MERCAPTO-ACYLAMINO ACID ANTIHYPERTENSIVES

Human hypertension represents a disease of multiple etiologies. Included among these is a sodium and volume dependent low renin form of hypertension. Drugs that act to control one aspect of hypertension will not necessarily be effective in controlling another.

A variety of mercapto-acylamino acids are known as enkephalinase inhibitors useful as analgesics and in the treatment of hypertension.

U.S. 4,513,009 to Roques et al discloses, inter alia, compounds of the formula.

H

wherein n is 0 to 1; R includes hydrogen, optionally substituted alkyl, optionally substituted phenyl,

2 cyclohexyl and thienyl; and R includes hydrogen optionally substituted alkyl, optionally substituted benzyl, phenyl, phenoxyalkyl and optionally substituted mercaptoalkyl. The compounds are disclosed as principally having enkephalinase activity, but also are said to be antihypertensives.

U.S. 4,401,677 to Greenberg et al and EPA

38,046 to Wilkinson disclose compounds of a scope similar

to Roques et al, the former disclosing analgesic activity and the latter disclosing a greater specificity for enkephalinase than ACE. U.S. 4,053,651 to Ondetti et al discloses the use of similar -compounds, in- the treatment of renin-angiotensin related hypertension.

It has recently been discovered that the heart secretes a series of peptide hormones called atrial natriuretic factors (ANF) which help to regulate blood pressure, blood volume and the excretion of water, sodium and potassium. ANF were found to produce a short-term reduction in blood pressure and to be useful in the treatment of congestive heart failure. See P. Needle an et al, "Atriopeptin: A Cardiac Hormone Intimately Involved in Fluid, Electrolyte and Blood-Pressure Homeostasis" , N. Engl. J. Med. , 314, 13 (1986) pp. 828- 834, and M. Cantin et al in "The Heart as an Endocrine Gland", Scientific American, 254 (1986) pg. 76-81.

A class of drugs known to be effective in treating some types of hypertension is.ACE inhibitors, which compounds are useful in blocking the rise in blood pressure caused by increases in vascular resistance and fluid volume due to the formation of angiotensin II from angiotensin I. For a review of ACE inhibitors,., see M. Wyvratt and A. Patchett, "Recent Developments in the Design of Angiotensin Converting Enzyme Inhibitors" in Med. Res. Rev. Vol. 5, No. 4 (1985) pp. 483-531.

The present invention relates to mercapto- acylamino acids useful in the treatment of various types of hypertension, particularly volume expanded hypertension, and congestive heart failure.

These novel compounds have been found to enhance both the magnitude and duration of the antihypertensive and natriuretic effects of endogenous _ ANF. Administration of a combination of a mercapto- acylamino acid and an ACE inhibitor provides an

antihypertensive effect greater than either the mercapto- acylamino acid or ACE inhibitor alone. Administration of a combination of a mercapto-acylamino acid of the present invention and an exogenous ANF or ACE inhibitor is therefore particularly useful in treating hypertension.

In addition to the compound aspect, the present invention therefore also relates to treating hypertension with a mercapto-acylamino acid or with a mercapto- acylamino acid in combination with an ANF or an ACE inhibitor, which methods comprise administering^to a mammal in need of such treatment an antihypertensive effective amount of the mercapto-acylamino acid or an antihypertensive effective amount of a combination of a mercapto-acylamino acid and ANF or ACE inhibitor. The drug or combination of drugs is preferably administered in a pharmaceutically acceptable carrier, e.g. for oral or parenteral administration. The combinations of drugs may be co-administered in a single composition, or components of the combination therapy may be administered separately. Where the components are administered separately, any convenient combination of dosage forms may be used, e.g. oral mercapto-acylamino acid/oral ANF, oral mercapto-acylamino acid/parenteral ACE inhibitor, parenteral mercapto-acylamino acid/oral ANF, parenteral mercapto-acylamino acid/parenteral ACE inhibitor.

When the components of a combination of a mercapto-acylamino acid and an ANF are administered separately, it is preferred that the mercapto-acylamino acid be administered first.

Another aspect of the invention relates to pharmaceutical compositions comprising a mercapto- acylamino acid of this invention, alone or in combination with an ANF or an ACE inhibitor, and to methods of treatment of hypertension and congestive heart failure comprising administering a mercapto-acylamino acid of

this invention, alone or in combination with an ANF or an ACE inhibitor to a mammal in need of such treatment.

