Probing the antiamoebic and cytotoxicity potency of novel tetrazole and triazine derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.12.033

A series of compounds bearing a Tetrazole and Triazine ring motif conjugated with a SO₂NH function were synthesized and investigated for their antiamoebic potency. Cytotoxicity of the compounds was checked on human hepatocellular carcinoma cell line HepG2. Incorporation of Triazine ring in place of tetrazole resulted in a precipitous increase in the antiamoebic activity of the compounds. Antiamoebic activity of the investigated compounds was found to be position and substituent dependent. In vitro cytotoxicity results revealed noncytotoxic nature of all the tested compounds up to a concentration of 25 μM. Compound 5c and 5d were obtained as least cytotoxic (IC₅₀ > 100 μM) and excellent Entamoeba histolytica inhibitors with IC₅₀ values of 1.05 μM and 1.02 μM respectively.

Full text of DOI:10.1016/j.ejmech.2011.12.033

European Journal of Medicinal Chemistry 48 (2012) 313e320
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Probing the antiamoebic and cytotoxicity potency of novel tetrazole and triazine
derivatives
,
Mohmmad Younus Wani a, Abdul Roouf Bhat b, Amir Azam c, Inho Choi b, Fareeda Athar a
*
a Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia (Central, University), New Delhi 110025, India
b School of Biotechnology, Yeungnum University, Gyeongshan, South Korea
c Department of Chemistry, Jamia Millia Islamia (Central University), 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:
A series of compounds bearing a Tetrazole and Triazine ring motif conjugated with a SO2NH function
were synthesized and investigated for their antiamoebic potency. Cytotoxicity of the compounds was
checked on human hepatocellular carcinoma cell line HepG2. Incorporation of Triazine ring in place of
tetrazole resulted in a precipitous increase in the antiamoebic activity of the compounds. Antiamoebic
activity of the investigated compounds was found to be position and substituent dependent. In vitro
cytotoxicity results revealed noncytotoxic nature of all the tested compounds up to a concentration of
Received 4 October 2011
Received in revised form
9 December 2011
Accepted 20 December 2011
Available online 30 December 2011
25
m
M. Compound 5c and 5d were obtained as least cytotoxic (IC50 > 100
M and 1.02 M respectively.
Ó 2011 Elsevier Masson SAS. All rights reserved.
mM) and excellent Entamoeba
Keywords:
Tetrazole
Triazine
histolytica inhibitors with IC50 values of 1.05
m
m
Entamoeba histolytica
Cytotoxicity
HepG2
1. Introduction
as the bioequivalent (bioisostere) of the carboxylic acid group, often
results in a dramatic enhancement in the in vitro and/or in vivo
activity [17,18], and had been extensively studied in pharmaceutical
industry [19,20]. Tetrazoles exhibit potential biological activities
and the core ring is an important constituent of number of modern
drugs [21e26]. Similarly 1,3,5-Triazines show diverse biological
activities, including antifungal, antibacterial, antimalarial, anti-
cancerous [27e30] and more importantly antiprotozoal activity
[31,32].
SO2N < moiety is an important function incorporated in many
chemotherapeutically important sulpha drugs, like sulphadiazine,
sulphathiazole, sulphamerazine, and sulfonamides [33]. Compounds
possessing this moiety have been reported to show antibacterial
activity [34,35], some of them are HIV protease inhibitors [36],
carbonic anhydrase inhibitors [37e39], antiepileptic agents [40], and
anticonvulsant agents [41,42], and are used as ETA, being a selective
antagonist [43,44]. These observations prompted the incorporation
of a sulfonamide moiety into the tetrazole and triazine ring to make
use of both functionalities in the potentiation of pharmacological
activities. Herein, the newsulfonamide derivatives ofpiperonalbased
tetrazole and triazines were evaluated for antiamoebic activity and
their toxicity profile was checked on human hepatocellular carci-
noma (HepG2) cell line. This work is an additional effort to the
development of new chemotherapeutic agents which are anti-
amoebic and non toxic to human cells.
A real advance in chemotherapy of protozoan infections was
made more than four decades ago with the introduction of metro-
nidazole. It was the first orally effective treatment for amoebiasis
[1,2], one of the most dreadful disease among the parasitic diseases
[3,4]. More than 50 million people are estimated to suffer from the
symptoms of amoebiasis such as hemorrhagic colitis and amoebic
liver abscess resulting in 100,000 deaths annually [5,6]. Metroni-
dazole is still the drug of choice to treat this disease, however, it is
associated with serious side effects [7e10], such as neurological
alterations and impairmentof cardiac rhythm due tothe chelation of
Metronidazole with calcium ions [11,12]. It is carcinogenic to man
and animals [13]. There are occasional reports of failure, recurrence
of amoebic liver abscesses even after treatment with metronidazole
and survival of parasites in spite of adequate treatment [14e16].
Such serious global health problem demands a renewed effort
seeking the development of new antiamoebic agents effective
against the pathogenic parasite and non toxic to the human cells.
Considering the need of a new molecule for the treatment of
amoebiasis, we had synthesized a series of tetrazole and triazine
based sulfonamide derivatives. The ability of tetrazole ring to serve
* Corresponding author. Tel.: þ91 11 26981717x4492; fax: þ91 11 26980164.
E-mail address: fathar@jmi.ac.in (F. Athar).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.12.033

314
M.Y. Wani et al. / European Journal of Medicinal Chemistry 48 (2012) 313e320
2. Results and discussion
been studied by MTT assay on human hepatocellular carcinoma cell
line (HepG2). The results of biological activity and cytotoxicity are
summarized in Tables 1a and b and Fig. 1a and b respectively.
