GB1572201A - Cephalosporins - Google Patents

Abstract

New amino-3-(2-carboxyalkyl-2,3-dihydro-3-oxo-s-triazolo[4,3-b]pyridazin-6 -ylthiomethyl)-3-cephem-4-carboxylic acids of formula I, their salts and their easily hydrolysed esters of the 4-carboxyl group are prepared. The synthesis consists in reacting a compound of formula III with a compound of formula IV and then treating the product obtained with an acylating agent R<2>-X. The compounds of the invention are powerful antibacterial agents which have a good solubility in water. <IMAGE>

Description

(54) CEPHALOSPHORINS (71) We, BRISTOL-MYERS COMPANY, a Corporation organised and existing under the laws of the State of Delaware, United States of America, having offices located at 345 Park Avenue, New York, New York 10022, United States of America, do hereby declare the invention for which we pray that a patent may be granted to us and the method by which it is to be performed to be particularly described in and by the following statement:- The cephalosporins of the present invention in general possess the usual attributes of such compounds and are particularly useful in the treatment of bacterial infections.

The cephalosporins are a well-known group of semisynthetic antibaterial agents made originally, for example, by acylatien of the 7-amino group of the nucleus 7-aminocephalosporanic acid (7-ACA) and later by similar acylation of nuclei derived therefrom, as by modification of its substituent at the 3-position.

Various reviews have appeared in the scientific literature (e.g. Cephalosporins and Penicillins - Chemistry and Biology, edited by Edwin H. Flynn, Academic Press, New York, 1972, and particularly pages 554569) and in the patent literature, e.g.

as in U.S. Patents Nos. 3,687,948; 3,741,965; 3,743,644; 3,759,904; 3,759,905; 3,766,175; 3,766,906; 3,769,281; 3,769,801; 3,799,923; 3,812,116; 3,813,388; 3,814,754 and 3,814,755 (all United States Class 26()-243C).

Issued patents on 34hiolated cephalosporins in which the 7-substituent is a) a-Amino-cx-phenylacetamido include U.S. 3,641,021; U.S. 3,734,907; U.S.

3,687,948; U.S. 3,741,965, U.S. 3,757,015, U.S. 3,743,644, Japan 71/24400 (Farmdoc 46374S), Belgium 776,222 (Farmdoc 38983T; U.K. 1,328,340 which includes various substituents on the benzene ring), Belgium 772,592 (Farmdock 19696T; U.S. 3,687,948, 3,734,907 and 3,757,012), West Germany 2,202,274 (Farmdoc 50428T) corresponding to U.S. 3,759,904, Netherlands 7205644 (Farmdock 76309T; U.S. 3,757,014); and b)- o-, m- or p-aminoethoxyphenylacetamido as Netherlands 72/13968 (Farmdoc 24740U) corresponding to U.S. 3,759,905 and c) o-aminomethylphenylacetamido as U.S. 3,766,176 and 3,766,175 (which also review of the older patent literature concerning substituted 7-phenylacetamidocephalosporanic acids) and d) N-(phenylacetimidoyl)aminoacetamido as U.S. 3,692,779; and e) a-amino-a-(1,4-cyclohexadienyl)acetamido as in, Belgium 776,222 (Farmdoc 38983T; U.K. 1,328,340).

Additional similar disclosures are found in U.S. 3,692,779 (Belgium 771,189;.

Farmdoc 12819T), Japan 72/05550 (Farmdoc 12921T), Japan 72/05551 (Farmdoc 12922T), U.S. 3,719,673 (Belgium 759,570; Farmdoc 39819S), Belgium 793,311 (Farmdoc 39702U) and Belgium 793,191 (Farmdoc 39684U).

Issued disclosures of 3-thiolated cephalosporins in which the 7-substituent is 7mandelamido (7-a-hydroxyphenylacetamido) are found, for example, in U.S.

3,641,021, France 73.10112, U.S. 3,796,801, Great Britain 1,328,340 Farmdoc 38983T), U.S. 3,701,775, Japan 48--44293 (Farmdoc 55334U) and in Hoover et al., J. Med. Chem. 17(1), 3441 (1974) and Wick et al., Antimicrobial Ag. Chemo., 1(3), 221-234 (1972).

U.S. 3,819,623 (and, for example, also U.K. 1,295,841 and West Germany 1,953,861) discloses specifically and with working details the preparation of 2-mercapto - 1,3,4 - thiadiazole - 5 - acetic acid and its conversion to 7 (lH - tetrazol - - yl - acetamido) - 3 - (5 - carboxymethyl - 1,3,4 - thiadiazol - 2 - ylthiomethyl) -3 - cephem - 4 - carboxylic acid which is also disclosed in West Germany Offenlegungsschrift 2,262,262.

U.S. 3,766,175 and 3,898,217 disclose A compound of the formula

wherein R is

or a nontoxic, pharmaceutically acceptable salt thereof, and A compound of the formula

wherein R is -H or lower alkyl; R1 is -H (lower)alkanoyloxy,

n is an integer from 4--7, inclusive; and the pharmaceutically acceptable addition salts thereof.

respectively.

U.S. 3,883,520 and 3,931,160 and Farmdoc Abstract 22850W make reference to 3-heterocyclicthio-methyl cephalosporins containing a number of substituents (including carboxyl) on the numerous heterocycles included but these references are completely general in nature and include no physical constants, yields, methods of synthesis or the like and do not even name any such compound containing a carboxyl substituent.

U.S. 3,928,336 provides a review of much of the older cephalosporin art.

U.S. 3,907,786 and 3,946,000 discloses cephalosporins containing various fused ring bicyclic thiols.

Farmdoc abstract 1 8830X discloses compounds of the formula

(where R' = acyl or H; R3 = H or methoxy; n = 1--9).

Our copending Application No. (Serial No. 1572202) 25885/79 describes and claims a compound having the formula

wherein R1 represents

wherein R is hydrogen, hydroxy or methoxy; R' is hydrogen or methyl; and R2 is hydrogen, or a conventional, pharmaceutically acceptable, easily hydrolyzed ester forming group; or a non-toxic, pharmaceutically acceptable salt thereof.

The present invention provides compounds having the structure

often written herein as

wherein R' is acyl or hydrogen and esters and non-toxic pharmaceutically acceptable salts thereof. The stereochemistry of the bicyclic nucleus is that found in cephalosporin C.

The esters of the compounds of formula I include, but are not limited to, those having the group

wherein, when W represents hydrogen, Z represents (lower)alkanoyl, benzoyl, naphthoyl, furoyl, thenoyl, nitrobenzoyl, methylbenzoyl, halobenzoyl, phenyl- benzoyl, N-phthalimido, N-succinimido, N-saccharino, N-(lower)alkylcarbamoyl, (lower)alkoxy, (lower)alkylthio, phenoxy, carbalkoxy, carbobenzoxy, carbamoyl, benzyloxy, chlorobenzyloxy, carbophenoxy, carbo-tert.-butoxy or (lower)alkylsulfonyl, and when W represents carbalkoxy, Z represents carbalkoxy and, when W represents phenyl, Z represents benzoyl or cyano or wherein W and Z taken together represent 2-oxocycloalkyl containing 4 to 8 carbon atoms inclusive.

Preferred embodiments of this invention include the pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl, fi -trichloroethyl, 3phthalidyl- or 5-indanyl- esters.

By the term "lower" as used herein we mean that the group so qualified has a carbon chain length of up to 12 carbon atoms.

Acyl (R') comprises the groups having the structures:

wherein R is hydrogen, hydroxy or methoxy and R' is hydrogen or methyl.

A preferred embodiment of the present invention consists of the compounds of Formula I wherein RI has the structure

Another preferred embodiment of the present invention consists of the compounds of Formula I wherein R1 has the structure

A preferred embodiment of the present invention consists of the compounds having the formula

wherein R1 represents

wherein R is hydrogen, hydroxy or methoxy; R' is hydrogen or methyl;

n is 0 to 4; R is hydrogen, alkyl having I to 8 carbon atoms, cycloalkyl of 3 to 6 carbon atoms, phenyl, C1-C4 phenylalkyl, pyridyl, thienyl, or pyrrolyl; R10 is hydrogen, methyl or ethyl; R2 and R3 are each hydrogen, alkyl having 1 to 6 carbon atoms, phenyl, pyridyl, or thienyl; R4 and R" are each hydrogen or alkyl of I to 4 carbon atoms; RS is alkyl having I to 4 carbon atoms, phenyl, phenalkyl having I to 4 carbon atoms, pyridyl, thiadiazolyl, amino or C1-C4 alkylamino; X is NH or oxygen; and each phenyl group is unsubstituted or substituted with one or two substituents selected from alkyl having I to 6 carbon atoms, alkoxy having 1 to 4 carbon atoms, hydroxy, amino, NHR1, N(R1)2, nitro, fluoro, chloro, bromo or carboxy, or a non-toxic, pharmaceutically acceptable salt thereof.

Another preferred embodiment of the present invention consists of the compounds having the formula

wherein R' represents

wherein R is hydrogen, hydroxy or methoxy; R' is hydrogen or methyl; and M is selected from

wherein Ras is a hydrogen atom, a methyl or ethyl group; X2 is -0-, -NH-; Re is a basic group such as -alkyl or aralkyl substituted with substituted or unsubstituted NH2, such as alkyl-NHCH3, aralkyl-NHCH3,

alhyt-NH- , Qralkyl- - CH2 s CH2NH2, - IH~CHz 2 NH R7 is an alkyl group such as a methyl, ethyl propyl, isopropyl, butyl, isobutyl, pentyl or 2-ethyl-hexyl group; a cycloalkyl group such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl; an aryl group such as phenyl or naphthyl; an aralkyl group such as benzyl or naphthylmethyl; a heterocyclic group and wherein the alkyl, cycloalkyl, aryl, aralkyl and heterocyclic groups may be substituted with one or more groups selected from the class consisting qf amino groups, substituted amino groups such as methylamino, diethylamino or acetamido groups, the halogen groups such as fluorine, chlorine or bromine, nitro groups, alkoxy groups such as methoxy, ethoxy, propyloxy, isopropyloxy, butoxy or isobutoxy; or a nontoxic, pharmaceutically acceptable salt thereof, There is further provided by the present invention a pharmaceutical composition comprising an antibacterially effective amount of a compound having the formula

wherein R1 represents

wherein R is hydrogen, hydroxy or methoxy; R' is hydrogen or methyl; and M is hydrogen. pivaloyloxymethyl, acetoxymethyl, methoxymethyl. acetonyl, Phenacyl, p-nitrobenzyl, p,p,p-tnchloroethyl, 3-phthalidyl or 5-indanyl and preferably is hydrogen or a non-toxic, pharmaceutically acceptable salt thereof.

