Synthesis, characterization and biological activity of Schiff bases derived from metronidazole

Article abstract of DOI:10.1007/s00044-011-9830-y

A series of novel Schiff bases; compounds 3a- j were prepared by reacting 1-(2-aminoethyl)-2-methyl-5-nitroimidazole dihydrochloride monohydrate (1) with different aldehydes. The structures of these compounds were confirmed through different spectroscopic methods such as ¹H-NMR, ¹³C-NMR and mass spectrometry and also by elemental analyses. The prepared compounds were evaluated in vitro for their antigiardial, antibacterial and antifungal activities. Compounds 3h, 3b and 3d showed remarkable antigiardial activities and were found to be more active than metronidazole with IC₅₀ of 0.83, 1.36 and 1.83 μM, respectively. Other compounds also exhibited antigiardial activity and were as good as or even more potent than metronidazole. Some of the newly synthesized Schiff bases exhibited more antifungal activities than the parent drug. In addition, a few of the prepared compounds exhibited modest antibacterial activity but were not as active as metronidazole. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9830-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:2969–2974
DOI 10.1007/s00044-011-9830-y
ORIGINAL RESEARCH
Synthesis, characterization and biological activity of Schiff bases
derived from metronidazole
•
Haythem A. Saadeh Eman A. Abu Shaireh
•
•
Ibrahim M. Mosleh Amal G. Al-Bakri
•
Mohammad S. Mubarak
Received: 31 May 2011 / Accepted: 20 October 2011 / Published online: 3 November 2011
Ó Springer Science+Business Media, LLC 2011
Abstract A series of novel Schiff bases; compounds 3a–
j were prepared by reacting 1-(2-aminoethyl)-2-methyl-5-
nitroimidazole dihydrochloride monohydrate (1) with dif-
ferent aldehydes. The structures of these compounds were
confirmed through different spectroscopic methods such as
1H-NMR, 13C-NMR and mass spectrometry and also by
elemental analyses. The prepared compounds were evalu-
ated in vitro for their antigiardial, antibacterial and anti-
fungal activities. Compounds 3h, 3b and 3d showed
remarkable antigiardial activities and were found to be
more active than metronidazole with IC50 of 0.83, 1.36 and
1.83 lM, respectively. Other compounds also exhibited
antigiardial activity and were as good as or even more
potent than metronidazole. Some of the newly synthesized
Schiff bases exhibited more antifungal activities than the
parent drug. In addition, a few of the prepared compounds
exhibited modest antibacterial activity but were not as
active as metronidazole.
Introduction
Schiff bases form an important class of organic compounds
with a variety of uses. They have been widely used as
ligands in the formation of transition metal complexes
(Gulco et al., 2005). In addition, Schiff bases were found to
exhibit biological activities including antibacterial, anti-
fungal and anti-inflammatory (Zhou et al., 2007). More-
over, Schiff bases containing heterocycles have attracted
much attention due to their diverse biological activity such
as anticancer, antiviral, fungicidal, bactericidal and anti-
HIV (Patel and Parmar, 2010). Alternatively, development
of new chemotherapeutic Schiff bases is now attracting the
attention of medicinal chemists (Khan et al., 2009).
