CA1049502A - 7-(substituted phenylglycinamido)-3-substituted-3-cephem-4-carboxylic acid derivatives and preparation thereof - Google Patents

Abstract

Abstract of the Disclosure A cephalosporin compound of the formula:

Description

1~95~2 The present invention rela-te~ to new cephalosporanic acid derivatives which possess an antibacterial activity, and their production and use.
There is described a class of cephalosporanic acid derivatives represented by the general formula:

R ~ 0~ N \ ~ CH2-R3 (I) ~OOH

wherein Rl is hydrogen or hydroxy, R2 is nitro, alkoxy, alkane-sulfonamidO, dialkanesulfonamido, alkylaminosulfonamido, alkyl-ureido or alkylthioureido, R3 is hydrogen or a heterocyclic-thio group (in which the heterocyclic group may be substitutedwith alkyl), X is free or protected amino and non-toxic, pharmaceutically acceptable salts or esters thereof, provided that Rl is hydroxyl when R2 is nitro alkoxy and R3 is hydrogen when Rl is hydroxyl and R2 is alkoxy. ~;
The invention is especially concerned with the compounds of formula (I) in which Rl is hydrogen or hydroxy, ~ `
R2 is alkanesulfonamido, dialkanesulfonamido or alkylamino- ~`
sulfonamido, X is amino or protected amino, and M is hydrogen, a non-toxic pharmaceutically acceptable cation or haloalkyl, provided that Rl is hydroxy when R2 is alkanesulfonamido.
In a further aspect of the invention there is provided a process for the preparation of the compounds of the invention.

, ,' ~

95(~2 The term "alkyl" is preferred to be the one having not more than 8 carbon atoms. Specific examples are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, hexyl, etc. The term "alkoxy" is also preferred to be the one having not more than 8 carbon atoms. Specific examples are methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, pentyloxy, hexyloxy, etc.
The term "alkanesulfonamido" is favored to have an alkane moiety of not more than 8 carbon atoms. Specific examples are mesylamino, ethanesulfonamido, propanesulfonamido, iso-~ - 2 -propanesulfonamido, butanesulfonamido, isobutanesulfonarnido, t-butanesulfGnamido, etc. The term "dialkanesulfonamido" is also favored to have an alkane moiety of not more than 8 carbon atoms. Specific examples are dimesylamino, diethane-sulfonamido, dipropanesu]fonamido, diisopropanesulfonamido, dibutanesulfonamido, diisobutanesulfonamido, di-t-butanesulfon-amido, dipentanesulfonamido, dihexanesul-~onamido, etc.
Similarly, the alkyl moiety in the terms "alkylamino-sulfonylamino", "alkylureido" and "alkylthioureido" is preferred to have not more than 8 carbon atoms.
The term "heterocyclic-thio group" is intended to mean the residue of a thiol compound having a heterocyclic group, ~hich is the residue of ~uran, thiophene, pyrrole, pyra- ~
~ole, imidazole, triazole, thiazole, isothiazole, oxazole, iso_ ~- :
xazole, thiadiazole, oxadiazole, thiatriazole, oxatriazole, tetrazole, pyridine, pyrazine, pyrimidine, pyridazine, benzo-thiophene, benzofuran, indole, indazole~ benzimidazole, benzo-thiazole, benzothiadiazole, benzoxazole~ purine, quinoline, isoquinoline, phthalazine, naphthyridine, quinoxaline, ~uina-zoline, pyrrolidine, imidazolidine, piperidine, piperazine or `~
the like. The heterocyclic moiety of the "heterocyclic-thio group" may be substituted with one or more alkyls, preferably having 1 to 8 carbon atoms.
The term "protected amino" represents an amino group protected by any conventional amino protecting group such as benzyloxycarbonyl, substituted benzyloxycarbonyl, alkoxycarbonyly substituted alkoxycarbonyl, aralkoxycarbonyl, adamantyloxy-carbonyl, trityl, substituted phenylthio, substituted aralkyl-idene~ subs-tituted alkylidene or substituted cycloalkylidene.
~ he ter~ "non-toxic, pharmaceutically acceptable ~L~49502 salts" means a salt o-f an alkali metal cation such as sodium or potassium.
The objective cephalosporin compound (I) may be produced by reacting a 7-amino-3-substituted-3-cephem-4-carboxylic acid of the forrnula:

, H2N I " :, .
~ N ~ - CH2-R3 (II) O I . .
COOH
wherein R3 is as defined above or its derivative at the amino group and/or the carboxyl group with an amino acid of the formula:

Rl ~ OEI-COOH ~III) wherein Rl and R2 and X are each as defined above or its reactive derivative at the carboxyl group.

The derivative at the carboxyl group of the compound (II) may be a salt such as magnesium salt, calcium salt, tri-ethylamine salt, etc., an ester such as methyl ester, ethyl ester, propyl ester, butyl ester, pentyl ester, trimethylsilyl ester, 2-mesylethyl ester, 2-iodoethyl ester, 2,2,2-trichloro-ethyl ester, benzyl ester, 4-methoxybenzyl ester, 4-nitrobenzyl ester, phenacyl ester, phenethyl ester, trityl ester, dipenyl-methyl ester, bis(methoxyphenyl)methyl ester, 3,4-dimethoxy-benzyl ester, (l-cyclopropyl)ethyl ester, ethynyl ester, 4-hydroxy-3,5-di-t-butylbenzyl ester, etc., an activated amide, ;
an acid anhydride, an acid halide, or the like.

The derivative at the amino group of the compound (II) B _ 4 _ , -,= . .
. .. ,~ . .

~ 49S~2 may be the reaction product of the compound [II] and a silyl compound such as bis(trimethylsilyl)acetamide.
The reactive derivative at the carboxyl group of the amino acid [III] may be an acid halide9 an acid anhydride, an aetivated amide, an activated ester, or the like. ~he ~
suitable examples may be an aeid ehloride; an aeid azide; -a mixed acid anhydride with an aeid sueh as dialkylphosphoric aeid, phenylphosphorie acid, diphenylphosphorie aeid, di- ;
benzylphosphorie aeid, halogenated phosphorie acid, dialkyl-phosphorous acid, sulfurous acid, thiosulfuric acid, sulfurie aeid, alkylcarbonie aeid, aliphatic earboxylie aeid (e.g.
pivalic aeid, pentanoic acid, isopentanoic aeid, 2-ethyl-butyrie aeid, triehloroaeetie aeid) or aromatie earboxylie -aeid (e.g. benzoic acid), or a symmetrieal aeid anhydride;
an aeid amide with imidazole, 4-substituted imidazole, di-methylpyrazole, triazole or tetrazole; or an ester (e.g.
eyanomethyl ester, methoxymethyl ester, vinyl ester, propargyl ester, p-nitrophenyl ester, 2,4 dinitrophenyl ester, trichloro-phenyl ester, pentaehlorphenyl ester, methanesulfonylphenyl ester, phenylazophenyl ester, phenyl thioester, p-nitrophenyl thioester, p-eresyl thioester, earboxymethyl thioester, pyranyl ester, pyridyl ester, piperidyl ester, 8-quinolyl thioester, or an ester with N,N-dimethylhydroxylamine, l~hydroxy-2-(lH)-pyridone, N-hydroxysueeinimide, N-hydroxyphthalimide), or the like. . --~
When the amino aeid [III] is used in the form of a free aeid or a salt, the reaetion may be preferably ear-ried out in the presenee of a eondensing agent appropriately seleeted from N,N'-dicyelohexylearbodiimide, N-cyclohexyl-N'- ~-morpholinoethylcarbodiimide, N-cyclohexyl-N'-(4-diethylamino-.. ~ - ................ , . :
.

~L0~95a\Z
cyclohexyl)carbodiimide, ~ diethylcarbodiimide~ N,N'-diiso-propylcarbodiimide, N~ethyl-N'-(3-dimethylaminopropyl)carbodi-imide, N,~'-carbonyldi(2-methylimidazole), pentamethylene-ketene-~-cyclohexylimine, diphenylketene-~-cyclohexylimine, alkoxyacetylene, l-alkoxy-l-chloroethylene, trialkyl phosphite, ethyl polyphosphate, isopropyl polyphosphate, phosphorus oxy-chloride, phosphorus trichloride, thionyl chloride, oxalyl chloride, triphenylphosphine, 2-ethyl-7-hydroxybenzisoxazolium salt, 2-ethyl-5-(m-sulfophenyl)isoxazolium hydroxide intra- -~
molecular salt, (chloromethylene)dim~thylammonium chloride and the li~e. The salt of the amino acid [III] may be an alkali salt, an alkaline earth metal salt, an ammonium salt, a salt with an organic base such as trimethylamine, dicyclohexylamine or the like.
The coupling reaction may be carried out in the presence of a base such as alkali metal bicarbonate, trialkyl-amine, ~,~-dialkylbenzylamine or pyridine, usually in an inert solvent. Examples of the solvent are acetone, dioxane, aceto-nitrile, chloroform, methylene chloride, ethylene chloride, ~ -tetrahydrofuran, ethyl acetate, dimethylformamide, pyridine, etc.
Among them, hydrophilic solvents may be used in a mixture with water~ When the base or the condensing agent is in liquid, it can be used al~o as a solvent. The reaction temperature is not restrictive, and the reaction is usually carried out under cool-ing or at room temperature.
Depending on its kind, the amino protective group in the protected amino group represented by the symbol X may be removed in the course of the coupling reaction or the post~
treatment~
When the reaction product has a protected amino ~495~2 : ~
group, the amino protective group rnay be eliminated therefrom, when desired, by applying a suitable procedure aæ mentioned below.
lhe elimination of the amino protective group may be accomplished by a conventional procedure such as decomposi-tion with an acid or ca~alytic reduction, which is appropriately selected depending on the kind of the amino protective group.
One of the typical elimination procedures is conducted by treatment with an acid which is usually applied to sub-stituted or unsubsti-tuted benzyloxycar-bonyl, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted aralkoxycarbon~l, adamantyloxycarbonyl, trityl, substituted phenylthio, substituted aralkylidene, substituted alkylidene, ~ubstituted cycloalkylidene or the like. As the acid, there may be used various ones, and particularly preferred is the one as can be readily distilled out under reduced pressure (e.g. ~ormic acid, trifluoroacetic acid). The elimination ~s sometimes effected in an inert solvent, usually in a water-miscible organic solvent or water or their rnixture.
Another typical procedure is catalytic reduction which is ordinarily applied to substituted or unsubstituted benzyloxycarbonyl, 2-pyridylmethoxycarbonyl, etc. The most frequently used catalyst is a palladium catalyst but any other catalyst may be used.
Among other typical procedures, there are included treatment with water applicable to trifluoroacetyl and treat-ment with a heavy metal (e.g. copper, zinc) applicable to haloalkoxycarbonyl and 8-quinolyloxycarbonyl.
The elimination of the amino protective group may be carried out with or without previous isolation or purifica-5~2 tion of the reaction product from the reaction mixture, The cephalosporin compound [I: R2 = nitro, alkane-sulfonamido, dialkanesulfonamido, alkylaminosulfonamido, alkylureido or alkylthioureido R3 = heterocyelie-thio (in which the heteroeyelic group may be optionally substituted with alkyl)] may be also produced by reacting the corresponding cephalosporin compound [I: R2 ~ nitro, alkanesulfonamid 9, dialkanesulfonamido, alkylaminosulfonamido, alkylureido or alkylthioureido; R3 = alkanoyloxy] with a thiol compound of the formula:
R3 H [IV]
wherein R3 is a heterocyclic-thio group, in whieh the hetero-eyelic group may be substituted with lower alkyl, eorresponding to the symbol R3 in the objective compound or its alkali metal salt.
The term "alkanoyloxy" hereinabove used may be the one having 2 to 9 earbon atoms such as aeetyloxy, propionyloxy or butyryloxy.
The alkali metal salt of the thiol eompound [IV] may be a sodium sal~, a potassium salt or the like.
The above reaction may be earried out in an inert sol-vent sueh as water, aeetone, chloroform, nitrobenzene, dimethyl-formamide, methanol, ethanol or dimethylsulfoxide. Among these solvents, hydrophilie solvents may be used in a mixture with water. The reaetion is preferably effected at a pH around 7 or in a nearly neutral medium. When the starting eephalosporin compound [I: R2 = nitro, alkanesulfonamido, dialkanesulfon-amido, alkylaminosulfonamido, alkylureido or alkylthloureido, R3 = alkanoyloxy] or the thiol eompound [IV] is used in a free form, the reaction is favourably eonducted in the presenee of .:

5~2 a base such as an alkali metal hydroxide, an alkali metal carbonate, an alkali metal bicarbonate, trialkylamine or pyridine base. The reaction temperature is not limitative, and the reaction is ordinarily performed at room temperature or under warming. The reaction product can be isolated from the reaction mixture by any conventional procedure.
When the reaction product is protected at the amino group, the protective group may be eliminated by applying an appropriate procedure as mentioned above.
In the above processes, any precipitate which forms -during the reaction may be separated from the reaction mixture by methods commonly used for this purpose, and the resulting reaction product may be subjected to routinely used purifica-tion procedures, for instance, to recrystallization from an appropriate solvent or a mixture of such solvents.
All the reactants to be employed in the above processes are commercially available or can be prepared by conventional methods well known to the art or by a variety of analogous methods thereto~
Among the reactants, the amino acid [III: R2 = alkane-sulfonamido, dialkanesulfonamido, alkylaminosulfonamido, alkyl-ureido or alkylthioureido], i~e. the amino acid of the formula:

l~CH-COOH [III ' ]
R4-Y--N~

wherein R4 is alkyl, R5 is hydxogen or alkanesulfonyl, Y is -S02-, -~HS02-, ~HCO- or -NHCS- and Rl and X are each as defined above, provided that R5 is hydrogen when Y is -NHS02-, ~HCO- or " '.-'.,i!,. .
~04~S~Z

-NHCS-, is novel and can be produced, for instance, by reacting a compound of the formula:

~ COOH [Vj wherein Rl is as defined above in a free or protected form on the earboxyl group and/or the a-amino group with an aeylat-ing agent of the formula:
4 ~VI~
wherein ~ is the residue of an aeid and R4 and Y are each as defined above.
`~ When the a-amino group is protected, one or two hydrogen atoms therein may be replaeed, for instanee, by aeyl sueh as substituted or unsubstituted ben2yloxycarbonyl (e.g.
benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxy~
earbonyl, 4-methoxybenzyloxycarbonyl, ~,4-dimethoxybenzyloxy- --;earbonyl, 4-~phenylaæo)benzyloxycarbonyl, 4-(4-methoxyphenyl-azo)benzyloxyearbonyl), sub~tituted or unsubstituted alkoxy-earbonyl or eycloalkoxycarbonyl (e.g. -t-butoxycarbonyl, t-pentyloxycarbonyl, isopropoxyearbonyl, diphenylmethoxycarbonyl,

2-pyridylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2,2,2-tribromoethoxycarbonyl, l-cyclopropylethoxycarbonyl, ~-iodo-propoxycarbonyl, 2-furfuryloxycarbonyl, l-adamantyloxycarbonyl), (heteroeyclic ring)oxycarbonyl (e.g. 8-quinolyloxyearbonyl) or substituted aikanoyl (e.g. trifluoroacetyl); trityl; trialkyl-silyl (e.g. trimethylsilyl, triethylsilyl); substituted phenyl-thio (e.g. 2-nitrophenylthio, 2,4-dinitrophenylthio), ete.
The a-amino group may be also protected in the form of a Schiff base and, in sueh case, the two hydrogen atoms therein are ~'~ ' . ' ' .

