On the search for potential antimycobacterial drugs: synthesis of naphthoquinoidal, phenazinic and 1,2,3-triazolic compounds and evaluation against mycobacterium tuberculosis

Abstract

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.

lapachol; phenazines; Mycobacterium tuberculosis ; triazoles

Introduction

Tuberculosis (TB), a disease caused mainly by Mycobacterium tuberculosis, is responsible for approximately 1.4 million deaths annually worldwide.¹ In 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.^2,3

The 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.⁴

Although, 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.⁷ Phenazine-1-carboxamides have also recently been described as having potent activity against M. tuberculosis (Figure 1).⁸ Naphthoquinoidal compounds are considered to be privileged structures with remarkable pharmacological potential.⁹ Lapachol (1) and β-lapachone (2) are quinones with notable activities and are representative of a select group of bioactive substances, which, for instance, exhibit antitumor¹⁰ and trypanocidal¹¹activities, and can be used as models to obtain new drugs. Recently, our research group investigated the leishmanicidal,¹²trypanocidal,¹³ and antimalarial¹⁴ activities of a large number of substances prepared from lapachol (1) as part of our search for new potential drugs against neglected diseases.^15,16 Within this context, we have described the synthesis and evaluation of quinonoid, naphthoimidazole, naphthoxazole,¹⁷ and phenazine¹⁸ compounds from lapachol (1) with marked activity against M. tuberculosis (Figure 1).

Figure 1
Clofazimine, phenazine-1-carboxamides, and examples of compounds obtained from lapachol (1) with activity against M. tuberculosis.

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.¹⁹ Recently, 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.^20,21 In 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 H₃₇Rv (ATCC 27294).

Results and Discussion

Initially, β-lapachone-based 1,2,3-triazoles 6-10 were prepared from lapachol (1) (Scheme 1). As previously described,²² lapachol (1) underwent cyclisation to β-lapachone (2) in the presence of H₂SO₄. Compound 2 was reacted with N-bromosuccinimide (NBS) in CCl₄, with benzoyl peroxide as an initiator, to produce 3,4-dibromo-β-lapachone (3).²⁰ In the next step, 3 was stirred overnight with sodium azide in dichloromethane, to prepare 4 as previously described.²⁰ Two major products were obtained: 3-bromo-4-azide-α-lapachone and 3-bromo-4-azide-β-lapachone (4). Key intermediate 4 was isolated by column chromatography and used to prepare the respective β-lapachone-based 1,2,3-triazoles 6-10.²⁰ Cu-catalyzed azide-alkyne cycloaddition (CuAAC),²³ a well-established click chemistry reaction that employs a Cu(I) source, was used to produce the desired 1,2,3-triazoles, as shown in Scheme 1. The final step involved reaction of 6-10 with ortho-phenylenediamine in the presence of sodium acetate and acetic acid to obtain the phenazine compounds 11-15. The same conditions were applied to key intermediate 4, and phenazine 5 was generated in good yield. Compounds 5-15 were obtained in racemic form, but the trans-stereochemistry was confirmed by X-ray crystallography analysis of compound 12 and by comparison with previously reported data.²⁰

Scheme 1
Synthetic route used to prepare novel phenazine compounds 5, 11-15.

The second group of compounds was obtained from 3-azido-nor-β-lapachone (17) following a previously described procedure.²⁴ Initially, 1,2,3-triazolic naphthoquinone derivatives possessing either aryl groups 19-24^13,24 or alkyl groups 31-34²⁵ were prepared (Scheme 2). To evaluate redox centre modification, the respective phenazines were prepared by the methodology described above, and novel compounds 25-30 and 35-38 were obtained in high yields (Scheme 2).

Scheme 2
Synthetic route used to prepare nor-β-lapachone-based 1,2,3-triazoles and their phenazine derivatives.

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.²⁶ This reaction generates two regioisomeric products (ortho and para isomers), which were separated by column chromatography; only ortho isomer 40was 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, CuSO₄^.5H₂O as catalyst and sodium ascorbate as the reducing agent, in CH₂Cl₂:H₂O (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.

Scheme 3
Synthesis of phenazine 43 from C-allyl lawsone (39).

In the last few years, the synthesis and evaluation against TB of nitrophenyl-triazoles were described and these compounds exhibited remarkable activities.²⁷ Bearing 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. tuberculosisH₃₇Rv (ATCC 27294). Compounds 45-47 and 51-53are described herein for the first time. Spectroscopic data for all previously published 1,2,3-triazoles 48-50 and 54-59 are in agreement with the data reported in the literature.^28-32

Scheme 4
Synthesis of 1,2,3-triazoles from azide derivative 44.

The structures of all novel compounds were determined by infrared (IR) and ¹H and ¹³C 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 ¹H NMR spectra were obtained due to the low solubility of these derivatives.

Figure 2
ORTEP-3 projections of molecules 12, 19 and 29showing the atom-numbering and displacement ellipsoids at the 20% (for 12 and 19) and 50% (29) probability level.

Our research group has recently described quinoidal compounds with potent activity against neglected diseases, such as leishmaniasis,¹² tuberculosis,^17,18 and Chagas disease.^13,24 As 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 H₃₇Rv.

The structures were designed based on the activities previously reported for β-lapachone and their phenazine derivatives against H₃₇Rv 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 TB³⁴ 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).³⁴ Based 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.^12,13,33 Another important example of molecular hybridization³⁵was recently reported by Pyta et al.³⁶ Hybrids were prepared by the coupling of triazole to 3-formylrifamycin moieties and the reported compounds presented antibacterial and antitubercular properties.

Scheme 5
Structures of the compounds evaluated against M. tuberculosis.

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

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 these structures were considered important prototypes to further studies against TB.

