SI8310101A8 - Process for obtaining z-2-acylamino-3-monosubstituted propenoates - Google Patents

Description

PROCEDURE FOR OBTAINING Z-2-ACIIAMINO-3-MONOSUPSTITOISED

PKOFENOMAA

Preparation of Dipeptidase Inhibitory Compounds which Can Be Used in Canning with Antibacterial Compounds Named Thienamycin Class of Compounds

Technically problan

A new class of antibodies with a fused--lactam ring has been described, including thienamycin and its semi-synthetic derivatives, epitienamycin and olivic acids. These compounds, called thienamycin compounds, have a high level of antibacterial activity but are subject to extensive metabolism by the Mammalian side.

There was a problem in the preparation of a compound that would act as dipeptidase inhibitors and which would be used in combination with thienamycin compounds.

The state of the art

A kidney was identified as the primary site of metabolism of thienamycin compounds and purified from renal extracts by an enzyme that catalyzes the inactivation of thienamycin by P-lactam hydrolysis.

Based on such criteria as cytologic localization and substrate specificity, as well as sensitivity to enzyme inhibitors, this enzyme is very similar to almost identical to the widely studied renal dipeptidase (E.C.3.4.13.11), also referred to as dehydrcpeptidase-I in the literature. However, the action of / 2> -lactamase

- 2 only prana to the thienamycin class of compound is expressed. There is no early example of metabolism in p-lactam cleavage patterns in the macaw representative of β -lactam antibiotics, penicillin and cephalosporins.

There is a description of a solution to a technical problem with an example

The present invention provides for the preparation of dipeptidase inhibitors that can be used in carbination with thienamycin compounds.

The chania substances which are inventively obtained selectively inhibit the metabolism of dipeptidase (E.C.3.4.13.11) are called dipeptidase inhibitors and are reacted by reacting the acid chloride of the formula

with the t-butyl ester-amino acid of the formula

R 4 -CH 1 -CH-COO-C (CH ₃ ) ₃ in the presence of a base such as triethylamine in a solvent such as methylene chloride at room temperature produces an N-acylated product of the formula

R ³ -CH _o -CH-C00-C (CH _Q ), ikm? ² which is oxidized by butyl hypochlorite with the addition of sodium methoxide at room temperature and then treated with anhydrous hydrochloric acid to form the desired ¢ (, ^ -acid, which is optionally converted into an acceptable sodium or potassium salt, of the formula

R ³ 'c ^z \ 1 ₂ R CONH COOR in which R ^x is lower alkyl, hydrogen or a pharmaceutically acceptable cation,

R and R are C _3-1 g or C ₁ _ ₁ ^ hydrocarbon radicals in which up to 6 hydrogen may be replaced by halogens or non-terminal methylene may be replaced by oxygen or sulfur.

The terminal hydrogen in R may also be substituted for a hydroxyln or thioln group, which may be arylated, for example with alkanoyl acid of 1-8 carbon atoms, or may be carbamoylated, including alkyl and dialkyl carbamate derivatives; or hydrogen may be replaced by amino groups, which may be a derivative such as acylamino, ureido, amidino, guanidino or alkyl or a substituted alkylamino group, including quaternary nitrogen groups; or alternatively there may be a substitution with acid groups such as carboxylic, phosphonic or sulfonic acid groups or their esters or amides, as well as cyano; or combinations thereof, such as terminal amino acid groups

R is preferably a branched alkyl or cycloalkyl radical (C

3-10), with the limitation that carbon adjacent to the carbonyl cannot be tertiary to the pit. R ¹ is hydrogen, lower alkyl (C 1-10) or dialkylaminoalkyl (e.g., -Cl 2 CH 1 CH 2 N (CH 3 · ·

Some compounds of formula II above have asymmetric forms. Racemic Z-2- (2,2-dimethylcyclopropanecarboxamido) -2-acetic acid is decomposed. The activity lies in the dextrogated isomer having the S-configuration.

Within the definition, R, the following sub-groups are included:

-R ⁴ IA wherein R is a straight, branched or cyclic hydrocarbon radical of 3-10 carbon atoms which may be substituted as specified above in definition R above;

-R ⁵ R ⁶ IB

6 where R is cycloalkyl of 3-6 carbon atoms and R is either 1 or 2 alkyl substituents which may be linked together to form a 5 6 second ring on a cycloalkyl group, or R and R may be substituted 2 as specified above in the definition of R;

-R ⁷ R ⁸

8 where R is an alkylene group of 1-3 carbon atoms and R is cycloalkyl of 3-6 carbon atoms which may be substituted

- 4 2 3 as specified above in the definitions R and R.

Within these sub-groups, the following specific compounds are included:

I A: Z-2-Isovaleramido-2-pentenoic acid; methyl

Z-2-from ovaleramido-2-butenoate; Z-2-isovaleramido-2-butenoic acid; Z-2-benzamido-2-butenoic acid; Z-2- (3,5,5 trimethylhexanamido) -2-butenoic acid; Ζ-2-cyclobutanecarbcksamido-2-butenoic acid; Ζ-2-cyclopropanecartocksamido2-butenoic acid; Z-2-cyclopropanecarboxamido-2-pentanoic acid; Z-2-cyclicheptanecarboxamido-2-butenoic acid; Z-2-nonamido-2-butenoic acid; N-2-cyclohexanecarboxamido2-butenoic acid; Z-2- (4-methylvaleramido) -2-butenoic acid; Z-2-t-butylacetamido-2-butenoic acid; Z-2-cctanamido-2-butenoic acid; Z-2-butyramido-2-butenoic acid; Z-2valeramido-2-butenoic acid; Z-2-valeramido-2 ~ pentenoic acid; Z-2-cyclopentanecarboxamido-2-butenoic acid;

Z-2- (6-methylheptanamido) -2-butenoic acid; Ζ-2-hexanamidote ¹ 2-butenoic acid; Z-2- (3,7-dimethylactanamido) -2-butenoic acid; Z-2- (3,7-dimethyl-6-cecenamido) -2-butenoic acid; Z-2- (5-chlorovaleramido) -2-butenoic acid; Z-2- (3-chlorobenzoylamido) -2-butenoic acid; Z-2- (3-chlorobenzoylamido) 2-butenoic acid; Z-2- (chlorobenzamido) -2-butenoic acid; Z-2-nonanamido-2-butenoic acid; Z-2- (6-Brnoxhexanamido) -2-butenoic acid; Z-2- (3,3-dimethylpropenamido) 2-butenoic acid; Z-2-benzamido-2-cinnamic acid; Z-2ben2amido-2-pentanoic acid; Z-2-benzamido-5-methoxy-2pentenoic acid; Z-2-benzamido-2-hexanedioic acid;

