CH627184A5 - Process for the preparation of antibacterial agents - Google Patents

Description

The present invention relates to the production of cephalosporins, which have the general virtues of compounds of this kind and which are particularly advantageous to use in the treatment of bacterial infections.

Cephalosporins constitute a well-known group of semisynthetic antibacterial agents prepared initially, for example, little: acylation of the 7-amino group of the nucleus of 7-aminocephalosporanic acid (7-ACA) then by a similar acylation of nuclei derived from this acid , for example by modification of its substituent in position 3. These compounds have been reviewed in various publications published in the scientific literature (see, for example, "Cephalosporins and Penicillins - Chemistry and Biology", edited by Edwin H. Flynn, Academic Press, New York, 1972, in particular pages 554-569) and in the patent literature, for example in the patents of the United States of America No. 3,687,948, No. 3,741,965, No. 3,743,644 , N ° 3 759 904, N ° 3 759 905, N ° 3 766 175, N ° 3 766 906, N ° 3 769 281, N ° 3 769 801, N ° 3 799 923, N ° 3 812 116, N ° 3 813 388, N ° 3 814 754 and N ° 3 814 755 (all belonging to class 260-243C).

Patents have been filed on thiolated cephalosporins in position 3, in which the substituent in position 7 represents:

a) an a-amino-a-phenylacetamido group; these are the United States patents No. 3,641,021, No. 3,734,907, No. 3,687,948 cited above, No. 3,741,965 cited above, No. 3,757,015, No. 3,743,644 cited above; Japanese patent N ° 71/24,400 (Farmdoc 46374S), Belgian patent N ° 776,222 (Farmdoc 38983T; British patent N ° 1,328,340, with various substituents on the benzene nucleus), Belgian patent N ° 772,592 ( Farmdoc 19696T; U.S. Patents No. 3,687,948 supra, No.

3,734,907 and No. 3,757,012), the patent of the Federal Republic of Germany No. 2,202,275 (Farmdoc 50428T) corresponding to the aforementioned United States patent No. 3,759,904; Netherlands Patent No. 7,205,644 (Farmdoc 76309T; United States Patent No. 3,757,014);

b) an o-, m- or p-aminoethoxyphenylacetamido group; this is the Netherlands patent No 72/13 968 (Farmdoc 24740U) corresponding to the United States patent No

3,759,905 supra);

c) an o-aminomethylphenylacetamido group; these are United States Patent Nos. 3,766,176 and No.

3,766,175 cited above (which also review the prior patent literature relating to substituted 7-phenylacetami-docephalosporanic acids);

d) an N- (phenylacetimidoyl) aminoacetamido group; this is United States Patent No. 3,692,779; and e) an a-amino-a- (1,4-cyclohexadienyl) acetamido group; it is the Belgian patent N ° 776 222 cited above (Farmdoc 38983T; British patent N ° 1 328 340 cited above).

Other similar descriptions are given in U.S. Patent No. 3,692,779 (Belgian Patent No. 771,189; Farmdoc 12819T), Japanese Patent No. 72/05 550 (Farmdoc 12921T), the patent Japanese No. 72/05 551 (Farmdoc 12922T), U.S. Patent No. 3,719,673 (Belgian Patent No. 759,570; Farmdoc 39819S), Belgian Patent No. 793,311 (Farmdoc 39702U) and Belgian Patent No. 793,191 (Farmdoc 39684U).

Descriptions of thiolated cephalosporins in position 3, in which the substituent 7 is a 7-mandelamido- (7-a-hydroxyphenylacetamido) group, are given, for example, in United States Patent No. 3,641,021 , French Brevet No. 7,310,112, United States Patent No. 3,796,801, British Patent No. 1,328,340 (Farmdoc 38983T), United States Patent N No. 3,701,775, Japanese Patent No. 48-44,293 (Farmdoc 55334U), and in Hoover et al., "J. Med. Chem. " 17 (1), 34—41 io (1974) and in Wiek et al., “Antimicrobial Ag. Chemo.”, 1 (3), 221-234 (1972).

U.S. Patent No. 3,819,623 (as well as, for example, British Patent No. 1,295,841 and the Federal Republic of Germany Patent No. 1,953,861) i5 describes in particular , with practical details, the preparation of 2-mercapto-l, 3,4-thiadiazole-5-acetic acid and its transformation into acid 7- (1H-tetrazol-l-yl-acetamido) -3- (5 -carb-oxymethyl-1,3,4-thiadiazol-2-ylthiomethyl) -3-cephem-4-carb-oxyl which is also described in the patent application of the Federal Republic of Germany DOS No. 2,262,262.

U.S. Patents 3,776,175 supra and 3,898,217, respectively, describe a compound of the formula:

25

30

CSiNHi

O 8

CHr-ê — NH — CH— CH CH »

0 = è N Ì — CHr-S— a

V

èooH

in which R is a group of formula:

35

TT

W

N — CEU N N = N

• -u - u

40 or a pharmaceutically acceptable non-toxic salt of this compound and a compound of formula:

55

CHA

wherein R is a hydrogen atom or a lower alkyl group; R 'is a hydrogen atom, a lower alkanoyloxy group or a group of formula:

N — N

-O • -s4U-1Uciv.

60

OR

v -, - Ç

65

(where n is an integer of 4-7) and their pharmaceutically acceptable acid addition salts.

627184

U.S. Patents 3,883,520 and 3,931,160 and Farmdoc 22850W describe 3-heterocyclo-thiomethyl cephalosporins carrying multiple substituents (including a carboxyl group) on the many heterocycles they contain , but these references are of a very general nature and do not indicate physical constants, yields, methods of synthesis, etc., and do not even name a compound containing a carboxyl substituent.

U.S. Patent No. 3,928,336 reviews much of the prior knowledge of cephalosporins.

U.S. Patents 3,907,786 and 5,946,000 describe cephalosporins containing various bicyclic thiols with condensed nuclei.

We find in "Farmdoc" 18830X the description of compounds of formula:

not:

-s- \

-n li,

c00h n- \

(ch2) n-c00h in which R1 is an acyl group or a hydrogen atom, R3 is a hydrogen atom or a methoxy group and n is equal to 1-9).

The present invention provides a process for the production of compounds of formula I as defined in claim 1.

The esters of the compounds of formula I include, without limitation, those which carry the group:

W

-CH

in which, when W is a hydrogen atom, Z represents a lower alkanoyl, benzoyl, naphthoyl, furoyl, thenoyl, nitrobenzoyl, methylbenzoyl, halobenzoyl group,

25

phenylbenzoyl, N-phthalimido, N-succinimido, N-saccharino, N- (lower alkyl) carbamoyl, lower alkoxy, (lower alkyl) thio, phenoxy, carbalkoxy, carbobenzoxy, carbamoyl, benzyloxy, chlorobenzyloxy, carbophenoxy, carbo-tertio -but-oxy or (lower alkyl) sulfonyl; when W represents a carbalkoxy group, Z is a carbalkoxy group; when W represents a phenyl group, Z is a benzoyl group or a cyano group; or W and Z combine to form a 2-oxocycloalkyl group containing 4 to 8 carbon atoms. Advantageous examples of compounds of the invention include the esters of pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl, ß, ß, ß-trichlorethyl, 3 -phthalidyle and 5-indanyl.

The acyl group (R1) is a group corresponding to one of the 35 formulas:

ch2nhr «

r- / vch2c0-

y ^ C ^ NHR1

u-

ch2c0-

and laughed ch2nhr '

S 'CH2C0-

in which R is a hydrogen atom, a hydroxy group An advantageous embodiment of the present invention or methoxy and R 'is a hydrogen atom or a methyl group comprises the production of the compounds of formula I in thyle.

which R1 is a group of formula:

ch2nh2

ch2co-

CHgNHg ch2nh2

chgco-

and

^ ch2nh2

ch2c0-

Another advantageous embodiment of the present invention comprises the compounds of formula I in which R1 is one of the following groups:

5

627,184

CHgNHCH ^

ch2co-

CHgNHCH ^

ch „nhch-

.ch2nhch3

and

An advantageous embodiment of the present invention comprises the compounds which correspond to the formula:

R1 -NH-CH-CH CH I I | 2

/ -% ^ c-0h2s

0 \

C-om

If

O

in which R1 is a group of formula:

^ CHJTHR '

vchsco-

where R is a hydrogen atom, a hydroxy or methoxy radical; R 'is a hydrogen atom or a methyl radical; n is 1 or 2 and M is a group of formula:

LqJ-CH2C0 '

"

N ~ ^ CH2 ^ nC00H

or aCH2ttKR '

ch2c0-

or

CH2nhr1

chgco-

0

ii

0

-ckoc (cha) nr,

II

-chcc (cha) nc

'R:

r-

10

0

he .

-ckxcor r10

or

^ r nr4r5

0

ii r

1 0

'-ch-s-c-r6 r10

where n is 0-4; R is a hydrogen atom, an alkyl group having 1 to 8 carbon atoms, a cycloalkyl group having 3 to 6 carbon atoms, phenyl, phenylalkyl having a C 4 to C 4 alkyl radical, pyridyl, thienyl or pyrrolyl; R10 is a hydrogen atom, a methyl or ethyl group; R2 and R3 each represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, phenyl, pyridyl or thienyl; R4 and R5 each represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms; R6 is an alkyl group having 1 to 4 carbon atoms phenyl, phenalkyl with a C1-C4 alkyl radical, pyridyl, thiadiazolyl, amino or Q-C4 alkylamino; X is an NH group or an oxygen atom;

65 and each phenyl group is devoid of substituents or carries one or two substituents chosen from alkyl radicals having 1 to 6 carbon atoms, alkoxy having 1 to 4 carbon atoms, hydroxy, amino, NHR1, N (R1) 2, nitro , fluoro, chloro, bromo or

627 184 6

carboxy; these compounds also exist in the form of salts. Another advantageous embodiment of the present pharmaceutically acceptable. invention includes the compounds of formula:

R1 -NH-CH-

, //

YOUR

0

in which R1 is a group of formula:

-vs

~ ck2 c — cHa: C-OM

ii

0

-M "

Ï- (CH 3) nCCŒ

CHoMR1

ch2co-

or

CKpHrIR1

-CH2C0-

or

He ji

CHpNHR

CHa-CO-

where R is a hydrogen atom, a hydroxy or methoxy group; R 'is a hydrogen atom or a methyl group; n is 1 or 2 and M is a group of formula:

CH3 O C2Hs O I II I 11

-CH-O-C-R6, -CH-C-R6,

25

R5

o alkyl aralkyl or -CH-X2-C-OR7

wherein R5 is a hydrogen atom, a methyl or ethyl group; X2 represents -O- or -NH-; R6 is a basic group such as an alkyl or aralkyl group carrying a substituted or unsubstituted NH2 radical, for example an alkyl-NHCH3, aralkyl-NHCH3 group,

'T "®

-CHgNHgOu-CH-CHg nh „

R7 is an alkyl group such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl or 2-ethylhexyl; a cy-cloalkyl group such as cyclopropyl, cyclobutyl, cyclopentyl, cyclo-hexyl or cycloheptyl; an aryl group such as phenyl or naphthyl; an aralkyl group such as benzyl or naphthylmethyl; or a heterocyclic group, the alkyl, cycloalkyl, aryl, aralkyl and heterocyclic groups being able to carry one or more substituents chosen from amino groups, substituted amino groups such as methylamino, diethylamino

45 or acetamido groups, halogens such as fluorine, chlorine or bromine, nitro groups, alkoxy groups such as methoxy, ethoxy, propyloxy, isopropyloxy, butoxy or iso-butoxy; these compounds also exist in the form of non-toxic pharmaceutically acceptable salts.

With the compounds obtained according to the invention, a pharmaceutical composition can be formulated containing an antibacterial effective amount of a compound of formula:

R1- ÏÎH-CH-CH CH Y ^

C — N C — CHpS-J ^ vN - ^ / N-fCHg ^ COOH

o c \

C-OM

not

0

in which R1 is a group of formula:

7

627184

nyïHR1

R "\ ^ CE2C ° -

or

C.CH2NHR 'l 0h2c0-

where

ch2nhr *

ch2co-

where R is a hydrogen atom or a hydroxy or methoxy radical; R 'is a hydrogen atom or a methyl radical; n is equal to 1 or 2 and M is a hydrogen atom, a pivaloyloxymethyl group, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl, ß, ß, ß-trichIorethyIe, 3-phta-lidyl or 5 -indanyl, preferably a hydrogen atom or a non-toxic salt acceptable from the pharmaceutical point of view of this compound.

r ^ -nh -ch-ch ch! I | 2 c — n c — ch ^ s

0 \

c-om

II

0

(in which R1 is a group of formula:

The compounds obtained according to the invention allow the treatment of bacterial infections, treatment which consists in administering by injection to a warm-blooded animal or a human being infected with an effective but non-toxic dose of 250 to 1000 mg of a compound of formula:

, N ^^ n- (ch2) nc0 ° h

: h2nhr '

ch, c0-

or l! l — ch2c0-

or

U-

CH ^ NHR

ch2co-

where R is a hydrogen atom or a hydroxy or methoxy radical; R 'is a hydrogen atom or a methyl radical; n is 1 or 2 and M is a hydrogen atom or a pivaloyloxymethyl, acetoxymethyl, methoxymethyl, aceto-nyl, phenacyl, p-nitrobenzyl, ß, ß, ß-trichlorethyl, 3-phta- group

lidyl or 5-indanyl) or a non-toxic pharmaceutically acceptable salt of this compound.

Shig infections. dvsenteriae can be combated by administering to a mammal suffering from such an infection an amount effective for the treatment of this infection of a composition containing a compound of formula:

(ch2) nc0 ° h

(in which R1 is a group of formula:

O

chpco-

or ch3CO-

OR

ch2nhr *

,co-

where R is a hydrogen atom or a hydroxy or methoxy radical; R 'is a hydrogen atom or a methyl group; n is 1 or 2 and M is a hydrogen atom, a pivaloyloxymethyl group, acetoxymethyl, methoxymethyl, aceto-

65 nyle, phenacyl, p-nitrobenzyl, ß, ß, ß-trichlorethyl, 3-phta-lidyl or 5-indanyl, preferably a hydrogen atom) or a pharmaceutically acceptable non-toxic salt of this compound.

627 184 8

The compounds of the invention also make it possible to be effective in the treatment of such an infection, a compound

beat B. anthracis infections by administration to a tion containing a compound of the formula:

mammal with B. anthracis infection of a quantity,

i h1 - ìth-ch-ch ch2

c — n ^ c ~ ch2s

0 C \

c-om

II

0

"- (ch ^) ncooh

(in which R1 is a group of formula:

^ H2NHR '

ch3co-

or

CH ^ WR 'chaco-

or

CH ^ THH

ch2co-

where R is a hydrogen atom or a hydroxy or mididyl or 5-indanyl radical, preferably hydrogen) or a non-thoxy salt; R 'is a hydrogen atom or a methyl radical; n is toxic acceptable from a pharmaceutical point of view

equal to 1 or 2 and M is a hydrogen atom or a pivaloyloxymethyl, acetoxymethyl, methoxymethyl, aceto-nyl, phenacyl, p-nitrobenzyl, ß, ß, ß-trichIorethyle, 3-phta- radical

30

pose.

The present invention relates to the process for producing the antibacterial agents of formula:

v

, n- (chs) nc0c: -i

(I)

cocr as defined in claim 1.

r1 -nh-ch-

i-

^ S \

■ ch ch2

O

I!

s

■ n x-ch-o-c-chs

) * (HI)

cooh

(in which R1 is an acyl group) (including the cephalosporin C itself) with a compound of formula:

H S-

NOT

■ N

f (IV)

n- (ch3) nc00h

0

The salts of the compounds obtained according to the invention include the salts of non-toxic carboxylic acids of these compounds, among which mention is made of non-toxic metal salts such as the sodium, potassium, calcium and aluminum salts, the ammonium salt and substituted ammonium salts

such as non-toxic amine salts such as trialkylamines including triethylamine, procaine, dibenzylamine, N-benzyl-beta-phenethylamine, 1-ephenamine, N, N'-di-benzylethylenediamine, dehydroabiethylamine, N, N'-bis-45 dehydroabiethylethylenediamine, an N- (lower alkyl) -pi-peridine such as N-ethylpiperidine as well as other amines which have been used to form salts with the benzylpenicil-line; and their non-toxic acid addition salts (i.e., amine salts) including the addition salts of 50 mineral acids such as hydrochlorides, hydrobromides, iodhydrates, sulfates, sulfamates and phosphates and the addition salts of orange acids such as maleates, acetates, citrates, Oxalates, suc-cinates, benzoates, tartrates, fumarates, malates, mandelates, ascorbates, etc.

