CA1056814A - Formyl-(p-acyloxyphenyl) acetamidocephalosporanic acid derivatives - Google Patents

Abstract

AsSTRACT 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 i5 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

~C~ ~Y ec~

This invention relates to certain novel a-amino- or a-formy~ (p-acyloxyphenyl)acetamidocephalosporanic acids which are useful as antibacterial agents.
The novel cephalosporin derivatives of this invention comprise the D-(-) compounds of the formula R-C-O~ 1' 5~

2 --N~ ~H 2Y

COOH
wherein Y is hydrogen or S-Het, in which ~et represents a 5 or 6 membered heterocyclic ring containing 1 to 4 atoms selected from M, O or S, said heterocyclic ring being optionally sub-stituted by Cl-C~ 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 C1-C4 alkyl, halogen, nitro, amino or trifluoromethyl: R' is hydrogen, hydroxy, Cl-C4 alkyl, Cl-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) l~ -CO-NH ~ ~ ~

COOH
I(a) wherein Y is S-Het, in which Het i5 1, 2,3-triazol-5-yl, l-N-methyl-tetrazol-5-yl or 2-methyl-l,3,4-thiadiazol-5-yl; R is hydrogen, Cl-Clo alkyl optionally substituted by carboxylic ~ s ~3~ 4 acid, or phenyl optionally substituted by Cl-C4 alkyl, halo-ge~, nitro, amino or trifluoromethyl; R' is hydrogen, hydroxy, Cl-C~ alkyl, Cl-C~ alkoxy or halogen, and pharmaceutically acceptable salts thereof, when substantially free of the L~ isomer.
In the above compounds, the substituent halogen is preferably selected from fluorine, chlorine or bromine.
The pharmaceutically acceptable salts referred to above include the nontoxic carboxylic acid salts, e.g., nontoxic metallic salts such as sodium, potassium, calcium and alumi nium, the ammonium salt and salts with nontoxic amines, e.g., trialkylamines, procaine~ dibenzylamine, N-benæyl-~-phene-thylamine, l-ephenamine, N,N'-dibenzylethyleneaiamine, N-alkylpiperidine and other amines which are used to form salts of penicillins and cephalosporins. Also încluded within the definition of pharmaceutically acceptable salts are the nontoxic acid addition salts (amine salts), e.g., salts with mineral acids such as hydrodhloric, hydrobromic, hydroiodic, phosphoric, sulfuric and salts with organic acids such as 20 maleic, acetic,citric, oxalic, succinic, benzoic, tartaric, ~-fumaric, mandelic, asoorbic and malic.
Examples of suit~ble heterocyclic groups included within the definition of "Het" in formula I are such heterocyclic -~
radicals as thienyl, pyrazolyl, imidazolyl, isoimidazolyl, 25~ triazolyl, tetrazolyl, thiazolyl, thiadiazolyl, thiatriazolyl, oxazolyl, oxadiazolyl, isothiazolyl, isoxa201yl, pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl and triazinyl. The hetero-cyclic ring may be unsubstituted or substituted with one or more of the substituents as mentioned above.
3Q Pxeferred D-(~) oompounds of formula I are tho~e wherein - , , ~

Y is hydrogen or S-Het, in which Het represents 1,2,3-triaæolyl, 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 optionally substituted by Cl-C4 alkyl, fluorine, chlorine, nitro, amino or txifluoromethyl; 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-thiadiazol-5-yl, or 1-N-methyl-tetrazol-5-yl; R is hydrogen or Cl-C4 alkyl; and R' is hydrogen.
More preferred D-~-) compounds 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 includes a process for the ~5 preparation of a compound of the formula R_~_o~
2 N ~ H2Y

COOH
wherein Y i hydrogen or S-Het, in which Het represents a 5 or 6 membered heterocyclic ring contai.ning 1 to 4 atoms selected from N, O or S, said heterocyclic ring being op-tionally substituted by Cl-C4 alkyl which may be further op-~5 tionally substituted by carboxylic acid, or alk.oxyalkyl of up to 4 carbon atoms; R is hydrogen Cl-Clo alkyl optionally substituted by carboxylic acid, or phenyl optionally substi-tuted by Cl-C4 alkyl, halogen, nitro, amino or trifluoromethyl;
R' is hydrogen, hydroxy, C1-C4 alkyl, Cl-C4 alkoxy or halosen, and pharmaceutically acceptable salts thereof when substantially ~s~

free of the L- (+) isomer, which process comprises reacting a compound of the formula S

