Synthesis, characterization and in vitro anticancer evaluation of novel 1,2,4-triazolin-3-one derivatives

Article abstract of DOI:10.1016/j.ejmech.2013.01.004

A series of novel 2-(4-chlorophenyl)-5-methyl-4-(2-amine/oxy-ethyl)-2,4- dihydro-[1,2,4]triazol-3-one (5a-t) were synthesized and in vitro anticancerous action of the resulting compounds was studied against NCI-60 Human Tumor Cell Line at a single high do

Full text of DOI:10.1016/j.ejmech.2013.01.004

Accepted Manuscript
Synthesis, characterization and in vitro anticancer evaluation of novel 1,2,4-
triazolin-3-one derivatives
Pramod P. Kattimani, Ravindra R. Kamble, Mahadevappa Y. Kariduraganavar,
Atukuri Dorababu, Ravindra K. Hunnur
PII:
S0223-5234(13)00012-3
10.1016/j.ejmech.2013.01.004
EJMECH 5927
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 September 2012
Revised Date: 31 December 2012
Accepted Date: 3 January 2013
Please cite this article as: P.P. Kattimani, R.R. Kamble, M.Y. Kariduraganavar, A. Dorababu, R.K.
Hunnur, Synthesis, characterization and in vitro anticancer evaluation of novel 1,2,4-triazolin-3-one
derivatives, European Journal of Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.01.004.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Synthesis, characterization and in vitro anticancer evaluation of
novel 1,2,4-triazolin-3-one derivatives
Pramod P. Kattimani, Ravindra R. Kamble, Mahadevappa Y. Kariduraganavar, Atukuri
Dorababu, Ravindra K. Hunnur
A series of novel 1,2,4-triazol-3-one appended to different heterocyclic/aryl moieties (5a-t)
were synthesized and in vitro anticancerous action was studied against NCI-60 Human
Tumor Cell Lines. The compound 5g comprising 1,2,4-triazolin-3-one appended to 4-
methylcoumarin ring has shown potent anticancer activity against various cell lines.
O
R
H
R
N
N
heterocycle/
aryl group
N
N
N
O
O
5a-t
1

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Highlights
• Novel 1,2,4-triazolin-3-one derivatized with various heterocycles/aryl groups were
synthesized.
• In vitro anticancer activity against NCI-60 human tumor cell lines was done.
• The compound 5g comprising 4-methylcoumarin ring has shown potent anticancer activity.