- 5 Z-2-isovaleramido-2-octanoic acid; Z-2-isovaleramido-2-cinnamic acid; Z-2-isovaleramido-2-hexanedioic acid; Z-2-cyclopropanecarboxamido-2-cinnamic acid; N-2-cyclopropanecarboxamido-2-hexanedioic acid; Z-2- (5-Methyl-3-methylvaleraraido) -2-butenoic acid; N-2-ethylthioacetamido 2-butenoic acid; H-2- (2-diblorocyclopropanecarboxylic) 2-butenoic acid; Z-2- (2-ethylhexanamido) -2-butenoic acid; Z-2-di-n-propylacetamido-2-butenoic acid;

IB; Z-2- ( _2f 2-2,2-dimethylcyclopropanecarboxamido) -2-butenoic acid; O) -Z-2- (2,2-diraethylcyclopropanecarboxanido) -2-butenoic acid; Z-2- ( _2f 2-Diethylethylcyclopropanecarboxyacido) -2-pentenoic acid; Z-2- (2,2-dimethylcyclopropanecarboxamido) -2-octenoic acid; Z-2- ( _2f 2-dimethylcyclopropanecarboxyido) -2-hexanoic acid; Z-2- (2, 2-Diethylethylcyclopropanecarboxarido) -2-cinnamic acid; Z-2- (2,2-dimethylcyclopropanecarboxylamido) -5-methyl-2-pentanoic acid; Z-2- (2,2-Diethylcyclopropanecarboxamido) 4,4,4-trifluoro-2-butenoic acid; Z-2- (2,2-dimethylcyclopropanecarboxarido) -3- (2-chlorophenyl) -propionic acid; Z-2- (2,2- dimethylcyclopropanecarboxamido) -2-hexanedioic acid; Z2- (2-ethylcyclopropanecarboxamido) -2-butenoic acid; Z-2 (2 _r 2-diethylcyclopropanecarfidccaraido) -2-butenoic acid;

Z-2- ( _2f 2-diethylcyclopropanecarboxamido) -2-pentenoic acid;

Z-2- (2-Isopropyl-2-methylcyclopropanecarboxamido) -2-butenoic acid; Z-2- (2-methylcyclohexanecarboxamido) -2-butenoic acid Z-5-cyano-2- (2,2-dinethylcyclopropanecarboxamido) -2pentenoic acid; Z-5- (N, N-dimethylcarbamoyl) -2-t2,2-dimethylcyclopropanecarbamoxy) -2-pentenoic acid; Z-2 (2,2-dimethylcyclopropanecarboxyanido) -5-methanesulfonyl-2pentenoic acid; Z-2- (2,2-dimethylcyclopropanecarboxamido) -5-6 ethoxycarbonyl-2-pentanoic acid; Z-2- (2-methylcyclopropanecarboxamido) -2-butenoic acid; methyl Z-2- (2,2-dimethylcyclopropanecarboxamido) -2-butenoate; ethyl Z-2- (2,2-dimethylcyclopropanecarboxamido) -2-butenoate; Z-2- (2,2-dimethylcyclopropanecarboxyamido) -2-butenoic acid 2-dimethylaminoethyl ester;

Z-2- (2,2-dimethylcyclopropanecarboxamido) -2-pentenoic acid 3- diethylaminopropyl ester; Z-2- (2,3-dimethylcyclopropanecarboxamido) -2-butenoic acid; Z-2- (3,3-dimethylcyclobutanecarboxamido) -2-butenoic acid; Z-2- (2-Spirocyclopentanecarboxamido) -2-butenoic acid; Z-2- (2-t-butyl-3,3-dimethylcyclopropanecarboxyamido) -2-butenoic acid; Z-2- (2,2dimethylcyclopropanecarboxamido) -4-methyl-2-pentanoic acid; Z-2- (2-t-Butylcyclopropylcarbamoxy) -2-butenoic acid;

Z-2- (2-Phenylcyclopropanecarboxamido) -2-butenoic acid /? Z-3-cyclohexyl-2- (2,2-dimethylcyclopropanecarboxyamido) propionic acid; Z-5-carboxy-5- (2,2-dimethylcyclopropanecarboxamido) 4- pentanamidine; Z-5-dimethylamino-2- (2,2-dimethylcyclopropanecarboxamido) -2-pentanoic acid; Z-3-cyclopropyl-2- (2,2dimethylcyclopropanecarboxamido) -propionic acid; Z-2- (2,2- dimethylcyclopropenecarboxamido) -2,5-hexanediic acid;

Z-2- (2,2-dimethylcyclopropanecarboxamido) -4-phenyl-2-butenoic acid; Z-2- (2,2-dimethylcyclopropanecarboxamido) -6-mercapto2-hexanoic acid; Z-2- (2,2-dimethylcyclopropanecarboxanido) 5-methylthio-2-pentenoic acid; Z-2- (2,2-dimethylcyclopropanecarboxamido) -5-phosphono-2-pentanoic acid; Z-2- (2,2-dimethyl cyclopropanecarboxamido) -2-heptenoic acid; Z-2- (2,2dimethylcyclopropanecarboxamido) -5-phenyl-2-pentanoic acid;

- 7 N-5- (2,2-dimethylcyclopropanecarboxamido) -2-nonenoic acid;

Z-2- (2,2-dimethylcyclopropanecarboxamido) -2-decenoic acid;

Z-2- (2,2-dimethylcyclopropanecarboxamido) -2-triacetic acid;

Z-2- (2,2-dimethylcyclopropanecarboxamido) -6-methoxy-2-hexanoic acid and (5-methoxy-2-pentenoic acid); Z-2- (2,2dimethylcyclopropanecarboxamido) -6-methyl-2-heptenoic acid; Z-2-4-Clecheloxy 1-2- (2,2-dimethylcyclopropanecarboxamido) -2-butenoic acid;

I C: Z-2-Cyclooctylthioethylamido-2-butenoic acid; N-2-cyclopentylacetamido-2-butenoic acid; N-2-cyclohexylacetamido 2-butenoic acid; Z-2- (4-cyclohexylbutyramido) -2-butenoic acid; Z-2-cyclopropylaoetamido-2-butenoic acid; Z-2 cyclopropylacetamido-2-pentanoic acid; Z-2- (3-cyclopentylpropicamido) -2-butenoic acid; Z-2- (3-cyclohexylpropionamido) -2-butenoic acid; Z-2- (3-cyclohexylpropionamido) 2-butenoic acid; Z-2- (4-thienyl) -butyramido) -2-butenoic acid; Z-2- (4-phenylbutyramido) -2-butene (D, L -? - lipoamido) 2-pentenoic acid; Z-2- (D, L-tX-lipoamido) -2-cinnamic acid;

Z-2- (3- (2-Tetrahydrofuryl) -propionamido) -2-butenoic acid.

