Thiosemicarbazone fragment embedded within 1,2,4-triazole ring as inhibitors of Entamoeba histolytica

Article abstract of DOI:10.1016/j.bmcl.2012.02.084

A series of 1,2,4-triazole derivatives containing thiosemicarbazone linkage was synthesized and evaluated for their in vitro antiamoebic activity against HM1:IMSS strain of Entamoeba histolytica. All the compounds were capable of inhibiting the growth of

Full text of DOI:10.1016/j.bmcl.2012.02.084

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2768–2771
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Thiosemicarbazone fragment embedded within 1,2,4-triazole ring as
inhibitors of Entamoeba histolytica
⇑
Shadab Miyan Siddiqui, Attar Salahuddin, Amir Azam
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:
A series of 1,2,4-triazole derivatives containing thiosemicarbazone linkage was synthesized and evaluated
Received 4 February 2012
Revised 23 February 2012
Accepted 24 February 2012
Available online 2 March 2012
for their in vitro antiamoebic activity against HM1:IMSS strain of Entamoeba histolytica. All the compounds
were capable of inhibiting the growth of E. histolytica out of which four compounds (IC50 = 0.28–1.38
were found to have better efficacy than the standard drug Metronidazole (IC50 = 1.8 M). Cytotoxicity of
the active compounds was assessed by MTT assay using human breast cancer MCF-7 cell line, which
revealed that all the compounds were low cytotoxic in the concentration range of 2.5–250 M.
lM)
l
l
Keywords:
1,2,4-Triazole
Ó 2012 Elsevier Ltd. All rights reserved.
Entamoeba histolytica
Antiamoebic activity
MTT assay
H
S
Invasive amoebiasis is an emerging parasitic disease in HIV-
infected patients in an area endemic for amoebic infection.1 This
awful gastrointestinal infection is caused by an anaerobic parasitic
protozoa Entamoeba histolytica,2 which lyses host cells by direct
contact using surface lectins and releases cysteinase proteinases.3
It is more virulent than other pathogens as it can cause severe
infections, both in immunocomponent and immunodeficient hosts.
Infection from this parasite occurs in a number of countries of the
world, resulting in 50 million cases of invasive disease and up to
100,000 fatalities per year.4–7 Nitroimidazoles such as metronida-
zole, ornidazole, and tinidazole are main drugs used for the
treatment of this protozoan disease. Cross resistance exists among
the nitroimidazoles8 and long-term use of these medicaments pro-
duces plenty of perilous side effects in patients.9 Furthermore,
Metronidazole, the first-line medication for amoebiasis chemo-
therapy, is potentially carcinogenic to humans because it is
genotoxic to human cells.10 Therefore, the treatment of this dan-
gerous disease is demanding and attracting scientists to develop
novel antiamoebic agents.
Thiosemicarbazones, a very promising class of compounds
show a broad spectrum of therapeutic properties.11–21 These
compounds exhibit thione-thiol tautomerism (Fig. 1). It has been
observed that the drugs containing a thiosemicarbazone fragment
(e.g. Thioacetazone) is often the source of acute toxicity such as
Stevens–Johnson Syndrome (SJS) and Toxic Epidermal Necrolysis
(TEN) caused through allergic drug reaction.22 Over the years, we
have synthesized a range of thiosemicarbazone derivatives that
S
Thione-thiol
R
N
Ar
R
N
Ar
N
R
N
H
N
R
N
Figure 1. Thione-thiol tautomerism in thiosemicarbazones.
N
N
N
N
N
HN
S
N
N
N
HN
S
TSC-1, IC50 = 1.0µM
Cl
TSC-2, IC50 = 0.84 µM
S
N
N
N
N
N
N
N
H
S
O2N
S
N
Cl
TSC-3, IC50 = 0.72 µM
TSC-4, IC50 = 1.71 µM
Figure 2. Antiamoebic compounds containing TSC fragment embedded in various
heterocycles.
have exhibited potent in vitro antiamoebic activity23 comparable
or superior to metronidazole, the reference drug.