55 The amino group in the compounds (used as intermediates or as metabolic precursors) can be "protected" by substituents such as 2-iodethoxycarbonyl groups (British Patent No. 1,349,673), tert-butoxycarbonyl, carbobenzyloxy , formyl, o-nitrophenylsulfenyl, 60 ß, ß, ß-trichlorethoxycarbonyl, 4-oxo-2-pentenyl-2,

l-carbomethoxy-1-propenyl-2, etc. Among these protecting groups are in particular ketones (in particular acetone) and aldehydes (in particular formaldehyde and acetal-dehyde) described, for example, in United States patents 65 of America No. 3,198,804 and No. 3,347,851, and the ß-cötoesters and P-diketones described, for example, in United States Patent No. 3,325,479 and the ß-cötoamides described in Japanese Patent No. 71 / 24,714 (Farmdoc 47321S).

9

627,184

The preferred esters of the cephalosporins of the present invention are the esters of pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl and phenacyl. All are useful intermediates for use in the production of free carboxyl group cephalosporin.

As indicated above, these five esters of 7-amino-cephalosporanic acid are each prepared by known methods. An excellent process is that described in U.S. Patent No. 3,284,451, in which the sodium salt of cephalothin is esterified by reaction with the corresponding chlorine or bromine compound (e.g. bromide phenacyl, chloroacetone, chloromethyl ether, pivaloyloxymethyl chloride (also called chlo-romethyl pivalate), acetoxymethyl chloride), then the side chain of thienylacetic acid is eliminated enzymatically as described in this same patent or chemically as described in U.S. Patent No. 3,575,970 and in the journal "Journal of Antibiotics", XXIV (11), 767-773 (1971). In another suitable method, the triethylamine salt of 7-aminocephalosporanic acid is reacted directly with the compound carrying an active halogen, as described in British Patent No. 1,229,453.

These 7-aminocephalosporanic acid esters are then reacted with the nucleophile HSR3 as described herein for 7-aminocephalosporanic acid itself. The 3-thiol ester of 7-aminocephalosporanic acid is then combined with the organic carboxylic acid R'-OH, as above. Before or after the elimination of any protective group carried, for example, by an amino group of the side chain in position 7, the cephalosporin ester thus obtained is transformed, insofar as it is not used under this form, in corresponding free acid, including its zwitterion (and, where appropriate, any salt) by elimination of the esterifying group, for example by aqueous or enzymatic hydrolysis (as in the case of human serum or of animal serum ) or by acid or alkaline hydrolysis or by treatment with sodium thiophenoxide, as described in United States Patent No. 3,284,451 or, in the penicillin series, by Sheehan et al in "J. Org. Chem. 29 (7), 2006-2008 (1964).

In another synthetic variant, the 7-aminocephalosporanic acid thiolated in position 3 is prepared as described in the present specification then acylated on the amino group in position 7 and finally esterified, for example by reaction of the corresponding alcohol with the chloride of acid prepared, for example, by reaction of the final cephalosporin with thionyl chloride or by other esterification operations in an essentially acid medium.

In the treatment of bacterial infections in humans, the compounds of the invention can be administered parenterally in an amount of about 5 to 200 mg / kg / day and preferably about 5 to 20 mg / kg / day in several doses, for example 3 to 4 times a day. They are administered as dosage units containing, for example, 125, 250 or 500 mg of active ingredient, with suitable physiologically acceptable vehicles or excipients. The dosage units can be in the form of liquid preparations such as solutions or suspension.

The other reactants used to prepare the compounds of the present invention can be obtained synthetically, either as described in practice (for example as described in the patents and in the abovementioned publications), or by strictly analogous operations . However, for convenience and by way of illustration, some of these particular syntheses are indicated below, for example for preparing carboxylic acids containing a free amino group which is "protected" by a tert-butoxycarbonyl group.

2- (tert-butoxycarbonylaminomethyl) -1,4-cyclohexadi-enylacetic acid

Diluted slowly with 500 ml of anhydrous tert-butanol, a solution of 16.5 g (0.1 mole) of o-aminomethyl-5-nylacetic acid in 1.5 liters of liquid ammonia (which has been treated with 50 mg of lithium to remove traces of moisture). 3.4 g (0.5 gram atom) of lithium are added to the solution in small portions over a period of 4 hours and the mixture is stirred for 16 hours at room temperature, removing the liquid ammonia in a hood. and finally evaporated to dryness below 40 ° C. The residue is dissolved in 500 ml of water and the solution is chromatographed on a column of "IR-120" resin (H + form, 700 ml) and eluted with a 1% solution of ammonia. The eluate fractions which give a positive reaction in the ninhydrin test are combined and evaporated to dryness. The residue is washed with four 50 ml portions of hot acetone and recrystallized from 500 ml of 1: 1 mixture of ethanol and water to give 11.2 g (67%) of colorless acid needles o - (2-aminomethyl-1,4-cyclohexadienyl) acetic acid 2-odant at 183 ° C.

Infrared spectrum: 'v 1630,1520,1380,1356e111 "1.

Nuclear magnetic resonance: ôD20 + K2C03

25

2.72 (4H, singlet, H2C <^), 3.01 (2H, singlet,

CHzCO), 3.20 (2H, singlet, CH2-N), 5.78 (2H, singlet, H ^> C =).

30

C% H% N% 64.65 7.84 8.38 64.77 8.06 8.44

35 Improved process for the preparation of α- (2-aminomethyl-1,4-cyclohexadienyl) acetic acid

Analysis:

Calculated for C9H13N02: Found:

Li, tert.-BuOH

*

NH, -TEA • HCl

(/ \ —CH2C02H LiCl + (C2H5) 5N

The method of Example 1 of US Patent No. 3,720,665 is adapted to the preparation of D-2-amino-2- (1,4-cyclohexadienyl) acetic acid. A solution of 830 ml of distilled liquid ammonia is dried with 40 mg of lithium under an argon atmosphere. To this solution is added with stirring, 11.0 g (0.07 mol) of 2-aminomethylphé-60 nyl-acetic acid and 340 ml of tert-butanol. A total amount of 1.6 g (0.225 mol) of lithium is added to the solution with energy stirring over a period of 2 hours. The gray mixture is then treated with 35 g (0.215 mole) of triethylamine hydrochloride (TEA) and stirred for approximately 65 hours at room temperature. The tert-butanol is removed at 40 ° (15 mm), leaving a white residue which is dried under vacuum over phosphorus pentoxide for about 18 hours. The solid is dissolved in 30 ml of a 1: 1 mixture of

627,184

10

methanol and water and the solution is added with stirring to 3.5 liters of 1: 1 mixture of chloroform and acetone at 5 ° C. The mixture is stirred for 20 minutes and the amino acid, namely α- (2-aminomethyl-1,4-cyclohexadienyl) acetic acid, is collected and dried for 16 hours under vacuum over phosphorus pentoxide, giving 6.3 g (58%) of white crystals melting at 190 ° as they decompose. The infrared spectrum and the nuclear magnetic resonance spectrum agree with the formula.

A solution of 19.31 g (0.135 mole) of tert-butoxycarbonylazide in 152 ml of tetrahydrofuran (THF) is added to a stirred solution of 14.89 g (0.09 mole) of 2-aminomethyl-1 acid, 4-cyclohexadienyl) acetic acid and 7.20 g (0.18 mole) of sodium hydroxide in 281 ml of water. The solution is stirred for 18 hours at 25 °, then filtered through diatomaceous earth ("Super-cel"). The tetrahydrofuran is removed at 40 ° (15 mm) and the residual solution is washed with twice 175 ml of ether and then acidified with 6N hydrochloric acid. The mixture is stirred in an ice bath and the precipitate is collected and then left for 18 hours under vacuum on phosphorus pentoxide at 25 ° to obtain 17.3 g (72.6%) of 2- (tertiary- butoxycarbonylaminomethyl) -1,4-cyclohexadienylacetics in the form of a white powder. The infrared spectrum and the nuclear magnetic resonance spectrum agree with the formula.

Preparation of 3-aminomethyl-2-thiophene-acetic acid

CH2-NH2

then it is extracted correctly with ether (twice 200 ml). The extraction phases are combined with ether, dried over magnesium sulfate and concentrated. The residue is distilled under reduced pressure and the pale yellow oil is collected at 100-5125 ° C under a vacuum of 0.7 mm of mercury. Yield 277 g (56%).

Infrared spectrum: vjj ^ 3110,1660,1100 cm "1, io Nuclear magnetic resonance: ò PFjm 3.40 (6H,

singlet, OCH3), 5.86 (1H, singlet, CH <^ J, 7,

27

not

S CH2-C02H

A) Thiophene-3-carboxaldehyde dimethylacetal (2a)

A mixture of 322 g (2.9 moles) of thiophene-3-carboxaldehyde (S. Gronowitz, "Arkev. Kemi.", 8.411 (1955), 636 g (6 moles) of trimethoxymethane and 6 g of resin "IR-120" (H +) in 200 ml of methanol The resin is separated and the filtrate is evaporated under reduced pressure to obtain a colorless oil which is distilled under reduced pressure.

Yield 423 g (94%); Eb. 90-95 ° C, 13 mm of mercury.

Infrared spectrum: v j ^ x 3150,1045,1025 cm "1

Nuclear magnetic resonance: ô 3.21 (6H,

O—

singlet, OCH3), 5.43 (1H, singlet, CH <^^ Y

^ -O- '

7.0-7.4 (3H, multiplet, thiophene-H); n.d. = without dilution

B) 2-Formylthiophene-3-carboxaldehyde dimethylacetal (3a)

A freshly prepared solution of 27 moles of n-butyl lithium in the glass is added dropwise over 1 hour to a stirred solution of 423 g (2.68 moles) of compound 2a in 1 liter of anhydrous ether. ether while maintaining moderate reflux, under a nitrogen atmosphere. Continuing reflux for 0.5 hour, a solution of 432 g (6 moles) of dimethylformamide in 0.8 liters of anhydrous ether is added dropwise to the mixture over 0.75 hours with vigorous stirring. When the additive is finished, the mixture is stirred for approximately 18 hours, it is poured onto 1 kg of crushed ice without interrupting the agitation and it is allowed to return to room temperature. The organic phase is separated and the aqueous sodium chloride phase is saturated,

* 0- '

15 (IH, doublet, J = 6Hz, thiophene-HP), 7.81 (1H, doublet of doublets, J = 1.5 and 6Hz, thiophene-Ha), 10.34 (1H, doublet, J = 1.5 Hz, -CHO).

C) 1-methylsulfinyl-1 -methylthio-2- (3-carboxaldehyde-ethylene-20 neacetal-2-thienyl) ethylene (4b)

The preparation of compound 4b is carried out by following a procedure similar to that which has been described by K. Ogura et al. "Triton B" (40% in methanol, 2 ml in 5 ml of tetrahydrofuran) is added to a solution of methyl methylthiomethyl sulfoxide2) (2.5 g, 20 milli-moles) and ethylene acetal 2-formyl-3-thiophene-carboxal-dehyde3) (3b). The mixture is refluxed for about 1 hour and concentrated under reduced pressure. The residue is dissolved in 150 ml of benzene and the solution is extracted with three times 20 ml of water. The organic phase is dried over magnesium sulfate and evaporated to dryness under reduced pressure. The residue is chromatographed on a column of silica gel (80 g), eluting successively with 500 ml of benzene and 500 ml of chloroform. 4.9 g (85%) of product 4b are isolated from chlorofor-35 mic eluate as a pale yellow oil.

Infrared spectrum:, v | j ^ x 3110,1600 cm "1.

Nuclear magnetic resonance:? P? '3 2.42 (3H, singu-

40 ""

let, S-CH3), 2.78 (3H, singlet, SO-CH3), 4.15 (4H, multiplet,

CH2CH2-), 6.12 (1H, singlet, CH <^ Y 7.34 (1H, doublet,

O- '

45 J = 4.5 Hz, thiophene-Hg), 7.40 (1H, doublet, J = 4.5 Hz, thiophene-Ha), 8.28 (1H, singlet, -CH =).

The semicarbazone of compound 4 is prepared in the usual manner and crystallized from a mixture of ethanol and dimethylformamide. Melting point 212-213 ° C.

50

Analysis: C% H% N% S%

Calculated for C10H13N3O2S2: 39.58 4.32 13.85 31.70 Found: 39.46 4.24 14.05 31.63

55

2) K. Ogura et al., “Bull. Chem. Soc. " (Japan), ", 2203 (1972)

3) D. W. McDowell et al., "J. Org. Chem. ”, 31, 3592 (1966)

60 4) K. Ogura et al., "Tetrahedron Letters", 1383 (1972).

D) l-methylsulfinyl-1-methylthio-2- (3-carboxaldehydedime-thylacetal-2-thienyl) ethylene (4a)

The compound 4a is prepared by following a procedure similar to that which is used for the compound 4b. "Triton B" (50 ml, 40% in methanol) is added to a solution of methyl methyl thiomethyl sulfoxide (72 g, 0.58 mole) and

11

627,184

108 g (0.58 mole) of compound 3a in 300 ml of tetrahydrofuran and the mixture is refluxed for 4 hours. Separation by column chromatography on silica gel (400 g), eluting with 5 liters of chloroform, gives 130.5 g (78%) of compound 4a as a pale yellow oil.

Infrared spectrum: v | j ^ x 3100,1580,1100,1050 cm- '. Nuclear magnetic resonance: ò p CI4 2.42 (3H, singlet, S-CH3), 2.70 (3H, singlets, SO-CH3), 3.34 (6H, singlet,

OCH3), 5.56 (1H, singlet, CH '

O

), 7.20 (1H,

doublet,

J = 6Hz, thiophene-HP), 7.40 (1H, doublet, J = 6Hz, thio-phene-Ha), 8.12 (1H, singlet, -CH =).

E) ethyl 3-formyl-2-thienylacetate4) (5)

33 g of anhydrous hydrochloric gas are absorbed in 500 ml of anhydrous ethanol. 130 g (0.45 mole) of compound 4a are added to this solution and the mixture is heated to reflux for 5 minutes. The reaction mixture is diluted with water and evaporated under reduced pressure. The residue is extracted with twice 100 ml of benzene and the benzene extracts are combined, washed with 50 ml of water, dried over magnesium sulfate and evaporated to dryness. The oily residue is chromatographed on a column of silica gel (400 g), eluting with 5 liters of chloroform. The fractions containing the desired product are combined and concentrated. The residual oil (60 g) is distilled under reduced pressure and 23 g (23%) of compound 5 boiling at 120-126 ° C / 1 mm of mercury are thus obtained.

Infrared spectrum: vj | ^ x 3110,1730,1670 cm "1.

Nuclear magnetic resonance: ô p ^ p'3 1.30 (3H, tri-

plet, J = 6Hz, -CH2CH3), 4.25 (2H, quadruplet, J = 6Hz, -CH2CH3), 4.26 (2H, singlet, -CH2CO), 7.25 (1H, doublet, J = 5Hz, thiophene-HP), 7.48 (1H, doublet, J = 5Hz, thiophene-Ha), 10.15 (1H, singlet, CHO).

The analytical sample of compound 5 is studied in the form of 2,4-dinitrophenylhydrazone which is crystallized from chloroform. Melting point 178-179 ° C.

Infrared spectrum: v '

1,720,1610,1570 cnT

Analysis: C% H% N% S%

Calculated for C15H14N406S: 47.62 3.73 14.81 8.47 Found: 47.33 3.47 14.77 8.68

Following a similar procedure, 2.2 g (7.6 millimoles) of ethylene acetal 4b are treated with 1.1 g of anhydrous hydrochloric gas in 800 ml of anhydrous ethanol to obtain compound 5 as purified by column chromatography of 30 g of silica gel. 663 mg (44%) of compound 5 is obtained by elution with chloroform of 5 in the form of a pale yellow oil.

F) Ethyl 3-formyl-2-thienylacetate oxime (6)

1.7 g (16 millimoles) of sodium carbonate are added to a solution of aldehyde 5 (3.14 g, 16 millimoles) and 2.2 g (32 millimoles) of hydroxylamine hydrochloride in 40 ml of 50% aqueous ethanol at 5 ° C with stirring. The reaction mixture is allowed to warm to room temperature. After 2.5 hours, this mixture is concentrated under reduced pressure. The residue is extracted with three times 50 ml of benzene. The benzene extracts are washed with 10 ml of water, dried over magnesium sulphate and evaporated under reduced pressure. Separation by column chromatography of 60 g of silica gel gives 2.7 g (80%) of compound 6 as a colorless oil.

Infrared spectrum: v J | ^ x 3400,1730,1620 cm "1.

Nuclear magnetic resonance: ô ppf ^ one "^ 6 1 = 23 (3H,

1C triplet, J = 7.5Hz, -CH2CH3), 4.01 (2H, singlet, -CH2CO), 4.14 (2H, quadruplet, J = 7.5Hz, -CH2CH3), 7.31 (2H, singlet , thiophene-H), 8.26 (1H, singlet, -CH = N), 10.15 (1H, singlet, NOH, disappears by addition of D20).

