Click chemistry inspired synthesis and bioevaluation of novel triazolyl derivatives of osthol as potent cytotoxic agents

Article abstract of DOI:10.1016/j.ejmech.2014.07.069

A new series of diverse triazoles linked through the hydroxyl group of lactone ring opened osthol (1) were synthesized using click chemistry approach. All the derivatives were subjected to 3-(4,5-Dimethylthiazol-yl)-diphenyl tetrazoliumbromide (MTT) cytot

Full text of DOI:10.1016/j.ejmech.2014.07.069

European Journal of Medicinal Chemistry 84 (2014) 545e554
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Click chemistry inspired synthesis and bioevaluation of novel triazolyl
derivatives of osthol as potent cytotoxic agents
,
Saleem Farooq a *, Shakeel-u-Rehman b, Aashiq Hussain c, Abid Hamid c,
,
*
Mushtaq A. Qurishi d, Surrinder Koul a
a Bioorganic Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India
b Bioorganic Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Sanatnagar, Srinagar 190005, India
c Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India
d 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 new series of diverse triazoles linked through the hydroxyl group of lactone ring opened osthol (1)
were synthesized using click chemistry approach. All the derivatives were subjected to 3-(4,5-
Received 18 March 2014
Received in revised form
17 July 2014
Accepted 19 July 2014
Available online 21 July 2014
Dimethylthiazol-yl)-diphenyl tetrazoliumbromide (MTT) cytotoxicity screening against
a panel of
seven different human cancer cell lines viz. colon (colo-205), colon (HCT-116), breast (T47D), lung (NCI-
H322), lung (A549), prostate (PC-3) and Skin (A-431) to check their cytotoxic potential. Interestingly,
among the tested molecules, most of the analogs displayed better cytotoxic activity than the parent
osthol (1). Of the synthesized triazoles, compounds 8 showed the best activity with IC50 of 1.3, 4.9, 3.6,
41.0, 35.2, 26.4 and 7.2 mM against colon (Colo-205 and HCT-116), breast (T47D), lung (NCI-H322 and
A549), prostate (PC-3) and Skin (A-431) cancer lines respectively. Compound 8 induced potent apoptotic
Keywords:
Osthol
Click chemistry
Triazole
effects in Colo-205 cells. The population of apoptotic cells increased from 11.4% in case of negative control
to 24.1% at 25
mM of 8. Compound 8 also induced a remarkable decrease in mitochondrial membrane
Cytotoxic
Cell cycle analysis
Mitochondrial membrane potential loss
potential (L m) leading to apoptosis of cancer cells used. The present study resulted in identification of
J
broad spectrum cytotoxic activity of analogs bearing electron withdrawing substituents, besides the
enhanced selective activity of analogs with electron donating moieties.
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
interesting pharmacological properties [11,12] has intrigued
chemists for decades to explore the natural coumarins or their
Natural products (NPs) have been used to treat human disease
for thousands of years and play an increasingly important role in
drug discovery and development. In fact, the majority of anticancer
and anti-infectious agents are of natural origin [1,2]. NPs with a
coumarinic moiety have been reported to have multiple biological
activities including anticancer [3,4], antioxidant [5,6], anti-
inflammatory [7], antimicrobial [8], antiviral [9] and enzymatic
inhibitory activities [10]. Natural coumarins and their derivatives
are of great interest due to their widespread pharmacological
properties, and this has attracted many medicinal chemists for
further derivatization and screening them as novel therapeutic
agents. The coumarin ring system, present in a large number of
natural products (such as the anticoagulant, Warfarin), having
synthetic derivatives for their applicability as drugs.
Many intersecting molecules having coumarin based ring sys-
tems have been synthesized utilizing novel synthetic methodolo-
gies. Some new derivatives bearing coumarin ring including the
furanocoumarins (imperatorin), pyranocoumarins (seselin) and
coumarin sulfamates (coumates) have been found to be useful in
photochemotherapy, antitumour and anti-HIV therapy [13,14].
Among the diverse biological activities of coumarins, the notable
one being their effect against breast cancer and sulfatase and aro-
matase inhibitory activity [15,16].
The naturally occurring coumarin osthol 1 (Fig. 1) has been
thoroughly investigated during the past years for its promising
pharmacological properties, particularly in the field of cancer
[17,18]. It is clinically ingested as an important component of
medicinal plants and herbs in Tradition Chinese Medicine (TCM)
[19e21], and it exhibits a host of biological activities [22e26].
Osthol has been found to be a promising agent for the treatment of
* Corresponding authors.
E-mail addresses: farooqprince100@yahoo.in (S. Farooq), skoul@iiim.ac.in
(S. Koul).
http://dx.doi.org/10.1016/j.ejmech.2014.07.069
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

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S. Farooq et al. / European Journal of Medicinal Chemistry 84 (2014) 545e554
2. Result and discussion
2.1. Chemistry
Osthol exhibits comparatively weak activity, low water solubi-
lity and limited permeability, and these properties may lower its
absorption and bioavailability upon oral administration [19,20,47].
To overcome these lacunas it becomes imperative to introduce
more hydrogen bond donors through its lactone ring opening and
simultaneous introduction of heterocyclic ring system to improve
its solubility and activity for better drug-likeness. The incorpora-
tion of heterocyclic moieties either as substituent group or as a
fused component into parent coumarin nucleus alters its properties
and converts it into a more useful product [48]. Keeping in view the
interesting pharmacological activities of coumarins, click chemistry
inspired approach involving union of terminal alkynes with organic
azides has been taken up for the synthesis of regiospecific novel OH
linked triazolyl derivatives of osthol in excellent yields. These click
reactions exhibit remarkably broad scope and exquisite selectivity
and have contrasting applications in chemistry, biology, and ma-
terials science. The target compounds 3e22 were synthesized as
depicted in Scheme 1.
In the present study, osthol (1), isolated from the root parts of
Prangos pabularia was used as the starting material. It was sub-
jected to lactone ring opening in DMSO using NaOH as base yielding
a cis (Z) product which simultaneously undergoes alkylation at OH
group in presence of propargyl bromide to form (E)-3-(4-methoxy-
3-(3-methylbut-2-en-1-yl)-2-(prop-2-yn-1-yloxy)phenyl)acrylic
acid 2. The proposed structure (2) was confirmed by spectral data
analysis. From elemental analysis and HR-ESIMS (m/z 301.1459)
data, this compound was assigned the molecular formula C18H20O4.
Fig. 1. Structure of osthol (1).
osteoporosis [27] and in reproductive system improvement
properties by activation of the central cholinergic neuronal system
[28]. Physiological [29], bacteriostatic and antitumour activities
make these compounds attractive for further backbone derivati-
zation and screening as novel therapeutic agent/s [30]. From the
literature scan, both in vitro and in vivo studies revealed that
osthol possesses anticancer effect by inhibiting human cancer cell
growth and inducing apoptosis [31]. The studies on cytostatic
activity in human cancer cell line MCF-7 (breast carcinoma)
revealed that osthol displays some estrogenic activity by pre-
venting the synthesis and action of estrogens (ER antagonists)
indicating its potential to become a breast cancer treatment re-
agent [32]. This compound reportedly possesses anticancer effects
by inhibiting cancer cell growth, metastasis and inducing cell
apoptosis [33]. It induces G2/M arrest and apoptosis by modu-
lating the PI3K/Akt pathway and suppresses migration and inva-
sion through inhibition of matrix metalloproteinase-2 and matrix
metallopeptidase-9 in human lung adenocarcinoma A549 cells
[34]. Osthol has also been found to be effective in inhibiting the
migration and invasion of breast cancer cells by wound healing
and transwell assays [35]. The biological studies of osthol carried
out in last few years have provided an additional dimension to the
bioactivity profile of the title compound. The potential of osthol
has not been fully exploited despite its biological importance;
therefore, more efforts are invested towards the building of
diverse libraries around its chemical structure and their biological
profiles are in demand.
