Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
  • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
  • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
  • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
  • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D215/38—Nitrogen atoms
  • C07D215/42—Nitrogen atoms attached in position 4
  • C07D215/44—Nitrogen atoms attached in position 4 with aryl radicals attached to said nitrogen atoms

Definitions

• the present invention relates to the novel N -(3-((diethylamino)methyl)-4-hydroxyphenyl)-N-(quiholin-4 yl)sulfonamide compounds of formula A and a process of preparation thereof.
• the present invention further relates to pharmaceutical compositions useful in the prevention or treatment of tuberculosis.
• R alkyl, aryl, aralkyl, heteroaryl, mono- and/or bicyclic substituted and/unsubstituted aryl or heteroaryl.
• Tuberculosis is a life-threatening chronic infection primarily caused by Mycobacterium tuberculosis that continues as global epidemic, with more than 9 million new cases each year and nearly 2 million deaths in every year [WHO report 2010].
• LTBI latent TB infection
• DOTS direct observed therapy short-course
• INF isoniazid
• RMP rifampicin
• PZA pyrazinamide
• EMB ethambutol
• SM alternatively streptomycin
• MDR-TB multidrug-resistant TB
• XDR-TB extensively drug-resistant TB
• the MDR-TB is resistant to the most common first-line anti-TB drugs, i.e., INH and RMP
• XDR-TB is also resistant to the fluoroquinolones and at least one of the intravenous second-line drugs, i.e., kanamycin, capreomycin, or amikacin.
• MDR-TB is responsible for approximately 500,000 new cases per year and for the infection in 740 thousand new patients by both Mtb and HIV/AIDS [WHO report 2010].
• XDR-TB emphasizes the urgent need for development of new therapeutics for TB to prevent the emergence LLS268 of drug-resistant Mycobacterium tuberculosis strains, and to shorten the long-term therapies lasting for 6-12 months period.
• TMC207 also known as R207910 or the 'J' compound
• TMC207 is especially promising due to its novel mechanism of action, potent activity against drug- sensitive and drug-resistant TB, and potential for treatment shortening in preclinical studies [Future Microbiol. 2010, 5, 849-858. Nature 2011, 469, 483-490].
• the TMC207 is a diarylquinoline that inhibits FoFi-ATP synthase (or ATPase) of M. tuberculosis, which is a novel mechanism of action.
• N, ⁇ - dicyclohexylcarbodiimide (DCCD) can bind covalently to subunit c and thereby abolish the ATPase activity.
• DCCD binds to a specific glutamate or aspartate residue which is located in the middle of the second hydrophobic region near the C terminus of the ATPase. This entry represents the site that includes the DCCD-binding residue.
• the main object of the present invention is to provide novel N-(3-((diethylamino)methyl)-4- hydroxyphenyl)-N-(quinolin-4-yl)sulfonamides compounds of general formula A and pharmaceutical acceptable salts thereof.
• Another object of the present invention is to provide a process of preparation of N-(3- ((diethylamino)methyl)-4-hydroxyphenyl)-N-(quinolin-4-yl)sulfonamides compounds of general formula A and pharmaceutical acceptable salts thereof useful as therapeutic agents, particularly in the prevention or treatment of tuberculosis targeting F 0 F ATP synthase enzyme of M. tuberculosis.
• the present invention relates to a compound of general formula A and pharmaceutically acceptable salts thereof:
• R is methyl
• Ri, R 2 , and R 3 may be same or different present at any position(s) and are groups selected the group of hydrogen, halogen, alkyl (C1-C3), nitro, cyano, trifluoromethyl;
• R is a group of the structure wherein X may be CH or N, and the attachment point of sulfonyl may be at the position 1 or 2, (or) R is a group of the structure LLS268
• Ri is hydrogen or halogen (or) R is a group of the structure
• the pharmaceutical acceptable salts may be selected from the group consisting from hydrochloride, tartarate, sodium, potassium.