Novel mercapto-acylamino acid- antihypertensive compounds of .the present invention are ^" represented by the following formula:

wherein R ¹ is Y-C ₈ H ₄ -, Y-C ₈ H ₄ S-, Y-CgH ₄ 0-,

α-naphthyl, ø-naphthyl, furyl, thienyl, benzofuryl, benzothienyl, H2N(CH ₂ ) _m -, diphenylmethyl,

R ² is R ¹⁴ (CH ₂ ) _k S(O) ₀ _ ₂ (CH ₂ ) _q - or R ⁶ OCO(CH ₂ ) _q -

R ' ^J i"sis --OunR. ⁷ ,» -— ;

R is hydrogen, alkyl or γ ¹ -CgH ₄ - _;

R 14 is mono-unsaturated lower alkyl, hydroxy, alkoxy or alkylthio, provided that when R 14 is hydroxy or alkoxy, k is 2 or 3 and when R 14 is mono-unsaturated alkykl or alkylthio, k is 1, 2 or 3;

R" is dihydroxyalkyl, dialkoxyalkyl, alkoxyalkoxyalkyl, haloalkyl, (haloalkoxy)alkyl or alkyl substituted with a 5-6 membered saturated ring comprising 1-2 oxygen atoms as ring members wherein the ring carbon atoms may be substituted with 0-2 alkyl substituents;

R ⁷ and R are independently R , H, alkyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl or arylalkyl, or R ⁷ and R ⁸ together with the nitrogen to which they are attached complete a 5-7 membered ring, wherein one of the 4-6 ring members comprising R and R° may be a nitrogen atom, an alkyl-substituted nitrogen atom or an oxygen atom, and wherein the ring may be substituted on the ring carbon atoms with substituents chosen from alkyl and hydroxy groups;

R ⁹ is hydrogen, alkyl, carboxyalkyl, mercaptoalkyl, alkylthioalkyl, aminoalkyl, hydroxyalkyl, phenylalkyl, hydroxyphenylalkyl, guanidinoalkyl, imidazolylalkyl, indolylalkyl or carbamoylalkyl; n is 0-2; and k are independently 0-3; q is 1-4;

X is a bond, -0-, -S-, or -CH ₂ -

Q is hydrogen or R CO-;

R is alkyl, hydroxyalkyl, alkoxyalkyl, dialkylaminoalkyl,

Y ² -C ₆ H ₄ -alkyl, alkoxy, Y ² -CgH ₄ ~, naphthyl, furyl, thienyl or pyridyl;

Y, Y~ and Y ² independently represent one or more substituents selected from H, alkyl, cycloalkyl, alkoxy, OH,

F, Cl, Br, CN, -CH ₂ NH ₂ , -C0 ₂ H, -C0 ₂ alkyl, -CONH ₂ and phenyl; the pharmaceutically acceptable addition salt thereof.

As used herein the term "alkyl" means straight or branched alkyl chains of 1 to 6 carbon atoms, and "alkoxy"

similarly refers to alkoxy groups having 1 to 6 carbon atoms. "Cycloalkyl" means cyclic alkyl groups of 3-6 carbon atoms.

"Aryl" means mono-cyclic or fused ring bi-cyclic carbocyclic aromatic groups having 6 to 10 ring members or mono-cyclic or fused ring bycyclic aromatic groups wherein 1-2 ring members may independently be nitrogen, oxygen or sulfur, wherein the carbon ring members of the aryl group are substituted by 0-3 substituents as defined above by Y. Examples of carbocyclic aryl groups are phenyl, α-naphthyl and 3-naphthyl, and examples of heterocyclic aryl groups are furyl, thienyl, benzofuryl, benzothienyl, indolyl and pyridyl. All positional isomers, e.g. 2-pyridyl, 3-pyridyl, are contemplated.

"Halo" refers to fluorine, chlorine, bromine or iodine radicals. The term "poly", when used to describe substitution in a phenyl, alkylphenyl or alkoxyphenyl group, means 2 to 5 substituents.

3 Groups R comprising the partial

structure -NHCH-C-

R ⁹ are derived from amino acids of formula H-NCHCOOH. Examples of such amino acids are alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine and valine.

