Title of Invention

NOVEL ANTI-FUNGAL AGENTS

Abstract

The invention relates to novel antifungal compounds and its pharmaceutically acceptable salts, having as part of its structure, imidazolyl or benzimidazolyl derivatives. The imidazolyl derivative may be substituted at 2 position from among the group consiting of aroyl, p-chlororoyl, phenyl hydroxy methine or p-chloro phenyl hydroxy metheine or with n-butyl at 2 position and chlorine at 4 and 5 position. In the alternative, whem it is a benzimadazoyl derivative, with hydrogen at 6 poaition, the substituent at 2 position may be selected from among methyl.ethyl,isopropyl,2-oxoprop-1-yl, n-propyl, methoxy methyl, propen-1-yl, phenyl, p-chlorophenyl,p-toluyl,benzyl,4-pyridyl, p-methoxy phenyl, 3-pyridyl, o-methoxy phenyl, styryl, 2-cyano methyl, p-hydroxy phenyl, p-amino phenyl, p-toluyl sulfonyl methyl or p-(t-butyl) phenyl group and if hydrogen is at 2 position, the substituent at the 6 poaition is selected from among nitro, trifluoromethyl or methoxy group.

Full Text

FORM 2 PATENT ACT, 1970 (39 of 1970) COMPLETE SPECIFICATION (Section 10) TITLE: Novel Anti-fungal agents APPLICANTS: FDC Limited, a Company incorporated under the Company"s Act 1956, and having their registered office at B, 8 MIDC Industrial Estate, Waluj, Maharashtra 431 135,India The following specification describes the nature of the invention: FIELD OF INVENTION:The invention relates to a novel anti-fungal compound which are derivatives of l-(lH-l,2,4-triazol-l-yl>-2-(2",4"-difluorophenyl)-3-(2-a%l-benzimdidazol-l-yi)propan -ol useful in the treatment of fungal infections in animals, including humans and as agricultural fungicides. BACKGROUND OF INVENTION: In the past two decades the frequency and types of life-threatening fungal infections have increased dramatically in immuno-compromised patients. Several factors have contributed to this rise such as the expansion of severely ill and or immuno-compromised patient populations with HTV infection, with chemotherapy induced neutropenia, and receiving immunosuppressive therapy; more invasive medical procedures, such as extensive surgery and the use of prosthetic devices and vascular catheters; treatment with broad-spectrum antibiotics or glucocorticosteroids; and peritoneal dialysis or hemodialysis This problem of increased fungal infections is accentuated by the emergence of fungal strains which are resistant to currently used antifungal agents. Major opportunistic fungal pathogens include Candida albicans, Aspergillus, Fusurian spp. Other species of Candida such as C. krusei, C tropicalis, C. glabrata are major causative agents of candidiasis. Invasive pulmonary aspergillosis is a leading cause of mortality in bone marrow transplant recipients. HIV-infected patients are particularly susceptible to mucosal candidiasis, cryptcoccal meningitis. Fluconazol is the preferred broad spectrum anti-fungal agent used in treatment of fungal infections. In recent times resistance of Candida albicans the most common cause of mucosal candidiasis in HIV-infected patients, after long-term suppressive therapy, to azoles, particularly fluconazole, is a cause of increasing concern. Resistance to fluconazole in other Candida species and in Cryptococcus neoformans has also been reported. Also, fluconazole appears to be less active against the two emerging Candida species, C glabratta and C. krusei. Infection with Aspergillus, although not common, is frequently Ufe-threatening, and fluconazole has only moderate activity against this fungi This has necessitated the need for new antifungal agents with broad spectrum of antifungal activity, which this invention seeks to provide. SUMMARY; (I) In its main aspect, the invention consists of a novel anti-fungal compound being a derivative of l-(lH-l,2,4-triazol-l-yl)-2-(2",4"-difluorophenyl)-3-(2-all^l-benzimdidazol-l-yl)propan -ol of formula (I), and its pharmaceutically acceptable salts When R is Imidazolyl (n), R2 and R3 are hydrogen and Rl is selected from among aroyl, p-chloroaroyl, phenyl hydroxy methine or p-chloro phenyl hydroxy methine, and when Rl is n-butyl , R2 and R3 are chlorine. In the alternative , when R is a benzimadazolyl (HI) , R5 is hydrogen and R4 is selected from among methyl, ethyl, isopropyl, 2-oxoprop-l-yl, n-propyl, methoxy methyl, propen-1-yi, phenyl, p-chlorophenyl, p-toluyl, benzyl, 4-pyridyl, p-methoxy phenyl, 3-pyridyl, o-methoxy phenyl, styryl, 2-cyano methyl, p-hyrdroxy phenyl, p-amino phenyl, p-toluyl sulfonyl memyl or p-(t-butyl) phenyl group . In a further aspect, in benzimadazole(IH) R4 is hydrogen and R5 can be selected from among nitro, trifluoromethyl or methoxy group. In another aspect of this invention, the compound of Formula 1 is used along with pharmaceuticafly acceptable excipients. In yet another aspect of the invention, the compound of Formula 1 is along with agriculturally acceptable diluants. In the final aspect, the invention relates to a process whereby an oxirane (TV) is reacted with a imidazolyl derivative (II) or a benzimidazolyl derivative (in) as described above, in a solvent and in the presence of a base at elevated temperatures over a period of time, resulting in the synthesis of a compound which is an anti-fungal agent wrfli broad spectrum activity. DESCRIPTION: The main embodiment of the invention is given by the following description. Novel anti¬fungal compounds, being derivatives of l-(lH-l,2,4-triazol-l-yl)-2-(2\4"-chfluorophenyi)-3-(2-alkyl-benzimdidazol-l-yi)propan -ol of the formula (I) according to the invention can be obtained by the reaction of the oxirane of the formula (TV) or its methane sulphonate salt with substituted imidazoles (II) -R3 R2 HN R1 (II) 4 Where, R1 is selected from among aroyl, p-chloroaroyl, phenyl hydroxy methine or p-chloro phenyl hydroxy methine and R2 and R3 are hydrogen or where R1 is n-butyl and R2 and R3 are chlorine or with substituted benzimidazoles (III) where R4 is selected from among methyl, ethyl, isopropyl, 2-oxoprop-l-yl, n-propyl, methoxy methyl, propen-1-yl, phenyl, p-chlorophenyl, p-toluyl, benzyl, 4-pyridyl, p-methoxy phenyl, 3-pyridyl, o-methoxy phenyl, styryl, 2-cyano methyl, p-hyrdroxy phenyl, p-amino phenyl, p-toluyl sulfonyl methyl or p-(t-butyl) phenyl group and R5 is hydrogen or where R4 is hydrogen and R5 is selected from among nitro, trifluoromethyl or methoxy group The reaction is carried out in the presence of a base, where by the epoxide is opened to form the compound of formula (1). The base used is selected from among sodium hydride, potassium carbonate, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium methoxide, potassium t-butoxide. The reaction is carried out in a solvent selected from dimethyl sulfoxide, dimethyl formamide, tetrahydrofuran, dichloromethane, toluene, acetone or mixtures thereof. The choice of base and solvent used is not limited by the cited examples and the person skilled in art can select any other base in combination with any other solvent to get the desired results. In a typical procedure oxirane (IV) or its methane sulphonate salt and substituted imidazole (II) or benzimidazole (HI) are reacted together in a suitable solvent eg. dry dimethyl formamide preferably with heating from 60°-140°C for 8-24 hours. The progress of the reaction is monitored by Thin Layered Chromatography (TLC). The product is isolated and purified by conventional procedures. The pharmaceutically acceptable salts of free bases which are acid addition salts is obtained by conventional procedures Such as, mixing solutions containing equimolar amounts of free base and the desired acid together, followed by filtration to collect the required salt, if insoluble; or else by evaporation of the solvent from the system in