Synthesis, characterization, antiamoebic activity and toxicity of novel bisdioxazole derivatives

Article abstract of DOI:10.1016/j.ejmech.2009.06.016

Cyclization of benzene-1,4-dicarbaldehyde dioxime 1 with different aromatic aldehydes in inert atmosphere yielded the corresponding new bisdioxazoles 2-11. The structure of 2-11 was elucidated by spectral data. In vitro antiamoebic activity was performed

Full text of DOI:10.1016/j.ejmech.2009.06.016

European Journal of Medicinal Chemistry 44 (2009) 4747–4751
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, characterization, antiamoebic activity and toxicity
of novel bisdioxazole derivatives
,
Prince Firdoos Iqbal a, Humaira Parveen a, Abdul Roouf Bhat a, Faisal Hayat a, Amir Azam a
*
a Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Cyclization of benzene-1,4-dicarbaldehyde dioxime 1 with different aromatic aldehydes in inert atmo-
sphere yielded the corresponding new bisdioxazoles 2–11. The structure of 2–11 was elucidated by
spectral data. In vitro antiamoebic activity was performed against HM1:IMSS strain of Entamoeba his-
Received 21 February 2009
Received in revised form
11 June 2009
Accepted 12 June 2009
Available online 21 June 2009
tolytica. The results showed that the compounds 3 (IC50 ¼ 1.22
and 10 (IC50 ¼ 1.01 M) exhibited better antiamoebic activity than the standard drug metronidazole
(IC50 ¼ 1.80 M). The compounds 3, 4, 7 and 10 were tested for toxicity by MTT assay on H9c2 cardiac
myoblasts and the results showed that the compounds 3, 4, 7 and 10 offered remarkable viability of
96.2%, 83.5%, 82% and 89%, respectively at a concentration of 12.5 g/ml.
m
M), 4 (IC50 ¼ 1.41
m
M), 7 (IC50 ¼ 1.05
mM)
m
m
Keywords:
Benzene-1,4-dicarboxaldehyde
Dioxime
m
Ó 2009 Elsevier Masson SAS. All rights reserved.
Bisdioxazoles
Entamoeba histolytica
MTT assay
1. Introduction
properties like cognitive-enhancing and anxiolytic-like activity
[10–13]. The antifungal agents of azoles are useful drugs and are
widely used for the treatment of topic or inner mycoses and AIDS-
related mycotic pathologies [14–16]. Imidazoles representing one
class of antifungal azole derivatives have shown a broad spectrum
of antifungal activities both in vitro and in vivo [17,18]. Oxazole an
important member of the azole family also contains a number of
biologically active molecules, which play an important role in the
drug chemistry. A number of compounds were screened for anti-
tuberculosis agents including dihydrophenazines [19], indoles and
ureas [20]. Oxybenzylglycine, possessing an oxazoline group, is
currently in clinical development for the treatment of type II dia-
betes and dyslipidemia [21]. Recently a number of dioxazole
derivatives were synthesized and their potential antiamoebic
activity has been studied in our laboratory [22].
Considering the facts that nearly all the classes of the azole
family are biologically active and as a part of our continuous efforts
towards the development of more potent amoebicidal agents, we
herein report the synthesis, characterization, antiamoebic activity
and toxicity of a new series of bisdioxazoles 2–11 to find an efficacy
better than metronidazole, a member of azole family and the
commercially available drug for amoebiasis.
Amoebiasis is the most aggressive protozoal disease and
considered to be the second or third leading cause of death amongst
the parasitic diseases [1]. Entamoeba histolytica, a protozoan parasite,
is the causative agent of amoebiasis and amoebic dysentery. Though
ubiquitous in distribution, this parasite is more prevalent in tropical
and subtropical regions [2]. It can invade extra intestinal tissues such
as liver and brain and result in the formation of abscesses which
could be life threatening [3]. Metronidazole is known to be highly
effective amoebicide and is considered to be the drug of choice for
the treatment of amoebiasis, but this drug has been shown to be
mutagenic in a microbiological system and carcinogenic to rodents
[4–6]. In addition, this drug has several adverse effects for which the
most common are gastrointestinal disturbances, especially nausea,
vomiting and diarrhoea or constipation may also occur [7]. Due to its
adverse effects and the emergence of drug resistance [8,9], it is
desirable to search for discovering and developing newer anti-
amoebic agents so that the limitation as presented by available
therapeutic agents can be minimized and more safer, effective
antiamoebic drugs or vaccines can come into existence.
Dioxazoles belong to the azole family which has long been
targets of synthetic investigation because of their known biological
2. Chemistry
The synthesis of the bisdioxazole derivatives (2–11) was per-
formed in a manner as outlined in Scheme 1 [22]. The reaction of
* Corresponding author. Tel.: þ91 11 2698 1717; fax: þ91 11 2698 0229.