Table 1
MIC of the tested compounds against Mycobacterium tuberculosis H₃₇Rv

Compound	Classes of the evaluated compound	MIC / (μg mL–1)
5	Phenazine (dihydropyran ring)	> 200
6	β–Lapachone–based 1,2,3–triazole (quinone)	≤ 6.25
7	β–Lapachone–based 1,2,3–triazole (quinone)	≤ 6.25
8	β–Lapachone–based 1,2,3–triazole (quinone)	≤ 6.25
9	β–Lapachone–based 1,2,3–triazole (quinone)	≤ 6.25
10	β–Lapachone–based 1,2,3–triazole (quinone)	≤ 6.25
11	Phenazine (dihydropyran ring)	100
12	Phenazine (dihydropyran ring)	> 200
13	Phenazine (dihydropyran ring)	> 200
14	Phenazine (dihydropyran ring)	> 200
15	Phenazine (dihydropyran ring)	> 200
18	Phenazine (dihydrofuran ring)	25
19	nor–β–Lapachone–based 1,2,3–triazole (quinone)	≤ 6.25
20	nor–β–Lapachone–based 1,2,3–triazole (quinone)	nd
21	nor–β–Lapachone–based 1,2,3–triazole (quinone)	12.5
22	nor–β–Lapachone–based 1,2,3–triazole (quinone)	12.5
23	nor–β–Lapachone–based 1,2,3–triazole (quinone)	nd
24	nor–β–Lapachone–based 1,2,3–triazole (quinone)	12.5
25	Phenazine (dihydrofuran ring)	100
26	Phenazine (dihydrofuran ring)	> 200
27	Phenazine (dihydrofuran ring)	> 200
28	Phenazine (dihydrofuran ring)	> 200
29	Phenazine (dihydrofuran ring)	> 200
30	Phenazine (dihydrofuran ring)	100
31	nor–β–Lapachone–based 1,2,3–triazole (quinone)	25
32	nor–β–Lapachone–based 1,2,3–triazole (quinone)	25
33	nor–β–lapachone–based 1,2,3–triazole (Quinone)	12.5
34	nor–β–Lapachone–based 1,2,3–triazole (quinone)	12.5
35	Phenazine (dihydrofuran ring)	100
36	Phenazine (dihydrofuran ring)	100
37	Phenazine (dihydrofuran ring)	100
38	Phenazine (dihydrofuran ring)	100
43	Phenazine (dihydrofuran ring)	> 200
45	Nitrophenyl triazole	> 200
46	Nitrophenyl triazole	> 200
47	Nitrophenyl triazole	> 200
48	Nitrophenyl triazole	> 200
49	Nitrophenyl triazole	> 200
50	Nitrophenyl triazole	> 200
51	Nitrophenyl triazole	200
52	Nitrophenyl triazole	200
53	Nitrophenyl triazole	200
54	Nitrophenyl triazole	100
55	Nitrophenyl triazole	100
56	Nitrophenyl triazole	50
57	Nitrophenyl triazole	> 200
58	Nitrophenyl triazole	> 200
59	Nitrophenyl triazole	100
Etambutol		4
Streptomycin		1

• nd: not determined.

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 24showed 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).

In the case of naphthoquinoidal compounds, the importance of the dihydropyran ring for biological activitity was highlighted, as compounds 6-10 (MIC value < 6.25 µg mL^-1) showed generally stronger activities compared to the respective nor-β-lapachone derivatives 21, 22, 24, 33 and 34, with MIC values < 12.5 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 H₃₇Rv 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.

Finally, we also undertook the pharmacological evaluation of 1,2,3-triazoles (45-59), which do not possess a quinoidal system. Unfortunately, these compounds were inactive with MIC values > 100 µg mL^-1 (Table 1).

Conclusions

Twenty five novel compounds are described herein, and forty-six substances were evaluated against M. tuberculosis H₃₇Rv. 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.

Experimental

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, 0.035-0.070 mm, pore diameter ca. 6 nm). Infrared spectra were recorded on a Shimadzu IR Prestige-21 Fourier transform infrared (FTIR) spectrometer. ¹H and ¹³C 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 (δ) 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.²⁰ From lapachol (1), nor-lapachol (16) was prepared in two steps using Hooker oxidation.³⁸

Synthetic 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 Hooker³⁹ 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 A 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.

1-Azido-2-bromo-3,3-dimethyl-2,3-dihydro-1H-benzo[a] pyrano[2,3-c]phenazine (5)

The reaction of compound 4 (181 mg, 0.5 mmol), ortho-phenylenediamine (60 mg, 0.55 mmol) in the presence of sodium acetate (78 mg, 0.95 mmol) in 3 mL of acetic acid yielded product 5, (167 mg, 0.360 mmol, 72% yield), as a yellow solid; mp 154-156 ºC; IR (KBr) ν / cm^-1 2102 (N₃), 1633 (C=N), 3061 (C=C), 760 (Ar-CH₃), 2975 (C-H); ¹H NMR (200 MHz, CDCl₃) δ 9.36 (d, 1H, J 7.2 Hz), 8.43-8.19 (3H, m), 7.94-7.72 (4H, m), 5.80 (d, 1H, J 5.5 Hz), 4.48 (d, 1H, J 5.5 Hz), 2.08 (3H, s), 1.80 (3H, m); ¹³C NMR (50 MHz, CDCl₃) δ 143.2, 142.1, 140.8, 140.6, 140.0, 131.6, 129.8, 129.4, 129.8, 128.8, 125.2, 122.0, 107.9, 107.3, 83.2, 79.0, 66.9, 61.4, 57.0, 26.2, 24.2; ESI-MS m/z, calcd. for [C₂₁H₁₆BrN₅OH⁺]: 434.0616; found: 434.1029.

2-Bromo-3,3-dimethyl-1-(4-phenyl-1H-1,2,3-triazol-1-yl)-2,3-dihydro-1H-benzo[a]pyrano[2,3-c]phenazine (11)

The reaction of compound 6 (232 mg, 0.5 mmol), ortho-phenylenediamine (60 mg, 0.55 mmol) in the presence of sodium acetate (78 mg, 0.95 mmol) in 3 mL of acetic acid yielded product 11, (219 mg, 0.410 mmol, 82% yield), as a yellow solid; mp 249-250 ºC; IR (KBr) ν / cm^-1 1603 (C=N), 3133 (C=C), 760 (Ar-CH₃), 2912 (C-H); ¹H NMR (200 MHz, CDCl₃) δ 9.26 (d, 1H, J 6.5 Hz), 8.30 (d, 1H, J 8.8 Hz), 8.17-8.12 (1H, m), 8.08 (1H, s), 7.89-7.71 (5H, m), 7.67-7.51 (2H, m), 7.34-7.18 (3H, m), 6.60 (d, 1H, J 8.1 Hz), 5.16 (d, 1H, J 8.1 Hz), 1.70 (3H, s), 1.65 (3H, s); ¹³C NMR (50 MHz, CDCl₃) δ 153.2, 146.5, 142.3, 142.1, 140.7, 140.2, 132.2, 131.0, 130.3, 130.2, 129.7, 129.6, 129.3, 128.9, 128.8, 128.1, 125.9, 125.5, 123.4, 122.6, 106.6, 80.8, 61.2, 57.2, 27.7, 21.3; ESI-MS m/z, calcd. for [C₂₉H₂₂BrN₅OH⁺]: 536.10861; found: 536.1193.

