CA1049503A - 7-acylamine substituted cephalosporin compounds and their preparation - Google Patents

Abstract

Abstract of the Disclosure Cephalosporin derivatives are provided of the formula:

(IXb) wherein R2 is lower alkyl and R4 is hydrogen, lower alkanoyl-oxy, triazolyl or thiadiazolylthio which is unsubstituted or substituted with lower alkyl, a halo(lower)alkyl ester thereof or non-toxic, pharmaceutically acceptable salts thereof:
processes for preparing the novel derivatives are similarly provided. The novel derivatives exhibit a strong antimicrobial activity against a wide variety of microorganisms including gram-negative and gram-positive bacteria; in particular their antibiotic potency is maintained at a high level for a long period of time on oral administration.

Description

-- q~l4~503 The present invention relates to cephalosporin anti-biotic compounds having an amino acid side chain at the 7-position, and processes for preparing them. There is also disclosed a novel class of amino acids for the formation of the side chain. -In this specification, the term "lower" used in con-nection with an alkyl group or moiety is intended to mean the one having 1 to 8 carbon atoms, preferably 1 to 5 carbon atoms.
Hitherto, there have been produced a great number of cephalosporin compounds. ~mong them, however, only a few have been used in practice, and there is a continuing need to develop novel cephalosporin antibiotics which can be used in practice.
As the result of the extensive study, it has now been found that the cephalosporin compounds having at the 7-position an acylamino group of the formula:

R~ CH-Co~ [a]
R~-Y-I 2 wherein Rl is hydrogen or hydroxyl, R~ is lower alkyl (e.g.
methyl, ethyl, propyl, isopropyl, butyl, pentyl), R3 is hydrogen or lower alkanesulfonyl (e.g. methanesulfonyl, ethanesulfonyl) and Y is -SO2- exhibit a strong antimicrobial activity against a wide variety of microorganisms including gram-positive and gram-negative bacteria. It is particularly notable that their antimicrobial potency is higher than that of cephalexin, which is one of the well known ~ephalosporin antibiotics, in some species of pathogenic microorganisms. It is also notable that, when orally administered to mammals, their antibiotic potency is maintained at a high level for a long period of time. Accord-ingly, they are useful as orally-administerable, long-acting antibiotics.

~495~3 The amino acids [ I ] used for the formation of the acylamino group [A] at the 7-position of the cephalosporin nucleus are fundamentally representable by the formula:

~3CH-COOH [ I ]

wherein Rl, R2~ R3 and Y are each as defined above.
When the symbol R3 represents a lower alkanesulfonyl group, it corresponds to the group represented by the formula:

In particular the invention is concerned with cephalo-sporin derivatives in which in the acylamino group at the 7-position, Rl and R3 are hydrogen:and Y is -S02-.
According to the invention there is provided compounds --of the formula:

Z 2 2 ~ ~ 2 4 (IX) OOH
wherein R2 is lower alkyl and R4 is hydrogen, lower alkanoyloxy :;
.
or thiadiazolylthio which is unsubstituted or su~stituted with ; ~:.
lower alkyl, a halo(lower)alkyl ester thereof, or non-toxic pharmaceutically acceptable salts thereof.
The term "derivative(s)" herein is used in a broad sense and may include any modified form of the amino acid ~I]
which is employed for enhancement of the reactivity of the ::
carboxyl group (the so-called "reactive derivative") or for protection of the carboxyl group and/or the ~-amino group therein from the influence of any reaction (the so-called '

- 2 - : .

~ L95C~3 "protective derivative"). Examples of the derivative on the carboxyl group are salts, esters, halides, amides, anhydrides, etc. Examples of the derivative on the ~-amino group are salts, acylamino, Schiff bases, ~tc.
The amino acids _ `) 95~3 ~I~ can be produced in a variety o~ procedures, o~ which a typical one comprises reacting a compound of the formula:

~ CH-COOH [II]

~herein Rl is as defined above in a free or protected form on the carboxyl group and/or -the a-amino group ~ith an acylat-ing agent of the formula:
R2-Y-Z [III~
wherein Z is the re~idue o~ an acid and R2 and Y are each as defined above.
When the a-amino grou~ is protected, one or two hydrogen atoms therein may be replaced, for instance, by ac~1 such as substituted or unsubstituted benzyloxycarbonyl (e.g.
ben~yloxycarbonyl, 4-nitrobenz~loxycarbonyl9 4-oro~oben~yloxy-carbonyl, 4-methoxybenzyloxycarbonyl, 3,4-dimetho~ybenzylo~y-carbonyl, 4-(phenylazo)benzylo~ycarbonyl, 4-(4-methoxyphenyl- - -~
azo)benzyloxycarbonyl), substituted or unsubstituted alXoxy-carbonyl or cycloalkoxycarbonyl (e.g. t-butoxycarbonyl, t-pentyloxycarbonyl, isopropoxycarbonyl, diphenylmetho~ycarbonyl, 2-pyridylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2,2,2 tribromoetho~ycarbonyl, l-cyclopropyletho~ycaroonyl, ~-Lodo-propoxycarbonyl, 2-furfuryloxycarbonyl, 1-adamantylo~ycarbonyl), (heterocyclic ring)oxycarbonyl (e.g. 8-quinolyloxycarbonyl~ or sub~tituted alkanoyl (e.g. trifluoroacetyl); trityl; trialkyl-8ilyl (e.g. trimethylsilyl, triethylsilyl); substituted phenyl-thio (e.gD 2-nitrophenylthio, 2,4-dinitrophenylthio), etc.
The a-amino group may be also protected in the form of a Schiff ba8e and, in such case, the two hydrogen atoms therein are 4 ~

.

~L(l 49503 replaced by substituted alkylidene (e.g. 2-hydroxybenzylidene, 2-hydroxy-5-chloro~enzylidene, 2-hydroxy-1-naphthylmekhylene,

3-hydroxy-4-pyridylmethylene, 1-methoxycarbonyl-2-propylidene, l-ethoxycarbonyl-2-propylidene, 3-ethoxycarbonyl-2-butylidene, l-acetyl-2-propylidene~ l-propionyl-2-propylidene9 l-benzoyl-2-propylidene, 1,3-bis(ethoxycarbonyl)-2-propylidene, 1,~-bis-~ethoxycarbonyl)-2-propylidene, 1-(N-methylcarbamoyl)-2-pro-pylidene, l-(N,N-dimethylcarbamoylj-2-propylidene, 1-[N-(2-methoxyphen~l)carbamoyl]-2-propylidene, 1-[N-(4-methoxyphenyl)-carbamoyl]-2-propylidene, 1-(N-phenylcarbamoyl)-2-propylidene, 2-ethoxycarbonylcyclopen~ylider.e, 2-ethoxycarbonJrlcyclohexyli-denej 2-acetoxycyclohexylidene, 3,3-dimethyl-5-oxocyclohexyli-dene) or the like. The a-amino group may be further protected in the ~orm o~ an acid addition salt, for ins'ance, hydro-ohloride, hydrobromide, hydroiodide or the like. -The carboxyl group may be protected by any conventionalprotective ~roup. ~he partic~2rly preferred protected form on the carboxyl group is a silyl ester which is obtainable by ~he reaction with a silyl compound such as trialkyLhalosilane, dialkylhalosilane, alkyltrihalosilane, hexaalkylcyclotri-silazane, octaalkylcyclotetrasilazane, trialkylsilylacetamide or bis(trialkylsilyl)acetamide.
Further, the a-amino group a~d the carboxyl group may be protected simultaneously, for instance, m a chelating ~orm intervening a metal compound. E~amples of the metal compound may be anyone which can form a chelating bond wit-h ~he a-amino group and the carboxyl grou such a~ organic and inor~anic copper, cobalt, nickel and magnesium compoundsO More specifically, t~.ere m~y be exemplified cupric chloride, cupric bromide, cupric fluoride, copper nitrate, copper sulfate, copper .

_ 5 _ , .

~ L049503 borate, copper phosohate, copper cyanidc, copper formate, copper acetate, copper ~ropionate, copper citrate, copper tartrate, copper benzoate, copper salic~late, etc.
In the acylating agent [III], the residue of an acid represented by the symbol Z may be, for instance, the residue of an acid halide such a 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 solvent are acetone, dioxane, acetonitrile, chloroform 9 methylene chloride, èthylene chloride, tetrahydro-~uran, ethyl acetate, dimethylformamide, pyridine, etc. lw~en the solvent is water-miscible, it may be enployed in the form of a mixture with water. ~uny particular limitation is not present on the reaction temperature but, in most cases, the reaction may be performed while cooling or at room temperature.
Another typical procedure for the preparation of the amino acid [I] comprises (1) reacting a compound of the formula:

~ CHO -~
H~N ~ [IV]
' ' ' wherein Rl is as defined above with t~.e acylating agent [III] , (2) reactin~ t~e resul~ing product ~ the ~ormula:

1 ~ CHO
R2 ~ I [Y ]

~. :
wherein Rl, R2, R3 and Y are each a~ defined above with .

.. . . . : , ''`'~lF .: ' :: . . . .
. .