2.1. Chemistry
Present study was undertaken to synthesize some novel
Sulfonamide derivatives of tetrazole and triazine and investigate
their probable antiamoebic effects. Target compounds were ob-
tained in a four step reaction procedure as outlined in Scheme 1.
Piperonal (1,3-benzodioxole-5-carbaldehyde) was converted into
1,3-benzodioxole-5-carbonitrile (2) in a two step reaction proce-
dure via an oxime intermediate (1) using a standard procedure [45].
The nitrile group of 1,3-benzodioxole-5-carbonitrile (2) was
cyclized into 5-(1,3-benzodioxol-5-yl)-1H-tetrazole (3a) and 6-(1,3-
benzodioxol-5-yl)-1,3,5-triazine-2,4-diamine (3b) [46,47]. The
compounds (4ae4f) and (5ae5f) were obtained by (3a) and (3b)
with different arylsulfonylchlorides in presence of triethylamine
using dry CH2Cl2 as a solvent. All the synthesized compounds were
characterized by elemental analysis, IR, 1H, 13C NMR and ESI-MS
studies and their data are presented in experimental section.
2.2.1. Antiamoebic activity
Preliminary experiments were carried out to determine the in vitro
antiamoebic activity of all the compounds (4ae4f) and (5ae5f) by
microdilution method using HM1:IMSS strain of E. histolytica. The
results are summarized in Tables 1a and 1b. The data is presented in
terms of percent growth inhibition relative to untreated controls, and
plotted as probit values as a function of drug concentration. The
antiamoebic activity of the synthesized compounds was compared
with widely used antiamoebic medication, metronidazole with 50%
inhibitory concentration (IC50) of 1.80 mM in our experiments. The
target compounds under investigation were synthesized from
a simple terpene molecule; piperonal and two different pharmaco-
phore groups were incorporated to study their probable antiamoebic
effect. The compounds 4ae4f contain a tetrazole ring while as in
compounds 5ae5f a triazine ring was incorporated in the place of
tetrazole ring. Replacement of the tetrazole ring with a triazine ring
resulted in a 4 fold improvement in the activity of the key interme-
2.2. Pharmacology
diate 3b (IC50 ¼ 3.54
3a (IC50 ¼ 14.24 M). It is interesting to mention that none of the
tetrazole ring bearing derivatives 4ae4f showed any significant
activity (IC50 ¼ 3.75e7.56 M) against the test organism where as all
the triazine ring bearing derivatives 5ae5f showed moderate to
excellent activity (IC50 ¼ 1.02e2.85 M). This significant change in
mM) against Entamoeba histolyticaascompared to
The compounds (4ae4f) and (5ae5f) and their key intermedi-
ates 3a and 3b were screened in vitro against HM1:IMSS strain of
Entamoeba histolytica by microdilution method [48]. All the
experiments were carried out in triplicate at each concentration
level and repeated thrice. Cytotoxicity of all the compounds has
m
m
m
Scheme 1. Schematic representation of synthesis of different substituted tetrazole and Triazine derivatives.

M.Y. Wani et al. / European Journal of Medicinal Chemistry 48 (2012) 313e320
315
Table 1a
In vitro antiamoebic activity of compounds (4ae4f) against HM1:IMSS strain of
Entamoeba histolytica and toxicity profile.
Compound
R
Antiamoebic
activity
Toxicity profile
IC50
M)
S.D.a
(ꢀ)
IC50
M)
Safety
Index (SI)
(
m
(
m
4a
4b
4c
4d
4e
4f
H
4-Me
4-Cl
4-NO2
2,4-diCl
4-Isopropyl
7.56
6.10
4.85
3.75
6.90
6.28
1.80
0.14
0.20
0.18
0.23
0.16
0.14
0.20
>100
>100
>100
z84.7
z85
>13.22
>16.39
>20.61
22.58
12.31
>15.92
>55.55
>100
>100
MNZ
Fig. 1. (a): Percentage of viable cells after 48 h pre-treatment of HepG2 cell line with
Metronidazole, key intermediate 3a and compounds 4ae4f evaluated by MTT assay.
a
The value obtained in at least three separate assay done in triplicate, S.D. (ꢀ)
(b): Percentage of viable cells after 48
h pre-treatment of HepG2 cell line with
Standard deviation.
Metronidazole, key intermediate 3b and compounds 5ae5f evaluated by MTT assay.
activity of these compounds can be speculated with either the indi-
vidual effect of triazine ring and sulfonamide moieties or their
conjugated effect. The substituted phenyl groups of the sulfonamide
fragment also have a marked effect on the activity of the compounds.
It was observed that the compounds bearing a chloro or nitro group at
the para position of the phenyl ring showed better activity. The
presence of methyl group at para position decreased the activity (5b;
IC50 ¼ 2.15
m
M) and was further decreased with insertion of isopropyl
group (5f; IC50 ¼ 2.85
mM). The presence of chloro group at the ortho
and para position of the phenyl ring in compound 5e showed
moderate activity similar to the unsubstituted phenyl ring containing
compound 5a. It will be interesting to speculate the effect with drastic
conformational and steric changes at the ortho and para position of
the phenyl ring of the sulfonamide fragment. These findings indicate
that the presence of electronwithdrawing groupslike chloro and nitro
group at the para position of the phenyl ring of the sulfonamide
fragment of the triazine ring incorporated compounds, generally
increase the antiamoebic activity of the compounds under study than
the compounds bearing electron releasing groups. Tetrazole ring
bearing derivatives (4ae4f) also adopted a similar behaviour but not
that significant. The poor antiamoebic activity of the key intermedi-
ates and the enhancement of activity with the introduction of SO2NH
group suggest that the sulfonamide moiety was important to activity.
