FI63035B - FAR OIL FRAMSTAELLNING AV 7-ALPHA-AMINOACYL-3-CHLORCEPHALOSPORIN DERIVATIVES - Google Patents

Description

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Patent- och ragistaratyraltan 'Amekan utiafd och utUkrifUn pubiicond 31.12.82 (32) (33) (31) Posted «bringUcMi -8t | W priority 23.02.73 USA (US) 335381 (Tl) Eli Lilly and Company, 307 East McCarty Street , Indianapolis,

Indiana, USA (72) Robert Raymond Chauvette, Indianapolis, Indiana, USA (7 ^) Oy Kolster Ab (5I +) Process for the preparation of 7-o £ -aminoacyl-3-chlorocephalosporin derivatives - Förfarande för framställning av 7- The present invention relates to a process for the preparation of Ί-έλ-aminoacyl-3-chloro-cephalosporins and their non-toxic, pharmacologically acceptable salts, which are valuable oral antibiotics.

Several 7 - (/ - aminoacylcephalosporin antibiotics with various substituents at the 3-position of the molecule are known, for example the well-known antibiotic cephexin, 7- (D - (N-phenylglycylamido) -3-methyl-cephem-U-carboxylic acid, deacetoxycephalosporan) At the 3-position is a methyl group, the antibiotic cephaloglycine, 7- (D- (N-phenylglycylamido) -3-acetoxymethyl-3-cephem-β-carboxylic acid, a cephalosporanic acid having an acetoxymethyl group at the 3-position, and β-aminoacylcephalosporins); substituted with thiadiazolithiomethyl or tetrazolithiomethyl, all of which are valuable drugs, and several cephalosporin compounds having a 3-substituent as a 3-halomethyl group have been described, e.g. valuable intermediates in the preparation of cephalosporin antibiotics prepared in accordance with this invention chloro-substituted cephalosporin compounds are structurally unique compounds of the cephalosporin group because the chlorine atom is directly attached to the 2,63035 carbon at the 3-position of the dihydrothiazine ring. Compared to previous halogen derivatives of cephalosporins, for example those described above, the halogen atom is attached to the methylene group of the carbon atom in the 3-position of the dihydrothiazine ring. Thus, the new compounds combine the special advantages of the 7- <λ-aminoacyl attached to the cephalosporin nucleus and the chlorine atom substituting at the 3-position of the nucleus.

More particularly, the invention relates to a process for the preparation of 7 -? - aminoacyl-3-chlorocephalosporin derivatives useful as orally active antibacterial agents having the formula

HÖH Q

R-C-ί-Ν - / \ ^ 2 --N Cl (I)

COOH

wherein R is phenyl, hydroxyphenyl or halophenyl.

"Hydroxyphenyl" means mono- and dihydroxyphenyl groups, e.g., k-hydroxyphenyl, 3-hydroxyphenyl, 2-hydroxyphenyl, 3, 4-dihydroxyphenyl, and 2, U-dihydroxyphenyl. "Halophenyl" means mono- and halogen-substituted phenyl groups in which halogen is fluorine, chlorine or bromine, e.g. U-fluorophenyl, h-chlorophenyl, N, N-dichlorophenyl, 3-chlorophenyl, 2-chlorophenyl, 3-bromophenyl or U-bromophenyl.

The invention is characterized in that 7-amino-3-chloro-3-cephem-1-carboxylic acid or an ester thereof having the formula v-rY'j "-γΑ" ™ C00R1 having hydrogen or a carboxylic acid protecting group which is benzyl , p-methoxybenzyl, p-nitrobenzyl, diphenylmethyl, 2,2,2-trichloroethyl, trimethylsilyl or t-butyl, is reacted with a carboxylic acid of formula

B

R-C-COOH

NH

R 2 wherein R is as defined above and R 1 is hydrogen or an amino protecting group selected from the group consisting of t-butyloxycarbonyl, benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, trichloroethoxycarbonyl or trityl or methylacetoacetate and acetylacetate, provided that groups and cannot simultaneously denote hydrogen, or a reactive derivative thereof, and that the carboxylic acid and amino protecting groups are removed.

In the above formula, the ester groups represented by R 1 are all known esters commonly used in cephalosporin chemistry to protect the C 1-4 carboxyl group of a cephalosporin molecule when performing reactions that require other reactive sites on the molecule. Esters of formula I (R 2 other than hydrogen) are prepared by methods in which the same ester groups are used to protect the C 1-4 carboxyl groups of known cephalosporins.

"Pharmacologically acceptable non-toxic salts" means both C 1-4 carboxylic acid salts and acid addition salts of the amino group of the 7-glycylamide side chain. Pharmacologically acceptable salts having a C 1-4 carboxylic acid function are those formed with inorganic bases, for example the sodium, potassium and calcium salts, which can be prepared by means of sodium bicarbonate, potassium carbonate, calcium hydroxide or sodium hydroxide. Pharmacologically acceptable amine salts can also be prepared with organic amines, for example dicyclohexylamine, benzylamine, 2-aminoethanol, diethanolamine or diisopropylamine. Acid addition salts of the <7C-amino group include salts formed with mineral acids, for example hydrochlorides, hydrobramides and sulphates, and with organic sulphonic acids, e.g. p-toluenesulphonate.

It is readily apparent that when R 1 is hydrogen, intramolecular salt formation may cause zwitterion formation of the compounds.

Because the cA aminoacyl group has an asymmetric carbon atom, the 3-chlorocephalosporins described above include the D, L, and DL forms. Form D is the preferred isomeric form of the present invention.

Examples of β-aminoacyl-3-chloro-cephalosporins of formula I described above are: 7- (D-phenylglycylamido) -3-chloro-3-cephem-U-carboxylic acid 7- (D-3-hydroxyphenylglycylamido) -3- Chloro-3-cephem-U-carboxylic acid 7- [D-2- (2-thienyl) -glycylamido] -3-chloro-3-cephem-U-carboxylic acid 7- [P-2- (3-thienyl) -glycylamide] 3-Chloro-3-cephem-U-carboxylic acid and zwitterionic forms and pharmacologically acceptable salts.

The compounds of formula I and their pharmacologically acceptable non-toxic salts are valuable antibiotics in the control of infections caused by 4,63035 gram-positive and gram-negative microorganisms in warm-blooded mammals. They are effective when administered parenterally, e.g., subcutaneously or intramuscularly, and equally well when administered orally.

7- (t-Aminoacyl-3-chlorocephalosporins have a broad spectrum of antibacterial activity, as shown by the in vitro spectrum of 7- (D-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid in the following tables. .

Table I shows the minimum inhibitory concentrations (MICs) in micrograms per milliliter (yg / ml) achieved with this compound in a standard agar dilution assay.