There is further provided by the present invention a method of treating bacterial infections comprising administering by injection to an infected non-human warmblooded animal, an effective but non-toxic dose of 250--1000 mgm. of a compound having the formula

wherein R' represents

wherein R is hydrogen, hydroxy or methoxy, R' is hydrogen or methyl; and M is hydrogen, pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl, p,p,p-trichloroethyl, 3-phthalidyl or 5-indanyl or a non-toxic, pharmaceutically acceptable salt thereof.

There is also provided by the present invention a method for combatting Shig.

dwsenteriae infections which comprises administering to a warm-blooded nonhuman mammal infected with a Shig. dysenteriae infection an amount effective for treating said Shig. dysenteriae infection of a composition comprising a compound having the formula

wherein R' represents

wherein R is hydrogen, hydroxy or methoxy; R' is hydrogen or methyl; and M is hydrogen, pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl p-nitrobenzyl, , "B-trichloroethyl, 3-phthalidyl or 5-indanyl and preferably is hydrogen or a non-toxic, pharmaceutically acceptable salt thereof.

There is also provided by the present invention a method for combatting B.

anthracis infections which comprises administering to a warm-blooded non-human mammal infected with a B. anthracis infection an amount effective for treating said B. anthracis infection of a composition comprising a compound having the formula

wherein R1 represents

wherein R is hydrogen, hydroxy or methoxy; R' is hydrogen or methyl; and M is hydrogen, pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl, p,p,p-trichloroethyl , 3-phthalidyl or 5-indanyl and preferably is hydrogen or a non-toxic, pharmaceutically acceptable salt thereof.

The present invention also provides the process for the production of the antibacterial agents having the structure

wherein R1 is acyl as defined above which comprises reacting a compound of the formula

or a salt or easily hydrolyzed ester of Schiff base as with benzaldehyde or salicylaldehyde thereof (including, but not limited to, those of U.S. 3,284,451 and U.K. 1,229,453 and any of the silyl esters described in U.S. patent 3,249,622 for use with 6-aminopenicillanic acid and used in Great Britain 1,073,530 and particularly the pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, pnitrobenzyl, jB, ,-trichloroethyl, 3-phthalidyl and 5-indanyl esters) with an organic monocarboxylic acid chloride or a functional equivalent thereof as an acylating agent.

Such functional equivalents include the corresponding acid anhydrides, including mixed anhydrides and particularly the mixed anhydrides prepared from stronger acids such as the lower aliphatic monoesters of carbonic acid, or alkyl and aryl sulfonic acids and of more hindered acids such as diphenylacetic acid. In addition, an acid azide or an active ester or thioester (e.g. with p-nitrophenyl, 2,4dinitrophenol, thiophenol, thioacetic acid) may be used or the free acid itself may be coupled with compound II after first reacting said free acid with N,N'dimethylchloroformiminium chloride [cf. Great Britain 1,008,170 and Novak and Weichet, Experientia XXI, 6, 360 (1965)] or by the use of enzymes or of an N,N'carbonyldiimidazole or an N,N'-carbonylditriazole [cf. South African patent specification 63/2684] or a carbodiimide reagent [especially N,N'-dicyclohexylcarbodiimide. N,N'-diisopropylcarbodiimide or N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide; cf. Sheehan and Hess, J. Amer. Chem. Soc., 77, 1967 (1955)], or of alkylylamine reagent [cf. R. Buijle and H. G. Viehe, Angew. Chem. International Edition 3, 582, (1964)] or of an isoxazolium salt reagent [cf. R. B. Woodward, R. A.

Olofson and H. Mayer, J. Amer. Chem. Soc., 83, 1010 (1961)], or of a ketenimine reagent [cf. C. L. Stevens and M. E. Munk, J. Amer. Chem. Soc., 80, 4065 )1958)] or of hexachlorocyclotriphosphatriazine or hexabromocyclotriphosphatriazine (U.S.

3,651,050) or of diphenylphosphoryl azide [DPPA; J. Amer. Chem. Soc., 94, 62036205 (1972)] or of diethylphosphoryl cyanide [DEPC; Tetrahedron Letters No. 18, pp. 1595-1598 (1973)] or of diphenyl phosphate [Tetrahedron Letters No.

49, pp. 5047--5050 (1972)]. Another equivalent of the acid chloride is a corresponding azolide, i.e., an amide of the corresponding acid whose amide nitrogen is a member of a quasiaromatic five membered ring containing at least two nitrogen atoms, i.e., imidazole, pyrazole, the triazoles, benzimidazole, benzotriazole and their substituted derivatives. As an example of the general method for the preparation of an azolide, N,N'-carbonyldiimidazole is reacted with a carboxylic acid in equimolar proportions at room temperature in tetrahydrofuran, chloroform, di methylformamide or a similar inert solvent to form the carboxylic acid imidazolide in practically quantitative yield with liberation of carbon dioxide and one mole of imidazole. Dicarboxylic acids yield diimidazolide. The by-product, imidazole, precipitates and may be separated and the imidazolide isolated, but this is not essential. The methods for carrying out these reactions to produce a cephalosporin and the methods used to isolate the cephalosporin so produced are well known in the art.

Mention was made above of the use of enzymes to couple the free acid with compound II. Included in the scope of such processes are the use of an ester, e.g.

the methyl ester, of that free acid with enzymes provided by various mlcroorganisms, e.g. those described by T. Takahashi et al., J. Amer. Chem. Soc., 94(11), 4035--4037 (1972) and by T. Nara et al., J. Antibiotics (Japan) 24(5), 321-323 (1971) and in U.S. 3,682,777.

For the coupling of the organic carboxylic acid as described above with compound II (or a salt or preferably an easily hydrolyzed ester of Schiff base, as with benzaldehyde, thereof) it is also convenient and efficient to utilize as the coupling agent phosphonitrilic chloride trimer (J. Org. Chem., 33(7), 2979-81, 1968) or N ethoxy-l,2-dihydroquinoline (EEDQ) as described in J. Amer. Chem. Soc., 90, 823-824 and 1652-1653 (1968) and U.S. Patent 3,455,929. The reaction is preferably carried out at 30-350C., in benzene, ethanol or tetrahydrofuran using about equimolar quantities of all three reagents followed by conventional isolation and removal by conventional methods of any blocking groups present.

An additional process of the present invention comprises the preparation of the compounds of the present invention the displacement of the 3-acetoxy group of a 7-acylaminocephalosporanic acid (prepared by substituting 7-aminocephalosporanic acid for the 3-thiolated-7-aminocephalosporanic acids in the acylation procedures described herein and elsewhere reported) with a thiol HSR3 having the formula

and then removing the protecting group if any is present, as on the aminomethyl or methylaminomethyl group or on the carboxyl group or both. The displacement of such a 3-acetoxy group with such a thiol may be accomplished in solution as in water or aqueous acetone at a temperature of at least room temperature and preferably within the range of 50 to 1000C. in the presence of a mild base such as sodium bicarbonate, e.g. preferably near neutrality such as at about pH 6. An excess of the thiol is preferably employed. The reaction product is isolated by careful acidification of the reaction mixture followed by extraction with a waterimmiscible organic solvent. As noted above, the preparation of many other 7acylamidocephalosporanic acids is described in the patent and scientific literature, e.g. in U.S. Class 260243C.

When the organic carboxylic acid contains a functional group such as amino or methylamino it is often desirable to first block (or protect) said group, then carry out the coupling reaction and finally subject the resulting compound to chemical removal of the protecting group, that is, subjecting the resulting compound to elimination reaction of the protecting group.

The present invention thus also provides the'process for the production of the antibacterial agents having the formula

wherein R' is acyl as defined above which comprises reacting a compound of the formula

wherein R' is acyl with a compound having the formula

The salts of the compounds of this invention include the non-toxic carboxylic acid salts thereof, including non-toxic metallic salts such as sodium, potassium, calcium and aluminum, the ammonium salt and substituted ammonium salts, e.g.

salts of such non-toxic amines as trialkylamines including triethylamine, procaine, dibenzylamine, N-benzyl-beta-phenethylamine, I-ephenamine, N,N'-dibenzylethylenediamine, dehydroabietylamine, N,N'-bis-dehydroabietylethylenediamine, N-(lower)-alkylpiperidine, e.g. N-ethylpiperidine, and other amines which have been used to form salts with benzylpenicillin; and the non-toxic acid addition salts thereof (i.e., the amine salts) including the mineral acid addition salts such as the hydrochloride, hydrobromide, hydroiodide, sulfate, sulfamate and phosphate and the organic acid addition salts such as the maleate, acetate, citrate, oxalate, succinate, benzoate, tartrate, fumarate, malate, mandelate, and ascorbate.

Also to be mentioned are the compounds (used as either intermediates or metabolic precursors) in which the amino group is "blocked" by substituents such as 2-iodoethoxycarbonyl (U.K. 1,349,673), t-butoxycarbonyl, carbobenzyloxy, formyl, o-nitrophenylsulfenyl, p,p,p-trichloroethoxycarbonyl, 4-oxo-2-pentenyl-2, l-carbomethoxy-l-propenyl-2- and the like. Particularly included in such blocking groups are the ketones (especially acetone) and aldehydes (especially formaldehyde and acetaldehyde) disclosed, for example, in U.S. patents 3,198,804 and 3,347,851 and the ,5-ketoesters and p-diketones disclosed, for example, in U.S.

patent 3,325,479 and the A-ketoamides disclosed in Japan 71/24714 (Farmdoc 47,321S).

The preferred esters of the cephalosporins of the present invention are the pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl and phenacyl esters.

All are useful intermediates in the production of the cephalosporin having a free carboxyl group.

As indicated above, these five esters of 7-aminocephalosporanic acid are each prepared by known methods. One excellent procedure is that of U.S. patent 3,284,451 in which sodium cephalothin is esterified by reaction with the corresponding active chloro or bromo compound (e.g. phenacyl bromide, chloroacetone, chloromethyl ether, pivaloyloxymethyl chloride [also called chloromethyl pivalate], acetoxymethyl chloride) and then the thienylacetic acid sidechain is removed enzymatically as in the same Datent or chemically as in U.S. patent 3,575,970 and in Journal of Antibiotics, XXIV (11), 767-773 (1971). in another good method the triethylamine salt of 7-aminocephalosporanic acid is reacted directly with the active halogen compound, as in United Kingdom 1,229,453.

