CA1037467A - 7-(.alpha.-(2-AMINOMETHYL-1-CYCLOHEXENYL AND 1,4-CYCLOHEXADIENYL)-ACETAMIDO)-3-HETEROCYCLICTHIOMETHYL-3-CEPHEM-4-CARBOXYLIC ACIDS AND DERIVATIVES THEREOF - Google Patents

Abstract

ABSTRACT OF DISCLOSURE

This invention relates to novel 7-(a-(2-Aminomethyll-cyclohexenyl and 1,4-cyclohexadienyl) -acetamido)-3-heterocyclic thiomethyl-3-cephem-4-carboxylic acids, and their nontoxic, pharmaceutically acceptable salts and their Schiff bases, as made by reaction of salicylaldehyde with the free amino group, which are valuable as antibacterial agents and are particularly valuable as therapeutic agents in poultry and animals, including man, in the treatment of infectious diseases caused by many Gram-positive and Gram-negative bacteria.

Description

Thi~ invention relates to novel 7-[~-(2-Aminomethyl-l-cyclohexenyl and 1,4-cyclohexadienyl)-acetamido]-3-heterocyclic thiomethyl-3-ee~hem-4-carbo~ylic acids, and their nontoxic, pharmaceutically acceptable salts and their Schiff bases, as made by reaction of salicylaldehyde with the free amino group, which are valuable as antibacterial agents and are particularly valuable as therapeutic agents in poultry and animals, including man, in the treatment of infectious diseases caused by many Gram-positive and Gram-negative bacteria.
- The novel cephalo~porin derivatives of this invention : compxise the compounds of the formula CH2NEi2 CH2CONH ~ S ~
O ~ ~ CH2-S-R
COOH

wherein R is OH
N -N

OH ~ N ~ 5 ~ CH , 3 ~ HN~-- S~ ~s~L CH20~

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., . . . ,. . , , , ., . , ~,. .. .

and their nontoxic, ph ~ Q3ceu~ically acceptable salts and Schiff bases, as with salicylaldehyde.
In the preerred embodiments o~ the present invention R is HO

N~ 01~ and ~sp~ci.~l.ly _~J ~ ,N

Cff3 Such ~alt~ include carbox~lic acid salts lnoluding nontoxlc metallic salk~ such a~ sodlum, potasslum, calcium and al~minum~ the ammoniu~ salt and ~ubstltuted ammonlum ~a.lt~, e.~. ~al~ Or such nontoxic amlne~ a~ trialkylamines~ includlng tri-ethylamine, procaine, dibenzylamine, N-benzyl-beta-phenethylamine, l-ephenamine, N,N'-dibenzyl-ethylenediamine, dehydroabietylamlne, N,N'-bis-dehydroa~ietylethylenediamine, N-(lower)-alkyl-piperidine, e.g. N-ethylpiperidine3 and ~ther amines which have been used to ~orm salts with benzsrlpeniclllirl, and ln all cases the nontoxic, aoid addition salts thereof (i.e., the amlne ~alts) includlng the mlneral acid addition salts such as the hydrochloride, hydrobromide, hydroiodide~ sulfate, ~ul~amate and pho~phate and the organic acid addition ~alts such as the maleate, acetate, citrate, oxalate, ~uccinate, benzoate, tartrate, fumarate~ malate, mandela~e, a~corbate and the lilce.

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The present invention also includes a process for the preparation of a compound of formula I which process comprises either A~ reacting a compound of the -Formula ~ ~ CH2~S-R II
O CooH

wherein R haq the meaning set out above or an easily cleavable ester or salt thereof with an acylating derivati~e of an acid having the formula ~ ~2N~
III

wherein B represents an amino-protecting group and removing said amino-protecting group to produce the des.ired compound of formula I or an easily cleavable ester or pharmaceutically acceptable salt thereof and, if desired, either before or after removal of B ea) converting by methods known per se the product in the for.m of the free acid or salt thereof to the corresponding Schiff base, easily cleavable ester or pharmaceutically acceptable salt thereof or (b) converting by methods known E~r se the product in the form of an easily cleavable ester or salt thereof to the corresponding free ac.id compound or pharma-ceutically acceptable salt thereof or B) reacting a compound of the formula ~ 7~
~ 2 2 ~--CH2C - NH ~f S
N ~ CH20COCH3 CO~H IV

or an easily cleavable ester or salt thereof with a thiol of the formula HS - R
V
wherein R is as defined above or a salt thereoE to ~o.rm a compoun~ of formula I or an easily cleava~le ester or pharmaceutically acceptable salt thereof and, .iE d~s:ired, (a) con~erting by methods known ~ e the product in l:he form of the free acid or salt thereof to the correspondin~
Schiff base, easily cleavable ester or pha~.aceut.ically acceptable salt thereof or (b) converting by methods known per se the product in the form of an easily cleavable ester or salt thereof to the correspondin~ free acid compound or pharmaceutically accepta~le salt thereof.
In one method of preparing the novel cephalosporin c~mpounds of th~ present invention, a 3-thiolated-7-amino-cephalosporanic acid compound of formula II or an easily cleavable ester or salt of said acid or ester is acylated with the appropriate a-(2-aminomethyl-1-cyclohexenyl or 1,4-cyclohexadienyl-acPtic acid acylat.ing derivativ~ of :~ormula III.

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The 3-thiolated-7-aminocephalosporanic acid intermediate of formula II may be prepared by displacement of the 3-acetoxy group of 7-aminocephalosporanic acid or a salt thereof with the appropriate heterocyclic thiol or a salt thereof. The displacement of an ester group with a thiol group is a known reaction and is preferably accomplished in aqueous solution with heating.
The claimed compounds may then be obtained by acylation according to known methods of the 7-amino group of intermediate II with the acylating agent of formula III.

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The intermediate II may, if desired, be canverted prior to the acylation reaction to an easily cleavable ester or acld addition salt thereof. The procedures ~or ~reparinq such esters are disclosed in the literature and are well~known to!
those skilled in the axt of penicillin and cephalosporin chemistry. One preferred method especially useful for pre-paring the most preferred easily hydrolyzed esters, i.e. the pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl and phenacyl esters, is disclosed in U.S. Patent 3,284,451.
This reference describes the esterification of sodium cepha-lothin with the appropriate active chloro or brorno compound (e.g. phenacyl bromide, chloroacetone, chloromethyl ether, pivaloyloxymethyl chloride, acetoxymethyl chloride) eollowed by enzymatic removal of the thienylacetic acid sidechain~
In another good method the triethylamine ~alt o~ 7-amino-cephalosporanic acid is reac~ed directly with the active halogen compound a~ in U. ~C. Patent 1,229,453. ~ more preferred method is the conversion of the compound of formula II to a silyl e~ter as by the methods de~cribed in the literature, e.g. U.S. Pat-ent 3,249,622~ The silyl ester group may be removed following the acylation reaction by hydrolysis or alcoholysis.
Prior to the acylation reaction the amino group of the acylating agent III is protected by a conventional amino-blocking group B of the type used in either peptide syntheses or in any o~ the numerous syntheses of a-amino-benzylpenicillin from

2-phenylglycine which may be readily removed at the conclusion of of the reaction.- Examples of suitable amino-protecting or blocking groups include t-butoxycarbonyl, carbobenzyloxy, 2-hydroxy-l-naphthcarbonyl, trichloroethoxycarbonyl, 2-ethoxy-carbonyl-l-methylvinyl and 2-methoxycarbonyl-1-methylvinyl.

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A particularly valuable blocking group is a proton, as in the compound of the formula (~I2NH2 HCl .. ' ~ 11 , ~2C-Cl rhe preferred amino~protecting groups are t-butoxycar~onyl, the proton and a B-d~ketone or a 3-~etoester as in U. K.
Patent 1,123/333 or U. S. Patents 3,325,479 and 3,316,247, e.g. methyl acetoacetate, or a B-ketoamide as in Ja~an 71/24714. When the t-butox~carhonyl, ~-ketoester, ~-~iXetone or ~-ketoamide protecting grouDs are em~loyed, it i5 ~referred to convert the acylatin~ acid containing the blocked a~.ino group to a mixed anhydricle, e.a with ~t~yl or isob-lt~l chloroformatel berore reaction with com~und II or an e.ster or salt thereof~ After the acvlation coilpl~ng reaction, the amino~~rotecting grouD B may be removed ~y methods known per se to form the desired ~roduct of formul~ I. Thus, for example, the t-butoxycarbonyl grou~ may he removed by use.of : formic ac;d~ the carbobenzylox~ grou~ by catalytic hydrogena-tion, the 2-hydroxy-1-na~hthcarbon~l grou~ by acid hy~rolysi the trichloroethoxycarbonyl grou~ by treatment with zinc dust in glacial aretic acid, the proton by neutralization, etc.
Obviously other functionally e~uivalent blocking groups for an amino grou~ can be used and such grol~s are consiaered within the scope of this invention.
Acylat.ion of a 7-amino grou~ of a cephalosDorin is a well-known reaction and any of the functional eauivalents of formula III commonly used as acyla~ing agents for primary .... . , , :
,~... .
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amino groups may be em~loyed. Exa~ples of suitable acylati~
derivatives of the ~ree aci~ include th~ correspon~in~ acid anhydrides, mixed anhydrides, e.g. alXoxyformic anhydrides, acid halides, acid azides, active esters and ~ctive thio-esters. The free acid may be coupled with`comDouna II after first reacting said free acia with N,N'-dimethylchloroformi-miniu~ chloride or by the use of enzymes or of an N,N'-carbonyldiimidazole or an N,N'-carbonylditriazole or a carbodiimide reagent, e.g. N,N'-diisopropylcarbodiimide.
N,N'-dicyclohexylcarbodiimide or N-cyclohexyl~N'-(2-morphilinoethyl) carbodiimide; cf. Sheehan and Hess, J. Amer.
Chem. Soc., 77, 1967 (1955)], or of alkylylamine reagent [cf~ R. Buijle and H. G. Viehe, Anqew.,Chem. International Edition 3, 582, (1964)~, or of an isoxa~olium salt reag~nt [cf. R. B. Woodwaxd, R. A. Olof~on and EI. Mayer, ~. Amer.
Chem. Soc. 83, 1010 (1961)~, or of a ketenimine reayent [cf. C. L. Stevens and M. F. Munk, J. Amer. Chem. Soc.
80, 4065 (1958)] or of hexachlorocyclotriphosphatriazine or hexabromocyclotriphosphatriazine (U.S. 3,651,050) or of diphenylphosphoryl azide [DPPA; J. Amer. chem. 50c., 94 6~03-6205 (1972)]. Another equivalent of the free acid is a corresponding azolide, i.e., an amide of the corresponding acid who~e amide nitrogen is a member of a quasiaromatic five m~mbered ri~g containirg at least two nitrogen atoms~ i.e., imida201de, pyrazole, the triazoles, benzimidazole, benzotriazole and their substituted derivatives. As an example of the general method for the preparation of an azolide. N,NI-carbonyl-diimidazole is reacted with a carbo~lic acid in equimolar proportions at room temperature in tetrahydrofuran, chloroform, dimethyl~ormamide or a ~imilar inert solvent to form the :: ,. ~ ..

--'`\ :
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carboxylic acid imidazolide in practically quantitative yield with liberation of carbon dioxide and one mole of imidazole.
Dicarboxylic acids yield dimidazolide. The by-product, imidazole, 'precipitates and may be separated and the imidazolide isolated, ' but this is not essential. A particularly preferred acylating ;' agent is the acid chloride hydrochloride of the formula ~' :
CH NH2 HCl ,' ~ .
CE2COCl ' 10 ~, which also serves a dual function of carboxyl activation and amino protection. Mention was made above of the use of enzymes to couple the free acid with its blocked amino group with compound II. Enclosed in the scope of such processes are the use of an ester, e.g. the methyl ester, o~ that Er~e acid with enzymes provided by ~arious microoxganisms, 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. An't'ibi'o'tics (Japan), 24(5), 321-323 (1971) and in West Germany 2,216,113.
The particular process conditions, e.g. temperature, solvent, reaction time, etc. selected for the coupling re-action are determined by the nature of the acylation method used and are known to those skilled in the artO Generally ' it is useful ~o add an organic tertiary amine, e.g. triethyl-amine, N,N-dimethylaniline, ethylpiperidine, 2,6-lutidine ' or quinoline, to serve as a proton acceptor or salt-forming agent.

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The compounds o~ the present invention may be isolated in any of the ways customarily employed for the isolation of similar cephalosporins. Thus, the product may be obtained ~- as the neutral molecule, although this is probably more accurately represented as the zwitterion, or it may be isolated as a salt. Formation of the desired pharmaceutically accept- .
able carboxylic acid or acid addition salt is carried out by known methods, e.g. reaction of the acid with an appropri-ate base or acid.
At the conclusion of the acylation reaction the pro-duct obtained may be converted (before or after removal of the amino-protecting group) by methods known per se to another desired product of formula I. Thus, the compound o~ formula I in the form o the free ac:id or a sal.t thereof ma~ be con-: verted by known methods to the corre~pon~ing Schi~ base, easily cleavable ester or pharmaceutically acceptable salt thereof.
Similarly, the product of formula I in the form of an easily cleavable ester of salt thereof may be converted to the free acid product or pharmaceutically acceptable salt thereof by removal of the esterifying group, e.g. by aqueous or enzymatic hydrolysis (as with human or animal serum) or acidic or alkaline hydrolysis or by cata-lytic hydrogenation or by treatment with sodium thiophenoxide as taught in U.S. Patent 3,284,451.
., In another method of preparing the compounds of the present invention, 7-aminocephalosporanic acid or a salt thereof ,~ is acylated with the acid of formula III or an acylating de-I rivative thereof to form a 7-acylated cephalosporin compound of i the formula ., ~ CH2NH2 CH~ - C - NH r s ~ N ~ CH20COCH3 IV

Compound IV in the form of the free acid or an easily cleav-able ester or salt thereof is then reacted according to the proceqs of the present invention with a heterocyclic th.iol of formula V or a salt thereof, most preferably the sodium or pota~sium salt. The displacement reaction of such an acetoxy group with ~uch a thiol is a well-k~own reaction and may be accomplished in ~olution as in water or a~ueous acetone at a temperature o at least room tempera-ture and preferably within the range of about 50 to about 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. The product of the displacement reaction may, .- if de~ired, be converted to a pharmaceutically acce~table salt m ~ by treatment with an appropriate acid or base. As in the case of the alternate process described above for preparatlon of the . ,.; .
compound~ of fo~mula I, the product .in the form of the ~free acid or salt thereof may be converted to the corresponding Schiff . base, easily cleavable ester or pharmaceutically acceptable salt : thereof Or, alternatively, the product in the form of an easlly .' ' ~.
.

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cleavable ester or salt thereof may be converted to the free acid or pharmaceutically acceptable salt thereof.
The Schiff bases of the compoundof formula I, parti-cularly the salicylaldehyde Schiff base, are also useful as active antibacterial agents. They are prepared by reacting the free amino compound of the formula I with an aldehyde and, prefer-ably, salicylaldehyde. The reaction is normally carried out at ambient or elevated temperakures in an inert solvent, e.g.
benzene or toluenel where water may be removed by azeotropic distillation or in the presence of a water scavenger, e.g.
potassium carbonate.
The easily cleavable esters of the compound o formula I are useful as intermediates in the production o~ the free acid product. The pivaloyloxymethyl, acetoxymethyl and me-thoxymethyl esters are also useful as active antibact~rial agents since on oral administration they are rapidly hydrolyzed to the active metabolite. These esters are of interest because they provide en oral administration different rates and amounts of absorption and give differing concentrations of the active antibacterial agent in blood and tissues.
The pharmaceutically active compounds of the present invention are potent antibacterial agents useful in the treat-ment of infectious diseases in poultry and animals, including -~
man, caused by many Gram-positive and Gram-negative bacteria .
The active compounds are also of value as nutritional supple-ments in animal feeds and as agents for the treatment of mastitis in cattle. The preferred compounds have also been unexpectedly ~, found to be efficiently absorbed upon oral administration.
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- The novel medicaments provided by the present invention ;
may be formulated as pharmaceutical compositions comprising, in addition to the active ingredient, a pharmaceutically accept-able carrier or diluent. The compounds may be administered both orally and parenterally. The pharmaceutical preparations may be in solid form such as capsules, tablets or dragees, or in liquid form such as solutions, suspensions or emulsions. In the treatment of bacterial infections in man, the compounds of this invention may be administered parenterally in an amount of from about 5 to 200 mg./kg./day and preferably about 5 to 20 mg./kg./day in divided dosage, e.g. 3 to 4 times a day. They are administered in dosage units containing e.y. 125, 250 or 500 mg. of active in~redient with suitable physiologically ;~
acceptable c~rri~rs or excipients. The dosacJ~ un:its are :ln the form of liquid preparations such as solutlon~ or suspensions.
Certain 3-substituted 7-[~-(2-aminomethylphenyl)-acetamido]cephalosporanic acid derivatives (A; see Netherlands 72/06326, Farmdoc 76374T, carresponding to U.S.S.N. 142,337 filed May 11, 1971) provide a series of parenteral-use cephalosporins which are very effective derivatives with a broad spectrum o activity. Their limited water solubility (~ 2mg./ml.) has, however, caused crystalluria formation even when the antibiotics had been administered parenterally as a readily-dissociable soluble derivative. The object of the present invention was, therefore, to obtain equally active compounds which show higher water solubility as the zwitterion form.

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CH NH

CH2C-Nr~ S~
O ~ N ~ CH2-S-R

(A) There is provided by the present invention certain 7-[~-(2-aminomethyl-1,4-cyclohexadienyl and 1,4-cyclohexadienyl)-acetamido]cephalosporanic acids (B). -CH N
, Ul FI2CO-N ~
N ~ ~-- CH~-S R

(B) C 2 Many of the new series were found to be more soluble than the corresponding phenyl derivatives and to have a solubilit~
of more than 7 mg./ml. determined routinely in 0.1 M pH 7.0 phosphate buffer, as shown below compared with that of certain . corresponding phenyl derivatives.

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(1) This compound ~also ca~lPd BB-S150) is disclosed else~here by the present applicant.

(2) This compound (also called BB-S226) is disclosed ;~
elsewhere by the present applicant, (3) This compound (also called MR-S94) is claimed in (see Netherlands 72/06326; Farmdoc 76,37AT) U. S. Patent No. 3,766,175, issued October 16, 1973.

(4) This compound (also called MR-S96) is also claimed in U. 5. Patent No. 3,766,175.
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The following examples are given in illustration of, but not in limitation of, the present invention~ ;
All temperatures are given in degrees Centigrade.
"Skellysolve B" is a petroleum ether fraction of B.P.
60-68 C. consisting essentially of n-hexane. IR-120 is also called Amberlite IR-120 and is a strong cation exchange resin containing sulfonic acid radicals.
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-linked with ; divinyl benzene obtained by the procedure given by Kunin, Ion Exchange Resins, 2nd. Edition (1958), John Wiley and Sons, Inc.

2,4-Dinitrophenol is represented as 2,4-D~P, N,N'-dicyclohexylcarbodiimide as DCC, trifluoroacetic aald as TFA, tetrahydrofuran as THF, t-butoxycarbonyl azide as t-BuOCON3 and t-butoxycarbonyl as t-BOC.

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Preparation of Starting Materials r~_ ( 2-Aminomethyl-1,4-cyclohexadienyl)aCetic- _id t2) A solution of 16.5 g. (0.1 mole) of _-aminomethyl-phenylacetic 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 10 evaporated to dryness below 40C. The residue was dissolved in 500 ml. o~ water and the solution was chromatographed on a column of IR-120 (H , 700 ml.) resin and eluted with 1% NH40H
solution. Ninhydrin positive fractions o~ khe eluate were combined and evaporated to dryness. The residue was washed with four 50 ml. portions o~ hot acetone and recrystallized ~rom 500 ml. of ethanol-water (1:1) to give 11.2 g. (67%) of colorless needles, 2. M.p. 183C.
IR: ~ mU~l 1630, 1520, 1380, 1356 cm 1.

NMR:S D2O + K2CO3 2-72 (4H, s, _2C ), 3.01 (2H, s, CH2CO), 3.20 (2H, s, CH2-N), 5.78 (2H, s, -,~C=).
Anal. Calcd. for CgH13NO2: C, 64.65; H, 7.84; N, 8.38.
Found: C, 64.77; H~ 8.06; N, 8.44.
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Alternative Procedure for the Prepara*ion of ~-(2-aminomethyl-1,4-cyclohexadienyl)-acetic acid 2 2 '`
-CH2c2H Li, tert. BuO~
NH3 - TEA-HCl 2CO2H ~ I~iCl + (c2H5)3N
. ~ CH2NH2 ; 10 The procedure used by Welch, Dolfini and Giarrusso in U.S. patent 3,720,665 (Example 1) to make D-2-amino-2-(l,~l-cyclohexadienyl)acetic acid was adapted. A solution of 830 ml. of distilled liquid ammonia was dried with 40 mg. of lithium under an argon atmosphere. To this stirred solution was added ll.0 g. (0.07 mole) of 2-ami~nomethylphenylacet:Lc acid a~d 3~0 ml.
o tert. but~l alcohol. A total of 1.6 g. (0.225 mole) o~
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 t'ert.
butyl alcohol was removed at ~0 (15 mm.) to yield a white residue which was dried in vacuo over P2O5 overnight. The solid was ddssolved in 30 ml. of 1:1 methanol-water and added with ~,:

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stirring to 3.5 1. of 1:1 chloroform-acetone at 5. The mixture was stirred for 20 min. and the amino acid 2, ~ -(2-aminomethyl-1,4-cyelohexadienyl)acetie aeid, was eollected and dried for 16 hours in vacuo over P2O to yield 6.3 g. (58%) ~ 5 of white crystals, m.p. 190 decomp. The IR and NMR spectra were consistent for the strueture.

