Technical field

The present invention relates to a compound of Formula I (as described herein), which is isolated from the microorganism belonging to Actinomycetes (PM1180133), having antibacterial activity; particularly against Mycobacterium tuberculosis strain. The present invention further relates to processes for the production of the compound of Formula I (as described herein) or its isomer(s) from the microorganism belonging to Actinomycetes. The present invention relates to a method for the treatment of tuberculosis comprising administering to a subject in need thereof, a therapeutically effective amount of the compound of Formula I or the compound of Formula la, or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof. The present invention also relates to a pharmaceutical composition comprising the compound of Formula I or the compound of Formula la.

Background of the invention

Tuberculosis (TB) is a common lethal infectious disease caused by various strains of mycobacteria, particularly Mycobacterium tuberculosis (MTB). The distribution of tuberculosis is not uniform across the globe. In 2012, an estimated 8.6 million people developed TB and 1.3 million died from the disease (including 320,000 deaths among HIV- positive people) (The World Health Organization Report 2013).

Tuberculosis primarily affects the lungs, but it may also affect various organs of the body. Tuberculosis of the lungs usually spreads through the air when a person having lung TB coughs, or sneezes or transmits respiratory fluids, the TB germs get propelled in the air. Other persons can get infected if a few of these TB germs are inhaled by them and the infected person has a lifetime risk of falling ill with TB. The common symptoms of TB are chronic cough with blood-tinged sputum, chest pain, fever, night sweats, and weight loss. Diagnosis relies on radiology (commonly chest X-rays), a tuberculin skin test, blood tests, as well as microscopic examination and microbiological culture of body fluids such as sputum. However, diagnosing MDR-TB (Multidrug-resistant TB) and HIV-associated TB can be more complex.

Effective tuberculosis treatment involves use of antibiotics, primarily referred to as anti-tubercular drugs. However, unusual structure and chemical composition of the mycobacterial cell wall, hinders the entry of the anti-tubercular (anti-TB) drugs and makes many antibiotics ineffective. Instead of the short course of antibiotics typically used to cure other bacterial infections, TB requires much longer period of treatment (around six to twenty four months) to entirely eliminate mycobacteria from the body. The first-line treatment of TB involves use of the anti-tubercular drugs namely rifampicin, isoniazid, pyrazinamide and ethambutol; in combination. Over the period it has been observed that patients infected with Mycobacterium tuberculosis harbor strains with naturally occurring mutations, which are resistant to one or more anti-TB drugs. The strains that are resistant to major anti-TB drugs have been identified. Multidrug-resistant tuberculosis (MDR TB) is a form of tuberculosis that is resistant to two or more of the primary drugs used in the treatment of tuberculosis. In view of the above discussion, it is evident that the currently available therapy for TB is no longer consistently effective as a result of the problems with treatment compliance, and these compliance problems contribute to the development of drug resistant mycobacterial strains.

Thus, there exists an absolute need for the development of a new or an improved regimen that would involve less treatment time, thus decreasing cost, increasing patient compliance, and slowing the emergence of multidrug -resistant TB (MDR-TB) strains.

In consideration of the need as indicated above, inventors of the present application directed their efforts to provide a novel treatment regimen for TB. These efforts led to the identification of compounds of Formula I that exhibit activity against Mycobacterium tuberculosis and multidrug resistant Mycobacterium tuberculosis. The compounds of Formula la encompass Netropsin, which is a known oligopeptide, isolated from the actinobacterium Streptomyces netropsis. Netropsin was reported to be active against both Gram-positive and Gram-negative bacteria (The Journal of American Chemical Society, 1951, 73, 341-343; The Journal of Molecular Biology, 1986, 47, 31-112).

The inventors of the present invention have found that the compound of Formula I (as described herein) and the compound of Formula la (as described herein) are active against both, Mycobacterium tuberculosis and multidrug resistant Mycobacterium tuberculosis, and hence, useful in the treatment of TB.

Summary of the invention

In one aspect, the present invention relates to a compound of Formula I (as described herein) or its isomer(s) isolated from the microorganism belonging to Actinomycetes (PM1180133). The compound of Formula I is selected from the compound of Formula lb (as described herein) or the compound of Formula Ic (as described herein).

In another aspect, the present invention relates to processes for the production of the compound of Formula I selected from the compound of Formula lb or the compound of Formula ic or its isomer(s) from the microorganism belonging to Actinomycetes (PM1180133).

In an aspect, the present invention relates to a compound of Formula I or its isomer(s) produced from the microorganism belonging to Actinomycetes (PM1180133) by a process comprising the steps of:

(a) growing the culture number PM1180133;

(b) isolating the compound of Formula I from the culture broth; and

(c) purifying the compound of Formula I.

In an aspect, the present invention relates to a method for the treatment of tuberculosis comprising administering to a subject in need thereof, a therapeutically effective amount of the compound of Formula I or the compound of Formula la, or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof.

In an aspect, the present invention relates to a method of inhibiting growth of Mycobacterium tuberculosis organism, comprising contacting the said organism in vitro or ex vivo with the compound of Formula I or the compound of Formula la, or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof, in an amount sufficient to inhibit growth of the said organism.

In another aspect, the present invention relates to the compound of Formula I or the compound of Formula la, or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof, for use in the treatment of tuberculosis.

In another further aspect, the present invention relates to a pharmaceutical composition comprising the compound of Formula I /or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof, and at least one pharmaceutically acceptable carrier.

In yet another aspect, the present invention also relates to use of the compound of

Formula I or the compound of Formula la, or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof, for the manufacture of a medicament for the treatment of tuberculosis.

In a further aspect of the present invention, there is provided a method for the treatment of tuberculosis comprising administering to a subject in need thereof, a therapeutically effective amount of the compound of Formula I or the compound of Formula la, or its isomer(s), a tautomer or a pharmaceutically acceptable salt or a solvate thereof; along with one or more further therapeutically active agents. These and other aspects and advantages of the present invention will be apparent to those skilled in the art from the following description

Description of the drawings

FIGURE 1 shows the ^J H NMR spectrum (500 MHz) of the compound of Formula la in DMSO d ₆ .

FIGURE 2 shows the 13 C NMR spectrum (125 MHz) of the compound of Formula la in DMSO d ₆ .

FIGURE 3 shows the ^J H NMR spectrum (500 MHz) of the compound of Formula I in DMSO d ₆ .

FIGURE 4 shows the ¹³ C NMR spectrum (125 MHz) of compound of Formula I in DMSO d ₆ .

FIGURE 5 a shows an effect of the compound of Formula I on incorporation of H Thymidine into DNA.

FIGURE 5b shows an effect of the compound of Formula la on incorporation of H Thymidine into DNA.

FIGURE 6a shows an effect of the compound of Formula I on incorporation of H Uridine into RNA.

FIGURE 6b shows an effect of the compound of Formula la on incorporation of ³ H Uridine into RNA. Detailed Description of the Invention

It should be understood that the detailed description and specific examples, while indicating embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art. One skilled in the art, based upon the definitions herein, may utilize the present invention to its fullest extent. The following specific embodiments are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

Except as defined herein, all the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Definitions

For the purpose of the disclosure, listed below are definitions of various terms used to describe the present invention. These definitions apply to the terms as they are used throughout the specification and the appended claims (unless they are otherwise limited in specific instances) either individually or as part of a larger group. They should not be interpreted in the literal sense. They are not general definitions and are relevant only for this application.

It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. It should also be noted that the term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise.

Also, use of "(s)" as part of a term, includes reference to the term singly or in plurality, e.g. the term isomer(s) may indicate one isomer or more than one isomer.

The term "pharmaceutically acceptable" refers to a compound or a carrier or an additive or a salt that is not biologically or otherwise undesirable. In other words a pharmaceutically acceptable compound or a salt must not cause any undesirable biological effects or interact in an undesirable manner with any of the other ingredients of the pharmaceutical composition in which it is contained.

The term "pharmaceutically acceptable carrier" as used herein means a diluent, excipient, encapsulating material or formulation auxiliary, which is non-toxic, and inert, which does not have undesirable effects on a subject, preferably a mammal, more preferably a human, and is suitable for delivering a therapeutically active agent (e.g. the compound of Formula I or the compound of Formula la, or its isomer(s), a tautomer, or a pharmaceutically acceptable salt thereof) to the target site without affecting activity of the agent.

The term "(Ci-C6)alkyl", as used herein either alone or as part of another group refers to unsubstituted or substituted straight or branched chain hydrocarbons containing 1 to 6 carbons selected from, but not limited to, methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl and hexyl. The substituted alkyl refers to a (Ci-Ce)alkyl substituted with one or more groups selected from, but not limited to, halogen, hydroxy, -0(Ci-Ce)alkyl, nitro, cyano, -COOH, -NH ₂ , -NH(d-C ₆ )aikyl, -N[(C ₁ -C ₆ )alkyl] ₂ , -NHC(0)0(d-C ₆ )alkyl, - NHC(O)O(C ₁ -C ₆ )alkyl(C ₆ -C ₁₀ )aryl, -NH-PEG, (C ₆ -C ₁₀ )aryl or the like groups.