1~49S03 hydrogen c~znide in the presence of ammonia and hydrolyzing the resultant product of the formula:

R13~3CHCN
R2-Y-N ~H2 [VI]

.
~herein Rl, R2, R3 and Y are each as defined above.
; The reaction (1) can be readily accomplished by treat-ing the compound ~IV] with the acylating agent [III] in _?
an inert solvent (e.g. water, dioxane, tetrahydrofuran, ether), if necessary, in the presence o~ a ba3e (e.g. pyridine, tri-ethylamine, dimethylaniline). The startin~ compound [rv] is :
sometimes available commercially in the polymerized ~orm and may be as such subjected to the reaction in the sa~e manner ~ -a~ ~n case of using the s~me in the monomeric form.
The reaction (2) is the so-called "Strecker mino acid synthesis" and may be carried out in a per se con~en~ional manner. ~or instance, it can be carried out by treating the compound ~VJ uith an alkali metal cyanide (e.g. sodiu~ cyanide, potassium cyanide) and an ammonium halide (e.g. a~monium chlo-ride, ammonium bromide) in aqueous ammonia.
The hydrolysis (-~) may be also carried out in a per se; conventional manner, for instance, by treatment of the compound [VI] 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 ~ingle step. On the other hand, the treatment under a 10~9503 comparatively mild 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 followin~ formula is produced as the main product:

Rl ~ IH_CONH2 [VII]
R -~-N NH

~ R3 whereln Rl, R2, R3 and Y are each as defined above. In the ;~ ~
latter case, the carbamoyl group can be readily hydrolyzed -to a carboxyl group by treatment under a relatively strong .
¢ondition as stated above. Thus, the hydrolysis may be ac-complished in two steps~
The protective group in the thus prepared amino acid [I] may be, if any, eliminated by a per se conventional procedure with or without previous isolation or purification of ~he product.
One of the typical elimination procedures for the protective grou~ on the a-amir.o group is conducted by treat-ment with an acid which is usually applied to benzyloxycarbonyl, ~ubstituted or tmsubstituted benzyloxycarbonyl, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted , i aralkoxycarbonyl, adamantyloxycarbonyl, trityl, substituted phenylthio9 substituted aralkylidene, substituted alkylidene, ~ubstituted cycloalkylide~e or the like. As the acid, there msy be used various ones, and particularly preferred is the one as can be readily distilled out under reduced pressure elimination ~e.g. formic acid, tri~luoroacetic acid). The ~ X~ Lc~ -- . , j .

~4~503 is sometimes effected in an inert solvent, usually in a water-miscible organic solvent or water or their mixture. -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 iAaloalkoxycarbonyl and 8-quinolyloxycarbonyl.
When the -amino group is protected in the form of an acid addition salt, the elimination may be performed by treatment with an organic base (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.
~ he elimination of the silyl ester to recover a carboxyl group can be accompli~shed quite easily in the presence of water. For instance, the working up of the reaction mix-ture containing the amino acid [I] 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 o~ the silyl group. Thus~ any particular operation for the ~09~95~3 elimination of the silyl group is usually not required.
When the carboxyl ~roup is protected in any form other than a silyl ester, the elimination of such protective group may be accomplished in any conventional procedure~
As stated above, however, the amino acid [I] is useful -for the formation of the acylamino group at the 7-position of cephalosporin compounds and, insofar as such use is intended,, ~ -the elimina~ion of the protective group on the a-amino group is ordinarily not required. ~ -The amino acid [I] can be present in the D-isomer or the L-isomer or in the racemic form. When obtained in the racemic form, it may be subjected to racemic resolution at any stage of the production. For instance, in the production of the amino acid LI ] by the said procedure as comprising three steps, i.e. the reaction (1), the reaction (2) and the hydrolysis (3), the final product is usually obtained in a racemic mixture, and the racemic resolution may be effected at the stage after the reaction (2) (i.e. on the compound [VI]) or at the stage after the hydrolysis (3) (i.e~ on the compound [I]). In general, however, the racemic resolution after the reaction (2) is favorable, because a better result is obtained.
In another aspect o~ the invention there is provided a process for the preparation of a cephalosporin derivative of formula (IXj, as described previously.
In one embodiment of this aspect of the invention a compound of the formula:

S
H2,~H2 -R4 ( VIII ) coo~ ; , . ~
- 10 - , , .

~49~
wherein R4 is as def.ined previously by reference to formula (IX), or a halo(lower)alkyl ester, or a salt thereof, is re-acted with a substituted phenylglycine of the formula: ~.

~ ~ H-COOH (I) R2 - S2 ~
~I2 wherein R2 is as defined previously by reference to formula (IX), ~;
or its lower alkoxycarbonyl-protected derivative at the ~-amino group, or a reactive derivative at the carboxyl group, and if desired, eliminating the ester and/or the protecting group at the ~-amino group from the reaction product.
Heterocyclic-thio groups other than thiadiazolylthio :
might also be employed in R4, for example residues of furan, ;
thiophene, pyrrole, pyrazole, imidazole, triazole, thiazole, isothiazole, oxazole, isoxazole, thiadiazole, oxadiazole, thia- :
triazole, oxatriazole~ tetrazole, pyridine, pyrazine, pyrimidine, pyridazine, benzothiophene,benzofuran, indole, indazole, benzimidazole, benzothiazole, benzothiadiazole, benzoxazole, purine, quinoline, isoquinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, pyrrolidine, imidazolidine, piperi-dine, piperazine or the like. The heterocyclic moiety of the "heterocyclic-thio group" may be substituted with one or more lower alkyls.
The derivative of the carboxyl group of the compound [VIII] may be a salt such a magnesium salt, calcium æalt, triethylamine 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, diphenyl-1. .

-;... .
. . .

~(~4~S(~3 methyl ester, bis(methoxyphenyl)methyl ester, 3,4-dimethylbenzyl ester, (l-cyclopropyl)ethyl ester, ethynyl ester, 4-hydroxy-3,4-di-t-butylbenzyl ester, etc., an amide, or the like.
The derivative at the carboxyl group and/or the amino group may be the reaction product of the compound with a silyl compound such as bis(trimethylsilyl)acetamide.
The derivative at the amino group of the amino acid [I] includes the one wherein one or two hydrogen atoms of the amino group are replaced by acyl such as substituted or un-substituted benzyloxycarbonyl (e.g. benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxy-benzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 4-(phenyl-azo)benzyloxycarbonyl, 4-(4-methoxyphenylazo)benzyloxycarbonyl), substituted or unsubstituted alko~ycarbonyl or cycloalkoxycar- ;
bonyl (eOg. t-butoxycarbonyl, t-pentyloxycarbonyl, isopropoxy-carbonyl, diphenylmethoxycarbonyl, 2-pyridylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2,2,2-tribromoethoxycarbonyl, l-cyclopropylethoxycarbonyl, 3-iodopropoxycarbonyl, 2-furfuryl-oxycarbonyl, l-adamantyloxycar~onyl),(heterocyclic ring)oxy-:, . .
carbonyl ~e.g. 8-quinolyloxycarbonyl) or substituted al~anoyl (e.g. trifluoroacetyl), trityl, trialkylsilyl (e.g. trimethyl-silyl, triethylsilyl) or substituted phenylthio (e.g. 2-nitro-phenylthio, 2,4-dinitrophenylthio). There is also included the one wherein the amino group is protected in the form of a ,, . : : , : . . -. ::

1~495~3 Schiff base and the two hydrogen atoms therein are replaced by substituted alkylidene (e.g. 2-hydroxybenzylidene, 2-hydroxy-5-chlorobenzylidene, 2-hydroxy-1-naphthylmethylene, 3-hydroxy-

4-pyridylmethylene, 1-methoxycarbonyl-2-propylidene, l-ethoxy-carbonyl-2-propylidene, ~-ethoxycarbonyl-2-butylidene, 1-acetyl-2-propylidene, 1-propionyl-2-propylidene, 1-benzoyl-2-propyli- -dene, 1,3-bis(ethoxycarbonyl)-2-propylidene, 1,3-bis(ethoxy-carbonyl)-2-propylidene, 1~(~-methylcarbamoyl)-2-propylidene, l-(N,N-dimethylcarbamoyl)-2-propylidene, 1-[N-(2-methoxyphenyl)- ~-carbamoyl]-2-propylidene, 1-[N-(4-methoxyphenyl)carbamoyl]-2-propylidene, l-(N-phenylcarbamoyl)-2-propylidene, 2-ethoxy-carbonylcyclopentylidene, 2-ethoxycarbonylcyclohexylidene, 2-acetoxycyclohexylidene, 3,3-dimethyl-5-oxocyclohexylidene).
There is further included the one wherein the amino group is protected in the form of an acid addition salt, for instance, hydrochloride, hydrobromide, hydroiodide or the like.

The reactive derivative at the carboxyl group of the amino acid [I] may be an acid halide, an acid anhydride, an activated amide, an activated ester, or the like. The suitable examples may be an acid chloride; an acid azide;
a mixed acid anhydride with an acid such as dialkylphosphoric acid, phenylphosphoric acid, diphenylphosphoric acid, di-benzylphosphoric acid, halogenated phosphoric acid, di~lkyl-phosphorous acid, sulfurous acid, thiosulfuric acid, sulfuric acid, alkylcarbonic acid, aliphatic carboxylic acid (e.g.
pivalic acid, pentanoic acid~ isopentanoic acid~ 2-ethyl~

butyric acid, trichloroacetic acid) or aromatic carboxylic acid (e.g. benzoic acid), or a symmetrical acid anhy~ride;

an acid amide with imidazole, 4-substituted imidazole, di~
methylpyrazole, triazole or tetrazole; or an ester (e.g.

.

~49503 :
cyanomethyl ester, me-thoxymethyl ester, vinyl ester, propargyl ester, p-nitrophenyl ester, 2,4-dinitrophenyl ester, trichloro-phenyl ester, pentachlorphenyl ester, methanesulfonylphenyl ester, phenylazophenyl ester, phenyl thioester, p-nitrophenyl : -thioester, p-cresyl thioester, carboxymethyl thioester, pyranyl ester, pyridyl ester, piperidyl ester, ~-quinolyl thioester, or an ester with N,N-dimethylhydroxylamine, l-hydroxy-2-(lH)-pyridone, ~-hydroxysuccinimide, N-hydroxyphthalimide), or the like. .
When the amino acid [I] is used in the form of a ~ree acid or a salt, the coupling reaction is preferably car-ried out in the presence of a condensing agent appropriately selected ~rom ~,N'-dicyclohexylcarbodiimide, N-cyclohexyl-N'-morpholinoethylcarbodiimide, ~-cyclohexyl-N'-(4-diethylamino-cyclohexyl)carbodiimide, N,N'-diethylcarbodiimide, N,N'-diiso- -propylcarbodiimide, N-ethyl-N'-(3-dimethylaminopropyl)carbodi-imide, N,~'~carbonyldi(2-methylimidazole), pentamethylene-ketene-N-cyclohexylimine, diphenylketene-N-cyclohexylimine, alkoxyacetylene, l-alkoxy-l-chloroethylene, trialkyl phosphite, .
~thyl 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)dimethylammonium chloride - .
and the like. The salt of the amino acid CI] may be an alkali salt, an al~aline 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- ~ :

- 14 - .

, ~
... ..

. . .

