An Efficient One-Pot Catalyzed Synthesis of 2,4-Disubstituted 5-Nitroimidazoles Displaying Antiparasitic and Antibacterial Activities

A one-pot regioselective bis-Suzuki-Miyaura or Suzuki-Miyaura/Sonogashira reaction on 2,4-dibromo-1-methyl-5-nitro-1H-imidazole under microwave heating was developed. This method is applicable to a wide range of (hetero)arylboronic acids and terminal alkynes. Additionally, this approach provides a simple and efficient way to synthesize 2,4-disubstituted 5-nitroimidazole derivatives with antibacterial and antiparasitic properties.


Introduction
Nitroimidazole drugs show a wide range of activities against parasites and anaerobic bacteria. These activities vary according to the nature of the substituents and the position of the nitro group [1][2][3][4][5][6][7]. Metronidazole (mtz) appears on the WHO Model List of Essential Medicines and is today the most commonly used 5-nitroimidazole compound worldwide. 5-Nitroimidazoles are prodrugs which need to be activated by reduction of their nitro group. Reduced metronidazole forms covalent bonds to DNA macromolecules, leading to the loss of helical structure and irreversible breaking [8]. The reduction of these compounds involves several anaerobic enzymatic pathways such as ferredoxin oxidoreductase [9], nitroreductase [10] and thioredoxin reductase [11].
However, higher doses and longer regimens may promote side effects and decrease compliance. Although the most common side effects of metronidazole treatment are mild (headache, vertigo, nausea, a metallic taste in the mouth and an Antabuse-like effect with alcohol) [25], other more serious adverse effects have been documented, such as mutagenicity on bacteria [26][27][28][29][30][31] and carcinogenicity on carcinogenicity on mice and rats [32]. Moreover, in recent decades, an increasing number of anaerobic bacterial pathogens have been observed to cause severe diarrhea and enterocolitis [33][34][35][36].
Palladium-catalyzed reactions, particularly Suzuki-Miyaura cross-coupling, are widely applied in the synthesis of bioactive complex molecules, offering mild reaction conditions and compatibility with a broad range of functional groups [51,52]. Moreover, some Suzuki-Miyaura cross-coupling conditions allow regioselective cross-coupling on polyhalogenated heterocycles [53,54], leaving a halogenated α position unchanged and able to react in a second step. This enables a wide range of compounds with the same starting material.
The regioselectivity in the Suzuki-Miyaura reaction is driven by the electrophilic properties of the halogenated carbon. During the oxidative addition step, the palladium catalyst which is a nucleophilic species, specifically links the most electrophilic halogenated carbon [53][54][55]. The nitro group, essential for medicinal properties, can also act as a chemical directing group during synthesis [56]. Thus, the nitro group in position 5 can significantly increase the electrophilic properties of the C-4, making this position the most reactive toward cross-coupling reactions, although on the naked imidazole ring, the C-2 position was described as the most reactive [57,58].
Finally, the antiparasitic activity of the obtained 5-nitroimidazole derivatives was assessed against T. vaginalis and anaerobic bacteria of the genus Clostridium and Bacillus.
We began our study by optimizing the regioselective coupling reaction of compound 2 with phenylboronic acid (1.1 equiv.) in the presence of Pd(PPh3)4 (0.04 equiv.) as catalyst, Na2CO3 (3 equiv.) as base, in a mixture of DME/EtOH/H2O (3/1/1) as solvent. First, the reaction was performed at room temperature, and after 1.5 h, no conversion of compound 2 was observed ( Table 1, entry 1). When the temperature was increased to 60 °C, after 48 h of reaction, a mixture of compounds 2/3a/4/5 was observed with both low conversion and selectivity (LC/MS ratio 25/28/26/21%, respectively) ( Table 1, entry 2). In these conditions, the reactivity of positions C-2 and C-4 was similar. The use of microwave irradiation at 80 °C for 1 h (Table 1, entry 4) allowed better conversion of compound 2 in the Suzuki-Miyaura reaction, with lower selectivity and improved yield of the dicoupled compound 5. Next, we examined the influence of other palladium sources such as PdCl2(PPh3)2, Pd(DPPF)Cl2 and Pd(OAc)2. With PdCl2(PPh3)2 and Pd(DPPF)Cl2, low conversion was observed and there was no improvement in terms of selectivity (Table 1, entries 5 and 6). To our satisfaction, good conversion and selectivity were observed with Pd(OAc)2 and compound 3a was obtained in 60% yield ( So, the best reaction conditions were 1 equivalent of imidazole 2, 1.3 equivalents of phenylboronic acid, 0.04 equivalents of palladium acetate and 3 equivalents of sodium carbonate in DME/EtOH/H2O (3/1/1) for 2 h at 60 °C under microwave irradiation. With the optimized reaction conditions in hand, we assessed the scope and limitations of the regioselective Suzuki-Miyaura reaction of 2,4-dibromo-1-methyl-5-nitro-1H-imidazole (2). A variety of substituted arylboronic acids (with both electron-donating or electron-withdrawing groups) were used to give the corresponding coupling products 3a-i in moderate to good yields (55-65%). However, the use of hydroxymethylphenylboronic acid (3i) and boronic acids substituted by withdrawing groups such as NO2 or CF3 required a temperature increase to 65 °C ( So, the best reaction conditions were 1 equivalent of imidazole 2, 1.3 equivalents of phenyl-boronic acid, 0.04 equivalents of palladium acetate and 3 equivalents of sodium carbonate in DME/EtOH/H 2 O (3/1/1) for 2 h at 60 • C under microwave irradiation. With the optimized reaction conditions in hand, we assessed the