The synthesis of novel hybrid compounds containing 5-nitrothiazole moiety as potential antiparasitic agents

Article abstract of DOI:10.1007/s00706-015-1511-y

A number of novel compounds containing 5-nitrothiazole moiety as potential antiparasitic agents have been synthesized through known chemical routes. The structures of the new compounds were confirmed by spectroscopic techniques, ¹H NMR, ¹³C NMR, and mass spectrometry, and by elemental analyses. The prepared compounds were evaluated in vitro for their antigiardial and antitrichomonal activities. All tested compounds exhibited remarkable antigiardial activity with IC₅₀ values ranging from 2.2 to 6.9 μg/cm³ as compared to the reference drug metronidazole (IC₅₀ = 7.3 μg/cm³). In addition, three of the prepared compounds exhibited significant antitrichomonal activity with IC₅₀ values of 4.3, 5.0, and 7.9 μg/cm³, respectively, as compared to the reference drug metronidazole (8.5 μg/cm³). Graphical abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00706-015-1511-y

Monatsh Chem
DOI 10.1007/s00706-015-1511-y
ORIGINAL PAPER
The synthesis of novel hybrid compounds containing
5-nitrothiazole moiety as potential antiparasitic agents
Yasmin R. H. Saleh1 Haythem A. Saadeh1,2 Hargobinder Kaur3 Kapil Goyal3
•
•
•
•
Rakesh Sehgal3 Mohammad S. Mubarak1
•
Received: 12 February 2015 / Accepted: 7 June 2015
Ó Springer-Verlag Wien 2015
Abstract A number of novel compounds containing
5-nitrothiazole moiety as potential antiparasitic agents have
been synthesized through known chemical routes. The
structures of the new compounds were confirmed by
Keywords 5-Nitrothiazole Á Metronidazole Á
Chloroquine Á Hybrid molecules Á Antigiardial activity Á
Antitrichomonal activity
1
spectroscopic techniques, H NMR, 13C NMR, and mass
Introduction
spectrometry, and by elemental analyses. The prepared
compounds were evaluated in vitro for their antigiardial
and antitrichomonal activities. All tested compounds
exhibited remarkable antigiardial activity with IC50 values
ranging from 2.2 to 6.9 lg/cm3 as compared to the refer-
ence drug metronidazole (IC50 = 7.3 lg/cm3). In addition,
three of the prepared compounds exhibited significant
antitrichomonal activity with IC50 values of 4.3, 5.0, and
7.9 lg/cm3, respectively, as compared to the reference
drug metronidazole (8.5 lg/cm3).
Parasitic diseases caused by helminthes and protozoa are
major causes of human misery in most countries of the
tropics. Giardia lamblia is a flagellated protozoan parasite
which causes an intestinal infection. Worldwide, there are
approximately one billion cases of giardiasis and among
the mortality due to diarrheal diseases, giardiasis causes 2.5
million deaths annually [1]. According to the World Health
Organization (WHO), three billion people reside in places
where giardiasis prevalence is around 30 %. It is mainly
prevalent in developing nations where sanitation conditions
are poor. However, in developed nations it is also an
important cause of diarrhea among the international trav-
elers and migrants from endemic countries [2]; in the
United States, approximately 20,000 new cases of giar-
diasis are reported annually [3]. Giardiasis has also been
included in the ‘Neglected Disease Initiative’ by the WHO
since 2004 due to its increasing global burden [4].
Graphical abstract
& Mohammad S. Mubarak
mmubarak@ju.edu.jo
The most commonly used antibiotics/antiparasitic
agents to treat giardiasis are 5-nitroimidazoles (5-NIs)
which include metronidazole, tinidazole, secnidazole, and
ornidazole; among these, metronidazole is the most widely
used drug [5]. Most of these drugs are easily available, as
the over the counter drugs, in developing nations and
metronidazole is commonly prescribed for other indica-
tions such as gingivitis, bacterial vaginitis, part of the
combination treatment for H. pylori, infections
with Clostridium difficile and other anaerobic bacteria,
amebiasis, and as prophylaxis in colorectal surgery [6, 7].
