7beta--[(Z)-2-(2-AMINO-4-THIAZOLYL)-2-HYDROXYIMINOACETAMIDO]-3- -(1,2,3-TRIAZOLE-4-YL)-THIOMETHYLTHIO-3-CEPHEM-4-CARBOXYLIC ACID HYDROCHLORIDE, ITS CRYSTAL HYDRATE AND METHOD OF THEIR SYNTHESIS

The invention relates to novel triazoliltiometiltiotsefalosporina useful as the active antibiotic agent. Specifically hydrochloride to 7β - [(Z) -2- (2-amino-4-thiazolyl) -2-hydroxyiminoacetamido] -3- (1,2,3-triazol-4-yl) thiomethylthio-3-cephem-4 carboxylic acid and its crystalline hydrate, which have strong antibiotic activity and low toxicity and, in addition, pharmaceutically stable.

The present inventors have developed effective on oral administration of cephalosporin derivatives having at the 3-position of the cephem nucleus thioalkylthio side chain substituted by a heterocyclic group (See. US application N 07 / 729.413 of 07.12.91 and EP 0,467,647 A N 2). Among these antibiotic compounds, a compound containing a thioalkylthio side chain substituted by 1,2,3-triazolilnoygruppoy, toest7β - [(Z) -2- (2-amino-4-thiazolyl) -2-hydroxyiminoacetamido] -3- (1,2 , 3- triazol-4-yl) thiomethyl-thio-3-cephem-4-carboxylic acid (hereinafter designated as S-1090), which was obtained as a pale yellow powder exhibits a remarkably potent antibacterial activity. S-1090 represented by the following formula: S-1090, however, it proved to be not stable enough for its entry into the therapeutic composition. The instability of S-1090 is attributed to the following properties: a) S-1090 generally contains water because hygroscopic. The problem is that the water content tends to vary during the cooking process such as pulverization, formulation and the like preparation that is accompanied by a change in the content of active ingredient in pharmaceutical compositions containing S-1090. This creates a problem of quality control and makes the product fragile in terms of maintaining the quality and content of the active ingredient.

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b) S-1090 obtained in the usual way, it is an amorphous powder insoluble in water and in an amount sufficient for its recrystallization from water, which makes it difficult to remove impurities contained in the amorphous powder of S-1090.

c) Even dried S-1090 contains residual solvent that is difficult to remove the pharmaceutically acceptable value.

The present inventors have conducted intensive studies to obtain improved clinical standpoint derivatives of S-1090, which are pharmaceutically stable and contain a small amount of contaminating solvent, and have found that S-1090 hydrochloride and crystalline hydrate thereof have the desired properties such as low toxicity, high solubility in water, pharmaceutical stability and high antibacterial activity.

Thus, the present invention provides S-1090 hydrochloride and a crystalline hydrate thereof. The latter gives X-ray diffraction pattern of the lattice, represented by the following Table 1 and Fig. 1, and a differential scanning calorimetry (Fig. 2).

Measurement conditions: tube Cu, voltage - 40 kV, current - 20 mA, the angle of the sample - 0,02o.

These X-ray diffraction 2θ value varies depending on the structure of the crystal lattice and the peak intensity change depends on the direction in which the growing crystal, and the crystallization rate. It is well known from the practice that the change does not mean only the intensity variation of the crystal lattice structure.

Crystalline Hydrate of S-1090 of the invention is stable to retain bound water showing a water content corresponding to 1 or 2 hydrates in a wide range of drying conditions as shown in the experiments below. Typically, the crystalline hydrate remains stable like crystalline hydrates showing the proportion of the water content corresponding to a hydrate number of about 1.1 to about 1.3. Although the compound may save combined water and converted into anhydrous crystalline form to drying conditions at low humidity and / or high temperature, the final anhydrous crystals, when kept in high humidity, rapidly absorb water until until it reaches a stable hydrate form showing a degree of water content corresponding to a hydrate number of about 1.2 to 1.3, or even 1.8 under certain conditions, even if they are placed in the usual atmospheric conditions.

FIG. 1 is a diffraction curve of crystalline hydrate of S-1090 hydrochloride, wherein the intensity is represented on the ordinate in units of 2θ value and - on the horizontal axis. The measurement conditions are: tube - Cu; voltage - 40 kV; Current - 20 mA; and the angle of the sample 0,02o.