~951~2 replaced by sub~tituted alkylidene (e.g. 2-hydroxybenzylidene, 2-hydroxy-5-chlorobenzylidene, 2-hydroxy-1-naphthylmethylene,

3-hydroxy-~-pyridylmethylene, 1-methoxycarbonyl-2-propylidene, l-ethoxycarbonyl-2 propylidene, 3-ethoxycarbonyl-2-butylidene, l-acetyl-2~propylidene, 1-propionyl-2-propylidene, l-benzoyl-2-propylidene, 1,3-bis(ethoxycarbonyl)-2-propylidene, 1,3-bis-(ethoxycarbonyl)-2-propylidene, 1-(N-methylcarbamoyl)-2-pro-pylidene, l-(N,~-dimethylcarbamoyl)-2 propylidene, 1-[N-(2-methoxyphenyl)carbamoyl]-2-propylidene, 1-[N-(4-methoxyphenyl)-carbamoyl]-2-propylidene, 1-(N-phenylcarbamoyl)-2-propylidene, 2-ethoxycarbonylcyclopentylidene, 2-ethoxycarbonylcyclohexyli-dene, 2-acetoxycyclohexylidene, 3,3-dimethyl-5-oxocyclohexyli-dene), or the like. The ~-amino group may be further protected in the form of an acid addition salt, for instance, hydro- ~-chloride, hydrobromide, hydroiodide or the like.
The carboxyl group may be protected by any conventional protective group. The particularly preferred protected form on the carboxyl group is a silyl ester which is obtainable by the reaction with a silyl compound such as trialkylhalosilane, dialkylhalo~ilane, alkyltrihalosilane, hexaalkylcyclotri-silazane, octaalkylcyclotetrasilazane, trialkylsilylacetamide or bi~(trialkylsilyl)acetamide.
Further, the ~-amino group and th~ carboxyl group may be protected simultaneously, for instance, in a chelating form intervening a metal compound. Examples of the metal compound may be anyone which can form a chelating bond with the a-amino group and the carboxyl group such as organic and inorganic copper, cobalt, nickel and magnesium compounds~ More specifically, there may be exemplified cupric chloride, cupric bromide, cupric fluoride, copper nitrate, copper sulfate, copper , ~4gsoz borate, copper phosphate, copper cyanide, copper ~ormate, copper acetate, copper propionate, copper citrate, copper tartrate, copper benzoate, copper salicylate, etc.
In the acylating agent [VI], the residue of an acid represented by the symbol Z may be, for instance, the residue of an acid halide such as acid chloride or acid bromide, an acid anhydride, an acid ester or an acid azide.
The reaction is usually carried out in an inert solvent, Examples of the inert so~vent are acetone, dioxane, acetronitrile, chloroform, methylene chloride, ethylene chloride, tetrahydro-furan, ethyl acetate, dimethylformamide, pyridine, etc. When the solvent is water-miscible, it may be employed in the form of a mixture with water. Any particular limitation is not present on the reaction temperature but, in most cases, the reaction may be performed while cooling or at room temperature.
In case of the symbol Y in the amino acid ~III'] repre-senting -~HCO- or -NHCS-, there may be used as the acylating agent a compound of the formula:

R4-Y' [VI']
wherein Y' is isocyanato or thioisocyanato and R4 is as defined above in place of the compound [VI]. The reaction may be car-ried out in the similar manner to the case of using the compound [VI].
The amino acid [III' 3 may be also produced by the stepq of (1) reacting a compound of the formula:

~ ~ C~O tVII]

wherein Rl is as defined abo~e with the acylating agent [VI]

3L~4~5~Z
or [VI'], (2) xeacting the resulting product of the formula:
Rl ~ CH0 [VIII]
~- R4-Y-N

wherein Rl, R4, R5 and Y are each as defined above with hydrogen cyanide in the presence of ammonia and hydrolyzing the resultant product of the formulao R
~ fHCN [IX]

4 1 2 wherein Rl, R4, R5 and Y are each as defined above The reaction (1) can be readily accomplished by treat-ing the compound [VII] with the acylating agent [VI] or [VI'] in an inert solvent (e.g. water, dioxane, tetrahydrofuran, ether), if necessary, in the presence of a base (e~g pyridine, tri-ethylamine, diI~thylaniline), The starting compound CVII ~ is sometime~ availabl~ commercially in the polymerized form and may be as such ~ubjected to the reaction in the same manner as in case of using the same in the monomeric form.
The reaction (2) is the so-called "Strecker amino acid synthe~is" and may be carried out in a per se conventional manner. For instance, it can be carried out by treating the compound [VIII] with an alkali metal cyanide (e~g. sodium cyanide, potassium cyanide) and an ammonium halide (e.g. ammonium chlo-ride, ammonium bromide) in aqueous ammonia.
The hydrolysis (~) may be also carried out in a per ' . ~ , , : , ~95~Z
se conventional manner, for instance, by treatment o~ the compound [IX] with an acid (e.g. hydrochloric acid, nitric acid, sulfuric acid) in an aqueous medium. When the treatment is carried out in a relatively strong condition, for instance, using a higher concentration of the acid and/or at a higher temperature, the cyano group is hydrolyzed to a carboxyl group - in a single step. On the other hand, the treatment under a comparatively muld condition, for instance, using a lower con-centration of the acid and/or at a lower temperature, the cyano group is hydrolyzed only up to a cabamoyl group, whereby a compound of the following formula is produced as the main product: R
3~H-CoNH2 R4-Y N NH2 [X]

wherein Rl, R4, R5 and Y are each as clefined above. In the latter case, the carbamoyl group can be readily hydrolyzed to a carboxyl group by treatment under a relatively strong condition as stated above. Thu~, the hydrolysis may be accomplished in two steps.
The protective group in the thus prepared amino acid [III'~ may be, if any, eliminated by a per se conventional procedure with or without previous isolation or purification of the product.
The elimination of the protective group on the ~-amino group may be accomplished in the manner as hereinbefore illustrated.
When the ~-amino group is protected in the form of - 14 _ :

~L~495~2 an acid addition salt, the elimination may be performed by ~ treatment with an organic ba~e (e.g. trimethylamine, tri-ethylamine, N-methylpiperazine, N,N-dimethylaniline or pyri-dine) or an inorganic base (e.g. sodium hydroxide~ potassium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate or ammonium carbonate).
In case of the a-amino group is protected together with the carboxyl group in a chelating form, the elimination of the metal compound used for chelation may be accomplished in a conventional method, for instance, treatment with hydrogen sulfide or an ion exchange resin.
The elimination of the silyl ester to recover a carboxyl group can be accomplished quite easily in the presence -~
of water. For instance, the working up of the reaction mixture containing the amino acid [III'] wherein the carboxyl group is protected in the form of a silyl ester in a conventional manner in the presence of water results in easy elimination of the silyl group. Thus, any partic~lar operation for the elimination of the silyl group is usually not required.
When the carboxyl group is protected in any form other than a ~ilyl ester, the elimination of such pro-tective group may be accomplished in any conventional procedure, The above product cephalosporin rompound [I] may be converted into its pharmaceutically acceptable, substantially non-toxic salts by a per se conventional procedure, for instance, by reacting with an inorganic base such as an alkali metal hydroxide, an alkali metal bicarbonate or an alkali metal carbonate or an organic base. The preferred procedure for pre-paration of the salts consists in dissolving the cephalosporin '' ' ~ . :

... "' ~ :' . ~ ' ' ' : -~4~5~Z
compound [I] in the free form into ~ solvent wherein the salt is insoluble and then adding a solution of the base thereto, whereby the salt is precipitated from the reaction mixture.
The cephalosporin compounds [I] exhibit an excellent antimicrobial activity. For instance, the cephalosporin compound ~I: Rl = hydrogen R2 ~ mesylamino; R3 = hydrogen, X = amino, M =
hydrogen] (hereinafter referred to as "Compound A") has a higher antimicrobial potency than cephalexin as shown in Table 1.
Table 1 Minimal inhibitory concentration of Compound A and cephalexin on microorganisms determined by the two fold agar plate dilution method.
~ ,_ _ _ , _ _ _ Test organism Minimal inhibitory concentration (mcg/ml) Compound A Cephalexin _ . , ._ Staph. aureus Terashima 1205 25 Staph. aureus Smith 3.13 6.25 B. subtilis ATCC-6633 0.78 1.56 B. subtilis PCI-219 0.78 1.56 Klebsiella NCTC-418 3.13 6.25 Sal. typhi T-~87 3.13 6.25 Sal. typhi 0-901 3.13 6O25 Sal. para A 1015 6.25 12O5 Sal. typhimurium 1406 3.13 12,5 Sal. enteritidis 1891 3.13 6.25 ._ -- _ Further, tha cephalosporin compound [I~ can main-tain their antibiotic potency at a high level for a long period of time when orally administered. For instance, the serum levels of Compound A and cephalexin with elapse of time when 0 administered orally to mice and rats are shown in Table 2.

.. ~ . , . . . .' .. :
:. . - ~ . , -~04951~2 Table 2 Serum levels of Compound A and cephalexin in mice and rats.
. __ ~ l Animal Test Serum level (mcg/ml) & compound Dose 1 hr- 2 hrs 4 hrs 6 hrs .. ~.~ .. ___ .
Mouse (ICR) Compound A37.6 25.0 14.7 8.4 100 mg/kg Cephalexin 10.44.6 _ _ . , .. __ _ . _ ~ . _ _ .
Rat (SD) Compound A 49.844.8 41.1 39.0 00 mg/kg Cephalexin 23.112 3 6.6 5.9 As stated above, the cephalosporin compounds [I]
of this invention are useful as orally administrable, long acting antibiotics. For therapeutic administration the cephalo-sporin compounds [I~ are used in the form of pharmaceutical preparations which contain said compounds in admixture with a pharmaceutically accep~able organic or inorganic solid or liquid excipient suitable for oral, parenteral, intestinal or percutaneous administration. The pharmaceutical preparations may be in a solid form such as capsules, ~ablets or dragees, or in a liquid form such as solutions, suspensions or emulsions.
They may be also in any other form such as suppositories and ointments, If desired, there may be included in the above preparations auxiliary substances, stabilizing agents, wetting or emulsifying agent~, buffers and other commonly used additives.
While the dosage of the cephalosporin compounds [I~
will vary from and also depend upon the age and condition of the patient, an average single dose of about 100 mg., 250 mg., and 500 mg. has proved to be effective in treating diseases caused by bacterial infection. In general, amounts between 10 mg~ and about 1000 mg~ or even more may be administered.
The following Examples are given solely for the - . . . .: : .

~)495~Z
purpose of illustrating the pre~ent invention, not of limiting the same thereto, Example 1 D-~-t-Butoxycarbonyl-2-(3-methoxy-4-hydroxyphenyl)-glycine (2.97 g.) and triethylamine (1.01 g.) were added to methylene chloride (50 ml.), and the mixture was cooled at -10 to -15C. A solution of pivaloyl chloride (1.17 g.) in methylene chloride (5 ml.) was added thereto with stirring, and the re ulting mixture was stirred at 0 to -10C. for 2 hours. After cooling the resultant mixture to -20C, a solution of 2,2,2-trichloroethyl 7-amino-3-methyl-3-cephem-4-carboxylate hydrochloride(3.34 g.) and 2,6-lutidine (1.94 g.
in methylene chloride (30 ml.) was added thereto all at once. Stirring was continued at -lO~C for 1.5 hours and at room temperature for an hour. After removal of the solvent from the reaction mixture under reduced pressure, ethyl acetate (80 ml.) and 5% sulfuric acid (80 ml.) were added to the residue, and the mixture was shaken. The ethyl acetate layer was separated out, washed in tuxn with 5% sulfuric acid (40 ml.), an aqueous saturated solution of sodium chloride, an aqueous saturated solution of sodium bicarbonate (40 ml.) and an aqueous saturated solution of sodium chloride, dried over magnesium ulfate and then treated with activated charcoal. Removing the solvent from the solution under re-duced pressure, the residue was pulverized with diisopropyl ether to give powder (4.86 g.). The powder was purified by column chromatography on silica gel (120 g.) using a mixture of benzene and acetone (5 : 1) as an eluent to give 2,2,2-tri- -chloroethyl 7-~D-N-t-buto~cycarbonyl-2-(3-methoxy-4-hydroxy-phenyl~glycinamido]-3-methyl-3~cephem-4-carboxylate (3.58 g.).

' - 18 _ . . . . . . . . .

- 1~415t5~2 I~Ro (Nujol)v cm 1 3370, 1788, 1683, 1740. M.M.R. ~CDC13) ~ ppm: 1.41 (9H,s), 2.17 (3H,s), 3.30 (2H,AB-q), 3.83 (3H,s), 4.88 (2H,AB-q), 4.94 (LH,d,J=4Hz), 5.15 (IH,d,J=6Hz), 5.6-6.0 (3H,m), 6.8-7.1 (3H,m). Nujol is a trademark. -~2a~ ~
2,2,2-Trichloroethyl 7-[D-N-t-butoxycarbonyl-2-(3-methoxy-4-hydroxyphenyl)glycinamido]-3-methyl-3-cephem~4-carboxylate (3.25 g.), obtained as in Example 1, zinc powder (3 g.) and glacial acetic acid (3 mL) were added to dimethyl-formarnide (30 ml.~, and the resulting mixture was stirredunder ice-cooling for an hour. Filtering the reaction mixture, the filtrate was added to a mixture of ethyl acetate (60 ml.) and 3% aqueous hydrochloric acid (60 ml.) with stirring.
The resultant mixture was thoroughly shaken, and the ethyl acetate layer was separated out, washed with an aqueous satu-rated solution of sodium chloride, dried over magnesium sul-fate and concentrated under reduced pressure to give 7-~D-N-t-butoxycarbonyl-2-(3-methoxy-4-hydroxyphenyl)glycinamido]-3-methyl-3-cephem~4-carboxylic acid ~2.31 g.). I.R. (Nujol) ~ cm : 3350, 1780, 1700 1518. N.M.R. (DMSo d6) ~ ppm:
1.3~3 (9H,s), 1.97 (3H,s), 3~40 (2H,ABq), 3.78 (3H,s), 4.99 (LH,d,J=4.5Hz), 5.21 (IH,d,J=8Hz), 5.62 (lH,dd,J=4Hz,7Hz), 6.6-702 (4H,m), 8.93 (LE,d,J=9Hz).
Example 3 7-[D-N-t-Butoxycarbonyl-2-(3-methoxy-4-hydroxy-phenyl)glycinamido3-3-methyl-3-cephem,4-carboxylic acid (2.43 g.), obtained as in Examples 1 and 2, was added to formic acid (20 ml.~, and the resulting mixture was stirred at room temperature for 3 hoursO After the formic acid was removed from the reaction mixture under reduced pressure, ' -~

-- 19 _ .. . .
. . , , , , , . ~ .
', . ' .