E-mail address: amir_sumbul@yahoo.co.in (A. Azam).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.06.016

4748
P.F. Iqbal et al. / European Journal of Medicinal Chemistry 44 (2009) 4747–4751
Table 1
benzene-1,4-dicarbaldehyde dioxime in the presence of sodium
hypochlorite and triethylamine in ethyl acetate gave the bisdiox-
azole derivatives. The solid compounds showed sharp melting
points and the elemental analysis was found in accordance with
ꢀ0.3%. The compounds were stable in solid state and were soluble
in DMSO, methanol and ethanol.
Dioxime (1) and bisdioxazoles (2–11), their in vitro antiamoebic activity against
HM1:IMSS strain of E. histolytica and toxicity profile of compounds 3, 4, 7, 10 and
metronidazole.
Compound
Antiamoebic
activity
Toxicity profile
Safety Index
IC50
(
mM)
S.D
IC50
(
m
M)
S.D
1
2
3
4
5
6
7
8
5.73
3.96
1.22
1.41
4.21
6.10
1.05
2.78
4.65
1.01
4.38
1.80
0.62
0.13
0.16
0.27
0.29
0.16
0.18
0.25
0.28
0.14
0.13
0.33
N.D
N.D
34
N.D
N.D
0.19
0.21
N.D
N.D
0.11
N.D
N.D
0.13
N.D
0.33
N.C
N.C
27.86
5.67
N.C
N.C
46.66
N.C
N.C
>16.83
N.C
3. Pharmacology
Benzene-1,4-dicarbaldehyde dioxime (1) and dioxazole deriva-
tives (2–11) were screened in vitro against HM1:IMSS strain of
E. histolytica by microdilution method. All the experiments were
carried out in triplicate at each concentration level and repeated
thrice. Toxicity of active compounds has been studied by MTT assay
on H9c2 cardiac myoblasts. The results of biological activity and
toxicity are summarized in Table 1 and Fig. 1.
8
N.D
N.D
49
N.D
N.D
>17
N.D
9
10
11
Metronidazole
>116
>64.44
S.D. ¼ Standard deviation, N.D. ¼ Not done, N. C. ¼ Not calculated.
4. Results and discussion
4.1. Synthesis
formation of dioxime 1. The absence of a band at 3250–3267 cmꢁ1
in all the bisdioxazoles 2–11 confirmed the conversion of dioxime
The bisdioxazoles 2–11 were prepared by treating benzene-1,4-
dicarbaldehyde dioxime 1 with different aldehydes as shown in
Scheme 1. The bisdioxazole derivatives 2–11 were recrystallized
from appropriate solvents and the yield was in the range of 23–60%.
All the compounds were highly soluble in methanol and DMSO. The
bisdioxazole derivatives were characterized by electronic, IR,1H and
13C NMR spectra. The purity of the compounds was confirmed by
elemental analysis and data were found in accordance with ꢀ0.3%.
Characteristic IR bands provide significant indications for the
formation of the dioxime 1 and bisdioxazoles 2–11. The absence of
a band at/or around 2670 cmꢁ1 due to aldehydic proton and the
appearance of characteristic bands at 3250–3267 cmꢁ1, 1623–
into bisdioxazole. The n(C]N) band in bisdioxazoles was in the
range of 1630–1664 cmꢁ1 and a new band in the range of 1125–
1193 cmꢁ1 arised due to (C–O–C) group, which was splitted in some
cases. In addition to n(C]C) of aromatic region, the respective
bands of substituted benzene were also present.
The electronic spectral data of the compounds 2–11 studied in the
UV region, exhibited three absorption bands at 317–356 nm, 299–
318 nm and 250–290 nm assigned to n / p* p* and n / s*
, p /
transitions respectively. The band at 317–356 nmwas assigned to the
transition involving the azomethine group (C]N). The two other
absorption bands at 299–318 nm and at 250–290 nm were due to
p
/
p* transition of dioxazole ring and n / s* transition of azo-
1634 cmꢁ1 due to
n(NO–H) and n(C]N) respectively, confirmed the
methine nitrogen respectively.
The structure of the dioxime 1 was further confirmed by 1H NMR
spectra. A singlet at 8.13 ppm due to (CH]N) proton showed the
occurrence of condensation between the (CH]O) and (H2N–OH)
groups. The signal at 11.13 ppm due to (N–OH) proton further
confirmed the formation of the dioxime. The structure of dioxime 1
was further supported by 13C NMR spectra. The absence of aldehydic
carbon signal at 191 ppm and the presence of a signal at 147.58 ppm
due to (C]N) confirmed the formation of dioxime. The formation of
bisdioxazoles was also supported by the absence of a signal at
Synthesis of bisdioxazoles:
H
H
O
H
HON
a
H2NOH.HCl
+
O
NOH
H
1
b
12
O
N
O
R
R
3
5
O
O
N
4
2-11
Compound
RC
Compound
R
2
7
CH3
Cl
3
8
C2H5
Cl
4
9
CH3
CH3
5
6
10
11
Cl
Cl
OMe
OMe
OMe
Cl
Scheme 1. Synthesis of bisdioxazoles: a ¼ Pyridine, C2H5OH, reflux 24 h, b ¼ aq. NaOCl,
Et3N, EtOAc, different aldehydes. Where R ¼ aryl group of different aldehydes.
