IE45613B1 - 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.

Description

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 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 554-569) 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 260-243C) Issued patents on 3“thiolated cephalosporins in which the 7-substituent is a) c.-Amino-o-phenylacetamido include U. S. 3,641,021; U. s. 3,734,907; U. S. 3,687,948; U. s. 5613 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 I9696T; 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 to) ο-, m- or p-aminoethoxyphenylacetamldo 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 7-phenylacetamidocephalosporsnic acids) and d) N-(phenylacetlmidoyl)aminoacetamldo as U.S. 3,692,779; and e) a-amino-a-(l,4-cyclohexadienyl)acetamido as in Belgium 776,222.(Farmdoc 38983T; U.K. 1,328,34ο).

Additional similar disclosures are found in U.S, 3,692,779 (Belgium 771,189; Farmdoc 12819T), Japan 72/05550 (Farmdoc 12$21T), 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). ri ri 613 Issued disclosures of 3“thiolated cephalosporins in which the 7-substituent Is 7-mandelamido (7-ahydroxyphenylacetamido) 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(l). 34-41 (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-l,3,4-thiadiazole-5-acetic acid and its conversion to 7-(lH-tetrazol-l-yla cetaraid0)-3”(5 ~carboxyme thyl-1,3,4-thiadia zo1-2ylthiomethyl)-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 or a aontoxlc, pharmaceutically acceptable salt thereof. .4 ./ -O η Is in bteger from 4-7, inclusive; the pharma· eeutically acceptable addition talta thereof. respectively.

U.S. 3,383,520 and 3,931,160 and Farmdoc Abstract 22850W make reference to 3-heterocyclicthiomathyl 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 disclose cephalosporins containing various fused ring bicyclic thiols.

Farmdoc abstract I883OX discloses compounds of the formula COOH IK {CH2)n-C°0H X 5 (vihere R => acyl or H; Rr = H or methoxy; n = 1-9).

Patent Specification No. f IL, describes and claims a compound having the formula wherein R is hydrogen, hydroxy or methoxy; R1 is hydrogen or 2 methyl; and R is hydrogen, or a conventional, pharmaceutically acceptable, easily hydrolyzed ester forming group; or a non-toxic, pharmaceutically acceptable salt thereof. ]_q The present invention provides compounds having the structure I δ is 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 W -CH 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, earbalkoxy, carbobenzoxy, carbamoyl, benzyloxy, chlorobenzyloxy, carbophenoxy, carbo-tert.-butoxy or (lower)alkylsulfonyl, and when W represents earbalkoxy, Z represents earbalkoxy 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, 8,8,8“ 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 (rS comprises the groups having the structures: R£H2NHR' V· ch2co'CI^KHR' CIi2C0 and 48613 CH„NHR wherein R is hydrogen, hydroxy or methoxy and R' or methyl.

A preferred embodiment of the present 5 consists of the compounds of Formula I wherein 3 structure is hydrogen invention ; has the '4301® and ^^.ck2nh2 CHgCQch2nh2 • s^-ch2coCX Another preferred embodiment of the present invention consists of the compounds of Formula I wherein has the structure CHgNHfflj CHgCO- CHgNHCHj CH C0“ CHoCOCH2NHCH3 and CX .CH2NHCH3 5613 A preferred embodiment of the present invention consists of the compounds having the formula wherein R1 represents CH2CO- OR aCH2W •CH2C0OR CH2NHRs CHgCOwherein R is hydrogen, hydroxy or methoxy; R' is hydrogen or methyl; and M is R10 j) /Rs -CHOC(CHe)nC^ NR4Rb I1 β -CHXCOR6 R10 or -CH-S-C-R6 310 n is 0 to 4; R is hydrogen, alkyl having 1 to 8 carbon 10 atoms, cycloalkyl of 3 to 6 carbon atoms, phenyl, phenylakyl, 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; R^ and r5 are each hydrogen or alkyl of 1 to 4 carbon atoms; R8 is alkyl having 1 to 4 carbon atoms, phenyl, phenalkyl having 1 to 4 carbon atoms, pyridyl, thiadiazolyl, amino or C1-C4 alkylamino; X is HH or oxygen; and each phenyl group is unsubstituted or substituted with one or two substituents selected from alkyl having 1 to 6 carbon atoms, alkoxy having 1 to 4 carbon 5 atoms, hydroxy, amino, NHR1, NtR1^, 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 Rl-NH-CH-Cfi^ ^ΟΙ8H C___^C—CHaS—^N^Nx.K_(CHa)2COOH σ ^c-om K s wherein R1 represents CH3NHR* ^2 GG” CHaCOOR CH3-CO wherein R is hydrogen, hydroxy or methoxy; R’ is hydrogen or methyl; and M is selected from CHq 0 I 3 II 6 -CH - 0 - C - R g2h5 -CH - C f52 ff 7 CH - X2 - C - OR' 43613 aralkyi-KH· -<2> ey R' is an alkyl group such as a methyl, ethyl, propyl isopropyl, butyl, isobutyl, pentyl or 2-ethyl-hexyl groupj a cycloalkyl group such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptylj an aryl group such as phenyl or naphthylj 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 isobutoxyj or a nontoxic, pharmaceutically acceptable salt thereof, SBQ13 There is a pharmaceutical effective amount further provided by the present invention composition comprising an antibacterially of a compound having the formula R1RH-CH-CK 1 J 2 AC—H , C—CHnS % ’ s-om wheeeln R“ represents R-Z ^-CHgCOOR .CH2NHR ’ , CHjiJHR ’ . ch2co- or (~JLch2cowherein R is hydrogen, hydroxy or methoxy; R' is hydrogen or methyl; and M is hydrogen, pivaloyloxy ’ ί methyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, ; p-nitrobenzyl, β,β,β-trichloroethyl, 5-phthalidyl or 5indanyl and preferably is hydrogen cr a nontoxic, pharmaceutically acceptable salt thereof. i There ls further provided by the present invention · a method of treating bacterial infections comprising ad- ministering by injection to an infected non-human warm-blooded ϊ animals, an effective but nontoxic dose of 250-10G0 mgm. of a compound having the formula ra, /-κχ s>c-cH2sJ:;ai'!,XI,'tCi^,2000itGx 0 g-om I 14. wherein R1 represents oa >^ch2nhr’ llJL-CHaCO_^CH2NHR’ °R QLch.cowherein R is hydrogen, hydroxy or methoxy; R' is hydrogen or methyl; and M is hydrogen, pivaioyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl, β,β,β-trichloroethyl, 3-phthalidyl or 5indanyl or a nontoxic, pharmaceutically 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 non-human 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, R1 represents !H2NHR‘ ch3coOR .CH2NKR' ' _, CH2NHR * -CHaCO- °R S.gJL- CHjCOwherein R is hydrogen, hydroxy or methoxy; R' is hydrogen or methyl; and M is hydrogen, pivaloyloxv2o methyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl. , 4 5 613 p-nitrobenzyl, β,β,β-triehloroethyl, J-phthalidyl or 5indanyl and preferably is hydrogen or a nontoxic, pharmaceutically acceptable salt thereof.

There is also provided by the present invention a 5 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 whesehs E1 represents 1 V/ CHsC0 QR ,ch2W -CHaCOOR wherein R is hydrogen, hydroxy or methoxy; R1 is hydrogen or methyl; and M is hydrogen, pivaloyloxy15 methyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl, β,β,β-trichloroethyl, 3-phthalidyl or 5indanyl and preferably is hydrogen or a nontoxic, pharmaceutically acceptable salt thereof. 43613 The present Invention also provides tne process for the production of the antibacterial agents having the structure R -NH-CH—CH CH„ ϊΝ N, χ ^Ο-ΟΗ^Λ^^^ΟΗ^ΟΟΟΗ ? 0 . .