~49SOz the residue was pulverized with ethex, and 5% aqueous acetonitrile (20 ml.~ was added thereto. The resultant mixture was stirred for 30 minutes and then filtered to give pale yellow powder. The powder was washed with acetonitrile and ether successively and dried to give 7-~D-2-(3-methoxy-4-hydroxyphenyl)glycinamido]-3-methyl-3-cephem~4-carboxylic ; acid. M.P. 183 to 185C (decomp,). I.R~ (Nujol) ~cm 1 3500, 2500, 1775, 1697, 1626, 1570. N.M.R. (D20 + DCl) ~
ppm: 2.10 (3H,s), 3~36 (2H,ABq), 3.93 (3H,s), 5.08 (lH,d,J=
4Hz), 5.32 (lH,s), 5~70 (IH,d,J=4Hz),7.0-7.3 (3H,m). U.V.

~max 237,3 m~, E = 274; APa64 phosphate buffer 266 n~, E = 193.
Example_4 DL-N-t-ButQxycarbonyl-2-(3-methoxy-4-hydrQxyphenyl)-glycine (2.97 g.) and triethylamine (1.11 g.) were added to tetrahydrofuran (25 ml.), and the resulting mixture was cooled to 0C. Adding a solution of pivaloyl chloride (1.36 gO) in tetrahydrofuran (5 ml.) thereto, the resultant mixture was stirred at 0C for 10 minutes. A solution of 7-amino-3-methyl-3-cephem~4-carboxylic acid (2.14 g.) and triethylamine (1.01 g ) in 5~/O aqueous acetone (30 ml.) was added to the mixture all at once at 0C and stirred at room temperature for 1.5 hours. Removing the organic solvent from the reaction mixture under reduced pressure, an aqueous saturated solution of sodium bicarbonate (30 ml.) and ethyl acetate (S0 ml.) were added to the residue. The aqueous layer was adjusted to pH
2 with l~/o aqueous hydrochloric acid, and then the ethyl acetate layer was separated. The aqueous layer was extracted with ethyl acetate (50 ml.) twice, and this extract was com~
bined with the previously obtained ethyl acetate layer.

_ 20 -.. ,, . . . . ; :

1~4~5~Z
After drying over magnesium sulfate, the ethyl acetate was removed under reduced pressure. The residue was pulverized with diisopropyl ether to give 7 [DL-N-t-butoxycarbonyl-2-(3-methoxy-4-hydroxyphenyl)glycinamido~-3-methyl-3-cephem~
4-carboxylic acid (3 g.). M.P. 127 to 130C (decomp.)O
I.R. (Nujol) ~cm 1 3350, 2650, 1770, 1718, 1695, 1680 1518, 1275, 1250, 1160, 1055, 1032, 860, 782, 720.
ExamPle 5 7-~DL-N-t-Butoxycarbonyl-2-(3-methoxy-4-hydro~y-phenyl)glycinamido]-3-methyl-3-cephem~4-carboxylic acid (200 g.), obtained as in Example 4, was dissolved in formic acid (20 ml.), and the resulting solution was stirred at 40C for 4 hours. Removing the formic acid from the solution under reduced pressure, the residue was pulverized with acetonitrile.
The powder was added to l~/o aqueous acetonitrile, triturated with a gla~s-bar and then filtered. Thus obtained powder was washed with acetonitrile and ether successively and dried to give 7-[DL-2-(3-methoxy-4-hydroxyphenyl)glycinamido]-3-methyl-3-cephem-4-carboxylic acid (1.10 g.). M.P. 193 to 197~C (decomp.). I.R. (Nujol) ~cm 1; 1760, 1690. U.V.
~pH6.~ phosphate buffer 237 ~ E = 275; ~pH6.4 phosphate buffer 265.5 ~1, E = 189.
Example 6 :
D-~-t-Butoxycarbonyl-2-(3-nitro-4-hydroxyphenyl)-glycine (5 g.) was dis~oIved in methylene chloride (100 ml.), and 2,6-lutidine (1.9 g.) was added thereto. The resulting mixture was cooled at -10 to -15C, pivaloyl chloride ~1.9 g.3 was dropwise added thereto, and the mixture was stirred at -15C for 3 hours. To this mixture, there was added all at once at -5 to -10C a solution prepared by adding N,N-bis--10~9S~Z
(trimethylsilyl)acetam~de (10 ml.) ~o a suspension o 7-amino-3-methyl-3-cephem 4-carboxylic acid (3.45 g.) in methylene chloride (50 ml.) and stirring at room temperature for 3 hours. Thus obtained rn~xture was stirred at -10 to -15C for 3 hours, and the solvent was removed under reduced pressure.
Ethyl acetate and 5~/3 sulfuric acid were added to the residue, and the ethyl acetate layer was separated out. To the ethyl acetate extract, an a~ueous solution of sodium bicarbonate was added, and the aqueous layer was separated out. The 10 aqueous solution was acidified with sulfuric acid and back-extracted with ethyl acetate, The ethyl acetate extract was washed with water and an aqueous saturated solution of sodium chloride successively, dried over magnesium sulfate and con-centrated under reduced pressure to give 7-[D-N-t-butoxy-carbonyl-2-(3-nitro-4-hydroxyphenyl)glycinamido~-3-methyl-3-cephem-4-carboxylic acid (6.0 g.) as powder. M.P. 150 to 155 C (decomp.). I.R. (Nujol) ~Jcm : 1755, 1700, 1690, -1670. NoM~R~ (DMSO-d6) o ppm: 1.40 (9H,s), 2.03 (3H,s), 3.38 (2H,broad s), 4.99 (lH,d,J=4.5Hz), 5.32 (lH,d,J=8.5Hz),

5,60 (H,dd,J-4.5,800Hz), 7.10 (lH,d,J=8Hz), 7.2-7.55 (lH,m), 7.65 (lH,dd,J=2.0,8.0Hz), 8.05 (~H,d,J=2Hz), 9.13 (lH,d,J=8Hz).
Example 7 7-[D-N-t-Butoxycarbonyl-2-(3-nitro-4-hydro~yphenyl)-glycinamido]-3-methyl-3-cephem-4-carbo~ylic acid (5 g.), obtained as in Example 6, was dissolved in formic acid (40 ml.), and the resulting solution was qtirred at room temperature for 4.5 hours. Removing the solvent under reduced pressure, the residue was pulverized with ether and filtered. Thus obtained powder was washed with e her, ethyl acetate and acetonitrile _ 22 --::

-~(~49~0Z
successively and then suspended in acetonitrile (40 ml.).
Water (4 ml.~ was dropwise added to the suspension under stirring, and the resultant mixture was stirred at room temperature overnight. The precipitated crystals were filtered, washed with acetonitrile and dried to give 7-[D-2-(3-nitro-4-hydroxyphenyl~lycinamido]-3-methyl-3-cephem~4-carboxylic acid (2.64 g.). M.P. 170 to 172C (decomp.). I.R. (Nujol) V cm 1 3200, 1763, 1702, 1628, 1546. N.M.R. ~D20 + DCl ~ ppm: 2.15 (3H,s), 3.42 (2H,d,J=~H~), 5.13 (lH,d,J=4.5Hz), 5.54 (LH,9), 5.70 (lH,d,J-4.5Hz), 7.38 (LH,d,J=8.5Hz), 7.91 (LH,dd,J=2.5,8.5Hz), 8.45 (IH,d,J=2.5Hz). U.V. ~PHa6 4 phos phate buffer 2~2 ml, E = 434.

Example 8 To a solution of D-N-t-butoxycarbonyl-2-(3-nitro-4-hydroxyphenyl)glycine (1 g.) in methylene chloride (20 ml.), there was added 2,6-lutidine (0.38 g.), and the resulting mix-ture was cooled at -15 to -20C, A solution of pivaloyl chloride (385 mg.) in methylene chloride (1 ml.) was dropwise added thereto, and the mixture was stirred at -20C for 2 houxs.
To this mixture, there was added at -20C all at once a solution prepared by adding 2,6-lutidine (0.64 g.) to a suspension of 2,2,2-trichloroethyl 7-amino-3-methyl-3-cephem-4-carboxylate p-toluenesulfonate (1.91 g.3 in methylene chloride (20 ml.) under ice-cooling and stirring for 15 minutes. The resultant mixture was ~tirred at -20C for 3.5 hours, the solvent was removed therefrom under xeduced pressure, ethyl acetate and water were added to the residue, and insoluble materials were collected by filtration and washed with ethyl acetate to give 2,2,2 trichloroethyl 7-[D-N-t-butoxycarbonyl-2-(3-nitro-4-hydroxyphenyl)glycinamido]-3-methyl-3-cephem-4-carboxylate , . , -~495~Z
(0.27 g.). The ethyl acetate layer after filtration o~ the product and the washings of ethyl acetate were combined, washed with 5% hydrochloric acid, an aqueou~ saturated solution of sodium bicarbonate and water in turn, dried over magnesium sulfate, and then concentrated under reduced pres-sure. The resultant gel-like residue was washed ether to give the same product as above (1.09 g.). Total yield, 1.36 g. IR. tNujol)~ cm 1 (3320, 3270, 1775, 1735, 1685, 1650, 1630. N.M.R. (DMS0-d6) ~ ppm: 1.40 (9H,s~, 2.10 (3H,s), 3.49 (2H,broad s), 5.05 (2~,AB-q), 5.07 tlH,d,J=
4.5Hz), 5.35 (lH,d,J=8Hz), 5.69 (IH,dd,J=4.5,8.0Hz), 7.10 (IH,d,J=8.5 Hz), 7.2-7.5 (lH,m), 7.63 (IH,dd,J=2.0,8.5Hz), 8.04 (LH,d,J=2Hz), 9.2 (LH,d,J=8Hz).
Exam~le 9 To a suspension of 2,2,2-trichloroethyl 7-amino-3-methyl-3-cephem~4-carboxylate hydrochloride (3820 mg.) in ab~olute methylene chloride (50 ml.), triethylamine (810 mg.) and N,N-dim~thylaniline (245 mg.) were added dropwise under ice-cooling with stirring, and stirring was continued at room ~emperature for 30 minutes. Separately, N-t-butoxycarbonyl-2-(3-m~sylaminophenyl)-D-glycine (3440 mg.), triethylamine (1010 mg.) and N,~-dimethylbenzylamine (4 drops) were added to absolute methylene chloride (50 ml.), and the mixture was stirred under dry ice-acetone cooling. A solution of ethyl chloroformate (1085 mg.) in absolute methylene chloride ~25 ml.) was dropwise added thereto at -25 to -30C in 10 minutes, and stirring was co~ducted at the same temperature for 15 minutes. To the result-ing mixture, the previously obtained mixture precooled at -15C
was added all at once. The resultant mixture was stirred at -25 to -30C for 4 hours and wa~hed with water, 3% hydrochloric 1~9L9502 acid and water in order. The organic layer was separated, washed with 3% aqueous solution of sodium bicarbonate and water in turn and then dried. The methylene chloride was removed under reduced pressure, the residue was dis~olved in ethyl acetate (10 ml.), and ether was added thereto. The precipitated cxystals were Eiltered to give 2,2,2-trichloroethyl 7- [D-N-t-butoxycarbonyl-2-(3-mesylaminophenyl)glycinamido]-3-methyl-3-cephe~[~4-carboxylate (4195 mg.3. M.P. 204 to 205C (decomp.).
The above obtained 2,2,2-trichloroethyl 7-[D-N-t-10 butoxycarbonyl-2-(3-mesylaminophenyl)glycinamido]-3-methyl-3-cephen~4-carboxylate (3985 r~ .) was dissolved in dimethylforlr~
amide (17 ml.). To the solution were added acetic acid (5.0 ml.) and zinc powder (3985 mg.) under ice cooling with stirring, and the mixture was 3tirred at the same temperature for 2 hours.
The insoluble material was collected by filtration and washed with dimethylformamide (3 ml.). The washings and the filtrate were conibined together and added to 50/0 hydrochloric acid (100 ml.) under ice cooling. Water (50 ml,) was added thereto, and the resultant mixture was extracted three times with ethyl 20 acetate (50 ml.)" The extracts were combined together, washed with water and back-extracted three times with 5% aqueous solution of sodium bicarbonate (50 ml.). The aqueous layer was acidified with hydrochloric acid and back-extracted again with ethyl acetate. The extract was washed with water, dried and concentrated under reduced pressure. The residue was dissolved in ethyl acetate (10 ml.) and allowed to stand at room tempera-ture for an hour, Ether waq added thereto, whereby 7- [D-N-t-butoxycarbonyl-2-(3-mesylaminophenyl)glycinamido]-3-methyl-3-cephem-4-carbo~ylic acid (2687 ng.) was obtained. M.P. 187 to 30 189C (decomp.).