accordance with the standard techniques. The oxirane (IV) or its methane sulphonate salt can be obtained by conventional methods, lypically from the corresponding acetophenone derivative (V) by reacting it with sulfur ylide viz. dimethyl oxo sulfonium methylide prepared from trimethyl oxosulfonium iodide and a base. The methane sulphonate salt of IV can be obtained by treating IV in toluene or acetone with methane sulphonic acid. The ketone (V) required for preparing the oxirane is synthesized by conventional methods. For the purpose of the preferred embodiment of this invention, the oxirane (IV) may be prepared by the following method: Preparation of 2-[(lH-l,2,4-triazol-l-yl)methyl]-2-(2,4-difluorophenyl)oxirane (IV) : An ylide is prepared from 2.19 gm (0.0099 mole) of trimethyloxosulfonium iodide, 0.43 gm (0.0099 mole, 55%) of sodium hydride, and 5 ml of dimethyl sulfoxide. To this, a solution of 2 gm (0.00896 mole) of acetophenone derivative (VH) in 5 ml of dimethyl sulfoxide is added. The reaction mixture is heated to 60°C for 3 hours and poured over crushed ice after cooling. The product is isolated by extracting with ethyl acetate followed by repeated washings with satureated brine solutioa The evaporation of solvent under reduced pressure gives titled compound as a dark brown oil (1.26 gm, 63%). For the purpose of the preferred embodiment of this invention, the imidazoles (IT) may be synthesized as per conventional methods as follows :- where, R , R and R are as defined earlier. In atypical procedure 0.20 mole of aroyl chloride is added dropwise to a solution of 0.10 mole of imidazole and 0.20 mole of triethyl amine in 30 ml. of pyridine at 0°-10°C under nitrogen atmosphere. The mixture is stirred for 3 hours at room temperature and then treated with aqueous sodium hydroxide. It is then heated to reflux for 1 hour, addition of 100 ml. of water and cooling lead to the precipitation of the product. The product is isolated by filtration and washed successively with water, ice-cold methanol and diethyl ether. The corresponding alcohols are prepared by reducing ketones with sodium borohydride. where, R and R are as defined earlier. For the purpose of the preferred embodiment of this invention the benzimidazoles (III) may be synthesized as follows:- 1 In a typical procedure equimolar amounts of o-phenylenediamine suitably substituted and the carboxylic acid (or its corresponding ester) are mixed with sufficient quantity of polyphosphoric acid (PPA) to give a stirrable paste. The mixture is slowly heated to 250°C, and the resulting solution is digested at 250°C (± 3°C) for 4 hours, cooled to about 100°C and poured in a thin stream into a large volume of rapidly stirred water. The insoluble residue is collected by filtration, washed with water and 10% sodium carbonate solution followed by water to remove residual alkaiinily. The crude product is purified by recrystaUization from alcohol or aq. alcohol mixture subsequent to treatment with a small amount of activated carbon or by column chromatography over silica gel (60-120) column. The following examples describe the preferred embodiment of mis invention for preparation of compound of Formula (I): Example 1 : Preparation of l-(lH-l,2,4-triazol-l-yl)-2-(2",4"-difluorophenyl)-3-[2-(p- chloroaroyl)imidazol-l -yl] propan-2-ol. lH-[2-(p-chloro)aroyl] imidazole (1.169 g, 0.00566 mol) and oxirane (TV) (1.2 gm, 0.00506 mol), potassium carbonate (0.938 gm, 0.0068 mol) and dry dimethyl formamide (25 ml.) are stirred and heated together at 90°C under nitrogen atmosphere. The progress of the reaction is monitored by TLC. After completion of reaction, reaction mixture is quenched in water and extracted with dichloromethane. Evaporation of solvent gives the titled compound (1.75 gm, 78%), melting point ( 193°C-195°C). The DR. and NMR spectra of the compound are consistent with the