WO2011006536A1

WO2011006536A1 - Pyridyl-substituted isoquinoline derivatives with activity againstmycobacteriaand especially myobacterium tuberculosis

Info

Publication number: WO2011006536A1
Application number: PCT/EP2009/059003
Authority: WO
Inventors: Frans Herwig Jansen
Original assignee: Dafra Pharma N.V.
Priority date: 2009-07-14
Filing date: 2009-07-14
Publication date: 2011-01-20

Abstract

The present invention relates to compounds with activity against mycobacteria, and especially Mycobacterium tuberculosis, pharmaceutical compositions comprising the present compounds, and the use of the present compounds for the treatment of tuberculosis or other mycobacteria caused diseases. Specifically, the present invention relates to compounds with bactericidal activity against Mycobacterium tuberculosis according to the general formula (I) wherein R₁ is selected from the group consisting of H, F, Cl, Br, I and NO₂; R₂ is selected from the group consisting of -NH₂ or an amide according to the general formula (II), wherein R₃ is a C₁ to C₈ alkyl, preferably selected from the group consisting of methyl, ethyl, propyl and butyl, under the condition that if R₂ is -NH₂ R₁ is not H.

Description

PYRIDYL-SUBSTITUTED ISOQUINOLINE DERIVATIVES WITH ACTIVITYAGAINST MYCOBACTERIA AND ESPECIALLY MYOBACTERIUM TUBERCULOSIS

The present invention relates to compounds with activity against mycobacteria, and especially Mycobacterium tuberculosis, pharmaceutical compositions comprising the present compounds, and the use of the present compounds for the treatment of tuberculosis or other mycobacteria caused diseases.

Tuberculosis, also abbreviated herein as TB for tubercle bacillus or tuberculosis, is a common, and often, deadly infectious disease caused by mycobacteria, in humans mainly Mycobacterium tuberculosis . Tuberculosis generally targets the lungs, for example pulmonary tuberculosis, but can also target other tissues or organs such as the central nervous system, the lymphatic system, the circulatory system, the genitourinary system, the gastrointestinal system, bones, joints, or even the skin.

Mycobacterium tuberculosis is an aerobic bacterium that divides every 16 to 20 hours. Since Mycobacterium tuberculosis has a cell wall but lacks a phospholipid outer membrane, it is classified as a Gram-positive bacterium. However, if a Gram stain is performed, Mycobacterium

tuberculosis either stains very weakly Gram-positive or does not retain dye due to the high lipid & mycolic acid content of its cell wall. Mycobacterium tuberculosis is a small rod- like bacillus that can withstand weak disinfectants and survive in a dry state for weeks. In nature, the bacterium can grow only within the cells of a host organism, but

Mycobacterium tuberculosis can be cultured in vitro.

Other species of mycobacteria, such as

Mycobacterium bovis, Mycobacterium africanum, Mycobacterium canetti, or Mycobacterium microti, although less common than Mycobacterium tuberculosis, can also cause tuberculosis in humans .

Other pathogenic mycobacteria include

Mycobacterium leprae, the cause of lepra, Mycobacterium avium and M. kansasii. The last two are part of the

nontuberculous mycobacteria (NTM) group. Nontuberculous mycobacteria cause neither tuberculosis nor leprosy, but they do cause pulmonary tuberculosis-like diseases.

The classic symptoms of tuberculosis are a chronic cough with blood-tinged sputum, fever, night sweats, and weight loss. Infection of other organs causes a wide range of symptoms. The diagnosis relies on radiology (commonly chest X-rays) , a tuberculin skin test, blood tests, as well as microscopic examination and microbiological culture of bodily fluids.

Symptoms vary depending on variants of

Mycobacterium tuberculosis and stages of the disease with many symptoms overlapping, while others are more (but not entirely) specific for certain variants.

Multiple variants may be present simultaneously. When the disease becomes active, 75% of the cases are pulmonary tuberculosis. Symptoms include chest pain, coughing up blood, and a productive, prolonged cough for more than three weeks. Systemic symptoms include fever, chills, night sweats, appetite loss, weight loss, pallor, and often a tendency to fatigue easily.

