Antileishmanial Activities of (Z)-2-(Nitroimidazolylmethylene)-3(2H)-Benzofuranones: Synthesis, In Vitro Assessment, and Bioactivation by NTR 1 and 2

ABSTRACT

The antileishmanial activity of a series of (Z)-2-(heteroarylmethylene)-3(2H)-benzofuranone derivatives, possessing 5-nitroimidazole or 4-nitroimidazole moieties, was investigated against Leishmania major promastigotes and some analogues exhibited prominent activities. Compounds with IC₅₀ values lower than 20 μM were further examined against L. donovani axenic amastigotes. Evaluated analogues in 5-nitroimidazole subgroup demonstrated significantly superior activity (~17-88-folds) against L. donovani in comparison to L. major. (Z)-7-Methoxy-2-(1-methyl-5-nitroimidazole-2-ylmethylene)-3(2H)-benzofuranone (5n) showed the highest L. donovani anti-axenic amastigote activity with IC₅₀ of 0.016 μM. The cytotoxicity of these analogues was determined using PMM peritoneal mouse macrophage and THP-1 human leukemia monocytic cell lines and high selectivity indices of 26 to 431 were obtained for their anti-axenic amastigote effect over the cytotoxicity on PMM cells. Further studies on their mode of action showed that 5-nitroimidazole compounds were bioactivated predominantly by nitroreductase 1 (NTR1) and 4-nitroimidazole analogues by both NTR1 and 2. It is likely that this bioactivation results in the production of nitroso and hydroxylamine metabolites that are cytotoxic for the Leishmania parasite.

INTRODUCTION

Leishmaniasis, a parasitic disease caused by Leishmania species, is transmitted to humans by the bite of infected female sandflies. Each year, 700,000 to 1 million new cases of this neglected disease are reported, mainly from underdeveloped and developing countries in tropical and subtropical areas (1). Visceral leishmaniasis, the most serious form of the disease with 50,000 to 90,000 annual outbreaks, is invariably fatal if left untreated (1). Cutaneous leishmaniasis, the most common form of leishmaniasis, leaves lifelong scars and serious disability of stigma, with an estimated prevalence of 600,000 to 1 million new cases each year worldwide (1).

Currently, according to guidelines for clinical management of leishmaniasis prepared by a panel of the Infectious Diseases Society of America (IDSA) and the American Society of Tropical Medicine and Hygiene (ASTMH), the most currently used medicines for treating visceral leishmaniasis include liposomal amphotericin B (AmBisome, treatment of choice) (1), miltefosine (2), sodium stibogluconate, Pentostam) (3), and meglumine antimonials (Glucantime) (2) (Fig. 1). For cutaneous disease, fluconazole, ketoconazole, or pentamidine may also be effective (2). However, some of these medicines have shown serious side effects which limit their broad use. Sodium stibogluconate (Pentostam), for example, can cause acute pancreatitis and fatal cardiac arrhythmia (3). Amphotericin B shows adverse effects such as nephrotoxicity, hepatotoxicity, and fatal immediate anaphylaxis (4).

FIG 1.

Representative examples of antileishmanial drugs and synthesized compounds (5 to 7).

Most of the drugs of choice for treating leishmaniasis are not orally available, have inconvenient routes of administration, require long periods of treatment, are prohibitively expensive, and are associated with severe side effects.

Miltefosine is an approved oral medicine for the treatment of leishmaniasis but has limited therapeutic efficacy and a long half-life (7 days), which both contribute to the development of resistant species (5, 6). Further, it is contraindicated during pregnancy because of its teratogenicity (7).

Due to increased numbers of drug-resistant cases, Leishmania-HIV coinfections, the changing epidemiology of leishmaniasis due to climate change, and the altered distribution of insect vectors and hosts in areas affected by drought, flood, famine, war, etc., in recent years, new investigations have factored into the development of new antileishmanial compounds. Some studies have focused on modifying existing compounds to increase their efficacy or/and decrease their toxicity, and others have targeted new molecules or pathways in the parasite. Some recently proposed compounds include ferrocenyl N-heterocyclic compounds (8), α-aminophosphonates (9), quinolinone-chalcone compounds (10), a new generation of 2H-benzimidazole 1,3-dioxide derivatives (11), and redox-active nitro-containing compounds (12). Redox-active nitroheterocyclic compounds such as metronidazole, benznidazole, and tinidazole have been widely used as antibacterial and antiprotozoal drugs.

From this class, fexinidazole has recently been approved as the first oral treatment for human African trypanosomiasis (sleeping sickness) and has also shown potential for use in the treatment of visceral leishmaniasis (13). Enzymes in parasites called nitroreductases (NTRs) are responsible for bioactivation of fexinidazole (14). NTRs, which bear a flavin cofactor, catalyze the 1- or 2-electron reduction of nitroaromatic derivatives to produce a range of electrophilic intermediates (15–17). These metabolites are cytotoxic and capable of disrupting DNA, proteins, and membranes. The selective toxicity of anti-parasitic nitroaromatic compounds is largely due to the absence of close NTR homologues in mammalian cells, meaning that these pro-drugs are only activated within the parasites. So far, two leishmanial nitroreductases have been characterized: an essential type 1 mitochondrial NTR1 and a cytosolic type 2 NTR2, which catalyze the 1-electron reduction of nitroaromatics (14, 18).

As part of our ongoing program to develop new active antimicrobial agents, we synthesized a new class of (Z)-2-(nitroimidazolylmethylene)-3(2H)-benzofuranone derivatives with MICs as low as 0.78 μg/mL against methicillin-resistant Staphylococcus aureus (MRSA) (19). These compounds were hybrids of nitroimidazole and 3(2H)-benzofuranone moieties. Nitroimidazolyl compounds, as mentioned earlier, are well-established antimicrobial agents and mainly act by disrupting DNA molecules in the pathogen via formation of active radical anions. There is limited data available on benzofuranone moieties; however, a series of 2-benzylidene-3(2H)-benzofuranones has shown antibacterial activity by inhibiting chorismate synthase, a key enzyme in the aromatic amino acid synthesis pathway in bacteria (20, 21). Moreover, these analogues have demonstrated prominent antiplasmodium activity against drug-sensitive (3D7) and chloroquine- and multidrug-resistant (K1) strains of Plasmodium falciparum, with some of them showing high selectivity indices against KB cells (22), making them interesting candidates for further investigations.

Considering the promising results from our previous studies, here we report the biological assessment of a library of (Z)-2-(nitroimidazolylmethylene)-3(2H)-benzofuranones against Leishmania major promastigotes. For more active compounds, we expanded our study to examine their efficacy against Leishmania donovani axenic amastigotes and also investigated their cytotoxic effects on PMM (peritoneal mouse macrophage) and THP-1 human leukemia monocytic cell lines to calculate their selectivity indices. Finally, to obtain better insights into the mechanisms of action of these promising compounds, the bioactivation routes of these analogues have been examined using genetically modified L. donovani cell lines which overexpress either NTR1 or NTR2.

RESULTS AND DISCUSSION

Chemistry.

As explained earlier (19), the compounds in question, substituted (Z)-2-(1-methyl-5-nitroimidazole-2-ylmethylene)-3(2H)-benzofuranones (5a to 5o) and (Z)-2-(1-methyl-4-nitroimidazole-5-ylmethylene)-3(2H)-benzofuranones (7a to 7m), were prepared by condensing the corresponding 3(2H)-benzofuranones with 1-methyl-5-nitroimidazole-2-carbaldehyde or 1-methyl-4-nitroimidazole-5-carbaldehyde in acidic media. Condensation reactions resulted in single isomers, as shown by nuclear magnetic resonance (NMR) experiments.

It is known from the literature that the thermodynamically more stable Z isomer always prevails (23). In this regard, the X-ray crystallographic data of the previously reported synthesized aurones are shown to be in accordance with the structures proposed based on the NMR data (23–25).

