DK153157B - METHOD OF ANALOGUE FOR THE PREPARATION OF 7-ALFA-AMINOACYL-3-HALOGEN-CEPHALOSPORINE DERIVATIVES OR PHARMACEUTICAL ACCEPTABLE ACID ADDITION SALTS. - Google Patents

Description

DK 153157 BDK 153157 B

Den foreliggende opfindelse- angår en analogifremgangsmåde til fremstilling af hidtil ukendte 7-a-aminoacyl-3-halogen-cephalo-sporiner med den i kravets indledning angivne formel I og ikke-toxiske farmaceutisk acceptable salte deraf, hvilke forbindelser kan anvendes som værdifulde, oralt effektive antibiotika. Fremgangsmåden ifølge opfindelsen er ejendommelig ved det i kravets kendetegnende del anførte.The present invention relates to an analogous process for the preparation of novel 7-α-aminoacyl-3-halo-cephalo-sporins of the formula I and the non-toxic pharmaceutically acceptable salts thereof as set forth in the preamble, which compounds can be used as valuable oral agents. effective antibiotics. The process according to the invention is characterized by the characterizing part of the claim.

Der kendes adskillige 7-a-aminoacyl-cephalosporinantiobiotika med forskellige substituenter i 3-stilling i molekylet. For eksempel kan nævnes det velkendte antibiotikum cephalexin, 7-(D-a-phenyl-glycylamido)-3-methyl-3-cephem-4-car.boxylsyre> der er en desace-toxvcephalosporansyre med en methylgruppe i 3-stillingen, anti-biotiket cephaloglycin, 7-(D-a-phenylglycylamido)-3-acetoxymethvl-Several 7-α-aminoacyl-cephalosporin antibiotics are known with various 3-position substituents in the molecule. For example, the well-known antibiotic cephalexin 7- (Da-phenyl-glycylamido) -3-methyl-3-cephem-4-carboxylic acid is a desacetoxycephalosporanoic acid having a methyl group at the 3-position, the anti-biotic Cephaloglycine, 7- (Da-phenylglycylamido) -3-acetoxymethyl

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3- cephem-4-carbo:xylsyre, der er en cephalosporansyre med en acet-oxymethylgruppe i 3-stillingen, οσ endelig a-aminoacylcephalospo-riner, hvori 3-stillingen er substitueret med en thiadiazol-thio-methyl· eller tetrazolthiomethyl-del, og de er alle værdifulde terapeutiske midler.3- cephem-4-carboxylic acid, which is a cephalosporanoic acid with an acetoxymethyl group at the 3-position, or finally α-aminoacylcephalosporins, wherein the 3-position is substituted with a thiadiazole-thiomethyl or tetrazolthiomethyl moiety , and they are all valuable therapeutic agents.

ligeledes er der beskrevet et antal cephalosporinforbindelser, hvori molekylet er substitueret i 3-stillingen med en 3-halogen-methylgruppe, f.eks. 3-brom-methyl-3-cephem-4-oarboxylsyreestere.Also disclosed are a number of cephalosporin compounds wherein the molecule is substituted at the 3-position by a 3-halo-methyl group, e.g. 3-bromo-methyl-3-cephem-4-oarboxylsyreestere.

De beskrevne 3-halogenmethylcephalosporinestere er nyttige mellemprodukter til fremstilling af cephalosporinantibiotika.The described 3-halo methyl cephalosporin esters are useful intermediates for the preparation of cephalosporin antibiotics.

Halogencephalosporinforbindelseme, der tilvejebringes ved fremgangsmåden ifølge opfindelsen, er strukturelt enestående forbindelser inden for cephalosporinerne, idet halogenatomet er knyttet direkte til carbonatomet i 3-stillingen i dihydrothiazinringen.The halo-cephalosporin compounds provided by the process of the invention are structurally unique compounds within the cephalosporins, the halogen atom being directly linked to the carbon atom at the 3-position of the dihydrothiazine ring.

I modsætning hertil har de kendte halogenderivater af eephalo-sporiner, såsom de, der er beskrevet ovenfor, et halogenatom på en methylengruppe knyttet til carbonatomet i 3-stillingen i dihydrothiazinringen. De omhandlede forbindelser har en kombination af karakteristika, der omfatter en 7-a-aminoacyldel knyttet til en cephalosporinkeme, der er substitueret i 3-stillingen med et chlor-eller bromatom. 1 den i kravet angivne almene formel I refererer hydroxyphenyl til mono- og dihydroxyphenylgrupper, såsom 4-hydroxyphenyl, 3-hydroxyphe-nyl, 2-hydroxyphenyl, 3,4-dihydroxyphenyl og 2,4-dihydroxyphenyl. Ha-logenphenyl refererer til mono- og di-halogen-substituerede phenyl-grupper, hvori halogen refererer til fluor, chlor og brom, såsom 4- fluorphenyl, 4-chlorphenyl, 3,4-dichlorphenyl, 3-chlorphenyl, 2-chlorphenyl, 3-bromphenyl og 4-bromphenyl. Methylphenyl refererer til mono- og dimethylerede phenylgrupper, såsom isomere 2-, 3- og 4-methylphenylgrupper og dimethylphenylgrupper, såsom 3,4-dimethylphenyl og 2,4-dimethylphenyl. Betegnelsen methoxy-phenyl refererer til mono- og di-methoxylerede phenylgrupper, såsom 4-methoxyphenyl, 3-methoxyphenyl, 2-metho'xyphenyl, 3}4-dimethoxyphenyl og 2,6-dimethoxyphenyl.In contrast, the known halogen derivatives of eephalo-sporins, such as those described above, have a halogen atom on a methylene group attached to the carbon atom at the 3-position of the dihydrothiazine ring. The compounds of the present invention have a combination of characteristics comprising a 7-α-aminoacyl moiety attached to a cephalosporin nucleus substituted at the 3-position by a chlorine or bromine atom. In the general formula I as claimed, hydroxyphenyl refers to mono- and dihydroxyphenyl groups, such as 4-hydroxyphenyl, 3-hydroxyphenyl, 2-hydroxyphenyl, 3,4-dihydroxyphenyl and 2,4-dihydroxyphenyl. Halogen phenyl refers to mono- and di-halogen-substituted phenyl groups, wherein halogen refers to fluorine, chlorine and bromine, such as 4-fluorophenyl, 4-chlorophenyl, 3,4-dichlorophenyl, 3-chlorophenyl, 2-chlorophenyl, 3-bromophenyl and 4-bromophenyl. Methylphenyl refers to mono- and dimethylated phenyl groups such as isomers 2-, 3- and 4-methylphenyl groups and dimethylphenyl groups such as 3,4-dimethylphenyl and 2,4-dimethylphenyl. The term methoxy-phenyl refers to mono- and di-methoxylated phenyl groups such as 4-methoxyphenyl, 3-methoxyphenyl, 2-methoxyphenyl, 3} 4-dimethoxyphenyl and 2,6-dimethoxyphenyl.

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Estergruppeme repræsenteret af R i ovennævnte formel er alle kendte esterdele, der sædvanligvis anvendes i cephalosporiner for at beskytte C^-carboxylsyregruppen i cephalosporinmolekylet, medens andre reaktioner, der omfatter andre reaktive steder i molekylet, udføres. Fremstillingen af estere med formel I (R1 = andet end bydrogen) udføres ved hjælp af følgende metode, ved hvilken der anvendes samme estergrupper til at beskytte carb-oxylsyregruppen som i andre cephalosporinforbindelser.The ester groups represented by R in the above formula are all known ester moieties commonly used in cephalosporins to protect the C1-carboxylic acid group in the cephalosporin molecule, while other reactions involving other reactive sites in the molecule are performed. The preparation of esters of formula I (R1 = other than the urban drug) is carried out by the following method, using the same ester groups to protect the carboxylic acid group as in other cephalosporin compounds.

Betegnelsen pharmaceutisk acceptable, ikke-toxiske salte refererer både til salte af C^-carboxylsyrefunktionen og syreadditionssalte af a-amino-gruppen af 7-glycylamido-sidekæden. Pharmaceutisk acceptable salte, der omfatter C^-carboxylsyrefunktionen, omfatter salte dannet med uorganiske baser, såsom natrium-, kalium og calciumsalte, der kan fremstilles med natriumbicarbonat, kaliumcarbonat, calciumhydroxid eller natriumhydroxid. Pharmaceutisk acceptable aminsalte kan også fremstilles, f.eks. med organiske aminer, såsom dieyclohexylamin, benzylamin, 2-amino-ethanol, diethanolamin eller diisopropylamin. Syreadditionssalte af α-aminogruppen omfatter salte dannet med mineralsyrer, såsom hydrogenchloridsyre, hydrogenbromidsyre og sulfater og salte dannet med organiske sulfonsyrer, såsom p-toluensulfonat.The term pharmaceutically acceptable, non-toxic salts refers to both salts of the C 1-6 carboxylic acid function and acid addition salts of the α-amino group of the 7-glycylamido side chain. Pharmaceutically acceptable salts comprising the C1-carboxylic acid function include salts formed with inorganic bases such as sodium, potassium and calcium salts which can be prepared with sodium bicarbonate, potassium carbonate, calcium hydroxide or sodium hydroxide. Pharmaceutically acceptable amine salts may also be prepared, e.g. with organic amines such as dieyclohexylamine, benzylamine, 2-aminoethanol, diethanolamine or diisopropylamine. Acid addition salts of the α-amino group include salts formed with mineral acids such as hydrochloric acid, hydrobromic acid and sulfates, and salts formed with organic sulfonic acids such as p-toluenesulfonate.

Det ses nemt, at når R er hydrogen, kan den amfotere form af forbindelsen eksistere, idet denne form fremkommer via intra-molekylær saltdannelse.It is readily seen that when R is hydrogen, the amphoteric form of the compound may exist, this form occurring via intra-molecular salt formation.

På grund af nærværelsen af det asymmetriske carbonatom i a-amino-acylgruppen omfatter de ovennævnte 3-halogen-cephalosporiner B-, I- og Dl-formeme. B-formen er den foretrukne isomere form.Due to the presence of the asymmetric carbon atom in the α-amino-acyl group, the aforementioned 3-halo-cephalosporins comprise the B, I and D1 forms. The B form is the preferred isomeric form.

Eksempler på a-aminoacyl-3-halogen-cephalosporiner repræsenteret ved den i kravet angivne formel er følgende: 7-(B-phenylglycylamido)-3~chlor-3-cephem-4-carboxylsyre, 7- (B-phenylglycylamido) -3-brom-3-cephem-4-carboxylsyre, 7~(B-3~hydroxyphenylglycylamido)-3-chlor-3-cephem-4-carboxylsyre, 7-[B-2-(2-thienyl)glycylamido]-3-chlor-3-cephem-4-carboxylsyre, 7~[B-2-(2-furyl)glycylamido]-3-chlor-3-cephem-4-carboxylsyre, 7- (D-4-chlorphenylgly c.ylamido) -3-brom-3-cephem-4-carboxylsyre,Examples of α-aminoacyl-3-halo-cephalosporins represented by the formula set forth in the claim are as follows: 7- (B-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid, 7- (B-phenylglycylamido) -3 -bromo-3-cephem-4-carboxylic acid, 7 ~ (B-3 ~ hydroxyphenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid, 7- [B-2- (2-thienyl) glycylamido] -3- chloro-3-cephem-4-carboxylic acid, 7- [B-2- (2-furyl) glycylamido] -3-chloro-3-cephem-4-carboxylic acid, 7- (D-4-chlorophenylglycylamido) - 3-bromo-3-cephem-4-carboxylic acid,

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7- (D- 3-me thylphenylglycylanildo) - 3-oIilor-3- c epiiem-4-carftoxylsyre, 7-(D-2,6-dime tiioxyplienylgly cylamido )-3-chlor-3-cephem-4-carboxylsyre og 7-[D-2-(3-thienyl)glycylamido]-3-chlor-3-cephem-4-carboxylsyre som amfotere ioner og farmaceutisk acceptable salte deraf.7- (D-3-methylphenylglycylanildo) -3-olilor-3-epilim-4-carftoxylic acid, 7- (D-2,6-dimethyl-oxyplienylglycylamido) -3-chloro-3-cephem-4-carboxylic acid and 7- [D-2- (3-thienyl) glycylamido] -3-chloro-3-cephem-4-carboxylic acid as amphoteric ions and pharmaceutically acceptable salts thereof.

Forbindelser med den i kravet angivne formel, hvor R1 er hydrogen, eller farmaceutisk acceptable ikke-toxiske salte deraf, er værdifulde antibiotika, der er anvendelige til at bekæmpe infektioner i varmblodede pattedyr, nærmere bestemt infektioner, der er forårsaget af gram-positive og gram-negative mikroorganismer. De er også effektive, når de indgives parenteralt, f.eks. subcutant eller intra-musculært, samt når de indgives oralt.Compounds of the formula as claimed in claim 1 wherein R 1 is hydrogen, or pharmaceutically acceptable non-toxic salts thereof, are valuable antibiotics useful in combating infections in warm-blooded mammals, in particular infections caused by gram-positive and gram. -negative microorganisms. They are also effective when administered parenterally, e.g. subcutaneously or intramuscularly, as well as when administered orally.

7-a-aminoacyl-3-halogen-cephalosporiner har et bredt spektrum af antibakteriel virkning som illustreret af in vitro spektret for 7-(D-phenylglycylamido)-3-chlor-3-cephem-4-carboxylsyre, der ses af følgende tabel:7-α-aminoacyl-3-halo-cephalosporins have a broad spectrum of antibacterial activity as illustrated by the in vitro spectrum of 7- (D-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid, as seen in the following table :

Tabel I nedenfor angiver den minimale hæmningskoncentration (MIC) i μg pr. ml, der er opnået med denne forbindelse ved standard-agar-fortyndingsforsøg.Table I below indicates the minimum inhibitory concentration (MIC) in μg per day. ml obtained with this compound by standard agar dilution tests.

Tabel_ITabel_I

7-(D-phenylglycylamido)-3-chlor-3-cephem-4-carboxylsyre in vitro spektrum.7- (D-Phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid in vitro spectrum.

* MIC* MIC

Organisme (ug/ml)Organism (µg / ml)

Staphylococcus aureus 3055 1Staphylococcus aureus 3055 1

Staphylococcus aureus 3074 1Staphylococcus aureus 3074 1

Streptococcus faecalis X66 16Streptococcus faecalis X66 16

Proteus morganii PR15 >128Proteus morganii PR15> 128

Salmonella typhisa SA12 <0,5Salmonella typhisa SA12 <0.5

Klebsiella pneumoniae K114 0,2Klebsiella pneumoniae K114 0.2

Enterobacter aerogenes EB17 8Enterobacter aerogenes EB17 8

Serratia marcescens SE3 >128Serratia marcescens SE3> 128

Escherichia coli EC14 2Escherichia coli EC14 2

Citrobacter freundii CE17 >128Citrobacter freundii CE17> 128

Pseudomonas aeruginosa X239 >128Pseudomonas aeruginosa X239> 128

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Tatel_I (forts.)Tatel_I (cont.)

* MIC* MIC

Organisme (pg/ml)Organism (pg / ml)

Salmonella typhimurium 1Salmonella typhimurium 1

Pseudomonas solanacearum X185 >128Pseudomonas solanacearum X185> 128

Erwinia amylovora 1 * lal og/eller "bogstaver efter navnet på organismen refererer til stammen.Erwinia amylovora 1 * lal and / or 'letters after the name of the organism refers to the strain.

Tabel II nedenfor angiver diameter i millimeter af hæmningszonen gældende for den angivne mikroorganisme i standardpladeforsøget opnået med 7-(l>-phenylglycylamido)-3-chlor-3-cephem-4-carboxyl-syre.Table II below indicates the diameter in millimeters of the inhibition zone applicable to the specified microorganism in the standard plate experiment obtained with 7- (1-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid.

Tabel_IITabel_II

Hæmningszone* (mm-diameter)Inhibition zone * (mm diameter)

Mikroorganisme Koncentration (mg/ml) 1,0 0,1 0,01Microorganism Concentration (mg / ml) 1.0 0.1 0.01

Staphylococcus aureus 35 28 20Staphylococcus aureus 35 28 20

Bacillus subtilis 44 32 21Bacillus subtilis 44 32 21

Sarcina lutea 48 27 27Sarcina lutea 48 27 27

Mycobacterium avium 24 TrMycobacterium avium 24 Tr

Proteus vulgaris 23 Tr —Proteus vulgaris 23 Tr -

Salmonella gallinarum 35 26 13Salmonella gallium 35 26 13

Escherichia coli 30 20 11Escherichia coli 30 20 11

Klebsiella pneumoniae 28 20 12Klebsiella pneumoniae 28 20 12

Pseudomonas solanacearcum 32 23 * Tr indicerer spor af en zonePseudomonas solanacearcum 32 23 * Tr indicates traces of a zone

En streg (—) angiver, at der ikke er observeret nogen zone. 1 tabel III nedenfor er vist den minimale hæmningskoncentration opnået med 7-(E-phenylglycylamido)-3-chlor-3--cephem-4-carboxyl~ syre over for et spektrum af gram-positive og gram-negative mikroorganismer ved aqar-fortvndinasforsøa.A dash (-) indicates that no zone has been observed. Table III below shows the minimum inhibitory concentration obtained with 7- (E-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid against a spectrum of gram-positive and gram-negative microorganisms in agar dilution experiments. .

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Tabel_IIITabel_III

In vitro antibiotisk spektrum af 7-(D-phenylglycylamido)-3~chlor- 3-cephem-4-carboxylsyre # Minimal hæmnings-In vitro antibiotic spectrum of 7- (D-phenylglycylamido) -3 ~ chloro-3-cephem-4-carboxylic acid # Minimal inhibition

Testorganisme koncentration MIC pg/mlTest organism concentration MIC pg / ml

Staphylococcus aureus 3055 1,0Staphylococcus aureus 3055 1.0

Staphylococcus aureus 3123 1,0Staphylococcus aureus 3123 1.0

Staphylococcus aureus 3074 2,0Staphylococcus aureus 3074 2.0

Streptococcus (gruppe D) 9901 64Streptococcus (Group D) 9901 64

Enterobaeter cloacae EB9 >128Enterobaeter cloacae EB9> 128

Enterobacter aerogenes EB17 64Enterobacter aerogenes EB17 64

Escherichia coli EC14 1,0Escherichia coli EC14 1.0

Escherichia coli EC35 2,0Escherichia coli EC35 2.0

Escherichia coli EC38 1,0Escherichia coli EC38 1.0

Klebsiella sp. K13 <0,5Klebsiella sp. K13 <0.5

Klebsiella sp. KI14 2,0Klebsiella sp. KI14 2.0

Klebsiella sp. K125 1,0Klebsiella sp. K125 1.0

Proteus mirabilis PR6 1,0Proteus mirabilis PR6 1.0

Proteus morganii PR1 128Proteus morganii PR1 128

Proteus rettgeri PR9 >128Proteus straightener PR9> 128

Proteus rettgeri PR2 >128Proteus Straightening PR2> 128

Salmonella SA12 <0,5Salmonella SA12 <0.5

Shigella sp. SH3 2,0 * Tal og/eller bogstaver efter organismens navn henfører til stammen.Shigella sp. SH3 2.0 * Numbers and / or letters after the organism's name refer to the strain.

I standard-agar-fortyndingsforsøg viste 7-(D-phenylglycylamido)-3“Chlor-3-cephem-4~carboxylsyre aktivitet over for Hemophilus influenzae med en MIC-værdi på 1 - 4 mg/ml over for et antal stammer.In standard agar dilution experiments, 7- (D-phenylglycylamido) -3- chloro-3-cephem-4-carboxylic acid showed activity against Hemophilus influenzae with a MIC of 1-4 mg / ml against a number of strains.

I tabel IY nedenfor er vist den orale absorption af 7-(D-phenyl-giycylamido)-3-chlor-3-cephem-4-carboxylsyre ved hjælp af de blod-Table IY below shows the oral absorption of 7- (D-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid by the blood

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og urinkoncentrationer, der opnåedes i mus. Ved udførelsen af forsøget blev schweiziske McAllister mus, der vejede fra 11 til 13 gram, fastet natten over og blev indgivet oralt 20 mg/kg af 7-(D-phenylglycylamido)-3-chlor-3-cephem-4-carboxylsyre. Blod-og urinprøver blev udtaget ved de anførte intervaller, og koncentrationen af antibiotikum i hver prøve bestemtes ved et mikrobiologisk forsøg under anvendelse af Sarcina lutea i en agar med pH 6,0 ved et pladeforsøg.and urine concentrations obtained in mice. In carrying out the experiment, Swiss McAllister mice weighing from 11 to 13 grams were fasted overnight and administered orally 20 mg / kg of 7- (D-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid. Blood and urine samples were taken at the indicated intervals, and the concentration of antibiotic in each sample was determined by a microbiological test using Sarcina lutea in a pH 6.0 agar in a plate test.

Tabel_IVTabel_IV

7-(D-phenylglycylamido)-3-chlor-3-eephem-4-carboxylsyre Koncentration i blod og urin M Koncentration (pg/ml) ius nr. efter følgende tider (min.) 5 15 50 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 1,07- (D-Phenylglycylamido) -3-chloro-3-eephem-4-carboxylic acid Concentration in blood and urine M Concentration (pg / ml) ius no after the following times (min) 5 15 50 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

Gennemsnit i blod 23,4 14,8 10,5 4,6 2,0 1,4 0,6Blood average 23.4 14.8 10.5 4.6 2.0 1.4 0.6

Gennemsnit i gennem- __ -1474 1754 843 305 308 226 snitsprøve af urinen ^ ^Average in average __-1474 1754 843 305 308 226 sample of urine ^^

Ben effektive dosis (ED^q) for 7-(B-phenylglycylamido)-3-chlor- 3-cephem-4-carboxylsyre, der er illustrativ for den effektive dosis af forbindelserne ifølge opfindelsen, er 0,74 mg/kg x 2 indgivet oralt og 0,48 mg/kg x 2 s.c. vs. Streptococcus pyogenes og 5,5 mg/kg x 2 oralt over for Escherichia coli og 17,6 mg/kg x 2 oralt over for Diplococcus pneumoniae bestemt i mus.Bone effective dose (ED ^ q) for 7- (B-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid illustrative of the effective dose of the compounds of the invention is 0.74 mg / kg x 2 administered orally and 0.48 mg / kg x 2 sc vs. Streptococcus pyogenes and 5.5 mg / kg x 2 orally against Escherichia coli and 17.6 mg / kg x 2 orally to Diplococcus pneumoniae determined in mice.