Preferred embodiments of compounds of formula I - are compounds wherein R ¹ is Y-CgH - especially wherein Y is hydrogen. Also preferred are compounds wherein R is -OR ⁷ or -NR 7R8, wherein R7 and R° ^~ are as defined above. Further preferred compounds of formula I are those-wherein Q is hydrogen or R CO- wherein R is alkyl, especially methyl, or phenyl. Also preferred are compounds wherein R is

R ¹⁴ (CH ₂ ) _k -(s(0)g_ ₂ (CH)-,-. Especially preferred R ¹⁴ groups are mono-unsaturated lower alkyl such as vinyl and alkylthio such as methylthio.

Compounds of this invention may, depending on the nature of functional groups, form addition salts with various inorganic and organic acids and bases. Such salts include salts prepared with organic and inorganic acids, e.g. HC1, HBr, H ₂ S0 ₄ , H ₃ P0 ₄ , methanesulfonic acid, toluenesulfonic acid, maleic acid, furmaric acid and camphorsulfonic acid. Salts prepared with bases include ammonium salts, alkali metal salts, e.g. sodium and potassium salts, and alkaline earth salts, e.g. calcium and magnesium salts.

The salts may be formed by conventional means, as by reacting the free acid or base forms of the product with one or more equivalents of the appropriate base or acid in a solvent or medium in which the salt is insoluble, or in a solvent such as water which is then removed in vacuo or by freeze-drying or by exchanging the cations of an existing salt for another cation on a suitable ion exchange resin.

Compounds of formula I have two or more asymmetrical carbon atoms and therefore include various stereoisomers. All stereoisomers are included within the scope of the present invention.

In general, compounds of the present invention can be made by an appropriate process selected from the following process A and B, wherein Q, R 1, R2, R3 and n are as defined in claim 1, including suitable protection:

Process A: condensation of a 3-thiopropionic acid of formula IV, or a reactive derivative thereof, with an amine of formula V

IV

Process B: for compounds of formula I wherein R ^~ is -NR ,7rR_ 8, condensation of a (3-thiopropionyl)amino acid of formula .VI, or a reactive derivative thereof^ with an amine of formula VII ^" ,

wherein both Process A and Process B are followed by isolating the preferred isomer if desired and removal of the protecting groups, if necesary, to yield the desired products, and if desired, preparing a salt thereof.

More particularly, compounds of the present invention may be prepared by using coupling reactions well known in the peptide art to join a 3-acetylthio-2- (substituted)-propionic acid of formula _1_ with an amino aci ester of formula _2_. The following reaction Scheme 1 is an example:

DEC,HOBT NM ,D F

In the above scheme, RP=R ¹ ; R ^r =R ² ; R~ is methyl, ethyl, t-butyl or aralkyl (e.g. benzyl); Ac is acetyl; n is 0-2; DEC is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide - hydrochloride; HOBT is 1-hydroxybenzotria^ole hydrate? ^' NMM is N-methylmorpholine; and DMF is dimethylformamide.

As Scheme 1 shows, an amino acid ester of formula _2_ and a 3-acetylthio propionic acid of formula _1_ are reacted at room temperature in an inert solvent such as DMF in the presence of coupling agents such as DEC and HOBT in the presence of a base such as NMM. The resultant isomers are separated by chromatography and the isomers are deprotected at the acid and mercapto positions.

Alternatively, a propionic acid of formula 1_ may be reacted with thionyl chloride to prepare the corresponding propionyl chloride, which may then be reacted with an amino acid ester of formula 2_ or with the corresponding free acid 2a in an inert solvent such as- acetonitrile in the presence of a base such as triethylamine to give isomers of formula _3_, which may be separated as in Scheme 1. The following Scheme 2 is an example:

wherein n, Ac, R^, R ^r and R are as defined above, and wherein R ^fc may also be hydrogen.

Other R esters of compounds of formula I are prepared by standard esterification techniques, for example N-(t-butoxycarbonyl)-S-allyl-(R)-cysteine is reacted with 2

( 2-chloroethoxy)ethanol in the presence of a coupling agent such as DEC and a base such as 4-dimethylaminopyridine, the amino function is deprotected and the resultant amino acid ester is reacted with a compound of formula 1 in a manner similar to that described in Scheme 2. In another example,

N-(t-butoxycarbonyl)-S-methylthiomethyl-(R)-cysteine is reacted with N,N-diethylbromoacetamide and a reagent such a cesium carbonate, the resultant ester is deprotected at the amino function and a reaction similar to that described in

Scheme 2 is carried out.