H2N T--~ ~

~ N ~ CH2Y
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 o~ the formula R' NHB
III
in which R and R' are as defined above and B is an amino-pro-tecting group, and xemoving the amino-protecting group to pro-duce the compound of formula I or a pharmac~utically acceptable salt thereof, and converting by methods known per se the compound of formula I, wherein Y is acetoxy, to the corre~pond-in~ pxoduct wherein Y is S-Het and, if desired, either beforP
or after removal of B, converting by methods known 2er se the product in the form of the free acid or silyl ester or salt th~reof to the corresponding free acid or pharmaceutically acceptable salt thereof.
In the preparation of the novel cephalosporin compounds o~ the present invention, a corresponding 7-amino-cephalosporanic acid compound of formula II or salt thereof is acylated by kno~n methods with the appropr:iate D-(-) acylating agent of formula III.
In the case of 3 thiolated-7-aminocephalosporanic acid ~56~

intermediate of formula II, when Y is S-Het, said intermediate may be prepared by displacement of the 3~acetoxy group of 7-aminoc~phalosporanic acid or a salt thereof with the appropriate heterocyclic thiol or a salt thereof. The displacement of an ester group with a thiol group is a known reaction and is preferably accomplished in aqueous solution with heating.
The intermediate II may, if de~ired, be converted prior to the acylation reaction to a silyl ester or acid addition salt hereof. The silyl 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-lS blocklng group B respectively, which may be re~dily rPmovedat the conclusion of the reaction by methods known ~ se. -Examples of suitable amino-protecting or blocking groups include t~butoxycarbonyl, carbobenzyloxy, 2-hydroxy-1-naphthGarbonyl, trichloroethoxycaxbonyl, 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' R-C-O ~f ~ coc 1 NH2 . HCl 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 groups are considered within the scope of this invention.
In addi~ion to the abo~e, the pr~sent invention also in-cludes a proce~s 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, l~N-methyl-tetraæol-5-yl or 2-m~thyl-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, halogen, ~itro, amino or trifluoromethyl; R' is hydro-gen, hydroxy, Cl-C4 alkyl, Cl-C4 alkoxy or halogen, and pharma-ceutically acceptable salts thereof, when 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-(-) acylating agent of an acid of the formula R' R-~- ~ H-COOH
OCHO

III(a) .
20 in which R and R' are as defined immediately above, to produce the compound of formula I(a) or a pharmaceutically acceptable sal~ thereof, and, if desired, converting by methods known per se the product in the form of the fxee acid or silyl ester or salt thereof to the corresponding free acid or pharma-ceutically acc~ptable salt th~reof.

Ln one aspect of t~is i`n~ention there is- provided a process for the preparation o~ a 7-D-(-)d-formyloxy-~(p-acyloxy-phenylacétamido)cephalosporanic acid of the formula fH~CO-NN ~ ~ S ~
OCHO ~ CH2Y

~0()~
I(a) : . , ., ~ \

wherein Y is S-Het, in which Het is l-N-methyl-tetrazol-5-yl;
R is hydrogen or Cl C10 alkyl; when substantially free of the L (~) isomer. The process comprises reacting a ompound of the formula S .
~2N~ ~ :
~N~C~2Y
COOR

or a silyl ester or salt thereof~ in which Y is as deined above, with a corresponding D-(-~ acylating a~ent of an acid of the formula ~ ~ ~ ~ H-COOH ,~
OCHO
III5a) in which R is as defin~d ab3ve.- ~ha~l the compoun* ~f formula II is in the form of the silyl ester or salt thereof, ~0 the thus-obtained compound ~e converted to the corresponding ~:
final product.
Acylation of ~ 7-~ino grt~up of ~ c:ephalo~porin 1$ a : .
well-known reae~ion and any of the functional equivalents :
of formula III or III (a) commonly used as acylating agents for 25 primary amino group~ may be employed. Examples of suitable ~cylating d rivat~ves c~f the free acid include th~ c:orr~ponaing ~ .
' .

- 6(a) - :
~ ,.

~56~
acid anhydrides, mixed anhydrides, e.g. alko~yformic c~nhydrid~s, acid halides, acid azides, acti~e esters and active thioesters.
The free acid may be coupled with compound II after fi~st re-acting said free acid with N,N'-dimethylchloroforminium chloride or by the use of enzymes or of an N,N'-carbonyldiimidazole or an N,N'-carbonylditriazole or a carbodiimide reagent, e.g.
N,N-diisopropyl carbodiimide. N,NI-di Gy clohexylcarbodiimide or N-cyclohexylcarbodiimide or N-cyclohexyl-N'-(2-morphilino-ethyl) carbodiimide or of an alkylyl~mine reagent or o~ an isoxasolium salt reagent. ~nother 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 memkered ring containing at least t~o nitrogen atoms, i.e. imidazole, pyrazole, the triazoles, benziimidazole, benzotriazole c~nd their substituted deriva-tives. A reactive derivative of the phenylglycine acid of formula III is the N-oarboxy c~hydride ~Leuch's anhydride). In ~his structure the group which activate the carboxyl group al~o serves to protect the ~mino group. A
particularly preferred acylating agent is the acid Ghloride hydrochloride of the ~ormula : R~

