IE39046B1 - Alpha-aminocyl-3-halo cephalosporins - Google Patents

Abstract

1461323 Silylated x-aminoacyl-3-halo-cephalosporins ELI LILLY & CO 22 Feb 1974 [23 Feb 1973] 8097/74 Heading C3P [Also in Division C2] Novel compounds of the Formula I wherein R is phenyl,mono- or dihydroxy phenyl, mono or dihalophenyl, mono or dimethylphenyl, mono- or dimethoxyphenyl, 2- or 3-thienyl or 2- furyl; R 1 is hydrogen, benzyl, p-methoxybenzyl, p-nitrobenzyl, diphenylmethyl, 2,2,2- trichloroethyl, trimethylsilyl or t-butyl; R 2 is hydrogen or an amino-protecting group; X is fluoro, chloro or bromo and pharmaceutically acceptable salts thereof when R 1 or R 2 is hydrogen, may be prepared by (i) halogenating and acylating in either order a compound of the Formula II wherein R 3 is a carboxylic acid protecting ester forming group optionally followed by removal of the carboxylic acid protecting ester forming group and or the amino-protecting group or (ii) fluorinating a 3-alkyl or aryl sulphonate of a compound II (R 1 #H) having a protected amino group, followed by acylation. In the above process (i) when halogenation is effected as the first step a protecting acyl group may be introduced on the 7-amino group prior to halogenation and thereafter removed. [GB1461323A]

Description

300 46 The present invention rotates to 7-a-aminoacyl-1-halo cephalosporins and the non-toxic pharmaceutically acceptable salts thereof, valuable, orally effective antibiotics.

Several 7-a-aialnoacyl cephalosporin antibiotics with varyinn suhstituents in the 3-position of the molecule are known. For example, the well-known antibiotic cephalexin 7-(D-a-phnnylglycylamino)-3-methyl-J-cephe«»-4-carboxylic acid adesacetoxycephalosporanie acid which has a methyl qroup in the 3-position; the antibiotic cephaloqlycine, 7-(n-a-phenyl-qlycyla«ino)-3-aeetoxymethyl-3-eephem-4-carhoxylic acid, a cephalosporanic acid havinq an acetoxymethyl qroup in the 3-position; and the o-amlnoacyl cephalosporins wherein the 3-position is substituted by a thiadiasoljlthioscthyl or tetnzo- ylthiomethyl moiety are all valuable therapeutic aqents.

Also a number of cephalosporin compounds have been described wherein the molecule is substituted in the 1-oosition by a 3-halomethyl qroup, for example, 3-hromomethyl-3-cephe»n-4-carboxylie acid esters. The 1-halomethyl cephalosporin esters which have been described are disclosed as useful intermediates for the preparation of cephalosporin antibiotics.

The halo cephalosporin compounds provided by this invention are structurally unique compounds n* the cephalosporin class in that the haloqon atom is attached directly to th<» carbon in the 1-p«sttion of the dihydrotM.^ttn^ rlnn. In contrast, previous halo derivatives of cephalosporins, such as those described above, bear a halogen a to™ on a methylene Troup attached to the carbon aton in the 3-posltion of the dihydrothiazine rinq. The novel compounds thu3 provided havn a combination of features which comprise a 7-a-aminoacyl 3 0 0 1 (. moiety attacluMl to a cephalosporin nucI«us which is sul>-stitated in the 3-position with a fluoro, chloro, or br The 7-° -aminoacyl-3-halo cephalosporin compounds of the invention are represented by the following formula I H 0 H wherein R is phenyl, monohydroxypheny I, dihydroxyphenyI , mortohalopheny J., d.ihalophcnyl , monomethy I plieoy I , dini-i ln I -phenyl, monome.thoxyphtnyl, di.acthoxyplic.ny1, 2-thienv 1 , ,!-1" thienyl or 2-furyl; Rj is hydrogen, benzyl, p-aethoxybenzyl, p-njt.ro-benzyl, diphenylmethyl, 2,2,2-trichloroethyI, trimethylsilyl or t-butyl; R^ i hydrogen or an amino protecting group; 15 X is fluoro, chloro, or bfomo; and when R^ or Rj is hydrogen the pharmaceutica 1 i v .icr.'ptabl, non-toxic sails thereof.

Preferably, R., is a hydrogen atom.

Mono and dihydroxyphenyl groups arc, for example, 20 4-hydroxyphenyl, 3-hydroxypheny1, 2-hydroxyphenyl, ',4- dihydroxyphenyJ , and 2,4-dihydroxypheny I. Mono and -25 phenyl, 3-bromopheny 1, or 4-hromopheny I. Mono methy 1 pheny I groups are, for example, the isomeric 2,3, and 4-metIt-/'.phenyl groups and the dimethylphenyl groups are, for example, -,4-di methylpheny 1., and 2 , 4-dimethy lphenyl . Mono and di methoxyphenyl groups arc, for example, 4-oethoxy- 30046 phenyl, 3-anthoxyphenyl, 2-metboxyphenyl, 3,4-dimethoxyphenyI, and 2,6-dimethoxyphenyl.

Preferably, R is phenyl. Further embodiments ot' the invention comprise compounds of formula 1 wherein (a) R is mono or dihydroxyphenjrl; (b) R is mono or 4ih*lopbanyl; or (c) R is 2-thienyl.

The ester groups represented by R^ in the above formula are all known ester moieties which are commonly employed in the cephalosporin art to protect the C. carboxyl 4 Kt-oup of the cephalosporin molecule while reactions involving other reactive sites in the molecule are carried out.

The term, "pharmaceutically acceptable non-toxic salts", refers to both the salts of the C. carboxylic acid 4 function and the acid addition salts of the a -amino group of tlx- 7-glycylamino side chain. Pharmaceutically acceptable salts involving the carboxylic acid function include the salts formed with inorganic bases such as the sodium, potassium and calcium salts which can be prepared with sodium bicarbonate, potassium carbonate, calcium hydroxide, or sodium hydroxide. Pharmaceutically acceptable amine salts can also be prepared for example with the organic amines such as dicyclohexylamine, benzylamine, 2-aminoethanol, diethanolaaine, or diisopropyiamine. Acid addition salts < It will be readily recognised that when and R2 are hydrogen the zwitterionic form of the compounds can exist which form arises via intramolecular salt formation. 39 0 1 G Owing to the preaoncu of the aaymmetric carbon atom in the a-aainoacyl group the 3-halo cephalosporins of the above formula include the D-, L- and DL- forn*-The D- for* is the preferred isomeric form of the present invention.

Exaaples of the o-aainoacyl-3-halo-c«phaloaporins represented by the above forauila arc the following: 7-(D-°-phenyl{lycyluu no)-3-fluoro-3-cephem-4-carbo*y I i • ac i d ?-(D- ii-ph«nyl glycy 1 ami no )-3-chloro-J-c< phero-4-carhmy lie acid 7-(D-cr-pheny 1 glycy 1 ami no)-3-brnao-3-cephem-4-carboxy 1 ic acid, 7- [l>- ot-( * -hydroxyphenyl )-(jlycyl amino ]] -3-chloro-3-cephein-4-carboxylic acid, 7— (J- rr-( 3-hydroxyphenyl )— glycylami r.ijj - 3-ch I i>o>- 3~c«-phem-4 -cartaoiyl i«: acid, 7- [D- 2-( 2-thi eny I)glycy I ami no} - 3-chloro - 7-c«;phem-4-carl>'.) - 'i-chlm-o-J-c ephi m- 4-carboxylic acid, 7-(D-»-(2, fr-d i metnuxypheny I )- gl ycy I am i. un) - l-«:hloro-3- ceptirm-4-carboxylic acid. 7- [D—2-( 3-thienyl )glycylaminoJ - 3-f luoro-3-cephera— 4-carhoxy-lic acid, 7-[D-2-(3-thienyl)-glycylaminoj -3-chloro-3-cephea-4-carboxy-lic acid, as the zwitterionic forms and the pharmaceutically acccptablc-salts thereof.

The term "a-" in connection with the rompounds listed above, and other phenyl derivatives of the invention, indicates the portion of attachment of the phenyl group, arid docs not refer to stereochemical features oi I.lie molecule. 30046 The compounds of tho formula T nhcwln Rj 1m hydrogen or fche pharmaceutically acceptable non-toxic salts thereof arc valuable antibiotics useful for combating infections in warm blooded mammals caused by graa-5 positive and gram-negative microorganisms. They are effective when administered parenterally, for example, subcutaneously or intramuscularly, as well as when administered orally.

Accordingly, in one aspect of the invention >0 there is provided a pharmaceutical formulation containing a cephalosporin of formula I where Rj is hydrogen, or a pharmaceutically-accoptable salt thereof, associated with a pharmaccutically-acceptable carrier therefur.

The 7-(r~*minoacyl 3-halo-cephalofipor ins possess a IS broad spectrum of anti-bacterial activity as illustrated I>y tho in vitro spectrun for 7-(D-a~pbenylglycylamino)-.3-chli>f> ■?-cephem-4-carboxyl ic ar.Lu presented in the following tabl<-s - 6 - 39 0 4 G Table I lists the minimum inhibitory concentrations (MIC) in micrograms per milliliters(wg./ml.) obtained with this compound in the standard agar dilation test.

Table I 7_(D_a_phenylqlycyla*ino)-3-chloro-3-caphem-4-carboxylic acid in vitro spectrum Organism* M*c — Staphylococcus aureus 3055 1 Staphylococcus aureus 3074 1 streptococcos faecalIs X66 16 Proteus morgan11 PR15 >128 Salmonella typhosa SA12 < .5 Klebsiella pneumoniae KL14 . 2 Rnterobacter aerogenes EB17 8 Serratla marcescens SH3 >128 Escherichia coli ECU 2 Cltrobacter freundil CF17 >128 Pseudoaonas aeruginosa X239 >128 Bordetella bronchiseptlea 16 64 Salmonella typhimurlura l Pseudomones solanacearum X1B5 >128 Erwinia amylovora l • Numbers and/or letters follovinq the organism name refer to strains. -7- 39046 Table IX below lists the dimeter in aillineters of tt:3 sone of inhibition of growth of the Indicated aicro-organisms in the standard disc-plate assay obtained with 7-(D-,rphcnyiglycylamino)-3-chloro-3-cephea-4-carboxylie acirf. 5 Table IX Sone of inhibition* Microorqanism (Ma-dianeter) Concentration (ag/al) 1.0 0.1 0.01 Staphylococcus aureus 35 28 20 Bacillus subtilis 44 32 21 Sarcina lutea 48 27 27 Mycobacterium avium 24 Tr — Proteus vulgaris 23 Tr -- Salmonella gallinarua 35 25 13 Escherichia coli 30 20 11 Klebsiella pneumoniae 28 20 12 Pseudomonas solanacearcum 32 23 #Tc indicates a trace zone. 20 A dash (—) indicates no observed *one. -8- 3 8 0 4 6 In Table III which follow* the NIC values obtained with 7-(D-a-phonylglycyl*miQo)-3-chloro-3-ccphem-4-carboxylic acid against a spectra* of gram-positive and gram-negative microorganisms in the agar dilution test is shown.

Table III In vitro Antibiotic Spectrum of 7-(D-a~phenylgly cylami&o)-3-chloro-3-cephem-4-carboxylic acid Test Organism* Hinimum Inhibitory Concentration MIC ug./ml.

Staphylococcus aureus 3055 1 .0 do 3123 1 .0 do 3074 2 .0 Streptococcus (Group D) 9901 64 Enterobacter cloacae BB9 >128 Enterobacter aeroqene S EB17 64 Escherichia coli BC14 1, .0 do EC35 2 .0 do EC 38 1, .0 Klebsiella sp.

XL 3 >0. . 5 do KL14 2, . 0 do KL25 2. . 0 Proteus mirnbilis PR6 1 . .0 Proteus morganii PR1 128 Proteus rettgeri PR9 >128 do PR2 >128 Salmonella SA12 < 0. 5 Shigella sp.

SH3 2. 0 •Humbers and/or letters following the organism name refers to strains.

In the standard agar dilution test, 7- (D- -^-phenyl— qlycylamino)-3-chloro-3-cephem-4-carboxylic acid detaonstra-ted activity against Haemophilus influenzae having MIC values of from 1 to 4 mg/ml against a number of strains. 39046 In T«iMo tv which follow* the oral absorption of 7_(D-T-phenylglycytami no )-3-chloro-3-cephem-4-cat-boxy lie acid is demonstrated by the blood and urine levels obtained in •■•ice. Tn carryina oat the test HcAllister Swiss mice weiqhi-q 5 from 11 to 13 qrams were fasted overnight and were then orally administered 20 mg./ka. of 7- (D- a-phonylglycylfuaino )-3-chloro-3-cephem-4-carboxylie acid. Blood and urine samples were taken at the indicated intervals and the concentration of the antibiotic in each sample was determined by microbioloqical 10 assay usinq Sarcina lutea in pH 6.0 aqar in the disc-plate assay.