The term, "therapeutically effective amount" as used herein means an amount of the compound of Formula I or the compound of Formula la or its isomers or pharmaceutically acceptable salts or solvates thereof; or a composition comprising said compound(s), sufficient to significantly induce a positive modification in the condition (e.g. tuberculosis or MDR tuberculosis or XDR tuberculosis or latent tuberculosis) to be treated, but low enough to avoid side effects, if any (at a reasonable benefit/risk ratio), within the scope of sound medical judgment. The requirement of therapeutically effective amount of the compound of Formula I or the compound of Formula la, or the composition containing said compound(s), for the treatment will vary with the severity of tuberculosis being treated, the age and physical condition of the subject (patient), the duration of the treatment, the nature of concurrent therapy, the particular pharmaceutically acceptable carrier utilized, and like factors.

As used herein the term "contacting" is meant to broadly refer to bringing

Mycobacterium tuberculosis organism or the multi-drug resistant Mycobacterium tuberculosis organism and the compound of Formula I or the compound of Formula la, or its isomers or pharmaceutically acceptable salts or solvates thereof; into sufficient proximity that the said compound(s) can exert an effect on the bacterial cell. The compound may be transported to the location of the Mycobacterium tuberculosis organism or the multi-drug resistant Mycobacterium tuberculosis organism, or the compound may be situated in a location to which the Mycobacterium tuberculosis organism or the multi-drug resistant Mycobacterium tuberculosis organism travels or is brought into contact. The skilled artisan will understand that the term "contacting" includes physical interaction between the compound and Mycobacterium tuberculosis organism or the multi-drug resistant Mycobacterium tuberculosis organism, as well as interactions that do not require physical interaction.

The term "in an amount sufficient" as used herein in relation to inhibiting the growth of Mycobacterium tuberculosis organism, means an amount of the compound of Formula I or the compound of Formula la, or its isomers or pharmaceutically acceptable salts or solvates thereof; or a composition comprising said compound(s), sufficient to significantly induce inhibition of the growth of Mycobacterium tuberculosis organism or the multi-drug resistant(MDR) Mycobacterium tuberculosis organism or extensively drug-resistant (XDR) Mycobacterium tuberculosis organism.

The term 'treating", "treat" or "treatment" as used herein generally have their ordinary or customary meanings, and include alleviating the disease, slowing the progression of, attenuation or cure of the existing disease, in this case tuberculosis or the multi-drug resistant (MDR) tuberculosis or extensively drug-resistant (XDR) tuberculosis or latent tuberculosis. The term "subject" as used herein refers to an animal, preferably a mammal, and most preferably a human. The term "mammal" used herein refers to warm-blooded vertebrate animals of the class mammalia, including humans, characterized by a covering of hair on the skin and, in the female, milk-producing mammary glands for nourishing the young. The term mammal includes without limitations animals such as cat, dog, horse, rabbit, bear, fox, wolf, monkey, deer, mouse, pig as well as human. The term "subject" may be used interchangeably with the term patient. In the context of the present invention the phrase "a subject in need thereof means a subject in need for the treatment of tuberculosis. Alternatively, the phrase "a subject in need thereof means a subject (patient) diagnosed having tuberculosis.

The term "pharmaceutically acceptable salt(s)" is meant to include salt(s) of the compound of Formula I or the compound of Formula la, which are prepared by treating the compound with an appropriate acid or a base. Examples of pharmaceutically acceptable base addition salts include, but are not limited to, sodium, potassium, calcium, magnesium, ammonium salts or an organic base salt. Examples of pharmaceutically acceptable organic base addition salts include, but are not limited to, those derived from organic bases such as lysine, arginine, guanidine, and the like. Examples of pharmaceutically acceptable acid addition salts include, but are not limited to, those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid and the like, as well as the salts derived from organic acids such as acetic acid, propionic acid, oxalic acid, maleic acid, benzoic acid, succinic acid, fumaric acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, methanesulfonic acid and the like.

As used herein, the term "solvate" refers to a compound formed by solvation, for example as a combination of solvent molecules with molecules or ions of a solute. Examples of solvate include water, alcohols and other polar organic solvents. Alcohols include methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, and t-butanol. Alcohols also include polymerized alcohols such as polyalkylene glycols (e.g., polyethylene glycol, polypropylene glycol).

As used herein, the term "isomer" is a general term used for all isomers of the compound of Formula I or the compound of Formula la that differ only in the orientation of their atoms in space. The term isomer includes optical isomers (pure enantiomers, diastereomers, and mixtures thereof) as well as conformational isomers (isomers that differ only in their angles of at least one chemical bond), position isomers (differ only in the position of a substituent), and geometric isomers (cis-trans isomers). Tautomerism may be defined as the phenomenon in which a single compound exists in two readily interconvertible structures that differ markedly in the relative position of at least one atomic nucleus, generally hydrogen. The term "tautomers" as used herein refers to the two readily interconvertible structures that differ markedly in the relative position of at least one atomic nucleus, generally hydrogen. When the two structures differ with respect to the relative position of hydrogen, the resulting tautomers are referred to as keto-enol tautomers.

The term "whole broth" may be used interchangeably with the terms "nutrient broth" or "culture broth".

As used herein, the term "mutant" refers to an organism or cell carrying a mutation, which is an alternative phenotype to the wild- type.

As used herein, the term "variant" refers to an individual organism that is recognizably different from an arbitrary standard type in that species.

As used herein, the term "nutrient" refers to a substance that provides nourishment for growth or metabolism.

In the context of the present invention, reference to "the compound of Formula I" may indicate reference to the compound of Formula lb or the compound of Formula Ic; or isomer(s) thereof, or a pharmaceutically acceptable salt or a solvate thereof.

The term "active ingredient" or "active compound" may be used interchangeably and as used herein, said term(s) refers to the compound of Formula I or the compound of Formula lb or the compound of Formula Ic or the compound of Formula la, or an isomer or a tautomer or a pharmaceutically acceptable salt or a solvate thereof.

In one aspect, the present invention relates to a compound represented by Formula I (referred to herein as the compound of Formula I);

Formula I wherein;

1 2

R and R independently represent hydrogen and Cj-C alkyl;

1 2

provided that both R and R are not H or Ci-Ce alkyl simultaneously;

or its isomer (s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof.

1 2

In an embodiment, in the compounds of Formula I, R and R independently represent

1 2

hydrogen and methyl; provided that both R and R are not H or methyl simultaneously.

In an embodiment, the compound of Formula I is represented by the following Formula lb (referred to herein as the compound of Formula lb),

Formula lb

or its isomer(s), a tautomer, or a pharmaceutical acceptable salt or a solvate thereof. In another embodiment, the compound of Formula I is represented by the following Formula Ic (referred to herein as the compound of Formula Ic),

Formula Ic

or its isomer(s), a tautomer, or a pharmaceutical acceptable salt or a solvate thereof. In another embodiment, the present invention relates to a compound represented by Formula la (referred to herein as the compound of Formula la),

Formula la

or its isomer(s), a tautomer, or a pharmaceutical acceptable salt or a solvate thereof, for use in the treatment of tuberculosis.

The compound of Formula la refers to Netropsin, which is a known oligopeptide, isolated from the actinobacterium Streptomyces netropsis. In the context of the present invention, Netropsin (the compound of Formula la) refers to a synthetically prepared compound and/or that obtained from other natural sources including microorganisms and/or that is commercially obtained.

In another embodiment, the compound of Formula I selected from the compound of

Formula lb or the compound of Formula Ic, was isolated from microorganism belonging to Actinomycetes strain (culture number PM 1180133)

In yet another embodiment, the compound of Formula I selected from the compound of Formula lb or the compound of Formula Ic was characterized by physicochemical and spectral techniques, such as mass spectra (MS) and nuclear magnetic resonance (NMR) spectra.

In an aspect, the present invention relates to a process for the production of the compound of Formula I from the culture number PM1180133 comprising the steps of:

(a) growing the culture number PM1180133;

(b) isolating the compound of Formula I from the culture broth; and

(c) purifying the compound of Formula I.

In an aspect, the present invention relates to a compound of Formula I or its isomer(s) produced from the microorganism belonging to Actinomycetes (PM1180133) by a process comprising the steps of:

(a) growing the culture number PM1180133;

(b) isolating the compound of Formula I from the culture broth; and

(c) purifying the compound of Formula I. In tne process for the production of the compound of Formula I; said compound of Formula I can be selected from the compound of Formula lb or the compound of Formula Ic.