Based upon the results it will also be necessary to optimize the
lead compound by substitution in C-4 position of phenyl ring of the
sulfonamidefragmentof thetriazine ringincorporated compoundsby
more polar groups, which seem to be very important for antiamoebic
effect, besides the position of the substituents seems to be an
important factor behind the antiamoebic activity of the
tested compounds. From the results it can be inferred that the
compound 5c (N, N0-6-(1,3-benzodioxol-5-yl)-(1,3,5-triazine-2,4-diyl)-
Table 1b
In vitro antiamoebic activity of compounds (5ae5f) against HM1:IMSS strain of
Entamoeba histolytica and toxicity profile.
Compound
R
Antiamoebic activity Toxicity profile
IC50 (
m
M) S.D.a (ꢀ) IC50 (
m
M) Safety Index(SI)
5a
5b
5c
5d
5e
5f
H
4-Me
4-Cl
4-NO2
2,4-diCl
4-Isopropyl 2.85
1.80
2.60
2.15
1.05
1.02
2.56
0.22
0.16
0.17
0.14
0.24
0.17
0.20
>100
>100
>100
>100
>100
>100
>100
>38.46
>46.51
>95.23
>98.03
>39.06
>35.08
>55.55
bis-4-nitrobenzene sulfonamide) IC50 ¼ 1.05
benzodioxol-5-yl)-(1,3,5-triazine-2,4-diyl)-bis-4-nitrobenzene sulfon-
M is a promising amoebicidal with high anti-
m
M, and 5d (N, N0-6-(1,3-
amide) IC50 ¼ 1.02
m
amoebic efficacy and least cytotoxicity on human cell line. The results
were statistically evaluated by analysis of variance. The null hypoth-
esis was tested using t-test. The significance of the difference between
the IC50 values of metronidazole and the compound 5c and 5d was
MNZ
The compounds with bold font IC50 values are more active than metronidazole.
a
The value obtained in at least three separate assay done in triplicate, S.D. (ꢀ)
Standard deviation.

316
M.Y. Wani et al. / European Journal of Medicinal Chemistry 48 (2012) 313e320
evaluated by t-test. The values of the calculated t were found higher
than the Table value of t at 5% level, thus concluding that the character
under study is said to be significantly influenced by the treatment.
place of tetrazole results in enhancement of antiamoebic activity.
These results also clearly documented that modification at position-
4 of the phenyl ring of the sulfonamide fragment of the triazine ring
incorporated compounds by some electron withdrawing substitu-
ents allows an optimization of these compounds for an effective and
probably selective antiamoebic therapy. More importantly, anti-
amoebic and cytotoxicity studies of these compounds resulted in the
binding of two compounds 5c (N, N0-6-(1,3-benzodioxol-5-yl)-(1,3,5-
2.2.2. In vitro cytotoxicity studies
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) is reduced by the succinate dehydrogenase system of mito-
chondrial living cells to produce water insoluble purple formazan
crystals [49,50] which, after solubilization, can be measured spec-
trophotometrically. Since the amount of formazan produced is
directly proportional to the number of active cells in the culture,
MTT has long been used to assess the cell viability in cell prolifer-
ation and cytotoxicity [51e53].
In the present study, some newly synthesized compounds were
screened for their antiamoebic activity and then evaluated for their
cytotoxicity against Human hepatocellular carcinoma cell line (HepG2)
to ensure their toxic effect. Metronidazole was used as a reference
drug. A sub-confluent population of HepG2 cells was treated with
increasing concentration of these compounds and the number of
viable cells was measured after 48 h by MTT cell viability assay. The
triazine-2,4-diyl)-bis-4-chlorobenzene sulfonamide) IC50 ¼ 1.05
and 5d (N, N0-6-(1,3-benzodioxol-5-yl)-(1,3,5-triazine-2,4-diyl)-bis-
4-nitrobenzene sulfonamide) IC50 ¼ 1.02 M as stronger E. histolytica
inhibitors with least cytotoxicity to Human cells (HepG2) than the
M, which gains some
mM,
m
standard drug metronidazole IC50 ¼ 1.80
m
insights into the synthesis or structure modifications of triazine
pharmacophore bearing derivatives for the purpose of discovering
new antiamoebic drug candidates.
4. Experimental Protocol
Solvents and organic reagents were purchased from Sigma
Aldrich, Merck (Germany) and Loba Chemie (India) and were used
without further purification. Melting points (mp) were performed
using a Mel-temp instrument, and the results are uncorrected.
Elemental analyses were performed on HeraeusVario EL III analyzer
at Central Drug Research Institute, Lucknow, India. The results were
within ꢀ0.4% of the theoretical values. IR spectra were recorded on
PerkineElmer model 1600 FT-IR RX1 spectrophotometer as KBr
discs/ATR mode. 1H NMR and 13C NMR spectra were recorded on
Bruker AVANCE 300 (300.13) MHz spectrometer using DMSO-d6/
CDCl3 as solvent with TMS as internal standard. Splitting patterns
are designated as follows; s, singlet; br s for broad 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. Reactions were
monitored using thin-layer chromatography (TLC) using commer-
cially available precoated plates (Merck Kieselgel 60 F254 silica).