Compounds of formula I wherein R ³ is -NR ⁷ R ⁸ are prepared by coupling reactions as described above in Scheme

1 and 2 by replacing the amino acid ester 2_ with an amide o substituted amide as shown in Scheme 3:

^• 3

Alternatively, compounds of formula I wherein R is -NR 7R8 may be prepared by coupling a propionyl chloride of formula _8_ with an amino acid of formula 2a in the presence of a base and then coupling the desired -NR ⁷ R ⁸ group to the carboxylic group using a typical peptide- coupling reaction. Scheme 4 shows an example of such a procedure:

2a

_4a

A third method for preparing compounds- of formula

^■ 3 η o

I wherein R is -NR R° comprises reacting a propionic acid of formula _1_ with an amino acid t-butyl ester of for ula:_2_, removing the t-butyl ester and coupling the -NR 7R8 group to the carboxylic acid

Compounds wherein R 3 R7 are prepared analogously to those wherein R 3 is -NR7R8.

Compounds wherein Q is R CO- may be prepared by known methods, for example by adding a mercaptoacid of formula R COSH to an acrylic acid to obtain a thio- substituted propionic acid analogous to compounds, of formul 1. Alternatively, an amide of formula I wherein Q is -SH may be reacted with a compound of formulae R ¹⁰ COC1 in the presence of a base to obtain the desired sulfur substituted derivative.

Compounds wherein R ² is R ¹ (CH ₂ ) _k -S(0) ₁ _ ₂ -(CH ₂ ) - are prepared by oxidizing with hydrogen peroxide the corresponding substituted alkylthioalkyl compound of formul I (e.g. those wherein R is, e.g. R ¹ (CH ₂ ) _k -S-(CH ₂ . -) .

Starting materials of formulae _1_ and _2_ are known in the art or may be prepared by methods well known, to thos skilled in the art. See for example U.S. 4,329,495 for the preparation of the R_ and S_ enantiomers of 3-benzoylthio-2- benzylpropionic acid.

A second aspect of the invention is the administration of a combination of a compound of formula I and an ANF.

As indicated by Needleman et al., a number of ANF have been isolated so far, all having the same core sequence of 17 amino acids within a cysteine disulfide bridge, but having different N-termini lengths. These peptides represent N-terminal truncated fragments (21-48 amino acids ⁾ of a common preprohormone (151 and 152 amino acids for man and rats, respectively). Human, porcine and bovine σarboxy- terminal 28-amino acid peptides are identical and differ from similar peptides in rats and mice in that the former contain a methionine group at position 12 while the latter contain isoleucine. Various synthetic analogs of naturally occurring ANFs also have been found to have comparable biological activity. Examples of ANFs contemplated for use in this invention are α human AP 21 (atriopeptin I), α human AP 28, α human AP 23 (atriopeptin II or APII), α human AP 24, α human AP 25, α human AP 26, α human AP 33, and the corresponding rat sequence of each of the above wherein Met 12 is _l ie. See Table 1 for a comparison of the peptides.

TABLE l

HUMAN

— PE——PT—ID—E to ' w„

I « I

D (β .o cpv.3 tτιD-ι-ι ^ n α) - .εr>4J αcpα) >ι«e s-ι >-.3 >•<-£ I-I ωσ ^*

AP 33 (IHH h U βHI W M β JI WHH μ jl β H r-(r-_-l 0> •— I <D ι-l>.W CUjChi > α<!3C_<.» ιJ<_.!βl_Uft ^, JU< _- <t>.H(. «CUlOI3JUU«:o)(-<t«

AP 26 ζ

< t*

AP 25 5 ¹

AP 24 - g &

03 H

AP 23 S S"

AP 21 ø v w w

* I la in the rst peptidβ

A third aspect of the invention in the administration of a combination of an ACE, inhibitor and a compound of formula I. .. ^■

Examples of ACE inhibitors are those disclosed in the article by Wyvratt et al., cited above, and in the following publications: U.S. Patents 4,105,776, 4,468,519, 4,555,506, 4,374,829, 4,462,943, 4,470,973, 4,470,972, 4,350,704, 4,256,761, 4,344,949, 4,508,729, 4,512,924, 4,410,520 and 4,374,847; and the following foreign patents or published patent applications: GB 2,095,682, EPA 50,800, EPA 79,522, EPA 79,022 and EPA 46,953.

