Anticancer screening and structure activity relationship study of some semicarbazides and 1,2,4-Triazolin-5-ones

Article abstract of DOI:10.2174/157018012800673047

In this paper some semicarbazide and 1,2,4-triazolin-5-one derivatives are evaluated in vitro for their anticancer activity towards human tumour cell line: ovary (TOV 112D), lung (A 549), breast (T47D) and uterus (Hela). Compounds 2-4 have been found to s

Full text of DOI:10.2174/157018012800673047

568
Letters in Drug Design & Discovery, 2012, 9, 568-572
Anticancer Screening and Structure Activity Relationship Study of Some
Semicarbazides and 1,2,4-Triazolin-5-ones
Monika Pitucha*,a and Jolanta Rzymowskab
aDepartment of Organic Chemistry, Faculty of Pharmacy, Medical University, Chodźki 4a, 20-093 Lublin, Poland
bDepartment of Biology and Genetics, Faculty of Pharmacy, Medical University, Chodźki 4a, 20-093 Lublin, Poland
Received January 30, 2012: Revised February 20, 2012: Accepted February 21, 2012
Abstract: In this paper some semicarbazide and 1,2,4-triazolin-5-one derivatives are evaluated in vitro for their anticancer
activity towards human tumour cell line: ovary (TOV 112D), lung (A 549), breast (T47D) and uterus (Hela). Compounds
2-4 have been found to show the most effective in vitro activity against ovary cancer cell line. For compound 4 the growth
inhibition (80% and 70%) is observed in two concentrations (100 µg/ml and 50 µg/ml). The cytotoxic effect of examined
compounds seems to be dose-dependent and time-dependent. Compound 7 has GI=80% values towards the breast cancer
cell line in concentration of 100 µg/ml. Structure-activity relationship (SAR) studies are carried out for all the compounds
of the series. Compounds 2-4 show an activity profile that can be improved through medicinal chemistry strategies.
Keywords: Anticancer activity, Cytotoxicity, Isocyanate, Semicarbazide, Synthesis, 1,2,4-Triazol-5-one.
1. INTRODUCTION
1-methylpyrrole-2-carboxamide
moieties
linked
to
guanidinoacetamidino and amidinopropyl [20].
Cancer is an important issue of clinical medicine and
pharmacology. At present, a wide range of cytotoxic drugs
with different mechanisms of action are used to treat human
cancer, either alone or in combination. Many compounds are
in different phases of clinical trials. The main drawback of
the cytotoxic drugs is that they do not discriminate between
cancerous and normal cell types and are accompanied by
toxic side effects that are often cumulative and are also
accompanied by doses limiting.
In the previous paper 1,6-bis(1,2,4-triazol-5-one)hexane
derivatives were screened in vitro for their antiproliferative
and anticancer activity in human tumor cell lines derived
from breast and lung carcinoma cells. Some of them were
found to be moderate effective against tested cell line in
concentration 50 µg/ml [21]. In continuation of our research
in the domain of heterocyclic compounds herein,
semicarbazide and 1,2,4-triazolin-5-one derivatives are
tested in vitro against human ovary, lung, breast and uterus
cancer cell lines. Additionally, the geometry of all
compounds is optimized by the semi-empirical (AM1)
method. Selected descriptors are applied for SAR studies
which provides indications for the synthesis of new
potentially active compounds.
Recently, 1,2,4-triazole and its derivatives have attracted
attention as anticancer agents [1-5]. It was reported that
compounds having triazole moieties, such as Verozole,
Letrozole and Anastrozole, appear to be very effective
aromatase inhibitors that are also very useful for preventing
breast cancer [6-10]. Some triazoles can inhibit HER2
tyrosine kinase phosphorylation in the breast cancer cell
[11,12]. Semicarbazides, linear analogues, commonly
applied in the 1,2,4-triazole synthesis, are also an important
class of compounds of diverse biological properties [13,14].
Some of them exhibit a promising cytotoxic activity profile
[15,16].
2. MATERIAL AND METHOD
2.1. Chemistry
Preparation, physicochemical and spectroscopic charac-
terization of the compounds 1-7 have been described
elsewhere. All compounds were characterized by elemental
analysis and spectroscopic methods and the data was
described in the previous papers [22,23].