Haythem A. Saadeh
h.saadeh@uaeu.ac.ae
1
Department of Chemistry, The University of Jordan, Amman
11942, Jordan
2
Department of Chemistry, College of Science,
United Arab Emirates University, P.O. Box 15551, Al-Ain,
United Arab Emirates
3
Department of Medical Parasitology, Postgraduate Institute
of Medical Education and Research, Chandigarh, India
160012
123

Y. R. H. Saleh et al.
The widespread use could pose a challenge of emerging
drug resistance in near future. There are several reports
which highlight the treatment failure among the patients
suffering from giardiasis and clinical resistance has been
observed in up to 20 % of cases [5, 8–11]. Giardia resis-
tance has been demonstrated in vitro towards common
antigiardials and cross resistance has also been shown
between metronidazole and tinidazole [12, 13]. A recent
study by Pasupuleti et al. [14] has shown that efficacy of
5-nitroimidazoles in the treatment of giardiasis is highly
heterogeneous. They have also highlighted that 5-NIs have
a slightly better efficacy and worse profile for mild harmful
outcomes in the treatment of giardiasis in comparison to
controls. Thus, there is a need for developing new drugs
which can be used in the treatment of giardiasis with better
efficacy and fewer side effects. In the present study, hybrid
compounds have been tested in vitro against Portland 1
strain of G. lamblia.
Another parasite which has been selected for in vitro
drug assay is the Trichomonas vaginalis. It is a causative
agent of trichomoniasis which is the most common non-
viral sexual infection. Trichomoniasis is considered as a
neglected parasitic infection which not only causes
vaginitis in women and urethritis in males but also
increases the risk of HIV transmission, pelvic inflammatory
disease, and spontaneous abortion [15–17]. Exact burden of
trichomoniasis is difficult to establish as most of the clin-
ical infections go undetected as symptoms are often mild or
absent, however, globally 170 million cases occur every
year [18]. The main course of treatment is metronidazole
but resistance has been reported in approximately 5 % of
clinical cases [19]. The lack of alternative therapies for T.
vaginalis means that higher doses of metronidazole are the
only option for patients with resistance diseases. Therefore,
there is a strong need to develop new drugs which can be
safely used in the treatment of trichomoniasis. With these
aims in view, the present study was planned to develop new
hybrid compounds and their efficacy was checked in vitro
against G. lamblia and T. vaginalis.
Fig. 1 Structures of some quinoline-containing antimalarial drugs
Fig. 2 Structures of nitazoxanide and artemisinin
antiprotozoal drug that has been in use for over 40 years. It
is particularly used for anaerobic bacteria and protozoa
[22] and for the treatment of several parasitic diseases such
as trichomoniasis, giardiasis, and amebiasis, and has
appeared in the essential drug list of the World Health
Organization [23]. On the other hand, thiazolides represent
a novel class of anti-infective drugs with the parent com-
pound, nitrothiazole nitazoxanide (2-acetyloxy-N-(5-nitro-
2-thiazolyl)benzamide) (5) (Fig. 2) [24]. Nitazoxanide,
also known as Alinia, is utilized broadly for the treatment
of the anaerobic intestinal parasites G. lamblia and Cryp-
tosporidium parvum and is also effective in the treatment
of other anaerobic bacteria and parasites residing in the
human gut [25]. It was first described in 1984 as a cesto-
cidal drug with the effectiveness in a single dose against T.
saginata and Hymenolepis nana; its anti-protozoal activity
was discovered in 1994.
Chemotherapy has been used in the treatment of indi-
vidual patients and in conjunction with public health and
vector-control measures, in reducing the transmission of
parasitic contagions [20]. The quinoline-containing anti-
malarial drugs, chloroquine (1), amodiaquine (AQ, 2),
mefloquine (3), and quinine (4) (Fig. 1) are a vital part of
chemotherapeutic armory against malaria. These drugs are
thought to act by interfering with the formation of
haemazoin in the blood stages of the malaria life cycle. For
several decades, the chloroquine (CQ) and the amodiaquine
(AQ, 2) were considered the most important drugs for the
control and eradication of malaria [21].
