CA1276929C - Cephalosporin antibacterial agents - Google Patents

Abstract

Abstract of the Disclosure 7-[2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-(substituted)-iminoacetamido]-3-[3-(quaternaryammonio)-1-propen-1-yl]-3-cephem-4-carboxylates of the formula I

in which R1 and R2 are as defined herein and -N?Q is a quater-nary ammonio group as defined herein, and salts, solvates, hydrates and esters thereof, are potent antibacterial agents.
Processes for their preparation and intermediates in such pro-cesses are described.

Description

~6~Z~

Summary of the Invention .

This application relates to 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(substituted)iminoacetamido~-3-[3-(quaternaryammonio)-1-propen-l-yl]-3-cephem-4-carboxylates of the formula C CON~ ~ ~

RlIIN~ oR2 'O ~CH=CHCH2-N_Q
O

in which Rl and R2 are as defined herein and -~ --Q is a quaternary ammonio group as defined herein, and to salts and esters thereof.
This invention also relates to processes for the preparation of the compounds of Formula I, to pharmaceutical compositions containing at least one compound of ~ormula I, and to intermediates in their preparation.

Back~round and Prior Art A) U.S. Patent 4,390,534, issued June 28, 1983 to Tsutomu Teraji _ al., discloses cephem and cepham compounds of the formula Rl ~ ~ C - CON~- ~

wheLein Rl is amino or protected amino; R~ is hydrogen, acyl, optionally substituted aryl, substituted alkyl, alkenyl, alkynyl, ~, . .

~2'~6~3Z~

optionally substituted cycloalkyl, cycloalkenyl or an O- or S-containing 5-membered heterocyclic ring substituted with oxo group(s); R3 is hydrogen or alkyl; R4 is hydrogen, acyloxyalkyl, acylthioalkyl, optionally substituted pyridinioalkyl, optionally substituted heterocyclylthioalkyl, alkyl, halogen, hydroxy or optionally substituted thiazolioalkyl; and R5 is carboxy or protected carboxy; provided that RS is COO when R4 is optionally substituted pyridinioalkyl or optionally substituted thiazolioalkyl;
and the dashed line indicates either a single or double bond.

European Patent ~pplication No. 13,762, published August 6, 1980 is concordant thereto and has a similar disclosure.

U.S. Patents 4,381,299 (issued April 26, 1983), 4,331,665 (issued May 25, 1982) and 4,332,798 (issued June 1, 1982) each issued on parent applications of U.S. 4,390,534, and have similar disclosures.

B) European Patent Application No. 62,321, published October 13, 1982, discloses cephem compounds of the formula R~ CONH_~
~R2 ~

wherein Rl is amino or pro~ected amino; R2 is an optionally subs~ituted lower aliphatic hydrocarbon group, or cycloalkenyl;
and the group of the formula --N ~3 ~, :~76~9 is an optionally substituted heterocyclic cation group containing more than one nitrogen atom; and pharmaceutically acceptable salts thereof. Also disclosed are intermediates of the formula R ~ ~ Il- CONH

CH N ~

wherein Rl and R2 are as defined above, R4 is a protected carboxyl group and X is an acid residue.

C) European Patent Application No. 74,653, published March 23, 1983, discloses cephem compounds of the formula Rl ~ I-CONH F ~ ~ 3 wherein Rl is amino or protècted amino;
R is an optionally substituted lower aliphatic hydrocarbon group, cyclo(lower)alkyl or cyclo~lower)alkenyl;
R3 is (lower)alkylamino, N-protected(lower)alkylamino, di(lower)alkylamino, suIfo(lower)alkylamino, hydroxy(lower~-alkylamino, N-protected hydroxy(lower)alkylamino, acyloxy(lower)-alkyl, (lower~alkoxy(lower)alkoxy(lower)alkyl, di(lower~alkylamino-(lower)alkyl, (lower)alkylthio(lower)alkyl, (lower)alkylthio, ~;~ 76~29 (lower)alkoxy, tlower)alkoxy(lower)alkoxy, hydroxy(lower)alkoxy, acyl(lower)alkyl, hydroxy(lower)alkylthio, di(lower)alkylamino-(lower)alkylthio, N-containing unsaturated S-membered heterocyclic group, N-containing unsaturated S-membered heterocyclicthio, or N-containing unsaturated 5 or 6-membered heterocyclicthio(lower)alkyl which may be substituted with suitable substituent(s); and R4 is hydrogen or (lower)alkyl; or a salt thereof.

D) U.S. Patent 4,332,800, iss~ed June 1, 1982 to Tsutomu Teraji et al., discloses inter alia compounds of the formula oR2 ~ ?--c~2~

wherein Rl is amino or protected amino; R2 is (lower)alkyl and X
is hydrogen or carbamoyl.
.

E) European Patent Application No. 47,977, published March 24, 1982, discloses cephem compounds of the formula (~)m Am- T ~11 - CONH

O-R ~ ~

2 O . ¦ CH2Rl COO~

., .

~Z'716~Z~ .

wherein m is 0 or 1; Am is optionally substituted amino; T is a thiadiazolyl moiety (attached to the other groups by two of its carbon atoms); R2 is hydrogen, optionally substituted alkyl, cycloalkyl or optionally substituted carbamoyl; and Rl is optionally substituted thiazolio, optionally substituted pyrazolio, tri(lower)-alkylammonio or a pyridinio group of the formula R

~ ~ b R

in which Ra is substituted (lower)alkyl [the substituent being cycloalkyl, phenyl, hydroxy, alkoxy, halogen, cyano, carbamoyl, carboxyl or sulfo], (lower)alkenyl or carboxy-substituted (lower)-alkenyl, (lower)alkylthio or carboxy-substituted (lower)alkylthio, ami.no or monosubsti~uted amino [the substituent being (lower)alkyl, (lower)alkanoyl or aminobenzenesulfonyl], di(lower)alkylamino, substituted carbamoyl [the substituent being (lower)alkyl, hydroxy(lower)alkyl, (lower)alkoxy, hydroxy or cyano], di(lower)-alkylcarbamoyl, thiocarbamoyl, cycloalkyl, phenyl, hydroxy, (lower)alkoxy, halogen, (lower)alkoxycarbonyl, (lower)alkanoyloxy, (lower)alkanoyl, carboxyl, sulfo, cyano, nitro or hydroxysulfo-(lower)alkyl; Rb is hydrogen or carbamoyl, or has the same meaning as Ra; and Rc is hydrogen or has the same meaning as Ra;
and salts thereof.

Although not formally related, European Patent Application No..25,017, published March 11, 1981, has a similar disclosure.

F) European Patent Application No. 30~630, published June 24, i981, discloses 3-vinyl cephem compounds of the formula ~7~;~2g~

. S
Rl _ A ~ CONH ~ ~
"~N~J~CH=CH2 wherein Rl is an optionally protected amino-substituted heterocyclic group which may also have halogen, or a group of the formula R SO2~N ~ ~

in which R3 is (lower)alkyl; R2 is carboxy or protected carboxy;
and A is lower alkylene which may have a substituent selected from amino, protected amino, hydroxy, oxo and a group of the formula =N~oR4, wherein R4 is hydrogen, cyclo(lower)alkenyl, ~lower)alkynyl, (lower)alkenyl [optionally substituted by carboxy or protected carboxy], (lower)alkyl [optionally substituted by one or more of carboxy, protected carboxy, amino, protected amino, cyano, phosphono, protected phosphono and a heterocyclic ~roup which itself may be substituted]; and salts thereof.

This application specifically discloses compounds of the formula Cl CONI~S

H2N S/ \OR ~J\CH=CH;~
COOH

~2769Z9 in which oR4 is methoxy, carboxymethoxy, tert-butoxycarhonyl-methoxy or l-tert-butoxycarbonylethoxy.

G) U.K. Patent Specification No. 1,399,0R6 published June 25, 1975, contains a generic disclosure encompassing a vast number of cephalosporins of the formula R-C-CO-N -COOH

wherein R is hydrogen or an organic group, Ra is an etherifying monovalent organic group linked to the oxygen through a carbon atom, B is ~ S or ~ S-~O, and P is an organic group. In one embodiment, P may be inter alia a vinyl group of the formula -CH=C

in which R3 and R4 independently may be hydrogen, nitrile, (lower)alkoxycarbonyl, or substituted or unsubstitu~ed aliphatic, cycloaliphatic, araliphatic or aromatic. However, the 5-amino-1,2,4-thiadiazol-3-yl group is not identified as a possible R
substituent and there is no disclosure or suggestion that P may be a ~uaternary ammonio-substituted propenyl group. ~.S. Patent 3,971,778 and its divisionals Nos. 4,024,133l 4,024,137, 4,064,346, 4,033,950, 4,079,178, 4l091,209, 4,092,477 and 4,093,303 have similar disclosures.

~76~9 H) European Patent Application No. 88,385, published September 14, 1983, discloses compounds of the formula R ~--CO~

in which Rl is (unsubstituted) ~hiadiazolyl; R2 is carboxy-(lower)alkyl or protected carboxy(lower)alkyl; R3 is hydrogen, halogen or (lower)alkenyl; and R4 is carboxy or protected carboxy.
Although l-propenyl is listed as one of the possible meanings of R3, the applicatian only exemplifies compounds where R3 is hydrogen, chloro or vinyl.

I) ~.S. Patent No. 4,307,233 issued to Daniel Farge et al. on December 22, 1981, discloses inter alia, 3-vinylcephalosporin derivatives of the formula S

N~_ CONH ~ ~ R3 H2 S \oR5 o// ~ CH=CH N \R4 COOH

in which R5 inter alia may be alkyl, vinyl, cyanomethyl or a protective group such as 2-methoxyprop-2-yl, and R3 and R4 are alkyl groups (optionally substituted by hydroxy, alkoxy, amino, alkylamino or dialkylamino) or phenyl groups, or R3 and R4, taken together with the nitrogen to which they are attached, may form a saturated heterocyclic ring of 5 or 6 members, optionally containing another hetero-atom selected from N, O and S, and optionally substituted by an alkyl group. The co~pounds are useful as intermediates in the preparation of 3-thiovinyl cephalosporin derivatives. There is no disclosure or suggestion of a 5-amino-76~9 1,2,4-thiadiazol-3-yl moiety in place of the 2-aminothiazol-4-yl substituent or of a quaternary ammonio-substituted propenyl moiety for the 3-substituent. Published United Xingdom Patent Application No. 2~051,062 is concordant thereto and has a similar disclosure.

J) European Patent Application No. 53,537, published June 9, 1982, discloses, inter alia, 3-vinylcephalosporin derivatives of the formula S

CONH

~32~ O~COOR5 ~ \f\CE~=C~-N~

in which Ra5 and Rb are the same or different and are hydrogen or alkyl, or taken together, form an alkylene group containing 2 or 3 carbon atoms, RC5 is an acid protecting group, R2 is an acid protecting group such as an ester, R3 and R4 are the same or different and are hydrogen, alkyl (optionally substituted by hydroxy, alkoxy, amino, alkylamino or dialkylamino) or phenyl groups, or R3 and R4, taken together with the nitrogen to which they are attached, may form a saturated heterocyclic ring of 5 or 6 members, optionally containing another hetero-atom selected from N, O and S, and optionally substituted by an alkyl group.
The compounds are useful as intermediates in the preparation of 3-thiovinyl cephalosporin derivatives. There is no disclosure or suggestion of a 5-amino-1,2,4-thiadiazol-3-yl moiety in place of the 2-aminothiazol-4-yl substituent or of a quaternary ammonio-substituted propenyl group for the 3-substituent.

U.S. 4,423,214 is concordant thereto and has a similar disclosure.

~Z'7~

~) European Patent Application No. 53,074, published ~une 2, 1982, generically discloses a vast number of 3-vinylcephalosporin derivatives of the formula (~)n R la ~ S ~

O CH=C-R
COOR2a wherein Rla (in one of several embodiments) may be N~ ICl _CO_ in which R5 inter alia may be hydrogen, alkyl, vinyl, cyanomethyl, an oxime-protecting group such as trityl, etc., or a group of the f ormula / C\OR 5 R S S

in which Ra5 and Rb5 are the same or different, and may be hydrogen, alkyl or, taken together, an alkylene radical of 2 or 3 carbon atoms, and RC5 is hydrogen or an acid-protecting radical;
R2a is hydrogen or an acid-protecting radical such as methoxymethyl;
R (in one of several embodiments) may be a methyl group substitu~ed by a 5- or 6-membered aromatic heterocyclic ring containing a single hereto atom, such as 2- or 3-pyridyl, 2- or 3-thienyl or 2- or 3-furyl; and t76~Z~ !

R3 is a group of the for~ula in which R4 may be alkyl, trihalomethyl or optionally substituted phenyl.

These compounds are stated to be intermediates in the preparation of compounds in which the 3-substituent is a group of the formula R

-C~=C-SR

which are stated to have antibacterial activity.

Although this patent includes the possibility of R being a methyl group substituted by an N-containing heterocyclic ring, in both the intermediates and final products (thus giving a heterocyclic-substituted propenyl moietyJ, it teaches only that the heterocyclic ring is attached via one of its carbon atoms. Thus, there is no suggestion of a quaternary ammonio-substituted propenyl group.
The reference exemplifies R~ in the intermediates and final products only as methyl. Further, in both the intermediates and final product, the propenyl group must contain a second substituent (-03SR or -SR, respectively). Also there is no disclosure or suggestion of a 5-amino-1,2,4-thiadiazol-3-yl moiety in place of the 2-aminothiazol-4-yl substituent.

L) European Patent Application No. 53,538, published June 9, 1982! discloses, inter alia, 3-vinylcephalosporin intermediates of the formula .

(~)n /~\~ /'/ ~H=CH-R
2~ O
t::OOH

in which n is O or 1, R5 is hydrogen, alkyl, vinyl, cyanomethyl or an oxime-protecting group, and R3 is halogen. There is no disclosure or suggestion of a 5-amino-1,2,4-thiadiazol-3-yl moiety in place of the 2-aminothiazol-4-yl substituent, and no disclosure or ~uggestion of a 3-halo-1-propen-1-yl substituent in the 3-position.

6~g Com~lete Dlsclosure This applica~ion relates to novel cephalosporin deriva-tives which are potent antibacterial agents. More particularly, it relates to compounds of the formula N ~ ~ CONH ~ ~ I
Rl~N S~ oR2 ~ N ~ C~=CH-CH2-N- Q
CO~

wherein ~1 is hydrsgen or a conventional amino-protecting group, R2 is hydrogen, a straight or branched chain alkyl group contain-ing from 1 to 4 carbon atoms, a cycloalkyl or cycloalkenyl ring containing from 3 to 6 carbon atoms, or a group of the formula -~-CH=C~-R , -~-C- C-R ' X or -C COOH

in which R3 is hydrogen, (lower)alkyl or carboxyl, X is halogen, hydroxy or (lower)alkoxy, and R4 and R5 are each independently hydrogen~ methyl or ethyl, or R4 and R5, taken together with the carbon atom to which they are attached, may be a cycloalkylidene ring containing from 3 to 5 carbon atoms, and -N - Q

~ILZ'7~!32~

is a quaternary ammonio group, and nontoxic pharmaceutically acceptable salts and physiologically hydrolyzable esters thereof.
Also included within the scope of the invention are the solvates (including hydrates) of the compounds of ~ormula I, as well as the tautomeric forms of the compounds of Formula I~ e.g. the 2-iminothiazolin-4-yl form of the 2-aminothiazol-4-yl moiety.

In another aspect, this application relates to a process for the preparation of the compounds of Formula I and to cer~ain intermediates in their ~reparation.

As shown in the structural formula, the compounds of Formula I have the ~syn" or HZ~ configuration with respect to the alkoxyimino group. Because the compounds are geometric isomers, some of the "antir isomer may also be present. This invention comprises compounds of ~ormula I containing at least 90% of the ~syn~ isomer. Pre~erably the compounds of Formula I are rsyn~
isomers which are essentially free of the corresponding ~anti~
isomers.

In addition to geometric isomers possible with respect to the alkoxyimino group, the compounds of Formula I (and the intermediates of Formulae VIII and IX) also form geometric (cis and trans) isomers about the double bond of the propenyl group.
Both the Ci5 ( rz~ ) and trans (rEr) isomers of these compounds are specifically included within the scope of this invention.

The nontoxic pharmaceutically acceptable salts of the compounds of Formula I include salts with mineral acids such as hydrochloric, hydrobromic, phosphoric and sulfuric, or with organic carboxylic acids or sulfonic acids such as acetic, trifluoroacetic, citric, formic, maleic, oxalic, succinic, benzoic, tartaric, fumaric, mandelic, ascorbic, malic, methane-sulfonic, benzenesulfonic, p-toluenesulfonic and other acids known and used in the penicillin and cephalosporin arts. Prepa-ration of these acid addition salts is carried out by convention al techniques.

.

Examples of physiologically hydrolyzable esters of the compounds of Formula I include indanyl, phthalidyl, methoxy-methyl, acetoxymethyl, pivaloyloxymethyl, glycyloxymethyl, phenylglycyloxymethyl, S-methyl-2~oxo-1,3-dioxolen-4-ylmethyl and other physiologically hydrolyzable esters known and used in the penicillin and cephalosporin arts. Such esters are prepared by conventional techniques known in the art.

The compounds of Formula I in which Rl is hydrogen exhibit high antibacterial acti~ity against various Gram-positive and Gram-negative bacteria, and are useful in the treatment of bacterial infections in animals, including man. The compounds of Formula I may be formulated for parenteral use in a conventional manner utiliziny known pharmaceutical carriers and excipients, and may be presented in unit dosage form or in multi-dosage containers. The compositions may be in the form of solutions, suspensions or emulsions in oily or aqueous vehicles, and may contain conventional dispersing, suspending or stabilizing agents. The compositions may also be in the form of a dry powder for reconstitution before use, e.g. witk s~erile, pyrogen-free water. The compounds of Formula I may also be formulated as suppositories utilizing conventional suppository bases such as cocoa butter or other glycerides. The compounds of this in-vention may, if desired, be administered in combination with other antibiotics such as penicillins or other cephalosporins.

When provided in unit dosage forms the compositions will preferably contain from about 5n to about 1500 mg of the active ingredient of ~ormula I. The dosage of the compounds of Formula I is dependent on such factors as the weight and age of the patient as well as the particular nature and severity of the disease, and is within the discretion of the physician. However, the dosage for adult human treatment will usually be in the range of from about 500 to about 5000 mg per day, depending on the frequency and route of administration. When administered intra-muscularly or intravenously to an adult human, a total dosage of from about 750 to about 3000 mg per day, in divided doses, normally will be sufficient, although higher daily doses of some ~L~'7~i~Z~ ~

of ~he compounds may be desirable in the case of Pseudomon.as infections.

The quaternary ammonio group of the formula -N - Q

may be acyclic, cyclic, or a combination of ~he two, and may contain one or more additional hetero atoms selected from nitro-genr sulfur and oxygen.

An example of an acyclic quaternary ammonio group is a group of the formula ~91 7 -I-R
R~

in which R , R and R may be the same or different and may, for example, be (lower)alkyl or substituted (lower)alkyl in which the substituents are, for example, halogen, amino with the provision that the amino group may not be on an a-carbon, hydroxy with the provision that the hydrsxy group may not be on an ~-carbon, (lower~alkoxy with the provision tha~ the alkoxy group may not be Qn an -carbon, (lower)alkylthio, (lower)alkylamino, di(lower)-alkylamino, carbamoyl, (lower)alkenyl, phenyl(lower)alkyl, phenyl or substituted phenyl (in which the substituents may be, for example, halogen, hydroxy, amino, (lower)alkylamino, di(lower)-alkylamino, acylamino, (lower)alkyl, (lower)alkylthio, (lower)-alkoxy or the like).

Examples of cyclic quaternary ammonio groups are fully unsaturated monocyclic heterocyclic ring systems, and bicyclic heterocyclic ring systems in which at least one N-containing ring is fully unsaturated. Suitable cyclic guaternary ammonio ring ~.~7~

systems include, for example, those of the formulae -N~10 ~R10 R9 ,j~R9 , ~ ~R9 SR109 RlD S R10 ~R~ ~10 oRltl 0 ~5 S

RlO~R9 R10 RlO~R9 -N~

N~VN
~O~R9 Rl and the like, in which R9 and R10 are the same or different and ~1~27~

may be, for example, hydrogen, halogen, amino, (lower)alkyl, (lower)alkenyl, (lower)alkylthio, carboxy, hydroxy, (lower)-alkoxy, ~lower)alkoxy(lower)alkyl, halo(lower)alkyl, hydroxy-(lower)alkyl, amino(lower)alkyl, (lower)alkylamino(lower)alkyl, di(lower)alkylamino(lower)alkyl, (lower)alkylamino, di(lower)-alkylamino, carboxy~lower)alkyl, carboxy(lower)alkylamino, carboxy(lower)alkylthio, carbamoyl, N-~lower)alkylcarbamoyl, formylamino, acylamino, acyloxy, phenyl, pyridyl, amidino, guanidino and the like. Where the structure of the heterocyclic ring permits, R9 and R10, taken~together, may be an alkylene group containing from 3 to 5 carbon atoms, e.g. propylene.

Examples of combined acyclic/cyclic quaternary ammonio groups include, for example, those of the formulae 12 ~ ~ 12 0 ~ 12 -N ~ R , -N ~ ~ R , -N
" Rll Rll ,Rll ~ 12 ~ R12 ~12 -N ~ O ~ -N S

Rl~ ~,11/ li/ \--/

~ 2 Rll ~ ~ Rll ~ ~ 1 ~ R

0 ~ 12 N N
-N N~ wer)alkyl, -N ~ t R , ~ ~ S ~ ~12 3LZ~
2~
and the like, in which Rll may be, for example, (lower)alkyl, (lower)alkoxy(lower)alkyl, hydroxy(lower)alkyl with the provision that the hydroxy may not be on an a-carbon, carboxy(lower)alkyl, amino(lower)alkyl with ~he provision that the amino may not be on an ~-carbon, (lower)alkenyl, halo(lower)alkyl, allyl and the like, and R12 may be, for example, hydrogen, hydroxy, halogen, (lower)alkyl, hydroxy(lower)alkyl, (lower)alkoxy(lower)alkyl, halo(lower)alkyl, amino(lower)alkyl/ (lower)alkoxy, (lower)-alkylthio, (lower)alkenyl, amino~ (lower)alkylamino, di(lower)-alkylamino, acylamino, acyloxy,-carbamoyl, amidino(lower)alkyl, phenyl, pyridyl, amidino, guanidino and the like.
.

Preferred quaternary-ammonio groups are those of the formulae R13 - ~ ~ 16 (3 ~ R16 ~ ~ 18 - (CN2)n - ~ R~

wherein R13, R14 and R15 are the same or different and are (lower)alkyl, (lower)alkenyl, amino(lower)alkyl with the pro-vision that the a~ino may not be on an a-carbon, or hydroxy-(lower)alkyl with the provision that the hydroxy group may not be on an ~-carbon;

R15 is hydrogen, ~lower)alkyl, (lower)alkoxy, (lower~-alkylthio, amino, (lower)alkylamino, di(lower)alkylamino, formyl-amino, (lower)alkanoylamino, carboxy, hydroxy, carboxy(lower)-alkyl, carboxy(lower)alkylthio, hydroxy(lower)alkyl, halo(lower)-;aZ'76~
`` 21 alkyl, amino(lower)alkyl, (lower)alkoxy(lower)alkyl, carbamoyl orN-~lower)alkylcarbamoyl, or R16 may represent a divalent alkylene group having 3 to 5 carbon atoms;

R17 is (lower)alkyl, (lower)alkoxy(lower)alkyl, halo-(lower)alkyl, allyl, hydroxy~lower)alkyl with the provision that the hydroxy group is not on the a-carbon, amino(lower)alkyl with the provision that the amino group is not on the ~-carbon, or phenyl(lower)alkyl;

R18 is hydrogen, (lower)alkyl, (lower)alkoxyl (lower)-alkoxy(lower)alkyl, (lower)alkyithio, amino, (lower)alkylamino, di(lower)alkylamino, carboxy, hydroxy, carboxy(lower)alkyll kydroxy(lower)alkyl, amino(lower)alkyl, formylamino, (lower)-alkanoylamino, carbamoyl or N-(lower)alkylcarbamoyl;

n is an integer of from 1 to 3, inclusive;

Z is CH2 or, when n is 2, Z also may be S, O or N-~l9, in which Rl is hydrogen or (lower)alkyl; and R20 and R~l are the same or different and are hydrogen, (lower)alkyl, (lower)alkoxy, (lower)alkylthio, amino, (lower)-alkylamino, di(lower)alkylamino, carboxy, hydroxy, hydroxy-(lower)alkyl, amino(lower)alkyl, (lower)alkoxy(lower)alkyl, carboxy(lower)alkyl, carboxy(lower)alkylamino, (lower)alkanoyl-amino, carboxy(lower)alkanoylamino, carbamoyl or N-(lower)alkyl-carbamoyl.

Particularly preferred quaternary ammonio groups are N-(lower)alkylpyrrolidinio (and especially N-methylpyrrolidinio), tri(lower)alkylammonio (and especially trimethylammonio), pyri-dinio, aminopyridinio, formylaminopyridinio, carbamoylpyridinio, amino(lower)alkylpyridinio, carboxypyridinio, hydroxy(lower)-alkylpyridinio, N-(lower)alkylcarbamoylpyridinio, (lower)-alkylenepyridinio, 2-methylthiazolio and 2-amino-5-thiazolo-[4,5-c]pyridinio.

