An efficient one-pot catalyzed synthesis of 2,4-disubstituted 5-nitroimidazoles displaying antiparasitic and antibacterial activities

Article abstract of DOI:10.3390/molecules22081278

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.

Full text of DOI:10.3390/molecules22081278

molecules
Article
An Efficient One-Pot Catalyzed Synthesis of
2,4-Disubstituted 5-Nitroimidazoles Displaying
Antiparasitic and Antibacterial Activities
Fanny Mathias 1, Youssef Kabri 1, Liliane Okdah 2, Carole Di Giorgio 3, Jean-Marc Rolain 2,
Cédric Spitz 1, Maxime D. Crozet 1 and Patrice Vanelle 1,
*
1
Aix Marseille University, Institut de Chimie Radicalaire ICR, UMR CNRS 7273, Laboratoire de
Pharmaco-Chimie Radicalaire, Faculté de Pharmacie, 27 Boulevard Jean Moulin—CS 30064,
13385 Marseille CEDEX 05, France; fanny.mathias@laposte.net (F.M.); youssef.kabri@univ-amu.fr (Y.K.);
cedric.spitz@univ-amu.fr (C.S.); maxime.crozet@univ-amu.fr (M.D.C.)
IHU Méditerranée Infection, Aix Marseille University, Unité de Recherche sur les Maladies Infectieuses et
Tropicales Emergentes, URMITE UMR 63, CNRS 7278, IRD 198, Inserm 1095, Faculté de Médecine de la
Timone, 19-21 Boulevard Jean Moulin, 13005 Marseille, France; liliane.okdah@hotmail.com (L.O.);
jean-marc.rolain@univ-amu.fr (J.-M.R.)
2
3
Aix Marseille University, CNRS, IRD, Avignon Université, IMBE UMR 7263, Laboratoire de Mutagénèse
Environnementale, 13385 Marseille, France; carole.di-giorgio@univ-amu.fr
*
Correspondence: patrice.vanelle@univ-amu.fr; Tel.: +33-4-9183-5580
Received: 20 July 2017; Accepted: 29 July 2017; Published: 3 August 2017
Abstract: 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.
Keywords: 5-nitroimidazole; Suzuki-Miyaura/Sonogashira; regioselectivity; microwave; antiparasitic;
antibacterial
1. 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 [1–7].
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].
Resistance to metronidazole has emerged, but despite studies on Trichomonas vaginalis [
9,12–15],
Giardia intestinalis [16 18], and anaerobic bacteria [19 23], the mechanism is not yet fully understood.
–
–
Such resistance leads physicians to use higher doses of metronidazole and longer courses of treatment
to improve activity [24].
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 [26–31] and carcinogenicity on
Molecules 2017, 22, 1278; doi:10.3390/molecules22081278
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Molecules 2017, 22, 1278
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mice and rats [32]. Moreover, in recent decades, an increasing number of anaerobic bacterial pathogens
have been observed to cause severe diarrhea and enterocolitis [33–36].
Therefore, there is an ongoing search for alternative 5-nitroimidazoles effective on
anaerobic-resistant strains without serious adverse effects [37–48]. Following the work of Walsh et al.
in 1987 [49], we focused our research on pharmacomodulation of the 2- and 4-positions of the
5-nitroimidazole scaffold [50].
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
allow regioselective cross-coupling on polyhalogenated heterocycles [53
,
52]. Moreover, some Suzuki-Miyaura cross-coupling conditions
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 [53–55]. 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].
As part of our research program centered on the design and synthesis of novel 5-nitroimidazole
compounds [40,43,48,50,59,60], we decided to exploit this particular reactivity of the 5-nitroimidazole
series toward cross-coupling reactions, seeking a rapid and versatile pathway for the synthesis
of 2,4-disubstitued 5-nitroimidazole derivatives. Therefore, we report herein a regioselective
Suzuki-Miyaura cross-coupling between 2,4-dibromo-1-methyl-5-nitro-1H-imidazole (2) and aryl or
heteroarylboronic acids resulting in the formation of 2-bromo-4-substituted-1-methyl-5-nitroimidazoles
which can then undergo a second (Suzuki-Miyaura or Sonogashira) cross-coupling reaction to obtain
2,4-disubstituted 5-nitroimidazoles during a sequential one-pot process.
Finally, the antiparasitic activity of the obtained 5-nitroimidazole derivatives was assessed against
T. vaginalis and anaerobic bacteria of the genus Clostridium and Bacillus.
2. Results
2.1. Chemistry
The synthesis of 2,4-dibromo-1-methyl-5-nitro-1H-imidazole (2) was realized from the commercial
product 4(5)-nitroimidazole in two sequential steps, as shown in Scheme 1. The first step consisted
of bromination with elemental bromine in water and in the presence of sodium hydrogen carbonate,
according to Pedada et al. [61]. The 2,4(5)-dibromo-5(4)-nitroimidazole mixture was then alkylated by
dimethyl sulphate (DMS) in DMF leading to two isomeric products 1 and 2 in 71% overall yield.
Scheme 1. Synthesis of 2,4-dibromo-1-methyl-5-nitro-1H-imidazole (2).

Molecules 2017, 22, 1278
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Our particular aim in exploring the reactivity of 2,4-dibromo-1-methyl-5-nitro-1H-imidazole
(2
) in Suzuki-Miyaura reactions was to test the regioselectivity of this type of reaction toward the
halogenated positions C-2 and C-4.
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
the temperature was increased to 60 C, after 48 h of reaction, a mixture of compounds
2
was observed (Table 1, entry 1). When
3a
◦
2/
/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 (Table 1, entry 7).
More importantly, the use of 1.3 equiv. of phenylboronic acid for a longer time afforded compound 3a
in better yield (64%, Table 1, entry 12). No yield improvement for compound 3a was observed when
we carried out the reaction in presence of Xantphos (Table 1, entry 8) or increased the temperature
(Table 1, entries 10 and 11). Moderate selectivity was obtained when PPh3 was added (Table 1, entry 9).
These trials with different catalysts revealed that regioselectivity depends on the catalytic system:
Pd(OAc)2 performed best, both on conversion and selectivity, in our reaction conditions.
Table 1. Optimization of the regioselective Suzuki-Miyaura reaction.
◦
Entry
Pd (0.04 equiv.)
Temp ( C)
Time (h)
2 (%)
3a (%)
4 (%)
5 (%)
1 a
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
25
60
1.5
48
100
25
29
12
48
42
20
18
16
5
-
-
26
23
27
16
14
traces
9
-
2 a
28
22
24
27
37
60
58
30
36
41
64
21
26
37
9
7
8
15
14
26
19
9
3 a
60 MW
80 MW
60 MW
60 MW
60 MW
60 MW
60 MW
80 MW
70 MW
60 MW
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
2.0
4 a
Pd(PPh3)4
5 a
PdCl2(PPh3)2
Pd(DPPF)Cl2
Pd(OAc)2
6 a
7 c
8 a
Pd(OAc)2/Xantphos
Pd(OAc)2/PPh3
Pd(OAc)2
9 c
24
10 c
11 c
12 b,c
traces
traces
traces
Pd(OAc)2
Pd(OAc)2
10
13
a
LC/MS ratio; b 1.3 equiv. of phenylboronic acid; c Isolated yields.
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/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 (
electron-donating or electron-withdrawing groups) were used to give the corresponding coupling
products 3a in moderate to good yields (55–65%). However, the use of hydroxymethylphenylboronic
2). A variety of substituted arylboronic acids (with both
–
i
acid (3i) and boronic acids substituted by withdrawing groups such as NO2 or CF3 required
◦
a temperature increase to 65 C (Table 2, Compounds 3c
,
3d). On the other hand, the use of

Molecules 2017, 22, 1278
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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.
Compound
R1
Yield (%)
3a
3b
C6H5
p-(OCH3)-C6H4
p-(CF3)-C6H4
m-(NO2)-C6H4
p-(CN)-C6H4
64
65
62
61
64
62
55
63
58
3c a
3d a
3e
3f
3g
p-(F)-C6H4
m, m, p-(OCH3)-C6H2
p-(CH3)-C6H4
p-(CH2OH)-C6H4
3h
3i a
Reaction conditions: boronic acids (1.3 equiv.), Pd(OAc)2 (0.04 equiv.), Na2CO3 (3 equiv.), DME/EtOH/H2O (3/1/1),
a
MW, 60 ◦C, 2 h. MW, 65 ◦C, 2 h.
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).
Figure 1. X-ray crystal structure of compound 3c.
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.), 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
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
to 30% (Table 3, entry 2). Performing the reaction at 110 ◦C
increased the yield of compound 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 isolated respectively in 60 and 64% yields (Table 3, entries 4
6 in moderate yield (22%, Table 3,
,
6
6
6