scope and limitations of the regioselective Suzuki-Miyaura reaction of 2,4-dibromo-1-methyl-5-nitro-1H-imidazole (2). A variety of substituted arylboronic acids (with both electron-donating or electron-withdrawing groups) were used to give the corresponding coupling products 3a-i in moderate to good yields (55-65%). However, the use of hydroxymethylphenylboronic acid (3i) and boronic acids substituted by withdrawing groups such as NO 2 or CF 3 required a temperature increase to 65 • C ( heteroarylboronic acids (pyridine-3-ylboronic acid, 5-methylthiophen-2-ylboronic acid) led to very low conversion under our optimal conditions. Increasing the temperature in these cases improved the yield of the corresponding dicoupled product. hand, the use of heteroarylboronic acids (pyridine-3-ylboronic acid, 5-methylthiophen-2-ylboronic acid) led to very low conversion under our optimal conditions. Increasing the temperature in these cases improved the yield of the corresponding dicoupled product.  The X-ray crystal structure ( Figure 1) was determined for compound 3c to confirm the regioselective Suzuki-Miyaura reaction on 4-position of 2,4-dibromo-1-methyl-5-nitro-1H-imidazole (2). Following optimization of the regioselective Suzuki-Miyaura reaction, we investigated the onepot regioselective bis-Suzuki-Miyaura reactions of 2,4-dibromo-1-methyl-5-nitro-1H-imidazole (2).
The sequential one-pot two-step bis-Suzuki-Miyaura reactions of compound 2 were studied using phenylboronic acid and 4-methoxyphenylboronic acid under microwave irradiation. Phenylboronic acid can be expected to react selectively at the C-4 position, with the subsequent addition of 4methoxyphenylboronic acid leading to reaction at the C-2 position of compound 2 ( Table 3).
The first step was performed in the presence of phenylboronic acid (1.3 equiv.), Pd(OAc)2 (0.04 equiv.), Na2CO3 (3 equiv.) in DME/EtOH/H2O (3/1/1) at 60 °C under microwave irradiation for 2 h. In the second step, the reaction was carried out without addition of a palladium catalyst and in presence of 4-methoxyphenylboronic acid (1.3 equiv.), Na2CO3 (3 equiv.) at 60 °C under microwave irradiation for 1 h, giving the expected disubstituted imidazole 6 in moderate yield (22%, Table 3, entry 1). These conditions did not allow a complete conversion of the intermediate compound 3a, which remains the majority product with a yield of 47%. Adding Pd(OAc)2 (0.04 equiv.) in the second step increased the yield of compound 6 to 30% (Table 3, entry 2). Performing the reaction at 110 °C increased the yield of compound 6 to 40% (Table 3, entry 3). Interestingly, using Pd(PPh3)4 (0.04 equiv.) or adding only PPh3 (0.1 equiv.) in the second step led to complete conversion of the intermediate 3a and the corresponding product 6 isolated respectively in 60 and 64% yields ( The X-ray crystal structure ( Figure 1) was determined for compound 3c to confirm the regioselective Suzuki-Miyaura reaction on 4-position of 2,4-dibromo-1-methyl-5-nitro-1Himidazole (2). hand, the use of heteroarylboronic acids (pyridine-3-ylboronic acid, 5-methylthiophen-2-ylboronic acid) led to very low conversion under our optimal conditions. Increasing the temperature in these cases improved the yield of the corresponding dicoupled product. Table 2. Scope of the regioselective Suzuki-Miyaura reaction.
The sequential one-pot two-step bis-Suzuki-Miyaura reactions of compound 2 were studied using phenylboronic acid and 4-methoxyphenylboronic acid under microwave irradiation. Phenylboronic acid can be expected to react selectively at the C-4 position, with the subsequent addition of 4methoxyphenylboronic acid leading to reaction at the C-2 position of compound 2 ( Table 3).
The first step was performed in the presence of phenylboronic acid (1.3 equiv.), Pd(OAc)2 (0.04 equiv.), Na2CO3 (3 equiv.) in DME/EtOH/H2O (3/1/1) at 60 °C under microwave irradiation for 2 h. In the second step, the reaction was carried out without addition of a palladium catalyst and in presence of 4-methoxyphenylboronic acid (1.3 equiv.), Na2CO3 (3 equiv.) at 60 °C under microwave irradiation for 1 h, giving the expected disubstituted imidazole 6 in moderate yield (22%, Table 3, entry 1). These conditions did not allow a complete conversion of the intermediate compound 3a, which remains the majority product with a yield of 47%. Adding Pd(OAc)2 (0.04 equiv.) in the second step increased the yield of compound 6 to 30% (Table 3, entry 2). Performing the reaction at 110 °C increased the yield of compound 6 to 40% (Table 3, entry 3). Interestingly, using Pd(PPh3)4 (0.04 equiv.) or adding only PPh3 (0.1 equiv.) in the second step led to complete conversion of the intermediate 3a and the corresponding product 6 isolated respectively in 60 and 64% yields ( Table 3, entries 4 and 5). Note that, when the reaction temperature was reduced to 90 °C, incomplete conversion of the intermediate 3a was observed (according to LC/MS analysis). Following optimization of the regioselective Suzuki-Miyaura reaction, we investigated the one-pot regioselective bis-Suzuki-Miyaura reactions of 2,4-dibromo-1-methyl-5-nitro-1H-imidazole (2).
The sequential one-pot two-step bis-Suzuki-Miyaura reactions of compound 2 were studied using phenylboronic acid and 4-methoxyphenylboronic acid under microwave irradiation. Phenylboronic acid can be expected to react selectively at the C-4 position, with the subsequent addition of 4-methoxyphenylboronic acid leading to reaction at the C-2 position of compound 2 ( Table 3).