_ 25 --~ ^\
~(~49soz Example 10 To a suspension of 2,2,2-trichloroethyl 7-amino-3-methyl-3-cephem~4-carboxylate hydrochloride (26.7 g.) in methyl-ene chloride (500 ml.), there were added successively a solution of triethylamine (7 g.) in methylene chloride (25 ml.) and a solution of 2,6-lutidine (2.14 g.) in methylene chloride (25 ml.) under ice cooling. N-t-Butoxycarbonyl-2-(3-mesylaminophenyl)-D-glycine (26.0 g.) and then dicyclohexylcarbodiimide (15 g.) were gradually added thereto, and the mixture was stirred under ice cooling for 1.5 hours. After removal of the insoluble material by filtration, the filtrate was washed four times with ice-cooled 5% hydrochloric acid (100 ml.), once with water, three times with l~/o aqueous solution of sodium bicarbonate and once with an aqueous saturated solution of sodium chloride and then dried over magnesium sulfate. The solution was treated with acti-vated charcoal, and the solvent was removed under reduced pressure. The residue was dissolvedin ethyl acetate (100 ml.) and allowed to stand at room temperature overnight. The pre-cipitated crystals were collected by filtration to give 2,2,2-trichloroethyl 7-[D-N-t-buto~ycarbonyl-2-(3-mesylaminophenyl)-glycinamido]-3-methyl-3-cephem~4-carboxylate (31.3 g.) as colorless plate3. M.P. 189 to 191C (decomp.).
The above obtained 2,2,2-trichloroethyl 7-[D-t-butoxycarbonyl-2-(3-mesylaminophenyl)glycinamido]-3-methyl-3- ;~
cephem-4-carboxylate (12.01 g.) was dissolved in dimethylform~
amide (40 ml.). Glacial acetic acid (15 ml.) and zinc powder (12 g.) were added to the solution under ice cooling, and the mixture was kept stirred at the same temperature for an hour.
After the reaction was over, the insoluble material was collected by filtration and washed with ethyl acetate. The filtrate and .. , , ,, . . : .
, ', ~495~2 the washings were added to 3% hydrochloric acid (300 ml.) under ice cooling. The mixture was extracted three times with ethyl acetate (150 ml.). The ethyl acetate extract was washed with water and then back-extracted three times with 5% aqueous solu-tion of sodium bicarbonate (150 ml.). The aqueous solution was washed with ethyl acetate and adjusted to pH 2 with l~/o hydro-chloric acid. The precipitated substance was extracted three times with ethyl acetate (lS0 ml.). The extract was washed with water and dried over magnesium sulfate, and the solvent was removed under reduced pressure. The resîdue was washed with ethyl acetate and ether to give 7-[D-N-t-buto~ycarbonyl-2-(3-mesyl-aminophenyl)glycinamido]-3-methyl-3-cephem-4-carboxylic acid ;
(8.38 g.). M.P. 188 to 189C (decomp.). I.R.(Nujol)~ cm 1 3330, 3290, 3240, 1769, 1720, 1690, 1668, 1524, 1308, 1220, 1146, 912, 890, 772. ~.M.R. (DMS0-d6) ~ ppm: 1.40 (9H,s), 2.00 (3H,s), 2.99 (3H,s), 3.37 (2H,broad s), 4.96 (lH,d,J=
404~z), 5.30 (LH,hroad d,J=8Hz); 5.63 (lH,d,d,J=4,7Hz), 6090-7.50 (4H,m).
The above obtained 7-[D-N-t--butoxycarbonyl-2-(3-20 mesylamino)glycinamido]-3-methyl-3-cephem~4-carbQxylic acid (8.28 g.) was added to formlc acid (140 ml.) under ice cooling, and the mixture was stirred at room temperature for 1.5 hours.
Removing the formic acid at 35C under reduced pressure, the residue was dissolved in 5% hydrochloric acid (30 ml.)~ The solution was washed with ethyl acetate (20 ml.), treated with activated charcoal and then adjusted to pH 3 with 10% aqueous solution of sodium hydroxide. The precipitated crystals were collected by filtration, washed with water and dried to give 7-[D-(3-mesylamir,ophenyl)glycinamido]-3-methyl-3-cephem~4-carbQxyliC
30 acid ~6.18 g.). M.P. 199 to 199.5C (decomp.). [~]D = +131 - , . . ~ . :

1~49S~2 (0.1~ HC1, C=l). I.R. (Nu~ol)~ cm 1 3510, 3510, 3330, 1750, 1700, 1600, 1526, 1380, 1366, 1328. N.M.R. (D20 ~
DCl) ~ ppm: 2.08 (3H,s), 3.19 (3H,s), 3.21, 3.44 (2H,AB-q,) J=18Hz), 5.04 (IH,d,J=5Hz), 5O37 (lH,s), 5.67 (IH,d,J=5Hz), 7.4-7,67 (4H,m), U.v.~PH6 4 phosphate buffer 263 5 172 ~pH6.4 phosphate buffer 228 m~ E 299 Example 11 A solution of ethyl chloroformate (1.32 g.) in methylene chloride (20 ml.) was cooled to -10C, and a solution of ~-t-butoxycarbonyl-2-(3-ethanesulfonamidophenyl)-D-glycine (4.4 g.) and triethylamine (1.22 g.) in methylene chloride (20 ml.) and N,N-dimethylbenzylamine (2 drops) was dropwise added thereto in 10 minutes. The resulting mixture was stirred at room tempera-ture for an hour. Separately, 2,2,2-trichloroethyl 7-amino-3-methyl-3-cephem-4-carboxylate (3.82 g.), triethylamine (0.9 g.) and N,N-bis(trimethylsilyl)acetamide (0.12 g.) were dissolved in methylene chloride (40 ml.). The solution was dropwise added to the above obtained mixture at -15C in 10 mQnutes. The resultant solution was kPpt stirred at the same temperature for 2 hours, washed twice with 2% hydrochloric acid, an aqueous solution of sodium ~icarbonate and an aqueous saturated solution of sodium chloride and dried over magnesium sul~ate. Removing the ~olvent under reduced pressure, the residue was pulverized with a small amount of ethanol to give 2,2,2-trichloroethyl 7-[D-~-t-butoxycarbonyl-2-(3-ethanesulfonamidophenyl)glycinamido]-3-methyl-3-cephem-4-carboxylate (4.2 g.) as colorless crystals.
M.P. 117C. I~Ro (Nujol) v cm 1 3400, 3230, 1762, 1735, 1690~ N.M.R. (CDC13) ~ ppm: 1.28 (3H,t,J=7Hz), 1.38 (9E,s), 2.16 (3H,s), 2.75-3.50 (4H,m), 4.82, 4,95 (2H,AB-q,J=12Hz), 4.95 (lH,d,J=5Hz), 5.24 (lH,d,J=6Hz), 5.81 (IH,d,d,J=5,8Hz), ~049S~
7.25 (4H,s1.
The above obtained 2,2,2-trichloroethyl 7-CD-2~-t-butoxycarbonylamino-2--(3-ethanesulfonamidophenyl)glycinamido]-3-methyl-3-cephem-4 carboxylate (4.2 g.) was dissolved in a mixture of dimethylformamide (15 ml.) and acetic acid (45 ml~).
Zinc powder (3.6 g.) was added to the solution under ice cooling, and the mixture was stirred for 2 hours. After the zinc powder was filtered off, the iltrate was poured into a mixture of 2%
hydrochloric acid (40 ml.) and ethyl acetate (40 ml.), and the ethyl acetate layer was separated out. The aqueous layer was extracted further with ethyl acetate (20 ml.). The ethyl acetate layer and the ethyl acetate extract were con~ined together, washed with 2% hydrochloric acid (20 ml.) and an aqueous saturated solution of sodium chloride (20 ml.) in turn and dried over mag-nesium sulfateO The solvent was removed under reduced pressure, and the residue was washed with diisopropyl ether to give 7-~D-~-t-butoxycarbonyl-2-(3-ethanesulfonamidophenyl)glycinamido]-3-methyl-3-cephe~4-carboxylic acid (3.2 g.) as an oil. I.R.
(film) ~ cm 1 3300, 1765, 1700. ~.M.R. (CD30D) ~ ppm: - ;~
1.22 (3H,t,J=7.5EIz), 2.09 (3H,s), 2.75-3.75 (4H,m), 4.96 (lH, d,J=4,5Hz), 5.26 (lH,s), S.67 (lH,d,J=4.5Hz), 7.25 (4H,s).
A solution of the above obtained 7- [D-~l-t-butoxycarbonyl-2-(3-ethanesulfonamidophenyl)glycinamido~-3-methyl-3-cephem-4-carboxylic acid (3.1 g.~ in formic acid (15 ml.) was stirred at room temperature for 2 hours. The formic acid was removed from the solution at room temperature under reduced pressureO The residue was pulverized with ether and filtered. The resultant powder was added to 10~/o aqueous acetonitrile (20 ml.) and stirred under ice cooling for an hour. The precipitated crystals were collected by filtration to give 7-[D-2-(3-ethanesulfonamido-- 29 ~

~4~Z
phenyl)glycinamido]-3-methyl-3-cephem-4-carboxylic acid (1.7 g.) as white crystals. M.P. 179 to 182C (decomp.). I.R. (Nujol) V cm : 1770, 1695, 1600. ~.M.R. (D20 ~ Na~CO3) ~ ppm: 1.29 (3H,t,J=7.5Hz), 1.90 (3H,s), 2.75-3.67 (~H,m), 4.96 (lH,d, J=4.5Hz), 5.25 (lH,s), 5.59 (lH,d,J=4.5Hz), 7.34 (4H,s).
Example 12 2,2,2-Trichloroethyl 7-[D-N-t-butoxycarbonyl-2-(3-mesylaminophenyl)glycinamido]-3-methyl-3-cephem-4-carboxylate (1.0 g.) was added to ice-cooled formic acid (20 ml.), and the mixture was stirred at room temperature for 2 hours. Removing the formic acid under reduced pressure, water was added to the oily residue. The mixture was adjusted to pH 8 to 9 with an aqueous saturated solution of sodium bicarbonate under ice cooling. The precipitated crystals were collected by filtration, ;
washed with water and then dried over phosphorus pentoxide to ~-give 2,2,2-trichloroethyl 7-[D-2-(3-mesylaminophenyl)glycinamido]-3-methyl-3-cephem-4-carboxylate (0.78 g.). M.P. 107 to 110C
(decomp.). I.R. (~ujol)~ cm 1 3350, 1785, 1740, 1685.
Exam~le 13 ~0 To a suspension of 2,2,2-trichloroethyl 7-amino-3-methyl-3-cephem-4-carboxylate hydrochloride (5.9 g.) in methylene chloride (100 ml.), there were added a solution of triethylamine (1.55 g.) in methylene chloride (10 ml.) and a solution of 2,6-lutidine (0.16 g.) in methylene chloride (10 ml.). ~-t-Butoxycarbonyl-2-(4-mesylaminophenyl)-D-glycine (5.8 g.) and dicyclohexylcarbodi-irnide (3.3 g.) were added to the resultant solution under ice coolingO The mixture was kept stirred at the same temperature for 3 hours and filtered. IThe filtrate was concentrated under reduced pressure, and ethyl acetate (200 ml.) was added to the residue. The mixture was washed with 5 % hydrochloric acid, ~049SOZ

water, an aqueous saturated solution of sodium bicarbonate and water in order, dried and then concentrated to give 2,2,2-trichloroethyl 7-[D-N-t-butoxycarbonyl-2-(4-mesylaminophenyl)-glycinamido]-3-methyl-3-c~phem-4-carboxylate (10.83 g.). M.P.
129 to 136C (decomp.). I.R. (Nujol) ~ cm 1 3290, 1770, 1680. N.M.R. (CDC13) ~ ppm: 1.43 (9H,s), 2.20 (3H,s), 3.32 (2H,AB-~), 4.8-5.0 (3H,m), 5.2-6.0 (3H,m), 7.0-7.7 (4H,m), 7.9 (lH, broad s).
Acetic acid (12.5 ml.) and zinc powder (lO g.) were added to a solution of 2,2,2-trichloroethyl 7-~D-N-t-~utoxy-carbonyl-2-(4-mesylaminophenyl)glycinamido]-3-methyl-3-cephem- -4-carboxylate (10 g.) in dimethylformamide (33 ml.) under ice cooling. The resultant mixture was stirred at the same temper-ature for an hour and filtered. The filtrate was added to a mixture of 5 % hydrochloric acid (100 ml.), ice water (50 ml.) and ethyl acetate (100 m-l.) and extracted three times with ethyl acetate (100 ml.). The extract was back-extracted twice with 5 /O aqueous solution of sodium bicarbonate (100 ml.). The aqueous layer was washed with ethyl acetate, adjusted to pH 2 with 10 % hydrochloric acid and extracted with ethyl acetate.
The ethyl acetate extFact was washed with water and dried, and then the solvent was removed under reduce pressure to give 7 [D-N-t-butoxycarbonyl-2-(4-mesylaminophenyl)glycinamido]-3-methyl-3-cephem-4-carboxylic acid (7.8 g.). M.P. 180 to 200C
(decomp.). I.R. (Nujol) V cm : 3300, 1770, 1710 (shoulder), 169S, 1680. N.M.R. (~MS0-d6) ~ ppm: 1.40 (9H,s), 2.02 (3H,s), 2.95 (3H,s), 3.2-3.7 (2H,m), 4.97 (lH,d), 5.2-5.8 (2H,m), 7.30 (4H,AB-q).
7-[~-N-t-Butoxycarbonyl-2-(4-mesylaminophenyl)glycin-amido]-3-methyl-3-cephem 4-carboxylic acid (4.6 g.) was added ;..., ;-~4950Z
to formic acid (70 ml.) under ice cooling. The resultant mix-ture was kepk stirred at the same temperature for 2 hours and concentrated under reduced pressure. To the residue, there was added water, and the resultant mixture was washed with ethyl acetate, adjusted to pH 6 with an aqueous solutian of sodium bicarbonate, concentrated to one half of its initial volume, ad-sorbed on a resin adsorbent (trade mark "Amberlite XAD-2") (460 g.), which was prewashed with methanoi and water, and then eluted with ;
water and methanol. The eluate was concentrated, and the separated crystals were collected by filtration and washed with methanol to give 7-~D-2-(4-mesylaminophenyl)glycinamido]-3-methyl-3-cephem-4-carboxylic acid (2.1 g.). M.P. 205 to 207C (decomp.). I.R. (Nujol) ~ cm 1 3175, 1760, 1670.
N.M.R. (D20) ~ ppm: 1.80 (3H,s), 3.05 (3H,s), 3.15 (2H,AB~q), 4.85 (lH,d), 5.12 (lH,s), 5.54 (lH,d), 7.35 (4H,AB-q).
Example 14 , ~-t-Butoxycarbonyl-2-(3-mesylaminophenyl)-D-glycine (2.066 g.), triethylamine (0.606 g.) and N,~-dimethylbenzylamine (15 ml.) were added to tetrahydrofuran (20 ml.),and the mixture was cooled to -10 to -12C. A solution of isobutyl chloro-formate (0.820 g.) in tetrahydrofuran (10 ml.) was dropwise added thereto at the same temperature in 2 minutes, and the resultant mixture was kept stirred at the same temperature for 30 minutes.
Separately, 7-amino-3-methyl-3-cephem-4-carboxylic acid (I.070 g.) and triethylamine (0.581 g.) were added to 50 % aqueous tetra-hydrofuran (30 ml.) under ice cooling, and the resulting solution - was added all at once to the above obtained mixture cooled to -6C. Thus obtained,mixture was stirred under ice cooling for an hour and additionally at room temperature for 2 hours, after which the tetrahydrofuran was removed under reduced pressure.

: .