2-Bromo-1-(4-(4-bromophenyl)-1H-1,2,3-triazol-1-yl)-3,3-dimethyl-2,3-dihydro-1H-benzo[a]pyrano[2,3-c]phenazine (12)

The reaction of compound 7 (271 mg, 0.5 mmol), ortho-phenylenediamine (60 mg, 0.55 mmol) in the presence of sodium acetate (78 mg, 0.95 mmol) in 3 mL of acetic acid yielded product 12, (201 mg, 0.375 mmol, 75% yield), as a yellow solid; mp 232-235 ºC; IR (KBr) ν / cm^-1 1633 (C=N), 3124 (C=C), 773 (Ar-CH₃), 2922 (C-H); ¹H NMR (200 MHz, CDCl₃) δ 9.35 (d, 1H, J 9.1 Hz), 8.41-8.36 (1H, m), 8.29-8.20 (1H, m), 8.19 (1H, s), 7.93-7.78 (3H, m), 7.75-7.62 (4H, m), 7.57-7.46 (2H, m), 6.66 (d, 1H, J 8.3 Hz), 5.21 (d, 1H, J 8.3 Hz), 1.80 (3H, s), 1.73 (3H, s); ¹³C NMR (50 MHz, CDCl₃) δ 154.2, 146.5, 143.3, 143.2, 141.7, 141.2, 133.2, 131.5, 131.3, 130.9, 130.8, 130.7, 130.3, 129.8, 129.1, 128.5, 126.7, 124.5, 123.8, 122.9, 107.6, 81.9, 62.4, 58.3, 28.8, 22.7.

2-Bromo-1-(4-(4-fluorophenyl)-1H-1,2,3-triazol-1-yl)-3,3-dimethyl-2,3-dihydro-1H-benzo[a]pyrano[2,3-c]phenazine (13)

The reaction of compound 8 (241 mg, 0.5 mmol), ortho-phenylenediamine (60 mg, 0.5 mmol) in the presence of sodium acetate (78 mg, 0.95 mmol) in 3 mL of acetic acid yielded product 13, (216 mg, 0.390 mmol, 78% yield), as a yellow solid; mp 263-264 ºC; IR (KBr) ν / cm^-1 1484 (C=N), 2915 (C=C-H), 776 (Ar-CH₃), 3127 (C-H);¹H NMR (400 MHz, CDCl₃/DMSO) δ 9.34 (d, 1H, J 9.0 Hz), 8.39 (d, 1H, J 9.0 Hz), 8.24 (1H, s), 8.22 (d, 1H, J 7.8 Hz), 7.89-7.81 (3H, m), 7.80-7.67 (4H, m), 7.07 (t, 2H, J 8.6 Hz), 6.67 (d, 1H, J 8.5 Hz), 5.20 (d, 1H, J 8.4 Hz), 1.82 (3H, s), 1.73 (3H, s); ¹³C NMR (100 MHz, CDCl₃/DMSO) δ 161.2 (246.5 Hz, d) 152.9, 145.3, 142.0, 141.8, 140.4, 139.9, 131.8, 130.1, 130.0, 129.6, 129.4, 129.1, 128.5, 127.8, 127.3 (J 8.0 Hz, d), 127.1 (J 3.0 Hz, d), 125.2, 123.2, 122.4, 115.7 (J 21.6 Hz, d), 106.5, 80.8, 61.0, 57.2, 27.7, 20.7; ESI-MS m/z, calcd. for [C₂₉H₂₁BrFN₅OH⁺]: 554.0991; found: 554.1006.

2-Bromo-1-(4-(4-methoxyphenyl)-1H-1,2,3-triazol-1-yl)-3,3-dimethyl-2,3-dihydro-1H-benzo[a]pyrano[2,3-c]phenazine (14)

The reaction of compound 9 (247 mg, 0.5 mmol), ortho-phenylenediamine (60 mg, 0.5 mmol) in the presence of sodium acetate (78 mg, 0.95 mmol) in 3 mL of acetic acid yielded product 14, (232 mg, 0.410 mmol, 82% yield), as a yellow solid; mp 248-249 ºC; IR (KBr) ν / cm^-1 1494 (C=N), 2981 (C=C), 1252 (Ar-O-CH₃), 3124 (C-H); ¹H NMR (400 MHz, CDCl₃/DMSO) δ 9.30 (d, 1H, J 6.1 Hz), 8.36 (d, 1H, J 7.2 Hz), 8.19-8.16 (1H, m), 8.16 (1H, s), 7.85-7.81 (3H, m), 7.73-7.63 (4H, m), 6.90 (d, 2H, J 8.6 Hz), 6.65 (d, 1H, J 8.3 Hz), 5.20 (d, 1H, J 8.3 Hz), 3.78 (3H, s), 1.78 (3H, s), 1.71 (3H, s); ¹³C NMR (100 MHz, CDCl₃/DMSO) δ 159.6, 153.0, 149.8, 146.3, 142.3, 142.1, 140.6, 140.1, 136.4, 130.0, 130.2, 129.7, 129.6, 129.2, 128.7, 128.1, 127.1, 125.5, 124.0, 123.7, 123.4, 122.1, 114.4, 106.8, 80.9, 61.1, 57.5, 55.5, 27.8, 21.1; ESI-MS m/z, calcd. for [C₃₀H₂₄O₂BrN₅H⁺]: 566.1299; found: 566.1226.

2-Bromo-3,3-dimethyl-1-(4-(p-tolyl)-1H-1,2,3-triazol-1-yl)-2,3-dihydro-1H-benzo[a]pyrano[2,3-c]phenazine (15)

The reaction of compound 10 (239 mg, 0.5 mmol), ortho-phenylenediamine (60 mg, 0.5 mmol) in the presence of sodium acetate (78 mg, 0.5 mmol) in 3 mL of acetic acid yielded product 15, (211 mg, 0.385 mmol, 77% yield), as a yellow solid; mp 246-248 ºC; IR (KBr) ν / cm^-1 1418 (C=N), 2915 (C=C), 770 (Ar-CH₃), 3127 (C-H);¹HNMR (400 MHz, CDCl₃/DMSO) δ 9.33 (d, 1H, J 8.5 Hz), 8.38 (d, 1H, J 7.0 Hz), 8.21 (d, 1H, J 7.6 Hz), 8.16 (1H, s), 7.94-7.81 (3H, m), 7.73-7.68 (4H, m), 7.18 (d, 2H, J 8.0 Hz), 6.65 (d, 1H, J 8.3 Hz), 5.21 (d, 1H, J 8.3 Hz), 2.34 (3H, s), 1.78 (3H, s), 1.72 (3H, s); ¹³C NMR (100 MHz, CDCl₃/DMSO) δ 153.1, 146.6, 142.3, 142.1, 140,7, 140.1, 137.9, 132.1, 130.3, 130.3, 129.7, 129.6, 129.3, 128.8, 128.2, 128.1, 125.8, 125.5, 123.4, 122.4, 106.8, 80.9, 61.2, 57.4, 27.8, 21.5, 21.1; ESI-MS m/z, calcd. for [C₃₀H₂₄BrN₅OH⁺]: 550.1242; found: 550.1207.

