Note: Descriptions are shown in the official language in which they were submitted.
~ S8'~
~he present invention relates to novel amino acids,
and their production and use. More particularly, it relates
to amino acids useful for the formation of side chains at
the 7-position of cephalosporin compounds and cephalosporin
antibiotics produced by the use of such amino acids, and
processes for preparing them.
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. Among them, however, only a few
`~ are practically used, and there is a ceaseless need for novel
cephalosporin antibiotics which are practically utilizable.
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:
1~
~ ~ CH-CONH- [A]
R2--Y-N~ J NH2
R3
wherein Rl is hydrogen or hydroxyl, R2 is lower alkyl (e.g.,
methyl, ethyl, propyl, isopropyl, butyl, pentyl), R3 is hydrogen
or lower alkanesulfonyl (e.g. methanesulfonyl, ethanesulfonyl)
and Y is -S02- exhibit a strong antimicrobial activity against
a wide variety of microorganisms including gram-positi-ve 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 cephalosporin antibiotics, in
some species of pathogenic microorganisms. It is also notable
~L~5~2~
that, when orally administered to mammals, their antibiotic
potency is maintained at a high level for a long period of
time. Accordingly, they are useful as orally_administerable,
long-acting antibiotics.
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:
R
~/ ~) CH-COOH ( I )
R2-Y-~1 ~H2
R3
wherein Rl, R2, R3 and Y are each as defined above.
lo When the symbol R3 represents a lower alkanesulfonyl
group, it corresponds to the group represented by the formula:
R2_y- .
An object of the present invention is to provide the
amino acids(I) and their derivatives, in which Rl and R3 are
hydrogen, Y is -SO2- and R2 is lower alkyl.
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
"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, etc.
In particular the present invention provides compounds
of the formula:
A
1(~58'~C)6
~ CH_COOH (I)
R2_S02_~H NH2
wherein R2 is lower alkyl, as well as the lower alkyl esters,
acid amides, loweralkoxycarbonyl and l-(lower)alkoxycarbonyl-
1-(lower)alken~2-yl protected derivatives at the ~-amino
group, and salts thereof.
In another aspect of the invention there is provided
a process of preparing the compound (I) comprising reacting
a compound (II) with a reagent (III), as hereinafter defined,
or reacting a compound (IV) with said reagent (III), reacting
the resulting product (V) with hydrogen cyanide in the
presence of ammonia and hydrolyzing the resulting product
(VI) under conditions to produce the free acid (I) or acid
amide or lower alkyl ester of the acid (I), as hereinafter
defined.
According to the present invention, the amino acids
- 3 -
~; ..,,~,
~L~5~
~I] can be produced in a variety of procedures, of which a
typical one comprises reacting a compound of the formula~
Rl ~ ~-COOH [II]
H2 ~H2
wherein Rl is as defined above in a free or protected form
on the carboxyl group and/or the ~-amino group with an acylat-
ing agent of the formula:
R2-Y-Z ~ ]
wherein Z is the residue of an acid and R2 and Y are each as
defined above.
- When the ~-amino group is protected, one or two
hydrogen atoms therein may be replaced, for instance, by acyl
such as substituted or unsubstituted benzyloxycarbonyl (e.g.
benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-bromobenzyloxy-
carbonyl, 4-methoxybenzyloxycarbonyl, 3,4-dimethoxybenzyloxy-
carbonyl, 4-(phenylazo)benzyloxycarbonyl, 4-(4-methoxyphenyl-
azo)benzyloxycarbonyl), substituted or unsubstituted alkoxy-
carbonyl or cycloalkoxycarbonyl (e.g., t-butoxycarbonyl, t-
pentyloxycarbonyl, isopropoxycarbonyl, diphenylmethoxycarbonyl,
2-pyridylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2,2,2-
tribromoethoxycarbonyl, l-cyclopropylethoxycarbonyl, 3-iodo-
propoxycarbonyl, 2-furfuryloxycarbonyl, l-adamantyloxycarbonyl~,
(heterocyclic ring)oxycarbonyl (e.g., 8-quinolyloxycarbonyl) or
substituted alkanoyl (e.g. trifluoroacetyl), trityl, trialkyl-
silyl (e.g. trimethylsilyl, triethylsilyl), substituted phenyl-
thio (e.g. 2-nitrophenylthio, 2,4-dinitrophenylthio), etc.
The ~-amino group may be also protected in the form of a Schiff
base and, in such case, the two hydrogen atoms therein are
~058Z~D6
replaced by substituted alkylidene (e.g. 2-hydroxybenzylidene,
2-hydroxy-5-chlorobenzylidene, 2-hydroxy-1-naphthylmethylene,
3-hydroxy-4-pyridylmethylene, 1-methoxycarbonyl-2-propylidenç,
l-ethoxycarbonyl-2-propylidene, 3-ethoxycarbonyl-2-butylidene,
l-acetyl-2-propylidene, 1-propionyl-2-propylidene, l-benz~oyl-
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 fbrm
on the carboxyl group is a silyl ester which is obtainable by
the reaction with a silyl compound such as trialkylhalosilane,
dialkylhalosilane, alkyltrihalosilane, hexaalkylcyclotri-
silazane, octaalkylcyclotetrasilazane, trialkylsilylacetamide
or bis~trialkylsilyl)acetamide.
Further,the a-amino group and the 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
~0513~
borate, copper phosphate, copper cyanide, copper formate, copper
acetate, copper propionate, copper citrate, copper tartrate,
copper benzoate~ copper salicylate, etc.
In the acylating agent (III), the residue of an acid
represented by the symbol Z is a leaving group in an SN sub-
stitution nucleophilic reaction displaceable by amino and
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. Thus the acylating agent (III)
lo includes an alkanesulfonic acid halide, for example, an acid
chloride or bromide; an alkanesulfonic acid anhydride; an
alkanesulfonic acid ester and an alkanesulfonic acid azide,
and the residue of an acid represented by the symbol Z is a
residue of the alkanesulfonic acid derivatives mentioned
above.