76~

In the compounds of Formula I, particularly preferred values of R2 are (lower)alkyl (and especially methyl), cycloalkyl containing from 3 ~o 5 carbon atoms, l-carboxycycloalk-l-yl containing from 3 to 5 carbon atoms, allyl, propargyl and carboxy(lower)alkyl (and especially 2-carboxyprop-2-yl)~ The most preferred compounds of the invention are 1) 7-[2-(5-amino-1,2,4-thiadiazol-3-yl~-2-methoxyiminoace~amido]-3-[3-(trime~hylammonio)-1-propen-1-yl]-3-cephem-4-carboxylate, 2) 7-[2-(5-amino-1,2,4-thiadiaz~1-3-yl)-2-methoxyiminoacetamido]-3-[3-(1-methylpyrrolidinio)-1-propen-1-yl]-3-cephem-4-car~oxy-late, 3) 7-[2-(5-amino 1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido~-3-[3-pyridinio-1-propen-1-yl]-3-cephem-4-carboxylate, 4) 7-[2-(5 amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-(3-aminopyridinio)-1-propen-1-yl]-3-cephem-4-carboxylate, 5) 7-[2-(S-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-(3-formylaminopyridinio)-1-propen-1-yl]-3-cephem-4-carboxylate, 6) 7-[2-(5-amino-1,2,4-thiadiazol-3-yl~-2-methoxyiminoacetamido]-3-[3-(3-aminomethylpyridinio)-1-propen-1-yl]-3-cephem-4-carboxylate, 7) 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-(3-carbamoylpyridinio)-1-propen-1-yl]-3-cephem-4-carboxylate, 8) 7-[2-(5-amino-1,2,4-thiadiaæol-3-yl)-2-methoxyiminoacetamido]-3-[3-(4-carbamoylpyridinio)-1-propen-1-yl]-3-cephem-4-carboxylate, 9) 7-[2-t5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-(2-methylthiazolio)-1-propen-1-yl]-3-cephem-4-carboxylate, 10) 7-~2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-(2-amino-5-thiazolo[4,5-cjpyridinio)-1-propen-1-yl]-3-cephem 4-carboxylate, 11) 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-(4-hydroxymethylpyridinio)-1-propen-1-yl]-3-cephem-4-carboxylate, 12) 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-"`` 1~6~

3-[3-(3-hydroxymethylpyridinio)~l-propen-1-yl]-3-cephem-4 carboxylate, 13) 7-[2-~5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-(4-{N-methylcarbamoyl}pyridinio)-l-propen-l-yl]-3-cephem-4-carboxylate, 14) 7-[2-~5-amino-1,2,4-~hiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-(2,3-propylenepyridinio)-1-propen-1-yl]-3-cephem-4-carboxylate, 15) 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-ethoxyiminoace~amido]-3-[3-(4-carbamoylpyridinio)-~-propen-1-yl]-3-cephem-4-carboxylate, 16) 7-[2-(5-amino-1,2,4- thiadiazol-3-yl)-2-cyclopentyloxyimino-acetamido]-3-[3-(4-carbamoylpyridinio)-1-propen-1-yll-3-cephem-4-carboxylate, 17) 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-allyloxyimino-acetamido]-3-[3-(4-carbamoylpyridinio)-1-propen-1-yl~-3-cephem-4-carboxylate, 18) 7-[2-(S-amino-1,2,4-thiadlazol-3-yl)-2-propargyloxyimino-acetamido]-3-[3-(4-carbamoylpyridinio)-1-propen-1-yl]-3-cephem-4-carboxylate, 19) 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-(4-carboxypyridinio)-1-propen-1-yl]-3-cephem-4-carboxylate, 20) 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-ethoxyiminoacetamido]-3-[3-(4-carboxypyridinio)-2-propen-1-yl]-3-cephem-4-carboxylate, 21~ 7-[2-(5-amino-1,2,4~-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-(3-carboxymethylpyridinio)-1-pEopen-l-yl]-3-cephem-4-carboxylate and 22) 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-(4-carboxym~thylthiopyridinio)-1-propen-1-yl]-3-cephem-4-carboxylate.

The numbering system utilized herein for the various reactants, intermediates and final products is as follows:

. .

~lLz76~!291 [Roman Numeral] - rArabic Numeral~ r Letter ~ f appropriate~ ~ f appropriate)J .

The Roman Numeral designates whether the compound is a final product [I] or an intermediate or other reactant [all other Roman Numerals]. The Arabic Numerals and Letters are not used in those instances where the overall class ~genus) of compounds is meant.
.

The Arabic Numeral designates the particular meaning of substituent R2. If the particular R2 group contains a carboxyl group which is protected by a conventional carboxyl-protecting group, a ~prime~ (') is used after the Arabic Numeral to indicate this fact. No ~prime~ is used if the carboxyl group is unpro-tected. A ~primer also is used with the generic R2 substituent ~i.e. R ) when generically referring to an R2 group containing a protected carboxyl group.

The Le~ter at the end of the compound number refers to the particular meaning of the quaternary ammonio group -N -- Q -For convenience, the Arabic Numerals and Letters assigned to some of the preferred R2 groups and quaternary ammonio groups are set forth below.

Arabic Numeral R2 1 = methyl 2 = ethyl 3 = allyl 4 = propargyl S = cyclopentyl . .

Letter ~N - Q
_ _ _ A 8 l-methylpyrrolidinio B = pyridinio C = 2-amino-5-thiazolo[4,5-c]pyridinio D = trimethylammonio E = 3-aminopyridinio F = 3-formylaminopyridinio G = 3-carbamoylpyridinio - H = 4-carbamoylpyridinio I = 3-amin~methylpyridinio J = 2-methylthiazolio K = 3--hydroxymethylpyridinio L = 4-hydroxymethylpyridinio M = 4-(N-methylcarbamoyl)pyridinio N = 4-carboxypyridinio O = 2,3-propylenepyridinio P - 3-carboxymethylpyridinio Q = 4-carboxymethylthiopyridinio In the primary evaluation of the compounds of this invention, the Minimum Inhibitory Concentrations (MIC's) of the compounds were determined by the two-fold serial agar dilution method in Mueller-Hinton agar agains~ 32 strains of test orga-nisms in six groups. The geometric means of the MIC's determined in these tests are shown in Table 1.

`` ~276~

Table i Config, Geometric Mean of MIC ~mcg/ml) ~mpd. double (G~)-Ia (G+)-Ib (G-)-Ia (G-~-Ib (G-)-II (G-)-III
No. bond (5) (5) ~5) (5) (5) (7) _ _ _ ~ _ _ _ I-lA E/Z=l/l 0.26 0.70 0.05 0.15 0.23 2.4 I-lA E/Z=7/1 0.13 0.35 0.029 0.05 0.17 1.4 I-lB E 0.20 0.40 0.016 0.044 0.11 1.6 I-IB E/Z=1/4 0.35 0.80 0.05 0.11 0.35 3.5 I-lC E 0.10 0.20 0.0071 0.033 0.087 3.8 I-lD E/Z=l/l 0.61 1.4 0.10 0.26 0.46 2.4 I-lD E/Z=10/1 0.30 0.53 0.05 0.076 0.26 1.3 I-lE E 0.20 0.40 0.0094 0.029 0.10 1.4 I-lF E 0.15 0.40 0.0094 0.033 0.099 1.2 I-lG E 0.20 0.35 0.0094 0.033 0. io 1 . 4 I-lH E 0.20 0.40 0.013 0.043 0.10 0.97 I-lI E 0.80 1.6 0.10 0.20 0.69 3.1 I-lJ E 0.17 0.35 0.025 0.076 0.15 1.6 I-lK E 0.35 0.80 0.029 0.044 0.20 3.5 I-lL E 0.26 0O61 0.029 0.088 0.15 2O6 I-lM E 0.35 0.70 0.029 0.10 0.17 2.3 I-lN E/2=7/1 1.2 1.6 0.013 0.066 0.30 5.7 I-lO E 0.17 0.35 0.029 0.033 0.11 14 I-2H E 0.20 0.40 0.014 0.057 0.15 1.4 I-2N E 1.2 2.1 0.016 0.11 0.35 4.7 I-2N . Z 1.4 3.1 0.044 0.15 0.69 10 I-3H E 0.23 0.40 0.057 0.10 0.52 1.9 I-4H E 0.26 0.46 0.06Ç 0.11 0.60 2.6 I-5H E 0.13 0.40 0.20 0.46 2.1 4.2 I-lP E 0.8 1.6 0.013 0.087 0.34 14 I-lQ 0.7 0.92 0.0095 0.044 0.23 14 ~G~)-Ia : Penicillin-sensitive S. aureus (5 strains) (G+)-Ib : Penicillin resistant S. aureus (5 strains) G-)-Ia : Cephalothin-sensitive E. coli (2 strains), Rl.
pneumoniae (1 str~in) and Pr. mirabilis (2 strains) (G-)-Ib : Cephalothin-resistant E. coli (3 strains) and Kl.
~neumoniae (2 strains) G-)-II : M. ~ (1 strain), Ent. cloacae (2 strains) and Ser. marcescens (2 strains) (G-)-III : Ps. aeru~inosa (7 strains~

Table 2, below, gives the Protective Dose50 (PD50) in mice for a number of the compounds of Formula I against selected microorganisms. Table 3 gives blood levels of various compounds of Formula I upon intramuscular administration of the test compounds to mice at a dosage of 20 mg/kg.

Table 2 PD50 (mg/kg) 5. aureus E._coli P. aeruginosa 5~ Smith Juhl A9843A
I-lB 0.44 0.028 7.7 I-lB 0.65 0.072 NT
. I-lC 0O22 0.013 NT
I-lG 0.96 0.021 5.92 I-lH 0.39 0.015 3.9 I-lJ 0.35 0O029 NT
I-lK 0.53 NT NT
I-lM 0.96 NT NT
I-lN 2.0 NT NT
I-lO 0.26 0.17 NT
I-2N 5.0 NT NT

NT = Not Tested 6~

Table 3 max Tl/2 AUC
~y~ (mcg/ml) (min) mcg hr/ml) I-lB 17 21 11 I-lC 21 32 18 I-lD 20 19 11 I-lH 23 16 14 I lJ 19 16 9.7 I-lR 24 ~ 14 14 I-lM 20 23 14 I-lN 24 19 18 I-lO 28 32 17 In another aspect, this invention relates to processes for the preparation of the compounds of Formula I. The preferred procedures are shown below in Reaction Schemes la, lb and lc, while an alternative procedure is shown in Reaction Scheme 2.
. The abbreviation "Ph" represents the phenyl group. Thus, the -CH(Ph)2 moiety is the benzhydryl group, which is a preferred carboxyl-protecting group. When R contains a carboxyl group, it is desirable to protect the carboxyl group with a conventional carboxyl-protecting group such as the t-butvl moiety. Y
represents chloro, bromo or iodo.

Reaction Scheme la ~N ~ ~ II

o~ N~CH;i!
CoocH(ph)2 ¦ H2N ~ ~ N \ 2 III

.. . ...

,7~9 ~ICI--CON~

COOC~I ( Ph ) 2 ~1, NaX or RI

N~;~C----CONEI~ ~ \

~2N S ~P ~bR2 ~ N~c~2 I

. v ~oc~c~(Ph)2 P(Ph)3 P (Ph~ 3 N ~C--C:ONH ~ S 1~ VI

~2N S ~ o:R2~ ~2P (Ph) 3 COOCH (Ph) 2 ~base N~ CONHF 5 ~ VII
H2 S ~ \oR2N~CH=P(Ph) 3 OOC H 5 Ph ) 2 ~ ClCH2CHO

~ t7~

N fi __--CONH T~ ~ VIII

~ S~N \oR2 ~N~C~I=CHCH2C
2 . COOCE~ (Ph ) 2 NaI or KI
~ ~ ' N.~C----CCN~ 1 H2 ~ C~ IC~2 I ~
IX CC)C)CH (Ph) 2 \

~R . Q ~N
J,~secondary am~n ) XI
~tert-N~ CON}~ ~ iary H22~ 5~ \oR2 ~ N~H CHCEI2 N\ amine) COOC~ h) 2 R' 'X
~ R . . .

N ~C;----CONH Tl ~ ~

X2N S oR2 o~ ~3=CHCE2 N~Q
XII COOCB ~Ph ) 2 oeblock R ~ ~ ~ ~=C~c~2~

Reaction Scheme la shows two alternate means of going from Compound IX to Compound XII. The direct route, utilizing a tertiary amine (XI), is applicable for the preparation of all compounds of Formula Io The indirect route, via Compound X, utilizes a secondary amine as reactant, and is quaternized in the following step. The secondary amine RR'NH may be acyclic (e.g.
dimethylamine) or cyclic (e.g. pyrrolidine), and this indirect procedure therefore is suitable for the preparation of compounds of Formula I in which the quaternary ammonio group is acyclic or "mixed~ acyclic/cyclic. This indirect route is not suitable for the preparation of compounds of Formula I wherein the quaternary nitrogen is in a fully unsaturated heterocyclic ring (e.g.
pyridinio, thiazolio, 2-amino-5-thiazolo[4,5-c]pyridinio, and the like).

Reac~ion Scheme lb o 7~9 ~3CH~N,~ S ~ XIII

N ~ CH2Cl COOCH (Ph ) 2 Na I or XI
' ~3CE~=N~ ~

XIV ~H2 I P t Ph ) 3 ., P (Ph ) 3 ~ /

~3 CH--NF,~S ~

N~ H2P(Ph)3 COOC:II ( Ph ) 2 ¦ ba~e <~CH-~S ~ XVI
N ~CH-P (Ph ) 3 COOCH (Ph~ 2 I ClCH2CHO
~ /

. ~ CH= ~ ~ XVII

~CH=C~ H2Cl oOCH(Ph)2 ¦ ~irard Reagent T
or / HCl H2N ~ ~ XVIII

0 ~ ~ CH=CHCH2Cl COOCH(Ph)2 ~ IIT

_ __~ ----------- I
VIII as in Schemela Reaction Scheme lb is a variation of Reaction Scheme la in that the 7-amino group of the starting material (II) is protected as a Schiff base during most of the reaction steps, and the desired 7-side chain acid is added later in the synthesis.
9therwise, the yeneral procedure is similar.

2~

Rea ct i on 5ch eme lc ~-CH=N~/ ~

N~ CH=CH-CH2Cl XVI I
COOCH ( Ph ) 2 NaI or KI

~CH=N F,'~, oCH=CH-CH2 I XIX
COOCH (Ph) 2 R
HN~R, (secondary amine) ~3--C~ CH=CHCH~ (terti.ry XX O R ~amine ) COOCH (Ph) 2 ~"Y

~H=N~.S ~

XXI O \~CR=CHCH2 ~--Q
~ COOCH (Ph) 2 ~ 76~

S~
H2N~
~ ~3 O I CH=CHCH2-N_ Q
XXII
COO ~' N-acylation \ with III

N ~ CONH

H2N ~ \oR2 ~ N ~
O ¦ CH=CHCH2-N - Q
. COO ~) Reaction Scheme lc is a further variation of Reaction Scheme lbo In Reaction Schemes la and lb~ quaternization of the 3-side Ghain is the last step, but in Reaction Scheme lc tbe last step is acylation of the 7-amino group. The relationship between Reaction Schemes la, lb and lc is shown in the following flowchart.

~'7i6~

H2NT~S~

0~ CH2cl la / II COOCH~Ph)2 \ lb \lc N ~ fi;CONH ~ ~ ~ H-N

IV COOCH(Ph) XIIX H2Cl 2 COOCH(Ph)2 la ~ ~ lb ~lc / ,~

C - CONH ~ ~ S
N~ 1 7-N-Acylation H~N~~r--f~ ~
H2N ~ S ~ \oR2 g ----N ~ CH2Cl lb ~ ~ CH=CKC~ Cl COOCH(Ph)2 2 YIII XVIII co Wl(Ph)2 .

Quaternization Quaternization lb Deblocking lC Deblocking 7-N-~cylation Comp ound I H ~5 ~CII
~r ~Q

XXII

. , .

~76~2~
~7 In Reaction Schemes la, lb and lc, the benzhydryl group was shown as the preferred carboxyl-protecting group. It will be appreciated by those skilled in the art that other carboxyl protecting groups, well-known in the art, may be used. The acylating acid III may be used in the form of a derivative such as its acid halide, activated ester, mixed acid anhydride, etc., all of which are well-known in the art. We prefer to utilize it in the form of its acid chloride. Acylating acid III also may have its amino group protected ~y any of the common amino-protecting groups, e.g. N-trityl, N-formyl, or the like. The base used to conveet the phosphonium iodide (VI or XV) to give the phosphorylide (VII or XVI) may be NaOH, Na2CO3l IRA-410 (OH ) resin, IRA(CO3 ) resin, or the like, or a mixture thereof. The chloroacetaldehyde used to convert the phosphorylide VII to the 3-chloropropenyl-3-cephem compound VIII (or Compound XVI to Com-pound XVII) may be the commercially available 40-50% aqueous solution, a distilled solution (e.g. 70%) or the anhydrous aldehyde.

We have found that Compound VIII prepared from Compound VII (Scheme la) typically had a Z:E ratio of about 2:1 at the propenyl double bond. Compound VIII prepared from Compound XVIII
(Scheme lb), on the other hand, typically was almost exclusively the Z isomer. The difference may not be in the route used, but in the conditions utilized in the Wittig reaction (VII to VIII or XVI to XVII). We have also found that the use of an appropriate silyl reagent such as N,O bis(trimethylsilyl)acetamide in the Wittig reaction (VII to VIII in Scheme la and XVI to XVII in Scheme lb) caused improYement of the yields and purity of VIII
and XVII. The reaction is preferably carried out with 2-5 equivalents of the silyl reagent. When the chloropropenyl cephem (VIII) was reacted with NaI in acetone to give the iodopropenyl cep~.em (IX), the double bond in the propenyl group WAS isomerized from Z to E during the iodination. A short reaction period retained the configuration of the parent Compound VIII to a large extent, while a long reaction period gave primarily the ~ isomer of Compound IX. However, an excessive reaction time at high ~2'7~9 3~
temperature gives a lower purity compound IX. We find that abo~t 10 minutes at 25C and 2 hours at 5C gives pure IX in good yield. When utilizing Reaction Scheme Ic, we have found that, when iodinating compound XIV with Nal, a purer compound is obtained if the acetone solution is diluted with CC14 when iodination is essentially completed, and the isomerization portion of the reaction is conducted in the acetone-CC14 mixture.
When iodination of the chloropropenyl cephem (XVII) to the iodopropenyl cephem (XIX) was performed with KI in DMF, the isomerization of the double bon* from Z to E proceeded as fast as the iodination did. The whole reaction completed within 45 minutes at room temperature to give pure XIX without dilution with CC14 in the course of the reaction.

Compound XII normally was deblocked without purifica-tion, and the final product (I) was purified by reverse phase column chromatography utilizing a glass column containing the packing removed from a Waters' ~ssociates PrepPAK-500/C18*car-tridge.

* Trade Mark ~.2~

Reection Scheme 2 N ~ I ~ CONH~

Rl-HNJ~ S ~ \oR2 ~ N~71`CH=CH-CH2I XXIII
COOCH (Ph) 2 R ~1 5 ~OR oF_~C~I=CH_ COOCH ~Ph) ;~

~CONH~ ~1 ~3 XXV
R --HN S ~ oR2 ~ H=CH-CH2N----Q
COOCH (Ph) 2 Reduc:tion Rl-H~l~C--CONH~ CH=CH-CH~N---Q XXVI
COOCH (Ph) 2 ~7~ 2~ i 4~

deblock 1 fi ~ CONH ~ ~ ~ I
H2N ~S N ~OR2 ~ ~ N ~--CH=CH2N--Q
CO~

Reaction Scheme 2, shown in brief outline form above, is similar to Reaction Scheme la except that Compound XXIII
(equivalent to Compound IX of Reaction Scheme la) is converted ~o its S-oxide prior to quaterniæation. Compound XXV is subsequently reduced, and the remainder of Reaction Scheme 2 is as Reaction Scheme la. In Reaction Scheme 2, it is preferred to protect the amino group of the 7-side chain with a known amino-protecting group such as the trityl group.
.
The acylating acids of Formula I~I herein are either known compounds or are readily prepared by published procedures.
European Patent Specification 7,470 published October 12, 1983 (application published February 6, 1980) exemplifies the preparation of compounds of Formula III wherein R2 is methyl, ethyl, propyl and isopropyl. U.S. Patent 4,390,534, referred to in the Prior Art section, above, exemplifies the preparation of a wide variety of compounds of Formula III wherein R is, for example, cyclopentyl, 2-cyclopenten-1-yl, allyl, 2-propynyl, l-tert. butyloxycarbonyl-l-methylethyl, l-tert. butyloxycarbonyl-l-cyclopentyl, l-ethoxy-carbonyl-l-methylethyl, tert. butyloxycarbonylmethyl, l-tert.
butyloxycarbonyl-2-methylpropyl, trityl, and the like.

Compound II herein (7-amino-3-chloromethyl-3 cephem-4-~2~7~

carboxylate), used as a starting material in Reaction Schemes la, lb and lc, is a known compound.

~ he tertiary amines of Formula XI (and the secondary amines RR'NH) utilized in preparing the ~uaternary ammonio compounds of this invention are either known compounds or are readily prepared by those of ordinary skill in the art. Many of the amines are commercially available.

The present invention also provides a process for the preparation of compounds of the formula _~ S ~
N~~CONBTl I I
~ A--N ~ ~E3 RlHN~S ~~oR2 C~ ~ ~CH--C~--C~I2-N~Q
~:od3 , wherein Rl is hydrogen or a conventional amino-protecting group, R2 is hydrogen, a straight or branched chain alkyl group contain-ing from 1 to 4 carbon atoms, a cycloalkyl or cycloalkenyl ring containing from 3 ~o 6 carbon atoms, or a group of the formula -C-C~-CH R3 , -C-C _C-R , ~ or in which R3 is hydrogen, (lower)alkyl or carboxyl, X is halogen, hydroxy or (lower)alkoxy, and R4 and R5 are each independently hydrogen, methyl or ethyl, or R4 and R5, taken together with the -~2~;~6~:~9 carbon atom to which ~hey are attached, may be a cycloalkylidene ring containing from 3 to 5 carbon atoms, and -N _ Q

is a quaternary ammonio group, and nontoxic pharmaceutically acceptable salts and physiologically hydrolyzable esters thereof, which process comprises reacting a compound of the formula ~)m 2 ~ / ~O~R~
O CH=CHCH2 Z
COOBl wherein R~ is the same as.R2 or is a group of the formula X COOBl 14 < > or -C--COOB
Y

in which X, R4 and R5 are as defined above, Bl is a conventional carboxyl-protecting group, B2 is hydrogen or a conventional amino-protecting group, Z is chloro, bromo or iodo, and m is zero or onet with a tertiary amine Q -~N (or sequentially with a secondary amine R2'NH and a compound of the formula R~Z), an~d, if m is 1, reducing the sulfoxide by conventional means, and subsequently removing all blocking groups by conventional means.

The.present invention also provides a process for the preparation of co~pounds of the formula ....

~;Z7~

~ ~ ~ ~ C~

Rl~N l S OR~ ~ N ~ HCCR-CH2-~--Q
cod~

wherein Rl is hydrogen or a conventional amino-protecting group, R2 is hydrogen, a straight or branched chain alkyl group contain-ing from 1 to 4 carbon atoms, a cycloalkyl or cycloalkenyl ring containing from 3 to 6 carbon atoms, or a group o~ the formula -C-CH-CH-R3 , -C-C -~C-R3 , ~ ~

-C-COOH
R~

i-n which R3 is hydrogen~ (lower)alkyl or carboxyl, X is halogen, hydroxy or (lower)alkoxy, and R4 and RS are each independently hydrogen, methyl or ethyl, or R4 and R5, taken together with the carbon atom to which they are attached, may be a cycloalkylidene ring containing from 3 to 5 carbon atoms, and -N -.Q

is a quaternary ammonio group, and nontoxic pharmaceutically acceptable salts and physiologically hydrolyzable esters thereof, which process comprises acylating a compound of the formula , 1~716~aZ9 s 2N~ ~

O ~ CH=CHC~2-N- Q
. COo ~ XXII
with an acid of the formula B HN ~ 5 ~ ICl - COOH III

or with an acylating derivative of said acid, wherein R is the same as R or is a group of the formula .

X
~ ~ or -C---COOBl r X RS

in which X, R4 and R5 are as defined above, Bl is a conventional carboxyl-protecting group and B2 is hydrogen or a conventional amino-protecting group~

The reactions are carried out in a non-aqueous organic solvent such as dimethyl sulfoxide, hexamethylphosphoramide, methylene chloride, chloroform, ethyl ether, hexane, ethyl a~etate, tetrahydrofuran, acetonitrile and the like, or mixtures of such solvents. The reactions are conveniently carried out a~
a temperature of from about -10C to about +50C; we normally prefer to conduct the reactions at room temperature. During the quaternization step, at least one mole of the tertiary amine should be used per mole of Compound IX, XIX, XXIII or XXIV; we .. . .

~2~2g normally prefer to utilize from about 25% to 100~ excess of the tertiary amine.

Carboxyl~protecting groups suitable for use as Bl in the above reactions are well-known to those skilled in the art and include aralkyl groups such as benzyl, p-methoxybenzyl, p-nitrobenzyl and diphenylmethyl (benzhydryl); alkyl groups such as t-butyl; haloalkyl groups such as 2,2,2-trichloroethyl, and other carboxyl protecting groups described in the literature, e.g. in V.K. Patent 1,399,086. We prefer to utili3e carboxyl-protecting groups which are readily removed by treatment with acid. Particularly preferred carboxyl-protecting groups are the benzhydryl and t-butyl moieties.

Amino-protecting groups suitable for use as B2 are also well-known in the art, and include the trityl group and acyl groups such as chloroacetyl, formyl and trichloroethoxycarbonyl.
Amino-protecting groups which are readily removed by treatment with acid, e.g. the trityl group, are preferred.
.

When the cephalosporin nucleus is utilized in the form of the 1-oxide (m = 1), the l-oxide is prepared by known procedures such as oxidation with m-chloroperbenzoic acid, peracetic acid, sodium tungstate, etc. The l-oxide subsequently may be reduced by known procedures, e.g. reduction of the corresponding alkoxysulfonium salt with iodide ion in an aqueous medium. The alkoxysulfonium salt itself is readily prepared by treatment of the l-oxide with, for example, acetyl chloride.

In another aspect, this invention relates to novel intermediates of the formula 76~29 H N 1 5 ~ \oR2 ~ ~ CH=C~CH2Z
COOH
. XXVIII

wherein Z is chloro, bromo, or iodo, R? is hydrogen, a straight or branched chain alkyl group containing from 1 to 4 carbon atoms, a cycloalkyl or cycloalkenyl ring containing from 3 to 6 carbon atoms, or a group of the formula -C-C~=C~-R , -C-C -- C-R3 , ~ ~

-C-COOH , in which R3 is hydrogen, (lower)alkyl or carboxyl, X is halogen, hydroxy-or (lower)alkoxy, and R4 and R5 are each independently hyd.rogen, methyl or ethyl, or R4 and R5r taken together with the carbon atom to which they are attached~ may be a cycloalkylidene ring containing from 3 to 5 carbon atoms, and salts and esters thereof. Also included are compounds of Formula XXVIII in which the amino and/or carboxyl groups are protec~ed by conventional amino-protecting or carboxyl-protecting groups.

In still another aspect, this invention relates to novel intermediates of the formula ~Z76~

R24~ /--CH=N ~S~

R25 \~ CH=CHC}1 COOR
XXIX

wherein R22 is hydrogen or a conventional carboxyl-protecting group, and R23, R24 and ~25 are the same or different and are hydrogen, hydroxy, (lower)alkyl or (lower)alkoxy; or a salt, solvate, hydrate or ester thereofO

In still another aspect, this invention relates to novel intermediates of the formula ~\~\ CH=CH--CH2-N_C;!
~0 ~
XXII

wherein ~ is a quaternary ammonio group; or a salt, ester solvate or hydrate thereof.

