Design and synthesis of (Z/E)-2-phenyl/H-3-styryl-2H-chromene derivatives as antimicrotubule agents

Article abstract of DOI:10.1007/s12039-018-1520-6

Abstract: Two new series of compounds (Z?/?E)-2-phenyl/H-3-Styryl-2H-Chromenes 9(a-r) and 10(a-s) were synthesized and evaluated in vitro cytotoxic activities against four cancer cell lines. One compound, (Z)-8-ethoxy-3-(4-methoxystyryl)-2-phenyl-2H-chrom

Full text of DOI:10.1007/s12039-018-1520-6

J. Chem. Sci. (2018) 130:127
© Indian Academy of Sciences
https://doi.org/10.1007/s12039-018-1520-6
REGULAR ARTICLE
Design and synthesis of (Z/E)-2-phenyl/H-3-styryl-2H-chromene
derivatives as antimicrotubule agents
a
a,∗
a
a
b
P PANDA , S NAYAK , S BHAKTA , S MOHAPATRA and T R MURTHY
a
Department of Chemistry, Ravenshaw University, Cuttack 753 003, Odisha, India
Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, Telangana,
b
India
E-mail: sabitanayak18@gmail.com; ramslin1312@gmail.com
MS received 29 March 2018; revised 4 July 2018; accepted 5 July 2018
Abstract. Two new series of compounds (Z/E)-2-phenyl/H-3-Styryl-2H-Chromenes 9(a-r) and 10(a-s) were
synthesized and evaluated in vitro cytotoxic activities against four cancer cell lines. One compound, (Z)-8-ethoxy-
3-(4-methoxystyryl)-2-phenyl-2H-chromene (9g) was found to be the most active among the tested compounds
in HeLa cell lines (IC 10μM). Compound 9g arrested cells at G2/M phase, disrupted microtubule network,
5
0
accumulated tubulin in the soluble fraction and manifested an increased expression of the G2/M marker, Cyclin
B1.
Keywords. Combretastatin; candenatenin E; chromene; resveratrol; Wittig reaction; cytotoxicity; antimitotic
activity; chromene.
1
. Introduction
chemistry, a significant source of inspiration for the
design of structural analogues with improved pharma-
cological profile of action acting in synergy to inhibit
cancer tumor growth. Therefore, the search for potent,
safe and selective hybrid anticancer drug is a crucial
aspect of modern cancer research. In this connection,
current literature report reveals that 2H-chromene, a
small molecule natural product and their derivatives
Cancer is a collection of different life-threatening
diseases, in which a group of cells display uncontrolled
growth in a body. According to the statistics, cancer
is the second most common cause of death world-
wide after cardiovascular diseases. The corresponding
incidence and mortality statistics shows that it is grow-
5
1,2
6
7
8
ing in developing as well as developed countries.
such as KCN1, S14161, BENC-511, Seselin, Xan-
9
10
11
Although the cancer research has led to a number of
new and effective solutions, the medicines used as treat-
ments have clear limitations and unfortunately, cancer
is projected as the primary cause of death. Existing
drugs are effective and cytotoxic and thus exhibit severe
side effects, particularly on normal proliferating tissues
such as the haematopoietic system. The use of novel
and improved chemopreventive and chemotherapeutic
agents for the prevention and treatment of cancer is
on the rise. Natural products have always afforded a
thylein, Lonchocarpin, Acronycine, and (Z)-(2H-
Chromene-3-yl) methylene azolidinones etc., also
show potent anticancer activities. Candenatenin E is a
chromene derivative isolated from the heartwood of D.
candenatensis exhibits potent cytotoxic activity against
HT-29 (colon cancer), KB (human oral cancer), MCF-
12
13
7
(breast adenocarcinoma), and HeLa (human cervical
7,14
cancer) cell lines.
A key aspect is that the lipophilic
nature of the benzopyran derivatives helps to cross the
cell membrane more easily.
3
rich source of such agents. Nowadays, there is a huge
scientific and commercial interest in the discovery of
new hybrid anticancer drugs, with their ability to inter-
act with more than one target, represent, in medicinal
From the literature survey we also found that cis
stilbenebasednaturalproductcombretastatinA4, exhib-
ited a significant role in clinical applications, par-
4
14
ticularly acting as anticancer agent. Various hybrid
*
For correspondence
Electronic supplementary material: The online version of this article (https://doi.org/10.1007/s12039-018-1520-6) contains
supplementary material, which is available to authorized users.
0123456789().: V,-vol

127 Page 2 of 15
J. Chem. Sci. (2018) 130:127
combretastatin analogs have been designed and
Table 1. Reaction time and yield of products.
synthesized; among them, cis restricted heterocycles,
Entry R R1 R2 Time(h) Producta Yield (%)
b
fused heterocycles and nonsubstituted aromatic ring
analogs of CA4 are most important.1
5–33
Not only cis
1
2
3
4
5
6
Ph
H
H
12
12
12
16
18
12
5a
5b
5c
5d
5e
5f
89
89
88
83
82
85
stilbene but also trans-stilbene based natural product
and synthetic analogs show pronounced anticancer and
Ph OMe H
Ph OEt
Ph
Ph
H
Cl
Br
H
H
antiapoptotic activities; among them, resveratrol and its
analogs are most important.14,34
Ph Cl Cl
Nowadays, the preparation of hybrid compounds that
possess important skeletons present in drugs/clinical
agents has become an important research area for medic-
35
inalchemists. Thiscanbeaneffectiveapproachtopos- coated (0.2 mm layer thickness) with Kieselgel 60 F254, with
visualization by ultraviolet light and product purification by
sibly avoid physicochemical/pharmacokinetic/toxicity
flash column chromatography. Mass spectra were determined
problems that appear in the later stages of devel-
36
on an Agilent Technology (HP) mass spectrometer operating
at an ionization potential of 70 eV. The elemental analysis
for C, H and N was carried out with an Elementary Analysen
system GmbH VarioEL. All reagents and solvents used in this
study were commercially available (from Sigma-Aldrich) and
were used without further purification. The intermediate com-
pounds were prepared according to the literature methods.
opment. All these observations and our interest in
chromene and stilbene derivatives prompted us to
explore a series of new compounds, (Z)-2-phenyl/H-
3
3
-styryl-2H-chromenes 9(a–r) and (E)-2-phenyl/H-
-styryl-2H-chromenes 10(a–s) as hybrid analogs of
candenatenin E 1, combretastatin A4(CA4) and resver-
atrol 2 as potential anticancer agents.
Herein we have designed the molecules on the basis
of SAR studies and explored their cytotoxicity in four
different cancer cell lines such as HeLa, MCF-7, A549
and DU145. Among the compounds tested, 9g displayed
the most potent antiproliferative activity against HeLa
2
.2 Synthesis of 2-phenyl-2H-chromene-3-
carboxaldehyde 5(a–f) (Table 1)
Salicylaldehyde (1 equiv.) and cinnamaldehyde (1.1 equiv.)
were taken in a round bottom flask and anhyd. DMSO (7 mL)
cells with IC 10.62 μM. Further, we have characterized
50
the antiproliferative mechanism of 9g. In addition, the was added to it followed by pyrrolidine (0.2 equiv.). Then
structural features of the compounds along with their it was stirred in an argon atmosphere at room temperature.
antiproliferative activity have been discussed.
The reaction was monitored by TLC and was found to be
completed after 12 h. 50 mL of water was added to it and
then extracted with ethylacetate. Organic layers were sepa-
rated out and dried over Na2SO4, filtered and concentrated in
rotavapour and purified by column chromatography on silica
gel (200–330 mesh) to afford the desired product.
2
. Experimental
2.1 Materials and methods
2.2.1 2-Phenyl-2H-chromene-3-carboxaldehyde(5a) Prepa
red from 3a (Salicylaldehyde) and 4 (Cinnamaldehyde).Yel
All reactions were carried out under a positive pressure of
argon and with oven-dried glassware. Melting points are
uncorrected and were determined with SMP10 digital melting
point apparatus using open capillary tubes. Proton magnetic
◦
−1
low solid, M.p. 72−74 C. IR(KBr) cm :3048, 2820, 2707,
1
1
670, 1570, 1457, 1216, 1102, 996, 768, 612, 520. H NMR
(
7
(
400 MHz, CDCl3): δ 9.66 (s, 1H), 7.42 (s, 1H), 7.37–
.35 (m, 2H), 7.30–7.25 (m, 5H), 6.96–6.87 (m, 2H), 6.35
s, 1H). 13C NMR (CDCl3, 100 MHz): δ 190.1, 154.9,
1
resonance ( H NMR) spectra were recorded using Bruker
400 spectrometers. Chemical shifts are reported in parts per
140.8, 139.1, 133.7, 129.5, 128.7, 126.8, 121.8, 120.0, 117.1,
million (ppm) relative to internal standards (tetramethylsi-
lane, δH = 0.00; CDCl3, δH = 7.26). Data are presented
as follows: chemical shift (δ, ppm), multiplicity (s = sin-
glet, d = doublet, t = triplet, q = quadruplet, m = multiplet,
7
4.2. Anal. Calculated for C6H12O2: C, 81.34; H, 5.12%.
+
Found: C, 81.35; H, 5.15%. ESI-HRMS [M + Na] : Calcd
for C16H12O2Na: 259.0730, Found: 259.0730.
dd = doublet of doublet, br = broad), coupling constant (J) 2.2.2 8-Methoxy-2-phenyl-2H-chromene-3-carboxaldehyde
values are presented in Hz, integration. Carbon magnetic res- (5b) Prepared from 3b (3-Methoxy salicylaldehyde) and 4
1
3
◦
onance ( C NMR) spectra were recorded using Bruker 400 (Cinnamaldehyde).Yellow solid, M.p. 117−119 C. IR (KBr)
−1
spectrometers. Chemical shifts are reported in parts per mil- cm :3051, 2908, 2811, 2720, 1657, 1631, 1573, 1378,
lion (ppm) relative to internal standard (tetramethylsilane, 1255, 1210, 1093, 964, 892, 763, 723, 691, 581, 510. H
1
δC = 0.00; CDCl3, δC = 77.00). Reactions were monitored NMR (400 MHz, CDCl3): δ 9.67 (s, 1H), 7.40 (s, 1H),
with analytical Thin Layer Chromatography (TLC) which 7.38-7.36 (m, 2H), 7.28–7.24 (m, 3H), 6.94–6.88 (m, 3H),
was carried out using Merck commercial aluminium sheets 6.44 (s, 1H), 3.84 (s, 3H). 13C NMR (CDCl3, 100 MHz): δ