The reaction is usually carried out in an inert
solvent. Examples of the inert solvent are acetone, dioxane,
acetonitrile, chloroform, methylene chloride, ethylene chloride,
tetrahydrofuran, 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.
Another typical procedure for the preparation of the
amino acid (I) comprises (1) reacting a compound of the
formula:
~ ~ (IV)
H2N
~ - 6 -
,,.. 1 ~
- ;l
~IDS~32~;
wherein Rl is as defined above with ~he acylating agent (III),
(2) reacting the resulting product of the formula:
Rl\~s;~
~ ~ CHO (V)
R2_Y--N~\=J
R3
wherein Rl, R2, R3 and Y are each as defined above with
_ 6a -
~05~32~
hydrogen cyanide in the presence of ammonia and hydrolyzing
the resultant product of the formula:
1 ~ CHCN ~VI]
R2-Y-N NEI;~
R3
wherein 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 CIII] inan inert solvent (e.g~ water, dioxane, tetrahydrofuran, ether),
if necessary, in the presence of a base (e.g. pyridine, tri-
ethylamine, dimethylaniline). The starting compound CIV] is
sometimes available commercially in the polymerized form and
may be as such subjected 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 1I Strecker amino
acid synthesis" and may be carried out in a per se conventional
manner. For instance, it can be carried out by treating the
compound [V] 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 (3) 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 single step. On the other hand, the treatment under a
~S8~
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 carbamoyl group, whereby
a compound of the following formula is produced as the main
product:
Rl ~ CH-CONH2 [VII]
R2--Y-N NH2
,.
wherein 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
condition 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 the product.
One of the typical elimination procedures for the
protective group on the ~-amino group is conducted by treat-
ment with an acid which is usually applied to benzyloxycarbonyl,
substituted or unsubstituted benzyloxycarbonyl, substituted or
unsubstituted alkoxycarbonyl, substituted or unsubstituted
aralkoxycarbonyl, adamantyloxycarbonyl, trityl, substituted
phenylthio, substituted aralkylidene, substituted alkylidene,
substituted 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
30 (e.g. formic acid, trifluoroacetic acid). The elimination
-- 8 --
~L~)582V6
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 tohaloalkoxycarbonyl and 8-quinolylo~ycarbonyl.
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 ca~e of the ~-amino group is protected together
with the carboxyl group!in a chelating form, the elimination
of the metal compound used for chelation rnay 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 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
of the silyl group. Thus, any particular operation for the
~s~z~
elimination of the silyl group is usually not required.
When the carboxyl group is protected in any
form other than a silyl ester, the elimination of such pro-
tective group may be accomplished in any conventional proce-
dure~
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 elimination of the protective group on the
~-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 [I] 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 [~I]) 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.
For preparation of the cephalosporin compound hav-
ing the acylamino group [A] at the 7-position by the use of
the above prepared amino acid [I], a 7-amino-3-substituted-
3-cephem-4-carboxylic acid of the formula:
2 ~ S
~ N ~ CH2-R4 [VIII~
COOH
-- 10 --
1(~58Z~6
wherein R4 is ~ydrogen, lower alkanoyloxy or a heterocyclic-
thio group, in which the heterocyclic group may be substituted
with lower alkyl or its derivative at the amino group and/or
the carboxyl group may be reacted with the amino acid [I] or
its reactive derivative at the carboxyl group by a per se
conventional coupling procedure employed for the formation of
an amide linkage to give a cephalosporin compound of the
formula~
~-CO~
COOH
wherein Rl, R2, R3, R4 and Y are each as defined above or its
derivative.
The term "heterocyclic-thio group" as hereinabove
used is intended to mean the residue of a thiol compound
having a heterocyclic group, which is the residue of furan,
thiophene, pyrrole, pyrazole, imidazole, triazole, thiazole,
~O isothiazole, oxazole, isoxazole, thiadiazole, oxadiazole, thia-
triazole, oxatriazole, tetrazole, pyridine, pyrazine, pyrirnidine,
pyridazine, benzothiophene, benzofuran, indole, indazole,
benzimidazole, benzothiazole, benzothiadiazole, benzoxazole,
purine, quinoline, isoquinoline, phthalazine, naphthyridina,
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 at the carboxyl group of the compound
[VIII~ may be a salt such as magnesium salt, calcium salt,
~ 0~Z~6
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-
methyl ester, bis(methoxyphenyl)methyl ester, 3,4-dimethylbenzyl
ester, (l-cyclopropyl)ethyl ester, ethynyl ester, 4-hydroxy-
3,5-di-t-butylbenzyl ester, etc.' an amide, or the like.
The derivative at the carboxyl group and/or the
amino group of the compound [VIII] may be the reaction product
of the compound ~VIII] with a silyl compound such as bis(tri-
methylsilyl)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-dimethoxy~enzyloxycarbonyl, 4-(phenyl-
azo)benzyloxycarbonyl, 4-(4-methoxyphenylazo)benzyloxycarbonyl),
substituted or unsubstituted alkoxycarbonyl or cycloalkoxycar-
bonyl (e.g. t-butoxycarbonyl, t-pentyloxycarbonyl, isopropoxy-
carbonyl, diphenylmethoxycarbonyl, 2-pyridylmethoxycarbonyl,
2,2,2-trichloroethoxycarbonyl, 2,2,2-tribromoethoxycarbonyl,
l-cyclopropylethoxycarbonyl, 3-iodopropoxycarbonyl, 2-fururyl-
oxycarbonyl, l-adamantyloxycarbonyl), (heterocyclic ring)oxy-
- carbonyl (e.g. 8-quinolyloxycarbonyl) or substituted alkanoyl
- (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
- 12 -
~05~3ZI)~
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, 3-ethoxycarbonyl-2-butylidene, l-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-(N-methylcarbamoyl)-2-propylidene,
l-(N,N-dimethylcarbamoyl)-2-propylidene, 1-[N-(2-methoxyphenyl)-
carbamoyl]-2-propylidene, 1-CN-(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, dialkyl-
phosphorous acid, sulfurous acid, thiosulfuric acid, sulfuric
acid, alkylcarbonic acid, aliphatic carboxylic acid (eOg.
pivalic acid, pentanoic acid, isopentanoic acid, 2 ethyl-
butyric acid, trichloroacetic acid) or aromatic carboxylic
acid (e.g. benzoic acid), or a symmetrical acid anhydride,
an acid amide with imidazole, 4-substituted imidazole, di-
methylpyrazole, triazole,or tetrazole, or an ester (e.g.
