Click chemistry inspired facile synthesis and bioevaluation of novel triazolyl analogs of Ludartin

Article abstract of DOI:10.1016/j.bmcl.2014.01.018

A convenient and modular synthesis involving diastereoselective Michael addition followed by regioselective Huisgen 1,3-dipolar cycloaddition reaction was carried out to furnish 1,4-disubstituted-1,2,3-triazoles of Ludartin. This reaction scheme involving Michael addition followed by regioselective Huisgen 1,3-dipolar cycloaddition reaction leading to the formation of triazolyl analogs is being reported for the first time. All the triazolyl products were characterised using spectral data analysis. Sulphorhodamine B cytotoxicity screening of the resulting products against a panel of five human cancerous cell-lines revealed that few of the analogs display promising broad spectrum cytotoxic effect. Among all the synthesized compounds, only 3q displayed the best cytotoxic effect with IC₅₀ values of 12, 11, 38, 39 and 8.5 μM but less than the standard Ludartin (1) with IC₅₀ values of 6.3, 7.4, 7.5, 6.9 and 0.5 μM against human neuroblastoma (T98G), lung (A-549), prostate (PC-3), colon (HCT-116) and breast (MCF-7) cancer cell lines, respectively. The present synthesis was designed based on the previous literature reports of Ludartin as an aromatase inhibitor. Our work provides an initial study on structure-activity relationship of triazolyl analogs of sesquiterpene lactones in general and Ludartin (1) in particular.

Full text of DOI:10.1016/j.bmcl.2014.01.018

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1047–1051
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Click chemistry inspired facile synthesis and bioevaluation of novel
triazolyl analogs of Ludartin q
b
b
c
Shabir H. Lone a, Khursheed A. Bhat a, , Rabiya Majeed , Abid Hamid , Mohd A. Khuroo
⇑
a Bioorganic Chemistry Division, Indian Institute of Integrative Medicine (CSIR), Srinagar 190005, India
b Cancer Pharmacology Division, Indian Institute of Integrative Medicine (CSIR), Jammu 180001, India
c Department of Chemistry, University of Kashmir, Srinagar 190006, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A convenient and modular synthesis involving diastereoselective Michael addition followed by regiose-
lective Huisgen 1,3-dipolar cycloaddition reaction was carried out to furnish 1,4-disubstituted-1,2,3-tri-
azoles of Ludartin. This reaction scheme involving Michael addition followed by regioselective Huisgen
1,3-dipolar cycloaddition reaction leading to the formation of triazolyl analogs is being reported for
the first time. All the triazolyl products were characterised using spectral data analysis. Sulphorhodamine
B cytotoxicity screening of the resulting products against a panel of five human cancerous cell-lines
revealed that few of the analogs display promising broad spectrum cytotoxic effect. Among all the syn-
thesized compounds, only 3q displayed the best cytotoxic effect with IC50 values of 12, 11, 38, 39 and
Received 26 October 2013
Revised 30 December 2013
Accepted 8 January 2014
Available online 15 January 2014
Keywords:
Ludartin
Michael-addition
Huisgen 1,3-dipolar cycloaddition
Cytotoxicity
8.5 lM but less than the standard Ludartin (1) with IC50 values of 6.3, 7.4, 7.5, 6.9 and 0.5 lM against
human neuroblastoma (T98G), lung (A-549), prostate (PC-3), colon (HCT-116) and breast (MCF-7) cancer
cell lines, respectively. The present synthesis was designed based on the previous literature reports of
Ludartin as an aromatase inhibitor. Our work provides an initial study on structure–activity relationship
of triazolyl analogs of sesquiterpene lactones in general and Ludartin (1) in particular.
Ó 2014 Elsevier Ltd. All rights reserved.
