GB2289674A - Antibacterial naphthyridine - Google Patents

Abstract

The compound 7-([1 alpha ,5 alpha ,6 alpha ]-6-acetylamino-3-azabicyclo [3.1.0]hex-3-yl)-1-(2,4-difluorophenyl)-6-fluoro-1,4-dihydro-4-oxo- 1,8-naphthyndine-3-carboxylic acid is an antibacterial agent of use in the treatment of bacterial infections, and gastric and duodenal ulcers.

Description

ANTIBACTERIAL NAPHTHYRIDINE This invention relates to a novel naphthyridine carboxylic acid, antibacterial compositions containing such compound and methods of treating bacterial infections using such compound. The compound also, in particular, inhibits Helicobacter infections.

U.S. Patent 5,164,402 discloses the antibacterial agent 7-([1 a, So, 6a]-6-amino-3- azabicyclo[3. 1 .O]hex-3-yl)-1 -(2,4-difluorophenyl)-6-fluoro-1 ,4-dihydro-4-oxo-1 ,8naphthyridine-3-carboxylic acid.

This invention relates to 7-([1a, 5a, 6a]-6-acetylamino-3-azabicyclo[3.1 .O]hex-3- yl)-1 -(2,4-difluorophenyl)-6-fluoro-1 ,4-dihydro-4-oxo-1 ,8-naphthyridine-3-carboxylic acid (the active compound), having antibacterial activity and also being an inhibitor of Helicobacter infections.

The invention also relates to an antibacterial composition comprising an antibacterially effective amount of the active compound and a pharmaceutically acceptable carrier.

The invention further relates to a method for the treatment of a bacterial infection by administering to a subject affected by a bacterial infection an antibacterially effective amount of the active compound; a method for the treatment of a Helicobacter infection by administering to a subject affected by such infection an effective amount of the active compound, and a method for the treatment of gastric and duodenal ulcers by administering to a subject in need of such treatment an effective amount of the active compound.

The active compound of the invention may be prepared by acetylation of the monomethanesulfonate of the amine of the formula

The preparation of this amine is disclosed in U.S. Patent 5,164,402. The acetylation is with a conventional acylating agent such as CH3-C(O)X wherein Xis chloro or acetoxy.

The reaction is conducted in a reaction-inert solvent such as dimethylformamide, in the presence of a base such as pyridine. The reaction temperature generally ranges from about 400C to about 11 00C and the reaction time generally ranges from about 3 hours to about 24 hours, usually from about 3 to 9 hours.

According to the invention, particularly for the treatment of ulcers, the active compound may be used in combination with a second antimicrobial agent, such as nitroimidazole antibiotics, e.g. tinidatole and metronidazole; tetracyclines, e.g.

tetracycline, doxycycline and minocycline; penicillins, e.g. amoxicillin, ampicillin and mezlocillin; cephalosporins, e.g. cefaclor, cefadroxil, cephadrine, cefuroxime, cefuroxime axetil, cephalexin, cefpodoxime proxetil, ceftazidime and ceftriaxone; carbapenems, e.g. imipenem and meropenem; aminoglycosides, e.g. paromomycin; macrolide antibiotics, e.g. erythromycin, clarithromycin and azithromycin; lincosamide antibiotics, e.g. clindamycin; rifamycins, e.g. rifampicin; and nitrofurantoin. Also included within the invention are combinations of the active compound with a pharmaceutical compound used in the treatment of acid-related disorders such as acid pump inhibitors, e.g. omeprazole and lansoprazole, or H2 antagonists, e.g. ranitidine, cimetidine, and famotidine.

The active compound is useful in the treatment of bacterial infections caused by Gram-positive or Gram-negative bacteria. The active compounds are also useful in the treatment of H. pylori in humans, and related Helicobacter infections in animals, e.g., H. felis in cats. H. pviori, a pathogenic bacterium discovered in 1982, has been established as the cause of gastric and duodenal ulcers in humans. In clinical trials, eradication of the organism has been shown to result in healing of the ulcer and a low incidence of relapse. The active compound is therefore useful in the treatment of gastric and duodenal ulcers. As used herein, Treatment is meant to include the cure and alleviation of bacterial infections, the eradication of the Helicobacter microorganism and the alleviation of the gastric and duodenal ulcers.

The active compound of the invention may be administered alone, but will generally be administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

For example, the active compound can be administered orally or in the form of tablets containing such excipients as starch or lactose, or in capsules either alone or in admixture with excipients, or in the form of'elixirs or suspensions containing flavoring or coloring agents. In the case of animals, the active compound is advantageously contained in an animal feed or drinking water in a concentration of 5-5000 ppm, preferably 25-500 ppm.