FIG. 2 shows a differential scanning calorimetry curve of crystalline hydrate of S-1090 hydrochloride, wherein heat flow is represented on the ordinate in milliwatts (mW) and the temperature on the abscissa. FIG. 2 indicates that heat absorption occurs at temperatures 70-120oC, with a maximum at 105oC, possibly due to the evaporation heat of water contained in crystalline hydrate.

Crystalline Hydrate of S-1090 product is especially useful in clinical use because it contains only pharmaceutically acceptable amount of solvent and is low in toxicity and stable quality.

Methods for preparing S-1090 hydrochloride and crystalline hydrate thereof will be described in detail below, that does not mean that it is limited to the scope of the invention.

Starting compound, 7β- [(Z) -2- (2-amino-4-triazolyl) -2-hydroxyiminoacetamido] -3- (1,2,3-triazol-4-yl) thiomethylthio-3-cephem-4- carboxylic acid can be prepared using any of known methods used in the cephalosporin, for example by neutralization of the sodium salt of S-1090 as described in sozayavke US 07 / 729.413 (especially in Example 7 (5)).

S-1090 hydrochloride can be prepared generally by reacting S-1090 with hydrochloric acid. Crystalline Hydrate of S-1090 was prepared by crystallization of S-1090 from an aqueous solution of S-1090 is acidified with hydrochloric acid according to the method of the present invention.

The preformed S-1090 was treated in any suitable manner, preferably with one or more equivalents, more preferably - the amount of hydrochloric acid sufficient to adjust to pH 0.1-5 at a temperature of about 0-50oC, 5-30oC preferably for 1-60 minutes, preferably 10-30 minutes, to obtain the hydrochloride of S-1090.

Crystalline Hydrate of S-1090 can be obtained by crystallizing S-1090 from an aqueous solution or suspension adjusted to acidic with hydrochloric acid, preferably after seeding crude S-1090 hydrochloride. Crystallization can generally be conducted in the presence of higher concentrations of hydrochloric acid. In particular, pH of the solution or suspension may be preferably about 1-5, more preferably from 0 to 3. Although the crystallization can be carried out from the water as the only solvent, in certain cases a mixture of water and one or more organic solvents may be preferred. Such solvents used in the present process can be selected from typical industrial solvents, for example lower alcohols, ketones, nitriles, esters, and the like.

Crystallization is generally conducted at a temperature between 0 and 90C, preferably 30 to 50C. Under these conditions, the crystalline product was precipitated for 10 minutes to 20 hours, preferably from 1 to 6 hours, though the time required for complete precipitation varies depending on the solvent composition.

The resulting S-1090 hydrochloride or a crystalline hydrate thereof can be dried using a conventional conditions such as elevated, room or cold; elevated, atmospheric or reduced pressure; Strong blowing air, wherein the air possibly heated; Use of the drying agent to remove moisture. It should be noted that in case of crystalline hydrate product should be selected appropriate conditions for drying the product to not lose water molecule. As noted above, anhydrous S-1090 hydrochloride can be converted into crystalline hydrate product through the reabsorption of water, i.e. by placing it in the appropriate conditions of high humidity, e.g., at a temperature between 10-30oC and humidity between 50-90% for a period sufficient for re-adsorption of water.

End gidrohlrorid S-1090 or a crystalline hydrate of S-1090 holds the combined water under various conditions as shown in Table 2 below, which indicates that the derivatives of S-1090 of the invention are stable enough to be used in pharmaceutical compositions and give reliable quality to the final product.

S-1090 hydrochloride or a crystalline hydrate thereof, when administered for the treatment, may be converted into the same active form as the original S-1090 in vivo, and show potent antibacterial activity as experimentally demonstrated in US Patent No. 07 / 729.413. Thus, in tests in vitro S-1090 provides an effective effect on gram-positive bacteria such as Staphylococcus aureus and Streptococcus pyogenes, as well as on gram-negative bacteria such as Escherichia coli, Enterobacter cloacar, Pceudomonas aeruginosa, Proteus vulgaris, Proteus mirabilis, Serratia marcescens, Haemophilus influenzae, Klebsiella pheumoniae, and Morgania morganii.

S-1090 is especially effective on gram-negative bacteria when evaluated Escherichia coli 7437 by Enterobacter cloacae and SR 233.

The following examples are intended to further illustrate the present invention is not limited thereto.

All the crystalline hydrate of S-1090 obtained in the following examples show the same x-ray diffraction and differential scanning calorimetry curves, they are shown in Fig. 1 and 2, respectively, on the basis of the same crystalline structure.

