FI64803B - EXAMINATION OF ANTIBACTERIAL VERIFICATION OF 7-2-ALCOXO-IMINO-2-FURYLACETAMIDO) -3- (1-CARBOXYKYLTETRAZOYL-5-THIOMETHYL) -3-CEFEM-4-CARBOXYL SYROR - Google Patents

Description

- Γβ,. lnt.CI.3 C 07 D 501/36 FINLAND-FINLAND (21) Puwttlhtt.mus - Pat.ntansöknlng 7725 ^ 9 (22) Hkmlsptlvi · —Ansöknlngsdag 26.08.77 (23) Alkupllvi —Glltighetsdsg 26.08.77 (41) Become Public - Blivlt offtntllg 01.03.78

National Board of Patents and Registration Nihttvtt.lp.non j. month, date and date. _

Patent-och registerstyreiaan Anseksn uttagd och uti.skrifwn public * ™ * 30.09.83 (32) (33) (31) Pyy ^ ttty stuoikeus —Begird prlorlt.t 30. Οθ. 76 USA (US) 719025 (71) Bristol-lfyers Company, 3 ^ + 5 Park Avenue, New York, N.Y. 10022, USA (72) William J. Gottstein, Fayetteville, New York, USA (7M Oy Kolster Ab (5M Method for antibacterial 7 '(2-alkoxyimino-2-furylacetamido) -3_ for the preparation of (1-carboxyalkyltetrazolyl-5-thiomethyl) -3-cephem - 1 + carboxylic acids - For the preparation of fractions of an antibacterial network 7 "(2-alkoxyimino-2-furylacetamido) -3- (1-carboxyalkyl tetra The present invention relates to a process for the antibacterial action of 7- (2-alkoxyimino-2-furylacetamido) -3- (1-carboxyalkyltetrazolyl), azoryl-5-thiomethyl) -3-cephem-1 * carboxylic acid. To prepare -5-thiomethyl) -3-cephem-4-carboxylic acids of the formula [i-jT-C - C-NH-CH-CH XCH2 fi Ί. Vn. 0 / _ \ c <iC'C "OS ' CS..2 CK OR 'OI j

COOH (CH-) COOH

i n 2 64803 wherein R 1 is a lower alkyl group and n is 1, 2 or 3, or a pharmaceutically acceptable salt thereof.

British Patent No. 1,399,086 discloses Kefalos porins of the formula n / R -C-CO-NH -

Il 1 N --CH2Y <B) \, _ b o '|

OR I

COOH

wherein RU is a phenyl, naphthyl, thienyl, furyl, benzothienyl, benzofuryl, pyridyl group or one of these groups substituted by a halogen atom (chlorine, bromine, iodine or fluorine atom), hydroxy, alkyl, nitro -, amino, alkylamino, dialkylamino, alkanoyl, alkanoylamino, alkoxy, alkylthio or carbamoyl, R * 3 is an alkyl, cycloalkyl group having 3 to 7 carbon atoms or a heterocyclic arylalkyl group or one of these groups substituted hydroxy, carboxy, esterified carboxy, amido, cyano, alkanoyl, amino, substituted amino, halogen or alkoxy; and Y is of the formula

Q

a group according to ** a> »(substituents as defined in claim 19); a group of the formula -SW wherein W is thiadiazolyl, diazolyl, triazolyl, tetrazolyl, thiazolyl, thiatriazolyl, oxazolyl, oxadiazolyl, benzimidazolyl, benzoxazolyl, triazolopyridyl, triazolopyridyl , pyridyl or pyrimidyl 9

group; An alkylthio group having 1 to 4 carbon atoms; of the formula -O-CO-R

9, wherein R is an alkyl or alkenyl group having 2 to 4 carbon atoms; a group -O-CO-NH- (CH2) wherein m is an integer from 1 to 4 and D is a chlorine, bromine, iodine or fluorine atom; and an azido group and 3 64803 their non-toxic salts and esters. Methods for preparing the starting acids used to form the 7-substituent, including separating them into syn and anti-isomers, are also described therein and in British Patent No. 1,404,221.

Recently published U.S. Patents 3,966,717 and 3,971,778 contain, at least in part, the material of English Patent 1,399,086 as well as U.S. Patent No. 3,974,153. See also Farmdoc Reference, 17270 X and 19177 X.

U.S. Patent No. 3,974,153 discloses compounds of the formula

H H

I! 1 '1.

R -C-CONH - - <V j-ivJ-CH 2 O-CO-NH 2

COOH

having a furyl, thienyl or phenyl group; and R a is a C 1-6 alkyl, C 1-6 cycloalkyl or phenyl group; and their physiologically acceptable salts.

For examples of publications in the scientific literature, see Ryan et al., Antimicrobial Agents and Chemotherapy, 9f 520-525 (1976), and O'Callaghan et al., Ibid. 9, 51 1-519 (1976) and Norby et al., Ibid. 9, 506-510 (1976).

U.S. Patent No. 3,819,623 discloses the conversion of 2-mercapto-1,3,4-thiadiazole-5-acetic acid to 7- (1H-tetrazol-1-yl-acetamido) -3- (5-carboxymethyl-1, 3,4-thiadiazol-2-ylthiomethyl)-cephem-4-carboxylic acid; and see also Farmdoc Reference 12921 T.

U.S. Patent Nos. 3,883,520 and 3,931,160 and Farmdoc Reference 22850 W refer to 3-heterocyclic thiomethyllephalosporins containing multiple substituents (including a carboxyl group) in several of the heterocycles contained therein, but these references are entirely common in nature. , and do not contain any physical constants, yields, synthetic methods, etc., and do not even name any such compound containing a carboxyl substituent. See also Farmdoc-00145 W.

U.S. Patent No. 3,928,336 provides an overview of the much older cephalosporin industry.

Farmdoc Reference 18830 X describes compounds of formula 4 64803 f3 1 ^ -

R NH - f η NN

1 i ii J-CH2S - COOH | (CH0) -COOH i n 1 3

In this case, R is an acyl group or a hydrogen atom, R is a hydrogen atom or a methoxy group, and n is 1 to 9. See also Farmdoc Reference 01981 X.

The current medical problem is described by Arnold L. Smith in an article entitled Antibiotics and Invasive Haemophilus influenzae, N. Engl. Med., 294 (1976), 1329-1331. The introductory sentence of the article reads as follows: "Recently, the Information Service of the Center for Disease Control, the Medical Letter and the American Academy of Pediatrics have warned that the spread of Haemophilus influenzae species , which have been isolated throughout the United States, have been found to be ampicillin - resistant! The end states: “The current situation predicts a bleak future for antibiotic treatment of the spreading H. influenzae disease. An H. influenzae species resistant to the second generation drug, chloramphenicol, has been described, and a very recently untyped H. influenzae species resistant to chloramphenicol and tetracycline was isolated from the throat of a four-year-old girl. Therefore, neither of these currently effective substances may be useful in the future. "

The process according to the invention is characterized in that a 7-aminocephalosporanic acid of the formula ΝΗ, -CH-f

2 I O

- ch2-o-c-ch3

COOH

or a salt thereof to react with 1-carboxyalkyl-5-mercaptotetrazole of the formula 5 64803

N-N

\\ Il

HS / N

OF

(CH-) COOH 2 n where n is 1, 2 or 3, or a salt of this compound, and the resulting 7-amino-3- (1-carboxyalkyltetrazol-5-yl-thiomethyl) -3-cephem-4-carboxylic acid is treated with an acylating agent with the formula

ry rL

o OR

wherein X is a halide or a reactive equivalent thereof and R 1 is as defined above, and optionally converting the resulting free acid or salt of a compound of formula I to its corresponding pharmaceutically acceptable salt, and optionally converting the resulting salt of a compound of formula I to the corresponding acid of formula I, wherein the compound of formula I or a salt thereof is present in the form of a mixture of isomers containing at least 75% by weight of syn isomer.

The stereochemistry of the bicyclic core of the compounds of the invention is the same as that found in cephalosporin C.

The compounds of this invention are syn isomers or mixtures of syn and anti isomers containing at least 75% syn isomer. Preferably, such mixtures of isomers contain at least 90% syn-isomer and at most 10% anti-isomer. Most preferably, the compounds are syn isomers that are substantially free of the corresponding anti-isomer. Preferred embodiments of this invention are the syn-isomers of the compounds of formula I wherein R is a methyl or ethyl group 64803 i and n is 1 or 2.