Table IV 7-(D-a-Phenylqlycylanino) -3-chloro-3-cephem-4-carboxylic acid , Blood and Urine ravels House No. Concentration uq./ml. at time intervals (win.) 5 15 30 60 90 120 240 1 29. 1 16.2 11.6 3.5 1.2 0.5 0.2 2 23. 7 19.2 11.0 5.2 3.? 2.0 0.6 3 17. 6 10.1 9.1 5.7 2.1 1.3 0.7 4 23. 3 13.6 10.1 4.0 1.6 1.6 1.0 Average Flood 23. 4 14. A 10.5 4.6 2.0 1.4 0.6 Average in "ooled urine — 1474 1764 843 305 308 226 The effective dose (F.D,.^) for 7-(D-a-phenylglycy lamin o) -3-chloro-3-cephe«-4-earboxylic acid which is illustrative of the effective dose for the compounds of the invention is 0.74 30 mq./kn. x 2 oral and 0.48 mo./kq. x 2 s.c. vs. Streptococcus pyogenes, -10- 3 9 0 1 t» and 5.5 mg./kq. x 2 orally vs. Escherichia col 1 anil 17.6 toq/kq x 2 orally vs. Oiplococcua pneumoniae as determined in mice.

In Table V, below, are presented the mininum Inhibitory concentrations (MIC) against representative ciram-neqative bacteria Cor illustrative 3-halo-1-ccphe«* compounds of the invention. The inhibitory concentration!* were determined by the Gradient-Plate method carried out essentially as described by Brijson and Szybalski, Science 116. 45 (1952). In the Table V the A terns have reference to the stroctural formula.

Table V Antibiotic Activity of Substituted 7-(^-Phenylqlycylamino)-1-chloro-3-cephem-4-carboxylic acids vs. f>ram-Neqative Bacteria • Test Minimum Inhibitory Concentration Organism (nq/ml) A 1 3-OH 4-OH 4-CI a Shigella sp. _ 1.0 2.0 13. 3 6.7 Escherichia coli 1.0 2.0 17.2 S.7 Klebsiella pneumoniae 0.9 1. 5 R . 0 0.9 Herobacter aeroqenes 0.8 i.O ^ 5 . 0 0.8 Salmonella heidelbero 0.5 0.8 10.7 0.2 Pseudonon.is aeruginosa >200 >200 >200 >200 In the followino Table VT, the activity of r<»pre- 3 9 0 4 6 sencative 3-halo-3-cephem compounds against several clinical isolates of penicillin-resistant staphylococci is presented. The activity is presented in minimun inhibitory concentrations of tha test compound. The minimum concentrations were dctor-5 mined by the Cradient-Plate method.

Table VI Antibiotic Activity of Substituted 7-( o-phenylglycylamino-3-chloro-3-cephera-4-carboxylio acids vs. 10 Penicillin Resistant Staphylococci9 0 M ii i CH-C-N-vr_rX"Sj NH. 0 C00M Staphylococcus Minimum Inhibitory Concentration Strain (vg/ml) A 3-OH 4-OH 4-CI H V41 3.0/ 5.0/ 10.5/>20 11.5/>20 V32 3.5/ 7.0/ 18/>20 17/>20 X400 >20/ >20/ >20/>20 >20/>20 V84 0.5/ 0.6/ 3.0/>20 2.7/15 XI.1 0.4/ 0.6/ 0.4/0.7 0.4/1.0 20 1/ The letter-numeral designations refer to strains of clinical isolates of penicillin-resistant staphylococcus. 2/ In the columns, the value above the slash is the MIC "in the absence of human serum. The value 25 below the slash, when present, is the MtC in the presence of human serum. -12- 390 46 The compounds of the invention can be prepared b> N-acylation of a 7-amino-3-halo-3-cephen-4-carboxyllc acid or an ester thereof, for example the benxyl, g_-rw»thoxy benzyl, g-nitrobensyl, diphenylmethyl, 2,2,2-trichloroethyl, trlmethyl-5 silyl or £-butyl ester, with an active derivative of a phenyl, thlenyl or furyl substituted glycine of the formula R-CH-COOH NHj wherein R has the same meanings as defined in Formula X. in carrying out the acylation the amino group of the glycine is 10 desirably protected a^ for example , a salt such as the hydrochloride or with one of the commonly used amino-protecting groups, for example,the t-butyloxycarbonyl, benxyloxycarbonyl, £-nltrobensyloxycarbonyl, trichloroethoxycarbonyl, or trityl group, or enamines formed with methyl acetoacotate and acetyl -15 acetone and like groups. Activated derivatives of the carhoxyl group of the substituted glycine can be the acid halides such as the acid chlorides, the activated eaters such as those forneo with pentachlorophenol, the aside, or the mixed anhydride formed with the glycine and methyl chlorc-2o formate and lsobutyl chloroformate. Also, the amino-pro-tected glycine can be used directly in the acylaticn of the desired 3-halo nucleus ester by employing a condensinq aaent such as N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline (SKDQ). Por example, g^nitrobentyl 7-amino-3-chloro-3-cephen-4-car-25 boxylate is reacted with m-(t-butyloxycarbonyl) D-phenyl- glycine in a dry inert solvent, for example tetrahydrofuran, to torn p^-nitrobenzyl 7- (D-a-phenylglycylM>ino)-3-chloro-3-cephem-4-carboxylate. -13 3 0 0 4 6 In general, any of the known amid* coupling method* can be used in th* aeylatlon of the 7-amino-3-halo-3-caphem-4-carboxylie aoid* or esters. When th* activated derivative is an acid halid* the aeylatlon 1* carried out in the preaence of a hydrogen halide acceptor such a* sodium bicarbonate, pyridine, sodium bisulfite, or any alkylene oxide «uch as propylene oxide. When a mixed anhydride is used in the aeylatlon the mixed anhydride may be formed with the us* of H-ethoxycarbonyl-2-ethoxyl,2-dihydroqulnoline (KEnQ). Mien an N-protected phenyl, thienyl or furylglyelne is used, the aeylatlon can be carried oat in the presence of a condensing agent such as )V,N'-dieyelohexylcarbodilnide.

Illustrative of the substituted glycines which can be used In the preparation of the compounds of the invention are D-phenylglycyl chloride hydrochloride, D-4-hydroxyphenyl-glycyl chloride hydrochloride, pentachlorophenyl D-phenyl-N-(t-hutyloxycarbonyl)glycinate, pentaehlorophenyl D-2-thlenyi-N-(2,2,2-trichloroethoxycarbonyl)-glycinate, N-(t-butyloxycarbonyl) -O-phenylglycine, N-(l-carbomethoxy-2-propenyl)-D-phenylglyclne, 3-thienylglycyl chloride hydrochloride, N-(t-butyloxycarbonyl)-2-thienylglyclne or W- (t-butyloxycarbonyl )-2-furylglycine.

The aeylatlon is carried out in an inert solvent for example in acetone, acetonitrile, dimethylformamide or methylene chloride and preferably at a temperature between about -20 and 20*C. Tor example, 7- Illustrative of the 7-amino-3-halo-3-cephem-4-carboxylic acids and eaters which are starting materials in the preparation of compounds of formula X are 7-amino-3-chloro-3-cephe»-4-carboxylic acid, 7-amino-3-fluoro-3-cephera-4-carboxylic acid, p-nitrobensyl 7-arnino-3-chloro-3-eeph«w-4-carboxylate, dlphenylniethyl 7-aaino-3-bro»o-3-cephem-4-carboxy late, diphenylmethyl 7-a»ino-3-chloro-3-eephem-4-carboxylate and p-methoxybenzyl 7-amino-3-flaoro-3-cephem-4-carboxylate.

The 7-amino-3-halo-3-cephem-4-carboxylie acids and esters can be prepared in tho following luimecA 7-acylamiQ) cephalosporanic acid or an ester thereof is converted to a 7-acylamino-3-exomethylenecepham-4-carboxylic acid or eater. An ester of the 3-exomethylenecepham compound is then oxidized with ozone to form an intermediate oxonide involving the 3-exoaethylene grouping, which ozonide on decomposition provides the 7-acylami 3-hydroxy-3-cephe™-4-car boxy 1 ic acid ester. The 3-:.t/4*oxy-3-cephem ester is then fluorinated, chlorinated, or brominated to provide the corresponding 3-halo-i ccphem e«ter. The 7-Acyl group of the 3-halo cephen is then removed by the well-known side chain cleavage reaction by employing phosphorus pentachloride in pyridine to obtain the imino chloride derivative of the side chain, the imino chloride is then reacted with an alcohol such as methanol to provide the imino ether, which can then be readily hydrolyzed to provide the 7-amlno-3-halo-3-cephem-4-carboxylic acid ester.

For example, a 7-acylaminocephalosporanic acid such as 7-phenoxyacetamidocephalosporanic acid 13 reacted with a sulfur-containing nucleophile according to known procedures -15- 39046 to effect a nucleophilic displacement of the aeetoxy group of the cephalosporanic acid and provide a 7-acylaml"o-3-thio-substituted-methyl-3-c*phain-4-carboxylic acid. The 3-thio-sufcstituted-eephem acid is then reduced with zinc/formic acid in the presence of dimethyl forinamide (DNF) or with Raney nickel in the presence of hydrogen to yield a 7-acylassio.o-3-exomethylcnecephaffl-4-carboxyllc acid. For example, 7-phenoxyacetajnido-3-aeetoxymethyl-3-cephen-4-carboxylie acid is reacted with potassium ethylxanthate to provide 7-phenoxy-ace tamido- 3-ethoxyth lonocar bony 1 th io«e thy 1-3 -cephem- 4 -carboxylic acid. Reduction of the latter compound with sine and formic acid in the presence of Off provides 7-phenoxy-aee tan ido-3-exome thy lenecepham- 4 -ca r boxy lie acid. Likewise, any of a wide variety of ?-aeylamlnocephalosporanic acids can bm reacted with a variety of sulfur-containing nueleophlles to provide the 3-thio-*ubatituted-T8«thyl-3-cephew compounds. For example the 7-acylamiao group can be a heterocyclic containing chain for example 2-thlenylacetamido, or 2-furyl-acetaaldo, or it can be an *lkanoyl side chain as exemplified by aeataraido or a wide variety of other side chains.

Also, the sulfur-containing nucleophile can be any of a wide variety of compounds such as thiourea and substituted thioureas which react with the cephalosporanic acids to provide isothlouronium salts, the thlobenzoates, mercaptopyridlne N-oxide, l-methyl-tetrazol-5-thiol, 5-methyl-l,3,4-thiadla-zol-2-thiol, and other sulfur-containing nueleophlles. Following the reductive displacement reaction described above the 3-exomethylenecepham-4-carboxylic acid thus prepared is then esterified for example with p-nitrobensyl bromide, p-methoxybenzyl bromide, diphenyldiasomethane, or 2,2,2-tri- -16- 3 9 0 l«. chloroethyl chloroformate or other ester forminq compounds, and the 3-exomothylenecepham ester is reacted with ozone to provide the 3-hydroxy-3-cephetn eater. The ozonolysis reaction of a 3-exomethylenecepham ester is carried out in an inert 5 solvent at a temperature between about -10 and 0*C. and preferably between -80 and -50*C. to form an Intermediate ozonide. The ozonide is then decomposed. In situ, in the cold to provide the corresponding 3-hydroxy-3-cophem ester as illustrated in the followinq reaction scheme. 0 0a 10 [ozonI do] 0 H In the above formula R is H or the residue of the 4 group as described above for example R can be benzyl, phenoxy- 4 methyl, methyl, 2-thienylmethyl or 2-furylmethyl, *nd is as defined in the formula I. 15 The ozonolysis of a 3-exom#»thyleneccpha!n ester i<; carried out by bubblinq ozone into a solution of th« astor in an inert solvent until ozonide formation is complete. -17- 39046 Inert solvents which can be used in the ozonolysis method are those solvents in which tha 3-exomethylenecephan esters are at least partially soluble and which are unreactive with ozone under the described conditions. Comoonly used solvents such as methanol, ethanol, ethyl acetate, methyl acetate, iso-amyl acetate, and methylene chloride are satisfactory solvents.

