-phenγlpropyl] disulfide (0.74 g ⁾ . mp : 146°C

IR (Nujol) : 1750, 1695, 1580, 1550 cm ^"1

NMR (DMSO-dg, δ) : 2.5-3.1 (10H, m) , 5.57 (4H, s),

7.0-7.4 (10H, m), 11.30 (2H, s) FAB-MASS (m/z) : 641