1-(4-(4-Fluorophenyl)-1H-1,2,3-triazol-1-yl)-2,2-dimethyl-1,2-dihydrobenzo[a]furo[2,3-c]phenazine (26)

The reaction of compound 20 (194 mg, 0.5 mmol), ortho-phenylenediamine (60 mg, 0.5 mmol) in the presence of sodium acetate (78 mg, 0.5 mmol) in 3 mL of acetic acid yielded product 26, (189 mg, 0.410 mmol, 82% yield), as a yellow solid; mp 291-293 ºC; IR (KBr) ν / cm^-1 1596 (C=N), 3091 (C=C), 1428 (C-F), 3412 (C-H); ¹H NMR (400 MHz, CDCl₃) δ 9.45 (d, 1H, J 8.0 Hz), 8.29-8.24 (1H, m), 8.21 (d, 1H, J 7.7 Hz), 8.03-7.98 (1H, m), 7.93 (t, 1H, J 7.5 Hz), 7.86 (t, 1H, J 7.2 Hz), 7.76-7.71 (2H, m), 7.65-7.59 (2H, m), 7.28 (1H, s), 6.95 (t, 2H, J8.5 Hz), 6.70 (1H, s), 1.83 (3H, s), 1.36 (3H, s); ¹³C NMR (100 MHz, CDCl₃) δ 162.5 (d, J 246.7 Hz), 160.2, 146.5, 142.8, 141.6, 140.9, 140.5, 133.4, 130.3, 130.0 (d, J 6.8 Hz), 129.7, 129.0, 128.8, 127.4, 127.3, 126.7 (d, J 3.2 Hz), 126.2, 124.1, 123.2, 118.6, 115.5 (d, J 21.7 Hz), 108.8, 92.8, 68.7, 27.9, 21.5; ESI-MS m/z, calcd. for [C₂₈H₂₀FN₅OH⁺]: 462.1730; found: 462.1697.

1-(4-(4-Bromophenyl)-1H-1,2,3-triazol-1-yl)-2,2-dimethyl-1,2-dihydrobenzo[a]furo[2,3-c]phenazine (27)

The reaction of compound 21 (225 mg, 0.5 mmol), ortho-phenylenediamine (60 mg, 0.5 mmol) in the presence of sodium acetate (78 mg, 0.5 mmol) in 3 mL of acetic acid yielded product 27, (177 mg, 0.340 mmol, 68% yield), as a yellow solid; mp 297-299 ºC; IR (KBr) ν / cm^-1 1596 (C=N), 2988 (C=C), 1047 (C-Br), 3081 (C-H); ¹H NMR (400 MHz, CDCl₃) δ 9.48 (d, 1H, J 7.5 Hz), 8.29 (d, 1H, J 5.1 Hz), 8.23 (d, 1H, J 7.7 Hz), 8.11-8.06 (1H, m), 7.93 (dt, 2H, J 26.2, 7.2 Hz), 7.82-7.70 (2H, m), 7.55 (d, 2H, J 8.3 Hz), 7.41 (d, 2H, J 8.3 Hz), 7.33 (1H, s), 6.72 (1H, s), 1.83 (3H, s), 1.35 (3H, s). ESI-MS m/z, calcd. for [C₂₈H₂₀BrN₅OH⁺]: 522.0798; found: 522.0800.

2,2-Dimethyl-1-(4-(4-nitrophenyl)-1H-1,2,3-triazol-1-yl)-1,2-dihydrobenzo[a]furo[2,3-c]phenazine (28)

The reaction of compound 22 (208 mg, 0.5 mmol), ortho-phenylenediamine (60 mg, 0.5 mmol) in the presence of sodium acetate (78 mg, 0.5 mmol) in 3 mL of acetic acid yielded product 28, (158 mg, 0.325 mmol, 65% yield), as a yellow solid; mp 289-291 ºC; IR (KBr) ν / cm^-1 1336 (NO₂), 1517 (C-NO₂), 3073 (C=C), 756 (Ar-NO₂), 3412 (C-H); ¹H NMR (400 MHz, CDCl₃) δ 9.46 (d, 1H, J 8.3 Hz), 8.28-7.84 (8H, m), 7.78-7.58 (3H, m), 7.48 (1H, s), 6.76 (1H, s), 1.38 (3H, s), 1.27 (3H, s); ¹³C NMR (100 MHz, CDCl₃/DMSO) δ 160.2, 146.8, 144.8, 142.4, 140.2, 129.8, 129.5, 128.6, 128.5, 128.5, 126.0, 125.9, 123.9, 123.8, 123.1, 113.8, 108.3, 92.5, 92.5, 68.7, 68.4, 55.0, 29.4, 27.4, 21.4; ESI-MS m/z, calcd. for [C₂₈H₂₀N₆O₃H⁺]: 489.1675; found: 489.1655.

1-(4-(4-Methoxyphenyl)-1H-1,2,3-triazol-1-yl)-2,2-dimethyl-1,2-dihydrobenzo[a]furo[2,3-c]phenazine (29)

The reaction of compound 23 (200 mg, 0.5 mmol), ortho-phenylenediamine (60 mg, 0.5 mmol) in the presence of sodium acetate (78 mg, 0.5 mmol) in 3 mL of acetic acid yielded product 29, (184 mg, 0.39 mmol, 78% yield), as a yellow solid; mp 269-270 ºC; IR (KBr) ν / cm^-1 1597 (C=N), 3094 (C=C), 1247 (Ar-O-CH₃), 3413 (C-H); ¹H NMR (400 MHz, CDCl₃) δ 9.46 (d, 1H, J 8.0 Hz), 8.34-8.19 (2H, m), 8.08-7.84 (4H, m), 7.80-7.70 (2H, m), 7.66-7.54 (2H, m), 6.81 (d, 2H, J 8.5 Hz), 6.72 (1H, s), 3.74 (3H, s), 1.83 (3H, s), 1.36 (3H, s); ¹³C NMR (100 MHz, CDCl₃) δ 160.2, 159.4, 147.2, 142.6, 141.6, 140.9, 140.4, 130.2, 129.9, 129.8, 129.6, 128.9, 128.8, 126.9, 126.2, 124.1, 123.2, 118.0, 114.0, 108.9, 92.9, 68.6, 55.2, 27.9, 21.5; ESI-MS m/z, calcd. for [C₂₉H₂₃N₅O₂H⁺]: 474.1930; found: 474.1897.

