FI69309C - FOERFARANDE FOER FRAMSTAELLNING AV 3-TIOMETYLKEFALOSPORANSYRADERIVAT - Google Patents

Description

Ι «* Τ · Ί ΓΒ1« «NOTICE 7 _ Λ B 11 UTLÄGGNINGSSKRIFT 69309 5ζ> \ γ · C / AC \ Patent granted

Jtjpl ([-'e ~ 3 12 Cl 1230 (51) Kv.lk. '/ Lnt.a.' C 07 D 501/00, 501/36 FINLAND —FINLAND (21) Patent application - Patentansökning 780716 (22) Application date - Ansöknlngsdag 03 · 03.78 (ψ \\ VV (23) Start date —Giltlghetsdag 03.03.78 (41) Become iuli - Blivit offentllg 08.09.78

National Board of Patents and Registration of Finland Date of visit |

Patent- och registerstyrelsen '' Ansökan utlagd oeh utl.skrlften publlcerad 30. Oy .op (32) (33) (31) Privilege requested - Begärd prlorltet 07.03.77 27.12.77 USA (US) 773018, 862871 (71) Eli Lilly and Company, 307 East McCarty Street, Indianapolis,

Indiana 46206, USA (72) Lowell Deloss Hatfield, Bargersville, Indiana, USA (74) Oy Koi ster Ab (54) Process for the preparation of 3 * thiomethylcephalosporanic acid derivatives -Förfarande för framstallning av 3-thiomethylkephalosporansyraderivat

The invention relates to a process for the preparation of 3-thiomethylcephalosoranic acid derivatives of the formula I,

, R1 S

R + Y> 1 0> Νγ ^ -CH2Sr13

COOH

13 wherein R is an amidino, benzoyl, phenyl, lower alkyl or unsubstituted or lower alkyl, hydroxy, N-methylacetamido, phenylbenzyl or carboxymethyl-substituted 5- or 6-membered heterocyclic ring having 1 to 4 heteroatoms which: may be one of the following combinations a) 1 nitrogen and 1 oxygen or sulfur b) 2 nitrogen and 0 or 1 oxygen or sulfur 2 c) 3 nitrogen 6 9 309 d) 4 nitrogen, 13 or R is benzothiazolyl, R1 is hydrogen or methoxy, R is amino or a group of the formula R (-C-NH-, wherein R is lower alkyl, -CH 2 - lower chloroalkyl), lower cyanoalkyl, tert-butoxy, protected H-amino-4-carboxybutyl group, of the formula 0 R120-C-CH- (CHo) o-CH -I 1 2 2 2

AX

1. . .12 wherein A is a protected amino group and R is hydrogen, a group of formula a '. .

<TV <Z) m-CH 2 -V-. »'Wherein a is hydrogen, halogen, hydroxy or a group A ^ CH-- where 12 2 A is a protected amino group as above, Z is oxygen or sulfur and m is 0 or 1, a group of the formula * -, 1- / '' V- - CH-

Q

wherein α 1 is hydrogen or hydroxy, and Q is hydroxy, formyloxy, a protected amino group, an acylated amino group of the formula 0 tr -NH-CT, where T is amino, 1,3-dimethylureido, β-ethyl-2,3- 3 dioxo-1-piperazinyl, or R is thienyl or tetrazolylmethyl.

The process according to the invention makes it possible to replace the acetoxy group of Kephalosporanic acid with a sulfur nucleophile under practically anhydrous conditions.

Replacement of the acetoxy group of a cephalosporin with a sulfur nucleophile is known (U.S. Patent 3,278,531). U.S. Patent 3,69,309 to 3,278,531 and also other publications, e.g., J.D. Cocker, J. Chem. Soc. (1965), 5015, states that the reaction takes place only in aqueous medium. In practical substitution reactions, a salt of cephalosporanic acid in aqueous solution and a sulfur nucleophile or a salt thereof at a pH between 5-8 have been used. A large proportion of cephalosporin nuclei are usually destroyed when a combination of aqueous reaction medium, elevated temperature (35-70 ° C) and neutral and basic pH is used, and the purification of products prepared in this way is often complicated. When a substitution reaction of cephalosporanic acids is performed in water at a lower pH (pH 2-3), considerable lactone formation has occurred, and this side reaction is likely to greatly reduce the yield of the desired product.

It has now been found that the replacement of the acetoxy group of cephalosporanic acids (as well as other 3-acyloxy groups) with a sulfur nucleophile can be carried out in an organic solvent under virtually anhydrous conditions. Under these conditions, lactone formation does not complicate the reaction, yields are generally higher, and the products are easier to isolate. Some products precipitate from the reaction mixture themselves. This method is believed to be general in nature and applicable to virtually any sulfur nucleophile and any cephalosporanic acid.

The process according to the invention is characterized in that the compound of the formula II * 2-ί / Ξχ | cr '-ch2ocor

COOH

1. 2 '.2

where R is as defined above, R is as defined above

or a formylamino group, and R is lower alkyl or C 1 -C 6 cycloalkyl, is reacted under substantially anhydrous conditions with a sulfur nucleophile of formula III 13 14

R -S-R III

69309 13 14 ...

wherein R is as defined above] a R is hydrogen or the substituent 13 when R is amidino, the compound of general formula III is thiourea.

The products prepared by the process of the invention are antibacterial agents and many of them are intermediates in the preparation of other cephalosporins which are also antibacterial agents, cf. e.g., U.S. Patent 3,932,393.

In the above definitions, the term "alkyl" means. . . . . 3 both straight and branched chain alkyl groups. In the definition of the group R, "lower alkyl" is e.g. methyl, ethyl, propyl, iso-propyl, butyl, sec-butyl, amyl, isoamyl, hexyl, 2,3-dimethylbutyl or the like. The term "-CH 2 - (lower chloroalkyl)" means groups such as chloroethyl, 2-chloroethyl, 2-chloropropyl and 4-chlorobutyl. The term "lower cyanoalkyl" means groups such as cyanomethyl, 2-cyanoethyl, 3-cyanopropyl and 2-cyanopropyl.

In both the 3- (acyloxymethyl) cephalosporin of formula II and the sulfur nucleophile, the amino groups are preferably substituted. Protection of amino groups takes place in a known manner, cf. Protective Groups In Organic Chemistry, ed. J.T.W. McOmie (Plenum Press, London and New York, 1973). In general, protection of the amino group requires the possibility of deprotection, but this is not necessary, for example, in the case of a 7-acylamido group of the following formula 12 ° 0 R 2 OC-CH- (CHO) O-CHO-C-NH-2 2 2 cephalosporins. Suitable protecting groups for this group include e.g. C 1 -C 4 alkanoyl, C 1 -C 4 chloro or fluoroalkanoyl, benzoyl or substituted benzoyl. The term "C 1 -C 4 alkanoyl" means groups such as acetyl, propionyl, butyryl or the like. The term "C 1 -C 4 chloro or fluoroalkanoyl" means groups such as 3-chloropropionyl, 3-fluoropropionyl and the like. "substituted benzoyl" means e.g. halogen-substituted groups such as 4-chlorobenzoyl, 4-bromobenzoyl and 2,4-dichlorobenzoyl. These 69309 groups can also be used to protect amino groups located elsewhere in 3- (acyloxymethyl) cephalosporin and sulfur nucleophiles. However, if it is desired to restore the free amino group, the protecting group should be easily removable, cf. McOmie. Suitable protecting groups that are easily removed include benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, tert-butoxycarbonyl, p-methoxybenzyloxycarbonyl and diphenylmethoxycarbonyl. The amino group of the following formula) 0 R120-C-CH- (CHo) o-CH-C-NH-A1 can also be protected as described in BE patent 771,694.

As used herein, the terms "halogen" and "halo" each mean fluorine, chlorine, bromine or iodine. The term "lower alkoxy" means methoxy, ethoxy, isopropoxy, n-butoxy, etc. The term "alkali metal" preferably means sodium, potassium or lithium.

Many of the cephalosporanic acids used as starting materials in the process of this invention are known compounds, all of which can be prepared by methods known to those skilled in the art. As a reference, mention may be made of "Cephalosporins And Penicillins, Chemistry and Biology", ed. Edwin H.Flynn. (Academic Press,

New York, 1972). U.S. Patent 3,914,157 and ZA 71/3229 may also be mentioned herein.

Suitable five-membered heteroaromatic rings of said formula include e.g. the following: pyrrole, oxazole, isoxazole, thiazole, isothiazole, pyrazole, imidazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3, 4- oxadiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, 1,2,5-thiadiazole, 1,3,4-thiadiazole, 1,2,3-triazole, 1,2,4-triazole, oxatriazole and tetrazole.

Suitable six-membered tires include e.g. the following: pyridine, pyridazine, pyramidine, pyrazine, 1,2,3-triazine, 1,2,4-triazine, 1,3,5-triazine, 1,2,3,4-tetrazine and 1,2,4, 5-tetrazine.

6 69309

Some heteroaryl spores, alkylthiols and phenylthiols of formula III actually exist as thiones or as tautomeric mixtures of thiol and thione. For example, the compound 2-methyl-1,3, H-thiadiazole-5-thiol exists as the following tautomer:

B

N— N N-- N

i - ^ Ί ί ^ s h

HS____i -CH b 'CH

/ O o xs / xs

However, the reaction proceeds according to the type of sulfur nucleophile, despite the fact that the reactant is in thiol or thione form. Thus, by the process of the present invention, the reaction can be carried out using a thione or a thiol-thione tautomer as a reagent.

The heteroarylthiol may be unsubstituted or may have one or more substituents. Substituents are not critical to the reaction. To achieve good results, the primary and secondary amino groups should be protected. When the heteroarylthiol contains more than one thiol group, the group which is not desired to participate in the reaction should be protected. Methods for protecting thiols are known, cf. McOmie above. If such groups are protected, the reaction proceeds regardless of the nature of the substituents.

Very large substituents should be avoided so that the molecular weight of the substituents should not exceed about S00. The most common substituted heteroaromatic ring substituents have a total molecular weight of less than about 250.

Suitable substituents include e.g. C 1 -C 4 alkyl, C 1 -C 4 alkenyl, C 2 -C 4 alkynyl, C 1 -C 8 cycloalkyl, benzyl, phenethyl, aryl (phenyl and substituted phenyl as defined above), halogen, -CF, = O, -OH, protected amino or 2 ^. .

C 1 -C 4 alkylamino, -CO 2 H, -C00A, -CON 2, protected aminomethyl or C 1 -C 4 alkylaminomethyl, CH 2 SO 4 -methyl] -metal, -CH 2 CO 2, -CH 2 COOA 5 or - (CH 2) 2 N (CH 2 .

Most of the sulfur nucleophiles used in the method of this invention are known compounds and all can be prepared by methods known in the art. For heteroarylthiols, reference is made to "Heterocyclic Compounds" ed. Robert C. Elderfield (John Wiley and Sons, Inc., N.Y.) and, for specific heterocyclic systems, in "The Chemistry of Heterocyclic Compounds," ed. Weissberger et al. (John Wiley and Sons, N.Y.).

It is important for the present invention that the reaction is carried out in an organic solvent. Several organic solvents have proven to be satisfactory, so any particular solvent per se is not a critical factor. Solvents belonging to the following groups of solvents have been found to be satisfactory: hydrocarbons (both aliphatic and aromatic), alcohols, amides, ethers, ketones, carboxylic acids, carboxylic acid esters, halogenated hydrocarbons, nitro compounds, nitriles and thioethers. Because some nucleophiles are liquid, an excess of such reagents can be used as a solvent.

The solvent should be inert so that it does not compete with either reactant.

Suitable solvents for use in the reaction of this invention include e.g. pentane, cyclopentane, hexane, heptane, octane, benzene, toluene, o-, m- or p-xylene, cumene, mesethylene, p-cymene, 1-pentene, diethyl ether, butylethyl ether, diamyl ether, benzylethyl ether, acetone, methyl ethyl ketone, 2-butanone, 3-pentanone, methyl isobutyl ketone, cyclohexanone, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, methyl acetate, ethyl acetate, propyl acetate, ethyl acetate, isopropyl acetate, isopropyl acetate butyl acetate, amyl acetate, isoamyl acetate, ethyl isovalerate, methyl benzoate, benzyl acetate, methyl butyrate, diethyl carbonate, dimethyl maleate, diethyl oxalate, ethylene glycol diacetate, diethylbenzoate, diethyl malonate, fluorobenzene, fluorobenzene o-, m- or p-chlorotoluene, o-, m- or p-bromotoluene, 2-chloroethane, 1- or 2-chloropropane, 1- or 2-chlorobutane, 1-chloro-2-m ethylpropane, 1-, 2- or 3-chloropentane, 1-chloronaphthalene, methylene chloride, chloroform, carbon tetrachloride, 1,1-dichloroethane, 1,2-dichloroethane, 1,1,1-trichloroethane, 1.1 , 2-trichloroethane, 1,1,2,2-tetrachloroethane, o-, m- or p-dichlorobenzene, nitromethane, nitroethane, 1- or 2-nitropropane, nitrobenzene, acetonitrile, propionitrile, butyronitrile, isobutyronitrile, valeronitrile, benzonitrile, phenylacetonitrile and thiophene.

Preferred solvents are:

Hydrocarbons: benzene, toluene, o-, m- or p-xylene, cumene, mesitylene.

Halogenated hydrocarbons *, chloroethane, 1- or 2-chloropropane, 1- or 2-chlorobutane, isobutyl chloride, 1-, 2- or 3-chloropentane, methylene chloride, chloroform, 1,2-dichloroethane, carbon tetrachloride, 1.1, 1-trichloroethane, 1,1,2-trichloroethane, 1,1,2,2, -tetrachloroethane, fluorobenzene, chlorobenzene, bromobenzene, o-, m- or p-fluorotoluene, o-, m- or p-chlorotoluene, o -, m- or p-bromo-toluene, o-, m- or p-dichlorobenzene.

Acids: acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid.

Esters: methyl acetate, ethylene glycol diacetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate, sec-butyl acetate, pentyl acetate, ethyl propionate, methyl butyrate, n-butyl formate, propyl formate, propyl acetate

Nitro compounds: nitrobenzene, nitromethane, nitroethane, nitropropane.

Nitriles: acetonitrile, propionitrile, butyronitrile, isobutyronitrile, valeronitrile, benzonitrile, phenylacetonitrile.

Ketones: methyl isobutyl ketone, methyl ethyl ketone.

Highly preferred solvents are acetonitrile, 1,2-dichloroethane, methylene chloride, propionitrile, nitromethane, nitroethane, acetic acid, isopropyl acetate, butyl acetate and methyl isobutyl ketone.