Reference to the syn (cis) isomeric form refers to the configuration of the OR group with respect to the carboxamide group.

Salts of the compounds of this invention include their non-toxic carboxylic acid salts, including non-toxic metal salts such as sodium, potassium, calcium and aluminum salts, ammonium salt and substituted ammonium salts, e.g. salts of benzyl-β-phenethylamine, 1-ephenamine, Ν, Ν'-di-benzylethylenediamine, dehydroabietylamine, Ν, Ν'-bis-dehydroabiethylethylenediamine and other amines used to form salts of benzylpenicillin, with arginine and histidine.

Results of pharmacological tests

Bacteria.

Organisms of predominantly fresh clinical origin were obtained from several sources with a broad geographical distribution. Anaerobic organisms were maintained in egg-meat medium (Difco); Mycobacterium was maintained in Lowenstein's medium (Jensen variant; Difco). The preservation technique of all other organisms has been described previously (Leitner et al., BL-S640, A Cephalosporin with a Broad Spectrum of Antibacterial Activity: Properties in Vitro, Antimicrob. Agents Chemother. 7: 298-305 (1975)).

Antibiotic spectrum.

The growth inhibitory activity of the compounds was determined by the antibiotic dilution technique. The procedures were as follows:

Aerobic organisms (except Mycobacterium).

With the exception of Haemophilus and Neisseria species, the experiment was performed in Mueller.Hinton medium (Difco). Sensitive organisms, i.e. For Streptococcus, Listeria, Pasteurella, Bordetella and Vibrio species, the medium was supplemented with 4% defirinated sheep blood. The antibiotic susceptibility of Haemophilus and Neisseria species was determined in GC basal medium (BBL) supplemented with 1% hemoglobin (BBL) and 1 £ Isovitalex (BBL).

7 64803

Broth cultures grown overnight in an exponentially growing culture (Neisseria) served as a source of planting. An approximately 0.003 ml volume of undiluted or diluted culture was applied to the surface of antibiotic-containing agar plates according to Streers1 et al. with an implant, An Inocula Replicating Apparatus for Routine Testing of Bacterial Susceptibility to Antibiotics, Antibiot. Chemother, 9: 307-311 (1959). Cultures of Neisseria, Streptococcus pneumoniae, 5. viri-dans and S. pyogenes were used undiluted; cultures of all other organisms were diluted 100-fold. The assay contained about 10 viable cells of Neisseria species, 10 S.

6 4 pneumoniae and S. pyogenes species, 10 S. viridans species and 10 all other species. Culture plates were germinated at 37 ° C either overnight or for 24 hours (Haemophilus) and the minimum inhibitory concentration (MIC), i. the lowest concentration of antibiotic that inhibits visible growth was noted.

Antibiotic concentration in the blood.

Male Swiss-Webster mice weighing 19-22 g were administered 0.2 ml of antibiotic solutions at appropriate concentrations by intramuscular injection. The carrier was 0.01% phosphate buffer at pH 7.0. Eight animals were used for each dose amount. Blood samples (0.03 ml) were obtained from the bases of the orbit with heparinized capillary tubes (Clay Adams) at 0.25, 0.5, 1 and 1.5 hours after compound administration. 6.35 mm diameter paper discs were impregnated with blood and antibiotic activity was tested by diffusion technique using seed agar (BBL) planted with Bacillus subtilis ATCC 6633. A standard line was obtained for the ratio of inhibition zone diameter to drug concentration by testing compounds at known concentrations in heparinized mouse blood.

Treatment of organically infected mice.

The procedures were similar to those previously published (Leitner et al., BL-S640 a Cephalosporin with a Broad Spectrum of Antibacterial Activity: Bioavailability and Therapeutic Properties in Rodents, Antimicrob. Agents Chemother. 7: 306-310 (1975)) except that a ) porcine stomach mucus used to infect Staphylococcus aureus species 11a No. 2 was purchased from American Laboratories Inc., Omaha, Neb. (lot no. 154163) and b) the medium used to suspend all other organisms contained 3% (instead of 4%) porcine gastric mucosa (type 1701 W, Wilson Laboratories, Inc., Park Forest South, 111).

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Concentrations in mouse blood after intramuscular administration

Dose Blood concentration (pg / ml), hours of administration,,. . „After%

Compound_ (mg / kg) _0.25 0.5_1 ^ 0_V5_ BL-S1080 40 46.3 30.4 15 <8.9 20 21.1 16.4 <8.1 <8.1 BL-S1095 40 45, 8 45.1 27.3 <15.9 BL-S1081 40 30.5 20.7 8.4 <4.9 20 17.4 10.7 <4.7 <4.7

Cefuroxime 40 34.3 22.8 7.1 <1.8 20 16.7 10.4 2.4 <1.8 10 8.6 5.9 <1.8 <1.8 BL-S1105 40 39, 3 27.3 <10.9 <10.9 (* not all of these experiments have been performed simultaneously 10 64803

Efficacy of intramuscular treatment in mice self-infected with organs (‘’) by various organisms (*

Exposure PD ^ q / treatment (mg / ^ gj

(organs of BL-S Kerur- BL-s BT Organism_number) _1080 oxime_786 '** ^ L ~ S

H.influenzae 5 x 106 3.1 13 29 A9729 6 x 106 1, 4 33 25 E · c ° li 7 x 105 7.1 0.3 6i3 A15119 5 (*** '7x10 6.3 2.3' 0.4 P. mirabilis 3 x 106 3.1 5.4 0.8 A9900 _ fi 5x10 0.6 - 0.7 5 P. vulgaris 4 x 10 0.2 0.29 1.6 A9436 3 S.pyogenes 2x10 1.6 0.2 1.6 A9604 c in2, 5x10 1.6 - 1.6 4 S. pneumoniae 1x10 2.7 0.4 1.6 A9585 4 x 103 2.7 0.9 1.6 S. aureus 8x108 19 0.6 11 8.2 A96 ° 6 8 x 108 43 1.0 1 x 109 13 - 6.3 (* not all of these experiments were performed simultaneously (** BL-S786 is 7- (2- Aminomethylphenylacetamido) -3- (1-carboxymethyltetrazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid (*** Result of three or more previous experiments 11 64803

Microbiological screening of a compound

Compound BL-S1081 against Haemophilus influenzae. Secondary in vitro screening results - MIC (pg / ml)

Organism No BL-S1081 Cefuroxime BL-S1080 Δ9729 0.5 1 0.5 A9832 0.5 1 0.13 A20173 0.5 1 0.5 A20177 0.5 0.5 0.5 A20178 0.13 0 .5 0.13 A20181 0.25 1 0.13 A20188 0.25 1 0.13 A20189 0.25 1 0.13 A20192 0.13 0.5 0.063 A20193 0.25 1 0.5 A21515 0.25 0 .5 0.13 A21517 0.25 0.5 0.13 A21519 0.25 0.5 0.13 A21520 0.25 0.5 0.063 A21522 0.032 0.5 0.13 A21523 0.25 0.5 0 13 12 64803

Microbiological screening of a compound

Compound BL-S1080 against Haemophilus influenzae. Secondary in vitro screening results - MIC (pg / ml)

Organism No BL-S1080 Cefuroxime BL-S786 A9729 0.13 0.5 4 A9832 0.032 0.25 4 A9833 0.032 0.25 4 A20177 0.063 0.25 4 'A20178 0.063 0.5 4 A20183 0.063 0.25 4 A20188 0.063 0.25 4 A20189 0.063 0.25 4 A20191 0.063 0.25 8 A20192 0.032 0.25 4 A21515 0.063 0.13 2 A21517 0.063 0.13 4 A21518 0.032 0.13 4 A21519 0.032 0.25 8 A21522 0.032 0.13 4 A21523 0.032 0.25 4 13 64803

Microbiological screening of a compound

Compound BL-S1081 against Neisseria gonorrheae. Secondary in vitro screening results - MIC (jng / ml)

Organism No. BL-S1081 Cefuroxime BL-S1080 A20142 0.032 0.063 0.13 A20143 0.032 0.008 0.016 A21440 0.032 0.032 0.016 A21442 0.13 0.13 0.13 A21444 0.13 0.032 0.063 A21448 0.016 0.016 0.016 A21449 0.016 0.016 0.016 A21453 0.016 0.016 0.008 A21455 0.063 0.13 0.13 A21456 2 1 2 A21461 0.063 0.063 0.063 A21462 1 0.5 2 A21467 0.13 0.25 0.25 A21468 0.016 0.032 0.008 A21469 0.063 0.25 0.25 A21470 0 13 0.13 0.25 64803 14

