Synthesis of novel hybrid molecules from precursors with known antiparasitic activity

Article abstract of DOI:10.3390/molecules14041483

Three novel new compounds derived from antiparasitic precursors have been synthesized and tested for their antiamoebic and antigiardial activities. The condensation of 2-(2-methyl-5-1H-nitroimidazolyl)ethylamine (6) with 5-nitro-2-furylacrylic acid (7) gave 3-(5-nitrofuran-2-yl)-N-[2-(5- nitroimidazol-1-yl)ethyl]acrylamide (8). Condensation of 7 with 7-chloro-4-(piperazin-1-yl)quinoline (9) afforded 1-[4-(7-chloroquinolin-4-yl) piperazin-1-yl)-3-(5-nitrofuran-2-yl)propenone as a mixture of two isomers; 10-a (the E-isomer) and 10-b (the Z-isomer). In addition, the reaction of 9 with 1-(2-bromoethyl)-2-methyl-5-nitroimidazole (11) in the presence of K ₂CO₃ and NaI yielded 7-chloro-4-(4-[2-(5-nitroimidazol-1- yl)ethyl]-piprazin-1-yl)quinoline (12). On the basis of preliminary screening data for these new compounds, compound 12 exhibited potent lethal activities against Entamoeba histolytica and Giardia intestinalis; its IC₅₀ ( about 1 μM) was lower, at least by a factor of five, compared to the standard drug, metronidazole. In addition, the IC₅₀ of compound 12 against the tested parasites is 600 times below that against Hep-2 and Vero cells. Compounds 8 and 10-a also exhibited potent or moderate antiamoebic and antigiardial activities with IC_{50 values} of about 5.5 μM, and 140 μM, respectively, against the tested parasites. These two hybrid molecules, 8, 10-a, were also non-cytotoxic at the lethal concentrations against the parasites.

Full text of DOI:10.3390/molecules14041483

Molecules 2009, 14, 1483-1494; doi: 10.3390/molecules14041483
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of Novel Hybrid Molecules from Precursors With
Known Antiparasitic Activity
Haythem A. Saadeh 1,*, Ibrahim M. Mosleh 2 and Mohammad S. Mubarak 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
* Authors to whom correspondence should be addressed; E-mail: h.saadeh@ju.edu.jo (H-A.S.),
mmubarak@ju.edu.jo (M-S.M.); Tel.: +962 6 5355000 ext. 22159 (H-A.S.), +962 6 5355000 ext.
22168 (M-S.M.); Fax: +962 6 5348932 (H-A.S.), +962 6 5348932 (M-S.M.).
Received: 4 March 2009; in revised form: 2 April 2009 / Accepted: 7 April 2009 /
Published: 9 April 2009
Abstract: Three novel new compounds derived from antiparasitic precursors have been
synthesized and tested for their antiamoebic and antigiardial activities. The condensation of 2-
(2-methyl-5-1H-nitroimidazolyl)ethylamine (6) with 5-nitro-2-furylacrylic acid (7) gave 3-(5-
nitrofuran-2-yl)-N-[2-(5-nitroimidazol-1-yl)ethyl]acrylamide (8). Condensation of 7 with 7-
chloro-4-(piperazin-1-yl)quinoline (9) afforded 1-[4-(7-chloroquinolin-4-yl)piperazin-1-yl)-3-
(5-nitrofuran-2-yl)propenone as a mixture of two isomers; 10-a (the E-isomer) and 10-b (the
Z-isomer). In addition, the reaction of 9 with 1-(2-bromoethyl)-2-methyl-5-nitroimidazole (11)
in the presence of K2CO3 and NaI yielded 7-chloro-4-(4-[2-(5-nitroimidazol-1-yl)ethyl]-
piprazin-1-yl)quinoline (12). On the basis of preliminary screening data for these new
compounds, compound 12 exhibited potent lethal activities against Entamoeba histolytica and
Giardia intestinalis; its IC50 ( about 1 µM) was lower, at least by a factor of five, compared to
the standard drug, metronidazole. In addition, the IC50 of compound 12 against the tested
parasites is 600 times below that against Hep-2 and Vero cells. Compounds 8 and 10-a also
exhibited potent or moderate antiamoebic and antigiardial activities with IC50 values of about 5.5
µM, and 140 µM, respectively, against the tested parasites. These two hybrid molecules, 8, 10-
a, were also non-cytotoxic at the lethal concentrations against the parasites.