These esters of 7-aminocephalosporanic acid are then reacted with the nucleophile HSR3 in the same manner as illustrated herein for 7-aminocephalosporanic acid itself. The 3-thiolated ester of 7-aminocephalosporanic acid is then coupled with the organic carboxylic acid R'-OH as before. Before or after removal of any blocking group, e.g. on an amino group in the 7-sidechain, the ester of the cephalosporin so obtained is, if not used per se, converted to its free acid, including its zwitterion (and, if desired, any salt) by removal of the esterifying group, as by aqueous or enzymatic hydrolysis (as with human or animal serum) or acidic or alkaline hydrolysis or by treatment with sodium thiophenoxide as taught in U.S.

3,284,451 and, in the penicillin series, by Sheehan et al., J. Org. Chem. 29(7), 2006-2008 (1964).

In another alternative synthesis, the 3-thiolated 7-aminocephalosporanic acid is prepared as described herein and then acylated at the 7-amino group and finally esterified, as by reaction of the appropriate alcohol with the acid chloride prepared, for example, by reaction of the final cephalosporin with thionyl chloride or by other essentially acidic esterification procedures.

In the treatment of bacterial infections in man, the compounds of this invention are usually administered parentally in an amount of from 5 to 200 mg./kg./day and preferably 5 to 20 mg./kg./day in divided dosage, e.g. three to four times a day.

They are administered in dosage units containing, for example, 125, 250 or 500 mg.

of active ingredient with suitable physiplogically acceptable carriers or excipients.

The dosage units are in the form of liquid preparations such as solutions or suspensions.

The other reagents used to prepare the compounds of the present invention are synthesized either as described in the art (e.g. as in the patents and publications noted above) or by strictly analogous procedures. For convenience and purposes of illustration, however, there are given below some specific examples of such syntheses, e.g. to prepare carboxylic acids containing a free amino group which is "blocked" with tert.-butoxycarbonyl.

2-(tert.-Butoxycarbonylaminomethyl)- ,4-cyclohexadienylacetic acid A solution of 16.5 g. (0.1 mole) of o-aminomethylphenylacetic acid in 1.5 1 of liquid ammonia (which had been treated with 50 mg. of Li fo remove a trace of moisture) was slowly diluted with 500 ml. of dry t-BuOH. To the solution was added in small portions 3.4 g. (0.5 atom) of Li over a period of 4 hours and the mixture was stirred for 16 hours at room temperature removing the liquid ammonia in a hood and finally evaporated to dryness below 40"C. The residue was dissolved in 500 ml.

of water and the solution was chromatographed on a column of IR--120 (H+, 700 ml.) resin and eluted with 1% NH40H solution. Ninhydrin positive fractions of the eluate were combined and evaporated to dryness. The residue was washed with four 50 ml. portions of hot acetone and recrystallized from 500 ml. of ethanol-water (1:1) to give 11.2 g. (67 a) of colorless needles, o-(2-amino-methyl- 1,4cyclohexadienyl)acetic acid. M.p. 183"C.

IR: v nut 1630, 1520, 1380, 1356 cm~'.

NMR: D2O + K2CO3 2.72 (4H, s,

), 3.01 (2H, s, CH2CO), 3.20 (2H. s, CH2-N), 5.78 (2H, s,

Anal. Calcd. for CgHt3NO2 C, 64.65; H, 7.84; N, 8.38.

Found; C, 64.77; H, 8.06; N, 8.44.

Improved Procedure for the Preparation of -(2-aminomethyl-1,4-cyclo- hexadienyl)-acetic acid

The procedure used bv Welch, Dolfini and Giarrusso in U.S. patent 3,720,665 (Example 1) to make D-2-amino-2-(l,4-cyclohexadienyl)acetic acid was adapted. A solution of 830 ml. of distilled liquid ammonia was dried with 40 mg. of lithium under an argon atmosphere. To this stirred solution was added 11.0 g. (0.07 mole) of 2-aminomethylphenylacetic acid and 340 ml. of tert. butyl alcohol. A total of 1.6 g. (0.225 mole) of lithium was added to the vigorously stirred solution over a period of 2 hours. The grey mixture was then treated with 35 g. (0.215 mole) of triethylamine (TEA) hydrochloride and stirred overnight at room temperature for 18 hours. The tert. butyl alcohol was removed at 40 (15 mm.) to yield a white residue which was dried in vacuo over P205 overnight. The solid was dissolved in 30 ml. of 1:1 methanol-water and added with stirring to 3.5 1. of 1:1 chloroformacetone at 5 . The mixture was stirred for 20 min. and the amino acid, a-(2-amino methyl-I ,4-cyclohexadienyl)acetic acid, was collected and dried for 16 hours in wouo over P2O5 to yield 6.3 g. (58No) of white crystals, m.p. 1900 decomp. The IR and NMR spectra were consistent for the structure.

A solution of 19.31 g. (0.135 m) of tert.-butoxycarbonylazide in 152 ml. of tetrahydrofuran (THF) was added to a stirred solution of 14.89 g. (0.09 m) of 2 aminomethyl I,4-cyclohexadienylacetic acid and 7.20 g. (0.18 m) of sodium hydroxide in 281 ml. of water. The solution was stirred for 18 hr. at 250 and then filtered thru diatomaceous earth (Super-cel). The THF was removed at 400 (15 mm) and the residual solution was washed with ether (2 x 175 ml.) and acidified with 6 N hydrochloric acid (HCI). The mixture was stirred in an ice-bath and the precipitate was collected and dried for 18 hr. in vacuo over P206 at 259 yield 17.3 g.

(72.6%) of 2-(tert.-butoxycarbonylaminomethyl)-1 ,4-cyclohexadienylacetic acid as a white powder. The IR and NMR spectra were consistant for the structure.

Preparation of 3-Aminomethyl-2-thiophene Acetic Acid

A) Thiophene-3-carboxaldehyde Dimethyl Acetal (2a) A mixture of thiophene-3-carboxaldehyde" (322 g., 2.9 moles), trimethoxymethane (636 g., 6 moles) and IR--120 resin (H+, 6 g.) in methanol (200 ml.) was refluxed over a period of 4 hours. The resin was removed and the filtrate was evaporated under reduced pressure to give a colorless oil which was distilled under reduced pr

7.27 (1 H, d, J=6Hz, thiophene-Hss), 7.81 (1H, d-d, J=1.5 and 6Hz, thiophene-Ha), 10.34 (1H, d, J=1.5 Hz, -CHO).

C) 1 - Methylsulfinyl - I - methylthio - 2 - (3 - carboxaldehyde ethyleneacetal 2 - thienyl)ethylene (4b) Preparation of 4b was carried out according to the procedure similar to that reported by K. Ogura et al.4). Triton B (40% in methanol, 2 ml. in THF (tetrahydrofuran) (5 ml.) was added to a solution of methyl methylthiomethyl sulfoxide21 (2.5 g., 20 m. moles) and 2-formyl-3-thiophenecarboxaldehyde ethylene acetate (3b). The mixture was refluxed for about one hour and concentrated under reduced pressure.

The residue was dissolved in benzene (150 ml) and extracted with water (3 x 20 ml.) The organic layer was dried over MgSO4 and evaporated to dryness under reduced pressure. The residue was column-chromatographed on silica gel (80 g) eluting with benzene (500 ml) and chloroform (500 ml) successively. From the chloroform eluate 4.9 g (85%) of the product 4h was isolated as a pale yellow oil.

ir: p liq 3110, 1600 cm~'.

max nmr: 6 CDCl3 2.42 (3H, s, S-CM2), 2.78 (3H, s, SO--CH,), 4.15 (4H, m, ppm CH2CH2-), 6.12 (1H, s, ),

7.34 (1H, d, J=4.5Hz, thiophene-Hp), 7.40 (1H, d, J=4.5Hz, thiophene-Ha), 8.28 (lH, s, -CH=).

The semicarbazone of 4 was prepared by a usual manner and crystallized from ethanol-DMF. M.p. 212--2130C.

Anal. Calcd. for C,oHt3N302S2: C, 39.58; H, 4.32; N, 13.85; S, 31.70.

Found: C, 39.46; H, 4.24; N, 14.05; S, 31.63.

D) 1 - Methylsulfinyl - 1 - methylthio - 2 - (3 - carboxaldehyde dimethylacetal 2 - thienyl)ethylene (4a).

The compound 4a was prepared by the procedure similar to that for 4b. Triton B (40% in methanol, 50 ml) was added to a solution of methyl methylthiomethylsulfoxide (72 g., 0.58 mole) and 3a (108 g, 0.58 mole) in THF (300 ml) and the mixture was refluxed for 4 hours. Separation by column chromatography with silica gel (400 g) eluting with chloroform (5 L) gave 130.5 g (78%) of 4a as a pale yellow oil.

ir; u liq 3100, 1580, 1100, 1050-'.

max nmr; CCI4 2.42 (3H, s, S-CM3), 2.70 (3H, s, SO-CM3), 3.34 (6H, s, OCH2), ppm 5.56 (1H, s.

2) K. Ogura, et al. Bull. Chem. Soc. (Japan), 45, 2203 ('72) 3) D. W. McDowell et al., J. Org. Chem. 31, 3592 ('66) 4) K. Ogura, et al., Tetrahedron Letters, 1383(1972).

7.20 (1H, d, J=6Hz, thiophene-H,), 7.40 (1H, d, J=6Hz, thiophene-H), 8.12 (lH, s, --CH=).

E) Ethyl 3-formyl-2-thienylacetate4' (5) Dry hydrogen chloride (33 g) was absorbed in anhydrous ethanol (500 ml). To this solution 4a (130 g, 0.45 mole) was added and the mixture heated under reflux for 5 mins. The reaction mixture was diluted with water and evaporated under reduced pressure. The residue was extracted with benzene (2 x 100 ml) and the benzene extracts were combined, washed with water (50 ml), dried over MgSO and evaporated to dryness. The oily residue was column-chromotographed on silica gel (400 g) eluting with chloroform (5 L). Fractions containing the desired product were combined and concentrated. The residual oil (60 g) was distilled under reduced pressure to afford 23 g (23%) of 5, boiling at 120-126 C/1 mmHg.

ir: P liq 3110, 1730, 1670 cm~'.

max nmr: 6 CDCl3 1.30 (3H, t, J=6Hz, -CH2CH3), 4.25 (2H, q, J=6Hz, -CH2CH3), ppm 4.26 (2H, s, -CH2CO), 7.25 (1H, d, J=5Hz, thiophene-H > , 7.48 (1H, d, J=5Hz, thiophene-Ha), 10.15 (1H, s, CHO).

The analytical sample of 5 was submitted as the 2,4-dinitrophenylhydrazone which was crystallized from chloroform. M.p. 178-179 C.

ir: # nmuaixol 1720, 1610, 1570 cm-1.

Anal. Calcd. for C,,H 14N4O6S: C, 47.62; H, 3.73; N, 14.81; S, 8.47.