COOH wherein R is acyl as defined above which comprises reacting a compound of the formula ^C-CH2S-\nzN^xN-(CH2)2C00H °C o COOH II 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. 3,284351 and U.K. 1,229353 and any of the silyl esters described in U.S. patent 3,249,622 for use with 6-aminopenicillanie acid and used in Great Britain 1,073,530 and particularly the pivaloyloxymethyl, acetoxymethyl, methoxymethyl, . acetonyl, phenacyl, p-nitrobenzyl, β,β,β-trichloroethyl, 3-phthalidyl snd 5“indanyl esters) with an organic monocarboxylic acid chloride or a functional ·. equivalent thereof as an acylating agent.

Such functional equivalents include the corresponding aeid anhydrides, including mixed anhydrides and particularly the mixed anhydrides prepared from stronger acids such as the lower aliphatic monoestera of carbonic aeid, er 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, 2,4-dinitrophenol, thiophenol, lo thioacetlc acid) may be used or the free acid itself may be coupled with compound II after first reacting said free acid with Ν,Ν'-dimethylchloroformiminium chloride [cf. Great Britain 1,008,170 and Novak and Weichet, Exoerientia XXI, 6, 360 (1965)] or by the use of enzymes or of an N,N’-carbonyldiimidazole or an Ν,Ν'-carbonyIditrlazole (cf.

South African'patent specification 65/2684] or a carbodiimide reagent [especially N,N'-dicyclohexylcarbodiimide. Ν,Ν'-diisopropylcarbodiimide or N-cyclo20 hexyl-N'-(2-morphollnoethyl)carbodiimide; cf. Sheehan and Hess, jL Ama.r.^ Chem.. Soc... 22., 1967 (1955)], or of alkylylamine reagent [cf» R. Buijle and H. G.

Viehe, Angew, Chem. International Edition 3. 582, (1964)] or of an isoxazollum salt reagent [cf. R. B. Woodv/ard, R. A. Olofson and H. Mayer, J, Amer, Chem., Soc. 83, 1010 (1961)], or of a ketenimine reagent [cf. C. L. Stevens and Μ. E. Munk, J, Amer, Chem, Soc,, 80. 4o65 (1958)] or of hexachlorocyclotrlphosphatriazine ' or hexabromocyclotriphosphatrlazine (U.S. 3,651,050) or of diphenylphosphoryl azide [DPPA; J. Amer, Chem. Sec.. · 94. 6203-6205 (1972)] or of diethylphosphoryl cyanide [DEPC; Tetrahedron Letters No. 18, pp. 1595-1598 (1973)1 or of diphenyl phosphite [Tetrahedron Letters No. 49, pp. 5^47-5050 (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. §8613 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 5 that free acid with enzymes provided by various microorganisms, e.g. those described by T. Takahashi et al., J. Amer. Chem. Soc., QUll). 4035-^.037 (1572) and by T. Nara et al., J. Antibiotics (Japan) 24(6), 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-ethoxy1,2-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 SOBS °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-acylarainocephalosporanic acid (prepared by substituting 7-amino20 cephalosporanic acid for the 3-thiolated-7-aminocephalosporanic acids in the acylation procedures described herein and elsewhere reported) with a thiol 3 HSR having the formula HS .ΛΪ, rr ( N- (CHg)2COOH and then removing the protecting group if any is present, as on the aminornethyl or methylaminomethyl group or on the carboxyl group or both. The displacement of such a 3-acetoxy group with such s 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 of the reaction mixture followed by extraction with a water-immiscible organic solvent. As noted above, the preparation of many other 7-acylamidocephalosporanic acids is described in the patent and scientific literature, e.g. in U.S. Class 260-243C.

When the organic carboxylic acid contains a functional group such as amino or methylamino it is often 2i 5613 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 COOH wherein r^ is acyl as defined above which comprises reacting a compound of the formula R1 -NH-CH-CH CHa 0 i COOH wherein R1 is acyl with a compound having the formula 4£6i 3 The salts of the compounds of this invention include the nontoxic carboxylic acid salts thereof, Including nontoxic metallic salts such as sodium, potassium, calcium and aluminum, the ammonium salt and substituted ammonium salts, 5 e.g. salts of such nontoxic amines as trialkylamines including triethylamine, procaine, dibenzylamine, H-benzyl-beta-phenethylamine, 1-ephenamine,· HjM'-dibenzylethylenediamine, dehydroabietylamine, £3, Ν'“bis-dshydroabietylethylenedlamine, N-(lower)lo alkylpiperidlne, e.g. N-ethylpiperidine, and other amines which have been used to'form salts with benzylpenicillin; and the nontoxic acid addition salts thereof (i.e., the amine salts) Including. the mineral acid addition salts such as the hydro15. chloride, hydrobromide, hydroiodide, sulfate, eulfamate 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 A g 61 ® ’ ------- -. y ' *. b is blocked11 by substituents such as 2-iodoethoxyesrbonyl (U.K. 1^3^9,673)/ t-butoxyesrbonyl, carbobenzyloxy, formyl, q-hitrophenylsulfenyl, 6,6,p-trichioroethoxyearbonyl, 4-oxo-25 senteny1-2, l-carbomethoxy-X-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 β-ketoesters and (Wiketenes disclosed, for'example, in U.S. patent 3,325,479 and the β-ketoamides disclosed in Japan 71/24714 (Farmdoc 47,321S). ihe preferred esters of the cephalosporins ef the present invention- are the pivaioyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl and phenacyl esters. All are useful Intermediates in the production of the cephalosporin having a free carboxyl- groupk 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 cephaiothin is esterified by reaction with the corresponding active 2S chloro er bromo compound (e.g. phenacyl bromide, ehloroacetone, chloromethyl ether, pivaloyloxymetbyl chloride (also called ehloromethyl pivalatel, acetoxymethyl chloride) and then the thienylacetic acid sidechain is removed enzymatically as in tha same patent or chemically as in U.S, patent 3,575,970 and in Journal of Antibiotics, rav (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-aminocephalosporanie acid are then reacted -with the nucleophile HSR? in the same manner as is illustrated herein for 7-aminocephalosporanic acid itself. The 3-thloiated ester of 7-amlnocephalosporanic acid is then coupled with the organic carboxylic acid R*-OH as before. Before er 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 zwittetion (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 3thiolated 7“aminocephalosporanic acid is prepared as described herein and then acylated’at the 7SBiino 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 iaa, the compounds of Ehis invention are usually administered parenterally 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 physiologically acceptable carriers or excepients.

The dosage units are in the form of liquid prepara0 tions such as solutions or suspensions. 45013 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.-Butoxvcarbonvlaminopethvl)-!.4lo crclohexadisnylacetlc 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 to remove a trace of moisture) was slowly diluted with 500 ml. of dry t-BuOH. To the solution was added in small portions 3.¾ 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 4c° C. The residue was 2b dissolved in 500 ml. of water and the solution was ehromatographed 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 drynes3. The residue was washed with four 50 ml. portions of hot acetone and recrystallized from 500 ml. of ethanol-water (l:l) to give 11.2 g. (67$) of colorless needles, o-(2-aminomethyl-l,4-cyclohexadienyl)acetic acid. M.p. 183° C. l6jO, Ι52Ο., 1380, 1356 em1. max NMR: & °2° * V°3 2.72 (4H, s, HgCC ), 3.01 (2H, s, Q^CO), 3.20 (2H, a, Cgg-N), 5-7δ (2H, s, £>C=).

