On the Search for Potential Antimycobacterial Drugs : Synthesis of Naphthoquinoidal , Phenazinic and 1 , 2 , 3-Triazolic Compounds and Evaluation Against Mycobacterium tuberculosis

Fifteen naphthoquinones, sixteen phenazines and fifteen aryl triazoles were synthesized and evaluated against Mycobacterium tuberculosis. Twenty five substances are reported here for the first time and, among all of the compounds evaluated, six presented MIC (minimal inhibitory concentration) values < 6.25 µg mL-1. These substances are promising antimycobacterial prototypes.


Introduction
Tuberculosis (TB), a disease caused mainly by Mycobacterium tuberculosis, is responsible for approximately 1.4 million deaths annually worldwide. 1In the absence of an effective vaccine the best way to control dissemination of TB, is by treatment, but the length of this, typically six months, is a major problem faced by patients. 2,3he increasing problem of multidrug resistance (MDR) and associated treatment failure represents a threat for the control of this disease.This is driving the necessity to discover new anti-TB drugs that are effective against active or persistent infections, as well as against sensitive or resistant strains.This, in turn, would lead to a reduction of the treatment time and would therefore reduce toxic effects in comparison with current chemotherapy. 4lthough, in recent years, a large number of molecules have been identified as potential new anti-TB drugs, only a few examples have emerged for clinical use. 5 Moreover, an attractive strategy, from economical, pharmaceutical, and clinical viewpoints, is the development of new anti-TB drugs from known molecules, especially those for which anti-TB therapeutic use has already been demonstrated to be safe and effective. 6Clofazimine, a phenazine compound, was originally reported as a potent antituberculosis agent. 7henazine-1-carboxamides have also recently been described as having potent activity against M. tuberculosis (Figure 1). 8Naphthoquinoidal compounds are considered to be privileged structures with remarkable pharmacological potential. 9Lapachol (1) and β-lapachone (2) are quinones with notable activities and are representative of a select group of bioactive substances, which, for instance, exhibit antitumor 10 and trypanocidal 11 activities, and can be used as models to obtain new drugs.Recently, our research group investigated the leishmanicidal, 12 trypanocidal, 13 and antimalarial 14 activities of a large number of substances prepared from lapachol (1) as part of our search for new potential drugs against neglected diseases. 15,16Within this context, we have described the synthesis and evaluation of quinonoid, naphthoimidazole, naphthoxazole, 17 and phenazine 18 compounds from lapachol (1) with marked activity against M. tuberculosis (Figure 1).1,2,3-Triazoles are well known for their various biological activities, including anti-TB and antifungal activity, and these arise by inhibition of cell wall synthesis. 19ecently, our group has devoted efforts to the preparation of new triazoles with potent biological activities, 15 including, for instance, β-lapachone and nor-β-lapachone-based 1,2,3-triazoles as potent trypanocidal compounds. 20,21In this context, as a continuation of our programme to develop new antimycobacterial compounds, we describe herein the synthesis of lapachone-based 1,2,3-triazoles and their respective phenazine derivatives, besides aryl triazoles.We also outline the evaluation these new derivatives against pan-susceptible M. tuberculosis H 37 Rv (ATCC 27294).
A new class of naphthoquinoidal and phenazine compounds, containing a pendant 1,2,3-triazole motif, was prepared from C-allyl lawsone (Scheme 3).The first step involved iodination to provide compound 40 using methodology described by Pinto and co-workers. 26This reaction generates two regioisomeric products (ortho and para isomers), which were separated by column chromatography; only ortho isomer 40 was used for subsequent studies.Reaction of 40 with sodium azide in dimethylformamide gave the corresponding azide 41 in high yield.CuAAC was used to prepare the new naphthoquinone-based 1,2,3-triazole 42.The reaction was accomplished using classic click chemistry conditions with phenylacetylene, CuSO  In the last few years, the synthesis and evaluation against TB of nitrophenyl-triazoles were described and these compounds exhibited remarkable activities. 27earing in mind the potential antimicrobial activity of these structures, the last class of compounds described herein was obtained by reaction of azide derivative 44 with a range of alkynes (Scheme 4).Our aim was to obtain 1,2,3-triazoles that possess an aryl-nitro group at N-1 and diverse substituted aryl or alkyl groups at C-4. Compounds 45-59 were easily obtained as crystalline solids (Scheme 4) and evaluated against M. tuberculosis H 37 Rv (ATCC 27294).Compounds 45-47 and 51-53 are described herein for the first time.][30][31][32] The structures of all novel compounds were determined by infrared (IR) and 1 H and 13 C nuclear magnetic resonance (NMR) spectroscopies.Electrospray ionization mass spectra data was also obtained.Selected compounds were recrystallised, and crystals of 12, 19, and 29 obtained were suitable for X-ray crystallographic analysis.The ORTEP-3 projections are shown in Figure 2, and Table S1 (Supplementary Information) lists the main crystallographic parameters.For compounds 27 and 42, only 1 H NMR spectra were obtained due to the low solubility of these derivatives.
Our research group has recently described quinoidal compounds with potent activity against neglected diseases, such as leishmaniasis, 12 tuberculosis, 17,18 and Chagas disease. 13,24As a continuation of our screening programme for the discovery of novel antimycobacterial compounds, we describe here the synthesis of a series of naphthoquinones and their phenazine derivatives, as well as the evaluation of these compounds against Mycobacterium tuberculosis H 37 Rv.
The structures were designed based on the activities previously reported for β-lapachone and their phenazine ( compounds presented antibacterial and antitubercular properties.Finally, by using the strategy of redox centre modification, the phenazine derivatives were prepared (Scheme 5).As discussed before, this class of compounds presents activity against M. tuberculosis and is also a subject of our study.
The first class of compounds evaluated were phenazines 5 and 11-15 and their precursors quinones 6-10 (Table 1).Low activities were observed for the phenazine derivatives, with average MIC values = 100 μg mL -1 or > 200 μg mL -1 , indicating that incorporation of the 1,2,3-triazole in the presence of the phenazine moiety was not effective from the biological point of view.A hypothesis for observation is that the presence of the 1,2,3-triazole in the C-ring of phenazines could hinder the penetration of the compound into the lipid mycobacterial membrane.In contrast, potent activities were observed for β-lapachone-based 1,2,3-triazoles 6-10, with MIC values ≤ 6.25 μg mL -1 , and Scheme 2. Synthetic route used to prepare nor-β-lapachone-based 1,2,3-triazoles and their phenazine derivatives. ( 54% 99%