1~495~3 amine, ~ dialkylbenzylamine or pyridine, usually in an inert solvent. Example.s 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 waterO When the base or the condensing agent is in liquid, it can be used also as a solvent. The reaction temperature is not restrictive, and the reaction is usually carried out under cooling or at room temperature.
Depending on its kind, the protective group for the amino group may be removed in the course of the coupling re-action or the post-treatment. When the product has a protected amino group, the protective group may be eliminated therefrom, when desired, by applying a suitable procedure as mentioned above.
In another embodiment of the process of the invention the cephalosporin compound [IX: R4 is thiadiazolylthio, optionally substituted with lower alkyl] or its derivative may be also prepared by reacting the corresponding cephalosporin compound [IX: R4 = lower alkanoyloxy] or a halo(lower)alkyl ester thereof, or a salt thereof with a thiol compound of the formula:
R4-H [X]
wherein R4 is thiadiazolylthio which is unsubstituted or sub stituted with lower alkyl, corresponding to the symbol R4 in the ob~ective compound, or its alkali metal salt.
When desired the ester group is eliminated from the reaction product, and when desired the free acid of formula (IX) is converted to a corresponding non-toxic, pharmaceuti-cally acceptable salt thereof.
The alkali metal salt of the thiol compound [X~ may be a sodium salt, a potassium salt or the like.

. ~ " .

~49S03 The above reaction may be carried out in an inert sol-vent such as water, acetone, chloroform, nitrobenzene, dimethyl- ;
formamide, methanol, e-thanol or dimethylsul~oxide. Among these solvents, hydrophilic solvents may be used in a mixture with water. The reaction is preferably effected at a pH around 7 or in a nearly neutral medium. When the starting cephalosporin compound [IX: R4 = heterocyclic-thio (optionally substituted with lower alkyl)] or the thiol compound [X] is used in a free ~ -form, the reaction is favorably conducted in the presence of -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.
The cephalosporin compound [IX] thus produced may be converted into its pha~maceutically acceptable, substantially non toxic salts ~y a per seconventional procedure, for instance, by reacting with an inorganic base such as an alkali metal hydroxide, an alkali metal bicarbonate or an alkali metal car bonate or an organic base. The preferred procedure for prepara-tion of the salts consists in dissolving the cephalosporin compound [IX] in the free form into a solvent wherein the salt is insoluble and then adding a solution of the base thereto, whereby the salt is precipitated from the reaction mixture.
Broadly, the invention relates to a process for the preparation of a compound of the ormula:

, . .

~ - 16 -~al4~503 ~:EI-CONH ~ ~1 ( IX) R2 - S2 -N N~2 6~ ~ CH2--R4 COOH

wherein R2 is lower alkyl and R4 is hydrogen, lower alkanoyloxy or thiadiazolylthio which may be substituted with lower alkyl, or a non-toxic pharmaceutically acceptable salt thereof, which comprises (1) reacting a compound of the formula:

H2N--~S ~ . .
CH2-R4 (VIII) COOH

wherein.R4 is as defined above, or its halo(lower) alkyl ester, or a salt thereof with a substituted phenylglycine of the formula:

~ CH-COOH (I) R2-- S02- ~ ~
H ~H2 wherein R2 is as defined above, or its lower alkoxycarbonyl protected derivative of the ~-amino group or its reactive derivative at the carboxyl group, and if desired, eliminating the ester and/or the protecting group at the ~-amino group from the reaction product, or (2) reacting a compound of the formula: :

~ CH-CO~ S~
R2 S 2 1 ~H2 /~ ,~L CH2--R4" ( IX" ) COOH
..

~ - 16a -- , : .

~04g5i~3 wherein R2 is as defined above, and R4" is lower alkanoyloxy, or its halo(lower)alkyl ester, or a sait thereof with a thiol compound of the formula:
R4' - H (X) wherein R4' is thiadiazolylthio which may be substituted with lower alkyl, or an alkali metal salt thereof, and if desired, eliminating the ester from the reaction product.
The cephalosporin compounds ~IX] exhibit an excellent antimicrobial activity. For instance, the cephalosporin com- .
pound [IX: R2 = methyl, R4 = hydrogen, (the group R2-Y-~ being present at the R3 - 16b ~

., . :

.
1~49St)3 m-position on the benzene ring)] (hereinafter referred to as "Compound B") shows a higher antimicrobial potency than cepha-lexin as shown in Table 1.
Table 1 Minimal inhibitory concentration of Compound B and cephalexin on microorganisms determined by the two fold agar plate dilutlon method.

. _ _ .
Te~t organism Minimal inhibitory concentration (mcg/ml) Compound B Cephalexin , Staph. aureus Terashima 12.5 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 S.25 Sal. typhi T-287 3-13 6.25 Sal. typhi 0-901 3-13 6.25 Sal. para A 1015 6.25 12.5 Sal. typhimurium 1406 3-13 12.5 Sal. enteritidis 1891 3-13 6.25 Further, the cephalosporin compounds [IX] 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 B and cephalexin with elapse of time when adminstered orally to mice and rats are shown in Table 2.

, ~ . . . . .
.

~495~3 ~: -Tabl~e 2 Serum levels of Compound B and cephalexin in mice and rats.

. .. __ .. . . ... .. _ Animal Test Serum level (mcg/ml) & compound _ Dose 1 hr 2 hrs 4 hrs 6 hrs .~ .. _ .....
Mouse (I~R) Compound B ~7.6 25.0 14.7 8.4 100 mg/kg~ Cephalexin 10.~ 4.~ _ _ ..... _, _ _ ................................... ... ~ .
Rat (SD) Compound ~ 49.8 44.8 41.1 39.0 100 mg/kg Cephalexin 23.1 12.3 6.6 5.9 ~ s stated above, the cephalosporin compounds [IX]
of this invention are useful as orally administrable, long acting antibiotics. For therapeutic administration the cephalo-sporin compounds [IX] are used in the form of pharmaceutical ~;
preparations which contain said compouncls in admixture with a pharmaceutically acceptable 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, tablets 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 agents, buffers and other commonly used additives.
While the dosage of the cephalosporin compounds [IX]

will vary from and also depend upon the age and condition ofthe 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.

. .

i4~49S03 The following ~xamples are given solely for the purpose o~ illustrating the present invention, not of limit-ing the same thereto.

Example 1 (1) N-t-~utoxycarbonyl-2-(3-aminophenyl)-D-glycine (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 chloride (8.45 g.) in methylene chloride (35 ml.) was dropwise added thereto at a temperature of 3 to 5C. Stirring was continued at the same temperature for 30 minutes and then at room temperature for 4 hours. The reaction mi~ture was shaken with an aqueous solution of citric acid, washed with water, and then the methylene chloride layer was separated. The methylene chloride layer was shaken with 5 ~0 aqueous solution of sodium hydrogen carbonate, and the water 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 ~odium 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) o ppm:

1.38 (9H,s); 2.99 (3H,s); ~.09 (lH,d,J=7.5Hx); 7.00-7.60 (4H,m).
(2) The above prepared ~-t-butoxycarbonyl-2-(3-mesylaminophenyl)-D-glycine (4.0 g.) was dissolved in formic acid (100 ml.)J the solution was stirred at room temper-ature for 1 hour and the solvent was distilled off under reduced '~ ' . . .
: . , ., . , . :. . . :
-. . : , ~ , , . . :

~4951[~3 pressure, The residue was dissolved in 10 ~0 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 ~.7 g.) as colorless prisms. M.~. 196 to 198OCI
D I.R. (Nujo ~ v cm 1 3240, 2580, 1616, 1147. N.M.R. (D20 +
DCl~ ~ ppm: 3.1~ (3H,s); 5,32 (lH,s), 7,47 (4H,m), Example 2 As in Example 1, D-a-(3-mesylaminophenyl)-N~(l-methoxycarbonyl-l-propen-2-yl)glycine sodium salt was prepared from ~-(a-3-aminophenyl)-N-(l-methoxycarbonyl-l-propen-2-yl)-glycine sodium salt and methanesulfonyl chloride. M.P. 141 to 143C. I.R. (Nujo ~ ~ cm 1 3250, 1650, 1610, 1150.
N.M.R. (DMS0-d6) ~ ppm: 1.68 (3H,s), 2.92 (~H,s), 3.52 (3H,s), -4.31 (lH,s), 4.78 (IH,d,J=5.6Hz), 6.90-7.25 (4H,m), 9.58 (lH, ~ -d,J=5.6Hz).
Example 3 ~o a solution of D-a-(3~aminophenyl)-N-t-buto~y-carbonylglycine (1.34 g.) and sodium hydrogen carbonate (924 mg.) in water (25 ml.) cooled at -2C, methanesulfonyl chIoride (860 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 eliminated by filtration. The filtrate was adjusted to pH 3 with 10 % hydrochloric acid, salted out and extracted with ethyl acetate (40 ml.) twice. The extract was washed with an aqueous saturated solution of sodium chioride, , . .

~495(~3 dried over anhydrous magnesium sulfate and concentrated under reduced pressure to give a-(3-mesylaminophenyl)-N-t-butoxy~
carbonylglycine (1.4 g.). [a]D = -96 (methanol, C _ 1).

