CA1118413A - PROCESS FOR THE PREPARATION OF 7.alpha.-METHOXY- CEPHALOSPORANIC ACID DERIVATIVES - Google Patents
PROCESS FOR THE PREPARATION OF 7.alpha.-METHOXY- CEPHALOSPORANIC ACID DERIVATIVESInfo
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- CA1118413A CA1118413A CA000280218A CA280218A CA1118413A CA 1118413 A CA1118413 A CA 1118413A CA 000280218 A CA000280218 A CA 000280218A CA 280218 A CA280218 A CA 280218A CA 1118413 A CA1118413 A CA 1118413A
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- methoxy
- cephem
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- carboxylic acid
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Abstract
PROCESS FOR THE PREPARATION OF 7.alpha.-METHOXY CEPHALOSPORANIC
ACID DERIVATIVES
ABSTRACT
Novel 7.alpha.-methoxy-7.beta.-heterocyclic thioacetamido-3-heterocyclic thiomethyl-.DELTA.3-cephem-4-carboxylic acid derivatives and pharmaceu-tically acceptable salts thereof. These compounds show high anti-biotic activities against various microorganisms including gram positive and negative bacteria.
ACID DERIVATIVES
ABSTRACT
Novel 7.alpha.-methoxy-7.beta.-heterocyclic thioacetamido-3-heterocyclic thiomethyl-.DELTA.3-cephem-4-carboxylic acid derivatives and pharmaceu-tically acceptable salts thereof. These compounds show high anti-biotic activities against various microorganisms including gram positive and negative bacteria.
Description
4~3 The present invention relates to a process for the preparation of cephalosporins having a methoxy group at the 7a-position. More particularly, the invention relates to a process for the prepara-tion of 7a-methoxy-7~-heterocyclic thioacetamido-3-heterocyclic thiomethyl-~3-cephem-4-carboxylic acid represented by -the general formula R SCH2CONH ~ S ~ 2 ~ N ~ CH2S-R
COOH
wherein R represents R3 R4 ~ ~ R5 g ~n (wherein R and R , which may be the same or different, each rep-resents a hydrogen atom, a hydroxy group, an amino group or a lower alkyl group; n represents 0 or 1; R represents a hydroxy group, an amino group, a mercapto group, a lower alkylamino group, a lower alkanoylamino group, a lower alkoxycarbonylamino group, a carboxy -~
lower alkylthio group, or a 3-lower alkylureido group; and X rep-resents -C~= or -N= ) and R represents a 5-lower alkyl-1,3,4-thia-diazol-2-yl group or a l-lower alkyltetrazol-5-yl group, and the pharmaceutically acceptable salts thereof.
Cephalosporin derivatives having a methoxy group at the 7~-position, a heterocyclic acyl group at the 7~-position, and a heterocyclic thiomethyl group at the 3-position are known. For example, German Offenlegungsschrift Nr. 2,455,884 discloses 7~-methoxy-7~-(5-methyl-substituted or unsubstituted)l,3,4-thiadiazol-
COOH
wherein R represents R3 R4 ~ ~ R5 g ~n (wherein R and R , which may be the same or different, each rep-resents a hydrogen atom, a hydroxy group, an amino group or a lower alkyl group; n represents 0 or 1; R represents a hydroxy group, an amino group, a mercapto group, a lower alkylamino group, a lower alkanoylamino group, a lower alkoxycarbonylamino group, a carboxy -~
lower alkylthio group, or a 3-lower alkylureido group; and X rep-resents -C~= or -N= ) and R represents a 5-lower alkyl-1,3,4-thia-diazol-2-yl group or a l-lower alkyltetrazol-5-yl group, and the pharmaceutically acceptable salts thereof.
Cephalosporin derivatives having a methoxy group at the 7~-position, a heterocyclic acyl group at the 7~-position, and a heterocyclic thiomethyl group at the 3-position are known. For example, German Offenlegungsschrift Nr. 2,455,884 discloses 7~-methoxy-7~-(5-methyl-substituted or unsubstituted)l,3,4-thiadiazol-
2-yl-thioacetamido-3-(1-methyltetrazol-5-yl)thiomethyl-~ -cephem-4-carboxylic acid. Furthermore, Japanese Patent Application Laid Open ~o. 52,195/'76 discloses 7~-(2-amino-4-thiazolyl)acetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-~ -cephem-4-carboxy-lic acid. Other similar compounds are also described in, for example, U.S. Patent ~o. 3,887,549; Dutch Patent No.7,216,268;
German Offenlegungsschriften ~r. 2,440,790, Nr. 2,448,582, ~r.
2,445,341, ~r. 2,412,598, ~r. 2,432,415 and ~r. 2,455,884 and British Patent No. 1,412,886.
An object of this invention is to provide novel cephalosporin derivatives having excellent antibiotic activity against gram posi-tive and negative bacteria.
It has now been found that the aforesaid object of this inven-tion can be attained by the provision of 7a-methoxy-7~-heterocyclic thioacetamido-3-heterocyclic thiomethyl-~ -cephem-4-carboxylic acid shown by aforesaid general formula I and the pharmaceutically acceptable salts thereof.
The compounds of this invention are suggested as very broad and vague concepts such as 5-membered or 6-membered heterocyclic ring-substituted cephalosporin derivatives in the known art but have neither been prepared nor been investigated about the pharma-ceutical activities prior to the inventors' discovery.
Now, in the compounds of this invention represented by formula I, examples of the lower alkyl group include straight chain or hranched chain hydrocarbon groups having 1-4 carbon atoms, such as, methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, sec-butyl group, tert-butyl group, etc. Also, as the phar-maceutically acceptable salts of the aforesaid compounds of this invention, there are the alkali metal salts such as sodium salts, potassium salts, etc.; the alkaline earth metal salts such as cal-cium salts, magnesium salts, etc.; the organic amine salts such as 4i3 trimethylamine salts, ethanolamine salts, diethanolamine salts,lysine salts, alginine salts, ornithine salts, etc.
In cases where the substituents mean hydroxy group, mercapto group or amino group, it is possible for the substituents to exist in more than one tautomeric form, i.e. the hydroxy or oxo, mercapto or thioxo and substituted or unsubstituted amine or substituted or unsubstituted imino group. The compounds may exist exclusively as one tautomer or may be in equilibrium mixture between other forms.
The aimed compounds of this invention may be produced by several modes of reaction which vary with the selection of the starting materials, the availability of reaction reagents, the separation procedure of by-products at the reaction, the applica-bility of reaction condition, etc. The typical reaction modes of producing the aimed compounds of this invention will be illustrated below but not to limit it in any way.
In the first reaction mode, 7a-methoxy-3-heterocyclic thio-methyl-7-substituted or unsubstituted amino-~ -cephem-4-carboxylic acid represented by the general formula OCH
German Offenlegungsschriften ~r. 2,440,790, Nr. 2,448,582, ~r.
2,445,341, ~r. 2,412,598, ~r. 2,432,415 and ~r. 2,455,884 and British Patent No. 1,412,886.
An object of this invention is to provide novel cephalosporin derivatives having excellent antibiotic activity against gram posi-tive and negative bacteria.
It has now been found that the aforesaid object of this inven-tion can be attained by the provision of 7a-methoxy-7~-heterocyclic thioacetamido-3-heterocyclic thiomethyl-~ -cephem-4-carboxylic acid shown by aforesaid general formula I and the pharmaceutically acceptable salts thereof.
The compounds of this invention are suggested as very broad and vague concepts such as 5-membered or 6-membered heterocyclic ring-substituted cephalosporin derivatives in the known art but have neither been prepared nor been investigated about the pharma-ceutical activities prior to the inventors' discovery.
Now, in the compounds of this invention represented by formula I, examples of the lower alkyl group include straight chain or hranched chain hydrocarbon groups having 1-4 carbon atoms, such as, methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, sec-butyl group, tert-butyl group, etc. Also, as the phar-maceutically acceptable salts of the aforesaid compounds of this invention, there are the alkali metal salts such as sodium salts, potassium salts, etc.; the alkaline earth metal salts such as cal-cium salts, magnesium salts, etc.; the organic amine salts such as 4i3 trimethylamine salts, ethanolamine salts, diethanolamine salts,lysine salts, alginine salts, ornithine salts, etc.
In cases where the substituents mean hydroxy group, mercapto group or amino group, it is possible for the substituents to exist in more than one tautomeric form, i.e. the hydroxy or oxo, mercapto or thioxo and substituted or unsubstituted amine or substituted or unsubstituted imino group. The compounds may exist exclusively as one tautomer or may be in equilibrium mixture between other forms.
The aimed compounds of this invention may be produced by several modes of reaction which vary with the selection of the starting materials, the availability of reaction reagents, the separation procedure of by-products at the reaction, the applica-bility of reaction condition, etc. The typical reaction modes of producing the aimed compounds of this invention will be illustrated below but not to limit it in any way.
In the first reaction mode, 7a-methoxy-3-heterocyclic thio-methyl-7-substituted or unsubstituted amino-~ -cephem-4-carboxylic acid represented by the general formula OCH
3 S
Z-NH ~
N ~ CH -S-R
COOH
wherein X represents Y-CH2CO- group (wherein Y represents a halogen atom) or a hydrogen atom and R represents 5-lower alkyl-1,3,4-thiadiazol-2-yl group or l-lower alkyl-tetrazol-5-yl group, is made to react with the heterocyclic ring compound represented by the general formula Rl-S-B
wherein R represents n ~herein R and R , which may be the same or different, each repre-sents a hydrogen atom, a hydroxy group, an amino group, or a lower-alkyl group; n represents 0 or 1; R represents a hydroxy group, an amino group, a mercapto group, a lower alkylamino group, a lower alkanoylamino group, a lower alkoxycarbonylamino group, a carboxy lower alkylthio group, or a 3-lower alkylureido group;and X repre-sents -CH- or =N- ) and B represents a hydroyen atom or R -S- when Z is Y-CH2-CO- group or represents -CH2COOH or the reactive deri-vative of the carboxy group thereof when Z is a hydrogen atom.
The reaction of the first mode is explained below in more detail.
In one type of the first mode of reaction, 7~-haloacetamido-7a-methoxy-3-heterocyclic thiomethyl-~ -cephem-4-carboxylic acid represented by the general formula Y-CH2CONH ~ S ~ 2 II
O N ~ CH -S-R
COOH
wherein Y and R have the same meaning as above, is caused to react with the compound shown by R -S-A III
(wherein A represents a hydrogen atom or R -S- ) in the presence of a base.
That is, the reaction of compound of formula II and the 34i3 compound of formula III is usually performed in an organic solvent which does not affect the reaction, water, or a mixture thereof under cooling or at room temperature. As the base used in this reaction, there are aliphatic nitrogen bases, aromatic nitrogen bases, heterocyclic nitrogen bases, alkali metal carbonates, alkali metal hydrogencarbonates, etc. Suitable examples of these bases are triethylamine, N,~-dimethylaniline, N-ethylmorpholine, pyridine, collidine, 2,6-lutidine, potassium carbonate, sodium carbonate, potassium hydrogencarbonate, and sodium hydrogencarbonate.
As the halogen atom shown by Y in the above-described formula II, there are chlorine atom, bromine atom, fluorine atom, etc. The amount of the compound of formula III used in this reaction is equimolar amount or excessive molar amount to that of compound of formula II, preferably 1-2 mole times.
Typical examples of the organic solvent which is used without influence on the reaction are methanol, chloroform, methylene chloride, ethylene chloride, acetone, tetrahydrofuran, dimethyl-formamide, etc.
In other type of the first reaction mode, 7~-amino-7a-methoxy-3-heterocyclic thiomethyl-~ -cephem-4-carboxylic acid represented by the formula 21H ' ~ S ~
O ~ ~ ~ CH2-S-R IV
COOH
wherein R has the same meaning as above, is allowed to react with an e~uimolar or excessive molar amount, preferably 1-2 mole times of the heterocyclic thioacetic acid shown by 34~3 wherein R has the same meaning as above, or the reactive derivative of the carboxy group thereof in an organic solvent which does not effect the reaction.
When the carboxy group of the compound of formula V is not protected, the reaction is carried out in the presence of a con-densing agent such as dicyclohexylcarbodiimide, carbonyldiimida-zole, etc. Also, when the reactive derivative of the carboxy group is an acid halide such as acid chloride, etc., the reaction is performed in the presence of an almost equimolar amount of a base such as triethylamine, pyridine, etc. Furthermore, as the reactive derivative of the carboxy group, active esters such as p-nitro-phenyl ester, etc., may be employed.
In the second reaction mode of producing the aimed compounds of this invention, 7a-methoxy-7~-heterocylic-thioacetamido-3-acetoxymethyl-~ -cephem-4-carboxylic acid represented by the general formula R SCH2CONH I ~ ~
~ --N~ CH20COCH3 VI
COOH
wherein R has the same meaning as above, is caused to react with the compound shown by R -SM VII
wherein R has the same meaning as above and M represents a hydrogen atom or an alkali metal atom.
It is preferred to conduct the reaction at nearly neutral condition and the compound of formula VI is made to react with an equimolar amount or excessive molar amount of the compound of formula VII. As the alkali metal salt of the compound of formula 34~3 VII, there are the sodium salt, potassium salt, etc. When the com-pound of formula VII wherein M is hydrogen atom is used, it is pre-ferred to perform the reaction in the presence of a base such as an alkali hydroxide, an alkali metal carbonate, an alkali metal hydro~
gencarbonate~ a trialkylamine (e.g. triethylamine), pyridine, di-methylaniline, etc. The reaction is usually performed in an organic solvent, which does not affect the reaction, such as acetone, dimethylformamide, methanol, ethanol, etc., water, or a mixture thereof, or further a phosphate buffer solution at room temperature or under heating.
The carboxy group of at 3~position of the cephem ring of the starting material may also be protected by the known protecting group such as diphenyl methyl group, triphenyl methyl group, methyl group or ethyl group during the reaction steps and can be split in the final stage by a procedure known in the art.
The compounds of this invention thus produced can be converted into the nontoxic pharmaceutically acceptable or useful salts thereof. These salts may be formed according to an ordinary manner.
For example, the alkali metal salt of the aimed compound of formula I can be obtained by adding to the compound a n-butanol solution of an alkali metal 2-ethylhexanoate and then adding an organic solvent having low solubility of the formed salt, such as ether, ethyl acetate, etc. Also, by adding to the compound an equimolar or a slightly excessive amount of an organic base such as triethylamine, diethanolamine, alginine, lysine, etc., the salt of the organic base can be formed. Or, further, by adding to the compound aqueous ammonia or an organic solvent solution of ammonia, the ammonium salt of the compound can also be formed. These salts are inter-changeable with each other by an ordinary manner, if desired.
The compound of the formula I or the salt thereof of this 34~3 invention is isolated and purified by ordinary manners.
The compounds of this invention produced by the aforesaid methods are antibiotics showing excellent antimicrobial activity against various gram positive and negative bacteria and are useful for treatment and prophylaxis of various diseases caused by these bacteria, for preservatives of foods and chemical industrial pro-ducts, and as additives for feed. The minimum inhibiting concen-trations (MIC) (mcg/ml) thereof by in vitro test are shown in the following tables in detail.
Table I
~x.~o.
B. megatherium 10778 3.13 6.25 6.253.136.25 3.13 B. subtilis ATCC 6633 0.78 0.78 0.780.783.13 0.78 Micrococcus flavas0.19 0.19 0.19 0.780.390.19 Sarcina lutea ATCC 9341 0.09 0.09 0.090.190.39 0.19 Staph. aureus ATCC 6538P0.78 0.39 0.781.561.56 0.78 Staph. aureus Smith1.56 0.78 3.13 3.136.251.56 Staph. aureus Terashima 1.56 0.78 1.563.133.13 0.78 Corynebacterium xerosis 0.78 0.78 0.781.563.13 0.78 Mycobacterium 607 6.25 12.5 50 50 12.5 3.13 " phlei 6.25 12.5 50 12.512.53.13 15Staph. aureus Oonuma (JM. 1. 56 0.78 3.133.136.25 1.56 LM . EM . SPM . OI~l. SM .
