CA1056813A - .alpha.-AMINO- (P-ACYLOXYPHENYL) ACETAMIDOCEPHALOSPORANIC ACID DERIVATIVES - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

This specification discloses certain novel .alpha.-amino- or .alpha.-formyl-.alpha.-(p-acyloxyphenyl)acetamidocephalosporanic acids which are useful as antibacterial agents, and methods of making them. The novel cephalosporin derivatives of this invention comprise the D-(-) compounds of the formula I

wherein Y is hydrogen or S-Het, in which Het represents a 5 or 6 membered heterocyclic ring containing 1 to 4 atoms selected from N, O or S, said heterocyclic ring being optionally sub-stituted by C1-C4 alkyl which may be optionally substituted by a carboxylic acid group or hydroxy, or alkoxyalkyl of up to 4 carbon atoms; R is hydrogen, C1-C10 alkyl optionally sub-stituted by carboxylic acid, or phenyl optionally substituted by C1-C4 alkyl, halogen, nitro, amino or trifluoromethyl; R' is hydrogen, hydroxy, C1-C4 alkyl, C1-C4 alkoxy or halogen, and pharmaceutically acceptable salts thereof, when substantially free of the L-(+) isomer, and the D-(-) compounds of the formula I(a)

Description

This invention relates to cextain novel a-amino- or a-for~y~ (p-acyloxyphenyl)acetamidocephalosporanic acids which are useful as antibacterial agents.
The novel cephalosporin derivatives of this invention comprise the D-(-) compounds of the formula ~3NH2 F--N~H2Y

COOH
wherein Y is hydrogen or S-He~, in which Het represents a 5 or 6 membered heterocyclic ring containing 1 to 4 atoms selected from N, O or S, said heterocyclic ring being optionally 5ub-stituted by Cl-C4 alkyl which may be optionally substituted by a carboxylic acid group or hydroxy, or alkoxyalkyl of up to 4 carbon atoms; R is hydrogen, Cl-Clo alkyl optionally sub-stituted by carboxylic acid, or phenyl optionally substituted by Cl-C4 alkyl, halogen, nitro, amino or trifluoromethyl; R' is hydrogen, hydroxy, Cl-C4 alkyl, Cl-C~ alkoxy or halogen, and pharmaceutically acceptable salts thereof, when substantially free of the L-(~) isomer, and the D-(-) compounds of the formula I(a) R' R-~ H-CO-NH ~ S ~
CHO ~ N ~ CH2Y
COOH
I(a) wherein Y is S Het, in which Het is 1,2,3-triazol-5-yl, l-N-methyl-tetrazol-5-yl or 2-methyl-1,3,4-thiadiazol-5-yl; R is hydrogen, Cl-Clo alkyl optionally substituted by carboxylic acid, or phenyl optionally substituted by Cl-C4 alkyl, halo-gen, nitro, amino or trifluoromethyl; R' is hydrogen, hydrox~, Cl-C4 alkyl; Cl-C~ alko~y or halogen, and pharmaceutically acceptable salts thexeof, when substantially free of the 5 L-(~) isomer.
In the above compounds, the substituent halogen is preferably selected from fluorine, chlorine or bromine.
The pharmaceutically acceptable salts r~ferred to above include the nontoxic carboxylic acid salts, e.g., nontoxic metallic salts such as sodium, potassium, calcium and alumi-nium, ~he ammonium salt and salts with nontoxic amines, e.g., _ trialkylamines, procaine, dibenzylamine, N-benzyl-~-phene-thylamine, l-ephenamine, N,N'-dibenzylethylenediamine, N
alkylpiperidine and other c~nines which are used to form salts of penicillins and cephalosporins. Also included within the definition of pharmaceutically acceptable salts are the nontoxic acid addition salts (amine salts), e.gO, salts with mineral acids such as hydrochloric, hydrobromuc~ hydroiodic, phosphoric, sulfuric and salts with organic acids such as maleic, acetic,citric, oxalic, succinic, ben~oi~, tartaric, fumaric, mandelic, asoorbic and malic.
Examples o~ suitable heterocyclic groups included within the definition of "Het" in formula I are such heterocyclic radicals as thienyl, pyrazolyl, imidazolyl, isoimidazolyl, triazolyl, tetrazolyl, ~hiazolyl, thiadiazolyl, thiatriazolyl, oxazolyl, oxadiazolyl, isothiazolyl, isoxa~o~yl, pyridylt pyridazinyl, pyrazinyl, pyrimidinyl and triazinyl. The hetero-cyclic ring may be unsubstituted or substituted wi~h one or more of the substituents as mentioned above~
3Q Pre~erred D-(-) compounds of formula I are those wherein ~5~ 3 Y is hydrogen or S-Het, in which Het represents 1, 2, 3-triazolyl,

2~methyl-1,3,4-thiadiazol~5-yl, 2-methyl 1,3,4-oxadiazol-5-yl, l-N-methyl-tetrazol~5-yl and 1,2,3,4-tetrazolyl; R is hydrogen, Cl-C4 alkyl or phenyl vptionally substituted by Cl-C4 alkyl, fluorine, chlorine, nitro, amino or trifluoromethyl; and R' is hydrogen, or the above D (-) compounds of formula I(a) wherein Y is S-Het, in which Het is 1,2,3-triazolyl, 2-methyl-1,3,4-thiaaiazol-5-yl, or l-N-methyl-tetrazol-5-yl; R is hydrogen or Cl-C4 alkyl; and R' is hydrogen.
More preferred D-(-) com~unds of formula I and I(a) are those in which Y is hydrogen or S-Het, in which Het is 1,2,3-triazolyl, l-N--methyl-tetrazol-5-yl or 2-methyl-1,3,4-thiadiazol-5yl; R is hydrogen or methyl; and R' is hydrogen.
The present invention also incllld~s a process fox the preparation of a 7-D-(-)~-amino~d(p-ac~loxyphenylacetamido) cephalosporanic acid of the formula R-~-O ~ fH-~-NH

~2 ~ ~ H2Y
I ~ :

wherein Y is hydrogen or S-Het, in which Het is a 5 membered heterocyclic ring containing l to 4 N atoms; and R is Cl-ClO
alkyl, or phenyl, when substantially free of the L-(~) isomer, which :
.:

:

.- " .