~e~ .
(1) To a solution of N~t-butoxycarbonyl-2-(3-aminophenyl)-D-glycine (5.326 g.) in methylene chloride (100 ml.), 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 (~.165 g.) was added while ice cooling (2 to 3C) 9 and a solution of methane-sulfonyl chloride (4.58 g.) in methylene chloride (25 ml.) was dropwise added thereto while ice cooling in 30 minutei3.
Stirring was continued while ice cooling for 30 minutes and the~ at room temperature ~or 3 hours. The reaction mi:cture ~a~ concentrated under reduced pressure, and the residue was shaken with ethyl acetate (100 ml.) and 2M citric acid solution (50 ml.). ~he ethyl acetate layer was separated and washed w1th 2M citric acid solution (50 mlO). The washing was ex-tracted with ethyl acetate (~0 ml.). The extract was combined with the said ethyl acetate layer, washed wi-th aqueous saturated solution of sodium chloride and extracted three times with aqueous solution of sodium hydrogen carbonate (40 ml.). The extracts were combined together, washed with ethyl acetate (50 ml.), adjusted to pX 4 with 2~ citric acid solution and ;
salted out with sodium chloride. The precipitate was extracted four times with ether (50 ml.), and the extracts were washed with an aqueous ~3aturated 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. (DMS0-d6) ~ ppm: 1.40 (9H,s), ~ --, : : , . . . . .
. . ,. : .. . . . . ,:

1049S03 i 3000 (3H,s), 5.11 (lH,d,J=8H~) 9 7000-7~60 (5H,m).
(2) The above obtained N-t-butoxycarbonyl-2-(3-mesylaminophenyl)-D-glycine was treated -as in Example 1 (2) to give 2-(3-mesylaminophenyl)-D-glycine.
Example 5 Dw2-(3-Aminophenyl)glycine (16.6 g.) was dissolved in N sodium hydroxide solution (100 ~ and dioxane (40 ml.) wa~ 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. ~he resulting mi~ture was adjusted to pH 7.0 w th 20 ~o sodium hydroxide solution, and a solution of methane-~ulfonyl chloride (17.1 g.) in dioxane (20 ~1.) was dropwise added at 26 to 28C in 1 hour9 during which 20 ~O sodium hydroxide solution (47 ml.) was dropwise added thereto to maintain pH at 5.v to 6.5. The reaction mixture was stirred at room temperature for 1 hour and adjusted to pH 3.0 with 10 % hydrochloric acid. Removal of the organic solvent by dis-tillation under reduced pressure gave cupric 2-(3-mesylamino-phenyl)-D-glycinate, whi ~ was adsorbed on an ion e.~change D resin ("Amberlite IR 120B" manufactured by Rohm and ~aas, Co,) and washed with water until the eluate showed neutral. ~he ion exchange resin was then eluted with ~mmonia water (water : 30 %
ammonia water = 14 : 1 by weight~. The eluate was concentrated under reduced pressureO The residue was dissolved in a small amount of methanolic hydrochloric acid and adjusted to pH 5 with methanolic ~monia. The resulting solution was allowed to stand overnight. The precipiated crystals were collected by ~iltration and dried to gi~e 2-(3-mesylaminophenyl)-D-glycine (15.6 g.) as pale brown needles. M.P. 186.5 to 187C.

.. . . .

1~34~51~3 I.R. (Nujo~)v cm~l: 1605 (broad), 1150. N.~I.R. (DCl + D20) o ppm: 3.17 (3~I,s), 5.16 (l~,s), 7.43 (4H,m).
Example 6 To the ~ixture obtained by the use of D-2-(3-amino-phenyl)glycine (16.6 g.) and cupric sulfate pentahydrate (26.2 g.) as in Example 5, 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 mair.tain pH 8 to 9. The reaction mixture was treated as in Example 5 to give 2-(~-dimesylaminophenyl)-D-glycine as, ~colorless prisms. M.P. 171 to 17~C (decomp.). I.R. (Nujo~ v cm 1 1650, 1160. N.M.R. -(DCl + D20) o ppm: 3.50 (6H,s), 5.33 (lE~,s), 7.61 (4H,m).
Example 7 A solution of D-(a-4-aminophenyl)-N-t-butoxycar-bonylglycine (5 g.) and bis(trimethylsilyl)acetamide (11.0 g.) in methylene chloride (120 ml.) was stirred at room temperature for 2 hours, and pyridine (2.85 g.) and a solu-tion of 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 Ior 30 minutes and allowed to stand at room temper-ature overnight. A~ter 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 aqueous saturated solution - 2~ -'' ~' ' . ' ' ,. . ' . . ' .; .',1,, ' , ~, ' ' ' ' , . : : . . ... :
. . . :: :. : ~. : ..

-104~5(33 of sodium hydrogen carbonate, The aqueous layer was separated, 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.
After removal of the solvent by distillation under reduced pressùre, the resulting oil (6.0 g.~ was pulverized with ether and isopropyl ether to give D-a-(4-mesylaminophenyl)-N-t-butoxycarb ~ylglycine (5.5 g.) as crystals. M.P. 165 to 183C.
j ~ I.R. (Nujo ~ v cm 1 3325, 3125, 1747, 1673. N.M.R. ((CD3)2C0) ppm. 1.32 (9H,s), 2.84 (3H,s), 5.0 (lH,d), 6.90-7.3 (4H,m).
ExamPle~8 N-t-Butoxyc~rbonyl-2-(3-aminophenyl)-D-glycine (3.99 g.) and N,N-bis(trimethylsilyl)acetamide (6.12 g.) were added to methylene chloride (40 ml.), and the resulting mixture was stirred at room temperature for 2 hours. To the resulting mixture, pyridine (2.4 g.) was dropwise added while ice cooling, and ethanesulfonyl chloride (3.84 g.) was dropwise added thereto.
Stirring was continued at 0 to 5C for 2 hours and then at i room temperature ~or 1 hour. The reaction mixture was washed with 2 ~O hydrochloric acid (20 mI.) twice, water (20 ml.) twice and an aqueous saturated solution of sodium chloride once in order. The methylene chloride layer was separated, dried o~er magnesium sulfate and concentrated under reduced pressure. The i 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.~3 (4H,s), 7.71 (IH,broad s), 8.59 (lH, broad s).
Exam~le 9 - 24 _ ~04~503 To a solution of D-a-~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 wa~ stirred at room temperature for ~0 minutes. After cooling with ice, pyridine (4.5 g.) was added thereto9 and then methanesulfonyl chloride (6.5 g.) was drop-wise added to the resulting mixture, ~tirring 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 Wit}l ethyl acetate and water, made acidic with phosphoric acid and the ethyl acetate layer was separated from the ~Jater layer. The ethyl acetate layer was back-extracted with an a~ueous solution of sodium hydrogen carbonate, and the water layer was separated, made acidic with phosphoric acid and back-extracted a~ain with ethyl acetate. The ethyl acetate extract was washed with water, dried and treated with activated chzrco~l. The resultant solu-tion was then concentrated to give 3-a-(3-mesylamino-4-hydroxy-phenyl)-N-t-~utoxycarbon~l~lycine (6.0 g.). I.R. (Chloroform) v cm 1 1730 (shoulder), 1715~ N.M.R. (D20-~H~C3) ~ ppm: ;
1~4 (9H,s), 3.05 (3~,s), 4.83 (lF,9), 6.87 (l~.,d,~=8H~.) Example 10 (1) 3-Aminobenzaldehyde (polymerized form; water con-tent, 3 % by weight; 5.16 g.) and water (~.~ ml.) were added to tetrahydro~uran (80 ml.), and the resulting mixture was cooled to 5 to 10C. After the addition of pyridine (7.9 g.), methane-~ul~onyl chloride (11.45 g. ? was dropwise added thereto at the ~ame temperature in 15 minutes, and stirring was continued at room temperature for 3.5 hours. The reaction mixture was con-centrated under reduced pressure, 5 5~ hydrochloric acid (120 ml.), .

' -9~03 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 (1.8 g.) as crystals. 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 the ethyl acetate mother liquor, there was further obtained the ~ame prQduct as above (0.83 g.). Total yield, 6.99 g. I.R.
D ~Nujol ~ cm 1 ~120, 1670, 1600, 1580, 1320, 1242, 116~, 1143, 997, 970, 890, 788, 753, 670. N.M.R. (DMS0-d6) o ppm: 3.03 (~H,s), 7.4-7.8 (4H,m)J 9.95 (~I,s), 10.07 (lH,broad s).
t2) To a mixture of sodium cyanide (2.18 g~
ammonium chloride (2.19 g.) and 28 % ammonia water (17 ml.) cooled at 10C, 3-mesylaminobenzaldehyde (3.98 g.) was added thereto at 10 to 15C for 4 hours while stirringO 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 (30 ml) 5 times. ~he e~tract was washed with an aqueous saturated solu-tion of sodium chloride (30 ml), dried over anhydrous magnesium ~ulfate and concentrated to give D~-a-amino-a-(3-mesylamino-phenyl)acetonitrile (4.19 g.) as an oil. I.R. (film) v cm 1 3270, 1608, 1595, 1477, 1400, 1327, 1148, 972, 890, 795, 768, 697. N.M.R. (DMS0-d6) ô ppm: 3.00 (3H,s), 5.01 (lH,s), 7.0-7.7 (4H,m).
(3) To D~-a-amino-a-(3-mesylaminophenyl)acetonitrile (2.10 g.), there was added glacial acetic acid (7 ml.), and finely pulverized ~(+)-tartaric acid (1.56 g.) was added thereto.

- 26 - ;

