GB1572202A

GB1572202A - Cephalosporins

Info

Publication number: GB1572202A
Application number: GB2588579A
Authority: GB
Original assignee: Bristol Myers Co
Current assignee: Bristol Myers Co
Priority date: 1977-09-26
Filing date: 1977-09-26
Publication date: 1980-07-23

Description

(54) CEPHALOSPORINS (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 antibacterial agents made originally, for example, by acylation 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 review 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 26W243C).

Issued patents on 3-thiolated cephalosporins in which the 7-substituent is (a) a-Amino-a-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 (Farmdoc 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 (Farmdoc 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 the older patent literature concerning substituted 7phenylacetamidoc ephalosporanic acids) and (d) N-(phenylacetimidoyl)aminoacetamido as U.S. 3,692,779; and (e) a-amino-cr-( 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 (Begium 759,570; Farmdoc 39819S), Belgium 793,311 (Farmdoc 39702U) and Begium 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- I ,3,4-thaidiazole-5-acetic acid and its conversion to 7-(1H-tetrazol-l -yl- acetamido) - 3 - (5 - carboxymethyl - 1,3,4 - thiadiazol - 2 - ylthiomethyl) - 3cephem-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; R' is -H, (lower)alkanoyloxy,

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

U.S. 3,883,5 0 and 3,931,160 and Farmdoc Abstract 22850W make reference to 3-heterocyclicthiomethyl cephalosporins containing a numberof substituents (including carboxyl) on the numerous hereocycles 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 carboyxl 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 disclose cephalosporins containing various fused ring bicyclic thiols.

Farmdoc abstract 18830X discloses compounds of the formula

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

Our copending Application No. Serial No. 1572201 39951177 describes and claims a compound having 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.

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, phenylbenzoyl, N-phthalimido, N-succinimido, N-saccharino, N (lower)alkylcarbamoyl, (lower)alkoxy, (lower)alkylthio, phenoxy, carbalkoxy, carbobenzoxy, carbamoyl, benzyloxy, chlorobenzyloxy, carbophenoxy, carbotert.-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 cmbodiments of this invention include the pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, pnitrobenzyl, p,p,p-trichloroethyl, 3-phthalidyl- 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 R' has the structure

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

A 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

n is 0 to 4; R is hydrogen, alkyl having 1 to 8 carbon atoms, cycloalkyl of 3 to 6 carbon atoms, phenyl, C1C4 phenyrakyl, pyridyl, thienyl, or pyrrolyl; R'O is hydrogen, methyl or ethyl; R and R are each hydrogen, alkyl having 1 to 6 carbon atoms, phenyl, pyridyl, or thienyl; R4 and R5 are ech hydrogen or alkyl of 1 to 4 carbon atoms; R6 is alkyl having 1 to 4 carbon atoms, phenyl, phenylalkyl having 1 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, NHR', N(R')2, nitro, fluoro, chloro, bromo or carboxy, or a nontoxic, 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 the group

wherein R5 is a hydrogen atom, a methyl or an ethyl group; X is-O-, -NH-; R6 is a basic group such as alkyl or aralkyl substituted with substituted or unsubstituted NH2, such as alkyl-NHCH3, aralkyl-NHCH3,

R' 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 of 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 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, , -trichloroethyl, 3-phthalidyl or 5-indanyl and preferably is hydrogen or a nontoxic, pharmaccutically acceptable salt thereof.

There is further provided by the present invention a method of treating bacterial infections comprising administering by injection to an infected nonhuman warm-blooded animal, an effective but nontoxic dose of 25-1000 mgm. of a compound having the formula

wherein Rl represents

wherein R is hydrogen, hydroxy or methoxy; R' is hydrogen or methyl; and M is hydrogen, pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl, A, ,4-trichloroethyl, 3-phthalidyl or 5-indanyl or a nontoxic, pharmaceutical y acceptable salt thereof.

There is also provided by the present invention a method for combatting Shig. dysenteriae 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, p,p,p-trichloroethyl, 3-phthalidyl or 5-indanyl and preferably is hydrogen or a nontoxic, 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 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 "B-trichloroethyl, 3-phthalidyl or 5-indanyl and preferably is hydrogen or a nontoxic, pharmaceutically acceptable salt thereof.

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

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

or a salt or easily hydrolyzed ester or 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, p:P,P-trichloroethyl, 3-phthalidyl and 5-indanyl ester) 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'carbonyl-diimidazole 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;c.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)1, or of a ketenimine reagent [cf. C. L. Stevens and M. E. Munk, J. Amer. Chem. Soc..

80, 4065 (1958)] or of hexachlorocyclotriphosphatriazine or hexabromocyclotri phosphatriazine (U.S. 3,651,050) or of diphenyphosphoryl azide [DPPA; J. Amer.

Chem. Soc., 94, 6203205 (1972)] or of diethylphosphoryl cyanide [DEPC; Tetrahedron Letters No. 18, pp. 1595-1598 (1973)] or of diphenyl phosphite [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, dimethylformamide 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 microorganisms, 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-1,2-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 3W35 C. 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 by the displacement of the 3-acetoxy group of a 7-acylamino-cephalosporanic acid (prepared by substituting 7-aminocephalosporanic acid for the 3-thiolated-7-amino-cephalosporanic 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 100"C. 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 owt 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 26-243C.

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 with a compound having the formula

The salts of the compounds of this invention include the nontoxic 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 nontoxic amines as trialkylamines including triethylamine, procaine, dibenzylamine, N-benzyl-beta-phenethylamine, I-ephenamine, N,N'-dibenzyl- ethylenediamine, dehydroabietylamine, N,N'-bis-dehydroabietylethylenediamine, N-(lowertalkylpiperidine, e.g. N-ethylpiperidine, and other amines which have been used to form salts with benzylpemcillin; and the nontoxic 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-nitrophenyl-sulfenyl, 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 -ketoesters and p-diketones disclosed, for example, in U.S. patent 3,325,479 and the ,5-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 patent or chemically as in U.S. patent 3,575,970 and in Journal of Antibiotics, XXIV (if), 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 is illustrated herein for 7aminocephalosporanic acid itself. The 3-thiolated ester of 7-aminocephalosporanic acid is then coupled with the organic carboxylic acid R1-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 parenterally in an amount of from 5 to 200 mg./kg./day and preferably 5 to 20 mgJkg./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 physiologically 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.

2tert. -Butoxycarbonylaminomethyl)-1 , 4-cyclohexadienylacetic acid.

A solution of 16.5 g. (0.1 mole) of o-amino-methylphenylacetic acid in 1.5 1 of liquid ammonia (which had been treated with 50 mg. of Li to 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% NH,OH 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%) of colorless needles, o-(2-aminomethyl-l,4-cyclohexa- dienyl)acetic acid. M.p. 1830C.

Anal. Calcd. for CSHs3NO2: 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 a-(2-aminomethyl-1 .4-cyclohexadienyl). acetic acid

The procedure used by Welch, Dolfini and Giarrusso in U.S. patent 3,720,665 (Example 1) to make D-2-amino-2-(1,4-cyclonexadienyl)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 40O (15 mm.) to yield a white residue which was dried in vacuo over P2Op 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 50. The mixture was stirred for 20 min. and the amino acid, a-(2 aminomethyl-1 ,4-cyclohexadienyl)acetic acid, was collected and dried for 16 hours in vacuo over P,O, to yield 6.3 g. (58%) of white crystals, m.p. 1900 decomp. The IR and NMR spectra were consistent for the structure. in 152 ml. of A solutlon of 19.31 g. (0.135 m) of tert.-butoxycarbonylazide tetrahydrofuran (THF) was added to a stirred solution of 14.89 g. (0.09 m) of 2aminomethyl-1,4-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 25 and then filtered thru diatomaceous earth (Super-cel). The THF was removed at 40 (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 P,05 at 250 to 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 Aceric Acid

A) Thiophene3-carboxaldehyde Dimethyl A petal (2a) A mixture of thiophene-3-carboxaldehydett (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 pressure. Yield 423 g. (94%), b.p. 90-95 C. 13 mm Hg. ir: v liq 3150, 1045, 1025 cm1 nmr: 6 ppm 3.21 (6H, s, OCH), 5.43 (1H, s,

(3H, m, thiophene-H) 1) S. Gronowitz, Arkev. kemi., 8, 411(1955).