Anal. Calcd. for CgH^NOg: C, 64.65; H, 7.84; N, 8.38.

Found: C, 64.77; Η, 8.06; N, 8,44.

Improved Procedure for the Preparation of ,g° (g-aminomethvl-1.4-cvclohex'adlertyl·)-acetic acid 1° rt? WH Li, tert. BuOH NHj - TEA‘HC1 X’ // \_CH2C02H * L1C1 * {c2h5)5k The procedure used by Welch, Dolfini and Oiarrusso in U.S. patent 3,720,665 (Example 1) to make D-2-amIno-2“(l,4-cyclonexadienyl)acetie acid was adapted. A solution of 830 ml. of distilled liquid ammonia was dried with 40 mg. of lithium .9 under art'argon atmosphere. To this stirred solution was added 11.0 g. (0.07 mole) of 2-aminomethylphenylacetic acid and 3^0 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 4o° (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 lsl chloroform-acetone at 5°. The mixture was stirred for 20 min. and the amino acid, a-(2-aminomethyl-l,4-cyclohexadienyl)acetic acid, was collected and dried for 16 hours in vacuo over F.O.. 2 to yield 6,3 g. (58=3) of white crystals, m.p. 190° aecomp, The IR and NMR spectra were cqnsister.t for the structure.

' A solution of 19.51 g. (0,135 m) of tert.butoxycarbenylaside in 152 ml. of tetrahydrofuran (THF) was added to a stirred solution of 14.89 g. (0.09 m) of 2-aminomethyl-l,4-cyclohexadienylacetic acid and 7.20 g. (0.18 m) of sodium hydroxide in 28l ml. of water. The solution was stirred for 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 (HCl). The mixture was stirred in an ice-bath and the precipitate was collected and dried for 18 hr. in vacuo over P205 at 25° to yield 17.3 S· (72.6^) of 2-(tert.30 butoxycarbonylaminomethy1)-1,4-cyclohexadlenylscetic acid as a white powder. The IR and NMR spectra were consistent for the structure. ' 45 613 Preparation of 3-Amlnomethyl-2-thiophene Acetic Acid S CHg-COgH A) Thiophene-5-carboxaldehyde Dimethyl Acetal (2a) A mixture of thiophene^J-carboxaldehyde1) (?22 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 dislo tilled under reduced pressure. Yield 42? g. (94$), b.p. 90-95° C. 13 mmHg. ir; J 3150, 1045, 1025 cm'1 nmr: S 3-21 (6h, s, OCHj), 5-43 (IH, s, ΟΗ<θΖ), 7.0-7.4 (?H, m, thiophene-H) 1) S. Gronowitz, Arkev, kemi., 8, 411 (1955).

B. 2-Formylthiophene-3-carboxaldehyde Dimethylacetal (?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 (2? moles) in ether keeping a gentle reflux under dry Ng. Reflux being continued for 0.5 hour, a solution of DMF (dimethylformamide) (432 g ., 6 moles) in anhydrous ether (0.8 L) was added dropwise to the mixture over a period of 0.75 hour with vigorous stirring. After the complete addition the mixture was stirred overnight, poured onto crushed ice (1 Kg.) with stirring and allowed 43613 io rise to room temperature. The organic layer was separated and the water layer was saturated, with NaCl and extracted thoroughly with ether (2 x 200 ml.). The ether extracts were combined, dried over MgSO^ and con5 centrated. The residue was distilled under reduced pressure and the pale yellow oil' was collected at 100125° C., 0.7 mmHg. Yield, 277 g. (56$). ir: -) 3110, ΐ6βθ, 1100 cm1.

IU3.X nmr: ^ρρί 3·40 (6H> s> OCH3^ 5'·86 (¾ s> CH-^Zb 7.27 (IH, d, J=6Hz, thiophene-Ηβ), 7.8l (1 H, d-d, J=1.5 and 6Hz, thiophene-Ha), 10.34 (IH, d, J-1.5 Hz, -OHO).

G) l-Methylsulflnyl-l-methylthlo-g-(3-carboxaldehydeethyleneacetal-2-thienyl)ethylene (4b) Preparation of 4b was carried out according to the procedure similar to that reported by K. Ogura et al.1*). Triton B (40$ in methanol, 2 ml. in THF (tetrahydrofuran) (5 ml.) was added to a solution of methyl methylthiomethyl sulfoxide2) (2.5 g., 20 a. moles) and 2-formyl-3-thiophenecarboxaldehyde ethylene acetal'’) (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 MgSO^ and evaporated to dryness under reduced pressure. The residue was column-chromatographed 5 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; v 1*2 3110, 1600 cm1, max nmr:<5 £^3 2.42 (311, s, S-CHjJ, 2.78 (3H, s, SO-CH3), 4.15 (4H, m, CHjCl^-), 6.12 (ΪΗ, s, CH^qI ), 7.34 (III, d, J=4.5Hz, thiophene-Πβ) „ 7,40 (IH, a, 0=4.5(42, thiophene-Ho), 8.28 (III, s, -CH=).

The semicarbazone of £ was prepared by a usual manner and crystallized from ethanol-DMF. M.p. 212213eC.

Anal. Calcd. for ciQHi3N3°2S2i c' 39-S8i H« 4.32; N, 13.85; S, 31.70.

Found; C, 39.46; II, 4.24; N, 14.05; 5, 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 al., Tetrahedron Letters, 1383 (1972). 4561 D) l-Methylsulfinyl-l-methylthio-2-(3-carboxaldehyde dimethylacetaI-2-fchienyl)ethylene (4a), The compound 4a was prepared by the procedure similar to that for 4b. Triton B (40% in methanol, 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. irs v 3100' 1580» 110θ' 10501. ϊΡ<4Λ CCl nmr: 5 2.42 (3H, s, S-CH-j), 2.70 (3H, s, SO-CH3), 3.34 (611, s, OCH3), 5.56 (lH, s, CH^°), 7.20 (IH, d, d^SKz, thiophene-Πβ), 7.40 (IH, d, J=6IIz, thiophene-Ha) , 8.12 (IH, s, ~CH=).

S) Ethyl 3-formyl-2-thienylacetate^ (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. Tha 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). *·» 7 Fractions containing the desired product were combined and concentrated. The residual oil (SO g) was distilled under reduced pressure to afford 23 g (23¾) of 5, boiling, at 120-125° C/l mmHg. ir: v 3110, 1730, 1670 cm1, max nmr: δ ^1 3 1.30 (3H, t, J=SHz, -CH2CH3), 4.25 (2H, q, J«6Hz, -CH2CII3), 4.26 (2H, S, -CH^O) „ 7.25 (IH, d, J=5Hz, thiophene-HS), 7.48 (IH, d, J=5Hz, 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, ir: v ^ol 1.720, 1610, 1570 cm1.

Anal. Calcd. for C^H^4N4OgS: C, 47.62; H, 3.73;. H, 14.81; S, 8.47.