~ ~ 4 ~ ~ 0 3 To the resulting solution, ethyl acetate (4.7 ml.) was portion-wise added while stirring, and stirring was continued at room temperature overnight. ~he precipitated crystals were collected by filtration, washed with a mixture of ethyl acetate and acetic acid and ethyl acetate in order and dried to give D--amino-a-(3-mesylaminophenyl)acetonitrile ~(+)-tartrate (having one molecul~ of acetic acid) (3.25 g.). M.P. 97 to 98C.
I.R. (Nujol~ v cm 1 3220, 2670, 1735, 1692, 1593, 1542, 1520, 1422, 1405, 13107 1262, 1237, 1220, 1165, 1133, 1070, 978, 903, 800, 782, 663, 602. N.M.R. (D20-DCl) o ppm: 2.13 (3H,s), 3.20`(3H,s), 4.78 (2H,s), 5.87 (lH,s), 7.3-7.7 (4H,m).
j [~]D = ~31 (lN hydrochloric acid, C = 1).
(4) A solution of D-a-amino-a-(3-mesylaminophenyl)-acetonitrile ~(+)-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. ~he resulting solution was adjusted to pH 6 with methanolic ammonia and allowed to stand iIl a refrigerator over-nigh-t. The precipitated crystals were collected by filtration.
The collected crystals (132 mg.) was dissolved in water (0.5 ml.) while hot, methanol (2 ml.) was added thersto and the resulting mixture was allowed to stand in a refrigerator over-I night. The precipitated crystals were collected by filtrationand washed with methanol to give D-a-(3-mesylaminophenyl)-glycine (54.3 mg.). M.P. 193 to 194C (decomp.). I.R. (Nujol cm~l: 3230, 2720, 2550, 1610, 1508, 1400, 1330~ 1310, 1260, 1145, 985, 783, 695, 662. N.M.R. (D20-HCl) o ppm: 3.~0 (3H,s) 6.38 ~lH,s), 7.3-7.7 (4H,m). [a]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 35C under reduced pressure. The result-ing solution was adjusted to pH 7 with 10 ~o hydroc~.loric acid and ext~acted wlth ethyl acetate (15 ml.) 4 times. The extract was washed with an aqueous saturated solution of sodium chloride and conc~ntrated under reduced pressure to about 10 ml. The concentrated solution was extracted with 10 ~0 hydrochloric acid (5 ml.) 4 timesO The e~tract was washed with ethyl acetate (5 ml.), admixed with 35 % hydrochloric acid (8.6 ml.) and refluxed for 3 hours. The reaction mixture was treatea with activated charcoal, concentrated under reduced pressure to remo~e the hydrochloric acid, admixed with ethanol (5 ~1.) and then concentrated under reduced pressure to dryness. The re~idue was dissolved in methanol (20 nl.) by heating 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 dissol~ed 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 gi~e D~a-(3-mesylaminophenyl)gl~cine (3.98 g.).
D I.R. (Nujol ~v cm 1 3260, 1695, 1605, 1590, 1477, 1400, 1330, 1250, 1150, 973, 8909 790, 760. N.M.R. (D20 + DCl) .S ppm: 3.18 (3H,s? 5 38 (lH,s), 7-2-7-6 (4H,m).
~ample ll , 10 9L9 5(13 (1) D-a-Amino--(3-mesylaminophenyl)acetonitrile tartrate (having one molecule of acetic acid) (435 mg.) ~as dissolved in conc.hydrochloric acid (1.5 ml.), and the resulting solution was heated to reflux. ~rom the reaction ~lxture, hydrochloric acid was removed by distillation ur.der reduced pressureO The residue was added to a col~mn of an ion e~change resin ('`Amberlite IR-120B", H+ type, manufzc~ured by Rohm and Haas Co.) (10 ml.) and washed with water until the washings became neutralO ~hen, 7 ~ aaueous a~monia was poured on the columnO The eluate was collected and concentrated under reduced pressure. The residue was crystaIlized from methanol to give D~-(3-mesylaminophenyl)glyci~e (220 mg.) as crystals.
M.P. 207C (decomp.). ~a]D = -100 (IN hydrochloric acid, C = 1).
-(2) Into a suspension of the above prepar~d D-a-(3 mesylaminophenyl)glycine (3.0 gO) in anhydrous methanol (60 ~1.) cooled with ice water, dried hydro~en chloride ~as was introduced to make saturation. ~he resultant mixture ~as allowed to ~tand at 4 to 5~C for 40 hours. After 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-a-(3-mesylaminophenyl)glycine methyl ester hydrochloride (having 1/2 molecule o~ acetone) (3.8 g.). N.M.R. (CD30D-D20~ ~ ppm: 2.16 (3H,s), 3.05 (3H,s), 3.81 (3H,s)~ 5.23 (lH,s), 7.16-7.5 (4H,m).
Example 12 ~ solution of D--amino-a-(3-mesylaminophenyl)aceto-nitrile L(~)-tartrate thaving 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 ~acuo. The obtained yellow viscous solid was washed with acetone (10 ml.) and collected to give D-a-(3-mesylaminophenyl)glycinamide hydrochloride (580 ~.) as greenish white powder. M.P. ~36 to D 240C I.R. (Nujol ~v cm 1 3450_3350, 3240, 3180, 1695, 1605, 1590, 1150.
Example 13 To a methanol solution saturated with hydrogen chloride (10 ml.), there were added water (one drop) and D-a-amino-~-(3-mesylaminophenyl)acetonitrile ~ tartrate (having one molecule of acetic acid) (435 mg.), and the resulting mi~ture was reflu~ed for 1 hour. The reaction mixture was con~entrated ~ncler reduced pressure, water (20 ml.) was added thereto, and the resultant solution was neutralized with an aclueous solution of sodium hydrogen carbonate. The neutralized .. .
~olution was then extracted with chloroform. The chloroform e~ctract was concentrated un~er reduced pressure to gi~e D-a-(3-mesylaminophenyl ~lycine methyl ester (2~0 mg.). M.P. 101 to 153~. I.R. (~ujol ~v cm 1 3370, 3300, 1740, 1605, 1590.
Exam~le A
; To a suspenæion of 2,2,2-trichloroethyl 7-amino-3-methyl-3-cephem-4-carboxylate hydroc~loride (~820 mg.) in absolute methylene chloride (50 ml.), triethylamine ~810 mg.) and N,N-dimethylaniline t245 mg.) were added dropwise under ice-coolin~ with stirring, and stirring was continued at room temperature for 30 minutes. Separately, N-t-buto2ycarbonyl-2-(3-mesylaminophenyl)-D-glycine ~3440 mg.~, triethylamine (1010 mg.~ and N,N-dimethylbenzylamine t4 drops) were added to absolute methylene chloride (50 ml.), and the mixture was stirred under ~ 3~ -.

~ 0~5~3 dry ice-acetone cooling. A solution of e-thyl chlorofor ate ~1085 m_.~ in absolute methylene chloride (25 ml;) ~Jas dropwise added thereto at -25 to -30C in 10 minu-tes, and stirrin~ ~as conducted æt the same temperature for 15 minutes. mO 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 washed with water, 3 % hydrochloric ~ -acid and water i~ order. ~he organic layer was ~eparated, washed with 3 ~0 aqueous solution of sodium bicarbonate and water in turn and then dried. ~he methylene chloride ~æs removed under reduced pressure, the residue was dissolved in ethyl acetate (10 ml.), and ether was added thereto. The precipitated crystals were filtered to give 2,2,2-trichloroethyl 7-[D-N-t-butoxycarbonyl-2~ mesylaminophenyl)glycinarnido~-3-methyl-~-cephem-4-carboxylate (4195 mg.). M.Y. 204 to 205C (decomp.).
The above obtained 2,2,2-trichloroethyl 7-[D-N-t-but~xyc2rbonyl~ -mesylam.inophenyl)~rlycinar;.ido]-3-methyl-3-cephem-4-carboxylate (~985 mg.) was di~solved in dimethylform-amide (17 ml.). To the solution were added acetic acid (5.0 ml.)and zinc powder (~985 mg.) under ice coolin~ with stirrin~, and the mixture was stirred at the same temperature for 2 hours.
The insolu~le material was collected by ~ ration and washed with dimethylformamide (~ ml.). The washings and the filtrate were combined together and added to 50 ~0 hydrochloric acid (100 ml.) under ice cooling. ~iater (50 ml.) was added thereto, and the resultant mixture was extracted three times with ethyl acetate (50 ml.). The extracts were combined together, washed with water and back-extracted three tir1es with 5 ~0 aq~eous solution of sodium bicarbonate (50 rnl.). The aqueous layer was acidified with hydrochloric acid and back-extrac~ed a~ain with ''`~

. . -. . .

ethyl acetate. The extract ~as washed with water~ dried and concentr~ted under reduced pressureO The residue was dissolved in ethyl acetate (10 ml.) and allo~Jed to stand at room tempera-ture for an hour. Ether was added thereto, whereby 7-[D-N-t-butoxycarbonyl-2-(3-mesylaminophenyl)glycinamido~-~-methyl 3-cephem-4-carboxylic acid (2687 mg.) ~ras obtained. M.P. 187 to 189C (decomp.).
Exam~)le ~3 ' . ~' To a susper~sion of 2,2,2-trichloroethyl 7-amino-3-methyl-3-cephem-4-carboxylate hydrochloride (26.7 g.) in methyl-ene ~hloride (500 ml.), there were added successively a solution of triethylaDIine (7 ~.) in methylene chloride (25 ml.) and a solu~ion of 2,6-lutidine (2.14 ~.) in methylene chloride (25 ml.) under ice coolingc N-t-Butoxycarbonyl-2-(3-mesylaminophenyl)-D~glycine (26.0 g.) and then dicyclohexylcarbodiimide (15 ~.) were gradual y added +hereto, and the mixtuL~e was stirred under ice coolin,7 for 1.5 hours. After removal of the insoluble material by filtration, the filtrate was washed four ti~es with ice-cooled -~

5~% hydrochloric acid (100 ml.), once with water, three times with 10 ~0 aqueous solution o~ sodium bicarbonzte and once with an aqueous saturated soiution of sodium chloride and then dried over magnesium sul~ate. The solution was treated with acti-~ated charcoal, and the solvent ~!as removed under reduced pressureO The residue was dissolved in ethyl acetate (100 ml.) and allowed to stand at room temperature overni~ht. The pre-cipitated crystals were collected by filtration to gi~e 2,2,2-trichloroethyl 7-[D-~I-t-butoxycarbonyl-2-(3-me~ylaminophenyl) glyciIlamidol-3-lllethyl-3-cephem-4-ca~rboxyl~te (~ilo~ g- ) as colorless plates. ~I.P. 189 to 191C (decomp.)0 ~he above obtained 2,2,2~trichloroethyl 7-[D_t-.