On the other hand, pyrrole and its derivatives have shown
to possess antitumor activity [17]. 2,4-Disubstituted
brominated pyrroles, which are brominated at position 3 and
5 along with 2,3,4-trisubstituted pyrroles have demonstrated
potent cytotoxic activity in vitro against a variety of murine
and human suspended and solid tumor models [18].
Distamycin is a well known antibiotic, which contains three
pyrrole rings and can selectively recognize AT region in
DNA minor groove [19]. Netropsin is a basic oligopeptide
isolated from Streptomyces netropsis which is composed of
2.1.1. 4-Ethyl-1[(1-methylpyrrol-2-yl)acetyl]semicarbazide
(1)
Yield: 78%; M.p: 165-166 °C [22].
2.1.2. 4-(4-Bromophenyl)-1-[(1-methylpyrrol-2-yl)acetyl]
semicarbazide (2)
Yield: 86%; M.p: 200-201 °C [22].
2.1.3. 4-Ethyl-3-[(1-methylpyrrol-2-yl)methyl]-1,2,4-triazo-
lin-5-one (3)
*Address correspondence to this author at the Department of Organic
Chemistry, Faculty of Pharmacy, Medical University, Chodźki 4a Str., 20-
093 Lublin, Poland; Tel: 48-81-535-73-74; Fax: 48-81-535-73-74;
E-mail: monika.pitucha@umlub.pl
Yield: 90%; M.p: 150-151 °C [22].
1875-628X/12 $58.00+.00
© 2012 Bentham Science Publishers

Anticancer Screening of Semicarbazides and Triazoles
Letters in Drug Design & Discovery, 2012, Vol. 9, No. 6 569
2.1.4. 4-(4-Bromophenyl)-3-[(1-methylpyrrol-2-yl)methyl]-
1,2,4-triazolin-5-one (4)
incubated for 24, 48 and 72 h. End-point determinations
were made with 5-bromo-2-deoxy-uridine (BrdU) labeling
and detection kit III (Roche) on Elisa reader (BIO-TEC
Instruments USA). The growth percentage was evaluated
spectrophotometrical versus untreated controls using cell
Yield: 89%; M.p: 180-181 °C [22].
2.1.5. 4,4’-Bis(3-methyl-1,2,4-triazolin-5-on-4-yl)diphenyl-
methane (5)
viability
of
growth
assay.
Results
for
each
spectrophotometrical measure were reported as percent of
growth inhibition in comparison to untreated ones. All
experiments were done in triplicate.
Yield: 83%; M.p: 308-310 °C [23].
2.1.6. 4,4’-bis[3-(1-methylpyrrol-2-yl)methyl-1,2,4-triazo-
lin-5-on-4-yl]diphenylmethane
Yield: 81%; M.p: 225-227 °C [23].
2.3. Computational Methods
2.1.7. 4,4’-Bis(3-benzyl-1,2,4-triazolin-5-on-4-yl)diphenyl-
methane (7)
The molecular modeling was performed using
HYPERCHEM software packet and VEGA ZZ [24,25]. The
energy and geometry of the derivatives were optimized by
the AM1 semi-empirical method [26]. In order to perform
structure-activity relationship (SAR) studies, some
descriptors were calculated: the highest occupied molecular
orbital energy (EHUMO), the lowest occupied molecular
orbital energy (ELUMO), dipole moment (µ), Polar Surface
Area (PSA), polarizability (P), refractivity (R) and logarithm
of n-octanol/water partition coefficient (logP).
Yield: 69%; M.p: 120-121 °C [23].
2.2. Inhibition of Tumour Cell Growth Assay
The semicarbazides 1, 2 and 1,2,4-triazol-5-ones 3-7
were evaluated in vitro for their anticancer activity in human
tumour cell line: human ovary carcinoma (TOV 112D -
ATCC CRL-11731), human lung carcinoma (A 549 -
ECACC 86012804 ), human breast carcinoma (T47D -
ECACC 85102201) and human uterus carcinoma (Hela -
ECACC 93021013). Additionally, the primary cell line to
normal human skin fibroblasts (HSF) was used in this
experiment.