structure. Example 2: Preparation of l-(lH-l,2,4-triazol-l-yl)-2-(2,,4"-difluorophenyl)-3-(2-methyl- benzimidazol-1 -yl)-propan-2-ol. lH-(2-memyl)-benzimidazole (0.748 gm, 0.00566 mol), oxirane (IV) (1.2 gm, 0.00506 mol), potassium carbonate (0.938 gm, 0.0068 mol) and dry dimethyl formamide (25 ml.) are stirred and heated together at 90°C under nitrogen atmosphere. The progress of the reaction is monitored by TLC. After completion of reaction, reaction mixture is quenched in water and extracted with dichloromethane. Evaporation of solvent gives the titled compound (1.53 gm, 82%), melting point (207°C-210°C). The BR and NMR spectra of the compound are consistent with the structure. Following the procedure as substantially described by above examples the various azole compounds of Formula (I) as mentioned herein are prepared. The compound of Formula (I) and their pharmaceutically acceptable salts are antifungal agents effective to a greater or lesser extent, and useful in treating fungal infections in animals and humans, especially those caused by C.albicans, Aspergillus and Fusarium. In vitro evaluation of antifungal activity can be performed by determining the minimum inhibitory concentratioa Anti-fungal susceptibility testing of these anti-fungal compounds was done by conventional method using soyabean casein digest broth. Known anti-fungal agents like Fluconazole and amphotericin-B were used as positive Control. End points were determined after 48 hours visually and by using Spectrophotometer wherever necessary. Different dilutions were hied and the set of experiments were repeated to confirm the end points. Standard cultures of Candida, Aspergillus and Fusarium were used for screening of these drugs. Substituents and in-vitro anti-fungal activity (Calbicans) of the compounds expressed as MIC* (ug/ml). Reference standard IRS) Fluconazole. Table I Compounds (I) formed from Imidazolyl derivatives (II) No: Rl substitution MIC ug/ml 1 Aroyl 2. p-chloro aroyl 3 Phenyl hydroxy methine 25-50 4. p-chlorophenyl hydroxy methine 25-50 9 I Table II Compounds (I) formed from benzimidazolyl derivatives (EI) No: R4 substitution R51 MIC ug/ml 1. methyl Hydrogen 6.25-12.50 2. Ethyl Hydrogen 6.25-12.50 3. isopropyl Hydrogen 6.25-12.50 4. 2-oxoprop-l-yl Hydrogen 6.25-12.50 5. n-propyl hydrogen 25-50 6. methoxymethyl Hydrogen 25-50 7. Propen-1-yl Hydrogen 25-50 8. phenyl Hydrogen 6.25-12.50 9. p-chloro phenyl Hydrogen 6.25-12.50 10. p-toluyl Hydrogen 100-200 11. -Benzyl Hydrogen 100-200 12. -4-pyridyl Hydrogen 100-200 13. p-methoxy phenyl Hydrogen 50-100 14. 3-pyridyl Hydrogen 100-200 15. o-methoxy phenyl Hydrogen 50-100 16. styryl Hydrogen 3.125-6.25 17. Hydrogen Nitro 100-200 18. Hydrogen trifluoromethy 50-100 19. Hydrogen methoxy 100-200 • Minimum Inhibitory Concentration (MIC) measured in terms of ug/ml The activity of these compounds were tested against the reference standard of Fluconazole which showed minimum inhibitory concentration at Antifungal activity of these compounds also extends to Aspergillus and Fusarium. The activity seen in compound of Formula (1) as against these strains suggests mat it exhibits broad spectrum antifungal activity I For human use, the antifungal agents of formula (I) or a pharmaceutically acceptable salt may be administered alone, but is generally administered in an admixture with a pharmaceutically acceptable carrier, which is selected depending on the intended route of administration and standard medical practice. Thus it can be administered orally in the form of tablets, capsules or ovules or they can be injected intradermally, intramuscularly or subcutaneously. For parenteral administration the compound is in the form of aqueous solution, along with other excipients. Alternatively, the antifungal compound of formula (I) can be administered in the form of supository or can be applied topically