Preparation. 7β - [(Z) -2- (2-tert-butoxycarbonylamino-4-thiazolyl) -2-trityloxyiminoacetamido] -3- (1,2,3-triazol-4-yl) thiomethylthio-3- cephem-4- carboxylate, diphenylmethyl ester.

To a solution of 4-acetylthiomethylthio-1,2,3-triazole (11.50 g, 61 mmol in DMF - 300 ml) was added dropwise a solution of sodium methoxide (1,28n., 94 ml) in methanol at -60C ... -50oC. After stirring for 20 minutes a solution of diphenylmethyl ester 7β - [(Z) -2- (2-t-butoxycarbonylamino-4- 4-carboxylic acid (48.55 g, 50 mmol) in dimethylformamide (190 ml) was added dropwise to the mixture over 7 minutes at the same temperature. After 50 minutes, the reaction mixture was diluted with acetic acid (10 ml) and water (2 L ) and extracted with ethyl acetate. The extract was washed with brine, dried over sodium sulfate and concentrated under reduced pressure. The residue was crystallized from toluene and recrystallized from ethyl acetate - toluene to give diphenylmethyl ester acetamido] -3- (1,2,3-triazol-4-yl) thiomethylthio-3-cephem-4-carboxylic acid as colorless crystals (29.14 g), yield 57%, m.p. 190 -200oC (with decomposition).

NMR δ (CDCl3 -CD3OD) ppm .: 1.53 (s, 9H), 3,45, 3.63 (AB q. J = 17,2 Hz, 2H), 4,12, 4,15 ( AB q. J = 14,2 Hz, 2H), 5.08 (d, J = 5 Hz, 1H), 5.88 (d, J = 5 Hz, 1H), 6.98 (c, 1H), 7.08 (s, 1H), 7.2-7.5 (m, 25H), 7.60 (s, 1H).

IR ν (KB r) cm-1: 3390, 3210, 1800, 1725, 1688, 1555, 1495, 1449, 1375, 1275, 1245, 1225, 1155.

Example 1 S-1090 hydrochloride.

1) The hydrochloride of S-1090.

Diphenylmethyl ester 7β - [(Z) -2- (2-t-butoxycarbonylamino-4- thiazolyl) -2-trityloxyiminoacetamido] -3- (1,2,3-triazol-4-yl) thiomethylthio-3-cephem-4-carboxylate (26.0 g) was suspended in a mixture of 270 ml of dichloromethane and 51 ml anisole. To the suspension was added dropwise a solution of 17.0 g aluminum chloride in 60 ml of anisole at 0-5oC with stirring and the mixture was stirred for 1 hour and 50 minutes. The reaction mixture, containing the final S-1090 is poured into 220 ml of methanol and 114 ml of water and 29.8 g of 36% hydrochloric acid under stirring at 0-15oC. The aqueous layer containing the end of S-1090 hydrochloride, was collected and washed twice with 100 ml dichloromethane. The solution is seeded with 30 mg of crystals of crude S-1090 hydrochloride, stirred for 1.5 hours at 20-28oC, and concentrated under reduced pressure to 150 ml. The resulting crystalline precipitate was collected, washed with 260 ml of water to yield 24.5 g of S-1090.

2) Hydrate of S-1090 hydrochloride.

The hydrochloride of S-1090 in the form as prepared in 1) above was suspended in 228 ml of water to which was added dropwise 46 g of 4% aqueous solution of sodium hydroxide to obtain a solution. The solution was treated with 1.5 g of activated charcoal. The mixture was filtered and the precipitate was washed with 20 ml of water. The filtrate and washings were containing sodium salt of S-1090 are combined and adjusted to pH 2 by adding dropwise 4N. HCl at 5-10oC stirring. The acidic solution is seeded with 50 mg of crystals and the mixture adjusted to pH 1 by the dropwise addition of 4N. HCl for 6 hours (the total amount of added 4N. HCl equal to 63 g). If the mixture is stirred for 16 hours at 60oC and cooled to 0C, then the precipitated crystalline product which was collected by filtration, washed with 160 ml of water and dried to yield 11.0 g of hydrate of S-1090 hydrochloride as a light brown powder. Mp 177,6-181,0oC (with decomposition). The drying process is carried out in a fluidized bed apparatus (305p FBD 68 L) for 2 hr using the following conditions: air temperature 20oC and air velocity 1200 cm3 / min.

Example 2 Crystalline Hydrate of S-1090.