Molecules 2009, 14
1484
Keywords: Hybrid Molecules; Metronidazole; Chloroquine; Entamoeba Histolytica; Giardia
Intestinalis.
Introduction
The concept of “hybrid drugs” has been gaining popularity in medicine. Since a single drug is not
always able to adequately control the illness, the combination of drugs with different pharmaco-
therapeutic profile may be needed [1]. Drugs involving the incorporation of two drug pharmacophores in a
single molecule with the intention of exerting dual drug action have been described [2]. For example, one
of the hybrid parts may be incorporated to counterbalance the known side effects associated with the other
hybrid part, or to amplify its effects through action on another biological target. Ultimately, no matter how
familiar the building blocks may be, hybrid drug molecules may, at their core, become new molecules
with identities independent of their precursors.
Encouraging examples of hybrid drug use on systemic heart disease and malaria were recently
published in the literature. Bisi and coworkers [1] reported the synthesis and pharmacological profile of
some hybrid compounds bearing both the benzazepinone moiety present in Zatebradine (a drug that is
used to reduce the heart rate without concomitant negative inotropic or hypertensive effects) and typical β-
blocker aryloxypropanolamine groups; the new compounds proved to be endowed with negative
chronotropic and inotropic activity and are weak vasorelaxant agents.
Chloroquine (1) had been used as the prime therapy for treating malaria for nearly half a century [3],
but Plasmodium falciparum, the cause of the most deadly variety of malaria, has now become chloroquine
resistant in all malaria-endemic regions of the globe [3]. Some strains have also developed resistance to
mefloquine (2) [4] and even to the naturally occurring and highly efficient antimalarial quinine (3) [3,5].
NEt2
N
HN
N
H
HO
N
HO
H
MeO
Cl
N
CF3
N
CF3
1
2
3
One of the generally accepted options to inhibit or delay the emergence of resistance to new
antimalarial agents is the combination therapy [2,4,6]. The principle of combination drug therapy can be
achieved by either using concomitant administration of two or more single active drugs or by drugs in
which the single active agents are combined in one molecule, i.e., hybrid molecules; the two drug-like
portions (pharmacophores) have independent modes of action that make the emergence of drug resistance
less likely to occur.
Recently, Peyton and his team [3] synthesized a class of hybrid molecules termed “reversed
chloroquines”; these drugs were found to be effective against both chloroquine-sensitive and chloroquine-
resistant strains of Plasmodium falciparum. In their work, they combined chloroquine with the so-called

Molecules 2009, 14
1485
reversal agent 4, which counters resistance by inhibiting a membrane channel that pumps chloroquine out
of the parasite's digestive vacuole, the site at which the drug acts against the parasite. The best of Peyton's
hybrid drugs were about 10 times more effective against drug-sensitive malaria than chloroquine itself.
Adding to this hybrid's promise is its ability to kill a chloroquine-resistant strain of P. falciparum. More
recently, Walsh and coworkers [7] combined fast-acting artemisinin and slow-acting quinine into a hybrid
drug 5 for malaria, for which drug resistance is a barrier to effective treatment. In vitro assays showed that
the hybrid 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. Walsh suggested that the hybrid drug may
increase cellular uptake which improves the treatment's efficacy.
O
O
O
O
CH3
N
HN
N
O
H
O
N
N
N
Cl
HO
MeO
4
5
As part of our ongoing research in the synthesis of new compounds of pharmacological interest [8-11],
we describe herein the synthesis, characterization, and antiamoebic and antigiardial activities of three new
hybrid molecules assembled from precursors with known antiparasitic activity, namely, metronidazole
(Flagyl), chloroquine (CQ), and 5-nitrofuranacrylic acid (Scheme 1). The three hybrid molecules
displayed good antiamoebic and antigiardial activity in vitro; one exhibited higher activity than the
standard drug, metronidazole.
Results and Discussion
Chemistry
Syntheses of the hybrid molecules 8, 10-a, 10-b, and 12 were carried out via the route shown in
Scheme 1. Condensation of 2-(2-methyl-5-1H-nitroimidazolyl)ethylamine (6) with 7 in DMF using
carbonyl diimidazole (CDI) as a coupling agent and in the presence of triethylamine gave the desired
compound 8 as the E-isomer, as confirmed by its 1H-NMR spectrum where the coupling constant, J, of the
1
vinylic protons resonating at  6.62 and 7.28 ppm was 15.7 Hz; the Z-isomer was not detected. The H-
13
NMR and C-NMR spectra of 8 confirmed the formation of an amide bond; the corresponding proton
appeared at  8.58 ppm and the amide carbonyl carbon at 164.7 ppm. In addition, the presence of N-H,
C=O (amide), and conjugated C=C absorption bands in the IR spectrum of 8 at 3362, 1694 and 1605 cm-1,
respectively, confirmed that compound 8 was obtained. Condensation of 7 with 9 in DMF, using CDI as a
coupling reagent, and in the presence of triethylamine afforded 10-a (the E-isomer) and 10-b (the Z-
1
13
isomer) as a mixture of two isomers with identical M+ and elemental analysis. The H-NMR and C-