~L~495C1 2 -An aqueous satur~ted solution of sodium bicarbonate (15 ml.) was added to the residue, and the resultant mixture was washed twice with ethyl acetate (10 ml.). The washings were extracted with an aqueous saturated solution of sodium bicarbonate (10 ml.).
The aqueous extract was combined with the above obtained aqueous solution, and ethyl acetate (30 ml.) was added thereto. The resulting mixture was adjusted to pH 2 with 10 o/O hydrochloric acid and shaken thoroughly. The insoluble material was filtered ~
off. The ethyl acetate layer was separated out, the aqueous layer was extracted twice with ethyl acetate (20 ml.), and the ethyl acetate extract and the said ethyl acetate layer were combined together. The mixture was washed with water (10 ml.) and an aqueous saturated solution of sodium chloride (10 ml.), dried over magnesium sulfate and treated with activated charcoal~
- Removal of the solvent gave p~sty residue (3.47 g.). The residue (3.42 g.) was added to ether (30 ml.), and the mixture was stirred at room temperature overnight. The separated crystals were collected by filtration, washed with ether and dried to give 7-~D-N-t-butoxycarbonyl-3-(3-mesylaminophenyl)glycinamido~-3-methyl-3-cephem-4-carboxylic acid (2.326 g.). M.P. 174C
~decomp.). ~]D = 57 (methanol, C ~l).
Example lS
~-t-Butoxyearbonyl-2-(3-mesylaminophenyl)-D-glycine (3.44 g.) and triethylamine (1.01 g.) were dissolved in methylene chloride (~5 ml.~, and the solution was dropwise added to a solution of isobutyl chloroformate (1.36 g.) in methylene chloride (35 ml.) at -10 to -15C in 5 minutes and stirxed at the same temperature for 15 minutes. Separately, N,0-bistrimethyl~ilyl acetamide (3.5 g.~ was dissolved in a suspension of 7-amino-3-(5-methyl-1,3,4-thiadiazol-2-yl)-~o~9s~ :
thiometh~l-3-cephe~-4 carboxylic acid (3.44 g.) in methylene chloride (30 ml.), and the resulting solution was drop~,rise added to the above prepared solution of mixed anhydride at -15C and stirred at the same temperature for 1.5 hours and at 10C for 3 hours. The resultant mixture was washed with 5 hydrochloric acid and water, dried and then the solvent ~as removed. The oily residue was purified by column chrcmatography on silica gel (eluent: chloroform) to give 7-[D-N-t-butoxy-carbonyl-2-(3 mesylaminophenyl)glycinamido]-3-(5-methyl-1,3,4-thiadiazol-2-yl)thiomethyl-3-cephem-4-carboxylic acid (3.8 g.) as an oil. I.R. (film) v cm 1 33oo, 1780, 1725, 1685, 1670.
The thus obtained 7-[~-N-t-butoxycarbonyl-2-(3-methylaminophenyl)glycinamido]-3-(5-methyl-1,3,4-thiadiazol-2-yl)thiomethyl-3-cephem-4-carboxylic acid (2.23 g.) was dissolved in ~ormic acid (35 ml.) and stirred at 18 to 20C
for 4 hours. The resultant mixture was concentrated under reduced pressure, pulverized with ethyl acetate and then filtered to give 7-[D-2-(3-mesylaminophenyl)glycinamido]-3-(5 methyl-1,3 9 4-thiadiazol-2-yl)thiomethyl-3-cephem-4-carboxylic acid (1.95 g.). The product was added to acetone (30 ml.), ~tirred at 15 to 200a for an hour and allowed to stand. After removal of the supernatant solution by decantation, acetone (30 ml.) was added to the residue and stirred at 15 to 20C for 3 hours.
The precipitate was collected by filtration and washed with ,~cetone and ether to give the objective compound as pale yellow powder. I.R. (Nujol) v cm 1 1765, 1690, 1605.
N.M.R. (D20-DCl) ~ ppm: 2.97 (3H,s), 3.01 (3H,s), 3.38, 3.78 (2H,AB-q,J-18Hz), 4.26) 4.47 (2H,AB-q,J=14Hz), 5.12 (lH,d, J=4Hz), 5.35 (lH,s), 5.71 (~f,d,J=4Hz)-xample 16 :.

-: . . ~ . .. . . .

9~0Z
(1) Dicyclohexylcarbodiimide (2.0 g.) ~las added under ice cooling with stirring to a solution of N-t-butoxy-carbonyl-2-(3-mesylamino-4-hydroxyphenyl)-D-glycine (5.2 g.), 2,2,2-trichloroethyl 7-amino-3-methyl-3-cephem-4-carboxylate hydrochloride (5.52 g.) and 2,6-lutidine (1.74 g.) in methylene chloride (180 ml.). The resultant mixture was kept stirring at the same temperature for an hour and at room temperature for 3 hours. After removal of the insoluble material, the filtrate was condensed under reduced pressure. Ethyl acetate was added to the residue, and the solution was adjusted to about pH 2 with phosphoric acid. The ethyl acetate layer was separated, washed with water and dried over magnesium sulfate. Removing -the ethyl acetate under reduced pressure, the residue was pulverized with isopropyl ether to give 2,2,2-trichloroethyl 7-[N-t-butoxycarbonyl-2-(3-mesylamino-4-hydroxyphenyl)-D-glycinamido]-3-methyl-3-cephem-4-carboxylate (6.75 g.)~ The product was crystallized from ethyl acetate to give the objective ;~
compound. M.P. 1~5 to 188.5C (decomp.). I.R. (Nujol) v cm 1 1766. ~.M.R. (¦~D3)2CO) o ppm: 1.40 (9H,s), 2.16 (3~1,s), 3.01 (3H,s), 3.34, 3.59 (2H,AB-q,J=18Hz), 4.87, 5.07 (2H,AB-q,J=12.5Hz), 5.07 (lH,d,J=4.5Hz), 5.35 (lH,d,J=8Hz), 5.80 (lH,d,d,J=4.5,8Hz), 6.3-6.5 (IH,m), 6.90 (lH,d,J=8Hz), 7.20 (lH,d,d,J=2,8E~z), 7.52 (lH,d,J=2Hz), 8.0-8.3 (2H,m).
(2) Acetic acid (3.5 ml.) and zinc powder (2.6 g.)' were added under ice cooling to a solution of 2,2,2-trichloro-ethyl 7-[N-t-buto~ycarbonyl-2-(3-mesylamino-4-hydroxyphenyl)-D-glycinamido]-3-methyl-3-cephem 4-carbox~late (3.0 g.) in dimethylformamide (9 ml.), and the resulting mixture was kept stirred at the same temperature for 40 minutes. After the reaction was over, the zinc powder was collected by filtration ..

95~Z
and washed ~ith dimethylformamide. The filtrate and the ~ash-ings were combined together, and ethyl acetate and dilute phosphoric acid were added thereto. The ethyl acetate layer was separated, washed with water and back-extracted with an aqueous solution of sodium bicarbonate. The aqueous solution was aeidified with hydrochlorie acid and extracted w th ethyl aeetate. The ethyl aeetate extract was washed with water, dried over magnesium sulfate and eoncentrated under reduced pressure. The residue was washed with ether to give 7-[N-t-butoxycarbonyl-2-(3-mesylamino-4-hydroxyphenyl)-D-glyeinamido]-3-methyl-3-eephem-4-carboxylic acid (1.58 g.) as powder. I.R.
(Nujol) v em 1 1765. N.M.R. (D20 + NaHC03) o ppm: 1.40 (9H,s), 1.90 (3H,s), 3.05 (3H,s), 3.00, 3.39 (2H,~B-q,J=18Hz), 4.90 (lH,d,J=4.5Hz), 5.08 (lH,s), 5.52 (IH,d,J=4.5Hz), 6.8-7.4 (3H,m), (3) A solution of 7-[N-t-butoxyearbonyl-2-(3-mesylamino-4-hydroxyphenyl)-D-glyeinamido]-3-methyl-3-cephem-4-earboxylie aeid (1.45 g.) in formie aeid (6 ml.) wa3 stirred at 40C for 1.5 hours. After removal of the acetic acid under ;
redueed pressure, acetonitrile (30 ml.) and water (0.~ ml.) were added to the residue with stirring, and stirring was continued for 30 minutes. The preeipitated powder was filtered and washed with acetonitrile and ether to give 7-[2-(3-mesylamino-4-hydroxyphenyl)-D-glyeinamido]-3~methyl~3-cephem-4-carboxylic acid (1.13 g.). M.P. 186 to 192~ (decomp.). I.R. (Nujol) em 1 1760. N.M.R. (D20 + D~l) o ppm: 2.12 (3H,s), 3.20 (3H,s), 3.25, 3.50 (2H,AB-q,J=18Hz), 5.09 (~I,d,J=4.5Hæ), 5.33 (l~,s), 5.66 (~19d,J=4.5Hz), 7.15 (IH,d,J=8Hz), 7.40 (lH,d,d,J=2,8Hz), 7.53 (lH,d,J=2Hz).
Example 17 -- ~6 --. . .
.
. .
:

10~91 50Z 1 ~
(1) A solution of triethylamine (140 mg.) in methylene chloride (5 ml.) and 2,6-lutidine (15 mg.) were added to a solution of 2,2,2-trichloroethyl 7-amino-3~methyl-3-cephem-4-carboxylate hydrochloride (540 mg.) in methylene chloride (10 ml.) under ice cooling, and a solution of N-t-butoxycarbonyl-2-(3-ethylaminosulfonamidophenyl)-D-glycine (530 mg.) in methylene chloride (5 ml.) was added thereto while stirring under ice cooling. After the addition of N,N'-dicyclohexylcarbodiimide (320 mg.), the resultant mixture was stirred at room tempera-ture for 3 hours. The insoluble material in the resultant mixture was filtered and washed with methylene chloride. The filtrate and the washings were combined together, and the mixture was concentrated under reduced pressure. Ethyl acetate was added to the residue~ and the precipitate was filtered off.
The ethyl acetate layer was washed with cold 5 ~ hydrochloric acid, water, an aqueous saturated solution of sodium bicarbonate and an aqueous saturated solution of sodium chloride in order and then dried. Ethyl acetate was added to the solution, the insoluble material was filtered off, and then the resultant solution was concentrated under reduced pressure to give 2,2,2-trichloroethyl 7-~N-t-butoxycarbonyl-2-(3-ethylaminosulfon-amidophenyl)-D-glycinamido]-3-methyl-3-cephem-4-carboxylate (1.07 g.) as an amorphous product. M.P. 70 to 73C (decomp.).
I.R. (Nujol) v cm 1 3290, 1770, 16~0.
(2) Acetic acid (1.2 ml.) and zinc powder (0.96 g.) were added un~er ice cooling to a solution of 2,2,2-trichloro-ethyl 7-~N-t-butoxycarbonyl-2-(3-ethylaminosulfonamidophenyl)-D-glycinamido]-3-cephem-4-carboxylate (0.94 g.) in dimethyl-formamide (5 ml.). The mixture was kept stirring at the same temperature for an hour. After the reaction was over, zinc ~L~4950Z j;~ ~

powder was filtered and washed with ethyl acetate. The wa~hings were combined together with the filtrate, poured into cold 5 % , hydrochloric acid (20 ml.) and extracted twice with ether acetate The extract was washed with an aqueous saturated solu-tion of sodium chloride and back-extracted with 5 ~ aqueous solution oi sodium bicarbonate. The aqueous layer was separated out, adjusted with 10 % hydrochloric acid to pH 1 to 2 and extracted twice with ethyl acetate. The extract was washed with an aqueous saturated solution of sodlum chloride, dried and concentrated under reduced pressure to give 7-[~-t-butoxycarbonyl-2-(3-ethylaminosulfonamidophenyl)-D-glycinamido]-3-methyl-3-cephem-4-carboxylic acid (0.68 g.) as an amorphous product. M.P. 122 to 128C (decomp.). I.R. (Nujol) v cm 1 3300, 1765, 1690. N.~l.R. (D20 + NaHC03) o ppm: 1.03 (7~H,t), i ~-1.40 (9~,s), 1.93 (3H,s), 2.6-3.5 (4~,m)9 5.0 (~I,d), 5.30 (~I,s), 5.60 (IH,d), 7.30 (4H,m). --(3) A mixture of 7-[N-t-butoxycarbonyl-2 (3-ethyl-aminosulfonamidophenyl)-D-glycinamido3-3-methyl-3-cephe~-4-carboxylic acid (590 mg.) in formic acid (8 ml.) was s~irred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in water and shaken with ethyl acetate. The aqueous layer was separated and concentrated. The residue was adjusted to pH 5 with 5 C/~ aqueous solution of sodium hydroxide, adsorbed on a `
resin ("XAD-2" manufacted by Rohm and Haas Co.), washed with water and eluted with methanol. The eluate was concentrated under reduced pressure, and the residue was washed with aceto-nitrile to give 7-~2-(3-ethylaminosulfonamidophenyl)-D-glycin-amido]-3-methyl-3-cephem-~-carboxylic acid (350 mg.). M.P.
16- to 166C. I.R. (Nujol) ~ cm 1 3200, 1760, 1690. ~.M.R.

' , : i . . . :::
, .