• the compound of general formula A is useful as anti- tubercular agent.
• the compound of general formula A and pharmaceutical acceptable salts thereof is useful in the treatment of multi-drug resistant tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB).
• MDR-TB multi-drug resistant tuberculosis
• XDR-TB extensively drug resistant tuberculosis
• the compound of general formula A exhibits an in vitro anti-tuberculosis activity against Mycobacterium tuberculosis H37Rv and Mycobacterium smegmatis. In yet another embodiment of the present invention, the compound of general formula A exhibits mycobacterial ATPase inhibitatory activity against Mycobacterium smegmatis.
• the compound of general formula A exhibits MIC in the range of 5 to 50 ⁇ causing 90% growth inhibition.
• concentration of the compound of general formula A, exhibiting 50% inhibition of mycobacterial ATPase, (IC 50 ) is ranging between 0.3 to 9.5 ⁇ .
• step (b) dissolving compound of formula 3 as obtained in step (a) in an aprotic solvent; (c) adding a base to the solution of compound 3 as obtained in step (b) followed by stirring under inert atmosphere;
• step (d) adding substituted / unsubstituted aryl /alkyl sulfonyl chloride to the mixture as obtained from step (c) followed by stirring under inert atmosphere to obtain the substituted compound;
• step (e) purifying the substituted compound as obtained in step (d) by chromatographic methods to obtain the compounds of general formula A.
• the substituted / unsubstituted aryl /alkyl sulfonyl chloride compounds are selected from the group consisting of 2,3-dichlorobenzenesulfonyl chloride, 4- chlorobenzene-sulfonyl chloride, 2,4-dichlorobenzenesulfonyl chloride, 2,5-dichlorobenzenesulfonyl LLS268 chloride, 4-riitrobenzenesulfonyl chloride, 4-cyanobenzenesulfonyl chloride, 7,7-dimethyl-2- oxobicyclo[2.2.1]heptane-l-sulfonyl chloride, 3-(trifluoromethyl)benzenesulfonyl chloride, naphthalene-2- sulfonyl chloride, quinoline-8-sulfonyl chloride, 2,4,6-triisopropylbenzenesulfon
• the acid in step (a) is selected from the group consisting of hydrochloric acid, glacial acetic acid and sulfuric acid.
• the aprotic solvent in step (b) is selected from the group consisting of tetrahydrofruan (THF), dichloromethane (DCM) and acetone.
• the base in step (c) is selected from the group consisting of triethylamine, NN-diisopropylethylamine (NN-DIPEA) and NN-dimethylaminopyridine (DMAP).
• N-DIPEA NN-diisopropylethylamine
• DMAP NN-dimethylaminopyridine
• stirring under inert atmosphere in step (c) is done for a period ranging between 10 to 30 minutes.
• stirring under inert atmosphere in step (d) is done for a period ranging between 0.5 to 24 hrs.
• the chromatographic method in step (e) is column chromatography using EtOAcHexane as eluent.
• Yet another embodiment of the invention provides a compound of formula 3
• a pharmaceutical composition comprising therapeutically effective amount of a compound of general formula A and pharmaceutically acceptable salts thereof, optionally along with one or more pharmaceutically acceptable carriers, additives, lubricants and diluents.
• Figure 1 outlines the scheme for the synthesis of compounds represented by formula A [4a - 1].
• Figure 2 outlines the scheme for the synthesis of the intermediate compound 1.
• Figure 3 outlines the synthesis of the intermediate compound 2.
• Figure 4 outlines in vitro Vero monkey kidney cell toxicity data for compound 4b (A) and for compound 4b (B) using cytotoxicity assay 1.
• Figure 5 outlines in vivo results of compounds against M. tuberculosis H37Rv in mouse model. ABBREVIATIONS
• the present invention relates to novel N-(3-((diethylamino)methyl)-4-hydroxyphenyl)-JV-(quinolin-4- yl)sulfonamides, their preparation, and to their use as therapeutic agents, particularly in the prevention or treatment of tuberculosis.