78%
derivatives against H 37 Rv strain of M. tuberculosis, 34 as illustrated in the Scheme 5.The compound β-lapachone (2) presented activity against both susceptible and resistant strains of TB 34 and this molecule represents an important starting point for the synthesis of new compounds.
As recently published by our group, 15 redox centre and C-ring modifications in prototype 2 are important strategies used in the preparation of compounds with diverse biological activities.These strategies were used with success in the synthesis of anti-TB compounds from β-lapachone (2) as previously described (Scheme 5). 34ased on these principles, 1,2,3-triazole heterocyclic rings were coupled to the C-ring of lapachones (Scheme 5).The incorporation of this class of heterocyclic ring was successful in the preparation of trypanocidal and leishmanicidal compounds. 12,13,33Another important example of molecular hybridization 35 was recently reported by Pyta et al. 36 Hybrids were prepared by the coupling of triazole to 3-formylrifamycin moieties and the reported these structures were considered important prototypes to further studies against TB.Differences between the β-lapachone (dihydropyran ring) versus nor-β-lapachone derivatives (dihydrofuran ring) were also investigated.Initially, phenazines 25-30 were prepared which possess an aryl ring with either electron withdrawing or donating groups.As observed for phenazines 11-15, compounds 25-30 were also inactive, with a majority having MIC values > 200 μg mL -1 (Table 1).Finally, quinones 21, 22, and 24 showed moderate activity against TB, with MIC values of 12 μg mL -1 ; the value observed for 19 was ≤ 6.25 μg mL -1 , highlighting the potential of this structure (Table 1).
With the aim of improving bacillus membrane lipid penetration, we planned the synthesis of phenazines 35-38, which are modified with linear aliphatic substituents.Unfortunately, our strategy was not successful, and compounds 35-38 were also inactive against M. tuberculosis, with MIC values = 100 μg mL -1 .As observed for the β-lapachone derivatives, the quinones used to prepare the respective phenazines, 35-38, presented MIC values in the range of 12.5-25 μg mL -1 (Table 1).
Lapachones prepared from C-allyl lawsone (39) represent an unexplored class of substances with potential activities.Recently, we described the antitumor properties of iodinated and methylated naphthoquinones synthesized from 39. 37 Coelho et al. 34 reported that the phenazine obtained from 40 has an MIC value > 100 μg mL -1 (Scheme 6).Herein, we described the synthesis and evaluation against M. tuberculosis H 37 Rv of compound 43, obtained by C-ring modification of compound 40.Our strategy, based on the molecular hybridization by appendage of a 1,2,3-triazole, was not effective since the new phenazine 43 was also inactive (MIC > 200 μg mL -1 ) (Scheme 6).
Although the phenazines were not active against TB, the quinones described herein showed potential as effective anti-TB agents and these structures represent an important starting point for the development of new drugs.For instance, strategies for structural modification, such as molecular hybridization with potent antimycobacterial moieties, could be considered to improve the activity of the naphthoquinones 5-10, which have MIC values ≤ 6.25 μg mL -1 .

Conclusions
Twenty five novel compounds are described herein, and forty-six substances were evaluated against M. tuberculosis H 37 Rv.Among these structures, six compounds are considered to be potential antimycobacterial agents, with MIC values ≤ 6.25 μg mL -1 .These compounds were obtained from lapachol, an abundant natural product, using a simple synthetic route, representing a facile strategy to obtain anti-TB drugs.As part of our programme to identify novel drugs, the compounds described here will be modified further to obtain compounds that are more active than drugs currently used in the therapeutic fight against tuberculosis.