Found: C, 47.33; H, 3.47; N, 14.77; S, 3.68. 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 (44¾) of 5 as a pale yellow oil. 5613 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 n mole) and hydroxylamine hydrochloride (2.2 g, 32 m mole) in 50% aq. ethanol (4 0 ml) at 5’ C 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 SO ml). The benzene extracts were washed with water (10 ml), dried over MgSO^, and evaporated under reduced pressure. Separation by column chromatography on silica gel (60 g) gave 2.7 g (80%) of colorless oil 6. irs v 3400, max ' 1730, 1620 cm . nmr: δ Aceton-d ppm <3H, t, J=7.5Hz, -ch2ch3), 4.01 (2H, s, -CHjCO), 4.14 (2H, q, J=7.5Hz, -ch2ch3), 7.31 (2H, s, thiophene-H), 8.26 (IH, s, -CH; =N), 10.15 (IH, s, NOH, disappeared by addition of DjO). 6) The 6-lactam of 3-aminomethyl-2-thienylacetic acid (7).

Method A: Catalytic reduction A mixture of the oxime £ (2.S5 g, 12.4 ra moles), % palladium on charcoal, dry hydrogen chloride (1.4 g, 37.2 m moles) in anhydrous ethanol (68 ml) was hydrogenated ovori.ighv under atmospheric pressure at room temperature. The catalyst was exchanged twice . 4S6l3and the reaction was carried out over a period of 3 \ days. The catalyst was removed and the filtrate was i ’ 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 ElgSO^, treated with charcoal, and evaporated under reduced pressure. Recrystallization from ethyl acetate gave 417 mg (22%) of colorless needles 7, melting· at 19<-195°C. irs ν 3200' 1650, 1480 era”1, max nmr:6 3·53 <2»» 4, J=3Hz, ,-CH„CO~)., 4.3S pp«t & (211, d-t, J=3, 1.5Kz, changed to a triplet by addition of Q,0(, J=3Hz, CSIjN), 6.95 (111, d, J=4.5Hz, thiophene% Ηβ), 7O45 (111, d, 0=4.5Hz, thiophene-Ηα), 8.0 (IK, m, disappeared by addition of DjO, HU).

Anal. Calcd. for C^NOS; C, 54.88; K, 4.61» H, 2o 9-14» S, 20.93.

Founds C, 55.04; H, 4.45; N, 9.13;‘s, 20.50.

Method Bs Zn-dust reduction To a solution of the oxime £ (18.3 g, 86 a moles) in acetic acid (200 ml), zinc dust (17 g, 25S in moles) was added portionwise over a period of 1 hr. at 40-50° C with vigorous stirring. The reaction mixture was stirred i i 613 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 SO 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 MgSO^, and evaporated under reduced pressure. The residual solid was triturated vzith benzene. Crystallization from ethyl acetate gave 2.7 g (21%) of the lactam 7_ which was identical to, Method Λ in the IR and the tlMR 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) vzas heated under reflux for 3 hrs. The reaction mixture was con20 centrated 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 £ hydrochloride (3.72 g, 95%; m.p. 171-172° C; ir (KBr) cm-1: 3450, 3000, 1700, 1200; nmr (D2O)ppttu 4.80 (2H, s, -CH2CO), 4.27 (2H, s, CHj-N), 7.26 (111, d, J=6Hz, thiophene-H8), 7.53 (lH, d, J=6Hz, thiophene-Ηα). The hydrochloride (3.71 g, 17.9 mmoles) was dissolved in water (10 ml) chromatographed on a column of IR-120 (H , 30 ml) and developed successively with water (100 ml) and SM-NH^OH (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 3000, 1620, 1520 cm'1. jararsfi p|°Na2CO3 3.20 (2H, s, -CK^CO), 4.13 (2H, s, CHgN), 7.04 (IH, d, J=SHz, thiophene-Ηβ), 7.30 (lH, d, Jo6Hz, thiophene-Ηα).

Anal. Calcd. for CyHgNOjS: 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-thienylacefcic 15 acid (9) A mixture of 3-aminomethyl-2-thienylaeetic acid 8. (3.1 g, l8 m. moles) and triethylamine (8 g. m moles) in 50% aqueous acetone (80 ml) was to added dropwise/t-butoxycarbonyl azide (5.7 g, 40 2Q m moles) over a period of 20 mins, at 0° C with vigorous sitrring. 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 cone.

HCl and extracted with ethyl acetate (2 x 50 ml).

The ethyl acetate extracts vzere washed with saturated aqueous sodium chloride, dried over MgSO^, 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 '5 (92¾) of colorless needles £, melting at 62 - 63’C. ir: v ^°1 3350, 1700 cm1.

CDCl nmr: δ 3 1.43 (9H, s, BOC-H), 3.27 (2H, s, ppm ~ CH-jCO), 4.16 (2H, d, J=6Uz, CHj-N, a singlet when was added), 5.00 (IH, br, -NH-, disappeared by addition oi' D2O), 6.30 (III, broad s, -COOH, disappeared by addition of D2O), 6.86 (IH, d, J=6Hz, thiophene-Ηβ), 7.06 (IH, d, J=6Hz, thiophene-Ηα).

Anal. Calcd. for ci2H17NO4S: C, 52.89; H, 6.29; II, 5.14; S, 11.77.

Found: C, 53.30; II, 6.39; K, 5.13; S, 11.72. 613 Preparation of g-N-Mcthylaminomethyl^-methoxy-(and 4-hydroxy-)phenylacetic Acids CH2NH2 CHgCOgH Ts-Cl > a HgNH-Ts h2co2h CH,I CH, H3C°Tii?X?rCH2i'r“TS l^Ji-CHgCOgH CHjNH-CHj ch2co2h 4b g-N-To3ylamlnomethyl-4-hydroxyphenylacetlc Acid (2) To a solution of 14.56 g. (0.08 mol.) of 2-aminomethyl-4-hydroxyphenylacetic acid (1) (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 65-70° C. a solution of 18.5 g. (0.097 lo 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 vzas separated, washed with ether (2 x 50 ml.), acidified with 6n HCl and extracted with 400 ml. of ethyl acetate. The extract was washed with v/ater and a saturated aqueous NaCl solution, dried v/ith NagSOjj and treated v/ith active carbon (1 g.). The filtrate was concentrated to dryness and the residue v/as crystallized from ethyl acetate to give 11.0 g. 00.5$) of 2 melting at 212-215° C. ir: V KBr 3240, 1700, I38O, 1330, 1150 cm1 IuclX UV: ^22°°5 230nMs; 7/750) nmr: <5 ppS°d6 2Λ7 <5Η’ s’ Ar-CH3b 2·60 (2H, s, CH2C0), 3.93 (2H, d, J=6.0 Hz, CHgN), 6.6-8.2 (7H, m, phenyl-H). 2- (' N-Methyl -N- tosy lamino) me thyl -4-methoxyphenylac et ic Acid (3) A mixture of 11 g. (0,033 mol.) of 2, 10.3 ml. (0.I7 mol,) of methyl iodide and 9·2 g. (0,24 mol.) of sodium hydroxide in 100 ml. of water v/as heated at 8Ο-9Ο0 C. for 45 minutes in a sealed tube with occasional shaking. The mixture v/as washed with ethyl acetate (30 ml.) and the water layer was acidified with 6n HC1 and extracted·with ethyl acetate (3 x 30 ml.). The com20 bined extracts were washed with water (30 ml.) and a saturated aqueous NaCl solution (30 ml.) treated with active carbon (1 g.) and dried over NagSO^. The filtrate was evaporated to dryness and the residue was · crystallized from benzene to give 8 g. (66.5)() of the' Ν,Ο-dimethyl derivative 3 melting at 146-150° C. ir: 1/ KB*? 1690, 1500, 1340, 1280, 1150 cm1.