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~-[2-(~-Butoxycarbonylaminomethyl)-1,4-cyclohexadienyl]acetic acid (1) ; --To a stirred solution of 8.0 g. (0.048 mole) of ~-(2-aminomethyl-1,4-cyclohexadienyl)acetic acid and 3.8 g.
(0.096 mole) of NaOH in 150 ml. of water was added a solution of 10.3 g. (0.072 mole) of t-butoxycarbonylazide in 80 ml. of THF and the mixture was stirred for 18 hours at room temperature.
The THF was removed under reduced pressure and the residual solution was washed with ether (2 x 100 ml.), acidified with 6 N HCl and extracted with ether (3 x 100 ml.). The combined extracts were washed with water (2 x 100 ml.) and a s~turated NaCl solution (100 ml.), dried with Na2SO4 and evaporated to dryness. The oily residue was triturated with n-hexan~ to give 10.5 g. (82~) of colorless powder 1 melting at 113 C.
IR:~ nllj 3370, 1715, 1640, 1530, 1230, 1160 cm 1.
NMR:~¢ pCpCL3 1-45 (9H, s, t-Bu-_), 2-73 l4H, s~
H2C ) 3.16 (2H, s, CH2CO), 3.76 (2H, d, 6Hz, CH2N) 4.90 (lH, m, NH), 5.66 (2H, s, - > C=), 10.6 (lH, br-s, COOH).
Anal- Calcd- for C14H21N4 C~ 62.90; H~ 7.92; N~ 5-24 Found: C, 63.13; H, 8.21; N, 5.26.

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6~' [2-(N-t-Butoxycarbonylamino)methyl-l-cyclohexen-l-yl]- acetic acid (2) A solution of [2-(N-t-butoxycarbonylamino)methyl-1,4-cyclohexadien-1-yl]acetic acid (1), (1.33 g., 5 mmoles) in 3% ammonium hydroxide (10 ml.) was hydrogenated at 40 psi with palladium on charcoal (10%, 0.2 g~). A theoretical amount of hydrogen was taken up in 3 hours. The catalyst was removed and the filtrate was acidified to pH 2 with dil. HCl and ex-tracted with ethyl acetate (2 x 50 ml.). The combined extracts were washed with water (20 ml.), dried with Na2SO4 and evaporated under reduced pressure to afford an oil (1.34 g.) which solid-ified on standing for several days. Recrystallization from n-hexane - ethyl acetate gave 1.2 g. (90~) of 2 as colorless prisms melting at 118-119C.
IR: ~ ax 3450~ 1730, 1660, 1510 ~m NMR ~ CDC13 1.58 (9H, s, t-butyl H), l-50 - 1.90 (4H, m, -CH2-), 1.90 - 2.20 (4H, m, allylic methylene-H), 3.18 (2H, s, CH2-CO), 3.78 (2H, d, 6 Hz, CH2-N), 5.00 (lH, br-s, NH), 8.98 (lH br-s, COOH).
Anal. Calcd. for C14H23NO4: C, 62.43; H, 8.61; N, 5.20.
Found: C, 62.12; H, 8.77; N, 5.37 . . .:
, . , . . .

~L~37g~
~-[2-(t-Butoxycarbonylaminomethyl)-1,4-cyclohexadienyl]acetic acid (3) To a stirred solution of 8.0 g. (0.048 mole) o~
2 and 3.8 g. (0.096 mole) of NaOH in 150 ml. of water was added a solution of 10.3 g. (0.072 mole) of t-butoxycarbonyl-azide in 80 ml. of THF and the mixture was stirred ~or 18 hours ; at room temperature. The THF was removed under reduced pressure and the residual solution was washed with ether (2 x 100 ml.), acidified with 6 N HCl and extracted with ether (3 x lQ0 ml.).
The combined extracts were washed with water (2 x 100 ml.) and a saturated NaCl solution (100 ml.), dried with Na2SO4 and evaporated to dryness. The oily residue was triturated with n-hexane to give 10.5 g. (82%) of colorless powder 3 melting at 113C.
IR:~(maUx 3370, 1715 " 640, 1530, 1280, 1160 cm " NMR:~ppDml3 1.45 (9M, s, t-Bu-H), 2.73 (4H, s/ H2C / ), 3.16 (2H, s, CH2CO), 3.76 (2H, d, 6Hz, CH2N) 4.90 (lH, m, NH),

5.66 (2H, s, H~ C=), 10.6 (lH, br-s, COOH.) Anal- Ca]~d- for C14N21N4 C~ 62-90; H~ 7-92;
N, 5.24.
Found: C, 63.13; H, 8.21; N, 5026.
Sodium ~2-[N-(l-carbethoxypropen-2-yl)aminomethyl]-1,4-cyclo-hexadienyl3 acetate (4) To a stirred solution of 460 mg. (0.02 mole) of metallic sodium in 100 ml. of absolute EtOH was added 3.34 g.
(0.02 mole) of 2 and 3.1 g. (0.024 mole) of ` 30 .~, '. '' ~ - 24 - 25 -~7~
ethyl acetoacetate and the mlxture ~as heated to re~lux for 4 hours with stirring. The hot reaction mixture was flltered and the fl~trate was allowed to keep cold overnight to give 2.0 g~ Or colorless needles 4 meltin~ at 264 C. The additior~l product (3.3 g.) was o~alned by concentration of the mother llqulcl. The kotal ~ield ~/as 5.3 g. (88~).
IR:~ ma~ 33~ 1635~ 1600J 1570~ 1~00~ 1275, 1170, lOgo cm~l NMR: ~ ~m 1.23 (3H, t, 7Hz, ~2~1~), 1.96 & 2-25 (3H, s, C=C-C~3, cis & trans)~ 2.70 (!~H, s, ~T2C'~), 3.04 (2H~ SJ C~2CO) ~ 3.66 & 3.95 (2~ t CT 2~N~ C1S &;
trans), 4.07 (21I, q, 7Hæ, ~ ), 4,45 ~ 1~.56 (lI~I, F:I, -~ J CiS & trans), 5.76 (2~I, s~ iI ~ ).

Anal; Calcd- for C15~20N4Na C~ 59-79; H, 9;
N, 4.64.
Found: CJ 59.69; H, 6.76; N, 4.75.

.

~3~
7-Amino-3-(3-hydroxypyridazin-6 ylthiomethyl)-3-cephem-4-.
; carboxylic acid (4a) -N ~ S
N-N
N ~ ~ C~2-S -- ~ ~ OH

3-Chloro-6-hydroxypyridazine A mixture of 22.47 g. (0.15 mole) of 3,6-dichloro-pyridazine and 50 ml. of acetic acid was refluxed for two hours.
; The reaction mixture was cooled and diluted with 50 ml. of water and then concentra-ted to dryness under reduced pressure.
The residue was crys;tallized from wa-ter to give 15.8 g. (80~) of 3-chloro-6-hydroxypyridazine as colorless prisms which m~lted at 133-7 C. (lit. 138-lA0C.). See N. Takabayash:;, Yakugaku ; Zasshi, 75, 778 (1955).
3-Hydroxy-6-mercaptopyridazine A mixture of 2.6 y. (0.02 mole) of 3-chloro-6-hydroxy-pyridazine and 5.0 g. (0.07 mole) of freshly prepared potassium i~
hydrogen sulfide in 30 ml. of e-thanol was heated at 130-140 C.
in a sealed tube for 6 hours. The reaction mixture was cooled and diluted with 200 ml. of water. Almost all organic solvent was removed by distillation under reduced pressure. The residual aqueous solution was acidified with dilute hydrochloric acid :
.' `. .

,;, : . . -- i.... .

~7~7 to pH 3 and extracted with ethyl acetate (6 x 50 ml.). The combined extracts were evaporated to dryness and the residue was reprecipitated from 30 ml; of ethanol-ligroin (1:1) to give 2.2 g. (87%) of amorphous 3-hydroxy-6- mercaptopyridazine.
m.p. 158 - 159 C. (lit. 157-158 C.).
See J. Druey et al., ~Ielv. Chem. Acta. 37, 121 (1954).
3-Hydroxy-6-mercaptopyridazine To a solution of 5.6 g. (0.05 mole) o~ 3,6-di hydroxypyridazine in 150 ml. o~ pyridine was added portion-wise 2~70 g. (0.012 mole) of phosphorus pentasul~ide with vigorous stirring under refluxing. The refluxing was continued for one hour and then the reaction mixture was diluted with 200 ml. of water and concentrated to remove the pyridine. The resulting oily res:idue was suspended in water ancl cxtractecl w:ith ethyl acetate. The oryanic extracts were combined and concen-trated again to give oily material which was triturated with a small amount of water to give 3-hydroxy-6-mercaptopyridazine as a yellow solid. Recrystallization from water afforded 0.62 g.
(12%) o~ the product which was identical with that prepared above.
',,.

~ , 9L~ 6'7 3,6-Dihydroxypyridazine To a boiling solution of 315 g. (3 moles) of hydrazine dihydrochloride in 2 L of water was added portionwise 295 g.
(3 moles) of finely ground maleic anhydride with stirringO After the addition was completed the heating was continued for 4 hours and then allowed to stand overnight in a refrigerator to give 285 g. (85%) of 3,6 dihydroxypyridazine as massive pillars.
; m.p. ~ 290C.
3,6-Dichloropyridazine A mixture of 150 g. (1.33 moles) of 3,6-dihydroxy-pyridazine and 250 g. of phosphorus oxychloride was refluxed for 3 hours under proteetion from moisture. The axcess of phosphorus oxychloride was removed under reduced pressure and ; the dark residue wa9 poured into one Kg. O:e crughccl ice. The resulting precip:itate wa~ collectecl by Eiltration. The s~eond ; crop of the product was obtained from the mother liquor by extraction with five 300 ml. portions of chloroform followed by treating with 1~. of eharcoal and evaporating the solvent. The first and seeond crops were eombined, dissolved in 500 ml. of chloroform and treated again with one g. of charcoal and eon-centrated to give 165 g. (83%) of 3,6-dichloropyridazine as fine needles melting at 60-61C. (in a sealed tube).

- A suspen3ion of 60 g. (0.4 mole) of 3,6-dichloro-~; pyridazine in 200 ml. of 10% hydrochloric acid was refluxed :, ' .

i' 30 ~

.

. . .

for 2 hours until a clear solution was obtained. The clear solution was treated with ca. 1.5 g. of active carbon and filter-ed. The filtrate was concentrated under reduced pressure to give

6-chloro-3-hydroxypyridazine as colorless needles. Yield 49.5 g. (98%). M.p. 137-139C.
3-Hy~o~,- -~ ~ yridazine A mixture of 50 g. (0.38 mole) of 6-chloro-3-hydroxy-pyridazine, 60 g. (0.33 mole) of potassium hydrogen sulfide in 250 ml. of ethanol was heated in a 500 ml. autoclave at 140C.
for 14 hours with magnetic skirring. The pressure reached to 15-20 Kg./cm2. The mixture was evaporated to dryness and the residue was dissolved in 300 ml. of water. The aqueous solution was acidified with dil. hydrochloric acid to pII 3 and extracted with ten 200 ml. portions of ethyl acetate. Th~ combined extracts were concentrated to gi~e 34.3 g. (70~) of amorphous 3-hydroxy-6-mercaptopyridazine. M.p. 151-152C.

7-Amino-3-(6-hydroxypyridazin-3-ylthiomethyl)-3-cephem-4-carboxylic acid ,.
A mixkure of 0.60 g.(0.0047 mole) of 3-hydroxy-6-mercaptopyridazine, 1.27 g. (0.0047 mole) of 7-amino-cephalosporanic acid, 0.73 g. (0.0094 mole) of sodium bicarbonate in 25 ml. of O.I M phosphate buffer ~pH 6.4) was heated at 60~C.

. , .

,, for 5 hours. The reaction mixture was filtered to remove a trace of insoluble material and the filtrate was adjusted to plI
5 with acetic acid to give brown precipitates, which were collected by filtration, washed with water and acetone successively and dried in vacuo to give 7-amino-3-(6-hydroxypyridazin-3-., ylthiomethyl)-3-cephem-4-carboxylic acid; 1.03 g. t71%). M.p.
` 290-300C. (decomp.).

; IR: ~ KBx 1805, 1680, 1650, 1580, 1415 cm 1.
UV ~ 1% NaOH 249 nm (~ 19400)-max NMR:~ ppm K2C3 3.22 (lH, d, l9Hz), 3.37 (lH, d, 14Hz), 3.65 (lH, d, 14 Hz), 3.72 (lH, d, 19 Hz), 4.90 (lH, d, 4 Hz), 5.30 (lH, d, 4 Hz), 6.75 (lH, d, 10 Hz), 7.30 (lH, d~
10 Hz).
Anal Calcd- for C12H12N4O4S2 1/2EI2 H, 3~75; N, 16.04; S, 1~.36.
~ 'ound: C, 41.45; ~, 3.70; N, 15.83; S, 18.03.
7-Amino-3`-(3-hydroxypyridazine-6-ylthiomethyl)-3-cephem-4-carboxylic acid.
A mixture of 141 g. (0.52 moles) of 7-ACA, 92 g.
(1.1 moles) of sodium bicarbonate and 73 g. (0.57 moles) of 3-hydroxy-6-mercaptopyridazine in 1.5 L of 0.1 M phosphate buffer (pH 6.4) was heated at 60-65C. under nitrogen atmosphere for 4 hours. The hot mixture was treated with 2 g. of charcoal and filtered. The filtrate was cooled to room -temperature and adjusted to pH 4.5 with glacial acetic acid to give the precipitate, which was collected by filtration, washed with : ; , .

, :

-31-, -32-one L of acetone and air-dried at room temperature to yield 125 g. ::
(70~) of 7-amino-3-(3-hydroxypyridazine-6-ylthiomethyl~-3-cephem-4-carboxylic acid. M.p. 240-250C~ (dec.).

:,;

~L~3~
7-Amino-3-(tetrazolo[1,5_b~pyridazin-6-ylthiomethyl)-3-cephem-4 carboxylic acid (4b) .

N ~ ~ CH -S

Preparation of 6-Mercaptotetrazolo[4,5-b]pyridazine O "' ' ~ NH NH //N-N~ POC13 N -N
~2 2 ~ HO ~ OH ~ >C1 ~ Cl O ~ .

8 9 10 NH NH /N- N NaN02_ /N~IN
2 2~ Cl~ NHNH2~ Cl ~ ~ ~

11 .l2 .
' :'' ~, N- N/ ~IN
alc. KSHHS ~ N
. :.
13 :
.' ' .:

' 30 .

' . .

:
- \
~9397~
3,6-Dihydroxypyridazine (9) To a boiling solution of 315 g. (3 moles) of hydrazine dihydrochloride in 2 L of water was added portionwise 295 g. (3 moles) of finely ground maleic anhydride 8 with stirring. After the addition was completed the heating was continued for 4 hours and then allowed to stand overnight in a refri~erator to give 285 g. (85~) of 9 as massi~e pillars.
m.p. ~ 290C.
3,6-Dichloropyridazine (10) ... . . . . _ _ A mixture of 150 g. (1.33 moles) of 9 and 250 g.
of phosphorus oxychloride was refluxed for 3 hours under pro-tection from moisture. The excess of phosphorus oxychloride was removed under reduced pressure and the dark residue was poured into one Kg. of crushed ice. The resulting precipitate ~; was collected by filtration. The second crop of the product was obtained rom the mother li~uor by the extraction with five 300 ml. portions of chloroform followed by treating with 1 g.
of charcoal and evaporating the solvent. The first and second crops were combined, dissolved in 500 ml. of chloroform and treated again with one g. of charcoal and concentrated to give 165 g. ~83%) of 10 as fine needles melting at 60-61C. (in a sealed tube~.

.~

3-Chloro-6-hydrazinopyridazine (11) A mixture of 40 g. (0.27 mole) of 3,6-dichloro-pyridazine (10) and 40 ml. of 80% hydraæine hydrate in 80 ml. ~-of ethanol was refluxed for one hour. The reaction mixture was evaporated to dryness and the residue was recrystallized from benzene to give 39 g. (100%) of 11 melting at 114-115C.
6-Chlorotetrazolo[4,5-b]pyridazine ~12) To a solution of 25.7 g. (0.174 mole) of 11 in 100 ml. of 1S% acetic acid was added dropwise a solution of 13.8 g. (0.2 mole) of sodium nitrite in 50 ml. of wa-ter with vigorous s-tirring at 5-lO~C. Stirring was continued for one hour at the same temperature. The precipitate which separated was filtered, washed with 20 ml. oE water and air-clried to give 17.02 g. of 12. ~clditional product was obtained b~ ~vaporation of the filtrate. Total yielcl 18.32 g. (64%). M.p. 104-105C.
6-Mercaptotetrazolo[4,5-b]pyridazine (13) mixture of 21.3 g. (0.137 mole) of 12 and 20 g.
(0.25 mole) of potassium hydrosulfide in 200 ml. of ethanol was refluxed for 2 hours and evaporated to dryness. The residue was dissolved in lO0 ml. of water and filtered to remove a small amount of insoluble material. The filtrate was acidified to pH 1 with dil. hydrochloric acid to precipitate 13 as colorless needles which were collected by filtration, washed with 20 ml. of water and dried. Yield 9.80 g. (47%). M.p.

:, :;

..
.`

.

. .

140-141C. (dec.).
IR:~ E`Bax 2500, 1540, 1445, 1295, 840 cm NMR:~ p2m 2 3 7.44 (lH, d, 10 Hz, pyridazine-H), - 7.77 (lH, d, 10 Hz, pyridazine-H).
Anal. Calcd. for C4H3N5S: C, 31.37; H, 1-97; N, 45-72;
S, 20.94.
Found: C, 31.52; 31.66; H, 1.70, 1.69; N, 46.01;
46.01; S, 20.95.
Preparation of 7-amino-3~(te*razolo[4,5-b]pyridazin-6-ylthio-methyl)-3-cephem-4-carboxylic acid El N -- ~ S ~I / ~N
N ~ --~ CH2-0-COCH3 -~ HS ~ N

H2N ~~ ~ ~ N
1 N ~ CH -S- -O

4b 7-Amino-3-~tetrazolo[4,5-b]pyridazin 6-ylthiomethyl?-3-cephem-4-carboxylic acid ~4b) ~i) To a hot solution (50-60C.) of 9. 5 6 g.
(0.062 mole) of 13 and 10. 42 g. (0 .124 mole) of sodium ~, , .
.: . - ~ . :

~L~337~
bicarbonate in 300 ml. of water was added care~ully 16.86 g.
(0.062 mole) of 7-ACA and the mixture was heated at 80-85C.
for 30 minutes. About 7 g. of sodium bicarbonate was added to the reaction mixture to dissolve insoluble material. The solution was treated with active carbon, filtered and the filtrate was acidified to pH 5 with dil. hydrochloric acid.
The precipitate was collected by filtration, washed with water, .
air-dried and finally in vacuo on P2O5 to give 14.47 g. (64%) , of 4b. M.p. 248-250C. (dec.).
10(ii) A stirred solution of 16.8 g. (0.11 mole) of 13 and 18.48 g. (0.22 mole) of NaHCO3 in 1 L of 0~1 M phosphate buffer (pH 6.~) was heated at 50C. and to the solution was added portionwise 30 g. (0.11 mole) of 7-ACA. The mi~ture was ; heated at 80C. for 2.5 hours, during which periocl insoluble material still remained. The reaction mi~ture was cooled to room temperature and the precipitated 4b was collected by filtra-tion, washed thoroughly with 200 ml. of water and air-dried.
Additional 4b was obtained from the filtra-te and the washings by acidifying to pH 5 with dil. HCl. Total yield 32.9 g. (83~). M.p. 245-250C. (dec.).
'' '~' ' :;"
' : ~ .
`,~'"

j, , , . :

~C~3t~1L$~
IR ~ 1800, 1615, 1533, 1360 cm max UV:~ max 3 237 nm (~ 19500), 275 nm (~ 12000), 310 nm (sh) ( 5700). ;~

NMR:~ DpO K2C3 3.35 (lH, d, 18 Hz, 2-_), 3-76 (lH, d, 18 Hz, 2-_), 4.00 (lH, d, 10 Hz, 3-CH2), 4.48 (lH, d, 10 Hz, 3-CH2), 4.93 (lH, d, 4 Hz, 6-_), 5.32 (lH, d, 4 Hz, 7-H), 7.46 (lH, d, 10 Hz, pyridazine-H), 8.18 (lH, d, 10 Hz, pyri-dazine-H).
Anal. Calcd. for C12HllN703S2 N, 26.83; 5, 17.55.
Found: C, 39.19; H, 2.71; N, 26.84; S, 17.35.

:
.

.

~3'7~
7-Amino-3-(5-methyl-1,3,4-t'hi'adi'azo'1-'2~y1thiomethyl)-3-cephem-; 4-carboxylic acid (4d) ., : .

~ N ~ C~2 5 5 C02H : .

2-Mercapto-5-methyl-1,3,4-thiadiazole.
,:
Lit. ref. U.S. patent 3,516,997 (1970); J. Antibiotics, 23, 131-36 (1970).
11.5 g. (0.1 mole) of 2-amino-5-methyl-1,3,4-thia-diazole was carefully ground together with 32 g. ~0.45 mole) of sodium nitrite and slowly added to 160 ml. oE 48% IIBr containing 50 mg. o powdered copper at -10C. with stirrin~. ~Eter the addition was completed, the solution was stirred at -5C. for one hour and then at 20C. ~or one and a half hours. The pH
; was adjusted to 9.5 by addition of 50~ KOH and the solution was heated to 60C. At 60C. the pH was readjusted to 9.5 by ''' 20 addition of 50% KOH. The solution was cooled and filtered.
~ The precipitate was dissolved in ether and the filtrate was - extracted with 2 x 200 ml. ether. The combined ether solutions were dried over sodium sulfate and evaporated to dryness. '-; The product was recrystallized from benzene-"Skellysolve B".