• the present invention provides the molecules useful for the treatment or prevention of tubercular infections caused by Mycobacterium species.
• the present invention particularly relates to compound of general formula A: LLS268
• the compound 3 is prepared by the reaction of the N-(3-((diethylamino)methyl)-4- hydroxyphenyl)acetamido (2) with the 4,6-dichloro-2-methylquinoline (1) using 20% hydrochloric acid (HC1) followed by the refluxing in absolute ethanol.
• HC1 hydrochloric acid
• the compound 1 may be prepared by the scheme shown in Figure 2. This involves the following steps:
• the compound 6 is prepared by the reaction of m-chloroaniline (5) with the ethylacetoacetate in the presence of catalytic amount of HC1, followed by heating under reflux using phenyl ether.
• the compound 2 may be prepared by the scheme outlined in Figure 3. This involves the following steps:
• Example 1 4,7-dichloro-2-methylquinoline (1)
• the compound 2 (20g, 85 mM) was refluxed with 20% HC1 (30 mL) for 2 hrs for complete N-deacylation. After the completion of the deacetylation reaction, a solution of 4,7-dichloro-2-methylquinoline (18g, 85mM) in ethanol (50mL) was added and reaction mixture was refluxed for 6 hrs. After the completion of the reaction, ethanol was evaporated under reduced pressure and residue was dissolved in distilled water. Careful addition of dilute (2%) ammonium hydroxide solution to the diluted reaction mixture liberated the free base as bright yellow solid, which was filtered and washed with distilled water, and then, dried.
• Example 3 7-chloro-2-methylquinolin-4-ol (6)
• Concentrated HC1 0.5 mL was added to a mixture of m-chloroaniline (3.2g, 25 mM) and ethyl acetoacetate (4g, 28mM) and then stirred overnight at 25°C.
• the mixture was then diluted with dichloromethane (DCM) (50 mL), washed with water, dried over sodium sulfate, and then DCM layer was evaporated under vacuum.
• the carbonate formed as a crude oil was suspended in the boiling phenyl ether (70 mL) and refluxed for half an hour.
• Example 4 p-methoxy-acetanilide (8) p-Anisidine (4, ImM) was acylated with acetic anhydride (2mM) by heating under reflux in toluene for about 1 hr. After the completion of the reaction (monitored using TLC), the toluene was removed under reduced pressure to yield the acylated product for the use in the next reaction.
• Example 5 p-hydroxy-acetanilide (9) BBr 3 (1M in DCM, 16.8 mM) was added to a stirring suspension of p-methoxy-acetanilide (5) in dry DCM (80 mL) under inert gas environment, and the resulting mixture was allowed to stir at room temperature (RT) overnight. Water was added to hydrolyze the excess of BBr3, and the suspension was further stirred for additional 30 mins. Then, DCM layer was separated and the aqueous layer was extracted three times with ethyl acetate (EtOAc). The combined organic layer was dried over sodium sulfate and evaporated to yield the product.
• EtOAc ethyl acetate
• Method A The compound 3 (lg, 2.7mM) was dissolved in dry acetone and stirred for 5 mins.
• the 4-chlorobenzene-sulfonyl chloride (0.7 g) was then added to the reaction mixture dropwise under inert nitrogen gas environment.
• the reaction mixture was stirred under inert gas environment for 3 hrs. The completion of the reaction was monitored using TLC method.
• the compound 3 (lg, 2.7mM) was dissolved in dry DCM and stirred for 10 mins.
• the ⁇ , ⁇ - diisopropylethyl amine (NN-DIPEA) (0.7 mL) was then added dropwise to the stirring mixture and stirred for an additional 30 mins.
• the 4-chlorobenzene-sulfonyl chloride (0.7 g) was then added to the reaction mixture dropwise, and stirred for 10 hrs.