Sesquiterpene lactones (SLs) form one of the largest biogeneti-
cally homogenous group of secondary metabolites known. SLs have
been a subject of vast number of phytochemical/biological studies
in the past four decades, mainly due to the fact that many of them
display various conspicious biological activities.1–3 These biological
activities are attributed to the presence of electrophilic structure
elements in SLs which undergo covalent reaction with functional
biological molecules resulting in their deactivation.1–3 The
alkylation of free sulphur moieties of enzymes and other functional
proteins by SLs is responsible for SL bioactivity.1–3 This is in har-
mony with the pearsons hard-soft acid base principle (HSAB) that
soft nucleophiles and electrophiles react more readily with each
other than with reactants classified as hard electrophiles and
nucleophiles, for example, non-conjugated carbonyl groups and
amino or hydroxyl groups, respectively.4,5
approach has been developed to improve their pharmacokinetic
potential.7 Following the amino prodrug approach effective
structure–activity relationships have been developed for various
guaianolides (Fig. 1A) including, Ludartin8 (1), arglabin9 and other
non-guaianolide sesquiterpene lactones (Fig. 1B) like alantolac-
tone,10 isoalantolactone,10 costunolide,11 parthenolide,12–15
a
-san-
tonin,16 helenalin17 and ambrosin.18 As a result few synthetic
derivatives which include (11R)-13-(dimethyl-amine)-11,
13-dihydroarglabin and (11R)-13-(dimethyl amine)-11,13-dihy-
droparthenolide have reached to clinical trials.6,9
Arglabin along with its dimethylamine analog, (11R)-13-(di-
methylamine)-11,13-dihydroarglabin is an approved anticancer
agent in several countries for treatment of lung, liver, breast and
ovarian cancers. 9 (11R)-13-(dimethylamine)-11,13-dihydro-argla-
bin has less side effects than other chemotherapeutic agents.9
Ludartin (1), position isomer of Arglabin, shows gastric cytopro-
tective effect19 and also inhibits aromatase enzyme which is in-
volved in hormone-dependent breast cancer.20,21 Earlier, we
reported the structure–activity relationship of amino analogs of
Among the sesquiterpene lactones only a few have reached clin-
ical trials which include artemisinin from Artemisia annua L,
thapsigargin from Thapsia garganica and parthenolide from Tanace-
tum parthenum.6 However owing to the relatively non selective
mechanism of action, most of the SLs are not suitable for drug
development. To address this non specificity issue amino prodrug
Ludartin (1) at its highly reactive a-methylene-c
-lactone moiety.8
Some of the amino derivatives of Ludartin (1) were found to be po-
tent and selective cytotoxic agents and the results were in fine
tune with those reported for Arglabin by R. Csuk and coworkers
that the Michael addition at the exocyclic double bond leads to
derivatives with reduced/or equal cytotoxic effect but cell line
q
Institute’s Publication No.: IIIM/1607/2013.
⇑
Corresponding author. Tel.: +91 194 2431253x123; fax: +91 194 24441331.
E-mail address: kabhat@iiim.ac.in (K.A. Bhat).
0960-894X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2014.01.018

1048
S. H. Lone et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1047–1051
of two diagnostic proton resonances at d 5.38 ppm (d, J = 3.5 Hz)
O
O
and d 6.21 ppm (d, J = 3.5 Hz) of
Ludartin (1).
a-methylene-protons (13-H2) of
N
O
O
Such reaction creates one more chiral center at C-11 position
whose configuration was determined as R on the basis of correla-
tions deduced from NOESY described earlier.8 On the other hand
aromatic azides were prepared from their corresponding aromatic
amines by diazotisation with sodium nitrite in acidic conditions
followed by displacement with sodium azide. These aromatic
azides were allowed to undergo 1, 3-dipolar cycloaddition reaction
typically called Huisgen cycloaddition with the terminal acetylene
bearing Michael adduct (2) under sharpless click chemistry condi-
tions (CuSO4Á5H2O and sodium ascorbate in t-BuOH/H2O (2:1)) to
afford regioselectively 1,4-disubstituted-1,2,3-triazoles in good to
excellent yields22 (Scheme 1B, Table 1). Under these conditions a
series of such analogs was synthesized to look for structure–activ-
ity relationship studies. All the triazolyl products were character-
ised using spectral data analysis. Formation of products could
easily be confirmed by a very down field H-5 proton signal (almost
around 8.0 Hz) and other proton resonances in the aromatic region.
Further characterisation of the products was done using 13C, DEPT-
NMR and HRMS as well as ESI-MS.
O
O
O
O
1
O
(11R)-13-(Dimethylamine)
Ludartin ( )
Arglabin
11,13-dihydroarglabin
O
O
NH
O
N
N
O
O
O
O
(11R)-13-(Morpholine)-11,
(11R)-13-(Piperzine)-11,
13-dihydroludartin
13-dihydroarglabin
Figure 1A. Some bioactive guaianolides that have been studied for SAR and their
active analogs.