. .
,., . , . : .

process comprises ~ea~tln~ ~ compound of the formula H2 N T~S

,~N~7~C~2Y :
COOH II
or a silyl ester or salt thereof in which Y is as defined above or acetoxy with a corresponding D-(-) acylating agent of an acid of the formula ~ .
: ' lQ R-~-O ~ fH-COO~
NHB ~ .
III -in which R is as defined above and B is an amino-pro-tecting group, and removing the amino-prote~ting group, and ;
when Y is acetoxy converting the thus-obtained compound to the corresponding final compound wherein Y i~3 S-Het and, when the .
compound of formula II is in the form of the silyl ester or .
salt thereof, either before or after removal of B, converting the thus-obtained compou~d to the corresponding final product.
In a preferred embodiment, the acylating agent is of the formula R~C O ~ ~ CH - COCl NH2JHCl wherein R ~s ~s dP~ined above.
In the preparation of the novel cephalosporin compounds of the present invention, a corresponding 7-amino-cephalosporanic acid compound of formula II or salt thereof is acylated by known me~hods with the appropriate D-(-) acylating agent of formula In the case o$ 3-thiolated-7-aminocephalosporanic acid . ,~ ~ . .

intermediate of formula II, when Y is S-Het, said intermediate may he prepared by dlsplacement of the 3 acetoxy group of 7-aminocephalosporanic acid or a salt thereof with th~ appropriate heterocyclic thiol or a salt thereof. The displacement of an ester group with a thiol group is a known reaction and i5 preferably accomplished in aqueous solution wit~ heating.
The intermediate II may, if desired, be converted prior to the acylation reaction to a silyl ester or acid addition salt thereof. The ~ilyl esters may be prepared by the methods described in the literature, e.g. U. S. Patent 3,249,622. The silyl ester group may be removed following the acylation re-action by hydrolysis.
Prior to the acylation reaction the amino group of the acylating agent III may be protected by a conventional amino-blocking group B respectively, which may be readily removedat the conclusion of the reaction by methods known per se.

.
Examples of suitable amino-protecting or blocking groups include t-butoxycarbonyl, carbobenzyloxy, 2-hydroxy-1 naphthcarbonyl, trichloroethoxycarbonyl, 2-ethoxycarbonyl-1-methylvinyl and 2~methoxycarbonyl-1-methylvinyl. A particularly valuable blocking group is a proton, as in the compound of the formula ~' R~-O ~ ICH - COCl NH2 . ~Cl ~5 For example, following the acylation coupling reaction, it can be easily removed by neutralization. Obviously other func-tionally equivalent blocking groups for an amino group can be used and such grollps are considered within the scope of this invention.
In addition to the above, the present invention also in-cludes a process for the preparation of a compound of the formula I(a) wherein Y is S-Het, in which Het is 1,2,3-triazol-5-yl, 1-N-methyl-tetrazol-5-yl or 2-methyl-1,3,4 thiadiazol-5-yl; R is hydrogen, Cl-C10 alkyl optionally substituted by carboxylic acid, or phenyl optionally substituted by Cl-C4 alkyl, halogen, nitro, amino or trifluoromethyl; R' is hydro-gen, hydroxy, Cl-C~ alkyl, Cl-C~ alkoxy or halogen, and pharma-ceutically acceptable salts thereof, whsn substantially free of the L- (~) isomer, which process comprises reacting a com-pound of the formula II, in which Y is as immediately defined above or a silyl ester or salt thereof with a corresponding D~t~) acylating agent of an acid of the formula R' R--~--o~H-COOr:~
OCHO

~ a~
in which R and R' are as defined immediately above, to produce the co~pound of formula Ita) or a pharmaceutically acceptable salt thereof, and, if desired, converting by methods known se the product in the form of the free acid or silyl ester or salt thereof to the corresponding free acid or pharma-ceutically acceptable salt thereof.
Acylation of a 7-amino group of a cephalosporin is a well-known reaation and any of the functional equivalents of ~ormula III or III(a) commonly used as acylating agents for primary amino groups may be employed. Examples of suitable acylating derivatives of the free acid include the correspondin~

~5~ L3 acid c~nhydrides, mixed anhydrides, e.g. alko~yformic anhydrides, acid halides, acid azides, active esters and active thioesters.
The free acid may be coupled with compound II after first re-acting said free acid with N,N'-dimethylchloroformunium chloride or by the use of enzymes or of an N,N'-carbonyldiimudazole or an N,N'-carbonylditriazole or a carbodiimide reagent, e.g.
N,N-diisopropylcarbodiimide. N,N'-dic~clohexylcarbodiimide or N-cyclohexylcarbodiimide or N-cyclohexyl-N'-~2-morphilino-ethyl) carbodiimide or of an alkylylamine reagent or of an isoxasolium salt reagent. Another equivalent of the free acid is a corresponding azolide, i.e., an amide of the corresponding acid whose amide nitrogen is a member of a quasiaromatic five member~d ring containing a~ least two nitrogen atoms, i.e. imidazole, pyrazole, ~he triazoles, ben~iimidazole, benzotriazole and their substituted deriva-tives. A reactive derivative of the phenylglycine acid of formula III is the N-carboxy c~hydride tLeuch's anhydride~. In this structure the group which activate the carboxyl group also serves to protect th~ amino group. A
particularly preferred acylating agent is the acid chloride hydrochloride of the formula : R~

R-C-O ~ C5 - COCl ~S NH2~Cl which also ser~es a dual function of carboxyl activation and amino protection. Mention was made above of the use of enzymes to couple the free acid wi~h its blocked amino group with compound III. Included in the scope of such processes _ 7 _ :
..
,,. ,.:: , .,, ,. , ., ; . .... ; .:, . .. : : . :
- . . . . , ,. , ,, . .. , . : .