J. Chem. Sci. (2018) 130:127
Page 3 of 15 127
1
1
90.3, 148.6, 144.2, 141.1, 139.1, 134.1, 128.7, 128.6, 126.7,
21.7, 121.4, 120.9, 116.2, 74.3, 56.4. Anal. Calculated for
Table 2. Reaction time and yield of products.
Entry R R1 R2 Time(h) Producta Yield (%)
b
C17H14O3: C, 76.68; H, 5.30%. Found: C, 76.69; H, 5.33%.
+
ESI-HRMS [M + Na] : Calcd for C17H14O3Na: 289.0835,
ꢀ
1
2
H OMe H
H OEt
12
12
5 b
89
87
Found: 289.0837.
ꢀ
H
5 c
2.2.3 8-Ethoxy-2-phenyl-2H-chromene-3-carboxaldehyde
(
5c) Prepared from 3c (3-Ethoxy salicylaldehyde) and 4
◦
(
Cinnamaldehyde).Yellow solid, M.p. 98−100 C. IR (KBr)
−1
cm :2974, 2807, 1748, 1664, 1627, 1609, 1469, 1376, 1256,
2.3 Synthesis of 2H-chromene-3-carbaldehydes
1
1
218, 1098, 1005, 902, 754, 689, 642, 615, 521. H NMR
5
’(b–c) (Table 2)
(400 MHz, CDCl3): δ 9.70 (s, 1H), 7.41 (s, 1H), 7.40–7.37
(
m, 2H), 7.30–7.26 (m, 3H), 6.96–6.88 (m, 3H), 6.46 (s,
3
-methoxysalicylaldehyde (1 equiv.) in dioxane (10 mL)
1
H), 4.08 (q, J = 8.0 Hz, 2H), 1.40 (t, J = 8.0 Hz, 3H).
C NMR (CDCl3, 100 MHz): δ 190.4, 147.9, 144.7, 141.3,
39.1, 134.1, 128.6, 128.5, 126.6, 121.7, 121.5, 121.2, 118.1,
3.9, 65.1, 14.9.Anal. Calculated for C18H16O3: C, 77.12; H,
was taken to it K2CO3(0.2 equiv.) and acrolein (2.2 equiv.)
was added and refluxed. The reaction was monitored by
TLC. After 2 h, the reaction mixture was poured into water
1
3
1
7
5
(100 mL). The solution was extracted with ethylacetate
+
.75%; Found: C, 77.15; H, 5.77%. ESI-HRMS [M + Na] :
(
30 mL × 3). The combined organic layers were washed
Calcd for C18H16O3Na: 303.0992, Found: 303.0985.
2
with NaOH (30 mL) and water (30 mL) successively. Then
the organic layers were dried over anhydrous Na2SO4. The
solvent was evaporated under vacuum; the crude residue
was purified by column chromatography on silica gel (100–
200 mesh) to afford the desired product.
.2.4 6-Chloro-2-phenyl-2H-chromene-3-carboxaldehyde
(
5d) Prepared from 3d (5-Chloro salicylaldehyde) and 4
◦
(
(
Cinnamaldehyde). Faint yellow solid, M.p. 129−131 C. IR
−1
KBr) cm :3058, 2934, 2824, 2714, 1891, 1819, 1677, 1631,
1
586, 1560, 1482, 1411, 1384, 1307, 1203, 1158, 1132, 1067, 2.3.1 8-methoxy-2H-chromene-3-carbaldehyde (5’b) Yellow
1
◦
−1
9
57, 814, 691, 626, 522. H NMR (400 MHz, CDCl3): δ 9.67 solid, M.p. 82−84 C. IR(KBr) cm : 2974, 2883, 2818,
(
s, 1H), 7.36 (s, 1H), 7.34–7.22 (m, 7H), 6.82 (d, J = 8.0 Hz, 2733, 2364, 2325, 1663, 1560, 1475, 1339, 1216, 1093, 1002,
H), 6.34 (s, 1H). 13C NMR (CDCl3, 100 MHz): δ 189.8, 886, 782, 723, 704, 594, 490. H NMR (400 MHz, CDCl ):
1
1
1
1
7
3
53.3, 139.3, 138.5, 134.6, 133.1, 128.9, 128.7, 128.5, 126.8, δ 9.59 (s, 1H), 7.26 (s, 1H), 6.97–6.92 (m, 2H), 6.86 (dd,
1
3
26.7, 121.2, 118.6, 74.5. Anal.Calcd for C16H11ClO2: C,
J = 8.0 Hz, 4.0 Hz, 1H), 5.11 (s, 2H), 3.90 (s, 3H). C NMR
0.99; H, 4.10%. Found: C, 71.02; H, 4.13%.ESI-HRMS (100 MHz, CDCl ): δ 189.9, 148.1, 145.0, 141.3, 131.8,
3
+
[
M + Na] : Calcd for C16H11ClO2Na: 293.0340, Found: 121.7, 121.3, 121.1, 115.5, 63.7, 56.2. Anal.Calcd for C H
1
1
10
2
93.0338.
3
O : C, 69.46; H, 5.30%. Found: C, 69.49; H, 5.32%.GCMS
m/z: Calcd for C11H10O3: 190.0, Found: 190.2.
2
.2.5 6-Bromo-2-phenyl-2H-chromene-3-carboxaldehyde
(
5e) Prepared from 3e (5-Bromo salicylaldehyde) and 4 (Cin- 2.3.2 8-Ethoxy-2H-chromene-3-carbaldehyde (5’c) Yellow
◦
◦
−1
namaldehyde). Yellow solid, M.p. 137−139 C. IR (KBr) solid, M.p. 89−91 C. IR (KBr) cm : 2974, 2883, 2818,
−1
cm : 3058, 2934, 2824, 2714, 1891, 1819, 1677, 1631, 1586, 2733, 2364, 2325, 1663, 1560, 1475, 1339, 1216, 1093, 1002,
560, 1482, 1411, 1384, 1307, 1203, 1158, 1132, 1067, 957, 886, 782, 723, 704, 594, 490. H NMR (400 MHz, CDCl ): δ
1
1
8
1
1
1
1
3
1
14, 691, 626, 522. H NMR (400 MHz, CDCl3): δ 9.66 (s, 9.60 (s, 1H), 7.26 (s, 1H), 6.96 (dd, J = 8.0Hz, 4.0 Hz, 1H),
H), 7.39–7.27 (m, 8H), 6.78 (d, J = 8.0 Hz, 1H), 6.34 (s, 6.91 (t, J = 8.0 Hz, 1H), 6.85 (dd, J = 4.0 Hz, 4.8Hz, 1H),
1
3
H). C NMR (CDCl3, 100 MHz): δ 189.9, 153.9, 139.2, 5.11 (s, 2H), 4.12 (q, J = 4.0 Hz, 2H), 1.47 (t, J = 4.8 Hz,
38.6, 136.2, 134.7, 131.6, 129.1, 128.8, 126.9, 121.9, 119.2, 3H). 13C NMR (100 MHz, CDCl ): δ 189.9, 147.4, 145.4,
3
13.8, 74.6. Anal. CalcdforC16H11BrO2:C, 60.98;H, 3.52%. 141.5, 131.7, 121.6, 121.3, 117.0, 64.7, 63.6, 14.8. Anal.
+
Found: C, 61.01; H, 3.54%.ESI-HRMS [M + Na] : Calcd for Calcd for C H O : C, 70.57; H, 5.92%. Found: C, 70.59;
1
2
12
3
C16H11BrO2Na: 336.9835, Found: 336.9833.
.2.6 6, 8-Dichloro-2-phenyl-2H-chromene-3-carboxalde
12 12 3
H, 5.95%. GCMS m/z: Calcd for C H O : 204.0, Found:
204.1.
2
hyde (5f) Prepared from 3f (3,5-dichloro salicylaldehyde)
◦
and 4 (Cinnamaldehyde).Yellow solid, M.p. 136−138 C.
−1
IR(KBr) cm : 3071, 2811, 1683, 1631, 1462, 1398, 1333, 2.4 Synthesis of (Z/E)-2-phenyl/H-3-Styryl-2H-chro-
242, 1171, 1086, 88, 883, 723, 652, 555, 451. H NMR
1
1
menes 9(a–r) and 10(a–s) (Table 3)
(
400 MHz, CDCl3): δ 9.73 (s, 1H), 7.35–7.27 (m, 7H),
7
1
1
6
.17 (s, 1H), 6.48 (s, 1H). 13C NMR (CDCl3, 100 MHz): δ Benzyltriphenylphosphonium bromide (3 equiv.) was taken
89.8, 149.3, 138.7, 138.0, 135.4, 133.0, 129.1, 128.8, 127.1, in anhydrous THF (10 mL) to it BuLi (1.6 M in Hexane)
◦
26.6, 123.3, 122.4, 74.8. Anal. Calcd for C16H10Cl2O2: C, (3 equiv.) was added slowly at −78 C and stirried in
2.97; H, 3.30%. Found: C, 62.99; H, 3.28%.ESI-HRMS argon atmosphere for 1h. Substituted 2-phenyl-2H-chromene
+
[
M + Na] : Calcd for C16H10Cl2O2Na: 326.9950, Found: aldehyde (1 equiv.) in THF was added and stirred at
◦
3
26.9947.
−78 C. Reaction was monitored by TLC and observed to