Ozone gas is prepared by means of an ozone generator of the type commonly used in synthetic and analytical chemical work. Such generators produce ox one by the action of an electric discharge on oxygen. One such ozone generator is that manufactured by the wellsback Corporation. The osone is generated in a stream of oxygen which is then passed directly into the reaction vessel. The percentage of osone contained in the oxygen stream can be varied as desired, for example, by varyinq the rate of flow of oxygen through the ozonizer as well as by varying the intensity of the electric discharge.

The concentration of the 3-exomethylenecapham ester starting material in the inert solvent is not critical and it is preferred to use a solvent volume sufficient to form a complete solution.

When ozonide formation is complete, any excess ozone present in the reaction mixture is purged from the mixture by bubbling nitrogen, oxygen or an inert gas such as argon through the mixture. Pollowing the removal of any excess ozone, the ozonide is decomposed by adding to the reaction mixture a reducinq agent selected from the qroup consisting of sodium bisulfite, sulfur dioxide, and trimethylphosphite to provide the 3-hydroxy-3-cephejn-4-carboxylic acid ester. -18- 3 9 0 1 C The decomposition is carried out by addinn an exces* of thr« decomposition reaqont and then stirrinq the reaction mixture until the reaction mixture is negative to the potassium iodide starch test. A preferred reaqent for decomposinq the intermediate ozonide is qaseous sulfur dioxide.

The 3-hydroxy-3-cephem esters are recovered from the reaction mixture by evaporating off the volatile solvents from the mixture to obtain the reaction product mixture as a residue and thereafter recrystallising the reaction product from the residue.

The 7-acylamino-3-hydroxy-3-cephem-4-carboxylic a«-id estern thus prepared are then haloqenated to provide the 7-acylami»o-3-halo-3-cephem-4-carboxylic acid ester.

The compounds represented by the formula I wherei-. X is chloro or bromo can be prepared by reacting a ?-acylAmi.>o 3-hydroxy-3-cephem ester or «i 3-hydroxy-3-cophert nucleus ester in dimethylformamide (n.HF) with a react!' e chloro o" bromo compound whic^ forms with DMF the chloro or bromo dimethyl-iminium chloride or bromide as represented by the formula H CM* - I X- ^=CN CH3 nX wherein X and X represent chloro or bromo and chloride or bromide respectively. The reactive halo iminium halide of the above formula is formed in situ and is a hiqhly reactive chlorinating or brominatinq intermediate. Chloro and bro">o compounds which form the above iminium halide include the commonly used chlorinatinq agents such as phosqene (carbonyl chloride), oxalyl chloride, thionyl chloride, and the phosphorus chlorides, for example, phosphorus trichloride and -19- 390 4 6 phosphorus oxychloride (phosphoryl chloride). Brorain*ting reagents which can be employed in the present invention include carbonyl dibroraide, oxalyl bromide, thionyl bromide (sulfurous oxybromide), and tha phosphorus bromides, pho»-5 phorus oxybromide, and phosphorus tribromide. Phosphorus pentachloride can be employed in the preparation of the 3-chloro-3-cephem compounds of tha invention, however this reagent concurrently reacts with the 7-acylamino side chain of the starting material to form the imino chloride, the 10 reactive intermediate in the well known ceohalosporln side chain cleavage reaction. Accordingly, it is preferable to us« one of the other named chlorinatinq agents.

The chlorination and bromination can be carried out by adding the halogenatinq reagent to a solution of the 3-hydroxy-15 3-cephem ester in dry DHP at a temperature of about 5* to 15*C. and allowing the reaction mixture to stand at roon temperature for between 4 and B hours or longer. The reaction is initially exothermic and accordingly the reaction vessel is initially cooled in an ice-water bath and then maintained about 25*C. 20 during the remainder of the reaction. The DMT is preferably dried prior to use over a molecular sieve. Although the reaction can be carried out in DMP as the solvent, a cosolvent may also be employed along with the DMP. For example, a cosolvent such as tetrahydrofuran, dioxan, methylene chloride, 25 dimethylacetamide or dimethylsulfoxide can be used along with OMP. Other solvents capable of forming the Iminium halide can also be used in conjunction with or instead of DMF.

The 3-chloro or 3-bromo-3-cephem eaters are recovered from the reaction product mixture by pouring the mixture into 30 a water-ethyl acetate mixture and separating the organic phase containing the product. The organic phase is washed, dried - 20 - 390 I (i . iIM I in t I it *1 ( *•* ('(I I n p i t » v i t It - I-I it • ! I i.i I « i i-l .'lllli'lli * * >. I • • i * . i -* .in .iin> ■ ■ plioti i*f-,itliif. rin* |ntitliiil i-. . ■). i .i i 11. 11 Thu compounds reprcsentud t»y the formula 1 wlirit-in X is fluoro and i» oilier than hydrouoti imh be prepared from th«- alky I sul t'onatc or a ry Isu 1 ft mate cst.i'r.t of t lie liytl»-o*y- j-ccph(;ni i:i»tiM"rt t>y tin- following Sonera I i /et! i--.o I ium stlicoe : II to I It ■ I t • i II >1 i |ml .iS.siui, -.1 »d i um III' s i I VI • I , K . I •> it . • >. ;. > • itill / i C j ^ .i i k \ I ' i c .in n pt 11 tn.i I I y 1 i 1 ii I • - d pi ii ft; I ; i • •(!( i .

Crown 1't.hiTS are ile.si rilifd in To' ralii'drnn l.'-t I ■ i , I - , 1 7*>3- Tlie reaction ran he- carried out. in .hi inert Mil v- m , spci: L f ica lly a nitrile or a ni troa Ikane, pn-i erab I y at. tool' -i-i I *"» propionitrile, nLtromuthano, or n i t rot^t hano , under substantial ly anhydrous conditions at a tenpfratun- f ft em about -2l>°C. to about, room temperature and preferab Ly .it ihoot. room temperature. The j-^ul t'onatr estfr « eph.i 1 ospo r t n pjf.^.u! ill the so]ectod solvent is mixed with a maximum «• I" one equiv alont of the fluoride source ba>ed on tin -.u I i oiiati- e:,i.i unl at least an equivalent amount of < rown ether ha.-iod _ . 39046 upon the fluoride which la present. Tha reaction la completed generally within a period of from 30 minutes to 3 hours, with time generally being dependent upon the temp-erature of reaction. Typical crown ethers include 18-crown-5 6-ether as well as corresponding substituted 18-crown-6-ether, such as dicyclohexyl-18-crown-6-ether. Preferably, the fluoride source is an Inorganic fluoride salt, H*T~ hereinabove being, for example, sodion fluoride, or silver fluoride. 10 The product which is obtained from the reaction will correspond structurally in every respect to the sulfonate ester starting material with the exception that the group -o-SOj-* will have been displaced by a fluorine atom.

Xt has been discovered that the initial 3-sulfonate 15 ester cephalosporin reactant can be either a ^-cephalosporin, a cephalosporin, or a mixture of the two. The active reactant is the A^-cephalosporin. However, it has been discovered that, under the conditions of reaction, any ^-cephalosporin which may be present is isoaetized to the A*-cephalo»porin, 20 and thus the active reactant is formed in situ if not present initially in the reaction mixture.

Isolation of the resulting product can be accomplished employing generally recognized techniques. Preferably, the product is isolated by means of preparative thin 25 layer chromatography employing customary procedures.

The preparation of the alkylsulfonate and aryl-sulfonate esters of the 3-hydroxy-3-cephem esters is illustrated in the following generalised reaction scheme. -22- 3 9 0 11; H I Z-S0»-CI H I .5 'H-Y ^ S J M ^'0-W 0 s COORi In the above formula, the terns R^and R^ have the same i-eaninqs as previously defined and 7. is alkyl, such as methyl, ethyl, propyl, n-butyl or n-hoxyi, <»r /. is a phenyl group or a substituted ph.-nyi group such as methylphenyl, nitrophenyl, or halophenyl. Representatives <»t" the sulfonyl halides which can he employed in the preparation of these sulfonate enters are methanesulforyi chloride, toluenesulfonyl chlorid"., p-fluorobenzene 3ulfonyl chloride, ethanesulfonyl chlori.ia, or butanesulfonyl chloride.

The reaction can bo carried out in an inert solvent at a temperature between about -5 and 35*C., and preferably between about 15 and 25*C. Inert solvents which can be employed in the sulfonation reaction are generally aprotic solvents, and those solvents which are unreactive with the sulfonyl halide. Solvents which can be employed include the ether solvents such as tetrahydrofuran and dioxan. A preferred solvent for the sulfonation reaction i3 di-ethylace-tamide. The reaction is carried out in the presence of a 39046 hydrogen halid* acceptor, and the conmonly employed tertiary amines such as pyridine and triethylamine can be used, however, the preferred hydrogen halide acceptor in this process is propylene oxide. Other alkylene oxidea can be employed, however* propylene oxide ia much preferred. The tertiary amine hydrogen halide acceptors are leas desirable than the alkylene oxides in that the doable bond in the delta 3 position of the cephem ring is susceptible to iso-merixation to the delta 2 position in tha presence of these aaines. However, should isoraerixation to the delta 2 isomer occur daring sulfonate ester formation, the double bond can be isomerixed back to the delta 3 position in the following manner. The delta 2 isomer of the sulfonate ester is first oxidixed with a peracid, such as ra-chloroperbenxoic acid or peracetic acid to the sulfoxide. During sulfoxide formation the double bond shifts from the delta 2 to the delta 3 position and thereafter the sulfoxide is reduced, for example, with phosohorus trichloride, according to the well known method to provide the 3-cephem «"tfonate ester.

A preferred sulfonate ester for the conversion of the 3-hydroxy-3-cephc*-4-carboxylic acid esters to the corresponding 3-fluoro-3-cephem compounds is the methylsulfonate ester (mesylate). Another preferred ester is that formed with p-toluenesulfonyl chloride (tosylate ester). A preferred carboxylic acid protecting ester group in the above described process is the g-nitrobenxyl ester.

Alternatively, the fluorination of a 3-hydroxy-3-cephem ester is carried out by reacting a 7-acylamino-3-hydroxy-3-cephen ester in an inert solvent with the fluori-nated teritary amine, N-(2-chloro-l,l,2-trifluoroethyl)di- -24- 39 0 I G ethylamine. The f luorir.ated tertiary amine can hi- pr••■•par«*«l i>> reactinq diethylamide with chlorotrifluoroethylene as described in J. Org. Chew. 29, 2187 (19M); Tetrahedron T.ett. 22, 1065 (1962) and Tetrahedron Lett. 26, 124") (1962). An eguivalent amount of the fluorinated tertiary amine is employed in an inert solvent, for example methylene chloride, chloroform, tetrahydrofuran, or any unreactive solvent in which the starting material and fluorinating reaoent are substantially soluble. The reaction mixture containing the 3-hydroxy ester and the fluorinatinq reactent is heated in the inert solvent at a temperature between about 30 and ahout ftn*i for about 1 hour. The.fluoro cephem ester is recovered by following recovery procedures analogous to those described for the recovery o? the 3-chloro and 3-bromo compounds.

Following the preparation of the 7-acyl3mino-3-vi1i!o 3-cophem-4-carboxylie acid estirs as described above thn respective 7-*mino-3-halo-3-cephem-4-carboxylic acid esters are obtained by c.- -ryino out the well-known N-'leacy Lation r>c the 7-acylami--w. side chain. For example the 7-acyJ.amino-3-halo cephalosporin eater is reacted with phosphorus pentachlT ride in methyler chloride in the presence of pyridine to far' the intermediate inino chloride. The imino chloride is th*n reacted with an alcoholic solvent such as methanol or iso-butanol to provide the correspondina imino ether. The inino ether is then hydrolyzed to provide the 7-amino-3-halo-3-cephem-4-carboxylic acid ester as a hydrochloride salt.

