MC1259A1 - ACYL DERIVATIVES - Google Patents

Abstract

Compounds of the general formula <IMAGE> [wherein X represents the 1,2,5,6- tetrahydro-2-methyl-5,6-dioxo-as- triazin-3-yl group, the 2,5-dihydro-6- hydroxy-2-methyl-5-oxo-as-triazin-3- yl group of the 1,4,5,6-tetrahydro-4- methyl-5,6-dioxo-as-triazin-3-yl group] and readily hydrolysable esters, readily hydrolysable ethers and salts thereof and hydrates of these compounds have broad-spectrum anti-bacterial activity.

Description

—I —

The present invention relates to novel acylated compounds, i.e., cephalosporin derivatives of the general formula h h ch3on=c—conh n \

-O

I

10

h2n cooh in which X represents the 1,2,5-tetrahydro-2-methyl-5,6-dioxo-as-triazine~3-yl group, the corresponding tautomeric form, the 2,5-dihydro-6-hydroxy-2-methyl-5-oxo-as-triazine-3-yl group or the 1,4,5,6-tetrahydro-4~methyl-5,6-dioxo~as-tria-zine-3-yl group;

The invention also includes the easily hydrolyzable esters, easily hydrolyzable ethers and salts of the above formula I compounds as well as the hydrates of the formula I compounds, their esters, their ethers and their salts.

which the carboxy group is in the state of an ester group easily hydrolyzed. As particular examples of these esters which may be of known types, we will mention the lower alkanoyloxyalkyl esters, for example acetoxymethyl, pivaloyloxymethyl, 1-acetoxyethyl and 1-pivaloyloxyethyl esters; the lower alkoxycarbonyloxyalkyl esters, for example methoxycarbonyloxymethyl, 1-ethoxycarbonyloxyethyl and 1-isopropoxycarbonyloxyethyl esters; lactonyl esters, for example phthalidyl and thiophthalidyl esters; the lower alkoxymethyl esters, for example methoxymethyl esters; and the lower alkanoylaminomethyl esters, for example acetamidomethyl esters. However, it could be other esters, for example benzyl and cyanomethyl esters.

The easily hydrolyzable esters of compounds of formula I are compounds that correspond to formula I in the-

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The easily hydrolyzable ethers of compounds of formula I are compounds that correspond to the formula I in which X represents the 2,5-dihydro-6-hydroxy-2-methyl-5-oxo-as-triazine-3-yl group in which the enolic OH group is in the easily hydrolyzable ether group state. The etherifying groups are the same as those mentioned above with reference to the easily hydrolyzable ester groups. Particular examples of these ethers include, for example, the lower alkanoyloxyalkyl ethers, e.g., acetoxy-10 methyl, pivaloyloxymethyl, 1-acetoxyethyl, and 1-pivaloyloxyethyl ethers; lower alkoxycarbonyloxyalkyl ethers, for example methoxycarbonyloxymethyl, 1-ethoxycarbonyloxyethyl and 1-isopropoxycarbonyloxyethyl ethers; lactonyl ethers, for example 15-phthalidyl and thiophthalidyl ethers; lower alkoxymethyl ethers, for example methoxymethyl ethers; and lower alkanoylaminomethyl ethers, for example acetamidomethyl ethers.

The salts of compounds with formula I are, for example, salts of alkali metals such as sodium and potassium salts; ammonium salt; alkaline earth metal salts such as calcium salt; salts of organic bases, for example, amine salts such as N-ethylpiperidine, procaine, dibenzylamine, N,N'-dibenzylethylethyl-25-lenediamine, alkylamines or dialkylamines, but also amino acid salts, for example, arginine or lysine salts. These can be single or double salts. The second salification can occur in compounds with the hydroxyl group, such as 2,5-α-6-hydroxy-2-30-methyl-5-oxo-as-triazine-3-yl.

Compounds of formula I also form salts by addition with organic or mineral acids. Particular examples of these salts include halogenated hydrocarbons, such as hydrochlorides, hydrobromides, and iodide hydrates; salts of other mineral acids such as sulfates, nitrates, phosphates, and similar salts; and alkyl sulfonates and monoaryl sulfonates such as ethanesulfonates, toluenesulfonates, and benzenesulfonates.

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10

analogous salts and salts of other organic acids such as acetates, tartrates, maleates, citrates, benzoates, salicylates, ascorbates and analogous salts.

Compounds of formula I, their salts, esters, and readily hydrolyzable ethers may be in a hydrated state. Hydration may occur during preparation or develop gradually due to the hygroscopic properties of a compound that was originally anhydrous.

The compounds according to the invention can be found in the isomeric form syn

15

CONH-

och3

or in the anti isomeric form

20

25

30

35

CONH-

CH3.0

or in the form of mixtures of these two forms. The syn isomeric form and mixtures in which this form is predominant are preferred.

The most appreciated compounds are the following: (6R,7R)-7-[2-(2-amino-4-thiazolyl)-2-(Z-methoxyimino)-acetamido]-3-^[ (2,5-<iiiiyâ.ro-6-hydroxy-2-methyl-5-oxo-as-triazine-3-yl)-thio]-methyl^-8-oxo-5-thia-1-azabicyclo[4-.2.0]octa-2-ene~ 2-carboxylic acid, its salts and the corresponding hydrates.

