Design and synthesis of novel 1,2,3-triazole derivatives of coronopilin as anti-cancer compounds

Article abstract of DOI:10.1016/j.ejmech.2014.05.053

A series of 1,2,3-triazole coronopilin congeners have been designed and synthesized by employing click chemistry approach starting from parthenin and evaluated for their cytotoxicity against a panel of six human cancer cell lines (PC-3, THP-1, HCT-15, HeLa, A-549 and MCF-7). While many compounds exhibited significant anticancer activity, compound 3a, was found to be the most promising analogue in this series with IC₅₀ values of 3.1 μM on PC-3 cell line. Flow-cytometric studies showed that 1,2,3-triazole derivative-3a induce dose dependent apoptosis in the sub G1 phase. This lead molecule-3a was further studied for NF-κB (p65) transcription factor inhibitory activity using Elisa and western blotting analysis which confirmed concentration dependent inhibitory activity against NF-κB, p65 with 80% inhibition in 24 h at 100 μM.

Full text of DOI:10.1016/j.ejmech.2014.05.053

European Journal of Medicinal Chemistry 82 (2014) 255e262
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of novel 1,2,3-triazole derivatives of coronopilin
as anti-cancer compounds
Jabeena Khazir b, Irfan Hyder a, J. Laxmi Gayatri a, Leela Prasad Yandrati a, Naresh Nalla a,
Gousia Chasoo b, Ajay Mahajan b, A.K. Saxena b, M.S. Alam c, G.N. Qazi c,
,
*
Halmuthur M. Sampath Kumar a
a Natural Products Chemistry Division, CSIR e Indian Institute of Chemical Technology, Hyderabad 500007, India
b Medicinal Chemistry Division, CSIR e Indian Institute of Integrative Medicine, Jammu 180001, India
c Department of Chemistry, Jamia Hamdard University, New Delhi 110062, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 1,2,3-triazole coronopilin congeners have been designed and synthesized by employing click
chemistry approach starting from parthenin and evaluated for their cytotoxicity against a panel of six
human cancer cell lines (PC-3, THP-1, HCT-15, HeLa, A-549 and MCF-7). While many compounds
exhibited significant anticancer activity, compound 3a, was found to be the most promising analogue in
Received 7 September 2013
Received in revised form
22 May 2014
Accepted 23 May 2014
Available online 24 May 2014
this series with IC50 values of 3.1
derivative-3a induce dose dependent apoptosis in the sub G1 phase. This lead molecule-3a was further
studied for NF- B (p65) transcription factor inhibitory activity using Elisa and western blotting analysis
which confirmed concentration dependent inhibitory activity against NF- B, p65 with 80% inhibition in
24 h at 100 M.
mM on PC-3 cell line. Flow-cytometric studies showed that 1,2,3-triazole
k
Keywords:
Sesquiterpenoid lactone
Parthenin
k
m
© 2014 Elsevier Masson SAS. All rights reserved.
Coronopilin
1,2,3-Triazole
NF-kB (p65) inhibitors
1. Introduction
prevention is enormous [4]. Compared with chemical synthesis,
plant derived natural products represent an attractive source of
biologically active agents since they are natural and available at
affordable prices [5]. Also plants derived agents may have different
mechanisms than conventional drugs, and could be of clinical
importance in health care improvement [6]. Plant materials might
be bioactive secondary metabolites that have the potential to treat
different afflictions. In this study we focussed our attention towards
parthenin[I, Fig. 1], a major constituent of the aggressive and
obnoxious herb, Parthenium hysterophorus L. (Compositae) that
grows wild in different regions of India. Parthenin-a sesquiterpene
lactone (SL) isolated from this plant, is reported to be responsible
for plethora of pharmacological effects of this plant viz., anticancer,
antibacterial, antiamoebic, anti-inflammatory, lipid peroxidation
inhibition, and trypanocidal activity [7e15]. Of particular interest
would be the anticancer activity of this bifunctional psuedogua-
nolide structure, which is attributed to the presence of two un-
saturated double bonds on the scaffold that might act as Michael
Cancer is the most feared disease second only to heart disease as
a leading cause of death in most of the developed countries
including India. It is characterized by uncontrolled growth and
spread of abnormal cells [1]. Cancer development includes a multi-
step process such as induction of genetic instability, abnormal
expression of genes, abnormal signal transduction, angiogenesis,
metastasis, and immune evasion [2]. The high mortality rate caused
by this group of diseases is an indication of the limited efficiency of
the current therapies including radiation, chemotherapy and sur-
gery [3]. Therefore development of new anticancer agents is the
major focus for scientists across the world. In last few decades,
research has been focussed on chemically synthesised or natural
product derived compounds as anticancer entities. Although
chemical synthesis is currently the dominant means to discover
and produce drugs, but still the potential of bioactive plants or their
extracts to provide novel products for disease treatment and
acceptor for the cysteine residues of NF-kB, thereby permanently
inhibiting the transcription factors leading to apoptosis. In this di-
rection, our group generated novel spiro derivatives of parthenin at
its exocyclic unsaturation and found that some of the spiro analogs
having free cyclopentenone moiety as Michael acceptor, were
* Corresponding author.
E-mail addresses: sampath@iict.res.in, hmskumar@yahoo.com (H.M. Sampath
Kumar).
http://dx.doi.org/10.1016/j.ejmech.2014.05.053
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

256
J. Khazir et al. / European Journal of Medicinal Chemistry 82 (2014) 255e262
found to be the better cytotoxic entities [17]. We hypothesized that
the presence of one double bond may suffice the purpose on in-
hibition of NF-kB, whereas, the other olefinic bond can be conve-
niently utilized as site of structural modification in order to derive
novel semi synthetic entities with improved cytotoxicity and drug-
likeness.1,2-Dihydroparthenin also known as coronopilin [II, Fig.1],
exhibit potential anticancer activity against leukaemia to induce
apoptosis [16]. In this context, herein we attempted preparation of
various analogs of coronopilin by modifying endocyclic double
bond of parthenin to obtain some potent derivatives, while keeping
the exocyclic methylene unsaturation as possible site of attack by
Scheme 1. Synthesis of 2a-azido Coronopilin.
NF-kB cystiene residues. Such an effort should explain unequivo-
cally, the role of individual double bonds in retaining the cytotox-
icity with more clarity and also should unequivocally prove the
mode of action of the pseudoguanolides as inhibitors of tran-
scription factor. Herein, we describe the preparation and cytotox-
icity of a focused library of novel derivatives at of coronopilin where
Table 1
Optimization of reaction conditions for Michael addition.
