Heterocyclic-2-carboxylic Acid (3-Cyano-1,4-di-N-oxidequinoxalin-2-yl)amide Derivatives as Hits for the Development of Neglected Disease Drugs

Neglected diseases represent a major health problem. It is estimated that one third of the world population is infected with tuberculosis (TB). Besides TB, Chagas disease, affects approximately 20 million people. Quinoxalines display great activities against TB and Chagas. Forty new quinoxaline 1,4-di-N-oxide derivatives have been prepared and tested against M. tuberculosis and T. cruzi. Carboxylic acid quinoxaline 1,4-di-N-oxides (CAQDOs) 5 and 17 showed MIC values on the same order as the reference antituberculosis drug, rifampicin. Meanwhile, CAQDOs 12 and 22 presented IC50 values in the same order as the anti-chagasic drug, nifurtimox.


Introduction
Mycobacterium tuberculosis (M. tuberculosis), and to a lesser extent M. bovis and M. africanum, can cause a chronic and fatal condition in humans known as tuberculosis (TB). Until about 50 years ago, this disease was considered virtually incurable. The discovery of several active anti-TB agents heralded a new age of anti-TB chemotherapy. Therefore, TB was considered to be a curable disease. Unfortunately, in only a few years, it became apparent that the use of these drugs as single agents led to rapid drug resistance and treatment failures among a substantial number of patients. It was quickly realized, however, that the development of resistance could be forestalled or prevented through treatment with several active agents in a combination regimen. Of particular concern is the development of multi-drug-resistant forms of the disease (MDR-TB), defined as forms resistant to two or more of the front line anti-TB agents. These forms of the disease are most often fatal and are difficult and expensive to treat. It is estimated that one third of the world's population is infected with TB, with about eight million new cases annually. Of these cases, 3.1 million die annually, more deaths than those caused by any other single infectious disease. TB is the leading killer of youths, women, and AIDS patients in the world [1,2]. HIV-infected patients have an elevated risk of tuberculosis, and such active infectious process may enhance HIV replication and increase the risk of death. It has been estimated that up to 50 million people are infected with drug-resistant forms of TB. Due to the fact that the current frontline therapy for TB consists of administering three different drugs (the antibiotic rifampicin, RIF, and the azaheterocycles isoniazid and pyrazinamide, Isnz and Pyzd, Scheme 1) over an extended period of time as well as the problems that arise due to MDR-TB, it is necessary to develop new, potent, fast-acting anti-tuberculosis drugs with low-toxicity profiles for treating drug resistant forms of the disease that can be given in conjunction with drugs used to treat HIV infections [3,4].
Besides TB, the parasitic diseases represent a major health problem in Third World countries. More specifically, Chagas disease, or American trypanosomiasis, caused by the protozoan Trypanosoma cruzi (T. cruzi), is the largest parasitic disease burden in the American continents. It affects approximately 20 million people from the southern United States to southern Chile. Even though the enforcement of public health programs towards vector elimination in some Latin American countries has decreased the incidence of new infections, the disease is still endemic in large areas. Every year, 21,000 people die from this parasitosis and over 200, 000 new cases arise [5]. Currently, there are only two clinically used drugs, nifurtimox (Nfx, Scheme 1) and benznidazole. Both are nitroheterocyclic compounds that possess important toxic effects and relative clinical efficacy; therefore, the pharmacotherapy of Chagas disease is very deficient and there is an urgent need for the development of safe and effective drugs [6].
Galactofuranose is an essential component of the mycobacterial cell wall, not found in man; UDPgalactofuranose is biosynthesized from UDP-galactopyranose using the enzyme UDP-galactose mutase (Glf). In 2004, Tangallapally et al. discovered that nitrofuryl derivatives have the requirements for optimum inhibition of Glf activity (i.e. compound 4, Scheme 1) [4]. In addition, the nitrofuryl moiety is present in a large number of anti-T. cruzi agents acting via a nitroreduction process, generating redox cycling at different levels [21]. Based on these structural features, we have designed a new series of quinoxaline 1,4-di-N-oxide derivatives containing a nitrofuryl side chain as potential anti-neglected diseases agents. More specifically, we have designed new hybrid QDO with potential anti-tubercular activity combining some previous structural features, amide and cyano QDO substituents, and the heteroaryl retro-amide moieties. In order to determine the action of the 5nitrofuryl moiety, we synthesized another analogue series by substituting this group by a 5-nitrothienyl one, and in order to determine the influence of the nitro group, another two series were designed, furyl and thienyl side chains (Table 1). Moreover, the same designed compounds could also act as hybrid potential anti-T. cruzi agents because they are QDO that have maintained the 3-cyano and 2-NH, as retro-amide moieties, with the extra-heteroaryl substituents (5-nitrofuryl, 5-nitrothienyl