127 Page 4 of 15
J. Chem. Sci. (2018) 130:127
Table 3. Reaction time and yield of products.
R5 Time(h) Producta Yield (%)
b
Entry R R1 R2 R3
R4
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
Ph
Ph
H
H
H
H
H
H
H
H
H
H
H
H
OMe
H
H
OMe
OMe
OMe
OMe
H
H
H
H
H
H
H
H
H
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9p
9q
9r
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
10q
10r
10s
38
35
39
38
35
36
39
40
41
40
39
40
41
40
39
39
35
35
32
34
33
33
31
35
34
31
30
32
32
32
30
32
30
32
31
35
31
Ph OMe H
Ph OMe H
Ph
Ph
Ph OEt
Ph OEt
Ph OMe H OMe OMe OMe
Ph OEt H OMe OMe OMe
H
H
Br
Cl
H
H
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
Ph OEt
Ph OMe H
H
H
H
OMe OMe
OMe OMe
H
OEt H OMe OMe OMe
H OMe H OMe
H OMe H OMe OMe OMe
H
H
H OMe H
H
H
Ph
Ph
Ph Cl Cl
Ph Cl Cl
Ph OMe H
Ph
Ph
Ph OEt
Ph OEt
Ph OMe H OMe OMe OMe
Ph OEt H OMe OMe OMe
H
H
H
H
H
H
H
H
H
H
H
H
OMe OMe
OMe
OMe OMe
OMe
H
H
OMe
H
OMe
OMe
OMe
H
OEt
OEt
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
Cl
H
H
Ph OEt
Ph OMe H
H
H
H
OMe OMe
OMe OMe
H
OEt H OMe OMe OMe
H OMe H OMe
H OMe H OMe OMe OMe
H
H
H OMe H
H
H
H
H
H
OMe OMe
OMe
OMe OMe
OEt
OEt
H
H
H
be completed in 6h. Reaction mixture was quenched by 55.3.Anal.Calcd for C24H20O2: C, 84.68; H, 5.92%. Found:
saturated NH4Cl solution and stirred in room temperature C, 84.70; H, 5.95%.
for 1–2 h. Reaction mixture was extracted thrice with ethyl
2
.4.2 (Z)-2-phenyl-3-styryl-2H-chromene (9b) Prepared from
acetate and separated out. The combined organic layers were
dried over anhydrous sodium sulphate (Na2SO4), filtered and
concentrated. The crude reaction mixture was purified by
flash column chromatography on silica gel (230–330 mesh)
to afford the desired product.
◦
1
5
a and 6a. White solid, M.p. 83−85 C. H NMR (400 MHz,
CDCl3): δ 7.46–7.44 (m, 2H), 7.35 (d, J = 8.0 Hz, 2H), 7.30–
7.24 (m, 6H), 7.09–7.07 (m, 1H), 7.05–7.04 (m, 1H), 6.93 (d,
J = 12.0 Hz, 1H), 6.87–6.83 (m, 1H), 6.78 (s, 1H), 6.75 (d,
J = 4.0 Hz, 1H), 6.38 (d, J = 12.0 Hz, 1H), 6.23 (s, 1H).
1
3
C NMR (100 MHz, CDCl3): δ 152.0, 139.4, 137.3, 132.2,
31.5, 129.4, 128.6, 128.3, 127.4, 126.7, 124.0, 122.1, 121.2,
16.0, 78.1. Anal.Calcd for C23H18O : C, 89.00; H, 5.85%.
2
.4.1 (Z)-3-(4-methoxystyryl)-2-phenyl-2H-chromene (9a)
1
1
1
Prepared from 5a and 6b. Yellow liquid, H NMR (400 MHz,
CDCl3): δ 7.46 (d, J = 8.0 Hz, 1H), 7.31–7.18 (m, 10H),
Found: C, 89.04; H, 5.89%.
7.07–7.03 (m, 1H), 6.95–6.93 (m, 1H), 6.63 (s, 1H), 6.39 (d,
J = 12.0 Hz,1H), 5.91 (d, J = 12.0 Hz, 1H), 5.89 (s, 1H), 2.4.3 (Z)-8-methoxy-2-phenyl-3-styryl-2H-chromene (9c)
.80 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 159.4, 151.8, Prepared from 5b and 6a. Yellow liquid, H NMR (400MHz,
38.6, 132.6, 129.7, 129.1, 128.8, 128.5, 128.4, 127.7, 127.6, CDCl3): δ 7.31–7.20 (m, 10H), 6.76 (d, J = 8.0 Hz, 1H),
27.6, 126.5, 125.2, 123.5, 123.0, 121.3, 116.4, 114.1, 77.2, 6.72–6.70 (m, 1H), 6.61 (s, 1H), 6.60–6.58 (m, 1H), 6.49 (d,
1
3
1
1

J. Chem. Sci. (2018) 130:127
Page 5 of 15 127
J = 12.0 Hz, 1H), 6.08 (d, J = 12 Hz, 1H), 5.97 (s, 1H), 3.75 13C NMR (100 MHz, CDCl3): δ 147.1, 141.6, 139.2, 137.3,
(
s, 3H). 13C NMR (100 MHz, CDCl3): δ 147.9, 141.0, 139.2, 132.5, 131.4, 128.6, 128.3, 128.1, 127.7, 127.4, 127.3, 124.3,
137.3, 132.5, 131.5, 128.5, 128.4, 128.2, 127.6, 127.4, 127.3, 123.3, 120.8, 119.3, 114.8, 77.5, 64.9, 14.8. Anal.Calcd for
1
24.1, 122.9, 120.9, 119.2, 112.5, 77.7, 56.1. Anal.Calcd for C H20O2: C, 84.72; H, 6.26%. Found: C, 84.75; H, 6.27%.
2
5
C24H20O2 : C, 84.68; H, 5.92%. Found: C, 84.64; H, 5.89%.
2
.4.9(Z)-8-methoxy-2-phenyl-3-(3,4,5-trimethoxystyryl)-2H-
chromene (9i) Prepared from 5b and 6d. Deep yellow solid,
2.4.4 (Z)-8-methoxy-3-(4-methoxystyryl)-2-phenyl-2H-chro
◦
1
M.p. 129−131 C. H NMR (400 MHz, CDCl3): δ 7.51 (d,
J = 4.0 Hz, 2H), 7.30–7.25 (m, 3H), 6.90 (d, J = 12.0 Hz,
mene (9d) Prepared from 5b and 6b. Yellow solid, M.p.
◦
1
1
8
6
29−131 C. H NMR (400 MHz, CDCl3): δ 7.29–7.21 (m,
H), 6.81 (d, J = 8.0 Hz, 2H), 6.75 (d, J = 8.0 Hz, 1H),
.70 (d, J = 8.0 Hz, 1H), 6.62–6.59 (m, 2H), 6.43 (d,
1H), 6.85–6.82 (m, 1H), 6.81–6.79 (m, 1H), 6.73 (s, 1H),
6
(
.71 (s, 1H), 6.60 (s, 2H), 6.35 (d, J = 12.0 Hz, 1H), 6.31
s, 1H), 3.87 (s, 6H), 3.83 (s, 3H), 3.79 (s, 3H). 13C NMR
J = 12.0 Hz, 1H), 5.99 (s, 1H), 3.80 (s, 3H), 3.77 (s, 3H).
1
3
3
(100 MHz, CDCl ): δ 152.9, 148.0, 140.9, 139.2, 137.2,
C NMR (100 MHz, CDCl3): δ 158.9, 147.9, 140.9, 139.4,
32.9, 131.2, 129.9, 129.7, 128.2, 127.2, 126.1, 123.5, 123.4,
20.9, 119.1, 113.8, 112.4, 77.7, 56.1, 55.2. Anal.Calcd for
1
1
32.7, 132.5, 131.3, 128.2, 127.6, 127.2, 124.5, 123.0,
21.0, 119.1, 112.6, 105.4, 103.5, 77.3, 60.9, 56.1, 55.8.
1
1
Anal. Calcd for C27H26O : C, 75.33; H, 6.09%. Found:
C H22O3 : C, 81.06; H, 5.99%. Found: C, 81.08; H, 5.97%.
5
2
5
+
+
C, 75.35; H, 6.07%. ESI-HRMS [M + Na] : Calcd for
ESI-HRMS [M + Na] : Calcd for C H22O3Na: 393.14612,
2
5
C27H26O Na: 453.16725, found: 453.16680.
Found: 393.14671.
5
2
.4.10 (Z)-8-ethoxy-2-phenyl-3-(3,4,5-trimethoxystyryl)-2H-
2
.4.5 (Z)-6-bromo-3-(4-methoxystyryl)-2-phenyl-2H-chro
1
chromene (9j) Prepared from 5c and 6d. Pale yellow solid,
mene (9e) Prepared from 5e and 6b. Yellow liquid, H NMR
400 MHz, CDCl3): δ 7.28–7.24 (m, 8H), 7.20 (d, J = 8.0Hz,
H), 7.14–7.11 (m, 1H), 7.04 (d, J = 4.0 Hz, 1H), 6.60 (d,
◦
1
M.p. 120−122 C. H NMR (400 MHz, CDCl3): δ 7.32–
.21 (m, 5H), 6.75–6.71 (m, 2H), 6.68 (s, 1H), 6.62–6.60
m, 1H), 6.49 (s, 2H), 6.45 (d, J = 12.0 Hz, 1H), 6.15 (d,
J = 12.0 Hz, 1H), 5.98 (s, 1H), 3.97 (q, J = 8.0 Hz, 2H),
(
2
7
(
J = 8.0 Hz, 1H), 6.56 (s, 1H), 5.91 (d, J = 8.0Hz, 1H),
.87 (s, 1H), 3.82 (s, 3H). 13C NMR (100 MHz, CDCl3): δ
59.1, 158.5, 138.9, 133.8, 132.0, 131.6, 130.0, 129.9, 128.9,
28.5, 128.4, 128.3, 127.3, 125.4, 122.1, 117.8, 113.8, 113.6,
8.3, 55.2. Anal.Calcd for C24H19BrO2: C, 68.75; H, 4.57%.
5
1
1
7
1
3
3
.85 (s, 3H), 3.65 (s, 6H), 1.29 (t, J = 8.0 Hz, 3H). C NMR
(100 MHz, CDCl3): δ 152.8, 147.1, 141.6, 139.3, 137.2,
1
1
32.7, 132.5, 131.2, 128.1, 127.8, 127.1, 124.7, 123.4,
21.0, 119.2, 114.8, 105.5, 77.2, 64.9, 60.9, 55.8, 14.8.
Found: C, 68.78; H, 4.55%.
Anal. Calcd for C28H28O : C, 75.65; H, 6.35%. Found:
5
+
2.4.6
(Z)-6-chloro-3-(4-methoxystyryl)-2-phenyl-2H-chro C, 75.62; H, 6.37%. ESI-HRMS [M + Na] : Calcd for
1
mene (9f) Prepared from 5d and 6b. Yellow liquid, H NMR C H O Na: 467.18290, found: 467.18315.
2
8
28
5
(
1
(
5
400MHz, CDCl3): δ 7.29–7.25(m, 7H), 6.99(d, J = 8.0 Hz,
2.4.11 (Z)-3-(3,4-dimethoxystyryl)-8-ethoxy-2-phenyl-2H-
H), 6.91 (d, J = 4.0 Hz, 1H), 6.83 (d, J = 8.0 Hz, 2H), 6.63
chromene (9k) Prepared from 5c and 6c. White solid, M.p.
d, J = 8.0 Hz, 1H), 6.57 (s, 1H), 6.45 (d, J = 12.0 Hz, 1H),
◦
1
1
3
105−107 C. H NMR (400 MHz, CDCl3): δ 7.33–7.31 (m,
.92 (d, J = 12.0 Hz, 1H), 5.87 (s, 1H), 3.82 (s, 3H).
C
2H), 7.23–7.21 (m, 3H), 6.90–6.87 (m, 2H), 6.79–6.73 (m,
3H), 6.65 (s, 1H), 6.60–6.58 (m, 1H), 6.45 (d, J = 8.0 Hz,
1H), 6.06 (d, J = 8.0 Hz, 1H), 5.99 (s, 1H), 3.98 (q, J =
8.0 Hz, 2H), 3.88 (s, 3H), 3.64 (s, 3H), 1.31 (t, J = 8.0 Hz,
3
NMR (100 MHz, CDCl3): δ 159.1, 150.5, 138.9, 133.8, 132.0,
1
1
29.9, 129.5, 128.7, 128.5, 128.4, 127.3, 126.0, 125.4, 123.6,
22.3, 117.3, 113.8, 78.3, 55.3.Anal. Calcd for C24H19ClO2:
C, 76.90; H, 5.11%. Found: C, 76.88; H, 5.15%.
1
3
H). C NMR (100 MHz, CDCl3): δ 148.4, 147.1, 141.6,
2.4.7 (Z)-8-ethoxy-3-(4-methoxystyryl)-2-phenyl-2H-chro 139.5, 132.8, 131.3, 129.9, 128.2, 128.1, 127.0, 126.6, 123.9,
mene (9g) Prepared from 5c and 6b. White solid, M.p. 123.3, 121.6, 120.9, 119.2, 114.7, 111.2, 110.9, 77.4, 64.9,
◦
1
1
7
6
13 − 115 C. H NMR (400 MHz,CDCl3): δ 7.31–7.20 (m, 55.8, 55.6, 14.8. Anal.Calcd for C27H26O4: C, 78.24; H,
+
H), 6.82–6.79 (m, 2H), 6.75–6.73 (m, 2H), 6.63 (s, 1H), 6.32%. Found: C, 78.26; H, 6.35%. ESI-HRMS[M + Na] :
.61–6.58 (m, 1H), 6.43 (d, J = 12.0 Hz, 1H), 6.03 (d, Calcd for C27H26O4Na: 437.17233, found: 437.17231.
J = 12.0 Hz, 1H), 5.98 (s, 1H), 3.98 (q, J = 8.0 Hz, 2H),
1
3
2.4.12 (Z)-3-(3,4-dimethoxystyryl)-8-methoxy-2-phenyl-2H-
chromene (9l) Prepared from 5b and 6c. Yellow liquid, H
3
.80 (s, 3H), 1.30 (t, J = 8.0 Hz, 3H). C NMR (100 MHz,
1
CDCl3): δ 158.9, 147.1, 141.5, 139.4, 132.9, 131.1, 129.9,
NMR (400 MHz, CDCl3): δ 7.53–7.50 (m, 2H), 7.28–7.26
1
1
8
29.7, 128.1, 127.2, 126.3, 123.7, 123.4, 120.8, 119.3, 114.7,
13.8, 77.5, 64.9, 55.2, 14.8. Anal. Calcd for C26H24O3: C,
1.22; H, 6.29%. Found: C, 81.25; H, 6.27%.
(m, 3H), 6.93–6.91 (m, 3H), 6.85–6.80 (m, 2H), 6.78 (d,
J = 12.0 Hz, 1H), 6.72 (d, J = 4.0 Hz, 1H), 6.39 (d,
J = 12.0 Hz, 1H), 6.31 (s, 1H), 3.90 (s, 3H), 3.87 (s, 3H),
2
.4.8 (Z)-8-ethoxy-2-phenyl-3-styryl-2H-chromene (9h) 3.79 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 148.5, 147.9,
◦
Prepared from 5c and 6a. Pale yellow solid, M.p. 127−129 C. 140.9, 139.5, 132.8, 131.4, 129.9, 128.2, 127.1, 126.4, 123.6,
1
H NMR (400 MHz, CDCl3): δ 7.31–7.20 (m, 8H), 6.76– 123.0, 121.6, 120.9, 119.1, 112.5, 111.2, 110.9, 77.6, 56.2,
6
.70 (m, 2H), 6.62 (s, 1H), 6.59–6.57 (m, 1H), 6.50 (d, 55.8, 55.6. Anal. Calcd for C26H24O4: C, 77.98; H, 6.04%.
+
J = 12.0 Hz, 1H), 6.10 (d, J = 12.0 Hz, 1H), 5.95 (s, Found: C, 77.96; H, 6.05%. ESI-HRMS [M + Na] : Calcd
1H), 3.96 (q, J = 8.0 Hz, 2H), 1.28 (t, J = 8.0 Hz, 3H). for C26H24O4Na: 423.15668, found: 423.15675.