H
H
H
O
O
O
O
H
O
O
Alantolactone
Costunolide
Isoalantolactone
Ludartin(1)anditstriazolylanalogswerethenstudiedina colori-
metricSulphorhodamine B (SRB) cytotoxicity assay23 against a panel
of five human cancer cell lines viz. neuroblastoma (T98G), lung (A-
549),prostate(PC-3),colon(HCT-116)andbreast(MCF-7).Preliminary
O
O
H
O
O
O
O
OH
cytotoxicityscreeningoftheanalogswascarriedoutat50 lMconcen-
O
O
O
Parthenolide
Santonin
trationandcelldeathwasdetermined.Ludartin(1)servedasapositive
controlinthisassay.Theanalogswhichexhibitedsignificantcytotoxic
effect,greaterthan50%growthinhibitionatthepreliminaryscreening
concentration were further assayed at different concentrations (5–
50 lM)togeneratetheIC50 valuesgiveninTable2. Thevaluesarethe
averageofthetriplicateanalysis.
From the cytotoxicity profile it is clear that the parent molecule,
Ludartin (1) demonstrated to be cytotoxic not only against human
leukaemia (THP -1), lung (A-549), colon (HCT-116), and prostate
(PC-3) as described earlier8 but proved to be effectively cytotoxic
against breast (MCF-7) and neuroblastoma (T98G) cancer cells
Helenalin
N
H
O
O
O
O
O
O
(11R)-13-(dimethylamine)-11,
Ambrosin
13-dihydroparthenolide
Figure 1B. Some bioactive Sesquiterpene lactones apart from guaianolides.
dependent selectivity.9 Keeping in view the fact that Ludartin
inhibits aromatase enzyme involved in hormone-dependent breast
cancer and that the triazole based aromatase inhibitors in general
represent the third generation frontline therapy for early and even
advanced cases of breast cancer in postmenopausal women21, we
designed a reaction strategy, to develop effective triazole based
analogs to rationalise lead properties of Ludartin (1) especially
against breast cancers, involving a combination of Michael addition
and Huisgen 1,3-dipolar cycloaddition reaction.
In view of the broad spectrum cytotoxic potential of triazoles in
general, the triazole analogs of Ludartin were screened against
other cancer cell-lines available, apart from breast cancer cells
(MCF-7). An exhaustive literature survey revealed that such triaz-
olyl formation has not been reported on any sesquiterpene lactone
so far using click chemistry approach and represents the first of its
kind on any sesquiterpene lactone and thus constitutes an initial
structure–activity relationship of triazolyl derivatives of sesquiter-
pene lactone class of natural products in general and Ludartin (1)
in particular.
with IC50 values of 0.5 and 6.3 lM respectively. Among the synthe-
sized triazolyl analogs, only five compounds 3a, 3d, 3e, 3q and 3r
exerted broad spectrum cytotoxic effects against the tested cancer
cell-lines at their preliminary screening concentration (50 lM) and
hence IC50 values of these analogs were evaluated. Compound 3q
NH2
ACN, Reflux, 12 hrs
H
O
N
O
O
O
2
1
O
O
Scheme 1A. Preparation of amino propargyl of Ludartin.8
a) H2SO4 b) NaNO2 c) NaN3
1,4-dioxane, -15 C
N3
Aromatic azides
NH2
R
R
Aromatic amines
Ludartin (1) was subjected to Michael addition using propargyl
N
R
amine in acetonitrile under reflux at its highly reactive
lene- -lactone motif described previously (Scheme 1A).
a-methy-
N
N
c
H
N
Sod. Ascorbate, CuSO4.5H2O,
O
O
HN
This reaction serves two important purposes: one it gives ad-
ducts that react in a retro-michael’s fashion at the target site as
is hypothesized for amino analogs of sesquiterpene lactones8 and
simultaneously acts as a template (terminal triple bond) for the
second step of the reaction. Formation of propargylated product
(2) in the first step could easily be confirmed by the disappearance
,
t-BuOH:H2O (2:1), rt
R
N3
O
O
2
O
O
3a-3r
where R is any substituted
aromatic group
Scheme 1B. Preparation of aromatic azides and the triazoles of 2.

S. H. Lone et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1047–1051
1049
Table 1
Preparation of different 1,4-disubstituted-1,2,3-triazole analogs of Ludartin (1)
S.No.