Molecules 2017, 22, 1278
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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).
Table 3. Optimization of the second step of the one-pot regioselective bis-Suzuki-Miyaura reaction.
◦
Entry
Catalyst
Temp ( C)
3a (%)
5 (%)
6 (%)
7 (%)
1
2
3
4
5
-
60
60
110
110
110
47
35
21
-
6
6
6
8
12
22
30
40
60
64
13
16
15
18
10
Pd(OAc)2 0.04 equiv.
Pd(OAc)2 0.04 equiv.
Pd(PPh3)4 0.04 equiv.
PPh3 0.1 equiv.
-
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-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%).
In view of these encouraging results, we then turned our attention to the sequential one-pot
regioselective Suzuki-Miyaura/Sonogashira reaction of 2,4-dibromo-1-methyl-5-nitro-1H-imidazole
(2). Having optimized reaction conditions for the regioselective Suzuki-Miyaura coupling, we decided
to develop the one-pot Suzuki-Miyaura/Sonogashira sequence.
Thus, based on the already optimized regioselective Suzuki-Miyaura reaction conditions and
the classical conditions for Sonogashira reaction, we prepared the disubstituted 5-nitroimidazole
derivative 8a from 4-methoxyphenylboronic acid and ethynylbenzene (Scheme 3). The first step was
performed in presence of 4-methoxyphenylboronic acid (1.3 equiv.), Na2CO3 (3 equiv.), Pd(OAc)2
(0.04 equiv.) in a mixture of DME/EtOH/H2O (3/1/1) under microwave irradiation for 2 h at 60 ◦C.
After cooling, ethynylbenzene (1.3 equiv.), PPh3 (0.1 equiv.), CuI (0.1 equiv.) and Et3N (2 equiv.) were
added and the mixture was heated again under microwave irradiation for 1 h at 70 ◦C. This sequence
led to the desired compound 8a in good yield (67%) and compounds 7 and 9 in 11% and 10% yields,
respectively (Scheme 3).
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

Molecules 2017, 22, 1278
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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).
a
Table 4. Scope of one-pot regioselective bis-Suzuki-Miyaura reaction.
H3CO
Br
1) 4-CH3OPhB(OH)2 (1.3 equiv)
N
N
O2N
Br
O2N
6a-j
N
R2
N
2) R2B(OH)2 (1.3 equiv)
CH3
CH3
2
Compound
R2
Yield (%)
6a
6b
6c
6d
6e
6f
6g
6h
6i
C6H5
p-(F)-C6H4
p-(Cl)-C6H4
71
70
60
65
50
58
56
61
52
69
m-(NO2)-C6H4
m, m, p-(OCH3)-C6H2
m-(CF3)-C6H4
p-(CH3)-C6H4
naphthalen-2-yl
5-methylthiophen-2-yl
3-pyridine
6j
a
Reaction conditions: Step 1: 4-methoxyphenylboronic acid (1.3 equiv.), Pd(OAc)2 (0.04 equiv.), Na2CO3 (3 equiv.),
DME/EtOH/H2O (3/1/1), MW, 60 ◦C, 2 h; Step 2: boronic acid (1.3 equiv.), PPh3 (0.1 equiv.), Na2CO3 (3 equiv.),
MW, 110 ◦C, 1 h.
Scheme 3. One-pot regioselective Suzuki-Miyaura/Sonogashira reaction.
a
Table 5. Scope of the one-pot Suzuki-Miyaura/Sonogashira reaction.
H3CO
Br
O2N
N
1) 4-Methoxyphenylbronic acid
2) R3
N
Br
O2N
N
N
H
R3
CH3
CH3
8a-j
2
Compound
R3
Yield (%)
8a
8b
8c
8d
8e
8f
8g
8h
8i
C6H5
m-(CH3)-C6H4
m-fluoro-C6H4
67
74
59
53
60
63
51
61
52
63
o-(CH3)-p-(OCH3)-C6H3
cyclopentyl
p-(tert-butyl)-C6H4
CH2OH
m-chloro-C6H4
o-chloro-C6H4
cyclopropyl
8j
a
Reaction conditions: Step 1: 4-methoxyphenylboronic acid (1.3 equiv.), Pd(OAc)2 (0.04 equiv.), Na2CO3 (3 equiv.),
DME/EtOH/H2O (3/1/1), MW, 60 ◦C, 2 h; Step 2: alkyne (1.3 equiv.), PPh3 (0.1 equiv.), Et3N (2 equiv.), CuI
(0.1 equiv.), MW, 70 ◦C, 1 h.

Molecules 2017, 22, 1278
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2.2. 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.
2.2.1. 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
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.
–3i in Table 6, for compounds 6a–6j in Table 7,
–
Table 6.
Antibacterial activity of molecules 3a–3i obtained with the first regioselective
Suzuki-Miyaura reaction.
Compounds
Bacterial Species
3a
3b
3c
3d
3e
3f
3g
3h
3i
mtz
C. butyricum Nec 0
C. perfringens
C. neonatale
C. difficile
14
>20
25
>20
0
38
0
29
31
0
0
0
14
15
0
26
0
>20
10
0
18
0
24
25
0
14
0
28
28
0
27
0
30
23
0
21
0
25
29
0
0
0
22
32
0
>30
<20
32
>20
<20
0
B. circulans
E. coli
0
0
0
0
0
0
0
0
0
Inhibition zone diameter (mm).
Table 7. Antibacterial activity of molecules 6a–j obtained with the one-pot regioselective
bis-Suzuki-Miyaura reaction.
Compounds
Bacterial Species
6a
6c
6d
6e
6g
6h
6i
6j
mtz
C. butyricum Nec 0
C. perfringens
C. neonatale
C. difficile
24
0
22
23
0
20
0
>20
12
0
29
0
>20
14
0
14
0
>20
14
0
0
0
>20
0
13
0
0
25
0
>20
0
0
0
>20
0
>20
15
0
>30
<20
32
>20
<20
0
0
14
16
0
B. circulans
E. coli
0
0
0
0
0
0
Inhibition zone diameter (mm).
Table 8. Antibacterial activity of molecules 8a
–
j
obtained with the one-pot Suzuki-Miyaura/
Sonogashira reaction.
Compounds
Bacterial Species
8a
8b
8c
8d
8e
8g
8h
8i
8j
mtz
C. butyricum Nec 0
C. perfringens
C. neonatale
C. difficile
0
0
11
0
0
0
25
0
>20
13
0
0
0
29
0
0
0
0
24
0
0
0
17
0
15
17
0
17
0
23
20
0
8
>20
0
0
0
18
0
13
0
0
0
23
0
18
19
0
>30
<20
32
>20
<20
0
B. circulans
E. coli
0
0
0
0
0
0
Inhibition zone diameter (mm).

Molecules 2017, 22, 1278
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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 [
would suggest another mechanism of action.
For the anaerobic bacteria, it was observed that compounds 3b
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 (Table 7) with an aryl group in
2-position had a lower activity than compounds 3a . Only compound 6a was active on C. butyricum
Nec 0, C. neonatale and C. difficile. Compounds 8a , the last series, with an alkyne in 2-position, had a
2]. If the molecules were active on aerobic bacteria, this
,
3e–i (Table 6), with a bromine in
–j
–j
–
j
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
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,
2 with two bromine atoms in 2 and 4-position. Unfortunately, this last
,
–
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.
Table 9. Antibacterial activity of the most promising molecules.
Compounds
Bacterial Species
2
3b
3e
3f
3g
3h
3i
mtz
B. cereus
B. beijerenckii
C. butyricum Nec 0
C. butyricum Nec 1
C. butyricum Nec 8
C. tetani
0
0
0
0
0
0
0
5.8
10.9
0
29.4
32.8
31.3
0
9.6
0
24.6
27
24.8
0
20
9.6
9
26.7
29.8
27.8
0
10.9
0
30.4
32.2
34.5
0
10.7
0
27.6
29.3
27.8
0
11.5
0
30.2
34.1
32
0
29
32.8
0
11
31
34
30
56
30
37
C. perfringens
C. difficile O27
0
32.4
0
30.6
26
33.7
0
29.7
35
Inhibition zone diameter (mm).
2.2.2. 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% (CC50). 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.