Fig. 1. Percentage of viable cells after 48 h pre-treatment of H9c2 myoblasts with
metronidazole (MNZ), compounds 3, 4, 7 and 10, evaluated by MTT assay.

P.F. Iqbal et al. / European Journal of Medicinal Chemistry 44 (2009) 4747–4751
4749
11.13 ppm and 8.13 ppm due to (N–OH) and (N]CH) respectively in
6. Experimental protocol
all the compounds 2–11. The singlet at 5.96–6.24 ppm, which arised
due to (CH) group present at C-5 of the dioxazole ring, confirmed the
condensation of dioxime with different aromatic aldehydes. For
methyl group, a singlet at 2.21–2.43 ppm appeared in compound 7
while for ethyl group, a quartet at 1.87–2.66 ppm for (CH2) protons
and a triplet at 1.11–1.19 ppm for (CH3) protons appeared for
compound 8. For methine proton, a multiplet at 3.01–3.11 ppm
appeared for compound 9. In addition, the signals for aromatic
region appeared in the range of 6.67–7.99 ppm for all the
compounds. The structures of the compounds 2–11 were further
supported by 13C NMR spectra. The (C]N) signal in the range 158.1–
164.6 ppm and the characteristic signal for (–OCO–) found in the
range of 88.4–98.7 ppm clearly favored the formation of dioxazole
rings. The signals due to the aromatic and aliphatic carbons resonate
at their usual positions and the values are given in Experimental
section.
All the chemicals were purchased from Aldrich Chemical
Company (USA). Precoated aluminium sheets (silica gel 60 F254
,
Merck Germany) were used for thin-layer chromatography (TLC)
and spots were visualized under UV light. Elemental analyses were
performed on Heraeus Vario EL III analyzer at Central Drug
Research Institute, Lucknow, India. The results were within ꢀ0.3%
of the theoretical values. Electronic spectra were recorded on
a Shimadzu UV 1601 PC UV–vis spectrophotometer. IR spectra were
recorded on Perkin–Elmer model 1600 FT-IR RX1 spectrophotom-
eter as KBr discs. 1H NMR and 13C NMR spectra were recorded on
Bruker AVANCE 400 spectrometer using DMSO-d6 as solvent with
TMS as internal standard. Splitting patterns are designated as
follows; s, singlet; d, doublet; dd, double doublet; t, triplet; m,
multiplet. Chemical shift values are given in ppm. ESI-MS was
recorded on a MICROMASS QUATTRO II triple quadrupole mass
spectrometer.
4.2. Antiamoebic activities
6.1. Preparation of benzene-1,4-dicarbaldehyde dioxime (1)
Preliminary experiments were carried out to determine the in
vitro antiamoebic activity of all the compounds 1–11 by micro-
dilution method using HM1:IMSS strain of E. histolytica. The results
are summarized in Table 1. The data are present in terms of percent
growth inhibition relative to untreated controls, and plotted as
probit values as a function of drug concentration. The antiamoebic
effect was compared with the most widely used antiamoebic
medication metronidazole which had a 50% inhibitory concentra-
A solution of terephthalaldehyde (0.12 mol) and hydroxylamine
hydrochloride (0.26 mol) in a solution of ethanol and pyridine (2:1)
was refluxed with stirring for 24 h. After cooling, the mixture was
concentrated and then poured on 600 ml of ice cold water. The solid
mass was collected, washed with water, crystallized from methanol
and gave the corresponding dioxime.
Yield 86%; cream coloured crystals; m.p.: 190–192 ꢂC; Anal. calc.
For C8H8N2O2: C 58.53, H 4.87, N 17.07%; found: C 58.51, H 4.89,
N 17.06%; IR nmax (cmꢁ1): 3263 (NO–H), 3033 (Ar–H), 2864 (C–H),
tion (IC50) of 1.80
the compounds 3 (IC50 ¼ 1.22
chloro group at ortho and meta positions respectively, compound 7
(IC50 ¼ 1.05 M) having methyl group at para position and
compound 10 (IC50 ¼ 1.01 M) having methoxy group at para
mM in our experiments. The results showed that
1624 (C]N); 1H NMR (DMSO-d6)
d
(ppm): 11.13 (s, 1H, N–OH), 8.31
m
M) and 4 (IC50 ¼ 1.41 M) having
m
(s, 1H, CH]N), 7.40–7.98 (m, 4H, Ar–H); 13C NMR (DMSO-d6)
m
d
(ppm): 147.58 (C]N),133.65 (2 ꢃ Ar–C),126.48 (4 ꢃ Ar–C); ESI-MS
m/z: [Mþ þ 1] 165.
m
position exhibited better antiamoebic activity than the standard
drug metronidazole. Therefore out of eleven compounds screened
in vitro for antiamoebic activity, four compounds 3, 4, 7 and 10 were
more active than the standard drug metronidazole.