Microbiological screening of a compound

Compound BL-S1080 against Neisseria gonorrheae. Secondary in vitro screening results - MIC (pg / ml)

Organism No. BL-S1080 Cefuroxime BL-S786 A20142 0.002 0.0005 0.008 A20143 0.001 0.0005 0.008 A20144 0.063 0.063 1 A21440 0.008 0.002 0.5 A21442 0.004 0.001 0.5 A21 444 0.008 0.001 1 ^ .s' A21446 0.016 0.008 1 A21447 0.008 0.002 0.5 A21448 0.004 0.001 0.5 A21449 0.008 0.001 0.5 A21450 0.032 0.008 2 A21455 0.13 0.063 1 A21456 0.25 0.13 1 A21461 0.008 0.008 0.5 A21462 0.25 0, 25 4 A21469 0.032 0.13 2 15 64803

Microbiological Compound Screening Compound BL-S1081

Primary in vitro screening results - MIC (μg / ml)

Organisms BL-S BL-S Cefuroxime _1081 1080_

Diplococcus pneumoniae A9585 0.004 - 0.004

Streptococcus pyogenes A9604 0.004 - 0.004

Staphylococcus aureus A9537 8 8 0.25

Staph.aureus ^ -50% Serum A9537 16 63 1 -3

Staph.aureus at 10 Dii A9606 4 - 0.5 -2

Staph.aureus at 10 Dii A9606 4 - 0.5

Salmonella enteritidis A9531 0.06 - 0.06

Escherichia coli A15119 4 - 4

Escherichia coli A9675 4 4

Klebsiella pneumoniae A9977 1 - 1

Klebsiella pneumoniae A15130 32 - 32

Proteus mirabilis A9900 0.13 - 0.13

Proteus morganii A15153 63 - 63

Pseudomonas aeruginosa A9843A> 125 -> 125

Serratia marcescens A20019 32 - 125

St.aur. Meth-Res.10-3 Dii A15097 32 4

Enterobacter cloacae A9656> 125 -> 125

Enterobacter cloacae A9657 8 2

Enterobacter cloacae A9659 63 - 125 64803

Microbiological Compound Screening Compound BL-S1105

Primary in vitro screening results - MIC (pg / ml) BL-S BL-S BL-S Kefur

Organic 1150 1080 786 oxime

Diplococcus pneumoniae A9585 0.016 0.016 0.016 0.004

Streptococcus pyogenes A9604 0.13 0.03 0.03 0.004

Staphylococcus aureus A9537 4 4 1 0.5

Staph, aureus + 50% Serum A9537 63 63 4 1 -3

Staph, aureus at 10 Dii A9606 481 1

Staph, aureus at 10 2 Dii A9606 482 1

Salmonella enteritidis A9531 0.5 0.5 0.016 0.13

Escherichia coli A15119 8 2 0.5 8

Escherichia coli A9675 4 2 8 63

Klebsiella pneumoniae A9977 2 1 0.016 2

Klebsiella pneumoniae A15130 63 32 1 32

Proteus mirabilis A9900 0.25 0.016 0.03 0.03

Proteus morganii A15153 63 63 63 32

Pseudomonas aeruginosa A9843A 125> 125> 125> 125

Serratia marcescens A20019 63 125 125> 125

St.aur.Meth.Res. 10-3 Dil A15097 32 32 4 4

Enterobacter cloacae A9656> 125 125 125> 125

Enterobacter cloacae A9657 4 2 0.5 2

Enterobacter cloacae A9659> 125> 125> 125> 125 17 64803

In the treatment of bacterial infections in humans, the compounds of the invention are administered parenterally in an amount of about 10-90 mg / kg / day, and preferably about 14-50 mg / kg / day in divided doses, e.g. 2-4 times daily. They are administered in dosage units containing, for example, 125, 250 or 500 mg of active ingredient with suitable physiologically acceptable carriers or diluents. Dosage units are in the form of liquid preparations, such as solutions or suspensions, and are preferably aqueous solutions of the sodium or potassium salt, injected intravenously or intramuscularly or as a continuous or intermittent liquid injection at concentrations of about 125 to 500 mg / ml and more preferably 250 mg / ml -losporin antibiotics.

Preparation of starting materials: 1-carboxymethyl-5-mercaptotetrazole

N-N

Il II

HS-C „N

'•'OF"'

CH2C00H

a) Recrystallization of 1-methyl-5-mercaptotetrazole

Procedure: 1. 110 g of 1-methyl-5-mercaptotetrazole are slurried in 350 ml of boiling chloroform. An almost finished solution is obtained.

2. Quickly filter the hot solution (50-60 ° C) through a vacuum-heated Biichner funnel (11 cm SS No. 604 paper containing 1/4 to 1/3 inch of concentrated filter aid ("Supercel")).

3. Cool the filtrate to approximately 0-6 ° C and maintain at 0-6 ° C for 2 hours. The formed crystals are collected by filtration at 0 to 6 ° C and washed with 60 ml of 0 to 6 ° C chloroform, which is added to the filtrate. The crystals (fraction A) are air dried at 37-45 ° C for 18 hours.

4. Concentrate the filtrate on a rotary evaporator (60 ° C bath) to approximately half volume. This slurry is cooled to 0-6 ° C and kept at 0-6 ° C for 2 hours. The crystals are collected by filtration at 18-80 ° C, washed with 40 ml of 0-6 ° C chloroform, which is added to the filtrate. The crystals (fraction B) are air-dried at 37-45 ° C for 18 hours. The crystal fractions A and B are combined to give a yield of approximately 65% by weight.

5. The filtrate from fraction B from step 4 can be reprocessed twice as described in step 4 to give an additional 15% yield.

b) Preparation of the disodium salt of 1-carboxymethyl-5-mercaptotetrazole 1. 500 ml of essentially dry and pure tetrahydrofuran in a 2-liter 3-neck flask equipped with a stirrer are cooled in a salt-acetone ice bath to approximately -10 ° C. Dry nitrogen gas is blown onto the surface of the liquid.

2. 500 ml of a 15.06% (1,6-n) butyllithium hexane solution (Foote Mineral Co.) is added over ten minutes under dry nitrogen and with stirring in tetrahydrofuran. The nearly finished solution is cooled to -5 to -10 ° C.

3. Dissolve 46.4 g of 1-methyl-5-mercaptotetrazole (recrystallized as above) in 200 ml of substantially pure and dry tetrahydrofuran. If the solution is cloudy, it is filtered and then cooled to 5-10 ° C. y "'4. The cooled solution of step 3 is added to the butyllithium solution under dry nitrogen under stirring for 10 minutes. The temperature must be kept between -5 ° C and not more than + 10 ° C.

5. The mixture is stirred under dry nitrogen and at 0-10 ° C for half an hour.

6. Anhydrous carbon dioxide gas is blown through the mixture at high speed with rapid stirring for 15-30 minutes from approximately ambient temperature (0-10 ° C) to a maximum of + 20 ° C. until.

7. The resulting white precipitate is suitably collected by filtration under low humidity. The precipitate is washed with approximately 7 5 ml of tetrahydrofuran.

8. Dissolve the precipitate in 250 ml of water (pH 8.5-9.5). A second layer of tetrahydrofuran may be present. It can be removed on a vacuum rotary evaporator (50 ° C bath).

19 64803 9. The aqueous solution is adjusted to pH 1.6-2.0 with concentrated hydrochloric acid.

10. The acidic aqueous solution is extracted twice with 250 ml portions of ethyl acetate. Each 250 ml of ethyl acetate extract is back-extracted with 100 ml portions of water. The aqueous extracts are discarded. The ethyl acetate extracts (without any aqueous layer) are filtered and combined.

11. Concentrate the combined ethyl acetate extracts to dryness on a vacuum rotary evaporator (60 ° C bath).

12. Boil the crystals in the flask with 300 ml of chloroform for about 2 minutes. The hot slurry (50-60 ° C) is vacuum filtered through a heated Buchner funnel (11 cm - SS - 604 paper). The crystals are washed with about 75 ml of 50% chloroform. The crystals are air dried at room temperature for about 3 hours and ground to a size of about 100-200 mesh.