As used herein, the terms acylamino and acyloxy refer to an acylated amino or acylated hydroxy group in ~hich the acyl moiety is (lower)alkanoyl (e.g. formyl, acetyl, propionyl, butyryl, isobutyryl, isovaleryl, etc.), aroyl ~e.g~ benzoyl, etc.), (lower)alkanesulfonyl (e.g. mesyl, ethanesulfonyl, etc.) or arylsulfonyl (e.g. benzenesulfonyl, tosyl, etc.).

.

7~9~ , As used herein, the terms ~(lower)alkyl~, ~(lower)alkoxy~, ~(lower)alkylthio~ (or the like) mean straight or branohed chain alkyl, alkoxy, alkylthio (or the like) groups containing from 1 to 6 carbon atoms, inclusive. Similarly, the terms (lower)alkenyl and (lower)alkynyl mean alkenyl or alkynyl groups containing from 2 to 6 carbon atoms.

... .

6~9 ~9 ExamPle 1 N ~ ~ ~ CONH

J~ N~ ~ N,~ C~--CH~,N~
C}13 I-lA *Z/l =l/l 7-[2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxYiminoacetamido]-3-[3-(1-methylPyrrolid _io)-l- ~open-l-yl]-3-cephem-4-car_ xYlate (I-lA) To a solution of diphenylmethyl 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3~(3-iodo-1-propen-1-yl)-3-cephem-4-carboxylate (IX-l~ (Z/E=2/1, 150 mg, 0.21 mmole) in ethyl acetate (2 ml) was added a solution of l-methylpyrrolidine (36 mg, 0.42 mmole) in ethyl acetate (1 ml) in one portion with stirring. The mixture was stirred for 15 minutes and diluted with isopropyl ether (10 ml) to form a precipitate, which was collected b~ filtration. A mixture of the solid (130 mg), formic acid (1 ml) and concentrated HCl (0.1 ml) was stirred at room temperature. Af~er 1 hour, the reaction mixture wa~ concentrated under reduced pressure, diluted with water (20 ml) and filtered. The aqueous solution was passed through a reverse phase column (the packing of PrepPAK-500/C18 cartridge, 100 ml), elu~ing with water and 10% CH30H. The desired fractions were collected, and concentrated in vacuo to a small volume and ~reeze-dried to give 13 mg (12~) of the title compound (I-lA) (Z/E=lfl), melting at ~280C (dec.).

IR : ~K~r cm 1 3400, 1760, 1660, 1610.
max UV ~Phosphate buffer (pH 7~ nm (El~ ) 236 (372), 288 (322)-max 1 cm NMR :~D2O 2.31 (4~1, m, ~ )~ 3.12 (3H, s, N-CH3), 3.6 (5H, ppm ,~
m, 2-H & N ~ ~, 3.79 (lH, s, 2-H), 4.1 (2H, d, J=8, CH2N)~ 4.2 ~3H, s, OCH3), 5.36 (lH, d, J=4.5, 6-H), 5.95 (3H, m, 7-H ~ 3-CH=CH), 6.66 (1/2H, d, J=10, 3-CH cis), 7.0 (1/2H, d, J=16, 3-C~ trans).

Example 2 ~ ~ C ~ CONH ~ S

H2 S ~ \ OC~3 ~ N ~ CH=CH-CH2-N

I-lB *E

7-[2-(5-Amino-1,2,4~thiadiazol-~yl)-2-methoxyiminoac_tamido]-3-[3-pyridinio-1-propen-1-yl]-3-cephem-4-carboxylate (I-lB) A mixture of diphenylmethyl 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-(3-iodo-1-propen-1-yl)~3-cephem-4-carboxylate ~IX-l) (E, 716 mg, 1 mmole~, pyridine 1158 mg, 2 mmoles) in dimethylsulfoxide (DMSO) ~1 ml) was stirred for 1 hour at room temperature. To the mixture was added ethyl acetate (20 ml) to precipitate a solid (620 mg), which was added to formic acid (6 ml) containing sodium bisul~ite (60 mg). The mixture was stirred for 30 minutes at 40JC and concentrated to dryness. The residue was dissolved in H2O (40 ml) and some insolubles were removed. The aqueous solution was charged on a column of reverse phase (PrepPAK-500/C18, 100 ml~ eluting with ., , ~ - . . .

~L~7~i~9 1 H2O (300 ml) and 5% aqueous CH30~ ~800 ml), and the eluate was monitored by uv (254 nm) and HPLC. The fractions (5% aqueous CH30H) containing the desired product were combined, concentrated to a small volume and lyophilized to yield 40 mg (8%) of the title compound ~I-lB), melting at >200C (dec.).

IR : v~Br cm 1 3350, 1760, 1660, 1600.
max UV ~Phosphate buffer (pH 7~ nm (El% ) 240 (352)~ 258 (366)~
max 1 cm 267 (279), ~90 (469)-NMR : ~ 2 D 6 3.74 (2H, br-s, 2-H), 4.20 (3~, s, OCH3), ppm 5.92 (lH, d, J-4.5~ 7-H), 6.15 (lH, m, 3-CH=CH), 7.04 (lH, d, J=16, 3-CH ~rans), 8.2 (2~, m, Py-H3 5), 8.62 (lH, m, Py-H4), 8.97 (2H, m, Py-H2 6) Example 3 N ~ C ~ CONH

H2N 5 OC~3 o ~ ~ C~-CH-o~3 I-lB *Z/E=4/1 7-lk~ 1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-Pyridinio-l-propen-1-~1]-3-cephem-4-carboxylate (I-lB) The chloropropenyl compound, diphenylmethyl 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2~methoxyiminoacetamido]-3-(3-chloxo-l-propen-l-yl)-3-cephem-4-carboxylate (VIII-l) (Z, 937 mg, 1.5 mmoles) was added to a s~irred sslution oP pyridine (237 mg, ;76~

3 mmoles) in DMSO (3 ml) containins NaI (11 mg, 0.075 mmole).
The mixture was allowed to stand overnight at room temperature in the dark. The mixture was diluted with ethyl acetate (30 ml) to separate the precipitate which was then collected by filtration, washed with ethyl acetate (10 ml) and dried to give 350 mg of the blocked product. The precipitate was ~reated with formic acid ~3.4 ml) containing sodium bisulfite (34 mg) for 30 minutes at 40C. After removal of the formic acid, the residue was purified by reverse phase column chromatography (packiny of PrepPAK-500/C~8 cartridge, 100 ml) by eluting with 5% aqueous CH30H. The fractions containing the desired product were combined on the basis of HPLC analysis, evaporated under reduced pressure and lyophilized to give 41 mg (5.5~) of the title compound (I-lB) (Z/E=4/1). Mp. >200C (dec.).

IR : vKBr cm 1 3300, 1760, 1660, 1600-max UV ~phosphate buffer (pH 7) nm (El% ) 237 (386), 250 (377), max 1 cm 258 (369), 265 (347)~
280 (311).

N~R : ~D2O 3.45 & 3.76 (each lH, d, J=16, 2-H), 4.18 (3H, s, ppm OCH3), 5.34 (3H, m, CH=CH-CH2 & 6-H), 5.92 (lH, d, J=4.5, 7-H), 6.5R (4/5H, d, J=ll, 3-CH cis), 7.03 (l/SH, d, J=16, 3-CH trans), 8.12 (2H, m, Py-H3 5), 8.56 (lH, m, Py-H4), 8.82 (2H, m, Py-H2 6) 7~i~Z~

S

~ CON~ ~ ~
H2N S OCH3 N ~ CH=CHCII~- ~ S ~ NH2 I-lC *~

7-~2-(5-Amino-1,2,4-thiadiazol-3-~1~-2-methoxyiminoacetamido]-3-[3-(2-amino-5-thiazolo~4,5-c]pyridinio)-1-propen-1-yl]-3-cephem-4-carboxylate (I-lC) A stirred solutlon of diphenylmethyl 7-[2-(5-amino~
1,2,4-thiadiazol 3-yl) 2-methoxyiminoacetamido]-3 (3-iodo-1-pr~pen-l-yl)-3-cephem-4-carboxylate (IX-l) (E isomer, 714 mg, 1 mmole), 2-aminothiazolo[4,5-c]pyridine [prepared according to the procedure of T. Takahashi et al., Pharm. Bull (Japan), 2, 34 (1954)] and dry DMSO ~1 ml) was kept for 1 hour at room tempera-ture. To the reaction mixture was added ethyl acetate (20 ml) to give a yellow powder (710 mg). Formic acid (7 ml) and sodium bisulfite (70 mg) were added to the powder (700 mg), and the mixture was stirred for 30 minutes at 40-45C. After evapo-ration, the residue was triturated with ~2 (40 ml). Insolubles were filtered off, and the filtrate was chromatographed over a reverse phase column (PrepPA~-500/C18, 100 ml), with H2O and 10%
CH30H as eluant. The fractions containing the desired product were combined, and the solvent was removed under reduced pres-sure. Lyophilization gave the desired product (I-lC) as a colorless amorphous powder of the E isomer. Yield 110 mg (19~.
Mp. ~200C (dec.).

, .

76;~329 I~ : vK r cm 1 3300, 1760, 1660, 1630, 1600.
max UV ~Phosphate buffer (pH 7) nm (El% ) 245 (499)~ 285 (286)-max 1 cm NMR ~ SO d6 2 3.86 (3H, s, OC~3), 4~98 (lH, d, J-4.5, ppm 6-H), 5.2 (2H, m, CH=CH-CH2), 5.57 (lH, m, 3-CH=CH), 5.96 (lH, m~ 7-H), 7.16 (lH, d, J=16, 3-C~ trans), 8.36 & 8.45 (each lH, d, J=7, Py-~), 8.~2 (lH, s, Py-H).

Example S

N ~ ~ ~ CONH ~ ~
H2N S \ OCH3 ~ CH=C~-CH2-N(~H3)3 ., COO~) I-lD *Z~E ~ 1~1 7 [2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-(3-trimethylammonio-1-propen-1-~1)-3-cephem-4-carboxyl te (I-lD) To a solution of diphenylmethyl 7-[2-(5-amino-1,2,4-thiadiaæol-3-yl)-2-methoxyiminoacetamido]-3-(3-iodo-1-propen-1 yl)-3-cephem-4-carboxylate (IX-l) (Z/E=2/1, 490 mg, ~.68 mmole~
in ethyl acetate (14 ml) was added a 0.1 M trimethylamine so-lution in ether (13.6 ml) in one portion. The mixture was stirred for 10 minutes and evaporated to dryness, and the residue was triturated with ether (20 ml). The resulting solid (490 mg) was added to trifluoroacetic acid (0.2 ml) containing one drop of anisole. After 1.5 hours' stirring, the mixture was evaporated to dryness under reduced pressure and the residual oil was triturated with ether (20 ml). The resulting precipitate was collected by filtration and dissolved in ~2 (20 ml). Some insolubles were removed, and the aqueous solution was eluted on a ~27~

C18 reverse phase column (the packing of PrepPAK-500/C18 car-tridge, Waters' 30 ml) using wa~er as eluant. Fractions contain-ing the desired c~mpound were combined and concentrated to a small volume and lyophilized to afford 30 mg (9.2%) of the title compound (I-lD) ~/E = 1/1) a~ a colorless amorphous powder, melting at >150C ~dec.).

IR : vKBr cm 1 3300, 1770, 1670, 1605.
max UV ~Phosphate buffer (pM 7) nm (El~ ) 236 (389), 287 (343)-max 1 cm NMR : ~D2O 3.45 & 3.7 (lH, d, J=16, 2-H), 3.81 (lH, s, 2-H), ppm 4.1 (2H, d, J=8, -CH2N)~ 4.21 (3H, s, OCH3), 5.39 (lH, d, J=4.5, 6-H), 5.95 (2H, m, 3-CH=CH ~ 7-H), 6.61 (1/2H, d, J=ll, 3-C~ cis), 7.05 (1/2H, d, J=16, 3-CH trans~.

Example 6 N _ ----CON~

3 ~ C~ CHC32-I-lE *E

7-[2-(5-Amino-1,2,4-thaadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-(3-amino~yridinio)-1-propen-1-yl]-3-cephem-4-carboxylate (I-lE) Diphenylmethyl 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-(3 iodo-1-propen-1-yl)~3-cephem-4-carboxylate (IX-l) (E, 716 mg, 1 mmole) was added to a stirred solution of 3-aminopyridine (188 mg, 2 mmoles) in DMSO (1 ml).

~6~32~3 The mixture was stirred for 1 hour and diluted with ethyl acetate (20 ml). The resulting precipitate was collected by filtration, washed with ethyl acetate and dried to give 520 mg of yellow powder. A mixture of the powder (500 mg), formic acid (5 ml) and sodium bisulfite (50 mg) was stirred for 30 minutes at 40C. The ixture was concentrated in vacuo, dissolved in H O (40 ml) and filtered to remove insolubles. The aqueous solution was chromatographed on a column of reverse phase (packing of PrepPAK-500/C18, 100 ml), with 7.5% aqueous CH30H elution. The fractions containing the desired compound were evaporated and lyophilized to give the title compound (I-lE) (7 mg, 1.4~), melting at >185C (dec.).

IR : v cm 1 3400, 1765, 1675, 1620, 1600.
max UV ~Pho~phate buffer (pH 7) nm (El% ) 246 (403), 290 (468)-max lcm NMR : ~D2O 3.72 (2H, m, 2-H), 4.14 (3H, s, OCH3), 5.35 (3H, m, ppm 6-H & CH=CH-CH2), 5.9 (lH, d, J=4.5, 7-H), 6.1 (lH, m, 3-CH=CH), 7.05 (lH, d, J=16, 3-CH, trans), 8.1 (lH, m, Py-H5), 8.54 (lH, br-s, Py-H6), 8.68 (lH, m, Py-H4), 9.4 (lH, m~ PY H2) Treatment of IX-l (716 mg, 1 mmole) with 3-t-butoxy-carbonylaminopyridine (324 mg, 2 mmolesl by a procedure similar to that described above gave 12 mg (2.3%) of I-lE.

~Z 76~Z9 5~' ExamPle ?

N fi - CON
~ ~\ ~ 1 * ~, ,7~ NH2 H2 S ~ OCH3 ~ CH=CHCH2-N

I-lE *Z/E G 1/1 7-[2-(S-Amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-(3-amino-1-pyridinio)-1-propen-1-yl]-3-cephem-4-carboxylate (I-lE) ~ mixture of diphenylmethyl 7-~2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido] 3-(3-iodo-1-propen-1-yll-3-cephem-4-carboxylate (IX-l) (Z/E=2/1, 500 mg, 0.7 mmole) and 3-aminopyridine (66 mg, 0.7 mmole) in dimethylsulfoxide (1 ml) was stirred for 20 minutes at room temperature. The mixture was diluted with ethyl acetate (10 ml) and ether (10 ml), and the resulting precipitate was collected by filtration, washed with ether (10 ml) and dried. The quaternized salt was dissolved in formic acid (3 ml) containing concentrated HCl (0.3 ml) and stirred for 1.5 hours at room temperature. The mixture was concentrated to dryness under reduced pressure. The residue was dissolved in 2% HCl (10 mlj and filtered. The aqueous layer was chromatographed on a reverse phase column (PrepPAK-500/C18, 100 ml). After washing with water (500 ml), the column was eluted with 5~ aqueous CH30H. The fractions containing the title compound were combined, concentrated ln vacuo and freeze-dried to give 15 mg (4.2%) of the title compound (I-lE) (Z/E=l/l) as a colorless amorphous powder. Mp. >160C (dec.).

~ 76~
5~

IR : ~KBr cm 1 3400~ 1765, lG75, 1620, 1600.
max UV ~Phosphate bUffer (pH 7~ nm (El% ~ 244 (434), 287 ~333)-max 1 cm NMR ~DMS-d6+D2 3.73 (2H, m), 4.14 ~3~, s, OCH3), ppm m, 6-H & CH-CH-CH2), 6.0 (2H, m, 7 H &
3-CH=CH), 6.6 (1/2H, d, J=ll, 3-CH cis), 7.05 (1/2H, d, J=16, 3-CH trans), 8.08 (lH, m, Py-H5), 8.6 (2H, m, Py-H4 6)~ 9 4 (lH, m, Py-~12 ) .
S

H2N ~ ~ \ OC~3 O ~ ~ C~-CHCH2-I-lF *F

7-~2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[.3-(3-fo~ylaminopyridinio)-1-propen-1-yl]-3-cephem-4-carboxylate (I-lF) A mix~ure of diphenylmethyl 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-(3-iodo-1-propen-1-yl)-3-cephem-4-carboxylate (IX-l) (E, 716 mg, 1 mmole) and 3-formylaminopyridine [prepared according to the procedure of N.
Enomoto et al., Bull. Chem. Soc. Japan, 45, 2665 (1972)] (244 mg, 2 mmoles) in DMSO (2 ml) was stirred at room temperature for 1 hour, and poured into ethyl acetate (200 ml). The precipitate was oollec~ed by filtration, washed well with ethyl acetate and dried A mix~ure of the quaternized salt (500 mg) and sodium bisulfite (50 mg) in HCOOH (5 ml) was stirred at 40-50~C for 80 minutes and evaporated to dryness in vacuo. The residue was .~. ,, , ~ .

6~

dissolved in water (40 ml), neutralized with NaHCO3 and then filtered to remove insoluble material~ The clear filtrate was chromatographed on a reverse phase column, PrepPAK-500/C18 (100 ml), with water and 5~ CH30H, 10~ CH30H, 20% CH30H and 30% CH30H.
The frac~ions containing the desired compound were combined, concentrated in vacuo and lyophilized to give 16 mg (2.9~) of the title compound (I-lF) (E) as a tan powder. Mp. ~170C (dec.).

IR : ~K r cm 1 3340(br)l 1760, 1670, 1620(br), 1590.
max UV ~Phosphate buffer (pH 7) nm (El% ) 218 (428), 248 (362), max 1 cm 290 (474).

NMR : ~D2O+NaHCo3 3.68 (2H, br, 2-H), 4.15 (3H, s, OCH3), 5.~1 ppm ~lH, d, J=4.5, 7-H), 6.25 (lH~ m, CH=CH-CH2), 6.98 (lH, d, J=16, 3-CH trans), 8.8-7.9 (4H, m, Py-H), 9.38 (lH, br, NHCHO).
.
' Example 9 N ~ CONH

H~N S ~ ~r~3 ~ ~ CH CHCH2-I-lG *E

7-[2-(5~Amino-1,2,4-thiadiazol-3-yl)-2-methoxyimin acetamido]-3-[3-(3-carbamoylpyridinio~-1-propen-1 yl~-3-cephem-4-carboxylate ~I-lG~

To a solution of diphenylmethyl 7-[2-(5-amino-1,2,4-thiadiazol~3-yl)-2-methoxyiminoacetamido]-3-(3-iodo-1-propen-1-yl)-3-cephem-4-carboxylate (IX-l) (E, 716 mg, 1 mmole) in DMSO (2 ml) was added nicotinamide (244 mg, 2 mmoles), and the mixture was stirred at ambient temperature for 1.5 hours and poured into ~o ethyl acetate (200 ml) with stirring. The resulting precipitate was collected by filtration. The quaternized salt (500 mg) was dissolved in HCOOH (5 ml) in the presence of sodium bisulfite (50 mg), and the mixture was heated at 40-50C for 40 minutes, with stirring, and evaporated to dryness. The residue was dissolved in water (40 ml) and an insoluble solid was filtered off and washed with a small amount of water. The filtrate and wash were combined and chromatographed on a reverse phase column, PrepPAK-500/ClB ~100 ml). After elution with water and 5%, 10% and 20%
aqueous CH30H, successively, the fractions containing the desired material were combined, concentrated in vacuo and freeze-dried to yield 21 mg (3.8%) of the title compound (I-lG) (E) as a yellow powder. Mp. >175C (dec.3.

IR : vKBr cm 1 3340(br), 1760, 1670, 1600.
max UY ~Phosphate buffer (pH 7~ nm (El~ ) ~35 (326), 274 (sh, max 1 cm 405), 290 (446).

MR :~D2O NaHC8 3.68 (2H, br, 2-H), 4.15 (3H, s, OCH3), 5.32 ppm (lH, d, J=4.5, 6-H), 5.45 (lH, d, J=7, CH=CH CH2), 5.88 (lH, d, J-4.5, 7-H~, 6.15 (lH, d-t, J=16 & 7, 3-CH=CH), 7.00 (lH, d, J=16, 3-CH trans), 8.23 (lH, m, Py-H5), 9.03 (2H, m, Py-H4&6), 9.34 (lH, s, Py-H2).

6~

ExamPle 10 - ~ ~ CONH ~ ~
\ ~ N ~ CH-CHCH2- ~ CONH2 I~lH *E

7-~2-(5-Amino-1,2,4-thiadiazo~ y~ methoxyiminoacetamido]-3-[3-(4-carbamoylpyridinio?-1-propen-1-yl]-3-cephem 4-carboxylate (I-lH) To a stirred solution of diphenylmethyl 7-[2-(5-amino 1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-(3-iodo-1-pr~pen-l-yl)-3-cephem-4-carboxylate (IX-l) (E~ 716 mg, 1 mmole) in dry DMSO (2 ml) was added isonicotinamide (244 mg, 2 mmoles).
The mixture was stirred at room temperature for 1 hour and then poured into ethyl acetate (200 ml). The resulting precipitate was collected by fil~ration, washed well with ethyl acetate and dried. A stirred mixture of the quaternized material (400 mg) and sodium bisulfite (40 mg) in HCOOH (4 ml) was heated at 40-$0C for 1 hour, and evaporated to dryness under reduced pressure. The crude solid was dissolved in water (40 ml). After filtration of an insoluble material, the filtrate was chromato-graphed on a reverse phase column (packing of PrepPAK/Cl~, 100 ml) using water and 5~, 10~, 20% and 30% aqueous CH30H as eluant.
The fractions containing the desired compound were combined, evaporated and lyophilized to ~ive 21 mg (3.8%) of the title compound (I-lH) (E) as a pale yellow powder. Mp. ~180C (dec.).

IR : ~ cm 1 3340(br), 1760, 1670, 1500.
max UV ~Phosphate buffer (pH 7) nm (El% ) 222 (362), 285 (452)-max 1 cm N~lR : ~D2O NaHCO3 3.68 (2H, br, 2-H), 4.15 (3H, s, OCH3), 5.33 ppm(1~, d, J=4.5, 6-H), 5.46 (2~, d, J=7~
CH=CH-CH2), 5.90 (1~, d, J=4.5~ 7~~)l 6.17 (1~, d-t, J=16 ~ 7r 3-CH-C~), 7.02 (lH~ d, J=16, 3-CH trans), 8.43 & 9.09 (each 2H, d, Ja7~ py H).

Example 11 N ~ N ~ CONH ~ ~ C~2N~2 ~2~ ~ S ~ \ OCH3 ~ N ~ CH-CHCHz-N
COO~) ,. .
I-lI *E

7-[2-(S-Amino-1,2 ! 4-thiadiazol-3-yl)-2-methoxyimlnoacetamido]-3-[3-(3-aminometh~lpYrldinio)-l-propen-l-yl]-3-cephem-4-carbo~ylate (I-lI) A mixture of diphenylmethyl 7-[2-(5-amino-1,2,4-thiadiazol-3-yl3-2-methoxyiminoacetamido]-3-(3-iodo-1-propen-1-yl)-3-cephem-4-carboxylate (IX-1) (E, 716 mg, 1 mmole) and 3-(t-butyloxycarbonylaminomethyl)pyridine (516 mg, 2 mmoles) in DMSO
(2 ml) was stirred at ambient temperature for 30 minutes. The mixture was poured into ethyl acetate ~200 ml), and the precipi-tate was collected by filtration, washed well with ethyl acetate and dried. A mixture of the quaternized salt (500 mg), sodium bisulfite (50 mg) in HCOOH (5 ml) was ~tirred at 40-50C for 30 minutes and evaporated to dryness under reduced pressureO The residual solid was dissolved in water (40 ml), and the mixture ' was neutralized with NaHCO3. Insoluble material was filtered off, and the filtrate was chromatographed on a reverse phase column (packing of PrepP~K-500/C18 cartridge, 100 ml), eluting with water, 5%, 10%, 20% and 30~ aqueous CH30H, successively.
The fractions containing the desired compound were combined, evaporated and lyophilized to provide 10 mg (l.e%) of the title compound (I-lI) (E) as a tan powder.

IR : vK3r cm 1 3380(br), 1760, 1650(sh), 1620(sh).
max UV ~Phosphate buffer (pH 7) nm (El% ) 235 (sh, 260)~ 286 max 1 cm (370).

NMR :~ 2 3 3.68 (2H, br, 2-H), 4.16 (3~, s, OCH3), 6.98 PP (lH, d, J=16, 3-CH trans), 8.05 (lH, m, Py-H5), 8.50 (lH, m, Py-H4), 8.80 (2H, m, Py-H2 6) Ex~ple 12 CON~
- N ~,~ * ~3 ~
N' ` S ~ OCH3 ~ ~ CH=CHCH2- ~ -CONH2 ~ *E

7-[2-(5-Amino-1,2,4-thiadia~ol-3-yl)-2-methoxyim noacetamido]-3-[3-(4-carbamoylpyridinio)-1-propen-1-yl]-3=cephem-4-carboxylate ~I-lH) A mixt~re of diphenylmethyl 7-[2-(5-amino~ ,4-thiadiazol-3-yl)-2-methoxyiminoacetamidoJ-3-(3-iodo-1-propen-1-yl)-3-cephem-4-carboxylate (IX-l~ (E isomer, 4.1 g, 5.7 mmoles) and ~sonicotinamide (1.4 g, 11 mmoles) in dry DMSO (6 ml) was 12~ 29 ?

stirred for 2 hours at room temperature while monitoring by TLC
(silica gel plate, CHC13:CH30H = 3:1). The reaction mixture was diluted with ethyl acetate (100 ml) to separate a yellow gum, which was treated with formic acid (40 ml) and sodium bisulfite (390 mg) at 45~C for 30 minutes. The resulting solution was concentrated to dryness. The residue was dissolved in H20 (100 ml) and insolubles were removed by filtration. The combined filtrate and water wash was applied to the top of a column containing reverse phase packing (PrepPAK-500/C18j 120 ml). The column was eluted with H20. The eluate was collected in 300 ml fractions and monitored by uv (254 nm) and HPLC (Lichrosorb RP-18, 4 x 300 mm, 0.01 M ammonium phosphate buffer, pH 7.2 containing 20% CH30H). Fraction Nos. 4 and 5 were combined and concentrated to a small volume. Lyophilization gave 250 mg (8.1~) of the title compound I-lH, melting at >180~C (dec.).

The spectra indicated that tbe product was identical to that obtained in Example 10.

Preparation of the hydrochloride - To a suspension of Compound I-lH (98 mg, 0.18 mmole) in CH30H (1 ml~ was added 10%
HCl (0.1 ml), and the mixture was stirred for 5 minutes. To the resulting yellow solution was added acetone (100 ml) to give a precipitate, which was collected by filtration, washed with acetone (2 x 10 ml) and dried in vacuo to give the hydrochloride salt of I-lH as a colorless powder. Yield 88 mg (79%). Mp.
>l90~C (dec.).

