New dioxazole derivatives: Synthesis and effects on the growth of Entamoeba histolytica and Giardia intestinalis

Article abstract of DOI:10.1016/j.ejmech.2009.12.051

Cyclization of oxime with different aldehydes and ketones under basic condition led to the formation of new dioxazole derivatives and the structure was elucidated by spectral data. The effects of diaoxazoles on the inhibition of growth of Entamoeba histol

Full text of DOI:10.1016/j.ejmech.2009.12.051

European Journal of Medicinal Chemistry 45 (2010) 1648–1653
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
New dioxazole derivatives: Synthesis and effects on the growth of Entamoeba
histolytica and Giardia intestinalis
,
Iram Irfan a, Nongyao Sawangjaroen b, Abdul R. Bhat a, Amir Azam a
*
a Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi-110025, India
b Natural Products Research Center and Department of Microbiology, Faculty of Science, Prince of Songkla University, 90112 Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
Cyclization of oxime with different aldehydes and ketones under basic condition led to the formation of
new dioxazole derivatives and the structure was elucidated by spectral data. The effects of diaoxazoles on
the inhibition of growth of Entamoeba histolytica and Giardia intestinalis in vitro have been determined,
and selected compounds further investigated for their toxicity. SAR showed that the compounds with
5-nitrothiophene group at the 3-postion of the diaoxazole ring were more active than those with the
p-toluene group at the same position. It is interesting to note that the compounds found active against
E. histolytica were not found active against G. intestinalis. Toxicity studies showed that the compound
8 and 9 were non-toxic against Vero cell line ATCC CCL-81.
Received 22 January 2009
Received in revised form
19 December 2009
Accepted 23 December 2009
Available online 13 January 2010
Keywords:
Dioxazoles derivatives
G. intestinalis
Ó 2010 Elsevier Masson SAS. All rights reserved.
E. histolytica
Toxicity studies
1. Introduction
present in wide variety of natural and unnatural biologically active
compounds and is useful reagent in the synthesis of a range of
Intestinal parasitic infections are among the major public health
problems in the developing countries [1]. Especially amoebiasis
(Entamoeba histolytica) and giardiasis (Giardia intestinalis) have
high morbidity and mortality indexes due to the effects of severe
diarrhea and invasive infections. E. histolytica causes approximately
50 million cases of infection resulting approximately 100,000
deaths annually [2–5]. G. intestinalis is the most prevalent proto-
zoan parasite worldwide with about 200 million people being
currently infected [6,7]. Metronidazole is the drug of choice for the
treatment of amoebiasis and giardiasis. Recent studies have shown
that this drug have several toxic effects such as genotoxicity, gastric
mucus irritation, and spermatozoid damage [8,9]. Furthermore,
failures in the treatment of several intestinal protozoan parasites
may result from drug resistant to parasites [10,11]. Considering
these problems, there is a pressing need for new effective drug to
treat these infections.
biologically active scaffold [13–15]. Isoxazole had been screened for
antiviral agents because of their good tolerability and potent anti-
viral activity [16]. As part of our search for basic information about
the requirements for antiprotozoal activity [17], we have synthe-
sized 3,5-Substituted-1,4,2-dioxazole derivatives (1–10). The in
vitro antiparasitic activities of these compounds on two intestinal
protozoa (E. histolytica and G. intestinalis) were screened to develop
an effective drug than metronidazole, a member of azole family.
2. Chemistry
The synthesis of the dioxazole derivatives (1–10) was performed
in a manner as out lined in Scheme 1 [18]. The reaction of aldo-oxime
in the presence of sodium hypochlorite and triethylamine in
dichloromethane gave the dioxazole deriatives. All the compounds
showed sharp melting points and the elemental analysis was found
in accordance with ꢀ0.3%. These compounds were stable in the solid
state. The compounds are soluble in DMSO, methanol and ethanol.
