New synthesis and antiparasitic activity of model 5-aryl-1-methyl-4- nitroimidazoles

Article abstract of DOI:10.3390/molecules14082758

A number of 5-aryl-1-methyl-4-nitroimidazoles 5a-f have been synthesized in good yields by the Suzuki coupling reaction between 5-chloro-1-methyl-4- nitroimidazole (3) and arylboronic acids 4a-f, aided by dichlorobis- (triphenylphosphine)palladium(II), K<

Full text of DOI:10.3390/molecules14082758

Molecules 2009, 14, 2758-2767; doi:10.3390/molecules14082758
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
New Synthesis and Antiparasitic Activity of Model 5-Aryl-1-
methyl-4-nitroimidazoles
Haythem A. Saadeh 1,*, Ibrahim M. Mosleh 2 and Mustafa M. El-Abadelah 1
1
Chemistry Department, Faculty of Science, University of Jordan, Amman 11942, Jordan
2
Department of Biological Sciences, Faculty of Science, University of Jordan, Amman 11942,
Jordan
* Author to whom correspondence should be addressed; E-mail: h.saadeh@ju.edu.jo;
Tel. +962 6 5355000, ext. 22159; Fax: +962 6 5348932
Received: 3 July 2009; in revised form: 19 July 2009 / Accepted: 23 July 2009 /
Published: 27 July 2009
Abstract: A number of 5-aryl-1-methyl-4-nitroimidazoles 5a-f have been synthesized in
good yields by the Suzuki coupling reaction between 5-chloro-1-methyl-4-nitroimidazole
(3) and arylboronic acids 4a-f, aided by dichlorobis-(triphenylphosphine)palladium(II),
K2CO3, and tetrabutylammonium bromide in water at 70-80 °C. Compounds 5a-f were
characterized by elemental analysis, NMR and MS spectral data. On the basis of in vitro
screening data, 5-(3-chlorophenyl)-1-methyl-4-nitro-1H-imidazole (5f) exhibited potent
lethal activity against Entamoeba histolytica and Giardia intestinalis with
IC50 = 1.47 µM/mL, a value lower by a factor of two than that of the standard drug,
metronidazole. The boosted activity of 5f was not accompanied by any increased
cytotoxicity. The rest of the series also exhibited potent antiparasitic activity with IC50
values in the 1.72-4.43 µM/mL range. The cytotoxicity of the derivatives 5c and 5e was
increased compared to the precursor compound, metronidazole, although they remain
non-cytotoxic at concentrations much higher than the antiparasitic concentration of the
two derivatives.
Keywords: 5-chloro-1-methyl-4-nitroimidazole; arylboronic acids; Suzuki coupling;
5-aryl-1-methyl-4-nitroimidazoles; antiparasitic activity

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Introduction
The imidazole nucleus occurs naturally in L-histidine, histamine (a vasodelator hormone), thiamine
(vitamin B1), and in several other biomolecules [1-4]. An example of an imidazole-containing
synthetic drugs is cimetidine (1, Figure 1), widely used for the treatment of peptic ulcers [5-8].
Nitroimidazoles have wide applications in the drug synthesis due to their biological activity. Examples
include natural azomycin (antibiotic, 2-nitroimidazole) [9-12] and synthetic metronidazole (2, anti-
amoebic dysentry) [13-16] (Figure 1).
Figure 1. Model imidazole-based synthetic drugs.
H
N
H
N
N
N
S
Me
O2N
Me
N
N
NH
OH
Me
(CH2)2OH
(1)
(2)
(Substituted)-4-nitroimidazoles also showed activity against various microbes and found
applications in chemotherapy [17-19]. Thus, a series of 1-methyl-4-nitro-5-substituted imidazoles were
reported to exhibit antileishmanial, antiamebic and anthelmintic activities [17]. Also, a number of
synthetic 5-(substituted azolyl)-1-methyl-4-nitroimidazoles were reported to exhibit anti-parasitic
activity [20-22].
Herein, we wish to report a one-pot synthesis of 5-aryl-1-methyl-4-nitroimidazoles 5a-f by reaction
of 5-chloro-1-methyl-4-nitroimidazole (3) with arylboronic acids 4 under Suzuki reaction conditions
[25-30] (Scheme 1), and the evaluation of their antiparasitic activity.
Results and Discussion
Chemistry
Previously, the synthesis of 5-aryl-1-methyl-4-nitroimidazoles (exemplified by 5a/Scheme 2) was
achieved via N(1)-methylation of the respective 5(4)-aryl-4(5)-nitroimidazole (6A
step led to a mixture of 5a and its isomeric 1-methyl-4-aryl-5-nitroimidazole 7A, the isolation of which
required extra separation and purification efforts. Additionally, the required synthon (6A 6B) was
6B) [23]. This
prepared by condensation of (substituted)phenacyl chloride with formamide, followed by acidification
and then neutralization with aqueous ammonium hydroxide [24].
In the present work, reaction of the commercially available 5-chloro-1-methyl-4-nitroimidazole (3)
with arylboronic acids 4 under Suzuki reaction conditions provided an efficient route towards the
formation of 5-aryl-1-methyl-4-nitroimidazoles 5a-f, as outlined in Scheme 1.