In the current protocol each cell line was inoculated at l04
cells per ml density on a microlitre plate at RPMI medium.
Test compounds were then added at three examined
concentrations (10, 50 and 100 µg/ml) and cultures were
3. RESULTS AND DISCUSSION
The investigated semicarbazides 1 and 2 were obtained in
the reaction of 1-methylpyrrol-2-ylacetic acid hydrazide with
ethyl and 4-bromophenyl isocyanate respectively. The
cyclization reaction of these compounds led to 1,2,4-
triazolin-5-ones 3, 4 [22]. Compounds 5-7 were obtained in
the intermolecular dehydrative cyclization of appropriate bis-
R1
O
O
R1NCO
a
H
H
N
b,c
N
NH2
N
R
O
R
N
R
N
R1
H
H
N
NH
3,4
O
1,2
MDI a
O
O
H
H
H
H
N
N
N
N
R
N
N
R
H
H
O
O
b,c
R
R
N
N
HN
NH
N
N
O
O
5-7
For 1, 3: R= 1-methylpyrrol-2-ylmethyl, R1= C2H5
For 2, 4: R= 1-methylpyrrol-2ylmethyl, R1= 4-BrC6H4
For 5: R= CH3
For 6: R= 1-methylpyrrol-2-ylmethyl
For 7: R= CH2C6H5
Scheme 1. Synthesis of semicarbazide and 1,2,4-triazolin-5-one derivatives.
Reagents and conditions: a) diethyl ether, room temperature; b) 2% NaOH, reflux: c) 3M HCl.

570 Letters in Drug Design & Discovery, 2012, Vol. 9, No. 6
Pitucha and Rzymowska
Table 1. Inhibition of Cancer Cell Line In Vitro by Selected Compounds at Concentrations 100 µg/ml and 50 µg/ml
Time of incubation (h)
Growth inhibition factor
GI (%)
4
Compound
2
3
6
7
Doses
Cell line
I
II
0
I
II
0
I
II
0
I
II
0
I
II
0
Normal cell line
HSF
24
0
0
10
0
10
48
72
0
0
0
0
0
0
1
1
0
0
0
0
0
0
0
0
10
10
0
0
Cancer cell line
TOV 112D
24
48
72
24
48
72
24
48
72
20
30
30
0
50
75
85
5
10
15
50
0
45
50
70
0
50
75
80
10
25
25
3
25
50
70
5
0
0
15
15
5
0
0
5
25
50
10
25
50
10
15
25
50
50
5
5
A549
20
2
0
5
0
5
10
15
1
5
5
5
5
0
5
15
0
10
10
20
80
10
0
T 47D
5
0
5
0
5
0
10
10
5
5
1
0
0
0
0
5
10
2
0
0
I-Concentration of 100 µg/ml, which corresponds to concentration of 0.28 mM for compound 2, 0.48 mM for compound 3, 0.30 mM for compound 4, 0.19 mM for compound 6, and
0.24 mM for compound 7.
II-Concentration of 50 µg/ml, which corresponds to concentration of 0.14 mM for compound 2, 0.24 mM for compound 3, 0.15 mM for compound 4, 0.09 mM for compound 6, and
0.12 mM for compound 7.
semicarbazides [23]. The synthetic pathway followed for the
preparation of the title compounds was presented in Scheme
1.
1and 5 proved to be inactive against tested cell line in vitro
(GI < 50%). Additionally, all compounds exhibited no
cytotoxic activity against human uterus carcinoma (Hela)
cell lines (GI≤25%) and no or low toxicity against normal
fibroblast cultures (HSF) with GI≤15%.
All compounds were evaluated in vitro for their cytotoxic
activity against four cell lines: human ovary carcinoma
(TOV 112D), human lung carcinoma (A 549), human breast
carcinoma (T47D) and human uterus carcinoma (Hela).
Beside human skin fibroblast (HSF) was included in the
cytotoxicity study. Compounds were tested at three
concentrations (100, 50, 10 µg/ml). Results for each test
compound were reported as the percentage of growth (GI %)
of the treated cells compared to the untreated control cells.