6.2. General procedure for the synthesis of bisdioxazoles (2–11)
A 13% aqueous solution of NaOCl (3.2 equiv) was added to
a solution of aldehydes (2 equiv) and triethylamine (0.2 equiv) in
ethyl acetate under argon atmosphere. The dioxime (1 equiv) in
ethyl acetate was added dropwise (over a period of 1 h) at 0 ꢂC to the
above solution and stirred at room temperature for 24 h and
refluxed for additional 24 h, water was added to the reaction
mixture and the aqueous layer was extracted with ethyl acetate. The
combined organic layers were washed with water, brine, dried over
MgSO4, filtered and concentrated under vacuo. The compounds
were crystallized using ethyl acetate–hexane (85:15) solution [22].
4.3. Toxicity profile
To ensure the toxicity of the compounds 3, 4, 7 and 10 with
better IC50 values than metronidazole, they were tested against
H9c2 cardiac myoblasts. A subconfluent population of H9c2 cells
was treated with increasing concentrations of compounds 3, 4, 7
and 10 and the number of viable cells was measured after 48 h by
MTTcell viability assay. The concentration range of compounds 3, 4,
7 and 10 was 3.125–200
compounds 3, 4, 7 and 10 exhibited >80% viability at the concen-
tration of 12.5 g/ml. The toxicity IC50 values along with the safety
mg/ml. Fig. 1 depicted that metronidazole,
6.2.1. 5-Phenyl-3-(4-(5-phenyl-1,4,2-dioxazol-3-yl)phenyl)-1,4,2-
dioxazole (2)
Yield 60%; Oil; Anal. calc. For C22H16N2O4: C 71.73, H 4.34,
N 6.52%; found: C 71.70, H 4.33, N 6.54%; UV/vis lmax (nm): 319, 299,
261; IR nmax (cmꢁ1): 3030 (Ar–H), 2840 (C–H),1632 (C]N); 1H NMR
m
index values of compounds 3, 4, 7 and 10 are given in Table 1.
5. Conclusion
(DMSO-d6)
d
(ppm): 7.86 (s, 4H, Ar–H), 7.13–7.19 (m,10H, Ar–H), 6.12
(ppm): 162.4
(s, 2H, C–H (dioxazole ring)); 13C NMR (DMSO-d6)
d
This research involves the synthesis of benzene-1,4-dicarbalde-
hyde dioxime, which was cyclized to give novel bisdioxazoles 2–11.
The antiamoebic activity was examined using HM1:IMSS strain of E.
histolytica and results showed that the compounds 3 and 4 having
chloro group at ortho and meta positions respectively, compound 7
having methyl group at para position and compound 10 having
methoxy group at para position exhibited better antiamoebic
activity than the standard drug metronidazole. The MTT assay
results showed that thecompounds 3, 4, 7 and10 offered remarkable
(C]N), 142.2, 136.2, 132.3, 131.1, 129.3, 128.1 (Ar–C), 94.7(O–C–O);
ESI-MS m/z: [Mþ þ 1] 369.
6.2.2. 5-(2-Chlorophenyl)-3-(4-(5-(2-chlorophenyl)-1,4,2-dioxazol-
3-yl)phenyl)-1,4,2-dioxazole (3)
Yield 30%; Oil; Anal. calc. For C22H14N2O4Cl2: C 60.41, H 3.20, N
6.40%; found: C 60.39, H 3.17, N 6.43%; UV/vis lmax (nm): 333, 308,
251; IR nmax (cmꢁ1): 3041 (Ar–H), 2853 (C–H),1624 (C]N); 1H NMR
(DMSO-d6)
d
(ppm): 7.85 (s, 4H, Ar–H), 7.01–7.23 (m, 8H, Ar–H), 5.89
(ppm): 158.9
viability (>80% at 12.5
mg/ml).
(s, 2H, C–H (dioxazole ring)); 13C NMR (DMSO-d6)
d

4750
P.F. Iqbal et al. / European Journal of Medicinal Chemistry 44 (2009) 4747–4751
(C]N), 142.6, 135.4, 134.9, 132.2, 131.9, 130.8, 129.2, 127.6 (Ar–C),
96.3 (O–C–O); ESI-MS m/z: [Mþ þ 1] 438.