Chemistry
Melting points were obtained on Thomas Hoover apparatus and are uncorrected.Analytical grade solvents were used.Column chromatography was performed on silica gel (Acros Organics, 0.035-0.070mm, pore diameter ca.6 nm).Infrared spectra were recorded on a Shimadzu IR Prestige-21 Fourier transform infrared (FTIR) spectrometer. 1H and 13 C NMR spectra were recorded at room temperature using a Varian Unity Plus 300, Bruker AVANCE DPX200 and AVANCE DRX400, in the solvents indicated, with tetramethylsilane (TMS) as internal reference.Chemical shifts (d) are given in ppm and coupling constants (J) in Hertz.Mass spectra (electrospray ionization) were obtained using a MicroTOF Ic (Bruker Daltonics).For the mass spectra analysis, methanol was used as solvent.Some parameters: positive ion mode, acquisition m/z: 200.0000-700.0000,event time: 300 ms and ion accumulation: 10.00 ms.

Preparation of the synthetic precursors 1, 2, 3 and 16
Lapachol (1) was extracted from the heartwood of Tabebuia sp.(Tecoma) and purified by a series of recrystallizations.The compound β-lapachone (2) was prepared from 1, by reaction with sulfuric acid, and used to prepare the respective bromine derivative, 3,4-dibromo-β-lapachone (3), as previously reported. 20rom lapachol (1), nor-lapachol (16) was prepared in two steps using Hooker oxidation. 38nthetic procedure to prepare the phenazine compounds All the phenazines 5, 11-15, 25-30, 35-38 and 43 were prepared according to the classical methodology described by Hooker 39 from the reaction of the appropriated quinones with ortho-phenylenediamine.All compounds are described here for the first time with exception of compound 25. 33 mixture of the quinone (0.5 mmol), sodium acetate (0.95 mmol), ortho-phenylenediamine (0.55 mmol) in 3 mL of glacial acetic acid was stirred and monitored by silica gel thin-layer chromatography (TLC).After, the crude reaction product was poured into water and the precipitate formed was filtrate and then purified in silica gel chromatography, using a mixture of hexane-ethyl acetate, increasing polarity.In the case that did not form precipitate, the residue was extracted with organic solvent, dried over anhydrous sodium sulfate, followed by concentration under vacuum and purification by column chromatography.

X-ray analysis
X-ray data were collected at 150 K using MoKα (0.71073 A) on an Agilent Gemini diffractometer equipped with a charge-coupled device (CCD) area detector.The CrysAlisPro software package 40 was used for data collection and data reduction.The data were corrected empirically for absorption using spherical harmonics using the SCALE3 ABSPACK 41 scaling algorithm.The structure was solved by direct methods using SHELXS-97 42 and refined by full-matrix least squares on F 2 using SHELXL-97. 43All non-hydrogen atoms were successfully refined using anisotropic displacement parameters.Hydrogen atoms were found in the Fourier difference synthesis and fixed.

Isolates and strain preparation
The antimicrobial activity of the compounds 5-15 and 18-38 were evaluated against M. tuberculosis H 37 Rv (ATCC 27294) pan-susceptible.The bacterial suspensions were prepared in sterile water containing beads of glass of 3 mm.The suspension was homogenized by vortex agitation and the turbidity was adjusted in agreement with tube one of the scale of McFarland (3.2 × 106 cfu mL -1 ).The inoculum was prepared diluting the bacterial suspension in the proportion of 1:20 in medium 7H9 Middlebrook (Difco, Becton-Dickinson) enriched with 10% oleic acid, albumin, dextrose and catalase (OADC-BBL ® ).

Minimum inhibitory concentration determination
The resazurin microtiter assay (REMA) was performed adapting the technique proposed by Palomino et al. 44 Briefly, 200 μL of sterile water was added to all outer-perimeter wells of the sterile 96 well plates to minimize evaporation of the medium in the test wells during incubation.Hereafter, 100 μL of Middlebrook 7H9 broth (Difco Laboratories) enriched with 10% OADC was placed in each tests well of a plate, and a two-fold serial dilution of the compounds (prepared in dimethyl sulfoxide (DMSO)) was made on the plate, with range concentrations from 200 to 6.25 μg mL -1 .A volume of 100 μL of the inoculum was added to each well and incubated at 37 °C for seven days.After these, 30 μL of resazurin solution were added to each well and the plate returned to the incubator for two more days.The alteration in the oxidized state (blue color) to reduced state (pink) was scored as bacterial growth.The minimal inhibition concentration (MIC) was defined was defined as the lowest compound concentration that inhibited bacterial growth, which prevented a color change from blue to pink.

Scheme 6 .Scheme 5 .
Scheme 6. Strategy used for the design of the compound 43.

4 .
5H 2 O as catalyst and sodium ascorbate as the reducing agent, in CH 2 Cl 2 :H 2 O (1:1).The final step was the preparation of the respective phenazine by the reaction of 42 with ortho-phenylenediamine in the presence of sodium acetate and acetic acid.