The study of Azoles has become of much interest on account of
their diverse biological properties. Azoles as antifungal agents
prevent synthesis of ergosterol, a major component of fungal
membranes, by inhibiting the cytochrome P-450-dependent
3. Pharmacology
enzymes 14-a lanosterol demethylase [12]. The oxazole nucleus is
All the dioxazole derivatives (1–10) were screened in vitro
against HM1:IMSS strain of E. histolytica by microdilution method
[19] and G. intestinalis on local Thai strain as described previously
[20]. All the experiments were carried out in triplicate at each
* Corresponding author. Tel.: þ91 11 26981717/3250; fax: þ91 11 26980229.
E-mail address: amir_sumbul@yahoo.co.in (A. Azam).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.12.051

I. Irfan et al. / European Journal of Medicinal Chemistry 45 (2010) 1648–1653
1649
a
R
CHO
NH2OH.HCl
R
N
OH
R''
i-ii
b
N O
O
C
R
R'
1-10
H3C
O2N
S
Where R=
,
and (R'-CO-R'') represents different aldehydes
and ketones. (a) Pyridine, C2H5OH, refulx 24 hours, (b) aq.NaOCl2, Et3N, CH2Cl2, (R'-CO-R'')
Scheme 1. General method for the synthesis of dioxazole derivatives.
concentration level and repeated thrice. Toxicity of active
4. Result and discussion
compounds have been studied against the Vero cell line ATCC CCL-
81 (kidney fibroblast of an African green monkey) [21]. The
observed data of biological activities of the compounds and the
control drug are given in Table 1.
A series of 10 new compounds were synthesized. Scheme 1
illustrated the way used for the preparation of target compounds.
The structure of the dioxazole derivatives (1–10) was elucidated by
Table 1
In vitro activity of Dioxazole derivatives against E. histolytica, G. intestinalis and cytotoxicity profile.
N
O
N
O
O
R''
R'
C
H3C
C
R''
O
S
O2N
R'
(1-5)
(6-10)
Comp. No.
R0
H
H
R00
Antiamoebic
Antigiardia
Toxicity profile
IC50 M)
Safety Index
IC50
(
m
M)a
S.D.b
MIC(
m
M)a
(m
1.
2.
9.97
0.19
0.01
5.65
<180
<180
ndc
ndc
OH
2.64
5.33
3.
4.
H
1.60
3.77
0.08
0.03
20.40
10.78
<180
<180
ndc
ndc
OMe
CH3
5.
6.
CH3
6.10
7.82
0.03
0.13
4.55
<180
ndc
ndc
Cl
H
>1500
>1500
ndc
OH
7.
H
8.61
0.07
ndc
ndc
(continued on next page)

1650
I. Irfan et al. / European Journal of Medicinal Chemistry 45 (2010) 1648–1653
Table 1 (continued)
Comp. No.
R0
H
R00
Antiamoebic
Antigiardia
MIC(
M)a
Toxicity profile
IC50 M)
Safety Index
IC50
(
m
M)a
S.D.b
0.02
m
(m
8.
1.80
>1500
>180
>100
OMe
9.
CH3
CH3
H
1.80
7.30
1.80
0.01
0.03
0.04
>1500
>1500
14.61
>180
ndc
>100
ndc
10.
Cl
(MNZ)
ndc
ndc
a
The values obtained in at least three separate assays done in triplicate.
Standard deviation.
nd not determined.
b
c
IR, 1H NMR, 13C NMR, electronic spectra and was further supported
by FAB MAS.
table value of t at 4% level, thus concluding that the character under
study is said to be influenced by the treatment [23].
All the compounds were evaluated for their antiamoebic and anti-
giardial properties. The oxime of 5-nitrothiophenecarboxaldehyde
and p-tolualdehyde (i–ii) were found inactive on both strains. The
oxime of 5-nitrothiophenecarboxaldehyde (i) and p-tolualdehyde
The compounds with less (IC50) value than metronidazole were
evaluated for their toxicity against Vero cell line ATCC CCL-81
(kidney fibroblast of an African green monkey) and the results are
shown in Table 1. It was found that only compounds 8 and 9 are non
(ii) were found inactive (i, IC50 ¼ 10.28
against E. histolytica and G. intestinalis (i, MIC ¼ 48.44
M). Two classes of 3,5 subsituted dioxazoles were
synthesized and their structural activity relationship (SAR) estab-
lished on the basis of substituted molecules at position-3 and 5 of the
dioxazole ring. The dioxazole derivatives with substituted and
unsubstituted phenyl ring attached at carbon-5 of the dioxazole ring
m
M, ii, IC50 ¼ 2.57
m
M)
cytotoxic (IC50 ꢁ 180
mM). The safety index: Toxicity IC50/Protozoal
IC50 was therefore calculated for compounds 8 and 9. It is
remarkable that all compounds that were active against G. intesti-
m
M, ii,
MIC ¼ >1500
m
nalis were toxic to normal cell line (IC50 ꢂ 180
mM). This is because
that the nitro group of 5-nitrothiophene converted into it’s reduced
form to nitroso group and this reduced form is toxic as it was in the
case of metronidazole [22].