PC.SA-R) E. coli Kauffmann 0.1 0.39 1.56 6.251.560.78 0.78 E . " NIHJ O. 39 0.78 3.13 0.780.780.78 E. " alkalescens 0.39 1.56 3.13 1.560.780.78 Kleb. pneumoniae ATCC 0.39 0.78 0.780.780.39 0.78 Vibrio Hy 133 0.78 0.39 0.78 1.560.781.56 Sal. cholerae-suis 1348 0.78 6.25 25 6.25 0.78 0.78 Sal. typhi H9OlW 0.39 0.39 0.78 1.560.780.39 Sal. euteritidis 1891 0.19 0.19 0.780.780.39 0.39 Shigella flexneri 2a 1675 0.78 6.25 25 3.13 0.78 1.56 " sonnei II 37148 0.78 3.13 25 6.250.780.78 Prot. vulgaris OXK US 0.78 0.78 1.56 1.563.131.56 " mirabilis IFM OM-9 1. 56 3.13 25 6.256.253.13 ~L8413 Ex. ~o.
B . megatherium 10778 6.2512.53.13 6.25 6.25 3.13 B. subtilis ATCC 6633 3.136.253.13 1.56 3.13 1.56 Micrococcus flavas 0.39 0.390.190.09 0.39 0.04 Sarcina lutea ATCC 9341 0.390.390.39 0.09 0.39 0.04 Staph. aureus ATCC 6538P 1.561.561.56 0.78 1.56 0.78 Staph. aureus Smith 3.13 6.253.131.56 3.13 1.56 10 Staph. aureus Terashima 3.133.133.13 1.56 3.13 1.56 Corynebacterium xerosis 3.136.253.13 1.56 3.13 1.56 Mycobacterium 607 12.5 12.56.253.13 12.5 3.13 " phlei 12.5 12.56.253.13 12.5 3.13 Staph. aureus Oonuma (JM. 3.13 6.25 3.13 3.13 3.13 1.56 LM.EM.SPM.OLM.SM.
15PC.SA-R) E. coli Kauffmann 0-1 1.56 3.130.783.13 1.56 1.56 E. " NIHJ 0.78 3.130.780.78 0.78 0.78 E. " alkalescens 0.78 3.130.783.13 1.56 1.56 20 Kleb. Pneumoniae ATCC 0.783.130.78 0.19 0.78 0.39 Vibrio Hy 133 3.13 3.131.561.56 1.56 1.56 Sal. cholerae-suis 1348 1.566.250.39 6.25 0.39 1.56 Sal. typhi H901W 0.78 1.560.780.19 0.78 0.78 Sal. euteritidis 1891 0.390.780.19 0.09 0.39 0.39 25Shigella flexneri 2a 1675 3.1312.5 0.78 3.13 3.13 3.13 " sonnei II 37148 3.13 6.250.786.25 3.13 3.13 Prot. vulgaris OXK US 1.563.130.78 0.39 0.78 1.56 " mirabilis IFM OM-9 6.256.251.56 1.56 3.13 3.13 E~. ~o.
s. megatherium 10778 6.25 6.25 3.13 6.253.13 3.13 B. subtilis ATCC 6633 1.56 1.56 1.56 3.131.56 3.13 Micrococcus flavas 0.390.090.19 0.39 0.090.19 Sarcina lutea ATCC 93410.39 0.04 0.09 0.190.09 0.19 Staph. aureus ATCC 6538P1.560.78 0.78 1.561.56 1.56 Staph. aureus Smith 3.131.563.13 3.13 3.133.13 10 Staph. aureus Terashima3.13 1.56 1.56 3.133.13 3.13 Corynebacterium xerosis1.56 1.56 1.56 1.563.13 3.13 Mycobacterium 607 50 3.13 6.25 6.25 50 12.5 " phlei 25 3.13 3.13 6.25 25 6.25 Staph. aureus Oonuma (JM. 3.13 1.56 1.563.13 3.13 3.13 LM.EM.SPM.OLM.SM.
PC.SA-R) E. coli Kauffmann 0-1 12.5 6.25 3.13 3.133.13 0.78 E. " ~IHJ 3.131.560.78 0.78 1.560.78 E. " alkalescens 6.253.131.56 3.13 3.130.78 20 Kleb. pneumoniae ATCC 1.56 0.39 0.39 0.781.56 0.78 Vibrio Hy 133 3.131.561.56 3.13 3.133.13 Sal. cholerae-suis 1348 12.5 12.5 6.25 6.251.56 0.78 Sal. typhi H9OlW 3.130.390.39 0.78 1.560.78 25 Sal. euteritidis 1891 0.78 0.09 0.19 0.390.39 0.19 Shigella flexneri 2a 1675 256.25 6.25 6.256.25 1.56 " sonnei II 37148 25 12.5 6.25 6.256.25 3.13 Prot. vulgaris OXK US 3.13 0.78 0.39 1.560.78 0.78 " mirabilis IFM OM-9 12.5 3.13 1.56 6.253.13 1.56 Ex. No.
B. megatherium 10778 3.13 3.13 3.13 6.25 1.56 6.25 B . subtilis ATCC 6633 1.56 0.78 0.78 0.78 0.39 3.13 Micrococcus flavas0.19 0.19 0.09 0.19 0.10 0.39 Sarcina lutea ATCC 93410.09 <0.04 <0.040.09 <0.05 0.20 Staph. aureus ATCC 6538P1.560.39 0.39 0.39 0.39 1.56 Staph. aureus Smith3.130.78 0.78 1.56 0.78 6.25 Staph. aureus Terashima1.56 0.78 0.78 0.78 0.39 3.13 Corynebacterium xerosis1.56 0.78 0.78 0.78 0.20 1.56 Mycobacterium 607 6.25 3.13 6.25 12.5 1.56 6.25 " phlei 3.13 3.13 6.25 12.5 1.56 6.25 Staph. aureus Oonuma (JM. 3.13 0.78 0.78 1.56 0.78 6.25 LM.EM.SPM.OLM.SM.
15PC.SA-R) E. coli Kauffmann 0-1 1.56 1.56 1.56 1.56 0.39 3.13 E. " NIHJ 0.39 0.39 0.39 0.78 0.39 1.56 E. " alkalescens 1.56 0.78 0.78 1.56 0.20 1.56 KLeb. pneumoniae ATCC 0.19 0.19 0.19 0.39 0.20 1.56 Vibrio Hy 133 3.13 0.78 0.39 0.78 0.39 1.56 Sal. cholerae-suis 1348 6.25 3.13 1.56 3.13 0.78 3.13 Sal. typhi H9OlW 0.19 0.39 0.19 0.78 0.20 1.56 Sal. euteritidis 1891 0.09 0.19 0.19 0.39 0.10 0.78 Shigella flexneri 2a 1675 1.56 0.78 0.78 3.13 0.39 6.25 Shiyella sonnei II 371486.251.56 1.56 3.13 0.39 3.13 Prot. vulgaris OXK US 0.39 0.78 0.78 1.56 0.78 6.25 " mirabilis IFM OM-9 0.78 1.56 1.56 3.13 0.78 6.25 Ex. No.
CEZ
B. megatherium 10778 3.13 0.39 B. subtilis ATCC 6633 6.25 0.19 5 Micrococcus flavas 0.19 0.19 Sarcina lutea ATCC 9341 0.39 0.19 Staph. aureus ATCC 6538P 1.56 0.19 Staph. aureus Smith 3.13 1.56 10 Staph. aureus Terashima 3.13 0.78 Corynebacterium xerosis 3.13 0.19 Mycobacterium 607 6.25 ~100 I! phlei 6.25 ~100 Staph. aureus Oonuma (JM. 3.13 1.56 LM.EM.SPM.OLM.SM.
PC.SA-R) E. coli Kauffmann 0-1 25 1.56 E. " NIHJ 3.13 3.13 E. " alkalescens I 12.5 3.13 20 Kleb. pneumoniae ATCC 1.56 1.56 Vibrio Hy 133 1.56 0.78 Sal. cholerae-suis 1348 25 1.56 Sal. typhi H9OlW 0.78 1.56 25 Sal. euteritidis 1891 0.78 1.56 Shigella flexneri 2a 1675 50 1.56 " sonnei II 37148 50 1.56 Prot. vulgaris 0XK US 3.13 3.13 " mirabilis IFM OM-9 6.25 6.25 (CEZ: Cefazolin) 34~3 Table 2 Ex. No.
Staph. aureus 209P 0.780.78 1.56 1.56 1.56 E. coli NIHJ 0.390.78 1.56 1.56 1.56 E. " Ebara 0.786.25 25 6.25 1.56 E. " Takeda 1.5612.5 50 25 1.56 Ent. cloacae 0.7&0.39 0.78 1.56 1.56 Ent. " V-8 100~100 ~100 >100 100 10 Ent. aerogenes 0.780.39 25 1.56 1.56 Ent. " NY-2 ~100~100 50 ~100 >100 Kleb. pneumoniae Y-113.133.13 12.5 3.13 0.78 Kleb. " ~-17 0.783.13 100 6.25 1.56 Serratia marcescens 50 100 >100 ~100 6.25 " " No. 10 25 100 ~100 >100 12.5 Prot. morganii Kono 25 100 ~100 100 12.5 Prot. rettgeri Y-l >100>100 ~100 >100 25 Citrobacter freundii25 100 >100 ~100 100 Pseud. pyocyanea NCTC >100 >100 >100 ~100 >100 ~:184i3 Ex. No.
Staph. aureus 209P 1.56 3.13 3.13 1.560.78 E. coli NIHJ 1.56 1.56 1.56 1.560.78 5 E. " Ebara 1.56 6.25 6.25 1.56 6.25 E. " Takeda 6.25 100 12.5 3.1325 Ent. cloacae 3 .13 3 .136.25 3 .13 1. 56 Ent. " V-8 >100 ~100 >100 100>100 Ent. aerogenes 3 .13 3 .136.25 3 .13 1. 56 Ent. " ~Y-2 ~100 ~100 >100 ~100>100 Kleb. pneumoniae Y-ll 1.56 3 .133 .13 O. 78 6.25 Kleb. " V-17 1.56 3.13 6.25 0.786.25 Serratia marcesceus 100 50 100 50 50 " " No.10 50 50 100 50 50 Prot. moryanii Kono 25 50 >100 25 25 Prot. rettgeri Y-l >100 ~100 >100 100 >100 Citrobacter freundii 25 100 50 50 25 Pseud,pyocyanea NCTC ~100 >100 >100 ~100~100 Ex. ~o.
Staph. a~reus 209P 1.56 1.56 1.56 1.56 1.56 E. coli NIHJ 0.78 0.78 1.56 3.13 1.56 5 E. " Ebara 1. 56 3.13 12.5 12.5 3.13 E. " Takeda 6.25 25 50 50 25 Ent. cloacae 3.13 1.56 3.13 1.56 1.56 Ent. " V-8 >100 >100 >100 >100 ~ 100 Ent. aerogenes l. 56 1.56 1.56 1.56 1.56 10 Ent. " NY-2 >100 >100 >100 >100 >100 Kleb. pneumoniae Y-ll 3.13 3.13 12.5 25 6.25 Kleb. " V-17 3.13 3.13 12.5 25 6.25 Serratia marcesceus 25 25 ~100 100 50 " " No.10 50 25 ?loO 100 50 15 Prot. morganii Kono 12.5 12.5 >100 100 25 Prot. rettgeri Y-l 50 )100 ~100 >100 >100 Citrobacter freundii 50 6.25 100 100 50 P~eud pyocyanea ~CTC ~lO0 >lO0 lO0 ~lO0 ~lO0
Z-NH ~
N ~ CH -S-R
COOH
wherein X represents Y-CH2CO- group (wherein Y represents a halogen atom) or a hydrogen atom and R represents 5-lower alkyl-1,3,4-thiadiazol-2-yl group or l-lower alkyl-tetrazol-5-yl group, is made to react with the heterocyclic ring compound represented by the general formula Rl-S-B
wherein R represents n ~herein R and R , which may be the same or different, each repre-sents a hydrogen atom, a hydroxy group, an amino group, or a lower-alkyl group; n represents 0 or 1; R represents a hydroxy group, an amino group, a mercapto group, a lower alkylamino group, a lower alkanoylamino group, a lower alkoxycarbonylamino group, a carboxy lower alkylthio group, or a 3-lower alkylureido group;and X repre-sents -CH- or =N- ) and B represents a hydroyen atom or R -S- when Z is Y-CH2-CO- group or represents -CH2COOH or the reactive deri-vative of the carboxy group thereof when Z is a hydrogen atom.
The reaction of the first mode is explained below in more detail.
In one type of the first mode of reaction, 7~-haloacetamido-7a-methoxy-3-heterocyclic thiomethyl-~ -cephem-4-carboxylic acid represented by the general formula Y-CH2CONH ~ S ~ 2 II
O N ~ CH -S-R
COOH
wherein Y and R have the same meaning as above, is caused to react with the compound shown by R -S-A III
(wherein A represents a hydrogen atom or R -S- ) in the presence of a base.
That is, the reaction of compound of formula II and the 34i3 compound of formula III is usually performed in an organic solvent which does not affect the reaction, water, or a mixture thereof under cooling or at room temperature. As the base used in this reaction, there are aliphatic nitrogen bases, aromatic nitrogen bases, heterocyclic nitrogen bases, alkali metal carbonates, alkali metal hydrogencarbonates, etc. Suitable examples of these bases are triethylamine, N,~-dimethylaniline, N-ethylmorpholine, pyridine, collidine, 2,6-lutidine, potassium carbonate, sodium carbonate, potassium hydrogencarbonate, and sodium hydrogencarbonate.
As the halogen atom shown by Y in the above-described formula II, there are chlorine atom, bromine atom, fluorine atom, etc. The amount of the compound of formula III used in this reaction is equimolar amount or excessive molar amount to that of compound of formula II, preferably 1-2 mole times.
Typical examples of the organic solvent which is used without influence on the reaction are methanol, chloroform, methylene chloride, ethylene chloride, acetone, tetrahydrofuran, dimethyl-formamide, etc.
In other type of the first reaction mode, 7~-amino-7a-methoxy-3-heterocyclic thiomethyl-~ -cephem-4-carboxylic acid represented by the formula 21H ' ~ S ~
O ~ ~ ~ CH2-S-R IV
COOH
wherein R has the same meaning as above, is allowed to react with an e~uimolar or excessive molar amount, preferably 1-2 mole times of the heterocyclic thioacetic acid shown by 34~3 wherein R has the same meaning as above, or the reactive derivative of the carboxy group thereof in an organic solvent which does not effect the reaction.
When the carboxy group of the compound of formula V is not protected, the reaction is carried out in the presence of a con-densing agent such as dicyclohexylcarbodiimide, carbonyldiimida-zole, etc. Also, when the reactive derivative of the carboxy group is an acid halide such as acid chloride, etc., the reaction is performed in the presence of an almost equimolar amount of a base such as triethylamine, pyridine, etc. Furthermore, as the reactive derivative of the carboxy group, active esters such as p-nitro-phenyl ester, etc., may be employed.
In the second reaction mode of producing the aimed compounds of this invention, 7a-methoxy-7~-heterocylic-thioacetamido-3-acetoxymethyl-~ -cephem-4-carboxylic acid represented by the general formula R SCH2CONH I ~ ~
~ --N~ CH20COCH3 VI
COOH
wherein R has the same meaning as above, is caused to react with the compound shown by R -SM VII
wherein R has the same meaning as above and M represents a hydrogen atom or an alkali metal atom.
It is preferred to conduct the reaction at nearly neutral condition and the compound of formula VI is made to react with an equimolar amount or excessive molar amount of the compound of formula VII. As the alkali metal salt of the compound of formula 34~3 VII, there are the sodium salt, potassium salt, etc. When the com-pound of formula VII wherein M is hydrogen atom is used, it is pre-ferred to perform the reaction in the presence of a base such as an alkali hydroxide, an alkali metal carbonate, an alkali metal hydro~
gencarbonate~ a trialkylamine (e.g. triethylamine), pyridine, di-methylaniline, etc. The reaction is usually performed in an organic solvent, which does not affect the reaction, such as acetone, dimethylformamide, methanol, ethanol, etc., water, or a mixture thereof, or further a phosphate buffer solution at room temperature or under heating.
The carboxy group of at 3~position of the cephem ring of the starting material may also be protected by the known protecting group such as diphenyl methyl group, triphenyl methyl group, methyl group or ethyl group during the reaction steps and can be split in the final stage by a procedure known in the art.
The compounds of this invention thus produced can be converted into the nontoxic pharmaceutically acceptable or useful salts thereof. These salts may be formed according to an ordinary manner.