Particularly preferred substituents in the definition of R above include 2,2-dimethylcyclopropyl and 2,2-dichlorocyclopropyl groups.

In the definition of R, particularly preferred groups of compounds include N-alkyl (1-9 carbons) and N-methyl (1-9 carbons), which have a terminal substituent which is a quaternary nitrogen, an amine derivative or a ____ ... amino acid derivative group.

Tenminan quaternary nitrogen means tetrasubstituted or heteroarctic nitrogen which is positively charged. Significant is the ammonium group, substituted by hydrocarbon groups having 1-7 carbons, which may be the same or different.

The term amino derivative is intended to mean a group such as amino, acylamino, ureido, amidino, guanidino and its alkyl derivatives.

The term amino acid derived group is understood to mean such a group as cysteinyl (-SC3 ^ CH (NI ^) OOOH) and or sarcosylL 't-NiCHgJCJ ^ OOOH) in which hydrogen is fused to 0, N or S of known amino acids by the substitution of en.

Particularly suitable compounds of the preferred groups of 2 2 substituents R and R are those wherein R is 2,2-dimethylcyclopropyl or 2,2-dichlorocyclopropyl / and R is a hydrocarbon series having 3 to 7 carbon atoms, without the tenninal substituent. or having a tertiary substituent which is trimethylammonium, amidino, guanidino, 2-amino-2-carboxyethylthio or ureido.

Names of specific examples include:

Internal are Z-2-C2,2-dimethylcyclicpropanecarboxamido) -8-trimethylammonium hydroxy-2-octenic acid;

the internal salt of Z-2- (2,2-dichlorocyclicpropanecarboxamido) -8-trinethylammonium hydroxide-2-octenoic acid;

Z-2- (2,2-dimethylcyclopropanecarboxamido) -8-amidino-2-octanoic acid;

Z-2- (2,2-dimethylcyclopropanecarboxamido) -8-guanidino-2-octanoic acid;

Z-2- (2,2-dimethylcyclopropanecarboxamido) -8-ureido-2-octoic acid;

9Z-8- (L-2-amino-2-carboxyethylthio) -2- (2,2-dimethylcyclopropanecarboxyido) -2-octenoic acid;

Z-2- (2,2-dimethylcyclopropanecarboxamido) -2-octenoic acid;

(Rahoe and right-handed form); i

Z-2- (2,2-Dichlorocyclopropanecarboxamido) -2-acetic acid.

Z configuration (JE Blackwood et al., J. Am. Chem. Soc. 90, p. 509 (1968 attributed to the above compounds on the basis of their NMR spectra by analogy to the work of A. Srinavasan et al. / Tetrahedron Lett. 891 (1976).

Although these compounds of Formula I, where R ¹ H is described and referred to as free acid, it will be apparent to those skilled in the art that various acceptable pharmaceutical derivatives such as alkali and alkaline earth metal salts, ammonium or amine salts or the like may be used as their equivalents. Suitable salts are sodium sulfate, potassium, calcium or tetramethylammonium salts.

THE usefulness of FINDING

As indicated above, the compounds of the present invention are dipeptidase inhibitors (E; C.3.4.13.11) and can be used in combination with antibacterial compounds that are subject to kidney degradation. The group of antibiotics of the present primary importance for use in combination with the Z-2-acylamino-3monosubstituted propenoates of the present invention are the thienamycin class of compounds.

The term thienamycin class of compound is used to identify more than a few natural, semi-synthetic

- 10 or synthetic derivatives or analogous compounds having a common ^ -lactam nucleus with a fused ring.

These compounds can be generically classified as 6- and (optionally) 2-substituted pen-2-em-3-carboxylic acids and 1-carbadetiapen-2-em-3-carboxylic acids or 1-azabicyclo / 3.2.0 / hept2- en-7-cn-2-carboxylic acids.

Specific compounds which are particularly useful in the σναη invention are represented structurally by the following formula II: X .6

II //

Oh

N-eooH · 3 where X may be or S; R may be hydrogen; -S-Cl ^ CHB ^^ HR z where R ^ is hydrogen, aoethyl, formimidoyl, acetimidoyl; -S (O) -CH = CHNHCOCH. and -S-CR ^ CHNHCOCH- ,; and R ⁶ is -CHCH. where R ⁷ □ j, 7 j

R '£

hydrogen, hydroxy or sulfanyloxy or R is H. All possible stereoisemic forms are included in the above structure defihition.

All these compounds within Formula II have been described in the 2 6 literature. When X is CH ₂ , and R is SOI ^ Cl ^ Ni ^ and R is CH (CH) CH ₃ , the compound is known as thienamycin, an antibiotic produced by fermentation of S. cattleya, described and protected in the US

Patent 3,950,357, published April 13, 1976. N-substituted thienamycin derivatives, i.e., in formula II above, wherein 3

R differs from hydrogen are described and protected in undecided U.S. Pat. applications and their published foreign equivalents.

- 11 θ

The fermentation product Nhacetylthienamycin (R is CH (CH) CH.