Table I

In vitro spectrum of 7- (D-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid

Organisms4 ^ MIC

___ (yq./ml.)

Staphylococcus aureus 3055 1

Staphylococcus aureus 3074 1

Streptococcus faecalis X66 16

Proteus morganii PH15> 128

Salmonella typhosa SA12 <.5

Klebsiella pneumoniae KL14 .2

Enterobacter aerogenes EB17 8

Serratia marcescens SE3> 128

Escherichia coli EC14 2

Citrobacter freundii CF17> 128

Pseudomonas aeruginosa X239> 128

Bordetella bronchiseptica 16 64

Salmonella typhimurium 1

Pseudomonas solanacearum X185> 128

Erv; inia amylovora 1 + Numbers and / or letters following the name of the organism indicate strains.

5,63035

Table II below shows a standard rotary plate test for 7- (D-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid. the diameter of the growth inhibition zone in millimeters achieved with microorganisms.

Table II

blocking zone

Microorganism (diameter mm)

Concentration (mg / ml)

Staphylococcus auraus 35 28 20

Bacillus subtilis 44 32 21

Sareina lutea 48 27 27

Mycobacterium avium 24

Proteus vulgaris 23 -

Salmonella gallinarum 35 25 13

Escherichia coli 30 20 11

Klebsiella pneumoniae 28 20 12

Pseudomonas solanacearcum 32 23 + Traces of the zone with a dash (-) el detectable zone.

63035 6

M1C values for 7- (D-phenrylglycylanido) -3-chloro-3-cephene-4-carboxylic acid in an agar expansion experiment in Table III oa for a spectrum of graphitopositive and graphanegative microorganisms *

Table III

In vitro antibiotic spectrum of 7- (β-phenylglycyl) -3-chloro-3-ceph. -4-carboxylic acid.

Minimum inhibitory concentration

Tissue Organisms *) MIC Jig./ml.

Staphylococcus aureus 3055 1.0 gets 3123 1.0 gets * 3074 2.0

Streptococcus (ryha D) 9901 64

Enterobacter cloacae EB9> 128

Enterobacter aerogenes EB17 64

Escherichia coli EC14 1.0 the word EC35 2.0 gets EC38 1.0

Klebsiella sp. KL3 <0.5 eaaa KL · 14 2.0 and KL25 2.0

Proteus mirabilis PR6 1.0

Proteus morganii PRl 128

Proteus rettgeri PR9. > 128 can * PR2> 128

Salmonella SA12 <0.5

Shigellr. mp. SH3 2.0 + Post-organ differences and / or lettering mean strains.

7 63035

In a standard agar dilution experiment, 7 ”(D-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid with MiC values of 1-4 pg / ml was effective against many strains of Hemophilus indluenzae.

The following Table IV shows the oral absorption of 7- (D-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid by the blood and urine levels of mice. · In the experiment, McAllister Swiss mice weighing 11-13 g were fasted overnight and they were then orally administered 20 mg / kg of 7- (D-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid. Blood and urine samples were taken at the intervals described and the antibiotic concentration of each sample was determined microbiologically using Sareina lute at pH 6.0 in a round plate agar assay.

Table IV

7- (D-fenyyliglysyyliamido) -3-chloro-3-cephem-4-carboxylic acid

Blood and urine content

Mouse No. Concentration} μg / ml at time intervals (min.) 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 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 blood 23.4 14.8 10.5 4.6 2.0 1.4 0.6

Mean combined urine - 1474 1764 843 305 308 226

A typical effective dose of the compounds of the invention for (ED 2) 7- (H-phenyl-glycylamido) -3-chloro-3-cephem-4-carboxylic acid for Streptococcus pyogenes is 0.74 mg / kg twice oral and 0.46 mg / kg twice daily. kg subcutaneously, 5 x 5 ®g / kg twice oral for Escherichia coli and 17 to 6 mg / kg twice oral for Diplococcus pneumoniae in rats.

Table V below shows the minimum inhibitory concentrations (MICs) of the 3-chloro-3-cephem compounds of the invention for some typical bacteria. Inhibition concentrations were determined by the gradient plate experiment 8 63035 avails, which essentially followed the method described by Brijson and Szybalski, Science, 116, 45 (1952). In Table V R refers to the structural formula.

Table V

Antibiotic activity of substituted 7- (phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acids against gram-negative bacteria.

rvcH-IL-f "S

COOH

Minimum inhibitory concentration

Tissue organs (pg / ml)

R 3 -OH 4-OH 4-Cl H

Shigella sp. 1.0 2.0 11.36.7

Escherichia coli 1.0 2.0 17.2 5.7

Klebsiella pneumoniae 0.9 1.5 8.0 0.9

Acrobacter aerogens 0.8 1.0 5.0 0.8

Salmons13 and Heidelberg 0.5 0.8 10.7 0.2

Pseudomonas aeruginosa> 200> 200> 200> 200

The following Table VI shows the efficacy of typical 3'-chloro-3-cephem compounds on various clinically isolated penicillin-resistant staphylococci. Efficacy is expressed as lower inhibitory concentrations of test compound. The minimum inhibitory concentration was determined by a gradient plate test.

9 63035

Table VI

Antibiotic activity of substituted 7- (phenylglycylamido) -3-chloro-3-cephem-1 + carboxylic acids against penicillin-resistant staphylococcus.

/ - \ 2H

\ V_ CH-C-N - ^ _ 'nh2 Jci

COOH

Staphylococcus - Minimum inhibitory concentration in Kantal) (μg / ml)

R 3 -OH 1 + -OH 1 + -C 1 H

Vl + 1 3.0 / 5.0 / 10.5 /> 20 11.5 /> 20 V32 3.5 / 7.0 / 18 /> 20 17 /> 20 XU00> 20 /> 20 /> 20 /> 20> 20 /> 20 V8U 0.5 / 0.6 / 3.0 /> 20 2.7 / 15

Xl.l 0.1 + / 0.6 / 0.1 + / 0.7 0.1 + / 1.0 1) Letters and numbers refer to clinically isolated, penicillin-resistant staphylococcal strains.

2) In the columns, the value before the slash indicates the lowest inhibitory concentration without human blood. A possible value after the slash indicates the lowest inhibitory concentration in the presence of human blood serum.

10 63035

Table VII

Biological body tests

Oblique culture MIC pg / ml

Shigella E. K. A. Sai. Ser. Penicillin m.p. coli pneu- aero- heidel- mar- resistant to manye genes Berg cescens Staph.

Cefaclor 0.7 0.8 0.6 0.6 0.5 21.5 3.0 Cephalexin 15.0 13.5 6.9 5.2 6.3 200 6.2

The novel compounds are prepared by N-acylation of 7-amino-3-chloro-3-cephem-U-carboxylic acid or its ester, e.g. benzyl, p-methoxybenzyl, p-nitrobenzyl, diphenylmethyl, 2,2,2 -trichloroethyl, trimethylsilyl or tert-butyl ester.