For example, the alkali metal salt of the aimed compound of formula I can be obtained by adding to the compound a n-butanol solution of an alkali metal 2-ethylhexanoate and then adding an organic solvent having low solubility of the formed salt, such as ether, ethyl acetate, etc. Also, by adding to the compound an equimolar or a slightly excessive amount of an organic base such as triethylamine, diethanolamine, alginine, lysine, etc., the salt of the organic base can be formed. Or, further, by adding to the compound aqueous ammonia or an organic solvent solution of ammonia, the ammonium salt of the compound can also be formed. These salts are inter-changeable with each other by an ordinary manner, if desired.
The compound of the formula I or the salt thereof of this 34~3 invention is isolated and purified by ordinary manners.
The compounds of this invention produced by the aforesaid methods are antibiotics showing excellent antimicrobial activity against various gram positive and negative bacteria and are useful for treatment and prophylaxis of various diseases caused by these bacteria, for preservatives of foods and chemical industrial pro-ducts, and as additives for feed. The minimum inhibiting concen-trations (MIC) (mcg/ml) thereof by in vitro test are shown in the following tables in detail.
Table I
~x.~o.
B. megatherium 10778 3.13 6.25 6.253.136.25 3.13 B. subtilis ATCC 6633 0.78 0.78 0.780.783.13 0.78 Micrococcus flavas0.19 0.19 0.19 0.780.390.19 Sarcina lutea ATCC 9341 0.09 0.09 0.090.190.39 0.19 Staph. aureus ATCC 6538P0.78 0.39 0.781.561.56 0.78 Staph. aureus Smith1.56 0.78 3.13 3.136.251.56 Staph. aureus Terashima 1.56 0.78 1.563.133.13 0.78 Corynebacterium xerosis 0.78 0.78 0.781.563.13 0.78 Mycobacterium 607 6.25 12.5 50 50 12.5 3.13 " phlei 6.25 12.5 50 12.512.53.13 15Staph. aureus Oonuma (JM. 1. 56 0.78 3.133.136.25 1.56 LM . EM . SPM . OI~l. SM .
PC.SA-R) E. coli Kauffmann 0.1 0.39 1.56 6.251.560.78 0.78 E . " NIHJ O. 39 0.78 3.13 0.780.780.78 E. " alkalescens 0.39 1.56 3.13 1.560.780.78 Kleb. pneumoniae ATCC 0.39 0.78 0.780.780.39 0.78 Vibrio Hy 133 0.78 0.39 0.78 1.560.781.56 Sal. cholerae-suis 1348 0.78 6.25 25 6.25 0.78 0.78 Sal. typhi H9OlW 0.39 0.39 0.78 1.560.780.39 Sal. euteritidis 1891 0.19 0.19 0.780.780.39 0.39 Shigella flexneri 2a 1675 0.78 6.25 25 3.13 0.78 1.56 " sonnei II 37148 0.78 3.13 25 6.250.780.78 Prot. vulgaris OXK US 0.78 0.78 1.56 1.563.131.56 " mirabilis IFM OM-9 1. 56 3.13 25 6.256.253.13 ~L8413 Ex. ~o.
B . megatherium 10778 6.2512.53.13 6.25 6.25 3.13 B. subtilis ATCC 6633 3.136.253.13 1.56 3.13 1.56 Micrococcus flavas 0.39 0.390.190.09 0.39 0.04 Sarcina lutea ATCC 9341 0.390.390.39 0.09 0.39 0.04 Staph. aureus ATCC 6538P 1.561.561.56 0.78 1.56 0.78 Staph. aureus Smith 3.13 6.253.131.56 3.13 1.56 10 Staph. aureus Terashima 3.133.133.13 1.56 3.13 1.56 Corynebacterium xerosis 3.136.253.13 1.56 3.13 1.56 Mycobacterium 607 12.5 12.56.253.13 12.5 3.13 " phlei 12.5 12.56.253.13 12.5 3.13 Staph. aureus Oonuma (JM. 3.13 6.25 3.13 3.13 3.13 1.56 LM.EM.SPM.OLM.SM.
15PC.SA-R) E. coli Kauffmann 0-1 1.56 3.130.783.13 1.56 1.56 E. " NIHJ 0.78 3.130.780.78 0.78 0.78 E. " alkalescens 0.78 3.130.783.13 1.56 1.56 20 Kleb. Pneumoniae ATCC 0.783.130.78 0.19 0.78 0.39 Vibrio Hy 133 3.13 3.131.561.56 1.56 1.56 Sal. cholerae-suis 1348 1.566.250.39 6.25 0.39 1.56 Sal. typhi H901W 0.78 1.560.780.19 0.78 0.78 Sal. euteritidis 1891 0.390.780.19 0.09 0.39 0.39 25Shigella flexneri 2a 1675 3.1312.5 0.78 3.13 3.13 3.13 " sonnei II 37148 3.13 6.250.786.25 3.13 3.13 Prot. vulgaris OXK US 1.563.130.78 0.39 0.78 1.56 " mirabilis IFM OM-9 6.256.251.56 1.56 3.13 3.13 E~. ~o.
s. megatherium 10778 6.25 6.25 3.13 6.253.13 3.13 B. subtilis ATCC 6633 1.56 1.56 1.56 3.131.56 3.13 Micrococcus flavas 0.390.090.19 0.39 0.090.19 Sarcina lutea ATCC 93410.39 0.04 0.09 0.190.09 0.19 Staph. aureus ATCC 6538P1.560.78 0.78 1.561.56 1.56 Staph. aureus Smith 3.131.563.13 3.13 3.133.13 10 Staph. aureus Terashima3.13 1.56 1.56 3.133.13 3.13 Corynebacterium xerosis1.56 1.56 1.56 1.563.13 3.13 Mycobacterium 607 50 3.13 6.25 6.25 50 12.5 " phlei 25 3.13 3.13 6.25 25 6.25 Staph. aureus Oonuma (JM. 3.13 1.56 1.563.13 3.13 3.13 LM.EM.SPM.OLM.SM.
PC.SA-R) E. coli Kauffmann 0-1 12.5 6.25 3.13 3.133.13 0.78 E. " ~IHJ 3.131.560.78 0.78 1.560.78 E. " alkalescens 6.253.131.56 3.13 3.130.78 20 Kleb. pneumoniae ATCC 1.56 0.39 0.39 0.781.56 0.78 Vibrio Hy 133 3.131.561.56 3.13 3.133.13 Sal. cholerae-suis 1348 12.5 12.5 6.25 6.251.56 0.78 Sal. typhi H9OlW 3.130.390.39 0.78 1.560.78 25 Sal. euteritidis 1891 0.78 0.09 0.19 0.390.39 0.19 Shigella flexneri 2a 1675 256.25 6.25 6.256.25 1.56 " sonnei II 37148 25 12.5 6.25 6.256.25 3.13 Prot. vulgaris OXK US 3.13 0.78 0.39 1.560.78 0.78 " mirabilis IFM OM-9 12.5 3.13 1.56 6.253.13 1.56 Ex. No.
B. megatherium 10778 3.13 3.13 3.13 6.25 1.56 6.25 B . subtilis ATCC 6633 1.56 0.78 0.78 0.78 0.39 3.13 Micrococcus flavas0.19 0.19 0.09 0.19 0.10 0.39 Sarcina lutea ATCC 93410.09 <0.04 <0.040.09 <0.05 0.20 Staph. aureus ATCC 6538P1.560.39 0.39 0.39 0.39 1.56 Staph. aureus Smith3.130.78 0.78 1.56 0.78 6.25 Staph. aureus Terashima1.56 0.78 0.78 0.78 0.39 3.13 Corynebacterium xerosis1.56 0.78 0.78 0.78 0.20 1.56 Mycobacterium 607 6.25 3.13 6.25 12.5 1.56 6.25 " phlei 3.13 3.13 6.25 12.5 1.56 6.25 Staph. aureus Oonuma (JM. 3.13 0.78 0.78 1.56 0.78 6.25 LM.EM.SPM.OLM.SM.
15PC.SA-R) E. coli Kauffmann 0-1 1.56 1.56 1.56 1.56 0.39 3.13 E. " NIHJ 0.39 0.39 0.39 0.78 0.39 1.56 E. " alkalescens 1.56 0.78 0.78 1.56 0.20 1.56 KLeb. pneumoniae ATCC 0.19 0.19 0.19 0.39 0.20 1.56 Vibrio Hy 133 3.13 0.78 0.39 0.78 0.39 1.56 Sal. cholerae-suis 1348 6.25 3.13 1.56 3.13 0.78 3.13 Sal. typhi H9OlW 0.19 0.39 0.19 0.78 0.20 1.56 Sal. euteritidis 1891 0.09 0.19 0.19 0.39 0.10 0.78 Shigella flexneri 2a 1675 1.56 0.78 0.78 3.13 0.39 6.25 Shiyella sonnei II 371486.251.56 1.56 3.13 0.39 3.13 Prot. vulgaris OXK US 0.39 0.78 0.78 1.56 0.78 6.25 " mirabilis IFM OM-9 0.78 1.56 1.56 3.13 0.78 6.25 Ex. No.
CEZ
B. megatherium 10778 3.13 0.39 B. subtilis ATCC 6633 6.25 0.19 5 Micrococcus flavas 0.19 0.19 Sarcina lutea ATCC 9341 0.39 0.19 Staph. aureus ATCC 6538P 1.56 0.19 Staph. aureus Smith 3.13 1.56 10 Staph. aureus Terashima 3.13 0.78 Corynebacterium xerosis 3.13 0.19 Mycobacterium 607 6.25 ~100 I! phlei 6.25 ~100 Staph. aureus Oonuma (JM. 3.13 1.56 LM.EM.SPM.OLM.SM.
PC.SA-R) E. coli Kauffmann 0-1 25 1.56 E. " NIHJ 3.13 3.13 E. " alkalescens I 12.5 3.13 20 Kleb. pneumoniae ATCC 1.56 1.56 Vibrio Hy 133 1.56 0.78 Sal. cholerae-suis 1348 25 1.56 Sal. typhi H9OlW 0.78 1.56 25 Sal. euteritidis 1891 0.78 1.56 Shigella flexneri 2a 1675 50 1.56 " sonnei II 37148 50 1.56 Prot. vulgaris 0XK US 3.13 3.13 " mirabilis IFM OM-9 6.25 6.25 (CEZ: Cefazolin) 34~3 Table 2 Ex. No.
Staph. aureus 209P 0.780.78 1.56 1.56 1.56 E. coli NIHJ 0.390.78 1.56 1.56 1.56 E. " Ebara 0.786.25 25 6.25 1.56 E. " Takeda 1.5612.5 50 25 1.56 Ent. cloacae 0.7&0.39 0.78 1.56 1.56 Ent. " V-8 100~100 ~100 >100 100 10 Ent. aerogenes 0.780.39 25 1.56 1.56 Ent. " NY-2 ~100~100 50 ~100 >100 Kleb. pneumoniae Y-113.133.13 12.5 3.13 0.78 Kleb. " ~-17 0.783.13 100 6.25 1.56 Serratia marcescens 50 100 >100 ~100 6.25 " " No. 10 25 100 ~100 >100 12.5 Prot. morganii Kono 25 100 ~100 100 12.5 Prot. rettgeri Y-l >100>100 ~100 >100 25 Citrobacter freundii25 100 >100 ~100 100 Pseud. pyocyanea NCTC >100 >100 >100 ~100 >100 ~:184i3 Ex. No.
Staph. aureus 209P 1.56 3.13 3.13 1.560.78 E. coli NIHJ 1.56 1.56 1.56 1.560.78 5 E. " Ebara 1.56 6.25 6.25 1.56 6.25 E. " Takeda 6.25 100 12.5 3.1325 Ent. cloacae 3 .13 3 .136.25 3 .13 1. 56 Ent. " V-8 >100 ~100 >100 100>100 Ent. aerogenes 3 .13 3 .136.25 3 .13 1. 56 Ent. " ~Y-2 ~100 ~100 >100 ~100>100 Kleb. pneumoniae Y-ll 1.56 3 .133 .13 O. 78 6.25 Kleb. " V-17 1.56 3.13 6.25 0.786.25 Serratia marcesceus 100 50 100 50 50 " " No.10 50 50 100 50 50 Prot. moryanii Kono 25 50 >100 25 25 Prot. rettgeri Y-l >100 ~100 >100 100 >100 Citrobacter freundii 25 100 50 50 25 Pseud,pyocyanea NCTC ~100 >100 >100 ~100~100 Ex. ~o.
Staph. a~reus 209P 1.56 1.56 1.56 1.56 1.56 E. coli NIHJ 0.78 0.78 1.56 3.13 1.56 5 E. " Ebara 1. 56 3.13 12.5 12.5 3.13 E. " Takeda 6.25 25 50 50 25 Ent. cloacae 3.13 1.56 3.13 1.56 1.56 Ent. " V-8 >100 >100 >100 >100 ~ 100 Ent. aerogenes l. 56 1.56 1.56 1.56 1.56 10 Ent. " NY-2 >100 >100 >100 >100 >100 Kleb. pneumoniae Y-ll 3.13 3.13 12.5 25 6.25 Kleb. " V-17 3.13 3.13 12.5 25 6.25 Serratia marcesceus 25 25 ~100 100 50 " " No.10 50 25 ?loO 100 50 15 Prot. morganii Kono 12.5 12.5 >100 100 25 Prot. rettgeri Y-l 50 )100 ~100 >100 >100 Citrobacter freundii 50 6.25 100 100 50 P~eud pyocyanea ~CTC ~lO0 >lO0 lO0 ~lO0 ~lO0
4~3 Ex. ~o.
Staph. aureus 209P3.133.13 3.13 1.56 0.39 E. coli ~IHJ 0.78 3.13 1.56 ~0.2 ~0.2
Staph. aureus 209P3.133.13 3.13 1.56 0.39 E. coli ~IHJ 0.78 3.13 1.56 ~0.2 ~0.2
5 E. " Ebara 3.13 6.25 3.13 0.78 3.13 E. " Takeda 25 25 6.25 6.25 12.5 Ent. cloacae 3.13 6.25 3.13 1.56 0.78 Ent. "- V-8 ~100 >100 ~100 100 >100 Ent. aerogenes 3.13 6.25 3.13 3.13 0.78 10 Ent. " ~Y-2 ?loO >100 >100 >100 >100 Kleb. pneumoniae Y-ll 3.13 3.13 3.13 6.25 3.13 Kleb. " V-17 12.5 6.25 3.13 3.13 6.25 Serratia marcesceus >100 >100 25 100 25 " " No.10 50 >100 50 25 25 15 Prot. morganii Kono 50 100 25 25 25 Prot. rettgeri Y-l~100 100 ~100 >100 >100 Citrobacter freundii 50 >100 50 50 100 P~eud pyocyanea ~CTC ~100 >100 >100 >100 >100 ~118413 Ex. No.
Staph. aureus 209P0.390.78 0.39 3.13 ~0.2 E. coli NIHJ 0.391.56 ~0.2 1.56 1.56 E. " Ebara 1.566.25 0.39 6.25 50 E. " Takeda 6.2512.5 0.78 100 12.5 Ent. cloacae 1000.78 ~0.2 3.13 ~0.2 Ent. " V-8 >100~100 100 >100 ~100 Ent. aerogenes 0.780.78 ~0.2 3.13 ~0.2 Ent. " NY-2 ~100>100 25 >100 100 Kleb. pneumoniae Y-ll 3.13 6.25 0.78 50 3.13 Kleb. " V-17 3.136.25 0.39 25 ~100 Serratia marcesceus100 100 6.25 ?100 >100 " " No.10 100~100 3.13 100 ?100 Prot. morganii Kono100 100 12.5 ~100 ~100 Prot. rettgeri Y-l >100?100 ~100 >100 ~100 Citrobacter freundii 100 >100 12.5 50 12.5 Pseud pyocyanea NCTC ~100 >100 100 >100 ?100 (CEZ: Cefazolin) 4~3 The compounds of this invention are formed into formulation in similar manners as other known cephalosporin derivatives and are administered orally or parenterally as, for example, tablets, powder, granules, troches injections, suppositories, suspensions, etc., and the dose thereof differs according to the severity of patients' diseases and the general conditions, ages, weights, etc, of patients but is usually 10-50 mg/Kg.
The invention will further be illustrated by the following examples.