R
Product
Yielda (%)
S.No.
10
R
Product
Yielda (%)
1
3a
72
3j
74
90
NO2
2
3b
76
11
3k
NO2
CN
CN
Cl
Br
3
4
3c
68
60
12
13
3l
76
71
3d
3m
5
6
7
8
9
3e
3f
92
90
68
60
68
14
15
16
17
18
3n
3o
3p
3q
3r
85
82
77
86
74
Br
CN
OCH3
OCH3
NO2
F
F
3g
3h
3i
OH
OCH3
Cl
Cl
Cl
a
Refers to isolated yields, ‘R’ refers to any substituted aromatic group.
was the most effective analog depicting IC50 values of 8.5, 11 and
12 M against breast (MCF-7), lung (A-549) and neuroblostoma
Persual of the literature revealed a good agreement between
our results of Ludartin (1) against breast cancer cells (MCF-7, IC50
l
(T98G) cancer cell lines respectively. Compound 3d, showed
reduced but a bit selective cytotoxic effect against human lung
of 0.5 lM) with those of earlier reports of this molecule, that it
inhibits the aromatase20 enzyme involved in breast cancer leading
(A-549) and colon (HCT-116 cell-lines) (IC50 values of 24 and 26
lM
us to propose that the most potent triazolyl analogs of Ludartin (1)
respectively), with no such cytotoxic effect against neuroblastoma
(T98G), prostate (PC-3) and breast cancer cells (MCF-7). Contrary
to 3d, 3e exhibited decreased cytotoxicity with IC50 values ranging
that is, compounds 3q and 3r (IC50 values of 8.5 and 12 lM)
respectively against this particular cell line (MCF-7) may also be
acting in the same fashion. However, in vivo studies are warranted
to investigate the molecular mechanisms of action responsible for
the antiproliferative activity of the most active compounds. The
work has already been initiated in this direction.
between 32-52
3a was little bit potent against neuroblastoma (T98G) and breast
(MCF-7) (IC50 of 21 M against both) as compared to that of lung
(A-549), prostate (PC-3) and colon (HCT-116) cancer cells
displaying IC50 values of 24, 54 and 37 M respectively. However
2,4,5-trichloro-analog (3r) showed better cytotoxicity against
breast (MCF-7) (IC50 of 12 M) followed by neuroblastoma
(T98G), colon (HCT-116), lung (A-549) and prostate (PC-3) with
IC50 of 17, 18, 21 and 33 M respectively.
lM against the tested cancer cell lines. Compound
l
Comparing the results of our earlier study8 with that of the cur-
rent work made us to conclude that the amino analogs (previously
reported) were better than the triazolyl analogs both in terms of
activity as well as selectivity towards particular cell-line. Among
the amino analogs (Table 3) morpholino (11), 6-nitro- (17) and
5-nitroindazole (18) derivatives exhibited potent and selective
cytotoxic effect against leukemia (THP-1), prostate (PC-3) and lung
(A-549) cancer cell lines respectively. Similarly the diethyl (2) and
piperidine (10) analogs exerted the cytotoxic effect against both
l
l
l
However among all the tested compounds, none of them exhib-
ited a selective cytotoxic effect against a particular cell line as was
true of the amino analogs described earlier.8

1050
S. H. Lone et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1047–1051
Table 2
Cytotoxicity profile of Ludartin and its triazolyl analogs against five human cancer cell lines
Compound
T98G
A-549
PC-3
HCT-116
MCF-7
% GIa
IC50
% GIa
IC50
% GIa
IC50
% GIa
IC50
% GIa
IC50
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
1
73
32
37
17
58
34
00
00
00
00
00
42
00
00
12
00
99
77
97
21
nd
nd
nd
52
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
12
17
6.3
77
21
32
68
74
21
08
20
17
00
26
46
10
00
19
47
73
91
97
24
nd
nd
24
39
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
11
21
7.4
56
08
28
00
28
40
00
00
00
00
00
22
00
00
21
00
46
73
97
54
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
38
33
7.5
58
48
06
79
72
38
00
00
00
00
00
20
00
00
00
11
49
74
97
37
nd
nd
26
35
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
39
18
6.9
77
00
12
22
79
00
06
00
00
00
00
12
00
00
29
13
84
81
100
21
nd
nd
nd
32
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
8.5
12
0.5
IC50 values are expressed in
‘nd’ means not determined in the given concentration range.