:, :
: ' :
.

:

, I

~ .

. - 40 -'.':
:.. ' ~ , , ~ ` ' .: ., . '.
:, . , , ' . : .

Yield 12 g., m.p. 105-107C.
12 g. (.07 mole) of 2-bromo-5-methyl-1,3,4-thiadiazol and 5 g. (.07 mole) of thiourea were dissolved in 40 ml. of 100~ ethanol and refluxed for one and a half hours on a steam bath. This solution was added to 4.5 g. (.08 mole) of KOH
in 65 ml. H2O and the mixture heated to boiling for five minutes.
The ethanol was removed under vacuum and the pH of the aqueous solution adjusted to 3 by addition of 3 _ IICl. The product crystallized out and after cooling at 0C. for one hour was collected by filtration, washed with cold water and recrystallized from 100% ethanol. Yield 5 g., m.p. 186-187C.
Anal- Calcd- for C3H4N2S2: C, 27.25; H, 3.05; N, 21.19;
S, 48.51.
Pound: C, 27.20; H, 3.34; N, 21.18; S, 48.48.
7-Amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylic ~cid.
To a stirred suspension of 2.72 g. (0.01 mole) of 7-ACA in 50 ml. of 0.1 M, pH 6.4 phosphate buffer, was added 1.68 g. (0.02 mole) of NaHCO3 followed by 1.45 g. (0.011 mole) of 2-mercapto-5-methyl-1,3,4-thiadiaæole and the mixture heated and stirred at 60C. for five hours. The resulting slurry was .
.~

~7 :-~
- then allowed to cool to about 22C. over a one hour period.
The cyrstalline precipitate was collected by filtration, washed with water and air dried. Yield 1.3 g., dec~ pt. 206C. Scaling up the reaction lOX gave 18.0 g.

11 12 4 3 3 ~ i , ; ;
S, 27.96.
Found: C, 39.06; H, 3.91; S, 26.67.
7-Amino-3-(1-methyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid (4c) ', 10 ~, o/F~ c~l2-s ~ !1 ~

; C2 3 The above preparation was repeated us:iny l-methyl~
1,2,3,4-tetrazole-5-thiol instead of the thiadiazole. There was obtained 25 g. (76%) of 7-amino-3-(1-methyl-1,2,3,4-tetrazole-; 5-thiomethyl)-~ 3-cephem-4-carboxylic acid. The preparation of this same compound i5 also described in U.S. patent 3,516,997 in column 6 under the heading "Preparation 7".

... .

:

.' .
,' .

.

. ~

: . . : . ~ : :
"

374~7, 7-Amino-3-(3-hydroxypyridazino[3,2-c]-s-triazol-6-ylthiomethyl)-3-cephem-4-carboxylic acid (4e).
- OH

: H N S
2 ¦ CH2-S
O
C02~I

.
'" .

. ., "
'. ', ."
,''~'.' .
... :

,; ;~"
,.

. 30 '~;'' ,; - 43 -.`, .
'."" ~

7~
6-Mercapto-2,3-dihydro-s-triazolo~4,3-b]-pyridazin-3-one A mixture of 6-chloro-2,3-dihydro-s-triazolo[4,3-b]-pyridazin-3-one [P. Francavilla and F. Lauvia, J. Het.Chem., 8. 415 (1971)~ (1.70 g., 0.01 mole) and potassium hydrosulfide (1~44 g., 0.02 mole) in ethanol (30 mlO) was heated in a sealed tube for 8 hours at 140. After cooling, water (50 ml.) was added to the reaction mixture and a small amount of insoluble material was removed by filtration. The filtrate being con-centrated under reduced pressure, the concentrate was acidified to pH 2 with dil. HCl to afford yellow precipitate of 6-mercapto-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-one which was collected by filtration, washed with water (lO ml.) and dried ln vacuo on P2O5. Yield 1.43 g. (84%)- M.p. ~ 300C-~ KBr 2500, 1710, 1495, 1350 cm l.

" ~

' .

.
.

UV:A 3 261 nm (~ 90000), 320 nm ( 2700) NMR:~ D2O K2C3 6.96 (lH, d, 10 Hz, 7-H or 8 H).
7.12 (1 H, d, 10 Hæ, 7-_ or 8-H).
Anal. Calcd. for C5H4N40S: C, 35.71; H, 2.40; N, 33.31;
S, 19.07.
Found: C, 35.17; H, 2.28; N/ 33.38; S, 19.76 7-Amino-3-[2,3-dihydro-s~triazolo[4,3-b]pyridazin-3-one-6-.. . . _ _ ylthiomethyl]-3-cephem-4-carboxylic acid (4e) 7-ACA (1.36 g., 5 mmoles) was added a-t 50C. to a solution of 6-mercapto-2,3-dihydro-s-triazolo[4,3-b] pyridazin-3-one (0.84 g., 5 mmoles) and sodium bicarbonate (0.84 g., 10 mmoles) in 20 ml. of 0.1 M phosphate buEf~r solut:ion (pll 6.4) and the mixture was heated eor 2 hours at 70C. ~ small amourlt of insolub:le matcrial was removcd by eiltration ancl acidification of the ~iltrate to pH 5 with dil. HCl afforded the product 4e which was collected by filtration, washed with water (30 ml.) and dried ln vacuo on P2O5. Yield of 7-amino-3-[2,3-dihydro-3-triazolo(4,3-b)pyridazin-3-one-6-ylthiomethyl~-3-cephem-4-carboxylic acid 1.25 g. (66%), M.p. ~ 300C.
IR: ~ KB 1805, 1720, 1620, 1550 cm UV: ~ 1%NaHC3 257 nm (~ 17700) NMR: ~ p~m 2 3 3 40 (lH, d, 20 Hz, 2-H), 3.78 (lH, d, 20 Hz/ 2-H), 4.00 tlH, d, 13 Hz, 3-CH ), 4.35 (lH, d, 13 Hz, ; - -2 3-C_~), 5.02 (lH! d, 4 Hz, 6-H), 5.40 (lH, d, 4 Hz, 7-_), 6.70 tlH, d, 9 ~Iz, pyridazine-_), 7.40 (1~, d, 9 Mz, pyridazine-H).

." ~ .
'':

r C13H12N604S2 H200 C, 39019; H, 3.54; ~:
N, 21. 09; S, 16. 09. ~.
Found: C, 39.40; H, 3.39; N, 20.36; S, 15.89.

~', .

.
' :, ., ` 30 - ~6 -7-Amino-3-(pyrido~2,1-c]-s-*riazol-3-ylthiomethyl)-3-cephem-4-carboxylic acld (_) o ~L CH2 ~ ¦

~ ' To a hot (60) solution of 3-mercapto-s-triazolo-[4,3-a]pyridine ~D. 5. Tarhell et al., J. Am. Chem. Soc. 70, 1381 (1948)~ (1.51 g., 10 mmole) and NaHCO3 (1.68 g., 20 mmole) in 0.1 M pH 7.4 phosphate buffer (50 ml.) was added portionwi.se 7-ACA (2.72 g., 10 mmole) and the mixture was heated at 80-85 for 30 min~ The reaction mixture being treated w:ith carbon, the .. . .filtrate was acidi~ied to pH 5 with dil. HCl to give 7-amino-3-(s-triazolo[4,3-~]pyridin-3-ylthiomethyl)-3-cephem-4-carboxylic acid which was collected by filtration, washed with water (10 ml.) and dried ln vacuo on P2O5. Yield 1.40 g. (39%). M.p.
215-220 (dec.).
IR: ~ mBx 1805, 1620, 1530, 1410, 1545 cm 1.

UV: ~ max 3 280 nm (~ 13200).
:`
.' . -;, ~;

;', .
. ~ .
- ~7 -' 3~

- NMR:~ ppm 2 3 3.25 (1 H, d, 18 Hz, 2-~), 3.63 (lH, d, 13 Hz, 3-H), 3.68 (lH, d, 18 Hz 2-H), 4.18 (lH, d, 13 Hz, 3-H), 4.7-5.3 (2H, m, 6-H & 7-_).
Anal. Calcd- for C14H13N5O3S2 N, 19.27; S, 17.65.
Found: C, 45.81; 45.74; H, 3.58j 3.69; N, 18.21, 18.13; S, 17.08.
7-Amino-3-pyridazino[2,1_c]-s-triazol-3-ylthiomethyl)-3-cephem-4-carboxylic acid (4g) , 10 . S ~ N _ Nl ~ N ~ C~I2-S
O /
' C2H ", ~

3-Mercapto-s-triazolo[4,3-b~pyridazine A mixture of 1.20 g. (8 mmole) of 3-chloro-s-triazolo-[4,3-b]pyridazine (P. Francavilla and F. Lauvia, J. Het. Chem., 8, 415 (1971)] and 1.20 g. (16 mmole) of KSH in 20 ml. of ethanol was heated for 8 hours at 130 in a sealed tube. After cooling the mixture was evaporated to dryness and the residue was dissolved in 100 ml. of water, treated with active carbon, ~ '.
.' ' .' ,.

- ~8 -~3~ 7 acidified to pH 1 with dil. IICl to precipitate 3-mercapto-s-triazolo[4,3-b]pyridazine which was collected, washed with 10 ml. of water and dried in vacuo over P2O5 to yield 0.75 g.
(62~), m.p. 260-270 (dec.).
- IR:~ max 3080, 2940, 2760, 1520, 1500, 1280, 1055 cm ~ DMS-d6 6.99 (lH, d-d, 4 & 10 Hz. 7 ~
7.67 (lH, d-d, 2 & 10 Hz, 8-H), 8 15 (lH, d-d, 2 & 4 Hz, 6-H), 12.3 (lH, br-s, disappear by addition of D2O).

Anal. Calcd. for C5H4N~S 1/2H2O: C, 37-26; H, 3-13;
N, 34.76.
Found: C, 37.35; H, 2.32; N, 34.81.
7-Amino-3-(s-triazolo[4 ! 3-b]pyridazin=3~1 hiomethyl)~3-ce~ ~m-4-carboxylic acid (4g) 7-ACA (1.36 g., 5 mmole) was added portionwise to a solution of 0.68 g. (4.5 mmole) of 3-mercapto-s-triazolo-[4,3-b]pyridazine and 0.84 g. (10 mmole) of NaHCO3 in 20 ml. of 0.1 M pH 7.4 phosphate buffer at 40-50. The mixture was heated at 80-85 for 40 min., treated with a small amount of active carbon and acidified with dil. HCl to pH 4 to precipitate 7-amino-3-(s-triazolo[4,3-b]pyridazin-3-ylthiomethyl~-3-cephem--4-carboxylic acid which was collected, washed with 20 ml. of ; , ~7~
; water and dried in vacuo on P2O5 to yield 0081 g. (49%), M.p. > 300.
IR:~ KBar 1705, 1620, 1540, 1415, 1350 cm UV:~ m%NaHCO3 275 nm ( 13100) NMR:~ p~m 2 3 3.35 (lH, d, 18 Hz, 2-H), 3086 (lH, d, 18 Hz, 2-H), 4.80 (lH, d-d, 4 & 2 Hz, 6-_), 5.24 (lH, d, 4 Hz, 7-H), 7.1-8.6 (3H, m, pyridazine-~I).
Anal. Calcd. for C13H12N6O3S2 ~I2 N, 21.96; S, 16078.
Found: C, 41.15; H, 3.24; N, 20.37; S, 17.90.

"

~

;

.~ ~
:'' ~r~9~ (~) S C~I N
H2Nn~ ~
0// ~ CH2 S ~ / N
COOH

s~n~ c~llLs~ lum 112~-tr~azo~e-~-thiolat,e ~C-N=C=S ~ CH2N2-~ ~ NHCO~
16~.19 42~04 205.24 ' S~ SK

. X
. 101013 l~g.23 , ~
The synthe~i3 o~ the thiol was accomplished by procedure es~entially identical to that de~crlbed-in the literature [J. Goerdler and G. Gnad, Chem. Ber.
, 1618 (~6)].

.~ , .
~. -Sl-- \

3~
~enzamido-1~2~-thiadlazole To a ~tlrred ~olution o~ benzoylisothiocyanate (50.6 g., 310 mmole~) in commercial anhydrous ether (400 ml.), maintained at 0 and in a nitrogen atmosphere, was added dropwise with vigorou~ stirring, o.685 N
ethereal dlazomethane (45~ ml.~ ~lO mmoles). When the additlon was completed, the mixture was stlrred f`or 1 hour at 0, the ~olid was collected by ~iltratlon and drled 1n vacuo. The meltlng polnt o~ the crude materlal (2~,~ g.) thus obtained was observed somewhere in the reglon 232 to 257~. Goerdler reported ~.p. 267 for the pure materlal. A smal] 3econd crop (2.1 g.) was obtalned by evaporation o~ the mother llquor in vacuo.
m e total yield was there~ore 4 ~ .
1~2.~-Tria'zole-~-thlol A ~olutlon o~ the above benzamido compound (8.2 g., 40 mmoles~ ln 2 N ~odlum hydroxide (80 ml., 160 ~molês) was heated under reflux temperature in a nitrogen atmosph~re ~or 24 hour~. The ~olution wa~ ~ooled to 0 ln ice, and concentrated hydrochloric acid (26 ml.) wa~ added~ while a continous stream o~
nitrogen was passed through the solutlon. The benzoic acld which precipltated was collected b~ ~iltration~
the ~iltrate was saturated with sodium chloride and the additional benzolc acid which separated was removed by ~iltratlon. The ~iltrate was immedlately ! 52--.

~LqJ3~
extracted with ethyl acetate~ the extract was washed wlth ~aturated salt solution, dried over magnesium sul~ate and then evaporated in vacuo. The vlscous oil whlch remained was immediately evapora~ively diatllled in vacuo (70-75/0.001 mm.) to give an oil (2.84 g ~ 7~? which solidl~ied (m.p. 52-59; Goerdler reported m.p. 60) spontaneously.

To a ~olution of the above thiol (2~84 g., 28.1 mmole~) in ahsolute ethanol (28 ml.) was added 1.93 ~ alcoholic potas3ium hgdroxide ~olution (14.5 ml.). m e solut~on wa~ then diluted with anhgdrous ether until cr~talllæation of the salt was oompleted. The ~olid was collected by f`lltratlon, wa~hed wit~ ether, and dried ln vacuo. The ~alt obtained in thi~ manner (3.65 g., 93~) had m.p. 225 with decomposition.
,'' ''' ~
o.ephet;~4-carb.ox~li,c ac~d (,~
Ten grams (0.075 mole) o~ 5-mercapto-lj2,3-triazole pota~sium ~alt W~B added to a stirred slurry o~ 19 g. (0.07 mole) of ~uri~ied 7-aminocephalosporanic acid and 5.9 g. (0.07 mole) of NaHC03 in 350 ml. of O.lM pho~pha~e buf~er (pH 6.4) and the mixture heated and ~tirred at 55 C~ ~or 3 1/2 hours under a nltrogen atmo~phere. The re~ulting ~olution was cooled to 22 C.
'' ' .

, -53-' .
. .
, - - ~

9L~3~4~ .
and the pH ad~usted to 5.5 wlth 40~ H~P04. The re ulting precipitate wa~ filtered o~, washed with cold water (50 ml.) and alr dried. The yield of 7-amlno-3-[S-(1,2,~-triazole-5~yl)thiomethyl]-3-cephem-4-carboxylic acld wa~ 8 g., dec. pt 230 C
IR analy~l~ showed some decomposition of the ~-lactam ring but it wa~ used "as is" ~or the next step.
; Anal. Calcd. ~or CloHllN50~S2 Found: C, 38.36; H, 3.78. ---~_ mino~ ~th~ :;c aci~,, (4h) Crude 7-amlno-3~(1,2,3-trlazol-5-ylthiomethyl)-~-~ephem-4-oarboxyllc acld (16.1 g.) containing approximately 20 mole % o~ 7-amlnocephalosporanic acid a~ an ~mputity, wa3 brought into 801utlon with 600 ml. o~ methanol and 40 ml. o~ conc. HCl. After carbon treatment, the solu~lon was diluted with 1.5 1~ of ice water and extracted once with!eth~l acekate. The aqueous phase was concentrated at reduced pressure to remove methanol. The cold aqueous conc.~ntrate was ~hen adjusted slo-rly to pH 4.0 with 20 ~odium hydroxide causlng the product to crys.tallize.
The product was collected by flltratlon, washed with ~ater and methanol and dried in vacuo over phosphoru~
pentoxlde; 11.4. g. q~he N~ spectrum lndlcated that-thls product contained about 7 mole % of 7-amino-oephalo3poranlc acid ~ 3 an impurity~

~, .

~3~
The above purification procedure was repeated on 11.4 g. of' the product using 425 ml. of methanol, 28 ml. o~ conc. HCl and 1 1. o~ ice water yielding 800 g. Or product. The NMR spectrum was fully con~istent for the de~ired product and indlcated no trace of' 7-aminocephalosporanlc acid as an impurity.
Anal. Calcd. ~or CloH15N503S2: , 3.55; N, 22.40, Found: C, 39.o6; 38.53; ~I7 ~56, 3.51,-N, 22.05J 21.60; H20, 1.78.

I~A~ ~~,~4~-thladiazol~rl~iometh~ cePhersl-,b,~ a;,rbQXVliC aolq (~) , oF~ c H2-S-~ ,C H
COOH

2~
The prooedure o~ J . Go~rde ler, J O Ohm and O.
Te~;tmeyer, Berîchte 8C1, 15~4 . ~19~6) was ~ollowed. To a ~olution- o~ 160 ml. of 48% hgdrobromic acld and 100 mg. o~ powdered copper ~t -7~ wa~ ~dded 810wlg wlth alternation 13.6 g.

~.~3~
(0 1 mole) of 2-amino-133,4-thiadiazole (Eastman) and 32 g. of ~odlu~ nitrite in small portions over a perlod o~ 1/2 hour. The mixture wa~ stirred for 1 1/2 hours at 0 and for 1 hour at room temperature. Th~e mixture was then neutralized with 50~ pota~ium hydroxide to p~ 9.5.
The mixture wa~ filtered and the filtrate was continuously extracted with ether for 6 hour~. The ether was evaporated to 15 mm (25) to a solld which wa~ di~solved ln 40 ml~ o~ ethgl alcohol and treated with 5 g. o~
thiourea. ~le ~olution wa~ heated at re~lu~ ~or 1 1/2 hour~. A solutlon o~ 4.5 g o~ pota~ium h~droxicle ln 65 ml. o~ water wa~ added and the mixture wa~ heated at re~lux for an additional 1 1/2 hours. The alcohol was evaporated at 15 mm. (32) and the aqueou~ re~ldue wa~
neutralized with concentrated hydrochloric acid to pH 7.5. A~ter cooling for 2 hour~ in an ice bath ~.5 g. o~ 2-mercapto-1,3,4-thiadiazole as yellow crystal~
were col~ected and weighed 3.5 g. M.p. 125-127~.
m~ ~R and NMR ~pectra were consl~tent for the structure.
. ' a 4-v~a~boxvlic acid To a ~u~pension o~ 8.1 g. (.03 mole) of 7-amino-cephalosporanic acld and 3.5 g. (.03 mole) 2-m~rca~to-1,3,4-thiadiazole in 200 ml. o~ .1 M pho~phate bu~er r ~E;6 ~3~
(pH 6.5) wa~ added wlth ~tirring 5.4 g. (.o64 mole) o~ ~odlum bicarbonate. The mixture was stirred at 55 under nltro~en and all of the solid di~ olved.
m e ~tirring wa~ continued for ~ hour~ an~ the solution wa~ cooled to 5 and adJusted to pH 5 wlth ~lacial acetlc acid. The mixkurè was ~tored ~or 2 hour~ and the product, 7-amino-3-[2~ ,4-thiadiazolyl?-thio-methyl]-3-cephem-4-carboxylic acid~ wa~ collected and weighed 9 g.~ M.p. ~ 1'40 (~low decomp.). The IR and NM~ ~pectra were con~i~tent for the ~tructure.

~Amlno-~[2~ h,vdroxvmethvl-1,3~4-thladlazolvl)thio-~kh~ 3,-,Qe~h~m-4,-carboxvllc acid (~ ) H2N ~ ~ N - N
~CH2-S-C~ S~C-C~20X
,~OH

A mixture o~ 18.2 g~ (0.2 mole) of thio~emicarbazide and ~0.4 g, (0.4 mole) o~ gly¢ollic acid was heated tog~ther at 80-90 ~or 2 hour~ with good ~tirring. The ' '' .

' -57-~ 3~4~
mlxture was cooled at room temp. and washed with cold abs. alcoholO The ~olid wa3 collected and air dried to welgh 40 g. The product wa~ recry~allized from 100 ml. o~ boiling 5 ~ alcohol to yield 23 g. o~ the crg3talline l-hydroxyacetylthlo~emicarbazide. M.p.
189-190; Anal~ Calcd. for C3H7N30S~ C~ 24-18; HJ 4-74 N, 28.21; S, 21~51.
Found: C, 24.08; H~ 4.50; N~ 27.67; SJ 21.35.
The IR and NMR spectra were consistent for the ~tructure.
çQko,x~m~thyl-~-amino-1,~,4-thiadiazole A mixture o~ ~244 g. (0.15 mole) o~ l-hydro~y-acetylthlo~emicarbazide and 50 ml. o~ acetgl chlorlde was stirred at room kemperature ~or 2 hour~
and then heated to 40 until all the ~olid had di~solved (oa. 2 hour~). The acetyl chloride was removed under reduced pPe~ure 40 (15 min.) and the residue ~as stlrr~d with 100 ml. of lce water. The mi~ture was adJu~ted to p~ 9.2 wlth 10% KOH and the product ~as .~
~ollec~d. ~he crude solld wa~ recrystalli2ed from aDsolute alcohol to yield after air drying 6 g. of crystalline 2-acetoxymethyl-5-amino-1~3,4-thiadiazole, M.p. 191-192 C.
Anal. Calcd. ~or C5X7N302S: C~ 34.63; ~I~ 4.07; N~
24.26.
Found: CJ 35.16; HJ 11.35; NJ 24.~58.