Several research groups have been involved in the
synthesis and biological screening of Schiff bases (Gulco
et al., 2005; Zhou et al., 2007; Patel and Parmar, 2010;
Khan et al., 2009; Ronad et al., 2008; Bertinaria et al.,
2003). Zhou et al. (2007) synthesized a range of Schiff
bases derived from 2-aminothiazoles and substituted
benzaldehyde; the in vitro antitumor activity of the pre-
pared compounds with three human tumour cell lines was
evaluated. On the other hand, Ronad et al. (2008) have
synthesized a series of Schiff bases from 7-amino-4-
methylcoumarin and benzaldehydes and studied their anti-
inflammatory and analgesic activity. They discovered that
the anti-inflammatory and analgesic activities of some of
the prepared Schiff bases were either comparable or more
potent than the reference drug. In addition, Bertinaria and
his colleagues (Bertinaria et al., 2003) have prepared a
number of Schiff bases through the condensation of aro-
matic and heteroaromatic aldehydes with coumarin acet-
ohydrazides under conventional and microwave conditions;
the prepared compounds were tested for their antimicrobial
activity and were found to display moderate to potent
Keywords Metronidazole Á Schiff bases Á
Antigiardial activity
H. A. Saadeh Á E. A. A. Shaireh Á M. S. Mubarak (&)
Department of Chemistry, The University of Jordan,
Amman 11942, Jordan
e-mail: mmubarak@ju.edu.jo
I. M. Mosleh
Department of Biological Sciences, The University of Jordan,
Amman 11942, Jordan
A. G. Al-Bakri
Department of Pharmaceutics and Pharmaceutical Technology,
Faculty of Pharmacy, The University of Jordan,
Amman 11942, Jordan
123

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Med Chem Res (2012) 21:2969–2974
activity against different bacterial strains. Recently, the
synthesis and antiglycation activity of bis-Schiff bases of
isatins have been reported by Khan and colleagues (2009);
a remarkable effect on the antiglycation activity due to the
presence of electron withdrawing groups at isatin was
observed. Very recently, Patel and Parmar (2010) have
reported on the synthesis and antimicrobial activities of
novel optically active Schiff bases derived from substituted
benzaldehyde with different amines and discovered that the
presence of nitro, methoxy and halogen group in the phenyl
ring increases the activity of the prepared compounds
against bacteria.
Results and discussion
Chemistry
The Schiff bases 3a–j were prepared by reacting 1-(2-
aminoethyl)-2-methyl-5-nitroimidazole dihydrochloride
monohydrate (1) with different aldehydes in methanol as
shown in Scheme 1. The prepared compounds were
checked for purity by TLC using glass plates precoated
with silica gel 60 GF254, supplied by Fluka as stationary
phase and suitable solvent system as mobile phase and was
also confirmed by melting point determination. The struc-
tures of the prepared compounds were confirmed by NMR,
mass spectrometry and elemental analysis. The 1H and 13C-
NMR spectra of all prepared compounds are in total
agreement with the suggested structures. DEPT experi-
ments were employed to differentiate secondary and qua-
ternary carbons from primary and tertiary ones. Additional
supports of the proposed structures come from mass
spectral data; mass spectra of the prepared compounds
showed the correct molecular ions as suggested by their
molecular formulas.
Similarly, mertonidazole, 2-(2-methyl-5-nitro-1H-imi-
dazol-1-yl)ethanol (1) and its derivatives have a wide range
of biological activity (Bertinaria et al., 2003; Martino
et al., 2005; Hay et al., 2003; Gu¨nay et al., 1999). They are
highly effective against trichomoniasis, various forms of
amoebiasis and infections with anaerobic bacteria and
protozoa (Goldman and Wuest, 1981); it can kill or inhibit
the majority of anaerobic bacteria when the metronidazole
concentration in serum is in the range from 2 to 8 lg/ml
(Salimi et al., 1997). Recently (Abu-Shaireh et al., 2009), a
number of novel metronidazole derivatives have been
synthesized and their anti-parasitic activity has been eval-
uated. In view of the wide interest in the activity and profile
of Schiff bases and metronidazole, and as part of our
ongoing research in the synthesis of new compounds of
pharmacological interest (Abu-Shaireh et al., 2009; Al-
Zghoul et al., 2005; Saadeh et al., 2009, 2010), we describe
herein the synthesis and characterization of a number of
new Schiff bases derived from metronidazole which, to the
best of our knowledge, have not previously been described
in the literature. The antigiardial, antibacterial and anti-
fungal activities of the newly prepared compounds were
evaluated.