Molecules 2017, 22, 1278
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Table 10. Antiparasitic evaluation on T. vaginalis.
Compounds
Activity T. vaginalis MIC (µM)
Cytotoxicity CC50 (µM)
Selectivity Index a
3b
3e
3f
3g
3h
3i
4.00
4.07
1.04
0.84
1.05
0.99
7.30
2.58
-
0.64
-
1.72
2.59
3.59
1.13
557.34
1.65
3.08
3.42
1.13
18.65
mtz
a
Selectivity index calculated according to the formula: IC50/MIC.
2.2.3. 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.
Table 11. Cytotoxicity evaluation on Chinese Hamster Ovary cells.
Compounds
Cytotoxicity CC50 (µM)
3a
3b
3d
3f
3g
3h
3i
1.89
2.58
1.87
1.72
2.59
3.59
1.13
6a
6c
6d
6e
6g
6h
6i
3.21
1.41
6.94
0.90
2.97
0.83
1.48
1.24
6j
8a
8b
8c
8d
8e
8g
8h
8i
2.65
1.33
1.35
0.43
2.39
0.27
4.57
2.73
1.17
8j
mtz
557.34
3. Experimental Section
3.1. General Information
Melting points were determined on Büchi B-540 (BÜCHI Labortechnik AG, Flawil, Switzerland)
and are uncorrected. Elemental analyses and HRMS were carried out at the Spectropole, Faculté
des Sciences et Techniques de Saint-Jérôme, Marseille. Elemental analyses were performed on
a EA1112 instrument (Thermo Finnigan, San Jose, CA, USA). HRMS spectra were recorded on a

Molecules 2017, 22, 1278
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QStar Elite (Applied Biosystems SCIEX, Foster City, CA, USA) spectrometer. PEG was a matrix for
HRMS. LC/MS analyses were performed at the Faculté de Pharmacie de Marseille on an Accela High
System® chain U-HPLC coupled with a Thermo MSQ Plus® simple quadrupole. A Thermo Hypersil
Gold® 50
×
2.1 mm chromatographic column was used (SiO2 C18) with 1.9
Analysis is 8 min running, with an MeOH/H2O eluent gradient from 50/50 to 95/05. Both 1H-NMR
spectra (250 MHz, reference CDCl3 = 7.26, [d6]DMSO
= 2.50) and 13C-NMR spectra (62.5 MHz,
reference CDCl3 = 77.0, [d6]DMSO
= 39.7) were recorded at 24 ◦C on an ARX 250 spectrometer
µm diameter particles.
δ
δ
δ
δ
(Bruker, Wissembourg, France) in CDCl3 and [d6]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.
3.2. Chemistry
3.2.1. 2,4-Dibromo-1-methyl-5-nitro-1H-imidazole (2)
First step: to a solution of 4(5)-nitro-1H-imidazole (20 g, 177 mmol), NaHCO3 (44.6 g, 3 equiv.,
◦
531 mmol) in water (120 mL) was added d◦ropwise at 0 C dibromine (27 mL, 3 equiv., 531 mmol)
and the reaction mixture was heated at 65 C for 6 h. After cooling, the solution was poured into
bath of ice. A yellow precipitate appeared and was filtered, washed with water (3
dried in vacuum drying oven (dessicator cabinet). The aqueous layer was extracted with ethyle
acetate (3 100 mL) and the organic layer was washed with brine (3 100 mL), dried over Na2SO4
×
100 mL) and
×
×
and evaporated. Yellow solid was obtained and added to the precipitate to give 36.3 g (80%) of
2,4(5)-dibromo-5(4)-nitroimidazole.
Second step; to a solution of 2,4(5)-dibromo-5(4)-nitroimidazole (34 g, 126 mmol) in DMF (80 mL)
◦
was added dropwise at 0 C dimethyl sulfate (13.1 mL, 1.1 equiv., 138 mmol) and the reaction mixture
◦
was heated at 100 C for 2 h. After cooling, the solution was treated with a saturated solution of
sodium hydrogen carbonate at 0 ◦C. A white precipitate appeared and was filtered. One part of the
precipitate was solubilised in ethyl acetate and evaporated after filtration. The precipitate not dissolved
was the 2,5-dibromo-1-methyl-4-nitro-1H-imidazole (
1H-NMR (CDCl3): = 3.75 (s, 3H, NCH3) ppm. 13C-NMR (CDCl3):
120.4 (CAr) ppm (CNO2 was not visible under these conditions). The aqueous layer was extracted
with ethyl acetate (3 100 mL) and the organic layer was washed with brine (3 100 mL), dried
over Na2SO4 and evaporated. The crude product was purified by column chromatography [silica
gel, petroleum ether/ethyl acetate (9/1) and the 2,4-dibromo-1-methyl-5-nitro-1H-imidazole ( ) was
= 4.02 (s, 3H,
= 37.2 (NCH3), 119.8 (CAr), 126.4 (CAr) ppm (CNO2 was not visible
1
, 38%, 14 g), M.p. 192 ◦C (lit. [26] 204–205 ◦C).
= 35.0 (NCH3), 106.7 (CAr),
δ
δ
×
×
2
obtained in 33% in yield (12.1 g). M.p. 167 ◦C (lit. [42] 160–161 ◦C). 1H-NMR (CDCl3):
δ
NCH3) ppm. 13C-NMR (CDCl3):
under these conditions).
δ
3.2.2. General Procedure for the Regioselective Suzuki-Miyaura Coupling Reaction
A solution of 2,4-dibromo-1-methyl-5-nitro-1H-imidazole ( , 0.1 g, 0.35 mmol), boronic acid
2
(0.46 mmol, 1.3 equiv.), Pd(OAc)2 (3 mg, 0.014 mmol, 0.04 equiv.), Na2CO3 (0.11 g, 1.05 mmol, 3 equiv.),
in a DME/EtOH/H2O mixture (3/1/1, 5 mL) was heated at 60 ◦C under microwave irradiation for
2 h. After cooling, 60 mL of water were added and the solution was extracted with dichloromethane
(3
×
50 mL). The organic layer was washed with water (3
×
100 mL), dried over Na2SO4 and evaporated.
The crude product was purified by column chromatography [silica gel, petroleum ether/ethyl acetate
(9/1), (1/1 for 3i), cyclohexane/ethyl acetate (9/1 for 3a)] and recrystallized from propan-2-ol.