• the compound 3 (lg, 2.7mM) was dissolved in dry acetone and stirred for 5 mins. ⁇ , N-DIPEA (0.7 mL) was then added drop-wise to the stirring mixture during 10 mins inert gas environment and stirred for an additional 10 mins.
• the 2,3-dichlorobenzenesulfonyl chloride (0.8 g) was then added to the reaction mixture drop-wise under inert nitrogen gas environment.
• the reaction mixture was stirred under inert nitrogen gas environment for 3.5 hrs.
• the completion of the reaction was monitored using TLC method. After the completion of the reaction, the solvent was evaporated under reduced pressure and the residue was dissolved in ethyl acetate and washed with distilled water.
• Example 8 N-(7-chloro-2-methylquinolin-4-yl)-N-(3-((diethylamino)methyl)-4-hydroxyphenyl)-2,4- dichlorobenzenesulfonamide (4c)
• the compound 3 (lg, 2.7mM) was dissolved in dry acetone and stirred for 5 mins.
• N, N-DIPEA (0.7 mL) was then added drop-wise to the stirring mixture during 10 mins inert nitrogen gas environment and stirred for an additional 10 mins.
• the 2,4-dichlorobenzenesulfonyl chloride (0.8 g) was then added to the reaction mixture drop-wise under inert nitrogen gas environment.
• the compound 3 (lg, 2.7mM) was dissolved in dry acetone and stirred for 5 mins. N, N-DIPEA (0.7 mL) was then added drop-wise to the stirring mixture for 10 mins under inert nitrogen gas environment and stirred for an additional 10 mins. The 2,5-dichlorobenzenesulfonyl chloride (0.8 g) was then added to the reaction mixture drop-wise under inert nitrogen gas environment. The reaction mixture was stirred under inert nitrogen gas environment for 3 hrs. The completion of the reaction was monitored using TLC method. After the completion of the reaction, the solvent was evaporated under reduced pressure and the residue was dissolved in ethyl acetate and washed with distilled water.
• the compound 3 (lg, 2.7mM) was dissolved in dry acetone and stirred for 5 mins.
• the NN-DIPEA (0.7 mL) was then added drop-wise to the stirring mixture for 10 mins under inert nitrogen gas environment and stirred for an additional 10 mins.
• the 4-nitrobenzenesulfonyl chloride (0.72 g) was then added to the reaction mixture drop-wise under inert nitrogen gas environment.
• the reaction mixture was stirred under inert nitrogen gas environment for 5 hrs.
• the completion of the reaction was monitored using TLC method. After the completion of the reaction, the solvent was evaporated under reduced pressure and the residue was dissolved in ethyl acetate and washed with distilled water.
• the compound 3 (lg, 2.7mM) was dissolved in dry acetone and stirred for 5 mins.
• the NN-DIPEA (0.7 mL) was then added drop-wise to the stirring mixture for 10 mins under inert nitrogen gas environment and stirred for an additional 10 mins.
• the 4-cyanobenzenesulfonyl chloride (0.66 g) was then added to the reaction mixture drop-wise under inert nitrogen gas environment.
• the reaction mixture was stirred under inert nitrogen gas environment for 3 hrs.
• the completion of the reaction was monitored using TLC method. After the completion of the reaction, the solvent was evaporated under reduced pressure and the residue was dissolved in ethyl acetate and washed with distilled water.
• the compound 3 (lg, 2.7mM) was dissolved in dry acetone and stirred for 5 mins.
• the NN-DIPEA (0.7 mL) was then added drop-wise to the stirring mixture during 10 mins inert nitrogen gas environment and stirred for an additional 10 mins.
• the (lR)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-l-sulfonyl chloride (1.36 g) was then added to the reaction mixture drop-wise under inert nitrogen gas environment.
• the reaction mixture was stirred under inert nitrogen gas environment for 6 hrs depending on nature of sulphonyl chloride. The completion of the reaction was monitored using TLC method.