~L~5~3 are the use of an ester, e.g. the methyl ester, of that free acid with enzymes provided by various microorganisms, ~ e.g. those described ~y T. Takahashi et al., J. Amer. Chem. Soc., 94(11), 4035-4037 ~1~72) and by T. Nara et al., J Antibio$ics (Japan), 24(5), 321-323 (1971) and in West Germany 2,216,113.
The particular process conditions, P.g. temperature, solvent, reaction time, etc. selected for the coupling re action are determined by the nature of the acylation method u~ed and are known to those skilled in the art. Generally it is useful to add an organic tertiary amine, e.g. triethyl-amine, N,N-dimethylaniline, ethylpiperidine, 2,6-lutidine or quinoline, to s~rve as a proton acceptor or salt-forming agent.
The compounds of the present invention may be isolated in any of the ways customarily employed for the isolation of similar cephalosporins. Thus, the product may be obtained as the neutral molecule and, in the case of compounds of formula I, this is probably more accurately represented as the zwitterion, or it may be isolated as a salt. Formation of the desired pharmacautically acceptable carbox~lic acid or acid addit~on salt is carried out by known methods, e.g. reaction of the acid with an appropriate base or acid.
At the conclusion of the acylation reaction the product obtained may be converted tbefore or after removal of the amino-protecting group1 by methods known ~ se to another desired product of formula I. For example; the product of formula I
or I(a~ in the form of a silyl ester or salt thereof may be converted to the free acid product or pharmaceutically acceptable sal$ thereof by removal of thé silyl ester group, e.g. by hydroly~is. :
The pharmaceutically active compounds of the present inven-tion are potent antibacteri~l agents useful in the treatment o~

. ... . .... : ' ' ' ', ~, ',' ', ,

3~5~ 3 inf~ctious diseases in poultry and animals, including man, caused by many Gram positive and Gram-negative bacterial The active compounds are also of value as nutritional supplements in animal feeds and as agents for the treatment of mastitis in cattle. The preferred compounds have also been unexpectedly found to be efficiently absorbed upon oral administration.
The novel medicaments provided by the present invention may be formulated as pharmaceutical compositions comprising, in addition to the active ingredient, a pharmaceutically acceptable carrier or diluent. The compounds may be administered both orally and parenterally. The pharmaceutical preparations may be in solid form such as capsules, tablets or emulsions.
In the treatment of bacterial infections in man, the compounds of this invention may be administered parenterally in an amount of from about 5 to 200 mg./kg./day in divided dosage, e.g. 3 to 4 times a day. They are administered in dosage units containing e.g. 125, 250 or 500 mg~ of active ingxedient with suitable physiologically acceptable carriers or excipients.
The following illustrates the preparation of staxting materials used in the production of the novel compounds of the invention.

,,. ~
.. . .

_ g - " ' ~

: .' :

~: . . ,, .: ....... , :. . . . . . . ~

L3 ~:
Star*in~ Materials .

Synthesis of potassium 1,2,3-triazole-~-thiolate . o Il N
~C-N=C=S ~ CH2N2 ~ OH-~
N~ ~ -~HCO
163.1g ~.0~ 205.24 .... ....
n N ~N ~ ~
N N
H H
-101~13 139.23 The synthesis of the thiol was accomplished by a pro-cedure essentially identical to that described in the litera-IS ture [~. Gverdler and G. Gnad, Chem. Ber. 99, 1618 (1966)].
: 5 Benzamide~1,2,3-thiadiazole To a stirred solution of benzoylisothiocyanate (50.6 g., 310 mmoles) in commercial anhydrous ether (400 ml.)~ main-tained at 0 and in a nitrogen atmosphere, was added dropwise wi~h vigorous stirring, 0.685 N e~hereal diazomethane (453 :~
~1., 310 mmoles). When the addition was completed, the mix-t~r~ was stirred for 1 hour at 0, the solid was collected by filtration and dried in vacuo. The melting point of the ~ crude material (23.3 g.) thus obtained was observed somewhere in the region 232 to 257C. Goerdler reported m.p. 267 for the pure material. A small second crop (2.1 g.~ was nbtained by evaporation of the mothex liquor in vacuo. The total . : :
yield was therefore 40%.
' . .

,.

~i6~3L3 1,2,3-Triazole-5-thiol A solution of the above benzamido compound (8.2 ~O~ 40 mmoles) in 2N sodium hydroxide (80 ml'., 160 mmoles) was heated under reflux temperature in a nit~ogen atmosphere for 24 hours.
The solution was cooled to 0 in ice, and concentrated hydro-chloric acid (26 mla) was added, while a continuous stream of nitrogen was passed through the solution. The benzoic acid which precipitated was collected by filtration; the filtrate was saturated with sodium chloride and the additional benzoic acid which separated was removed by filtration. The filtrate was immediately extracted with ethyl acetate~ the extract was washed with saturated salt solution, dried over magnesium ~' -sul~ate and then evaporated'ln vacuo. The viscous oil which remained ~as immediately evaporatively distilled ln vacuo ~70- ' 75/0.001 mm.) to give an oil (2.84 g., 70%) which solidified ~' (m.p. 52-59; Goerdler reported m.p. 60) spontaneously.
Potassium '1',2,3-Tr'ia'zole~5-thi'o'late ~ ' To a solution of the above thiol (2.84 g., 28.1 mmoles) in absolute ethanol (28 ml.) was added 1.93 N alcoholic potas- ;' sium hydroxide solution (14.5 ml.). The solution was then diluted with anhydrous ether until crystallization of the salt was completed. The solid was collected by filtration, washed with ether~ and dried' in vacuo. The salt obtained in this manner (3.65 g., 93%) had m.p. 225 with decomposition.
It is important to note that the conversion of the benzamido thiadiazole to the triazole thiol is known to proceed v'ia 5-amino-1~2,3-thiadiazole [G. Goerdler and G. Gnad, Chem.
:: -Ber.''99, 1618 (1966)~
'' ' ' . ', . ," , ' .
: ': ' . .

~35~ 3 .. _ N~ ~

NHCO0 ~ ~ l~I2 N OH ~
I/ ~ ~ N N 87%
N N N~ S ~ ~ N
H 93%
5-Amino-1,2,3-thiadiazole can be prepared by an alternative :~ .
route, not involving diazomethane [D. L. Pain and R. Slack, ~ :
J. Chem. Soc. 5166 (1965)]~
O `~

~N-CH2CH + H2NNHC2Et -~

o ,: ~

: ~ N-CH2CH-N-NHCO2Et O 93%

N

O ~N :
89%
N ~

30 . ~ ~ H ~ ~:
- 12 - 77%
~ . .