Easy availability and overuse of drugs can lead to
emergence of drug resistance pathogens at a significant
Metronidazole, [1-(2-hydroxyethyl)-2-methyl-5-nitroimi-
dazole], also known as ‘‘Flagyl’’ is an antibacterial and
123

The synthesis of novel hybrid compounds containing 5-nitrothiazole moiety as potential…
level. Therefore, there is a desperate need for new drugs to
counteract the alarming increase in evolution of antimi-
crobial resistance to existing antibiotics [22]. Chloroquine
for example was a very effective, safe, and inexpensive
antimalarial agent. However, in the early 1960s, resistance
to chloroquine developed at two foci, in Southeast Asia and
in Latin America, then it has spread to all areas where
malaria is present. There are indications, however, that
resistance is developing against the last remaining class of
antimalarial drugs, artemisinin (6) (Fig. 2) [26]. Similarly,
nitazoxanide-resistant G. lamblia trophozoites were gen-
erated using parasites with the same genetic background
(i.e. WB C6). A simultaneous increase of resistance to
metronidazole was attributed to nitazoxanide resistance
formation; nitazoxanide and metronidazole differentially
affect Giardia in terms of morphological alterations,
despite their structural similarities.
on the other hand, was prepared by combining the fast-
acting artemisinin and slow-acting quinine into a hybrid
drug for malaria, for which drug resistance is a barrier to
effective treatment [28]. In vitro assays showed that the
hybrid 8 is more effective against drug-sensitive and drug-
resistant malaria than the individual drugs alone or a
cocktail made of a 1:1 molar ratio of the two. These hybrid
drugs were more effective against drug-sensitive malaria
than chloroquine itself. In addition, they have the ability to
kill a chloroquine-resistant strain of P. falciparum [28].
Recently, Saadeh et al. [28] synthesized a set of hybrid
molecules assembled from precursors with known
antiparasitic activities. Bioassay of these compounds indi-
cated significant antiparasitic activities against Entamoeba
histolytica and Giardia intestinalis. The hybrid molecule 9
(Fig. 4) was found to be approximately five times more
active against those parasites than the standard drug
metronidazole.
The concept of ‘‘hybrid drugs’’ has been attracting
attention in medicine. A hybrid molecule can be as a
chemical that consists of two active components linked
with either a resistant or a labile linker unit that brings
distinct biological properties into one molecule. This
hybrid can have several advantages in addressing issues
associated with the rapid emergence of drug resistance,
potency, and solubility, route of administration, pharma-
cokinetics, metabolism, and safety over simple drug
combinations [27]. For example, one component of the
hybrid may amplify effects of the other hybrid part through
action on another biological target or to counterbalance the
known side effects associated with the other hybrid com-
ponent. Ultimately, no matter how familiar the building
blocks may be, hybrid drug molecules may become new
molecules with identities independent of their precursors
[28]. Examples on hybrid drugs are compounds 7 and 8
shown below (Fig. 3).
As part of our ongoing research in the synthesis of new
compounds of pharmacological interest [22, 28], we
describe herein the synthesis, characterization, and anti-
giardial and antitrichomonal activities of new hybrid
molecules, which, to the best of our knowledge, have not
previously been described in the literature. The new hybrid
molecules incorporate, in addition to 5-nitrothiazole, the
active sites of metronidazole (Flagyl), and chloroquine
(CQ) (Scheme 1). The antigiardial and antitrichomonal
activities of the newly prepared compounds were
evaluated.
Results and discussion
Chemistry
Drug hybrid 7 for example was found to be effective
against both chloroquine-sensitive and chloroquine-resis-
tant strains of Plasmodium falciparum. The hybrid drug 8,
Synthesis of the hybrid molecules 18–23 was carried out
via the route shown in Scheme 1. The prepared compounds
were checked for purity by TLC using glass plates
Fig. 3 Hybrid drugs
123

Y. R. H. Saleh et al.
exhibited less antiparasitic activity than metronidazole for
Trichomonas. The antigiardial (18–23) and antitrichomonal
activity (19, 20, and 22) exhibited by the new compounds
suggests that the derivatives may be used as new lead
compounds in the development of new antiparasitic drugs.
This cidal activity of the prepared compounds could extend
to cover other related anaerobic protozoal parasites such as
Entamoeba histolytica. Further, these compounds can also
be tested for anti-Plasmodium activity.