S. No
Base
Time e (h)
Yield (%)
1
2
3
4
5
NaOAc
DIEA
Et3N
C5H5N
Pyrolidine
6
6
3.5
6
6
49
50
85
40
40
1,2,3-triazole moiety is appended at the b-position of a,b-unsatu-
rated cyclopentenone ring of parthenin. Using Huisgen [3 þ 2]
cycloaddition between a terminal alkyne and an azide a series of
novel regioselective 1,2,3-triazole coronopilin derivatives have
been synthesized and screened for anticancer activity against a
panel of human cancer cell lines. From the IC50 values it is found
that most of the compounds retained anticancer activity with
improved cytotoxicity as compared to parent compound parthenin.
Table 2
Various 1,2,3-triazole derivatives of Coronopilin.
2. Results and discussion
2.1. Chemistry
As illustrated in Scheme 1, azido group was generated on
unsaturated cyclopentonone ring through Michael addition reac-
tion of trimethylsilylazide selectively at -position. Reaction was
optimised using different bases as given in Table 1 and best results
were obtained using triethylamine as base. Through this method
a,b-
b
Compound
R
X
Yield (%)
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
2Cl ꢀ C6H4ꢀ
O
O
O
O
O
O
O
O
O
O
N
N
N
N
N
N
95
92
95
90
95
95
96
95
97
90
96
97
95
94
96
97
98
93
95
2F ꢀ C6H4ꢀ
yields for the synthesis of 2
compared to those reported in the literature [18]. Addition of azido
group at
-position was confirmed by 1H NMR, mass and IR. In 1H
a-azido coronopilin were improved
2Br ꢀ C6H4ꢀ
4Br ꢀ C6H4ꢀ
4CF3C6H4ꢀ
b
4Cl ꢀ C6H4ꢀ
NMR disappearance of the alkene protons of cyclopentonone ring
was observed. In IR absorption signal at 2100 cm-1 corresponding to
azido group was observed. Stereochemistry at C-2 position was
confirmed through 1H NMR by observation of the position and
2 ꢀ C10H9ꢀ
4 Isopropyl ꢀ 2CH3ꢀ C6H3ꢀ
2CH3 ꢀ C6H4ꢀ
4 ꢀ CH3 ꢀ C6H4ꢀ
2 ꢀ CH3 ꢀ C6H4ꢀ
2 ꢀ F ꢀ C6H4ꢀ
2 ꢀ Br ꢀ C6H4ꢀ
5-Bromo indole
4 ꢀ CF3 ꢀ OCH2 ꢀ C6H4ꢀ
4F ꢀ C6H4ꢀ
splitting pattern of the signal for proton (
the characteristic J value of orientation [19]. While for
tation characteristic J value is 15.4 Hz [20]. Also another reason for
d
3.3, t, J ¼ 9.6 Hz) which is
a
b
orien-
a
attack is the steric hindrance posed by the methyl group and the
possible hydrogen bonding between the hydroxyl group and ni-
2 ꢀ OCH3 ꢀ C6H4ꢀ
N
CH2
CH2
trogen of azide group which makes the
(Table 2).
a orientation stable
C6H5ꢀ
CH3ꢀ
As illustrated in Scheme 2 triazole derivatives of coronopilin
were synthesised through [3 þ 2] cycloaddition reaction of azide
generated on parthenin and terminal alkynes generated on phenols
and anilines. The synthesis of triazole ring was confirmed through
1H NMR, 13C and mass spectrometry data.
2.2. Biological results
2.2.1. Evaluation of in vitro anticancer activity
All the compounds were screened for in vitro cytotoxicity
against a panel of six human cancer cell lines including PC-3
(prostrate), HCT-15 (colon), THP-1 (leukaemia), HeLa (cervix), A-
549 (lung) and MCF-7 (breast). Mitomycin, Adriamycin and 5-FU
were taken as reference compounds and the results are reported
in terms of IC50 values (Table 3). From the IC50 values, it is clear that
most of the compounds have significant cytotoxic activity against
Fig. 1. Structures of parthenin (I) and coronopilin (II).

J. Khazir et al. / European Journal of Medicinal Chemistry 82 (2014) 255e262
257
Scheme 2. Synthesis of 1,2,3-triazole on ring A of Coronopilin.
prostrate, cervix, lung and breast derived cancer cell lines. How-
ever, the activity shown by these compounds was highest against
HeLa, a cervix derived cancer cell line. Compound-3a, showed
significant cytotoxicity against PC-3, HeLa and THP1 cell lines,
however, its activity was found to be maximum against PC-3 and
Thus, our results clearly indicate that compound- 3a has a good
inhibitory effect on the activity of NF-kB (p65).
3. Conclusion
HeLa cell line with IC50 value of 3.1, 3.6
mM respectively. Com-
In summary a focussed library of novel 1, 2, 3-triazole de-
rivatives were synthesised through alkyne-azide click reaction on
coronopilin. All the compounds have been then screened for their
anti cancer activity. Majority of the compounds exhibited signifi-
cant cytotoxicity when tested against a panel of human cancer cell
lines with compound-3a being most active within the series of
derivatives studied. This derivative also showed 80% binding with
pounds 3b, 3c, 3e, 3f, 3h, 3i and 3l were also found to have
promising activity against the above mentioned cell lines. How-
ever, compounds 3m, 3n, 3o, 3p, 3q, 3r did not show significant
cytotoxicity against THP and PC3 cell lines (IC50 100
the cytotoxicity exhibited by the majority of the derivatives were
below 10 M when compared to the cytotoxicity of the parent
compound parthenin (IC50 35.3e60 M), there by indicating that
mM). Overall,
m
m
DNA at 100
mM. This reinforces the view, that compounds of this
triazolyl coronopilin derivatives prepared from parthenin could
make better ligands with improved cytotoxicity against cancer cell
lines.
type exert their biological effects by inhibiting the NF-
k
B/p65. Thus,
novel leads from parthenin can be further developed into potential
chemotherapeutic agents in cancer therapy. Many of the new tri-
azolyl coronopilin analogues derived from parthenin scaffold
exhibited superior activity as compared to the precursor natural
product-parthenin.
The present study also generated some important information
about SAR of semi-synthetic sesquiterpene lactones. Various in-
ferences drawn from the study include the requirement of single
unsaturation on the scaffold should be sufficient for the inhibition
2.2.2. Cell cycle analysis of compound 3a
To address the cell death caused by compound-3a the extent of
apoptotic death was assessed using FACS flow cytometry through
the determination of sub-G1 cell population by propidium iodide
(PI) staining. Since most of the compounds showed good cyto-
toxicity on PC-3 cell line, therefore this cell line was chosen for
further studies. The DNA cell cycle analysis of compound-3a
(Fig. 2) revealed a concentration dependent increase in the sub G1
of NF-
k
B. Cyclopentenone moiety can be utilized for structural
-methylene-
-lactone moiety un-altered to be available as Michael acceptor for
modifications to fine tune the activity while keeping
g
a
phase of cell cycle being 38.8, 39.9 and 49.2 at 1, 5 and 10
m
M
respectively.