:. ; , ,::, ~ .
., . ~ I

Synthesis of 7-Ami~o-3-(1,2,3~triazol-5-ylthiomethyl)-3-cep~em-4-carboxylic acld The reactions were conducted under a nitrogen atmosphere in a reaction vessel protected from light. The water and phosphate buffer were gassed vigorously with nitrogen prior to use to displace oxygen.
5-Amino-1,2,3~thiadiazole (10.3 g., 0.102 mole) was added to a solution of 8.16 g. of sodium hydroxide in 100 ml. of water. The mixture was heated rapidly to reflux and then re-fluxed or 10 min. to rearrange 5-amino-1,2,3-thiadiazole to 5-mercapto-1,2,3-triazole. To the reaction mixture containing 5-mercapto-1,2,3-triazole cooled in an ice bath was added 1100 ml. of ice cold O.lM pH 6.4 phosphate buffer. The solution, which was at pH 10.5t was adjusted to pH 8.5 with 42~ phosphoric acid. 7-Aminocephalosporanic acid (21.8 g., 0O08 mole) was added and the mixture heated at 50~ for 4 hours~
The clear solution was cooled in an ice bath and adjusted to p~ 4.5 with conc. HCl. The precipitated product was collected by filtration, washed with water and air dried; 16.2 g.
The crude product (15.2 g.) wa-~ brought in o solution with 600 ml. of methanol and 40 ml. of conc. ~C1. After carbon treatment the solution was diluted with 1.5 1. of ice water and extxacted once with ethyl acetate. The aqueou~
phase was concentrated at reduced pressure to remove methanol.
The cold aqueous concentrate was adjusted slowly to pH 4.0 with 20% sodium hydroxide causing crystallization of the product. The product was collected by filtration, washed with water and methanol and dried in vacuo over phosphorus pentoxide; 11.4 g. The IR and NMR spectra were fully consistent for the desired product.

-13- ;

Anal. Calcd- for CloHllN53S2 C, 38-42;
Found: C, 38.27, 38.26; H~ 3.76, 3.40; N, 21.02, 21.00; H2O, 1070.

Purific tion of 7-Amino-3 (1,2,3-t ~ thiom~ 3-~ .
Crude 7-amino 3-(1,2,3-triazol-5-ylthiomethyl3-3- -cephem-4-carboxylic acid (16.1 g.) containing approximately 20 mole % of 7-aminocephalosporanic acid as an impurity, was brought into solution with 600 ml. of methanol and 40 ml.
of conc. ~Cl. Ater carbon treatment, the solution was diluted with 1.5 lo of ice water and extracted once with ethyl acetate. The aqueous phase was concentrated at reduced pressure to remove methanol. rhe cold aqueous concentrate was then adjusted slowly to pH ~.0 with 20% sodium hydroxide causing the product to crystallize. The product was collected by filtration, washed with water and methanol and dried in vacuo over phosphorus pentoxide; 11.4 g. The NMR spectrum indicated that this product contained about 7 mole ~ of 7 aminocephalo~poranic acid as an impurity.
~ he above purification procedure was repeated on 11.4 g.
of the product using 425 ml. of methanol, 28 ml. of conc. HCl and 1 1. of ice water yielding 8.0 g. of product. The NMR
spectrum was fully consistent for the desired product and indicated no trace of 7-aminocephalosporanic acid as an impurity.
Anal. Calcd. for CloH15N5O3S2: C, 38.42; X, 3.55; N, 22.40.
Found: C, 39.06, 38.53; H, 3.56, 3.51; N, 22.05, 21,60; H2O, 1.780 3n :
-14~

~6~3 7-Amino-3-(1,2,3-t_iazole-5- lthlometh ~ hem-4-carboxylic acid (II) .
Ten grams (0.075 mole) of 5-mercapto-1,2,3-triazole potassium salt was added to a stirred slurry of 19 g. (0.07 mole) of purified 7-aminocephalosporanic acid and 5.9 g.
(0.07 mole~ of NaHCO3 in 350 ml. of 0.1 M phosp~ate buffer (pH 6.4) and the mixture heated and stirred at 55 C~ for 3 1/2 hours under a nitrogen atmosphere. The resulting solution was cooled to 22 C. and adjusted to pH 5.5 with 40~ H3PO4. The resulting precipitate was filtered off, washed with cold water (50 ml.) and air dri~d. The yield of 7-amino-3-(1,2,3-triazole-5-ylthiomethyl)-3-cephem~4-carboxylic acid was 8 g., dec. pt. 230 C. IR
analysis showed some decomposition of the ~-lactam ring but it was used "as is" for the next step.
~nal. Calcd. for ClOHllN5O3S2 C, 38-39; ~, Found: C, 38.36; ~, 3~78.

? ~ hiomethyl)_3-cephem-4-carbo~_ic acld (II) Two hundred seventy-two g. (1.0 mole) of 7-amino~
caphalosporanic acid was suspended in 3000 ml. of O.lM phosphate buffer, pH 6.4, and 150 ml. of methyl isobutyl ketone followed by 84 g. (1.0 mole) of sodium bicarbonate tNote: The sodium bicarbonate was added in portions). Then 143 g. (1.0 mole) of 5-mercapto~ 1,2~3-triazole potassiwm salt was added and the mixture stirred at 55 C. 1~ C. under a nitrogen atmosphere for 4 hours. After 1 hr. the pH was readjusted to 6.4 by addition of a small amount of 40% H3PO4. At the end of the 4 hr. heating period, 50 g. of "Darco KB" decolorizing *Trade Mark ~5~3 charcoal was added and, after stirred for 15 min. at 55 C., the slurry was filtered hot through a diatomaceous earth ("Celite") pad. The pad was washed with 3 x 100 ml. water.
The pH of the combined filtrates was adjusted while hot to