IilcLX uv: λ^χΗ 229 (C: 2°5θ0)/ 27θ nm (£: 2400). nmr: 2.52 (3H, s, N-CH?), 2,,47 (3H, s, Ar-CIL), 3.6? (211, s, CH„CO), 3-74 (3H, s, OCIL), 4.10 s > (2H, s, CH2N), 6.7-7.8 (7H, m, Ar-H), 11.5 (HI, br-s, COOH).

' Anal. Calc'd. for C^H^NOgS: C, 59-49; H, .82; N, 3.84; S, 8.82. Found: C, 59.48; II, 5-68; N, 3-37; s, 9.22. 2-N-Methylaminomethyl-4-methoxyphenylacetic Acid (4a) To a solution of liquid ammonia (300 ml.) was 10 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 S· (0.14 g. atom) of Na in small pieces at -40° G. and the mixture was stirred for 2 hours. Ammonia was eva15 porated 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 chromato graphed with a column of IR-120 ion-exchange resin (H+, 100 ml.) by eluting With 5“10(i 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. (8l£) of 4a, m.p. 225-227° C. ir:V 1590, 13θ0, 1260, 1035 cm1. 48613 nmr: 2.77 (JH, s, Ν-CHj), J.6 (2H, s, CHgCO), 3.87 (3H, s, OCHj), 4.18 (2H, s, CHgN), 6.8-7/ (311, m, phenyl-H).. 2-N-Methylaminomethyl-4-hydroxyphenylacetic Acid (4b) 5 A mixture of 2.9 g. (0.014 mol.) of 4a in JO ml. of 48# hydrobromic acid was refluxed for 5 hours and the solution was evaporated to dryness. The residue was dissolved in 5θ 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 vzas 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 2 2000-3400, l6l0, 1540, 1460, I380, 1270 cm1. uv: ΛίηΧ2°°3 243 nm 47θ0)> 297 11111 &· 135°)· nmr: J p|^Na0H 2.64 (3H, s, N-CH^), J.4? (2H, s, CHgCO), 3.94.(2H, S, N-CHg), 6.5-7.2 (3H, m, phenyl-H).

Anal. Calc'd. for CjqH^NO^: C, 61.53; η, 6.71; Ν, 7.I7. Found: C, 6l.44; Η, 6.8l; N, 7.20. 2-K-t-Butoxycarbonyl-N-methylaminomethyl-4-methoxyphenylacetlc Acid (5., R = CH^) A mixture of 1.05 g. (5 m.mol.) of 4a, I.43 g. (6 m.mol.) of t-butyl 4,6-dimethylpyrimidin-2-ylthiolcarbonate and 1.4 ml. of triethylamine in 40 ml. of 5θ# THF was stirred at room temperature for 20 hours. Most of the THF was evaporated arid the resulting aqueous solution (ca. 20 ml.) was washed with ether. The water layer vras acidified with 6n HCl and extracted.with ether (3 χ 10 ml.). The ethereal extracts were washed with water (10 ml.) 'and a saturated aqueous HaCl solution (10 ml.), treated with a small amount of active carbon and dried over HagSOjj. The filtrate was evaporated to dryness to give 1.0 g. (77-5$) of £ (R = CH^) as an oil. nmr: ύ ^3 1.'47 (9H, s, BOC-H), 2.77 (3H, s, N-CH^), io 3.60 (2H, s, CH2C0), 3.79 (3H, s, 0-0Η?), 4.49 (2H, s, CHgN), 6.1-7.3 (3H, m, phenyl-H). 2-N-t-Butoxycarbonyl-N-methylaminomethyl-4-hydroxyphenylacetic Acid (5, R =H) A mixture of 1 g. (4·7θ m.mol.) of 4b, 1.5 g. (6.3 m.mol.) of t-butyl 4,6-dimethylpyrimidin-2-ylthiolcarbonate and 2.1 ml·, of triethylamine in 50 ml. of 50$ 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 HCl and extracted with ethyl acetate (2 x 100 ml.). The combined extracts were washed with water (30 ml.) and a saturated aqueous NaCl solution (2 x 30 ml.), treated with a small amount of active carbon and dried over anhydrous NagSO^. The filtrate was evaporated to dryness to give 1.3 g. (92$) of 5 (R = H) as an oil. ir: 3000-3600, 1670, I26O, 1150 cm1, nmr: S 1.44 (9H, s, 0(0^), 2.75 (3H, s, N-CH^), 3.54 (2H, s, CH2C0), 4.38 (2H, s, CHgN), 6.5-7.3 (3H, ‘ m, phenyl-H).

Preparation of Ortho-H-methylaminomethyl-phenylacetic Acid.

CHgNHg CHgCOgH CH SOgCl T3 CHgHH CH2C02H HBr --> or Na/liq-NH^ CH, I 3 CtigN-BOC CHgCOgH CH BOC : CH,-C-OCO- 3 ι CH o-(p-Toluenesulfonylamlnomethyl)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 6o° 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, lo 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. 155156° C.

Ir: D £ 5300, 17°5' 1555' 1170 cnf1· nmr: $ 2..38 (3H, s, CK^), 3.65 (2H, s, CH2C0), 3.97 (2H, d, J=5 Hz, CH2N), 7.1-8.2 (9H, m, pheny1-H & NH).

Anal. Calc'd. for C^H^NO^S: C, 60.17; H, .37; N, 4.39; s, 10.10. Found: C, 60.11, 60.15; H, .43, 5.40; N, 4.28, 4.30; S, 9-72, 9-80. o-(N-p-Toluenesulfonyl-N-methylamihomethyl)phenylacetic Acid (3) · A mixture of 2 (9-0 g.,28 m.mol.) sodium hy25 droxide (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 to separate pale yellow precipitate which was crystallized from ethyl acetate-n-hexane to give colorless prisms, 5- Yield, 8.5 g. (91#)· M.p. 162-165 ’ C. ir: 2700-2300, 1700, 1600, 1345, 1200, 925 cm1. nmr: J pf£+K°H 2·37 s> 2Λ9 s, CHj), 3.8Ο (2H, s, CHgCO), 4.18 (2H, s, CHgN), 7.0-8.0 (8H, m, phenyl-H).

Anal. Calc'd. for C^H^NOgi G, 61.24; H, .74; S, 4.20; S, 9-61- Found;· c, 61.31, 61.36; h, -73, 5-71; N, 4.51, 4.29; S, 9.63, 9-55N-Methylaminomethylphenylacetic Acid (£) Method A (using hydrobromic acid) - A mixture of 28.6 g. (Ο.Ο86 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 ehromatographed 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 ethan'ol 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. (Ο.57θ atom) 45S613 - .. ; 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 filteredoff and to the filtrate was added an aqueous 1 M solution of barium acetate until no 10 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$) of 4. o-(N-me thyl-H-t-butoxyc arb onylaminome thyl)phenylac etic Acid (5,) t-Butyl 4,6-dimethylpyrimidin-2-ylthiolcarbanate (11 g., 0.048 mol.) was added in one portion to a mixture of 4, (7.2 g., 0.04 mol.) and 1,1,3,3-tetramethylguanidine (6.9 g., 0.06 mol.) in 50$ 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 25 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-hexane-ether to afford 9.2 g. (83$) of 5 as colorless prisms. M.p. 96-98° C. 45S13 ir: J/ 1730, 1^30, 1430, 1830, 1250 cm1. ΓΠ3.Λ nmr: 6 1.49 (9H, s, t-butyl), 2.78 (3H, s, K-Cri3), 3.72 (2H, s, CHgCO), 4.25 (2H, S, CHgN), 7.28 (4h, s, phenyl), 9·δ3 (IH, s, -COOH).

