Design, Synthesis, and in vitro and in vivo Evaluations of (Z)-3,4,5-Trimethoxystyrylbenzenesulfonamides/sulfonates as Highly Potent Tubulin Polymerization Inhibitors

Article abstract of DOI:10.1002/cmdc.201600643

Newer therapeutics can be developed in drug discovery by adopting the strategy of scaffold hopping of the privileged scaffolds from known bioactive compounds. This strategy has been widely employed in drug-discovery processes. Structure-based docking studies illustrate the basic underlying concepts and reveal that interactions of the sulfonamide group and hydrophobic interactions are crucial. On the basis of this strategy, over 60 synthetic analogues were synthesized and evaluated for their cytotoxicity against the NCI panel of 60 human cancer cell lines; the majority of these compounds exhibited promising cytotoxicity with GI₅₀ values ranging between 18 and 50 nm. Among these compounds, (Z)-N-[2,3-dimethoxy-5-(3,4,5-trimethoxystyryl)phenyl]-4-methoxybenzenesulfonamide (7 a) and (Z)-N-[2-hydroxy-3-methoxy-6-(3,4,5-trimethoxystyryl)phenyl]-4-methoxybenzenesulfonamide (9 a) were found to be potent. Similar results were obtained against three human cancer cell lines with IC₅₀ values ranging between 0.04 and 3.0 μm. Studies aimed at elucidating the mechanism of action of these new analogues revealed that they inhibited the in vitro polymerization of tubulin and disorganized the assembly of microtubules in HeLa and MCF-7cancer cells. Lead compounds 7 a and 9 a displayed notable in vivo antitumor activity in a HeLa tumor xenograft model. Our studies have resulted in the identification of a scaffold that can target tubulin polymerization, which should have significant potential toward the development of new antitumor drugs.

Full text of DOI:10.1002/cmdc.201600643

Accepted Article
Title: Design, Synthesis, in vitro and in vivo Evaluation of (Z)-3,4,5-
Trimethoxystyryl Benzenesulfonamides/Sulfonates as Highly
Potent Tubulin Polymerization Inhibitors
Authors: Ahmed Kamal
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To be cited as: ChemMedChem 10.1002/cmdc.201600643
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A Journal of
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ChemMedChem
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FULL PAPER
Design, Synthesis, in vitro and in vivo Evaluation of (Z)-3,4,5-
Trimethoxystyryl Benzenesulfonamides/Sulfonates as Highly
Potent Tubulin Polymerization Inhibitors
Rasala Mahesh,[a] V. Lakshma Nayak,[a] Korrapati Suresh Babu,[a] Riyaz, Sd.,[a] Thokhir B. Shaik,[a] G.
Bharth Kumar,[a] Mallipeddi. P. L.,[d] Ch. Ratna Reddy, [a] Kunta Chandra Shekar,[a] Jedy Jose,[c]
Narayana Nagesh,*[c] Ahmed Kamal,*[a,b]
[
a]
Rasala Mahesh, V. L. Nayak, K. S. Babu, Riyaz, Sd., Thokhir B. Shaik, G. B. Kumar, C. R. Reddy, K. Chandra Shekar, Ahmed Kamal,*
Medicinal Chemistry and pharmacology division,
CSIR-IICT, Hyderabad, India
E-mail: ahmedkamal@iict.res.in
Ahmed Kamal,*
[
b]
Department of Medicinal Chemistry,
NIPER-Hyderabad, India
[
c]
d]
Jedy Jose, Narayana Nagesh*
CSIR-CCMB Hyderabad. India
Mallipeddi. P. L.,
[
University of Houston USA
Abstract: Newer therapeutics can be developed in drug discovery
by adopting the strategy of scaffold hopping of the privileged
scaffolds from known bioactive compounds. This strategy has been
widely employed in drug discovery process. Structure based docking
studies illustrates the basic underlying concepts, which have been
carried out, reveal interactions of sulfonamide group and
hydrophobic interactions are crucial. Based on this strategy over 60
the polymerization of tubulin by interacting at the colchicine-
binding site.[9] The potent activity and the simple chemical
structure has made it a popular starting point for the design of
new anticancer agents.[10] A water soluble pro-drug (CA-4P, 2b)
that is currently undergoing phase I/II clinical trials is a notable
and closely related CA-4 analogue.[11] Numerous synthetic
derivatives and analogues of 2a have been developed in the
recent past to address various drawbacks such as unstable
nature of the olefinic unit, poor solubility and toxicity.
synthetic analogues were
synthesized and evaluated for their
cytotoxicity against the NCI panel of sixty human cancer cell lines,
majority of these compounds exhibited promising cytotoxicity with
GI50 values ranging between 18-50 nM, among them compounds 7a
and 9a were found to be potent. Similar results were obtained
against three human cancer cell lines with IC50values ranging
between 0.04-3.0 µM. Further studies aimed at elucidating the
mechanism of action of these new analogues revealed that they
inhibited the in vitro tubulin polymerization and disorganized the
microtubule assembly in HeLa and MCF-7cancer cells. The lead
compounds 7a and 9a displayed notable in vivo antitumor activity in
HeLa tumor xenograft model. Our studies resulted in the
identification of a scaffold that can target tubulin polymerization
having significant potential towards the development of new
antitumor drugs.
Figure 1. Novel combretastatin-sulfonamide (sulfonate) conjugates designed
for the present study.
Introduction
The beneficial influence of sulfonamide substituents on the
anticancer potential of designed compounds have been
demonstrated earlier.[12,13] The antitubulin agents such as E7010
(
constitute a few illustrative examples that are pertinent to the
present work. The sulfonamide moiety is easy to install as well
as it generally imparts stability and crystallinity to the attached
The aggregation of α- and β-tubulins to form microtubules and
its disassembly, generally known as microtubule dynamics,
constitutes a vital process that aids in the proper functioning and
division of eukaryotic cells.[1-3] The pivotal role of microtubule
dynamics in mediating cell division makes it an attractive target
for the design of cancer chemotherapeutics. A number of natural
and manmade molecules that cause mitotic arrest by disrupting
the microtubule dynamics are known. They are generally
3),[14] E7070 (indisulam, 4),[15] and the biarylsulfonamide 5 [16]
pharmacophore.
Our
approach
of
hybridizing
two
pharmacophoric units to design improved anticancer agents has
led to the development of a variety of potent, small molecule
tubulin binders.[17] In this context, we surmised that the anchoring
of sulfonamide groups on the combretastain scaffold may
improve its general anticancer potential and stability. The
[
4]
classified into microtubule stabilizers (e.g., paclitaxel) and
microtubule destabilizers such as vinca alkaloids,[5,6] colchicine
(
1)[7] and combretastain A-4 (2a)[8] (Figure 1). The latter inhibits
1
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ChemMedChem
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docking process involves two basic steps: prediction of the
ligand conformation as well as its position and orientation within
the sites and assessment of the binding affinity, the binding
modes of these compounds are discussed.[18-19] Considering this
fact, a collection library of combretastatin-sulfonamide (and
sulfonate) conjugates were designed as depicted in Figure 2.
The trimethoxyphenyl ring of CA-4 has not been tinkered as it
has shown to be essential for the biological activity. Attachment
of the sulfonamide/sulfonate groups on the remaining aryl ring
would result in 6, 7, 8 and 9, whereas the fictionalization of the
olefinic unit with N-sulfonyl carboxamide groups would afford
compounds 10 and 11 (Figure 2). This study, therefore, is aimed
at the synthesis of combretastatin-sulfonamide (and sulfonate)
conjugates and later the antiproliferative activity. The cytotoxicity
designed and synthesized (Figure 1).[28] Previous studies
enabled to rationalize the key pharmacophore elements from a
diverse set of colchicine site inhibitors, which included a
hydrogen bond acceptor, two hydrophobic centers and a planar
group.Trimethoxystyryl series has an additional hydrogen bond
donor and a hydrophobic feature. Trimethoxystyryl series has
been designed to test the optimal position to add
phenylsulfonalamide moiety and the synthesized analogues are
divided into the following four groups (Figure 3):
(i) Sulfonamide phenyl moiety at meta position of ring B of CA-4
(6a-j, 7a-i series),
(ii) Replace ‘NH’ of sulfonylamide moiety with a ‘O’ from group 1
(8a-8iseries),
(iii) Sulfonamide phenyl moiety at ortho position of ring B of CA-
4 (9a-9k) and
(iv) Sulfonamide phenyl moiety on the olefinic bond (10a-m,
11a-f series).
depicted in this article has been previously described in patent
20]
3
076DEL2014 (PCT/IN2015/050148).[ Further, tubulin-binding
potential and the in vivo antitumor activity of the designed
compounds were examined. It is noteworthy to mention that
most all the compounds exhibited excellent antiproliferative
activity and two representative compounds (7a and 9a)
displayed promising in vivo activity against HeLa tumor in mice.
Figure 2: Novel combretastatin-sulfonamide (sulfonate) conjugates designed
for the present study.
Figure 3. Compound 9a docked into the αβ interface of tubulin (1SA0).
Residues within 3.5 A of ligands are shown. Site 1 binds with trimethoxy
phenyl moiety (backbone is coloured in magenta), Site 2 is occupied by 4-
methoxy phenyl moiety (backbone is shown in blue) and site 3 involves 3,4-
substituted phenyl moiety (backbone is shown in blue) and site 3 involves 3,4-
substituted phenyl moiety (backbone is shown in green). H-bonding interaction
with Thr179α is represented by a dotted line.
Design of newer tubulin inhibitors based on pharmacophore
features.
Pharmacophore may be defined as a set of key chemical
features contributing to biological activity of a compound. Based
on the strategy of scaffold hoping of the privileged scaffolds from
known bioactive compounds, new therapeutics can be
Molecular docking analysis.
[
21-
Docking studies were performed to understand binding modes of
trimethoxystyryl benzene series. Following observations are
based on binding mode predicted by docking. Trimethoxy
phenyl binds in a pocket surrounded by residues Leu255β,
Leu248β and Ala250β (site 1, Figure 3). Where These studies
also suggest the water mediated hydrogen bonding interactions
between Leu248β and Ala250β and the methoxy groups, and
this observation, however needs further investigation. Predicted
binding mode revealed the presence of a olefin bond which
developed and has been widely employed in drug discovery.
2
2]
Trimethoxy phenyl and tropolone methyl ester rings of
colchicine are essential features contributing to the binding of
colchicine. Specifically, the keto group on the tropolone ring has
been known to exhibit interactions with binding site residues.23]
The hydroxyl and methoxy groups on the second phenyl ring of
CA-4 is known to exhibit interactions with colchicine binding site
similar to functional groups on tropolone of colchicines.[24]
Olefinic bond is known to be important in maintaining the
compound in the right configuration and its modification is
reported to reduce the activity of CA-4 analogues.[25]
Pharmacophore of sulfonamide focused libraries (including
E7010) targeting tubulin have been evaluated in an array-based
structure and gene expression relationship study. The study
revealed N-(2-aminophenyl sulfoniamide moiety as the key
pharmacophore.[26-27] Novel series of tubulin inhibitors including
plays
a key role in maintaining the compounds in 'cis'
conformation, consistent with prior observations. Addition of a
bulky group onto this bond thus weakens the activity of group 4
series. The amino group from sulfonyl amido group acts as H-
bond donor to Thr179α. The H-bonding interaction of colchicine
binding site inhibitors with Thr179α, has been widely discussed
in literature. Lack of H-bond donor in this position explains the
difference in activities of group 3 series versus group 1 and 2.
Methoxy phenyl occupies a pocket formed by Ala180α, Val181α,
key pharmacophore features from E7010 and CA-4 were
2
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ChemMedChem
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Lys352β and Asn258β (site 2, Figure 3). Mutational studies
established equivalent residue (Lys350β) as one of the key
interacting residues.[29,30] Where as 4-methoxy of phenyl
exhibited hydrophobic interactions with side chains of Val 181
and Met b259 and is consistent with the pharmacophore
discussed previously. The residues Gln11α and Tyr224α are
within 3.5 Å of 3-hydroxy, 4-methoxy phenyl moiety (site 3,
Figure 3). The 3-hydroxy and 4-methoxy phenyl moiety
represents novel hydrophobic feature among colchicine
inhibitors that target novel binding pocket (site 3, Figure 3).
Mutational studies of Gln11α and Tyr224α could be interesting
to further establish this new binding pocket. Extending or
replacing 4-methoxy with hydrogen bond donors for the
trimethoxystyryl series, could also contribute to favorable
interactions in this binding site and the predicted docking model
agrees well with the earlier experimental studies.[33,34] However,
some experimental studies will further establish crucial roles
played by various residues and encourage structure-based
optimization of suggested leads. The docking studies also reveal
there is ample scope for further optimization of the
trimethoxystyryl series.
prostate cancer and breast cancer) as per the NCI protocol.
Over sixty compounds, eighteen (6a–c, 7a-d, 7f, 7g-i, 9a-c 9f-i
and 9k) were selected for the preliminary screening at a single
dose concentration (10 μM) which exhibited significant mean
growth inhibition. Eleven compounds were further evaluated in
the secondary screening at five dose concentrations with 10-fold
dilutions which displayed remarkable antiproliferative efficacy on
most of the NCI cell lines. The primary objective of the present
study was to develop new combretastatin sulfonamides bearing
the key pharmacophore such as E7010 and CA-4. In addition,
the SAR studies disclosed that the sulfonamide group situated
between two aromatic rings (B, C rings) need to be eternal as a
basic design accompanied by a methoxy group at the para-
position as observed in E7010 which is crucial for maximum
cytotoxicity.
The SAR studies have been examined for these diverse
combrestatin sulfonamide analogs. In particular, the aryl
sulphanamide has been attached on B-ring and cis- double bond.
Mostly B-ring linked sulphonamides (6a-j, 7a-i, 8a-I and 9a-k)
exhibited remarkable antiproliferative activity as compared to the
double bond linked sulphonamides (10a-m and 11a-f).
Specifically, the compound 7a and 9a possess broad spectrum
of cytotoxicity within nanomolar range against most of cell lines
with GI50 values below 0.05 µM, wherein, eight different types of
tumor cell lines displayed GI50 values below 0.03 µM and
relatively twenty five cell lines showed GI50 values less than 0.04
µM. This is reliable with pharmacophore studies and docking. In
detail, the compound 7a demonstrated remarkable cytotoxicity
against HL-60 (TB), K-562, SR (leukemia), NCI-H552 (lung),
HCC-2998, HCT-116 (colon), SF-539, SNB-75 (CNS), MDA-MB-
Results and Discussion
Chemistry
Compounds (6a-j, 7a-i) and (9a-k) were prepared by coupling of
properly substituted (Z)-(3,4,5-trimethoxystyryl)anilines (2c, 21
and 22) with various commercially available substituted
benzenesulfonyl chlorides (23a-k) in a 1:4 (v/v) mixture of
pyridine in anhydrous dichloromethane13 and (Z)-3-styrylphenyl
benzenesulfonates (8a-i) prepared by the reaction of
combretastatin (2a) with the similar benzenesulfonyl chlorides by
4
(
0
35 (melanoma), SK-OV-3 (ovarian), A498 (renal) and MCF-7
breast) cancer cell lines with GI50 values of 0.02, 0.03, 0.03,
.02, 0.04, 0.02, 0.02, 0.02, 0.04, 0.02 and 0.03 µM, respectively.