For acylation, the amino group of glycine is protected, for example, as the hydrochloride salt or with conventional amino protecting groups, e.g. tert-butyloxycarbonyl, benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, trichloroethoxycarbonyl or trityl Activated derivatives of the carboxyl group of the substituted glycine may be acid halides, for example acid chlorides, activated esters, for example esters with pentachlorophenol, an azide or a mixed anhydride formed from glycine, methyl chloroformate or isobutyl chloroformate. Amino-protected glycine can also be used directly in the acylation of the desired 3-chloroester using, for example, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDK) as a condensing agent. For example, p-nitrobenzyl 7-amino-3-chloro-3-cephem-carboxylate is reacted with N- (tert-butyloxycarbonyl) -D-phenylglycine 63035 11 in a dry, inert solvent, e.g. tetrahydrofuran, to form p-nitrobenzyl 7- (D-fenyyliglysyyliamido) -3-chloro-3-cephem-U-carboxylate.

When acylating 7-amino-3-chloro-3-cephem-U-carboxylic acids or esters, any known amide coupling method can generally be used. When the activated derivative is an acid halide, the acylation is carried out in the presence of a hydrogen halide acceptor, for example sodium bicarbonate, pyridine, sodium bisulphite or an alkylene oxide, for example propylene oxide. When mixed anhydride is used in the acylation, such can be prepared using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDK). When N-protected phenyl or thienylglycine is used, the acylation can be carried out in the presence of a condensing agent, for example Ν, Ν'-dicyclohexylcarbodiimide.

Examples of substituted glycines used in the preparation of the compounds of the invention include D-phenylglycyl chloride hydrochloride, D-4-hydroxyphenylglycyl chloride hydrochloride, pentachlorophenyl-D-phenyl- (N-tert-butyloxycarbonyl) glycine, pentachloro, pentachloro 2-trichloroethoxycarbonyl) glycinate, N- (tert-butyloxycarbonyl) -D-phenylglycine, N- (1-carbomethoxy-2-propenyl) -D-phenylglycine, 3-thienylglycyl chloride hydrochloride, N- (tert-butyloxycarbonyl) 2-thienylglycine.

The acylation is performed in an inert solvent, for example acetone, acetonitrile, dimethylformamide or methylene chloride, and preferably at a temperature between about -20 ° and + 20 ° C. For example, 7- (E> -phenylglycylamido] -3-chloro-3-cephem-H-carboxylic acid is prepared by reacting D-phenyl-glycyl chloride hydrochloride with p-nitrobenzyl 7-amino-3-chloro-3-cephem-k-carboxylate. with acetonitrile in the presence of propylene oxide, then the ester group is removed by hydrogenolysis.

Examples of 7-amino-3-chloro-3-cephem-U-carboxylic acids and esters of the formula II used as starting materials are 7-amino-3-chloro-3-cephem-β-carboxylic acid, p-nitrobenzyl-7 "-amino-3 -chloro-3-cephem-U-carboxylate and diphenylmethyl 7-amino-3-chloro-3-cephem-U-carboxylate.

63035

7-Amino-3-chloro-3-cefine-n-carboxylic acids and esters of formula II can be prepared as follows. 7-Acylamidocephalosporanic acid or its ester is converted to 7-acylamido-3-exomethylenecepham-U-carboxylic acid or ester. The ester of the 3-exomethylene cefme compound is then oxidized with ozone to form the intermediate ozonide containing the 3-exomethylene group, which upon decomposition forms the 7-acylamido-3-hydroxy-3-cephem-U-carboxylic acid ester. The 3-hydroxy-3-cephem ester is then chlorinated to give the corresponding 3-chloro-3-cephem. The 7-acyl group of 3-chlorocephem is removed by a well-known side chain cleavage reaction using phosphorus pentachloride in pyridine to give a side chain iminochloride derivative. The iminochloride is reacted with an alcohol, for example methanol, to give the imino ether, which is easily hydrolysed to the 7-amino-3-chloro-3-cephem-1-carboxylic acid ester.

The chlorination is carried out by adding a halogenating agent to a solution of 3-hydroxy-3-cephem ester in dry DMF at a temperature of about 5 ° -15 ° C and allowing the reaction mixture to stand at room temperature for U-8 hours and longer. Initially, the reaction is exothermic, and initially the reaction vessel is cooled in an ice-water bath and the remainder of the reaction takes place at a temperature of about 25 ° C. Prior to use, the DMF is preferably dried using a molecular sieve. Although the reaction can be carried out in the presence of DMF as a solvent, another solvent can be used in addition to DMF. In addition to DMF, for example, tetrahydrofuran, dioxane, methylene chloride, dimethylacetamide or dimethyl sulfoxide can be used.

The 3-chloro-3-cephem esters are isolated from the reaction mixture by pouring the mixture into water-ethyl acetate and isolating the organic phase containing the product. The organic phase is washed, dried and evaporated to dryness to give 3-chloro-3-cephemester as an amorphous residue. In many cases, the product is made to crystallize by triturating the residue in ether or n-hexane.

Following the preparation of 7-acylamido-3-chloro-3-cephem-U-carboxylic acid esters described above, the corresponding 7-amino-3-chloro-3-cephem-U-carboxylic acid esters are obtained by performing the well-known N-deacylation of the 7-acylamide side chain. For example, 7-acylamido-3-chlorocephalosporin ester is reacted with phosphorus pentachloride in methylene chloride in the presence of pyridine to give the iminochloride intermediate. The iminochloride is then reacted with an alcoholic solvent, for example methanol or isobutanol, to give the corresponding iminoether. The iminoether is hydrolyzed to give 7-amino-3 '<' chloro-3-cephem-1 + carboxylic acid ester as its hydrochloride salt.

As previously mentioned, the 7-amino-3-chloro starting material can be acylated as either the free acid or the corresponding ester. The acylation of the 7-amino-3-chloro nucleus takes place as described above.