In the other 25% of active cases, the infection migrates from the lungs to other organs or tissues, causing other symptoms of tuberculosis, collectively denoted

extrapulmonary tuberculosis. These subsequent infections are more common in immunosuppressed persons and young children. Extrapulmonary infection sites include the pleura in tuberculosis pleurisy, the central nervous system in

meningitis, the lymphatic system in scrofula of the neck, the genitourinary system in urogenital tuberculosis, and bones and joints in Pott's disease of the spine. An

especially serious form is disseminated tuberculosis, more commonly known as military tuberculosis. Although

extrapulmonary tuberculosis is generally not contagious, it may co-exist with contagious forms of pulmonary

tuberculosis .

Tuberculosis treatment is difficult and requires long courses of multiple antibiotics. Contacts are also screened and treated if necessary. Antibiotic resistance is a growing problem in (extensively) multi-drug-resistant tuberculosis. Prevention relies on screening programs and vaccination, usually with Bacillus Calmette-Guerin (BCG vaccine) . Tuberculosis is spread through the air mainly when people suffering from the disease cough, sneeze, or spit.

One-third of the world's current population has been infected with Mycobacterium tuberculosis, and new infections occur at a rate of one per second. Generally, the majority of infections do not initially result in the development of the active disease, i.e., showing, or

suffering from, one or more symptoms of tuberculosis;

asymptomatic and latent infection is common.

About one in ten of the latent infections will eventually progress into active disease, which, if not treated, results in the death of about half of this

population.

The number of people in the general population suffering from tuberculosis each year is stable or falling worldwide but, mainly because of population growth, the absolute number of new cases is still increasing. In 2004, mortality and morbidity statistics for tuberculosis included 14.6 million chronic active cases, 8.9 million new cases, and 1.6 million deaths, mostly in

developing countries. In addition, a rising number of people in the developed world are infected with Mycobacterium tuberculosis because their immune system is compromised by immunosuppressive drugs, substance abuse, or AIDS.

The distribution of tuberculosis is not uniform across the globe with about 80% of the population in many Asian and African countries testing positive in tuberculin tests, while only 5 to 10% of the US population test

positive. It is estimated that the US has 25,000 new cases of tuberculosis each year, 40% of which occur in immigrants from countries where tuberculosis is endemic.

Twin studies in the 1940s showed that

susceptibility to tuberculosis was heritable. If one of a pair of identical twins suffered from tuberculosis, then and the other was more likely to also suffer from tuberculosis. This relation was not observed in non-identical twins. After the first reports, the genetic susceptibility for

tuberculosis has been linked to gene polymorphisms in IL12B. Also patients with diabetes mellitus are at increased risk of contracting tuberculosis, and they have a poorer response to treatment, possibly due to poorer drug absorption.

Other conditions that identify populations with an increased risk for the development of tuberculosis include intravenous drug abuse; a recent tuberculosis infection or a history of inadequately treated tuberculosis; chest X-ray suggestive of previous tuberculosis showing fibrotic lesions and nodules; silicosis; prolonged corticosteroid therapy and other immunosuppressive therapy; head and neck cancers;

hematologic and reticulo endothelial diseases, such as leukemia and Hodgkin's disease; end-stage kidney disease; intestinal bypass or gastrectomy; chronic malabsorption syndromes; vitamin D deficiency; and low body weight.

Some drugs, including rheumatoid arthritis drugs that work by blocking tumor necrosis factor-alpha (an inflammation-causing cytokine) , raise the risk of activating a latent infection due to the importance of this cytokine in the immune defence against tuberculosis.

Treatment of tuberculosis mainly involves a bactericidal approach, for example the use of antibiotics. Medicaments most commonly used as a first line treatment of tuberculosis are rifampicin, pyrazinamide, ethambutol, and isoniazid.

However, treatment using these first line medicaments is not risk-free. The Centers for Disease

Control and Prevention (CDC) notified healthcare

professionals of revised recommendations against the use of rifampin plus pyrazinamide for treatment of latent

tuberculosis infection, due to high rates of hospitalization and death from liver injury associated with the combined use of these drugs.

