CA1200544A

CA1200544A - Crystalline cephalosporin

Info

Publication number: CA1200544A
Application number: CA000451713A
Authority: CA
Inventors: Byron W. Daugherty
Original assignee: Eli Lilly and Co
Current assignee: Eli Lilly and Co
Priority date: 1983-07-22
Filing date: 1984-04-11
Publication date: 1986-02-11
Also published as: PH18429A; US4474780A

Abstract

Abstract 7-(D-2-Naphthylglycylamido)-3-methyl-3-cephem-4-carboxylic acid tetrahydrate is a stable crystal form of a potent orally active gram positive antibiotic.

Description

~30S4~

X-6283~

CRYSTALLINE CEP~ALOSPORIN

Background of the Invention 7-(D-2-Naphthylglycylamido)-3-methyl-3-cephem-4-carboxylic acid recently has been discover~d to be a potent orally active antibiotic, displaying favorable pharmacokinetics and excellent gram positive activity.
The com~ound is synthesized by reacting 7-amino-3-methyl-3-cephem-4-carboxylic acid (7-ADCA) with an N-protected 2-naphthylglycine acyla~ing agent, rollowed by removal of the protecting group.
An object of this invention is to provi~e a new compound that is a stable crystalline form of 7-(D-2-naphthylglycylamido)-3-methyl-3-cephem-4 car-boxylic acid.

Summary of the Invention This invention provides a crystalline com-position of matter which is 7-(D-2-naphthylglycylamido)-3-methyl-3-cephem-4-carboxylic acid tetrahydrate. The crystals are large, dense and stable, and readily lend themselves to milling and grinding for adaptation to pharmaceutical formulation, particularily into solid dosage forms such as filled capsules and the like. The tetrahydrate of this invention is prepared by isolating 7-(D-2-naphthylglycylamido)-3-methyl-3-cephem~a-carbo~ylic acid from an aqueous medium.

: ~ 2()Q~4~

Detailed Description of the Invention The crystalline compound provided by this in-vention has the following unique x-ray powder diffraction properties when measured with a 114.6 mm Debye-Scherrer camera contal n; ng a nickel filtered copper radiation of 1.5405A:

Spacing, d: Relative intensities, I/I

13.29 .44 10.53 .22 .44 1 .00 6.09 .22 5.~4 .33 5.36 .88 5.14 .28 4.86 .17 4.66 .11 4.44 .33 4.12 .28 3.92 .17 ~ZQ/~

3.75 .~2 3.65 .94 3.51 .38 3,34 .72 ~.94 .39

2.78 .17 2.69 .14 2.58 .11 2.46 .17 2.42 .17 2.32 .11 2.25 .03 2.18 .14 2.10 .06 2.0~ .06 The compound provided by this invention can be prepared by reacting an acid addition salt of 7-~D-2-naphthylglycylamido)-3-methyl-3-cephem-4-carboxylic acid with a base such as sodium hydroxide or triethyl-amine so as to form the corresponding zwitterion, and crystallizing the zwitterion from water. For example, a salt such as the trifluoroacetic acid salt or the hydrochloric acid salt can be dissolved in water or a l~æ~)s4~

X-6~83 -4-mixture of water and an organic solvent such as acetone or acetonitrile. A base such as aqueous ammonium hydroxide is added to adjust the p~ to about 3 to about 5. The precipitate that forms is the tetrahydrate of this invention and is readily recrystallized from water.
The compound of this invention alternatively can be prepared by isolating the product of acylation of 7-amino-3-methyl-3-cephem-4-carboxylic acid (7-ADCA) from a solvent cont~lning water. For example, 7-ADCA, typically as a silylated derivative, can be acylated with an N-protected 3-2-naphthylglycine acid halide or mixed anhydride. The acylation generally is carried out in an organic solvent such as acetonitrile. Once the acylation is complete, the protecting groups can be removed by standard procedures and the solution of 7-(D-2-naphthylglycylamido)-3-methyl-3-cephem-4-car-boxylic acid can be diluted wi~h water so that it con-tains abo-lt 10 to S0% by volume of water, and the pH of the solution can be adjusted to about pH 3 to about 5.
The crystalline product that forms is 7-(D-2-naphthyl-glycylamido)-3-methyl-3-cephem-4-carboxylic acid tetra-hydrate.
The preparation of the compound of this in-vention is more fully described in the following detailed examples.