. .
'.~
, .
~58 - ~, `;
; 2~Ldr-o-x~methtJ~ mercapto-l~3~4-thl-a-diazole m e procedure Or G~erdler et al. was followed uslng 34.6 g. grams (0.2 mole) of 2-acetoxymethyl-5-amino-1,~,4-thiadlazole and 64 grams (0.9 mole) of sodlum nitrite ln ~20 ml. of 48% hydrobromlc ac~d and 0.1 gram of powdered copper. The bromo compound was treated wlth 7.6 grams ~0.1 mole) of thiourea and 11.2 grams o~
pota~slum hydroxlde in 25 ml. of water. Up-o~ acidlficat-lon with 6 hydrochlorlc acid the mercaptan was extracted in ethyl acetate and treated with 17 gram~ o~ pota~sium ~-ethylhexanoate. The potassium salt was collected, ~aahed with ethyl acetate and dr~ed to we~gh 12.2 grams.
The ~alt wa~ recr~tallizea from acetone-water to glve 7.6 grams of cry~talline potassium 2-hydroxymethyl-5-mercapto~ ,4-thladlazole, M.p. 130-131 C.
Anal. Calcd. ~or C~H3KN2S~O H20: C, 17.64;
2.46; ~ .70.
Fouffd: C, 17.81; H, 2.43, N, 13.66 -~he IR and NMR spectra were conslstent for the $~ructure.

: c _59_ ,7 m e procedure followed wa~ the ~ame as the procedure ~or the unsub~tituted thiadiazole using 2.25 g (0.012 mole~ of mercaptan, 3.3 g. (0.012 mole) of 7-amino-cephalo~poranic acid and 1 g. (0.012 mole) of sodium blcarbonate in 100 ml., 1 M phosphate buffer to yield 3.15 g. of tan, solid 7-amino-~-[2-(5-hydroxymethyl-1,~,4-thiadlaæolyl)thiomethyl]-~-cephem-4-carboxylic acid, M.p. 170-175 C. decomp. The IR and NMR spectra were consistent for the structure.

.' ~.' '^.', .

, ' .

,.
.
, . .
.

` : :
;

~o~
Description of the Preferred Embodiments Example 1 ~-~H2 N_ M
J_ N ~ H2-S~ - OH
' O I __ 6a 7-[~-(2-t-Butoxycarbonylamlnomethyl-l-c~clohexen~l)-acetamido]-3-(6-hydroxypyridazin-3-ylthiometh~1)-3-cephem-4-c _boxylic acid (5a) A mixture o~ 2 (1.08 y., ~ mmoles), 2,4-dinitrophenol (0.74 g., 4 mmoles) and DCC (0.82 g., 4 mmoles) in THF (20 ml.) was stirred for 1 hr. at room temperature and filtered to remove the precipitated dicyclohexylurea which was washed with THF
(10 ml.). The combined filtrate and washings were cooled at 5 C. and poured in one portion into a cold solu-tion of 7-amino-3-(6-hydroxypyridazin-3-ylthiomethyl)-3-cephem-4-caxboxylic acid (1.02 g., 3 mmoles) and triethylamine (0.81 g., 8 mmoles) in 50% aqueous THF (20 ml.). The mi-xture was stirred overnight at ~ room temperature and washed with ether (2 x 50 ml.). The aqueous .,..... ~ .
layer was acidified to pH 2 with dil. HCl to precipitate crude 5a which was dissolved in THF (100 ml.) and filtered to remove insoluble material. The Eiltrate was treated with a small ~, amount of carbon and dried over anhydrous sodium sulEate.
Evaporation oE the solvent under reduced pressure afEorded 5a '`;' .' .: ' .

~ - 61 -.,.. ~. :

7~
as an oily residue which was solidified by trituration in ether (100 ml.). Yield 0.84 g. (47~). M.p. 185-195C. (dec.).

IR: ~ a~ 3250, 1780, 1660, 1580, 1530, 1370, 1250, 1160 cm 1.

~ D S-d6 1 40 (9H, s, t-Bu-H), 5.

4Hz, 6-H), 5.70 (lH, d-d, 4 and 8 H~, 7-H), 6.88 (lH, d, 10 Hz, pyridazine-H), 7.45 (lH, d, 10 Hz, pyridazine-H), 9.0 (lH, d, 8 Hz, CONH), 13.2 (lH, br-s, -OH).

Anal. Calcd- for C26H33N507S2 1/2 H2 H, 5.70; N, 11.65; S, 10.67.

Found: C, 51.53; 51.56; H, 5 63, 5.80; M, 11.28, 11.34; S, 11.4~, 11.31.

7-[~-(2-aminomethyl-1-c~clohexenyl_ac tamiclol-3-(6-h~droxk-pyridazin-3-ylthiomethyl ~ oxylic acld (6a) _ To tri~luoroacetic acid (1.5 ml.) was added 5a (0.80 g., 1.4 mmoles) and the mixture was stirred for 30 min~
at room temperature and diluted with ether (50 ml.) to pre-cipitate the trifluoroacetate o~ 6a which was collected by filtration and slurried in water (2 ml.). The mixture was adjusted at pH 6 with ammonium hydroxide and diluted with acetonitrile (200 ml.) to precipitate 6a. Yield 0.64 g. (93%).

~3~
M.p. 200-208 (decO~.
IR: ~ maUx 1780, 1680 - 1640, 1580 cm UV: ~ m~x2C3 255 nm (, 14100).
Anal- Calcd- for C21H25N55S2 3 1/2 H2 H, 5.82; N, 12.63; S, 11.56.
Found: C, 45.73, 45.83; H, 4.50, 4.47; N, 12.45, 12.59; S, 11.83, 12.06.
Example 2 . 10 ~--NH2 ....
; ~ ~50NH r S ~ /N N
O /~ N ~ ~ CM2-S
6b 2 7-[~-(2-t-Butoxycarbonylaminomethyl-l-cyclohexenyl)-acetamido]-.
' 3-(pyridazino[2,3-d]tetrazol-6-ylthiomethyl)-3-cephem-4-carboxylic :
acid (5b) A mixture of 2 (1.08 g., 4 mmoles), 2,4-dinitro-phenol (0.74 g.~ 4 mmoles) and DCC (0.82 g., 4 mmoles) in l'HF
(20 ml.) was stirred for 1 hr. at room temperature and filtered to remove the dicyclohexylurea which was washed with THF (10 ml.).
The combined filtrate and washing were cooled at 5C. and poured into a solution of 7-amino-3-(pyridazino[2,3-d]tetrazol-6-ylthiomethyl)-3-cephem-4-carboxylic acid (4b) (1.08 g., 3 mmoles) . :

7~L~7 and triethylamine (0.81 g., 8 mmoles) in 50% aqueous THF (20 ml.) at 5C. The mixture was stirred overnight at room temperature and washed with ether (2 x 50 ml.). The aqueous layer was acidified to pH 2 with dil. HC1 and extracted with ethyl acetate (3 x 50 ml.). The combined extracts were washed with water (50 ml.), treated with carbon and dried over anhydrous Na2SO4.
Removal of the solvent under reduced pressure gave 5b as an oil which was solidified by trituration with ether (50 ml.). The product was collected by filtration, washed with ether and dried. Yield 0.94 g. (51%). M.p. 125-134C. (dec.).
IR:~ max 1780, 1680, 1520, 1370, 1250, 1160 cm 1.

NMR:~ ppm 6 1.40 (9H, s, t-Bu-H), 4.23 (lH/ d, 14 Hz, 3-CH2S), 4.70 (lH, d, 15 Hiz, 3-CM~S), 5.15 (lH, d, ~ Hz, 6-_), 5.78 (lH, d-d, 4 and 8 ~Iz, 7-H), 7.88 (l~I, d, 10 Hz, pyridazine-H), 8.75 (lH, d, 10 Hz, pyridazine-_), 9.0 (lH, d, , 8 Hz, 7-CONH).

Anal. Calcd. for C26H32N8O6 2 ; S, 10.40.

Found: C, 50.36, 50.42; H, 5.11, 5.21; S, 9.60.

:. .
..,~

`.

.;,,,,,"
'i ' ''~' :
.;~:.' :" ';:
~ 30 ' ::
.

~37~
7~[~-~2-aminomethyl~l-cy'clohexeny'l')ace'tamido]-3-'(pyridazino-[2,3-d]tetrazol-6-ylthiomethy1~-3'-cephem-4-carboxylic acid (6b) To trifluoroacetic acid (1.5 ml.) was added 5b (0.90 g., 1.5 mmoles) at 0C. and the mixture was stirred for 30 min. at room temperature. The mlxture was diluted with ether (50 ml.) to precipitate the trifluoroacetate which was collected by ~iltration and suspended in water (2 ml.). The suspension was adjusted at pH 6 with ammonium hydroxide and diluted with acetonitrile (200 ml.) to precipitate 6b which was collected by ~iltration and dried. Yield 0.65 g. (83%).
M.p. 184-188C. (dec.).
IR: ~ maU~ 1780, 1620, 1570 cm 1.

UV: ~ ~x2c3 245 nm (~, 18500), 255 nm (sh) (~, 16300), ; 275 nm (sh) (~, 10700), 315 nm (sh) (E , 4700).
Anal. Calcd- for C21~I24N84 2 / 2 H, 4.79; N, 21.32; S, 12.20.
Found: C, 48.21, 47.90; H, 4.61, 4.67; N, 20.68, 20.63; S, 11.47.

' ' 30 Example 3 f \~ ' NH2 ~ CNHr~/S'~

6c~ N ~ CH2-S N
I

7-{2-[(N-t-Butoxycarbonylamino)methyl-l-cyclohexen-l-yl]-acetamido}-3-(1-me*hyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid (5c) A mixture of 2 (1.30 g., 4.8 mmoles), 2,4-dinitro~
phenol (0.88 y.~ 4.8 mmoles) and DCC (0.99 g., 4.8 mmoles) in THF (20 ml.) was stirred Eor l hr. at room t~mp~ratur~ to precipitate the dicyclohexylurea which was removed by filtration and the bed was washed with THF (5 ml.). The combined filtrate and washings were poured into a cold solution of 7-amino-3-(l-methyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid (4c) (1.31 g., 4 mmoles) and triethylamine (1.01 g., lO mmo:Les) in 50% aqueous THF with stirring at 5C. The mixture was stirred overnight at room temperature, evaporated under reduced pressure below 40C. and washed well with ether (3 x 30 ml.). The aqueous layer was acidified to pH 2 with dil. ~Cl and extracted with ethyl acetate (4 x 500 ml). The combined extracts were treated ' with a small amount of carbon and dried. Removal of the solvent ;
below 40C. under reduced pressure afforded an oily residue ' ,: .

~ID3~7 which was solidified by trituration with ether (100 ml.) to give 1.33 g. of the product 5c. M.p. 146-159C. (dec.).

IR: ~ aUx 1780, 1700 (sh), 1680, 1520, 1240, 1155 cm 1.

NMR: ~ ppm 6 1.42 (9H, s, t-butyl-H), 1.5 - 1.7 (4H, m, aliphatic methylene-H), 1.8 - 3.3 (4H, m, allylic methylene-H), 4.12 (3H, s, N-CH3), 5.15 (lH, d, 5 Hz, 6-H), 5.80 (lH, d-d 5 and 3 Hz, 7-H), 6.35 (lH, br-s, NHBOC), 9.00 (lH, d, 8 Hz, CONH).

Anal. Calcd. for C24H33N7O6S2 C, 49.73; H, 5.74;
N, 16.91; S, 11.06.

Found: C, 49.46, 49.46; H, 5.53, 5.62; N, 16.00, 15.97; S, 11.01.

7-[(2-aminomethyl-1-cyclohexen-1-yl)acetamido]-3-(1-meth~l-. _~
tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acid (6c) A mixture of trifluoroacetic acid ( 2ml.) and 5c (1.30 g., 2.4 mmoles) was stirred magnetically for 30 min. at room temperature. The mixture was diluted with ether (50 ml.) to precipitate the trifluoroacetate of 6c. The trifluoro-acetate was suspended in a small amount of water (2 ml.) ancl the20 suspension was adjusted at pH 6 with ammonium hydroxide and diluted with acetonitrile (200 ml.) to give 6c which was washed with acetonitrile (50 ml.) and dried.

~:.

' M.p. 194-207C. (dec.). Yield 0.80 g. (69 IR ~ maU~ 1770, 1630, 1590, 1370 cm Uv:~ 1%K2C3 270 nm (, 10000) .
max 19 25 7 4 2 2 ' 8 ; H, 5.47;
N, 19.70; S, 12.89.
Found: C, 45.89, 46.18; H, 5.26, 5.28; N, 19.74, 19.82; S, 12.49.
Example 4 ;

,'' ~/--NH2 ~ .

~ CH2-s - ~ S~ CE13 O :
; C02H
6d 7-[~-(2-t-Butoxycarbonylaminomethyl-l-cyclohexenyl)-acetamido]-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylic acid (5d) A mixture of 2 (1.08 g., 4 mmoles), 2,4-dinitro- ~ -phenol (0.74 g., 4 mmoles) and DCC (0.82 g., 4 mmoles) in THF
(20 ml.~ was stirred for 1 hr. at room temperature and filtered to remove the dicyclohexylurea which was washed with THF (10 ml.). The combined filtra~e and washings were cooled at 5C. and poured in one portion into a cold (5C.) solution of 7-amino-3-(5-methyl-1,3,4-thiadiazol~2-ylthiomethyl)-3- `

cephem-4-carboxylic acid (4d) (0.94 g., 3 mmoles) and triethyl-amine (0.81 g., 8 mmoles) in 50% aqueous THF (20 ml.). The reaction mixture was stirred overnight at room temperature and washed with ether (2 x 50 ml.). The aqueous layer was acidified to pH 2 with dil. HCl and extracted with ethyl acetate (3 x 50 ml.).
The combined extracts were washed with water (50 ml.), treated with a small amount of carbon, dried with Na~SO~ and evaporated under reduced pressure to give an oily residue which was solidified by trituration with ether - n-hexane (1:1, 100 ml.).
The product 5d was collected by filtration, washed with n-hexane (50 ml.) and dried. Yield 1.29 g. (72%). M.p. 95 - 102C.
(dec.).
IR:~ a~ 1780, 1680, 1520, 1245, 1160 cm 1.

NMR:~ ppm 6 1.30 (9E~, s, t-Bu-H), 2.58 (3H, s, CH3), 4.03 (lH, d, 14 Hz, 3-CH2S), 4.38 (lH, d, 14 Hz, 3-CH~S), 4.88 (lEI, d, 4 Hz, 6-H), 5.46 (lH, d-d, 4 and 8 Hz, 7-_), 8.54 (lH, d, 8 Hz, CONH).
Anal. Calcd. for C25H33N5O6S3: C, 50.40; H, 5.58;

N, 11.76.

Found: C, 50.76, 50.98; H, 5.67, 5.67; N, 11.27 11.28.

~3~7 .
7-[~-(2-aminomethyl-1-cyclohexenyl)acetamido]-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylic acid (6d) A mixture of trifluoroacetic acid ~2 ml.) and 5d (1.20 g., 2 mmoles) was stirred for 30 min. at room temperature and diluted with ether (S0 ml.) to precipitate the trifluoro-acetate of 6d. The trifluoroacetate was slurried in water (2 ml.), adjusted at pH 6 with ammonium hydroxide and diluted with acetonitrile (200 ml.) to precipitate 6d. Yield 0.75 g. (76%).
M.p. 215-220C. (dec.).
IR: ~ maUx 1760, 1640, 1580 cm UV: ~ 1%K2C03 276 nm (~, 13400).
Anal- Calcd. for C20H2SN54S3 1/2~I2 H, 5.19; N, 13.87; S, 19.06.
Found: C, 47.14, 47.23; H, 4.96, 5.07; N, 13.61, 13.70; S, 18.66.

. . .
. . , ,: j -~

~, .

- 70 - ~

:

Ex'ample''5 -CONN ~
O 2 ~ > -N

6e 7-[~-(2-t-Butoxycarbonylamin'ome'thyl-l-cyclohexenyl)-acetamidO

3-(3-hydroxypyridazino-~3,2-c]-s-triazol-6-ylthiomethyl)-3-- ''--------'' cephem-4-carboxylic acid (5e) A mix-ture o 2 (1.08 g., 4 mmoles), 2,4-dinitro-phenol (0~74 g., 4 mmoles) and DCC (0.82 g., 4 mmoles) in 'r~lF
(20 ml.) was stirred for 1 hr. at room temperature and ~iltered to remove the precipitated dicyclohexylurea which was washed with THF (10 ml.). The combined ~iltrate and washings were cooled at 5C. and poured in one portion into a cold (5C.) solution of 7-amino-3-(3~hydroxypyridazino[3,2-c]-s-triazol-~
6-ylthiomethyl)-3-cephem-4-carboxylic acid (1.14 g., 3 mmoles) ;-and triethylamine (0.81 g., 8 mmoles) in 50% aqueous THF (20 , 20 ml.)~ The m~xture was stirred overnight at room temperature and washed with ether (2 x 50 ml~). The aqueous layer was acidified to pH 2 with dil. HCl~ The precipitate was dissolved at 50C. in THF (100 ml.), treated with a small amount of carbon and dried with anhydrous Na2SO~. Evaporation of the solvent under reduced pressure a~forded an oily residue which , :

was solidified by trituration with ether (50 ml.) to give 5e.
Yield 0.85 g. (45~). M.p. 190~198C. (dec.).
IR: ~ max 1780, 1700, 1520, 1350, 1250, 1160 cm ~ DMS~d6 1 40 (9H, s, t-Bu-H), 5.2 4 Hz, 6-H), 5.80 (lH, m, 7-H), 7.10 (lH, d, 10 Hz, pyridazin-H), 7.80 (lH, d, 10 Hz, pyridazin-H).
Anal. Calcd. for C27H33N7O7S2 1/ 2 H, 5.35; S, 10.01.
Found: C, 50.59, 50.68; H, 5.40, 5.59; S, 9.57, 9.55.
7-[u-(2-Aminomethyl-l-cyclohexenyl)acetamido]-3-(3-hydroxy-pyridaz o-[3,2-c]-3-triazol-6-ylthiomethyl)-3-cephem-4-carboxylic acid (6e) ... . . _ . _ A mixture of trifluoroacetic acid (2 ml.) and 5e (0.81 g., 1.3 mmoles) was stirred Eor 30 min. at room temperature and diluted with ether (50 ml.) to precipitate the trifluoro-acetate of 6e. The trifluoroacetate was slurried in water (2 ml.). The mixture was adjusted at pH 6 with ammonium hydroxide and diluted with acetonitrile (200 ml.) to give 6e.
Yield 0.57 g. (82%). M.p. 225-234C. (dec.).
IR: ~ maU~ 1770, 1710, 1630, 1545, cm UV: ~ m%X2 3 260 nm (~, 18100~, 305 nm ~sh) (~, 6300).

`~ , ''"' . ~ , .. . ..

Anal. Calcd. for C22H25N7O5S2 3H2 H, 5.34, N, 16.74; S, 10.94.
Found: C, 45.28, 45.44; H, 4.35, 4.45; N, 16.56, 16.66; S, 11.75.
~- Example 6 '`CH CO-N r~ S~ N -Nl /~ N ~ CH2-S

CO2H N ~
6f ~_ _ Y

7-[(2-N-t-Butoxycarbonylaminomethyl-l-cyclohexenyl)-acetamido]-;3-~pyrido[2,1-c]-s-triazol-3-ylthiomethyl)-3-cephem-4-carboxylic acid (5f) DDC (740 mg., 3.6 mmoles) is added to a solution of 950 mg. (3.3 mmoles) of a-[2-(t-butoxycarbonylaminomet~yl)-l-cyclohexenyl]acetic acid (2), and 660 mg. (3.6 mmoles) of 2,4-dinitrophenol in 30 ml~ of THF and the mixture is stirred for one hour at room temperature and the urea filtered off.
To the filtrate is added a solution of 1.1 g. (3 mmoles) of 7-amino-3-(pyrido[2,1-c]-s-triazol-3-ylthiomethyl)-3-ylthio-mentyl)-3-cephem-4-carboxylic acid (4E) and 1.25 ml. (9 mmoles) of triethylamine in 30 ml. of wa~er and the mixture is stirred .~

for 18 hours at room temperature. The THF is removed under reduced pressure below 40C. and the residue is washed with ether (2 x 10 ml.), acidified with 6 N HCl and extracted with ethyl acetate (5 x 10 ml.). The combined extrac-ts are washed with water (2 x 10 ml.) and a saturated NaCl solution (10 ml.) and evaporated to dryness below 40C. Trituration of the residue with ether yives about 1.2 g. o~ solid 5~.
7- E (2-Aminomethyl-l-cyclohexenyl)acetamido]-3-(pyrido[2,1-c]-s-triazol-3-ylthiomethyl)-3-cephem-4-carboxylic acid (6f) Trifluoroacetic acid (2.5 ml.) is added to the BOC-blocked cephalosporin 5~ (1.1 g., 1.8 mmoles) with stirring for 1.5 hours under cooling and the mixture is diluted with 100 ml. o~ ether to separate the trifluoroacetate oE 6E whiah is dissolved in 5 ml. of water, adjusted to pl~ 6 with conc.
NH40H and diluted with 100 ml. oE acetonitrile. The resulting `~ yellow precipitate is collected by ~iltration and washed with acetonitrile to give about 700 mg. (76~) of solid 6f.
" '' ' ''' "`

.:
, - 7~ -.