The acylated compounds defined above can be prepared according to the invention by one of the following processes:

a) in a compound corresponding to the general formula

-J+-

cooh

10 in which X has the meanings indicated above,

H represents a separable protecting group, the carboxy group being able to be in a protected state,

we eliminate the protecting group R and, if applicable, any possible protecting group of the carboxy group;

15 b) to prepare an ester or an ether easily hydrolyzed from a compound of formula I, a carboxylic acid or an enol of formula I is subjected to esterification or etherification respectively;

(c) to prepare salts or hydrates of a compound 20 of formula I or hydrates of these salts, a compound of formula I is converted into salts or hydrates or hydrates of salts.

If desired, the carboxy group present in the starting compound of formula II can be protected, for example, by esterification as an easily cleaved ester such as a silyl ester, for example, trimethylsilyl ester. It can also be one of the easily hydrolyzed esters mentioned above. The carboxy group can also be protected by salification with a tertiary mineral or organic base such as triethylamine. Protecting groups (H) include, for example, groups easily removed by acid hydrolysis such as tert-butoxycarbonyl or triphenylmethyl, or groups easily removed by basic hydrolysis such as trifluoracetyl. Preferred protecting groups (R) are chloroacetyl, bromacetyl, and iodacetyl, particularly the first. These latter protecting groups can be removed by treatment with thiourea.

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The starting compounds corresponding to formula II can themselves be prepared, for example, by nitrogen acylation of the corresponding compound bearing an amino group at position 7 -> by reacting a compound corresponding to the general formula

10

H H

III

COOH

15 in which X has the meanings indicated above, the carboxy group and/or the amino group being able to be in a protected state,

with an acid corresponding to the general formula

20

25

30

35

CH3ON=C—COOH J

N

TV

in which R has the meanings indicated above, or with a reactive functional derivative of such an acid, then removing, where appropriate, a possible protecting group of the group^earboxy.

If desired, the carboxy group present in the compound of formula III, amined at position 7, can be protected, for example as described above with reference to the starting compound of formula II, which is the subject of this preparation. The amino group of the compound of formula III can be protected, for example, by a silyl group such as trimethylsilyl.

Reactive functional derivatives of acids with the formula IY include, for example, halides such as chlorides, bromides, and fluorides; azides; and anhydrides.

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in particular mixed anhydrides of stronger acids; reactive esters, for example N-hydroxysuccinimide esters; and amides, for example imidazolides.

The reaction of the compound of formula III bearing a 5-amino group at the 7-position with the acid of formula IV or a reactive functional derivative of such an acid can be carried out in a manner known per se. Thus, for example, a free acid corresponding to formula IV can be condensed with one of the esters of formula III mentioned above in the presence of a carbodiimide such as dicyclohexylcarbodiimide, in an inert solvent such as ethyl acetate, acetonitrile, dioxane, chloroform, methylene chloride, benzene, or dimethylformamide, and the ester group can then be cleaved. Instead of carbodiimides, oxazolium salts, for example N-ethyl-5-phenylisoxazolium-31-suifonate, can be used as condensing agents.

In another embodiment, a salt of an acid of formula III, for example a trialkylammonium salt such as triethylammonium salt, is reacted with a reactive functional derivative of an acid of formula IV as mentioned above in an inert solvent*, for example one of those mentioned above.

According to another embodiment, an acid halide, preferably the chloride of an acid of formula IV, is reacted with an amine of formula III. The reaction is preferably carried out in the presence of an acid-neutralizing agent, for example, an aqueous alkali, preferably sodium hydroxide, or an alkali metal carbonate such as potassium carbonate, or a lower alkylamine such as triethylamine. The solvent used is preferably water, possibly mixed with an inert organic solvent such as tetrahydrofuran or dioxane. It can also be carried out in aprotonic organic solvents such as dimethylformamide, dimethyl tallow oxide, or hexamethylphosphorotriamide. If the starting compound of formula III is silylated, the reaction is carried out in an anhydrous medium.

The reaction of the compound of formula III bearing an amino group in the 7- position with the acid of formula IV or a

functional reactive derivative of such an acid, is preferably carried out at a temperature between -40°C and room temperature and for example between 0 and 10°C approximately.

Starting compounds corresponding to formula II can also be prepared by thiolation, by reacting a compound corresponding to the general formula

CH30N=C—CONH-

Nl 1

H H

U/\

J \\

rhnTX^X

-CH2—Y

COOH

in which R has the meanings indicated previously, Y represents an eliminable group, the carboxy group being able to be in a protected state,

with a thiol corresponding to the general formula

HS—X VI

in which X has the meanings indicated above, eliminating where appropriate a possible protecting group of the carboxy group.

The removable group Y of a compound of formula V may consist, for example, of a halogen such as chlorine, bromine, or iodine; an acyloxy group, for example, a lower alkanoyloxy group such as acetoxy; a lower alkylsulfonyloxy or arylsulfonyloxy group, for example, methanesulfonyloxy or toluenesulfonyloxy; or the azido group. The compound of formula V may be protected on the carboxy group as described with reference to the starting compound of formula II.

The reaction of the compound of formula V with the thiol of formula VI can be carried out in a manner known per se, for example at a temperature between approximately 40 and 80°C, preferably around 60°C, in a polar solvent.

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for example in an alcohol such as a lower alkanol (ethanol, propanol and similar alcohols), in dimethylformamide or dimethyl sulfoxide, but preferably in water or in a buffer solution at a pI of about 6 to 7 and preferably of 5-6.5.

In variant a) of the process according to the invention, the protecting group R of the amino group of a starting compound of formula II is removed. Protecting groups that can be removed by acid hydrolysis are preferably removed using a lower alkanoic acid, which may optionally be halogenated.