J ¼ 6.2 Hz, Ar–H), 7.12 (d, 4H, J ¼ 6.2 Hz, Ar–H), 5.96 (s, 2H, C–H
(dioxazole ring)), 3.09 (m, 2H, 2 ꢃ C–H), 1.02 (d, 12H, 4 ꢃ CH3); 13C
NMR (DMSO-d6)
d (ppm): 163.8 (C]N), 148.9, 138.4, 134.6, 132.3,
6.2.3. 5-(3-Chlorophenyl)-3-(4-(5-(3-chlorophenyl)-1,4,2-dioxazol-
3-yl)phenyl)-1,4,2-dioxazole (4)
Yield 27%; Oil; Anal. calc. for C22H14N2O4Cl2: C 60.41, H 3.20, N
6.40%; found: C 60.42, H 3.23, N 6.39%; UV/vis lmax (nm): 340, 301,
255; IR nmax (cmꢁ1): 3054 (Ar–H), 2880 (C–H),1660 (C]N); 1H NMR
129.9,126.7 (Ar–C), 92.6 (O–C–O), 34.4 (C–H), 21.7 (CH3); ESI-MS m/
z: [Mþ þ 1] 457.
6.2.9. 5-(4-Methoxyphenyl)-3-(4-(5-(4-methoxyphenyl)-1,4,2-
dioxazol-3-yl)phenyl)-1,4,2-dioxazole (10)
(DMSO-d6)
d
(ppm): 7.89 (s, 4H, Ar–H), 7.03–7.34 (m, 8H, Ar–H), 5.99
(ppm): 162.4
Yield 33%; Oil; Anal. calc. for C24H20N2O6: C 66.66, H 4.62, N
6.48%; found: C 66.65, H 4.64, N 6.50%; UV/vis lmax (nm): 340, 302,
277; IR nmax (cmꢁ1): 3036 (Ar–H), 2929 (CH3), 2880 (C–H), 1639
(s, 2H, C–H (dioxazole ring)); 13C NMR (DMSO-d6)
d
(C]N), 138.3, 136.2, 132.3, 129.5, 128.2, 127.2 (Ar–C), 96.3 (O–C–O);
ESI-MS m/z: [Mþ þ 1] 438.
(C]N); 1H NMR (DMSO-d6)
d (ppm): 7.97 (s, 4H, Ar–H), 7.64 (d, 4H,
J ¼ 7.1 Hz, Ar–H), 6.88 (d, 4H, J ¼ 7.1 Hz, Ar–H), 5.98 (s, 2H, C–H
(dioxazole ring)), 3.79 (s, 6H, 2 ꢃ OCH3); 13C NMR (DMSO-d6)
6.2.4. 5-(4-Chlorophenyl)-3-(4-(5-(4-chlorophenyl)-1,4,2-dioxazol-
3-yl)phenyl)-1,4,2-dioxazole (5)
d
(ppm): 162.8 (C]N), 158.01, 133.6, 132.1, 131.4, 129.1, 128.2 (Ar–C),
Yield 42%; m.p.: 150–152 ꢂC; Anal. calc. for C22H14N2O4Cl2:
C 60.41, H 3.20, N 6.40%; found: C 60.42, H 3.19, N 6.43%; UV/vis lmax
(nm): 347, 305, 265; IR nmax (cmꢁ1): 3026 (Ar–H), 2867 (C–H), 1664
95.8 (O–C–O), 55.25 (OCH3); ESI-MS m/z: [Mþ þ 1] 433.
6.2.10. 5-(3,4-Dimethoxyphenyl)-3-(4-(5-(3,4-dimethoxyphenyl)-
1,4,2-dioxazol-3-yl) phenyl)-1,4,2-dioxazole (11)
(C]N); 1H NMR (DMSO-d6)
d
(ppm): 7.97 (s, 4H, Ar–H), 7.19 (d, 4H,
J ¼ 7.6 Hz, Ar–H), 7.14 (d, 4H, J ¼ 7.6 Hz, Ar–H), 6.24 (s, 2H, C–H
(dioxazole ring)); 13C NMR (DMSO-d6)
(ppm): 164.8 (C]N), 139.1,
Yield 26%; Oil; Anal. calc. for C26H24N2O8: C 63.41, H 4.91, N
5.69%; found: C 63.43, H 4.85, N 5.70%; UV/vis lmax (nm): 322, 306,
287; IR nmax (cmꢁ1): 3049 (Ar–H), 2960 (CH3), 2857 (C–H), 1663
d
137.5, 134.7, 132.9, 130.7, 128.2 (Ar–C), 96.4 (O–C–O); ESI-MS m/z:
[Mþ þ 1] 438.
