INTRODUCΉQN

This invention relates to new cephalosporin esters, which compounds are useful as antibi¬ otics, primarily for treating valuable warm-blooded animals to resist, ward off or combat pathoge- nic infections caused by bacteria susceptible to these cephalosporin compounds.

BACKGROUND OF THE INVENTION The cephalosporin antibiotic ceftiofur, named as 7-[2-(amino-l,3-thiazol-4-yl)-2- (methoxyimino)acetamido]-3-[( ιr-2-ylcarbonyl)thiomethyl]-3-cephem-4-carboxylic acid, its alkali metal, alkaline earth metal and amine salts of the carboxylic acid group and easily hydrolyzable ester groups thereof are described and claimed in Labeeuw et al U.S. Patent No. 4,464,367.

A hydrohalide salt of ceftiofur, particularly the hydrochloride salt thereof, is described and claimed in Amin et al. U.S. Patent No. 4,902,683.

Ochiai U.S. Patent No. 4,278,671 and related patents 4,510,138 and 4,520,194 disclose some 7-[2-(2-aminothiazol-4-yl)-2-(syn)-methoxyiminoacetamido]cep halosporirιs . Many groups are described for positioning in the R ₃ or 3-(Ry _2~) cephalosporin molecule position. Among the many such R ₃ groups are mentioned hydroxy and mercapto at column 1, lines 67 and 68.

Desacetyl cefotaxime, 7-[2-(2-amino-l ,3-thia2X)l-4-yl)-(syn)-2-methoxyiminoacetamido]-3- hydroxymethylceph-3-em-4-caτboxylic acid, is disclosed in a publication entitled "Disposition of Cefotaxime in Rat, Dog and Man" by C. M. Macdonald, et al in Arzneimittel Forschung Drug Research, 34 (Tl), No. 12 (1984), pp. 1719 to 1723.

Published PCT application WO82/03395 published October 14, 1982 discloses some therapeutically active organic compounds which exhibit at least one group comprising the structure -S'S"-R.

INFORMATION DISCLOSURE Y. Yoshimura et al., International Journal of Pharmaceutics, 38: 179-190 (1987), discloses

Synthesis and oral absorption of acyloxymethyl esters of 7β-(2-(2-aminothiazol-4-yl)acetamido)-3-

(((l-(2-dime ylaminoemyl)-lH-tetrazol-5-yl)thio)-methyl)ceph-3-em-4-carbo xylicacid(cefotiam).

Y. Yoshimura, N. Hamaguchi and T. Yashiki, The Journal of Antibiotics, Vol. XXXIX,

No. 9, 1329-1342 (Sept. 1986), disclose preparation of 1-acyloxy ethyl esters of 7-[2-(2- amino±iazol-4-yl)acetamido]-3-[[[l-(2-dimeΛylaminoeώyl)-l H-tetrazol-5-yl]thio]-methyl]ceph-3- em-4-carboxylic acid (cefotiam) and their oral absorption in mice.

T. Nishimura et al., The Journal of Antibiotics, Vol. XL, No. 1, 81-90 (Jan. 1987), disclose orally active l-(cyclohexyloxycarbonyloxy)alkyl ester prodrugs of cefotiam.

SUMMARY OF THE INVENTION

The present invention particularly provides:

A compound of the formula I wherein R _j is

wherein n is one to five, inclusive. The carbon atom content of various hydrocarbon-containing moieties is indicated by a prefix designating the minimum and maximum number of carbon atoms in the moiety, i.e., the prefix C _j -C indicates a moiety of the integer "i" to the integer "j" carbon atoms, inclusive. Thus C-*-C ₄ alkyl refers to alkyl of one to 4 carbon atoms, inclusive, or methyl, ethyl, propyl, butyl, and isomeric forms thereof. The compound of Formula I is useful as an active antibiotic drug compound in pharmaceutical dosage forms for treating valuable warm-blooded animals or humans. Presently, it is contemplated that this compound will be especially useful as a veterinary antibiotic drug to treat valuable warm-blooded animals such as cattle, horses, sheep, monkeys, goats, dogs, cats and the like to fight the effects of bacterial infections caused by organisms such as Pasteurella hemolyt- ica, P.multocida, Haemophilus pleuropneumoniae, H.somnus, Escherichia coli, Salmonella spp., Staphylococcus aureus, Streptococcus agalactiae, Strep, bovis, Strep, dysgalactiae, Strep, faecatis, Strep, uberis, Salmonella typhimurium, E.coli, Staphyloccus aureus, and the like, some of which are commonly associated with infections referred to as "shipping fever" in animals.