Formic acid or trifluoroacetic acid are commonly used. Generally, the process is carried out at room temperature, but it can also be performed at slightly higher or lower temperatures, for example, in the range of approximately 0 to 15 to 40°C. Protecting groups that can be removed by alkalis are generally hydrolyzed using a diluted aqueous alkaline solution at a temperature of 0 to 30°C. Chloracetyl, bromacetyl, and iodacetyl protecting groups can be removed using thiourea in acidic, neutral, or al-20 solutions at a temperature of approximately 0 to 30°C. Separation by hydrogenolysis (and, for example, the separation of a benzyl group) is not suitable in this case because, during hydrogenolysis, the oxime function would be reduced to an amino group.

When the reaction has been carried out as described above in (a), a protecting group of the carboxy group may, if desired, be removed from the reaction product. If the protecting group is a silyl group (silyl esters), it is removed particularly easily by treating the reaction product with water. Lower alkanoyloxyalkyl, alkoxy-30-carbonyloxyalkyl, lactonyl, alkoxymethyl, and alkanoylaminomethyl esters are preferably cleaved by enzymatic treatment with a suitable esterase (at a temperature of approximately 20 to 40°C). If the carboxy group is protected by salification (for example, with 35-triethylamine), the salifying protecting group can be removed by treatment with an acid. Hydrochloric acid, sufuric acid, phosphoric acid, or citric acid may be used for this purpose.

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The protecting group of the carboxy group can also be eliminated before the protecting group R as just described.

To prepare the readily hydrolyzable esters of the 5 carboxylic acids of formula I according to variant b), the carboxylic acid is preferably reacted with the corresponding halide containing the ester group, and preferably with iodide. The reaction can be accelerated with a base such as an alkali metal hydroxide or carbonate, or an amine such as triethylamine. If the compound contains the 2,5-clikydro-6-hydroxy-2-methyl-5-oxo-as-triazine-3-yl group X, the enol function of the latter is etherified with the formation of a corresponding readily hydrolyzable ether. Preferably, an excess of the corresponding halide is used. The esterification/etherification reaction is preferably carried out in an inert organic solvent such as dimethyl-

acetamide, hexamethylphosphorotriamide, "the" "dimé- ~

thyl sulfoxide or, preferably, dimethylformamide. The temperature is preferably from approximately 0 to 40°C.

20 To prepare the salts and hydrates of compounds of formula I and the hydrates of their salts, one can proceed in a manner known per se, for example by reacting the carboxylic acid of formula I with the equivalent amount of the desired base, preferably in a solvent such as water or an organic solvent such as ethanol, methanol, acetone, and others. If a second equivalent of the base is used, there is also salification of a possible tautomeric enolic form (group 2,5-α-1ihydro-6-hydroxy-2-methyl-5α-oxo-as-triazine-3-yl 2) and formation of a double salt. The temperature of salification is not a critical factor. It is generally room temperature, but one can also proceed at a slightly higher or slightly lower temperature, for example, in the range of approximately 0 to +50°C.

In most cases, the preparation of hydrates 35 is automatic: it occurs during the preparation or as a result of the hygroscopic properties of an initially anhydrous product. If one wishes to intentionally prepare a hydrate, one exposes a compound that is wholly or partially anhydrous.

<€1

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(carboxylic acid of formula I, ester, ether or salt of such an acid) in a humid atmosphere, for example at a temperature between approximately 10 and 40°C.

The compounds of formula III bearing an amino group in position 7- that we have used as described above can themselves be prepared from a compound corresponding to the formula

10

15

h h h2n-

-CH2—Y

VII

cooh in which Y represents an eliminable group, the carboxy group being able to be in a protected state,

with a thiol of formula VI. The reaction can be carried out under the same conditions as the reaction of the starting compounds of formulas V and VI. On the other hand, compounds of formula V can be prepared from a compound of formula VII and an acid of formula IV or a reactive functional derivative of such an acid under the same conditions as for the reaction of compounds of formulas III and IV.

When a compound of formula I is obtained in the state of a syn/anti mixture, the syn and anti forms can be separated in the usual way, for example by recrystallization or by chromatographic techniques using a suitable solvent or solvent mixture.

The compounds corresponding to formulas I and II, the corresponding readily hydrolyzable esters and ethers, and the salts and hydrates of these compounds have antibiotic activity and, in particular, bactericidal activity. They have a broad spectrum of activity against Gram-positive and Gram-negative microorganisms, including beta-lactamase-forming staphylococci and various beta-lactamase-forming Gram-negative bacteria such as Pseudomonas aeruginosa.

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Eaemophilus influenzae, Escherichia coli, Serratia marcescens, Proteus and Klebsiella species.

The compounds corresponding to formulas I and II, the corresponding readily hydrolyzable esters and ethers, the salts, and the hydrates of these compounds can be used for the treatment and prophylaxis of infectious diseases. For adults, the daily dosage is approximately 0.1 to 2 g. The preferred route of administration for the compounds according to the invention is parenteral administration.

10. To study the antimicrobial activity of the compounds according to the invention, representative compounds were subjected to tests:

Compound A: (6R,7R)-7-[2-(2-amino-4-thiazolyl)-2-(Z-

methoxyimi no)-ac etami do]-3-^[(2,5-ài hydro-6~hydroxy-15 2-methyl-5-oxo-as-triazine-3-yl)thioj-methyl^-8-oxo-

5-thia-1-azabicyclo[4.2.0]octa-2-ene-2-carboxylic acid Compound B: (6R,7R)-7-(2-[2-(2-chloracetamido)-4—thiazolyl] 2-(Z-methoxyimino)-acetamido^-3-^[(2,5-dihydro-6-hydroxv-2-methyl-5-oxo-as-triazine-3-yl)-thio]-20 methyl^-8-oxo-5-thia-1-azabicyclo[4.2.0]octa-2-ene-

2-carboxylic.