To reassess the geometrical stereochemistry of the synthesized compounds based on ¹H NMR data, the E isomers of 5a and 7a were obtained by UV irradiation at 366 nm of the respective Z isomers. The spectra of the mixtures of Z and E stereoisomers are presented in Fig. 2. The new photoisomers were assigned to have the E configuration on the basis that β-protons (vinylic C-H) were deshielded (7.01 and 7.09 ppm) than in the Z isomers (6.86 and 7.07 ppm) in 5a and 7a, respectively (26). The high field signals of the β-protons in (Z)-isomers should be due to the influence of the ring oxygen atom. Olefinic proton trans to the ether ring oxygen gives a signal at a higher field than in the cis arrangement. The lone pair on the ring oxygen atom cause more deshielding of the β-protons in the E isomer than in the Z isomer (26) and more deshielding of imidazole C-H and N-CH₃ signal in the Z isomer than in the E isomer.

FIG 2.

¹H-NMR spectra of a mixture of Z and E isomers of 5a and 7a.

In vitro assays.

The antileishmanial activities of compounds 5 to 7 were investigated in vitro against L. major promastigotes (MHOM/Pk/88/DESTO) (27, 28). The 50% inhibitory concentrations (IC₅₀, μM) were determined in comparison to the reference drugs amphotericin B, pentamidine, and nifurtimox (4) (Fig. 1), and the results are shown in Table 1.

TABLE 1.

Anti-Leishmania major promastigote activities of nitroimidazolylmethylene-3(2H)-benzofuranones (5 to 7)

Compound	Ra	In vitro anti-promastigote activity, IC50 ± SDb (μM)
5a	H	1.29 ± 1.1
5b	5-Cl	63.33 ± 0.50
5c	5-Br	46.2 ± 0.53
5d	5-CH3	11.86 ± 0.04
5e	5-OCH3	7.44 ± 0.10
5f	5-I	66.33 ± 0.92
5g	5-NO2	42.79 ± 0.57
5h	6-Cl	21.13 ± 0.28
5i	6-CH3	53.68 ± 0.18
5j	6-OCH3	39.51 ± 0.01
5k	6-OH	66.0 ± 1.1
5l	7-Cl	26.94 ± 1.22
5m	7-CH3	30 ± 0.35
5n	7-OCH3	1.18 ± 0.9
5o	6,7-(OCH3)2	38.95 ± 0.23
6	H	84.48 ± 0.04
7a	H	>100
7b	5-Cl	>100
7c	5-Br	13.03 ± 0.53
7d	5-CH3	>100
7e	5-OCH3	51.82 ± 0.85
7f	5-I	>100
7g	5-NO2	65.0 ± 1.25
7h	6-Cl	>100
7i	6-CH3	44.36 ± 1.055
7j	6-OCH3	>100
7l	7-CH3	>100
7m	7-OCH3	>100
Amphotericin B	n.t.	0.29 ± 0.05
Pentamidine	n.t.	5.09 ± 0.04
Nifurtimox		14.11 ± 0.06

^aThe R group is shown in Figure 1, structures 5 to 7. n.t., not tested.

^bData represent mean ± SD of three independent experiments.

Generally, most 5-nitroimidazole analogues revealed considerable in vitro activity against L. major, while most of the compounds bearing 4-nitroimidazole moiety exhibited no effect or moderate antileishmanial activity up to a concentration of 100 μM.

The subgroup bearing 5-nitroimidazole moiety (5a to 5o) showed moderate to prominent in vitro antileishmanial activity against L. major promastigotes, with IC₅₀ values of 1.18 to 66.33 μM. L. major exhibited noticeable susceptibility to the activity of the unsubstituted benzofuranone analogue (5a), (Z)-2-(1-methyl-5-nitroimidazole-2-ylmethylene)-3(2H)-benzofuranone, with an IC₅₀ of 1.29 ± 1.10 μM compared to the amphotericin B, pentamidine, and nifurtimox IC₅₀s of 0.29 ± 0.05, 5.09 ± 0.04, and 14.11 ± 0.06 μM, respectively. Next, we studied substitution of the benzofuranone moiety with indanone (6), which resulted in a significantly less active compound (IC₅₀ = 84.48 ± 0.04 μM). This observation showed the importance of the oxygen of benzofuranone moiety in antileishmanial activity, similar to previous results obtained for antimicrobial and antimalarial activities (19, 22).

Next, we investigated the effects of different substituents on the benzofuranone ring on anti-promastigote activity. The results showed that the presence of any substituent (except for 7-OCH₃) on this moiety diminished activity. This might suggest that there is limited space available for this part of the ligand at the target binding site. Of the different compounds, 5- and 7-methoxy substituted benzofuranones (5e and 5n) exhibited superior activity, with IC₅₀s of 7.44 ± 0.10 and 1.18 ± 0.9 μM, respectively.

Even though most of the analogues possessing 4-nitroimidazole were shown to be inactive against promastigotes of L. major, the compounds bearing 5-OCH₃ (7e), 5-NO₂ (7g), and 6-CH₃ (7i) exhibited moderate activity, with IC₅₀s of 44.36 to 65.0 μM, and the compound possessing 5-Br substituent (7c) revealed noticeable activity with an IC₅₀ of 13.03 ± 0.53 μM.

The compounds showing promising anti-promastigote activity against L. major with IC₅₀s lower than 20 μM (5a, 5d, 5e, 5n, and 7c) were further examined for their activity against axenic amastigote (MHOM/ET/67/L82) (29–31). As shown in Table 2, compounds in the 5-nitroimidazole subgroup (5a, 5d, 5e, and 5n) presented significantly superior activity (~17- to 88-fold) against L. donovani axenic amastigote compared to that against L. major promastigote, with IC₅₀s of 0.016 to 0.435 μM. Among these compounds, 7-methoxy substituted benzofuranone (5n) demonstrated the highest anti-axenic amastigote activity against L. donovani with an IC₅₀ of 0.016 μM, being more active than standard antileishmanial agents.

TABLE 2.

Anti-promastigote activity, anti-axenic amastigote activity, and cytotoxicity of selected compounds^a

Compound	Inhibitory activity, IC50 (μM) ± SD		Cytotoxicity, CC50 (μM) ± SD		Selectivity index, CC50/anti-amastigote L. donovani IC50
	Anti-promastigote (L. major)	Anti-axenic amastigote (L. donovani)
			PMM	THP-1	PMM	THP-1
5a	1.29 ± 1.1	0.063 ± 0.007	6.694 ± 1.897	0.451 ± 0.021	107.70	7.17
5d	11.86 ± 0.04	0.284 ± 0.017	20.674 ± 2.642	0.894 ± 0.044	72.79	3.60
5e	7.44 ± 0.10	0.435 ± 0.269	18.488 ± 5.004	0.769 ± 0.024	42.50	1.77
5n	1.18 ± 0.9	0.016 ± 0.002	6.900 ± 0.786	0.152 ± 0.016	431.25	9.5
7c	13.03 ± 0.53	2.704 ± 0.417	70.940 ± 14.51	44.46 ± 1.13	26.23	16.44
Amphotericin B	0.29 ± 0.05	n.t.	n.t.	n.t.	n.t.	n.t.
Nifurtimox	14.11 ± 0.06	4.386 ± 1.497	>350	n.t.	n.t.	n.t.
Delamanid	n.t.	n.t.	n.t.	n.t.	n.t.	n.t.
Miltefosine	n.t.	0.40 ± 0.039	n.t.	n.t.	n.t.	n.t.

^aData represent means ± SD of at least two independent experiments. IC₅₀, 50% inhibitory concentration; PMM, peritoneal mouse macrophage; n.t., not tested.

4-Nitroimidazole (7c) also demonstrated considerable anti-axenic amastigote activity (IC₅₀ = 2.70 ± 0.42 μM).