showed IC50 forantiamoebic activity in the range of 1.80–9.97
MIC for anti-giardial in the range of 4.55–ꢁ15 M. The compound
3(IC50 ¼ 1.60 M), in this series showed better antiamoebic activity,
vs. IC50 value of MNZ (IC50 ¼ 1.80 M), but the compounds
8(IC50 ¼ 1.80 M) and 9(IC50 ¼ 1.80 M) showed as active as metro-
mM and
m
N
N
m
-
O2N
CH3
ON
CH3
N
N
m
CH3
CH3
m
m
nidazole. The compounds 3 and 8 showed activity due to the pres-
ence of methoxy substituted phenyl ring at the posistion-5 of the
dioxazole ring, but 3 showed better activity because of the presence
on nitro group in dioxazole ring [22]. With respect to the activity
against G. intestinalis four compounds were found more active than
Metronidazole
Nitroso free radical
Based on this experiment, compounds 8 and 9 may consider to
be the greatest potential for future use as an antiamoebic drug,
since they were non cytotoxic and their IC50 was similar to that of
metronidazole. Further study to pursue more experiments trying to
find potential compounds for use as therapeutic agents against
both E. histolytica and G. intestinalis infections may carry out. The
numerical results of each compound are given in Table 1.
metronidazole. Compound 1 (MIC ¼ 5.65
(MIC ¼ 10.78 M) and 5 (MIC ¼ 4.55
(MIC ¼ 14.61 M) with a 5-nitrothiophene group at position-3 of the
m
M), 2 (MIC ¼ 9.45
mM), 4
M) vs. metronidazole
m
m
m
dioxazole ring. The activity of these compounds (1, 2, 4 and 5) may be
due to nitro group at position-5 of the thiophene ring as in the case of
metronidazole, the activity is due to the nitro group present at
position-5 of the imidazole ring, indicating that toluene group at
3-position drastically decrease the activity against this parasite [22].
Therefore, it is concluded that good anti-giardial activity is limited to
those compounds in which 5-nitothiophene group is attached at
3-position of the dioxazole ring. It was further interesting to note that
compound found active against G. intestinalis did not show activity
against E. histolytica. The null hypothesis was tested using t-test and
the significance of the difference between the IC50 value of metro-
nidazole vs. 3 and the MIC value of metronidazole vs.1, 2, 4 and 5 was
evaluted. The values of the calculated t were found higher than the
5. Conclusion
The antiamoebic and anti-giardial activities of new dioxazole
derivatives having different aldehyde and ketone substitutions
were examined. The biological behavior revealed that the
compounds containing nitrothiophene ring are more active than
the compounds containing simple toluene ring. Moreover, the
compounds 1, 2, 4 and 5 were found to be more active than
the reference drug, metronidazole against G. intestinalis in vitro. The

I. Irfan et al. / European Journal of Medicinal Chemistry 45 (2010) 1648–1653
1651
compound 3, 8 and 9 showed significant less/at par IC50 value than
metronidazole against HM1:1MSS strain of E. histolytica. Toxicity
assay showed that compound 8 and 9 are non-toxic against Vero
cell line ATCC CCL-81.