Molecules 2009, 14
Scheme 1. Synthesis of 5-aryl-1-methyl-4-nitroimidazoles 5a-f via Suzuki coupling.
2760
O2N
4
O2N
Cl
N
N
2'
5
1'
+
2
(i)
3'
4'
B(OH)2
N
N
X
CH3
CH3
X
5a-f (57-75 %)
3
4a-f
(compounds 4a-f and 5a-f)
a
b
c
d
e
f
entry
X
4'-Cl
3'-Cl
4'-OMe
4'-F
4'-Me
H
(i) Pd(PPh3)2Cl2, Bu4NBr (TBAB), K2CO3 / H2O, 75-80 oC, 5-8 h.
Scheme 2. N-Methylation of 5(4)-aryl-4(5)-nitroimidazole [23].
O2N
O2N
Ph
H
CH3
O2N
Ph
O2N
Ph
N
N
CH3I
base
N
N
+
Ph
N
N
H
N
N
CH3
6A
6B
7A (minor)
5a (major)
Compared to the multistep synthetic route noted above, the direct Suzuki coupling reaction provides
a more convenient and efficient route for the preparation of 5-aryl-1-methyl-4-nitroimidazoles 5a-f.
Thus, treatment of 5-chloro-1-methyl-4-nitroimidazole (3) with arylboronic acids 4a-f using 3 mole %
of dichloro bis-(triphenylphosphin)palladium(II), K2CO3, and tetrabutylammonium bromide in water at
70-80 °C, gave the corresponding 5-arylimidazole derivatives 5a-f [31]. These Pd-catalyzed cross-
coupling reactions proceeded nicely in the presence of the ‘additive’ (TBAB). The latter is known to
stabilize colloidal palladium nanoparticles that act as catalysts in the Suzuki coupling of aryl halides
[32], and thus enhances the rate of the coupling reaction. The presence of the strongly electron-
withdrawing nitro group on the imidazole ring facilitates the coupling process which results in good
yields of 5a-f.
The microanalytical and spectral (HRMS and NMR) data, given in the Experimental part, are in
accordance with the assigned structures for compounds 5a-f. Thus, the observed high resolution MS
data for M+ are in good agreement with the values calculated for the respective molecular formulas.
1
The NMR spectra displayed H- and 13C- signals characteristic of the respective aryl moieties
1
13
introduced at the C-5. Assignments of the H- and C- signals to the different respective protons and
carbons are based on DEPT and 2D (COSY, HMQC, HMBC) experiments which showed correlations
1
consistent with these assignments. The H-NMR spectra for compounds 5a-f show aromatic protons
for the aryl group introduced by Suzuki coupling besides the imidazole C-2 proton at ~ 7.45 ppm. The
CH3 and OCH3 aryl substituents for compounds 5b and 5c appear at 2.41 and 3.85 ppm, respectively.