The relative cytotoxicities of these compounds were assessed against PMM peritoneal mouse macrophage and THP-1 human leukemia monocytic cell lines (32, 33) (Table 2). According to the results, these analogues presented high selectivity indices of 26 to 431 for their anti-axenic amastigote effects over their toxicity against PMM cells and moderate selectivity indices of 2 to 16 against THP-1 cells.

The activity of these compounds was also assessed against L. donovani intracellular amastigotes, and the results are shown in Table 3 (32). Most of these analogues did not show promising results at concentrations lower than their cytotoxic levels on host PMM cells, except for 5e which reduced the infection rate by 39.2% and the number of intracellular amastigotes by 58.3% at a concentration of 10.96 μM, compared to nifurtimox as the reference drug which reduced the infection rate by 50% at 105.12 μM and the number of intracellular amastigotes by 50% at 92.07 μM.

TABLE 3.

Intracellular activities of selected compounds against L. donovani amastigotes in peritoneal mouse macrophage host cells

Compound	Infection rate (μM)b		No. of intracellular amastigotes (μM)		Cytotoxicity, PMMa IC50 (μM)c
	IC50a	IC90	IC50	IC90
5a	>4.06	>4.06	>4.06	>4.06	6.694
5d	>11.58	>11.58	>11.58	>11.58	20.674
5e	>10.96	>10.96	10.15	>10.96	18.488
5n	>3.65	>3.65	>3.65	>3.65	6.900
7c	>31.43	>31.43	>31.43	>31.43	70.940
Miltefosine	5.40	18.89	5.45	16.79	>24.5
Nifurtimox	105.12	>350	92.07	>245	>350

^aIC, inhibitory concentration; PMM, peritoneal mouse macrophage.

^bIC₅₀s are calculated based on infection rate.

^cIC₅₀s are calculated based on number of intracellular amastigotes.

The high antileishmanial activity of these series of compounds coupled with their high selectivity indices encouraged us to pursue further studies on their modes of action.

Bioactivation by Leishmania-specific nitroreductases (NTR1 and NTR2).

In order to gain insights into the basis for the antileishmanial activity of the evaluated analogues, and in view of the fact that the mechanism of action of most nitroimidazole derivatives depends upon bioactivation by parasite-specific nitroreductases (NTRs), the potency of the most promising developmental compounds (5a, 5d, 5e, 5n, and 7c) was determined against three strains of L. donovani promastigotes: a wild-type strain (MHOM/SD/62/1S-CL2D), a strain genetically engineered to overexpress NTR1, and a strain overexpressing NTR2 (14, 15, 34, 35). The IC₅₀ values of each compound were evaluated and compared with the established NTR1 substrate nifurtimox and the NTR2 substrate delamanid. According to the results shown in Table 4, parasites overexpressing NTR1 were found to be hypersensitive (9- to 14.1-fold more susceptible) to 5-nitroimidazole compounds (5a, 5d, 5e, and 5n) in comparison to wild-type cells, while parasites overexpressing NTR2 demonstrated similar sensitivities to compounds 5a, 5d, 5e, and 5n to those of wild-type parasites. In contrast, parasites overexpressing both NTR1 and NTR2 demonstrated hypersensitivity to 4-nitroimidazole compound (7c) and 51.2- and 9-fold greater susceptibility to 7c than that of the wild type, respectively. Collectively, these data suggest that 5-nitroimidazole derivatives were predominately bioactivated via NTR1, while 4-nitroimidazole derivatives could be activated by both NTR1 and NTR2 enzymes.

TABLE 4.

Anti-promastigote activities of selected compounds against the clonal Leishmania donovani cell line LdBOB and transgenic cell lines LdBOB NTR1^OE and NTR2^OEa

Compound	IC50 (μM) ± SDa
	Wild type	NTR1OE	Fold-change	NTR2OE	Fold-change	N
5a	0.046 ± 0.002	0.005 ± 0.001	9.2	0.064 ± 0.001	n.t.	3
5d	0.134 ± 0.007	0.013 ± 0.001	10	0.135 ± 0.002	n.t.	2
5e	0.085 ± 0.005	0.006 ± 0.001	14.1	0.099 ± 0.003	n.t.	2
5n	0.018 ± 0.002	0.002 ± 0.001	9	0.007 ± 0.001	2.6	2
7c	13.946 ± 2.032	0.269 ± 0.019	51.2	1.497 ± 0.021	9.2	3
Nifurtimox	2.694 ± 0.265	0.064 ± 0.003	42	0.177 ± 0.003	15.2	3
Delamanid	0.0025 ± 0.00004	NA	n.t.	0.0002 ± 0.00001	12.5	3

^aCompounds were tested for 72 h against the clonal Leishmania donovani cell line LdBOB and transgenic cell lines LdBOB NTR1^OE and NTR2^OE. Data are weighted means and weighted standard errors for at least two independent experiments. Each individual experiment is derived from a 10-point dilution curve where each individual sample was measured in duplicate. IC, inhibitory concentration; SD, standard deviation; NA, not active; N, number of independent experiments; n.t., not tested.

The superior antileishmanial activity of the compounds against L. donovani compared to L. major could be attributed to the different susceptibilities of the studied microorganisms to the biological activities of the synthesized analogues or their different bioactivation potential by nitroreductases (NTR1 and NTR2), as this superiority could also be observed for the reference drug nifurtimox, which acts through a similar mechanism of action. Furthermore, previous studies on miltefosine have shown that among the different Leishmania species, L. donovani is the most sensitive to this antileishmanial drug, while L. major was the least sensitive (36, 37).

The bioactivation of these compounds with NTRs strengthens the hypothesis that these compounds destroy the Leishmania parasite by disrupting DNA and inhibiting cell wall protein synthesis by producing reactive oxygen species.

Conclusion.

A library of (Z)-2-heteroarylmethylene-3(2H)-benzofuranones possessing 1-methyl-5-nitroimidazole (5a to 5o) and 1-methyl-4-nitroimidazole (7a to 7m) were assessed for their activity against L. major promastigotes. The 5-nitroimidazole analogues showed moderate to significant inhibition of L. major growth in vitro, whereas the 4-nitroimidazoles showed none to moderate activity against promastigotes. The compounds showing promising activity against L. major promastigotes were further examined on L. donovani axenic amastigotes and demonstrated significantly superior activity against this strain. Cytotoxicity assessment of these analogues on PMM and THP-1 cells exhibited high selectivity indices. To better understand antileishmanial mechanism of action of these compounds, we studied their bioactivation by NTR1 and NTR2. While 5-nitroimidazoles were activated solely by NTR1, 4-nitroimidazole (7c) proved to be a substrate of both nitroreductases (NTR1 and NTR2). Therefore, the high antileishmanial activity of these series of compounds, along with their high selectivity indices, could help them serve as a novel scaffold for developing new antileishmanial drug candidates.

MATERIALS AND METHODS

Chemistry.

Although we have published the antimicrobial and antimalarial activities of these compounds in previous research studies, their spectral information has not been reported. For this reason, their spectral information is reported here (19, 22).

Melting points were determined using a Reichert-Jung hot-stage microscope and were uncorrected. Infrared spectra were recorded on a Nicolet Magna 550-FT spectrometer. NMR spectra were measured on a Bruker spectrometer (500 MHz for ¹H NMR, 125 MHz for ¹³C NMR spectra) in CDCl₃ or DMSO (dimethyl sulfoxide)-d₆ with TMS (tetramethylsilane) as the internal standard, where J (coupling constant) values were estimated in Hertz. Spin multiples are given as s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), m (multiplet), and br (broad). Mass spectra were obtained using a Finnigan Mat TSQ-70 spectrometer. Elemental microanalyses were carried out with a Perkin-Elmer 240-C apparatus and were within ±0.4% of the theoretical values for C, H, and N.

General procedure for the preparation of substituted (Z)-2-(1-methyl-5-nitroimidazole-2-ylmethylene)-3(2H)-benzofuranones (5a to 5o).