6.1.2.1. 3-(5-Nitro-2-thienyl)-5-phenyl-1,4,2-dioxazole (1): Black
solid(DMSO). Yield 52%, Mp. 276 ꢃC, Anal (C12H8N2O4S) calc. C 52.17,
H 2.80, N 10.10, found C 52.19, H 2.72, N 10.00; UV:lmax (nm) 311,
318, 290. IR: nmax(cmꢄ1): 1656.41 (C]N), 2879 (CH), 1167 (C–O–C);
1H NMR (DMSO-d6), (
d
, ppm) 5.94 (s, 1H, CH), 7.76 (d, 1H,
J ¼ 2.92 Hz, Ar-H), 8.10 (d, 1H, J ¼ 2.92 Hz, Ar-H), 7.72–8.12 (m, 5H,
Ar-H); 13C NMR (DMSO-d6): (
, ppm) 159.73 (C]N), 116.80 (O–C–
6. Experimental protocols
d
O), 152, 127, 128, 138, 141, 127, 129 (Ar-C); FAB MAS: m/z
(Mþþ1)277.13.
Reaction was monitored on Merck pre-coated aluminum plate
60
silica gel
F
thin layer plates. All chemicals were purchased
254
from Sigma–Aldrich Chemical Company (USA). Melting points
were recorded on KSW melting point apparatus. Elemental anal-
ysis (C, H, N) was carried out on Heraeus Vario EL III analyzer by
Central Drug Research Instituted, Lucknow, India and the results
were within ꢀ0.3 of the theoretical values. The electronic spectra
were recorded in DMSO on a Shimadzu UV-1601 PC UV–Visible
spectrophotometer. IR spectra on KBR disks were recorded on
a Perkin Elmer model 1620 FT-IR spectrophotometer. 1H NMR
spectra were obtained at ambient temperature using a Brucker
spectroscopic DPX-300 MHZ spectrometer in DMSO using Tetra-
methylsilane (TMS) as an internal standard. Splitting pattern are
designated as; s, singlet; d, doublet; m, multiplet, coupling
constant J is given in hertz. Chemical shift value is given in (ppm).
The FAB mass spectra of the compounds were recorded on JEOL SX
102/DA-6000 mass spectrometer using Argon/Xenon 6 kV, 10 mA
as the FAB gas and m-nitro benzyl alcohol (NBA) was used as the
matrix.
6.1.2.2. 2-[3-(5-nitro-2-thienyl)-1,4,2dioxazol-5-yl]phenol (2): Brown
solid (methanol:DMSO). Yield 40%, Mp. 165 ꢃC, Anal (C12H8N2O5S)
calc. C 49.31, H 2.76, N 10.14, found C 49.32, H 2.76, N 10.14; UV:lmax
(nm) 253, 299, 367; IR: nmax(cmꢄ1): 1611 (C]N), 2845 (CH), 1191
(C–O–C); 1H NMR (DMSO-d6), (
d
, ppm) 5.91 (s, 1H, CH), 7.83 (d, 1H,
J ¼ 2.70 Hz, Ar-H), 8.10 (d, 1H, J ¼ 2.70 Hz, Ar-H), 8.40–7.71 (m, 4H,
Ar-H). 13C NMR (DMSO-d6): (
, ppm) 161.72 (C]N), 113.60 (O–C–O),
d
151, 121, 128.81, 136, 142.21, 128.60, 121.62, 129.11 (Ar-C), 158
(C–OH); FAB MAS: m/z (Mþ þ 1) 293.64.
6.1.2.3. 5-(4-Methoxy-phenyl)-3-(5-nitro-2-thienyl)-1,4,2-dioxazole
(3): Brown solid (DMSO). Yield 45%; Mp. 93 ꢃC; Anal (C12H8N2O4S)
calc. C 50.90, H 3.26, N 9.15, found C 50.81, H 3.27, N 9.16; UV:Vis
lmax(nm): 321, 297, 250; IR: nmax(cmꢄ1): 1675 (C]N), 2870 (CH),
1153 (C–O–C); 1H NMR (DMSO-d6), (
d, ppm) 5.89 (s, 1H, CH), 8.16 (d,
1H, J ¼ 2.91 Hz, Ar-H), 7.90 (d, 1H, J ¼ 2.90 Hz, Ar-H), 8.12 (d, 2H,
J ¼ 6.60 Hz, Ar-H), 8.51 (d, 2H, J ¼ 6.60 Hz, Ar-H), 2.42 (s, 3H, CH3);
13C NMR(DMSO-d6) (
d, ppm) 163.23 (C]N), 112.90 (O–C–O) 151,
6.1. Chemistry
129, 128, 135, 133, 128, 114, 159.62 (Ar-C), 27.7 (CH3); FAB MAS: m/z
(Mþ þ 1) 307.29.