Fig. 4 Structure of an antiparasitic hybrid molecule
As has previously been mentioned, compounds 19, 20,
and 22 exhibited potent anti-parasitic activity against both
parasites, i.e. G. lamblia and T. vaginalis. These com-
pounds were found to be more active as compared to the
standard drug metronidazole. Thus, the new hybrid com-
pounds prepared by combining 5-nitrothiazole, the active
sites of metronidazole (Flagyl), and chloroquine (CQ)
with different linkers render these compounds more
active. However, in vivo studies are required to investi-
gate their pharmacodynamic and pharmacokinetic
activities. Antiparasitic activity also differs among dif-
ferent parasites; this has been observed in the present
study. Although the newly synthesized compounds were
found to be active against both parasites, however, these
compounds exhibited greater anti-giardial activity as
compared to anti-trichomonal activity. Metronidazole is
the most commonly used drug in the treatment of giar-
diasis and trichomoniasis. It is listed among the essential
drug list of WHO and is available over the counter.
However, clinical resistance has been reported in
10–20 % [30] and 2–4 % [31] of patients suffering from
giardiasis and trichomoniasis, respectively. Empirical use
of metronidazole can lead to emergence of drug resistance
in the near future. The present study highlights the
importance of formulation of new compounds from the
existing known compounds and can provide an alternate
treatment option in the future. The limitation of the pre-
sent study is that compounds were tested against two
parasites only, in future work the same compounds can
also be tested against other parasites such as Entamoeba
histolytica and Plasmodium spp. In addition, cytotoxicity
studies are also required so as to check the toxic effects on
host cells before these compounds can further be evalu-
ated in clinical trials.
precoated with silica gel 60 GF254, supplied by Fluka as a
stationary phase and suitable solvent system as mobile
phase and was also confirmed by melting point determi-
nation. The newly synthesized compounds 18–23 were
characterized by MS and NMR spectral data and by ele-
mental analysis. The 1H NMR and 13C NMR spectra of all
prepared compounds and precursors are in total agreement
with the proposed structures. DEPT experiments were
employed to differentiate secondary and quaternary car-
bons from primary and tertiary ones. These data, detailed in
the experimental part, are consistent with the suggested
structures. Thus, the mass spectra display the correct
molecular ion peaks for which the measured high-resolu-
tion (HRMS) data are in good agreement with the
calculated values.
Antigiardial and anti-trichomonal activity
The antigiardial activity of the newly synthesized com-
pounds was investigated using in vitro MTT assays against
metronidazole-sensitive Portland 1 strain of G. lamblia
[29]. Their bioactivity was compared with the standard
antigiardial drug, metronidazole. The IC50 values of the
compounds against Portland 1 strain of G. lamblia are
given in Tables 1 and 2. As shown in the tables, com-
pounds 18–23 exhibited remarkable biological activity
against Giardia with IC50 values ranging from 2.2 to
6.9 lg/cm3 as compared to an IC50 of 7.3 lg/cm3 for
metronidazole. Compounds 18, 20, and 23 displayed anti-
giardial activity with IC50 values of 2.2, 2.3, and 2.9 lg/
cm3, respectively, and were 3.3, 3.1, and 2.5 times,
respectively, more active than metronidazole. The prepared
compounds were further tested for their antitrichomonal
activity by in vitro MTT assay against metronidazole-
sensitive T. vaginalis strain. As indicated in Table 1, 19,
20, and 22 showed significant antitrichomonal activity with
IC50 values of 4.3, 5.0, and 7.9 lg/cm3, respectively, and
compounds 19 and 20 were 2.0 and 1.7 times more potent
than metronidazole. The other compounds 18, 21, and 23
exhibited anti-trichomonal activity but with a higher IC50
values as compared with the reference drug. The molecular
modifications on our derivatives did not render any of the
compounds inactive; however, compounds 18, 21, and 23
Conclusions
A series of new hybrid compounds containing 5-nitroth-
iazole as the parent moiety were prepared. Structures of the
newly synthesized compounds were confirmed by means of
1H NMR, 13C NMR, and mass spectrometry and by ele-
mental analysis. The prepared compounds were tested
in vitro for their antigiardial and antitrichomonal activity.