The active compound can be injected parenterally, for example, intramuscularly, intravenously or subcutaneously. For parenteral administration, the active compound is best used in the form of a sterile aqueous solution which can contain other solutes, for example, enough salt or glucose to make the solution isotonic. In the case of animals, the active compound can be administered intramuscularly or subcutaneously at dosage levels of about 0.1-50 mg/kg/day, advantageously 1-5 mg/kg/day given in a single daily dose or up to 3 divided doses.

The active compound of the invention can be administered to humans by either the oral or parenteral routes, and may be administered orally at dosage levels of about 0.1 to 50 mg/kg/day, advantageously 1-5 mg/kg/day given in a single dose or up to 3 divided doses. For intramuscular or intravenous administration, dosage levels are about 0.1-50 mg/kg/day, advantageously 1-5 mg/kg/day. While intramuscular administration may be a single dose or up to 3 divided doses, intravenous administration can include a continuous drip. Variations will necessarily occur depending on the weight and condition of the subject being treated and the particular routes of administration chosen as will be known to those skilled in the art.

The antibacterial activity of the active compound is shown by testing according to the Steer's replicator technique which is a standard in vitro bacterial test described by E. Steers et al., Antibiotics and Chemotherapy, 9, 307 (1959).

The activity of the active compound in vitro against H. nvlori may be shown by the following procedure.

Agar Dilution of Antimicrobial 6 mg. of the active compound is solubilized in 0.6 ml 100% dimethylsulfoxide (DMSO) and then brought up to 6 ml with sterile brucella broth and the solubility is noted. The final concentration of DMSO is 10% of the total volume. Serial 2-fold dilutions (3 ml compound + 3 ml brucella broth) are then made in sterile brucella broth.

A 2 ml aliquot of each broth dilution within the series is placed in separate sterile petri dishes, to which 18 ml of melted and cooled (approx. 500C) brucella agar supplemented with 7% horse blood is added. This yields a final 1:10 dilution of compound in agar, and a final concentration of DMSO of 1%. For example, if the highest concentration (1st broth dilution) contains 1000 ug/ml, and is diluted 1:10 in agar, the final concentration of drug in agar is 100 ug/ml. Agar plates can be prepared one day prior to inoculating, and refrigerated ovemight.

Inocula Preparation Helicobacter Dvlori cultures are maintained on trypticase soy-% sheep blood agar plates, and are transferred every 48 hours. Plates are incubated at 370C in GasPak jars with water-activated (10 ml) CampyPak Plus (BBL Microbio. Systems) envelopes with palladium catalyst.

Helicobacter cultures can be grown in brucella broth supplemented with 10% fetal calf serum in 10 ml volumes in deep petri dishes. The plates are incubated for 1820 hours at 370C in GasPak jars with water-activated (10 ml) CampyPak Plus envelopes with palladium catalyst on a shaker at 50 rpm.

Overnight cultures (approx. 108 CFU/ml) are diluted 10-fold in brucella broth (no FCS) in screw-capped tubes for use as the standard inoculum. The wells of a Steere replicator are filled with 0.8 ml of the diluted organism, and approximately 2 x 104 cells in 0.002 ml are placed on the agar surface. Inoculated plates are placed in a GasPak jar to which water-activated (10 ml) Campy Pak Plus envelopes with palladium catalyst have been added, and incubated at 370C for 48 hours.

Interpretation of Results Following incubation, all test plates are compared to a compound-free growth control plate. The MIC is the concentration which inhibits growth compared to the control plate. A thin film of growth might be visible at higher concentrations but this is discounted, and not considered the true MIC. Control organisms are also inoculated on each plate, and these are diluted 1000-fold for use as inocula. The control organisms include CamDvlobacter ieiuni (joe6686), and the screening cultures of E. coli (#51A266 and #51A470), Enterobacter aeropenes (#67A040), E. cloacae (#678009), Providencia stuartii (#77A013) and P. rettoeri (#77C025). Plates and/or inocula transfers should not be out of the microaerophilic environment longer than 2 hours. It is also recommended that all manipulations involving Helicobacter cultures be performed in a laminar flow hood to decrease the chance of contaminating the cultures with mold.

The mouse model of Lee et al., Gastroenterology, 99,1315-23(1990) is used to predict the in vivo activity of a compound against H. Dvlori in humans.