Click chemistry of natural products has acquired great impor-
tance in recent years. Some of the molecules studied include al-
kaloids [36,37], coumarins [38], saponins [39], steroids [40] and
triterpenes such as betulinic acid [41e43]. Triazoles and their de-
rivatives are of great importance in medicinal chemistry and can be
used for the synthesis of numerous heterocyclic compounds with
different biological activities such as antiviral, antibacterial, anti-
fungal, anti-tuberculosis, anticonvulsant, antidepressant, anti-
inflammatory and anticancer [44,45]. Triazole based compounds
such as anastrozole, letrozole and vorozole are very important
antineoplastic drugs (Fig. 2) [46]. Thus, the design and synthesis of
novel triazole derivatives is the prospective direction for the
development of novel anticancer agents with better curative effect,
lower toxicity as well as higher selectivity.
Proton singlets at
d 2.52 and 4.45 (integrating for one and two
protons respectively) were assigned to terminal alkyne proton and
two methylenic protons of propargylic moiety respectively. The cis
(Z) behaviour of the protons of
depicted by the presence of two doublets at
a
,
b
-unsaturated system was
5.92 and 7.26 with the
d
coupling constant (J) of 12.4 Hz each. Compound 2 was allowed to
undergo 1,3-dipolar cycloaddition reaction typically called Huisgen
cycloaddition with various aromatic azides under sharpless click
chemistry conditions (CuSO4.5H2O and sodium ascorbate in t-
BuOH:H2O (1:1)) to afford regioselectively 1,4-disubstituted-1,2,3-
triazoles (3e22) in good to excellent yields (Scheme 1). Under
click conditions a series of such analogs was synthesized to look for
structureeactivity relationship studies. The structures of all the
synthesised triazolyl derivatives were characterised by analytical
and spectral data analysis. Formation of products could easily be
confirmed by a downfield H-5 proton singlet (almost around
8.0 ppm) and other proton resonances in the aromatic region.
Further characterisation of all the products was done using 13C
NMR-DEPT and HR-ESIMS as well as HRESI-MS.
2.2. Biology
Based on the above cited findings and inspiration from the
potential anticancer activity of triazoles, we directed this work
towards the synthesis of a diverse series of novel triazolyl de-
rivatives of biological interest using osthol (1) as a key starting
material. All the newly synthesized compounds were subjected
to MTT [3-(4,5-Dimethylthiazol-yl)-diphenyl tetrazolium-
bromide] assay against a panel of seven different human cancer
cell lines viz. prostate colon (Colo-205 and HCT-116), breast
(T47D), lung (NCI-H322 and A549), prostate (PC-3) and skin (A-
431) to check their cytotoxic potential. This work provides the
initial report on structureeactivity relationship of triazolyl ana-
logs of lactone ring opened coumarins in general and osthol (1)
in particular.
2.2.1. Anti-proliferative activity
There are innumerable number of synthetic drugs to treat the
diseased condition, but the treatment is not satisfactory, often due
to their severe adverse effects, making provision for the synthesis of
new and safer ones. Presently, scientists are keen to evaluate drugs
from plant origin, due to their specific curative properties, healthy
action, and safe and non-toxic effects. The biological studies of
coumarins and related molecules containing coumarin ring; carried
out in last few years have provided an additional dimension to the
bioactivity. The potential of coumarins has not been fully exploited
despite their biological importance; therefore, more efforts towards
building the diverse libraries around its chemical structure and

S. Farooq et al. / European Journal of Medicinal Chemistry 84 (2014) 545e554
547
Fig. 2. Some representative anticancer triazoles.
their biological profiles are in demand. Thus, based on the reported
anti-cancer activity of coumarins, it was envisaged to carry out
structural modifications of osthol (1), for improved anti-cancer
activity.
MTT cytotoxicity assay was used to screen the newly synthe-
sized osthol analogs against a panel of seven human cancer cell
lines; prostate colon (Colo-205 and HCT-116), breast (T47D), lung
(NCI-H322 and A549), prostate (PC-3) and skin (A-431). The cyto-
toxicity profile indicates that the parent molecule, osthol (1)
demonstrated cytotoxic effect against all the cancer cell lines. Most
of the synthesized analogs displayed broad spectrum cytotoxic ef-
fect in a dose dependent manner. The analogs which exhibited
significant cytotoxic effect, greater than 50% growth inhibition at
active against colon (HCT-116) and lung (A549) cancer cell line with
IC50 of 8.7 and 10.2 M respectively. Among 7, 20 and 21 containing
electron withdrawing group (cyano) in R moiety at different posi-
tions, 20 having cyano group at ortho position displayed better
results in comparison to corresponding meta and para analogs and
displayed superior potency to BEZ-235 against T47D and A431 cells
m
with IC50 of 8.8 and 10.3 mM respectively. These results demonstrate
the broad spectrum cytotoxic efficiency of compounds bearing
electron withdrawing groups, besides the selective activity of an-
alogs with electron donating moieties. Among all the tested cancer
cell lines, colon (HCT-116) cancer cell line was sensitive towards
most of these derivatives, while as, prostate (PC-3) cancer cell line
was least affected cell line by these derivatives.
the preliminary screening concentration (50
using MTT assay at different concentrations (0.04e60
m
M) were assayed
From these results, we come to the conclusion that it is not only
the effect of a particular group but also its position plays a signifi-
cant role on the bioactivity profile. The most active compounds
could become a better arsenal towards the corresponding sensitive
cell lines after further polishing and fine tuning.
mM) to
generate the IC50 values (Table 1). BEZ-235 was used as positive
controls in this assay. The values are the average of triplicate
analysis. Among all the tested triazolyl analogs, compound 8
exhibited the best results against colon (colo-205), colon (HCT-116),
breast (T47D) and skin (A-431) cancer cell lines with IC50 of 1.3, 4.9,
2.2.2. Cell cycle analysis of compound 8
3.6 and 7.2 mM respectively. Additionally, compound 8 exhibited
The effect of the most active osthol analog, 8 on the DNA content
by cell cycle phase distribution was evaluated by flow cytometry
using colon (Colo-205) cancer cell line. Propidium iodide (PI)
staining of Colo-205 cell line (5 ꢀ 105 cells/ml/6-well plates)
superior potency to BEZ-235 against T47D and A431 cells. Com-
pound 13, bearing OH group in place of CN group as in case of
compound 8, is moderately active against all the tested cancer cell
lines. Among compounds 5, 6, 9 and 12 containing a heterocyclic
ring system, compound 5 displayed slightly better activity than
exposed to different concentrations (1, 5,10 and 25 mM) of 8 for 48 h
induces apoptotic induction with apoptotic cell population of 12.5,
15.2, 15.7 and 24.1% respectively. For control cultures (untreated
cells, DMSO) the apoptotic DNA content as reflected by apoptotic
cell percentage of Colo-205 was 9.8%.
Also, it was found that 8 caused G1 phase arrest as percentage of
cells in G1 phase increases from 45.7% in negative control to 46.8%
BEZ-235 against A549 cancer cell line with IC50 of 5.6 mM while as it
was weakly active towards the other cancer cell lines. Compounds
6, 9 and 12 were moderately active against all the tested cancer cell
lines. Compound 14 bearing an iodo group at ortho position of R
moiety exhibited 3-fold increase in cytotoxic activity against A549
cancer cell line (IC50: 2.2
mM) as compared to BEZ-235 (IC50:
at 5 mM. Camptothecin (1 mM) for Colo-205, used as positive con-
6.5 M), while as, compound 19 having a fluoro at the same posi-
m
trols, showed 69.4% cells in apoptotic phase (Fig. 3). Thus the
molecule 8 exhibited significant apoptosis inducing effects in colon
(Colo-205) in a dose dependent manner.
tion was inactive towards lung A549 cancer cell line but strongly
active towards T47D and A431 cells. These observations demon-
strate the role of a particular substituent or a group in determining
the biological activity against a particular target.