~5 IR : v cm 3300, 1770f 1680, 1620.
max UV ~Phosphate buffer (pH 7) nm (E 1% ) 227 (385), 286 max 1 cm (374).

N~IR : ~D20 2.32 (lH, s, acetone-H), 3.79 (2H, br-s, 2-H), 4.17 ppm 3H, s, OCH3), 5.34 (lH, d, J=4.5, 6-H), 5.49 (2H, d, J=7, CH=CH-CH2~, 5.93 (lH, d, J=4.5, 7-H), 6.28 (lH, d-t, J=16 & 7, 3-CH=CH), 7.15 (lH, d, J=16, 3-CH), 8.43 & 9.1 (each 2H, d, J=7, Py-H).

. S
I ~ ~ ~ CONH

H2N ~ S~' OCH3 ~ ~ CH-CHCH

I-lJ *E

7-12-(5-Amino--1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-(2-methylthiazolio)-1-propen-1-yl]-3-cephem-4-carboxylate (I-lJ) To a mixture of diphenylmethyl 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-(3-iodo-1-propen-1-yl)-3-cephem-4-carboxylate ~IX-l) (E, 714 mg, 1 mmole) and 2-met~ylthiazole EPrePared according to the procedure of R. P.
Kurkjy, E. V. Brown, J. Am. Chem. Soc., 74, 5778 (1952)] (198 mg, 2 mmoles) in dry CH2C12 (10 ml) was added AgBF4 (90~ pure, 217 mg, 1 mmole) at 20C. The mixture was stirred for 30 minutes at room temperature and filtered. The precipitate was extracted with 10% CH30H-CHC13 (3 x 20 ml). The combined extracts were washed with brine (2 x 5 ml), dried over MgS04 and evaporated to dryness to give a yellow residue, which was triturated with isopropyl e~her and riltered to yield 350 mg of the quaternized ~L27~ 9 product. A mixture of this solid, sodium bisulfite (35 mg) and formic acid (3.5 ml) was stirred at 40C for 30 minutes. The mixture was concentrated to remove the formic acid, and the residue was diluted with H2O (40 ml). Some insolubles were removed by filtration. The filtrate was placed on a reverse phase column (PrepPAK-500/C18, 100 ml). The column was eluted with H2O (200 ml), 5% aqueous CH30H (400 ml) and 10% aqueous CH30EI (300 ml), successively. The fractions containing the desired product were pooled on the basis of HPLC analysis (Lichrosorb RP-18, 4 x 300 mm, 0.01 M ammonium pbosphate buffer pH 7.2, containing 20% CH30H). The combined solution was concen-trated to a small volume and lyophilized to give 40 mg (7.7%) of the title compound (I-lJ) (E). Mp. >195C (dec.).

IR : v cm 3300, 1760, 1660, 1600.
max UV ~Phosphate buffer (pH 7) nm (El% ) 238 (442), 292 (421)-max 1 cm NMR : ~ 2 6 3.06 (3H, s, thiazole-CH3), 3.74 (2H, br-s, ppm 2-H), 4.19 (3H, s, OCH3), 5.92 (lH, d, J=4.5, 7-H), 6.1 (lH, m, 3-CH=CH), 6.8 (lH, d, J=16, 3-CH trans), 8004 & 8.23 (each lH, d, Ja4~ thiazole-H).

~276~;~9 .

CON~

,~ ~CH3 ~ CH=C~2--I-lL *E

7-[2-(5-Amino-1,2,4-thiadiazol-3-yl)-2~methoxyiminoacetamido]-3-ate ~I-lL) A mixture of diphenyl 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-(3-iodo-1-propen-1-yl)-3-cephem-4-carboxylate (IX-l) (E isomer,l.07 g, 1.5 mmole), 4-hydroxy-methylpyridine (818 mg, 7.5 mmole) in CH3CN (4.5 ml) and CH30H (3 ml) was stirred at room temperature under N2 atmosphere for one hour. After removal of ~he solvents by evaporation, the residual oil was triturated with isopropyl ether, collected by filtration, and washed with a mixture of isopropyl ether and methanol (3 : 1, 10 ml) to give 1.28 9 of the quaternized cephem ester as a yellow powder. A solution of the ~uaternized ester (1.25 g) and sodium bisulfite (600 mg) in B5 ~ HCOOH (10 ml) was stirred a~ room temperature under N2 atmosphere for one hour. After the addition of 85 % ~COOH (5 ml), the mixture was stirred under the same conditions for an additional hour. Toluene was added and the reaction mixture was evaporated azeotropically under reduced pre sure. The residue was triturated with acetone to yield 1.17 g of the crude formate of the title compoundO A suspension of this compound tl.l5 g) in water (100 ml) was filtered to remove insolubles, which were washed wi~h water (10 ml x 2)o The filtrate and the washes were combined and subjected to reverse phase column chromatography. The column, which was packed with the packing taken out of a prepPAK-500/C18 cartridge column 1L;276~29 (Waters) 60 ml), was developed with water, 5 % methanol and 10 %
methanol, successively. The fractions containing the desired compound were combined, concentrated under reduced pressure, and precipitated by the addition of acetone to give 100 mg of the title compound (I-lL)as a pale yellow powder. To a suspension o, the powder (90 mg) in methanol (9 ml) was added 1 M HCl in CH30H
(0.5 ml) and the mixture was stirred at room temperature and concentrated in vacuo. To the concentrate was added isopropanol to precipitate 77 mg of the hydrochloride of the title compound.
Pale yellow powder. M. p. >190C(dec.).

KBr IR : vmax cm 1, 1775, 1670, 1635, 1530.

Phosphate buffer (pH7) UV ~max nm (), 230 (22600), 264 (sh, 163~0) MR : ~D20 ppm 3.83 (2H, br. 2-CH), 4.17 (3H, s, OCH3~, 5.06 (2H, s, ~ CH2OH), 5.36 (lH, d, J=4.5 Hz, 6-H), 5.41 (2H, d, J=7 Hz, CH=CH-CH2), 5.94 (lH, d, J=4.5 Hz, 7-H), 6.36 (lH, d-t, J=16 and 7 Hz, CH=CHCH2), 7.13 (lH, d, J=16 Hz, CH=CH-CH2), 8.08 and 8.83 (each 2H, d, J=7 Hz, P~-H).

~L27~g N C ~ CON~ ~ S ~

H2N ~ CC H ~ ~ CH=C~H2--~~ oNH2 I-2H ~E

7-[2-(5-Amino-1,2,4-thiadiazol-3-~1)-2-ethoxYiminoacetamido]-3-(I-2H) To a solution of 200 mg of 7-amino-3-[3-(4-carbamoyl-pyridinio)- l-propen-l-yl~-3-cephem-4-carboxylate hydrochloride (E isomer) in 5 ml of 50 ~ aqueous acetone was added portionwise 190 mg of 2-ethoxyimino-2-(5-amino-1,2,4-thiadiazol-3-yl)acetyl chloride hydrochloride Lprepared according to the procedure described in published Japan patent application (Rokai) 57-24389 (2/8/82)], and the mixture was adjusted ~o pH 6.5-7.0 with 2 N
Na~CO3 (about 1 ml). The reaction mixture was stirred at 10 C
~or an hour, acidi ied to pH 2 with 1 N HCl and evaporated in vacuo. The residue was filtered and the filtrate was chroma-tographed on a column of HP-2~, which was eluted with 500 ml of water and 25 4 aqueous isopropanol, successively. ~ractions containing the desired product were combined and evaporated under reduced pressure. The oily residue was treated wi~h isopropanol (20 ml) to give 263 mg ~93 ~) of the title compound (I 2H). M.
p. 170 C ~dec.~

To a stirred suspension of 225 mg ~0.40 mmole) of ~he above zwitterion in 10 ml of methanol was added 1 ml of 1 N HCl in CH30H and the mixture was stirred at room temperature for 30 minutes. The solution was filtered and concentrated under * Trade ~ark ~,~

3L27~

reduced pressure. To the residue was added 15 ml of isopropyl alcohol, and the resulting precipitate was collected by filtration and dried in vacuo to give the title compound as its hydrochloride. Yield 146 mg (57 %). M. p. 160 C (dec.).
Estimated purity 65 ~.
KBr IR : vma~ cm 1, 3300, 1780, 1680, 1620.
~V : ~phosphate buffer tPH7) nm (E), 227 max (22300), 288 (22800).

NMR : ~D2 1.44 (3H, t, J=7 Hz, OCH2-CH3), ppm 3.74 (2~, br. s, 2-H) 4.45 (2H, q, J-7 Hz, OCH2-CH3), 5.36 (1~, d, J=4.5 Hz, 6-H), 5.46 (2H, d, J=7 Hz, 3-CH-CH-CH2),5.92 (lH, d, J=4.5 Hz, 7-H), 6.20 (lH, m, 3-C~=CH), 7.04 (lH, d, J=16 Hz, 3-CH=CH), 8.43 (2H, d, J=7 H~, Py-HA), 9.10 (2H, d, J=7 Hz, Py-HB).

Example 16 7-[2-(5-Amino-1,2,4-thiadlazol-3-~)-2-meth~ mlnoacetamido ? -3-[3-(4-carbamoylpyridinio?-1-propen-1-yl]-3-cephem-4-ca_boxylate (I-lH) (E isomer) This Example shows the preparation of Compound I-lH via the last few steps of ~eaction Scheme la or lb, wherein the intermediate benzhydryl 7-[2-(5-amino-1,2,4-thiadia201-3-yl)-2-methoxyiminoaceta~ido]-3-[3-(4-carbamoylpyridinio)-1-propen-1-yl]-3-cephem-4-carboxylate formate (XXVII-lH) is isola~ed.

z9 A. Benzhydryl _ 12-~5-Amlno-1~2,4-thiadiazol-3-yl)-2-methoxy-imlnoacetamido]-3-[3-(4-ca~ ~ o)~ ropen-1-~1]-3-ce~hem-4-carboxylate Formate (E isomer) ~XXVII-lH) A solution of XI~-lH ( ~ = ~ , E isomer) (34 g, 75%
pure) in a mixtuee of acetone and C~30H tl/l, 200 ml) was placed on a column of Amberlite*IRA-410 (formate form 340 ml). The column was eluted with the same solven~ system. The first fraction (1 L) was evaporated to about 100 ml of the volume and the brown residue was triturated wi~h isopropyl ether (400 ml).
The resulting powder was collected by filtration and dried under vacuum to afford 29 9 (75~ pure by HPLC) of the title compound XXVII-lH (E isomer) as a brown powder melting at >150C 5dec.).

IR : vKBr cm 1 3300, 1780~ 1680, 1630, 16GO.
max UV ~EtOH nm (El% ) 282 (186).
max 1 cm I~IR ~acetone-d6/CH30H~d4(1/1) 4.0 (3H, s, OCH3), 5.26r (lH~ d~
ppm J=4.5 Hz, 6-H), 5.43 (2H, d, J=7 Hz, CH2N ), 5.99 (lH, d, J=4.5 Hz, 7-~), 6.5 (lH, m, 3-CH=C~), 6.92 (lH, s, CHPh2~, 7.1 (lH, d, J=16 Hz, 3-CH), 7.35 ~lOH, m, Ph-H), 8.36 (lH, s, HCOO), 8.46 & 9.12 (2H each, d, J=8 Hz, Py-H).

B. 7-[2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacet-amido]-3-[3-(4-carbamoyl-1-pyridinio?-1-pro~en-1-yl]-3-cephem-4-carboxYlate (I-lH) (E lsomer) A mixture of XXVII-lH (E isomer) from Step A (29 g, 75%
pure) and 85% formic acid (290 ml1 was stirred for 2 hours at room temperature. Evaporation of the mixture gave a brown oil which was triturated with acetone (500 ml). The powder was collected by filtration, washed with acetone (2 x 100 ml) and * Trade Mark ~27~i~3Z~
. ., ~

dried in vacuo to give 24 g (50~ pure by HPLC) of the crude title compound. The brown solid was treated twice with 2 N HCl ~l L
and 0.5 L). The aqueous extracts were combined and placed on a column packed with Diaion*~P-20 ll.5 L). The column was washed with water (8 L) and eluted with 30~ CH30H (5 L). The fraction containing the desired produc~ was evaporated to about 30 ml.
The concentrate was treated with acetone (200 ml) to give a precipitate, which was collected by filtration and dried in vacuo to give lO.l 9 (85~ pure) of the title compound (zwitterion form) as a yellow powder. To a suspension of this product in CH30H
(lOO ml) was added N HCl in CH30H (55 ml) at room temperature and the mixture was stirred for 30 minutes. The resulting clear solution was filtered to remove insolubles, concentrated to about 50 ml of the volume and precipitated with isopropanol (200 ml).
The resulting powder was collected, washed with isopropanol (50 ml) and dried _ vacuo to give 1005 g (85% pure) of the title compound I-lH (E isomer) (HCl salt), melting at >l80C (dec.~.
Pale yellow powder.

Example 17 7-1~2-(5-Amino-l,2,4-thiadiazol-3-yl)~2-methoxyiminoaceta~ido]-3-[3-(4-carbamoylpy~idinlo)-l-propen-l~yl]-3-cephem-4-carboxylate (I-lH) (E isomer) This example shows the preparation of Compound I-lH via the last few steps of Reaction Scheme la or lb, wherein inter-mediate XXVII-lH (the formate) is not isolated.

A solution of IX-l (E isomer) (27.6 9, 38.5 mmole) and isonicotinamide (22.8 9, 187 mmole) in a mixture of CB3CN (120 ml) and C~30H (lOQ ml) was stirred at room temperature for l hour under a nitrogen atmosphere. After evaporation of the organic solvents, the oily residue was triturated with isopropyl ether to give 50.5 g of a mixture of the quaternized salt and isonicotin-amide. A solution of the mixture (50.3 g) and sodium bisulfite ~l6 g) in 85% HCOOH (160 ml) was stirred at room temperature for 40 minutes and subsequently at 40C for l hsur under N2. The * Trade Mark T ' , ~

~ ;Z!76i~2~

mixture was evaporated in vacuo. The residual oil was mixed wi~h toluene (50 ml), evaporated azeotropically and triturated with acetone (400 ml~ to give 27.8 g of the crude title compound.
This material was treated twice with 2 N HCl (1 L and 0.5 L).
The acid extrac~s were combined and placed on a column of HP-20 resin (1.5 L). The column was eluted with water (9L) and 30%
methanol (lOL). The fractions containing the desired compound were combined and concentrated to give a yellow oil, which was triturated with acetone (300 ml) to yield 9.35 g of the zwitterion form of the title compound.

To a suspension of the product (9.3 g) in CH30H (180 ml) was added 1 N HC1 in CH30H (55 ml) to obtain a clear solution. The solution was concentrated to about 100 ml and diluted with isopropanol to precipitate 9.50 g (pueity 754) of the title compound I-lH (E isomer) as its hydrochlorid.e. Pale yellow amorphous powder. M.p. >195C (dec.).

.
Example 18 7-[2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxy_minoacetamido]-3-[3-(4-carbamoylpyridlnio)-1-propen-1-yl]-3-cephem-4-carboxylate (I-lH) (E isomer?

Tkis example shows the preparation of Compound I-lH via the last step (7-N-acylation) of Reaction Scheme lco To an ice-chilled suspension of the 7-amino-cephem hydrochloride XXII-H (E isomer) ~S.O g, 12.6 mmole) in 50~
aqueous acetone (100 ml) was added sodium bicarbonate in small portions. The pH of the mixture was monitored by a pH meter throughout the reaction. To the cold neutralized solution (pH
about 7) was added 2-~5-amino 1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetyl chloride hydrochloride (4.02 g, 15.6 mmole) in small portions over a period of an hour, and the pH of the reaction mixture was maintained in the range of 6.8-7.5 by occasional additions of sodium bicarbonate. The reaction was also monitored by tlc. After all of Compound XXII-H had been . . ~ .

~76~Z~
.

consumed, the mixture was acidified to pH 3 by the addition of 2 N hydrochloric acid. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was diluted with acetone ( 4bo ml) to separate the precipitate, which was collected by filtration to afford 9.5g g of the crude title compound as a light yellow powder. Estimated purity 40% by HPLC.
A suspension of the crude product (9.S g) in 2 N hydrochloric acid (150 ml) was filtered, and the filtrate was adsorbed on a column of HP-20 resin (S00 ml~. After washing with water (1.5 L), the column was eluted with 25% aqueous isopropyl alcohol and the eluate was collected in 100-ml fractions. The desired fractions were pooled, acidified with 2 N hydrochloric acid (10 ml) and concentrated. The residual oil was triturated with isopropyl alcohol (200 ml), and the precipitate was collected by filtration. After drying over phosphorus pentoxide, 5.18 g of the title compound I-lH (E isomer) hydrochloride was obtained as a yellow amorphous powder. M.p. >19ODC (dec.). Estimated purity 75%.

Example 19 Purification and crystallization of Compound I-lH (E isomer) Compound I-lH hydrochloride obtained in Example 16 was a pale yellow amorphous powder of 85~ purity.

Procedure 1 Six grams of the 854 purity hydrochloride was dissolved in 20 ml of H2O and filtered through a celite pad. The amber-colored filtrate ~pH 2~ was passed through a reverse phase column (the packing of prepPAK-500/C18 cartridge, Waters; 120 ml), which was eluted with water. The eluate was collected in 120-ml fractions with monitoring by ~PLC*. Fraction No. 3 through fraction No. 5 were combined and concentrated to about 10 ml, and precipitated by acetone (100 ml) to give 3.3 y of the zwitterion form of I-lH (pale yellow amorphous powder; estimated purity 95~).

~2~6~2g To a suspension of the 95~ purity powder (3.2 9) in CH30H (32 ml) was added N HCl in CH30H (18 ml), and the mixture was stirred at roo~ temperature until a clear solution was obtained. The solution was filtered and the filtrate was concentrated to about 10 ml. To the concentrate was added isopropanol (100 ml) to separate a pale yellow precipitate, which was collected by flltration, washed with isopropanol (5 ml) and dried to yield 2.6 g of ~he HCl salt (amorphous powder; estimated purity 95~).

A solution of the 95~ purity hydro~hloride (1 g) in water (4 ml) was adjusted to pH 6.5 with NaHCO3 (200 mg) and stirred for 30 minutes. The crystals which separated during stirrins were collected by filtration, washed with water (2 x 5 ml)- and dried in vacuo to give 710 mg of I-lH (zwitterion form) __ as pale yellow prisms. M.p. >185C (dec.)~ Microanalysis showed it to be the trihydrate.

IR : vKBr cm 1 1780, 1695, 1660, 1630, 1610.
max UV ~phOsphate buffer (pH7) nm (E) 227 (22000), 290 (23000)-max N~ DMSo-d6+D2O
ppm 3.45 (2H, br, s, 2-H), 3.9 (3H, s, OCH3), 4.99 (lH~ d, J=4.5 Hz, 6-H), 5.16 (2H, d, J=7 Hz, CH2N ), 5.61 (lH, d, J=4.5 Hz, 7-H), 5.8 (lH, d-t, J-16 h 7 Hz, 3-CH=CH), 6.93 (lH, d, J=16 Hz, 3-CH), 8.18 & 8.89 ~each 2H, d, J=7 Hz, Py-H).

Anal. Calc d for C21H20N8O652 2 ~ ; , N, 18.72; S, 10.71.
Found: C, 42.41; H, 4.35;
N, 18.86; S, ll.G0.

* Column, Lichrosorb RP-18, 4x300 mm: Mobile phase, 0.01 M

phosphate buffer (pH 7.2)~CH30H = 85/15: Detection, uv (254 nm);

Procedure 2 Once crystalline I-lH had been obtained from Procedure 1, it was possible to obtain the crystalline zwitterion form of I-lH directly from the crude I-lH hydrochIoride by seeding with a few crystals of the pure I-lH.

A solution of the 85~ pure hydrochloride (250 mg) in water (1 ml) was treated with charcoal. The solu~ion was adjusted to p~ 6.5 with NaHC03 (60 mg) and decolorized with charcoal. The filtrate was seeded with a few pieces of the crystals obtained from Procedure 1 and stirred overnight at room temperature. The separated crystals were collected by filtration, washed with water (2 x 2 ml) and dried under reduced pressure to give 170 mg (80% recovery) of pale yellow prisms of I-lH (zwitterion form), melting at >185C (dec.), which was identical with that obtained by Procedure 1, (as shown by IR, W, NMR).

The crystalline zwitterion form of Compound I-lH was slightly soluble in water (6 m~/ml in saline at 23C).

6~

Example.Z0 N CONH ~ 5 H2~ 1 ~ OCH3 ~ N ~ CH-C~-CHz-I-lR ~E
7-[2-(5-Amino-1,2,4- hiadiazol=3-yl)-2-methoxyiminoacetamido]-3-[3-(3-hydroxymethylpyrldinio)-1-1~r~ enyl]-3-cephem-4-carboxylate (I-lK) (E isomer) A. DiphenylmethYl 7-[2-(5-Am no=1,2,4-thiadiazol-3-yl)-2-ethoxyiminoacetamido]-3~[3-~3-hydroY.ymethylpyridinio)-l-prope~yl~-3-cephem-4-carboxylate iodide (E-isomer) (XII-lK) To a solution of IX-l (E-isomer, 1.79 g, 2.5 mmoles) in 2.5 ml of CH30~ and 7.5 ml of CH3CN was added 3-hydroxymethyl-pyridine (545 mg, 5 mmoles), and the mixture was stirred at room te~.perature for 3 hours. The reaction mixture was poured into ethyl acetate (100 ml) with vigorous stirringO The resulting precipitate was collected by filtration, washed with a small volume of ethyl acetate and dried to give 2.06 g (100%) of the title compound XII~lK as a tan powder. Mp. 170-180C (dec.).

IR : vmax (KBr) in cm 1 1780, 1725, 1675, 1615, 1530, 1385, 1225, 1040, 750, 700.

UV : ~ax (CzH50H) in nm ~E ) Z90 ~196).

7~

NMR : ~ (DMSO + D20) in ppm 3.7 (2H, br.s, 2-H), 3.91 (3H, s, OCH3), 4.70 (2H, s, Py C~2-OH), 5.28 ~2H, m, CH2 N+), 5.23 (lH, d, - J-5Hz, 6-H), 5.90 (lH, d, J=5Hz, 7-H), 6.34 (lH, m, 3-CH=C~), 6.86 (lH, d, J=16H~, 3-CH)l 6.89 (1~, s, CHPh2), 7.35 (lOH, m, Ph-H), 7.9-8.9 (4H~ m, Py-H).

B. 7-[2-(5 Amino-1,2,4-thiadiazol-3-yl)-2-methoxyimino-acetamido]-3-[3-(3-hydr~ymethylpyridinio)-1-pr~enyl]-3-cephem-4-carboxylate (I-lK) (E isomer) A mixture of XII-lK ~E isomer, 2.~ g, 2.4 ~noles) and sodium bisulfite (1 g) in 85% ~COOH (10 ml) was stirred for 2 hours at room temperature. The reaction mixture was concentrated to ca. 5 ml under reduced pressure~ The oily residue was poured into acetone (100 ml) with vigorous stirring. The precipitate was collected by filtration, washed with a small amount of acetone and dried to give 1.1 g of a tan powder, which was purified by column chromatography [using the packing o~ a PrepPAK-500/C18 cartridge (Waters)] to give 283 mg (22%) of I-lK
as an amorphous powder. The powder was crystallized from 4N
H2S04 and acetone to give 144 mg of the title compound I-l~ as colorless needles. Mp. 185~188~C (dec.).

IR : vmax (X~r) in cm 1 1775, 1680sh, 1660, 1630, 1225, 1045, B50.

UV : ~ma~ (Phosphate buffer, pH 7) in nm (El~ ) 236.5 (283);

292.5 (330).

~ ,~,.
, .

, ~76gZ~ I

NMR : ~ (D2O) in ppm 3.75 (2~, s, 2-H), 4.18 ~3H, s, OCH3), 4.97 (2H, s, Py-CH2OH), 5.35 (lH, d, J=4Hz, 6-H), 5.43 (2H, d, J=6.5HZ, CH2-N ), 5.92 (1~, d, J=4Hz, 7-H), 6.18 (1~, d-t, J=16Hz, J=6.5Hz, 3-CH=C9-), 6.97 (lH, d, J=16Hz, '-CH), 8.13 (lH, d-d, J=8Hz, J=6Hz, Py-H), 8.60 (lH, d, J=~Hz, Py-H), 8.84 (lH, d, J=6Hz, Py-H), 8.90 (1~, s, Py-H).

Example ~1 ~ CONH ~ ~ ~

H2N S ~ oc~3 CH=CH-~2- ~ 0NHCH3 I-lM *E

7-[2-(5-Amino-1,2l4-thiadiazol-3-yl)-2-(Z)-methoxyimino-acetamido]-3-[3-(4-N-meth~tlcarbamoYlPYridinio)-l-propenyl]-3-cephem-4-carboxylate (I-lM) (E isomer) A mixture of diphenylmethyl 7-[~-(5-amino-1,2,4-thiadiazol 3-yl)-2-methoxyiminoacetamido]-3-(3-iodo-1-propenyl)-3-cephem-4-carboxylate tIX-l) ~E isomer, 450 mg, 0.62 mmole) and 4-N-methylcarbamoylpyridine [prepared according to the procedure of M. Samejima, Yakugaku 2asshi, 80, 1706 (1960)] (215 mg, 1.58 mmoles) in acetonitrile (2 ml) was stirred under nitrosen atmosphere for 5 hours at room temperature. The mixture was evaporated under reduced pressuxe and the residue was triturated with ether to give 530 mg o the guaternary salt. A mixture of the solid and sodium bisulfite (150 mg) in 85~ formic acid (2 ml) was stirred for 4 hours and then heated at 40C for 30 minutes.
The mixture was evaporated under reduced pressure. The residue was triturated with acetone and the crude product was collected by filtration. The crude product was chromatographed on a column of HP-20 (1.5 x 18 cm) and the column was eluted with water and 30% aqueous methanol. The methanolic eluate was evaporated under ~2~Z9 ~o redueed pressure and ~he residue was freeze-dried to give 140 mg of an amorphous powder, which was further purified by HPLC
(Column: Lichrosorb RP-18, Solvent: 15~ CH3OH) and the eluate of HPLC was freeze-dried ~o give 60 mg (18%) of the title product I-lM. Mp. 180-183C (dec.). Estimated purity: 80~.

IR : ~max (KBr) in cm 1 17~0, 1660, 1600.

UV : ~max (Phosphate buffer, pH 7) in nm (~) 230 (22100), 286 (22100).