4.5 by slow addition of 6 N HCl. After cooling 30 min. at 0 C., the crude product was collected by filtration, washed with 2 x 200 ml. of cold water followed by 2 x 1000 ml. of methanol and air dried.
The crude product was suspended in 3000 ml. of 50~
methanol-water and 300 g. (1.5 mole) of ~-toluenesulfonic acid was added. The mixture was stirred for 15 min. and then 50 g. of "Darco KB" decolorizing charcoal was added. A~ter stirring for 15 min. at 22 C., the slurry was ~iltered through a "Celite~" pad and the pad washed with 2 x 100 ml.
o 50% methanol-water. The pH of the combined filtrates was adjusted to 4.0 by addition of apprc)ximately 210 ml. of tri-ethylamine. After cooling at 0 C. for 1 hour the product was collected by filtration, washed with 2 x 400 ml. of 50%
methanol~water and then 2-1000 ml. of methanol and air dried.
This material was suspended in 2000 ml. of water and 84 g. (1 mole) of sodium ~icarbonate was added. After stirring for 10 min. at 22 C., 50 g. of "Darco KB" charcoal was added and, after stirring for 15 min. at 22 C. the slurry was fil-ter~d thxough a "Celite" pad. The product was washed with ~5 2 x 100 ml. of water and the pH of the combined filtrates was adjusted to 3~5 by slow addition of 6 N HCl. After stir-ring for 10 min. at 22 C., the mixture was cooled to 0 C.
for 1 hr. The product was collected b~ filtration was washed with 2 x 200 ml. of cold water and 2 x 1000 ml. of acetone.
After drying over P2O5 in a vacuum desiccator for 14 hr. at *Trade Mark ~5~ 3 room temperature the yield wa~ lQ~,g.; dec. pt~ 230Co Th~ IR and NMR were consistent for the desired structure.
. . .
~ Preparation of D (-)-2-amino-2-~4-acetoxyphenyl3acetic acid Method A ~in acetic acid as solvent~
203.5 g ~1 Mole) of D~-)p-hydrox~phenylglycine chloride, 800 ml of acetic acid and 314 g( 4 Moles) of acetyl chloride are stirred 48 hours at room temperature. The solid i5 collected, washed three times with acetone (3 x 250 ml) and twice wi~h ethanol (2 x 250 ml) and dried at 40O. Yield 10 210 g (85.4%). This hydrochloride is dissolved in 3.0 1 of water; the solution is cooled to ~5 to 10C and the pH
adjusted to 4O5 with 2Q% NH40H. The suspension is stirred 1 hour at 5C and the solid collected, washed twice with water and twice with acetone, and dried at 40C. Yield 133 g~ ~4~ from D(-)p-hydroxy phenyl glycine).
~D (1~ HCl N/10) ~ -104.5 :' '''-' Me thod B (in methylene chloride~ r. .... .
4.07 g (0.02 Mole) of D(,-)p-hydroxyphenylglycine hydrochloride, 30 ml of methylene chloride and 6.28 g ( p.0$ Mole) of acetyl 2Q chloride are stirred 48 hours at room temperature. ~he solid is collected~ washed twice with acetone and twice with ethanol.
Yield 4.17 g (84.5~). Anal. cl = 14.8% (calculated 14.4%~

Method C (in trifluoroacetic acid) 1.67 g ~0.01 Mole) of D(-)p-hydroxyphenylglycine is added with stirring, to 10 1~ of tri~luoroacetic acid at room temperature. After dissolution, 1~57 gC~.02 Mole) of acetyl chloride is added. A~ter a slightly exothermic reaction, a . . ' ' . ,' ~ .

~!3 solid appPars. The suspension is stirred 1 1~2 hours at room temperature and th~ trifluoroacetic aci~ is remov d in ~ vacuum. The remaining solid is collected, washed with methylene chloride and with ethanol. The D(-~2-anuno-2-(4-acetoxyphenyl~acetic acid is identical to that prepared by method A or B.
Yield: 1.9 g(75~) Preparation of D (-) 2-amino-2-(4-pivalyloxyphenyl~ acetic acid hydrochloride 1.67 g ~0.01 Mole) of D(-~p-hydroxyphenylglycine is added - to 10 ml of trifluoroacetic acid, followed by 2.4 g (0.02 Mole~ of pivalyl chloride. The resulting solution is stirred 24 hours at room temperature and vacuum concentrated to dryness. The solid is collected and washed with ether.
Yield: 2.56 g (89%)~
This hydrochloride is recrystallized Erom isopropanol.
Anal. Cl = 11.8% (calculated 12.3~) W ,~,maxO 205 nm and 220 nm.

Preparation of D ~-),2-amino-2-(4-benzoyloxyphenyl)acetic _id hydrochloride This compound is prepared according to the same procedure as used for the pivalyloxy derivakive. Yield: 2.7 g ~87%).
25' An analytical sample is recrystallized from ethanol. ~ -Anal. cl. = 11.3% (calculated 11.5%) UV ~max. 205 nm and 234 nm ... . .. . ... .

~S~3 Preparation of D (-)2-amino-2-(4-aceto~henyl)acetx~ chloride hydrochloride 83.6 g. (0.40 mole) of D(-)2-amino-2-(4-acetoxyphenyl)-acetic acid and 1.25 1. of anhydrous methylene chloride are cooled to -5 C. with stirring. Then 152 gO of phosphorous pentachloxide are slowly added followed by 4 ml. of dimethyl formamide. The mixture is stirred 4 hours at 0 C. The solid is collected, washed with anhydrous methylene chloride and vacuum dried at room temperature. Yield: 61 g. (57O5~).
Anal. Total chlorine - 27.2% (Theory 26.9%) Pre aration of D~(-)2-form~__xy~2-(4-f_rmxloxy~h n P
acid ~ . _ A solution of 3.6 g~ (0.02 mole) of D-~-)-2-hydroxy-2-(4-hydroxyphenyl)acetic acid in 50 ml. of 97% aqueous formic acid was allowed to react at 22 C. for approximately 68 hours.
The excess formic acid was removed by distillation at 22 C.
under reduced pressure. The residue is extracted with diethyl ether; the ethereal layer is dried over sodium sul~ate, fil-tered and evaporated to afford the desired product.