~495(~3 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 ~inc powder (12 g.) were added ~o the solution under ice cooling, and the mixture was Icept 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 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 10 % hydro-chldric acid. The precipitated substance was extracted three times with ethyl acetate (150 ml.). The extract was washed with water and dried over magnesium sulfate, and the solvent was removed under reduced pressure. The residue was washed with ethyl acetate and ether to give 7-CD-N-t_butoxycarbonyl-2-(3-mesyl-aminophenyl)glycinamidol-3-methyl-3-cephem-~4-carboxylic acid (8.38 g.). "~M.P.188 to 189C (decomp.).
The above obtained 7~D-N-t-butoxycarbonyl-2-(3-mesylamino)glycinamido]-3-methyl-3-cephem-4-carboxylic acid (8,28 g.) was added to formic 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-mesylaminophenyl)glycinamido~-3-methyl~3_cephem-4-carboxylic acid (6.18 g.). M.P. 199 to 199.5 C (decomp.). ~ D2 = +131 (O.lN HCl, C=l)o ~.
Example C
.
A solution of etbyl chloroformate (1.32 g.) in methylene chloride (20 ml~) was cooled to -10C, and a solution of N-t-butoxycarbonyl-2-(3-e~hanesulfonamidophenyl)-D-glycine (4.4 g.) and triethylamine (1.22 gO) in methylene chloride (20 ml.) and N,N-dimethylbenzylamine (2 drops) was dropwise added thereto in ]0 minutes. The resulting mixture was stirred at room tempera-ture for an hour. Separately9 2,2,2~trichlo~oethyl 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 minutes. The resul~ant solution was kept stirred at the same temperature for 2 hours, washed twice with 2 % hydrochloric acid, an aqueous solution of sodium bicarbonate and an aqueous saturated solution of sodium chloride and dried over magnesium sulfate. Removing the solvent under reduced pressure, the res~due was pulverized with a small amount of ethanol to give 2,2,2-trichloroethyl 7-~D-N-t-butoxycarb onyl- 2-(3-ethanesulfonamidophenyl)glycinamid oJ-3-methyl-3-cephem-4-carboxylate (4.2 g~) as colorless crystals.
M.P. 117C.
The above obtained 2,2,2-trichloroethyl 7-~D-N-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 m.).
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 95~3 was filtered off, the filtrate 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 ith ethyl acetate (20 ml.). The ethyl acetate layer and the ethyl acetate extract were combined 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 sulfate. The solvent was removed under reduced pressure, and the residue was washed wi~h diisopropyl ether to give 7 ~D-N-t-butoxycarbonyl-2_(3-ethanesulfonamidophenyl)glycinamido~-3-methyl-3-cephem-4-carboxylic acid (3.2 g.) as an oil.
A solution of the above obtained 7-~D-N-t-butoxycarbonyl-2-(3-ethanesulfonamidophenyl)glycinamido~-3-methyl-3_cephem-4 carboxylic acid (3~1 g.) in ~ormic acid (15 ml.) was stirred at room temperature for 2 hours. The formic acid was removed from the solution at room temperature under reduced pressure~ The residue was pulverized with ether and filtered. The resultant powder was added to 10 % aqueous acetonitrile (20 ml.) and stirred under ice cooling for an hour. The precipieated crystals were collected by filtration to give 7-~D-2-(3-ethanesulfonamido-phenyl)glycinamido~ 3-methyl-3-cephem-4-carboxylic acid (1.7 g.) as white crystals. M'.'P. 179 to 182 C (decomp.).
Example D
292,2-Trichloroethyl 7-~D-N-t-butoxycarbonyl-2-(3-mesylaminophenyl)glycinamido~ methyl-3-cephem-4-carboxylate (1.0 g.) was added to ice-cooled formic acid (20 ml.)~ and the mixture was stirred a~ room temperature for 2 hoursr Removing the formic acid under reduced pressure~ wa~er was added to the oily residue. The mixture was ad~usted to pH ô to 9 with an aqueous saturated solution of sodium bicarbonate under ice .35-:
:

~495~3 cooling. The precipitated crystals were collected by filtratlon, washed with wa~er 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~.
Example E
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 solutlon 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.). N-t-butoxycarbonyl-2-(4-mesylaminophenyl)-D-glycine (5.8 g.) and dicyclohexylcarbodi-imide (3.3 g.) were added to the resulta~ solution under ice cooling. The mixture was kept stirred at the same temperature for 3 hours and filtered. The filtrate was concentrated under reduced pressure~ and ethyl acetate (200 mll) was added to the residue~ The mixture was washed with 5 % bydrochloric acid, water~ an aqueous saturated solution of sodlum bicarbonate and water in order~ dried and then concentrated to give 2~2~2-trichloroethyl 7-~-N-~qbutoxycarbonyl-2~(4-mesylaminophenyl)-glycinamidol-3-methyl-3_cephem-4-carboxylate (10.83 g.). M.P.
129 to 136C (decomp.).
Acetic acid (12.5 ml.) and zinc powder (10 8-) were added to a solution of 2,2,2-trichloroethyl 7-~D-N~t-butoxy-carbonyl-2-(4-mesylaminophenyl)glycinamido~-3-methyl-3-cephem-4-carboxylate (10 g.) inddimethylformamide (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 aceeate (100 ml.) and extracted three times with ethyl 5~3 acetate (100 ml.). The extract was back-extracted twice with 5 % 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 extrac~ was washed with water and dried~ and then the solvent was removed under reduced ~pressure to give 7- -D-N-t-butoxycarbonyl-2_(4-mesylaminophenyl)glycinamidol-3-methyl-3-cephem-4-carboxylic acid (7.8 g.). M.P. 180 to 200C
(decomp.).
7-~D-N-t-Butoxycarbonyl-2-(4-mesylaminophenyl)glycin-amido~ 3~methyl-3-cephem-4-carboxylic acid (4.6 g.) was added to formic acid (70 ml.) under ice cooling. The resultant mix-ture was kept 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 solution of sodium bicarbonate, concentrated to one half of its initial vo-lume~ ad-sorbed on a resin adsorbent (trade mark "Amberlite XAD-2") (460 g.)~
which was prewashed with methanol 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_ceph~m -4-carboxylic acid (2.1 g.). M.P. 205 to 207C (decomp.).
N-t-Butoxycarbonyl-2-(3-mesylaminophenyl)-D-glycine (2.066 g.), triethylamine (0.606 g.) and N~N-dimethylbenzylamine (15 ml.) were added to tetrahydrofuran (20 mlO), 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 :

.

~L~4~D5~3 thereto at the same tamperature 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 (1.070 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.
An aqueous saturated solution of sodium bicarbonate (15 ml.) was added to the residue, and the resultant mixture was washed ewi~e with ethyl acetate (10 ml.). The washings were extracted with an aqueous saturated solution of odium 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 % 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.l~, dried over magnesium sulfate and treated with activated charcoal.
Removal of the solvent gave pasty 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-2-(3-mesylaminophenyl)glycinamido~
3-methyl-3-cephem-4-carboxylic acid (2.326 g.). M.P. 174C
(decomp.).

10~9$03 , N-t-Butoxycarbonyl-2-(3-mesylaminophenyl)-D-glycine (3.44 g.) and triethylamine (1 oOl g~ ) were dissolved in methylene chloride (25 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 stirred at the same temperature for 15 minutes. Separately, N,0-bistrimethylsilyl acetamide (3.5 g.) was dissolved in a suspension of 7-amino-3-(5-methyl-1,3,4-thiadiazol-2-yl)-thiomethyl-3-cephem-4-carboxylic acid (3.44 g.) in methylene chloride (30 ml.), and the resulting solution was dropwise 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 was removed~ The oily residue was purified by column chromato~raphy on silica gel (eluent: chloroform) to give 7-[D-N-t-butoxy-carbonyl-2-(3-mes~laminophenyl)gl~cinamido]-3-~5-methyl-1,3,4-thiadiazol-2-yl)thiomethyl-3-cephem-4-carboxylic acid ~3.8 g.) as an oil.
The thus obtained 7-[D-~-t-butoxycarbonyl-2-(3-methylaminophenyl)glycinamido]-3-(5-methyl-19 3,4-thiadiazol-2-yl)thiomethyl-3-cephem-4-carboxylic acid (2~23 g.) was dissolved in formic acid (35 ml~) and stirred at 18 to 20C
~or 4 hours. The resultant mixture was concentrated ~nder reduced pressure, pulverized with ethyl acetate and then filtered to give 7-[D-2-(3-mesylaminophenyl)glycinamido]-3-(5-methyl-1,3,4-thiadia201-2-yl)thiomethyl-3-cephem-4-carboxylic acid (1.95 g.). The product was added to acetone (30 ml.), stirred at 15 to 20C for an hour and allowed to stand. After removal ' `''''lIF
.

10~9503 of the supernatant qolution by decantation, acetone (30 ml.) was added to the residue and stirred at 15 to 20C for ~ hour~.
~he precipitate was collected by filtration and washed with acetone and ether to give the objective compound as pale yellow powder.
Exam~le H
(1) Dicyclohexylcarbodiimide (2.0 g.) was added under ice cooling with stirring to a solut-on of ~-t-butoxy-carbonyl-2-(3-mesylamino-4-hydroxyphenyl)-D-glycine (~.2 g.), 2,2,2-trichloroethyl 7-amino-3-meth~1-3-cephem-4-carboxvlate hydrochloride (5.52 g.) and 2,6-lutidine (1.74 g.) in methylene chloride (130 ml.). The resultant mixture was kept stirring at the same temperature for an hour and at room temperature ~or 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 p~ 2 ~ith phosphoric acidO The ethyl acetate layer w~s separated, washed with water and dried over ma~nesium sulfate. Removing the ethyl acetate under reduced pressure, the residue was pulverized with isopropyl ether to give 2,2,2-tricrloroethyl 7-[~1-t-butoxycarbonyl-2-(3-mesy]amino-4-hydroxyphen~rl)-D-glycinamido]-3-methyl-3-cephem-4-carboxylate (6.75 g.). ~he product was crystallized from ethyl acetate to give the objective ~compound. M.P. 185 to 188.5C (decomp.).
(2) Acetic acid ~3.5 ml.) and zinc powder (2.6 g.)' were added under ice coolin~ to a solution of 2,2,2-trichloro-ethyl 7-[N-t-buto~ycarbonyl-2-(3-mesylamino-4-hydro~yphenyl)-D-glycinamido]-3-methyl-3-cephem-4-carboxylate (3.0 g.~ in dimethylformamide (9 ml.), and the resulting mixture was kept stirred at the same temperature for 40 minutes. After the f , ~