In similar cell lines, compound 9a exhibited GI50 values of 0.02,
.03, 0.03, 0.02, 0.03, 0.03, 0.02, 0.02, 0.03, 0.02 and 0.04 µM,
0
using
dichloromethane as a solvent in the presence of
respectively. Moreover, the other compounds with methyl and
fluoro substituents at the para-position of C-ring showed
significant activity in the sub-micromolar range against all the
tested cell lines. However, the addition of one methoxy group in
the C ring decreased the cytotoxicity. Furthermore, the presence
of electron withdrawing substituents like nitro and trifluoromethyl
groups at the para-position of the sulfonamide system resulted
in the decrease in cytotoxicity. These observations suggest that
by replacing the electron donating by withdrawing groups on the
C-ring showed a reduction in the antiproliferative activities.
triethylamine with outstanding yields. The intermediate
compounds 2a, 2c, 21 and 22 were obtained by the reduction
and deprotection of their respective nitro, hydroxyl stilbene
derivatives (17-20), which were synthesized by double bond
forming Wittig reaction between the suitable benzaldehydes (13-
16) with 3,4,5-trimethoxybenzyl triphenylphosphonium bromide
(
12) in presence of sodium hydride in anhydrous
dichloromethane, afforded a mixture (1:1, v/v) of nitro Z-
stilbenes (17-20) and E-stilbenes as depicted in Scheme 1 and
these isomers have been separated by column chromatography.
The compounds (10a-m and 11a-f) have been achieved by the
formation of amide bond between the stilbene-acrylic acids (27a-
f and 31a-c) with a variety of substituted benzene sulfonamides
Further, all of the compounds (6a-j, 7a-i, 8a-i, 9a-k, 10 a-m and
11a-f) of this series were evaluated for their antiproliferative
activities against three human cancer cells such as A549 (lung),
MCF-7 (breast) and HeLa (cervical) and the results to this
regard are summarized in Table 2. Interestingly, similar to the
NCI screening results, the compounds (Figure 2) possessing a
methoxy group at para-position of C ring showed superior
cytotoxicity with IC50 values ranging between 0.04 to 3 μM.
Correspondingly, the compounds 7a and 9a showed significant
cytotoxicity with respective IC50 values of 0.057 μM and 0.078
μM in MCF7 cell line. These compounds were quite promising in
cervical cancer cell line (HeLa) with respective IC50 values of
(
28a-f and 32a-c) by using EDCI/DMAP.39 Wherein, the
intermediates were prepared in good yield by the Claisen–
Schmidt condensation of different phenyl acetic acids with
appropriate benzaldehydes.
Biological studies
The synthesized derivatives (Z)-N-(3-styrylphenyl)benzene
sulfonamides/sulfonates (6a-j, 7a-i, 8a-i, 9a-k, 10a-m and 11a-f)
have been evaluated for their cytotoxicity against a panel of sixty
human tumor cell lines accomplished from different types of nine
cancer cell lines (leukemia, non-small cell lung cancer, colon
cancer, CNS cancer, melanoma, ovarian cancer, renal cancer,
0.044 μM and 0.047 μM. Based on these promising results, we
selected the HeLa cell line for further in vivo experiments.
3
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Scheme 1. Reagents and conditions: (a) NaH, dry CH
2
Cl
2 2 4
, 0 °C to rt, 12 h; (b) Zn, HCO NH , MeOH, rt, 4 h; (c) TBAF (1M in THF), THF 0 °C, 2 h (d)
2 2
Pyridine, 0-5 °C, 4 h; (e) TEA, CH Cl , 0-5 °C, 4 h.
4
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ChemMedChem
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2 2
Scheme 2 & 3. Reagents and conditions:(a) AC O, NaOH, 140 °C; (b) EDCI, DMAP, TEA, CH Cl2.
the cell cycle block and apoptosis.[35,36] The in vitro screening
results revealed that compounds 7a and 9a showed significant
antiproliferative activity against cervical (HeLa) and human
breast (MCF-7) cancer cell lines. In this context, HeLa and
MCF-7cells were treated with these compounds at 25 and 50
Nm for 48 h. The results clearly indicated that these compounds
arrested the cell cycle at G2/M phase when compared to
untreated control cells [Figures 5a and (Supplemental
Information 5b)]. Compounds 7a and 9a showed 26.30 and
2
6.22% of cell accumulation in G
2
/M phase at 25 nM, whereas
/M
they showed 28.43 and 31.85% of cell accumulation in G
2
Figure 4. Structure–activity relationship (SAR) of the (Z)-3,4,5-trimethoxystyryl
benzenesulfonamide /sulfonates derivatives.
phase at 50 nM concentration, respectively, in HeLa cells. In
case of MCF7 cells, these compounds showed 18.29 and
1
8.80% of cell accumulation in
concentration, whereas they showed 32.83 and 34.41% of cell
accumulation in G /M phase at 50 nM concentration respectively
Tables 5a and (5b in supplemental information)].
2
G /M phase at 25 nM
Cell cycle analysis
2
[
Many anticancer compounds exert their growth inhibitory effect
either by arresting the cell cycle at a particular checkpoint of cell
cycle or by induction of apoptosis or a combined effect of both
5
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ChemMedChem
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6
R=H
a
7a
R= OCH
7b
R= OCH
R -OCH
R =OCH
7c
7d
9
a
9b
R -OCH
R =OCH
9c
9g
9h
9k
1
3
3
R= OCH
3
R= OCH
3
1
1
1
1
1
1
1
1
1
1
R -OCH
3
3
R = Cl
R = F
R = t-Bu
R = CH
3
R =OCH
3
R =OCH
3
3
R = Cl
R = H
2
2
2
2
2
2
2
2
2
2
2
R =H
3
R =H
R =H
R =H
R =H
R =H
R =H
3
R =H
R = Cl
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
0.17
0.19
0.05
0.38
0.31
0.07
0.03
0.02
0.02
0.05
0.04
0.03
0.34
0.37
0.42
0.70
0.56
0.70
0.38
0.24
0.30
0.67
0.38
0.40
0.05
0.03
0.04
0.04
0.06
0.06
0.03
0.02
0.03
0.04
0.04
0.03
0.04
0.33
0.08
0.48
0.45
0.09
0.15
0.03
0.03
0.03
0.20
0.03
0.05
0.46
0.04
0.07
0.20
0.50
0.04
0.04
0.04
0.04
0.04
0.05
0.03
0.03
0.03
0.05
0.05
0.03
NSClung
A549/ATCC
HOP-62
HOP92
NCI-H226
NCI-H23
NCIH322M
NCI-H460
NCI-H522
0.15
0.04
0.10
12.8
0.48
0.43
0.18
0.02
0.05
0.04
17.5
NT
0.06
0.05
0.03
0.02
1.31
17.23
0.20
1.87
3.31
4.71
0.39
0.36
0.71
0.41
0.72
13.5
0.71
0.75
0.36
0.20
0.26
3.1
0.04
0.04
16.0
41.3
0.05
0.06
0.03
0.02
0.36
0.07
NT
0.14
0.27
0.74
0.25
0.06
0.27
0.67
23.6
NT
0.28
0.48
0.31
0.08
0.46
4.80
0.04
0.33
038
0.84
0.04
0.22
0.62
10.0
0.18
0.54
0.49
0.69
0.37
0.21
0.05
28.0
0.05
0.04
0.05
0.10
0.03
0.04
0.07
0.31
0.38
0.37
0.03
0.03
Colon
COLO-205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
CNS
0.03
0.44
0.07
0.06
0.08
0.07
0.08
0.02
0.04
0.03
0.03
0.03
0.04
0.04
1.28
1.83
0.46
0.67
0.48
0.63
0.47
0.25
1.98
0.46
0.39
0.34
0.57
0.34
0.26
0.31
0.04
0.14
0.04
0.04
0.04
0.41
0.03
0.03
0.04
14.0
0.03
0.04
0.47
0.33
0.13
0.33
0.41
0.07
0.11
17.4
0.42
0.11
0.05
27.6
0.12
0.06
3.02
0.37
0.04
0.14
2.53
0.07
0.04
3.32
1.50
0.36
0.23
3.51
0.58
0.32
1.46
0.09
0.04
0.05
1.28
0.04
0.04
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
0.40
0.04
0.12
0.03
0.07
0.10
0.06
0.03
0.02
0.06
0.03
0.03
20.8
0.47
0.51
1.11
0.40
0.68
1.10
0.31
0.30
0.74
0.22
0.46
0.20
0.04
0.03
0.21
0.03
0.05
0.08
0.03
0.03
0.06
0.02
0.04
2.70
0.27
0.08
0.52
0.30
0.34
0.38
0.25
0.04
0.80
0.03
0.28
0.63
0.34
0.15
0.41
0.04
0.47
1.71
1.37
0.15
0.56
0.23
0.59
0.73
0.07
0.02
0.06
0.02
0.05
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
0.27
0.05
0.10
0.03
0.08
0.14
0.05
2.98
0.04
0.05
NT
0.47
NT
0.56
NT
0.06
20.0
0.03
0.03
1.38
1.61
0.22
20.3
0.06
40.0
15.1
0.03
0.02
0.04
NT
0.03
NT
0.03
0.25
NT
0.15
NT
0.36
NT
0.03
0.02
0.6
3.07
0.27
NT
0.66
NT
0.06
NT
0.03
0.02
0.04
0.08
0.03
41.0
0.04
0.49
0.26
5.29
3.27
4.88
NT
0.39
0.18
0.61
1.14
0.29
12.5
0.43
0.17
0.03
6.01
1.85
0.06
NT
0.09
0.02
0.06
0.08
0.05
14.2
0.06
0.19
0.04
1.22
4.91
0.34
13.2
0.11
31.0
0.03
0.02
24.0
4.0
0.03
0.05
0.49
0.05
0.05
Ovarian
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-
RES
0
0
0
0
0
.16
.05
.58
.32
.27
0.06
0.03
0.08
0.05
0.04
4.58
0.41
3.12
6.57
4.08
0.91
0.36
0.15
0.72
0.60
0.09
0.03
0.24
0.64
0.42
0.05
0.03
0.07
10.20
0.04
0.49
0.21
0.93
0.65
0.38
0.37
0.06
0.10
28.6
0.29
0.71
0.04
1.48
0.46
0.34
0.98
0.22
0.18
0.62
0.44
0.11
0.04
0.62
0.16
0.06
0.07
.09
0.03
0.04
0.48
11.4
0.36
0.44
0.09
1.15
0.15
0.03
0.94
2.75
0.26
0.22
0.27
0.86
0.30
1.42
0.21
7.80
0
SK-OV-3
Renal
7
86-0
0.36
0.03
0.06
0.05
0.08
0.68
0.08
0.13
0.06
0.02
0.06
0.05
0.04
0.07
0.06
0.06
0.44
0.15
0.92
0.81
0.68
1.18
10.3
1.59
0.66
0.17
0.58
0.37
0.27
0.75
0.77
0.69
0.06
0.02
0.10
0.05
0.33
0.22
2.01
0.09
0.37
0.02
0.06
0.05
0.03
0.08
0.07
0.06
0.46
0.02
0.54
1.28
0.08
0.40
0.76
0.99
0.57
0.02
0.83
0.05
0.03
0.70
0.99
0.07
0.31
0.02
0.48
0.18
0.05
0.38
1.44
1.52
0.49
0.02
0.45
0.07
0.04
0.61
1.30
0.98
0.11
0.02
0.11
0.14
0.02
0.08
1.23
1.65
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
Prostate
PC-3
0.12
0.34
0.04
0.04
0.37
0.84
0.39
0.50
0.04
0.07
0.04
0.04
0.28
0.38
0.15
0.20
0.05
0.04
0.36
0.39
0.05
0.04
DU-145
Breast
MCF7
0.04
0.03
0.48
0.37
0.05
0.04
0.04
0.04
0.06
0.13
0.04
MDA-MB-
2
31/ATCC
0.39
0.22
0.19
NT
0.05
0.04
0.04
52.1
2.43
3.42
0.66
NT
1.16
0.35
0.96
0.38
0.25
0.08
0.03
39.5
0.05
0.05
0.05
NT
0.33
0.21
0.23
NT
0.20
0.29
0.35
NT
0.16
0.35
0.08
NT
35
0.06
0.05
0.03
42.5
HS 578T
BT-549
0.52
0.46
NT
T-47D
MDA-MB-
435
0.16
0.04
0.26
0.27
0.29
0.04
0.04
0.03
0.16
0.30
0.02
6
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Table 1. GI50 concentrations (µM) for in vitro cytotoxic effect induced by compounds (6a–c, 7a-d, 7f, 7g-i, 9a-c 9f-i and 9k) against the NCL panel of 60
human cancer cells. Data are reported as the GI50 value (concentration required to cause 50% inhibition of cell growth after an incubation time of 48 h).