123

The synthesis of novel hybrid compounds containing 5-nitrothiazole moiety as potential…
Scheme 1
Results revealed that most of the prepared compounds
displayed remarkable antigiardial activity and were more
potent than metronidazole itself. In addition, some of the
newly synthesized hybrids exhibited more antitrichomonal
activities than the parent drug, metronidazole. These
findings may suggest that the new compounds can be lead
candidates as anti-parasitic agents, however, more work
would be needed to test these compounds in vivo and may
be using animal models to evaluate their usefulness as lead
drugs.
123

Y. R. H. Saleh et al.
constants (J) are given in Hertz. High-resolution mass
spectra (HRMS) were obtained (in positive or negative
mode) using electrospray ion trap (ESI) technique by col-
lision-induced dissociation on a Bruker APEX-4 (7-Tesla)
instrument (Bremen, Germany). The samples were dis-
solved in acetonitrile, diluted in spray solution (methanol/
water 1:1 v/v 0.1 % formic acid) and infused using a syr-
inge pump with a flow rate of 2 mm3/min. External
calibration was conducted using arginine cluster in a mass
range m/z = 175–871. Elemental analyses were performed
on a Euro Vector Elemental Analyzer (EA 3000 A).
Compounds were checked for their purity by TLC using
glass plates, pre-coated with silica gel 60 GF254, supplied
by Fluka.
Table 1 Antigiardial and antitrichomonal activities of compounds
18–23 by MTT assay
Compound
Mean
IC50 SD(n)/lg cm-3
Giardia
Mean
IC50 SD(n)/lg cm-3
T. vaginalis
18
2.2 0.2
4.3 0.2
2.3 0.1
6.9 0.6
4.9 0.6
2.9 0.1
7.3 0.9
8.8 1.2
4.3 1.2
5.0 1.3
9.4 2.0
7.9 0.1
9.8 1.3
8.5 0.9
19
20
21
22
23
Metronidazole
SD(n) standard deviation
2-Chloro-N-(5-nitrothiazol-2-yl)acetamide
(17, C5H4ClN3O3S)
Table 2 IC50 ratios of the tested compounds 18–23
This compound was synthesized according to the following
modified procedure [36]. To a stirred solution of 4.4 g
2-amino-5-nitrothiazole (10, 30 mmol) in 333 cm3 ethyl
acetate in the presence of 70 cm3 saturated sodium
carbonate at 0 °C was added chloroacetyl chloride until
completion of reaction (around 30 cm3). The organic layer
was separated, washed with 40 cm3 of 3 N HCl solution
followed by brine, dried over sodium sulfate, and evapo-
rated to afford compound 17 as yellow to brown crystals.
Yield: 6.6 g (79.0 %); m.p.: 149–151 °C (Ref. [37]
146–147 °C).
Compound
IC50 ratio
(metronidazole/compound)
Giardia
IC50 ratio
(metronidazole/
compound)
T. vaginalis
18
19
20
21
22
23
3.3
1.7
3.2
1.1
1.5
2.5
0.97
1.96
1.7
0.91
1.1
0.87
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-N-(5-nitrothiazol-
2-yl)acetamide (18, C9H8N6O5S)
Experimental
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)acetic acid (11,
0.19 g, 1.0 mmol) was dissolved in 5 cm3 dry THF under
stirring with 0.13 g N,N0-diisopropylcarbodiimide (DIC,
1.0 mmol) and 0.012 g 4-dimethylaminopyridine (DMAP,
0.1 mmol) under nitrogen. 2-Amino-5-nitrothiazole (10,
0.15 g, 1.0 mmol) was added and the mixture was stirred
overnight at room temperature. Water was then added and
the product was extracted with chloroform (5 9 50 cm3).
The organic solvent was removed under reduced pressure
and the residue was purified by column chromatography
(eluent: chloroform) to yield 18. Yield: 0.19 g (61.0 %);
m.p.: 132–137 °C; 1H NMR (500 MHz, CDCl3): d = 2.40
(s, 3H, CH3), 3.35 (s, 2H, H-10), 8.07 (s, 1H, H-4), 8.50 (s,
1H, H-400), 9.24 (s, 1H, H-30) ppm; 13C NMR (125 MHz,
CDCl3): d = 14.0 (C2–CH3), 48.5 (C-10), 132.5 (C-500),
133.2 (C-4), 141.1 (C-5), 142.3 (C-400), 152.1 (C-2), 164.1
(C-100), 165.3 (C=O) ppm; HRMS (ESI): m/z calcd for
C9H8N6NaO5S [M?Na]? 335.01746, found 335.01691.