the attack by cystein sulfhydryl residue of NF-kB. The triazolyl ad-
ducts appended at cyclopentenone ring prove to be the better li-
gands as they may facilitate better orientation of the molecule for
attack by the cysteine residue of target protein and also might be
improvising the drug-likeness of the molecule by improving the
bioavailability of the molecule in in-vitro assay. Furthermore, the
new analogues have shown to induce apoptosis through concen-
tration dependent inhibition of NF-kB (p65) binding with DNA
response element. Overall the present study reveals the SAR of SQLs
with better clarity establishing the minimal requirement of unsa-
2.2.3. NF-
In literature, most of the
anti-cancer compounds works by blocking the NF-
factor [21]. In our study, we visualized that derivatives of parthenin
possess - unsaturated functionality and it might also work
through by inhibiting NF- B transcription factor. In this direction,
k
B (p65), transcription factor assay
- unsaturated moiety bearing potent
B transcription
a,b
k
a,b
k
compound-3a was taken for inhibitory NF-kB (p65) transcription
factor activity.
turation for NF-kB inhibition and thus, improved lead compounds
Mammals have five members of NF-
factors viz. Rel (c-Rel), Rel A (p65), RelB, NF-
cursor p105) and NF- B2 [22]. NF- B plays a central role in the
regulation of cancer cell proliferation and survival [23]. NF- B has
kB group of transcription
with far superior cytotoxicity can be derived from parthenin by
modifying the cyclopentenone double bonds while keeping the
exocyclic unsaturation unmodified.
k
B1 (p50 and its pre-
k
k
k
also been shown to play a vital role in the cancer cell progression by
regulating the process of angiogenesis [24], Therefore, many new
4. Experimental protocols
anti-cancer therapies are being developed against NF-kB. NF-kB
binding of compound-3a was checked through Elisa and Western
blot analysis as given in Fig. 3. Our data showed that PC-3 cells
treated with compound-3a displayed concentration dependent
Melting points were recorded on Buchi Melting point apparatus
D-545. NMR spectra were recorded on Bruker DPX500 instrument
in CDCl3 with TMS as an internal standard. Chemical shift values are
inhibition of NF-
where, cells treated with compound-3a exerted highly significant
(80%) effect on the binding of NF- B (p65) with DNA at 100 M.
k
B (p65) binding with DNA response element,
reported in d (ppm) and coupling constants in Hertz. Mass spectra
were recorded on Maldi TOFeTOF. The progress of all reactions was
monitored by TLC on 2e5 cm precoated silica gel 60 F254 plates of
k
m

258
J. Khazir et al. / European Journal of Medicinal Chemistry 82 (2014) 255e262
characterized on the basis of IR, 1H/13C NMR, DEPT and mass
spectrometry.
Table 3
IC50 values (mM) of 1,2,3- triazolyl derivatives of coronopilin against a panel of hu-
man cancer cell lines.
Compound
THP-1
3.8
PC-3
3.1
HCT-15
Hela
3.6
A549
MCF-7
4.1.3. Compound characterization
4.1.3.1. Compound 3a. Crystalline white solid; mp: 165e169 ꢁC;
½aꢂ2D5 þ29 (c 0.5, CHCl3); IR (KBr, cmꢀ1): 681.49, 752.84, 997.96,
1118.39, 1162.79, 1241.73, 1277.09, 1446.84, 1484.51, 1588.92,
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
5.3
23
12
48
100
27
4.2
6.3
8.8
14
3.8
4.1
9
21
6.2
21
9.7
4.3
3.4
16
13
7
24
32
3.7
4.2
15
5.1
4.2
4.5
17
15
28
16
14
40.2
e
25
4.3
5.6
4.7
6.4
5.2
3.6
7.5
3.9
4.8
6.5
3.5
3.6
7.3
6.8
8.4
19
10
48
12
25
33
3.8
26
17
100
100
100
100
100
100
100
100
1753.62, 2925.18, 3400.23; 1H NMR (200 MHz, CDCl3):
d 8.25 (d, 1H,
J ¼ 11.58 Hz), 7.45 (d,1H, J ¼ 7.99 Hz), 7.30e7.25 (m, 2H), 6.95 (d,1H,
J ¼ 1.29 Hz), 6.25 (d,1H, J ¼ 2.72 Hz), 6.00 (t,1H, J ¼ 9.77 Hz), 5.73 (d,
1H, J ¼ 2.35 Hz), 5.25 (t, 2H, J ¼ 6.78 Hz), 5.10 (d, 1H, J ¼ 8.28 Hz),
3.33e3.30 (m, 1H), 2.20 (d, 2H, J ¼ 4.87 Hz), 1.75e1.53 (m, 4H), 1.30
(d, 3H, J ¼ 5.6 Hz), 1.28 (s, 3H), 1.20 (m, 1H); 13C (100 MHz, CDCl3):
10.9, 11.5, 19.3, 27.7, 30.3, 38.0, 38.1, 42.4, 46.3, 60.3, 72.3, 73.6, 73.6,
83.4,115.7, 120.9, 123.7, 126.6, 129.9, 138.3, 142.4, 158.8, 170.1, 220.3;
ESI-MS: (MþNa) 494; Anal. Calcd for C24H26ClN3O5: C, 61.08; H,
5.55; N, 8.90. Found: C, 61.13; H, 5.58; N, 8.93.
20
45
10
5.5
39
8.5
100
20
7.6
4.6
3.3
100
3.9
3.7
4.8
7.2
23
34
100
100
100
23
100
23
5.8
12
37.6
e
100
100
100
100
100
100
26
100
6.5
15
4.3
5.5
35.3
0.5
e
17
15
28
16
8.4
60
3q
3r
3s
4.1.3.2. Compound 3b. Colourless solid; mp: 104 ꢁC; ½aꢂD25 þ32 (c
0.5, CHCl3); IR (KBr, cmꢀ1): 751.80, 816.88, 999.95, 1110.38, 1160.43,
1198.80, 1257.75, 1385.32, 1457.00, 1504.70, 1613.60, 1753.60,
Parthenin
Mitomycin
Adriamycin
5-FU
46.2
43.1
e
e
e
e
e
2928.37, 3400.10; 1H NMR (200 MHz, CDCl3):
d 7.75 (d, 1H,
6
2.2
e
e
0.5
e
J ¼ 10.4 Hz), 7.25e6.85 (m, 4H), 6.25 (d, 1H, J ¼ 2.72 Hz), 5.50 (t, 1H,
J ¼ 11.5 Hz), 5.25 (t, 2H, J ¼ 6.9 Hz), 5.00 (d, 1H, J ¼ 7.9 Hz), 4.60 (d,
1H, J ¼ 2.5 Hz), 3.75 (d, 2H, J ¼ 5.4 Hz), 3.25e3.00 (m, 1H), 2.00 (m,
1H), 1.71 (s, 3H), 1.25e1.00 (m, 7H), 0.92 (m, 1H); 13C (100 MHz,
CDCl3): 10.96, 17.44, 22.97, 28.92, 29.68, 30.36, 37.39, 38.73, 41.32,
43.50, 80.93, 82.28, 103.30, 109.54, 116.56, 123.91, 125.93, 130.89,
144.08, 149.46, 150.62, 154.88, 173.16, 186.05; ESI-MS: (MþNa) 478;
Anal. Calcd for C24H26FN3O5: C, 63.29; H, 5.75; N, 9.23. Found: C,
63.32; H, 5.71; N, 9.26.