Equimolar amounts of the appropriate substituted 3(2H)-benzofuranone and 1-methyl-5-nitroimidazole-2-carboxaldehyde (5 to 15 mmol) in acetic acid (5 to 15 mL) and sulfuric acid (96% W/W, 0.11 to 0.33 mL) were stirred for 6 to 8 h at 100°C. After cooling, 10 mL of methanol was added, and the insoluble product was filtered off and recrystallized from methanol.

(Z)-2-(1-Methyl-5-nitroimidazole-2-ylmethylene)-3(2H)-benzofuranone (5a).

Yield, 56%; mp (melting point) 239°C to 241°C; IR (KBr): 1,716 (CO), 1,481, 1,383 (NO₂) cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.34 (s, 1H),7.88 to 7.80 (m, 2H), 7.57 (d, J = 8.5 Hz, 1H), 7.35 (t, J = 8.5 Hz, 1H), 6.87 (s, 1H), 4.06 (s, 3H); ¹³C NMR (DMSO-d₆) δ 183.89, 166.30, 149.87, 144.65, 139.63, 138.53, 134.29, 124.82, 124.54, 120.34, 113.39, 96.13, 33.60; MS m/z (%) 271 (M⁺, 76), 225 (38), 184 (100), 155 (81), 127 (15), 101 (52), 76 (54), 50 (57).

Analysis calculated for C₁₃H₉N₃O₄: C, 57.57; H, 3.34; N, 15.49. Found: C, 57.70; H, 3.42; N, 15.33.

(Z)-5-Chloro-2-(1-methyl-5-nitroimidazole-2-ylmethylene)-3(2H)-benzofuranone (5b).

Yield, 54%; mp 276°C to 278°C; IR (KBr): 1,711 (CO), 1,465, 1,363 (NO₂) cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.34 (s, 1H), 7.91 (d, J = 2.0 Hz, 1H), 7.86 (dd, J = 2.0, 9.0 Hz, 1H), 7.63 (d, J = 9.0 Hz, 1H), 6.91 (s, 1H), 4.05 (s, 3H); ¹³C NMR (DMSO-d₆) δ 182.92, 164.79, 150.03, 144.48, 139.75, 137.90, 134.46, 128.82, 124.31, 122.20, 115.35, 97.02, 33.68; MS m/z (%) 307 (M⁺, 33), 305 (M⁺, 100), 275 (10), 259 (39), 218 (96), 190 (53), 163 (13), 135 (28), 110 (32), 99 (22), 75 (35), 52 (49).

Analysis calculated for C₁₃H₈ClN₃O₄: C, 51.08; H, 2.64; N, 13.75. Found: C, 50.89; H, 2.77; N, 13.82.

(Z)-5-Bromo-2-(1-methyl-5-nitroimidazole-2-ylmethylene)-3(2H)-benzofuranone (5c).

Yield, 62%; mp 254°C to 257°C; IR (KBr): 1,721 (CO), 1,465, 1,373 (NO₂) cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.26 (s, 1H), 7.94 (d, J = 2.6 Hz, 1H), 7.81 (dd, J = 2.6, 8.8 Hz, 1H), 7.37 (d, J = 8.8 Hz, 1H), 6.69 (s, 1H), 4.14 (s, 3H); ¹³C NMR (DMSO-d₆) δ 182.66, 165.09, 149.76, 144.40, 140.45, 139.0, 134.31, 127.09, 122.42, 116.38, 115.67, 96.89, 33.64; MS m/z (%) 351 (M⁺, 65), 349 (M⁺, 65), 304 (25), 261 (100), 233 (46), 180 (18), 150 (89), 107 (25), 74 (56), 52 (73).

Analysis calculated for C₁₃H₈BrN₃O₄: C, 44.60; H, 2.30; N, 12.00. Found: C, 44.72; H, 2.18; N, 11.89.

(Z)-5-Methyl-2-(1-methyl-5-nitroimidazole-2-ylmethylene)-3(2H)-benzofuranone (5d).

Yield, 61%; mp 251°C to 253°C; IR (KBr): 1,711 (CO), 1,475, 1,368 (NO₂) cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.33 (s, 1H), 7.65 (d, J = 9.0 Hz, 1H), 7.64 (s, 1H), 7.46 (d, J = 9.0 Hz, 1H), 6.84 (s, 1H), 4.05 (s, 3H), 2.38 (s, 3H); ¹³C NMR (DMSO-d₆) δ 183.96, 164.84, 150.30, 144.79, 139.49, 134.35, 134.16, 124.34, 124.26, 120.26, 113.10, 112.97, 95.90, 33.60, 20.23; MS m/z (%) 285 (M⁺, 17), 240 (18), 198 (22), 151 (22), 134 (47), 93 (28), 89 (37), 81 (46), 69 (81), 67 (90), 55 (100).

Analysis calculated for C₁₄H₁₁N₃O₄: C, 58.95; H, 3.89; N, 14.73. Found: C, 58.82; H, 4.02; N, 14.85.

(Z)-5-Methoxy-2-(1-methyl-5-nitroimidazole-2-ylmethylene)-3(2H)-benzofuranone (5e).

Yield, 59%; mp 268 to 270°C; IR (KBr): 1,706 (CO), 1,486, 1,322 (NO₂) cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.33 (s, 1H), 7.51 (d, J = 9.5 Hz, 1H), 7.42 (dd, J = 2.5, 9.5 Hz, 1H), 7.31 (d, J = 2.5 Hz, 1H), 6.86 (s, 1H), 4.05 (s, 3H), 3.82 (s, 3H); ¹³C NMR (DMSO-d₆) δ 184.00, 161.28, 156.25, 150.60, 144.70, 139.60, 134.31, 126.83, 120.60, 114.35, 106.03, 96.09, 56.04, 33.57; MS m/z (%) 301 (M⁺, 96), 255 (23), 214 (100), 185 (60), 130 (12), 116 (22), 79 (22), 63 (52).

Analysis calculated for C₁₄H₁₁N₃O₅: C, 55.82; H, 3.68; N, 13.95. Found: C, 55.70; H, 3.88; N, 13.81.

(Z)-5-Iodo-2-(1-methyl-5-nitroimidazole-2-ylmethylene)-3(2H)-benzofuranone (5f).

Yield, 58%; mp 283°C to 285°C; IR (KBr): 1,716 (CO), 1,450, 1,363 (NO₂) cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.32 (s, 1H), 8.14 (s, 1H), 8.09 (dd, J = 2.6, 9.6 Hz, 1H), 7.42 (d, J = 9.6 Hz, 1H), 6.89 (s, 1H), 4.06 (s, 3H); ¹³C NMR (DMSO-d₆) δ 182.48, 165.68, 149.48, 146.08, 144.45, 139.69, 134.31, 132.81, 122.88, 115.86, 96.74, 88.21, 33.63; MS m/z (%) 397 (M⁺, 0.5), 351 (2), 305 (100), 275 (12), 259 (27), 246 (11), 218 (98), 190 (30), 150 (47), 121 (28), 67 (21).

Analysis calculated for C₁₃H₈IN₃O₄: C, 39.32; H, 2.03; N, 10.58. Found: C, 39.20; H, 2.12; N, 10.43.

(Z)-5-Nitro-2-(1-methyl-5-nitroimidazole-2-ylmethylene)-3(2H)-benzofuranone (5g).

Yield, 45%; mp 244°C to 247°C; IR (KBr): 1,721 (CO), 1,527, 1,460, 1,367, 1,342 (NO₂) cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.63 (dd, J = 2.0, 9.0 Hz, 1H), 8.56 (d, J = 2.0 Hz, 1H), 8.37 (s, 1H), 7.82 (d, J = 9.0 Hz, 1H), 7.05 (s, 1H), 4.08 (s, 3H); ¹³C NMR (DMSO-d₆) δ 182.75, 169.00, 150.08, 146.06, 144.10, 140.06, 134.30, 132.80, 121.29, 120.50, 114.86, 33.80; MS m/z (%) 316 (M⁺, 100), 269 (33), 228 (64), 182 (60), 154 (46), 83 (33), 73 (60), 53 (72).