G) 3-aminomethyl-2-thienyl-acetic acid yl-lactam (7) Method A: catalytic reduction A mixture of 2.65 g (12.4 millimoles) of oxime 6 is hydrogenated for approximately 18 hours , 10% palladium fixed on charcoal and anhydrous hydrochloric gas (1.4 g, 37.2 millimoles) in 68 ml of anhydrous ethanol, at atmospheric pressure and at room temperature. The catalyst is replaced twice and the reaction is carried out for a period of 3 days. The catalyst is removed and the filtrate is concentrated under reduced pressure. 10 ml of water are added to the residue and the mixture is washed with twice 10 ml of ethyl acetate. The pH of the aqueous phase is adjusted to 9 by adding sodium carbonate, saturated with sodium chloride and extracted with three times 20 ml of ethyl acetate. The ethyl acetate extraction phases are dried over magnesium sulfate, treated with charcoal and evaporated under reduced pressure. By recrystallization from ethyl acetate, 417 mg (22%) of colorless needles of compound 7 are obtained, melting at 194-195 ° C.

20

25

35

Infrared spectrum: v ® | ®x 3200,1650,1480 cm 1n

Nuclear magnetic resonance: ô "^ 6 3.53 (2H,

triplet, J = 3Hz, -CH2CO-), 4.36 (2H, doublet-triplet, J = 3 and 1.5Hz, becomes a triplet by adding D20, J = 3Hz, CH2N), 6.95 ( 1H, doublet, J = 4.5Hz, thiophene-HP), 7.45 (1H, doublet, J = 4.5Hz, thiophene-Ha), 8.0 (1H, multiplet, disappears by addition of D20, NH) .

45

Analysis: C% H% N% S%

Calculated for CjHyNOS: 54.88 4.61 9.14 20.93

Found: 55.04 4.45 9.13 20.50

Method B: reduction to zinc powder s ”17 g (258 milli-gram atoms) of zinc powder are added in portions over 1 hour at 40-50 ° C. with vigorous stirring to a solution of 18.3 g ( 86 millimoles) of oxime 6 in 200 ml of acetic acid. The reaction mixture is stirred for about 18 hours at room temperature and heated to 60 ° C for 4 hours. The contents of the reactor are filtered and the filtrate is concentrated under reduced pressure. 100 ml of water are added to the residual oil and the mixture is washed with twice 50 ml of ether. 100 ml of ethyl acetate are deposited on the aqueous solution and the pH is adjusted to 10 by addition of sodium carbonate-60. The precipitate is separated by filtration. The filtrate is extracted with ethyl acetate. The extraction phases are washed with ethyl acetate with 10 ml of water, dried over magnesium sulphate and evaporated under reduced pressure. The solid residue is triturated with benzene. By crystallization 65 in ethyl acetate, 2.7 g (21%) of lactam 7 are obtained, which is identical to the compound of method A with regard to the infrared spectrum and the nuclear magnetic resonance spectrum.

627,184

12

H) 3-Aminomethyl-2-thienylacetic acid (8)

A mixture of 2.88 g (18.8 millimoles) of lactam 7 and 50 ml of 6N hydrochloric acid is heated at reflux for 3 hours. The reaction mixture is concentrated under reduced pressure. 20 ml of water are added to the residue and the mixture is treated with charcoal and then evaporated under reduced pressure. By trituration of the residue with tetrahydrofuran, the amino acid hydrochloride 8 is obtained (3.72 g, 95%; melting point 171—172 ° C; infrared spectrum (Kbr), cm-1: 3450.3000 , 1700.1200; nuclear magnetic resonance spectrum in D20, ppm: 4.80 (2H, singlet, -CH2CO), 4.27 (2H, singlet, CH2-N), 7.26 (1H, doublet, J = 6Hz, thiophene-Hp), 7.53 (1H, doublet, J = 6Hz, thiophene-Ha)). The hydrochloride (3.71 g; 17.9 millimoles) is dissolved in 10 ml of water, the solution is chromatographed on a column of "IR-120" (H +, 30 ml) and the elution is carried out successively. , 100 ml of water and 2 liters of 5N ammonia. The ammoniacal eluate is evaporated to dryness. The residue is cirstallized in aqueous acetone to give 3.0 g (98%) of compound 8, melting at 223-225 ° C.

Infrared spectrum: v

3000.1620.1520 cm-

Nuclear magnetic resonance: ô Na2C03 3 ^ 0

(2H, singlet, -CH2CO), 4.13 (2H, singlet, CH2N), 7.04 (1H, doublet, J = 6Hz, thiophene-Hp), 7.30 (1H, doublet, J = 6Hz, thiophene -Ha).

I) 3-tertio-butoxycarbonylaminomethyl-2-thienylacetic acid (9)

5.7 g (40 millimoles) of tert-butoxycarbonylazide are added dropwise over 20 minutes at 0 ° C. with vigorous stirring to a mixture of 3.1 g (18 millimoles) of 3-aminomethyl acid -2-thienylacetic 8 and 8 g (80 millimoles) of triethylamine in 80 ml of 50% aqueous acetone. The reaction mixture is stirred for about 18 hours at room temperature and concentrated under reduced pressure. The concentrate is washed with twice 20 ml of ether, its pH is adjusted to 2 by addition of concentrated hydrochloric acid and it is extracted with twice 50 ml of ethyl acetate. The ethyl acetate extraction phases are washed with a saturated aqueous sodium chloride solution, dried over magnesium sulfate, treated with charcoal and evaporated under reduced pressure. The residue is triturated in n-hexane and crystallized from a mixture of n-hexane and benzene to give 4.5 g (92%) of colorless needles of compound 9, melting at 62-63 ° C.

20

Infrared spectrum: v

3350.1700 cm-

Nuclear magnetic resonance: Ô j ^ P3 1.43 (9H, singlet, BOC-H), 3.27 (2H, singlet, CH2CO), 4.16 (2H, dou-25 blet, J = 6Hz, CH2-N , singlet when D2Ò is added), 5.00 (1H, broad, -NH-, disappears by addition of D20), 6.30 (1H, broad singlet, -COOH, disappears by addition of D20), 6.86 (1H, doublet, J = 6Hz, thiophene-HP), 7.06 (1H, doublet, J = 6Hz, thiophene-Ha).

30

Analysis:

Calculated for CjHçNO ^: Found:

C% H% 49.10 5.30 48.53 5.22

N% 8.18 7.98

S%

18.73

18.97

Analysis: C% H% N% S%

Calculated for C12H17N04S: 52.89 6.29 5.14 11.77 Found: 53.30 6.39 5.13 11.72

35 Preparation of 2-N-methylaminomethyl-4-methoxy- (and 4-hydroxy) -phenylacetic acids ch2nh2 ch2c02h

Ts-cl

HgNH-Ts h2co2h

CtUI

c h,

I 3

CHpN-Ts ch2co2h

Na / NH., Liq. H ^ CO r>

chgnh-ch ^ ch2co2h ka.

\ y

HBr

HO-i

CH ^ NH-CHj ch2c02h

4b

13

627,184

2-N-tosylaminomethyl-4-hydroxyphenylacetic acid (2)

A solution of 18.5 g (0.097 mole) of p-toluene sulfonyl chloride in 50 ml of anhydrous ether is added dropwise with stirring at 65-70 ° C to a solution of 14.56 g (0, 08 mole) of 2-aminomethyl-4-hydroxyphenylacetic acid (1) (U.S. Patent No. 3,823,141) and 13 g (0.32 mole) of sodium hydroxide in 200 ml d water and the mixture is kept at the same temperature for 1 hour. After cooling the mixture, the aqueous phase is separated, washed with twice 50 ml of ether, acidified with 6N hydrochloric acid and extracted with 400 ml of ethyl acetate. The extract is washed with water and with a saturated aqueous sodium chloride solution, dried over sodium sulfate and treated with 1 g of active carbon. The filtrate is concentrated to dryness and the residue is crystallized from ethyl acetate, which gives 11.0 g (40.5%) of compound 2, melting at 212-215 ° C.

Infrared spectrum: v 3240,1700,1380,1330,1150 cm-1.

Ultraviolet spectrum: X] ^ P3 al% 230 nm (e: 7750)

Nuclear magnetic resonance: ô 2.47 (3H,

singlet, Ar-CH3), 3.60 (2H, singlet, CH2CO), 3.93 (2H, doublet, J = 6.0 Hz, CH2N), 6.6—8.2 (7H, multiplet, H of phenyl).

2- (N-methyl-N-tosylamino) methyl-4-methoxy-phenylaceous acid (3)

A mixture of 11 g (0.033 mole) of compound 2 is heated, 10.3 ml (0.17 mole) of methyl iodide and 9.2 g (0.24 mole) of sodium hydroxide in 100 ml d water at 80-90 ° C for 45 minutes in a stoppered tube, shaking occasionally. The mixture is washed with 30 ml of ethyl acetate and the aqueous phase is acidified with 6N hydrochloric acid and then it is extracted with three times 30 ml of ethyl acetate. The combined extracts are washed with 30 ml of water and 30 ml of saturated aqueous sodium chloride solution, treated with 1 g of active carbon and dehydrated over sodium sulfate. The filtrate is evaporated to dryness and the residue is crystallized from benzene giving 8 g (66.5%) of the N.O-dimethyl derivative 3, melting at 146-150 ° C.

Infrared spectrum: v 1690,1500,1340,1280,1150 cm-1.

Ultraviolet spectrum: 229 nm (e: 20500), 278 nm

(e: 2400). maX '

Nuclear magnetic resonance: ô 2.52 (3H,

singlet, N-CH3), 2.47 (3H, singlet, Ar-CH3), 3.67 (2H, singlet, CH2CO), 3.74 (3H, singlet, OCH3), 4.10 (2H, singlet, CH2N), 6.7-7.8 (7H, multiplet, Ar-H), 11.5 (1H, broad singlet, COOH).

Analysis: C% H% N% S%

Calculated for Ci8H21NOsS: 59.49 5.82 3.84 8.82 Found: 59.48 5.68 3.37 9.22

2-N-methylaminomethyl-4-methoxyphenylacetic acid (4a) 9.4 g (0.026 mole) of compound 3 are added at -50 ° C to a solution of 300 ml of liquid ammonia and the mixture is stirred until that a clear solution was obtained at the same temperature. 3.3 g (0.14 atomgram) of sodium are added to the solution in small pieces at -40 ° C. and the mixture is stirred for 2 hours. The ammonia is evaporated and the residue is carefully dissolved in 100 ml of water. 100 ml of "Amberlite IR-C 50" (ammonium type) are added to the solution and the mixture is stirred for 30 minutes at room temperature. The resin is removed and the filtrate is treated with barium acetate until no more precipitation is observed. The precipitate is separated by filtration and the filtrate is chromatographed on a column of “IR-120” ion exchange resin (H + form, 100 ml), eluting with 5-10% ammonia. The eluate (2 liters) containing the desired product is evaporated to dryness below 50 ° C. and the residue is triturated with acetone, giving 4.4 g (81%) of compound 4a, melting at 225 -227 ° C.

Infrared spectrum: v 1590,1380,1260,

. Ä 4 ili Cl / k *

1035 cm

Nuclear magnetic resonance spectrum: ô

2.77 (3H, singlet, N-CH3), 3.6 (2H, singlet, CH2CO), 3.87 (3H, singlet, OCH3), 4.18 (2H, singlet, CH2N), 6.8- 7.4 (3H, multiplet, phenyl H).

20

2-N-methylaminomethyl-4-hydroxyphenylacetic acid (4b) A mixture of 2.9 g (0.014 mole) of compound 4a is refluxed in 30 ml of 48% hydrobromic acid for 5 hours and the solution is evaporated to dryness . The residue is dissolved in 50 ml of water. The solution is chromatographed on a 50 ml column of "Amberlite IR-120" (H + form), eluting with 5-10% ammonia. The eluate is collected in .fractions of 250 ml. The fractions which contain the product are combined and evaporated to dryness below 50 ° C. The residue is triturated with acetone to give 1.3 g (48.5%) of compound 4b which is crystallized from 80% ethanol. Melting point 218—221 ° C.

35 Infrared spectrum: v 2000-3400,1610,1540, 1460,1380,1270 cm-1.

Ultraviolet spectrum: X ** 1% 243 nm (e: 4700),

297 nm (e: 1350).

40

Nuclear magnetic resonance: ô + NaOH

2.64 (3H, singlet, N-CH3), 3.47 (2H, singlet, CH2CO), 3.94 (2H, singlet, N-CH2), 6.5-7.2 (3H, multiplet, H phenyl).

45

C% H% N% 61.53 6.71 7.17 61.44 6.81 7.20

2-N-tertio-butoxycarbonyl-N-methylaminomethyl-4-methoxyphenylacetic acid (5, R = CH3)

55 A mixture of 1.05 g (5 millimoles) of compound 4a, 1.43 g (6 millimoles) of 4,6-dimethylpyrimidin-2-ylthiolbutylcarbonate and 1.4 ml of triethylamin in 40 ml of 50% tetrahydrofuran. Most of the tetrahydrofuran is evaporated and the resulting aqueous solution (about 20 ml) is washed with ether. The ether phase is acidified with 6N hydrochloric acid and extracted with three times 10 ml of ether. The ether extraction phases are washed with 10 ml of water and 10 ml of saturated aqueous sodium chloride solution, treated with a small amount of active carbon and dehydrated over sodium sulfate. The filtrate is evaporated to dryness, giving 1.0 g (77.5%) of compound 5 (R = methyl) in the form of an oil.

Analysis:

Calculated for C10H13NO3: Found:

627,184

14

Nuclear magnetic resonance: ô j ^ P3 1.47 (9H,

singlet, BOC-H), 2.77 (3H, singlet, N-CH3), 3.60 (2H, singlet, CH2CO), 3.79 (3H, singlet, 0-CH3), 4.49 (2H, singlet, CH2N), 6.1-7.3 (3H, multiplet, phenyl H).

2-N-tertio-butoxycarbonyl-N-methylamino-methyl-4-hy-droxyphenylacetic acid (5, R = hydrogen)

A mixture of 1 g (4.78 millimoles) of compound 4b, 1.5 g (6.3 millimoles) of 4,6-dimethylpyrimidin-2-ylthiolbutylcarbonate is stirred at room temperature for 20 hours. and 2.1 ml of triethylamine in 50 ml of 50% aqueous solution of tetrahydrofuran. The mixture is concentrated to 20 ml under reduced pressure. The concentrate is washed with 10 ml of ether, acidified with 6N hydrochloric acid and extracted with twice 100 ml of ethyl acetate. The combined extracts are washed with 30 ml of water and twice 30 ml of saturated aqueous sodium chloride solution, treated with a small amount of active carbon and dried over anhydrous sodium sulfate. The filtrate is evaporated to dryness, giving 1.3 g

5 (92%) of compound 5 (R = hydrogen) in the form of an oil.

Infrared spectrum: vjjfjj3000-3600,1670,1260, 1150 cm "1.

10 Nuclear magnetic resonance: ô 1> 44 (9H,

singlet, C (CH3) 3), 2.73 (3H, singlet, N-CH3), 3.54 (2H, singlet, CH2CO), 4.38 (2H, singlet, CH2N), 6.5-7, 3 (3H, multiplet, phenyl H).

15 Preparation of ortho-N-methylaminomethylphenyl acetic acid ch2nh2 ch2c02h ch2nhs02 chgcogh ch, i?

ch-

CHgNSOg- ^ // ~ CU-

ch2co2h ^ '

hbr or

Na / NH-, liq.

->

ch.

CH-

chgnh CHgCOgH

chgn-boc ch2co2h ch.,

I ^

boc: ch -.- c-0c0- ~ 5 d ch.

P

15

627,184

O- (p-toluenesulfonylaminomethyl) phenylacetic acid (2)

7.64 g (40 millimoles) of p-toluene sulfonyl chloride are added in portions at 60 ° C. to a stirred solution of 7.50 g (37 millimoles) of o-aminomethylphenylacetic acid hydrochloride and 4 , 74 g (118 millimoles) of sodium hydroxide in 100 ml of water. The mixture is stirred for 1 hour at the same temperature and acidified with hydrochloric acid. The mixture is extracted with four times 50 ml of ethyl acetate. The combined extracts are washed with water, treated with a small amount of carbon and dehydrated. The solvent is evaporated under reduced pressure and the residue is crystallized from ethyl acetate, giving compound 2 in the form of colorless prisms. Yield 9.84 g (84%); melting point 155-156 ° C.

3300.1705.1335,

Infrared spectrum: v j - _ Ä i iliu / v •

1170 cm-1.

Nuclear magnetic resonance: ò ^^ SO-d6 2.38 (3H,

singlet, CH3), 3.65 (2H, singlet, CH2CO), 3.97 (2H, doublet, J = 5Hz, CH2N), 7.1-8.2 (9H, multiplet, phenyl H and NH).

Analysis:

VS%

H%

NOT %

S%

Calculated for C16Hi7N04S:

60.17

5.37

4.39

10.10

Find:

60.11

5.43

4.28

9.72

60.15

5.40

4.30

9.80

O- (N-p-toluenesulfonyl-N-methylaminomethyl) -phenyl-acetic acid (3)

A mixture of compound 2 (9.0 g, 28 millimoles), 6.0 g of sodium hydroxide and 6 ml of methyl iodide in 60 ml of water is heated for 30 minutes at 70 ° C in a closed tube. After cooling, the reaction mixture is acidified by addition of hydrochloric acid to separate a pale yellow precipitate which is crystallized from a mixture of ethyl acetate and n-hexane, which gives colorless prisms of the compound. 3. Yield 8.5 g (91%); melting point 162-163 ° C.