CAQDO
A name could not be generated for this structure.
The new developed CAQDOs were subjected to the following set of tests: i) determination of the MICs, in μg/mL, against M. tuberculosis H37Rv strain and, ii) determination of the percentage of growth inhibition, at 25 μM, and IC 50 values, in μM, against T. cruzi Tulahuen 2 strain (Table 1).
With regard to the anti-M. tuberculosis evaluations, the CAQDO 5 and 17 were identified as the most active derivatives against H37Rv strain, with MIC values on the same order as the reference compound, RIF (Table 1). Some structure-activity relationships could be established; in general, thienyl-derivatives are more active than furyl derivatives (cf. , with the 5-nitrothienyl-derivatives being more active than the unsubstituted ones. For this biological activity we were unable to find relationships between this and the electronic characteristics of benzo-substituent on the quinoxaline heterocycle. However, it could be pointed out, bearing in mind derivatives [29][30][31][32], that the mono-halogen substitution produce compounds more actives than the di-halogen substituted ones furthermore chlorine-substitution is better for the activity than fluorine-substitution. Considering the couple of derivatives 13 and 14, 23 and 24, 33 and 34, and 43 and 44 the 7-trifluoromethyl-substitution produces more active compounds. With regard to the anti-T. cruzi evaluations, the difluoro substituted CAQDOs 12 and 22 were identified as the most active derivatives against the Tulahuen 2 strain, with IC 50 values on the same order as the reference compound, Nfx. Moreover, compound 22 was found more active than the parent compound 3 (Scheme 1, Table 1). Similar to M. tuberculosis, the thienyl-derivatives are more active than furyl derivatives (compare anti-T. cruzi activities of 22 and 12), unlike in the case of M. tuberculosis, in which the influence of the 5-nitro substitution is clear, with the 5-nitro-substituted derivatives being less active than the un-substituted derivatives (compare anti-T. cruzi activities of 42 and 22). In T. cruzi findings, some relationships between the electronic characteristics of benzosubstituent, on the quinoxaline heterocycle, and the activity could be established; for example, when the electron-withdrawing property increases, the activity increases (compare activity of compound 10 with 8 or 7, 12 and 10, 22 and 21, or 40 and 39). The hybridization process, pharmacophore quinoxaline dioxide plus pharmacophore nitrofurane, does not produce active compounds.
a Minimum inhibitory concentration against M. tuberculosis H37Rv. b Percentage of growth inhibition at 25 μM doses in T. cruzi Tulahuen 2 strain. c NT: Not tested. d From reference [17].