Prevaration of D-(-)2-formvloxY-2-(4-acetoxY~henvl)acetic acid The D-(-)2-formyloxy 2-t4-formyloxyphenyl)acetic acid obtained above is dissolved in 10 ml. of acetyl chloride and the resulting mixture was allowed to stand at 22 C. for 20 hours. The excess acetyl chloride was distilled off under re-duced pressure; the residue was treated with benzene and the benzene then removed under vacuum to afford the desired prod-uct which analyzed as 60~ pure according to NMR.

The following examples are gi~en in illustration of,bii. not in limitation of, the prPsent invention. All temper-atures are in degrees Centigrage. 7-Aminocephalosporanic acid is a~breviated as 7-ACA and 7-aminodesacetoxycephalosporanic aci~ as 7-ADCA.

Exam~le 1 7-D(-)2-amino-2-(4 aceto~yphenylacet_mido)desacetoxy-cephalosporanic acid - (acetoxy ce~halexine) - RN 1394 15.27 g ~ poO714 Mole~ of 7~ADCA are stirred in 500 ml of anhydrous methylene chloride; 120 ml of methylene chloride are distilled off and 11.8 ml o hexamethyldisilazane are added. The mixture is stirred and refluxed 20 hours (after about 10-15 hours all the 7-ADCA is run in solution~. The above solution is cooled to 0C and 120 ml of methylene chloride followed by the addition of 9.5 ml of dimethylaniline and 7 ml of a solution of dimethyl~niline hydrochloride in methylene chloride (30%). Then 20 g (0.0756 Mole) of D(-)2-amlno-2~4 -acetoxyphenyl~acetyl chloride hydrochloride are added in small portions (~1 1/2 hours) a~ 0C. :
The mixtuxe is stirred 30 min. at +10C and 4 hours at ~20OC
and-allowed to stand overnight at ~5C. Then S ml of methanol followed by 240 ml of water are added~ The pH is adjusted at 2.5 with triethylamine and ~he mixture is filtered , through a celite pad; then the pH is checked and the aqueous phase is separated, washed twice 52 x 150 ml) with methylene c~loride and treated with charcoal. .~.
The solution i~ adjusted to pH 4~5 and vacuum concentrated to a volume of ~ 150 ml. m e suspension is allowed to stand - 20 ~

~ ~s~

ov~rrlight at f5C and th~ solid colleeted and washed with wa ~cer and ace tone, and dried at 40c C .
Yield: 15,1 g ( - 50~O of 75-80-^,' pure ma~erial) ¦ ~D ( 1% H2O) -- +107

5 14 g of this crude material is suspended in 30 ml of wat~3r (p~ = 3,2): hydrochloxic acid t~6%7 is added to p~
1, 3 and ~he resulting solutio;~ is charcoal treated and filt~red through a celite pæ~d and adjusted to pH 4 4, 5 otter stirring~ After 2 hours at û- ~5C the R~ 139~
10 collç~cted, i~ashed with wat~r and acetone and dried at ~'.O~C.

Yield: 7 g, ~D(l,-~ ~l2O) ~ +i33- :
T`ne infrared and nucl~ar magnetic resonance s~ectra are consistent with the d~sired product.

Biological Data Table I shows comparative MIC dlata for BL-S 578-4 (~-hydroxy analogue of cephalexin) and (p-acetoxycephalexin (RN 1394). Minimal inhibitory concentrations were detexmined by the 2-fold broth dilution method utilizing equimolar con-centrations o~ each compound.

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Example 7~ -12-Am~no-2-(4-acetoxyphenylacetamido]-3~ 2~3-tria - _-yl)-thiomethyl3-3-cephem-4-carbo~ylic acid: ~N 1396 62.6 g (0.2 Mole) of 7-amino-3-~(1,2,3-triazcl-5-yl)thiomathyl]-3-cephem-4-carboxylic acid (7-TACA), 1.5 1 of methylene chloride and 6Q.2 g (0.374 le~ of hexamethyl disilazane are stirred and refluxed 20 hours with a slight pressure o~ nitrogen (after about 2 hours all 7-TACA is run in solution~.
The solution is cooled to 0C and 30.4 ml of dimethylaniline, followed by 2n .4 ml of a solution of dimethylaniline hydrochloride in methylene chloride (30%) and 1.35 g of imidazole are added.
Then 60.5 g (0.22~ mole~ of 2-amino-2-t4-acetoxyphenyl)acetyl chloride hydrochloride are added in small portions (~ 1 1~
hour ) at 0C. The mixture is then stirred 3 hours at 20C
and let stand overnight at +5C. 25 ml of methanol followed by 75~ ml of water are then added. The pH is adjusted to 2.3 - 2.5 with triethylamine and the l~xture is filtered through a celite pad. The a~ueous phase is separated, washed twice with n~thyle~e chloridP ~nd charcoal treated.
The solutio~ is adjusted to p~ 4.3 and stirred 2 hours at ~ .
~5C. The solid is collected, washed twice with wat~r and dried at 40C. Yield: 53 g (about 5û%). ~:
This crude material is purified twice as follows:
The solid is treated with eight volumes of 0.5N hydrochloric acid and the suspension is decolorized with charcoal. An equal volume o~ methanol is adde~ to the solution and the pH
adjusted to 2 - 2.1; aftex 15 minutes a small amount of suspended solid is colle~ted and discarded. The filtrate is adjusted to pH 4. The precipitated solid is c~llected, washed with - ~3 -.
. . .. . , ~ :

MeO~/H20 (50/50) and pure methanol.
Yield: 25 g. (after two purifications).
- IR consistent with assigned structure - Moisture (KF) : 5.1%
- Chemical ascay - Iodometric assay 885 mcg/mg - Potentiometry Amine assay: 97 ~ ~: D~t~ .
Table II shows comparative MIC data for the above prepared 7-[~( )2~amino~2-(4-acetoxyphenylacetamido)]-3-[1,2,3-triazole-S~yl)-thiomethyl]-3~cephem-4-carboxylic acid, RN-1396, and its p-hydroxy analogue, BLWS640. Minimal inhibitory concentrations were determined by the 2-fold broth dilution method utilizing equimolar concentrations of each compoundO