Chart A Chart A illustrates the preparation of the esters of the present invention.

The compound of formula A-l, which is prepared by methods described in PCT

International Publication NO. WO 89/04313, published 18 May 1989, is reacted with the compound of formula A-2, which is prepared as described in 43 JACS 660 (1921), in the presence of dimethylformamide to obtain the compound of formula A-3. In these formula, the variables are as defined above. Additional compounds of the present invention, including the other esters of the present invention, are prepared by methods analogous to those described above which would be readily apparent to one of ordinary skill in the art of organic chemistry.

Chart B Chart B describes the preparation of the R _j -desfuroyl ceftiofur disulfide sodium salt which is used as the starting material for Chart A.

In the Chart B formula, the variables are as defined above.

The compounds of the present invention have the advantage of increased oral bioavailability over other cephalosporin compounds. For example, these compounds are more orally bioavailable than the known cephalosporin antiobiotic ceftiofur.

The compounds of the present invention are preferably orally administered in the form of, for example, tablets, capsules and soluble powders. Such means of administration would be readily known and available to one of ordinary skill in the art.

The compounds of this invention are administered in accordance with the preceding description to provide from about 1 mg to about 500 mg of me compound per dose. An effective amount of the compound provided per dose is that amount sufficient to obtain antibiotic effects within me aforesaid non-toxic range. Expressed otherwise, an effective amount of the compound is provided to a recipient within a range from about 0.2 mg/kg to about 100 mg/kg of body weight of the recipient. Preferred dosages for most applications are 0.2 mg/kg to 10.0 mg/kg of body weight of the compound depending upon the animal being treated. In a topical semi-solid ointment formulation the concentration of the compound may be l%-20%, preferably 5%-10% in a carrier, such as a pharmaceutical cream base.

Thus, the compounds of the present invention are useful for obtaining antibiotic effects in mammals, for example, valuable warm-blooded animals such as dogs, cats, horses, and other commercially valuable animals, by administering to the mammals an effective, non-toxic amount of the compounds of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Preparation 1 General Procedure for the Preparation of R _j -desfuroylceftiofur Disulfide Sodium (Refer to Chart B: wherein the variables are as defined above)

The R-desfuroylceftiofur disulfide is dissolved in tetrahydrofuran (10ml) and cooled to 0°C.

Sodium 2-ethylhexanoate in tetrahydrofuan solution (14ml, 1.6g) is added causing the precipitation of a white solid. It is filtered and washed with acetone to give a white solid. Example 1 General Procedure for the Synthesis of 1-Acyloxyalkyl Esters and 1- (Alkyloxycarbonyloxy)alkyl Esters of Methyl- and Furfuryl-desfuroylceftiofur Disulfide Sodium Salts (Refer to Chart A: wherein the variables are as defined above. The sodium salt is dissolved in dimethylformamide (10ml) and cooled to -5°C. Iodoalkyloxy acylate or alkyl-iodoalkyl carbonate (2.44mmol) is added with stirring, and die mixture is stirred at RT for 30 minutes. It is poured into a mixture of ethyl acetate (50ml) and ice cold H ₂ O (40ml) and the organic layer is separated. The aqueous layer is extracted with ethyl acetate (40ml). The combined ethyl acetate layer is washed with ice cold H ₂ O (3x40ml) and dried over magnesium sulfate. The organic layer is evaporated in vacuo, and me residue is taken up in methylene chloride and the undissolved portion is filtered. The filtrate is flash chromatographed on a silica gel column eluting with 1:1 ethyl acetate:methylene chloride. Appropriate fractions are combined and evaporated in vacuo. The residue is crystallized or recrystallized from e yl acetate- ethyl ether-hexane to give white crystals.