Molecules 2009, 14
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Compound 5d containing 4'- fluorine substituent showed additional coupling caused by the 19F nucleus
13
with a vicinal coupling 1H-19F (3J= 8.5 Hz). The C-NMR spectra of the prepared compounds 5a-f
showed carbon signals expected for aryl groups introduced by Suzuki coupling besides the imidazole
C-2, C-4 and C-5 carbons. In compound 5d, the skeletal carbons of the benzo ring at C-5 are readily
recognizable by their signal splitting 'as doublets' arising from spin-spin coupling with the nearby
fluorine atom at C-4' with varying JC-F values for the four different carbons C1'-C4' (1J = 250 Hz ; 2J =
3
4
22 Hz; J = 8.6 Hz ; J = 3.6 Hz). DEPT experiments were employed to differentiate primary and
tertiary carbons from quaternary carbons.
Antiamoebic and antigiardial activity
The antiamoebic and antigiardial activities of the compounds 5a-f were investigated using in vitro
bioassays that included the standard antiamoebic and antigiardial drug metronidazole as a control. The
cytotoxicity of the compounds on the two cell lines, Hep-2 and Vero cells, was also compared with
that of metronidazole. The IC50 values of the compounds against Entamoeba histolytica, Giardia
intestinalis, and the two cell lines are given in Table 1. As indicated in the Table, all the tested
compounds showed biological activities against Entamoeba and Giardia. Compounds 5c, 5e, and 5f
showed the highest activity against the parasites, with IC50 values ranging from around one to two
micromolar, compared to around four micromolar for the standard drug, metronidazole. When the
cytotoxicity of the prepared molecules is considered, compound 5f appears to be the best among the
prepared derivatives of metronidazole, as indicated in Table 2. This Table presents the IC50 ratio of
metronidazole over that of the derivative compounds 5a-f against the parasites and the two
investigated cell lines. Compound 5f was around two to three times more active than metronidazole,
against Entamoeba and Giardia, respectively. Fortunately, the boosted activity of this compound was
not accompanied with any increased cytotoxicity. In contrast, the cytotoxicity of the derivatives 5c and
5e has increased compared to the precursor compound, metronidazole, although they remain non-
cytotoxic at concentrations much higher than the antiparasitic concentration of the two derivatives. As
can be concluded from Table 1, the IC50 values of compounds 5c and 5e against the two cell lines were
≥ 230 times higher than that against the parasites under investigation.
Interestingly, the tested compounds exhibited an almost similar pattern of activity against both G.
intestinalis and E. histolytica (Table 1), indicating that each compound affects both parasites by a
similar mechanism of action. In addition, the molecular modifications on our derivatives did not render
any of the compounds inactive. The other molecules, 5a, 5b, and 5d remained as active as
metronidazole (Table 1 and 2).
The activities exhibited by the derivatives, especially compound 5f, suggest that the derivatives may
be used as new lead compounds in the development of new antiparasitic drugs. Although drug
resistance to Entamoeba and Giardia does not, so far, appear to be a serious problem, occasional
reports of failure with metronidazole [33] and the reported variations in drug sensitivities of isolates
[34] may be alarming. Therefore, the importance of such biologically active, non-cytotoxic
metronidazole derivatives, especially 5f, lies in their potential contribution to overcome the problem of
resistance of pathogens to the standard drugs. Additionally, because of the limited number of drugs
available in the market against anaerobic protozoal parasites and bacteria there is a serious need for