The following chemicals and materials were obtained from
commercial sources and were used without further purifi-
cation: 2-(2-methyl-5-nitroimidazolyl)ethylamine hydro-
chloride, 2-amino-5-nitrothiazole, 4,7-dichloroquinoline,
1,3-diaminopropane,
1,4-diaminobutane,
piperazine,
sodium bicarbonate, bromoacetyl chloride. The following
compounds were synthesized and purified according to
literature procedures: 2-(2-methyl-5-nitro-1H-imidazol-1-
yl)acetic acid (11) [32], 2-bromo-5-nitrothiazole (12)
[33–35], 7-chloro-4-(piperazin-1-yl)quinolone (13) [28],
N-(3-aminopropyl)-7-chloroquinolin-4-amine (14), and
N-(4-aminobutyl)-7-chloroquinolin-4-amine (15) [33]. Sil-
ica gel for column chromatography and silica gel plates
were purchased from Fluka and Macherey–Nagel, respec-
tively. Progress of reactions was monitored by thin layer
chromatography (TLC) using glass plates pre-coated with
silica gel (E. Merck Kiesegel 60 F254, layer thickness
0.25 mm). Melting points were measured with a Fischer-
1
Johns melting point apparatus. H and 13C NMR spectra
7-Chloro-4-[4-(5-nitrothiazol-2-yl)piperazin-1-yl]-
quinoline (19, C16H14N5O2S)
were acquired with the aid of a Bruker DPX 500 MHz
spectrometer (Germany) with TMS as the internal standard.
Chemical shifts are expressed in d (ppm) units; coupling
A mixture of 0.35 g 7-chloro-4-(piperazin-1-yl)quinoline
(13, 1.4 mmol), 0.29 g 2-bromo-5-nitrothiazole (12,
123

The synthesis of novel hybrid compounds containing 5-nitrothiazole moiety as potential…
1.4 mmol) and 0.12 g sodium bicarbonate (1.4 mmol) in
3 cm3 ethanol was stirred under reflux for 30 min. The
reaction mixture was then cooled to room temperature and
the precipitate was filtered, washed with ethanol, water,
glacial acetic acid, and then with small portions of water to
give a pure brown solid 19. Yield: 0.53 g (71.0 %); m.p.:
251–256 °C; 1H NMR (500 MHz, DMSO-d6): d = 3.30 (s,
8H, H-200), 7.10 (d, J = 5.0 Hz, 1H, H-3), 7.60 (dd,
J = 2.2, 9.0 Hz, 1H, H-6), 8.00 (d, J = 2.2 Hz, 1H, H-8),
8.10 (d, J = 9.0 Hz, 1H, H-5), 8.50 (s, 1H, H-40), 8.80 (d,
J = 5.0 Hz, 1H, H-2) ppm; 13C NMR (125 MHz, DMSO-
d6): d = 51.1 (C-200), 110.5 (C-50), 121.8 (C-3), 126.5 (C-
4a), 128.6 (C-6), 134.3 (C-8), 136.8 (C-40), 147.8 (C-7),
150.1 (C-8a), 152.7 (C-2), 156.1 (C-20), 172.9 (C-4) ppm;
HRMS (ESI): m/z calcd for C16H15N5O2S [M?H]?
376.06350, found 376.06295.
ethyl acetate as the developing solvent to give pure 21.