Tabel V nedenfor angiver den minimale hæmningskoncentration for illustrative 3-halogen-3-cephem-forbindelser fremstillet ifølge opfindelsen over for repræsentative gram-negative bakterier. Hæmningskoncentrationerne bestemtes ved gradient plade-metoden udført i det væsentlige som beskrevet i Science 116, 45 (1952). I tabel V refererer betegnelsen R til strukturformlen.Table V below indicates the minimum inhibitory concentration for illustrative 3-halogen-3-cephem compounds of the invention against representative gram-negative bacteria. The inhibition concentrations were determined by the gradient plate method performed essentially as described in Science 116, 45 (1952). In Table V, the designation R refers to the structural formula.

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Iabel_TIabel_T

Antibiotisk virkning af substitueret 7-(phenylglycyl-amido)-3-chlor-3-cephem-4-carboxylsyre over for gramnegative bakterier.Antibiotic action of substituted 7- (phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid against gram-negative bacteria.

COOHCOOH

Minimal hæmningskoncentration Organisme ^g/ml)Minimum inhibitory concentration Organism (g / ml)

R 3-OH 4-OH 4-C1 HR 3-OH 4-OH 4-C1 H

Shigella sp. 1,0 2,0 13,5 6,7Shigella sp. 1.0 2.0 13.5 6.7

Escherichia coli 1,0 2,0 17,2 5,7Escherichia coli 1.0 2.0 17.2 5.7

Klebsiella pneumoniae 0,9 1,5 8,0 0,9Klebsiella pneumoniae 0.9 1.5 8.0 0.9

Aerobacter aerogenes 0,8 1,0 5,0 0,8Aerobacter aerogenes 0.8 1.0 5.0 0.8

Salmonella heidelberg 0,5 0,8 10,7 0,2Salmonella heidelberg 0.5 0.8 10.7 0.2

Pseudomonas aeruginosa >200 >200 > 200 >200 1 følgende tabel VI er anført aktiviteten af repræsentative 3-halogen-3-cephem-forbindelser over for adskillige kliniske isolater af penicillin-resistente staphylococci. Aktiviteten er angivet i minimal hæmningskoncentrationer af den omhandlede forbindelse. Pen minimale koncentration bestemtes ved gradient-plade-metoden.Pseudomonas aeruginosa> 200> 200> 200> 200 The following Table VI lists the activity of representative 3-halogen-3-cephem compounds against several clinical isolates of penicillin-resistant staphylococci. The activity is indicated in minimal inhibition concentrations of the subject compound. Pure minimum concentration was determined by the gradient plate method.

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Tatel_YITatel_YI

Antibiotisk virkning af substitueret 7-(phenylglycyl-amido-3~chlor-3-cephem-4-carboxylsyrer over for penicillin-resistent Staphylococcus r—v o h // A it »Antibiotic effect of substituted 7- (phenylglycylamido-3 ~ chloro-3-cephem-4-carboxylic acids against penicillin-resistant Staphylococcus r-v o h // A it »

C SC S

nh2 i ]nh2 i]

COOHCOOH

Minimal hæmningskoncentrationMinimum inhibitory concentration

Staphylococcus1 _(ftg/ml)_Staphylococcus1 _ (ftg / ml) _

Stamme R 3-0Ξ 4-OH 4-01 HStrain R 3-0Ξ 4-OH 4-01 H

741 3,0/ 5,0/ 10,5/>20 11,5/>20 732 3,5/ 7,0/ 18 />20 17 />20 X400 >20 >20 >20 />20 >20 />20 784 0,5/ 0,6/ 3,0/ >20 2,7/ 15 XI.1 0,4/ 0,6/ 0,4/0,7 0,4/1,0 1/ Bogstav-tal-betegnelsen refererer til stammer af kliniske isolater af penicillin-resistent staphylococcus.741 3.0 / 5.0 / 10.5 /> 20 11.5 /> 20 732 3.5 / 7.0 / 18 /> 20 17 /> 20 X400> 20> 20> 20 /> 20> 20 /> 20 784 0.5 / 0.6 / 3.0 /> 20 2.7 / 15 XI.1 0.4 / 0.6 / 0.4 / 0.7 0.4 / 1.0 1 / The letter-number designation refers to strains of clinical isolates of penicillin-resistant staphylococcus.

2/ I kolonnerne er værdien ovenfor skråstregen den minimale hæmningskoncentration i fravær af humant serum. Værdien under stregen er, når den er til stede, den minimale hæmningskoncentration i nærværelse af humant serum.2 / In the columns, the value above the slash is the minimum inhibitory concentration in the absence of human serum. The value below the line, when present, is the minimum inhibitory concentration in the presence of human serum.

Forbindelserne ifølge opfindelsen fremstilles ved N-acylering af en 7-amino-3-halogen-3-cephem-4-carboxylsyre eller en ester deraf, f.eks. benzyl, p-methoxybenzyl, p-nitrobenzyl, diphenyl-methyl, 2,2,2-trichlorethyl, trimethylsilyl eller tert.-butylester, med et aktivt derivat af en phenyl, thienyl eller furyl substitueret glycin med formlen:The compounds of the invention are prepared by N-acylation of a 7-amino-3-halogen-3-cephem-4-carboxylic acid or an ester thereof, e.g. benzyl, p-methoxybenzyl, p-nitrobenzyl, diphenylmethyl, 2,2,2-trichloroethyl, trimethylsilyl or tert-butyl ester, with an active derivative of a phenyl, thienyl or furyl substituted glycine of the formula:

10 DK 153157 BDK 153157 B

R-CH-COOHR-CH-COOH

m2 hvor R har samme betydning som i formel I. Ved acyleringen beskyttes aminogruppen i glycinen, f.eks. som et salt, såsom et hy-drochlorid, eller med en af de sædvanligt anvendte aminobeskyt-tende grupper, som f.eks. tert.-butyloxycarbonyl, benzyloxycar-bonyl, p-nitrobenzyloxycarbonyl, trichlorethoxycarbonyl eller trithylgruppen, eller enaminer dannet med methylacetoacetat og acetylacetone og lignende grupper. Aktiverede derivater af carb-oxylsyregruppen i den substituerede glycin kan være syrehaloge-nider, såsom syrechlorider, aktiverede estre, såsom de, der dannes med pentachlorphenol, azid eller et blandet anhydrid dannet med glycin Og methylchlorformiat og isobutylchlorformiat. Ligeledes kan den aminobeskyttede glycin anvendes direkte i acyleringen af den ønskede 3-halogenkerneester ved at anvende et kondenseringsmiddel, såsom N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinolin (EEDQ). Eor eksempel omsættes p-nitrobenzyl-7-amino-3-chlor-3-cephem-4-carboxylat med N-(tert.-butyloxycarbonyl)-D-phenylgly-cin i et tørt inert opløsningsmiddel, f.eks. tetrahydrofuran, til dannelse af p-nitrobenzy1-7-(D-phenylglycylamido)-3-ehlor-3-cephem-4-carboxylat.m2 where R has the same meaning as in formula I. In the acylation, the amino group of the glycine, e.g. as a salt, such as a hydrochloride, or with one of the commonly used amino protecting groups, such as e.g. tert-butyloxycarbonyl, benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, trichloroethoxycarbonyl or trithyl group, or enamines formed with methyl acetoacetate and acetylacetone and the like. Activated derivatives of the carboxylic acid group in the substituted glycine may be acid halides such as acid chlorides, activated esters such as those formed with pentachlorophenol, azide or a mixed anhydride formed with glycine and methyl chloroformate and isobutyl chloroformate. Likewise, the amino-protected glycine can be used directly in the acylation of the desired 3-halogen core ester using a condensing agent such as N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). For example, p-nitrobenzyl-7-amino-3-chloro-3-cephem-4-carboxylate is reacted with N- (tert-butyloxycarbonyl) -D-phenylglycine in a dry inert solvent, e.g. tetrahydrofuran, to form p-nitrobenzyl-7- (D-phenylglycylamido) -3-ehloro-3-cephem-4-carboxylate.

Generelt kan man anvende enhver af de kendte amid-koblingsmetoder ved acyleringen af 7-amino-3-halogen-3-cephem-4-carboxylsyre eller -estere. Når det aktiverede derivat er et syrehalogenid, udføres acyleringen i nærværelse af et hydrogenhalogenidbindende middel, såsom natriumbicarbonat, pyridin, natriumbisulfit eller en alkylenoxid, såsom propylenoxid. Når et blandet anhydrid anvendes ved acyleringen, kan det dannede anhydrid dannes ved hjælp af N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinolin (EEDQ). Når en N-beskyttet phenyl, thienyl eller furylglycin anvendes, kan acyleringen udføres i nærværelse af et kondenseringsmiddel, såsom N,N'-dicyclohexylcarbodiimid.In general, any of the known amide coupling methods can be used in the acylation of 7-amino-3-halogen-3-cephem-4-carboxylic acid or esters. When the activated derivative is an acid halide, the acylation is carried out in the presence of a hydrogen halide binding agent such as sodium bicarbonate, pyridine, sodium bisulfite or an alkylene oxide such as propylene oxide. When a mixed anhydride is used in the acylation, the anhydride formed can be formed by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). When an N-protected phenyl, thienyl or furylglycine is used, the acylation can be carried out in the presence of a condensing agent such as N, N'-dicyclohexylcarbodiimide.

Som eksempler på substituerede glyciner, der kan anvendes til fremstilling af de omhandlede forbindelser, kan nævnes D-phenylglycyl-chlorid, hydrochlorid, D-4-hydroxyphenylglycyl-chlorid, hydrochlorid, pentachlorphenyl-D-phenyl-N-(tert.-butyloxycarbonyl)glycinat, pentachlorphenyl-D-2-thienyl-N-(2,2,2-trichlorethoxycarbonyl)-Examples of substituted glycines which can be used to prepare the subject compounds include D-phenylglycyl chloride, hydrochloride, D-4-hydroxyphenylglycyl chloride, hydrochloride, pentachlorophenyl-D-phenyl-N- (tert-butyloxycarbonyl) glycinate, pentachlorophenyl-D-2-thienyl-N- (2,2,2-trichloroethoxycarbonyl) -

DK 153157 BDK 153157 B

glycinat, N-(tert.-butyloxycarbonyl)-D-phenylglycin, N-(1-carbo-methoxy-2-propenyl) -D-phenylglycin, 3-thienylglycylchlorid, hy-drochlorid, N-(tert,-butyloxycarbonyl)-2-thienylglycin eller N-(tert. -butyloxycarbonyl) -2-furylglycin.glycinate, N- (tert-butyloxycarbonyl) -D-phenylglycine, N- (1-carbo-methoxy-2-propenyl) -D-phenylglycine, 3-thienylglycyl chloride, hydrochloride, N- (tert, butyloxycarbonyl) - 2-thienylglycine or N- (tert -butyloxycarbonyl) -2-furylglycine.

Acyleringen udføres i et inert opløsningsmiddel, f.eks. i acetone, acetonitril, dimethylformamid eller methylenchlorid og fortrinsvis ved en temperatur på -20 - +20° C. Por eksempel fremstilles 7-(D-phenylglycylamido)-3-chlor-3-cephem-4-carboxylsyre ved at omsætte D-phenylglycylchlorid, hydrochlorid med p-nitro-benzyl-7-amino-3-chlor-3-cephem-4-carboxylat i acetonitril i nærværelse af propylenoxid. Estergruppen fjernes dernæst ved hydrogenering.The acylation is carried out in an inert solvent, e.g. in acetone, acetonitrile, dimethylformamide or methylene chloride and preferably at a temperature of -20 - + 20 ° C. For example, 7- (D-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid is prepared by reacting D-phenylglycyl chloride , hydrochloride with p-nitro-benzyl-7-amino-3-chloro-3-cephem-4-carboxylate in acetonitrile in the presence of propylene oxide. The ester group is then removed by hydrogenation.

Illustrative eksempler på 7-amino-3-halogen-3-cephem-4-carboxyisyrer og -estere, der er udgangsmaterialer ved fremstillingen af de omhandlede forbindelser, er 7-amino-3-chlor-3-cephem-4-carboxylsyre, p-nitrobenzyl-7-amino-3-chlor-3-cephem-4-carboxylat, diphenyl-methyl-7-amino-3-brom-3-cephem-4-carboxylat eller diphenylmethyl-7-amin o-3-chlor-3-c ephem-4-carboxy lat.Illustrative examples of 7-amino-3-halogen-3-cephem-4-carboxylic acids and esters which are starting materials in the preparation of the compounds of the invention are 7-amino-3-chloro-3-cephem-4-carboxylic acid, p -nitrobenzyl-7-amino-3-chloro-3-cephem-4-carboxylate, diphenylmethyl-7-amino-3-bromo-3-cephem-4-carboxylate or diphenylmethyl-7-amine o-3-chloro 3-c ephem-4-carboxy lat.

7-amino-3-halogen-3-cephem-4-carboxylsyrer og -estere fremstilles på følgende måde: En 7-acylamido-cephalosporansyre eller en ester deraf omdannes til en 7-acylamido-3-exomethylencepham-4-carboxylsyre eller ester. Esteren af 3-exomethylencepham-forbin-delsen oxideres med ozon til dannelse af et mellemprodukt i form af et ozonid, der dekomponeres til opnåelse af 7-acylamido-3-hydroxy-3-cephem-4-carboxylsyreester. Denne chloreres eller bromeres til frembringelse af den tilsvarende 3-halogen-3-cephem-forbindelse. 7-acylgruppen i denne forbindelse fjernes ved velkendt sidekæde-spaltningsreaktion, idet der anvendes phosphor-pentachlorid i pyridin til opnåelse af iminochloridderivatet af sidekæden, der dernæst omsættes med en alkohol, såsom methanol, til opnåelse af iminoetheren, der herefter nemt hydrolyseres til opnåelse af 7-amino-3-halogen-3-cephem-4-carboxylsyreesteren.7-Amino-3-halogen-3-cephem-4-carboxylic acids and esters are prepared as follows: A 7-acylamido-cephalosporanoic acid or an ester thereof is converted to a 7-acylamido-3-exomethylene cepham-4-carboxylic acid or ester. The ester of the 3-exomethylene cepham compound is oxidized with ozone to form an intermediate in the form of an ozonide decomposed to give 7-acylamido-3-hydroxy-3-cephem-4-carboxylic acid ester. This is chlorinated or brominated to give the corresponding 3-halogen-3-cephem compound. The 7-acyl group in this connection is removed by well-known side chain cleavage reaction using phosphorous pentachloride in pyridine to give the iminochloride derivative of the side chain, which is then reacted with an alcohol such as methanol to give the imino ether which is then readily hydrolyzed to give 7-amino-3-halo-3-cephem-4-carboxylic acid ester.

Por eksempel omsættes en 7-acylamido-cephalosporansyre, såsom 7-phenoxyacetamidocephalosporansyre, med en svovlholdig nucleo-phil forbindelse ifølge velkendte metoder til udøvelse af en nu-cleophil substitution af acetoxygruppen i cephalosporansyren ogFor example, a 7-acylamido-cephalosporanoic acid, such as 7-phenoxyacetamidocephalosporanoic acid, is reacted with a sulfur-containing nucleophilic compound according to well-known methods for exerting a nucleophilic substitution of the acetoxy group in the cephalosporanoic acid and

12 DK 153157 B12 DK 153157 B

til frembringelse af en 3-thio-substitueret 7-acylamido-methyl- 3-cephem-4-carboxylsyre. Denne reduceres dernæst med zink/myre-syre i nærværelse af dime thylformamid eller med Raney-nikkel i nærværelse af hydrogen til opnåelse af en 7-acylamido-3-exomethyl-encepham-4--carboxylsyre. For eksempel omsættes 7-phenoxyacet-amido-3-acetoxymethyl-3-cephem-4-carboxylsyre med kaliumethyl-xanthat til opnåelse af 7-phenoxyacetamido-3-ethoxythionocar-bonylthiomethyl-3-cephem-4-carboxylsyre. Reduktion af sidstnævnte forbindelse med zink og myresyre i nærværelse af dimethylformamid giver 7-pbenoxyacetamido-3-exomethylencepham-4-oarboxylsyre.to produce a 3-thio-substituted 7-acylamido-methyl-3-cephem-4-carboxylic acid. This is then reduced with zinc / formic acid in the presence of dime thylformamide or with Raney nickel in the presence of hydrogen to give a 7-acylamido-3-exomethyl-encepham-4-carboxylic acid. For example, 7-phenoxyacetamido-3-acetoxymethyl-3-cephem-4-carboxylic acid is reacted with potassium ethyl xanthate to give 7-phenoxyacetamido-3-ethoxythionocarbonylthiomethyl-3-cephem-4-carboxylic acid. Reduction of the latter compound by zinc and formic acid in the presence of dimethylformamide affords 7-pbenoxyacetamido-3-exomethylene cepham-4-oreboxylic acid.

På lignende måde kan en lang række 7-acylamido-cephalosporansy-rer omsættes med en lang række svovlholdige nucleophile forbindelser til opnåelse af 3-thio-substituerede methyl-3-cephem-forbin-delser. Por eksempel kan 7-acylamidogruppen være en heterocyc-lisk kæde, f.eks. 2-thienylacetamido eller 2-furylacetamido, eller det kan være en alkanoylsidekæde som eksemplificeret af acetamido eller en lang række andre sidekæder. Ligeledes kan den svovlholdige nucleophile forbindelse være en lang række forbindelser, såsom thiourinstof og substituerede thiourinstoffer, der reagerer med cephalosporansyrer til opnåelse af isothiouroniumsalte, thiobenzoater, mercaptopyridin-N-oxid, 1-methyl-tetrazol-5-thiol, 5-methyl-1,3»4-thiadiazol-2-thiol og andre svovlholdige nucleophile forbindelser. Efter den reduktive substitutionsreaktion beskrevet ovenfor kan den således fremstillede 3-exomethylen-cepham-4-carboxylsyre esterificeres for eksempel med p-nitro-benzylbromid, p-methoxybenzylbromid, diphenyldiazomethan eller 2,2,2-trichlorethylchlorformat eller en anden esterdannende forbindelse, og 3-exomethylencephamesteren omsættes med ozon til opnåelse af 3-hydroxy-3-cephem-esteren. Reaktionen, hvorved man ozono-lyserer en 3-exomethyIencephamester udføres i et inert opløsningsmiddel ved en temperatur på mellem -80 og 0°C og fortrinsvis mellem -80 og -50°C til dannelse af det intermediære ozonid. Dette dekom-poneres in situ i kulden til opnåelse af den tilsvarende 3-hydroxy-3-cephem-ester som illustreret i følgende reaktionsskema:Similarly, a wide variety of 7-acylamido-cephalosporanoic acids can be reacted with a wide variety of sulfur-containing nucleophilic compounds to obtain 3-thio-substituted methyl-3-cephem compounds. For example, the 7-acylamido group may be a heterocyclic chain, e.g. 2-thienylacetamido or 2-furylacetamido, or it may be an alkanoyl side chain as exemplified by acetamido or a variety of other side chains. Likewise, the sulfur-containing nucleophilic compound may be a wide variety of compounds such as thiourea and substituted thioureas which react with cephalosporanoic acids to give isothiouronium salts, thiobenzoates, mercaptopyridine N-oxide, 1-methyl-tetrazole-5-thiol, 5-methyl-5 , 3 »4-Thiadiazole-2-thiol and other sulfur-containing nucleophilic compounds. Following the reductive substitution reaction described above, the 3-exomethylene cepham-4-carboxylic acid thus prepared can be esterified, for example, with p-nitrobenzyl bromide, p-methoxybenzyl bromide, diphenyl diazomethane or 2,2,2-trichloroethyl chloroformate or another ester-forming compound, and 3 The exomethylenecephame ester is reacted with ozone to give the 3-hydroxy-3-cephem ester. The reaction whereby ozonolysing a 3-exomethylene polymer ester is carried out in an inert solvent at a temperature of between -80 and 0 ° C and preferably between -80 and -50 ° C to form the intermediate ozonide. This is decomposed in situ in the cold to give the corresponding 3-hydroxy-3-cephem ester as illustrated in the following reaction scheme:

13 DK 153157 B13 DK 153157 B

0 IT0 IT

R-C-N-,-SR-C-N -, - S

tf-CH2 COOR1 °5 Φ [ozonid] vtf-CH2 COOR1 ° 5 Φ [ozonide] v

0 H0 H

R-C-N-*-|^S'^ 0J- COOR1 I ovennævnte formel er R hydrogen eller resten af acylamido-gruppen som beskrevet ovenfor. For eksempel kan R være "benzyl,In the above formula, R is hydrogen or the remainder of the acylamido group as described above. For example, R may be "benzyl,

’ A'A

phenoxymethyl, methyl, 2-thienylmethyl eller 2-furylmethyl, og R har samme "betydning som defineret i formel I.phenoxymethyl, methyl, 2-thienylmethyl or 2-furylmethyl, and R has the same meaning as defined in formula I.

Ozonolysen af 3-exomethylencephamesteren udføres ved at lede ozon gennem en opløsning af esteren i et inert opløsningsmiddel, indtil ozoniddannelsen er tilendebragt. Inerte opløsningsmidler, der kan anvendes, er de opløsningsmidler, hvori 3-exomethylen-cephamestere i det mindste delvis er opløselige, og som ikke reagerer med ozon under de beskrevne betingelser. Sædvanligvis anvendte opløsningsmidler er methanol, ethanol, ethylacetat, methylacetat, isoamylacetat og methylenehlorid.The ozone analysis of the 3-exomethylenecephame ester is carried out by passing ozone through a solution of the ester in an inert solvent until the ozone formation is completed. Inert solvents which may be used are those solvents in which 3-exomethylene cephame esters are at least partially soluble and which do not react with ozone under the conditions described. Commonly used solvents are methanol, ethanol, ethyl acetate, methyl acetate, isoamyl acetate and methylene chloride.