As previously mentioned the 7-amino-3-halo starting material can be aeylated oither as the free acids or as an ester thereof. The acylation of the 7-amino-'J-halo nuih-nn is carried out as previously described. -25- 390 46 In a specific embodiment of the preparation of starting materials and the compounds of the invention, 7-phenoxyacetamidocephalosporanic acid is reacted with thiourea to provide the isothiouronium salt by the displacement of the acetoxy group in the 3-position of the cephalosporanic acid dihydrothiazine ring. The isothiouronium salt is then reacted with zinc and excess 90« formic acid in the presence of dimethylformamide at a temperature of about 25* to provide the 7-phenoxyacetamido-3-exomethylenecepham-»4-carboxylic acid. The 3-exomethylenecephan carboxylic acid is esterifled with £-nitrobenzy 1 bromide in tho presence of a hydrogan halide acceptor to provide the 3-exontethylenecepham-4-carboxylie acid £-nitrobenzyl ester. The 3-exomethylenecephan ester is then ozonized in methylene chloride at a temperature of about -70* and the ozonolysis mixture is treated with sulfur dioxide to decompose the intermediate ozonide to yield j^-nitrobenzyl 7-phenoxyacetaraido-3-hydroxy-3-CGphem-4-carboxylate. The 3-hydroxy ester is then reacted with phosphorus trichloride in dry OMF to provide £-nitr ^"nzyl 7-phenoxyacetamido-3-chloro-3-cephem-4-carboxylate. The 3-chloro ester is then reacted in methylene chloride with phosphorus pentachloride in the presence of pyridine to provide, in situ, the imino chloride intermediate, which is *-h»n reacted with methanol to provide the correspondinq imino ether intermediate. Upon addition of water to the reaction mixture the imino ether decomposes to provide the £-nitrobenzyl 7-amino-3-chloro-3-cephem-4-carboxylate. The 7-amino-3-chloro-3-cephem-4-carboxylic acid ester can then be acylated, as previously described herein, with D-phenylglycyl chloride hydrochloride or with an araino-protected D-phenylqlycine derivative to pro- -26- 3 9 0 10 wide the 7-(D-phenylqlycylamino)-3-chloro-3-cephoi«'-4-cj»rboxy 1 ic acid j>-nitroben»yl ester or an N-protectcd derivative thereof. After removal of the a-araino protectIna group and the carboxyl ester qroup, 7-(D-phenylqlycylamino)-l-chloro-3-cephem-4-carboxylic acid is obtained. It will bo recognized from the foreaoina description of the preparation of the startinq materials that a wide variety of known 7-aeylamino-cephalosporanic acids can be employed in the preparation of the 7-*mino-3-halo-3-cephem-4-carboxylic acids described herein.

A preferred qroup of compounds of this invention are those represented by the formula I wherein X represents fluoro or chloro, R^ beinq hydroqen, and 9 beinq phenyl and the pharmaceutically acceptable, non-toxic salts thereof. For example 7-(D-a-phenylqlycylamino )-3-chloro-3-cephem-4-carboxylic acid and 7-(n-a-phenylqlycylamino)-3-fluoro-3-cephem-4-carboxylic acid are preferred coeinounds.

A further preferred qroup of compounds of the present invention are ..hose represented by the formula I wherein R is hydroxyphenyl, P.^ is hydroqen and X is fluoro or chloro and the pharmace-.i ioilly acceptable, non-toxic s*lts thereof. Illustrative of the this preferred aroup of comoounds are 7-(n-a-4-hydroxyphenylqlycylamino)-3-chloro-3-ce?hem-4-carboxylic acid, 7-(D-a-4-hydroxyphenylaylcylami no)-3- f luoro-l-cephem^-carboxylic acid, and 7- (D-a-3-hydroxyphenylqlycyl.imi uo) -3-chloro- 3-cephem-4 -carboxylic acid.

An especially preferred antibiotic of this invention is 7-(D—' -phi-ny 1 ;il y< y I amiu )-.l-i-li I f> i! i-~■ phcm-.!-<•„ i - -27- 3 9 0 4 0 boxylie acid.

The following examples are provided to further Illustrate the present Invention and are not Intended to be limiting thereof. 5 Example 1 p-Nitrobemyl 7-amlno-3-f*ethylenecephaxi-4-oarboxylate hydrochloride To a solution of 965 mg. (2 otnole) of £-nitrobensyl 7-phenoxyacetamido-3-«ethylenecepham-4-carboxylate in 10 ml. 10 of methylene chloride were added 175 tag of dry pyridine and 450 mg. of phosphorus pentachloride and the mixture was stirred at room temperature for 6 hours. One ml. of iso-butanol was added to the mixture which was then stored at 0*C. overnight. The reaction product, £-nltroben*yl 7-amino-j5 3-wethylenecepham-4-carboxylate hydrochloride, which formed as a crystalline precipitate was filtered to yield 430 mg. (58% yield).

Elemental Analysis for C^jH^gNjOjSCl Theory: C, 46.69? ». 4.18; N, 10.09 20 Pound: C, 46.40; H, 4.20} N, 10.62 I.R. (Nujol Mull) Carbonyl absorption at 5.65 (B-lactam) and 5.75 (ester) microns.

H.M.R. (OMSO dg) signals at 6.34 (2d, 2H, Cj-Hj), 4.98 25 Cd, 1H, Cg-H) j 4.7-4.4 (ra, 6h, Cj-H, ester CHj, C4-CH2 and C?-H)> and 2.4-1.6 (m, 4H, aromatic H) tau. -28- 3 9 0 4 G Example 2 p-Nitrcbenzyl 7-amino-2-hydroxy-3-oepheri-4-carboxylate hydrochloride A solution of 4 g. of p-nltrob«nzyl 7-amino-3-methylenecepham-4-carboxylate hydrochloride in 620 ml. of methanol was cooled in a dry ico-acetone bath and ozone was bubbled through the cold solution for about 20 minutes. The reaction mixture was purged of the remaining ozone by pas3inq nitrogen through the solution and 10 g. of sodium bisulfite were added.

The reaction mixture was stirred for one hour at ice-bath temperature at which time the mixture gave a negative potassium iodide-starch test.

The mixture was evaporated in vacuo to yield tw<* reaction product as an amorphous yellow residue. The residue was crystallized in acetone to yield 3.4 g. of o-nitrobensyl 7-araino-3-hydroxy-3-cephem-4-carboxylate hydrochloride as « crystalline acetone solvate.

I.R. (N'Jlol Mull): Carbonyl absorption bands *t 5.60 (S-lactam) and 6.04 (ester carbonyl hydroqen bonded to 3 hydroxy) microns.

N.M.R. (HMSO dg): signals at 7.92 (s, 3H, 1/2 mole acetone), 6.22 (2d, 211, C2-H2) , 5.07 (d, 1H, CfiH) , 4.8-4.5 (m, 3H, ester CH2 and C7«), 2.4-1.6 (m, 4H, aromatic H) tau. 39046 example 3 _p-Methoxyben zyl 7-phenoxyace tamido-3-hyd roxy-3-cephem-4-carboxylate. h solution of 2.5 g. of p-methoxytoensyl 7-phenoxy-5 acetamido-3-methylenecephai»-4-earboxylate In 350 ml. of ethyl aeetate was cooled *.n an acetone-dry ice bath, ozone was babbled through the cold eolation for 0 minates and then oxygen was passed through the osonlxed reaction mixture to expel excess osone. The intermediate osonide was decomposed 10 by adding to the reaction mixture 25 g. of sodium bisulfite with stirring at a temperature of about 0*C. The reaction, solution was decanted and was washed successively with water, 5% hydrochloric acid and a saturated solution of sodium chloride. The washed mixture was dried and evaporated to 15 yield the reaction product, g-methoxybenxyl 7-phenoxyacetamido-3-hydroxy-3-cephem-4-carboxylate as an amorphous solid. n.m.r. (CDClj): signals at 6.73 (s, 2H, C2H2), €.23 (s, 3H, £-metv-oxy), 20 5.53 (s, 2H, side-chain CH2), 5.03 (d, 1H, C6H>, 4.87 (s, 2H, ester CH-j), 4.47 (q, 1H. C?H), 3.40-2.50 (m, 9H, aromatic R), 25 2.33 (d, 1H, amide NH), and 1.53 (broad s, 1H, 3 OH) tau. 3 9 0 1 G Example 4 £-Mitrobenxyl 7-{2- (2-thionyl)acetamido) -1-hydroxy-1-cephem-4-c»rh©xylat® To a solution of 1.55 q. of £-nitroben*yl 7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride in 30 ml. of acetone containing 364 mq. (0.5 ml., 3.6 mmole) of triethyl-amine was added 962 mq. of urea, with stirrinq at room temperature, a solution of 730 rnq. (4.4 nnole) of 2-thiophene acetyl chloride in 20 ml. of acetone was added dropwise to the mixture. After 2.5 hours the reaction mixture was filtered and evaporated. The residue was dissolved in ethyl acetate and the solution was washed successively with water, a 5% solution of sodium bicarhonate, 5* hydrochloric aci-1. and a saturated solution of sodium chloride. The washed solution was dried and then was concentrated hy evaporation in vacuo to yield 1.2 a. of the reaction product as a crystalline residue. The product was recrystallized fror* or'wl acetate to yield p''re p-nitrobenzyl 7-f2-(2-thienyi} sceta-ii^ol 3-hydroxy-3-c. :-^em-4-carboxylate havinq the following snectral properties.

T.R. (Mujol Mull): absorption peaks at 3.0 (amfrlo •'!!), 5.68 (3-lactam carbonyl), and 6.1 (amid*. *n<1 ■"iter hydrogen bonded to 3 Off) microns.

►S.M.R. (PDCl-j/DMSO dg): signals at 6.54 (2d, 2H, CjPj), 6.16 (s, 2H, 3ide-chain C'?2) , 4.90 (d. Iff, C^H) 4.60 (d, 2H, nster CHj) , 4.43 (a, l!f, C?H) , 3.1-1.6 (m, 7H, aromatic H) and 1.30 (d, ltf, amide TR) tau. 39 0 46 F.xamplo 5 2^ N11 roNsnzy 1 ?-ac*tamido-3 -hydroxy-3-cephem- 4-carboxy lata A solution of 10 mmole of £-nitrobentyl 7-*mlno»3-hy Elemental analysis for: Theory: C, 48.85; H, 3.84; V, 10.68 Found: C, 48.97; « 3.96; N, 10.42.

I.R. (CHClj): absorption bands at 2.9 and 3.0 (amide NH and OH), 5.63 (B-lactan carbonyl) and 5.95 (broad, amide, and ester carbonyl hydrogen bonded to 3 OH) microns.

H.M.R. (CDClj): signals at 7.90 (s, 3H, 7-acetamido CHj) 6.55 (s, 2R, C2»T2), 4.92 (d, 1H, CgH) , 4.63 (m, 2H, ester ar2), 4.30 (q. Iff, C?H) , 2.81 (d, 1H, amide NH) , 2.5-1.8 (m, 4H, aromatic H), and 2.8 (s, 1H, C3 OH) tau.

Plectrometric titration (66% aqu<*ous DMF) pKa 5.9 -32- 390 10 Example 6 Diphonylmcthyl 7-f2-(2-thiGnyl)acetamido}-3-chLoro-3-eepher-4-carhoxylate a) To a solution of 34 <-». (100 mmole) of 7- [2 - (2-thienyl) acetamido) - 3-nothy lenecepham-4-carboxyl Ic acid in 500 ml. of methylene chloride was added 21.4 cj. (110 mmole) of diphenyl diazomethane and the resulting mixture was stirred for 2 hours at room temperature. The solvent was evaporated under reduced pressure and the residue was dissolved in ethyl acetate. The ethyl acetate solution was washed with a 5% solution of sodiun bicarbonate, then with water and was dried over magnesium sulfate. The dried solution was concentrated to a small volume. On standing 40 q. of diphenylnethyl 7-[2-(2-thienyl)acetamido]-3-methylenecepham-4-carboxylate neltina at about 132-133°C. precipitated as a crystalline solid.

IP (chloroform): absorption p^aks at 2.9 fa-ide M-l!) , 5.65, 5.75 and 5.93 (!3-lactam, ester md amide car1"—vis respectively) and 6.62 (amide IT) nicrons •MR (cnci3): sinnals at 6.72 l\*n. 2H, C,-'!,) , (s, 2'!. 4.83-4.65 in, 4!(, and Cj-CM,), 4.""* (q, 1H, C?-w) , 3. 4 - - . 6 5 1 ester Ct! and aromatic H) tan. b) To the solution of 8.1 q. (16 rr-.olo) of the aw-o-"* ester in 80 mi. of methylene chloride wore add*d 1.57 <3. (1.6 ml, 19.6 rnolo) of dry pyridine and 3.8 q. (1s.. 1 rf-olc» of phosphorus pentachloride. The reaction nixture was stirred for 2 hours at room temperntiire and was thereafter cooled in an ic?e-water bath. The cold mixture was treated with 9 ml. of isobutancl with stirrino. Stirrina was continued fcr 2 39046 hours during which time 3 g. of diphenylmethyl 7-amino-3-flMthylenecepham-4-carboxylate hydrochloride formed as a crystalline precipitato. The product wu filtered and washed with methylene chloride and vacuum dried.

Elemental analysis (percent) for Cj^Hj^NjOjSCI.