Molecules 2009, 14
1486
NMR spectra suggested an E-Z isomerization of the double bond during the course of the reaction, with
1
the E-isomer being the major product. The H-NMR spectra of the products confirmed their
stereochemistry. The vinylic protons of the double bond at  6.66 and 7.30 ppm couple most strongly,
with a J- value of about 15.7 Hz, for the E isomer, 10-a and for the Z isomer, 10-b, a J-value of about 7.8
Hz was found for the corresponding vinylic protons at  6.72 and 7.36 ppm. Furthermore, in 10-b, the
piperazine CH2 protons adjacent to the amide bond are not equivalent; that was not the case in 10-a. The
former has two different CH2 protons (3.85 and 4.0 ppm) corresponding to two hydrogens each, and two
different carbons that resonate at 42.2 and 45.7 ppm, whereas the latter has just one CH2 peak at 3.70-
3.80 ppm corresponding to four hydrogens and has just one carbon that resonates at 46.2 ppm. The IR
spectra of compounds 10a and 10b, showed the characteristic C=O, and C=C stretching vibrations at 1676
and 1637 cm-1, respectively, in addition to the absence of a N-H absorption band. Alkylation of 9 with 1-
(2-bromoethyl)-2-methyl-5-nitroimidazole (11) in DMF and in the presence of K2CO3 and NaI yielded the
hybrid molecule 12.
Scheme 1. Preparation of hybrid molecules 8, 10-a, 10-b and 12.
N
N
O2N
CH3
N
i
O2N
CH3
NH2
H
N
+
O2N
COOH
N
O
NO2
O
O
6
7
8
Cl
Cl
N
N
N
NO2
O
O
i
10-a
N
N
NH
+
O2N
COOH
O
+
O2N
Cl
N
7
9
O
N
N
O
10-b
Cl
N
Cl
N
N
O2N
CH3
ii
N
+
O2N
CH3
Br
N
NH
N
N
N
N
9
11
12
Reagents and conditions (i) CDI, TEA, DMF, 25 oC (ii) K2CO3, NaI, DMF, 70 oC, 4 h.

Molecules 2009, 14
1487
1
13
The H-NMR and C-NMR spectra of all prepared compounds and precursors are in total agreement
with the suggested structures. DEPT experiments were employed to differentiate secondary and
quaternary carbons from primary and tertiary carbons. Additional support of the proposed structures
comes from mass spectral data; low resolution or high resolution mass spectra of the prepared compounds
showed the correct molecular ions, (M+·), as suggested by their molecular formulas or fragments that stem
from the molecular ion. Analyses of the molecular ions and the fragmentation pattern are used in the
identification and characterization of these compounds.
Antiamoebic and antigiardial activity
The antiamoebic and antigiardial activities of the three compounds 8, 10-a, and 12 were investigated
using in vitro bioassays that included their precursors and their combinations as controls. The cytotoxicity
of the three novel hybrids on the two cell lines, Hep-2 and Vero cells, was also investigated and compared
with that of the standard antiamoebic and antigiardial drug, metronidazole. The IC50
of the
values
compounds against Entamoeba histolytica, Giardia intestinalis, and the two cell lines are given in Table 1.
Table 1. In vitro antiamoebic and antigiardial activities of compounds 8, 10-a, 12, their
precursors, and the standard drug, metronidazole, and the cytotoxic activity of 8, 10-a, 12,
and metronidazole.
Mean IC50 ± SD (n)
(µM)
Entamoeba
histolytica
Giardia
Compound
Hep-2 cells
Vero cells
intestinalis
5.56 ± 0.37
144.82 ± 5.37
0.97 ± 0.32
5.70 ± 0.78
18.50 ± 1.43
88.42 ± 7.10
67.51 ± 8.67
8
10-a
12
5.32 ± 0.21
139.96 ± 6.37
0.96 ± 0.23
5.20 ± 1.03
18.20 ± 2.28
94.26 ± 9.76
55.70± 5.00
108.46 ± 8.02
574.03 ± 7.77
604.52 ± 12.69
90.55 ± 7.83
593.04 ± 8.71
616.25 ± 16.94
Metronidazole
1460.86 ± 18.31 1495.13 ± 17.79
6
7
9
Chloropip/furan
Chloropip/metro
Furan/metroamine
22.53/22.53 ±1.07 25.75/25.75 ± 2.23
1.04/1.04 ± 0.21
3.19/3.19 ± 0.21
1.17/1.17 ± 0.26
3.77/3.77 ± 0.33
6 = 2-(2-Methyl-5-1H-nitroimidazolyl)ethylamine; 7 = 5-Nitro-2-furylacrilic acid; 9 = 7-Chloro-4-
(piperazine-1-yl) quinoline; Chloropip/metro = 1:1 mixture of 9 and metronidazole; Furan/
metroamine = 1:1 mixture of 6 and 7; Chloropip/furan = 1:1 mixture of 7 and 9; (n) = six
determinations
As indicated in the table, the three hybrid molecules 8, 10-a, and 12 showed biological activities
against Entamoeba and Giardia. Compound 12, with an IC50 of around one micromolar, was the most
active against Entamoeba and Giardia compared with the other compounds. In terms of molar
concentration, this hybrid is at least five times more active than metronidazole. In addition, the