~495~Z
(D20) ~ ppm 1.03 (3H,t), 3.03 (2H,q), 3.0, 3.45 (2H,d,d, J=19Hz), 4.95 (lH,d), 5.27 (lH,broad s), 5.61 (lH,d~, 7.30 (4H,m).
Example 18 (1) To a suspension o~ 2,2,2-trichloroethyl 7-amino-3-methyl~3-cephem-4-carboxylate in methylene chloride (50 ml.), there was added triethylamine (0.95 g.~ all at once at 5C.
N-t-Butoxycarbonyl -2-C3-(3-methylureido)phenyl~-D-glycine (3.37 g.) and N,~'-dicyclohexylcarbodiimide (2.88 g.) were successively added to the mixture and stirred at room tempera-ture for an hour. The insoluble material was filtered off, and the filtrate was concent'rated under reduced pressure. The residue was dissolved in ethyl acetate, washed with 5 % hydro-chloric acid (three times), water, an aqueous solution of sodium bicarbonate (three times), water and an aqueous solution of sodium chloride (three times) in turn, dried and then ~ -~
concentrated~ The concentrate was allowed to stand at a cold place, and the precipitated crystals were collected by filtra-tion to give 2,2,2~trichloroethyl 7-[N-t-butoxycarbonyl-2-~3-(3-methylureido)phenyl~-D-glycinamido]-3-methyl-3 cephem-4-carboxylate (3.96 g.). M.P'. 152 to 160C (decomp.). I.R.
(~uiol) V cm : 3320, 1782, 1731. ~.M.R. (DMS0-d6) ~ ppm-1.42 (9H,s), 2.08 (3H,s), 2.63 (3H,d,J=5Hz), 3.47 (2H,t,J=
20Hz), 4.8-5~4 (4H,m), 5.95 (lH,d,J=5Hz), 6.7-7,6 (4H,m), 8.37 (lH,s), 9.05 (lH,d,J=7Hz).
(2) Zinc powder (5.7 g.) and glacial acetic acid (5.7 ml.) were added to a solution of 2,2,2-trichloroethyl 7-~-t-butoxycarbonyl-2-~3-(3-methylureido)phenyl~-D-glycinamido~-3-methyl-3-cephem-4-carboxylate (5.7 g.) in dimethylformamide (57 ml~) at 5C. The mixture was stirred at room temperature ;'-95~2 ~or an hour and filtered. The filtrate was shaken with a mixture of ice, 5 % hydrochloric acid and ethyl acetate. The organic layer was separated, washed twice with water and back-extracted with an aqueous solution of sodium bicarbonate. The aqueous layer was separated, extracted with ethyl acetate, acidified with 5 % hydrochloric acid and then extracted with ethyl acetate.
The extract was washed with water, dried over magnesium sulfate and concentrated to give 7-[N-t-butoxycarbonyl-2-~3-t3-methyl ureido)phenyl~-D-glycinamido]-3-methyl-3-cephem-4-carboxylic -~
acid (3.29 g.). M.P. 143 to 150C (decomp.). I.R. (~ujol) y cm : 3370, 1780. N.M.R. (DMS0-d6) ~ ppm: 1.38 (9H,s), 1.99 (3H,s), 2.63 (3H,d,J=5Hz), 3.28, 3.45 (2H,AB-q,J=18Hz), -4.96 tlH,d,J=5HZ), 5.28 (lH,broad d,J=8Hz), 5.62 (lH,d,d,J=
5,8Hz), 5.97 (lH,broad d,J=5Hz), 6.8-7.6 (4H,m), 8.44 (lH,s), 9.02 (lH,broad d,J-8Hz).
(3) 7-[N-t-Butoxycarbonyl-2-~3-(3-methylureido)-phenyl~-D-glycinamido~-3-methyl-3-cephem-4-carboxylic acid (3.18 g.) was added to formlc acid (60 ml.) at 10C, and the mixture was stirred at room temperature ~or 2 hours and con-centrated under reduced pressure. The residue was pulverized with ether ~70 ml.), and the collected powder was added to 5 %
aqueous acetonitrile (10 ml.), stirred for 30 minutes and then filtered to give 7-~2-~3-(3-methylureido)phenyl~-D-glycinamido~-3-methyl-3-cephem-4-carboxylic acid (2.2 g.) as crystals.
M.P. 215 to 218C (decomp.). I.R. (~ujol) Ycm : 3350, 1760.
.M.R. (D20 + DCl) ~ ppm: 2.2 (3H,s), 2.85 (3H,s), 3.23, 3.51 (2H,AB-~,J=19Hz), 4.95 (lH,d,J=SHz), 5.33 (lH,s), 5.7 (lH,d, J=5Hz), 7.2-7.7 (4H,m).
Example 19 (1) A solutionofethyl chlorocarbonate (1.79 g.) :

1~4~50Z

in methylene chloride (10 ml.) was added to a solution of N-t-butoxycarbonyl-2-~3-~3-methylthioureido)phenyl~-D-glycine (6.45 g.) and triethylamine (1.82 g.) in methylene chloride ( 75 ml.) at -25 to -30~C in 10 minutes, and the mixture was kept stirred at the same temperature for 15 minutes. A solu-tion of 2,2,2-trichloroethyl 7-amino-3-methyl-3-cephem-4-carboxylate hydr~chloride (5.73 g.) and triethylamine (1.52 g.) in methylene chloride (75 ml.) was added to the above obtained mixture at -40C all at on¢e. The resultant mixture was gra-dually warmed to 0C in 4 hours under stirring and concentrated under reduced pressureO The residue was dissolved in ethyl acetate (150 ml.), washed with 5 % hydrochloric acid, an aque-ous saturated solution of sodium bicarbonate and an aqueous saturated solution of sodium chloride in order and then con-centrated under reduced pressure~ The residue was purified by column chromatography on silica gel (eluant: benzene:
acetone - 5 : 1 by weight) to give 2,2,2-trichloroethyl 7-[N-t-butoxycarbonyl-2-~3-(3-methylthioureido)phenyl7-D-glycin-amido]-3-methyl-3-cephem-4-carboxylate (3.83 g.). M.P. 123 to 130C I.R. (Nujol) Vcm 1 3320, 1778, 1680, 1535, 1367, 1306, 1243, 1217, 1162, 1110, 1050, 787, 718. N.M.R. (DMS0-d6) ~ ppm: 1.39 (9H,s), 2.08 (3H,s), 2.91 (3H,d,J=4Hz), 3.41 (2H,broad s), 4.8-5.1 (2H,m) 5.28 (lH, broad d,J=7Hz), 5.5-5.8 (lH;q), 7.0-7.75 ~5H,m), 9.10 (lH,broad d,J=7Hz), 9.48 (lH,s).
- (2) To a solution of 2,2,2-trichloroethyl 7-[~-t-butoxycarbonyl-2-~3-(3-methylthioureido)phenyl~-D-glycinamido]-3-methyl-3-cephem-4-carboxylate (3.55 g.) and glacial acetic acid (3 ml.) in dimethylformamide (15 ml.), there was added zinc powder (3.0 g.), and the mixture was stirred under ice cool-9S/L~
ing for 1.5 hours. After the reaction was over, the zinc powder was collected by filtration and washed with ethyl acetate (50 ml.), and the washings and the Eiltrate were combined together.
After the mixture was washed with water, the aqueous layer was separated and extracted twice with ethyl acetate (30 ml.).
The extract was combined together with the above obtained ethyl acetate layer. The ethyl acetate layer was washed with an aqueous saturated solution of sodium chloride and then back-extracted four times with an aqueous saturated solution of sodium bicarbonate (40 ml.). The aqueous layer was washed with ethyl acetate, adjusted to pH 2 with 10 % hydrochloric acid and then extracted five times with ethyl acetate (60 ml.).
The extract was washed with water and dried over magnesium sulfate, and then the solvent was removed under reduced pressure to give 7-[N-t-butoxycarbonyl-2-~3-(3-methylthioureido)phenyl~-D-glycinamido]-3-methyl-3-cephem-4-carboxylic acid (2.45 g.~
as powder. I.R. (Nujol) ~ cm : 3320, 1700, 1670, 1540, 1370, 1250, 1165, 1110, 1056, 866, 718. N.M.R. (DMS0-d6) ~ ppm:
1.40 (9H,s), 2.00 (3H,broad s), 2.92 (3H,s), 3.1-3.7 (2H, --~
broad s), 4.99 (lH,d,J=5Hz), 5.33 (lH, broad d,J=8Hz), 5.5-5.8 (lH,m), 7.0-7.7 (4H,m), 9.07 (lH,broad d,J=8Hz), 9.50 (lH,broad s).
(3) A solution of 7-[N-t-butoxycarbonyl-2-~3-(3-methylthioureido)phenyl~-D-glycinamido]-3-methyl-3-cephem-4-carboxylic acid (2.1 g.) in formic acid (20 ml.) was stirred at room temperature for 3 hours. After the reaction was over, the formic acid was removed under reduced pressure, and the residue was stirred in 10 % aqueous acetonitrile (20 ml.). The precipitate was collected by filtration, washed with aceto-nitrile and dried to give 7-[2-~3-(3-methylthioureido)phenyl~-D-~g50Z
glycinamido]-3-methyl-3-cephem-4-carboxylic acid (1.21 g.).
M.P. 198 to 199C (decomp.). I.R. (Nujol) V cm : 3500, 3430, 3230, 2600-2700, 1767, 1700, 1608, 1550, 1412, 1328, 1280, 1230, 1178, 1160, 1130, 978, 812, 788, 757, 711.
N.M.R. (D20 + DCl) ~ ppm: 2.07 (3H,s), 3.02 t3H~S)~ 3~34, 3.37 (2H,AB-q,J=18Hz), 5.09 (lH,d,J=4.5~), 5.37 (lH,s), 7.52 (4H,broad s).
Example 20 (1) To a solution of N-t-butoxycarbonyl~2-(3-mesyl-aminophenyl)-D-glycine (9.93 g~ in a mixture of tetrahydrofuran (140 ml) and triethylamine (3.22 g) kept at -10C, isobutyl chloroformate (3.95 g) was added, and the resultant mixture was stirred at --10C for 20 minutes. A cold solution of 7-aminocephalosporanic acid (7.87 g) in a mixture of triethyl-- amine (3.5 g), tetrahydrofuran (40 ml) and water (40 ml) was added thereto all at once, and the resulting mixtu~e was stirred while ice cooling for 1 hour and at room temperature for 2 hours.
After the reaction was comple~ed, the tetrahydrofuran was removed by distillation, and water (100 ml) was added thereto. The resulting solution was adjusted to pH 2.5 with 10 % hydrochloric acid while ice cooling and shaken with ethyl acetate. The ethyl acetate layer was separated, washed with water, dried and ~oncentrated under reduced pressure. The residual oil was treat-ed with ether to give 7-[D-N-t-butoxycarbonyl-2-(3-mesylamino-phenyl)glyci~namido]-3-acetoxymethyl-3-cephem-4-carboxylic acid (10.2 g.) as yellow powder. M.P. 83 to 84C (decomp.). I.R.
(~ujol) ~ cm : 1780. N.M~R. (DMS0-d6) ~ ppm: 1.43 (9H,s)-, 2.05 (3H,s), 3.03 (3H,s), 3.30-3.70 (2H), 5.06 (lH,d,J-5Hz), 5.55-5.97 (lH), 9.13-9.43-(lH), 9~82 (lH), (2) A solution o~ 7-[D-~-t-butoxycarbonyl-2-(3-!

. ., - : .

~49S~2 mesylaminophenyl)glycinamido]-3-acetoxymethyl-3-cephem-4-carboxylic acid (1 g) in formic acid (15 ml) was stirred at roorn temperature for 3 hours. After removal of the formic acid by distillation, water (10 ml) was added thereto, and the resultant solution was washed with ethyl acetate and stirred with a solution of an-ion exchange resin ("Amberlite LA-l"
manufactured by Rohm and Haas Co.) in methylisobutylketone (3 ml) for 1 hour. The water layer was separated, washed with ether and lyophilized to give 7-[D-2-(3-mesylaminophenyl)-glycinamido]-3-acetoxymethyl-3-cephem-4-carboxylic acid (0.63 g) as colorless powder. M.P. 240C (decomp.). I.R. (Nujol~
~ cm 1 1770. N.M.R. (D20) ~ ppm: 207 (3H,s), 3.13 (3M,s), 5.03 (lH,d,J-5Hz), 5.23 (lH,s), 5.70 (lM,d,J=5Hz~, 7.30-7.63 (4H,m). "Amberlite LA - 1" is a trademark.
Example 21 Sodium bicarbonate (0.86 g.) was added to a stirred suspension of 7-[D-N-t-butoxycarbonyl-2-(3-mesylaminophenyl)-glycinamido]cephalosporanic acid (3 g.) in phosphate buffer (pH 6.4) (130 ml.), and acetone (80 ml.) was added thereto~
5-Methyl-1,3,4-thiadiazole-2-thiol (0.68 g.) was added to the solution and stirred at 60 to 65C for 6 hours. Acetone was remove* from the resultant mixture under reduced pressure, and the remaining aqueous layer was washed with ether. The aqueous solution was adjusted to pH 2 with dilute hydrochloric acid and then extracted with ethyl acetate. The extract was washed with water, dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified by coiumn chromato~raphy on silica gel (eluant: chloroform) to give 7-[D-N-t-butoxycarbonyl-2-(3-mesylaminophenyl)glycinamido~-3-(5-methyl-1,3,4-thiadiazol-2-yl)thiomethyl-3-cephem-4-carboxylic :,., . - ' :

~49S~Z

acid (1.8 g.). I.R. (Nujol) v cm 1 33oo, 1780, 1725, 1685, 1670.
Example 22 N-t-Butoxycarbonyl-2-(3-dimesylaminophenyl)-D-glycine, which was derived from 2-(3-dimesylaminophenyl)-D-glycine in a conventional method, was coupled with 2,2,2-trichloroethyl 7-amino-3-methyl-3-cephem-4-carboxylate to give 2,2,2-trichloroethyl 7-[N-t-butoxycarbonyl-2-(3-dimesyl-aminophenyl)-D-glycinamido]-3-methyl-3-cephem-4-carboxylate.
rrhe resultant substance~ 272,2-trichloroethyl 7-~N-t-butoxycarbonyl-2-(3-dimesylaminophenyl)-D-glycinamido]-3-methyl-3-cephem-4-carboxylate was subjected to elimination of 2,2,2-triehloroethyl group to give 7-[N-t-butoxycarbonyl-2-(3-dimesylaminophenyl)-D-glycinamido]-3-methyl-3-cephem-4-earboxylic acid, which was furthermore subjected to elimination of t-butoxycarbonyl group to give 7-[2-(3-dimesylaminophenyl)-D-glycinamido]-3-methyl-3-cephem-4-carboxylie aeid in a similar manner to ~xample 11.
~xample A
(1) N-t-Butoxyearbonyl-2-(3-aminophenyl)-D-glyeine (8.7 g.) and N-trimethylsilylacetamide (13.1 g.) were added to methylene chloride (166 ml.), and the resultant mixture was stirred at room temperature for 1.5 hours.~ To the resulting mixture, pyridine (13.1 g.) was dropwise added while ice cooling, and then a solution of methanesulfonyl ehloride (8.45 g.) in methylene chloride (35 ml.) was dropwise added thereto at a temperature of 3 to 5C. Stirring was eontinued at the same temperature for 30 minutes and then at room temperature for 4 hours. ~he reaction mix~ure was shaken with an aqueous solution of citric acid, washed with water, : .:

. .

~IL049~
and then the methylene chloride layer was separated. The methylene chloride layer was shaken with 5 % aqueous solution of sodium hydrogen carbonate, and the wa~er layer was separated.
The water layer was adjusted to pH 5 with an aqueous solution of citric acid and salted out to give a resinous substance.
The resinous substance was extracted with methylene chloride, -and the extract was washed with a dilute aqueous solution of sodium chloride, dried and concentrated under reduced pressure to give ~-t-butoxycarbonyl-2-(3-mesylaminophenyl)-D-glycine (6.3 g.) as amorphous powder. N.M.R. (DMS0-d6) ~ ppm:
1.38 (9H,s), 2.99 (3H,s), 5.09 (lH,d,J=7.5H~); 7.00-7.60 (4H,m).
- (2) T~e above prepared ~-t-butoxycarbonyl-2-(3-mesylaminophenyl)-D-glycine (4.0 g.) was dissolved in formic acid (100 ml.), the solution was stirred at room temper-ature for 1 hour and the solvent was distilled off under reduced pressure. The residue was dissolved in 10 % aqueous aceto-nitrile (10 ml.) and filtered. To the filtrate, acetonitrile (10 ml.) was added, and the precipitated crystals were collected by filtration and dried to give 2-(3-mesylaminophenyl)-D-glycine (2.7 g.) as colorless prisms. M.P. 196 to 198C.
I.R. (~ujol) V cm : 3240, 25~30, 1616, 1147. ~.M.R. (D20 +
DCl) ~ ppm~ 3.18 (3H,s); 5.32 (lH,s), 7.47 (4H,m).
Example B
As in Example A, D-~-t3-mesylaminophenyl)-~-(1-methoxycarbonyl-l-propen-2-yl)glycine sodium salt was prepared from D-(~-3-aminophenyl)-~-(1-methoxycarbonyl-1-propen-2 yl)-glycine sodium salt and methanesulfonyl chloride. M.P. 141 to 143C. I.R. (Nujol) y cm 3250, 1650, 1610, 1150.
N.M.R. (DMSO-d6) ~ ppm: 1.68 (3H,s), 2.92 (3H,s), 3.52 (3H,s), 4.31 (lH,s), 4.78 (lH,d,J=5.6Hz), 6.90-7.25 (4H,m), 9.58 (lH, .