Molecules 2017, 22, 1278
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2-Bromo-1-methyl-5-nitro-4-phenyl-1H-imidazole (3a): Yield 64% (63 mg); yellow solid; M.p. 96 ◦C.
1H-NMR (CDCl3):
13C-NMR (CDCl3):
δ = 4.01 (s, 3H, NCH3), 7.43–7.45 (m, 3H, 3 Ar-H), 7.73–7.77 (m, 2H, 2 Ar-H) ppm.
δ
= 36.5 (NCH3), 126.6 (CAr), 128.1 (2 CHAr), 129.5 (2 CHAr), 129.9 (CHAr), 130.7
(CAr), 136.0 (CAr), 144.3 (CAr) ppm. HRMS (ESI) m/z [M + H]+ calcd. for [C10H8BrN3O2]+: 281.9873;
found 281.9874.
2-Bromo-4-(4-methoxypheny)-1-methyl-5-nitro-1H-imidazole (3b): Yield 65% (71 mg); orange solid;
M.p. 125 ◦C. 1H-NMR (CDCl3):
δ
= 3.86 (s, 3H, NCH3), 4.00 (s, 3H, OCH3), 6.95 (d, 3JH-H = 8.9 Hz,
3
2H, 2 Ar-H), 7.77 (d, JH-H = 8.8 Hz, 2H, 2 Ar-H) ppm. 13C-NMR (CDCl3):
δ = 36.6 (NCH3), 55.3
(OCH3), 113.6 (2 CHAr), 123.0 (CAr), 126.8 (CAr), 131.3 (2 CHAr), 135.8 (CAr), 144.5 (CAr), 161.0 (CAr
ppm. HRMS (ESI) m/z [M + H]+ calcd. for [C11H10BrN3O3]+: 311.9978; found 311.9977.
)
2-Bromo-1-methyl-5-nitro-4-[4-(trifluoromethyl)phenyl]-1H-imidazole (3c): Yield 62% (76 mg); yellow
◦
solid; M.p. 102 C. 1H-NMR (CDCl3):
δ
= 4.03 (s, 3H, NCH3), 7.69 (d, 3JH-H = 8.2 Hz, 2H, 2 Ar-H), 8.87
= 36.6 (NCH3), 123.9 (q, 1JC-F = 272.4 Hz,
(d, 3JH-H = 8.2 Hz, 2H, 2 Ar-H) ppm. 13C-NMR (CDCl3):
δ
CF3), 125.1 (q, 3JC-F = 3.9 Hz, 2 CHAr), 126.9 (C), 129.9 (2 CHAr), 131.6 (q, 2JC-F = 32.5 Hz, CAr), 134.2
(CAr), 136.3 (CAr), 142.4 (CAr) ppm. HRMS (ESI) m/z [M + H]+ calcd. for [C11H7BrF3N3O2]+: 349.9746;
found 349.9744.
2-Bromo-1◦-methyl-5-nitro-4-(3-nitrophenyl)-1H-imidazole (3d): Yield 61% (70 mg); yellow solid;
1
3
M.p. 166 C. H-NMR (CDCl3):
δ = 4.06 (s, 3H, NCH3), 7.63 (t, JH-H = 8.2 Hz, 1H, Ar-H), 8.10
(d, 3JH-H = 7.7 Hz, 1H, Ar-H), 8.28-8.32 (m, 1H, Ar-H), 8.66 (s, 1H, Ar-H) ppm. 13C-NMR (CDCl3):
δ
= 36.8 (NCH3), 124.5 (CHAr), 124.8 (CHAr), 127.1 (CAr), 129.2 (CHAr), 132.4 (CAr), 135.4 (CHAr), 136.3
(CAr), 141.5 (CAr), 148.0 (CAr) ppm. Anal. Calcd. for C10H7BrN4O4 (327.09): C 36.72, H 2.16, N 17.13;
found C 36.82, H 2.06, N 17.13.
4-(2-Brom◦o-1-methyl-5-nitro-1H-imidazol-4-yl)benzonitrile (3e): Yield 64% (69 mg); yellow solid;
1
3
M.p. 155 C. H-NMR (CDCl3):
δ = 4.04 (s, 3H, NCH3), 7.73 (d, JH-H = 8.5 Hz, 2H, 2 Ar-H), 7.88
(d, 3JH-H = 8.5 Hz, 2H, 2 Ar-H) ppm. 13C-NMR (CDCl3):
δ = 35.8 (NCH3), 113.3 (C), 118.4 (CAr), 127.1
(CAr), 130.2 (2 CHAr), 131.9 (2 CHAr), 135.0 (CAr), 136.4 (CAr), 141.8 (CAr) ppm. HRMS (ESI) m/z
[M + H]+ calcd. for [C11H7BrN4O2]+: 306.9825; found 306.9824.
2-Bromo-4-(4-fluorophenyl)-1-methyl-5-nitro-1H-imidazole (3f): Yield 62% (65 mg); yellow solid; M.p.
◦
112 C. 1H-NMR (CDCl3):
δ = 4.01 (s, 3H, NCH3), 7.08–7.15 (m, 2H, 2 Ar-H), 7.74–7.80 (m, 2H, 2 Ar-H)
ppm. 13C-NMR (CDCl3):
δ
= 36.6 (NCH3), 115.2 (d, 2JC-F = 21.5 Hz, 2 CHAr), 126.7 (CAr), 126.8 (CAr),
131.7 (d, 3JC-F = 8.8 Hz, 2 CHAr), 135.9 (CAr), 143.3 (CAr), 163.6 (d, 1JC-F = 250.9 Hz, CAr) ppm. Anal.
Calcd. for C10H7BrFN3O2 (300.08): C 40.02, H 2.35, N 14.00; found C 39.86, H 2.23, N 13.80.
2-Bromo-1-methyl-5-nitro-4-(3,4,5-trimethoxyphenyl)-1H-imidazole (3g): Yield 55% (72 mg); yellow
solid; M.p. 134 ◦C. 1H-NMR (CDCl3):
δ = 3.89 (s, 9H, NCH3, 2 OCH3), 4.00 (s, 3H, OCH3), 7.07 (s, 2H,
2 Ar-H), ppm. 13C-NMR (CDCl3δ = 36.7 (NCH3), 56.2 (2 OCH3), 60.9 (OCH3), 107.0 (2 CHAr), 107.4
(CAr), 125.7 (CAr), 126.6 (CAr), 139.6 (CAr), 144.0 (CAr), 152.8 (2 CAr) ppm. HRMS (ESI) m/z [M + H]+
calcd. for [C13H14BrN3O5]+: 372.0190; found 372.0187.
2-Bromo-1-methyl-5-nitro-4-p-tolyl-1H-imidazole (3h): Yield 63% (65 mg); yellow solid; M.p. 160 ◦C.
1H-NMR (CDCl3):
δ
= 2.40 (s, 3H, CH3), 4.01 (s, 3H, NCH3), 7.24 (d, 3JH-H = 8.2 Hz, 2H, 2 CH ar), 7.66
(d, 3JH-H = 8.2 Hz, 2H, 2 CH ar) ppm. 13C-NMR (CDCl3):
δ = 21.4 (CH3), 36.5 (NCH3), 125.4 (CAr),
126.6 (CAr), 127.8 (CAr), 128.9 (2 CHAr), 129.4 (2 CHAr), 140.2 (CAr), 144.5 (CAr) ppm. Anal. Calcd. for
C11H10BrN3O2 (296.12): C 44.62, H 3.40, N 14.19; found C 44.63, H 3.27, N 13.95.
[4-(2-Bromo-1-methyl-5-nitro-1H-imidazol-4-yl)phenyl]methanol (3i): Yield 58% (65 mg); yellow solid;
M.p. 132 ◦C. 1H-NMR (DMSO-d6):
δ
= 3.91 (s, 3H, NCH3), 4.55 (d, 3JH-H = 5.8 Hz, 2H, CH2), 5.29 (t,
3JH-H = 5.7 Hz, 1H, OH), 7.40 (d, 3JH-H = 8.2 Hz, 2H, 2 Ar-H), 7.63 (d, 3JH-H = 8.2 Hz, 2H, 2 Ar-H) ppm.
13C-NMR (DMSO-d6):
= 36.7 (NCH3), 62.8 (CH2), 126.1 (2 CHAr), 127.4 (CAr), 129.1 (2 CHAr), 129.6
δ