Yield: 0.087 g (23.0 %); m.p.: 151–153 °C; 1H NMR
(500 MHz, DMSO-d6): d = 1.50 (m, 4H, H-300 ? H-400),
2.80 (m, 4H, H200 ? H-500), 3.40 (s, 2H, H-100 ? H-600),
6.70 (d, J = 8.6 Hz, 1H, H-3), 7.30 (d, J = 5.0 Hz, 1H,
H-6), 7.60 (s, 1H, H-8), 8.00 (d, J = 5.0 Hz, 1H, H-5),
8.30 (s, 1H, H-40), 8.50 (d, J = 8.6 Hz, 1H, H-2) ppm; 13
C
NMR (125 MHz, DMSO-d6): d = 27.1 (C-300 ? C-400),
42.8 (C-200), 42.9 (C-500), 109.9 (C-50), 116.9 (C-3), 124.5
(C-4a), 124.6 (C-5), 127.8 (C-6), 127.9 (C-8), 133.9 (C-7),
137.4 (C-8a), 149.4 (C-40), 150.1 (C-2), 152.2 (C-4), 158.5
(C-20) ppm; HRMS (ESI): m/z calcd for C16H15ClN5O2S
[M-H]- 376.06350, found 376.06405.
N-[2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl]-5-
nitrothiazol-2-amine (22, C9H10N6O4S)
A mixture of 0.17 g 2-(2-methyl-5-nitro-1H-imidazol-1-
yl)ethanamine (16, 1.0 mmol), 0.21 g 2-bromo-5-nitroth-
iazole (12, 1.0 mmol), and 0.14 g potassium carbonate
anhydrous (1.0 mmol) was dissolved in dry DMF and
stirred at 70 °C overnight under nitrogen. The mixture was
then cooled to room temperature, treated with 50 cm3
water, and extracted with ethyl acetate (5 9 50 cm3). The
solvent was removed under reduced pressure and the
residue was purified by preparative silica gel plates using
ethyl acetate to give pure 22. Yield: 0.075 g (25 %); m.p.:
204–207 °C; 1H NMR (500 MHz, DMSO-d6): d = 1.24 (s,
3H, CH3), 3.85 (m, 2H, H20), 4.51 (m, 2H, H30), 8.02 (s,
1H, H400), 8.22 (s, 1H, H4), 9.50 (s, 1H, NH10) ppm; 13C
NMR (125 MHz, DMSO-d6): d = 14.3 (C-200-CH3), 45.5
(C-20 ? C-30), 133.6 (C-4 ? C-400), 136.1 (C-5), 139.1 (C-
500), 147.1 (C-200), 151.8 (C-2) ppm; HRMS (ESI): m/
z calcd for C9H11N6O4S [M ? H]? 299.05625, found
299.05570
N-[3-(5-Nitrothiazol-2-ylamino)propyl]-7-chloroquinolin-
4-amine (20, C15H14ClN5O2S)
A mixture of 0.14 g N-(3-aminopropyl)-7-chloroquinolin-
4-amine (14, 1.0 mmol) and 0.21 g 2-bromo-5-nitrothia-
zole (12, 1.0 mmol) was dissolved in dry DMF in presence
of 0.42 g anhydrous sodium carbonate (4.0 mmol). The
reaction mixture was kept under nitrogen and stirred at
70 °C overnight. The reaction mixture was then cooled to
room temperature, treated with 50 cm3 water, and was then
extracted with ethyl acetate/THF (4 9 50 cm3). The sol-
vent was removed under reduced pressure and the residue
was purified by preparative silica gel plates using ethyl
acetate to give pure 20. Yield: 0.20 g (56.0 %); m.p.:
1
148–150 °C; H NMR (500 MHz, CDCl3): d = 1.40 (m,
2H, H-300), 2.00 (m, 4H, H-200 ? H-400), 3.50 (s, 2H,
H-100 ? H-500), 6.60 (d, J = 5.2 Hz, 1H, H-3), 7.50 (d,
J = 9.0 Hz, 1H, H-6), 7.80 (s, 1H, H-8), 8.20 (d,
J = 9.0 Hz, 1H, H-5), 8.30 (s, 1H, H-40), 8.50 (d,
J = 5.2 Hz, 1H, H-2) ppm; 13C NMR (125 MHz, CDCl3):
d = 29.5 (C-300), 39.1 (C-200), 39.6 (C-400), 103.1 (C-50),
117.8 (C-3), 124.7 (C-4a), 124.8 (C-5), 127.2 (C-6), 129.1
(C-8), 134.3 (C-7), 135.6 (C-8a), 149.4 (C40), 150.9 (C-2),
151.6 (C-4), 158.1 (C-20) ppm; HRMS (ESI): m/z calcd for
C15H13ClN5O2S [M-H]- 362.04785, found 362.04822.