B. 2-Formalthiophene-3-carboxaldehyde Dimethylacetal (3 a) To a stirred solution of 2a (423 g., 2.68 moles) in anhydrous ether (1 L) was added dropwise in 1 hour a freshly prepared solution of n-butyllithium (27 moles) in ether keeping a gentle reflux under dry N2. Reflux being continued for 0.5 hour, a solution of

C) 1-Methylsulfinyl-1-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/D in methanol, 2 ml. in THF (tetrahydrofuran) (5 ml.) was added to a solution of methyl methylthiomethyl sulfoxide21 (2.5 g., 20m. moles) and 2-formyl-3-thiophenecarboxaldehyde ethylene acetal3) (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 lyer 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 4b was isolated as a pale yellow oil. ir: P liq 3110, 1600 cm-1. max nmr: 6 CDCl3 2.42 (3H, s, S-CH3), 2.78 (3H, s, SO-CH3), 4.15 (4H, m, CH2CH2-), 6.12 (1H, s,

7.34 (1H, d, J=4.5 Hz, thiophene-Hss), 7.40 (1H, d=4.5 Hz, thiophene-Hα), 8.28 (1H, s, -CH=).

The semicarbazone of 4 was prepared by a usual manner and crystallized from ethanol-DMF. M.p. 212-213 C.

Anal. Calcd. for C10H13N3O2S2: 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.

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 a!., Tetrahedron Letters, 1383 (1972).

D) I -Methylsulf nyl-l -methylthio-2X3-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) gavie 130.5 g (78%) of 4a as a pale yellow oil. ir. ,, liq 3100 1580 1100 1050-1 max nmr: 6 CCl4 2.42 (3H, s, S-CH2), 2.70 (3H, s, SO-CH3) 3.34 (6H, s,OCH3), 5.56 (IH, s,

7.20 (1H, d,J=6 Hz, thiophene-Hss), 7.40 (1H, d, J=6 Hz, thiophene-Hα), 8.12 (1H, 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 MgSO4 and evaporated to dryness. The oily residue was column-chromatographed 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/l mm Hg. ir: a' liq 3110, 1730, 1670 cm~'. max nmr: 6 ppmCDCl3 1.30 (3H, t, J=6 Hz, -CH2CH3), 4.25 (2H, q, J=6 Hz, -CH2CH3), 4.26 (2H, 5, -CH2CO), 7.25 (1H, d, J=5 Hz, thiophene-Hss), 7.48 (lH, d, J=5 Hz, thiophene-Ha), 10.15 (IH, s, CHO).

The analytical sample of 5 was submitted as the 2,4-dinitrophenylhydrazone which was crystallized from chloroform. M.p. 178-179 C. nujol ir: # max 1720, 1610, 1570 cm~'.

Anal. Calcd for C,5Hr4N40 S: C, 47.62; H, 3.73; N, 14.81; S, 8.47.

Found: C, 47.33; II, 3.47; , 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, 16m 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 maxliq 3400, 1730, 1620 cm-'. nmr: a Acetone 1.23 (3H, t, J=7.5 Hz, -CH2CN3), 4.01 (2H, 5, -CH2CO), ppm 4.14 (2H, q, J=7.5 Hz, -CH2CH3), 7.31 (2H, s, thiphene-H), 8.26 (1H, s, -CH=N), 10.15 (1H, s, NOH, disappeared by addition of D2O).

G) The 6-lactam of 3-aminomethyl-2-thienylacetic acid (7) Method A: Catalytic reduction A mixture of the 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 417 mg (22%) of colorless needles 7 melting at 194-1950C. ir: # max KBr 3200, 1650, 1480 cm-1. nmr:# ppm DMSO-d6 3.53 (2H, t, J=3Hz, -CH2CO-), 4.36 (2H, d-t, J=3, 1.5 Hz, changed to a triplet by addition of D2O, J=3 Hz, CH2N), 6.95 (1H, d, J=4.5 Hz, thiophene-Hss), 7.45 (1H, d, J=4.5 Hz, thiophene-Hα), 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 40-50 C with vigorous stirring. The reaction mixture was stirred overnight at room temperature and heated at 600C 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 reflux 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-1; 3450, 3000, 1700, 1200; nmr (D2O) ppm; 4.80 (2H, s, -CH2CO), 4.27 (2H, s, CH2-N), 7.26 (1H, d, J=6 Hz, thiophene-Hss), 7.53 (1H, d, J=6 Hz, thiophene-Hα). The hydrochloride (3.71 g, 17.9 m moles) was dissolved in water (10 ml) chromatographed on a column of IR-120 (H, 30 ml) and developed successively with water (100 ml) and 5N-NH4OH (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. 223-2250C. ir: # KBr 3000, 1620, 1520 cm~'. max nmr:# ppm D2O-Na2CO3 3.20(2H, s, -CH2CO), 4.13 (2H, s, CH2N), 7.04 (1H, d, J=6 Hz, thiophene-Hss) 7.30 (1H, d, J=6 Hz, thiophene-Ha).

Anal. Calcd. for C,H,NO2S: C, 49.10; H, 5.30; N, 8.18; S, 18.73.

Found: C, 48.53; H, 5.22; N, 7.98; S, 18.97.

I. 3-t-Butoxycarbonylaminomethyl-2-thienylacetic acid (9) A mixture of 3-aminomethyl-2-thienylacetic acid 8 (3.1 g, 18 m. moles) and triethylamine (8 g. 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 romm 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 nhexane and benzene to give 4.5 g (92%) of colourless needles 9, melting at 62-63 C. ir: #maxnujol 3350, 1700 cm-1. nmr:#ppmCDCl3 1.43 (9H, s, BOC-H), 3.27 (2H, s, CH2CO), 4.16 (2H, d, J=6 HZ, CH2-N, a singlet when D2O was added), 500 (1H, br, -NH-, disappeared by addition of D2O), 6.30 (1H, broad s, -COOH, disappeared by addition of D2O), 6.86 (1H, d, J=6 Hz, thiophene-Hss), 7.06 (1H, d, J=6 Hz, thiophene-Hα).

Anal. Calcd. for C,2H,7NO4S: 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-(1nad 4-hydroxy-)phenylacetic Acids

2-N- Tosylam inomethyl4-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-toluenesulonylchloride 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 (I 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: # max KBr 3240, 1700, 1380, 1330, 1150 cm-1 uv: # max 1%K2CO3 230 nm (#: 7,750) nmr: O DMSO-ds .47(3H,s, Ar-CH3), 3.60 (2H, s, CH2CO), 3.93 (2H, d, J=6.0 Hz, CH2N), 6.6M8.2 (7H, m, phenyl-H).

2-(N-Methyl-N-toxylamino)methyl4-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. (024 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 (I g.) and dried over Na 504 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 146-150 C.

KBr ir: v max1690, 1500, 1340, 1280, 1150 cm-1. uv: # max @@UM 229 nm (#: 20500), 278 nm (#: 2400).

DMSO @ nmr: a DMSO-dss 2.52 (3H, s, NCH3), 2.47 (3H, s, Ar-CH3), 3.67 (2H, s, CH2CO), 3.74 (3H, s, OCH3), 4.10 (2H, s, CH2N), 6.7-7.8 (7H, m, Ar-H), 11.5 (lH, br-s, COQH).

Anal. Calc'd. for C",H2,NO5S: 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-Methylaminomethyl4-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 90"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 1R-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 1R-120 ionexchange resin (H+, 100 ml.) by eluting with 5-f0% ammonia. The eluate (2 L) containing the desired product was evaporated to dryness below 500C. and the residue was triturated with acetone to give 4.4 g. (81%) of 4a, m.p. 225-227 C. ir: v KmBr 1590, 1380, 1260, 1035 cm-1. nmr: a ppmD2O 2.77 (311, s, N-CH3), 3.6 (2H, s, CH2CO), 3.87 (3H, s, OCH3), 4.18 (2H, s, CH2N), 6.8-7.4 (3H, m, phenyl-If).

2-N-Methylaminomethyl-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 5-10% ammonia. The eluate was collected in 250 ml. fractions. Fractions containing the product were combined and evaporated to dryness below 50 C. The residue was triturated with acetone to give 1.3 g. (48.5%) of 4b, which was crystallized from 80% ethanol. M.p. 218-221 C. ir: v max 2000-3400, 1610, 1540, 1460, 1380, 1270 cm-'. uv: A 1%K2CO3243 nm (E: 4700), 297 nm (E: 1350). nmr: # ppm D2O+NaOH 2.64 (3H, s, N-CH3), 3.47 (2H, s, CH2CO), 3.94 (2H, s, N-CH2), 6.5-7.2 (3H, m, phenyl-H).

Anal. Calc'd. for C10H13NO3: C, 61.53; H, 6.71; N, 7.17. Found: C, 61.44; H, 6.81; N, 7.20.

2-N-t-Butoxycarbonyl-N-methylaminomeihyl4-methoxyphenylacetic Acid (5, R=CH3) A mixture of 1.05 g. (5 m mol.) of 4a, 1.43 g. (6 m mol.) of t-butyi 4,6dimethylpyrimidin-2-ylthiolcarbonate 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 NaCl 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=CH2) as an oil. nmr: # ppmCDCl3 1.47 (9H, s, BOC-H), 2.77 (3H, s, N-CH3), 3.60 (2H, s, CH2CO), 3.79 (3H, s, O-CH3), 4.49 (2H, s, CH2N), 6.1-7.3 (3H, m, phenyl-H).