Ozongassen fremstilles ved hjælp af en ozongenerator af den type, der sædvanligvis anvendes ved syntese og analytisk arbejde. Sådanne generatorer fremstiller ozon ved indvirkning af en elektrisk spænding på oxygen. En sådan ozongenerator sælges af Wells- η DK 153157 Β back Corporation. Ozon frembringes i en strøm af oxygen, der ledes direkte ind i reaktionsbeholderen. Det procentvise indhold af ozon i oxygenstrømmen kan varieres efter ønske, f.eks. ved at variere strømningshastigheden af oxygen igennem ozongeneratoren samt ved at variere intensiteten af den elektriske ladning.The ozone gas is produced by an ozone generator of the type commonly used in synthesis and analytical work. Such generators produce ozone by the action of an electrical voltage on oxygen. Such an ozone generator is sold by Wells η DK 153157 Β back Corporation. Ozone is generated in a stream of oxygen which is fed directly into the reaction vessel. The percentage content of ozone in the oxygen stream can be varied as desired, e.g. by varying the flow rate of oxygen through the ozone generator, and by varying the intensity of the electric charge.

Koncentrationen af 3-exomethylencephamester-udgangsmaterialet i det inerte opløsningsmiddel er ikke kritisk, og det foretrækkes at anvende et opløsningsmiddel-volumen, der er tilstrækkeligt til at danne en fuldstændig opløsning.The concentration of the 3-exomethylene cephame 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.

Når ozoniddannelsen er tilendebragt, fjernes ethvert overskud af ozon i reaktionsblandingen ved at boble nitrogen, oxygen eller en inert gas, såsom argon, igennem blandingen. Efter fjernelse af ethvert overskud af ozon dekomponeres ozonidet ved at sætte et reduktionsmiddel til reaktionsblandingen, hvilket middel vælges fra gruppen bestående af natriumborsulfit, svovldioxid og trims thylphosph.it til opnåelse af 3-hydroxy-3-cephem-4-carboxylsyre-esteren. Dekomponeringen udføres ved at sætte et overskud af dekompositionsmidlet til blandingen og omrøre denne, indtil der opnås en negativ kaliumiodid-stivelsesprøve. Et foretrukkent middel til brug ved dekomponeringen er gasformigt svovldioxid.When ozone formation is complete, any excess ozone in the reaction mixture is removed by bubbling nitrogen, oxygen or an inert gas, such as argon, through the mixture. After removal of any excess ozone, the ozonide is decomposed by adding a reducing agent to the reaction mixture, which agent is selected from the group consisting of sodium borosulfite, sulfur dioxide and trimethylphosphite to give the 3-hydroxy-3-cephem-4-carboxylic acid ester. The decomposition is carried out by adding an excess of the decomposition agent to the mixture and stirring it until a negative potassium iodide starch test is obtained. A preferred agent for use in the decomposition is gaseous sulfur dioxide.

3-hydroxy-3-cephem-estere udvindes fra reaktionsblandingen ved at afdampe de flygtige opløsningsmidler fra blandingen, hvorved der opnås en reaktionsproduktblanding som remanens, hvorefter der omkrystalliseres. Den således dannede 7-acetamido-3-hydroxy-3-cephem-4-carboxylsyreester halogeneres dernæst til opnåelse af 7-acylamido-3-halogen-3-cephem-4“Carboxylsyreesteren.3-Hydroxy-3-cephem esters are recovered from the reaction mixture by evaporating the volatile solvents from the mixture to give a reaction product mixture as residue, and then recrystallize. The 7-acetamido-3-hydroxy-3-cephem-4-carboxylic acid ester thus formed is then halogenated to give the 7-acylamido-3-halogen-3-cephem-4 “carboxylic acid ester.

Eorbindelser repræsenteret ved formel I, hvori X er chlor eller brom fremstilles ved at omsætte en 7-acylamido~3-hydroxy-3-cephem-ester eller en 3-hydroxy-3-cephem-ester i dimethylformamid med en reaktiv chlor- eller bromforbindelse, der med dimethylformamid danner chlor eller brom-dimethyliminiumchlorid eller bromid med følgende formel:Compounds represented by Formula I wherein X is chlorine or bromine are prepared by reacting a 7-acylamido ~ 3-hydroxy-3-cephem ester or a 3-hydroxy-3-cephem ester in dimethylformamide with a reactive chlorine or bromine compound forming chloro or bromo-dimethyliminium chloride or bromide of the following formula with dimethylformamide:

OH* , HOH *, H

3+ t -V-— N=C x3+ t -V-— N = C x

CH^ ZCH 2 Z

hvor X og X repræsenterer henholdsvis chlor eller brom og chloridwhere X and X represent chlorine or bromine and chloride, respectively

15 DK 153157 BDK 153157 B

eller bromid. Det reaktive halogeniminiumhalogenid med ovennævnte formel dannes in situ og er et meget reaktivt chlorerings- eller bromeringsmiddel. Chlor- og bromforbindelser, der danner ovennævnte iminiumhalogenid, indbefatter sædvanligvis anvendte chlo-reringsmidler, såsom phosgen (carbonylchlorid), oxalylchlorid, thionylchlorid og phosphorchlorider, såsom f.eks. phosphortri-chlorid og phosphoroxychlorid (phosphorylchlorid). Bromerings-midler, der kan anvendes i nærværende opfindelse, omfatter carbo-nylbromid, oxalylbromid, thionylbromid (svovloxybromid) og phos-phorbromider, phosphoroxybromid og phosphortribromid. Phosphor-pentachlorid kan anvendes ved fremstilling af 3-chlor-3-cephem-forbindelser ifølge opfindelsen. Imidlertid reagerer dette middel samtidigt med 7-acylamido-sidekæden i udgangsmaterialet til dannelse af et iminochlorid, der er det reaktive mellemprodukt i den velkendte reaktion til fraspaltning af cephalosporin-sidekæden. Derfor foretrækkes det at anvende et af de andre chloreringsmidler.or bromide. The reactive halogeniminium halide of the above formula is formed in situ and is a highly reactive chlorinating or brominating agent. Chlorine and bromine compounds which form the above-mentioned iminium halide include commonly used chlorinating agents such as phosgene (carbonyl chloride), oxalyl chloride, thionyl chloride and phosphorus chlorides such as e.g. phosphorus trichloride and phosphorus oxychloride (phosphoryl chloride). Brominating agents which can be used in the present invention include carbonyl bromide, oxalyl bromide, thionyl bromide (sulfur oxybromide) and phosphorus bromides, phosphorus oxybromide and phosphorus tribromide. Phosphorus pentachloride can be used in the preparation of 3-chloro-3-cephem compounds of the invention. However, this agent reacts simultaneously with the 7-acylamido side chain in the starting material to form an imino chloride, which is the reactive intermediate in the well-known reaction to cleave the cephalosporin side chain. Therefore, it is preferred to use one of the other chlorinating agents.

Chloreringen og bromeringen udføres ved at sætte halogenerings-midlet til en opløsning af 3-hydroxy-3-cephem-esteren i tør di-methylformamid ved en temperatur på 5 - 15° C og lade reaktionsblandingen stå ved stuetemperatur i 4 - 8 timer eller længere. Reaktionen er til at begynde med exotherm,og derfor køles reaktionsbeholderen først i et is-vandbad og holdes dernæst ved 25° 0 under resten af reaktionen. Dimethylformamidet tørres fortrinsvis over en molekylsi før anvendelsen. Selv om reaktionen kan udføres i dimethylformamid som opløsningsmiddel, kan der sammen med dimethylformamidet anvendes et andet opløsningsmiddel.The chlorination and bromination are carried out by adding the halogenating agent to a solution of the 3-hydroxy-3-cephem ester in dry dimethylformamide at a temperature of 5 - 15 ° C and leaving the reaction mixture at room temperature for 4 - 8 hours or longer. . The reaction is initially exothermic and therefore the reaction vessel is first cooled in an ice-water bath and then maintained at 25 ° C for the remainder of the reaction. The dimethylformamide is preferably dried over a molecular sieve before use. Although the reaction can be carried out in dimethylformamide as a solvent, another solvent can be used together with the dimethylformamide.

Por eksempel kan der anvendes tetrahydrofuran, dioxan, methylen-chlorid, dimethylacetamid eller dimethylsulfoxid som yderligere opløsningsmiddel.For example, tetrahydrofuran, dioxane, methylene chloride, dimethylacetamide or dimethylsulfoxide may be used as additional solvent.

3-chlor- eller 3-brom-3-cephemesterne udvindes fra reaktionsproduktblandingen ved at udhælde denne i en blanding af vand og ethyl-acetat og fraseparere den organiske fase indeholdende produktet. Den organiske fase vaskes, tørres og inddampes til opnåelse af 3-halogen-3-cephemesteren som en amorf remanens. Dette produkt opnås ofte i krystallinsk form ved behandling af remanensen med ether eller med n-hexan. Ved at følge metoden til fremstilling af 7-acylamido-3-halogen-3-cephem-4-carboxylsyreestere som beskrevet ovenfor opnås de respektive 7-amino-3-halogen-3-cephem-4-carb-The 3-chloro or 3-bromo-3-cephemester is recovered from the reaction product mixture by pouring it into a mixture of water and ethyl acetate and separating the organic phase containing the product. The organic phase is washed, dried and evaporated to give the 3-halo-3-cephemester as an amorphous residue. This product is often obtained in crystalline form by treating the residue with ether or with n-hexane. Following the method of preparing 7-acylamido-3-halo-3-cephem-4-carboxylic acid esters as described above, the respective 7-amino-3-halo-3-cephem-4-carb

16 DK 153157 B16 DK 153157 B

oxylsyreestere ved at udføre den velkendte N-deacylering af 7-acylamido-sidekæden. For eksempel omsættes 7-aeylamido-3-halo-gen-cephalosporinesteren med phosphorpentachlorid i methylenchlorid i nærværelse af pyridin til dannelse af iminochloridet. Dette omsættes med et alkoholisk opløsningsmiddel, såsom methanol eller isobutanol til opnåelse af den tilsvarende iminoether. Denne hydrolyseres til opnåelse af 7-amino-3-halogen-3-cephem-4-earb-oxylsyre som et hydrogenchloridsalt.oxylic acid esters by performing the well-known N-deacylation of the 7-acylamido side chain. For example, the 7-aeylamido-3-halo gene cephalosporin ester is reacted with phosphorus pentachloride in methylene chloride in the presence of pyridine to form the imino chloride. This is reacted with an alcoholic solvent such as methanol or isobutanol to give the corresponding imino ether. This is hydrolyzed to give 7-amino-3-halogen-3-cephem-4-earb oxylic acid as a hydrochloride salt.

Som tidligere nævnt kan 7-amino-3-halogen-udgangsmaterialet acyleres enten som frie syrer eller som estre deraf. Acyleringen af 7-amino-3-halogen-kernen udføres som tidligere beskrevet.As previously mentioned, the 7-amino-3-halogen starting material can be acylated either as free acids or as esters thereof. The acylation of the 7-amino-3-halogen nucleus is carried out as previously described.

I en særlig udførelsesform for fremgangsmåden ifølge opfindelsen omsættes 7-phenoxyacetamido-cephalosporansyre med thiourinstof til opnåelse af St isothioroniumsalt ved substitution af acetoxygruppen i 3-stilling i cephalosporansyrens dihydrothiazinring. Iso-thioroniumsaltet omsættes dernæst med zink og overskud af 90 $ myresyre i nærværelse af dimethylformamid ved en temperatur på 25° C til opnåelse af 7-phenoxyacetamido-3-exomethylencepham-4-carboxylsyre. Denne esterificeres med p-nitrobenzylbromid i nærværelse af et hydrogenhalogenidbindende middel til opnåelse af 3-exomethylencepham-4-carboxylsyre-p-nitrobenzylester. Denne ozoniseres i methylenchlorid ved en temperatur på -70° C,og ozonolyseblandingen behandles med svovldioxid for at dekomponere ozonidet til opnåelse af p-nitrobenzyl-7-phenoxyacetamido-3-hydroxy-3-eephem-4-carboxylat. 3-Hydroxy-esteren omsættes dernæst med phosphortrichlorid i tørt dimethylformamid til opnåelse af p-nitrobenzyl-7-phenoxyacetamido-3“Chlor-3-cephem-4-carboxy-lat. 3-chlor-esteren omsættes dernæst i methylenchlorid med phos-phorpentachlorid i nærværelse af pyridin til opnåelse af imino-chlorid-mellemproduktet in situ, der dernæst omsættes med methanol til opnåelse af den tilsvarende iminoether. Ved tilsætning af vand til reaktionsblandingen dekomponeres iminoetheren, hvorved der opnås p-nitrobenzyl-7-amino-3-chlor-3-cephem-4-carboxylat. 7-Amino-3-chlor-3-cephem-4-carboxylsyreesteren kan dernæst acyleres som tidligere beskrevet med D-phenylglycylchlorid, hydrochlo-rid eller med et aminobeskyttet D-phenylglycylderivat til opnåelse af 7-(D-phenylglycylamido)-3-chlor-3-cephem-4-carboxylsyre, p-nitrobenzylester eller et N-beskyttet derivat deraf. EfterIn a particular embodiment of the process of the invention, 7-phenoxyacetamido-cephalosporanoic acid is reacted with thiourea to give St isothioronium salt by substitution of the 3-position acetoxy group in the dihydrothiazine ring of the cephalosporanoic acid. The iso-thioronium salt is then reacted with zinc and excess 90 $ formic acid in the presence of dimethylformamide at a temperature of 25 ° C to give 7-phenoxyacetamido-3-exomethylene cepham-4-carboxylic acid. This is esterified with p-nitrobenzyl bromide in the presence of a hydrogen halide binding agent to give 3-exomethylene cepham-4-carboxylic acid p-nitrobenzyl ester. This is ozonized in methylene chloride at a temperature of -70 ° C and the ozonolysis mixture is treated with sulfur dioxide to decompose the ozonide to obtain p-nitrobenzyl-7-phenoxyacetamido-3-hydroxy-3-eephem-4-carboxylate. The 3-hydroxy ester is then reacted with phosphorus trichloride in dry dimethylformamide to give p-nitrobenzyl-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 give the imino chloride intermediate in situ, then reacted with methanol to give the corresponding imino ether. By adding water to the reaction mixture, the imino ether is decomposed to give p-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 with D-phenylglycyl chloride, hydrochloride or with an amino-protected D-phenylglycyl derivative to give 7- (D-phenylglycylamido) -3-chloro -3-cephem-4-carboxylic acid, p-nitrobenzyl ester or an N-protected derivative thereof. After

17 DK 153157 B17 DK 153157 B

fjernelse af den α-amino-beskyttende gruppe og C^-carboxylsyre-estergruppen opnås der 7-(I>-phenylglycylainido)-3-chlor-3-cephem- 4-carboxylsyre. Det er klart fra den foregående omtale af fremstillingen af udgangsmaterialerne, at der kan anvendes en lang række kendte 7-acylamidocephalosporansyrer ved fremstilling af 7-amino-3-halogen-3-cephem-4-carboxylsyrer.removal of the α-amino-protecting group and the C1-carboxylic acid ester group yields 7- (1H-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid. It is clear from the foregoing discussion of the preparation of the starting materials that a wide variety of known 7-acylamidocephalosporanoic acids can be used in the preparation of 7-amino-3-halogen-3-cephem-4-carboxylic acids.

En foretrukken gruppe af forbindelser ifølge opfindelsen er de, der er repræsenteret ved formel I, hvor X betegner chlor, R er hydrogen, og R er phenyl, og farmaceutisk acceptable, ikke-toxi-ske salte deraf. Eor eksempel er 7-(D-a-phenylglycylamido)-3-chlor-3-cephem-4-carboxylsyre en foretrukken forbindelse.A preferred group of compounds of the invention are those represented by Formula I wherein X represents chloro, R is hydrogen and R is phenyl, and pharmaceutically acceptable, non-toxic salts thereof. For example, 7- (D-α-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid is a preferred compound.

En anden foretrukken gruppe af forbindelser ifølge opfindelsen består af forbindelser med formel I, hvor R er hydroxyphenyl, R·*" er hydrogen, og X er chlor, samt farmaceutisk acceptable, ikke-toxiske salte deraf. Som illustrative eksempler på sådanne forbindelser kan nævnes 7-(Ε-α-4-hydroxyphenylglycylamido)-3-chlor-3-cephem-4-carboxylsyre og 7-(D-α-3-hydroxyphenylglycylamido)-3-chlor-3-cephem-4-carboxylsyre.Another preferred group of compounds of the invention consists of compounds of formula I wherein R is hydroxyphenyl, R R is hydrogen and X is chlorine, as well as pharmaceutically acceptable, non-toxic salts thereof. Illustrative examples of such compounds may be mentioned. 7- (Ε-α-4-hydroxyphenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid and 7- (D-α-3-hydroxyphenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid.

Et særligt foretrukkent antibiotikum fremstillet ifølge opfindelsen er 7-(D-phenylglycylamido)-3-chlor-3-cephem-4-carboxylsyre.A particularly preferred antibiotic prepared according to the invention is 7- (D-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid.

Eølgende eksempler illustrerer nærmere fremgangsmåden ifølge opfindelsen.The following examples further illustrate the process of the invention.

Eksemgel_2 p-nitrobenzyl-7-amino-3-methylencepham-4-carboxylat1^hydrochloridExample Gel p-nitrobenzyl-7-amino-3-methyleneepham-4-carboxylate hydrochloride

Til en opløsning af 965 mg (2 mmol) p-nitrobenzyl-7-phenoxyacet-amido-3-methylencepham-4-carboxylat i 10 ml methylenchlorid sattes 175 mg tør pyridin og 460 mg phosphorpentachlorid, og blandingen omrørtes ved stuetemperatur i 6 timer. 1 ml isobutanol sattes til blandingen, der henstod ved 0° -C natten over. Reaktionsproduktet , p-nitrobenzyl-7-amino-3-methylencepham-4-carb-oxylat, hydrochlorid, der dannedes som et krystallinsk bundfald, 18 DK 153157 S' filtreredes til opnåelse af 430 mg (58 % udbytte) af titelforbindelsen.To a solution of 965 mg (2 mmol) of p-nitrobenzyl-7-phenoxyacetamido-3-methylene cepham-4-carboxylate in 10 ml of methylene chloride was added 175 mg of dry pyridine and 460 mg of phosphorus pentachloride and the mixture was stirred at room temperature for 6 hours. 1 ml of isobutanol was added to the mixture which was left at 0 ° C overnight. The reaction product, p-nitrobenzyl-7-amino-3-methylene cepham-4-carb oxylate, hydrochloride formed as a crystalline precipitate, was filtered to give 430 mg (58% yield) of the title compound.

Grundstof-analyse for C^H^gN^O^SOl: teoretisk: C 46,69 - H 4,18 - N 10,89.Elemental analysis for C ^H ^ gNNO₂SOl: theory: C 46.69 - H 4.18 - N 10.89.

fundet: 0 46,40 - H 4,20 - N 10,62.found: 0 46.40 - H 4.20 - N 10.62.

I.R. (Nujol Mull): C ar b onylab sorption ved 5,65 (β-lactam) og 5,75 (ester) γ..I.R. (Nujol Mull): C ar b onylab sorption at 5.65 (β-lactam) and 5.75 (ester) γ.

N.M.R. (DMS0 dg):N.M.R. (DMS0 dg):

Signaler ved 6,34 (2d, 2H, 02-H2), 4,98 (d, 1H, Cg-H), 4,7 - 4,4 (m, 6H, C^-H, ester CH2, C^-CH2 og Ογ-Η) og 2,4 - 1,6 (m, 4H, aromatisk H) tau.Signals at 6.34 (2d, 2H, 02-H2), 4.98 (d, 1H, Cg-H), 4.7-4.4 (m, 6H, C1-H, ester CH2, C1 -CH2 and Ογ-Η) and 2.4 - 1.6 (m, 4H, aromatic H) tau.

Eksempel_2Eksempel_2

El^iii2^®S^yizZz2?i22z^I^Z^£25Zr|-ce£liem-4-carbox2;la;ti_liydrocklori:dEl ^ III 2 ^ ^ ®S yizZz2? I22z ^ I ^ Z ^ £ 25Zr | -CE £ liem-4-carbox2; la; ti_liydrocklori: d

En opløsning af 4 g p-nitrobenzyl-7-amino-3-methylencepliam-4-carboxylat, hydrochlorid i 620 ml methanol afkøledes i et tøris-acetone-bad, og ozon bobledes igennem den kolde opløsning i 20 minutter. Reaktionsblandingen befriedes for den tiloversblevne ozon ved at lede nitrogen igennem opløsningen, og der tilsattes 10 g natriumbisulfit.A solution of 4 g of p-nitrobenzyl-7-amino-3-methylenecepliam-4-carboxylate, hydrochloride in 620 ml of methanol was cooled in a dry ice-acetone bath and ozone was bubbled through the cold solution for 20 minutes. The reaction mixture was liberated for the remaining ozone by passing nitrogen through the solution and 10 g of sodium bisulfite was added.

Reaktionsblandingen omrørtes i en time ved 0° C, hvorefter blandingen gav en negativ kaliumiodid-stivelsesprøve.The reaction mixture was stirred for one hour at 0 ° C, after which the mixture gave a negative potassium iodide starch sample.

Blandingen inddampedes i vakuum til opnåelse af et reaktions-produkt som et amorft gult fast stof. Remanensen omkrystalliseredes i acetone til opnåelse af 3,4 g p-nitrobenzyl-7-amino-3-hydroxy-3-cepbem-4-carbo2ylat, hydrochlorid som et krystallinsk acetonesolvat.The mixture was evaporated in vacuo to give a reaction product as an amorphous yellow solid. The residue was recrystallized in acetone to give 3.4 g of p-nitrobenzyl-7-amino-3-hydroxy-3-cepbem-4-carboxylate hydrochloride as a crystalline acetone solvate.

DK 153157 BDK 153157 B

I.R. (Nujol Mull):I.R. (Nujol Mull):

Carbonyl-absorptionsbånd ved 5,60 (β-lactam) og 6.04 (estercarbonylhydrogen bundet til 3-hydroxy) mp..Carbonyl absorption band at 5.60 (β-lactam) and 6.04 (ester carbonyl hydrogen bonded to 3-hydroxy) mp.

N.M.R, (DMSO dg):N.M.R, (DMSO dg):

Signaler ved 7,92 (s, 3H, 1/2 mol acetone), 6,22 (2d, 2H, Cg-Hg), 5.07 (d, 1H, CgH), 4.8 - 4,5 (m, 3H, ester CHg og ΟγΗ), 2.4 - 1,6 (m, 4H, aromatisk H) tau.Signals at 7.92 (s, 3H, 1/2 mol acetone), 6.22 (2d, 2H, Cg-Hg), 5.07 (d, 1H, CgH), 4.8 - 4.5 (m, 3H, ester) CHg and ΟγΗ), 2.4 - 1.6 (m, 4H, aromatic H) tau.