Pyrazoline compounds24 (TSC-1) containing hydrazine (N–N)
part (in blue) of TSC-Fragment embedded within the pyrazoline ring
and 3,7-dimethyl-4H-pyrazolo[3,4-e][1,2,4]triazine derivative
⇑
Corresponding author.
E-mail address: amir_sumbul@yahoo.co.in (A. Azam).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.02.084

S. M. Siddiqui et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2768–2771
2769
R
R
S
[5,4-b]quinoxaline derivatives (TSC-3) having full thiosemicarba-
zone fragment (in blue) present within the heterocyclic ring system
were found as most effective inhibitor of E. histolytica.26 Linear TSC-
fragment containing compounds such as nitrothiophene derivative
(in red) (TSC-4) also showed good antiamoebic activities27 (Fig.2).
In view of the number of pharmacological significances of 1,2,4-
triazole derivatives28–33 and to confirm the importance of thiosem-
icarbazone fragment for the display of antiamoebic activity, we
designed compounds containing this fragment by using thiourea
(in blue) part embedded within the 1,2,4-triazole ring (Fig. 3).
The combination of these two biologically important structural
features is expected to exhibit synergistic effects to display anti-
amoebic activity. To the best of our knowledge, this is the first
report of 1,2,4-triazole derivatives showing promising in vitro
activity against E. histolytica.
The route employed for synthesis of target compounds is shown
in Scheme 1. 4-Amino-5-phenyl-4H-1,2,4-triazole-3-thiol (1), the
key intermediate for the synthesis of desired compounds, was
obtained by the reported method.34 Finally, condensation of com-
pound (1) with various substituted aromatic aldehydes in the pres-
ence of catalytic amount of conc. hydrochloric acid, under reflux in
ethanol for 2 h furnished the title compounds (2–11) in reasonable
yields (62–70%).
N
NH
N
N
N
Thione-thiol
S
N
N
HN
N
SH
N
N
Ar
Ar
Ar
TSC
1, 2, 4-triazole ring
embeded TSC
Figure 3. Thiourea (blue) part of thiosemicarbazone fragment embedded in 1,2,4-
triazole ring and tautomerism.
N
N
N
N
(a)
SH
N
N
SH
N
NH2
Ar
(2-11)
1
Scheme 1. The synthetic route for obtaining the TSC–embedded 1,2,4-triazoles.
Reagents: (a) Ar–CHO, EtOH Reflux.
(TSC-2) having only one terminal nitrogen (in blue) of the hydrazine
part embedded in the triazine ring25 displayed appreciable activity.
2-(3-(3-chlorophenyl)-4,5-dihydro-4-methylpyrazol-1-yl)thiazolo-
Table 1
In vitro antiamoebic activity of 1,2,4-Triazole derivatives (2–11) against HM1: IMSS strain of E. histolytica and cytotoxicity profile of compounds 2, 3, 6, 7 and metronidazole
Compound no.
Ar
Antiamoebic activity
M)
Cytotoxicity profile
M)
IC50
(
l
SDa
IC50
ND
(l
SDa
ND
1
2
NH
10.81
0.022
1.38
0.018
0.016
>250
>250
0.21
0.27
O
3
4
0.322
O
8.79
0.008
0.006
N.D.
N.D.
N.D.
N.D.
O
O
O
O
5
2.70
NO2
6
7
0.280
0.309
0.007
0.008
>250
>250
0.23
0.25
N
Cl
OH
8
9.55
10.16
4.68
0.006
0.014
0.013
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
9
F3C
10
OH
O
11
12
3.05
0.007
N.D.
N.D
S
NH2
2.29
0.031
ND
ND
H2N
HO
N
H
N
NO2
MNZ
1.80
0.029
>250
0.22
N
Bold values indicates the activity of the compounds is better than the standard drug metronidazole.
a
Standard Deviation, N.D. Not determined.