The 3- (acyloxymethyl) cephalosporin of formula II is defined as an acid. It is probable that an acid is involved in the reaction because the cephalosporin salts are generally insoluble in organic solvents. An exception is the solubility of cephalosporin salts in carboxylic acids such as acetic acid. Thus, the reaction contained in this invention can be carried out by dissolving a 3- (acyloxymethyl-3,630,309) cephalosporin salt such as a metal salt in a carboxylic acid.

Whatever solvent is used, it is important that the reaction be carried out under virtually anhydrous conditions. In general, the reaction mixture should contain less than 5% water, and preferably less than 1%. Even more preferred is if the water content is less than 0.5%. If the commercially available reagents and solvents are not sufficiently dry, the water can be removed by known methods such as azeotropic drying or desiccants (alumina, silica gel, anhydrous calcium sulfate, etc.).

The temperature range of the reaction according to the invention is wide. In general, reaction temperatures in the range of 50 to 1 + 0 ° C can be used, but a better temperature range for the results is 70 to 120 ° C. The reaction can be carried out at even higher temperatures, but this has not been found to bring any advantages. Thus, for simplicity, atmospheric pressure is preferably used.

Reactive amounts are not critical to the reaction. In general, it is preferred to use about 1.0 to 5.0 molar equivalents of sulfur nucleophile per molar equivalent of 3- (acyloxymethyl) cephalosporin.

For example, the following known and important cephalosporin compounds of formula I can be prepared by the process of this invention: 0 s

, ~ * L-CH-C-NH - «- /> N- -N

1 V

COOH & 3, _ 2 s

cefamandolina naphthaate: · ^ • -CH-C-NH- f "f ^ N - - -N

V- · 'i Lii 1> 1 ".

OCHO n. ^ 9-CH2-S- N

i I

COOH CH3 ... N -'- ·: N 0 kefazolxini: I ji „ySv N _n N. N-CH0-C-NH-f f T T ·,

COOH

69309 Ceftazole: 0 g

Λ Λ -CH2-C-NH> _ / \ N- N

.L - .. N ICH-S-Vl V

v r 2 N. o, 0 'V ^

COOH

The invention is illustrated by the following examples, which are easy to repeat for those skilled in the art.

Example 1 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (thienyl) acetamidio) -3-cephem-4-carboxylic acid in acetonitrile 7- (2- ( 2-thienyl) acetamido) cephalosporanic acid (2.2 g, 5.5 mol) and 1-methyl-1H-tetrazole-5-thiol (1.0 g, 8.6 mmol) were added to 25 mL of acetonitrile in a flask was equipped with a condenser and a drying tube containing anhydrous calcium sulfate ("Drierite"). The reaction mixture was cooled to reflux with stirring and the reaction was monitored by thin layer chromatographic (TLC) analysis. After refluxing for 90 minutes, TLC gave about 2/3 of the cephalosporin starting material and 1/3 of the desired product. After refluxing for 3 hours, TLC gave about 1/3 cephalosporanic acid starting material and 2/3 product. After 6 hours, TLC analysis indicated that the reaction was virtually complete and the reaction mixture was cooled to room temperature and allowed to stand overnight.

Removal of the solvent on a rotary evaporator gave a foam which was dissolved in 10 ml of ethanol. When a solution of 1 ml of dicyclohexylamine in 10 ml of ethanol was added dropwise, the product precipitated as a dicyclohexylamine salt which, after stirring for 15 minutes, was filtered off. After washing the separated precipitate with 15 ml of ethanol and drying in vacuo at 40 ° C for 2 hours, 2.50 g (yield 76.1%) of product were obtained, m.p. 184-184 ° C (dec.). The product was subjected to NMR, IR and TLC analysis, and all analytical results were identical to those obtained for the product prepared by a previously known method. NMR (DMSQ-d 6) δ 3.52 (m, 2, 2-CH 2), 3.76 (s, 2, -CH 2 ONH-), 3.92 (s, 3, tetrazoline -CH), 4.35 (s, 2,3-CH 2 S), 5.00 (d, 1.1 Cg-H, J = 5 Hz), 5.55 69309 11 (q, 1, C 6 H -H, J = 5 Hz) , J = 9 Hz), 6.95 (d, 2, thiophene 3- and 4-H, J = 3 Hz), 7.35 (t, 1, thiophene 5-H, J = 3 Hz) and 8, 75 (d, 1, -CH 2 CONH-, J = 9 Hz).

Example 2 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid in acetonitrile 7- (2- (2-thienyl) acetamido) cephalosporanic acid (4.0 g, 10 mmol) and 1-methyl-1H-tetrazole-5-thiol (5.8 g, 50 mmol) were cooled to reflux in 50 mL of dry acetonitrile ( treated with anhydrous sulfonic acid resin "Amberlite 15") in a dry atmosphere for 8 3/4 hours. The reaction was monitored by TLC analysis and was almost complete in 8 3/4 hours.

The solvent was evaporated off and the residue was added to 125 ml of ethanol. When dicyclohexylamine (6 ml) in 75 ml of ethanol was added, the product precipitated as the dicyclohexylamine salt. The precipitate was collected by filtration, washed and dried to give 4.50 g (71.0%) of product. The results of IR, NMR and TLC analysis were similar to those of the sample obtained by the reaction in aqueous solution. The NHR spectrum was identical to the NMR spectrum of the product of Example 1.

Example 3 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid in 1,2-dichloroethane 7- (2- (2-thienyl) acetamido) cephalosporanic acid (2.0 g, 5 moles) and 1-methyl-1H-tetrazole-5-thiol (1.2 g, 10 mmol) were refluxed in 25 mL 1, 2-dichloroethane in a dry atmosphere for 5.5 hours. The solvent was removed on a rotary evaporator, first 25 ml of ethanol was added to the filtrate, followed by dropwise addition of a solution of 2 ml of dicyclohexylamine in 25 ml of ethanol.

After stirring for 20 minutes at room temperature, the reaction mixture was filtered, washed with 25 ml of ethanol and dried at 40 ° C under vacuum to give 2.34 g (73.8%) of the product as a hard dicyclohexylamine salt. The IR and NMR analysis results were similar to those of the sample obtained by the reaction in aqueous solution. The NMR spectrum was also identical to the NMR spectrum of the product of Example 1 ____ to 12,690,309.

Example 4 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7-amino-3-cephem-4-carboxylic acid in 1,2-diclocriethane.

7-Formamidocephalosporanic acid (3.0 g, 10 mmol) and 1-methyl-1H-tetrazole-5-thiol (2.4 g, 20 mmol) were refluxed in 50 mL of 1,2-dichloroethane for 7 hours with stirring. The reaction mixture was cooled to room temperature and allowed to stand at room temperature overnight. A rubbery precipitate formed. The 7-formyl group was removed from the product as follows:

The solvent was removed on a rotary evaporator and the residue was dissolved in a mixture of 25 ml of methanol and 2.8 ml of concentrated hydrochloric acid, and the resulting solution was allowed to stand overnight at room temperature. The solution was diluted with 50 ml of water and the pH was adjusted from 0.9 to 3.6 by dropwise addition of triethylamine.

When the light brown crystals were collected by filtration, washed with water and dried, 2.10 g (64%) of product were obtained. The NMR spectra of this product and the same product prepared in aqueous solution were identical. NMR (D 2 O, NaHCO 3> δ 3.65 (q, 2, 2-CH 2, JAB = 16.5 Hz), 4.4 (s, 3, -CH 3 in tetrazole), 4.16 (q, 2, 3 -CH2S-, JAB = 12.5 Hz), 5.05 (d, 1, C8-H, J = 5 Hz) and 5.45 (d, 1, C2-H, J = 5 Hz).

Example 5 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid in isopropyl acetate 7- (2 - (2-thienyl) acetamido) cephalosporanic acid (2.0 g, 5 mmol) and 1-methyl-1H-tetrazole-5-thiol (1.2 g, 10 mmol) were cooled to reflux in 25 mL of isopropyl acetate (90 ° C) 23 hours while stirring. The reaction mixture was then cooled to room temperature. TLC analysis showed that some cephalosporanic acid starting material remained. After the cream precipitate was collected, washed with isopropyl acetate and dried, 1.60 g (70.8%) of product were obtained. The product was identified by NMR analysis, which also showed that less than 1% of the cephalosporan starting material was present.

NMR (DMSO-d 6) δ - 3.72 (s, 2, 2-CH 2), 3.80 (s, 2, -CH 2 CONH-), 3.95 (s, 3, tetrazoline -CH 2), 4, 30 (s, 2,3-CH 2 S-), 5.10 (d, 1, C 6 -C 8 -H, J = 5 Hz), 5.70 (q, 1, C 8 -H, J = 5 Hz, J = 8 Hz), 6.92 (d, 2, thiophene 13 69309 3- and 4-H, J = 3 Hz), 7.35 (t, 1, thiophene 5-H, J = 3 Hz) and 8.78 (d, 1, -CH 2 CONH, J = 8 Hz).

Example 6 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid in propionitrile 7- (2 - (2-Thienyl) acetamido) cephalosporanic acid (2.0 g, 5 mmol) and 1-methyl-1H-tetrazole-5-thiol (1.2 g, 10 mmol) were cooled to reflux in 25 mL of dry propionitrile (97 ° C) until TLC analysis showed complete disappearance of the cephalosporanic acid starting material (4.5 hours). The reaction mixture was cooled to room temperature and the solvent was removed on a rotary evaporator, but some solution was lost due to overboiling. The residue was dissolved in 35 ml of warm ethanol and a solution of 2 ml of dicyclohexylamine in 10 ml of ethanol was added. The mixture was stirred for 10 minutes at room temperature, filtered, washed with ethanol and dried to give 1.24 g (39% yield except for rotary evaporator loss). The NMR spectra of this product and the product of Example 1 were identical.

Example 7 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid in acetonitrile, in addition 3,5-Dichlorophenyl dihydrogen phosphate 7- (2- (2-thienyl) acetamido) cephalosporanic acid (2.0 g, 5 mmol), 1-methyl-1H-tetrazole-5-thiol (0.87 g, 7.5 mmol) and 3,5-dichlorophenyl dihydrogen phosphate (0.122 g, 0.5 mmol) was heated overnight in 25 mL of dry acetonitrile at 70 ° C. The mixture was then evaporated on a rotary evaporator to 8-10 ml. The product began to crystallize. The mixture was stirred for 1/2 hour and then 25 ml of isopropyl acetate was added dropwise over 1/2 hour to enhance crystallization. After stirring for an additional 1/2 hour, the precipitate was collected by filtration, washed with isopropyl acetate and dried to give 0.98 g (43.4%) of product. The NMR spectra of the obtained product and the product of Example 5 were identical.

69309 14

Example 8 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (2-thienyl) -acetamido) -3-cephem-4-carboxylic acid in acetic acid 7 - (2- (2-thienyl) acetamido) cephalosporanic acid (2.0 g, 5 mmol) and 1-methyl-1H-tetrazole-5-thiol (0.87 g, 7.5 mmol) were added to glacial acetic acid (25 mL). The reaction mixture was heated to 60 ° C and maintained at this temperature for 1 hour. TLC analysis showed that there was only a small amount of product. The reaction mixture was heated to 80 ° C and maintained at this temperature for 5 hours. After allowing the reaction mixture to stand overnight, the precipitate was collected by filtration, washed with acetic acid and dried to give 0.71 g (31%) of product. The NMR spectra of this product and the product of Example 5 were identical.

Example 9 3 - (((4,5-dihydro-6-hydroxy-4-methyl-5-oxo-1,2,4-triazin-3-yl) thio) methyl) -7- (2- (2- Preparation of thienyl) acetamido) -3-cephem-4-carboxylic acid in acetonitrile 7- (2- (2-thienyl) acetamido) cephalosporanic acid (2.0 g, 5 mmol) and 4,5-dihydro-6-hydroxy-4 -methyl-5-oxo-1,2,4-triazine-3-thiol (1.2 g, 7.5 mmol) was added to 25 mL of dry acetonitrile in a reaction vessel immersed at 84-85 ° C. oil bath. The reaction vessel was equipped with a condenser with a drying tube containing anhydrous calcium sulfate ("Drierite"). The reaction mixture was kept in this state and stirred slowly for 16 hours with a magnetic stirrer. The product crystallized from the hot solution. When the reaction mixture was cooled to room temperature, filtered, washed first with acetonitrile and then with acetone and dried in vacuo to give 1.81 g (72.6%) of product. The product was obtained as heavy crystals, m.p. 161 ° C (dec.).

Another batch of crystals was obtained from the filtrate on standing.

This batch was collected by filtration, washed with acetonitrile and dried to give 0.26 g (10%) of product. This product sp. was 161 ° C (dec.). The total yield thus became 83.1%.

Example 10 Preparation of 3 - (((5-methyl-1,3,4-oxadiazol-2-yl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid 1,2-dichloroethane.

7- (2- (2-thienyl) acetamido) cephalosporanic acid (2.0 g, m.p. 69309 5 mmol) was added to any 1,2-dichloroethane flask equipped with a magnetic stirrer and condenser equipped with a drying tube containing anhydrous calcium sulfate. ( "Drierite"). The flask was immersed in an oil bath at 84-85 ° C for 8 hours and then kept in the refrigerator for 2 days.

A portion of the product had crystallized, and when filtered, washed with 1,2-dichloroethane and dried, 1.10 g (48.7%) of product were obtained. The product was dissolved in 15 ml of acetone and the insoluble matter was filtered off. When 75 ml of deionized water was added dropwise to the filtrate, the precipitate was collected by filtration and dried to give 0.61 g of product, m.p. > 114 ° C (dec.).

When the solvent was evaporated from the filtrate, 0.23 g of a mixture of product and starting material was obtained.

Example 11 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid in nitromethane 7- (2 - (2-thienyl) acetamido) cephalosporanic acid (2.0 g, 5 mmol) and 1-methyl-1H-tetrazole-5-thiol (0.87 g, 7.5 mmol) were added to 25 mL of dry nitromethane in a flask and the flask was immersed in an oil bath at 100-101 ° C for 4.5 hours and then cooled to room temperature. TLC analysis showed the reaction to be complete. The solvent was removed on a rotary evaporator and the residue was dissolved in 75 mL of warm ethyl acetate. A small amount of insoluble matter was filtered off from the solution, followed by extraction twice with 25 ml of 5% aqueous sodium bicarbonate solution. The combined extracts were layered with 50 ml of ethyl acetate and the pH was adjusted to about 1.0 with 70% aqueous methanesulfonic acid. The ethyl acetate layer was separated and the aqueous layer was extracted with 2 x 5 ml of ethyl acetate. The ethyl acetate layers were combined, dried over anhydrous calcium sulfate and concentrated to 25 mL on a rotary evaporator. The product crystallized when 50 ml of diethyl ether was added dropwise. When the precipitate was filtered, washed with diethyl ether and dried, 1.27 g (56.2%) of product were obtained as hard crystals, m.p. 156-159 ° C (dec.). The product was identified by NMR analysis.