^J a R is acetyl, also called 924A, is protected by Belgian Patent No. 4,910. No. 848,346, published May 16, 1977. N-imidoyl derivatives are covered by Belgian Patent No. 848,346. No. 848,545, published May 20, 1977. The compound containing the unsaturated side sequence, so-called N-acetyl-dehydrothienamycin or 924A ^, is a fermentation product protected in US SN 788,491, filed April 18, 1977, .Case 16022, now ...... .. and also in

Belgian Patent No. No. 866,035, published October 17, 1978. Eplemic forms of N-acetylthienamycin, also called 890A and 890A ^, as well as desacetyl 890A and desacetyl 89OA3, are described, in published France application 763,887, filed Nov. 19, 1976. US SN 634,300, reported US Prior 634,300, reported. November 21, 1975, Hours 15745, and Belgian Patents 848,349, published May 16, 1977. Epidemic forms of unsaturated thienamycin, also called 890A2 and 890A ^, are protected in published French Patent of April 28, 1976, Hours 15839. N-acetyl compounds containing 6-sulfonyloxy, also called 890Αθ or 890A _{lQZ, are} protected in published France application 7,734,456, filed November 16, 1977, with US priority of November 17, 1976, Time 15935, respectively, in published French application no. No. 7,734,457, filed Nov. 16, 1977,

U.S. priority of November 17, 1976, Deacacetyl analogs 890Αθ and θδΟΑ ^ θ are protected in U.S. Pat. SN 767,723, filed Feb. 11, 1977, Cse 15975, now abandoned, and in its continuation U.S. Pat. SN

860,665, filed December 15, 1977, now ....... and also in France, application 7,803,665, filed February 9, 1978; i

- 12 U.S. SN 767,920, filed Feb. 11, 1977, Chase 15976, now abandoned, and its U.S. continuation. SN 006,959, filed Jan. 25, a.

1979, now ...... and, also, application to France 7,803,667, filed February 9, 1978. Some of these last compounds in the 890Α ^ and δΟΟΑ ^ θ series are also known as olivic acid derivatives (see Corbett et al., J _L Chem, Soc, Chan Ccnitiun.

1977, No. 1 24, pp. 953-54). The compounds of Formula I above when 2

The R hydrogen, also called descysteaminyl thienamycin, is protected by U.S. Pat. SN 668,898, filed March 22, 1976, Chas 15866, now abandoned, and n jencnr continued, U.S. Pat. SN 847,297, filed October 31, 1977, now abandoned, and also in Belgian Patent 867,227, published November 20, 1978.

When R ^ is hydrogen and X is Cf ^, these compounds are described in Times 15902, U.S. Pat. SN 843,171, filed January 1, 1977, and its published German equivalent Off. No. 2,751,624.1, filed Nov. 18, 1977.

The thienamycin type antibiotic in which R is -SO ^ CT ^ NHAc and r6 is C2H5, was named PS-5 and was published in K. Ckaimura et. al., J. Antibiotics 31 p. 480 (1978), see also Belgian Patent 865,578.

Compounds in which X S, also called penemi, are described by R.B. Woodward in Recent Advances in Chemistry by / ^ - Lactam Antibiotica ”, J. Elke (ed), The Chemical Society, London,

1977, p. 167; R. B. Woodward, Abstracts of the uppsala University 500 Years Symposium of Current Tcpics in Drug Research, Uppsala, Sweden. October 1921, 1977. Acta Pharm. Suecica, Vol 14, Supplement, p. 23, and U.S. Pat. Patent 4,070,477, published January 24,

1978.

- 13 Particularly preferred members in the thienamycin class of compounds are Nhformimidoyl and N-acetamidoyl thienamycin derivatives. The crystalline form of N-formimidoyl thienamycin recently described is also successful in the practice of the present invention. The following is an example that describes a preferred method of making this compound.

HOUSED EXAMPLE

N — Formimidoylthienamycin, crystalline

Phase A, Benzylformimidate chloride, crystalline

Triple bubble balloon of 3 1. Washed with levccm for addition, stirred front and condensed with reflux, batch with a mixture of benzyl alcohol (125 g, 1.15 mol), formamide (51 g, 1.12 mol) and anhydrous ether (1200 ml. ). The mixture was stirred vigorously at room temperature (20-25 ° C) under atmospheric nitrogen and added dropwise with benzoyl chloride (157 g, 1.12 moal) in 50 ml. of anhydrous ether using a funnel to add. The addition takes approximately 50 minutes.

The reaction mixture was stirred for an additional 60 minutes at room temperature. The ether was separated by decantation and 300 ml of acetanhydride in 500 ml was added. of anhydrous ether. The mixture was stirred for 30 minutes at room temperature. The precipitate was allowed to settle and the etaracethanhydride mixture was decanted again. The solid was collected by filtration, washed with 500 ml of ether and dried in vacuo over KOH at 25 ° C for 2 hours to give 130 g.

- 14 (67%) benzylfoimimidate hydrochloride as a white solid.

The product was tested for NMR c $ ~ (DMSO) 5.7 (s, 2H, 7.5 (s, 5h, 0), 9.0 (s, 1H, HC = N). The product is thermally unstable. It decomposes to formamide and benzyl chloride to 0 ° C and above, however, no noticeable decomposition was detected by staking at -20 ° C for 2 months.

Phase B, Thienamycin derivatization

Tienamycin (form 6 1. aqueous solution, pH = 6.5, fermentation broth concentrate containing 28 g of thienamycin) was placed in a large beaker t 12 1.) and cooled to 0 ° C. The beaker is equipped with a pH meter and a high-speed mixer. The pH was increased to 8.5 by carefully adding 3n KOH (KOH was added dropwise over a syringe to the mixed solution). The solution was treated with 6 equivalents of solid benzylformimidate hydrochloride (100 g part) in batches while maintaining the pH at 8.5 - 0.3 by adding 3N KOH (200 ml) using a syringe. Addition requires 3-5 minutes. The reaction mixture was stirred for 6 min. at 0 ° C and then subjected to liquid chromatography to ensure completion of the reaction. The solution was adjusted to pH 7 with IN HCL. The volume of the reaction mixture was measured and the solution tested for UV. The neutralized reaction mixture was concentrated to 15 g / l. on the reverse osmosis unit, the man is 10 ° C. The volume of the concentrate was measured and the pH adjusted to 7.2 - 7.4 as needed. The concentrate was filtered through a sintered funnel of average porosity to remove the solids present after concentration.

- 15 Phase C. Dowex 50W x 2 Chromatography

The concentrate (750-1000 ml., 15-20 g) was applied at 0 ° C. to a pre-cooled column of 18 1. Dowex x 2 in a potassium cycle (200 - 400 mesh. resin) and the column was eluted at 0 - 5 ° C with distilled actual water at a flow rate of 90 ml / min and a front pressed from 0 - 45 psig.