Molecules 2017, 22, 1278
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(CAr), 136.1 (CAr), 143.0 (CAr), 144.3 (CAr) ppm. Anal. Calcd. for C11H10BrN3O3 (312.12): C 42.33, H
3.23, N 13.46; found C 42.34, H 3.11, N 13.33.
1-Methyl-5-nitro-2-4-diphenyl-1H-imidazole (
regioselective Suzuki-Miyaura coupling reaction (9 mg); yellow solid; M.p. 129 ◦C. 1H-NMR (CDCl3):
= 4.00 (s, 3H, NCH3), 7.44–7.47 (m, 3H, 3 Ar-H), 7.54–7.56 (m, 3H, 3 Ar-H), 7.69–7.72 (m, 2H, 2 Ar-H),
7.83–7.87 (m, 2H, 2 Ar-H) ppm. 13C-NMR (CDCl3):
= 36.2 (NCH3), 128.1 (2 CHAr), 128.3 (CAr), 128.9
(2 CHAr), 129.5 (CHAr), 129.7 (4 CHAr), 130.7 (CHAr), 131.7 (CAr), 135.9 (CAr), 144.2 (CAr), 150.5 (CAr
5): Yield 9% using general procedure for the
δ
δ
)
ppm. Anal. Calcd. for C16H13N3O2 (279.29): C 68.71, H 4.69, N 15.05; found C 68.43, H 4.62, N 14.85.
3.2.3. General Procedure for the One-Pot Regioselective Bis-Suzuki-Miyaura Coupling Reaction
A
solution of 2,4-dibromo-1-methyl-5-nitro-1H-imidazole
(
2
,
0.1 g, 0.35 mmol),
4-methoxy-phenylboronic acid (72 mg, 0.46 mmol, 1.3 equiv.), Pd(OAc)2 (3 mg, 0.014 mmol,
0.04 equiv.), Na2CO3 (0.11 g, 1.05 mmol, 3 equiv.), in a DME/EtOH/H2O mixture (3/1/1, 5 mL)
◦
was heated at 60 C under microwave irradiation for 2 h. After cooling, boronic acid (0.45 mmol,
1.3 equiv.), PPh3 (9 mg, 0.035 mmol, 0.1 equiv.), Na2CO3 (0.11 g, 1.05 mmol, 3 equiv.), were introduced
under argon. The mixture was heated at 110 ◦C for 1 h under microwave irradiation. After cooling,
60 mL of water were added and the solution was extracted with dichloromethane (3
×
50 mL). The
organic layer was washed with water (3 100 mL), dried over Na2SO4 and evaporated. The crude
×
product was purified by column chromatography [silica gel, petroleum ether/ethyl acetate (9/1), (7/3
for 6e), (5/5 for 6j)] and recrystallized from propan-2-ol.
4-(4-Methoxyphenyl)-1-methyl-5-nitro-2-phenyl-1H-imidazole (6a): Yield 71% (77 mg); yellow solid;
M.p. 120 ◦C. 1H-NMR (CDCl3):
δ
= 3.86 (s, 3H, NCH3), 3.99 (s, 3H, OCH3), 6.98 (d, 3JH-H = 8.8 Hz,
2H, 2 Ar-H), 7.53–7.56 (m, 3H, 3 Ar-H), 7.68–7.72 (m, 2H, 2 Ar-H), 7.87 (d, 3JH-H = 8.8 Hz, 2H, 2 Ar-H)
ppm. 13C-NMR (CDCl3):
= 36.3 (NCH3), 55.3 (OCH3), 113.5 (2 CHAr), 123.9 (CAr), 128.3 (CAr), 128.9
δ
(2 CHAr), 129.7 (2 CHAr), 130.7 (CHAr), 131.4 (2 CHAr), 135.6 (CAr), 144.3 (CAr), 150.6 (CAr), 160.7 (CAr
ppm. HRMS (ESI) m/z [M + H]+ calcd. for [C17H15N3O3]+: 310.1186; found 310.1185.
)
2-(4-Fluorophenyl)-4-(4-methoxyphenyl)-1-methyl-5-nitro-1H-imidazole (6b): Yield 70% (80 mg);
◦
1
yellow solid; M.p. 132 C. H-NMR (CDCl3):
δ = 3.87 (s, 3H, NCH3), 3.97 (s, 3H, OCH3), 6.98
3
(d, JH-H = 9.0 Hz, 2H, 2 Ar-H), 7.21–7.28 (m, 2H, 2 Ar-H), 7.68–7.73 (m, 2H, 2 Ar-H), 7.85
(d, JH-H = 9.0 Hz, 2H, 2 Ar-H) ppm. 13C-NMR (CDCl3):
3
δ = 36.3 (NCH3), 55.4 (OCH3), 113.6
2
(2 CHAr), 116.3 (d, JC-F = 21.6 Hz, 2 CHAr), 123.9 (CAr), 123.6 (CAr), 131.4 (2 CHAr), 131.9 (d,
3JC-F = 8.7 Hz, 2 CHAr), 144.3 (CAr), 148.9 (CAr), 149.6 (CAr), 160.0 (d, 1JC-F = 272.1 Hz, CAr), 160.8 (CAr
ppm. HRMS (ESI) m/z [M + H]+ calcd. for [C17H14FN3O3]+: 328.1092; found 328.1093.
)
2-(4-Chlorophenyl)-4-(4-methoxyphenyl)-1-methyl-5-nitro-1H-imidazole (6c): Yield 60% (72 mg);
◦
1
yellow solid; M.p. 134 C. H-NMR (CDCl3):
(d, 3JH-H = 9.0 Hz, 2H, 2 Ar-H), 7.53 (d, 3JH-H = 8.7 Hz, 2H, 2 Ar-H), 7.66 (d, 3JH-H = 8.7 Hz, 2H, 2 Ar-H),
7.84 (d, 3JH-H = 8.9 Hz, 2H, 2 Ar-H) ppm. 13C-NMR (CDCl3):
= 36.3 (NCH3), 55.3 (OCH3), 113.6
δ = 3.87 (s, 3H, NCH3), 3.98 (s, 3H, OCH3), 6.98
δ
(2 CHAr), 123.8 (CAr), 126.8 (CAr), 129.3 (2 CHAr), 131.0 (2 CHAr), 131.4 (2 CHAr), 134.8 (CAr), 137.1
(CAr), 144.3 (CAr), 149.4 (CAr), 160.8 (CAr) ppm. Anal. Calcd. for C17H14ClN3O3 (343.76): C 59.40, H
4.10, N 12.22; found C 59.16, H 3.82, N 12.31.
4-(4-Methoxyphenyl)-1-methyl-5-nitro-2-(3-nitrophenyl)-1H-imidazole (6d): Yield 65% (81 mg); yellow
1
solid; M.p. 192 ◦C. H-NMR (CDCl3):
δ = 3.87 (s, 3H, NCH3), 4.03 (s, 3H, OCH3), 6.99 (d,
3JH-H = 8.9 Hz, 2H, 2 Ar-H), 7.76 (t, 3JH-H = 8.0 Hz, 1H, Ar-H), 7.85 (d, 3JH-H = 8.9 Hz, 2H, 2 Ar-H),
8.06 (d, 3JH-H = 7.7 Hz, 1H, Ar-H), 8.42 (d, 3JH-H = 8.2 Hz, 1H, Ar-H), 8.59 (t, 4JH-H = 1.7 Hz, 1H, Ar-H)
ppm. 13C-NMR (CDCl3):
δ = 36.3 (NCH3), 55.4 (OCH3), 113.6 (2 CHAr), 123.5 (CAr), 124.6 (CHAr),
125.2 (CHAr), 130.2 (CHAr), 131.3 (2 CHAr), 135.3 (CHAr), 135.9 (CAr), 144.2 (CAr), 147.7 (CAr), 148.4
(CAr), 156.1 (CAr), 160.9 (CAr) ppm. Anal. Calcd. for C17H14N4O5 (354.32): C 57.63, H 3.98, N 15.81;
found C 57.38, H 3.79, N 15.59.