In case the first line medicaments provide

insufficient, or inadequate results, second line medicaments are available and include, aminoglycosides, polypeptides such as capreomycine, isoquinolines, thioamides, cycloserine and PAS.

However, despite the availability of a number of medicaments available to treat diseases caused by

mycobacteria, and especially tuberculosis, there is still a need in the art for further compounds providing a more effective treatment of the diseases, for example, compounds causing less side effects, less toxic compounds allowing higher dosages or compounds with a higher activity against mycobacteria and especially Mycobacterium tuberculosis . It is an object of the present invention, amongst other objects, to provide such compounds.

This object, amongst others, is met by the present invention through compounds with activity against

Mycobacterium tuberculosis as defined in the appended claim 1.

Specifically, this object is met by providing compounds with bactericidal activity against Mycobacterium tuberculosis according to the general formula (I)

wherein

Ri is selected from the group consisting of H, F, Cl, Br, I and NO₂;

Ra is selected from the group consisting of -NH2 or an amide according to the general formula (II)

H

^NγP wherein R₃ is a Ci to C₈ alkyl, preferably selected from the group consisting of methyl, ethyl, propyl and butyl, under the condition that if R₂ is -NH₂ Ri is not H.

The compound, being not according to the present invention, wherein R₂ is -NH₂ and Ri is H is known in the prior art for its bactericidal activity against

mycobacteria, and especially Mycobacterium tuberculosis. The present inventors surprisingly found that the bactericidal activity of this compound could be further improved by the present substitutions on either the Ri and/or R₂ positions.

The observed improvement was found in the finding of a better efficacy/toxicity ratio of the present compounds as compared to the parent compound. Specifically, the present inventors found that the present substitutions did not substantially affect the bactericidal activity, but the toxicity was significantly reduced resulting in a beneficial efficacy/toxicity . Amongst others, the reduced toxicity of the present compounds allows for higher dosages, or

concentrations, of the present compounds thereby allowing a higher overall efficacy for the treatment of tuberculosis.

Considering the reduced toxicity, also less side effects can be expected in this treatment.

According to a preferred embodiment, the present invention relates to a compound having the chemical

structure of formula (II)

According to another preferred embodiment, the present invention relates to a compound having the chemical structure of formula (III) .

According to yet another preferred embodiment, the present invention relates to a compound having the chemical structure of formula (IV) .

According to still another preferred embodiment, the present invention relates to a compound having the chemical structure of formula (V) .

According to a further preferred embodiment, the present invention relates to a compound having the chemical structure of formula (VI) .

Considering the beneficial effects of the present compounds on the treatment of mycobacterial infections, and especially tuberculosis, the present invention relates according to another aspect to pharmaceutical compositions comprising:

a compound accorind to the present invention; and

pharmaceutically acceptable carries and excipients.

Still another aspect of the present invention relates to the present compounds for the treatment of tuberculosis and methods for treating tuberculosis

comprising administering to an individual in need of treatment of a Mycobacterium tuberculosis infection a therapeutically effective amount of a compound according to the present invention.

The present invention will be further described in the examples below of preferred embodiments of the

invention. In the examples, reference is made to figures, wherein: Figure 1: is a schematic representation of chemical

synthetesis schemes available for providing the compounds according to the present invention.

Figure 2 : shows the chemical structure of 5 preferred

compounds according to the present invention compounds

Examples

Example 1 Synthesis of the present compounds

I : Synthesis of substituted l-amino-3(2- pyridyl) isoguinolines

A suitable synthesis scheme for the synthesis of substituted l-amino-3 (2-pyridyl) isoquinolines is presented in Figure l(la-c). Potassium amide, freshly prepared in 500 ml ammonia, was stirred continuously into a three-neck flask, equipped with a mechanical stirrer. The mixture was cooled at -78 ⁰C. Then, 0.5 mole of substituted 2- methylbenzonitril in 200 ml anhydrous diethylether was slowly added while maintaining the temperature at -78⁰C.