According to the data listed in Table 1 compounds 2-4, 6 and
7 have potential to reduce the growth of the ovary cancer cell
line (TOV 112D). For compounds 2-4 growth inhibitory
values towards ovary cancer cell line at two concentrations
(100 and 50 µg/ml) were found to be the most effective
against this cancer line and their factors were in the range
50-85% (Fig. 1). Cytotoxic effect of the compound 4
towards cancer ovary cell line seemed to be time and dose-
dependent (Fig. 2). The highest inhibition was observed for
compound 2 (GI=85%) after 72h on incubation. Only for
compounds 4 inhibition in concentration 10 µg/ml (GI=10%)
was observed. Compounds 6 and 7 show 50% inhibition at
concentration 100 µg/ml. Compound 6 show tumor cell
growth inhibition against lung cancer line (A 549) in
concentration 100 µg/ml (GI=50%). Only compound 7 was
found the most active against breast cancer line (T 47D); its
GI was 80% in concentration 100 µg/ml. Whereas compound
For studies of structure-activity analysis some descriptor
were calculated (Table 2). An important descriptor is the
lipophilicity log P which determines the capability of
biological active substances to pass through biological
membranes. Lipohilicity is directly connected with the Polar
Surface Area (PSA). Molecules with a polar surface area
greater than 140 Å2 permeate cell membranes to the lesser
extent. The tested compounds had PSA values ranged from
52.68 Å2 to 115.26 Å2. For compounds 3 and 4 showing the
highest proliferative activity against ovarian cancer cells,
these values were the lowest (52.68 Å2 and 52.98 Å2).
However, the biologically active compound
2
was
characterized by a high PSA values (115.26 Å2). The log P
were in the range from -1.95 (for compound 2) to 2.30 (for
compound 6). It seems that the log P and PSA are not
decisive factors for the antitumor activity of tested
compounds, similarly as volume, molar mass and dipole
moment (Table 2). Analysis of LUMO and HUMO energies
indicates that the studied compounds are stable. The SAR
study revealed that anticancer activity against ovary
carcinoma cell line of tested compounds depends on the
nature of a substituent. Among the semicarbazides the
4-bromophenyl moiety was more favorable than the ethyl

Anticancer Screening of Semicarbazides and Triazoles
Letters in Drug Design & Discovery, 2012, Vol. 9, No. 6 571
90
80
70
60
50
40
30
20
10
100 µg/ml
50 µg/ml
10 µg/ml
0
2
3
4
6
7
Compound
Fig. (1). Inhibition of in vitro human ovary cell line (TOV 112D) growth (expressed as GI percentage) by the most active compounds in three
examined concentrations (100 µg/ml, 50 µg/ml and 10 µg/ml) after 72h incubation.
80
70
60
50
100 µg/ml
50 µg/ml
10 µg/ml
40
30
20
10
0
24
48
72
Time of incubation (h)
Fig. (2). Inhibition of in vitro human ovary cell line (TOV 112D) growth (expressed as GI percentage) by tested compound 4 in three
examined concentrations (100 µg/ml, 50 µg/ml and 10 µg/ml) and time of incubation.
Table 2. Physicochemical and Topological Parameters of Compounds 1-7
No
PSA [Å2]
V [Å3]
M [amu]
µ [D]
logP
P [A3]
R [A3]
ELUMO [eV]
EHOMO [eV]
DELH
[eV]
1
2
3
4
5
6
7
77.49
115.26
52.98
52.68
98.33
96.69
103.78
267.96
210.64
247.84
193.68
318.58
463.37
469.21
351.20
224.26
333.19
206.25
362.39
514.59
520.59
1.92
2.03
4.34
3.65
3.65
4.12
5.99
-1.75
-1.95
1.66
32.28
23.67
30.88
22.26
38.84
58.16
57.70
88.88
61.51
-0.278
-0.605
-0.453
0.485
-8.688
-8.565
-8.735
-8.892
-8.991
-8.968
-8.603
8.410
7.960
8.282
9.377
8.693
8.697
8.329
82.32
-0.47
0.19
59.67
107.50
165.06
159.83
-0.298
-0.271
-0.274
2.30
-0.20
group. In case of cyclic derivatives mono-triazoles turned
out to be more active than bis-triazoles.