4.5
4.9
e
* Mitomycin, Adriamycin and 5-FU used as positive control for this study.
thickness 0.25 mm (Merck). The chromatograms were visualized
under UV 366 nm and iodine.
4.1. Synthesis
4.1.1. Synthesis of 2a-azido coronopilin
4.1.3.3. Compound 3c. Colourless solid; mp: 126 ꢁC; ½aꢂD25 þ54 (c
0.5, CHCl3); IR (KBr, cmꢀ1): 629.95, 751.99, 816.02, 995.37, 1051.82,
1163.06, 1239.75, 1276.08, 1478.92, 1585.52, 1753.37, 2929.66,
In a typical procedure, to a solution of parthenin (0.177 g,
1.2 mmol) in MeOH (10 mL) stirred over a period of 10 min,
maintaining the temperature between 0 and 5 ꢁC, was added tri-
methylsilylazide (0.262 g, 1 mmol), then triethylamine was added
until the pH of solution was slightly alkaline, stirred the reaction
mixture at same temperature for 15 min followed by stirring at
ambient temperature for 4 h. The solvent was evaporated in vacuo
and the crude was subjected for flash chromatography and product
was obtained with 90% yield. The pure product was characterized
on the basis of 1H NMR, 13C NMR and mass spectrometry.
3150.25, 3400.57; 1H NMR (200 MHz, CDCl3):
d 8.25 (d, 1H,
J ¼ 5.326 Hz), 7.55 (d, 1H, J ¼ 7.9 Hz), 7.50e7.25 (m, 2H), 6.96 (d, 1H,
J ¼ 7.8 Hz), 6.24 (d,1H, J ¼ 2.6 Hz), 6.00 (t,1H, J ¼ 8.5 Hz), 5.75 (d,1H,
J ¼ 2.4 Hz), 5.25 (s, 2H), 5.00 (d, 1H, J ¼ 8.4 Hz), 3.00 (d, 2H,
J ¼ 9.7 Hz), 2.25 (s, 3H), 2.00e1.95 (m, 1H), 1.62e1.60 (m, 4H), 1.12
(d, 3H, J ¼ 5.9 Hz), 0.95 (m, 1H); 13C (100 MHz, CDCl3): 10.9, 11.5,
20.2, 27.7, 30.3, 38.0, 38.1, 42.4, 44.3, 46.3, 60.3, 71.6, 73.1, 73.4,
112.2, 116.5, 123.3, 123.7, 128.8, 132.7, 138.3, 142.4, 170.8, 220.0; ESI-
MS: (MþNa) 538; Anal. Calcd for C24H26BrN3O5: C, 55.38; H, 5.04;
N, 8.10. Found: C, 55.87; H, 5.11; N, 8.12.
4.1.1.1. Compound 2. Crystalline white solid; mp: 176e178 ꢁC;
½aꢂ2D5 þ29 (c 0.5, CHCl3); IR (KBr, cmꢀ1): 3456.39, 2958.98, 2927.09,
2857.01, 2103.89, 1749.39, 1726; 1H NMR (200 MHz, CDCl3): 6.20 (d,
1H, J ¼ 1.29 Hz), 5.57 (d,1H, J ¼ 2.72 Hz), 5.00 (d,1H, J ¼ 8.8 Hz), 3.30
(t, 1H, J ¼ 9.6 Hz), 2.28 (m, 1H), 2.40 (d, 2H, J ¼ 7.5), 2.48 (m, 1H),
2.20e1.64 (m, 4H), 1.18 (d, 3H, J ¼ 7.0 Hz), 1.10 (s, 3H); 13C (100 MHz,
CDCl3): 10.12, 16.30, 28.21, 31.23, 34.51, 38.54, 41.21, 50.45, 65.45,
70.98, 80.21, 123.31, 140.10, 170.01, 220.11; Maldi mass: (MþNa)
328.
4.1.3.4. Compound 3d. Crystalline white solid; mp: 186 ꢁC;
½aꢂ2D5 þ28 (c 0.5, CHCl3); IR (KBr, cmꢀ1): 658.00, 752.38, 997.27,
1030.72, 1051.18, 1162.43, 1237.60, 1384.32, 1478.16, 1628.75,
1751.16, 2927.10, 3367.93; 1H NMR (200 MHz, CDCl3):
d 8.25 (d, 1H,
J ¼ 5.36 Hz), 7.55 (d, 1H, J ¼ 7.9 Hz), 7.25 (dd, 2H, J ¼ 0.79, 6.87 Hz),
6.95 (d, 1H, J ¼ 7.8 Hz), 6.25 (d, 1H, J ¼ 2.75 Hz), 5.95 (t, 1H,
J ¼ 8.1 Hz), 5.52 (d, 1H, J ¼ 2.5 Hz), 5.25 (s, 2H), 5.00 (d, 1H,
J ¼ 7.8 Hz), 3.25e3.00 (m, 2H), 2.00 (m, 4H), 1.95 (m, 3H), 1.25e1.00
(m, 4H), 0.95 (m, 1H); 13C (100 MHz, CDCl3): 7.22, 12.63, 20.3, 26.34,
31.40, 33.82, 34.30, 42.42, 50.00, 64.43, 69.31, 72.31, 82.3, 110.2,
115.43, 116.56, 120.4, 123.32, 127.00, 128.34, 132.00, 138.33, 159.46,
170.34, 220.32; ESI-MS: (MþNa) 538; Anal. Calcd for C24H26BrN3O5:
C, 55.82; H, 5.07; N, 8.34. Found: C, 55.47; H, 5.16; N, 8.53.
4.1.2. Synthesis of triazoles
In a typical procedure, to a solution of coronopilin azide (0.01 g,
1 mmol) in tBuOH: H2O taken in 1:1 proportion was added 1-
chloro-3-(prop-2-ynyloxy)benzene
(0.025
g, 1.2
mmol),
CuSO4.5H2O (2 mmol) and sodium ascorbate (2 mmol). The reac-
tion mixture was then stirred at room temperature for 3 h, then
filtered and extracted with ethyl acetate and water. The ethyl ace-
tate layer was then separated. The solvent was evaporated in vacuo
and the crude was subjected for column chromatography (silica gel,
100e200 mesh, elution; n-hexane/EtOAc gradient) to afford pure
product as colourless solid (0.3 g, 90%). The pure product was
4.1.3.5. Compound 3e. Off-white crystalline solid; mp: 132 ꢁC;
½aꢂ2D5 þ43 (c 0.5, CHCl3); IR (KBr, cmꢀ1): 506.02, 771.27, 1063.08,
1116.65, 1160.56, 1273.90, 1327.00, 1615.87, 1750.39, 2924.60,
3400.14; 1H NMR (200 MHz, CDCl3):
d
7.78 (s, 1H), 7.43 (d, 2H,
J ¼ 8.2 Hz), 6.73 (d, 2H, J ¼ 8.4 Hz), 6.25 (d, 1H, J ¼ 6.02 Hz), 5.50 (t,

J. Khazir et al. / European Journal of Medicinal Chemistry 82 (2014) 255e262
259
Fig. 2. Flow cytometric analysis of compound (3a) treated with PC-3 cell lines.