• the compound 3 (lg, 2.7mM) was dissolved in dry acetone and stirred for 5 mins.
• the NN-DIPEA (0.7 mL) was then added drop-wise to the stirring mixture during 10 mins inert nitrogen gas environment and stirred for an additional 10 mins.
• the 3-(trifluoromethyl)benzenesulfonyl chloride (0.8 g) was then added to the reaction mixture drop-wise under inert nitrogen gas environment.
• the reaction mixture was stirred under inert nitrogen gas environment for 3 hrs.
• the completion of the reaction was monitored using TLC method. After the completion of the reaction, the solvent was evaporated under reduced pressure and the residue was dissolved in ethyl acetate and washed with distilled water.
• the compound 3 (lg, 2.7mM) was dissolved in dry acetone and stirred for 5 mins.
• the NN-DIPEA (0.7 mL) was then added drop-wise to the stirring mixture during 10 mins inert nitrogen gas environment and LLS268 stirred for an additional 10 mins.
• the naphthalene-2-sulfonyl chloride (0.76 g) was then added to the reaction mixture drop-wise under inert nitrogen gas environment.
• the reaction mixture was stirred under inert nitrogen gas environment for 2 hrs.
• the completion of the reaction was monitored using TLC method. After the completion of the reaction, the solvent was evaporated under reduced pressure and the residue was dissolved in ethyl acetate and washed with distilled water.
• the compound 3 (lg, 2.7mM) was dissolved in dry acetone and stirred for 5 mins.
• the NJV-DIPEA (0.7 mL) was then added drop-wise to the stirring mixture during 10 mins inert nitrogen gas environment and stirred for an additional 10 mins.
• the quinoline-8-sulfonyl chloride (0.75 g) was then added to the reaction mixture drop-wise under inert nitrogen gas environment.
• the reaction mixture was stirred under inert nitrogen gas environment for 4 hrs.
• the completion of the reaction was monitored using TLC method. After the completion of the reaction, the solvent was evaporated under reduced pressure and the residue was dissolved in ethyl acetate and washed with distilled water.
• the compound 3 (lg, 2.7mM) was dissolved in dry acetone and stirred for 5 mins.
• the NJV-DIPEA (0.7 mL) was then added drop-wise to the stirring mixture during 10 mins inert nitrogen gas environment and stirred for an additional 10 mins.
• the 2,4,6-triisopropylbenzenesulfonyl chloride (1.0 g) was then added to the reaction mixture drop-wise under inert nitrogen gas environment.
• the reaction mixture was stirred under inert nitrogen gas environment for 2 hrs.
• the completion of the reaction was monitored using TLC method. After the completion of the reaction, the solvent was evaporated under reduced pressure and the -LS268 residue was dissolved in ethyl acetate and washed with distilled water.
• the compound 3 (lg, 2.7mM) was dissolved in dry acetone and stirred for 5 mins.
• the iViV-DIPEA (0.7 mL) was then added drop-wise to the stirring mixture during 10 mins inert nitrogen gas environment and stirred for an additional 10 mins.
• the 2,4,6-trimethylbenzenesulfonyl chloride (0.72 g) was then added to the reaction mixture drop-wise under inert nitrogen gas environment.
• the reaction mixture was stirred under inert nitrogen gas environment for 1.5 hrs depending on nature of sulphonyl chloride. The completion of the reaction was monitored using TLC method.
• the compound 3 (lg, 2.7mM) was dissolved in dry acetone and stirred for 5 mins.
• the JVN-DIPEA (0.7 mL) was then added drop-wise to the stirring mixture during 10 mins inert nitrogen gas environment and stirred for an additional 10 mins.
• the naphthalene- 1-sulfonyl chloride (0.75 g) was then added to the reaction mixture drop-wise under inert nitrogen gas environment.
