Patent Application
20050209248
The invention relates to phenyl oxazolidinone derivatives. More particularly, it relates to polymorphic forms of (S)-N-[[3-fluoro-4-[N-1[4-{2-furyl-(5-nitro)methyl}]piperazinyl]-phenyl]-2-oxo-5-oxazolidinyl]-methyl]acetamide hydrochloride having the Formula I.
Further, the invention relates to methods of using such compounds as antimicrobials, pharmaceutical compositions containing the novel polymorphic forms, and processes for the preparation of the polymorphic forms.
S. epidermidis is the causative agent in many incidents of infection of implanted medical devices such as catheters, pacemakers, prosthetics joints, cardiac valves and central venous system shunts. These infections often recur and tend to be difficult to treat with antibiotics agents. Removal of the devices with concurrent administration of antibiotics is usually the only method of eradicating the focus of infection.
The compound of Formula I, namely, (S)-N-[[3-fluoro-4-[N-1 [4-{2-furyl-(5-nitro)methyl}] piperazinyl]-phenyl]-2-oxo-5-oxazolidinyl]-methyl]acetamide hydrochloride is a phenyl oxazolidinone derivative, as disclosed in PCT application WO 02/06278. It is said to be useful as antimicrobial agent, effective against a number of human and veterinary pathogens, including gram-positive aerobic bacteria, such as multiply resistant staphylococci, streptococci and enterococci as well as anaerobic organisms such as Bacterioides spp. and Clostridia spp. species, and acid fast organisms such as Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium spp.
The PCT application WO 02/06278 describes the preparation of compounds of Formula I. The products of Formula I obtained by following the cited methods tend to be hygroscopic and difficult to filter. These types of disadvantageous properties have proven to be serious obstacles to the large-scale manufacture of a compound. Further, handling problems are encountered during the preparation of pharmaceutical compositions comprising the hygroscopic compound of Formula I obtained by following the method disclosed in WO 02/06278.
Provided herein is means to prepare a compound of Formula I in a form, which is non-hygroscopic, permits large scale, synthesis and which can overcome the handling problems encountered during the preparation of pharmaceutical compositions. There is a need to discover and develop a new agent active against all anaerobes including drug resistant strains.
Herein are provided new polymorphic forms of S)-N-[[3-fluoro-4-[N-1 [4-{2-furyl-(5-nitro)methyl}]piperazinyl]-phenyl]-2-oxo-5-oxazolidinyl]-methyl]acetamide hydrochloride (Formula I) designated as ‘Form A’and ‘Form B.’ Processes for the preparation of new polymorphic forms are also provided. Additionally, pharmaceutical formulations comprising polymorphic forms A and/or B and methods of using them as antimicrobial agents, agents for treating or preventing anaerobic infections, catheter infections and foreign body or prosthesis infections in mammals are provided. Further, ‘Form A’ is very active against slime-producing bacteria and retains activity against adherent bacteria, making it useful for the prevention and treatment of catheter infections and foreign body or prosthesis infections.
The polymorphic forms of the compound of Formula I designated as ‘Form A’ and ‘Form B’ can be characterized by their X-ray powder diffraction patterns (XRPD), infrared spectra and differential scanning calorimetry (DSC) characteristics.
Accordingly, polymorphic ‘Form A’ of the compound of Formula I and a process for the preparation of polymorphic ‘Form A’ are provided. This process comprises:
In another aspect, there is provided a polymorphic ‘Form B’ of the compound of Formula I and a process for the preparation of polymorphic ‘Form B’. This process comprises:
According to another embodiment, there is provided a process for the preparation of polymorphic ‘Form A’ of the compound of Formula I, which comprises:
According to another embodiment, there is provided a process for the preparation of novel polymorphic ‘Form A’ of the compound of Formula I, which comprises:
According to another embodiment, there is provided a process for the preparation of novel polymorphic ‘Form A’ of the compound of Formula I, which comprises:
Embodiments of the invention are explained in greater detail by way of the accompanying figures:
Data were collected as follows:
Data collection parameters: Scanning rate: 10° C./min; Temperature: 50° C.-300° C.
Biological Activity
Activity Against Anaerobes and microbacterium
Agar dilution method for anaerobic bacteria:
MICs were determined by the NCCLS agar dilution method with Wilkins Chalgren Agar (Difco). The plates were incubated in an anaerobic jar containing an atmosphere of 85% nitrogen, 10% hydrogen and 5% carbon dioxide for 48 hour. MIC values are presented in Table II.