[
7
6a] NSC774998/1. [7a] NSC775010/1. [7b] NSC 780186/1. [7c] NSC 775011/1 [7d] NSC780185/1. [9a] NSC775014/1 [9b] NSC780182/1 [9c] NSC
75015/1. [9g] NSC780183/1 [9h] NSC780176/1 [9j] NSC780184/1.
code
A549b
MCF-7c
HeLad
code
A549b
MCF-7c
HeLad
6
a
0.86±0.05
1.17±0.04
1.85±0.10
0.07±0.003
1.30±0.09
2.61±0.10
0.06±0.002
1.07±0.06
1.49±0.06
9a
9b
9c
0.09±0.0005
0.69±0.08
0.64±0.06
0.07±0.001
0.07±0.05
0.07±0.05
0.04±0.001
0.06±0.05
0.07±0.05
6
b
c
6
6
d
e
1.13±0.04
0.84±0.007
0.71±0.005
9d
1.08±0.11
1.14±0.05
0.80±0.05
6
2.12±0.07
5.06±0.10
4.26±0.17
2.14±0.18
3.71±0.05
2.13±0.05
1.93±0.05
2.31±0.18
1.62±0.11
9e
9f
2.2±0.18
1.21±0.08
1.06±0.07
3.12±0.05
0.09±0.05
0.95±0.05
2.02±0.05
0.08±0.05
0.76±0.05
6
f
6
g
h
9g
6
1.40±0.08
1.47±0.07
2.81±0.05
0.08±0.003
2.08±0.12
1.61±0.08
0.70±0.007
1.35±0.04
2.00±0.02
1.20±0.06
1.82±0.07
2.13±0.13
1.81±0.11
2.50±0.15
1.22±0.09
1.25±0.13
2.4±0.15
1.18±0.08
1.11±0.04
0.93±0.01
0.04±0.001
0.16±0.01
0.19±0.05
0.09±0.05
1.10±0.07
1.30±0.05
0.95±0.03
1.44±0.07
1.78±0.05
1.21±0.09
0.09±0.005
1.09±0.06
9h
9i
2.89±0.14
1.32±0.05
1.86±0.04
1.02±0.008
3.12±0.14
1.67±0.05
6.30±0.24
2.80±0.12
11.74±0.31
2.31±0.15
2.30±0.14
4.80±0.20
2.88±0.19
2.87±0.28
2.20±0.05
1.54±0.05
0.86±0.05
0.72±0.05
0.08±0.007
1.8±0.05
1.17±0.05
0.76±0.05
0.624±0.05
0.072±0.003
1.21±0.05
0.35±0.05
9.95±0.18
1.12±0.04
5.14±0.10
0.56±0.008
0.41±0.01
1.12±0.05
1.64±0.11
2.59±0.22
0.58±0.008
6
i
6
j
1.05±0.06
0.05±0.002
0.66±0.006
0.57±0.008
0.08±0.05
0.90±0.006
2.08±0.10
2.89±0.15
1.53±0.06
1.83±0.05
1.28±0.04
0.09±0.004
1.23±0.08
9j
7a
7b
7c
7d
7e
9k
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
0.53±0.05
12.6±0.32
0.88±0.05
4.25±0.22
0.92±0.009
0.85±0.03
0.88±0.10
1.29±0.06
0.48±0.05
0.61±0.04
7
f
7
g
h
7
7
i
7
j
8
a
b
8
8c
0.98±0.015
0.85±0.009
0.70±0.08
10l
3.80±0.25
3.25±0.18
1.23±0.015
8
d
e
1.90±0.07
1.92±0.05
3.63±0.25
1.28±0.15
1.99±0.09
3.34±0.12
0.03±0.008
1.23±0.05
1.79±0.10
1.28±0.08
2.56±0.05
0.78±0.008
1.29±0.07
1.45±0.05
0.04±0.003
1.96±0.08
1.54±0.12
1.16±0.04
4.34±0.14
0.74±0.008
1.15±0.05
1.13±0.08
0.04±0.003
2.42±0.12
10m
11a
11b
11c
11d
11e
11f
7.91±0.28
2.93±0.12
1.04±0.009
1.38±0.05
1.57±0.08
1.99±0.02
3.34±0.15
3.97±0.22
1.73±0.08
1.36±0.04
0.74±0.04
0.84±0.07
0.39±0.004
1.29±0.09
1.45±0.04
2.46±0.13
0.63±0.055
2.16±0.045
0.49±0.009
0.80±0.08
0.38±0.002
1.15±0.08
1.13±0.052
2.05±0.09
8
8
f
8
g
h
8
8
i
(
2a)
2c)
(
24
17a
2.14±0.05
3.75±0.05
3.04±0.05
E7010
1.22±0.03
1.97±0.10
1.98±0.05
Table 2. IC50 concentrationsa (µM) for in vitro cell growth inhibition by compounds (6a-j, 7a-i, 8a-i, 9a-k, 10a-m and 11a-f) treated against selected human
cancer cell lines for 48 h. [a] Concentration required to inhibit 50% cell growth following 48 h treatment with the tested drug. [b] A549: non-small cell lung
cancer; [c] MCF-7: breast cancer cell line; [d] HeLa: cervix cancer.
Wherein the inhibition was demonstrated by a decreased
Effect of 7a and 9a on tubulin polymerization.
The G /M cell cycle arrest is mostly associated with tubulin
fluorescence at emission wavelength of 420 nm against
excitation wavelength of 360 nm (Figures 6). Tubulin was co-
incubated with or without compounds (7a, 9a and CA ) and
4
2
polymerization inhibition [38] and since the compounds 7a and 9a
arrested the cell cycle at G2/M phase as compared to the
untreated control cells, it was important to understand the
microtubule inhibitory function of compounds 7a and 9a. To
evaluate these compounds on tubulin polymerization, the
fluorescent-based tubulin polymerization assay was conducted.
fluorescence readings were recorded at emission wavelength
420 nm against excitation at 360 nm using Infinite 200M
multimode reader (Tecan). The IC50 of tubulin by both the
compounds was determined at five different concentrations (0.1,
0.5, 1, 2 and 4 µM). It was observed that the tubulin IC50 with
compound 9a and CA-4 was 0.6 µM and 0.7 µM, respectively,
which was comparable (Table 4).
7
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Figure 6. Effect of conjugates 7a and 9a on tubulin polymerization, the tubulin
polymerization was monitored by the increase in fluorescence at 360 nm
(
excitation) and 420 nm (emission) for 1 h at 37 °C. All the compounds were
incubated at a final concentration of 3 µM. CA-4 was used as a positive control.
Compound
Tubulin (IC50, µM)
7
a
a
1.6 ±0.08
0.6 ±0.02
0.7 ±0.03
9
CA-4
Table 4. a Concentration of drug to inhibit 50% of tubulin assembly. Values
indicated are the mean ± SD of two different experiments performed in
triplicates.
Competitive colchicine binding assay.
Compounds 7a and 9a revealed parallel inhibitory effects on
tubulin polymerization which was comparable to CA-4 and this
prompted us to explore whether these compounds bind to the
colchicine site of tubulin using fluorescence based assay.
Tubulin (3 µM) was incubated with the various concentrations of
compounds 7a and 9a (0–20 µM) in the presence of colchicine
(
3 µM) with CA-4 as positive control at 37 °C for 60 min and the
fluorescence of tubulin–colchicine complex was monitored at
35 nm when excited at 350 nm. We noticed an increase in
4
Figure 5a. Cell cycle analysis of compounds 7a and 9a treated on HeLa cells
for 48 h. (A-F) Response of mitotic spindle checkpoints on HeLa cells to
compounds 7a and 9a. Flow cytometric analysis showed that the majority of
fluorescence of tubulin–colchicine complex in the presence of
tested CA-4 analogues (7a and 9a) and CA-4. This fact
corroborates with the earlier observation that CA-4 generates
increased fluorescence upon binding at the colchicine site
(Figures 7).[37] Consequently, the experiment was carried out
both in the presence and absence of colchicine to obtain
fluorescence values of the desired tubulin–colchicine complex,
the fluorescence values of tubulin–test compounds complex was
deducted from the tubulin–test compounds–colchicine complex.
It was found that at low concentrations the tested compounds 7a
and 9a and CA-4 showed significant affinity towards colchicine
binding site, whereas at higher concentration 7a and 9a
exhibited a steady increase, while CA-4 showed lower binding
affinity. Compounds 7a and 9a exhibited more affinity towards
colchicine site, when compared to the positive control CA-4.
Vinblastine was used as a negative control, which is known to
bind at a different site and did not show any effect on tubulin–
colchicine complex. Therefore, this study suggests that
compounds 7a and 9a binds at the colchicine site on tubulin.
the cells were arrested in G
Control or untreated HeLa cells, (B): treatment with 25 nM of compound CA-4;
C): treatment with 25 nM of compound 7a; (D): treatment with 50 nM of
compound 7a; (E): treatment with 25 nM of compound 9a; (F): treatment with
0 nM of compound 9a. According to figure 5a; P-1 represents Sub G1 phase,
2
/M phase of the cell cycle in HeLa cells; (A):
(
5
P-2 represents G0/G1 phase, P-3 represents S phase and P-4 represents
G2/M phase.
Sample
Sub G
1
%
G
0
/G
1
%
S %
0.83
0.32
0.46
0.52
0.30
0.31
2
G /M %
A: Control (HeLa)
B: CA-4 (50 nM)
C: 7a (25 nM)
D: 7a (50 nM)
E: 9a (25 nM)
F: 9a (50 nM)
1.30
0.72
1.18
1.16
1.01
1.50
94.43
58.74
64.43
59.96
66.22
62.42
3.29
27.46
26.30
31.85
26.22
28.43
Table 3a. Distribution of HeLa cells in various phases of cell cycle.
Immunohistochemistry studies on tubulin.
2
1
1
1
1
1
0000
8000
6000
4000
2000
0000
In addition, we investigated the alterations in the organization of
the cellular microtubule network in HeLa and MCF-7cells
induced by conjugates 7a and 9a by using fluorescence
microscopy, as most antimitotic agents affect microtubules.[ In
this context, the HeLa and MCF-7cells were treated with
compounds 7a and 9a at a concentration of 25 nM for 48 h. The
results demonstrated a well-organized microtubular network in
control cells. However, the cells treated with these compounds
showed disrupted microtubule organization as depicted in
Control
7
a
a
9
CA4
39]
8
6
4
2
000
000
000
000
0
[
Figures 8a and (8b in Supplemental Information)], thus
confirming the inhibition of tubulin polymerization.
0
5
10
15
20
25
30
35
-2000
Time (min)
8
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death.[40] It was considered interesting to investigate the
apoptotic inducing effect of conjugates 7a and 9a by Hoechst
staining (H-33258) in HeLa and MCF-7cells. Therefore, HeLa
1
.2
1
and
MCF-7cells were treated with these compounds at
concentrations of 25 and 50 nM for 48 h. After treatment with
these compounds, marked morphological changes, such as
nuclear fragmentation, condensation of chromatin, and formation
of apoptotic bodies were observed as seen in Figures 9a and
0
0
0
0
.8
.6
.4
.2
0
7a
9a
CA4
(
9b in Supplemental Information).
Vinblastin
0
5
10
15
20
25
Concentration (µM)
Figure 7. Fluorescence-based colchicine competitive binding assay of
conjugates 7a and 9a were carried out at various concentrations containing 3
µM of tubulin and colchicine for 60 min at 37 °C. CA-4 was used as a positive
control and vinblastine was used as negative control which binds at the vinca
domain site. Fluorescence values were normalized to DMSO (control).
Figure 9a: Hoechst staining of HeLa cells. A: Untreated control cells (HeLa),
B: CA-4 (25 nM), C: 7a (25 nM), D: 9a (25 nM).
Measurement of mitochondrial membrane potential (∆Ψm).
The maintenance of mitochondrial membrane potential ((∆Ψm)
is significant for mitochondrial integrity and bioenergetic
function.[
41]
Mitochondrial changes, including loss of
mitochondrial membrane potential (∆Ψm), are key events that
take place during drug-induced apoptosis. Mitochondrial injury
by 7a and 9a was evaluated by detecting a drop in mitochondrial
membrane potential (∆Ψm). In this study, we have investigated
the involvement of mitochondria in the induction of apoptosis by
these compounds. HeLa and MCF-7cells were treated with 7a
and 9a at 25 and 50 nM concentrations for 48 h. After 48 h of
drug treatment, it was observed that reduced mitochondrial
membrane potential (∆Ψm) in HeLa and MCF-7cells was
assessed by JC-1 (5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenz
imidazolocarbo cyanine iodide) staining [Figures 10a and (10b in
Supplemental Information)].
Figure 8a: Immunohistochemistry analysis of the effects of conjugates on the
organization of cellular microtubule network. HeLa cells were treated with
conjugates 7a and 9a (25 nM) for 48 h followed by staining with an anti-α-
tubulin antibody. After reaction with FITC-conjugated secondary antibody, the
cellular microtubules were observed by fluorescence microscopy. The
microtubule organization was clearly observed as tubulin network-like
structures in control cells and was found to be disrupted in cells treated with
these conjugates.
Annexin V–FITC for apoptosis.
The apoptotic effect of these compounds as studied by Hoechst
staining, reduced mitochondrial membrane potential ((∆Ψm) and
increased cytochrome c level suggested that these compounds
induced apoptosis. Hence, the apoptotic effect of compounds 7a
and 9a were further evaluated by using Annexin V–
Hoechst staining for apoptosis.
Apoptosis is one of the major pathways of programmed cell
death. Chromatin condensation, nuclear shrinking, and
fragmented nuclei are some of the salient characteristics of
apoptotic cells. Disruption of microtubule formation leads to cell-
cycle arrest in the G2/M phase, followed by apoptotic cell
[
43]
FITC/propidium iodide (AV/PI) dual staining assay to examine
the occurrence of phosphatidylserine externalization and also to
understand whether it is due to physiological apoptosis or non-
specific necrosis. In this study, HeLa and MCF-7cells were
9
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treated with compounds 7a and 9a at concentrations of 25 and
proliferation and tumorigenesis. [44,45] The PI3K/Akt pathway is
regulated by several critical upstream factors, e.g., tumor
suppressor, PTEN.[46] The tumor suppressor gene PTEN is one
of the most common targets of mutation in human cancers, with
a mutation frequency approaching that of p53. In this study,
HeLa and MCF-7cells were treated with compounds 7a and 9a
at a concentration of 25 nM for 48 h and Western blotting was
performed. The results revealed that the expression levels of p-
Akt and p-PTEN were effectively suppressed in both HeLa and
MCF-7cells [Figures 12a and (12b in Supplemental Information)].
50 nM for 48 h. After 48 h treatment, it was observed that these
conjugates significantly induced apoptosis in HeLa and MCF-7
cells as shown in Figures 11a and 11b, respectively. Results
indicate that this conjugate induced 64.14 and 69.07% apoptosis
at 0.5 and 1 µM, respectively, and nocodazole caused 62.90%
of cells to become apoptotic at 1 µM, in the untreated control,
only 9.1% of cells were undergoing apoptosis [Figures 11a and
(
11b in Supplemental Information)].
Figure 10a: Compounds 7a and 9a triggers mitochondrial injury. Drops in
membrane potential (ΔΨm) was assessed by JC-1 staining of HeLa cells
treated with test compound and samples were then subjected to flow
cytometry analysis on a FACScan (Becton Dickinson) in the FL1, FL2 channel
to detect mitochondrial potential. (A): HeLa untreated control cells; (B): treated
with CA-4 at 50 nM; (C): treated with compound 7a at 25 nM; (D): treated with
compound 7a at 50 nM; (E): treated with compound 9a at 25 nM; (F): treated
with compound 9a at 50 nM.
Figure 11a: Annexin V-FITC staining assay in HeLa cells. (A): HeLa untreated
control cells; (B): treated with CA-4 at 50 nM; (C): treated with compound 7a at
25 nM; (D): treated with compound 7a at 50 nM; (E): treated with compound
9a at 25 nM; (F): treated with compound 9a at 50 nM.
Sample
UL %
UR %
LL%
LR %
A: Control (HeLa)
B: Ca-4 (50 nM)
C: 7a (25 nM)
D: 7a (50 nM)
E: 9a (25 nM)
F: 9a (50 nM)
4.29
0.64
0.37
0.70
0.33
5.37
1.35
5.49
4.63
6.09
3.42
7.20
94.17
53.09
5.61
48.41
65.89
48.73
0.20
Cytochrome C release.