05~9~

Preparation 1 Preparation of D,L-2-naphthylglycine (also named ~-amino-~-(2-naphthyl)acetic acid) A solution of 1~.6 g (0.1 m) of 2-n~phAlde-hyde in 700 ml of 50% ethanol-water cont~ln-ng 14.7 g (0.3 m) of sodium cyanide and 3~.4 g (0.4 m) of ammonium carbonate was hea~ed at 50C for twenty hours. The reaction mixture was cooled and concentrated to a~out 400 ml by evaporation under reduced pressuxe, and then the solution was made acidic to p~ 2.0 by the addition of conc. hydrochloric acid. The solid precipitate that formed was collected by filtration, washed witn dilute hydrochloric acid, and then dried to afford 22.1 g of lS 4-(2~naphthyl)-2,4-imidazolidindione.
A solution of ~.O g (22 ~I) of ~he 4~
naphthyl)-~,4-imidazolidindione in 100 ml of 1S% (v/v) aqueous sodium hydroxide was heated at reflu~ for two and one-half hours. The reaction mixture was then filtered, cooled, and washed with ethylacetate. The aqueous solution was next diluted with 6N hydrochloric acid to p~ 5.1 and filtered to provide D,L-2-naphthyl~
glycine. The reaction was repeated several times to produce larger quantities of the product.
A 10 . O g sample of D,L-2-naphthylglycine was purified by dissolving it into 125 ml of methanol con-t~l nl ng 3.9 ml of acetyl chloride. The reaction mix-ture was filtered and the filtrate cont~ nlng D,L-2-naphthylglycine hydrochlolide was diluted with 5 ml of aniline. The precipitated product was collected by filtration and dried to give 7.0 g of D,L-2-naphthyl-glycine. m.p. 219-221C.

lz~5~

Preparation 2 Resolution of 2-naphthylglycine A mixture of D and L 2-naphthylglycine (from Preparation 1) was reacted with di-tert.-bu-tyl carbonate and sodium hydroxide to provide D,L-N tert.-butoxy-carbonyl-2-naphthylglycine. The t.Boc naphthylglycine was re-acted with optically pure a-aminoethylbenzene in the presence of N,N'-dicyclohexylcarbodiimide to provide N~ phenylethyl)-~-tert.-butoxycarbonylamino-a-(2-naphthyl)acetamide. Separation of the diastereomers by chromatography over silica gel afforded, following acid hydrolysis with 6N hydrochloric acid, D-2-naphthyl-glycine (O~ -lg0 + 3) and L-2-naphthylglycine (OR = +190 + 3).

Preparation 3 Preparation of D-N-tert.-butoxycarbonyl-2-naphthyl-glycine To a stirred solution of 10 g (50 mM) of D-2-naphthylglycine (from Preparation 2) in 100 ml o lN
sodium hydroxide were added 50 ml of tetrahydrofuran followed by 30 g (1~0 m~l) of di-tert.-butyl carbonate.
The reaction mixture was stirred at 24C for four hours.
The product was isolated by first washing the reaction mixture three times with 50 ml portions of diethyl ether, and then the mixture was made acidic to pH 2.0 by the addition of conc. hydrochloric acid. The agueous ~'~Q(~4 X-6283 _7_ acid mixture was extracted several times with ethyl acetate, and the extracts were combined, washed with water, dried and the solvent was removed by evap-oration under reduced pressure to provide 12.8 g (85% yield) of D N-tert.-butoxycarbonyl-2-naphthylglycine.
NMR (DMSO): ~ 2.5 (s, 9H); ~ 6.85 (s, lH); ~ 7.28-7.9 (m, 7H~.