Following are preferred ACE inhibitors for use in the combination of this invention: spirapril, enalapril, ramipril, perindopril, indolapril, lysinopril, pentopril, cilazapril, captopril, zofenopril, pivalopril, fosinopril and L-(5)-2-[N-[1-(ethoxycarbony1)-3-phenylpropyl1alanyl]- 1,2,3,4-tetrahydro-6,7-dimethoxyisoquinoline-3-carboxylic acid monohydrochloride.

The antihypertensive effect of mercapto-acylamino acids alone and in combination with ACE.inhibitors is . determined according to the following procedure:

Male Sprague Dawley rats weighing 100-150 ^' g are anesthetized with ether and the right kidney is removed. Three pellets containing Doc acetate (desoxycorticosterone acetate, DOCA, 25 mg/pellet) are implanted subcutaneously. Animals recovered from surgery are maintained on normal rat chow and are allowed free access to a fluid of 1% NaCl and 0.2% KC1 instead of tap water for a period of 17-30 days. This procedure results in a sustained elevation in blood- pressure and is a slight modification of published procedures (e.g. Brock et al., 1982) that have been used to produce DOCA salt hypertension in the rat.

On the day of study, animals are again anesthetized with ether and the caudal artery is cannulated for blood pressure measurement. Patency of the caudal

artery cannula is maintained with a continuous infusion of dextrose in water at a rate of 0.2 ml/hr. Animals are placed into restraining cages where they recover consciousness. Blood pressure is measured from caudal artery catheter using a Statham pressure transducer attache to a Beckman oscillographic recorder. In addition, a cardiovascular monitoring device (Buxco Electronics, Inc.) and a digital computer are used to calculate average blood pressures.

After an equilibration period of at least 1.5 hr., animals are dosed subcutaneously (1 ml/kg) with vehicle (methylcellulose, hereinafter MC) or mercapto-acylamino acid and blood pressure is monitored for the next 4 hours.

The antihypertensive effect of mercapto-acylamino acids in combination with ANF is determined according to the following procedures:

Male spontaneously hypertensive rats (SHR), 16-18 weeks old, 270-350 g, are anesthetized with ether and the abdominal aorta is cannulated through the tail artery. The animals were then placed into restrainers to recover from anesthesia (in less than 10 min.) and remain inside throughout the experiments. Through a pressure transducer (Gould P23 series) analog blood pressure signals are registered on a Beckman 612 recorder. A Buxco digital computer is used to obtain mean arterial pressures. Patency of the arterial cannula is maintained with a continuous infusion of 5% dextrose at 0.2 ml/hr. Animals are allowed a 90-min equilibration period. The animals first undergo a challenge with an ANF such as atriopeptin II (AP II) or AP28 30 vg/kg iv and at the end of 60 min. are treated with drug vehicle or a mercapto-acylamino acid subcutaneously. A second ANF challenge is administered 15 min. later and blood pressure is monitored for the next 90 min.

The antihypertensive effect in SHR of mercapto- acylamino acids and ACE inhibitors, alone and in combination, is determined as follows:

— -

Animals are prepared for blood pressure measurement as described above. After stabilization, animals are dosed subcutaneously .o orally with test drugs or placebo and blood pressure is monitored for the next 4 hr.

The compositions of this invention comprise a mercapto-acylamino acid or a mercapto-acylamino acid and an ANF or a mercapto-acylamino acid and an ACE inhibitor in combination with a pharmaceutically acceptable carrier, for administration to mammals. . A variety of pharmaceutical forms is suitable, preferably for oral or parenteral administration, although mechanical delivery systems such as transdermal dosage forms are also contemplated.

The daily antihypertensive dose of the compound or combinations of this invention is as follows: for mercapto-acylamino acids alone the typical dosage is 1 ^' to 100 mg/kg of mammalian weight per day administered in single or divided dosages; for the combination of mercapto-acylamino acid and an ANF, the typical dosage is 1 to 100 mg of mercapto-acylamino acid/kg mammalian weight per day in single or divided dosages plus 0.001 to 0.1 mg ANF/kg of mammalian weight per day, in single or_ divided dosages, and for the combination of mercapto- acylamino acid and an ACE inhibitor, the typical dosage, is 1 to 100 mg of mercapto-acylamino acid/kg mammalian weight per day in single or divided dosages plus 0.1 to 30 mg ACE inhibitor/kg of mammalian weight per day in single or divided dosages. The exact dose of any component or combination to be administered is determined by the attending clinician and is dependent on the potency of the compound administered, the age, weight, condition and response of the patient. Compounds of this invention are not toxic at the therapeutically effective dose.