R-C-O ~ CH - COCl ~5 N~2~HCl which also serves a dual function of carboxyl activation and amino protection. Mention was made above of the use of enzymes to couple the free acid with its blocked c~mino group with compound III. Included in the scope of such processes . . . ' ' ' , ' '' . .. ' ... ' . , " ~ .
,,, :, . . .
,, . . . .. . : .
,, ,:, , , ~ . :
' ' . '', ,': :. , , . : .

are the use of an ester, e.g. the methyl ester, of that free acid with en~ymes provided by various microorganisms, ~ e.g. those described ~y T. Takahashi et al., J. Amer. Chem. Soc., 94(11), 4035-4037 Clq72) and by T. Nara et al., J. Antibiotics (Japan), 24(5), 321-323 ~1971) and in West Germany 2,216,113.
The particular process conditions, e.g. temperature, solvent, reaction time, etc. selected for the coupling re-action are determined by the nature of the acylation method used and are known to ~hos~ skilled in the art. Generally it is useful to add an organic tertiary amine~ e.g. triethyl-amine, N,N-dime~hylaniline, ethylpiperidine~ 2,6~1utidine or quinoline, to serve as a proton acceptor or salt-forming agent.
The compounds of ~he presen~ invention may be isolated in any o the ways customaxily employed for the isolation of similar cephalosporins. Thus, the product ma~ be obtained as the neutral molecule and, in the case of compounds of formula I, this i5 probably more accurately represented as the zwitterion, or it may be isolated as a salt. Formation of the desired pharmaceutically accepta~le carboxylic acid or acid addit~ion salt is carxied out by known methods, e.g. xeaction of the acid wi~h an appropriate base o~ acid~
At the conclusion o the acylation reaction the product obtained may be converted (before or after removal of the amino-protecting group) by methods known per se to another desired product of formula I. For example, th~ product of formula I
or I(a) in the form of a silyl ester ox salt thereof may be converted to the free acid product or pharmaceutically acceptable salt thereof by removal of the silyl ester group, e.g. by hydrolysis.
The pharmaceutically active compounds of the present inven-~ion are potent antibacterial agents useful in the txeatment of .. . . .
.. .. . . . . ..

~s~

infectious diseases in poultry and animals, including man, caused by many Gram-positive and Gram-negative bacteria. The active compounds are also of value as nutrition~l supplements in animal feeds and as agents for ~he treabment of mastitis in cattle.
The preferred compounds have also been unexpectedly round to be e~ficiently absorbed upon oral administration.
The novel medicaments provided by the present invention ~ay ~e formulated as pharmaceutical compositions comprising, in addition to the ~ctive ingredientO a ph?rmaceutically ac~eptable carrier or diluent. The compounds may be administered ~o h orally and parenterally. The pharmaceutical preparations may be in solid form such as ~apsules, tablets or emulsions.
In the tr~atment of bacterial infections in.man, the compoun~s of this invention may ~e administered parenterally in an amount of ~rom about 5 to 200 mg./kg.~day in divided dosage, e.g~ 3 to ~
times a day. They ar~ administered in dosage units co~tainins eOg. 125, 2$0 or 500 mg. of active ingredient with suitable physiologically accepta~le carriers or excipients.
The following illustrates the preparation of starting 2~ materials used Ln the production of the novel compounds of the invention.

/ ,~' /

,:

~;tartin~ ~aterials Synthesis of po*assium_l,2,3-~riazole-~-thiolate O
Il N~
~bc-~a=c=s~ CH2N~ OH
`S
163~19 42.04 ~05.24 ~ SH ~ SK
10 N / ~ N
N N
H H
101.13 13g.23 .

The synthesis of the thiol was accomplished by a pro-cedure essentially identical to that described in the litera-ture [J. GoPrdler and G. Gnad, Chem. Ber. ~9, 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 2~ with vigorous stirri~g, 0.685 N ethereal diazomethane (453 ml., 310 mmoles~. When the addition was completed, the mix-t~re 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 - ~he pure material. A small second crop ~21,1 g~) was obtained .
by evaporation of the mother liquor in vacuo. The total : -yield w~s therefore 4Q%.

- 10 - ' ';' ' ,.' ' - . ' ' ', ; ,~'''' "', . ' :' ' ' ' . ' ; ' , ' ' .,~ , .

