Compounds containing 2-substituted imidazole ring for treatment against human African trypanosomiasis

Article abstract of DOI:10.1016/j.bmcl.2010.12.040

A series of compounds containing 2-substituted imidazoles has been synthesized from imidazole and tested for its biological activity against human African trypanosomiasis (HAT). The 2-substituted 5-nitroimidazoles such as fexinidazole (7a) and 1-[4-(1-methyl-5-nitro-1H-imidazol-2-ylmethoxy)-pyridin-2- yl-piperazine (9e) exhibited potent activity against T. brucei in vitro with low cytotoxicity and good solubility. The presence of the NO₂ group at the 5-position of the imidazole ring in 2-substituted imidazoles is the crucial factor to inhibit T. brucei.

Full text of DOI:10.1016/j.bmcl.2010.12.040

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1015–1018
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Compounds containing 2-substituted imidazole ring for treatment
against human African trypanosomiasis
Bhupesh S. Samant a, , Mugdha G. Sukhthankar b,
⇑
a Natural Product and Medicinal Chemistry (NPMC) research group, Division of Pharmaceutical chemistry, Faculty of Pharmacy, Rhodes University, Grahamstown 6140, South Africa
b Laboratory of Environmental Carcinogenesis, Department of Pathobiology, College of Veterinary Medicine, The University of Tennessee, Knoxville, TN 37996, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of compounds containing 2-substituted imidazoles has been synthesized from imidazole and
tested for its biological activity against human African trypanosomiasis (HAT). The 2-substituted 5-nitro-
imidazoles such as fexinidazole (7a) and 1-[4-(1-methyl-5-nitro-1H-imidazol-2-ylmethoxy)-pyridin-2-
yl-piperazine (9e) exhibited potent activity against T. brucei in vitro with low cytotoxicity and good
solubility. The presence of the NO2 group at the 5-position of the imidazole ring in 2-substituted
imidazoles is the crucial factor to inhibit T. brucei.
Received 16 October 2010
Revised 2 December 2010
Accepted 6 December 2010
Available online 10 December 2010
Keywords:
Fexinidazole
Ó 2010 Elsevier Ltd. All rights reserved.
Sleeping sickness
African trypanosomiasis
2-Substituted 5-nitroimidazole
Human African trypanosomiasis (HAT), commonly known as
sleeping sickness, is one of the most neglected diseases in the
Tropics.1 HAT is a vector-borne disease caused by the parasitic
protozoa belonging to the Genus Trypanosoma.2–4 The protozoan
is transmitted to humans by bites of blood sucking tsetse flies
(Glossina Genus) infected from biting human beings or animals
harboring the human pathogenic parasites.5 Sleeping sickness is
a threat to millions of people around 36 countries of sub-Saharan
Africa.
The drugs used for the treatment of HAT have various draw-
backs; for example, an intravenous drug melarsoprol causes a
life-threatening syndrome.6,7 Hence, an oral drug without harmful
side effects, acting against acute and chronic stages of the disease
and which can be synthesized easily and cost effectively is always
appealing. In this respect, the members of azole class of com-
pounds like 2-substituted-5-nitroimidiazoles (e.g., fexinidazole)
qualify as promising drug candidates. Their advantages, especially
of fexinidazole, include oral administration and curing of chronic
sleeping sickness in the brain within two weeks. The phase I hu-
man trials of fexinidazole began in 2009.8,9 The synthesis processes
in the literature for various 2-substituted-5-nitroimidiazoles de-
scribe the use of either costly starting materials,10,11 catalysts,12
and reaction conditions,13 or compounds that are not easily avail-
able. Thus, there is a demand for a synthesis process, which uses
simple, inexpensive reaction conditions, and starting compounds.
As a part of our ongoing research on novel chemical entities with
antitrypanosomal activities, we hereby like to introduce a synthesis
of different derivatives of 2-substituted 5-nitroimidazoles from eas-
ily available, economically and ecologically viable starting com-
pounds (i.e., imidazole), and evaluate their biological activity
against HAT. This series of compounds contain 2-substituted imid-
azole ring as a backbone structure; by substituting different func-
tional groups on this backbone, we studied structural–activity
relationship with respect to their ability to fight against HAT.