First, the previous fractions of 4 1, 2 1 and 1 are collected

1. and then 18 fractions of 450 ml each. each, and a kenak-final fraction of 2 1. Each test fraction is plated with UV (diluted 1/100. NI ^ OH extinction is omitted) and the total amount in each fraction is calculated. The initial and final fractions are tested for purity by liquid chromatography and the desired rich fractions are combined. The pH of the combined rich fractions was determined by the pH meter panel and the indicator solution panel with bromothymol blue and adjusted to pH 7.2 - 7.4 as appropriate. The combined rich fractions (3-4 1) were then tested for a UV panel and the total formamidine content, 15-16 g, 75% of the yield from the husk was determined. Rich fractions are concentrated on the reverse osmosis unit at a 10 ° C rental as high as possible, then the concentration is up to 33 g / l. end on a circular evaporator at 28 ° C. A total volume of about 500 ml is obtained.

Phase D. Crystallization of N-formimidoyl thienamycin

The concentrate from the previous phase was adjusted to X3, washed as needed and the content of N-formimidoylthienamycin was assayed with UV, and was about 85-9%. The concentrate is filtered through a sintered glass funnel (average porosity) into a large Erlenmeyer

- 16 balloons. Five volumes (approx. 220 ml) of 3A ethanol were filtered into concentrate and the solution was stirred at room temperature for 10 minutes and at oC for 12 - 24 hours.

The crystals were filtered off by vacuum filtration and washed with 0.1 volume (about 250 ml) of 80% 3A ethanol at oC and then with 1/25 volume (100 ml) of 3A ethanol at room temperature. The crystals were dried in vacuo for 12-24 hours to give approximately 40% total yield of N-phonnimidoyl thienamycin (10-12 g).

Analytical results at 50 g. mixtures of N-formimidoyl thienamycin made as above are as follows:

theoretically 45.42%; found, 45.82% H, theoretically 6.03%; found, 5.72% N, theoretically 13.10%; found, 13.10% Sr theoretically 10.10%; found, 10.14%

ignition residue, predicted 0.5, over 0.47%;

/ 4X? ⁵ = 89.4 °, TG = 6.8%, UV?) 300 MM, E% = 328.

D 'max

PROCEDURE FOR USING FINDINGS

As discussed above, thienamycin-type compounds are used in combination with a dipeptidase inhibitor.

The product of the combination is not part of the invention, but is protected in a pending application, Time 16174, US SN 927,213, filed July, 1978, now ......, and in Time 16174IA, U.S. SN ........

which was filed competitively with σναη.

The combination of novel chemical inhibitors of the present invention

- 17 and thienamycin class compounds may be in the form of a pharmaceutical composition containing two compounds in a pharmaceutically acceptable carrier. The two compounds can be used in amounts such that the weight ratio of the thienamycin class compounds to the inhibitor is 1: 3 to 30 µl, and preferably 1: 1 to 5: 1.

The components can also be added separately. For example, a thienamycin class compound may be administered intramuscularly or intravenously in quantities of 1-100 mg / kg / day, preferably

1- 20 mg Ag / day, or 1-5 mg Ag / day, in divided dosage forms, e.g., three or four times daily. The inhibitor may be administered separately, orally, intramuscularly, or IV (intravenously), in amounts of 1-100 mg / kg / day, or preferably 1-30 mg Ag / day, or 1-5 mg Ag / day. The amounts of two kcmpcents given during the day are ideally within the ratios indicated above.

The most preferred dosage levels currently known to applicants are one dose of two crystalline compounds, one being N-formimidoyl thienamycin and the other being (+) Z-2- (2,2-dimethylcyclopropanecarboxamido) -2-octanoic acid, which is given together in form for sterile aqueous IV injection (sodium salt), in the level of 150 mg. thienamycin and either 75 or 150 mg. of acetic acid. This dose is given to humans (each based on an estimated weight of about 80 kg) 1 to 4 times daily, or

2-8 mg / kg / day compounds of the thienamycin class and 1-8 mgAg / day of inhibitors.

The compositions, whether administered separately or in combination, are used in pharmaceutically acceptable carriers adapted for oral administration such as capsules, tablets or liquid solution or suspensions. The components, either individually or collectively, can also be dissolved in a vehicle adapted for injection. Suitable oral formulations may include diluents, odors, granulating agents, preservatives, binders and coating agents. For example, a preparation for oral use in the combination of the active ingredients, or the acid component alone, is mixed in a dry pulverized state with gelatin, starch, magnesium stearatam and alginic acid and compressed into a tablet.

As indicated above, the currently known preferred form is parenteral administration of a thienamycin class compound and any co-parenteral administration or oral administration of an inhibitory compound.

PROCEDURE FOR TESTING ANTIBACTERIAL AGENT

As indicated, studies on the absorption of thioiamycin, its convenient analogues and its semi-synthetic derivatives revealed that the major metabolic degradation pathways for elimination in the various species tested (mouse, pao, dog, chimpanzee, rhesus monkey), Cfoim metabolism reflected in niche isolation in urine and short plasma half-lives.

The nature of this degradation has been demonstrated to be lactam cleavage by renal dipeptidase (E.C. 3.4.13.11), as first described by Bergmann, M. and Schleich, H., Z. Physiol. Chem., 205 65 (1932); see also Greenstein, J.P., Advances in Enzymology, Vol. VIII, Wiley-InterScience, (1948), New York, and Campbell, B.J.; Lin, Υ-C., Davis, R.V. and Ballew, E.,

Ur '

The Purification and Properties of Particulate Renal Dipeptidase, Biochem. Biophys. Acta., 118, 371 (1966).

To demonstrate the ability of the compound of Formula I to suppress the action of the renal dipeptidase enzyme, an in vitro test procedure was followed. This measures the ability of the compounds to inhibit the hydrolysis of glycyldehydrophenylalanine (GDP) by a panel of a dissolved dipeptidase preparation isolated from the kidney of a kernel. The procedure is as follows: into a 1 ml system. containing 50 mM M3PS (3- (n-morpholino) propanesulfonic acid) buffer, pH 7.1, 5 mg of lyophilized enzyme was added, and the compound tested to a final concentration of 0.1 mM. After several minutes of inoculation at 37 ° C, GDP was added to a final concentration of 0.05 mM. The incubation was continued for 10 minutes at 37 ° C and the hydrolysis of GDP was measured by changing the optical density from temporal to 275 nm. Enzyme inhibition is measured by comparison with a standard experiment containing no enzyme and is expressed as the inhibitor binding constant, K ^. This is the concentration of inhibitor that achieves 50% inhalation of the enzyme.

The GDP substrate is used more preferably than thienamycin in this assay because it has a much higher maximum hydrolysis rate at night of renal dipeptidase, thus reducing the amount of enzyme required. Both GDP and thienamycin have a similar affinity for renal dipeptidase; further, the IC values of the inhibitors tested were identical for the two substrates.