In one embodiment, the step (c) involving purification of the compound of Formula lb or the compound of Formula Ic is carried out by purification procedures generally used in the related art.

In an embodiment, in step (b) of the process, the compound of Formula I is the compound of Formula lb.

In an embodiment, in step (b) of the process, the compound of Formula I is the compound of Formula Ic.

In an embodiment, in step (c) of the process, the compound of Formula I is the compound of Formula lb.

In an embodiment, in step (c) of the process, the compound of Formula I is the compound of Formula Ic.

In an aspect, the present invention relates to a process for the production of the compound of Formula lb from the culture number PM1180133 comprising the steps of:

(a) growing the culture number PM1180133;

(b) isolating the compound of Formula lb from the culture broth; and

(c) purifying the compound of Formula lb.

In an aspect, the present invention relates to a process for the production of the compound of Formula Ic from the culture number PM1180133 comprising the steps of:

(a) growing the culture number PM1180133;

(b) isolating the compound of Formula Ic from the culture broth; and

(c) purifying the compound of Formula Ic.

Accordingly, the compound of Formula I selected from the compound of Formula lb or the compound of Formula Ic produced according to the process of the present invention is substantially pure compound.

In another embodiment, the present invention relates to the compound of Formula I selected from the compound of Formula lb or the compound of Formula Ic obtained by the process described herein.

In an embodiment, the compound of Formula I selected from the compound of

Formula lb or the compound of Formula Ic is an isolated pure compound.

Typically, the compound of Formula lb and the compound of Formula Ic are purified by column chromatographic techniques. According to an embodiment of the invention, the compound of Formula I selected from the compound of Formula lb or the compound of Formula Ic has molecular formula C ₁₇ H24N ₁₀ O ₃ and molecular weight 416.4.

One microorganism from which the compound of Formula I selected from the compound of Formula lb or the compound of Formula Ic, is isolated, belongs to Actinomycetes strain (PM1180133/MTCC 5850) (herein after referred to as culture number PM1180133) which in turn is isolated from the soil sample collected from the land with dried leaves foliage located in Nagaur district of Rajasthan State, India.

Preliminary identification of the culture number PM1180133, from which the compound of Formula I selected from the compound of Formula lb or the compound of Formula Ic, is produced, was performed by examination of its colony characteristics. The colonies were picked up with sterile needle by viewing the colony morphology through stereo microscope (SM). Culture number PM1180133 is identified as a microorganism belonging to Actinomycetes.

Culture number PM1180133 has been deposited with Microbial Type Culture

Collection (MTCC), Institute of Microbial Technology, Sector 39-A, Chandigarh - 160 036, India, a World Intellectual Property Organization (WIPO) recognized International Depository Authority (IDA). The culture number PM1180133 has been given the accession number MTCC 5850.

In addition to the specific microorganism described herein, it should be understood that mutants of PM1180133, such as those produced by the use of chemical or physical mutagens including X-rays, U.V. rays etc. and organisms whose genetic makeup has been modified by molecular biology techniques, can also be cultivated to produce the compound of Formula I selected from the compound of Formula lb or the compound of Formula Ic.

The screening for suitable mutants and variants which can produce the compound of

Formula I selected from the compound of Formula lb or the compound of Formula Ic, according to the invention were confirmed by HPLC, NMR, MS and/or determination of biological activity of the active compounds accumulated in the culture broth, for example by testing the compounds for anti-mycobacterial activity, or by a combination thereof.

The medium and/or nutrient medium used for isolation and cultivation of culture number PM1180133, from which the compound of Formula I selected from the compound of Formula lb or the compound of Formula Ic, is produced, preferably contains sources of carbon, nitrogen and nutrient inorganic salts. The carbon sources are, for example, one or more of starch, glucose, sucrose, dextrin, fructose, molasses, glycerol, lactose, or galactose. A preferred carbon source is soluble starch and glucose. The sources of nitrogen are, for example, one or more of soyabean meal, casein, peanut meal, yeast extract, beef extract, peptone, malt extract, corn steep liquor, gelatin, tryptone or casamino acids. Preferred nitrogen source is casein, peptone, beef extract and yeast extract. The nutrient inorganic salts are, for example, one or more of sodium chloride, potassium chloride, calcium chloride, manganese chloride, magnesium chloride, strontium chloride, cobalt chloride, potassium bromide, sodium fluoride, sodium hydrogen phosphate, potassium hydrogen phosphate, dipotassium hydrogen phosphate, disodium phosphate, calcium carbonate, sodium bicarbonate, sodium silicate, sodium nitrate, ammonium nitrate, potassium nitrate, sodium sulphate, ammonium sulphate, ammonium heptamolybdate, ferric citrate, copper sulphate, magnesium sulphate, ferrous sulphate, zinc sulphate or boric acid. Calcium carbonate, sodium chloride and potassium nitrate are the preferred inorganic salts.

Typically, culture number PMl 180133 was purified and maintained on slants of agarified soyabean casein digest medium (SCDM); [Hi Media, Mumbai, Maharashtra, India Catalogue number MO 11 -500G] .

For long term preservation, the culture is preserved in lyophilized vials and is stored at -20 °C.

Seed culture cultivation of culture number PMl 180133 can be carried out at a temperature ranging from 29 °C to 30 °C and a pH of about 6.5 to 7.5, for 70 to 72 hours at 230-250 rpm (revolutions per minute).

The production of the compound of Formula I selected from the compound of Formula lb or the compound of Formula Ic, can be carried out by cultivating the culture number PMl 180133 by fermentation at a temperature ranging from 29 °C to 30 °C and a pH of about 5.6 to 5.9, for 30 minutes at 335 rpm.

The production of the compound of Formula I selected from the compound of

Formula lb or the compound of Formula Ic, can be carried out by cultivating culture number PMl 180133 in a suitable nutrient broth under conditions described herein. The progress of fermentation and production of the compound of Formula I selected from the compound of Formula lb or the compound of Formula Ic, can be detected by high performance liquid chromatography (HPLC) and by measuring the bioactivity in anti-infective screening test models such as Staphylococcus aureus, Enterococcus faecium, Candida albicans, Escherichia coli, Aspergillus fumigates, Mycobacterium smegmatis, Pseudomonas aeruginosa and Acinetobacter baumannii. Fermentation is a process of growing microorganisms for the production of various chemical or therapeutically active compounds. Microbes are incubated under specific conditions in the presence of nutrients. Whole broth is obtained after completing the process of fermentation. The whole broth is subjected to centrifugation which results in the formation of cell mass and culture filtrate, which can be processed further by processes, described herein.

The compound of Formula I selected from the compound of Formula lb or the compound of Formula Ic, produced by the fermentation process as described above, can be isolated from the culture broth using different extraction methods and chromatographic techniques.

Thus, the compound of Formula I selected from the compound of Formula lb or the compound of Formula Ic, can be recovered from the culture filtrate by extraction with a water immiscible solvent such as petroleum ether, dichlorome thane, chloroform, ethyl acetate, diethyl ether or butanol, or by hydrophobic interaction chromatography using polymeric resins such as "HP-20 ^® " (Kitten Enterprises Private Limited), "Amberlite XAD ^® " (Rohm and Haas Industries, USA) or adsorption on activated charcoal. These techniques can be used repeatedly, alone or in combination.

The organic extract of the cells containing the compound of Formula I selected from the compound of Formula lb or the compound of Formula Ic can be recovered from the cell mass by treatment with a water miscible solvent such as methanol, acetone, acetonitrile, n- propanol, or iso-propanol or by extraction with a water immiscible solvent such as petroleum ether, dichlorome thane, chloroform, ethyl acetate or butanol.

Alternatively, the organic extract of the cells containing the compound of Formula I selected from the compound of Formula lb or the compound of Formula Ic can be recovered by extracting the whole broth with a solvent selected from petroleum ether, dichlorome thane, chloroform, ethyl acetate, methanol, acetone, acetonitrile, n-propanol, iso- propanol, or butanol.