ACCEPTED MANUSCRIPT
Synthesis, characterization and in vitro anticancer evaluation of
novel 1,2,4-triazolin-3-one derivatives
Pramod P. Kattimania, Ravindra R. Kamblea*, Mahadevappa Y. Kariduraganavara, Atukuri
Dorababua, Ravindra K. Hunnurb
a Department of Studies in Chemistry, Karnatak University, Pavate Nagar, Dharwad-580003,
India
b APL Research Ltd., Batchupally, Hyderabad-500090, India
Corresponding author: Tel: +91-836-2215286, Extn-34; Fax: +91-836-2771275
E-mail: kamchem9@gmail.com
A B S T R A C T
Keywords:
A series of novel 2-(4-chlorophenyl)-5-methyl-4-(2-amine/oxy-ethyl)-2,4-
Cancer, 1,2,4- dihydro-[1,2,4]triazol-3-one (5a-t) were synthesized and the in vitro
triazole,
sydnone,
anticancer
activity
anticancerous action of the resulting compounds was studied against NCI-60
Human Tumor Cell Line at a single high dose (10-5 M) concentration for
primary cytotoxicity assay. Among the tested compounds (5a-e, 5g-h, 5k, 5p),
the compound 5g (NSC: 761736/1) was further evaluated for five dose criteria
at five different minimal concentrations against the full panel of 60 human
tumor cell lines which exhibited activity against Leukemia (GI50: 1.10 ꢀM),
Non-Small Cell Lung Cancer (GI50: 1.00 ꢀM), Renal Cancer (GI50: 1.00 ꢀM),
Colon Cancer (GI50: 1.66 ꢀM), CNS Cancer (GI50: 1.36 ꢀM), Melanoma (GI50:
1.82 ꢀM), Ovarian Cancer (GI50: 1.64 ꢀM) and Breast Cancer (GI50: 1.69
ꢀM).
1. Introduction
Cancer still remains a potentially life threatening disease and the number of cancer
related deaths are increasing alarmingly. Literature clearly indicated that more than 90% of
cancer patients die due to chronic tumor metastases - the spread and invasion of other organs
[1]. Considering this, the development of newer chemotherapeutic scaffolds which selectively
act on the target without side effects has became a primary objective of medicinal chemists.
Recently, 1,2,4-triazole derivatives have been incorporated into wide variety of therapeutically
interesting molecules to transform them into better drugs and exhibited significant biological
activity for wide range of therapeutic properties viz., antimicrobial [2], anti-tumour [3],
antiinflammatory [4], antihypertensive [5], analgesic [6], anticonvulsant [7], antiviral [8],
antidepressant [9], antitubercular [10], sedative [11] and plant growth regulatory activities [12].
Several compounds possessing 1,2,4-triazole nucleus are proved as drugs such as ribavirin
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(antiviral agent) [13], terconazole and flucanozole (antifungal agent) [14]. The heterocyclic
derivatives of 1,2,4-triazole exhibit a broad spectrum of pharmacological properties, for
example 1,2,4-triazoles bearing coumarin, quinoline and morpholine [15-17] Fig. 1, have been
reported as potential antimicrobial agents. Itraconazole, a potent antifungal agent, possess
1,2,4-triazole bearing piperazine substituent [18].
We opted to synthesize 1,2,4-triazolin-3-one derivatives (5a-t) from 3-arylsydnone (1)
as it is proved to be useful precursor since it undergoes ring transformation via 1,3-dipolar
cycloaddition reaction into various pharmacologically active heterocycles [19-22]. Also, we
have recently demonstrated the anticancer activity of carbazole derivatives synthesized from 3-
arylsydnone as synthon [23]. All these factors prompted us to synthesize 1,2,4-triazolin-3-ones
appended to different bio-potent moieties in order to explore their in vitro anticancer activity
against 60 human cancer cell lines at National Cancer Institute (NCI), National Institute of
Health (NIH), Bethesda, USA.
2. Results and discussion
2.1. Chemistry
Initially, N-(4-chlorophenyl)sydnone (1) was ring transformed into 3-(4-chlorophenyl)-
5-methyl-2-oxo-∆4-1,3,4-oxadiazole (2) which was further refluxed with ethanolamine to get 4-
(2-hydroxy-ethyl)-2-(4-chlorophenyl)-5-methyl-2,4-dihydro-3H-1,2,4-triazol-3-one (3) through
ring insertion. This (3) upon reaction with methanesulphonyl chloride in presence of
triethylamine gave the mesilate 4. Finally, the title compounds (5a-t) were obtained by reacting
mesilate (4) with various secondary amines and/or hydroxyl substituted heterocycles/aromatic
hydroxy compounds (a-t, Scheme 1) under mild basic condition. All the newly synthesized
compounds were characterized by IR, 1H and 13C NMR, mass spectral and elemental analyses.
In case of IR studies, all the compounds have shown a sharp intense band at 1698-1716 cm-1
which corresponds to carbonyl group of 1,2,4-triazolin-3-one ring. A medium intense band
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appeared around 1595-1624 cm-1 was attributed to C=N stretching. In case of 1H NMR, the C5
methyl of 1,2,4-triazolin-3-one ring appeared at 1.86-2.68 ppm. The protons of ethyl chain
resonated as two triplets in the upfield region viz., 2.60-4.60 ppm. The signals appeared at
6.61-8.95 ppm were attributed to aromatic protons. For 13C NMR, the C5 methyl carbon of
1,2,4-triazolin-3-one ring resonated in the range 10.86-12.39 ppm. The ethyl carbons showed
signals at 37.70-66.58 ppm. A signal resonated at 144-145 ppm was attributed to C5 carbon of
1,2,4-triazolin-3-one ring. The carbonyl carbon of 1,2,4-triazolin-3-one ring was resonated in
the range 149-152 ppm. The other rings attached to alkyl chain have shown signals at their
respective positions. The mass spectral analyses of all the title compounds have shown the m/z
values which corresponds to their molecular mass.
2.2. Pharmacology
2.2.1. Anticancer activity at a single high dose concentration (10-5M)
All the newly synthesized compounds were subjected to in vitro anticancer screening in
a single high dose (10-5 M) concentration against full 60 human cancer cell lines at NCI under
DTP drug discovery programme. Output from the single dose screen is reported as a graph of
mean growth percent of the treated cells. This allows detecting both growth inhibition values
(between 0 and 100) and cytotoxicity values (less than 0). The results of the single dose
screening were analyzed by COMPARE program [24]. Compounds 5a (NSC: 761735/1), 5b
(NSC: 761741/1), 5c (NSC: 761737/1), 5d (NSC: 761740/1), 5e (NSC: 761739/1), 5g (NSC:
761736/1), 5h (NSC: 761742/1), 5k (NSC: 765439/1), 5p (NSC: 765438/1) have been
screened for single high dose (10-5 M) concentration on all the 60 human cancer cell lines
organized into nine sub-panels derived from nine different human cancer types: leukemia, lung,
colon, CNS, melanoma, ovarian, renal, prostate and breast cancer cell lines. The following are
the percentage growth inhibition (GI%) of the treated cells at 10-5 M concentration with the
compound 5b (comprising 1,2,4-triazolin-3-one bearing morpholine substituent): CNS Cancer
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SNB-75 (GI% 92.35); Colon cancer (GI% 70.54); compound 5g (comprising 1,2,4-triazolin-3-
one bearing 4-methylcoumarin substituent, Table 1): Colon Cancer HT 29 (GI% 99.30),
COLO 205 (GI% 89.42), HCT 116 (GI% 84.88), HCT 15 (82.26); Melanoma SK-MEL-2
(GI% 96.45), SK-MEL-5 (GI% 89.24), M14 (GI% 85.28), UACC-257 (GI% 77.48); Leukemia
CCRF-CEM (GI% 86.59), K-562 (GI% 83.73), SR 21.22 (GI% 78.78); Non-Small Cell Lung
Cancer NCI-H460 (GI% 82.79); CNS Cancer SF-295 (GI% 85.97); Ovarian Cancer OVCAR-
3 (GI% 85.18), OVCAR-8 (GI% 82.32); Renal Cancer CAKI-1 (GI% 80.52); Prostate Cancer
DU-145 (GI% 78.36) and Breast Cancer MCF7 (GI% 84.21), MDA-MB-468 (GI% 83.92) and
has shown cytotoxic effect against Leukaemia HL-60 (TB) and Melanoma MDA-MB-435
cancer cell lines. All other compounds (5a, 5c-e, 5h, 5k, 5p) have shown negligible GI%.
2.2.2. Anticancer activity at five dose concentrations
The methodology followed comprised in vitro screening of the compounds against full
60 cell lines divided into nine sub panels at five different minimal concentrations (0.01, 0.1, 1,
10 and 100 ꢀM). The percentage of growth was evaluated spectrophotometrically versus
controls not treated with test agents. The 48 h continuous drug exposure protocol was followed
and SRB (sulforodamine B) protein assay was used to estimate cell viability or growth. The
results were represented by graph of log concentration vs GI% and three dose response
parameters (GI50, TGI and LC50) were calculated for each cell line. The GI50 value (growth
inhibitory activity) corresponds to the molar concentration of the compound that inhibits 50%
net cell growth; TGI value (cytostatic activity) is the molar concentration of the compound
resulting in total growth inhibition and LC50 value (cytotoxic activity) is the molar
concentration of the compound causing 50% net cell death. Furthermore, mean graph
midpoints (MGMID) were calculated for GI50 parameter, giving an average activity parameter
over all cell panels for tested compounds.
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Among the tested compounds viz., 5a-e, 5g-h, 5k, 5p the derivative possessing 4-
methylcoumarin ring (5g) has shown significant growth inhibition against variety of cell lines
at a single high dose (10-5 M) concentration and has been further evaluated for five dose
screening at five different minimal concentrations. The compound 5g has exhibited broad
spectrum of growth inhibition activity against eight tumor cell lines with average GI50 values
(MGMID) 2.97-3.65 ꢀM namely, Leukemia K-562 (GI50: 1.29 ꢀM), RPMI-8226 (GI50: 1.10
ꢀM); Non-Small Cell Lung Cancer EKVX (GI50: 1.00 ꢀM), HOP-92 (GI50: 1.61 ꢀM); Colon
Cancer KM 12 (GI50: 1.66 ꢀM); CNS Cancer SF-268 (GI50: 1.36 ꢀM); Melanoma SK-MEL-2
(GI50: 1.82 ꢀM), UACC-62 (GI50: 1.99 ꢀM); Ovarian Cancer (GI50: 1.64 ꢀM); Renal Cancer
A498 (GI50: 1.13 ꢀM) CAKI-1 cell line (GI50: 1.00 ꢀM), TK-10 (GI50: 1.34 ꢀM), UO-31
(GI50: 1.27 ꢀM) and Breast Cancer BT-549 (GI50: 1.69 ꢀM) cell lines. The dose response curve
of eight sub-panel cell lines for compound 5g is represented in Fig 2.
3. Conclusions
In this communication, we report the synthesis of heterocyclic derivatives of 1,2,4-
triazolin-3-one (5a-t) and their in vitro anticancer activity at NCI/NIH, Bethesda, USA. Out of
twenty compounds, nine compounds (5a-e, 5g-h, 5k, 5p) have been screened at a single high
dose (10-5 M) concentration. Among these, the 1,2,4-triazolin-3-one bearing 4-methylcoumarin
ring (5g) has shown nearly 50-99% growth inhibition of the tumor cells against various cell
lines at 10-5 M concentration. It has also exhibited marked anticancer activity even at micro
molar (ꢀM) concentration against Leukemia, Non-Small Cell Lung Cancer, Renal Cancer,
Colon Cancer, CNS Cancer, Melanoma, Ovarian Cancer, Renal Cancer and Breast Cancer cell
panels. Structural modifications of these title compounds may lead to the discovery of more
effective anticancer agents in future.
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4. Experimental
4.1. Materials and methods
The purity of the compounds was checked by TLC on a silica gel plate using ethyl
acetate and hexane (30%) as eluent. Melting points were determined in open capillaries. IR
1
spectra (KBr) were recorded on a Nicolet Impact-410 FTIR spectrometer. H (300 MHz) and
13C (100 MHz) NMR spectra were recorded on a Bruker Avance FT NMR spectrometer with
TMS as an internal standard. Mass spectra were recorded using a Finnegan MAT (Model MAT
8200) spectrometer and elemental analysis was carried out using a Heraus CHN rapid analyzer.
Compound 3 and corresponding mesilate 4 from N-(4-chlorophenyl)sydnone (1) were prepared
according to the reported methods [25-27].
4.2. General procedure for the preparation of title compounds (5a-t)
A mixture of compound 4 (0.331 g, 0.001 M), appropriate secondary amine (a-f, 0.001
M) or hydroxyl substituted compound (g-t, 0.001 M) and anhydrous potassium carbonate
(0.002 M) in dry DMF (20 ml) was stirred at 100 ºC. The completion of the reaction was
checked by TLC using ethyl acetate and hexane (30%) as eluent. The reaction mixture was
then poured into ice cold water and neutralized with dil. HCl (few drops). The precipitate thus
formed was filtered and dried. All the newly prepared compounds were purified by column
chromatography using ethyl acetate and hexane (10%) as eluent.
4.2.1. 4-(2-(1H-1,2,4-Triazol-1-yl)ethyl)-2-(4-chlorophenyl)-5-methyl-2H-1,2,4-triazol-3(4H)-
one (5a)
1
Yield: 85%; m.p: 170-172 ºC; IR (KBr, cm-1): 1710 (C=O), 1615 (C=N); H NMR (CDCl3, δ
ppm): 1.86 (s, 3H, C5-CH3), 4.04 (t, 2H, N-CH2), 4.50 (t, 2H, N-CH2), 7.47-7.50 (d, 2H, J = 9
Hz, Ar-H), 7.86-7.89 (d, 2H, J = 9 Hz, Ar-H), 7.98 (s, 1H, triazole C3-H), 8.52 (s, 1H, triazole
C5-H); 13C NMR (CDCl3, δ ppm): 12.39, 43.05, 48.38, 120.98, 130.50, 132.11, 137.55, 145.52,
151.38, 153.17, 154.45; MS (m/z): 306 (M+2), 304 (M+), 238, 236, 211, 209, 127, 125; Anal.
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Calcd. for C13H13N6ClO (304.08): C, 51.24%; H, 4.30%; N, 27.58%; Found: C, 51.23%; H,
4.28%; N, 27.56%.
4.2.2. 2-(4-Chlorophenyl)-5-methyl-4-(2-morpholinoethyl)-2H-1,2,4-triazol-3(4H)-one (5b)
1
Yield: 60%; m.p: 110-112 ºC; IR (KBr, cm-1): 1703 (C=O), 1624 (C=N); H NMR (CDCl3, δ
ppm): 2.36 (s, 3H, C5-CH3), 2.53 (t, 4H, morpholine CH2-N-CH2), 2.64 (t, 2H, N-CH2), 3.74 (t,
4H, morpholine CH2-O-CH2), 3.76 (t, 2H, N-CH2), 7.35-7.39 (d, 2H, J = 12 Hz, Ar-H), 7.92-
13
7.96 (d, 2H, J = 12 Hz, Ar-H), C NMR (CDCl3, δ ppm): 12.02, 39.11, 53.90, 57.04, 66.94,
119.46, 129.22, 130.24, 136.55, 144.63, 152.04; MS (m/z): 324 (M+2), 322 (M+), 238, 236,
211, 209, 127, 125; Anal. Calcd. for C15H19N4ClO2 (322.12): C, 55.81%; H, 5.93%; N,
17.36%; Found: C, 55.79%; H, 5.90%; N, 17.38%.
4.2.3. 4-(2-(1H-Benzo[d][1,2,3]triazol-1-yl)ethyl)-2-(4-chlorophenyl)-5-methyl-2H-1,2,4-
triazol-3(4H)-one (5c)
1
Yield: 82%; m.p: 168-170 ºC; IR (KBr, cm-1): 1702 (C=O), 1612 (C=N); H NMR (CDCl3, δ
ppm): 1.92 (s, 3H, C5-CH3), 4.28 (t, 2H, N-CH2), 5.07 (t, 2H, N-CH2), 7.40-7.42 (m, 2H, Ar-
H), 7.44-7.49 (d, 2H, J = 15 Hz, Ar-H), 7.80-7.85 (d, 2H, J = 15 Hz, Ar-H), 7.88-7.91 (m, 2H,
Ar-H); 13C NMR (CDCl3, δ ppm): 10.86, 41.84, 53.90, 118.02, 119.52, 126.61, 126.93, 128.94,
130.37, 136.30, 143.89, 144.66, 151.69; MS (m/z): 356 (M+2), 354 (M+), 238, 236, 211, 209,
127, 125; Anal. Calcd. for C15H19N4ClO2 (354.10): C, 57.55%; H, 4.26%; N, 23.69%; Found:
C, 57.58%; H, 4.23%; N, 23.66%.
4.2.4. 3-(2-(1-(4-Chlorophenyl)-3-methyl-5-oxo-1H-1,2,4-triazol-4(5H-yl)ethyl)thiazolidine-
2,4-dione (5d)
1
Yield: 62; m.p: 278-280 ºC; IR (KBr, cm-1): 1739 (C=O), 1705 (C=O), 1611 (C=N); H NMR
(DMSO, δ ppm): 2.50 (s, 3H, C5-CH3), 2.73 (t, 2H, N-CH2), 2.89 (t, 2H, N-CH2), 3.63 (s, 1H,
C5-H of thiazolidine), 7.47-7.50 (d, 2H, J = 9 Hz, Ar-H), 7.90-7.93 (d, 2H, J = 9 Hz, Ar-H);
13C NMR (CDCl3, δ ppm): 11.31, 33.85, 37.70, 41.60, 119.00, 128.36, 128.92, 136.66, 145.74,
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151.50, 164.51, 167.90; MS (m/z): 354 (M+2), 352 (M+), 281, 278, 238, 236, 254, 251, 211,
209, 127, 125; Anal. Calcd. for C14H13N4ClO3S (352.04): C, 47.66%; H, 3.71%; N, 15.88%;
Found: C, 47.68%; H, 3.74%; N, 15.86%.
4.2.5. 4-(2-(1H-Benzo[d]imidazol-1-yl)ethyl)-2-(4-chlorophenyl)-5-methyl-2H-1,2,4-triazol-
3(4H)-one (5e)
1
Yield: 76%; m.p: 78-80 ºC; IR (KBr, cm-1): 1699 (C=O), 1598 (C=N); H NMR (CDCl3, δ
ppm): 2.16 (s, 3H, C5-CH3), 4.05 (t, 2H, N-CH2), 4.59 (t, 2H, N-CH2), 7.27-7.29 (m, 2H, Ar-
H), 7.32-7.37 (d, 2H, J = 15 Hz, Ar-H), 7.39-7.81 (m, 2H, Ar-H), 7.83-7.88 (d, 2H, J = 15 Hz,
13
Ar-H), 7.90 (s, 1H, C2-H of benzimidazole); C NMR (CDCl3, δ ppm): 11.20, 40.80, 44.92,
112.21, 115.41, 119.11, 125.66, 125.91, 128.71, 128.89, 131.46, 131.96, 136.16, 142.19,
145.25, 151.34; MS (m/z): 355 (M+2), 353 (M+), 237, 235, 211, 209, 127, 125; Anal. Calcd. for
C18H16N5ClO (353.10): C, 61.10%; H, 4.56%; N, 19.79%; Found: C, 61.12%; H, 4.53%; N,
19.77%.
4.2.6. 2-(4-Chlorophenyl)-5-methyl-4-(2-(4-methylpiperazin-1-yl)ethyl)-2H-1,2,4-triazol-
3(4H)-one (5f)
1
Yield: 65%; semisolid; IR (KBr, cm-1): 1706 (C=O), 1599 (C=N); H NMR (CDCl3, δ ppm):
2.24 (s, 3H, C5-CH3), 2.32 (s, 3H, N-CH3), 2.52-2.56 (m, 8H, piperazine), 2.66 (t, 2H, N-CH2),
3.68 (t, 2H, N-CH2), 7.25-7.28 (d, 2H, J = 9 Hz, Ar-H), 7.85-7.88 (d, 2H, J = 9 Hz, Ar-H); 13C
NMR (CDCl3, δ ppm): 12.05, 45.60, 52.95, 54.76, 119.34, 128.78, 130.08, 136.37, 145.32,
151.85; MS (m/z): 337 (M+2), 335 (M+), 238, 236, 211, 209, 127, 125; Anal. Calcd. for
C16H22N5ClO (335.15): C, 57.22%; H, 6.60%; N, 20.85%; Found: C, 57.23%; H, 6.58%; N,
20.87%.
4.2.7. 2-(4-Chlorophenyl)-5-methyl-4-(2-(4-methyl-2-oxo-2H-chromen-7-yloxy)ethyl)-2H-
1,2,4-triazol-3(4H)-one (5g)
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1
Yield: 76%; m.p: 238-240 ºC; IR (KBr, cm-1): 1712 (C=O), 1615 (C=N); H NMR (CDCl3, δ
ppm): 2.34 (s, 3H, coumarin CH3), 2.38 (s, 3H, C5-CH3), 4.11 (t, 2H, N-CH2), 4.30 (t, 2H, O-
CH2), 6.14 (s, 1H, C4-H of coumarin), 6.77-7.26 (m, 2H, Ar-H), 7.34-7.37 (d, 2H, J = 9 Hz,
Ar-H), 7.47-7.49 (m, 1H, Ar-H), 7.89-7.91 (d, 2H, J = 9 Hz, Ar-H); 13C NMR (CDCl3, δ ppm):
12.05, 18.61, 41.26, 65.78, 101.84, 106.22, 111.76, 112.55, 119.61, 125.81, 128.99, 130.44,
136.28, 144.79, 151.81, 152.26, 155.31, 160.72, 160.95; MS (m/z): 413 (M+2), 411 (M+), 238,
236, 211, 209, 127, 125; Anal. Calcd. for C21H18N3ClO4 (411.10): C, 61.24%; H, 4.41%; N,
10.20%; Found: C, 61.25%; H, 4.44%; N, 10.17%.
4.2.8. 2-(4-Chlorophenyl)-4-(2-(5-chloroquinolin-8-yloxy)ethyl)-5-methyl-2H-1,2,4-triazol-
3(4H)-one (5h)
1
Yield: 72%; m.p: 308-310 ºC; IR (KBr, cm-1): 1710 (C=O), 1599 (C=N); H NMR (CDCl3, δ
ppm): 2.68 (s, 3H, C5-CH3), 4.26 (t, 2H, N-CH2), 4.46 (t, 2H, O-CH2), 6.94-6.97 (d, 1H, J = 9
Hz, Ar-H), 7.33-7.36 (d, 1H, J = 9 Hz, Ar-H), 7.48-7.51 (d, 2H, J = 9 Hz, Ar-H), 7.54-7.56 (m,
13
1H, Ar-H), 7.88-7.91 (d, 2H, J = 9 Hz, Ar-H), 8.51-8.92 (m, 2H, Ar-H); C NMR (CDCl3, δ
ppm): 12.06, 41.43, 66.62, 108.95, 119.53, 122.50, 126.28, 128.95, 130.26, 132.98, 136.43,
144.58, 149.73, 152.37, 158.91; MS (m/z): 418 (M+4), 416 (M+2), 414 (M+), 238, 236, 206,
204, 127, 125; Anal. Calcd. for C20H16N4Cl2O2 (414.07): C, 57.84%; H, 3.88%; N, 13.49%;
Found: C, 57.85%; H, 3.87%; N, 13.47%.
4.2.9. 2-(4-Chlorophenyl)-5-methyl-4-(2-(2-oxo-2H-chromen-4-yloxy)ethyl)-2H-1,2,4-triazol-
3(4H)-one (5i)
1
Yield: 81%; m.p: 230-232 ºC; IR (KBr, cm-1): 1716 (C=O), 1605 (C=N); H NMR (CDCl3, δ
ppm): 2.41 (s, 3H, C5-CH3), 4.22 (t, 2H, N-CH2), 4.49 (t, 2H, O-CH2), 5.95 (s, 1H, C4-H of
coumarin), 7.36-7.38 (m, 2H, Ar-H), 7.50-7.53 (d, 2H, J = 9 Hz, Ar-H), 7.62-7.81 (m, 2H, Ar-
13
H), 7.92-7.95 (d, 2H, J = 9 Hz, Ar-H); C NMR (CDCl3, δ ppm): 11.53, 40.01, 62.48, 91.11,
115.45, 116.47, 119.04, 122.74, 124.21, 128.95, 129.04, 132.81, 137.45, 145.01, 150.09,
9