Example 7 OJ

CO-N ~ S ~ N -N

O /~ ~ 2 ~ N

~ ,;
7-[(2-N-t-Butoxycarbonylaminome*hyl-l-cyclohexenyl)-acetamido]-.- 1 0 , ,, , ~
3-pyridazino[2,1-c]-s-triazol~3-ylthiomethyl)-3-cephem-4-carboxylic acid (~) To a solution o~ ~-[2-(t-butoxycarbonylam:inomethyl)-l-cyclohexenyl~acetic acid (2) (1.07 g., 4 mmoles) and 2,~-dinitrophenol (0.74 CJ., 4 mmoles) :Ln dry TE:IF (10 ml.) is add~d DCC (0.82 g., 4 mmoles) and the mixture is stirred for one hour at room temperature to precipitate the urea which is :
-~ removed by filtration. To the filtrate is added in one portion a cold solution of 7-amino-3-pyridazino[2,1-c]-s-triazol-3-ylthiomethyl)-3-cephem-4-carboxylic acid (4g) (1.09 g., 3 mmoles) and triethylamine (0.81 gO, 8 mmoles) in 50% aqueous T~F
(20 ml.) and the mixture is stirred for 20 hours at room ; ::
- temperature. The reaction mixture is washed with ether (2 x 50 ml.), acidified to pH 2 with diluted hydrochloric acid and extracted with ethyl acetate (6 x 50 ml.). The combined extracts are washed with water (2 x 30 ml.), treated with carbon and dried on anhydrous sodium sulfate. Evaporation of the solvent -~379~6~
under reduced pressure affords an oily residue which is solidified by trituration with ether (50 ml.) to give about 0.6 g. of solid 5g.
7-(2-Aminomethyl-l-cyclohexenylacetamido)-3-(pyridazino-[2,1-c]-s-triazol-3-ylthiomethyl)-3-cephem-4-carboxylic acid (6g) A mixture of 5g (0.53 y., 0.86 mmole) in trifluoro-acetic acid (1 ml.) is stirred for one hour at O - 5C. and diluted with ether (30 ml.) to precipitate the trifluoroacetate of 6g which is collected by filtration and dissolved in water (4 ml.). The solution is adjusted to pH 6 with ammonium hydroxide and diluted with acetonitrile (100 ml.) to precipitate 6g which is collected by filtration, washed with acetonitril~
(30 ml.) and dried in vacuo over P205 to y:ielcl abou~t 0.3 g.
solid 6g.
Example 8 ., :.
..

: 1 11 ' .
`I ~ CH2CQNH ~ I ~ N N

~ N ~ CH -S-C CH
6i ~ .

r~

7-[(2-tert.-Butoxycarbonylaminomethyl-'l-cyclohexenyl)-acetamido]--3-(1,3,4-thiadiazo~-2-me'rcap*ome'thy'l)-3'-cephem-4-carb'oxylic acid (5i) : -- .
To a stirred solution of 1.10 g. (0.0041 mole) of 2 and 0.80 g. (0.0044 mole) o~ 2,4-dinitrophenol in 40 ml. of ethyl acetate is added in one portion 0.90 g. (0.0044 mole) of N,N'-dicyclohexylcarbodiimide and -the mixture is stirred for 3 hr. at 25. The dicyclohexylurea is collected and the ~iltrate is evaporated at 45 (15 mm) to give the activated ester as an oil. The oil is dissolved in 30 ml. o~ THF. A
solution of 1.22 g. (0.0037 mole) of 7-amino-3-(1,3,~-thia-diazole-2-mercaptomethyl)-3-cephem-4-carboxylic ac:Ld and 1.03 ml. (0.0074 mole) of triethylamine in 20 ml. of 50'~ 'rHF-water is added to the solution O:e the activated ester and stirred for 18 hr. at 25. The THF is removed at 40 (15 mm.) and the concentrate (10 ml.) is washed with ether (5 x 100 ml.) and ` acidified to pH 2 with 40~ phosphoric acid. The mixture is extracted with ethyl acetate (6 x 100 ml.) and the combined extracts are washed with water and finally with a saturated sodium chloride solution. The ethyl acetate solution is evaporated at 40 (15 mm.) to a volume of 20 ml. and diluted with 30 ml. of Skellysolve B to precipitate the solid product 5i which is collected and dried for 16 hr. in vacuo over P2O5 at 25 to yield about 1.4 g.

~ 77 -3~qDI;317~
7-[(2-Amlnomethyl-1-cyclohexenyl')acetamido]-3-(1,3,4-thiadiazole-2-mercaptomethyl)-'3-cephem-'4-carboxylic~ ac'id (6i) A solution of 1.30 g. (0.00226 mole) of (5i) and 3.0 ml. trifluoroacetic acid is stirred for 1 hr. at 0. The 501uti:0n i5 diluted with 200 ml. of ~ther and the precipitate ,' eolleeted by filtration. The trifluoroacetate salt is sus-pended in 40 ml. of water and adjusted to pH 6.0 with dilute ammonium hydroxide. The gummy residue is then triturated with ~!',' 25 ml. of aeetonitrile to give about 350 mg. of 6i as a white powder. The produet is dried for 16 hr. in vaeuo over P2O5 at 25. ,, Example 9 ~/ CH2NH2 ' ~ .
\~CE2CON~ N -- N

1~ s COOH
'" 20 7-[(2-Aminomethyl-l-eyelohexenyl)aeetamido]-3-(5-hydroxymethyl-~ 1,3,4-thiadiazo~e-2_mereaptome*hy'1)-3-eephem-4-earboxylie aeid '~, (6j) ; ' ; The proeedure is the same as that of Example 8, exeept ~, that the produet 6j is eolleeted from the water to obtain about 340 mg. as a tan powder. A seeond fraetion is obtained from :, - 78 ~

. ~ -~L~3~
the filtrate to give about 200 mg. of 6j as a yellow crystalline solid.
Example 10 :`

C~'~ CONHr,/ S ~ ~
' ~ ~ CH2-S ,,N

6h 7-[~-(2-t-Butoxycarbonylaminometh~l-l-cyclohexen~l)-acetamido]-3-(lH-1,2,3-triazol-4-ylthiometh~1)-3-cephem-4-carbox~lic a _ (5h) A mixture of 2 (1.08 g., 4 mmoles), 2,4-dinitro-phenol (0.74 g., 4 mmoles) and DCC (0.82 g., 4 mmoles) in THF
(20 ml.) was stirred for 1 hr. at room temperature and ~iltered to remove the dicyclohexylurea which was washed with THF (10 ml.).
The combined filtrate and washings were poured in one portion at 5C. into a solution of 7-amino-3-(lH,1,2,3-triazol-4-ylthio-methyl)-3-cephem-4-carboxylic acid (4h) (1.24 g., 4 mmoles) and triethylamine (0.81 g., 8 mmoles) in 50% aqueous THF (20 ml.).
The reaction mixture was stirred overnight at room temperature and washed with ether (2 x 50 ml.). The aqueous layer was acidified to pH 2 with dil. HCl and extracted with ethyl acetate (3 x 50 ml.). The combined extracts were washed with water ; (50 ml.), treated with a small amount o~ carbon and dried over ` 30 :

anhydrous Na2SO4. Removal of the solvent under reduced pressure afforded an oily residue which solidified by trituration with ether - n-hexane (1:1, 100 ml.) to give 0.60 g. (26%) of 5h.
M.p. 120-128C. (dec.).
IR: ~ maUx 1780, 1720, 1680, 1520, 1250, 1160 cm NMR: ~ DMSO d6 1.38 (9H, s, t-su-H), 5.08 (lH, d, 4 Hz, 6-H), 5.65 (lH, d-d, ~ and 8 Hz, 7-H), 8.00 (lH, s, triazol-H), 8.92 (lH, d, 8 Hz CONH).

Anal CalCd- for C24H32N66S2 1/2H2 H, 5.80; N, 14.65; S, 11.18.
Found: C, 50.51, 50.69; H, 5.73, 5.65; N, 14.57, 1~.25; S, 10.05, 10.16.
7-[u-t2-~minomethyl-1-cyclohexenyl)acetamido~-3-(~ -1,2,3--triazol-4-ylthiomethyl)-3-cephem-4-carboxvlic acicl (6h) , ., , . . _ . . .... ...
A mixture of trifluoroacetic acid (1 ml~) and 5h t0.55 g., 0.98 mmoles) was stirred for 30 min. at room temperature and diluted with ether (50 ml.) to give the precipitate which was collected by filtration and slurried in a small amount of water (2 ml.). The mixture was adjusted to pH 6 with ammonium hydroxide and diluted with acetonitrile (200 ml.) to pre-cipi~ate 6h which was washed with acetonitrile (50 ml.)O
Yield 0.36 g. (80~)~ M.p. 203 - 215C. (dec.).
; IR: ~ max 1760, 1630, 1570 cm ~' .!
, .

; - 80 -;, . . .

UV: ~ %K2C3 266 nm (~, 8000).
max Anal. Calcd. for ClgII24N6O4S2- 1 1/2 H2O: C, 46.42;
H, 5.54; N, 17.10; S, 13.04.
Found: C, 46.06, 46.16; H, 5.18, 5.28; N, 18.06, 18.02; S, 12.37.
Example 11 The salicylaldehyde adduct o~ 7-[(2-aminomethyl-1-cyclohexenyl)-acetamido]-3-(3-hydroxypyridazine[3,2-c]-s-triazol-6-ylthio-methyl)-3-cephem-4-carboxylic acid (_) HO
~ ~ N=CH
6c ~ S .

~ _ ~ 7c CO2K ~H3 To a stirred suspension of 6c (Example 3, 766 mg., 1.6 mmoles) and triethylamine (300 mg., 3 mmoles) in methanol (8 ml.) is added salicylaldehyde (370 mg., 3 mmoles) and the - mixture is stirred for 30 min~ at room temperature to make a clear dark yellow solution. The solution is treated with a small amount of carbon and KEH (2 ml., 1 M solution in ethyl acetate of potassium 2-ethylhexanoate) is added to the filtrate.
~ The mixture is diluted with a large amount of ether (100 ml.) ': "

.. :

~3~
to precipitate the solid product 7c which is collected by filtration; washed wi-th ether (30 ml.) and dried. Yield about 750 mgm.
Example 12 The sallcylaldehyde adduct of 7-[(2-aminomethyl-1-cyclohexenyl)-_ acetamido]-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl) 3-cephem-4-carboxylic acid (7d) HO

~ N=CH- ~
6d ~ ,CONH-- ~ S ~ N - N

~ N ~ CH2-S ~ S ~ C 3 7d O

To a s-tirred mix-ture of 6d (E'xample 4, 778 mg., 1.6 mmoles) and triethylamine (300 mg., 3 mmoles) is added ; salicylaldehyde (370 my., 3 mmoles) and the suspension is stirred for 15 min. at room temperature to make a clear dark yellow solution which is treated with a small amount of carbon. KEH
(2 ml., 1 M solution in ethyl acetate) is added to the filtrate.
The mixture is diluted wlth a large amount of ether (100 ml.) to precipitate the product 7d which is collected by filtration, washed with ether (30 ml.) and dried. Yield about 850 mg.

~3~
Example '13 The salicyialdehyde adduct of 7-[(2'-aminomethyl-1-cyclohexenyl)-acetamido]-3-(3-hydroxypyridazino-['3,2-c]-s-triazol-6-ylthio-. _ .
methyl ? - 3-cephem-4~carboxylic acid (7e) HO

6e _~ ~ N=CH ~ OH
~ CONH - r~ s ~ N N
7e ~ ~ ~ CH2-S- ~ GN

` 10 C02K

To a s-tirred susp~nsion of 6e (Example 5, 754 mg., 1.42 mmoles) and triethylamine (300 mg., 3 mmoles) in N,N-dimethylformamide (7 ml.) is added salicyla.ldehydc (370 mg., 3 mmoles) and the suspension is stirred for 1~5 hr. at room temperature to be a clear dark yellow solution which is treated with a small amount of carbon. KEH (1.5 ml., 1 _ solution in ethyl acetate) is added to the filtrate. The mixture is diluted ' with a large amount of ether (100 ml.) to precipitate the product 7e which is washed with ether (30 ml.) and dried.
Yield about 900 mg.

Example 14 ; .
The potassium salt of the compound of Example 3 is prepared by adding a soluiton of potassium 2-ethylhexanoate (KEH) in ethyl acetate to a solution of the cephalosporin "
tzwitterion) (6c) in DMSO to precipitate the desired potassium salt.

., .

-3~
Example 15 -The sodium salt of -the compound of ExampIe 3 is pre-pared by pre-formation of its diethylammonium salt in methanol followed by addition of a solution of sodium 2-ethylhexanoate (SEH) in ethyl acetate and then dilution with isopropanol to precipitate the desired sodium salt which has a solubility in water greater than 250 mgm./ml.
Example 16 '- :,,, : ~/ NH2 ~\~ CONH-I / S ~
z~- N ~ ~ CH2-OCOCH3 , 6w CO2~l 7-[d-t2-t-Butoxycarbonylaminomethyl~l-cyclohexenyl)-acetamido]-cephalosporanic acid (5w) A mixture of 2 (1.08 g., 4 mmoles), 2,4-dinitrophenol (0.74 g., 4 mmoles) and DCC (0.82 g., 4 mmoles) in THF (20 ml.) was stirred for 1 hr. at room temperature and filtered to ~; remove the dicyclohexylurea which was washed with THF (10 ml.).
The combined filtrate and washings were poured in one portion at 5C~ into a solution of 7-ACA (4w) (0.82 g., 3 mmoles) and triethylamine (0.81 g., 8 mmoles) in 50% aqueous THF (20 ml.).
The reaction mixture was stirred overnight at room temperature .~ .
' '~

~ - ~4 -~g! 3~
and washed with; ether (2 x 50 ml.). The aqueous layer was acidified to pH 2 with dil. HCl and extracted with ethyl acetate (3 x 50 ml.). The combined ex*racts were washed with water (50 ml.) and dried over anhydrous Na2SO4. Evaporation of the solvent und~r reduced pressure afforded 5w as an oily residue which was solidified by trituration with ether - n-hexane (1:1, 100 ml.) and washed with n-hexane (50 ml.). Yield 1.24 g.
(79~). M.p. 100-110C.
IR:~ nu~ 1780, 1730 - 1640, 1520, 1355, 1225 cm NMR:~ DMSO d6 1.23 (9H, s, t-Bu-_), 1.96 (3H, s, 3-OAc), 4.48 (lH, d, 13 Hz, 3-CH2), 4.83 (lH, d, 13 Hz, 3-CH2~, 4.89 (lH, d, 4 Hz, 6-E[), 5.48 (lH, d-d, 4 and 8 Elz, 7-EI), 8~5 (lH, d, 8Hz, CONH).
Anal- Calcd- for C24~I33N38S ~/2 ~I2 H, 6.43; N, 7.88; S, 6.02.
Found: C, 54.16, 54.07; H, 6.19, 6.22; N, 8.36, 8.34; S, 5.61, 5.79.
7-[~-(2-Aminomethyl-l-cyclohexenyl)acetamido]cephalosporanic acid (6w) A mixture of trifluoroacetic acid (2 ml.) and 5w (1.20 g., 2.3 mmoles) was stirred for 30 min. at room temperature and dilu-ted with ether (50 ml.) to precipitate the trifluoroacetate which was slurried in water (2 ml.). The ~3~
mixture was adjusted to pH 6 with ammonium hydroxide and diluted with acetonitrile (200 ml.) to precipitate 6w. Yield 0.68 g.
~69~). M.p. 250 - 260C (dec.).
IR:~ nUaj 1800, 1735, 1625, 1570, 1230 cm UV:~ maX2C3 255 nm (~, 7100~.
~nal- CalCd- for C19H25N36S 1/2 ~I2O
' H, 6.05; N, 9.71; S, 7.41.
Found: C, 52.36, 52.15; H, 6.09, 6.06; N, 9.74,

9.71; S, 7.71, 7.81.

The solubility was 5.4 (BA) and 5.3 (VV).

Example 17 ,. .. .

, 1 ~1 ~\, CONH~ S ~
~ N ~ CH3 " O I

6x 7-[~-(2-t-Butoxycarbonylaminomethyl~'1-cyclohexenyl')'-acetamido]- ;
- desacetoxy-cephalosporanic acid (5x) A mixture of 2 (1~08 g., 4 mmoles), 2.4-dinitro-; phenol (0.74 g., ~ mmoles) and DCC (0.8~ g., 4 mmoles) in THF
(20 ml.) was stirred for 1 hr. at room temperature and filtered ` to remove the precipitated dicyclohexylurea which was washed with THF (10 ml.). The combined filtrate and washings were poured in one portion at 5C. into a solution of 7-ADCA (4x) .
~'' ..

~3~
(0.64 g., 3 mmoles) and triethylamine (~0.81 g., 8 mmoles) in 50%
aqueous THF (20 ml.). The reaction mixture was stirred at room temperature for 18 hr. and washed with ether (2 x 50 ml.). The aqueous layer was acidified to pH 2 with dil. HCl and extracted with ethyl acetate (2 x 50 ml.). The combined extracts were washed with water (50 ml.), treated with a small amo~mt of carbon and dried over anhydrous Na2SO4. Evaporation o~ the solvent under reduced pressure afforded an oily residue which was solidified by trituration in ether - n-hexane (1:1~ 100 ml.) to give 5x. Yield 0.88 g. (63%). M.p. 120 - 127 (dec.).
max 1780, 1680, 1520, 1250, 1160 cm NMR: (~ DpmO d6 1.35 (9H, s, t-Bu-H), 1.98 (3H, s, ; 3-CH3), 4.86 (lH, d, 4 HY.~ 6-H), S.40 (lH, d-d, 4 ~nd 8 Hz, 7-H), 8.50 (lH, d, 8 Hz, CONH).
Anal. Calcd. for C22H31N3O6S: C, 56.76; H, 6.71;
N, 9.03; S, 6.89.
Found: C, 56.21, 56.21; H, 6.65, 6.77; N, 8.98, 9.04; S, 6.71; 7.00.
7-[~-(2-Aminomethyl-l-cyclohexenyl)acetamido]--desacetoxycephalo-~ .
~oranic acid (6x~
To a cooled (0C.) trifluoroacetic acid (2 ml.) was added 5x (0.84 g., 1.8 mmoles) and the mixture was stirred for 1/2 hr. at room temperature. The mixture was diluted .

~L037~
; with ether (50 ml.) to precipitate the trifluoroacetate which -~ was slurried in water (2 ml.). The mixture was adj~sted at pH 6 with ammonium hydroxide and diluted with acetonitrile (200 ml.) to give 6x which was washed with acetonitrile (20 ml.). Yield 0.48 g. (73%). M.p. 240-245C. (dec.).
IR: ~ maU~ 3580, 3300, 1750, 1640, 1525 cm 1.

UV: ~ maK2C3 260 nm (~, 6500).
Anal. Calcd. for C17H23N3O4S 2 ` N, 10.95; S, 8.36.
~; Found: C, 53.83, 53.87; H, 6.06, 6.23; N, 11.08,

10.99; S, 8.75, 9.01.
The solllbility was 4.1 (BA) and 3.3 (UV).

,'~ ' .~, ", , , . ,, ~,~.. .... . .

'' ~3~8 ~C~2NH2 C H2C-~s I2--,s~y ,~ ~ .
7~-[,~ (2-t-Butoxvca"rbon,vlaminorneth,vl-1, 4-c.~rclohexa-di~n,vl)ac,et~mido~ -h!tdroxA~rPyridaz:ln-6-~rlth ~ethvl)-3-c~ephem,-4~-carboxA~lic acid (6a), To a stirred solution of 1.1 g. (4.1 mmoles) o~
a-[2-(t~but.oxycarbonylaminomethyl)-1,4-c~clohexadienyl]-acetic aci~(~) and 80o mg. (4.4 mmoles) of 2,4-dini~ro-phenol in 40 ml. of ethyl acetate was added in one portion ~.9 g. (4.4 mmoles) o~ N,N'-dicyclohexyl-oarb~diimide (DCC). The reaction mixture was ~tirred at room temperature ~or 3 hours~ m e separated di-c~clo~exylurea was riltered o.~f. The ~iltrate was evaporated to dryness to give the oily activated e~ter whlch wa~ dlssolved in 30 ml. o~ l~. To the ~olution was added a solution o~ 1.26 g. (3.7 mmol~s) o~ 7-amino-3-(3-hydrox~pyridazin 6~ylthiometh~ 3-cephe~l-4-carboxyllc acid (~a) ar.d 1.03 ml. (7.4 mmoles) ., ' ' `
. I

~.
.~ ,.

:3Le337~L~ii7 o~ trlethylamlne ln 20 ml. of 50~ THF. The mixture wa~ ~tirred at room temperature for 18 hours and concentrated in vacuo to 10 ml. o~ the volume. The concentrate wa~ washed wlth flve 100 ml. ~rtions Or ether, acidi~ied with 6 N hydrochloric acid and extracted wlth six 100 ml. portions o~ ethyl acetate. The combined extract~ were washed with water and a saturated NaCl solution. m e drled solution wa~ concentrated to dryne~s to give 720 mg. (~3%) o~ the N-BOC protected cephalo~porln meltlng at 170 C, (deo.), IR: YKaB~ 1780) 1710, 1690, 1670, 1580, 1520, 1~70, 1255, 1170 cm~~, NMR ~ DppmO d6 1.~4 (9H9 ~J ~13-C~), 2-57 t4HJ 3 ~ 3.~3 (2H, ~, ~ CO~, 3.4 - 3.7 (4H, m, 2-H & C~ -N), 3.9 - ~.2 (2H, m, 3-t~ ~ J
4.98 (lH, dJ 4.5Hz, 6-H), 4.9 - 5.2 (3H, m, ~ ( &
C~ 9 5~.29 (lH, d~ 9.5Hz, pyridazlne-H~, 7.23 (lH, d, 9.5Hz~ pyridazine-H) J 8.77 (lH, d, 7.5Hz, CO~) .
Anal, Calcd. for ~26H31N507S2 H2 ~J 5.16; N, 11.56; SJ 10.59.
~ ound: C, 51.43; X, 5.13; N, 11.66; S, 10.79.