Eksempel 5Example 5

En opløsning af 2,5 g p-meth.oxybenzyl-7-ph.enoxyacetamido-3-methylencepham-4-car'bo2ylat i 350 ml ethylacetat afkøledes i et acetone-tøris'bad. Ozon bobledes igennem den kolde opløsning i 8 minutter, og dernæst ledtes oxygen igennem den ozoniserede reaktionsblanding for at fjerne overskud af ozon. Mellemproduktet dekomponeredes ved at sætte 25 g natriumbisulfit til reaktionsblandingen under omrøring ved en temperatur på 0° 0. Reaktionsopløsningen dekanteredes og vaskedes successivt med vand, 5 % saltsyre og en mættet opløsning af natriumchlorid. Vaskeblandingen tørredes og afdampedes til opnåelse af et reaktionsprodukt, nemlig p-meth.oxybenzyl-7-plienoxyacetainido-3-hydroxy- 3-ceph.em-4-carboxylat som et amorft fast stof.A solution of 2.5 g of p-methoxybenzyl-7-phenoxyacetamido-3-methylencepham-4-carboxylate in 350 ml of ethyl acetate was cooled in an acetone-dry ice bath. Ozone was bubbled through the cold solution for 8 minutes and then oxygen was passed through the ozonized reaction mixture to remove excess ozone. The intermediate was decomposed by adding 25 g of sodium bisulfite to the reaction mixture with stirring at a temperature of 0 ° 0. The reaction solution was decanted and washed successively with water, 5% hydrochloric acid and a saturated solution of sodium chloride. The wash mixture was dried and evaporated to give a reaction product, namely, p-methoxybenzyl-7-plienoxyacetamido-3-hydroxy-3-cephem-4-carboxylate as an amorphous solid.

N.M.R. (CDClj):N.M.R. (CDCl):

Signaler ved 6,75 (s, 2H, C2-H2), 6,23 (s, 3H, p-methoxy), 5.53 (d, 1H, CgH), 4,87 (s, 2H, ester CHg), 4,47 (q, 1H, C7H), 3,40 - 2,50 (m, 9H, aromatisk H), 2,33 (d, 1Ξ, amid NH) og 1.53 (bred s, 1H, 3 OH) tau.Signals at 6.75 (s, 2H, C2-H2), 6.23 (s, 3H, p-methoxy), 5.53 (d, 1H, CgH), 4.87 (s, 2H, ester CHg), 4 , 47 (q, 1H, C7H), 3.40 - 2.50 (m, 9H, aromatic H), 2.33 (d, 1Ξ, amide NH) and 1.53 (broad s, 1H, 3 OH) tau.

2020

DK 153157 BDK 153157 B

Eksemgel_4 p-nitrobenzyl-7-[2-(2-thienyl)acetamido]-3-hydro:xy-3-cephem-4-__car^bozylat_________ 5Dil en opløsning af 1,55 g p-nitrobenzyl-7-amino-3-hydro:xy-3-cephem-4-carboxylat, hydrochlorid i 30 ml acetone indeholdende 364 mg (0,5 ml, 3»6 mmol) triethylamin sattes 962 mg urinstof.Example Gel 4 p-Nitrobenzyl-7- [2- (2-thienyl) acetamido] -3-hydroxy-3-cephem-4-carbozylate -hydro: xy-3-cephem-4-carboxylate hydrochloride in 30 ml of acetone containing 364 mg (0.5 ml, 3 »6 mmol) of triethylamine was added 962 mg of urea.

Under omrøring ved stuetemperatur tilsattes dråbevis en opløsning af 730 mg (4f4 mmol) 2-thiophenacetylchlorid i 20 ml acetone. Efter 2,5 timer blev reaktionsblandingen filtreret og inddampet. Remanensen opløstes i ethylacetat, og opløsningen vaskedes successivt med vand, en 5 % opløsning af natrium-bicarbonat, 5 % saltsyre og en mættet opløsning af natrium-chlorid. hen vaskede opløsning tørredes og koncentreredes dernæst ved inddampning i vakuum til opnåelse af 1,2 g af reaktionsproduktet som et krystallinsk fast stof. Produktet omkrystalliseredes fra ethylacetat til opnåelse af rent p-nitrobenzyl-7-[2-(2-thienyl)acetamido]-3-hydroxy-3-cephem-4-carhoxylat, der har følgende spektrale egenskaber: I.R. (Nujol Mull):While stirring at room temperature, a solution of 730 mg (4f4 mmol) of 2-thiophenacetyl chloride in 20 ml of acetone was added dropwise. 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 bicarbonate, 5% hydrochloric acid and a saturated solution of sodium chloride. The washed solution was dried and then concentrated by evaporation in vacuo to give 1.2 g of the reaction product as a crystalline solid. The product was recrystallized from ethyl acetate to give pure p-nitrobenzyl 7- [2- (2-thienyl) acetamido] -3-hydroxy-3-cephem-4-carhoxylate having the following spectral properties: I.R. (Nujol Mull):

Absorptionstoppe ved 3.0 (amid NH), 5,68 (β-lactam-carbonyl) og 6.1 (amid og ester hydrogen bundet til 3 OH) mu.Absorption peaks at 3.0 (amide NH), 5.68 (β-lactam carbonyl) and 6.1 (amide and ester hydrogen bonded to 3 OH) mu.

N.M.R. (ChCl^/hMSO d6):N.M.R. (ChCl3 / hMSO d6):

Signaler ved 6,54 (2d, 2H, C2H2), 6,16 (s, 2H, sidekæde CH2), 4,90 (d, 1H, C6H), 4,60 (d, 2H, ester CH2), 4,43 (g, 1H, C?H), 3.1 - 1,6 (m, 7H, aromatisk H) og 1,30 (d, 1H, amid NH) tau.Signals at 6.54 (2d, 2H, C2H2), 6.16 (s, 2H, side chain CH2), 4.90 (d, 1H, C6H), 4.60 (d, 2H, ester CH2), 4, 43 (g, 1H, C? H), 3.1 - 1.6 (m, 7H, aromatic H) and 1.30 (d, 1H, amide NH) tau.

21 DK 153157 B21 DK 153157 B

Eksemgel^ £-nitrobenzYl-7-acetamido-3-h2drox2i3-2eE£em-4-carbox2latExample Gel-Nitrobenzyl-7-acetamido-3-hydroxyl-2,3-eem-4-carboxylate

En opløsning af 10 mmol p-nitrobenzyl-7-amino~3-hydroxy-3-cephem- 4-carboxylat, hydrochlorid i en blanding af 325 ml acetone og 125 ml vand afkøledes i et is-vandbad. Under omrøring ledtes en strøm af ketengas igennem opløsningen i 30 minutter. Herefter inddampedes reaktionsblandingen for at fjerne acetonen, og den vandige remanens opslemmedes i ethylacetat. Ethylacetat-fassn frasepareredes og vaskedes med 5 % saltsyre og en mættet opløsning af natriumchlorid. Een vaskede ekstrakt tørredes og inddampedes i vakuum til opnåelse af et reaktionsprodukt som et krystallinsk fast stof. Eette behandledes med diethylether og vakuumtørredes til opnåelse af 3,55 g p-nitro-benzyl-7-acetamido-3-hydroxy-3-cephem-4-carboxylat, der smeltede ved 146 - 152° C under dekomponering.A solution of 10 mmol of p-nitrobenzyl 7-amino-3-hydroxy-3-cephem-4-carboxylate, hydrochloride in a mixture of 325 ml of acetone and 125 ml of water was cooled in an ice-water bath. With stirring, a stream of ketene gas was passed through the solution for 30 minutes. Then, the reaction mixture was evaporated to remove the acetone and the aqueous residue was slurried in ethyl acetate. The ethyl acetate phase was separated and washed with 5% hydrochloric acid and a saturated solution of sodium chloride. A washed extract was dried and evaporated in vacuo to give a reaction product as a crystalline solid. The ether was treated with diethyl ether and vacuum dried to give 3.55 g of p-nitro-benzyl-7-acetamido-3-hydroxy-3-cephem-4-carboxylate, which melted at 146 DEG-152 DEG C. during decomposition.

Grundstof-analyse for C^gH^N^OyS: teoretisk: C 48,85 - H 3,84 - N 10,68.Elemental analysis for C ^ gH ^N ^O OyS: theory: C 48.85 - H 3.84 - N 10.68.

fundet: C 48,97 - H 3,96 - N 10,42.Found: C 48.97 - H 3.96 - N 10.42.

I.R. (CHC15):I.R. (CHC15):

Absorptionsbånd ved 2,9 og 3,0 (amid NH og OH), 5.63 (β-lactam-carbonyl)og 5,95 (bredt amid og estercarbonylhydrogen bundet til 3 OH) ιημ.Absorption bands at 2.9 and 3.0 (amide NH and OH), 5.63 (β-lactam carbonyl) and 5.95 (broad amide and ester carbonyl hydrogen bonded to 3 OH) ιημ.

N.M.R. (CBOlj):N.M.R. (CBOlj):

Signaler ved 7,90 (s, 3H, 7-acetamido-CH^), 6,55 (s, 2H, C2H2), 4,92 (d, 1H, C6H), 4.63 (m, 2H, ester CH2), 4,30 (q, 1H, C7H), 2,81 (d, 1H, amid NH), 2,5 - 1,8 .(m, 4H, aromatisk H) og 2,8 (s, 1H, C3 OH) tau.Signals at 7.90 (s, 3H, 7-acetamido-CH 2), 6.55 (s, 2H, C 2 H 2), 4.92 (d, 1 H, C 6 H), 4.63 (m, 2H, ester CH 2), 4.30 (q, 1H, C7H), 2.81 (d, 1H, amide NH), 2.5 - 1.8 (m, 4H, aromatic H) and 2.8 (s, 1H, C3 OH) ) tau.

22 DK 153157 B22 DK 153157 B

Elektrometrisk titrering (66 fo vandig DMP) pKa 5,9.Electrometric titration (66 fo aqueous DMP) pKa 5.9.

Eksempel 6Example 6

Diphenyl-7-[ 2-(2-thienyl)acetamidoJ-5-chlor-5-cephem.-4-carboxylat (a) Til en opløsning af 34 g (100 mmol) 7-[2-(2-thienyl)acet-amido ]-3-methylencepham-4-carboxylsyre i 500 ml methylenchlorid sattes 21,4 g (110 mmol) diphenyldiazome than, og den fremkomne blanding omrørtes i 2 timer ved stuetemperatur. Opløsningsmidlet inddampedes under reduceret tryk, og remanensen opløstes i ethyl-acetat. Ethylacetatopløsningen vaskedes med en 5 opløsning af natriumbicarbonat, dernæst med vand og tørredes over magnesiumsulfat. Den tørrede opløsning koncentreredes til et lille volumen. Yed henstand udfældedes 40 g diphenylmethyl-7-[2-(2-thienyl)-acetamido]-3-methylencepham-4-carboxylat som et krystallinsk fast stof, der smeltede ved 132 - 133° 0.Diphenyl 7- [2- (2-thienyl) acetamido] -5-chloro-5-cephem-4-carboxylate (a) To a solution of 34 g (100 mmol) of 7- [2- (2-thienyl) acetate -amido] -3-methylenecepham-4-carboxylic acid in 500 ml of methylene chloride was added 21.4 g (110 mmol) of diphenyl diazomene and the resulting mixture was stirred for 2 hours at room temperature. The solvent was evaporated under reduced pressure and the residue dissolved in ethyl acetate. The ethyl acetate solution was washed with a solution of sodium bicarbonate, then with water and dried over magnesium sulfate. The dried solution was concentrated to a small volume. Upon standing, 40 g of diphenylmethyl-7- [2- (2-thienyl) acetamido] -3-methylene cepham-4-carboxylate precipitated as a crystalline solid, melting at 132-133 °.

I.R. (chloroform):I.R. (Chloroform):

Absorptionstoppe ved 2,9 (amidN-H), 5,65} 5,75 og 5,95 (β-lactam, ester og amidcarbonyl) og 6,62 (amid II) mp., N.M.R. (CDCl^):Absorption peaks at 2.9 (amide N-H), 5.65} 5.75 and 5.95 (β-lactam, ester and amide carbonyl) and 6.62 (amide II) mp., N.M.R. (CDCl ^):

Signaler ved 6,72 (ABq., 2H, Og-Hg), 6,21 (s, 2H, a-0H2), 4,83 - 4,65 (m, 4H, O^-H, Cg-H og C3-CH2), 4,39 (cl, 1H, C7-H), 3,4 - 2,65 (m, 15H, Ογ-NH, ester CH og aromatisk H) tau.Signals at 6.72 (ABq., 2H, Og-Hg), 6.21 (s, 2H, α-OH), 4.83 - 4.65 (m, 4H, O C3-CH2), 4.39 (cl, 1H, C7-H), 3.4 - 2.65 (m, 15H, Ογ-NH, ester CH and aromatic H) tau.

(b) Til en opløsning af 8,1 g (16 mmol) af ovennævnte ester i 80 ml methylenchlorid sattes 1,57 g (1,6 ml, 19,6 mmol) tør pyridin og 3,8 g (18,1 mmol) phosphorpentachlorid. Reaktionsblandingen omrørtes i 2 timer ved stuetemperatur og afkøledes derefter i et is-vandbad. Den kolde blanding behandledes med 8 ml isobutanol under omrøring. Omrøring fortsattes i 2 timer, hvorunder 3 g diphenylmethyl-7-amino-3-methylencepham-4-carb02ylat, hydrochlorid(b) To a solution of 8.1 g (16 mmol) of the above ester in 80 ml of methylene chloride was added 1.57 g (1.6 ml, 19.6 mmol) of dry pyridine and 3.8 g (18.1 mmol) phosphorus pentachloride. The reaction mixture was stirred for 2 hours at room temperature and then cooled in an ice-water bath. The cold mixture was treated with 8 ml of isobutanol with stirring. Stirring was continued for 2 hours, during which 3 g of diphenylmethyl-7-amino-3-methylenepepham-4-carboxylic acid hydrochloride

23 DK 153157 B23 DK 153157 B

dannedes som et krystallinsk bundfald. Produktet filtreredes fra og vaskedes med methylenchlorid og vakuumtørredes.formed as a crystalline precipitate. The product was filtered off and washed with methylene chloride and vacuum dried.

Grundstof-analyse ($) for C21H21W2°3SC1: teoretisk: C 60,50 - H 5,08 - N 6,72 - Cl 8,50.Elemental analysis ($) for C 21 H 21 W 2 ° 3 SC1: theoretical: C 60.50 - H 5.08 - N 6.72 - Cl 8.50.

fundet: 0 60,70 - H 5,02 - N 6,71 - Cl 8,80.found: 0 60.70 - H 5.02 - N 6.71 - Cl 8.80.

N.M.R. (DMSO d6):N.M.R. (DMSO d6):

Signaler ved 6,45 (ABq., 2H, C2-H2), 5.00 (d, 1H, C6-H), 4,68 (d, 1H, Ογ-Ξ), 4.60 (s, 2H, 3-CH2), 4,44 (s, 1H, C4-H), 3,10 (s, 1H, ester CH) og 2.61 (s, 10H, aromatisk H) tau.Signals at 6.45 (ABq., 2H, C2-H2), 5.00 (d, 1H, C6-H), 4.68 (d, 1H, Ογ-Ξ), 4.60 (s, 2H, 3-CH2) , 4.44 (s, 1H, C4-H), 3.10 (s, 1H, ester CH) and 2.61 (s, 10H, aromatic H) tau.

(c) Hydrochloridsaltet af 7-amino-3-exomethylencephamester, i alt 2,1 g (5 mmol), opløstes i 200 ml methanol, og opløsningen afkøledes i et acetone-tørisbad. Ozon bobledes igennem den kolde opløsning i 7 minutter til dannelse af ozonidet. Dette dekompo-neredes ved at lede en strøm af svovldioxidgas igennem reaktionsblandingen i 2 minutter. Herefter inddampedes reaktionsblandingen, og remanensen behandledes med diethylether til opnåelse af 1,6 g diphenylmethyl-7-amino-3-hydroxy-3-cephem-4-carboxylat, hydrochlorid som et krystallinsk fast stof.(c) The hydrochloride salt of 7-amino-3-exomethylene cephame ester, a total of 2.1 g (5 mmol), was dissolved in 200 ml of methanol and the solution was cooled in an acetone-dry ice bath. Ozone was bubbled through the cold solution for 7 minutes to form the ozonide. This was decomposed by passing a stream of sulfur dioxide gas through the reaction mixture for 2 minutes. The reaction mixture was then evaporated and the residue treated with diethyl ether to give 1.6 g of diphenylmethyl-7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride as a crystalline solid.

N.M.R. (CDC15):N.M.R. (CDC15):

Signaler ved 6,4 (ABq., 2H, C2—H2), 5.0 - 4,5 (m, 2H, Cg-H og Ογ-Η), 3,2 - 2,4 (m, 11H, ester CH og aromatisk H) tau.Signals at 6.4 (ABq., 2H, C2-H2), 5.0 - 4.5 (m, 2H, Cg-H and γγ-Η), 3.2 - 2.4 (m, 11H, ester CH and aromatic H) rope.

I.R. (chloroform):I.R. (Chloroform):

Carbonylabsorptionstoppe ved 5,57 og 5,70 (henholdsvis β-lactam og estercarbonyl) ιημ.Carbonyl absorption peaks at 5.57 and 5.70 (β-lactam and ester carbonyl, respectively) ιημ.

U.Y. (pH 7 buffer): X max 275 ιημ, 7550.U.Y. (pH 7 buffer): X max 275 ιημ, 7550.

u DK 153157 Bu DK 153157 B

Elektrometrisk titrering (60 $ ag. DMP): titrerhare grupper ved 4,5 og 6,5.Electrometric titration ($ 60 ag DMP): titrate groups at 4.5 and 6.5.

(d) Til en opløsning af 840 mg diphenylmethyl-7-amino--3--hydro:xy- 3-cephem-4-carBoxylat i 10 ml vand og 10 ml acetone sattes 1 g natriumhisulfit. Blandingen omrørtes, og 800 mg thiophen-2-acetylchlorid i 10 ml acetone tilsattes dråBevis. Blandingen omrørtes i 4?5 timer ved stuetemperatur og inddampedes dernæst under reduceret tryk. Remanensen opløstes i en Blanding af ethyl-acetat og en vandig 5 $ opløsning af natriumBicarBonat. Ethyl-acetatfasen frasepareredes, vaskedes med vand og tørredes. Den tørrede opløsning inddampedes, og remanensen Behandledes med ether til opnåelse af 500 mg diphenylmethyl-7-[2-(2-thienyl)acetamido]- 3-hydroxy-3-cephem-4-carBoxylat.(d) To a solution of 840 mg of diphenylmethyl-7-amino-3-hydro: xy-3-cephem-4-carboxylate in 10 ml of water and 10 ml of acetone was added 1 g of sodium bisulfite. The mixture was stirred and 800 mg of thiophene-2-acetyl chloride in 10 ml of acetone was added dropwise. The mixture was stirred for 4-5 hours at room temperature and then evaporated under reduced pressure. The residue was dissolved in a mixture of ethyl acetate and an aqueous 5 $ solution of sodium bicarbonate. The ethyl acetate phase was separated, washed with water and dried. The dried solution was evaporated and the residue Treated with ether to give 500 mg of diphenylmethyl-7- [2- (2-thienyl) acetamido] -3-hydroxy-3-cephem-4-carboxylate.

N.M.R. (CDClj):N.M.R. (CDCl):

Signaler ved 6,79 (s, 2H, C2-H2), 6,16 (s, 2H, a-CH2), 5,0 (d, 1H, C6-H), 4,32 (cl, 1H, C7-H), 3,05 - 2,46 (m, 15H, Ογ-ΝΗ, ester CH og aromatisk H) tau.Signals at 6.79 (s, 2H, C2-H2), 6.16 (s, 2H, a-CH2), 5.0 (d, 1H, C6-H), 4.32 (cl, 1H, C7 -H), 3.05 - 2.46 (m, 15H, Ογ-ΝΗ, ester CH and aromatic H) tau.

I.R. (chloroform): ABsorptionstoppe ved 2,9 (amid NH), 5,6, 5,73 og 5,95 (henholdsvis β-lactam, ester og amidcarBonyler) og 6,65 (amid II) mp.I.R. (chloroform): ABsorption peaks at 2.9 (amide NH), 5.6, 5.73 and 5.95 (β-lactam, ester and amide Carbonyls respectively) and 6.65 (amide II) mp.

(e) Til en opløsning af 4,2 g diphenylmethyl-7-[2-(2-thienyl)-acetamido]-3-hydroxy-3-oephem-4-carBoxylat i 44 ml tør dimethyl-formamid sattes 865 mg phosphortrichlorid. Blandingen omrørtes 1,5 timer ved stuetemperatur og udhældtes i en Blanding af ethyl-acetat og 5 i° vandig saltsyre. Ethylacetatfasen inddampedes, vaskedes med 5 % saltsyre og med vand og tørredes. Den tørrede opløsning koncentreredes i vakuum, hvorved produktet udkrystalliserede. 3-chloresteren filtreredes fra, vaskedes med kold ethylacetat og tørredes til opnåelse af 2,2 g af titelforbindelsen.(e) To a solution of 4.2 g of diphenylmethyl 7- [2- (2-thienyl) acetamido] -3-hydroxy-3-oephem-4-carboxylate in 44 ml of dry dimethyl formamide was added 865 mg of phosphorus trichloride. The mixture was stirred for 1.5 hours at room temperature and poured into a mixture of ethyl acetate and 5 in aqueous hydrochloric acid. The ethyl acetate phase was evaporated, washed with 5% hydrochloric acid and with water and dried. The dried solution was concentrated in vacuo to crystallize the product. The 3-chloro ester was filtered off, washed with cold ethyl acetate and dried to give 2.2 g of the title compound.

25 DK 153157 B :;DK: 153157 B :;

Grundstof-analyse {i») for C26H21N2°4^2C1: teoretisk: C 59,48 - H 4,05 - N 5,34 - Cl 6,75.Elemental analysis (i) for C26H21N2 ° 4 ^ 2Cl: theory: C 59.48 - H 4.05 - N 5.34 - Cl 6.75.

fundet: C 59,77 - H 4,25 - N 5,40 - Cl 6,91.Found: C 59.77 - H 4.25 - N 5.40 - Cl 6.91.