2770
S. M. Siddiqui et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2768–2771
group (2, IC50 = 1.38
tro group (6, IC50 = 0.280
IC50 = 0.309 M) at para position of phenyl ring showed better
antiamoebic activity than the reference drug metronidazole
(IC50 = 1.80 M). It was clear from the IC50 values that none of
the derivatives without a 4-position substituent displayed good
antiamoebic activity. The thiosemicarbazide showed
IC50 = 2.29 M. As the thiosemicarbazone fragment is present in
all the synthesized molecules they showed good inhibition ranging
from (IC50 = 0.280–10.16 M). Compound 2 (IC50 = 1.38 M), com-
pound 3 (0.322 M), compound 6 (0.28 M), and compound 7
(0.309 M) were found to be the most potent inhibitors, while thi-
osemicarbazide (IC50 = 2.29 M) showed activity close to that of
metronidazole (IC50 = 1.8 M). A comparison of relative antiamoe-
lM), methoxy group (3, IC50 = 0.322
lM), ni-
lM) and dimethylamino group (7,
l
l
l
l
l
l
l
l
l
l
bic activity is shown in the Figure 4.
The cytotoxicity of active compounds (2, 3, 6 and 7) was studied
by MTT assay36 on human breast cancer MCF-7 cell line A confluent
population of MCF-7 cells was treated with increasing concentra-
tions of compounds and the number of viable cells was measured
after 48 h by MTT cell viability assay based on mitochondrial
reduction of the yellow MTT tetrazolium dye to a highly colored
blue formazan product. The concentration range for all the com-
pounds (2, 3, 6, 7 & MNZ) is mentioned in Figure 5, which illus-
trates that all the active compounds and metronidazole were non
Figure 4. Relative antiamoebic activity of compound 2, 3, 6, 7, TSC and
metronidazole.
toxic in the concentration range of 2.5–100
lM, while they showed
cytotoxic effects above 250 M. Comparatively at 250
l
l
M concen-
tration all the four compounds were more cytotoxic than metroni-
dazole showing 70% viability. These results demonstrate that the
compounds 2, 3, 6, 7 and metronidazole were non-cytotoxic
against the human breast cancer MCF-7 cell line. At concentration
of 250
lM, compounds 2 and 7 showed 65% viability. On the other
hand, compounds 3 and 6 showed 60% viability at the concentra-
tion of 250
viability.
lM whereas metronidazole (MNZ) exhibited >70%
Figure 5. Cytotoxicity assessment by MTT assay.
In conclusion, a series of 1,2,4-triazole derivatives embedded
with thiosemicarbazone fragment were synthesized with an aim
to find novel antiamoebic agents. Incorporation of thiosemicarba-
zone fragment within the triazole ring enhances the antiamoebic
activity. Substitution at the fourth position of the aromatic ring
such as methyl, methoxy, nitro and dimethylamino group were
more potent inhibitors of E. histolytica than the reference drug met-
ronidazole (IC50 = 1.80 lM). Four compounds 2, 3, 6 and 7 were
more active than the standard drug metronidazole and all the ac-
tive compounds were non cytotoxic in the concentration range of
2.5–250 lM.
All the synthesized compounds gave satisfactory spectral data
for the proposed structures. The IR spectrum of compound (1)
exhibited characteristic strong absorptions at 3420 cmÀ1 and
2355 cmÀ1 due to NH and SH groups respectively. The absence of
NH stretching and presence of C@N stretching in the IR spectra of
compounds (2–11) established the conversion of –NH into its Schiff
base. Further, evidences in support of the proposed structures were
obtained by recording the proton NMR. In the 1H NMR spectra of
compounds (2–11), N@CH proton resonated as a singlet at d 9.21–
10.65 ppm, while the signal due to SH proton appeared as a singlet
at d 14.04–14.32 ppm. The signals due to the aromatic protons ap-
peared in the expected region. Although, 1,2,4-triazoles may exist
in thione-thiol tautomeric forms, yet our investigations demon-
strate that in this case all the synthesized compounds exist in thiol
form, as indicated by IR and 1H NMR data of compounds (1–11). All
the structures were further confirmed by 13C NMR and mass
spectral studies.
Acknowledgements
This work was supported by Council of Scientific and Industrial
Research (Grant # 01(2278)/08/EMR-II New Delhi, India) We
acknowledge the financial support for Shadab Miyan Siddiqui from
the University Grant Commission (UGC) and Attar Salahuddin from
CSIR India.