40.78
36.39
44.80
30.36
38.71
flow cytometric analysis of mitochondrial membrane potential
(
(∆Ψm) measurement, it was observed that reduced
mitochondrial membrane potential (∆Ψm) of MCF-7 as well as
HeLa cells after treatment with compound 7a and 9a. Thus,
HeLa and MCF-7cells were treated with these compounds at 25
nM concentration for 48 h to examine the effect on cytochrome
C level, and it was observed that there was an increase in
Table 5a: Distribution of apoptotic cells in Annexin-V FITC experiment of HeLa.
In table 5a, Upper left (UL) represents necrotic cells, Upper right (UR)
represents late apoptotic cells, Lower left (LL) represents live cells (viable
cells) and Lower right (LR) represents early apoptotic cells.
cytochrome
C protein levels [Figures 12a and (12b in
In vivo studies.
Supplemental Information)] suggesting that the cytosolic
cytochrome C release from mitochondria might be responsible
for apoptosis inducible capacity of these compounds.
MTT assay with normal and tumor cell lines indicate that these
two compounds, namely 9a and 7a, have the potential to
terminate cell proliferation and induce apoptosis in tumor cells.
However, the effect of synthetic molecules under in vivo
situation provides unfeigned data on the actual potential of these
molecules as anti-proliferative agents. Therefore, it would be
Effect of compounds on p-Akt and p-PTEN.
The phosphatidylinositol 3-kinase or (PI3K /AKT) pathway is a
pivotal signaling pathway, which controls cell growth, survival,
1
0
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interesting to observe variations in tumor volume among tumor-
induced mice after treatment with 7a and 9a molecules. The
graph showing variation in tumor volume (Figure 13a) indicated
that among the group of mice treated with 7a and 9a molecules,
the tumor volume did not grow to the maximum extent in 7a
treated nude mice when compared to 9a treated mice. In 7a
treated nude mice, there was sluggish growth of tumor and
better reduction in tumor volume when compared to 9a treated
Figure 13a. Tumor growth inhibition curves for 9a and 7a in nude mice during
2 months period. About 2 mg/Kg of each compound was injected to the nude
mice in the intra peritoneal region. Before starting the experiment each mice
was weighing about 22-25 gm. But after the treatment, the nude mice weight
has reduced about 2-3 gm.
3
mice. The tumor volume has increased only up to ≈ 500 mm (in
2
8 days) in 7a treated nude mice. Whereas the tumor volume
3
has increased approximately up to 600 mm in 9a treated nude
mice (in 20-24 days). On the other hand, tumor volume has
3
increased nearly three times (approximately > 1500 mm ) in the
case of control nude mice, to which neither 7a nor 9a was
injected. In about 2 months time period, the variation in tumor
volume before and after the treatment of 7a and 9a is shown in
Figure 13b.
Figure 13b.
Figure showing the volume of tumor, before and after the
treatment with 7a and 9a. Every time, three sets of nude mice (each set
consisting of 4 mice in the age group of 6–8 weeks and weighing about 22-25
gm) were considered. Figure A, C and E represent nude mice after tumor
induction. Figure D and F represent the mice after the treatment with 7a and
9a for 60 days respectively. Similarly, the figure B, represent the mice to which
neither 7a nor 9a was given (vehicle control) during the same period.
CONCLUSIONS
In summary, we have presented data that supports the potential
for two molecules 7a and 9a with methoxy group at para position
of C-ring of the trimethoxystyrylbenzene sulfonamide /sulfonates
scaffold as novel anticancer agents. All the evidences gathered
suggest that these trimethoxystyrylbenzene sulfonamide/
sulfonate analogues exert promising antiproliferative by
effectively inhibiting the tubulin polymerization and disrupt the
microtubule dynamics in cancer cells. Compounds 7a and 9a
also exhibited promising efficacy in HeLa tumor xenograft model
and have shown an excellent therapeutic window and we
believe that these molecules have significant potential for clinical
development as new anticancer agents.
Figure 12a: Western blot analysis with antibodies specific for p-Akt (Ser473),
p-PTEN (Ser380), cytochrome c and β-actin in HeLa cells.
Thus, the in vivo data corroborates with the earlier experimental
results and indicates that 7a has better tumor controlling
capability as compared to 9a. Further, in another set of
experiments, the survival of mice treated with 7a and 9a
molecules was checked and compared with the control group.
The details of survival data obtained from three different sets of
mice indicate that among the mice treated with small synthetic
molecules, 7a and 9a, the mice treated with 7a survived for
longer period (approximately 8-9 weeks months) indicating that
Experimental Section
Chemistry.
Experimental All chemicals and reagents were obtained from Aldrich
(
Sigma-Aldrich), St. Louis, MO, USA), Lancaster (Alfa Aeser, Johnson
Matthey Company, Ward Hill, MA, USA), or Spectrochem Pvt. Ltd.
Mumbai, India) and were used without further purification. Reactions
(
were performer by TLC performed on silicagel glass plate containing 60
GF-254, and visualization was achieved by UV light or iodine indicator.
1H and 13C NMR spectra were determined in DMSO-d
7a was efficient in controlling tumor cell proliferation without
developing harmful effects in tumor-induced nude mice.
Whereas among the control and 9a treated nude mice, most of
them died little earlier (in about 5-6 weeks and 7-8 weeks).
Further, among 7a and 9a treated nude mice, the mice treated
with 9a have become weak and less active when compared to
the mice that are treated with 7a. This indicates that 7a followed
by 9a is efficient in reducing the tumor volume in the nude mice.
6
by using Varian
and Avance instruments. Chemical shifts are expressed in parts per
million ( in ppm) downfield from internal TMS and coupling constants
1
are expressed in Hz. H NMR spectroscopic data are reported in the
following order: multiplicity (s, singlet; brs, broad singlet; d, doublet; dd,
doublet of doublets; t, triplet; m, multiplet), coupling constants in Hz,
number of protons. ESI mass spectra were recorded on a Micro mass
Quattro LC using ESI+ software with capillary voltage 3.98 kV and an ESI
mode positive ion trap detector. High-resolution mass spectra (HRMS)
were recorded on QSTAR XL Hybrid MS/MS mass spectrometer. Melting
points were determined with an Electro thermal melting point apparatus,
and are uncorrected The purity of tested compounds was P95% as
determined by HPLC performed on a Shimadzu LCMS-2020 apparatus
equipped with a SPD-M20A diode array detector and a Shimadzu SIL-
2
0AC auto injector using C18 column (Phenomenex luna 5 µM C18, 4.6
mm 250 mm column). Elution conditions: mobile phase (75%)-
acetonitrile; mobile phase B(25%)-water containing 0.1% formic acid + 10
A
1
1
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ChemMedChem
10.1002/cmdc.201600643
FULL PAPER
mmol NH
4
OAc. The flow rate was 1.0 mL/min and the injection volume
calculated for C25
HPLC: t
H
27
O
7
N Na S[M+Na]+ 508.14004; found: 508.13847.
was 5 µL at 25 °C and detection at 254 nm.
R
6.70 min, purity 98.0%.
(Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol (2a).
(Z)-3,4-dimethoxy-N-(2-methoxy-5-(3,4,5-
trimethoxystyryl)phenyl)benzenesulfonamide(6b). Compound 6b was
prepared according to the method described for compound 6a,
employing (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)aniline (2c, 100 mg,
Solution of 3-((tert-butyldimethylsilyl)oxy)-4-methoxy benzaldehyde (15;
.0 g, 0.0075 mol) and triphenyl(3,4,5-trimethoxybenzyl)phosphonium
2
0
.317 mmol), and 3,4-dimethoxybenzene-1-sulfonyl chloride (23b, 89.9
bromide (12, 4.71 g, 0.009 mol) in dry CH2Cl2 (100 ml) was stirred under
a nitrogen atmosphere. Sodium hydride (0.517 g, 0.0225 mol) was added
to the mixture at 0°C. Then, the temperature of the mixture was slowly
increased to RT, and the mixture was stirred for 18 h. The progress of
the reaction was monitored by TLC (EtOAc/hexane= 3:7), and water was
added after completion of reaction (until foaming stopped). The organic
layer was separated, and the aqueous layer was extracted with CHCl3.
mg, 0.38 mmol) to obtain the pure product 6b as a white colour solid.
1
(149mg, 91% yield); mp 193.0−186.0 °C; H NMR (CDCl3): δ 3.61 (s, 6H),
3.70 (s, 6H), 3.84 (s, 3H), 3.87 (s, 3H) 6.43 (s, 2H), 6.43 (s, 2H), 6.41-
6.44 (d, J = 12.10 Hz, 1H), 6.45-6.48 (d, J = 12.10 Hz, 1H), 6.73-6.75 (d,
J = 8.43 Hz, 2H), 6.90-6.93 (d, J=9.04, 2H), 7.06 (s, 1H), 7.10-7.13 (d,
J=8.43, 2H), 7.72-7.75 (d, J=8.9Hz, 2H) ppm; 13C NMR (CDCl
, 75
MHz) (ppm): 55.7, 56.2, 60.7, 105.6, 110.5, 120.0, 120.8, 125.6, 125.7,
3
2
The organic layer was washed with brine, dried with anhydrous Na SO4,
1
1
O
9
27.6, 128.9, 129.8, 130.7, 131.7, 132.8, 134.5, 137.3, 140.6, 142.6,
46.8, 152.8; MS(ESI): 516 [M+H]+ ; HRMS (ESI) calculated for C26
N S[M+H]+ 516.16866; found: 516.17081. HPLC: t 6.89 min, purity
8.0%.
and concentrated under reduced pressure to afford the crude compound,
which was purified by column chromatography (15% EtOAc/hexanes).
H
30
Yield: 1.7 g. 54%; 1H NMR (CDCl
8
R
3
): δ 3.69 (s, 6H), 3.84(s, 3H), 3.86 (s,
3H), 6.39-6.43 (d, J = 12.08 Hz, 1H), 6.49-6.53 (d, J = 12.08, 1H), 6.52
(s, 2H), 6.71-6.74 (d, J = 8.30 Hz, 1H), 6.78-6.81 (d, J=8.30Hz, 1H) 6.92
(d J=1.7 Hz, 1H) ppm.
(Z)-4-chloro-N-(2-methoxy-5-(3,4,5-
trimethoxystyryl)phenyl)benzenesulfonamide (6c).
(Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)aniline (2c).
Compound 6c was prepared according to the method described for
compound 6a, employing (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)aniline
Compound 2c was prepared according to the method described for
compound 2a a by employing 4-methoxy 3-nitrobenzaldehyde (13; 2.0 g,
(
2c, 100 mg, 0.317 mmol), and 4-chlorobenzene-1-sulfonyl chloride (23c,
8
0.3 mg, 0.38 mmol) to obtain the pure product 6c as a white colour
0
(
.013 mmol), triphenyl(3,4,5-trimethoxybenzyl) phosphonium bromide
12; 8.15 g, 0.0156 mol), and sodium hydride, which was employing (1
mmol) in Methanol was added Zn (3 mmol) HCOONH (3 mmol) to obtain
the pure product 22 as a white colour solid (1.5g, 53% yield); 1H NMR
1
solid. (145mg, 93% yield); mp 162.0−165.0 °C; H NMR (CDCl
3
): δ 3.64
(
s, 3H), 3.68 (s, 6H), 3.84 (s, 3H), 3.83 (s, 3H) 6.44 (s, 2H), 6.46 (s, 2H),
6.61 (d, J=8.3Hz 1H), 6.93 (s, 1H), 6.98(d, J = 8.3 Hz, 1H), 7.02-7.03 (m,
.06Hz 2H), 7.40 (d J=2.26Hz, NH), 7.65-7.70 (d, J=12.086Hz, 2H)ppm;
FABMAS:(M+H)=489 13C NMR (CDCl
, 75 MHz) (ppm): 55.6, 55.9, 60.8,
06.0, 110.2, 115.7, 116.0, 121.6, 125.4, 126.2, 128.8, 129.7, 129.9,
30.3, 132.5, 135.3, 137.3, 148.5, 152.0 . MS(ESI): 490 ; HRMS (ESI)
N Cl S[M+H]+490.10856; found: 490.11091.
8.01 min, purity 99.0%.
4
9
(CDCl
3
): δ 3.65 (s, 3H), 3.81(s, 6H), 3.86 (s, 3H), 5.50 (s, 1H), 6.29-6.33
3
(d, J = 11.33 Hz, 1H), 6.30-6.34 (d, J = 11.33, 1H), 6.36-6.38 (d, J = 6.04
1
1
Hz, 2H), 6.70-6.73 (d, J = 8.30Hz, 1H) 6.79-6.82 (d, J = 8.30Hz, 1H),
.91- 6.92 (d J = 2.2 Hz, 1H) ppm.
6
calculated for C24
HPLC: t
25 6
H O
R
(Z)-2-((tert-butyldimethylsilyl)oxy)-3-methoxy-6-(3,4,5-
trimethoxystyryl)aniline (22).
(Z)-3-chloro-N-(2-methoxy-5-(3,4,5-
trimethoxystyryl)phenyl)benzenesulfonamide (6d).
Compound 22 was prepared according to the method described for
compound 2c, employing (Z)- (Z)-tert-butyl(6-methoxy-2-nitro-3-(3,4,5-
trimethoxystyryl)phenoxy)dimethylsilane (1 mmol) in Methanol was
Compound 6d was prepared according to the method described for
compound 6a, employing (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)aniline
(2c, 100 mg, 0.317 mmol), and 3-chlorobenzene-1-sulfonyl chloride (23d,
added Zn (3 mmol) HCOONH
4
(3 mmol) to obtain the pure product 22 as
a white colour solid. (1.8g, 52% yield); 1H NMR (CDCl3): δ 0.17 (s, 6H),
8
0.3 mg, 0.38 mmol) to obtain the pure product 6d as a white color solid.
1
1
.00 (s, 9H), 3.63 (s, 6H), 3.75(s, 6H), 3.81 (s, 3H), 6.26-6.29 (d, J = 8.49
H), 6.39-6.45 (d, J = 11.89 Hz, 1H), 6.41-6.47 (d, J = 11.89, 1H), 6.51
1
(
(
(
142mg, 91% yield); mp 165.0−168.0 °C; H NMR (CDCl
ppm): 3.64 (s, 3H), 3.66 (s, 6H), 3.84 (s, 3 H), 6.43 (s, 2H), 6.45-6.46
d, J=12.207 Hz, 1H), 6.46-6.47 (d, J=12.207 Hz, 1H), 6.61-6.63 (d, 1H),
3
, 300 MHz) δ
(s, 2H), 6.70-6.73 (d J = 8.49 Hz, 1H) ppm.
6
.99-7.01 (d, 1H), 7.26-7.32 (m, 1H), 7.40 (s, 1H), 7.45-7.47 (d, J= 8.08
(
Z)-4-methoxy-N-(2-methoxy-5-(3,4,5-
Hz 1H), 7.52-7.53 (d, J= 7.9 Hz 1H), 7.72 (t, 1H), ppm; 13C NMR (CDCl
3
,
trimethoxystyryl)phenyl)benzenesulfonamide (6a).