Molecules 2009, 14
2762
new active compounds. The molecular modification on the original drugs, therefore, offers alternatives
that may bypass the already developed mechanisms adopted by the anaerobic pathogens against the
standard drugs. Our derivatized compounds (5c, 5e, 5f) are good drug candidates to be tested against
metronidazole-resistant parasites and bacteria.
Table 1. Antiparasitic activities of compounds 5a-f.
Mean IC50 ± SD(n)
(μM)
Giardia
intestinalis
Entamoeba
histolytica
Compound
Hep-2 cells
Vero cells
5a
5b
5c
5d
5e
5f
4.43 ± 1.97
4.04 ± 0.28
3.10 ± 0.41
1.16 ± 0.19
4.39 ± 0.71
1.56 ± 0.156
1.89 ± 0.14
4.10 ± 0.78
1040.27 ± 19.18 1748.28 ± 18.38
1610.74 ± 22.23 1633.32 ± 13.61
4.01 ± 0.75
1.72 ± 0.57
3.76 ± 0.2
1.90 ± 0.14
1.47 ± 0.14
568.80 ± 22.71
1894.12 ± 21.13 1918.41 ± 13.37
437.19 ± 16.39 725.05 ± 11.79
868.24 ± 22.02
1780.21 ± 15.71 1783.16 ± 19.66
2044.20 ± 26.36 2071.35 ± 16.37
Metronidazole 4.39 ± 0.59
SD(n): Standard deviation
Table 2. IC50 ratios.
IC50 Ratio
(metronidazole/compound)
Entamoeba
Giardia
Compound
Hep-2 cells
Vero cells
intestinalis
histolytica
1.0
5a
5b
5c
5d
5e
5f
1.0
1.1
3.5
1.2
2.6
2.2
2.0
1.3
3.6
1.1
4.7
1.2
1.2
1.3
2.4
1.1
2.9
1.2
1.3
2.6
0.93
2.3
2.9
Experimental
General
The following chemicals, employed in this study, were purchased and used without further
purification: 5-chloro-1-methyl-4-nitroimidazole, phenylboronic acid, 4-tolylboronic acid, 4-methoxy-
phenylboronic acid, 4-flourophenylboronic acid, 4-chlorophenylboronic acid, 3-chlorophenylboronic
acid, tetrabutyl ammonium bromide and dichloro bis-(triphenylphosphin)palladium(II). Melting points
1
(uncorrected) were determined on a Gallenkamp electrothermal melting temperature apparatus. H-
and 13C-NMR spectra were recorded on a Bruker DPX-300 instrument with TMS as internal reference.
High-resolution mass spectra (HRMS) were measured in positive ion mode using electrospray ion trap
(ESI) technique by collision-induced dissociation on a Bruker APEX-4 (7 Tesla) instrument. The
samples were dissolved in acetonitrile, diluted in spray solution (methanol/water 1:1 v/v + 0.1% formic
acid) and infused using a syringe pump with a flow rate of 2 µL/min. External calibration was