Anal. Calc'd. for C-^HgjNO^: C, 64.50; H, 7·5θ; N, 5.01. Found: C, 64.69'; H, 7.66; N, 4.89.

Preparation of .5-N-methylaminomethyl-2-thlenylacetic Acid.

CH=N0H CH2C02C2H ->> · . .2 NaH, CHjI benzene 3-Aminomethyl-2-thienylacetic Acid Glacial acetic acid (140 ml.) was added, dropwise with stirring to a mixture of 2-ethoxycarbonylmethylthiophene-3-carboxaldehyde oxime (1,) (41 g., 0.19 mole) and zinc dust (65· 4 g., 1 mole) in methanol, and. the mixture was stirred under reflux for 4 hours. The lo mixture was cooled and insolubles were removed by filtration and washed with methanol (5 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 pit 10 with Νη^ΟΟ^ 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 (JO 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 (7-7 g., 26#), melting at 195-196° C.

UV: XjJeOK 232 nm (f, 6500)· Anal. Calc'd. for CyHyNOS: C, 54.88; H, 4.6l; N, 9.14; s, 20.93. Found: C, 55-04; H, 4.45; N, 9-13j s, 20.50. 3-N-Methylaminomethyl-2-thlenylacetic Acid To a suspension of sodium hydride (50# in paraffin, 1.82 g., 3θ m.moles) in absolute benzene (500 ml.) was added the lactam 2 (4.85 B·, 32 m.moles) with stirring under nitrogen atmosphere and the mixture was refluxed for 2 hours. Methyl iodide (22.7 g., I60 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 5ί β13 was evaporated and the residue was crystallized from benzene-n-hexane to afford colorless plates 3- Yield; 2.7 g. (5l£). M.p. 9S-1OO0 C. ir· D nuJo1 1620 cm1. . nmr; 6 ^3 3-15 (3H, s, N-CH^), 3-72 (2H, t, J=3Hz, CHgCO), 4.53 .(2H, t, J=3Hz, -CHg-N), 6.87 (IH, d, J=4.5Hs, thiophene-Ηβ), 7-30 (IH, d, J=4.5 Hz, thiophene-Hc). uv;λ 232 nm (¢, 6700) Anal. Calc'd. for CQHgNOS: C, 57.46; H} 5-42; N, 8.385 S, 19.17. Found: C, 57,5^5 fi, 5.26; Η, 8.3I; s, 19.13. 3-^N-Methylaminomethyl)-2-thienylacetlc Acid (£) A mixture of the lactam £ (3-5 g., 21 m.moles) and 6n HCl (100 ml.) was heated under reflux for 12 hours. 15 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 NH^OH (> L·.). The amino acid £ (5.0 g., 77$) was isolated by evaporation of the ammonia eluates followed by crystallization from aqueous acetone.

M.p. 181-1820 C. ir; 1570, 1360 cm1. luSX nmr: S 2.21 (3H, s, N-CHj), 3-80 (2H, s, CHgCO), 4.20- (2H, s, CHg-N), 7-19 (IH, d, J=6Hz, thiophene-Ηβ), 7.46 (IH, d, J=6hz, thiophene-Ηα). uv:^mi£ 237 nm (£, 7600) 5813 Anal, Calc'd. for CgH^NOgS: C, 51.87; Ή, 5·99) Η, 7·5δ; S,· 17-51- Found: c, 51.67; H, 6.50; N, 7-28} S, 16.69. g-(M-t4butoxycarbonyl-N-methylanilnoinethyl)-2-thienylacetic Acid (£) To a mixture of 3N-methylaminomethyl-2thienylacetic 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° 0. 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 KC1 and extracted with ethyl acetate (2 x 50 ml.). The ethyl acetate extracts were washed with a saturated aqueous NaCl solution, dried on MgSO^, treated with charcoal and evaporated under reduced pressure. The residue was triturated with n-hexane and crystallised from n-hexane-benzene to give 5-68 g. (88$) of colorless needles melting at 82-85° C. ΐΓί max01 175°5 l6!|° cm”1, nmr: <5 1.47 (9H, s, BOC-H), 2.78 (3H, s, N-CHj), 3.87 (2H, s, CHg-CO), 4.48 (2H, s, CHg-N), 6.91 (1H, d, J=»6hz, thiophene-Ηβ), 7.20 (IH, d, J=6hz, thiophene-Ha), IO.63 (IH, s, COgH, disappeared by addition of DgO).

Anal. Calc'd. for C^H^NO^S: C, 54.72; H, 6.71; N, 4.91; S, 11.24. Found: C, 54-91; H, 6.85; N. 4.92; S, II.I9. 3 613 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--.fl-carbethoxvoropen-2-vl)anti.nomgthyl]-1.4evclohexadienvl acetate (4) To a stirred solution of 460 mg. (0.02 g. atom) of metallic lo sodium in 100 ml. of absolute EtOH was added 3.34 g. (0.02 mole) of 2-aminomethyl-l,4-cyclohexadienylacetic acid and 3.1 g. (0.024 mole) of ethyl acetoacetate ahd the mixture was heated to reflux for 4 hours with stirring. The hot reaction mixture was filtered and fche filtrate was allowed to keep cold overnight to give 2,0 g. of colorless needles 4. melting at 264° c. The additional product (3.3 g.) was obtained by concentration of the mother liquid. The total yield was 5.3 g. (88$).

XK:/ 2 33OO> 1635' 1600 ’ 1570' 13°°> 1S75, 1170, 1090 cm1. 1.23 (3H, t, 7Hz, CHgCHg), I.96 & 2.25 (3H, s, CsC-CHp Cis & trans), 2.70 (4h, s, 3.04 (2H, s, CHgCO), 3.66 & 3-95 (2H, s, CHg-Ν, cis & trans), 4,07 (2K, q, 7Hz, OUCH ), 4.45 & 4.56 (IH, s, / , els & trans), 5.76 (2H, s, ).

Anal. Calcd. for C^H^NO^Na: C, 59.79; H, 6.69; H, 4.64.

Pound: C, 59.69; H, 6.76; N, 4.75. 501 2-t-Butoxycarbonylaminomethyl-4-hyaroxyphenylacetic acid is prepared, for example, according to U.S. 3,823,141. o-(N-Methylaminomethyl)phenylacetic acid δ-lactam '3 N-CH, Sodium hydride (57% in paraffin, 4.3 g.; 0.11 mol.) was washed with dry n-hexane and suspended in dry benzene (100 ml.). To the suspension was added a solution of o-aminomathylphenylacetic acid δ-lactam (U.S. 3,796,716) (14.7 g., 0.1 mol.) in dry benzene or xylene (200 ml.) with stirring lo 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 I 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 CHCl^ (2 x 50 ml.). The extracts were combined with the organic layer and dried on MgSO^. The solvent was removed and the oily residue was distilled in vacuo to afford 14.9 g. (92%) of o-(N-methylaminomethyl)phenylacetic acid 2o δ-lactam, boiling at 130-135°C/2 mmHg., m.p. 35-37*C. ir: v ir: vKBr 3300, 1620, 1490 cm'1, max nmr: 0CDC13 3.12 (3H, s), 3.59 (2H, t, J=1.5 Hz), 4.48 ppm (2H, t, J=1.5 Hz), 7.21 (4H, br-s). $ ΰ 613 Anal. Calc'd. for 0^^0.1/4^0: 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 CH, ‘NHCH, ch2cooh A mixture of the above-produced o-(N-raethylaminomethyl)phenylacetic acid δ-lactam (5.0 g., 0.031 mol) and cone hydrochloric acid (.500 ml.) was refluxed for 40 hours. The mixture was evaporated under reduced pressure, and the re10 sidual 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-methylamino15 methylphenylacetic acid hydrochloride (4.5 g., 67%). Anal. Calc'd. for 01θΗ13Ν02·Η01; C, 55.69; H, 6.54; tf, 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. 140-143flC/2mmHg). 4(j613 An aqueous solution of o-N-methylaminomethylphenylacetic acid hydrochloride (5 g.) was column chromatographed with IR-120 ion-exchange resin (H*, 70 ral.) and eluted with 3N NH^OH (2 1) to afford 3,9 g, (937«) of o-N-methylarainomethyl5 phenylacetic acid as needles. 613 The following examples are given in illustration of, but not in limitation of, the present invention. All temperatures are in degree Centigrade. 7-Aminocephalosporanic acid is abbreviated as 7-ACA: -ACA represents the moiety having the structure -ΝΗ-ψ--Xs·I 0 COOH II and thus 7-ACA can be represented as H-ACA-O-C-CH^ 4!ί6ί! 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 divinylbenzene 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 THR, thin layer chromatography as TLC, p15 toluenesulfonyl as Ts and methanol as MeOH.