Found: C 47.33: H. 3.47: N 14.77: S 8.68.

According to the similar procedure 2.2 g (7.6 m moles) of the ethylene acetal 4b was treated with 1.1 g of dry hydrogen chloride in 800 ml of anhydrous ethanol to afford 5 which was purified by column chromatography on silica gel (30 g).

Elution with chloroform gave 663 mg (44%) of 5 as a pale yellow oil.

F) Ethyl 3-formyl-2-thienylacetate oxime (6) Sodium carbonate (1.7 g, 16 m mole) was added to a solution of the aldehyde 5, (3.14 g, 16 m mole) and hydroxylamine hydrochloride (2.2 g, 32 m mole) in 50% aq.

ethanol (40 ml) at 50C with stirring. The reaction mixture was warmed up to room temperature, After 2.5 hrs., the reaction mixture was concentrated under reduced pressure. The residue was extracted with benzene (3 x 50 ml). The benzene extracts were washed with water (10 ml), dried over MgSO4, and evaporated under reduced pressure. Separation by column chromatography on silica gel (60 g) gave 2.7 g (80%) of colorless oil 6.

ir: v liq 3400, 1730, 1620 cm~'.

max nmr: 6Aceton-d6 1.23 (3H, t, J=7.5Mz, -CH2CH3), 4.01 (2H, s, -CH2CO), ppm 4.14 (2H, q, J=7.5Hz, -CH2CH3), 7.31 (2H, s, thiophene-H), 8.26 (1H, s,-CH=N), 10.15 (1H, s, NOH, disappeared by addition of D2O).

G) The 8-lactam of 3-aminomethyl-2-thienylacetic acid (7).

Method A: Catalytic reduction A mixture of oxime 6 (2.65 g. 12.4 m moles), 10% palladium on charcoal, dry hydrogen chloride (1.4 g, 37.2 m moles) in anhydrous ethanol (68 ml) was hydrogenated overnight under atmospheric pressure at room temperature. The catalyst was exchanged twice and the reaction was carried out over a period of 3 days. The catalyst was removed and the filtrate was concentrated under reduced pressure. To the residue was added water (10 ml) and the mixture washed with ethyl acetate (2 x 10 ml). The aqueous layer was adjusted to pH 9 with sodium carbonate, saturated with sodium chloride, and extracted with ethyl acetate (3 x 20 ml). The ethyl acetate extracts were dried over MgSO4, treated with charcoal, and evaporated under reduced pressure. Recrystallization from ethyl acetate gave 41? mg (22 ó) of colorless needles 7 melting at 194--1950C.

ir: p KBr 3200, 1650, 1480 cm-'.

max nmr 6 ppm DMSO-d6 3.53 (2H, t, J=3Mz, -CH2CO-), 4.36 (2H, d-t, J=3, 1.5Hz, changed to a triplet by addition of D2O, J=3Hz, CH2N), 6.95 (1H, d, J=4.5Hz, thiophene-ff,l) 7.45 (1H, d, J=4.5Hz, thiophene-Ha), 8.0 (1H, m, disappeared by addition of D2O, NH).

Anal. Calcd. for C,H,NOS: C, 54.88; H, 4.61; N, 9.14; S, 20.93.

Found: C, 55.04; H, 4.45; N, 9.13; S, 20.50.

Method B: Zn-dust reduction To a solution of the oxime 6 (18.3 g, 86 m moles) in acetic acid (200 ml), zinc dust (17 g, 258 m moles) was added portionwise over a period of 1 hr. at 40--500C with vigorous stirring. The reaction mixture was stirred overnight at room temperature and heated at 60 C for 4 hours. The contents were filtered and the filtrate was concentrated under reduced pressure. To the residual oil was added water (100 ml) and the mixture washed with ether (2 x 50 ml). The aqueous solution was layered with ethyl acetate (100 ml) and adjusted to pH 10 with sodium carbonate. The precipitate was filtered off. The filtrate was extracted with ethyl acetate. The ethyl acetate extracts were washed with water (10 ml), dried over MgSO4, and evaporated under reduced pressure. The residual solid was triturated with benzene. Crystallization from ethyl acetate gave 2.7 g (21%) of the lactam 7 which was identical to Method A in the IR and the NMR spectra.

H) 3-Aminomethyl-2-thienylacetic acid (8) A mixture of the lactam 7 (2.88 g, 18.8 m moles) and 6N hydrochloric acid (50 ml) was heated under refluxed for 3 hrs. The reaction mixture was concentrated under reduced pressure. To the residue was added water (20 ml) and the mixture treated with charcoal and evaporated under reduced pressure. The trituration of the residue with THF gave the amino acid 8 hydrochloride (3.72 g, 95%; m.p.

171--172"C; ir (KBr) cm~': 3450, 3000, 1700, 1200; nmr (D2O)ppm: 4.80 (2H, s, -CH2CO), 4.27 (2H, s, CH2-N), 7.26 (1H, d, J=6Hz, thiophene-HO, 7.53 (1H, d, J=6Hz, thiophene-Hcr). The hydrochloride (3.71 g, 17.9 m moles) was dissolved in water (10 ml) chromatographed on a column of IR-l20 (H, 30 ml) and developed successively with water (100 ml) and 5N-NHdOH (2 L). The ammonia eluate was evaporated to dryness. The residue was crystallized from aqueous acetone to give 3.0 g (98%) of 8, m.p. 223--225"C.

ir: v max KBr 3000, 1620, 1520 cm~'.

nmr: 6 D29-Na2CO3 3.20 (2H, s, -CH2CO), 4.13 (2H, s, CH2N), 7.04 (lH, d, ppm J=6Hz, thiophene-Hss), 7.30 (H, J=6Hz, thiophene-Ha).

Anal. Calcd. for CtHgNO2S C, 49.10; H, 5.30; N, 8.18; S, 18.73.

Found: C, 48.53; H, 5.22; N, 7.98; S. 1.8.97.

I) 3-t-B utoxycarbonylaminomethl-2-thienylacetic acid (9) A mixture of 3-aminomethyl-2-thienylacetic acid 8 (3.1 g, 18 m moles) and triethylamine (8g, 80 m moles) in 50% aqueous acetone (80 ml) was added dropwise t-butoxycarbonyl azide (5.7 g, 40 m moles) over a period of 20 mins. at 0 C with vigorous stirring. The reaction mixture was stirred overnight at room temperature and concentrated under reduced pressure. The concentrate was washed with ether (2 x 20 ml), adjusted to pH 2 with conc. HCI and extracted with ethyl acetate (2 x 50 ml). The ethyl acetate extracts were washed with saturated aqueous sodium chloride, dried over MgSO4, treated with charcoal and evaporated under reduced pressure. The residue was triturated with n-hexane and crystallized from n-hexane and benzene to give 4.5 g (92%) of colorless needles 9, melting at 62630C.

ir: v muaiol 3350, 1700 cm-'.

nmr: a pCpDmcl3 1.43 (9H, s, BOC-H), 3.27 (2H, s, CH2CO), 4.16 (2H, d, J=6Hz, CM2-N, a singlet when D2O was added) 5.00 (1H, br, -NM-, disappeared by addition of D2O), 6.30 (1H, broad s, -COOH, disappeared by addition of D2O), 6.86 (1H, d, J=6Hz, thiophene-Hp), 7.06 (1H, d, J=6Hz, thiophene-Ha).

Anal. Calcd. for C,2H,NO4S: C, 52.89; H, 6.29; N, 5.14; S, 11.77.

Found: C, 53.30; H, 6.39; N, 5.13; S, 11.72.

Preparation of 2 - N - Methylaminomethyl - 4 - methoxy - (and 4 - hydroxy -) phenylacetic Acids

2-N-Tosylaminomethyl-4-hydroxyphenylacetic Acid (2) To a solution of 14.56 g. (0.08 mol.) of 2-aminomethyl-4-hydroxyphenylacetic acid (I) (U.S. 3,823,141) and 13 g. (0.32 mol.) of sodium hydroxide in 200 ml. of water was added dropwise with stirring at 65-700C a solution of 18.5 g. (0.097 mol.) of p-toluenesulfonyl chloride in 50 ml. of dry ether and the mixture was kept at the same temperature for one hour. The mixture being cooled, the aqueous layer was separated, washed with ether (2 x 50 ml.), acidified with 6N HCI and extracted with 400 ml. of ethyl acetate. The extract was washed with water and a saturated aqueous NaCI solution, dried with Na2SO4 and treated with active carbon (1 g.).

The filtrate was concentrated to dryness and the residue was crystallized from ethyl acetate to give 11.0 g. (40.5%) of 2 melting at 212-2150C.

ir: v mKB:r 3240, 1700, 1380, 1330, 1150 cm-l uv: A 1%K2CO3 230 nm ( 7,750) max nmr: Ô DpMpSm ~d3 2.47 (3H, s,-Ar-CH3), 3.60 (2H, s, CH2CO), 3.93 (2H, d, J=6.0 Hz, CH2N), 6.6-8.2 (7H, m, phenyl-H).

2-(N-Methyl-N-tosylamino)methyl-4-methoxyphenylacetic Acid (3) A mixture of 11 g. (0.033 mol.) of 2, 10.3 ml. (0.17 mol.) of methyl iodide and 9.2 g. (0.24 mol.) of sodium hydroxide in 100 ml. of water was heated at 80-90 C for 45 minutes in a sealed tube with occasional shaking. The mixture was washed with ethyl acetate (30 ml.) and the water layer was acidified with 6N HCI and extracted with ethyl acetate (3 x 30 ml.). The combined extracts were washed with water (30 ml.) and a saturated aqueous NaCI solution (30 ml.) treated with active carbon (1 g.) and dried over Na2SO4. The filtrate was evaporated to dryness and the residue was crystallized from benzene to give 8 g. (66.5%) of the N,O-dimethyl derivative 3 melting at 146--1500C.

ir: v KBr 1690, 1500, 1340, 1280, 1150 cm~'.

uv: A mEtOxH229 nm (E: 20500), 278 nm (E: 2400).

nmr: 6 DMSO-d, 2.52 (3H, s, N-CM3), 2.47 (3H, s, Ar-CM3), 3.67 (2H, s, CH2CO), 3.74 (3M, s, OCH3), 4.10 (2h, s, CH2N), 6.7-7.8 (7H, m, Ar-H), 11.5 (1H, br-s, COOH).

Anal. Calcd. for C18H2,NOsS: C, 59.49; H, 5.82; N, 3.84; S, 8.82.

Found: C, 59.48; H, 5.68; N, 3.37; S, 9.22.