2700-2300,1700,1600,

Infrared spectrum: v J® *

1345,1200.925 cm "1.

Nuclear magnetic resonance: ô 2.37

(3H, singlet, CH3), 2.49 (3H, singlet, CH3), 3.80 (2H, singlet, CH2CO), 4.18 (2H, singlet, CH2N), 7.0-8.0 (8H , multiplet, phenyl H).

Analysis:

VS%

H%

NOT%

S%

Calculated for C17H19N02:

61.24

5.74

4.20

9.61

Find:

61.31

5.73

4.51

9.63

61.36

5.71

4.29

9.55

N-methylaminomethylphenylacetic acid (4)

Method A (using hydrobromic acid) - A mixture of 28.6 g (0.086 mole) of compound 3 and 20 g (0.213 mole) of phenol in 260 ml of 48% hydrobromic acid are refluxed for 30 minutes. The mixture is cooled, diluted with the same volume of water and washed with twice 50 ml of ethyl acetate. The aqueous phase is evaporated to dryness under reduced pressure to obtain an oil which is chromatographed on a column of "Amberlite IR-120" (form H +, 200 ml), killing the elution with a solution of 5% ammonium hydroxide. The eluate is collected (2.5 liters) and evaporated to dryness under reduced pressure. The residue is triturated with acetone and crystallized from ethanol to give 6.7 g (43.5%) of compound 4 in the form of colorless needles, melting at 168-170 ° C. decomposing.

Method B (using metallic sodium in liquid ammonia) - 13.3 g (0.578 gram atoms) of sodium are added in small pieces with vigorous stirring over 2 hours to a mixture of compound 3 (35 g, 0.105 mol) in 1000 ml of liquid moniac friends. The ammonia is evaporated with stirring in a water bath in a suitably ventilated hood and, at the end, under reduced pressure to completely eliminate this gas. The residue is dissolved in 400 ml of ice water and the solution is stirred with 400 ml of ion exchange resin "IRC-50" (form H +) pen-2odant 0.5 hours at room temperature. The resin is separated by filtration and a 1M aqueous solution of barium acetate is added to the filtrate until no precipitate is formed (approximately 50 ml of barium acetate solution is required. mix and the filtrate is chromatographed on a column of "IR-120" resin (H + form, 400 ml) as in method A to obtain 13.6 g (72%) of compound 4.

O- (N-methyl-N-tertio-butoxycarbonylaminomethyl) phe-nylacetic acid (5)

11 g (0.048 mole) of tert-butyl 4,6-dimethylpyrimidin-2-ylthiolcarbonate are added in a single portion to a mixture of compound 4 (7.2 g 0.04 mole) and 1.1.3, 3-tetramethyl-guanidine (6.9 g, 0.06 mole) in 50% aqueous tetrahydrofuran and the mixture was stirred for a further 18 hours at room temperature. After having evaporated the tetrahydrofuran under reduced pressure, the aqueous phase is acidified to a pH equal to 2 by addition of dilute hydrochloric acid and it is extracted with twice 20 ml of ethyl acetate. The combined extracts are washed with water, treated with a small amount of active carbon and evaporated under reduced pressure. The residue is triturated with hexane and crystallized from a mixture of n-hexane and ether, giving 9.2 g (83%) of compound 5 in the form of colorless prisms melting at 96-98 ° C.

45

Infrared spectrum: v 1730,1630,1430,1830,

1250 cm

-1

Nuclear magnetic resonance spectrum: ô | ^ P3

1.49 (9H, singlet, tert-butyl), 2.78 (3H, singlet, N-CH3), 50 3.72 (2H, singlet, CH2CO), 4.25 (2H, singlet, CH2N), 7 , 28 (4H, singlet, phenyl), 9.83 (1H, singlet, -COOH).

Analysis: C% H% N%

55 Calculated for C15H21N04: 64.50 7.58 5.01 Found: 64.69 7.66 4.89

Preparation of 3-N-methylaminomethyl-2-thienyl-acetic acid t

s "

-ch = n0h

-ch2co2c2h5 "

O

nah, ch, i benzene

627,184

16

h ch2n-ch5 ch2c02h

œ

CH,

I 3

chon-boc cooh h-lactam 3-aminomethyl-2-thienylacetic acid (2)

140 ml of crystalline-acetic acid are added dropwise, with stirring, to a mixture of 41 g (0.19 mole) of 2-ethoxycarbonylmethylthiophene-3-carboxaldehyde oxime £ 1) and 65.4 g ( 1 mol) of zinc powder in methanol and the mixture is stirred at reflux for 4 hours. The mixture is cooled and the insoluble material is separated by filtration and then washed with three times 50 ml of methanol. The filtrates and the washing liquors are combined and the mixture is evaporated to dryness under vacuum, extracting the residue with five times 100 ml of methanol. The methanolic extracts are combined and evaporated under reduced pressure. 50 ml of water are added to the residue and the mixture is adjusted to pH 10 by adding sodium carbonate, then this mixture is extracted with three times 100 ml of chloroform. The combined chloroform extracts are washed with 10 ml of water, dried over magnesium sulfate and evaporated under reduced pressure. The residual oil (30 g) is triturated with 150 ml of hot benzene. The colorless needles are collected by filtration and recrystallized from ethyl acetate; 7.7 g (26%) of lactam 2 are thus obtained, melting at 195-196 ° C.

Ultraviolet spectrum: X 232 nm (e, 6500)

Analysis: C% H% N% S%

Calculated for QHyNOS: 54.88 4.61 9.14 20.93 Found: 55.04 4.45 9.13 20.50

h-lactam 3-N-methylaminomethyl-2-thienyl-acetic acid

(3)

4.85 g (32 millimoles) of lactam 2 are added with stirring under a nitrogen atmosphere to a suspension of 1.82 g (38 millimoles) of 50% sodium hydride in paraffin in 500 ml of absolute benzene and the mixture is refluxed for 2 hours. 22.7 g (160 millimoles) of methyl iodide are added in a single portion at room temperature and the mixture is again refluxed for 2 hours. 50 g of ice water are added to the mixture and the organic phase is separated. The aqueous phase is extracted successively with twice 50 ml of benzene and 50 ml of chloroform. The extracts are combined and dried over magnesium sulfate. The solvent is evaporated under reduced pressure. A warm mixture of

100 ml of benzene and n-hexane at 1: 1 to collect compound 2 in the form of needles (2.02 g, 42%). The filtrate is evaporated and the residue is crystallized from a mixture of benzene and n-hexane; colorless strips of compound 3 are thus obtained. Yield: 2.7 g (51%); melting point 98-100 ° C.

Infrared spectrum: v 1620 cm ~ 1.

35 Nuclear magnetic resonance: ò ^ HC13 3 ^ 5 (3H,

singlet, N-CH3), 3.72 (2H, triplet, J = 3Hz, CH2CO), 4.53 (2H, triplet, J = 3Hz, -CH2-N), 6.87 (1H, doublet, 1 = 4.5Hz, thiophene-HP), 7.30 (1H, doblet, J = 4.5Hz, thiophene-Ha).

40

Ultraviolet spectrum: X 232 nm (e, 6700)

45,

Analysis:

Calculated for C8H9NOS: "Found:

C% H% 57.46 5.42 57.56 5.26

N% 8.38 8.31

S%

19.17

19.13

Acid 3- (N-meth ylaminométh yl} -2-thien en y lacé tick (4)

Is heated under reflux for 12 hours, a mixture of 3.5 50g (21 millimoles) of lactam 3 and 100 ml of 6N hydrochloric acid. The mixture is treated with carbon and concentrated to dryness under reduced pressure. The residual oil is dissolved in 10 ml of water and the solution is chromatographed on a 50 ml column of "IR-120" (H +). The column is eluted with 200 ml of water 55 and 3 liters of 5N ammonia. Amino acid 4 (3.0 g, 77%) is isolated by evaporation of the ammoniacal eluates followed by crystallization from aqueous acetone. Melting point 181-182 ° C.

60

Infrared spectrum: v 1570.1360 cm

Nuclear magnetic resonance: ô ^ 2® 2.21 (3H,

singlet, N-CH3), 3.80 (2H, singlet, CH2CO), 4.20 (2H, sin-65gulet, CH2-N), 7.19 (1H, doublet, J = 6Hz, thiophene-H (3 ), 7.46 (1H, doublet, J = 6Hz, thiophene-Ha).

Ultraviolet spectrum: X 237 nm (e, 7600)

17

627,184

Analysis: C% H%. N% S%

Calculated for CgHuN02S: 51.87 5.99 7.56 17.31 Found: 51.67 6.50 7.28 16.69

3- (N-tertio-butoxycarbonyl-N-methylaminomethyl) -2-thienylacetic acid (5)

4.2 g (29.2 millimoles) of tert-butoxycarbonylazide are added dropwise over 20 minutes at 0 ° C. with vigorous stirring to a mixture of 2.7 g (14.6 millimoles) of 3-N- acid me-thylaminomethyl-2-thienylacetic 4 and 6 g (60 millimoles) of triethylamine in 60 ml of 50% aqueous acetone. The reaction mixture is stirred for about 18 hours at room temperature and concentrated under reduced pressure. The concentrate is washed with twice 20 ml of ether, its pH is adjusted to 2 by addition of concentrated hydrochloric acid and it is extracted with twice 50 ml of ethyl acetate. The ethyl acetate extraction phases are washed with a saturated aqueous solution of sodium chloride, dried over magnesium sulfate, treated with charcoal and evaporated under reduced pressure. The residue is triturated with n-hexane and crystallized from a mixture of n-hexane and benzene to give 3.68 g (88%) of colorless needles of compound 5 melting at 82-83 ° C.

Infrared spectrum: v 1730.1640 cm-1.

Nuclear magnetic resonance: ô 1.47 (9H,

singlet, BOC-H), 2.78 (3H, singlet, N-CH3), 3.87 (2H, singlet, CH2-CO), 4.48 (2H, singlet, CH2-N), 6.91 ( 1H, doublet, J = 6Hz, thiophene-H ^), 7.20 (1H, doublet, J = 6Hz, thiophene-Ha), 10.63 (1H, singlet, C02H, disappears by addition of D20).

Analysis: C% H% N% S%

Calculated for C13H19N04S: 54.72 6.71 4.91 11.24 Found: 54.91 6.85 4.92 11.19

The use of an “enamine” protective group with a future side chain in position 7 carrying a free amino group before the acylation of a nucleus such as II as described in the present specification is well known (see US Patents United States of America No. 3,223,141, No. 3,813,390, No. 3,813,391 and No. 3,823,141 and Belgian Patent No. 773,773).

2- [N- (1-carbethoxypropen-2-yl) aminomethyl] -1,4-cyclohexa-dienylacetate (4)

3.34 g (0.02 mole) of 2-aminomethyl-1,4-cyclohexadienylacetic acid and 3.1 g (0.024 mole) of ethyl acetylacetate are added to a stirred solution of 460 mg (0, 02 gram atom) of metallic sodium in 100 ml of absolute ethanol and the mixture is heated to reflux for 4 hours with stirring. The hot reaction mixture is filtered and the filtrate is kept in the refrigerator for about 18 hours to obtain 2.0 g of colorless needles of compound 4, melting at 264 ° C.

Another 3.3 g of product are obtained by concentration of the mother liquor. The total yield is 5.3 g (88%).

Infrared spectrum: v 3300,1635,1600,1570,

1300,1275,1170,1090 cm "1.

Nuclear magnetic resonance: ô 1.23 (3H, triplet, 7Hz, CH2CH3), 1.96 and 2.25 (3H, singlet, C = C-CH3, eis and trans), 2.70 (4H, singlet, H2C <^), 3.04 (2H, singlet,

CH2CO), 3.66 and 3.95 (2H, singlet, CH2-N, eis and trans), 4.07 (2H, quadruplet, 7Hz, CH2CH3), 4.45 and 4.56 (1H, singlet,

„H

■ IX))

, eis and trans), 5.76 (2H, singlet.

Analysis: C% H% N%

Calculated for C15H20NO4Na: 59.79 6.69 4.64 0 Found: 59.69 6.76 4.75

2-Tertiary-butyoxycarbonylaminomethyl-4-hydroxyphenyl-acetic acid is prepared, for example, in accordance with United States Patent No. 3,823,141.

b-lactam o- (N-methylaminomethyl) phenylacetic acid n-ch.

15

Sodium hydride (4.3 g; 0.11 mole, 57% paraffin) was washed with anhydrous n-hexane and suspended in 100 ml of anhydrous benzene. To the suspension is added a solution of 14.7 g (0.1 Mole) of o-lactam o-aminomethylphenylacetic acid (United States Patent No. 3,796,716) in 200 ml of benzene or anhydrous xylene with stirring under a nitrogen atmosphere. The mixture is refluxed for 1 hour and allowed to cool to room temperature. 18 ml of methyl iodide are added to the mixture in a single portion and re-refluxed for 1.5 hours. The reaction mixture is allowed to cool to room temperature and poured into 100 ml of ice water. The aqueous phase is separated from the organic phase and extracted with twice 50 ml of chloroform. The extracts and the organic phase are combined and dried over magnesium sulfate. The solvent is removed and the oily residue is distilled under vacuum; 14.9 g (92%) of o-lactam of o- (N-me-40 thylaminomethyl) phenylacetic acid boiling at 130-135 ° C / 2 mm of mercury are obtained; melting point 35-37 ° C.

Infrared spectrum: v 3300,1620,1490 cm "1

45

Nuclear magnetic resonance: ô pjJnP3 3.12 (3H, singlet), 3.59 (2H, triplet, J = 1.5Hz), 4.48 (2H, triplet, J = 1.5Hz), 7.21 (4H , large singlet).

50 Analysis:

Calculated for CioHn N0 1 / 4H20: Found:

H% N%

55

72.49 72.78 72.70

6.84 6.76 6.81

8.45 8.49 8.51

60

O-N-methylaminomethylphenylacetic acid

UL

nhch,

ch2cooh

65

A mixture of 5.0 g (0.031 mole) of the o- (N-methylaminomethyl) -phenylacetic acid produced above and 500 ml of chlorhy-

627184

18

concentrated drique. The mixture is evaporated under reduced pressure and the residual oil is dissolved in 20 ml of water and then the solution is treated with a small amount of active carbon. The filtrate is washed with 50 ml of benzene and evaporated to dryness. The residual oil is crystallized by trituration with tetrahydrofuran (or acetone) to obtain colorless needles of o-N-methylaminomethylphenylacetic acid hydrochloride (4.5 g, 67%).

Analysis: C% H% N% CI%

Calculated for CI0H13NO2-HCl: 55.69 6.54 6.49 16.44

Found: 55.65 6.62 6.53 16.36

55.74 6.60 6.53

A little unreacted raw material is recovered in the benzene phase and the washing liquors with tetrahydrofuran (1.2 g, 24%, bp 140-143 ° C / 2 mm of mercury).

An aqueous solution of oN-methyl-aminomethylphenylacetic acid hydrochloride (5 g) is chromatographed on a column of ion exchange resin "IR-120" (form H +, 70 ml) and eluted with 2 liters of 3N ammonia as yielding 3.9 g (93%) oN-methylaminomethylphenyiacetic acid in the form of needles.

Infrared spectrum: v 1650.1470 cm ~ l.

The invention is illustrated by the following examples, given without limitation. All temperatures are expressed in degrees Celsius. 7-aminocephalosporanic acid is designated by the abbreviation 7-ACA; the radical -ACA-corresponds to the formula:

-nh-ch-

• ch l

ck2

^ C-CH -

c cooh and therefore 7-ACA can be represented by the for-O

II

mule H-ACA-O-C-CHy Methyl isobutyl ketone is designated by the abbreviation MIBC. "Skellysolve B" is a fraction of petroleum ether boiling at 60-68 ° C and mainly formed of n-hexane.

The "LA-1" resin is a mixture of secondary amines, each of which corresponds to the formula:

R1

I

CH3 (CH2) 10CH2NHC -R2

I

R3

in which each of the symbols R1, R2 and R3 is a monovalent aliphatic hydrocarbon radical and these radicals total 11 to 14 carbon atoms. This particular mixture of secondary amines, which is sometimes called “mixture of liquid amines 5 No. II” in the examples, is a clear liquid of amber color having the following physical characteristics: viscosity at 25 ° C = 70 centipoise , density at 20 ° C - 0.826; refractive index at 25 ° C = 1.4554; distillation range under a vacuum of 10 mm: up to 170 ° C = 0.5%, 170-220 ° C = 3%, 220-io230 ° C = 90% and above 230 ° C = 6, 5%.