ACCEPTED MANUSCRIPT
154.21, 160.10, 164.23; MS (m/z): 399 (M+2), 397 (M+), 238, 236, 211, 209, 127, 125; Anal.
Calcd. for C20H16N3ClO4 (397.08): C, 60.38%; H, 4.05%; N, 10.56%; Found: C, 60.35%; H,
4.04%; N, 10.59%.
4.2.10. 2-(4-Chlorophenyl)-5-methyl-4-(2-(o-tolyloxy)ethyl)-2H-1,2,4-triazol-3(4H)-one (5j)
1
Yield: 81%; m.p: 138-140 ºC; IR (KBr, cm-1): 1711 (C=O), 1608 (C=N); H NMR (CDCl3, δ
ppm): 2.15 (s, 3H, C5-CH3), 2.41 (s, 3H, CH3), 4.09 (t, 2H, N-CH2), 4.23 (t, 2H, O-CH2), 6.61-
6.99 (m, 4H, Ar-H), 7.09-7.11 (d, 2H, J = 6 Hz, Ar-H), 7.88-7.90 (d, 2H, J = 6 Hz, Ar-H); 13C
NMR (CDCl3, δ ppm): 12.11, 24.51, 40.71, 66.05, 114.78, 119.72, 121.23, 124.60, 126.04,
128.65, 130.54, 136.61, 144.81, 151.65, 155.68; MS (m/z): 345 (M+2), 343 (M+), 238, 236,
211, 209, 127, 125; Anal. Calcd. for C18H18N3ClO2 (343.11): C, 62.88%; H, 5.28%; N,
12.22%; Found: C, 62.84%; H, 5.26%; N, 12.19%.
4.2.11. 2-(4-Chlorophenyl)-5-methyl-4-(2-(p-tolyloxy)ethyl)-2H-1,2,4-triazol-3(4H)-one (5k)
1
Yield: 80%; m.p: 156-158 ºC; IR (KBr, cm-1): 1714 (C=O), 1611 (C=N); H NMR (CDCl3, δ
ppm): 2.19 (s, 3H, C5-CH3), 2.43 (s, 3H, CH3), 3.90 (t, 2H, N-CH2), 4.27 (t, 2H, O-CH2), 6.65-
6.69 (d, 2H, J = 12 Hz, Ar-H), 7.11-7.14 (d, 2H, J = 12 Hz, Ar-H), 7.48-7.52 (d, 2H, J = 12 Hz,
13
Ar-H), 7.88-7.92 (d, 2H, J = 12 Hz, Ar-H); C NMR (CDCl3, δ ppm): 11.98, 24.63, 40.74,
66.35, 113.64, 119.76, 128.86, 129.63, 130.65, 131.29, 136.52, 144.73, 152.09, 156.32; MS
(m/z): 345 (M+2), 343 (M+), 238, 236, 211, 209, 127, 125; Anal. Calcd. for C18H18N3ClO2
(343.11): C, 62.88%; H, 5.28%; N, 12.22%; Found: C, 62.89%; H, 5.27%; N, 12.25%.
4.2.12. 2-(4-Chlorophenyl)-5-methyl-4-(2-(m-tolyloxy)ethyl)-2H-1,2,4-triazol-3(4H)-one (5l)
1
Yield: 78%; m.p: 162-164 ºC; IR (KBr, cm-1): 1701 (C=O), 1598 (C=N); H NMR (CDCl3, δ
ppm): 2.15 (s, 3H, C5-CH3), 2.35 (s, 3H, CH3), 4.11 (t, 2H, N-CH2), 4.26 (t, 2H, O-CH2), 6.75-
6.85 (m, 4H, Ar-H), 7.38-7.41 (d, 2H, J = 9 Hz, Ar-H), 7.88-7.91 (d, 2H, J = 9 Hz, Ar-H); 13C
NMR (CDCl3, δ ppm): 12.01, 24.58, 40.78, 66.26, 111.49, 113.67, 119.69, 120.37, 128.63,
130.31, 136.74, 139.09, 144.87, 152.06, 156.79; MS (m/z): 345 (M+2), 343 (M+), 238, 236,
10