13. 100-200 mesh crystals are treated with boiling chloroform exactly as described in step 12 (hot chloroform removes most of the unreacted 1-methyl-5-mercaptotetrat-Sol). Yield: Approximately 4-5-50 g of crystalline 1-carboxymethyl-5-mercaptotetrazole. These crystals may contain 0.02 to 0.05 moles of 1-methyl-5-mercaptotetrazole.

14. The crystals of step 13 are slurried in 250 mL of ethyl ether at room temperature for 3-5 minutes. The mixture is filtered. The insoluble matter (0.5-5%) may be an impurity symmetrical mercaptotetrazole ketone with the following experimental structure: N = N n, T—

,, 0 N-N

N. .N-CH0-C-CH0-N .N

i i

SH SH

Warning: This compound will explode at approximately 205-210 ° C.

15. The ether filtrate of step 14 is evaporated to dryness under vacuum rotary evaporation 11 a (50 ° C bath). The largest amount of 42-48 g of crystalline 1-carboxymethyl-5-mercaptotetra containing about 0.0 1-0.05-2080803 moles of 1-methyl-5-mercaptotetrazole , is recovered.

16. Dissolve the crystals in 420 ml of absolute ethanol (approximately 100 mg / ml). The solution is heated to 50-60 ° C.

17. To the hot solution of step 16 is added 310 mL of a 41% solution of sodium 2-ethylhexanoate (SEH) in isopropanol with very rapid stirring for Ί0 minutes. A crystalline precipitate forms. Mix the slurry at 50-60 ° C for 20 minutes.

18. Filter the mixture through a heated (50-60 ° C) heated Buchner funnel (11 cm SS-No. 64 paper). The crystals are washed with 75 ml of 50 ml of ethanol.

19. The wet crystals from the ethanol of step 18 are slurried in 200-300 ml of ethanol. The slurry is passed through a 200 mesh screen. The slurry is kept at 50-60 ° C for 5 minutes with rapid stirring (unreacted monosodium 1-methyl-5-mercaptotetrazole is highly soluble in hot ethanol).

20. The crystals are collected at 50-60 ° C in an 11 cm SS No. 604 paper in a heated Biichner funnel. The crystals are washed with 75-100 ml of ethanol and vacuum dried at 50-60 ° C for 24-48 hours. Yield: 40-48 g of disodium 1-carboxymethyl-5-mercaptotetrazole (not containing 1-methyl-5-mercaptotetrazole; NMR analysis).

7-Amino-3- (1-carboxymethyltetrazol-5-ylthiomethyl) -3-cephem-9-carboxylic acid + .Ljl J.ch2-o-c-ch, N “CHgCOgNe * ^ 2 5

N8S COOH

v —pf3 '!

-s -— il -ra cOgH

0 I

COOH

1. Into a three-necked flask equipped with a stirrer, a temperature 11, a thermometer and a nitrogen supply pipe! a, 21 64803 18 g (0.066 mol) of 7-aminocephalosporanic acid (preferably crystallized by the toluenesulphonic acid procedure) and 300 ml of 0.1 M phosphate buffer pH 6.4 (20.7 g of sodium phosphate, monobasic, one crystal water) are placed, 8.5 g sodium phosphate, dibasic, anhydrous, + water to 2 liters).

2. While stirring the mixture described in step 1, 1.5 g of sodium bisulfite and 16 g (0.078 mol) of 1-carboxymethyl-5-mercaptotetrazolidisodium are added.

3. While stirring, nitrogen is blown through the mixture for 10 minutes.

4. Maintain stirring and nitrogen flow, heat the slurry to 56 ° C over 20 minutes. During this period, 6.5 g of sodium bicarbonate are added in small portions.

5. With continued stirring and nitrogen flow, maintain the temperature of the solution at 56 ° C for 4 hours. The pH should remain between 6.2 and 6.6.

6. Cool the reaction mixture with an ice bath to 5 ° C.

7. Add 50 ml of a 1: 1 phosphoric acid / aqueous solution or concentrated hydrochloric acid to a pH of 2.0 to 3.0.

8. The product is collected by filtration. The filter cake is washed with 20 ml of cold water and then with 200 ml of cold methanol.

9. The solid is air dried to constant weight. (A typical run yielded 14.5 g of product). This product can vary in color from yellow to dark brown.

10. The product is passed through a 200 mesh stainless steel screen.

11. 10 g of 200 mesh powder is suspended in 200 ml of n-Oro panol with rapid stirring.

12. Add 2.0 mL of concentrated hydrochloric acid and stir vigorously for 0.5 h at room temperature.

13. Filter the slurry. The brown solid is washed with 20 ml of n-propanol and the washing solution is added to the filtrate (the filter cake is saved for possible recovery of the additional product).

14. Add 1.5 g of charcoal ("Darco G-60") to the n-propanol filtrate of step 13. Slurry for 0.5 hours. The carbon is removed by filtration. The carbon is washed with 20 ml of n-propanol and the washing solution is added to the filtrate.

15. With rapid stirring, triethylalminin is added to the n-propanol filtrate to an apparent pH of 3.0. Crystals are formed. Slurry for 10 minutes.

22 64803 16. The white crystals are collected by filtration and washed with 30 ml of n-propano, 50 ml of methanol and dried in vacuo at 40 ° C for 2 hours.

Yield: 4-8 g of 7-amino-3- (1-carboxymethyltetrazol-5-ylmethyl) -3-cephem-4-carboxylic acid.

17. An alternative procedure for purifying 7-amino-3- (1-carboxymethyl-tetrazol-5-ylthio) -3-cephem-4-carboxylic acid is as follows: a) Slurry 10 g of 200 mesh product (from step 10) in 75 ml: 1N hydrochloric acid for 10-15 minutes at room temperature. Filter to remove a dark brown solid.

(b) Add 3,5 g of charcoal ("Darco G-ti0") and soak in 0,5 * unn in aj an.

c) Remove carbon by filtration. The charcoal is washed with 15 ml of water and the washing solution is added to the filtrate.

d) With rapid stirring, concentrated ammonium hydroxide is added to the filtrate to a pH of 2.5-3.0. Crystals are formed.

e) Slurry the crystalline mass for 25 minutes. The crystals are removed by filtration. The crystals are washed with 30 ml of water and 50 ml of methanol and dried in vacuo at room temperature. Yield: 4-7 g of off-white crystals.

Preparation of 1-carboxyethyltetrazole-5-thiol

N N

| i »

HS kN / N

(CH2) 2-COOH

A) 2-Carboethoxyethyl isocyanate 45-alanine ethyl ester hydrochloride (93.6 g), triethylamine (123.5 g) and methylene chloride (400 ml) were mixed together and cooled to -10 ° C. Sulfur carbon (46.5 g) dissolved in 150 ml of chloroform was added to the above-mentioned V ahd <'* n during turbidity p ί I.' Mists I 1a a lärnpöt i 1. a about -10 Omana, hi - ' ; i y 3 m m | .’Ί, ‘i I v I I yä temperature. ι! ιn <> I I i ι n i n> n n t a 1 0 C: seen m.innni! I m 1 n 10 mi nuu t i. hi u <> :; '| .'iälnly t «· I! i in '| ä I 1 <n -10' C to large and (. i., go I νγ- i. 1 k chlorine i. The temperature was allowed to rise to room temperature over 3 minutes and cooled again to 0 ° C, a further 1 g of triethylamine was added at 0 ° C and the subsequent solution was stirred at room temperature for 3 hours.

The mixture was treated with water and the organic phase was collected, washed with 2 x 250 ml of 2N hydrochloric acid, then 2 x 250 ml of sodium bicarbonate and then 2 x 250 ml of water. The organic phase was dried over sodium sulfate and the solvent was removed in vacuo to give 93.7 g of an oil which was found to be the desired product (IR and NMR analysis).