6.1.1. General procedure for the synthesis of oxime i and ii
A solution of 5-nitro–2-thiophenecarboxyldehyde or p-tol-
ualdehyde (1 equiv.) and hydroxylamine hydrochloride (1.08 equiv.)
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 into 600 mL of ice water. The precipitated solid was collected
and recrystallized from methanol, which gave corresponding
oxime.
6.1.2.4. 5-Methyl-3-(5-nitro-2-thienyl)-5-phenyl-[1,4,2]dioxazole
(4): Black solid (DMSO). Yield 43%; Mp. 120 ꢃC; Anal (C12H8N2O4S)
calc. C 53.79, H 3.47, N 9.65, found C 53.78, H 3.47, N 9.66; UV:Vis
(nm): 337, 296, 261; IR:nmax(cmꢄ1): 1678 (C]N), 2973 (CH3), 1176
(C–O–C); 1H NMR(DMSO-d6), (
, ppm) 7.91 (d, 1H, J 42.51 Hz, Ar-H),
8.12 (d,1H, J ¼ 2.51 Hz, Ar-H), 2.72 (s, 3H, CH3), 7.91–8.20 (m, 5H, Ar-
H); 13C NMR(DMSO-d6), (
, ppm), 159.31 (C]N), 115.74 (O–C–O), 25
l
d
d
(CH3), 154, 124, 127.80,134, 141, 127.21, 129, 127 (Ar-C); FAB MAS: m/
z (Mþþ1) 291.11.
6.1.1.1. 5-Nitrohiophene-2-carbaldehyde oxime (i): Yellow solid
(dichloromethane). Yield 85%, Mp. 130 ꢃC, Anal (C5H4N2O3S) calc. C
34.87, H 2.26, N 16.21, found C 34.88, H 2.34, N 16.27; IR:
nmax(cmꢄ1): 1624.31 (C]N), 2817 (C–H), 1500 (NO2); 1H
NMR(CDCl3) (d, ppm)11.28 (s, 1H, N–OH), 6.81 (s, 1H, CH]N),
7.80(d, 1H, 2.91 Hz, Ar-H), 7.70(d, 1H, 2.91 Hz, Ar-H).
6.1.2.5. 5-(4-Chloro phenyl)-5-methyl-3-(5-nitro-2-thienyl)-1,4,2-
dioxazole (5): Brown solid (methanol:DMSO). Yield 39%; Mp. 150 ꢃC;
Anal (C13H9ClN2O4S) calc. C 48.08, H 2.79, N 8.63, found C 48.09, H
2.77, N 8.64; UV:Vis
(C]N), 3816 (Ar-C–H), 2361 (CH3), 1191 (C–O–C); 1H NMR (DMSO-
d6), (
, ppm) 2.51 (s, 1H, CH3), 7.74 (d, 1H, J ¼ 2.81 Hz, Ar-H), 8.11 (d,
l
(nm): 317, 274, 318; IR:nmax(cmꢄ1): 1693
6.1.1.2. 4-Methylbenzaldehyde oxime (ii): White solid (dichlor-
omethane). Yield 76%, Mp. 80 ꢃC, Anal (C8H9NO) calc. C 71.06, H
d
1H, J ¼ 2.81 Hz, Ar-H),7.58 (d, 2H, J ¼ 6.42 Hz, Ar-H), 7.90 (d, 2H,
6.62, N 10.41, found C 71.09, H 6.71, N 10.36; 1H NMR (CDCl3) (
d
,
J ¼ 6.42 Hz, Ar-H); 13C NMR(DMS-d6), (
d, ppm) 160.2 (C]N), 111.92
(O–C–O), 28.13 (CH3), 151, 126, 128, 135, 139, 128, 129 (Ar-C); FAB
ppm) 11.31 (s, 1H, N–OH), 7.40 (s, 1H, CH]N), 2.40 (s, 3H, CH3), 7.94
(d, 2H, J ¼ 2.60 Hz, Ar-H), 7.98 (d, 2H, J ¼ 2.60 Hz, Ar-H).