- 13 -
~582(~6
cyanomethyl ester, methoxymethyl ester, vinyl ester, propargyl
ester, p-nitrophenyl ester, 2,4-dinitrophenyl ester, trichloro-
phenyl ester, pentachlorophenyl ester, methanesulfonylphenyl
ester, phenylazophenyl ester, phenyl thioester, p-nitrophenyl
thioester, p-cresyl thioester, carboxymethyl thioester, pyranyl
ester, pyridyl ester, piperidyl ester, 8-quinolyl thioester,
or an ester with N,N-dimethylhydroxylamine, l-hydroxy-2-(lH)-
pyridone, N-hydroxysuccinimide, N-hydroxyphthalimide), or the
like.
When the amino acid ~I] is used in the form of a
free acid or a salt, the coupling reaction is preferably car-
ried out in the presence of a condensing agent appropriately
selected from N,N'-dicyclohexylcarbodiimide, ~-cyclohexyl-N'-
morpholinoethylcarbodiimide, ~-cyclohexyl-N'-~4-diethylamino-
cyclohexyl)carbodiimide, N,N'-diethylcarbodiimide, N,N'-diiso-
propylcarbodiimide, N-ethyl-N'-(3-dimethylaminopropyl)carbodi-
imide, N,N'-carbonyldi(2-methyl.imidazole), pentamethylene-
ketene-N-cyclohexylimine, diphenylketene-N-cyclohexylimine,
alkoxyacetylene, l-alkoxy-l-chloroethylene, trialkyl phosphite,
ethyl pol.yphosphate, 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 [I] 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-
- 14 -
~05~ZI~
amine, N,N-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 also 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 protective group for the
amino group may be removed in the course of the coupling
reaction or the post-treatment. When the product has a pro-
tected amino group, the protective group may be eliminated
~~~ ~ -~ therefrom, when desired, by applying a suitable procedure as
mentioned above.
The cephalosporin compound [IX: R4 = heterocyclic-
thio (optionally substituted with lower alkyl)] or its deri-
vative may be also prepared by reacting the corresponding
cephalosporin compound [IX: R4 = lower alkanoyloxy] or its
derivative with a thiol compound of the formula:
-- R4 ~X]
wherein R4 is a heterocyclic-thio group, in which the hetero-
cyclic group may be substituted with lower alkyl, corresponding
to the symbol R~ in the objective compound or its alkali metal
salt.
The alkali metal salt of the thiol compound [X] may
be a sodium salt, a potassium salt or the like.
The above reaction may be carried out in an inert eol-
vent such as water, acetone, chloroform, nitrobenzene, dimethyl-
formamide, methanol, ethanol or dimethylsulfoxide. Among these
- 15 -
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 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 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 theretog
whereby the salt is precipitated from the reaction mixture.
Broadly, the invention relates to a process for
preparation of a compound of the formula:
~ CIH-COOH (I)
R2-Y-r NH2
, R3
~1
~ - 16 -
~L()582~)6
wherein Rl is hydrogen, R2 is lower alkyl, R3 is hydrogen and Y
is -SO2-, or its lower alkyl ester, acid amide or a lower
alkoxycarbonyl- or l-(lower)alkoxycarbonyl-l-(lower)-alken-2-yl-
protected derivative at the ~-amino group, or a salt thereof,
which comprises (1) reacting a compound of the formula:
~ CH-COOH (II)
H2 NH2
wherein Rl is as defined above, or its lower alkyl ester, acid
amide, a lower alkoxycarbonyl- or l-(lower)alkoxy-carbonyl-l-
(lower)alken-2-yl-protected derivative at the ~-amino group, or a
salt thereof with a reagent of the formula:
R2 Y Z (III)
wherein Z is a residue of an acid, and R2 and Y are each as
defined above, and if desired, eliminating the protecting group
at the ~-amino group from the reaction product, or (2) (i)
reacting a compound of the formula:
~ CHO (IV)
R2N
wherein Rl is as defined above with a reagent of the formula
R2 Y Z (III)
wherein R2, Y and Z are each as defined above, (ii) reacting the
resultant product of the formula:
R13~CHO ( V)
R2-Y-N
R3
wherein R1, R2, R3 and Y are each as defined above, with hydrogen
~ - 16a -
1~
10~ 6
cyanide in the presence of ammonia, and then (iii) hydrolyzing
the resultant product of the formula:
1 ~ OEI-CN (VI)
R2-Y-N
NH2
R3
wherein Rl, R2, R3 and Y are each as defined above.
The cephalosporln compounds [IX] exhibit an excellent
antimicrobial activity. For lnstance~ the cephalosporin com-
pound [IX: Ri = hydrogen; R2 = methyl; R3 = hydrogen; R4 =
hydrogen, Y = -S02- (the group R2-Y-IN- being present at the
R3
- 16b -
~58Z06
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.
. ..
Test organism Minimal inhibitory concentration
(mcg/ml)
Compound B ~ C~ephalexin
Staph. aureus Terashima 12.5 25
Staph. aureus Srnith 3-13 6.25
B. subtilis ATCC-6633 0.78 1.56
~. subtilis PCI-219 0.78 1.56
Klebsiella NCTC-418 3.13 6.25
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 5 ~,
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 ins-tance, the serum
levels of Compound B and cephalexin with elapse of time when
adminstered orally to mice and rats are shown in Table 2.