., : ~3746~7 7~ (2-Aminomethvl-1.4.-cvclohexadlen~l)acetamido~

carboxylic acid. (7a) (A) A ~olution o~ 670 mg. (1.13 mmol~) of the BOC-protected cephalosporin 6a in 1.5 ml. of tri-~luoroacetlc acid wa~ stlrred at 0 C. for one hour.
To the solutlon was added 100 ml. o~ dry ether. The reaultlng preclpitate wa~ collected by ~iltration5 ~uspended in 20 ml. of water and ad~u~ted to pH 6 with dllute ammonium hydroxide to give a vi9cou9 oil, which was trlturated with acetonitrile to give 380 mg. (75.5~) Or the de~ired product ~ meltlng at Z00-205~ C. (dec.).
I~: KBr 1765, 16ll0, 1580, 1390, 1350 cm 1.
.'; .
Anal. Calcd- for C21H23N55$2 1/ 2 ~, 4.85; N, 14.05.
Fouhd: CJ 50.95; ~, 4.79, NJ 13.10.
(B) To a stirred suspension o~ 1.1 g. (3.6 mmoles) o~ sodium~2-[N~ carbethoxynropen-2-yl)aminomethylJ-1~4 cyclohexadienyl3ace~ate (4) in 20 ml. o~ dry tetra-h~dro~uran (~HF) containing one drop of N,N-dimethyl-benzylamlne was added 0.38 ml. (~.95 mmole~j o~ ethyl ~hloro~ormate at -10 to ~5 C. To the mixed anhydride solutlon wa~ added ln one portion a solution o~ 1~02 g.
(3 mmoles) of 7-~a-(2-aminomethyl-1,4 cyclohexadienyl)-acetamldoJ-3-(3-hydroxypyridazin-6-ylthiomethyl)-3 , -91-: . . 1, : .

~ J379L~i7 cephem-4-carboxyllc acid (~) in 10 mlO of 50% aqueou~ THF
containlng 0.42 ml. (3 mmoles) o~ triethylamlne. The m~xture wa~ stirred for 30 mln. at room temperature, treated wlth active carbon and filtered~ To the ~iltrate was added 3 ml. o~ ~ormic acld a~d the mLx-ture wa~ ~tirred for ~0 min. at room temperature. The re~ultlng preclpitate wa3 filtered, washed with water and acetonitrile to glve 570 mg. o~ 7a xamPle L9 ~ oH2N~2 C~C0-N ~ ~ N--N
2-S ~ - N

1- ~r2- tN-t-~u't~carbon~lamin~omethvl~-1.4-c~clohexadien~l~-n-6-Ylthiomethvl~

To a ~tlrred olution o~ 3.2 g. (0.012 mole) of a-[2-~t butoxycarbonylaminomethyl)-1~4-c~clohexadienyl]acetic ~c~d ~) and 0.~ gO (0.0125 mole) of ~,4-dinltrophenol ~n 50 ml. o~ THF wa~ added 2.6 g. (0.0125 mole) of DCC and the mixture wa~ stirred for 1.5 hours at room temperature.
The precipitated urea wa~ removed b~ riltration. A
~olution o~ 3.65 g. (0.01 mole) o~ 7-amino-3-(tetrazolo-[1,5-b~pyrldazin-6-ylthiomethyl-3-cephem-4-carboxylic aold (5b) and 2.8 ml~ (0.02 moles)of trlethylamlne ln 100 mla o~ water was added to the active ester solution.
The mixture was ~tirred for 18 hours at room temperature.

~37~i7 Mo~t of THF wa3 removed under reduced pre~sure below 40c C, and the re~idual solution was washed with ether (3 x 50 ml.~ and acidified with 6 N HCl and xtracted with ethyl acetate (8 x 100 ml.)~. The combined extracts were wa~hed with water (~ x 100 ml.) and saturated NaCl ~olution (2 x 100 ml.) and evaporated to dr~nes~. The oily residue was trlturated with ether to give 2,2 g. (52%) of 6b melting at 165 C. (dec.).
IR: ~na~ 1770, 1710, 1670, 1510, 1240, 1150 cm 1.
An~l. Calcd. ~or C26~30N806S2 ' 5 4.92; N, 18.~; S, 10.43.
Found: C, 51.05; H, 1~.97; N, 17.24; S, 9.09.

Amino~cyclohexadienyl~ac~etamidoJ~
(te~,razol,o[l ~bJP~rridazin-6-Ylthiometh~ 3-cephem A mixture of 3.1 g. (5.05 mmoles) of 6b and 6 ml~ o~ tri~luoroacetic acid was stirred for one hour under cooling and dlluted with 200 ml. o~
ether to ~eparate the trl~luoroacetate of 7b which Wa8 dis~olved in 5 ml. o~ water, ad~usted to pH 6 with conc. NH40H and diluted with 900 ml. of a~etonitrile. m e yellow precipitate wa~ collected by filtration and wa~hed with acetonitrile to give 2.3 g. (88.5~) of 7b melting at 210 C. (Dec.).
Recr~talllzation of 2 g. o~ 7b from 350 ml. of .

. ` ..

50% THF gave 1.19 g. of pale yellow needles melting at 220-230C. (dec.).

IR: ~ ax 1785, 1640, 1610, 1570 cm ... .
Anal. Calcd- for C21H22N84S2 1/2C4 8 N, 4.76; N, 20.35; S, 11.64.

' Found: C, 50.17; H, 4.56; N, 19.59; S, 11.30.

Example 20 :

~ CH2NH2 H

~H2~O~N ~ ~ ~ N - N
N ~ ~-- CH2 S ~ N N
~13 7c 7-[(2-N-t-Butoxycarbonylaminomethyl-1',4-cyclohexadienyl)-acetamido]-3-(1-methyltetrazol-5-ylthiomekhyl)-3-cephem-4--carboxylic acid (c) To a stirred solu~ion of 3.2 g. (0.012 mole) of ~-[2-(t-butoxycarbonylaminomethyl)-1,4-cyclohexadienyl]-acetic acid (3) and 2.3 g. (0.012 mole) of 2,4-dinitrophenol ~
in 50 ml. of T~IF was added in one portion 2.6 g. (0.012 mole) ' ' ' of DCC and the mixture was stirred for 1.5 hours at room :~ .
'~ temperature. The precipitated urea was ' ':

:,.
. ,~.

.

~ L~3791~7 removed by ~iltratlonO To the ~lltrate wa~ adde~ a solut~on Or 3.28 g. (0.01 mole) of 7-amino-3~
methyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylic acld (~) and 2.8 ml. (0.02 mole) o~ triethylamine ln 100 ml. o~ water and the mixture wa~ stirred for 18 hours at room temperature. Mo~t of THF wa~ removed under reduced pre~sure below 40 C~ and the resiclual aqueou~ ~olutlon wa~ wa~hed with -ether (2 x-50 ml.), acldified with 6 N HCl and extracted with eth~l acetate (4 x 50 ml.). The combined e~tracts were wa~hed with water (2 x 50 ml.) and a saturated NaCl 801utlon (2 X 50 ml.), dried wlth Na2SOI~ an~ evaporated to dryne~. The olly re~idue was triturated with ether (100 ml.) to give 2.3 g. (48~3 Or 6c, melting at 145 C.
; (dec.).
IR:~ maJ 1780, 1700, 15109 1240, 1150 cm 1.
Anal. Calcd. for C2~H3}N706S2: C, ~9.90; H, 5.41;
N, 16.97;~S, 11.10.
.Found: C, 50.23; H~ 5.2~; N~ 15.80; S, 12.4 7-[(?- Aminomethyl ~,_ ,~_,~
:, ' ~Q,~ (~Q)-A mixture o~ 2.2 g. (~.8 mmole~) o~ 6c and 5 ml.
Or tri~luoroacetic acid was ~tirred ~or one hour under ~oollng and diluted with 200 ml. ar ether to separate ~ . .

_95 ,. . :

3~
the tri~luoroacetate of ~Q which was dls~olved in 5 ml. o~ water. The ~olutlon was adJusted to pH 6 wlth conc. NH40H and dlluted wlth 200 mlO o~ aceto-nltrlle to glve the yellow preclpitate wh~h was collected by f'iltratlon, washed with acetonitrile to yield 1.5 g. (82.5~) of ~ melting at 200 C.
(dec.), which was recrystallized ~rom 50~ THF to ~lve pale yellow needles, M.p. 210 C. (dec.).
IR:~ ma~ 1785~ 1640, 1615, 1570 ~m 1.

Anal- Calad, ~or C19~l~3N704S2~1/2C4~8 C, 49.11; H, 5.30; N, 19.09; S, 12.48.
Found: C, 49.85; H, 5.20; N, 18.5~; S, 12.26.

, xam~le_~
''.
. " _~ " ~H2NH2 ~ CH2C-N ~--N
: ~ ~ ~ C~-S S ~ CH3 ~O~I
7d 7-LL2-N~ utox~carb.onylaminometh~yl-1,4-cvclohexadien~
a~etamiq~o~ -m~ethgl~ ~th~ lazol-2 ~ thiometh~
; ~cephem-4-carbox~llc~acld. (6d) To a ~tirred solutlon.of 900 mg. (~.3 mmoles) of a~[2-(t-butox,ycarbon~lamlnomethyl)-1,4-cyclohexadienylJ-acetio acid (~) and 660 mg. (3.6 mmole~) o~ 2J4-dinitro~
~' .
.

37~6~7 phenol in 40 ml. o~ THF was added in one portlon 740 mg. (3.6 mmoles) of DCC and the m~xture wa~ ~tlrred ~or 2 hour~ at room temperature. I'he preclpitated urea wa~ removed by flltratlon. To the ~iltrate was added a solution of 1.03 g. (3 mmole~) c~ 7-amlno-3-(5~mekhyl-1,3,4-thladiazol-2-ylthiomethyl)-3-cephem-4-carboxylic acid (~d) and 1.~ ml. (9 mmoles) o~
t~lethylamine in ~0 ml. of water in one portlon and the mlxture wa~ stirred for 18 hour~ at room temperature.
The THF was remov~d under reduaed pre~sure below 40~ C.
and the concentrat~ was wa~hed wlth ether (~ x 10 ml.) and acidifled with 6 N HCl and extracted wlth eth~l acetate (5 x 10 ml.)~ qhe comblned extracts were washed ~uc¢e~ ely with water (2 x 10 ml,) and a saturated NaCl solu~ion (10 ml.). Evaporation ~ollowed by trituratlon with e~her gave 980 mg. (55~) of 6d, IR:Y~nauJ 1770, 1710, 16609 1610, 1510, 1240 ,' ':i 1150 cm AnalO Calcd. ~or C25H~lN506S3: C~ 50.57; H, 5.26~ N~ 11.80.
Found: C, 50.51; H, 5.20; N, 11.~6.
7-~2-Aminomethyl-1,4 cyclohexadlenyl)acetamido]-3-(5-methyl~ ,4-thiadiazol-2-ylthlomethgl)-3-cephem~4-oarboxyllc acid (7d) -g7-~ 37~i7 (A) A mixture o~940 mg. (1.58 mmoles) Or 6d ~nd 2 ml. of trlfluoroacetlc acid wa~ stirred for one hour under coollng and diluted with 100 ml.
of ether to separate the trifluoroacetate of X~
wh~ h wa~ di~olved in 5 ml. of water and 5 ml. of acetonitrile, m e solution was ad~usted to pH 6 wlth aonc. NH40H and diluted with 100 ml. o~_ acetonitrile. The resulting pale yellow precipitate wa~ ¢ollected by flltration and wa~hed with acetonltrile (5 ml.) to give 600 mg. (77~) of 7d J
melting at 230 - 240 ~, (dec.).
TR:~ maJ 179OJ 1650, 1580 cm ~.

, 20 2~ 5 4 ~ ' ; J 7 ;
N, 1~.19; S, 19,~9"
Found: C, 49.24; *, 4.59; N, 1~.88; S~ 20.16 (B~ To a st~rred suspen~ion o~ 1.1 g. (~.6 mmole~) o~ sodium~2-[N-~ carbethox~propen-2 yl)aminomethyl]-1,4-cgolohexadien~l~acetate (4) ln 20 ml. Or dry ~HF containing one drop of NgN-dimethyl-benzylamlne was added 0.~8 ml. (3.95 mmole~) of ethyl ohloroformate for 10 min. at -10 to -15 C. q'o the mixed anhydride solution wa~ added ln one portion a ~olution Or 1.03 g. (3 mmoles) o~ n 10 ml. Or 50 aqueou~ THF containing 0~42 ml. (3 mmole~) o~ tri-ethylamine and the mi~ture was ~tlrred for ~0 min.

. -98-..

-~37~67 at room temperature. A ~ew drops of formlc acid wa~ added and the mixture was filtered. To the filtrate was added 200 ml. of ether and 3 ml. o~
~ormic acid. The precipltate was collectffd by ~iltration, washed wlth water and acetonitrile ~o give 1.1 g. (62%) of 7d.
xample 2~

~ CH2NH2 OH

2C N ~ N
0 ~02}1 .
~ZQ ' ' ~[ (~-N-t-Butox~çarbonvlaminomethvl-1.4-cvclohexa-dienYllasetam-ido]-~ hydrox~yridazino[3~2-c]
~azo'1-6-glthiomethvl) -~.ce~hem-4-carboxYlic (6e) To a stirred mixture o~ 900 mg. (3.3 mmole~) o~ 2-(t-butoxycarbonylaminometh~ 1/4-cyclo-hexadienyl]acetic acid (~) and 660 mg. (3.6 mmoles) of 2,4-dlnitrophenol in 30 ml. of mF was -added in one portlon 740 m~. (3.6 mmoles) o~ DCC
and the mixture was stirred for 2 hours at room temperature. The ~eparated urea wa~ filtered .
_99_ 37~7 O~r. ~0 the activated ester solution was added in one portlon a solution Or 1.1~ g. (3 mmoles) of 7-amino-~-(3-h~droxyp~ridazino[3,2-c]-s-triazol-6-yl~hlomethyl)-~-cephem-4-carboxylic acid (Se) and 1.25 ml. (9 mmoles) o~ trlethylamine in ~0 ml. of water and the mixture was stirred for 18 hours at room temperature. The THF was distilled o~f~under reduced pre~sure below 40 C. and the residue was wa~hed with ether (2 x 10 ml.) and acidified with 6 N HCl and extracted with e~hylacetate (5 x 10 ml.). The combined e~tract~ were wa~hed wi~h water (2 x 10 ml.) and a ~aturated NaCl solutlon (10 ml.), and evaporated to dryness. The residue wa~ triturated with ether to give 505 mg. (27~) o~ 6~9 melting at 175 - 180 C. (dec.).
IR:~ naJ 1770, 1700, 1510~ 1200, 1150 cm 1.
Anal~ Calcd. ~or C27H31N707S2 C~ 51-50;
H~ 4.96; N~ 15.57; S~ 10.18.
Found: CJ 51.61; H~ 4.80; N~ 14.47; S~ 10.25 3-~3-h~drox~pvridazino-[~,2-c~-s-triazol-6-~lthio-~ç~kYl~ hem-4-carbox~lic acid ~Z~) ~ ri~luoroacetic acid (1 ml.) wa~ added to 470 mg.
(0.747 mmole~) o~ 6'e and the mixture wa~ stirred ~or one hour under oooling and d~lute~ with 100 ml. o~

, . --100--ether to ~eparate the tri~luoroacetate o~ 7e which was dlssolved in 5 ml. o~ water and adJusted to pH 6 with conc. NH~OH. An addition o~ 100 ml.
Or acetonitrlle to the mixture gave yellow~
precipitate whlch wa~ collected by filtration and wa~hed with acetonitrile to give 360 mg. (91%) o~
7e, melting at 210-220 C. (dec.).
IR:~ mnaJ 1760, 1710, 1640, 1580 cm 1.

Ana 1. Calcd. ~or C22H23N705S2 2 H, 4.60; N, 17.91; S, 11.71.
Found: C, ~8.75; H, 4.60; N, 16.56; S, 12.17.

EXample 23 C~2CO-N ~ ~ N- N

~ ~ H

acQtamido]-~- (p~rido[~,l-cl-s-triazo.l-~-~Tlth:l ometh~
cephem-4-carbox~lic acid (6e).
DCC (74C mg., 3.6 mmole~) wa~ added to a ~olution o~ 950 mg. (3.3 mmoles) o~ a-~2-(t-butoxycarbonylamlno-methyl)-1,4-cycloh~xadlenyl~acetic acid (~) J and 660 mg.

-~01--~ 6~
(3.6 mmole~) o~ 2~4-dinitrophenol in 30 ml. o~ THF and the mixture wa~ ~tirred for one hour at room temperature and the urea wa~ filtered o~. To the ~iltrate was added a ~olution of 1.1 g. (3 mmole~) of 7-amin~-3-(pyrido[2,1-c]-s-triazol-3-ylthiomethyl)-3-ylthiomethyl)-3-cephem-4-carboxylic acid (~f) and 1.25 ml. (9 mmoles) of tri-ethylamine ln 30 ml. o~ water and the mixture was stirred ~or 18 hourc at room temperature. me THF wa~ removed under reduced pre~ure below 40 C, and the residue wa~ wa~hed wlth ether (2 x 10 ml.) 7 acldi~ied with 6 ~ HCl and extracted with ethyl acekake (5 x 10 ml.).
me combined extraots were ~a~hed with water (2 x 10 ml.) and a saturated NaCl ~olution (10 ml.~ and evaporated to dryne~ below 40 C. Trlturation of the residue with ethe~ gave 1.15 g. (63~) o~ 6~, melting at 145 -150 C. (dec.).
IR:~ ma~ 1770, 1680, 151~, 1240, 1150 cm 1 A~a~l. Calcd. for C28H3~N606S2 C, 5 N, 13.72; S, 10.47.
Found: C, 53.84; ~, 5~13; N, 13.12; S~ 9.26.
7,~ 2-Aminometh~1-1,4-c~clohexadienvl)acetamido]~

YC~ II (7f) ~ rl~luoroacetic acid (2~5 ml.) wa~ added to the BOC-blocked cephalo~porin 6~ (1.1 g.~ 1.8 mmole~) -~2~

1~93~67 wlth ~tirrlng ~or 1.5 hours under cooling and the mixture wa3 diluted wlth 100 ml. o~ ether to separate the trl~luoroacetate of 7f which ~as dissolved ln 5 ml.
o~ water, ad~u~ted to pH 6 wlth conc. NH4~H and diluted wlth 100 ml. o~ acetonitrile. The resultlng yellow preclpltate wa~ collected by filtrationJ washed with acetonitrlle to glve 700 mg. (76%) of 7~ melting ak 200-210 C . (dec . ) .
, . IR~ aJ 179OJ 1660, 1630, 1580 cm 1.
. , A~al Calcd. rOr C22~I2~N60l~S2 1/2 ~I20 C~ 52-96;
H, 4.8~; N~ 16.11; S, 12.29~
Found: C, 53.08; H, 4.65; N, 15.16; S, 12.29.

ExamPle~ ~4 ~ ~H2NH2 . ~H2CO-N ~ N--N
CH
.,' ' ' ~

7~L(2-N-t-:3utox~carbonvlaminometh~ l ,,4-cYclohexad~ enyl~ --~-Q~ daæl~Q~.l-c]-~-tri_z~ 2-LLll~a~sohyll:
~ÇEb~m=3=93~box~1ic acld tl~) To a solution of a-[2~(t-butoxycarbonylaminomethyl)-1,4-cyclohexadienyl3acetic acld (~) ~1.07 g., 4 mmoles) and 2,4-dinitrophenol ~0.74 g.J4 ~moles) in dry THF
(10 ml.) wa~ added pCC (0.82 g., 4 mmoles) and the ~ ~37~
mixture was stirred for one hour at room temperature at preclpitate the urea whicn was removed by filtratlon.
To the ~lltrate wa~ added in one portion a cold solution of 7-amino-~-pyridazlno[2,1-c]-s-trlazol-3-ylthiomethyl)-~ phem 4-carboxylic acid (~) (1.09 g./ 3 mmoles) and triethylamlne (0.81 g., 8 mmoles) in 50% aqueous T~F (20 ml.) and the mixture wa~ stlrred for 20 hours at room temperature. The reactlon mixture was washed with ether (2 x 50 ml.), acidified to pH 2 with diluted hydrochloric acid and extracted wlth ethyl acetate (6 x 50 ml.). I~le combined extracts were wa~hed with water (2 x 30 ml~), treated with carbon and drle~ on anhydrous ~odium ~ul~ate. Evaporation Or the 801vent under reduced pre~sure af~orded an olly re~idue which wa~ ~olidi~ied by trlturation with ether (50 ml.) to glve o,63 g. (34%) of 6~, M,p. 202-213 C. (dec.).
IR ~ na~ 1785, 1780, 15209 1250, 1160 cm 1.
NMR:~ ppm 6 1.~9 (9Hg s, CH3-C~), 3.89 (4H, 8J H2 ~ ), 5.0~ (lH, d, 4Hzg 6-H), 5.67 (,~H, br-s, 7 H & ~`C~), 7.20 - 8.90 (3H, m9 pyridazin-H), 9.05 (lH, d, 8Hz, NHC0~.
Anal. Calcd. for C27H31N706S2 H2 H, 4.95; N, 17.83; S~ 11.67.
Found: Cg 48.32; H, 3.94; N, 17.19; S7 11.90.