After addition of the 2-methylbenzonitril, a solution of 0.5 mole of 2-cyanopyridine in 250 ml THF was added in 20 minutes. The cooling was then stopped and the mixture was stirred continuously overnight to allow

evaporation of the ammonia. Subsequently, the mixture was hydrolysed by adding a small volume of water. The compound was then extracted with diethylether and recrystallised from methanol. This procedure was used to synthesize the

following compounds:

6-fluoro-3- (pyridine-2-yl) isoquinoline-1-amine :

Yield 65%; ¹H NMR: 5,34 (br s, 2H, NH₂), 7, 12 (m,

IH, ArH), 7,21 (dd, J = 7,8 Hz, 1 Hz, IH, ArH), 7,35 (d, J = 1 Hz, IH, ArH), 7,65 (m, IH, ArH), 7,85 (d, J = 7,8 Hz, IH, ArH), 8,11 (s, IH), 8,50 (dd, IH), 8,58 (dd, IH). 6-chloro-3- (pyridine-2-yl) isoquinoline-1-amine :

Yield 60%; ¹H NMR: 5,30 (br s, 2H, NH₂), 7,12 (m, IH, ArH), 7,52 (dd, J = 7,8 Hz, 1 Hz, IH, ArH), 7,66-7,68 (m, 2H, ArH), 7,81 (d, J = 7.8 Hz, IH, ArH), 8,10 (s, IH), 8,50 (dd, IH) , 8,58 (dd, IH) .

6-nitro-3- (pyridin-2-yl) isoquinoline-1-amine :

Yield 62%; ¹H NMR: 5,35 (br s, 2H, NH₂), 7,12 (m, IH, ArH), 7,65 (m, IH, ArH), 8,04 (d, J = 7,8 Hz, IH, ArH) 8,32 (dd,J = 7,8 Hz, 1 Hz, IH, ArH), 8,38 (s, IH), 8,50 (dd, IH), 8,59 (dd, IH), 8,64 (d, J = I Hz, IH, ArH),

II : Synthesis of amides of l-amino-3- (2- pyridyl) isoguinolines A solution of 0.002 mole of 1 -amino-3- (2- pyrydil) isoquinoline in 40 ml anhydrous THF was stirred under nitrogen atmosphere and cooled at -1O⁰C.

12.5 ml of 1.6 M n-butyllithium in hexane was slowly added. The solution was left under continuous

stirring for 10 minutes. Subsequently, 0.02 mole of n- methylbenzoyl chloride (in a small volume of THF) was added and stirred for 1 hour. Then, the mixture was brought to room temperature and hydrolyzed with water. The resulting mixture was extracted with chloroform. The combined chloroform layers were washed with sodium bicarbonate, dried with anhydrous potassium

carbonate, and, after filtration, evaporated to dryness.

This procedure was used to synthesize the following

compounds :

N- (6~fluoro-3- (pyridine-2-yl) isoquinoline-1-yl) -3- methylbenzamide :

Yield 35%; ¹H NMR: 2,50 (s, 3H, CH₃), 7,27-8,13 (m,

8H), 8,34 (m, 2H), 8,87 (d, J = 4,5 Hz, IH, H-6' ) , 9,05 (d J = 7,2 Hz, IH, H-3' ) .

N- (6~chloro-3- (pyridine-2-yl) isoquinoline-1-yl) -3- methylbenzamide :

Yield 38%; ¹H NMR: 2.50 (s, 3H, CH₃), 7,27-8,13 (m, 8H), 8,34 (m, 2H), 8,87 (d, J = 4,5 Hz, IH, H-6'), 9,05 (d J = 7,2 Hz, IH, H-3' ) . N- (6-nitro-3- (ρyridin-2-yl) isoquinoline-1-yl) -3- methylbenzamide :

Yield 34%; ¹H NMR: 2,50 (s, 3H, CH₃), 7,30-7,53 (m, 6H) , 8, 6-9,05 (m, 6H) . III Synthesis of the amidines from the respective amides obtained from l-amino-3- (2-pyridyl) isoguinolines

A suitable synthesis scheme for the synthesis of the respective amidines obtained from l-amino-3- (2- pyridyl) isoguinolines is presented in Figure l(3a-c). A solution of 0.02 mole of phosphor pentachloride in 50 ml freshly distilled chloroform was stirred under nitrogen atmosphere at room temperature. A solution of 0.01 mole of the amide in 50 ml chloroform was slowly added and the resulting mixture was refluxed for 30 minutes. Then, the mixture was cooled in an ice bad and anhydrous ammonia was flushed through the mixture for 1 hour.