Molecules 2017, 22, 1278
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4-(4-Methoxyphenyl)-1-methyl-5-nitro-2-(3,4,5-trimethoxyphenyl)-1H-imidazole (6e): Yield 50%
(70 mg); yellow solid; M.p. 158 ◦C. 1H-NMR (CDCl3):
= 3.86 (s, 3H, NCH3), 3.91 (s, 9H, 3 OCH3),
3.98 (s, 3H, OCH3), 6.87 (s, 2H, 2 Ar-H), 6.97 (d, 3JH-H = 8.8 Hz, 2H, 2 Ar-H), 7.86 (d, 3JH-H = 8.8 Hz,
2H, 2 Ar-H) ppm. 13C-NMR (CDCl3):
= 36.4 (NCH3), 55.3 (OCH3), 56.4 (2 OCH3), 61.0 (OCH3), 107.0
δ
δ
(2 CHAr), 113.5 (2 CHAr), 123.6 (CAr), 123.9 (CAr), 131.4 (2 CHAr), 135.6 (CAr), 140.2 (CAr), 144.3 (CAr),
150.7 (CAr), 153.5 (2 CAr), 160.7 (CAr) ppm. Anal. Calcd. for C20H21N3O6 (399.40): C 60.14, H 5.30, N
10.52; found C 59.67, H 5.05, N 10.67.
4-(4-Methoxyphenyl)-1-methyl-5-nitro-2-[3-(trifluoromethyl)-phenyl]-1H-imidazole (6f): Yield 58%
◦
(76 mg); yellow solid; M.p. 117 C. 1H-NMR (CDCl3):
δ
= 3.87 (s, 3H, NCH3), 4.00 (s, 3H, OCH3),
6.99 (d, JH-H = 9.0 Hz, 2H, 2 Ar-H), 7.69 (t, JH-H = 7.7 Hz, 1H, Ar-H), 7.81–7.90 (m, 4H, 4 Ar-H),
8.00 (s, 1H, Ar-H). 13C-NMR (CDCl3):
= 36.2 (NCH3), 55.3 (OCH3), 113.6 (2 CHAr), 123.5 (q,
3
3
δ
1JC-F = 272.9 Hz, CF3), 123.6 (CAr), 126.7 (q, 3JC-F = 3.7 Hz, CHAr), 127.4 (q, 3JC-F = 3.7 Hz, CHAr), 129.3
(CAr), 129.6 (CHAr), 131.3 (2 CHAr), 131.7 (q, 2JC-F = 33.0 Hz, CAr), 132.8 (CHAr), 135.8 (CAr), 144.2
(CAr), 148.8 (CAr), 160.9 (CAr) ppm. HRMS (ESI) m/z [M + H]+ calcd. for [C18H14F3N3O3]+: 378.1060;
found 378.1058.
4-(4-Methoxyphenyl)-1-methyl-5-nitro-2-p-tolyl-1H-imidazole (6g): Yield 56% (64 mg); yellow solid;
M.p. 136 ◦C. 1H-NMR (CDCl3):
δ
= 2.45 (s, 3H, CH3), 3.86 (s, 3H, NCH3), 3.97 (s, 3H, OCH3), 6.98 (d,
3JH-H = 8.9 Hz, 2H, 2 Ar-H), 7.35 (d, 3JH-H = 7.9 Hz, 2H, 2 Ar-H), 7.58 (d, 3JH-H = 8.2 Hz, 2H, 2 Ar-H),
7.86 (d, 3JH-H = 8.9 Hz, 2H, 2 Ar-H) ppm. 13C-NMR (CDCl3):
= 21.5 (CH3), 36.3 (NCH3), 55.3 (OCH3),
δ
113.5 (2 CHAr), 124.1 (CAr), 125.5 (CAr), 129.5 (2 CHAr), 129.6 (2 CHAr), 131.4 (2 CHAr), 135.6 (CAr),
141.1 (CAr), 144.5 (CAr), 150.8 (CAr), 160.7 (CAr) ppm. Anal. Calcd. for C18H17N3O3 (323.35): C 66.86,
H 5.30, N 13.00; found C 66.74, H 5.22, N 12.81.
4-(4-Methoxyphenyl)-1-methyl-2-(naphthalen-2-yl)-5-nitro-1H-imidazole (6h): Yield 61% (77 mg);
◦
1
yellow solid; M.p. 143 C. H-NMR (CDCl3):
δ = 3.87 (s, 3H, NCH3), 4.05 (s, 3H, OCH3), 6.99 (d,
3JH-H = 8.9 Hz, 2H, 2 Ar-H), 7.56–7.64 (m, 2H, 2 Ar-H), 7.77 (dd, 4JH-H = 1.6 Hz, 3JH-H = 8.5 Hz, 1H,
3
3
Ar-H), 7.90 (d, JH-H = 8.9 Hz, 2H, 2 Ar-H), 7.92–7.96 (m, 2H, 2 Ar-H), 8.01 (d, JH-H = 8.5 Hz, 1H,
Ar-H), 8.22 (s, 1H, Ar-H) ppm. 13C-NMR (CDCl3):
= 36.4 (NCH3), 55.3 (OCH3), 113.6 (2 CHAr), 124.0
(CAr), 125.7 (CAr), 125.9 (CHAr), 127.1 (CHAr), 127.8 (CHAr), 127.9 (CHAr), 128.6 (CHAr), 128.8 (CHAr),
130.2 (CHAr), 131.4 (2 CHAr), 132.8 (CAr), 134.0 (CAr), 135.8 (CAr), 144.4 (CAr), 150.7 (CAr), 160.8 (CAr
δ
)
ppm. Anal. Calcd. for C21H17N3O3 (359.38): C 70.18, H 4.77, N 11.69; found C 70.23, H 4.69, N 11.69.
4-(4-Methoxyphenyl)-1-methyl-2-(5-methylthiophen-2-yl)-5-nitro-1H-imidazole (6i): Yield 52% (60 mg);
yellow solid; M.p. 151 ◦C. 1H-NMR (CDCl3):
δ
= 2.57 (s, 3H, CH3), 3.86 (s, 3H, NCH3), 4.09 (s, 3H,
OCH3), 6.86-6.88 (m, 1H, Ar-H), 6.97 (d, 3JH-H = 9.0 Hz, 2H, 2 Ar-H), 7.37 (d, 4JH-H = 3.6 Hz, 1H, Ar-H),
7.85 (d, 3JH-H = 9.0 Hz, 2H, 2 Ar-H) ppm. 13C-NMR (CDCl3):
= 15.4 (CH3), 36.0 (NCH3), 55.3 (OCH3),
δ
113.5 (2 CHAr), 124.0 (CAr), 126.4 (CHAr), 127.7 (CAr), 129.9 (CHAr), 131.5 (2 CHAr), 135.5 (CAr), 136.8
(CAr), 145.0 (CAr), 145.3 (CAr), 160.8 (CAr) ppm. HRMS (ESI) m/z [M + H]+ calcd. for [C16H15N3O3S]+:
330.0907; found 330.0907.
3-[4-(4-Methoxyphenyl)-1-methyl-5-nitro-1H-imidazol-2-yl]pyridine (6j): Yield 69% (75 mg); yellow
1
solid; M.p. 152 ◦C. H-NMR (CDCl3):
δ = 3.86 (s, 3H, NCH3), 4.01 (s, 3H, OCH3), 6.98 (d,
3JH-H = 9.0 Hz, 2H, 2 Ar-H), 7.48–7.53 (m, 1H, Ar-H), 8.84 (d, 3JH-H = 8.9 Hz, 2H, 2 Ar-H), 8.04–8.08
(m, 1H, Ar-H), 8.79–8.80 (m, 1H, Ar-H), 8.96 (s, 1H, Ar-H) ppm. 13C-NMR (CDCl3):
= 36.2 (NCH3),
δ
55.3 (OCH3), 113.6 (2 CHAr), 123.7 (CHAr), 125.0 (CAr), 131.3 (2 CHAr), 135.9 (CAr), 137.2 (CHAr), 144.4
(CAr), 147.5 (CAr), 149.9 (CHAr), 151.4 (CHAr), 160.8 (2 CAr). HRMS (ESI) m/z [M + H]+ calcd. for
[C16H14N4O3]+: 311.1139; found 311.1136.
2-(4-Methoxyphenyl)-1-methyl-5-nitro-4-phenyl-1H-imidazole (
6
): Yield 64% (69 mg); yellow solid;
= 3.88 (s, 3H, NCH3), 3.99 (s, 3H, OCH3), 7.04 (d, 3JH-H = 8.9 Hz, 2H,
2 Ar-H), 7.43–7.48 (m, 3H, 3 Ar-H), 7.65 (d, 3JH-H = 8.7 Hz, 2H, 2 Ar-H), 7.82–7.86 (m, 2H, 2 Ar-H) ppm.
13C-NMR (CDCl3):
= 36.1 (NCH3), 55.5 (OCH3), 114.6 (2 CHAr), 121.0 (CAr), 128.0 (2 CHAr), 129.4
◦
M.p. 128 C. 1H-NMR (CDCl3):
δ
δ

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(CHAr), 129.7 (2 CHAr), 131.3 (2 CHAr), 132.1 (CAr), 136.1 (CAr), 144.2 (CAr), 150.6 (CAr), 161.7 (CAr
)
ppm. Anal. Calcd. for C17H15N3O3 (309.32): C 66.01, H 4.89, N 13.58; found C 65.98, H 4.75, N 13.42.
2,4-bis(4-methoxypheny)-1-methyl-5-nitro-1H-imidazole ( ): Yield 9% using general procedure for
7
the one-pot regioselective bis-Suzuki-Miyaura coupling reaction (11 mg); yellow solid; M.p. 121
◦C. 1H-NMR (CDCl324 ◦C):
δ = 3.86 (s, 3H, NCH3), 3.89 (s, 3H, OCH3), 3.97 (s, 3H, OCH3), 6.98 (d,
3JH-H = 7.6 Hz, 2H, 2 Ar-H), 7.02 (d, 3JH-H = 8.1 Hz, 2H, 2 Ar-H), 7.65 (d, 3JH-H = 7.9 Hz, 2H, 2 Ar-H),
3
7.86 (d, JH-H = 7.6 Hz, 2H, 2 Ar-H) ppm. 13C-NMR (CDCl3):
δ = 36.5 (NCH3), 55.3 (OCH3), 55.5
(OCH3), 113.6 (2 CHAr), 114.5 (2 CHAr), 120.5 (CAr), 124.0 (CAr), 131.3 (2 CHAr), 131.5 (2 CHAr), 135.6
(CAr), 144.5 (CAr), 150.8 (CAr), 160.8 (CAr), 161.6 (CAr) ppm. HRMS (ESI) m/z [M + H]+ calcd. for
[C18H17N3O4] 340.1292; found 340.1292.
3.2.4. General Procedure for the One-Pot Regioselective Suzuki-Miyaura/Sonogashira
Coupling Reaction
A solution of 2,4-dibromo-1-methyl-5-nitro-1H-imidazole (2, 0.1 g, 0.35 mmol), 4-methoxy-
phenylboronic acid (72 mg, 0.46 mmol, 1.3 equiv.), Pd(OAc)2 (3 mg, 0.014 mmol, 0.04 equiv.), Na2CO3
(0.11 g, 1.05 mmol, 3 equiv.), in a DME/EtOH/H2O mixture (3/1/1, 5 mL) was heated at 60 ◦C under
microwave irradiation for 2 h. After cooling, terminal alkynes (0.45 mmol, 1.3 equiv.), PPh3 (9 mg,
0.035 mmol, 0.1 equiv.), CuI (7 mg, 0.035 mmol, 0.1 equiv.), Et3N (0.1 mL, 0.7 mmol, 2 equiv.), were
introduced under argon. The mixture was heated at 70 ◦C for 1 h under microwave irradiation. After
cooling, 60 mL of water were added and the solution was extracted with dichloromethane (3
×
50 mL).
The organic layer was washed with water (3 100 mL), dried over Na2SO4 and evaporated. The crude
×
product was purified by column chromatography [silica gel, petroleum ether/ethyl acetate (10%), (50%
for 8g)] and recrystallized from propan-2-ol.
4-(4-Methoxyphenyl)-1-methyl-5-nitro-2-(phenylethynyl)-1H-imidazole (8a): Yield 67% (78 mg); yellow
1
solid; M.p. 156 ◦C. H-NMR (CDCl3):
δ
= 3.87 (s, 3H, NCH3), 4.14 (s, 3H, OCH3), 6.97 (d,
3JH-H = 8.0 Hz, 2H, 2 Ar-H), 7.39–7.51 (m, 3H, 3 Ar-H), 7.64 (d, JH-H = 6.6 Hz, 2H, 2 Ar-H), 8.82
(d, JH-H = 8.8 Hz, 2H, 2 Ar-H) ppm. 13C-NMR (CDCl3):
3
3
δ = 35.8 (NCH3), 55.3 (OCH3), 77.2 (C),
96.5 (C), 113.5 (2 CHAr), 120.3 (CAr), 123.6 (CAr), 128.7 (2 CHAr), 130.3 (CHAr), 131.2 (2 CHAr), 132.2
(2 CHAr), 134.1 (CAr), 134.4 (CAr), 144.7 (CAr), 160.8 (CAr) ppm. Anal. Calcd. for C19H15N3O3 (333.34):
C 68.46, H 4.54, N 12.61; found C 68.39, H 4.44, N 12.53.
4-(4-Methoxyphenyl)-1-methyl-5-nitro-2-(m-tolylethynyl)-1H-imidazole (8b): Yield 74% (90 mg);
◦
1
yellow solid; M.p. 138 C. H-NMR (CDCl3):
3H, OCH3), 6.97 (d, 3JH-H = 9.0 Hz, 2H, 2 Ar-H), 7.20–7.39 (m, 3H, 3 Ar-H), 7.60 (d, 3JH-H = 8.4 Hz, 1H,
Ar-H), 8.81 (d, 3JH-H = 9.0 Hz, 2H, 2 Ar-H) ppm. 13C-NMR (CDCl3):
= 20.9 (CH3), 35.7 (NCH3), 55.3
δ = 2.56 (s, 3H, CH3), 3.86 (s, 3H, NCH3), 4.14 (s,
δ
(OCH3), 81.0 (C), 95.6 (C), 113.5 (2 CHAr), 120.2 (CAr), 123.6 (CAr), 126.0 (CHAr), 129.8 (CHAr), 130.3
(CHAr), 131.2 (2 CHAr), 132.8 (CHAr), 134.1 (CAr), 134.6 (CAr), 141.1 (CAr), 144.7 (CAr), 160.8 (CAr) ppm.
Anal. Calcd. for C20H17N3O3 (347.37): C 69.15, H 4.93, N 12.10; found C 68.94, H 4.79, N 11.89.
2-[(3-Fluorophenyl)ethynyl]-4-(4-methoxyphenyl)-1-methyl-5-nitro-1H-imidazole (8c): Yield 59%
◦
(73 mg); yellow solid; M.p. 148 C. 1H-NMR (CDCl3):
δ = 3.86 (s, 3H, NCH3), 4.13 (s, 3H, OCH3),
3
6.97 (d, JH-H = 8.8 Hz, 2H, 2 Ar-H), 7.13–7.21 (m, 1H, Ar-H), 7.30–7.42 (m, 3H, 3 Ar-H), 8.80 (d,
3JH-H = 8.8 Hz, 2H, 2 Ar-H) ppm. 13C-NMR (CDCl3):
δ = 35.9 (NCH3), 55.3 (OCH3), 77.9 (C), 94.8 (d,
4JC-F = 3.2 Hz, C), 113.5 (2 CHAr), 117.7 (d, 2JC-F = 21.1 Hz, CHAr), 119.0 (d, 2JC-F = 23.9 Hz, CHAr),
122.1 (d, 3JC-F = 9.7 Hz, CAr), 123.4 (CAr), 128.1 (d, 4JC-F = 3.2 Hz, CHAr), 130.4 (d, 3JC-F = 8.7 Hz, CHAr),
131.2 (2 CHAr), 133.9 (CAr), 134.2 (CAr), 144.6 (CAr), 160.8 (CAr), 162.2 (d, 1JC-F = 248.2 Hz, CAr) ppm.
Anal. Calcd. for C19H14FN3O3 (351.33): C 64.95, H 4.02, N 11.96; found C 64.67, H 3.84, N 11.79.
2-[(4-Methoxy-2-methylphenyl)ethynyl]-4-(4-methoxyphenyl)-1-methyl-5-nitro-1H-imidazole (8d):
Yield 53% (70 mg); yellow solid; M.p. 182 ◦C. 1H-NMR (CDCl3):
δ
= 2.53 (s, 3H, CH3), 3.84 (s, 3H,
NCH3), 3.86 (s, 3H, OCH3), 4.13 (s, 3H, OCH3), 6.74–6.81 (m, 2H, 2 Ar-H), 6.97 (d, 3JH-H = 8.9 Hz, 2H, 2