Some of the MICs obtained are presented in Table III.
Activity Against Catheter Related Infections
In device-related infections, the correlation between MIC levels and clinical efficacy is poor, leading to the situation that infected implants have to be removed in order to achieve cure. The main characteristics of such infections are the microbial adherence affected by the biofilm and the low growth rate of surface-adherent microorganisms. The discrepancy between the results of routine antibiotic susceptibility testing and treatment success in device-related infections may therefore be due to the fact that bacterial biofilms have different resistance patterns compared with planktonic bacteria. It has been demonstrated that the cure rate in experimental device-related infections can be predicted by the in vitro bactericidal effect of antibiotics on non-growing and adherent bacteria.
The most important anaerobes clinically are the genera of gram negative rods. Bacteroides, especially the B. fragilisgroup is particularly important. The other principal gram negative genera are Prevotella, Fusobacterium, Porphyromonas, Bilophila and Sitterella. Among the gram positive anaerobes, there are cocci (primarily Peptostreptococcus) and spore forming (clostridium) and non spore forming bacilli (Actinomyces and Propionibacteria).
Treatment of anaerobic infections may be difficult. Failure to provide coverage for anaerobes in mixed infections may lead to a poor response or to no response. Many antibacterial agents including aminoglycosides, trimethoprim-sulphamethoxazole, most quinolones and monobactams have poor activity against many or most anaerobes. Four groups of drug are active against majority of anaerobic bacteria of clinical significance: these are nitroimidazole such as metronidazole, carbepenems such as imipenem, chloramphenicol and a combination of 0 lactam and βlactamase inhibitors.
Non spore forming, anaerobic, gram positive bacilli (e.g. Actinomyces, Eubacterium and Propionibacterium) are commonly resistant to metronidazole. Of late, there have been reports of resistance to all the above agents in small number of strains of B. fragilis group. Cefoxitin, clindamycin and braod spectrum penicillins such as ticarcillin or piperacillin also have some anti anaerobic activity. But 15-25% of B. fragilis isolated in the U.S. hospitals are resistant to these drugs. Cefoxitin and clindamycin have relatively weak activity against clostridia other than C. peringens (20-35% of such strains re resistant) and some anaerobic cocci are resistant to clindamycin. Penicillin G is not reliable for treating serious infections involving any of these anaerobic gram negative bacilli because the incidence of β lactamase production among these organisms is high.
To demonstrate the usefulness of novel polymorphic ‘Form A’ in device related infections two tests of experiments have been performed:
To study the effect of polymorphic ‘Form A’ on the inhibition of biofilm production, the following study was carried out as set forth in Blake et al. J. Clinical Microbiol. 2001; 39:544-550; and Polonio et al. Chemother. 2001; 45:3262-3266. Since Mueller Hinton broth does not support the formation of biofilm, trypticase soy broth with 2% glucose was used to stimulate biofilm formation by MRSA 1029/99 and MRSE 879/247 (both recent clinical isolates collected from tertiary care hospital). Bacterial suspensions (in triplicate) were exposed a doubling dilution of antibiotics and incubated overnight at 37° C. with constant shaking (100 rpm). The next day, after aspirating the medium, the biofilm was stained with safranin (0.1%) for 1 hour at room temperature, washed with distilled water, tapped dry and stain-extracted into 200 μl of 0.2M NaoH and the OD measured at 544 nm. The relative inhibition was determined by using the formula:
% inhibition=100-[(OD of treated well/OD of Reference well)×100]
Inhibition of Biofilm formation occurs at a lower concentration for polymorphic ‘Form A’ as depicted in Graphs A to D.
Polymorphic ‘Form A’ is active against adherent bacteria:
Linezolid has been shown to be active against nearly all clinically relevant gram positive pathogens, with MIC90 of 2 to 4 μg/ml, while the Cmax is 12 to 16 μg/ml. Linezolid is active against all gram positive bacteria, irrespective of their susceptibility to other antibiotics. Though the action is bacteriostatic, it has proven difficult to generate resistant mutants in the laboratory. However, within months of clinical use, resistance in Vancomicin Resistant Enterococci (VRE) and Methicillin Resistant Staphylococcus Aureus (USA) has been reported. The common feature in both reports is the presence of foreign body (catheter) in these patients leading to treatment failure and development of resistant mutants.