Typically, the organic extract of the cells containing the compound of Formula I selected from the compound of Formula lb or the compound of Formula Ic is obtained from the whole broth using methanol for extraction, followed by concentration and chromatography using HP-20 column. Concentration and lyophilization of the eluates of the HP-20 column provides the crude for isolation of the compound of Formula lb or the compound of Formula Ic. According to the present invention, the compound of Formula I selected from the compound of Formula lb or the compound of Formula Ic, can be isolated from the crude material by fractionation using any of the following techniques: dilution with water and extraction with a water immiscible solvent selected from petroleum ether, dichloromethane, chloroform, ethyl acetate, diethyl ether or butanol; normal phase chromatography (using alumina or silica gel as stationary phase; eluents selected from petroleum ether, ethyl acetate, dichloromethane, acetone, chloroform, methanol, or a mixture thereof; and additions of amines such as triethylamine); reverse phase chromatography (using reverse phase silica gel such as dimethyloctadecylsilylsilica gel (RP-18) or dimethyloctylsilyl silica gel (RP-8), as stationary phase; and eluents such as water, buffers (for example, phosphate, acetate, citrate (pH 2-8)), and organic solvents (for example methanol, acetonitrile, acetone, tetrahydrofuran, or a mixture thereof)); gel permeation chromatography (using resins such as Sephadex LH-20 ^® (Pharmacia Chemical Industries, Sweden), TSKgel ^® Toyopearl HW (TosoHaas, Tosoh Corporation, Japan) in solvents such as methanol, chloroform, acetone, ethyl acetate, or a mixture thereof); or by preparative high performance liquid chromatography (HPLC) (using a eluent system made up of two or more solvents water, trifluoroacetic acid, methanol, ethanol, iso-propanol, n-propanol, tetrahydrofuran, acetone, acetonitrile, methylene chloride, chloroform, ethyl acetate, petroleum ether, benzene or toluene). These techniques may be used repeatedly, alone or in combination. Typically, pure compound (e.g. the compound of Formula lb or of Formula Ic) is obtained by dilution with water and extraction with a water immiscible solvent; followed by reverse phase chromatography using acetonitrile and water; and chromatography using reverse phase silica gel (RP-18).

In another embodiment, the compound of Formula I selected from the compound of Formula lb or the compound of Formula Ic, or its isomer(s), may be converted into their pharmaceutically acceptable salts, which are all contemplated by the present invention.

The salts may be prepared by standard procedures known to one skilled in the art, for example, salts like sodium and potassium salts, can be prepared by treating the compound of Formula I selected from the compound of Formula lb or the compound of Formula Ic, or its isomer(s), with a suitable sodium or potassium base, for example sodium hydroxide or potassium hydroxide. Similarly, salts like hydrochloride and sulphate salts, can be prepared by treating the compound of Formula I (as described herein), or its isomer(s), with a suitable acid, for example hydrochloric acid, or sulphuric acid. Ail the derivatives of the compounds of Formula I, particularly of the compound of Formula lb or the compound of Formula Ic, are encompassed within the scope of the present invention. The derivatives of the compound of Formula I of particular interest according to the present invention are those wherein one or more of the functional group present in the compound of Formula I are derivatised. The derivatives of the compound of Formula I can be prepared by conventional methods known to persons of skill in the art.

In an aspect, the present invention relates to a method for the treatment of tuberculosis caused by Mycobacterium tuberculosis organism comprising administering to a subject in need thereof, a therapeutically effective amount of the compound of Formula I or the compound of Formula la or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof.

In an aspect, the present invention relates to a method for the treatment of tuberculosis caused by Mycobacterium tuberculosis organism comprising administering to a subject in need thereof, a therapeutically effective amount of the compound of Formula I, or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate or thereof.

In an aspect, the present invention relates to a method for the treatment of tuberculosis caused by Mycobacterium tuberculosis organism comprising administering to a subject in need thereof, a therapeutically effective amount of the compound of Formula la, or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof.

Accordingly, in an embodiment the Mycobacterium tuberculosis organism is sensitive Mycobacterium tuberculosis organism.

Accordingly, in an embodiment the Mycobacterium tuberculosis organism is extensively drug-resistant (XDR) Mycobacterium tuberculosis organism.

Accordingly, in an embodiment the Mycobacterium tuberculosis organism is multi- drug resistant (MDR) Mycobacterium tuberculosis organism. Accordingly, the compound of Formula I or the compound of Formula la or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof; is/are provided for use in the treatment of multi-drug resistant tuberculosis.

In another embodiment, the present invention relates to a method for the treatment of latent tuberculosis.

In an embodiment, the present invention relates to a method of inhibiting growth of the Mycobacterium tuberculosis organism, comprising contacting the Mycobacterium tuberculosis organism with the compound of Formula I or the compound of Formula la or its isomer(sj, a tautomer, or a pharmaceutically acceptable salt or a solvate thereof; in an amount sufficient to inhibit growth of the organism.

In another embodiment, the method of inhibiting growth of the Mycobacterium tuberculosis organism, may be in vitro or ex vivo, comprising contacting the organism in vitro or ex vivo with the compound of Formula I or the compound of Formula la, or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof, in an amount sufficient to inhibit the growth of the organism.

In another embodiment, the present invention relates to a method of inhibiting growth of Mycobacterium tuberculosis organism comprising contacting the organism in vitro with the compound of Formula I or the compound of Formula la, or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof, in an amount sufficient to inhibit the growth of the organism.

In yet another embodiment, the present invention relates to a method of inhibiting growth of Mycobacterium tuberculosis organism comprising contacting the organism ex vivo with the compound of Formula I or the compound of Formula la, or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof, in an amount sufficient to inhibit the growth of the organism.

In another embodiment, the compound of Formula I or the compound of Formula la, has activity against Mycobacterium tuberculosis organism which is sensitive organism.

In another embodiment, the compound of Formula I, has activity against

Mycobacterium tuberculosis organism which is sensitive organism.

In another embodiment, the compound of Formula la, has activity against Mycobacterium tuberculosis organism which is sensitive organism.

In another embodiment, the compound of Formula I or the compound of Formula la, has activity against Mycobacterium tuberculosis organism which is multi drug resistant organism such as Mycobacterium tuberculosis H37Rv or Mycobacterium tuberculosis Clinical isolate R (Rifampicin) resistant.

In another embodiment, the compound of Formula I, has activity against Mycobacterium tuberculosis organism which is multi drug resistant organism such as Mycobacterium tuberculosis H37Rv or Mycobacterium tuberculosis Clinical isolate R (Rifampicin) resistant.

In another embodiment, the compound of Formula la, has activity against Mycobacterium tuberculosis organism which is multi drug resistant organism such as Mycobacterium tuberculosis H37Rv or Mycobacterium tuberculosis Clinical isolate R (Rifampicin) resistant.

According to an embodiment, the compound of Formula I or the compound of Formula la, or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof is used, for the treatment of tuberculosis which is caused by Mycobacterium tuberculosis organism. The Mycobacterium tuberculosis organism is a sensitive Mycobacterium tuberculosis organism or a multi drug resistant Mycobacterium tuberculosis organism or a combination thereof.

The term "Mycobacterium" or "Mycobacterium species" refers to Gram-positive, non-motile, pleomorphic rods related to the actinobacteria. MDR-TB (multi-drug resistant tuberculosis) describes strains of tuberculosis that are resistant to anti-TB drugs/agents. For instance, strains of tuberculosis that are resistant to the two first-line anti-TB drugs, Isoniazid and Rifampicin

In another embodiment, the compound of Formula I or the compound of Formula la, or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof; is found to be active against Mycobacterium tuberculosis. Accordingly, the compound of Formula la or the compound of Formula lb or the compound of Formula Ic or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof; is useful in the treatment of tuberculosis.

Accordingly in an embodiment, the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of the compound of Formula I or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof, together with a pharmaceutically acceptable carrier.

Accordingly in an embodiment, the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of the compound of Formula I, as described herein, for use in the treatment of tuberculosis.

Accordingly in an embodiment, the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of the compound of Formula la, or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof, together with a pharmaceutically acceptable carrier; for use in the treatment of tuberculosis. In another embodiment, the effective amount of the compound of Formula I or the compound of Formula la, or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof; as the active ingredient in the pharmaceutical compositions ranges from about 0.1 mg to 2000 mg. In another embodiment, the effective amount of the compound of Formula I selected from the compound of Formula lb or the compound of Formula Ic, or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof; as the active ingredient in the pharmaceutical compositions ranges from about 0.1 mg to 2000 mg.

In another embodiment, the effective amount of the compound of Formula la, or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof; as the active ingredient in the pharmaceutical compositions ranges from about 0.1 mg to 2000 mg.

In another embodiment, the effective amount of the compound of Formula I or the compound of Formula la, or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof; as the active ingredient in the pharmaceutical compositions ranges from about 0.1 mg to 1000 mg.

In another embodiment, the effective amount of the compound of Formula I or the compound of Formula la, or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof; as the active ingredient in the pharmaceutical compositions ranges from about 0.1 mg to 500 mg.

In another embodiment, the present invention relates to a method for the treatment of tuberculosis comprising administering to a subject in need thereof, a therapeutically effective amount of the composition recited above.