Molecules 2009, 14
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conducted using Arginine cluster in a mass range m/z 175-871. IR spectra were recorded as KBr discs
on a Nicolet Impact-400 FT-IR spectrophotometer. Elemental analyses were preformed at the
Microanalytical Laboratory of the Hashemite University, Zarqa-Jordan, and the results were found to
be in good agreement (± 0.4%) with the calculated values.
General procedure for the synthesis of 5-aryl-1-methyl-4-nitroimidazoles 5a-f
A mixture of 5-chloro-1-methyl-4-nitroimidazole 3 (4 mmol), the particular arylboronic acid 4a-f (4
mmol), Pd(PPh3)2Cl2 (3 mol%, 80 mg), powdered K2CO3 (1.4 g, 10 mmol) and Bu4NBr (1.3 g, 4
mmol) in water (3 mL) was heated with stirring at 75-80 ºC for 5-8 h. Thereafter, the reaction mixture
was cooled, poured into water (25 mL) and extracted with dichloromethane (2 x 30 mL). The
combined organic extracts were dried over anhydrous sodium sulfate and the solvent was removed
under reduced pressure. The residue was purified by column chromatography with chloroform-
methanol (95:5 v/v) to afford the respective compounds 5a-f.
1-Methyl-4-nitro-5-phenyl-1H-imidazole (5a): Yield 0.73 g (70%); mp 177-179 °C (Lit.[23] 178-180
oC). 1H-NMR (CDCl3): δ 3.51 (s, 3H, N-CH3), 7.36 (m, 2H, 2'-H + 6'-H ), 7.46 (s, 1H, 2-H), 7.50 (m,
3H, 3'-H + 4'-H+ 5'-H ); 13C-NMR (CDCl3): δ 33.1 (CH3), 126.5 (C), 128.9 (C-2'+ C-6'), 130.1 (C-3'+
C-5'), 130.2 (C-4'), 132.6 (C-5), 135.6 (C-2), 140.1 (C-4); HRMS ((+ve)-ESI): m/z calcd. for
C10H9N3O2Na [M+Na]+: 226.05870, found: 226.05870; Anal. Calcd. for C10H9N3O2 (203.20): C,
59.11; H, 4.46; N, 20.68. Found: C, 59.50; H, 4.77; N, 20.54.
1
1-Methyl-5-(4-methylphenyl)-4-nitro-1H-imidazole (5b): Yield 0.57 g (65%); mp 179-181 °C. H-
NMR (CDCl3): δ 2.41 (s, 3H, 4'-CH3), 3.50 (s, 3H, N-CH3), 7.25 (d, J = 8 Hz, 2H, 2'-H+ 6'-H), 7.31 (d,
J = 8 Hz, 2H, 3'-H+ 5'-H), 7.45 (s, 1H, 2-H); 13C-NMR (CDCl3): δ 21.6 (4'-CH3), 33.1 (N-CH3), 115.0
(C-1'), 123.4 (C-4'), 129.6 (C-3'+ C-5'), 129.9 (C-2'+ C-6'), 130.6 (C-5), 135.6 (C-2), 140.5 (C-4);
HRMS ((+ve)-ESI): m/z calcd. for C11H11N3O2Na [M+Na]+: 240.07435, found: 240.07435; Anal.
Calcd. for C11H11N3O2 (217.22): C, 60.82; H, 5.01; N, 19.34. Found: C, 61.03; H, 5.15; N, 19.05.
5-(4-Methoxyphenyl)-1-methyl-4-nitro-1H-imidazole (5c): Yield 0.70 g (75%); mp 182-184 °C. 1H-
NMR (CDCl3): δ 3.51 (s, 3H, N-CH3), 3.86 (s, 3H, O-CH3), 7.01 (d, J = 8.8 Hz, 2H, 3'-H+ 5'-H ), 7.31
(d, J = 8.8 Hz, 2H, 2'-H+ 6'-H), 7.45 (s, 1H, 2-H); 13C-NMR (CDCl3): δ 33.1 (N-CH3), 55.5 (O-CH3),
114.4 (C-3'+ C-5'), 118.2 (C-1'), 131.6 (C-2'+ C-6'), 132.6 (C-5), 135.4 (C-2), 141.0 (C-4), 160.9 (C-
4'); ); HRMS ((+ve)-ESI): m/z calcd. for C11H12N3O3 [M+H]+: 234.08732, found: 234.08732; Anal.
Calcd. for C11H11N3O3 (233.22): C, 56.65; H, 4.75; N, 18.02. Found: C, 56.72; H, 4.82; N, 17.78.
1
5-(4-Fluorophenyl)-1-methyl-4-nitro-1H-imidazole (5d): Yield 0.65 g (73%); mp 137-139 °C. H-
NMR (CDCl3): δ 3.52 (s, 3H, N-CH3), 7.20 (m, 2H, 2'-H + 6'-H), 7.38 (m, 2H, 3'-H + 5'-H), 7.47 (s,
13
2
1H, H-2); C-NMR (CDCl3): δ 33.1 (CH3), 116.3 (d, JC-F = 22 Hz, C-3'+ C-5'), 122.4 (d, 4JC-F = 3.6
Hz, C-1'), 131.0 (C-5), 132.3 (d, 3JC-F = 8.6 Hz, C-2'+ C-6'), 135.6 (C-2), 141.0 (C-4), 163.7(d, 1JC-F
=
250 Hz, C-4'); HRMS ((+ve)-ESI): m/z calcd. for C10H8FN3O2Na [M+Na]+: 244.04928, found:
244.04928; Anal. Calcd. for C10H8FN3O2 (221.19): C, 54.30; H, 3.65; N, 19.00. Found: C, 53.98; H,
3.49; N, 18.80.