In vitro activity: minimum inhibitory concentration (micrograms/ml)

HAS

B

Haemophilus influenzae Strain 1

0,08

1.2

Strain 2

0.005

0.3

Strain 3

0.005

0.16

Strain 4-

0.005

'0.16

Strain 5

0.0025

0.08

Strain 6

0.0025

0.16

Strain 7

0.0025

0.16

Klebsiella pneumoniae

1.2

10

Escherichia coli Strain 1

0.02

0.16

Strain 2

0.6

5

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Proteus mirabilis

Strain

1

4o,oi

0.08

Strain

2

4.0.01

0.16

Proteus vulgaris

£0.01

0.16

Proteus rettgeri

£0.01

0.16

Staphylococcus aureus

Strain

ATCC 6538

2.5

2.5

Penicillium-resistant strain

2.5

5

Pseudomonas aeruginosa

Strain

1

0.3

1.2

Strain

2

10

>80

Strain

3

2.5

40

Strain

4

5

80

Strain

5 •

5

80

Strain

6

10

80

Strain

7

5

80

Serratia marcescens

0.08

2.5

in vivo activity

Groups of 5 mice were infected with an aqueous suspension of Escherichia coli via intraperitoneal administration. Three times, at 1 hour, 2.5 hours, and 4 hours post-infection, the active substance in physiological saline was administered by subcutaneous injection. Surviving animals were counted on the fourth day. Various doses were used, and the dose at which 50% of the experimental animals survived (DO₂q, mg/kg) was determined by interpolation. The results are reported below:

Active substance A B

DC^0, mg/kg 40.005 0.16

Toxicity

Active substance A B

LD^0 , mg/kg i.v.

s.c. p.o.

250-500 250-500 >4000 2000-4000 >5000 >5000

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The compounds according to the invention can be used as medicinal products, for example in the form of therapeutic compositions containing them in the form of free acid or salt in a mixture with an appropriate inert organic or mineral vehicle for pharmaceutical use for enteral or parenteral administration, for example water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oils, polyalkylene glycols, petrolatum, etc. The therapeutic compositions can be in the solid state, for example in the form of tablets, coated tablets, suppositories, capsules, or in the liquid state, for example in the form of solutions, suspensions or emulsions. Where appropriate, they are sterilized and/or contain auxiliary products such as preservatives, stabilizers, wetting or emulsifying agents, salts for modifying osmotic pressure, anesthetics, or buffers. Therapeutic compositions may also contain other useful drugs. Formula I compounds, their salts, and their hydrates are particularly suitable for parenteral administration, and for this purpose, they are preferably in lyophilized or dry powder form, which is diluted with common liquids such as water or isotonic saline. The easily hydrolyzed esters and ethers of formula I compounds, their salts, and their hydrates can also be used for enteral administration.

The following examples illustrate the invention without however limiting it; in these examples, indications of parts and % are understood to be by weight unless otherwise stated.

Example 1

30 Preparation of the disodium salt of (6R,7R)-7-[2-[2-] acid

amino-4-thiazolyl)-2-(Z-methoxyimino)-acetamido]-3-^[(2,5" dihydro-6-hydroxy-2-methyl-5-oxo-as-triazine-3-yl)~thio]-methyl^-8-oxo-5-thia-1~azabicyclo[4.2.0]octa-2-ene-2-carboxylic.

35 15.3 g of acid (6R,7R)-7-(2-[2-) are suspended.

(2-chloracetamido)-4-thiazolyl]-2-(Z-methoxyimino)-acetamido^-• 3-([(2,5-dihydro-6-hydroxy-2-methyl-5-oxo-as-triazine-3-yl)'

thio]-methyl^-8-oxo-5-thia-1-azabicyclo[4.2.0]octa-2-ene-2~

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carboxylic acid (fraction xr see below) with 5 g <ie thiourea in 150 ml of water. Under good injection of nitrogen and stirring, the fold is adjusted to 6.8-7.0 using a saturated sodium bicarbonate solution; an orange-colored solution is obtained.

5. Using an autotitrator and a sodium bicarbonate solution, the pH of the reaction solution is maintained at a constant 6.8 for 6 hours. An additional 2.5 g of thiourea is added, and the solution is stirred for 3 hours, maintaining the pH at 6.8 by adding a saturated sodium bicarbonate solution. 10. The red-colored solution is then stored overnight in the refrigerator; it darkens. The pH of the solution is adjusted to 2.0–2.5 by adding 100% formic acid. The substance precipitates. It is drained and washed with 100 mL of 10% formic acid. The mother liquors are discarded. The brownish filter cake is resuspended in 200 mL of water; the pH is adjusted to 7 with triethylamine; a brown-colored solution is obtained. This solution is stirred for 30 minutes with 2 g of activated charcoal. The charcoal is filtered out, and the filtrate, which is still brown at pH 3-5 with thorough stirring, is adjusted with 100% formic acid. The precipitated substance is drained, washed with 50 mL of 10% formic acid, and discarded. The dark yellow filtrate, now at pH 2-2.5, is adjusted with 100% formic acid, and the precipitated substance is drained. It is drained, washed with ice-cold water, and dried. The cephalosporic acid obtained is converted to the disodium salt by suspending it in a mixture of 40 mL of acetone and 40 mL of water and adding 20 mL of a 2 N solution of sodium 2-ethylcaproate in ethyl acetate. An orange-colored solution is obtained, to which 30-50 ml of acetone is added; a brown resin precipitates, which is separated by filtration. The yellow filtrate is stirred for 30 minutes; the disodium salt crystallizes. 50 ml of acetone is added to the mixture in portions, and it is kept overnight in the refrigerator. The crystals are drained, washed successively with an acetone-water mixture (85:15), pure acetone, and light petroleum ether, and dried overnight at 40°C under vacuum.