General
All of the synthesized compounds were chemically characterized by thin layer chromatography (TLC), infrared (IR), proton nuclear magnetic resonance ( 1 H-NMR), mass spectra (MS) and elemental microanalyses (CHN). Alugram SIL G/UV254 (Layer: 0.2 mm) (Macherey-Nagel GmbH & Co. KG., Düren, Germany) was used for TLC and Silica gel 60 (0.040-0.063 mm, Merck) was used for Flash Column Chromatography. The 1 H-NMR spectra were recorded on a Bruker 400 Ultrashield instrument (400 MHz), using TMS as internal standard and with DMSO-d  General procedure for the synthesis of cyanoamines II Malononitrile (18.0 mmol) was added to a solution of the appropriate benzofuroxane (I, 15.0 mmol) in DMF (10 mL). The mixture was allowed to stand at 0 °C. Triethylamine was added dropwise (1.5 mL), and the reaction mixture was stirred at room temperature in darkness for 1-3 days. The precipitate was filtered off and washed by adding diethyl ether affording the target compound. The obtained red solid was used in the next step without further purification.

In vitro evaluation of antituberculosis activity
In vitro evaluation of the antituberculosis activity was carried out at the GWL Hansen's Disease Center within the Tuberculosis Antimicrobial Acquisition & Coordinating Facility (TAACF) screening program for the discovery of novel drugs for the treatment of tuberculosis. The Southern Research Institute coordinates the overall program under the direction of the U.S. National Institute of Allergy and Infectious Disease (NIAID). The purpose of the screening program is to provide a resource whereby new experimental compounds can be tested for their capability to inhibit the growth of virulent M. tuberculosis [26].

Determination of growth inhibition percentage via MABA:
The initial screen is conducted against Mycobacterium tuberculosis H37Rv (ATCC 27294) in BACTEC 12B medium using the Microplate Alamar Blue Assay (MABA) [27]. The fluorescence changes due to the reduction of Alamar blue dye during the growth of Mycobacterium were monitored by the BACTEC 460-radiometric system. Compounds effecting <90% inhibition in the primary screen (MIC >6.25 g/mL) were not further evaluated.
Determination of minimum inhibitory concentration (MIC) via MABA: Compounds demonstrating at least 90% inhibition in the primary screen were re-tested against M. tuberculosis H37Rv at lower concentrations in order to determine the actual minimum inhibitory concentration (MIC) in the MABA. The MIC was defined as the lowest concentration effecting a reduction in fluorescence of 90% relative to controls. RIF was used as the reference compound (RIF MIC = 0.015-0.125 g/mL).

In vitro evaluation of trypanocidal activity
Trypanosoma cruzi epimastigotes (Tulahuen 2 strain) were grown at 28 ºC in an axenic medium (BHI-Tryptose) as previously described [28][29][30], supplemented with 5% fetal bovine serum (FBS). Cells from a 10-day-old culture (stationary phase) were inoculated into 50 mL of fresh culture medium in order to give an initial concentration of 1  10 6 cells/mL. Cell growth was followed by measuring the absorbance of the culture at 600 nm daily. Before inoculation, the medium was supplemented with the indicated amount of the drug from a stock solution in DMSO. The final concentration of DMSO in the culture medium never exceeded 0.4%, and the control was run in the presence of 0.4% DMSO and in the absence of any drug. No effect on epimastigote growth was observed by the presence of up to 1% DMSO in the culture medium. The percentage of growth inhibition (PGI) was calculated as follows: PGI (%) = {1 -[(Ap -A0p)/(Ac -A0c)]}  100, where Ap = A 600 of the culture containing the drug at day 5; A0p = A 600 of the culture containing the drug just after addition of the inocula (day 0);Ac = A 600 of the culture in the absence of the drug (control) at day 5; A0c = A 600 in the absence of the drug at day 0. In order to determine IC 50 values, 50% inhibitory concentrations, parasite growth was observed in the absence (control) and presence of increasing concentrations of the corresponding drug. At day 5, the absorbance of the culture was measured and related to the control. The IC 50 value was considered to be the concentration of drug needed for reducing the absorbance ratio to 50%.

Conclusions
New structural modifications on the QDO skeleton were performed, and promising biological results against M. tuberculosis and T. cruzi were obtained. The biological evaluation showed a broad range of activities, thereby showing new structural hits for future chemical pharmacomodulations of QDO for the development of new drugs against tuberculosis and Chagas disease.