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, Example 3 7-D-(-)[2-(4-acetoxyphenyl)-2-ormyloxyacetamido]-3~ methy~
1,2l 3,4-tetrazol-S-yl)thiomethyl-3-cephem-4 carboxylic acid To a solution of 4.8 g. (0~02 mole) of crude D-(-)2-formyloxy-2-(4-acetoxyphenyl)acetic acid and 25 ml. of diethyl ether was added one drop of dimethylformamide and 5 ml. of oxalyl chloride. After stirring at 22 C. for 1 hour, the solvent was removed and the residue dissolved in 25 ml. of acetone, the resulting solution was added dropwis~ to a solu-tion of 6.3 gO (0.02 mole) of 7 amino-3~ methyl-1,2,3l4-tetrazol-5-yl~thiomethyl-3 ceph~m-4-carboxylic acid, 5.6 g.
of sodium bicarbonate, 300 ml. of water and 80 ml. of acetone at approximately 3 C. The reaction mixture was stirred for 1 hour at 3 to 5 C. followed by removal of the acetone. The pH of the residue wa~ adjusted to 2.0 by the addition of 40%
aqueous phosphoric acid under a layer of ethyl acetate~ The aqueous layer was extracted with 2 x 100 ml. of ethyl acetate and the combined organic layers dried over sodium sulfate.
The organic layers were filtered and the filtrate evaporated under vacuum to an oil~ Trituration of the oil with diethyl ... .
ether afforded 8 g. of solid product which analyzed by NMR
to have 85 90~ acetyl and 50-60% formyl.

Example 4 7-D-_(-)[2-(4-~orm~ x~phenyl) 2-formyloxyacetamidol-3~ methyl-1,2,3,4--tetrazol 5-yl)thiomethyl-3-c~phem-4-carboxylic acid The same procedure as in Example 3 was followed to react 2.S g. (0.01 mole) of D-(-)2-formyloxy-2-(4-formyloxyphenyl)-acetic acid with 3.28 g. ~0.01 mole) of 7 amino-3-(1-me~hyl-1,2,3,4-tetrazol-5yl~thiomethyl-3-cephem 4-carboxylic acid to -2~-~L~5~
afford after trituration with diethyl ether 4.2 g. of desired product which solids had a decomposition point of 160-5 C., an infrared and NMR spectrum consistent with its structure but containing a by-product.
Microanalysis of the desired product gave:
Calc'd. for C20H18N6O8S2 % The~ %_Found C ~4.86 46.47 H 3.38 4.14 N 15.70 13.50 K.F. (H2O) 1.78 lS

~5~
In addition to the above, the compounds of the instant invention are also valuable as intermediates for ~he prepara-tion of other pharmaceutically active compounds. For example, the instant a-formyloxy or a-amino-a-(p-acyloxyphenyl)acetamido cephalosporanic acids may be converted to the corre~ponding p~hydroxy compounds which are known to be potent antibacterial agents useful in the treatment of infectious diseases in poultry and animals, including man, caused by many Gram-positive and Gram-negative bacteria. The conversion can be carried out chemically by simple acid or base hydrolysis in an aqueous medium in most circumstances.
We have found that 7~D~(-ja-amino-a-(p-acetoxyphenyl-acetamido)desacetoxycephalosporanic acid, although stable in normal saline, is hydrolyzed enzymatically to the known and potent 7-D-(-)a-amino-a-(p-hydroxyphlenylacetamido)desacetoxy-cephalosporanic acid.
Accordingly, the present invention also provides for a novel process for preparing 7-D~ amino-a-(p-hydroxy-phenylacetamido)de~acetoxycephalosporanic acid, hydrate or a pharmaceukically acceptable salt thereof, which process comprises treating in an aqueous solution 7-D-(-)a-amino -(p acetoxy-phenylacetamido~des~cetoxycephalosporanic acid with an esterase at a pH between about 5.0 and about 7.5; isolating the product by m~thods known ~ se, and, if desired, converting by methods kno~m ~ se the product in the form of the free acid or hydrate to the corresponding phaxmaceutically acceptable salt thereof.
A preferred embodiment is the preparation of 7-D~
- amino-a-(p-hydroxyphenylacetamido)desacetoxycephalosporanic acid, hydrate or a pharmaceutically acceptable salt which process comprises treating in aqueous solution 7 D-(-)-a-amino--2~-:~5~ 3 a-(p~acetoxyphenylacetamido)desaceto~ycephalosporanic acid with an esterase selected from human serum, animal serum, citrus esterase, wheat bran, wheat germr and bacillus subtilis at a pH between about 5.0 and about 7.5 and at a concentration of about 5 to about 10 mg./ml. of esterase per total volume of the aqueous solution; isolating the product by methods known s , and, if desired, converting the product in the form of free acid or hydrate to the corresponding pharmaceutically ; acceptable salt thereof.
A commercially preferred embodimPnt of the present inven-tion is the preparation of 7~D-(-)a-amino-a-~p-hydroxyphenyl-acetamido)desacetoxycephalosporanic acid, hydrates or pharma-ceutically acceptable salts thereof, which process comprises:
treating in an aqueous solution 7 D-(-)-a-amino-a-(p-acetoxyphenylacetamido)cephalosporanic acid with an esterase selected rom citrus esterase, wheat bran, and wheat germ at a pH between about 5.0 and about 7.5 and at a concentration of about 5 to about 10 mg./ml. of esterase per total volume of the'aqueous solution; and isolating the product by methods known per se, and, if desired, converting the product in the form of the free acid or hydrate to the corresponding pharmaceutically acceptable salt ~hereof.
. Of special commercial interest is the process for prepar ing 7~ )a-amino-a-(p-hydroxyphenylacetamido)desacetoxy-cephalosporanic acid, hydrate or pharmaceutically acceptable salt the,reof comprising:
treating in an aqueous solution 7-D~( )-a-amino-a-(p~acetoxyphenylacetamido)desacetoxycephalosporanic acid with the commercially available esterase, coarse wheat bran, at a i6~3 pH between 5.5 and 6.0 or optionally in the presence of a buf-fer at a pH of 7.0 at a concentration of about 10 mg./ml. of ~sterase per total volume of solution; and isolating the product by methods known per se, and, if desired, converting the product in the form of a free acid or hydrate to the corresponding pharmaceutically acceptable salt thereofu The 7-D-(-)a-~mino-~-(p-hydroxyphenylacetamido~desacetoxy-cephalosporanic acid prepared by the instan~ inven~ion is known to be a potent antibacterial agent useful in the treat-ment of infectious diseases in poultry and animals, including man, caused by many Gram-positive and Gram-negative bacteriaO
~ he following example illustrates the preparation of p-acetoxycephalexin aceording to the invention.