The resin "IR-120" is also called resin "Amberlite IR-120" and it is a strong cation exchange resin containing sulfonic acid radicals. The resin "Amberlite 15IR-120" is a commercial cation exchange resin of the polystyrene-sulfonic acid type; it is therefore a sulfonated polystyrene resin in the nucleus, crosslinked with divinylben-zene, obtained by the process described by Kunin in "Ion Exchange Resins", second edition (1958), John Wiley and Sons, 2C Inc. (see, in this book, for example pages 84 and 87).

The resin "Amberlite IRC-50" is a commercial cation exchange resin, of the carboxylic type; it is a co-mother of methacrylic acid and divinylbenzene.

Dicyclohexylcarbodiimide is designated by the abbreviation DCC, tetrahydrofuran by the abbreviation THF, the para-toluenesulfonyl radical by Ts and methanol by MeOH.

When the following instrumental measures are indicated; the naked Greek letter (v) is used for the infrared spectrum, the Greek letter X is used for the ultraviolet spectrum, the molecular extinction coefficient is designated by the letter (e) and the letters ô and x are used for the nuclear magnetic resonance (ô = 10-t).

Summary (Diagrams 1,2 and 3)

35 2-carboxymethyl-2,3-dihydro-s-triazolo [4,3-b] pyridazine-3-one-6-ylthio (3), (side chain thiol in position 3), is prepared by N-ethoxycarbonylmethylation 6-chloro-2,3-dihydro-s-triazolo [4,3-b] pyridazine-3-one (1) with sodium hydride and ethyl chloroacetate in dimethylformamide, 40 with subsequent thiolation in presence of sodium hydrosulfide (diagram 1). By condensation of 7-ACA acid (7-amino-cephalosporanic acid) with compound 3 effected by reflux in a phosphate buffer (pH 7), 7-ACA with thio function substituted in position 3 is obtained (compound 4 ) that are combined with an amino acid with amino function protected by a BOC group by the activated ester method using 2,4-dinitrophenol (DNP). The cephalosporins 7 and 11 obtained, with an amino function protected by a BOC group, are freed from the protective group by means of TFA (trifluor-50 acetic acid) and transformed into a monosodium salt with sodium hydroxide IN ( diagrams 2 and 3).

Diagram 1

Preparation of 7-amino-3- (2-carboxymethyl-2,3-di-55 hydro-s-triazolo [4,3-b] pyridazine-3-one-6-ylthiomethyl) -3-c- acid phem-4-carboxylic

NaH / 'DMF

cich2cooc2h5

> Cl-

' not'

,not

V

o n-ch2c00c2h5

1

2

19

627,184

NaSH

hs

•not

-NOT

-N i n-chgcooh

7-aca y

o

H2N-

CH2S

■ n

^ n-cr * cooh co ^ h o

h

Diagram 2

do] -3- (2-carboxymethyl-2,3-dihydro-s-triazolo [4,3-b] pyrida-

Preparation of 7 - [(o-aminomethylphenyl) -acetaminizin-3-one-6-ylthiomethyl) -3-cephem-4-carboxylic acids

, 1

2,4-dnp / dcc

+ he r

r r

^ i

—N-BOC

corn r "

kA / CONH-J-f A

0

a: R1 = H, R2 = H b: R1 = CH3, R2 = H c: R1 = CH3, R2 = OH

n ch2-s-

co2h 7

not"

"Y

tpa

■ n i n-ch2c00h ch2s-

^ -chocooh N> r d co2h

8

0

8a: BB-S469 8b: BB-S479 8c: BB-S478

627 184 20

Scheme 3 do) -3- (2-carboxymethyl-2,3-dihydro-2-triazolo [4,3-b] pyrida-

Preparation of 7- (3-aminomethyl-2-thienylacetaminizin-3-one-6-ylthiomethyl) -3-cephem-4-carboxylic acid r I

n-boc cooh

2,4-dnp / dcc g:

r j

n-boc

COO

ff \

no,

no,

+ Ì

• 10

11

dd ^ Nv ^ N-CKgCOOH

ce-, cooh

ch „s r = F

.N ^^ N-CHpCOOH

T

a: R = H b: R = CH3

12

12a: BB-S483 12b: BB-S472

6-chloro-2,3-dihydro-2-ethoxycarbonylmethyl-s-triazolo [4,3-bJpyridazine-3-one (2)

0.3 g (6.3 millimoles) of sodium hydride (50% in paraffin) is added while stirring to a solution of 6-chloro-2,3-dihydro-s-triazolo [4,3-b ] pyridazine-3-one [P. Francavilla and F. Lauria, “J. Het. Chem. ", 8.415 (1971)] (compound 1.1.00 g, 5.9 millimoles) in 30 ml of anhydrous dimethylformamide; yellow crystals are formed. 1.4 ml (13

60 millimoles) of ethyl chloroacetate and the mixture is heated at 90 ° C for 8 hours with stirring. After cooling, the reaction mixture is poured into 50 ml of water and extracted with five times 40 ml of toluene. The organic extracts are combined, dried over anhydrous sodium sulfate and evaporated under reduced pressure. The residue is crystallized from a mixture of benzene and n-hexane to obtain yellow needles of compound 2 (1.16 g, 77%) melting at 114-115 ° C (the literature indicates a melting point of 110 ° C ).

21

627,184

Infrared spectrum: v 1735.1710 cm

Ultraviolet spectrum: X ^ 31 nm (e, 26,000)

Nuclear magnetic resonance: ô 7.58 (1H,

doublet, J = 10Hz, H of pyridazine), 6.98 (1H, doublet, J = 10Hz, H of pyridazine), 4.80 (2H, singlet, -CH2CO), 4.27 (2H, quadruplet, J = 7.5 Hz, CH2CH3), 1.29 (3H, triplet, J-7.5 Hz, CH2CH3).

Analysis:

Calculated for C9H9N403C1: Found:

C% H%

42.12 3.53

41.54 3.22

41.46 3.49

N% Cl%

21.83 13.81

21.51 13.88

21.53 13.99

Ultraviolet spectrum: 1% aqueous X 260 nm

(e 19,500), 313 nm (ô, 7000).

Nuclear magnetic resonance: ô ~ ^ 6 7.88

(1H, doublet, J = 10Hz, H of pyridazine), 7.45 (1H, doublet, J = 10Hz, H of pyridazine), 4.72 (2H, singlet, CH2CO).

Analysis:

Calculated for C7H6N403S: Found:

C% H%

37.17 2.67

37.35 2.26

37.23 2.28

N% S%

24.77 14.17

23.58 14.32 23.69

2-carboxymethyl-2,3-dihydro-6-mercapto-s-triazolo- [4,3-b] py-ridazine-3-one (3)

45.9 g (0.36 mole) of NaSH.2 HzO (purity equal to 70%) is added to a solution of 30 g (0.12 mole) of 6-chloro-2,3-dihydro-2-ethoxycarbonylmethyl -s-triazole [4,3-b] pyrida-zine-3-one (compound 2) and the mixture is refluxed for 0.5 hour. The reaction mixture is evaporated under reduced pressure. The residue is dissolved in water (200 ml) and concentrated hydrochloric acid is added to the solution to adjust the pH to 2. The precipitate (3) is collected by filtration and washed with water. Yield 18.3 g (69%).

Infrared spectrum: v 2900,2450,1750,

1660 cm-1. X-

7-Amino-3- (2-carboxymethyl-2,3-dihydro-s-triazo-lo [4,3-b] pyridazine-3-one-6-ylthiomethyl) -3-cephem-4-carb-oxylic acid (4)

io 8.14 g (97.0 millimoles) of NaHCO3 and 7.30 g (32.2 millimoles) of thiol 3 are added with stirring to a suspension of 8.79 g (32.2 millimoles) of acid 7- aminocephalosporanic in 149 ml of 0.1 M phosphate buffer (pH 7). The mixture is heated at 80 ° C for 0.5 hour under a stream of nitrogen.

The mixture is treated with active charcoal and its pH is adjusted to 3 by the addition of concentrated hydrochloric acid. The resulting precipitate is collected by filtration and washed with water, giving 7.59 g (54%) of compound 4.

20 Infrared spectrum: v 1470 cm-1.

1800,1720,1600,1540,

Ultraviolet spectrum: X (pH7) 252 nm (e, 19,500),

25,298 nm (e, 8400).

Nuclear magnetic resonance: + K2C03 7 5g ^ H,

rr "

doublet, J = 9Hz, H of pyridazine), 7.05 (1H, doublet, J = 9Hz, H of pyridazine), 5.45 (1H, doublet, J = 5Hz, 6-H), 5.05 (1H , doublet, J = 5Hz, 7-H), 4.43 (1H, doublet, J = 14Hz, 3-CH2), 4.04 (1H, doublet, J = 14Hz, 3-CH2), 3.88 ( 1H, doublet, J = 18Hz, 2-H), 3.45 (1H, doublet, J = 18Hz, 2-H).

35

Example 1

Preparation of BB-S469 compound

ch2nh2 chgconk n-ch2c00h

8a, BB-S469

o- (N-butoxycarbonylaminomethyl) 2,4-dinitro-phenyl phenylacetate (6a)

10.1 g (49 millimoles) of dicyclohexylcarbodi-imide (DCC) is added while cooling with water (5-15 ° C) to a mixture of 13 g (49 millimoles) of o- (N-butoxycarbonylaminomethyl) acid phenylacetic (5a) and 9.02 g (49 millimoles) of 2,4-dinitrophé-nol in 123 ml of ethyl acetate and the mixture is stirred for 30 minutes at the same temperature and then for 40 minutes at room temperature. The resulting precipitate is separated by filtration and the filtrate is evaporated to give 23.9 g of the active ester 6a which is used in the following acylation reaction, without further purification.

Infrared spectrum: v 1775,1700,1600,1530 cm-Acid 7- (o- [N-butoxycarbonylaminomethyl) phenylacetamido] -

3- (2- carboxymethyl-2,3-dihydro-s-triazolo [4,3-b] pyridazine-3-one-6-ylthiomethyl) -3-cephem-4-carboxylic (7a)

Add a solution of 4.79 g of 2,4-dinitrophenyl (6a) o- (N-butoxycarbonyl-55 aminomethyl) phenylacetate (6a) in 20 ml of THF (tetrahydrofuran) to a cold mixture (0 ° C) of 4, 38 g (10 millimoles) of the compound 4.4.5 ml (30 millimoles) of Et3N, 20 ml of CH3CN and 20 ml of water. After stirring at room temperature for approximately 18 hours, the THF and CH3CN are removed from the reaction mixture under reduced pressure and the pH of the resulting aqueous solution is adjusted to 2 by addition of dilute hydrochloric acid, then it is extracted. with ten times 30 ml of ethyl acetate. The organic extracts are dried over sodium sulfate and evaporated. The residue is 65 chromatographed on a column of silica gel (60 g) and eluted successively with chloroform and a 3% mixture of methanol and chloroform, to obtain 2.40 g (37%) of compound 7a which melts above above 161 ° C when decomposing.

627,184

22

Infrared spectrum: v 1780.1720 cm 1

Ultraviolet spectrum: X ^ ™ Pon P ** 7,252 nm (e, 19,800), 298 nm (e, 8900).

Nuclear magnetic resonance: ô + D20 7 ^ 7

doublet, J = 9.0 Hz, pyridazine H), 7.10 (4H, singlet, phenyl H), 7.05 (1H, doublet, J = 9.0 Hz, pyridazine H), 5.66 (1H , doublet, J = 4.5 Hz, 7-H), 5.07 (1H, doublet, J = 4.5 Hz, 6-H), 4.71 (2H, singlet, N-CH2-CO), 4.4-4.0 (4H, multiplet, 3-Œ2 and CH2-N), 3.8-3.5 (4H, multiplet, 2-H and CH2CO), 1.42 (9H, singlet, H of tertio-butyl).

Analysis:

VS%

H%

NOT %

S%

Calculated for

CaH ^ NrOrSî '2H20:

49.62

5.20

14.46

9.46

Find:

49.97

4.79

14.00

9.37

49.95

4.62

13.84

9.32

BB-S469; acid 7 - [(o- (aminomethylphenyl) acetamido] -3- (2-carboxymethyl-2,3-dihydro-s-triazolo [4,3-b] pyridazine-3-one-6-ylthiomethyl) -3-cephem -4-carboxylic (8a)

3.4 ml of trifluoroacetic acid are added to 2.33 g of compound 7a at 0 ° C and the mixture is stirred for 15 minutes at room temperature. 100 ml of anhydrous ether are added to the mixture and the precipitate is collected by filtration and then washed with twice 50 ml of anhydrous ether. The solid is dissolved in a mixture of 100 ml of CH3CN and 14 ml of water and the pH of the solution is adjusted to 4-5 by addition of concentrated ammonia to obtain a precipitate which is collected by filtration and washed with twice 50 ml of CH3CN, which gives 1.75 g (82%) of compound 8a in the form of the ammonium salt melting above 160 ° C. as it decomposes.

Infrared spectrum: v j _ A Ä H IlldAl

1590 cm-1.

1765,1710,1640,

Ultraviolet spectrum: X ~ ™ Pon PH 7 252 nm (e, 23 900), 298 nm (e, 10 900).

Analysis:

Calculated for

C24H22N707S2 • NH4 + • H20: Found:

C% H% N% S%

46.51 3.96

46.52 4.15 46.26 4.19

18.08 10.35 17.60 10.89 17.48 10.41

Preparation of monosodium salt of BB-S469

To a suspension of 1.58 g of compound 8a in 10 ml of 50% mixture of acetone and water is added a 10% solution of sodium hydroxide to adjust the pH to 7.7. Acetone is further added and the precipitate is collected by filtration and then washed with acetone to give 1.38 g (87%) of the monosodium salt of compound 8a, melting above 170 ° C. as it decomposes. 15 posing.

Infrared spectrum: v 1765,1710,1640,1600,

1540.1485 cm-1.

20 Ultraviolet spectrum: X 7 252 nm (e, 20 600),

298 nm (e, 9200).

Nuclear magnetic resonance: ô ^ K2CO3 7 ^ 52

(1H, doublet, J = 9Hz, pyridazine H), 7.27 (4H, singlet, phenyl H), 7.03 (1H, doublet, J = 9Hz, pyridazine H), 5.62 (1H, doublet , J = 4.5 Hz, 7-H), 5.07 (1H, doublet, J = 4.5 Hz, 6-H7).

Analysis:

Calculated for C24H22N707S2Na • 2H20: Found:

25

35

40

VS%

H%

NOT%

S%

44.79

4.07

15.23

9.96

44.44

3.68

16.50

10.38

44.95

3.90

16.67

10.45

Example 2 Preparation of BB-S472

J-

CHgNHCH ^ CHgCONH

12b, BB-S472

N / N ^ ^ N-CHoCOOK

7- (3-N-tertio-butoxycarbonyl-N-methylaminomethyl-2-thienylacetamido) -3- (2-carboxymethyl-2,3-dihydro-s-triazo-lo [4,3-b] pyridazine-3- acid one-6-ylthiomethyl) -3-cephem-4-car-boxylic (11b)

A mixture of 513 mg (1.8 millimole) of amino acid 10b with a protective BOC group, 400 mg (2.16 millimoles) of 2,4-dinitrophenol and 445 mg (2 mg) is stirred at room temperature for 12 hours. , 16 millimoles) of DCC in 5 ml of THF. The precipitated urea is collected and the filtrate is added to a mixture of 800 mg (1.8 millimole) of 7-amino-3- (2-carboxymethyl) -2,3-dihydro-s-triazolo acid [4, 3-b] pyridazine-3-one-6-ylthiomethyl) -3-cephem-4-carboxylic 4 and 0.76 ml (5.4 millimoles) of triethylamine in 5 ml of water at 0 ° C with stirring. Stirring is continued until the active ester has disappeared from thin layer chromatography (silica gel plate; Rf 0.95;

solvent mixture = CHCl3: MeOH 3: 1). The reaction mixture is diluted with 20 ml of water, a layer of 50 ml of AcOEt is superimposed on it and its pH is adjusted to 2 by addition of concentrated hydrochloric acid, at 5 ° C. The organic phase is separated and the aqueous phase is extracted with three times 50 ml of AcOEt. The ethyl acetate extraction phases are combined, washed with a saturated aqueous NaCl solution, dried over magnesium sulfate and concentrated under reduced pressure. The residual oil (1.9 g) is chromatographed on 40 g of silica gel. The column is successively eluted with 400 ml of chloroform, 100 ml of 3% methanol in chloroform 65 and 10% methanol in chloroform, by carrying out controls by thin layer chromatography (silica gel plate; mixture of solvents = MeOH: CHCl3 at 1: 2, detection with iodine). A mixture is collected in the chloroform eluate.

23

627,184

2,4-DNP and amino acid 9 protected by a BOC group and 50 mg of compound 9 are collected in the eluate at 3% methanol in chloroform. The desired product (11b) (Rf 0, 4, mixture of solvents CHCl3: MeOH at 3: 1) is obtained by evaporation of the eluate with 10% methanol in chloroform. Yield 490 mg (39%). Melting point 215-220 ° C.