Regioselective nitration at 5-position of imidazole (1), using
nitrating mixture (nitric acid and sulfuric acid) in an aqueous sur-
factant solution to form 5-nitroimidazole (2a) as
a product
(Scheme 1) was the first step of the synthesis process.14,15 The spa-
tial orientation of substrate 1 in micelles increased the selectivity
towards 5-nitroimidazole. The use of surfactant as a catalyst in
the reaction media to increase the reaction rate towards particular
NO2
R
NO2
HNO3,
x/y/z
N
NH
N
NH
N
NH
H2SO4,
H2O+SDS
3a, R= Cl (61%),
3b, R= Br (58%),
3c, R= SO3H (60%)
1
2a (55%)
⇑
Corresponding author. Tel.: +27 46 6038395; fax: +27 46 6361205.
E-mail addresses: B.Samant@ru.ac.za, bhupesh.samant@gmail.com (B.S. Samant).
Present address: Pharmaceutical Sciences Department, St. Jude Children’s
Research Hospital, Memphis, TN 38105, USA.
Scheme 1. Synthesis of 4-substituted-5-nitro-1H-imidazole. xOxychlorination;
yoxybromination; zsulfonation. Values in brackets indicate isolated yields.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.040

1016
B. S. Samant, M. G. Sukhthankar / Bioorg. Med. Chem. Lett. 21 (2011) 1015–1018
product is well known.16 Nitration reaction of 1 in pure water was
carried out to form 4-nitroimidazole (2b); in order to substitute a
NO2 group at the 4-position of imidazole (to obtain 1-methyl-2-(4-
methylsulfanyl-phenoxymethyl-)-4-nitro-1H-imidazole 18 as the
final product of total synthesis).
azole (6a–d) as product. For this reaction, a solution of compound
5 in dichloromethane was added drop wise into pre cooled (0 °C)
mixture of thionyl chloride and dichloromethane. This mixture
was then heated to reflux for half an hour. The concentrate, which
was obtained after evaporation of solvent, was dissolved in ethanol
and again refluxed for 15 min to afford product 6. Williamson ether
synthesis reaction was used to generate fexinidazole (7a), its deriv-
atives (7b–d), and other 2-substituted 5-nitro-imidazoles (9a–c)
by the condensation of compound 6 with 4-(methylmercapto)-
phenol or (p-hydroxyphenyl methyl sulfide) (Table 1); and the
compound 6a with corresponding substituted alcohols (Table 2),
respectively.17
Three different methods such as Zn metal catalysis reaction in
the microwave, in normal oil bath, and using Cs2CO3 and KI as
the catalysts were used to synthesize the final 2-substituted 5-
nitro-imidazoles (7a–d and 9a–e). Though, the percentage yield
for microwave method is less than oil bath method, the reaction
time for microwave is shorter (1.45 h) compared to other methods.
Lower yield in the microwave method was due to the formation of
impurities in the reaction media. There was no significant
improvement in the product yield after the change in temperature,
or reduction in time for the microwave reaction. We also synthe-
sized the analogues of fexinidazole utilizing the same protocol to
study the changes in the biological activity against HAT, after
replacing NO2 group with other functional groups (Me, Cl, Br and
SO3H).
Different substitution reactions on 5-nitroimidazole (2) were
carried out as exemplified in Scheme 1. A cost effective oxyhalo-
genation method was applied to obtain 4-chloro-5-nitro-1H-imid-
azole (3a) and 4-bromo-5-nitro-1H-imidazole (3b) by using
hydrogen peroxide and corresponding acid.16 Hydrochloric acid-
hydrogen peroxide and sulfuric acid-hydrogen peroxide-sodium
bromide systems were used for chlorination and bromination,
respectively. Oxidation of hydrochloric acid by hydrogen peroxide
produced hypochlorous acid (HOCl), which then formed the
electrophilic Cl+ species in the reaction mixture. In the case of
bromination, hydrogen peroxide and sodium bromide formed
hypobromous acid (HOBr), which ultimately gave Br+ species and
NaOH. The generated Br+ species on reaction with aromatic com-
pound formed brominated aromatic compound, while sulfuric acid
neutralized NaOH. The oxyhalogenation reaction conditions were
simple, with no hazardous side products, and with satisfactory
yields (61% for 3a and 58% for 3b). Sulfonation of 2 by Sulfuric acid
formed 5-nitro-4-sulfoimidazole (3c) with satisfactory yield (60%).