In addition to this in vitro test procedure, in vivo testing was violated to measure the ability of the test compound to inhibit metabolism, which is reflected in the increased isolation of thienamycin from the bladder of mice. The process involves co-giving

- 20 compounds to be tested by intravenous or subcutaneous route at a rate of 10-100 mg / kg with 10 mg / kg of thienamycin. The isolation of thienamycin in the urine over a period of 4 hours was compared with its isolation in the control group to which the test compound was not co-administered.

Urinary thienamycin isolation was measured in all cases using a cylinder or disc diffusion test performed as described in U.S. Pat. Patent 3,950,357, -This bioassay, with Staphylococcus aureus ATCC 6538 as a professional organism, has a useful reaction interval of 0.04 pg / ml to 3.0 pg / ml.

The following are examples that illustrate this invention.

0DEL3AK 1. EXAMPLES TO ILLUSTRATE ACTIVITY

EXAMPLE 1

In vitro testing data

A system of 1 ml of 50 mM M3PS buffer, pH 7.1 was used. To this, 5% of the porcine renal enzyme was added and the amount of the compound to be tested leading to its final concentration of 0.1 mM. After five minutes of incubation at 37 ° C, such amount of GDP was added to bring the final concentration to 0.05 mM. The system was incubated again for 10 minutes, at 37 ° C. GDP hydrolysis is measured by pranenan optical density with time at 275 nm. Enzyme inhibition is overestimated by comparison with a standard inhibitor-free experiment and presented as percentage inhibition. is a constant that is required to produce 50% enzyme inhalation. This is the calculated value obtained by performing more in vitro pr both, as above, at concentrations leading to lower inhibition and above the 50% inhibition point. The results are shown in Table I.

TABLE I

COOH ______R ^J —C = ξ c —_nhcor;

_R 3 _R 2 dipeptidase% Inhibitor

-4 ' ^K i at 10 .i! _____ Ih «1 _______ i ch ₂ ch ₃

2® ch ₃

2a * c « ₃

2b 'CHj

CU ₃

CH, with 3

100

0.18

0.39

0.12

19.8

1.7

CHjCHj

CH - CH \

3.2

CH,

CH,

-ch ₂ ch-ch ₂ c (ch ₃ ) ₃

CH,

4.4

CH,

A ~

CH,

CH,

4.6

Compounds 2, 2a and 2b are Raoemic, dextrogyric and left-handed forms, respectively.

Tab! T, continued

CH ₃ - -CHj-CH * \ 91 6 CH ₃ ch ₃ o 80 6.2 ch ₃ -¾ A 83 ' 6.6 cn ₃ Λ 97 9 CH ₃ -CH ₂ -CH-CH ₂ CH ₃ 82 10 J ·, • 12 - (ch ₂ ) ₄ ch ₂ .03 13 - <ch ₂ ) ₅ n ⁺ (ch ₃ ) ₃ A; " Cl 14 15 - (CH ₂ ) ₅ N ⁺ (ch ₃ ) ₃ «3 - (CHj) ₅ ~ NH-C» NH tv ” ³ • ch ₃ Ά ^ ί ^ <* 3 1.11 16 - (CH,) _c -NH-CN ⁺ * -> 1 NH (CH ₃ ) ₂ A ^ ^CH 3 ch ₃ 17 H ”(CH,) -S-CH, -Ci ^{2 2} 1 NH co ° - ^ ch ₃ 3 ^ 3 0.21

- 23 τάρτ.τγα and continued

ia ch ₃ -CH ₂ C (CH ₃ ) ₃ 75 20 19 ch ₃ - (CHjJgGHj 72 26 20 ch ₃ - (CH ₂ ) ₂ CH ₃ 69 30 21 ch ₃ - ^<C “? 3 o 68 30 22 ch ₃ .c ₂ .Δ 64 22 23 CHj (CH ₂ ) ₃ CH ₃ 64 32 24 CH ₃ 59 30 25 ch ₃ - <ch ₂ ) ₄ ch <ch ₃ ) ₂ 57 ' 26 CHj -ch ₂ ch ₂ 56 27 CH ₃ -ch ₂ ch ₂ About 54 28 ch ₃ -CH ₂ - {CH ₂ ) ₃ CH ₃ 54 39 29 ch ₃ - (CH ₂ ) _with CH ₃ 49 30, ch ₃ -CH (CH ₂ CH ₃ ) CHjCHjCHjCHj 33 31 ch ₃ -CH (CH ₂ CH ₂ CH ₃ ) ₂ 13 32 ch ₃ -CH (CH ₃ ) ₂ 31

- 24 TABLE I, continued

33 HOO-CHjCHj 90 5 34 ch ₃ -CH, -CH-CH, CH, OCH, L 88 9 35 CHj CH ₃ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ Art 70 19 36 «3 CHjCHjCHjCHjCI 64 20 37 ch ₃ ch ₂ ch ₂ ch ₂ 72 11 38 ch ₃ <1 C (CK ₃ ) ₃ 90. 6.5 39 CH ₃ (CH ₂ ) ₄ CH ₂ -CH (CH ₃ ) ₂ 95 2.6 40 ch ₃ - CH ₂ CH ₃ ch ₃ 100 0.45 41 (CH ₃ , ₂ CH ch ₃ 98 0.54 42 ch ₃ ch ₂ ch ₃ CH ₂ CH ₃ 98 0.86 43 ch ₃ - <1 96 1.6

CH ₂ CH ₃

- 25 TABLE I nozzle

44 ^CH 3 ch (ch ₃ ) ₂ ch ₃ 45 CHjCHj 5 “ ^ch i * CH ₃ 46 Ph ca ₃ ch ₃ 47 CHjCHjCHj CH, ch ₃ CH ₃ 48 \ ³ CHCH, / CH ₃ CH ₃ 49 CHj (CH ₂ ) ₃ ^CH 3 50 ch ₃ (ch ₂ ) ₄ ca ₃ ch ₃ 51 HOOCC8 ₂ CH ₂ ^< j ~ ^CT 3 ca ₃ 52 O · ** » ^< j— “3 ch ₃ 53 PhCH ₂ CH ₂ ch ₃ ch ₃

Χ00

100

0.18

0.62

0.11

0.23

0.11

100 0.17

0.145

100 0.15

0.33

CHjSCHjCHj

CH ₃ SO ₂ CH ₂ CH ₂

5 «ch ₃ (ch ₂ ) ₅ ch ₃ <ch ₂ ) ₆ ch ₃ (ch ₂ ) ₉

PhCH ₂

CH ₃ O (CH ₂ ) ₃

CH ₃ OCH ₂ CH ₂

TABLE AND CONTINUED

CH ₃ <μ

CH,

CH,

CH,

0.12

0.5

0.149

0.092

0.14

0.44

0.28

0.32

- 27 TABLE I Continued (CHjJjCCHj (CH ₃ ) ₂ CHCH ₂ CH ₂

H ₂ OC (CH ₂ ) ₃ s0.34

0.15

0.048

0.39

- 28 EXAMPLE 2

In vivo test data

Tests were performed in vivo in mice as follows: 20 g heavy Chrales River CD mice (female) were treated subcutaneously with injections with a selected dose of chemical inhibitor.