S-1090 hydrochloride (10.7 g) in the form as prepared in Example 1 (1) above is dissolved in 128 ml of water and 20 ml of 1N. aqueous sodium hydroxide solution and the resulting solution was adjusted to pH 4 with hydrochloric acid. Then the solution was adjusted to pH 0.5 by adding 5% hydrochloric acid over 1 hour. If the mixture is stirred for 1-5 hours at 40oC and cooled to 5-10oC, crystalline product precipitates. The crystals were collected by filtration, washed with 70 ml of water and dried to give 5.0 g of crystalline hydrate of S-1090 hydrochloride as white crystalline powder. The drying process was carried out for 5 hours according to the method described in Example 1 using the following conditions: air temperature 30oC and air flow rate 400 cm3 / min.

Example 3 Crystalline Hydrate of S-1090.

S-1090 hydrochloride (51 g) as prepared in Example 1 (1) above is dissolved in 200 ml of water and 72 ml of 1N. aqueous sodium hydroxide solution under ice cooling to obtain an aqueous solution with pH 6,1. The solution was treated with activated carbon, and the precipitate was separated by filtration and washed with 200 ml of water. The filtrate and washings were combined and parts added 400 ml of 6N. HCl and 50 mg of seed crystals. If the solution stirred at 0C, then the precipitated crystalline product. The crystals were collected by filtration, washed with water and dried to obtain 16.7 g of crystalline hydrate of S-1090 hydrochloride as white crystalline powder. The drying process is carried out for 1 hr according to the method described in Example 1 using the following conditions: air temperature 40oC and air velocity 1200 cm3 / min.

Example 4 Crystalline Hydrate of S-1090.

S-1090 hydrochloride (25.8 g) as prepared in Example 1 (1) above is dissolved in 190 ml of water and 46 g of 4% aqueous sodium hydroxide solution. The solution was adjusted to pH 4 with the addition of 3.1 g of 20% hydrochloric acid and poured into an excess of a mixture of 148 ml of 36% hydrochloric acid and 128 ml of water with stirring at 40oC for 40 minutes, adding at this time the seed crystal 10 minutes after the solution was poured into the mixture. The precipitate was collected by filtration, washed with 130 ml of water and dried to obtain 11.0 g of crystalline hydrate of S-1090. Mp 178,1-181,2oC (with decomposition).

Example 5 Crystalline Hydrate of S-1090.

The sodium salt of S-1090 hydrochloride (28.7 g) as it was obtained in Example 1 (2) above is dissolved in 296 ml of water. The solution was adjusted to pH 5 by adding dropwise 4N. HCl at 5 - 10oC with stirring. The solution was poured into a mixture of 164 g of 36% hydrochloric acid and 284 ml of water at once with stirring at 15oC. The solution is seeded with 50 mg of crystals and precipitates are collected by filtration, washed with 145 ml of water and dried to obtain 12.2 g of crystalline hydrate of S-1090 hydrochloride as white crystalline powder.

Example 6 Crystalline Hydrate of S-1090.

The sodium salt of S-1090 hydrochloride (15.2 g) as it was obtained in Example 1 (2) above is dissolved in 157 ml of water. To the solution was added 36% hydrochloric acid to obtain an aqueous solution with pH 2. The solution is poured into a mixture of 187 g of 36% hydrochloric acid and 50 ml of water over 4 min under ice cooling. If the solution is allowed to stand for 1 hour under ice-cooling, a crystalline precipitate which was collected by filtration, washed with 75 ml of water and dried to give 6.6 g of crystalline hydrate of S-1090 hydrochloride as white crystalline solid.

Example 7 Crystalline Hydrate of S-1090.

S-1090 hydrochloride (5 g) as prepared in Example 1 (1) above is dissolved in a mixture of 20 ml of methanol, 4 ml of water and 0.3 ml of 6N. HCl. The solution is seeded with 10 mg of crystals and concentrated under reduced pressure to obtain 32 g in weight. The crystalline precipitate was collected by filtration, washed with water and dried to give 3.0 g of crystalline hydrate of S-1090 hydrochloride as white crystalline powder.

Example 8 Crystalline Hydrate of S-1090.

S-1090 hydrochloride (5 g) as prepared in Example 1 (1) above is dissolved in 15 ml of methanol. The solution was dropwise added to 100 ml of 1N. hydrochloric acid at 40oC. The crystalline precipitate was collected by filtration, washed with water and dried to yield 4.6 g of crystalline hydrate of S-1090 hydrochloride as white crystalline powder.

Example 9 Crystalline Hydrate of S-1090.