127 Page 6 of 15
J. Chem. Sci. (2018) 130:127
2
.4.13 (Z)-8-ethoxy-3-(3,4,5-trimethoxystyryl)-2H-chromene 2.4.18 (Z)-3-(3,4-dimethoxystyryl)-8-ethoxy-2H-chromene
◦
(
9m) Prepared from 5’c and 6d. Yellow solid, M.p. 75−77 C. (9r) Prepared from 5’c and 6c. Yellow solid, M.p. 109−
1
◦
1
H NMR (400 MHZ, CDCl3): δ 6.81 (d, J = 8.0 Hz, 1H), 111 C. H NMR (400 MHz, CDCl3): δ 6.82–6.77 (m, 4H),
.77–6.75 (m, 1H), 6.67 (d, J = 8.0 Hz, 1H), 6.55–6.52 6.76–6.74 (m, 1H), 6.66–6.64 (m, 1H), 6.55 (d, J = 8.0 Hz,
6
(
2
m, 2H), 6.46 (s, 2H), 6.21 (d, J = 12.0 Hz, 1H), 4.58 (s, 1H), 6.50 (s, 1H), 6.18 (d, J = 8.0 Hz, 1H), 4.55 (s, 2H),
H), 4.05 (q, J = 8.0 Hz, 2H), 3.87 (s, 3H), 3.82 (s, 6H), 4.05 (q, J = 8.0 Hz, 2H), 3.88 (s, 3H), 3.83 (s, 3H), 1.40
13 13
1
.41 (t, J = 8.0 Hz, 3H). C NMR (100 MHz, CDCl3): (t, J = 4.0 Hz, 3H). C NMR (100 MHz, CDCl3): δ
δ 152.9, 146.9, 142.9, 137.5, 133.7, 131.2, 130.8, 127.9, 148.6, 148.5, 146.9, 142.9, 131.6, 130.9, 127.2, 125.2, 124.2,
1
5
25.7, 124.1, 121.2, 119.1, 113.5, 105.7, 67.1, 64.5, 61.0, 121.4, 121.1, 119.1, 113.5, 111.5, 110.8, 67.2, 64.5, 55.9,
6.1, 14.9. Anal.Calcd for C22H24O : C, 71.72; H, 6.57%. 55.8, 14.9. Anal. Calcd for C21H22O4: C, 74.54; H, 6.55%.
5
+
+
Found: C, 71.76; H, 6.54%. ESI-HRMS [M + Na] : Calcd Found: C, 77.58; H, 6.56%. ESI-HRMS [M + Na] : Calcd
for C22H24O Na: 391.15160, found: 391.15185.
for C21H22O4Na: 361.14103, found: 361.14111.
.4.19 (E)-3-(4-methoxystyryl)-2-phenyl-2H-chromene (10a)
5
2
2.4.14(Z)-8-methoxy-3-(4-methoxystyryl)-2H-chromene(9n)
◦
1
◦
1
Prepared from 5a and 6b. White solid, M.p. 135−137 C; H
Preparedfrom5’band6b. Yellowsolid, M.p. 106−108 C. H
NMR (400 MHz, CDCl3): δ 7.47–7.44 (m, 2H), 7.31–7.25
NMR (400 MHz, CDCl3): δ 7.16 (d, J = 8.0 Hz, 2H), 6.82
(m, 5H), 7.08–7.01 (m, 2H), 6.93–6.84 (m, 4H), 6.83–6.80
(
6
1
d, J = 8.0 Hz, 3H), 6.76–6.74 (m, 1H), 6.67–6.65 (m, 1H),
(
m, 1H), 6.78–6.74 (m, 1H), 6.35(d, J = 24.0 Hz,1H), 6.22
.56 (d, J = 12.0 Hz, 1H), 6.48 (s, 1H), 6.17 (d, J = 12.0 Hz,
1
3
H), 4.52 (s, 2H), 3.83 (s, 3H), 3.80 (s, 3H). 13C NMR
(s, 1H), 3.79 (s, 3H). C NMR (100 MHz, CDCl3): δ 159.4,
151.8, 138.6, 132.6, 129.7, 129.1, 128.8, 128.5, 128.4, 127.7,
127.6, 126.5, 125.2, 123.5, 123.0, 121.3, 116.4, 114.1, 77. 2,
55.3.Anal. Calcd for C24H20O2: C, 84.68; H, 5.92%. Found:
(100 MHz, CDCl3): δ 159.0, 147.6, 142.4, 131.9, 130.9,
1
6
6
30.5, 129.8, 127.1, 124.8, 124.1, 121.1, 119.0, 113.6, 111.7,
7.3, 56.0, 55.3. Anal. Calcd for C19H18O3: C, 77.53; H,
.16%. Found: C, 77.56; H, 6.14%. ESI-HRMS [M + Na] :
+
C, 84.71; H, 5.94%.
Calcd for C19H18O3Na: 317.11482, found: 317.11456.
2
.4.20 (E)-2-phenyl-3-styryl-2H-chromene (10b) Prepared
◦
1
from 5a and 6a. Yellow solid, M.p. 131−133 C. H NMR
2
.4.15
mene (9o) Prepared from 5’b and 6d. Yellow liquid, H NMR
400 MHz, CDCl3): δ 6.89–6.82 (m, 4H), 6.68–6.65 (m, 1H),
(Z)-8-methoxy-3-(3,4,5-trimethoxystyryl)-2H-chro
(400 MHz, CDCl3): δ 7.34–7.23 (m, 10H), 7.05–7.02 (m, 1H),
1
6
6
1
.93 (dd, J = 4.0 Hz, J = 8.0 Hz, 1H), 6.84–6.81 (m, 1H),
(
.72 (d, J = 8.0 Hz, 1H), 6.62 (s, 1H), 6.47 (d, J = 20.0 Hz,
6
3
.65–6.64 (m, 2H), 6.62 (d, J = 12.0 Hz, 2H), 6.48–6.39 (m,
13
H), 6.02 (d, J = 20.0 Hz, 1H), 5.86 (s, 1H). C NMR
H), 6.34 (s, 2H), 5.01 (s, 2H), 3.77 (s, 12 H). 1 C NMR
3
(100 MHz, CDCl3): δ 152.0, 138.5, 136.9, 132.4, 129.5,
(100 MHz, CDCl3): δ 153.4, 147.6, 142.3, 138.2, 132.6,
1
1
29.3, 128.6, 128.5, 127.8, 127.7, 127.2, 126.8, 126.4, 124.6,
22.8, 121.4, 116.5, 76.5.Anal. Calcd for C23H18O: C, 89.00;
1
6
7
30.6, 128.0, 126.0, 123.7, 123.4, 121.2, 119.3, 112.0, 103.5,
5.9, 60.9, 56.1, 56.1, 56.0. Anal.Calcd for C21H22O : C,
1.17; H, 6.26%. Found: C, 71.16; H, 6.24%. ESI-HRMS:
5
H, 5.85%. Found: C, 89.02; H, 5.87%.
.4.21 (E)-6,8-dichloro-2-phenyl-3-styryl-2H-chromene
10c) Prepared from 5f and 6a. Yellow solid, M.p. 140−
2
(
Calcd for C21H22O : 353.13835, found: 353.13886.
5
◦
1
2.4.16 (Z)-3-(3,4-dimethoxystyryl)-8-methoxy-2H-chromene 142 C. H NMR (400 MHz, CDCl3): δ 7.48–7.46 (m, 2H),
◦
(
9p) Prepared from 5’b and 6c. Yellow solid, M.p. 97−99 C. 7.33–7.25 (m, 5H), 7.07 (d, J = 4.0 Hz, 1H), 6.93 (d, J =
1
H NMR (400 MHz, CDCl3): δ 6.83–6.79 (m, 4H), 6.76–6.74 4.0 Hz, 1H), 6.86–6.82 (m, 3H), 6.63 (s, 2H), 6.42 (d, J =
(
(
m, 1H), 6.67–6.65 (m, 1H), 6.55 (d, J = 8.0 Hz, 1H), 6.50 16.0 Hz, 1H), 6.35 (s, 1H). 13C NMR (100 MHz, CDCl3): δ
s, 1H), 6.18 (d, J = 8.0 Hz, 1H), 4.55 (s, 2H), 3.88 (s, 3H), 159.9, 146.3, 137.6, 134.9, 130.7, 129.2, 128.8, 128.5, 128.0,
3
1
1
.83 (s, 6H). 13C NMR (100 MHz, CDCl3): δ 148.6, 148.5, 127.5, 126.1, 125.6, 124.4, 122.2, 121.7, 114.2, 76.7.Anal.
47.6, 142.4, 131.7, 131.0, 130.8, 127.1, 125.0, 124.0, 121.4, Calcd for C23H Cl2O: C, 72.83; H, 4.25%. Found: C, 72.82;
21.2, 119.0, 111.8, 111.5, 110.8, 67.2, 56.0, 55.9, 55.8. Anal. H, 4.27%.
1
5
Calcd for C20H20O4: C, 74.06; H, 6.21%. Found: C, 74.08;
H, 6.24%.ESI-HRMS: Calcd for C20H24O4N: 342.16998,
found: 342.17097.
2
.4.22 (E)-6,8-dichloro-3-(4-methoxystyryl)-2-phenyl-2H-ch
romene (10d) Prepared from 5f and 6b. Yellow solid, M.p.
◦
1
1
2
72−174 C. H NMR (400 MHz, CDCl3): δ 7.48–7.46 (m,
H), 7.33–7.25 (m, 5H), 7.07 (d, J = 4.0 Hz, 1H), 6.93 (s,
2
.4.17 (Z)-8-ethoxy-3-(4-methoxystyryl)-2H-chromene (9q)
1
1H), 6.86-6.82 (m, 3H), 6.63 (s, 1H), 6.43 (d, J = 16.0 Hz,
Prepared from 5’c and 6b. Yellow liquid, H NMR (400 MHz,
CDCl3): δ 7.15 (d, J = 8.0 Hz, 2H), 6.83–6.80 (m, 3H), 6.75–
1
H), 6.35 (s, 1H), 3.79 (s, 3H). 13C NMR (100 MHz, CDCl3):
δ 159.9, 146.3, 137.6, 134.9, 130.7, 129.2, 128.8, 128.5,
6
6
.73 (m, 1H), 6.65–6.63 (m, 1H), 6.55 (d, J = 12.0 Hz, 1H),
.48 (s, 1H), 6.18 (d, J = 12.0 Hz, 1H), 4.52 (s, 2H), 4.05
1
5
28.0, 127.5, 126.1, 125.6, 124.4, 122.2, 121.7, 114.2, 76.7,
5.3.Anal. Calcd for C24H18Cl2O2: C, 70.43; H, 4.43%.
(q, J = 8.0 Hz, 2H), 3.81 (s, 3H), 1.40 (t, J = 8.0 Hz, 3H).
1
3
Found: C, 70.42; H, 4.45%.
C NMR (100 MHz, CDCl3): δ 159.5, 146.8, 142.7, 130.9,
1
6
29.7, 127.7, 124.5, 123.8, 122.9, 121.0, 119.2, 114.2, 113.5, 2.4.23 (E)-8-methoxy-2-phenyl-3-styryl-2H-chromene (10e)
5.8, 64.6, 55.3, 14.9.Anal. Calcd for C20H20O3: C, 77.90; Prepared from 5b and 6a. Yellow solid, M.p. 140−142 C. H
NMR (400 MHz, CDCl3): δ 7.52–7.50 (m, 2H), 7.38–7.20 (m,
◦
1
H, 6.54%. Found: C, 77.88; H, 6.56%.