Methylation of these substituted imidazoles (2 and 3a–c), by
the action of dimethyl sulfate in dioxane, was carried out to obtain
80% average yield of methylated products 4a–d (Scheme 2). These
methylated derivatives of substituted imidazoles (4a–d), on reac-
tion with formaldehyde in DMSO, gave the corresponding methyl-
ated imidazolyl methanol derivatives (5a–d) as products in
excellent yield. This reaction provided substitution on the 2-posi-
tion of the imidazole ring.
The series of 2-substituted imidazoles has been tested for its
biological activity against HAT. These compounds were evaluated
on the basis of their ability to inhibit cell proliferation of T. brucei
rhodesiense in culture. The growth inhibitory activity against L-6
rat skeletal muscle myoblast cells was determined to establish a
cellular therapeutic index. The solubility of each compound at
acidic and basic pH was determined.
Compounds 5a–d reacted with thionyl chloride in chloroform to
give corresponding 2-chloromethyl-1-methyl-5-substituted imid-
Fexinidazole (7a) showed effective T. brucei rhodesiense inhibi-
tory activity and low cytotoxicity (Table 3, entry 1). The T. brucei
cell proliferation inhibition ability by three derivatives of fexini-
dazole was also tested. 4-Chloro-1-methyl-2-(4-methylsulfanyl-
phenoxymethyl)-5-nitro-1H-imidazole (7b), 4-bromo-1-methyl-
2-(4-methylsulfanyl-phenoxymethyl)-5-nitro-1H-imidazole (7c),
and 1-methyl-2-(4-methylsulfanyl-phenoxymethyl)-5-nitro-
1H-imidazole-4-sulfonic acid (7d) showed higher inhibition
activity with higher solubility than fexinidazole; however, their
cytotoxicity was also high. The presence of an additional electron
R
NO2
R
NO2
Me2SO4
Dioxane
N
N
N
NH
Me
4a, R= H (82%),
4b, R= Cl (80%),
4c, R= Br (78%),
4d, R= SO3H (75%)
Table 1
Isolated yields of fexinidazole and its derivatives by three different methods17
R
NO2
HCHO,
DMSO,
140 OC
OH
Method A/
Method B/
Method C
N
N
Me
6
R
NO2
R
NO2
O
S
C2H5OH
N
N
N
N
SOCl2,
CH2Cl2
Me
Me
HO
Cl
S
5a, R= H (62%),
5b, R= Cl (65%),
5c, R= Br (60%),
5d, R= SO3H (58%)
6a, R= H (74%),
6b, R= Cl (62%),
6c, R= Br (64%),
Entry
Product
Method A (%)
Method B (%)
Method C (%)
1
2
3
4
7a, R = H
25
20
17
19
55
52
49
47
60
55
57
52
6d, R= SO3H (61%)
7b, R = Cl
7c, R = Br
7d, R = SO3H
Scheme 2. Synthesis of 4-substituted 2-chloromethyl-1-methyl-5-nitro-1H-imida-
zoles (6a–d). Values in brackets indicate isolated yields.

B. S. Samant, M. G. Sukhthankar / Bioorg. Med. Chem. Lett. 21 (2011) 1015–1018
1017
Table 2
The importance of NO2 group position may be related to the
structural arrangement of the five-member imidazole ring with
the carbon atom containing NO2 (5-position), which is adjacent
to the electron donating tertiary amine group (1-position) as in
compound 7a. This distinctive feature in the structure may be
the cause of T. brucei inhibitory activity. This is in accordance with
an earlier study of 2-substituted nitro imidazoles for their biolog-
ical activity against HAT.18,19
Isolated yields of various 2-substituted 5-nitro-imidazoles (9a–e) by three different
methods17
NO2
NO2
Method A/
Method B/
Method C
N
R1OH
N
N
N
Me
Me
Further, to study the effect of an electron donating group
at 2-position of imidazole on T. brucei inhibitory activity of
2-substituted 5-nitro imidazoles, we analyzed compounds 9a–e.