The desired substance is obtained in the form of cou- crystals

PO

their beige; [a]p = -144° (c = 0.5 in water). The spectrum of

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Nuclear resonance and microanalysis confirm the indicated structure.

L1acid (6R,7R)-7~(2-[2-(2-chloracetamido)-4—thiazolyl] -2-(Z-methoxyimino)-acetamido^-3-^[(2,5-dihydro-6-hydroxy-2-5-methyl-5-oxo-as-triazine-3-yl)-thio]-methyl^-8-oxo-5-thia-1-

azabicyclo[4-.2.0]octa-2-ene-2-carboxylic acid used as a starting compound can itself be prepared in the following manner:

22.24 g of 2-(2-chloroacetamide-do-thiazole-4-yl)-2-(Z-methoxyimino)acetic acid is suspended in 240 mL of 10% methylene chloride. To this suspension, 1339 mL of triethylamine is added, yielding a light brown solution. The solution is cooled to 0-5°C and 16.72 g of phosphorus pentachloride is added; the solution is stirred for 5 minutes at 0-5°C and 20 minutes without cooling. The yellow solution is evaporated at 35°C under vacuum. The evaporation residue is stirred twice with n-heptane, separating the latter by decantation. The resinous residue is treated with 240 mL of tetrahydrofuran, and the undissolved triethylamine hydrochloride is filtered out. The yellow filtrate contains the acid chloride.

20 22 g of (7R)-7-amino-3- acid are suspended

desacetoxy-3-[(2,5-dihydro-6-hydroxy-2~methyl-5-oxo-as-triazine~

3-yl)-thio]-cephalosporan in a mixture of 300 ml of eatax and 150 ml of tetrahydrofuran. To this suspension, drop by drop is added, under good nitrogen injection, using

25. The autotitrator is used to stir a 2.1T sodium hydroxide solution until a reddish-brown solution is obtained at pH 8. This solution is cooled to 0-5°C, and over 15 minutes, the acid chloride solution in tetrahydrofuran obtained above is added dropwise. The mixture is stirred for another 2 hours 30 minutes at 25°C. The pH of the acylation mixture is maintained constant at 8 by the addition of a 2H sodium hydroxide solution. The nearly black solution is freed of tetrahydrofuran.

4-0°C under vacuum. 100 ml of 2 N sulfuric acid is added. The precipitating substance is drained, washed with water, and dried.

35. Carefully sift again. The brown, wet filter cake is redissolved in 1.5 liters of acetone. A small amount of dark-colored insolubles is removed by filtration through Hyflo filter media, activated carbon* is added, and the mixture is stirred for

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After 50 minutes, the mixture is filtered again through Hyflo filtering medium. The orange-red filtrate is dried over sodium sulfate, concentrated under vacuum, and evaporated with ethyl acetate. A black resin precipitates, which is then filtered and discarded. The two-phase filtrate, which still contains water, is dried azeotropically by three distillations with benzene at 40°C under vacuum. The precipitating substance is drained and dried under vacuum at 40°C. It is agitated twice, each time with one liter of acetone; a brown resin is obtained as a residue and is discarded. The combined acetone extracts, orange in color, are concentrated at 40°C under vacuum to a final volume of approximately 150 ml; a brown resin is filtered and discarded. One liter of ethyl acetate is added to the filtrate, and it is concentrated under vacuum at 40°C. The precipitating substance is drained, washed with ethyl acetate, and then with ether. This is the acid (6R,71O-7-(2-[2-(2-chloracetamido)-4-thiazolyl]-2-(Z-methoxyimino)acetamido^-3-^[(2,5-dihydro-6-hydroxy-2-methyl-5-oxo-as-triazine-3-yl)-thio]-methyl j-8-oxo-r5-thia-1-azabicyclo[4-.2.0]octa-2-ene-2-carboxylic, 20 fraction I, beige amorphous acid. This fraction I can be used directly for the preparation of the desired final product.

ethyl at 40°C under vacuum, diluted with ether and extracted. tion II, amorphous acid of light beige color, a little more pure than fraction I in thin layer chromatography.

of the acid (fraction II) in a mixture of 20 mL of acetone and 11 mL of water. To this solution, 7 mL of a 2 H solution of sodium 2-ethylcaproate in ethyl acetate is added; the disodium salt crystallizes. 25 mL of acetone is added in portions, and the mixture is kept in the freezer for 2 hours. The crystals are drained, then washed successively with 25 mL of an ice-cold 80:20 acetone-water mixture, with pure acetone, and with

The mother liquors of acetate are highly concentrated

To prepare disodium salt, dissolve 3*5 g

light petroleum ether; it is dried overnight at 4-0°C under high vacuum. The disodium salt of the acid (6R,7R)-7-(2-[2-(2-chloracetamido)-4-thiazolyl]-2-(Z-methoxyimino)-acetamido-3-(2,5-Cl-6-hydroxy-2-methyl-5-oxo-as-triazine-3-yl)-thio]- is obtained

methyl-8-oxo-5-thia-1-azabicyclo[4-.2.0]octa-2-ene-2-carboxy-

/ 20

liquid in the form of light beige colored crystals. [a]D =

-142.7° (c = 1 in water). The nuclear resonance spectrum and microanalysis confirm the indicated structure.