-1,2,3-Triazole-5-thiol A solution of the above benzamido compound (8.2 ~., 40 mmoles~ in 2N sodium hydroxide (80 ml.~ 160 mmoles) w~s heated under reflux temperature in a nitrogen atmosphere for 24 hours.
The solution ~as cooled to 0 in ice, ~nd concentrated hydro-chloric acid (26 ml. ) was added, while a continuous stream of nitrogen was passed through the solution. The benæoic 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 fil-trate was --immediately extracted with ethyl acetate, the extract was washed with saturated salt solution, dried over magnesium sulfate and then evaporated 'in vacuo. The viscous oil which remained was immediately evaporatively distilled in vacuo (70-75/0.001 mm. ?
to give an oil (2.84 g., 70~ which solidified (m.p. 52-59;
Goerdler reported mOp. 60) spontaneously. -' Potassi'um 1','2~3-Triaz'o'l'e`-`5`-'th'io'la-te 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 thendiluted with anhydrous ether until crystallization of the salt was completed. The solid was collected by ~iltration, 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 ~' via 5-amino-1,2,3-thiadiazole [G. Goerdler and G. Gnad, Chem.
Ber. ~9, 1618 (1966)~.

~5~

É~
NEICO,0 L ~ ~H2 ~

S-S
OH J~ ..
El~ ~ N 8 7 %
~N ~ 93% M~ S ~NH N

5-Amino-1, 2, 3-thiadiazole can be prepared by an alternative route~ . .
not involvin~ diazomethane [D. L. Pain and R. Slack, J. Chem.
Soc. 5166 (1965)].

O . :

~\N--CEIzCE10 + ElzNNE~CO2Et ~ ' O '. '. ~' ' '. ' '.
2C3 Q !!~ ... ~ .
~,~N-CH2CEI--N--NHC02Et .....
8 93% ~;
O

r\ N

o ~ S ~ '' '' "' ' ' 8g% " ;"~
N~ . .

3 0 7 7 %

Synthesis of 7-Amino-3-(lr2!3-triazol-5-ylthiomethyl)-3-cephem 4 r carboxylic_acid 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 for 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-txiazole 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 10.5, was adju~ted to pH 8.5 with 42% phosphoric acid. 7-Aminocephalosporanic acld (21.8 g., 0.08 mole) was added and the mixture heated at 50 for 4 hours.
The clear solution was cooled in an ice bath and adjusted to pH 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.) was brought into solution with 600 ml. of methanol and 40 ml. of conc. HCl. After carbon treatment the solution was diluted with 1.5 1. of ice water and extracted once with ethyl acetate. The aqueous 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 ~iltration, washed with water and methanol and dried in vacuo over phosphorus pentoxide; 11~4 g. The IR and NMR spectra were ~ully consistent for the de~ired product.

~5~

Anal. Calcd. for CloHllN53S2 C, 38.42; H~ ; ~
Found: C, 38.27, 38.25; H, 3.76, 3.40; N, 21.02, 21.00; H2O, 1.70.

Purification of 7-Amino-3-( ~ hiomethyl)-3-_ _ .
cephem-4-carbox~lic acid (II~
Crude 7-amino~3-(1,2,3-triazol-5-ylthiomethyl)-3-cephem 4-carboxylic acid (16.1 g.) containing approximately 20 mole % of 7-aminocephalosporanic acid as an impurity, was 1~ brought into solution with 600 ml. of methanol and 40 ml.
of conc. HCl~ After carbon treatment, the solution was diluted with 1.5 1. of ice water and extracted once with ethyl acetate. m e aqueous phase was concentrated at reduced pressure to ramove methanol. The cold aqueous concentrate was then adjusted slowly to pH 4.0 with 20% sodium hydroxide causing the product to crystallize. The product was collected by filtrati~n, washed with water ancl methanol and dried in vacuo over phosphorus pentoxide; 11.4 g. The NMR spectrum indicated that this product contained about 7 mole % of 7-aminocephalosporanic acid as an impurity.
The 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 C10~15N503S2 C, 38-42; H~
Found: C, 39.06, 38.53; H, 3.56, 3.Sl; N, 22.05, 21,60; H2O, 1.78, ~L~5~

7-Amino-3-tl,2 ~ t ~ omethyl)-3-cephem-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 phospha~e 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% H3PO~. The resulting precipitate was filtered off, washed with cold water (50 ml.) and air dried. 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.
Anal. Calcd. for CloHllN503S2: C, 38~39; H, Found: C, 38.36; ~, 3.78.

acid (II) Two hundxed seventy-two g~ (1.0 mole) of 7-amino-cephalosporanic-acid was su~pended 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 (Note: The sodium bicarbonate was added in portions). Then 143 g. (1.0 mole) of 5 mercapto-1~ 1,2,3-triazole potassium salt was added and the mixture stirred at 55 C. + 1~ C. under a nitrogen atmosphere for 4 hou~s. After 1 hr. the pH was readjusted to 6.4 ~y addition of a small amoun~ of 40% H3PO4. At the end *

of the 4 hr. heating period, 50 g. of "Darco KB" decolorizing *~rade Mark room tem~era~ure the yield ~as lQ~ g.; dec. pt. 230C.
The IR and NMR were consistent for the desired structure.