B) 1-Carboxy or tetrazole-5-thiol

Sodium azide (29.7 g) was dissolved in MQO in ml of water and heated to 60 ° C under a nitrogen blanket, 2-carboethoxyethyl isocyanate (46.9 g) dissolved in 50 ml. Skellysolve B product (essentially n-hexane) was added to the heated sodium azide solution. The solution was stirred for about 150 minutes at about 70-72 ° C and then cooled to 30 ° C with an ice bath. A 50% sodium hydroxide solution was added until the pH was 12. The mixture was heated at 70 ° C for 40 minutes and cooled to 15 ° C with an ice bath. The pH was adjusted to 2 using concentrated hydrochloric acid and then extracted with ethyl acetate (M x 150 mL). The ethyl acetate extracts were washed with water and then dried over sodium sulfate. The solvent was evaporated in vacuo and the product was collected as crystals from methylene chloride to give 19.5 g of the title compound.

Alternative synthesis of 1-carboxymethyl-5-mercaptotetrazole CII2CO2C2H5 + CS2 + HaN3

, W2 a <* l, a0H

N = »

| -CH 2 CO 2 H + Ua 2 S + C 2 H 5 OH

N =

SH

To a stirred mixture of 13.95 g (0.10 moles) of glycine ethyl ester hydrochloride, 8.0 g (0.20 moles) of sodium hydroxide and 8.37 g (0.11 moles) of carbon disulphide was added to the solution. containing 7.47 g (0.115 mol) of sodium azide in 125 ml of water. The solution was refluxed for 6 1/2 hours and stored at 25 ° C for 16 hours. The dark brown mixture was filtered and the filtrate was acidified to pH 1.5 with concentrated hydrochloric acid. The solution was treated with charcoal and the yellow filtrate was extracted with 4 x 100 mL of ethyl acetate. The ethyl acetate was washed with water, dried over magnesium sulfate and evaporated at 40 ° C (15 Torr) to an oil, the oil triturated in methylene chloride and the product filtered off. The sample was dried in vacuo over phosphorus pentoxide for 16 hours at 25 ° C.

Reference: German Patent 106,645.

Preparation of 5-mercaptotetrazole-1-acetic acid from 2-carbethoxymethyl isothiocyanate

S

II

? H2 "NH2 © 2 (C2H5), N CH2-NH-C-SH

C-OCpHg- * HCl —-— »C-OC2H5 * (C H) N

Il 2 5 © csP II ° 2 5 3 0 p

1 L J

? 2H5 O-C-C1

II

i o

M

CH0CNS CHp -NH-C-S-C- OC * Ης | 2 I 2 II II 2 5 C-OC2H5 c (CaH5) 3M C-OC2H5 S 0 o 'o 2 L -1

NaN, *

lj: -N - CHgCOgCgH ^ 0H ~ Nj-N - CHgCOgH

—SH -SH

25 64803

Carboethoxymethyl isothiocyanate 2

Sulfur carbon (22.8 g, 0.3 mol) dissolved in chloroform (40 ml) was added over one hour to a stirred suspension of glycine ethyl ester hydrochloride (41.7 g, 0.3 mol) and triethylamine (60.7 g, 0 mol). 6 moles) in methylene chloride (300 mL) at -10 ° C. The reaction mixture was allowed to warm to 10 ° C and stirred for 10 minutes at this temperature. The mixture was treated dropwise with ethyl chloroformate (32.6 g, 0.3 mol) in chloroform (50 mL) at 0-5 ° C over 0.5 h. The mixture was stirred for an additional 40 minutes at room temperature and triethylamine (30.4 g, 0.3 mol) was added dropwise over 15 minutes. The mixture was stirred for 1/4 hour at 0 ° C and another 1/2 hour at room temperature and finally stored overnight at 25 ° C. The mixture was washed with water (250 mL), 2N hydrochloric acid (2 x 300 mL) and 5% sodium bicarbonate solution (4 x 300 mL) and dried over anhydrous sodium sulfate. The solvent was evaporated at 40 ° C (15 Torr) to give 38.1 g of crude isothiocyanate. After standing overnight, a red dye formed on the product. It can be removed by distillation of the isothiocyanate. The isothiocyanate can be distilled at 104-106 ° C (7 Torr); T. B. Johnson and A. G. Renfrew JACS 47 240-245 (1925).

5-mercaptotetrazole-1-acetic acid 3

To a solution of sodium azide (4.9 g, 0.075 mol) in water (100 mL) heated to 60 ° C under nitrogen was added isothiocyanate (7.3 g, 0.5 mol) over 1/4 hour. The mixture was heated at 70-75 ° C for 2 hours, cooled to 5 ° C and 50% sodium hydroxide was added until pH 12. The solution was heated to 75 ° C for 1 hour, cooled to 5 ° C and adjusted to pH 2 with concentrated hydrochloric acid and filtered through diatomaceous earth ("Supercel"). The solution was extracted with ethyl acetate (4 x 120 ml), treated with charcoal and evaporated to an oil at 30 ° C (15 Torr). The residue was slurried in chloroform to give 1 g of acid.

1-carboxypropyl-2-mercaptotetrazole

To a solution of 40.8 g (0.63 moles) of sodium azide in 400 ml of water at 60 ° C was added 66.8 g (0.42 moles) of methoxycarbonyl 6 6 4 8 0 3 nylpropyl isothiocyanate (DL Garmaise et al., J. Amer. Chem. Soc., 80, 3332 (1958)) dropwise. The mixture was heated at 78 ° C for 2 hours, cooled to room temperature and adjusted to pH 12 with 50% sodium hydroxide solution. The solution was then refluxed for 1 1/2 hours, cooled to 27 ° C and adjusted to pH 26 with 6N hydrochloric acid. The mixture was filtered through diatomaceous earth ("Dicalite") and the cake was washed with 100 ml of ethyl acetate. The filtrate was extracted with 4 x 100 mL of ethyl acetate. The extracts were combined, washed with water and dried by azeotropic distillation to precipitate an oil which crystallized on cooling in an ice bath to give 22 g of 1-carboxypropyl-2-mercaptotetrazole.

7-amino-3- (1-karboksipropyylitetratsol-5-ylthiomethyl) -3-cephem-4-carboxylic acid

To a suspension of 22 g (0.081 mol) of 7-aminocephalosporanic acid in 350 ml of 0.1 M phosphate buffer pH 6.4 was added 16.9 g (0.089 mol) of 1-carboxypropyl-5-mercapto- tetrazole and 1.5 g of sodium bisulfite. The mixture was heated to 55 ° C under nitrogen and solid sodium bicarbonate was added until the mixture became clear (pH 7.5). The solution was heated for 3.5 hours, cooled to 10 ° C and adjusted to pH 26 with 6N hydrochloric acid. The precipitate was collected, washed with cold water and finally with methanol and air-dried to give 17.5 g of 7-amino-3- (1-carboxypropyltetrazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid. The sample was recrystallized from 100 ml of methanol, to which concentrated hydrochloric acid was added dropwise until the mixture was clear. The solution was adjusted to pH 5 with concentrated ammonium hydroxide and the precipitate was collected in a yield of 6.7 g.

Preparation of 7-amino-3- (1-carboxypropyltetrazolyl-5-thiomethyl) -3-cephem-4-carboxylic acid 2 7 6 4 3 0 3 o

Il CS

H2NCH2CH2CH2C-OCH3'HCl + (C2H5) 3N --ch ^ -> 90 CH2CH2C - OCH3 CoHcO-C-Cl • (2 \ - = - 2- \ CHO-NH ~ - C - '2 2 | l Θ L s J (c2h5)? nh oo

Il II

CH, CH „C-OCH, CH„ CH, C-OCH,

I 2 2 3 (C2H) N | 2 2 5 Na [J

CH 2 NH-C-S-C-OC-Hj- - ± -2-2 .- * CHO-N = C = S - ^ - 4 18

@NaOH

|| ~ || OiEi_> liT "

NaS-C ^ N ^ N Δ 7 HS - C ^ N> (ch2) 3co2ch3 ((!: H2) 3co2h

SH

N /> L_ CHg-O-C-CH2 _ COgH Δ »2N-rΐΠΓ J — CH2S-C ^ n7n co2h (ch2) 3co2h 28 6 4 8 0 3 N-butyrylmethoxyisothiocyanate

To a mixture of 153.6 g (1.0 mol) of 4-aminobutyric acid methyl ester hydrochloride, 202.4 g (2.0 mol) of triethylamine and 600 ml of methylene chloride cooled to -15 ° C was added 76.1 g (1 mol) of .0 moles) of carbon disulphide dissolved in 200 ml of chloroform over 60 minutes. The mixture was warmed to 10 ° C, stirred for 10 minutes, cooled to 0 ° C, and 108.5 g (1.0 mol) of ethyl chloroformate dissolved in 80 ml of chloroform was added over 20 minutes (0-5 ° C). The mixture was stirred without an ice bath for 70 minutes and warmed to 18 ° C. The reaction mixture was again cooled to 0 ° C and 101.2 g (1.0 mol) of triethylamine were added over 20 minutes. The reaction mixture was stirred for 15 minutes at 0 ° C and then for 1.5 hours without an ice bath. The mixture was washed with 250 mL of water, 2 x 300 mL of 2N hydrochloric acid, 2 x 300 mL of 5% sodium bicarbonate and 2 x 300 mL of water. The organic phase was dried over anhydrous magnesium sulfate and reduced in volume at 15 Torr to a yellow oil. Yield: 126 g of oil.