Helicobacter felis is grown in brucella broth with 10% fetal bovine serum. A frozen culture is quickly thawed; the culture is checked for motility and 0.5 cc. of the thawed frozen culture is inoculated into a deep tissue culture dish containing 9.5 cc.

of the brucella/serum mix. The dishes are put into a Campy Pak jar [BBL] to insure a microaerophilic atmosphere. The jar is put on a rotary shaker at 60 RPM in a 370C incubator. After 18 hours there should be visible turbidity. The culture is checked for purity and motility under a (phase) microscope and then pooled into a flask. Swiss Webster female mice (1 8-20g) are fasted for 18 hours before infection. The mice are infected three times on alternate days during a single week. Dosing begins two weeks after the last dose of organism. Treatments are given once per day for fourteen consecutive days. Sacrifice is about three weeks after completion of therapy. For each mouse, the stomach is excised and opened along the greater curvature. A plug (a 3 mm. tissue section) is taken from the antrum region of the stomach. The plug surface is washed, minced, and dropped into a tube with 100 microliters of urease reagent.

The urease reagent is the reagent of Hazell et. al., Am. J. Gastroenterology, 82,292-296 (1982). The urease reagent (pH 6.3-6.5) contains urea and phenol red. If Helicobacter is present, urease will break down urea producing a change of pH. Purple (alkaline) is positive for Helicobacter; red/yellow (no change) is negative. Any.color change is recorded after 18 hours. There are usually twenty mice per treatment group; the percent positive for each group is recorded.

There are several methods used clinically to determine whether Helicobacter Dvlori is present in a human subject. These are employed for initial diagnosis of infection prior to treatment, as well as for determining the success of treatment in eradicating the organism from the patient.

The urea breath test involves ingestion of radiolabelled urea. H. Pylori produces a urease enzyme which degrades urea; mammalian gastric cells do not contain this enzyme. Exhalation of labeled carbon dioxide (analyzed by mass spectrometry or radioactivity, depending on the isotope employed) therefore indicates that H. pylori is present.

Serology can also be used to assess infection with H. pylori. Detection of serum antibodies to H. pylori, such as IgG and IgA, is carried out using enzyme-linked immunosorbent assay (ELISA). Numerous different H. pylori proteins can be employed as antigens.

Endoscopy of the patient provides samples of tissue which can be cultured in a microaerophilic environment to diagnose H. pylori infection. Alternatively, the sample can be examined histologically by employing one of a number of stains such as Giemsa or hematoxylin-eosin. A urea test, which again takes advantage of the production of urease by H. Pviori, can also be applied. This test relies on the formation of ammonia from the urea hydrolysis, which results in an observable change in pH.

Example 7-(l1 a. sa. 6α]-6-Acetylamino-3-azabicyclo[3.10]hex-3-yl)1-(2,4-difluorophenyl)-6- fluoro-1.4-dihvdroRoxo-1.8-naPhthvridine-3-carboxylic acid A mixture of 7-([1 a, So, 6a]-6-amino-3-azabicyclo[3.i .0]hex-3-yl)-1 -(2,4difluorophenyl)-6-fluoro-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxylic acid monomethanesulfonate (1.25 g, 2.44 mmol), acetyl chloride (0.34 ml, 4.7 mmol) and pyridine (0.42 ml, 5.2 mmol) in dimethylformamide (3 ml) was heated at 800C for 8 hours. Removal of solvent in vacuo provided an orange residue, which was recrystallized from methanol. The title product was obtained as a solid, mp > 2750C (461 mg, 1.0 mmol, 41% yield).

1H NMR (DMSO-d6): 15.13 (s, 1H), 8.81 (s, 1H), 8.05 (d, J=13 Hz, 1H), 8.01 (s, 1H), 7.81 (m, 1H), 7.63 (m, 1H), 7.35 (m, 1H), 3.6 (vbm, 4H), 2.31 (bs, 1H), 1.75 (bs, 5H).

Claims (9)

1. 7-([1 a,5o,6a]-6-Acetylamino-3-azabicyclo [3.1.01 h ex-3-yl) -1 -(2,4 difluornphenyl)6fluorn-1 ,4-dihydroxo-1 ,S-naphthyridine-3carboxylic acid.

2. An antibacterial composition comprising an antibacterially effective amount of the compound of claim 1, and a pharmaceutically acceptable carrier.

3. A composition according to claim 2 which comprises in addition another antibacterial agent.

4. A composition according to claim 3 wherein said other antibacterial agent is amoxicillin, tetracycline or omeprazole.

5. A method for the treatment of a bacterial infection which comprises administering to a subject affected by a bacterial infection an anti bacterially effective amount of the compound of claim 1.

6. A method for the treatment of a Helicobacter infection which comprises administering to a subject affected by such infection an effective amount of the compound of claim 1.

7. A method according to claim 6 wherein said treatment in addition comprises administration of another antibacterial agent.

8. A method for the treatment of gastric and duodenal ulcers by administering to a subject in need of such treatment an effective.amount of the compound of claim 1.

9. A method according to claim 8 wherein said treatment in addition comprises administration of another antibacterial agent.