Molecules 2009, 14
1488
cytotoxicity of this hybrid molecule against Hep-2 and Vero cells, represented by the IC50, was slightly
above 600 µM. In other words, the IC50 of this compound against the tested parasites is about 600 times
less than that against Hep-2 and Vero cells. If the IC50 against the parasites and the cell lines are
considered, compound 12 is then about two times better than metronidazole with IC50 ratio of around 270
times higher.
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 results in Table 1 reveal that compound 12 is more active than its
precursors, 7-chloro-4-(piperazin-1-yl)quinolines (9) and metronidazole when tested separately.
Additionally, the activity of one molar fraction of 12 is almost the same as the activity of a mixture
fraction of about one molar from each of its precursors. The fact that the mixture of the two precursors has
a synergistic effect over the standard drug alone actually confirms the boosted activity of the hybrid 12
and the necessity of having the hybrid as a drug candidate. The hybrid molecule 8 is more potent than its
precursors (6 and 7) and is almost equivalent to metronidazole, but the hybridization turned it relatively
more cytotoxic (Table 1). On the other hand, although compound 10-a remained moderately active, but it
is still less potent than any of its precursors (7 and 9) or their mixture (Table 1).
The activities exhibited by the three hybrids, especially compound 12, suggest that the hybrids may be
used as new lead compounds in the development of new antiparasitic drugs. Moreover, the importance of
such biologically active, non-cytotoxic hybrid molecules, especially 12, lies in their potential contribution
to overcome the problem of resistance of pathogens to the standard drugs. This problem is arising now and
may become serious in the future [12-14]. The widespread prophylactic use of the standard drugs may
support the spread of resistant strains potentially developing through different proposed mechanisms [15-
18]. Therefore, and because of the limited number of drugs available in the market against anaerobic
protozoal parasites and bacteria, there is a serious need for new active compounds. The molecular
modification on the original drugs, offers alternatives that may bypass the already developed mechanisms
adopted by the anaerobic pathogens against the standard drugs. Our three hybrid compounds are good
drug candidates to be tested against metronidazole-resistant parasites and bacteria.
Experimental
General
Melting points were measured with a Fischer-Johns melting point apparatus and are uncorrected.
Infrared (IR) spectra were recorded as KBr discs on a Nicolet-400 Fourier transform infrared
spectrophotometer from 400 to 4000 cm-1. All new compounds were analyzed for C, H, and N, and the
observed results agreed with the calculated percentages to within ±0.4%. 1H- and 13C-NMR were obtained
with the aid of a Bruker-DPX 300 MHz spectrometer with CDCI3 or DMSO-d6 as solvents, chemical
shifts are reported in δ ppm relative to TMS as internal standard. Solid-probe high-resolution mass
spectral data were acquired with the aid of a Thermo Electron Corporation MAT 95XP-Trap instrument,
operated in the positive chemical-ionization (CI) mode with methane as the reagent gas. Low resolution

Molecules 2009, 14
1489
mass spectra were measured using a Finnegan MAT TSQ-70 spectrometers at 70 eV (Finnegan MAT,
USA); ion source temperature = 200 ºC. Solid-probe high-resolution mass spectral data were acquired
with the aid of a Thermo Electron Corporation MAT 95XP-Trap instrument, operated in the positive
chemical-ionization (CI) mode with methane as the reagent gas. Elemental analyses were obtained with a
Eurovector Euro EA3000, CHNS-O elemental analyzer. Thin-layer chromatography was carried out using
glass plates, precoated with silica gel 60 GF254, supplied by Fluka and with silica gel plates ((Macherey-
Nagel). The following compounds were obtained from Acros Organics and were used without further
purification: carbon tetrabromide, N,N´-carbonyldiimidazole, 1-(2-aminoethyl)-2-methyl-5-nitroimidazole
dihydrochloride monohydrate (6), 4,7-dichloro-quinoline, metronidazole, and 2-furylacrylic acid. 5-Nitro-
2-furylacrilic acid (7) was synthesized and purified according to published procedures [19].
1-(2-Bromoethyl)-2-methyl-5-nitro-1-imidazole (11)
The title compound was synthesized according to the following general procedure: to a mixture of
metronidazole (3.42 g, 20 mmol) and carbon tetrabromide (8.0 g, 30 mmol) in dry THF (20 mL) was
added triphenylphosphine (5.8 g, 21 mmol), portionwise over 15 min at 0 ºC and the mixture was stirred
for 1.5 h at room temperature. Water was then added and the reaction mixture was extracted with CH2Cl2
(2 x 20 mL). The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced
pressure, and the residue was purified using column chromatography with CH2Cl2 -hexane (3:1 v/v), to
1
give pale-yellow crystals (5.43 g, 91%, mp. 81-82 ºC; Lit. mp = 80-81 ºC [20]). H-NMR (CDCl3):  =
13
2.50 (s, 3H, CH3), 3.64 (t, 2H, J = 6.1 Hz), 4.61 (t, 2H, J = 6.1 Hz), 7.90 (s, 1H). C-NMR:  = 14.03
(CH3), 39.2, 44.6, 132.8, 138.8, 152.1.
7-Chloro-4-(piperazine-1-yl) quinoline (9)
This compound was synthesized according to published procedures [21] that involved stirring a
mixture of piperazine (10.88 g, 126.45 mmol), potassium carbonate (1.05 g, 7.58 mmol), triethylamine
(5.28 mL, 37.88 mmol) and 4,7-dichloroquinoline (5.00 g, 25.25 mmol) in N-methyl-2-pyrrolidinone
(17.7 mL) under nitrogen at 135 °C for 2 h. After cooling to room temperature, the mixture was diluted
with CH2Cl2 (200 mL). The reaction mixture was then washed with brine (2 × 50 mL), dried (MgSO4) and
concentrated under reduced pressure. The resulting oil was purified by column chromatography on silica
gel using CH2Cl2/MeOH (4:1 v/v) as the eluent to afford the desired product (5.40 g, 86%, mp 112–114;
lit. mp. 113-115 °C [21]). 1H NMR (CDCl3) δ = 8.62 (d, J = 5.1 Hz, 1H), 8.04 (d, J = 2.1 Hz, 1H), 7.96 (d,
J = 9.0 Hz, 1H), 7.35 (dd, J = 2.1 and 9.0 Hz, 1H), 6.76 (d, J = 5.0 Hz, 1H), 3.16-3.20 (m, 4H), 1.65 (s,
1H); 13C-NMR (CDCl3) δ = 151.9, 150.2, 134.9, 128.9, 126.1, 125.2, 121.9, 108.9, 53.6 (2C), 46.1 (2C).