NMR : ~ (D2O) in ppm 3.08 (3H, s, CONHCH3), 3.72 (2H, s, 2-H), 4.16 (3H, s, OCH3), 5.35 (lH, d, J=4.5Hz, 6-~), 5.95 (lH, d, J=4.5Hz, 7-H), 7.00 ~lH, d, J=16Hz, 3-CH), 8.35 (2~, d, J-6Hz, pyridine~H), 9.05 (2H, d, J=16Hz, pyridine-H).

Example 22 N ~ N CONH ~ S ~

H2N S ~ OC~3 ~ ~ CH-CH-CH2- ~ OOH

I-lN *E/Z = 7/l 7-12-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-~3-(4-carboxypyridini_)-1-propenyl]-3-cephem-4-carboxylate (I-lN) To a stirred suspension of isonicotinic acid (340 mg, 2.8 ~moles) in dry DMF (3.5 ml) was added N,O-bis(trimethyl-silyl)acetamide (0.7 ml, 2.8 mmoles) under nitrogen atmosphere.
To the resulting clear solution was added diphenylmethyl t-[2-(5-amino-1,2,4-thiadiazol-3-yl3-2-methoxyiminoacetamido]-3-~3-iodo-l-propenyl)-3-cephem-4-carboxylate (IX-l) (E isomer, 720 mg, l mmole) in one portion, and the red solution was stirred for 1.5 hours at room temperature. The reaction mixture was added ~ , .
,.. .

~` ~
~27~9 dropwise to a stirred saturated sodium chloride solution (50 ml) containing sodium thiosulfate (150 mg). The yellow precipitate was collected by filtration, washed with water, and dried to obtain 722 mg of a pale yellow powder. The powder (700 mg) and sodium bisulfite (70 mg~ were dissolved in 85~ formic acid (S
ml), and the solution was allowed to stand at room temperature for 1.5 hours. The mixture was suspended in toluene (50 ml) and concentrated. T~e residue was triturated with acetone (70 ml), and the precipitate was isolated by filtration to afford 421 mg of a yellow powder. This crude powder (400 mg) was suspended in water (2 ml) and to the suspension was added sodium bicarbonate.
The resulting dark solution was adsorbed on a column of the packing (50 ml) of a PrepPAK/C18 cartridge (Water's Sys~em 500)r and the column was eluted by water (200 ml). The eluent was fractionated into 10 fractions (20 ml of each), and the desired fractions (Fractions Nos. 4-7) were combined, acidified to pH 3 with 2N hydrochloric acid, and concentrated. The residue was triturated with acetone (30 ml) and the precipitate was collected by filtration to give 201 mg (37~ of the title compound I-lN as a yellow powder. E/Z = 7/1; 80~ pure. Mp. >lB9C (dec.).

XR : v~ax (KBr) in cm 1 1770, 1665, 1600.

UV : ~max (Phosphate buffer, pH 7) in nm () 227 (22500), 290 (22100).

NMR : ~ (D2O ~ NaHCO3) in ppm 3.7 (2H, br.s), 4.15 (3H, s), 5.32 (lH, d, J=4Hz), 5.39 (2H, d, J=6Hz), 6.14 (lH, d-t, J=15.5 and 6Hz), 7.03 (lH, d, J=15.5Hz), 8.31 (2H, d, J=7Hz), 8.94 ~2H, d, J=7Hz).

~27~

Examp_e 23 S

N ~T--C ~ CONH

H2NJ~S~ ~OC~13 ~N ~C~I-CH-CH;2-~
coo~3 )~
I-lO *E
?-[2-(5-~mino-1,2,4-thiadiazol-3-yl)-2-(Z)-methoxyimino-acetamido]-3-[3-(2,3~c~cLopentenopyridinio~-l-propenyl]-3-cephem-4-carboxylate (I-lO) (E isomer) A mixture of diphenylmethyl 7-[2-(5-amino-1,2,4-thiadia~ol-3 yl)-2-methoxyiminoacetamido~-3-(3-iodo~l-propenyl)-3-cephem-4-carboxylate (IX-l) (E isomer, 450 mg, 0.62 mmole) and 2,3-cyclopentenopyridine (217 mg~ 1.83 mmole) in acetonitrile (2 ml) was stirred under nitrogen atmosphere for 4 hours at room temperature. After evaporation under reduced pressure, the mixture was triturated with ether to give 560 mg of the quaternary salt. A mixture of the solid and 85% formic acid (2 ml) was stirred under nitrogen for 3 hours at room temperature and then ~eated at 40C for 30 minutes. The mixture was evaporated under reduced pressure and trituration of the residue afforded 391 mg oX the crude product, which was purified by chromatography on a column of HP-20 (1O5 x 18 cm). The column was eluted with water and 30~ aqueous methanol. Evaporation of the methanolic eluate under reduced pressure, followed by freeze-drying afforded 160 mg of an amorphous powder, which was further purified by HPLC (Column: Lichrosorb, Solvent: 10~
CH30H). The eluate of HPLC was freeze-dried to give 5~ mg (15~) of the title product I-lO. Mp. ~190C (dec.). Estimated purity:
75%.

IR : vmax (KBr) in cm 1 1765, 1670, 1600.

W : ~max (Phosphate buffer, pH 7) in nm (~) 235 (20000), 283 (25000).

~,~

1~76~;ZY) I
~3 MR : ~ (D2O ~ NaHCO3) in ppm 2.2-2.6 (2H, m, -CH2-), 3.1-3.6 (4H, m, -CH2-)~ 3.72 (2H, s, 2-H~, 4.17 (3H, OCH3), 5.33 (lH, d, J=4.5~z, 6-H), 5.90 (lH, d, J=~.5Hz, 7-H), 6.75 (1~, d, J=16Hz, 3-CH), 7.65-8.2 (3H, m, pyridine-H).

~ ple 24 N - CONH ~ ~

2 ~ O ~ ~ C~=C~-CN2--~ OOH

7-[2-(5-Amino-1~2,4-th_ diazol-3-yl)-2-(z)-ethoxyimino-acetamido]-3-[3-(4~carboxY~Yridinio)-l-~roPenYl]-3-cephem-4 carboxylate (I-2N, E isomer) and 7-[2-(5-Amino-1~2,4_thiadiazol-3-yl)-2-(Z)-ethoxyimino-acetamido~-3-[3-(4-carboxypyridinio)-1-~ropenyl]-3-cephem-4-carboxYlate ~I~2N, Z isomer) To a chilled mixture of BSA (1.0 ml, 4.12 mmoles) and isonicotinic acid (506 mg, 4.12 mmoles) was added IX-2 (from Preparation No. 21~ (1.0 g, 1.37 ~moles), and the mixture was stirred under nitrogen at room te~perature for 2 hours. The mixture was poured into 10~ Na2S2O3 (20 ml) to precipitate 1.3 9 of the quaternary salt, which was collected by filtration, washed with water and driedO A mixture of the solid and sodium bisulfite (0.3 g) in formic acid (98%, 5 ml) was heated at 40C
for 1 hour and evaporated under reduced pressure. The residue was triturated with acetone and iltered to give 900 mg of the crude product (E-propenyl isomer:Z-propenyl isomer = 2:1).
Separation of the isomers was carried out by HPLC (Column:

~2~ 91 . 84 Lichrosorb, Solvent: 15% CH30H). The faster moving fractions of HPLC were colle~ted, evaporated under rsduced pressure and ~reeze-dried to give the E-propenyl isomer of I-2N (44 mg, yield 6%). The slower moving fractions gave the Z-propenyl isomer of I-2N (32 mg, yield 4%~ in a similar procedure.

I-2N, ~ isomer Mp.: >2D0C (dec.).

IR : vmax (KBr) in cm 1 1765, 1660, 1620, 1380.

UV : ~max (Water) in nm (E) 228 (22200), 291 (23600).

N~ (D2O) in ppm 1.45 (3H, t, J=6Hz, CH2CH3), 3.72 (2H, s, 2-H), 4.45 (2H, q, CH2CH3), 5.4.0 (lH, d, J=4Hz, 6-H), 5.90 (lH, d, J=4Hz, 7-H), . 7.05 (lH, d~ J=15Hz, 3-CH), 8.30 ~2H, d, J=6Hz, Py-H), 8.95 (2H, d, J=6Hzr Py-H).

I-2N, Z isomer Mp.: >200C (dec.).

I-R : vmax (KBr) in cm 1 1760, 1660(sh), 1620, 1370.

UV : )~max (Phosphate buffer, pH 7) in nm (E) 225 (22400), 275 (sh, 16000).

NMR : ~ (D~O) in ppm 1.45 (3H, t, J=7Hz, CH2CH3), 3.50 (lH, d, J=17Hz, 2-H), 3.75 (lH, d, J-17Hz, 2-H), 5.38 (lH, d, J-4Hz, 6-H), 5.95 (lH, d, J=4Hz, 7 H), 6.62 (lH, d, J=llHz, 3-CH), 8.35 (2H, d, J=6Hz, Py-H), 8.92 (2H, d, J=6Hz, Py-H).

- ~2~ 9 Example 25 S
CONH

H2N ~ S ~ \O ~ N ~ CH-C~-CH2-N ~ CONH2 ~H2-CEI=C~2 COO~
I-3~ *E
7-[2-(Amino-1,2,4-thiadiazol-3-yl)-2-~prol?en-3-yloxyimino)-acetamid ~ io)-l-pr~p~enyl]-3-ce~h_m-4-carboxylate (I-3~ (E isomer) To a solution of 35 mg (0.08 mole) of 7-amino-3-13-(4-carbamoylpyridinio)-l~(E)-propenyl]-3-cephem-4-carboxylate hydrochloride in 2 ml of 50% aqueous acetone was added 52 mg of 2-[5-amino-1,2,4-thiadiaæol-3-yl)]-2-(propen-3-yloxyimino)acetyl chloride ~.ydrochloride (from Preparation No. 25~ and ~he mixture was adjusted ~o pH 6O5-7~0 with 21~ Na2CO3. The mix~ure was stirred at room temperature for 1 hour, acidified to pE3 2 with lN
HCl and concentrated under reduced pressure. The residue was chromatographed on a column of HP-20 resin which was eluted with 300 ml of water and 30~ CH3OH-H2O. Fractions containing the product were combined and evaporated under reduced pressure. The residue, 73 mg, was purified by a column of reverse phase carrier which was taken out of a PrepPAK-500/C18 cartridge (Waters, 30 ml). The column was eluted with water, 5~ CH30EI, 10% CH30H and 20~ CH30H, successively. Fractions containing the product were combined and lyop~ilized to afford 26 mg (62~) of the title product I-3H. Mp. 160C (dec.).

IR : vmax (K~r) in cm 3400, 1765, 1680, 1605, 1400.
V : AmaX (Phosphate buffer, pH 7) in nm (~) 226 (24600), 288 (22800).

~7~

NMR : ~ (D20) in ppm 3.75 (2H, s, 2 H), 5.41 ~lH, d, J=SHz, 6-H), 5.50 (4H9 m, CH2N & CH=CH2), 5.~8 ~lE, d, J=SHz, 7-H), 6.20 (lH/ m, 3-CH=CH), 7.09 (lH, d, J=17~z, 3-CH), 8.50 (2H, d, J=7Hz, Py-H), 9.16 (2H, d, J-7Hz, Py-H).

Example 26 N C ~ CONH ~ ~

~2N 1 ~ ~H C ~C~ ~ N ~ CH--CH-CH2- ~ CONH2 I-4H *E
7-[2-(5-Amino-1~2~4-thiadiazol-3-yl)-2-prO~argylOXyiminO-acetamido~-3-~3-(4-carbamo,ylpyridinio)-1-propenyl]-3-ce~hem-4-carboxylate (I-4H) (E isomer) To a solution of 86 mg (0.19 mmole) of 7-amino-3-[3-(4-carbamoylpyridinio)-1-(E)-propenyl]-3-cephem-4-carboxylate hydrochloride (XXII-H) in 2 ml of 50% aqueous acetone was added 63 mg of 2-propargyloxyimino-2-~5-amino-1,2,4-thiadiazol-3-yl)-acetyl chloride hydrochloride (from Preparation No. 26). The suspension was maintained at pH 6.5-7.0 with 2N Na2C03 and then stirred at rosm temperature for 1 hour. The reaction mixture was acidified to pH 2 with lN HCl and concentrated in vacuo. The residue was diluted with 30 ml of water, neutralized with NaHC03 and filtered. The filtrate was transferred on the top of a column which was packed with reverse phase carrier (30 ml) taken from a PrepPAK-500/Cl8 cartridge (Waters). The column was elu~ed with water, 5~ CH30H, 10% CH30H and 20~ CH30H, successively.
Fractions containing the product were combined and lyophilized to afford 13 mg (12%~ of the title product I-4H. Estimated purity 7096~ ~p~ 160Co IR : vmax (KBr~ in cm l 3400, 2120, 1765~ 1680~ 1610 UV : ~max (Phosphate buffer, pH 7) in nm (~) 229 (24000), 288 (21200).

NMR : ~ (D2O) in ppm 3.78 (2H, s, 2-H), 5.15 (2H, d, J=lHz, -CH -C - CH), 5.40 (lH, d, J=5~z, 6-H), 5.50 (2H, ~, CH-N ), 5.98 (lH, d, J=5Hz, 7-H), 6.20 ~lH, m, 3-CH=CH), 7.05 (1~, d, J=17Hz, 3-CH), 8.50 (2H, d, J=7Hz, Py-H), 9.16 (2H, d, J-7Hz, Py-H).
.

Example 27 S
~ CONH ~
H2N S ~ ~ ~ N ~ CH=CH-CH2-N ~ CONH2 I-5H *E
7-[2-(5-Amino-1,2,4-thiadiazol-3 yl)-2-cyclopentyloxyimino-acetamido]-3-[3-(4-carbamoylp~ridinio)-1-propenyl]-3-cephem-4-carboxylate (I-5H) ~E i~omer) To a stirred solution of 139 mg (0.31 mmole) of 7-amino-3-[3-(4-carbamoylpyridinio)-1-propenyl]-3-cephem-4-carboxylate hydrochloride in 3.5 ml of 50% aqueous acetone in an ice-cooling ~ath was added portionwise 120 mg (0.44 mmole) of 2-(S-amino-1,2,4-thiadiazol-3-yl)-2-cyclopentyloxyiminoacetyl chloride hydrochloride (from Preparation No. 27). The mixture was adjusted to pH 6.5-7.0 with 2N Na2CO3 (0.9 ml) and stirred for 1 hour at 10C. The reaction mixture was acidified to pH 2 with lN
HCl and evaporated under reduc~ed pressure. The residue was chromatographed on a column of HP-20 resin ~20 ml) and was eluted with 300 ml of water and 30~ CH3OH-H2O, successively. Fractions containing the product were combined and concentrated in vacuo.
The residue was treated with 60 ml of acetone to give 111 mg ( R3%) of the title compound I-5H. Mp. 160C (dec.). Estimated purity 70%.

B~ 1276~32 !3 .

IR : vmax (K8r) cm 1 8400, 1770, 1680, 1605, 1530.

UV : ~'ma~ (Phosphate buffer, pH 7) in nm (~) 224 (23300), 286 (24600)~

NMR: ô (DMSO-d6) in ppm 1.70 (8H, br.s, H~ ), 4.68 (lH, br.s, )~ )/ 5O05 (lH, d, J=5Hz~

6 H), 5.30 (2H, m, C~2N ), 5.67 ~
d-d, J=5Hz ~ 7Hz, 7-H), 6.20 (lH, m, 3-CH=CH), 7.08 (lH, d, J=17~z, 3-CH), R.34 (2H, d, J=7Hz, Py-Hl, 9.11 (2H, d, J=7Hz, Py-H), 9.38 (lH, d, J=7Hz, 7-NH).

H2N ~ OC 3 ~ * (~) CO~
I-lP *E
ï-[2-(5-Amino-1,2,4-thiadiazol-3-ylJ-2-methoxyiminoacetamido]-3-[3-(3-carboxymethylpyridinio)-1-propenyl]-3-cephem-4-carboxylate (I-lP) (E isomer) A. Diphenylmethyl 7-[2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamldo~-3-[3-(3-carboxymethylp ridinio)-l-proPenyl]-3-cephem-4-carboxylate (XII lP, iodide, E isomer) To a suspension of 3-carboxymethylpyridine hydro-chloride (0.89 g, 5 mmoles) in 10 ml of CH2C12 was added N,O-bis(trimethylsilyl)acetamide (4.97 ml, 18 mmoles), and the mixture was stirred at room temperature until a clear solution was obtained. To the solution was added IX-l (1.79 g, 2.5 mmoles), and the mixture was allowed to stand at room . ~

1~ 7~ 9 !
8~
temperature. After 3 hours, 3 ml of CH3OH was added to the cooled mixture and the solution was evaporated 1n vacuo to give an oil which was triturated with ethyl acetate to afford 2.28 g of the title compound XII-lP as a tan powder. Mp. 161C (dec.).
R : vmax (XBr) ln cm 1 1780, 1720, 1675, 1630, 1530, 1385, 1225, 1045, 755, 700.

UV ~max (C2HsoH) in nm (El% ) 295 (188).
1 cm NMR : ~ (DMSO + D2O) in ppm 3.70 (2H, br.s, 2-~), 3.90 (5H, s, OCH3 & Py-CH2CO), 5.25 (3H, m, -CH2N & 6-H), 5.92 (lH, d, J=4.5Hz, 7-H), 6.35 (lH, m, 3-CH=CH-), 6.90 (lH, d, J=16~, 3-CH), 6.92 (lH, s, CHPh2), 7.35 (lOH, m, Ph-H), 8.8-9.0 (4H, m, P H) B. 7-[2-(5-Amino-1,2~4-thiadiazol-3-yl)-2 methoxyimino-acetamido]-3-[3-~3-carboxymethylpyridinio)-1 Pro~enyl]-3-cephem-4~carb_xylate (I-lP) (E isomer) A mixture of XII-lP ~iodide) (2.28 g) and sodium bisulfite (1.1 g) in 85% HCOOH (10 ml) was stirred at room temperature ~or 2 hours. The reaction mixture was concentrated to ca. 5 ml under reduced pressure. The oily residue was triturated with acetone (100 ml) to give 1.22 9 of the crude pro~uctt which was purified by column chromatography (HP-20, 420 ml) to afford 533 mg of the title compound I-lP (38%, from IX-l) as a pale yellow amorphous powder. Mp. 165~C (dec.).

IR : vmax (KBr) in cm 1 1770, 1670, 1600, 1530, 1385, 1140, 1040.

Zg 9o UV : ~max (Phosphate buffer, pH 6.2) in nm (El% ) 234 (374), 1 cm 277sh (390)~
290 (402).
MR : ~ (D2O + NaHCO3) in ppm 3.78 (2H, s, 2-H), 3.92 (2H, s, Py-CH2CO), 4.22 (3H, s, OCH3), 5~40 (lH, d, J=4~z, 6-H~, 5.44 (2H, d, J=6.5Hz, -CH2-N ), 5.97 (lH, d, J=4Hz, 7-H), 6.20 (lH, d-t, J=16 ~ 6.5Hz, 3-CH~CH), 7.08 (1~, d, J=16Hz, 3-CH), 8.11 (lH/ d-d, J-8 & 7~z, Py H5), 8.53 (lH, d, J=8Hz, Py-H4), 8~82 (lH, d, J=7Hz, Py-H6), 8.86 (lH, s, Py-H2).

Example 29 N ~ ~ CONH

~2N ~ S ~ H3 ~ ~ H-CH-C~2-N ~ S-CH2C~

I-lQ *E
7.-[2-(5-Amino-1,2,4-thiadiazol-3-Yl)-2-methoxyiminoacetamido]-3-[3-(4-carboxymeth~lthio~yridinio)-1-propenyl~-3-cephem-4-carboxylate (I-lQ) (E isomer) A. Diphenylmethyl 7-~2-(5-Amino-1/2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido~-3-[3-(4-carboxymethylthiopyridinio)-l-propenyl]-3-cephem-4-carboxylate ~XII-lQ, iodide,_E
isomer) To a suspension of 4-carboxymethylthiopyridine (0.88 9, 5 mmoles) in 10 ml of CH2C12 was added N,O-bis(trimethylsilyl)-acetamide (5 ml, 18 mmoles), and the mixture was stirred at room temperature until a clear solution was obtained. To the solution was added IX-l (E isomer 1.79 g, 2.5 mmoles), and the mixture was ~L~7~Z9 allowed to stand at room temperature. After 3 hoursl 3 ml of CH30}~ was added to the cold mixture and the solution was evaporated in vacuo to give oily residue which was triturated with ethyl acetate to give 2.43 g of the title compound XII-lQ
(iodide) as a tan powder. Mp. 155C (dec.).

IR : vmax (KBr) in cm 1 1789, 1720, 1670, 1625, 1525, 1385, 122~, 1115, 1040, 755, 700.

max (C2HsoH) in nm (E ) 312 (299).

NIIR: ~ (DMSO-d6 ~ D2O) in ppm 3.70 (2H, br.s, 2-H), 3.93 (3H, s, OCH3), 5.07 (2H, m, CH2-N ), 5.23 (lH, d, J=5Hz, 6-H), 5.90 (lH, d, J=5Hz, 7-H), 6.29 (lH, m, 3-CH~CH), 6.87 (lH, d, J=16Hz, 3-CH), 6.91 (lH, s, CHPh2), 7.35 (10H, m, Ph-H), 7.88 & 8.58 (each 2~, d, J=6Hz, Py-H).

B. 7-[2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxyimino-acetamido]-3-[3-(4-carboxymethylthiopyridinio)-1-propenyl]-3-cephem-4-carboxylate (I-lQ) (E isomer) A mixture of XII-lQ (iodide, 2.43 g~ and sodium bisulfite (1.1 g) in 85% HCOOH (10 ml) was stirred at room temperature for 2 hours. The reaction mixture was concentrated to ca. 5 ml under reduced pressure. The oily residue was tri~urated with acetone (100 ml)r filtered and dried to give a crude product (1.39 ~), which was purified by column chrohlatography (HP-20, 20 ml) to afford 577 mg of 'che title compound I-lQ (39~ from IX-l) as a pale yellow amorphous powder.
Mp. 188C (dec.).

IR : vll~ax (KBr) in cm 1765, 1670, 1625~ 1530, 1380, 1110, 1035.

UV : AmaX (Phosphate buffer, pH 6.2) in nm (El% ) 234 (459), l cm 310 (678)-MR : ~ (D20 ~ NaHC03) in pp~ 3.79 (2H, br.s, 2-H), 4.10 (2H, s, S-CH2), 4.23 ~3H, s, OCH~), 5.25 (2H, d, J=6.5Hz, CH2-N ), 5.39 (lH, d, J=4.0Hz, 6-H), 5.97 (lH, d, J=4Hz, 7-H), 6.18 (lH, d-t, J=15.5Hz & 6.5Hz, 3-CH=CH), 7.05 (lH, d, J=lS.5Hz, 3-CH), 7.84 & 8.55 (each 2H, d, J=7Hz, Py~

E~le 30 N -- CONH~

H2N1 5~0CH O~N~CH CH--C~2\--N~

*E/Z -- 7/1 7-[2-(5 Amino-1,2,4-thiadiazol-3-Yl)-2-methoxyiminoacetamido]-3-[3~ methYlpyrrolidinio~ propenyl]-3-cephem-4-carboxylate sulfate (I-lA, sulfate) A. ~iphenylmethyl 7-12-(5-Amino-lL2,4-thiadiazol-3 yl)-2-~ethoxyiminoacetamido]-3-[3-(1-methylpyrrolidinio)-1-propenyl]-3-cephem-4-carboxylate (XII-lA, iodide~

To a cold solution of diphenylmethyl 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-(3-iodo-propenyl)-3~cephem-4-carboxylate (IX-l) (from Preparation No. 14 (21.5 g, 30 mmoles) in ethyl acetate (300 ml) was added dropwise a solution of l-methylpyrrolidine (2.55 g, 30 mmoles) in ethyl acetate (30 ml) over a period of 1 hour at -5 to 0C, with stirring. After stirring for an additional lO minutes, the resulting precipitate was collected by filtration and washed with ~z~s~

chloroform (200 ml) to give 23.0 g ~95.8%) of the title compound (IX-lA, iodide), melting at >175C (dec.).

IR : vmax (KBr) in cm 1 3300, 1780, 1730, 1685, 1615.

max (C2H5oH) in nm (E ~ 218 (435), 295 (188~.

B. Diphenylmethyl 7-[2-(5-Amino-1,2,4-thiadiazol-3-~_)-2-methoxyiminoacetamido]-3-[3-(1-methylpyrrolidinio)-1-p~ropenyl]-3-cephem-4-carboxylate_(XII-lA, chloride) The compound (XII-lA, iodide) (23 g, 28.7 mmoles) was dissolved in a mixture of acetone and methanol (1:1, 230 ml~ and applied on an Amberlite IRA-410 (chloride form, 230 mll column which was pretreated with the same mixed solvent. The column was developed with the solvent and the fractions containing the desired compound were combined and concentrated to an oily residue, which was triturated with ethyl acetate (300 ml) to yield 17.9 9 (87.7~) of the ~itle compound (XII-lA, chloride), melting at l90~C Idec.).

I~ : vmax (KBr) in cm 1 3380, 1780, 1680, 1620.
1%
max (C2H5H) in nm (E ) 220 (369), 290 (232).

6~Z~' . 7~ (5-Amino-lr2~4-thiadia~ol~3-y~ S~
acetamido~-3 [3~ methylpyrrolidinio)-1-proPenyl]-3--ce~hem-4-carboxylate su fa~e (I-lA, sulfate) A mixture of the compound (XII-lA, chloride) (17.8 g, 25 mmoles) in 85% formic acid (178 ml) was stirred at room temperature for 2 hours under a nitrogen atmosphere. The mixture was evaporated in vacuo and the oily residue was triturated with acetone to give 9.80 g of crude I-lA. Concentration of the filtrate and the acetone washings yielded additional 2.95 g of crude I-lA. Two crops of the crude material were combined and extracted with 2N ~Cl (1 L and O.S L). The combined extracts were adsorbed on a Diaion HP-20 resin (1.5 L column), which was eluted with water and 30% aqueous methanol. The desired fractions were collected and evaporated ln vacuo to an oily residue, which was tri~urated with isopropanol (200 ml) and acetone (200 ml), successively, to yield 7.09 g of a light yellow powder. This material (6.80 g) was dissolved in water (20 ml) and then subjected to column chromatography over the packing of PrepPAK-500~C18 cartridge (90 ml), using water and 10~ aqueous methanol as an eluent. The eluate was collected in 20-ml fractions with monitoring by HPLC. [Column, Nucleocil SSC-ODS-262~ 8 x 100 mm; Mobile phase, O.OlM phosphate buffer (pH
7.2)/CH30H = 90:10; Detection, UV (254 nm)]. Fraction No. 4 through Fraction No. 10 were combined, evaporated under reduced pressure and lyophilized to give 2. 28 9 of a yellow powder (E/Z =
7/1, 70% pure) [Crop 1~. Fraction No. 11 through Fraction No. 85 were worked up in the same manner as described above to give 3.27 g of yellow powder (E/Z = 5/lr 70% pure) [Crop 2]. A portion of rop 1 (1.0 g~ was purified by rechromatography on the packing of PrepPAK-500/C18 cartridge (90 ml). The column was eluted with water and S~ aqueous methanol, successively. The eluate containing the desired compound was concentrated and lyophilized to yield 638 mg (E/Z = 7/1, 80~ pure) of yellow powder. Another portion of Crop 1 tl.l4 g) was worked up the same way to give 880 mg (E/Z = 7/1, 80% pure) of yellow powder. The two purified samples were combined and a portion (1.4S g) dissolved in lN
sulfuric acid (S ml). The solution was diluted with acetone (315 ~Z7~i~2~
g5 ml), with stirring. The creamy precipitate was collected by filtration to obtain 1.48 g of the title compound (I-lA, sulfate) (E/Z = 7/1, 80~ pure), melting at >185C (dec.).