1-carboxypropyl-5-mercaptotetrazole

To a solution of 40.8 g (0.63 mol) of sodium azide in 400 ml of water at 60 ° C under a strong stream of nitrogen was added dropwise 66.8 g (0.42 mol) of N-butylmethoxyisothiocyanate. The reaction mixture was heated at 78 ° C for 2 hours under nitrogen. The reaction mixture was cooled to 25 ° C, 50% sodium hydroxide was added to pH 12, and the mixture was refluxed for 1.5 hours and then cooled. The mixture was adjusted to pH 2 using 6N hydrochloric acid (warning: hydrogen azide). The mixture was filtered through a pad of Supercel and the cake was washed with ethyl acetate. The washings were added to the filtrate and the phases were separated. The aqueous solution was extracted with 4 x 100 mL of ethyl acetate and the organic fractions were combined. The organic phase was washed with water, dried over anhydrous magnesium sulfate and reduced in volume at 35 ° C (15 Torr) to an oil which was allowed to stand in an ice bath. The product was collected and dried in vacuo over phosphorus pentoxide at 25 ° C. Yield 22 g of an off-white solid.

7-Amino-3- (1-carboxypropyltetrazolyl-5-thiomethyl) -3-cephem-4-carboxylic acid -2 29 64803

To a nitrogen purged solution of 1.5 g (1.4 x 10 moles) of sodium bisulfite in 350 mL of pH 6.4 phosphate buffer, -2 was added 22 g (8.1 x 10 moles) of 7-aminocephalosporanic acid, -2 16, 85 g (9.0 x 10 moles) of 1-carboxypropyl-5-mercaptotetrazole and sufficient sodium bicarbonate to form a clear solution. The reaction mixture was heated for 3.5 hours at 56 ° C under a strong stream of nitrogen and then cooled to 10 ° C. The pH was adjusted to 2.5 using 6N hydrochloric acid and the mixture was stirred in an ice bath to aid precipitation. The product was collected and then washed with cold water, methanol and acetone. The product was recrystallized from methanol-hydrochloric acid and dried in vacuo over phosphorus pentoxide at 25 ° C. Yield: 6.7 g.

2-furoyylisyanidi

To a suspension of 26.1 g (0.4 moles) of ground potassium cyanide in 300 mL of acetonitrile at 5 ° C was added 26.1 g (0.2 moles) of CX-furoyl chloride while maintaining the temperature below 8 ° C. :in. The mixture was stirred in the cold for 15 minutes and then refluxed for 30 minutes. The reaction mixture was cooled, filtered and the acetonitrile removed at 15 Torr (steam bath) to leave 24.5 g of a dark oil which was used without further purification. The infrared spectrum showed a nitrile band at 2265 cm.

2-furaaniglyoksyylihappo

The obtained 24.5 g of crude 2-furoyl cyanide was mixed with 160 ml of concentrated hydrochloric acid at 25 ° C with occasional stirring. The reaction mixture was stored at 25 ° C for 24 hours and diluted with 80 ml of water. The reaction mixture was stirred for 5 minutes and filtered. The filtrate was saturated with sodium chloride and extracted with 5 x 120 mL of 1: 1 ether-ethyl acetate solution. The extracts were combined, dried over anhydrous magnesium sulfate and evaporated at 30 ° C (15 Torr) to give a brownish orange solid. The solid was dissolved in methanol, treated with charcoal and evaporated to dryness under reduced pressure (15 Torr) to give 17 g of acid. The product was recrystallized from toluene to give 11.5 g (m.p. 76 ° C).

30 64803 2-Methoxyimino-2-furylacetic acid

To a solution of 4.5 g (0.032 mol) of 2-furanglyoxylic acid in 40 ml of 50% alcohol and 3.1 g (0.037 mol) of methoxyamine hydrochloride in 6 ml of water at 20 ° C was added dilute sodium hydroxide solution. to pH 4-5. The solution was stirred at pH 4-5 at 25 ° C for 24 hours. The alcohol was removed under reduced pressure (15 min) and the solution was adjusted to pH 7-8 with 50% sodium hydroxide solution. The reaction mixture was extracted with 3 x 50 mL of ether and the aqueous layer was adjusted to pH 1.9 using concentrated hydrochloric acid. The mixture was extracted with 5 x 50 mL of ethyl acetate. The organic fractions were combined, washed with brine, dried over anhydrous magnesium sulfate and evaporated under reduced pressure (15 Torr) to an oil which was cooled for 1 hour in an ice bath. The product was slurried in "Skellysolve B" ("Skellysolve B is a petroleum ether fraction, b.p. 60-68 ° C; consists mainly of n-hexane), and collected to give 3.1 g of yellow crystals, m.p. 78 ° C. The analytical sample was recrystallized from toluene, dried under vacuum with phosphorus pentoxide at 25 ° C for 16 hours.

Analysis:

Calculated from C 18 H 18 NO: C 49.65 H 4.17 N 8.28

Experimental: C 49.30 H 4.21 N 8.37 7-Amino-3- (1-carboxyethyltetrazol-5-ylthiomethyl) -3-cephem-4-carboxylic acid

To a mixture of 5 g (0.0286 moles) of 5-mercaptotetrazolylpropionic acid and 7.8 g (0.0286 moles) of 7-aminocephalosporanic acid in 150 ml of 0.1 M phosphate buffer (pH 6.4) was added with stirring solid sodium bicarbonate until the solution became clear (pH 7.5). The solution was heated at 55 ° C under nitrogen for 4 hours and acidified with phosphoric acid in a ratio of 1: 1. The precipitate was collected, washed with water and finally with a small volume of methanol. The solid was then slurried in 150 mL of methanol and concentrated hydrochloric acid was added dropwise until the mixture became clear. The solution was treated with charcoal, filtered and neutralized with concentrated ammonium hydroxide to pH 4.5. The solid was collected and washed with methanol; yield 5.2 g, m.p. > 130 ° C (decomposed).

2-Ethoxyiminofurylacetic acid 7.85 g (0.056 mol) of furyl-2-glyoxylic acid were dissolved in 3 to 64803 x 100 ml of water and adjusted to pH 7 with 50% sodium hydroxide. 6.83 g (0.070 mol) of ethoxyamine hydrochloride in 10 ml of water were added while maintaining the pH at 4-5. The reaction mixture was diluted with 25 mL of alcohol, stirred for 3 hours at room temperature and then filtered. The alcohol was removed at 35 ° C (15 Torr) and the aqueous portion was adjusted to pH 7-8 with dilute sodium hydroxide solution and then washed with ether and the washings were discarded. The aqueous fraction was adjusted to pH 1.5 with 6N hydrochloric acid and extracted with 3 x 80 mL of ethyl acetate. The acetate fractions were combined, washed with brine and evaporated at 35 ° C (15 Torr) to an oil, the oil cooled in an ice bath, triturated in Skellysolve B solvent, collected and dried over phosphorus pentoxide in vacuo at 25 ° C; yield: 4.8 g, m.p. 83-85 ° C.

Analysis for C 9 H 9 NO 2: C 52.46 H 4.95 N 7.65

Experimental: C 52.22 H 4.94 N 7.60

Sodium <? -Ethoxyimino-O- (2-furyl) acetate To 50 μl of methanol was added 250 mg (0.0109 moles) of metallic sodium and stirred until all the sodium was dissolved. This sodium methoxide solution was treated with 2.0 g (0.0109 moles) of CX-ethoxyimino-O- (2-furyl) acetic acid dissolved in 10 ml of methanol and stirred at room temperature for 1 hour. The methanol was removed at 40 ° C (15 Torr) and the product was dried in vacuo over phosphorus pentoxide to give 2.22 g of a white solid; mp. > 240 ° C (decomposed).