Example 1 7-(D-2-~aphthylglycylamido)-3-methyl-3-cephem-4-carboxylic acid trifluoroacetate salt To a stirred suspension of 1.0 g (4.7 mM) of 7-amino-3-methyl-3-cephem-~-carboxylic acid (7-ADCA) in 25 ml of acetonitrile were added in one portion 3.7 ml (14.0 mM) of bis(trimethylsilyl)trifluoro-acetamide. The reaction mixture was stirred at room temperature until all solids had dissolved, thus indicating complete formation of the trimethylsilyl ester of 7-.~DCA.
In a separate flask a solution of 1.35 g (4.5 mM) of D-N-tert.-butoxycarbonyl-2-naphthylglycine (from Preparation 3) in 20 ml of acetonitrile containing 1.1 g (4.5 mM) of N-ethoxycarbonyl~2-ethoxy-1,2-di-hydro-guinoline (EEDQ) was stirred at room temperature for fifteen minutes. This solution was then added in one portion to the cold (0C) acetonitrile solution contAi ni ng the trimethylsilyl ester of 7-ADCA from above. The reaction mixture was stirred for one hour at 0C, and then warmed to room temperature. The solvent )Sg~4 ~-6283 -8-was removed by evaporation under reduced pres-sure to give an oil, and the oil was dissolved in ethyl acetate, washed two times with lN hydrochloric acid, dried, and the solvent was removed by evaporation to provide 7-(D-N-tert.-butoxycarbonyl-2-naphthylglycyl-amido)-3-methyl-3-cephem-4-carboxylic acid as a foam.
The N-pror.ected naphthylglycyl cephalosporin thus produced was dissolved in 5 ml of trifluoroacetic acid, and then the trifluoroacetic acid was removed by evaporation under reduced pressure to provide, followiny precipitation from diethyl ether, 5.7 g of 7-(D-2-naphthylglycylamido)-3-methyl-3-cephem-4-carboxylic acid trifluoroacetate salt.

Example 2 7-(D-2-Naphthylglycylamido)-3-methyl-3-cephem-a-car~oxylic acid tetrahydrate A mixture of 5.7 g of the trifluoroacet.ic acid addition salt from Example 1 in 55 ml of 10% (v/v) water and acetonitrile was warmed to about 50~C and then filtered to remove the undissolved solids. The filtrate was diluted with 1.3 molar ammonium hydroxide to pH 4.5.
The precipitate that formed W2S collected by filtration and dried to sive 3.15 g (72% yield) of 7-(D-2-naphthyl-glycylamido)-3 methyl-3-cephem-~-carboxylic acid tetra-hydrate.

'-~` 120~

X~6283 -9-Example 3 The procedure of Example 1 was repeated using S.0 g of optically active D-N-tert.-butoxycarbonyl-2-naphthylglycine and 5.6 g of 7-ADCA to provide, fol-lowing removal of the protecting groups, 6.8 g (80%
yield) of D-7-(2-naphthylglycylamido)-3-methyl-3-cephem-4 carboxylic acid trifluoroacetic acid salt. The salt thus formed was dissolved in 90 ml of aceto-nitrile and 10 ml of water. The pH of the solution was adjusted to 4.0 by addition of triethylamine, and the reaction mixture was stirred at 25C for twenty minutes. The mixture was filtered and the filtrate was concen-trated to dryness and the product was crystallized from water to give 2.9 g of 7-(D-2-naphthylglycylamido)-

3-methyl-3-cephem-4-carboxylic a~id tetrahydrate.
Analysis calc. for C20H19N3O4S 4H20 Theory: C, 51.16; H, 5.80; N, 8.95; S, 6.93;
Found: C, 52.52; H, 5.47; N, ~.73; S, 6.83.
NMR (DMSOd6~: ~ 1.9 (s, 3H~; ~ 4.8 (s, lH); ~ 4.9 (dd, lH); ~ 5.6 (dd, lH); ~ 7.49-7.99 (m, 7~).

Example 4 Preparation of 7-(D-2-naphthylglycylamido)-3-methyl-3 cephem-4-carboxylic acid tetrahydrate D-2~Naphthylglycine sodium salt was protected as an en~;ne by reaction with methyl acetoacetate. A
suspension of 102 g (317.7 mM) of the protected D-2~
naphthylglycine sodium salt in 1000 ml of acetonitrile and 500 ml of N,N-dimethylformamide was cooled to -30C