2H, J ¼ 9.6 Hz), 5.00 (d, 1H, J ¼ 11.3 Hz), 4.50e4.25 (m, 3H), 3.50 (m,
1H), 3.00 (d, 2H, J ¼ 10.3 Hz), 2.00e1.96 (m, 3H), 1.55e1.25 (m, 4H),
1.12 (d, 3H, J ¼ 5.9 Hz), 0.95 (m, 1H); 13C NMR (100 MHz, CDCl3):
10.12, 13.63, 13.85, 14.00, 20.23 26.68, 29.00, 29.21, 36.90, 38.13,
38.17, 43.62, 58.90, 60.47, 78.25, 80.40, 84.42, 111.71, 111.82, 121.10,
130.32, 139.71, 149.38, 169.73, 210.73; ESI-MS: (MþNa) 528; Anal.
Calcd for C25H26F3N3O5: C, 59.40; H, 5.18; N, 8.31. Found: C, 59.43;
H, 5.24; N, 8.29.
1H, J ¼ 2.75 Hz), 5.50 (m, 2H), 5.25 (s, 2H), 5.00 (d, 1H, J ¼ 7.9 Hz),
3.75 (m, 1H), 3.25 (m, 1H), 3.00 (t, 2H, J ¼ 10 Hz), 1.75 (s, 3H), 1.52 (s,
3H), 1.25e1.20 (m, 4H), 0.95 (m, 1H); 13C (100 MHz, CDCl3): 10.9,
11.5, 20.2, 25.3, 27.7, 30.3, 38.0, 38.1, 42.4, 46.3, 60.2, 72.3, 73.6, 73.6,
115.7, 120.9, 123.7, 126.6, 129.9, 138.3, 142.4, 158.8, 170.1, 220.3; ESI-
MS: (MþNa) 494; Anal. Calcd for C24H26ClN3O5: C, 61.08; H, 5.5; N,
8.90. Found: C, 61.13; H, 5.56; N, 8.83.
4.1.3.7. Compound 3g. Colourless solid; mp: 105 ꢁC; ½aꢂD25 þ56 (c
0.5, CHCl3); IR (KBr, cmꢀ1): 772.31, 1049.86, 1097.17, 1237.97,
1269.19, 1384.72, 1580.25, 1752.35, 2923.22, 3400.10; 1H NMR
4.1.3.6. Compound 3f. Crystalline white solid; mp: 138 ꢁC; ½aꢂD25 þ46
(c 0.5, CHCl3); IR (KBr, cmꢀ1): 666.78, 770.24, 825.21, 991.52,
1097.34, 1162.13, 1237.79, 1271.49, 1393.05, 1462.42, 1490.94,
(200 MHz, CDCl3):
d
8.25 (d, 1H, J ¼ 7.8 Hz), 7.85 (m, 2H), 7.50 (m,
1633.33, 1750.85, 3435.23; 1H NMR (200 MHz, CDCl3):
d
7.84 (d, 1H,
4H), 7.00 (d, 1H, J ¼ 5.6 Hz), 6.25 (d, 1H, J ¼ 2.75 Hz), 5.53e5.25 (m,
J ¼ 9.6 Hz), 7.50 (d, 2H, J ¼ 4.5 Hz), 6.98 (d, 2H, J ¼ 8.8 Hz), 6.25 (d,
3H), 5.00 (d, 1H, J ¼ 8.0 Hz), 4.20 (d, 1H, J ¼ 4.3 Hz), 3.75 (s, 1H),

260
J. Khazir et al. / European Journal of Medicinal Chemistry 82 (2014) 255e262
Fig. 3. NF-kB, p65 transcription factor assay.
3.50e3.25 (m, 1H), 3.00 (t, 2H, J ¼ 9.4 Hz), 1.54 (s, 3H), 1.25 (m, 3H),
1.20e1.00 (m, 4H), 0.95 (m, 1H); 13C (100 MHz, CDCl3): 14.10, 18.21,
19.11, 20.3, 27.37, 29.71, 30.91, 38.10, 38.83, 42.48, 44.56, 59.59,
60.83, 62.46, 79.69, 84.97, 120.96, 121.30, 123.52, 124.34, 125.37,
125.67, 125.78, 134.61, 140.32, 144.60, 153.84, 210.80; ESI-MS:
(MþNa) 510; Anal. Calcd for C28H29N3O5: C, 68.98; H, 6.00; N,
8.62. Found: C, 69.02; H, 5.95; N, 8.67.
30.31, 32.11, 38.34, 39.10, 45.05, 51.96, 59.25, 60.38, 61.54, 79.20,
83.54, 110.31,118.23, 120.12, 131.15, 140.35, 155.63, 170.51, 210.54;
ESI-MS: (MþNa) 474; Anal. Calcd for C25H29N3O4: C, 66.50; H, 6.47;
N, 9.31. Found: C, 66.54; H, 6.43; N, 9.22.
4.1.3.11. Compound 3k. Crystalline white solid; mp: 123e125 ꢁC;
½aꢂ2D5 þ13 (c 0.5, CHCl3); IR (KBr, cmꢀ1): 566.89, 666.53, 753.88,
988.09,1075.48,1118.96,1162.27,1218.56,1272.55,1391.03,1476.23,
1512.25, 1605.48, 1752.30, 2104.36, 2930.87, 3419.76; 1H NMR
4.1.3.8. Compound 3h. Colourless solid; mp: 137 ꢁC; ½aꢂD25 þ46 (c
0.5, CHCl3); IR (KBr, cmꢀ1): 754.37, 815.66, 1033.51, 1125.94,
1160.82, 1250.87, 1383.35, 1611.03, 1752.43, 2925.17, 3391.67; 1H
(200 MHz, CDCl3):
d
7.65 (s, 1H), 7.10 (dd, 2H, J ¼ 6.2, 3.4 Hz), 6.75
(dd, 2H, J ¼ 8.3, 2.6 Hz), 6.25 (d, 1H, J ¼ 2.5 Hz), 5.65 (d, 1H,
J ¼ 2.4 Hz), 5.53 (t, 1H, J ¼ 8.5 Hz), 5.00 (d, 1H, J ¼ 8.4 Hz), 4.52 (s,
3H), 3.75 (s,1H), 3.54e3.50 (m, 2H), 3.00 (d, 2H, J ¼ 9.6),1.63 (s, 6H),
1.25e1.20 (m, 4H); 13C (100 MHz, CDCl3): 10.90, 11.52, 15.55, 20.32,
27.73, 30.31, 38.00, 38.14, 42.45, 42.70, 46.35, 60.73, 73.12, 113.42,
117.17, 123.22, 123.70, 126.23, 126.65, 129.98, 143.20, 146.52, 170.22,
220.23; ESI-MS: (MþNa) 473; Anal. Calcd for C25H30N4O4: C, 66.65;
H, 6.71; N, 12.44. Found: C, 66.68; H, 6.74; N, 12.33.