In one embodiment, the starting materials and novel compounds used in the invention are reacted with 7-phenoxyacetamidocephalosporanic acid to form an isothiouronium salt by removal of the acetoxy group at the 3-position of the dihydrotiazine ring of the cephalosporanic acid ring. The isothiouronium salt is then reacted with zinc and a 90% excess of formic acid in the presence of dimethylformamide at about 25 ° C to give 7-phenoxyacetamido-3-exomethylenecepham-U-carboxylic acid. 3-Exomethylenecefam carboxylic acid is esterified with p-nitrobenzyl bromide in the presence of a hydrogen halide acceptor to give 3-exo-methylenecepham-1-carboxylic acid p-nitrobenzyl ester. The 3-exo-methylene cefester is then ozonated in methylene chloride at about 7070 ° C and the ozonolysis mixture is treated with sulfur dioxide to decompose the ozonide intermediate to give p-nitrobenzyl 7-phenoxyacetamido-3-hydroxy-3-cephem-1 + carboxylate. The 3-hydroxy ester is then reacted with phosphorus trichloride in dry DMF to give p-nitrobenzyl 7-phenoxyacetamido-3-chloro-3-cephem-U-carboxylate. The 3-chloroester is then reacted in methylene chloride with phosphorus pentachloride in the presence of pyridine to give the iminochloride intermediate which is reacted with methanol to give the corresponding iminoether intermediate. Upon addition to the reaction mixture, the iminoether decomposes to form p-nitrobenzyl 7-amino-3-chloro-3-cephem carboxylate. The 7-amino-3-chloro-3-cephem-U-carboxylic acid ester can then be acylated as described above with D-phenylglycyl chloride hydrochloride or an amino-protected D-phenylglycine derivative to give 7- (L-phenyl-glycylamido) -3-chloro-3-cephem-3-cephem. After removal of the N-amino-protecting group and the N-carboxylic ester group, 7- (D-phenylglycylamido) -3-chloro-3-cephem-H-carboxylic acid is obtained after removal of the N-carboxylic acid p-nitrobenzyl ester or its N-protected derivative. From the above description of the preparation of the starting material, it is found that a number of known 7-acylamidocephalosporanic acids can be used in the preparation of the 7-amino-3-chloro-3-cephem-U-carboxylic acids described herein.

. lh 63035 A preferred compound prepared in accordance with this invention is one in which R is phenyl and its pharmacologically acceptable, non-toxic salts. 7- (D- [* -phenylglycylamido) -3-chloro-3-cephem-U-carboxylic acid is the preferred compound.

In another preferred group of compounds of this invention, R is hydroxyphenyl, as well as pharmacologically acceptable, non-toxic salts of the compounds. Examples of this group of compounds are: 7- (D- [7c-U-hydroxyphenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid and 7- (Dc? -3-hydroxyphenylglycylamido) -3-chloro-3-cephem -U-karhoksyyli acid.

A highly preferred antibiotic prepared in accordance with this invention is 7- (D-phenylglycylamido) -3-chloro-3-cephem-U-carboxylic acid.

Preparation of starting materials p-nitrobenzyl 7-amino-3-methylenecepham-U-carboxylate hydrochloride To a solution of 965 mg (2 mmol) of p-nitrobenzyl 7-phenoxyacetamido-3-methylenecepham-U-carboxylate in 10 ml methylene chloride was added 175 mg of dry pyridine and U60 mg of phosphorus pentachloride and the mixture was stirred for six hours at room temperature. To the mixture was added 1 ml of isobutanol and allowed to stand at 0 overnight. Reaction product p-Nitrobenzyl 7-amino-3-methylene cefb: m-1 + carboxylate hydrochloride precipitated as crystals, yield U30 mg (58%).

Elemental analysis C 1 H 2 N 2 O 2 SCl

Calculated: C, 1 * 6.69; H, H, 18; N, 10.89

Found: C, U6, 1 * 0; H, U, 20; N, 10.62 IR (in Nujol): carbonyl absorption 5.65 (β-lactam) and 5.75 (ester) microns.

NMR (DMSO d 6): signal 6.3 ** (2d, 2H, C 6 H 8), U, 98 (d, 1H, C 6 H 6), 1 * »7-1t, 1t (m, 6h, Cu H, ester CH 2, C 2 -C 6 and C 2 -H 2, 2, U-1.6 (m, Uh, aromatic H) tau.

p-Nitrobenzyl 7-amino-3-hydroxy-3-cephem-U-carboxylate hydrochloride

A solution of H g of p-nitrobenzyl 7-amino-3-methylenecepham-U-carboxylate hydrochloride in 620 ml of methanol was cooled in a dry ice-acetone bath and ozone was bubbled through the cold solution for about 20 minutes. Excess ozone was removed by bubbling nitrogen through the solution and 10 g of sodium bisulfite was added.

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

The mixture was evaporated in vacuo and the reaction product was obtained as a yellow, amorphous residue. This was crystallized from acetone to give 3.1 g of p-nitrobenzyl 7-amino-3-hydroxy-1-cephem-1-carboxylate hydrochloride as a crystalline acetone solvate.

IR (in Nujol): carbonyl absorption 5 »6θ β-lactam) and 6.0U (ester carbonyl hydrogen coupled to 3-hydroxy) microns.

NMR (DMSO d 6): signals 7.92 (s, 3H 1/2 mole acetone), 6.22 (2d, 2H, C 2 H 2), 5.07 (d, 1H, C 9 H) 1 +, 8-1 +.5 (m, 3H, ester CH2 and CH2 H), 2.1 + -1.6 (m, 1 + H, aromatic H) tau.

p-Nitrobenzyl 7-amino-3-chloro-3-cephem-1-carboxylate hydrochloride

To a solution of 500 mg of p-nitrobenzyl 7- [2- (2-thienyl) -acetamido] -3-chloro-3-cephem-1-carboxylate in 6 ml of methylene chloride were added 95 mg of dry pyridine and 237 mg of phosphorus pentachloride. The reaction mixture was stirred for 1.5 hours at room temperature, then cooled in an ice-water bath to about Sose and 0.6 ml of isobutyl alcohol was added. With continuous cooling and stirring, the reaction product p-nitrobenzyl 7-amino-3-chloro-3-cephem-1'-carboxylate hydrochloride crystallized from the reaction mixture. The product was filtered, washed with cold methylene chloride and dried to give 200 mg of crystalline product, m.p. about 168 ° C with decomposition.

Elemental analysis C ^ H ^ CIN ^ O ^ S. HC1

Calculated: C, Ui, 39; H, 3.20; N, 10.3U; Cl, 17, U5 Found: C, U.11 +; H, 3.31; N, 10.1 + 1 +; Cl, 17.29 IR (in Nujol): absorption 5> 55 (β-lactam carbonyl) and 5.78 (ester carbonyl) microns.

UV (pH 7 buffer): max. = 268 T '13 800 16 63035 NMR (DMSO d 6): signals 5.97 (s, 2H, C 6 H 8), (m, i + H, C 6 H, C 1 H and ester CH 2) and 2, 35-1.6 (q, 1 + H, aromatic H) tau.