When the following instrumental readings are given, for infrared nu if used is written ν, for ultraviolet lambda is written as λ, with molar absorptivity as epsilon (ε) and for nuclear magnetic resonance (nmr) delta is written as δ and tau as r (5=10-r). The word Nujol is a registered Trade Mark.

Additional Starting Matei'ials /~4 3 613 / / Ά ΐ 6-Chloro-2-(2-cyanoethyl)-2,3-dihydro-3-triazolo[4,5-b]pyrldazln-3-on.

To a solution of 6-chloro-2,3-dihydro-s-tri5 azolo[4,3-b]pyridazin-3-on [P. Prancabilla and P. Lauria, J. Het. Chem. .8, 415 (1971)] (17 g·, O.l 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 io mixture was stirred at 100-110° C. 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 NagSO^ and evaporated. The residue was crystallized from ethyl acetate to give light yellow needles of’ 6-chloro-2-(2-cyanoethyl)-2,5-dihydro-s-triazolo[4,3-b]pyridazin-3-on (2.5 g., 11$). M.p. 166-168° C. ir; 2230, 1720, 1550, 1500 cm1. uv: 373 nm (e 2000). nmr: 3-03 (2H, t, 1=6.0 Hz, CHg), 4.21 (2H, t, J=6.0 Hz, CHg), 7.23 (IH, d, J=10.0 Hz, pyridazine-H), 7-93 (IH, d, JfelO.O Hz, pyridazine-H).

Anal. Calc'd. for CgHgl^OCl: 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-'iihydro-s-trlazolor4,3-'b1pyrldazini-5-Qft.

A solution of 6-chloro-2-(2-cyanoethyl)-2,3dihydro-s-triazolo[4,3-b]pyridazin-3-on (724 mg.) in 6n-HC1 (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 NagSO^ and evaporated to give light yellow;solid 2-(2-carboxyethyl)-6-chloro-2,3dihydro~s-triazolo[4,3-b]pyridazin-3-on (567 mg., 72#).

M.p. >170° C. (sublimation). ir: ν®* 3400-2400, 1730, 1710, 1540 cm-1. lueUt uv: dioxane max 377 nm (ε 1500). nmr; 0D2O*^HC°3 2.70 (2H, t, J=7.0 Hz, CHg), 4,24 (2H, t, J=7.0 Hz, CHS), 7.17 (IH, d, J=10.0 Hz, pyridazine-H), 7.70 (IH, d, J=10.0 Hz, pyridazine-H).

Anal. Calc'd. for CgHyN^Cl: C, 39.60; H, 2.91; N, 23.09; Cl, 14.61. Found; C, 39-62, 39-48; H, 2.97, 2.67; N, 23.05, 22.70; Cl. 13.93, 14.12. 4(2613 2-(2-Carboxyethyl)-2,3-dihydro-6-mercapto-s-triazolof4,3-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$.sodium hydrosulfide dihydrate (924 mg., 7.02 m.mole) in water (10 ml.) was stirred at room temperature for two hours. The reaction mixture was adjusted successively to.pH 1 with c. HCl, to pH 10 with NaOH and then to pH 1 with c. HCl. The resulting precipitate of 2-(10 carboxyethyl)-2,3-dihydrO-6-mercapto-s-triazolo[4,3-b]pyridazln-3-on .was collected by filtration and washed with water. Yield: 4l8 mg. (74$). M.p. 174-176° c. ir:vmax 3600-2600, 2440, 1730, 1720 (sh) cm-1. uv: λρΗ 7wayf,fer 2δ2 1111 (ε ^-7000),318 nm (e 66ΟΟ). 15 nmr: dDMppad6 2-73 (2H, t, J=7.0 Hz, CHg), 4.07 (2H, t, J=?.O Hz, CHg), 7.30 (IH, d, J=10.0.Hz, pyridazine-H), 7.74 (IH, d, J=10.0 Hz, pyridazine-H).

Anal. Calc'd. for CgHgN^O^S: C, 40.00; H, 3.36; N, 23.32; S, 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-triazolor4,5-b1pyrldazin-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)-2,3-dihydro-6-mercapto-s25 triazolo(4,3-b]pyridazln-3-on (357 mg., 1.49 m.moles) and NaHCO^ (375 mg., 4.47 m.moles) in 0.1 M phosphate buffer (pH 7, 8 ml.) was stirred at 8oe C. for 30 minutes. The reaction mixture was cooled and filtered to remove insolubles.

/ The filtrate was adjusted to pH 1-2 with c. HCl. The resulting precipitate, 7-antino-3-[2-(2-carboxyethyl)-2,3-dihydros-triazolo[ 4,3-b]pyridazin-3-on-6-ylthiomethyl]-3-cephem4-carboxylic acid, was collected by filtration and washed with water. Yield: 519 og. (77#). ir: 3000-2200, 1800, 1725, 1620, 1550, l480 cm1, uv: λρΗ naxUffer 253 (ε 20θ°°), 298 nm (ε 10000). nmr: SD2°^gC°3 2.20 (2H, t, J=7<0 Hz, CHg), 3/0 (IH, d, J=17.5 Hz, 2-H), 3-85 (IH, d', J=17-5 Hz, 2-H), 4.0010 4.50 (4H, m, 3-CHg and N-CHg-), 5-01 (IH, d, J=4.0 Hz, 6-H), .40 (IH, d, J=4.0 Hz, 7-H), 6.94 (IH, d, J=10.0 Hz, pyridazine-H), 7.44 (IH, d, J=10.0 Hz, pyridazine-H).

Anal. Calc'd. for C-^H-^NgOgSg^HgO: C, 40.09; H, 3.99; N, 17.52; S, 13.37. Found: C, 40.06, 40.12; H, 3.33, 3-34; N, 16.96, 16.98; S, 13.87, 13.98. 7-ACA refers to 7-aminocephalosporanic acid and DMF to dimethylformamide.