ACCEPTED MANUSCRIPT
211, 209, 127, 125; Anal. Calcd. for C18H18N3ClO2 (343.11): C, 62.88%; H, 5.28%; N,
12.22%; Found: C, 62.86%; H, 5.27%; N, 12.21%.
4.2.13. 2-(4-Chlorophenyl)-5-methyl-4-(2-(4-nitrophenoxy)ethyl)-2H-1,2,4-triazol-3(4H)-one
(5m)
Yield: 72%; m.p: 292-294 ºC; IR (KBr, cm-1): 1709 (C=O), 1595 (C=N), 1498, 1340 (NO2); 1H
NMR (CDCl3, δ ppm): 2.37 (s, 3H, C5-CH3), 4.06 (t, 2H, N-CH2), 4.27 (t, 2H, O-CH2), 6.86-
6.88 (d, 2H, J = 6 Hz, Ar-H), 7.28-7.30 (d, 2H, J = 6 Hz, Ar-H), 7.82-7.84 (d, 2H, J = 6 Hz,
Ar-H), 8.12-8.14 (d, 2H, J = 6 Hz, Ar-H); 13C NMR (CDCl3, δ ppm): 12.08, 40.65, 66.37,
115.32, 121.72, 119.81, 128.85, 130.56, 136.42, 138.69, 144.92, 151.78, 160.03; MS (m/z):
376 (M+2), 374 (M+), 238, 236, 211, 209, 127, 125; Anal. Calcd. for C17H15N4ClO4 (374.08): C,
54.48%; H, 4.03%; N, 14.95%; Found: C, 54.46%; H, 4.05%; N, 14.97%.
4.2.14. 4-(2-(4-Bromophenoxy)ethyl)-2-(4-chlorophenyl)-5-methyl-2H-1,2,4-triazol-3(4H)-one
(5n)
1
Yield: 74%; m.p: 178-180 ºC; IR (KBr, cm-1): 1711 (C=O), 1610 (C=N); H NMR (CDCl3, δ
ppm): 2.18 (s, 3H, C5-CH3), 4.02 (t, 2H, N-CH2), 4.12 (t, 2H, O-CH2), 6.66-6.70 (d, 2H, J = 12
Hz, Ar-H), 7.38-7.42 (d, 2H, J = 12 Hz, Ar-H), 7.44-7.48 (d, 2H, J = 12 Hz, Ar-H), 7.79-7.83
(d, 2H, J = 12 Hz, Ar-H); 13C NMR (CDCl3, δ ppm): 12.06, 41.42, 65.56, 113.80, 116.06,
119.53, 128.97, 130.37, 132.48, 136.35, 144.91, 151.82, 157.02; MS (m/z): 411 (M+4), 409
(M+2), 407 (M+), 238, 236, 211, 209, 155, 153, 127, 125; Anal. Calcd. for C17H15N3BrClO2
(407.00): C, 49.96%; H, 3.70%; N, 10.28%; Found: C, 49.93%; H, 3.73%; N, 10.30%.
4.2.15. 4-(2-(4-Chlorophenoxy)ethyl)-2-(4-chlorophenyl)-5-methyl-2H-1,2,4-triazol-3(4H)-one
(5o)
1
Yield: 68%; m.p: 166-168 ºC; IR (KBr, cm-1): 1702 (C=O), 1596 (C=N); H NMR (CDCl3, δ
ppm): 2.46 (s, 3H, C5-CH3), 4.08 (t, 2H, N-CH2), 4.23 (t, 2H, O-CH2), 6.65-6.69 (d, 2H, J = 12
Hz, Ar-H), 7.27-7.31 (d, 2H, J = 12 Hz, Ar-H), 7.41-7.45 (d, 2H, J = 12 Hz, Ar-H), 7.81-7.85
11