2,2-Dimethyl-1-(4-(p-tolyl)-1H-1,2,3-triazol-1-yl)-1,2-dihydrobenzo[a]furo[2,3-c]phenazine (30)

The reaction of compound 24 (192 mg, 0.5 mmol), ortho-phenylenediamine (60 mg, 0.5 mmol) in the presence of sodium acetate (78 mg, 0.5 mmol) in 3 mL of acetic acid yielded product 30, (164 mg, 0.360 mmol, 72% yield), as a yellow solid; mp 269-271 ºC; IR (KBr) ν / cm^-1 1633 (C=N), 3095 (C=C), 756 (Ar-CH3), 3413 (C-H); ¹H NMR (400 MHz, CDCl₃) δ 9.48 (d, 1H, J 6.9 Hz), 8.30-8.21 (2H, m), 8.08-7.83 (3H, m), 7.79-7.69 (2H, m), 7.62-7.51 (2H, m), 7.31 (1H, s), 7.08 (d, 2H, J 6.3 Hz), 6.72 (1H, s), 2.28 (3H, s), 1.83 (3H, s), 1.35 (3H, s); ¹³C NMR (100 MHz, CDCl₃) δ 147.4, 142.7, 141.6, 140.8, 140.4, 137.8, 133.3, 130.2, 129.9, 129.8, 129.6, 129.3, 129.0, 128.8, 127.7, 126.2, 125.4, 124.1, 123.2, 118.5, 108.8, 92.8, 68.7, 29.7, 27.5, 21.5, 21.1; ESI-MS m/z, calcd. for [C₂₉H₂₃N₅OH⁺]: 458.1902; found: 458.1950.

2,2-Dimethyl-1-(4-propyl-1H-1,2,3-triazol-1-yl)-1,2-dihydrobenzo[a]furo[2,3-c]phenazine (35)

The reaction of compound 31 (168 mg, 0.5 mmol), ortho-phenylenediamine (60 mg, 0.5 mmol) in the presence of sodium acetate (78 mg, 0.5 mmol) in 3 mL of acetic acid yielded product 35, (147 mg, 0.360 mmol, 72% yield), as a yellow solid; mp 278-280 ºC; IR (KBr) ν / cm^-1 1596 (C=N), 2925 (C=C), 753 (Ar-CH₃), 3107 (C-H); ¹H NMR (400 MHz, CDCl₃) δ 9.44 (d, 1H, J 6.5 Hz), 8.32-8.24 (1H, m), 8.21 (d, 1H, J 6.6 Hz), 8.04-7.84 (3H, m), 7.80-7.72 (2H, m), 6.98 (1H, s), 6.62 (1H, s), 2.61-2.48 (2H, m), 1.81 (3H, s), 1.55 (dt, 2H, J 14.7, 7.3 Hz), 1.29 (3H, s), 0.80 (t, 3H, J 7.3 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 159.5, 147.1, 142.1, 141.2, 140.4, 139.8, 132.6, 129.7, 129.5, 129.3, 129.2, 128.2, 128.2, 125.6, 123.6, 122.7, 119.9, 108.4, 92.2, 67.9, 27.1, 27.0, 21.9, 20.8, 13.0. ESI-MS m/z, calcd. for [C₂₅H₂₃N₅OH⁺]: 410.1980; found: 410.1897.

1-(4-Butyl-1H-1,2,3-triazol-1-yl)-2,2-dimethyl-1,2-dihydrobenzo[a]furo[2,3-c]phenazine (36)

The reaction of compound 31 (175 mg, 0.5 mmol), ortho-phenylenediamine (60 mg, 0.5 mmol) in the presence of sodium acetate (78 mg, 0.5 mmol) in 3 mL of acetic acid yielded product 36, (154 mg, 0.36 mmol, 73% yield), as a yellow solid; mp 269-270 ºC; IR (KBr) ν / cm^-1 1596 (C=N), 2955 (C=C), 763 (Ar-CH₃), 3071 (C-H);¹HNMR (400 MHz, CDCl₃) δ 9.43 (d, 1H, J 7.9 Hz), 8.28 (dd, 1H, J 3.5, 7.3 Hz), 8.21 (d, 1H, J 7.6 Hz), 8.00-7.87 (3H, m), 7.81-7.76 (2H, m), 7.05 (s, 1H), 6.61 (s, 1H), 2.65-2.51 (2H, m), 1.82 (3H, s), 1.57-1.44 (2H, m), 1.29 (3H, s), 1.21 (2H, m), 0.79 (t, 3H, J 7.3 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 159.5, 147.2, 142.0, 141.2, 140.3, 139.7, 132.5, 129.7, 129.5, 129.3, 129.1, 128.2, 123.6, 122.7, 125.6, 125.4, 119.9, 108.6, 92.1, 67.2, 30.8, 27.1, 24.7, 21.5, 20.8, 13.2; ESI-MS m/z, calcd. for [C₂₆H₂₅N₅OH⁺]: 424.2137; found: 424.2099.