Theory: C, 60.50; H, 5.08; N, 6.72; CI, 8.50; Found: C, 60.70; H, 5.02; «, 6.71; CI, 8.80.

N*(R (OMSO dfi)t signals at i6.45 (ABq, 2H, Cj-Hj) , 5.00 (d, 1H, Cj-R), 4.68 (d, 1H. C?-H), 4.60 (s# 2H, 3-CHj), 4.44 (s, 1H, C|-R), 3.10 (s, In, ester CH), and 2.61 (s, 10H, aromatic R) tau. c) The 7-amino-3-exomethylenecepham ester hydrochloride salt product, 2.1 9. (5 mnole) was dissolved in 200 ml. of methanol and the solution was cooled in an acetone-dry ice bath. Ozone was bubbled into the cold solution for 7 minutes to form the internediatn ozonide. The ozonide was decomposed by passing a stream of sulfur dioxide gas through the reaction mixture for 2 minutes. Thereafter the reaction mixture was evaporates- and th« residue was triturate* with diethyl ether to yield 1.6 g. of diphenylmethyl 7-amino-3-hydroxy-3-cephen-4-carboxylate hydrochloride as a crystalline solid.

NMR (CDClj): signal* at 6.4 (ABg, 2H, Cj-Hj), 5.0-4.5 (m, 2H, Cg-H and C?-H), 3.2-2.4 (m, 11H, ester CR and aromatic H) tau.

IR (chloroform): carbonyl absorption peaks at 5.57 and 5.70 (B-lactam and e3ter carbonyl respectively) microns.

UV (pll7 buffer): X max 275 mu, e=7550. 3 9 0 11.

Electromotrlc titration (60% aq. OMF): titratable qroups at 4.5 and 6.S. d) To a solution of 940 mg. of diphenylmethyl 7-amino-3-hydroxy-3-cephem-4-carboxylate in 10 ml. of water 5 and 10 ml. of acetone was added ono gram of sodium bisulfite. The mixture was stirred and 800 mg. of thiophene-2-acetyl chloride in 10 ml. of acetone were added dropwise. The mixture was stirred for 4.5 hours at room temperature and was then evaporated under reduced pressure. The residue was 10 dissolved in a mixture of ethyl acetate and an aqueous 5* solution of soditn bicarbonate. The ethyl acetate layer wis separated, washed with water and dried. The dried solution was evaporated and the residue triturated with ether to yield 500 mq. of diphenylmethyl 7-[2- (2-thienylJacetamido)-3-hvdroxy-15 3-cephem-4-carboxylate.

NMR (CDC1j): signals at 6.79 (s, 2H, Cj-Hj), 6.16 (s, 2H, a-CH2), 5.0 (d, 1H, Cg-H) , 4.32 (q, 1H. C?-H:. 3.05-2.46 (in, 15H, ester CM and aronatic H) tau. 20 I.P. (chloroform): absorption peaks at 2.9 (an:d« , 5.6, 5.73 and 5.95 (3-lactam, ester an-i carbonvls respectively) and 6.65 (amide TT) microns, e) To a soluticr: of 4.2 g. of diphenylmethyl 7-f2-(2-thienyl)acetamido]-3-hydroxy-3-eeohen-4-carboxylate 25 in 44 ml. of dry dimethylformamide was added 865 m. of phosphorus trichloride. The mixture was stirred for 1.5 hour* at room temperature and was poured Into an ethyl actatp-5* aqueous hydrochloric acid mixture. The ethyl acetate layer was evaporated, was washed with 5* hydrochloric acid, water 30 and was dried. The dried solution was concentrated in vacuo -35- 39046 and the product crystallite*. The 3-chloro ester was filtered, washed with cold ethyl acetate and dried to yield 2.2 g. Elemental analysis (percent) for C26*f21^2®4®2C^' Theory: C, 59.48; H, 4.03; N, 5.14; CI, 6.75 Found: C, 59.77, H, 4.25; *, 5.40; CI, 6.91. NMR (CDClj): signals at 6.49 (Artq, 2H, C2-n2), 6.22 (S, 2H, a-CH2), 5.08 (d, 10, C6-H), 4.19 (q, 1H, C7-H), 3.13-2.5 (», 15H, C?-WH, ester CH, and aromatic H) tau.

X.R. (CHClj): absorption peaks at 2.9 (amide NH) , 5.55, 5.72 and 5.90 (8-lactam, ester and amide'carhonyIs) and 6.60 (amide II) microns.

U.V. (dioxan ): * max 275 mu, e»8700.

Example 7 p-Nitrobenzyl 7-f2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylate. (via thionyl chloride) To a solution o? 1.9 g. (4 mmole) of jfc-nitrobenzyl 7-{2-(2-thienyl)acetami^ -3-hydroxy-3-cephem-4-carboxylate in 10 ml. of DMF (dried over a molecular sieve) was added 950 mg. (0.58 ml., 8 mmole) of freshly distilled thionyl chloride. The mixture was stirred at room temperature for 6.5 hours and was then poured into 100 ml. of ethyl acetate. The mixture was extracted three times with 30 ml. portions of 51 hydrochloric acid and with a saturated solution of sodium chloride. The washed ethyl acetate solution was filtered and evaporated to dryness in vacuo. The residue was triturated with ether to yield 1.2 g. of £-nitrobenzyl 7-[2-(2-thienyl)acetamido)-3-chloro-3-cephem-4-carboxylate as a brown crystalline solid meltina at about 164-166*c.

Elemental analysis (percent) for C20H16*3°6S2C1: -36- 3 9 0 16 Theory: C. 48.63; H, 3.27; M, 8.51; CI, 7.19 Found: C, 48.47; H, 3.29; N, 8.78; Cl, 6.9*! IR (Chloroform) showed absorption bands at 2.9 (amide vn), 5.59 (8-lactam carbonyl), 5.75 (ost«r carbonyl) and ^ 5.92 microns (amido carbonyl).

IJV absorption spectrum (acetonitrile) showed maxima at * max 235 mu, e • 12,100 and X max 268 mu, c • 15,800.

The mass spectrum of the product showed a molecular ion of 10 493 m/e. *1MR (COClj) showed signals at 6.^9 (ABq, 2H, C2~H2), 6.17 (S, 2H, a CH2), 4.99 (d, 1H, Cg-H), 4.64 (s, 2X, ester C«2) , 4.19 (q, l», C7-H) , 3.45 (d. 1H, C?-rn>) , 3.1-1.67 (m, 711, aromatic H) tau. l5 Pxample 9 p-Nitrobenryl 7-(2-(2-Thienyl)acetamido)-3-chloro-3-cephem-4-carboxylate (via phosphorus trichloride) To a cr->ted solution of 439 r.g. (0.13 mole) of p-nitrobenzyl 7- (2- (2-thienyl) acetamidol - 3-hydr^xy-3-ceph^,r.-20 4-carboxylate in 4.4 ml. of DMT was 3lowly added 85 ng. (0.0r> ml., 0.63 mmole) of phosphorus trichloride. The reaction mixture was allowed to 3tand for 4 hours at room tpmoeratnr" and thereafter the reaction product mixture was worked-up by following the recovery procedures described in Example 6 to 25 provide 374 mg. of g^nitrobentyl 7-(2-(2-thienyl)ac^tanidoj-3-chloro-3-cephem-4-carhoxylate. The NMR spectrun of the product was consistent with the expected product and •-»!th that of the product- of ~xar»ple 7. -37- 3 0 0 4 0 Example 9 7-Phenoxyacetamido-3-chloro-3-cephem-4-carboxylic acid Following the ehlorinwtion procedure of ftxample € p-nitrobenzyl 7-phenoxyacotamldo-3-chloro-1-c«phem-4-carboxy-5 late was prepared with phosphorus trichloride. The ji-nltro- benzyl ester group was removed by hydrogenolysls with hydrogen and 5% Pd/C to provide tha 3-chloroceohAlosporanlc acid antibiotic compound.

Kxample 10 10 E>-Mitrobenxyl 7-t2-(2-thlenyl)acotamidoJ-3-chloro-3-cophe«»- 4-carboxylate (via oxalyl chloride) To a solution of 439 mg. (0.93 mmole) of j>-nitro» benzyl 7-[2-(2-thienyl)acetamidol-3-hydroxy-3-cephem-4-carboxy-late in 4.4 ml. of DM? cooled in an ice bath was added drop-15 wise 118 mg. (0.07 ml., 0.93 mmole) of oxalyl chlorldc. The reaction mixture wan allowed to stand for 4 hours at room temperature and was then poured into a mixture of aqueous 5* hydrochloric acid and ethyl acetate. The organic layer was separated and was washed sequentially with 5% hydrochloric 20 acid, water and a saturated solution of sodium chloride. The washed layer was dried and evaporated to dryness to yield the reaction product, £-nitrobenzyl 7-(2-(2-thienyl)acetamido)-3-chloro-3-cepha"»-4-carboxylate as an amorphous solid. The product was obtained crystalline by trituratinq the amorphous 25 residue with ether. Yield 360 mg. The infrared spectrum and NMR spectrum of the crystalline product were consistent with the spectra of authentic material. 3 9 0 4 n Example 11 7-l2-(2-Thier.yl)acetamldol-3-bromn-3-e«ph«st,i-4-carboxylic acid To a solution of 19 n (40 mmole) of £-nitrob«snzyl 7 [2-(2-thlenyl) acetamido]-3-hydroxy-3-eephe"»-4-earhoxylnte In 100 ml of dry HMP was added IS a (56 mmole) of phosphorus trlhr^wide and the reaction mixture was stirred at room temperature overnight. The reaction mixture was poured into a mixture o* ethyl acetate and water and the orqanic phase was separated and washed repeatedly with water and dried over •nagnesium sulfate. The dryed oraanic phase was evaporated in vacuo to dryness. The crude reaction product residue weiohlno about 9 q. was purified by chromatography over 500 o of silica qel using ethyl acetatc-hexane (55:45 v:v) as eluent. The eluate was evanorated to dryness under reduced pressure and tho product, o-nitrobenzyl 7-f2-(2-thienyl)ac«-tamidol-l-bromo-l-cephem^-carhoxy'ate wa«* ohtain^d crystalline by trituratinq the dry residue with diethyl ether, n.v. (ethanol) X max. 270 nu (e = 13,V*"») and X max. 241 my Cc=12,700) Elemental analvsis calculated for r20!,l ^ Theory: C, 44.61; H, 3.00: M. 7.«1; »r, 14.S4.

Pound: C, 44.78: M, 3.01; *1, 7.65: Dr. 14.91. •Juclear nametic resonance spectrum Cwnd^) showed sianal3 at 6.21 (s, 2H a-C!?^) , 5.'"' (A*q, 211, C2~V?.) ' 4-72^»1hC6-H) , 451 (s , 2t!, ester-r-H2) , 420(q, 151, C?-K) , 1.04-1.74 |m, 7H: aromatic 10 and n.6« in, c?-CM) tau.

T»>e above 3-hromo ester was de-esterifie^ in the follovinq manner. The pstor, 545 *vt (1.0 mmole) w,is hydro-genated at room temperature in ethanol in the presence pre-reduced 5 percent palladinm-on-carhon catalyst. The catalyst was filtered and the filtrate evaporated under reduced pressure t.o dryness. The residual product was triturated with diethyl ether tn yield 1*0 mo (44 percent) of crystallino product, 7-f2-(2-thlenyi)aceta«ldo1-3-hromo-3-eephr*«>-4-carboxylic acid.

Rlectrometrlc titration (*6 percont aqueous 0M7) showed a pKa of 4.4 and an appraent molecular weiqht of 193.

The calculated molecular weight " 401.

Plemental analysis calculated for C13Rlinrn2°4S2* 1/2 diethyl etherate: Theory: C, 40.91; H, 3.<56; W, 6.3K: Br, 19.IS. Found: C, 41.29; H, 3.20; *T, 6.29; Br, 18.50. Vuclear magnetic resonance spectrum (CDOl^) showed signals at 8.8 (t, diethyl ether-CHj), 6.58-5.86 (m, C^-n^, !*-C?2 anrt diethyl ether-CJ^), 4.90(d, 1H, Cg-R), 3.0-2.63 (m, 3H, aromatic-fl), and 1.9 (d, 1H, amide NH) tau.