N.M.R. (CDOlj):N.M.R. (CDOlj):

Signaler ved 6,49 (ABq., 2H, , 6,22 (s, 2H, a-0H2), 5.08 (d, 1H, 06-H), 4,19 (G, 1H, C?-H), 3,13-25, (m, 15H, Ογ-ΝΗ, ester CH og aromatisk H) tau.Signals at 6.49 (ABq., 2H,, 6.22 (s, 2H, α-OH), 5.08 (d, 1H, 06-H), 4.19 (G, 1H, C? -H), 3.13-25, (m, 15H, Ογ-ΝΗ, ester CH and aromatic H) tau.

I.R. (CHOlj):I.R. (CHOlj):

Absorptionstoppe ved 2.9 (amid NH), 5,55, 5,72 og 5,90 (β-lactam, ester og amidcarbonyler) og 6,60 (amid II) mu.Absorption peaks at 2.9 (amide NH), 5.55, 5.72 and 5.90 (β-lactam, ester and amide carbonyls) and 6.60 (amide II) mu.

U.V. (dioxan): λ max 275 mu, ε=87Ο0.U.V (dioxane): λ max 275 mu, ε = 87Ο0.

Eksempel_7 p-nitrobenzyl-7-[ 2-( 2-thienyl)acetamido] -5-chlor-3-cephem-4--carb-__oxylat__iyia_tbionylcblorid2___________________________________Example 7 p-Nitrobenzyl-7- [2- (2-thienyl) acetamido] -5-chloro-3-cephem-4-carboxylic acid oxyylthiobionyl chloride

Til en opløsning af 1,9 g (4 mmol) p-nitrobenzyl-7-[2-(2-thienyl)-acetamido]-3-hydroxy-3-cephem-4-carboxylat i 10 ml dimethylformamid (tørret over en molekylsi) sattes 950 mg (0,58 ml, S mmol) frisk destilleret thionylchlorid. Blandingen omnørtes ved stuetemperatur i 6,5 timer og udhældtes i 100 ml ethylacetat.To a solution of 1.9 g (4 mmol) of p-nitrobenzyl-7- [2- (2-thienyl) acetamido] -3-hydroxy-3-cephem-4-carboxylate in 10 ml of dimethylformamide (dried over a molecular sieve) 950 mg (0.58 ml, S mmol) of freshly distilled thionyl chloride was added. The mixture was stirred at room temperature for 6.5 hours and poured into 100 ml of ethyl acetate.

Blandingen ekstraheredes tre gange med 50 ml portioner :5 f° hydro-genchloridsyre og med en mættet opløsning af natriumchlorid. Den vaskede ethylacetatopløsning filtreredes fra og inddampedes til tørhed i vakuum. Remanensen behandledes med ether til opnåelse af 1,2 g p-nitrobenzyl-7-[2-(2-thienyl)acetamido]-3-chlor-3-cephem- 4-carboxylat som et brunt krystallinsk fast stof med et smeltepunkt på 164 - 166° C.The mixture was extracted three times with 50 ml portions: 5 µl of hydrochloric acid and with a saturated solution of sodium chloride. The washed ethyl acetate solution was filtered off and evaporated to dryness in vacuo. The residue was treated with ether to give 1.2 g of p-nitrobenzyl 7- [2- (2-thienyl) acetamido] -3-chloro-3-cephem-4-carboxylate as a brown crystalline solid, m.p. - 166 ° C.

26 DK 153157B26 DK 153157B

Grundstof-analyse (<fo) for C20H16N306S2C1: teoretisk: C 48,63 - H 3,27 - N 8,51 - 01 7,18.Elemental analysis (<fo) for C20 H16 N3 O6 S2 Cl: theory: C 48.63 - H 3.27 - N 8.51 - 01 7.18.

fundet: C 48,47 - H 3,29 - N 8,78 - Cl 6,96.Found: C 48.47 - H 3.29 - N 8.78 - Cl 6.96.

I.R. (chloroform):I.R. (Chloroform):

Absorptionsbånd ved 2,9 (amidNH), 5,59 (β-lactamcarhonyl), 5,75 (estercarhonyl) og 5,92 mp (amidcarbonyl).Absorption bands at 2.9 (amide NH), 5.59 (β-lactamcarhonyl), 5.75 (estercarhonyl) and 5.92 mp (amide carbonyl).

U.B, ahsorptionsspektrum (acetonitril) viste maxima ved X max 235 mp, ε 12.100 X max 268 mp, e=15.800.U.B, ahsorption spectrum (acetonitrile) showed maxima at X max 235 mp, ε 12,100 X max 268 mp, e = 15,800.

Massespektret af produktet viste en molekylærion på 493 m/e.The mass spectrum of the product showed a molecular ion of 493 m / e.

N.M.R. (CDOl^) viste signaler ved 6,39 (ABq., 2H, Cg-Hg), 6,17 (s, 2H, a-CH2), 4,99 (d, 1H, C6-H), 4,64 (s, 2H, ester CH2), 4,19 (q, 1H, C?-H), 3,45 (d, 1H, C7-NH), 3,1 - 1,67 (m, 7H, aromatisk H) tau.N.M.R. (CDO1) showed signals at 6.39 (ABq., 2H, Cg-Hg), 6.17 (s, 2H, α-CH2), 4.99 (d, 1H, C6-H), 4.64 (s, 2H, ester CH 2), 4.19 (q, 1H, C?-H), 3.45 (d, 1H, C7-NH), 3.1 - 1.67 (m, 7H, aromatic H ) tau.

Eksemgel_8 p-nitrobenzyl-7-[2-(2-thienyl)acetamido]-3-chlor-3-cephem-4-carb-__oxylat (via phosphortrichlorid)_________________________Example Gel 8 p-Nitrobenzyl 7- [2- (2-thienyl) acetamido] -3-chloro-3-cephem-4-carb-oxylate (via phosphorus trichloride)

Til en afkølet opløsning af 439 mg (0,93 mmol) p-nitrobenzyl-7-[2-(2-thienyl)acetamido]-3-hydroxy-3-cephem-4-carhoxylat i 4,4 ml dimethylformamid sattes langsomt 85 mg (0,05 ml, 0,63 mmol) phosphortrichlorid. Reaktionsblandingen henstod 4 timer ved stuetemperatur ,og derefter oparbejdedes reaktionsproduktet ved at følge metoden beskrevet i eksempel 6 til opnåelse af 374 mg p-nitro-To a cooled solution of 439 mg (0.93 mmol) of p-nitrobenzyl 7- [2- (2-thienyl) acetamido] -3-hydroxy-3-cephem-4-carhoxylate in 4.4 ml of dimethylformamide was slowly added. phosphorus trichloride (mg, 0.05 ml, 0.63 mmol). The reaction mixture was allowed to stand for 4 hours at room temperature and then the reaction product was worked up following the method described in Example 6 to obtain 374 mg of

27 DK 153157 B27 DK 153157 B

benzyl-7-[ 2- (2-thienyl) acetamido ] -3-chlor-3-cephem-4-carboxylat. NMR-spektret af produktet stemte overens med det forventede produkt og med forbindelsen i eksempel 7.benzyl 7- [2- (2-thienyl) acetamido] -3-chloro-3-cephem-4-carboxylate. The NMR spectrum of the product was consistent with the expected product and with the compound of Example 7.

Eksempel_9 7-phenoxyacetamido-3~chlor-3-cephem-42carbox2lsyreExample 9 9 7-Phenoxyacetamido-3-chloro-3-cephem-42-carboxylic acid

Ved at følge chloreringsmetoden i eksempel 6 fremstilledes p-nitrobenzyl-7-phenoxyacetamido-3-chlor-3-cephem-4-carboxylat med phosphortrichlorid. p-nitrobenzylestergruppen fjernedes ved hydrogenering med hydrogen og 5 i° palladium-på-carbon til opnåelse af 3-chlorcephalosporansyre.Following the chlorination method of Example 6, p-nitrobenzyl-7-phenoxyacetamido-3-chloro-3-cephem-4-carboxylate was prepared with phosphorus trichloride. The p-nitrobenzyl ester group was removed by hydrogenation with hydrogen and 5 ° palladium-on-carbon to give 3-chlorocephalosporanoic acid.

Eksempel 10 p-nitrobenzyl-7-[2-(2-thienyl)aeetamido]-3-chlor-3-cephem-4-carb-__oxylat__iyia^oxalylchlorid)____________________________________Example 10 p-Nitrobenzyl-7- [2- (2-thienyl) ethylamido] -3-chloro-3-cephem-4-carb-oxylate-oxalylchloride)

Til en opløsning af 439 mg (0,93 mmol) p-nitrobenzyl-7-[2-(2-thienyl)acetamido]-3-hydroxy-3-cephem-4--carboxylat i 4»4 ml dime'thylformamid afkølet i et isbad sattes dråbevis 118 mg (0,07 ml, 0,93 mmol) oxalylchlorid. Reaktionsblandingen henstod i 4 timer ved stuetemperatur og udhældtes i en blanding af vandig 5 fo hydrogenchloridsyre og ethylacetat. Den organiske fase separeredes fra og vaskedes dernæst sekventielt med 5 % saltsyre, vand og en mættet opløsning af natriumchlorid.To a solution of 439 mg (0.93 mmol) of p-nitrobenzyl-7- [2- (2-thienyl) acetamido] -3-hydroxy-3-cephem-4-carboxylate in 4/4 ml of dimethylformamide cooled In an ice bath, 118 mg (0.07 ml, 0.93 mmol) of oxalyl chloride was added dropwise. The reaction mixture was allowed to stand for 4 hours at room temperature and poured into a mixture of aqueous 5 µl of hydrochloric acid and ethyl acetate. The organic phase was separated and then washed sequentially with 5% hydrochloric acid, water and a saturated solution of sodium chloride.

Den vaskede fase tørredes og inddampedes til tørhed, hvorved der opnåedes p-nitrobenzyl-7-[2-(2-thienyl)acetamido]-3-chlor-3-cephem-4-carboxylat som et amorft fast stof. Produktet blev opnået i krystallinsk form ved behandling med ether. Udbytte 360 mg. Det infrarøde spektrum og IMR-spektret af det krystallinske produkt stemte med spektret for et autentisk materiale.The washed phase was dried and evaporated to dryness to give p-nitrobenzyl 7- [2- (2-thienyl) acetamido] -3-chloro-3-cephem-4-carboxylate as an amorphous solid. The product was obtained in crystalline form by ether treatment. Yield 360 mg. The infrared spectrum and the IMR spectrum of the crystalline product matched the spectrum of an authentic material.

Eksempel^! 7-[2-(2-thienyl)acetamido]-3vbrom-3-cephem-4-garbo^lsyreExample ^! 7- [2- (2-thienyl) acetamido] -3vbrom-3-cephem-4-garbo ^ L acid

Til en opløsning af 19 g (40 mmol) p-nitrobenzyl-7-[2-(2-thienyl)-acetamido]-3-hydroxy-3-cephem-4-carboxylat i 300 ml tør dimethyl-To a solution of 19 g (40 mmol) of p-nitrobenzyl-7- [2- (2-thienyl) acetamido] -3-hydroxy-3-cephem-4-carboxylate in 300 ml of dry dimethyl acid

28 DK 153157 B28 DK 153157 B

formamid sattes 15 g (56 mmol) phosphortribromid, og reaktionsblandingen omrørtes ved stuetemperatur natten over. Reaktionsblandingen udhældt es i en blanding af ethylacetat og vand, og den organiske fase frasepareredes og vaskedes derefter gentagne gange med vand og tørredes over magnesiumsulfat. Den tørrede organiske fase inddampedes i vakuum til tørhed. Det rå reaktionsprodukt, der vejede ca. 9 g, oprensedes ved kromatografi over 500 g silicagel under anvendelse af ethylacetat-hexan (55:45 v:v) som elueringsmiddel. Eluatet inddampedes til tørhed under reduceret tryk, og produktet p-nitrobenzyl-7-[2-(2-thienyl)acetamido]-3-brom-3-cephem-4-carboxylat opnåedes i krystallinsk forir. ved at behandle den tørre remanens med diethylether.formamide was added 15 g (56 mmol) of phosphorus tribromide and the reaction mixture was stirred at room temperature overnight. The reaction mixture was poured into a mixture of ethyl acetate and water and the organic phase was separated and washed repeatedly with water and dried over magnesium sulfate. The dried organic phase was evaporated in vacuo to dryness. The crude reaction product, which weighed approx. 9 g, was purified by chromatography over 500 g of silica gel using ethyl acetate-hexane (55:45 v: v) as the eluent. The eluate was evaporated to dryness under reduced pressure and the product p-nitrobenzyl-7- [2- (2-thienyl) acetamido] -3-bromo-3-cephem-4-carboxylate was obtained in crystalline feed. by treating the dry residue with diethyl ether.

U.V. (ethanol): λ max. 270 mp (£=13.500) λ max. 245 mp (6=12.700).U.V (ethanol): λ max. 270 mp (£ = 13,500) λ max. 245 mp (6 = 12,700).

Grundstof-analyse beregnet for °20H16’BrN306S2: teoretisk: C 44,61 - H 3,00 - N 7,81 - Br 14,84.Elemental analysis calculated for ° 20H16'BrN306S2: theory: C 44.61 - H 3.00 - N 7.81 - Br 14.84.

fundet: 0 44,78 - H 3,03 - N 7,65 - Br 14,91.found: 0 44.78 - H 3.03 - N 7.65 - Br 14.91.

NMR-spektrum (DMS0 dg):NMR Spectrum (DMSO dg):

Signaler ved 6,21 (2H, a-CH2), 5,98 (ABq., 2H, C2-H2), 4,72 (d, 1H, C6-H), 4,51 (s, 2H, ester-CH2), 4,20 (q, 1H, C7-H), 3,04 - 1,74 (m, 7H; aromatisk H) og 0,66 (d, 1H, C7-CH) tau.Signals at 6.21 (2H, α-CH2), 5.98 (ABq., 2H, C2-H2), 4.72 (d, 1H, C6-H), 4.51 (s, 2H, ester CH2), 4.20 (q, 1H, C7-H), 3.04 - 1.74 (m, 7H; aromatic H) and 0.66 (d, 1H, C7-CH) tau.

Ovennævnte 3-bromester deesterificeredes på følgende måde: 545 mg (1,0 mmol) af esteren hydrogeneredes ved stuetemperatur i ethanol i nærværelse af præreduceret 5 procent palladium-på-car-bon katalysator. Katalysatoren filtreredes fra, og filtratet inddampedes under reduceret tryk til tørhed. Remanensen behandledes med diethylether til opnåelse af 180 mg (44 i«) af det krystallinske produkt, 7-[2-(2-thienyl)acetamido]-3-brom-3-cephem-4-' carboxylsyre.The above 3-bromoester was deesterified as follows: 545 mg (1.0 mmol) of the ester was hydrogenated at room temperature in ethanol in the presence of pre-reduced 5 percent palladium-on-carbon catalyst. The catalyst was filtered off and the filtrate was evaporated under reduced pressure to dryness. The residue was treated with diethyl ether to give 180 mg (44 i) of the crystalline product, 7- [2- (2-thienyl) acetamido] -3-bromo-3-cephem-4- 'carboxylic acid.

29 DK 153157 B29 DK 153157 B

Elektrometrisk titrering (66 $ vandig dimethylformamid) viste en pKa-værdi på 4*4 og en tilsyneladende molekylvægt på 393.Electrometric titration (66 $ aqueous dimethylformamide) showed a pKa value of 4 * 4 and an apparent molecular weight of 393.

Beregnet molekylvægt = 405.Calculated molecular weight = 405.

Grundstof-analyse for C^H·}-jBrNgO^S^ 1/2 diethyletherat: teoretisk: C 40,91 - H 3*66 - N 6,36 - Br 18,15.Elemental analysis for C ^H ·}-BBrNgO ^S₂ 1/2 diethyl etherate: theoretical: C 40.91 - H 3 * 66 - N 6.36 - Br 18.15.

fundet: C 41,29 - H 3,20 - N 6,29 - Br 18,,50.found: C 41.29 - H 3.20 - N 6.29 - Br 18,, 50.

NMR (CDC13) viste signaler ved 8.8 (t, diethylether-CH^), 6,68 - 5,86 (m, G^-R^, a-CIL, og diethylether-CB^), 4,90 (d, 1H, C6-H), 3,0 - 2,63 (m, 30, aromatisk-H) og 1.9 (d, 1H, amid NH) tau.NMR (CDCl 3) showed signals at 8.8 (t, diethyl ether-CH 2), 6.68 - 5.86 (m, G 1 -R 4, α-CIL, and diethyl ether-CB 2), 4.90 (d, 1H, C6-H), 3.0 - 2.63 (m, 30, aromatic-H) and 1.9 (d, 1H, amide NH) tau.

Eksempel 12 (a) p-nitrobenzyl-7-[2-(2-tMenyl)acetamido]-3-metliylsulfonyl- __________________________________Example 12 (a) p-Nitrobenzyl-7- [2- (2-phenyl) acetamido] -3-methylsulfonyl

Til en opløsning af 4,75 g (10 mmol) p-nitrobenzyl-7-[2-(2-thienyl)-acetamido]-3-bydroxy-3-cephem-4-carboxylat i 50 ml tør dimethyl-acetamid sattes 2 ml propylenoxid. Til opløsningen sattes under omrøring en ækvivalent mængde methansulfonylchlorid,· og omrøringen fortsattes i 3 timer. Reaktionsblandingen opløstes i ethylaeetat, og opløsningen vaskedes med en mættet opløsning af natriumcblorid.To a solution of 4.75 g (10 mmol) of p-nitrobenzyl 7- [2- (2-thienyl) acetamido] -3-hydroxy-3-cephem-4-carboxylate in 50 ml of dry dimethyl acetamide was added. ml of propylene oxide. To the solution was added stirring an equivalent amount of methanesulfonyl chloride, and stirring was continued for 3 hours. The reaction mixture was dissolved in ethyl acetate and the solution washed with a saturated solution of sodium chloride.

Den vaskede organiske fase inddampedes i vakuum til tørhed, hvorved der opnåedes en reaktionsproduktblanding som en remanens.The washed organic phase was evaporated in vacuo to dryness to give a reaction product mixture as a residue.

Denne oprensedes ved præparativ tyndtlagskromatografi på silica-gel under anvendelse af 65 $ ethylacetat/hexan som eluerings-middel.This was purified by preparative thin layer chromatography on silica gel using 65 $ ethyl acetate / hexane as eluent.

Det oprensede produkt gav følgende procentvise sammensætning ved mikroanalyse:The purified product gave the following percent composition by microanalysis:

Beregnet for C^H^N^OgS^: teoretisk: C 45,56 - H 3,46 - N 7,59 - S 17,38.Calculated for C C ^H NN ^O₂S: theory: C 45.56 - H 3.46 - N 7.59 - S 17.38.

fundet: C 45.74 - H 3.56 - N 7.30 - S 17.06.found: C 45.74 - H 3.56 - N 7.30 - S 17.06.

?0 DK 153157 B? 0 DK 153157 B

NMR-spektret og det infrarøde absorptionsspektrum stemte med strukturen af det dannede produkt.The NMR spectrum and the infrared absorption spectrum corresponded to the structure of the product formed.

(b) p-nitrobenzyl-7-[ 2-(2-thienyl)acetamido]-3-fluor-3-ceph.em- ______________________________________________(b) p-Nitrobenzyl-7- [2- (2-thienyl) acetamido] -3-fluoro-3-cephem - ______________________________________________

Til 93 mg dicyclohexyl-18-krone-6-et]ier i 15 ml acetonitril tørret over en molekylsi sattes 25 mg kaliumfluorid, der var tørret i vakuum ved 90° C. Blandingen omrørt es i 10 minutter, og 138 mg p-nitrobenzyl-7-[2-(2-thienyl)acetamido]-3-methylsulfonyloxy-3-ceph.em-4-carboxylat i 4 ml acetonitril tilsattes. Blandingen om-rørtes i 1 time. Blandingen blev gjort sur ved tilsætning af fortyndet (5 %) saltsyre, og den fremkomne sure blanding eks-traheredes med ethylacetat. Det rene produkt opnåedes fra ethyl-acetatekstrakten ved præparativ tyndtlagskromatografi på silica-gel, idet der som elueringsmiddel anvendtes ethylacetat:benzen (1:1). Der opnåedes 10 mg p-nitrobenzyl-7-[2-(2-thienyl)acetami'doJ-3-fluor-3-cephem-4-carboxylat.To 93 mg of dicyclohexyl-18-crown-6-ethylene in 15 ml of acetonitrile dried over a molecular sieve was added 25 mg of potassium fluoride which was dried in vacuo at 90 ° C. The mixture was stirred for 10 minutes and 138 mg of p-nitrobenzyl -7- [2- (2-thienyl) acetamido] -3-methylsulfonyloxy-3-cephem-4-carboxylate in 4 ml of acetonitrile was added. The mixture was stirred for 1 hour. The mixture was acidified by adding dilute (5%) hydrochloric acid and the resulting acidic mixture was extracted with ethyl acetate. The pure product was obtained from the ethyl acetate extract by preparative thin layer chromatography on silica gel, using as the eluent ethyl acetate: benzene (1: 1). 10 mg of p-nitrobenzyl 7- [2- (2-thienyl) acetamido] -3-fluoro-3-cephem-4-carboxylate was obtained.

I.R.:I. R .:

Absorptionstoppe ved 1792, 1740 og 1685 cm"1.Absorption peaks at 1792, 1740 and 1685 cm -1.

NMR (CDC15) viste signaler ved 6,15 (s, 2H, o-CH2), 4,97 (d, IH, J = 4H2, Cg-H), 4,20 (q, 1H, C?-H), 3,52 (d, 1H, Cy-NH), 2,32 - 1,7 (m, 2H, C2-H2) tau.NMR (CDCl15) showed signals at 6.15 (s, 2H, o -CH₂), 4.97 (d, 1H, J = 4H₂, Cg-H), 4.20 (q, 1H, C?-H) , 3.52 (d, 1H, Cy-NH), 2.32 - 1.7 (m, 2H, C2-H2) tau.

Pluor NMR — (d, J = 10 Hz).Fluorine NMR - (d, J = 10 Hz).

M.S.: Beregnet — 477.0465M.S .: Calculated - 477.0465

Pundet — 477.0455.Pound - 477.0455.