T98G, A-549, PC-3, HCT-116 and MCF-7 are the human neuroblastoma, lung, prostate, colon and breast cancers cells, respectively.
lM concentration and represent the average of the triplicate analysis.
a
% GI refers to growth inhibition measured at 50 lM concentration.
Table 3
Comparision between the potent amino and triazolyl analogs of Ludartin
Amino analogs of Ludartin
Triazolyl analogs of Ludartin
IC50
Compound
IC50
Compound
A549
HCT-116
PC-3
THP-1
A549
HCT-116
PC-3
MCF-7
0.5
21
8.5
12
T98G
1
6
10
11
17
18
7.4
—
—
—
—
6.9
3.7
4.0
—
—
—
7.5
—
—
—
2.2
—
3.1
3.5
3.9
2.8
—
1
7.4
24
11
6.9
—
—
—
7.5
—
—
6.3
—
12
3a
3q
3r
21
18
17
2.6
—
Compound 1 refers to the parent molecule Ludartin, 6, 10, 11, 17 and 18 refers to diethyl, piperidine, morpholine, 6-nitro and 5-nitroindazole analogs respectively described
in earlier study8 while as 3a, 3q and 3r refer to variously substituted triazolyl analogs of Ludartin.
colon (HCT-116) and leukemia (THP-1) cell-lines. However among
the triazolyl analogs only one compound (3q) depicted a good
cytotoxic potential against the breast cancer cell line (MCF-7) with
data, based on triazolyl synthesis of sesquiterpene lactones in gen-
eral and Ludartin (1) in particular.
IC50 of 8.5
ing IC50 of 0.5
l
M but that too less than the parent Ludartin (1) show-
M. Despite low bioactivity of the triazolyl analogs
Acknowledgments
l
synthesized this study provides new insight with regard to the bio-
activity of parent Ludartin (1) against breast (MCF-7) and neuro-
blastoma (T98G) cell-lines making us to conclude that Ludartin is
highly active against breast cancer cells and that the modification
One of the authors Shabir H. Lone is thankful to CSIR India for
providing Senior Research Fellowship. The authors also thank
DST (Govt of India) for financial grant (Project No. GAP-2100).
at the highly active
leads to analogs with reduced cytotoxic effect compared to Ludar-
tin (1) itself, thereby making the presence of -methylene- -lac-
tone moiety essential for the desired biological effect.
In summary, a range of triazolyl analogs were synthesized using
a diastereoselective Michael addition followed by regioselective
Huisgen’s cycloaddition reaction and studied for their behaviour
against a panel of five human cancerous cell lines. Compound 3q
a-methylene-c-lactone moiety of Ludartin (1)
Supplementary data
a
c
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
01.018.
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line after standard Ludartin (1) with IC50 of 0.5
l
M against MCF-7 cell
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l
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S. H. Lone et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1047–1051
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(0.12 mmol) was added and the reaction mixture was sonicated till its
completion. The crude mixture was extracted with ethyl acetate (3 Â 20 ml)
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data of compound 3e: Yield 92%: 1H NMR (400 MHz, CDCl3) d 8.02 (s, 1H), 7.65
(s, 4H), 5.30 (s, 1H), 4.06 (s, 2H), 3.70 (t, J = 10.8, 10.7 Hz, 1H), 3.38 (s, 1H),
3.11–2.83 (m, 3H), 2.76–2.62 (m, 1H), 2.42 (m, 2H), 2.21–1.83 (m, 4H), 1.68 (s,
3H), 1.63 (s, 3H), 1.25 (s, 1H). 13C NMR (125 MHz, CDCl3) d 177.20, 146.94,
135.38, 133.36, 132.96, 132.61, 122.32, 121.23, 121.83, 119.92, 111.36, 80.76,
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19. Giordano, O. S.; Guerreiro, E.; Pestchanker, M. J.; Guzman, J.; Pastor, D.;
Guardia, T. J. Nat. Prod. 1990, 53, 803.
20. Blanco, J. G.; Gil, R. R.; Alvarez, C. I.; Patrito, L. C.; Genti-Raimondi, S.; Flury, A.
FEBS Lett. 1997, 409, 396.