2-N-t-Butoxycarbonyl-N-methylaminom ethyl4-hydroxyphenylaceticA cid (5, R=H) A mixture of I g. (4.78 m.mol.) of 4b, 1.5 g. (6.3 m.mol.) of t-butyl 4,6dimethylpyrimidin-2-ylthiolcarbonate and 2.1 ml. of triethylamine in 50 ml. of 50% aqueous T11F 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 100 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 liq 3000-3600, 1670, 1260, 1150 cm-1. max nmr: # ppm CDCl3 1.44 (9H, s, C(CH3)3), 2.73 (3H, s, N-CH3), 3.4 (2H, s, CH2CO), 4.38 (211, s, CH2N), 6.57.3 (311, m, phenyl-ll).

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 60 C. The mixture was stirred for 1 hour at the same temperature and acified 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-l560C.

@@@ nuj 2300 1705 1225 1170 @@@@ max nmr: #ppm @2.38 (311, s, CH3), 3.65 (211, s, CH2CO), 3.97 (211, d, J=5 Hz, CH2N), 7.1-8.2 (9H, m, phenyl-H & NH).

Anal. Calc'd. for C,H,7NO4S: C, 60.17; H, 5.37; N, 4.39; S, 10.10. Found: C, 60.11, 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 70 C. After cooling, the reaction mixture was acidified with hydrochloric acid rto separate pale yellow precipitate which crystallized from ethyl acetate-n-hexane to give colorless prisms, 3. Yield, 8.5 g. (91%). M.p. 162-l630C. ir: v KBr 2700-2300, 1700, 1600, 1345, 1200, 925 cm-1. max nmr: S pDp2mH+K H 2.37 (311, s, CH3), 2.49 (311, s, CH3), 3.80 (211, s, CH2CO), 4.18 (211, s, CH2N), 7.0-8.0 (811, m, phenyl-H).

Anal. Calc'd. for C17H,9NO2: 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 R-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 168-170 C. (dec.).

Method B (using metallic sodium in liquid ammonia) -- To a mixture of 3 (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 pfIR-12p (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-ylthiocarbonate (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 THF 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 pressure. The residue was triturated with hexane and crystallized from n-hexaneether to afford 9.2 g. (83%) of S as colorless prisms. M.p. 96--98"C. ir: # maxKBr 1730, 1630, 1430, 1839, 1250 cm-1. nmr: O CDCl3 1.49 (9H, s, t-butyl), 2.78 (3H, s, N-CH3), 3.72 (2H, s, CH2CO), 4.25 (2H, s, CH2N), 7.28 (4H, s, phenyl), 9.83 (IH, s, -COOH).

Anal. Calc'd. for C15H2tNO4: 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 a-lactam Glacial acetic acid (140 ml.) was added dropwise with stirring to a mixture of 2ethoxycarbonylmethylthiophene-3-carboxaldehyde oxide (1) (41 g., 0.19 mole) and zinc dust (65.4., 1 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 Na2CO, and extracted with chloroform (3 x 100 ml.). The combined chloroform extracts were washed with water (10 ml.), dried over MgSO,, and evaporated under reduced pressure. The residual oil (30 g.) was triturated with hot benzene (150 ml.).

The colourless needles were collected by filtration and recrystllized from ethyl acetate to give the lactam 2 (7.7 g., 26%), melting at 195-196 C.

MeOH UV: A max 232 nm (E, 6500).

Anal. Calc'd. 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.

3-N-Methylaminomethyl-2-thienylacetic Acid a-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 MgSO . 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. 9l000C. ir: v maxnujol 1620 cm-1. nmr: # maxCHCl3 3.15 (3H, s, N--CH,), 3.72 (2H, t, J=3 Hz, CH2CO), 4.53 (2H, t, J=3 Hz, -C112-N), 6.87 (1H, d, J=4.5 Hz, thiophene-H ), 7.30 (1H, d, 5=4.5 Hz, thiophene-Ha). uv: A maxMeOH 232 nm (E, 6700) Anal. Calc'd. for C8H9NOS: C, 57.46; H, 5.42; N, 8.38; S, 19.17. Found: C, 57.56; H, 5.26; N, 8.31; S, 1913.

3-(N-Methylaminomethyl)-2-thienylacetic Acid (4) A mixture of the lactam 3 (3.5 g., 21 m. moles) and 6N HCl (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 NH4OH (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. 181-182 C.

IR: # maxKBr 1570, 1360 cm-1. nmr: a D2O 2.21 (3H, s, N--CH,), 3.80 (2H, s, CH2CO), 4.20 (2H, s, ppm CH2-N), 7.19 (1H, d, J=6 Hz, thiphene-H ), 7.46 (1H, d, J=6 Hz, thiophene-Hα).

A A H2O 237 nm (E, 7600). max Anal. Calc'd. for C8H11NO2S: C, 51.87; H, 5.99; H, 7.56; S, 17.31. Found: C, 51.67; H, 6.50; N, 7.28; S, 16.69.

3XN-t-butoxycarbonyl-N-methylaminomethyl)-2-thienylacetic 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 OOC. 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 HCI 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: v nujol 1730, 1640 cm-1. max nmr: 8 pCpDmcl3 1.47 (9H, s, BOC-H), 2.78 (3H, s, NCH3), 3.87 (2H, s, C112-CO), 4.48 (2H, s, C112-N), 6.91 (1H, d, J=6 Hz, thiophene-Hss), 7.20(1 H, d, J=6 Hz, thiophene-Ha), 10.63 (1H, s, CO2H, disappeared by addition of D2O).

Anal. Calc'd. for C,3H,9NO4S: C, 54.72; H, 6.71; methylaminomethyl)phenylacetic acid #-lactam, boiling at 130-135 C/2 mm Hg., m.p. 35-37 C. ir: v KBr 3300, 1620, 1490 cm-1 max nmr:d O CDCl3 3.12 (311, s), 3.59 (2H, t, J=1.5 Hz), 4.48 (2H, t, J=1.5 Hz), 7.21 (4H, br-s).

Anal. Calc'd. for C1oH"NO-I/4H2O: 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 #-lactam (5.0 g., 0.031 mol) and conc hydrochloric acid (500 ml.) was refluwed for 40 hours. The mixture was evaporated under reduced pressure, 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. Calc'd. for C10H12NO2.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/2 mm Hg).

An aqueous solution of o.N-methylaminomethylphenylacetic acid hydrochloride (5 g.) was column chromatographed with 1R-120 ion-exchange resin (H+, 70 ml.) and eluted with 3N NH4OH (21) to afford 3.9 g. (93%) of o-N-methylaminomethylphenylacetic acid as needles. ir: v max KBr 1650, 1470 cm-1-. 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

Methyl isobutyl ketone is represented as MIBK. "SHELLYSOLVE B" is a petroleum ether fraction of B.P. 60-68 C. consisting essentially of n-hexane.

LA-1 resin is a mixture of secondary amines wherein each secondary amine has the formula

wherein each of R', R2 and R3 is a monovalent aliphatic hydrocarbon radical and wherein R', R2 and Ra contain in the aggregate from eleven to fourteen carbon atoms. This particular mixture of secondary amines, which is sometimes referred to in these examples as "Liquid Amine Mixture No. II," is a clear amber liquid having the following physical characteristics: viscosity at 25"C. of 70 cpd., specific gravity at 20 C. of 0.826; refractive index at 25 C. of 1.4554; distillation range at 10 mm., up to 170 C -0.5%, 170-220 C. -3%, 22-230 C. -90% and above 230 C.

-6.5%.

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 abbreviated as DCC, tetrahydrofuran as THF, 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 a and tau as T (a = 10 - t). The word "Nujol" is a registered Trade Mark.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Synthesis (Schemes 1, 2 and 3) The 3-side chain thiol, 2.carboxymethyl-2,3-dihydro-s-triazolo[4,3- b]pyridazin-3-on-6-ylthiol (3), was prepared by N-ethoxycarbonylmethylation of 6 chloro-2,3-dihydro-s-triazolo[4,3-bipyridazin-3-one (I) with sodium hydride and ethyl chloroacetate in DMF (dimethylformamide) and subsequent thiolation with sodium hydrosulfide (Scheme 1). Condensation of 7-ACA (7-amino-cephalosporanic acid) with 3 carried out by refluxing in phosphate buffer (pH 7) to give the 3-substituted-thio 7-ACA (4), which was coupled with an appropriate N-BOCprotected amino acid by the active ester method using 2,4-dinitrophenol (DNP).