J. Chem. Sci. (2018) 130:127
Page 7 of 15 127
8
H), 6.95 (d, J = 20.0 Hz, 1H), 6.83–6.69 (m, 4H), 6.42 (d, 7.31–7.28 (m, 2H), 7.23–7.21 (m, 3H), 6.77 (d, J = 8.0 Hz,
1
3
J = 20.0 Hz, 1H), 6.32 (s, 1H), 3.77 (s, 3H). C NMR 1H), 6.73–6.71 (m, 1H), 6.68 (s,1H), 6.62 (d, J = 8.0 Hz,
(
100 MHz, CDCl3): δ 148.3, 141.0, 138.5, 136.9, 132.7, 1H), 6.48 (s, 2H), 6.45 (d, J = 16.0 Hz, 1H), 6.15 (d,
1
1
8
29.4, 128.6, 128.4, 127.8, 127.6, 127.3, 126.4, 124.5, 123.6,
21.1, 119.2, 112.8, 76.3, 56.3.Anal. Calcd for C24H20O2: C, 3.66 (s, 6H). 13C NMR (100 MHz, CDCl3): δ 153.3, 148.3,
4.68; H, 5.92%. Found: C, 84.65; H, 5.93%. 140.9, 138.4, 138.1, 132.6, 132.5, 129.2, 128.4, 127.6, 126.9,
24.5, 123.7, 121.1, 119.2, 112.7, 106.0, 103.5, 76.1, 60.9,
J = 16.0 Hz, 1H), 6.00 (s, 1H), 3.85 (s, 3H), 3.76 (s, 3H),
1
2
.4.24 (E)-6-bromo-3-(4-methoxystyryl)-2-phenyl-2H-chro 56.2, 56.1, 56.0. Anal. Calcd for C27H26O : C, 75.33; H,
5
+
mene (10f) Prepared from 5e and 6b. Yellow solid, M.p. 6.09%. Found: C, 75.36; H, 6.12%. ESI-HRMS [M + Na] :
◦
1
1
53−155 C. H NMR (400 MHz, CDCl3): δ 7.43–7.41 (m, Calcd for C27H26O Na: 453.16725, found: 453.16680.
5
2
H), 7.30–7.25 (m, 5H), 7.17 (d, J = 4.0 Hz,1H), 7.11–7.09
(m, 1H), 6.82 (d, J = 4.0 Hz, 2H), 6.78 (d, J = 16.0 Hz, 1H), 2.4.29 (E)-8-ethoxy-2-phenyl-3-(3,4,5-trimethoxystyryl)-2H-
6.65 (s, 1H), 6.62 (d, J = 8.0 Hz, 1H), 6.36 (d, J = 16.0 Hz, chromene (10k) Prepared from 5c and 6d. Pale yellow solid,
1
H), 6.21 (s, 1H), 3.78 (s, 3H). 13C NMR (100 MHz, CDCl3): M.p. 118 − 120 C. H NMR (400 MHz, CDCl3): δ 7.54–
◦
1
δ 159.7, 159.0, 138.1, 133.8, 131.5, 129.9, 128.8, 128.7, 7.53 (m, 1H), 7.52–7.51 (m, 1H), 7.28–7.26 (m, 3H), 6.92
1
1
4
28.6, 127.8, 127.7, 125.0, 124.7, 122.1, 118.2, 114.2, 113.8, (d, J = 16.0 Hz, 1H), 6.79–6.76 (m, 2H), 6.74–6.70 (m,
13.4, 76.6, 55.3.Anal. Calcd for C24H19BrO2: C, 68.75; H, 2H), 6.61 (s, 2H), 6.35 (d, J = 16.0 Hz, 1H), 6.32 (s, 1H),
.57%. Found: C, 68.76; H, 4.59%.
4.00 (q, J = 8.0 Hz, 2H), 3.87 (s, 6H), 3.84 (s, 3H), 1.35
13
(
t, J = 8.0 Hz, 3H). C NMR (100 MHz, CDCl3): δ 153.4,
2.4.25 (E)-6-chloro-3-(4-methoxystyryl)-2-phenyl-2H-chro 147.5, 141.5, 138.4, 138.1, 132.6, 132.6, 129.1, 128.4, 127.6,
mene (10g) Prepared from 5d and 6b. Yellow solid, M.p. 127.1, 124.7, 124.0, 121.2, 119.3, 114.8, 103.5, 75.8, 65.0,
◦
1
1
8
6
89−191 C. H NMR (400 MHz, CDCl3): δ 7.42 (d, J = 61.0, 56.1, 14.9. Anal. Calcd for C28H28O : C, 75.65; H,
5
+
.0 Hz, 2H), 7.30–7.25 (m, 5H), 7.03 (d, J = 4.0 Hz, 1H), 6.35%. Found: C, 75.67; H, 6.38%. ESI-HRMS[M + Na] :
.98–6.95 (m, 1H), 6.83 (d, J = 8.0 Hz, 2H), 6.78 (d, Calcd for C28H28O Na: 467.18290, found: 467.18315.
5
J = 16.0 Hz, 1H), 6.68-6.66 (m, 2H), 6.37 (d, J = 16.0 Hz,
H), 6.21 (s, 1H), 3.79 (s, 3H). 13C NMR (100 MHz, CDCl3):
δ 159.7, 150.3, 138.1, 133.8, 129.9, 129.4, 128.7, 128.6,
2.4.30 (E)-3-(3, 4-dimethoxystyryl)-8-ethoxy-2-phenyl-2H-
1
chromene (10l) Prepared from 5c and 6c. Yellow solid,
◦
1
M.p. 102−104 C. H NMR (400 MHz, CDCl3): δ 7.52 (d,
1
7
27.8, 127.7, 126.1, 125.9, 124.7, 124.4, 122.3, 117.7, 114.2,
6.6, 55.3.Anal. Calcd for C24H19ClO2: C, 76.90; H, 5.11%.
Found: C, 76.89; H, 5.13%.
J = 4.0 Hz, 2H), 7.29–7.23 (m, 3H), 6.93–6.84 (m, 3H),
6
.79 (d, J = 4.0 Hz, 1H), 6.77–6.70 (m, 4H), 6.37 (d,
J = 16.0 Hz, 1H), 6.31 (s, 1H), 3.99 (q, J = 8.0 Hz, 2H),
1
3
3
.90 (s, 3H), 3.86 (s, 3H), 1.34 (t, J = 8.0 Hz, 3H).
C
2
.4.26 (E)-8-ethoxy-3-(4-methoxystyryl)-2-phenyl-2H-chro
NMR (100 MHz, CDCl3): δ 149.1, 147.5, 141.4, 138.5, 132.8,
mene (10h) Prepared from 5c and 6b. White solid, M.p.
◦
1
130.0, 129.0, 128.3, 127.6, 125.7, 124.2, 123.9, 121.8, 121.1,
1
2
6
6
32−134 C. H NMR (400 MHz, CDCl3): δ 7.52–7.49 (m,
H), 7.32 (d, J = 8.0 Hz, 2H), 7.29–7.23 (m, 3H), 6.88–
.83 (m, 3H), 6.79–6.70 (m, 4H), 6.38 (d, J = 16.0 Hz, 1H),
.31 (s, 1H), 3.98 (q, J = 4.0 Hz, 2H), 3.79 (s, 3H), 1.33
119.9, 119.3, 114.7, 111.2, 110.9, 108.7, 75.9, 65.0, 55.9,
5
5.8, 14.9. Anal. Calcd for C27H26O4: C, 78.24; H, 6.32%.
+
Found: C, 78.25; H, 6.35%. ESI-HRMS [M + Na] : Calcd
1
3
for C27H26O4Na: 437.17233, found: 437.17231.
(
t, J = 4.0 Hz, 3H). C NMR (100 MHz, CDCl3): δ 156.9,
1
1
5
44.9, 138.9, 136.1, 130.4, 127.2, 126.3, 125.8, 125.2, 125.1,
22.9, 121.6, 121.1, 118.5, 116.7, 112.2, 111.6, 73.5, 62.5,
2.8, 12.3.Anal. Calcd for C26H24O3: C, 81.22; H, 6.29%.
2
.4.31 (E)-3-(3,4-dimethoxystyryl)-8-methoxy-2-phenyl-2H-
1
chromene (10m) Prepared from 5b and 6c. Yellow liquid, H
NMR (400 MHz, CDCl3): δ 7.32–7.21 (m, 5H), 6.91–6.88
Found: C, 81.24; H, 6.32%.
(m, 2H), 6.81–6.71 (m, 3H), 6.65 (s, 1H), 6.62–6.59 (m,
1
H), 6.45 (d, J = 16.0 Hz, 1H), 6.05 (d, J = 16.0 Hz,
2
.4.27 (E)-8-ethoxy-2-phenyl-3-styryl-2H-chromene (10i)
1
H), 6.01 (s, 1H), 3.88 (s, 3H), 3.78 (s, 3H), 3.65 (s, 3H).
1
Prepared from 5c and 6a. Yellow liquid. H NMR (400 MHz,
CDCl3): δ 7.50(d, J = 4.0 Hz, 2H), 7.37 (d, J = 8.0 Hz,
13
C NMR (100 MHz, CDCl3) δ 149.1, 148.3, 140.8, 138.5,
132.8, 130.0, 129.1, 128.4, 127.6, 126.6, 125.6, 123.8, 123.7,
121.1, 119.9, 119.5, 119.1, 112.5, 111.2, 108.7, 76.3, 56.3,
55.9, 55.8. Anal.Calcd for C26H24O4: C, 77.98; H, 6.04%.
2
6
6
3
1
1
H), 7.31–7.19 (m, 6H), 6.98 (d, J = 16.0 Hz, 1H), 6.79–
.76 (m, 2H), 6.73–6.71 (m, 2H), 6.43 (d, J = 16.0 Hz, 1H),
.32 (s, 1H), 3.98 (q, J = 4.0 Hz, 2H), 1.33 (t, J = 4.0 Hz,
+
Found: C, 77.97; H, 6.09%. ESI-HRMS [M + Na] : Calcd
1
3
H). C NMR (100 MHz, CDCl3): δ 147.5, 141.6, 138.5,
for C26H24O4Na: 423.15668, found: 423.15675.
36.9, 132.7, 129.3, 128.6, 128.4, 127.8, 127.6, 126.4, 124.7,
24.0, 121.1, 119.4, 115.0, 76.0, 65. 0, 14.9.Anal. Calcd for 2.4.32 (E)-8-ethoxy-3-(3,4,5-trimethoxystyryl)-2H-chromene
1
C H22O2: C, 84.72; H, 6.26%. Found: C, 84.73; H, 6.29%.
(10n) Prepared from 5’c and 6d. Yellow liquid, H NMR
400 MHz, CDCl3): δ 6.84–6.81 (m, 3H), 6.80–6.75 (m, 1H),
2
5
(
2
.4.28 (E)-8-Methoxy-2-phenyl-3-(3,4,5-trimethoxystyryl)- 6.69–6.67 (m, 2H), 6.51 (s, 1H), 6.37 (d, J = 16.0 Hz, 1H),
2
H-chromene (10j) Prepared from 5b and 6d. Pale yellow 5.14 (s, 2H), 4.13 (q, J = 8.0 Hz, 2H), 3.91 (s, 6H), 3.86
◦
1
13
solid, M.p. 104−106 C. H NMR (400 MHz, CDCl3): δ (s, 3H), 1.47 (t, J = 8.0 Hz, 3H). C NMR (100 MHz,