Example 1 A mixture of 100 mg. of 7~-bromoacetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl~ -cephem-4-carboxylic acid, 30 mg.
of 2-hydroxy-4-mercaptopyridine, 40 mg. of sodium hydrogencarbonate 5 ml. of water, and 10 ml. of methanol was stirred for 4.5 hours at room temperature. After distilling off methanol from the reac-tion mixture, the pH of it was adjusted to 9 with sodium hydrogen-carbonate and the mixture was filtered. The filtrate was acidified with 1~5% hydrochloric acid and extracted with 100 ml. of a mixture of n-butanol and ethyl acetate of 1 : 1 by volume ratio. The extract was washed with a saturated aqueous solution of sodium chloride, water, and then a saturated aqueous solution of sodium chloride and dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure. The residue obtained was subjected to a gradient elution column chromatography using the initial solution of a mixture of chloroform, isopropanol, and for-mic acid of 90 : 10 : 3 by volume ratio and subsequent increase of the % methanol as elution proceeds.
Then, the fractions containing the aimed compound were collec-ted and the solvent was distilled off from the combined fractions to provide 57 mg. of 7~-[4-(2-hydroxypyridyl)] thioacetamido-7a-~8413 methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-~ -cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D6-DMSO) ~j(p.p.m.): 3.38 (s, 3H), 3.66 (q, 2H), 3.84 (s, 3H), 3.92 (s, 3H), 4.30 (q, 2H), 5.06 (s, lH),
Staph. aureus 209P0.390.78 0.39 3.13 ~0.2 E. coli NIHJ 0.391.56 ~0.2 1.56 1.56 E. " Ebara 1.566.25 0.39 6.25 50 E. " Takeda 6.2512.5 0.78 100 12.5 Ent. cloacae 1000.78 ~0.2 3.13 ~0.2 Ent. " V-8 >100~100 100 >100 ~100 Ent. aerogenes 0.780.78 ~0.2 3.13 ~0.2 Ent. " NY-2 ~100>100 25 >100 100 Kleb. pneumoniae Y-ll 3.13 6.25 0.78 50 3.13 Kleb. " V-17 3.136.25 0.39 25 ~100 Serratia marcesceus100 100 6.25 ?100 >100 " " No.10 100~100 3.13 100 ?100 Prot. morganii Kono100 100 12.5 ~100 ~100 Prot. rettgeri Y-l >100?100 ~100 >100 ~100 Citrobacter freundii 100 >100 12.5 50 12.5 Pseud pyocyanea NCTC ~100 >100 100 >100 ?100 (CEZ: Cefazolin) 4~3 The compounds of this invention are formed into formulation in similar manners as other known cephalosporin derivatives and are administered orally or parenterally as, for example, tablets, powder, granules, troches injections, suppositories, suspensions, etc., and the dose thereof differs according to the severity of patients' diseases and the general conditions, ages, weights, etc, of patients but is usually 10-50 mg/Kg.
The invention will further be illustrated by the following examples.
Example 1 A mixture of 100 mg. of 7~-bromoacetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl~ -cephem-4-carboxylic acid, 30 mg.
of 2-hydroxy-4-mercaptopyridine, 40 mg. of sodium hydrogencarbonate 5 ml. of water, and 10 ml. of methanol was stirred for 4.5 hours at room temperature. After distilling off methanol from the reac-tion mixture, the pH of it was adjusted to 9 with sodium hydrogen-carbonate and the mixture was filtered. The filtrate was acidified with 1~5% hydrochloric acid and extracted with 100 ml. of a mixture of n-butanol and ethyl acetate of 1 : 1 by volume ratio. The extract was washed with a saturated aqueous solution of sodium chloride, water, and then a saturated aqueous solution of sodium chloride and dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure. The residue obtained was subjected to a gradient elution column chromatography using the initial solution of a mixture of chloroform, isopropanol, and for-mic acid of 90 : 10 : 3 by volume ratio and subsequent increase of the % methanol as elution proceeds.
Then, the fractions containing the aimed compound were collec-ted and the solvent was distilled off from the combined fractions to provide 57 mg. of 7~-[4-(2-hydroxypyridyl)] thioacetamido-7a-~8413 methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-~ -cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D6-DMSO) ~j(p.p.m.): 3.38 (s, 3H), 3.66 (q, 2H), 3.84 (s, 3H), 3.92 (s, 3H), 4.30 (q, 2H), 5.06 (s, lH),
6.06 (d, lH), 6.20 (d, lH), 6.20 (s, lH),
7.22 (d, lH).
Example 2 a). To 6 ml. of methylene chloride were added 39 mg. of tri-10 ethylamine and 164 mg. of 7~-bromoacetamido-7a-methoxycephalospor-anic acid, the mixture was stirred at room temperature and after adding the~eto 2.8 mg. of 4-mercaptopyridine under stirring, the resultant mixture was further stirred for 2 hours. After the reac-tion was over, the reaction mixture was cooled with ice for 30 15 minutes and the precipitates formed were recovered by filtration, washed with methylene chloride, and dried to provide 97 mg. of 7a-methoxy-7~-(4-pyridyl)thioacetamidocephalosporanic acid.
Nuclear magnetic resonance spectra (D6-DMSO) ~(p.p.m.): 2.02 (3H, -C0~3), 3.40 (3H, -OCH3), 3, 97 (2H, 20 -SCH2CO-), 5.14 (lH, ~ ), 7.38 ~2H, ~ ), ~ O ~LN ~/
Example 2 a). To 6 ml. of methylene chloride were added 39 mg. of tri-10 ethylamine and 164 mg. of 7~-bromoacetamido-7a-methoxycephalospor-anic acid, the mixture was stirred at room temperature and after adding the~eto 2.8 mg. of 4-mercaptopyridine under stirring, the resultant mixture was further stirred for 2 hours. After the reac-tion was over, the reaction mixture was cooled with ice for 30 15 minutes and the precipitates formed were recovered by filtration, washed with methylene chloride, and dried to provide 97 mg. of 7a-methoxy-7~-(4-pyridyl)thioacetamidocephalosporanic acid.
Nuclear magnetic resonance spectra (D6-DMSO) ~(p.p.m.): 2.02 (3H, -C0~3), 3.40 (3H, -OCH3), 3, 97 (2H, 20 -SCH2CO-), 5.14 (lH, ~ ), 7.38 ~2H, ~ ), ~ O ~LN ~/
8.42 (2H, b). In a mixture of 5 ml. of methylene chloride and 19.5 mg.
of triethylamine was dissolved 92 mg. of 7-bromoacetamido-713-25 methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-~3-cephem-4-carboxylic acid with stirxing at room temperature and after further adding thereto 22 mg. of 4-mercaptopyridine, the resultant mixture was ~tirred for about 40 minutes. The precipitates formed were reco-vered by filtration, washed with ethylene chloride and dried to 30 provide 58 mg. of 7a-methoxy-3-(1-methyltetraæol-5-yl)thiomethyl-4~3 7~-(4-pyridyl)thioacetamido-Q3-cephem-4-carboxyllc acid.
Nuclear magnetic resonance spectra (D6-DMS0) ~(p.p.m.): 3,33 (3H~ -OCH3), 3,93 (5HJ -SCH2C0-, and \N' ), 5.09 (lH, ~ ~ ) 7.35 (2H, S ~ ), ~ _ ~
8.40 (2H, -S- ~ ) Example 3 ~
In a mixture of 5 ml. of methylene chloride and 22.0 mg. of triethylamine was dissolved 100 mg. of 7~-bromoacetamido-7a-methoxy-3-(5-methyl-1,3,4-thiadiazol-2-yl)thiomethyl~3-cephem-4-carboxylic acid with stirring at room temperature and then the solution was treated as in Example 2-b) to provide 60 mg. of 7~-methoxy-3-(5-methyl-1,3,4-thiadiazol-2-yl)thiomethyl-7~-(4-pyridyl)-thioacetamido~Q -cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D6-DMS0) ~ (p-p-m.): 2,70 (3H, ~ ~ ), 3-41 (3H, 3), 3.94 (2H, -SCH2CO)- ~__ -Exam~le 4 A mixture of 200 mg. of 7~-bromoacetamido-7~-methoxy-3-(5-Z0 methyl-1,3,4-thiadiazol-2-yl)thiomethyl-~3-cephem-4-carboxylic acid 65 mg. of 2-hydroxy-4-mercaptopyridine, 8 ml. of water, and 16 ml.
of methanol was stirred on ice bath and after further adding to the mixture 80 mg. of sodium hydrogencarbonate, the resultant mixture was stirred for 2 hours at rOom temperature. Then, methanol was distilled off under reduced pressure, and after filtering, the fil-trate was acidified with diluted hydrochloric acid and extracted with 50 ml. of a mixture of n-butanol and ethyl acetate of 1 : 1 by volume ratio. The extract was washed with saturated aqueous solution of sodium chloride, dried over anhydrous magnesium sulfate, and the solvent was distilled off from the residue to provide 210 mg. of 7~-(2-hydroxy-4-pyridyl)thioacetamido-7a-methoxy-3-(5-methyl-1,3,4-thiadiazol-2-yl)thiomethyl~3-cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D6-DMS0) ,~i(p.p.m.): 2.70 (s,3H), 3.42 (s, 3H~, 3~72 (~, 2H), 3,84 5 (s,2H), 4,36 (q, 2H), 5, 12 (s, lH), 6.10 (d, lH), 6.24 (s, lH), 7.26 (d, lH).
ExamPle 5 To a mixture of 120 mg. of 3-hydroxy-4-mercaptopyridine, 140 mg. of sodium hydrogencarbonate, 15 ml. of methanol, and 7.5 ml.
10 of water was added 360 mg. of 7,~-bromoacetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl~ -cephem-4-carboxylic acid and the mixture was stirred under ice-cooling and after raislng the tempera-ture to room temperature, was further stirred for 2 hours.
After distilling off methanol, the reaction mixture was fil-15 tered and the filtrate was acidified with 2 ml. of 1 N hydrochloric acid. The precipitates thus formed were recovered by filtration, washed with water and dried to provide 200 mg. of 7~-(3-hydroxy-4-pyridyl)thioacetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thio-methyl-l~ -cephem-4-carboxylic acid as a crude product.
Meanwhile, the aqueous layer formed in the aforesaid procedure was extracted with 50 ml. of a mixture of n-butanol and methanol of 1: 1 by volume ratio and the organic layer was recovered, washed twice with saturated aqueous sodium chloride, dried over anhydrous magnesium sulfate, and then the solvent was distilled off 25 to provide 280 mg. of the crude product shown above.
By applying silica gel column chromatography to the crude pro-duct obtained from the aqueous layer using a mixture of chloroform, isopropanol, formic acid, and methanol at 30: 3: 1: 8 by volume ratio as the developing solvent, 100 mg. of the pure product was 30 obtained.
~uclear magnetic resonance spectra (D6-DMSO) ~(p.p.m.): 3,40 (s, 3H, 7-OCH3), 3.94 (s, 3H, N-CH3), 4.06 (s, 2H, -S-CH2Co), 4.30 (q, 2H), 5.10 (s, lH, 6-H), ~ S-- ~ _ 7 64 (d 1 H ~ OEI ) 8 24 ( 2H ~ ,OH
ExamPle 6 ~ H ~ ~ H
To a solution of 100 mg. of 7~-bromoacetamido-7a-methoxy-3-(l-methyltetrazol-5-yl)thiomethyl~3-cephem-4-carboxylic acid in 3 ml. of methanol was added a solution of 62.5 mg. of 2-amino-5-mer-capto-1,3,4-thiadiazole and 0.066 ml. of triethylamine in 3 ml. of methanol and the solution was stirred for 30 minutes at room tem-perature. The solvent was distilled off from the reaction mixture under reduced pressure and the residue formed was subjected to ~ilica gel solumn chromatography using a mixture of methylene chloride, methanol, formic acid at 80 : 20 : 2 by volume ratio as the developing solvent. The fractions containing the aimed com-pound were combined and the solvent was distilled off to provide 60 mg. of 7~-(5-imino-4,5-dihydro-1,3,4-thiadiazol-2-yl)thioaceta-mido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl~3-cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D6-DMSO) ~(p.p.m.): OCH
3 N _N
3.38 (3H,H2N ~ ~), 3.92 (3H, ~ N,~ + 2H, -~S~ SCH2-CO-), 4.29 (2H, ~ H S ), 5-06 (lH, C6-H).
25 Exam~le 7 To a solution of lOO mg. of 7~-bromoacetamido-7a-methoxy-3-(l-methyltetrazol-5-yl)thiomethyl-~3-cephem-4-carboxylic acid in 5 ml. of methanol was added a solution of sodium salt of 5-mercapto-2-oxo-2,3-dihydro-1,3,4-thiadiazole in 3 ml. of methanol prepared by dissolving 60 mg. of 5-mercapto-2-oxo-2,3-dihydro-1,3,4-thiadiazole and 0.59ml. of a 1 ~ aqueous sodium hydroxide solution in 2 ml. of methanol, and distilling off the solvent therefrom.
After stirring the mixture for 30 minutes at room temperature, the solvent was distilled off under reduced pressure and the residue 5 obtained was subjected to a column chromatography using a mixture methanol and ethyl acetate of 1: 4 by volume ratio as a developing solvent for removing impurities and then a mixture of methanol and ethyl acetate of 1: 2 by volume ratio as a developing solvent for the product.
10 The fractions containing the aimed compound were combined and ther the solvent was distilled off from the combined solution to provide 70 mg. of 7~3-(5-oxo-4,5-dihydro-1,3,4-thiadiazol-2-yl)thio-acetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl~ -cephem-4-carboxylic acid.
15 ~uclear magnetic resonance spectra (D6-DMS0) ~;(p.p.m.):
3,40 (3H, C7-OCH3), 3-94 (3H, ~ ~S--SCH2CO-4.32 (2H, ~C S )~ 5.02 (lH, C6-H).
20ExamPle 8 ~
a). A solution of 226 mg. of 4-thioxo-2-thiazolidinone in 5 ml. of methanol was cooled to -10C. and after adding thereto 344 mg. of triethylamine, the resultant solution was added to a solution of 718 mg of 7~-bromoacetamido-7a-methoxycephalosporanic acid in 5 ml. of methanol at temperatures below -5C. After carry-ing out the reaction for 2 hours at temperatures of between -5C.
and 0C., the solvent was distilled off at low temperature under reduced pressure and the residue formed was subjected to a silica gel column chromatography using a mixture of chloroform, isopropanol, and formic acid of 90: 10: 2 by volume ratio as a developing J~18413 solvent.
The fractions containing the aimed compound were combined and the solvent was distilled off to provide 860 mg. of 7a-methoxy-7~-(2-oxo-~ -thiazolin-4-yl)thioacetamidocephalosporanic acid.
Nuclear magnetic resonance spectra (D6-acetone) S(p.p.m.) 2.04 (3H), 3.52 (3H, C7-OCH3), 3,31-3.62 (2H, 2-position), 3.81 (2H, -S-CH2-CO-), 4,97 (2H, C3-CH20COCH3), 5.15 (lH, 6-position), 6.51 (lH, H ~ ), 8.62 (lH, -CO~H-), 10.33 (lH, ~ S ) ~
b). To 27 ~1. of a phosphate buffer solution having pH of 6.86 were added 484 mg. of 7a-methoxy-7~-(2-oxo-~4-thiazolin-4-yl)-thioacetamidocephalosporanic acid, 118.4 mg. of 5-mercapto-1-methyltetrazole, and 85.7 mg. of sodium hydrogencarbonate and then the reaction was carried out for 18 hours at 58-60C. After the reaction was over, the pH of the reaction mixture was adjusted to pH 1-2 by adding thereto a 40% aqueous phosphoric acid solution under ice-cooling and then the product was extracted twice each with 30 ml. of ethyl acetate. The extracts were combined, washed with water, dried over anhydrous sodium sulfate, and then the sol-vent was distilled off under reduced pressure. The residue formedwa~ ~ubjected to a silica gel column chromatography using a mixture of chloroform, isopropanol, and formic acid of 80 : 20 : 2 by volume ratio as a developing solvent.
The fractions containing the aimed compound were combined and the solvent was distilled off to provide 60 mg of 7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-7~-(2-oxo-~4-thiazolin-4-yl)thio-acetamido~ -cephem-4-carboxylic acid.