~9~o~
d,J=5.6Hz).
ExampleC
To a solution of D-a-(3-aminophenyl)-N-t-butoxy-carbonylglycine (1.34 g.) and sodium hydrogen carbonate (924 mg.) in water (25 ml.) cooled at -2~C, methanesulfonyl chIoride (~60 mg.) was dropwise added in 10 minutes, and the resultant mixture was stirred at the same temperature for 1 hour. After the addition of sodium hydrogen carbonate (164 mg.), stirring was continued at the same temperature for 2 hours and then at room temperature ~or 1 hour. The reaction mixture was washed with ether (30 ml.) twice and a trace amount of insoluble materials was eli~inated by filtration. The filtrate was adjusted to pH 7) with 10 ~ hydrochloric acid, salted out and extracted with ethyl acetate (40 ml.) t~ice. The extract was washed with an aqueous saturated solution of sodium chloride, dried over anhydrous magnesium sulfate and concentrated under i; reduced pressure to give a-(3-mesylaminophenyl)-N-t-butoxy carbonylglycine (1.4 g.). [a]D = -96 (methanol, C = 1).
Example (1) To a solution of N-t-butoxycarbonyl-2-(3-aminophenyl)-D-glycine (5.326 g.) in methylene chloride (100 ml.), N,N-bis(trimethylsilyl)acetamide (8.20 g.) was added while stirring at room temperature. Stirring was continued at room temperature for 1 hour. Then, pyridine (3.165 g.) was added while ice cooling (2 to 3C), and a solution of methane-sulfonyl chloride (4.58 g.) in methylene chloride (25 ml.) was dropwise added thereto while ice cooling in 30 minutes.
Stirring was continued while ice cooling for 30 minutes and then at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure, and the residue was ~..

1~495~
shaken with ethyl acetate (100 ml.) and 2M cltric acid solution (50 ml.). The ethyl acetate layer was separated and washed with 2M citric acid solution (50 ml.). The washing was ex-j tracted with ethyl acetate (30 ml.). The extract was combined with the said ethyl acetate layer, washed with aqueous saturated solution of sodium chloride and extracted three time with aqueous solution of sodium hydrogen carbonate (40 ml.). The extracts were combined together, washed with ethyl acetate (50 ml.), adjusted to pH 4 with 2M citric acid solution and salted out with sodium chloride. The precipitate was extracted ~our times wi-th ether (50 ml.), and the extracts were washed with an aqueous saturated solution of sodium chloride, dried over magnesium sulfate and concentrated under reduced pressure to give N-t-butoxycarbonyl-2-(3-mesylaminophenyl)-D-glycine (6.15 g.) as powder. N.M.R. (DM~0-d6) ô ppm: 1.40 (9H,s), .00 (~H,s), 5.11 (lH,d,J=8Hz), 7.00-7.60 (5H,m).
(2) The above obtained N-t-~utoxycarbonyl-2-(3-mesylaminophenyl)-D-glycine was treated as in Example ~ (2) to give 2-(3-mesylaminophenyl)-D-glycine. ~ -D-2-(3-Aminophenyl)glycine (16.6 gO) was dissolved in N sodium hydroxide solution (100 ml.j, and dioxane (40 ml.) was added thereto. To the resulting solution, a solution of cupric sulfate pentahydrate (26.2 g.) in water (50 ml.) was added at room temperature, and stirring was continued for 40 minutes. The resulting mixture was adjusted to pH 7.0 with 20 % sodium hydroxide solution, and a solution of methane-sulfonyl chloride (17.1 g.) in dioxane (20 ml.) was dropwise added at 26 to ?8C in 1 hour, during which 20 ~0 sodium hydroxide solution (47 ml.) was dropwise added thereto to - 4~ -', ' '~

maintain p~I at 6.0 to ~.5. The reaction mixture was stirred at room temperature for 1 hour and adjusted to pH 3.0 with 10 hydrochloric acid. Removal of the oreanic solvent by dis-tillation under reduced pressure gave cupric 2-(3-mesylamino-phenyl)-D-glycinate, which was adsorbed on an ion exchange resin ("Amberlite IR 120B" manufactured by Rohm and Haas, Co~) and washed with water until the eluate showed neutral. The ion exchange resin was then eluted with ammonia water (water : 30 ammonia water = 14 : 1 by weight). The eluate was concentrated under reduced pressure. The residue was dissolved in a small amount of methanolic hydrochloric acid and adjusted to pH 5 with methanolic ammonia. The resulting solution was allowed to stand overnight. The precipiated crystals were collected by filtration and dried to give 2-(3-mesylaminophenyl)-D-glycine (15.6 g.) as pale brown needles. M.P. 186.5 to 187C.
I.R. (Nujol) v cm 1 1605 (broad), 1150. N.M.R. (DCl + D20) o ppm: 3.17 (3H,s), 5.16 (IH,s), 7.43 (4HJm).
Exam~le F
To the mixture obtained by the use of D-2-(3-amino-phenyl)~lycine (16.6 g.) and cupric sul~ate pentahydrate (26.2 g.) as in ~xample ~, a solution of methanesulfonyl chloride (27.5 g.) in dioxane (27 ml.) was dropwise added at 26 to 28C in an hour, during which 20 % sodium hydroxide solution dropwise added thereto to mairtain pH 8 to 9. The ;
reaction mixture was treated as in Example ~ to give 2-(3 -~
dimesylaminophenyl)-D-glycine as colorless prisms. M.P. 171 to 173C (decomp.). I.R. (Nujol) v cm 1 1650, 1160. N.M.R.
(DCl + D20) ~ ppm: 3.50 (6H,s), 5.33 (lH,s), 7.61 (4~I,m).

Example G

A solution of D-(a-4-aminophenyl)-N-t-butoxycar-- ' '' -- 49 -- ~:

.. . .

-S~2 bonylglycine (5 g.) and bis(trimethylsilyl)acetamide (11.0 g.) in methylene chloride (120 ml.) ~Jas stirred at room temperature for 2 hours, and pyridine (2.85 g.) and a solu-tion o-f methanesulfonyl chloride (4.12 g~) in methylene chloride (40 ml.) were dropwise added thereto at 2 to 3C in 30 minutes. The resultant mixture was stirred at the same temperature for 30 minutes and allowed to stand at room temper-ature overnight. After removal of the solvent under reduced pressure, ethyl acetate and 1 M citric acid solution were added to the residue. The ethyl acetate layer was separated and washed with an aqueous solution of citric acid. The washings and the water layer were combined together and ex-tracted with ethyl acetate. The ethyl acetate extract and the previously obtained ethyl acetate layer were combined together, washed with water and shaken with an aaueous saturated solution of sodium hydrogen carbonate. The aqueous layer was separated, adjusted to pH 4 with 2 M citric acid solution and extrac-ted with ethyl acetate. The ethyl acetate extract was washed with an aqueous saturated solution of sodium chloride and dried.
After removal of the solvent by distillation under reduced pressure, the resulting oil (6.0 g.) was pulverized ~ith ether and isopropyl ether to give D-a-(4-mesylaminophenyl)-~-t-butoxycarbonylglycine (5.5 g.) as crystals. M.P. 165 to 183C.
I.R. (Nujol) ~1 cm 1 3325, 31259 1747, 1673. N.M.R. ((CD3)2C0) o ppm: 1.32 (9H,s), 2.84 (3H,s), 5.0 (lH,d), 6.90-7.3 (4H,m).
~xample H
.
N-t-Butoxycarbonyl-2-(3-aminophenyl)-D-glycine (3.99 g.) and ~,N-bis(trimethylsilyl)acetamide (6.12 g.) ~ere added to methylene chloride (40 ml.), and the resulting mixture ~as stirred at room temperature for 2 hours. To the resulting , .

~4g5~2 mixture, pyridine (2.4 g.) was dropwise added while ice cooling, and ethanesulfonyl chloride (3O84 g.) was dropwise added thereto.
Stirring was continued at 0 to 5C for 2 hours and then at room temperature for 1 hour. The reaction mixture was washed with 2 % hydrochloric acid (20 ml.) twice, water (20 ml.) twice and an aqueous satruated solution of sodium chloride once in order. The methylene chloride layer was separated, dried over magnesium sulfate and concentrated under reduced pressure. The residue was recrystallized from diisopropyl ether to give N-t-butoxycarbonyl-2-(3-ethanesulfonamidophenyl)-D-glycine (4.4 g.) as powder. I~R. (film) V cm 1 3230, 1720, 1680, N.M.R. (CDC13) ~ ppm: 1.18 (3H,t,J=7.5 Hz), 1.36 (9H,s), 3.05 (2H,q,J=7.5 Hz), 3.16 (lH,broad s), 7.23 (4H,s), 7.71 (lH,broad s), 8.59 (lH, ~road s).
Example I
A solu~ion of D-~-(3-aminophenyl)-N-t-butoxycarbonyl-glycine (0.5 g.) and bis(trimethylsilyl)acetamide (1015 g.) in methylene chloride t20 ml.) was stirred at room temper~ture for 2 hours and cooled to 2 to 3C. Pyridine (0.3 g.) and a solution of ethylaminosulfonyl chloride (0.54 g.) in methylene chloride (6 ml.) were dropwise added thereto. The resulting mixture was stirred at the same temperature for 30 minutes and then at room temperature for 15 hours. -After removal of the solvent, ethyl acetate and 1 M citric acid solution were added to the residue. The ethyl acetate layer was separated and washed with an aqueous solution of citric acid. The washings and the water layer were combined together, salted out and extracted with ethyl acetate. The ethyl acetate extract and the previously obtained ethyl acetatç layer were combined together, and an aqueous solution of sodium hydrogen ~.~4L9502 carbonate was added thereto. The water layer was separated, washed with ethyl acetate, adjusted to pH 4 with 2 M citric acid solution and extracted with ethyl acetate. The ethyl acetate extract was washed with an aqueous saturated solution of sodium chloride and dried. Removal of the solvent by distil-lation under reduced pressure afforded D-~-(3-ethylaminosulfon-amidophenyl)-N-t-butoxycarbonylglycine (0.54 g.) as an oil.
I.R. (film) ~ cm 1 3250, 170~ (broad). N.M.R. (CDC13) ~
ppm: 0.97 (3H,t), 1.35 (9~I,s), 3.00 (2H,q), 5.20 (lH,broad s), 7.20-(4H,m).
Example J
To a solution of D-~-(3-amino-4-hydroxyphenyl)-N-t-butoxycarbonylglycine (8.0 g.) in methylene chloride (150 ml.), bis(trimethylsilyl)acetamide (11.5 g.) was added, and the resultant solution was stirred at room temperature for 30 minutes. After cooling with ice, pyridine (4.5 g.) was added thereto, and then methanesulfonyl chloride (6.5 g.) was drop-wise added to the resulting mixture. Stirring was continued for 30 minutes while ice cooling and then at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure. The residue was admixed with ethyl acetate and water, made acidic with phosphoric acid and the ethyl -acetate layer was separated from the water layer. The ethyl acetate layer was back-extracted with an aqueous solution of sodium hydrogen carbonate, and the water layer was sepaxated, made acidic with phosphoric acid and back-extracted again with ethyl acetate. The ethyl acetate extract was washed with water, dried and treated with activated charcoal. The resultant solu tion was then concentrated to give D-~-(3-mesylamino-4-hydroxy-phenyl)-~-t butoxycarbonylglycine ~6.0 g.). I.R. (Chloroform) .

951~Z
V cm : 1730 (shoulder), 1715, N.M.R. (D20-NaHC03) ~ ppm:
1.4 (9H,s), 3.05 (3H,s), 4.83 (lH,s), 6.87 (lH,d,J=8Hz) Example K_ To a solution of D-~-(3-aminophenyl)-N-t-butoxy-carbonylglycine (7.0 g.) and triethylamine (2.92 g.) in methylene chloride (100 ml.) cooled at 5C, a solution of methyl isocyanate (3.0 g.)~in methylene chloride (25 ml.) was dropwise added in 5 minutes, and the resulting mixture was stirred at room temperature for 5 hours. After removal of the solvent by distillation, the residue was dissolved in ethyl acetate and washed with 2Mcitric acid solution. The organic solvent layer was washed with water and extracted with an aqueous solution of sodium hydrogen carbonate. The water layer -was separated, washed with ether and adjusted to pH 6 with 10 %
sulfuric acid. The resulting solution was adjusted to pH 3 to 4 with citric acid, salted out and extracted with ether. The ether extract was washed with water, dried over anhydrous mag-nesium sulfate and concentrated under reduced pressure to give D~ 3-t3-methylureido)phenyl7-N-t-butoxycarbonylglycine (5.7 gO) as a foamy material. ~.M.R. (DMS0-d6) ~ ppm: 1.37 t9H,s), 2.54 (3H,d,~=4.5Hz), 4.97 (lH,d,J=8Hz), 5.96 (lH,d,J--4.5Hz),