NMR (200 MHz, CDCl3):
d
8.12 (s,1H), 7.10 (d,1H, J ¼ 3.8 Hz), 6.92 (d,
1H, J ¼ 8.5 Hz), 6.75 (d, 1H, J ¼ 7.6 Hz), 6.22 (d, 1H, J ¼ 7.19 Hz), 6.00
(t, 1H, J ¼ 9.59 Hz), 5.65 (d, 1H, J ¼ 2.28 Hz), 5.25 (s, 2H), 5.00 (d, 1H,
J ¼ 8.2 Hz), 3.00 (t, 2H, J ¼ 2.85 Hz), 2.10 (m, 1H), 1.84 (m, 3H), 1.75
(m, 4H), 1.25e1.20 (m, 9H), 0.95 (m, 1H); 13C (100 MHz, CDCl3):
14.18, 16.28, 27.30, 29.70, 37.44, 38.70, 44.55, 50.83, 59.55, 60.97,
62.29, 81.66, 84.97, 111.60, 111.82, 112.07, 121.38, 122.12, 124.21,
126.83, 126.91, 127.66, 130.66, 136.97, 140.34, 156.11,170.14, 210.97;
ESI-MS: (MþNa) 516; Anal. Calcd for C28H35N3O5: C, 64.36; H, 6.48;
N, 12.02. Found: C, 64.34; H, 6.40; N, 12.12.
4.1.3.12. Compound 3l. Light brown solid; mp: 153e156 ꢁC;
½aꢂ2D5 þ27 (c 0.5, CHCl3); IR (KBr, cmꢀ1): 666.56, 751.28, 991.34,
1019.59, 1118.30, 1162.16, 1237.31, 1273.62, 1384.25, 1456.95,
1504.14, 1595.87, 1752.76, 2925.51, 3400.00; 1H NMR (200 MHz,
4.1.3.9. Compound 3i. Crystalline white solid; mp: 128 ꢁC; ½aꢂD25 þ24
(c 0.5, CHCl3); IR (KBr, cmꢀ1): 668.84, 754.80, 815.70, 989.38,
1121.27, 1162.34, 1189.07, 1237.92, 1273.08, 1336.93, 1391.95,
1494.14, 1601.60, 1752.83, 2941.26, 3434.86; 1H NMR (200 MHz,
CDCl3):
d
7.75 (s, 1H), 7.50 (d, 1H, J ¼ 8.5 Hz), 7.20 (t, 1H, J ¼ 8.4 Hz),
6.70 (dd, 2H, J ¼ 8.3, 2.6 Hz), 6.25 (d, 1H, J ¼ 2.6 Hz), 5.50 (d, 1H,
J ¼ 8.5 Hz), 5.45 (t, 1H, J ¼ 2.4 Hz), 5.00 (d, 1H, J ¼ 8.4 Hz), 4.95 (m,
1H), 4.53 (d, 2H, J ¼ 5.4 Hz), 3.75 (s, 1H), 3.50 (m, 1H), 3.00 (t, 2H,
J ¼ 3.6 Hz), 1.58 (s, 3H), 1.25 (d, 3H, J ¼ 3.4 Hz), 1.00 (m, 4H), 0.95 (m,
1H); 13C (100 MHz, CDCl3): 10.91, 11.53, 27.75, 30.33, 38.00, 38.15,
42.47, 42.52, 46.35, 60.00, 73.00, 73.12, 115.15, 116.83, 118.86,
123.76, 125.21, 130.55, 130.77, 138.36, 143.20, 154.92, 170.56,
220.23; ESI-MS: (MþNa) 477; Anal. Calcd for C24H27FN4O4: C,
63.42; H, 5.99; N, 12.33. Found: C, 63.46; H, 5.90; N, 12.28.
CDCl3):
d
7.75 (s, 1H), 7.25 (d, 2H, J ¼ 5.8 Hz), 7.00 (d, 2H, J ¼ 8.3 Hz),
6.25 (d, 1H, J ¼ 8.5 Hz), 5.63 (t, 1H, J ¼ 9.8 Hz), 5.25 (d, 2H,
J ¼ 9.7 Hz), 5.00 (d, 1H, J ¼ 8.1 Hz), 3.75 (t, 1H, J ¼ 5.6 Hz), 3.54e3.52
(m, 1H), 3.10 (t, 2H, J ¼ 10.1 Hz), 2.55 (m, 1H), 2.25 (s, 3H), 1.52 (s,
3H), 1.25 (d, 3H, J ¼ 3.4 Hz), 1.00 (m, 4H), 0.95 (m, 1H); 13C
(100 MHz, CDCl3): 14.18, 16.28, 22.20, 29.70, 37.44, 38.70, 44.55,
50.83, 60.90, 62.32, 84.97, 111.60, 111.82, 121.07, 121.38, 122.12,
124.23, 126.83, 126.94, 127.06, 130.06, 140.34, 156.11, 170.84, 210.97;
ESI-MS: (MþNa) 474; Anal. Calcd for C25H29N3O4: C, 68.95; H, 6.71;
N, 9.65. Found: C, 68.79; H, 6.75; N, 9.69.