The resulting N-BOC-protected cephalosporins 7 and 11 were deblocked with TFA (trifluoroacetic acid) and converted to the monosodium salt with N sodium hydroxide (Schemes 2 and 3).

Scheme 1.

Preparation of 7-Amino-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4.3-bipyridazin-3 on-6-ylthiomethyl)-2-cephem-4-carboxylic Acid.

Schem 2.

Preparation of 7-[(o-Aminomethylphenyl)acetamido-3-(2-carboxymethyl-2,3-dihydro-s triazolo[4,3-b1pyridazin-3-on-6-ylthiomethylJ-3-cephem4 Acids.

a: R'=H, R2=H 8a: BB-S469 b: R'=CH3, R2=H 8b: BB-S479 c: R=CH3, R2=OH 8c: BB-S478 Scheme 3.

Preparation of 7-(3-Aminomethyl-2-thienylacetamido)-3-(2-carboxymethyl-2.3-dihydro s-triazolo[4.3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic Acids

a: R=H 12a: BB-5483 b: R=CH3 12b: BB-5472 6-Chloro-2,3-dihydro-2-ethoxycarbonylmethyl-s-triazolo[4.3-b]pyridazin (2) To a solution of 6-chloro-2,3-dihydro-2-triazolo[4.3-b]pyridazin-3-one [P.

Francavilla and F. Lauria, J. Het. Chem., 8,415 (1971)] (1, 1.00 g., 5.9 m.mole) in dry DMF (30 ml.) was added sodium hydride (50% in paraffin, 0.3 g., 6.3 m.mole) under stirring with formation of yellow crystals. To the mixture was aded ethyl chloracetate (1.4 ml., 13 m.mole) and the mixture was heated at 90 C. for 8 hours with stirring. After cooling, the reaction mixture was poured into water (50 ml.) and extracted with toluene (5 x 40 ml.). The organic extracts were combined, dried over anhydrous sodium sulfate and evaporated at reduced pressure. The residue was crystallized with benzene-n-hexane to give yellow needles of 2 (1.16 g., 77%), m.p. 114-115 C. (lit. 110 C.) KBr ir: # maxKBr 1735, 1710 cm-1. uv: A EtOH 231 nm (e, 26000) max nmr: 5 CDCl3 7.58 (IH, d, J=10 Hz, pyridazine-H), 6.98 (1H; d, J=10 Hz, pyridazine-H), 4.80 (2H, s, -CH2CO), 4.27 (2H, q, J=7.5 Hz, CH2CH3), 1.29 (3H, t, J=7.5 Hz, CH2CH3).

Anal. Cal'd. for C8H9N4O3Cl: C, 42.12; H, 3.53; N,21.83;Cl, 13.81. Found: C, 41.54; 41.46; H, 3.22, 3.49; N, 21.51, 21.53; Cl, 13.88, 13.99.

2-Carboxymethyl-2.3-dihydro-6-mercapto-s-triazolo[4.3-b]-pyridazin-3-one (3) To a solution of 6-chloro-2,3-dihydro-2-ethoxy-carbonylmethyl-s-triazolo[4,3b]pyridazin-3-one (2, 30 g., 0.12 mole) in ethanol (900 ml.) was added NaSH.2H2O (70% pure, 45.9 g., 0.36 mole) and the mixture was refluxed for 0.5 hour. The reaction mixture was evaporated at reduced pressure. The residue was dissolved in water (200 ml.) and concentrated HCI was added to the solution to adjust to pH 2.

The precipitate (3) was collected by filtration and washed with water. Yield 18.3 g.

(69%). ir: v KBr 2900, 2450, 1750, 1660 cm-'. max uv: # max1%NaHCO3aq. 260 nm (#, 19500), 313 nm (#, 7000) nmr: # DMSO-d6ppm .88 (1H, d, J=10 Hz, pyridazine-H), 7.45 (1H, d, J=10 Hz, pyridazine-H=, 4.72 (211, s, CH2CO).

Anal. Calc'd. for C7HeN,O,S: C, 37.17; H, 2.67; N, 24.77; S, 14.17. Found: C, 37.35, 37.23; H, 2.26, 2.28; N, 23.58, 23.69; S, 14.32.

7 - Amino -3 - (2 - carboxymethyl - 2,3 - dihydro - s - triazolo[4.3-b]pyridazin -3 - on - 6- ylthiomethyl)-3-cephem-4-carboxylic Acid (4) To a suspension of 7-aminocephalosporanic acid (8.79 g., 32.2 m.mole) in 0.1 M phosphate buffer (pH 7, 149 ml.) were added NAHCO3(8.14 g., 97.0 m. mole) and the thiol 3 (7.30 g., 32.3 m.mole) with stirring. The mixture was heated at 80 C. for 0.5 hour under N2 stream. The mixture was treated with active carbon and adjusted to pH 3 with concentrated HCI. The resulting precipitate was collected by filtration and washed with water to give 7.59 g. (54%) of 4. ir: v KBr 1800, 1720, 1600, 1540, 1470 cm1. max uv: A Buffer (pH 7) 252 nm (e, 19500), 298 nm (#, 8400). nmr:# ppmD2O+K2CO3 7.56 (1H, d, J=9 Hz, pyridazine-H), 7.05 (1H, d, J=9 Hz, pyridazine-H=, 5.45 (1H, d, J=5 Hz, 6-H), 5.05 (1H, d, 5 Hz, 7-H), 4.43 (1H, d, J=14 Hz, 3-CH2), 4.04 (1H, d, J=14 Hz, 3-CH2), 3.88 (1H, d, J=18 Hz, 2-H), 3.45 (IH, d, J=18 Hz, 2-H).

EXAMPLE 1.

Preparation of BB-S469

8a, BB-S469 2,4-Dinitrophenyl o-(N-Butoxycarbonylaminomethyl)phenylacetate (6a) To a mixture of o-(N-butoxycarbonylaminomethyl)phenylacetic acid (5a, 13 g., 49 m.mole) and 2,4-dinitrophenol (9.02 g., 49 m.mole) in dry ethyl acetate (123 ml.) was added dicyclohexylcarbodiimide (DCC) (10.1 g., 49 m.mole) under water cooling (5-15 C.), and the mixture was stirred for 30 minutes at the same temperature and then for 40 minutes at room temperature. The resulting precipitate was filtered off and the filtrate was evaporated to give 23.9 g. of the active ester 6a, which was used in the next acylation reaction without further purification. ir: # maxKBr 1775, 1700, 1600, 1530 cm-1.

7-[o-(N-Butoxycarbonylaminomethyl)phenylacetamido]-3-(2-carboxymethyl-2,3dihydro-s-triazolo[4.3-b]pyridazin-3-on-6-ylthiomethy)-3-cephem-4-carboxylic Acid (7a) To a cold (0 C.) mixture of 4 (4.38 g., 10 m.mole), Et3N (4.5 ml., 30 m.mole).

CH3CN (20 ml.) and water (20 ml.) was added a solution o?2,4-dinitrophenyl o-(Nbutoxycarbonylaminomethyl)phenylacetate (6a, 4.79 g.) in THF (tetrahydrofuran) (20 ml.). After stirring at room temperature overnight, THF and CH3CN in the reaction mixture were removed at reduced pressure and the resulting aqueous solution was adjusted to pH 2 with dilute HCI and extracted with ethyl acetate (10 x 30 ml.). The organic extracts were dried over sodium sulfate and evaporated.

The residue was chromatographed on a column of silica gel (60 g.) and eluted with CHCl3 and 3% MeOH-CHCl2 successively to give 2.40 g. (37%) of 7a. mp. > 161 C.

(dec.): ir: #maxKBr 1780, 1720 cm-1. uv: #maxpH 7 Buffer 252 nm (#, 19800), 298 nm (#, 8900). nmr: e7 ppm D2O 7.67 (1H, d, J=9.0 Hz, pyridazine-H), 7.10 (411, s, phenyl-H), 7.05 (1H, d, J=9.0 Hz, pyridazine-H), 5.66 (1H, d, J=4.5 Hz. 7-H). 5.07 (1H, d, J=4.5 Hz, 6-H), 4.71 (2H, s, N-CH2-CO), 4.4-4.0 (4H, m, 3-CH2 & CH2-N), 3.8-3.5 (411, m, 2-H & CH2CO), 1.42 (911, s, t-Butyl-H).

Anal. Calc'd. for C28H31N7O7S2.2H2O: C, 49.62; H, 5.20; N, 14.46; S, 9.46.

Found: C, 49.97, 49.95; H, 4.79, 4.62; N, 14.00, 13.84; S, 9.37, 9.32.