Infrared spectrum: v 3400,1780,1720,1680,1550

cm

Ultraviolet spectrum: X ÎSÏÏP011 pH 7 245 nm (e, 23 000), 260 nm (e, 18 000), 300 nm (e, 7900).

Nuclear magnetic resonance: ö 1 ^ 42 (9H,

singlet, BOC-H), 2.75 (3H, singlet, N-CH3), 3.80 (4H,

broad singlet, 2-H and h

4.35 (4H, large singlet,

H H

CO),

H

NOT

Analysis:

Calculated for CANA ^ -HzO: Found:

C% H% N% S%

46.46 4.60 46.67 4.71

13.55 12.79

13.29 12.81

times 5 ml of acetonitrile and dried at 60 ° C under a vacuum of 1mm of mercury for 7 hours to obtain 310 mg (90%) of compound 12b, melting at 188—191 ° C while decomposing.

5 Infrared spectrum: v 3400,1770,1720,

1680.1550 cm-1.

Ultraviolet spectrum: X j ^ ^ Pon pH 7 245 nm (e, 22 400), io260 nm (e, 18 700), 300 nm (e, 8600).

Analysis:

Calculated for 15 C23H23N707S3 • 3H20: Found:

C% H% N% S%

41.87 4.43 14.86 14.58 42.03 3.59 14.79 14.35

Preparation of monosodium salt of BB-S472

IN sodium hydroxide is added to a suspension of 20230 mg (0.38 millimole) of compound 12b in 0.5 ml of deionized water to adjust the pH to 8.9. 15 ml of acetone are added to the solution. The precipitate is collected by filtration, washed twice with 5 ml of acetone and dried at 60 ° C / 1 mm of mercury for 7 hours to obtain 170 mg (71%) of the monosodium salt 25 of BB-S472 melting above 210 ° C when decomposing.

and 3-CH2), 4.72 (2H, singlet, BOC-N-CH2), 5.10 (1H, doublet, J = 4.5 Hz, 6-H), 5.70 (1H, double doublet, J = 4.5 and 10.5 Hz, changing into a doublet J = 4.5 by addition of D20, 7-H), 6.85 (1H, doublet, J = 4.5 Hz, Hß of thiophene) , 7.19 (1H, doublet, J = 9 Hz, H of pyridazine), 7.34 (1H, doublet, j = 4.5, Ha of thiophene), 7.72 (1H, doublet, J = 9 Hz , H of pyridazine), 9.11 (1H, doublet, J = 10.5 Hz, disappears by addition of D20, NH).

30

Infrared spectrum: v 3400,1765,1710,1680,

1600 cm- '.

CompoundBB-S472; 7- (3-methylaminomethyl-2-thienyl-acetamido) -3- (2-carboxymethyl-2,3-dihydro-s-triazolo [4,3-b] -pyridazine-3-one-6-ylthiomethyl acid) - 3-cephem-4-carboxylic (12b)

0.4 ml of trifluoroacetic acid is added to 400 mg (0.57 millimole) of compound 11b at 0 ° C. and the mixture is stirred at room temperature for 15 minutes. 10 ml of anhydrous ether are added to the reaction mixture to separate a precipitate which is collected by filtration, which is washed with twice 10 ml of anhydrous ether and which is suspended in 10 ml of acetonitrile. The pH of the suspension is adjusted to 4 by the addition of concentrated ammonia and the mixture is stirred for 10 minutes. The solid is collected by filtration, washed with two

Ultraviolet spectrum: X j ^^ Pon P ^ 7 245 nm (e, 21 800), 260 nm (e, 18 500), 300 nm (e, 7800).

35 Nuclear magnetic resonance: ô 2.72 (3H, singlet, N-CH3), 3.45 (1H, doublet, J = 18 Hz, 2-H), 3.75 (1H,

X

doublet, J = 18 Hz, 2-H), 3.95 (2H, singlet, S ^ xCH2CO), 4.18 (4H, multiplet,

j ^ ch2N

40

and 3-CH2), 4.57 (2H, singlet, N-CH2),

5.00 (1H, doublet, J = 4.5 Hz, 6-H), 5.53 (1H, doublet, J = 4.5 Hz, 7-H), 6.97 (1H, doublet, J = 9 Hz, H of pyridazine), 7.03 45 (1H, doublet J = 4.5 Hz, Hß of thiophene), 7.34 (1H, doublet, J = 4.5 Hz, Ha of thiophene), 7, 48 (1H, doublet, 3 = 9Hz, H of pyridazine).

50 Analysis:

VS%

H%

NOT%

S%

Calculated for

C23H22H707S3Na • 1/2 H20:

43.40

3.64

15.40

15.11

Find:

43.26

4.08

14.18

13.91

55. Example 3

Preparation of BB-S478 compound

chgnh-ch ^

CHgCONH

nJy N-CH2C00H

0

8c, BB-S478

627,184

24

7- [2- (N-tertiobutoxycarbonyl-N-methylaminomethyl) -4-hydroxyphenylacetamido] -3- (2-carboxymethyl-2,3-dihydro-s-triazolo [4,5-b] pyridazine-3- one- acid 6-ylthiomethyl) -3-cephem-4-carboxylic (7c)

A mixture of 708 mg (2.4 millimoles) of 2-N-tert-butoxy-carbonyl-N-methylaminomethyl-4-hydroxyphenylacetic acid (5c), 478 mg (2.6) is stirred at room temperature for 2 hours. millimoles) of 2,4-dinitrophenol and 536 mg (2.6 millimoles) of DCC in 20 ml of anhydrous tetrahydrofuran. The precipitated urea is removed by filtration. The filtrate is added to a solution of 876 mg (2 millimoles) of 7-amino-3- (2-carboxymethyl-2,3-dihydro-s-triazolo [4,5-b] pyridazine-3-one- acid 6-ylthiomethyl) -3-cephem-4-carboxylic acid (4) in 20 ml of water containing 0.84 ml (6 millimoles) of triethylamine and the mixture is stirred at room temperature for 18 hours. After concentration to 20 ml, the aqueous solution is washed with ether, acidified with 6N hydrochloric acid and extracted with 200 ml of ethyl acetate. The extract is filtered to remove insoluble matter, washed with water and with a saturated aqueous solution of sodium chloride and dried. The solution is evaporated to dryness and the oily residue is chromatographed on silica gel ("Wakogel C-200", 25 g), eluting with chloroform and 3% chloroform in methanol. The fractions containing the desired product are combined (checked by thin layer chromatography; Rf 0.3; mixture of solvents, CHCl3: MeOH at 2: 1) and they are evaporated to dryness. The oily residue is triturated with a mixture of ether and n-hexane to obtain 630 mg (44%) of product 7c, melting at 200-210 ° C (slow decomposition).

1780,1720,1660,1400,

Infrared spectrum: v 1240.11 150 cm-1.

Ultraviolet spectrum: X J5 ??? 100 7 252 nm (e, 13000), 300 nm (e, 5400).

Nuclear magnetic resonance: ô 1> 39 (9H,

singlet, C-CH3), 2.73 (3H, singlet, N-CH3), 3.3-3.9 (4H, multiplet, CH2CO and 2-H), 4.35 (4H, multiplet, CH2N and 3 -H), 4.48 (2H, singlet, NCH2CO), 5.03 (1H, doublet, 4.5Hz, 6-H), 5.61 (1H, double doublet, 8 and 4.5Hz, 7-H) , 6.4-7.2 (3H, multiplet, phenyl H), 6.98 (1H, doublet, 10Hz, pyridazine H), 7.61 (1H, doublet, 10Hz, pyridazine H), 8.87 (1H, doublet, 8 Hz, NH).

A mixture of 570 mg (0.8 millimole) of compound 7c and 1.5 ml of trifluoroacetic acid is stirred at 10 ° C. for 30 minutes and the mixture is diluted with 50 ml of ether to separate the trifluoroacetate 8c which is collected by filtration and then dissolved in a mixture of 10 ml of acetonitrile and 5 ml of water, then filtering the solution. The pH of the filtrate is adjusted to 6 by adding concentrated ammonium hydroxide and the mixture is diluted with 100 ml of acetonitrile. The resulting precipitate is collected by filtration, washed with acetonitrile and dried under vacuum over phosphorus pentoxide, giving 370 mg (75%) of compound 8c, melting at 215-220 ° C while decomposing.

Infrared spectrum: v KB? 1770,1710,1600,1380,

^ 1 HICIA »

1350 cm 1.

15 Ultraviolet spectrum: X S ^ fon P ^ 7 252 nm (e, 19000), 300 nm (e, 9100).

Nuclear magnetic resonance: ô + K2C03 2.75

2o (3H, singlet, N-CH3), 2.9—3.3- (4H, multiplet, CH2CO and 2-H), 4.0-4.3 (4H, multiplet, CH2N and 3-H), 4.57 (2H, singlet, NCH2CO), 4.81 (1H, doublet, 4.5-Hz, 6-H), 5.53 (1H, doublet, 4.5 Hz, 7-H), 6, 6-7.5 (5H, multiplet, phenyl H and pyridazine H).

25

Analysis:

Calculated for ÇhHJBNTOA- 5/2 H20: 3o Found:

C% H% N% S%

45.45 45.69

4.58 4.21

14.84 15.03

9.71 9.46

Preparation of the sodium salt of BB-S478

0.3-0.4 ml of 1N NaOH is added to a suspension of 308 mg (0.5 millimole) of compound 8c in 2 ml of water and the mixture is stirred at room temperature; the pH of the resulting solution is 9.2. 20 ml of acetone are slowly added to the solution. The resulting precipitate is collected by filtration, washed with 10 ml of acetone and dried under vacuum over phosphorus pentoxide giving 290 mg (91%) of the monosodium salt of 40 BB-S478 melting at 230-235 ° C while decomposing .

Infrared spectrum: v ^ Br 1770,1700,1600,1390,

-1

1350 cm

Ultraviolet spectrum: X. ÎB ™ Pon 7 250 nm (e, 18,000),

300 nm (E, 8400).

Analysis: C% H% N% S%

Calculated for C30H33N7O10S2: 50.34 4.65 13.70 8.96 Found: 50.98 5.36 11.88 7.60

Compound BB-S478; 7- (2-N-methylaminomethyl-4-hydr-oxyphenylacetamido) -3- (2-N-carboxymethyl-2,3-dihydro-s-triazolo [4,5-b] pyridazine-3-one-6- acid ylthiomethyl) -3-cephem-4-carboxylic (8c)

Analysis: 50 Calculated for C25H24N708S2Na 'Found:

C% H% N% S%

5/2 H20:

43.98 43.96

4.28 4.14

Example 4 55 Preparation of compound BB-S479

14.36 13.51

9.39 9.34

ch2nh-ch3

ch2conh

^ ^ N-CK2C00H

8b, BB-S479

25

627,184

Acid 7- [o- (N-butoxycarbonyl-N-methylaminomethyl) -phenyl-acetamido] -3- (2-carboxymethyl-2,3-dihydro-s-triazolo [4,3-b] -pyridazine-3-one -6-ylthiomethyl) -3-cephem-4-carboxylic (7b)

Add a solution of 2,4-dinitrophenyl (6b) o- (N-butoxycarbonyl-N-methyl-aminomethyl) phenylacetate in tetrahydrofuran [prepared from 3.48 g (13.5 mmol) of acid o - (N-butoxycarbonyI-N-methylaminomethyl) -phenyI-acetic (5b), 2.49 g (13.5 millimoles) of 2,4-dinitrophenoide and 2.79 g (13.5 millimoles) of DCC in 37 ml anhydrous tetrahydrofuran] to a cold (0 ° C) mixture of 5.4 g (12 millimoles) of the compound 4.5.5 ml of Et3N, 25 ml of CH3CN and 25 ml of water. The mixture is stirred at room temperature for about 18 hours. The THF and CH3CN are removed from the reaction mixture by evaporation under reduced pressure and the resulting aqueous solution is washed with three times 30 ml of ether, its pH is adjusted to 2-3 by addition of dilute hydrochloric acid and it is extracted with four times 30 ml of ethyl acetate. The organic extracts are combined, dried over sodium sulfate and evaporated. The residue is chromatographed on a column of 100 g of SiO2. After washing with chloroform, the column is eluted with 3% methanol in chloroform to obtain a desired fraction containing compound 7b. Yield 3.8 g (45%); melting point above 200 ° C (decomposition).

Infrared spectrum: v 1780,1720,1680 cm-1.

Ultraviolet spectrum: X J ^ Pon 7 250 nm (e, 18 800), 297 nm (e, 8400).

Nuclear magnetic resonance: ô + DzO 7 ^ 2

(1H, doublet, J = 10.5Hz, pyridazine H), 7.14 (4H, singlet, phenyl H), 6.98 (1H, doublet, J-10.5Hz, pyridazine H), 5.61 (1H, doublet, J = 4.5 Hz, 7-H7.5.03 (1H, doublet, J = 4.5Hz, 6-H), 4.67 (2H, singlet, N-CH2), 4, 42 (2H, singlet, CH2-N), 4,4,4,0 (2H, multiplet, 3-CH2), 3,8-3,4 (4H, multiplet, 2-H and CH2-CO), 2 , 72 (3H, singlet, N-CH3), 1.38 (9H, singlet, BOC-H).

Analysis: Calculated for C3 () H33N709S2 Found:

C% H% N% S%

5/2 H, 0:

48.38 48.25 48.23

5.14 4.52 4.46

13.16 12.93 12.86

8.61 8.68

Compound BB-S479; 7- [o- (methylaminomethyl) -phenyl-acetamido] -3- (2-carboxymethyl-2,3-dihydro-s-triazolo [4,3-b] -pyridazine-3-one-6-ylthiomethyl) acid 3-cephem-4-carboxylic (8b)

7 ml of trifluoroacetic acid is added to 3.8 g (5.5 millimoles) of the tertio-BOC derivative 7b at 0 ° C and the mixture is stirred for 20 minutes at room temperature. Then added 100 ml of anhydrous ether. The precipitate formed is collected by filtration and washed with three times 100 ml of anhydrous ether. The precipitate is dissolved in a mixture of 120 ml of CH3CN and 18 ml of water and the pH of the solution is adjusted to 5-6 by addition of concentrated ammonia to obtain an oily precipitate which is triturated with l acetonitrile to form a solid. The product 8b is collected by filtration, it is washed with acetonitrile and dried. Yield 2.55 g (77%).

cm

Infrared spectrum: v

1,770,1710,1600,1550

20

Preparation of monosodium salt of BB-S479

Cooling approximately 3 ml of IN sodium hydroxide is added to a solution of 2.54 g (4.3 millimoles) of BB-S479 (8b) in 25 ml of water (the pH of the solution is equal to 10 ). A large amount of acetone is added to the solution and the precipitate is collected by filtration, then washed with acetone to give 1.94 g (84%) of the monosodium salt of BB-S479 melting above 200 ° C when decomposing.

30

Infrared spectrum: v

1550 cm

KBr max.

1770,1710,1600,

Ultraviolet spectrum: X ™ ïfon ^ 7 250 nm (e, 19 400), 297 nm (e, 8700).

35

40

Analysis: Calculated for C25H24N707S2 'Found:

C% H% N% S%

V2H20:

45

Preparation of BB-S483

47, '61 47.43 47.43

Example 5

4.00

4.67

4.68

15.55 15.97 15.70

10.17 9.25 9.84

-chgnhg -CHgCONH

n-ck ^ cooh

12a, BB-S483

7- (3-N-tertio-butoxycarbonylamino methyl-2-thienylacet- 683 mg (1.56 millimole) acid 7-amino-3- (2-carboxymethyl-

amido) -3- (2-carboxymethyl-2,3-dihydro-s-triazolo [4,3-b] pyri-2,3-dihydro-s-triazolo [4,3-b] pyridazine-3-one- 6-ylthiomethyl) -

dazine-3-one-6-ylthiomethyl) -3-cephem-4-carboxylic (lia) 3-cephem-4-carboxylic 4 and 0.62 ml (4.68 millimoles) of

A triethylamine in 5 ml of water at 0 ° C. is stirred at room temperature for 12 hours with stirring. A mixture of 410 mg (1.56 millimoles) of group 65 amino acid is continued to stir at room temperature until the active ester has

Protective BOC (9), 313 mg (1.7 millimole) of 2,4-dinitrophed - disappeared according to thin layer chromatography (nolet plate 353 mg (1.7 millimole) of DCC in 5 ml of THF. silica gel; Rf 0.95; mixture of solvents, CHCl3: MeOH at

collect the precipitated urea and add the filtrate to a 3: 1 mixture. The reaction mixture is diluted with 20 ml of water, it is

627,184

26

superimpose a layer of 50 ml of ethyl acetate and its pH is adjusted to 2 by adding concentrated hydrochloric acid, at 5 ° C. The organic phase is separated and the aqueous phase is extracted with three times 50 ml of ethyl acetate. The ethyl acetate extraction phases are combined, washed with a saturated aqueous solution of sodium chloride, dried over magnesium sulfate and concentrated under reduced pressure. The residual oil (1.8 g) is chromatographed on 40 g of silica gel. The column is eluted successively with 400 ml of CHCl3 and 500 ml of 3% MeOH in CHCl3. The eluate is checked by thin layer chromatography (silica gel plate, mixture of solvents, CHCl3: MeOH at 2: 1, detection with iodine). The desired product IIa (Rf 0.2) is obtained by evaporation of the methanolic-chloroform eluate. Yield 450 mg (42%), melting point 155-160 ° C.