• the reaction mixture was stirred under inert nitrogen gas environment for 2 hrs.
• the completion of the reaction was monitored using TLC method. After the completion of the reaction, the solvent was evaporated under reduced pressure and the residue was dissolved in ethyl acetate and washed with distilled water.
• the compound 3 (lg, 2.7mM) was dissolved in dry acetone and stirred for 5 mins.
• the NN-DIPEA (0.7 mL) was then added drop-wise to the stirring mixture during 10 mins under inert nitrogen gas environment and stirred for an additional 10 mins.
• the 3,5-dichlorobenzenesulfonyl chloride (0.81 g) was then added to the reaction mixture drop-wise under inert nitrogen gas environment.
• the reaction mixture was stirred under inert nitrogen gas environment for 1.5 hrs.
• the completion of the reaction was monitored using TLC method. After the completion of the reaction, the solvent was evaporated under reduced pressure and the residue was dissolved in ethyl acetate and washed with distilled water.
• the compound 3 (lg, 2.7mM) was dissolved in dry acetone and stirred for 5 mins.
• the NiV-DIPEA (0.7 mL) was then added drop-wise to the stirring mixture during 10 mins inert nitrogen gas environment and stirred for an additional 10 mins.
• the 3,4-dichlorobenzenesulfonyl chloride (0.8 g) was then added to the reaction mixture drop-wise under inert nitrogen gas environment.
• the reaction mixture was stirred under inert nitrogen gas environment for 1 hrs.
• the completion of the reaction was monitored using TLC method. After the completion of the reaction, the solvent was evaporated under reduced pressure and the residue was dissolved in ethyl acetate and washed with distilled water.
• the compound 3 (lg, 2.7mM) was dissolved in dry acetone and stirred for 5 mins.
• the NN-DIPEA (0.7 mL) was then added drop-wise to the stirring mixture during 10 mins inert nitrogen gas environment and stirred for an additional 10 mins.
• the thiophene-2-sulfonyl chloride (0.98 g) was then added to the reaction mixture drop-wise under inert nitrogen gas environment.
• the reaction mixture was stirred under inert nitrogen gas environment for 2.5 hrs.
• the completion of the reaction was monitored using TLC method. After the completion of the reaction, the solvent was evaporated under reduced pressure and the residue was dissolved in ethyl acetate and washed with distilled water.
• the compound 3 (lg, 2.7mM) was dissolved in dry acetone and stirred for 5 mins.
• the NN-DIPEA (0.7 mL) was then added drop-wise to the stirring mixture during 10 mins inert nitrogen gas environment and stirred for an additional 10 mins.
• the 2,4-dinitrobenzenesulfonyl chloride (0.87 g) was then added to the reaction mixture drop-wise under inert nitrogen gas environment.
• the reaction mixture was stirred under inert nitrogen gas environment for 3 hrs.
• the completion of the reaction was monitored using TLC method. After the completion of the reaction, the solvent was evaporated under reduced pressure and the residue was dissolved in ethyl acetate and washed with distilled water.
• the compound 3 (lg, 2.7mM) was dissolved in dry acetone and stirred for 5 mins.
• the NN-DIPEA (0.7 mL) was then added drop-wise to the stirring mixture during 10 mins inert nitrogen gas environment and stirred for an additional 10 mins.
• the 5-chlorothiophene-2-sulfonyl chloride (0.72 g) was then added to the reaction mixture drop-wise under inert nitrogen gas environment.
• the reaction mixture was stirred under inert nitrogen gas environment for 4 hrs.
• the completion of the reaction was monitored using TLC method. After the completion of the reaction, the solvent was evaporated under reduced pressure and the residue was dissolved in ethyl acetate and washed with distilled water.
• Example 24 N-(7-chloro-2-methylquinolin-4-yl)-N-(3-((diethyIamino)methyl)-4-hydroxyphenyl)-3,5- dimethylbenzenesulfonamide (4s)
• the compound 3 (lg, 2.7mM) was dissolved in dry acetone and stirred for 5 min.