Analysis calculated for C₁₃H₈N₄O₆: C, 49.38; H, 2.55; N, 17.72. Found: C, 49.20; H, 2.39; N, 17.91.

(Z)-6-Chloro-2-(1-methyl-5-nitroimidazole-2-ylmethylene)-3(2H)-benzofuranone (5h).

Yield, 46%; mp 266°C to 268°C; IR (KBr): 1,706 (CO), 1,465, 1,378 (NO₂) cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.35 (s, 1H), 7.86 (d, J = 8.5 Hz, 1H), 7.84 (d, J = 1.5 Hz, 1H), 7.41 (dd, J = 1.5, 8.5 Hz, 1H), 6.92 (s, 1H), 4.06 (s, 3H); ¹³C NMR (DMSO-d₆) δ 182.63, 166.58, 149.96, 144.41, 142.56, 139.94, 134.28, 126.06, 125.05, 119.53, 113.97, 96.84, 34.32; MS m/z (%) 305 (M⁺, 3), 287 (17), 173 (23), 170 (16), 157 (7), 111 (14), 85 (32), 68 (100).

Analysis calculated for C₁₃H₈ClN₃O₄: C, 51.08; H, 2.64; N, 13.75. Found: C, 50.96; H, 2.73; N, 13.89.

(Z)-6-Methyl-2-(1-methyl-5-nitroimidazole-2-ylmethylene)-3(2H)-benzofuranone (5i).

Yield, 54%; mp 263°C to 266°C; IR (KBr): 1,716 (CO), 1,470, 1,363 (NO₂) cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.33 (s, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.41 (s, 1H), 7.18 (d, J = 8.0 Hz, 1H), 6.83 (s, 1H), 4.05 (s, 3H), 2.49 (s, 3H); ¹³C NMR (DMSO-d₆) δ 183.18, 166.82, 150.59, 150.33, 144.72, 139.55, 134.26, 125.72, 124.44, 117.95, 113.34, 95.76, 33.54, 22.19; MS m/z (%) 285 (M⁺, 64), 239 (32), 198 (52), 151 (32), 134 (56), 90 (37), 87 (43), 69 (62), 67 (81), 55 (100).

Analysis calculated for C₁₄H₁₁N₃O₄: C, 58.95; H, 3.89; N, 14.73. Found: C, 58.85; H, 3.81; N, 14.88.

(Z)-6-Methoxy-2-(1-methyl-5-nitroimidazole-2-ylmethylene)-3(2H)-benzofuranone (5j).

Yield, 56%; mp 223°C to 225°C; IR (KBr): 1,711 (CO), 1,464, 1,373 (NO₂) cm⁻¹; ¹H NMR (DMSO-d6) δ 8.23 (s, 1H), 7.71 (d, J = 8.8 Hz, 1H), 6.88 (dd, J = 2.6, 8.8 Hz, 1H), 6.74 (d, J = 2.6 Hz, 1H), 6.58 (s, 1H), 4.12 (s, 3H), 3.92 (s, 3H); ¹³C NMR (DMSO-d₆) δ 181.58, 168.99, 168.11, 151.04, 144.80, 139.52, 134.24, 126.06, 113.22, 113.07, 97.56, 95.12, 56.68, 33.56; MS m/z (%) 301 (M⁺, 27), 290 (21), 262 (16), 213 (41), 160 (19), 116 (12), 108 (23), 88 (32), 72 (100).

Analysis calculated for C₁₄H₁₁N₃O₅: C, 55.82; H, 3.68; N, 13.95. Found: C, 55.96; H, 3.79; N, 13.79.

(Z)-6-Hydroxy-2-(1-methyl-5-nitroimidazole-2-ylmethylene)-3(2H)-benzofuranone (5k).

Yield, 52%; mp 242°C to 244°C; IR (KBr): 3,130 (OH), 1,704 (CO), 1,524, 1,369 (NO₂) cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.33 (s, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.46 (s, 1H), 7.14 (d, J = 8.0 Hz, 1H), 6.88 (s, 1H), 4.05 (s, 3H); ¹³C NMR (DMSO-d₆) δ 182.60, 168.58, 167.01, 158.05, 150.25, 144.52, 139.67, 134.28, 125.86, 118.11, 98.91, 96.34; MS m/z (%) 287 (69), 257 (23), 241 (79), 229 (15), 200 (30), 185 (16), 169 (19), 150 (20), 139 (13), 115 (21), 105 (17), 89 (46), 77 (26), 69 (47), 63 (70), 43 (100).

Analysis calculated for C₁₃H₉N₃O₅: C, 54.36; H, 3.16; N, 14.63. Found: C, 54.20; H, 3.09; N, 14.41.

(Z)-7-Chloro-2-(1-methyl-5-nitroimidazole-2-ylmethylene)-3(2H)-benzofuranone (5l).

Yield, 42%; mp 255°C to 257°C; IR (KBr): 1,721 (CO), 1,470, 1,373 (NO₂) cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.39 (s, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.35 (t, J = 8.0 Hz, 1H), 6.97 (s, 1H), 4.06 (s, 3H); ¹³C NMR (DMSO-d₆) δ 183.60, 162.04, 149.89, 144.75, 140.20, 138.11, 134.81, 126.00, 123.93, 123.08, 117.71, 97.75, 34.15; MS m/z (%) 305 (M⁺, 100), 260 (40), 219 (92), 190 (51) 135 (26), 50 (55).

Analysis calculated for C₁₃H₈ClN₃O₄: C, 51.08; H, 2.64; N, 13.75. Found: C, 51.22; H, 2.81; N, 13.57.

(Z)-7-Methyl-2-(1-methyl-5-nitroimidazole-2-ylmethylene)-3(2H)-benzofuranone (5m).

Yield, 55%; mp 229°C to 231°C; IR (KBr): 1,706 (CO), 1,465, 1,368 (NO₂) cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.37 (s, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.26 (t, J = 8.0 Hz, 1H), 6.84 (s, 1H), 4.06 (s, 3H), 2.41 (s, 3H); ¹³C NMR (DMSO-d₆) δ 184.23, 164.90, 149.87, 144.81, 139.61, 139.11, 134.41, 124.28, 122.96, 122.05, 119.91, 96.07, 33.64, 13.81; MS m/z (%) 285 (M⁺, 77), 238 (37), 198 (100), 151 (37), 134 (41), 90 (49), 51 (49).

Analysis calculated for C₁₄H₁₁N₃O₄: C, 58.95; H, 3.89; N, 14.73. Found: C, 58.82; H, 3.81; N, 14.95.

(Z)-7-Methoxy-2-(1-methyl-5-nitroimidazole-2-ylmethylene)-3(2H)-benzofuranone (5n).

Yield, 48%; mp 235°C to 237°C; IR (KBr): 1,711 (CO), 1,527, 1,373 (NO₂) cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.35 (s, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.38 (d, J = 8.0 Hz, 1H), 7.26 (t, J = 8.0 Hz, 1H), 6.67 (s, 1H), 4.06 (s, 3H), 3.98 (s, 3H); ¹³C NMR (DMSO-d₆) δ 184.09, 155.58, 149.70, 145.54, 144.57, 139.64, 134.42, 125.16, 121.64, 120.50, 115.47, 96.57, 56.28, 33.62; MS m/z (%) 301 (M⁺, 5), 285 (12), 254 (18), 198 (13), 150 (100), 122 (89), 92 (11), 68 (48).

Analysis calculated for C₁₄H₁₁N₃O₅: C, 55.82; H, 3.68; N, 13.95. Found: C, 55.74; H, 3.49; N, 13.83.

(Z)-6,7-Dimethoxy-2-(1-methyl-5-nitroimidazole-2-ylmethylene)-3(2H)-benzofuranone (5o).