127 Page 8 of 15
J. Chem. Sci. (2018) 130:127
CDCl3): δ 152.3, 145.8, 141.7, 137.1, 131.6, 129.5, 126.9, 1H), 6.47 (s, 1H), 6.38 (d, J = 16.0 Hz, 1H), 5.13 (s,
1
5
6
25.0, 122.8, 122.5, 120.1, 118.3, 112.6, 102.4, 64.7, 63.5, 2H), 4.11 (q, J = 8.0 Hz, 2H), 3.93 (s, 3H), 3.89 (s, 3H),
1
3
9.9, 55.1, 13.9. Anal. Calcd for C22H24O : C, 71.72; H, 1.46 (t, J = 8.0 Hz, 3H). C NMR (100 MHz, CDCl3): δ
5
+
.57%. Found: C, 71.74; H, 6.59%.ESI-HRMS [M + Na] : 149.1, 146.8, 142.7, 130.8, 130.0, 128.7, 127.8, 124.7, 123.7,
Calcd for C22H24O Na: 391.15160, found: 391.15185.
123.0, 121.1, 119.9, 119.2, 113.5, 111.2, 108.7, 65.8, 64.6,
5
5
5.9, 14.9. Anal. Calcd for C21H22O4: C, 74.54; H, 6.55%.
2.4.33
(E)-8-methoxy-3-(4-methoxystyryl)-2H-chromene
+
Found: C, 77.56; H, 6.57%. ESI-HRMS [M + Na] : Calcd
(
10o) Prepared from 5’b and 6b. Yellow solid, M.p. 124−
for C21H22O4Na: 361.14103, found: 361.14111.
◦
1
1
2
6
1
26 C. H NMR (400 MHz, CDCl3): δ 7.38 (d, J = 8.0 Hz,
H), 6.87 (d, J = 4.0 Hz, 2H), 6.82 (d, J = 4.0 Hz, 1H),
.78–6.76 (m, 1H), 6.73 (d, J = 16.0 Hz, 1H), 6.69–6.67 (m,
H), 6.45(s, 1H), 6.40(d, J = 16.0 Hz, 1H), 5.13(s, 2H), 3.88
3
. Results and Discussion
1
3
(
s, 3H), 3.82 (s,3H). C NMR (100 MHz, CDCl3): δ 159.5,
3.1 Chemistry
1
1
47.5, 142.3, 131.0, 129.7, 127.7, 127.1, 124.5, 123.6, 122.7,
21.1, 119.2, 114.2, 111.8, 65.9, 56.0, 55.3. Anal. Calcd for
Compounds (Z)-2-phenyl/H-3-styryl-2H-chromenes
C19H18O3: C, 77.53; H, 6.16%. Found: C, 77.57; H, 6.15%.
9
1
(a–r) and (E)-2-phenyl/H-3-styryl-2H-chromenes
0(a–s) derivatives involves two steps. The first step
+
ESI-HRMS [M + Na] : Calcd for C19H18O3Na: 317.11482,
found: 317.11456.
involves the formation of 2H-chromene-3-
2
.4.34 (E)-8-methoxy-3-(3,4,5-trimethoxystyryl)-2H-chrom carbaldehydes 5(a–f) and 5’(b–c) and the second step
ene (10p) Prepared from 5’b and 6d. Yellow liquid, H NMR involves the reaction of chromene aldehyde 5(a–f) and
1
(
(
1
400 MHz, CDCl3): δ 6.84 (d, J = 8.0 Hz, 1H), 6.80–6.70 5’(b–c) with the phosphonium ylide to form the tar-
m, 3H), 6.67 (s, 2H), 6.52 (s, 1H), 6.38 (d, J = 24.0 Hz,
get molecules (Z)-2-phenyl/H-3-styryl-2H-chromenes
1
3
H), 5.15 (s, 2H), 3.91 (s, 9H), 3.87 (s, 3H). C NMR
9
1
(a–r) and (E)-2-phenyl/H-3-styryl-2H-chromenes
0(a–s). The synthesis of 2H-chromene-3-carbaldehy
(100 MHz, CDCl3): δ 153.1, 152.2, 147.6, 142.3, 136.8,
1
6
7
32.0, 130.8, 127.6, 126.0, 124.9, 123.7, 121.2, 119.3, 105.1,
5.8, 61.1, 60.8, 56.1, 56.0. Anal. Calcd for C21H22O : C,
1.17; H, 6.26%. Found: C, 71.18; H, 6.28%.ESI-HRMS:
de 5(a–f) and 5’(b–c) started from o-hydroxybenzaldeh
yde 3(a-f). The o-hydroxybenzaldehyde 3(a–f) reacted
with pyrrolidine 4 in DMSO at room temperature for
1
5
dehyde 3(b–c) was allowed to react with acrolein 4’
using K CO in dioxane under reflux condition for 2 h
to afford 2H-chromene-3-carbaldehyde 5’(b-c) in 87–
8
5
Calcd for C21H22O : 353.13835, found: 353.13886.
5
2 h to provide 2-phenyl-2H-chromene-3-carbaldehyde
(a–f) in 82–89% yield. Similarly, the o-hydroxybenzal
2
(
.4.35 (E)-3-(3,4-dimethoxystyryl)-8-methoxy-2H-chromene
10q) Prepared from 5’b and 6c. Yellow solid, M.p. 124−
◦
1
1
6
6
26 C. H NMR (400 MHz, CDCl3): δ 7.02–6.99 (m, 2H),
.86–6.82 (m, 2H), 6.79–6.76 (m, 2H), 6.69–6.67 (m, 1H),
.48 (s, 1H), 6.40 (d, J = 16.0 Hz, 1H), 5.15 (s, 2H), 3.93
2
3
37,38
9% yield (Schemes 1 and 2).
(
s, 3H), 3.90 (s, 6H). 13C NMR (100 MHz, CDCl3): δ 149.1,
All the synthesized chromene aldehyde molecules
(a-f) and 5’(b-c) were characterised by H, C NMR,
IR and Mass spectroscopy. Melting points of the solid
compounds were also taken.
1
1
5
47.5, 142.2, 130.9, 130.0, 127.9, 126.6, 124.7, 123.6, 122.9,
21.1, 119.8, 119.2, 111.8, 111.2, 108.7, 65.9, 56.0, 55.9,
5.8. Anal. Calcd for C20H20O4: C, 74.06; H, 6.21%. Found:
1
13
5
C, 74.09; H, 6.23%. ESI-HRMS: Calcd for C20H24O4N:
3
42.16998, found: 342.17097.
O
O
O
2.4.36 (E)-8-ethoxy-3-(4-methoxystyryl)-2H-chromene (10r)
R2
R2
◦
H
i
H
Prepared from 5’c and 6b. White solid, M.p. 110−112 C.
H
1
H NMR (400 MHz, CDCl3): δ 7.38 (d, J = 8.0 Hz, 2H),
OH
O
Ph
Ph
R1
R1
6
6
6
.87 (d, J = 4.0 Hz, 2H), 6.80 (d, J = 8.0 Hz, 1H), 6.77–
.75 (m, 1H), 6.72 (d, J = 16.0 Hz, 1H), 6.68–6.66 (m, 1H),
.45 (s, 1H), 6.40 (d, J = 16.0 Hz, 1H), 5.13 (s, 2H), 4.12
3
(a-f)
4
5 (a-f)
Scheme 1. Synthesis of compounds 5(a–f). Reagents and
conditions: i) pyrrolidine, DMSO, rt, 12 h.
(
q, J = 8.0 Hz, 2H), 3.82 (s, 3H), 1.46 (t, J = 8.0 Hz, 3H).
1
3
C NMR (100 MHz, CDCl3): δ 159.5, 146.8, 142.7, 130.9,
29.7, 127.7, 127.6, 124.5, 123.8, 122.9, 121.0, 119.2, 114.2,
13.5, 65.8, 64.6, 55.3, 14.9.Anal. Calcd for C20H20O3: C,
7.90; H, 6.54%. Found: C, 77.93; H, 6.57%.
1
1
7
O
O
O
R2
R2
H
H
i
H
OH
O
2
.4.37 (E)-3-(3,4-dimethoxystyryl)-8-ethoxy-2H-chromene
R1
R1
(
10s) Prepared from 5’c and 6c. Yellow solid, M.p. 130−
3 (a-f)
4
'
5' (b-c)
◦
32 C. H NMR (400 MHz, CDCl3): δ 7.01–6.99 (m, 2H),
1
1
6
6
.82 (d, J = 8.0 Hz, 1H), 6.80 (d, J = 4.0 Hz, 1H), 6.78– Scheme 2. Synthesis of compounds 5’(b–c). Reagents and
.76 (m, 1H), 6.74 (d, J = 16.0 Hz, 1H), 6.67 (d, J = 8.0 Hz, conditions: i) K CO , dioxane, reflux, 2 h.
2
3