Generally, in treating humans having hyperten¬ sion, the compounds or combinations of this invention may be administered to patients in a dosage range as follows: for treatment with mercapto-acylamino acids alone, about 10 to about 500 mg per dose given 1 to 4 times a day, giving a total daily dose of about 10 to 2000 mg per day; for the combination of mercapto-acylamino acid and ANF, about 10 to about 500 mg mercapto-acylamino acid per dose given 1 to 4 times a day and about 0.001 to about 1 mg ANF given 1 to 6 times a day (total daily dosage range of ^' 10 to 2000 mg day and .001 to 6 mg/day, respectively); and for the combination of a mercapto-acylamino acid and an ACE inhibitor, about 10 to about 500 mg mercapto- acylamino acid per dose given 1 to 4 times a day and about 5 to about 50 mg ACE inhibitor given 1 to 3 times a day (total daily dosage range of 10 to 2000 mg/day and 5 to 150 mg/day, respectively). Where the components of a combination are administered separately, the number of doses of each component given per day may not necessarily be the same, e.g. where one component may have a greater duration of activity, and will therefore need to be administered less frequently.

Typical oral formulations include tablets, capsules, syrups, elixirs and suspensions. Typical injectable formulations include solutions and suspensions. Typical pharmaceutically acceptable carriers may be used in said formulations.

Examples of formulations follow. "Drug" refers to any mercapto-acylamino acid of the present invention. "ACE inhibitor" refers to any of the ACE inhibitors listed on page 14, especially those in the list of preferred ACE inhibitors. "Atrial peptide" refers to any antihypertensive atrial peptide, especially those listed in Table 1.

EXAMPLE 1

Tablets

Formula

No. In redient m tablet m /tablet

1 2 3

4 5

Method of Manufacture

Mix Item Nos . 1 and 2 in a suitable mixer for 10-15 _ minutes. Granulate the mixture with Item No. 3. Mill the damp granules through a coarse screen (e.g., 1/4") if needed. Dry the damp granules. Screen the dried - granules if needed and mix with Item No. 4 and mix for ^* 10-15 minutes. Add Item No. 5 and mix for 1-3 minutes.

Compress the mixture to appropriate size and weight on a suitable tablet machine.

EXAMPLE 2

Drug for Injection: (per vial) g/vial g/vial

Drug: Sterile Powder 0.5 1.0

Add sterile water for injection or bacteriostatic water for injection for reconstitution.

EXAMPLE 3

Disodium Edetate 0.1 0.1

Sodium Sulfate 2.6 2.6

Water for Injection q.s. ad 1.0 ml 1.0 ml

Method of Manufacture

Dissolve parabens in a portion ( 85% of the final volume) of the water for injection at 65-70°C. Cool to 25- 35°C. Charge and dissolve the sodium bisulfite, disodium edetate and sodium sulfate. Charge and dissolve the drug. Bring the solution to final volume by adding water for injection. Filter the solution through 0.22 μ membrane and fill into appropriate containers. Terminally sterilize the units by autoclaving.

EXAMPLE 4

Drug for Injection: (per vial) g/vial Drug 1.0

Sodium Citrate 0.05

pH is adjusted to 6.2 using 0. IN citric acid solution.

Add sterile water for injection or bacteriostatic water for injection for reconstitution.

EXAMPLE 5 Tablets Formula

Method of Manufacturing

Prepare - the tablets as in Example -I .- ^"*

EXAMPLE 6

Tablets

Formula

No. 1 2 3

4 5

Method of Manufacturing

Prepare the tablets as in Example 1.

EXAMPLE 7

Tablets

Formula

No. 1 2 3 4

5 6

Method of Manufacturing

Prepare the tablets as in Example 1.

EXAMPLE 8

Tablets

Formula

No. Ingredient mg/tablet mg/tablet 1 Atrial Peptide 0.1 1 2 Lactose 61 121

Method of Manufacture

Prepare tablets as in Example 1.

Following are descriptions of preparations of typical starting materials and examples of procedures for preparing compounds of formula I.

PREPARATION 1

S-ALLYL-(R)-CYSTEINAMIDE HYDROCHLORIDE

Step 1: N-t-Butyloxycarbonyl-S-allyl-(R)-cysteine: Treat S-allyl-(R)-cysteine (1.42g) in THF (20 ml) and MeOH (5 ml) with di-t-butyl dicarbonate (2.1g) and triethylamine (2.5 ml) and stir the resulting mixture at room temperature for 20 hr. Concentrate the mixture in vacuo, dilute with water, and extract with hexane. Acidify the aqueous solution with KHSO. solution and extract with EtOAc. Concentrate the dried (MgS0 ) EtOAc solution in vacuo to give the title compound, a clear oil .