2-[4-(7-Chloroquinolin-4-yl)piperazin-1-yl]-N-
(5-nitrothiazol-2-yl)acetamide (23, C18H17ClN6O3S)
2-Chloro-N-(5-nitrothiazol-2-yl)acetamide (17, 0.22 g,
1.0 mmol) was dissolved in 10 cm3 dry THF and stirred
in presence of excess triethylamine. To the stirred solution
0.25 g 7-chloro-4-(piperazin-1-yl)quinoline (13, 1.0 mmol)
was added and the mixture was stirred at room temperature
overnight. Water was then added and the reaction mixture
was extracted several times with a mixture of ethyl acetate/
THF (3:1). The organic layer was separated, the solvent
was removed under reduced pressure, and the residue was
purified by column chromatography, eluting with ethyl
acetate to afford 23. Yield: 0.10 g (23.0 %); m.p.:
165–167 °C; 1H NMR (500 MHz, DMSO-d6): d = 2.90
(s, 4H, H-200), 3.40 (s, 4H, H-300), 3.60 (s, 2H, H-30), 7.05
(d, J = 5.1 Hz, 1H, H-3¢¢¢), 7.58 (dd, J = 2.2, 9.0 Hz, 1H,
H6¢¢¢), 7.99 (d, J = 2.2 Hz, 1H, H-8¢¢¢), 8.04 (d,
J = 9.0 Hz, 1H, H-5¢¢¢), 8.64 (s, 1H, H-4), 8.72 (d,
J = 5.1 Hz, 1H, H-2¢¢¢), 12.00 (s, 1H, H-10) ppm; 13C
N-[4-(5-Nitrothiazol-2-ylamino)butyl]-7-chloroquinolin-
4-amine (21, C16H16ClN5O2S)
A mixture of N-(4-aminobutyl)-7-chloroquinolin-4-amine
(15, 1.0 mmol) and 2-bromo-5-nitrothiazole (12,
1.0 mmol) was dissolved in dry DMF in the presence of
anhydrous sodium carbonate (4.0 mmol). The reaction
mixture was kept under nitrogen and stirred at 70 °C
overnight. The reaction mixture was then cooled to room
temperature, treated with 50 cm3 water, and extracted
several times with a mixture of ethyl acetate/THF (3:1).
The solvent was removed under reduced pressure and the
residue was purified by preparative silica gel plates using
123

Y. R. H. Saleh et al.
NMR (125 MHz, DMSO-d6): d = 52.0 (C-300), 53.0 (C-
200), 60.0 (C-30), 109.8 (C-5), 121.6 (C-3¢¢¢), 126.3 (C-4a¢¢¢),
126.7 (C-5¢¢¢), 128.1 (C-6¢¢¢), 134.3 (C-8¢¢¢), 141.9 (C-7¢¢¢),
143.3 (C-4), 149.6 (C-8a¢¢¢), 152.1 (C-2¢¢¢), 156.8 (C-4¢¢¢),
162.9 (C-2), 172.5 (C=O) ppm; HRMS (ESI): m/z calcd for
C18H18ClN6O3S [M?H]? 433.08496, found 433.08441.
presented in Tables 1 and 2, and was calculated with a
sigmoid dose–response curve using the following equation:
Percentage of inhibition ¼ ½ðO:D: of blank À O:D: of testÞ=
O:D: of blankŠ  100:
Statistical analysis
Antigiardial and antitrichomonal activity
Data presented are expressed as the mean SD of dupli-
cate samples from three independent assays. The IC50
values were calculated using dose–response curves in
Graph Pad Prism 6.0 demo software.
Test organism
Details of the parasites, T. vaginalis and G. lamblia used in
this investigation and the protocol followed for checking
anti-parasitic activity can be found elsewhere [38]. In brief,
the parasites were grown in standard TYI-S33 medium (pH
6.8) supplemented with 10 % horse serum, vitamin mix-
ture, and penicillin/streptomycin mixture at 37 °C in
10 cm3 screw-stoppered glass tubes [39]. The cultures
routinely attained a concentration of 2 9 107 cells/cm3 in
48 h and an inoculum of 1 9 104 cells per tube was used
for maintenance of the culture.
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