• the NN-DIPEA (0.7 mL) was then added drop-wise to the stirring mixture during 10 min inert nitrogen gas environment and stirred for an additional 10 min.
• the 3,5-dimethylbenzenesulfonyl chloride (0.68 g) was then added to the reaction mixture drop-wise under inert nitrogen gas environment.
• the reaction mixture was stirred under inert nitrogen gas environment for 3 hrs.
• Method A The compound 3 (lg, 2.7mM) was dissolved in dry acetone and stirred for 5 min.
• the NN- DIPEA (0.7 mL) was then added drop-wise to the stirring mixture during 10 min inert nitrogen gas environment and stirred for an additional 10 min.
• the methane sulfonyl chloride (0.4 g) was then added to the reaction mixture drop-wise under inert nitrogen gas environment.
• the reaction mixture was stirred LLS268 under inert nitrogen gas environment for 5 hrs. The completion of the reaction was monitored using TLC method. After the completion of the reaction, the solvent was evaporated under reduced pressure and the residue was dissolved in ethyl acetate and washed with distilled water.
• ADA In vitro Anti-TB activity evaluation Agar Dilution Assay (ADA): Compounds were dissolved in dimethylsulfoxide (DMSO) to make 5mg/mL stock solutions. Serial dilutions from stocks were also made in DMSO. Standard antitubercular drugs (isoniazid and rifampicin) were used as positive control and the vehicle (DMSO) was used as negative control. An amount of O.lmL of serially diluted test compounds or standard drugs were added to 1.9mL Middlebrook 7H10 agar medium (with OADC supplement, in glass tubes). O.lmL/tube DMSO was used as vehicle control. The contents were mixed and allowed to solidify as slants. Three-week old culture of M.
• DMSO dimethylsulfoxide
• tuberculosis H37Rv was harvested from Lowenstein-Jensen medium and its suspension (lmg/mL, equivalent to 10 8 bacilli) was made in normal saline containing 0.05% Tween-80. 10 ]i of 1:10 dilution of this suspension (-10 5 bacilli) was inoculated into each tube and incubated at 37°C for 4 weeks. The lowest concentration of the compound up to which there was no visible growth of bacilli was its minimum inhibitory concentration (MIC). [ J. K. McClachy. Lab. Med. 1978, 9, 47] BACTEC Assay: The in vitro anti-TB activity of compounds was measured by BACTEC-460TB radiometric method for determination of the minimum inhibitory concentration (MIC). The M.
• tuberculosis H37Rv strain was used to grow in 7H12 medium containing 14 C labeled palmitic acid as substrate during which 14 C0 2 was liberated.
• the amount of 14 C0 2 detected by the BACTEC system reflected the growth of the organism and was expressed in terms of the "Growth Index” (GI).
• GI Crowth Index
• MABA method This method, the microplate Alamar Blue assay (MABA), is based on the detection of colorimetric changes caused by the oxidation and reduction capabilities of Alamar Blue dye. Briefly, the MABA involved the addition of Alamar Blue solution to microplates containing antibiotics in increasing concentrations and pure cultures of M. tuberculosis. After an incubation period of 5 days, the growth of M. tuberculosis was observed as a change in the coloration of the Alamar Blue solution due to reduction of the dye. MICs of each antibiotic tested was determined by this change of color in the wells. [Franzblau et al. J. Clin. Microbiol. 1998, 36, 362] Cytotoxicity Studies:
• Resazurin assay using Vero monkey kidney cells (Cytotoxicity Assay 1): The compounds for cytotoxicity were tested in an in vitro model for toxicity with Vero monkey kidney cells using Resazurin assay.