Molecules 2017, 22, 1278
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Ar-H), 7.53 (d, 3JH-H = 8.4 Hz, 1H, Ar-H), 7.81 (d, 3JH-H = 8.9 Hz, 2H, 2 Ar-H) ppm. 13C-NMR (CDCl3):
= 21.1 (CH3), 35.7 (NCH3), 55.3 (OCH3), 55.4 (OCH3), 80.1 (C), 96.2 (C), 111.8 (CHAr), 112.3 (CAr),
δ
113.5 (2 CHAr), 115.5 (CHAr), 123.7 (CAr), 131.3 (2 CHAr), 134.0 (CAr), 134.4 (CHAr), 135.1 (CAr), 143.2
(CAr), 144.8 (CAr), 160.7 (CAr), 161.1 (CAr) ppm. Anal. Calcd. for C21H19N3O4 (377.39): C 66.83, H 5.07,
N 11.13; found C 66.56, H 4.91, N 11.10.
2-(Cyclopentylethynyl)-4-(4-methoxyphenyl)-1-methyl-5-nitro-1H-imidazole (8e): Yield 60% (69 mg);
yellow solid; M.p. 98 ◦C. 1H-NMR (CDCl3):
δ = 1.59–1.68 (m, 2H, CH2), 1.73–1.82 (m, 4H, 2 CH2),
3
2.00–2.10 (m, 2H, CH2), 2.92 (q, JH-H = 7.5 Hz, 1H, CH), 3.84 (s, 3H, NCH3), 4.01 (s, 3H, OCH3),
3
3
6.94 (d, JH-H = 9.0 Hz, 2H, 2 Ar-H), 7.77 (d, JH-H = 9.0 Hz, 2H, 2 Ar-H) ppm. 13C-NMR (CDCl3):
δ
= 25.2 (2 CH2), 30.5 (CH), 33.4 (2 CH2), 35.5 (NCH3), 55.3 (OCH3), 68.9 (C), 103.5 (C), 113.4 (2 CHAr),
123.7 (CAr), 131.2 (2 CHAr), 133.6 (CAr), 134.9 (CAr), 144.4 (CAr), 160.7 (CAr) ppm. Anal. Calcd. for
C18H19N3O3 (325.36): C 66.45, H 5.89, N 12.91; found C 66.21, H 5.76, N 12.88.
2-[(4-tert-Butylphenyl)ethynyl]-4-(4-methoxyphenyl)-1-methyl-5-nitro-1H-imidazole (8f): Yield 63%
1
(86 mg); yellow oil. H-NMR (CDCl3):
δ = 1.34 (s, 9H, 3 CH3), 3.86 (s, 3H, NCH3), 4.12 (s, 3H, OCH3),
6.96 (d, 3JH-H = 8.8 Hz, 2H, 2 Ar-H), 7.43 (d, 3JH-H = 8.4 Hz, 2H, 2 Ar-H), 7.56 (d, 3JH-H = 8.4 Hz, 2H,
2 Ar-H), 7.81 (d, 3JH-H = 8.8 Hz, 2H, 2 Ar-H) ppm. 13C-NMR (CDCl3):
δ
= 31.0 (3 CH3), 35.0 (C), 35.7
(NCH3), 55.3 (OCH3), 76.8 (C), 96.9 (C), 113.5 (2 CHAr), 117.2 (CAr), 123.6 (CAr), 125.7 (2 CHAr), 131.2
(2 CHAr), 132.0 (2 CHAr), 134.0 (CAr), 134.6 (CAr), 144.7 (CAr), 153.9 (CAr), 160.7 (CAr) ppm. HRMS
(ESI) m/z [M + H]+ calcd. for [C23H23N3O3]+: 390.1812; found 390.1813.
3-[4-(4-Methoxyphenyl)-1-methyl-5-nitro-1H-imidazol-2-yl]prop-2-yn-1-ol (8g): Yield 51% (51 mg);
yellow solid; M.p. 177 ◦C. 1H-NMR (DMSO-d6):
δ
= 3.81 (s, 3H, NCH3), 3.97 (s, 3H, OCH3), 4.43 (d,
3JH-H = 5.7 Hz, 2H, CH2), 5.67 (t, 3JH-H = 5.8 Hz, 1H, OH), 7.01 (d, 3JH-H = 8.8 Hz, 2H, 2 Ar-H), 7.67 (d,
3JH-H = 8.8 Hz, 2H, 2 Ar-H). 13C-NMR (DMSO-d6):
= 35.9 (NCH3), 49.8 (CH2), 55.7 (OCH3), 72.7 (C),
97.5 (C), 113.9 (2 CHAr), 124.1 (CAr), 131.2 (2 CHAr), 133.6 (CAr), 134.5 (CAr), 143.2 (CAr), 160.6 (CAr
δ
)
ppm. Anal. Calcd. for C14H13N3O4 (287.27): C 58.53, H 4.56, N 14.63; found C 58.64, H 4.51, N 14.40.
2-[(3-Chlorophenyl)ethynyl]-4-(4-methoxyphenyl)-1-methyl-5-nitro-1H-imidazole (8h): Yield 61%
◦
(79 mg); yellow solid; M.p. 139 C. 1H-NMR (CDCl3):
δ = 3.86 (s, 3H, NCH3), 4.13 (s, 3H, OCH3),
3
6.97 (d, JH-H = 8.9 Hz, 2H, 2 Ar-H), 7.32–7.38 (m, 1H, Ar-H), 7.42–7.47 (m, 1H, Ar-H), 7.51 (dt,
4JH-H = 1.4 Hz, 3JH-H = 7.4 Hz, 1H, Ar-H), 7.61 (t, 4JH-H = 1.4 Hz, 1H, Ar-H), 8.80 (d, 3JH-H = 8.9 Hz,
2H, 2 Ar-H) ppm. 13C-NMR (CDCl3):
δ = 35.8 (NCH3), 55.3 (OCH3), 78.2 (C), 94.7 (C), 113.6 (2 CHAr),
122.0 (CAr), 123.4 (CAr), 130.0 (CHAr), 130.3 (CHAr), 130.6 (CHAr), 131.2 (2 CHAr), 131.9 (CHAr), 133.8
(CAr), 134.2 (CAr), 134.6 (CAr), 144.6 (CAr), 160.8 (CAr). Anal. Calcd. for C19H14ClN3O3 (367.79): C
62.05, H 3.84, N 11.43; found C 61.90, H 3.77, N 11.35.
2-[(2-Chlorophenyl)ethynyl]-4-(4-methoxyphenyl)-1-methyl-5-nitro-1H-imidazole (8i): Yield 52%
◦
(67 mg); yellow solid; M.p. 171 C. 1H-NMR (CDCl3):
δ
= 3.87 (s, 3H, NCH3), 4.19 (s, 3H, OCH3),
6.98 (d, 3JH-H = 8.5 Hz, 2H, 2 Ar-H), 7.29–7.42 (m, 2H, 2 Ar-H), 7.49 (d, 3JH-H = 7.9 Hz, 1H, Ar-H), 7.67
(d, 3JH-H = 7.4 Hz, 1H, Ar-H), 7.80 (d, 3JH-H = 8.7 Hz, 2H, 2 Ar-H) ppm. 13C-NMR (CDCl3):
= 35.8
δ
(NCH3), 55.3 (OCH3), 82.1 (C), 92.8 (C), 113.6 (2 CHAr), 120.7 (CAr), 123.6 (CAr), 126.9 (CHAr), 129.6
(CHAr), 131.2 (CHAr), 131.3 (2 CHAr), 133.9 (CHAr), 134.0 (CAr), 134.1 (CAr), 136.6 (CAr), 144.6 (CAr),
160.9 (CAr) ppm. Anal. Calcd. for C19H14ClN3O3 (367.79): C 62.05, H 3.84, N 11.43; found C 61.86, H
3.66, N 11.31.
2-(Cyclopropylethynyl)-4-(4-methoxyphenyl)-1-methyl-5-nitro-1H-imidazole (8j): Yield 63% (66 mg);
◦
yellow solid; M.p. 124 C. 1H-NMR (CDCl3):
δ
= 0.95–1.03 (m, 4H, 2 CH2), 1.50–1.60 (m, 1H, CH), 3.85
(s, 3H, NCH3), 4.02 (s, 3H, OCH3), 6.94 (d, 3JH-H = 8.8 Hz, 2H, 2 Ar-H), 7.77 (d, 3JH-H = 8.9 Hz, 2H,
2 Ar-H) ppm. 13C-NMR (CDCl3):
= 0.2 (CH), 9.3 (2 CH2), 35.5 (NCH3), 55.3 (OCH3), 64.4 (C), 77.2
δ
(C), 102.4 (CAr), 113.5 (2 CHAr), 123.7 (CAr), 131.3 (2 CHAr), 134.7 (CAr), 144.3 (CAr), 160.8 (CAr) ppm.
Anal. Calcd. for C16H15N3O3 (297.31): C 64.64, H 5.09, N 14.13; found C 64.46, H 4.92, N 14.22.