Compounds 4, 16 and 17 having an electron donating OMe
functionality in R group at different positions were inactive towards
prostate (PC-3) cancer cell line. Compounds 16 and 17 were almost
equally toxic towards the other tested cancer cell lines while as,
compound 4 bearing a para OMe group in R moiety was selectively
2.2.3. Mitochondrial membrane potential (L
Further studies were carried out to find the effect of 8 on the
mitochondrial membrane potential (L m) for the apoptotic in-
Jm) loss
J
duction in Colo-205 cells caused due to the loss of mitochondrial
membrane potential. Colon (Colo-205) cells (5 ꢀ 105 cells/ml) were
treated with concentrations of 1, 5, 10 and 25 mM and LJm was

548
S. Farooq et al. / European Journal of Medicinal Chemistry 84 (2014) 545e554
Where R is a substituted aromatic system
Scheme 1. Reagents and conditions: (a) NaOH/DMSO, propargyl bromide, rt, 2 h (b) RN3, t-butanol:H2O (1:1), CuSO4, sodium ascorbate, rt, 0.5e1 h.
measured by flow cytometry. In case of untreated cells having
intact mitochondria, almost all the cells were bio-energetically
active as evident by high rhodamine-123 (Rh-123) uptake
compared to damaged mitochondria in positive control campto-
processes, cytochrome c, which is normally found in the mito-
chondrial inter chamber, is released into the cytosol of apoptotic
cells. This released cytochrome c through a cascade of events
eventually leads to cleavage of a number of cellular proteins and the
final cell death.
thecin (1
increase in L
was found to be 24.4%, 26.9%, 29.7% and 29.9% at concentrations of
1, 5, 10 and 25 M respectively (Fig. 4).
mM) for Colo-205. Compound 8 induced a remarkable
Jm (mitochondrial membrane potential loss) which
3. Conclusion
m
The results suggested that the apoptotic induction occurred due
to the loss of mitochondrial membrane potential in Colo-205 cancer
cell line. Loss of MMP indicates that mitochondrial membrane is
leaky and permeable which represents a crucial stage in apoptosis
in cancer cell death as it allows release of factors such as cyto-
chrome c and the apoptotic inducing factor (AIF) that trigger the
final and degradative stage of apoptosis. As a result of above
In conclusion, the current study demonstrates the cytotoxic
activity of a novel library of triazolyl derivatives of osthol (1)
anchored through the hydroxyl group of cis-ring opened lactone
created through regioselective click chemistry approach and char-
acterized by spectral data analysis. All the derivatives displayed
significant broad-spectrum cytotoxic activity against the tested
cancer cell lines. The most active derivative 8 exhibited potent

S. Farooq et al. / European Journal of Medicinal Chemistry 84 (2014) 545e554
549
Table 1
IC50 M) values of analogs against colon (colo-205), colon (HCT-116), breast (T47D), lung (NCI-H322), lung (A549), prostate (PC-3) and Skin (A-431) cancer lines using MTT
assay.
(
m
Entry
Colon (Colo-205)
Colon (HCT-116)
Breast (T47D)
Lung (NCI-H322)
Lung (A549)
Prostate (PC-3)
Skin (A-431)
IC50 (mM)
1
3
4
5
6
7
8
9
>60
32.3
nd
30.2
26.0
8.7
23.0
16.4
21
4.9
32.0
nd
45.3
20.4
31.7
23.1
16.4
21.6
32.2
35.5
nd
38.3
35.3
27.3
0.044
42.4
34.1
Nd
30.9
29.2
nd
42.2
23.0
18.6
41.0
nd
45.7
35.4
22.7
27.1
42.3
17.2
25.2
41.5
nd
45.6
14.4
26.5
43.2
10.3
46.2
40.5
10.2
5.6
42.1
42.2
35.2
nd
nd
nd
19.4
nd
2.2
42.7
42.7
43.0
nd
nd
38.6
nd
24.8
35.0
nd
nd
nd
23.2
21.2
nd
32.6
20.8
23.0
7.2
31.5
23.4
nd
23.1
16.4
32.7
21.2
23.2
32.6
43.3
12.4
10.3
22.9
18.4
12.5
nd
Nd
20.2
32.0
1.3
25.4
22.6
43.0
nd
19.3
nd
20.3
nd
12.2
nd
14.5
3.2
26.8
6.9
0.085
17.2
10.2
3.6
13.4
17.4
17.3
13.2
40.0
Nd
23.3
20.4
Nd
30.6
11.0
8.8
nd
26.4
36.2
nd
14.2
32.0
28.3
nd
nd
nd
nd
36.2
24.2
nd
45.5
42.1
12.3
10
11
12
13
14
15
16
17
18
19
20
21
22
BEZ-235
19.2
15.4
11.2
nd
6.5
IC50 values are expressed in
nd: not determined.
mM concentration.
BEZ-235 was used as positive control.
anticancer activity by inducing apoptosis in Colo-205 cell line
through the disruption of mitochondrial membrane potential
sulphate and purified through column chromatography to give
pure product 2 in 96% yield. 1H NMR (CDCl3, 400 MHz)
: 1.63 (3H,
d
(L
J
m). However, in vivo studies are warranted to investigate the
s),1.78 (3H, s), 2.52 (s,1H), 3.38 (d, J ¼ 6.81 Hz, 2H), 3.86 (s, 3H), 4.45
mechanisms of action responsible for the cytotoxic activity of the
most active derivatives.
(m, 2H), 5.17 (t, J ¼ 6.82 Hz, 1H), 5.92 (d, J ¼ 12.4 Hz, 1H), 6.67 (d,
J ¼ 8.7 Hz, 1H), 7.26 (d, J ¼ 12.8 Hz, 1H), 7.58 (d, J ¼ 8.64 Hz, 1H). 13
C
NMR (CDCl3, 100 MHz) d: 17.91, 22.69, 23.40, 55.74, 62.16, 75.55,
78.89, 106.64, 117.69, 120.99, 122.31, 129.54, 131.77, 132.15, 141.65,
155.96, 159.79, 170.89. IR (KBr) nmax cmꢂ1: 2941, 1682, 1608, 1593,
1486, 1425, 1384, 1366, 1314, 1266, 1162, 1118, 1088. HR-ESIMS m/z:
calcd for C18H20O4 [MþH] þ 301.1452, found 301.1459.
4. Experimental
4.1. Chemistry
All reagents for chemical synthesis were obtained from Sigma
Aldrich and the solvents used in reactions were distilled and dried
prior to use. All the chemical reactions were monitored by TLC on
0.25 mm silica gel 60 F254 plates (E. Merck) using 2% ceric
ammonium sulphate solution for detection of the spots. Purifica-
tion of compounds was carried out by column chromatography
using Silica gel 60e120 mesh stationary phase. 1H NMR and 13C
4.1.2. General procedure for synthesis of azides
To a solution of particular aromatic amine in 1,4-Dioxane (at the
rate of 50 mg/ml) at ꢂ15.0 ꢁC, 5 equivalents of 2 M Sulphuric acid
was added in small instalments while stirring. After 5 min 2
equivalents of 3 M sodium nitrite was added drop wise and after
30 min 3 equivalents of 3 M sodium azide was added drop wise
carefully. Reaction was brought to room temperature and extracted
with diethyl ether for at least three times. Organic layers were
washed with saturated sodium bicarbonate solution two times,
dried over anhydrous sodium sulphate and concentrated to a
minimum volume under reduced pressure on Rotary evaporator
without making use of heating from water bath.