All the synthesized compounds (1–11) and thiosemicarbazide
were screened in vitro against HM1: IMSS strain of E. histolytica
by microdilution method.35 All the experiments were carried out
in triplicate at each concentration level and repeated thrice. Cyto-
toxicity of active compounds has been studied by MTT assay on hu-
man breast cancer MCF-7 cell line. The results of antiamoebic
activity and cytotoxicity are summarized in Table 1. The anti-
amoebic effect was compared with thiosemicarbazide and the
most widely used antiamoebic medication metronidazole (MNZ),
Supplementary data
Supplementary data (synthetic procedures, spectral data, anti-
amoebic assay and MTT assay) associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2012.02.084.
References and notes
which had a 50% inhibitory concentration (IC50) of 1.80
our experiments. The results showed that the triazole derivatives
(2–11) displayed IC50 values in the range 0.280–10.16 M. Biolog-
ical behavior revealed that the compounds which have methyl
lM in
1. Hung, C. C.; Chen, P. J.; Hsieh, S. M.; Wong, J. M.; Fang, C. T.; Chang, S. C.; Chen,
M. Y. AIDS 1999, 13, 2421.
2. Loftus, B.; Anderson, I.; Davies, R.; Alsmark, U. C.; Samuelson, J.; Amedeo, P.;
Roncaglia, P.; Berriman, M.; Hirt, R. P.; Mann, B. J.; Nozaki, T.; Suh, B.; Pop, M.;
l

S. M. Siddiqui et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2768–2771
2771
Duchene, M.; Ackers, J.; Tannich, E.; Leippe, M.; Hofer, M.; Bruchhaus, I.;
Willhoeft, U.; Bhattacharya, A.; Chillingworth, T.; Churcher, C.; Hance, Z.;
Harris, B.; Hance, D.; Jagels, K.; Moule, S.; Mungall, K.; Ormond, D.; Squares, R.;
Whitehead, S.; Quail, M. A.; Rabbinowitsch, E.; Norbertczak, H.; Prince, C.;
Wang, Z.; Guillen, N.; Gilchrist, C.; Stroup, S. E.; Bhattacharya, S.; Lohia, A.;
Foster, P. G.; Sicheritz-Ponten, T.; Weber, C.; Singh, U.; Mukherjee, C.; El-Sayed,
N. M.; Petri, W. A.; Clark, C. G.; Embley, T. M.; Barrell, B.; Fraser, C. M.; Hall, N.
Nature 2005, 24, 865.
21. (a) Sherman, L.; Edelstein, F.; Schtacher, G.; Avramoff, M.; Teitz, Y. J. Gen. Virol.
1980, 46, 195; (b) Ronen, D.; Nir, E.; Teitz, Y. Antiviral Res. 1985, 5, 249; (c) Biot,
C.; Pradines, B.; Sergeant, M. H.; Gut, J.; Rosenthal, P. J.; Chibale, K. Bioorg. Med.
Chem. Lett. 2007, 17, 6434.
22. (a) Rieder, H. L.; Arnadottir, T.; Trebucq, A.; Enarson, D. A. Int. J. Tuberc. Lung Dis.
2001, 5, 1; (b) Nunn, P.; Porter, J.; Winstanley, P. Trans. R. Soc. Trop. Med. Hyg.
1993, 87, 578; (c) Suzuki, Y.; Inagi, R.; Aono, T.; Yamanishi, K.; Shiohara, T. Arch
Dermatol. 1998, 134, 1108; (d) Descamps, V.; Valance, A.; Edlinger, C.; Fillet, A.
M.; Grossin, M.; Lebrun-Vignes, B.; Belaich, S.; Crickx, B. Arch. Dermatol. 2001,
137, 301; (e) Roujeau, J. C.; Kelly, J. P.; Naldi, L.; Rzany, B.; Stern, R. S.; Anderson,
T.; Auquier, A.; Bastuji-Garin, S.; Correia, O.; Locati, F. N. Engl. J. Med. 1995, 333,
1600; (f) Fujita, Y.; Yoshioka, N.; Abe, R.; Murata, J.; Hoshina, D.; Mae, H.;
Shimizu, H. J. Am. Acad. Dermatol. 2011, 65, 65; (g) Wolf, R.; Orion, E.; Marcos,
B.; Matz, H. Clin Dermatol 2005, 23, 171; (h) Roujeau, J. C. Toxicology 2005, 209,
123.