Example 2

Preparation of the sodium salt of (6E,7R)-7-[2-(2-amino-) acid

thiazolyl)-2-(methoxyimino)-acetamido] -8.-oxo-3-| f ( 1,4,5,6-tetra-hydro-4-methyl-5,6-dioxo~as-triazine-3-yl)-thio]-methyl thia-1-azabicyclo[4.2.0]octa-2-ene-2-carboxylic.

19 g of (6R,7R)-7-(2-[2-(2-chloracetamido)-4-thiazolyl]-2-(methoxyimino)acetamido]-8-oxo- acid are suspended

3-([(1,4-,5j6-tetrahydro-4-methyl-5-6-dioxo-astriazine-3-yl)-

thio-methyl-5-thia-1-azabicyclo[4-2.0J octa-2-ene-2-carboxylic acid with 9>5 S <3-e thiourea in 150 mL of water. Under nitrogen injection and stirring, the pH is adjusted to 6.8 using a 5% sodium bicarbonate solution, yielding an orange-yellow solution. Using an auto-titrator and a sodium bicarbonate solution, the pH of the reaction solution is maintained constant at 6.8–7.0 for 6 hours. 100% formic acid is added to the orange-colored solution until the pH is 3.5. The precipitating substance is drained and washed with 100 mL of 10% formic acid. The filter cake is hereinafter referred to as product (1). The filtrate is adjusted to pH 2.5 by adding 100% formic acid; a substance precipitates again. The mixture is maintained in an ice bath for 1 hour, then the precipitated substance is drained and washed with a little ice water. This product is fraction I. The orange-brown filter cake (1) is resuspended in 250 ml of water. The suspension is adjusted to pH 7 with a 2 N KaOH solution; an orange-brown solution is obtained. 100% formic acid is added to this solution until pH 3.5 is reached. The precipitated substance is drained and discarded. The filtrate is adjusted to pH 2.5 with 100% formic acid; a substance precipitates again. The

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After soaking the mixture for one hour in an ice bath, the precipitated substance is drained and washed with a little ice water. This product is fraction II. Fractions I and II are suspended together in 500 ml of ethanol, and the water is removed by evaporation using a rotary evaporator. After adding ether, the mixture is drained and washed successively with ether and then with light petroleum ether. This yields the desired substance in the form of a yellowish solid, which will be referred to hereafter as substance A.

10. The mother liquors and wash waters from fractions I and II are concentrated from a volume of approximately 1.7 L to a final volume of 250 mL. The pH is adjusted to 2.5 with 100% formic acid, and the solution is kept overnight in the refrigerator. A substance re-crystallizes, which is then drained and washed with a little water. The filter cake is dried azeotropically with ethanol. The desired substance is obtained as a solid, practically colorless compound (substance B). Thin-layer chromatography shows substance B to be purer than substance A.

To obtain the desired substance in its pure form, acid B is suspended in 150 mL of methanol, and 10 mL of a 2 N solution of sodium 2-ethylcaproate in ethyl acetate is added while stirring. After approximately 10 minutes, a solution is obtained, to which 100 mL of ethanol is added. The mixture is then highly concentrated at 40°C under vacuum. After the addition of ethanol, the sodium salt precipitates in an amorphous state.

It is wrung out, washed successively with ethanol and then with light petroleum ether and dried for 24 hours at 40°C under high vacuum. The desired substance is obtained in the form of a practically colorless amorphous powder, [ct]p = -42.9° (c = 1 in 30 1' water).

The nuclear resonance spectrum shows that the substance is in a Z/E mixture state, 90:10. Microanalysis also confirms the indicated structure.

The (6R,7R)-7-^2-[2-(2-chloracetamido)-4-thiazolyl]-35 2-(methoxyimino)-acetamido]-8-0x0-3-^[(1,4,5,6-tetrahydro-4-methyl-5 > 6-dioxo-as-triazine-3-yl)-thio]-methyl^-5-thia-1-aza-bicyclo[4.2.0]octa-2-ene-2-carboxylic acid used as a starting compound can be prepared as follows:

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44 g of (7R)-7-amino-3-deacetoxy-3-[(1,4,5,6-tetrahydro-4-methyl-5,6-dioxo-as-triazine-3-yl)-thio]-cephalosporanic acid is suspended in a mixture of 600 ml of water and 300 ml of tetrahydrofuran. To this suspension, 5 drops of the solution are added, under good nitrogen injection, using...