Biological activities
Antigiardial activity The antigiardial activity of the
reported compounds was investigated using in vitro bio-
assays. Their bioactivity was compared with the standard
antigiardial drug, metronidazole. The IC50 values of the
compounds against Giardia intestinalis are given in
Table 1. As shown in the table, all the tested compounds
exhibited biological activity against Giardia. Compounds
3a–e, 3i and 3h have IC50 values ranging from 0.83 to
2.8 lM compared to 3.74 lM for metronidazole. Com-
pound 3h showed the highest antigiardial activity with IC50
Scheme 1 Synthesis of
compounds 3a–j, 6
N
N
Et3N / CH3OH
Ar-CHO
+
O2N
O2N
CH3
CH3
N
N
2a-j
N=CH-Ar
NH2 HCl
3a-j
1
a
b
c
d
e
g
i
j
f
h
OH
OH
Cl
Ar
O
S
Cl
N
CH3
F
OCH3
NO2
123

Med Chem Res (2012) 21:2969–2974
2971
variations in drug sensitivities of isolates may be alarming
(Majewska et al., 1991; Aley et al., 1994). Therefore, the
importance of such biologically active metronidazole
derivatives lies in their potential contribution to overcome
the problem of resistance of pathogens to the standard drugs
(Elizondo et al., 1996; Hager and Rapp, 1992). In addition,
because of the limited number of drugs available in the
market against anaerobic protozoan parasites and bacteria
there is a serious need for 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 new compounds, especially 3h, 3b and
3d are good drug candidates to be tested against metroni-
dazole-resistant parasites and bacteria.
Table 1 Antigiardial activities of the tested compounds
Mean IC50 SD (n) (lM)
Compound
G. intestinalis
3a
2.39 0.18
1.36 0.11
2.26 0.17
1.83 0.30
2.15 0.07
4.32 0.49
7.29 0.51
0.83 0.08
2.78 0.12
11.20 0.77
3.74 0.29
3b
3c
3d
3e
3f
3g
3h
3i
3j
Metronidazole
Antimicrobial activity The antibacterial and antifungal
activities of the prepared compounds are presented in
Table 2. Results revealed that the Schiff bases derived
from metronidazole were significantly less active in their
antimicrobial activity towards anaerobic culture of Clos-
tridium sporogenes relative to the parent drug, metroni-
dazole. In addition, as shown in Table 2, the relatively
weak activity displayed by metronidazole against the tested
gram positive and gram negative bacteria is weak, how-
ever, Schiff base formation led to the increased activity of
3f towards both gram positive and gram negative species
tested. Interestingly, Schiff base formation of metronida-
zole increased the antifungal activity, where the parent
drug was devoid of any anti-candida effect under the
experimental conditions. The tested compounds exhibited
MIC values ranging from 0.17 to 2.0 mM.
of 0.83 lM and was around five times more potent than
metronidazole. The molecular modifications on our deriva-
tives did not render any of the compounds inactive, but 3f, 3g
and 3j exhibited less antigiardial activity than metronida-
zole. The giardicidal activity exhibited by the derivatives,
especially compounds 3h, 3b and 3d suggest that the
derivatives may be used as new lead compounds in the
development of new antiparasitic drugs. This cidal activity
of the prepared Schiff bases could extend to cover other
related anaerobic protozaol parasites such as Entamoeba
histolytica and Trichomonas vaginalis; this, however, needs
further investigations. Although, drug resistance of Giardia
and other target pathogens such as E. histolytica does not, so
far, appear to be a serious problem, occasional reports of
failure with metronidazole (Knight, 1980) and the reported
Table 2 Minimum inhibitory concentrations (MIC) (mM) of the prepared compounds and reference substances against tested microorganisms
Compound
MIC (mM)
C. sporogenes
E. coli
S. aureus
C. albicans
Metronidazole
0.0456
0
5.48
0.0023
–
2.05
NA
Ampicillin (mg/ml)
–
–
0.000036
–
Miconazole (mg/ml)
0.0029
3a
3b
3c
3d
3e
3f
1.133 0.493
1.438 0.500
4.30
4.37
0
0
5.09 2.93
1.99 1.30
1.00 0.66
0.34 0.12
0.45 0.29
0.36 0.12
0.44 0.29
0.17 0.06
2.07 1.36
NA
6.89
0
0.203
0
4.34 1.88
NA
4.34 1.88
NA
0.257 0.112
0.285 0.124
1.013 0.438
0.267 0.116
0.369 0.129
2.24 1.02
2.00 1.31
0.76
2.14 0.92
7.10
0
0.76
2.67 0.92
7.10
0
3g
3h
3i
0
0
0.236
0
8.85 1.09
8.46 1.03
8.85 1.09
8.46 1.03
3j
0.376 0.131
NA
NA not achieved at concentration B1.25 mg/ml
123

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Med Chem Res (2012) 21:2969–2974
Conclusions
corresponding aldehyde (2.60 mmol) in methanol (15 ml)
was stirred at room temperature for 24 h. The desired
products were collected by filtration, washed with water and
then recrystallized form methanol. Using the aforemen-
tioned general procedure, the following compounds were
synthesized.