16 69309

Example 12 Preparation of 3 - (((1-methyl-β-tetrazol-5-yl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid in methylene chloride

A mixture of 7- (2- (2-thienyl) acetamido) cephalosporanic acid (11.9 g, 30 mmol) and 1-methyl-1H-tetrazole-5-thiol (7.0 g, 60 mmol) 300 in 1 ml of cyclohexane-stabilized methylene chloride was placed in a 1-liter stainless steel autoclave equipped with a heating device. The reaction mixture was stirred and heated at 83-86 ° C under an evolved pressure of 290 kPa for 16 hours. The reaction mixture was then cooled to room temperature. TLC analysis showed that the starting material had formed the product and only a small amount of cephalosporanic acid starting material remained. After allowing the reaction mixture to stand overnight at room temperature, the product crystallized, and then the mixture was concentrated to 200 ml. The crystals were collected by filtration and washed with methylene chloride to give 7.37 g (54.3%) of product as white crystals, m.p. 163.5-164 ° C (dec.).

When the filtrate was diluted with 100 ml of diethyl ether, filtered, washed with diethyl ether and dried, a second crop of 1.40 g (10.3%) of light brown crystals was obtained.

When the filtrate was diluted with isopropyl acetate, a third crop of 1.18 g (8.7%) of product was obtained.

The total yield was 73.3%.

Example 13 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid in fluorobenzene 7- (2 - (2-thienyl) acetamido) cephalosporanic acid (2.0 g, 5.04 mmol) and 1-methyl-1H-tetrazole-5-thiol (1.2 g, 10 mmol) were mixed with 75 mL of fluorobenzene (b.p. 85.1 ° C) in a flask equipped with a condenser and a drying tube containing anhydrous calcium sulfate ("Drierite"). The mixture was refluxed and the reaction was monitored by TLC analysis (ethyl acetate / acetic acid 7: 1). In the reaction mixture, which remained heterogeneous throughout, the reaction was virtually complete in 72 hours. The reaction mixture was then cooled to room temperature 1769309 and the precipitated product was filtered off under vacuum at 40 ° C for 5 hours to give 2.13 g (93.4%) of product as hard crystals, m.p. 161-162 ° C (dec.). The product was identified by NHR analysis.

Example 14 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid in thiophene 7- (2 - (2-thienyl) acetamido) cephalosporanic acid (2.0 g, 5 mmol) and 1-methyl-1H-tetrazole-5-thiol (0.87 g, 7.5 mmol) were refluxed in 25 mL of thiophene for 7 hours. , during which the product crystallized. The reaction mixture was cooled to room temperature, stirred for 30 minutes to allow complete crystallization, and filtered. After washing the precipitate with 0.5 ml of thiophene and drying in vacuo for 2 hours, 1.82 g (80.2%) of product were obtained as white crystals, m.p. 162-163 ° C (dec.). TLC analysis showed that the filtrate still contained product.

Example 15 3 - (((5-methyl-1,3,4-thiadiazol-2-yl) thio) methyl) -7- (2- (1H-tetrazol-1-yl) acetamido) -3-cephem- Preparation of 4-carboxylic acid in acetonitrile

A mixture of 7- (2- (1H-tetrazol-1-yl) acetamido) cephalosporanic acid (0.75 g, 2 mmol) and 5-methyl-1,3,4-thiadiazole-2-thiol (0 , 33 g, 2.5 mmol) in 10 mL of reagent grade acetonitrile, was refluxed for 2 hours 40 minutes. The product crystallized. After cooling the reaction mixture with an ice bath, the precipitate was collected by filtration, washed with 3 ml of acetonitrile and dried at 40 ° C under vacuum to give 0.61 g (67%) of product. The product was identified by NMR analysis: δ 2.68 (s, 3, tetrazole -CH 3), 3.72 (s, 2, β-CH 2), 4.40 (q, 2,3-CH 2 S-, JAB = 13 Hz ), 5.12 (d, 1.1Cg-H, J = 5 Hz), 5.38 (s, 2, -CH 2 CONH-), 5.72 (q, 1.1C-H, J = 5 Hz) , J = 9 Hz), 9.00 (s, 1, tetrazole 5-H) and 9.17 (d, 1, -CH 2 CONH-).

Example 16 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2-formyloxy-2-phenylacetamido) -3-cephem-4-carboxylic acid 1,2- in dichloroethane 7- (2-formyloxy-2-phenylacetamido) cephalosporanic acid (2.17 g, 5 mmol) and 1-methyl-1H-tetrazole-5-thiol (0.87 g, 18 69309 7.5 mmol) were added 25 mL to 1,2-dichloroethane and the resulting mixture were refluxed for 6 hours. Samples were taken after 3 and 6 hours for TLC analysis. The reaction mixture was heated for an additional 1 hour and then allowed to cool overnight. The solvent was evaporated off. When diethyl ether was added to the residue, it first became gummy and then solid.

When the precipitate was collected by filtration, washed with diethyl ether and dried, 1.90 g (77.0%) of product were obtained. NMR (DMSO-d 6) δ 3.52 (s, 2, 2-CH 2), 3.88 (s, 3, tetrazoline -CH 2), 4.10 (s, 2,3-CH 2 S-), 4.92 (d, 1, Cg-H), 5.62 (q, 1, Cy-H, J = 5 Hz, J = 9 Hz), 6.06 (s, 1, -CHCONH-), 7.26 ( s, 5, phenyl-H) and 8.28 (s, 1,?, -CH).

Example 17 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (5-carboxy-5- (2,4-dichlorobenzamido) valeramido) -3-cephem-4 preparation of carboxylic acid in (A) acetonitrile and (B) 1,2-dichloroethane (A) A mixture of 7- (5-carboxy-5- (2,4-dichlorobenzamido) valeramido) cephalosporanic acid (2.9 g, 5 mmol), 1-methyl-1H-tetrazole-5-thiol (1.2 g, 10 mmol) and 50 mL of acetonitrile were refluxed overnight (18 hours). TLC analysis showed that the reaction was about 90% complete. When the reaction mixture was evaporated and the residue was taken up in ethyl acetate and filtered, 1.51 g (48.3%) of product were obtained.

(B) A mixture of 7- (5-carboxy-5- (2,4-dichlorobenzamido) valeramido) cephalosporanic acid (2.9 g, 5 mmol), 1-methyl-1H-tetrazole-5-thiol (1, 2 g, 10 mmol) and 50 mL of 1,2-dichloroethane, refluxed for 5 1/2 hours. TLC analysis showed that the reaction was about 90% complete. The solvent was decanted from the reaction mixture and the remaining stretchable mass was triturated back to chilled ether. The product was filtered to give 2.83 g (89.3%) of brown crystals. NMR analysis confirmed the identity of the product but also showed ether. NMR (DMSO-dc) δ δ, δ δ and 2.2 δ (each m, adipoyl side ju), 3.66 (m, 2,2-CH 2), 3.95 (s, 3, tetrazoline -CH 3), 4.30 (m, 2,3-CH 2 S-), 5.08 (d, 1, C 6 H 6, J = 5 Hz), 5.68 (q, 1, Cy-H, J = 5 Hz), 7.50 and 7.62 (s, 2,4-dichlorophenyl each) and 9.00 (m, 2, two -CONH).

69309 19

Example 18 Preparation of 3 - (((1-methyl-1H-tetrazol-5 * yl) thio) methyl) -7 '(2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid in 1,2-dichloroethane 7- (2- (2-thienyl) acetamido) cephalosporanic acid (0.99 g, 2.5 mmol) and 1-methyl-1H-tetrazole-5-thiol (0.58 g, 5 mmol) were added in 12.5 mL to 1,2-dichloroethane and the reaction mixture was cooled to reflux. On reflux, the acetic acid product was removed by passing 1,2-dichloroethane through the calcium oxide. Refluxing was continued for 5 3/4 hours and then the reaction mixture was cooled to room temperature and filtered. When the fluffy needles were washed in 10 ml of 1,2-dichloroethane and dried, 0.77 g (68.1%) of product was obtained.

Evaporation of the filtrate gave an additional 0.04 g of product (3.5%).

The product was identified by TLC analysis and the major product by NMR analysis. The NMR spectra of this product and the product of Example 5 were identical.

Example 19 Preparation of 3 - (((5-methyl-1,3,4-thiadiazol-2-yl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid in acetonitrile

A mixture of 7- (2- (2-thienyl) acetamido) cephalosporanic acid (2.0 g, 5 mmol) and 5-methyl-1,3,4-thiadiazole-2-thiol (2.64 g, 20 mmol) in 25 mL of acetonitrile, was refluxed overnight at 79 ° C. TLC analysis showed that some cephalosporan acid starting material remained. The oily residue was filtered off from the reaction mixture and the solvent was removed on a rotary evaporator. After the residue was crystallized from acetonitrile-isopropyl acetate (1: 1), the precipitate was collected by filtration, washed and dried in vacuo to give 1.59 g (33.9%) of product, m.p. . 166 ° C. The product was identified by IR, UV, NMR, MS and elemental analysis.

NMR (DMSO-d 6): δ 2.68 (s, 3, tetrazoline -CH 3), 3.68 (s, 2, 2-CH 2), 3.76 (s, 2, -CH 2 CONH-), 4.38 (q, 2,3-CH 2 S-, J = 13 Hz), 5.10 (d, 1, C 6 H 6, J = 5 Hz), 5.70 (q, 1, C 2 -H, J = 5 Hz, J = 9 Hz), 6.92 (d, 2, thiophene 3- and 4-H), 7.37 (t, 1, thiophene 5-H) and 9.10 (d, 1, -CH 2 CONH, J = 9 Hz).

20 6 9 3 0 9

Example 20 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid in 1, 2-trichloroethane 7- (2- (2-thienyl) acetamido) cephalosporanic acid (2.0 g, 5 mmol) and 1-methyl-1H-tetrazole-5-thiol (1.2 g, 10 mmol) were added to 25 mL of 1, 1,2-Trichloroethane and the mixture thus obtained were heated to 100-101 ° C. The progress of the reaction was monitored by TLC analysis and, after stirring for 4 hours at 100-101 WC, TLC analysis showed no sign of cephalosporanic acid starting material. The reaction mixture was cooled to room temperature with stirring. The mixture was seeded and stirring was continued overnight. The product crystallized.

The solvent was evaporated off and 25 ml of 1,2-dichloroethane were added. The solvent was again filtered off and the product was washed and dried in vacuo. There was thus obtained 0.98 g of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid , sp. 158 ° C (dec.) (43.9%).

Example 21 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid in (A) methyl ethyl ketone and (B) In 1,1,2-trichloroethane (A) 7- (2- (2-thienyl) acetamido) cephalosporanic acid (2.0 g, 5 mmol) and 1-methyl-1H-tetrazole-5-thiol (1, 2 g, 10 mmol) was added to 25 ml of methyl ethyl ketone and the resulting mixture was refluxed for 48 hours. TLC analysis showed that only a small amount of cephalosporanic acid remained. The reaction mixture was washed with a solution of 2.5 g of sodium bicarbonate and 50 ml of water. The aqueous layer was treated with 1 g of carbon and 50 ml of ethyl acetate was added. The pH was adjusted to 1.6 with a solution of 4 ml of methanesulfonic acid in 30 ml of water. The ethyl acetate layer was dried over sodium sulfate and evaporated on a rotary evaporator to an oily residue. The product was crystallized by dropwise addition of 50 ml of ether. The precipitate was collected by filtration, washed and dried in vacuo to give 0.94 g (41.6%) of product. The NMR spectra of this product and the product of Example 5 were identical.

II 21 69309 (B) The same starting materials were used in the reaction in the same amounts as in (A), but 50 ml of 1,1,2-trichlorethylene was used as the solvent. After refluxing the reaction mixture for 16 hours, TLC analysis indicated that no cephalosporanic acid starting material remained. 0.47 g (20.8%) of product was obtained when the separation was carried out as described in (A). The NMR spectra of this product and the product of Example 5 were identical.

Example 22 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2-phenylacetamido) -7-methoxy-3-cephem-4-carboxylic acid in 1,2-dichloroethane 7 - (2-phenylacetamido) -7-methoxycephalosporanic acid (210 mg, 0.5 mmol), 1-methyl-1H-tetrazole-5-thiol (87 mg, 0.75 mmol) and 15 ml of 1,2-dichloroethane were mixed and at the same time, it was refluxed for 6 hours under a nitrogen atmosphere, and after this time, TLC analysis showed that only a small amount of cephalosporanic acid starting material remained. Additional 1-methyl-1H-tetrazole-5-thiol (29 mg, 0.25 mmol) was added to the reaction mixture and the mixture was refluxed for an additional 3 hours. TLC analysis showed no significant difference. The reaction mixture was washed 4 times with saturated sodium bicarbonate solution, the bicarbonate layer was washed three times with ethyl acetate, fresh ethyl acetate was added, cooled to 0 ° C and the pH was adjusted to 2.2 with 20% hydrochloric acid. The layers were separated and the aqueous layer was washed with ethyl acetate. When the acetate layers were combined, washed with brine, dried over magnesium sulfate, filtered and evaporated to give 199 mg (83.6%) of a light green foamy residue. The product was identified by TLC and NMR analysis. NMR (CL) C 13 + Id acetone-d 6) δ 3.45 (s, 3, -OCH 2), 3.55 (s, 2, 2-CH 2), 3.75 (s, 2, OCH 2 CO-) , 3.9 (s, 3, CH 2 in tetrazole), 4.4 (s, 2,3-CH 2 S-), 5.15 (s, 1, C 6 H 6), 7.35 (s, 5.0) , 8.0 (s, 1, -CH 2 CONH-) and δ 11.2 (s, 1, J = 0 Hz, -C00H).