Example 12 7-[2- '2-Thlenyl)acetamido!-3-fluoro-3-cephem-4-carhoxylie acid To a solution of 325 «<7. (0.7 mmole) of jj-nitro-benzyl 7-f2-(2-thienyl)asctamidol-3-hydro«y-3-cephem-4-carboyy late in 10 ml. of methylene chloride was slowly added an equivalent amount of N-(2-chloro-l,I,2-trifluoroethyl) diethyl amine. The reaction mixture w-ii heated for 30 minutes under gentle reflux and was then evaporated to dryness in vacuo. The residue was dissolved in a mirture of ethyl acetate-water and the orqanic layer was separated. **he oroanic layer was washed with 5* hydrochloric acid, water and brine and was then dried. The dried reaction product solution was concentrated -40- 3 9 0 4 0 to a snail volume and n-hexane was add»d to nrecioitate, £-nitroben*yl 7- [2- (2-thienyl)acetamidol -3-fluoro-3-cephe~»-4-carhoxylate.

The ester qroup was reeved by catalytic hydroeen-5 olysis to yield the 3-Cluoro cephalosporin free aeid antibiotic.

Rxanolc 13 a) £-Wltrobon*yl 7-(2-(2-thienyl)acetamido)-3-m«thylsul-fonyloxy-3-cephem-4-earboxy1ate. 10 To a solution of 4.75 g. (10 mmole) of j£-nitro- benty1 7-[2-(2-thienyl)acetamidol-3-hydroxy-3-cephem-4-carhoxylate in 50 ml. of dry dinethvlacetamide wore added 2 ml. of propylene oxide, ^o the solution was added with stirring one equivalent of -nothanesulfonyl chloride and stir-jj ring was continued for 3 hours. The reaction mixture w*s then taken up in ethyl acetate and the solution w*s washed with a saturated solution of sodium chloride. The washe* organic phase was evaporated in vacuo to dryness to obtain the reaction pr-4uc. nixture as a residue. The rotation 20 product was purified by preparative thin layer chronatc?rj">hy on silica gel usi"i for elution 65* ethyl aeetitc/h<*xane- The purified title compound gave tho following [M-n ■ elemental composition o" microanalysis.

Calculated for C21H19*,3°9 Pound: C. 45.74; 1?, 3.56; •!, 7.10; S, 17.06. """he nuclear nacnpti^: resonance sncctrui and the infrared absorption spectrun wore in aareenont with the structure of the product former*. -41- 39040 b) p-witrobe.nzyl 7-t2-(2-thienyl)acetamido]-3-fluoro-3-cephem-4-carboxylate.

To 93 mg. of dicyclohexyl-18-crown-*-ether in 15 ml. of acetonitrile dried over a moleculei- nicro were added 25 ma. i of pntas.iium fluoride which had been ilried in vac»o at 90*C. The mixture was stirred for ten minutes, and 138 mq. of j»-nitrobenzyl 7-[2-(2-thienyl)acetamido]-3-mathylsulfonyloxy-3-cephem-4-carboxylate in 4 ml. of acetonitrile were then added. The mixture was stirred for 1 hour. The mixture was acidified 10 by addition of dilute (St) HC1, and the resultinq acidified mixture was extracted with ethyl acetate. Pure product was obtained from the ethyl acetate extract by preparative thin layer chromatography on silica qel usinn ethyl acetate: benzele (1:1). Ten mq. of j>-nitrobenzyl 7-12-(2-thienyl)-If acetamidoJ-3-fluoro-3-cephem-4-carboxylate were obtained.

T.R.: absorption peaks at 17<»2, 1740, 1695 cn.~l HMR (CDClj) showed sinnals at <5.15 (s, 2'!, n C"2) , 4.97 (d, 11!, J - 41*2» Cg-H), 4.20 (q, 1H, C?-H), 3.52 (d, 1H, C7-'. ; , 2.12 - 1.7 (m. 2H, Cj-Hj) tv:. 20 Plnorine WMR — (d, J » 10Hz).

M.S.: Calculated -- 177.04*5 found — 477.045"; -42- 38u »i; Fraq»cnt COOPHD Calculated -- 297.0145 Pound — 207.0144 ^xamnlc 14 5 7-f2-(2-thienyl)acetamido}-1-fluoro-l-cephen-4-carboxylic acid.

To 110 ml. of methanol and 83 ma. of pr' -reduced 5 percent pallidlun on carbon were added S3 nq. of n-nitro-benzyl 7- r2-(2-thienyl)acet/»,^i'lo1 -3-f luoro-l-ceohem^-cnrb'^v-)() late. The mixture was hydrooonated at 51 psio. for one ho-ir.

The resulting mirturc was filtered, and the filtered catalys" was washed with r^ethanol, and the ncthimol washlnar; were a-ld-d to the filtrate. The €iltrate was then evaporate* in /ar-io. The residue %n\s dissolved in ethyl acetate, and the ethvl 15 acetate solution was extracted with dilute aqueous sodium bicarbonate. The sodium bicarbonate solution was washed with ethyl acetate, layered with ethyl acetate, and di)iit« aq\i'=" HC1 was added. The layered ethyl acetate was serrated and evaporated to recover 7- [2-(2-thionyl!acetairidol-1-fluoro-1-20 cenhe«*-4-carboxylic acid, show by bioautonrar» to b<-> bio- lonically active.

Example 15 p-Witrobenzyl 7-a*ino-1-chloro-3-ce,>he'*-4-carboxyl;»te hydrochloride. 25 To a solution of 500 "ig. of _p-nitrobon7.yl 7— (2—(?- thienyl)acetamido!-l-chloro-l-cephen-4-carboxvlate in 6 nl. of methylene chloride was added 95 nq. of dry pyridine and -41- 39046 237 ma. of. phosphorus pentachloride. The reaction mixture was stirred at room temperature for 1.5 hours, was thereafter cooled in an ice-water bath to about 5*C. and 0,6 al. of isobutyl alcohol wero added. On continued coolln NMR (o2o-»MHro3>: slonals at 6.25 (**q, 2«f, C2-H,) 15 (d, 1H, Cg-H) and 4.54 (d. IM, C7-!t) tvi.

".V. (r>M 7 buffer) : absorption maximum at V max 26 j T.U , e ■ T550 Example 17 20 Piphenylmethyl ?-Tiino-3-rhloro-3-c^-'ho^-4-carb<->Yv] -it"" To a sa ition of mfj. oc diohenylmcthy i 7- {2- (2-thienyl 5 ic ~!t ani'io) -1 -chloro- 3-ceohem-A-carboxy 1 at<» i i'O ml. of methylene chloride w.is added 0.1 nl. of dry oyridme and 237 mq. of phosnhorus pentachloride. The reaction -ixture 2 5 was stirred for 2 hours at room temperature and was then cooled in an ice-water mixture. To the cold mixture was added O.fi ml. of isobutanol and after 30 minutes the reaction mixture was evaporated. The residue was dissolved in ethyl acetate and the solution was washed with 5* s«^dium bicarbonate ->rs-i 30 with water and was dried. The dried solution was e-raoorat^d - 45 - 39046 to dryness and the residue was triturated with ether to yield 190 mg. of 3-chloro nucloas ester# diphenylmethyl 7-ariino-l-chlorc-3-cephem-4-carboxyl*t«.

T.w. (tnull)t absorption peaks at 5.7 and 5.9 (ft-lactam and ester carbonyl) microns.

«WR (COClj): signals at 6.35 (KBq, 2H, Cj-Hj), 4.78 (2d, 2H, Cg-H and C7~H), 3.05 (s, 111, ester CH) and 2.€S (s, 10R, aromatic W).

Rxamole 16 Following the 7-acyl side chain cleavage reaction conditions described in Example 17 £-nitroben*yl 7-anino-l-fluoro-3-cephe«n-4-carboxylate is prepared from tho interned late of Kxanple 13, £-nitrobensyl 7-(2-(2-thienyl)acetamido] -3-fluoro-3-cephem-4-carboxylate.

Example 19 Pnllowino the 7-acvl side chain cleavaae reaction procedure described by Kxanple 15 diphenylmethyl 7-*mino-1-brofo-3-cephfm-4-carboxylat* is pren*red froa diphenylmethyl 7-phenoxyacetanido-3-bromo-'*-'*eph«m-4-carhoxylate.

Example 20 7-(r>-i-Phenylglycylamino) -3-chloro-3-cephem-4-carboxylic acid To a suspension of 280 mer. (1.2 mole) of 7-amino-3-chloro-3-oenhem-4-carboxylic acW in 14 ml. of acetonitrile was added with stirring at room temperature 0.5 nl. of M,0-bis-(trimethylsilyl)acetamitfe to fom the soluble disilyl-methyl derivative thereof. The solution was cooled to 0*P. and was slowly added to a solution of the mixed anhydride formed by reacting 408 mg. (1.5 mmole) of methyl 3-a-carboxy-benzylaminocroton*te sodium s-ilt with 16) mg. (1.7 nmole) of methyl chloroformate in the presence of 2 drops of N,N-dimethyl- 39046 benzyl amine in 7 ml. of acetonitrile. The -nixttire wa# atirrc-* at ice bath tomperaturo for 2 hours, 1 ml. of methanol was added an* tha fixture was filtered to r<*wove insoluble Impurities. Two millilltors of wat«r wero added to the filtrate and the p»» was adjusted momentarily to oH 1.5, to effect removal of the enamine block, and then to pH 4.5 with triethylamine. After stirrin? for an additional hoar at ice-hath temperature the reaction product, 7-(n-a-p»i*nylalyeyl*mia«)-1-chloro-l-cephe«-4-carboxylic acid (zwitterion) precipitated from th® reaction nixtare as a crystalline solid. The product was filtered, was washed with acetonitrile and dried JLn vacuo to yield 200 mg.

The product has the following physical characteristics: rie-ental analysis for ClsH14M^o4Sd*l/2 Theory: C, 47.80: B, 4.01; M, 11.15; Cl, 9.401 Pound: C, 47.55: n, 4.12: N, 19.. 2S (carboxylate) microna. ♦jmr spectrtm (n^n/nci): sicmals at ^.S—ft.7 ("Bq, 2H, C2-W2), 4.B4 (d, IH, Cf-It), 4.2K ( UV Spectrum (pH 7 buffer): X max 2fi5 nu (e»6,800).

Rxawple 21 To a solution of 500 irg. (1.85 mmole) of methyl 3-a-c-irboxybensylaminocrotors^te sodium salt (formed with phenylalycine and methyl aeotoacetate) in 20 ml. of aceto-nitril"! was added 4 drops of N(Nr-dimcthylbcnzylaniine and the -47- 39046 solution was cooled in a dry ice-carbon tetrachloride nixture with stirring. To the cold solution was slowly added 184 *n. ;i.95 mole) of methyl chloroformate to form the mixed anhydride. After 20 minutes • pre-cooled solution of 750 rag. (1^85 mmole) of p-nitrobensyl 7-amino-3-chloro-3-ceph«n-4-carboxylate and 188 mg. (1.85 mole) of triethylamine in 40 ml. of acetone was added. The addition took place over 3 minutes and thereafter the reaction mixture was stirred in the cold for 30 minutes and then at room temperature for 2 hours. The reaction mixture was filtered to remove insoluble impurities and was evaporated in vacuo. The reaction product residue was dissolved in a mixture of ethyl acetate-water and the pH of the solution was adjusted to pH 7. The organic layer was separated and washed with water. After drying over magnesium sulfate the organic layer was concentrated under vacuum to a snail volume. Upon addition of n-hexane to the concentrate, 620 mg. of £-nitrobenzyl 7-[N-(l-carbomethoxy-2-propenyl)-D-<»-phenylglycylamin^l-3-chloro-3-cephem-4-carboxylate precipitated fren. the reaction mixture.

The product gave the following elemental analysis and nuclear magnetic resonance spectrum: Elemental analysis for C27H26N409SC*: Theory: C, 53.87; H, 4.35; N, 9.31* Pound; C, 54.05; H, 4.13; N, 9.36% NMR (DNSO dg): signals at 8.20 (s, 3H, enanine CH^), 6.60 (ABq, 2R, C-j-Hj) , 6.45 (s, 3H, ester CHj), 5.48 (s, 1H, enamine vinyl H), 4.90-4.1 (m, 5H, Cg-H, Cy-H, a-CH and ester CHj) and 3.10-1.5 (m, 9H, aromatic H) tau.

The product, 540 mg. (0.9 mmole) was dissolved in -4S- 3804C 40 "il, of acetonitrile containing 20 ml. of water and the solution was first cooled in an ice-water mixture and then acidified momentarily to pH 1.5 and thereafter adjusted to pi! 2.5. The mixture was evaporated and the residue was 5 dissolved in 40 ml. of tetrahydrofuran and 80 ml. of methanol.