4.1.3.13. Compound 3m. Colourless solid; mp: 169e170 ꢁC;
½aꢂ2D5 þ31 (c 0.5, CHCl3); IR (KBr, cmꢀ1): 581.37, 770.36, 990.46,
1053.05, 1118.05, 1219.31, 1331.19, 1394.30, 1467.22, 1751.06,
ꢁ
4.1.3.10. Compound 3j. Colourless solid; mp: 136 C; ½aꢂ2D5 þ16 (c
0.5, CHCl3); IR (KBr, cmꢀ1): 514.99, 565.72, 754.27, 815.98, 990.45,
1052.85, 1273.53, 1392.88, 1450.56, 1510.34, 1753.97, 2927.74,
2926.80, 3400.12; 1H NMR (200 MHz, CDCl3):
d 7.75 (s, 1H),
7.35e7.25 (m, 4H), 6.54 (d, 1H, J ¼ 3.2 Hz), 6.33 (d, 1H, J ¼ 2.69 Hz),
5.65 (d, 1H, J ¼ 2.4 Hz), 5.52 (s, 2H), 5.00 (d, 1H, J ¼ 8.5 Hz), 3.65 (s,
1H), 3.41e3.35 (m, 1H), 3.00 (t, 2H, J ¼ 9.8 Hz), 2.00 (m, 1H), 1.63 (s,
3H), 1.25e1.20 (d, 3H, J ¼ 3.4 Hz), 0.95 (m, 1H); 13C NMR (100 MHz,
CDCl3): 14.18, 16.20, 27.41, 28.31, 37.40, 38.66, 42.78, 44.18, 59.52,
60.41, 79.74, 84.90, 101.91, 110.96, 113.18, 122.17, 123.64, 124.77,
129.08, 130.52, 134.56, 140.20, 144.31, 170.21, 211.03; ESI-MS:
3400.66; 1H NMR (200 MHz, CDCl3):
d
7.80 (d, 1H, J ¼ 7.8 Hz), 7.24
(d, 2H, J ¼ 8.2 Hz), 6.75 (d, 2H, J ¼ 8.4 Hz), 6.25 (d, 1H, J ¼ 12 Hz),
5.50 (t, 2H, J ¼ 2.3 Hz), 5.25 (s, 2H), 5.00 (d, 1H, J ¼ 7.9 Hz), 3.75 (t,
1H, J ¼ 10.08 Hz), 3.50e3.25 (m, 1H), 3.00 (t, 2H, J ¼ 9.58 Hz), 2.25
(d, 3H, J ¼ 6.4 Hz), 1.50 (s, 3H), 1.25 (d, 3H, J ¼ 3.4 Hz), 1.00 (m, 4H),
0.95 (m, 1H); 13C (100 MHz, CDCl3): 10.12, 14.11, 14.31, 20.49, 27.42,

J. Khazir et al. / European Journal of Medicinal Chemistry 82 (2014) 255e262
261
(MþNa) 537; Anal. Calcd for C26H27BrN4O4: C, 57.89; H, 5.05; N,
3.03 (d, 3H, J ¼ 10.1 Hz), 1.75e1.70 (m, 3H), 1.25e1.20 (m, 4H); 13C
(100 MHz, CDCl3): 10.94, 11.50, 15.56, 27.73, 30.35, 38.05, 38.10,
42.46, 42.75, 46.36, 60.34, 73.54, 73.10,115.23,115.14,116.30,116.35,
123.23, 123.70, 138.36, 143.27, 143.30, 170.21, 220.09; ESI-MS:
(MþNa) 430; Anal. Calcd for C23H25N3O4: C, 55.82; H, 5.07; N,
8.14. Found: C, 55.86; H, 5.13; N, 8.20.
10.39. Found: C, 57.85; H, 5.10; N, 10.43.
4.1.3.14. Compound 3n. Crystalline white solid; mp: 171 ꢁC;
½aꢂ2D5 þ35 (c 0.5, CHCl3); IR (KBr, cmꢀ1): 581.37, 770.36, 990.46,
1053.05, 1118.05, 1219.31, 1331.19, 1394.30, 1467.22, 1751.06,
2926.80, 3400.12; 1H NMR (200 MHz, CDCl3):
d 7.85 (s, 1H),
7.25e7.35 (m, 5H), 6.54 (d, 1H, J ¼ 3.2 Hz), 6.33 (d, 1H, J ¼ 2.69 Hz),
5.65 (d, 1H, J ¼ 2.4 Hz), 5.52 (s, 2H), 5.05 (d, 1H, J ¼ 8.5 Hz), 3.65 (s,
1H), 3.41e3.35 (m, 1H), 3.05 (t, 2H, J ¼ 9.8 Hz), 2.00 ( d, 3H,
J ¼ 6.2 Hz), 1.63 (s, 3H), 1.25e1.20 (m, 4H); 13C (100 MHz, CDCl3):
14.18, 16.20, 20.31, 27.41, 28.31, 37.40, 38.66, 42.78, 44.18, 59.52,
60.41, 79.74, 84.90, 101.91, 110.96, 113.18, 122.17, 123.64, 124.77,
129.08, 130.52, 134.56, 140.20, 144.31, 170.21, 211.03; ESI- MS:
(MþNa) 561; Anal. Calcd for C26H27BrN4O4: C, 57.89; H, 5.05; N,
10.39. Found: C, 57.80; H, 5.10; N, 10.48.
4.1.3.19. Compound 3s. Crystalline white solid; mp: 141e143 ꢁC;
½aꢂ2D5 þ32 (c 0.5, CHCl3); IR (KBr, cmꢀ1): 720.46, 1024.13, 1124.26,
1156.30, 1249.23, 1334.53, 1524.46, 1634.37, 1756.43, 2945.21,
3370.13; 1H NMR (200 MHz, CDCl3):
d 7.95 (s, 1H), 6.23 (d, 1H,
J ¼ 2.65 Hz), 5.91 (t, 1H, J ¼ 9.71 Hz), 5.75 (d, 1H, J ¼ 2.24 Hz), 5.00
(d, 1H, J ¼ 8.28 Hz), 3.31 (d, 1H, J ¼ 1.51 Hz), 3.13 (d, 2H, J ¼ 9.58 Hz),
2.75 (t, 2H, J ¼ 7.76 Hz), 2.00 (q, 2H, J ¼ 8.4 Hz), 1.67e1.75 (m, 6H),
1.351e1.41 (m, 9H); 13C (100 MHz, CDCl3): 11.01, 17.43, 24.93, 37.28,
41.37, 41.76, 54.62, 80.59, 86.04, 125.93, 127.12, 128.33, 129.18,
131.65,135.07,150.35,153.97, 154.86,171.73,186.00; ESI-MS: (MþK)
412; Anal. Calcd for C20H27N3O4: C, 68.13; H, 7.15; N, 8.51. Found: C,
68.16; H, 7.20; N, 8.57.
4.1.3.15. Compound 3o. Colourless solid; mp: 122e126 ꢁC;
½aꢂ2D5 þ22 (c 0.5, CHCl3); IR (KBr, cmꢀ1): 758.05, 1048.58,
1118.82,1160.05, 1254.15, 1384.85, 1515.99, 1611.44, 1751.28,
2928.55, 3394.42; 1H NMR (200 MHz, CDCl3):
d 7.81 (s, 1H), 6.75 (d,
2H, J ¼ 7.8 Hz), 6.51 (d, 2H, J ¼ 8.2 Hz), 6.25 (s, 1H), 5.62 (s, 1H), 5.53
(t, 1H, J ¼ 9.8 Hz), 5.10 (d, 1H, J ¼ 8.4 Hz), 4.51 (s, 2H), 3.75 (s, 2H),
3.52e3.48 (m, 2H), 3.05 (d, 2H, J ¼ 11 Hz), 2.00 (d, 3H, J ¼ 6.2 Hz),
1.75e1.70 (s, 3H), 1.25e1.20 (m, 4H); 13C (100 MHz, CDCl3): 10.95,
11.54, 15.50, 27.73, 30.35, 38.23, 38.12, 42.46, 42.77, 46.32, 60.54,
73.56, 73.10, 82.52, 114.11, 114.25, 116.36, 116.26, 122.47, 123.78,
138.34, 139.20, 143.26, 145.94, 170.45, 220.34; ESI-MS: 557; Anal.