~ Pre~aration o~ D (~-2-amino-2-t4-acetoxyphenyl)acetic acid Method A ~in-acetic acid as solvent~
203.5 g (1 Mole) of D(-)p-hydroxyphenylglycine chloride, 800 ml of acetic acid and 314 g( 4 Moles~ of acetyl chloride are stirred 48 hours at room temperature. The solid is collected, washed three times with acetone ~3 x 250 ml) and twice with ethanol (2 x 250 ml) and dried at 40. Yield 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 4.5 with 20% 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 glycine3.
~D (1~ HCl N/10) ~ -104.5 Method B cin methylene chloride~

4.07 g ~0.02 Mole~ of D(-)p-hydroxyphenylglycine hydrochloride, 30 ml of methylene chloride and 6.28 g t p.08 Mole~ of acetyl ;~
chloride -are stirred 48 hours at room temperature. The solid is collected, washed ~wice with acetone and twice with ethanol. ;~
Yield 4.17 g (84.5%3. 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 ml of tri*luoroace~ic acid at room temperature. After dissolution, 1.57 g( 0.02 Mole3 of acetyl chloride is added. After a slightly exothermic reaction, a ,, : . .,. . , , ~ ., , , .
,,:, , ~igii8~

charcoal was added and, after stirred for 15 min. at 55 C., the slurry was filtered hot through a diatomaceous ear~h ["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 ~Cl. 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. ~f methanol and air dried.
The crude product was suspended in 3000 ml. of 50%
methanol-water and 300 gO (1.5 mole) of ~-toluenesulfonic acid was added. The mixture was stirred for 15 min. and then 50 g. of "Darco XB" decolorizing charcoal was added. A~ter stirring for 15 min. at 22 C., the slurry was filtered through a 'ICelite" pad and the pad washed with 2 x 100 ml.
of 50% methanol;water. The pH of the combined filtrates was adjusted to 4.0 by addition of approximately 210 ml. of tri-ethylamine. After cooling at O 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 a~d air dried.
This material was suspended in 2000 ml. of water and 84 g. (1 mole) of sodium bicarbonate 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 tered through a "Celite" pad. The product was washed with 2 x 100 ml. of water and the pH of the combined filtrates was adjusted to 3O5 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 by filtration was washed with 2 x 200 ml. of cold water and 2 x 1000 ml. of acetone.
After drying over P2Os in a vacuum desiccator for 14 hr. at *Trade Mark solid appears. The suspension is stirred 1 lf2 hours at roo~
temperature and t~e trifluoroacetic acid is removed in ~ vacuum. The remaining solid is collec~ed, washed with methylene chloride and with ethanol. The DC-]2-amino-2-(4-acetoxyphenyl~acetic acid is identical to that prepared by method A or B.
Yield~ g(75%) Preparation of D (-)2-amino-2-~4-pivalyloxyphenyl2acetic ~. .
acid hydro~hloride 1.67 g (0.01 Mole) of D(-~p-hydroxyphenylglycine is added - to 10 ml of txiEluoroacetic 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 from isopropanol.

Anal. Cl = 11.8% (calculated 12.3%) W ~max. 205 nm and 220 nmO

Preparation of D ~-),2~amino-2~(4-benzoyloxyphenyl)acetic_ acid hyd_och oride This compound is prepared according to the same procedure as used for the pivalyloxy derivative. Yield: 2.7 g (87%).

25~ An analytical sample is recrystallized from ethanol.

Anal. cl. = 11.3% (calculated 11.5%) W ~max. 205 nm and 234 nm ~: :

, hydrochloride 83~6 g. (0.40 mole) of D(-)2-amino-2-(4-acetoxyphenyl)-acetic acid and 1.25 1. of anhydrous me~hylene chloride are cooled to -5 C. with stirring. Then 152 g. of phosphorous pentachloride are slowly added followed by 4 ml. of dimethyl formamide. The mixture is stirred 4 hours at 0 C. The solid is collected, wa~hed with anhydrous methylene chloride and vacuum dried at room temperature. Yield: 61 g. (57.5~).
Anal. Total chlorine = 27.2% (Theory 26.9%) Preparation of D-t-)2-formyloxy-2-~4-formyloxyphenyl)acetic a _ A solution of 3.6 g. (0.02 mol~) of D-5-)-2-hydroxy-2-(4-hydroxyphenyl)acetic acid in 50 ml. of 97% aqueous formic acidwas 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 sulfate, fil-~ tered and evaporated to aford the desired product.