NMR spectra (with chemical shifts expressed in
d and coupling
constants in Hertz) were recorded on Bruker DPX 200, 400 and DPX
500 instruments using CDCl3 or CD3OD as the solvents with TMS as
internal standard. High resolution mass spectra (HRMS) were
recorded on Agilent Technologies 6540 instrument and IR recorded
on an FT-IR Bruker (270-30) spectrophotometer. Melting points of
compounds were recorded on Buchi melting point apparatus B-
542.
4.1.3. General procedure for synthesis triazolyl derivatives (3e22)
Compound 2 (3 mmol) and different freshly prepared organic
azides (3 mmol) were suspended in 10 ml of a 1:1 water:t-butanol
4.1.1. Synthesis of (Z)-3-(4-methoxy-3-(3-methylbut-2-enyl)-2-
(prop-2-ynyloxy)phenyl)acrylic acid 2 (Scheme 1)
To a solution of osthol (1) (2 g, 12.3 mmol) in DMSO (15 ml),
crushed NaOH pellets (1.87 g, 13.1 mmol) were added and stirred
for 30 min at 25 ꢁC. Solution of propargyl bromide (1.1 ml,
14.8 mmol) in DMSO was then added slowly to the mixture and the
suspension was stirred for 2e3 h. Progress of reaction was moni-
tored using TLC at regular intervals. After the completion of reac-
tion, the reaction mixture was extracted with ethyl acetate
(3 ꢀ 30 ml) and the combined organic layer was dried over sodium
mixture. Sodium ascorbate (0.3 mmol, 300
1 M solution in water) was added, followed by copper (II) sulphate
pentahydrate (0.03 mmol, in 100 l of water). The heterogeneous
mixture was stirred vigorously till the completion of reaction. The
reaction mixture was extracted with ethyl acetate (30 ꢀ 3 ml) and
the combined organic layer was dried over sodium sulphate and
purified through column chromatography to give pure 3e23 in
excellent yields of 90e95%.
mL of freshly prepared
m

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S. Farooq et al. / European Journal of Medicinal Chemistry 84 (2014) 545e554
Fig. 3. Effect of 8 on cell cycle phase distribution of colon cancer cell line (Colo-205). Flow cytometric analysis of Colo-205 cells after propidium iodide staining. Cells were incubated
for 48 h in presence of 8 at (0, 1, 5, 10 and 25 M) concentration. Figures show the representative staining profile of one of two similar experiments. P1 (Sub-G1) is the population of
apoptotic cells, which increases from 12.0% in case of negative control to 24.1% at 25 M of 8. At lower concentrations of 1 and 5 M G1 phase cell cycle arrest of 46.4% and 46.8%
m
m
m
respectively as compared to negative control of 45.7% was found. Camptothecin (1
mM) was used as positive control. P1, P2, P3 and P4 represents Sub-G1, G1, S and G2 phases of cell
cycle in the figure respectively.
4.1.3.1. (Z)-3-(2-((1-phenyl-1H-1,2,3-triazol-4-yl)methoxy)-4-
(d, J ¼ 7.95 Hz, 1H), 8.08 (s, 1H), 8.55 (d, J ¼ 6.40 Hz, 1H). 13C NMR
methoxy-3-(3-methylbut-2-enyl)phenyl)acrylic
acid
3.
(CDCl3, 100 MHz) d: 17.93, 23.17, 25.73, 33.05, 55.73, 62.15, 67.33,
Colourless liquid. Yield: 92%. 1H NMR (CDCl3, 400 MHz)
d
: 1.67 (s,
106.59, 117.84, 122.59, 123.50, 125.15, 129.51, 131.71, 131.99, 136.02,
141.51, 144.07, 144.88, 150.25, 155.93, 159.80, 171.05. IR (KBr) nmax
cmꢂ1: 2953, 2791, 1725, 1608, 1437, 1393, 1304, 1276, 1196, 1032.
3H), 1.73 (s, 3H), 3.38 (d, J ¼ 6.5 Hz, 2H), 3.8 (s, 3H), 5.12 (s, 2H), 5.30
(t, J ¼ 6.82 Hz, 1H), 5.93 (d, J ¼ 12.17 Hz, 1H), 6.71 (d, J ¼ 8.6 Hz, 1H),
7.24 (d, J ¼ 12.29 Hz, 1H), 7.31 (m, 2H), 7.52 (m, 2H), 7.83 (m, 2H),
þ
HR-ESIMS m/z: calcd for C23H25ClN4O4 [MþH] 455.1458, found
8.02 (s, 1H). 13C NMR (CDCl3, 100 MHz)
d: 17.89, 23.09, 25.71, 29.60,
455.1452.
55.71, 67.51, 106.47, 120.53, 120.55, 120.57, 121.56, 122.76, 123.21,
123.75, 128.71, 129.26, 129.75, 129.79, 131.37, 136.98, 138.41, 155.48,
159.29, 171.07. IR (KBr) nmax cmꢂ1: 2856, 1725, 1608, 1455, 1393,
1304, 1276, 1196, 1113, 1032, 826, 762, 615. HR-ESIMS m/z: calcd for
4.1.3.4. (Z)-3-(2-((1-(benzo[d]thiazol-2-yl)-1H-1,2,3-triazol-4-yl)
methoxy)-4-methoxy-3-(3-methylbut-2-enyl)phenyl)acrylic acid 6.
Brown liquid. Yield: 94%. 1H NMR (CDCl3, 400 MHz)
d: 1.67 (s, 3H),
C
24H25N3O4 [MþH] þ 420.1917, found 420.1919.
1.73 (s, 3H), 3.40 (d, J ¼ 6.75 Hz, 2H), 3.83 (s, 3H), 4.46 (s, 2H), 5.17 (t,
J ¼ 6.92 Hz, 1H), 5.92 (d, J ¼ 12.00 Hz, 1H), 6.78 (d, 1H, J ¼ 8.16 Hz),
7.23 (d, J ¼ 12.40 Hz,1H), 7.41 (d, J ¼ 8.59 Hz,1H), 7.33e7.62 (m, 4H),
4.1.3.2. (Z)-3-(2-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)
methoxy)-4-methoxy-3-(3-methylbut-2-enyl)phenyl)acrylic acid 4.
7.96 (s, 1H). 13C NMR (CDCl3, 100 MHz)
d: 17.92, 25.75, 29.11, 31.93,
Colourless liquid. Yield: 93%. 1H NMR (CDCl3, 400 MHz)
d: 1.67 (s,
55.73, 62.15, 75.55, 78.93, 106.61, 121.04, 121.87, 122.66, 122.92,
123.30, 123.44, 125.65, 125.80, 126.18, 126.74, 129.56, 131.73, 141.31,
155.94, 159.70, 160.03, 171.06. IR (KBr) nmaxcmꢂ1: 3477, 2922, 1770,
1472, 1346, 1049, 778, 688, 644, 558, 548, 490. HR-ESIMS m/z: calcd
for C25H24N4O4S [MþH] þ 477.1591, found 477.1580.
3H), 1.73 (s, 3H), 3.35 (d, J ¼ 6.62 Hz, 2H), 3.83 (s, 6H), 5.08 (s, 2H),
5.14 (t, J ¼ 6.67 Hz,1H), 5.90 (d, J ¼ 12.17 Hz,1H), 6.67 (d, J ¼ 8.53 Hz,
1H), 7.01 (d, J ¼ 8.74 Hz, 2H), 7.23 (d, J ¼ 12.16 Hz, 1H), 7.53 (d,
J ¼ 8.38 Hz, 1H), 7.61 (d, J ¼ 8.76 Hz, 2H), 7.89 (s, 1H). 13C NMR
(CDCl3, 100 MHz) d: 17.94, 23.14, 25.74, 25.74, 29.69, 55.61, 55.70,
106.49, 114.77, 121.24, 121.58, 122.14, 122.66, 123.38, 123.80, 128.9,
129.46, 129.47, 130.35, 131.67, 141.37, 155.62, 159.79, 159.85, 172.01.