2,2-Dimethyl-1-(4-pentyl-1H-1,2,3-triazol-1-yl)-1,2-dihydrobenzo[a]furo[2,3-c]phenazine (37)

The reaction of compound 33 (182 mg, 0.5 mmol), ortho-phenylenediamine (60 mg, 0.5 mmol) in the presence of sodium acetate (78 mg, 0.5 mmol) in 3 mL of acetic acid yielded product 37, (185 mg, 0.425 mmol, 85% yield), as a yellow solid; mp 261-263 ºC; IR (KBr) ν / cm^-1 1597 (C=N), 2958 (C=C), 760 (Ar-CH₃), 3072 (C-H);¹HNMR (400 MHz, CDCl₃) δ 9.43 (d, 1H, J 7.8 Hz), 8.28 (dd, 1H, J 6.6, 3.1 Hz), 8.21 (d, 1H, J 7.6 Hz), 8.05-7.84 (3H, m), 7.77 (dd, 2H, J 6.6, 3.1 Hz), 7.01 (1H, s), 6.62 (1H, s), 2.67-2.47 (2H, m), 1.82 (3H, s), 1.63-1.43 (2H, m), 1.30 (3H, s), 1.24-1.11 (4H, m), 0.75 (t, 3H, J 6.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 159.8, 147.6, 142.3, 141.5, 140.6, 140.0, 132.9, 129.9, 129.8, 129.6, 129.4, 128.5, 125.9, 125.8, 123.9, 123.0, 120.2, 108.7, 92.5, 68.2, 30.9, 28.6, 27.4, 25.3, 21.9, 21.1, 13.7; ESI-MS m/z, calcd. for [C₂₇H₂₇N₅OH⁺]: 438.2293; found: 438.2210.

1-(4-Hexyl-1H-1,2,3-triazol-1-yl)-2,2-dimethyl-1,2-dihydrobenzo[a]furo[2,3-c]phenazine (38)

The reaction of compound 34 (189 mg, 0.5 mmol), ortho-phenylenediamine (60 mg, 0.5 mmol) in the presence of sodium acetate (78 mg, 0.5 mmol) in 3 mL of acetic acid yielded product 38, (182 mg, 0.405 mmol, 81% yield), as a yellow solid; mp 259-261 ºC; IR (KBr) ν / cm^-1 1612 (C=N), 2955 (C=C), 763 (Ar-CH₃), 3071 (C-H);¹HNMR (400 MHz, CDCl₃) δ 9.45 (d, 1H, J 7.8 Hz), 8.33-8.24 (1H, m), 8.20 (d, 1H, J 7.5 Hz), 8.04 (dd, 1H, J 6.2, 3.6 Hz), 7.90 (dt, 2H, J24.8, 7.0 Hz), 7.76 (dd, 2H, J 6.5, 3.4 Hz), 6.84 (1H, s), 6.64 (1H, s), 2.68-2.44 (2H, m), 1.79 (3H, s), 1.53-1.42 (2H, m), 1.28 (3H, s), 1.19-1.06 (6H, m), 0.73 (t, 3H, J 6.5 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 160.0, 148.0, 142.7, 141.7, 140.8, 140.4, 133.2, 130.1, 129.9, 129.7, 129.6, 128.9, 128.7, 126.1, 124.1, 123.2, 119.9, 109.0, 92.8, 68.4, 31.3, 29.1, 28.6, 27.5, 25.6, 22.3, 21.3, 13.8; ESI-MS m/z, calcd. for [C₂₈H₂₉N₅OH⁺]: 452.2450; found: 452.2400.

2-((4-Phenyl-1H-1,2,3-triazol-1-yl)methyl)-1,2-dihydrobenzo[a]furo[2,3-c]phenazine (43)

The reaction of compound 42 (80 mg, 0.29 mmol), ortho-phenylenediamine (60 mg, 0.55 mmol) in the presence of sodium acetate (100 mg, 1.22 mmol) in 4 mL of acetic acid yielded product 43, (88.5 mg, 0.206 mmol, 71% yield), as a yellow solid; mp 211-213 ºC; IR (KBr) ν / cm^-1 1638 (C=N), 763 (Ar-CH₃), 2924 (C-H); ¹H NMR (400 MHz, CDCl₃) δ 9.44-9.37 (1H, m), 8.33 (dd, 1H, J 8.1, 1.4 Hz), 8.28 (d, 1H, J 7.5 Hz), 8.12-8.04 (1H, m), 8.01 (1H, s), 7.90-7.76 (6H, m), 7.42 (t, 2H, J7.4 Hz), 7.33 (t, 1H, J 7.4 Hz), 5-75-5.66 (1H, m), 4.97 (dd, 1H, J 14.5, 3.6 Hz), 4.83 (dd, 1H, J 14.5, 7.6 Hz), 4.06 (dd, 1H, J 15.9, 10.0 Hz), 3.67 (dd, 1H, J15.9, 6.8 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 163.00, 148.19, 141.39, 140.34, 131.91, 130.69, 130.46, 130.19, 130.16, 130.14, 130.10, 129.92, 128.91, 128.87, 128.82, 128.75, 128.74, 128.27, 126.21, 125.84, 122.10, 120.72, 83.15, 54.13, 32.06; ESI-MS m/z, calcd. for [C₂₇H₁₉N₅OH⁺]: 430.1667; found: 430.1598.

2-(Azidomethyl)-2,3-dihydronaphtho[1,2-b]furan-4,5-dione (41)

Compound 40 (255 mg, 1.0 mmol) was stirred in presence of sodium azide (120 mg, 1.85 mmol) in 2 mL of dimethylformamide (DMF), generating product 41, (238 mg, 0.930 mmol, 93% yield), as a red solid; mp 172-174 ºC; IR (KBr) ν / cm^-1 2923 (CH), 2106 (N₃), 1660 (C=O); ¹H NMR (200 MHz, CDCl₃) δ 8.05 (d, 1H, J 7.3 Hz), 7.92-7.32 (3H, m), 5.45-5.21 (1H, m), 3.84-3.49 (2H, m), 3.25 (dd, 1H, J 15.6, 10.1 Hz), 2.92 (dd, 1H, J15.6, 7.0 Hz); ¹³C NMR (50 MHz, CDCl₃) δ 180.9, 175.4, 169.4, 134.9, 132.3, 130.7, 130.3, 129.7, 127.2, 124.7, 85.8, 54.3, 29.6; ESI-MS m/z, calcd. for [C₁₃H₉N₃O₃H⁺]: 256.0722; found: 256.0606.