2-N-Methylaminomethyl-4-methoxyphenylacetic Acid (4a) To a solution of liquid ammonia (300 ml.) was added 9.4 g. (0.026 mol.) of 3 at -50 C and the mixture was stirred until a clear solution was obtained at the same temperature. To the solution was added 3.3 g. (0.14 g. atom) of Na in small pieces at -40 C and the mixture was stirred for 2 hours. Ammonia was evaporated and the residue was dissolved in 100 ml. of water carefully. To the solution was added 100 ml. of Amberlite IR-C 50 (ammonium type) and the mixture was stirred for 30 minutes at room temperature. The resin was removed and the filtrate was treated with barium acetate until no more precipitate was observed. The precipitate was filtered off and the filtrate was chromatographed with a column of IR-120 ionexchange resin (H+, 100 ml.) by eluting with 510% ammonia. The eluate (2 L) containing the desired product was evaporated to dryness below 50 C and the residue was triturated with acetone to give 4.4 g. (81%) of 4a, m.p. 225-2270C.

ir: v K Br 1590, 1380, 1260, 1035 cm-1.

max nmr: 6 D2O 2.77 (3H, s, N-CM3), 3.6 (2H, s, CH2CO), 3.87 (3H, s, OCH3), ppm 4.18 (2H, s, CH2N), 6.8-7.4 (3H, m, phenyl-H).

2-N-M ethylaminomethyl-4-hydroxyphenylacetic Acid (4b) A mixture of 2.9 g. (0.014 mol.) of 4a in 30 ml. of 48% hydrobromic acid was refluxed for 5 hours and the solution was evaporated to dryness. The residue was dissolved in 50 ml. of water. The solution was chromatographed on a column of Amberlite IR-120 (H+, 50 ml.) eluting with 510% ammonia. The eluate was collected in 250 ml. fractions. Fractions containing the product were combined and evaporated to dryness below 500 C. The residue was triturated with acetone to give 1.3 g. (48.5%) of 4b, which was crystallized from 80% ethanol. M.p.

2l8-2210C.

ir: P K 2000--3400, 1610, 1540, 1460, 1380, 1270 cm~'.

uv: 1 %K2CO3 243 nm (#: 4700), 297 nm (E: 1350).

max nmr 6 D2O+NaOH 2.64 (3H, s, N-Ch3), 3.47 (2H, s, Ch2CO), 3.94 (2H, s, ppm N-CM2), 6.5-7.2 (3H, m, phenyl-H).

Anal. Calcd. for C10H,3NO3: C? 61.53, H, 6.71; N, 7.17.

Found: C, 61.44; H, 6.81; N, 7.20.

2 - N - t - Butoxycarbonyl - N - methylaminomethyl - 4 - methoxyphenylacetic Acid (5, R = CH3) A mixture of 1.05 g. (5 m. mol.) of 4a, 1.43 g. (6 m. mol.) of t-butyl 4,6dimethylpyrimidin-2-ylthiol-carbonate and 1.4 ml. of triethylamine in 40 ml. of 50% THF was stirred at room temperature for 20 hours. Most of the THF was evaporated and the resulting aqueous solution (ca. 20 ml.) was washed with ether.

The water layer was acidified with 6N HCI and extracted with ether (3 x 10 ml.).

The ethereal extracts were washed with water (10 ml.) and a saturated aqueous NaCI solution (10 ml.), treated with a small amount of active carbon and dried over Na2SO4. The filtrate was evaporated to dryness to give 1.0 g. (77.5%) of 5 (R = CH3) as an oil.

nmr: 6 CDCI3 1.47 (9H, s, BOC--H), 2.77 (3H, s, N-CM3), 3.60 (2H, s, ppm CH2CO), 3.79 (3H, s, O--CH,), 4.49 (2H, s, CH2N), 6.1-7.3 (3H, m, phenyl-Il).

2 - N - t - Butoxycarbonyl - N - methylaminomethyl - 4 - hydroxyphenylacetic Acid (5, R = H) A mixture of 1 g. (4.78 m.mol.) off, 1.5 g. (6.3 m.mol.) of t-butyl 4,6-dimethylpyrimidin-2-ylthiolcarbonate and 2.1 ml. of triethylamine in 50 ml. of 50% aqueous THF solution was stirred at room temperature for 20 hours. The mixture was concentrated to 20 ml. under reduced pressure. The concentrate was washed with ether (10 ml.), acidified with 6N HCI and extracted with ethyl acetate (2 x 1UO ml.). The combined extracts were washed with water (30 ml.) and a saturated aqueous NaCI solution (2 x 30 ml.), treated with a small amount of active carbon and dried over anhydrous Na2SO4. The filtrate was evaporated to dryness to give 1.3 g. (92%) of 5 (R = H) as an oil.

ir: v mqax 3000-3600, 1670, 1260, 1150 cm~'.

nmr: 6 CpDpCrn3 1.44 (9H, s, C(CH3)3), 2.73 (3H, s, N-CM3), 3.54 (2H, s, CH2CO), 4.38 (2H, s, CH2N), 6.5-7.3 (3H, m, phenyl-H).

Preparation of Ortho-N-methylaminomethyl-phenylacetic Acid.

o-(p-Toluenesulfonylaminomethyl)phenylacetic Acid (2) To a stirred solution of o-aminomethylphenylacetic acid hydrochloride (7.50 g., 37 m.mol.) and sodium hydroxide (4.74 g., 118 m.mol.) in water (100 ml.) was added p-toluenesulfonyl chloride (7.64 g., 40 m.mol.) in portions at 600 C. The mixture was stirred for 1 hour at the same temperature and acidified with hydrochloric acid. The mixture was extracted with ethyl acetate (4 x 50 ml.) The combined extracts were washed with water, treated with a small amount of carbon and dried. The solvent was evaporated under reduced pressure and the residue crystallized from ethyl acetate to afford 2 as colorless prisms. Yield, 9.84 g. (84%).

M.p. 155--1560C.

ir: v may 3300, 1705, 1335, 1170 cm~'.

nmr; 6 DMppSmds 2.38 (3H, s, CH3), 3.65 (2H, s, CH2CO), 3.97 (2H, d, J=5 Hz, CH2N), 7.1-8.2 (9H, m, phenyl-H & NH).

Anal. Calcd. for C,H17NO4S: C, 60.17; H, 5.37; N, 4.39; S, 10.10.

Found: C, 60.i1, 60.15; H, 5.43, 5.40; N, 4.28, 4.30; S, 9.72, 9.80.

o-(N-p-Toluenesulfonyl-N-methylaminomethyl)phenylacetic Acid (3) A mixture of 2 (9.0 g., 28 m.mol.) sodium hydroxide (6.0 g.) and methyl iodide (6 ml.) in water (60 ml.) was heated in a sealed tube for 30 minutes at 700C. After cooling, the reaction mixture was acidified with hydrochloric acid to separate pale yellow precipitate which was crystallized from ethyl acetate-n-hexane to give colorless prisms, 3. Yield, 8.5 g. (91%). M.p. 162--163"C.

ir: v maBxr 27002300, 1700, 1600, 1345, 1200, 925 cm~'.

nmr: 6 D2O+KOH 2.37 (3H, s, CH3), 2.49 (3H, s, CH3), 3.80 (2H, s, CH2CO) ppm 4.18 (2H, s, CH2N), 7.08.0 (8H, m, phenyl-H).

Anal. Calcd. for C"H,s,NO2: C, 61.24; H, 5.74; N, 4.20; S, 9.61.

Found: C, 61.31, 61.36; H, 5.73, 5.71; N, 4.51, 4.29; S, 9.63, 9.55.

N-Methylaminomethylphenylacetic Acid (4) Method A (using hydrobromic acid) - A mixture of 28.6 g. (0.086 mol.) of 3 and 20 g. (0.213 mol.) of phenol in 260 ml. of 48% hydrobromic acid was refluxed for 30 minutes. The mixture was cooled, diluted with the same volume of water and washed with ethyl acetate (2 x 50 ml.). The aqueous layer was evaporated to dryness in diminished pressure to give an oil which was chromatographed on a column of Amberlite IR--120 (H+ form, 200 ml.) eluting with 5% ammonium hydroxide solution. The eluate (2.5 1.) was collected and evaporated to dryness under reduced pressure. The residue was triturated with acetone and crystallized from ethanol to afford 6.7 g. (43.5%) of 4 as colorless needles, melting at 168--1700C (dec.).

Method B (using metallic sodium in liquid ammonia) - To a mixture of3 (35 g., 0.105 mol.) in liquid ammonia (1000 ml.) was added 13.3 g. (0.578 atom) of sodium in small pieces under vigorous stirring over a period of 2 hours. The ammonia was evaporated with stirring on a water-bath in a well-ventilated hood and finally under reduced pressure to remove it completely. The residue was dissolved in ice water (400 ml.) and the solution was stirred with ion-exchange resin IRC-50 (H+ form, 400 ml.) for 0.5 hour at room temperature. The resin was filtered off and to the filtrate was added an aqueous 1 M solution of barium acetate until no more precipitate was formed (ca 50 ml. of the barium acetate solution was required). The mixture was filtered and the filtrate was chromatographed on a column of IR--120 (H+ form, 400 ml.) as in Method A to give 13.6 g. (72%) of 4.

o-(N-methyl-N-t-butoxycarbonylaminomethyl)phenylacetic Acid (5) t-Butyl 4,6-dimethylpyrimidin-2-ylthiolcarbonate (11 g., 0.048 mol.) was added in one portion to a mixture of 4 (7.2 g., 0.04 mol.) and 1,1,3,3-tetramethylguanidine (6.9 g., 0.06 mol.) in 50% aqueous TMF and the mixture was stirred overnight at room temperature. The THF being evaporated under reduced pressure, the aqueous solution was acidified to pH 2 with dil. hydrochloric acid and extracted with ethyl acetate (2 x 20 ml.) The combined extracts were washed with water, treated with a small amount of active carbon and evaporated under diminished pre ssure. The residue was triturated with hexane and crystallized from n-hexane-ether to affort 9.2 g. (83%) of 5 as colorless prisms. M.p. 96--98"C.

ir: v max KBr 1730, 1630, 1430, 1830, 1250 cm~'.

nmr: 6 CDCI, 1.49 (9H, s, t-butyl), 2.78 (3H, s, N--CH,), 3.72 (2H, s, CH,CO), 4.25 (2H, s, CH2N), 7.28 (4H, s, phenyl), 9.83 (1H, s, --COOH).

Anal. Calcd. for CrsH21NO4: C, 64.50; H, 7.58; N, 5.01.

Found: C, 64.69; H, 7.66; N, 4.89.

Preparation of 3-N-methylaminomethyl-2-thienylacetic Acid.