IR (KBr) nmax cmꢂ1: 2923, 2851, 1725, 1608, 1455, 1393, 1304, 1276,
1196, 1113, 1032, 826, 762, 615. HR-ESIMS m/z: calcd for C25H27N3O5
[MþH]þ 450.2023, found 450.2019.
4.1.3.5. 5.
methoxy)-4-methoxy-3-(3-methylbut-2-enyl)phenyl)acrylic acid 7.
Yellow liquid. Yield: 90%. 1H NMR (CDCl3, 400 MHz)
: 1.64 (s, 3H),
(Z)-3-(2-((3-(4-cyanophenyl)-3H-1,2,3-triazol-4-yl)
d
1.74 (s, 3H), 3.34 (d, 2H, J ¼ 6.49 Hz), 3.83 (s, 3H), 5.10 (s, 2H), 5.15 (t,
J ¼ 6.67 Hz, 1H), 6.68 (d, J ¼ 12.23 Hz, 1H), 7.10 (d, 1H, J ¼ 8.72 Hz),
7.25 (d, J ¼ 12.18 Hz, 1H), 7.48 (d, J ¼ 8.67 Hz, 1H), 7.82 (d, J ¼ 8.31,
2H), 7.90 (d, J ¼ 8.24 Hz, 2H), 8.05 (s, 1H). 13C NMR (CDCl3, 100 MHz)
4.1.3.3. (Z)-3-(2-((1-(2-chloropyridin-3-yl)-1H-1,2,3-triazol-4-yl)
methoxy)-4-methoxy-3-(3-methylbut-2-enyl)phenyl)acrylic acid 5.
Yellow liquid. Yield: 92%. 1H NMR (CDCl3, 400 MHz)
d: 1.67 (s, 3H),
d: 15.72, 20.94, 23.52, 53.52, 69.81, 79.69, 105.41, 110.22, 117.61,
1.73 (s, 3H), 3.34 (d, J ¼ 6.90 Hz, 2H), 3.84 (s, 3H), 5.14 (s, 2H), 5.17 (t,
J ¼ 7.01 Hz, 1H), 5.91 (d, J ¼ 12.01 Hz, 1H), 6.68 (d, J ¼ 8.0 Hz, 1H),
7.26 (d, J ¼ 14.40 Hz, 1H), 7.47 (m, 1H), 7.54 (d, J ¼ 8.04 Hz, 1H), 8.04
118.23, 118.24, 119.26, 120.24, 122.25, 127.45, 129.26, 131.73, 138.72,
139.41,143.32,158.45,159.81,170.06. IR (KBr) nmaxcmꢂ1: 3732, 3314,
2918, 1764, 1681, 1585, 1461, 1299, 1170, 1126, 1050, 994, 944, 899,

S. Farooq et al. / European Journal of Medicinal Chemistry 84 (2014) 545e554
551
Fig. 4. Effect of 8 on mitochondrial membrane potential loss (DJm). 8 induced loss of mitochondrial membrane potential (DJm) in colon cancer cell line (Colo-205) incubated with
the compound at different concentrations (0, 1, 5, 10 and 25 M) in 6 well plate for 48 h treatment. Figures show the representative of one of two similar experiments. P4 is the
m
percentage of loss of mitochondrial membrane potential (DJm), which increases from 12.4% in case of negative control to 29.9% in case of 25
used as positive control. P3 and P4 represents percentage of intact and loss mitochondrial membrane potential (DJm) respectively.
mM of 8. Camptothecin (1 mM) was
845, 775, 720, 654. HR-ESIMS m/z: calcd for C25H24N4O4 [MþH]
4.1.3.8. (Z)-3-(2-((N-phenylacetamide)-1H-1,2,3-triazol-4-yl)
445.1870, found 445.1869.
methoxy)-4-methoxy-3-(3-methylbut-2-enyl)phenyl)acrylic acid 10.
White solid. Mp: 166 ꢁC. Yield: 92%. 1H NMR (CDCl3, 400 MHz)
d:
1.49 (s, 3H), 1.57 (s, 3H), 2.03 (s, 2H), 3.18 (d, J ¼ 6.51 Hz, 2H), 3.68 (s,
3H), 4.96 (t, J ¼ 6.01 Hz, 1H), 5.18 (s, 2H), 6.55 (d, J ¼ 8.55 Hz, 1H),
6.97 (d, J ¼ 11.94 Hz, 1H), 7.40 (d, J ¼ 8.52 Hz, 1H), 7.46 (d,
J ¼ 13.89 Hz, 1H), 7.50 (d, J ¼ 8.55 Hz, 2H), 7.65 (d, J ¼ 8.59 Hz, 2H),
4.1.3.6. (Z)-3-(2-((1-(benzylnitrile)-1H-1,2,3-triazol-4-yl)methoxy)-
4-methoxy-3-(3-methylbut-2-enyl)phenyl)acrylic acid 8. Yellow
liquid. Yield: 92%. 1H NMR (CDCl3, 400 MHz)
d: 1.78 (s, 6H), 3.35 (d,
J ¼ 6.53 Hz, 2H), 3.68 (s, 2H), 3.82 (s, 3H), 5.13 (s, 2H), 5.17 (t,
J ¼ 6.65 Hz, 1H), 5.78 (d, J ¼ 12.45 Hz, 1H), 6.72 (d, J ¼ 8.23 Hz, 1H),
7.25 (d, J ¼ 12.20 Hz, 1H), 7.48 (d, J ¼ 7.91 Hz, 1H), 7.82 (d, J ¼ 8.31,
2H), 7.90 (d, J ¼ 8.24 Hz, 2H), 8.10 (s, 1H). 13C NMR (CDCl3, 100 MHz)
7.89 (s, 1H). 13C NMR (CDCl3, 100 MHz)
d: 17.82, 25.62, 27.14, 29.28,
55.65, 63.4, 69.68, 106.50, 118.58, 119.27, 120.77, 121.37, 121.55,
121.81, 122.65, 123.38, 129.18, 131.55, 132.44, 139.77, 144.20, 155.29,
159.52, 169.67, 171.40. IR (KBr) nmax cmꢂ1: 2967, 2851, 1767, 1634,
1455, 1393, 1304, 1276, 1196, 1113, 1032, 826, 762, 615. HR-ESIMS m/
z: calcd for C26H28N4O5 [MþH] þ 477.2132, found 477.2132.
d: 17.92, 23.17, 25.71, 29.67, 55.72, 67.15, 75.52, 78.9, 106.61, 120.52,
121.71, 122.62, 123.47, 127.36, 130.28, 131.10, 131.69, 140.70, 144.17,
144.52, 155.24, 155.86, 159.80, 170.07. IR (KBr) nmax cmꢂ1: 3480,
2932, 1764, 1700, 1601, 1478, 1298, 1208, 1167, 1124, 987, 949, 838,
4.1.3.9. (Z)-3-(2-((1-Phenylethanol)-1H-1,2,3-triazol-4-yl)methoxy)-
þ
733, 672, 571, 527. HR-ESIMS m/z: calcd for C28H26N4O4 [MþH]
4-methoxy-3-(3-methylbut-2-enyl)phenyl)acrylic
acid
d: 1.66 (s, 3H),
11.
459.2026, found 459.2022.