The first step was performed in the presence of phenylboronic acid (1.3 equiv.), Pd(OAc) 2 (0.04 equiv.), Na 2 CO 3 (3 equiv.) in DME/EtOH/H 2 O (3/1/1) at 60 • C under microwave irradiation for 2 h. In the second step, the reaction was carried out without addition of a palladium catalyst and in presence of 4-methoxyphenylboronic acid (1.3 equiv.), Na 2 CO 3 (3 equiv.) at 60 • C under microwave irradiation for 1 h, giving the expected disubstituted imidazole 6 in moderate yield (22%, Table 3, entry 1). These conditions did not allow a complete conversion of the intermediate compound 3a, which remains the majority product with a yield of 47%. Adding Pd(OAc) 2 (0.04 equiv.) in the second step increased the yield of compound 6 to 30% (Table 3, entry 2). Performing the reaction at 110 • C increased the yield of compound 6 to 40% (Table 3, entry 3). Interestingly, using Pd(PPh 3 ) 4 (0.04 equiv.) or adding only PPh 3 (0.1 equiv.) in the second step led to complete conversion of the intermediate 3a and the corresponding product 6 isolated respectively in 60 and 64% yields ( Table 3, entries 4 and 5). Note that, when the reaction temperature was reduced to 90 • C, incomplete conversion of the intermediate 3a was observed (according to LC/MS analysis).  In addition, the use of 4-methoxyphenylboronic acid in the first step and phenylboronic acid in the second step gave the corresponding product 6a in better yield (71%, Scheme 2). Scheme 2. One-pot regioselective bis-Suzuki-Miyaura reaction.
With optimized conditions established, the scope of the one-pot regioselective bis-Suzuki-Miyaura reaction was explored using a variety of (hetero)arylboronic acids. We chose 4-methoxyphenylboronic acid for the first step and different boronic acids for the second step (Table 4). This approach enabled a broad range of (hetero)arylboronic acids bearing an electron-donating or withdrawing group to be used under standard conditions, and provided the disubstituted imidazole derivatives 6a-j in moderate to good yields (Table 4, 50-71%).
The scope of this Suzuki-Miyaura/Sonogashira sequence was then investigated using the optimized procedure. We were pleased to observe the formation of the disubstituted imidazole derivatives 8a-j in moderate to good yields when 4-methoxyphenylboronic acid was used in the first  In addition, the use of 4-methoxyphenylboronic acid in the first step and phenylboronic acid in the second step gave the corresponding product 6a in better yield (71%, Scheme 2).  In addition, the use of 4-methoxyphenylboronic acid in the first step and phenylboronic acid in the second step gave the corresponding product 6a in better yield (71%, Scheme 2). Scheme 2. One-pot regioselective bis-Suzuki-Miyaura reaction.
With optimized conditions established, the scope of the one-pot regioselective bis-Suzuki-Miyaura reaction was explored using a variety of (hetero)arylboronic acids. We chose 4-methoxyphenylboronic acid for the first step and different boronic acids for the second step (Table 4). This approach enabled a broad range of (hetero)arylboronic acids bearing an electron-donating or withdrawing group to be used under standard conditions, and provided the disubstituted imidazole derivatives 6a-j in moderate to good yields (Table 4, 50-71%).
The scope of this Suzuki-Miyaura/Sonogashira sequence was then investigated using the optimized procedure. We were pleased to observe the formation of the disubstituted imidazole derivatives 8a-j in moderate to good yields when 4-methoxyphenylboronic acid was used in the first Scheme 2. One-pot regioselective bis-Suzuki-Miyaura reaction.
With optimized conditions established, the scope of the one-pot regioselective bis-Suzuki-Miyaura reaction was explored using a variety of (hetero)arylboronic acids. We chose 4-methoxy-phenylboronic acid for the first step and different boronic acids for the second step (Table 4). This approach enabled a broad range of (hetero)arylboronic acids bearing an electron-donating or withdrawing group to be used under standard conditions, and provided the disubstituted imidazole derivatives 6a-j in moderate to good yields (Table 4, 50-71%).
The scope of this Suzuki-Miyaura/Sonogashira sequence was then investigated using the optimized procedure. We were pleased to observe the formation of the disubstituted imidazole derivatives 8a-j in moderate to good yields when 4-methoxyphenylboronic acid was used in the first step and various terminal alkynes in the second step (Table 5). Both electron-poor (compounds 8c, 8h and 8i) and electron-rich (compounds 8b, 8d and 8f) substituents on aryl alkynes are well tolerated. Interestingly, this strategy was also tolerant to cycloalkyl alkynes (compounds 8e and 8j) and to a hydroxyl moiety (compound 8g). step and various terminal alkynes in the second step (Table 5). Both electron-poor (compounds 8c, 8h and 8i) and electron-rich (compounds 8b, 8d and 8f) substituents on aryl alkynes are well tolerated. Interestingly, this strategy was also tolerant to cycloalkyl alkynes (compounds 8e and 8j) and to a hydroxyl moiety (compound 8g). Table 4. Scope of one-pot regioselective bis-Suzuki-Miyaura reaction. a naphthalen-2-yl 61 6i 5-methylthiophen-2-yl 52 6j 3  step and various terminal alkynes in the second step (Table 5). Both electron-poor (compounds 8c, 8h and 8i) and electron-rich (compounds 8b, 8d and 8f) substituents on aryl alkynes are well tolerated. Interestingly, this strategy was also tolerant to cycloalkyl alkynes (compounds 8e and 8j) and to a hydroxyl moiety (compound 8g). p naphthalen-2-yl 61 6i 5-methylthiophen-2-yl 52 6j 3   step and various terminal alkynes in the second step (Table 5). Both electron-poor (compounds 8c, 8h and 8i) and electron-rich (compounds 8b, 8d and 8f) substituents on aryl alkynes are well tolerated. Interestingly, this strategy was also tolerant to cycloalkyl alkynes (compounds 8e and 8j) and to a hydroxyl moiety (compound 8g).