J. Chem. Sci. (2018) 130:127
Page 9 of 15 127
PPh3+Br-
O
R2
R2
R3
R4
H
i
O
R
R3
R5
O
R
R1
R4
R1
R5
6
6
6
6
a: R = R = R = H
R = Ph: 5(a-f)
R = H: 5'(b-c)
3
4
5
Mixture of cis and trans stilbene
b: R = R = H; R = OMe
3
5
4
c: R = H; R = R = OMe
7: R = Ph
3
4
5
8
: R = H
d: R = R = R = OMe
3
4
5
R3
R4
R5
R2
R3
R4
R2
Flash chromatography
O
R
O
R
R1
R5
R1
9
(a-r)
10(a-s)
◦
Scheme 3. Synthesis of compound 9(a–r) and 10 (a–s). Reagents and conditions: i) BuLi, THF, −78 C, 6–8 h.
After successful synthesis of chromene aldehydes
5
(a–f) and 5’(b–c), they are then allowed to react with
O
OMe
OMe
O
OMe
OMe
Ph
H
the appropriate phosphonium ylide 6(a-d) using BuLi
OEt
OEt
◦
in THF at −78 C for 6–8 h to afford the cis and
9
g 9.36 (10 µM)
9q 93.41(10 µM)
trans mixture of stilbene in good yield. Flash column
chromatography purification provided the desired (Z)-
2
-phenyl/H-3-styryl-2H-chromenes 9(a–r) and (E)-2-
phenyl/H-3-styryl-2H-chromenes 10(a–s) in 35–41%
and 30–35% yields, respectively (Scheme 3). The syn-
thesized compounds were characterized by the use of
different spectroscopic techniques viz., H, C NMR
and Mass spectroscopy. The diastereomers are well dif-
O
Ph
O
H
OMe
OMe
9
d 21.51 (10 µM)
9n 39.21 (10 µM)
1
13
Figure 1. Comparison of antiproliferative activity of 9g/9q
and 9d/9n.
1
ferentiated in H NMR by the value of their coupling
constant, the vicinal alkenyl protons in 9(a–r) ranges
from 8.0 to 12.0 Hz whereas for 10(a–s) the range was
from 16.0 to 24.0 Hz (Scheme 3).
The SAR study demonstrated that the presence of
electron donating group (OEt) at the eighth position of
the benzopyran ring and phenyl group in the second
position of pyran ring improves anti-proliferative activ-
ity against tested cell line. But compound 9q though
quite similar to 9g except phenyl substituent at the
3.2 Biology
3
.2.1 In vitro anti-proliferative activity To evaluate the second position of pyran ring did not show any antipro-
anti-proliferativeactivitiesofthesynthesizedchromene- liferative activity. This indicates that phenyl group at
stilbenecompounds, fourhumancancercelllines, MCF- the second position of pyran ring has a key role in deter-
7
(Breast adenocarcinoma), A549 (Lung carcinoma), mining activity (Figure 1). Compound 9d, 9e and 9f also
DU145 (prostrate carcinoma) and HeLa (Cervical carci- showed good activity in HeLa cell line (Table S4).
noma) were tested using MTT (3-(4,5-dimethylthiazol- From Table S4 (Supplementary Information) we
-yl)-2,5-diphenyltetrazolium bromide) assay with CA- found that among the series only the Z-olefinic com-
, a known anticancer drug, was used as positive pounds 9d, 9e, 9f, 9g, 9m and 9n exhibited good
2
4
control. The concentrations for antiproliferative activ- cytotoxicity against the HeLa cell line. Particularly
ities (growth percentage of cells) of the synthesized compound 9g decreased the viability of HeLa cells to
compounds are shown in Table 4 in the supplementary 9.36% at 10μM concentration whereas CA-4 (1 μM)
information.
exhibited similar values in parallel experiments. Then