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(d, 2H, J = 12 Hz, Ar-H); 13C NMR (CDCl3, δ ppm): 12.03, 40.89, 65.48, 113.85, 115.75,
119.55, 128.79, 130.28, 132.56, 137.57, 144.98, 151.95, 155.62; MS (m/z): 367 (M+4), 365
(M+2), 363 (M+), 238, 236, 211, 209, 155, 153, 127, 125; Anal. Calcd. for C17H15N3Cl2O2
(363.05): C, 56.06%; H, 4.15%; N, 11.54%; Found: C, 56.03%; H, 4.12%; N, 11.56%.
4.2.16. 4-(2-(2-Chlorophenoxy)ethyl)-2-(4-chlorophenyl)-5-methyl-2H-1,2,4-triazol-3(4H)-one
(5p)
1
Yield: 65%; m.p: 188-190 ºC; IR (KBr, cm-1): 1705 (C=O), 1597 (C=N); H NMR (CDCl3, δ
ppm): 2.46 (s, 3H, C5-CH3), 4.06 (t, 2H, N-CH2), 4.25 (t, 2H, O-CH2), 6.93-7.38 (m, 4H, ArH),
13
7.46-7.49 (d, 2H, J = 9 Hz, Ar-H), 7.84-7.87 (d, 2H, J = 9 Hz, Ar-H); C NMR (CDCl3, δ
ppm): 12.11, 40.76, 65.78, 115.80, 119.74, 121.09, 122.56, 127.11, 128.81, 129.98, 130.29,
136.11, 144.88, 152.01; 155.68; MS (m/z): 367 (M+4), 365 (M+2), 363 (M+), 238, 236, 211,
209, 155, 153, 127, 125; Anal. Calcd. for C17H15N3Cl2O2 (363.05): C, 56.06%; H, 4.15%; N,
11.54%; Found: C, 56.05%; H, 4.18%; N, 11.52%.
4.2.17. 2-(4-Chlorophenyl)-4-(2-(2,3-dimethylphenoxy)ethyl)-5-methyl-2H-1,2,4-triazol-3(4H)-
one (5q)
1
Yield: 68%; m.p: 183-185 ºC; IR (KBr, cm-1): 1712 (C=O), 1610 (C=N); H NMR (CDCl3, δ
ppm): 2.17 (s, 3H, C5-CH3), 2.31 (s, 6H, CH3), 4.06 (t, 2H, N-CH2), 4.14 (t, 2H, O-CH2), 6.65-
7.06 (m, 3H, ArH), 7.47-7.51 (d, 2H, J = 12 Hz, Ar-H), 7.81-7.85 (d, 2H, J = 12 Hz, Ar-H);
13C NMR (CDCl3, δ ppm): 11.89, 11.97, 20.07, 41.71, 65.50, 109.04, 119.73, 123.21, 124.92,
126.01, 128.96, 130.30, 136.43, 138.22, 144.92, 151.87, 156.03; MS (m/z): 359 (M+2), 357
(M+), 238, 236, 211, 209, 155, 153, 127, 125; Anal. Calcd. for C19H20N3ClO2 (357.12): C,
63.77%; H, 5.63%; N, 11.74%; Found: C, 63.75%; H, 5.60%; N, 11.76%.
4.2.18. 2-(4-Chlorophenyl)-4-(2-(3,5-dimethylphenoxy)ethyl)-5-methyl-2H-1,2,4-triazol-3(4H)-
one (5r)
12