Two minutes later, a dose of thienamycin was given intravenously. Thienamycin catitrol was also performed above. The level of thienamycin in the urine as% of dze was measured using a bioassay technique. The results are in Table II. The tests were performed with two test compounds whose numbers are as in Table I. Compound 7 is 2-isovaleramido-2-butenoic acid; Compound 10 is Z-2-cyclopropylcarbamsamido-2-butenoic acid.

TABLE II

The compound Dose, mg / kg compounds Dose, mg / kg thienamycin % of insulation of urine cyanamycin 7 50 10 53 7 10 10 53 1Q 50 10 56 Control - 10 25-30

- 29 EXAMPLE 3

2-Isovaleramido-2-butenoic acid compound. Compound 7, and Z-2- (2,2-dimethylcyclopropanecarboxamido) -2-butenoic acid were studied in vivo in combination with thienamycin (THM) in mice in greater detail in mice. The general test of the procedure was similar to that of Example 2. The results are summarized in Table III and Table IV.

TABLE III: Effect of co-administration of 2-isovaleramidecbutenic acid (Compound 7) on isolated thienamycin from urine in mice

The way compound 7 THM mg / kg dose of compound 7 THM Isolation of THM from the urine - IV or SC - 10 30 - 5 SC SC 0.3 10 33 SC IV ^; 2 10 42 SC SC 2 10 47 SC IV 10 10 53 Ph.D. SC 50 10 54 Ph.D. IV 50 10 53 Ph.D. SC SC 10 59 Ph.D. SC 100 10 81

(a) 20 g Charles River, CD ^ female mice (b) co-administered

- 30 TABLE. IV: Effect of administration of Z-2- (2,2-dimethylcyclopropanecarboxamido) -butanoic acid (Compound 2) for the isolation of thienamycin from mouse mice ^

The way The compound 2 THM mgAg dose Compound 2 THM Urinary Isolation% - SC - 10 10 ί 5 SC SC 0.1 10 35 Ph.D. SC 0.3 10 40 Ph.D. SC 1 10 46 Ph.D. Ph.D. 10 10 60 Ph.D. Ph.D. SC 10 73

(a) 20 g Charles River, CD ^ female mice (b) co-administered

EXAMPLE 4

In another study in mice, the systemic antibacterial activity of thienamycin was enhanced by approximately three-fold by coding for 2-isovaleramido-2-butenoic acid, see Table V.

TABLE V: Effect of co-administration of 2-isovaleramido-2-butenoic acid on systemic activity of teinamycin during treatment of infection with Staphylococcus aureus

THM alone + 100 mgAg inhibitors

ED ₅₀ , mgAg 0.2 0.06

- 31 EXAMPLE 5

An unclean male dog was used to study the effect of dipeptidase inhibitors on the isolation of N-fQrmimidoyl thienamycin from the urine. In the control study, the dog was given 5 πκτ / kg IV N-formimidoyl thienamycin without inhibitor. Another experiment used the same amount of N-formimidoyl thienamycin, but Z-2-isovaleramido-2-butenoic acid was also administered in 3 doses, each providing 20 mg / kg of compound. The first dose was given just after injection of N-formimidoyl thienamycin, the second after 40 minutes and the third after 60 minutes. The third study used a single dose (2 mg / kg) of Z-2- (2,2-dimethylcyclopropanecarboxyamido) -2-butenoic acid given just before the injection of N-formimidoyl thienamycin. The results are in Table VI.

TABLE VI: Isolation of urine 3 hours after administration of N-formimidoylthienamycin (5 mg / kg IV) to dog whisker 'Excess compound% isolated in urine

N-formimidoyl thienamycin 7.8 plus Z-2-isovaleramido-2-butenoic acid 46 plus Z -2- (2,2-dimethylcyclopropanecarboxyamido) -2-butenoic acid 53

The process for novel inhibitory compounds utilizes the t-butyl ester of an alpha amino acid in reaction with acid chloride:

r ² -cci r ³ -ch2-c-cooc (CH ₃ ) ₃

NH ₂ in VI

This reaction is carried out in the presence of a base, such as triethylamine, in such a solvent as methylene chloride. The debilitated N-acylated product (VII) is then oxidized by treatment with t-butyl hypochloride and then addition of sodium methoxide. This yields 2-methoxy derivative (VIII) and / or its elimination product alpha, beta-unsaturated ester (II). Further treatment with anhydrous hydrochloric acid translates either VIII or IX (or a mixture of both) into the desired alpha, beta-unsaturated hydrochloric acid (II).

ch ₂ chcjo ^ c (ch ₃ )

NHCR i '

OCR,

I ³ and R C3i ₂ (jW ₂ C (CH ₃ ) ₃

NHCR ²

H

VII

VIII

R ³ CH = CCO _o C (CH _Q ) o | 2 3 3

NHCR

It

IX

EXAMPLE 6

2- (2,2-Dimethylcyclopropanecarboxamido) -2-hexanoic acid

Phase S: DIz-NOrleucine t-butyl ester

The general procedure is given in J. Org. Chan. 28, 1251 (1963)

- 33 In a suspension of 9.82 g (75 nmol) of DL-norleucine in 80 ml of dixane in a 500 ml large flask for pressurized cooling in an ice bath was added lightly with a mixture of 8 ml of concentrated H ₂ SO 4. The resulting mixture was cooled in a dry ice bath after 80 ml of liquid isefutylene was added. The mixture was allowed to warm to room temperature and stirred under autogenous pressure for about 23 hours. After most of the isobutylene is weathered, the slightly cloudy solution is cooled on ice and then 400 ml of 1H NaOH and 500 ml of Et ₂ O are added to the cold mixture. After stirring in a separatory funnel, the layers are separated and the aqueous fraction is washed with 100 more. ml EtjO.