127 Page 10 of 15
J. Chem. Sci. (2018) 130:127
Table 4. In vitro IC50 values of cis combretastatin analogs 9(d–g) and
(m–n) on HeLa, MCF-7, A549 and DU145 cancer cells.
9
Comps. HeLa MCF7 A549
DU145
9
9
9
9
9
9
d
g
e
f
m
n
20.73 ± 0.02 54.75 ± 0.15 66.66 ± 0.44 56.02 ± 0.09
10.62 ± 0.01 75.43 ± 0.06 60.56 ± 0.26 64.37 ± 0.10
22.63 ± 0.02 50.91 ± 0.11 72.76 ± 0.12 71.94 ± 0.16
28.54 ± 0.11 58.38 ± 0.04 78.65 ± 0.11 73.21 ± 0.11
61.45 ± 0.12 62.27 ± 0.05 58.37 ± 0.20 64.69 ± 0.07
62.84 ± 0.06 86.02 ± 0.05 56.12 ± 0.12 75.40 ± 0.02
CA-4
1.32 ± 0.01
1.03 ± 0.06
1.48 ± 0.07
1.70 ± 0.03
we planned to study the half maximal inhibitory microtubule polymer. Microtubule disrupting agents
concentrations (IC ) of these selective compounds (9d, target this dynamic equilibrium tubulin depolymeriza-
50
9
e, 9f, 9g, 9m and 9n) in the same cell lines (HeLa, tion agents (CA-4) inhibit polymerization. To further
MCF-7, A549 and DU145). The IC values are shown analyze whether the block in cell cycle at G2/M by
50
below in Table 4.
9g can be reflected even in the cellular levels of solu-
ble and polymerized tubulin (microtubules), we treated
HeLa cells with 9g at 10 μM concentration for 18 h
and CA-4 (1 μM) was employed as a positive control.
Following treatments, intracellular levels of soluble
3
.2.2 Antimitotic studies of compound 9g and combre-
tastatin (CA-4) in HeLa cell lines We further studied
the anti mitotic activity of most potent compound 9g in
HeLa cell lines. To examine the antimitotic effects, cell
cycle analysis of most active compound 9g (10 μM) and
combretastatin(CA-4) (1 μM) standard, flow cytome-
try was carried out. Cell cycle analysis in HeLa cell
lines revealed that treatment with 9g and combretas-
tatin (CA-4) exhibited 87.39% and 82.13% respectively.
Accumulation of cells in G2/M phase with a concomi-
tant decrease in the percentage of cells at G0/G1 phase
was observed (Figure 2).
(
free tubulin) and polymerized (tubulin from micro-
tubules) fractions of tubulin were analyzed by immuno-
blotting. Results indicate that DMSO treated cells (con-
trols) exhibited nearly equal distribution of tubulin in
soluble and insoluble fraction, cells treated with 9g and
CA-4 demonstrated complete shift into soluble fraction
(Figure 4A).
3
.2.5 Cell cycle related expression of cyclin B1 Cyclin
3
.2.3 Effects of compound 9g on cellular microtubules B1regulatestheprogressionofcellcycleatG2/Mphase.
network and nuclear morphology To examine the anti- Maximum expression of cyclin B1 expression will be
tubulin effects of compounds 9g and CA-4 at cellular seen during metaphase. Immunoblot analysis of cyclin
level, we treated HeLa cells at 10μM and 1μM concen- B1 following treatment of cells with 9g (10 μM) and
trations for 18 h and analyzed the cellular microtubule CA-4(1 μM)for18hresultedinanincreasedexpression
network by immunofluorescence followed by nuclear of cyclin B1 as compared to DMSO treated controls.
staining with DAPI. Results demonstrate that cells Therefore, the increased protein levels of cyclin B1 by
treated with 9g and CA-4 exhibited disrupted micro- 9g confirm that these compounds acts as anti-tubulin
tubule organization, as compared to the DMSO control agents and block the cells at mitotic phase (Figure 4B).
(
Figure 3).
HeLa cells were treated with 9g at 10 μM and CA-4 3.2.6 Materials and Methods: Cell Cultures, Mainte-
at 1 μM concentrations for 18 h. Following the termi- nance and Anti-proliferative Evaluation All cell lines
nation of the experiment, cells were fixed and stained used in this study were purchased from the Ameri-
for tubulin. DAPI was used as counterstain. The merged can Type Culture Collection (ATCC, United States).
images of cells stained for tubulin and DAPI are repre- HeLa, A549 and DU145 were grown in Dulbecco’s
sented. The photographs were taken using an Olympus modified Eagle’s medium (containing 10% FBS in a
◦
confocal microscope equipped with FITC and DAPI fil- humidified atmosphere of 5% CO
at 37 C). MCF-7
2
ter settings. Data is the representative of five different cells were cultured in Eagle’s minimal essential medium
fields of view.
(MEM) containing non-essential amino acids, 1 mM
sodium pyruvate, 10 mg/mL bovine insulin, and 10%
3
.2.4 Analysis of soluble versus polymerized tubulin FBS. The synthesized test compounds were evalu-
in cells A dynamic equilibrium exists between the ated for their in vitro antiproliferative activity in four
intracellular pool of α,β-tubulin heterodimers and the different human cancer cell lines. MTT cell proliferation

J. Chem. Sci. (2018) 130:127
Page 11 of 15 127
Figure 2. Anti-mitotic effects of compound 9g. Panel A. HeLa cells were harvested after treatment with
compound 9g (10μM) and CA-4 (1μM) for 18 h. Untreated and CA-4treated cells served as control and
standard. The percentage of cells in each phase of the cell cycle was quantified by flow cytometry. Panel B:
Distribution of cells at G0/G1, S and G2/M phase of cell cycle following treatment with compound 9g and
CA-4 in HeLa cells.
assay was used to estimate cell viability or growth. Cell
lines were grown in their respective media containing screened for IC50 in four concentrations (1, 10, 25 and
0% fetal bovine serum. Cells were trypsinized when 50 μM) of test compounds (2 μL) were evaluated in
Compounds with good inhibition were further
1
sub-confluent from T25 flasks/60 mm dishes and were triplicates, resulting in the required final drug concen-
seeded into 48-well microtiter plates in 500μL aliquots trations. Plates were incubated further for 48 h, and the
at plating densities depending on the doubling time of assay was terminated by the addition of 10μL of 5%
◦
individual cell lines. The microtiter plates were incu- MTT and incubated for 60 min at 37 C. Later, the plates
◦
bated at 37 C, 5% CO , 95% air, and 100% relative were air-dried. The bound stain was subsequently eluted
2
humidity for 24 h prior to addition of experimental with 100μL of DMSO and the absorbance was read on a
drugs. Aliquots of 1μL of the test compounds were multimode plate reader (Perkin Elmer) at a wavelength
added to the wells already containing 500μL of cells, of 565 nm. The sensitivity of the cancer cells to the test
resulting in the required final drug concentrations and compound was expressed as IC50 values which are indi-
each compound was tested in duplicate wells. Plates cated as mean ± SD of three independent experiments.
were incubated further for 48 h, and the assay was
terminated by the addition of 50 μL of 5% MTT and 3.2.7 Analysis of Cell Cycle HeLa cells grown in
◦
incubated for 60 min at 37 C. Later, the plates were 60 mm dishes were treated with compound 9g (10 μM)
air-dried. The bound stain was subsequently eluted with and CA-4(1 μM) for 18 h. Cells were harvested with
◦
2
50μL of DMSO. Per cent growth was calculated on Trypsin-EDTA, fixed with ice-cold 70% ethanol at 4 C
a plate by plate basis for test wells relative to control for 30 min, ethanol was removed by centrifugation and
wells.
cells were stained with 1 mL of DNA staining solution

127 Page 12 of 15
J. Chem. Sci. (2018) 130:127
Figure 3. Effect of compound 9g on microtubules and nuclear condensation.
[
containing 50μg of Propidium Iodide (PI), and 0.1 mg in medium containing DAPI. Images were captured
RNase A] for 30 min. The DNA contents of 20,000 using the Olympus confocal microscope and analyzed
events were measured by flow Cytometer (MoFlow). with Provision software.
3
.2.8 Immunofluorescence HeLa cells were seeded on 3.2.9 Western blot analysis Cells were seeded in
5
glass coverslips, treated for 18 h in the presence 6-well plates at 2 × 10 cells per well in complete
or absence of test compounds 9g (10 μM) and CA- growth medium. Following treatment with compound
4
(1 μM). Following treatments, cells were fixed in 3.5% 9g (10 μM) and CA-4(1 μM) for 18 h, cells were
formaldehyde in phosphate-buffered saline (PBS) pH washed with PBS and subsequently soluble and insol-
.4 for 10 min at room temperature. Cells were per- uble tubulin fractions were collected. To collect the
7
meabilized for 6 min in PBS containing 0.5% Triton soluble tubulin fractions, cells were permeabilized with
X-100 (Merck) and 0.05% Tween-20 (Merck).The per- 200 μL of pre-warmed lysis buffer [80 mM Pipes-KOH
meabilized cells were blocked with 2% BSA (Merck) in (pH 6.8), 1 mM MgCl2, 1 mM EGTA, 0.2% Triton
PBS for 1 h. Later, the cells were incubated with the pri- X-100, 10% glycerol, 0.1% protease inhibitor cocktail
◦
mary anti-α-tubulin antibody (Merck) (1:200) diluted in (Sigma Aldrich)] and incubated for 3 min at 30 C. Lysis
blocking solution for 4 h at room temperature. Subse- buffer was gently removed, and mixed with 100 μL
quently, cells were washed thrice with PBS and then of 3x Laemmli’s sample buffer (180 mMTris HCl pH
incubated with FITC labelled anti-mouse secondary 6.8, 6% SDS, 15% glycerol, 7.5% b-mercaptoethanol
antibody (Merck) (1:500) for 1 h at room temperature. and 0.01% bromophenol blue). Samples were immedi-
◦
Finally, cells were washed thrice with PBS and mounted ately heated to 95 C for 3 min. To collect the insoluble

J. Chem. Sci. (2018) 130:127
Page 13 of 15 127
8
2.13% respectively. Accumulation of cells in G2/M
phase with a concomitant decrease in the percentage of
cells at G0/G1 phase was observed. The result of the
effect of Compound 9g, was most evident, on cellu-
lar microtubule networks (immunofluorescence study).
Compound 9g increased the amount of tubulin in the sol-
uble fraction, a feature of microtubule depolymerizing
agents. Increased expression of cyclin B1 in cells treated
with compound 9g was found. Thus, these results sug-
gest that (Z)-2-phenyl/H-3-styryl-2H-Chromenes 9(a–
r) and (E)-2-phenyl/H-3-styryl-2H-Chromenes 10(a–
s) derivatives particularly compound 9g have potential
to develop as a new class of tubulin polymerization
inhibitors. The compounds of these series are further
amenable for structural modifications and will be useful
as templates for the design of new anticancer agents.
Figure 4. Effect of compound 9g on tubulin
polymerization and Cyclin-B1. A: HeLa cells were
treated with 10 μM of 9g and CA-4 at 1 μM
concentration for 18 h. The fractions containing
soluble and polymerized tubulin were collected;
tubulin was detected by immunoblot analysis. B:
HeLa cells were treated with 10 μM of 9g and
CA-4 at 1 μM concentration for 18 h. Subse-
quently, whole lysates were prepared and analyzed
for cyclin-B1. A non-specific (NS) band at 90 kDa
was used as loading control.
Supplementary Information (SI)
The characterization of the compounds 5(a–f), 5’(b–c), 9(a–
r) and 10(a–s) using 1H, C NMR, IR and Mass data
13
(Figures S1–S125) are given in the supplementary informa-
tion. Supplementary Information is available at www.ias.ac.
in/chemsci.
tubulin fraction, 300 mL of 1xLaemmli’s sample buffer
was added to the remaining pellet in each tube and the
samples were heated to 95 C for 3 min. Equal volumes
◦
Acknowledgements
of samples were run on an SDS-10% polyacrylamide gel
and were transferred to a nitrocellulose membrane by SN and SRM are thankful to DST India (SR/FT/CS-139/2011
&
SR/FT/CS-87/2012), UGC New Delhi (47-276/2013),
semidry transfer at 120 mA for 1 h. Blots were probed
with mouse anti-α-tubulin antibody (Merck) and fur-
ther incubated with horseradish peroxidise-conjugated
rabbit anti-mouse secondary antibody(Merck) at room
temperature for 1 h. Bands were visualized using an
enhanced chemiluminescence method (Merck).
UGC start-up-grant no. F.30-127/2015(BSR), CSIR New
Delhi [02(0134)/13/EMR-II, 02(0218)/14/ EMR-II] for pro-
viding research grant. SMiLE project (IICT, Hyderabad) from
CSIR, India is gratefully acknowledged.
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