23. (a) Singh, S.; Bharti, N.; Gonzalez Garza, M. T.; Delia, E.; Cruz-Vega, E.; Garza, J.
C.; Saleem, K.; Naqvi, F.; Azam, A. Bioorg. Med. Chem. Lett. 2001, 11, 2675; (b)
Singh, S.; Bharti, N.; Naqvi, F.; Azam, A. Bioorg. Med. Chem. Lett. 2003, 13, 689;
(c) Singh, S.; Bharti, N.; Naqvi, F.; Azam, A. Eur. J. Med. Chem. 2004, 39, 459; (d)
Sharma, S.; Athar, F.; Maurya, M. R.; Naqvi, F.; Azam, A. Eur. J. Med. Chem. 2005,
40, 557; (e) Sharma, S.; Athar, F.; Maurya, M. R.; Azam, A. Eur. J. Med. Chem.
2005, 40, 1414; (f) Budakoti, A.; Abid, M.; Azam, A. Eur. J. Med. Chem. 2006, 41,
63; (g) Singh, S.; Athar, F.; Maurya, M. R.; Azam, A. Eur. J. Med. Chem. 2006, 41,
592; (h) Budakoti, A.; Abid, M.; Azam, A. Eur. J. Med. Chem. 2007, 42, 544; (i)
Bhat, A. R.; Athar, F.; Van Zyl, R. L.; Chen, C. T.; Azam, A. Chem. Biodiversity 2008,
5, 764; (j) Husain, K.; Bhat, A. R.; Azam, A. Eur. J. Med. Chem. 2008, 43, 2016; (k)
Husain, K.; Abid, M.; Azam, A. Eur. J. Med. Chem. 2008, 43, 39.
3. Singh, D.; Naik, S. R.; Naik, S. Indian J. Med. Res. 2004, 119, 157.
4. Agarwal, S. M.; Jain, R.; Bhattacharya, A.; Azam, A. Int. J. Parasito. 2008, 38, 137.
5. WHO/PAHO/UNESCO, Weekly Epidemiological Report of the World Health
Organization, 1997, 72, 97.
6. Walsh, J. A. Rev. Infect. Dis. 1996, 8, 228.
7. Van, S. J. H. Med. J. Aust. 2007, 186, 412.
8. Shailendra; Bharti, N.; Naqvi, F.; Azam, A. Bioorg. Med. Chem. Lett. 2003, 13, 689.
9. Pichardo, C. O.; Shibayama, M.; Treviño, S. V.; Alba, M. A.; Angeles, E.
Antimicrob. Agents Chemother. 2005, 49, 1160.
10. Bendesky, A.; Menendez, D.; Ostrosky-Wegman, P. Mutat. Res. 2002, 11, 133.
11. Domagk, G.; Behnish, R.; Mietzch, F.; Schmidt, H. Naturwissenshaften 1946, 33,
315.
12. Hamre, D.; Bernstein, J.; Donovick, R. Proc. Soc. Exp. Biol. Med. 1950, 73, 275.
13. (a) Wagner, W. H.; Winkelmann, E. Arzneimittelforschung 1972, 22, 1713; (b)
Dobek, A. S.; Klayman, D. L.; Dickson, E. T.; Scovill, J. P.; Tramont, E. C.
Antimicrob. Agents Chemother. 1980, 18, 27; (c) Dobek, A. S.; Klayman, D. L.;
Dickson, E. T.; Scovill, J. P.; Oster, C. N. Drug Res. 1983, 33, 1583.
14. Jensen, K. A.; Jensen, C. L. Acta Chem. Scand. 1952, 6, 957.
15. Klayman, D. L.; Scovill, J. P.; Bartosevich, J. F.; Mason, C. J. J. Med. Chem. 1979, 22,
1367.