Example 1 Potassium salt of 7- (2-methoxyimino-2-furylacetamido) -3- (1-carboxyethyl-tetrazolyl-5-thiomethyl) -3-cephem-4-carboxylic acid (BL-S1095)

A suspension of 750 mg (0.0039 mol) of sodium 2-methoxyimino-2-furyl acetate in 25 ml of benzene and 2 drops of dimethylformamide was stirred vigorously while 0.35 ml (0.0039 moles) of oxalyl chloride was added dropwise. The suspension was stirred for 45 minutes and the salts formed were collected by filtration. Benzene was removed at 40 ° C (15 Torr) and the pale yellow oil was dissolved in 20 mL of acetone and added to a solution of 1.6 g (0.0039 mol) of 7-amino-3- (1-carboxyethyltetrazol-5-one). ylthiomethyl) -3-cephem-4-carboxylic acid in 25 ml of water and 32 6 4 8 0 3 500 mg of sodium carbonate at 5 ° C. The solution was stirred for 1/2 hour at 5 ° C and the acetone was evaporated under reduced pressure at 40 ° C (15 Torr), diluted with 25 ml of water and acidified with phosphoric acid in a ratio of 1: 1. The mixture was extracted with 3 x 50 ml of ethyl acetate and the organic layer was separated, washed with water and evaporated to give a gummy residue. The resulting residue was slurried in ether, the free acid was dissolved in acetone and treated with 750 mg of potassium 2-ethylhexanoate. The potassium salt was collected, washed with acetone and dried over phosphorus pentoxide to give 800 mg of product, m.p. > 130 ° C (slowly decomposing).

Analysis for G 1 H 7 K 2 N 2 O 2 S 2.1.1 For C 37.21 H 3.52 N 15.90

Experimental: C 37.17 H 3.31 N 13.89 NMR (D 2 O ppm S) 7.75, d, 1.1 -CH-OO; 6.95, d, 1, = CH-CH-; 6.6 - 6.8 m., 1 = CH-CH =; 5.83, d, 1, N-CH-; 5.25, d, 1, -CH-S; 4.6, T, 2, N-CH2-CH2-CO2H; 4.0 - 4.6 M2 = C-CH2-SO; 4.0, S, 3, N-OCH 3; 3.3 - 4.0 m, 2 S-CH 2 =; 2.85, T, 2, CH 2 -CO> 2H.

IR (KBr cm) 3100-2600, OH, NH, 1765-lactam carbonyl; 1675 NHC = 0 1600 CO. '* x

Example 2 7- (2-Methoxyimino-2-furylacetamido) -3- (1-carboxymethyltetrazolyl-5-thiomethyl) -3-cephem-4-carboxylic acid (BL-S1080) To 25 ml of methanol was added 100 mg (0.00425 moles) of metallic sodium and the mixture was stirred until all the sodium had dissolved. The sodium methoxide solution was cooled to 3 ° C and 0.179 g (0.00425 moles) of methoxyiminofurylacetic acid in 5 ml of methanol was added. The solution was stirred for 10 minutes at room temperature and the solvent was evaporated at 30 ° C (15 Torr) and dried by azeotropic distillation with 3 x 20 ml of benzene (15 Torr).

Sodium 2-methoxyimino-2-furyl acetate was suspended in 25 ml of benzene and treated with 4 drops of dry dimethylformamide. A total of 1.1 g (0.0085 mol) of oxalyl chloride was added and the solution was stirred for 40 minutes at 25 ° C. The benzene was removed at 35 ° C (15 Torr) and the acid chloride was dissolved in 10 mL of acetone.

A solution of 1.2 g (0.0032 mol) of 7-amino-3- (1-carboxymethyl-tetrazolyl-5-thiomethyl) -3-cephem-4-carboxylic acid in 20 ml of 33 64803 water and 0.68 g (0.008 moles) of sodium bicarbonate, was cooled to 3 ° C. An acetone solution of the above acid chloride was added and the reaction mixture was stirred for 40 minutes without an ice bath. The acetone was removed at 30 ° C (15 Torr) and the aqueous solution was adjusted to pH 1.8 using 6N hydrochloric acid. The product was extracted with 3 x 60 mL of ethyl acetate. The organic fractions were combined, washed with brine and azeotroped at 30 ° C (15 Torr) to an amorphous solid. The residue was treated with 50 ml of ether and the product was collected and dried in vacuo over phosphorus pentoxide to give 750 mg of a light brown solid, m.p. > 120 ° C (decomposed).

Analysis C1. 0.5 for C 42.17 H 4.06 N 17.20

Experimental: C 42.16 H 3.96 N 16.66 NMR spectra showed the compound to be a mixture of 75% syn and 25% anti-isomer with diethyl ether as impurity or solvate. The NMR spectrum of the syn isomer is as follows: NMR (DMSO ppm S) 9.75 d, 1, -NH-; 7.75, S 1, = CH-O-; 6.5 - 6.8 μm, 2, = CH-CH =; 5.6 - 5.9, m, 1, N-CH-; 5.3-5.2, N-CH2-CH2; 5.15, d, 1, -CH-S; 3.95 - 4.65 m, 2, CH 2 -S; 3.9, S, 3, OCH 3; 3.4 - 4.0, m, 2, -S-CH 2 -C.

Separate anti-patterns: 9.55 d, 1, -CO-NH-; d, 1, = CH-CH =; 4.0, S, 3, OCH 3.

Example 3 7- (2-Methoxyimino-2-furylacetamido) -3- (1-carboxypropyltetrazolyl-5-thiomethyl) -3-cephem-4-carboxylic acid (BL-S1081)

iMLc - 8 - NH-pA N — N

N-OCH 2 O - "> ^ - CHa's-Cv" x "COOH (CH5), I 5

C02H

34 64803 -3 To 25 ml of methanol was added 96 mg (4.16 x 10 moles) of metallic sodium, which was stirred until all the sodium had dissolved. This sodium methoxide solution was cooled to 3 ° C and 704 g (4.16 x 10 6 mol) of 2-methoxyimino-2-furylacetic acid in 5 ml of methanol were added. The reaction mixture was stirred for 10 minutes at room temperature, the methanol was removed at 35 ° C (15 Torr) and the sample was azeotroped with 3 x 30 ml of benzene at 35 ° C (15 Torr).

To a stirred suspension of the above sodium 2-methoxyiminofuryl acetate in 25 ml of benzene containing 3 drops of dimethylformamide was added 1.06 g (8.3 x 10 6 moles) of oxalyl chloride and stirred for 1 hour at room temperature. The benzene was removed at 35 ° C (15 Torr) and the acid chloride was dissolved in 10 mL of acetone.

The acid chloride solution was added to a solution of 1.28 g of -2 (3.2 x 10 moles) of 7-amino-3- (1-carboxypropyltetrazolyl-5- -3-thiomethyl) -3-cephem-4-carboxylic acid and 0.672 g (8 x 10 moles) of sodium bicarbonate in 25 ml of water at 3 ° C. The solution was stirred for 40 minutes with the ice bath removed. The reaction mixture was filtered, the acetone was removed at 30 ° C (15 Torr) and the aqueous fraction was adjusted to pH 1.6 with 6N hydrochloric acid. The sample was layered with ethyl acetate and then filtered through a Supercel mass. The phases were separated and the aqueous phase was further extracted with 3 x 30 ml of ethyl acetate. The organic fractions were combined, treated with charcoal, filtered through a Supercel mass and then azeotroped at 30 ° C (15 Torr) to an amorphous solid. This residue was triturated with excess ether and the product was filtered, dried over phosphorus pentoxide at 25 ° C to give 370 mg of an off-white solid, m.p. > 83 ° C (decomposed).

Example 4 7- (2-Ethoxyimino-2- (furyl) acetamido) -3- (1-carboxymethyl-tetrazolyl-5-thiomethyl) -3-cephem-4-carboxylic acid syn isomer (BL-S 1105) 64803 35 (TjL. G. 7j — i »

LoC2H5 o ^ N-Y ^ -CH2-S-C "iKN

COOH CH0 I 2 co2h _ ^

To a stirred suspension of 1.1 g (5.3 x 10 "moles) of sodium N-ethoxyimino-α- (2-furyl) acetate in 40 ml of benzene and 6 drops of dimethylformamide were added 706 mg (5 , 57 x 10 moles) of oxalyl chloride and stirred for 1 hour at room temperature The benzene was removed at 40 ° C (15 Torr) and the acid chloride was dissolved in 5 ml of acetone.