Visualization was achieved with UV light at 254 nm or I2 vapour
staining.
concentration range of all the compounds was 3.13e100 mM. The cell
viability (%) obtained with continuous exposure for 48 h are depicted
in Fig.1a and b. The cytotoxicity of all the compounds was found to be
concentration-dependent. Fig. 1a and
b depicts that all the
compounds including the reference compound metronidazole
showed viability ranging from 92.5 to 100% at the concentration
range of 3.13
compounds showed a viability of ꢁ70%. On increasing the concen-
tration range up to 50 and 100 M the compounds showed moderate
mM and up to a concentration of 25 mM all the
m
tohighcytotoxicityagainst theHepG2 cellline. Except forcompounds
4d and 4e all the compounds showed least cytotoxicity and have IC50
values greater than 100 mM as given in Tables 1a and 1b. To further
investigate the selectivity of the compounds, the “safety index” (SI),
defined as the toxicity IC50/protozoal IC50, was calculated. This allows
estimating the efficacy of compounds. The results are summarized in
Tables 1a and 1b. Compound 5c and 5d showed higher safety index
values, better than metronidazole. From the results of antiamoebic
activity and cytotoxicity it can be inferred that compound 5c and 5d
are least cytotoxic and excellent E. histolytica inhibitors as compared
to the reference drug metronidazole (Fig. 2). These results also
showed that the compounds 5c and 5d despite of being highly anti-
amoebic do not show any marked toxicity on human cell line and
have safety index values of ꢁ95 which is better than metronidazole.
4.1. Synthesis of 5-(1,3-benzodioxol-5-yl)-1H-tetrazole (3a)
5-(1,3-benzodioxol-5-yl)-1H-tetrazole was synthesized from
1,3-benzodioxole-5-carbonitrile by following a reported procedure
[46]. The requisite nitrile (2) was prepared from Piperonal (1,3-
benzodioxole-5-carbaldehyde) via oxime formation, followed by
dehydration with acetic anhydride, following a reported procedure
[45]. Nitrile (2.95 g, 20 mmol), sodium azide (1.43 g, 22 mmol) and
zinc bromide (4.50 g, 20 mmol), were put in 60 mL of water. 5 mL of
isopropanol was also added to stop the formation of clumps. The
reaction mixture was refluxed for 24 h and monitored by TLC;
vigorous stirring is essential. After 24 h HCl (3 N, 30 mL) and ethyl
acetate (100 mL) were added, and vigorous stirring was continued
until no solid was present and the aqueous layer had a pH of 1. If
necessary, additional ethyl acetate was added. The organic layer
was isolated and the aqueous layer extracted with 2 ꢂ 100 mL of
ethyl acetate. The combined organic layers were evaporated,
200 mL of 0.25 N NaOH was added, and the mixture was stirred for
30 min, until the original precipitate was dissolved and a suspen-
sion of zinc hydroxide was formed. The suspension was filtered, and
the solid washed with 20 mL of 1 N NaOH. To the filtrate was added
50 mL of 3 N HCl with vigorous stirring causing the tetrazole to
precipitate. The tetrazole was filtered and washed with 2 ꢂ 50 mL
of 3 N HCl and dried in a drying oven to furnish the tetrazole as
a white powder.
3. Conclusion
This study has achieved the efficient synthesis of novel Tetrazole
and Triazine ring containing derivatives and examined their in vitro
antiamoebic and cytotoxic activity. Incorporation of triazine ring in
Fig. 2. Comparison of antiamoebic activity and cytotoxicity profile of compounds 5d,
5c and reference drug metronidazole. Compound 3d has almost 100 times more
antiamoebic potency than its cytotoxicity which is better than Metronidazole.
Schemes.

M.Y. Wani et al. / European Journal of Medicinal Chemistry 48 (2012) 313e320
317
4.1.1. (E)-1-(1,3-benzodioxol-5-yl)-N-hydroxymethanimine (1)
4.2.3. 5-(1,3-Benzodioxol-5-yl)-1-[(4-chlorophenyl)sulfonyl]-1H-
White; Yield 95%; mp. 100e105 ꢃC; IR nmax cmꢄ1: 3225
(NOeH), 2921 (CeH), 1660 (C]N), 1608, 1500 (C]C, Ar), 937
tetrazole (4c)
White solid; Yield 67%; mp. 228e230 ꢃC; Anal. Calc. for
C14H9N4ClO4S: C 46.10, H 2.49, N 15.36; found: 46.24, H, 2.38, N,
15.47%. IR nmax cmꢄ1: 3065 (CeH, Ar), 1595 (C]C, Ar), 1645 (C]N),
(NeO stretch); 1H NMR (DMSO-d6)
d(ppm): 10.85 (broad s, 1H,
NeOH), 7.90 (s, 1H, CH ] NeOH), 7.30e6.50 (m, 3H, AreH), 5.90
(s, eOeCH2eOe); 13C NMR (DMSO-d6) d(ppm): 152.3, 148.1, 146.0,
132.6, 123.8, 115.8, 101.6 (eOeCH2eOe); ESI-MS m/z: [Mþþ1]
166.04.