Synthetic procedure to prepare quinone-based 1,2,3-triazoles

The substituted alkyne (0.83 mmol) was reacted with respective azide compound (0.83 mmol) in 12 mL of CH₂Cl₂/H₂O (1:1), CuSO₄^.5H₂O (9.3 mg, 0.04 mmol) and sodium ascorbate (22 mg, 0.11 mmol). The mixture was agitated at room temperature until formation of the product was complete and monitored by TLC. The organic phase was extracted with CH₂Cl₂, dried over sodium sulfate and concentrated under reduced pressure. The resulting residue was purified by column chromatography on silica gel using a gradient mixture of hexane/ethyl acetate with increasing polarity up to 100% ethyl acetate as an eluent. Compounds 6-9, 19-24 and 32-34 assayed against M. tuberculosis here were previously described.^12,13,20,25

3-Bromo-2,2-dimethyl-4-(4-(p-tolyl)-1H-1,2,3-triazol-1-yl)-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (10)

The alkyne employed was 1-ethynyl-4-methylbenzene (96 mg, 0.83 mmol) with the azide 4 (300 mg, 0.83 mmol) and the resulting compound 10 was a yellow solid (217 mg, 55% yield); mp 201-202 ºC; IR (KBr) ν / cm^-1 1650 (C=O), 1605 (C=O); ¹H NMR (200 MHz, CDCl₃) δ 8.11 (d, 1H, J 7.4 Hz), 8.04 (s, 1H), 7.93 (d, 1H, J 7.7 Hz), 7.76-7.71 (m, 3H), 7.65-7.61 (m, 1H), 7.27-7,20 (m, 1H), 5.71 (d, 1H J 8.8 Hz), 5.07 (d, 1H, J 8.9 Hz), 2.37 (s, 3H), 1.76 (s, 3H), 1.69 (s, 3H); ¹³C NMR (50 MHz, CDCl₃) δ 177.6, 176.3, 162.4, 146.6, 137.6, 134.9, 132.0, 130.4, 130.3, 129.1, 128.9, 127.3, 125.5, 124.9, 122.1, 110.2, 83.2, 58.7, 53.9, 27.12, 20.9, 20.5; ESI-MS m/z, calcd. for [C₂₄H₂₀O₃BrN₃H⁺]: 478.0766; found: 478.0579.

2,2-Dimethyl-3-(4-propyl-1H-1,2,3-triazol-1-yl)-2,3-dihydronaphtho[1,2-b]furan-4,5-dione (31)

The alkyne employed was pent-1-yne (56 mg, 0.83 mmol), with the azide 17(223 mg, 0.83 mmol) and the resulting compound 31 was a brown solid (260 mg, 93% yield); mp 173-176 ºC; IR (KBr) ν / cm^-1 1635 (C=O), 1678 (C=O); ¹H NMR (200 MHz, CDCl₃) δ 8.12-7.97 (1H, m), 7.93-7.45 (4H, m), 5.86 (1H, s), 2.55 (t, 2H, J 7.0 Hz), 1.67 (3H, s), 1.61-1.49 (2H, m), 1.09 (3H, s), 0.82 (t, 3H, J 7.1 Hz); ¹³C NMR (50 MHz, CDCl₃) δ 180.2, 174.6, 171.1, 148.0, 134.9, 133.2, 131.4, 129.7, 126.7, 125.6, 120.5, 111.4, 96.0, 66.6, 27.6, 27.5 22.5, 20.9, 13.7; ESI-MS m/z, calcd. for [C₁₉H₁₉N₃O₃H⁺]: 338.1504; found: 338.1517.

2-((4-Phenyl-1H-1,2,3-triazol-1-yl)methyl)-2,3-dihydronaphtho[1,2-b]furan-4,5-dione (42)

The alkyne employed was ethynylbenzene (84 mg, 0.83 mmol), with the azide 41 (211 mg, 0.83 mmol) and the resulting compound 42 was a red solid (281 mg, 95% yield); mp 187-188 ºC; IR (KBr) ν / cm^-12921 (C-H), 1621 (C=O), 1648 (C=O); ¹H NMR (400 MHz, CDCl₃) δ 8.10 (d, 1H, J 7.3 Hz), 8.06-7.92 (1H, m), 7.91-7.77 (2H, m), 7.71-7.65 (1H, m), 7.61 (t, 2H, J 7.1 Hz), 7.49-7.41 (2H, m), 7.40-7.34 (1H, m), 5.64-5.53 (1H, m), 4.86 (dd, 1H, J 14.1, 2.7 Hz), 4.77 (dd, 1H, J 14.1, 7.0 Hz), 3.40 (dd, 1H, J15.6, 10.1 Hz), 3.03 (dd, 1H, J 15.6, 6.9 Hz); ESI-MS m/z, calcd. for [C₂₁H₁₅N₃O₃H⁺]: 358.1191; found: 358.1092.

Synthetic procedure to prepare 1,2,3-triazoles (45-59)

A mixture of m-NO₂-aryl-azide 44 (0.6 mmol), the appropriate alkynes (0.9 mmol), copper iodide (I) (0.1 mmol) in 3 mL of acetonitrile was stirred and monitored by silica gel TLC. The solvent was then removed under reduced pressure and the reaction mixture was purified on a silica gel column, using a mixture of hexane/ethyl acetate of increasing polarity.

4-(1-(3-Nitrophenyl)-1H-1,2,3-triazol-4-yl)aniline (45)

The reaction of compound 44 (100 mg, 0.6 mmol) and 4-ethynylaniline (100 mg, 0.9 mmol) yielded product 45, (137 mg, 0.5 mmol, 80% yield), as an orange solid; mp 210-211 ºC; IR (KBr) ν / cm^-1 3450, 3367 (NH), 3102 (CH), 1567, 1522 (C=C), 1346 (N=O); ¹H NMR (400 MHz, DMSO-d₆) δ 9.25 (1H, s), 8.74 (1H, s), 8.42 (d, 1H, J 8.2 Hz), 8.31 (d, 1H, J 8.2 Hz), 7.90 (t, 1H, J 8.2 Hz), 7.60 (d, 2H, J 8.2 Hz), 6.65 (d, 2H, J 8.2 Hz), 5.31 (2H, s); ¹³C NMR (100 MHz, DMSO-d₆) δ 149.1, 148.8, 148.6, 137.4, 131.6, 126.4, 125.5, 122.9, 117.5, 117.3, 114.0, 112.4.; calcd. for (C₁₄H₁₁N₅O₂): C, 59.78; H, 3.94; N, 24.90; found: C, 59.56; H, 4.09; N, 24.85.

1-(3-nitrophenyl)-4-(4-nitrophenyl)-1H-1,2,3-triazole (46)

The reaction of compound 44 (100 mg, 0.6 mmol) and 1-ethynyl-4-nitrobenzene (134 mg, 0.9 mmol) yielded product 46, (176 mg, 0.57 mmol, 93% yield), as an orange solid; mp 268-270 ºC; IR (KBr) ν / cm^-1 3096 (CH), 1603, 1522 (C=C), 1344 (N=O); ¹H NMR (400 MHz, DMSO-d₆) δ 9.77 (1H, s), 8.79 (1H, s), 8.46 (d, 1H, J 7.8 Hz), 8.38 (3H, m), 8.22 (d, 2H, J 9.0 Hz), 7.95 (t, 1H, J 8.2 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ 149.1, 148.8, 148.6, 137.4, 131.6, 126.4, 125.5, 122.9, 117.5, 117.3, 114.0, 112.4.; calcd. for (C₁₄H₉N₅O₄): C, 54.02; H, 2.91; N, 22.50; found: C, 54.02; H, 3.33; N, 22.58.