According to an embodiment, pharmaceutical compositions which contains the compound of Formula I or the compound of Formula la or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof, can be prepared by mixing, the compound of Formula I or the compound of Formula la with one or more pharmacologically acceptable auxiliaries and/or excipients such as, wetting agents, solubilisers such as surfactants, vehicles, tonicity agents, fillers, colorants, masking flavors, lubricants, disintegrants, diluents, binders, plasticizers, emulsifiers, ointment bases, emollients, thickening agents, polymers, lipids, oils, cosolvents, complexation agents, or buffer substances, and converting the mixture into a suitable dosage form such as, for example, tablets, coated tablets, capsules, granules, powders, creams, ointments, gels, syrup, emulsions, suspensions, or solutions suitable for parenteral administration.

In the context of the present invention, the auxiliaries and/or excipients that can be used in the preparation of pharmaceutical composition containing the compound of Formula I or the compound of Formula la or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof, can be selected from: cremophor, poloxamer, benzalkonium chloride, sodium lauryl sulphate, dextrose, glycerin, magnesium stearate, polyethylene glycol, starch, dextrin, lactose, cellulose, carboxymethylcellulose sodium, talc, agar-agar, mineral oil, animal oil, vegtetable oil, organic and mineral waxes, paraffin, gels, propylene glycol, benzyl alcohol, dimethylacetamide, ethanol, polyglycols, Tween 80, solutol HS 15, and water. It may also be possible to administer the active substance (e.g. the compound of Formula I or the compound of Formula la) as such, without vehicles or diluents, in a suitable form, for example, in capsules.

In the context of the present invention, the excipients used in parenteral preparations containing the compound of Formula I or the compound of Formula la or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof, can be selected from, but not limited to, the group consisting of stabilizing agents (e.g. carbohydrates, amino acids and polysorbates, such as 5 % dextrose), solubilizing agents (e.g. cetrimide, sodium docusate, glyceryl monooleate, polyvinylpyrolidone (PVP) and polyethylene glycol (PEG)), surfactants (e.g. polysorbates, tocopherol PEG succinate, poloxamer and Cremophor™), buffers (e.g. acetates, citrates, phosphates, tartrates, lactates, succinates, amino acids and the like), antioxidants and preservatives (e.g. BHA, BHT, gentisic acids, vitamin E, ascorbic acid, sodium ascorbate and sulfur containing agents such as sulfites, bisulfites, metabisulfites, thioglycerols, thioglycolates and the like), tonicity agents (for adjusting physiological compatibility), suspending or viscosity agents, antibacterials (e.g. thimersol, benzethonium chloride, benzalkonium chloride, phenol, cresol and chlorobutanol), chelating agents, and administration aids (e.g. local anesthetics, anti-inflammatory agents, anti-clotting agents, vaso-constrictors for prolongation and agents that increase tissue permeability), and combinations thereof. Parenteral formulations using hydrophobic carriers include, for example, fat emulsions and formulations containing lipids, lipospheres, vesicles, particles and liposomes. Fat emulsions include in addition to the above-mentioned excipients, a lipid and an aqueous phase, and additives such as emulsifiers (e.g. phospholipids, poloxamers, polysorbates, and polyoxyethylene castor oil), and osmotic agents (e.g. sodium chloride, glycerol, sorbitol, xylitol and glucose). Liposomes include natural or derived phospholipids and optionally stabilizing agents such as cholesterol.

In one embodiment of the present invention, the compound of Formula I or the compound of Formula la or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof, or the pharmaceutical compositions containing the compound of Formula I or the compound of Formula la; can be used in combination with at least one further therapeutically active agent; for use in the treatment of tuberculosis. In another embodiment, the present invention relates to use of the compound of Formula I or the compound of Formula la or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof, for the manufacture of a medicament for use in the treatment of tuberculosis.

In another embodiment, the present invention relates to use of the compound of

Formula I or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof, for the manufacture of a medicament for use in the treatment of tuberculosis.

In another embodiment, the present invention relates to use of the compound of Formula la or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof, for the manufacture of a medicament for use in the treatment of tuberculosis.

In one embodiment, the compound of Formula I or the compound of Formula la, or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof, can be administered orally, nasally, topically, subcutaneously, intramuscularly, intravenously, or by other modes of administration.

The mode of administration which is suitable in a specific case depends on the type of tuberculosis to be treated and its severity. Further, the method of administration can be optimized by a medical practitioner using methods known in the art. A skilled medical practitioner can select the dosage level in the light of the relevant circumstances, including the disease (tuberculosis) to be treated, the chosen route of administration depending on a number of factors, such as age, weight and physical health and response of the individual patient, pharmacokinetics, severity of the disease and the like factors known in the medical art. Actual dosage levels of the active ingredients in the pharmaceutical composition can be varied so as to obtain an amount of the active ingredient, which is effective to achieve the desired therapeutic response for a particular patient (subject in need of the treatment), composition, and mode of administration without being toxic to the patient. On an average, the daily dose of the active compound (one or more of the compounds of the present invention) for a patient can range from about 0.5 mg per kg to about 200 mg per kg, or any dosage range that falls within the scope of the broader dose range as indicated herein. The desirable dose of the active compound i.e. the compounds of the present invention can be selected over a wide range. The daily dosage to be administered is selected to achieve the desired therapeutic effect in subjects being treated for tuberculosis. If required, higher or lower daily dosages can also be administered.

Accordingly, in an embodiment, the present invention relates to the compound of Formula I or the compound of compound of Formula la, or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof, for use in the production of medicaments for the treatment of tuberculosis.

Accordingly, in an embodiment, the present invention relates to the compound of Formula I, or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof, for use in the production of medicaments for the treatment of tuberculosis.

Accordingly, in an embodiment, the present invention relates to the compound of Formula la, or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof, for use in the production of medicaments for the treatment of tuberculosis.

Accordingly, in an embodiment, the present invention relates to use of the compound of Formula I or the compound of Formula la, or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof, for the treatment of tuberculosis.

Accordingly, in an embodiment, the present invention relates to use of the compound of Formula I, or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof, for the treatment of tuberculosis.

Accordingly, in an embodiment, the present invention relates to use of the compound of Formula la, or its isomer(s), a tautomer, or a pharmaceutically acceptable salt or a solvate thereof, for the treatment of tuberculosis.

In one or more embodiments of the present invention, the compound of Formula I is selected from either the compound of Formula lb or the compound of Formula Ic.

The following examples illustrate the present invention. They do not however, limit the invention in any way. In this regard, it is important to understand that the particular assay used in the examples section is designed only to provide an indication of anti-bacterial activity, particularly, anti-tubercular activity, and a negative result in any one particular way is thereof not determinative.

The following terms/abbreviations/chemical formulae are employed in the examples:

L : Litre rpm : Revolutions per minute

mL : Millilitre HPLC : High performance liquid chromatography μL· : Microlitre DMSO : Dimethyl sulfoxide

g : Gram w/v : Weight (of solute) per volume (of solvent) mg : Milligram v/v : Volume (of solute) per volume (of solvent) μ& : Microgram MIC : Minimum inhibitory concentration

h : Hours ISP2 : International Streptomyces Project medium number 2 min : Minutes CFU: Colony forming unit

NaOH : Sodium hydroxide FICI: fractional inhibitory concentrations index Example 1:

Isolation of culture number PM1180133 from soil sample

(a) Composition of the isolation medium:

Starch Casein Digest Medium, (SCDM) [contains % (w/v): Pancreatic Digest of

Casein 17 g/L, Papaic Digest of Soyabean Meal 3 g/L, Sodium Chloride 5 g/L, Dipotassium Hydrogen Phosphate 2.5 g/L, Dextrose 2.5 g/L, pH adjusted to 7.3 ± O.2.]

(b) Procedure:

The soil sample was collected from a land with dried leaves foliage in Nagaur District of Rajasthan State, India. The air dried soil (1 g) in saline (10 mL) was diluted and vortexed for 1 minute. The soil debris was allowed to settle and 1 mL of supernatant was diluted twice in distilled water (9 mL) to 1 : 1000 dilution. 100 μL· of this dilution was spread on Starch Casein Agar plates containing 40 μg/mL amphotericin B. The isolation plates were dried in Laminar Air Flow and incubated at 28 °C for a week and were observed periodically microscopically. Colonies were picked up with sterile needle under stereomicroscope by viewing the colony morphology and transferred to ISP2 media for purification and maintenance. PM1180133 was one such Actinomycetes strain isolated by this procedure.

Example 2:

Purification and preservation of producer strain culture number PM1180133

(a) The isolate was purified and maintained in ISP2 media. ISP2 media contains Yeast Extract 4 g/L, Malt Extract 10 g/L, Glucose 4 g/L and having a pH of 7.3 to 7.5.