Eksempel 13Example 13

EzSiiiH^^^illrSBiSSz^-cb.lor-^-cephem-^-carbox^iat^^^drochloridEzSiiiH ^^^ illrSBiSSz ^ -cb.lor - ^ - cephem - ^ - carboxylic ^ iat ^^^ hydrochloride

Til en opløsning af 500 mg p-nitrobenzyl-7-[2-(2-thienyl)acet-amido] -3-ch.lor-3-cephem-4-carboxylat i 6 ml methylenchlorid sattes 95 mg tør pyridin og 237 mg phosphorpentachlorid. ReakTo a solution of 500 mg of p-nitrobenzyl 7- [2- (2-thienyl) acetamido] -3-chloro-3-cephem-4-carboxylate in 6 ml of methylene chloride was added 95 mg of dry pyridine and 237 mg phosphorus. reak

51 DK 153157 B51 DK 153157 B

tionsblandingen omrørtes ved stuetemperatur i 1,5 timer og afkøledes derefter i et is-vandbad til ca. 5° C, og der filsattes 0,6 ml isobutylalkohol. Ved fortsat køling og omrøring udkrystalliseredes reaktionsprodukter, p-nitrobenzyl-7-amino-5-chlor-3-cephem-4-carboxylat, hydrochlorid, fra reaktionsblandingen. Produktet frafiltreredes, vaskedes med koldt methylenchlorid og tørredes til opnåelse af 200 mg af det krystallinske produkt, der smeltede under dekomponering ved ca. 168° C.The reaction mixture was stirred at room temperature for 1.5 hours and then cooled in an ice-water bath to ca. 5 ° C and 0.6 ml of isobutyl alcohol was added. Upon continued cooling and stirring, reaction products, p-nitrobenzyl-7-amino-5-chloro-3-cephem-4-carboxylate, hydrochloride, were crystallized from the reaction mixture. The product was filtered off, washed with cold methylene chloride and dried to give 200 mg of the crystalline product which melted during decomposition at ca. 168 ° C.

Grundstof-analyse for C^H^CIN^O^S.HCI: teoretisk: C 41,39 - H 3,20 - N 10,34 - Cl 1,7,45.Elemental analysis for C ^H ^ClN ^O ^SHCl: theory: C, 41.39; H, 3.20; N, 10.34; Cl, 1.7.45.

fundet: C 41,14 - H 3,34 - N 10,44 - Cl 17,29.found: C 41.14 - H 3.34 - N 10.44 - Cl 17.29.

I.R. (Nujol Mull): viste absorptionsbånd ved 5,55 (β-lactamearbonyl) og 5,78 (estercarbonyl) mu.I.R. (Nujol Mull): showed absorption bands at 5.55 (β-lactamearbonyl) and 5.78 (ester carbonyl) mu.

U.V. (pH 7 buffer): viste absorptionsmaximum λ max 268 mu (£=13.800).U.V (pH 7 buffer): showed absorption maximum λ max 268 mu (£ = 13,800).

N.M.R. (DMSO dg):N.M.R. (DMSO dg):

Signaler ved 5,97 (s, 2H, C2-H2), 4,8 - 4,5 (m, 4H, Cg-H, Ογ-H og ester-CH2) og 2,35 - 1,6 (1, 4H, aromatisk H) tau.Signals at 5.97 (s, 2H, C2-H2), 4.8 - 4.5 (m, 4H, Cg-H, Ογ-H and ester-CH2) and 2.35-1.6 (1, 4H, aromatic H) tau.

Eksempel_14Eksempel_14

IlSSiH2z2l£^i2iz5~cephem-4“earbox2;lsyreIlSSiH2z2l £ ^ i2iz5 ~ cephem-4 "earbox2; L acid

Til en opløsning af 750 mg {1,85 mmol) p-nitrobenzyl-7-amino-3-chlor-3-cephem-4-carboxylat, hydrochlorid i 20 ml tefrahydrofuran og 40 ml methanol sattes en suspension af 750 mg præreduceret 5 i° palladium-på-carbon katalysator i 20 ml ethanol, og suspensionen hydrogeneredes under et tryk på 50 psi (ca, 3.,5 kg/cm2) ved stuetemperatur i 45 minutter. Katalysatoren filtreredes fra- og vaskedes med tetrahydrofuran og vand. Filtratet og vaskevandetTo a solution of 750 mg (1.85 mmol) of p-nitrobenzyl-7-amino-3-chloro-3-cephem-4-carboxylate, hydrochloride in 20 ml of tefrahydrofuran and 40 ml of methanol was added a suspension of 750 mg of pre-reduced 5 ° palladium-on-carbon catalyst in 20 ml of ethanol, and the suspension was hydrogenated under a pressure of 50 psi (approx. 3.5 kg / cm 2) at room temperature for 45 minutes. The catalyst was filtered off and washed with tetrahydrofuran and water. The filtrate and the wash water

32 DK 153157B32 DK 153157B

kombineredes og inddampedes til tørhed. Remanensen opløstes i en vand-ethylacetatblanding, og pH-værdien indstilledes til 3. Det uopløselige produkt filtreredes fra og behandledes med acetone. Produktet tørredes til opnåelse af 115 mg 7-amino-3-chlor-3-cephem-4-carboxylsyre.were combined and evaporated to dryness. The residue was dissolved in a water-ethyl acetate mixture and the pH was adjusted to 3. The insoluble product was filtered off and treated with acetone. The product was dried to give 115 mg of 7-amino-3-chloro-3-cephem-4-carboxylic acid.

I.R. (Mull):I.R. (Mull):

Absorptionstoppe ved 5,61 (β-lactamcarbonyl) og 6,2 (carboxylsyre).Absorption peaks at 5.61 (β-lactam carbonyl) and 6.2 (carboxylic acid).

N.M.R. (D20-NaH003);N.M.R. (D20-NaH003);

Signaler ved 6,25 (ABq., 2H, Og-Hg), 4,88 (d, 1H, Og-H) og 4,54 (d, 1H, C7-H) tau.Signals at 6.25 (ABq., 2H, Og-Hg), 4.88 (d, 1H, Og-H) and 4.54 (d, 1H, C7-H) tau.

U.V. (pH 7 buffer):U.V (pH 7 buffer):

Absorptionsmaximum ved λ max 265 mu, e=7550.Absorption maximum at λ max 265 mu, e = 7550.

Piphgnylmethyl-7-amino~5-ghlor-5-cepliem-4-carboxylatPiphgnylmethyl-7-amino ~ 5-ghlor-5-cepliem-4-carboxylate

Til en opløsning af 525 mg diphenylmethyl-7-[2-(thienyl)acet-amido]-3-chlor-3-cephem-4-carboxylat i 20 ml methylenchlorid sattes 0,1 ml tør pyridin og 237 mg phosphorpentachlorid. Reaktionsblandingen omrørtes i 2 timer ved stuetemperatur og afkøledes dernæst i et is-vandbad. Den kolde blanding tilsattes 0,6 ml isobutanoljOg efter 30 minutter inddampedes reaktionsblandingen. Remanensen opløstes i ethylacetat, og opløsningen vaskedes med 5 io natriumbicarbonat og med vand, hvorefter den tørredes. Den tørrede opløsning inddampedes til tørhed, og remanensen behandledes med ether til opnåelse af 190 mg 3-chlor-kemeesteren, diphenyl-methyl-7-amino-3-chlor-3-cephem-4-carboxylat.To a solution of 525 mg of diphenylmethyl 7- [2- (thienyl) acetamido] -3-chloro-3-cephem-4-carboxylate in 20 ml of methylene chloride was added 0.1 ml of dry pyridine and 237 mg of phosphorus pentachloride. The reaction mixture was stirred for 2 hours at room temperature and then cooled in an ice-water bath. The cold mixture was added 0.6 ml of isobutanol oil and after 30 minutes the reaction mixture was evaporated. The residue was dissolved in ethyl acetate and the solution was washed with 5 µl sodium bicarbonate and with water and then dried. The dried solution was evaporated to dryness and the residue treated with ether to give the 190 mg 3-chloro chemester, diphenyl-methyl-7-amino-3-chloro-3-cephem-4-carboxylate.

I.R. (Mull):I.R. (Mull):

Absorptionstoppe ved 5,7 og 5,9 (β-lactam og estercarbonyl) mu.Absorption peaks at 5.7 and 5.9 (β-lactam and ester carbonyl) mu.

33 DK 153157 B33 DK 153157 B

N.M.R. (0Γ013):N.M.R. (0Γ013):

Signaler ved 6,35 (ΑΒα, 2H, Cg-Hg), 4.78 (2d, 2H, Gg-H og Ογ-Η), 3,05 (s, 1H, ester-CH) og 2,65 (s, 10H, aromatisk H).Signals at 6.35 (ΑΒα, 2H, Cg-Hg), 4.78 (2d, 2H, Gg-H and Ογ-Η), 3.05 (s, 1H, ester-CH) and 2.65 (s, 10H , aromatic H).

Eksempel 16Example 16

Ved at følge reaktionen til fraspaltning af 7-acyl-sidekæden beskrevet i eksempel 13 fremstilledes diphenylmethyl-7-amino-3-brom-3-cephem-4-carboxylat ud fra diphenylmethyl-7-phenoxyacetamido- 3- brom-3-cephem-4-carboxylat.Following the reaction for cleavage of the 7-acyl side chain described in Example 13, diphenylmethyl-7-amino-3-bromo-3-cephem-4-carboxylate was prepared from diphenylmethyl-7-phenoxyacetamido-3-bromo-3-cephem. 4-carboxylate.

Eksempel 17 7- (D-a-phenylgl^cyl^ido) -3-ciilor-5-cepliem-4-garbo^ylsyreExample 17 7- (D-α-Phenylglycylcido) -3-chloro-5-cephalim-4-garboxylic acid

Til en suspension af 280 mg (1,2 mmol) 7-amino-3-chlor-3-cephem- 4- carboxylsyxe i 14 ml acetonitril sattes under omrøring ved stuetemperatur 0,5 ml N,O-bis-(trimethylsilyl)acetamid til dannelse af det opløselige disilylmethylderivat. Opløsningen afkøledes til 0° C, og der tilsattes langsomt en opløsning af det blandede anhydrid dannet ved reaktion mellem 408 mg (1,5 mmol) af natriumsaltet af methyl-3-a-carboxybenzylaminocrotonat og 161 mg (1,7 mmol) methylchlorformiat i nærværelse af 2 dråber dimethylbenzylamin i 7 ml acetonitril. Blandingen omrørtes i et isbad i 2 timer, og der sattes 1 ml methanol til blandingen, der dernæst filtreredes for at fjerne uopløselige urenheder. Der sattes 2 ml vand til filtratet, og pH-værdien indstilledes i første omgang til 1,5 for at udvirke fjernelse af enamin-blokeringen og dernæst til 4,5 med triethylamin. Efter omrøring i yderligere 1 time ved isbadtemperatur udfældede reaktionsproduktet, 7-(D-a-phenylglycylamido)-3-chlor-3-cephem-4-carboxylsyre (zwdtterion) fra reaktionsblandingen som et krystallinsk fast stof.. Produktet filtreredes fra, vaskedes med acetonitril og tørredes i vakuum til opnåelse af et udbytte på 200 mg.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 with stirring at room temperature. to form the soluble disilylmethyl derivative. The solution was cooled to 0 ° C and a solution of the mixed anhydride formed by reaction between 408 mg (1.5 mmol) of the sodium salt of methyl 3-a-carboxybenzylaminocrotonate and 161 mg (1.7 mmol) of methyl chloroformate was slowly added. presence of 2 drops of dimethylbenzylamine in 7 ml of acetonitrile. The mixture was stirred in an ice bath for 2 hours and 1 ml of methanol was added to the mixture which was then filtered to remove insoluble impurities. 2 ml of water was added to the filtrate and the pH was initially adjusted to 1.5 to effect removal of the enamine block and then to 4.5 with triethylamine. After stirring for an additional hour at ice bath temperature, the reaction product precipitated, 7- (Da-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid (zwdtterion) from the reaction mixture as a crystalline solid. The product was filtered off, washed with acetonitrile and was dried in vacuo to give a yield of 200 mg.

34 DK 153157 B34 DK 153157 B

Produktet havde følgende fysiske egenskaber;The product had the following physical properties;

Grundstof-analyse for C^H^N^O^SOl· 1/2 H20: teoretisk; O 47,80 - H 4,01 - N 11,15 - Cl 9,40.Elemental analysis for C ^H ^N ^O₂SO · · 1/2H₂O: theoretical; O 47.80 - H 4.01 - N 11.15 - Cl 9.40.

fundet; C 47,55 - H 4,12 - N 10,98 - 01 9,21.found; C 47.55 - H 4.12 - N 10.98 - 01 9.21.

I.R.-spektrum (Mull);I.R. spectrum (Mull);

Absorptionstoppe ved 2,9 (amid NH), 5,70 (β-lactarnearbony1), 5,95 (amidcarbonyl) og 6,28 (carboxylat) mp.Absorption peaks at 2.9 (amide NH), 5.70 (β-lactarnebonyl), 5.95 (amide carbonyl) and 6.28 (carboxylate) mp.

NMR-spektrum (D20/I)C1);NMR Spectrum (D20 / I) C1);

Signaler ved 6,5 - 6,7 (ABq, 2H, 02-H2).Signals at 6.5 - 6.7 (ABq, 2H, 02-H2).

4,84 (d, 1H, Og-H), 4,26 (d, 1H, Cy-H) og 2,44 (s, 5H, aromatisk H) tau.4.84 (d, 1H, Og-H), 4.26 (d, 1H, Cy-H) and 2.44 (s, 5H, aromatic H) tau.

U.V.-spektrum (pH buffer); λ max 265 mp (e=6.800).U.V. spectrum (pH buffer); λ max 265 mp (e = 6,800).

Eksempel_18 I il en opløsning af 500 mg (1,85 mmol) af natriumsaltet af methyl-3-a-carboxybenzylaminocrotonat (dannet med phenylglycin og methyl-acetoacetat) i 20 ml acetonitril sattes 4 dråber dimethylbenzyl-amin, og opløsningen afkøledes i et tøris-carbontetrachlorid-bad under omrøring. Til den kolde opløsning sattes langsomt 184 mg (195 mmol) methylchlorformiat til dannelse af det blandede anhydrid. Efter 20 minutter tilsattes en på forhånd afkølet opløsning af 750 mg (1,85 mmol) p-nitrobenzyl-7-amino-3-chlor-3-cephem-4-carboxylat og 188 mg (1,85 mmol) triethylamin i 40 ml acetone. Tilsætningen forløb over 3 minutter, hvorefter reaktionsblandingen omrørte s i kulden i 30 minutter og dernæst ved stuetemperatur i 2 timer. Reaktionsblandingen filtreredes for at fjerne uopløselige urenheder og inddampedes i vakuum. Reaktionsproduktet opløstes i en blanding af ethylacetat og vand, og opløsningens pH indstilledes til 7. Den organiske fase separe-Example 18 In a solution of 500 mg (1.85 mmol) of the sodium salt of methyl 3-a-carboxybenzylaminocrotonate (formed with phenylglycine and methylacetoacetate) in 20 ml of acetonitrile, 4 drops of dimethylbenzylamine were added and the solution cooled in a dry ice. carbon tetrachloride bath with stirring. To the cold solution was slowly added 184 mg (195 mmol) of methyl chloroformate to form the mixed anhydride. After 20 minutes, a pre-cooled 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. acetone. The addition was carried out over 3 minutes, after which 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 evaporated in vacuo. The reaction product was dissolved in a mixture of ethyl acetate and water and the pH of the solution was adjusted to 7. The organic phase was separated.

35 DK 153157 BDK 153157 B

redes fra og vaskedes med vand. Efter tørring over magnesium-sulfat koncentreredes den organiske fase under vakuum til et lille volumen. Efter tilsætning af n-hexan til koncentratet udfældede 620 mg p-nitrobenzyl-7“[N-(l~carbomethoxy-2-propenyl)-D-a-ph.enylglycylamido] -3-dilor-3-cep]iem-4-carTDoxylat .‘fra reak-tionshlandingen.cleaned and washed with water. After drying over magnesium sulfate, the organic phase was concentrated in vacuo to a small volume. After addition of n-hexane to the concentrate, 620 mg of p-nitrobenzyl-7 "[N- (1-carbomethoxy-2-propenyl) -Da-phenylglycylamido] -3-dilor-3-cep] iem-4-carTDoxylate from reaction reactions.

Produktet gav følgende grundstof-analyse og NMR-spektrum: G-rundstof-analyse for C^H^U^OgSCl: teoretisk: C 53,87 - H 4,35 - N 9,31.The product gave the following elemental analysis and NMR spectrum: G-round material analysis for C ^ HH ^ UO OgSCl: theoretical: C 53.87 - H 4.35 - N 9.31.

fundet: C 54,05 - H 4,13 - N 9,36.Found: C 54.05 - H 4.13 - N 9.36.

NMR (EMSO dg):NMR (EMSO dg):

Signaler ved 8,20 (s, 3H, enamin CH^), 6,60 (ABq, 2H, C2-H2), 6,45 (s, 3H, ester-CH^), 5,48 (s, 1H, enamin-viny1-H), 4,90 - 4,1 (m, 5H, Cg-H, C^-H, α-CH og ester-CH2) og 3,10 - 1,5 (m, 9H, aromatisk H) tau.Signals at 8.20 (s, 3H, enamine CH 2), 6.60 (ABq, 2H, C 2 -H 2), 6.45 (s, 3H, ester-CH 2), 5.48 (s, 1H, enamine-vinyl-H), 4.90 - 4.1 (m, 5H, Cg-H, C ^-H, α-CH and ester-CH 2) and 3.10 - 1.5 (m, 9H, aromatic H) rope.

540 mg (0,9 mmol) af produktet opløstes i 40 ml acetonitril indeholdende 20 ml vand, og opløsningen blev først afkølet i isvand og dernæst gjort sur til pH 1,5, hvorefter pH indstilledes til 2,5. Blandingen inddampedes, og remanensen opløstes i 40 ml tetrahydrofuran og 80 ml methanol.540 mg (0.9 mmol) of the product was dissolved in 40 ml of acetonitrile containing 20 ml of water, and the solution was first cooled in ice water and then acidified to pH 1.5, then the pH was adjusted to 2.5. The mixture was evaporated and the residue was dissolved in 40 ml of tetrahydrofuran and 80 ml of methanol.

Til opløsningen sattes 540 mg 5 % palladium-på-carbon .katalysator (præreduceret i 20 ml ethanol i 45 minutter under et hydrogentryk „ 2 pa ca. 3,5 kg/cm ved stuetemperatur), og opløsningen-hydrogeneredes ved stuetemperatur i 2,5 timer under et hydrogentryk på 50 psi (ca.To the solution was added 540 mg of 5% palladium-on-carbon catalyst (pre-reduced in 20 ml of ethanol for 45 minutes under a hydrogen pressure "2 of about 3.5 kg / cm at room temperature) and the solution was hydrogenated at room temperature for 2 hours. 5 hours under a hydrogen pressure of 50 psi (approx.

2 3,5 kg/cm ). Katalysatoren filtreredes fra og vaskedes på filteret med methanol, tetrahydrofuran og vand. Filtratet og vaskevandet kombineredes og inddampedes til tørhed i vakuum. Reaktionsproduktet opløstes i en blanding af vand og ethylacetat, og pH i opløsningen indstilledes til 4,5. Den vandige fase separeredes fra, vaskedes med ethylacetat og inddampedes til et lille volumen på ca. 2 ml. Ved afkøling udfældede 65 mg af produktet, 7-(D-a-phenylglycylamido)-3-chlor-3-cephem-4-carboxylsyre, som et krystallinsk fast stof fra det kolde2 3.5 kg / cm). The catalyst was filtered off and washed on the filter with methanol, tetrahydrofuran and water. The filtrate and wash water were combined and evaporated to dryness in vacuo. The reaction product was dissolved in a mixture of water and ethyl acetate and the pH of the solution was adjusted to 4.5. The aqueous phase was separated, washed with ethyl acetate and evaporated to a small volume of ca. 2 ml. Upon cooling, 65 mg of the product, 7- (D-α-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid, precipitated as a crystalline solid from the cold

36 DK 153157 B36 DK 153157 B

koncentrat.concentrate.

Eksem-gel_19 fil en suspension af 3,0 g (8,1 mmol) p-nitrobenzyl-7-amno-3-chlor-3-ceph.em-4-*carboxylat i 200 ml tetrahydrofuran (tørret med molekylsi) sattes 2,1 g (8,3 mmol) N-(t-butyloxycarbonyl)-D-a-pbenylglycin og 2,0 g (8,3 mmol) N-ethoxycarbonyl-2-ethoxy- 1,2-dihydroguinolin (EEDQ). Reaktionsblandingen omrørtes natten over ved stuetemperatur, og opløsningsmidlet afdampedes under reduceret tryk. Remanensen opløstes i en blanding af etbylacetat og vand, og den organiske fase frasepareredes. Denne fase afkøledes og vaskedes successivt med en kold 5 i° vandig opløsning af natriumbicarbonat, kold 5 % saltsyre og vand. Den vaskede opløsning tørredes over magnesiumsulfat, filtreredes og koncentreredes ved inddampning under reduceret tryk til et volumen på ca. 50 ml. Fra koncentratet opnåedes 3,7 g (83 % udbytte) af reaktionsproduktet, p-nitrobenzyl-7-[D-2-(t-butyloxy-carbamido)-2-phenylacetamido]-3-chlor-3-cephem-4-carboxylat, som et krystallinsk produkt. Et andet udbytte af produktet på i alt 2 gram opnåedes ved yderligere at koncentrere filtratet.Example gel 19 µl of a suspension of 3.0 g (8.1 mmol) of p-nitrobenzyl-7-amino-3-chloro-3-cephem-4- * carboxylate in 200 ml of tetrahydrofuran (dried with molecular sieve) was added. , 1 g (8.3 mmol) of N- (t-butyloxycarbonyl) -Da-pbenylglycine and 2.0 g (8.3 mmol) of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroguinoline (EEDQ). The reaction mixture was stirred overnight 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. This phase was cooled and washed successively with a cold 5 in aqueous solution of sodium bicarbonate, cold 5% hydrochloric acid and water. The washed solution was dried over magnesium sulfate, filtered and concentrated by evaporation under reduced pressure to a volume of ca. 50 ml. From the concentrate was obtained 3.7 g (83% yield) of the reaction product, p-nitrobenzyl-7- [D-2- (t-butyloxy-carbamido) -2-phenylacetamido] -3-chloro-3-cephem-4-carboxylate , as a crystalline product. Another yield of the product totaling 2 grams was obtained by further concentrating the filtrate.