21. Muftuoglu, Y.; Mustata, Y. Bioorg. Med. Chem. Lett. 2010, 20, 3050.
22. Isolation of Ludartin (1): Ludartin was isolated from Artemisia amygdalina
Decne as described previously.24 Synthesis of (11R)-13-(propargyl amine)-11,
13-dihydroludartin (2): A solution of 1 (1000 mg, 4.65 mmol) in acetonitrile
(2 ml) and propargyl amine (0.121 mmol) was heated under reflux for 12 h in
presence of base DBU. After cooling the reaction mixture was evaporated under
vaccuo on a rotary evaporator and the residue obtained was subjected to
normal silica-gel column chromatography using Hexane-EtOAc as eluent to
furnish the pure product. Spectral data of compound (2). Yield 80%; Rf = 0.5 (5%
MeOH-DCM); IR (KBr cmÀ1) 3017, 2944, 1755; 1H NMR (400 MHz, CDCl3) d
3.69 (t, J = 10.8, 10,8 Hz, 1H), 3.46 (s, 1H), 3.38 (s, 1H), 3.06–2.97 (m, 2H), 2.89
(dd, J = 12.1, 6.7 Hz, 1H), 2.71 (d, J = 17.7 Hz, 1H), 2.51–2.33 (m, 2H), 2.21 (m,
3H), 2.10–1.97 (m, 1H), 1.89 (d, J = 4.4 Hz, 2H), 1.68 (s, 3H), 1.62 (s, 3H), 1.30–
1.18 (m, 1H). 13C NMR (100 MHz, CDCl3) d 176.84, 135.09, 133.33, 81.44, 80.50,
71.80, 66.90, 63.86, 52.80, 51.78, 46.87, 45.38, 38.66, 34.20, 33.52, 27.39, 22.55,
19.19. ESI-MS (m/z): 302 (M+H)+. Synthesis of triazole analogs of compound 2
(3a–3r): To a solution of compound 2 (25 mg, 0.083 mmol) in t-BuOH:H2O
(2:1, 3 ml), sodium ascorbate (2.0 mg, 0.012 mmol) and CuSO4Á5H2O (2 mg,
23. Sulphorhodamine B assay for % growth inhibition: The SRB assay was used to
screen the semisynthetic analogs of Ludartin for cell cytotoxicity. Various
human cancer cell lines, human breast cancer cell line (MCF-7, at a density of
7 Â 103 cells per mL per 100
lL per well), human lung carcinoma cell line
(A-549, at a density of 8 Â 103 cells per mL per 100
lL per well), human
prostate cancer cell line (PC-3, at a density of of 8 Â 103 cells per mL per 100
lL
per well), human colon cancer cell line (HCT-116, at a density of 1 Â 104 cells
per mL per 100 lL per well) and human neuroblastoma cell line (T98G, at a
density of 1 Â 104 cells per mL per 100
lL per well) used in this study were
purchased from European collection of cell culture (ECACC) USA) and seeded in
flat-bottomed 96-well plates. The cells were incubated at 37 °C for four hours
in a humidified atmosphere containing 5% CO2 and then media containing
samples at different concentrations were added. The plates were incubated for
the next 48 h. The cells were fixed by adding 50
to each well for 60 min maintained at 4 °C. The plates were washed five times
in running tap water and stained with 100 L per well SRB reagent (0.4% w/v
SRB in 1% acetic acid) for 30 min. The plates were washed five times in
1% acetic acid and allowed to dry overnight. SRB was solubilised with 100
lL per well of ice cold 50% TCA
l
lL
per well 10 mM Tris-base, shaken for 5 min and the OD was measured at
570 nm with reference wavelength of 620 nm.25 Further the IC50 values on the
cancer cells of different tissue origin used for screening were determined by
non-linear regression analysis using graph pad software.26
24. Lone, S. H.; Bhat, K. A.; Syed Naseer; Rather, R. A.; Khuroo, M. A.; Tasduq, S. A. J.
Chrom. B. 2013, 940, 135.
25. Majeed, R.; Reddy, M. V.; Chinthakindi, P. K.; Sangwan, P. L.; Hamid, A.;
Chashoo, G.; Saxena, A. K.; Koul, S. Eur. J. Med. Chem. 2012, 49, 55.
26. Chashoo, G.; Singh, S. K.; Mondhe, D. M.; Sharma, P. R.; Andotra, S. S.; Shah, B.
A.; Taneja, S. C.; Saxena, A. K. Eur. J. Pharmacol. 2011, 668, 390.