Example 23 Preparation of 3 - (((2-benzothiazolyl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid in 1,2-dichloroethane 7- (2- (2-thienyl ) acetamido) cephalosporanic acid (1.0 g, 2.5 mmol) and 2-mercaptobenzothiazole (0.625 g, 3.75 mmol) 6 9 3 0 9 22 were placed in a flask, deaerated with nitrogen, 25 mL of 1,2-dichloroethane was added, and the reaction mixture was refluxed for 24 hours with stirring. The Kdn reaction mixture was cooled and filtered to give 1.1 g (88%) of a harder product, m.p. 190.5-191 ° C (dec.). The product was dried overnight in vacuo at 40 ° C and analyzed. The product was identified by NMR, UV, MS, IR and elemental analysis. NMR (DMSO-d 6) δ 3.74 (s, 2, 2-CH 2), 3.80 (s, 2, -CH 2 CONH-), 4.58 (q, 1, C 2 -H, J = 5 Hz, J = 8 Hz), 6, 96, 7, 43 and 7.96 (each m, phenyl and thienyl rings), 9.10 (d, 1, -CH 2 CONH-, J = 8 Hz).

Example 24 3 - (((5- (N-methylacetamido) -1,3,4-thiadiazol-2-yl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3- Preparation of cephem-4-carboxylic acid in 1,2-dichloroethane 7- (2- (2-thienylacetamido) cephalosporanic acid (2.0 g, 5 mmol) and 5- (N-methylacetamido) -1,3,4-thiadiazole 2-Thiol (1.42 g, 7.5 mmol) was added to 50 mL of 1,2-dichloroethane and the reaction mixture was refluxed for 10 h.When the reaction mixture was cooled, filtered, washed with 1,2-dichloroethane and dried in vacuo, 2.06 g ( 78.3%) of product, mp 178-179 ° C. The product was identified by IR, UV, NMR, MS and elemental analysis NMR (DMSO-d 6) δ 2.42 (s, 3, -COCH 3 ), 3.74 (m, 7, 2-CH 2, tetrazoline -CH 3 and -CH 2 CONH-), 4.35 (q, 2,3-CH 2, 3-CH 2, J = 13 Hz), 5.10 (d, 1, C-H, J = 5 Hz), 5.70 (q, 1, C 2 -H, J = 5 Hz, J = 9 Hz), 6.96 and 7.36 (each m, 3, thiophene H) and 9.10 (d, 1, -CH 2 CONH-, J = 9 Hz).

Example 25 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (N-tert-butoxycarbonyl) -2-phenylglycylamino) -3-cephem-4-carboxylic acid preparation in 1,2-dichloroethane 1,2-Dichloroethane (45 ml) was refluxed to remove the water-solvent azeotrope, and then the volume was distilled to 30 ml and allowed to cool. 7- (N-tert-Butoxycarbonyl-2-phenylglycylamino) cephalosporanic acid (253 mg, 0.5 mmol) was added, the solution was distilled to 15 ml and cooled. 1-Methyl-1H-tetrazole-5-thiol (87 mg, 0.75 mmol) was added, 23,690,309 reaction mixtures were refluxed under nitrogen and the reaction was monitored by TLC analysis. After 16 hours, TLC analysis showed that only a small amount of starting material remained. The reaction mixture was washed 4 times with saturated sodium bicarbonate solution, the bicarbonate layers were combined and washed twice with ethyl acetate. Fresh ethyl acetate was added to the washed bicarbonate layers, the solution was cooled to 0 ° C and the pH was adjusted to 2.4 with 20% hydrochloric acid. The layers were separated. The aqueous layer was washed with ethyl acetate. When the ethyl acetate layers were combined, washed with brine, dried over magnesium sulfate, filtered and evaporated to give 224 mg (80%) of the product as a white foam. The product was identified by TLC and NMR analysis. NMR analysis showed little residual cephalosporan starting material (<0.10%).

The product was suspended in 10 ml of diethyl ether and triturated for 1 hour. The ether was decanted off, 10 ml of fresh diethyl ether was added and triturated for another 1 hour. After decanting the ether and evaporating to dryness, the product was obtained as a white powder. The product was analyzed by TLC and NMR. NMR (CDCl 3) δ 1.45 (3.9, -C00-tert-C 4 H 9), 3.6 (s, 2,2-CH 2), 3.95 (s, 3, CH 3 in tetrazole), δ , 3 (s, 2,3-CH 2 S-), 4.9 (d, 1, J = 6 Hz, C 6 H 6), 5.4 (d, 1, J = 8 Hz, OCH-), 5, 75 (q, 1, J = 4 Hz, on C7), b, 2 ia, 1, J = bf '

Hz, OCH-), 7.4 (s, 5.0), 7.65 (d, 1, J = 8 Hz), -CO (NH) and? 9.3 NH

(s, 1, C00H).

Example 26 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (N- (tert-butoxycarbonyl) -2- (p-hydroxyphenyl) glycylamido) -3-cephem- Preparation of 4-carboxylic acid in 1,2-dichloroethane 1,2-dichloroethane (45 ml) was refluxed and 15 ml of solvent was removed. The remaining 30 ml was cooled. 7- (N- (tert-butoxycarbonyl) -2- (p-hydroxyphenyl) glycylamido) cephalosporanic acid (260.5 mg, 0.5 mmol) was added and the solution was distilled to 15 ml and cooled again. 1-Methyl-1H-tetrazole-5-thiol (87 mg, 0.75 mmol) was added, the reaction mixture was heated to 65-70 ° C and the progress of the reaction was monitored by TLC analysis. TLC analysis showed that there was almost no starting material 69309 24 after 3 hours, and the reaction mixture was worked up after 4 hours. The treatment and purification took place essentially as in the previous example. 189 mg (65.5%) of crude product and 46 mg (about 16%) of purified product were obtained. NMR (CDCl 3 + 2d acetone dg) δ T45 (s, 9, -C00tertCl 2 Hg), 3.35 (s, 2, 2-CH 2), 3.85 (s, 3, tetracodyl CH 2), δ , 3 (s, 2, SC 2 -S-), 4.9 (d, 1, J = 6 Hz, C 6 -H), 5.3 (q, 1, J = 3 Hz, C 6 -H), 5.4 (s, 1, HO-OCH-), 5.75 (d, 1, J = 6 Hz, HOOCH-), 6.4 (s, 1, C00H), 6.8! f

NH

f <d, 2, J = 8 Hz, HO-ff -CH-), and 7.9 (d, 1, -CONH).

B

Example 27 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2-phenoxyacetamido) -3-cephem-4-carboxylic acid in 1,2-dichloroethane 1,2-dichloroethane ( 60 ml) was refluxed using a pad, 15 ml of solvent was removed to leave 4 5 ml and cooled. 7- (2-Phenoxyacetamido) cephalosporanic acid (406 mg, 1 mmol) was added, the solution was distilled to 30 ml and cooled. 1-Methyl-1H-tetrazole-5-thiol (174 mg, 1.5 mmol) was added and the reaction mixture was refluxed under nitrogen for 12 hours and, at this point, TLC analysis of 10: 3 diethyl ether / (3: 1 acetic acid / water). ), the product, excess thiol reagent, some cephalosporanic acid reagent and an unknown compound were observed. The reaction mixture was then treated in substantially the same manner as in the previous examples.

Examples 28-31 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid in fluorobenzene 3- (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid was prepared by four different reactions, each starting from a different cephalos- % ranoic acid as follows: 69309 2 8: 3- (Propionyloxymethyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid (41 mg, 0.1 mmol) 29: 3- (2- Methylpropionyloxymethyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid (42 mg, 0.1 mmol) 30: 3- (n-butyryloxymethyl) -7- (2- (2 -thienyl) acetamido) -3-cephem-4-carboxylic acid (42 mg, 0.1 mmol) 31: 3- (cyclobutylcarbonyloxymethyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4- carboxylic acid (43 mg, 0.1 mmol).

In each reaction, the compound was suspended in 10 ml of fluorobenzine (dried with aluminosilicate desiccant "4-A Linde Molecular sieve"). 1-Methyl-1H-tetrazole-5-thiol (18 mg, 0.15 mmol in each reaction) was added and the reaction mixture was refluxed for 24 hours. In each reaction, the product precipitated and was filtered and dried. TLC analysis in ethyl acetate-acetic acid showed that each substitution reaction had occurred quantitatively. The NMR spectra of all these products and the products of the substitution reactions in aqueous solutions were consistent.

Example 32 Preparation of 3 - (((3-methyl-1,2,4-oxadiazol-5-yl; thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid 1 In 1,2-trichloroethane 7- (2- (2-thienyl) acetamido) cephalosporanic acid (396 mg, 1 mmol) was suspended in 40 ml of 1,1,2-trichloroethane and then 3-methyl-1,2, 4-Oxadiazole-5-thiol (116 mg, 1 mmol) The reaction mixture was heated in an oil bath at 113 ° C for about 3 hours and then allowed to cool overnight and evaporated to an oil. the aqueous layers were combined and extracted with ethyl acetate, the aqueous layer was layered with fresh ethyl acetate, cooled in an ice bath and the pH was adjusted to 2.5 with 20% hydrochloric acid, the layers were separated and the aqueous layer was extracted again. hdutettiin. Addition of 1,1,2-trichloroethane to the residue gave 220 mg (48%) of solid product. The product was identified by NMR, IR and UV analysis and bioautogram. NMR (DMSO-d 6) δ 2.35 (s, 3, oxadiazoline CH 3), 3.7 (q, 2, J = 18 Hz, 2-CH 2), 3.8 (s, 2, -CH 2 CONH-), 4.4 (q, 2, J = 14 Hz, 3-CH 2 S-), 5.1 (d, 1, J = 5 Hz, C 6 -H), 5.6 (q, 1, J = 4 Hz, C (-H), 6.9, 7.3 (t, d, 3, thiophene H) and 9.1 (d, 1, J = 8 Hz, -CH 2 CONH-).

Example 33 Preparation of 3 - (((1H-1,3,4-triazol-5-yl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid 1 In 1,2-trichloroethane 7- (2- (2-thienyl) acetamido) cephalosporanic acid (396 mg, 1 mmol) was suspended in 30 ml of 1,1,2-trichloroethane and then 1H-1.3 was added. 4-triazole-5-thiol (100 mg, 1 mmol). The reaction mixture was heated to 105 ° C. Within 30 minutes, the product began to precipitate. Heating was continued for 6 hours at 100 ° C, after which the reaction mixture was cooled to room temperature. Filtration and rinsing with 1,1,2-trichloroethane gave 38 mg (87%) of product. The product was identified by IR, NMR and UV analysis and bioautogram. The bioautogram also showed the presence of the cephalosporanic acid starting material and the concentration was 6.8% by high performance liquid chromatography. NMR (DMSO-d 6) δ 3.7 (s, 2, 2-CH 2), 3.8 (3.2, -CH 2 CONH-), 4.2 (q, 2, J = 5 Hz, 3-CH 2 S -), 5.1 (d, 1, J = 5 Hz, Cg-H), 5.7 (q, 1, J = 4 Hz, Cy-H), 7.0, 7.4 (t, d , 3, thiophene H), 8.45 (s, 3, triazole> NH) and 9.13 (d, 1, J = 8 Hz, -CH 2 CONH-).

Example 34 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2-phenylacetamido) -3-cephem-4-carboxylic acid in chloroform 7- (2-phenylacetamido) cephalosporanic acid (195 mg, 0.5 mmol) was suspended in 75 mL of chloroform and then 1-methyl-1H-tetrazole-5-thiol (75 mg, 0.65 mmol) was added. The reaction mixture was heated in an oil bath at 80-85 ° C for 3 hours, and then 60 ml was distilled off. TLC analysis showed very little product and therefore 20 ml of 1,2-dichloroethane was added and heating was continued overnight. In the morning, TLC analysis showed little cephalosporanic acid starting material. The total reaction time became 26 hours, after which the reaction mixture was cooled to room temperature. When the reaction mixture was treated in substantially the same manner as in Example 32, 80 mg (35%) of purified product was obtained. The product was identified by NMR, IR and UV analysis and bioautogram. NMR (DMSQ-d 6) of 3.6 (s, 2, 2-O 2), 3.7 (s, 2, -C 2 CONH-), 4.0 (s, 3, tetrazoline CH 2), δ , 3 (s, 2,3-CH 2 S-), 5.1 (d, 1, J = 5 Hz, C 6 -H), 5.7 (q, 1, J = 4 Hz, C 2 -H), 7.3 (s, 5.0) and 9.13 (s, 1, J = 8 Hz, -C ^ CONH-).

Example 35 Preparation of 3 - (((1-methyl-β-tetrazol-5-yl) thio) methyl) -7-acetamido-3-cephem-4-carboxylic acid in 1,2-dichloroethane 7-acetamidocephalosporanic acid (314 mg, 1 mmol) ) and 1-methyl-1H-tetrazole-5-thiol (98 mg, 0.65 mmol) were stirred in 70 mL of 1,2-dichloroethane, 50 mL was distilled off, and then the reaction mixture was refluxed for 24 hours. When the reaction mixture was treated practically as in Example 32, 120 mg (32%) of product were obtained. The product was identified by NMR, IR and UV analysis and bioautogram. NMR (DMSO-d 6) -fl.97 (s, 3, CH 2 CONH-), 3.7 (s, 2,2-CH 2), 4.0 (s, 3, tetrazoline CH 3), 4.35 ( s, 2,3-CH 2 S-), 5.1 (d, 1, J = 5 Hz, C 6 H 5), 5.7 (q, 1, J = 4 Hz, C 8 H) and 8.8 (d , 1, J = 8 Hz, -CH 2 CONH).

Example 36 Preparation of 3 - (((3-methyl-1,2,4-thiadiazol-5-yl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid 1 In 2-dichloroethane 7- (2- (2-thienyl) acetamido) cephalosporanic acid (200 mg, 0.5 mmol) and 3-methyl-1,2,4-thiadiazole-5-thiol (85 mg, 0.65 mmol) ) was mixed with 50 ml of 1,2-dichloroethane and the mixture was heated in an oil bath to 95 ° C. The bath temperature was maintained at 90 ° C for 19 hours. The reaction mixture was then removed from the bath, allowed to cool and kept in the refrigerator for 24 hours. Three volumes of ethyl acetate were added and washed twice with 50 ml of saturated sodium bicarbonate solution. The combined aqueous washes were extracted with ethyl acetate, layered with fresh ethyl acetate and cooled in an ice bath. The pH of the mixture was adjusted to 2.0. The layers were separated and the aqueous layer was further extracted with ethyl acetate. When the combined ethyl acetate layers were washed with brine, dried over magnesium sulfate, filtered and evaporated to give 252 mg of a foam. Crystallization from acetone-diethyl ether gave 153 mg (65%) of product. The product was identified by NMR, UV and IR spectra and a bioautogram. NMR (DMSQ-d 6) δ 3.7 (s, 2, 2-CH 2), 3.8 (s, 2, -CH 2 CONH), 4.5 (q, 2, J = 15 Hz, 3-CH 2 S -), 5.1 (d, 1, J = 5 Hz, C 6 H 3), 5.7 (q, 1, J = 4 Hz, C 6 H), 6.96, 7.38 (t, d, 3, thiophene H) and 9.13 (d, 1, J = 8 Hz, -CH 2 CONH).