The autotitrator is used to titrate a 2 H sodium hydroxide solution until a brown solution with pH 7-8 is obtained. This is cooled to 0-5°C, and a solution of 2-(2-chloracetamidothiazole-4-yl)2-10 (Z-methoxyimino)acetyl chloride in tetrahydrofuran (prepared from 44.5 S of the corresponding acid as described in Example 1) is added dropwise over 15 minutes. The mixture is then stirred for 2 hours 30 minutes at 118 and 25°C. The pH of the acylation mixture is maintained constant at 7.8-8 using the autotitrator and a 2 N sodium hydroxide solution. The brown solution is then rid of the tetrahydrofuran at 40°C under vacuum. The solution is then diluted with water to a final volume of 2 L. The pH is adjusted to 2 with 2 N sulfuric acid. The precipitated substance is drained, washed with 1 liter of water, and dried for 2 days at 40°C under vacuum. For purification, the product is first dissolved in a mixture of 100 mL of water and 300 mL of acetone. The dark-colored solution is diluted with acetone to a final volume of 2 L. The dark-colored precipitated substance is filtered and discarded. 1 liter of ethyl acetate is added to the filtrate, and 1 liter of the solvent is evaporated at 40°C under vacuum. The solution is then diluted with 2 L of ethyl acetate. The brownish-beige precipitated substance is discarded. The filtrate is highly concentrated to 40°C under vacuum. The acid, which crystallizes, is then drained. For recrystallization, the acid is first dissolved in 800 mL of 30% methanol under reflux. The solution is cooled to 25°C, and a small amount of the orange-colored substance is filtered. The yellow filtrate is stirred for 1 hour 30 minutes in an ice bath; the acid crystallizes. It is then drained, washed successively with methanol and then light petroleum ether, and dried at 25°C under vacuum. The desired compound is thus obtained as beige-colored crystals. The nuclear resonance spectrum shows that this compound is in the form of a Z/E mixture, 75:25. [α] = -127.9° (c = 1 in dimethylformamide).

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Example 3

Preparation of methylene pivalate—(6R,7R)-7-[2-(2-amino-4~thiazolyl)-2-(Z-methoxyimino)-acetamido]-3-( £/~2,5~di-hydro-2-methyl-5-oxo-6-[(pivaloyloxy)-methoxy]-as-triazine-5 3-yl_7~thio^-methyl ^ -8-oxo-5-thia~1-azabicyclo[4.2.0]octa-2-ene-2-carboxylate.

1.85 S of the disodium cephalosporin salt prepared in Example 1 is suspended in 50 mL of dimethylformamide, and 1.35 S of pivaloyloxymethyl iodide is added under nitrogen injection at 0-5°C. The reaction mixture is stirred for 30 minutes at 0-5°C and then poured into 500 mL of ethyl acetate. The mixture is washed three times with water, twice with a 5% sodium bicarbonate solution, and finally with water. The solution is dried over sodium sulfate and highly concentrated to 35°C under vacuum. After the addition of ether, the desired substance precipitates in an amorphous state. It is dewatered, washed with ether and then with light petroleum ether, and dried overnight at 25°C under high vacuum. The desired substance is obtained as a beige, amorphous powder. Nuclear resonance spectroscopy and microanalysis confirm the indicated structure.

Example 4

Preparation of dry ampoules for intramuscular administration.

25. A 1 g lyophilized powder of the disodium salt of (6R,71O-7-[2-(2-amino-4-thiazolyl)-2-(Z-methoxyimino)-acetamido]-3-^[(2,,5-dihydro-6-hydroxy-2-methyl-5-oxo-as-triazine-3-yl)-thio]-methyl^-8-oxo-5-thia-1-azabicyclo[4.2.0]octa-2-ene-2-carboxylic acid is prepared in the usual manner and introduced into an ampoule. Before administration, the lyophilized powder is mixed with 2.5 ml of a 2% aqueous solution of lidocaine hydrochloride.

Example 5

We prepare 35 gelatin capsules with the following composition in the usual way:

methylene pivalate-(6R,71O-7-[2-(2-amino-4-thiazolyl)-2-(Z-methoxyimino)-acetamido]-3- ^/"~2,5-dihydro-2-methyl-

-21-

5-oxo-6-[(pivaloyloxy)-methoxy]-as-triazine-3-yl7-thiol-methyl -8-oxo-5-thia-1-azabi-cyclo[4.2.0]octa-2-ene-2-carboxylate Luviskol (water-soluble polyvinylpyrrolidone) 5-mannitol talc magnesium stearate

500 mg 20 mg 20 mg

15 mg

2 mg 557 mg

-b'V 4 410/ 12 8

Claims (1)