To the solution was added 540 mg. of S% palladium on carbon (prereduced in 20 ml. of ethanol for 45 tnin. under 50 psi hydroqen at room temperature) and the solution was hydrogenated at room temperature for 2.5 hr. under 50 psi 10 hydrogen pressure. The catalyst was filtered and was washed on the filter with methanol, TRF and with water. The filtrate and washes were combined and evaporated to dryness in vacuo. The reaction product residue was dissolved in water-ethyl acetate and the pH of the solution was adjusted to pH 4.5. 15 The aqueous phase was separated, was washed with ethyl acetate and was evaporated to a small volume of about 2 ml. On coolina, 65 mq. of the product, 7-(D-a-phenylqlycylamino)-3-chloro-l-cephem-4-carboxylic acid, precipitated as a crystalline sol 14 from the cold conee:rate. 20 Example 22 To a suspension of 3.0 g. (8.1 imole) of D-nitro-benzyl 7-amino-3-chloro-3-cephen-4-carboxylate in 200 r-l. of tetrahydrofuran (dried wi*h molecular siev«) were added 2.1 n. (8.3 mmole) of N-(t.-butyloxycarbonyl) D-a-nhenylqlycine and 25 2.0 q (8.3 wiole) of N-ethoxycarbonyl-2-ethoxy-l,2-dihydro- qulnoline (EBOQ). The reaction mixture was stirred overninht at room temperature and the solvent was evaporated under reduced pressure. The residue was dissolved in a mixture of ethyl acetate and water and the organic phase was separated. -49- 39046 The organic phase was cooled and successively washed with a cold 5 percent aqueous solution of sodium bicarbonate, a cold solution of 5 percent hydrochloric acid, and then with water. The washed solution was dried over m&cnesium sulfate, filtered, 5 and concentrated by evaporation undor reduced pressure to a volume of about 50 ml. Pron -chr: concentrate, 3.7 g. (63 percent ylsld) of the reaction product, £-nitr»benayi 7-fr>-2-(t-butyloxycarbrtmido) -2-ph«nylacetanido] -3-chloro-3-cephem-4-carboxylate, was obtained as a crystalline product, A second 10 crop of th® product waiqhing about 2 g. was obtained by further concentration of the filtrate obtained after filtering the first crop material.

Elemental analysis calculated for C27H27C1H408S; Theory: C, 53.78; H, 4.51; N, 9.29. IS Found: C, 52.66; H, 4.36; N, 8.98.

U.V. (acetonitrile ) showed a maximum at 268 mu (e«17,100).

I.R. The following sionifleant absorption peaks were observed in liit infrared spectra of the 20 prcduct. Amide HT1 absorption peak at 3.05 mu and carbonyl absorption peaks at 5.59, 5.75, and 6.0 mu.

NMR (CDC13): signals at 8.60 (s, 9H, t-ROC), 6.45 (ABq, 2H, C2-H2), 5.03 (d, 1H, Cg-H), 4.fi7 (s, 25 3H, a CH and ester C«2) , 4.12 (ra, 3H, C?-H and amide VH), and 2.72-1.74 (m, 10H, aromatic H and amide NH) tau.

To a solution of 3.0 a. (5.0 nmole) of the product in 15 ml. of dry tetrahydrofuran (dried with molecular sieve) 30 and 185 ml. of methanol were added 3 g. of pre-reduced 5 per-50- 39046 cent palladium on carbon. The catalyst was pre-reduosd In ethanol for 30 minutes at room temperature under a hydrogen pressure of 50 psi. Following the addition of the pre-re-duced catalyst, the product was hydrogenated at room temp-5 erature for one hour under a hydrogen pressure of 50 pal. The catalyst was filtered and washed on the filter with tetrahydrofuran and methanol. The filtrate and washes were combined and evaporated under reduced pressure. The residue was dissolved in ethyl acetate and water was added. The pH 10 of the mixture was adjusted to pH 7 by the addition of IN sodium hydroxide. The aqueous phase was separated and was washed with ethyl acetate. The aqueous phase was then layered with ethyl acetate and back titrated to pH 3.5 with IN hydrochloric acid. The organic phase was separated from tha aqueous phase and was washed with water and dried over magnesium sulfate. The dried organic phase was evaporated under reduced pressure dryness to obtain the reaction product, 7-[D-2-(t-butyloxycarbaraido)-2-phenylacetamido)-J-chloro-3-cephem-4-carboxyiic acid, as a dry solid residue. The 20 product was crystallized from 70 ml. of ether containing 2r ml. of petroleum • .her to yield 1.75 g. (75 percent) of crystalline product.

Elemental analysis calculated for C20H22C1N3°6S: Theory: C, 51.34; H, 4.74; N, B.9B: CI, 7.5R. 25 Found: C, 51.02; H, 4.06- 8.75: CI, 7.30.

U.V. (acetonitrile) maxinum at 268 mu (e«7400). NMR (CDClc): sionals at 9.55 (s, 9H, ' 6-48 (ABq, 2H, C2-H2), 5.0 (d, 1H, Cg-H) , 4.63 (d, 1H, a-CH) , 4.25 (q, 1H, Cy-H), 3.90 (d, 1H, anide VH), 30 and 2.59 (s, 5H, aromatic H) tau. -51- 39046 To a solution of 420 mg. (2.2 mnole) of £-toluene-sulfonic acid in 5 ml. of acetonitril* wem added 4(8 rag. (1 nnole) of the crystalline hydrogenolysis product. The solution was allowed to stand at room temperature for about S 16 hours and was then diluted with 0.5 ml. of water. The pH of the solution was adjusted to pH 4.8 at which 320 mq. (87 percent) of the crystalline deblocked product, 7-(n-«-phenylglycylamina)-3-chloro-3-caphem-4-carboxylie acid, precipitated from the solution. Tha product was collected by 10 filtration and dried.

Elemental analysis calculated for C15H14C1W 1/2 n2Ot Theoryi C, 47.30; H, 4.01; M, 11.15» CI, 9.40.

Found; C, 48.04; H, 3.92; N, 11.18; CI, 9.70. j 5 Example 23 The antibiotic compound 7-(n-a -phenylglycylanti»3)-3-chloro-3-cephem-4-carboxylic acid, was also prepared from ;3-nitrobcnzyl 7- (0-2- (t.-butyloxycarbamido) -2-phenylaeetamidol -3-chloro-3-cephem-4-carboxyl*te by a deblocking method alter-20 native to that described in the proceeding example. In this method, the fbutyloxycarhonyl amino protecting group is removed first and thereafter the Q.-nitrobenzyl ester group is cleaved with zinc and hydrochloric acid in dimethylfor-nanide. This alternative preparation is described in the 2 5 following paragraphs.

To a solution of 2.4 g. (12.6 rnnole) of p-toluene-sulfonic acid in 60 ml. of acetonitrile was added 3.6 g. (6 fvrtole) of g-nitrobenzyl 7- (D-2- (t-butyloxycarbamido) -2-phenylacetamido]-3-chloro-3-cephem-4-carboxylate. The 30 reaction solution was stirred at room temperature for about -52- 39046 15 hours during which tine the amino doblocJ'.od product, j?-nitrobenzyl 7-{0-phrnyl The product was filtered, washed with acetonitrile and vacuun dried. Yield: 3.1 g. (81 percent).

Elemental analysis calculated for CjjiijyClfJ^O^Sj: Theory: C, 51.58; II, 4.06. M, 8.29. CI. 5.25.

Found: C, 51.51; H, 4.14. it, 8.12, CI, 5.60.

Infrared absorption spectrun (chlorofom) : carbonyl absorption peaks at 5.61, 5.80, and S.95 nicrons. Tosylate salt absorption peak at 6.23 r.icrons. ffuclear magnetic resonance spectrun (DMCO dg) : si-;-nals at 7.70 (s, 3H, £-toluenesulfonic acid salt-CHj), 6.61 (s, 3U, side chain IJHj) , 6.20 (AB^, 211, C,-H2) , 4.94 (bron-sinolot. III, a-Ci:! , 4.80 (d, 1H, CQ-V.) , 4.51 (s, 2H, ester CIi2> , 4.08 (q, 1" C?-H) , 2.95-1.(52 In, 14H aror.atic hydrogen and anide :ni) , ami 0.32 (d, ill, anide :1M) .

The anino deblocked p-toluenosulfonic acid salt product obtained is described above was deesterifled to obtain the antil.^tic conpound as follows. A solution oi 1.5 g. (2.2 mnole) of the anine deblocked jj-toluenesulfor.ic acid salt, product obtained above in 10 ml. of dry dimethyl-fornanlde (dried with molecular sieve) was cooled in an i.c: alcohol bath. To the cold solution was added 2 ml. of concurs tratcd hydrochloric acid. 3inc dust, 400 -in. (C.l nnolc) was added to the cold solution portior.wiso over a period of about 15 minutes . Tlie rnction nixture was stirrnd in the cold for 30 mi nutos *nd v?.s thon a Hover* to \>'arr to roor, temperature with continued stirri.-sn. Thc. reaction nixture 3 0 0 i G was stirred at room temperature for about one hour and was filtered. The pH of the filtrate was adjusted to 6.8 with triethylanine. The product, 7-(D-phenylglycylaraino)-3-chloro- 3-cephem-4-carboxylic acid as the bis-dimcthylfoima-5 aide solvate of the 2witterion, fomed as a white crystalline precipitate. The product was filtered, washed with 10 ml. of cold dimethylformaaide and then with 6 ml. of diethyl ether. The washed product was vaeuum dried to yield 800 mg. (71 perccnt) . 10 Elemental analysis calculated for C2lH23C1M5°6S* 2DMP Theory: C, 49.07, H, 5.49; II, 14.63; CI. 6.90 Found: C, 48.84; H. 5.53; N, 13.48; CI. 7.18 Ultraviolet absorption spectrun (acetonitrile): 15 maximum at 265 mu (c*C,000).

Electrcxnetric titration (66 percent aqueous OMF): pKa at 4.55 and 7.2.

Nuclear magnetic resonance spectrun (DjO/DCl): signals at 6.34 (2s, 6It, OMF-CHj) . 6.33 (ADq, 211, Cj-Hj), 20 4.85 (d, 10, Cg-II) , 4.64 (s, IH, a-CH) , 4.27 (d. 111, C?-H), 2.41 (s, 5K, aronatic H) , and 1.C4 (s, 2H, DMF-Cli) tau.

Example 24 7- 3-hydroxyphfinyl )glycj i ami. no} -3-chloro-3-cephem-4- carboxylic acid. 25 H-(t^Butyloxycarbonyl) D-3-hydroxyphenylglycine, 2.9 g. (11 mmole) was reacted with 3.7 g. (10 nnole) of jj-nitrobenzyl 7-anino-3-chloro-3-cephem-4-carboxylate and 2.6 g. (10.5 nanole) of the coupling reagent EEDQ. The reaction was carried out and the product isolated by fol-30 lowing the procedures described in the acylation process of -54- 390 4 6 Example 22. The product (tec below) wu obtained a.i an n>n<.ri>li so l id after trituration with othor to yield 2.S g. (4C> percent.) Elemental analysis calculated for C27»27C1N4o9s: Theory: C, 52.39/ H, 4.40j N, 9.05. rounds C, 52.16) H, 4.59j N, 8.79.

Ultraviolet absorption spectrum (acetonitrile): maximum at 270 ew (e-17,200).

Nuclear maqnetie resonance spectrum (CDClj): ain-nals at 8.39 The product. £-nltroben*yl 7-fn-2-(t-hutyloxy-carhamido) -2- (3-hydroxyphenyl)-«cetamido) -3-chloro- 3 -ceohem-4 -carboxyl ate. 3.5 q. of (5.6 insole), was hydrogenatod in ethanol in the presence of ore-reduced 5 percent palladium cm carbon by followino the deesterificatlon procedure ^escribed in Example 22. The product, 7-(D-2-(t-butyloxycarbamido,-2-(3-hydroxyphenyD-aeetarildo] - 3-chloro-3-ceohe»«-4-carboxylic acid, was obt-n...o Oltraviolet -'_».<;orption spectrum (acetonitrile) : maximum at 272 mu (c-5,280).

Electroraetric titration (66 percent aqueous nM.F) : pKa at 4.5.

The above product, 1.3 g. (2.7 irmole) w.is reacted with 1.1 q. (5.9 mmole) of js-toluenesulfonic acid in 2fl ml. of acetonitrile to effect the removal of the £-butyloxycarbonyl protecting group. The procedure was carried out essentially accordina to the method described in Example 22. -55- 0040 Tha product, 7-Q)-a-(3-hydro:xyphanyl)glycylamiao]-3-chloro-3-cephem-4-carboxylic acid, crystallized from the reaction mixture, was filtered and then vacuum drlod. Yield, 700 tog. (64 percent).