- 17 -
--
~o~z~
Table 2
Serum levels o~ Compound B and cephalexin in mice
and rats.
Animal Test Serum level (mcg/ml)
~ cornpound l
Dose 1 hr 2 hrs 4 hrs 6 hrs
_ . .
Mouse (ICR) Compound ~ S7.6 25.0 14.7 8.4
100 m~/kg~ Cephalexin 10.4 4.~ _
_ . . . . _
Rat (SD) Compound ~ 49.8 44.8 41.1 39.0
100 mg/kg Cephalexin 2~.1 12.3 6.6 5.9
As stated above~ the cephalosporin compounds [IX]
of this invention are useful as orally administrable, long
acting antibiotics. ~or therapeutic adrninistration the cephalo
sporin compounds [IX] are used in the form of pharmaceutical
preparations which contain said compounds 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 cornmonly used additives.
While the dosage of the cephalosporin compounds [IX]
will vary frorn and also depend upon the age and condition o~
the patient, an average single dose of about 100 mg., 250 mg.
and 500 mg. has proved to be effec-tive in treating diseases
caused by bacterial infection. In general, amounts between
10 mg. and about 1000 mg. or even more may be administered.
- 18 -
~L~51~Q6
~ he ~ollowin~ Examples are given solel~ for the
purpose of illustrating the present invention, not of limit-
ing the same thereto.
Example 1
(1) N-t-Butoxycarbonyl-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 5~. Stirring was
continued at the same temperature for 30 minutes and then at
room temperature for 4 hours. The reaction mixture was shaken
with an aqueous solution of citric acid, washed with water,
and then the methylene chloride layer was separatea. mhe
methylene chloride layer was shaken with 5 ~0 aqueous solution
of sodium hydrogen carbona-te, and the water layer was separated.
The water layer was adjusted to pX 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 N-t-butoxycarbonyl-2-(3-mesylaminophenyl)-D-
glycine (6.3 g.) as amorphous powder. N.M.R. (DMSO-d6) ô ppm:
a (9H,s); 2.99 (3H,s); ~.09 (lH,d,J=7.5Hæ); 7.00-7.60 (4H,m).
(2) The above prepared N-t-butox~carbonyl-2-
(3-mesylaminophenyl)-D-glycine (4.0 g.) was dissolved in
formic acid (100 ml.), the solution was stirred at roorn temper-
ature for l hour and the solvent was distilled off under reduced
-- 19 --
~513;~V6
pressure. The residue l.ras dissolved in 10 c,~ aqueous aceto-
nitrile (10 ml.) and filtered. To the filtrate, acetonitrile
(10 ml.) was added, and the precipitated crystals were collected
by fil-tration and dried to give 2-(3-mesylaminophenyl)-
D-glycine (2.7 g.) as colorless prisms. ~.P. 196 to 198C.
I.R. (Nujol) v cm 1 3240, 25~0, 1616, 1147. N.M.R. (D20 +
DCl) o ppm: 3.18 (3H,s); 5.32 (lH,s), 7.47 (4H,m).
Example 2
As in Example 1, D-a-(3-mesylaminophenyl)-~-(1-
methoxycarbonyl-l-propen-2-yl)glycine sodium salt was prepared
from V-(~ rninophenyl)-N-(l-methoxycarbonyl-l-propen-2-yl)-
glycine sodium salt and methanesulfonyl chloride. l~l.P. 141
to 143C. I.R. (Nujol) v cm~l: 3250, ~650, 1610, 1150.
N.M.R. (DMS0-d6) o ppn: 1.68 (3~,s), 2.92 (3H,s), 3.52 (3H,s),
4.31 (lH,s), 4.78 (lH,d,J=5.6Hz), 6.90-7.25 (4~i,m), 9.58 (lH,
d,J=5.6~z).
Exam~le 3
To a solution of D-a-(3-amino~henyl)-N-t-butoxy-
carbonylglycine (1.34 g.) and sodium hydrogen carbonate (924
mg.) in water (25 ml.) cooled at -2C, methanesulfonyl chloride
(~60 mg.) was dropwise added in 10 minutes, and the resul-tant
mixture was stirred at the same temperature for 1 hour. ~fter
the addition of sodium hydrogen carbonate (164 mg.), stirring
was continued at the same temperature for 2 hours and then at
room temperature for 1 hour. The reaction mixture was washed
.~ith 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. 'rhe extract was
ashed with an aqueous saturated solution of sodium chloride,
- 20
~o~z~
dried over anhydrous magnesium sulfate and concentrated under
reduced pressure to give ~-(3-mesylaminophenyl)-~-t-butoxy-
carbonylglycine (1.4 g ) ~D = -96U (methanol, C = 1).
Example 4
(1) To a solution of ~-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 (3.165 g.) was
added while ice coolin~ (2 to 3UC), and a solution of methane-
sulfonyl chloride (4,58 gO) in methylene chloride (25 ml.)
was dropwise added thereto whilè 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
shaken with ethyl acetate (1~0~ ml.) and 2M citric acid solution
(50 ml.). The ethyl acetate layer was separated and washed
with 2M citric acid solution (50 ml.). The washing was ex-
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 times 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
four times with ether (50 ml.), and the extrac-ts 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. (DMS0-d6) ~ ppm: 1.40 (9H,s),
~58ZU~
3.00 (3H,s), 5.11 (lH,d,J=8Hz~, 7.00-7.60 (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
-
D-2-~3-Aminophenyl)glycine (16.6 g.) was dissolved
in ~ sodium hydroxide solution (100 ml.), and dioxane (40 ml.)
was added theretoO 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 p~ 7.0 with
20~/o sodium hydroxide solution, and a solution of methane-
sulfonyl chloride (17.1 g.) in dioxane (20 ml.) was dropwise
added at 26 to 28C in 1 hour, during which 20 % sodium
hydroxide solution (47 ml.) was dropwise added thereto to
maintain pH at 6.0 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, 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 precipitated 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.