Example 37 Preparation of 3 - (((2-pyrimidinyl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid in acetonitrile 7- (2- (2-thienyl) acetamido) cephalosporanic acid (2.0 g, 5 mmol) 2-mercaptopyrimidine (0.60 g, 5.5 mmol) was stirred in 25 mL of dry acetonitrile and the mixture was refluxed overnight (16 h) with stirring. TLC analysis indicated that the reaction was complete. The reaction mixture was cooled to room temperature and the product was filtered off. After washing the precipitate with 50 ml of acetonitrile and drying in vacuo at 50 ° C for 4 hours, 1.85 g (83.0%) of product were obtained as hard crystals, m.p. 217 ° C (dec.). NMR analysis in DMSO-d 6 did not show either starting material. The product was identified by IR, UV and NMR analysis.

NMR (DMSOg-d 6) - δ 3.55 (q, 2, 2-CH 2, J = 18 Hz), 3.74 (s, 2, -CH 2 CONH), 4.28 (q, 2,3-CH 2 S- , J = 13 Hz), 5.00 (d, 1, C 6 H 6, J = 5 Hz), 5.64 (q, 1, C 6 H, J = 5 Hz, J = 9 Hz), 7 .08 (m) and 8.52 (d) (6, thiophene and pyrimidine H) and 9.00 (d, 1, -CH2CONH-, J = 9 Hz).

Example 38 Preparation of 3 - (((2-pyrimidinyl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid in acetic acid

A mixture of 7- (2- (2-thienyl) acetamido] cephalosporanic acid (2.0 g, 5 mmol), 2-mercaptopyrimidine (0.6 g, 5.4 mmol) and sodium acetate (0.41 g, 5 mmol) was added. mmol) in 25 ml of glacial acetic acid, heated for 4 hours at 85 [deg.] C. The product crystallized during the reaction, and when the reaction mixture was cooled to room temperature and the precipitate was collected, washed with acetic acid and dried, 1.58 g (70.5%) of product were obtained. as white crystals, mp 218 ° C (dec.).

The product was identified by IR, UV, NMR and elemental analysis.

The NMR spectra of this product and the product of Example 37 were identical.

69309 29

Example 39 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (2-thienyl) acetamido) cephalosporanic acid in acetic acid

A mixture of 7- (2- (2-thienyl) acetamido) cephalosporanic acid (2.0 g, 5 mmol), 1-methyl-1H-tetrazole-5-thiol (0.81 g, 7 mmol) and sodium acetate (0.41 g, 5 mmol) in 25 mL of glacial acetic acid was heated at 75-77 ° C for 8 h. TLC analysis was performed once an hour, and after 8 hours, TLC analysis indicated that the reaction was complete. The reaction mixture was cooled to 40-45 ° C and the acetic acid was removed in vacuo. To the residue were then added 50 ml of ethyl acetate and 50 ml of water. The pH of the aqueous layer was adjusted to 1.5 with 1N sulfuric acid. The ethyl acetate layer was separated, dried over anhydrous sodium sulfate, and the ethyl acetate was removed on a rotary evaporator. The light oily residue was dissolved in 50 ml of ethanol and a solution of 2 ml of dicyclohexylamine (10.2 mmol) in 10 ml of ethanol was added. The product precipitated almost immediately as the dicyclohexylamine salt, but stirring was continued for another 15 minutes, and then the precipitate was filtered, washed with ethanol and dried to give 1.2 g (37.8%) of product, m.p. 185-186 ° C. The results of IR, NMR and UV analysis were identical to those of the same product previously prepared.

Example 40 Preparation of 3 - (((1-benzyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid in 1,2-dichloroethane 7- (2- (2-thienyl) acetamido) cephalosporanic acid (2.0 g, 5 mmol) and 1-benzyl-1H-tetrazole-5-thiol (1.44 g, 7.5 mmol) were added to 25 mL. 1,2-Dichloroethane and the reaction mixture were heated in an oil bath at 85 ° C overnight with stirring. TLC analysis showed no starting material remaining. When the solvent was removed on a rotary evaporator, a foamy residue remained. To the foam was added 25 ml of methanol and the mixture was heated on a water bath until dissolution occurred. The solvent was evaporated on a rotary evaporator and the product was crystallized. After stirring for 15 minutes, the precipitate was collected by filtration, washed with methanol and dried in vacuo to give 1.6 g (60.6%) of product, m.p. 171 to 171.5 ° C.

30

f ° ^ Ω Q

Example 41 Preparation of N'-3-amidinothiomethyl-7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid in acetonitrile

A mixture of 7- (2- (2-thienyl) acetamido) cephalosporanic acid (3.12 g, 8 mmol) and thiourea (912 mg, 12 mmol) in 15 mL of acetonitrile (dried with aluminosilicate desiccant "4-A

Linde Molecular sieve "), heated for 24 hours at 87 ° C with stirring. Within one hour, a precipitate began to form in the reaction mixture.

When the heating was complete, the precipitate was filtered off from the hot reaction mixture and dried to give 2.5 g (76%) of product. Elemental analysis: Theoretical C 43.68 H 3.91 N 13.58 Calculated C 43.50 H 4.03 N 1.329.

The product was identified by NMR analysis.

Example 42 Preparation of 3-benzoylthiomethyl-7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid in isopropyl acetate 7- (2- (2-thienyl) acetamido) cephalosporanic acid (1.0 g, 2.5 mmol) ) and thiobenzoic acid (510 mg, 3.7 mmol) were suspended in 20 mL of anhydrous isopropyl alcohol, and all reactants were allowed to dissolve when the suspension was refluxed for 31 hours. Evaporation of the solvent in vacuo gave a residue which, by TLC analysis, contained a mixture of product and starting material (60:40%).

Preparative thick layer chromatography on silica plates (acetone-acetic acid 16: 1 as eluent) gave a purified product sample. NMR (DMSO-d 6 / D, O) δ 3.50 ABq, 2H, 2-CH 2, J = 6 Hz, J = 19 Hz), 3.82 (s, 2H, -CH 2 CONH-), 4.20 (d, 2H, 3-CH 2 S-, J = 5 Hz), 5.03 (d, 1H, C 6 H 6, J = 5 Hz), 5.69 (d, 1H, C 8 H, J = 5 Hz) Hz), 6.89-8.0 (m, 8H, aromatic).

Example 43 Preparation of 3 - ((phenylthio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid in 1,2-dichloroethane 7- (2- (2-thienyl) acetamido) cephalosporanic acid (2.0 g, 5 mmol) and benzenethiol (0.75 mL, 7.5 mmol) were added to 25 mL of 1,2-dichloroethane. After refluxing the reaction mixture overnight, TLC analysis showed that the cephalosporanic acid starting material had disappeared. The solvent was evaporated on a rotary evaporator, 25 ml of ethanol was added to the ice and the mixture was heated so that partial dissolution occurred. Complete dissolution occurred when 25 ml of methanol was added and heated. The solution was filtered through cotton, treated with 2.0 g of carbon and filtered through a filter aid. The solvent was removed on a rotary evaporator. NMR analysis of the residue showed that some thiol starting material remained.

The residue was suspended in 25 ml of isopropyl acetate, filtered and kept in the refrigerator overnight. To remove the thiol, the filtrate was diluted with 25 mL of ethyl acetate and washed with 5 5 mL of dilute sodium bicarbonate solution. The ethyl acetate layer was added to 25 ml of water and the pH was adjusted to 1.4 with sulfuric acid. After drying the ethyl acetate layer over magnesium sulfate, filtering and evaporating on a rotary evaporator, 0.92 g (19.4%) of a foamy product was obtained. NMR (DMSO-d 6) δ 3.58 (m, 2, 2-CH 2 S), 3.75 (s, 2, -CH 2 CONH-), 4.12 (q, 2,3-CH 2 S-, J = 13 Hz ), 5.08 (d, 1, Cg-H, J = 5 Hz), 5.56 (q, 1, C2-H, J = 5 Hz, J = 9 Hz), 7.15 (m, 8, thiophene and phenyl H) and 9.10 (d, 1, -CH 2 CONH-, J = 9 Hz).

Example 44 Preparation of 3 - (((5-methyl-1,3,4-thiadiazol-2-yl) thio) methyl) -7- (2-phenylacetamido) -3-cephem-4-carboxylic acid 1,2- dichloro-ethane

A mixture of 7- (2-phenylacetamido) cephalosporanic acid (1.95 g, 5 mmol) and 5-methyl-1,3,4-thiadiazole-2-thiol (0.99 g, 7.5 mmol) in 25 mL in 1,2-dichloroethane, was refluxed for 16 hours. TLC analysis showed that some cephalosporan starting material remained. The product crystallized during the reaction. After cooling the reaction mixture to 0-5 ° C, the precipitate was collected by filtration, washed with cold 1,2-dichloroethane and dried in vacuo to give 1.82 g (78.8%) of product, 171-172 ° C. NMR (DMSO-d 6) δ 2.70 (broad s, 2,2-CH 2), 4.40 (q, 2,3-CH 2 S-, J = 13 Hz), 5.12 (d, 1, C H, J = 5 Hz), 5.72 (q, 1, C7-H, J = 5 Hz, J = 9 Hz), 7.28 (s, 5, phenyl H) and 9.08 (d, 1 , -CH 2 CONH-, J = 9 Hz).

69309 32

Example M-5 Preparation of 3 - ((methylthio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid in methylene chloride

A mixture of 7- (2- (2-thienyl) acetamido) cephalosporanic acid (24 g, 60 mmol), 7.0 mL methanethiol and 600 mL methylene chloride was heated at 84-86 ° C for 18 h: while stirring. The reaction mixture was then cooled to room temperature. A small amount of insoluble matter was filtered off and the filtrate was evaporated. The residue was taken up in ethyl acetate and filtered. About 150 ml of water was added to the filtrate, stirred and the pH was adjusted to 5.5 with 1N sodium hydroxide solution. The aqueous phase was separated and concentrated to about 75 ml, 75 ml of water were added and the pH was adjusted to 3.8 with glacial acetic acid. The mixture from which an amorphous solid began to precipitate was stirred for 3 hours in an ice bath and kept in the refrigerator overnight. Then, 100 ml of ethyl acetate was added to the filtrate, and the pH was adjusted to 2.0 with concentrated hydrochloric acid. The organic layer was separated and the aqueous layer was extracted with one 100 mL portion and one 150 mL portion of ethyl acetate. When the organic phases were combined, dried over magnesium sulphate and the solvent evaporated to give an amber foam. It was dissolved in 50 ml of ethyl acetate, seeded with the desired product and kept in the refrigerator overnight. The formed crystals were collected by filtration, washed with cold isopropyl acetate and dried in vacuo at 50 ° C to give 3.2 g (14%) of product, NMR Γ2.00 (s, 3,3-CH 2 SCH 3), 3.74 (m, 3-CH 2 S-, 2-CH 2 and -CH 2 CONH-), 5.14 (d, 1, Cg-H, J = 5 Hz), 6.64 (q, 1, C7-H, J = 5 Hz, J = 9 Hz), 7.15 (m, 3, thiophene H) and 9.12 (d, 1, -CH 2 CONH-, J = 9 Hz).

Example 46 Preparation of 7- (2-formyloxy-2-phenylacetamido) -3- (1-methyl-1H-tetrazol-5-yl) thio) methyl) -3-cephem-4-carboxylic acid sodium salt in benzene

A mixture of 7- (2-formyloxy-2-phenylacetamido) -cephalosporanic acid (2.17 g, 4.65 mmol) and 1-methyl-1H-tetrazole-5-thiol (0.87 g, 7, 5 mmol) in 25 ml of benzene, refluxed for 12 hours at about 80 ° C. During the reaction, a layer of soft mass formed in the glass vessel. Upon cooling, the mass cured to 69309 as a glassy solid. TLC analysis showed that the pulp contained only a small amount of product and most of it was the product formed with some deformylated product. The reaction mixture was evaporated with added acetone to a foam which was dissolved in 35 ml of dry acetone and treated with 10 ml of a solution of 1.25 g of sodium 2-ethylhexanoate (7.5 moles) in acetone. The product crystallized as the sodium salt.

After 1 hour, the precipitate was filtered, washed with 20 ml of acetone and dried to give 1.38 g (58%) of product. The product was identified by TLC and NMR analysis. (NMR (D 2 O) i (3.48 (q, 2, 2-CH 2, J = 18 Hz), 4.00 (s, 3, tetrazolyl-CH 3), 4.10 (m, 2,3-CH 2 S- ), 5.05 (d, 1, C8-H, J = 5 Hz), 5.70 (d, 1, C2-H, J = 5 Hz), 6.24 (s, 1, -CHCONH- ), 7.50 (m, 5, phenyl H) and 8.33 (s, 1, -O 2 H).

Example 47 Preparation of 7- (2-formyloxy-2-phenylacetamido) -3 - (((1-methyl-1H-tetrazol-1-yl) thio) methyl) -3-cephem-4-carboxylic acid in carbon tetrachloride.

A mixture of 7- (2-formyloxy-2-phenylacetamido) cephalosporanic acid (2.17 g, 4.65 mmol) and 1-methyl-1H-tetrazole-5-thiol (0.87 g, 7.5 mmol). mmol) in 25 mL of carbon tetrachloride was refluxed (about 77 ° C) for 12 hours. The reaction mixture was cooled to room temperature and the remaining solution was decanted from the semi-solid. To the solid was added 25 mL of 1,2-dichloroethane while heating. The product crystallized. After cooling to room temperature and stirring for about 1 hour, the precipitate was collected by filtration, washed with 10 ml of 1,2-dichloroethane and dried in vacuo overnight at 45 ° C to give 1.54 g (65%) of product as white crystals. The product was identified by NMR and TLC analysis, and the latter indicated that some de-formylated product was present. The NMR spectra of this product and the product of Example 16 were identical.

Example 48 Preparation of 3 - (((- 3-methyl-1,2,4-oxadiazol-5-yl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid 1,2-dichloro-ethane.