1328,1147 (S]O), 730 (CeCl); 1H NMR (DMSO-d6)
d(ppm): 7.98 (2H,
d, J ¼ 7.2 Hz), 7.77 (2H, m), 7.08 (1H, dd, J ¼ 2.2 Hz; 2J ¼ 4.8 Hz), 6.90
(1H, d, J ¼ 2.5 Hz), 6.85 (1H, d, J ¼ 8.5 Hz), 6.01 (2H, s, OeCH2eO);
13C NMR (DMSO-d6)
d(ppm): 154.6 (C]N), 149.8, 147.7, 139.2, 134.1,
4.1.2. 1,3-Benzodioxole-5-carbonitrile (2)
128.9,128.0,123.6,115.7,113.0,108.2 (AreC),101.5 (OeCH2eO); ESI-
White; Yield 92%; mp. 95e98 ꢃC; IR nmax cmꢄ1: 2928 (CeH, Ar),
2204 (CN), 1592 (C]C, Ar); 1H NMR (DMSO-d6) (ppm): 7.18e6.90
(m, 3H, AreH), 6.03 (s, 2H, eOeCH2eOe); 13C NMR (DMSO-d6)
MS m/z: [Mþþ1] 366.00.
4.2.4. 5-(1,3-Benzodioxol-5-yl)-1-[(4-nitrophenyl)sulfonyl]-1H-
tetrazole (4d)
d
(ppm): 153.1, 147.9, 123.1, 123.6, 118.2, 116.1, 113.8, 103.4
(eOeCH2eOe); ESI-MS m/z: [Mþþ1] 148.02.
Yellowish solid; Yield 62%; mp. 210e213 ꢃC; Anal. Calc. for
C14H9N5O6S: C 44.80, H 2.42, N 18.66; found: 44.92, H, 2.36, N,
18.74%; IR nmax cmꢄ1: 3068 (CeH, Ar), 1645 (C]N), 1590 (C]C,
Ar), 1545, 1350 (NO2), 1321, 1128 (S]O); 1H NMR (DMSO-d6)
4.1.3. 5-(1,3-Benzodioxol-5-yl)-1H-tetrazole (3a)
White solid; Yield 84%; mp. 190e193 ꢃC; Anal. Calc. for
C8H6N4O2: C 50.53, H 3.18, N 29.46%, found: C 50.43, H 3.08, N
29.58%; IR nmax cmꢄ1: 3280 (NeH br stretch), 2864 (CeH, Ar), 1632
d
(ppm): 8.13 (2H, d, J ¼ 7.8 Hz), 7.76 (2H, m), 7.12 (1H, dd,
J ¼ 2.8 Hz; 2J ¼ 5.8 Hz), 6.96 (1H, s), 6.93 (1H, d, J ¼ 8.7 Hz), 6.07
(C]N), 1595 (C]C, Ar); 1H NMR (DMSO)
d
(ppm): 7.62 (1H, d,
J ¼ 7.2 Hz), 7.54 (1H, s), 7.16 (1H, d, J ¼ 8.1 Hz), 6.15 (2H, s,
OeCH2eO), 4.45 (1H, NH, br s); 13C NMR (DMSO)
(ppm): 154.8
(2H, s, OeCH2eO); 13C NMR (DMSO-d6) (ppm): 13C NMR
d
(DMSO-d6) d(ppm): 156.4 (C]N), 152.5, 146.3, 143.9, 136.1, 128.6,
d
127.8, 122.4, 112.9 (AreC), 101.5 (OeCH2eO); ESI-MS m/z:
(C]N), 149.2, 147.6, 121.2, 118.5, 108.2, 106.7, 101.6 (eOeCH2eO);
ESI-MS m/z [Mþþ1] 191.07.
[Mþþ1] 376.03.
4.2.5. 5-(1,3-Benzodioxol-5-yl)-1-[(2,4-dichlorophenyl)sulfonyl]-
1H-tetrazole (4e)
4.2. General procedure for the synthesis of 5-(1,3-benzodioxol-5-
yl)-1-(phenyl/substituted phenyl sulfonyl)-2H-tetrazoles (4ae4f)
White solid; Yield 58%; mp. 228e230 ꢃC; Anal. Calc. for
C14H8Cl2N4O4S: C 44.80, H 2.42, N 18.66; found: 44.68, H 2.47, N
18.73%; IR nmax cmꢄ1: 3062 (CeH Ar), 1590 (C]C, Ar), 1640 (C]N),
To a solution of 5-(1,3-benzodioxol-5-yl)-1H-tetrazole (1 eq.)
and triethylamine (3 eq.) in dry CH2Cl2 at 0 ꢃC was added aryl
sulfonyl chlorides (1.2 eq.). The reaction mixture was stirred at
1321, 1128 (S]O), 742 (CeCl), 1H NMR (DMSO-d6)
d(ppm): 7.89 (1H,
d, J ¼ 7.5 Hz) 7.58 (1H, s), 7.42 (1H, d, J ¼ 7.8 Hz), 7.10 (1H, dd,
J ¼ 2.5 Hz; 2J ¼ 4.5 Hz), 6.94 (1H, d, J ¼ 2.4 Hz), 6.86 (1H, d,
0
ꢃC for about 2 h and stirring was continued at room tempera-
J ¼ 8.4 Hz), 6.02 (2H, s, OeCH2eO); 13C NMR (DMSO-d6)
d(ppm):
ture for about 4e5 h (completion of reaction was monitored by
TLC). After the completion of reaction the reaction mass was
quenched with distilled water and extracted with dichloro-
methane. Finally the combined organic layer was washed with
distilled water again and dried over anhydrous Na2SO4. After
removal of the solvent in vacuum, the residue was purified by
recrystallization.