Molecules 2017, 22, 1278
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1-Methyl-5-nitro-2,4-bis(phenylethynyl)-1H-imidazole (
9): Yield 10% using general procedure for the
one-pot regioselective Suzuki-Miyaura/Sonogashira coupling reaction (12 mg); yellow solid; M.p.
◦
1
166 C. H-NMR (CDCl3):
δ
= 4.14 (s, 3H, NCH3), 7.38–7.48 (m, 6H, 6 Ar-H), 7.61–7.66 (m, 4H, 4
= 35.6 (NCH3), 76.7 (C), 81.0 (C), 96.8 (C), 97.1 (C), 120.0 (CAr), 121.7
Ar-H) ppm. 13C-NMR (CDCl3):
δ
(CAr), 127.7 (CAr), 128.5 (2 CHAr), 128.7 (2 CHAr), 129.6 (CHAr), 130.5 (CHAr), 132.2 (2 CHAr), 132.3 (2
CHAr), 133.5 (CAr), 135.1 (CAr) ppm. HRMS (ESI) m/z [M + H]+ calcd. for [C20H13N3O2]+: 328.1081;
found 328.1081.
1
Figures S1–S66: H- and 13C-NMR of all compounds 3a–i, 5, 6a–j, 6, 7, 8a–j and 9.
3.2.5. Crystal Data for Compound 3c
2(C11H7BrF3N3O2), M = 700.21, a = 8.4376(4) Å, b = 12.5416(6) Å, c = 12.6319(5) Å,
= 73.324(4)◦, = 80.384(4)◦, V = 1257.48(10) Å3, T = 293 K, space group P
Z = 2, 14503 reflections
measured, 5058 independent reflections (Rint = 0.0184). The final R1 values were 0.0320 (I > 2 (I)).
The final wR(F2) values were 0.0689 (I > 2
(I)). The final R1 values were 0.0469 (all data). The final
α
= 82.406(4)◦,
β
γ
1
σ
σ
wR(F2) values were 0.0752 (all data). The goodness of fit on F2 was 1.007. CCDC 1542022 contains
the supplementary crystallographic data for this paper. These data can be obtained free of charge
at www.cdcc.cam.ac.uk/data_request/cif of from the Cambridge Crystallographic Data Centre, 12,
Union Road, Cambridge CB2 1EZ, UK; Fax: + 44 (1223) 336033; email: deposit@ccdc.cam.ac.uk.
3.3. Biology
3.3.1. 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 (3.108 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.
3.3.2. 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
was added to each well. Then, 10 L of each imidazole derivative with the primary concentration
200 g/mL was added to the first wells. After mixing, 100 L of this mixture was embedded into the
second well. A similar dilution procedure was carried out in the first wells line for metronidazole
µL of the LYI broth medium,
µ
µ
µ
as control group. 100
negative control, 200
µ
L of the trichomonas suspension (105 cells/mL) was added to each well. For
L of LYI broth medium were added to the border wells. The plates were
µ
incubated under anaerobic conditions 24 h at 37 ◦C.
3.3.3. In Vitro Cytotoxicity Evaluation
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

Molecules 2017, 22, 1278
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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.
Cell Line
CHO-K1 Chinese Hamster Ovary cells (ATCC CCL61), low passage number (<50).
Culture Medium
Mc Coy’s medium supplemented with penicillin 100 UI/mL and steptomycin 100
10% of inactivated calf serum, pH 7.2, freshly prepared, stored no longer than 1 week.
µg/mL, and
Test Procedure
Cells were seeded into two 96-well tissue culture plates (0.1 mL per well), at a concentration of
1.105 cells/mL, and incubated at 37 ◦C (5% CO2) 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% CO2)
in the dark for 24 h. At the end of the incubation period, cells were washed, placed into Neutral Red
medium (50 µ
g/mL Neutral Red in complete medium) and incubated for 3 h at 37 ◦C, 5% CO2. 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 CC50
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).
4. 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.

Molecules 2017, 22, 1278
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1
Supplementary Materials: Supplementary Materials are available online. Figures S1–S66: H- and 13C-NMR of
all compounds 3a–i, 5, 6a–j, 6, 7, 8a–j and 9.
Acknowledgments: This work was supported by the CNRS (Centre National de la Recherche Scientifique) and
Aix-Marseille Université. The authors thank V. Remusat for NMR spectra recording and the Spectropole team for
various analytical measurements.
Author Contributions: F.M. and Y.K. conceived and designed the experiments; F.M., Y.K., C.S., L.O. and C.D.
performed the experiments; F.M., Y.K., C.S, L.O., C.D., J.-M.R., M.D.C. and P.V. discussed the results. F.M., Y.K.,
L.O., C.D., J.-M.R., M.D.C. and P.V. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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