MAS: m/z (Mþ þ 1) 325.82.
6.1.2. General procedure for the preparation of 3, 5-substituted-
1,4,2-dioxazole (1–10)
6.1.2.6. 3-(4-methylphenyl)-5-phenyl-1,4,2-dioxazole (6): Light pink
(methanol:DMSO). Yield 33%; Mp. 127 ꢃC; Anal (C15H13NO2) calc. C
The 4% aqueous solution of NaOCl (1.6 equiv.) was added to
a solution of dipolarophile (respective aldehydes and ketones,
1 equiv.) and triethylamine (0.1 equiv.) in dichloromethane under
argon atmosphere. The oxime (1 equiv.) in dichloromethane was
added drop wise (over a period of 1 h) at 0 ꢃC, to an above solution.
After stirred at room temperature, water was added to the reaction
mixture and the aqueous layer was extracted with dichloro-
methane. The combined organic layer were washed with water,
brine, dried over (MgSO4), filtered and concentrated under vacuo.
The compounds were recrystallized using dichloromethane/hexane
solution.
75.31, H 5.43, N 5.85, found C 75.32, H 5.44, N 5.87; UV/Vis
315, 291, 240; IR: nmax(cmꢄ1): 1611 (C]N), 3046 (Ar-C–H), 2861
(CH), 1178 (C–O–C); 1H NMR(DMSO-d6), (
, ppm) 2.21 (s, 3H, CH3),
l (nm):
d
5.90 (s, 1H, CH), 7.53 (d, 2H, J ¼ 6.74 Hz, Ar-H), 7.52 (d, 2H,
J ¼ 6.74 Hz, Ar-H), 7.91–8.21 (m, 5H, Ar-H); 13C NMR(DMSO-d6), (
d,
ppm) 161.71(C]N), 116.14 (O–C–O), 21.92 (CH3), 140, 127, 129, 126,
141, 127.20, 129, 127 (Ar-C); FAB MAS: m/z (Mþ þ 1) 239.27.
6.1.2.7. 2-3(4-methylphenyl)-1,4,2-dioxazole-5-yl-phenol (7): Light
yellow (DMSO). Yield 46; Mp. 93 ꢃC; Anal (C15H13NO3) found C
70.59, H 5.09, N 5.47 calc. C 70.58, H 5.09, N 5.49; UV/Vis
l (nm):

1652
I. Irfan et al. / European Journal of Medicinal Chemistry 45 (2010) 1648–1653
310, 285, 312; IR:nmax(cmꢄ1): 1670 (C]N), 3166 (Ar-C–H), 2862
(CH), 1183 (C–O–C); 1H NMR(DMSO-d6), (
, ppm) 2.41 (s, 3H, CH3),
concentration (MIC) was recorded (the lowest concentration at
which >90% of the trophozoites rounded up). Each concentration
was tested in duplicate and at least three experiments were per-
formed on separate occasions.
d
6.19 (s, 1H, CH), 7.50 (d, 1H, J ¼ 6.90 Hz, Ar-H), 7.71 (d, 1H,
J ¼ 6.90 Hz, Ar-H),7.40–8.31(m, 1H, Ar-H); 13C NMR(DMSO-d6), (
d,
ppm) 159.95 (C]N), 112.19 (O–C–O) 26.11, (CH3), 140, 129.21, 125,
126.90, 122, 128.6, 116.11, 121.65, 129.11 (Ar-C); FAB MAS: m/z
(Mþ þ 1) 256.93.
6.2.2. In vitro testing against E. histolytica
All the dioxazole compounds (1–10) were screened in vitro for
antiamoebic activity against HM1:1MSS strain of E. histolytica by
using a microplate method [19]. E. histolytica trophozoites were
cultured in TYIS-33 growth medium in wells of 96 microtiter plate
6.1.2.8. 5-(4-Methoxy-phenyl)-3-(4-methyl phenyl)-1,4,2-dioxazole
(8): Pink solid (DMSO). Yield 40%, Mp. 110 ꢃC, Anal (C16H16NO3) calc.