Molecules 2009, 14
1490
3-(5-nitrofuran-2-yl)-N-[2-(5-nitroimidazol-1-yl)ethyl]acrylamide (8) and E-Z isomers of 1-[4-(7-
chloroquinolin-4-yl)piperazin-1-yl)-3-(5-nitrofuran-2-yl)propenone (10-a and 10-b)
To a stirred solution of 5-nitro-2-furylacrylic acid (7, 0.35 g, 1.90 mmol) in DMF (3 mL) under N2, was
added a solution of N,N´-carbonyldiimidazole, CDI, (0.3 g, 1.90 mmol) in DMF (4 mL). After stirring for
15 minutes at room temperature, a solution of the appropriate amine (6 or 9) (1.90 mmol) and
triethylamine (0.5 mL) in DMF (4 mL) was added and the mixture was stirred for 3 hours under N2. After
completion of reaction, the mixture was treated with water (20 mL) and extracted with ethyl acetate (2 x
20 mL). The organic layer was washed with water (3 x 30 mL), dried over anhydrous sodium sulfate and
the solvent was removed under reduced pressure. The residue was purified by means of thin layer
chromatography with chloroform-methanol (95:5 v/v) to afford compounds 8, 10-a and 10-b. The E and Z
isomers, 10-a and 10-b, respectively, were separated by preparative thin layer chromatography on silica
gel using chloroform-methanol (95:5 v/v) as eluent; the Z isomer has a higher Rf value than its E
counterpart.
Compound (8): Yield 0.45 g (63%). IR (cm-1): 3362 (NH), 1694 (C=O), 1605 (C=C); 1H-NMR (DMSO):
 = 2.35 (s, 3H), 3.60-3.70 (t, J = 5.6 Hz, 2H), 4.30-4.40 (t, J = 5.5 Hz, 2H), 6.62, 7.28 (dd, J1 = 15.7 Hz,
J2 = 2.8 Hz, 2H, C=C trans), 7.05, 7.68 (dd, J1 = 3.8 Hz, J2 = 3.8 Hz, 2H, furan), 7.95 (s, 1H), 8.58 (br,
13
NH). C-NMR (DMSO):  = 14.3 (CH3), 38.7 (CH2), 45.8 (CH2), 115.2 (CH), 116.9 (CH), 125.3 (CH),
125.6 (CH), 133.7 (CH), 139.0 (C), 151.9 (C), 152.1 (C), 153.5 (C), 164.7 (C). EIMS (probe) 70 eV, m/z
(rel. int.): 336 [M+1]+ (9), 289 [M-46]+ (67), 166 [M-169]+ (100); Anal. Calcd. for (C13H13N5O6): C,
46.57; H, 3.91; N, 20.89. Found: C, 46.63; H, 3.93; N, 20.68.
1
Compound (10-a): Yield 0.45 g (56%). IR (cm-1): 1676 (C=O), 1637 (C=C); H-NMR (DMSO): 3.20-
3.30 (m, 4H), 3.70-3.80 (m, 4H), 6.66, 7.30 (dd, J1 = 15.7 Hz, J2 = 2.8 Hz, 2H, C=C trans), 6. 78 (d, J =
3.8 Hz, 1H), 6.86, 7.68 (dd, J1 = 3.8 Hz, J2 = 3.8 Hz, 2H, furan), 7.44 (dd, J1 = 2.1 Hz, J2 = 9.0 Hz, 1H),
7.96 (d, J = 9.0 Hz, 1H), 8.04 (d, J = 2.1 Hz, 1H), 8.70 (d, J = 4.70 Hz, 1H). 13C-NMR (DMSO):  46.2
(CH2), 51.7 (CH2), 110.3 (CH), 119.2 (CH), 121.8 (C), 126.5 (CH), 128.6 (CH), 129.4 (CH), 134.2 (C),
137.7 (CH), 150.1 (C), 150.9 (C), 152.8 (CH), 156.3 (C), 163.7 (C). HRMS (CI) m/z: calcd for
C20H18ClN4O4 [M + H]+ 413.1004, found 413.1011.
1
Compound (10-b): Yield 0.12 g (15%). IR (cm-1): IR (cm-1): 1672 (C=O), 1628 (C=C); H-NMR
(DMSO): 3.10-3.20 (m, 4H), 3.85 (t, J = 3.7 Hz, 2H), 4.0 (t, J = 3.7 Hz, 2H), 6.72, 7.36 (dd, J1 = 7.8 Hz,
J2 = 2.6 Hz, 2H, C=C cis), 6.88 (d, J = 3.60 Hz, 1H), 6.82, 7.72 (dd, J1 = 3.60 Hz, 2H, J2 = 3.8 Hz, 2H,
furane), 7.42 (dd, J1 = 2.1 Hz, J2 = 9.0 Hz, 1H), 7.93 (s, 1H), 8.05 (d, J = 8.9 Hz, 1H), 8.68 (d, J = 4.7 Hz,
13
1H). C-NMR (DMSO):  42.3 (CH2), 45.7 (CH2), 52.1 (CH2), 52.6 (CH2), 110.2 (CH), 115.4 (CH),
116.4 (CH), 121.9 (C), 122.6 (CH), 126.4 (CH), 126.5 (C), 127.8 (CH), 128.6 (CH), 134.2 (C), 150.1 (C),
152.1 (C), 152.7 (CH), 154.2 (C), 156.4 (C), 163.7 (C). EIMS (probe) 70 eV, m/z (rel. int.): 414 [M+2]+
(10), 412 [M]+ (24), 248 [M-166]+ (11), 246 [M-166]+ (34), 219 [M-195]+ (32), 217 [M-195]+ (100);
Elemental anal. calcd. (%) for C20H17ClN4O4: C, 58.19; H, 4.15; N, 13.57. Found (%): C, 58.01; H, 4.27;
N, 13.34.