(C]N); 1H NMR (DMSO-d6)
d (ppm): 7.95 (s, 4H, Ar–H), 7.47 (d, 2H,
J ¼ 6.4 Hz, Ar–H), 7.25 (d, 2H, J ¼ 6.4 Hz, Ar–H), 6.88 (s, 2H, Ar–H),
6.2.5. 5-(3,4-Dichlorophenyl)-3-(4-(5-(3,4-dichlorophenyl)-1,4,2-
dioxazol-3-yl)phenyl)-1,4,2-dioxazole (6)
6.23 (s, 2H, C–H (dioxazole ring)), 3.75–3.76 (s, 12H, 4 ꢃ OCH3); 13
C
NMR (DMSO-d6)
d (ppm): 158.8 (C]N), 154.6, 149.6, 134.8, 133.0,
Yield 39%; m.p.: 131–133 ꢂC; Anal. calc. for C22H12N2O4Cl4:
C 52.17, H 2.37, N 5.53%; found: C 52.19, H 2.35, N 5.50%; UV/vis lmax
(nm): 330, 317, 291; IR nmax (cmꢁ1): 3032 (Ar–H), 2873 (C–H), 1666
129.4, 112.3, 111.5, 109.4 (Ar–C) 95.9 (O–C–O), 55.83 (OCH3), 55.50
(OCH3); ESI-MS m/z: [Mþ þ 1] 493.
(C]N); 1H NMR (DMSO-d6)
d
(ppm): 7.88 (s, 4H, Ar–H), 7.30 (d, 2H,
J ¼ 7.3 Hz, Ar–H), 7.16 (d, 2H, J ¼ 7.3 Hz, Ar–H), 7.13 (s, 2H, Ar–H),
6.17 (s, 2H, C–H (dioxazole ring)); 13C NMR (DMSO-d6)
(ppm):
6.3. In vitro antiamoebic assay
d
164.95 (C]N), 140.7, 136.9, 135.9, 133.5, 132.1, 131.1, 130.6, 128.4
(Ar–C), 98.2 (O–C–O); ESI-MS m/z: [Mþ þ 1] 507.
All the compounds 1–11 were screened in vitro for antiamoebic
activity against HM1:IMSS strain of E. histolytica by microdilution
method [23]. E. histolytica trophozoites were cultured in wells of
96-well microtiter plate by using Diamond TYIS-33 growth
medium [24]. The test compounds (1 mg) were dissolved in DMSO
6.2.6. 5-p-Tolyl-3-(4-(5-p-tolyl-1,4,2-dioxazol-3-yl)phenyl)-1,4,2-
dioxazole (7)
Yield 35%; m.p.: 115–117 ꢂC; Anal. calc. for C24H20N2O4: C
70.00, H 5.00, N 7.00%; found: C 70.03, H 5.01, N 7.03%; UV/vis
lmax (nm): 329, 309, 254; IR nmax (cmꢁ1): 3018 (Ar–H), 2930
(40 ml), level at which no inhibition of amoeba occurs [25,26]. The
maximum percentage of DMSO used in serial dilution of
compounds is 0.016%. The stock solutions of the compounds were
prepared freshly before use at a concentration of 1 mg/ml. Twofold
serial dilutions were made in the wells of 96-well microtiter plate
(costar). Each test includes metronidazole as a standard amoebi-
cidal drug, control wells (culture medium plus amoebae) and
a blank (culture medium only). All the experiments were carried
out in triplicate at each concentration level and repeated thrice.
The amoeba suspension was prepared from a confluent culture by
pouring off the medium at 37 ꢂC and adding 5 ml of fresh medium,
chilling the culture tube on ice to detach the organisms from the
side of the flask. The number of amoeba/ml was estimated with
a haemocytometer, using trypan blue exclusion to confirm the
viability. The suspension was diluted to 105 organisms/ml by add-
(CH3), 2858 (C–H), 1648 (C]N); 1H NMR (DMSO-d6)
d (ppm):
7.94 (s, 4H, Ar–H), 7.10 (d, 4H, J ¼ 7.4 Hz, Ar–H), 6.99 (d, 4H,
J ¼ 7.4 Hz, Ar–H), 6.19 (s, 2H, C–H (dioxazole ring)), 2.33 (s, 6H,
2 ꢃ CH3); 13C NMR (DMSO-d6)
d (ppm): 159.4 (C]N), 143.2, 142.9,
134.8, 131.4, 129.3, 128.1 (Ar–C), 96.7 (O–C–O), 24.9 (CH3); ESI-MS
m/z: [Mþ þ 1] 401.