~uclear magnetic resonance spectra (D6-acetone) S(p.p.m.): 3.52 (3H, C7-OCH3), 3.60~4.00 (4H, N/
2-position + N ~ SCH2CO ), 4.02 (3~, CH3 4.44 (2H, 3-position -CH2-S-), 5.12 (lH, 6-position), 6.52 (lH, ~ ), 8.66 (lH, -CONH-), 10.40 o O ~S~
Example 9 To 4 ml. of methylene chloride was added 21.1 mg. of triethyl-amine and then 100 mg. of 7~-bromoacetamido-7a-methoxy-3-(1-methyl-tetrazol-5-yl)thiomethyl-~3-cephem-4-carboxylic acid was dissolved with stirring at room temperature. To the solution was further added 34 mg. of 2,5-dimercapto-1,3,4-thiadiazole and after adding dropwise a few drops of methanol for dissolving the 2,5-dimercapto-1,3,4-thiadiazole, the resultant mixture was stirred for about 3 hours. After the reaction was over, the solvent was distilled off from the reaction mixture, and to the residue formed were added 40 ml. of ethyl acetate and diluted hydrochloric acid to acidify it, whereby an ethyl acetate layer and an aqueous layer formed.
The ethyl acetate layer formed was recovered, washed with water, dried over anhydrous sodium sulfate, and then the solvent was dis-tilled off under reduced pressure. The residue obtained was sub-jected to a silica gel column chromatography using a mixture of chloroform, methanol, and formic acid of 50 : 4 : 1 by volume ratio as a developing solvent. The fractions containing the aimed com-pound were combined and the solvent was distilled off to provide 60 mg. of 7~-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl)-thio-acetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-~3-cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D6-acetone) ~(p.p.m.): 3.49 (3H, CH30-), 4.00 (3H, H I ), ( ' ~ SCH2C0- )' ( ' ~ CH3 Example 10 3,413 In a mixture of 0.627 ml. of a 1 ~ (f=0.998) aqueous sodium hydroxide solution and 1.5 ml. of methanol was dissolved 110 mg.
of 5-acetamido-2-mercapto-1,3,4-thiadiazole followed by stirring for several minutes and then the solvent was distilled off. The white solid residue obtained was dissolved in 2.5 ml. of methanol and after adding to the solution a solution prepared by dissolving 200 mg. of 7~-bromoacetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)-thiomethyl~ -cephem-4-carboxylic acid in 2.5 ml. of methanol at room temperature, the resultant mixture was stirred for 30 minutes at room temperature. Then, the solvent was distilled off and the residue obtained was subjected to a silica gel column chromatogra-phy using a mixture of chloroform, isopropyl alcohol, and formic acid of ~0 : 10 : 3 by volume ratio to elute unchanged 5-acetamido-2-mercapto-1,3,4-thiadiazole. Thereafter, the product was eluted using an eluent, a mixture of chloroform, methanol, and formic acid of 90 : 10 : 2 by volume ratio.
The fractions containing the aimed compound were combined and the solvent was distilled off to provide 150 mg. of the light-yellow powder of 7~-(5-acetylimino-4,5-dihydro-1,3,4-thiadiazol-2-yl)thioacetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-~3-cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D6-DMSO) N--N
~(p.p.m.): 2.18 (s, 3H, CH3CO~H ~ ~ ), S H
3-41 (s, 3H, C7-OCH3), 3.56 (2H, 2-position, ~ 2 S
3.96 (s, 3H, ~ ~), 4.28 (d, 2H, 3-position~ ~ CH2S- )' ~H
~3 S
4.10 (2H, 1 ~ ), 5.08 (s, lH, 6-position, Lr 30 Example 11 ~18413 To a solution of 192 mg. of 7,3-bromoacetamido-7a-methoxy-3-(l-methyltetrazol-5-yl)thiomethyl-~ -cephem-4-carboxylic acid in 5 ml. of methanol was added a solution prepared by dissolving 89.5 mg. of l,3-thiazoline-2, 4-dithione and 113 ~ul. of triethylamine in 5 2 ml. of methanol at -5C. After performing the reaction for 2 hours at temperatures between -5C and 5C., the solvent was dis-tilled off under reduced pressure and the residue obtained was sub-jected to a silica gel column chromatography using a mixture of chloroform, methanol, and formic acid of 85: 15: 2 by volume 10 ratio as the eluent.
The fractions containing the aimed compound were combined and the solvent was distilled off. The residue thus obtained was homogenized by acetone and then solidified by the addition of ether to give a powdery material, which was recovered by filtration and 15 dried to provide 84.2 mg. of 7a-methoxy-3-(1-methyltetrazol-5-yl) thiomethyl-7~B-(2-thioxo-~ -thiazclin-4-yl)thioacetamido~ -cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D6-DMS0) H
~;(p.p.m.): 3-38 (3H, C7-OCH3), 3.40-3.90 (2H, C2<H, H SCH - N
2H, ~ 2 ), 3.93 (3H, 1H ), 4.28 (2H, C3-CH2-S-) 5.07 (lH, 6-position), 6.95 (lH,
of triethylamine was dissolved 92 mg. of 7-bromoacetamido-713-25 methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-~3-cephem-4-carboxylic acid with stirxing at room temperature and after further adding thereto 22 mg. of 4-mercaptopyridine, the resultant mixture was ~tirred for about 40 minutes. The precipitates formed were reco-vered by filtration, washed with ethylene chloride and dried to 30 provide 58 mg. of 7a-methoxy-3-(1-methyltetraæol-5-yl)thiomethyl-4~3 7~-(4-pyridyl)thioacetamido-Q3-cephem-4-carboxyllc acid.
Nuclear magnetic resonance spectra (D6-DMS0) ~(p.p.m.): 3,33 (3H~ -OCH3), 3,93 (5HJ -SCH2C0-, and \N' ), 5.09 (lH, ~ ~ ) 7.35 (2H, S ~ ), ~ _ ~
8.40 (2H, -S- ~ ) Example 3 ~
In a mixture of 5 ml. of methylene chloride and 22.0 mg. of triethylamine was dissolved 100 mg. of 7~-bromoacetamido-7a-methoxy-3-(5-methyl-1,3,4-thiadiazol-2-yl)thiomethyl~3-cephem-4-carboxylic acid with stirring at room temperature and then the solution was treated as in Example 2-b) to provide 60 mg. of 7~-methoxy-3-(5-methyl-1,3,4-thiadiazol-2-yl)thiomethyl-7~-(4-pyridyl)-thioacetamido~Q -cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D6-DMS0) ~ (p-p-m.): 2,70 (3H, ~ ~ ), 3-41 (3H, 3), 3.94 (2H, -SCH2CO)- ~__ -Exam~le 4 A mixture of 200 mg. of 7~-bromoacetamido-7~-methoxy-3-(5-Z0 methyl-1,3,4-thiadiazol-2-yl)thiomethyl-~3-cephem-4-carboxylic acid 65 mg. of 2-hydroxy-4-mercaptopyridine, 8 ml. of water, and 16 ml.
of methanol was stirred on ice bath and after further adding to the mixture 80 mg. of sodium hydrogencarbonate, the resultant mixture was stirred for 2 hours at rOom temperature. Then, methanol was distilled off under reduced pressure, and after filtering, the fil-trate was acidified with diluted hydrochloric acid and extracted with 50 ml. of a mixture of n-butanol and ethyl acetate of 1 : 1 by volume ratio. The extract was washed with saturated aqueous solution of sodium chloride, dried over anhydrous magnesium sulfate, and the solvent was distilled off from the residue to provide 210 mg. of 7~-(2-hydroxy-4-pyridyl)thioacetamido-7a-methoxy-3-(5-methyl-1,3,4-thiadiazol-2-yl)thiomethyl~3-cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D6-DMS0) ,~i(p.p.m.): 2.70 (s,3H), 3.42 (s, 3H~, 3~72 (~, 2H), 3,84 5 (s,2H), 4,36 (q, 2H), 5, 12 (s, lH), 6.10 (d, lH), 6.24 (s, lH), 7.26 (d, lH).
ExamPle 5 To a mixture of 120 mg. of 3-hydroxy-4-mercaptopyridine, 140 mg. of sodium hydrogencarbonate, 15 ml. of methanol, and 7.5 ml.
10 of water was added 360 mg. of 7,~-bromoacetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl~ -cephem-4-carboxylic acid and the mixture was stirred under ice-cooling and after raislng the tempera-ture to room temperature, was further stirred for 2 hours.
After distilling off methanol, the reaction mixture was fil-15 tered and the filtrate was acidified with 2 ml. of 1 N hydrochloric acid. The precipitates thus formed were recovered by filtration, washed with water and dried to provide 200 mg. of 7~-(3-hydroxy-4-pyridyl)thioacetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thio-methyl-l~ -cephem-4-carboxylic acid as a crude product.
Meanwhile, the aqueous layer formed in the aforesaid procedure was extracted with 50 ml. of a mixture of n-butanol and methanol of 1: 1 by volume ratio and the organic layer was recovered, washed twice with saturated aqueous sodium chloride, dried over anhydrous magnesium sulfate, and then the solvent was distilled off 25 to provide 280 mg. of the crude product shown above.
By applying silica gel column chromatography to the crude pro-duct obtained from the aqueous layer using a mixture of chloroform, isopropanol, formic acid, and methanol at 30: 3: 1: 8 by volume ratio as the developing solvent, 100 mg. of the pure product was 30 obtained.
~uclear magnetic resonance spectra (D6-DMSO) ~(p.p.m.): 3,40 (s, 3H, 7-OCH3), 3.94 (s, 3H, N-CH3), 4.06 (s, 2H, -S-CH2Co), 4.30 (q, 2H), 5.10 (s, lH, 6-H), ~ S-- ~ _ 7 64 (d 1 H ~ OEI ) 8 24 ( 2H ~ ,OH
ExamPle 6 ~ H ~ ~ H
To a solution of 100 mg. of 7~-bromoacetamido-7a-methoxy-3-(l-methyltetrazol-5-yl)thiomethyl~3-cephem-4-carboxylic acid in 3 ml. of methanol was added a solution of 62.5 mg. of 2-amino-5-mer-capto-1,3,4-thiadiazole and 0.066 ml. of triethylamine in 3 ml. of methanol and the solution was stirred for 30 minutes at room tem-perature. The solvent was distilled off from the reaction mixture under reduced pressure and the residue formed was subjected to ~ilica gel solumn chromatography using a mixture of methylene chloride, methanol, formic acid at 80 : 20 : 2 by volume ratio as the developing solvent. The fractions containing the aimed com-pound were combined and the solvent was distilled off to provide 60 mg. of 7~-(5-imino-4,5-dihydro-1,3,4-thiadiazol-2-yl)thioaceta-mido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl~3-cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D6-DMSO) ~(p.p.m.): OCH
3 N _N
3.38 (3H,H2N ~ ~), 3.92 (3H, ~ N,~ + 2H, -~S~ SCH2-CO-), 4.29 (2H, ~ H S ), 5-06 (lH, C6-H).
25 Exam~le 7 To a solution of lOO mg. of 7~-bromoacetamido-7a-methoxy-3-(l-methyltetrazol-5-yl)thiomethyl-~3-cephem-4-carboxylic acid in 5 ml. of methanol was added a solution of sodium salt of 5-mercapto-2-oxo-2,3-dihydro-1,3,4-thiadiazole in 3 ml. of methanol prepared by dissolving 60 mg. of 5-mercapto-2-oxo-2,3-dihydro-1,3,4-thiadiazole and 0.59ml. of a 1 ~ aqueous sodium hydroxide solution in 2 ml. of methanol, and distilling off the solvent therefrom.
After stirring the mixture for 30 minutes at room temperature, the solvent was distilled off under reduced pressure and the residue 5 obtained was subjected to a column chromatography using a mixture methanol and ethyl acetate of 1: 4 by volume ratio as a developing solvent for removing impurities and then a mixture of methanol and ethyl acetate of 1: 2 by volume ratio as a developing solvent for the product.
10 The fractions containing the aimed compound were combined and ther the solvent was distilled off from the combined solution to provide 70 mg. of 7~3-(5-oxo-4,5-dihydro-1,3,4-thiadiazol-2-yl)thio-acetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl~ -cephem-4-carboxylic acid.
15 ~uclear magnetic resonance spectra (D6-DMS0) ~;(p.p.m.):
3,40 (3H, C7-OCH3), 3-94 (3H, ~ ~S--SCH2CO-4.32 (2H, ~C S )~ 5.02 (lH, C6-H).
20ExamPle 8 ~
a). A solution of 226 mg. of 4-thioxo-2-thiazolidinone in 5 ml. of methanol was cooled to -10C. and after adding thereto 344 mg. of triethylamine, the resultant solution was added to a solution of 718 mg of 7~-bromoacetamido-7a-methoxycephalosporanic acid in 5 ml. of methanol at temperatures below -5C. After carry-ing out the reaction for 2 hours at temperatures of between -5C.
and 0C., the solvent was distilled off at low temperature under reduced pressure and the residue formed was subjected to a silica gel column chromatography using a mixture of chloroform, isopropanol, and formic acid of 90: 10: 2 by volume ratio as a developing J~18413 solvent.
The fractions containing the aimed compound were combined and the solvent was distilled off to provide 860 mg. of 7a-methoxy-7~-(2-oxo-~ -thiazolin-4-yl)thioacetamidocephalosporanic acid.
Nuclear magnetic resonance spectra (D6-acetone) S(p.p.m.) 2.04 (3H), 3.52 (3H, C7-OCH3), 3,31-3.62 (2H, 2-position), 3.81 (2H, -S-CH2-CO-), 4,97 (2H, C3-CH20COCH3), 5.15 (lH, 6-position), 6.51 (lH, H ~ ), 8.62 (lH, -CO~H-), 10.33 (lH, ~ S ) ~
b). To 27 ~1. of a phosphate buffer solution having pH of 6.86 were added 484 mg. of 7a-methoxy-7~-(2-oxo-~4-thiazolin-4-yl)-thioacetamidocephalosporanic acid, 118.4 mg. of 5-mercapto-1-methyltetrazole, and 85.7 mg. of sodium hydrogencarbonate and then the reaction was carried out for 18 hours at 58-60C. After the reaction was over, the pH of the reaction mixture was adjusted to pH 1-2 by adding thereto a 40% aqueous phosphoric acid solution under ice-cooling and then the product was extracted twice each with 30 ml. of ethyl acetate. The extracts were combined, washed with water, dried over anhydrous sodium sulfate, and then the sol-vent was distilled off under reduced pressure. The residue formedwa~ ~ubjected to a silica gel column chromatography using a mixture of chloroform, isopropanol, and formic acid of 80 : 20 : 2 by volume ratio as a developing solvent.
The fractions containing the aimed compound were combined and the solvent was distilled off to provide 60 mg of 7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-7~-(2-oxo-~4-thiazolin-4-yl)thio-acetamido~ -cephem-4-carboxylic acid.
~uclear magnetic resonance spectra (D6-acetone) S(p.p.m.): 3.52 (3H, C7-OCH3), 3.60~4.00 (4H, N/
2-position + N ~ SCH2CO ), 4.02 (3~, CH3 4.44 (2H, 3-position -CH2-S-), 5.12 (lH, 6-position), 6.52 (lH, ~ ), 8.66 (lH, -CONH-), 10.40 o O ~S~
Example 9 To 4 ml. of methylene chloride was added 21.1 mg. of triethyl-amine and then 100 mg. of 7~-bromoacetamido-7a-methoxy-3-(1-methyl-tetrazol-5-yl)thiomethyl-~3-cephem-4-carboxylic acid was dissolved with stirring at room temperature. To the solution was further added 34 mg. of 2,5-dimercapto-1,3,4-thiadiazole and after adding dropwise a few drops of methanol for dissolving the 2,5-dimercapto-1,3,4-thiadiazole, the resultant mixture was stirred for about 3 hours. After the reaction was over, the solvent was distilled off from the reaction mixture, and to the residue formed were added 40 ml. of ethyl acetate and diluted hydrochloric acid to acidify it, whereby an ethyl acetate layer and an aqueous layer formed.