Brown liquid. Yield: 91%. 1H NMR (CDCl3, 400 MHz)
1.78 (s, 3H), 3.39 (d, J ¼ 6.58 Hz, 2H), 3.81 (s, 3H), 4.48 (m, 2H), 4.86
(m, 1H), 5.01 (s, 2H), 5.16 (t, J ¼ 6.39 Hz, 1H), 6.63 (d, J ¼ 12.45 Hz,
1H), 7.25 (d, 1H, J ¼ 7.06 Hz), 7.35 (d, J ¼ 12.01 Hz, 1H), 7.38 (d,
J ¼ 7.63 Hz, 1H), 7.41e7.50 (m, 5H), 7.90 (s, 1H). 13C NMR (CDCl3,
4.1.3.7. (Z)-3-(2-((1(3-hydroxypyrrolidin-1-yl)(phenyl)methanone)-
1H-1,2,3-triazol-4-yl)methoxy)-4-methoxy-3-(3-methylbut-2-enyl)
phenyl)acrylic acid 9. Brown solid. Mp: 124 ꢁC. Yield: 94%. 1H NMR
(CDCl3, 400 MHz)
d: 1.30 (s, 3H), 1.72 (s, 3H), 1.83 (m, 2H), 2.04 (m,
100 MHz) d: 18.07, 25.74, 27.21, 31.93, 55.71, 62.14, 73.56, 73.63,
1H), 3.20 (m, 2H), 3.20 (d, J ¼ 6.91 Hz, 2H), 3.62 (m, 2H), 3.82 (s, 3H),
5.01 (s, 2H), 5.14 (t, J ¼ 7.01 Hz, 1H), 5.89 (d, J ¼ 12.07 Hz, 1H), 6.65
(d,1H, J ¼ 8.02 Hz), 7.10 (d, J ¼ 13.01 Hz,1H), 7.45 (d, J ¼ 8.01 Hz,1H),
78.93, 106.63, 118.53, 119.72, 120.96, 121.35, 121.53, 121.87, 122.66,
123.39, 129.33, 131.71, 132.43, 139.71, 144.22, 155.95, 159.69, 169.5.
IR (KBr) nmax cmꢂ1: 3423, 2956, 2867, 1725, 1608, 1455, 1393, 1304,
1276, 1196, 1113, 1032, 826, 762, 615. HR-ESIMS m/z calcd for
7.51e7.59 (m, 4H), 7.99 (s, 1H). 13C NMR (CDCl3, 100 MHz)
d: 17.90,
23.13, 25.72, 31.06, 33.76, 54.23, 55.71, 62.09, 67.20, 69.35, 70.28,
75.57, 106.52, 121.47, 122.68, 123.40, 124.65, 124.74, 127.4, 129.33,
129.83, 130.47, 131.65, 133.15, 155.41, 159.47, 159.52, 167.08, 170.01.
IR (KBr) nmax cmꢂ1: 3456, 2912, 2867, 1728, 1608, 1455, 1393, 1304,
1276, 1196, 1113, 1032, 826, 762, 615. HR-ESIMS m/z: calcd for
C
26H29N3O5 [MþH] þ 464.2179, found 464.2182.
4.1.3.10. (Z)-3-(2-((1-(benzo[d][1,3]dioxol-6-yl)-1H-1,2,3-triazol-4-
yl)methoxy)-4-methoxy-3-(3-methylbut-2-enyl)phenyl)acrylic acid
12. Brown solid. Mp: 133 ꢁC. Yield: 91%. 1H NMR (CDCl3, 400 MHz)
C
29H32N4O6 [MþH] þ 533.2394, found 533.2396.
d: 1.67 (s, 3H),1.70, (s, 3H), 3.33 (d, J ¼ 5.98 Hz, 2H), 3.81 (s, 3H), 5.06

552
S. Farooq et al. / European Journal of Medicinal Chemistry 84 (2014) 545e554
(s, 2H), 5.15 (t, J ¼ 6.09 Hz, 1H), 6.02 (s, 2H), 6.08 (d, J ¼ 13.42 Hz,
1H), 6.67 (d, J ¼ 8.51 Hz, 1H), 6.91 (d, J ¼ 12.42 Hz, 1H), 7.13 (d,
J ¼ 8.45 Hz, 1H), 7.22e7.30 (m, 3H), 7.87 (s, 1H). 13C NMR (CDCl3,
4.1.3.15. (Z)-3-(2-((3-(2-methoxyphenyl)-3H-1,2,3-triazol-4-yl)
methoxy)-4-methoxy-3-(3-methylbut-2-enyl)phenyl)acrylic acid 17.
Yellow liquid. Yield: 95%. 1H NMR (CDCl3, 500 MHz)
d: 1.64 (s, 3H),
100 MHz)
d
: 17.86, 23.07, 25.66, 29.62, 55.62, 67.46, 75.49, 77.31,
1.73 (s, 3H), 3.33 (d, J ¼ 6.70 Hz, 2H), 3.81 (s, 3H), 3.87 (s, 3H), 5.10 (s,
2H), 5.15 (t, J ¼ 6.67 Hz, 1H), 5.91 (d, J ¼ 12.30 Hz, 1H), 6.67 (d,
J ¼ 8.61 Hz,1H), 7.06e7.11 (m, 2H), 7.26 (d, J ¼ 11.45 Hz,1H), 7.42 (m,
101.56, 102.60, 106.44, 108.73, 114.11, 121.20, 122.57, 122.62, 123.27,
129.43, 131.56, 131.65, 147.96, 148.52, 155.53, 159.62, 169.12. IR (KBr)
nmax cmꢂ1: 3467, 2898, 2851, 1726, 1645, 1455, 1393, 1304, 1276,
1196, 1113, 1032, 826, 762, 615. HR-ESIMS m/z: calcd for C25H25N3O6
[MþH] þ 464.1816, found 464.1815.
1H), 7.55 (d, 1H, J ¼ 8.52 Hz), 7.78 (d, J ¼ 7.85 Hz,1H), 8.10 (s, 1H). 13
C
NMR (CDCl3, 100 MHz) d: 17.91, 22.70, 29.17, 55.70, 55.93, 66.23,
67.59, 106.45, 114.08, 117.99, 121.24, 122.72, 125.57, 128.85, 129.44,
130.12, 130.21, 130.93, 139.29, 141.09, 143.05, 151.11, 155.74, 159.74,
168.23. IR (KBr) nmax cmꢂ1: 3476, 2920, 1763, 1598, 1457, 1298, 1039,
947, 852, 808, 750, 699, 642. HR-ESIMS m/z calcd for C25H27N3O5
[MþH]þ 450.2023, found 450.2023.
4.1.3.11. (Z)-3-(2-((1phenylmethanol)-1H-1,2,3-triazol-4-yl)
methoxy)-4-methoxy-3-(3-methylbut-2-enyl)phenyl)acrylic acid 13.
Colourless liquid. Yield: 92%. 1H NMR (CDCl3, 400 MHz)
d: 1.64 (s,
3H), 1.78 (s, 3H), 3.33 (d, J ¼ 6.37 Hz, 2H), 3.78 (s, 3H), 4.45 (s, 2H),
5.08 (s, 2H), 5.13 (t, J ¼ 6.56 Hz, 1H), 6.60 (d, J ¼ 12.03 Hz, 1H), 7.13
(d, J ¼ 7.63 Hz, 1H), 7.34 (m, 1H), 7.43 (m, 2H), 7.60 (d, J ¼ 11.67 Hz,
1H), 7.63 (d, J ¼ 7.92 Hz, 1H), 7.66 (m, 1H), 7.93 (s, 1H). 13C NMR
4.1.3.16. (Z)-3-(2-((diphenylphosphonate)-1H-1,2,3-triazol-4-yl)
methoxy)-4-methoxy-3-(3-methylbut-2-enyl)phenyl)acrylic acid 18.