Antimicrobial Activity
The antimicrobial activity of the newly synthesized products was evaluated against anaerobic Gram positive bacteria of the genus Clostridium and Bacillus. They were also evaluated for their antiparasitic activity against T. vaginalis. Metronidazole (mtz) was used as control drug to evaluate the potency of the tested compounds under the same conditions.

Antibacterial Activity
Antibacterial activity was determined by the disc diffusion method, according to European committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations [62]. We first screened the 27 molecules synthesized for their antibacterial activity against C. butyricum, C. perfringens, C. neonatale, C. difficile, B. circulans and E. coli. The observed data on the antibacterial activity of the 3 series of compounds are reported for compounds 3a-3i in Table 6, for compounds 6a-6j in Table 7, and for compounds 8a-8j in Table 8. Molecules are considered active when the inhibition diameter is larger than 20 mm, and very active when it is larger than the inhibition diameter of metronidazole. Table 6.
Antibacterial activity of molecules 3a-3i obtained with the first regioselective Suzuki-Miyaura reaction.      Results show that none of the molecules tested, including metronidazole, were active on facultative anaerobic bacteria (B. circulans) and aerobic bacteria (E. coli). Indeed, to be active, 5-nitro-imidazole compounds must undergo a reduction of their nitro group which can only be carried out in an anaerobic environment [2]. If the molecules were active on aerobic bacteria, this would suggest another mechanism of action.