• the Vero cells (ATCC CCL-8 1) were seeded overnight at 1 x 10 4 - 3 xlO 6 cells per well in 96-well plates at 37°C in RPMI supplemented with 10% heat-inactivated fetal bovine serum and 5% C0 2 . Cells were exposed to dilutions of experimental and control drugs in triplicate for 2h with each compound at a range of concentrations from 100 -1.56 ⁇ g/ml. Rifampicin was used as a control at the same concentrations.
• MTS solution tetrazolium compound, Owen's reagent
• mice 8 weeks old inbred mice were infected /.v. with 10 6 CFU of M. tuberculosis H37Rv. Chemotherapy was initiated 4 days following infection. Mice treated with INH (25 mg/ kg) and Ethambutol (EMB, 100 mg/ kg) was used as positive controls and infected mice that were untreated served as negative controls. Test compounds were administered daily by gavage for 30 days (6 days/ week) at a dose of 100 mg/ kg.
• INH 25 mg/ kg
• EMB Ethambutol
• mice for every tested drug was sacrificed, and their lungs were removed and homogenized in sterile 2 ml PBS with 0.05% Tween 80. Lung homogenates were plated in 10-fold serial dilutions on MB 7H10 agar containing OADC and incubated at 37 ° C. CFU were calculated after 3 weeks of growth.
• Table 1 summarizes the in vitro M. tuberculosis growth inhibitory and mycobacterial ATP synthase inhibitory activities of the compounds represented by formula A.
• the claimed compounds are found to inhibit M. smegmatis ATP synthase (ATPase) enzyme potently at submicromolar concentrations.
• the compounds namely 4a-c, 4e, 4f, 4i, and 4r have ATPase IC 50 values lesser than 1 (Table 1).
• the exact IC 50 values of these compounds are summarized in Table 1.
• these compounds have exhibited potential M. tuberculosis growth inhibitory activity (Table 1).
• These compounds represent promising leads for the development of candidate drugs for the effective treatment of tuberculosis.
• the novel mechanism of action i.e. ATPase of mycobacterium, suggests their potential to cure the MDR-TB and XDR-TB.
• Cytotoxicity The selected potential compounds were also tested for any cytotoxicity in assay 1 and assay 2 using Vero cell lines, and in assay 3 utilizing macrophage systems.
• Figure 4(A) depicts the in vitro Vero monkey kidney cell toxicity data for compound (4b) while
• Figure 4(B) depicts in vitro Vero monkey kidney cell toxicity data for compound (4c) using cytotoxicity assay 1.
• compounds 4(b-e) were found to be non-toxic in the macrophage system (assay 3).
• these compounds have been found to be completely free from any cytotoxicity, thus suggesting their candidature for the effective anti-tubercular agents.
• Figure 5 depicts the in vivo data for the two compounds, namely (4b) and (4c) along with the current drugs ethambutol (EMB) and isoniazid (INH), against M. tuberculosis H37Rv in mouse model.
• the two compounds (4b) and (4c) showed reduction in CFU in lungs of mice as a result of chemotherapy.
• Compound 4b produced 1.19 logio reduction of the CFU in the lungs compared to the growth in the untreated control group (7.32 versus 8.51 logio CFU) while 4c produced 0.92 logio reduction (7.59 versus 8.51 log 10 CFU) that is indicative of anti-TB activity (Tuberculosis antimicrobial Acquisition and Coordinating Facility, www.tacf.org).
• Ethambutol (EMB) produced 1.94 log 10 reduction (6.57 versus 8.51 logio CFU) and isoniazid (INH) produced 2.8 log 10 reduction of the CFUs in the lungs of infected and
• the compound in the present invention represents a new class of potent anti-tuberculosis drugs targeting a novel M. tuberculosis target i.e. inhibition of mycobacterial ATP synthase (ATPase), and thus may be potentially effective for the treatment of MDR-TB and XDR-TB. Due to novel mechanism of action, the compounds in the present invention may be beneficial for TB programs that need to ensure optimal patient adherence throughout the entire treatment course.
• ATPase mycobacterial ATP synthase

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