Yellow solid. Mp: 172 ꢁC. Yield: 94%. 1H NMR (CDCl3, 500 MHz)
d:
(CDCl3, 100 MHz)
d: 17.71, 22.94, 25.34, 29.41, 55.43, 66.95, 75.29,
1.67 (s, 3H),1.78 (s, 3H), 3.39 (d, J ¼ 6.65 Hz, 2H), 3.81 (s, 3H), 4.45 (s,
2H), 5.15 (t, J ¼ 6.65 Hz, 1H), 5.93 (d, J ¼ 12.42 Hz, 1H), 6.64 (d,
J ¼ 8.64 Hz,1H), 7.25e7.31 (m, 5H), 7.38 (d, J ¼ 12.75 Hz,1H), 7.57 (d,
77.31, 106.38, 119.09, 119.81, 121.88, 122.47, 123.01, 123.20, 126.88,
128.95, 129.51, 131.36, 131.41, 136.73, 143.39, 155.04, 159.13, 169.21.
IR (KBr) nmaxcmꢂ1: 3432, 2923, 2851, 1745, 1608, 1455, 1393, 1304,
1276, 1196, 1113, 1032, 826, 762, 615. HR-ESIMS m/z: calcd for
1H, J ¼ 8.68 Hz), 8.01 (s, 1H). 13C NMR (CDCl3, 100 MHz)
d: 17.97,
23.41, 25.76, 55.74, 62.18, 106.65, 107.64, 112.85, 117.58, 120.23,
120.31, 122.33, 124.65, 126.15, 129.39, 129.52, 131.59, 131.94, 142.38,
155.76, 156.44, 159.57, 160.64, 170.01. IR (KBr) nmax cmꢂ1: 3436,
2927, 1766, 1600, 1460, 1314, 1127, 1041, 856, 787, 564. HR-ESIMS m/
z calcd for C25H27N3O5 [MþH] þ 576.1923, found 576.1927.
C
25H27N3O5 [MþH] þ 450.2023, found 450.2025.
4.1.3.12. (Z)-3-(2-((1-(2-iodophenyl)-1H-1,2,3-triazol-4-yl)
methoxy)-4-methoxy-3-(3-methylbut-2-enyl)phenyl)acrylic acid 14.
Brown liquid. Yield: 90%. 1H NMR (CDCl3, 400 MHz)
d: 1.63 (s, 3H),
1.75 (s, 3H), 3.33 (d, J ¼ 6.75 Hz, 2H), 3.83 (s, 3H), 5.13 (s, 2H), 5.20 (t,
J ¼ 5.96 Hz, 1H), 5.95 (d, J ¼ 12.95 Hz, 1H), 6.99 (m, 1H), 7.26 (d,
J ¼ 12.56 Hz, 1H), 7.28 (d, J ¼ 8.16 Hz, 1H), 7.48 (m, 1H), 7.55 (m, 2H),
4.1.3.17. (Z)-3-(2-((3-(2-fluorophenyl)-3H-1,2,3-triazol-4-yl)
methoxy)-4-methoxy-3-(3-methylbut-2-enyl)phenyl)acrylic acid 19.
Yellow liquid. Yield: 92%. 1H NMR (CDCl3, 400 MHz)
d: 1.65 (s, 3H),
7.98 (s, 1H), 7.99 (d, J ¼ 8.00 Hz, 1H). 13C NMR (CDCl3, 100 MHz)
d:
1.74 (s, 3H), 3.35 (d, J ¼ 6.67 Hz, 2H), 3.84 (s, 3H), 5.12 (s, 2H), 5.19 (t,
J ¼ 6.32 Hz, 1H), 5.94 (d, J ¼ 12.01 Hz, 1H), 6.69 (d, J ¼ 8.01 Hz, 1H),
7.33 (d, J ¼ 12.0 Hz, 1H), 7.36 (m, 1H), 7.45 (m, 2H), 7.54 (d,
J ¼ 8.0 Hz,1H), 7.98 (d, J ¼ 8.68 Hz,1H), 8.01 (s, 1H). 13C NMR (CDCl3,
17.14, 29.42, 22.93, 55.43, 66.91, 75.22, 77.33, 106.37, 119.01, 119.82,
121.80, 122.44, 123.01, 123.23, 126.87, 128.96, 129.54, 131.34, 131.47,
136.79, 143.35, 155.05, 159.17, 169.29. IR (KBr) nmax cmꢂ1: 2923,
2851, 1725, 1608, 1455, 1393, 1304, 1276, 1196, 1113, 1032, 826, 762,
615. HR-ESIMS m/z: calcd for C24H24IN3O4 [MþH] þ 456.0884, found
456.0855.
100 MHz) d: 17.94, 23.41, 25.73, 55.80, 68.18, 106.57, 108.57, 112.85,
117.04, 122.06, 122.34, 122.66, 124.48, 124.88,125.27, 129.42, 130.23,
130.39, 141.10, 142.14, 144.06, 159.54, 161.64, 169.64. IR (KBr) nmax
cmꢂ1: 3437, 2927, 2851, 1745, 1608, 1455, 1393, 1304, 1276, 1196,
1113, 1032, 826, 762, 615. HR-ESIMS m/z: calcd for C25H27N3O5
[MþH] þ 438.1723, found 476.1927.
4.1.3.13. (Z)-3-(2-((N-phenylbenzamide)-1H-1,2,3-triazol-4-yl)
methoxy)-4-methoxy-3-(3-methylbut-2-enyl)phenyl)acrylic acid 15.
Colourless solid. Mp: 142 ꢁC Yield: 95%. 1H NMR (CDCl3, 400 MHz)
d
: 1.66 (s, 3H), 1.78 (s, 3H), 3.37 (d, J ¼ 6.32 Hz, 2H), 3.83 (s, 3H), 4.45
4.1.3.18. (Z)-3-(2-((3-(2-cyanophenyl)-3H-1,2,3-triazol-4-yl)
(m, 2H), 5.12 (t, J ¼ 6.25 Hz, 1H), 5.94 (d, J ¼ 12.76 Hz, 1H), 6.67 (d,
J ¼ 7.65 Hz, 1H), 7.04 (d, J ¼ 12.31 Hz, 1H), 7.07 (d, J ¼ 7.89 Hz, 2H),
7.49e7.70 (m, 5H), 7.74 (d, J ¼ 8.31 Hz, 1H), 7.97 (d, J ¼ 8.01 Hz, 2H),
methoxy)-4-methoxy-3-(3-methylbut-2-enyl)phenyl)acrylic acid 20.
Colourless liquid. Yield: 90%. 1H NMR (CDCl3, 400 MHz)
d: 1.65 (s,
3H), 1.74 (s, 3H), 3.37 (d, J ¼ 6.49 Hz, 2H), 3.87 (s, 3H), 5.10 (s, 2H),
5.19 (t, J ¼ 6.67 Hz, 1H), 5.92 (d, J ¼ 12.03 Hz, 1H), 6.70 (d, J ¼ 8.0 Hz,
1H), 7.20 (d, J ¼ 12.0 Hz, 1H), 7.49 (d, J ¼ 8.01 Hz, 1H), 7.60 (t,
J ¼ 8.0 Hz, 1H), 7.78e7.80 (m, 3H), 8.24 (s, 1H). 13C NMR (CDCl3,
8.01 (s, 1H). 13C NMR (CDCl3, 100 MHz)
d: 17.99, 24.57, 24.85, 56.79,
59.78, 108.33, 113.69, 119.51, 121.89, 123.01, 123.92, 125.24, 128.03,
128.77, 129.10, 131.68, 132.48, 133.12, 134.93, 136.00, 159.98, 161.02,
169.58. IR (KBr) nmax cmꢂ1: 3446, 2956, 2851,1725,1608,1455,1393,
1304, 1276, 1196, 1113, 1032, 826, 762, 615. HR-ESIMS m/z: calcd for
100 MHz) d: 15.72, 20.94, 23.52, 53.52, 69.81, 79.69, 105.41, 110.22,
117.61, 118.23, 118.24, 119.26, 120.24, 122.25, 127.45, 129.26, 131.73,
132.42, 133.08,138.72, 139.41, 143.32, 158.45, 159.81, 170.06. IR (KBr)
nmax cmꢂ1: 3732, 3314, 2918, 1764, 1681, 1585, 1461, 1299, 1170,
1126, 1050, 994, 944, 899, 845, 775, 720, 654. HR-ESIMS m/z: calcd
for C25H24N4O4 [MþH] 445.1870, found 445.1872.