7-amino-3-chloro-3-cephem-it-carboxylic acid

To a solution of 750 mg (1.85 mmol) of p-nitrobenzyl 7-amino-3-chloro-3-cephem-carboxylate hydrochloride in 20 ml of tetrahydrofuran and 10 ml of methanol was added a suspension of 750 mg a pre-reduced, $ 5 palladium-on-carbon catalyst in 20 ml of ethanol and the suspension was hydrogenated under hydrogen pressure for 3.5 minutes at room temperature. The catalyst was removed by filtration and washed with THF and water. The filtrate and catalyst washes were combined and evaporated to dryness. The residue was dissolved in water-ethyl acetate and the pH was adjusted to 3. The insoluble product was isolated by filtration and triturated with acetone. The product was dried to give 115 mg of 7-amino-3-chloro-3-cephem-carboxylic acid.

IR (in Nujol): absorption 5.6l (β-lactam carbonyl) and 6.2 (carboxylic acid).

NMR (D 6 O-NaHCO 3): signals 6.25 (ABq, 2H, C 6 H 8), δ, 88 (d, 1H, C 6 H 4) and i +, (d, 1H, C 6 H -H) tau.

UV (pH 7 buffer): \ Λβ. = 265 ¥ · £ - 7550 ·

Diphenylmethyl 7-amino-3-chloro-3-cephem-U-carboxylate To a solution of 525 mg of diphenylmethyl 7- [2- (2-thienyl) -acetamide] -3-chloro-3-cephem-U- carboxylate in 20 ml of methylene chloride were added 0.1 ml of dry pyridine and 237 mg of phosphorus pentachloride. The reaction mixture was stirred for two hours at room temperature and then cooled in an ice-water bath. To the cold mixture was added 0.6 ml of isobutanol and after 30 minutes the reaction mixture was evaporated. The residue was dissolved in ethyl acetate, the solution was washed with 5% sodium bicarbonate solution and water, and dried. The dried solution was evaporated to dryness and the residue triturated with ether to give 190 mg of 3-chloro-diester, diphenylmethyl 7-amino-3-chloro-3-cephem-carboxylate.

IR (in Nujol): absorption of 5.7 and 5.9 (β-lactam and ester carbonyl) microns.

NMR (CDCl 3): signals 6.35 (ABq, 2H, C 6 H 8), δ, 78 (2d, 2H, C 6 H and C 6 H), 3.05 (s, 1H, ester CH) and 2 .65 (s, 10H, aromatic H).

IT

63035

The following examples illustrate the invention.

Example 1 7- (D-N-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid

To a suspension of 280 mg (1.2 mmol) of 7-amino-3-chloro-3-cephem-4-carboxylic acid in 14 ml of acetonitrile was added 0.5 ml of N, O-bis- (trimethylsilyl) - acetamide to form the above soluble disilylmethyl derivative. The solution was cooled to 0 ° C and added slowly to a mixed anhydride solution formed by reacting 40 mg (1.5 mmol) of sodium methyl 3-G ** -carboxybenzylaminocrotonate and 16 mg (1.7 moles) of methyl chloroformate. in the presence of two drops of dimethylbenzylamine in 7 ml of acetonitrile. The mixture was stirred for two hours at ice bath temperature, 1 ml of methanol was added and the mixture was filtered to remove insoluble impurities. 2 ml of water was added to the filtrate, and the pH was adjusted briefly to 1.5 to remove the enamine blocking group and then to 4.5 with triethylamine. After stirring for 1 hour at ice bath temperature, the reaction product 7- (D- <9t-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid (zwitterion) precipitated from the reaction mixture as crystals. The product was filtered, washed with acetonitrile and dried in vacuo, yield 200 mg.

The product has the following physical characteristics:

Elemental analysis C ^ H ^ N ^ O ^ SCl. 1/2 H 2 O.

Calculated: C, 47.80; H, 4.01; N, 11.15; Cl, 9.40

Found: C, 1 + 7.55; H, 4.12; N, 10.98; Cl, 9.21 IR (in Nujol): absorption 2.9 (amide NH), 5.70 (β-lactam carbonyl), 5.95 (amide carbonyl) and 6.28 (carboxylate) microns.

MR (DgO / DCl): signals 6.5-6.7 (ABq, 2H, CgH), 4.84 (d, 1H, Cg-H), 4.26 (d. 1H, Ογ-H) and 2 .44 (s, 5H, aromatic H) tau.

UV (pH 7 buffer): = 265 nyi, £ 6800.

Example 2

To a solution of 500 mg (1.85 mmol) of sodium methyl 3-cX-carboxybenzylaminocrotonate (formed from phenylglycine and methyl acetoacetate) in 20 ml of acetonitrile was added 4 drops of dimethylbenzylamine, and the solution was cooled with stirring under dry ice-carbon tetrachloride. To the cold solution was slowly added 184 mg (1.95 mmol) of methyl chloroformate to form mixed anhydride. After 20 minutes, a precooled solution of 750 mg (1> 85 mmol) of p-nitrobenzyl 7-amino-3-chloro-3-cephem-4-carboxylate and 188 mg (1.85 mmol) of triethylamine was added. In 40 ml of acetone. The addition took three minutes and then the reaction mixture was stirred for 30 minutes in the cold and for two hours at room temperature. The reaction mixture was filtered to remove insoluble impurities and evaporated in vacuo. The reaction product residue was dissolved in a mixture of ethyl acetate and water, and the pH of the solution was adjusted to 7. The organic layer was isolated and washed with water. After drying over magnesium sulfate, the organic layer was concentrated in vacuo to a small volume. After the addition of n-hexane to the concentrate, 620 mg of p-nitrobenzyl-7- [N- (1-carbomethoxy-2-propanyl) -Da-phenylglycylamido] -3-chloro-3-cephem-4 'was precipitated from the reaction mixture. The carboxyl-acetate.

Elemental analysis and NMR spectrum of the product were as follows:

Elemental analysis for C27H26N4O8SC1

Calculated! C, 53.87? H, 4.35? N, 9.31

Found: C, 54.05? H, 4.13? N, 9.36.

HMR (DMSO d 6): signals 8.20 (s, 3H, enamine CH 2), 6.60 (ABq, 2H, 6.45 (s, 3H, ester CH 2), 5.48 (s, 1H, enamine vinyl) H), 4.90-4.1 (m, 5H, Cg-H, Ογ-Η, oc-CH and ester CHg) and 3.10-1.5 (m, 9H »aromatic H) tau.

The product, 540 mg (0.9 mmol) was dissolved in 40 ml of acetonitrile containing 20 ml of water and the solution was cooled in an ice-water mixture, briefly acidified to pH 1.5 and then the pH was adjusted to 2.5. and the residue was dissolved in 40 no tetrahydrofuran and 80 any methanol.