EXAMPLE A
Solutions of 0.5 mg./ml. of 7-D-(-)a-amino-a-(p-acetoxy-phenylacetamido)desacetoxycephalosporanic acid (p-acetoxycepha-lexin) in normal saline and in human serum were prepared. Stan-dard solutions o~ 0.5 mg./ml. of 7-D~ amino-a-(p-hydroxy-phenylacetamido)desacetoxycephalosporanic acid (p-hydroxy- ;
cephalexin) were also prepared in both normal saline and human serum.
A11 the above solutions were incubated at 37 C. with shaking and sampled for chromatography a~ time intervals of 0, 2, 4, 8 and 24 hours. The solutions, approximately 5 micro-liters per strip, were spotted on Whatman No. 1 half-inch strips which were dried and developed in a solvent system containing 80 parts buty:Lacetate; 15 parts n-butanol; 40 parts acetic acid; and 24 parts water. The strips were then bio-*Trade Mark ~30-~5~ 3 autographed on plates seeded with bacillus subtilis at a pH
o~ 6Ø
The biochromatograms indicated that p-acetoxycephalexin is quickly hydrolyzed to thP p-hydroxy form in human serum but appears stable in normal saline.

Claims (15)

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 7-D-(-).alpha.-amino-.alpha.
(p-acyloxyphenylacetamido)-cephalosporanic acid of the formula I

wherein Y is hydrogen or S-Het, in which Het is a 5 membered heterocyclic ring containing 1 to 4 N atoms; and R is C1 - C10 alkyl or phenyl; when substantially free of the L-(+) isomer, which process comprises reacting a compound of the formula II

or a silyl ester or salt thereof, in which Y is as defined above or acetoxy, with A corresponding D-(-) acylating agent of an acid of the formula III

in which R is as defined above and B is an amino protecting group, and removing the amino-protecting group, and when Y is acetoxy, converting the thus-obtained compound to the corresponding final compound wherein Y is S-Het and, when the compound of formula II is in the form of the silyl ester or salt thereof, either before or after removal of B, converting the thus-obtained compound to the corresponding final product.

2. The process according to claim 1, wherein Y is hydrogen or S-Het, in which Het is 1,2,3-triazolyl; and R is C1 - C4 alkyl or phenyl.

3. The process according to claim 1 wherein Y is hydrogen or S-Het, in which Het is 1,2,3-triazol-5-yl; and R is methyl.

4. The process according to claim 1 wherein Y is hydrogen, and R is methyl.

5. The process according to claim 1 wherein Y is (1,2,3-triazol-5-yl)-thio, and R is methyl.

6. The process as claimed in claim 1, wherein the acylating agent is of the formula

7. The process according to claim 1 wherein the process further comprises converting the product to a pharmaceutically acceptable acid addition salt of the 7-D-(-)?-amino-?(p-acyloxy-phenylacetamido)-cephalosporanic acid of the formula (I).

8. The process as claimed in claim 2, 3 or 4 wherein the process further comprises converting the product to a pharmaceutically acceptable acid addition salt of the 7-D-(-)?-amino-?(p-acyloxyphenylacetamido)-cephalosporanic acid of the formula (I).

9. The process according to claim 5 or 6 wherein the process further comprises converting the product to a pharmaceutically acceptable acid addition salt of the 7-D-(-)?-amino-?(p-acyloxyphenylacetamido)-cephalosporanic acid of the formula (I).

10. A 7-D-(-)?-amino-?(p-acyloxyphenylacetamido)-cephalosporanic acid of the formula I

wherein Y is hydrogen or S-Het, in which Het is a 5 membered heterocyclic ring containing 1 to 4 N atoms; and R is C1 - C10 alkyl or phenyl; when substantially free of the L-(+) isomer, whenever prepared by the process of claim 1 or 6 or by an obvious chemical equivalent thereof.

11. A 7-D-(-)?-amino-?(p-acyloxyphenylacetamido)-cephalosporanic acid of the foxmula I

wherein Y is hydrogen or S-Het, in which Het is 1,2,3-triazolyl;
and R is C1 - C4 alkyl or phenyl; whenever prepared by the process of claim 2 or by an obvious chemical equivalent thereof.

12. A 7-D-(-)?-amino-?(p-acyloxyphenylacetamido)-cephalosporanic acid of the formula I

wherein Y is hydrogen or S-Het, in which Het is 1,2,3-triazol-5-yl; and R is methyl; whenever prepared by the process of claim 3 or by an obvious chemical equivalent thereof.

13. 7-D-(-)?-amino-?(p-acetoxyphenylacetamido)desacetoxy-cephalosporanic acid, whenever prepared by the process of claim 4 or by an obvious chemical equivalent thereof.

14. 7-D-(-)?-amino-?(p-acetoxyphenylacetamido)-3-[(1,2,3-triazol-5-yl)-thiomethyl]-3-cephem-4-carboxylic acid, whenever prepared by the process of claim 5 or by an obvious chemical equivalent thereof.

15. A pharmaceutically acceptable acid addition salt of a 7-D-(-)?-amino-?(p-acyloxyphenylacetamido)cephalosporanic acid of the formula I

wherein Y is hydrogen or S-Het, in which Het is a 5 membered heterocyclic ring containing 1 to 4 N atoms; and R is C1 - C10 alkyl or phenyl; when substantially free of the L-(+) isomer, whenever prepared by the process of claim 7 or by an obvious chemical equivalent thereof.