carcinoma (A 549), breast carcinoma (T47D) and uterus
carcinoma (Hela) cell lines. The preliminary results showed
that some of these excellent growth inhibition activity
against ovary, breast and lung cancer cell line. Compounds
2-4 were found be the most effective in vitro against ovary
cancer cell line in two examined concentrations. Compound
7 had comparable GI values towards the breast cancer cell
CONCLUSION
Some semicarbazides and 1,2,4-triazolin-5-ones were
tested in vitro against ovary carcinoma (TOV 112D), lung

572 Letters in Drug Design & Discovery, 2012, Vol. 9, No. 6
Pitucha and Rzymowska
[10]
[11]
[12]
Recanatini, M.; Vavalli, A.; Valenti, P. Nonsteroidal aromatase
inhibitors: recent advances. Med. Res. Rev., 2002, 22, 282-304.
Zwick, E., Bange, J., Ullrich, A. Receptor tyrosine kinases as
targets for anticancer drugs. Trends Mol. Med., 2002, 8, 17-23.
Cheng, Z.Y.; Li, W.J.; He, F.;. Zhou, J.M.; Zhu, X.F. Synthesis and
biological evaluation of 4-aryl-5-cyano-2H-1,2,3-triazoles as
inhibitor of HER2 tyrosine kinase. Bioorg. Med. Chem., 2007, 15,
1533-1538.
Holla, B.S.; Gonsalves, R.; Shenoy, S. Synthesis and antibacterial
studies of a new series of 1,2-bis(1,3,4-oxadiazol-2-yl)ethanes and
1,2-bis(4-amino-1,2,4-triazol-3-yl)ethanes. Eur. J. Med. Chem.,
2000, 35, 267-271.
El-Hawash, S.A.M.; El-Mallah, A.I. Synthesis of some novel
pyrazole derivatives as a potential antiinflammatory agents with
minimum ulcerogenic activity. Pharmazie, 1998, 53, 368-373.
El-Sadek, M.E.; Aboukull, M.E.; El-Sabbagh, O.I.; Shallal M.H.
Design, synthesis and cytotoxic activity of novel 1-aroyl-4(2-
chloroethyl)semicarbazides. Pharmaceutical Chemistry Journal,
2007, 41, 188-192
line in concentration of 100 µg/ml. All compounds showed
no or low cytotoxic activity against human utereus cell line
and human ski fibroblast cells-primary cancer cells. The
obtained results prove the necessity for further investigations
to clarify the features underlying the antitumor potential of
tested compounds. The most effective compounds have been
selected for further studies. These results provide useful
information about mechanisms of action of above-mentioned
compound. The preliminary study on the molecular
mechanism of activity of tested compounds shows that the
anticancer activity of derivatives results from the
competitive inhibition of receptor tyrosine kinases.
[13]
[14]
[15]
CONFLICT OF INTEREST
[16]
Zining, C.; Yun, L.; Baoju, L.; Yongqiang, L.; Changhui, R.;
Jingrong, C.; Yanxia, S.; Xinling, Y. Synthesis and bioactivity of
Declared none.
N-benzoyl-N'-[5-(2'-substitutedphenyl)-2-furoyl]
derivatives. Molecules, 2010, 15, 4267-4282.
Halazy, S.; Magnus, P. Tetrahedron Lett., 1984, 25, 1421-1424.
Blasko, A.; Browne, K.A.; Bruice, T.C. J. Am. Chem. Soc,. 1994,
116, 3726.
semicarbazide
ACKNOWLEDGEMENTS
[17]
[18]
Declared none.
[19]
[20]
Finlay, A.C.; Hochstein, F.A.; Sobin, B.A.; Murphy, F.X. J. Am.
Chem Soc. 1951, 73, 341.
Hahn, F.E. Antibiotics III. Mechanism of Action of Antimicrobial
and Antitumor Agents; Springer-Verlag, New York, 1975, pp. 79-
100.