- , ~

i i--~
~9S~3 reaction was over, the zinc powder was collected by filtration and washed with dimethylformamide~ The filtrate and the wash-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 ~a~ acid-fied with hydrochloric acid and extracted with ethyl acetate. The ethyl acetate extract was washed with water, dried over magnesium sulfate and concentrated under reduced pressure. The re~idue was washed with ether to give 7-LN-t-butoxycarbonyl-2-(3-mesylamino-4-hydroxyphenyl)-D-glycinamido]-3-methyl-3-cephem-4-carboxylic acid (1.58 g.) as powder.
(3) A solution of 7-[N-t-butoxycarbonyl-2-(3-mesylamîno-4-hvdroxyphenyl)-D-glycinamido]-3-methyl-3-cephem-4--carboxylic acid (1.45 g~) in formic acid (6 ml.) was stirred at 40C for 1.5 hours. After removal of the acetic acid under reduced pressure, acetonitrile (30 ml.) and water (0.5 ml.) were added to the residue with stirring, ancl stirring was continued for 30 minutesl The precipltated powder was filtered and washed ~th acetonitrile and ether to gi~e 7-~2-(3-mesylamino-4-hydroxyphenyl)-~-glycinamido]-3-methyl-3-cephem-4-carboxylic acid (1.13 g.). M.P. 186 to 192C (decomp.).
Exam~le I
(~) To a solution of N-t-butoxycarbonyl-2-(3-mesyl-aminophenyl)-D-glycine (9.93 g) in a mixture of tetrahydrofuran (140 ~1) and triethylamine (3.22 g) '~ept at -10C, isobutyl chloroformate (3.95 g) was added, and the resultant mixture ~as stirred at -10C for 20 minutes. A cold solution o~
7-aminocephalosporanic acid (7.87 g) in a mixture of triethyl-amine t3.5 g), tetrahydro uran (40 ml) and wa~er (40 ~1) was , 1~1t495~3 addcd thereto all at once, and the resulting mixture was stirred while ice cooling for 1 hour and at room temperature ~or 2 hours.
After the reaction was co~pleted, the tetrahydrofuran was removed by distillation, and water (100 ml) was added thereto, The resulting solution was adjusted to pH 2.5 with 10 ~0 hydrochloric acid while ice cooling and shaken ~rith ethyl acetate. The ethyl acetate layer was separated, washed ~rith water, dried and concentrated under reduced pressure. The residual oil was treat-ed with ether to give 7-[D-~J-t-butoxycarbonyl-2-(~-mesylamino-phenyl)~lycin2mido]-3-acetoxymethyl-3-ce~hem-4-carboxylic acid (10.2 g) as yellow powder, M.P. 83 to 84C (decomp.).
(2) A solution o~ 7-[D-~-t-buto~ycarbonyl-2-(3-mesylaminophenyl)glycinamido~-3-acetoxymethyl-3-cephem-4~
car~oxylic acid tl g) in formic acid (15 ml) was stirred at room temperature for 7 hours. After removal of the for~ic aci~ by distillation, water (10 ml) ~as added thereto, and the resultant solution ~as washed with ethyl acetate and stirred D with a solution o~ an ion exchange resin (`'Amberlite IA-l' manufactured by Rohm and Haas Co.) in methylisobutylketone (3 ml) for 1 hour. ~he water layer was separated, washed ~ith ether and lyophiliæed to give 7-[D-2~(3-mesylaminophenyl)~
glycinamido]-3-acetoxymethyl-3-cephem-4-carboxylic acid (0~63 g) as colorless powder. M.Pc 240C (decomp.).
j Example J
.
Sodium bicarbonate (0.86 g.) was added to a stirred - su~pension of 7-[D-~-t-butoxyc~rbonyl-2-(3-mesylaminophenyl)-glycina~ido]cephalo~poranic acid (3 g.) in phosphate buffer (pH 6.4) (130 ml.), and acetone (80 ml.) was added thereto.
5-Methyl 1,3,4-thiadia~ole-2-thiol (0.68 g.) was added to the solution and stirred at 60 to 65C for 6 hours. Acetone - 42 ~

.

1(~4~S03 was removed from the resultant mixtv.re under reduced pressure, and the remaining aqueous layer ~Jas washed with ether. ~he aqueous solu~ion was adjusted to pH 2 wi~h dilute h~drochloric acid and then extracted with ethyl acetate. The extract was washed with water, dried over magnesium sul~ate and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel~(eluant: chloroform) to give 7 [D-N-t-butoxycarbonyl-2-(3-mesylaminophenyl)glycinamido]-3-(5-methyl-1,3 9 4-thiadiazol-2-yl)thiomethyl-3-cephem-4-carboxylic acid (1.8 g.).
Example K ...

N-t-butoxycarbonyl-2-(3-dimesylaminophenyl)-D-glycine derived ~rom 2-(3-dimesylaminophenyl)-D-glycine in 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-butoxy- .
carbonyl-2-(3-dimesylaminophenyl)-D-glycinamido)-3-methyl-3-cephem-4-carboxylate. Thus obtained 2,2,2-trichloroethyl 7-(N-t-butoxycarbonyl-2-(3-dimesylaminophenyl)-D-glycinamido)-3-methyl-3 cephem-4-carboxylate was subjected to elimination of 2,2,2-tri-chloroethyl group to give 7-(N-t-butoxycarbonyl-2-(3-dimesyl-aminophenyl)-D-glycinamido)-3-methyl-3-cephem-4-carboxylic acid, which was further subjected to elimination of t-butoxycarbonyl group to give 7-(2-(3-dimesylaminophenyl)-D-glycinamido)-3-methyl-3-cephem-4-carboxylic acid in similar manner to Example C.

~ 43 ~

' " ' ~ ' " ' ' ' ' , 49S~3 SUPPLEMENTARY DISCLOSURE
This disclosure and the principal disclosure are con-cerned with novel cephalosporin compounds having an amino acid side chain at the 7-position.
It has been found that cephalosporin compounds of formula (IX), as defined in the principal disclosure, in which R4 is triazolyl display strong antimicrobial activity similar to the compounds described in the principal disclosure.
Thus, according to the invention there is provided a process for the preparation of a compound of the formula:

~ CH-CO~H ~ ~

R2a~S02-N NH2 COOH (IXa) wherein R2a is a lower alkyl and R4a is triazolyl, or a non-toxic pharmaceutically acceptable salt thereof, which comprises (1) reacting a compound of the formula:

H2N ~
~Ni~--CH2-R~ ( VIIIa ) O I ~:
COOH

wherein R4a is as defined above, or its halo(lower) alkyl ester, or a salt thereof with a substituted phenylglycine of the formula: -CH-CO~H
~ I (Ia) R2a S2 NH ~H
, ~49~;~D3 wherein R2a is as defined above or its lower alkoxycarbonyl-protected derivative at the ~-amino group or its reactive derivative at the carboxy group, and if desired, eliminating the ester and/or the protecting group at the ~-amino group from the reaction product, or t2) reacting a compound of the formula:

~ CHCONH S
R2a_sO2_N ~ C=J ~ ~1~ 2 4a (IX"a) COOH
wherein R2a is as defined above, and R4a''~ is lower alkanoyloxy, or its halo(lower)alkyl ester, or a salt thereof, with a thiol compound of the formula:

4a H (Xa) wherein R4a' is triazolylthio, or an alkali metal salt thereof, and if desired, eliminating the ester from the reaction product.
Preferably, a compound of the formula (IIa) or a salt thereof is reacted with a substitu~ed phenylglycine of the formula (IIIa), or its lower alkoxycarbonyl-protected deriva-tive at the ~-amino group, or its reactive derivative at the carboxy group, and if desired, the protecting group at the ~-amino group is eliminated from the reaction product.
For example, 7-[D-2-(3-mesylaminophenyl)glycinamido]-3 -(lH-1,2,3-triazol-5-yl)thiomethyl-3-cephem-4-carboxylic acid is obtained by reacting 7-amino-3-(lH-1,2,3-triazol-5-yl) thiomethyl-3-cephem-4-carboxylic acid with D-N-t-butoxycarbonyl-2-(3-mesylaminophenyl)glycine, and eliminating the t-butoxy-carbonyl group from the reaction product.
Example L
To a solution of N-t-butoxycarbonyl~2-(3-mesylamino-phenyl)-D-glycine (4.66 g), triethylamine (1.21 g) and N,N-dimethylbenzylamine (0.1 g) in dried tetrahydrofuran was added .. ' .: ' ~ '. . . ~ - ':

~lO49503 isobutyl chloroformate (1.64 g) at 10C to -13c with stirring and the solution was stirred at the same temperature for 30 minutes. A solution of 7-amino-3-( lH-l, 2,3-triazol-5-ylthio-methyl-3-cephem-4-carboxylic acid (3.13 g) and triethylamine (1.16 g) in tetrahydrofuran (30 ml) and water (30 ml) was added all at once to the solution obtained above at -10C to -13C
with stirring.
The mixture was stirred at -5C for an hour and then at room temperature for 2 hours. After removing tetrahydrofuran from the resultant solution, the residue was washed with ethyl acetate. After pouring ethyl acetate on the aqueous layer, the aqueous layer was ac~difie~ to pH 2 and shaken fully. After separating the ethyl acetate layer, the aqueous layer was ex-tracted with ethyl acetate twice. The ethyl acetate layer and extracts were combined together, washed with water and an aqueous solution of sodium chloride in turn, and then dried over magne-sium sulfate.
The solution was concentrated to give a pasty oil of 7-(D-~-t-butoxycarbonyl-2-(3-mesylaminophenyl~glycinamido)-3-(lH-1,2,3-triazol-5-yl)thiomethyl-3-cephem-4-carboxylic acid (6.8 g). The product was purified by column chromatography on silica gel (eluent: ethyl acetate (5) : benzene (5) : acetic acid (1)) to give pure product (1.4 g), mnp. above 160C. ~dec.). -7-(D-N-t-Butoxycarbonyl-2-(3-mesylaminophenyl)-glycin-amido)-3-(lH-1,2,3-triazol-5-yl)thiomethyl-3-cephem-4-carboxylic acid (1.35 g) was added into formic acid (13.5 ml) and stirred at room temperature for 2.5 hours. After removing formic acid ;~
from the resultant solution, the residue was pulverized with 95%
acetonitrile. The powder was collected by filtration and washed with acetonitrile to give 7-(D-2-(3-mesylaminophenyl3glycinamido) 3-(lH-1,2,3-triazol-5-yl)thiomethyl-3-cephem-4-carboxylic acid (1.13 g) m.p. above 175C~ (dec.).

.

.~ .