A series of new metronidazole-derived Schiff bases were
prepared by reacting 1-(2-aminoethyl)-2-methyl-5-nitroi-
midazole dihydrochloride monohydrate with different
aldehydes. Structures of the newly synthesized compounds
were confirmed by means of H-NMR, 13C-NMR, mass
1
(4-Fluoro-benzylidene)-[2-(2-methyl-5-nitro-imidazol-1-
yl)-ethyl]-amine (3a) Yield 77%; Mp 134–135°C. 1H-
NMR (CDCl3): d = 2.45 (s, 3H), 3.95 (t, J = 5.5 Hz, 2H),
4.65 (t, J = 5.5 Hz, 2H), 7.05 (m, J = 7.9 Hz, 2H), 7.60
(m, J = 7.9 Hz, 2H), 7.92 (s, 1H), 8.05 (s, 1H). 13C-NMR
(CDCl3): d = 14.9 (CH3), 47.0 (CH2), 60.6 (CH2), 116.0
(d, JC–F = 22 Hz, C), 130.1 (d, JC–F = 3.6 Hz, CH),
133.4 (CH), 138.5 (C), 151.6 (C), 162.2 (CH), 163.7 (d,
1JC–F = 250 Hz, C). HRMS (EIMS) m/z: [M ? Na]?
calcd. for C13H13FN4 NaO2 299.09202; found 299.09257.
Anal. Calcd. for C13H13FN4O2: C, 56.52; H, 4.74; N,
20.28. Found: C, 56.41; H, 4.69; N, 20.13.
spectrometry and elemental analyses. The prepared com-
pounds were tested in vitro for their antibacterial, antigiar-
dial and antifungal activity. Some of them displayed
remarkable antigiardial activity and were more potent than
metronidazole itself. Similarly, the newly synthesized Schiff
bases exhibited more antifungal activities than the parent
drug, metronidazole. In contrast, a modest antibacterial
activity was exhibited by some of the prepared compounds.
2
4
Experimental section
Chemistry
(4-Methyl-benzylidene)-[2-(2-methyl-5-nitro-imidazol-1-
yl)-ethyl]-amine (3b) Yield 86%; Mp 163–164°C. 1H-
NMR (CDCl3): d = 2.35 (s, 3H), 2.45 (s, 3H), 3.95 (t,
J = 5.5 Hz, 2H), 4.65 (t, J = 5.5 Hz, 2H), 7.15 (d,
J = 7.9 Hz, 2H), 7.50 (d, J = 7.9 Hz, 2H), 7.90 (s, 1H),
8.00 (s, 1H). 13C-NMR (CDCl3): d = 14.6 (CH3), 21.6
(CH3), 47.1 (CH2), 60.8 (CH2), 128.2 (CH), 129.5 (CH),
132.8 (C), 133.4 (CH), 141.8 (C), 151.6 (C), 163.5 (CH).
HRMS (EIMS) m/z: [M ? Na]? calcd. for C14H16N4NaO2
295.11709; found 295.12575. Anal. Calcd. for
C14H16N4O2: C, 61.75; H, 5.92; N, 20.58. Found: C, 61.68;
H, 5.87; N, 20.44.
All reagents were used as received from commercial
sources without further purification. 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-Johns melting point apparatus and
were uncorrected. 1H- and 13C-NMR spectra were acquired
with the aid of a Bruker DPX 300 MHz spectrometer
(Germany) with TMS as the internal standard. Chemical
shifts are expressed in d units; J values for 1H–1H, 1H–F
and 13C–F coupling constants are given in Hertz. High
resolution mass spectra (HRMS) were obtained (in posi-
tive/or negative mode) using electrospray ion trap (ESI)
technique by collision-induced dissociation on a Bruker
APEX-4 (7-Tesla) instrument (Bremen, Germany). 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 ll/min.
External calibration was conducted using arginine cluster
in a mass range m/z 175–871. Elemental analyses were
obtained using a Eurovector Euro EA3000, C, H, N and S
elemental analyzer and the obtained results agreed with the
calculated percentages to within 0.4%. Compounds were
checked for their purity by TLC using glass plates, pre-
coated with silica gel 60 GF254, supplied by Fluka.