154.8 (C]N), 148.2, 147.5, 138.4, 136.0, 128.6, 128.0, 122.6, 118.3,
112.0, 105.5, (AreC), 101.8 (OeCH2eO); ESI-MS m/z : [Mþþ1]
399.94, [Mþþ2] 400.96.
4.2.6. 5-(1,3-Benzodioxol-5-yl)-1-[(4-isopropylphenyl)sulfonyl]-
1H-tetrazole (4f)
White solid; Yield 60%; mp. 242e245 ꢃC; Anal. Calc. for
C17H16N4O4S: C 54.83, H 4.33, N 15.04%; found: 54.76, H 4.10, N
15.26%; IR nmax cmꢄ1: 3032 (CeH, Ar), 1595 (C]C, Ar), 1645 (C]N),
4.2.1. 5-(1,3-Benzodioxol-5-yl)-1-(phenylsulfonyl)-1H-tetrazole (4a)
Cream solid; Yield 65%; mp. 182e185 ꢃC; Anal. Calc. for
C14H10N4O4S: C 50.91, H 3.05, N 17.03; found: 50.82, H, 3.01, N,
17.18%; IR nmax cmꢄ1: 3068 (CeH, Ar), 1595 (C]C, Ar), 1690 (C]N),
1545, 1350 (NO2) 1321, 1128 (S]O); 1H NMR (DMSO-d6)
d(ppm):
7.95 (2H, d, J ¼ 7.8 Hz), 7.54 (2H, d, J ¼ 7.4 Hz), 7.11 (1H, dd,
J ¼ 1.8 Hz; 2J ¼ 4.8 Hz), 6.97 (1H, s), 6.85 (1H, d, J ¼ 8.1 Hz), 6.01 (2H,
s, OeCH2eO), 3.83e3.68 (1H, m), 1.89 (6H, s); 13C NMR (DMSO-d6)
1174 (S]O); 1H NMR (DMSO-d6)
d
(ppm): 7.93 (2H, d, J ¼ 7.2 Hz)
7.73 (1H, t, J ¼ 7.2 Hz), 7.62 (2H, m), 7.05 (1H, dd, J ¼ 1.8 Hz;
d(ppm): 156.8 (C]N), 152.4, 146.8, 143.5, 135.1, 128.5, 128.0, 126.7,
2J ¼ 4.8 Hz), 6.93 (1H, d, J ¼ 1.5 Hz), 6.81(1H, d, J ¼ 8.1 Hz), 6.04
125.2, 120.6, 115.0 (AreC), 101.5 (OeCH2eO), 42.6, 26.5 (Isopropyl);
(2H, s, OeCH2eO); 13C NMR (DMSO-d6)
d(ppm): 154.8 (C]N),
ESI-MS m/z: [Mþþ1] 373.06.
150.6, 147.3, 137.1, 134.1, 128.9, 128.0, 123.6, 122.6, 115.7, 113.0,
108.2, 107.8, (AreC), 101.5 (OeCH2eO); ESI-MS m/z: [Mþþ1]
331.05.
4.3. Synthesis of 6-(1,3-benzodioxol-5-yl)-1,3,5-triazine-2,4-
diamine (3b)
4.2.2. 5-(1,3-Benzodioxol-5-yl)-1-[(4-methylphenyl)sulfonyl]-1H-
tetrazole (4b)
6-(1,3-benzodioxol-5-yl)-1,3,5-triazine-2,4-diamine (3b) was
synthesized from 1,3-benzodioxole-5-carbonitrile (2) by following
a reported procedure [47]. The requisite nitrile (2) was prepared
from Piperonal (1,3-benzodioxole-5-carbaldehyde), via oxime
formation, followed by dehydration with acetic anhydride,
following a reported procedure [45]. Nitrile (5 m mol), dicyandia-
mide (5.5 m mol) and KOH (10 m mol) were put in 50 ml water. The
reaction mixture was heated at reflux for 12e48 h. The suspended
solid products were filtered and rinsed with Et2O to give a pure
diaminotriazine.
Yellow solid; Yield 60%; mp. 220e223 ꢃC; Anal. Calc. for
C15H12N4O4S: C 52.32, H 3.51, N 16.27; found: C 52.26, H, 3.48, N,
16.35%. IR nmax cmꢄ1: 3068 (CeH, Ar), 1598 (C]C, Ar), 1648 (C]N),
1158 (S]O); 1H NMR (DMSO-d6)
d
(ppm): 7.90 (2H, d, J ¼ 7.8 Hz),
7.65e7.60 (2H, m), 7.02 (1H, dd, J ¼ 1.2 Hz; 2J ¼ 5.2 Hz), 6.90 (1H, s),
6.83 (1H, d, J ¼ 8.1 Hz), 6.07 (2H, s, OeCH2eO), 2.83 (3H, s); 13C NMR
(DMSO-d6) d d(ppm): 156.4 (C]N),
(ppm): 13C NMR (DMSO-d6)
152.5, 146.3, 143.9, 136.1, 128.6, 127.8, 122.4, 116.0, 112.9 (AreC),
101.5 (OeCH2eO), 16.4 (CH3); ESI-MS m/z: [Mþþ1] 345.06.