The ethyl acetate layer formed was recovered, washed with water, dried over anhydrous sodium sulfate, and then the solvent was dis-tilled off under reduced pressure. The residue obtained was sub-jected to a silica gel column chromatography using a mixture of chloroform, methanol, and formic acid of 50 : 4 : 1 by volume ratio as a developing solvent. The fractions containing the aimed com-pound were combined and the solvent was distilled off to provide 60 mg. of 7~-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl)-thio-acetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-~3-cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D6-acetone) ~(p.p.m.): 3.49 (3H, CH30-), 4.00 (3H, H I ), ( ' ~ SCH2C0- )' ( ' ~ CH3 Example 10 3,413 In a mixture of 0.627 ml. of a 1 ~ (f=0.998) aqueous sodium hydroxide solution and 1.5 ml. of methanol was dissolved 110 mg.
of 5-acetamido-2-mercapto-1,3,4-thiadiazole followed by stirring for several minutes and then the solvent was distilled off. The white solid residue obtained was dissolved in 2.5 ml. of methanol and after adding to the solution a solution prepared by dissolving 200 mg. of 7~-bromoacetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)-thiomethyl~ -cephem-4-carboxylic acid in 2.5 ml. of methanol at room temperature, the resultant mixture was stirred for 30 minutes at room temperature. Then, the solvent was distilled off and the residue obtained was subjected to a silica gel column chromatogra-phy using a mixture of chloroform, isopropyl alcohol, and formic acid of ~0 : 10 : 3 by volume ratio to elute unchanged 5-acetamido-2-mercapto-1,3,4-thiadiazole. Thereafter, the product was eluted using an eluent, a mixture of chloroform, methanol, and formic acid of 90 : 10 : 2 by volume ratio.
The fractions containing the aimed compound were combined and the solvent was distilled off to provide 150 mg. of the light-yellow powder of 7~-(5-acetylimino-4,5-dihydro-1,3,4-thiadiazol-2-yl)thioacetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-~3-cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D6-DMSO) N--N
~(p.p.m.): 2.18 (s, 3H, CH3CO~H ~ ~ ), S H
3-41 (s, 3H, C7-OCH3), 3.56 (2H, 2-position, ~ 2 S
3.96 (s, 3H, ~ ~), 4.28 (d, 2H, 3-position~ ~ CH2S- )' ~H
~3 S
4.10 (2H, 1 ~ ), 5.08 (s, lH, 6-position, Lr 30 Example 11 ~18413 To a solution of 192 mg. of 7,3-bromoacetamido-7a-methoxy-3-(l-methyltetrazol-5-yl)thiomethyl-~ -cephem-4-carboxylic acid in 5 ml. of methanol was added a solution prepared by dissolving 89.5 mg. of l,3-thiazoline-2, 4-dithione and 113 ~ul. of triethylamine in 5 2 ml. of methanol at -5C. After performing the reaction for 2 hours at temperatures between -5C and 5C., the solvent was dis-tilled off under reduced pressure and the residue obtained was sub-jected to a silica gel column chromatography using a mixture of chloroform, methanol, and formic acid of 85: 15: 2 by volume 10 ratio as the eluent.
The fractions containing the aimed compound were combined and the solvent was distilled off. The residue thus obtained was homogenized by acetone and then solidified by the addition of ether to give a powdery material, which was recovered by filtration and 15 dried to provide 84.2 mg. of 7a-methoxy-3-(1-methyltetrazol-5-yl) thiomethyl-7~B-(2-thioxo-~ -thiazclin-4-yl)thioacetamido~ -cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D6-DMS0) H
~;(p.p.m.): 3-38 (3H, C7-OCH3), 3.40-3.90 (2H, C2<H, H SCH - N
2H, ~ 2 ), 3.93 (3H, 1H ), 4.28 (2H, C3-CH2-S-) 5.07 (lH, 6-position), 6.95 (lH,
9.48 (lH, C7-NH-).
25 Example 12 In 5 ml. of methanol was dissolved 200 mg. of 7,~-bromoaceta-mido-7a-methoxy-3-(1-methyltetrazo1-5-yl)thiomethyl ~ -cephem-4-carboxylic acid and then to the solution was added a solution of 110 mg. of 1-methylamino-5-mercapto-1,3,4-thiadiazole sodium salt 30 in 3 ml. of methanol.
4~3 The mixture was stirred for 30 minutes at room temperature, the solvent was distilled off under reduced pressure, and the resi-due obtained was subjected to a silica gel column chromatography to initially remOve excessive l-methylamino-5-mercapto-1,3,4-thiadiazole using a mixture of chloroform, isopropanol and formicacid of 90 : 10 : 2 by volume ratio and then elute the product using a mixture of chloroform, methanol, and formic acid of 80 :
20 : 10 as a developing solvent. The fractions containing the aimed material were combined and after distilling off the solvent under reduced pressure, the residue obtained was dried to provide 100 mg. of 7a-methoxy-7~-(5-methylamino-1,3,4-thiadiazol-2-yl)-thioacetamido-3-(1-methyltetrazol-5-yl)thiomethyl-~3-cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D6-DMS0) ~(p.p.m.): 2.86 (3H~H CNH~ 3 40 (3H, C7-OCH3) 3.95 (5H, ~ SC ), 4.28 (2H, ~ ~ CH -~-), 5.08 (lH, C6-H)~
ExamPle 13 In 5 ml. of methanol was dissolved 200 mg. of 7~-bromoacetamido-7a-methoxy-3-(5-methyl-1,3~4-thiadiazol-2-yl)thiOmethyl-~3-cephem-4-carboxylic acid and to the solution was added a solution of 100 mg. of l-methylamino-5-mercapto-1,3,4-thiadiazole sodium salt in 3 ml. of methanol.
Thereafter, the mixture was treated as in Example 12 to pro-vide 80 mg. of 7~-(5-methylamino-1,3,4-thiadiazol-2-yl)thioaceta-mido-7a-methoxy-3-(5-methyl-1,3,4-thiadiazol-2-yl)thiomethyl-~3-cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D6-DMSO) N__N
~ S ~ CH3 ), 2.84 (3H, 3.39 (3H~ C7-OCH3), 3.90 (2H, ~ ~ 2 4.14, 4.50 (2H, ~ 2 )~ 5.07 (lH, C6-H).
Example 14 In 5 ml. of methanol was dissolved 200 mg. of 7~-bromoaceta-mido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-~3-cephem-4-carboxylic acid and to the solution was added a solution of 150 mg.
of 1-ethoxycarboxamido-5-mercapto-1,3J4-thiadiazole sodium salt in 3 ml. of methanol. Then, the mixture was treated as in Example 12 to provide 110 mg. of 7~-(5-ethoxycarboxamido-1,3,4-thiadiazol-2-yl)thioacetamido~7a-methoxy-2-(1-methyltetrazol-5-yl)thiomethyl--cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D6-DMSO) ~(p-p.m-): 1-24 (3H, CH3CH20C-), 3.40 (3H, C7-OCH3), H
3.58 (2H~ -N~ ~ ), 3.92 (3H, ~ ~ ), 4.08 (2H, N~ .3 _ S
~ ~ SCH )~ 4-2-4- 3 (4H, CH3CH2-OC ~ CH )' 5.0B (lH, C6-H).
' Exam~le 15 In 5 ml. of methanol was dissolved 200 mg. of 7~-bromoaceta-mido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl~ -cephem-4-carboxylic acid and to the solution was added a solution of 140 mg.
o~ 1-methoxycarboxamido-5-mercapto-1,3,4-thiadiazole sodium salt in 3 ml. of methanol.
The mixture was, then, treated as in Example 12 to provide 150 mg. of 7a-methoxy-7~-(5-~ethoxycarboxamido-1 3,4-thiadiazol-2-yl)thioacetamido-3-(1-methyltetrazol-5-yl)thiomethyl~ -cephem-4-carboxylic acid.
~uclear magnetic resonance spectra (D6-DMSO) S (p.p.m.): 3-42 (3H, C7-oCH3), 3,78 (3H, CH30C-NH-), N~ N---N
3.95 (3H, ~`N ~ ), 4.10 (2H, ~S ~ CH2)' 4.31 (2H, ~ ~ CH2-S_)~ 5-09 (lH, C6-H).
-Exam~le 16 To a solution of 100 mg. of 7~-bromoacetamido-7a-methoxy-3-(l-methyltetrazol-S-yl)thiomethyl-~ -cephem-4-carboxylic acid in 5 ml. of methanol was added a solution of 27.5 mg. of 5-amino-2-mercaptothiazole and 0.058 ml. of triethylamine in 2 ml. of i 15 methanol under nitrogen gas stream. After stirring the mixture for 30 minutes at room temperature, the solvent was distilled off ' under reduced pressure and the residue obtained was subjected to , a silica gel column chromatography and then the product was devel-oped u~ing a mixture of chloroform, methanol, and formic acid of 20 85 : 15 : 2 by volume ratio. Then, the fractions containing the aimed compound were collected and the solvent was distilled off to provide 37 mg. of 7~-[(5-aminothiazol-2-yl)thioacetamido]-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-~ -cephem-carboxylic acid.
Nuclear magnetic resonance spectra (D6~MSO) S(p.p.m.): jOC~H3 HN !IN N
3.40 (3H, o ~ N ), 3.96 (3H, ~ N
r---N _S ~
~ S ~ S-CH2-CO~, 4.29 (2H, -~ CH2-S), 5.09 (lH, C6-H), 7.47 (lH, - ~ ~ S-~xample 17 In 15 ml. of methylene chloride were dissolved 50 mg. of tri-ethylamine, 70 mg. of 2,5-dimercapto-1,3,4-thiadiazole, and 1 ml.
of methanol and to the solution was added 200 mg. of 7~-bromoaceta-mido-7a-methoxy-3-(5-methyl-1,3,4-thiadiazol-2-yl)thiomethyl-~ -cephem-4-carboxylic acid followed by stirring for about 1 hour at room temperature. After the reaction was over, the solvent was distilled off under reduced pressure and after adding to the resi-due obtained a diluted aqueous hydrochloric acid solution, the product was extracted with 50 ml. of a mixture of n-butanol and ethyl acetate of 1 : 1 by volume ratio. The extract was washed with saturated aqueous sodium chloride solution, dried over anhy-drous magnesium chloride, and then the solvent was distilled off under reduced pressure. The residue obtained was subjected to a silica gel column chromatography and developed using a mixture of chloroform, methanol, and formic acid of 100 : 5 : 1 by volume ratio. Then, the fractions containing the aimed compound were col-lected and the solvent was distilled off to provide 120 mg. of 7~-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl)thioacetamido-7a-20 methoxy-3~(5-methyl-1,3,4-thiadiazol-2-yl)thiomethyl-~ -cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D6-DMS0) S(p.p.m.) 2.69 (3H, ~ S ~ CH3 ), 3.42 (3H, -OCH3), ~
25 4.06 (2H, -S-CH2C0-), 5.12 (lH, ~ ).
Example 18 In 3 ml. of water were dissolved 140 mg. of 2-mercapto-1,3,4-thiadiazol-5-yl)thioacetic acid and 72 mg. of sodium carbonate and to the solution was added a solution of 250 mg. of 7~-bromoaceta-30 mido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl~ -cephem-4-~1841;~
carboxylic acid in about 6 ml. of methanol with stirring under ice-cooling followed by further stirring for 2 hours.
After the reaction was over, the reaction mixture was concen-trated under reduced pressure to distill off methanol and after acidifying the concentrate by the addition of diluted ac[ueous hydro-chloric acid solution, the product was extra cted twice each with 30 ml. of a mixture of n-butanol and ethyl acetate of 1: 1 by volume ratio. The extracts were corr~ined, washed with saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and then the solvent was distilled off under reduced pressure. The residue formed was subjected to a silica gel column chromatography and developed with a mixture of chloroform, methanol, and formic acid of 100: 8: l by volume ratio. The fractions containing the aimed material were collected, the solvent was distilled off under reduced pressure, and the residue was dried to provide about 140 mg. of 7~-(5-carboxymethylthio-1,3,4-thiadiazol-2-yl)thioacetamido-7a-methoxy-3-(l-methyltetrazol-5-yl)thiomethyl~ -cephem-4-carboxy-lic acid.
Nuclear magnetic resonance spectra (D6-DMSO) ~(p.p.m.): 3.40 (3H, -OCH3), 3.93 (3H, >N-CH3), H
4.12 (4H, H02CCH2-S ~SCH , 5-08 (lH, F
ExamPle 19 In 5 ml. of methanol was dissolved 200 mg. of 7,~-bromoaceta-mido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl ~ -cephem-4-25 carboxylic acid and to the solution was added a solution of 130 mg.of 2-mercapto-5-(3-methylureido)-1,3,4-thiadiazole sodiurn salt in 3 ml. of methanol.
The mixture was stirred for 30 minutes at room temperature, the solvent was distilled off from the reaction mixture under 30 reduced pressure, and the residue obtained was subjected to silica gel column chromatography to initially remove excessive 2-mercapto-5-(3-methylureido)-1,3,4-thiadiazole using a mixture of chloroform, isopropanol, and formic acid of 90 : 10 : 2 by volume ratio and then develop the product using a mixture of chloroform, methanol, and formic acid of 80 : 20 : 1 by volume ratio. The fractions containing the aimed product were collected, the solvent was dis-tilled off under reduced pressure, and then the residue was solidi-fied by the addition of 20 ml. of ether to provide 140 mg. of 7-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-7~-~5-(3-methylureido)-1,3,4-thiadiazol-2-yl]thioacetamido-~ -cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D6-DMS0) ~(p.p.m.):
2.72 (3H, CH3-NH-C-NH - ~$ ~ )~ 3-44 (3H, C7-OCH3), about 3.60 (2H, C2-H), 3.97 (3H, ~N
CH
4.07 (2H, ~ ~ SCH ), 4.30 (2H, N~ ~ CH S )' 5.10 (lH, C6-H).
~xamPle 20 In about 4 ml. of water were dissolved 115 mg. of 2-hydroxy-4-mercapto-5-methylpyridine and 76 mg. of sodium hydrogencarbonate and to the solution was added a solution of 300 mg. of 7~-bromo-acetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-~3-cephem-4-carboxylic acid in 10 ml. of methanol with stirring under ice-cooling followed by further stirring for about 40 minutes.
After the reaction was over, the reaction mixture was concen-trated under reduced pressure to distill off methanol almost com-pletely and about 20 ml. of water was added to the concentrate.
After acidifying the solution with 1 N hydrochloric acid under ice-cooling, the product was extracted twice each with about 50 ml. of a mixture of n-butanol and ethyl acetate of 1 : 1 of volume ratio.
The combined extracts were washed with aqueous saturated sodium chloride solution and dried over anhydrous magnesium sulfate, and then the solvent was distilled off under reduced pressure. The residue formed was subjected to a silica gel column chromatography and developed with a mixture of chloroform, methanol, and formic acid of 100 : 8 : 1 by volume ratio. The fractions containing the aimed material were combined and 24 mg. of 7~-(2-hydroxy-5-methyl-4-pyridyl)thioacetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thio-methyl-~ -cephem-4-carboxylic acid was obtained.
Nuclear magnetic resonance spectra (D6-DMS0) ~(p.p.m.): 1.96 (3H ~ ~ ~3 ), 3.39 (3H, CH30- ), about 3.90 (2H, -SCH2C0- ), 3.93 (3H, CH3-~ ~ ), 5.09 (lH, ~ ), 6.20 (lH, H~
and 7.11 (lH, ExamPle 21 In 2 ml. of water was suspended 120 mg. of 2-hydroxy-4-mercap-to-3-methylpyridine and then 1.1 ml. of a 1 ~ aqueous sodium hydro-xide solution was added to the suspension to dissolve completelythe methylpyridine. Then, 300 mg. of 7~-bromoacetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl~ -cephem-4-carboxylic acid in
25 Example 12 In 5 ml. of methanol was dissolved 200 mg. of 7,~-bromoaceta-mido-7a-methoxy-3-(1-methyltetrazo1-5-yl)thiomethyl ~ -cephem-4-carboxylic acid and then to the solution was added a solution of 110 mg. of 1-methylamino-5-mercapto-1,3,4-thiadiazole sodium salt 30 in 3 ml. of methanol.