ACCEPTED MANUSCRIPT
1
Yield: 68%; m.p: 186-188 ºC; IR (KBr, cm-1): 1708 (C=O), 1598 (C=N); H NMR (CDCl3, δ
ppm): 2.20 (s, 3H, C5-CH3), 2.33 (s, 6H, CH3), 4.14 (t, 2H, N-CH2), 4.24 (t, 2H, O-CH2), 6.58-
6.77 (m, 3H, Ar-H), 7.61-7.65 (d, 2H, J = 12 Hz, Ar-H), 7.85-7.89 (d, 2H, J = 12 Hz, Ar-H);
13C NMR (CDCl3, δ ppm): 12.02, 24.87, 40.86, 66.01, 110.65, 119.81, 122.56, 128.94, 130.41,
136.57, 138.06, 144.89, 151.79, 157.03; MS (m/z): 359 (M+2), 357 (M+), 238, 236, 211, 209,
155, 153, 127, 125; Anal. Calcd. for C19H20N3ClO2 (357.12): C, 63.77%; H, 5.63%; N,
11.74%; Found: C, 63.74%; H, 5.65%; N, 11.75%.
4.2.19. 2-(4-Chlorophenyl)-5-methyl-4-(2-(naphthalen-1-yloxy)ethyl)-2H-1,2,4-triazol-3(4H)-
one (5s)
1
Yield: 71%; m.p: 163-165 ºC; IR (KBr, cm-1): 1700 (C=O), 1598 (C=N); H NMR (CDCl3, δ
ppm): 2.32 (s, 3H, C5-CH3), 4.05 (t, 2H, N-CH2), 4.20 (t, 2H, O-CH2), 6.75-8.25 (m, 11H,
ArH); 13C NMR (CDCl3, δ ppm): 12.13, 40.77, 66.41, 106.32, 119.67, 121.09, 122.27, 125.13,
125.98, 126.48, 127.01, 127.52, 128.96, 130.74, 135.61, 136.72, 144.87, 151.63, 156.71; MS
(m/z): 381 (M+2), 379 (M+), 238, 236, 211, 209, 155, 153, 127, 125; Anal. Calcd. for
C21H18N3ClO2 (379.11): C, 66.40%; H, 4.78%; N, 11.06%; Found: C, 66.42%; H, 4.80%; N,
11.03%.
4.2.20. 2-(4-Chlorophenyl)-5-methyl-4-(2-(naphthalen-2-yloxy)ethyl)-2H-1,2,4-triazol-3(4H)-
one (5t)
1
Yield: 68%; m.p: 157-159 ºC; IR (KBr, cm-1): 1698 (C=O), 1595 (C=N); H NMR (CDCl3, δ
ppm): 2.30 (s, 3H, C5-CH3), 4.03 (t, 2H, N-CH2), 4.16 (t, 2H, O-CH2), 6.81-8.22 (m, 11H,
ArH); 13C NMR (CDCl3, δ ppm): 12.12, 40.52, 66.58, 105.89, 117.68, 119.87, 124.41, 125.78,
126.31, 127.63, 128.14, 128.94, 129.54, 130.84, 134.80, 136.58, 144.77, 151.64, 157.30; MS
(m/z): 381 (M+2), 379 (M+), 238, 236, 211, 209, 155, 153, 127, 125; Anal. Calcd. for
C21H18N3ClO2 (379.11): C, 66.40%; H, 4.78%; N, 11.06%; Found: C, 66.42%; H, 4.77%; N,
11.04%.
13