(4-Methoxy-benzylidene)-[2-(2-methyl-5-nitro-imidazol-1-
1
yl)-ethyl]-amine (3c) Yield 80%; Mp 69–71°C. H-NMR
(CDCl3): d = 2.38 (s, 3H), 3.85 (s, 3H), 3.95 (t, J = 5.5 Hz,
2H), 4.65 (t, J = 5.5 Hz, 2H), 6.90 (d, J = 8.0 Hz, 2H),
7.55 (d, J = 8.0 Hz, 2H), 7.93 (s, 1H), 8.00 (s, 1H). 13C-
NMR (CDCl3): d = 14.8 (CH3), 47.2 (CH2), 60.7 (CH3),
60.7 (CH2), 114.2 (CH), 128.3 (C), 129.8 (CH), 133.3 (CH),
138.4 (C), 151.5 (C), 162.1 (C), 162.9 (CH). HRMS (EIMS)
m/z: [M - H]? calcd. for C14H15N4O3 287.11442, found
287.11496. Anal. Calcd. for C14H16N4O3: C, 58.32; H, 5.59;
N, 19.43 found C, 58.25; H, 5.56; N, 19.33.
[2-(2-Methyl-5-nitro-imidazol-1-yl)-ethyl]-(4-nitro-benzyl-
1
idene)-amine (3d) Yield 81%; Mp 190–191°C. H-NMR
(CDCl3): d = 2.45 (s, 3H), 4.00 (t, J = 5.6 Hz, 2H), 4.65
(t, J = 5.6 Hz, 2H), 7.80 (d, J = 8.0 Hz, 2H), 7.93 (s, 1H),
8.15 (s, 1H), 8.20 (d, J = 8.0 Hz, 2H). 13C-NMR (CDCl3):
d = 14.8 (CH3), 46.8 (CH2), 60.7 (CH2), 124.1 (CH),
128.9 (CH), 133.4 (CH), 138.4 (C), 140.6 (C), 151.3 (C),
161.4 (CH). HRMS (EIMS) m/z: [M]? calcd. for
Synthesis of imines 3a–j
Compounds 3a–j were synthesized and purified according to
the following general procedure: A mixture of 1-(2-amino-
ethyl)-2-methyl-5-nitroimidazole dihydrochloride mono-
hydrate 1 (2.50 mmol), triethylamine (5.50 mmol) and the
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Med Chem Res (2012) 21:2969–2974
2973
C13H13N5O4 303.09676; found 303.09730. Anal. Calcd. for
C13H13N5O4: C, 51.48; H, 4.32; N, 23.09 found C, 51.39;
H, 4.29; N, 23.98.
found 287.05732. Anal. Calcd. for C11H12N4O2S: C, 49.99;
H, 4.58; N, 21.20; S, 12.13 Found: C, 49.91; H, 4.55; N,
21.13; S, 12.04.
2-{[2-(2-Methyl-5-nitro-imidazol-1-yl)-ethylimino]-
Furan-2-ylmethylene-[2-(2-methyl-5-nitro-imidazol-1-yl)-
ethyl]-amine (3i) Yield 67%; Mp 74–76°C. 1H-NMR
(CDCl3): d = 2.50 (s, 3H), 3.90 (t, J = 5.6 Hz, 2H), 4.65 (t,
J = 5.6 Hz, 2H), 6.45 (dd, J = 3.5, 1.8 Hz 1H), 6.70 (d,
J = 3.5 Hz, 1H), 7.50 (d, J = 3.5 Hz, 1H), 7.85 (s, 1H), 7.95
(s, 1H). 13C-NMR (CDCl3): d = 14.9 (CH3), 47.0 (CH2), 61.0
(CH2), 111.9 (CH), 115.4 (CH), 133.4 (CH), 138.5 (C), 145.5
(CH), 150.9 (C), 151.6 (C), 152.0 (CH). HRMS (EIMS) m/z:
[M - H]? calcd. for C11H11N4O3 247.08312; found
247.08366. Anal. Calcd. for C11H12N4O3: C, 53.22; H, 4.87;
N, 22.57. Found: C, 53.17; H, 4.83; N, 22.46.