Example 2 5-Thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, 7-[[(2-amino-4-thiazolyl) (meΛoxyimino)acetyl]amino]-8-oxo-3-[φropyldithio) methyl], (acetyloxy) methyl ester, [6R- [6α,7B(Z)]]-(Formula A-3: R _χ is -S-(CH ₂ ) ₂ -CH ₃ ) Refer to Chart A. Propyl-desfuroylceftiofur disulfide sodium (445 mg),is dissolved in dimethylformamide (3.0 ml) and cooled to -10 to -20°C. A solution of iodomethyl acetate (0.2 g) in dimethylformamide (0.5 ml) is added slowly with stirring, and the mixture is stirred at room temperature for 30 minutes. The mixture is poured into a mixture of ethyl acetate (25 ml) and chilled water (10 ml) and the organic layer is separated. The aqueous layer is extracted with ethyl acetate (2 x 15 ml). The combined organic layer is washed with chilled water (3 x 15 ml) and saturated sodium chloride solution (15 ml) and dried over sodium sulfate. The organic layer is evaporated in vacuo to get a brown viscous oil. It is dissolved in methylene chloride and loaded onto 8 g silica gel column and eluted with 1:1 methylene chloride: e yl acetate. Those fractions containing pure product are combined and concentrated in vacuo to get a yellow viscous oil. It is crystallized from ethyl acetate-ethyl ether-hexane to give 81 mg of an off-white solid. Physical characteristics are as follows: HPLC of the product shows no peak in 1:1 methano water. Mass spec., FAB [m + H] ⁺ 576. TLC, RF 0.34 (SiO-,) 1:1 CH ₂ Cl ₂ :EtOAc. Example 3 5-Thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, 7-[[(2-amino-4-thiazolyl) (methoxyimino) acetyl]amino]-3-[[(4 methoxyphenyl)dithio]methyl]-8-

oxo-,(acetyloxy)methyl ester, [6R- [6α,7β(Z)]]-(Formula A-3: R _χ is -S-(4- methoxyphenyl)) Refer to Chart A. 4-Methoxphenyl-desfuroylceftiofur disulfide sodium (474 mg) is dissolved in dimethylformamide (3.0 ml) and cooled to -10 to -20°C. A solution of iodome yl acetate (0.2 g) in dimethylformamide (0.5 ml) is added slowly with stirring, and the mixture is stirred at room temperature for 30 minutes. The mixture is poured into a mixture of ethyl acetate (25 ml) and chilled water (10 ml), and the organic layer is separated. The aqueous layer is extracted with ethyl acetate (2 x 15 ml). The combined organic layer is washed with chilled water (3 x 15 ml) and saturated sodium chloride solution (15 ml), and dried over sodium sulfate. The organic layer is concentrated in vacuo to get a brown solid. It is dissolved in methylene chloride and loaded onto 8 g silica gel column and eluted with 1:1 methylene chloride: ethyl acetate. Those fractions containing pure product are combined and concentrated in vacuo to get a yellow viscous oil. It is crystallized from ethyl acetate-ethyl ether to give 106 mg of an off-white solid. Physical characteristics are as follows: HPLC of the product shows a small broad peak at about 20 minutes in 1 : 1 methanol : water.

Mass spec., FAB [m + H] ⁺ 640. TLC, Rf, 0.36 (SiO- _j ) 1:1 CH ₂ Cl ₂ :EtOAc. Example 4 3,4-Dimethoxyphenyl-desfuroylceftiofur disulfide acetyloxymethyl ester (FormulaA-3: R _χ is -S-(3,4-dimethoxyphenyl)) Refer to Chart A. 3,4-Dimet oxyphenyl-desfuroylceftiofur disulfide sodium (538 mg is dissolved in dimethylformamide (3.0 ml) and cooled to -10 to -20°C. A solution of iodomethyl acetate (0.2 g) in dimeώylformamide (0.5 ml) is added slowly with stirring, and the mixture is stirred at room temperature for 30 minutes. The mixture is poured into a mixture of ethyl acetate (25 ml) and chilled water (10 ml), and the organic layer is separated. The aqueous layer is extracted with ethyl acetate (2 x 15 ml). The combined organic layer is washed with chilled water (3 x 15 ml) and saturated sodium chloride solution (15 ml), and dried over sodium sulfate. The organic layer is concentrated in vacuo to get a brown solid. It is dissolved in methylene chloride and loaded onto 10 g silica gel column and eluted with 1:1 methylene chloride: ethyl acetate. Those fractions containing pure product are combined and concentrated in vacuo to get a yellow solid. It is recrystallized from ethyl acetate-ethyl ether to give 99 mg of an off-white solid. Physical characteristics are as follows:

HPLC of the product shows a small broad peak at about 16 minutes in 1 : 1 methanol: water. Mass spec, FAB [m + H] ⁺ 670. TLC, Rf 0.21 (SiO- _j ) 1:1 CH ₂ Cl ₂ :EtOAc. Example 5 5-Thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, 7-[[(2-amino-4- thiazolyl)(methoxyimino)acetyl]amino]-3-[(methyldithio) methyl] -8-oxo-