6.2.7. 5-(4-Ethylphenyl)-3-(4-(5-(4-ethylphenyl)-1,4,2-dioxazol-3-
yl)phenyl)-1,4,2-dioxazole (8)
Yield 35%; Oil; Anal. calc. for C26H24N2O4: C 72.28, H 5.60,
N 6.54%; found: C 72.30, H 5.58, N 6.55%; UV/vis lmax (nm): 347,
312, 250; IR nmax (cmꢁ1): 3040 (Ar–H), 2913 (CH3), 2893 (CH2),
2860 (C–H), 1655 (C]N); 1H NMR (DMSO-d6)
d
(ppm): 7.92 (s, 4H,
ing fresh medium and 170
test and control wells in the plate so that the wells were
completely filled (total volume, 340 l). An inoculum of
ml of this suspension was added to the
Ar–H), 7.76 (d, 4H, J ¼ 7.6 Hz, Ar–H), 7.34 (d, 4H, J ¼ 7.6 Hz, Ar–H),
6.12 (s, 2H, C–H (dioxazole ring)), 2.60 (q, 4H, 2 ꢃ CH2), 1.17 (t, 6H,
m
2 ꢃ CH3); 13C NMR (DMSO-d6)
d
(ppm): 164.5 (C]N), 148.6, 142.3,
1.7 ꢃ 104 organisms/well was chosen so that confluent, but not
excessive growth, took place in control wells. Plates were sealed
and gassed for 10 min with nitrogen before incubation at 37 ꢂC for
72 h. After incubation, the growth of amoeba in the plate was
checked with a low power microscope. The culture medium was
removed by inverting the plate and shaking gently. Plate was then
immediately washed with sodium chloride solution (0.9%) at 37 ꢂC.
This procedure was completed quickly and the plate was not
allowed to cool in order to prevent the detachment of amoebae.
The plate was allowed to dry at room temperature and the
134.2, 131.6, 129.2, 128.4 (Ar–C), 94.3 (O–C–O), 28.13 (CH2), 14.48
(CH3); ESI-MS m/z: [Mþ þ 1] 429.
6.2.8. 5-(4-Isopropylphenyl)-3-(4-(5-(4-isopropylphenyl)-1,4,2-
dioxazol-3-yl)phenyl)-1,4,2-dioxazole (9)
Yield 23%; Oil; Anal. calc. for C28H28N2O4: C 73.66, H 6.18, N
6.14%; found: C 73.31, H 5.90, N 6.36%; UV/vis lmax (nm): 337, 308,
269; IR nmax (cmꢁ1): 3046 (Ar–H), 2917 (CH3), 2881 (C–H), 1661
(C]N); 1H NMR (DMSO-d6)
d (ppm): 7.94 (s, 4H, Ar–H), 7.63 (d, 4H,

P.F. Iqbal et al. / European Journal of Medicinal Chemistry 44 (2009) 4747–4751
4751
amoebae were fixed with methanol and when dried, stained with
References
(0.5%) aqueous eosin for 15 min. The stained plate was washed
once with tap water, then twice with distilled water and allowed to
dry. A 200 ml portion of 0.1 N sodium hydroxide solution was added
to each well to dissolve the protein and release the dye. The optical
density of the resulting solution in each well was determined at
490 nm with a microplate reader. The % inhibition of amoebal
growth was calculated from the optical densities of the control and
test wells and plotted against the logarithm of the dose of the drug
tested. Linear regression analysis was used to determine the best
fitting line from which the IC50 value was found. The IC50 values in
[1] S. Ghosh, J.M.W. Chan, C.R. Lea, G.A. Meints, J.C. Lewis, Z.S. Tovian,
R.M. Flessner, T.C. Loftus, I. Bruchhaus, H. Kendrick, S.L. Croft, R.G. Kemp,
S. Kobayashi, T. Nozaki, E. Oldfield, J. Med. Chem. 47 (2004) 175–187.
[2] J. Acker, D. Mirelman, Exp. Parasitol. 110 (2005) 170–172.
[3] M. Espinosa-Cantellano, A. Martinez-Palmo, Clin. Microbiol. Rev. 13 (2000) 318–
331.
[4] M.C. Conde-Bonfil, C. De la Mora-Zerpa, Salud Publica Mex 34 (1992) 335–341.
[5] R. Cedillo-Rivera, A. Tapia-Contreras, J. Torres, O. Munoz, Arch. Med. Res. 28
(1997) S295–S297.
[6] K. Kapoor, M. Chandra, D. Nag, J.K. Paliwal, R.C. Gupta, R.C. Saxena, Int. J. Clin.
Pharmacol. Res. 19 (1999) 83–88.
[7] S.C. Swetman, Martindale, in: The Complete Drug Reference, thirty third ed.
Pharmaceutical Press, London, 2002 pp. 594.
[8] E. Orozco, D.G. Perez, M.C. Gomez, P. Ayala, Parasitol. Today 11 (1995) 473–475.
[9] P. Sharma, J.D. Sharma, Phytother. Res. 15 (2001) 1–17.
[10] Presented in part at the 217th American Chemical Society National Meeting,
Anaheim, CA, March 21–25, 1999, ORGN 415.