- 22 -
~58;~
r~
I.R. (Nujol~ y cm 1 1505 (broad), 1150. N.M.R. (DCl ~ D20)
p~n: 3.17 (3H,s), 5.16 (lH,s), 7.43 (4H,m).
Example 6
To the mixture 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 28UC in an hour, during which 20 % sodium hydroxide
solution dropwise added thereto to maintain pH 8 to 9. The
reaction mixture was treated as in Example 5 to give 2~(3-
dimesylaminophenyl)-D-glycine as~ olorless prisms. M.P. 171
to 173C (decomp.). I.R. (Nujo r cm : 1650, 1160. ~.M.R.
(DCl + D20) ~ ppm: 3.50 (6H,s), 5.33 (lH,s), 7.61 (4H,m).
Example 7
A solution of D-(~-4-aminophenyl)-~-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 mathanesulfonyl chloride (4.12 g.) in methylene
chloride (40 ml.) were dropwise added thereto at 2 to 3~C 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 o~ 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
- 23 -
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 e~tract was washed with
an aqueous saturated solu-tion of sodium chloride and dried.
After removal of the solvent by distillation under reduced
pressure, the resulting oil (6 0 g.) was pulverized with ether
and isopropyl ether to give D-~-(4-mesylaminophenyl)-N-t--
butoxycarbonylglycine (5.5 g.) as crystals. M.P. 165 to 183C.
I.R. (Nujol) v cm 1 3325, 3125, 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).
Example 8
N-t-~utoxycarbonyl-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
- ; room temperature for 1 hour. The reaction mixture was washed
with 2 o/O hydrochloric acid (20 ml.) twice9 water (20 ml.) twice
and an aqueous saturated 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 &ive N-t-
butoxycarbonyl-2-(3-ethanesulfonamidophenyl)-D-glycine (4.4 g.~
as powder. I.R. (film) v cm 1 3230, 1720, 1680. N.'~.R. (CDC13)
o 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 (IH,broad s)~ 8.59 (lH,
broad s).
Exam~le 9
- 24 -
~1~58~
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 eihyl 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 separated,
made acidic with phosphoric acid and back-extracted again with
ethylacetate. 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)-N-t-butoxycarbonylglycine (6.0 g.). I~R. (Chloroform)
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 10
(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 reaction mixture was con-
centrated under reduced pressure, 5 % hydrochloric acid (120 ml.),
- 25 -
~05~32~6
ethyl acetate (60 ml.) and sodium chloride ~rere added thereto,
and the precipitate was collected by filtration and recrystallized
from ethyl aceta-te to give 3-mesylar~inobenzaldehyde (1.8 g.) as
crysta].s. M.P. 142 to 144C. The ethyl acetate layer was
separated, washed with an aqueous saturated so]ution 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
same product as above (0.83 g.). Total yield, 6.99 g. I.R.
(Nujo ~ v cm 1 3120, 1670, 1600, 1580, 1320, 1242, 116~, 1143,
997, 970, 890, 788, 7539 670. N.i~I.R. (DMS0-d6) ~ ppm: ~.03
(3H,s), 7.4-7.8 (4H,m), 9.95 (IH,s), 10.07 (IH,broad s).
(2) To a mixture of sodium cyanide (2.18 g.j,
ammonium chloride (2.19 g.) and 28 % arnmonia water (17 ml.)
cooled at 10~, 3-mesylaminobenzaldehyde (3.98 g.) ~as added
thereto at 10 to 15C for 4 hours ~Ihile stirringu 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. The extract was washed with an aqueous saturated solu-
tion of sodium chloride (30 rnl), dried over anhydrous magnesiurn
sulfate and concentrated to give D~-a-arnino-a-(3-mesylamino-
phenyl)acetonitrile (4.19 g.) as an oil. I.R. (film) v cm 1
3270, 1608, 1595, 1477, 1400, 1327, 114~, 972, 890, 795, 768,
697. N.M.R. (DMS0-d6) o ppm: 3.00 (3H,s), 5.01 (lH,s),
7.0-7.7 (4H,m).
(3) 'r o DL-a-amino-a-(3-mesylaminophenyl)acetoni-trile
(2.10 g.), there was added glacial acetic acid (7 ml.), and
finely pulverized L(-~)-tartaric a~id (1.56 g.) was added thereto.
- 26 -
- -
~QS~
To the resulting solution, ethyl acetate (4.7 ml.) was portion-
wise added while stirring, and stirring was continued at room
temperature overnight. The precipita-ted crystals ~rere collected
by ~iltration, washed with a mixture of ethyl acetate and
acetic acid and ethyl acetate in order and dried to give D-a
amino--(3-mesylaminophenyl)acetonitrile ~(+)-tartrate (having
one molecule of acetic acid) (3.25 g.). M.P. 97 to 98C.
I.R. (Nujol) v cm 1 3220, 2670, 1735, 1692~ 1593, 15~2, 1520,
1422, ]405, 1~10, 1262, 1237, 1220, 1165, 1133, 1070, 978, 903,
800, 782, 663, 602. N.M.R. (D20~DCl) o p~m: 2.13 (3H,s),
3.20 (3H,s), 4.78 (2H,s), 5.37 (lH,s), 7.3-7.7 (4H,m).
._
[a]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 ~0 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 a~nonia and allowed to stand in a refrigerator over-
night. The precipitated crystals were collected by filtration.