REFERENCES
[1]
Demirbaş, A.; Ugurluoglu, R.; Demirbaş, A. Synthesis of 3-
alkyl(aryl)-4-alkylidenamino-4,5-dihydro-1H-1,2,4-triazol-5-ones
and 3-alkyl-4-alkylamino-4,5-dihydro-1H-1,2,4-triazol-5-ones as
antitumor agents. Bioorg. Med. Chem., 2002, 10, 3717-3723.
Holla, B.S.; Poojary, K.N.; Rao, B.S.; Shivananda, M.K. New bis-
aminomercapto triazoles and bis-triazolothiadiazoles as possible
anticancer agents. Eur. J. Med. Chem., 2002, 37, 511-517.
Al-Soud, Y.A.; Al-Dweri, M.N.; Al-Masoudi, N.A. Synthesis,
antitumor and antiviral properties of some 1,2,4-triazole
derivatives. Il Farmaco, 2004, 59, 775-783.
[21]
Pitucha, M.; Rzymowska, J.; Olender, A.; Grzybowska-
Szatkowska, L. Synthesis of 1,6-hexanediyl-bis(semicarbazides)
and
1,6-hexanediyl-bis(1,2,4-triazol-5-ones)
and
their
[2]
[3]
[4]
antiproliferative and antimicrobial activity. J. Chem. Serb. Soc.
2012, 77(1), 1-8.
Pitucha, M.; Polak, B.; Świeboda, R.; Kosikowska, U.; Malm, A.
Determination of the lipophilicity of some new derivatives of
semicarbazide and 1,2,4-triazol-5-one with potential antibacterial
activity. Z. Naturforsch. 2009, 46b, 570-576.
Pitucha, M.; Borowski, P.; Karczmarzyk, Z.; Fruziński, A.
Synthesis, experimental and theoretical investigations of some new
4,4’-bis(3-substituted-4,5-dihydro-1H-1,2,4-triazol-5-one-4-
yl)diphenylmethane. J. Mol. Struct., 2009, 919, 170-177.
HyperChem 7.0.3 for Windows, HyperCube, Inc. Gainsville, FL,
2007.
Pedretti, A.; Villa, L.; Vistoli, G. VEGA – an Open Platform to
Develop Chemo-Bio-Informatics Applications, Usig Plug in
Architecture nad Script Programming. J.C.A.M.D. 2004, 18, 167-
173.
[22]
[23]
Chen S.; Guo C.; Shi S.; Shi Y.; Fang D.; Fan H. Design, synthesis
and pharmacokinetic evaluation of a Novel series of triazole-based
Src kinase Inhibitors with anti-proliferative activity. Letters in
Drug Design & Discovery, 2011, 8(1), 9-13.
[5]
Murty, M.S.R.; Ram, K.R.; Rao, R.V.; Yadav, J.S.; Rao, J.V.,
Velatooru, L.R. Synthesis of new S-alkylated-3-mercapto-1,2,4-
triazole derivatives bearing cyclic amine moiety as potent
anticancer agents. Lett. Drug Des. Discov., 2012, 9(3), 276-281.
Goss, P.E.; Strasser, W.K. Aromatase inhibitors in the treatment
and prevention of breast cancer. J. Clin. Oncol, 2001, 19, 881-894.
Santen, J.R. Inhibition of aromatase: insights from recent studies.
Steroids, 2003, 68, 559-567.
[24]
[25]
[6]
[7]
[8]
[26]
Dewar, M.J.S; Zoebisch, E.G.; Healy, E.F.; Stewart J.J.P.
Development and use of quantum mechanical molecular models.
76. AM1: a new general purpose quantum mechanical molecular
model. J. Am. Chem. Soc., 1985, 107(13), 3902–3909.
Clemons, M.; Coleman, R.E.; Verma, S. Aromatase inhibitors in
the adjuvant setting: bringing the gold to a standard? Cancer Treat.
Rev., 2004, 30, 325-332.
[9]
Chen, S.; Kao, Y.C.; Laughton, C.A. Binding characteristics of
aromatase inhibitors and phytoestrogens to human aromatase. J.
Steroid Biochem., 1997, 61, 107-115.