. Example l 7-[o- (N-Butoxycarbonyl-N-me thylaminome thyl)phenylac etamid o ] 3-C2-(2-caΓboXyethyl)-2,5-dihydro-s-triazolo[4^5-b]pyΓidaztnTo a mixture of 7-a-mino-3-[2-(2-carboxyethyl)-2,5dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl]-3cephem-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 THP solution (3ml·) of 2,4-dinitro10 phenyl o-(Ii-t150°C. (dec.). d, J=5 Hz, 6-H), 5-60 (IH, d-d, J=5 & 8 Hz, the 8 Hz coupling disappeared by addition of DgO, 7-H), 6.95 (IH, d,. J=10 Hz, pyridazine-H). 7.58 (IH, d, J=10 Hz, pyridazine-H). 12.52, 12.53; S, 8.44, 8.43.

BB-S 525; 7-[o-(H-Methylaminotaethyl)phenylacetaiaido]-3-[2-(2carboxyethyl)-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6ylthiomethyl]-3-cephem-4-carb0xylic 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 fcr 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 (υ ml.) and acetonitrile (3 ml.). The stirred solution vzas adjusted at pH 4 vzith 1 II-MaOH (O.pS ml.) and diluted vzith acetonitrile (100 ml.) to give the precipitate (187 mg.), whieh vzas suspended in water (4 ml.) and adjusted at pH 9 vzith sodium hydroxide (1 H, 0.3 ml.). The solution vzas treated with a small amount of active carbon and freeze-dried to leave the monosodium salt of BB-S 525· Yield 10ό mg. (3S$) · i-i.c. > l80°C. (dec.). 3.4 (10H, m), 4.92 (IH, d, J=4.0 Hz, 6-H), 5-55 (IH, d, J=4.0 Hz, 7-H), β.95 (IK, ci, J=9-5 Hz, pyridazine-H), 7-23 (‘-MI, 3, Ph-Hy, 7.40 (IH, d, J=9.5 Hz, pyridazine-H).

Anal. Calcd. for C26H26N7°7S2' 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 oefamandole (determined by Steers' agar dilution method on Mueller-Hinton agar plate) .BB—S 479 is the compound 7-£θ-(N-butoxycarbonyl-Nmethylaminomethyl)phenylacetamidoJ-3-(2-carboxymethyl-2,310 dihydro-5-triazolo [[4,3-6jpyridazin-3-on-6-ylthiomethyl)3-cephem-4-oarboxylic acid whose preparation is given in Example 4 of Patent Specification No. Organism MIC (meg./ml) BB-S 525 BB-S 479 Cefamandole 15 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. faeoalis >100 >100 >100 E. coli NIHJ 0.2 0.1 0.1 20 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 25 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 30 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 35 Pr. morganii 0.2 0.2 0.8 Pr. rettgeri 0.8 0.8 0.1 5613 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 5 Shig. sonnei O.l O.l 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 O.l 10 B. anthracis 0.2 0.2 0.05 X» 455613 EXAMPLE 2 Substitution in the procedure of Example 1 for the 2-N-t-butoxycatbonyl-N-methylaminoniethyl-4-hy HO, ,ch2w? ch9conh-ch-ch ch. 2 I I 1 2 C—N . C0 cx CHgS ^ST^^N-CHgCHg COOH C-OH II Y and ^ziT^xM-CHgCHgCOOH 0 and CHjO .CH-NKCH, CH2C0NH-CH-GH CH. 0Z Xcx 0 C-OH respectively.

Claims (13)

1. wherein R represents CH 2 NHR* λ /~CH 3 COOR CHgWHR* OR -CH 2 C0_^CH 2 NHR ! l^ sz LcH a COWherein R is hydrogen, hydroxy or methoxy; R* is hydrogen or methyl; and is hydrogen, or a conventional, pharmaceutically acceptable, easily hydrolyzed ester forming group; or a non-toxic, pharmaceutically acceptable salt thereof.

2. , The pivaioyloxymethyl-, acetoxymethyl-, methoxymethyl», acetonyl-, phenacyl-, ρ-nitrobenzyl-, β,β,β-trichloroethyl-,

3. -phthalidyl- or 5-indanyl- esters of a compound as claimed in Claim 1. 3. A compound of Claim 1 or 2, wherein R is t 2 NHR> CHgCO· 43613 wherein R is hydrogen, hydroxy or methoxy and S' is hydrogen or methyl, ‘

4. A compound of Claim 1 or 2 wherein R 1 is CH 2 C05 wherein R' is hydrogen or methyl. A compound of Claim 1 or 2, wherein R 1 is -CH 2 NHR' CH 2 C0wherein R' is hydrogen or methyl. 6. A compound of Claim 3, wherein R is hydrogen. 10 7. A compound of Claim 3, wherein R is hydroxy. 8. A compound of Claim 3, wherein R is methoxy. g. A compound of any one of Claims 1-8, wherein R 1 is hydrogen. 10. A compound of any one of Claims 1 to 8, wherein R 1 15 is methyl. ll. A process for the preparation of a compound of the formula wherein R 1 represents σΟΗ 2 ΝΗΚ' CH,COOR ch 2 nhr· OR -CH 2 C0CH 2 NHR' CHgCO10 , - or methyl wherein S is hydrogen, hydroxy or methoxy; R is hydrogen/and R is hydrogen, ora conventional pharmaceutically acceptable, easily hydrolysed ester forming group or a non-toxic pharmaceutically acceptable salt thereof characterized by reacting a compound of the formula s Y-NH-CH-CH 'CHp 0 I i I II // <;i> C-CH 2 0-C-CH3 COOH in which Y is H or R 1 , as defined above, or a salt or easily hydrolyzable ester thereof with a compound of the formula HS N n-(ch 2 ). 2 cogh and, if Ϊ is H, treating the resulting compound with an acylating agent of the formula R 1 — X in which X is halogen or a functional equivalent thereof and R 1 is as defined above, and, if desired, converting the resulting free acid, salt or easily hydrolyzable ester of a compound of lo 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 1 to the corresponding free acid, of the formula I. 12. The process according to Claim 11, wherein a resulting 15 free acid of formula I is converted to an ester selected from the pivaioyloxymethyl-, acetoxymethyl, methoxymethyl-, acetonyl-, phenacyl-, ρ-nitrobenzyl-, β,β,β-trichloroethyl-, 3phthalidyl- or 5-indanyl-esters. 13. The process of Claims llorl2, wherein R 1 is JJHpHHR' ‘'χρν»· wherein Ή is hydrogen, hydroxy or methoxy and R' is hydrogen or methyl. 14, The process of Claims 11 or 12, wherein R 1 is aCH 2 NHR' ch 2 co5 wherein R' is hydrogen or methyl. 15. The process of Claims ll or 12, wherein R 1 is .CH 2 NHR' ®2C°wherein R' is hydrogen or methyl. lg,· The process of Claim 13, wherein R is hydrogen. 10 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. 43613 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

5. 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 non-toxic dose of 250-1000 mg. of a compound according to any one of Claims 1 to 10 or 21; or

6. 10 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

7. 15 composition comprising a compound according to any one cf Claims I to 10 or 21; or a non-toxic pharmaceutically acceptable palt thereof. 24. A method for combatting B. anthracis infections which comprises administering to a non-human warm-blooded mammal

8. 20 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 1 to 10, or 21; or a non-toxic, pharmaceutically acceptable salt thereof.

9. 25 25. A compound according to Claim 1 substantially as described hereinbefore with particular reference to either of the accompanying Examples.

10. 26. A compound according to Claim 1 which is any of the compounds specifically identified hereinbefore other than a 30 compound according to Claim 25. 43 613

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

12. 28. A compound according to Claim 1 when prepared by a 5 process according to any-of Claims 11 to 20 or 27.

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