Molecules 2009, 14
1491
Synthesis of 7-chloro-4-(4-[2-(5-nitroimidazol-1-yl)ethyl]piperazin-1-yl)quinoline (12)
A mixture of 7-chloro-4-(piperazin-1-yl)quinolines (9, 0.3 g, 1.3 mmol), 1-(2-bromoethyl)-2-methyl-5-
nitroimidazole (11, 0.23 g, 1.3 mmol), K2CO3 (0.21 g, 1.4 mmol) and NaI (0.25 g, 1.4 mmol) in DMF (5
mL) was heated at 70 oC for 4 h. The reaction mixture was cooled, treated with water, and extracted with
CHCl3. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced
pressure. The crude product was purified by silica gel plates using ethyl acetate-hexane (3:1 v/v) to give
the desired product as off white crystals. Yield 0.20 g (46%); mp 58-61 ºC. 1H-NMR (DMSO):  2.46 (s,
3H), 2.70-2.80 (m, 6H), 3.10-3.20 (m, 4H) 4.40 (t, J = 5.5 Hz, 2H), 6.75 (d, J = 4.3 Hz, 1H) 7.35 (d, J =
13
8.8 Hz, 1H), 7.80 (d, J = 9.3 Hz, 1H), 7.85 (s, 1H), 7.95 (s, 1H), 8.63 (d, J = 4.3 Hz, 1H). C-NMR
(DMSO):  14.7 (CH3), 43.9 (CH2), 52.1 (CH2), 53.5 (CH2), 57.7 (CH2), 109.1 (CH), 121.8 (C), 125.1
(CH), 126.2 (CH), 128.8 (CH), 132.8 (CH), 134.9 (C), 138.9 (C), 145.0 (C), 150.5 (C), 151.9 (CH), 156.7
(C), . EIMS (probe) 70 eV, m/z (rel. int.): 400.6 [M]+ (2), 354.5 [M-46]+ (8), 260.4 [M-140]+ (100);
HRMS (CI) m/z: calcd for C19H22ClN6O2 [M + H]+ 401.1487, found 401.1479. Elemental anal. calcd. (%)
for C19H21ClN6O2: C, 56.93; H, 5.28; N, 20.96. Found (%): C, 56.74; H, 5.25; N, 20.72.
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 cultivated in 15-mL screw-capped
borosilicate glass tubes containing 13 mL medium. The tubes were incubated on a 15º horizontal slant at
(36-37) ºC. Culture maintenance and subculturing was performed as described [22] Entamoeba and
Giardia were harvested from confluent cultures by chilling of the tubes on ice for 5-10 min. to detach
cells, followed by centrifugation at 800 x g for 5 min.
Antiamoebic and antigiardial activity
The antiamoebic and antigiardial activities of the prepared hybrid molecules 8, 10-a, 12, their
precursors, and 1:1 mixtures of these precursors were tested as described [23], with some modifications.
Due to insolubility in DMSO-aqueous medium, the bioactivity of 10-b was not determined. Two
milligrams of a test compound or a mixture were dissolved in 10 µL of dimethyl sulfoxide (DMSO) and
completed with 1 mL growth medium. The solutions were filter sterilized using 0.22 µm syringe filters
and the appropriate volumes of the solutions were taken to prepare the concentrations of each compound
or reference drug in 15-mL screw-capped borosilicate glass tubes. For each preparation, concentrations of
240, 120, 60, 30, 15, 7.5, 3.5, 1.7, 0.8, 0.4, 0.2, 0.1 µg/mL medium were prepared in a final volume of 15
mL to exclude air from the tube. For the control mixtures of the precursor compounds, 1:1 molar ratios
were prepared. Each tube was inoculated with 20,000 cells of the parasite under testing (Entamoeba or
Giardia). Each compound or mixture was assayed in duplicate in each of three independent experiments.