Elemental analysis calculated for CjjH^ClMjOjS.li^O: Theory: C, 44.83; H, 4.01; N, 10.46.

Foundi C, 45.12; H, 4.06; H, 10.31.

Ultraviolet absorption spectrum (ptl 6 buffer): maximum at 268 mu (e-9,750).

Nuclear magnetic resonance spectrum (DjO/DCl): signals at 6.31 (ABq, 211, Cj-Ilj) , 4.81 (d, 1U, Cfi-H) , 4.52 (•, IB, o-CH), 4.26 (d, 1H, C?-H), and 3.1-2.5 (», 411, arociatic H) tau.

Example 25 7-(D-4-Chlorophenylglycylaaino)-3-chloro-3-cephesn-4-carboxylic acid.

By following the procedures described in Example 22, 850 mg. (2.3 mmole) of p-nitrobenzyl 7-araino-3-chloro-3-cephea-4-carboxylate was acylated with 700 mg. (2.5 mmole) of N-t-butyloxycarbonyl D-4-chlorophenylglycine and 567 mg. (2.3 mmole) of EEDQ to provide 1.2 g. of £-nitrobenzyl 7-[D- 2-(t-butyloxycarbamido)-2-(4-chlorophenylacetarido)J-3-chloro- 3-cephem-4-carboxylate.

The product, 1.2 g. (1.9 mmole) was hydrogenated at room temperature in the presence of pre-reduccd 5 percent palladiua on carbon to effect the removal of the £-nitrobenzyl ester group and provide 450 mg. of 7-(D-2-(t-butyloxycarb-amido) -2-(4-chlorophenyl)-acetaiiiido) - 3-chloro-3-cephen-4-carboxylic acid as a crystalline product. The product was obtained crystalline by trituration of the amorphous reduc-56- 3 9 0 1 <; tion product with diethyl ether.

Elemental analysis calculated Cor C^H^CijUjOgS: Theory: C, 47.82; H, 4.21; II, 8.36; CI, 14.11.

Found: C. 47.75; H, 4.43; !i, 8.11; CI. 14.15.

Electrometric titration (66 percent aqueous DMF) : pKa at 4.4; apparent molecular weight - 508; calculated MW-502.

Tha deesterification product, 450 09. (0.9 :nmole), was reacted with j^-toluenesulfonic acid in acetonitrile by following the procedure described in previous examples to effect the removal of the t-butyloxycarbonyl protecting group and provide 160 mg. of crystalline 7-(D-»-(4-chlorophenylglycylamino)-3-chloro-3-cephem-4-carboxylic acid in the zwitterionic form.

Elemental analysis calculated for .

IHjO: Theory: C, 42.86; H, 3.59; U, 9.99; Cl, 16.37.

Found: C, 43.07; H, 3.63; N, 9.69; Cl, 16.75.

Ul"-wiolet absorption spectrum (pH 6 buffer): maxima at 265 mu Ce«8,100) and 2.25 mu (e-13,900).

Electrometric titration (66 percent aqueous DMF) : pKa ■ 4.15 and 6.8; apparent molecular weight ■* 407, calculated HW 403.

Example 26 7- (D- a -4-Hydroxyphenylglycylami no )-3-chloro- .Vcephein-4-cArb sxy-lic acid.

II- (t_-butyloxycarbonyl) D-4-hydroxyphenvlnlycine, 2.9 g. (11 mmole) was reacted with 3.7 g. (10 natole) £-nitrobenzyl 7-amino-3-chloro-3-cephem-4-carboxylate and 2.6 g. (10.5 mmole) of the coupling reagent EEDO in dry tetra- -57- 9040 hydrofuran. The reaction was carried out and the product isolated by following the procedures described in the acylation process of Example 22. The product, 3.7 a (60 percent yield) was obtained crystalline from cold diethyl other.

The product, j>-nitrobonzyl 7-(D-2-(t-butyloxy-carbamido) -2- (4-hydroxyphenyl)-acetamido] -3-chloro-3-cephen-4-car boxy late qave the following percent ele«tental analysis and nuclear magnetic resonanco spectrun. elemental analysis calculated for C27H27N^OgSCl5 Theory: C, 52.39; H, 4.40; H, 9.05.

Found: C, 52.12; B, 4.26; N, 8.91.

Nuclear magnetic resonance spectrun (dmso d^) showed siqnals at 8.62 (s, 911, t-BOC-Ofj) , 6.16 (ARq, 2H, C2"R2>' 4*81 (d' lH* C6'n) ' 4,75 ,d' 1H' °"CH) • 4*53 (8' 2H, ester-0»2), 4.18 (q, 1H, C?-H), 7.04 and 2.0 (2q, 8H, aromatic H), 0."»o (d, 1H, C?-MH) and 0.58 (s, 1H, n-OH) , tau.

The product, 2.2q (3.5 mmole) was hydroqenated in ethanol in the presence of ore-reduced 5 percent pnlladium-on-carbon catalyst to affect the removal of the o-nitro-benzyl trrouo. The deesterified product, 7- [0-2-(t^butyl-oxycarbamido) -2-(4-hydroxy)phenyl-acetai*ido) - 3-chloro- 3-cephem 4-carboxylic acid, was obtained crystalline from a mixture of diethyl ether and n-hexane. One gram of the product was obtained.

Elemental analysis calculated for C20H22C1N3°7S: Theory: C, 49.<54; H, 4.58: N, 8.08.

Found : C, 48.92; H, 4.40; N', 8.24.

Nuclear maqnetic resonance spectrum (pm«;o d^) showed sianals at 8.61 (s, 9H, t_-BOC-CHj) , 6.26 (ABq 2H, 30046 C2»2), 4.89 (d, 1H. C6-»), 4.78 (d, 1U, a-CH), 4.23 (q, 1H, C7"H), 3.06 (q, 411, aromatic-It) and 1.20 (d, IK, tau.

Tha t-DOC protecting group was removed from the doeosterification product by reacting the product in ace-tonitrile with £-toluenesulfonieacid. Prom the 1 g. of product ims obtained 330 mg (40 porcent) of the final product 7-C0»M4-hydroxyphenyl )-glycylajninqJ -3-chloro-3-i-f;plu-m-4-carboxylic acid having the following elemental analysis, nuclear magnetic spectrun and titration values.

Elemental analysis calculated for Cj^Hj^CIHjOjS. 1H20: Theory: C, 44.83; II, 4.01; N, 10.46.

Found : C, 44.92; II, 3.45; N, 10.63.

Electrometric titration in 66 percent aqueous DMP gave pKa values of 4.2, 7.7 and 12.4. The apparent molecular weigh*- calculated as 384; the calculated molecular weight is 333.8.

Nuclear magnetic spectrum (D20/DC1) showed signals at 6.32 (ABq, 2H, Cj-Hj), 4.84 (d, 1H, Cg-H), 4.27 (d, 111, C?-H) , and 3.79 (q, 4H, aromatic-H) tau.

Example 27 Following the acylation procedure and by employing the acylating reagent of Example 20, the following con-pounds are prepared by the acylation of the indicated 3-halo nucleus: 7-(D-a-phenylglycylamino)-3-fluoro-3-cephen-4-carboxylic acid is prepared from 7-amino-3-fluoro-3-cephem-4-carboxylic acid; 7-(D-a-phenylglycylamino)-3-bromo-3-cephen-4-carboxylic acid is prepared with 7-aiainc-3-bror.o-3-cephe.^-4-carboxylic acid; and 7-(0-:i-(4-hydroxyphenyl)glycylaniir.o] -3-chloro-3-cephem-4-carboxylic acid is prepared from the 39040 enanine protected 4-hydroxyphenylglycine mixed anhydride derivative formed with methyl chloroformato via the acylation of 7-amino-3-chlor o-3-caphcm-<-carboxylic acid.

Example 28 5 7-CD-2-(2-Tbienyl)«jlycylax»inq) -3-chloro-3-cephem-4- carboxylic acid is prepared by the N-acylation of 7-amino- 3-chloro-3-cepnem-4-carboxylic acid with 2-thienyl

Claims (2)

1. claims; 1. A com|K>und of generaL formula ii 0 h lit S R-c-C-nv "X f1 j—«Ls^-x R2 0 COOR, wherein R is phenyl, aonohydroxyphenyI, dihydroxyphony1, 5 monohalophenyl, dihalophenyl, monomcthoxyphenyl, dlmethoxy- phonyl, monomethylphenyl, dimethylphenyl , 2-thionvl, J-thi.c-tn or 2-furyl; Rj is hydrogen, benzyl, p-methoxybenzyI , p-nitrobenzyl, diphenylmethyl, 2,2,2-trichloroethyl, trimothylsiLy1, jq or t-butyl; R is hydrogen or amino-protecting pnup; 2 X is t'luiro, chloro, or bromo; or, when R or R, is hydrogen, a phartrvto-utical ly-acceptable, no.anoxic salt thereof.

2. A compound as claimed in Claim l, wherein R„ hydrogen. 3- A compound as claimed in Clairi 1 nr 2, wheroin is chloro. 4- A compound as claimed in Claire 1, 2 or <, v.hfr--2o R is phenyl. 5. 7 — (D—ft-Pheny1glycylamiao) — 3-chIoro-3-cephem-4-carboxylic acid. 6. A compound as claimed in any one of Claims J to 3, wh«;rein R is mono or dihydroxyphenyl. 25 7. 7-CD -a-(4-Hydroxyphenyl )-glvcyl .iminrj -3-ch lot 3-c(;phcm-4-carboxy 1 i c acid. S. 7-f0—.t-( 3-Hydroxypheuy I )-gl ycy I ami -3-chlund as claimed In any tine <»1' Claims I to 3, who rein K in mono or dAhaloph<-nyI.v 10. 7-CD-a-{4-Chloroplicnjrl) -glycy 1 ami mi] -.Vcliloro- 3-cepJiem-4-carboxylic acid. 11. A compound as claimed in any ono of Claims 1 to 3, wherein R is 2-thienyl. 12. 7-0>-2-(2-t'hienyl)-glycylajnino3 -3-chloro-3-cephem-4-carboxylic acid. 13- A compound as claimed in any one of Claims 1, 2»4»6,9, and VI, wherein X is a fluorine atom. 14 • 7-( D-a-Pheny IgLycyJ amino uori>- l-cit|ilica- 4-carb»»*ylie acid. IS- 7-p>-a-(4-Hydroxyphenyl )-glycylamino) -3-fluoro-3-cephem-4-carboxylic acid. l6. A compound as claimed in any one <>£' Claims t, 2,4 » and 11, whe roii. X is a bromint? atom. 17- A process for preparing a compound of the formut H O H when*in R, Rj and R^ are as defined in any one of Claims I, 2»4»6>9, or 11, and X is chloro or bromo, which comprises a) reacting in any sequence a 3-hydroxy-3-cephem est wherein is a carboxylic acid protecting ester forming group with a chlorinating or brominating agent in a solvent capabl. 3 0 0 4 (! of I'urniiig an imi.ni.uai chloride or brtNtide, ami with an acylating reagent of the formula HO I D R— C— C —OH I NH I R2 or a reactive derivative thereof, to provide the correspond.! ng 5 7-(a-amino) or 7-(fi-protected-an»ino)-«cyJai!ilno-3-halo-3-c<:phera- 4-curboxylic acid ester; and b) optionally removing the carboxylic acid protecting est<*r forming group or the xrtao protecting group to provide the corresponding 7-(a-aaino)acyl«®iw -3-H»lo-3-cephem-4-carboxy-10 lie acid. 18. A process according to Claim 17, wherein the solvent is dimethyl-fornamide. 19. A process according to Claim 17, of' Cl.i Lm 1-, wherein the ha 1 ^genating agent is phosphorus trichloride. IS 20. A process according the Claim 17 or Cl.ti • I- wherein the halo^etiat ing agent is thionyl chloric!-- 21. A process according to Claim 17 oi Clr«ii;i lS, wherein the halogenating agent is phosphorus tribromivle. 22. A process for preparing a compound according to 20 Claim 1, substantially as hereinbefore described with part n.iilar reference to any one of Examples 20 to 29. 23- A compound according to Claim 1 substantially as hereinbefore described with particular referenc to any -me of Examples 20 to 29-25 24. A compound according to Claim 1 whenever pre pared by a method as claimed in any one of Claims 17 to 22- -63- 39040 25* A phar*aeeuti«al formulation comprising a compound sa claimed in any one of Claima 1 to lt>, in which Rj is hydrogen, or a pharmaceutically-acceptablo aalt thereof, associated with a phamaceutieally-acceptable carrier therefor. ;F. R. KELLY A CO., AGENTS FOR TIIE APPLICANTS. -64-