BB-S469; 7-[(o-(Aminomethylphenyl)acetamido]-3-(2-carboxymethyl-2,3-dihydro-2triazolo[4.3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic Acid (8a) Trifluoroacetic acid (3.4 ml.) was added to 7a (2.33 g.) at 0 C and the mixture stirred for 15 minutes at room temperature. To the mixture was added dry ether (100 ml.), and the precipitate was collected by filtration and washed with dry ether (2 x 50 ml.). The solid was dissolved in a mixture of CH3CN (100 ml.) and water (14 ml.) and the solution was adjusted to pH 4-5 with concentrated NH4OH to afford a precipitate which was collected by filtration and washed with CH3CN (2 x 50 ml.) to give 1.75 g. (82%) of 8a as the ammonium salt. M.p. > 1600C. (dec.). ir. #maxKBr 1765, 1710, 1640, 1590 cm-1. uv: #maxpH 7 Buffer 252 nm (#, 23900), 298 nm (#, 10900).

Anal. Calc'd. for C24H22N7O7S2.NH+4.H2O: C, 46.51; H, 3.96; N, 18.08; S, 10.35.

Found: C, 46.52, 46.26; H, 4.15, 4.19; N, 17.60, 17.48; S, 10.89, 10.41.

Preparation of BB-S469 Monosodium Salt To a suspension of 8a (1.58 g.) in 50% acetone-water (30 ml.) was added a 10% solution to adjust the pH to 7.7. An additional amount of acetone was added and the precipitate was collected by filtration and washed with acetone to give 1.38 g.

(87%) of monosodium salt of 8a, m.p. > 170 C. (dec.) ir: #maxKBr 1765, 1710, 1640, 1600, 1540, 1485 cm-1. uv: #maxpH 7 Buffer 252 nm (#, 20500), 298 nm (#, 9200). nmr: #ppmD2O+K2CO3 7.52 (1H, d, J=9 Hz, pyridazine-H), 7.27 (4H, s, phenyl H), 7.03 (1H, d, J=9 Hz, pyridazine-H), 5.62(111, d, J=4.5 Hz, 7-H), 5.07 (111, d, J=4.5 Hz, 6-11).

Anal. Calc'd. for C24H22N7O7S2Na.2H2O: C, 44.79; H, 4.07; N, 15.23; S, 9.96.

Found: C, 44.44, 44.95; H, 3.68, 3.90; N, 16.50, 16.67; S, 10.38; 10.45.

EXAMPLE 2.

Preparation of BB-S472

12b, BB-S472 7 - (3 - N - t - Butoxycarbonyl - N - methylaminomethyl - 2 - thienylacetamido) - 3 - (2carboxymethyl-2.3-dihydro-2-triazolo[4.3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4carboxylic Acid (11b) A mixture of the BOC-protected amino acid (lob, 513 mg., 1.8 m.mole), 2.4 dinitrophenol (400 mg., 2.16 m.mole) and DCC (445 mg., 2.16 m. mole) in THF (5 ml.) was stirred at room temperature for 12 hours. The precipitated urea was removed and the filtrate was added to a mixture of 7-amino-3-(2-carboxymethyl2,3 - dihydro - s - triazolo[4,3-bl - pyridazin - 3 - on - 6 - ylthiomethyl) - 3 - cephem -4- carboxylic acid (4, 800 mg., 1.8 m.mole) and triethylamine (0.76 ml., 5.4 m.mole) in water (5 ml.) at 0 C. with stirring. Stirring was continued until active ester had disappeared on tic (silica gel plate; Rf 0.95; solvent system, CHCI3:MeOH=3:1).

The reaction mixture was diluted with water (20 ml.), layered with AcOEt (50 ml.) and adjusted to pH 2 with concentrated HCI at 50C. The organic layer was separated and aqueous layer was extracted with AcOEt (3 x 50 ml.). The AcOEt extracts were combined, washed with sat. aq. NaCI, dried over MgSO, and concentrated under reduced pressure. The residual oil (1.9 g.) was chromatographed on silica gel (40 g.). The column was eluted successively with CHCI, (400 ml.), 3% MeOH-CHCIJ (100 ml.) and 10% MeOH-CHCI3 while monitoring with tic (silica gel plate, solvent system MeOH-CHCI3=1:2, detected with I2). From the CHCl2 eluate was recovered a mixture of 2,4-DNP and the BOCprotected amino acid 9 and from 3% MeOH-CHCl3 eluate 50 mg. of 9. The desired product (11b) (Rf 0.4, solvent system CHCl3:MeOH=3:1) was obtained by evaporation of the eluate with 10% MeOH-CHCl3. Yield 490 mg. (39%). M.p.

21 5-2200C.

KBr ir: v max 3400, 1780, 1720, 1680, 1550 cm-1. uv: # maxpH7 245 nm (, 23000), 260 nm (#, 18000), 300 nm (s, 7900). nmr: # ppmDMSO-d6.42 (911, s, BOC-H), 2.75 (3H, s, N-CH3), 3.80(411, br-s,

& 3-CH2), 4.72(2H,s, BOC-N-Cfl2), 5.10(1H, d, J=4.5 Hz, 6-H), 5.70(111, dd, J=4.5 & 10.5 Hz, changed to a doublet J=4.5 by addition of D2O, 7-H), 6.85 (1H, d, J=4.5 Hz, thiophene H ), 7.19 (1H, d, J=9 Hz, pyridazine H), 7.34 (1H, d, J=4.5 thiphene Hα), 7.72 (1H, d, J=9 Hz, pyridazine-H), 9.11 (1H, d, J=10.5 Hz, disappeared by addition of D2O, NH).

Anal. Calc'd. for C26H21N7O8S2.H2O: C, 46.46; H, 4.60; N, 13.55; S, 13.29.

Found: C, 46.67; H, 4.71; N, 12.79; S, 12.81.

BB-S472; 7-(3-Methylaminomethyl-2-thienylacetamido)-3-(2-carboxymethyl-2.3dihydro-s-triazolo[4.3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic Acid (12b) Trifluoroacetic acid (0.4 ml.) was added to 11b (400 mg., 0.57 m.mole) at OOC. and the mixture was stirred at room temperature for 15 minutes. To the reaction mixture was added anhydrous ether (10 ml.) to separate a precipitate which was collected by filtration, washed with anhydrous ether (2 x 10 ml.) and suspended in acetonitrile (10 ml.). The suspension was adjusted to pH 4 with concentrated NH4OH and stirred for 10 minutes. The solid was collected by filtration, washed with acetonitrile (2 x 5 ml.) and dried at 60 C./1 mmHg for 7 hours to afford 310 mg. (90%) of 12b melting at 188-191 C. (dec.). ir: vmaxKBr 3400, 1770, 1720, 1680, 1550 cm-1. uv: #maxpH 7 Buffer 245 nm (#, 22400), 260 nm (#, 18700), 300 nm (#, 8600).

Anal. Calc'd. for C23H23N7O7S2.3H2O: C, 41.87; H, 4.43; N, 14.86; S, 14.58.

Found: C, 42.03; H, 3.59; N, 14.79; S, 14.35.

Preparation of BB-5472 Monosodium Salt To a suspension of 12b (230 mg., 0.38 m.mole) in 0.5 ml. of deionized water was added NNaOH to adjust to pH 8.9. Acetone (15 ml.) was added to the solution. The precipitate was collected by filtration, washed with acetone (2 x 5 ml), and dried at 60 C/1 mmHg for 7 hours to afford 170 mg. (71%) of monosodium salt of BB-S472. m.p. > 2100C(dec.). ir: v maBxr 3400, 1765, 1710, 1680, 1600 cm~'. uv: A pH 7 Buffer 245 nm (E, 21800), 260 nm (E, 18500), 300 nm (, 7800). max nmr: # ppm D2O 2.72 (3H, s, N-CH3), 3.45 (1H, d, J=18 Hz, 2-H), 3.75 (1H, d, J=18 hz, 2-H),

& 3-CH2), 4.57 (211, s, N-CH2), 5.00(111, d, J=4.5 Hz, 6-H), 5.53 (1H, d, J=4.5 Hz, 7-H), 6.97 (IH, d, J=9 Hz, pyridazine-H), 7.03 (1H, d, J=4.5 Hz, thiphene-H ), 7.34 (1H, d, J=4.5 Hz, thiophene-Ha), 7.48 (1H, d, J=9 Hz, pyridazine-H).

Anal. Calc'd. for C23H22N7O7S3Na.1/2H2O: C, C, 43.40; H, 3.64; N, 15.40; S, 15.11. Found: C, 43.26; H, 4.08; N, 14.18; S, 13.91.

EXAMPLE 3.