4~3 The mixture was stirred for 30 minutes at room temperature, the solvent was distilled off under reduced pressure, and the resi-due obtained was subjected to a silica gel column chromatography to initially remOve excessive l-methylamino-5-mercapto-1,3,4-thiadiazole using a mixture of chloroform, isopropanol and formicacid of 90 : 10 : 2 by volume ratio and then elute the product using a mixture of chloroform, methanol, and formic acid of 80 :
20 : 10 as a developing solvent. The fractions containing the aimed material were combined and after distilling off the solvent under reduced pressure, the residue obtained was dried to provide 100 mg. of 7a-methoxy-7~-(5-methylamino-1,3,4-thiadiazol-2-yl)-thioacetamido-3-(1-methyltetrazol-5-yl)thiomethyl-~3-cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D6-DMS0) ~(p.p.m.): 2.86 (3H~H CNH~ 3 40 (3H, C7-OCH3) 3.95 (5H, ~ SC ), 4.28 (2H, ~ ~ CH -~-), 5.08 (lH, C6-H)~
ExamPle 13 In 5 ml. of methanol was dissolved 200 mg. of 7~-bromoacetamido-7a-methoxy-3-(5-methyl-1,3~4-thiadiazol-2-yl)thiOmethyl-~3-cephem-4-carboxylic acid and to the solution was added a solution of 100 mg. of l-methylamino-5-mercapto-1,3,4-thiadiazole sodium salt in 3 ml. of methanol.
Thereafter, the mixture was treated as in Example 12 to pro-vide 80 mg. of 7~-(5-methylamino-1,3,4-thiadiazol-2-yl)thioaceta-mido-7a-methoxy-3-(5-methyl-1,3,4-thiadiazol-2-yl)thiomethyl-~3-cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D6-DMSO) N__N
~ S ~ CH3 ), 2.84 (3H, 3.39 (3H~ C7-OCH3), 3.90 (2H, ~ ~ 2 4.14, 4.50 (2H, ~ 2 )~ 5.07 (lH, C6-H).
Example 14 In 5 ml. of methanol was dissolved 200 mg. of 7~-bromoaceta-mido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-~3-cephem-4-carboxylic acid and to the solution was added a solution of 150 mg.
of 1-ethoxycarboxamido-5-mercapto-1,3J4-thiadiazole sodium salt in 3 ml. of methanol. Then, the mixture was treated as in Example 12 to provide 110 mg. of 7~-(5-ethoxycarboxamido-1,3,4-thiadiazol-2-yl)thioacetamido~7a-methoxy-2-(1-methyltetrazol-5-yl)thiomethyl--cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D6-DMSO) ~(p-p.m-): 1-24 (3H, CH3CH20C-), 3.40 (3H, C7-OCH3), H
3.58 (2H~ -N~ ~ ), 3.92 (3H, ~ ~ ), 4.08 (2H, N~ .3 _ S
~ ~ SCH )~ 4-2-4- 3 (4H, CH3CH2-OC ~ CH )' 5.0B (lH, C6-H).
' Exam~le 15 In 5 ml. of methanol was dissolved 200 mg. of 7~-bromoaceta-mido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl~ -cephem-4-carboxylic acid and to the solution was added a solution of 140 mg.
o~ 1-methoxycarboxamido-5-mercapto-1,3,4-thiadiazole sodium salt in 3 ml. of methanol.
The mixture was, then, treated as in Example 12 to provide 150 mg. of 7a-methoxy-7~-(5-~ethoxycarboxamido-1 3,4-thiadiazol-2-yl)thioacetamido-3-(1-methyltetrazol-5-yl)thiomethyl~ -cephem-4-carboxylic acid.
~uclear magnetic resonance spectra (D6-DMSO) S (p.p.m.): 3-42 (3H, C7-oCH3), 3,78 (3H, CH30C-NH-), N~ N---N
3.95 (3H, ~`N ~ ), 4.10 (2H, ~S ~ CH2)' 4.31 (2H, ~ ~ CH2-S_)~ 5-09 (lH, C6-H).
-Exam~le 16 To a solution of 100 mg. of 7~-bromoacetamido-7a-methoxy-3-(l-methyltetrazol-S-yl)thiomethyl-~ -cephem-4-carboxylic acid in 5 ml. of methanol was added a solution of 27.5 mg. of 5-amino-2-mercaptothiazole and 0.058 ml. of triethylamine in 2 ml. of i 15 methanol under nitrogen gas stream. After stirring the mixture for 30 minutes at room temperature, the solvent was distilled off ' under reduced pressure and the residue obtained was subjected to , a silica gel column chromatography and then the product was devel-oped u~ing a mixture of chloroform, methanol, and formic acid of 20 85 : 15 : 2 by volume ratio. Then, the fractions containing the aimed compound were collected and the solvent was distilled off to provide 37 mg. of 7~-[(5-aminothiazol-2-yl)thioacetamido]-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-~ -cephem-carboxylic acid.
Nuclear magnetic resonance spectra (D6~MSO) S(p.p.m.): jOC~H3 HN !IN N
3.40 (3H, o ~ N ), 3.96 (3H, ~ N
r---N _S ~
~ S ~ S-CH2-CO~, 4.29 (2H, -~ CH2-S), 5.09 (lH, C6-H), 7.47 (lH, - ~ ~ S-~xample 17 In 15 ml. of methylene chloride were dissolved 50 mg. of tri-ethylamine, 70 mg. of 2,5-dimercapto-1,3,4-thiadiazole, and 1 ml.
of methanol and to the solution was added 200 mg. of 7~-bromoaceta-mido-7a-methoxy-3-(5-methyl-1,3,4-thiadiazol-2-yl)thiomethyl-~ -cephem-4-carboxylic acid followed by stirring for about 1 hour at room temperature. After the reaction was over, the solvent was distilled off under reduced pressure and after adding to the resi-due obtained a diluted aqueous hydrochloric acid solution, the product was extracted with 50 ml. of a mixture of n-butanol and ethyl acetate of 1 : 1 by volume ratio. The extract was washed with saturated aqueous sodium chloride solution, dried over anhy-drous magnesium chloride, and then the solvent was distilled off under reduced pressure. The residue obtained was subjected to a silica gel column chromatography and developed using a mixture of chloroform, methanol, and formic acid of 100 : 5 : 1 by volume ratio. Then, the fractions containing the aimed compound were col-lected and the solvent was distilled off to provide 120 mg. of 7~-(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl)thioacetamido-7a-20 methoxy-3~(5-methyl-1,3,4-thiadiazol-2-yl)thiomethyl-~ -cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D6-DMS0) S(p.p.m.) 2.69 (3H, ~ S ~ CH3 ), 3.42 (3H, -OCH3), ~
25 4.06 (2H, -S-CH2C0-), 5.12 (lH, ~ ).
Example 18 In 3 ml. of water were dissolved 140 mg. of 2-mercapto-1,3,4-thiadiazol-5-yl)thioacetic acid and 72 mg. of sodium carbonate and to the solution was added a solution of 250 mg. of 7~-bromoaceta-30 mido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl~ -cephem-4-~1841;~
carboxylic acid in about 6 ml. of methanol with stirring under ice-cooling followed by further stirring for 2 hours.
After the reaction was over, the reaction mixture was concen-trated under reduced pressure to distill off methanol and after acidifying the concentrate by the addition of diluted ac[ueous hydro-chloric acid solution, the product was extra cted twice each with 30 ml. of a mixture of n-butanol and ethyl acetate of 1: 1 by volume ratio. The extracts were corr~ined, washed with saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and then the solvent was distilled off under reduced pressure. The residue formed was subjected to a silica gel column chromatography and developed with a mixture of chloroform, methanol, and formic acid of 100: 8: l by volume ratio. The fractions containing the aimed material were collected, the solvent was distilled off under reduced pressure, and the residue was dried to provide about 140 mg. of 7~-(5-carboxymethylthio-1,3,4-thiadiazol-2-yl)thioacetamido-7a-methoxy-3-(l-methyltetrazol-5-yl)thiomethyl~ -cephem-4-carboxy-lic acid.
Nuclear magnetic resonance spectra (D6-DMSO) ~(p.p.m.): 3.40 (3H, -OCH3), 3.93 (3H, >N-CH3), H
4.12 (4H, H02CCH2-S ~SCH , 5-08 (lH, F
ExamPle 19 In 5 ml. of methanol was dissolved 200 mg. of 7,~-bromoaceta-mido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl ~ -cephem-4-25 carboxylic acid and to the solution was added a solution of 130 mg.of 2-mercapto-5-(3-methylureido)-1,3,4-thiadiazole sodiurn salt in 3 ml. of methanol.
The mixture was stirred for 30 minutes at room temperature, the solvent was distilled off from the reaction mixture under 30 reduced pressure, and the residue obtained was subjected to silica gel column chromatography to initially remove excessive 2-mercapto-5-(3-methylureido)-1,3,4-thiadiazole using a mixture of chloroform, isopropanol, and formic acid of 90 : 10 : 2 by volume ratio and then develop the product using a mixture of chloroform, methanol, and formic acid of 80 : 20 : 1 by volume ratio. The fractions containing the aimed product were collected, the solvent was dis-tilled off under reduced pressure, and then the residue was solidi-fied by the addition of 20 ml. of ether to provide 140 mg. of 7-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-7~-~5-(3-methylureido)-1,3,4-thiadiazol-2-yl]thioacetamido-~ -cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D6-DMS0) ~(p.p.m.):
2.72 (3H, CH3-NH-C-NH - ~$ ~ )~ 3-44 (3H, C7-OCH3), about 3.60 (2H, C2-H), 3.97 (3H, ~N
CH
4.07 (2H, ~ ~ SCH ), 4.30 (2H, N~ ~ CH S )' 5.10 (lH, C6-H).
~xamPle 20 In about 4 ml. of water were dissolved 115 mg. of 2-hydroxy-4-mercapto-5-methylpyridine and 76 mg. of sodium hydrogencarbonate and to the solution was added a solution of 300 mg. of 7~-bromo-acetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-~3-cephem-4-carboxylic acid in 10 ml. of methanol with stirring under ice-cooling followed by further stirring for about 40 minutes.
After the reaction was over, the reaction mixture was concen-trated under reduced pressure to distill off methanol almost com-pletely and about 20 ml. of water was added to the concentrate.
After acidifying the solution with 1 N hydrochloric acid under ice-cooling, the product was extracted twice each with about 50 ml. of a mixture of n-butanol and ethyl acetate of 1 : 1 of volume ratio.
The combined extracts were washed with aqueous saturated sodium chloride solution and dried over anhydrous magnesium sulfate, and then the solvent was distilled off under reduced pressure. The residue formed was subjected to a silica gel column chromatography and developed with a mixture of chloroform, methanol, and formic acid of 100 : 8 : 1 by volume ratio. The fractions containing the aimed material were combined and 24 mg. of 7~-(2-hydroxy-5-methyl-4-pyridyl)thioacetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thio-methyl-~ -cephem-4-carboxylic acid was obtained.
Nuclear magnetic resonance spectra (D6-DMS0) ~(p.p.m.): 1.96 (3H ~ ~ ~3 ), 3.39 (3H, CH30- ), about 3.90 (2H, -SCH2C0- ), 3.93 (3H, CH3-~ ~ ), 5.09 (lH, ~ ), 6.20 (lH, H~
and 7.11 (lH, ExamPle 21 In 2 ml. of water was suspended 120 mg. of 2-hydroxy-4-mercap-to-3-methylpyridine and then 1.1 ml. of a 1 ~ aqueous sodium hydro-xide solution was added to the suspension to dissolve completelythe methylpyridine. Then, 300 mg. of 7~-bromoacetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl~ -cephem-4-carboxylic acid in
10 ml. of methanol was added to the solution prepared above with stirring under ice-cooling and the mixture was stirred for about 40 minutes under the same condition. After the reaction was over, the reaction mixture was concentrated under reduced pressure to di~till off methanol almost completely and then about 20 ml. of water was added to the residue. The residue was then acidified with 1 N hydrochloric acid under ice-cooling and extracted twice each with about 50 ml. of a mixture of n-butanol and ethyl acetate of 1 : 1 by volume ratio. The extracts were combined, washed with saturated aqueous sodium chloride solution, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. The residue obtained was subjected to a silica gel column chromatography using a mixture of chloroform, methanol, and formic acid of 100:10:1 by volume ratio as a developing solvent. The fractions containing the aimed material were combined to provide 200 mg. of 7~-(2-hydroxy-3-methyl-4-pyridyl)thioacetamido-7a-methoxy-3-(1-methyltetrazol-5-yl~thiomethyl~3-cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D6-DMSO) ~(P-P m ):
1.96 (3H, ~N ~ ~ ), 3.38 (3H, CH3-O-), about 3.87 (2E, -S-CH2-CO-), 3.93 (3H, CH3-N ~ ), H ~~
5.09 (lH, ~ ), 6.25 (lH, ~ ), 7.21 (lH, ~ )-H
ExamPle 22 In the manner of Example 20, 4-mercapto-3-methylpyridine N-oxide was used instead of 2-hydroxy-4-mercapto-5-methylpyridine, and as developing solvent of silica gel column chromatography a mixture of chloroform, methanol and formic acid of 100 : 20 : 1 of volume ratio was used, and the other reaction conditions were same as Example 20. Whereby, 220 mg. of 7~-methoxy-7~-(3-methyl-4-pyridyl)thioacetamido-3-(1-methyltetrazol-5-yl)thiomethyl-~ -cephem-4-carboxylic acid l'-oxide was obtained.
Nuclear magnetic resonance spectra (D6-DMS0) 5 (p.p.m.): 2.17 (3H, ~ 3 ), 3.37 (3H, CH30- ) 36 ~
$~ 3 about 3.9~ (5H, CH3N ~ , -SCH2C0- ), 5.09 (lH,o ~
~ .~
7.38 (lH, ~ ) and 8.10 (2H, O O
Example 23 In about 1 ml. of water was dissolved 33 mg. of sodium carbo-nate and to the aqueous solution was added a solution of 300 mg. of 7~-bromoacetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-~3-cephem-4-carboxylic acid in about 6 ml. of methanol with stirring under ice-cooling. Thereafter, a solution of 105 mg. of 2-amino-4-mercaptopyridine sodium salt in about S ml. of methanol was added dropwise over a period of about 20 minutes to the mixture prepared in the above step with stirring under ice-cooling and then the mix-ture was stirred for about 10 minutes to complete the reaction.
Then, the reaction mixture was concentrated under reduced pressure, about 10 ml. of water was added to the residue to dissolve the product, and undissolved matters were filtered off. The filtrate was neutralized with 1 N hydrochloric acid under ice-cooling and the precipitates formed were recovered by filtration, washed with water and then methanol, and dried over phosphorus pentoxide under reduced pressure to provide about 200 mg. of 7~-(2-amino-4-pyridyl) thioacetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-A3-cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D2O + ~a2CO3) S(p~p~m~) 3.46 (3H, CH3 ~ , about 3-96 (SH~ CH3~ ~ S' -SCH2C0), 5.07 (lH, 0 ~ N~ ), 6.50 (lH, ~ N~
S --6.58 (lH, ~ ~ ), 7.74 (lH, H ~ N <
Example 24 To the mixture of 40 mg. of 2,6-dihydroxy-4-mercaptopyridine, 4~3 ,0 mg. of sodium hydrogencarbonate, 5 ml. of water and 10 ml. of methanol was added 140 mg. of 7~-bromoacetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-~ -cephem-4-carboxylic acid with stirring under ice-cooling. The resultant mixture was heated to room temperature and stirred for 2 hours. After distilling off methanol, the residue was acidified with 2 ml. of 1 N hydrochloric acid and the product was extracted with 30 ml. of a mixture of n-butanol and ethyl acetate of 1 : 1 of volume ratio, washed three times with aqueous saturated sodium chloride solution, and dried over anhydrous magnesium sulfate. After distilling off the solvent, crude 7~-(2,6-dihydroxy-4-pyridyl)thioacetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl~ -cephem-4-carboxylic acid was obtained. This crude product was subjected to a silica gel column chromatgraphy using a mixture of chloroform, isopropyl alcohol and formic acid of 90 : 10 : 3 of volume ratio as developing solvent, whexeby 50 mg. of pure product was obtained.