Molecules 2009, 14
2764
5-(4-Chlorophenyl)-1-methyl-4-nitro-1H-imidazole (5e): Yield 0.54 g (57%); mp 237-239 °C (Lit.[24]
°C
238-240 ). 1H-NMR (CDCl3): δ 3.52 (s, 3H, N-CH3), 7.32 (d, J = 8.6 Hz, 2H, 2'-H + 6'-H), 7.48 (s,
1H, 2-H), 7.49 (d, J = 8.6 Hz, 2H, 3'-H + 5'-H); 13C-NMR (CDCl3): δ 33.2 (CH3), 116.6 (C-1'), 124.9
(C-4'), 129.3 (C-2'+ C-6'), 130.2 (C-5), 131.5 (C-3'+ C-5'), 135.8 (C-2), 141.1 (C-4); HRMS ((+ve)-
ESI): m/z calcd. for C10H9ClN3O2 [M+H]+: 238.03778, found: 238.03778; Anal. Calcd. for
C10H8ClN3O2 (237.64): C, 50.54; H, 3.39; N, 17.68. Found: C, 50.33; H, 3.28; N, 17. 32.
1
5-(3-Chlorophenyl)-1-methyl-4-nitro-1H-imidazole (5f): Yield 0.60 g (63%); mp 170-172 °C. H-
NMR (CDCl3): δ 3.50 (s, 3H, N-CH3), 7.26 (m, 1H), 7.36 (m, 1H), 7.45 (s, 1H, 2-H), 7.48 (m, 2H);
13C-NMR (CDCl3): δ 33.2 (CH3), 120.3 (C-1'), 128.4 (C-6'), 130.1 (C-2'), 130.3 (C-4'), 130.5 (C-5'),
130.9 (C-5), 134.9 (C-3'), 135.8 (C-2), 141.4 (C-4); HRMS ((+ve)-ESI): m/z calcd. for C10H9ClN3O2
[M+H]+: 238.03778, found: 238.03778; Anal. Calcd. for C10H8ClN3O2 (237.64): C, 50.50; H, 3.39; N,
17.68. Found: C, 50.90; H, 3.18; N, 17.75.
Biological Activity
Test organisms
Entamoeba histolytica HK-9 strain (ATCC number 30015) was cultured in LYI-S-2 medium
supplemented with antibiotics. Giardia intestinalis WB strain (ATCC number 30957) was grown in a
modified YI-S medium with antibiotics. Both parasites were grown as described [35]. Briefly, the
parasites were cultivated and maintained in 15-mL screw-capped borosilicate glass tubes. Entamoeba
and Giardia were harvested from confluent cultures by chilling of the tubes on ice, followed
by centrifugation.
Antiamoebic and antigiardial activity
The antiamoebic and antigiardial activities of the prepared molecules and metronidazole as the
standard antiamoebic and antigiardial drug were tested as described [36]. Briefly, the tested
compounds and metronidazole were dissolved in dimethyl sulfoxide (DMSO) then in medium and
filter-sterilized. Two-fold dilutions starting at 15 μg/mL were prepared in a final volume of 15 mL to
exclude air from the tube. Each tube was inoculated with 20,000 cells of the parasite under testing
(Entamoeba or Giardia). Each compound was assayed in duplicate in each of three independent
experiments. In each assay, the appropriate controls were performed, including the one without any
compound and another with metronidazole as the positive control. The biological activity of the
compounds was evaluated by counting the parasites in each tube using the standard hemacytometer. In
each count, trypan blue was employed to distinguish live from dead parasites [37].
Cytotoxicity assay
The cytotoxicity of the reported compounds and the reference drug, metronidazole, was investigated
on Hep-2 and Vero cells using the standard cytotoxicity assay and the trypan blue exclusion method as
described before [36]. Briefly, 100 μL portions of each cell suspension were added to the wells of 96-
well plates, incubated for 24 h, and the medium in each well was then replaced with 150 μL fresh

Molecules 2009, 14
2765
medium. Solutions of the compounds or the reference drug were dissolved in DMSO, prepared in
medium, and filter sterilized. Then, 150 μL-two fold serial dilutions of each of the compounds and the
reference drug starting at a concentration of 2,000 μg/mL in culture medium were prepared in the
plates. After 48 hour incubation, the number of cells in each well was determined a hemacytometer.
Each compound was assayed in duplicate in each of three independent experiments. In each assay the
negative controls (without any compound or reference drug) were included in duplicates.
Conclusions
In conclusion, we have described the synthesis of a number of 5-aryl-1-methyl-4-nitroimidazoles
5a-f with promising antiparasitic activity. Bioassay of these compounds indicated significant
antiparasitic activities against Entamoeba histolytica and Giardia intestinalis that they could be used as
lead structures for the development of antiparasitic drugs. The IC50 of the hybrid molecule 5f was
found to be about three times lower than that of the standard drug metronidazole against those
parasites and it could therefore be considered as a good drug candidate to be tested against
metronidazole-resistant parasites and possibly anaerobic bacteria.
Acknowledgement
H. A. Saadeh and M. M. El-Abadelah are grateful to the Deanship of Scientific Research at the
University of Jordan for financial support.
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Sample Availability: Samples of compounds 5a-f are available from the authors.
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