Preparation of BB-S478

8c, BB-S478 7-[2-(N-t-Butoxycarbonyl-N-methylaminomethyl)-4-hydroxyphenylacetamido]-3-(2carboxymethyl-2,3-dihydro-s-triazolo[4.5-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4carboxylic Acid (7c) A mixture of 2-N-t-butoxycarbonyl-N-methylaminomethyl-4-hydroxyphenyl- acetic acid (Sc), (708 mg., 2.4 m.mole), 2,4-dinitrophenol (478 mg., 2.6 m.mole) and DCC (536 mg., 2.6 m.mole) in dry THF (20 ml.) was stirred at room temperature for 2 hours. The precipitated urea was removed by filtration. The filtrate was added to a solution of 7-amino-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,5-b]pyridazin-3- on-6-ylthiomethyl)-3-cephem-4-carboxylic acid (4) (876 mg., 2 m.mole) in 20 ml. of water containing triethylamine (0.84 ml., 6 m.mole) and the mixture was stirred at room temperature for 18 hours. After concentrating to 20 ml. the aqueous solution was washed with ether, acidified with 6N HCI and extracted with 200 ml. of ethyl acetate. The extract was filtered to remove insolubles, washed with water and a saturated aqueous NaCI solution and dried. The solution was evaporated to dryness and the oily residue was chromatographed on a silica gel (Wakogel C-200, 25 g.) eluting with chloroform and 3% of chloroform-methanol. The fractions containing the desired product (monitored by talc; Rf 0.3; solvent system, CHCI3:MeOH=2:1) were collected and evaporated to dryness. The oily residue was triturated with ether-n-hexane to give 630 mg. (44%) of the product 7c melting at 202100C.

(slow dec.). ir: # maBxr 1780, 1720, 1660, 1400, 1240, 1150 cm-1. pH 7 Buffer uv: A max 252 nm (#, 13000), 300 nm (s, 5400). nmr; # ppmDMSO-d81.39 (9H, s, C-CH3), 2.73 (3H, s, N-CH3), 3.3-3.9 (4H, m, CH2CO & 2-11), 4.35 (4H, m, CH2N & 3-H), 4.48 (2H, s, NCH2CO), 5.03(111, d, 4.5 Hz, 6-11), 5.61 (1H, d-d, 8 & 4.5 Hz, 7-H), 6.4-7.2 (3H, m, phenyl-H), 6.98(111, d, 10 Hz, pyridazine-H), 7.61 (1H, d, 10 Hz, pyridazine-H), 8.87 (111, d, 8 Hz, NH).

Anal. Calc'd. for C30H33N7O10S2: C, 50.34; H, 4.65; N, 13.70; S, 8.96. Found: C, 50.98; H, 5.36; N, 11.88; S, 7.60.

BB-S478; 7-(2-N-Methylaminomeihyl-4-hydroxyphenylacetamido )-3-(2-N- carboxymethyl-2,3-dihydro-2-triazolo[4.5-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4carboxylic Acid (8c) A mixture of 7c (570 mg., 0.8 m.mole) and trifluoroacetic acid (1.5 ml.) was stirred at 10 C. for 30 minutes and the mixture was diluted with ether (50 ml.) to separate the trifluoroacetate of 8c which was collected by filtration and then dissolved in a mixture of 10 ml. of acetonitrile and 5 ml. of water and then filtered.

The filtrate was adjusted to pH 6 with concentrated ammonium hydroxide and the mixture was diluted with acetonitrile (100 ml.). the rsulting precipitate was collected by filtration, washed acetonitrile and dried in vacuo over P2O5 to give 370 mg. (75%) of 8c, melting at 215-220 C. (dec.) ir: # maxKBr 1770, 1710, 1600, 1380, 1350 cm-1. uv: # maxpH 7 Buffer 252 nm (#, 19000), 300 nm (#, 9100).

D2O+K2COs nmr: ppm 2.75 (311, s, N-CH3), 2.9-3.3 (4H, m, CH2CO & 2- H), 4.0-4.3 (4H, m, CH2N & 3-H), 4.57 (211, s, NCH2CO), 4.81 (1H, d, 4.5 Hz, 6 H), 5.53 (1H, d, 4.5 Hz, 7-11), 6.6-7.5 (5H, m, phenyl-H & pyridazin-H).

Anal. Calc'd for C25H25N7o8S2.5/2H2O: C, 45.45; H, 4.58; N, 14.84; S, 9.71.

Found: C, 45.69; H, 4.21; N, 15.03; S, 9.46: Preparation of Monosodium Salt of BB-S478 To a suspension of 8c (308 mg., 0.5 m.mole) in water (2 ml.) was added 0.3-0.4 ml. of N NaH and the mixture was stirred at room temperature; the pH of the resulting solution was 9.2. Acetone (20 ml.) was slowly added to the solution. The resulting precipitate was collected by filtration, washed with acetone (10 ml.) and dried in vacuo over P2O8 to give 290 mg. (91%) of the monisodium salt of BB-S478, melting at 230-235 C (dec.). ir: v maxr 1770, 1700, 1600, 1390, 1350 cm-1. pH 7 Buffer uv: A max 250 nm (#, 18000), 300 nm (#, 8400).

Anal. Calc'd. for C25H24N7O8S2Na.5/2H2O: C, 43.98; H, 4.28; N, 14.36; S, 9.39.

Found: C, 43.96; H, 4.14; N, 13.51; S, 9.34.

EXAMPLE 4.

Preparation of BB-S479

8b, BB-S479 7-[o -(N-Butoxycarbonyl- N-methylaminomethyl)phenylacetamidoj-3 -(2-carboxymethyl2.3-dihydro-s-triazolo[4.3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic Acid (7b) To a cold (0 C.) mixture of 4 (5.4 g., 12 m.mole), Et3N (5.5 ml.), CH3CN (25 ml.) and water (25 ml.) was added a solution of 2,4-dinitrophenyl o-(Nbutoxycarbonyl-N-methylaminomethyl)phenylacetate (6b) in THF [prepared from o-(N-butoxycarbonyl-N-methylaminomethyl)-phenylacetic acid (5b) (3.48 g., 13.5 m.mole), 2,4-dinitrophenol (2.49 g., 13.5 m.mole) and DCC (2.79 g., 13.5 m.mole) in dry THF (37 ml.)]. The mixture was stirred at room temperature overnight. THF and CH3CN were removed from the reaction mixture by evaporation under reduced pressure and the resulting aqueous solution was washed with ether (3 x 30 ml.), adjusted to pH 2-3 with dilute HCl and extracted with ethyl acetate (4 x 30 ml.). The organic extracts were combined, dried over sodium sulfate and evaporated. The residue was chromatographed on a column of SiO2 (100g.). After washing with CHCl3, the column was eluted with 3% MeOH in CHCl3 to afford a desired fraction containing 7b. Yield 3.8 g. (45%). m.p. > 200 C. (dec.).

KBr ir: v max 1780, 1720, 1680 cm-1. uv: A PmHa 7 Buffer 250 nm (E, 18800), 297 nm #, 8400). nmr a pDpMSO+D2O 7.62 (1H, d, J=10.5 Hz, pyridazine-H), 7,14 (4H, s, phenyl-H), 6.98 (1H, d, J=10.5 Hz, pyridazine-H), 5.61 (1H, d, J=4.5 Hz, 7-H), 5.03 (1H, d, J=4.5 Hz, 6-H), 4.67 (2H, s, N-CH2), 4.42 (2H, s, CH2-N), 4.4-4.0 (2H, m, 3-CH2), 3.8-3.4 (4H, m, 2-H & CH2-CO), 2.72 (3H, s, N-CH3), 1.38 )9H, s, BOC-H).

Anal. Calc'd. for C30H33N7O9S2.5/2H2O: C, 48.38; H, 5.14; N, 13.16; S, 8.61.

*Found: C, 48.2, 48.23; H, 4.52, 4.46; N, 12.93, 12.86; 8.68.

BB-S479; 7-(o-(Methylaminomethyl)phenylacetamido)-3-(2-carboxymethyl-2.3-dihydros-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic Acid (8b) Trifluoroacetic acid (7 ml.) was added to the t-BOC-derivative 7b (3.8 g., 5.5 m.mole) at 0 C., and the mixture was stirred for 20 minutes at room temperature.

Dry ether (100 ml.) was added to the mixture. The resulting precipitate was collected by filtration and washed with dry ether (3 x 100 ml.). The precipitate was dissolved in a mixture of CH3CN (120 ml.) and water (18 ml.) and the solution was adjusted to pH 5-6 with concentrated NH4OH to give an oily precipitate which with triturated with CH3CN to form solid material. The product 8b was collected by filtration, washed with CH3CN and dried. Yield 2.55 g. (77%). ir: v max 1770, 1710, 1600, 1550 cm~'.

Preparation of Monosodium Salt of BB-S479 To a solution of BB-S479 (8b) (2.54 g., 4.3 m.mole) in water (25 ml.), N NaOH (ca. 3 ml.) was added under cooling (the pH of the solution was 10). A large amount of acetone was added to the solution and the precipitate was collected by filtration and washed with acetone to give 1.94 g. (84/o) of monosodium salt of BB-S479.