DEMANDS 1 - Process for the preparation of acylated derivatives corresponding to the general formula 5 10 h h ch3on=c—conh ch2—s- cooh in which X represents the 1,2,5,6-tetrahyaro-2~methyl-5,6-dioxo-as-triazine-3-yl group, the 2,5-dihydro-6~hydroxy-2~ 15 raethyl-5-oxc-as-triazine-3-yl group or the 1,4,5,6-tetrahydro-4-methyl-5,6-dioxo-as-triazine-3-yl- group? the process is characterized in that, in a compound corresponding to the general formula 20 25 h h ch3on=ç—conh n rhn ^ CH9—S—X cooh in which X has the meaning indicated above, R is an eliminable protecting group, the carboxy group being able to be in the protected state, the protecting group R is eliminated and, if necessary, a possible protecting group of the carboxy group. 2 - A process for preparing readily hydrolyzable esters and ethers of compounds of formula I according to claim 1, the process being characterized in that a carboxylic acid or an enol, respectively, of formula I is subjected to esterification 35 or etherification respectively. 3 ~ Process for preparing salts and hydrates of compounds of formula I according to claim 1 and hydrates of these salts, the process being characterized in that a compound of formula I is converted into a salt or hydrate or hydrate of salt. -■23- FV 4410/128 4- - Process according to claim 1, characterized in that a starting compound corresponding to formula II in which R represents the chloroacetyl group is treated with thiourea. 5 3 - Process according to claim 1 or 4-, for the preparation of a compound of formula I in which X represents the group 1,2,5-tetrahydro-2-methyl-5i6-dioxo-astriazine-3-yl or the group 2,5-(3.ihydro-6-hydro-2-methyl-5-oxo-astriazine-3-yl, of salts of these compounds and of hydrates of these compounds and of their salts, the process being characterized in that it starts from a compound of formula II in which X has the meaning indicated. 6 - A process according to claim 1 or 4, for preparing compounds of formula I in which X represents the 15 group 1,4-,516-tetrahydro-4-methyl-5,6-dioxo-as-triazine-3-yl, of salts of these compounds and of hydrates of these compounds and of their salts, the process being characterized in that one starts from a compound of formula II in which X has this meaning. 7 - A process according to any one of claims 20 1, 3 or 4-, for the preparation of (6R,7R)-7-[2-(2-amino- 4—thiazolyl)-2-(Z~methoxyimino)-acetamido]-3-^[(2,5-dihydroxy-6-hydroxy-2-methyl-5-oxo-as-triazine-3-yl)-thio-methyl^-8-oxo- 5-thia~1-azabicyclo[4.2.0]octa~2-ene~2-carboxylic, of salts of this compound and of hydrates of this compound and its salts, the pro- 25 ceded being characterized in that one starts from a compound bearing the corresponding substituents. 8 - A process according to claim 2 or 4 for the preparation of readily hydrolyzable esters of compounds of formula I, salts of these esters, and hydrates of these esters and salts, the 30 process characterized in that a carboxylic acid of formula I is subjected to corresponding esterification and, where appropriate, the resulting compound is converted into a salt or a hydrate or a hydrate of the salt. 9 - Process according to claim 2 or 4 - for the preparation of readily hydrolyzable ethers of compounds of formula I, of salts of these ethers and hydrates of these ethers and its salts, the process being characterized in that an enol of formula I is subjected to a corresponding etherification and, the case FV 4410/128 If necessary, the compound obtained is converted into a salt or hydrate, or into a hydrate of the salt. 10 - A process according to claim 8 for the preparation of pivaloyloxymethyl esters of a compound of formula 5 I, of salts of these esters and of hydrates of these esters and of these salts, the process being characterized in that a carboxylic acid of formula I is reacted with a pivaloyl-oxymethyl halide, preferably with iodide and, where appropriate, the compound obtained is converted into a salt or a hydrate or into a hydrate of the salt. 11 - Process according to claim 8 or 9i for the preparation of methylene-(6R,7R)-7"-[2-(2-amino-4-thiazolyl)-2-(Z-methoxyimino)acetamido]-3-£ ^/_2,5-dihydro-2~methyl-5-oxo-6~[(pivaloyloxy)-methoxy]-as-triazine-3-yl7~thio^- 15 methylj-8-oxo-6-thia-1~azabicyclo[4.2.0]octa-2-ene-2-carboxylate, of salts of this ester and of hydrates of this ester and of its salts, the process being characterized in that the acid (6R,7R)-7-[2-(2-amino-4~thiaz olyl)-2-(Z-methoxyimino)-acetamido]-(2,5-dihydro-6-hydroxy-2-methyl-5-oxo-as-triazine~3-yl)-thio] 20 -methyl^-8-oxo-5~thia-1-azabicyclo[4.2.0]octa-2-ene-2-carboxylic, preferably in the disodium salt form, is reacted with a pivaloyloxymethyl halide, preferably iodide, and where appropriate is converted the compound obtained in a salt or hydrate or in a hydrate of that salt. 25 12 - A process for preparing therapeutic compositions useful, particularly due to their antibiotic activity, in the prophylaxis and treatment of infectious diseases, characterized in that the compounds of formula I according to claim 35 12, their readily hydrolyzable esters and ethers, their salts, the hydrates of compounds of formula I, their esters, ethers and salts, are mixed, as an active substance, with non-toxic, inert and therapeutically compatible solid or liquid carriers commonly used in such preparations, and/or excipients. 35 13 - As intermediate products, the acylated derivatives of general formula /D - J?'V 44iU / A. z a 10 15 ch3on=c c o ni- II rhn cooh in which X represents the 1,2,5,6-tetrahydro~2~ methyl-5,6-dioxo-as-triazine-3~yl group, the 2,5-dihydro-6-hydroxy-2-methyl~5-oxo-as~triazine~3-yl group or the 1,4,5,6-tetrahydro-4-methyl~5,6-dioxo-as-triazine-3~yl group, R represents a separable protecting group, the carboxy group being able to be in the protected state. 14 - Acylated derivatives according to claim 13, characterized in that X represents the 1,2,5>6-tetrahydro-2-methyl-5,6~dioxo-as-triazine-3-yl group or the 2,5-dihydro-6-hydroxy-2-methyl-5-oxo-as~triazine-3-yl group. 15 - Acylated derivative according to claim 15? (6R,7R)-7-(2-[2-(2-chloracetamido)-4-thiazolyl]-2~(Z-Eieth.oxy~ imino)-acetamido^-3-[(2,5-dihydro-6~hydroxy-2-methyl-5-oxo-as~ triasine-5-yl)-thio]-niethyl^-8-oxo-5-thia-1-azabicyclo[4.2.0]— o c t a-2-ene-2-c arb oxy1i qu e. ORIGINAL containing the word siooté ..aumoî mvé mil Jo^é CURAU Industrial Property Consultancy 26k|s, Princess Charlotte Boutique, Monte-Carlo P&r tiMrurïrttrt* do*