IR : vmax (KBr) in cm 1 3380, 3000, 1765, 1675, 1630, 1535, 139~, lllS.

UV : ~max (Phospha~e buffer, pH 7) in nm (~) 236 (19900), 291.5 (22500).
~ H
NMR : o (D20 + NaHC03) in ppm 2.36 (4H, br., N ~ ), 3.15 (3H, s, CH3+N ~ ), 3.62 (SH, br., 2-H and N ~ ), 3. 83 (lH, b~., 2-H), 4.13 (2H, d, J=8Hz, CH2N ), 4.22 (3H, s, OCH3), 5. 39 (lH, d, J=4.5Hz, 6-H), 5.96 (lH, d, J=4.5Hz, 7-H), 6.00 (lH, m, 3-CH=CH), 6.67 (1/8H, d, J=lOHz, 3-CH, cis), 7.04 (7/8H, d, J=16Hz, 3-CH, trans).

9~9 Example 31 ~ CON~ ~ ~

H2N S ~ \ OC~ o ~ ~ C~=CH-CH2-N(CH3)3 CO
I-lD *E/Z = 10/1 7-[2-(5-Amino-1~2,4-thiadiazol-3-yl)-2-methoxyiminoacetamidoi-3-[3 trimethylammonio-1-propen~l]-3-cephem-4-carboxylate ~I-lD) A. DiPhenylmethyl 7-[2-(S amino-1,2,4-thiadiazol-3-yl)-2-metboxyiminoacetamido]-3-(3-t~ o-1-propenyl)-3-cephem-4-carboxylate (XII-lD, iodide) To a solution of 13.0 g (19 mmoles) of diph~nylmethyl 7-[2-~5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-~3-iodopropenyl)-3-cephem-4-carboxylate (IX-l, from Preparation No. 10) in 38 ml of dry ethyl acetate was added 1.75 ml (19.1 - mmoles) of l.lN trimethylamine in ethyl acetate at -5C, and the mixture was stirred for 1 hour at -5C. The resulting precipitate was filtered off, washed well with CHC13 and dried to give 12.5 g (88%) of the title compound (XII-lD) as the iodide.

IR : vmax (KBr) in cm 1 3300, 1765, 1720, 1665.

~ax (C2~50H) in nm (~) 300 (18400) B. ~ ylme~yl 7-[2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-_inoacetamido]-3-(3-trimeth~ammonio-1-propeny~-3-cephem-4-carboxylate (XII-lD, chloride) The iodide (XII-lD, 12.5 g) was dissolved in 60 ml of C~30H-acetone (1:1~ and passed through a column of ion-exchange resin [IRA-410 (Cl ), 125 ml]. The colum~ was eluted with 300 ml of CH30H-acetone (1:1), and the eluate was evaporated in vacuo and triturated with 300 ml of isopropyl-ether to afford 10.4 9 (91~) of the quaternary salt (XII-lD, chloride).

6~29 i IR : vmax (KBr) in cm 1 8300, 1765, 1710, 1665.

UV : Amax (C2H5OH) in nm (e) 298 (15100).

C. 7-[2-(5-Amino-1,2,_-thiadiazol-3-yl)-2-methoxyimino-acetamido]-3-~3-trimethylammonio-1-propenyl]-3-cephem-4-carboxylate (I-lD, sulfate, E isomer) A solution of 10.4 g (16.0 mmoles) of XII-lD (chloride) in 20.8 ml of 85% formic acid was allowed to stand for 3 hours at room temperature and concentrated in vacuo. The residue was treated with 210 ml of isopropanol and the precipitate was filtered offO The solid (10.1 g) was triturated with 210 ml of water and neutralized with sodium bicarbonate. The suspension was filtered off and ~he filtrate was chromatographed on a column of HP-20 (300 ml) which was eluted with water (1000 ml), 10~
CH30H (200 ml) and 30~ CH30H (150 ml), successively. Fractions containing the desired product were combined and concentrated under reduced pressure. The residue was purified by reverse phase chromatography. The column was packed with a packing obtained from a PrepPAX-500/C18 cartridge (Waters, 200 ml).
Elution with water (600 ml) and 30% CH30H (200 ml), successively, followed by concentration of fractions containing the desired product gave 2.52 g (18%) of the title compound. A solution of the zwitterionic product (1.5 g) in lN H2SO4 (5 ml) was added portionwise to 300 ml of acetone and the resulting precipitate was filtered and dried. Yield o I-lD sulfate was 1.42 5 (80~).
The ratio of E/Z was approximately 10/1 based on HPLC.

IR : vmax (KBr) in cm 1 3380, 1765, 1665.
V : ~max (Phosphate buffer, pH 7) in nm (E) 237 (19500), 293 (22400).

6~

NMR : ~ ~D2O) in ppm 3.25 (9H, s~ N+-CH3), 3.94 (2H, s, 2-H), 4.14 (2H, d, J=7Hz, CH2N~), 4.23 (3H, s, O-CH3), 5.42 ~lH, d, J=4.5Hz, 6-H), 6.00 (lH, d, J=4.5Hz, 7-H), 6.23 (lH, d-t~ J=7 & 16Hz, 3-CH=CH), 7.23 (lH, d, J=16Hz, 3-CH).

NC----CON~I~

H2N~3 ~ ~ `CH--C~_CH2_N~CONH2 cod9 ~ E
7-[2-(S-Amino-1,2,~-thiadia~g~ 3~ ~L ~acLe~8~amido]-3-~3-(4-carbamoylpyridinio)-1-propenyl]-3-cephem-4-carboxylate (I-lH, E isomer) To a mixture of 7-amino-3-[3-(4-carbamoylpyridinio)-l-(E)-propenyl]-3-cephem-4-carboxylic acid hydrochloride (XXII-~, 397 mg, 1 mmole) and NaHC03 (168 mg, 2 mmoles) in aqueous DMF
(water, 3.5 ml and DMF, 7.5 ml) was added benzotriazol-1-yl~2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetate (479 mg, 1.5 mmoles) (from Preparation No. 28). The mixture was stirred at room temperature for 3.5 hours. The reaction mixture was adjusted to pH 3-9 with 3N HCl and diluted with ?00 ml of acetone to give a precipitate, which was collected by filtration. The crude product was dissolved in a small volume of aqueous THF and the solution was adjusted to pH 6.8 with NaHCO3, treated with decolorizing carbon, concentrated to ca. 1 ml and seeded with a few pieces of crystalline I-lH. After stirring overnight, the crystalline precipitate was collected by filtration to afford the title compound I-lH (zwitterion form). Yield 83 mg (16%). Mp.
>185C (dec.). Physico-chemical data of this product were identical to those of the compound in Example 10.

.

6~29 Preparation No. 1 Diphenylmethyl-7-~2-(5-Amlno-l~2~4-thiadiazol-3-~l)-2 methoxyiminoacetamldo]-3-chloromethyl-3-cephem-4-carboxylate (IV-l) To a ~tirred suspension of 2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetic acid (III-l) (2.1 g, 10 mmole) in dry CH2C12 (50 ml) was added PC15 (2.09 9, 10 mmole) at -30C, and the mixture was stirred for 20 minutes at -15 to -20 C. To the above acid chloride solution was added a solution of diphenyl-methyl 7-amino-3-chloromethyl-3-cephem-4-carboxylate hydrochloride (II) (4.5 g, 10 mmole~ in C~2C12 (50 ml) containing N,O-bis-(trime~hylsilyl3acetamide (10 g, 50 mmole) at -30 DC.
After stirring at -10 C for 1 hour, the mixture was c,oncentrated to,remove the CH2Cl~ and diluted with ethyl acetate (200 ml).
The mixture was washed with 10 ~ aqueous NaHC03 (2 x 40 ml), H20 (2 x 20 ml) and brine (10 ml), successively, and dried over MgS04. The solvent waC evaporated ~n vacuo and the resulting oily residue tlO g) was dissolved in CHC13 (20 ml) and chromato-graphed on a silica gel (Wak~ gel C-200, 100 g containing 10 ml of 1/1.5 M pH 7 phosphate buffer) using 1 - 3 ~ CH30H-CHC13.
Fractions containing the title compound were evapora~ed to give 5.7 g (95 %) of IV-l as a yellow amorphous powder. M. p. >140 DC
(dec.).

IR : vKBrcm 1 3300, 1780, 1720, 1680, 1620.
max V ~EtHnm () 245 (1800), 280 ~9900).
max NMR : ~DMSO d6 3.S3 (2~, AB~, 2~ 3.94 (3H, s, OCH3), 4.42 ppm (2H, s, 3 CH2~, 5.22 (1~, d, J~4.5, 6-H), 5.32 (lH, d-d, J=4.5 ~ 6, 7-H), 6.93 (lH, s, CHPh2), 7.36 (lOH, m, Ph-H), 8.1 (2H, br~s, NH2), 9.58 ~lH, d, J-6, 7-NH).

* Trade Mark Preparation No. 2 Diphenylmethyl 7-[2-(5-Amlno-1,2~4-thiadiazol-~-yl)-2-methoxyiminoacetamido]-3-iodomethyl-3-cephem-4 carboxylate (V-l) A mixture of IV-l from Preparation No. 1 (5.7 g, 9.5 mmole) and NaI (4.3 g 29 mmole) in dry acetone (50 ml) was stirred for 5 minutes at room temperature. The mixture was concentrated under reduced pressure and the resulting oil was shaken with a mixture of ethyl acetate (100 ml) and H20 (10 ml).
The organic layer was separated and washed with 10 % w/v sodium thiosulfate and brine, successively. After drying, the ethyl acetate was removed in vacuo to give 6.1 9 (93 %) of the title compound (V-l) as a yellow amorphous powder melting at > 120 C
(dec.).

IR : vK rcm 1 8300, 1780, 1725, 1680, 1620.
max UV : ~E Hnm (~) 245 (17000), 282 (12000).
max NMR : ~DMSO d6 3.72 (2H, ABq, 2-H), 3.94 ~3H, s, OCH3~, 4.23 ppm (2H, s, 3-CH2), 5.21 (1~, d, J=4.5, 6-H), 5.89 (lH, d-d, J=4.5 & 6, 7-H), 6.94 (lH, s, CHPh2), 7.35 (lOH, m, Ph-H), 8.12 (2H, br-s, NH2), 9.65 (lH, d, J=6, 7-NH).

Preparation No. 3 Diphenylmethyl 7-[2-(5-Amino-1,2,4-thiadiazol-~y~-2-methoxyiminoacetamido]-3~triphenylphosphoniomethyl-3-cephem-4-ca_boxylate iodide (VI-lJ

A mixture of Y-l from Preparation No. 2 (690 mg, 1 mmole) and triphenylphosphine (786 mg, 3 mmole) in ethyl acetate (20 ml) was stirred overnight at room temperature. The solid which separated was collected, washed with ethyl acetate (2 x 10 10 1 ~76q3~9 ml) and dried to give 950 mg (100 ~) of the phosphonium iodide VI-1. M. p. 186 C (dec.).

IR : vKBrcm 1 3300, 1780, 1710, 1680, 1610.
max UV : ~E Hnm (e) 268 (15000~, 275 (13000), 300 (7300).
max NMR : ~DMSO d6 3.52 (2H, br-s, 2H), 3.94 (3H, s, OCH3), 5.34 ppm (lH, d, J~4.5, 6-H), 5.9 (lH, m, 7-H), 6.3 (lH, s), 7~3 (lOH, m, Ph-H), 7.8 (lSH, m, Ph-H).

~ ~ e ~ 3 ~

Diphenylmethyl 7-[2-(5-Amino-1,2,4-thiadiazol-3 yl)-2-methoxy-iminoacetamido]-3-~(tri~henylphosphoranylidene~methyl]-3-ce~hem-4-carboxylate (VII-l) .
A mixture of VI-1 from Preparation No. 3 (952 mg, 1 mmole), Amberlite IRA-410 (OH form, 500 mg) and N NaO~ (4 ml) in CH2C12 (10 ml) was stirred for 1 hour at room temperature. The mixture was filtered and the separated organic layer was dried over MgSO4 and concentrated under diminished pressure. The resul~ing oil was triturated with ethyl acetate and the resulting yellow precipitate was collected by filtration to give 740 mg (90%) of the title compound VII-l. M.p. ~180C (dec.).

IR : vKBr c~ 1 3400, 1750, 1630 max UV : ~EtOH nm ~) 268 (1200D), 276 (10000), 384 (23000).
max ~6~9 Pre~aration No. 5 Diphen~methyl 7-[2-tS-Amino-1,2,4-'thiadiazol-3~1)-2-methoxy-iminoacetamido]-3-(3-chloro-1-propen-1-yl)-3-cephem-4-carboxYlate (VIII-l) To a solution of VII-l from Preparation No. 4 (6.9 g, 8.4 mmole) were added MgS04 (3 g) and 40~ chloroacetaldehyde (810 mg, 8.4 mmole). The mix~ure was stirred for 1.5 hours at room temperature and ~hen filtered. The filtrate was eluted on silica gel (Wakogel C-200, 100 g containing 10 ml of 1/1.5 M phosphate buffer) column by using CHC13, and CHC13 containing CH30H.
Fractions containing the desired product (0.5 - 1% CH30H-CHC13) were evaporated in vacuo to give 1.6 9 (30~) of the title compound VIII-l as a yellow amorphous powder, which was a mixture of the Z and E isomers ~ith respect to the chloroprope~yl moiety (Z/E=2/1, by nmr). M.p. ~130~C (dec.).

IR: vKBr cm 1 3300, 1780, 1725, 1680, 1620.
max UV : ~E OH nm ( ~ ) 240 (20000), 286 (12000).
max NMR ~DMSO-d6~D20 3.56 ~ 3.8 (m, 2-H), 3.94 (3H, s, OCH3), ppm 4.16 (d, J=7.5, CH2Cl), 5.26 (lH, d, J=4.5, 6-H), 5.87 (lH, d, J=4.5, 7-H), 6.28 (2~3H, d, J=ll, 3-CH cis-H), 6.72 (1/3H, d, J=16, 3-CH trans-H), 6.81 (2/3H, s, CHPh2~, 6.92 (1/3H, s, CHPh2), 7.4 (lOH, m, Ph-H).

P~epara~ion No. 6 Diphenvlmethyl 7-~enzylideneamino-3-[(tri~henylphosphoranyli-dene)methyl~-3-ce~hem-4-carbox~late (XVI) To a solution of diphenylmethyl 7-kenzylideneamino-3-[(triphenylphosphonio)methyl]-3-cephem-4-carboxylate iodide (XV) [prepared according to the procedure of Japan published patent application (Kokai) 56-86187 (7/31/81)] (60 g, 70 mmole) in CH2C12 (350 ml) were added N NaOH (140 ml) and Amberlite IRA-410 (OH form, 35 g) at 5C. The mixture was stirred for 1 hour at 5C and filtered. The organic layer was separated, dried over MgSO4, concentrated to ca. 100 ml of volume and precipitated with ethyl acetate (500 ml). The resulting yellow solid was collected ~y filtration and dried in vacuo to afford 48 9 (94~) of the title compound XVI, melting at 195~8C (dec.).

IR : vKBr cm 1 1770 1620 max Preparat on No 7 ~iP~ylmeth~ 7-Benzylideneamino-3-(3-chloro-l-pro~en-l-yl)-3 cephem-4-carboxylate (XVII) To a stirred solution of XVI from Preparation No. 6 (2.9 g, 4 mmole) in a mixture of CH2C12 (40 ml) and H2O (10 nl), was added anhydrous chloroacetaldehyde (800 mg) at room tempera-ture. ~o the mixture was added additional 800 mg of chloro-acetaldehyde in three portions over a period of 1 hour, while the p~ of the mixture was kept between 6 to 9 by addition of N NaOH.
After 15 minutes, the aqueous layer was removed and the organic layer was dried over MgSO4. Evaporation of the solvent gave a red oil which was dissolved in a mixture of ethyl aceta~e and isopropyl ether ~1/2, 80 ml~. The solution was washed with saturated aqueous NaHCO3 (l0 ml) and H2O (10 ml), successively.
After drying over MgSO4, removal of the solvent afforded 3.3 g of yellow oil. A solution of the oil in CH2C12 (50 ml) was filtered ~ Z76~2~
10~
with aid of silica gel (12 9, Wakogel C 200) containing 1/1.5 M
phosphate buffer (1.2 ml, pH 6.4) and the silica gel was washed with CH2C12 (50 ml). The filtrate and washing were combined and evaporated to dryness. The residue was triturated with n-hexane to give 1.7 g (80~) of ~:he title compound (XVII) as a yellow powder. The nmr spectrum indicated that the ch.loropropenyl moiety had the Z configuration. M.p. >50C (dec.).

IR: vKBr cm 1 3400, 1775, 1720, 1630.
max UV:~E OH nm (E) 253 (11000), 258 (llOûO), 265 (looao), 273 max(8300), 281 (7000), 290 (6300).

Nr~ DMSO d6 3.63 (2H, br-s, 2~H), 4.0 (2H, m, CH2-Cl), 5.42 ppm (2H, m, 6-H & 3-CH=CH), 5.72 ~lH, d, J=A.5, 7-H), 6.27 (lH, d, J=ll, 3-CH), 6.85 (lH, s, C~lPh2), 7.33 (lOH, m, Ph-H).

Preparation of anhydrous chloroac_taldehyde Anhydrous calcium chloride was added to a chilled solution of 50~ aqueous chloroacetaldehyde (50 ml), with stirring, to separate it into two layers. The chloroacetaldehyde hydrate layer (1) (the upper layer) was separated and diluted with CHC13 (100 ml), mixed with MgS04 (20 g), heated to reflux for 5 min~
utes, and filtered. The solvent and water were removed azeo-tropically (b.p. 56-64C) (2), and the residue was distilled to give anhydrous chloroacetaldehyde (3), b.p. 70-82C/760 mm.

IR vfilm cm 1 1720.
max (1) R.P. Kurkjy, E. V. Brown, J. Amer. Chem. Soc., 74, 5778 (1952).

(2) S. Trippett, D. M. Walker, J. Chem. Soc., 1961 1266.

31L;2~69~9 (3) H. G. House, V. X. Jones, G. A. Frank, J. Org. Chem., 29, 3327 (1964).

Preparation No. 8 Diphenylmethx~_7-Amino-3-(3-chloro-1-proeen-1-yl)-3-cephem-4-carboxylate (XYIII) A solution of XVII from Preparation No. 7 (180 mg, 0.34 mmole) in ethyl acetate (10 ml) was added to a solution of Girard Reagent T [(carboxymethyl)trimethylammonium chloride hydrazide]
(251 mg, 1.5 mmole) in CH30H (10 ml) containing acetic acid (0.25 ml), at 5C. After stirring for 30 minutes at 5C, the mixture was concentrated to remove the CH30~ and then ethyl acetate (20 ml) was added. The ethyl acetate solution was washed with H20 (2 x 5 ml), saturated aqueous NaHC03 (5 ml) and brine (5 ml), successively and dried over MgS04. Evaporation of the solvent gave 145 mg (97%) of the title compound XVIII (Z isomer) as a yellow powder. M.p. >100C (dec.).

IR : vK r cm 1 3400, 1770, 1720.
max UV : ~E nm (E) 252 (3700), 258 (3800), 260 (4000), 274 max (4000), 285 (4000).

Preparation No. 9 Di~enylme~yl 7-[2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxy-iminoacetamido]-3-(3-chloro-1-Propen-l-yl)-3-cephem-4-sarboxylate (VIII-l?

A mixture of 2-(S-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetic acid (III-l) (10.1 g, 50 mmole) and PC15 (10.4 g, 50 mmole) in dry CH2C12 (100 ml) was stirred at -7 to -15C
for 2 hours. The clear solution was poured into n-hexane (500 ~1) to give a precipitate. The organic layer was discarded by ~276~Z9 decantatlon and the remaining solid was triturated with n-hexane (100 ml). The yellow precipitate was collected by filtration and dried 1n vacuo to give 12.5 9 (99~) of the acid chloride, melting at 80C (deo.).

IR : v i cm 1 1770 max The acid chloride (25 mg, 0.1 mmoleJ was added to a solution of XVIII (Z isomer) from Preparation No. 8 (44 mg, 0.1 mmole) in dry CH2C12 (5 ml) at room temperature, with stirring.
After 30 minutes, ~he mixture was concentrated under reduced pressure and diluted with ethyl acetate ~20 ml) and saturated aqueous NaHC03 (5 ml). The organic layer was washed with sat-urated aqueous NaHC03 (S ml), brine (5 ml), 10% ~Cl (5 ml) and brine (5 ml). The solvent was dried over MgS04 and then evaporated to dryness to give the product as a yellow foam. The foam was purified by silica gel (Wakogel C-200, 1 g, containing 0.1 ml of 1/1.5 M phosphate buffer pH 6.4) column chromatography by elution with CH2C12-CH30H (100 : 1), to give 31 mg (50%) of the ti~le compound VIII-l (Z isomer) as a yellow powder. M.p.
>150C (dec.).

IR : vKBr cm 1 3400, 1775, 1720, 1675, 1630.
. max UV : ~EtOH nm () 240 (17000), 280 (10000).
max NMR : ~DMSO ~6 3.6 (2H, m, 2-H), 3.92 (3H, s, O-CH3), 4.0 (2H, ppm m, CH2ClJ, 5.27 (2H, m, 6-H & 3-CH=CH), 5.83 (lH, d-d, J=4.5 ~ 10, 7-H), 6.25 (lH, d, J=ll, 3-CH), 6.83 ~lH,s, CHPh2), 7.33 (lOH, m, Ph-H), 8.0 (2~, br-s, NH2), 9.57 (lH, d, J=10, 7-N~).

92~

Preparation No. 10 Diphenylmethyl 7-[2-(5-Amino-lJ2,4-thiadia2ol-3~ -2-methoxy-iminoacetamido] ~ ephem-4-carboxylate (IX-l) A solution of VIII-l from Preparation No. 5 (Z/E=2/1, 480 mg, 0.77 mmole) in dry acetone (10 ml) containing NaI (346 mg, 2.3 mmole) was stirred for 30 minutes at ambient temperature.
The reaction mixture was evaporated under reduced pressure. The resulting oil was partitioned between ethyl acetate (50 ml) and wa~er (10 ml). The upper layer was washed with 10~ w/v aqueous sodium thiosulfate solution (10 ml) and brine (10 ml) successively, and dried over MgS04. Evaporation of the solvent gave 540 mg (98~) of the title compound IX-l (Z/E=l/l) as a reddish amorphous solidt melting at >120C (dec.).

IR : vKBr cm 1 3300, 1780, 1720, 1680, 1620.
max ~EtOH n~ (~) 240 (21000), 290 (12000) max NMR : ~DMSO D20 3.67 (2H, m, 2-H), 5.29 (lH, d, J=4.5, 6-H), ppm 5.95 (lH, d, J=4.5, 7-H), 6.27 (1/2H, d, - J=ll, 3-CH cis), 6.72 (1/2H, d, J=16, 3-CH
trans), fi.87 h 6.96 (each 1/2H, s, CHPh2), 7.4 (lOH, m, Ph-H).

7~t~3 Z9 Preparation No. 11 Diphenylmethyl 7 [2-(5-Amlno-1,2,4-thiadiazol~ oxy-iminoacetamido]-3-(3-iodo-1-propen-1-yl)-3-cephem-4-carboxylate (IX-l) .

A mixture of VIII-l (Z isomer) from Preparation No. 9 (5.6 g, 9 mmole) and NaI (4 g, 27 mmole) in dry acetone (100 ml) was stirred for 1.5 hours at room temperature. The mixture was evaporated and the resulting oil was diluted with ethyl acetate (90 ml). The ethyl acetate layer was washed with 10% w/v aqueous sodium thiosulfate solution (10 ml) and H2O (10 ml). Removal of tke dried (MgsO4) solvent gave a yellow oil, which was solidified by trituration with isopropyl ether. Filtration of the precipitate gave 4.3 g (67%) of the title compound IX-l as the E
isomer. M.p. >165C (dec.).

IR : vRBr cm 1 3400, 17B0, 1725, 1680, 1610.
max UV : ~E OH nm t~) 240 (18000), 297 (11000).
max NMR : ~D SO 6 2 3.90 (3H, s, OCH3), 5.25 (lH, m, 6-H), 5.95 ppm (1~, m, 7-H), 6.72 (d, J=16, 3-CH trans), 6.96 (lH, s, CH-Ph2), 7.4 (lOH, m, Ph-H).

'~_ Benzhydryl 7-Amino-3-[3-cbloro-1-propen-1-yl~-3-cephem-4 carboxylate (Z-isomer) ~XVIII) Compound XVIII is the common intermediate u~ilized in Reaction Schemes lb and lc.

A. Benzhydryl 7-Benzylideneamino-3-triphenylphosphoniomethyl-3-cephem-4-carboxylate chloride (XV) To a suspension of benzhydryl 7-amino-3-chloromethyl-3-cephem 4-carboxylate hydrochloride (II hydrochloride) (200 g, 0.44 mole) in CH2C12 (940 ml~ was added 1 N NaOH (440 ml) at room temperature. ~he mixture was shaken for 10 minutes and the organic layer was separated. To the organic layer were added MgSO4 (75 g) and benzaldehyde (51 g, 0.48 mole) and the mixture was allowed to stand for 3 hours. The reaction mixture was filtered and the insolubles were washed with CH2C12 (200 ml). To the combined filtrate and washings was added triphenylphosphine ~126 g, 0.48 mole). The mixture was concentrated to about 400 ml and allowed to stand for 4 days. The resulting viscou~ oil was di~uted with ethyl acetate (1 L) and triturated to separate the title compound XV a pale yellow crystalline powder which was collected by filtration and dried in vacuo. Yield 322 g (96~).
M.p. 185-190C (dec.).