7
1
1
5 MHz) (ppm): 55.6,55.8, 60.8, 105.8, 110.1, 122.0, 125.1, 125.3, 126.4,
27.1, 128.7, 129.7, 129.9, 130.3, 132.4, 132.8, 134.8, 137.2, 140.9,
48.6, 152.9; MS(ESI): 490; HRMS (ESI) calculated for C24 NCl
6.89 min, purity 96.0%.
To a solution of (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)aniline (2c) (100
mg, 0.317 mmol) in a 1:4 mixture of pyridine and anhydrous CH Cl (10
2 2
ml), 4-methoxybenzene-1-sulfonyl chloride ( 23a, 79 mg, 0.380 mmol)
was slowly added at 0°C. After 5 min stirring remove ice both and stirred
at room temperature 3 h, then the reaction mixture was evaporated to
25 6
H O
S[M+H]+490.10856; found: 490.10786. HPLC: t
R
(Z)-3,4-dichloro-N-(2-methoxy-5-(3,4,5-
trimethoxystyryl)phenyl)benzenesulfonamide (6e).
2 2
dryness in vacuum and the residue was taken up with CH Cl (10 ml).
The organic solution was washed with water, 10% aqueous HCl, water
and brine, dried over MgSO4, and concentrated in vacuum to give 143
Compound 6e was prepared according to the method described for
compound 6a, employing (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)aniline
(2c, 100 mg, 0.317 mmol), and 3,4-dichlorobenzene-1-sulfonyl chloride
mg (92%) of analytically pure compound obtained from a 3:1 mixture of
1
hexan and ethyl acetate. mp 182.0−185.0 °C; H NMR (CDCl
3
): δ 3.64 (s,
(
23e, 93.3 mg, 0.38 mmol) to obtain the pure product 6e as a white
6
H), 3.66 (s, 3H), 3.81 (s, 3H), 3.83 (s, 3H) 6.43 (s, 2H), 6.45 (s, 2H),
1
colour solid. (152mg, 91% yield); mp 173.0−176.0 °C; H NMR (CDCl
500 MHz) δ (ppm): 3.48 (s, 3H), 3.72 (s, 6H), 3.82 (s, 3H), 6.43 (s, 2H),
3
,
6
.59-6.62 (d, J = 8.49 Hz, 1H), 6.81-6.84 (d, J = 9.1 Hz, 2H), 6.92-6.96
m, 2H), 7.42 (d J=1.8, 1H), 7.60-7.63 (d, J=8.87Hz, 2H) ppm; 13C NMR
(
(
6
6
7
5
1
.45-6.49 (d, J=12.086 Hz 1H), 6.53-6.57 (d, J=12.086 Hz 1H), 6.78-
.81 (d, J= 8.49Hz 1H), 7.0 (s, 1H), 7.34-7.37 (d, J=8.68 Hz 1H), 7.55-
.59 (m, 3H), 7.96 (s, 1H) ppm; 13C NMR (CDCl
, 75 MHz) (ppm): 55.6,
CDCl
3
, 75 MHz) (ppm): 55.46, 55.72, 55.77, 60.82, 105.88, 110.11,
13.81, 120.86, 125.61, 126.01, 129.05, 129.28, 129.52, 130.28, 130.80,
32.51, 137.10, 148.23, 152.87, 162.92; MS(ESI): 485; HRMS (ESI)
1
1
3
5.8, 60.8, 105.7, 110.2, 122.1, 124.7, 126.3. 126.7, 128.6, 129.0. 129.8,
30.3, 130.6, 132.4, 133.2, 137.1, 137.5, 138.9, 148.6, 152.9; MS(ESI):
1
2
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201600643
FULL PAPER
5
23[M+Na]+; HRMS (ESI) calculated for
S[M+Na]+546.05153; found: 546.05069. HPLC: t
9.0%.
C
24
H
23
O
6
N
Cl
2
Na
(m, 2H), 7.39 (s, 2H), 7.44-7.46 (d, J= 8.69 Hz 2H) ppm; 13C NMR (CDCl
75 MHz) (ppm): 55.69, 55.89, 60.8, 106.01, 110.2, 115.7, 116.0, 121.6,
125.4, 126.2, 128.8, 129.7, 129.9, 130.3, 132.5, 135.3, 137.3, 148.5,
3
,
R
10.31 min, purity
9
1
H
53.0; MS(ESI): 470 [M+Na]+; HRMS (ESI) calculated for
NaS[M+Na]+ 493.14038; found: 493.1392. HPLC: t 8.19 min,
purity 95.0%.
C
24
O N
26 6 2
R
(Z)-4-fluoro-N-(2-methoxy-5-(3,4,5-
trimethoxystyryl)phenyl)benzenesulfonamide (6f).
(
(
Z)-N-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenyl)-3-
trifluoromethyl)benzenesulfonamide (6j).
Compound 6f was prepared according to the method described for
compound 6a, employing (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)aniline
(
2c, 100 mg, 0.317 mmol), and 4-fluorobenzene-1-sulfonyl chloride (23f,
7
3.9 mg, 0.38 mmol) to obtain the pure product 6f as a white colour
Compound 6j was prepared according to the method described for
compound 6a, employing (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)aniline
(2c, 100 mg, 0.317 mmol), and 3-(trifluoromethyl) benzene-1-sulfonyl
chloride (23j, 92.5 mg, 0.38 mmol) to obtain the pure product 6j as a
white colour solid. (147mg, 88% yield); mp 193.0−196.0 °C; 1H NMR
1
solid. (137mg, 91% yield); mp 188−191.0 °C; H NMR (CDCl
δ (ppm): 3.64 (s, 3H), 3.68 (s, 6H), 3.84 (s, 3H), 6.44 (s, 2H), 6.42-6.46
3
, 500 MHz)
(
(
7
1
1
d, J=12.086 Hz 1H), 6.46-6.50 (d, J=12.086 1H), 6.60-6.63 (s, 1H), 6.93
s, 1H), 6.9-7.00 (d, J=8.30 Hz 1H), 7.02-7.08 (t, 2H), 7.40-7.41 (d, 1H),
.65-7.70 (d, 2H) ppm; 13C NMR (CDCl
3
, 75 MHz) (ppm): 55.7, 55.9, 60.8,
3
(CDCl , 300 MHz) δ (ppm): 3.58 (s, 3H), 3.66 (s, 6H), 3.84 (s, 3 H), 6.43
06.0, 110.2, 115.7, 116.0, 121.6, 125.4, 126.2, 128.8, 129.7, 129.8,
30.3, 132.5, 135.3, 137.3, 148.5, 153.0; MS(ESI): 475 ; HRMS (ESI)
(s, 2H), 6.43-6.46 (d, J=12.05 Hz, 1H), 6.47-6.50 (d, J=12.05 Hz, 1H),
6.58-6.60 (d, J=8.69 Hz 1H), 6.94 (s, 1H), 7.0-7.03 (d, J=9.9 Hz 1H),
7.43 (s, 1H), 7.49-7.52 (t, 1H), 7.74-7.76 (d, J= 7.6 Hz 1H), 7.80-7.82 (d,
calculated for C24
HPLC: t 6.00 min, purity 98.0%.
24 7
H O N F S[M+H]+475.12213; found: 475.12404.
J= 7.7 Hz 1H), 8.0 (s, 1H) ppm; 13C NMR (CDCl
5
1
, 75 MHz) (ppm): 55.5,
5.8, 60.8, 105.8, 110.1, 122.8, 124.1, 124.2, 124.7, 126.8, 128.6, 129.2,
29.3, 129.4, 129.8, 130.4, 130.5,132.4, 137.2, 140.3, 148.8, 152.9;
HRMS (ESI) calculated for
S[M+H]+524.13492;found: 524.13366. HPLC: t 3.28 min, purity 98.0%.
R
3
(Z)-4-(tert-butyl)-N-(2-methoxy-5-(3,4,5-
MS(ESI): 524
;
C
25
H
25
O
6
N F
3
trimethoxystyryl)phenyl)benzenesulfonamide (6g).
R
Compound 6g was prepared according to the method described for
compound 6a, employing (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)aniline
(
Z)-N-(2,3-dimethoxy-5-(3,4,5-trimethoxystyryl)phenyl)-4-
methoxybenzenesulfonamide(7a).
(
(
2c, 100 mg, 0.317 mmol), and 4-(tert-butyl)benzene-1-sulfonyl chloride
23g, 88.1 mg, 0.38 mmol) to obtain the pure product 6g as a white
1
colour solid. (150mg, 92% yield); mp 182.0−185.0 °C; H NMR (CDCl
3
,
Compound 7a was prepared according to the method described for
compound 6a, employing (Z)-2,3-dimethoxy-5-(3,4,5-trimethoxystyryl)
aniline (21, 100 mg, 0.289 mmol), and 4-methoxybenzene-1-sulfonyl
chloride (23a, 71.3 mg, 0.34 mmol) to obtain the pure product 7a as a
white colour solid. (138mg, 92% yield); mp 187.0−190.0 °C; H1 NMR
(CDCl , 300 MHz) δ (ppm):3.57 (s, 3H), 3.58 (s, 3H), 3.63 (s, 6H), 3.81 (s,
3
3H), 6.42 (s, 2H), 6.45-6.49 (d, J = 12.275 Hz, 1H), 6.50-6.52 (d, J
=12.275 Hz, 1 H) 6.53 (s, 1H), 6.83-6.86(d, J =8.87 Hz 1H) 7.12(s, 2 H),
300 MHz) δ (ppm): 1.29 (s, 9H) 3.60 (s, 3H), 3.66 (s, 6H), 3.83 (s, 3H),
6.46 (s, 2H), 6.47 (s, 2H), 6.59-6.60 (d, 1H), 6.94 (s,1H), 6.96-6.98 (d,
J=8.50 Hz 1H), 7.38-7.39 (d, J=8.69 2H), 7.46-7.47 (d, J=8.50 Hz 1
H),7.59-7.60 (d, J=8.50 Hz, 2H), ppm; 13C NMR (CDCl
, 75 MHz) (ppm):
0.9, 35.0, 55.6, 55.8, 60.8, 105.7, 110.0, 121.3, 125.6, 125.8, 126.0,
26.9, 129.0, 129.4, 130.2, 132.5, 136.3, 137.0, 148.4, 152.9, 156.5;
512 HRMS (ESI) calculated for
S[M+H]+;512.21013 found: 512.21023. HPLC: t 11.38 min, purity 98.0%.
3
3
1
MS(ESI):
;
28 34 6
C H O N
7.65-7.68 (d, J =9.06 Hz, 2 H). FABMAS:(M+H)=51513C NMR (CDCl
MHz) (ppm): 55.49, 55.53, 55.79, 60.7, 60.8, 105.9, 108.6, 112.4, 114.0,
R
3
, 75
1
1
29.2, 129.3, 130.3, 130.6, 130.7, 132.3, 133.3. 137.1, 137.2, 151.5,
52.8, 163.0.; MS(ESI): 516 ; HRMS (ESI) calculated for C26
6.66 min, purity 99.0%.
(
Z)-N-(2-methoxy-5-(3,4,5-trimethoxystyryl)phenyl)-4-
29 8
H O N
nitrobenzenesulfonamide (6h).
S[M+H]+516.16710; found: 516.16724. HPLC: t
R
Compound 6h was prepared according to the method described for
compound 6a, employing (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)aniline
(
Z)-N-(2,3-dimethoxy-5-(3,4,5-trimethoxystyryl)phenyl)-3,4-
dimethoxybenzenesulfonamide (7b).
(
2c, 150 mg, 0.476 mmol), and 4-nitrobenzene-1-sulfonyl chloride (23h,
1
26.3 mg, 0.57 mmol) to obtain the pure product 6h as a yellowcolour
1
solid. (220 mg, 92% yield); mp 191.0−194.0 °C; H NMR (CDCl
3
, 500
Compound 7b was prepared according to the method described for
compound 6a, employing (Z)-2,3-dimethoxy-5-(3,4,5-trimethoxystyryl)
aniline (21, 100 mg, 0.289 mmol), and 3,4-dimethoxybenzene-1-sulfonyl
chloride (23b, 80.2 mg, 0.34 mmol) to obtain the pure product 7b as a
white colour solid. (143mg, 90% yield); mp 205.0−208.0 °C; 1H NMR
MHz) δ (ppm): 3.65 (s, 3 H), 3.70 (s, 6 H), 3.84 (s, 3 H), 6.42-6.45 (d,
J=12.05 Hz, 1H), 6.44 (s, 1H), 6.24 (s, 2H), 6.48-6.51 (d, J=12.05 Hz,
1H), 6.63-6.65 (s, J=8.5, 2H), 6.98 (s, 1H), 7.00-7.02 (d, J=8.54 1H), 7.38
(
s, 1H), 7.80-7.82 (d, J = 8.80 Hz, 2H), 8.21-8.23 (d, J= 9.00 Hz 2H) ppm;
1
3C NMR (CDCl
3
, 75 MHz) (ppm): 55.6, 55.9, 60.8, 105.8, 112.4, 123.9,
24.0, 127.8, 129.1, 129.5, 130.4, 130.5, 132.1. 137.3, 137.7, 142.2,
50.3, 150.7, 153.0; MS(ESI): 501[M+Na]+ ; HRMS (ESI) calculated for
S[M+H]+501.13261; found: 501.13269. HPLC: t 3.17 min,
purity 100.0%.
3
(CDCl , 500 MHz) δ (ppm): 3.56 (s, 3H), 3.58 (s, 3H), 3.63 (s, 6H), 3.81
1
1
C
(s, 3H), 3.82 (s, 3H), 3.89, (s, 3H), 6.42 (s, 2H), 6.45-6.48 (d, J = 12.05
Hz, 1H), ), 6.49-6.52 (d, J = 12.05 Hz, 1H), 6.54 (s, 1H) 6.80-6.81 (d, J
= 8.54 1H), 7.10 (s, 1H), 7.15 (s, 1H), 7.24(s, 1H), 7.37-7.34 (dd, J = 8.54,
24
H O N
25 8 2
R
J = 8.54 1H), 6.73 (d, 1H), 6.82(d,1H), ppm; 13C NMR (CDCl
3
, 75 MHz)
(ppm): 55.5, 55.8, 56.06, 60.7, 60.8, 106.0, 108.6, 109.6, 110.3, 112.6,
1
1
C
21.1, 129.2, 130.3, 130.6, 130.7, 132.3, 133.3, 137.3, 137.4, 148.8,
51.5, 152.7, 152.9; MS(ESI): 546; HRMS (ESI) calculated for
NS [M+H]+546.17923; found: 546.18117. HPLC: t 6.02 min,
purity 98.0%.
(
Z)-4-amino-N-(2-methoxy-5-(3,4,5-
trimethoxystyryl)phenyl)benzenesulfonamide (6i).