We observed that T. brucei inhibitory activity increased from
compound 9a to 9e and surprisingly 9e showed higher T. brucei
inhibitory activity than fexinidazole (7a). The compound 9e also
had low cytotoxicity and good solubility. The reason for this
enhancement in T. brucei inhibitory activity may be the balance
between the electron donating group at the 2-position and strong
electron withdrawing group at 4-position (NO2) of the imidazole
ring in 9e.
Concurrently, we replaced NO2 with other electron withdraw-
ing groups (Cl, Br and SO3H) in fexinidazole and also substituted
two halo groups on 4- and 5-positions of an imidazole ring to test
the effect of different and/or more number of electronegative
groups on T. brucei inhibitory activity (compounds 11–17,
Supplementary data Table S1). However, these attempts failed to
give any enhancement in T. brucei inhibitory activity for these
compounds. Only the solubility of these compounds increased
effectively (ꢀ9-fold) due to the presence of halogen functional
groups in the structure.
The exact mechanism of action for this series of compounds is
yet to be elucidated. However, the above results indicate that the
compounds with 2-substituted-5-nitroimidiazole as the backbone
structure have an effective T. brucei inhibitory activity. This might
be due to their electrophilic center and the presence of a phenyl
ring, which is considered to be a good source of electron density,
and can react with nucleophile (i.e., cysteine and lysine) favorably
in the target. The substitution of Cl, Br and SO3H groups along with
the presence of the NO2 moiety on the imidazole ring causes an in-
crease in electrophilicity of this ring. These groups also act as good
leaving groups in the binding step of the cysteine thereby, confer-
ring activity against T. brucei. It seems that these compounds act as
a suicide inhibitor of the enzyme which regulates cell division by
catalysing the first step in polyamine biosynthesis. As the inhibitor
has short half-life in humans, it is rapidly degraded while the par-
asite cannot metabolise it quick enough to survive.
O
Cl
R1
6a
Entry Product
9
Method
A
(%)
Method
B
(%)
Method
C
(%)
1
2
3
4
5
9a, R1 = Ph
33
28
22
25
24
62
57
52
55
56
72
61
58
57
55
9b, R1 = Pyridine
9c, R1 = 2-Methyl-pyridine
9d, R1 = 2-Ethyl-pyridine
9e, R1 = 1-Pyridine-2-yl-
piperizine
withdrawing group (like Cl, Br and SO3H) on the imidazole ring in-
creased the T. brucei inhibitory activity. On comparing the T. brucei
inhibitory activities of the compounds 7b–d (Table 3, entries 2–4),
it was observed that compound 7c showed less potent T. brucei
inhibitory activity than compounds 7b and 7d. This may be due
to the steric and less electronegative nature of Br (which is present
on the imidazole ring in compound 7c) than Cl and SO3H. However,
there is no significant difference between cytotoxicity and solubil-
ity of the compounds 7b–d. The substitution of Cl, Br and SO3H
groups along with the presence of the NO2 moiety on the imidazole
ring causes an increase in the electrophilicity of this ring and con-
fers activity against T. brucei.
To evaluate the importance of the NO2 group on an imidazole
ring with respect to the inhibition of T. brucei cell proliferation,
we compared the activities of compounds 7a and 8. Compound 8
had weak T. brucei inhibitory activity (Table 3, entry 5) indicating
an important factor with respect to the required position of NO2
group on the imidazole ring. The only difference between the
structures of compounds 7a and 8 is the position of NO2 group
on an imidazole ring. However, there is a remarkable difference
in their T. brucei inhibitory activities. This proves that an electron
withdrawing NO2 group on the imidazole ring is an essential factor
to inhibit the T. brucei and should be present at the 5-position of
the imidazole ring.