The collected crystals (132 mg.) was dissolved in water (0.5
ml.) ~rhile hot, methanol (2 ml.) was added thereto and the
resultin~ mixture was allowed to stand in a refri~era-tor over-
night. The precipitated crystals were collected by filtration
and ~lashed ~lith methanol to give D-a-(3-mesylaminophenyl)-
glycine (54.3 mg.). M.P. 193 to 194C (decomp.). I.R. (Nujol~
v cm 1 3230, 2720, 2550, 1610, 15C8, 1~00, 1330, 1310, 1260,
1145, 9~5, 783, 695, 662. N.l~.R. (D20-HCl) o ppm: 3.20 (jH,s)
6.3~ ,s), 7.3-7.7 (4H,m). [a]D = -100 (1~ hydrochloric
acid, C = 1).
- ~7 -
8;~06
(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 % hydrochloric acid
and extracted with ethyl acetate (15 ml.) 4 times. The-extract
was washed with an aqueous saturated solution of sodium chloride
and concentrated under reduced pressure to about 10 ml. The
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
r-efluxed for 3 hours. The reaction mixture was treated with
activated charcoal, concentrated under reduced pressure to
remove the hydrochloric acid, admixed with ethanol (5 ml.) and
then concentrated under reduced pressure to dryness. The
residue was dissolved in methanol (20 ml.) by heating and
adjusted to pH 5 with methanolic ammonia. The precipitated
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 dr,~ed to give D~X-(3-mesylaminophenyl)glycine (0.98 g.).
I.R. (Nujo ~ ~ cm : 3260, 1695, 1605, 1590, 1477, 1400, 1330,
- 1250, 1150, 973, 890, 790, 760. N.M.R. (D20 + DCl) ~ ppm: 3.18
(3H,s) 5.38 (lH,s), 7.2-7.6 (4H,m).
Example 11
- 28 -
~s~
(1) D-a-Amino-a-(3-mesylaminophenyl)acetonitrile
I.(~)-tartrate (having one molecule of acetic acid) (435 mg.)
was dissolved in conc.hydrochloric acid (1.5 ml.), and the
resulting solution was heated to reflux. ~'rom the reaction
mi~ture, hydrochloric acid ~ras removed by distillation ur.der
reduced pressure. The residue was added to a colu~n of an ion
exchange resin ("~mberlite IR-120~", H+ type, manufactured by
Rohm and Haas Co.) (10 ml.) and washed with water until the
~ashings became neutral~ Then, 7 ~ aqueous ammonia las poured
on the column. The eluate was collected and concentra'~ed under
reduced pressure. The residue was c~ystallized from methanol
to give D-a-(3-mesylaminophenyl)Olycine (220 mg.) as crystals.
M.P. 207C (decomp.). [a]D = -100 (IN hydrochloric acid,
C= 1).
(2) Into a suspension of the above prepared D-a-(3-
mesylaminophenyl)glycine (3.0 g.) in anhydrous metnanol (60
ml.) cooled with ice water9 dried hydrogen chloride ,;as was
introduced to make saturation. The resultant mixture was
allowed to stand at 4 to 5C for 40 hours. Af-ter removal of
the methanol by'distillation under reduced pressure, the
precipitate was collected by ~iltration, 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 of acetone) (3.8 g.). N.M.R. (CD30D-
D20) o ppm: 2.16 (3H,s), 3.05 (3H,s), 3.81 (3H,s), 5.23 (lH,s),
7.16-7.5 (4H,~).
Example 12
A solution of D-a-amino-a-(3-mesylaminophenyl)aceto-
nitrile ~(+)-tartrate (having one molecule of acetic acid) (1.3
g.) in conc. hydrochloric acid (10 ml.) ~las ~llo~red to stand
- 2~ -
1~5~3Z()6
at room temperature for 1.5 hours. The reaction mixture was
concentrated under re~uced pressure and dried in vacuo. The
obtained yellow viscous solid was washed with acetone (10 ml~)
and collected to give D'X~(3-mesylaminophenyl)glycinamide
hydrochloride (580 ~g.) as greenish white powder. M.P~ 236 to
240UC. I.R. (Nujol) ~ cm : 3450-3350, 3240, 3180, 1695,
16~5, 15gO, llS0.
Example 13
To a methanol solution saturated with hydrogen
chloride (10 ml.3, there were added water (one drop) and DJ~-
amino-~-(3-mesylaminophenyl)acetonitrile L(+)-tartrate (having
one molecule of acetic acid) (435 mg.), and the resulting
mixture was refluxed for 1 hour, The reaction 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-~-
(3-mesylaminophenyl~glycine methyl ester (230 mg.). M.P. 101 to
153UC. I.R. (Nujo ~ cm : 3370, 3300, 1740, 1605, 1590.
Example A
To a suspension of 2,2,2-trichloroethyl 7-amino-3-
methyl-3-cephem-4-carboxylate hydrochloride (3820 mg~) in
absolute methylene chloride (50 ml.), triethylamine (810 mg.)
and N,N-dimethylaniline (245 mg.) were added dropwise under
ice-cooling with stirring, and stirring was continued at room
temperature for 30 minutes. Separately, N-t-butoxycarbonyl-2-
(3-mesylaminophenyl)-D-glycine (3440 mg.), triethylamine (1010
mg.) and N,N-dimethylbenzylamine (4 drops) were added to absolute
methylene chloride (50 ml.), and the mixture was stirred under
- 30 -
~)58~
dry ice-acetone cooling. A solution of ethyl chloroformate
(1085 mg.) in absolute methylene chloride (25 ml.) was dropwise
added thereto at -25 to -30~C in 10 minutes, and stirring was
conducted at the same temperature for 15 minutes. To the result-
ing mixture, the previously-obtai~ed mixture precooled at -15~C
was added all at once. The resultant mixture was stirred at
-25 to -30UC for 4 hours and washed with water, 3 % hydrochloric
acid and water in order. The organic layer was s~parated,
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 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-(3-mesylaminophenyl)glycinamido]-3-methyl-3-
cephem-4-carboxylate (4195 mg.). M.P. 204 to 205~C (decomp.).