27 32
H O
9
R
Compound 6i was prepared according to the method described for
compound 2c, employing (Z)-N-(2-methoxy-5-(3,4,5-trimethoxystyryl)
phenyl)-4-nitrobenzenesulfonamide (6i, 80 mg, 0.16 mmol), in Methanol
(Z)-4-chloro-N-(2,3-dimethoxy-5-(3,4,5-
trimethoxystyryl)phenyl)benzenesulfonamide (7c).
4
was added Zn (20.6 mg, 0.32 mmol) HCOONH (20.6 mg, 0.32 mmol) to
obtain the pure product 6i as a white colour solid. (70mg, 93% yield); mp
1
1
(
1
99.0−202.0 °C; H NMR (CDCl
3
, 300 MHz) δ (ppm): 3.63 (s, 6 H), 3.69
Compound 7c was prepared according to the method described for
compound 6a, employing (Z)-2,3-dimethoxy-5-(3,4,5-trimethoxystyryl)
aniline (21, 100 mg, 0.289 mmol), and 4-chlorobenzene-1-sulfonyl
s, 3 H), 3.83 (s, 3 H), 4.09 (s, 2 H), 6.41 (s, 2 H), 6.44 (s, 1H), 6.46 (s,
H), 6.48-6.50 (d, J=8.85 Hz, 1H), 6.61-6.63 (d, J=8.54, 2H), 6.91-6.94
1
3
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201600643
FULL PAPER
chloride (23c, 71.3 mg, 0.34 mmol) to obtain the pure product 7c as a
white colour solid. (140mg, 93% yield); mp 182.0−185.0 °C; 1H NMR
Compound 7g was prepared according to the method described for
compound
6a,
employing
(Z)-2,3-dimethoxy-5-(3,4,5-
(
CDCl
3
, 500 MHz) δ (ppm): 3.57 (s, 6H), 3.59 (s, 3H), 3.67 (s, 6H), 3.82
trimethoxystyryl)aniline (21, 100 mg, 0.289 mmol), and 4-nitrobenzene-1-
sulfonyl chloride (23h, 75.1 mg, 0.34 mmol) to obtain the pure product
7g as a white colour solid. (138mg, 90% yield); mp 198.0−201.0 °C; H
(s, 3H), 6.43 (s, 2H) 6.44-6.46 (d, J = 12.05 Hz, 1H), 6.51-6.54 (d, J =
1
1
8
2.05 Hz, 1H), 6.55 (s, 1H), 7.08 (s, 1H), 7.13 (s, 1H), 7.37-7.38 (s, J =
.54 Hz, 2H), 7.65-7.67 (s, J = 8.54 Hz, 2H)ppm; 13C NMR (CDCl
, 75
3
3
NMR (CDCl , 500 MHz) δ (ppm): 3.61 (s, 3H), 3.63 (s, 3H), 3.70 (s, 6H),
MHz) (ppm): 55.5, 55.8, 60.8, 106.0, 109.2, 112.7, 128.5, 129.2, 129.9,
3.82 (s, 3H), 6.43(s, 1H), 6.43-6.45 (d, J = 12.05 Hz, 1H), 6.53-6.55 (d, J
= 12.05 Hz, 1H), 6.58 (s, 1H), 7.04 (s, 1H), 7.22 (s, 1H), 7.86-7.87 (s, J =
1
30.5, 132.3, 133.3, 137.3, 137.4, 137.6, 139.4, 151.5, 153.0. MS(ESI):
19[M+Na]+; HRMS (ESI) calculated for NClNaS
3.20 min, purity 100.0%.
5
C H O
25 26 7
9.00 Hz 2H), 8.23-8.26 (s, J = 8.54 Hz, 2H) ppm; 13C NMR (CDCl
MHz) (ppm): 55.6, 55.9, 60.8, 60.9, 105.9, 109.6, 112.5, 124.2, 128.3,
3
, 75
[M+Na]+542.10107; found: 542.09981. HPLC: t
R
1
1
29.0, 129.3, 130.7, 132.3, 133.3, 137.3, 137.5, 144.7, 150.1, 151.6,
53.0. . MS(ESI): 548 [M+H]+; HRMS (ESI) calculated for C25 H26 O9
(
Z)-3-chloro-N-(2,3-dimethoxy-5-(3,4,5-
N2 S[M+H]+ 548.16929; found: 548.16930. HPLC: t
9.0%.
R
6.61 min, purity
trimethoxystyryl)phenyl)benzenesulfonamide (7d).
9
Compound 7d was prepared according to the method described for
compound 6a, employing (Z)-2,3-dimethoxy-5-(3,4,5-trimethoxystyryl)
aniline (21, 100 mg, 0.289 mmol), and 3-chlorobenzene-1-sulfonyl
chloride (23d, 71.3 mg, 0.34 mmol) to obtain the pure product 7d as a
white colour solid. (137mg, 91% yield); mp 193.0−196.0 °C; 1H NMR
(
Z)-4-amino-N-(2,3-dimethoxy-5-(3,4,5-
trimethoxystyryl)phenyl)benzenesulfonamide (7h).
Compound 7h was prepared according to the method described for
compound 2c, employing (Z)-N-(2,3-dimethoxy-5-(3,4,5-trimethoxystyryl)
phenyl)-4-nitrobenzenesulfonamide (7h, 80 mg, 0.094 mmol), in
Methanol was added Zn (13 mg, 0.18 mmol) HCOONH (13 mg, 0.32
4
mmol) to obtain the pure product 7h as a white colour solid. (70mg, 92%
(
(
1
CDCl
s, 3H), 6.43(s, 2H) , 6.45-6.48 (d, J = 12.05 Hz, 1H), 6.51-6.54 (d, J =
2.05 Hz, 1H), 6.56 (s, 1H), 7.08 (s, 1H), 7.32-7.35 (t, 1H), 7.46-7.48 (s,
J = 7.9 Hz, 1H) 7.58-7.60 (s, J = 7.9 Hz, 1H), 7.78 (t, 1H), 13C NMR
CDCl , 75 MHz) (ppm): 55.5, 55.8, 60.8, 105.9, 109.2, 112.8, 125.2,
27.1, 129.1, 129.8, 130.2, 130.5, 132.3, 133.0, 133.3, 135.1, 137.3,
37.5, 140.8, 151.5, 152.9; MS(ESI): 520 [M+H]+; HRMS (ESI)
3
, 500 MHz) δ (ppm): 3.57 (s, 3H), 3.60 (s, 3H), 3.66 (s, 6H), 3.82
1
(
1
1
3
yield); mp 210.0−213.0 °C; H NMR (CDCl
3
, 300 MHz) δ (ppm): 3.57 (s,
3H), 3.59 (s, 3H), 3.67 (s, 6H), 3.83 (s, 3H), 6.43(s, 1H), 6.45-6.47 (d, J =
12.05 Hz, 1H), 6.52-6.54 (d, J = 12.05 Hz, 1H), 6.56 (s, 1H), 7.08 (s, 1H),
7.15 (s, 1H), 7.37-7.38 (s, J = 8.54 Hz 2H), 7.66-7.67 (s, J = 8.54 Hz, 2H)
ppm; 13C NMR (CDCl
1
1
calculated for C25 H26 O7 N Cl S[M+H]+ 520.11762; found: 520.11782.
HPLC: t 7.95 min, purity 97.0%.
R
3
, 75 MHz) (ppm): 55.5, 55.7, 60.7, 60.8, 105.9,
08.2, 112.1, 113.7, 126.8, 126.9, 129.2, 129.4, 130.1, 131.0, 132.3,
33.4, 137.0, 150.9, 151.5, 152.8; MS(ESI): 500[M+Na]+; HRMS (ESI)
(Z)-N-(2,3-dimethoxy-5-(3,4,5-trimethoxystyryl)phenyl)-4-
calculated for C25 H28 O7 N2 Na S[M+Na]+523.1594; found: 523.14958.
HPLC: t 5.09 min, purity 99.0%.
fluorobenzenesulfonamide (7e).
R
Compound 7e was prepared according to the method described for
compound 6a, employing (Z)-2,3-dimethoxy-5- (3,4,5-trimethoxystyryl)
aniline (21, 100 mg, 0.289 mmol), and 4-fluorobenzene-1-sulfonyl
chloride (23a, 65.2 mg, 0.34 mmol) to obtain the pure product 7e as a
white colour solid. (133mg, 91% yield); mp 173.0−177.0 °C; 1H NMR
(
(
Z)-N-(2,3-dimethoxy-5-(3,4,5-trimethoxystyryl)phenyl)-3-
trifluoromethyl)benzenesulfonamide (7i).
Compound 7i was prepared according to the method described for
compound 6a, employing (Z)-2,3-dimethoxy-5-(3,4,5-trimethoxystyryl)
aniline (21, 100 mg, 0.289 mmol), and 3-(trifluoromethyl)benzene-1-
sulfonyl chloride (23j, 84.8 mg, 0.34 mmol) to obtain the pure product 7i
(CDCl
3
, 300 MHz) δ (ppm): 3.58 (s, 3H), 3.59 (s, 3H), 3.67 (s, 6H), 3.82
(s, 3H), 6.43 (s, 1H), 6.44-6.47 (d, J = 12.08 Hz, 1H), 6.51-6.55 (d, J =
1
2.08 Hz, 1H), 6.56 (s, 1H), 7.05-7.10 (m, 4H), 7.71-7.76 (m, 2H) ppm;
1
3C NMR (CDCl
1
3
, 75 MHz) (ppm): 55.5, 55.8, 60.8, 106.0, 109.1, 112.7,
as a white colour solid. (146mg, 91% yield); mp 203.0−206.0 °C; H
1
1
16.0, 116.3, 129.2, 129.8, 129.9, 130.1, 130.4, 132.3, 133.3, 135.2,
37.4, 151.5, 152.9; MS(ESI): 504 [M+H]+; HRMS (ESI) calculated for
3
NMR (CDCl , 300 MHz) δ (ppm): 3.56 (s, 6H), 3.66 (s, 6H), 3.82 (s, 3 H),
6.43 (s, 2H), 6.45-6.47 (d, J=12.05 Hz, 1H), 6.52-6.54 (d, J=12.05 Hz,
1H), 6.57 (s, 1H), 7.10 (s, 1H), 7.19 (s, 1H), 7.53-7.56 (t, 1H), 7.76-7.78
(d, J= 7.9 Hz 1H), 7.87-7.89 (d, J= 7.9 Hz 1H), 8.06 (s, 1H), ppm; 13C
C25 H26 O7 F N S[M+H]+ 504.14708; found: 504.14720. HPLC: t
min, purity 99.0%.
R
6.84
NMR (CDCl
3
, 75 MHz) (ppm): 55.5, 55.7, 56.0, 60.7, 105.9, 108.5, 109.5,
10.3, 112.6, 121.1. 129.2, 130.3, 130.5, 130.6, 132.9, 133.2, 137.2,
37.3, 148.8, 151.8, 152.7, 152.8; MS(ESI): 571 [M+H]+; HRMS (ESI)
1
1
(
Z)-4-(tert-butyl)-N-(2,3-dimethoxy-5-(3,4,5-
trimethoxystyryl)phenyl)benzenesulfonamide (7f).
calculated for C26 H30 O7 N2 F3 S[M+H]+ 571.17203; found: 571.17135.
HPLC: t 5.09 min, purity 99.0%.
R
Compound 7f was prepared according to the method described for
compound 6a, employing (Z)-2,3-dimethoxy-5-(3,4,5-trimethoxystyryl)
aniline (21, 100 mg, 0.289 mmol), and 4-(tert-butyl)benzene-1-sulfonyl
chloride (23g, 78.8 mg, 0.34 mmol) to obtain the pure product 7f as a
(Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenyl
4-
methoxybenzenesulfonate (8a).
1
brown colour solid. (143mg, 91% yield); mp 194.0−196.0 °C; H NMR
(
(
(
CDCl
3
, 300 MHz) δ (ppm): 1.28 (s, 9H) 3.50 (s, 3H), 3.56 (s, 3H), 3.65
To a solution of (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol (2a) (100
mg, 0.316 mmol) in a tryethylamine (TEA) (0.632 mmol) and anhydrous
CH Cl (10 ml), 4-methoxybenzene-1-sulfonyl chloride ( 23a, 79 mg,
2 2
0.380 mmol) was slowly added at 0°C. After 5 min stirring remove ice
both and stirred at room temperature 3 h, then the reaction mixture was
evaporated to dryness in vacuum and the residue was taken up with
s, 6H), 3.81 (s, 3H), 6.45 (s,1H), 6.47-6.49 (d, J=11.476 1H), 6.51-6.53
d, J=12.476 1H), 6.54 (s, 1H), 7.08 (s,1H), 7.17(s, 1H), 7.41-7.42 (d,
J=8.80 2H),7.65-7.67 (d, J=8.80 Hz, 2H), ppm;13C NMR (CDCl
3
, 75
MHz) (ppm): 35.0, 30.9, 55.5, 55.8, 60.6, 105.7, 108.7, 112.8, 125.9,
1
1
26.9, 129.3, 130.2, 132.2, 132.4, 136.1, 137.1, 137.3, 151.4, 152.9,
56.8; MS(ESI): 542 [M+H]+; HRMS (ESI) calculated for C29 H35 O7 N
2 2
CH Cl (10 ml). The organic solution was washed with water and brine,
S[M+H]+ 542.21953; found: 542.21970. HPLC:
00.0%.
t
R
3.17 min, purity
dried over MgSO4, and concentrated in vacuum to give 139 mg (89%) of
1
analytically pure compound (8a) obtained from a 3:1 mixture of hexan
1
and ethyl acetate. mp 193.0−196.0 °C; H NMR (CDCl
3
) δ (ppm): 3.62 (s,
3
1
2
7
H), 3.70 (s, 3H), 3.84 (s, 6H), 3.87 (s, 3H) 6.42 (s, 2H), 6.41-6.44 (d, J =
(
Z)-N-(2,3-dimethoxy-5-(3,4,5-trimethoxystyryl)phenyl)-4-
2.2 Hz, 1H), 6.46-6.49 (d, J = 12.2 Hz, 1H), 6.73-6.76 (d, J = 8.9 Hz,
H), 6.90-6.93 (d, J = 8.4 Hz, 2H), 7.05 (d, NH), 7.12 (d, J = 1.71 Hz, 1H),
nitrobenzenesulfonamide (7g).