Infrared spectrum: v KB? 1680 cm-1.

3300,1775,1720,

Analysis Calculated for C22H21N707S3-3H20 Found:

40.90 4.21 40.39 3.62

15.17 15.87

14.89 14.35

Preparation of monosodium salt of BB-S483

IN sodium hydroxide is added to a suspension of 280 mg (0.47 mmol) of compound 12a in 0.5 ml of deionized water to adjust the pH to 9.5 and the material is collected by filtration. insoluble. 15 ml of acetone are added to the filtrate to separate the precipitate which is collected by filtration, it is washed with twice 5 ml of acetone and it is dried at 70 ° C / 1 mm of mercury for 7 hours to obtain 220 mg (76%) of monosodium salt of compound 12a, melting above 210 ° C. in 15, decomposing slowly.

Infrared spectrum: v J®? 3400,3250,1760,1710,

Ultraviolet spectrum: X ^ ™ Pon P ** 7 245 nm (e, 23 900), 260 nm (e, 19 200), 300 nm (e, 8700).

Nuclear magnetic resonance: ô 1> 39 (9H,

singlet, BOC-H), 3.76 (4H, large singlet, 2-H and S

CH2CO), 4.05 (2H, doublet, 3 = 6 Hz, changed to a singlet by addition of D20, BOCNH-CH), 4.20 (2H, multiplet, 3-CH2), 4.69 (2H, singlet , N-CH2C02), 5.06 (1H, doublet, J = 4.5 Hz, 6-H), 5.62 (1H, double doublet, 1 = 4.5 and 9 Hz, changed to a doublet 3 = 4.5 Hz by addition of D20.7-H), 6.83 (1H, doublet, J = 4.5 Hz, Hß of thiophene), 7.00 (1H, multiplet, disappears by addition of DzO, NHBOC) , 7.04 (1H, doublet, 3 = 9 Hz, H of pyridazine), 7.12 (1H, doublet, 1 = 4.5 Hz, Ha of thiophene), 7.65 (1H, doublet, J = 9 Hz, pyridazine H), 8.97 (1H, doublet, 3 = 9 Hz, disappears by addition of D20.7-NH).

Analysis: C% H% N% S%

Calculated for C27H29N709S3: 46.88 4.23 14.17 13.90 Found: 46.42 4.37 13.49 13.61

20

1650.1600.1550 cm- •.

)

Ultraviolet spectrum: X ^ ™ Pon P ^ 7 245 nm (e, 19900), 260 nm (e, 16400), 300 nm (e, 6900).

25 Nuclear magnetic resonance: ô 3.60 (2H, m,

2-H), 3.91 (2H, s, CH2CO), 4.12 (2H, s, CH2-NH2), 4.20 (2H, m, 3-CH2), 4.55 (2H, s, N-CH2CO), 4.95 (1H, d, J = 4.5 Hz, 6-H), 5.50 (1H, d, J = 4.5 Hz, 7-H), 6.94 (1H , d, J = 9 Hz, H of pyridazine), 6.99 (1H, d, J = 4.5 Hz, thiophene Hß), 7.28 (1H, d, J = 4.5 Hz, Ha thiophene), 7.32 (1H, d, J = 9 Hz, H of pyridazine).

35 Analysis: C% H% N% S%

Calculated for

C22H2nN707S3

Na • 5 / 2ch3coch3 Found:

40

Solubility:

All the monosodium salts in this series have a solubility in water greater than 10%.

43.92 3.61 15.26 14.97 43.48 4.56 15.28 13.91

Compound BB-S483; 7- (3-Aminomethyl-2-thienyl-acet-amido) -3- (2-carboxymethyl-2,3-dihydro-s-triazolo [4,3-b] pyri-dazine-3-one-6- acid ylthiomethyl) -3-cephem-4-carboxylic (12a)

0.4 ml of trifluoroacetic acid is added to 410 mg (0.59 mmol) of compound IIa at 0 ° C and the mixture is stirred at room temperature for 15 minutes. 10 ml of anhydrous ether are added to the reaction mixture to separate a precipitate which is collected by filtration, which is washed with twice 10 ml of anhydrous ether and which is suspended in 10 ml of acetonitrile. The pH of the suspension is adjusted to 4 by the addition of concentrated ammonia and the mixture is stirred for 10 minutes. The precipitate is collected by filtration, washed with twice 5 ml of acetonitrile and dried at 60 ° C. under a vacuum of 1 mm of mercury for 7 hours to obtain 310 mg (88%) of compound 12a, melting at -above 200 ° C, slowly decomposing.

Infrared spectrum: v KBr 3400, 3150,1760,1700, 1680,1600 cm-1

Ultraviolet spectrum: X ^} ^ Pon P ^ 7: 245 nm (e, 17100), 260 nm (e, 14100), 300 nm (e, 6500).

45 Nephrotoxicity:

A preliminary nephrotoxicity study was carried out by administration of the test compound to a group of two rabbits intravenously at a dose of 100 mg / kg. The results obtained with BB-S469 and BB-S479 indicate that these compounds 50 could have a low nephrotoxic potential.

In vitro activity: (Table I)

The minimum inhibitory concentrations were determined by serial dilution methods using Mueller-Hinton agar 55, against 51 Gram-positive bacteria and 96 Gram-negative bacteria. The 147 test organisms were classified into 16 groups according to the genera and types of antibiotic resistance, 5 groups for Gram-positive bacteria and 11 groups for Gram-negative bacteria. Table 60 indicates the in vitro activity expressed by the geometric mean of the minimum inhibitory concentrations. Compounds BB-S472 and BB-S479 have better overall activity than their respective analogs BB-S483 and BB-S469 which are not methylated. Compound BB-S479 is superior to BB-S472 for 65 some species of Gram-negative bacteria. Compared to cefamandole, BB-S479 is more active against most test organisms, except against species of the genus Providentia and staphylococci.

27

627,184

Table I

In vitro activity against 147 test organisms

Geometric mean of minimum inhibitory concentrations *

([ig / ml)

Test organism

Number

BB-S469

BB-S472

BB-S478

BB-S479

BB-S483

Cefam-

BL-S786

of

(ex. 1)

(ex. 2)

(ex. 3)

(ex. 4)

(ex. 5)

andole

**

strains

S. aureus (sensitive)

4

0.4

0.4

0.4

0.3

0.3

0.1

1.3

S. aureus (penicillinase +)

13

1.2

0.9

1.0

0.9

1.1

0.4

3.0

S. aureus (Methicillino-R)

15

30

58

50

35

48

2.4

48

S. pyogenes

10

0.05

0.05

0.04

0.04

0.08

0.04

0.2

D. pneumoniae

9

0.03

0.05

0.03

0.04

0.04

0.2

0.08

E. coli (sensitive)

13

0.3

0.2

0.2

0.2

0.3

0.3

0.4

E. coli (penicillinase +)

7

11

15

10

2.6

21

5.7

9.3

Enterobacter (sensitive)

3

1.0

1.3

0.8

0.5

1.3

1.0

1.3

Enterobacter (cephalosporinase '+)

7

4.7

3.2

2.6

2.1

5.2

4.7

7.7

Proteus (indole -)

6

0.6

0.3

0.4

0.5

0.7

0.6

0.9

Proteus (indole +)

14

0.5

0.3

0.3

0.2

0.4

0.5

0.7

Proteus (indole +, cephalospirinase +)

5

11

8.3

7.3

4.8

17

3.6

29

Providencia sp.

5

4.2

4.8

4.8

4.2

2.1

0.7

1.4

Klebsiella sp.

12

0.9

0.8

0.6

0.6

1.1

2.4

0.8

S. marcescens

16

130

200

40

35

81

34

180

Miscellaneous (Salmonella, Shigella,

Citrobacter)

8

0.7

0.4

0.3

0.4

0.9

0.3

0.7

* Mueller-Hinton agar (size of the inoculum: dilution to 104)

Interruption for a minimum inhibitory concentration of 200 [ig / ml.

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

Example 6 xyphenylacetic acid 2-N-tertiobutoxycarbonylaminomé-

By replacing, in the procedure of Example 3, α-4-methoxyphenylacetic acid and 2-N-cide2-N-tertio-butoxycarbonyl-N-methylaminomethyl-4-hydr-tert-butoxycarbonyl-N-methylaminomethyl acid, respectively -4-methoxyphé-oxyphenylacétique used in this example by an equinylacetic weight, the respective compounds corresponding to the molars of 2-N-tert-butoxycarbonylaminomethyl-4-hydr are obtained:

ch2nh2

choc0iffl-ch-ch

2! i c — n // ^ 0

c, h2

^ C-CHgS

c-oh ii

0

= n

.n - ^ "\ ^ n" gh2c00h and ch2nh2

choc0nh-ch-ch ch_

2 t I 1 2

0

c — n c — ch0s

// 2

c-oh ii

0

.n ^ \ ^ n ~ ch2c00h and

627,184

28

CH-, 0 D

ch2nhch3

ch7c0nh-ch-ch ch * II I 2

f-Kr * c-cn2s 0 \

c-oh n-ch ^ cooh

Preparation of BB-S493

Example 7

O

"nh-boc cooh

'nh-boc conh r

■ n.

. ^ JK ^ -N-CHgCOgH

co2h

O

nh2 conh-

y

• r

-n ch2s-

■ n

^ n ^ i-ch2co2H

Y

c02h

0

3, BB-S493

Acid 7 - [(2-N-tertiobutoxycarbonylaminomethyl-1,4-cyclo

hexadienyl) acetamido] -3- (2-N-carboxymethyl-2-triazolo [4,5-

b] pyridazine-3-one-6-ylthiomethyl) -3-cephem-4-carboxylic

(2)

A mixture of 640 mg (2.4 mmol) of 2-N-tert-butoxycarbonylaminomethyl-1,4-cyclohexadienylacetic acid 1, of 422 mg (2.4 mmol) is stirred for 1.5 hours at room temperature. 2,4-dinitrophenol and 494 mg (2.4 mmol) of DCC in 10 ml of anhydrous THF. The urea precipitate is removed by filtration. The filtrate is added in a single portion to a solution of 7-amino-3- (2-N-carboxymethyl-s-triazole- (4,5-b] pyridazin-3-on-6-ylthiomethyl) -3 acid. -céphem-4-carboxyli-that in 10 ml of water containing 0.56 ml (4 mmol) of triethyl-

55 amine and the mixture is stirred at room temperature for 3 hours. The reaction mixture is concentrated to 10 ml under reduced pressure, washed with three times 10 ml of ether, acidified with 6N hydrochloric acid and extracted with five times 10 ml of acetate. ethyl. The combined extracts are washed with a saturated solution of sodium chloride and dehydrated with anhydrous sodium sulfate. The solvent is evaporated off and the residue is chromatographed on silica gel (30 g of "Wako-gel C-200"), eluting with a mixture of chloroform and methanol (0-50%). The fractions which contain the desired product are combined. The solvent is removed and the residue is triturated with a mixture of ether and n-hexane, giving 410 mg (30%) of product 2, melting at 110 ° C. while decomposing.

29

627,184

Infrared spectrum, v 1780,1730,1610,1530, 1250,1160 cm-1.

Ultraviolet spectrum: on 7,252 nm (e, 19,000),

300 nm (e, 8600), shoulder.

Analysis:

VS%

H%

NOT%

S%

Calculated for

C29H33N7O9S2 * 2H20

48.12

5.15

13.54

8.86

Find:

48.07

4.64

12.70

8.39

BB-S493; acid 7 - [(2-aminomethyl-1,4-cyclohexadienyl) -acet-amido] -3- (2-N-carboxymethyl-s-triazolo [4,5-b] pyridazine-3-one-6-ylthiomethyl) -3-cephem-4-carboxylic (3).

350 mg (0.51 mmol) of cephalosporin 2 with amino function protected by a BOC group are treated with 1 ml of tri-fluoracetic acid (TFA) for 30 min at room temperature. 50 ml of ether are added to the mixture to obtain the TFA salt of compound 3 which is collected by filtration and which is then dissolved in a mixture of 5 ml of acetonitrile and 2 ml of water. The solution is treated by adding a small amount of

Active carbon organism and its pH is adjusted to 6 by the addition of concentrated ammonium hydroxide. The precipitate is collected, washed with 5 ml of acetonitrile and dried under vacuum to obtain 235 mg (78%) of compound 3, melting at 220-230 ° C as it decomposes.

Infrared spectrum: v ^ Br 1770,1740,1710,1650, 1600,1550 cm-1.

Ultraviolet spectrum: X j ^? Pon 7,252 nm (e, 20,000), 300 nm (e, 9000, shoulder).

Analysis:

vs%

H%

not%

s%

Calculated for

c24h25n7o7s2h2o

47.60

4.49

16.19

10.59

Find:

47.77

4.06

16.49

10.64

20

Antibacterial activity in vitro of the BB-S493 compound compared to the activity of BB-S479 and cefamandole (determined by the Steers agar dilution method on Mueller-Hinton agar plate)

Minimum inhibitory concentration Og / ml)

BB-S493 BB-S479 Cefamandole

S. aureus Smith S. aureus

S. aureus BX-1633 St. faecalis

A9537 0.4

A9497 0.2

A9606 0.4

A9536 100

0.4 0.1

0.1 0.05

0.4 0.2

100 50

E. coli NIHJ

0.1

0.05

0.025

E. coli ATCC 8739

0.2

0.05

0.05

E. coliJuhl

A15119

0.2

0.1

0.4

E. coli BX-1373

0.4

0.2

0.4

E. coli

A15810

0.2

0.1

0.2

E. coli

A9660

0.1

0.05

0.1

E. coli

AI 5147

6.3

3.1

3.1

Kl. pneumoniae

A9678

0.4

0.4

1.6

Kl. pneumoniae

A9977

0.2

0.1

0.4

Kl. pneumoniae

A15130

0.2

0.1

0.4

Kl. pneumoniae

A9867

0.2

0.1

0.8

Pr. Vulgaris

A9436

0.4

0.1

0.2

Pr. Vulgaris

A9699

6.3

0.8

25

Pr. Mirabilis

A9554

0.2

0.1

0.8

Pr. Mirabilis

A9900

0.2

0.1

0.8

Pr. Morganii

A9553

> 100

> 100

> 100

Pr. Morganii

A20031

0.4

0.1

0.8

Pr. Rettgeri

A15167

0.1

0.1

0.1

Ps. Aeruginosa

A9930

> 100

> 100

> 100

Ps. Aeruginosa

A9843

> 100

> 100

> 100

Shig. dysenteriae

0.05

0.025

0.2

Shig. flexnery

A9684

25

12.5

3.1

Shig. sonnei

A9516

0.05

0.025

0.05

Serr. marcescens

A20019

100

25

50

Enterob. cloacae

A9656

6.3

1.6

3.1

Sai. enteritidis

A9531

0.1

0.05

0.1

Sai. typhosa

A9498

0.1

0.05

0.1

B. anthracis

A9504

0.0125

0.025

0.2

In vivo activity of BB-S479 and related compounds (mouse, subcutaneous)

627,184

30

(DP50, mg / kg)

Organization BB- BB- BB- BB- BB- BB- Céfa- BL-

S469 S472 S478 S479 S483 S493 mandolin S786

S. aureus Smith 0.19 0.12 0.08 0.16 0.34 0.29 0.93 0.53

0.16 0.12 0.74 0.55

0.29 0.2 0.6 0.94

0.2 0.8 0.46

0.8 0.6

E. coli Juhl 0.19 0.19 0.08 0.19 0.15 0.27 0.95 0.43

0.24 0.15 1.7 0.55

0.15 0.12 1.8 0.46

2.2 0.39 0.8

Other raw materials:

6-chloro-2- (2-cyanethyl) -2,3-dihydro-s-triazolo [4,3-b] pyri-dazine-3-one

0.5 g (4.5 mmol) of potassium tert-butoxide is added with stirring to a solution of 17 g (0.1 mole) of 6-chloro-2,3-dihydro-s-triazolo [4.3 -b] pyridazine-3-one [P. Francabilla and F. Lauria, “J. Het. Chem. 8.415 (1971)] in 300 ml of anhydrous dimethylformamide. 6.6 g (0.12 mol) of acrylonitrile in 10 ml of anhydrous DMF are added to the mixture. The mixture is stirred at 100-110 ° C for 24 hours then poured into 700 ml of water and extracted with five times 400 ml of ethyl acetate. The organic extracts are combined, dried over sodium sulfate and evaporated. By crystallization of the residue in ethyl acetate, light yellow needles of 6-chloro-2- (2-cyanethyl) -2,3-dihydro-2-triazo-lo [4,3-b are obtained ] pyridazine-3-one (2.5 g, 11%), melting at 166-168 ° C.