methyl}-phenol (3e) Yield 91%; Mp 105–106°C. 1H-
NMR (CDCl3): d = 2.45 (s, 3H), 4.00 (t, J = 5.6 Hz, 2H),
4.70 (t, J = 5.6 Hz, 2H), 6.85 (t, J = 7.1 Hz, 1H), 6.95 (d,
J = 7.7 Hz, 1H), 7.15 (dd, J = 7.6, 1.5 Hz, 1H), 7.35 (dt,
J = 7.7, 1.7 Hz, 1H), 7.95 (s, 1H), 8.20 (s, 1H). 13C-NMR
(CDCl3): d = 14.7 (CH3), 46.8 (CH2), 59.4 (CH2), 117.1
(CH), 118.1(C), 119.2 (CH), 131.9 (CH), 133.2 (CH), 133.6
(CH), 138.5 (C), 151.2 (C), 160.7 (C), 167.9 (CH). HRMS
(EIMS) m/z: [M ? H]? calcd. for C13H15N4O3 275.11442;
found 275.11387. Anal. Calcd. for C13H14N4O3: C, 56.93;
H, 5.14; N, 20.43. Found: C, 56.85; H, 5.11; N, 20.34.
[2-(2-Methyl-5-nitro-imidazol-1-yl)-ethyl]-pyridin-4-ylme-
1
4-Chloro-2-{[2-(2-methyl-5-nitro-imidazol-1-yl)-ethylimi-
1
thylene-amine (3j) Yield 90%; Mp 109–110°C. H-NMR
no]-methyl}-phenol (3f) Yield 86%; Mp 160–161°C. H-
(CDCl3): d = 2.45 (s, 3H), 4.00 (t, J = 5.5 Hz, 2H), 4.70 (t,
J = 5.5 Hz, 2H), 7.50 (d, J = 5.8 Hz, 2H), 7.90 (s, 1H), 8.10
(s, 1H), 8.65 (d, J = 5.8 Hz, 2H). 13C-NMR (CDCl3):
d = 14.8 (CH3), 46.7 (CH2), 60.7 (CH2), 121.2 (CH), 133.4
(CH), 138.4 (C), 141.9 (CH), 150.7 (C), 151.3 (C), 161.9
(CH). HRMS (EIMS) m/z: [M - H]? calcd. for C12H12N5O2
258.09910; found 258.09972. Anal. Calcd. for C12H13N5O2:
C, 55.59; H, 5.05; N, 27.01. Found: C, 55.35; H, 5.01; N,
26.74.
NMR (CDCl3): d = 2.45 (s, 3H), 4.00 (t, J = 5.6 Hz, 2H),
4.70 (t, J = 5.6 Hz, 2H), 6.90 (t, J = 8.6 Hz, 1H), 7.20 (s,
1H), 7.30 (d, J = 6.2, 1H), 7.95 (s, 1H), 8.10 (s, 1H), 12.6 (s,
1H). 13C-NMR (CDCl3): d = 14.6 (CH3), 46.7 (CH2), 59.3
(CH2), 118.8 (CH), 118.9 (C), 123.8 (C), 130.9 (CH), 133.1
(CH), 133.6 (CH), 138.5 (C), 151.0 (C), 159.3 (C), 166.8
(CH). HRMS (EIMS) m/z: [M - H]?calcd. for
C13H12ClN4O3 307.05979; found 307.06034. Anal. Calcd.
for C13H13ClN4O3: C, 50.58; H, 4.24; N, 18.15. Found: C,
50.52; H, 4.19; N, 18.06.
Antigiardial activity
(2-Chloro-benzylidene)-[2-(2-methyl-5-nitro-imidazol-1-
yl)-ethyl]-amine (3g) Yield 80%; Mp 142–143°C. 1H-
NMR (CDCl3): d = 2.50 (s, 3H), 3.85 (t, J = 5.5 Hz, 2H),
4.65 (t, J = 5.5 Hz, 2H), 7.38 (m, 3H), 7.58 (d,
J = 7.7 Hz, 1H), 7.15 (dd, J = 7.6, 1.5 Hz, 1H), 7.55 (t,
J = 7.7, 1H), 7.90 (s, 1H), 8.00 (s, 1H). 13C-NMR
(CDCl3): d = 14.8 (CH3), 46.9 (CH2), 60.6 (CH2), 126.5
(CH), 127.8 (CH), 130.1 (CH), 131.3 (CH), 133.4 (CH),
135.0 (C), 137.0 (C), 138.6 (C), 151.5 (C), 162.2 (C).