Example 3:

Fermentation of the culture number PM1180133 in shake flasks.

a) Composition of seed medium [274(1)]:

The seed medium, 274 (1) [contains % (w/v): Glucose 15 g, corn steep liquor 5 g, peptone 7.5 g, yeast extract 7.5 g, calcium carbonate 2 g, sodium chloride 5 g; demineralised water 1.0 L, and pH adjusted to 6.5-7.5].

b) The above medium was distributed in 200 mL amounts in 1000 mL capacity Erlenmeyer flasks and was autoclaved at 121 °C for 20 minutes. Each flask was inoculated with a loopful of well-grown slant culture (culture number PM1180133). The flasks were shaken for 70-72 hours on rotary shaker at 230-250 rpm and at 29 °C - 30 °C to obtain seed culture. c) Composition of the production medium [SMI 2 (1)]:

The production medium, SM12 (1) [contains % (w/v): Glucose 50 g, yeast extract 11 g, sodium chloride 2.5 g, calcium carbonate 5 g, peptone 4 g, beef extract 4 g; demineralised water 1.0 L, and pH adjusted to 7.4-7.6].

20 L of the production medium in 30 L fermenter with 0.04% of desmophen was sterilized in situ for 30 minutes at 121 °C, cooled to 29 °C-30 °C; and seeded with 600 mL of the seed culture.

d) Fermentation parameters:

Temperature 29-30 °C; Agitation 335 rpm; aeration 0.5 vvm & harvest time 96 hours. The production of the compound of Formula la or the compound of Formula lb or the compound of Formula Ic in the fermentation broth was determined by HPLC at 48 hours, 72 hours, and 96 hours. The pH of the culture broth at the time of harvest was in the range of 5.6-5.9. The 16 L culture broth was harvested and centrifuged at 5000 rpm for 30 minutes, and the culture filtrate was subjected to further purification.

The bioactivity was tested for anti-infective activity in agar diffusion assay in the gram positive and gram negative bacterial test cultures.

Example 4:

Isolation and purification of the active compounds (as described herein)

Step 1

The whole broth (20 L) of the microbial culture was centrifuged to separate cell mass and culture filtrate. The culture filtrate was subjected to HP-20 resin chromatography. The filtrate was stirred for 3 hours, washed with demineralized water and filtered. The desired compound was eluted with mixture of methanol and water (8:2, 1.8 L). The aqueous methanol washing was dried under reduced pressure and temperature to obtain a crude extract. Yield: 20 g.

Step 2

The crude extract (5 g) (as obtained in step 1) was subjected to flash chromatography using reverse phase silica (RP-18, 40-60 microns) pre equilibrated with water. The fraction containing compound of Formula I (as described herein) was eluted with 30 % methanol in water, active eluates were pooled based on thin layer chromatography and dried to obtain semi pure compound. Yield: 1 g.

Step 3 The semi pure compound (as obtained in step 2) was further purified using reverse phase (RP-18) preparative HPLC.

Preparative HPLC conditions:

Column : RP-18 column (Waters Xbridge 250 X 10 mm, 5 micron);

Eluent : Acetonitrile and water (0.1% TF A) starts from 02:98 to 25:75 in

30min then 100% acetonitrile for another 5 min;

Flow rate : 7 mL/minute;

Detection (UV): 298 nm;

The eluent obtained at RT 25 minute was dried and analyzed by analytical HPLC. The eluent obtained was a pure compound of Formula la. Yield: 55 mg.

The eluent obtained at RT 19 minute was dried and analyzed by analytical HPLC. The eluent obtained was a pure compound of Formula lb or the compound of Formula Ic. Yield: 50 mg.

Analytical HPLC condition:

Column : Ascentisl 150 x 4.6 mm, 5 μπι;

Solvent system : Gradient (0% acetonitrile to 100% acetonitrile in 10 minutes

against Water (0.1% TFA) followed by 100% acetonitrile for additional 5 minutes.

Flow rate : 1 mL/minute;

Detection (UV) : 298 nm;

Retention time for Formula la: 2.8 min.

Retention time for Formula lb or the compound of Formula Ic: 2.5 min.

Physical & spectral properties of the compound of Formula la:

Appearance : White powder

Solubility : Soluble in chloroform, ethyl acetate, methanol and water.

HRMS (m z) : 430.2190 (M+H) ⁺

Molecular weight : 430.2

UV : 254 nm, 298 nm

HPLC Purity : > 98%

Molecular formula : Ci ₈ ¾6 Njo O3

IR (KBr) : 3306.02, 2209.39, 1672, 1437.49, 1407.69, 1254.80, 1203.06,

1140.11, 1064.14, 1018.73, 963.52

^J H NMR (DMSO-d ₆ , 500 ΜΗζ):δ (ppm) 2.6, 3.5, 3.8, 4.0, 6.9, 7.0, 7.1, 7.2, 7.3, 7.6, 8.2,

8.7, 8.9, 9.9, 10.1 C NMR (DMSO-d ₆ , 125 ΜΗζ):δ (ppm) 33.2, 36.3, 36.4, 36.6, 44.0, 104.4, 104.9, 105.1, 118.7, 121.8, 122.5, 122.9, 123.4, 158.1, 158.7, 161.9, 164.9, 169.6

Physical & spectral properties of the compound of Formula lb or the compound of Formula Ic:

Appearance : White powder

Solubility : Soluble in chloroform, ethyl acetate, methanol and water.

HRMS (m/z) : 417.2091 (M+H) ⁺

Molecular weight : 416.4

UV : 254nm, 298 nm

HPLC Purity : > 99%

Molecular formula : Cn H24 Njo O3

IR (KBr) : 3311.29, 1675.39, 1440.81, 1406.61, 1252.97, 1202.5,1132.73,

1018.42, 989.71, 968.65, 841.63, 799.33

JH NMR (DMSO d ₆ , 500 ΜΗζ):δ (ppm) 2.5, 3.5, 3.8, 4.0, 6.8, 6.9, 7.1 , 7.2, 7.4, 7.6, 8.2,

8.8, 8.9, 9.8, 10.1, 11.1

¹³ C NMR (DMSO d ₆ , 125 ΜΗζ):δ (ppm) 33.0, 36.2, 36.6, 44.0, 103.0, 104.3, 104.9, 112.3,

121.8, 123.5, 124.4, 158.1, 158.7, 161.2, 164.9, 169.6

The compound of Formula la was characterised to be Netropsin by comparing physical and spectral data with standard Netropsin (procured from Sigma Aldrich). Example 5:

The biological activity against Mycobacterium tuberculosis organism was determined by using BACTEC MGIT 960 System from Becton Dickinson (BD), USA. The BACTEC 960 instrument is an automated system that exploits the fluorescence of an oxygen sensor to detect growth of Mycobacteria in culture. It is specially designed to accommodate Mycobacteria Growth Indicator Tube (MGIT) that contains a fluorescent compound embedded in silicone on the bottom of a tube. The fluorescent compound is sensitive to the presence of oxygen dissolved in the broth. The initial concentration of the dissolved oxygen quenches the emission of fluorescence from the compound, and little fluorescence can be detected. Later, actively growing and respiring microorganisms consume oxygen, which allows compound to fluoresce.

Two strains, Mycobacterium tuberculosis complex (Non-MDR strain) and Mycobacterium tuberculosis complex (MDR strain) were used in the study. These clinical strains are maintained at Super Raligare Laboratories Ltd, India. The terms resistance and susceptible are designated based on the interpretation of B ACTEC-960.

If the test compound (the compound of Formula la / the compound of Formula I / rifampicin) added to the medium is bacteriostatic or bactericidal to the test mycobacteria, it would inhibit growth of the mycobacteria and therefore, there would be little or no oxygen consumption, and ultimately there would be little or no fluorescence of the sensor. Whereas, the growth control will grow uninhibited and will have increasing fluorescence. Growth is monitored by the BACTEC MGIT 960 instrument which automatically interprets results as resistant (R) (growing) or susceptible (S) (not growing).

To determine the 1 % proportion of resistance, the bacterial inoculum used in the control vial is 100 fold less than that used for the drug-containing vial.

Results were interpreted and are presented in the following Table 1 to Table 4.

Table 1 : Activity of the compound of Formula la against Mycobacterium tuberculosis

S: Sensitive, R: Resistant

Table 2: Activity of Rifampicin against Mycobacterium tuberculosis

S: Sensitive, R: Resistant Table 3: Activity of the compound of Formula I against Mycobacterium tuberculosis.

S: Sensitive, R: Resistant

Table 4: Activity of Rifampicin against Mycobacterium tuberculosis.

S: Sensitive, R: Resistant Conclusion: By this method the minimum inhibitory concentration (MIC) of the compound of Formula la required to exhibit its effect against both Non-MDR-TB strain and MDR-TB strain was between 1 μg/mL and 2 μg/mL. MICs against drug-susceptible and MDR-TB isolates were, 1 μg/mL and 2 μg/mL for the compound of Formula la.