(acetyloxy)methyl ester, [6R- [6α,7β(Z)]]-(Formula A-3: R _χ is -S-CH ₃ ) Refer to Chart A. Methyl-desfuroylceftiofur disulfide sodium (0.50 g) is dissolved in dimethylformamide (3.7 ml) and cooled to -10 to -20°C. A solution of iodometiiyl acetate (0.25 g) in dimethylformamide (1.0 ml) is added slowly with stirring, and the mixture is stirred for another 10 minutes. The mixture is poured into a mixture of ethyl acetate (30 ml) and chilled water (15 ml) and Λe organic layer is separated. The aqueous layer is extracted with ethyl acetate (2 x 20 ml). The combined organic layer is washed with chilled water (3 x 20 ml) and saturated sodium chloride solution (20 ml) and dried over sodium sulfate. The organic layer is evaporated in vacuo to get a light yellow solid. It is dissolved in methylene chloride and loaded onto 6 g silica gel column and eluted with 1:1 methylene chloride: ethyl acetate. Those fractions containing pure product are combined and concentrated in vacuo to get a light yellow solid. It is recrystallized from methanol-ethyl acetate- ethyl ether to give 92 mg of a white solid.

Physical characteristics are as follows: HPLC of the sample shows a single peak in 1:1 methanol: water.

Mass spec., FAB [m + H] ⁺ 548. TLC, Rf 0.34 (SiO-2), 1:1 CH ₂ Cl ₂ :EtOAc. Example 6 In Vivo Mouse protection tests

For the mouse protection tests, 12-14 g weanling female mice 21-25 days old Crl:CD-l (ICR) BR Swiss mice are used. For the infection portion of the tests, bacteria (Pasteurella hemolytica or Salmonella typhimurium are mixed with 2% brewer's yeast (as adjuvant for infection) and administered intraperitioneally at approximately 100 LD50 doses. Antibiotics (0.1 ml) are administered per os into the stomach, using a 22G oral dosing needle, immediately after and at post-challenge exposure hours 24 and 48. Groups of 6 to ten mice are injected at each dosage level of antibiotic and at least 5, 2-fold serial dilutions of antibiotic are used for a single determination. Measurement of antibiotic effectiveness is calculated on post-challenge exposure day 6 and is reported as the ED50, the amount of antibiotic in milligrams per kilogram per day required to protect 50% of infected mice. This value is estimated by the statistical method of probit analysis. As mentioned, in all experiments the challenge-exposure dose is approximately 100 times the LD50. The LD50 for the bacteria is titrated for each experiment. Also, in each experiment, a nontreated, saline-dosed group is used to prove the lethality of the 100 LD50 dose.

In all experiments 90-100% of the nontreated mice died of infections as a result of inoculated bacteria.

The test compounds are dissolved in 95% ethanol and diluted in sterile vehicle 122 (0.25% methyl cellulose) to a final concentration of 10% ethanol before final dilutions for the test in vehicle 122.

The results of this testing of the compounds of the present invention are given in Table 1. Preparation 2 Desfuroylceftiofur

Ceftiofur sodium (25.0g) is dissolved in 500ml H ₂ O and the pH of this solution, which is around 5, is adjusted to 9 with ammonium hydroxide. Then a solution of dithioerythritol (10. lg) in 250ml H ₂ O is added and the white mixture is stirred at RT for 1 hr. The mixture is brought to between pH 3.0-3.5 with 85% phosphoric acid resulting in the precipitation of white solids. The solids are filtered and washed with H ₂ O. After drying the solids in vacuo in a dessicator , the yield of the title product is about 16g. Preparation 3 Methyl-desfuroylceftiofur Disulfide Ceftiofur sodium of Preparation 2 (5.00g) is dissolved in 100ml H ₂ 0, and me pH of this solution is adjusted to 9 with NH OH. Then a solution of dithioerythritol (2.25g) in 5ml H ₂ O is added resulting in a cloudy solution, and the mixture is stirred at RT for 1 hr. MeOH (140ml) is added to the mixture to make it clear, then methyl methanetiiiolsulfonate (2.65g) in MeOH (30ml) is added slowly to the mixture causing the mixture to become opaque. The mixture is stirred at RT for 1 hr. The liquid is decanted into another flask to leave behind an orange viscous material. The decanted MeOH is evaporated in vacuo to precipitate yellow viscous semi-solid material sticking to the sides of the flask. After discarding the remaining H ₂ O in the flask, the residue in the flask is dissolved in a minimal volume of MeOH, then enough ice H ₂ O is added to precipitate off-white solid clumps in white liquid. The liquid is discarded, and the solid is washed with ice H ₂ O and dried in vacuo in a dessicator to give 4.65g of off-white crystals. Physical characteristics are as follows:

HPLC, about 9 min. in TBA (1:1 MeOH:H ₂ O with 0.005M tetrabutylarnmonium hydroxide solution adjusted to pH 7 with acetic acid). MS, FAB [m + H] ⁺ 476. Preparation 4 Methyl-desftiroylceftiofur Disulfide Sodium

Methyl-desfuroylceftiofur disulfide of Preparation 3 (4.65g) is dissolved in tetrahydrofuran (10ml) and cooled to O°C. Sodium 2-ethylhexanoate in tetrahydrofuran solution (14ml, 1.6g) is added causing the precipitation of a white solid. It is filtered and washed with acetone to give 2.91g of the title product. Example 7 Methyl-desfuroylceftiofur Disulfide Acetyloxypropyl Ester

The sodium salt of Preparation 4 (l.OOg) is dissolved in dimethylformamide (10ml) and cooled to -5°C. 1-Iodomethyl propionate (0.55g) is added with stirring, and the mixture is stirred at RT for 30 minutes. It is poured into a mixture of ethyl acetate (50ml) and ice cold H ₂ O (40ml) and the organic layer is separated. The aqueous layer is extracted with ethyl acetate (40ml). The combined ethyl acetate layer is washed with cold H ₂ O (3x40ml) and dried over magnesium sulfate. The organic layer is concentrated in vacuo to get a light yellow solid. It is taken up in methylene

chloride and the undissolved portion is filtered. The filtrate is flash chromatographed on a silica gel column eluting with 1:1 ethyl acetate:methylene chloride. Appropriate fractions are combined and evaporated in vacuo to yield a yellow oil. It is crystallized from ethyl acetate-ethyl ether- hexane to give 75mg of white crystals. Physical characteristics are as follows:

HPLC, no peak in 1:1 MeOH:H ₂ O or TBA

MS, FAB [m + H] ⁺ 576

TLC, Rf 0.39 (SiO- _j ) 1:1 CH ₂ Cl ₂ :EtOAc. Examples 8 - 11 The following additional compounds of the present invention are prepared by methods analogous to those described above which would be readily apparent to one of ordinary skill in the art of organic chemistry: Example 8 Memyl-desfuroylceftiofur disulfide l-(isopropyloxycarbonyloxy)ethyl ester.

Physical characteristics are as follows: HPLC, no peak in 1:1 MeOH:H ₂ 0 or TBA

MS, FAB [m + H] ⁺ 606

TLC, Rf 0.28 (SiOz) 1:1 CH ₂ Cl ₂ :EtOAc. Example 9 Furfuryl-desfuroylceftiofur disulfide l-(isopropyloxycarbonyloxy)ethyl ester.

Physical characteristics are as follows: HPLC, no peak in 1:1 MeOH:H ₂ 0 or TBA

MS, FAB [m + H] ⁺ 672

TLC, Rf 0.39 (SiO-2) 1:1 CH ₂ Cl ₂ :EtOAc. Example 10 Methyl-desfuroylceftiofur disulfide l-(cyclohexylacetoxy)ethyI ester.

Physical characteristics are as follows: HPLC, no peak in 1:1 MeOH:H ₂ 0 or TBA

MS, FAB [m + H] ⁺ 645

TLC, Rf 0.45 (SiO _j ) 1:1 CH ₂ Cl ₂ :EtOAc. Example 11 Furfuryl-desfuroylceftiofur disulfide l-(cyclohexylacetoxy)ethyl ester.

Physical characteristics are as follows: HPLC, no peak in 1:1 MeOH:H ₂ 0 or TBA

MS, FAB [m + H]+ 711

TLC, Rf 0.44 (SiO-,) 1:1 CH ₂ Cl ₂ :EtOAc.

Table 1

10

15

F RMULA CHART

Chart A

OGI- ₃

10

15

R ₂ -I

A-2

20

ChartB

0

10

4-

B-3