, ' ç -104-~37~7 lpqrldazino[2 ,l-c ]-~triazol-~-vlthi omethyl) -~ce~hem-4-carbox~lic a cld (~;) A mixture o~ 612 (0.53 g., o.86 mmole) in trl-rluoroacetlc acid (l ml.) was stirred ~or one hour at 0 - 5~ C . and dlluted wlth ether (30 ml . ) to precipltate the trifluoroacetate of 7~ whlch was collected by ri.ltration and di~olved in water (4 ml.).
~he solution wa~ ad~u~ted to pH 6 with ammonium hydroxide and dilute with acetonitrlle (lO0 ml . ) to pre¢ipitate ~; which wa~ collected by ~iltration, washed wlth acetonitrile (~0 ml.) and drled ln,vacuo on P205. Yleld 0.38 g. (86~). M.p. 208 - 216 C.
(dec.).
IR: ~ maJ 1780, 1620, 1570, 1~40 cm l.
U~ C03 276 nm (~ 5600).
max ~ nal. Calcd. ~or C22H2~N704S2 H20 ' 5 ~, 5.27.;^ N, 15.52; S, 10.15.
Found: C, 51.00; Hg 5~10; N~ 15.00; S~ 10.32.

~3~7~
~7 ~H2NH2 l 3R

~CH2COOH - t-- CH3~ C-O-C-N;~
C~H3 O CH

O-C-CH , _ 3 ~ HO~N02 _~_ <~N_C_N
DCC
~2 NH ,C, O C ( CH3 ) 3 --( O N02 ~_ ~CH2-1CI-- ~3No2 ., O

.' ' O
il A ~ ~ S ,~ 2 NH-C-O-C(C:H3)~5 o~LCH~ S-R~ ~CH -C-NH~F
. ~)OH o ~ ~L CH2-S-R
~OH

,.

~37~

~ NH
6 ~ ~ ~ 2 2 ~LCH -S-R

z COOH

H ._ R~ S~ C~I20H ~N
N--N N N
h ' ~107-- .
~.

Examp~l~e 25 ~~H2NH2 S N--N
CH -S-C CH
COOH

~108--- , ~g~3~9L6~7 .' ~ 1~_ me~h~l2.-3-ce~hem-4-carboxvlic acid (6i).
To a ~tlrred ~olutlon o~ 1.10 g. (0.0041 mole) of and 0.80 g. (0.0044 mole) of 2,4-dinitrophen~l ln 40 ml. o~ ethyl acetate wa~ added in one portion 0.90 g. (0.00~ mole~ o~ N,N'-dicyclohexylcarbodiimide and the mi~ture was stirred for ~ hr. at 25. The dicyclo-hexylurea wa~ collected and the filtrate wa~ evaporated at 45 (15 mm) to give the activated e~ter a~ an oil.
The oll wa~ di~solved in ~0 ml. of ~. A ~olution o~
1.22 g.(0.0037 mole) o~ 7-amlno-3-(1,3J4-thiadiazole-2-mercaptometh~ -cephem-4-carboxyllc aaid and 1.0 ml. (0.0074 mole) of triethglamlne in 20 ml. o~ 50%
~HF-water wa~ added ko the solutlon of the activated e~ter and.'~tirred for 18 hr. at 25. The THF wa~
r~oved at l~o (1~ mm.) and th~ concentrate (10 ml.) ~a~ wa~hed with ether (5 X 100 ml.) and acldified to pH 2 with 40% pho3phoric acld. ~he ml~ture wa~
e~traoted with ethyl aGetate (6 X 100 ml.) and the ~omblned extracts were washed with water and ~inall~
~th a ~aturated sodium chloride ~olution. The eth~l a~etate solution wa~ evaporated at 40 (15 mm.) to a volume o~ 20 ml. and diluted with 30 ml. of Skell~olve B. The produot ~ wa~ oolleoted and dried ~or 16 hr~

.

~i37~

~ Q over P205 at 25 to ~ield 1.4 g. (65.4%) o~whlte powder. m e IR and NMR ~pectra were con3i~tent ~or the ~tructure. M.p. 1~5 slow dec.
7~(?~Aminometh~1-1,4-c~clohexadien~l)acetamldo]-3-~1,3,4-thiadlazole-2-mercaptometh~ 3-cephem-4 5~LXYllC acid (I~) A ~olution o~ 0 g. (0.00226 mole) o~ (6i) and 3.0 ml. trlfluoroacetic acid was stirred for-l hr. at 0. The solutlon was diluted with 200 ml. o~ ether and the preclpitate collected by riltra tion . The tri-rluoroacetal;e salk was suspended in 40 ml. of water and ad~u~ted to pH 6.o with dilute ammonlum hydro~ide. Th~
gu~ residue wa~ then triturated w:l th 25 m:L ~, o~
a~etonitrile to glve ~70 mg. (34.5~) of` white powder.
The product was dried for 16 hr. in vacu,o over P205 at 25. The IR and NMR ~pectra were consi~tent ~or the 3tructure. M.p. 135 810w dec. Anal: Calcd.
~or Cl~lNs4S3'H2: C~ 45-85; H, 4.65; N, 14.070 .~ound: C, 45.71; H, ~.52; N, 14.12.
m e equillbrium ~olubillty of this compound ln mgm./ml. was 4~28 in dlstllled water and 5085 in pH 7~0 phosphate bu~fer.

,~

~al3~7~6~7 ..

CH2N~I2 CH2CON~ ~ ~ N--N
0/~ N ~ 2 S C ~S ~ C-cH2o~I
COOH

7~ AmlnQmçtb~ 4-c~cloh~xadlen~ etami~o~-3 U~æ__ h~
carbox,~ic acid. (~,) The procedure wa~ the same as that o~ Example 8, except the product wa~ collected from the water to obtai~ 340 mg. (29. ~) o~ a tan powder. A second . rrac~ion wa~ obtained rrom th~ filtrate to give 2~0 mg. (20. ~9 of ~1 as a yellow crysta~line hemi hydrate.
: ~he I~ and NMR spectra were conslstent ror the ~tructure.
M.p. l50 slow dea7 . Anal. Calcd ~or C~oH2~N505S~- 1 l/2 H20: C, ~4.75;
- ~, 4088; N, 13~05. Found: C, 44.84; H, ~.63; N~ 12.75~
m e second rraction was found to be o~ equal purlty.
Ihe IR and NMR spectra were consistent ~or the ~trueture.

' .--3~6~7 -:

~H2NH2 ~2Co-~r~s~ C C~ 7 ~COOH 2 C~N~N

7h ~lnomethyl-1,4-c~clohexadl~ rn~ol '-(1,2~3-trlaæole-5-mercaptomethyl)-3-cephem 4 ~arbox~llc aald (~) m e procedure was the ~ame as that o~ Example 8, except the product Wa8 collected ~rom 15 ml. o~ , a¢0tonitrile to obtain 100 mg. (19.1%) o~ 7h as a i .llght tan powder. ~rhe IR and N~ spectra were con- j ~lstant ~or the structure. M.p. 160 ~low dec.
~nal- Calcd- ~or C~ 22N604S2 ~2~ ~ ¦
~, 4.88; N~ 16.380 ~ound: C3 45 .76; H, 5.25; N, 16085.

~ .
.. . " ' ..
.' , ' '.

, r~ --112--.

~7 ~' ~parat~on o~ 7-~2_Amlnometh~vl-1~4-c~lohexadienYl)-a~tamido]~ -meth~ltetrazol-s-~lthiome~h~ ~cephem .4~rbox.~11c acid (7c~ Lot ?

NH-BOC ~ ~ ~ ME-BOC
COOH l COO- ~ N2 J

,~ .

.

"

o~F~cH2-s~N ~
" ~ ~ o2H bH~ .

. . " ~ , CO~ N N
., . ~LCH2-SJ~
: ~02H ~H~

:~ 7Ç
.
Dlc~clohexylcarbodiimide (DCC) (9.0 g.~ 0.044 mole) was added to a mlxture of 11.2 g. (0.042 mole) o~ (2-N-t-butox~-~arbonylaminow1,4-cyclohexadienyl)acetic acid (2) and 8.0~ g.
(O .044 mole) o~ 2, 4-dînltrophenol in 250 ml . of IHF . The miJcture wa~ stirred at room temperature :Eor 1.5 hr. and the .' ;
.

~3~7 preclpltated urea was filtered of~. To the flltrate wa~ added a olutlon o~ 11.5 g. (0.035 mole) o~ 7-amino~3~ methyltetrazol-5 ylthiomethyl)-3-cephem-4-carboxyllc ac~d and 14.7 ml. (ca. 0.105 mole) of tr~ethylamine in 250 ml. of water. The mixture wa~ stirred at room temperature for 20 hr. and the ~HF was removed under reduced pre~sure below 40 C..
The aqueous solutlon wa~ washed with ether (-200 ml.), acldlfied wikh 6 N }ICl and extracted with ethyl acetate (~ ~ 200 ml.). The extract~ were washed with ~ater and a saturated aqueou~ NaCl ~olutlon, dried with Na2S04 and evaporated to dryne~s. The olly resldue was triturated with ether (500 ml.) to give 8.05 g. (4 ~) of the N-blocked 7c (that is, 6c).
IR: ~ ~aJol 1770, 1710~ 1650, 1510, cm 1.
A mlxture of 8.o g. (0.01~8 mole) of 6ç and 16 ml. o~ trl~luoroacetlc ~cid (TFA) was stirred at 0-5 C. for one hour and then diluted with 300 ml.
o~ ether to separate the tri~luoroacetate o~ ~J
~hich was di~solved in 30 mlO o~ 50% aceton~trile, ad~u~ted to pH 6 with conc. NH40H and diluted wlth 500 ml. Or acetonitrile. The ~ellow precipitate ~hich sepzrated wa~ wa~hed well with acetonitrile (200 ml-) ~o give 5.? g. (7 ~) amorphous ~ (lot 2~.

~L~3~
M.p, 195-205 C . (dec . ) .
IR: ~naJl 1780, 1650, 1620, 1560 cm 1.
W: ~ P~X7 bu~f ~ z74 nm ( , 9100) .

Anal. Calcd. f`or C19H2~jN704S2-H20: G, 46.05;
}I, 5.08; N, 19.78; S, 12.94.
Found: C, 46.64; H, 4.60; N, 18.63; S, 12.59.
Solubllity ln 0.1 M pho~phate buf~er (pH 7):
9<,4 mg./ml. ~~

~x~mple 2 Pre,~ar~ Qr~ o~ 7-~ (?~-A~ nomethyl-~,4-~:vc~oh~xad~
~Q~do~-meth~ 4-~liadla7ol-2~-ylthiomethyl) NH-BOC ~ ~ NH-BOC 1~~
~COOH L COO-Ç~N02 -- . , .~

-BOC S

~ ~CH2~S~s CH3 ~:o2H

I
~Y .

~_ ~o~
~2}~ S :5 ~3~4~7 To a ~'cirred ~olutlon o~ 8.02 g. (o.o~ mole3 o:~
2-(N-t-butoxycarbonylamlnomethyl) -1,4-cyclohexadienyl~
acetic acid (23 and 5.6 g. (0.0304 mole) o~ 2,4-dl nltrophenol in 80 ml. o~ dry THF wa~ added 6.2 g.
(0,0304 mole) o~ ~CC at 5 - 10 C. in one ~ortion and the m~xture wa~ ~tlrred for 2 hr. at room temperature.
A~ter removing the precipitated urea, the reaction m~xture wa~ mixed at 0 - 5 C. with a solutlon o~
9.4 g. (0.027 mole) o~ 7-amino-3-(5-methyl-lJ3,4-thiadlazol-2-ylthiomethy~ -cephem-4 carboxylic acid and 7.7 ml. (0.055 mole) o~ triethylamine in 30 ml.
o~ water and 50 ml. o~ ~, The mi~ture wa~ ~tirred ~or 20 hr. at room tempe.rature and conc~ntrated under reduced pre~ure to remove most o~ . The a~ueou~
~olution was treated with 2 g. o~ active carbon. The ~iltrake ~as covered wlth 100 ml. o~ ethyl acetate and acidl~led wlth 10~ HCl. The aqueou~ layer wa~
extracted with two 50 ml. portion~ o* ethyl acetate.
The eth~l acetate layer wa~ combined wlth the ethyl . ~cet~te extracts, waæhed wlth two 100 ml. portions o~
water, dried with anhydrou~ Na~S04 and concentrated to dr~nes~ below 40 ~. to give 6d as a vi~cou~ oil ~hl~h solidi~ied b~ trit~ration with ether to glve . Yleld 12.5 gu (70%). IR: ~ Bx 1780~ 1700, 1680, 1510 ¢m 1.

.

, ..
, . ~. .

~3~

The BOC-protected cephalo~porin 6d (12.5 g., 0.021 mole~ wa~ treated wlth 25 ml. of TFA at 0 - 5 C. with ~tlrring. Stirring w~ continued ~or one hour at the ~ame temperature. Th~ mlxture waa diluted with ~00 ml. o~ ether to g~ve white precipitate which was di~solved ln ~0 ml. of 5 ~ aqueou~ acetonitrile and treated with 1 g. o:~
actlve carbon. The filtrate wa~ ad~u~ted to p~I 6 wlth conc. Nff40H and diluted with 250 ml. of aceto-nltrlle to glve 8 g~ (77~) o~ 7d. (lot 2) a~ pale ~ellow powder. M.p. 230-2~5 C. (dec~).
IR: ~m~ar 1790, 1650, 1620, 1570 c~
W ~ ~a}~C03 - pH 7 Buf~- 225 rlm (~h) ( -11800), 277 nm ( f 12400).
Arlal ' Calcd . ror C20H23N504S3 H, ~.70; N, 14.19; S, 19.49.
Fou~d: C, 48.55; H, 4 .34; N, 13 .74; S 9 19 .00 ~lubilltg ln 0.1 M pho~phate buffer (pH 7~:
3.4 mg./mlO
: ~ . ' ' . '' .

' ' .''', ." " ' ' ' .

"
. ~ -117-` ' , ' ' ~. .

~037~
Exampl~ ~Q
PreParat-lo-n o~ 7-~ L2-aminomethyl 1.4-c~clohexadien~yl)-ace~çamldo ] -~- (3-h,vdrox~yridazino- [~ ,2-c ]-s-triazol-6-~,,thiomethYl)-3-cePhem-4-carboxylic acid (7e.), Lot 2 .
~N~I-BOC ~ ~ NH-BOC 1 3 COOH l coo-~N2 J
; NO~

~
~ NH-B0C S ~L f . . O~LCH2 S~N
COOH
.
~6~

J~ OH
. ~ I .
~--CO~
,, , , . ~LCEr2-S~N3N
CO~

: ZQ .

. !

--118~
.

. ~ , . .

To a ~tirred ~olution of 10.1 g. ~0.038 mole) of a-[2-(t-bu~ox~carbonylaminomethyl~-1,4-cyclohexadienyl3-aoetlc acld (2) and 8.1 g. (0,044 mole~ o~ 2~4-dinitro-phenol in 300 ml. o~ THF wa~ added 9~1 g. ~0.044 mole) oP DCC ln one portion and the mixture ~-as ~tlrred ~or 2 hr. at room temperature. The ~eparated urea wa~
filtered off. To the filtrate was added a ~olution of 14.4 g. to-0~8 mole) o~ 7-amino 3-(3 hydroxypyridazino-~3,2-c]-s-triazol-6 ylthiomethyl)-~-cephem-4-carbox~lic acld and 12.2 g. (0.12 mole) o~ trlethglamlne in 250 ml.
o~ ~aker and the m~xture wa~ stirred ror 16 hr. at room temperature. The reaction mixture wa~ evaporated under reducbd pres~ure below 40 C. and the aqueous concentrate wa~ washed with ether (5 ~ 200 ml.) and treated wl~h active carbon. The riltrate wa~ acidlried with dll, .: , HCl to pH 2 to give a precipikate which wa~ wa~hed ~ith 200 ml. o~ waker and extracted wlth hot THF
(5 ~ 500 ~1.)0 The ~HF e~tract~ were evaporated under reduoed pre~ure below 40 C. and the residue wa~
triturated wlkh 500 ml. o~ ether to give the N-blocked (thak 1~, 6e~. IR: y~KBr 1765, 1700, 1~40, 1240 1150 cm ~.
To the BOC-blocked cephalo~porln 6e (708 g,, 0.01~4 mole) wa~-added dropwi~e 16 ml. o~ TFA at 5~ C, ~nd the mi~ture was ~tirred ~or 1.2 hr. at room t~mperature. The mlxture Wa9 dil~ted wlth 1000 ml. o~

--119-- , , ~, ~3~
eth~r and the re~ulting precipitate was dissolved in 50 ml. o~ 50~ acatonitrile. The ~olution was ad~usted at pH 4 5 wlth conc. NH40H and diluted with 200 ml.
vr acetonitrlle to give 5.55 g. (84%) o~ 7e (Lot 2).
M.p. 220 - 230 C. (deo.).
IR: yr Im~aBx 1760, 1710, 1640, 1540, 1460 cm 1.
W ~ K2C03 - pH 7 Buf'f 253 nm ( ~ ~ 20~00) ~nal. Calcd. ~or C22H~3N705S2 2~I2 H, 5,03; N, 17.46.
Found: C, 46.28; 46.~7; H, 3.7~; ~5.71; Nl 17~20;
16.g3.
Solubility ln 0.~ M phosphate bu~er (pH 7):
1.1 mg./ml.
Repreclpltat~on ~ Tri~luoroacetic acid (0 15 ml. ) wa~ added to ~ (lot 2~ 100 mg.) at 5 C. and the mixture wa~ stirred for 5 min. at room temperature. The ~olution was dilu~ed with 50 ml. o~ ether. The re~ultlng tri-.
~luoroa~etate wa~ di~ol~ed in 10 ml. o~ 50~ aqueousao~tonitrile ~olutlon. ~he solutlon wa~ ad~u~ted at pH 6.o wlth 10~ NH40H to give a light brown precipitate (91 mg.~, m~p~ 224-2~4 C. (dec.).
IR: Y maJl 176OJ 1710, 1~30, 1540, 1460 cm 1 ~V: ~ K2C0~ p~ 7 buPPer 253 nm ( , 19,000).
. . .

7~

Anal. Calcd- ~or C22H23N705S2 2H2 ~, 5.03; N9 17.~6.
Found: C, 47.~0; 47. 25; H, 402~; 4.21; N, 17 .89; 17 .82 .
Solubllity in 0.1 M phosphate bu~fer (pH 7):
1.1 mg./ml.

. _ .

'''' ., . .
.' ' ,; , ~ .
.

.. I .

.

I i , .

-12 1~

.

~7~W7 ~he ~allc~laldeh7de add.uct o~ 7-[(2-amlnomethyl-1,4-Q~clohexadienYrl ¦acetamido L-~- Ll-methyltetX~2:0~
3~3-cephem-4-ca~bo~ylic acid (7c~

HQ _ NYCH~

>~ //~ 2-S~N~
C02K l I3 ~ 8c To a ctirred ~uspen3ion of ~ (E~cample 11, lo., 2, 766 mg., 1.6 mmoles) and triethylamine (300 mg., 3 mmoles) in methanol (8 mlO) wa~ added ~alicylaldehyde (370 mg., 3 mmole~)' and the mixture wa~ skirred ~or 30 min. at room temperature to make a clear dark yellow ~olution. Iqle solution wa ~ trea~ed with a ~rna 11 amount o~ carbon and ~I (2 ml., l.~L ~olutlon ln ethyl acetate o~ potassium 2~rethylhexanoate) wa~ added to the ~iltrate. The mix-ture wa~ diluted with a large amount o~ ether (100 ml.) to precipitate the product 8~, which wa~ collec~ed ~lltratlon, washed with ether (30 mlO) and drled.
Yield 788 mg. (78,¢) . M.p. 165-175 C. (dec . ) .
IR: ~ maJ 1760, 1660, 1625J 1605, 1280 cm 1, , , .

~3~91L6~7 U~ ~ PHx7 buf~er 256 nm ~ 19500), 275 nm ( 14000, 3h3, ~25 nm (~ 2600).
NMR: ~ ppm 6 3.95 (3H~ ~? N-G~3), 4-96 (lH~
d, 4 Hz, 6-~), 5.50 (1 H, m, 7-H), 5.69 (2H, ~, C_C-~)) 6.7 - 7~6 (4 H, m, phenyl-H), 8.6~ (1 HJ 3 -CH~N-), 9.00 (1 H, d, 8 Hz, 7-COMH).
Anal. Calcd. ~or C26~2605N7S2K ~2 ' 9 H, 4~43; NJ 15.~7; S~ 10.06.
Found: C, 48.96; 48.85; H, 4.01, 4.10; N, 1~.33, .
14.26; S, 9.31~ 9.70.