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4.3. Methodology of the in vitro anticancer screen
The human tumor cell lines of the cancer screening panel were grown in RPMI 1640
medium containing fetal bovine serum (5%) and L-glutamine (2 mM). For a typical screening
experiment, cells were inoculated into 96 well microtiter plates in 100 µL at plating densities
ranging from 5,000 to 40,000 cells/well depending on the doubling time of individual cell
lines. After cell inoculation, the microtiter plates are incubated at 37 °C, 5% CO2, 95% air and
100 % relative humidity for 24 h prior to addition of experimental drugs. After 24 h, two plates
of each cell line are fixed in situ with TCA to represent a measurement of the cell population
for each cell line at the time of drug addition (Tz). Experimental drugs were solubilized in
dimethyl sulfoxide at 400 fold the desired final maximum test concentration and stored frozen
prior to use. At the time of drug addition, an aliquot of frozen concentrate was thawed and
diluted to twice the desired final maximum test concentration with complete medium
containing 50 µg/ml gentamicin. Additional four, 10 fold or ½ log serial dilutions were made to
provide a total of five drug concentrations plus control. Aliquots of 100 µl of these different
drug dilutions were added to the appropriate microtiter wells already containing 100 µl of
medium, resulting in the required final drug concentrations. Following drug addition, the plates
were incubated for an additional 48 h at 37 °C, 5% CO2, 95% air, and 100% relative humidity.
For adherent cells, the assay was terminated by the addition of cold TCA. Cells were fixed in
situ by the gentle addition of 50 µl of cold 50% (w/v) TCA (final concentration, 10% TCA)
and incubated for 60 minutes at 4 °C. The supernatant was discarded, and the plates were
washed five times with tap water and air dried. Sulforhodamine B (SRB) solution (100 µl) at
0.4% (w/v) in acetic acid (1%) was added to each well, and plates were incubated for 10
minutes at room temperature. After staining, unbound dye was removed by washing five times
with acetic acid (1%) and the plates were air dried. Bound stain was subsequently solubilized
with trizma (10 mM) base, and the absorbance was read on an automated plate reader at a
14

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wavelength of 515 nm. For suspension cells, the methodology was the same except that the
assay was terminated by fixing settled cells at the bottom of the wells by gently adding 50 µl of
80% TCA (final concentration, 16% TCA). Using the seven absorbance measurements [time
zero (Tz), control growth (C) and test growth in the presence of drug at the five concentration
levels (Ti)], the percentage growth is calculated at each of the drug concentrations levels.
Percentage growth inhibition was calculated as:
[(Ti-Tz)/(C-Tz)] × 100 (for concentrations for which Ti ≥ Tz)
[(Ti-Tz)/Tz] × 100 (for concentrations for which Ti < Tz)
Three dose response parameters were calculated for each experimental agent. Growth
inhibition of 50% (GI50) was calculated from [(Ti-Tz)/(C-Tz)] × 100 = 50, which was the drug
concentration resulting in a 50% reduction in the net protein increase (as measured by SRB
staining) in control cells during the drug incubation. The drug concentration resulting in total
growth inhibition (TGI) is calculated from Ti = Tz. The LC50 (concentration of drug resulting
in a 50% reduction in the measured protein at the end of the drug treatment as compared to that
at the beginning) indicating a net loss of cells following treatment was calculated from [(Ti-
Tz)/Tz] × 100 = 50. Values were calculated for each of these three parameters if the level of
activity was reached; however, if the effect was not reached or was exceeded, the value for that
parameter was expressed as greater or less than the maximum or minimum concentration tested
[28-30].
Acknowledgements
Authors wish to thank the University Grants Commission (UGC), New Delhi [F. No.
14-3/2012 (NS/PE) Dated: 14-03-2012] for providing financial support under “Antitumor
activity an integrated approach” a focused area of UPE programme. The authors acknowledge
the University Scientific Instrumentation Centre (USIC), Karnatak University, Dharwad, NMR
Research Centre, Indian Institute of Science (IISc), Bengaluru, India for carrying out the
15