M.p. > 200 C (dec.). ir: # maxKBr 1770, 1710, 1600, 1550 cm-1. uv: A pH 7 Buffer 250 nm (F, 19400), 297 nm (f, 8700). max Anal. Calc'd. for C25H24N7O7S2Na.1/2H2O; 47.61; H, 4.00; N, 15.55; S, 10.17. Found: C, 47.43, 47.43; H, 4.67, 4.68; N, 15.97; 15.70; S, 9.25, 9.84.

EXAMPLE 5.

Preparation of BB-S483

12a, BB-S483 7-(3-N-t-Butoxycarbonylaminomethyl-2-thienylacetamido]-3-(2-carboxymethyl-2.3dihydro-s-triazolo[4.3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem.4.carboxylic Acid (11A) A mixture of the BOC-protected amino acid (9, 410 mg., 1.56 m.mole), 2,4dinitrophenol (313 mg., 1.7 m.mole) and DCC (353 mg., 1.7 m.mole) in THF (5 ml.) was stirred at room temperature for 12 hours. The precipitated urea was removed and the filtrate was added to a mixture of 7-amino-3-(2-carboxymethyl-2,3-dihydros-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid (4, 683 mg., 1.56 m.mole) and triethylamine (0.62 ml., 4.68 m,mole) in water (5 ml.) ar 0 C. with stirring. Stirring was continued at room temperature un and adjusted to pH 2 with concentrated HCl at 5 C. The organic layer was separated and the aqueous layer was extracted with AcOEt (3 x 50 ml.) The AcOEt (ethyl acetate) extracts were combined, washed with saturated aqueous NaCl, dried over MgSO4 and concentrated under reduced pressure. The residual oil (1.8 g.) was chromatographed on silica gel (40 g.). The column was eluted successively with CHCl3 (400 ml.) and 3% MeOH-CHcl3 (500 ml.). The eluate was monitored with tlc (silica gel plate, solvent system CHCl3:MeOH)=2;1, detected with I2). The desired product IIa (Rf 0.2) was obtained by evaporation of the MeOH-CHCl, eluate.

Yield 450 mg. (42%, melting at 155-160 C. ir: # maxKBr 3300, 1775, 1720, 1680 c,-1. uv: A pH 7 Buffer 245 nm (E, 23900), 260 nm (E, 19200), 300 nm (E, 8700). max # ppmDMSO-d61.39 (9H, s, BOC-H), 3.76 (4H, br-s, 2-11 & 4.05 (2H, d, J=6 Hz, changed to a singlet by addition of D2O, BOCNH-CH2), 4.20 (2H, m 3-CH2), 4.69 (2H, s, N-CH2CO2), 5.06 (1H, d, J=4.5 Hz, 6-H). 5.62 (1H, d-d, J=4.5 & 9Hz, changed to a doublet J=4.5 Hz by addition of D2O, 7-H), 6.83 (1H, d, J=4.5 Hz, thiphene-Hss), 7.00 )1H, m, disappeared by addition of D2O, NHBOC), 7.04 (1H, d, J=9 Hz, pyridazine-H), 7.12 (1H, d, J=4.5 Hz, thiophene Hα), 7.65 (1H, d, J=9 Hz, pyraidazine-H), 8.97 (1H, d, J=9 Hz, disappeared by addition of D2O, 7-NH).

Anal. Calc'd. for C27H29N7O9S3: C, 46.88; H, 4.23; N, 14.17;S, 13.90. Found: C, 46.42; H, 4.37; N, 13.49, S, 13.61.

BB-S 483; 7-(3-Aminomethyl-2-thienylacetamido)-3-(2-carboxymethyl-2,3-dihydro-striazolo[4.3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic Acid (12a) Trifluoroacetic acid (0.4 ml.) was addec to 11a(410 mg., 0.59 m.mole) at 0 C. and the mixture was stirred at room temperature for 15 minutes. To the reaction mixture was added anhydrous ether (10 ml.) to separate a precipitate which was collected by filtration, washed with anhydrous ether (2 x 10 ml.) and suspended in acetonitrile (10 ml.). The suspension was adjusted to pH 4 qith concentrated NH4OH and stirred for 10 minutes. The precipitate was collected by filtration, washed with acetonitrile (2 x 5 ml.) and dried at 60 C./1 mmHg for 7 hours to afford 310 mg. (88%) of 12a, melting at above 200 C. (slow dec.).

KBr ir: # max 3400, 3150, 1760, 1700, 1680, 1600 cm uv: # maxpH / Buffer 245 nm (#, 17100), 260 nm (#, 14100), 300 nm (#, 6500).

Anal. Clac'd. for C22H21N7O7S2.3H2O: C, 40.90; H, 4.21; N, 15.17; S, 14.89.

Found: C, 40.39, H, 3.62; N, 15.87; S, 14.35.

Preparation of Monosodium Salt of BB-5483 To a suspension of 12a (280 mg., 0.47 m.mole) in 0.5 ml. of deionized water was added N NaOH to adjust to pH 9.5 and insoluble material was collected by filtration. Acetone (15 ml.) was added to the filtrate to separate the precipitate which was collected by filtration, washed with acetone (2 x 5 ml.) and dried at 70 C./1 mmHg for 7 hours to afford 220 mg. (76%) of monosodium salt of 12a. M.p.

> 210 C. (slow dec.) KBr ir: v max 3400, 3250, 1760, 1710, 1650, 1600, 1550 cm-1. uv: A PmHax7 Buffer 245 nm (E, 19900), 260 nm (E, 16400), 300 nm (E, 6900). nmr: # ppmD2O 3.60 (2H, m, 2-H), 3,91 (2H, s, CH2CO), 4.12 (2H, s, CH2-NH2), 4.20 (2H, m, 3-CH2), 4.55 (2H, s, N-CH2CO), 4.95 (1H, d, J=4.5 Hz, 6-H), 5.50 (1H, d, J=4.5 Hz 7-H), 6.94 (1H, d, J=9 Hz, pyridazine-H), 6.99 (1H, d, J=4.5 Hz, thiophene-Hss), 7.28 (1H, d, J=4.5 Hz, thiophene-Hα), (1H, d, J=9 Hz, pyridazine-H).

Anal. Calc'd. for C22H20N,O,S,Na-l/2CH,COCH,: C, 43.92; H, 3.6l;N, 15.26; S, 14.97. Found: C, 43.48; H, 4.56; N, 15.28; S, 13.91.

Solubility All of the monosodium salts in this series showed more than 10% solubility in water.

Nephrotoxicity A preliminary nephrotoxicity study was carried out by administration of the test compound to a group of two rabbits at 100 mg./kg. intravenously. The result obtained with BB-S469 and BB-S479 indicated that they might have little nephrotoxic potential.

In Vitro Adiwty (Table 1) The MIC's were determined by the serial dilution method using Mueiler Hinton agar against 51 gram-positive and 96 gram-negative bacteria. The 147 test organisms were classified into 16 groups according to the genera and the types of antibiotic resistance, 5 groups for gram-positive and 11 for gram-negative bacteria.

In Table I is shown the in vitro activity in terms of geometric mean of MIC's. BB S472 and BB-S479 showed better overall activity than their non-N-methylated analogs, BB-S483 and BB-S469, respectively. BB-S479 was superior to BB-S472 in some species of gram negative bacteria. Comparing with cefamandole, BB-S479 was more active against most of the test organisms except against Pro viden cia species and Staphylococci.

TABLE 1 In vitro Activity Against 147 Test Organisms Geometric Mean MI#* (mcg./ml.) No. of BB-S469 BB-S472 BB-S478 BB-S479 BB-S483 Cefam Test Organism Strains (Ex. 1) (Ex. 2) (Ex. 3) (Ex. 4) (Ex. 5) andole BL-S786** S. aureus (sensitive) 4 0.4 0.4 0.4 0.3 0.3 0.1 1.3 S. aureus (penicillinase+) 13 1.2 0.9 1.0 0.9 1.1 0.4 3.0 S. aureus (Methicillin-R) 15 30 58 50 35 48 2.4 48 .