Preparation of D~(-)2-formvloxy-2-(4~acetoxY~henvl)acetic acid The D-~-)2-formyloxy-2-(4-formyloxyphenyl)acetic acid obtained above is dissolved in 10 ml. of acetyl chloride and the xesulting mixture wa6 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 ac~ording to NMR.

The follo~ing examplec are given in illustration o~,but not in limitation of, the present in~ention. All temper-atures are in degrees Centigrage. 7-Aminocephalosporanic acid is a~breviated as 7-ACA and 7-aminodesacetoxycephalOspOraniC
acid as 7-~D Q.

Example 1 7~ 2-amino-2-~4-acetoxyphenylacetamudo)desaceto~y-cephalospor nic acid - (acetoxy cephalexine) - RN 1394 15.27 g C P .0~14 Mole) of 7 ADCA are stirred in 500 ml of anhydrous methylene chloride; 120 ml o~ methylene chloride are distilled 10 off and 11.8 ml of hexamethyldisilazane are added. The mixture is stirred and re~luxed 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 ~.5 ml o~ dimethylaniline and 7 ml of a solution of dimethylaniline hydrochloride in methylene chloride (30%). Then 20 g (0.0756 Mole) of D~-~2-amino-2~4 -acetoxyphenyl)acetyl chloriae hydrochlorid~
are added in small portions (~1 1/2 hours) a~ O~C.
The mixture is ~tirred 30 min. at +lO~C and 4 hours at ~20OC
and allowed to stand overniyht at ~5CO Then 5 ml of methanol followed by 240 ml of water are added. ~he pH is adjusted at 2.S with triethylamine and the mixture is filtered , through a celite pad; then the pH is checked and the aqueous phase is separated, washed twice (2 x 15n m~) with methylene chloride and treated with charcoal.
The solution is adjusted to pH 4.5 and vacuum concentrated to ~:
a volume of ~ lS0 ml. The suspension is allowed to stand ' ov~r~ight a ~5C: arld the solid c911ected and washed wit~
water and acetone, and dried at 40Co ~ield: 15,1 g (~ so^æ of 75-80-~,' pure material3 (~D (1% H;20) = ~107

5 14 g of this crude material is suspended în 30 ml o wa'~:er (p~ ~ 3,2) hydrochloric acid (36~o) is added to pH
1, 3 and the resulting solutio;~ is charcoal treated and filt~red through a celite pad and 2djusted to pH 4-4, 5 over stirring. ~fter 2 hours at 0- 1 5C the RN 139~ i~
10 collected, ~shed wit~ water and acete~ne and dried at ~.'0C.
Yield: 7 g, ~ D t~ R20) 3 t 133.
~e infrared and nuclear magnetic resonance s~ectra are consistent wi~h the desired product.
siological Data - Table I sh~ws comparative MIC data for sL-s 578-4 (~-hydroxy analogue of cephalexin) and (~-acetoxycephalexin (RN 1394). Minimal inhibitory concentrations were determined by the 2-fold broth dilution method utilizing equimolar con-centrations of each compound.

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Example 2 7-[DC-)2-Amino-2~4-acetoxyphenylacetamido]-3-l(1,2,3-triazol-5~ thiomethyl]-3-cephem-4-carbo~y~ic acid: RN 139Ç
62.6 g (0.2 Mole~ of 7-amino-3-l(1,2,3-triazol-5-yl)thiomethyl]--3-cephem-4-carboxylic acid (7-TACA~, 1.5 1 of methylene chloride and 60~2 g (0O374 mole) o hexamethyl disilazane are stirred and refluxed 20 hours with a slight pressure of nitrogen ~after about 2 h~urs all 7-TACA is run in solution).
The s~lution is cooled to 0C and 3Q . 4 ml of dimethylaniline followed by 20.4 ml of a solution of dimethylaniline hydrochloride in methylene chloride (30%3 and 1. 35 g of .
imidazole are addedO
Then 60.5 g ~0.22~ mole) of 2-amino-2-~4-acetoxyphenyl)acetyl chloride hydrochloride are added in small portions (~ 1 1~2 hours) at 0C. The mixture is then stirred 3 hours at 20C
and let stand overnight at ~5C. 25 ml of methanol ~ollowed by 75Q ml of water are th~n added. The pH is adjusted to 2.3 - 2.5 with triethylamine and the l~xture is filtered through a celite pad. The aqueous phase is separated, washed twice with methylene ~hloride and charcoal treated.
The solution is adjusted to pH 4.3 and stirred 2 hours at ~5C. The solid is colleoted, washed twice with water and -~
dried at 40C. Yield: 53 g (about 5Q~)O
This crude material is purified twice as follows: ~ ~
The solid is treated with eight volumes of 0.5N hydrochloric ~ ~-acid and the suspen ion is decolorized with charcoal. An :~ :
equal volume of methanol is added to the solution and the pH
adjusted to 2 - 2.1; after 15 minut~s a small amount of suspended solid is collected and discarded. The ~iltrate is adjusted to pH 4. The precipitated solid is collected, washed with . .