HRMS (EIMS) m/z: [M ? H]? calcd. for C13H14ClN4O2
293.08053; found 293.07998. Anal. Calcd. for
C13H13ClN4O2: C, 53.34; H, 4.48; Cl, 12.11; N, 19.14.
Found: C, 53.25; H, 4.46; N, 19.07.
Test organism
Giardia intestinalis WB strain (ATCC number 30957) was
grown in a modified YI-S medium with antibiotics. The
parasite was cultivated in 15 ml screw-capped borosilicate
glass tubes containing 13 ml medium. The tubes were
incubated on a 15° horizontal slant at 36–37°C. Culture
maintenance and sub-culturing was performed as described
in a previous publication (Saadeh et al., 2009). Giardia was
harvested from confluent cultures by chilling of the tubes
on ice for 5–10 min to detach cells, followed by centrifu-
gation at 8009g for 5 min.
Antigiardial assay
[2-(2-Methyl-5-nitro-imidazol-1-yl)-ethyl]-thiophen-2-
ylmethylene-amine (3h) Yield 89%; Mp 88–90°C. 1H-
NMR (CDCl3): d = 2.40 (s, 3H), 3.85 (t, J = 5.4 Hz, 2H),
4.55 (t, J = 5.4 Hz, 2H), 6.95 (t, 1H), 7.15 (d, J = 5.0 Hz,
1H), 7.30 (d, J = 5.0 Hz, 1H), 7.80 (s, 1H), 8.05 (s, 1H).
13C-NMR (CDCl3): d = 14.8 (CH3), 47.0 (CH2), 60.3
(CH2), 127.7 (CH), 129.7 (CH), 131.6 (CH), 133.3 (CH),
138.5 (C), 141.4 (C), 151.8 (C), 156.7 (C). HRMS (EIMS)
m/z: [M ? Na]? calcd. for C11H12N4NaO2S 287.05786;
The antigiardial activity of the prepared molecules and
metronidazole as the standard antigiardial drug were tested
as described (Saadeh et al., 2009). In brief, the tested
compounds and metronidazole were dissolved in dimethyl
sulfoxide (DMSO) then in medium and filter-sterilized.
Twofold dilutions starting at 15 lg/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 Giardia. Each
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Broth microdilution method in 96 microtitre plates (Cell-
starÒ, Greinerbio-one, Germany) was employed to assess the
antimicrobial activity of the prepared Schiff bases. In brief,
stock solution (10 mg/ml) of each substrate was prepared in
DMSO under aseptic conditions. The first experimental well
was filled with the sabauroud dextrose broth (190 ll) and the
other wells were filled with 100 ll of the sabauroud dextrose
broth. A volume of 10 ll of each substance stoke solutions
was added to the first well and a twofold serial dilution was
then carried out across the plate. The antimicrobial activity
of the reference substances of ampicillin and miconazole
(both obtained as a gift from Dar Al Dawa Pharmaceutical
Company, Naour-Jordan) and metronidazole was deter-
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6538P, C. sporogenes ATCC 19404 and Candida albicans
ATCC 10231 were used to inoculate the wells to achieve a
final inoculum size of 1 9 106 cfu/ml and the plate was
incubated for 24 h at 37°C. Growth media, conditions and
positive and negative controls were performed according to
published procedures (Saadeh et al., 2010). MIC was
expressed as the mean concentration between the well
showing growth and the well showing no growth. Growth
was detected as turbidity (630 nm), relative to an un-inoc-
ulated well using a microtitre plate reader (Biotek, USA).
Each MIC determination was carried out in triplicate.
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Acknowledgments I.M. Mosleh was supported by a joint grant
from the Deanship of Scientific Research, The University of Jordan,
and Hamdi Manko Center for Scientific Research (HMCSR), The
University of Jordan. We also wish to thank Miss Lina Al-Natsheh for
the technical assistance and the maintenance of cultures.
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