Molecules 2009, 14
1492
In each assay, the appropriate controls were performed, including the one without any compound or
mixture and another with metronidazole as the positive control. The caps of Entamoeba and Giardia tubes
were tightly screwed and wrapped with Parafilm. The tubes were incubated on a 15o horizontal slant at 36-
37 oC for 72 hours.
The parasites in each tube were counted using the standard hemacytometer at the 10X objective. In
each count, trypan blue was employed to distinguish live from dead parasites [24]. To permit detachment
of Entamoeba and Giardia, the tubes were placed on ice for few minutes and the parasites were then
centrifuged at 1000 x g for 10 min. The supernatant was discarded and 1 mL fresh medium was added to
each tube. The final suspension was prepared by mixing 25 µL of the parasite suspension in each tube
with 100 µL of 0.4 % trypan blue in phosphate buffered saline (PBS). The 50% inhibitory concentration
(IC50) was employed as a parameter for biological activity. The IC50 is the concentration of compounds
which cuts the number of parasites to half that in the negative control (growth medium + DMSO +
parasites).
Cytotoxicity assay
The cytotoxicity of the hybrid 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. A cell
suspension (105 cells/mL RPMI medium-10% inactivated foetal calf serum) was prepared from confluent
cultures and 100 µL portions of the suspension were added to the wells of 96-well plates. The cells were
incubated for 24 h at 37 ºC and 5 % CO2 and the medium in each well was then replaced with fresh 150
µL medium. Solutions of the compounds or the reference drug were prepared and sterilized as described
in “Antiamoebic and antigiardial activity” section above. Then, 150 µL-two fold serial dilutions of each of
the compounds and the reference drug starting at a concentration of 2000 µg/mL in culture medium were
prepared in the plates. After 48 hour incubation at 37 ºC and 5 % CO2, the number of cells in each well
was determined as follows: The medium in each well was gently replaced with 100 µL of 0.25% (w/v)
trypsin-0.53 mM EDTA solution and the plates were incubated for 5 min. at 37 ºC to allow cell
detachment. Two-hundred microliters of trypan blue solution were added to each well and the plates were,
then, placed on ice. The cells were counted in a hemacytometer at the 10X objective. 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 three new hybrid drugs from precursors with known
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 12 was found to be about five times
less than that of the standard drug metronidazole against those parasites and could be considered as a good
drug candidate to be tested against metronidazole-resistant parasites and possibly anaerobic bacteria.