Yield, 57%; mp 272°C to 274°C; IR (KBr): 1,710 (CO), 1,490, 1,375 (NO₂) cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.34 (s, 1H), 7.57 (d, J = 8.5 Hz, 1H), 7.05 (d, J = 8.5 Hz, 1H), 6.80 (s, 1H), 4.04 (s, 3H), 4.01 (s, 3H), 3.95 (s, 3H); ¹³C NMR (DMSO-d₆) δ 181.63, 159.77, 158.0, 150.90, 144.81, 142.75, 135.08, 124.62, 119.87, 115.12, 109.83, 95.22, 61.15, 57.20, 33.50; MS m/z (%) 331 (M⁺, 22), 303 (100), 285 (8), 257 (76), 244 (44), 242 (42), 214 (34), 189 (15), 158 (25), 122 (39), 107 (54), 95 (46), 83 (32), 76 (45), 69 (63), 55 (55), 41 (33).

Analysis calculated for C₁₅H₁₃N₃O₆: C, 54.38; H, 3.96; N, 12.68. Found: C, 54.20; H, 3.79; N, 12.81.

(Z)-2-(1-Methyl-5-nitroimidazole-2-ylmethylene)-1-indanone (6).

Yield, 56%; mp 270°C to 272°C; IR (KBr): 1,695 (CO), 1,369, 1,528 (NO₂) cm⁻¹; ¹H NMR (CDCl₃) δ 8.19 (s, 1H), 7.91 (d, J = 7.2 Hz, 1H), 7.67 (t, J = 7.2 Hz, 1H), 7.58 (d, J = 7.2 Hz, 1H), 7.46 (t, J = 7.2 Hz, 1H), 7.43 (s, 1H), 4.27 (s, 2H), 4.16 (s, 3H); ¹³C NMR (DMSO-d₆) δ 172.37, 158.67, 148.34, 142.33, 136.35, 134.89, 133.37, 127.28, 126.39, 123.22, 114.74, 41.47, 32.74.

Analysis calculated for C₁₄H₁₁N₃O₃: C, 62.45; H, 4.12; N, 15.61. Found: C, 62.31; H, 4.29; N, 15.45.

General procedure for the preparation of substituted (Z)-2-(1-methyl-4-nitroimidazole-5-ylmethylene)-3(2H)-benzofuranones (7a-m).

Equimolar amounts of the appropriate substituted 3(2H)-benzofuranone (5 to 15 mmol), 1-methyl-4-nitroimidazole-5-carboxaldehyde in acetic anhydride (5 to 15 mL), and anhydrous sodium acetate (0.07 to 0.21 g) were stirred for 90 min at 100°C. After cooling, 10 mL of methanol was added, and the insoluble product was filtered off and recrystallized from methanol.

(Z)-2-(1-Methyl-4-nitroimidazole-5-ylmethylene)-3(2H)-benzofuranone (7a).

Yield, 60%; mp 261°C to 264°C; IR (KBr): 1,716 (CO), 1,501, 1,347 (NO₂) cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.04 (s, 1H), 7.82 (d, J = 7.5 Hz, 1H), 7.81 (t, J = 7.5 Hz, 1H), 7.46 (d, J = 7.5 Hz, 1H), 7.34 (t, J = 7.5 Hz, 1H), 7.07 (s, 1H), 3.80 (s, 3H); ¹³C NMR (DMSO-d₆) δ 182.82, 165.59, 148.68, 139.30, 139.02, 138.70, 124.78, 124.65, 120.67, 113.24, 96.94, 33.97; MS m/z (%) 271 (M⁺, 4), 225 (26), 157 (9), 151 (89), 120 (100), 105 (20), 92 (72), 68 (97).

Analysis calculated for C₁₃H₉N₃O₄: C, 57.57; H, 3.34; N, 15.49. Found: C, 57.69; H, 3.44; N, 15.26.

(Z)-5-Chloro-2-(1-methyl-4-nitroimidazole-5-ylmethylene)-3(2H)-benzofuranone (7b).

Yield, 46%; mp 273°C to 276°C; IR (KBr): 1721 (CO), 1,496, 1,347 (NO₂) cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.07 (s, 1H), 7.93 (d, J = 2.2 Hz, 1H), 7.86 (dd, J = 2.2, 9.0 Hz, 1H), 7.54 (d, J = 9.0 Hz, 1H), 7.12 (s, 1H), 3.87 (s, 3H); ¹³C NMR (DMSO-d₆) δ 182.08, 164.05, 148.70,145.70, 139.41, 137.99, 128.86, 124.55, 124.11, 122.18, 115.34, 97.89, 34.04; MS m/z (%) 305 (M⁺, 4), 259 (29),152 (100), 111 (12), 69 (21), 66 (48).

Analysis calculated for C₁₃H₈ClN₃O₄: C, 51.08; H, 2.64; N, 13.75. Found: C, 51.16; H, 2.50; N, 13.83.

(Z)-5-Bromo-2-(1-methyl-4-nitroimidazole-5-ylmethylene)-3(2H)-benzofuranone (7c).

Yield, 45%; mp 267°C to 269°C; IR (KBr): 1,721 (CO), 1,496, 1,342 (NO₂) cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.07 (s, 1H), 8.04 (d, J = 2.2 Hz, 1H), 7.97 (dd, J = 2.2, 9.0 Hz, 1H), 7.49 (d, J = 9.0 Hz, 1H), 7.13 (s, 1H), 3.81 (s, 3H); ¹³C NMR (DMSO-d₆) δ 181.21, 163.86, 147.65, 140.01, 138.68, 126.48, 123.76, 122.06, 115.69, 115.01, 97.17, 33.42; MS m/z (%) 350 (M⁺, 7), 200 (28), 121 (9), 93 (32), 68 (71).

Analysis calculated for C₁₃H₈BrN₃O₄: C, 44.60; H, 2.30; N, 12.00. Found: C, 44.77; H, 2.41; N, 11.85.

(Z)-5-Methyl-2-(1-methyl-4-nitroimidazole-5-ylmethylene)-3(2H)-benzofuranone (7d).

Yield, 65%; mp 256°C to 257°C; IR (KBr): 1,721 (CO), 1,496, 1,347 (NO₂) cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.05 (s, 1H), 7.64 (s, 1H), 7.63 (d, J = 9.5 Hz, 1H), 7.36 (d, J = 9.5 Hz, 1H), 7.05 (s, 1H), 3.80 (s, 3H), 2.37 (s, 3H); ¹³C NMR (DMSO-d₆) δ 182.40, 163.45, 148.32, 138.64, 138.48, 133.54, 124.10, 123.63, 119.90, 112.29, 96.03, 33.33, 19.60; MS m/z (%) 285 (M⁺, 31), 239 (25), 198 (73), 151 (33), 134 (100), 89 (43), 52 (53).

Analysis calculated for C₁₄H₁₁N₃O₄: C, 58.95; H, 3.89; N, 14.73. Found: C, 58.82; H, 3.75; N, 14.86.

(Z)-5-Methoxy-2-(1-methyl-4-nitroimidazole-5-ylmethylene)-3(2H)-benzofuranone (7e).

Yield, 50%; mp 286°C to 288°C; IR (KBr): 1,716 (CO), 1,491, 1,347 (NO₂) cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.06 (s, 1H), 7.51 (d, J = 9.0 Hz, 1H), 7.42 (dd, J = 2.2, 9.0 Hz, 1H), 7.22 (d, J = 2.2 Hz, 1H), 7.09 (s, 1H), 3.96 (s, 3H), 3.85 (s, 3H); ¹³C NMR (DMSO-d₆) δ 183.13, 160.55, 156.29, 149.27, 145.79, 139.09, 126.93, 124.80, 120.85, 114.27, 106.05, 96.89, 56.07, 33.93; MS m/z (%) 301 (M⁺, 10), 254 (20), 228 (11), 211 (31), 150 (100), 122 (35), 107 (56), 93 (37), 79 (52), 68 (70).