The above obtained 2,2,2-trichloroethyl 7-[D-N-t-
butoxycarbonyl-2-(3-mesylaminophenyl)glycinamido]-3-methyl-3~
cephem-4-carboxylate (3985 mg.) was dissolved in dimethylform-
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 stirred 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 combined together and added to 50 % hydrochloric acid
(100 ml.) under ice cooling. Water (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 times with 5 % aqueous
solution of sodium bicarbonate (50 ml.). The aqueous layer was
acidified with hydrochloric acid and back-extracted again with
3LOS~3~Q~
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 was added thereto, whereby 7-[D-N-t-
butoxycarbonyl-2-(3-mesylaminophenyl)glycinamido]-3-methyl-3-
cephem-4-carboxylic acid (2687 mg.) was obtained. M.P. 187 to
189~C (decomp.~.
~xample B
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 fl tration, the filtrate was washed four times with ice-cooled
5 % hydrochloric acid (100 ml~), once with water, three times
with 10 % 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 dissolved in 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-~-t-butoxycarbonyl-2~3-mesylaminophenyl)-
glycinamido]-3-methyl-3-cephem-4-carboxylate (31.3 g.) as
colorless plates. M.P. 189 to l91~C (decomp.~.
The above obtained 2,2,2-trichloroethyl 7-[D-t-
- 32 -
~1058Z0~
butoxycarbonyl-2~3-mesylaminophenyl)glycinamido]-3-methyl-3-
cephem-4-carboxylate t12.01 g.~ was dissolved in dimethylform-
amide (40 ml.1. Glacial acetic acid (15 ml.) and zinc powder
(12 ~.) 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
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 pEI 2 with 10 % hydro-
chloric 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-[D-N-t-butoxycarbonyl-2-(3-mesyl-
aminophenyl)glycinamido]-3-methyl-3-cephem-4-carboxylic acid
(8.38 g.). M.P. 188 to 189~C (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.
Removins 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 p~I 3 with 10 % aqueous
solution of sodium hydroxide. The precipitated crystals were
collected by filtration, washed with water and dried to give 7-
- 33 -
~s~z~
[D-(3-mesylaminophenyl)glycinamido]-3-methyl-3-cephem-4-carboxylic
acid (6~18 g.). M.P. 199 to 199.5~C (decomp.). [~]D = +131
(O.lN HCl, C=l).
Example C
A solution of ethyl chloroformate (1.32 g.) in methylene
chloride ~20 ml.) was cooled to -lO~C, and a solution of N-t-
butoxycarbonyl-2~3-ethanesulfonamidophenyl)-D-glycine (4.4 g.)
and triethylamine (1.22 g.) in methylene chloride (20 ml.) and
N,~-dimethylbenzylamine (2 drops) was dropwise added thereto in
10 minutes. The resulting mixture was stirre,d 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-bis(trimethylsilyI)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
resultant 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 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~o117~C~
The above obtained 2,2,2-trichloroethyl 7-~D-N-t-
butoxycarbonylamino-2-(3-ethanesulfonamidophenyl)glycinamido]-
3-methyl-3-cephem-4-carboxylate (402 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
- 34 -
~v~
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 with 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
solutlon of sodium chloride (20 ml.) in turn and dried over mag-
nesium sulfate. The solvent was removed under reduced pressure,
and the resldue was washed with 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-CD-N-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 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 precipitated 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 182C (decomp.).
Example D
2,2,2-Trichloroethyl 7-CD-~-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 p~I 8 to 9 with an
aqueous saturated solution of sodium bicarbonate under ice
~582~
cooling. The precipitated crystals were collected by filtration,
washed with water and then dried over phophorus pentoxide to give
2,2,2-trichloroethyl 7-[D-2-(3-mesylaminophenyl)glycinamido]-
3-methyl-3-cep~em-4-carboxylate (0.78 g.). M.P. 107 to 110C
(decomp.).
Example E --
To a suspension o 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-Butoxy-
carbonyl-2-(4-mesylaminophenyl)-D-glycine (5.8 g.) and dicyclo-
hexylcarbodiimide (3.3 g.) were added to the resultant solution
under ice cooling. The mixture was kept stirred at the same
~temperature for 3 hours and filtered. The filtrate was con-
centrated under reduced pressure, and ethyl acetate (200 ml.~ was
added to the residue. The mixture was washed with 5 % hydro-
chloric acid, water, an aqueous saturated solution of sodium
bicarbonate and water in order, dried and then concentrated to
give 2,2,2-trichloroethyl 7-~D-~-t-butoxycarbonyl-2-(4-mesylamino-
phenyl)glycinamido]-3-methyl-3-cephem-4-carboxylate (10.83 g.).
M.P. 12g to 136UC (decomp.).
Acetic acid (12.5 ml.) and zinc powder (10 g.) were
added to a solution of2,2,2-trichloroethyl 7-[D-N-t-butoxy-
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 ml.) and extracted three times with ethyl
- 36 -
~S~ 6
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 extract was washed with water and dried, and
then the solvent was removed under reduced pressure to give 7-
CD-N-t-butoxycarbonyl-2-(4-mesylaminophenyl)glycinamido]-3-
methyl-3-cephem-4-carboxylic acid (7.8 g.). M.P. 180 to 200UC
(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 volume, 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-~-(4-mesylaminophenyl)glycinamido~-
3-methyl-3-cephem-4-carboxylic acid (2.1 g.). M.P. 205 to
207UC (decomp.).
Example F
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 ml.), and the mixture
was cooled to -10 to -12UC. A solution of isobutyl chloro-
formate (0.820 g.) in tetrahydrofuran (10 ml.) was dropwise added
- 37 -
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 (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
-6~C. Thus obtained mixture was stirred under ice cooling for
an hour and additionally at room tempera-ture 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 twice
with ethyl acetate (10 ml.). The washings were extracted with
an aqueous saturated solution of sodium bicarbonate (10 ml.~.