24. Abid, M.; Azam, A. Eur. J. Med. Chem. 2005, 40, 935.
16. (a) French, F. A.; Blanz, E. J., Jr. J. Med. Chem. 1966, 9, 585; (b) Klayman, D. L.;
Scovill, J. P.; Mason, C. J.; Bartosevich, J. F.; Bruce, J.; Lin, A. J. Drug Res. 1983, 33,
909; (c) Karali, N. Eur. J. Med. Chem. 2002, 37, 909.
17. (a) Wilson, H. R.; Revankar, G. R.; Tolman, R. L. J. Med. Chem. 1974, 17, 760; (b)
Casero, R. A., Jr.; Klayman, D. L.; Childs, G. E.; Scovill, J. P.; Desjardins, R. E.
Antimicrob. Agents Chemothe. 1980, 18, 317.
18. (a) Teitz, Y.; Ronen, D.; Vansover, A.; Stematsky, T.; Riggs, J. L. Antiviral Res.
1994, 24, 305; (b) Mishra, V.; Pandeya, S. N.; Pannecouque, C.; Witvrouw, M.;
De Clercq, E. Arch. Pharm. Pharm. Med. Chem. 2002, 5, 183; (c) Bal, T. R.; Anand,
B.; Yogeeswari, P.; Sriram, D. Bioorg. Med. Chem. Lett. 2005, 15, 4451.
19. (a) Brockman, R. W.; Sidwell, R. W.; Arnett, G.; Shaddix, S. Proc. Soc. Exp. Biol.
Med. 1970, 133, 609; (b) Shipman, C., Jr.; Smith, S. H.; Drach, J. C.; Klayman, D. L.
Antimicrob. Agents Chemother. 1981, 19, 682; (c) Shipman, C., Jr.; Smith, S. H.;
Drach, J. C.; Klayman, D. L. Antiviral Res. 1986, 6, 197; (d) Altun, A.; Kumru, M.;
Dimoglo, A. J. Molec. Struct. (Teochem.) 2001, 535, 235.
25. Shailendra, S.; Husain, K.; Athar, F.; Azam, A. Eur. J. Pharm. Sci. 2005, 25, 255.
26. Abid, M.; Azam, A. Bioorg. Med. Chem. Lett. 2006, 16, 2812.
27. Bharti, N.; Husain, K.; Gonzalez Garza, M. T.; Cruz-Vega, D. E.; Castro-Garza, J.;
Mata-Cardenas, B. D.; Naqvi, F.; Azam, A. Bioorg. Med. Chem. Lett. 2002, 12,
3475.
28. Shujuan, S.; Hongxiang, L.; Gao, Y.; Fan, P.; Ma, B.; Ge, W.; Wang, X. J. Pharm.
Miomed. Anal. 2004, 34, 1117.
29. Barbuceanu, S.; Almajan, L. G.; Saramet, I.; Draghici, C.; Socoteanu, R.;
Barbuceanu, F. J. Serb. Chem. Soc. 2009, 74, 1041.
30. Castellano, S.; Stefancich, G.; Chillotti, A.; Poni, G., II Farmaco 2003, 58, 563.
31. Clemons, M.; Coleman, R. E.; Verma, S. Cancer Treat. Rev. 2004, 30, 325.
32. Johnston, G. A. R. Curr. Top. Med. Chem. 2002, 2, 903.
33. Mandrioli, R.; Mercolini, L.; Raggi, M. A. Curr. Drug Metab. 2008, 9, 827.
34. Reid, J. R.; Heidel, N. D. J. Het. Chem. 1976, 13, 925.
35. Wright, C. W.; O’Neill, M. J.; Phillipson, J. D.; Warhurst, D. C. Antimicrob. Agents
Chemother. 1988, 32, 1725.
36. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
20. (a) Sidwell, R. W.; Arnett, G.; Dixon, G. J.; Schabel, F. M., Jr. Proc. Soc. Exp. Biol.
Med. 1969, 131, 1223; (b) Sidwell, R. W.; Arnett, G.; Schabel, F. M., Jr.
Chemotherapy 1972, 17, 259.