The Et ₂ O solution was stirred with 150 ml of 0.5 N HCl. The acidic aqueous fraction was treated with 2.5 N NaDi until it was very basic and then stirred with 250 ml Et ₂ O. The solution was dried (MgSO4), filtered and concentrated in a evaporator. After prolonged high-vacuum pumping in the steam bath, the final yield of clear, colorless residual oil = 9.0 g (65 S). NMR now only shows traces of dickans. TLC (9: 1 CH CHCl3-NaOH) showed one stain.

Phase B: N- (2,2-Dmethylcyclopropanecarbonyl) -DL-norleucine 1 t-butyl ester

A solution of 8.98 g (48 nmol) of DL-norleucine t-butyl ester and 5.05 gc of 5Q nmol) of triethylamine in 100 ml of ice-stirring C.sub.4 of the ice under a drying tube was added dropwise CU over a period of 75 minutes) solution of 6.39 g (40 nmol) 2,2-dimethylcyclopropanecarbonyl chloride C M. Elliot and

- 34 Н.Р. James, British Pat. 1,260,847 (1972) in 50 ml CH2Cl2. Et ^ N.HCl precipitation occurs during addition, especially at the end. As the ice gradually melts, the mixture is allowed to warm to room temperature. After 16 hours, the mixture was shaken with 200 ml of 0.5 N HCl. The CI ^ C ^ fraction was washed with another 200 ml of 0.5 N NaOH and finally with 200 ml of t ^ O.

The CH2Cl2 fraction was dried over MgSO4, treated with charcoal and filtered through celite. The filtrate was concentrated on a rotary evaporator (finally under high vacuum). Yield of light orange residual oil = 11.93 g (88%). TLC (2: 1 hexane-BtCftc) showed one stain. NMR and IR are consistent with the proposed structures. After standing for several days, this material crystallizes; m.p. 52 over 65 °.

Phase G: t-Butyl 2 ^ (2/2-dimethylcyclopropanecarboxamido) “2methexyhexanoate

The Best Based Processes by H. Poisel and V. Schnidt, Chem. Ber. 108 2547 (1975).

To a solution of 6.37 g (22.5 mmol) of N- (1,2-dimethylcyclopropyl carbonyl) -DD-norleucine t-butyl ester in 35 ml of Et ₂ O stirred darkly at room temperature under N2 was added 2.69 ml (2.45 g of 22.5 mmol) t- of buitlhipochlorite. After 15 minutes, a solution of sodium methoxide made by dissolving 0.52 g (22.6 mmol) of sodium in 35 ml of MeOH was added. Stirring is continued at usually j temperature under N ₂ in the dark. After 16.5 hours, the precipitated NaCl was filtered. The filtrate was diluted with Et2O and washed successively with 3 x 50 ml of 0.5 N HCl, 50 ml of saturated Na ₂ CO ₃

- 35 and 2 χ 50 ml I ^ O. The Et 2 O phase was dried over MgSO 4 and filtered. The filtrate was concentrated on a rotary evaporator. The light, golden yellow residual oil (6.45 g) was subjected to preparative liquid chromatography under cisocane pressure, resulting in the isolation and isolation of 273 mg and 496 mg of the two diastereoisers of t-butyl 2- (2,2diroethylcyclopropanecarboxyamido) -2-methoxyhexanoate (corresponding to 114-methoxyhexanoate). -118 ° and 124-125.5 °) as well as 1.97 g of one isaner (apparently Z) t-butyl 2- (Z ^ -dimethylcyclopropanecarboxylidoido Z-hexenoate (colorless oil).

Phase D; 2- (2,2-Dimethylcyclopropanecarfocksamido) -2-hexanoic acid

A solution of 0.84 g (3.0 ranol) t-butyl 2- (2,2-dimethylcyclopropanecarboxamido) -2-hexenoate in 10 ml of Et2O saturated with anhydrous HCl was allowed to stand at room temperature under a drying tube. After 5 h, the solution was evaporated and the residual gum was dissolved in 10 ml of saturated NaHCO3. This solution was washed with another 15 ml of 0.5 N HCl. It is then dried (MgSO4), filtered and concentrated to give a viscous oil. The oil crystallizes it toluene. Yield of white crystals = 0.32 g (47%), m.p. 119-122 ?. TLC (4: 1 toluene ^ -AcOH) pycases one stain. NMR shows an essentially pure Z-isoner. (Note: Treatment of the methanolic adduct of t-butyl 2- (2, 2-dimethylcyclopropanecarboxyainido) -2 ^ non-oxyhexenoate with anhydrous HCl and Et ^ O yields the same product under similar conditions.

Claims (2)

PATENT APPLICATION P-101/83 Hours 16140ΥΆ (10445) isolated from P-1788/79 A process for the preparation of Z-2-acylamino-3-monosubstituted propenoates of the formula wherein R is hydrogen, C 1-6 alkyl, or a pharmaceutically acceptable cation of Na or K; R is a C ₄ _ _1Q alkyl radical with a forked or reduced-cyclo C ^ alkyl, Cj_ ₃ radical koji knives that Buddha substituted manometric or di- with a C ₁ _ ₃ alkyl group or a halogen; R is a hydrocarbon series having 1 to 15 carbon atoms, which reacts with an acid chloride of the formula 2 * ' R - C - C1 V where R has the above meaning, with the alpha-amino acid t-butyl ester of the formula R ' -ch ₂ -ch-coo-c (ch ₃ ) ₃ ML · VI - 2 3 where R has the above meaning, in the presence of a base such as triethylamine and a solvent such as methylene chloride at room temperature, and produces an N-acylated product of the formula r ³ -ch ₂ -ch-ooo-c (ch ₃ ) ₃ NHCOR ² VII 2 3 in which R and R have the meanings given above, which is oxidized by t-butyl hypochlorite and the addition of sodium methacid at room temperature to give the derivative OCIL 3 ¹ R - ^ - C-COO-C (CH ₃ ) ₃ 2 NHCOR VIII in two stereoisomers and one isomer ZR ³ -CB = C-COO-C (CH _q ) _? I 2 NHCOR IX Wherein R and R have the meanings given above, it is treated with anhydrous hydrochloric acid to form Z-alpha, beta-unsaturated acid of formula I wherein R ¹ is hydrogen, which is optionally converted into a pharmaceutically acceptable sodium or potassium salt.