In a similar manner, using the appropriate amino acid, prepare:

N-t-Butyloxycarbonyl-S-methylthiometyl-(R)- cysteine, a white solid, m.p. 79-80°C, [α]§ ⁶ = -33.0° (MeOH) .

Step 2: N-t-Butyloxycarbonyl-S-allyl-(R)-cysteinamide: React N-t-butyloxycarbonyl-S-allyl-R-cysteine (2.02g) with triethylamine (2.5 ml) in THF (25 ml). Cool the

mixture to 0-5°C. Add ethyl chloroformate (1.4 ml) in THF (5 ml) dropwise over 5 min. and stir the reaction mixture for 15 min. Add ammonium: hydroxide (29%, ^" 0.8,ml) in THF (5 ml) dropwise. Allow the reaction -mixture to warm to room temperature and stir for 18 hr. Filter the reaction mixture and concentrate the filtrate in vacuo. Dissolve the resultant residue in EtOAc and extract with H ₂ 0 and brine. Concentrate the dried (MgS0 ₄ ) EtOAc solution in vacuo to give a white solid, m.p. 105-108°C, [α] _D ²⁶ = -13.9° (MeOH).

In a similar manner, prepare N-t- butyloxycarbonyl-S-methylthiomethyl-(R)-cysteinamide, a white solid," m.p. 108-9°C, [ α]ξ ⁶ = -21.6° (MeOH).

Step 3: S-Allyl-(R)-cysteinamide hydrochloride: Treat the product of Step 1 (1.52g) with 9% HCl in dioxane (25 ml) at 0° and keep the resulting mixture at 0° for 2.0 hr. Concentrate the reaction mixture in vacuo. Triturate with Et ₂ 0 and dry in vacuo to give the title compound, a tan solid, m.p. 163-7°C, [α] ⁶ = 0.0° (MeOH).

In a similar manner, prepare S- methylthiomethyl-(R)-cysteinamide hydrochloride, a tan. solid, m.p. 166°C, [ α]§ ⁶ = -3.6° (MeOH).

EXAMPLE 1

N-[3-BENZOYLTHIO-2(S)-BENZYLPROPIONYL]-S- ALLYL-(R)-CYSTEINAMIDE Add 3-benzoylthio-2(S)-benzylpropionic acid (0.92 g) to S-allyl-(R)-cysteinamide hydrochloride (0.6 g), l-(3-dimethylaminopropyl)-3-ethylcarbodiimide . hydrochloride (DEC) (0.64 g), 1-hydroxybenzotriazole (HOBT) (0.47 g) and N-methylmorpholine (NMM) (0.7 ml) in dimethylformamide (DMF) (5 ml) and stir the resulting mixture at room temperature for 20 hours. Concentrate

the reaction mixture in vacuo and partition the residue between ethyl acetate and water. Concentrate the dried (MgS0 ₄ ) ethyl acetate solution in vacuo. Chromatograph the resultant residue on flash silica gel (200 ml) using ethyl acetate:hexane (7:13) (2L), then 1:1 as eluent to give the title compound, a white solid, m.p. 152-6°C, [ α] ² - ⁶ = -88.0° (MeOH).

In a similar manner, substitute S-methyl- thiomethyl-(R)-cysteinamide for the allyl compound to obtain N-[3-benzoylthiomethyl-2(S)-benzylpropionyl]-S- methylthiomethyl-(R)-cysteinamide, a white solid, m.p. 146-50°C, [α]f3 ⁶ = -101.1° (MeOH).

In a similar manner, substitute 3-acetylthio- 2(S)-benzylpropionic acid or the corresponding 2(R) compound for the 3-benzoylthio compound to obtain:

N-[3-acetylthio-2(S)-benzylpropionyl]-S-allyl- (R)-cysteinamide, a white solid, m.p. 116-20°C, [ α]β° = -61.6° (MeOH);

N-[3-acetylthio-2(R)-benzylpropionyl]-S-allyl- (R)-cysteinamide, a tan solid, m.p. 95-9°C, [ α] ² ° = -1.6° (MeOH) ;

N-[3-acetylthio-2(S)-benzylpropionyl]-S- methylthiomethyl-(R)-cysteinamide, a white solid, m.p. 106-8°C, [α]f5 ⁶ = -59.1° (MeOH); and

N-[3-acetylthio-2(R)-benzylpropionyl]-S- methylthiomethyl-(R)-cysteinamide, a light orange solid, m.p. 84-5°C [α] ²⁶ = -8.6° (MeOH).