7- (2- (2-thienyl) acetamido) -3- (carbamoyloxymethyl) -3-cephem-4-carboxylic acid (100 mg, 0.25 mmol) was suspended in 25 ml of 1,2-dichloroethane and the mixture was stirred. When 3-methyl-1,2,4-oxadiazole-5-thiol (35 mg, 0.30 mmol) was added, the reaction mixture was passed through an oil bath at 110 ° C to distill 5 mL with any a small amount of water, the temperature of the oil bath was lowered to 90-95 ° C and the reaction mixture was kept at this temperature for 19 hours and then cooled to room temperature. The insoluble matter was filtered off and washed with 1,2-dichloroethane and diethyl ether. TLC analysis showed that most was unreacted starting material and there was only a small amount of product.

The filtrate was evaporated and the oily residue partitioned between sodium carbonate solution and ethyl acetate. The aqueous solution was layered with fresh ethyl acetate, cooled in an ice bath and the pH was adjusted to 2.5 with 20% hydrochloric acid. The organic layer was separated. The aqueous layer was extracted with fresh ethyl acetate, the ethyl acetate solutions were combined, washed with brine, dried over magnesium sulfate, filtered and evaporated to an oily residue. Crystallization from a mixture of hexane and diethyl ether (1: 1) gave 13 mg (11.5%) of product. The product was identified by NMR and IR analysis, and it was found that it was the same product as in Example 32.

Example 49 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (tert-butoxycarbonyl) -2-phenylacetamido) -3-cephem-4-carboxylic acid in nitromethane .

A mixture of 7- (2- (tert-butoxycarbonyl) -2-phenylacetamido) cephalosporanic acid (490 mg, 1 mmol) and 1-methyl-1H-tetrazole-5-thiol (145 mg, 1.25 mmol) ) In 15 ml of dry nitro-methane, heated for 8 hours at 85-90 ° C under a nitrogen atmosphere. Excess thiol reagent and decarboxylated product were found by TLC analysis, but no cephalosporanic acid starting material. The nitromethane was evaporated off. The orange foamy residue was dissolved in 10 ml of saturated sodium bicarbonate solution, 20 ml of water was added and the mixture was washed successively with ethyl acetate until the washings were clear. The ethyl acetate washings were combined, 20 ml of water were added, the mixture was cooled to 0 ° C and the pH was adjusted to 2.2 with 20% hydrochloric acid. The layers were separated and the aqueous layer was washed with ethyl acetate. When the ethyl acetate solutions were combined, washed with saturated sodium chloride, dried over magnesium sulfate, filtered and evaporated to give 455 mg (83%) of the product as a foamy residue. TLC analysis showed excellent product, some thiol and some decarboxylated product.

359309 455 mg (0.833 mmol) of product were dissolved in 7 ml of ethyl acetate, and when lithium acetate (0.833 ml) was added dropwise with stirring, a brown precipitate formed which was the lithium salt. The precipitate was collected by filtration, washed with ethyl acetate and dried overnight in vacuo at room temperature to give 368 mg (80%) of product. The product was identified by TLC, NMR, elemental, IR and UV analysis as well as bioautogram. TLC analysis gave some decarboxylated material. NMR (CDCl 3) δ 1.4 (s, 9, -COOtertCl 2 H 2), 3.6 (3.2, 2-CH 2), 3.85 (s, 3, tetrazoline CH 3), 4.3 ( s, 2,3-CH 2 S-), 4.44 and 4.45 (2s, 1,0CH-), 4.9 (d, 1, J = 6 Hz, C 6 H 8), 5.8 (q, 1, J = 6 Hz, C 2 -H), 7.35 (s, 5.0), 8.2 and 7.8 (2d, 1, J = 9 Hz, -CON-) and 9.3 ( s, 1, -C00H).

Example 50 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (N - ((1,3-dimethylureido) carbonyl) -2-phenylglycylamido) -3-cephem Preparation of -4-carboxylic acid in nitromethane.

7- (N - ((1,3-dimethylureido) carbonyl) -2-phenylglycylamido) cephalosporanic acid (130 mg, 0.25 mmol) was suspended in 5 mL of nitromethane and 1-methyl-1H-tetrazole-5- thiol (43.5 mg, 0.375 mmol). The reaction mixture was stirred under nitrogen for 12 hours at 85 ° C and then at room temperature over the weekend.

The reaction mixture was then filtered and the solid was washed with a small amount of nitromethane and dried in a vacuum oven at 35 ° C to give 83 mg (58%) of product. The product was identified by TLC, NMR, IR, UV and elemental analysis and bioautogram. NMR (DMSO-d 6) δ 2.65 (d, 3, J = 4 Hz, -CONHCH 6), 3.15 (s, 3, CONCH 2 CO 2), 3.6 (s, 2, 2-CH 2), 3.9 (s, 3, tetrazoline CH3), 4.3 (s, 2,3-CH2S-), 5.0 (d, 1, J = 5 Hz, C8-H), 5.5 (d, 1, J = 7 Hz, OCHCO), 5.7 (q, 1, C'-NH), 5.8 (q, 1, -CNHCH'), 7.4 r'3 (s 5 N-O) 9 (3, J, 8 Hz, OCHCONH-) and 10 (d, 1, J = 8 Hz, OCH-CO-).

t

NH

t

Example 51 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid in acetonitrile, tetrabutylammonium iodide added.

A mixture of 7- (2- (2-thienyl) acetamido) cephalosporanic acid (2.0 g, 5 mmol), 1-methyl-1H-tetrazole-5-thiol (1.2 g, 10 mmol) and tetrabutylammonium iodide ( 0.2 g) in 25 ml of dry acetonitrile, refluxed for 8 hours. The reaction mixture was then cooled to room temperature and the solvent was removed on a rotary evaporator. The residue was treated with a hot mixture of 25 ml of 36 6 9 309 isopropyl acetate and 5 ml of acetonitrile, filtered and allowed to slowly cool. After the precipitate was collected by filtration, washed with isopropyl acetate and dried, 1.30 g (57.5%) of product were obtained as light cream crystals. The NMR spectra of this product and the product of Example 5 were identical.

Example 53 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (2-thienyl] acetamido) -3-cephem-4-carboxylic acid 1,2-di- in chloroethane, tetrabutylammonium iodide added

The reaction of Example 52 was repeated, but 1,2-dichloroethane was used as the solvent. Addition of 2 mL of dicyclohexylamine gave 1.55 g (48.9%) of the product as the dicyclohexylamine salt. The NMR spectra of this product and the product of Example 1 were identical.

Example 54 Preparation of 3 - ((phenylthio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephenyl-4-carboxylic acid in 1,2-dichloroethane, 1-methyl-5- (methylthio) -1H -tetrazole added

A mixture of 7- (2- (2-thienyl) acetamido) cephalosporanic acid (2.0 g, 5 mmol), benzenethiol (0.75 mL, 75 mmol) and 0.65 g of 1-methyl-5 - (methylthio) -1H-tetrazole in 25 ml of 1,2-dichloroethane, was refluxed. TLC analysis indicated that the reaction was complete in 14 hours. The solvent was removed on a rotary evaporator and the residue was extracted several times with diethyl ether. Removal of the solvent gave 1.66 g (74%) of a brown solid. The IR and NMR spectra of this product and the same product prepared in the previous examples were identical.

Example 55 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (2-thienylacetamido) -3-cephem-4-carboxylic acid in isopropanol

A mixture of 7- (2- (2-thienyl) acetamido) cephalosporanic acid (2.0 g, 5 mmol) and 1-methyl-1H-tetrazole-5-thiol (0.87 g, 7.5 mmol) ) In 25 ml of isopropanol was placed in a flask (equipped with a condenser with a drying tube containing anhydrous "Drierite" calcium sulfate). The flask was immersed in an oil bath at 84-85 ° C. The progress of the reaction was monitored by TLC analysis. After 40 hours at 82-83 ° C, only half of the cephalosporanic acid had reacted.

Il 69309 37

Example 55 Preparation of 3 - ((2-oxazolylthio) methyl) -7- (2- (1H-tetrazol-1-yl) acetamido) -3-cephem-4-carboxylic acid

Suspension of 7- (2- (ΙΗ-tetrazol-1-yl) acetamido) -cephalosporanic acid (0.38 g, 1.0 mmol) 2-oxazolithiol (0.11 g, 1.1 mmol) S mL: nitromethane was immersed in an oil bath at 90-91 ° C. The reaction mixture was kept under a nitrogen atmosphere. After 20 minutes all the reactant had dissolved and after 35 minutes the product began to crystallize. After cooling to room temperature after 6 hours, the precipitate was collected by filtration, washed with 7 ml of nitromethane, air dried and then under vacuum for 3 hours at 40 ° C to give 0.36 g (85.7%) of product as hard crystals, m.p. 196 ° C (dec.). NMR analysis showed that the product contained 70% of the desired compound and 30% of the phosphorosporanic acid starting material.

After recrystallization from a mixture of 5 ml of DMSO-dc

D

and 10 ml of water, filtered and the precipitate was washed with 5 ml of aqueous DMSO-dc

O

mixture (2: 1) and dried first in air and then in vacuo at 50 ° C for 6 hours to give 0.26 g of product. NMR analysis showed that the product contained 87% of the desired compound and 13% of a cephalosporanic acid starting material. Further recrystallization from a mixture of 3 ml of DMSO-dg and 6 ml of water with stirring for 1 hour gave 0.21 g of product. NMR analysis (DMSO-d 6) showed that the amount of cephalosporanic acid starting material had decreased to 5% and the values of the desired compound were as follows: <i3.7S (s, 2, 2-CH 2), 4.34 (q, 2, 3 -CH2S-, J = 14 Hz), 5.16 (d, 1, C8-H, J = 5 Hz), 5.76 (q, 1, C2-H, J = 5 Hz, J = 9 Hz) ), 5.44 (s, 2, -CH 2 CONH-), 7.28 (s, 1, oxazoline C 6 H), 8.14 (s, 1, oxazoline C 6 H), 9.37 (s, 1, tetrazole H) and 9.53 (d, 1, -CONH-, J = 9 Hz).

Example 56 Preparation of 3 - ((2-oxazolylthio) methyl) -7- (2-formyloxy-2-phenylacetamido) -3-cephem-4-carboxylic acid in 1,2-dichloroethane

A solution of 7- (2-formyloxy-2-phenylacetamido) -cephalosporanic acid methylene chloride solvate (0.52 g, 1 mmol) and 2-oxazolithiol (0.12 g, 1.1 mmol) in 20 mL of 1,2- dichloroethane, refluxed for 15 hours and then cooled to room temperature. TLC analysis indicated that the reaction was complete. The reaction mixture was concentrated on a rotary evaporator to ___ - τ 38 69309 to IQ ml, and when allowed to stand, jelly-like crystals formed. When the crystals were collected by filtration, washed with 1,2-dichloroethane and dried, 0.12 g of a gray solid was obtained.

When the solvent was removed from the filtrate, 0.45 g of a pale yellow foam was obtained. When the foam was mixed with 25 ml of diethyl ether, filtered, washed with diethyl ether and dried to give 0.21 g of a pale yellow powder. The NMR values of the product were 3.56 (m, 2, 2-CH 2), 4.24 (q, 2,3-CH 2 S-, J = 13 Hz), 5.00 (d, 1, C 6 -H, J = 5 Hz), 5.70 (q, 1, C? -H, J = 5 Hz), 6.14 (s, 1, ^ ^ CH-), 7.25 (s, 1, oxazoline C H), 7.45 (m, 5, phenyl H), 8.12 (s, 1, oxazoline C 1 H), 8.35 (s, 1, -CHO) and 9.40 (d, 1, -C0H-, J = 9 Hz).

Example 57 Preparation of 3 - (((4-phenyl-2-thiazolyl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid in acetonitrile

A mixture of 7- (2- (2-thienyl) acetamido) cephalosporanic acid (2.0 g, 5 mmol) and 4-phenyl-2-thiazolithiol (1.44 g, 7.5 mmol) in 35 mL: acetonitrile, refluxed for 16 hours to protect the mixture from moisture in the air using a drying tube containing anhydrous calcium sulfate (“Drierite”) TLC analysis indicated that the reaction was complete.

After the reaction mixture was cooled to room temperature, stirred for 2 hours, the crystals were filtered off, washed with acetonitrile and dried to give 2.25 g (85.6%) of product, m.p. 180 ° C (dec.). The product was identified by NMR analysis.

Example 58 3 - (((5-methyl-1,3,4-thiadiazol-2-yl) thio) methyl) -7- (2- (1H-tetrazol-1-yl) acetamido) -3-cephem-4 Preparation of carboxylic acid in nitromethane 7- (2- (ΙΗ-tetrazol-1-yl) acetamido) cephalosporanic acid (1.92 g, 5 mmol) and 5-methyl-1,3,4-thiadiazole-2-thiol (0.79 g, 6 mmol) was added to 25 ml of alumina-treated nitromethane and the reaction mixture was heated in an oil bath to 95 ° C and stirred for a total of 4 hours at this temperature. TLC analysis showed the reaction to take place except for a small amount of residual cephalosporanic acid starting material (less than 2%). After cooling to room temperature, washing with nitromethane and drying in vacuo, 2.11 g (92.5%) of product were obtained, m.p. 183.5 ° C (dec.). The product was identified by NMR analysis.

Example 59 3 - (((5-methyl-1,3,4-thiadiazol-2-yl) thio) methyl) -7- (2- (1H-tetrazol-1-yl) acetamido) -3-cephem-4 -carboxylic acid preparation. in propionitrile 7- (2- (1H-tetrazol-1-yl) acetamido) cephalosporanic acid (1.92 g, 5 mmol) and 5-methyl-1,3,4-thiadiazole-2-thiol (0.99 g, 7 mmol). .5 mmol) was added to 25 ml of propionitrile (treated with neutral alumina) and the mixture was heated to reflux (97 ° C). The reaction mixture was refluxed for 9 hours with stirring. TLC analysis showed only a small amount of cephalosporanic acid starting material. After cooling to room temperature, the reaction mixture was filtered, washed with propionitrile and dried in vacuo to give 2.04 g (89.5%) of product, m.p. 186.5 ° C (dec.).

The product was identified by NMR analysis.