318
M.Y. Wani et al. / European Journal of Medicinal Chemistry 48 (2012) 313e320
4.3.1. 6-(1,3-Benzodioxol-5-yl)-1,3,5-triazine-2,4-diamine (3b)
Cream solid; Yield: 80%; mp. 220e223 ꢃC; Anal. Calc. for
C10H9N5O2: C 51.95, H 3.92, N 30.29%; found: C 51.82, H 4.12, N
30.37%; IR nmax cmꢄ1: 3408 (NH2), 2865 (CeH, Ar), 1662 (C]N),
16.23%; IR nmax cmꢄ1: 3258 (NH), 3042 (CeH, Ar), 1586 (C]C, Ar),
1545, 1350 (NO2), 1314, 1127 (S]O); 1H NMR (CDCl3)
(ppm): 8.30
d
(2H, NH, br s), 7.99e7.82 (4H, m, AreH), 7.60e7.48 (4H, m, AreH),
7.25 (1H, d, J ¼ 7.6 Hz), 6.98 (1H, d, J ¼ 8.2 Hz), 6.20 (1H, d,
1587 (C]C, Ar); 1H NMR (CDCl3)
d
(ppm): 7.26e6.77 (3H, m), 5.87
J ¼ 8.4 Hz), 6.01 (2H, s, OeCH2eO); 13C NMR (CDCl3)
d(ppm): 178.2,
(2H, s, OeCH2eO), 4.70 (4H, NH2 br s); 13C NMR (CDCl3)
d(ppm):
176.2 (C]N), 152.5, 151.5, 149.4, 148.6, 145.8, 128.2, 124.8, 121.7,
169.8, 167.9 (C]N), 149.5, 148.6, 125.2, 121.0, 115.8, 113.2 (AreC),
102.2 (OeCH2eO); ESI-MS m/z: [Mþþ1] 232.07.
115.6,112.0 (AreC),100.8 (OeCH2eO); ESI-MS m/z: [Mþþ1] 602.05.
4.4.5. N, N0-6-(1,3-benzodioxol-5-yl)-(1,3,5-triazine-2,4-diyl)-bis-
(2,4-dichlorobenzene sulfonamide) (5e)
4.4. General procedure for the synthesis of N, N0-6-(1,3-
benzodioxol-5-yl)-1,3,5-triazine-2,4-diyl di/bisbenzene/substituted
benzene sulfonamides (5ae5f)
White solid; Yield 60%; mp. 210e213 ꢃC; Anal. Calc. for
C22H13N5Cl4O6S2: C 40.69, H 2.02, N 10.79%; found: C 40.58, H 2.08,
N 10.68%; IR nmax cmꢄ1: 3283 (NH), 3015 (CeH, Ar), 1595 (C]C, Ar),
To
a
solution of 6-(1,3-benzodioxol-5-yl)-1,3,5-triazine-2,4-
1556, 1343 (NO2), 1322, 1117 (S]O); 1H NMR (CDCl3)
d(ppm): 8.38
diamine (1.0 eq.) and triethylamine (5.0 eq.) in dry CH2Cl2 at 0 ꢃC
was added aryl sulfonyl chlorides (2.0 eq.). The reaction mixture
was stirred at 0 ꢃC for about 2 h and stirring was continued at room
temperature for about 4e5 h (completion of reaction was moni-
tored by TLC). After the completion of reaction the reaction mass
was quenched with distilled water and extracted with dichloro-
methane. Finally the combined organic layer was washed with
distilled water again and dried over anhydrous Na2SO4. After
removal of the solvent in vacuum, the residue was purified by
recrystallization.
(2H, NH, br s), 7.98e7.83 (2H, m, AreH), 7.70e7.58 (2H, m, AreH),
7.30 (2H, s), 6.80 (1H, d, J ¼ 5.4 Hz), 6.65 (1H, d, J ¼ 6.5 Hz), 6.56
(1H, d, J ¼ 5.8 Hz), 5.95 (2H, s, OeCH2eO); 13C NMR (CDCl3)
d(ppm):
176.8, 174.9 (C]N), 149.4, 148.6, 138.8, 137.8, 132.6, 130.6, 128.2,
124.8, 120.7, 115.4, 112.3 (AreC), 101.8 (OeCH2eO); ESI-MS m/z:
[Mþþ1] 647.86, [Mþþ2] 648.91.
4.4.6. N,N0-6-(1,3-benzodioxol-5-yl)-(1,3,5-triazine-2,4-diyl)-bis-
(4-sopropylbenzenesulfonamide) (5f)
White solid; Yield 65%; mp. 233e235 ꢃC; Anal. Calc. for
C28H29N5Cl4O6S2: C 56.46, H 4.91, N 11.96%; found: C 56.54, H 5.02,
N 11.86%; IR nmax cmꢄ1: 3256 (NH), 3020 (CeH, Ar), 1598 (C]C, Ar),
4.4.1. N, N0-6-(1,3-benzodioxol-5-yl)-(1,3,5-triazine-2,4-diyl)-
dibenzenesulfonamide (5a)
1533, 1320 (NO2), 1300, 1125 (S]O); 1H NMR (CDCl3)
d(ppm): 8.80
White solid; Yield 68%; mp. 233e235 ꢃC; Anal. Calc. for
C22H17N5O6S2: C 51.66, H 3.35, N 13.69%; found: C 51.54, H 3.42, N
13.76%; IR nmax cmꢄ1: 3295 (NH), 3025 (CeH, Ar), 1598 (C]C, Ar),
(2H, NH, br s), 7.85e7.43 (8H, m, AreH), 6.95 (1H, d, J ¼ 5.8 Hz), 6.55
(1H, d, J ¼ 7.5 Hz), 6.20 (1H, d, J ¼ 7.2), 5.95 (2H, s, OeCH2eO),
3.30e3.10 (2H, m), 1.93 (12H, s, CH3); 13C NMR (CDCl3)
d(ppm):
1322, 1147 (S]O); 1H NMR (CDCl3)
d(ppm): 7.68 (2H, NH, br s), 7.52
179.0, 172.6 (C]N), 150.8, 149.8, 148.4, 138.2, 128.2, 128.0, 124.8,
120.7,115.6,112.5 (AreC),101.8 (OeCH2eO), 38.5, 24.0; ESI-MS m/z:
[Mþþ1] 596.18.