The minimum concentration of the compound of Formula I required to exhibit its effect against both Non-MDR-TB strain and MDR-TB strain was between 0.5 μg/mL and 1 μg/mL. MICs against drug-susceptible and MDR-TB isolates were, 0.5 μg/mL and 1 μg/mL for compound of Formula lb. Thus, the three test compounds displayed their inhibitory activity against both Non-MDR-TB and MDR-TB strains.

Example 6:

Biological evaluation of the compound of Formula I or the compound of Formula la.

In-vitro assay

The in-vitro potency was established by minimum inhibitory concentration (MIC) determinations of PM1180133 against bacterial strains, by using the Microplate Alamar Blue Assay (MABA).

Antitubercular Testing of the compound of Formula I & the compound of Formula la: A) Minimal Inhibitory concentration (MIC) of the compound of Formula la and that of the compound of Formula I using Microplate Alamar Blue Assay (MABA) i. Minimal Inhibitory concentration (MIC) of the compound of Formula la and that of the compound of Formula I against sensitive strain (H^Rv) of Mycobacterium tuberculosis (ATCC Number 25678).

Principle: Alamar Blue is a proven cell viability indicator that uses the natural reducing power of living cells to convert resazurin, a non-fluorescent indicator dye to the bright red fluorescent molecule, resorufin. The active ingredient of Alamar Blue (resazurin) is a nontoxic, cell permeable compound that is blue in color and virtually non-fluorescent. Upon entering cells, resazurin is reduced to resorufin, which produces very bright red fluorescence. Viable cells continuously convert resazurin to resorufin, thereby generating a semiquantitative measure of viability and cytotoxicity. The amount of fluorescence produced is proportional to the number of living cells. Methodology:

The Assay was performed under aseptic condition using sterile clear bottom 96 well plates. The outer perimeter wells were filled with 200μΙ. sterile distilled water to prevent dehydration in the experimental wells. In each well ΙΟΟμΙ. Middlebrook broth (7H9/ADC) was added. ΙΟΟμΙ. of highest concentration of the compounds listed in Table 5 was added to all the wells of the first column. The compounds listed in Table 5 were serially diluted from the first well to the 7 ^th well. ΙΟΟμΙ. of the Mycobacterium culture was added to all wells, making total volume of each well to 200μΙ.. Column 9 was maintained as media control wells (negative control) and Column 10 containing ΙΟΟμΙ. of Mycobacterium tuberculosis culture media as culture control wells (positive control). The plate was sealed and incubated in the C0 ₂ incubator at 37 °C for 5 days. On the 5 ^th day, 50μΙ. of 1 : 1 Alamar blue + 10% Tween 80 was added to all the wells. The plates were re-incubated at 37 °C for 24 hours. The color change from blue to pink was observed. The least concentration resulting in blue colour determines the MIC of the drug (the test compound(s)).

Table 5: Comparison of the compound of Formula la, and the compound of Formula I with first line anti-tubercular drugs for the treatment of drug sensitive tuberculosis. Compounds MIC ^g/mL) for H ₃₇ Rv

Compound of Formula I 2-8

Test Compounds

Compound of Formula la 2-8

No anti-mycobacterial

Vehicle Control DMSO

activity

Pyrazinamide (PZA) 8-16

Isoniazid (INH) 0.125

First line antitubercular Rifampicin (RIF) 2

Drugs

Ethambutol (EMB) 2-4

Kanamycin (KAN) 2

The MIC achieved by the compound of Formula la and the compound of formula I was compared to the MIC of first line anti-tubercular agents used in treatment of tuberculosis.

Results & Conclusion: The MIC of the compound of Formula la and that of the compound of Formula I was in the range of 2-8 μg/mL against Mycobacterium tuberculosis H ₃₇ RV (ATCC number 25678) as determined using Microplate Alamar Blue Assay (MABA). Isoniazid, an anti-tubercular drug was used as a positive control. The anti- mycobacterial activity of the compound of Formula la and that of the compound of Formula I is comparable to the anti-mycobacterial activities of first line anti-tubercular drugs namely kanamycin, ethambutol, pyrazinamide (4-8 μg/mL) and rifampicin (2-4 μg/mL) used for the treatment of drug sensitive tuberculosis. The MIC achieved by the compound of Formula la and the compound of Formula I is comparable to the MIC of the first line anti-tubercular agents used in the treatment of tuberculosis and hence, suggestive of potent anti- mycobacterial activity of the compound of Formula la and that of the compound of formula I. ii. Minimal Inhibitory concentration (MIC) of the compound of Formula I or the compound of Formula la against rifampicin resistant strain (H37Rv-R) of Mycobacterium tuberculosis ( ATCC strain number 700457).

The MIC of the compound of Formula I against the rifampicin resistant Mycobacterium tuberculosis H ₃₇ RV-R was determined using Microplate Alamar Blue Assay (MABA). Table 6: iviIC of the Compound of Formula I and that of the Compound of Formula la against rifampicin resistant strain of Mycobacterium tuberculosis H ₃₇ RV-R

The MIC achieved by compounds of Formula la and the compound of Formula I was significant for the rifampicin resistant strain which exhibits absence of anti-mycobacterial activity upon treatment with rifampicin at concentrations at 32 μg/mL.

Table 7: Comparison table for anti-mycobacterial activity of the compound of Formula la and the compound of Formula I:

Results & Conclusion: The compound of Formula la and the compound of Formula I exhibited anti-mycobacterial activity against Mycobacterium tuberculosis H ₃₇ RV-R in the range of 2-8 μg/mL. Isoniazid was used as a positive control. The anti-mycobacterial activity of the compound of Formula la, and that of the compound of Formula I is comparable to the anti-mycobacterial activities of second line anti-tubercular drugs like capreomycin, kanamycin, ethionamide, para-aminosalicyclic acid (4-8 μg/mL) used for the treatment of drug resistant tuberculosis. The MIC achieved by the compound of Formula la and the compound of Formula I is comparable to the MIC of the second line anti-tubercular agents used in the treatment of tuberculosis and hence, suggestive of potent anti-mycobacterial activity of the compound of Formula I and the compound of Formula la against Mycobacterium tuberculosis H ₃₇ RV-R. Example 7:

Postantibiotic effect (PAE) of the compound of Formula I, and that of the compound of Formula la against sensitive strain (H ₃₇ RV) of Mycobacterium tuberculosis.

Q

Method: Mid-log-phase aerobic M. tuberculosis H3 ₇ R _V cultures at a density of 3*10 CFU/mL were diluted to 100-fold in Middlebrook's 7H9 (Difco, BD India) medium flask in 50 mL volumes. Freshly inoculated cultures were exposed to each of the individual anti-TB drugs at given concentrations in Erlenmeyer flasks for 2 hours at 37°C in C(¾ incubator. Before addition of drug, 0.1 mL culture from the culture flasks was inoculated onto 7H11/OADC agar plates (Difco, BD India) to determine the baseline load. After 2 hours incubation the cell suspension was centrifuged at 4000 rpm for 10 minutes at 4°C. The supernatant obtained was discarded and the pellet was dissolved in 50 mL sterile IX PBS. The incubation steps were repeated three times to completely washed off the drugs from the cultures. The pellet was dissolved in 10 mL pre-warmed Middlebrook's 7H9 (Difco, BD India) medium. 0.1 mL culture was drawn and inoculated onto 7H11/OADC agar plates (Difco, BD India) to determine To time point CFU load.

The flasks were incubated for 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 days treatment time point at 37°C under shaking conditions at 200 rpm. At a given time point 0.1 mL culture was drawn from the culture flasks and inoculated it onto 7H11/OADC agar plates (Difco, BD India). The plates were incubated at 37°C and CFU was counted after 4 weeks. The entire assay was conducted under aseptic conditions.

Table 8: PAE of compounds for sensitive strain of Mycobacterium tuberculosis H3 ₇ R _V

The PAE of the compound of formula I and that of the compound of formula la exhibited longest PAE in dose dependent increase compared to Isoniazid, standard first line anti tuberculosis drug.

Example 9:

Anti-mycobacterial activity of the compound of formula I, and that of the compound of formula la in combination with standard anti-TB drugs (Rifampicin, Moxifloxacin, Levofloxacin & Kanamycin) against sensitive strain of Mycobacterium tuberculosis H ₃₇ RV.