in the form of lotion, gel solution, cream, ointment or dusting powder. Volume 22 M27-A2 An NCCLS global consensus standard ©NCCLS. All rights reserved 19 Appendix C. Interpretive Guidelines for In Vitro Susceptibility Testing of Candida Species Shown are the breakpoints (µg/mL) for Candida species against the indicated agents. If MICs are measured using a scale that yields results felling between categories, the next higher category is implied. Thus, an isolate with a fluconazole MIC of 12.5 mcg/mL would be placed in the S-DDcategory.f Footnotes * For fluconazole, these guidelines are based substantially on experience with mucosal infections, but are consistent with the limited information for invasive infections due to Candida spp. Isolates of C. krusei are assumed to be intrinsically resistant to fluconazole, and their MICs should not be interpreted using this scale. It is also pertinent that the 8-mcg/mL upper boundary for the susceptible range of fluconazole is not known with certainty—the data would permit selection of either 4 or 8 mcg/mL for this cut-off. f For itraconazole, the data are based entirely on experience with mucosal infections, and data supporting breakpoints for invasive infections due to Candida spp. are not available. % Susceptibility is dependent on achieving the maximal possible blood level. For fluconazole, doses of 400 mg/day or more may be required in adults with normal renal function and body habitus. For itraconazole, measures to assure adequate drug absorption and plasma itraconazole concentrations of >0.5 mcg/mL may be required for optimal response. § The susceptibility of these isolates is not certain, and the available data do not permit them to be clearly categorized as either "susceptible" or "resistant. " f These breakpoints were adopted at a meeting of the subcommittee held June 1, 1996 in Reston, VA. These breakpoints are considered tentative for one year and are open for comments. WE CLAIM: and its pharmaceutically or agriculturally acceptable salts, where R is imidazolyl (II) 1. A novel anti-fungal compound, being derivatives of 1 -(1H-1.2,4-triazol-1 -yl)-2 -(2\4"-difluorophenyl)-3-(2-alk} l-benzimdidazol-l-yI)propaii -ol. of the fonnula (n) and Rl is selected from among aroyl, p-chloroaroyl, phenyl hydroxy methine or p-chloro phenyl hydroxy methine and R2 and R3 are hydrogen, or where Rl is n-butyl and R2 and R3 are chlorine, and where R is benzimidazolyl (HI), R4 is selected from among methyl, ethyl, isopropyl, 2-oxoprop-l-yl, n-propyl, methoxy methyl, propen-l-yl, phenyl, p-chlorophenyl, p-toluyl, benzyl, 4-pyridyl, p-methoxy phenyl, 3-pyridyl, o-methoxy phenyl, styryl, 2-cyano methyl, p-hyrdroxy phenyl, p- r% amino phenyl, p-toluyl sulfonyl methyl or p-(t-butyl) phenyl group and R5 is hydrogen or where R4 is hydrogen and R5 is selected from among nitro, trifluoromethyl or methoxy group. 2. A compound as described in claim 1, where in the compound is along with pharmaceuiicaHy acceptable excipients or agriculturally acceptable dUumts. 3. A compound as described in claim (1). prepared by a process comprising of : reacting oxirane (IV) or its methane sulphonate salt with substituted Imidazole (II) or substituted benzimidazole (III) in a solvent in the presence of a base, at a temperature of 60° C to 140T . for a period of 8-24 hours. 4. A compound as described in claim 5 where in the base is selected from among sodium hydride, potassium carbonate, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium methoxide, potassium f-buloxide. 5. A compound as described in claim 6 where in the solvent is selected from among from dimethyl sulfoxide, dimethyl formamide, tetrahydrofuran, dichloromethane, toluene, acetone or mixtures thereof. Dated this 10th day of February, 2003 For FDC Limited Mr.Mohan Chandavarkar Managing Director

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