.72-7.75 (d, J=8.8Hz, 2H) ppm; 13C NMR (CDCl
3
, 75 MHz) (ppm): 55.6,
1
4
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201600643
FULL PAPER
5
1
4
5
5.7, 55.8, 60.8, 105.9, 112.2, 113.8, 124.2, 127.8, 128.1, 128.4, 130.0,
30.1, 130.5, 132.2, 137.2, 138.1, 150.8, 152.9, 163.3; MS(ESI):
300 MHz) δ (ppm): 3.60 (s, 3H), 3.71 (s, 6H), 3.85 (s, 3 H), 6.43 (s, 2H),
6.42-6.45 (d, J=11.98 Hz, 1H), 6.49-6.52 (d, J=11.92 Hz, 1H), 6.74-6.76
(d, J=8.55 1H), 7.10-7.11 (s, 1H), 7.14-7.16 (dd, J=8.55 1H), 7.54-7.56
(d, J=8.43 1H), 7.60-7.63 (dd, J= 8.43 Hz 1H), 7.98 (s, 1H), ppm; 13C
86[M+H]+; HRMS (ESI) calculated for
25 26 8
C H O NaS [M+Na]+
09.12406; found: 509.12278. HPLC: t 8.15 min, purity 98.0%.
R
NMR (CDCl
3
, 75 MHz) (ppm): 55.5, 55.8, 60.8, 105.8, 112.2, 124.4,
27.3, 127.8, 129.0, 130.2, 130.4, 130.6, 132.1, 133.4, 136.0, 137.3,
37.6, 138.8, 150.3, 153.0. MS(ESI): 525[M+H]+; HRMS (ESI) calculated
1
1
(
Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenyl
3,4-
dimethoxybenzenesulfonate (8b).
23 7
for C24H O Cl
2
S [M+H]+525.05361; found: 525.05607. HPLC: t
R
11.24
min, purity 96.0%.
Compound 8b was prepared according to the method described for
compound 8a, employing (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol
(
Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenyl
4-
(
2a, 100 mg, 0.316 mmol), and 3,4-dimethoxy benzene-1-sulfonyl
fluorobenzenesulfonate (8f).
chloride (23b, 89.9 mg, 0.38 mmol) to obtain the pure product 8b as a
white colour solid. (144mg, 88% yield); mp 173.0−176.0 °C; 1H NMR
(
CDCl
3
) δ (ppm): 3.64 (s, 3H), 3.70 (s, 6H), 3.84 (s, 3H), 3.88 (s, 3H),
Compound 8f was prepared according to the method described for
compound 8a, employing (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol
(2a, 100 mg, 0.316 mmol), and 4-fluorobenzene-1-sulfonyl chloride (23f,
73.9 mg, 0.38 mmol) to obtain the pure product 8f as a white colour
3
=
1
.94 (s, 3H), 6.40-6.43 (d, J = 12.10 Hz, 1H), 6.43 (s, 2H), 6.45-6.48 (d, J
12.10 Hz, 1H), 6.75-6.77 (d, J=8.43 Hz 1H), 6.85-6.87 (d, J = 8.55 Hz,
H), 7.05 (d, J = 2.07 Hz, 1H), 7.11-7.14 (d J=8.43, 1H), 7.33 (d,
J=2.20Hz, 1H), 7.35-7.39 (d J=8.43, 1H)ppm; 13C NMR (CDCl
solid. (135mg, 90% yield); mp 194.0−196.0 °C; 1H NMR (CDCl
3
, 75 MHz)
3
, 500
(
1
1
ppm): 55.8, 55.86, 56.17, 60.8, 105.7, 110.0, 110.4, 112.2, 122.6, 124.1,
27.7, 128.0, 128.5, 130.0, 130.1, 132.1, 137.1, 138.1, 148.8, 150.9,
52.9, 153.5; MS(ESI): 516[M+H]+; HRMS (ESI) calculated for
MHz) δ (ppm): 3.60 (s, 3H), 3.71 (s, 6H), 3.85 (s, 3H), 6.43 (s, 2H), 6.42-
6.44 (d, J=12.054 Hz 1H), 6.46-6.50 (d, J=12.086 1H), 6.74-6.76 (s,
J=8.54 Hz 1H), 7.04 (s, 1H), 7.12-7.17 (m, 3H), 7.81-7.84(m, 2H) ppm;
C
26
H
28
O
7
NaS [M+Na]+539.13462; found: 539.13289. HPLC: t
R
7.16 min,
13C NMR (CDCl
124.0, 127.8, 129.1, 129.5, 130.4, 130.5, 132.1, 137.2, 137.6, 142.1,
50.4, 150.6, 153.0. MS(ESI): 492[M+H]+; HRMS (ESI) calculated for
3
, 75 MHz) (ppm): 55.6, 55.8, 60.8, 105.7, 112.3, 123.9,
purity 98.0%.
1
24 27
C H O
7
NFS [M+H]+492.1486; found: 492.14917. HPLC: t
R
8.18 min,
(Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenyl
4-chlorobenzene
purity 99.0%.
sulfonate (8c).
(
Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenyl
4-(tert-
Compound 8c was prepared according to the method described for
compound 8a, employing (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol
butyl)benzenesulfonate (8g).
(
2a, 100 mg, 0.316 mmol), and 4-chlorobenzene-1-sulfonyl chloride (23c,
8
0.3 mg, 0.38 mmol) to obtain the pure product 8c as a white colour
Compound 8g was prepared according to the method described for
compound 8a, employing (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol
(2a, 100 mg, 0.316 mmol), and 4-(tert-butyl)benzene-1-sulfonyl chloride
1
solid. (140mg, 90% yield); mp 188.0−191.0 °C; H NMR (CDCl3, DMSO )
δ (ppm): 3.64 (s, 3H), 3.68 (s, 6H), 3.84 (s, 3H), 3.83 (s, 3H) 6.44 (s, 2H),
6
.46 (s, 2H), 6.61 (d, J=8.3Hz 1H), 6.93 (s, 1H), 6.98(d, J = 8.3 Hz, 1H),
.02-7.03 (m, 9.06Hz 2H), 7.40 (d J=2.26Hz, NH), 7.65-7.70 (d,
(23g, 88.1 mg, 0.38 mmol) to obtain the pure product 8g as a white
1
7
colour solid. (153mg, 94% yield); mp 192.0−194.0 °C; H NMR (CDCl
3
,
J=12.086Hz, 2H)ppm; 13C NMR (CDCl
, 75 MHz) (ppm): 55.6, 55.9, 60.9,
05.8, 112.2, 124.3, 129.0, 128.0, 128.8, 129.7, 130.3, 132.2, 134.9,
37.3, 137.9, 140.5, 150.6, 153.0. MS(ESI): 491[M+H]+; HRMS (ESI)
300 MHz) δ (ppm): 1.33 (s, 9H) 3.53 (s, 3H), 3.70 (s, 6H), 3.84 (s, 3H),
6.44 (s, 2H), 6.41-6.44 (d, J=12.08 Hz 2H), 6.47-6.50 (d, J=12.08 Hz 1H),
6.71-6.73 (d, J=8.30 Hz 1H), 7.09-7.14 (m, 2H), 7.46-7.49 (d, J=8.68 1
3
1
1
calculated for C24 H24 O7 Cl S[M+H]+491.09258; found: 491.09246.
HPLC: t 10.77 min, purity 99.0%.
H),7.72-7.75 (d, J=8.68 Hz, 2H), ppm; 13C NMR (CDCl
30.97, 35.07, 55.66, 55.81, 60.87,105.82, 110.09, 121.39, 125.68,
3
, 75 MHz) (ppm):
R
1
1
25.83, 125.91, 126.96, 129.06, 129.53, 130.28, 132.57, 136.36,
37.097, 48.41, 152.94 156.56; MS(ESI): 530[M+H]+; HRMS (ESI)
(
Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenyl
3-
calculated for C28 H36 O7 N S[M+H]+530.22070; found: 530.22153.
HPLC: t 3.21 min, purity 100.0%.
chlorobenzenesulfonate (8d).
R
Compound 8d was prepared according to the method described for
compound 8a, employing (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol
(
Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenyl
4-
nitrobenzenesulfonate (8h).
(
8
(
(
(
2a, 100 mg, 0.316 mmol), and 3-chlorobenzene-1-sulfonyl chloride (23d,
0.3 mg, 0.38 mmol) to obtain the pure product 8d as a white color solid.
143mg, 93% yield). mp 183.0−186.0 °C; 1H NMR (CDCl
, 300 MHz) δ
3
Compound 8h was prepared according to the method described for
compound 8a, employing (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol
(2a, 150 mg, 0.476 mmol), and 4-nitrobenzene-1-sulfonyl chloride (23h,
ppm): 3.57 (s, 3H), 3.70 (s, 6H), 3.84 (s, 3 H), 6.44 (s, 2H), 6.42-6.44
d, J=12.05 Hz, 1H), 6.48-6.51 (d, J=12.05 Hz, 1H), 6.73-6.75 (d, J=8.54
1
H), 7.13-7.11 (s, 1H), 7.13-7.14 (d, J=8.39 1H), 7.41-7.44 (t, 1H), 7.58-
126.3 mg, 0.57 mmol) to obtain the pure product 8h as a yellow colour
1
7
.60 (d, J= 9.00 Hz 1H), 7.68-7.70(d, J= 7.78 Hz 1H), 7.87 (s, 1H), ppm;
solid. (210 mg, 88% yield); mp 198.0−201.0 °C; H NMR (CDCl
3
, 500
1
3C NMR (CDCl
3
, 75 MHz) (ppm): 55.87, 60.8, 105.7, 112.0, 122.6,
22.8, 124.5, 125.6, 127.8, 127.9, 129.0, 129.5, 130.3, 137.2, 137.6,
38.9, 143.9, 153.0 MS(ESI): 491[M+H]+; HRMS (ESI) calculated for
ClS [M+H]+491.09258; found: 491.09251. HPLC: t 10.91 min,
purity 99.0%.
MHz) δ (ppm): 3.61 (s, 3H), 3.71 (s, 6H), 3.85 (s, 3H), 6.41 (s, 2 H),
6.42-6.45 (d, J=12.05 Hz, 1H), 6.49-6.52 (s, J=12.05, 1H), 6.78-6.70 (d,
J=8.54 1H), 7.01 (d, J=2.13 1H), 7.15-7.17 (d, J=8.54 1H), 7.99-8.01 (d,
1
1
C
H O
24 24 7
R
J = 9.00 Hz, 2H), 8.31-8.33 (d, J= 9.00 Hz 2H) ppm; 13C NMR (CDCl
MHz) (ppm): 55.70, 55.91, 60.88, 105.84, 123.92, 124.04, 127.87,
3
, 75
1
1
29.15, 129.59, 130.00, 130.47, 130.56, 132.12, 137.33, 137.74, 142.23,
50.44, 150.70, 153.09; MS(ESI): 502[M+H]+; HRMS (ESI) calculated for
(
Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenyl 3,4-dichlorobenzene
C24 H24 O9
calculated 524.09857;found: 524.09808. HPLC: t
N S[M+H]+502.11663; found: 502.11672; [M+Na]+;
sulfonate (8e).
R
3.41 min, purity 97.0%.
Compound 8e was prepared according to the method described for
compound 8a, employing (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol
(
Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenyl
3-(trifluoromethyl)
benzenesulfonate (8i).
(
(
2a, 100 mg, 0.316 mmol), and 3,4-dichloro benzene-1-sulfonyl chloride
23e, 93.3 mg, 0.38 mmol) to obtain the pure product 8e as a white
1
colour solid. (148mg, 89% yield). mp 193.0−196.0 °C; H NMR (CDCl
3
,
1
5
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201600643
FULL PAPER
Compound 8i was prepared according to the method described for
compound 8a, employing (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol
(Z)-4-chloro-N-(2-hydroxy-3-methoxy-6-(3,4,5-
trimethoxystyryl)phenyl)benzenesulfonamide (9c).
(
2a, 100 mg, 0.316 mmol), and 3-(trifluoromethyl) benzene-1-sulfonyl
chloride (23i, 92.5 mg, 0.38 mmol) to obtain the pure product 8i as a
white colour solid. (152mg, 91% yield). mp 200.0−203.0 °C; 1H NMR
The compound 9c was prepared according to the general procedure by
employing
(Z)-2-((tert-butyldimethylsilyl)oxy)-3-methoxy-6-(3,4,5
(
(
6
(
CDCl
s, 2H), 6.42-6.45 (d, J=12.05 Hz, 1H), 6.49-6.52 (d, J=12.05 Hz, 1H),
.71-6.72 (d, J=9.15 1H), 7.13-7.15 (m, 2H), 7.63-7.66 (t, 1H), 7.88-7.89
d, J= 7.93 Hz 1H), 8.00-8.01 (d, J= 7.93 Hz 1H), 8.17 (s, 1H), ppm; 13C
NMR (CDCl , 75 MHz) (ppm): 55.4, 55.8, 60.8, 105.8, 112.1, 124.6,
25.5, 125.6, 127.9, 129.0, 129.5, 130.3, 131.3, 131.5, 131.6, 132.1,
37.3, 137.6, 137.7, 150.2, 153.0. MS(ESI): 542[M+H]+; HRMS (ESI)
N F S[M+H]+542.14548; found: 542.14633.
3.27 min, purity 94.0%.
3
, 300 MHz) δ (ppm): 3.51 (s, 3H), 3.71 (s, 6H), 3.85 (s, 3 H), 6.44
trimethoxystyryl)aniline (22, 100 mg, 0.227 mmol) and 4-
methoxybenzene-1-sulfinyl chloride (23c, 57.4 mg, 0.27 mmol) to afford
24c, which on deportation with TBAF (1.0M in THF, 2.5mmol) to obtain
the pure product 9c as a white colour solid. (105mg, 92% yield). mp
3
1
1
83.0−186.0 °C; H NMR (CDCl
3
, 300 MHz) δ (ppm): 3.63 (s, 6H), 3.82(s,
1
1
3H), 3.85 (s, 3H), 6.02-6.06(d, J= 12.08 1H), 6.19 (s, OH), 6.27-6.30 (d,
J= 12.086 1H), 6.31 (s, 2H), 6.72 (s, 2H), 7.35-7.38 (d, J= 8.30 2H),
calculated for C25
HPLC: t
H
27
O
7
3
.65-7.68 (s, J= 9.06 2H), ppm; 13C NMR (CDCl
5.8, 60.8, 106.0, 109.2, 112.7, 128.5, 129.2, 129.9, 130.5, 132.3, 133.3,
37.3, 137.4, 137.6, 139.4, 151.5, 153.0; MS(ESI): 506[M+H]+; HRMS
7
5
1
3
, 75 MHz) (ppm): 55.5,
R
General procedure for the synthesis of (Z)-N-(2-hydroxy-3-methoxy-
-(3,4,5-trimethoxystyryl)phenyl) benzenesulfonamides (9a-k).