Grundstof-analyse beregnet for ^yHgyCIN^OgS: teoretisk: C 53,78 - H 4,51 - N9,29.Elemental analysis calculated for γγHgyCIN Og OgS: theoretical: C 53.78 - H 4.51 - N9.29.

fundet: C 52,66 - H 4,36 - N 8,88.Found: C 52.66 - H 4.36 - N 8.88.

U.Y. (acetonitril) viste et maximum ved 268 ιημ (£=17.100).U.Y. (acetonitrile) showed a maximum at 268 ιημ (£ = 17,100).

I.R.:I. R .:

Ami-d NH absorptionstop ved 3,05 ιημ og carbonylabsorptionstop ved 5,59, 5,75 og 6,0 ιημ.Ami-d NH absorption peak at 3.05 ιημ and carbonyl absorption peak at 5.59, 5.75 and 6.0 ιημ.

NMR (CDC13):NMR (CDCl3):

Signaler ved 8,60 (s, 9H, t-BOC), 6,45 (ABcl, 2H, C2-H2), 5,03 (d, 1H, C6-H), 4,67 (s, 3H, α-CH og ester-CH2), 4,12 (m, 3H, Cy-H og amid NH) og 2,72 - 1,74 (m, 10H, aromatisk H og amid NH) tau.Signals at 8.60 (s, 9H, t-BOC), 6.45 (ABcl, 2H, C2-H2), 5.03 (d, 1H, C6-H), 4.67 (s, 3H, α -CH and ester-CH2), 4.12 (m, 3H, Cy-H and amide NH) and 2.72 - 1.74 (m, 10H, aromatic H and amide NH) tau.

37 DK 153157 B37 DK 153157 B

Til en opløsning af 3,0 g (5,0 mmol) af produktet i 15 ml tør tetra-hydrofuran tørret med molekylsi og 185 ml methanol sattes 3 g præ-reduceret 5 procent palladiua-på-carbon. Katalysatoren præreducere-des i ethanol i 30 minutter ved stuetemperatur under et hydrogentryk på 50 psi (ca. 3,5 kg/cm ). Efter tilsætning af den præreducerede katalysator hydrogeneredes produktet ved stuetemperatur 1 time under et hydrogentryk på 50 psi (ca- 3,5 kg/cm ). Katalysatoren filtreredes fra og vaskedes på filteret med tetrahydrofuran og methanol. Filtratet og vaskevandet kombineredes og inddampedes under reduceret tryk. Remanensen opløstes i ethylacetat, og der tilsattes vand. Blandingens pH-værdi indstilledes til 7 ved tilsætning af :1N natriumhydroxid. Den vandige fase separeredes og vaskedes med ethylacetat. Den vandige fase behandledes dernæst med ethylacetat og filtreredes til pH 2,5 med IN saltsyre. Den organiske fase separeredes fra den vandige fase, vaskedes med vand og tørredes over magnesiumsulfat. Den tørrede organiske fase inddampedes under reduceret tryk til tørhed, hvorved der opnåedes et reduktie^sprodukt, 7-(D-2-(t-butyloxycarbamido-2-phenylacetamido)-3-chlor-3-cephem-4-carboxylsyre, som et tørt fast stof. Produktet krystalliseredes fra 70 ml ether indeholdende 20 ml petroleumsether til opnåelse af 1,75 g (75 %) krystallinsk produkt.To a solution of 3.0 g (5.0 mmol) of the product in 15 ml of dry tetrahydrofuran dried with molecular sieve and 185 ml of methanol was added 3 g of pre-reduced 5 percent palladiua-on-carbon. The catalyst was pre-reduced in ethanol for 30 minutes at room temperature under a hydrogen pressure of 50 psi (about 3.5 kg / cm). After addition of the pre-reduced catalyst, the product was hydrogenated at room temperature for 1 hour under a hydrogen pressure of 50 psi (about 3.5 kg / cm). The catalyst was filtered off and washed on the filter with tetrahydrofuran and methanol. The filtrate and wash water were combined and evaporated under reduced pressure. 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. The aqueous phase was separated and washed with ethyl acetate. The aqueous phase was then treated with ethyl acetate and filtered to pH 2.5 with 1N hydrochloric acid. The organic phase was separated from the aqueous phase, washed with water and dried over magnesium sulfate. The dried organic phase was evaporated under reduced pressure to dryness to give a reducing product, 7- (D-2- (t-butyloxycarbamido-2-phenylacetamido) -3-chloro-3-cephem-4-carboxylic acid, as a The product was crystallized from 70 ml of ether containing 20 ml of petroleum ether to give 1.75 g (75%) of crystalline product.

Grundstof-analyse beregnet for C20H22C1N3°6S: teoretisk: C 51,34 - H 4,74 - N 8,98 - Cl 7,58.Elemental analysis calculated for C 20 H 22 ClN 3 O 6 S: theory: C 51.34 - H 4.74 - N 8.98 - Cl 7.58.

fundet: C 51,02 - H 4,96 - N 8,75 - Cl 7,30.Found: C, 51.02; H, 4.96; N, 8.75; Cl, 7.30.

U.V. (acetonitril) maximum ved 268 mp (£=7400).U.V (acetonitrile) maximum at 268 mp (£ = 7400).

KMR (CDClc): Signaler ved 8,55 (s, 9H, t-BOC), 6,48 (ABq, 2H, C2-H2), 5,0 (d, IH, Cg-H), 4,63 (d, IH, cc-CH), 4,25 (q, IH, C?-H), 3,90 (d, IH, amid NH), 2,59 (s, 5H, aromatisk H) tau.NMR (CDClc): Signals at 8.55 (s, 9H, t-BOC), 6.48 (ABq, 2H, C2-H2), 5.0 (d, 1H, Cg-H), 4.63 ( d, 1H, cc-CH), 4.25 (q, 1H, C? -H), 3.90 (d, 1H, amide NH), 2.59 (s, 5H, aromatic H) tau.

Til en opløsning af 420 mg (2,2 mmol) p-toluensulfonsyre i 5 ml acetonitril sattes 468 mg (1 mmol) krystallinsk hydrogeneringsprodukt. Opløsningen henstod ved stuetemperatur i 16 timer og fortyn-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 hydrogenation product. The solution was left at room temperature for 16 hours and diluted.

38 DK 153157 B38 DK 153157 B

4,8, hvorefter 320 mg (87 %) af det krystallinske produkt, 7-(D-phenylglycylamido)-3-chlor-3-cephem-4-carboxylsyre, udfældede fra opløsningen. Produktet opsamledes ved filtrering og tørredes.4.8, after which 320 mg (87%) of the crystalline product, 7- (D-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid, precipitated from the solution. The product was collected by filtration and dried.

Grundstof-analyse beregnet for C15Hi4C1N304S.1/2 H20: teoretisk: C 47,80 - H 4,01 - N 11,15 - Cl 9,40.Elemental analysis calculated for C 15 H 14 ClN 3 O 4 S / 2 H2 O: theoretical: C 47.80 - H 4.01 - N 11.15 - Cl 9.40.

fundet: C 48,04 - H 3,82 - N 11,18 - Cl 9,70.found: C 48.04 - H 3.82 - N 11.18 - Cl 9.70.

Eksempel_20Eksempel_20

Den antibiotiske forbindelse 7-(D-phenylglycylamido)-3-chlor-3-cephem-4-carboxylsyre fremstilledes ud fra p-nitrobenzyl-7-(D-2-(t-butyloxycarbamido)-2-phenylacetamido)-3-chlor-3-cephem-4-carb-oxylat ved en deblokeringsmetode alternativ til den i det foregående eksempel beskrevne. Ved denne metode fjernes den aminobeskyt-tende t-butyloxycarbonyl-gruppe først, og derefter fraspaltedes p-nitro-benzylestergruppen med zink og saltsyre i dimethylformamid. Denne alternative fremstilling er beskrevet i de følgende afsnit.The antibiotic compound 7- (D-phenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid was prepared from p-nitrobenzyl-7- (D-2- (t-butyloxycarbamido) -2-phenylacetamido) -3-chloro -3-cephem-4-carb oxylate by a deblocking method alternative to the one described in the previous example. In this method, the amino-protecting t-butyloxycarbonyl group is first removed and then the p-nitrobenzyl ester group is separated off with zinc and hydrochloric acid in dimethylformamide. This alternative preparation is described in the following sections.

Til en opløsning af 2,4 g (12,6 mmol) p-toluensulfonsyre i 60 ml acetonitril sattes 3,6 g (6 mmol) p-nitrobenzyl-7-(D-2-(t-butyloxycarbamido )-2-phenylacetamido)-3-chlor-3-cephem-4-carboxylat. Reaktionsblandingen omrørtes ved stuetemperatur i 15 timer, hvorunder det aminodeblokerede produkt, p-nitrobenzyl-7-(D-phenylgly-cylamido)-3-chlor-3-cephem-4-carboxylat, dannedes som et krystallinsk bundfald i form af p-toluensulfonsyresaltet.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- (D-2- (t-butyloxycarbamido) -2-phenylacetamido ) -3-chloro-3-cephem-4-carboxylate. The reaction mixture was stirred at room temperature for 15 hours during which the aminodeblocked product, p-nitrobenzyl-7- (D-phenylglycylamido) -3-chloro-3-cephem-4-carboxylate, formed as a crystalline precipitate in the form of the p-toluenesulfonic acid salt .

Produktet frafiltreredes, vaskedes med acetonitril og vakuumtørre des. Udbytte 3,1 g ( 81 96).The product was filtered off, washed with acetonitrile and vacuum dried. Yield 3.1 g (81 96).

Grundstof-analyse for C2^H2yClN40gS2: teoretisk: C 51,58 - H 4,06 - N 8,29 - Cl 5,25.Elemental analysis for C₂ ^H₂yClN40O₂S₂: theoretical: C 51.58 - H 4.06 - N 8.29 - Cl 5.25.

fundet: C 51,51 - H 4,14 - N 8,12 - Cl 5,60.found: C 51.51 - H 4.14 - N 8.12 - Cl 5.60.

IR-spektrum (chloroform):IR spectrum (chloroform):

Carbonylabsorptionstoppe ved 5,61, 5,80 og 5,95. Tosylatsaltabsorptionstop ved 6,29 mp.Carbonyl absorption peaks at 5.61, 5.80 and 5.95. Tosylate salt absorption peak at 6.29 mp.

39 DK 153157B39 DK 153157B

NMR-spektrum (DMSO dg):NMR Spectrum (DMSO dg):

Signaler ved 7,70 (s, 3H, p-toluensulfonsyre-salt-CH^), 6,61 (s, 3H, sidekæde NH^), 6,20 (ABq, 2H, C2~H2), 4,51 (s, 2H, ester CH2), 4,08 (q, IH, C?-H), 2,95 - 1,62 (m, 14H, aromatisk hydrogen og amid NH), 0,32 (d, IH, amid NH).Signals at 7.70 (s, 3H, p-toluenesulfonic acid-salt-CH 2), 6.61 (s, 3H, side chain NH 2), 6.20 (ABq, 2H, C2 ~ H2), 4.51 ( s, 2H, ester CH2), 4.08 (q, 1H, C? -H), 2.95 - 1.62 (m, 14H, aromatic hydrogen and amide NH), 0.32 (d, 1H, amide NH).

Det aminodeblokerede p-toluensulfonsyresalt, der opnåedes som beskrevet ovenfor, deesterificeredes til opnåelse af følgende antibiotiske forbindelse: En opløsning af 1,5 g (2,2 mmol) af p-tolu- ensulfonsyresaltet i 10 ml tørt dimethylformamid (tørret med mole-kylsi) afkøledes i et is-alkoholbad. Den kolde opløsning tilsattes 2 ml koncentreret saltsyre. 400 mg (6,1 mmol) zinkstøv sattes til den kolde opløsning i løbet af 15 minutter. Reaktionsblandingen omrørtes i kulden i 30 minutter og opvarmedes dernæst ved stuetemperatur under stadig omrøring.The aminodeblocked p-toluenesulfonic acid salt obtained as described above was deesterified to give the following antibiotic compound: A solution of 1.5 g (2.2 mmol) of the p-toluenesulfonic acid salt in 10 ml of dry dimethylformamide (dried with molecular sieve) ) cooled in an ice-alcohol bath. To the cold solution was added 2 ml of concentrated hydrochloric acid. 400 mg (6.1 mmol) of zinc dust was added to the cold solution over 15 minutes. The reaction mixture was stirred in the cold for 30 minutes and then heated at room temperature with still stirring.

Reaktionsblandingen omrørtes ved stuetemperatur i 1 time og filtreredes dernæst. Filtratets pH-værdi indstilledes til 6,8 med tri-ethylamin. Produktet, 7-(D-phenylglycylamido)-3-chlor-3-cephem-4-carboxylsyre som bis-dimethylformamidsolvatet af den amfotere ion, dannedes som et hvidt krystallinsk bundfald. Produktet frafiltrere-des, vaskedes med 10 ml koldt dimethylformamid og dernæst med 6 ml diethylether. Det vaskede produkt vakuumtørredes til opnåelse af et udbytte på 800 mg (71 %).The reaction mixture was stirred at room temperature for 1 hour and 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 as the bis-dimethylformamide solvate of the amphoteric ion, formed as a white crystalline precipitate. The product was filtered off, washed with 10 ml of cold dimethylformamide and then with 6 ml of diethyl ether. The washed product was vacuum dried to give a yield of 800 mg (71%).

Grundstof-analyse for C^H^ClN^OgS.2DMF: teoretisk: C 49,07 - H 5,49 - N 14,63 - Cl 6,90.Elemental analysis for C ^H ^ClN ^O₂S.2DMF: theory: C 49.07 - H 5.49 - N 14.63 - Cl 6.90.

fundet: C 48,84 - H 5,53 - N 13,48 - Cl 7,18.Found: C 48.84 - H 5.53 - N 13.48 - Cl 7.18.

U.V. spektrum (acetonitril): maximum ved 265 πιμ (£=6.000).U.V spectrum (acetonitrile): maximum at 265 πιμ (£ = 6,000).

Elektrometrisk titrering (66 % vandig DMF): pKa ved 4,55 og 7,2.Electrometric titration (66% aqueous DMF): pKa at 4.55 and 7.2.

40 DK 153157 B40 DK 153157 B

MR-spektrum (D20/DC1):MRI spectrum (D20 / DC1):

Signaler ved 6,34 (2s, 6H, DMF-CH3), 6.33 (ABq, 2H, C2-H2), 4,85 (d, IH, Cg-H), 4,64 (s, IH, a-CH), 4,27 (d, IH, C7-H), 2,41 (s, 5H, aromatisk H) og 1,84 (s, 2H, DMF-CH) tau.Signals at 6.34 (2s, 6H, DMF-CH3), 6.33 (ABq, 2H, C2-H2), 4.85 (d, 1H, Cg-H), 4.64 (s, 1H, a-CH ), 4.27 (d, 1H, C7-H), 2.41 (s, 5H, aromatic H) and 1.84 (s, 2H, DMF-CH) tau.

Eksempel_21Eksempel_21

ZiiDl2z4ydrox^henYlglYCYlamido)=322^l2rr3i2§Bhem-4-carboxYlsYre 2,9 g (11 mmol) N-(t-butyloxycarbonyl) -D-3-hydroxyphenylglycin omsattes med 3»7 g (10 mmol) p-nitrobenzyl-7-amino-3-chlor-3-ce-phem-4-carboxylat og 2,6 g (10,5 mmol) af koblingsreagenset EEDQ. Produktet isoleredes ved at følge metoden beskrevet i eksempel 18. Produktet opnåedes som et amorft fast stof efter behandling med ether i et udbytte på 2,8 g (46 %).ZiiDl2z4ydroxy (henylglYCYlamido) = 322 l2rr3i2§Bhem-4-carboxylic acid 2.9 g (11 mmol) of N- (t-butyloxycarbonyl) -D-3-hydroxyphenylglycine was reacted with 3 »7 g (10 mmol) p-nitrobenzyl -amino-3-chloro-3-ce-phem-4-carboxylate and 2.6 g (10.5 mmol) of the coupling reagent EEDQ. The product was isolated following the method described in Example 18. The product was obtained as an amorphous solid after treatment with ether in a yield of 2.8 g (46%).

Grundstof-analyse for C27H27C*N409S: teoretisk: C 52,39 - H 4,40 - N 9>05.Elemental analysis for C27H27C * N409S: theory: C 52.39 - H 4.40 - N9> 05.

fundet: C 52,16 - H 4,59 - N 8,79.Found: C, 52.16; H, 4.59; N, 8.79.

U.V. spektrum (acetonitril): maximum ved 270 πμ (£=17.200).U.V spectrum (acetonitrile): maximum at 270 πµ (£ = 17,200).

MR-spektrum (CDCl^) :MRI spectrum (CDCl3):

Signaler ved 8,59 (s, 9H, t-BOC), 6,50 (ABq, 2H, C2-H2) 5,06 (d, IH, Cg-H), 4,66 (s, IH, a-CH), 4,09 (m, 2H, C?-H), 3.34 - 1,70 (m, 9H, aromatisk H og amid NH) tau.Signals at 8.59 (s, 9H, t-BOC), 6.50 (ABq, 2H, C2-H2) 5.06 (d, 1H, Cg-H), 4.66 (s, 1H, α- CH), 4.09 (m, 2H, C?-H), 3.34 - 1.70 (m, 9H, aromatic H and amide NH) tau.

41 DK 153157 B41 DK 153157 B

3,5 g (5,6 mmol) af produktet, p-nitrobenzyl-7-(D-2-(.t-butyloxy-carbamido)-2- (3-hydroxy)phenylacetamido)-3-chlor-3-cephem-4-carb-oxylat, hydrogeneredes i ethanol i nærværelse af præreduceret 5 % palladium-på-carbon ved at følge deesterificeringsmetoden beskrevet i eksempel 18. Produktet, 7-(D-2-(t-butyloxycarbamido) - 2-(3~hydroxy)phenylacetamido)-3-chlor-3-cephem-4-carboxylsyre, opnåedes krystallinsk ved behandling af det amorfe stof med en opløsning af hexan i diethylether. Udbytte 1,5 g (55 %)· U.V. spektrum (acetonitril: maximum ved 272 mp (£=8.280).3.5 g (5.6 mmol) of the product, p-nitrobenzyl-7- (D-2 - (t-butyloxy-carbamido) -2- (3-hydroxy) phenylacetamido) -3-chloro-3-cephem -4-carb oxylate, hydrogenated in ethanol in the presence of pre-reduced 5% palladium-on-carbon following the deesterification method described in Example 18. The product, 7- (D-2- (t-butyloxycarbamido) - 2- (3 ~ hydroxyphenylacetamido-3-chloro-3-cephem-4-carboxylic acid was obtained crystalline by treatment of the amorphous substance with a solution of hexane in diethyl ether. Yield 1.5 g (55%) · U.V. spectrum (acetonitrile: maximum at 272 mp (δ = 8,280).

Elektrometrisk titrering (66 % vandig DMF): pKa på 4,5.Electrometric titration (66% aqueous DMF): pKa of 4.5.

1,3 g (2,7 mmol) af ovennævnte produkt omsattes med 1,1 g (5,9 mmol) p-toluensulfonsyre i 28 ml acetonitril for at fjerne den tert.-butyloxycarbonylbeskyttende gruppe. Metoden udførtes som beskrevet i eksempel 18.1.3 g (2.7 mmol) of the above product was reacted with 1.1 g (5.9 mmol) of p-toluenesulfonic acid in 28 ml of acetonitrile to remove the tert-butyloxycarbonyl protecting group. The method was carried out as described in Example 18.

Produktet, 7-(D-3-hydooxyphenylglycylamido)-3-chlor-3-cephem-4-carboxylsyre, krystalliseredes fra reaktionsblandingen, filtreredes fra og vakuumtørredes. Udbytte 700 mg (64 %).The product, 7- (D-3-Hydooxyphenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid, was crystallized from the reaction mixture, filtered off and vacuum dried. Yield 700 mg (64%).

Grundstof-analyse for C15H14C1N3°5S*1H20: teoretisk: C 44,83 - H 4,01 - N 10,46.Elemental analysis for C 15 H 14 ClN 3 ° 5S * 1H 2 O: theory: C 44.83 - H 4.01 - N 10.46.

fundet: C 45,12 - H 4,06 - N 10,31.Found: C 45.12 - H 4.06 - N 10.31.

Ultraviolet absorptionsspektrum (pH buffer): maximum ved 268 mp (£=9.750).Ultraviolet absorption spectrum (pH buffer): maximum at 268 mp (£ = 9,750).

NMR-spektrum (D20/DC1):NMR Spectrum (D20 / DC1):

Signaler ved 6,31 (ÅBq, 2H, C2-H2), 4,81 (d, IH, Cg-H), 4,52 (s, IH, a-CH), 4,26 (d, IH, Cy-H) og 3,1 - 2,5 (m, 4H, aromatisk H)tau.Signals at 6.31 (ÅBq, 2H, C2-H2), 4.81 (d, 1H, Cg-H), 4.52 (s, 1H, a-CH), 4.26 (d, 1H, Cy -H) and 3.1 - 2.5 (m, 4H, aromatic H) tau.

42 DK 153157 B42 DK 153157 B

Eksempel_22Eksempel_22

Ved at følge metoden beskrevet i eksempel 18 acyleredes 850 mg (2,3 mmol) p-nitrobenzyl-7-amino-3-chlor-3-cephem-4-carboxylat med 700 mg (2,5 mmol) N-t-butyloxycarbonyl-D-4-chlorphenylglycin og 567 mg (2,3 mmol) EEDQ til opnåelse af 1,2 g p-nitrobenzyl-7-(D- 2-(t-butyloxycarbamido)-2-(4-chlorphenylacetamido)-3-chlor-3-ce-phem-4-carboxy1at.Following the method described in Example 18, 850 mg (2.3 mmol) of p-nitrobenzyl 7-amino-3-chloro-3-cephem-4-carboxylate was acylated with 700 mg (2.5 mmol) of Nt-butyloxycarbonyl-D -4-chlorophenylglycine and 567 mg (2.3 mmol) of EEDQ to give 1.2 g of p-nitrobenzyl-7- (D-2- (t-butyloxycarbamido) -2- (4-chlorophenylacetamido) -3-chloro 3-ce-PHEM-4-carboxy1at.