Calcd for C26H29F3N3O5: C, 66.50; H, 6.47; N, 9.31. Found: C, 66.55;
H, 6.54; N, 9.36.
5. Biological assays
5.1. Evaluation of in vitro anti-cancer activity
The effect of 1,2,3-triazole derivatives of coronopilin on the
growth of cancer cell lines was evaluated according to the pro-
cedure adopted by the National Cancer Institute for in vitro anti-
cancer drug screening that uses the protein-binding dye Sul-
phorhodamine B to estimate cell growth [25]. Briefly, cells in
their log phase of growth were harvested, counted and seeded
(104 cells/well in 100 mL medium) in 96-well microtitre plates.
After 24 h of incubation at 37 ꢁC and 5% CO2 to allow cell
attachment, cultures were treated with varying concentrations
4.1.3.16. Compound 3p. Crystalline white solid; mp: 125 ꢁC;
½aꢂ2D5 þ36 (c 0.5, CHCl3); IR (KBr, cmꢀ1): 737.25, 1019.29, 1128.80,
1154.25, 1269.45, 1365.34, 1534.85, 1627.47,1759.20, 2956.52,
3378.46; 1H NMR (200 MHz, CDCl3):
d 7.73 (s, 1H), 7.00 (t, 2H,
(0.1e10 mM) of test samples made with 1:10 serial dilutions. Four
J ¼ 6.5 Hz), 6.72e6.68 (m, 2H), 6.25 (s, 1H), 5.65 (s, 1H), 5.53 (t, 1H,
J ¼ 9.8 Hz), 5.15 (d, 1H, J ¼ 8.4 Hz), 4.52 (s, 2H), 3.75 (s, 1H),
3.53e3.48 (m, 1H), 3.00 (d, 3H, J ¼ 10.1 Hz), 2.05 (t, 2H, J ¼ 6.2 Hz),
1.65e1.60 (m, 3H), 1.25e1.20 (m, 4H); 13C (100 MHz, CDCl3): 10.90,
11.53, 15.55, 27.70, 30.34, 38.00, 38.13, 42.40, 42.75, 46.37, 60.33,
73.28, 73.15, 115.14, 115.16, 116.24, 116.33, 123.28, 123.71, 138.32,
143.21, 143.45, 170.13, 220.34; ESI-MS: (MþNa) 477; Anal. Calcd for
replicate wells were set up for each experimental condition. Test
samples were left in contact with the cells for 48 h under same
conditions. Thereafter cells were fixed with 50% chilled TCA and
kept at 4 ꢁC for 1 h, washed and air-dried. Cells were stained
with Sulphorhodamine B dye. The adsorbed dye was dissolved in
tris-buffer and the plates were gently shaken for 10 min on a
mechanical shaker. The optical density (OD) was recorded on
ELISA reader at 540 nm. The cell growth was calculated by
subtracting mean OD value of the respective blank from the
mean OD value of experimental set. Percentage of growth in the
presence of test material was calculated considering the growth
in the absence of any test material as 100% and the results are
reported in terms of IC50 values.
C
24H27FN4O4: C, 63.42; H, 5.99; N,12.33. Found: C, 63.46; H, 5.90; N,
12.41.
4.1.3.17. Compound 3q. Pale yellow solid; mp: 146e147 ꢁC;
½aꢂ2D5 þ18 (c 0.5, CHCl3); IR (KBr, cmꢀ1): 727.16, 1019.45,
1110.76,1167.32, 1256.25, 1358.53, 1551.23, 1620.27, 1767.23,
2956.51, 3370.26; 1H NMR (200 MHz, CDCl3):
d 8.00 (d, 1H,
J ¼ 5.5 Hz), 7.95 (d, 2H, J ¼ 6.6 Hz), 7.50 (dd, 3H, J ¼ 7.55, 10.01 Hz),
6.35 (d, 1H, J ¼ 2.6 Hz), 5.76 (q, 2H, J ¼ 5.92 Hz), 5.15 (d, 1H,
J ¼ 8.2 Hz), 3.83 (s, 1H), 3.45e3.55 (m, 1H), 3.24 (t, 2H, J ¼ 8.8 Hz),
1.75e1.69 (m, 3H), 1.25e1.20 (m, 8H); 13C (100 MHz, CDCl3):
11.00,14.32, 17.41, 20.31, 22.97, 37.24, 39.35, 41.36, 54.34, 85.36,
110.21, 116.08, 116.30, 124.38, 125.97, 128.97, 129.06, 129.21, 150.29,
154.77, 155.66, 162.97, 165.47, 172.18, 185.97; ESI-MS: (MþNa) 489;
Anal. Calcd for C25H30N4O4: C, 64.32; H, 7.29; N, 11.25. Found: C,
64.36; H, 7.34; N, 11.34.
5.2. DNA cell cycle analysis
Effect of Compound-3a on DNA content by cell cycle phase
distribution was assessed using PC-3 cells by incubating the cells
1 ꢃ 106 mL/well with compound-3a (1, 5 and 10
mM each) for
24 h. The cells were then washed twice with ice-cold PBS, har-
vested, fixed with ice-cold PBS in 70% ethanol and stored at
20 ꢁC for 30 min [26]. After fixation, these cells were incubated
with RNase A (0.1 mg/mL) at 37 ꢁC for 30 min, stained with
propidium iodide (50 mg/mL) for 30 min on ice in dark, and
then measured for DNA content using BD-LSR flow cytometer
(Becton Dickinson, USA) equipped with electronic doublet
discrimination capability using blue (488 nm) excitation from
Argon laser. Data were collected in list mode on 10,000 events
for FL2-A vs FL2-W.
4.1.3.18. Compound 3r. Colourless solid; mp: 122e123 ꢁC; ½aꢂD25 þ43
(c 0.5, CHCl3); IR (KBr, cmꢀ1): 746.15, 1034.26, 1123.73, 1154.25,
1260.32, 1378.96, 1510.90,1617.35, 1759.24, 2934.05, 3386.56; 1H
NMR (200 MHz, CDCl3):
d
7.76 (s, 1H), 6.74 (d, 2H, J ¼ 8.5 Hz), 6.65
(d, 2H, J ¼ 8.4 Hz), 6.23 (s, 1H), 5.63 (s, 1H), 5.55 (t, 1H, J ¼ 9.8 Hz),
5.10 (d, 1H, J ¼ 8.4 Hz), 4.53 (s, 2H), 4.75 (s, 3H), 3.52e3.48 (m, 2H),

262
J. Khazir et al. / European Journal of Medicinal Chemistry 82 (2014) 255e262
5.3. NF-
kB (p65), transcription factor assay
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Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.05.053.