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spectral analyses. One of the authors (PPK) acknowledges the UGC, New Delhi, for awarding
RFSMS fellowship.
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19

ACCEPTED MANUSCRIPT
Scheme 1 Synthetic route for title compounds 5a-t
Table 1 Growth percent and growth inhibition (GI%) in single dose assay (10-5
concentration) for compound 5g (NSC: 761736/1)
M
Table 2 GI50, TGI and LC50 values against 60 human cancer cell lines for compound 5g
Fig. 1 Reported 1,2,4-triazole derivatives bearing heterocyclic substituent
Fig. 2 Dose response curve of eight sub-panel cell lines for compound 5g
20

ACCEPTED MANUSCRIPT
Scheme 1 Synthetic route for the title compounds 5a-t
R
O
R
O
R
H
N
Ac2O, Br2
0-60 0C
N
N
NH2CH2CH2OH
150-160 0C
N
N
OH
N
O
N
O
O
CH3
CH3
2
3
1
TEA
CH3SO3Cl
0-25 0C
R
O
N
R
N
O
N
R1H, K2CO3
O
N
DMF, 100 0C
-CH3SO3H
N
N
O
S CH3
O
R1
5a-t
CH3
4
CH3
R = p-ClC6H4
O
N
N
N
S
R1 = a;
c;
e;
N
b;
N
O
N
d;
N
N
N
N
O
O
O
O
Cl
O
f;
N
N CH3
g;
h;
N
O
O
j;
k;
i;
O
O
O
O
l;
O
Cl
p;
Cl
o;
m;
q;
O
O
n;
O
Br
NO2
O
O
r;
s;
O
t;
O
21

ACCEPTED MANUSCRIPT
Table 1 Growth percent and growth inhibition (GI%) in single dose assay (10-5
concentration) for compound 5g (NSC: 761736/1)
M
Panel/ Cell line
Growth percent
Growth inhibition
(GI%)
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
13.41
-5.70
16.27
37.58
31.00
21.22
86.59
cytotoxic
83.73
62.42
69.00
78.78
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H522
30.22
58.73
27.35
47.38
50.09
45.82
58.72
17.21
69.78
41.27
72.65
52.62
49.91
54.18
41.28
82.79
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
10.58
46.66
15.12
17.74
0.70
89.42
53.34
84.88
82.26
99.30
68.58
HT29
KM12
31.42
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
50.30
14.03
29.47
54.49
20.38
31.40
49.70
85.97
70.53
45.51
79.62
68.60
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
48.02
32.87
14.72
-38.55
3.55
40.80
10.76
22.52
43.33
51.98
67.13
85.28
cytotoxic
96.45
59.20
89.24
77.48
56.67
22

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Continued Table 1
Panel/ Cell line
Growth percent
Growth inhibiton
(GI%)
Ovarian Cancer
IGROV-1
59.69
14.82
67.10
38.80
17.68
24.67
41.12
40.31
85.18
32.90
61.20
82.32
75.33
58.89
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal Cancer
786-D
A498
32.86
25.70
41.87
19.48
37.43
51.82
59.63
42.80
67.14
74.30
58.13
80.52
62.57
48.18
40.37
57.20
ACHN
CAKI-1
RXF-393
SN12C
TK-10
UO-31
Prostate Cancer
PC-3
DU-145
48.50
21.64
51.50
78.36
Breast Cancer
MCF-7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
15.79
84.21
52.52
65.18
43.34
52.57
83.92
47.48
34.82
56.66
47.43
16.08
MDA-MB-468
23

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Table 2 GI50, TGI and LC50 values against 60 human cancer cell lines for compound 5g
Panel/ Cell line
GI50
MGMID
TGI
LD50
ꢀM
ꢀM
ꢀM
ꢀM
Leukemia
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
2.80
1.29
4.71
1.10
4.97
-----
>1.00
>1.00
>1.00
6.67
>1.00
>1.00
>1.00
>1.00
2.97
3.03
Non-Small Cell Lung
Cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H23
NCI-H460
NCI-H522
6.07
1.00
3.05
1.61
2.83
2.80
3.85
>1.00
>1.00
3.29
>1.00
>1.00
>1.00
3.82
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
2.39
3.59
3.57
3.20
3.82
1.66
2.20
7.34
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
2.91
3.65
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
1.36
4.73
4.06
5.37
2.21
4.18
>1.00
>1.00
4.74
>1.00
1.51
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
7.23
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
4.83
3.85
2.36
2.69
1.82
3.13
8.48
-----
1.99
>1.00
>1.00
>1.00
8.37
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
3.64
24

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Continued Table 2
Panel/ Cell line
GI50
MGMID
TGI
LD50
ꢀM
ꢀM
ꢀM
ꢀM
Ovarian Cancer
IGROV-1
3.50
3.25
2.68
2.19
5.17
1.64
3.36
>1.00
1.52
----
>1.00
>1.00
>1.00
4.88
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
3.11
Renal Cancer
786-D
A498
5.70
1.13
5.59
1.00
2.76
5.54
1.34
1.27
>1.00
3.19
>1.00
>1.00
3.00
>1.00
8.84
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
ACHN
CAKI-1
RXF-393
SN12C
TK-10
3.04
UO-31
Prostate Cancer
PC-3
DU-145
8.13
3.90
6.01
3.65
>1.00
>1.00
>1.00
>1.00
Breast Cancer
MCF-7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
2.70
4.53
2.82
1.65
7.55
2.69
>1.00
>1.00
>1.00
9.94
>1.00
5.76
>1.00
>1.00
>1.00
>1.00
>1.00
>1.00
MDA-MB-468
25

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Fig. 1 Reported 1,2,4-triazole derivatives bearing heterocyclic substituent
N
N
NH
N
O
O
O
O
N
N
N
a
b
O
Cl
S
O
O
O
N
N
N
N
c
26

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Fig. 2 Dose response curve of eight sub-panel cell lines for compound 5g
27