1,2 g (1,9 mmol) af produktet hydrogeneredes ved stuetemperatur i nærværelse af præreduceret 5 % palladium-på-carbon for at fjerne p-nitrobenzylestergruppen og til frembringelse af 450 mg 7-(D-2-(t-butyloxycarbamido)-2-(4-chlorphenylacetamido))-3-chlor-3-cephem~ 4-carboxylsyre som et krystallinsk produkt. Produktet opnåedes i krystallinsk form ved at behandle den amorfe remanens med diethylether.1.2 g (1.9 mmol) of the product was hydrogenated at room temperature in the presence of pre-reduced 5% palladium-on-carbon to remove the p-nitrobenzyl ester group and to give 450 mg of 7- (D-2- (t-butyloxycarbamido)) -2- (4-chlorophenylacetamido)) - 3-chloro-3-cephem-4-carboxylic acid as a crystalline product. The product was obtained in crystalline form by treating the amorphous residue with diethyl ether.

Grundst of-analyse for ^20^21^2^3^6^1 teoretisk: C 47,82 - H 4,21 - N 8,36 - Cl 14,11.Fundamentals of analysis for ^ 20 ^ 21 ^ 2 ^ 3 ^ 6 ^ 1 theory: C 47.82 - H 4.21 - N 8.36 - Cl 14.11.

fundet: C 47,75 - H 4,43 - N 8,11 - Cl 14,15.found: C 47.75 - H 4.43 - N 8.11 - Cl 14.15.

Elektrometrisk titrering (66 % vandig DMF): pKa ved 4,4; tilsyneladende molekylvægt = 508; beregnet MW = 502.Electrometric titration (66% aqueous DMF): pKa at 4.4; apparent molecular weight = 508; calculated MW = 502.

450 mg (0,9 mmol) af deesterificeringsproduktet omsattes med p-toluensulfonsyren i acetonitril ved at følge metoden beskrevet i de tidligere eksempler for at fjerne den beskyttende tert.-butyl-oxycarbonyl-gruppe og til opnåelse af 160 mg (44 %) af det krystallinske 7- (D-4-chlorphenylglycylamido) -3-chlor-3-cephem-4-carboxyl-syre i form af den amf ot er e ion.450 mg (0.9 mmol) of the deesterification product was reacted with the p-toluenesulfonic acid in acetonitrile following the method described in the previous examples to remove the protective tert-butyl-oxycarbonyl group and to obtain 160 mg (44%) of the crystalline 7- (D-4-chlorophenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid in the form of the amphoteric ion.

Grundstof-analyse for C-^H-^C^N^O^S. lH^O: teoretisk: C 42,86 - H 3,59 - N 9,99 - Cl 16,87.Elemental analysis for C- HH- ^C ^N ^O ^S. 1 H 2 O: theory: C 42.86 - H 3.59 - N 9.99 - Cl 16.87.

fundet: C 43,07 - H 3,63 - N 9,69 - Cl 16,75.Found: C 43.07 - H 3.63 - N 9.69 - Cl 16.75.

U.V. spektrum (pH buffer): maxima ved 265 ωμ (2=8.100) og 2,25 mp (6=13.900).U.V spectrum (pH buffer): maxima at 265 ωμ (2 = 8,100) and 2.25 mp (6 = 13,900).

DK 153157BDK 153157B

Elektrometrisk titrering (66 % vandig DMF): pKa = 4,15 og 6,8; tilsyneladende molvægt = 407, beregnet molvægt = 403.Electrometric titration (66% aqueous DMF): pKa = 4.15 and 6.8; apparent molecular weight = 407, calculated molecular weight = 403.

Eksempel_23 7;(D-4-hydro^ghenylglycylamido)-3ichlor-3-cephem-42carboxylsyre N-(t-butyloxycarbonyl)-D-4-hydroxyphenylglycin (2,9 g, 11 mmol) omsattes med 3,7 g (10 mmol) p-nitrobenzyl-7-amino-3-chlor-3-cephem- 4-carboxylat og 2,6 g (10,5 mmol) af koblingsreagenset EEDQ i tør tetrahydrofuran. Produktet isoleredes ved at følge metoden beskrevet i eksempel 22. Produktet, i alt 3,7 g (60 %) udbytte), opnåedes i krystallinsk form fra koldt diethylether.Example 237 (D-4-hydroxyphenylglycylamido) -3ichloro-3-cephem-42-carboxylic acid N- (t-butyloxycarbonyl) -D-4-hydroxyphenylglycine (2.9 g, 11 mmol) was reacted with 3.7 g (10 g). p-nitrobenzyl-7-amino-3-chloro-3-cephem-4-carboxylate and 2.6 g (10.5 mmol) of the coupling reagent EEDQ in dry tetrahydrofuran. The product was isolated following the method described in Example 22. The product, total 3.7 g (60%) yield), was obtained in crystalline form from cold diethyl ether.

Produktet, p-nitrobenzyl-7(D-2-(t-butyloxycarbamido)-2-(4-hydroxy)-phenylacetamido)-3-cblor-3-cepbem-4-carboxylat gav følgende procentvise grundstof-analyse og NMR-spektrum:The product, p-nitrobenzyl-7 (D-2- (t-butyloxycarbamido) -2- (4-hydroxy) phenylacetamido) -3-chloro-3-cepbem-4-carboxylate gave the following percentage elemental analysis and NMR spectrum :

Grundstof-analyse for C^H^N^OgSCl: teoretisk: C 52,39 - H 4,40 - H 9,05.Elemental analysis for C ^H ^N ^O₂SCl: theory: C 52.39 - H 4.40 - H 9.05.

fundet: C 52,12 - H 4,26 - N 8,91.Found: C 52.12 - H 4.26 - N 8.91.

NMR-spektrum (DMSO dg):NMR Spectrum (DMSO dg):

Signaler ved 8,62 (s, 9H, t-BOC-CH^), 6,16 (ABq, 2H, C2-H2), 4,81 (d, IH, C6-H), 4,75 (d, IH, a-CH), 4,53 (s, 2H, ester-CH2), 4,18 (q, IH, C?-H), 7,04 og 2,0 (2q, 8H, aromatisk H), 0,76 (d, IH, C?-NH) og 0,58 (s, IH, p-OH), tau.Signals at 8.62 (s, 9H, t-BOC-CH 2), 6.16 (ABq, 2H, C2-H2), 4.81 (d, 1H, C6-H), 4.75 (d, 1H, α-CH), 4.53 (s, 2H, ester-CH 2), 4.18 (q, 1H, C?-H), 7.04 and 2.0 (2q, 8H, aromatic H), 0.76 (d, 1H, C?-NH) and 0.58 (s, 1H, p-OH), tau.

2,2 g (3,5 mmol) af produktet hydrogeneredes i ethanol i nærværelse af præreduceret 5 % palladium-på-carbon katalysator for at udvirke fjernelse af p-nitrobenzylgruppen. Det deesterificerede. produkt, 7-(D-2-(t-butyl-oxycarbamido)-2-(4-hydroxy)phenyl-acetamido)-3-chlor-^-cenham-h-nar’bnyv! svrs. ormåedes i krvstallinsk form fra en bian-2.2 g (3.5 mmol) of the product was hydrogenated in ethanol in the presence of pre-reduced 5% palladium-on-carbon catalyst to effect removal of the p-nitrobenzyl group. It deesterified. product, 7- (D-2- (t-butyl-oxycarbamido) -2- (4-hydroxy) phenyl-acetamido) -3-chloro-cenham-h-nar'nybyl svrs. was rendered in crystalline form from an intermediate form.

44 DK 153157 B44 DK 153157 B

ding af diethylether og hexan. Der opnåedes 1 gram af produktet svarende til et udbytte på 59 %.diethyl ether and hexane. 1 gram of the product was obtained corresponding to a yield of 59%.

Grundstof-analyse for ^20^22^^3^7^ teoretisk: C 49,64 - H 4,58 - N 8,08.Elemental analysis for ^ 20 ^ 22 ^^ 3 ^ 7 ^ theory: C 49.64 - H 4.58 - N 8.08.

fundet: C 48,92 - H 4,40 - N 8,24.Found: C 48.92 - H 4.40 - N 8.24.

NMR-spektrum (DMSO dg):NMR Spectrum (DMSO dg):

Signaler ved: 8,61 (s, 9H, t-BOC-CKj), 6.26 (ABq, 2H, C2H2), 4,89 (d, IH, Cg-H), 4.78 (d, IH, a-CH), 4,28 (q, IH, C?-H), 3,06 (q, 4H, aromatisk-H) og 1,20 (d, IH, C?-NH) tau.Signals at: 8.61 (s, 9H, t-BOC-CKj), 6.26 (ABq, 2H, C2H2), 4.89 (d, 1H, Cg-H), 4.78 (d, 1H, a-CH) , 4.28 (q, 1H, C? -H), 3.06 (q, 4H, aromatic-H), and 1.20 (d, 1H, C? -NH) tau.

Den beskyttende tert-30C-gruppe fjernedes fra deesterificerings-produktet ved at omsætte dette i acetonitril med p-toluensulfon-syre. Fra 1 g af produktet opnåedes 330 mg (40 %) af slutproduktet, 7-(D-4-hydroxyphenylglycylamido)-3-chlor-3-cephem-4-carboxylsyre med følgende grundstof-analyse og NMR-spektrum:The protective tert-30C group was removed from the deesterification product by reacting it in acetonitrile with p-toluenesulfonic acid. From 1 g of the product, 330 mg (40%) of the final product, 7- (D-4-hydroxyphenylglycylamido) -3-chloro-3-cephem-4-carboxylic acid, was obtained with the following elemental analysis and NMR spectrum:

Grundstof-analyse beregnet for C15Hl4C1N3°5S*1H20i teoretisk: C 44,83 - H 4,01 - N 10,46.Elemental analysis calculated for C 15 H 14 ClN 3 O 5 S * 1H 2 O in theory: C 44.83 - H 4.01 - N 10.46.

fundet: C 44,92 - H 3,45 - N 10,63.Found: C 44.92 - H 3.45 - N 10.63.

Elektrometrisk titrering i 66 % vandig DMG gav v dier på 4,2, 7,7 og 12,4. Den tilsyneladende molvægt = 384; den beregnede molvægt = 383,8.Electrometric titration in 66% aqueous DMG yielded values of 4.2, 7.7 and 12.4. The apparent molecular weight = 384; the calculated molecular weight = 383.8.

NMR-spektrum (D20/DC1) viste signaler ved 6,32 (ABq, 2H, C2-H2), 4,84 (d, IH, Cg-H), 4.27 (d, IH, C7-H) og 2.79 (q, 4H, aromatisk-H) tau.NMR spectrum (D20 / DC1) showed signals at 6.32 (ABq, 2H, C2-H2), 4.84 (d, 1H, Cg-H), 4.27 (d, 1H, C7-H) and 2.79 ( q, 4H, aromatic-H) tau.

45 DK 153157B45 DK 153157B

Eksemgel_24 A. p-Nitrobenzyl-7-[N-(t-butyloxycarbonyl)-oc-D-(2-thienyl)-glycyiamido]-3-chlor-3-cephem-4-carboxylat.Example 24 A p-Nitrobenzyl 7- [N- (t-butyloxycarbonyl) -oc-D- (2-thienyl) glyciamido] -3-chloro-3-cephem-4-carboxylate.

N-t-butyloxycarbonyl-2-thienyl-D-glycin (3,2 g, 12,5 mM) opløstes i 300 ml tørt tetrahydrofuran. Der tilsættes p-nitrobenzyl-7-amino- 3-chlor-3-cephem-4-carboxylat (4,6 g, 12,5 mM) og EEDQ (3,4 g, 13,8 mM), og blandingen blev omrørt ved stuetemperatur natten over. Opløsningsmidlet blev fjernet i vacuum, og remanensen opløst i ethylacetat. Ethylacetatopløsningen blev vasket med syre og base og inddampet til tørhed. Produktet krystalliserede ved udrivning med ether.N-t-butyloxycarbonyl-2-thienyl-D-glycine (3.2 g, 12.5 mM) was dissolved in 300 ml of dry tetrahydrofuran. P-Nitrobenzyl 7-amino-3-chloro-3-cephem-4-carboxylate (4.6 g, 12.5 mM) and EEDQ (3.4 g, 13.8 mM) are added and the mixture is stirred. at room temperature overnight. The solvent was removed in vacuo and the residue dissolved in ethyl acetate. The ethyl acetate solution was washed with acid and base and evaporated to dryness. The product crystallized by rubbing off with ether.

Udbytte 5,7 g (78%) I.R. (CHC13)Yield 5.7 g (78%) I.R. (CHC13)

Carbonylbånd ved 5,59 pm (β-lactam), 5,72 pm (ester), 5,89 pm (bred) og 6,55 pm (amid) U.V. (MeCN) X max = 269 nm, £ =17 769Carbonyl bands at 5.59 pm (β-lactam), 5.72 pm (ester), 5.89 pm (wide) and 6.55 pm (amide) U.V. (MeCN) λ max = 269 nm, δ = 17 769

Analyse for 025^5^0^8201: beregnet: C 50,63; - H 4,25 - N 9,45 - Cl 5,98 fundet : C 50,42 - H.4,39 - N 9,22 - Cl 6,10.Analysis for C25 ^H₂01O01: calculated: C, 50.63; - H 4.25 - N 9.45 - Cl 5.98 found: C 50.42 - H.4.39 - N 9.22 - Cl 6.10.

B. 7- [N- (t-butyloxycarbonyl) -oc-D- (2-thienyl) -glycyiamido ]-3-chlor-3-cephem*-4-carboxylsyre.B. 7- [N- (t-Butyloxycarbonyl) -oc-D- (2-thienyl) -glyciamido] -3-chloro-3-cephem * -4-carboxylic acid.

p-Nitrobenzyl-7- [N-(t-butyloxycarbonyl) - oc-D- (2-thienyl) -glycyl-amido]-3-chlor-3-cephem-4-carboxylat (6,0 g, 10,2 mM) blev hydrogenolyseret ved konventionelle metoder under anvendelse af en lige så stor vægtmængde forreduceret palladium-på-kul. Pro-p-Nitrobenzyl-7- [N- (t-butyloxycarbonyl) oc-D- (2-thienyl) glycylamido] -3-chloro-3-cephem-4-carboxylate (6.0 g, 10.2 mM) was hydrogenolyzed by conventional methods using an equal amount of weight pre-reduced palladium-on-charcoal. pro-

DK 153157 BDK 153157 B

duktet krystalliserede ved udrivning med en blanding af ether og hexan i volumenforholdet 1:1.the product crystallized by tearing off with a mixture of ether and hexane in the volume ratio of 1: 1.

Udbytte 2,9 g (64%) U.V. (MeCN) X max = 270 nm = 7500Yield 2.9 g (64%) of U.V. (MeCN) λ max = 270 nm = 7500

Analyse for O-j^E^qN^O^^CI *1/2 mol ether beregnet: C 47,01 - H 4,93 - N 8,22 - Cl 6,94 fundet : C 46,58 - H 4,85 - N 8,19 - Cl 7,13- C. 7-D-2-thienylglycylamido-3“Chlor-3-cephem-4-carboxylsyre 7- [N- (t-butyloxycarbonyl) -oc-D- (2-thienyl)-glycylamido ]-3-chlor- 3-cephem-4-carboxylsyre (2,7 g, 5,3 mM) opløstes i 150 ml aceto-nitril og behandledes med p-toluensulfonsyre (2,2 g, 11,5 mM). Blandingen blev omrørt ved stuetemperatur natten over, fortyndet med 15 ml vand, og pH-værdien indstillet til 5,7 med koncentreret ammoniumhydrogencarbonatopløsning. Produktet udfældedes i krystal-form.Analysis for Cj ^H E ENNO₂Cl₂ 1/2 mol of ether calculated: C 47.01 - H 4.93 - N 8.22 - Cl 6.94 found: C 46.58 - H 4.85 - N 8.19 - Cl 7.13 C. C. 7-D-2-thienylglycylamido-3 “Chloro-3-cephem-4-carboxylic acid 7- [N- (t-butyloxycarbonyl) -oc-D- (2- thienyl) -glycylamido] -3-chloro-3-cephem-4-carboxylic acid (2.7 g, 5.3 mM) was dissolved in 150 ml of acetonitrile and treated with p-toluenesulfonic acid (2.2 g, 11.5 mM). The mixture was stirred at room temperature overnight, diluted with 15 mL of water, and the pH adjusted to 5.7 with concentrated ammonium hydrogen carbonate solution. The product precipitated in crystal form.

Udbytte 1,7 g (86%).Yield 1.7 g (86%).

I.R. (Nuøol-suspension)I.R. (Nuøol suspension)

Carbonylbånd ved 5,65 jum (β-lactam) og 5,91 pm (amid) U.V. (pH 6 puffer) Ά max = 265 nm, 8 = 9800Carbonyl bands at 5.65 µm (β-lactam) and 5.91 µm (amide) U.V. (pH 6 buffer) Ά max = 265 nm, 8 = 9800

Analyse for C-^H-j^N^O^S^CI j beregnet: C 41,77 - H 3,24 - N 11,24 - Cl 9,48 fundet : C 42,01 - H 3,02 - N 11,24 - Cl 9,34.Calcd for: C 41.7 - H 3.24 - N 11.24 - Cl 9.48 Found: C 42.01 - H 3.02 - N 11 , 24 - Cl 9.34.

47 DK 153157 B47 DK 153157 B

Eksemgel_25 p-Nitrobenzyl-7-ohenylglycylamido-3-chlor-3-cephem-4-carboxylat p-Nitrobenzyl-7-[N-(t-butyloxycarbonyl)-D-a-phenylglycylamido]-3-hydroxy-3-cephem-4-carboxylat (3,9 g, 6,7 mM) opløstes i 40 ml tørt dimethylformamid. Der tilsattes phosphortrichlorid (7 ml, 7 mM), og reaktionsblandingen blev omrørt i 3 timer ved stuetemperatur. Den hældtes derpå ud i en blanding af ethylacetat og vand. Ethylacetatlaget blev skilt fra, vasket flere gange med vand, tørret over magnesiumsulfat og inddampet til tørhed i vacuum.Example gel 25 p-Nitrobenzyl-7-ohenylglycylamido-3-chloro-3-cephem-4-carboxylate p-Nitrobenzyl-7- [N- (t-butyloxycarbonyl) -Da-phenylglycylamido] -3-hydroxy-3-cephem-4 carboxylate (3.9 g, 6.7 mM) was dissolved in 40 ml of dry dimethylformamide. Phosphorus trichloride (7 mL, 7 mM) was added and the reaction mixture was stirred for 3 hours at room temperature. It was then poured into a mixture of ethyl acetate and water. The ethyl acetate layer was separated, washed several times with water, dried over magnesium sulfate and evaporated to dryness in vacuo.

%%

Udbytte 1,95 g (58%)Yield 1.95 g (58%)

Analyse for C22H19N4°6SC1 beregnet: C 52,54 - H 3,81 - N 11,14 - Cl 7,05 fundet: C 52,52 - H 4,29 - N 10,73 - Cl 7,55*Analysis for C 22 H 19 N 4 ° 6 SCl calcd: C 52.54 - H 3.81 - N 11.14 - Cl 7.05 found: C 52.52 - H 4.29 - N 10.73 - Cl 7.55 *

Eksempel_26 p-Nitrobenzyl-7-(D-a-phenylglycylamido)-3-brom-3-cephem-4-carboxylat p-Nitrobenzyl-7-[N-(t-butyloxycarbonyl)-D-a-phenylglycylamido]-3-hydroxy-3-cephem-4-carboxylat (2,3 g, 4 mM) blev opløst i 25 ml dimethylformamid og behandlet med 3 ml phosphorpentabromid. Blandingen blev omrørt ved stuetemperatur natten over. Tyndtlagschro-matografi over silicagel, udviklet med ethylacetat/benzen 7:3, viste næsten fuldstændig omsætning af udgangsmaterialet til produktet. Reaktionsblandingen blev dispergeret i vand/ethylacetat.Example_26 p-Nitrobenzyl-7- (Da-phenylglycylamido) -3-bromo-3-cephem-4-carboxylate p-Nitrobenzyl-7- [N- (t-butyloxycarbonyl) -Da-phenylglycylamido] -3-hydroxy-3- cephem-4-carboxylate (2.3 g, 4 mM) was dissolved in 25 ml of dimethylformamide and treated with 3 ml of phosphorus pentabromide. The mixture was stirred at room temperature overnight. Thin layer chromatography over silica gel, developed with ethyl acetate / benzene 7: 3, showed almost complete reaction of the starting material to the product. The reaction mixture was dispersed in water / ethyl acetate.

Det organiske lag blev skilt fra, vasket med saltvand, tørret over magnesiumsulfat og inddampet til tørhed i vacuum. Den amorfe remanens blev chromatograferet over silicagel til opnåelse af det ovennævnte produkt.The organic layer was separated, washed with brine, dried over magnesium sulfate and evaporated to dryness in vacuo. The amorphous residue was chromatographed over silica gel to give the above product.

N.M.R. (CDCl-j): 6,24 (Abq, 2H, C2-H2), 4,80 (d, IH, C6-H),N.M.R. (CDCl3): 6.24 (Abq, 2H, C2-H2), 4.80 (d, 1H, C6-H),

Claims (1)

49 DK 153157 B 2 hvor R har den ovennævnte betydning, og R er t-butyloxycarbonyl, benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, trichlorethoxycarbonyl eller trityl, eller enaminer dannet med methylacetoacetat og acetylacetone eller et anhydrid, et syrehalogenid eller en aktiv ester deraf, hvorefter den carboxylsyrebeskyttende, esterdannende gruppe eller den amino-beskyttende gruppe fjernes til opnåelse af den tilsvarende 7-(α-amino)acylamido-3-halogen-3-cephem-4-carboxyl-syre, som om ønsket overføres i et farmaceutisk acceptabelt salt.Wherein R is as defined above and R is t-butyloxycarbonyl, benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, trichloroethoxycarbonyl or trityl, or enamines formed with methylacetoacetate and acetylacetone or an anhydride, an acid halide or an active ester thereof or the carboxylic acid protecting ester-forming group or the amino-protecting group is removed to give the corresponding 7- (α-amino) acylamido-3-halo-3-cephem-4-carboxylic acid, which if desired is transferred into a pharmaceutically acceptable salt.