[11] P. Gru¨nanger, P. Vita-Finzi, in: E.C. Taylor (Ed.), The Chemistry of Heterocyclic
Compounds, vol. 49, John Wiley & Sons, New York, 1991.
[12] (a) S. Ryng, T. Glowiak, J. Chem. Crystallogr. 28 (1998) 373–378;
(b) D.N. Nicolaides, K.C. Fylaktakidou, K.E. Litinas, J. Heterocycl. Chem. 33
(1996) 967–971;
(c) G. Nkusi, R. Neidlein, J. Prakt. Chem. (1992) 278–280;
(d) R.R. Bartlett, Int. J. Immunopharmacol. 8 (1986) 199–204.
[13] R.E. Sammelson, R.B. Miller, M.J. Kurth, J. Org. Chem. 65 (2000) 2225–2228.
[14] P.F. D’Arcy, E.M. Scott, Prog. Drug Res. 22 (1987) 94–147.
[15] D. Kerridge, Drugs Today 24 (1988) 705–715.
mM are reported in Table 1.
6.4. MTT assay
H9c2 rat cardiac myoblasts were cultured and maintained as
monolayer in Dulbecco’s modified Eagle’s medium (DMEM), high
glucose, supplemented with 10% fetal bovine serum (heat inacti-
vated), 100 units/ml penicillin, 100 mg/ml streptomycin, and 2.5 mg/
ml amphotericin B, at 37 ꢂC in humidified incubator with 5% CO2
[27].
[16] Y. Koltin, Annu. Rep. Med. Chem. 25 (1990) 141–148.
[17] A. Polak, Arzneim. Forsch. 32 (1982) 17–24.
[18] A. Polak, Sabouradia 21 (1983) 205–213.
[19] M. Kikuchi, H. Ishikawa, H. Horimoto, H. Tsubouchi, T. Shitsuta, H. Sasaki, M.
Itotani, World Patent WO199732859, 1997.
[20] M. Matsumoto, H. Hasizume, H. Tsubouchi, H. Sasaki, M. Itotani, H. Kuroda,
T. Tomishige, M. Kawasaki, M. Komatsu, Curr. Top. Med. Chem. 7 (2007) 499–
507.
[21] P.V. Devasthale, S. Chen, Y. Jeon, F. Qu, C. Shao, W. Wang, H. Zhang, M. Cap,
D. Farrelly, R. Golla, G. Grover, T. Harrity, Z. Ma, L. Moore, J. Ren, R. Seethala,
L. Cheng, P. Sleph, W. Sun, A. Tieman, J.R. Wetterau, A. Doweyko,
G. Chandrasena, S.Y. Chang, G. Humphreys, I.G. Sasseville, S.A. Biller,
D.E. Ryono, F.S.N. Hariharan, P.T.W. Cheng, J. Med. Chem. 48 (2005) 2248–
2250.
[22] A.R. Bhat, A. Fareeda, A. Azam, Eur. J. Med. Chem. 44 (2009) 926–936.
[23] C.W. Wright, M.J. O’Neill, J.D. Phillipson, D.C. Warhurst, Antimicrob. Agents
Chemother. 32 (1988) 1725–1729.
Cells were incubated with different concentrations of metroni-
dazole, compounds 3, 4, 7 and 10 for 48 h at 37 ꢂC in 5% CO2
humidified incubator together with untreated control sample. At
appropriate time points, cells were washed in PBS, treated with
600 ml MTT solution (5 mg/ml, tetrazolium salt) and incubated for
45 min at 37 ꢂC. After 45 min of incubation at 37 ꢂC, the cell
supernatants were discarded, MTT crystals were dissolved with
acid isopropanol and the absorbance measured at 570 nm. All
assays were performed in triplicate. Percent viability was defined as
the relative absorbance of treated versus untreated control cells.
Plates were analyzed in an ELISA plate reader (Labsystems Mul-
tiskan RC, Helsinki, Finland) at 570 nm with a reference wavelength
of 655 nm.
[24] L.S. Diamond, D.R. Harlow, C.C.R. Cunnick, Trans. R. Soc. Trop. Med. Hyg. 72
(1978) 431–432.
[25] F.D. Gillin, D.S. Reiner, M. Suffness, Antimicrob. Agents Chemother. 22 (1982)
342–345.
[26] A.T. Keene, A. Harris, J.D. Phillipson, D.C. Warhurst, Planta Med. 52 (1986)
278–284.
[27] M.K. Gupta, T.V. Neelakantan, M. Sanghamitra, R.K. Tyagi, A. Dinda, S. Mualik,
C.K. Mukhopadhyay, S.K. Goswami, Antioxid. Redox Signal. 8 (2006) 1081.
Acknowledgements
This work was supported by Department of Science and Tech-
nology (Grant no. Vll-PRDSF/44/2004-05/TT), New Delhi, India.