~h Q ~aliQ~la~ 4o~L __ _ f 7-~A?-~am~i~n~o~meth l-1,4-~ o,~xad~e.n.~l)ac,e,tamido]-~ me~h~ .4-thiadiazol-2-~lthlome~h~ ePhem-4-carbox~lic acid (7d~
r IO

~cO~r~ ~--N
~ C0 H 2 S ~ 3 ~d ~ o a ~tirred m~cture o~ xample 12, lot ~,-778 mg.~ 1.6 mmoleB) and trieth~lamlne (300 mg'J ~ 3 mmole~) in methanol (8 ml) was added salicylaldehyde (370 mg., 3 . mmolesL and the su~pen~ion wa~ ~tirred for 15 min. at room temperature to make a ~lear dar}c yellow ~olutlon ~hloh was treated with a ~nall amount of carbon.
-123- ~

2 ml., 1 M ~olution in ethyl aceta~e) wa~
added to 'che flltrate. The mixture was dlluted ~lth a large amount of ether (100 ml. ) to precipitate th~ product 8d wh~ ch wa~ collected by ~il~atîon) washed wlth ether (30 ml. ) and dried . Yield 855 mg.
(84,~;) . M.p. 163~170 C. (dec . ) .
na ~ 1765, 1630, 1610, 1280 cm 1, , pH 7 bu~er 257 nm ( 19800) J 275 nm (~ 14200, ~h), 325 nm (~ 2700).

ppm 6 2, 68 (3~ C~13 ), 4 . 94 ( ~I, d, 4 HZJ 6-~;,), 5.50 (1~, m, 7-H), 5.68 (2~ J
e_C-E), 6.70 - 7,60 (4H, m, phenyl-H), 8.65 (1 H, ~, C~_N) " 8~92 (lH, d, 8 Hz, -CONH-) .
Anal~ Calcd. ~or C27H2~jO5N5S3K-3/2 H20: C, 48.92;
H, 4 a41; N, 10.57; S . 14 .51 ..
l?ound: C~ 48.91, 49~24; H, 3.74, 4.0~, N, 10.159 10.34, S; 1~.75, 13.~7.

.
.

, :' '' .

- --124-- .

( ~ --~3~

Fxample 33 The ~aliçvlaldehyde adduct of 7-[(2-amlnomethvl-1.4-c~vclohexadlenYl~acetamldo~ -hYdroxvpvridazino-~3.2-cJ-~-triazol-6-~lthiometh~ -cePhe~-4-carboxvlic nclq (7e).
.

,_-OH

~ -C ~ OH

8e To a,~tirred ~u~pen~ion of 7e (Example 1~, lot 2, 754 mg., 1.42 mmole~) and triethylamine (300 mg., 3 mmole~) ln N,N-dlmeth~lformamide (7 ml.) wa~ added salicylaldehyde (~70 mg~, 3 mmoles) and the ~uspenslon wa~ ~tirred for 1.5 - hr. at room temperature to be a clear dark yellow ~olution whlch was treated wlth a small amount o~ car-bon. KE~ (1.5 ml., 1 M solution in ethyl acetate) was added to the filtrate. The mixture wa~ diluted wlth a large amount of ether (100 ml.) to precipitate the product 8e which wa~ wa~hed with ether (30 ml.) and dried. Yield 906 mg. (95~3. M.p. 215-222 C.
(deo.).

~ --125_ ~'~

~ . .

-~L~D37~

IR: ~ma~ 1760, 17209 1660, 1615, 1600D 1~45, 1275 cm 1.
UV:a~ pH 7 bUffer 255 nm ( 28800) 310 nm ( ~ 8500, ~h) .
NMR: 3DMSO-d6 5.00 (lH, dg ~ HZJ 6-H), 5.50 J m, 7-a) J 5 72 (2HJ EIJ C-C_H) ~ 6 .7 ~ 7.6 (6H~ mJ
phen~1-}I) J 8.70 (lX, 8, C~I_N) J 9.00 (1H, d~ 8~IZ~

AnR 10 Ca lcd rOr C29~266N7S2K H2 H~ 4.09; N~ 14.21; SJ 9.30.
Found: CJ 50.~54~ 50,30; H~ 4~15~ 4.00; NJ

11~.02, 14~,03; S, 9~6, 9.~1.

Ihe potassium ~alt of the compound of Example 1 wa~ prepar,ed by adding a ~olut~on o~ potassium 2-ethyl-he~anoate (KEH) in ethyl acetate to a ~olutlon o~ the cephalo~porin (zwitterion) (1~) in DMS0 to precipitate the de~^red potas~ium ~al~.
~!ml~
~ h~ sodium ~alt o~ the compound o~ Example ~
~as prepared by pre ~orm~tion o~ lt~ diethylammonium ~lt in methanol ~ollowed by add~tion o~ a solution o~
~odium 2-ethylhexanoate (SE~I) in ethyl acetake and then dllution with isopropanol to precipitate the desired aodium ~alt which had a ~olubility in water greater than 250 mg~./ml.
., , .~ ' ,, .

--12~-- ~

1. .

, A i~

The corresponding compounds containing a 3-acetoxy-methyl group and a 3-methyl group were prepared and compared in vitro with the compounds of Examples 3 and 18 with the following results // ~ ~ ~ CH R

COOH
N - N ~ -- N
`. 11 11 11 ll M.I.C. (~-c~. ml.) --S~~ ,N -S~~ N~ N

Organism R= -OCOCH3 -H C~13 CH3 ~ 6w 6x6c (E.x. 3) (~x. 13) S. aureus Smith-~ A9537 0.8 >3.1, 250.2~0.~ 0.8 S. aureus Smith-~ A9537 3~1 >6.3, 250.8~1.6 1.6 -~ 50~ serum S. aureus Russel 0.8-1.6 >12.5,12.5 0.4-0.8 0.8 S. aureus BX1633 A9606 3.1 ~12.5, 25 1.6 1.6 Str. pyogenes* A9604 0.8-0.16 1.25-2.5 0.4-0.08 0.16 -~ 5% serum D. pneumoniae* A9585 0.16-0.31>2.5,10 0.04-0.08 0.08 + 5% serum Mycobacterium 607 >100>100 >100 >100 E. coli NIHJ25-50 >1001.6-3.1 3.1 E. coli ATCC+8739 25 >100 3.1-6.3 3.1 E. coli Juhl++A15119 25 >1003.1-6.3 6.3 K. pneumoniae+ A9977 12.5-25 >1000.8-3.1 3.1 K. pneumoniae+ A15130 12.5-50 >100 3.1-6.3 3.1 Pr. mirabilis+ A9900 50 >100 100 25 - Pr. morganii+ + A15153 >100>100 >100 >100 Sal. enteritidis-~ A9531 12.5 >1001.6-3.1 l.h Ser. marcescens+ A20019 >100 >100>100 >100 Ps. aeruginosa-~ A9843 ~100 >100~100 >100 *50% Nutrient Broth - 45% Antibiotic Assay Broth -~-~ at 10 4 dilution.

~ - 127 -~.~3~
The above samples after solution in Nutrient Broth .
were found to exhibit the indicated Minimum Inhibitory Con-centrations (M.I.C.) in mc~./mlO versus the indicated micro-organisms as determined by overnight incubation at 37C. by Tube Dilution.

- 127a -~L~3~
The exC~llent ~n vivo activity of the preferred compounds of the pre~ent inventio~ for example, (7c, Ex. 20, BB-S311) and ~6c, Ex, 3, BB-S336) were demonstrated by comparing it with MR-S94 in determination~ (by subcutaneous administration of the drug to infected mice) o~ the minimum curative dose for 50%
of the mice (CD50) in mgm./kg. versus lethal doses of the indicated microorganism~ w.ith the following results:

~hallen~e CD o ~n mgm./kg.
~ _ . _ Organi~m BB-S311 BB-S336 MR-~94 ~ r_ __ 8 ~ aureu8 :E~X-163 ~5 1~8 2 ~ ~ 3 ~ 4 S'c. psrog~nes Ag6~4 1.0 _ 0.
E . coli Juhl 6 .25 5 . 4 4 . 4 E. coll Juhl 5.6 4.2 Pr. mlr~llis AggOO 9.4 _ 8 K. pneumoniae D-ll 8.5 _ 4,6 . aureus ~mlth 1 . 8 _ ~ . :l ;
, , In rat urinary recovery studies the cephalosporins are adm~n~ered ~ub~utaneously at a dose of 200 mgm./kg.
wi~h the æwitterion being given as an aqueous suspension (40 mg./ml., DM~O ~ 0.5% MaHC03- H20 ~ 1:1:8) and the salicylaldehyde derivatives, which are equipotent on a molar ba~i~, in a~ueou~ ~olution~

' .

.
. -128-.. . . .

~Lel 3~

In rat crystalluri~ ~tudles BB-S311 gave ~atl~-~actoril~ high serum and urlne concentrations wlthout any evidence of crystalluria. Studie~ ln dogs show that ~B-S~ll glve~ hlgh crystal-free urin~ concentra-tion~. Parenteral (IM) ab~orption studie~ in mice lndicate that the peak level of ~B-S311 is hlgher than that of cephalothin and that thl~ antiblotic m~g per~i~t ln the blood longer than cephalothin.
me amount o~ the IM-admlni~tered dose of BB-S311 recovered ~rom the urine of rat~ wa~ 31~, a value ver~
81milar to that obtalned wlth M~-S94 (3$.5~).
Cephaloridine recovery wa~ 43.1~ whlle that of cephalothln wa~ 18.1%. ~B-S311 appeared in the urlne a~ a single bloactive entity wlth an Rf identical to that found with an aqueous ~olutlon of the com-pound itsel~.
q~he ~rltlbacterial acti~rity of ~ S~ll wa~ also co~pare~ wlth that of MR-S94 in an agar dilukion t~8t y~tem. In e~periment~ on Mueller-Hinton In~olving 18 gram-po3itlve and 58 gram-negative organ~m~, ~B-S311 wa~ found to have activity o~
the sam~ order a~ MR-S94. In an additional Reries o~ experiment~, the relatlve actlvlty of MR-S94 and ~B-S311 wa~ determined a~alnst 32 ~train~ each of ' ' -129~
.

( ~C

~r~tia mL~9~, E! ~P
iJ a~d Proteus ~p. The e compounds had out3tandlng actiYlty again~t mo~t repre~entative~
0~ En~erQ~aÇt~ 8p., ~_ ~tuar~til, and all Proteu~
~p. except P. mor~anll.
Compounds of the present lnvention a~ter aolutlon, e.g. in DMS0 or water at 2 mgm./ml., followed b~ dllution wlth Nutrient Broth were ~ound to exhiblt the ~ollowing Mlnimum Inhibitory Con¢entrations (M.I.C.) in mcg~/ml. versus the lndicated m~croorgani~ms a~ determined by overnlght ~ncubation at 37~ C. by ~ube Dllution. Re~ult~ with ~arlou~ old cephalo~porins are also givcn. In th~s and other tests the ~alicylaldehyde derlvative~ are equlpotent, to the correspondlng zwltterion and are al~o very ~oluble ~n water.

.:' ,, 37~
~, N
CU ~ 0~ 0 ~D
~ _ ~ ~1 0 ~ O C~
u~ ~a O o o o ~1 -1 N ~ tf~ r-l ~ O O ~
_ C) O O O O O O ~ N O ~I N N O O N N
.
_ -- - _ ... .. _ ~O ~ ~O
~ ~1 ~1 ~I L~ I
U O O N N CU r~
. . O o o o~ C~ o o CU o CU

~_ O .- _ .
r ~1 ~) ~O
U O ~ O O ~\ N ~ In ~ ~ ~f)/~
_~ O O O O O O O O N N O O O N ~ O O N N
~, '. _ .
' ,.

C ~
U O O ~ U~
!~ ,.- o o o o ~1 O O C~
_ ~c ~-- ~ ~
OP O ~ 1~ 0 0 0 711~ *- _ _ ~_ * ~-- i~ C I ~
~ . I
I O C) O
O r~ ,~
O O ~ n ~
a) I O O
s~ ~
*
~> ~ ~ ~ ~ rl ~ ~ ~ a~
~ ~ ~ ~ 1 0 ~ ~ ~ ~ ~ 10 ~ ~
E ~ ~ ~--' ~ E
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The excellent in vivo activity of a preferred compound of the present invention (6c, Ex. 3, BB-S336) was demonstrated by comparing it with results previously obkained by substantially the same procedure for MR-S94 in determinations (by subcutaneous administration of the drug to infected mice) of -the m.inimum curative dose for 50% oE the mice (CD50) in mgm./kg. versus lethal doses of the i.ndicated microorganisms with the following results:
Challenge CD50 in mgm./kg.
. 10 Organism _ _ BB-S336 MR-S94 _ . S. aureus BX-1633 2.4 3.4 ; E. coli_J hl ¦5.4 __ _4 4 _ __ _ .

:~ 30 74~

Solubilities o~ cephalosporin derivati~es ln 0.1 M pH 7.0 phosphate buffer are deter~lned by bioa~ay and also by ~pectroscopic methodo(when the sample shows W absorption) each with lts own standard whlch has been completely solubilized. The tost ~olution and the standard ~olutlon are prepared a~ described below.
~Y~L~_tlnn o~ ~he tç~ ~olution About 10 mg. (1) of a sample is placed in a vlal and mixed with 1 ml. o~ 0.1 ~ pho~phate buf~er (pH 7.0). me vial is stoppered, ~ealed with a metal cap, p~aoed on a rotary ~haker and agitate.d a~ 160 rpm Por 4 hr. at 25 C. The ~olution 1~ ~lltered throu~h a,Milli~ore ~ilter (HAWP 01~00, mean pore ~ize 0045 ~m). m e filtrate i~ diluted to u~e for bloas~ay and W a~say as follow~:
iltrate (ca. 1 ml.) .5 ml. (4~
di~et~2~00 ml.( ~ with the 100 ml.
~ ~~ ~~~ (4) u~ed ~or W ~assay 1 ml.
dil. t~ ~ ml.(4 20 ml.
ml.(4) ~ . dll. to : ~ 20 ml. (4 20 ml.
" '' ' , used ~or bioa~say .
. -138-~3~4~7 ~3~ n of thç ~an~axd ~l.ution About 2 mg. of the ~ame ~ample 1~ weighed preclsely and dis~olved in 4 ml. o~ 1% K2C0~ ~olution(3) (u~ually within one mlnute). The solutlon i~ diluted exactly to 50 ml. ln a fla~k with 0.1 M pH 7.0 pho~phate bu~fer and u~ed ~or the preparation o~
; the ~tandard solution~ for bioa~say and W -assay a8 follow~:

50 ml.
' - 1, ror UV-a~sa~ 1 ml. ~

(40 mcg./ml.) 10 ml.(4) ¦ dil. to 20 ml.(4) ~ dll. to 20 ml.(4) 20 ml.
(20 mcg /ml.) (2 mcg./ml.~ 5 ml.(4) ~ ~ 20 ml.~4) used for W -assay a~ the standard ~olution (0.5 mcg./ml.) , .. , ~/ , I
u~ed ~or bloa~ay . as the standard solution , ., ' .

~ -13~-79~7 (1) A 20 mg. or more sample is used when the sample is expected to be more soluble.
(2) 0.1 M pH 7.0 phosphate buf'fer is used as a diluent in -the present test and also as a reference solution in W -assay.
(3) It was previously confirmed that almost the same result is obtained when the sample is solubilized in DMSO (dimethyl-sulfoxide) and then diluted similarily with the buffer.
(4) Volume of the solution is measured precisely with an appropriate pipette or flask.

. 10 ~37~7 :~ Solubilities (mg.~ml.) in 0 1 M phosphate buffer (pH 7_0) I .. .. ___ _ __ .___ . , .... . .. _. . _ _ ~_ . _ , . _ .
7-sidechain -t1~ 5 ~ ~ [ ~ H2 ( ~ E2 ~ [ ~ H2 ;,'', ._. . . .... -N-- - N MR-S94 BB-S336 (6C) BB-S311 (7C) . .II ll _ . _ - ~ N ,N BA UV BA UV BA UV
' ~ 2.0 1.9 25.6 2~.6 9.7 9.7 CH3 23.0 26.0 9.4 1 0 _ .. _ . _ . _ . ".
N N MR-S96 BB-S341 (6d) BB-S312 (7d) - ~ S CH3 1.2 0.9 4.3 4.6 8.8 7 6 ,' _ __ _ ._ BB-S150 BB-S340 (6a) BB-S180 (7a) , , N- N 4.4 4.4 16.8 16.03.1 OM 4.1 ~ _ ,~ :
,~ BB-S226 BB S338 (6b) BB-S269 (7b) ~ =~ 2.5 3.0 3.0 12.5 9.0 -3.8 3,5 1 1 1.7 ' I , `' :' :.

^ -` gL~3~
` ______ . t -N -N ~ ~ BB-S207 ~I BB~9339 (6e~ BB-9313 (7e~ BA UV I BA UV BA W
4.2 33 9 ~ 8.0 8- 5 9 1-7 __ _ . 3.7 4.4 ~ BB-5314 (7 e ) 1l -BB-5324 (~

i BA; Bioassay, VV: UV-aF.~aY
Results given for the same compound on diEferent lines were obtained using samples for different lots.

~, ~ 30 ;' :

.'` . ~

~37~

The corre~ponding compounds containlng a 3-acetoxymethyl group and a 3-methyl group were pre-pared and compared ln vi~ro with the compound of :Example 1 with the Yollowing result~:

~ CH2NH2 Il 11 .
~CE -C-~S ~

0// ~CH2R ,,._"
COOH

~ N~
O~r~ni~m P~- -OCOCH3 -H -S~N,N

S. aureu~ Smlth A9537 1.6 > 3.1 o.8 S. aureus Smith A9537 6.3 > 6.:~ l.o ~ 50% serum S. aureus Ru~el ~5.1 >12.5 0.8 ~. aureu~ BX1633 A9606 3.1 ~12.5 1.6 SkrO pyogena~ A96040.~1 ~ 2.5 0.16 ~ 5% ~.e~um D. pr~eumoniae A95850.31 > 2.5 o.o8 ~5~ ~erum ~cobacterium 607 >lOO ~>100 ~100 E. coli NIHJ 100 > 100 3.1 E. coli ATCC 87~59 25 >lt~O 3.1 E. coll Juhl A1511950 ,~100 6.3 K. pneumon~ ae A9g77 25 ? ~oo 3 .1 K. pneumonlae A1513025 > 100 ~ .1 Pr. mlrabili~ A9900 100 > 100 25 Pr. morganii A15}53 >100 ~100 100 5al. er~eritidi~ A9531 25 >100 1.6 Ser. marce~cen~ A20019 >100 ~ 100 ~100 P~. aeruginosa A9843 ~100 > 100 >100 , . ~

Claims (18)

CLAIMS:

1. A process for the preparation of a compound of the formula ( I) wherein R is , , , , , , , , , and easily cleavable esters, pharmaceutically acceptable salts and salicylaldehyde Schiff base thereof; which process comprises either A) reacting a compound of the formula ( II) wherein R has the meaning set out above or an easily cleavable ester or salt thereof with an acylating derivative of an acid having the formula ( III ) wherein B represents an amino-protecting group and removing said amino-protecting group to produce the desired compound of formula I or an easily cleavable ester or pharmaceutically acceptable salt thereof and, if desired, either before or after removal of B (a) converting the product in the form of the free acid or salt thereof to the corresponding salicylaldehyde Schiff base, easily cleavable ester or pharmaceutically acceptable salt thereof or (b) converting the product in the form of an easily cleavable ester or salt thereof to the corresponding free acid compound or pharmaceutically acceptable salt thereof or B) reacting a compound of the formula ( IV) or an easily cleavable ester or salt thereof with a thiol of the formula HS - R
( V ) wherein R is as defined above or a salt thereof to form a compound of formula I or an easily cleavable ester or pharmaceutically acceptable salt thereof and, if desired, (a) converting the product in the form of the free acid or salt thereof to the corresponding salicylaldehyde Schiff base, easily cleavable ester or pharmaceutically acceptable salt thereof or (b) converting the product in the form of an easily cleavable ester or salt thereof to the corresponding free acid compound or pharmaceutically acceptable salt thereof.

2. The process according to Claim 1 wherein the acylating agent of formula III is in the form of a mixed anhydride.

3. The process according to Claim 1 wherein the acylating agent of formula III has the formula

4. The process according to Claim 1 wherein the amino-protecting group is t-butoxycarbonyl or 1-carbomethoxy-1-propenyl-2.

5. The process of Claim 1, 2 or 3 wherein the compound of formula II is used in the form Of its silyl ester.

6. The process of Claim 1, 2 or 3 wherein the acylating agent of formula III is reacted with a pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl or phenacyl ester of a compound of formula II.

7. The process of Claim 1 wherein a pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl or phenacyl ester of the compound of formula IV or a salt thereof is reacted with the thiol of formula V or a salt thereof.

8. The process of Claim 1, 2 or 3 wherein the product of the formula wherein R is as defined in Claim 1, or a salt thereof, is converted to the corresponding pivaloyloxymethyl, acetoxymethyl, methoxy methyl, acetonyl or phenacyl ester or a pharmaceutically acceptable salt thereof.

9. The process of Claim 1 wherein the product of the formula wherein R is as defined in Claim 1 or a salt thereof is converted to the corresponding salicylaldehyde Schiff base thereof.

10. The process of Claim 2, 3 or 4 wherein the product of the formula wherein R is as defined in Claim 2, 3 or 4 or a salt there-of is converted to the corresponding salicylaldehyde Schiff base thereof.

11. A process as in Claim 1 wherein R is , , or .

12. A process as in Claim 1 wherein R is .

13. A process as in Claim 1 wherein the product is recovered as a sodium or potassium salt.

14. A compound of the formula I

wherein R is , , , , , , , , , or an easily cleavable ester, pharmaceutically acceptable salt or salicylaldehyde Schiff base thereof, whenever prepared or produced by the process of Claim 1, 2 or 3, or by an obvious chemical equivalent.

15. A compound of the formula (I) wherein R is , , or whenever prepared or produced by the process of Claim 11 or by an obvious chemical equivalent.

16. A compound of the formula (I) wherein R is whenever prepared or produced by the process of Claim 12, or by an obvious chemical equivalent thereof.

17. The salicylaldehyde Schiff base of a compound, as in Claim 9, whenever prepared or produced by the process of Claim 9, or by an obvious chemical equivalent thereof.

18. The sodium or potassium salt of a compound as in Claim 13, whenever prepared or produced by the process of Claim 13, or by an obvious chemical equivalent thereof.