Analysis calculated for C₁₄H₁₁N₃O₅: C, 55.82; H, 3.68; N, 13.95. Found: C, 55.75; H, 3.52; N, 13.75.

(Z)-5-Iodo-2-(1-methyl-4-nitroimidazole-5-ylmethylene)-3(2H)-benzofuranone (7f).

Yield, 62%; mp 242°C to 244°C; IR (KBr): 1,716 (CO), 1,460, 1,352 (NO₂) cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.05 (s, 1H),7.70 to 7.62 (m, 2H), 7.36 (d, J = 9.0 Hz, 1H), 7.05 (s, 1H), 3.81 (s, 3H); ¹³C NMR (DMSO-d₆) δ 181.64, 164.94, 148.14, 146.21, 145.89, 139.24, 132.77, 124.57, 123.07, 115.78, 97.54, 88.21, 34.01; MS m/z (%) 397 (M⁺, 0.8), 351 (2), 305 (62), 259 (29), 218 (58), 190 (47), 149 (43), 135 (28), 73 (42), 66 (49), 51 (100).

Analysis calculated for C₁₃H₈IN₃O₄: C, 39.32; H, 2.03; N, 10.58. Found: C, 39.48; H, 1.95; N, 10.71.

(Z)-5-Nitro-2-(1-methyl-4-nitroimidazole-5-ylmethylene)-3(2H)-benzofuranone (7g).

Yield, 39%; mp 255°C to 257°C; IR (KBr): 1,721 (CO), 1,527, 1,460, 1,367, 1,342 (NO₂) cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.65 (dd, J = 2.0, 9.2 Hz, 1H), 8.59 (d, J = 2.0 Hz, 1H), 8.10 (s, 1H), 7.73 (d, J = 9.2 Hz, 1H), 7.24 (s, 1H), 3.83 (s, 3H); ¹³C NMR (DMSO-d₆) δ 181.70, 168.00, 148.72, 146.06, 144.14, 139.45, 133.11, 124.18, 121.48, 120.66, 114.54, 98.96, 34.06; MS m/z (%) 316 (M⁺, 100), 269 (33), 228 (64), 182 (60), 154 (46), 83 (33), 73 (60), 53 (72).

Analysis calculated for C₁₃H₈N₄O₆: C, 49.38; H, 2.55; N, 17.72. Found: C, 49.24; H, 2.68; N, 17.60.

(Z)-6-Chloro-2-(1-methyl-4-nitroimidazole-5-ylmethylene)-3(2H)-benzofuranone (7h).

Yield, 26%; mp 283°C to 285°C; IR (KBr): 1,711 (CO), 1,491, 1,337 (NO₂) cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.07 (s, 1H), 7.86 (d, J = 8.0 Hz, 1H), 7.75 (d, J = 1.0 Hz, 1H), 7.41 (dd, J = 1.0, 8.0 Hz, 1H), 7.11 (s, 1H), 3.80 (s, 3H); ¹³C NMR (DMSO-d₆) δ 181.21, 165.40, 147.96, 145.90, 144.41, 141.94, 138.68, 126.12, 125.49, 124.49, 119.09, 113.32, 97.03, 33.44; MS m/z (%) 305 (M⁺, 73), 259 (50), 218 (100), 190 (38), 138 (20), 135 (49), 110 (34), 75 (39), 52 (94).

Analysis calculated for C₁₃H₈ClN₃O₄: C, 51.08; H, 2.64; N, 13.75. Found: C, 51.20; H, 2.80; N, 13.69.

(Z)-6-Methyl-2-(1-methyl-4-nitroimidazole-5-ylmethylene)-3(2H)-benzofuranone (7i).

Yield, 43%; mp 277°C to 278°C; IR (KBr): 1,706 (CO), 1,491, 1,337 (NO₂) cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.05 (s, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.31 (s, 1H), 7.18 (d, J = 8.0 Hz, 1H), 7.04 (s, 1H), 3.79 (s, 3H), 2.47 (s, 3H); ¹³C NMR (DMSO-d₆) δ 183.02, 166.62, 15.93, 150.02, 144.45, 139.69, 134.31, 125.75, 124.55, 118.25, 113.45, 96.56, 22.55; MS m/z (%) 285 (M⁺, 3), 239 (50), 171 (12), 151 (100), 134 (100), 106 (56), 93 (62), 89 (82), 66 (98).

Analysis calculated for C₁₄H₁₁N₃O₄: C, 58.95; H, 3.89; N, 14.73. Found: C, 58.77; H, 4.02; N, 14.96.

(Z)-6-Methoxy-2-(1-methyl-4-nitroimidazole-5-ylmethylene)-3(2H)-benzofuranone (7j).

Yield, 34%; mp 254°C to 257°C; IR (KBr): 1,711 (CO), 1,501, 1,327 (NO₂) cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.05 (s, 1H), 7.75 (d, J = 8.5 Hz, 1H), 7.08 (s, 1H), 6.99 (s, 1H), 6.89 (d, J = 8.5 Hz, 1H), 3.90 (s, 3H), 3.82 (s, 3H); ¹³C NMR (DMSO-d₆) δ 181.16, 168.67, 168.56, 150.16, 139.37, 126.49, 125.31, 113.92, 113.62, 97.81, 96.30, 57.06, 34.29; MS m/z (%) 301 (M⁺, 4), 255 (11), 198 (12), 150 (100), 120 (78), 68 (47), 66 (64).

Analysis calculated for C₁₄H₁₁N₃O₅: C, 55.82; H, 3.68; N, 13.95. Found: C, 55.98; H, 3.78; N, 14.08.

(Z)-7-Methyl-2-(1-methyl-4-nitroimidazole-5-ylmethylene)-3(2H)-benzofuranone (7l).

Yield, 52%; mp 266°C to 268°C; IR (KBr): 1,716 (CO), 1,501, 1,342 (NO₂) cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.05 (s, 1H), 7.67 (d, J = 7.5 Hz, 1H), 7.65 (d, J = 7.5 Hz, 1H), 7.26 (t, J = 7.5 Hz, 1H), 7.10 (s, 1H), 3.83 (s, 3H), 2.35 (s, 3H); ¹³C NMR (DMSO-d₆) δ 183.68, 164.50, 148.76, 146.67, 139.49, 137.91, 124.79, 124.65, 123.24, 122.45, 120.56, 96.94, 34.32, 13.98; MS m/z (%) 285 (M⁺, 3), 240 (39), 150 (100), 134 (100), 107 (57), 89 (44), 68 (61), 66 (61).

Analysis calculated for C₁₄H₁₁N₃O₄: C, 58.95; H, 3.89; N, 14.73. Found: C, 58.80; H, 3.77; N, 14.96.

(Z)-7-Methoxy-2-(1-methyl-4-nitroimidazole-5-ylmethylene)-3(2H)-benzofuranone (7m).

Yield, 41%; mp 265°C to 267°C; IR (KBr): 1,706 (CO), 1,501, 1,352 (NO₂) cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.05 (s, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.28 (t, J = 8.0 Hz, 1H), 7.11 (s, 1H), 3.91 (s, 3H), 3.80 (s, 3H); ¹³C NMR (DMSO-d₆) δ 183.16, 154.85, 148.44, 145.85, 145.40, 139.22, 125.21, 124.71, 121.86, 120.61, 115.52, 97.42, 56.40, 34.00; MS m/z (%) 301 (M⁺, 2), 283 (53), 237 (29), 196 (100), 168 (33), 150 (39), 120 (54), 87 (52), 52 (32).

Analysis calculated for C₁₄H₁₁N₃O₅: C, 55.82; H, 3.68; N, 13.95. Found: C, 55.95; H, 3.58; N, 13.80.

In vitro antileishmanial assays.

(i) Activity against L. major promastigotes. The anti-promastigote activity of the compounds against L. major (MHOM/Pk/88/DESTO) was evaluated by slight modifications to the assay described by Habtemariam et al. (27, 28). L. major parasite promastigotes were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, USA) supplemented with 10% heat-inactivated fetal calf serum (PAA Laboratories GmbH, Pasching, Austria). Parasites at log phase were centrifuged at 2,000 rpm for 10 min and washed three times with saline.