S-1090 hydrochloride (8 g) as prepared in Example 1 (1) above is dissolved in a mixture of 130 ml of ethanol, 50 ml water and 4 ml of 6N. HCl. The solution is seeded with 10 mg of crystals and concentrated under reduced pressure to 105 g in weight. The crystalline precipitate was collected by filtration, washed with water and dried to yield 5.9 g of crystalline hydrate of S-1090 hydrochloride as white crystalline powder.

Example 10 Crystalline Hydrate of S-1090.

S-1090 hydrochloride (5 g) as prepared in Example 1 (1) above is dissolved in 100 ml of ethanol water (1: 1). The solution was dropwise added to 100 ml of 1N. hydrochloric acid at 40oC. The crystalline precipitate was collected by filtration, washed with water and dried to give 4.5 g of crystalline hydrate of S-1090 hydrochloride as white crystalline powder.

Example 11 Crystalline Hydrate of S-1090.

S-1090 hydrochloride (5 g) as prepared in Example 1 (1) above is dissolved in a mixture of 20 ml of methanol and 0.2 ml of 6N. HCl. The solution was concentrated under reduced pressure to give 10.5 g in weight. To the residue was added 5 ml of ethanol and 10 mg of seed crystals. The crystalline precipitate was collected by filtration, washed with water and dried to give 2.5 g of crystalline hydrate of S-1090 hydrochloride as white crystalline powder.

Example 12 Crystalline Hydrate of S-1090.

S-1090 hydrochloride (5 g) as prepared in Example 1 (1) above is dissolved in a mixture of 30 ml of methanol, 30 ml of isopropanol and 0.5 ml of 6N. HCl. To the solution were added as seed crystals, and 10 mg of the resulting crystalline precipitate was collected by filtration, washed with water and dried to obtain 1.9 g of crystalline hydrate of S-1090 hydrochloride as white crystalline powder.

Example 13 Crystalline Hydrate of S-1090.

S-1090 hydrochloride (5 g) as prepared in Example 1 (1) above is dissolved in a mixture of 160 ml of acetone, 36 ml of water and 10 ml of 3N. HCl. To the solution was added to 10 mg of seed crystals and concentrated under reduced pressure to obtain 85 g in weight. The resulting crystalline precipitate was collected by filtration, washed with water and dried to yield 0.9 g of crystalline hydrate of S-1090 hydrochloride as white crystalline powder.

Example 14 Crystalline Hydrate of S-1090.

S-1090 hydrochloride (5 g), prepared as described in Example 1 (1) above is dissolved in a mixture of 20 ml of methanol, 80 ml of methyl ethyl ketone and 2 ml of 6N. HCl. After adding 10 mg of seed crystals, the solution is concentrated under reduced pressure to give 62 g in weight. To the residue was added 4 ml of methanol and 10 mg of seed crystals. The resulting crystalline precipitate was collected by filtration, washed with water and dried to give 4.4 g of crystalline hydrate of S-1090 hydrochloride as white crystalline powder.

Example 15 Crystalline Hydrate of S-1090.

S-1090 hydrochloride (5 g) as prepared in Example 1 (1) above is dissolved in a mixture of 20 ml of methanol, 30 ml of acetonitrile and 0.5 ml of 6N. of hydrochloric acid. After adding 10 mg of seed crystals, the solution is concentrated under reduced pressure to obtain 40 g in weight. The resulting crystalline precipitate was collected by filtration, washed with water and dried to give 2.0 g of crystalline hydrate as a white crystalline powder.

Example 16 Crystalline Hydrate of S-1090.

S-1090 hydrochloride (5 g) as prepared in Example 1 (1) above is dissolved in a mixture of 20 ml of methanol, 20 ml of ethyl acetate and 0.5 ml of 6N. of hydrochloric acid. After adding 10 mg of seed crystals, the solution is concentrated under reduced pressure to obtain 30 g in weight. The resulting crystalline precipitate was collected by filtration, washed with water and dried to yield 3.5 g of crystalline hydrate of S-1090 hydrochloride as white crystalline powder.

Experiment. Stability of S-1090 hydrochloride and crystalline hydrate thereof.

Stability of S-1090 hydrochloride and crystalline hydrate thereof as prepared in Examples are evaluated by an accelerated stability test conditions of high temperature, humidity and light. Thus, the free amine S-1090, S-1090 hydrochloride and crystalline hydrate of S-1090 hydrochloride were placed under conditions as indicated in Table 2 below and the change in titer and color was evaluated after 0.5 and 1 month from the beginning of the test. The results are shown in Table 2 in which titer is expressed in% of titer remaining after 0.5 and 1 month from the beginning of the test.