Again in a similar manner, substitute 3- acetylthio-2(S)-benzylpropionic acid or the corresponding 2(R) compound for the 3-benzoylthio compound and substitute S-methylthiomethyl-(R)-cysteine ethyl ester for the corresponding amide to obtain:

N-[3-acetylthio-2(S)-benzylpropionyl]-S- methylthiomethyl-(R)-cysteine ethyl ester, a white solid, m.p. 52-3°C, [α] ²⁶ = -76.4° (MeOH); and

N- [ 3-acetylthio -2 ( R) -benzylprop ionyl ] -S- methylthiomethyl-( R) -cysteine ethyl ester, a white solid , m .p . 56-7 °C , [ α] £ ⁶ = -7 . 7° (MeOH ) .

EXAMPLE 2

N-[2(S)-ACETYLTHIOMETHYL-3-PHENYLPROPIONYL)-S- ALLYL-(R)-CYSTEINE 2-(2-CHLOROETHOXY)ETHYL ESTER 1

Step 1: Combine N-(t-butoxycarbonyl)-S-allyl-(R)- cysteine (5.0 g), 2-(2-chloroethoxyJethanol (2.38 g) and 4-dimethylaminopyridine (20 mg) in THF (25 ml). Add DEC (4.02 g) . Add DMF (10 ml), stir overnight, concentrate, and partition between EtOAc and IN HCl. Dry (MgS0 ₄ ) and concentrate to obtain the 2-(2-chloroethoxy)ethyl .ester as an amber oil, [ α] ²⁶ = -22.4° (MeOH).

Step 2: Treat the ester of Step 1 with 9% HCl/dioxane (10 ml). Store at 0° overnight, concentrate and. triturate the residue with Et ₂ 0 to obtain the hydrochloride as a tan solid, m.p. 84-6°C, [α]^ ⁶ = -0.6° (MeOH) .

Step 3: In a manner similar to that described in - Example 1, step 1, condense the hydrochloride of Step 2 (1.8 g) with 2(S)-acetylthiomethyl-3-phenylpropionic acid (1.41 g) and chromatograph to obtain the title compound as a colorless oil, [ α]- _Q ⁶ = -52.8° (MeOH).

In a similar manner, substituting 2(R)- acetylthiomethyl-3-phenylpropionyl chloride in Step 3, obtain:

N-[2(R)-acetylthiomethyl-3-phenylpropionyl)-S- allyl-(R)-cysteine 2(2-chloroethoxy)ethyl ester, a colorless oil, [α] ² . ⁶ = +9.8° (MeOH).

EXAMPLE 3

N-[2(S)-BENZOYLTHIO-3-PHENYLPROPIONYL)-S-ALLYL- (R)-CYSTEINE 2-(2-CHLOROETHOXY)ETHYL ESTER Combine the product of Example 2, Step 2 (0.704 g) with 2(S)-benzoylthiomethyl-3-phenylpropionic acid (0.7 g), HOBT (0.36 g) and NMM (0.49 g) in DMF (5 ml). Add DEC (0.49 g) and stir 18 hours. Concentrate and partition between EtOAc and H ₂ 0. Wash with IN HCl, brine, then dry and concentrate. Chromatograph the resultant residue on flash silica gel (200 ml)

(EtOAc:hexane 1:4 as eluant) to obtain the title ccoommppoouunnid, a white solid, m.p. 71-4°C, r«ι ²⁶ = -55.2'

(MeOH).

Using a procedure similar to that described in Steps 1 and 2 of Example 2, prepare N-t-(butoxy- carbonyl)-S-methylthiomethyl-(R)-cysteine 2-(2- chloroethoxy)ethyl ester hydrochloride, and then following a procedure similar to that immediately above, prepare:

N-[2(S)-benzoylthio-3-phenylpropionyl)-S- methylthiomethyl-(R)-cysteine 2-(2-chloroethoxy)ethyl ester, a white solid, m.p. 70-l°C, [ α]ξ ⁶ = -63.5° (MeOH).