6.7-7.6 (6H,m), 8.47 (lH,s). [~]D = -111 (99 % ethanol).
Example L
A mixture of D-~-~3-aminophenyl)-~-t-butoxycarbonyl-glycine (9.00 g.), triethylamine (3.41 g.) and methyl thio-isocyanate (3.70 g.) in methanol (45 ml.) was re~luxed for 2 hours. After removal of the solvent by distillation, the --residue was dissolved in ethyl acetate (100 ml.), and the resulting solution was washed with 2 M citric acid solution (40 ml.) four times and an aqueous saturated solution of sodium 1~4~51;~2 chloride once and then extracted with an aqueous saturated solution of sodium hydrogen carbonate (40 ml.) four times.
The water layer was separated, washed with ethyl acetate, ad-justed to pH 1 with 10 % hydrochloric acid and extracted with ether (50 ml.) three times. The extract was dried over anhydrous magnesium sulfate and treated with activated charcoal. After removal of the activated charcoal by filtration, the filtrate was concentrated under reduced pressure to give D~ 3-(3-methylthioureido)phenyl~-N-t-butoxycarbonylglycine ~8.60 g.).
I.R. (Nujol)~ cm 1 3350, 1740-1670 (severals), 1370, 1250, 1163, 1052. N~M.R. (DMS0-d6) ~ ppm: 1.37 (9H,s), 2~93 (3H,d), 5.07 (lH,d,~=9~z), 6.9-7.9 (6H,m), 9.21 (lH,s). [~D = -101 (methanol, C = 1).
Example M
(1) 3-Aminobenzaldehyde (polymerized form; water con-tent, 3 % by weight, 5.16 g.) and water (3.3 ml.) were added to tetrahydrofuran (80 ml.), and the resulting mixture was cooled to 5 to 10C. After the addition of pyridine (7.9 g.), methane-sulfonyl chloride (11.45 g.) was dropwise added thereto at the same temperature in 15 minutes, and stirring was continued at room temperature for 3.5 hours. The reac~ion mixture was con-centrated under reduced pressure, 5 % hydrochloric acid (120 ml.), ethyl acetate (60 ml.) and sodium chloride were added thereto, and the precipitate was collected by filtration and recrystallized from ethyl acetate to give 3-mesylaminobenzaldehyde (1,~ g.) as crystalsO M.P. 142 to 144C. The ethyl acetate layer was separated, washed with an aqueous saturated solution of sodium chloride, dried over anhydrous magnesium sulfate~and concentrated under reduced pressure. The residue was recrystallized from ethyl acetate to give the same product as above (4.36 g.). From ,~

-~0~9S~Z:
the ethyl acetate mother liquor, there was further obtained the same product as above (0.83 g.). Total yield, 6.99 g. I.R.
(~ujol) ~ cm~l: 3120, 1670, 1600, 1580, 1320, 1242, 1160, 1143, 997, 970, 890, 788, 753, 670. ~.M.R. (DMS0-d6) ~ ppm: 3.03 (3H,s), 7.4-7.8 (4H,m), 9.95 (l~,s), 1Q~7 (lH, broad s).
(2) To a mixture of sodium cyanide (2.18 g.), ammonium chloride (2.19 g.) and 28 % ammonia water (17 ml.) cooled at lO~C, 3-mesylaminobenzaldehyde (3.98 g.) was added thereto at 10 to 15C ~or 4 hours while stirring. After removal of excess of ammonia from the reaction mixture at 15C under reduced pressure, the residue was adjusted to pH 7 with conc. ~ -hydrochloric acid and extracted with ethyl acetate t30 ml) 5 times. The extract was washed with an aqueous saturated solu-tion of sodium chloride (30 ml), dried over anhydrous magnesium sulfate and concentrated to give DL-~-amino-~-(3-mesylamino-phenyl)acetonitrile (4.19 g.~ as~an oil. I.R. (film) ~ cm 1 3270, 1608, 1595, 1477, 1400, 1327, 1148, 972, 890, 795, 768, 697, ~.M.R. ~DMSO-d6) ~ ppm: 3.00 (3H,s), 5.01 (lH,s),

7.0-7.7 (4H,m).
(3~ To DL-~-amino-~-(3-mesylaminophenyl)acetonitrile (2.10 g.), there was added glacial acetic acid (7 ml.), and finely pulverized L(+)-tartaric acid (1.56 g.) was added thereto.
To the resulting solution, ethyl acetate (4.7 ml.) was portion- ;
wise added while stirring, and stirring was continued at room temperature overnight. The precipitated crystals were collected by filtration, washed with a mixture of ethyl acetate and acetic acid and ethyl acetate in order and drled to give D-~-amino-~-(3-mesylaminophenyl)acetonitrile L(+)-tartrate (having one molecule of acetic acid) (3.25 g.)~ M.P. 97 to 98~C.
I.R. (Nujol) V cm 1 3220, 2670, 1735, 1692, 1593, 1542, 1520, ,, . . , ~ .

~9~oz 1422, 1405, 1310, 1262, 1237, 1220, 1165, 1133, 1070, 978, 903, 800, 782, 663, 602. N.M.R. (D20-DCl) ~ ppm: 2.13 (3H,s), 3.20 (3H,s), 4.78 (2H,s), 5.87 (lH,s), 7.3-7.7 (4H,m).
[~]D = +31 (L~ hydrochloric acid, C-= 1).
(4) A solution of D-~-amino-~-(3-mesylaminophenyl)-acetonitrile L(+)-tartrate (having one molecule of acetic acid) (434 mg.) in 23.2 % hydrochloric acid (1.9 ml.) was refluxed for 4 hours. After removal of the hydrochloric acid under reduced pressure, methanol (about 5 ml.~ was added to the residue. The resulting solution was adjusted to pH 6 with methanolic ammonia and allowed to stand in a refrigerator over-night. The precipitated crystals were collected by filtration.
The collected cr~stals (132 mg.) was dissolved in water (0~5 -~
ml.) while hot, methanol (2 ml.) was added thereto and the resulting mixture was allowed to stand in a refrigerator over-- night. The precipitated crystals were collected by filtration and washed with methanol to give D-~-(3-mesylaminophenyl)-glycine (54.3 mg.). M.P. 193 to 194C (decomp.). I.R. (Nujol) v cm : 3230, 2720, 2550, 1610, 1508, 1400, 1330, 1310, 1260, 1145, 985, 783, 695, 662. M.M.R. (n2o - HCl) ~ ppm: 3.20 (3H,s) 6.38 (lH,s), 7.3-7.7 (4H,m). [~]D = -100 (lN hydrochloric acid, C = 1).
(5) To a solution of sodium cyanide (purity, 90%;
1.08 g.) and ammonium chloride (purity, 98.5 %; 1.18 g) in 30 %
aqueous ammonia (17 ml.), m-mesylaminobenzaldehyde (1.99 g.) was added thereto at 15C, and the resultant mixture was stir-red at the same temperature for 4.5 hours and excess of ammonia was distilled off at 3SC under reduced pressure. The result-ing solution was adjusted to pH 7 with 10 % hydrochloric acid and extracted with ethyl acetate (15 ml.) 4 times. The extract ~0~9S~2 was washed with an aqueous saturated solution of sodiu~ c~oride and concentrated under reduced pressure to about 10 ml. ~he concentrated solution was extracted with 10 ,~ hydrochloric acid (5 ml.) 4 times. The extract was washed with ethyl acetate ¦-(5 ml.), admixed with 35 % hydrochloric acid (8.6 ml.) and refluxed for 3 hours. The reaction mixture was treated with activated charcoal, concentrated under reduced pressure to remove the hydrochloric acid. admixed with ethanol (5 l~ll.) and then concentrated under reduced pressure to dryness. The residue was dissolved in methanol (20 ml.) by heatin~ and adjusted to pH 5 with methanolic ammonia. The precipi~ated -crystals were, after allowing to stand in a refrigerator over-night, collected by filtration, washed with methanol and dried under suction. The resulting crystals (1.93 g.) were dissolved in water (4 ml.) by heating, ethanol (8 ml.) was added thereto and the resultant mixture was allowed to stand in a refrigerator overnight. The precipitated crystals were collected by filtra-tion and dried to give D-a-(3-mesylaminophenyl)glycine (0.98 g.).
I.R. (Nujol) v cm 1 3260, 1695, 1605, 1590, 1477, 1400, 1330, 1250, 1150, 973, 890, 790, 760. N.M.R. (D20 + DCl) o ppm: 3.18 (3H,s? 5-38 (lH,s), 7.2-7.6 (4H~m)-; Example N
(1) D-a-Amino-a-(~-mesylaminophenyl)acetonitrile ~(+)-tartrate (having one molecule of acetic acid) (~35 mg.) ~-was dissolved in conc.hydrochloric acid (1.5 ml.), and the resulting solution was heated to reflux. From the reaction mixture, hydrochloric acid was removed by distillation under reduced pressure. The residue was added to a column of an ion exchange resin ("Amberlite IR-120B", ~+ type, manufactured by Rohm and Haas Co.) (10 ml.) and washed with water until the s~z washings became neutral. Then, 7 % aqueous ammonia was poured on the column. The eluate was collected and concentrated under reduced pressure. The residue was crystallized from methanol to give D-~-(3-mesylaminophenyl)glycine (220 mg.) as crystals.
M.P. 207C (decomp.). [~]D = -100 (1~ hydrochloric acid, C = 1). "Amberlite IR - 120B" is a trademark.
~2) Into a suspension of the above prepared D-~-(3-mesylaminophenyl)glycine (3.0 g.) in anhydrous methanol (60 ml.) cooled with ice water, dried hydrogen chloride gas was introduced to make saturation. The resultant mixture was allowed to stand at 4 to 5C for 40 hours. AEter removal-of the methanol by distillation under reduced pressure, the precipitate was collected by filtration, dried under reduced pressure, washed with acetone and then dried again to give D-~-(3-mesylaminophenyl)glycine methyl ester hydrochloride (having 1/2 molecule of acetone) (3.8 g.). N.M.R. (CD30D-D2O) ~ ppm: 2.16 (3~,s), 3.05 (3H,s), 3.81 (3H,s), 5.23 (lH,s), ~7.16-7.5 (4H,m). ; -Example O
A solution of D-~-amino-~-(3-mesylaminophenyl)aceto-nitrile L(+)-tartrate (having one molecule of acetic acid) (1.3 g.) in conc. hydrochloric acid (10 ml.) was allowed to stand at room temperature for 1.5 hours. The reaction mixture was concentrated under reduced pressure and dried in vacuo. The obtained yellow viscous solid was washed with acetone (10 ml.) and collected to give D-~-(3-mesylaminophenyl)glycinamide hydrochloride (580 mg.) as greenish white powder. M.P. 236 to 240C. I.R. (~ujol) V cm 1 3450_3350, 3240, 3180, 1695, 1605, 1590, 1150.
Examp~le P
!

~L~495~2 To a methanol solution saturated with hydrogen chloride (10 ml.), there were added water (one drop) and D-a- -amino-a-(3-mesylaminophenyl)acetonitrile ~ tartrate (having one molecule of acetic acid) (435 mg.), and the resulting mixture was refluxed for 1 hour. The reac-tion mixture was concentrated under reduced pressure, water (20 ml.) was added thereto, and the resultant solution was neutralized with an aqueous solution of sodium hydrogen carbonate. The neutralized solution was then extracted with chloroform. The chloroform extract was concentrated under reduced pressure to give D-a-(3-mesylaminophenyl)glycine methyl ester (230 mg,). I~.P. 101 to 153C. I.R. (Nujol) v cm 1 3370, 3300, 1740, 1605, 1590. ~--, ' . . , - . , : -

Claims (17)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:-

1. A process for the preparation of the compound of the formula:

(I) wherein R1 is hydrogen or hydroxy, R2 is alkanesulfonamido, dialkanesulfonamido or alkylaminosulfonamido, X is amino or protected amino and M is hydrogen, a nontoxic, pharmaceuti-cally acceptable cation or haloalkyl, provided that R1 is hydroxy when R2 is alkanesulfonamido, which comprises react-ing a compound of the formula:

(II) wherein M is as defined above, or its derivative at the amino and/or carboxy group, with an amino acid of the formula:

(III) wherein R1, R2 and X are each as defined above, or its reactive derivative at the carboxy group, optionally followed by elimina-tion of any protective group, if present in the resulting product.

2. A process according to claim 1, which comprises reacting a compound of the formula (II), its trichloroethyl ester or a salt thereof, with an amino acid of the formula (III) in the presence of a condensing agent, or its acid anhydride with lower alkyl chloroformate.

3. A process according to claim 1, in which R2 is alkanesulfonamido in which the alkane moiety has not more than 8 carbon atoms; dialkanesulfonamido in which the alkane moiety has not more than 8 carbon atoms; or alkylaminosulfonamido in which the alkyl moiety has not more than 8 carbon atoms.

4. A process according to claim 3, in which M is tri-chloroethyl.

5. A process according to claim 2, wherein R1 is hydroxy in the 4-position, R2 is alkanesulfonamido, and M
is hydrogen or a non-toxic, pharmaceutically acceptable cation.

6. A process according to claim 5, wherein R2 is mesylamino in the 3-position and M is hydrogen.

7. A process according to claim 2, wherein R1 is hydrogen and R2 is dialkanesulfonamido or alkylaminosulfon-amido, and M is hydrogen or a non-toxic, pharmaceutically acceptable cation.

8. A process according to claim 7, wherein R2 is di-mesylamino in the 3-position and M is hydrogen.

9. A process according to claim 7, wherein R2 is ethylaminosulfonamido in the 3-position and M is hydrogen.

10. A compound of the formula:

(I) wherein R1 is hydrogen or hydroxy, R2 is alkanesulfonamido, dialkanesulfonamido or alkylaminosulfonamido, X is amino or protected amino and M is hydrogen, non-toxic, pharmaceutically acceptable cation or haloalkyl, provided that R1 is hydroxy when R2 is alkanesulfonamido, whenever prepared by the process of claim 1 or 2, or by an obvious chemical equivalent thereof.

11. A compound of formula (I), as defined in claim 1, in which R2 is alkanesulfonamido in which the alkane moiety has not more than 8 carbon atoms; dialkanesulfonamido in which the alkane moiety has not more than 8 carbon atoms; or alkylaminosulfonamido in which the alkyl moiety has not more than 8 carbon atoms, whenever prepared by the process of claim 3 or by an obvious chemical equivalent thereof.

12. A compound of formula (I), as defined in claim 1, in which R2 is alkanesulfonamido in which the alkane moiety has not more than 8 carbon atoms; dialkanesulfonamido in which the alkane moiety has not more than 8 carbon atoms;
or alkylaminosulfonamido in which the alkyl moiety has not more than 8 carbon atoms, and M is trichloroethyl, whenever prepared by the process of claim 4, or by an obvious chemical equivalent thereof.

13. A compound of the formula (I) as defined in claim 5, whenever prepared by the process of claim 5, or by an obvious chemical equivalent thereof.

14. A compound of formula (I) as defined in claim 7, whenever prepared by the process of claim 7, or by an obvious chemical equivalent thereof.

15. 7-[2-(3-Mesylamino-4-hydroxyphenyl)-D-glycinamido]-3-methyl-3-cephem-4-carboxylic acid, whenever prepared by the process of claim 6, or by an obvious chemical equivalent thereof.

16. 7-[2-(3-Dimesylaminophenyl)-D-glycinamido]-3-methyl-3-cephem-4-carboxylic acid, whenever prepared by the process of claim 8, or by an obvious chemical equivalent thereof.

17. 7-[2-(3-Ethylaminosulfonamidophenyl)-D-glycinamido]-3-methyl-3-cephem-4-carboxylic acid, whenever prepared by the process of claim 9, or by an obvious chemical equivalent thereof.