Example 60 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2-formyloxy-2-phenylacetamido) -3-cephem-4-carboxylic acid 1,2- in dichloroethane 7-aminocephalosporanic acid (7-ACA) (13.62 g, 0.05 mol) was suspended in 100 ml of 1,2-dichloroethane and then trimethylsilylacetamide (26.25 g, 0.200 mol) was added. The reaction mixture was heated to 40 ° C to form a cloudy solution which was then cooled to 20 ° C. A solution of 2-formyloxy-2-phenylacetyl chloride (10.92 g, 0.055 mol) in 25 ml of 1,2-dichloroethane was added dropwise over 20 minutes while allowing the temperature to rise to 30 ° C. The reaction mixture was stirred for 2 hours, 100 ml of 1,2-dichloroethane was added and the reaction mixture was then washed three times with 100 ml of water. The aqueous solutions were combined and washed twice with 50 ml of 1,2-dichloroethane and these in turn were washed with 40 ml of water. The 1,2-dichloroethane layers were combined, stirred for 20 minutes with activated carbon (2.0 g) _____ - ίγ; ___ 40 69309 ("Darco G-60") and filtered through diatomaceous earth ("Hyflo"). The solution totaled 375 ml and contained the desired 7- (2-formyloxy-2-phenylacetamido) cephalosporanic acid. This solution was evaporated at 30 ° C to 336 g, containing an estimated 250 ml of 1,2-dichloroethane and 22 g of 7- (2-formyloxy-2-phenylacetamido) cephalosporanic acid. A mixture of 1-methyl-1H-tetrazole-5-thiol (6.39 g, 55 mmol) in 250 mL of 1,2-dichloroethane was added and the reaction mixture was heated to reflux. The water (apparently left over from previous washes) was azeotroped into a condenser equipped with a trap where water accumulated. After 12 hours at reflux, TLC analysis showed an almost normal reaction.

When the reaction mixture was cooled to room temperature and seeded, 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2-formyloxy) -2-phenylacetamido) -3-cephem-4-carboxylic acid precipitated. .

After 2 hours, the precipitate was filtered and washed with 63 ml of 1,2-dichloroethane to give 13.90 g (56.7% based on 7-ACA). The TLC of the product was pure. The NMR spectra of this product and the product of Example 16 were identical.

Example 61 Preparation of 3 - (((1-methyl-β-tetrazol-5-yl) thio) methyl) -7- (2-phenyloxy-2-phenylacetamido) -3-cephem-4-carboxylic acid in 1,2-dichloroethane 7 - (2-formyloxy-2-phenylacetamido) cephalosporanic acid (2.33 g, 5 mmol) and 1-methyl-1H-tetrazole-5-thiol (0.64 g, 5.5 mmol) were added to 25 ml 1, The 2-dichloroethane and mixture was refluxed for 12 hours. The reaction mixture was cooled to room temperature and then reheated to reflux, and 10 ml of solvent was distilled off. Then 10 ml of carbon tetrachloride was added dropwise near reflux (77 ° C). The resulting mixture was allowed to cool to room temperature while stirring was continued for 1 hour. After the precipitate was collected, the mixture was washed with 14 ml of a 40% solution of carbon tetrachloride in 1,2-dichloroethane and dried at 50 ° C under vacuum to give 2.12 g (86.5%) of a pale solid. The NMR spectra of this product and the product of Example 16 were identical.

69309 41

Example 62 Preparation of 3 - (((5-methyl-1,3,4-thiadiazol-2-yl) thio) methyl) -7- (3-chloropropionamido) -3-cephem-4-carboxylic acid in nitromethane.

A suspension of 7- (3-chloropropionamido) cephalosporanic acid (1.0 g, 2.5 mmol) and 5-methyl-1,3,4-thiadiazole-2-thiol (0.4 g, 3 mmol) In 20 ml of nitromethane, stirred in an oil bath at 95-96 ° C. TLC analysis showed that the reaction was complete in 3 hours. When the reaction mixture was cooled to room temperature, filtered and evaporated in vacuo to give a light red oil which crystallized on standing at room temperature for 2 hours.

When the crystals were stirred in ethyl acetate (15 ml), filtered, washed with ethyl acetate and dried, 0.64 g (55.2%) of product was obtained. The product was identified by IR, UV, NMR and microanalysis and titration. Analysis: Calculated for C 41 H 19 ClN 3 O 2: C 41.51 H 4.14 N 12.10 S 20.78 Cl 7.66 Found C 41.70 H 4.23 N 11.84 S 20.51 Cl 7.88

Examples 63-68 Replacement of 7-methoxy-7- (2- (1H-tetrazol-1-yl) acetamido) cephalosporanic acid with various thiols in nitromethane.

7-Methoxy-7- (2- (1H-tetrazol-1-yl) acetamido) cephalosporanic acid was reacted with various thiols in nitromethane.

The preparation was essentially the same as in the previous example, but the product was purified from the unreacted starting material by high pressure liquid chromatography. The products and identification data were as follows: 42 69309 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7-methoxy-7- (2-1H-tetrazol-1-yl) acetamido ) -3-cephem-4-carboxylic acid, UVmax ethanol (273 ml) (£ 9,082).

3 - (((5-methyl-1,3,4-thiadiazol-2-yl) thio) methyl) -7-methoxy-7- (2- (1H-tetrazol-1-yl) acetamido) -3-cephem -4-carboxylic acid, (ethanol) 273 m 2 (1 12.785).

Analysis: c 15 H 16 N 8 O 5 S 3: C 37.18 H 3.33 N 23.13 calculated C 36.99 H 3.31 N 22.86 found.

3 - (((5-methyl-1,3,4-oxadiazol-2-yl) thio) methyl) -7-methoxy-7- (2- (1H-tetrazol-1-yl) -3 -Cephem-4-carboxylic acid UVmax (ethanol) 233 m / U (δ 8,910)

Analysis: c 15 H 16 N 8 O 6 S 2 calculated C 37.46 H 3.44 N 23.92 found C 38.58 H 3.69 N 23.91 3 - (((1- (carboxymethyl) -1H-tetrazol-5-yl) thio) methyl) -7-methoxy-7- (2- (ΙΗ-tetrazol-1-yl) acetamido) -3-cephem-4-carboxylic acid UVmakg (ethanol) 269 m 2 (E, 7.679).

Analysis: cp5H16NiqO2S2 calculated for C 35.16 H 3.15 N 27.33 found C 35.42 H 3.38 N 27.39.

3 - (((4,5-dihydro-6-hydroxy-4-methyl-5-oxo-1,2,4-triazin-3-yl) thio) methyl) -7-methoxy-7- (2- ( 1H-tetrazol-1-yl) acetamido) -3-cephem-4-carboxylic acid, UVmakg (ethanol) 274 (C 11.967). Analysis: c16H17NgO7S2 Calculated C 37.57 H 3.35 N 24.65 Found C 36.96 H 3.80 N 27.63 3 - (((1,3,4-triazol-5-yl) thio) methyl) -7-methoxy-7- (2- (1H-tetrazol-1-yl) acetamido) -3-cephem-4-carboxylic acid Analysis: c1i + H-L5NgO5S2 calculated C 37.08 H 3.33 N 27.80 found C 36.98 H 3.43 N 27.79.

Example 69 3 - (((1-Methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (4-ethyl-2,3-dioxo-1-piperazinylcarbonylamino) -2-phenylacetamido ) Preparation of -3-cephem-4-carboxylic acid in nitromethane 7- (2- (4-ethyl-2,3-dioxopiperazinylcarbonylamino) -2-phenylacetamido) cephalosporanic acid hydrate (0.3 g, 0.5 mmol) and 1-methyl-1H -tetrazole-5-thiol (0.0725 g, 0.625 mmol) was added to 6 mL of nitromethane dried over alumina and the mixture was heated at 85 ° C under nitrogen for 12 hours. The nitromethane was then evaporated off and the brown foamy residue was dissolved in sodium bicarbonate solution and washed twice with ethyl acetate. Fresh ethyl acetate was added, the mixture was cooled and the pH was adjusted to 2.3. The layers were separated and the aqueous layer was washed with ethyl acetate. When the ethyl acetate layers were combined, washed with saturated sodium chloride solution, dried over anhydrous magnesium sulfate, filtered and evaporated to give 130 mg (40%) of the product as a pale yellow powder. NMR δ 1.2 (t, 3, -NCH 2 CH 3, J = 7 Hz), 3.65 (m, 6, N-CH 2 CH 3 and piperazinyl H), 4.0 (s, 3, tetrazoline -CH 2), 4, 4 (s, 2,3-CH 2 S-), 5.1 (d, 1, C 6 H 6, J = 6 Hz), 5.8 (d, 1, C 8 H, J = 8 Hz), 6 , Q (d, 1, r -;% (f y-CH-, J = 6 Hz), 7.4

OF

8m, 5, phenyl-H), 8.4 (d, 1, -CHCONH-, J = 8 Hz) and 10.0 (d, 1, -CHCONH-, J = 6 Hz).

NH

Example 70 3 - (((1-carboxymethyl) -1H-tetrazol-5-yl) thio) methyl) -7- (2- (4-ethyl-2,3-dioxo-1-piperazinylcarbonylamino) -2 Preparation of (phenylacetamido) -3-cephem-4-carboxylic acid in 1,2-dichloroethane 7- (2- (4-ethyl-2,3-dioxopiperazinylcarbonylamino) -2-phenylacetamido) cephalosporanic acid hydrate (0.3 g, 0.5 mmol) was dissolved in 45 ml of 1,2-dichloroethane dried over alumina and the solution was azeotroped with water at 95 ° C. After collecting 40 mL of solvent, 10 mL of nitromethane and 1- (carboxymethyl) -1H-tetrazole-5-thiol (0.160 g, 1 mmol) were added and the reaction mixture was maintained at 90 ° C under nitrogen for 12 hours.

After the reaction mixture was filtered and the solvent was evaporated, a gum was obtained as a residue, to which were added ethyl acetate and saturated sodium bicarbonate solution. The mixture was washed twice with ethyl acetate, then fresh ethyl acetate was added, the mixture was cooled to 0 ° C and the pH was adjusted to 2.3 with 20% hydrochloric acid. The layers were separated and the aqueous layer was washed with ethyl acetate. When the ethyl acetate layers were combined, washed with saturated sodium chloride solution, dried over anhydrous 69309 44 magnesium sulfate, filtered, evaporated and dried at room temperature to give 123 mg of product. NMR δ 1.2 (t, 3, -NCt 2 CH 2, J = 7 Hz), 3.6 (m, 4, N-CH 2 CH 3 and piperazinyl 5-H), 4.1 (m, 2, piperazinyl 4- H), 4.4 (s, 2,3-CH 2 S-), 5.1 (d, 1,

C 6 H 8, J = 5 Hz), 5.3 (s, 2, 1-CH 2 CO 2 tetrazole), 5.75 (d, 1, C 2 H, J = 6 Hz), 5.9 (d, 1 , - CH-), 7.4 (m, 5, t of phenyl H) and 9.9 (d, 1, \ / _CH "> J = 7 Hz> · \ = y nh!

Example 71 3 - (((4,5-dihydro-4-methyl-6-hydroxy-5-oxo-1,2,4-triazin-3-yl) thio) methyl) -7- (2- (4- Preparation of ethyl 2,3-dioxo-1-piperazinylcarbonylamino) -2-phenylacetamido) -3-cephem-4-carboxylic acid in nitromethane 7- (2- (4-ethyl-2,3-dioxo-1-piperazinylcarbonylamino) - 2-phenylacetamido) cephalosporanic acid hydrate (0.3 g, 0.5 mmol) and 4,5-dihydro-4-methyl-6-hydroxy-5-oxo-1,2,4-triazine-3-thiol (0.111 g, 0.625 mmol) was dissolved in 10 ml of nitromethane under a nitrogen atmosphere and at room temperature and the reaction mixture was heated at 85 ° C for about 12 hours. A brown rubbery precipitate formed which was discarded. When the solvent was slowly evaporated off, a yellow precipitate formed. When the residue was cooled, filtered and washed with diethyl ether, two batches of off-white solid were obtained which were shown by TLC analysis to be the same compound. Total yield 80 mg (24%). NMR ε 1.1 (t, 3, -NCH 2 CH 3, J = 6 Hz), 3.3 (s, 3, triazine CH 3), 3.65 (m, 4, N-CH 2 CH 3 and piperazinyl 5-H) , 4.0 (m, 2, piperazinyl 6-H), 4.6 (s, 2,3-CH 2 S-), 5.1 (d, 1, J = 5 Hz), 5.75 (m, 2,

O-CH- and C, -H), 7.1 (5 -N

(m, 5, phenyl H), 9.5 (d, 1, -CHCONH-, J = 8 Hz) and 10.0 (d, 1, (f y-CH-, J = Hz)).

\ = f -NH

69309 45

Examples 72-79 3 - (((5-methyl-1,3,4-thiadiazol-2-yl) thio) methyl) -7- (2- (1H-tetrazol-1-yl) acetamido) -3-cephem Preparation of -4-carboxylic acid in various solvents.

7- (2- (1H-Tetrazol-1-yl) acetamido) cephalosporanic acid was reacted with 5-methyl-1,3,4-thiadiazole-2-thiol in various solvents. The required reaction details are summarized in the following table (otherwise the method is similar in type to the above examples).

69309 46

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69309 47

Example 80

Preparation of lithium 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2-hydroxy-2-phenylacetamido) -3-cephem-4-carboxylate in glacial acetic acid

A mixture of 7- (2-hydroxy-2-phenylacetamido) cephalosporanic acid (0.48 g, 1 mmol) and 1-methyl-1H-tetrazole-5-thiol (0.5 g, 4.3 mmol) In 15 ml of glacial acetic acid, was allowed to react for 8 hours at 84-86 ° C. The reaction mixture was cooled to room temperature and iodine (0.4 g, 1.6 mmol) was added to convert the unreacted thiol to a disulfide. The mixture was stirred for 20 minutes at room temperature, then poured into a mixture of 100 ml of ethyl acetate and 50 ml of water, and the excess iodine was removed by adding sodium sulfite. The layers were separated and the ethyl acetate layer was washed twice with 100 ml of water and once with 50 ml of 20% sodium chloride solution and dried over anhydrous sodium sulfate. The ethyl acetate was removed on a rotary evaporator and the residue was dissolved in 10 ml of methanol. Lithium acetate dihydrate (0.21 g, 2 mmol) was added and the mixture was stirred for 20 minutes at room temperature. After the formed crystals were collected by filtration, washed with 5 ml of methanol and dried, 0.34 g (69.4%) of the product were obtained as white crystals. NMR δ 3.50 (ABq, J 1 17 Hz), 3.92 (s, tetrazoline -CH 2), 4.20 (s, 3-CH 2 S-), 5.04 (d, C 6 H 8, J = 5 Hz), 5.24 (s, OCHClNH-), 5.64 (d, C 1 H -H, J = 5 Hz) and 7.44 (s, O-H). The NMR spectra of this product and the sample obtained by the reaction in aqueous solution were identical.