4-(4-Methoxyphenyl)-1-(3-nitrophenyl)-1H-1,2,3-triazole (47)

The reaction of compound 44 (100 mg, 0.6 mmol) and 1-ethynyl-4-methoxybenzene (121 mg, 0.9 mmol) yielded product 47, (143 mg, 0.5 mmol, 79% yield), as a yellow solid; mp 210-211 ºC; IR (KBr) ν / cm^-1 3096 (CH), 1603, 1522 (C=C), 1344 (N=O); ¹H NMR (400 MHz, DMSO-d₆) δ 9.39 (1H, s), 8.76 (1H, s), 8.44 (dd, 1H, J 8.4, 1.2 Hz), 8.33 (dd, 1H, J 8.0, 1.6 Hz), 7.92 (t, 1H, J 8.0 Hz), 7.88 (d, 2H, J 8.8 Hz), 7.08 (d, 2H, J 8.4 Hz), 3.82 (3H, s); ¹³C NMR (100 MHz, DMSO-d₆) δ 159.4, 148.5, 147.5, 137.2, 131.4, 126.7, 125.7, 122.8, 122.3, 118.8, 114.4, 114.3, 55.1.

2-((1-(3-Nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl) isoindoline-1,3-dione (51)

The reaction of compound 44 (100 mg, 0.6 mmol) and 2-(prop-2-yn-1-yl)isoindoline-1,3-dione (169 mg, 0.9 mmol) yielded product 51, (198 mg, 0.57 mmol, 93% yield), as a pink solid; mp 208-209 ºC; IR (KBr) ν / cm^-1 3138 (CH), 3093 (CH), 1705 (C=O), 1530, (C=C), 1346 (N=O); ¹H NMR (400 MHz, DMSO-d₆) δ 8.99 (1H, s), 8.67 (1H, s), 8.36 (dd, 1H, J 7.6, 1.2 Hz), 8.29 (dd, 1H, J 8.4, 1.6 Hz), 7.93-7.84 (5H, m), 4.96 (2H, s); ¹³C NMR (100 MHz, DMSO-d₆) δ 167.1, 148.3, 143.9, 136.9, 134.3, 131.5, 131.2, 125.8, 123.0, 122.9, 121.6, 114.5, 32.7; calcd. for (C₁₇H₁₁N₅O₄ × 0.6 H₂O): C, 56.68; H, 3.42; N, 19.45; found: C, 56.25; H, 3.04; N, 19.39.

1-(3-Nitrophenyl)-4-propyl-1H-1,2,3-triazole (52)

The reaction of compound 44 (100 mg, 0.6 mmol) and pent-1-yne (62 mg, 0.9 mmol) yielded product 52, (126 mg,

0.54 mmol, 89% yield), as a yellow solid; mp 109-110 ºC; IR (KBr) ν / cm^-1 3147, 3098 (CH), 1537, (C=C), 1347 (N=O); ¹H NMR (400 MHz, CDCl₃) δ 8.56 (1H, s), 8.27 (d, 1H, J 7.2 Hz), 8.19 (d, 1H, J 8.0 Hz), 7.83 (1H, s), 7.73 (t, 1H, J 7.6 Hz), 2.80 (t, 2H, J 6.8 Hz), 1.78 (2H, m), 1.03 (t, 3H, J 7.2 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ 149.2, 148.3, 137.3, 130.2, 125.1, 122.2, 118.0, 114.3, 27.0, 21.9, 13.1; calcd. for (C₁₁H₁₂N₄O₂): C, 56.89; H, 5.21; N, 24.12; found: C, 56.69; H, 5.13; N, 23.91.

4-Butyl-1-(3-nitrophenyl)-1H-1,2,3-triazole (53)

The reaction of compound 44 (100 mg, 0.6 mmol) and hex-1-yne (75 mg, 0.9 mmol) yielded product 53, (135 mg, 0.55 mmol, 90% yield), as a yellow solid; mp 101-102 ºC; IR (KBr) ν / cm^-1 3146 (CH), 3094 (CH), 1533, (C=C), 1346 (N=O); ¹H NMR (400 MHz, CDCl₃) δ 8.56 (1H, s), 8.27 (d, 1H, J 8.0 Hz), 8.19 (d, 1H, J 8.0 Hz), 7.83 (1H, s), 7.73 (t, 1H, J 8.0 Hz), 2.83 (t, 2H, J 8.0 Hz), 1.48-1.38 (2H, m), 0.98-0.93 (2H, m), 1.0 (t, 3H, J 8.0 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ 150, 148.9, 137.9, 130.8, 125.7, 122.8, 118.5, 114.9, 31.3, 25.3, 22.3, 13.8.

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 package⁴⁰ was used for data collection and data reduction. The data were corrected empirically for absorption using spherical harmonics using the SCALE3 ABSPACK⁴¹ scaling algorithm. The structure was solved by direct methods using SHELXS-97⁴² and refined by full-matrix least squares on F² using SHELXL-97.⁴³ All non-hydrogen atoms were successfully refined using anisotropic displacement parameters. Hydrogen atoms were found in the Fourier difference synthesis and fixed.

Antimycobacterial activity

Isolates and strain preparation

The antimicrobial activity of the compounds 5-15 and 18-38 were evaluated against M. tuberculosisH₃₇Rv (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.

Acknowledgments

This research was funded by grants from the Brazilian National Council for Technological and Scientific Development (CNPq) Project Universal-MCTI/CNPq No. 14/2012 (480719/2012-8), FACEPE-PRONEM (1232.1.06/10), FAPEMIG, CAPES, FURG, UFRPE and UFMG. The authors would like also to thank Prof Carlos B. Pinheiro and the student Willian Xerxes by the X-ray analysis of compound 29 and Prof Janaína V. dos Anjos (DQF-UFPE) by the collaboration in several aspects. We would like to thank Prof John Bower from University of Bristol for reviewing the manuscript.

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Publication Dates

• Publication in this collection
  May 2015

History

• Received
  01 Sept 2014
• Accepted
  20 Mar 2015