The acid chloride solution was added to a solution of 1.89 g. -3- (5.08 x 10 moles) 7-amino-3- (1-carboxymethyltetrazolyl-5S-thiomethyl) -3-cephem-4-carboxylic acid and 1.28 g (1.52 x 10-2 moles) sodium carbonate in 40 ml of water at 3 ° C. The reaction was stirred for 40 minutes, filtered and the acetone removed under reduced pressure at 35 ° C (15 TOrr). The aqueous solution was layered with ethyl acetate, adjusted to pH 1.9 by 40%. phosphoric acid and filtered. The phases were separated and the aqueous portion was extracted with 2 x 60 mL of ethyl acetate. The ethyl acetate fractions were combined, washed with brine, dried over anhydrous magnesium sulfate and reduced in volume at 35 ° C (15 Torr) to give an oil. excess ether was added to the oil and allowed to stand at room temperature for 16 hours. The product was collected and dried in vacuo over phosphorus pentoxide at 25 ° C to give 800 mg of a brown solid, m.p. 75 ° C (decomposed).

Analysis for C 18 H 19 N 2 O 8 S 2, 0.5 (C 2 H 5) 2 O C 43.90 H 4.29 N 17.06

Experimental: C 43.90 H 4.24 N 16.21 36 6 4 8 0 3

Example 5

The products of Examples 1-3 are prepared as syn isomers that are substantially free of the corresponding anti-isomers using the purified syn isomers of the appropriate 2-alkoxyimino-2- (furyl) acetic acid in the procedures of these examples. The partial conversion of syn isomers to the anti-isomer during the preparation of the acid chloride from the acid is substantially avoided by minimizing its exposure to hydrogen chloride, for example by first converting the acid to its anhydrous sodium salt and treating this salt with oxalyl chloride under anhydrous conditions in the presence of hydrogen ion acceptor such as dimethylformamide. Example 6 Potassium salt of 7- (2-methoxyimino-2-furylacetamido) -3- (1-carboxymethyltetrazolyl-5-thiomethyl) -3-cephem-4-carboxylic acid (BL-S1080) 3.0 g: To 6 ml of sodium 2-methoxyimino-2-furyl acetate suspended in 60 ml of benzene were added 6 drops of dimethylformamide and 1.98 g (0.0156 moles) of oxalyl chloride. The resulting solution was stirred at 25 ° C for 45 minutes. The benzene was removed at 40 ° C (15 Torr) and the acid chloride was dissolved in 4 mL of acetone. A solution of 5.8 g (0.0156 mol) of 7-amino-3- (1-carboxymethyltetrazolyl-5-thiomethyl) -3-cephem-4-carboxylic acid and 2.5 g (0.03 mol) moles) of sodium bicarbonate in 80 ml of water, cooled to 3 ° C and the acid chloride solution was added. The reaction mixture was stirred for 20 minutes at 3 ° C and for 20 minutes with the ice bath removed. pH 7 was maintained during stirring. The mixture was filtered and the acetone was removed at 35 ° C (15 Torr). The aqueous portion was layered with ethyl acetate, adjusted to pH 1.8 with 6N hydrochloric acid and extracted with 2 x 80 mL of ethyl acetate. The organic fractions were combined, washed with 2 x 40 mL brine, dried over anhydrous magnesium sulfate and evaporated at 35 ° C (15 Torr) to an amorphous solid which was triturated in ether. The free acid was collected and air dried to give 4.5 g of product. The acid was dissolved in acetone and treated with 1.2 g (0.0065 moles) of potassium 2-ethylhexanoate dissolved in acetone. The product was collected and dried in vacuo over phosphorus pentoxide at 25 ° C to give 2.49 g of potassium salt. Sp.

130 ° C with slow decomposition. NMR spectra showed the compound to be a mixture of 37 64803 with 95% syn and 5% anti-isomer.

Analysis for gH 2 N 6 N 2 O 2 S 2 C 38.49 H 2.87 K 6.96 N 17.45

Experimental: C 38.06 H 2.68 K 6.65 N 17.34

Example 7 7- (2-Methoxyimino-2-furylacetamido) -3- (1-carboxymethyltetrazolyl-5-thiomethyl) -3-cephem-4-carboxylic acid 2.5H-Ordisodium salt 1. 0.97 g 7- (2-Methoxyimino-2-furylacetamido) -3- (1-carboxymethyltetrazolyl-5-thiomethyl) -3-cephem-4-carboxylic acid was suspended in 10 ml of water and 1N sodium hydroxide was added. until a constant pH of 7 was reached and all starting material was dissolved. The solution was then lyophilized for 18 hours to give 0.93 g of the disodium salt. The product contained about 10% anti-isomer, about 10% furoic acid and traces of dimethylformamide by NMR analysis. The bioautograph showed only one round spot.

2. An alternative method for preparing the title disodium salt.

7- (2-Methoxyimino-2-furylacetamido) -3- (1-carboxymethyl-tetrazolyl-5-thiomethyl) -3-cephem-4-carboxylic acid (0.5 g) was dissolved in 5 ml of ethanol, filtered, 5 ml of water was added and titrated to pH 7 with 1 N sodium hydroxide. The ethanol was evaporated at 50 ° C (15 Torr) and the remaining solution was lyophilized for 13 hours to give 0.44 g of the title salt. The product contained about 10% anti-isomer and about 10% furoic acid (NMR analysis).

Analysis for gH17N70gS2Na2 C 38.1 H 3.01 N 17.28 Experimental (corrected for water content) C 38.28 H 3.29 N 16.41 Analysis gH17N70gS2Na2. 2.5 H 2 O KF (H 2 O) 7.33 Experimentally KF (H 2 O) 8.06.

Claims (2)

64803 38 A process for the preparation of antibacterial 7- (2-alkoxyimino-2-furylacetamido) -3- (1-carboxyalkyltetrazolyl-5-thiomethyl) -3-cephem-4-carboxylic acids of the formula li-r-N-NH -CH-CH "bi2 I nl! J \ ^ -N C-CH -SC n 1 ^ orI <T 2 COOH (CHJ COOH 2 n wherein R is a lower alkyl group and n is 1, 2 or 3, or a pharmaceutically acceptable for the preparation of a salt, characterized in that 7-aminocephalosporanic acid of the formula NH - CH- <10 ° C - CH2-O-C-CH3 COOH or a salt thereof is reacted with 1-carboxyalkyl-5- with mercaptotetrazole of the formula J i Ϊ (CH_) COOH 2 n where n is 1, 2 or 3, or with a salt of this compound to give 7-amino-3- (1-carboxyalkyltetrazol-5-yl-thiomethyl) -3-Cephem-4-carboxylic acid is treated with an acylating agent of the formula ^ 39 6 4 803 8 nrr OR wherein X is a halide or its reactive equivalent and is as defined above, and ha the resulting free acid or salt of a compound of formula I is converted to its corresponding pharmaceutically acceptable salt, and if desired, the resulting salt of a compound of formula I is converted to the corresponding acid of formula I, wherein the compound of formula I or a salt thereof is present in at least 75% by weight of the syn isomer. in the form of a mixture of isomers. 40 6 4 8 0 3 For the preparation of antibacterial meshes of 7- (2-alkoxyimino-2-furylacetamido) -3- (1-carboxyalkyltetrazolyl-5-thiomethyl) -3-cephem-4-carboxylic acid with a formulation of li- - c '«h-ch-ch ch2 ^ ti l» IN -N C-CH - SC JA 1 ^ 0R1 0 ^ 2 Y COOH (CH0) COOH 2 n color R1 är en lägre alkylgrupp och n är 1, 2 or 3, or a pharmaceutical product containing the same, 7-amino-cephalosporan compounds in the form of __ ^ NH, -CH-r i 2. I o - ch2-oc-ch3 COOH if a mixture is obtained, with 1-carboxyalkyl-5-mercaptotetrazole of formula J II HS-IL N Ϊ (CH_) COOH zn color is not 1, 2 or 3, or to give 7-amino-3- (1-carboxyalkyltetrazol-5-yl-thiomethyl) -3-cephem-4-carboxylicyran as an acylating agent with a formulation