: . :
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.

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MeOH/H2O (50/50) and pure methanol.
Yield: 25 g. (after two purifications)n - IR consistent with assigned structure - Moisture (KF) : 5.1 - Chemical assay - Iodometric assay 885 mcg/mg - Potentiometry Amine assay: 97%

Bioloyical Data Table II shows comparative MIC data for the above prepared 7-lD( )2-amlno 2-(4-acetoxyphenylacetamido)]-3-[1,2,3-triazole-5-yl)-thiomethyl]-3-cephem-4-carboxylic acid, RN-1396, and its p hydroxy analogue, BL-S640. Minimal inhibitory concentrations were determined by the 2-fold broth dilutio~ method utilizing equimolar concentrations of each compound.

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Example 3 7-D-(-)[2-(4-aceto~y~ n~ 2-for~yl$ey3g~t~a~ thyl-, 2 ~ 3 ! 4-tetrazol-5-yl~thiom th~1 3-ce~hem-4-carboxylic acid To a solution of 4.8 g. (0.02 mole) of crude D-(-)2-form~loxy-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 dropwise to a solu-tion of 6.3 g. (0.02 mole) of 7 amino-3-(1-methyl-1,2,3,4-tetrazol-5-yl)thiomethyl-3-cephem-4-carboxylic acid, 5.6 g.
of sodium bicarbo~ate, 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 was 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 sulfateO
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 ha~e 85-90% acetyl and 50-60% formyl.

7-D-(-)[2-(4-form~loxyphenyl)-2-form~l~ cetamido]-3~ methyl 1,2,3,4-ketrazol-5~yl)thiomethyl-3-cephem-4-carboxylic acid The same procedure as in Example 3 was followed to react 2.5 g. (0.01 mole) of D-~-)2-formyloxy-2-(4-formylox~phenyl)-acetic acid with 3.28 g. (0.01 mole) of 7-amino-3~ methyl-1,2,3,4-tetrazol-5yl)thiomethyl-3-cephem-4-carboxylic acid to -26~-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-pr~duct.
Microanalysis ~f the desired product gave:
Calc'd. for C20~1gN6O8S2 ~ The~y % Found .
C 44.86 46.47 H 3.38 4.14 N 15~70 .13.50 K.F. (H2O) 1.78 2~ -.

- :

In addition to the above, the compounds of the instant invention are also valuable as intermediates for the prepara-tion of other pharmaceutically active compounds. For example, the instant a-formyloxy or a-amino-a-(p-acyloxyphenyl)ac tamido cephalosporanic acids may be converted to the corresponding 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 circum~tances.
We have f~und 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-~-(p-hydroxyphenylacetamido~desacetoxy-cephalosporanic acid.
Accordingly t the present invention also provides for a novel process for preparing 7-D-~-)a-amino-a-(p-h~dro~y-phenylacetamido)desacetoxycephalosporanic acid, hydrate or a pharmaceutically acceptable salt thereof, which process comprises treating in an aqueous solution 7-D-(~ amino-a-(p-acetoxy-phenylacetamido)desacetoxycephalosporanic acid with an esterase at a pH between about 5.0 and about 7.5; isolating the product by methods known ~ se, and, if desired, converting by methods known ~ se the product in the form of the free acid or hydrate to the corresponding pharmaceutically acceptable salt thereof.
A preferred embodiment is the preparation of 7-D~ a-amino-a-(p-hydroxyphenylacetamido)desacetoxycephalosporanic acid, hydxate or a pharmaceutically acceptable salt which pxocess comprises treating in aqueous solution 7-D (-)-a-amino-~5~
~-(p-acetoxyphenylacetamido)desacetoxycephalosporanic acid with an esterase selected from human serum, animal serum, citrus esterase, wheat bran, wheat germ, and bacillus subtilis at a pH between about 5.0 and about 7.5 and at a concentration of 5 about 5 to about 10 mg./ml. of esterase per total volume of the aqueous solution; isolating the product by methods known se, and/ if desired, converting the product in the form of free acid or hydrate to the corresponding pharmaceutically . acc~ptable salt thereof.
A commercially preferred embodiment of the present inven-tion is the preparation of 7~D-(-)a-amino-~-(p-hydr~xyphenyl-acetamido)desacetoxycephalosporanic acid, hydrates or pharma-ceutically acceptable salts thereof, which process comprises:
treating in an aqueous sollltion 7 D-(-)-a-amino ~-(p-acetoxyphenylacetami~o)c phalosporanic acid with an esterase selected from citrus esterase, wheat bran, and wheat gexm at a p~ between about 5.0 and about 7.5 and at a concentration of abou~ 5 to about 10 mg./mlO of esterase per total volume of the aqueous solution; and isolating the product by methods known per se, a~d, if desir~d, converting the product in the form of the free acid or hydrate to the corresponding pharmaceutically acceptable salt thexeof.
. Of special commercial intexest is the process for prepar-25 ing 7 D- (-)a-amino-a ~p-hydroxyphenylacetamido)desacetoxy-cephalosporanic acid, hydrat~ or pharmaceutically acceptable salt thereof comprising:
treating in an agueous solution 7-D~ amino~
(p-acetoxyphenylacetamido)desacetoxycephalosporanic acid with the commercially available esterase, coarse wheat bran, at a --2g-8~