Molecules 2009, 14
1493
Acknowledgement
I.M. Mosleh was supported by a joint grant from the Deanship of Scientific Research and Hamdi
Manko Center for Scientific Research (HMCSR, University of Jordan. We wish to thank Miss Lina Al-
Natsheh for the technical assistance and the maintenance of cultures.
References
1. Bisi, A.; Rampa, A.; Budriesi, R.; Gobbi, S.; Belluti, F.; Ioan, P.; Valoti, E.; Chiarini, A.; Valenti, P.
Cardiovascular hybrid drugs: New benzazepinone derivatives as bradycardic agents endowed with
selective beta1-non-competitive antagonism. Bioorg. Med. Chem. 2003, 11, 1353-1361.
2. Schellenberg, D.; Abdulla, S.; Roper, C. Current issues for anti-malarial drugs to control P.
falciparum malaria. Curr. Mol. Med. 2006, 6 , 253-260.
3. Burgess, S.J.; Selzer, A.; Kelly, J.X.; Smilkstein, M.J.; Riscoe, M.; Peyton, D.H.A Chloroquine-like
Molecule Designed to Reverse Resistance in Plasmodium falciparum. J. Med. Chem. 2006, 49, 5623-
5625.
4. Greenwood, B.M.; Bojang, K.; Whitty, C.J.; Targett, G.A. Malaria. Lancet. 2005, 365, 1487-1498.
5. O’Neill, P.M.; Posner, G.H. A Medicinal Chemistry Perspective on Artemisinin and Related
Endoperoxides. J. Med. Chem., 2004, 47, 2945-2964.
6. Ashley, E.A.; White, N.J. Artemisinin-based combinations. Curr. Opin. Infect. Dis. 2005, 18, 531-
536.
7. Walsh, J.J.; Coughlan, D.; Heneghan, N.; Gaynor, C.; Bell, A. A novel artemisinin- quinine hybrid
with potent antimalarial activity. Bioorg. Med. Chem. Lett. 2007, 17, 3599-3602.
8. Al-Zghoul, K.H.A.; Salih, K.S.M.; Ayoub, M.T.; Mubarak, M.S.A Convenient Procedure for the
Synthesis of Substituted 4-Methylaminocoumarin. Heterocycles 2005, 65, 2937-2947.
9. Salih, K.S.M.; Al-Zghoul, K.H.A.; Mubarak, M.S.; Ayoub, M.T. Synthesis of Coumarinsulfonamides
With Potential Pharmacological Interest. J. Saudi Chem. Soc. 2006, 9, 623-630.
10. Salih, K.S.M.; Ayoub, M.T.; Saadeh, H.A.; Al-Masoudi, N.A.; Mubarak, M.S. Synthesis,
Characterization, And Biological Activities Of New Benzofuran Derivatives. Hetrocycles 2007, 71,
1577-1587.
11. Al-Soud, Y.A.; Al-Sa’doni, H.H.; Amajaour, H.A.S.; Salih, K.S.M.; Mubarak, M.S.; Al-Masoudi,
N.A.; Jaber, I.H.Z. Synthesis, Characterization and anti-HIV and Antitumor Activities of New
Coumarin Derivatives. Naturforsch. B 2008, 63, 83-89.
12. Knight, R. The chemotherapy of amebiasis. J. Antimicrob. Chemother. 1980, 6, 577–593.
13. Majewska, A.C.; Kasprzak, W.; De Jonckheere, J.F.; Kaczmarek, E. Heterogeneity in the sensitivity
of stocks and clones of Giardia to metronidazole and ornidazole. Trans. R. Soc. Trop. Med. Hyg.
1991, 85, 67-69.
14. Farbey, M.D.; Reynoldson, J.A.; Thompson, R.C. In vitro drug susceptibility of 29 isolates of
Giardia duodenalis from humans as assessed by an adhesion assay. Int. J. Parasitol. 1995, 25, 593-
599.

Molecules 2009, 14
1494
15. Johnson, P.J. Metronidazole and drug resistance. Parasitol. Today 1993, 9, 183-186.
16. Orozco, E.; Lopez, C.; Gomez, C.; Perez, D.G.; Marchat, L.; Banuelos, C.; Delgadillo, D.M.
Multidrug resistance in the protozoan parasite Entamoeba histolytica. Parasitol. Int. 2002, 51, 353-
359.
17. Samarawickrema, N.A.; Brown, D.M.; Upcroft, J.A.; Thammapalerd, N.P.; Involvement of
superoxide dismutase and pyruvate:ferredoxin oxidoreductase in mechanisms of metronidazole
resistance in Entamoeba histolytica. J. Antimicrob. Chemother. 1997, 40, 833-840.
18. Wassmann, C.; Hellberg, A.; Tannich, E.; Bruchhaus, I. Metronidazole resistance in the protozoan
parasite Entamoeba histolytica is associated with increased expression of iron-containing superoxide
dismutase and peroxiredoxin and decreased expression of ferredoxin 1 and flavin reductase. J. Biol.
Chem. 1999, 274, 26051-26056.
19. Pozas, R.; Carballo, J.; Castrob, C.; Rubio, J. Synthesis and in vitro antitrypanosomal activity of
novel Nifurtimox analogues. Bioorg. Med. Chem. Lett. 2005, 15, 1417-1421.
20. Benkli, K.; Karaburun, A.; Du-Karaburun, N.; Demi, E. Synthesis and antimicrobial activities of
some new nitroimidazole derivatives. Arch. Pharm. Res. 2003, 26, 773-777.
21. Musonda, C.C.; Little, S.; Yardleyb, V.; Chibalea, K. Application of multicomponent reactions to
antimalarial drug discovery. Part 3: Discovery of aminoxazole 4- aminoquinolines with potent
antiplasmodial activity in vitro. Bioorg. Med. Chem. Lett. 2007, 17, 4733-4736.
22. Clark, C.G.; Diamond, L.S. Methods for cultivation of luminal parasitic protists of clinical
importance. Clin. Microbiol. Rev. 2002, 15, 329-341.
23. Neal, R.A. Antiamoebic activity of drugs given singly and in combination against axenically grown
Entamoeba histolytica. Arch. Invest. Med. 1978, 9, 387-392.
24. Aley,S.B.; Zimmerman, M.; Hetsko, M.; Selsted, M.E.; Gillin, F.D. Killing of Giardia lamblia by
cryptdins and cationic neutrophil peptides. Infect. Immun. 1994, 62, 5397-5403.
Sample Availability: Samples of the compounds 8-12 are available from the authors.
© 2009 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland. This
article is an open-access article distributed under the terms and conditions of the Creative Commons
Attribution license (http://creativecommons.org/licenses/by/3.0/).