Claims (29)

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 a compound of the formula:

(IX) wherein R2 is lower alkyl and R4 is hydrogen, lower alkanoyloxy or thiadiazolylthio which is unsubstituted or substituted with lower alkyl, a halo(lower)alkyl ester thereof, or a nontoxic pharmaceutically acceptable salt thereof, which comprises (1) reacting a compound of the formula:

(VIII) wherein R4 is as defined above, or a halo(lower)alkyl ester, or a salt thereof with a substituted phenylglycine of the formula:

(I) wherein R2 is as defined above, or its lower alkoxycarbonyl-protected derivative at the .alpha.-amino group, or a reactive derivative at the carboxyl group, and if desired, eliminating the ester and/or the protecting group at the .alpha.-amino group from the reaction product, or (2) reacting a compound of the formula:

(IX") wherein R2 is as defined above, and R4" is lower alkanoyloxy, or a halo(lower)alkyl ester thereof, or a salt thereof with a thiol compound of the formula:
R4' - H (X) wherein R4' is thiadiazolylthio which is unsubstituted or sub-stituted with lower alkyl, or an alkali metal salt thereof, and if desired, eliminating the ester from the reaction product:
and when desired converting a free acid of said formula (IX) to a corresponding non-toxic, pharmaceutically acceptable salt thereof.

2. A process according to claim 1, which comprises reacting a compound of the formula (VIII), or its trichloro-ethyl ester, or a salt thereof with a substituted phenyl-glycine of the formula (I) in the presence of a condensing agent, or its acid anhydride with lower alkyl chloroformate.

3. A process according to claim 2, wherein R4 is hydrogen.

4. A process according to claim 3, in which 7-[D-2-(3-mesylaminophenyl)glycinamido]-3-methyl-3-cephemm-4-carboxylic acid is obtained by reacting 7-amino-3-methyl-3-cephem-4-carboxylic acid, or its trichloroethyl ester, or a salt thereof with N-t-butoxycarbonyl-2-(3-mesylamino-phenyl)-D-glycine, and if desired, eliminating the t-butoxy-carbonyl group and trichlo-roethyl group from the reaction product.

5. A process according to claim 3, in which 7-[D-2-(3-ethanesulfonamidophenyl)glycinamido]-3-methyl-3-cephem-4-carboxylic acid is obtained by reacting 7-amino-3-methyl-3-cephem-4-carboxylic acid or its trichloroethyl ester with N-t-butoxycarbonyl-2-(3-ethanesulfonamidophenyl)-D-glycine, and if desired, eliminating the t-butoxycarbonyl group and trichloro-ethyl group from the reaction product.

6. A process according to claim 3, in which 7-[D-2-(4-mesylaminophenyl)glycinamido]-3-methyl-3-cephem-4-carboxylic acid is obtained by reacting 7-amino-3-cephem-4-carboxylic acid or its trichloroethyl ester with N-t-butoxycarbonyl-2-(4-mesylaminophenyl)-D-glycine, and if desired, eliminating the t-butoxycarbonyl group and trichloroethyl group from the reaction product.

7. A process according to claim 1, wherein R4 is thiadiazolyl which may be substituted with lower alkyl.

8. A process according to claim 7, in which 7-[D-2-(3-mesylaminophenyl)glycinamido]-3-(5-methyl-1,3,4-thiadiazol-2-ylthio)methyl-3-cephem-4-carboxylic acid is obtained by reacting 7-amino-3-(5-methyl-1,3,4-thiadiazol-2-ylthio)methyl-3-cephem-4-carboxylic acid or its trichloroethyl ester with N-t-butoxycarbonyl-2-(3-mesylaminophenyl)-glycine, and if desired, eliminating the t-butoxycarbonyl group and trichloro-ethyl group from the reaction product.

9. A process according to claim 1, wherein R4 is lower alkanoyloxy.

10. A process according to claim 9, in which 7-[D 2-(3-mesylaminophenyl)glycinamido]-3-acetoxymethyl-3-cephem-4-carboxylic acid is obtained by reacting 7-amino-cephalosporanic acid or its trichloroethyl ester with N-t-butoxycarbonyl-2-(3-mesylaminophenyl)glycine, and if desired, eliminating the t-butoxycarbonyl group and trichloroethyl group from the reaction product.

11. A process according to claim 1, which comprises reacting a compound of the formula (IX") with a compound of the formula (X).

12. A process according to claim 11, in which 7-[D-2-(3-mesylaminophenyl)glycinamido]-3-(5-methyl-1,3,4-thiadiazol-2-ylthiolmethyl-3-cephem-4-carboxylic acid is obtained by reacting 7-[D-2-(3-mesylaminophenyl)glycinamido]-cephalospora-nic acid with 5-methyl-1,3,4-thiadiazole-2-thiol.

13. A compound of the formula:

(IX) wherein R2 is lower alkyl, and R4 is hydrogen, lower alkanoyl-oxy or thiadiazolylthio which may be substituted with lower alkyl, or a halo(lower)alkyl ester thereof, or a nontoxic pharmaceutically acceptable salt thereof, whenever prepared by the process of claim 1, or by an obvious chemical equivalent thereof.

14. A compound of the formula (IX) as defined in claim 3, whenever prepared by the process of claim 3, or by an obvious chemical equivalent thereof.

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

16. A compound of the formula (IX) as defined in claim 9, whenever prepared by the process of claim 9, or by an obvious chemical equivalent thereof.

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

18. 7-[D-2-(3-Ethanesulfonamidophenyl)glycinamido]-3-methyl-3-cephem-4-carboxylic acid, whenever prepared by the process of claim 5, or by an obvious chemical equivalent thereof.

19. 7-[D-2-(4-Mesylaminophenyl)glycinamido]-3-methyl-3-cephem-4-carboxylic acid, whenever prepared by the process of claim 6, or by an obvious chemical equivalent thereof.

20. 7-[D-2-(3-Mesylaminophenyl)glycinamido]-3-(5-methyl-1, 3,4-thiadiazol-2-ylthio)methyl-3-cephem-4-carboxylic acid, whenever prepared by the process of claim 8 or 12, or by an obvious chemical equivalent thereof.

21. 7-[D-2-(3-Mesylaminophenyl)glycinamido]-cephalospora-nic acid, whenever prepared by the process of claim 10, or by an obvious chemical equivalent thereof.

CLAIMS SUPPORTED BY THE SUPPLEMENTARY DISCLOSURE

22. A process for the preparation of a compound of the formula:

(IXa) wherein R2a is lower alkyl and R4a is triazolyl, a halo(lower)-alkyl ester thereof or a nontoxic pharmaceutically acceptable salt thereof, which comprises (1) reacting a compound of the formula:

(VIIIa) wherein R4a is as defined above, or its halo(lower)alkyl ester, or a salt thereof with a substituted phenylglycine of the formula:

(Ia) wherein R2a is as defined above, or a lower alkoxycarbonyl-protected derivative thereof at the .alpha.-amino group, or a reactive derivative at the carboxyl group, and if desired, eliminating the ester and/or the protecting group at the .alpha.-amino group from the reaction product, or (2) reacting a com-pound of the formula:

(IX"a) wherein R2a, is as defined above, and R4a" is lower alkanoyloxy, or a halo(lower)alkyl ester, or a salt thereof, with a thiol compound of the formula:
R4a' - H (Xa) wherein R4a' is triazolylthio, or an alkali metal salt thereof, and if desired, eliminating the ester from the reaction product;
and when desired, converting a free acid of said formula (IXa) to a corresponding non-toxic pharmaceutically acceptable salt thereof.

23. A prooess according to claim 22, which comprises reacting a compound of the formula (VIIIa) or a salt thereof with a substituted phenylglycine of the formula (Ia), or its lower alkoxycarbonyl-protected derivative at the ?-amino group, or its reactive derivative at the carboxy group, and if desired, eliminating the protecting group at the ?-amino group from the reaction product.

24. A process according to claim 23, in which 7-[D-2-(3-mesylaminophenyl)glycinamido]-3-(1H-1,2,3-triazol-5-yl) thiomethyl-3-cephem-4-carboxylic acid is obtained by reacting 7-amino-3-(1H-1,2,3-triazol-5-yl)thiomethyl-3-cephem-4-carboxylic acid with D-N-t-butoxycarbonyl-2-(3-mesylaminophenyl)glycine, and eliminating the t-butoxy-carbonyl group from the reaction product.

25. A compound of the formula (IXa) as defined in claim 22, a halo(lower)alkyl ester thereof or a nontoxic pharma-ceutically acceptable salt thereof, whenever prepared by the process of claim 22, or by an obvious chemical equivalent thereof.

26. A compound of formula (IXa), as defined in claim 22, whenever prepared by the process of claim 23, or by an obvious chemical equivalent thereof.

27. 7-[D-2-(3-Mesylaminophenyl)glycinamido]-3-(1H-1,2,3-triazol-5-yl)thiomethyl-3-cephem-4-carboxylic acid, whenever prepared by the process of claim 24, or by an obvious chemical equivalent thereof.

28. A process for the preparation of a compound of the formula:

(IXb) wherein R2 is lower alkyl and R4 is hydrogen, lower alkanoyl-oxy, triazolyl or thiadiazolylthio which is unsubstituted or substituted with lower alkyl, a halo(lower)alkyl ester thereof or a nontoxic, pharmaceutically acceptable salt thereof, which comprises (1) reacting a compound of the formula:

(VIIIb) wherein R4 is as defined above, or its halo(lower)alkyl ester, or a salt thereof with a substituted phenylglycine of the formula:

(Ib) wherein R2 is as defined above, or a lower alkoxycarbonyl-protected derivative thereof at the .alpha.-amino group or a reactive derivative at the carboxyl group, and if desired, eliminating the ester and/or the protecting group at the .alpha.-amino group from the reaction product, or (2) reacting a compound of the formula:

(IX"') wherein R2 is as defined above and R4"' is lower alkanoyl-oxy, or a halo(lower)alkyl ester thereof or a salt thereof with a thiol compound of the formula:
R4' - H (Xb) wherein R4' is thiadiazolylthio which is unsubstituted or substituted with lower alkyl, or an alkali metal salt thereof, and if desired, eliminating the ester from the reaction product;
and when desired, converting a free acid of said formula (IXb) to a corresponding nontoxic, pharmaceutically acceptable salt thereof.

29. A compound of the formula:

(IXb) wherein R2 is lower alkyl and R4 is hydrogen, lower alkanoyl-oxy, triazolyl or thiadiazolylthio which is unsubstituted or substituted with lower alkyl, or a halo(lower)alkyl ester thereof or a nontoxic, pharmaceutically acceptable salt there-of, whenever prepared by the process of claim 28, or by an obvious chemical equivalent thereof.