To the solution was added 540 mg of 5 * palladium-on-carbon catalyst (pre-reduced for 43 minutes at room temperature in 20 ml of ethanol under a hydrogen pressure of 3.5 aty) and the solution was hydrogenated for 2.5 hours at room temperature under a hydrogen pressure of 3.5 aty. The catalyst was removed by filtration and washed as a filter with methanol, THF and water. The filtrate and washings were combined and evaporated to dryness in vacuo. The reaction product residue was dissolved in a mixture of water and ethyl acetate and the pH of the solution was adjusted to 4.3. The aqueous phase was isolated, washed with ethyl acetate and evaporated to a volume of about two ml. On cooling, 65 mg of 7- (D-α-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid precipitated from the cold concentrate.

Example 3

To a suspension of 3.0 g (8.1 mmol) of p-nitrobenzyl 7-amino-19,63035 3-chloro-3-cephem-4-carboxylate in 200 ml of tetrahydrofuran (dried through a molecular sieve) was added 2.1 g (8.3 mmol) N- (tert-butyloxycarbonyl) -D- (x-phenylglycine) and 2.0 g (8.3 mmol) N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline ( EEDK). The reaction mixture was stirred at room temperature overnight and the solvent was evaporated in vacuo. The residue was dissolved in a mixture of ethyl acetate and water, and the organic phase was isolated. The organic phase was cooled and washed successively with cold 5% aqueous sodium bicarbonate solution, cold, $ 3 hydrochloric acid solution and water. The washed solution was dried over magnesium sulfate, filtered and concentrated by evaporation in vacuo to a volume of about 30 ml. 3.7 g of (63 #) p-nitrobenzyl 7 '[D-2- (tert-butyloxycarbamido) -2-phenylacetamido] -3-chloro-3-cephem-4-carboxylate were obtained as crystals from the concentrate. A second crop of product weighing about 2 g was obtained by concentrating the filtrate further after filtration of the first crop.

Elemental analysis C0 „H_„ C1N.0QS

<(40

Calculated: C, 53 * 78; H, 4.51; N, 9.29

Found: C, 52.66; H, 4 * 36; N, 8.88.

UV (acetonitrile): \ - 268 sec, e - 17 100.

IR. The following characteristic absorption peaks were observed in the IR spectra of the product: amide RH absorption peak 3 * 05 mu and carbonyl absorption peaks 5 * 59 »5 * 75 and 6.0 mp.

NMR (CDCl 3): signals 8.60 (s, 9H, t-BOC), 6.45 (ABq, 2H, C 6 H 6), 5.03 (d, 1H, C 6 H), 4.67 (s , 3H, α-CH, ester CH 2), 4.12 (m, 3H, C 7 -H, amide RH) and 2.72-1.74 (m, 10H, aromatic H, amide HH) -tau.

To a solution of 3.0 g (5 x 0 mmol) of the product in 15 mL of dry tetrahydrofuran (dried through a molecular sieve) and 185 mL of methanol was added 3 S of a pre-reduced 5% palladium on carbon catalyst. The catalyst was pre-reduced for 30 minutes at room temperature in ethanol under 3 * 5 aty hydrogen pressure. After addition of the reduced catalyst, the product was hydrogenated for one hour at room temperature under a hydrogen pressure of 3 * 5 aty. The catalyst was removed by filtration and washed as a filter with tetrahydrofuran and methanol. The filtrate and washings were combined and evaporated in vacuo. The residue was dissolved in ethyl acetate and water was added. The pH of the mixture was adjusted to 7 by the addition of 1N sodium hydroxide solution. The aqueous phase was isolated and washed with ethyl acetate. Ethyl acetate was then poured onto the aqueous phase and returned to pH 2.5 with 1 N hydrochloric acid. The organic phase was isolated from the aqueous phase, washed with water and dried over magnesium sulfate. The dried organic phase was evaporated to dryness in vacuo to give the reaction product 7- [D-2- (tert-butyloxycarbamido) -2-phenylacetamido] -3-chloro-3-cephem-4-carboxylic acid as a dry solid. The product was crystallized from 70 mL of ether and 20 mL of petroleum ether to give 1.75 g ($ 75) of crystalline product.

Elemental analysis for C20H22ClNj0gS

Calculated: C, 51 * 34 »H, 4» 74f N, 6.98} Cl, 7 * 58

Found: C, 51.02? H, 4.96; N, 8.75? Cl, 7.30.

UV (acetonitrile): \ maicB “268 mp * ε - 7400.

NMR (CDCl 3): signals 8.55 (e, 9H, t-BOC), 6.48 (ABq, 2H, C 6 H 8), 5.0 (d, 1H, C 6 H), 4.63 (d , 1H, α-CH), 4.25 (q, 1H, C2-H), 3.90 (d, 1H, amide NH) and 2.59 (β, 5H, aromatic Ξ) tau.

To a solution of 420 mg (2.2 mmol) of p-toluenesulfonic acid in 5 mL of acetonitrile was added 468 mg (1 mmol) of crystalline hydrogenolysis product. The solution was allowed to stand for about 16 hours at room temperature and then diluted with 0.5 ml of water. The pH of the solution was adjusted to 4.8, whereupon 329 mg (87%) of crystalline, liberated product 7- (D-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid precipitated from solution. The product was isolated by filtration and dried.

Elemental analysis C, C .CIN ^ O.S. l / 2 H „0 • O i4? 4 2

Calculated: C, 47.80; H, 4.01; N, 11.15? Cl, 9.40

Found: C, 48.04? H, 3.82? N, 11.18? Cl, 9.70.

Example 4

The antibiotic compound 7- (D-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid was also prepared from p-nitrobenzyl-7- [1 -2- (tert-butylocarbamido) -2-phenylacetamido] -3 -chloro-5-cephem-4-carboxylate by a method alternative to the release method of the previous example. In this method, the tert-butyloxycarbonylamino protecting group is first removed and then the p-nitrobenzyl ester group is cleaved with zinc and hydrochloric acid to give dimethylformamide. This alternative preparation is described in the following chapters.

To a solution of 2.4 g (12.6 mmol) of p-toluenesulfonic acid in 60 mL of acetonitrile was added 3.6 g (6 mmol) of p-nitrobenzyl-7- [B-2- (tert-butyloxycarbamido) -2 phenylacetamido] -3-chloro-3-cephem-4-carboxylate. The reaction mixture was stirred for about 15 hours at room temperature, during which time the amino-liberated product p-nitrobenzyl 7- (L-phenylglycylamido) -3-chloro-3-cephem-4-carboxylate precipitated as a crystalline p-toluenesulfonic acid salt.

21 63035

The product was filtered, washed with acetonitrile and dried in vacuo, yield 3.1 g ($ 81).

Elemental analysis C ^ Hg ^ ClN ^ O ^ S ,,

Calculated: C, 51.58? H, 4.06? N, 8.29? Cl, 5.25

Found: C, 51.51 * H, 4.14 »E, 8.12? Cl, 5.60.

IE (chloroform): carbonyl absorption peaks 5.61, 5.80 and 5 * 95 mu, toeylate salt absorption peak 6.29 ml.