Bacterial Species
For the anaerobic bacteria, it was observed that compounds 3b, 3e-i (Table 6), with a bromine in 2-position, possessed an activity on C. butyricum Nec 0, C. neonatale and C. difficile equivalent or higher than the standard metronidazole. On the other hand, compounds 6a-j (Table 7) with an aryl group in 2-position had a lower activity than compounds 3a-j. Only compound 6a was active on C. butyricum Nec 0, C. neonatale and C. difficile. Compounds 8a-j, the last series, with an alkyne in 2-position, had a lower activity than the molecules of the first two series ( Table 8).
The second step consisted in testing the most active molecules, 3b, 3e-i on other particularly virulent bacteria such as B. cereus, C. beijerenckii, C. tetani and C. difficile 027 ( Table 9). The first results showed that a bromine in 2-position could improve the activity of our compounds, so, we decided to test the starting compound 2 with two bromine atoms in 2 and 4-position. Unfortunately, this last compound did not exhibit any activity (Table 9). Whatever the nature of the aryl group in 4-position, the molecules with a bromine in 2-position (3b, 3e-3i) had promising activity on C. butyricum Nec 0, Nec 1, Nec 8 and on C. difficile O27. One of them, compound 3g, with a trimethoxyphenyl group in 4-position, exhibited higher activity against C. butyricum Nec 8 than metronidazole. It was thus observed from the investigation on anaerobic bacteria screening data that the combination of a bromine atom in 2-position and an aryl group in 4-position enhanced the compounds' antibacterial activities.

Antiparasitic Activity
We selected the six molecules 3b, 3e-3i showing the most promising activities against anaerobic bacteria, and assessed their therapeutic interest on T. vaginalis by determining minimum inhibitory concentration (MIC, Table 10). We also performed a cytotoxicity evaluation via the Neutral Red Uptake (NRU) toxicity test [63] on CHO-K1 Chinese Hamster Ovary cells (ATCC CCL61) to detect cytotoxic concentrations of 50% (CC 50 ). This procedure enabled us to determine the selectivity index of these 6 compounds (Table 10), all of which are shown to have a significantly higher activity, with lower MICs, than metronidazole. On the other hand, they have a significant and higher toxicity than metronidazole, especially for compounds 3b, 3f and 3i with methoxyphenyl, fluorophenyl or hydroxymethylphenyl groups in the 4-position.

Cytotoxicity Evaluation
The molecules with an aryl (6a-j) or alkyne (8a-j) group at 2-position show cytotoxicity equivalent to that of molecules with a bromine in 2-position (3a-i) ( Table 11). The homogeneity of the cytotoxicity of our molecules in the three different series shows the importance of the substituent at the 2-position of the imidazole nucleus.  6 ]DMSO solvents at the Faculté de Pharmacie de Marseille. Solvents were dried by conventional methods. The following adsorbent was used for column chromatography: silica gel 60 (Merck, Darmstadt, Germany, particle size 0.063-0.200 mm, 70-230 mesh ASTM). TLC was performed on 5 cm × 10 cm aluminium plates coated with silica gel 60F-254 (Merck) in an appropriate eluent. Microwave reactions were performed with an Initiator microwave oven (Biotage ® , Charlotte, NC, USA) using 2-5 mL sealed vials; temperatures were measured with an IR-sensor and reaction times are given as hold times.