We investigated the change in MIC of Linezolid, Vancomycin, Synercid and polymorphic ‘Form A’ in a sintered glass adherent bacteria model with MRSE 879 bacteria and found that though the broth MICs were Linezolid (2 μg/ml), Vancomycin (1 μg/ml), Synercid (0.5 μg/ml) and polymorphic ‘Form A’ (0.5 μg/ml), the concentration which would kill adherent bacteria were Linezolid (32 μg/ml), Vancomycin (8 μg/ml), Synercid (2 μg/ml) and polymorphic ‘Form A’ (2 μg/ml). The change in MIC in broth and on sintered glass adherent bacteria is presented in Graph E.
Agar Dilution Method for M. tuberculosis:
Antibiotics were incorporated at concentrations of 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.06 and 0.03 μg/ml into plate of Middlebrook 7H10 agar medium supplemented with OADC enrichment (Difco) Test organisms were grown in 7H9 medium (Difco) containing 0.05% Tween 80. After 7 days of incubation at 37° C., the broths were adjusted to 1 MacFarland, the organisms were then diluted 10 fold in sterile water containing 0.05% of Tween 80. The resulting bacterial suspensions were spotted on predried supplemented 7H10 plates. After 21 days of incubation at 37° C., the MICs were recorded as the lowest concentration of the drug that completely inhibited the growth of the organism, and are presented in Tables IV and V.
Mycobacterium avium intracellulare
Examples given below are presented by way of illustration only, and do not limit the scope of the invention.
The free base of Formula I (S)-N-[[3-fluoro-4-[N-1 [4-{2-furyl-(5-nitro)methyl}] piperazinyl]-phenyl]-2-oxo-5-oxazolidinyl]-methyl]acetamide, can be prepared by, for example, following the procedure as described in WO 02/06278.
50 gm of free base of Formula I was dissolved in ethanol (750 ml) by heating at about 60° C. and to this solution was added ethanolic HCl (13.36 ml, 8.9 N) at about 45-50° C. The reaction mixture was cooled to about 10° C., and stirred for about 4 hours. The separated solid was filtered off and dried under vacuum at 60° C. The solid was then digested in ethanol (150 ml) at 70-80° C. for about 4 hours. It was then cooled to about 10° C., the solid was filtered and dried under vacuum at 60-65° C. to give 30 gm of the pure polymorphic ‘Form A’ of compound of Formula I.
7.3 gm of free base of Formula I was dissolved in hot ethanol (130 ml) and cooled to about 20° C. Ethanolic. HCl (2.60 ml, 8.9 N) was added to it. The reaction mixture so obtained was stirred at 20° C. for about 15 minutes. The solid separated was filtered washed with ethanol (30 ml) and dried to give 5.9 gm of pure polymorphic ‘Form B’ of the compound of Formula I.
A solution of free base of Formula I (365 mg, 0.75 mmol, dissolved in 7 ml of ethanol) was heated to about 60-80° C., and then cooled to about 5° C. HCl dissolved in ethanol (0.30 ml, 2.6 N, 0.75 mmol) was added to the reaction mixture at about 5° C. The reaction mixture so obtained was stirred at 5-10° C. for about 2 hours. Solvent was removed completely under vacuum and the residue was digested with dichloromethane, the solid was filtered and crystallized from a mixture of methanol/isopropyl alcohol. The solid obtained was then digested in ethanol (4 ml) at about 80° C. for a time period of about 4 hours. The reaction mixture was cooled to 25-30° C., the solid was filtered and dried under vacuum at about 60° C. to give ‘Form A’ of compound of Formula I.
10 gm of (S)-N-[[3-fluoro-4-[N-1 [4-{2-furyl-(5-nitro)methyl})piperazinyl]-phenyl]-2-oxo-5-oxazolidinyl]-methyl]acetamide hydrochloride of Formula I was dissolved in 70 ml of de-mineralized water by heating at about 50° C. for few minutes. The solution was cooled to about 40-45° C., and filtered through 0.2 micron filter paper, and washed with water (10 ml). Ethanol (400 ml) was added slowly to the filtrate at room temperature (25-30° C.). Stirred at room temperature for about 30 minutes, solid separated out. Cooling was continued to about 10-15° C. and kept for 3 hours. The solid was filtered, washed with ethanol (10 ml) and dried under vacuum for 24 hours at about 60° C. to yield 9 gm of the pure polymorphic ‘Form A’ of compound of Formula I.
| Number | Date | Country | Kind |
|---|---|---|---|
| 60380606 | May 2002 | US | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB03/01853 | 5/15/2003 | WO | 5/20/2005 |