~Z~4~

and stirred while 0.88 ml of methane sulfonic acid was added in one portion, followed by the addition of 0.90 ml of N,N-dimethylbenzylamine and 24.7 ml of methyl chloroformate. The reaction mixture was stirred at -30C for two hours, and was the~ diluted by dropwise addition of a solution of 68.5 g (302.8 mM) of 7-amino-3-methyl-3-cephem-4-carboxylic acid in 460 ml of acetonitrile cont~ln~ng 118.8 ml hexamethyldisilazane.
The reaction mixture was stirred at -30C for about two hours following com-plete addition and then warmed to 0C. The reaction mix~ure was diluted by addition of 320 ml of lN hydro-chloric acid, followed by addition of 35.7 g of semi-carba2ide hydrochloride~ Ammonium hydroxide was added to adjust and maintain the pH at 3.0 while the mixture was warmed to 22C. The reaction mi~ture was further diluted by addition of 430 ml of watex, and ~hen de-colorized by stirring for fifteen minutes with 10.0 g of charcoal. The reaction mixture was filtered through hyflo filter aid and the filtrate was warmed to 40C. The pH was adjusted to 4.0 by addition of lN ammonium hydroxide, whereupon crystal-lization started. Crystallization continued for about thirty minutes, and then the p~ W2S raised to S.~ by addition of lN ammonium hydroxide. The mixture was cooled to 20C and stirred for one hour and filtered.
The ilter cake was washed twice with 50 ml portions of water and air dried to give 110.8 g of 7-(D-2-naphthyl-glycylamido)-3-methyl-3-cephem-4-carboxylic acid tetrahydrate.

.. .. ... .. .. .. . . . . ~ .

~Z~OS4~

Analysis calc. for C2oH1gN304S-4H20 Theory: C, 51.16; H, 5.80; N, 8.97; S, 6.83;
Found: C, 50.31; H, 5.62; N, 8.87; S, 6.89.
Karl Fisher water analysis:
Theory: 15.3 Found: 13.73%
NMR (TFA): ~ 2.2 (s, 3H); 3.21 ~g, 2H); 5.08 (d,~lH);
5.68 (m, 2H); 7.4-8.3 ~m, 7H); 10.5 (s, 12H).
IR (KBr): 1766, 1751, 1696 cm 1 W (C~30H) Amax 227, ~, 78,000 AmaX ~65, , 12,000, Titra~ion (66~ N,N-dime~hylformamide/water) pKa 5.5, 7.5, 11.2.
The compound of this invention is useful as an oral antibiotic, particularly in the treatment of in-fections caused by gr~l-positive organisms such as S.
aureus, S. pyo~enes and H. influenza. The compound is also effective against anaerobic cocci such as Pepto-streptococcus anaerobius and Peptostrept. intermedius.
The compound is very well absorbed following oral admin istration, and its favorable pharmacokinetics make it particularly attractive as an oral treatment for upper respiratory infections.
The compound will be administered at a dose of about 0.5 to about 50 mg/kg of ~nl ~1 body weight, and more normally at a dose of a~out 1 to about 10 mg/kg.
Such amounts will be administered to a human from once to about four times a day for th~ effecti~e control and prevention of bacterial infections. A typical daily ~ . , . , ~ . . .
`:

5~4 adult dose will be from about 200 to about 500 mg. per day.
The crystal form provided by this invention will be formulated for convenient oral or parenteral a~ml nl stration, and such ormulation will contain about O.1 to about 95 percent by weight of active ingredient.
The compound will be mixed with typical pharmaceu~ical carriers and excipients such as corn starch, sucrose, microcrystalline cellulose, gelatin, and the like. The formulations will be molded into tablets or placed into gelatin capsules, or made into solutions or suspensions for convenient oral a~mi nl stration. The compound can also be ormulated as a slow release long term dosage form employing conventional technology.
The crystal form provided by this invention is very s~able for prolonged periods of time, yet is very well absorbed following oral administration. This is somewhat suxprising since the compound is only ml nl ~a1 ly solu~le in water. For example, the compound forms a saturated solution in water at 37C according to the following table:
pH Solubility, ms/ml 1.2 2.0 3.0 ~Q0~44

4.0 0.

5.0 0.2

6.0 0-3

7.0 0.~
s

8.Q 4.1 8.5 - . `

.~

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for preparing 7-(D-2-Naphthyl-glycylamido)-3-methyl-3-cephem-4-carboxylic acid tetra-hydrate which comprises crystallizing 7-(D-2-naphthyl-glycylamido)-3-methyl-3-cephem-4-carboxylic acid from an aqueous medium.

2. 7-(D-2-Naphthylglycylamido)-3-methyl-3-cephem-4-carboxylic acid tetrahydrate whenever prepared by a process according to claim 1, or by an obvious chemical equivalent thereof.