Infrared spectrum: v 1500 cm-1.

2230.1720.1550,

Ultraviolet spectrum: X ^ j ° £ anne

373 nm (e, 2000).

Nuclear magnetic resonance: ô j ^ MpO-d6 3 93 (2H, t,

J = 6.0 Hz, C2H), 4.21 (2H, t, J = 6.0Hz, CH,), 7.23 (1H, d, J = 10.0 Hz, H of pyridazine), 7, 93 (1H, d, J = 10.0 Hz, H of pyridazine).

Analysis:

Calculated for C8H6N5OCl Found:

C% H%

42.97 2.70

42.73 2.57

42.56 2.50

N% Cl%

31.32 15.86

31.36 15.96

31.68 15.81

Infrared spectrum: K®?

3400-2400,1730,1710,

20

Ultraviolet spectrum: X Roxanne 377 nm (e, 1500). Nuclear magnetic resonance: ô ^ 2® + NaHC03

2.70

25

(2H, t, J = 7.0 Hz, CH2), 4.24 (2H, t, J = 7.0 Hz, CH2), 7.17 (1H, d, J = 10.0 Hz, H of pyridazine), 7.70 (1H, d, J = 10.0 Hz, H of pyridazine).

Analysis:

Calculated for C8H7N403C1 Found

C% H%

39.60 2.91

39.62 2.97

39.48 2.67

N% Cl%

23.09 14.61

23.05 13.93

22.70 14.12

2- (2-carboxyethyl) -2,3-dihydro-6-mercapto-s-triazolo [4,3-b] -pyridazine-3-one

A mixture of 567 mg (2.34 mmol) of 2- (2-carboxyethyl) -6-chloro-2,3-dihydro-s-triazolo [4,3-b] is stirred at room temperature for 2 hours. pyridazine-3-one and 924 mg (7.02 mmol) sodium hydrosylfide dihydrate 70% in 10 ml of water. The pH of the reaction mixture is adjusted successively to 1 by addition of concentrated hydrochloric acid, to 4010 by addition of sodium hydroxide and then to 1 by addition of concentrated hydrochloric acid. The precipitate formed of 2- (2-carboxyethyl) -2,3-dihydro-6-mercap-to-s-triazolo [4,3-b] pyridazine-3-one is collected by filtration and washed with water. Yield 418 mg (74%); melting point 174-176 ° C.

45

50

Infrared spectrum: v 3600-2600,2440,1730, 1720 (shoulder) cm-1.

Ultraviolet spectrum: X 7 262 nm (e, 17000),

318 nm (e, 6600).

2- (2-carboxyethyl) -6-chloro-2,3-dihydro-s-triazolo [4,3-b] pyri-dazine-3-one

A solution of 724 mg of 6-chloro-2- (2-cyanethyl) -2,3-dihydro-s-triazolo [4,3-b] pyrid-azine-3-one is refluxed for 6 hours in 15 ml 6N hydrochloric acid. The reaction mixture is extracted with ten times 20 ml of ethyl acetate. The combined extracts are washed with 50 ml of saturated sodium chloride solution, dried over sodium sulfate and evaporated to give 567 mg (72%) of 2- (2-carb-oxyethyl) -6-chloro-2, 3-dihydro-s-triazolo [4,3-b] pyridazine-3-one light yellow solid melting above 170 ° C while sublimating.

Nuclear magnetic resonance: p ^ ®® ~ (* 62.73 (2H, t,

J = 7.0 Hz, CH2), 4.07 (2H, t, J = 7.0 Hz, CH2), 7.30 (1H, d, J = 10.0 Hz, H of pyridazine), 7, 74 (1H, d, J = 10.0 Hz, H of pyridazine).

60

Analysis:

VS%

H%

NOT %

S%

Calculated for CgH8N403S

40.00

3.36

23.32

13.35

Find

39.08

3.12

22.65

14.23

39.06

3.20

22.70

14.29

65

1540 cm

7-amino-3- [2- (2-carboxyethyl) -2,3-dihydro-s-triazo-lo [4,3-b] pyridazine-3-one-6-ylthiomethyl] -3-cephem-4 acid -car-boxylic

31

627184

A mixture of 405 mg (1.49 mmol) of 7-ACA, 357 mg (1.49 mmol) of 2- (2-car-boxyethyl) -2,3-dihydro- is stirred at 80 ° C. for 30 minutes. 6-mercapto-s-triazolo [4,3-b] pyri-dazine-3-one (thiol) and 375 mg (4.47 mmol) of NaHC03 in 8 ml of 0.1 M phosphate buffer (pH 7) . The reaction mixture is cooled and filtered to remove insoluble matter. The pH of the filtrate is adjusted to 1-2 by adding concentrated hydrochloric acid. The resulting precipitate, which consists of 7-amino-3- [2- (2-carboxyethyl) -2,3-dihydro-s-triazo-lo [4,3-b] -pyridazine-3-one-6- acid ylthiomethyl] -3-cephem-4-carb-oxyle, is collected by filtration and washed with water. Yield 519 mg (77%).

Infrared spectrum: v 3600-2200,1800,1725,

Ultraviolet spectrum: X ^^ Pon P ^ 7 253nm (e, 19800), 298 nm (e, 9400).

5 Nuclear magnetic resonance: ô 1.37 (9H, s,

tert-butyl hydrogen), 2.70 (3H, s, N-CH3), 2.70 (2H, t, J = 7.0 Hz, -CH2-), 3.2-4.5 (10H, m) , 5.01 (1H, d, J = 5 Hz, 6-H), 5.60 (1H, dd, J = 5 and 8 Hz d, the 8 Hz coupling disappears by addition of D20.7-H), 6.93 (1H, d, J = 10 Hz, H of pyri-io dazine), 7.58 (1H, d, J = 10 Hz, H of pyridazine).

-1

1620.1550.1480 cm

Ultraviolet spectrum: X ^ ™ Pon PH 7 253 nm (e, 20000), 298 nm (s, 10000).

Nuclear magnetic resonance: ô 2.20

(2H, t, J = 7.0 Hz, CH2), 3.40 (1H, d, J = 17.5 Hz, 2-H), 3.85 (1H, d, J = 17.5 Hz, 2-H), 4.00-4.50 (4H, m, 3-CH2 and N-CH2-), 5.01 (1H, d, J = 4.0 Hz, 6-H), 5.40 (1H, d, J = 4.0 Hz, 7-H), 6.94 (1H, d, J = 10.0 Hz, H of pyridazine), 7.44 (1H, d, J = 10.0 Hz, H of pyridazine).

Analysis:

Calculated for 15 C3IH35N709S2 • 5 / 2H20 Found

C% H% N% S%

49.08 5.31 12.92 8.45

49.32 4.70 12.52 8.44

49.36 4.63 12.53 8.43

Analysis:

Calculated for C16H16N606S2.3 / 2H20 Found

C% H% N% S%

40.09 40.06 40.12

3.99

3.33

3.34

17.52 16.96 16.98

13.37 13.87 13.98

It will be recalled that 7-ACA denotes 7-aminocephaIo-sporanic acid and that DMF denotes dimethylformamide.

Example 8

7- [o- (N-butoxycarbonyl-N-methylaminomethyl) phenyl- acid

acetamidai] -3- [2- (2-carboxoyethyl) -2,3-dihydro-s-triazolo [4,3-

b] pyridazine-3-one-6-ylthiomethyl] -3-cephem-4-carboxylic.

452 mg (1 mmol) of 7-amino-3- [2- (2-carboxyethyl) -2,3-dihydro-s-triazolo [4,3-b] -pyridazine-3 are added to a mixture -one-6-ylthiomethyl] -3-cephem-4-carboxylic acid and 0.46 ml (3.3 mmol) of triethylamine in 4 ml of 50% aqueous acetonitrile, 3 ml of o-tetrahydrofuran solution ( N-tertiobutoxycarbonyl-N-methylaminomethyl) 2,4-dinitrophenyl phenyl acetate obtained from 283 mg (1.1 mmol) o- (N-tertiobutoxycarbonyl-N-methylaminomethyl) phenylacetic acid, 202 mg ( 1.1 mmol) of 2,4-dinitrophenol and 227 mg (1.1 mmol) of DCC. The mixture is stirred at room temperature for about 18 hours and concentrated under reduced pressure to remove the organic solvents. The aqueous concentrate is washed with three times 20 ml of ether, acidified to a pH equal to 2-2 by addition of concentrated hydrochloric acid and extracted five times with 20 ml of ethyl acetate. The combined extracts are dried over anhydrous sodium sulfate and evaporated to dryness. The residue is chromatographed on a column of silica gel (“Wako” gel C-200.10 g) by elution with a mixture of methanol and chloroform (MeOH- 0 to 3%). The combined eluates which contain the desired product are evaporated to give 359 mg (50%) of the title compound, melting above 150 ° C as it decomposes.

Infrared spectrum: v 3600-2400,1780,1720, 1680,1550,1490 cm-1.

BB-S525; 7- [o- (N-methylaminomethyl) phenylacetami-20 do] -3- [2- (2-carboxyethyl) -2,3-dihydro-s-triazolo [4,3-b] pyri-dazine-3- acid one-6-ylthiomethyl] -3-cephem-4-carboxylic

A mixture of 1 ml of trifluoroacetic acid and 302 mg (0.42 mmol) of the protective BOC group cephalosporin prepared as indicated above is left standing at room temperature for 15 minutes, then diluted. mixture with 10 ml of ether. The resulting precipitate is collected by filtration and washed with twice 10 ml of anhydrous ether, which gives 263 mg of a solid substance which is dissolved in a mixture of 6 ml of water and 3 ml of acetonitrile. . The pH of the stirred solution is adjusted to 4 by adding 0.36 ml of IN sodium hydroxide and this solution is diluted with 100 ml of acetonitrile, which gives 187 mg of precipitate which is suspended. in 4 ml of water, adjusting the pH of the suspension to 9 by adding 0.3 ml of IN sodium hydroxide. The solution is treated with a small amount of active carbon and lyophilized to obtain the monosodium salt of BB-S525. Yield: 106 mg (39%), melting point, above 180 ° C (decomposition).

“O Infrared spectrum: v ™? 3600-2400,1770,1710, 1600,1490,1400 cm "

Ultraviolet spectrum: X buffer pH7 253 nm (e, 19800),

298 nm (e, 8800).

Nuclear magnetic resonance: ô 2.70 (2H, m,

-CH2-), 2.75 (3H, s, N-CH3), 4.4-3.4 (10H, m), 4.92 (1H, d, n / a J = 4.0 Hz, 6-H ), 5.55 (1H, d, J = 4.0 Hz, 7-H), 6.93 (1H, d, J = 9.5 Hz, H of pyridazine), 7.28 (4H, s, Phenyl H), 7.40 (1H, d, J = 9.5 Hz, pyridazine H).

55 Analysis:

Calculated for C26H26N707S2.Na.3H20 Found

C% H% N% S%

45.28 4.68 14.22 9.30

45.34 4.01 14.14 9.76

44.84 3.85 14.08

60

65

In vitro antibacterial activity of BB-S525 compared to the activity of BB-S479 and cefamondole (determined by the Steers agar dilution method on Mueller-Hin-ton agar plate).

627184

32

Organization

Minimum inhibitory concentration (ixg / ml)

BB-S525 BB-S479 Sfamandole

S. aureus Smith

0.4

0.4

0.2

S. aureus

0.2

0.2

0.05

S. aureus BX-1633

0.8

0.8

0.2

St. faecalis

> 100

> 100

100

E. coli NIHJ

0.2

0.1

0.1

E. coli ATCC 8739

6.3

3.1

6.3

E. coli Juhl

0.4

0.2

0.4

E. coli BX-1373

0.4

0.2

0.2

E. coli

0.2

0.1

0.1

E. coli

0.2

0.05

0.05

E. coli

6.3

3.1

1.6

Kl. pneumoniae

1.6

0.8

3.1

Kl. pneumoniae

0.2

0.1

0.2

KI. pneumoniae

0.2

0.2

0.8

Kl. pneumoniae

0.2

0.2

0.8

Pr. Vulgaris

0.2

0.2

0.2

Pr. Vulgaris

3.1

0.8

50

Pr. Mirabilis

0.4

0.1

0.4

Pr. Mirabilis

0.2

0.1

0.2

Pr. Morganii

> 100

> 100

3.1

Pr. Morganii

0.2

0.2

0.8

Pr. Rettgeri

0.8

0.8

0.1

Ps. Aeruginosa

> 100

> 100

> 100

Ps. Aeruginosa

> 100

> 100

> 100

Shig. dysenteriae

0.1

0.1

0.1

Shig. flexneri

12.5

12.5

3.1

: Shig. sonnei

0.1

0.1

0.2

Serr. marcescens

25

12.5

50

Enterob. Cloacae

3.1

3.1

1.6

Sai. enteritidis

0.2

0.1

0.1

Sai. Typhosa

0.2

0.1

0.1

B. anthracis

0.2

0.2

0.05

15

Example 9

By replacing in the procedure of Example 8 the 2-N-tertiobutoxycarbonyl-N-aminomethyl-4-hydroxyphé-20nyl-acétique acid used in this example by an equimolar weight of 2-N-tertiobutoxycarbonylaminomethyl-4- acid hydroxyphenyl-acetic acid, 2-N-tertiobutoxycarbonylaminomethyl-4-methoxyphenylacetic acid and 2-N-tertio-butoxycarbonyl-N-methyl-4-methoxyphenylacetic acid respectively, the compounds corresponding to the respective formulas are obtained following:

HO.

CH2NH2

C H -, C QNH -C K - C K CH_ 1 II I 2

/ - ^^ C-CK2S

0

C-OH

ii

0

■ K Ì

dd ^ r ^^ N-CH-jCI ^ COOH 0

and here.

, CH2NH2

CKoCONK-CH-CH 2 I I C — N

c, h2

^ c-cs2s

C-OH

II

0

-K i

and

CH ^ O CH2NKCH3

^^ ch ....,. ,

II i 2 II I

^ C-CH2S

CH, CCNH-CH-CK CK, f ^^ Y 'K

0

C-CH

II

0

VS

Claims (10)

627184 2 CLAIMS 1. Process for the preparation of a compound of formula ï ^ -KK-CK-CH k- (ck2) nc0ck in which n is equal to 1 or 2 and R1 represents a group of formula (I) ch2nhr1 ch-> co- or ch2nhr1 ch2co- or CHpKHR1 ch2co- where R is a hydrogen atom or a hydroxy or meta group. 3. The process according to claim 1, characterized by the fact thoxy; R 'is a hydrogen atom or a methyl group and R2 that the easily hydrolyzable ester obtained of formula I is trans-is a hydrogen atom, the cation of a salt or an ester group' formed into the acid free correspondent of formula I. hydrolyzable acceptable from a pharmaceutical point of view, or 4. Process according to claim 1 and 2, characterized by the use of a non-toxic salt which is acceptable from a pharmaceutical point of view so that the free acid obtained of formula I is converted into one of this. compound, process characterized in that it consists in reacting a compound of formula y-nh-ch- // -ck ch2 l i -not 0 11 c-ch20-c-ch3 (n) cocr 30 ester selected from the esters of pivaloyloxymethyl, acetoxy-methyl, methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl, ß, ß, ß-trichloride, 3-phthalidyl and 5-indanyl. 5. Method according to one of claims 1 to 4, characterized in that R1 is a group of formula H2INKPJ 40 \\ CÏI2C0- in which Y is a hydrogen atom or a group R1 and R2,, „_ ^, ,,,,,, not . . ,,, ", in which R is a hydrogen atom, a hydroxy group are as defined above, with a compound of formula iT ,, _ x. r or methoxy and R is a hydrogen atom or a me- 45 thyle. 6. Method according to one of claims 1 to 4, characterized r N in that R1 is a group of formula N. ^ N- (CH2) nC0CH (in) hs- so in which n is equal to 1 or 2 and Y is a hydrogen atom to treat the resulting compound with an acylating agent of formula R1 — X in which X is a halogen atom or a functional equivalent and R1 has the definition given above and, where appropriate, in converting the free acid or the salt thus obtained from the compound of formula I into an acceptable non-toxic salt from the corresponding pharmaceutical point of view, and optionally transforming a salt resulting from a compound of formula I into the corresponding free acid of formula I. 2. Method according to claim 1, characterized in that the free acid or the easily hydrolysable ester obtained from the compound of formula I is transformed into a non-toxic and pharmaceutically acceptable ester. .ch2rjhr ' 55 ch2co— in which R 'is a hydrogen atom or a methyl group. 7. Method according to one of claims 1 to 4, characterized in that R1 is a group of formula 60 ! Uf, co- 65 in which R 'is a hydrogen atom and a methyl group. 3 627,184 8. Method according to claim 5, characterized in that R is hydrogen, hydroxy or methoxy. 9. Method according to claims 5 to 7, characterized in that R 'is hydrogen or a methyl group. 10. The method of claim 1, wherein n is one or two.