C
31H30N4O5 [MþH] þ539.2288, found 588.2289.
4.1.3.14. (Z)-3-(2-((3-(3-methoxyphenyl)-3H-1,2,3-triazol-4-yl)
methoxy)-4-methoxy-3-(3-methylbut-2-enyl)phenyl)acrylic acid 16.
Yellow liquid. Yield: 94%. 1H NMR (CDCl3, 500 MHz)
d: 1.63 (s, 3H),
1.73 (s, 3H), 3.35 (d, J ¼ 6.62 Hz, 2H), 3.80 (s, 3H), 3.89 (s, 3H), 5.08
(s, 2H), 5.14 (t, J ¼ 6.67 Hz, 1H), 5.90 (d, J ¼ 12.13 Hz, 1H), 6.67 (d,
J ¼ 8.53 Hz, 1H), 7.32 (m, 3H), 7.01 (d, J ¼ 8.74 Hz, 1H), 7.23 (d,
J ¼ 12.16 Hz, 1H), 7.5 (m, 1H), 7.97 (s, 1H). 13C NMR (CDCl3, 100 MHz)
4.1.3.19. (Z)-3-(2-((3-(3-cyanophenyl)-3H-1,2,3-triazol-4-yl)
methoxy)-4-methoxy-3-(3-methylbut-2-enyl)phenyl)acrylic acid 21.
Yellow liquid. Yield: 95%. 1H NMR (CDCl3, 400 MHz)
d: 1.66 (s, 3H),
1.78 (s, 3H), 3.37 (d, J ¼ 6.49 Hz, 2H), 3.87 (s, 3H), 5.10 (s, 2H), 5.19 (t,
J ¼ 6.67 Hz, 1H), 5.91 (d, J ¼ 12.34 Hz, 1H), 6.70 (d, J ¼ 8.0 Hz, 1H),
7.20 (d, J ¼ 12.0 Hz, 1H), 7.49 (d, J ¼ 8.01 Hz, 1H), 7.60 (t, J ¼ 8.0 Hz,
d
: 17.99, 24.57, 25.85, 56.04, 56.79, 59.78, 59.78, 108.07, 108.33,
113.21, 113.69, 118.30, 119.51, 121.89, 123.01, 129.10, 131.11, 131.61,
132.48, 133.10, 136.00, 137.84, 159.25, 161.02, 170.08. IR (KBr) nmax
cmꢂ1: 2923, 2851, 1725, 1608, 1455, 1393, 1304, 1276, 1196, 1113,
1032, 826, 762, 615. HR-ESIMS m/z: calcd for C25H27N3O5 [MþH] þ
450.2023, found 450.2026.
1H), 7.68e7.74 (m, 3H), 8.04 (s, 1H). 13C NMR (CDCl3, 100 MHz)
d:
15.72, 20.94, 23.52, 53.52, 69.81, 79.69, 105.41, 110.22, 117.61, 118.23,
118.24, 119.26, 120.24, 122.25, 127.45, 129.26, 131.73, 132.42,
133.08,138.72, 139.41, 143.32, 158.45, 159.81, 170.06. IR (KBr) nmax

S. Farooq et al. / European Journal of Medicinal Chemistry 84 (2014) 545e554
553
cmꢂ1: 3738, 3317, 2915,1756,1681,1585,1461,1309,1178,1047, 994,
944, 899. HR-ESIMS m/z: calcd for C25H24N4O4 [MþH] 445.1870,
found 445.1870.
Aria). Data from 10,000 cells were collected for each data file. All
the histograms were analysed using FACS Diva software.
4.2.3. Mitochondrial membrane potential (LJm) for cellular
energy status
4.1.3.20. (Z)-3-(2-((1-(3-nitrophenyl)-1H-1,2,3-triazol-4-yl)
Mitochondrial membrane potential (L
Jm) was measured using
methoxy)-4-methoxy-3-(3-methylbut-2-enyl)phenyl)acrylic acid 22.
Brown liquid. Yield: 92%. 1H NMR (CDCl3, 400 MHz)
d: 1.72 (s, 3H),
flow cytometry with 2 M rhodamine-123 (Rh-123), cellpermeable
m
cationic dye that preferentially enters mitochondria based on the
highly negative mitochondrial membrane potential. Depolarization
of the membrane results in the loss of Rhodamine-123 from the
mitochondria and a decrease in intracellular fluorescence. Colon
(Colo-205) cancer cell line was seeded in 6-well culture plate
(5 ꢀ 105 cells/ml/6-well plates). Cells were incubated for 24 h and
1.82 (s, 3H), 3.37 (d, J ¼ 6.67 Hz, 2H), 3.91 (s, 3H), 5.10 (s, 2H), 5.20 (t,
J ¼ 6.67 Hz, 1H), 6.21 (d, J ¼ 12.56 Hz, 1H), 6.94 (d, J ¼ 8.67 Hz, 1H),
7.45 (d, J ¼ 12.18 Hz, 1H), 7.75 (d, J ¼ 8.78 Hz, 1H), 7.82 (m, 2H), 8.01
(s, 1H), 8.21 (m, 2H). 13C NMR (CDCl3, 100 MHz)
d: 17.99, 24.57,
27.85, 56.79, 59.85, 108.33, 113.69, 119.51, 119.90, 121.49, 121.89,
123.01, 127.96, 129.10, 131.09, 131.73, 135.69, 145.64, 146.86, 157.98,
159.02, 170.60. IR (KBr) nmax cmꢂ1: 3732, 2947, 1764, 1681, 1598,
1457, 1298, 1047, 994, 944, 899. HR-ESIMS m/z: calcd for
then treated with different concentrations (1, 5, 10 and 25
mM) of
compound 8 for 48 h treatment. Rh-123 (10
mg/ml) was added 1 h
before the termination of experiment, incubated at 37 ꢁC for 30 min
and thereafter washed with PBS. The pellet collected by centrifu-
C
25H24N4O4 [MþH] 465.1770, found 465.1772.
4.2. Biology
RPMI-1640 medium, Penicillin, streptomycin, foetal calf serum,
gation, was resuspended in 300 mL of PBS. The florescence intensity
of Rh-123 in cells was analysed using flow cytometer set at 485 nm.
Appendix A. Supplementary data
sodium bicarbonate, phosphate buffer saline, sulphorhodamine,
trypsin, gentamycin sulphate, tryphan blue, ethanol, DMSO, para-
formaldehyde were purchased from Sigma Chemicals Co. Glacial
acetic acid from Fischer scientific, PBS and trichloroactetic acid
(TCA) from Merck specialties private limited. All the human cancer
cell lines colon (colo-205), colon (HCT-116), breast (T47D), lung
(NCIeH322), lung (A549), prostate (PC-3) and Skin (A-431) were
obtained from National Center for Cell Science, Ganeshkhind, Pune-
4111007 (India) and National Cancer Institute, Biological Testing
Branch DTP/DCTD/NCI, Frederick Cancer Research and Develop-
ment Center, Fairview Center, Suite 205, 1003 West 7th Street,
Frederick, MD 21701-8527 (USA).
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.07.069.
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