The drug susceptibility assay was carried out in a 96-well microtiter plate. The test compounds (20 μL dissolved in DMSO in stock concentrations of 5 μg/mL) were diluted in the culture medium (to 100 μL) in each well and serially diluted. Parasites were diluted with fresh culture medium to a final density of 1 × 10⁶ cells/mL and were added (100 μL) to each well. Two rows were left for negative (DMSO) and positive controls for the reference drugs amphotericin B (MP Biomedical Inc., Santa Anna, CA, USA), pentamidine (ICN Biomedical Inc., Costa Mesa, CA, USA), and nifurtimox. The plate was incubated at 22°C to 25°C for 72 h. At the end of incubation, cell viability was measured by counting the number of motile cells using an improved Neubaure counting chamber, and the IC₅₀ values of tested compounds were calculated by Software Ezfit 5.03 Perella Scientific. All assays were run in triplicate.

(ii) Activity against L. donovani axenic amastigotes. Amastigotes of L. donovani strain MHOM/ET/67/L82 were grown in axenic culture at 37°C in SM medium (29) (pH 5.4) supplemented with 10% heat-inactivated fetal bovine serum (FBS) under an atmosphere of 5% CO₂ in air. A 100-μL volume of culture medium with 10⁵ amastigotes from axenic culture with or without a serial drug dilution was seeded in 96-well microtiter plates. Serial drug dilutions of 11 3-fold dilution steps ranging from 100 to 0.002 μg/mL were prepared. After 70 h of incubation, the plates were inspected under an inverted microscope to ensure the growth of the controls and sterile conditions. Ten μL of alamarBlue (12.5 mg resazurin dissolved in 100 mL distilled water) (38) was then added to each well and the plates were incubated for another 2 h. Next, the plates were read with a Spectramax Gemini XS microplate fluorometer (Molecular Devices Cooperation, Sunnyvale, CA, USA) using an excitation wavelength of 536 nm and emission wavelength of 588 nm. Data were analyzed using the software Softmax Pro (Molecular Devices Cooperation, Sunnyvale, CA, USA). Decrease in fluorescence (= inhibition) was expressed as a percentage of the fluorescence of control cultures and plotted against the drug concentrations. The IC₅₀ values were calculated from the sigmoidal inhibition curves (30, 31).

(iii) Activity against L. donovani intracellular amastigotes. Peritoneal mouse macrophages (4 × 10⁴ in 100 μL RPMI 1640 medium with 10% heat-inactivated FBS) were seeded into the wells of a 96-well plate (32). After 24 h, 2 × 10⁵ amastigotes of L. donovani (in 100 μL) were added. The amastigotes were taken from an axenic amastigote culture grown at pH 5.4. At 24 h later, the medium containing free amastigote forms was removed and replaced by fresh medium. The washing step was repeated once more and, then the serial drug dilution was prepared with at least 6 steps. Compounds were dissolved in DMSO at 10 mg/mL and further diluted in medium. After 96 h of incubation at 37°C under a 5% CO₂ atmosphere, the medium was removed, and cells were fixed by adding 50 μL of 4% formaldehyde solution followed by staining with a 5 μM DRAQ5 solution. Plates were imaged using an ImageXpress XLS (MD) microscope with a 20× air objective (635 nm excitation, 690/50 emission). Nine images were collected per well. Automated image analysis was performed using a script developed on MetaXpress Software (MD). Three outputs were provided for each well: (i) number of host cell nuclei, (ii) number of infected and noninfected cells, and (iii) number of intracellular amastigotes per cell. The IC₅₀ values were calculated based on the infection rate and the numbers of intracellular amastigotes.

Measuring antileishmanial activity of compounds against L. donovani promastigotes, wild-type, overexpressing NTR1 and NTR2 cell lines.

The clonal Leishmania donovani cell line LdBOB (derived from MHOM/SD/62/1S-CL2D) was grown as promastigotes at 26°C in modified M199 medium as previously described (34). LdBOB promastigotes overexpressing NTR1 (LinJ.05.0660) (14) and NTR2 (LinJ.12.0730) (18) were grown in the presence of nourseothricin (100 μg mL⁻¹).

In vitro drug sensitivity assays.

To examine the effects of the test compounds on growth, triplicate promastigote cultures were seeded with 5 × 10⁴ parasites mL⁻¹. Parasites were grown in 10-mL cultures in the presence of the tested compounds for 72 h, after which 200-μL aliquots of each culture were added to 96-well plates, 50 μM resazurin was added to each well, and fluorescence (excitation 528 nm, emission 590 nm) was measured after a further 2 h of incubation (39). Data were processed using GRAFIT (version 5.0.4; Erithacus software) and fitted to a 2-parameter equation, where the data were corrected for background fluorescence, to obtain the effective concentration inhibiting growth by 50% (EC₅₀):

$$y=\frac{100}{1+{\left(\frac{\left[I\right]}{{\text{EC}}_{50}}\right)}^{\text{m}}}$$

In this equation, I represents inhibitor concentration and m is the slope factor. Experiments were repeated at least two times, and the data are presented as means ± standard deviation.

In vitro cytotoxicity assay in PMM cells.

(i) Isolation of peritoneal mouse macrophages. One or two mice were inoculated i.p. with 200 to 300 μL each of a 2% starch solution in distilled water (0.2g/10 mL). After 24 to 48 h, the macrophages were isolated by flushing the peritoneal cavity with 10 mL RPMI medium. The isolated macrophage suspension was centrifuged for 10 min at 1,500 rpm. The supernatant was discarded, and the pellet resuspended in 5 mL RPMI medium. Cell density was determined by counting with a hemocytometer. The macrophage concentration was adjusted using RPMI +10% FCS +1% MäserMix to 4 × 10⁵ cells/mL (4 × 10⁴/well) and seeded in the well plates.

(ii) In vitro cytotoxicity assay. Peritoneal mouse macrophages (4 × 10⁴ in 100 μL RPMI 1640 medium with 10% heat-inactivated FBS) were seeded into the wells of a 96-well plate (32). Serial drug dilutions were prepared with at least 6 steps. Compounds were dissolved in DMSO at 10 mg/mL and further diluted in medium. After 96 h of incubation, the plates were inspected under an inverted microscope to ensure sterility. Alamar Blue (20 μL of a solution consisting of 12.5 mg resazurin [Sigma-Aldrich] dissolved in 100 mL phosphate-buffered saline) was added to each well and the plates incubated for a further 4 h. The plates were then read with a Spectramax Gemini XS microplate fluorometer (Molecular Devices Cooperation, Sunnyvale, CA, USA) using an excitation wavelength of 536 nm and an emission wavelength of 588 nm. The IC₅₀ values were calculated by linear regression from the sigmoidal dose inhibition curves using SoftmaxPro software (Molecular Devices Cooperation, Sunnyvale, CA, USA).

In vitro cytotoxicity assay in THP-1 cell lines.

To determine the toxicity of experimental compounds, THP-1 cells were seeded in sterile flat-bottomed 96-well culture plates (Orange Scientific) containing 4 × 10⁴ cells/mL and incubated for 24 h with a supply of 5% CO₂ at 37°C (33). Experimental compounds at concentrations ranging from 10 to 20 μM (dissolved in DMSO) were added to the plates to provide a concentration range of 50 to 0.1 μM. The THP-1 cells were also incubated without compounds and in the presence of 0.5% DMSO. Plates were incubated for 72 h and an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was performed to assess cell viability. To determine the viability of THP-l cells, the plates were centrifuged (1,500 rpm, 20 min) and the optical density (OD) of each well was read in the presence and absence of compounds at 570 and 630 nm. Results were expressed as the percentage reduction of THP-1 cells compared with nontreated control samples and are shown as 50% inhibitory concentration THP-1 cells (CC₅₀).