(2H, d, J ¼ 7.8 Hz), 7.36e7.30 (4H, m), 7.02 (1H, d, J ¼ 8.1 Hz),
6.91e6.75 (4H, m), 6.49 (2H, t, J ¼ 7.5 Hz), 5.95 (2H, s, OeCH2eO);
13C NMR (CDCl3)
d(ppm): 174.8, 171.0 (C]N), 150.2, 149.6, 135.7,
134.4, 130.6, 129.8, 126.3, 115.5, 113.6 (AreC), 101.2 (OeCH2eO);
4.5. In vitro antiamoebic assay
ESI-MS m/z: [Mþþ1] 512.06.
All the test compounds (4ae4f and 5ae5f) and the key inter-
mediates 3a and 3b were screened in vitro for antiamoebic activity
against HM1:IMSS strain of E. histolytica by microdilution method
[48]. E. histolytica trophozoites were cultured in wells of 96-well
microtiter plate by using Diamond TYIS-33 growth medium [54].
4.4.2. N, N0-6-(1,3-benzodioxol-5-yl)-(1,3,5-triazine-2,4-diyl)-bis-
4-methylbenzene sulfonamide (5b)
Yellow solid; Yield 63%; mp. 230e233 ꢃC; Anal. Calc. for
C24H21N5O6S2: C 53.42, H 3.92, N 12.98%; found: C 53.48, H 3.84, N
13.15%; IR nmax cmꢄ1: 3285 (NH), 3012 (CeH, Ar), 1589 (C]C, Ar),
The test compounds (1 mg) were dissolved in DMSO (40 mL, level
1329, 1131 (S]O); 1H NMR (CDCl3)
d
(ppm): 8.41 (2H, NH, br s), 7.95
at which no inhibition of amoeba occurs) [55,56]. The stock
solutions of the compounds were prepared freshly before use at
a concentration of 1 mg mLꢄ1. Two-fold serial dilutions were made
in the wells of 96-well microtiter plate (costar). Each test includes
metronidazole as a standard amoebicidal 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
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 mLꢄ1 was estimated with a haemocytometer, using try-
pan blue exclusion to confirm the viability. The suspension was
(1H, d, J ¼ 7.8 Hz), 7.58e7.49 (4H, m), 7.10e7.00 (4H, m), 6.95 (1H, d,
J ¼ 8.1 Hz), 6.87 (1H, d, J ¼ 8.1 Hz), 6.05 (2H, s, OeCH2eO), 2.69 (6H,
s); 13C NMR (CDCl3)
d(ppm): 176.4, 172.8 (C]N), 150.6, 149.1, 138.2,
130.2, 128.5, 126.3, 124.7, 115.8, 112.4 (AreC), 101.2 (OeCH2eO),
26.8 (CH3); ESI-MS m/z: [Mþþ1] 540.08.
4.4.3. N, N0-6-(1,3-benzodioxol-5-yl)-(1,3,5-triazine-2,4-diyl)-bis-
4-chlorobenzene sulfonamide (5c)
White solid; Yield 67%; mp. 215e218 ꢃC; Anal. Calc. for
C22H15N5Cl2O6S2: C 45.52, H 2.60, N 12.07; found: C 45.43, H 2.54, N
12.25%; IR nmax cmꢄ1: 3262 (NH), 3052 (CeH Ar), 1594 (C]C, Ar),
1310, 1115 (S]O), 732 (CeCl); 1H NMR (CDCl3)
d(ppm): 8.96 (2H,
NH, br s), 8.15e7.89 (4H, m, AreH), 7.45e7.28 (4H, m, AreH), 7.17
(1H, d, J ¼ 7.8 Hz), 6.72 (1H, d, J ¼ 7.8 Hz), 6.43 (1H, d, J ¼ 8.5 Hz),
diluted to 105 cells mLꢄ1 by adding fresh medium and 170
mL of
this suspension was added to the test and control wells in the
plate so that the wells were completely filled (total volume,
6.02 (2H, s, OeCH2eO); 13C NMR (CDCl3)
d(ppm): 176.8, 172.0 (C]
N), 152.5, 148.6, 137.2, 129.1, 128.2, 125.0, 121.7, 118.0, 116.3 (AreC),
340
m
L). An inoculum of 1.7 ꢂ 104 organisms/well was chosen so
101.8 (OeCH2eO); ESI-MS m/z: [Mþþ1] 580.98.
that confluent, but not excessive growth, took place in control
wells. Plates were sealed and gassed for 10 min with nitrogen
before incubation at 35.5 ꢃC 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
4.4.4. N, N0-6-(1,3-benzodioxol-5-yl)-(1,3,5-triazine-2,4-diyl)-bis-
4-nitrobenzene sulfonamide (5d)
White solid; Yield 68%; mp. 205e208 ꢃC; Anal. Calc. for
C22H15N7O10S2: C 43.93, H 2.51, N 16.30%; found: C 44.06, H 2.57, N

M.Y. Wani et al. / European Journal of Medicinal Chemistry 48 (2012) 313e320
319
chloride solution (0.9%) at 35.5 ꢃC. This procedure was completed
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