C 71.11, H 5.92, N 7.03, found C 71.11, H 5.91, N 7.02; UV/Vis
317, 270; IR: nmax(cmꢄ1): 1667 (C]N), 3029 (Ar-C–H), 2849 (CH),
1158 (O–C–O); 1H NMR(DMSO-d6), (
, ppm) 6.19 (s, 1H, CH), 7.31 (d,
l
(nm):
[25]. DMSO (40 mL) was added to all the samples (1 mg) followed by
enough culture medium to obtain concentration of 1 mg/mL. The
maximum concentration of DMSO in the test did not exceed 0.1%, at
which level no inhibition of amoebal growth occurred [26,27].
Sample were dissolved or suspended by mild sonication in a son-
iclearner bath for few minutes and then further dilution with
medium to concentration of 0.1 mg/mL. Two fold serial dilutions
were made in the wells of 96-well microtiter plate (Costar) in
d
2H, J ¼ 6.91 Hz, Ar-H), 7.39 (d, 2H, J ¼ 6.91 Hz, Ar-H), 2.33 (s, 1H,
CH3), 7.41(d, 2H, J ¼ 6.62 Hz, Ar-H), 7.45 (d, 2H, J ¼ 6.62 Hz, Ar-H),
6.56 (s, 1H, CH3); 13C NMR (DMSO-d6), (
d, ppm) 163.29 (C]N),
115.31 (O–C–O), 24.63 (CH3), 140, 129.21, 129, 126.90, 141, 127.21,
129 (Ar-C); FAB MAS: m/z (Mþþ1)271.73.
170 mL of the medium. Each test included metronidazole as the
6.1.2.9. 5-Methyl-3-(4-methly phenyl)-5-phenyl-1,4,2-dioxazole (9):
Creamy white solid (DMSO). Yield 37%; Mp. 115 ꢃC; Anal
(C16H15NO2) calc. C 75.8, H 5.81, N 5.53, found C75.90, H 5.82, N
5.52; IR:nmax(cmꢄ1): 1669 (C]N), 3163 (Ar-C–H), 1187 (O–C–O),
standard amoebicidal drug, control wells (culture medium plus
amoebae) was prepared from a confluent culture by pouring off the
medium, adding 2 mL of medium and chilling the culture on ice to
detach the organisms from the side of the flask. The number of the
amoeba per mL was estimated with a heamocytometer and trypan
blue exclusion was used to confirm viability. The cell suspension
used was diluted to 105 organism/mL by adding fresh medium and
839.31, 2861 (CH3), 2981 (CH3); 1H NMR (DMSO-d6), (
d
, ppm) 2.34
(s, 1H, CH3), 7.2 (d, 1H, J ¼ 6.81 Hz, Ar-H), 7.75 (d, 1H, J ¼ 6.81 Hz, Ar-
H), 7.92–8.21 (m, 5H, Ar-H); 13C NMR (DMSO-d6), (
, ppm) 162.13
d
(C]N), 118.71 (O–C–O), 27.81 (CH3), 26.90 (CH3), 143,129.28,
140.75, 126.9, 141.12, 127.22, 129, 127.75 (Ar-C); FAB MAS: m/z
(Mþ þ 1)254.11.
170 ml of this suspension was added to the test and control well in
the plate so that the wells were completely filled (total volume,
340
m
L). An inoculum of 1.7 ꢅ 104 organisms/well was chosen so
that confluent, but not excessive growth took place in control wells.
Plate was sealed with expended polystyrene (0.5 thick). Secured
with tape, placed in a modular incubating chamber (flow labora-
tories, High Wycombe, UK), and gassed for 10 min with nitrogen
before incubation at 37 ꢃC for 72 h.