Methodology: The mycobacterial activity of compound of formula I, and that of the compound of formula la in combination with rifampicin, moxifloxacin, levofloxacin & kanamycin was individually evaluated against M. tuberculosis H ₃₇ RV by the checker board titration assay in sterile 96-well microtitre plates. Twofold serial dilutions of all drugs were prepared in 96-well microtitre plates, and 0.01 mL Mycobacterium tuberculosis suspension (3xl0 ⁶ cfu/mL) was added to each well. Each drug was tested at 4, 2, 1, 0.5, 0.25, 0.125 times their respective MIC. Following 7 days of incubation at 37°C, MICs of drug combinations were read as colorimetric change. Fractional inhibitory concentration (FIC) indices for M. tuberculosis H ₃₇ RV were calculated.

Analysis: The formula for fractional inhibitory concentration (FIC) index was used to calculate combination index. The fractional inhibitory concentrations index was calculated as follows:

FIC index = FIC _A + FIC _B

FIC _A = (MIC _A B of drug A in presence of B/MIC of drug A (Compound of formula I or la)) FICB = (MICB _A of drug B in presence of A/ MIC of drug B (Anti tuberculosis drug))

The combination index of < 0.5 is considered synergistic, an index of >0.5 and < 4 considered additive and an index of > 4 indicates antagonistic activity.

Table 9a: FICI between the compound of formula I and rifampicin, moxifloxacin, levofloxacin and kanamycin in two drug combinations.

Drug combinations MIC ^g/mL) MIC ^g/mL) FICI Activity drug combination

MIC _A MIC _B MIC _AB MICBA

Compound of formula I + 8 2 16 2 3 Additive Rifampicin

Compound of formula I + 8 0.2 4 0.125 1.125 Additive Moxifloxacin

Compound of formula I + 32 1 4 1 1.125 Additive Levofloxacin

Compound of formula I + 8 1 4 1 1.5 Additive Kanamycin

Conclusion: Rifampicin, Kanamycin, Levofloxacin and Moxifloxacin exhibited additive anti-mycobacterial effect with the compound of formula I. The study suggests that compound of formula I can be combined with rifampicin, moxifloxacin, levofloxacin & kanamycin as potential new therapy for drug sensitive patients.

Table 9b: FICI between the compound of formula la and ethambutol, pyrazinamide, moxifloxacin, levofloxacin and amikacin in two drug combinations.

Conclusion: Ethambutol, moxifloxacin, amikacin, levofloxacin and pyrazinamide exhibited additive anti-mycobacterial effect with the compound of formula la. The study suggests that compound of formula la can be combined with Ethambutol, moxifloxacin, amikacin, levofloxacin and pyrazinamide as potential new therapy for drug sensitive patients.

Example 10:

Anti-mycobacterial activity of the compound of formula I and that of the compound of formula la in combination with Moxifloxacin (second line anti-TB drug) against rifampicin resistant strain of Mycobacterium tuberculosis H ₃₇ RV-R.

Test Material: 1. Compound of formula I (Cone. Tested: 32-0.06μg/mL)

Standard Drug Control: Moxifloxacin (2 ^nd line anti-TB drug) Methodology: The mycobacterial activity of compound of formula I, and that of the compound of formula la in combination with moxifloxacin was individually evaluated against M. tuberculosis H ₃₇ RV-R by the checker board titration assay in sterile 96-well microtitre plates. Twofold serial dilutions of all drugs were prepared in 96-well microtitre plates, and O.OlmL M. tuberculosis suspension (3xl0 ⁶ cfu/mL) was added to each well. Each drug was tested at 4, 2, 1, 0.5, 0.25, 0.125 times their respective MIC. Following 7 days of incubation at 37°C, MICs of drug combinations were read as colorimetric change. Fractional inhibitory concentration (FIC) indices for M. tuberculosis H ₃₇ RV-R were calculated.

Analysis: The formula for fractional inhibitory concentration (FIC) index was used to calculate combination index. The fractional inhibitory concentrations index was calculated as follows:

FIC index = FIC _A + FIC _B

FIC _A = (MIC _A B :MIC of drug A in presence of B/MIC of drug A alone) FICB = (MICB _A :MIC of drug B presence of A/ MIC of drug B alone)

The combination index of < 0.5 is considered synergistic, an index of >0.5 and < 4 considered additive and an index of > 4 indicates antagonistic activity.

Table 10: FICI between the compound of formula I and the compound of formula la in drug combination with Moxifloxacin, Levofloxacin, Cycloserine and Amikacin.

Conclusion: Moxifloxacin, Levofloxacin, Cycloserine and Amikacin exhibited additive anti-mycobacterial effect with the compound of formula la. Moxifloxacin exhibited additive anti-mycobacterial effect with the compound of formula I. Example 11:

The potential of the compound of formula I and that of the compound of formula la to inhibit DNA synthesis in Mycobacterium tuberculosis H ₃₇ RV sensitive strain by macromolecule incorporation assay.

Test Material: 1. Compound of formula I (Concentration Tested: 10X of MIC (8μg/mL))

2. Compound of formula la (Concentration Tested: 10X of MIC (4μg/mL)) Positive Control: Moxifloxacin (Concentration Tested: 10X of MIC (lμg/mL))

Negative Control: para-Amino salicylic acid (Concentration Tested: 10X of MIC (lμg/mL)) Test System: Virulent M. tuberculosis H ₃₇ Rv (A.TCC No.25678)

Methodology: Mid-log-phase aerobic Mycobacterium tuberculosis H37Rv cultures were diluted 100-fold in Middlebrook's 7H9/ADC (Difco, BD India Ltd) medium and transferred to 150 mL flask. Freshly inoculated cultures were incubated at 37°C at 200 rpm till mid- exponential phase with OD5 ₆₀ of 0.5 corresponding to approximately 3x10 cfu/mL was achieved. Cultures were then centrifuged at 3000 rpm and 4°C for 10 minutes. The pellet was vortexed and diluted with sterile phosphate-buffered saline to obtain 3x10 cfu/mL.

In sterile 96-well microtitre plates, O.lmL of the above M. tuberculosis suspension was added to each well followed by the addition of all the drugs at concentrations 10-fold above their MIC values. Cultures were then radio labeled by adding the 0.1 μθ of radio labelled [ H] Thymidine into DNA synthesis. 96-well microtitre plates were then incubated at 37°C for 24 hours. 0.1 mL of cold 10% Trichloro acetic acid (TCA) was added to all sample for terminating the reaction. The assay plates were then harvested onto a 96-well microfilter plate using cell harvester and kept for overnight drying at 37°C. After drying 50 μL· of scintillation fluid was added to each well of microfilter plates. The radioactivity was measured by using scintillation counter.

Conclusion: The compound of formula I and the compound of formula la inhibited incorporation of [ H] Thymidine into DNA indicating that both the compounds are DNA synthesis inhibitor. The results are depicted in Figure 5a and Figure 5b.

Example 12:

The potential of the compound of formula I and the compound of formula la to inhibit RNA synthesis in Mycobacterium tuberculosis H ₃₇ RV sensitive strain by macromolecule incorporation assay.

Test Material: 1. Compound of formula I (Concentration Tested: 10X of MIC (8μg/mL))

2. Compound of formula la (Concentration Tested: 10X of MIC (4μg/mL)) Positive Control: Rifampicin (Concentration Tested: 10X of MIC (2μg/mL))

Negative Control: Ethambutol (Concentration Tested: 10X of MIC (8μg/mL))

Test System: Virulent M. tuberculosis H ₃₇ Rv (ATCC No.25678)

Methodology: Mid-log-phase aerobic Mycobacterium tuberculosis H37Rv cultures were diluted 100-fold in Middlebrook' s 7H9/ADC (Difco, BD India Ltd) medium and transferred to 150 mL flask. Freshly inoculated cultures were incubated at 37°C at 200 rpm till mid- exponential phase with OD5 ₆₀ of 0.5 corresponding to approx 3x10 cfu/mL was achieved. Cultures were then centrifuged at 3000 rpm and 4°C for 10 minutes. The pellet was vortexed and diluted with sterile phosphate-buffered saline to obtain 3x10 cfu/mL.

In sterile 96-well microtitre plates, 0.1 mL of the above M. tuberculosis suspension was added to each well followed by the addition of all the drugs at concentrations 10-fold above their MIC values. Cultures were then radio labeled by adding the 0.1 μθ of radio labelled [ H] Uridine for RNA synthesis. 96-well microtitre plates were then incubated at 37°C for 24 hours, 0.1 mL of cold 10% Trichloro acetic acid (TCA) was added to all sample to terminate the reaction. The assay plates were then harvested onto a 96-well microfilter plate using cell harvester and kept for overnight drying at 37°C. After drying 50 μL· of scintillation fluid was added to each well of microfilter plates. The radioactivity was measured by using scintillation counter.

Conclusion: The compound of formula I and the compound of formula la inhibited incorporation of [ H] Uridine into RNA indicating that both the compounds are RNA synthesis inhibitor. The results are depicted in Figure 6a and Figure 6b.

The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.