Technical Field The invention relates to the compounds or pharmaceutically acceptable salts thereof, that exhibit anti-tuberculosis activity.

Prior Art

According to the data of the World Health Organization, 9,2 million people suffer from tuberculosis annually and tuberculosis constitutes 2,5% of all of the diseases around the world and 26% of preventable deaths. Tuberculosis is usually caused by Mycobacterium tuberculosis (MTB). The bacteria Mycobacterium avium complex (MAC) (Mycobacterium avium and Mycobacterium intracellulare) results in the development of atypical tuberculosis infection. In particular, in the case of AIDS or cancer diseases, where the immune system weakens, the geriatric population and patients suffering from COPD (Chronic Obstructive Pulmonary Disease) may have a higher risk of death.

Medications that are used in tuberculosis treatment are mainly divided into two groups. The first group consists of; isoniazid (INH), rifampicin (RFM), pyrazinamide (PZA), ethambutol (ETM) and streptomycin (SM). The second group includes; more toxic and less tolerable medications such as cycloserine, ethionamide, kanamycin, capreomycin, and para-aminosalicylic acid. One of the most unfavorable cases in controlling tuberculosis is the development of drug resistance due to incorrect treatment or any other reasons. As MAC micro bacteria are naturally resistant to both of the groups, the development of novel compounds is seriously required.

The two compounds subject to the present invention and synthesis methods of these compounds have been disclosed in the articles “Some new 4-aryl-2-thiazolylhydrazones Turan-Zitouni, Gulhan; Kaplancikli, Z. Asim; Chevallet, PierreFarmaco (1997), 52(10), 635-638” and “Synthesis and anti-Candida activity of novel 2-hydrazino-l,3-thiazole derivatives Maillard, Ludovic T.; Bertout, Sebastien; Quinonero, Ophelie; Akalin, Gulsen; Turan-Zitouni, Gulhan; Fulcrand, Pierre; Demirci, Fatih; Martinez, Jean; Masurier, Nicolas Bioorganic & Medicinal Chemistry Letters (2013), 23(6), 1803-1807”

However, the prior art fails to clarify the anti-tuberculosis activities of these compounds and how it is ensured that Compound 2, which is water-insoluble can be dissolved in water.

Brief Description and Aims of the Invention

It has been surprisingly observed in an in vitro anti-microbial activity assessment carried out for two compounds with the chemical structures thereof presented here below that these two compounds were at least five times more active on Mycobacterium tuberculosis H37Rv than Rifampicin. Compound 2 exhibited activity on Mycobacterium avium as well.

After the determination of activity, Compound 1, which is soluble in water, was subjected to in vivo animal assays and it has determined that this compound didn’t exhibit toxicity. Compound 2, which is practically water- insoluble, was not subjected to in vivo tests.

Solubility ratios of Compound 2 in various organic solvents were determined and it has been observed in solubility assays carried out with solubility enhancing agents that this compound was dissolved in water to an extent with the use of hydroxypropyl-beta-cyclodextrin (HPPCD) or Polysorbate 80 (Tween 80).

Figures

Figure 1: Shows the chemical structures of Compound l:2-[l-(2,3-dihydro-lH-inden-5- yl)ethylidene]hydrazinecarbothioamide and Compound 2:2-[2-(2-(l-(2,3-dihydro-lH-inden-5- yl)ethylidene)hydrazinyl)thiazol-4-yl]-4-methoxy phenol. Figure 2: Shows the chemical structures of Compound 3: 4-(4-Chlorophenyl)-2-[2-(l-(2,3- dihydro-lH-inden-5yl)ethylidene)hydrazinyl]thiazole and Compound 4: 2-[2-(2-(l-(2,3-Dihydro- lH-inden-5-yl)ethylidene)hydrazinyl)thiazol-4-yl]phenol

Figure 3: Shows the chemical structures of Compound 5: 4-Chloro-2-[2-(2-(l-(2,3-dihydro-lH- inden-5 yl)ethylidene)hydrazinyl)thiazol-4-yl]phenol and Compound 6: 4-(4-Bromophenyl)-2-[2- (l-(2,3-dihydro-lH-inden-5-yl)ethylidene)hydrazinyl]thiazole

Figure 4: Shows the chemical structures of Compound 7: 2-[2-(l-(2,3-Dihydro-lH-inden-5- yl)ethylidene)hydrazinyl)-4-(p-tolyl) ]thiazole and Compound 8: 2-[2-(2-(l-(2,3-Dihydro-lH- inden-5-yl)ethylidene)hydrazinyl)thiazol-4-yl]-5-methoxyphen ol

Figure 5: Shows the chemical structures of Compound 9: 2-[2-(2-(l-(2,3-Dihydro-lH-inden-5- yl)ethylidene)hydrazinyl)thiazol-4-yl]-5-methylphenol and Compound 10: 2-[2-(l-(2,3-Dihydro- lH-inden-5-yl)ethylidene)hydrazinyl)-4-(4-nitrophenyl)]thiaz ole

Figure 6: Shows the chemical structures of Compound 11: 2-[2-(l-(2,3-Dihydro-lH-inden-5- yl)ethylidene)hydrazinyl]-4-phenylthiazole

Detailed Description of the Invention

Chemical structures and chemical names of said compounds are given below.