6.1.2.10. 5-(4-Chloro-phenyl)-5-methyl-3-(4-methyl)-1,4,2-dioxazole
(10): Creamy white solid (DMSO). Yield 31%; Mp. 130 ꢃC; Anal
(C16H14NO2Cl) calc. C 66.80, H 4.90, N 4.87, found C 66.80, H 4.88, N
4.86; UV/Vis
l
(nm): 355, 318, 297; IR:nmax(cmꢄ1): 1653 (C]N),
3046 (Ar-C–H), 2982 (CH3), 1189 (O–C–O); 1H NMR(DMSO-d6), (
d
,
ppm) 2.46 (s, 3H, CH3), 7.64 (d, 2H, J ¼ 6.70 Hz, Ar-H), 7.81 (d, 2H,
6.2.2.1. Assessment of antiamoebic activity. After incubation, the
growth of amoebae in the plate was checked with a low power
microscope. The culture medium was removed by inverting the
plate and shaking gently. The plate was then immediately washed
once in sodium chloride solution (0.9%) at 37 ꢃC. 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 the room temperature, and the amoebae was fixed
with methanol, when dry, stained with (0.5%) aqueous eosin for
15 min. Stained plate was washed once with tap water and then
J ¼ 6.70 Hz, Ar-H), 2.27 (s, 3H, CH3), 8.11 (d, 2H, J ¼ 6.44 Hz, Ar-H),
8.36 (d, 2H, J ¼ 2.44 Hz, Ar-H); 13C NMR (DMSO-d6) (
d, ppm) 160.3
(C]N),1 11.90 (O–C–O), 28.91 (CH3), 25.64 (CH3), 140.40, 129,
124.96, 139.21, 128.63, 129.11, 133.22 (Ar-C); FAB MAS: m/z (Mþ þ 1)
288.18.
6.2. Antiprotozoal activity
6.2.1. In vitro testing against G. intestinalis
The anti-giardial activity of the compounds i–ii and 1–10 were
performed according to the standard methods as described else-
where [20]. Briefly, trophozoites were harvested by chilling the
tube on ice for 15 min to detach the monolayer and centrifuged at
300g for 5 min. The supernatant was decanted and cells were re-
suspended in fresh medium. The numbers of viable cells were
calculated using a haemocytometer and 0.4% (w/v) trypan blue. The
criteria used for viability were motility and dye exclusion.
G. intestinalis, at a density of 2 ꢅ 105 trophozoites per mL of culture
twice with distilled water and allowed to dry. A 200 mL portion of
0.1 N sodium hydroxide solution was added in 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-fitted straight line from
which the IC50 value was found.
medium, were incubated in 96-well tissue culture plates (200
ml/
well) in the presence of two fold serial dilutions of compound, with
6.3. Toxicity assay
maximum concentration of 1500
maximum final concentration of 116.88
m
m
M. Metronidazole with
M and complete medium
The cytotoxicity of the active compounds 1–5 and 8–9 were
determined calorimetrically as modified against a normal cell lines
(Vero cell lines) [27]. The test samples (maximum final concen-
with added DMSO were used as negative and positive controls
respectively. After 24 h of incubation at 37 ꢃC under anaerobic
conditions, the trophozoites from each well were examined and
counted using an inverted microscope. The appearance and
numbers of trophozoites were scored from 1 to 4 with 1 showing
the most inhibition of growth and 4 showing no inhibition
according to Upcroft et al. [24] and the minimum inhibitory
tration 180 mM) were put into wells of microtiter plate together
with 105 cell/mL of Vero cell line ATCC CCL-81 (kidney fibroblast of
an African green monkey), that was cultured in Eagle’s minimum
essential medium with the addition of 10%/mL heat-inactivation
fetal bovine serum and 1 mM sodium pyruvate. The positive control

I. Irfan et al. / European Journal of Medicinal Chemistry 45 (2010) 1648–1653
1653
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was an antineoplastic agent, ellipticine (IC50 of 2.5
mM). DMSO
(0.05%) was used as negative control. After incubation at 37 ꢃC for
72 h in a 5% CO2 incubator, the viability of the Vero cell was
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Acknowledgements
The authors are thankful to Prof. Alok Bhattacharya and Prof
Sudha Bhattacharya, School of Environmental Science, Jawaharlal
Nehru University, New Delhi, respectively for providing Laboratory
facilities for antiamoebic activities.
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