Δ (DMSO dg): signals 7.70 (s, 3H, p-toluenesulfonic acid salt-CH 2), 6.61 (s, 3H, side chain BH 2), 6.20 (ε q, 2H, C 6 H 8), 4.94 (broad singlet, 1H, α-CH), 4.80 (d, 1H, Cg-H), 4.51 (s, 2H, ester CH2), 4.08 (q, 1H, Ογ-Ξ ), 2.95-1.62 (m, 14H, aromatic H and amide EH) and 0.32 (d, 1H, amide MH).

The amino-liberated p-toluenesulfonic acid salt obtained as described above was de-esterified as follows to obtain an antibiotic compound. A solution of 1.5 g (2.2 mmol) of the amino-liberated p-toluenesulfonic acid salt obtained above in 10 ml of dry dimethylformamide (dried through a molecular sieve) was cooled in an ice-alcohol bath. To the cold solution was added 2 ml of concentrated hydrochloric acid. To the cold solution was added portionwise 400 mg (6.1 mmol) of zinc dust over about 15 minutes. The reaction mixture was stirred in the cold for 30 minutes and then allowed to warm to room temperature while stirring. The reaction mixture was stirred for about an hour at room temperature, then filtered. The pH of the filtrate was adjusted to 6.8 with triethylamine. The product 7- (D-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid precipitated as white crystals of the zwitterionic bisdimethylformamide solvate. The product was filtered, washed with 10 ml of cold dimethylformamide and 6 ml of diethyl ether. The washed product was dried in vacuo, yield 800 mg ($ 71).

Elemental analysis G21S28C1N5 ° 6S *

Calculated: C, 49.07? H, 5.49? E, 14.63? Cl, 6.90

Found: C, 48.84? H, 5.53? E, 13.48? Cl, 7.18.

UV (acetonitrile): γ-265 mji e-6000.

Electrometric titration (66 DMF aqueous solution) pKft 4.55 and 7.2.

EMS (D 6 O / DCl): signals 6.34 (2β, 6H, DMP-CH 2), 6.33 (ABq, 2H, C 6 H 8), 4.85 (d, 1H, C 6 H 6), 4.64 (s, 1H, α-CH), 4.27 (d, 1H, O 2 -Η), 2.41 s, 5Ξ, aromatic H) and 1.84 (e, 2H, LMF-CH) tau.

22 63035

Example 5 7- (1H-Hydroxy-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid

2 * 9 g (1 mmol) of N- (tert-butyloxycarbonyl) -D-3-hydroxyphenylglycine, 3.7 g (10 mmol) of p-nitrobenzyl-7-amino-3-chloro-3-cephem were reacted. -4-carboxylate and 2.6 g (10.5 mmol) of coupling reagent EEDK. The reaction was performed and the product was isolated by the methods described in connection with the acylation procedure of Example 3. After trituration with ether, 2.8 g (46 l) of an amorphous solid were obtained.

Elemental analysis Cg ^ Eg ^ ClN ^ O ^ S

Calculated: C, 52.39; H, 4 * 40 $ N, 9.05

Found: C, 52.16; E, 4.59; N, 8.79 * UV (acetonitrile): λmax <- 270 ap, ε - 17,200.

NMR (CDCl 3): signals 8.59 (s, 9H, T-BOC), 6.50 (ABq, 2H, C 6 -G 6), 5.06 (d, 1H, C 6 -H), 4.66 (s , 1H, α-CH), 4.09 (m, 2H, C 2 -B), 3.54-1.70 (m, 9H, aromatic H, amide NE).

3.5 g (5.6 mmol) of p-nitrobenzyl-7- [D-2- (tert-butyloxycarbamido) -2- (3-hydroxy) phenylacetamido] -3-chloro-3-cephem The 4-carboxylate was hydrogenated in ethanol in the presence of a pre-reduced, 5 # * eta palladium carbon catalyst following the de-esterification procedure of Example 3. The product 7- [D-2- (tert-butyloxycarbamido) -2- (3-hydroxy) -phenylacetamido] -3-chloro-3-cephem-4-carboxylic acid was obtained as crystals by trituration of the amorphous crude product in hexane-diethyl ether solution, yield 1, 5 g (55 °).

UV (acetonitrile): γ Tna ^ g ^ 272 myi, ε - 8280

Electrometric titration ($ 66 IMF-water solution) pK 4.5 ·

1.3 g (2.7 mmol) of the above product were reacted with 1.1 g (5.9 mmol) of p-toluenesulfonic acid in 28 ml of acetonitrile to remove the tert-butyloxycarbonyl protecting group. Removal was performed essentially according to the method of Example 3.

The product 7- (D-3-hydroxyphenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid crystallized from the reaction mixture and was filtered and dried in vacuo, yield 700 mg ($ 64),

Elemental analysis C ^ B ^ ClNjO ^ S. 1 EgO

Calculated: C, 44.63; H, 4.01; N, 10.46.

Found: C, 45.12; H, 4.06; N, 10.31 * 63035 23 UV (pH 6 buffer): ^ malrp * ° 268 myi, ε »9750.

HMR (D 6 O / DCl): signals 6.31 (ABq, 2H, C 6 H 8), 4.81 (d, 1H, C 6 H 8), 4.52 (s, 1H, α-CH), 4.26 (d, 1H, C

Example 6 7- (D-4-Chloro-phenylglycylamido) -3-chloro-3-chloro-4-carboxylic acid

Following the procedure of Example 3, 830 mg (2.3 mmol) of p-nitrobenzyl 7-affino-3-chloro-3-cephem-4-carboxylate was acylated with 700 mg (2.5 mmol) of N-tert-butyloxycarbonyl. -D-4-chlorophenylglycine and 367 mg (by 2.3 mmol) of EEDK to give 1.2 g of p-nitrobenzyl-7- [D-2- (tert-butyloxycarbamido) -2- (4-chlorophenylacetamido) ] -3-chloro-3-cephem-4-carboxylate.

1.2 g (1.9 mmol) of the product were hydrogenated at room temperature in the presence of a pre-reduced 3 * palladium-on-carbon catalyst to remove the p-nitrobenzyl ester group to give 450 mg of 7- [D - 2- (tert-butyloxylurea) as crystals. ) -2- (4-chlorophenylacetamido)] - 3-chloro-3'-cephem-4-carboxylic acid. The product was obtained as crystals by trituration of the amorphous reduction product in diethyl ether.

Elemental analysis ^ Q ^ g ^ ClgNjOgS

Calculated: C, 47.82; H, 4.21; H, 8.36; 01, 14.11.

Found: C, 47.75; H, 4.43l N, 8.11; Cl, 14.15 ·

Electrometric titration (66 56 aqueous DMF) pK 4.4, apparent molecular weight 508, calculated molecular weight 302.