3-Aminomethyl-2-thienylacetic Acid 6-lactam (2) Glacial acetic acid (140 ml.) was added dropwise with stirring to a mixture of 2ethoxycarbonlymethylthiophene-3-carboxaldehyde oxime (1) (41 g., 0.19 mole) and zinc dust (65.4 g., I mole) in methanol, and the mixture was stirred under reflux for 4 hours. The mixture was cooled and insolubles were removed by filtration and washed with methanol (3 x 50 ml.). The filtrate was combined with the washings and evaporated in vacuo to dryness, the residue being extracted with methanol (5 x 100 ml.). The methanol extracts were combined and evaporated under reduced pressure. To the residue was added water (50 ml.) and the mixture was adjusted to ph 10 with Na2CO3 and extracted with chloroform (3 x 100ml.). The combined chloroform extracts were washed with water (loll.), dried over MgSO4, and evaporated under reduced pressure. The residual oil (30 g.) was triturated with hot benzene (150 ml.). The colorless needles were collected by filtration and recrystallized from ethyl acetate to give the lactam 2 (7.7g., 26%), melting at 195--196"C.

UV: i MeOM 232 nm (e, 6500) max Anal. Calcd. for C,H7NOS: C, 54.88; H, 4.61; N, 9.14; S, 20.93.

Found: C, 55.04; H, 4.45; N, 9.13; S, 20.50.

3-N-Methylaminomethyl-2-thienylacetic Acid 6-lactam (3) To a suspension of sodium hydride (50% in paraffin, 1.82 g., 38 m.moles) in absolute benzene (500 ml.) was added the lactam 2 (4.85 g., 32 m.moles) with stirring under nitrogen atmosphere and the mixture was refluxed for 2 hours. Methyl iodide (22;7 g., 160 m.moles) was added in one portion at room temperature and the mixture was again refluxed for 2 hours. Ice-water (50 g.) was added to the mixture and organic layer was separated. The aqueous layer was extracted successively with benzene (2 x 50 ml.) and chloroform (50 ml.). The extracts were combined and dried on MgSO4. The solvent was evaporated under reduced pressure To the residue was added a hot mixture of benzene-n-hexane (1:1, 100 ml.) to recover 2 as needles (2.02 g., 42%). The filtrate was evaporated and the residue was crystallized from benzene-n-hexane to afford colorless plates 3. Yield; 2.7 g. (51%). M.p.

98-1000C.

ir: v nujol 1620 cm~'.

max nmr: 6CmMf:l3 3.15 (3H, s, N-CM3), 3.72 (2H, t, J=3Hz, CH2CO), 4.53 (2H, t, J=3Mz, -CM2-N), 6.87 (lem, d, J=4.5Hz, thiophene-N,B), 7.30 (1H, d, J=4.5Hz, thiophene-Ha).

MeOM uv: A ma

3-(N-Methylaminomethyl)-2-thienylacetic Acid (4) A mixture of the lactam 3 (3.5 g., 21 m.moles) and 6N HCI (100 ml.) was heated under reflux for 12 hours. The mixture was treated with carbon and concentrated to dryness under reduced pressure. The residual oil was dissolved in water (10 ml.) and chromatographed on a column of IR-120 (H+, 50 ml.). The column was eluted with water (200 ml.) and 5N NH40H (3 L.). The amino acid 4 (3.0 g., 77%) was isolated by evaporation of the ammonia eluates followed by crystallization from aqueous acetone. M.p. 181--1820C.

ir: p KBr,570, 1360 cm~'.

max nmr: 6 D2O 2.21 (3H, s, N-CM3), 3.80 (2H, s, CH2CO), 4.20 (2H, s, CH2-N), ppm 7.19 (1H, d, J=6Hz, thiophene-Hss), 7.46 (1H, d, J=6Hz, thiophene-Ha).

uv: AMm2aO: 237 nm (E, 7600) Anal. Calcd. for C8H,,NO2S: C, 51.87; H, 5.99; N, 7.56; S, 17.31.

Found: C, 51.67; H, 6.50; N, 7.28; S, 16.69.

3-(N-t-butoxycarbonyl-N-methylaminomethyl)-2-thienyl-acetic Acid (5) To a mixture of 3-N-methylaminomethyl-2-thienylacetic acid 4 (2.7 g., 14.6 m.moles) and triethylamine (6 g., 60 m.moles) in 50% aqueous acetone (60 ml.) was added dropwise t-butoxycarbonyl azide (4.2 g., 29.2 m.moles) over a period of 20 minutes at 0 C with vigorous stirring. The reaction mixture was stirred overnight at room temperature and concentrated under reduced pressure. The concentrate was washed with ether (2 x 20 ml.), adjusted to pH 2 with concentrated MCI and extracted with ethyl acetate (2 x 50 ml.). The ethyl acetate extracts were washed with a saturated aqueous NaCI solution, dried on MgSO4, treated with charcoal and evaporated under reduced pressure. The residue was triturated with n-hexane and crystallized from n-hexane-benzene to give 3.68 g. (88%) of colorless needles 5 melting at 82-830C.

ir: # nujol 1730, 1640 cm~'.

max nmr: 6 CDCl3 1.47 (9H, s, BOC-M), 2.78 (3H, s, N-CM3), 3.87 (2H, s, ppm CH2-CO), 4,48 (2H, s, CH2-N), 6.91 (lH, d, J=6Hz, thiphene-Hp), 7.20 (1H, d, J=6Hz, thiophene-Ha), 10.63 (1H, s, CO2H, disappeared by addition of D2O).

Anal. Calcd. for C,3H,9NO4S: C, 54.72; H, 6.71; N, 4.91; S, 11.24.

Found: C, 54.91; H, 6.85; N, 4.92; S, 11.19.

The use of an "en-amine" blocking group with a prospective 7-side chain containing a free amino group prior to acylation of a nucleus such as II herein is well known as from U.S. 3,223,141, U.S. 3,813,390, U.S. 3,813,391, U.S. 3,823,141 and Belgium 773,773.

Sodium 2 - [N - (1 - carbethoxypropen - 2 - yl)aminomethyl] - 1,4 - cyclo hexadienyl acetate (4) To a stirred solution of 460 mg. (0.02 g. atom) of metallic sodium in 100 ml. of absolute EtOH was added 3.34 g. (0.02 mole) of 2-aminomethyl-l,4-cyclo- hexadienylacetic acid and 3.1 g. (0.024 mole of ethyl acetoacetate and the mixture was heated to reflux for 4 hours with stirring. The hot reaction mixture was filtered and the filtrate was allowed to keep cold overnight to give 2.0 g. of colorless needles 4 melting at 264cC. The additional product (3.3 g.) was obtained by concentration of the mother liquid. The total yield was 5.3 g. (88%).

IR: p muajx 3300, 1635, 1600, 1570, 1300, 1275, 1170, 1090 cm~'.

NMR: 6Dp2pO 1.23 (3H, t, 7Hz, CH2CH3), 1.96 & 2.25 (3H, s, C=C-CH3, cis & trans), 2.70 (4H, s,

3.04 (2H, s, CH2CO), 3.66 & 3.95 (2H, s, CH2-N cis & trans), 4.07 (2H, q, 7Hz, CH2CH3), 4.45 & 4.56 (1H, s,

cis & trans), 5.76 (2H, s,

Anal. Calcd. for C,5H20NO4Na: C, 59.79; H, 6.69; N, 4.64.

Found: C, 59.69; H, 6.76; N, 4.75.

2 - t - Butoxycarbonylaminomethyl - 4 - hydroxyphenylacetic acid is prepared, for example, according to U.S. 3,823,141.

o-(N-Methylaminomethyl)phenylacetic acid 8-lactam

Sodium hydride (57% in paraffin, 4.3 g., 0.11 mol.) was washed with dry nhexane and suspended in dry benzene (100 ml.). To the suspension was added a solution of o-aminomethylphenylacetic acid 8-lactam (U.S. 3,796,716) (14.7g., 0.1 mol.) in dry benzene or xylene (200ml.) with stirring under a nitrogen atmosphere. The mixture was refluxed for one hour and cooled to room temperature. To the mixture was added methyl iodide (18 ml.) in one portion and the mixture'was refluxed again for 1.5 hours. The reaction mixture was cooled to room temperature and poured into ice-water (100 ml.). The aqueous layer was separated from the organic layer and extracted with CHCI3 (2 x 50ml.). The extracts were combined with the organic layer and dried on MgSO4. The solvent was removed and the oily residue was distilled in vacuo to afford l4.9g. (92%) of o (N-methylaminomethyl)phenylacetic acid 6-lactam, boiling at 13W135 C/2 mmHg., m.p. 35--37bC.

ir: v max KBr 3300, 162Q, 1490 cm-1.

nmr: 6 ppm3 3.12 (3H, s), 3.59 (2H; t, J=1.5 Hz), 4.48 (2H, t, J=1.5 Hz), 7.21 Anal. Calcd. for C10M11NO.l/4M2O: C, 72.49; H, 6.84; N, 8.45.

Found: C, 72.78, 72.70; H, 6.76, 6.81; N, 8.49, 8.51.

o-N-Methylaminomethylphenylacetic acid

A mixture of the above-produced o-(N-methylaminomethyl)-phenylacetic acid 6-lactam (5.0g., 0.031 mol) and conc. hydrochloric acid (500 ml.) was refluxed for 40 hours. The mixture was evaporated under reduced pressured, and the residual oil was dissolved in water (20 ml.) and treated with a small amount of active carbon.

The filtrate was washed with benzene (50 ml.) and evaporated to dryness. The residual oil was crystallized by trituration with THF (or acetone) to give colorless needles of o-N-methylaminomethylphenylacetic acid hydrochloride (4.5 g., 67%).

Anal. Calcd. for C1oHl3NO2-HCl: C,- 55.69; H, 6.54; N, 6.49; Cl, 16.44.

Found: C, 55.65, 55.74; H, 6.62, 6.60; N, 6.53, 6.53; Cl, 16.36.

Some unreacted starting material was recovered from the benzene layer and the THF washings (1.2 g., 24%, b.p. 140-143 C/2mmHg).

An aqueous solution of o-N-methylaminomethylphenylacetic acid hydrochloride (5g.) was column chromatographed with IR--120 ion-exchange resin (H+, 70 ml.) and eluted with 3N NH40H (2 1) to afford 3.9 g. (93%) of o-Nmethylaminomethylphenylacetic acid as needles.

ir: V KBr 1650, 1470 cm-'.

max The following examples are given in illustration of, but not in limitation of, the present invention. All temperatures are in degrees Centigrade. 7-Aminocephalosporanic acid is abbreviated as 7-ACA: -ACA represents the moiety having the structure

and thus 7-ACA can be represented as

IR--120 is also called Amberlite IR--120 and is a strong cation exchange resin containing sulfonic acid radicals. ("Amberlite" is a registered Trade Mark).