Subsequently, ice cold bicarbonate was slowly added. The chloroform layer was separated, washed 3 times, dried with anhydrous potassium carbonate, and evaporated to dryness. This procedure was used to synthesize the following compounds :

N' - (6-fluoro-3- (pyridine-2-yl) isoquinoline-1-yl) -3- methylbenzamidine :

Yield 40%; ¹H NMR: 2,50 (s, 3H, CH₃), 5,1 (br s, 2H, NH₂), 7,32 (m, IH, H-5' ) 7,39-8,05 (m, 9H), 8,24 (d, J = 8.0 Hz, IH, H-8), 8,42 (s, IH, H-4), 8,78 (d, J = 4,8 Hz, IH, H-6'), 9,05 (m, IH, H-3' ) .

N'- (6-chloro-3- (pyridine-2-yl) isoquinoline-1-yl) -3- methylbenzamidine :

Yield 42%; ¹H NMR: 2,50 (s, 3H, CH₃), 5,1 (br s,

2H, NH₂), 7,32 (m, IH, H-5') 7,39-8,05 (m, 9H), 8,26 (d, J = 8.0 Hz, IH, H-8), 8,42 (s, IH, H-4), 8,78 (d, J = 4,8 Hz, IH, H-6'), 9,05 (m, IH, H-3'). N ' - (6-chloro-3- (pyridine-2-yl) isoquinoline-1-yl) -3- methylbenzamidine :

Yield 45%; ¹H NMR: 2,50 (s, 3H, CH₃), 5,4 (br s, 2H, NH₂), 7,32-7,51 (m, 6H), 8,5-9,05 (m, 6H). IV: Procedure for amide formation from the respective

amidines A suitable procedure for amide formation from the respective amidines is presented in Figure l(4a-c). 0.015 M of the respective anhydrides and 1 gram of potassium carbonate were added to a solution of 0.01 mole of the respective amidines in dichloromethane . The resulting mixture was stirred under nitrogen atmosphere for 6 hours. Subsequently, the reaction was quenched with water, extracted with dichloromethane, and recrystallised from dichloromethane/hexane . This procedure was used to

synthesize the following compounds:

N- (- (6-fluoro-3- (ρyridine-2-yl) isoqu±noline-1-ylimino) (intoIyI)methyl) acetamide :

Yield 95%; ¹H NMR: 2,25 (s, 3H, COCH₃), 2,50 (s, 3H, CH₃), 5,1 (br s, 2H, NH₂), 7,32 (m, IH, H-5' ) 7,39-8,05 (m, 9H), 8,24 (d, J = 8,0 Hz, IH, H-8), 8,42 (s, IH, H-4), 8,78 (d, J = 4,8 Hz, IH, H-6' ) , 9,05 (m, IH, H-3' ) .

N- (- (β-chloro-3- (pyridine-2-yl) isoquinoline-1-ylimino) (m- toly1)methyl) acetamide) :

Yield 98%; ¹H NMR: 2,25 (s, 3H, COCH₃), 2,50 (s,

3H, CH₃), 5,1 (br s, 2H, NH₂), 7,32 (m, IH, H-5') 7,39-8,05

(m, 9H), 8,26 (d, J = 8,0 Hz, IH, H-8), 8,42 (s, IH, H-4),

8,78 (d, J = 4,8 Hz, IH, H-6'), 9,05 (m, IH, H-3').

N- (- (6-nitro-3- (pyridin-2-yl) isoquinoline-1-ylimino) (m- tolyl)methyl) acetamide :

Yield 92%; ¹H NMR: 2,25 (s, 3H, COCH₃), 2,50 (s,

3H, CH₃), 5,4 (br s, 2H, NH₂), 7,32-7,51 (m, 6H), 8,5-9,05 (m, 6H) . Example 2 Effectivity of the present compounds against Mycobacterium tuberculosis

The compound DF152 is made of a 2-pyridyl isoquinoline moiety attached to an amidine group, making it a novel class of anti-mycobacterial compounds. The compound is found to be very active, especially against Mycobacterium tuberculosis strains. Chemical optimization of the parent molecule was initiated with the aim of improving its

physical and anti-microbial properties, and to identify its active pharmacophore.