S. pyogenes 10 0.05 0.05 0.04 0.04 0.08 0.04 0.2 D. pneumoniae 9 0.03 0.05 0.03 0.04 0.04 0.2 0.08 E. coli (sensitive) 13 0.3 0.2 0.2 0.2 0.3 0.3 0.4 E. coli (Penicillinase +) 7 11 15 10 2.6 21 5.7 9.3 Enterobacter (sensitive) 3 1.0 1.3 0.8 0.5 1.3 1.0 1.3 Enterobacter (Cephalosporinase +) 7 4.7 3.2 2.6 2.1 5.2 4.7 7.7 Proteus (indole -) 6 0.6 0.3 0.4 0.5 0.7 0.6 0.9 Proteus (indole +) 14 0.5 0.3 0.3 0.2 0.4 0.5 0.7 Proteus (indole +), cephalosporinase +) 5 11 8.3 7.3 4.8 17 3.6 29 Providencia sp. 5 4.2 4.8 4.8 4.2 2.1 0.7 1.4 Klebsiella sp. 12 0.9 0.8 0.6 0.6 1.1 2.4 0.8 S. marcescens 16 130 200 40 35 81 34 180 Miscellaneous, (Salmonella, Shigella, Citrobacter) 8 0.7 0.4 0.3 0.4 0.9 0.3 0.7 *Mueller-Hinton Agar (inoculum size: 104 dilution) MIC cut-off: 200 mcg./ml.

**BL-S786 is sodium 7-(2-aminomethylphenylacetamido)-3-(1-carboxymethyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylate EXAMPLE 6.

Substitution in the procedure of Example 3 for the 2-N.t-butoxycarbonyl-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.

EXAMPLE 7.

Preparation of BB-S493

3, BB-5493 7 -1(2 - N - t - Butoxycarbonylaminomethyl -1,4 - cyclohexadienyl)acetamidol - 3 - (2 N- carboxymethyl- 5 - triazolo[4,5 - bipyridazin - 3 - on - 6 - ylthiomethyl) - 3 cephem - 4 - carboxylic Acid (2) A mixture of 2-N-t-butoxycarbonylaminomethyl-1,4-cyclohexadienylacetic acid (l, 640 mg., 2.4 m.mole), 2,4-dinitrophenol (422 mg., 2.4 m.mole) and DCC (494 mg., 2.4 m.mole) in 10 ml. of dry THF was stirred for 1.5 hours at room temperature. The precipitated urea was removed by filtration. The filtrate was added in one portion to a solution of 7-amino-3-(2-N-carboxymethyl-s-triazol[4,5b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid in 10 ml. of water containing triethylamine (0.56 ml., 4 m.mole) and the mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated to 10 ml. under reduced pressure, washed with ether (3 x 10 ml.), acidified with 6N hydrochloric acid and extracted with ethyl acetate (5 x 10 ml.). The combined extract were washed with a saturated saline solution and dried with anhydrous Na2SO4. The solvent was evaporated and the residue was chromatographed on silica gel (Wakogel C-200, 30 g.) eluting with chloroform-methanol (0-50%). The fractions containing the desired product were collected. The solvent was removed and the residue was triturated with ether-n-hexane to give 410 mg. (30%) of the product 2.

M.p. I l00C. (decomp.). ir: v maxKBr 1780, 1730, 1610, 1530, 1250, 1160 cm-1. uv: A maxBuff (pH 7) 252 nm (e, 19000), 300 nm (e, 8600, sh).

Anal. Calc'd. for C29H33N7O9S2.2H2O: C, 48.12; H, 5.15; N, 13.54; S, 8.86.

Found: C, 48.07; H, 4.64; N, 12.70; S, 8.39.

BB-S493: 7-[(2-Aminomethyl-1,4-cyclohexadienyl)acetamido]-3-(2-N-carboxymethyl-striazolo[4,5-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic Acid (3) The N-BOC-protected cephalosporin 2 (350 mg., 0.51 m.mole) was treated with trifluoroacetic acid (TFA) (1 ml.) for 30 minutes at room temperature. To the mixture was added ether (50 ml.) to give the TFA salt of 3, which was collected by filtration and then dissolved in a mixture of acetonitrile (5 ml.) and water (2 ml.).

The solution was treated with a small amount of active carbon, adjusted to pH 6 with concentrated ammonium hydroxide. The precipitate was collected, washed with acetonitrile (5 ml.) and dried in vacuo to afford 235 mg. (78%) of 3. M.p.

220-230 C. (decomp.). ir: v KBr 1770, 1740, 1710, 1650, 1600, 1550 cm-'. max uv: A Buff (pH 7) 252 nm (e, 20000), 300 nm #, 9000, sh).

Anal. Calc'd. for C24H25N7O7S2.H2O: C, 47.60; H, 4.49; N, 16.19; S, 10.59.

Found: C, 47.77; H, 4.06; N, 16.49; S, 10.64.

In vitro Antibacterial Activity of BB-5493 Compared with BB-S479 and Cefamandole (Determined by Steers' Agar Dilution Method on Mueller-Hinton Agar Plate) MIC (mcg. ml.) Organism BB-S493 BB-5479 Cefa mandole S. aureus Smith A9537 0.4 0.4 0.1 S. aureus A9497 0.2 0.1 0.05 S. aureus BX-1633 A9606 0.4 0.4 0.2 St. faecalis A9536 100 100 50 E. coli NIHJ 0.1 0.05 0.025 E. coli ATCC 8739 0.2 0.05 0.05 E. coli Juhl A15119 0.2 0.1 0.4 E. coli BX-1373 ' 0.4 0.2 0.4 E. coli A15810 0.2 0.1 0.2 E. coli A9660 0.1 0.05 0.1 E. coli A15147 6.3 3.1 3.1 Kl. pneumoniae A9678 0.4 0.4 1.6 Kl. pneumoniae A9977 0.2 0.1 0.4 Kl. pneumoniae A15130 0.2 :0.1 0.4 Kl. pneumoniae A9867 0.2 0.1 0.8 Pr. vulgaris A9436 0.4 0.1 0.2 Pr. vulgaris A9699 6.3 0.8 25 Pr. mirabilis A9554 0.2 0.1 0.8 Pr. mirabilisi A9900 0.2 0.1 0.8 Pr. morganii A9553 > 100 ) 100 > 100 Pr. morganii A20031 0.4 0.1 0.8 Pr. rettgeri A15167 0.1 0.1 0.1 Ps. aeruginosa A9930 > 100 > 100 > 100 Ps. aeruginosa A9843 > f.00 \ 100 > 100 Shig. dysenteriae 0.05 0.025 0.2 Shig. flexneri A9684 25 12.5 3.1 Shig. sonnei A9516 0.05 0.025 0.05 Serr, marcescens A20019 100 25 50 Enterob. cloacae A9656 6.3 1.6 3.1 Sal enteritidis A9531 0.1 0,05 0.1 Sal. typhosa A9498 0.1 0.05 0.1 B. anthracis A9504 0.0125 0.025 0.2 In vivo Activity of BB-S479 and Related Compounds (Mice, sc) (PD5 0 (mgm./kg.) Organism BB-S469 BB-S472 BB-S478 BB-S479 BB-S483 BB-S493 Cefamandole BL-S786 S. aureus Smith 0.19 0.12 0.08 0.16 0.34 0.29 0.93 0.53 0.16 0.12 0.74 0.55 0.29 0.2 0.6 0.94 0.2 0.8 0.46 0.8 0.6 E. coli Juhl 0.19 0.19 0.08 0.19 0.15 0.27 0.95 0.43 0.24 0.15 1.7 0.55 0.15 0.12 1.8 0.46 2.2 0.39 0.8

Claims

WHAT WE CLAIM IS:- I. A compound having the formula

wherein R' represents

wherein R is hydrogen, hydroxy or methoxy; R' is hydrogen or methyl; and R' is hydrogen, or a conventional, pharmaceutically acceptable, easily hydrolyzed ester forming group; or a non-toxic, pharmaceutically acceptable salt thereof.
2. The pivaloyloxymethyl-, acetoxymethyl-, methoxymethyl-, acetonyl-, phenacyl-, p-nitrobenzyl- Ass,-trichloroethyl-, 3-phthalidyl- or 5-indanyl- esters of a compound as claimed in Claim I.
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 I or 2 wherein R' 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 I to 8, wherein R' is hydrogen.
10. A compound of any one of Claims I to 8, wherein R' is methyl.
I I. A process for the preparation of a compound of 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 characterized by reacting a compound of the formula

in which Y is H or R', 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 11 wherein a resulting free acid of formula l is converted to an ester selected from the pivaloyloxymethyl-, acetoxymethyl, methoxymethyl-, acetonyl-, phenacyl, p-nitrobenzyl-, ss,ss,ss-trichloroethyl-, 3phthalidyl- 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 II or 12, wherein RX 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 to 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 250-1000 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 combatting B. anthracis infections which comprises administering to a non-human warm-blooded mammal infected with a B. anthracis infection an amount effective for treating said B. anthracis infection of a composition comprising a compound according to any one of Claims I 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 any 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 any of the accompanying Examples.
28. A compound according to Claim 1 when prepared by a process according to any of Claims II to 20 or 27.
29. A pharmaceutical composition comprising a compound according to any of Claims I to 10, 21, 25, 26 or 28 in admixture wit a pharmaceutically acceptable carrier therefor.