Amberlite IR--120 is a commercially available cation exchange resin of the polystyrene sulfonic acid type; it is thus a nuclear sulfonated polystyrene resin cross-lined with divinyl benzene obtained by the procedure given by Kunin, Ion Exchange Resins, 2nd Edition (1958), John Wiley and Sons, Inc. Therein see Pages 84 and 87 for example.

Amberlite IRC-50 is a commercially available cation exchange resin of the carboxylic type; it is a copolymer of methacrylic acid and divinylbenzene.

Dicyclohexylcarbodiimide is abbrevited as DCC, tetrahydrofuran as THR, thin layer chromatography as TLC, p-toluenesulfonyl as Ts and methanol as MeOH.

When the following instrumental readings are given, for infrared nu if used is written v, for ultraviolet lambda is written as A, with molar absorptivity as epsilon (E) and for nuclear magnetic resonance (nmr) delta is written as 6 and tau as T (6= lO-T). The word "Nujol" is a registered Trade Mark.

Additional Starting Materials 6-Chloro-2-(2-cyanoethyl)-2,3-dihydro-s-triazolo[4,3-b]-pyridazin-3-on.

To a solution of 6-chloro-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on [P.

Francabilla and F. Lauria, J. Het. Chem. 8, 415 (1971)1 (17g., 0.1 mole) in dry DMF (300 ml.) was added potassium tert.-butoxide (0.5 g., 4.5 m.moles) with stirring. Acrylonitrile (6.6 g., 0.12 mole) in dry DMF (10 ml.) was added to the mixture.

The mixture was stirred at 100--1100C for 24 hours, then poured into water (700 ml.) and extracted with ethyl acetate (5 x 400 ml.). The organic extracts were combined, dried over Na7SO, and evaporated. The residue was crystallized from ethyl acetate to give light yellow needles of 6-chloro-2-(2-cyanoethyl)-2,3-dihydros-triazolo[4,3-b]pyridazin-3-on (2.5 g., 11%). M.p. 166--1680C.

ir: V nKBr 2230, 1720, 1550, 1500 cm-1.

uv: A dimoxaaxne 373 nm (E 2000).

nmr: 6 DMSO-d, 3.03 (2H, t, J=6.0 Hz, CH3), 4.21 (2H, t, J=6.0 Hz, CH2), 7.23 (1H, d, J=10.0 Hz, pyridazine-H), 7.93 (1H, d, J=10.() Hz, pyridazine-H).

* Anal. Calcd. for C8H6N5OCl: C, 42.97; H, 2.70; N, 31.32; Cl, 15.86.

Found: C, 42.73, 42.56; H, 2.57, 2.50; N, 31.36, 31.68; Cl, 15.96, 15.81.

2-(2-Carboxyethyl)-6-chloro-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on .

A solution of 6-chloro-2-(2-cyanoethyl)-2,3-dihydro-s-triazolo[4,3-b]pyridazin- 3-on (724 mg.) in 6N-HCl (15 ml.) was refluxed for 6 hours. The reaction mixture was extracted with ethyl acetate (10 x 20 ml.). The combined extracts were washed with saturated aqueous sodium chloride (50 ml.), dried over Na2SO, and evaporated to give light yellow, solid 2-(2-carboxyethyl)-6-chloro-2,3-dihydro-s- triazolo[4,3-b]pyridazin-3-on (567 mg., 72%). M.p. > 170"C. (sublimation).

ir: # max KBr 3400-2400, 1730, 1710, 1540 cm'.

dioxane uv: 2 max 377 nm ( 1500).

nmr: 6 D2O+NaMCO3 2.70 (2H, t, J=7.0 Hz, CH3), 4.24 (2H, t, J=7.0 Hz, CH2), ppm 7.17 (1H, d, J=10.0 Hz, pyridazine-H), 7.70 (1H, d, J=10.0 Hz, pyridazine-H).

Anal. Calcd. for C8H7N4O3Cl: C, 39.60; H, 2.91; N, 23.09; CI, 14.61.

Found: C, 39.62, 39.48; H, 2.97, 2.67; N, 23.05, 22.701 Cl. 13.93, 14.12.

2-(2-Carboxyethyl)-2,3-dihydro-6-mercapto-s-triazolo[4,3-b]pyridazin-3-on.

A mixture of 2-(2-carboxyethyl)-6-chloro-2,3-dihydro-s-triazolo[4,3- b]pyridazin-3-on (567 mg., 2.34 m.moles) and 70% sodium hydrosulfide dihydrate (924 mg., 7.02 m.mole) in water (10 ml.) was stirred at room temperature for two hours. The reaction mixture was adjusted succesively to pH 1 with c. HCl, to pH 10 with NaOH and then to pH 1 with c. HCI. The resulting precipitate of 2-(carboxy ethyl)-2,3-dihydro-6-mercapto-s-triazolo[4,3-b]pyridazin-3-on was collected by filtration and washed with water. Yield: 4.18 mg (74%). M.p. 1741760C.

ir: P KmBxr 3600-2600, 2440, 1730, 1720 (sh) cm~'.

uv: A max pH 7 buffer 262 nm (E 17000), 318 nm (# 6600) nmr: # ppm DMSO-d6 2.73 )2M, t, J=7.0 Hz, CH2), 4.07 (2H, t, J=7.0 Hz, CH3), 7.30 (1H, d, J=10.0 Hz, pyridazin-H), 7.74 (1H, d, J=10.0 Hz, pyridazin-H).

Anal. Calcd. for C1H1N4O3S: C, 40.00; H, 3.36; N, 23.32; $, 13.35.

Found: C, 39.08, 39.06; H, 3.12, 3.20; N, 22.65, 22.70; S, 14.23, 14.29.

7 - Amino - 3 - [2 - (2 - carboxyethyl) - 2,3 - dihydro - s - triazolo[4,3-b] pyridazin - 3 - on - 6 - ylthiomethyl] - 3 - cephem - 4 - carboxylic Acid.

A mixture of 7-ACA (405 mg., 1.49 m.moles), the thiol 2-(2-carboxyethyl)-2,3dihydro-6-mercapto-s-triazolo[4,3-b]pyridazin-3-on (357 mg., 1.49 m.moles) and NaHCO3 (375 mg., 4.47 m.moles) in 0.1 M phosphate buffer (pH 7, 8 ml.) was stirred at 80 C for 30 minutes. The reaction mixture was cooled and filtered to remove insolubles. The filtrate was adjusted to pH 1-2 with c. MCI. The resulting precipitate, 7-amino-3-[2-(2-carboxyethyl)-2,3-dihydro-s-triazolo[4,3-b]pyridazin- 3-on-6-ylthiomethyl]-3-cephem-4-carboxylic acid, was collected by filtration and washed with water. Yield; 519 mg. (77%).

ir: p KBr 3600-2200, 1800, 1725, 1620, 1550, 1480 cam-1.

max uv: # max pH 7 buffer 253 nm (E 20000), 298 nm (E 10000).

nmr: 6 D2O+K2CO3 2.20 (2H, t, J=7.0 Hz, CH2), 3.40 (1H, d, J=17.5 Hz, ppm 2-H), 3.85 (IH, d, J=17.5 Hz, 2-H), 4.00-4.50 (4H, m, 3-CH2 and N-CH2-), 5.01 (1H, d, J=4.0 Hz, 6-H), 5A0 (111, d, J=4.0 Hz, 7-H), 694 (111, d, J=10.0 Hz, pyridazine-H), 7.44 (1H, d, J=10.0 Hz, pyridazin-H).

Anal. Calcd. for C,,H,,N,O,S,.3/2H,O: C, 40.09; H, 3.99; N, 17.52; S, 13.37.

Found: C, 40.06, 40.12; H, 3.33, 3.341 N, 16.96, 16.98; S, 13.87, 13.98.

7-ACA refers to 7-aminocephalosporanic acid and DMF to dimethylformarnide.

Example 1 7 - [o - (N - Butoxycarbonyl - N - methylaminomethyl)phenylacetamido] - 3 [2 - (2 - carboxyethyl) - 2,3 - dihydro - s - triazolo[4,3-b]pyridazin - 3 - on 6 - ylthiomethyl] - 3 - cephem - 4 - carboxylic acid.

To a mixture of 7-amino-3- [2-(2-carboxyethyl)-2,3-dihydro-s-triazolo [4,3- b]pyridazin-3-on-6-ylthiomethyl]-3-cephem-4-carboxylic acid (452 mg., 1 m.mole) and triethylamine (0.46 ml., 3.3 m.mole) in 50% aqueous acetonitrile (4 ml.) was added a THF solution (3 ml.) of 2,4-dinitrophenyl o-(N-t-butoxycarbonyl-N methylaminomethyl)phenylacetate prepared from o-(N-t-butoxycarbonyl-Nmethylaminomethyl)phenylacetic acid (283 mg., 1.1 m.mole), 2,4-dinitrophenol (202 mg. 1.1 m.mole) and DCC (227 mg., 1.1 m.mole). The mixture was stirred at room temperature overnight and concentrated under reduced pressure to remove the organic solvents. The aqueous concentrate was washed with ether (3 x 20 ml.), acidified with c. MCI to pH 1-2 and extracted with ethyl acetate (5 x 20 ml.). The combined extracts were dried with anhydrous Na2SO4 and evaporated to dryness.

The residue was chromatographed on a column of silica gel (Wako gel, C-200, lOg.) by eluting with a mixture of MEOH-CHCl3 (MeOH: 0 to 3%). The combined eluates which contained the desired product were evaporated to give 359 mg.

(50%) of the title compound. M.p. > 1500C (dec.).

ir: # maxKBr 3600-2400, 1780, 1720, 1680, 1550, 1490 cm~'.

uv: # pHmax 7 Buffer253 nm (E 19800), 298 nm (E 9400).

nmr: 6 D MSO-d6 1.37 (9H, s, t-Bu-H), 2.70 (3H, s, N-CH3), 2.70 (2H, t, J=7.0 ppm Hz, -CH2-), 3.2-4.5 (10H, m), 5.01 (1H, d, J=5 Hz, 6-H), 5.60 (1H, d-d, J=5 & 8 Hz, the 8 Hz coupling disappeared by addition of D2O, 7-H), 6.93 (1H, d, J=10 Hz, pyridazine-H). 7.58 (1H, d, J=10 Hz, pyridazine-H).

Anal. Calcd. for C31H35N7O9S2#5/2H2O: C, 49.08; H, 5.31; N, 12.92; S, 8.45.

Found: C, 49.32; 49.36; H, 4.70, 4.63; N, 12.52, 12.53; S, 8.44, 8.43.