In Vitro Antibacterial Activity
Bacterial strains: the bacteria tested were obtained from the bacteriology laboratory of IHU Mediterranée Infection (Marseille, France). Each bacterium was cultured in anaerobic condition on 5% sheep blood-enriched Columbia agar (Biomérieux, Marcy l'Etoile, France), incubated at 37 • C for 48 h. The derivatives were dissolved in DMSO stock solution: 500 µg/mL. Thereafter, in a sterile medium and in a logarithmic growth phase, a concentration of 1 McFarland (3.10 8 CFU/mL) was obtained using a spectrophotometer. First, in the 5% sheep blood-enriched Columbia agar medium, the superficial bacterial culture was performed with a swab impregnated with the bacterial suspension. Then, 10 µL of each derivative were deposited on blank sterile discs. For the negative control, the DMSO-impregnated disc was used. After 48 h of incubation, the growth inhibition zone diameter was measured.

In Vitro Antiparasitic Activity
For Trichomonas vaginalis the MIC was carried out in a sterile 96-well plate by broth microdilution according to EUCAST guidelines, and using LYI medium. First, 190 µL of the LYI broth medium, was added to each well. Then, 10 µL of each imidazole derivative with the primary concentration 200 µg/mL was added to the first wells. After mixing, 100 µL of this mixture was embedded into the second well. A similar dilution procedure was carried out in the first wells line for metronidazole as control group. 100 µL of the trichomonas suspension (10 5 cells/mL) was added to each well. For negative control, 200 µL of LYI broth medium were added to the border wells. The plates were incubated under anaerobic conditions 24 h at 37 • C.

Principle of the Assay
The NRU toxicity test is based on concentration-dependent reduction of the uptake of the vital dye Neutral Red measured 24 h after chemical treatment. Neutral red is a weak cationic dye that readily penetrates cell membranes by non-diffusion, accumulating intracellularly in lysosomes. Alterations of the cell surface or the sensitive lysosomal membrane lead to lysosomal fragility and other changes that gradually become irreversible. Such changes brought about by the action of xenobiotics result in a decreased uptake and binding of NR in non-viable cells.

Test Procedure
Cells were seeded into two 96-well tissue culture plates (0.1 mL per well), at a concentration of 1.10 5 cells/mL, and incubated at 37 • C (5% CO 2 ) for 24 h until confluent. The culture medium was decanted and replaced by 100 µL of fresh medium containing the appropriate concentrations of the test substances (8 different concentrations in triplicate), then cells were incubated at 37 • C (5% CO 2 ) in the dark for 24 h. At the end of the incubation period, cells were washed, placed into Neutral Red medium (50 µg/mL Neutral Red in complete medium) and incubated for 3 h at 37 • C, 5% CO 2 . Then, the medium was removed and cells were washed three times with 0.2 mL of PBS to remove excess dye. The Neutral Red medium was removed and the distaining solution (50% ethanol, 1% acetic acid, 49% distilled water; 50 µL per well) was added into the wells. Then, the plates were shaken for 15-20 min at room temperature in the dark. The degree of membrane damage (i.e., the increase of released Neutral Red) was measured by a fluorescence-luminescence reader. The Optical Density (OD) of each well was read at 540 nm. The results obtained for wells treated with the test material were compared to those of untreated control wells (100% viability) and converted to percentage values.

Calculation of CC 50
The mean OD value of blank wells (containing only Neutral Red desorbed solution) was subtracted from the mean OD value of three treated wells (dilutions of the test material, positive control or HBSS). The percentages of cell viability were calculated as: (Mean OD of test wells − mean OD of blanks) × 100 Mean OD of negative control − mean OD of blanks The concentration of the test substances causing a 50% release of Neutral Red as compared to the control culture was calculated by non-linear regression analysis using the Phototox Version 2.0 software (Zebet, Berlin, Germany).

Conclusions
In summary, we describe here a novel and efficient method to access 2,4-disubstituted 5-nitro-imidazole derivatives under microwave irradiation. We have developed original one-pot regioselective bis-Suzuki-Miyaura or Suzuki-Miyaura/Sonogashira reactions of 2,4-dibromo-1-methyl-5-nitro-1H-imidazole (2) allowing the functionalization at both C-2 and C-4 positions of this scaffold. Moreover, this method tolerates a wide range of boronic acids and terminal alkynes and provides versatile and rapid access to 2,4-disubstituted 5-nitroimidazole derivatives in moderate to good yields. The synthesized products were tested for their antimicrobial activities and compared with the standard metronidazole. Compounds obtained with the regioselective Suzuki-Miyaura reaction, bearing a bromine in 2-position and aryl or hetero aryl group in 4-position (compounds 3b, 3e-i), showed an equivalent activity to metronidazole against anaerobic bacteria, and higher activity against T. vaginalis.