430 mg (0.9 mmol) of the de-esterified product were reacted with p-toluenesulfonic acid in acetonitrile following the procedure of the previous examples to remove the tert-butyloxycarbonyl protecting group to give 160 mg (44 #) of crystalline 7- (D'4-chlorophenylglylamido). 3-Chloro-3-cephem-4-carboxylic acid in zwitterionic form.

Elemental analysis ° 15H15012W · 1 H2 °

Calculated: C, 42.86; H, 4 * 59; N, 9.99; 01, 16.87 *

Found: C, 43-07; H, 3.63; N, 9.69f Cl, 16.75 * UV (pH 6 buffer): γ Wfl1rff? 265 myi, ε - 8100 \ max. - 225> μ, ε - 13 900.

Electrometric titration (66 i »aqueous DMF) pKft 4.15 μm 6.8, apparent molecular weight 407, calculated molecular weight 403 * 2k 63035

Example 7 7- (D-4-Hydroxy-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid

2.9 g (11 mmol) of N- (tert-butyloxycarbonyl) -D-4-hydroxyphenylglycine were reacted with 5 * 7 g (10 mmol) of p-nitrobenzyl-7-amino-5 'chloro-5 -cephem-4-carboxylate and 2.6 g (10.5 mmol) of coupling reagent EEM in dry tetrahydrofuran. The reaction was performed and the product was isolated by the methods described in connection with the acylation procedure of Example 3. Product 5 »7 g (yield 60 i» 5 was obtained as crystals from cold diethyl ether.

The product p-nitrobenzyl 7- [D-2- (tert-butyloxycarbamido) -2- (4-hydrocyano) phenylacetamido] -3-chloro-5-cephem-4-carboxylate had the following percentage Elemental analysis and HMR- spectrum.

Elemental analysis .0nDCl £ (cf 47

Calculated: C, 52.39} H, 4.40} N, 9.05 ·

Found: C, 52.12} H, 4.26} N, 8.91.

NMR (DMSO d 6): signals 8.62 (s, 9Hf t-BOC-CH 2), 6.16 (ABq, 2H, C 2 -H 2), 4.81 (d, 1H, C 6 H 6), 4.75 (d, 1H, α-CH), 4.55 (β, 2H, ester CH2), 4.18 (q, 1H, C1-H), 7.04 and 2.0 (2q, 8H, aromatic H ), 0.76 (d, 1H, C 1 H-NH) and 0.58 (s, 1H, p-OH) tau.

2.2 g (5.5 mmol) of product were hydrogenated in ethanol in the presence of a pre-reduced 5% palladium on carbon catalyst to remove the p-nitrobenzyl group. De-esterified product 7- [D-2- (tert-butyloxycarbamido) -2- (4-hydroxy) -phenylacetamido] -3-chloro-5-cephem-4-carboxylic acid was obtained as crystals from a mixture of diethyl ether and hexane. gram, corresponding to a yield of $ 59>,

Elemental analysis for C20H22ClN3 ° 7®

Calculated: C, 49.64} H, 4.58} N, 8.08.

Found: C, 48.92} H, 4.40} N, 8.24 * NMH (DMSO d 6): signals 8.61 (s, 9H, t-BOC-CH 2), 6.26 (ABq, 2H, 4.89 (*, 1H, cg, H), 5.06 (q, 4H, aromatic H) and 1.20 (d, 1H, C1-NH) tau.

The t-BOC protecting group was removed from the de-esterification product by reacting the product with p-toluenesulfonic acid in acetonitrile * One gram of product gave 350 mg (40,966) of the final product 7- (D-4-hydroxy-phenylglycylamido) -3-chloro-3 * cephem -4-Carboxylic acid with elemental analysis, UMR spectrum and titration value as follows: 63035 25

Elemental analysis C ^ H ^ ClN ^ Oj-S. 1 HgO

Calculated: C, HL, 83; H, H, 01; N, 10, Π6.

Found: C, ΠΠ.92 ;, H, 3.5; N, 10.63.

Electrometric titration (66% aqueous DMF) pK values H, 2, 7.7 and 12, H, Apparent molecular weight 3H, calculated molecular weight 383.8.

NMR (D 2 O / DC 1): signals 6.32 (ABq, 2H, C 6 H 8), H, 8H (d, 1H, C 6 H), H, 27 (d, 1H, C-H) and 2.79 (q, Hh, aromatic H) tau.

Claims (1)

26 63035 Patenting: Process for the preparation of 7- (N-aminoacyl-3-chlorocephalosporin derivatives useful as orally active antibacterial agents of the formula HÖH 1 11 1 RCCN ---- XN, 1 n NH JN J-Cl (I) COOH wherein R is phenyl, hydroxyphenyl or halophenyl, characterized in that 7-amino-3-chloro-3-cephem-1-carboxylic acid or an ester thereof having the formula vh- --- KJ-Cl (II) ο'C00R1 wherein R1 is hydrogen or a carboxylic acid protecting group which is benzyl, p-methoxybenzyl, p-nitrobenzyl, diphenylmethyl, 2,2,2-trichloroethyl, trimethylsilyl or t-butyl is reacted with a carboxylic acid which the formula is H RC-COOH NH * 2 wherein R is as defined above and R 1 is hydrogen or an amino protecting group selected from t-butyloxycarbonyl, benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, trichloroethoxyoxybonyl or trityl or methylacetoacetate and acetylacetate provided that both R 1 and R 2 cannot simultaneously represent hydrogen, or a reactive derivative thereof, and that the carboxylic acid and amino protecting groups are removed. 27 Patentkrav: ^ ^ O 3 5 Forfar and e för frstalstalining av 7 - ^ - ami noacyl-3-chlororeef alospori n-derivat, vilka är användbara bäsom oralt aktiva antibacteriella ämnen oc vilka har formeln HOE I I! is RCCN - S \ II NH2 -N-C1 (I) COOH color R is phenyl, hydroxyphenyl or halophenyl, the title compound is treated with 7-amino-3-chloro-3-cephem-U-carboxylic acid or ester ester ν- | -γ \ - “ύ ^ -01 (II) C00R1 color Rj är väte eller en skyddsgrupp for carboxylsyra, vilken utgörs av benzyl, p-methoxybenzyl, p-nitrobenzyl, diphenylmethyl, 2,2,2-trichloroethyl, trimethylsilyl or t-butyl, amylated with carboxylic acid in the form of H RC-C00H NH R2 color R is an angylic acid and R2 is used as an amino acid in the form of t-butyloxycarbonyl, benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, trichloroethylcarbonyl, trichloro and most preferably with methyl acetoacetate and acetylacetate, under the action of the group R or octyl group, the same or more reactive derivatives of carboxylic acid and amino acid *