Compound 1: 2-[l-(2,3-dihydro-lH-inden-5-yl)ethylidene]hydrazinecarbothi oamide

Compound 2: 2-[2-(2-(l-(2,3-dihydro-lH-inden-5-yl)ethylidene)hydrazinyl) thiazole-4-yl]-4- methoxy phenol

Compound 3: 4-(4-Chlorophenyl)-2-[2-(l -(2,3 -dihydro- lH-inden-5yl)ethylidene)hydrazinyl] thiazole

Compound 4: 2-[2-(2-(l -(2,3-Dihydro-lH-inden-5-yl)ethylidene)hydrazinyl)thiazol-4- yl]phenol Compound 5: 4-Chloro-2-[2-(2-(l-(2,3-dihydro-lH-inden-5-yl)ethylidene)hy drazinyl)thiazol-4- yl]phenol

Compound 6: 4-(4-Bromophenyl)-2-[2-(l -(2,3-dihydro- lH-inden-5-yl)ethylidene)hydrazinyl] thiazole

Compound 7: : 2-[2-(l-(2,3-Dihydro-lH-inden-5-yl)ethylidene)hydrazinyl)-4- (p-tolyl) ]thiazole Compound 8: 2-[2-(2-(l -(2,3-Dihydro-lH-inden-5-yl)ethylidene)hydrazinyl)thiazol-4- yl]-5- methoxyphenol Compound 9: 2-[2-(2-(l -(2,3-Dihydro-lH-inden-5-yl)ethylidene)hydrazinyl)thiazol-4- yl]-5- methylphenol

Compound 10: 2-[2-(l-(2,3-Dihydro-lH-inden-5-yl)ethylidene)hydrazinyl)-4- (4-nitrophenyl)] thiazole Compound 11: 2-[2-(l-(2,3-Dihydro-lH-inden-5-yl)ethylidene)hydrazinyl]-4- phenylthiazole Compound 4

Compound 11

When both of the compounds were tested on Mycobacterium tuberculosis H37Rv (ATCC27294) with the BACTEC 460 Radiometric system used in the primary screening carried out for in vitro assessment of antimicrobial activity of Compounds in the TAACF research center (Tuberculosis Antimicrobial Acquisition & Coordinating Facility), Alabama/USA, they exhibited an inhibition effect higher than 90% ( Compound 1 99%, Compound 2 95%). Following this, to determine the actual minimum inhibitory concentration in the second phase, both of the compounds were retested to the lowest concentration of Mycobacterium tuberculosis H37Rv with the technique of MABA (Microplate Alamar Blue Assay) and MIC value for the 1 ^st compound was determined <0.03 pg/ml while this value was determined <0.05 pg/ml for the second compound. The result of Rifampicin subjected to the assay under the same conditions was determined as 0.25 pg/ml. The results obtained show that both of the Compounds are more active than Rifampicin. Compound 2 also exhibited the MIC concentration of 0.39 pg/ml on Mycobacterium avium with BACTEC.

Any conditions concerning toxicity have not been observed in 1000 mg/kg determined as the maximum tolerable dose in in vivo tests carried out for Compound 1.

Anti -tuberculosis activities of Compounds subject to the invention have been disclosed. The results of the activity obtained and presented below show that both of the Compounds were determined to be very active.

Supersaturated solutions of Compound 2 in various organic solvents were left to rest in an ultrasonic bath for 15 minutes at room temperature and thus solubility ratios thereof were quantitatively determined by the use of an analytical method and the results obtained were presented below Table 1.

Table 1

To ensure that Compound 2 is dissolved in water, in the assays carried out with solubility enhancing cyclodextrin molecules, different cyclodextrins [hydroxypropyl beta-cyclodextrin (HR-b-CD), sulfobutyl ether beta-cyclodextrin (SBE-P-CD)], different methods [kneading, evaporation, lyophilization], different solvents (dimethyl sulfoxide, methanol, ethanol) were studied at the molar ratio of 1 : 1 compound: cyclodextrin. In these assays, only in the evaporation method with HR-b-CD within ethanol, it has been determined that Compound 2 was dissolved in water at the ratio of 0.59 pg/ml and any significant increase in solubility was not observed.

In the assays carried out with the solubility enhancing Polysorbate 80 (Tween 80) in order to ensure water solubility of Compound 2, firstly solubility of the compound was determined in an aqueous solution prepared with a mass ratio of 1:6 (compound: Tween 80) and then the same mass ratio was studied with different solvents (ethanol, methanol) and different drying methods (evaporation, evaporation+lyophilization). In light of the results of solubility obtained and presented below Table 2, it has determined that the solution of the compound that was prepared with the Tween 80 in methanol medium had the highest solubility rate after evaporation.

Table 2

Details of the method where high solubility was determined with Polysorbate 80 (Tween 80) are presented below:

After the substances at the mass ratio of 1:6 (compound: Tween 80) were dissolved in 7 ml methanol, they were evaporated in a rotating evaporator at 175 RPM, at a temperature of 40°C and a vacuum of 140 atm. The thin film obtained was firstly blended with vortex adding 3 ml water, and then was left to rest for 15 minutes in an ultrasonic bath. The mixture obtained was filtered out with a plastic filter having a pore diameter of 0.2 pm and afterward solubility ratio of the mixture was determined with HPLC. It has been determined with the following results that the compounds were at least five times more active than Rifampicin that is an antibiotic used in tuberculosis treatment (Table 3).

Table 3

These compounds provide an important advantage as they can be used in lower doses than the drugs used in current treatments, as these compounds exhibit very high activity.