IR : v cm 1780, 1720, 1630.
max .
UV ~CH2C12 nm (e) 260 (24100).
max B. Benzhydryl 7-~enzylideneamino-3-[~triphenyl~hosphoran-ylidene)methyl]-3-cephem-4-carbox~_ate (XVI) A mixture of XV (322 g, 0.42 mole) and 5 N Na2CO3 (252 ml) in CH2Cl~ (1.6 L) was stirred vigorously for 15 minutes at room temperature. The organic layer was separated, dried over MgSO4 and concentrated to about 500 ml of volume. The concentrate was added to acetone (1 L), with stirring, to give a light yellow crystalline powder which wa~ collected by filtration to yield 237 g (78%) of XVI, melting at 195-198C (dec.).

~769~9 IR vXBr cm~l 1770 1620 max UV : ~CH2C12 nm (E) 2S4 (23000), 389 (22000).
max NMR : ~CDC13 2.56 & 3.16 (2H, ABq), 5.00 (lH, d, J=4 Hz), 5.23 ppm (lH, d, J=4 ~z), 5.47 (lH, d, J=22 ~z), 6.95 (lH, s), 7.2-7.8 (30H, m), 8.55 (lH, s~.

C. Benzb~dryl ?~Amino-3-[chloro-1-propen-1-yl]-3-cephem-4-carboxylate hydrochloride (Z isomer) (XVIII Hydrochloride) To a refluxing solution of XVI (214 g, 0.294 mole) and N,O-bis-(trimethylsilyl)acetamide (40 ml, 0.15 mole) in dry CH2C12 (2.9 L) was added dropwise, with stirring, a 50% solution of chloroacetaldehyde (93 g, 0.59 mole) in CHC13 over a period of 15 minutes. After standing for 30 minutes, the mixture was concentrated to dryness. To the residual oil were added CH2C12 (1.5 L), Girard Reagent T (99 g, 0.59 mole) and 10% aqueous HCl (300 ml), and the mixture was stirred for 1 hour at roo~ tempera-ture. The organic layer was washed with water (200 ml) and a saturated NaCl solution (200 ml), dried over MgSO4, tre~ted with charcoal (5 g) and filtered. The filtrate was cooled to -10C
and treated with 1 N HCl in CH30H (300 ml). The mixture was stirred for 30 minutes at room temperature and concentrated ~o about 300 ml. The concentrate was diluted with ethyl acetate (400 ml) and seeded with a few crystals of XVIII hydrochloride.
After 2 hours the separated crystals were collected by filtration, washed with ethyl acetate (200 ml) and dried in vacuo to give ~4 g (53%) of the title compound XVIII as its bydrochloride, melting at ~185C (dec.). Pale yellow needles.

~2~6~9 IR : vKB~ cm 1 28309 1780, 1720.
max UV ~EtOH nm (~) 286 (8800).
max NMR : ~DMSO d6 3.73 (2H, br, s, 2-H), 3.97 (2H, m, C~2Cl), 5.22 ppm (lH, d, J=4.5 Hz, 6-H), 5.37 (lH, d, J=4.5 Hz, 7-H), 5.77 (lH, m, 3-CH=CH), 6.45 (lH, d, J=ll Hz, 3-CH), 6.88 (lH, s, CHPh2), 7.33 (lOH, br, s, Ph-H~.

Anal- Calc'd for C23H21N23SCl HCl C, 57-87; H, 4-65;
N, 5.87; S, 6.72;
C1, 14.85.
Found: C, 57.62; H, 4.53;
N, 5.70; S, 6.64;
C1, 14.89.

Preparation No. 13 Benzhydry~ 5~Amino-1,2,4-thiadiazol-3-yl)-2-methoxyimino-acetamido]-3-[3-chloro-1-Pro~e -~yl]-3-cephem-4-carboxylate (Z isomer) (VIII-l) To a stirred solution of XVIII (Z isome}) (20 g, 42 mmole) in CH2C12 (420 ml) containing N,O-bis(trimethylsilyl)-acetamide (34 ml, 125 mmole) was added 2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetyl chloride hydrochloride (15.2 9, 59 mmole) in three portions over a period of 30 minutes at -10 to 0C. The mixture was stirred for 30 minutes at 0-5C
and concentrated under reduced pressure. The residual brown oil was dissolved in ethyl acetate (420 ml) and the solution was washed successively with saturated aqueous NaHC03 (3 x 15 ml), saturated aqueous NaCl (15 ml), 10% HCl (15 ml) and saturated aqueous NaCl (15 ml), and concentrated to about 50 ml of the volume. To the concentrate was added n-hep~ane !200 ml) to give ~7~i9~
il2 28.5 g t90~ pure) of the title compound VIII-l (Z-isomer) as a colorless powder. M.p. >150C (dec.)O

IR : vKBr cm 1 3400, 1780, 1720, 1680, 1620.
max UV : ~E QH nm ~E) 240 (20000), 283 (12000).
max NMR ~acetne-d6 3.6 (2H, m, 2-H), 3.95 (3H, s, OCH3~, ppm (2H, m, CH2Cl), 5.32 (1~, d, J=4.5 ~z, 6-H), 5.62 (1~, m, 3-CH=CH), 6.03 (1~, d, J=4.5 Hz, 7-~), 6.32 ~lH, d, J=11 Hz, 3;CH), 6.87 (lH, s, CHPh2), 7.33 (lOH, br, s, Ph-H).

Preparation No. 14 Benzhydryl 7-[2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxyimino-acetamido]-3-[3-iodo-1-propen-1-yl]-3-cephem-4-carboxylate (E isomer) (IX-l) A mixture of VIII-l (Z isomer) (28.5 g, 90% pure) and sodium iodide (19 g) in dry acetone (420 ml) was stirred for 10 minutes at room temperature and allowed to stand at 5C for 2 ~ours. The mixture was concentrated under reduced pressure. To the residue were added ethyl acetate (420 ml) and 10~ w/v aqueous sodium thiosulfate solution (30 ml), and the mixture was shaken .
The organic layer was washed with water (30 ml), dried over MgS04 and evaporated to about 50 ml of volume. The concentrate was diluted with n-heptane (200 ml) to yield 30.5 g (9S~ pure) of the title compound IX-l (E isomer~ as a yellow powder, melting at > 12 0 C ( dec . ) .

~. 27 ~ 9 IR : vKBr cm 1 3400, 1780, 1725, 1680, 1620.
max UV ~EtOH nm () 306 (15000).
max NMR ~acetone-d6 ppm 3.71 (2H, m, 2-H), 3.97 (3H, s, OCH3), 4.0 (2H, d, J=8 Hz, CH2I), 5.26 (lH, d, J-4.5 Hz~
6-H), 6.03 (lH, d-d, J=4.5 & 8 Hz, changed to doublet J=4O5 Hz by D2O, 7-H), 6.32 ~lH, d-t, J=15 & 8 Hz, 3-CH=CH), 6.79 (lH, d, J=15 Hz, 3-CH~, 6.98 (lH, s, CHPh2), 7.35 (10~, ~, Ph-H), 7.63 (2B, br, s, disappeared by D2O7 NH2), 8.52 (lH, d, J=8 Hz, disappeared by .
Preparation No. 15 Benzhydry~7-E2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxvlmino-acetamido]-3-~3-(4-carbamoyl-1-pyr1dinio)-l-propen-1-ylJ-3-cephem-4-carboxylate Iodide (E isomer) (XII-lH) To a suspension of IX-1 (E isomer) (30.5 g) and isonicotinamide (26 g, 212 mmole) in CH3CN (120 ml) was added CH30H ~100 ml) until the mixture became clear. The solution was stirred for 2 hours under nitrogen atmosphere at room temperature and concentrated ~o about 100 ml under reduced pressure. The residual semi-solid was triturated with isopropyl ether (200 ml).
The solvent was removed by decantation and the residual yellow powder was washed with a mixture of isopropyl ether and CH30H
(3/1, 120 ml). The powder was collected by filtration and dried in vacuo to give 36 g (75% pure estimated by HPLC) of the title compound XII-lH (E isomer) as a light yellow powder mel~ing at >150C (dec.).

~ ~ ,.

'~.276~

IR : ~ cm 1 3300, 1780, 1720, 1680, 1620.
max UV : AEtOH nm ~El%H ) 282 (170) max 1 cm N~ ~DMSO-d6 ppm 3.72 (2H, m, 2-H), 3.90 (3H, s, OCH3), 5.25 (3H, m, 6-~ & CH2N ), 5.9 (lH, d-d, J=4.5 & 8 Hz, changed to a doublet J=4.5 ~z by D20 addition, 7-H), 6.35 (lH, m, 3-CH=CH), 6.89 (lH, s, CHPh2), 6.9 (lH, d, J=16 Hz, 3-CH), 7.35 (lOH, m, Ph-H), 8~06 (2H, br, ~, disappeared by D20, ~H2), 8.21 (2H, br, s, disappeared by D20 addition, NH2), 8.36 & 9.07 (each 2H, d, J=6 ~z, Py-H), 9.57 ~lH, d, J=8 Hz, disappeared by D20 addition, 7-NH).

Preparation No. 16 Benzkydryl 7-Benzylideneamino-3-[3-chloro-1-~ropen-1-yl]-3-cephem-4-carboxYlate (XVII) (Z isomer) To an ice-cooled mixture of the crystalline 7-amino-cephem intermediate XVIII (Z isomer) (13.4 9, 28 mmole) and benzaldehyde (3.3 g, 31 mmole) in ethyl acetate (150 ml) was added dropwise 0.5 N sodium hydroxide (56 ml, 28 mmole) over a period of 20 minutes~ to maintain the temperature of the reaction mixture below 10C. The mixture was stirred with cooling for another 15 minutes, and the organic layer was separated, washed with sa~urated aqueous sodium bicarbonate (100 ml x 2) and dried over magnesium sulfate. To the dried solution was added a small amount o charcoal and the mixture was filtered. The filtrate was concentrated to dryness. The residual oil was dissolved in carbon tetrachloride ~50 ml), and concentrated again. This procedure was repeated 3 times, and the mixture was monitored by reverse pha~e tlc to confirm that all of the starting 7-amino-.

" ~'Z~7Ç~29 cephalosporin was converted to the Schiff's base. Removing thesolvent ln vacuo gave 16.45 g of the title compound XVII (Z
isomer) as a pale yellow powder (estimated purity 85~; M.p. 74C
(dec.), which was ussd for the next step without purification.

IR : v r cm 1 1780, 1725, 1635.
max UV : ~CH2C12 nm (El% ) 257 ( max 1 cm NMR : ~CDC13 6.18 (lH, d, J-ll Hz).
ppm Preparation No. 17 Benzhydryl 7-Benzylideneamino-3-[3-(4-carbamoyl-1-pyridinio)-1-propen-l-yl]-3-cephem-4-carboxylate Iodide (XXI-~) (E isomer) To a chilled mixture of the 3-chloropropenylcephem XVII
(Z isomer3 (16.4 g) in acetone (5 ml), was added dropwise a solution of sodium iodide (6.3 g, 42 mmole) in acetone (~0 ml) over 10 minutes under nitrogen atmosphere, and the mixture was stirred at room temperature. The reaction was monitored by the ratio of uv absorption ~El ~ (255 nm)/El % (320 nm)]. When 1 cm 1 cm the ratio reached below 1.30 (after 45 minutes), the mixture was diluted with carbon tetrachloride (400 ml), and allowed to stand at room tempera~ure. When the ratio came to below 1.10 (after 3 hours), the mixture was concentrated to a half its volume. The concentrate was treated with a small amount of charcoal and diatomaceous earth, and filtered. The filter cake was washed with a 1:1 mixture (100 ml) of methylene chloride and carbon tet~achloride. To the combined solution of the filtrate and washings, was added a solution of isonicotinamide (3.5 g, 28.7 mmole) in dimethylformamide (20 ml) and the mixture was concen-trated under reduced pressure. The concentrate was allowed to stand at room temperature for 1.5 hours and washed with isopropyl .

e~her (100 ml x 3). The residual brown semi-solid was dissolved in methylene chloride ~50 ml) and the solution was added dropwise, with stirring, to ethyl acetate (1.5 L). ~he resulting precipitate was collected by filtration and washed with ethyl acetate (200 ml). After drying over phosphorous pentoxide in vacuo, 17 g of the title compound XXI-H (E isomer) was obtained.
Yellow amorphous powder. M.p. 150-155C (dec.). Estimated purity 80% by nmr.

IR : v cm 1775, 1725, 1690, 1635.
max ~C12 nm (El% ) 258 (335)~ 298 (255)-max 1 cm NMR ~DMSO-d6 ppm 3.4-3.8 (2H, br.), 5.35 (2H, br.), 5.41 (lH, d, J=4 Hz), 5.73 (lH, d, J=4 Hz), 6.93 (lH, s), 6.97 (lH, d, J=16 Hz), 7O3-7.5 (15H, br. s), 8.40 (2H, d, J=6.5 Hz), 9.15 (2H, d, J=6.5 Hz).

Preparation No. 18 7-Amino-3-[3-(4-carbamoyl-1-pyridinio)-1-propen-1-yl]-3-c~phem-4-carbox~late (XXII-H) (E isomer) To a suspension of the quaternized cephem XXI-H (17 g) in 85% formic acid (25 ml) was added dropwise concentrated hydrochloric acid (5 ml), and the mixture was stirred at room temperature for l.S hours and treated with a small amount of charcoal. The mixture was filtered and washed with 854 formic acid (5 ml). The filtrate was combined with the wash and poured into acetone (1 L), with stirring. The resulting precipitate was collected by filtration to give 9.52 g of yellow-colored crude product. To a suspension of the crude material (9.5 9) in water (50 ml) was added a small amount of charcoal, and the mixture was filtered. The filtrate was added dropwise, with stirring, to isopropyl alcohol (700 ml). The resulting precipitate was ~ ~ ~6$~9 collected by filtration, washed with a small a~ount of rnethanol (30 ml), and dried to give 7.58 g of the title compound XXII-H (E
isomer) as the hydrochloride. Light yellow powder. ~stimated purity 85% ~y VV. M.p. 173-188C (dec.).

IR : vKBr cm 1 1795, 1680, 1620, 1575, 1540.
max UV ~Phosphate buffer (pH 7) nm (El ~ ) 294 (457)-max 1 cm N~ D2o+D~l ppm 3.82 (2~, s), 5.17 (lH, d, J=5 H2), 5.33 (2H, d, J=7 Hz), 5.43 (lH, d, J=S Hz), 6.37 (lH, d-t, J=16 & 7 Hz), 7.23 (lH, d, J=16 Hz), 8.34 (2H, d, J=7 Hz), 9.00 (2H, d, J=7 Hz).

PreParation No._l9 2 (5-Amino-1,2,4-thiadiazol~x1)-2-methoxyiminoacetyl Chloride Hy~ochloride ~ l as its acid chloride hydrochloride) A. 2-Cyano-2-methoxyiminoacetamide To a stirred mixture of ~-cyanoacetamide (252 g, 3 mole) and sodium nitrite (414 g, 6 mole) in water (600 ml) was added acetic acid (371 ml, 10 mole) at 5-10C over 1.5 hours.
The mixture was allowed to stir for another 1.5 hours and adjusted to pH 8.5 with 6 N NaOH. To the mixture was added dimethyl sulfate (568 ml, 6 mole) at 15-20C and the mixture was stirred at 45C for 1.5 hours. The reaction mixture was adjusted to pH 8.5 with 6 N NaO~ and allowed to stand at 5C overnight to separate the precipitate, which was collected by filtration, washed with cold water and air-dried to give 292 g (77%) of ~he title compound as brown needles melting at 170-172C.

IR : vKB~ cm 1 3400, 3180, 1720(sh), 1715, 1690, max1615, 1570.

UV : ~H2O nm (~) 238.5 (8290), 268 (sh, 3870~.
max NMR: ~DMSO d6 4.20 (3H, s, OCH ), 7.85 (28, br. NH2).
ppm 3 Anal. Calc'd. for C4H5N3O2: C, 37.80; H, 3.97; N, 33.~
Found: C, 37.43: H, 3.75; N, 32.51.

B. 2-Methoxyiminopro~anedinitrile A stirred mixture of 2-cyano-2-methoxyiminoacetamide (88.9 g, 0.7 mole), sodium chloride t70 g) and phosphorus oxy-chloride (97 ml, 1.05 mole) in dry 1,2-dichloroethane (350 ml) was refluxed for 16 hours. ~he insolubles were filtered off through a dicalite pad and washed with dichloroethane. The filtrate and t~e wash were combined, and poured into stirred ice-water (1.5 L) to decompose the excess of phosphorus oxychloride. The organic phase was washed with 10~ NaHCO3 (500 ml), water (500 ml x 3) and a saturated NaCl solution (500 ml), and dried over MgSO4. The filtrate was distilled under diminished pressure to give 61.5 g (81%) of the title compound boiling at 62C/24 mm Hg. (Lit., b.p. 47-48C/12 mm Hg).

IR vLiquid Film cm~l 3020, 2960, 2245, 2020, 1530, 1455, max1080.

NMR: ~CD 3 ppm 4.35 (3H, s, OCH3).

.: . . . . .

~.2~;~6 ~G3~2~

C. 2-Cyano~2-~ethox~iminoacetamidinium Acetate To a solution of ammonium chloride (28.4 g 0.53 mole) in 28~ aqueous ammonia (355 ml) and ethanol (180 ml) was added dropwise a solution of 2-methoxyiminopropanedinitrile (58.0 g, 0.53 mole) in ethanol (120 ml) at -15 to -10C over a period of 30 minutes, with stirring. The mixture was stirred at -10C
overnight and then at ambient temperature (20-25C) for one day.
T~e reaction mixture was partitioned between water (350 ml) and CH2C12 (350 ml~, and the aqueous phase was saturated with sodium chloride, and extracted again with CH2C12 (300 ml~. The organic extracts were combined, dried over MgSO4 and evaporated in vacuo.
A solution of the residue in ethyl acetate (1.6 L) was adjusted to pH 3-4 with acetic acid to precipitate the title compound as crystals, which were collected by filtration and washed with ethyl acetate. Yield 67.6 g (69%). M.p. 152-4C (dec.). [Lit., m.p. 150-155C (dec.)~.

IR : vK cm 1 3160, 2900, 2360, 2235, 2000, 1665, 1555, max 1495, 1415.

UV : ~E nm (~) 243 (8500), 265 (sh, 5380), 305 (sh, 1400).
max NMR ~DMSO-d6 ppm 1.88 (3H, s, CH3COOH), 4.15 (3H, s, OCH3), 7.60 (4H, br.).

Anal. Calcld for C4H~N40-CH3COOH: C, 38.71; H, 5.41; N, 30O09 Found: C, 38.71; H, 5.59; N, 29.51.

D. 2-(5-Amino-1,2,4-thiadiazol-3-~1)-2-methoxyimino-acetonitrile To a su~pension of 2-cyano-2-methoxyiminoacetamidinium ace~ate (125 9, 0.672 mole) in CH30H (1.25 L) were added dropwise triethylamine (234 ml, 1.68 mole) at -10C, and subsequently Br2 . ~

(41.6 ml, 0.806 mole) ovee 20 minutes at -15 to -10C, and the mixture was stirred for 20 minutes. To the mixture was added dropwise a solution of KSCN (78.3 9, 0.806 mole) in CH30H (550 ml) over 1 hour at -15 to -10C. After stirring at 0-5C for 1 hour, the mixture was poured into ice-water (12 L) to form a crystalline precipitate, which was collected by filtration, washed with water and air dried to give 120 g ~98%) of the title compound. M.p. 263-5C (dec.). The m.p. o the compound prepared by us is higher by about 60C than that given in the literature* [m.p. 210-15C (dec.)~, but our spectral and microanalytical data are consistent for the structure.

IR : vK~r cm 1 3435, 3260, 3120, 2960, 2245, 2020, 1630, max 1545, 1455, 1415.

UV : ~E OH nm ~E~ 248 (13300), 310 (3470).
max Nl~ ~DMSO~d6 ppm 4.21 (3H, s, OCH3), 8.30 (2H, br. NH2).
nal. Calc'd for C5H5N50S: C, 32.?8; H, 2.75; N, 38.23; S, 17.50 Found: C, 32.76; H, 2.51; N, 38.02;
S, 17.50.

E. 2 (5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxYiminoacetic Acid (III-l) A mixture of 2-(5-amino-1,2,4-thiadiazol-3-yl-2-methoxyiminoacetonitrile (18.3 g, 0.1 mole) in 4 N NaOH (250 ml) was heated at 50-55C with stirring for 3 hours. The reaction mixture was adjusted to pH 1 with H3PO4, and washed with ethyl acetate (100 ml), saturated with NaCl, and extracted three times with a mixture of ethyl acetate and tetrahydrofuran (3 : 1, 300 ml x 2, and 200 ml x 1). The extracts were combined, dried over MgSO4 and concentrated under reduced pr-essure. The residue was triturated with isopropyl ether to afford pale yellow crystals of the title acid. Yield 16.8 g (83~). M~po 184-5C (dec.).
~Lit.*, m.p. 180-182C (dec.)].

IR O vKBr cm 1 3460, 3260, 3140, 1725, 1620, 1605, 1545.
max UV : ~2 nm (E) 234 (13200), 288 (sh, 3620).
max F. 2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetyl Chloride H~drochloride To a suspension of 2-(5-amino-1,2,~-thiadiazol-3-yl)-2-methoxyiminoacetic acid (III-l) (40.4 g, 0.2 mole) in dry CH2C12 (400 ml~ was added PC15 (41.6 g, 0.2 mole) in one portion at -S0C. The mixture was stirred for 4 hours at 20 to -5C, and poured into a mixture of n-heptane and isopropyl ether (2 : 1, 2 L). The yellow precipitate was collected by filtration, washed with the same solvent mixture, and dried with KOH under reduced pressure to give 46.0 g (90%) of the title acid chloride.

IR vNujol cm-l 1775 max *Japan Kokai 57-158769 published September 30, l9B2, to Fujisawa (Brit. appl., 3/6/81) ~L2~6~9 .

1 ~ C - CONH ~
H~N S ~ CH=CH-C~2-Cl C2H5 COOCH (Ph ) 2 VIII-2 *Z
Diphenylmethyl 7-[2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-(Z)-ethoxyiminoacetamido]-3-[3-chloro-l-propenyl]-3-cephem-4 carboxylate (VIII-2, Z isomer) To a mixture of N,O-bis(trimethylsilyl)acetamide (2.3 ml, 9 m~oles) and crystalline diphenylmethyl 7-amino-3-[3 chloro-l-(Z)-propen-l-yl]-3-cephem-4-carboxylate hydrochloride (XVIII) (1.338 g, 2.8 mmoles) (from Preparation No. 12) in methylene chloride (10 ml) was added 2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(Z)-ethoxyiminoacetyl chloride hydrochloride (800 mg, 2.95 mmoles) portionwise, with stirring, at -10C and the mixture was allowed to stand at 0C for 2 hours. The mixture was diluted with ethyl acetate (200 ml), washed with water and evaporated under reduced pressure. Trituration of the residue with isopropyl ether afforded the title product VIII-2 as an amorphous powder. Yield 1.70 9 (95%). Mp. >150DC ~dec.).

IR : vmax (KBr) in cm 1 8300, 1780, 1720, 1690, 1380, 1220.
max (C2H5OH) in nm (E) 285 (llooo) N~ (DMSO-d6) in ppm 1.26 (3H, t, J=7Hz, CH~CH3), 4.25 (2H, ~, J=7Hz, CH2CH3), 5.90 (lH, d-d, J=4 & 8Hz, 7-H), 6.26 (lH, d, J=llHz, 3-CH), 6.85 (lH, s, CHPh2), 9.53 (lH, d, J=8Hz, 7-NH3.

~:7~2~

Preparation No. 21 ,. . .
~ CONH~

H2 S ~ ~ CH=C~--CH2 c~5 COocHlph)2 IX-2 *A mixture of E and Z isomers DiPhenylmethyl- 7-[2-(5-Amino-1!2,4-thiadiazol-3~ 2-1Z)-ethoxY-iminoacetamido]-3-[3-iodo-1-propenyl]-3-c~phem-4-carboxylate (IX-2) A mixture of VIII~2 (1.9b g, 3 mmoles) (from Preparation No. 20) and sodium iodide (1.4 g, 9 mmoles) in acetone (20 ml) was stirred for 10 minutes at room temperature and then allowed to stand at 5C for 3 hours. The mixture was evaporated under reduced pressure, diluted with ethyl acetate (100 ml), washed with 10~ sodium thiosulate and water, and evaporated under reduced pressure. Trituration of the residue with isopropyl ether gave 1.82 g (84~) of the title product IX-2 as a light brown amorphous powder.
R : vmax (KBr) in cm 3290, 177û, 1720, 1670, 1530, 1370, 1220.

nmax (C2H5H) in nm (E ) 304 (199).

7~g2~ l Preparation No. 22 ~ r.
~CH=N~ ~ ~3 --CH=CH--C:H2 _~:ONH2 CoocH(ph~2 XXI~ dide *E
Diphenxlmethyl 7-Benzylideneamino-3-~(E)-3-(4-carbamoAy~-~y~ri io)-l-propenyl]-3-ce~em-4-carbo~ylate (XXI-H iodide) (E
isomer) To a chilled solution of the 3-chloropropenylcephem (XVII, Z isomer, 42.8 g, 90 mmoles) (from Preparation No. 16) in dry DMF (80 ml), was added KI (20 g, 120 mmoles) in one portion, and the mixture was stirred at room temperature. The reaction was monitored by the ratio of UV absorption [El% (255 nm)/
1 cm El% (320 nm)]. When the ratio became below 1.10 (after 45 1 cm minutes), the mixture was diluted with 800 ml of methylene chloride~ treated with active carbon (4 g), and filtered. The filter cake was washed with 100 ml of CH2C12. To the combined filtrate and washings was added isonicotinamide (14.64 g), and the mix~ure was concentrated under reduced pressure. The concentrate was kept at room temperature for 1.5 hours and washed with a mixture of toluene and n-heptane (1:1, 600 ml). ~he residual brown semi-solid was dissolved in CH2C12 (100 ml) and the solution was added dropwise to ethyl acetate (3 L) with vigorous stirring. After drying over P2O5 in vacuo, 57.37 g (88~ of the quaternized title product XXI-H was obtained as the iodide. Yellow amorphous powder. Mp. 150-155C (dec.). This product was identical to that obtained by iodination with NaI
(Preparation No. 17).

, ~76~

Preparat on No. 23 HCl-~2N ~ S ~

N ~ C~CH-CH2-Cl ~OOCH(Ph)2 XVIII *Z
Diphenylmeth ~ _7-Amino-3-(3-chloro-1-propen~l)-3-cephem-4 A 25~ solution of chloroacetaldehyde (69 g, 0.22 mmoles) in CHC13 was added to a solution of XVI (80 9, 0.11 mole) in CH2C12 ll.l L3 containing N,O-bis(trimethylsilyl)acetamide (16.2 ml, 0.06 mole) at -10C in one portion, and the mixture was allowed to stand overnight at 5C. The mixture was concentrated to ca. 0.3 L, diluted with a mixed solvent of ethyl acetate and isopropyl ether (1/2, 0.6 L), treated with silica gel (Wakogel C-100, 60 g) and filtered through a dicalite pad. The filter cake was washed with the same solvent system (0.2 L). The combined filtrate and washing were concentrated to ca. 0.2 L, treated with Girard Reagent T (60 g, 0.26 mole) and 4N ~Cl (220 ml), and seeded with a few crystals of XVIII hydrochloride.
~fter stirring for 3 hours, the resulting crystals were collected by filtration, washed with water (0.5 L) and ethyl acetate (0.5 L) and dried in vacuQ to give 37 g (70~3 of the title compound XVIII hydrochloride, melting at >185C (dec.3. Pale yellow needles. This product was identical to that obtained in Preparation No. 12.