In summary; a series of compounds containing 2-substituted
imidazole ring has been synthesized from imidazole and tested
for its biological activity against HAT including its cytotoxicity
and solubility. Compound 9e with an electron donating group at
Table 3
Pharmacological properties of 2-substituted imidazoles
Entry
Compound
IC50 versus T. bruceia
(l
M)
IC50 versus L-6 rat skeletal
Aq solution solubilityb
(lM)
myoblast cellsa
(lM)
pH 1.5
pH 7.5
1
2
3
4
5
6
7
8
9
7a
7b
7c
7d
8
9a
9b
9c
9d
9e
0.70 0.1
0.25 0.1
0.55 0.1
0.37 0.1
>30
377 25
0.2 0.05
0.8 0.1
0.2 0.1
>400
>400
>400
286 15
243 10
255 10
5.5 0.4
17.1 0.8
16.7 0.5
22.8 1.5
5.1 0.5
5.7 0.5
4.8 0.5
4.3 0.5
5.9 0.5
5.6 0.5
2.5 0.1
5.9 0.2
5.8 0.2
7.9 0.5
2.5 0.1
2.1 0.1
2.7 0.1
2.2 0.1
3.1 0.1
3.5 0.1
>30
>30
27
25
3
3
10
0.67 0.1
a
IC50 values are means standard deviations; three independent experiments were done with replicates of three.
Solubility of compounds was determined at 25 °C.
b

1018
B. S. Samant, M. G. Sukhthankar / Bioorg. Med. Chem. Lett. 21 (2011) 1015–1018
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the 2-position, and the strong electron withdrawing group at
4-position (NO2) seems to be the crucial factor to show an effective
T. brucei inhibitory activity along with low cytotoxicity and good
solubility. Substituting other electron withdrawing groups Cl, Br,
and SO3H on 4-position of imidazole in fexinidazole seems to
increase T. brucei inhibitory activity and solubility; however,
cytotoxicity also increased for these compounds (7b–d). Although
an exact mechanism of action is still unknown for this series of
compounds, it is observed that the presence of NO2 group at
5-position of an imidazole ring is an essential factor to illustrate
T. brucei inhibitory activity.
Acknowledgements
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Authors are thankful to Rhodes University Joint Research Com-
mittee (Rhodes University JRC grant number 35047) for providing
financial support for this work. Authors thank, Prof. S. S. Bhagwat
(ICT, University of Mumbai, India) for feedback on nitration reac-
tion in micellar media.
Supplementary data
17. Method A: p-Hydroxyphenyl methyl sulfide (0.8 g, 5.7 mmol), 2-chloromethyl-
1-methyl-5-substituted imidazoles (6a–d) (1 g, 5.7 mmol, 6a), Zn powder
(0.09 g, 1.42 mmol) in DMF (10 mL), microwave 150 °C, 1.45 h; Method B: p-
Hydroxyphenyl methyl sulfide (0.8 g, 5.7 mmol), 2-chloromethyl-1-methyl-5-
substituted imidazoles (6a–d) (1 g, 5.7 mmol, 6a), THF (5 mL), 55 °C, 14 h;
Method C: 2-Chloromethyl-1-methyl-5- substituted imidazoles (6a–d) (1 g,
5.7 mmol, 6a), p-hydroxyphenyl methyl sulfide (0.8 g, 5.7 mmol), Cs2CO3
(6.6 g, 34.2 mmol), KI (0.095 g, 0.57 mmol) in MeCN under inert atmosphere
at 80 °C reflux for overnight. Please refer supporting information for detail
procedures and analytical spectroscopic information of products.
18. Raether, W.; Ann, H. Trop. Seidenath Med. Parasitol. 1983, 77, 13.
19. Jennings, F. W.; Urquhart, G. M. Z. Parasitenkd. 1983, 69, 577.
Supplementary data (detail synthesis process and characteriza-
tion of all synthesized products (2–10), detail procedure for adap-
tation of bloodstream trypanosomes to liquid in vitro culture,
trypanosoma brucei proliferation assay, general cytotoxicity assay
and pharmacological properties of compounds 11–17 (Table S1))
associated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2010.12.040.
References and notes
1. Simarro, P. P.; Jannin, J.; Cattand, P. PLoS Med. 2008, 5, 174.