BB-S 525; 7 - [o - (N - Methylaminomethyl)phenylacetamido] - 3 - [2 -(2 carboxyethyl) - 2,3 - dihydro - s - triazolo[4,3-b]pyridazin - 3 - on - 6 -ylthio methyl] - 3 - cephem - 4 - carboxylic acid.

A mixture of trifluoroacetic acid (1 ml.) and the BOC-protected cephalosporin prepared above 302 mg., 0.42 m.mole) was allowed to stand at room temperature for 15 min. and then diluted with ether (10 ml.). The resulting precipitate was collected by filtration and washed with dry ether (2 x 10 ml.) to afford 263 mg. of solid which was dissolved in a mixture of water (6 ml.) and acetonitrile (3 ml.). The stirred solution was adjusted at pH4 with 1 N-NaOH (0.36 ml.) and diluted with acetonitrile (100 ml.) to give the precipitate (187 mg.), which was suspended in water (4ml.) and adjusted at pH9 with sodium hydroxide (1 N, 0.3 ml.). The solution was treated with a small amount of active carbon and freeze-dried to leave the monosodium salt of BB-S 525. Yield 106 mg. (39%). M.p. > 1800C (dec.).

ir: v mKBxr 36002400, 1770, 1710, 1600, 1490, 1400 cm~'.

uv: A max pH 7 Buffer 253 nm (E 19800), 298 nm (E 8800).

nmr: # max 010D2O 2.70 (2H, m, -CM2-), 2.75 (311, s, N-CH3), 4.4-3.4 (10H, m), 4.92 (1H, d, 5=4.0 Hz, 6-H), 5.55 (1H, d, 5=4.0 Hz, 7-H), 6.95 (1H, d, J=9.5 Hz, pyridazin-H), 7.28 (4H, s, Ph-H), 7.40 (1H, d, J=9.5 Hz, pyridazin-H).

Anal. Calcd. for C26H28N7O7S2.Na.3H2O: C, 45.28; H, 4.68; N, 14.22; S, 9.30.

Found: C, 45.34, 44.84; H, 4.01, 3.85; N, 14.14, 14.08; S, 9.76.

In vitro antibacterial activity of BB-S 525 compared with BB-S 479 and cefamandole (determined by Steers' agar dilution method on Mueller-Hinton agar plate) BB-S 479 is the compound 7 - [o - (N - butoxycarbonyl - N - methyl aminomethyl)phenylacetamideo] - 3 - (2 - carboxymethyl - 2,3 - dihydro - s triazolo[4,3-b]pyridazin - 3 - on - 6 - ylthiomethyl) - 3 - cephem -4 - carboxylic acid whose preparation is given in Example 4 of copending Application No. 25885179 Serial No. 1572202.

MIC (mcg./ml) Organism BB-S 525 BB-S 479 Cefamandole S aureus Smith 0.4 0.4 0.2 S. aureus 0.2 0.2 0.05 S. aureus BX-1633 0.8 0.8 0.2 St. faecalis > 100 > 100 100 E. coli NIHJ 0.2 0.1 0.1 E. coli ATCC 8739 6.3 3.1 6.3 E. coli Juhl 0.4 0.2 0.4 E. coli BX-1373 0.4 0.2 0.2 E. coli 0.2 0.1 0.1 E. coli 0.2 0.05 0.05 E. coli 6.3 3.1 1.6 Kl. pneumoniae 1.6 0.8 3.1 Kl. pneumoniae 0.2 0.1 0.2 Kl. pneumoniae 0.2 0.2 0.8 Kl. pneumoniae 0.2 0.2 0.8 Pr. vulgaris 0.2 0.2 0.2 Pr. vulgaris 3.1 0.8 50 Pr. mirabilis 0.4 0.1 0.4 Pr. mirabilis 0.2 0.1 0.2 Pr. morganii > 100 > 100 3.1 Pr. morganii 0.2 0.2 0.8 Pr. rettgeri 0.8 0.8 0.1 Ps. aeruginosa > 100 > 100 > 100 Ps. aeruginosa > 100 > 100 > 100 Shig. dysenteriae 0.1 0.1 0.1 Shig. flexneri 12.5 12.5 3.1 Shig. sonnei 0.1 0.1 0.2 Serr. marcescens 25 12.5 50 Enterob. cloacae 3.1 3.1 1.6 Sal. enteritidis 0.2 0.1 0.1 Sal. typhosa 0.2 0.1 0.1 B. anthracis 0.2 0.2 0.05 Example 2 Substitution in the procedure of Example I - for the 2-N-t-butoxyvarbonyl*N- methylaminomethyl-4-hydroxyphenylacetic acid used therein of an equimolar weight of 2-N-t-butoxycarbonylaminomethyl-4-hydroxyphenylacetic acid and of 2 N-t-butoxycarbonylaminomethyl-4-methoxyphenylacetic acid and of 2-N-t butoxycarbonyl-N-methylaminomethyl-4-methoxyphenylacetic acid, respectively, produces the compounds having the structures

respectively.

WHAT WE CLAIM IS: 1. A compound having the formula

wherein R1 represents

wherein R is hydrogen, hydroxy or methoxy; R' is hydrogen or methyl; and R2 is hydrogen, or a conventionl, pharmaceutically acceptable, easily hydrolyzed ester forming group; or a non-toxic, pharmaceutically acceptable salt thereof.

2. The pivaloyloxymethyl-, acetoxymethyl-, methoxymethyl-, acetonyl-, phenacyl-, p-nitrobenzyl-, P,P,P-trichloroethyl-, 3-phthalidyl- or 5-indanyl- esters of a compound as claimed in Claim 1.

3. A compound of Claim I or 2, wherein R' is

wherein R is hydrogen, hydroxy or methoxy and R' is hydrogen or methyl.

4. A compound of Claim 1 our 2 wherein R1 is

wherein R' is hydrogen or methyl.

5. A compound of claim I or 2, wherein R' is

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (29)

**WARNING** start of CLMS field may overlap end of DESC **. respectively. WHAT WE CLAIM IS:

1. A compound having the formula

wherein R1 represents

wherein R is hydrogen, hydroxy or methoxy; R' is hydrogen or methyl; and R2 is hydrogen, or a conventionl, pharmaceutically acceptable, easily hydrolyzed ester forming group; or a non-toxic, pharmaceutically acceptable salt thereof.

2. The pivaloyloxymethyl-, acetoxymethyl-, methoxymethyl-, acetonyl-, phenacyl-, p-nitrobenzyl-, P,P,P-trichloroethyl-, 3-phthalidyl- or 5-indanyl- esters of a compound as claimed in Claim 1.

3. A compound of Claim I or 2, wherein R' is

wherein R is hydrogen, hydroxy or methoxy and R' is hydrogen or methyl.

4. A compound of Claim 1 our 2 wherein R1 is

wherein R' is hydrogen or methyl.

5. A compound of claim I or 2, wherein R' is

wherein R' is hydrogen or methyl.

6. A compound of Claim 3, wherein R is hydrogen.

7. A compound of Claim 3, wherein R is hydroxy.

8. A compound of Claim 3, wherein R is methoxy.

9. A compound of any one of Claims 1--8, wherein R' is hydrogen.

10. A compound of any one of Claims I to 8, wherein R' is methyl.

I 1. A process for the preparation of a compound of the formula

wherein R' represents

wherein R is hydrogen, hydroxy or methoxy; R' is hydrogen or methyl; and R2 is hydrogen, or a conventional, pharmaceutically acceptable, easily-hydrolyzed ester forming group; or a non-toxic pharmaceutically acceptable salt thereof characterized by reacting a compound of the formula

in which Y is H or R1, as defined above, or a salt or easily hydrolyzable ester thereof with a compound of the formula

and, if Y is H, treating the resulting compound with an acylating agent of the formula R1-X in which X is halogen or a functional equivalent thereof and R' is as defined above, and, if desired, converting the resulting free acid, salt or easily hydrolyzable ester of a compound of formula I to the corresponding ester or non-toxic pharmaceutically acceptable salt thereof, and, if desired, converting a resulting salt or easily hydrolyzed ester of a compound of the formula I to the corresponding free acid of the formula I.

12. The process according to Claim Il, wherein a resulting free acid of formula I is converted to an ester selected from the pivaloyloxymethyl-, acetoxymethyl-, methoxymethyl-, acetonyl-, phenacyl-, p-nitrobenzyl-, ,"ss- trichloroethyl-, 3-phthalidyl- or 5-indanyl- esters.

13. The process of Claims 11 or 12, wherein R' is

wherein R is hydrogen, hydroxy or methoxy and R' is hydrogen or methyl.

14. The process of Claims 11 or 12, wherein R' is

wherein R' is hydrogen or methyl.

15. The process of Claims 11 or 12, wherein R' is

wherein R' is hydrogen or methyl.

16. The process of Claim 13, wherein R is hydrogen.

17. The process of Claim 13, wherein R is hydroxy.

18. The process of Claim 13, wherein R is methoxy.

19. The process of any of Claims 11-18, wherein R' is hydrogen.

20. The process of any of Claims 11 to 18, wherein R' is methyl.

21. A compound of formula I, or a pharmaceutically acceptable salt thereof, when prepared by the process of any one of Claims 11 to 20.

22. A method of treating bacterial infections comprising administering by injection to an infected non-human warm-blooded animal, of an effective but nontoxic dose of 2501000 mg. of a compound according to any one of Claims 1 to 10 or 21; or a non-toxic, pharmaceutically acceptable salt thereof.

23. A method for combatting Shig. dysenteriae infections which comprises administering to a non-human warm-blooded mammal infected with a Shig.

dysenteriae infection an amount effective for treating said Shig. dysenteriae infection of a composition comprising a compound according to any one of Claims 1 to 10 or 21: or a non-toxic pharmaceutically acceptable salt thereof.

24. A method for combating B. anthracis infections which comprises administering to a non-human warm-blooded mammal injected with a s. antracis infection an amount effective for treating said B. anthracis infection of a composition comprising a compound according to any one of Claims 1 to 10, or 21; or a non-toxic, pharmaceutically acceptable salt thereof.

25. A compound according to Claim I substantially as described hereinbefore with particular reference to either of the accompanying Examples.

26. A compound according to Claim I which is any of the compounds specifically identified hereinbefore other than a compound according to Claim 25.

27. A process according to Claim 11 substantially as described hereinbefore with particular reference to either of the accompanying Examples.

28. A compound according to Claim I when prepared by a process according to any of Claims 11 to 20 or 27.

29. A pharmaceutical composition comprising a compound according to any of Claims 1 to 10, 21, 25, 26 or 28 in admixture with a pharmaceutically acceptable carrier therefor.