This resulted in the production of a series of five derivatives shown in Figure 2, including amide prodrugs (at position R2) , which are expected to be more soluble in pharmaceutical solvents and for which the

mechanism of action might slightly differ. Further

derivatives with halogen substitutions on the isoquinoline moiety (at position Rl) have also been made hoping that they would show increased activity or safety compared to the parent molecule.

In vitro activity of the parent molecule DF152 against different mycobacterial species was assessed using the highly sensitive fluorometric BACTEC MGIT 960 system. The drug concentrations necessary for complete inhibiting (i.e. 100% inhibition) of the growth of several strains of Mycobacterium tuberculosis and Mycobacterium avium were determined.

The spectrum of Mycobacterium tuberculosis strains initially tested covered a total of 41 strains including the reference strain H37Rv, 8 fully susceptible strains, 14 multidrug resistant (MDR) strains and 2 extensively drug resistant (XDR) strains (Table 1) . Table 1: MJCs of DF152 inhibiting growth of different mycobacterial apeciea

Minimal concentration 100% growth inhibition, except for

M.ulcerans, for which MC90 is shown.

MDR=Resistant to DINH+RMP or 2) INH+RMP+EMB or 3) INH+RMP+SM or 4 (INH+RMP+EMB+SM.

XDR=Resistant to INH, RMP, SM, Ofloxaxin, Capreonycin.

Interestingly, median MIC values were found to be lower than 0.125 μg/ml for 31 of the 41 strains tested regardless of their resistance profile. DF152 was also found to be active against the 10 strains of Mycobacterium avium tested but with a higher median MIC value (1.0 μg/ml) . Very high efficacy was observed against Mycobacterium ulcerans (MIC90 of 0.02 μg/ml; Table 1).

The lack of cross-resistance between DF152 and currently used anti-TB agents (Isoniazid, Streptomycin, Ethambutol, Rifampicin, Ofloxaxin, Capreomycin) is of critical interest as it suggests that DF152 has a distinct mechanism of action (target) , and could be used to treat drug resistant mycobacterial species. Additional work on the elucidation of the mechanism of action of DF152 including the determination of whether the parent molecule has a cidal or a static activity.

Based on this initial screen, another series of experiments were performed aiming to test the efficacy of the parent molecule at concentrations lower than 0.1 μg/ml

(lowest concentration tested 0.063 μg/ml) , and to test the five derivatives of DF152 for in vitro efficacy. One susceptible strain and two MDR strains were also used to test the six compounds of the DF152 series and the results and presented in Table 2.

Table 2 Comparison of MICa between DFl52 and its

derivatives.

Minimal concentration for 100% growth inhibition.

Chelating properties of DF152 have previously been described in the literature and are expected to be conserved in DF152dl and DF152d2 due to the presence of the amine group (Figure 2) .

These three chelating molecules share the same range of MICs, and little to no difference were observed between the parental molecule and the two substitutions on the isoquinoline group (position Rl) , although the exact MICs for those three molecules still remain to be exactly defined.

The other three molecules substituted at position R2 (DF152d3, DF152d4 and DF152d5, representing the amide derivatives of DF152,. DF152dl and DF152d2, respectively) show lower, but still overall good efficacy compared to their amine counterparts with MICs ≤ 0.5 μg/ml for the

Mycobacterium tuberculosis strains tested, and ≤ 4 μg/ml for the Mycobacterium avium strains tested.

The nature of the Rl substitutions showed little impact on efficacy within the three amide derivatives. For all derivatives, the efficacy against Mycobacterium

tuberculosis was generally higher than the one seen against Mycobacterium avium, as observed for the parent molecule.

Considering the above, the present derivatives showed a comparable bactericidal activity against

mycobacteria, and especially Mycobacterium tuberculosis. Example 3 Cytotoxicity of the present compounds

The general in vitro cytotoxicity of the DF152 series was tested in a series of three independent

experiments performed in HepG2 cells (Table 3) . The

objective was to assess the cell viability/number after exposure to 7 concentrations of the 6 test items, compared to control conditions, and identified the concentration of each compounds necessary to kill 50% of the HepG2 cells (CC50) .