pH be~ween 5.5 and 6.0 or optionally in the presence of ~ buf-fer at a pH of 7.0 at a concentration of about 10 mg./ml. of esterase 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 thereof.
The 7-D-(-)a-amino-a-(p-hydroxyphenylacetamido)desacetoxy-cephalosporanic acid prepared by the instant invention is known to be a potent antibacterial agent useful in the treat-ment of infectious diseases in poultry and animals, including man, caused by ~any Gram-positive and Gram--negative bacteria.
The following example illustrates the preparation of p-acetoxycephalexin according to the invention.

Ex~Mæ~E A
Solutions o~ 0.5 mg./ml. of 7-D-(-) a-amino-a- ~p-acetoxy-phsnylacetamido)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- ~-) a-amino-a-(p-hydroxy-phenylacetamido)desacetoxycephalosporanic acid (p-hydroxy-cephalexin) were also prepared in both normal saline and human serum.
Rll the above solutions were incubated at 37~ CO with shaking and sampled for chxomatography at 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 dxied and developed in a solvent system containing 30 parts butylacetate; 15 parts n-butanol; 40 parks acetic acid; and 24 parts water. The strips were then bio-*~rade Mark l~S6~4 autographed on plates seeded with bacillus subtilis at a pH
of 6Ø
The biochromatograms indicated that p-acetoxycephalexin i5 quickly hydrolyzed to the p-hydroxy form in human serum but appears stable in normal saline.

.

Claims (6)

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-(-)?-formyloxy-?(p-acyloxyphenylacetamido)cephalosporanic acid of the formula I(a) wherein Y is S-Het, in which Het is 1-N-methyl-tetrazol-5-yl;
R is hydrogen or C1 - C10 alkyl; 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, with a corresponding D-(-) acylating agent of an acid of the formula III(a) in which R is as defined above and, when the compound of formula II is in the form of the silyl ester or salt thereof, converting the thus-obtained compound to the corresponding final product.

2. The process according to claim 1 wherein R is hydrogen or methyl.

3. The process according to claim 1 wherein said process further comprises converting the product to a pharmaceutically acceptable salt of the 7-D-(-)?-formyloxy-?
(p-acyloxyphenylacetamido)cephalosporanic acid.

4. A 7-D-(-)?-formyloxy-?(p-acyloxyphenylaecetamido)-cephalosporanic acid of the formula I(a) wherein Y is S-Het, in which Het is 1-N-methyl-tetrazol-5-yl;
R is hydrogen or C1 - C10 alkyl; when substantially free of the L-(+) isomer, whenever prepared by the process of claim 1 or by an obvious chemical equivalent thereof.

5. A 7-D-(-)?-formyloxy-?(p-acyloxyphenylacetamido)-cephalosporanic acid as defined in claim 4, of the formula I(a) wherein R is hydrogen or methyl, whenever prepared by the process of claim 2 or by an obvious chemical equivalent thereof.

6. A pharmaceutically acceptable salt of a 7-D-(-)?-formyloxy-?(p-acyloxyphenylacetamido)cephalosporanic acid as defined in claim 4, of the formula I(a) wherein Y is S-Het, in which Het is 1-N-methyl-tetrazol-5-yl;
R is hydrogen or C1 - C10 alkyl; when substantially free of the L-(+) isomer, whenever prepared by the process of claim 3 or by an obvious chemical equivalent thereof.