Example 81 3 - (((1-methyl-1H-tetrazol-5-yl) thienyl) methyl) -7- (2- (o- (tert-butoxycarbonylaminomethyl) phenyl) acetamido) -3-cephem-4-carboxylic acid manufacture in nitromethane

When a mixture of 7- (2- (o- (tert-butoxycarbonylaminomethyl) phenyl) acetamido) cephalosporanic acid (0.2 g, 0.386 mmol) and 1-methyl-1H-tetrazole-5-thiol (0.047 g , 0.405 mmol) in 5 mL of nitromethane, was allowed to react for 12 hours under nitrogen at 85 ° C to give 3 - (((1-methyl-1H-tetrazol-5-yl) thiomethyl-7- (2- (o- ( tert-butoxycarbonylaminomethyl) phenyl) acetamido) -3-cephem-4-carboxylic acid NMR δ 1.45 (s, 9, -CiCH 2) δ, 3.65 48 69309 (s, 4, 2-CH 2 and -CH 2 CONH -), 3.85 (s, 3, tetrazoline -CH 3), H, 3 (m, 4, 3-CH 2 S- and -CH 2 NHCl-tert-C 6 H 9), 5.0 (d, 2, C 6 - H, J = 4 Hz), 5.7 Cq, 1, Cy-H), 7.25 (s, 5, phenyl H and -CON-) and 8.9 (s, 1, -C00H).

Example 82 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2-ureido-2-phenylacetamido) -3-cephem-4-carboxylic acid in nitromethane

A mixture of 7- (2-ureido-2-phenylacetamido) cephalosporanic acid (Q, 224 g, 0.5 mmol) and 1-methyl-1H-tetrazole-5-thiol (Q, Q61 g, 0.525 mmol) In 8 ml of nitromethane, reacted for 12 hours at 85 ° C to give 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2-ureido-2-phenylacetamido ) -3-cephem-4-carboxylic acid. NMR cf 3.5 (d, 2, 2-CH 2, J = 3 Hz), 3.83 (s, 3, tetrazoline -CH 2), 4.17 (d, 2,3-CH 2 S-, J = 3 Hz) , 4.9 (d, 1.1Cg-H, J = 5 Hz), 5.4 (d, 1,1-CH 2 CONH-, J = 8 Hz), 5.6.

(m, 3, Cy-H and -NH 2), 6.7 (d, 1.0CH-, J = 8 Hz), 7.25 (m, 5,

NH

phenyl H) and 9.2 (d, 1, -CH 2 CONH-, J = 8 Hz).

Example 8 3 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2-cyanoacetamido) -3-cephem-4-carboxylic acid in nitromethane

When a mixture of 7- (2-cyanoacetamido) cephalosporanic acid (0.339 g, 1 mmol) and 1-methyl-1H-tetrazole-5-thiol (0.122 g, 1.05 mmol) in 10 mL of nitromethane , was allowed to react for 12 hours under nitrogen at 85 ° C to give 3 - (((1-methyl---tetrazol-5-yl) thio) methyl) -7- (2-cyanoacetamido) -3-cephem-4. -karboks.yylihappoa. NMR δ 3.5 (s, 2, 2-CH 2), 3.6 δ (s, 2, CNCH 2 CONH-), 3.95 (s, 3, tetrazoline -CH 3), 5.35 (s, 2, 3-CH 2 S-), 5.1 (d, 1,

C8-H, J = 6 Hz), 5.75 (q, 1, Cy-H, J = 4 Hz), 7.8 (s, 1, -C00H) and 8.7 (d, 1, -CH2CONH -, J = 9 Hz).

Example 84 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (m-chlorophenylthio) acetamido) -3-cephem-4-carboxylic acid in nitromethane

When a mixture of 7- (2- (m-chlorophenylthio) acetamido) -cephalosporanic acid (0.390 g, 0.855 mmol) and 1-methyl-1H-tetrazole-5-thiol (0.104 g, 0.9 mmol) in 10 mL: nitromethane was reacted for 12 hours under nitrogen at 85 ° C to give 69309 49 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (m-chloro- phenylthio) acetamido) -3-cephem-4-carboxylic acid. NMR cT3.6 (s, 2, 2-CH2)> 3.75 (s, 2, -CH2ClNH-), 3.9 (s, 3, tetrazoline -CH2), i +, 4 (s, 2, 3- CH2S-), 5.0 (d, 1, C8-H, J = 5 Hz), 5.8 (q, 1, Cy-H, J = 4 Hz), 7.15 (m, 4, phenyl H ), 7.7 (d, 1, -CH 2 CONH-, J = 8 Hz) and 8.5 (s, 1, C00H).

Example rs Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (phenylthio) acetamido) -3-cephem-4-carboxylic acid in nitromethane

When a mixture of 7- (2-phenylthio) acetamido) cephalosporanic acid (Q, 422 g, 1 mmol) and 1-methyl-1H-tetrazole-5-thiol (0.122 g, 1.05 mmol) in 10 mL of nitromethane , was allowed to react for 12 hours under nitrogen at 85 ° C to give 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2-phenylthio) acetamido) -3-cephem-4. - carboxylic acid. NMR δ f3.7 (s, 4, -CH 2 CONH- and 2-CH 2), 3.9. (s, 3, tetrazoline -CH 2), 4.35 (s, 2,3-CH 2 S-), 4.9 (d, 1,

C 6 H 8 J = 6 Hz), 5.8 (q, 1, C 1 H, J = 4 Hz), 7.25 (s, 5, phenyl H), 7.7 (d, 1, - CH 2 CONH-, J = 8 Hz) and 9.4 (s, 1, -C00H).

Example 86 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid in n-butyl formate

When a mixture of 7- (2- (2-thienyl) acetamido) cephalosporanic acid (3.96 g, 10 mmol) and 1-methyl-1H-tetrazole-5-thiol (1.28 g, 11 mmol) moles) in 98 ml of n-butyl formate, reacted for 48 hours at 84-86 ° C to give 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid, m.p. 167.5-168 ° C, yield 79.9%. The NMR spectra of this product and the product of Example 5 were identical.

Example 87 Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (2-thienyl) acetamido) -3-cephem-4-carboxylic acid in toluene

The reaction of Example 91 was repeated using 15 ml of toluene and the reaction time was 136 hours at 84-86 ° C. The product had a melting point of 163-163.5 ° C and a yield of 88.5%. The NMR spectra of this product and the product of Example 5 were identical.

__-----— - ..._____ 50 69309

Example Preparation of 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (tert-butoxycarbonylamino) -3-cephem-4-carboxylic acid in nitromethane

When a mixture of 7- (tert-butoxycarbonylamino) cephalosporanic acid (0.372 g, 1 mmol; and 1-methyl-1H-tetrazole-5-thiol (Q, 122 g, 1.05 mmol) in 10 mL nitromethane, was allowed to react for 12 hours at 80 ° C to give 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (tert-butoxycarbonylamino) -3-cephem-4- NMR δ = 1.45 (s, 9, -C (CH3) 3, 3.7 (s, 2, 2-CH2>, 3.95 (s, 3, tetrazole -CH3>, 4, 4 (s, 2,3-CH 2 S-), 5.0 (s, 1,

C8-H), 5.6 (s, 2, C2-H and -CH2CONH) and 13 (s, 1, -C00H).

Example 89 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (4-ethyl-2,3-dioxopiperazinylcarbonylamino) -2- (p-hydroxyphenyl) preparation of acetamido) -3-cephem-4-carboxylic acid

When a mixture of 7- (2- (4-ethyl-2,3-dioxopiperazinylcarbonylamino) -2- (p-hydroxyphenyl) acetamido) cephalosporanic acid (0.180 g, 0.3Q 6 mmol) and 1-methyl 1H-Tetrazole-5-thiol (Q, Q374 g, 0.322 mmol) in 6 mL of nitromethane was allowed to react for 12 hours at 80 ° C to give 3 - (((1-methyl-1H-tetrazole-5- yl) thio) methyl) -7- (2- (4-ethyl-2,3-dioxopiperazinylcarbonylamino) -2- (p-hydroxyphenyl) acetamido) -3-cephem-4-carboxylic acid. NMR d "1.2 (5, 3, N-CH 2 CH 3), 3.6 (m, 6, 2-CH 2, piperazinyl 6-H and N-CH 2 CH 3), 3.9 (m, 2, piperazinyl 5 -H), 3.95 (s, 3, tetrazoline -CH 2), 4.4 (s, 2,3-CH 2 S-), 5.0 (d, 1, C 6 H 6, J = 6 Hz), δ .6 (d, 1, C 2 -H, J = 6 Hz), 5.8 (d, 1, CHCONH-, J = 8 Hz), 6.8 (d, 2, phenyl H, J = 8 Hz) ), 7.3 (d, 2, phenyl H, J = 8 Hz), 8.3 (d, 1, -CHCONH-, J = 8 Hz) and 9.9 (d, 1, -CHCONH-, J = 6 Hz).

t

-NH

Example 90_ Preparation of 3 - (((1,3,4-thiadiazol-2-yl) thio) methyl) -7- (2- (1H-tetrazol-1-yl) acetamido) -3-cephem-4-carboxylic acid acetonitrile -lissä

When a mixture of 7- (2- (1H-tetrazol-1-yl) acetamido) -cephalosporanic acid (0.38 g, Γ mmol) and 1,3,4-thiadiazole-2-thiol (0.15 g, 1.27 mmol) in 15 ml of acetonitrile, refluxed under nitrogen for 24 hours, gave 3 - (((1,3,4-6,9309 thiadiazol-2-yl) thio) methyl) -7- (2- (1H- tetrazol-l-yl) acetamido) -3-cephem-4-carboxylic acid. NMR δ 3.74 (m, 2,2-CH 2 Cl 2), 4.48 (q, 2,3-CH 2 S-, J = g = 14), 5.16 (d, 1, C = 5), 5.42 (s, 2, tetrazolyl -CH 3), 5.76 (q, 1, C 7 H, J 9? = 5, J 7 NH = 8)> 8.87 (s, 1, tetrazole ring), 9.40 (s, 1, thiadiazolithiol starting material), 9.53 (d, 1, -CP 2 CONH, J = 8 Hz) and 9.57 (s, 1, thiadiazole ring).

Example 91

Preparation of lithium 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (1H-tetrazol-1-yl) acetamido) -3-cephem-4-carboxylate in acetonitrile .

A mixture of 7- (2- (1H-tetrazol-1-yl) acetamido) cephalosporanic acid (1.91 g, 5 mmol) and 1-methyl-1H-tetrazole-5-thiol (0.7 g, 6 mmol) in 50 ml of acetonitrile, refluxed for 24 hours and then cooled to room temperature. The solvent was evaporated on a rotary evaporator to about 10 ml. 40 ml of ethanol were then added, followed by a solution of 0.3 g of lithium hydroxide in 10 ml of methanol. 3 - (((1-methyl-1H-tetrazol-5-yl) thio) methyl) -7- (2- (1H-tetrazol-1-yl) acetamido) -3-cephem-4-carboxylate crystallized. After stirring the reaction mixture for 45 minutes at room temperature, the crystals were collected by filtration, washed with ethanol and dried in vacuo at 40 ° C to give 1.6 g (72%) of product. The product was identified by NMR analysis.

Claims (3)

69309 52 1. A process for the preparation of 3-thiomethylcephalosporanic acid derivatives of the formula I, r1 S. rhV \ 13 i-CH.SR 0 ^ \, · '2 COOH 13. . . . wherein R is araidmo, benzoyl, phenylx, lower alkyl or unsubstituted or lower alkyl, hydroxy, N-methylacetamido, phenyl, benzyl or carboxymethyl-substituted 5- or 6-membered heterocyclic ring having 1-4 heteroatoms which may be one of the following combinations a) 1 nitrogen and 1 oxygen or sulfur b) 2 nitrogen and 0 or 1 oxygen or sulfur c) 3 nitrogen d) 4 nitrogen, 13 or R is benzotlazolyl, R1 is hydrogen or methoxy, 0 2 . . 3 "3 R is amino or a group of the formula R -C-NH- (where R is lower alkyl, -CH 2 -Lower chloroalkyl), lower cyanoalkyl, tert-butoxy, protected 4-amino-4- a carboxybutyl group of the formula 0 R120-C-CH- (CH) -CH-1 2 2 l AX 1 12 wherein A is a protected amino group and R is hydrogen, a group of the formula a, ---> 'v V) ^ <Z> m-CH2- wherein a is hydrogen, halogen, hydroxy or a group A ^ CH---, where 1.1 ... A is a protected amino group as above, Z is oxygen or sulfur and m is 0 or 1, a group of formula li 69309 53 a1- ^ y - p.-Q wherein a ^ is hydrogen or hydroxy, and Q is hydroxy, formyloxy, a protected amino group, an acylated amino group of the formula 0 if -NH-CT, wherein T is amino, 1,3-dimethylureido, 4-ethyl-2,3-dioxo-1-piperazinyl, or R is thienyl or tetrazolylmethyl, characterized in that the compound of formula II R10 is R2- '—Y N II 0> - \> - CH 2 OCO C00H 1. 2' wherein R is as defined above, R is as defined above for 2 R or formylamino groups, and R is lower al alkyl or C 1 -C 6 cycloalkyl, is reacted under substantially anhydrous conditions with a sulfur nucleophile of formula III r 1 -s-r 11 + III 13. 14 wherein R is as defined above and R is hydrogen or a substituent. . When R is amidino, the compound of general formula III is thiourea. Process according to Claim 1, characterized in that the compound of the formula I prepared is cefamandole. Process according to Claim 1, characterized in that the compound of the formula I prepared is cefazoline. 69309 54 A process for the preparation of 3-thiomethylcephalosporan syraders with the formula I, 5 r1, S \ R2-4— <· 'N I I 13 I> ~ N i CH ^ SR-1 · σ' 2 C00H 13 color R is enidino, benzoyl, phenyl, alkyl or other substituents or alkyl, hydroxy, N-methylacetamido, phenyl, benzyl or carboxymethyl substituted heterocyclic ring with 5 or 6 medium and 1-4 heteroatoms, each of which can be combined with the following combinations: a) 1 quaternary and 1 quaternary or svavel b) 2 quaternary or 0 or 1 quaternary or svavel (c) 3 quaternary d) 4 quaternary, 13 or or methoxy, 0 2 3 "3 R is amino or a group of the formula R -C-NH-, color R is en (alkyl, (-CH2-chloroalkyl), not cyanoalkyl, tert-butoxy, and is not 4-amino-4- carboxybutyl group with formula 0 R12 0-C-CH- (CH9) 9-CH9-Ax 1. 12 color A is a shielded group and R is a group with the formula X ft ---- * v · ✓ 'x V '> - (Z) -CH - / m2 color A is a halogen, hydroxy, or group A ^ CH ,, -, color A is an amino acid such as, is halogen, hydroxyl, or