The MTT assay was used to determine the viability/number of cells in culture (Mosmann, 1983). MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide) is a yellow water-soluble tetrazolium salt which is taken up into cells and reduced by mitochondrial succinic dehydrogenase to the blue insoluble dye formazan. The product accumulates within the cell, due to the fact that it cannot pass through the plasma membrane. Upon solubilisation of the cells, the product is liberated and can readily be detected and quantified by a simple colorimetric method. The ability of cells to reduce MTT provides an indication of mitochondrial integrity and activity which, in turn, may be interpreted as a measure of viability and/or cell number. The assay was adapted for use with cultures of exponentially growing cells. Determination of their ability to reduce MTT to the formazan product after exposure to test compounds, compared to the control situation, enables the relative cytotoxicity of test chemicals to be assessed.

HepG2 cells were plated for 48 hours in 96-well plates, then the medium was changed and the cells were exposed for about 44 hours to the test chemicals, which corresponds to the doubling time of the cells. The MTT assay was subsequently performed on the exposed cells to evaluate the cytotoxic effect of the different molecules of the series (Table 3) .

Table 3 Cytotoxicity of DF152 series on HepG2 cells

NT: not tested; NDA: no data available due to technical problem.

Examination of the HepG2 cytotoxicity profiles demonstrated the following. The DF152 parent molecule as well as the two derivatives having chelating properties showed relatively high toxicity when applied to the HepG2 cells (CC50 ranging from 0,1 to 0,6 μg/ml) when compared to the non chelating ones (CC50 ranging from 0.2 to 1.9 μg/ml).

This result shows that the amide substitution at position R2 beneficially affects both the efficacy and the toxicity of the DF152 parent molecule. In addition, the nature of the substitution at position Rl has an influence on the safety profile, with the Cl- substitution (DF152dl, DF152d4) an overall lower cytotoxicity is observed within the amine and the amide series (Table 3, experiment 3) .

Summarizing, the present experiments show that the safety profile of the DF152 parent molecule is improved by additional modifications at the Ri and R₂ positions without affecting the general efficacy, i.e., bactericidal activity against mycobacteria. This is the most strongly demonstrated for DF152d4 and DF152d5 showing the highest safety/efficacy ratio .

Example 4 Cytotoxicity testing of the present compounds

using HeK293 cells

The cytoxicity of the present compounds was also tested using an adenosine A3 displacement assay (Salvatore, PNAS 1993) using HEK293 cells. The results are presented in Table 4.

Table 4 Adenosine A3 displacement

a Displacement of specific [125] AB-MECA binding at human adenosine A3 receptors expressed in HEK 293 cells

As can be clearly seen in Table 4, the cytotoxicity of the compounds according to the present inventions is

significantly reduced as compared to the parent compound DF152.

Claims

1. Compound with bactericidal activity against

Mycobacterium tuberculosis according to the general formula (I)

wherein

Ri is selected from the group consisting of H, F, Cl, Br, I and NO₂;

R2 is selected from the group consisting of -NH₂ or an amide according to the general formula (II)

wherein R₃ is a Ci to C₈ alkyl, preferably selected from the group consisting of methyl , ethyl , propyl and butyl, under the condition that if R₂ is -NH₂ R_x is not H.

2. Compound according to claim 1, wherein said compound has the formula (II)

3. Compound according to claim 1, wherein said compound has the formula (III)

4. Compound according to claim 1, wherein said compound has the formula (IV)

5. Compound according to claim 1, wherein said compound has the formula (V)

6. Compound according to claim 1, wherein said compound has the formula (VI)

7. Pharmaceutical composition comprising:

a compound as defined in any of the claims 1 to 6; and

pharmaceutically acceptable carries and excipients.

8. Compound as defined in any of the claims 1 to 6 for the treatment of tuberculosis.

9. Method for treating tuberculosis comprising administering to an individual in need of treatment of a

Mycobacterium tuberculosis infection a therapeutically effective amount of a compound according to any of the claims 1 to 6.