~uclear magnetic resonance spectra (D6-DMSO) ~(p.p.m.): 3.36 (3H, s, -OCH3), 3.92 (3H, s, ~-CH3), 5.06 (lH, s, 6-H) and 6.60 (2H, s, H~ ~ ~ )-Example 25 H ~ H
In about 2 ml. of water was suspended 120 mg. of 3-hydroxy-2-mercapto-6-methylpyridine and then 1.2 ml. of a 1 ~ aqueous sodium hydroxide solution was added to the suspension to dissolve the methylpyridine. To the solution was added a solution of 300 mg.
of 7~-bromoacetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thio-methyl-~ -cephem-4-carboxylic acid in about 10 ml. of methanol with stirring under ice-cooling followed by further stirring for about 40 minutes under ice-cooling to ensure the reaction. After dis-tilling off methanol almost completely from the reaction mixture under reduced pressure, about 10 ml. of ~ater was added to the 41~
residue and after acidifying the mixture by adding l N hydrochloric acid under ice-cooling, the product was extracted with about 150 ml.
of ethyl acetate. The extract was dried over anhydrous magnesium sulfate and the solvent was distilled off under reduced pressure.
Then, the residue formed was subjected to a silica gel column chro-matography and developed using a mixture of chloroform, methanol, and formic acid of lO0 : 7 : l by volume ratio. The fractions con-taining the aimed material were combined to provide about 200 mg. of 7~-(3-hydroxy-6-methyl-2-pyridyl)thioacetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-~ -cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D6-DMS0) S(P.p.m.):
2.33 (3H, ~ ), 3.34 (3H, CH 0-), H3C N ~3 about 3.87 (2H, -S-CH2C0-), 3.91 (3H, CH3-N < )~
H H
5.08 (lH,o ~ ~ ), 6.82-6.98 (2H, H ~ )
Nuclear magnetic resonance spectra (D6-DMSO) ~(P-P m ):
1.96 (3H, ~N ~ ~ ), 3.38 (3H, CH3-O-), about 3.87 (2E, -S-CH2-CO-), 3.93 (3H, CH3-N ~ ), H ~~
5.09 (lH, ~ ), 6.25 (lH, ~ ), 7.21 (lH, ~ )-H
ExamPle 22 In the manner of Example 20, 4-mercapto-3-methylpyridine N-oxide was used instead of 2-hydroxy-4-mercapto-5-methylpyridine, and as developing solvent of silica gel column chromatography a mixture of chloroform, methanol and formic acid of 100 : 20 : 1 of volume ratio was used, and the other reaction conditions were same as Example 20. Whereby, 220 mg. of 7~-methoxy-7~-(3-methyl-4-pyridyl)thioacetamido-3-(1-methyltetrazol-5-yl)thiomethyl-~ -cephem-4-carboxylic acid l'-oxide was obtained.
Nuclear magnetic resonance spectra (D6-DMS0) 5 (p.p.m.): 2.17 (3H, ~ 3 ), 3.37 (3H, CH30- ) 36 ~
$~ 3 about 3.9~ (5H, CH3N ~ , -SCH2C0- ), 5.09 (lH,o ~
~ .~
7.38 (lH, ~ ) and 8.10 (2H, O O
Example 23 In about 1 ml. of water was dissolved 33 mg. of sodium carbo-nate and to the aqueous solution was added a solution of 300 mg. of 7~-bromoacetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-~3-cephem-4-carboxylic acid in about 6 ml. of methanol with stirring under ice-cooling. Thereafter, a solution of 105 mg. of 2-amino-4-mercaptopyridine sodium salt in about S ml. of methanol was added dropwise over a period of about 20 minutes to the mixture prepared in the above step with stirring under ice-cooling and then the mix-ture was stirred for about 10 minutes to complete the reaction.
Then, the reaction mixture was concentrated under reduced pressure, about 10 ml. of water was added to the residue to dissolve the product, and undissolved matters were filtered off. The filtrate was neutralized with 1 N hydrochloric acid under ice-cooling and the precipitates formed were recovered by filtration, washed with water and then methanol, and dried over phosphorus pentoxide under reduced pressure to provide about 200 mg. of 7~-(2-amino-4-pyridyl) thioacetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-A3-cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D2O + ~a2CO3) S(p~p~m~) 3.46 (3H, CH3 ~ , about 3-96 (SH~ CH3~ ~ S' -SCH2C0), 5.07 (lH, 0 ~ N~ ), 6.50 (lH, ~ N~
S --6.58 (lH, ~ ~ ), 7.74 (lH, H ~ N <
Example 24 To the mixture of 40 mg. of 2,6-dihydroxy-4-mercaptopyridine, 4~3 ,0 mg. of sodium hydrogencarbonate, 5 ml. of water and 10 ml. of methanol was added 140 mg. of 7~-bromoacetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-~ -cephem-4-carboxylic acid with stirring under ice-cooling. The resultant mixture was heated to room temperature and stirred for 2 hours. After distilling off methanol, the residue was acidified with 2 ml. of 1 N hydrochloric acid and the product was extracted with 30 ml. of a mixture of n-butanol and ethyl acetate of 1 : 1 of volume ratio, washed three times with aqueous saturated sodium chloride solution, and dried over anhydrous magnesium sulfate. After distilling off the solvent, crude 7~-(2,6-dihydroxy-4-pyridyl)thioacetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl~ -cephem-4-carboxylic acid was obtained. This crude product was subjected to a silica gel column chromatgraphy using a mixture of chloroform, isopropyl alcohol and formic acid of 90 : 10 : 3 of volume ratio as developing solvent, whexeby 50 mg. of pure product was obtained.
~uclear magnetic resonance spectra (D6-DMSO) ~(p.p.m.): 3.36 (3H, s, -OCH3), 3.92 (3H, s, ~-CH3), 5.06 (lH, s, 6-H) and 6.60 (2H, s, H~ ~ ~ )-Example 25 H ~ H
In about 2 ml. of water was suspended 120 mg. of 3-hydroxy-2-mercapto-6-methylpyridine and then 1.2 ml. of a 1 ~ aqueous sodium hydroxide solution was added to the suspension to dissolve the methylpyridine. To the solution was added a solution of 300 mg.
of 7~-bromoacetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thio-methyl-~ -cephem-4-carboxylic acid in about 10 ml. of methanol with stirring under ice-cooling followed by further stirring for about 40 minutes under ice-cooling to ensure the reaction. After dis-tilling off methanol almost completely from the reaction mixture under reduced pressure, about 10 ml. of ~ater was added to the 41~
residue and after acidifying the mixture by adding l N hydrochloric acid under ice-cooling, the product was extracted with about 150 ml.
of ethyl acetate. The extract was dried over anhydrous magnesium sulfate and the solvent was distilled off under reduced pressure.
Then, the residue formed was subjected to a silica gel column chro-matography and developed using a mixture of chloroform, methanol, and formic acid of lO0 : 7 : l by volume ratio. The fractions con-taining the aimed material were combined to provide about 200 mg. of 7~-(3-hydroxy-6-methyl-2-pyridyl)thioacetamido-7a-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-~ -cephem-4-carboxylic acid.
Nuclear magnetic resonance spectra (D6-DMS0) S(P.p.m.):
2.33 (3H, ~ ), 3.34 (3H, CH 0-), H3C N ~3 about 3.87 (2H, -S-CH2C0-), 3.91 (3H, CH3-N < )~
H H
5.08 (lH,o ~ ~ ), 6.82-6.98 (2H, H ~ )
Claims (22)
PROVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process of producing the 7.alpha.-methoxy-7.beta.-heterocyclic thioacetamido-3-heterocyclic thiomethyl-.DELTA.3-cephem-4-carboxylic acid derivative represented by the general formula:
wherein R1 represents or (wherein R3 and R4, which may be the same or different, each represents a hydrogen atom, a hydroxy group, an amino group, or a lower alkyl group; n represents 0 or 1; R5 represents a hydroxy group, an amino group, a mercapto group, a lower alkylamino group, a lower alkanoylamino group, a lower alkoxycarbonylamino group, a carboxy lower alkylthio group, or a 3-lower alkylureido group; and X represents -CH= or =N-) and R2 represents 5-lower alkyl-1,3,4-thiadiazol-2-yl group or 1-lower alkyltetrazol-5-yl group; and the pharmaceutically acceptable salts thereof, which comprises selecting a process from the group of processes consisting of:
(a) reacting 7.alpha.-methoxy-3-heterocyclic thiomethyl-7-substituted or unsubstituted amino-.DELTA.3-cephem-4-carboxylic acid represented by the general formula:
wherein R2 has the same meaning as in the above general formula and Z represents Y-CH2-CO- group (wherein Y
represents a halogen atom) or a hydrogen atom with the heterocyclic compound represented by the general formula:
wherein R1 has the same meaning as in the above general formula and B represents a hydrogen atom or R1-S- when Z
is Y-CH2CO- group or represents -CH2COOH or the reactive derivative of the carboxy group thereof when Z is hydrogen atom; and (b) reacting the 7.alpha.-methoxy-7.beta.-heterocyclic thioacetamido-cephalosporanic acid represented by the general formula:
wherein R1 has the same meaning as in the above general formula with the heterocyclic thiol compound or the alkali metal salt thereof represented by the general formula:
wherein R2 has the same meaning as in the above general formula and M represents a hydrogen atom or an alkali metal atom.
wherein R1 represents or (wherein R3 and R4, which may be the same or different, each represents a hydrogen atom, a hydroxy group, an amino group, or a lower alkyl group; n represents 0 or 1; R5 represents a hydroxy group, an amino group, a mercapto group, a lower alkylamino group, a lower alkanoylamino group, a lower alkoxycarbonylamino group, a carboxy lower alkylthio group, or a 3-lower alkylureido group; and X represents -CH= or =N-) and R2 represents 5-lower alkyl-1,3,4-thiadiazol-2-yl group or 1-lower alkyltetrazol-5-yl group; and the pharmaceutically acceptable salts thereof, which comprises selecting a process from the group of processes consisting of:
(a) reacting 7.alpha.-methoxy-3-heterocyclic thiomethyl-7-substituted or unsubstituted amino-.DELTA.3-cephem-4-carboxylic acid represented by the general formula:
wherein R2 has the same meaning as in the above general formula and Z represents Y-CH2-CO- group (wherein Y
represents a halogen atom) or a hydrogen atom with the heterocyclic compound represented by the general formula:
wherein R1 has the same meaning as in the above general formula and B represents a hydrogen atom or R1-S- when Z
is Y-CH2CO- group or represents -CH2COOH or the reactive derivative of the carboxy group thereof when Z is hydrogen atom; and (b) reacting the 7.alpha.-methoxy-7.beta.-heterocyclic thioacetamido-cephalosporanic acid represented by the general formula:
wherein R1 has the same meaning as in the above general formula with the heterocyclic thiol compound or the alkali metal salt thereof represented by the general formula:
wherein R2 has the same meaning as in the above general formula and M represents a hydrogen atom or an alkali metal atom.
2. The process as claimed in claim 1, wherein Z is Y-CH2CO-group wherein Y is a halogen atom and B is a hydrogen atom or R1-S-, wherein R1 is as defined in claim 1.
3. The process as claimed in claim 1, wherein Z is a hydrogen atom and B is -CH2COOH or the reactive derivative of the carboxy group thereof.
4. The process as claimed in claim 1, wherein lower alkyl moiety is a methyl group or an ethyl group.
5. The process as claimed in claim 1, wherein R1 is and R2 is 5-lower alkyl-1,3,4-thiadiazol-2-yl group.
6. The process as claimed in claim 1, wherein R1 is and R2 is 1-lower alkyltetrazol-5-yl group.
7. The process as claimed in claim 1, wherein R1 is and R2 is 5-lower alkyl-1,3,4-thiadiazol-2-yl group.
8. The process as claimed in claim 1, wherein R1 is and R2 is 1-lower alkyltetrazol-5-yl group.
9. The process as claimed in claim 1, wherein R1 is 2-hydroxy-4-pyridyl group and R2 is 1-methyltetrazol-5-yl group.
10. The process as claimed in claim 1, wherein R1 is 3-hydroxy-4-pyridyl group and R2 is 1-methyltetrazol-5-yl group.
11. The process as claimed in claim 1, wherein R1 is 5-amino-1,3,4-thiadiazol-5-yl group and R2 is 1-methyltetrazol-5-yl group.
12. The process as claimed in claim 1, wherein R1 is 2-amino-4-pyridyl group and R2 is 1-methyltetrazol-5-yl group.
13. A 7.alpha.-methoxy-7.beta.-heterocyclic thioacetamido-3-heterocyclic thiomethyl-.DELTA.3-cephem-4-carboxylic acid compound represented by the formula:
wherein R1 represents or (wherein R3 and R4, which may be the same or different, each represents a hydrogen atom, a hydroxy group, an amino group, or a lower alkyl group; n represents 0 or 1; R5 represents a hydroxy group, an amino group, a mercapto group, a lower alkylamino group, a lower alkanoylamino group, a lower alkoxycarbonylamino group, a carboxy lower alkylthio group, or a 3-lower alkylureido group; and X represents -CH= or =N-) and R2 represents 5-lower alkyl-1,3,4-thiadiazol-2-yl group or a 1-lower alkyltetrazol-5-yl group and the pharmaceutically acceptable salts thereof, when prepared by the process of claim 1.
wherein R1 represents or (wherein R3 and R4, which may be the same or different, each represents a hydrogen atom, a hydroxy group, an amino group, or a lower alkyl group; n represents 0 or 1; R5 represents a hydroxy group, an amino group, a mercapto group, a lower alkylamino group, a lower alkanoylamino group, a lower alkoxycarbonylamino group, a carboxy lower alkylthio group, or a 3-lower alkylureido group; and X represents -CH= or =N-) and R2 represents 5-lower alkyl-1,3,4-thiadiazol-2-yl group or a 1-lower alkyltetrazol-5-yl group and the pharmaceutically acceptable salts thereof, when prepared by the process of claim 1.
14. A compound according to claim 13, wherein lower alkyl moiety is a methyl or ethyl group, when prepared by the process of claim 4.
15. A compound according to claim 13, wherein R1 is and R2 is 5-lower alkyl-1,3,4-thiadiazol-2-yl group, when prepared by the process of claim 5.
16. A compound according to claim 13, wherein R1 is and R2 is 1-lower alkyltetrazol-5-yl group, when prepared by the process of claim 6.
17. A compound according to claim 13, wherein R1 is and R2 is 5-lower alkyl-1,3,4-thiadiazol-2-yl group, when prepared by the process of claim 7.
18. A compound according to claim 13, wherein R1 is and R2 is 1-lower alkyltetrazol-5-yl group, when prepared by the process of claim 8.
19. 7.beta.-(2-hydroxy-4-pyridyl)thioacetamido-7.alpha.-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-.DELTA.3-cephem-4-carboxylic acid when prepared by the process of claim 9.
7.beta.-(3-hydroxy-4-pyridyl)thioacetamido-7.alpha.-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-.DELTA.3-cephem-4-carboxylic acid, when prepared by the process of claim 10.
21. 7.beta.-(5-amino-1,3,4-thiadiazol-5-yl)thioacetamido-7.alpha.-methoxy-3-(1-methyltetrazol-5-yl)thiomethyl-.DELTA.3-cephem-4-carboxylic acid, when prepared by the process of claim 11.
22. 7.beta.-(2-amino-4-pyridyl)thioacetamido-7.alpha.-methoxy-3-(1-methyl-tetrazol-5-yl)thiomethyl-.DELTA.3-cephem-4-carboxylic acid, when prepared by the process of claim 12.
Applications Claiming Priority (9)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP51076209A JPS5854156B2 (en) | 1976-06-28 | 1976-06-28 | New cephalosporin derivatives and their production method |
| JP76209/1976 | 1976-06-28 | ||
| JP8065976A JPS537695A (en) | 1976-07-07 | 1976-07-07 | Novel cephalosporin derivatives and their preparation |
| JP80659/1976 | 1976-07-07 | ||
| JP121143/1976 | 1976-10-08 | ||
| JP12114376A JPS5346994A (en) | 1976-10-08 | 1976-10-08 | Novel 7-(5-amino-1,3,4-thiadiazol-2-yl)thioacetamide-d3-cephem-4-carboxylic acid derivatives and their preparation |
| JP123601/1976 | 1976-10-15 | ||
| JP159908/1976 | 1976-12-27 | ||
| JP26835/1977 | 1977-03-11 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1118413A true CA1118413A (en) | 1982-02-16 |
Family
ID=27302090
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000280218A Expired CA1118413A (en) | 1976-06-28 | 1977-06-09 | PROCESS FOR THE PREPARATION OF 7.alpha.-METHOXY- CEPHALOSPORANIC ACID DERIVATIVES |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1118413A (en) |
-
1977
- 1977-06-09 CA CA000280218A patent/CA1118413A/en not_active Expired
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