The aqeuous 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.),
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.)~
- 38 -
1~5~tZQ6
Example G
N-t-Butoxycarbonyl-2-(3-mesylaminophenyl)-D-glycine
(3 44 g.) and triethylamine (1.01 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 mlO) at -10 to -15UC in 5 minutes and
stirred at the same temperature for 15 minutes. Separately,
~,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
-15UC and stirred at the same temperature for 1.5 hours and at
10UC for 3 hours. The resultant mixture was washed with 5 %
hydrochloric acid and water, dried and then the solvent was
removed. The olly residue was purified by column chromatography
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.
The thus obtained 7-~D-~-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 formic acid (35 ml.) and stirred at 18 to 20UC
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-
1,3,4-thiadiazol-2-yl)thiomethyl-3-cephem-4-carboxylic acid
(1.95 g.). The product was added to acetone (30 ml.), stirred
at 15 to 20UC for an hour and allowed to stand. After removal
- 3g -
U~;
o the supernatant solution by decantation, acetone ~30 ml.)
was added to the residue and stirred at 15 to 20~C for 3 hours.
The precipitate was collected by filtration and washed with
acetone and ether to give the objective compound as pale
yellow powder,
Example H
(1) Dicyclohexylcarbodiimide (2.0 g.) was added
under ice cooling with stirring to a solution of N-t-butoxy-
carbonyl-2-(3-~esylamino-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.3. 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,
~ashed 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-L~-t-butoxycarbonyl-2-(3-mesylaTnino-4~hydroxyphenyl)-D-
glycinamido]-3-methyl-3-cephem-4-carboxylate (6075 g.~. The
product was crystallized from ethyl acetate to give the objective
compound. M.P. 185 to 188.5~C (decomp.).
(2) Acetic acid (3.5 m~.) and zinc powder (2.6 g.~
were added under ice cooling to a solution of 2,2,2-trichloro-
ethyl 7-~-t-butoxycarbonyl-2-(3-mesylamino-4-hydroxyphenyl)-
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
- 40 -
~OSl~6
reactlon was over, the zinc powder ~as collected by filtration
and ~ashed with dimethylformamide. ~he filtrate and the ~7ash-
ings were combined together, and ethyl acatate and dilute
phosphoric acid were added thereto. The ethyl acetate layer
was separated, ~rashed with ~ater and back-extracted with an
aqueous solution of sodium bicarbonate. The aqueous solution
~as acidified with hydrochloric acid and extracted ~ith ethyl
acetate. The ethyl acetate extract was washed ~ith water,
dried over rnagnesium sulfate and concentrated under reduced
pressure. The residue was washed wit'n ether to give 7-[~-t-
butoxycarbonyl-2-(3-mesylamino-4-hydroxyphenyl)-D-glycina~ido]-
3-methyl-3-cep'ne~-4-carboxylic acid (1.58 g.) as powder.
~ (3) A solution of 7-[N-t-butoxycarbonyl-2-(3-
mesylamino-4-hydroxyphenyl)-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 t,later (0.5 ml.) were
added to the residue with stirring, and stirrin~ was continued
for 30 minutes. The precipitated powder was filtered and washed
with acetonitrile and ether to give 7-[2-(3-mesyla~ino-4-
~ hydroxyphenyl)-D-glycinamido]-3-methyl-3-cephem-4-carboxylic
acid (1.13 g.). M.P. 186 to 192C (decomp.).
Exam~le I
(1) ~o a solution of N-t-butoxycarbonyl-2-(3-mesyl-
aminophenyl)-D-glycine (9.93 g) in a mixture OL tetrahydrofuran
(140 ml) and triethylamine (3.22 g) ~ept at -10C, iso~utyl
chlorofor~ate (3.95 g) ,ras added, and the resultant mixture
~ras stirred at -10C for 20 minutes. A cold solution o
7-aminocephalosporanic acid (7.87 g3 in a mixture of trieth~Jrl-
amine (3.5 g), tetrahy(irofura~ (40 ml) and ~rater (40 ml) ~laS
- 41 -
~5~2~6
added thereto all at once, and the resulting mixture was stirred
while ice cooling for 1 hour and at room temperature for 2 hours.
After the reaction was completed, 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
concentrated under reduced pressure. The residual oil was treat-
ed with ether to give 7-[D-N-t-butoxycarbonyl-2-(3-mesylamino-
phenyl~glycinamido]-3-acetoxymethyl-3-cephem-4-carboxylic acid
(10.2 g) as yellow powder. M.P. 83 to 84C (decomp.).
(2) A solution of 7-[D-N-t-butoxycarbonyl-2-(3-
mesylaminophenyl)glycinamido]-3-acetoxymethyl-3-cephem-4-
carboxylic acid (1 g) in formic acid (15 ml) was stirred at
room 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.J 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. 240UC (decomp.).
Example J
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 5.4) (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 65 UC for 6 hours. Acetone
- 42 -
~0~2~6
was remo~e~ fro~ the re3ultant mixture under reduced pressure,
and the remaining aqueou~ layer ~ras washed with eth~rO The
aqueous solu~ion ~as aàjusted to pH 2 with di.lut~ hydrochloric
acid and then extract~d ~Jith ethyl acetate. The extra.ct was
washed with water, dried over magnesium sulfa-te and conc~n-trated
under reduced pre~sure. The residue was ~urified ~y column
chromatography on silica gel (eluant: chloroform) to give 7-
[D-N-t-butoxycarbonyl-2-(3-mesylaminophenyl),~lycinamido~-3-
(5-methyl-1,3,4-thiadia~ol-2-yl)thio~ethyl-3-cephem-4-carboxylic
acid (1.8 g.).
E~ampl~ K
N-t-butoxycarbonyl-2-(3-dimesylaminophenyl)-D-glycine
derived.from 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-dimesylaminophenylt-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.
This application is a divi~on of Canaclian application
serial nv~ber 204,0169 filed July 4, 1974.
~ 4-S ~