.

g Preparation No. 24 S
HCl H2~ ~ ~

N ~ CH-CH-CH2~Cl COOCH(Ph)2 XVIlI *Z
Diphenylme~yl 7-Amino-3-(3-chloro-1-propenyl) 3-cephem-4-carboxylate hydrochloride (Z_isomer) (XVIII, Hydrochloride) To a solution of chloroacetaldehyde (25% solution in CHC13, 628 mg, 2 mmoles) in CH2C12 (10 ml) were added N,O-bis~
(trimethylsilyl)acetamide (0.135 ml, 0.5 mmole) and XVI (728 mg, 1 mmole), successively, at 5C. The mixture was allowed to stand overnight at 5C. The mixture was evaporated and diluted with a mixture of ethyl acetate and isopropyl ether (1/2, 10 ml~.
Insolubles were removed by filtration and the filtrate was concentrated to ca. 5 ml. The concentrate was treated with 4N
HCl (2 ml3, seeded with XVIII hydrochloride and stirred for 1 hour at room temperature. The crystals were collected by filtration, washed ~ith ethyl acetate (10 ml) and water ~10 ml) and dried in vacuo to give 384 mg (80%~ of the title compound XVIII hydrochloride, melting at >185C (dec.). Pale yellow needles. This product was identical to that obtained by Preparation No~ 12.

PreParation No. 25 2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-(propen-3-yloxvlmlno)-acetyl chloride hvdrochloride (III-3 as its_acid chloride hydrochlor de A. Methyl 2-(5-t-butoxycarbonylamino-1,2,4-thiadlazol-3-yl)-2-(propen-3~ yimino)acetate A mixture of 635 mg (3.37 mmoles) of N-(propen-3-yloxy)phthalimide ~prepared according to the procedure of E.

.

.~7 ,~
Grochosaki & J. Jurczak, Synthesis 1976 682] and 175 mg (3.35 mmoles~ of hydrazine hydrate in 5 ml of C2H50H was stirred for 1 hour at room temperature. ~he resulting precipitate was filtered off and the filtrate and washings were combined. To the solution was added 967 mg ~3.37 mmoles) of methyl 2-~5-t-butoxycarbonyl-amino-1,2,4-thiadiazol-3-yl)-2-oxoacetate, and the mixture was allowed to s~and for 1 hour at room temperature and concentrated by a rotary evaporator. The residue was purified by silica gel chromatography. The column was eluted with n-hexane/ethyl acetate (401) and fractions containing the major product were combined and evaporated under reduced pressure. Yield 514 mg (46%). Mp. 83-86C.

IR : vmax tRBr) in cm 1 3100, 1745, 1710, 1610.

max (C2H50H) in nm (E) 223 (9700) ~42 (10000) N~ (CDC13) in ppm 1.55 (9H, s, BOC-H), 4.40 (2H, d, J=5~z, 0-CH2~l 5.21 (2H, m, CH2=CH), 5.90 (lH, m, -CH=CH2), 9.50 51H, br.s, NH).

B. 2 (5-t-Butoxycarbonylamino-1,2,4-thiadiazol-3-yl)-2-, ~
(propen-3-yloxyimino)acetic acidl' A solution of 770 mg (2.3 mmoles) of methyl 2-(5-t-butoxycarbonylamino-1,2,4-thiadiaæol-3-yl)-2-(propen-3-yloxy-imino)acetate and 3.5 ml of 2N NaOH solution (7.0 mmoles) in 15 ml o~ CH30H was refluxed for 30 minutes. The reac~ion mixture was concentrated in vacuo and dilut0d with 10 ml of ethyl acetate-H20 (1:1). The water layer was separated, acidified to pH 2 with 6N HCl and extracted with ethyl acetate (10 ml x 2).
The ethyl acetate solution was dried over MgS04 and concentrated by a rotary evap~rhtor to afford 596 mg (81%) of the title compound. ~p. 134-135C (litl): mp. 135-136C.

IR : vmax (Nujol*) in cm 1 3150, 1745, 1710, 1550.
* Trade Mark '~7'' .... ~

~ 69~9 UV: )~max (C2H50H) in nm (E) 223 (11000), 242 (113t)0).

N~ (DMSO-d6) in ppm 1.55 (9H, s, BOC-H), 4.77 (2H, d, J=SHz, ~CH2), 5.22 (2Hr m, CH2=CH), 6.0 (lH, m, CH=CH2).

1)I. Csendes, et al., J. Antibiotics, 36, 1020 (1983).

C. 2-(5-Am_n_1,2,4-thiadiazol-3-~-2-(prol?en-3-Yloxyimino)-acetic acid (III-3)1) A solution of 570 mg (1.74 mmoles) of 2-(5-t-butoxy-carbonylamino-1,2,4-thiadiazol-3-yl~-2-(propen-3-yloxyimino)-acetic acid in 6 ml of trifluoroacetic acid was allowed to stand for 1 hour at ambient temperature. Evaporation followed by trituration with 30 ml of isopropyl ether gave 376 mg (95%) of the title compound. Mp. 109C ~dec.).

IR : v~l~ax (Nujol) in cm 1 3180, 1710, 1545, 1460.

max (C2H5OH) in nm (f) 245 (13500) NMR: ~ (DMSO-à6) in ppm 4.77 (2H, d, J=5Hz, ~CH2), 5.20 (2E~, m, CH=CH), 6.0 (lH, m, CH=CH2).

1) Japan Kokai 57-112396 (7/13/82, Fujisawa) Brit. applO 7935538 (10/12/79).

D. 2-(5~Amino-1,2,4-thiadiazol-3-yl)-2-(propen-3-yloxyimino)-acetyl chloride hydrochloride A solution of 350 rng (1.54 mmoles) of III-3 and 410 mg (1.97 mmoles) of phosphorous pentachloride in.dichloromethane (5 ml) was stirred for 1 hour at 25C. The reaction mixture was poured into 60 ml of n-hexane and the precipitate was iltered off. ~ield 323 mg~

IR : vmaX (Nujol) in cm 1 1755.

.. ~ .

~L276'~Z9 Preparation No. 26 2-(5-Amino-1,2,4-~hiadiazol-3-vl)-2-propargy~oxyiminoacetyl chloride hYdrochloride (III-4 as its acid chloride hydrochloride) A. ~5ethyl 2-(S-t-Butoxycarbonylamino-1,2,4-thiadiazol-3-yl)-2-propargyloxyiminoacetate A suspension of 870 mg (4.32 mmoles) of N-propargyl-oxyphthalimidel) and 290 mg (4.0 mmoles) of hydrazine hydrate in 5 ml of ethanol was stirred at 25C for 1 hour and filtered. To the combined filtrate and washings was added 1.0 g (3.86 mmoles) of methyl 2-(S-t-butoxycarbonylamino 1,2,4-thiadiazol-~-yl)-2-oxoacetate2). The solu~ion was allowed to stand for 1 hour and concentrated under reduced pressure. Purification by silica gel chromatography followed by evaporation afforded 319 mg (27%) of the title product. Mp. 72-75C.

IR : vmax (KBr) in cm 1 3200, 2380, 1745, 1710, i61b.

: ~m,x (C2H5OH) in nm (E~ 235 (12200) DMSO-d6) in ppm 1.56 (9H, 5, BOC-H), 3.55 (lH, t, J=2Hz, C ~C~), 4.85 (2H, d, J=2Hz, -CH2-C_ CH), 8.9 (lH, br.s, NH).

1) Commercially available, Aldrich.

2) I. Csendes et al., J. Antibiotics 36, 1020 (1983).

B. 2-(S-t-Butoxycarbonylamino-1,2~4-thiadiazol-3-yl)-2-propargyloxyiminoacetic acid A solution of 490 mg (1.4 mmoles) of methyl 2-(5-t-butoxycarbonylamino-1,2,4-thiadiazol-3-yl)-2-propargyloxyimino-acetate and 2.2 ml of 2N aqueous NaOH solution (4.4 mmoles) in 14 ml of CH3OH was refluxed for 30 minutes. The reaction mix~ure was concentrated under reduced pressure and 10 ml of ethyl acetate~H2O (1:1) was added to the solution. The separated water layer was acidified to pH 2 with 6N HCl and extracted with ethyl acetate (2 x lO ml). Drying over MgSO4 followed by evaporation of the organic layer gave 149 mg (89~) of the title product. Mp.
135C (dec.).

IR : vmax (Nujol) in cm 1 3350, 1720, 1670, 1550.

max (C2H5OH) in nm (~) 233 (11500) N~ (DMSO-d6) in ppm 1.55 (9H, s, BOC-H), 3.55 (iH, t, JG2Hz, C~CH), 4.89 (2H, d, J=2Hz, CH2C _ CH), 9.0 (lH, s, NH).

C. 2-(5-Amino-1,2,4-th adiazol-3-yl)-2-proparg~loxyimino=
acetic acid (III-4) A solution o~ 410 mg (1.26 mmoles) of 2-(5-t-butoxy-carbonylamino-1,2,4-thiadiazol-3-yl)-2-propargyloxyiminoacetic acid in S ml of trifluoroacetic acid was allowed to stand for 1 hour at 25~C. Evapo~ation followed by trituration of the residue with 25 ml of isopropyl ether gave 204 mg (72%) of the title compound. Mp. 156-158C (dec.).

IR : vmax lNuiol) in cm 1 3300, 2480, 1730, 1610.

~ax (C2H5OH) in nm (~) 234 (12000) MR : ~ (DMSO-d6) in ppm 3.52 (lH, t, J=2Hz, C~H), 4.86 (2H, d, J=2Hz, CH2-C _CH), 8.10 (2H, br.s, NH2).

3) Japan Kokai 57-112396 ~7/13/82, Fujisawa) Brit. appl. 7935538 (10/12/79).

D.
acetyl chloride ~drochloride A mixture of 175 mg ~0.07 mmole) of III 4 and 182 mg (D.88 mmole) of phosphorous pentachloride in dichloromethane ~2 ml) was stirred for 1 hour at -5C. The reaction mixture was poured into 30 ml of n-hexane and the precipitate was filtered off. Yield 65 mg (34%).

IR : vmax (~ujol) in cm 1 1770.

PreParation No. 27 2-(5-Amino~1,2,4-thiadiazol 3-yl)-2-cyclopentyloximinoacetyl chloride hydrochloride (III-5 as its acid chloride hydrochloride) .
A. Methyl 2-(5-t-butox carbonylamino-1,2,4-thiadiazol-3-yl)-2-cyclopen~xyiminoacetate A suspension of 860 mg (3.7 mmoles) of N-(cyclo-pentyloxy)phthalimidel) and 185 mg (3.7 mmoles) of hydrazine hydrate in S ml o~ C2H5OH was stirred for 1 hour at ambient temperature and filtered. The filtrate and washings were combined and added to 1.06 g (3.7 mmoles) of methyl 2-(5-t-butoxycarbonylamino-1,2,4-thiadiazol-3-yl)-2-oxoacetate2). The solution was allowed to stand for 1 hour at room temperature and concentrated in vacuo. The residue was purified by silica gel column chromatography. Elution with n-hexane-ethyl acetate (4:1) followed by evaporation gave the title product. Yield 906 mg (81%). Mp. 115-118C.

IR : ~max (KBr) in cm 3200, 1745~ 1710, 1550.

UV : ~ax (C2H5OH~ in nm (~) 217 (1800), 252 (7600).

;9~ :

NMR : ~ (CDC13) in ppm 1.51 (9H, s, BOC H), 1.60 (8H, br.s, H ~ ), 3.88 (3H, s, OCH3), 4.90 (lH, br.s, ~ ), 8.70 (lH, br.s, I~H).

1) U.S. Patent 3,371,778 (7/27/76; Glaxo), Brit. appl. 49255 (10~25/72)o 2) I. Csendes et _ ., J. Antibiotics 36, 1020 (1983~.

B. 2-(5-t-Butoxycarbonylamino-1,2,4-thiadiaæol-3-yl)-2-cYclo-pentyloxx_minoacetic acid A solution of 500 mg (1.34 mmoles) of methyl 2-(5-t-butoxycarbonylamino-1,2,4-thiadiazol-3-yl)-2-cyclopentyloxy-iminoacetate and 2N NaO~ solution ~2 ml, 4 mmoles ) in 15 ml of CH30H was refluxed for 30 minutes. The reaction mixture was evaporated and 10 ~1 of ethyl acetate-H2O (1:1) wa~ added to the solution. The water layer was separated, acidified to pH 2 with 6N HC1 and extracted with ethyl acetate (10 ml x 2~. The organic layer was washed with brine, dried over MgSO4 and concentrated under reduced pressure to give 377 mg (78~) of the title compound. Mp. 185DC (dec.).

IR : vmax ( KBr ) in cm 3160, 1710, 1550.

max (C2H5OH) in nm (e) 238 (13300) NMR : ~ (DMSO) in ppm 1.51 (9H, s, BOC-H), 1.70 (8~, br.s., H ~ ), 4.82 (lH, m, ~ ).

C. 2-!5-Amino-1,2,4-thiadiazol-3-yl)-2-cyclopentyloxyimino-acetic acid (III-5, Z isomer) _ _ __ A solution of 348 mg ~0.37 mmoles) of 2-(5-t-butoxy-carbonylamlno-1,2,4-thiadiazol-3-yl)-2-cyclopentyloxyiminoacetic ~ ~17~

acid in 2 ml of trifluoroacetic acid was allowed to stand for 1 hour at room temperature. The reaction mixture was concentrated under reduced pressure. The residue was triturated with 5 ml of isopropyl ether and 10 ml of hexane to give 215 mg (86%~ of the title compound. Mp. 162-165C (dec.) [lit3): mp. 160-165C
(dec.)].

IR : vmax (Nujol) in cm 1 3290, 3200, lilO, 1615, 1600.

max (C2H50H) in nm (E) 238 (13300) N~ (DMSO-d6) in ppm 1.17-2.10 (8H, m), 4.60-4.98 (lH, m), 8.22 (2H, S).

3) Japan Rokai 57-15R769 (9/30/82, Fujisawa) Brit. appl. 8107134 (3/6/81) .

D. 2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-cy~lopentyloxy-lmin acetyl chloride hydrochloride A solution of 190 mg (O. 74 mmole) of III-S and 219 mg (1.0 mmole) of phosphorous pentachloride in dichloromethane (5 ml) was stirred for 1 hour at room temperature. The reaction mixture was poured into 50 ml of n-hexane. The resulting precipitate was collected by filtration. Yield 122 mg (60~).

IR : vmax (Nujol) in cm 1 1760.

Preparation No. 28 Benzotriazol-l-yl-2-~5-amino-1,2,4-thiadiazol-3-yl)-2-methoxy-iminoacetate A mixture of l-hydroxybenzotriazole (207 g, 20 mmoles) and dicyclohexylcarbodiimide (4.12 g, 20 mmoles) in 65 ml of DMF
was stirred at room temperature. After 15 minutes, IIX-l (4.04 g, 20 mmoles~ was added to the stirring mixture at 0C, and stirring was continued for 3 hours. The reaction mix~ure was ~769i~g filtered to remove the insoluble urea, and the filter cake was washed with a small volume of DMF. The filtrate and washings were combined and poured into 800 ml of ice water. The precipitate was collected by filtration to give 5.24 g (82%) of the title compound as a light grey powder. Mp. 189-192C (dec. ) .

IR : vmax (KBr) in cm 1 1815, 1620, 1540, 1415, 1090, 1060, 1005, 945, 865, 740, max (C2~5O~) in nm (El ) 246 (580), 283sh (228).

Claims (27)

1. A compound of the formula I

wherein R1 is hydrogen or a conventional amino-protecting group, R2 is hydrogen, a straight or branched chain alkyl group contain-ing from 1 to 4 carbon atoms, a cycloalkyl or cycloalkenyl ring containing from 3 to 6 carbon atoms, or a group of the formula , ; or , in which R3 is hydrogen, (lower)alkyl or carboxyl, X is halogen, hydroxy or (lower)alkoxy, and R4 and R5 are each independently hydrogen, methyl or ethyl, or R4 and R5, taken together with the carbon atom to which they are attached, may be a cycloalkylidene ring containing from 3 to 5 carbon atoms, and is a quaternary ammonio group; or a nontoxic pharmaceutically acceptable salt, solvate, hydrate or physiologically hydrolyzable ester thereof.

2. A compound of Claim 1 wherein is selected from , , , , and wherein R13, R14 and R15 are the same or different and are (lower)alkyl, (lower)alkenyl, amino(lower)alkyl with the pro-vision that the amino may not be on an .alpha.-carbon, or hydroxy-(lower)alkyl with the provision that the hydroxy group may not be on an .alpha.- carbon;

R16 is hydrogen, (lower)alkyl, (lower)alkoxy, (lower)-alkylthio, amino, (lower)alkylamino, di(lower)alkylamino, formyl-amino, (lower)alkanoylamino, carboxy, hydroxy, carboxy(lower)-alkyl, carboxy(lower)alkylthio, hydroxy(lower)alkyl, halo(lower)alkyl, amino(lower)alkyl, (lower)alkoxy(lower)alkyl, carbamoyl or N-(lower)alkylcarbamoyl, or R16 may represent a divalent alkylene group having 3 to 5 carbon atoms;

R17 is (lower)alkyl, (lower)alkoxy(lower)alkyl, halo-(lower)alkyl, allyl, hydroxy(lower)alkyl with the provision that the hydroxy group is not on the .alpha.-carbon, amino(lower)alkyl with the provision that the amino group is not on the .alpha.-carbon, or phenyl(lower)alkyl;

R18 is hydrogen, (lower)alkyl, (lower)alkoxy, (lower)-alkoxy(lower)alkyl, (lower)alkylthio, amino, (lower)alkylamino, di(lower)alkylamino, carboxy, hydroxy, carboxy(lower)alkyl, hydroxy(lower)alkyl, amino(lower)alkyl, formylamino, (lower)-alkanoylamino, carbamoyl or N-(lower)alkylcarbamoyl;

n is an integer of from 1 to 3, inclusive;

Z is CH2 or, when n is 2, Z also may be S, O or N-R19, in which R19 is hydrogen or (lower)alkyl; and R20 and R21 are the same or different and are hydrogen, (lower)alkyl, (lower)alkoxy, (lower)alkylthio, amino, (lower)-alkylamino, di(lower)alkylamino, carboxy, hydroxy, hydroxy-(lower)alkyl, amino(lower)alkyl, (lower)alkoxy(lower)alkyl, carboxy(lower)alkyl, carboxy(lower)alkylamino, (lower)alkanoyl-amino, carboxy(lower)alkanoylamino, carbamoyl or N-(lower)alkyl-carbamoyl.

3. A compound of Claim 2 wherein R2 is (lower)alkyl, cycloalkyl of 3 to 5 carbon atoms, 1-carboxycycloalk-1-yl of 3 to 5 carbon atoms, allyl, propargyl or carboxy(lower)alkyl.

4. The compound of Claim 3 which is 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-(trimethyl-ammonio)-1-propen-1-yl]-3-cephem-4-carboxylate, or a nontoxic pharmaceutically acceptable salt, solvate, hydrate or physiologically hydrolyzable ester thereof.

5. The compound of Claim 3 whioh is 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-(1-methyl-pyrrolidinio)-l-propen-l-yl]-3-cephem-4-carboxylate, or a nontoxic pharmaceutically acceptable salt, solvate, hydrate or physiologically hydrolyzable ester thereof.

6. The compound of Claim 3 which is 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-pyridinio-1-propen-l-yl]-3-cephem-4-carboxylate, or a nontoxic pharmaceutically acceptable salt, solvate, hydrate or physiologically hydrolyzable ester thereof.

7. The compound of Claim 3 which is 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-(3-amino-pyridinio)-1-propen-1-yl]-3-cephem-4-carboxylate, or a nontoxic pharmaceutically acceptable salt, solvate, hydrate or physiologically hydrolyzable ester thereof.

8. The compound of Claim 3 which is 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-(3-formylamino-pyridinio)-1-propen-1-yl]-3-cephem-4-carboxylate, or a nontoxic pharmaceutically acceptable salt, solvate, hydrate or physiologically hydrolyzable ester thereof.

9. The compound of Claim 3 which is 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-13-(3-aminomethyl-pyridinio)-1-propen-1-yl]-3-cephem-4-carboxylate, or a nontoxic pharmaceutically acceptable salt, solvate, hydrate or physiologically hydrolyzable ester thereof.

10. The compound of Claim 3 which is 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-(3-carbamoyl-pyridinio)-1-propen-1-yl]-3-cephem-4-carboxylate, or a nontoxic pharmaceutically acceptable salt, solvate, hydrate or physiologically hydrolyzable ester thereof.

11. The compound of Claim 3 which is 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-(4-carbamoyl-pyridinio)-1-propen-1-yl)-3-cephem-4-çarboxylate, or a nontoxic pharmaceutically acceptable salt, solvate, hydrate or physiologically hydrolyzable ester thereof.

12. The compound of Claim 3 which is 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-(2-methyl-thiazolio)-1-propen-1-yl]-3-cephem-4-carboxylate, or a nontoxic pharmaceutically acceptable salt, solvate, hydrate or physiologically hydrolyzable ester thereof.

13. The compound of Claim 3 which is 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-(2-amino-5-thiazolo[4,5-c]pyridinio)-1-propen-1-yl]-3-cephem-4-carboxylate, or a nontoxic pharmaceutically acceptable salt, solvate, hydrate or physiologically hydrolyzable ester thereof.

14. The compound of Claim 3 which is 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-(4-hydroxymethyl-pyridinio)-1 propen-1-yl]-3-cephem-4-carboxylate, or a nontoxic pharmaceutically acceptable salt, solvate, hydrate or physiologically hydrolyzable ester thereof.

15. The compound of Claim 3 which is 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-(3-hydroxymethyl-pyridinio)-1-propen-1-yl]-3-cephem-4-carboxylate, or a nontoxic pharmaceutically acceptable salt, solvate, hydrate or physiologically hydrolyzable ester thereof.

16. The compound of Claim 3 which is 7-[2-(5-amino-1,2,4-tbiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-(4-{N-methyl-carbamoyl}pyridinis)-1-propen-1-yl]-3-cephem-4-carboxylate, or a nontoxic pharmaceutically acceptable salt, solvate, hydrate or physiologically hydrolyzable ester thereof.

17. The compound of Claim 3 which is 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-(2,3-propylene-pyridinio)-1-propen-1-yl]-3-cephem-4-carboxylate, or a nontoxic pharmaceutically acceptable salt, solvate, hydrate or physiologically hydrolyzable ester thereof.

18. The compound of Claim 3 which is 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-ethoxyiminoacetamido]-3-[3-(4-carbamoyl-pyridinio)-1-propen-1-yl]-3-cephem-4-carboxylate, or a nontoxic pharmaceutically acceptable salt, solvate, hydrate or physiologically hydrolyzable ester thereof.

19. The compound of Claim 3 which is 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-cyclopentyloxyiminoacetamido]-3-[3-(4-carbamoylpyridinio)-1-propen-1-yl]-3-cephem-4-carboxylate, or a nontoxic pharmaceutically acceptable salt, solvate, hydrate or physiologically hydrolyzable ester thereof.

20. The compound of Claim 3 which is 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-allyloxyiminoacetamido]-3-[3-(4-carbamoyl-pyridiniol)-l-propen-l-yl]-3-cephem-4-carboxylate, or a nontoxic pharmaceutically acceptable salt, solvate, hydrate or physiologically hydrolyzable ester thereof.

21. The compound of Claim 3 which is 7-[2-(5-amino 1,2,4-thiadiazol-3-yl)-2-propargyloxyiminoacetamido]-3-[3-(4-carbamoylpyridinio)-l-propen-l-yl]-3-cephem-4-carboxylate, or a nontoxic pharmaceutically acceptable salt, solvate, hydrate or physiologically hydrolyzable ester thereof.

22. The compound of Claim 3 which is 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-(4-carboxy-pyridinio)-1-propen-1-yl]-3-cephem-4-carboxylate, or a nontoxic pharmaceutically acceptable salt, solvate, hydrate or physiologically hydrolyzable ester thereof.

23. The compound of Claim 3 which is 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-ethoxyiminoacetamido]-3-[3-(4-carboxy-pyridinio)-2-propen-1-yl]-3-cephem-4-carboxylate, or a nontoxic pharmaceutically acceptable salt, solvate, hydrate or physiologically hydrolyzable ester thereof.

24. The compound of Claim 3 which is 7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-( 3-carboxymethyl-pyridinio)-l-propen-l-yl]-3-cephem-4-carboxylate, or a nontoxic pharmaceutically acceptable salt, solvate, hydrate or physiologically hydr olyzable ester thereof.

25. The compound of Claim 3 which is 7-[2-(5-amino-1, 2, 4-thiadiazol-3-yl)-2-methoxyiminoacetamido]-3-[3-(4-carboxymethyl-thiopyridinio)-l-propen-l-yl]-3-cephem-4-carboxylate, or a nontoxic pharmaceutically acceptable salt, solvate, hydrate or physiologically hydrolyzable ester thereof.

26. A process for the preparation of compounds of the formula I

wherein R1 is hydrogen or a conventional amino-protecting group, R2 is hydrogen, a straight or branched chain alkyl group con-taining from 1 to 4 carbon atoms, a cycloalkyl or cycloalkenyl ring containing from 3 to 6 carbon atoms, or a group of the formula , , or , in which R3 is hydrogen, (lower)alkyl or carboxyl, X is halogen, hydroxy or (lower)alkoxy, and R4 and R5 are each independently hydrogen, methyl or ethyl, or R4 nnd R5, taken together with the carbon atom to which they are attached, may be a cyclo-alkylidene ring containing from 3 to 5 carbon atoms, and is a quaternary ammonio group, and nontoxic pharmaceutically acceptable salts and physiologically hydrolyzable esters thereof, which process Comprises reacting a compound of the formula with an acid of the formula or with an acylating derivative of said acid, wherein R2' is the same as R2 or is a group of the formula or in which X, R4 and R5 are as defined above, B1 is a conventional carboxyl-protecting group and B2 is hydrogen or a conventional amino-protecting group.

27. A pharmaceutical composition which comprises an effective antibacterial amount of at least one compound of formula I as defined in Claim 1, or a salt, hydrate or solvate thereof, together with a pharmaceutically acceptable carrier or excipient.