(ESI) calculated for C24 H25 O7 N Cl S[M+H]+506.10348; found:
506.10510. HPLC: t 3.02 min, purity 99.0%.
6
R
(a) Compounds (24a-k) were prepared according to the method
(Z)-3-chloro-N-(2-hydroxy-3-methoxy-6-(3,4,5-
described for compound 6a, employing (Z)-2-((tert-
trimethoxystyryl)phenyl) benzenesulfonamide (9d).
butyldimethylsilyl)oxy)-3-methoxy-6-(3,4,5-trimethoxystyryl) aniline (22, 1
mmol), and substituted benzene-1-sulfonyl chlorides (23-a-k, 1.2 mmol )
to obtain the pure products (24a-k) which were used without purification
in the next step. (b) To a solution of compounds (24a-k, 1 mmol) in dry
tetrahydrofuran, TBAF (1.0M in THF, 2.5mmol) was added at 0 °C and
stirred at room temperature for 1 h. After completion of reaction, the
reaction mixture was diluted with ethyl acetate, washed with water and
The compound 9d was prepared according to the general procedure by
employing
(Z)-2-((tert-butyldimethylsilyl)oxy)-3-methoxy-6-(3,4,5-
trimethoxystyryl)aniline (22, 100 mg, 0.227 mmol) and 4-
methoxybenzene-1-sulfinyl chloride (23d, 57.4 mg, 0.27 mmol) to afford
24d, which on deportation with TBAF (1.0M in THF, 2.5mmol) to obtain
the pure product 9d as a white colour solid. (102mg, 90% yield). mp
brine solution, dried over anhydrous Na
2 4
SO . The solution was filtered
1
1
3
1
7
7
93.0−196.0 °C; H NMR (CDCl
3
) δ (ppm): 3.64 (s, 6H), 3.82 (s, 3H),
and the solvent was removed under reduced pressure followed by the
purification of reside by column chromatography using ethyl acetate and
hexane as eluents to afford respective desired products (9a-k).
.85(s, 3H), 6.07-6.10 (d, J= 12.086 1H), 6.13 (s, 1H), 6.29-6.32 (d, J=
2.086 1H), 6.33 (s, 2H) 6.73 (s, 2H), 7.33-7.36 (t, 1H), 7.50-7.52 (d, J=
.62 1H), 7.61-7.63 (d, J= 7.62 1H), 7.71 (s, 1H) ppm; 13C NMR (CDCl
3
,
5 MHz) (ppm): 55.8, 56.1, 60.8, 105.8, 110.0, 110.5, 112.2, 122.6,
(
Z)-N-(2-hydroxy-3-methoxy-6-(3,4,5-trimethoxystyryl)
phenyl)-4-
124.2, 127.7, 128.1, 128.5, 130.0, 130.1, 132.1, 137.2, 138.1, 148.8,
150.9, 152.9, 153.5. . MS(ESI): 506[M+H]+; HRMS (ESI) calculated for
methoxybenzenesulfonamide (9a).
24 25 7 R
C H O NCl S[M+H]+506.10348; found: 506.10533. HPLC: t 5.60 min,
purity 98.0%.
The compound 9a was prepared according to the general procedure by
employing (Z)-2-((tert-butyldimethylsilyl)oxy)-3-methoxy-6-(3,4,5-
trimethoxystyryl)aniline (22, 100 mg, 0.2275 mmol) and 4-
methoxybenzene-1-sulfinyl chloride (23a, 55.2 mg, 0.27 mmol) to afford
(Z)-3,4-dichloro-N-(2-hydroxy-3-methoxy-6-(3,4,5-
trimethoxystyryl)phenyl)benzene sulfonamide (9e).
24a, which on deportation with TBAF (1.0M in THF, 2.5mmol) to obtain
the pure product 9a as a white colour solid. (103mg, 91% yield). mp
The compound 9e was prepared according to the general procedure by
employing (Z)-2-((tert-butyldimethylsilyl)oxy)-3-methoxy-6-(3,4,5-
trimethoxystyryl)aniline (22, 100 mg, 0.227 mmol) and 4-
methoxybenzene-1-sulfinyl chloride (23e, 66.6 mg, 0.27 mmol) to afford
1
2
3
6
2
1
1
03.0−207.0 °C; H NMR (CDCl
3
, 300 MHz) δ (ppm): 3.61(s, 6H), 3.81 (s,
H), 3.82 (s, 3H), 3.86 (s, 3H), 5.87 (d, J = 12.086 Hz, 1H), 6.04 (s, 1H)
.27 (s,2H), 6.31 (d, J = 12.086 Hz, 1H) 6.72 (m, 3H), 7.62 (d, J = 8.498,
H) ppm; 13C NMR (CDCl
3
, 75 MHz) (ppm): 55.5, 55.7, 56.2, 60.8, 105.7,
24e, which on deportation with TBAF (1.0M in THF, 2.5mmol) to obtain
10.6, 113.8, 120.5, 120.6, 125.4, 128.4, 129.8, 129.9, 131.2, 131.4,
37.6, 142.9, 147.6, 152.9, 163.3; MS(ESI): 502[M+H]+; HRMS (ESI)
the pure product 9e as a white colour solid. (107mg, 88% yield). mp
1
1
3
1
1
93.0−186.0 °C; H NMR (CDCl3) δ (ppm): 3.60 (s, 3H), 3.71 (s, 6H),
.85(s, 3H), 6.42-6.45 (d, J= 12.086 1H), 6.43 (s, 2H), 6.49-6.52 (d, J=
2.086 1H), 6.74-6.76 (s, J= 8.55 1H) 7.10 (s, 1H), 7.14-7.16 (d, J= 8.55
H), 7.54-7.57 (d, J= 8.43 1H), 7.61-7.63 (d, J= 7.62 1H), 7.71 (s, 1H)
calculated for C25
H
28
O
8
N
S[M+H]+502.15301; found: 502.15404.
HPLC: t 4.96 min, purity 98.0%.
R
(
Z)-N-(2-hydroxy-3-methoxy-6-(3,4,5-trimethoxystyryl) phenyl)-3,4-
ppm; 13C NMR (CDCl
120.0, 120.8, 125.6, 127.7, 129.0, 129.8, 130.9, 131.7, 132.9, 134.6,
37.5, 140.7, 142.6, 146.9, 152.9. MS(ESI): 540[M+H]+; HRMS (ESI)
calculated for C24 H24 O7 N Cl2 S[M+H]+540.06450; found: 540.06412.
HPLC: t 11.06 min, purity 98.0%.
3
, 75 MHz) (ppm): 55.8, 56.2, 60.8, 105.7, 110.6,
dimethoxybenzenesulfonamide (9b).
1
The compound 9b was prepared according to the general procedure by
employing
R
(Z)-2-((tert-butyldimethylsilyl)oxy)-3-methoxy-6-(3,4,5-
trimethoxystyryl)aniline (22, 100 mg, 0.227 mmol) and 4-
methoxybenzene-1-sulfinyl chloride (23b, 64.2 mg, 0.27 mmol) to afford
(Z)-4-fluoro-N-(2-hydroxy-3-methoxy-6-(3,4,5-
24b, which on deportation with TBAF (1.0M in THF, 2.5mmol) to obtain
trimethoxystyryl)phenyl) benzenesulfonamide (9f).
the pure product 9b as a white colour solid. (108mg, 91% yield). mp
1
2
10.0−213.0 °C; H NMR (CDCl
3
, 300 MHz) δ (ppm): 3.61 (s, 6H), 3.76
The compound 9f was prepared according to the general procedure by
(
6
(
(
1
s, 3H), 3.81 (s, 3H), 3.86 (s, 3H), 3.91 (s, 3H), 5.86 (d, 12.086 Hz 1H),
.26 (d, J = 12.086 Hz 1H), 6.27 (s, 2H), 6.63(s, OH), 6.70 (d, 1H), 6.73
d, 1H), 6.82(d,1H), 7.12 (d,1H), 7.63 (d, 1H). 13C NMR (CDCl
, 75 MHz)
ppm): 55.5, 55.7, 56.1, 60.7, 60.8, 105.9, 108.5, 109.5, 110.2, 112.6,
21.1, 129.2, 130.3, 130.5, 130.6, 132.3, 133.3, 137.1, 137.3, 148.8,
51.5, 152.7, 152.8; MS(ESI): 532[M+H]+; HRMS (ESI) calculated for
employing
(Z)-2-((tert-butyldimethylsilyl)oxy)-3-methoxy-6-(3,4,5-
trimethoxystyryl)aniline (22, 100 mg, 0.227 mmol) and 4-
methoxybenzene-1-sulfinyl chloride (23a, 66.6 mg, 0.27 mmol) to afford
3
24a, which on deportation with TBAF (1.0M in THF, 2.5mmol) to obtain
the pure product 9a as a white colour solid. (102mg, 89% yield). mp
1
C
1
1
3
6
94.0−197.0 °C; H NMR (CDCl
.86 (s, 3H), 5.98-6.01 (d, J=12.05 Hz 1H), 6.30-6.32 (d, J=12.05 3H),
.31 (s, 2H), 6.70-6.74 (m, 2H), 7.07-7.10 (t, 2H), 7.72-7.75 (m, 2H),
3
) δ (ppm): 3.63 (s, 6H), 3.82 (s, 3H),
26 2
H O
9
NS [M+H]+ 532.16123; found: 532.16137. HPLC: t
R
4.65 min,
purity 95.0%.
1
6
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201600643
FULL PAPER
ppm; 13C NMR (CDCl
1
1
, 75 MHz) (ppm): 55.7, 56.2, 60.8, 105.5, 110.5,
19.9, 120.8, 125.6, 127.7, 128.9, 129.8, 130.8, 132.9, 134.6, 137.4,
40.6, 142.6, 146.9, 152.9. MS(ESI): 490[M+H]+; HRMS (ESI) calculated
1H NMR (CDCl
, 300 MHz) δ (ppm): 2.89 (s, 3H), 3.80 (s, 3H), 3.88 (s,
3
3
3H), 3.88 (s, 6H), 6.43 (s, 1H), 6.45 (s, 1H), 6.78-6.81 (d, J=11.99 Hz,
1H), 6.83-6.86 (d, J=11.99 Hz, 1H), 5.57 (s, 1H), 5.88 (s, 1H), 6.89(s, 1H),
6.95-6.97 (d, J=7.99 Hz, 2H), 7.12 (s, 1H), 7.25 (s, 1H) ppm; 13C NMR
for C24 H25 O7 N F S[M+H]+490.13303; found: 490.13201. HPLC: t
.11 min, purity 97.0%.
R
5
(CDCl
3
, 75 MHz) (ppm): 30.8, 56.4, 56.6, 61.1, 106.1, 113.3, 126.1,
27.8, 128.1, 129.1, 130.0, 136.2, 136.6, 138.2, 138.4, 139.2, 140.7,
53.6, 155.0; MS(ESI): 486[M+H]+; HRMS (ESI) calculated for
NS[M+H]+486.15810; found: 486.15722. HPLC: t 4.94 min,
purity 97.0%.
1
1
(Z)-4-tert-butyl-N-(2-hydroxy-3-methoxy-6-(3,4,5-
25 28
C H O
7
R
trimethoxystyryl)phenyl) benzenesulfonamide(9g).
The compound 9g was prepared according to the general procedure by
employing (Z)-2-((tert-butyldimethylsilyl)oxy)-3-methoxy-6-(3,4,5-
trimethoxystyryl)aniline (22, 100 mg, 0.227 mmol) and 4-(tert-
butyl)benzene-1-sulfonyl chloride (23g, 55.2 mg, 0.28 mmol) to afford
(
E)-N-((4-fluorophenyl)sulfonyl)-3-(4-methoxyphenyl)-2-(3,4,5-
trimethoxyphenyl)acrylamide (10a).
2
4g, which on deportation with TBAF (1.0M in THF, 2.5mmol) to obtain
To a vigorously stirred mixture of 4-fluorobenzenesulfonamide (50 mg,
0.285 mmol) and potassium iodide (KI) (50 mol%) in acetonitrile (ACN),
(E)-3-(4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acryloyl chloride (155
mg, 0.428 mmol) was added under reflux condition. The progress of the
reaction was monitored by TLC. After completion of the reaction,
quenched with HYPO solution and extracted in to ethyl acetate (20 ml).
The solvent was washed with water and dried over MgSO4. After
evaporation of the solvent, the crude product was purified by column
chromatography on silica gel (60–120 mesh, EtOAc–petroleum ether) or
by recrystallization (toluene or ethyl acetate–n-hexane) to afford the
corresponding N-acyl sulfonamide 130mg (91%).
the pure product 9g as a white colour solid. (118mg, 88% yield). mp
1
203.0−205.0 °C; H NMR (CDCl
3
, 300 MHz) δ (ppm): 1.30 (s, 9H) 3.62 (s,
6H), 3.81 (s, 3H), 3.87 (s, 3H), 5.76 (d, J=11.897 1H), 6.22 (d,
J=11.897 1H), 6.26(s, 2H), 6.68(d,1H), 6.72(d, 1H), 7.40 (d, J=8.498
2
3
1
H),7.63 (d, J=8.498 Hz, 2H), ppm; 13C NMR (CDCl
, 75 MHz) (ppm):
3
0.9, 35.1, 55.7, 56.2, 60.8, 105.7, 110.6, 120.4, 120.5, 125.3, 125.6,
27.5, 128.5, 131.1, 131.4, 135.3, 137.5, 143.0, 147.5, 152.8, 157.2;
+
FABMAS:(M+H)=527. MS (ESI): 528[M+H] ; HRMS (ESI) calculated for
C
purity 95.0%.
28 7
H33NO S[M+H]+528.20388; found: 528.20379. HPLC: t
R
7.28 min,
(
Z)-N-(2-hydroxy-3-methoxy-6-(3,4,5-trimethoxystyryl)phenyl)-4-
1H NMR (CDCl
3H), 6.22 (s, 2H), 7.03-7.06 (d, J=8.49 Hz 2H), 7.16-7.19 (d, J=8.49 Hz
H), 7.51-7.54 (d, J=8.49 Hz 2H), 7.69 (s, 1H), 7.94 (s, 1H), ), 8.01-8.04
d, J=8.49 Hz 2H), ppm; mp 193.0−186.0 °C; 13C NMR (CDCl
, 75 MHz)
ppm): 55.4, 55.6, 60.7, 108.1, 108.3, 115.7, 115.9, 116.2, 15.9, 129.0,
3
, 300 MHz) δ (ppm): 3.53 (s, 3H), 3.79 (s, 6H), 3.86 (s,
nitrobenzenesulfonamide (9h).
2
(
(
3
The compound 9h was prepared according to the general procedure by
employing
trimethoxystyryl)aniline (22, 100 mg, 0.227 mmol) and 4-
trifluoromethyl)benzene-1-sulfonyl chloride (23h, 59.2 mg, 0.29 mmol) to
(Z)-2-((tert-butyldimethylsilyl)oxy)-3-methoxy-6-(3,4,5-
1
30.0, 131.3, 131.5, 131.6, 134.4, 134.5, 141.3, 152.6, 160.4. MS(ESI):
01.5; HRMS (ESI) calculated for C25 N F S[M+H]+ 502.13303;
4.15 min, purity 95.0%.
5
H O
24 7
(
found: 502.13378. HPLC: t
R
afford 24h, which on deportation with TBAF (1.0M in THF, 2.5mmol) to
obtain the pure product 9h as a white colour solid. (118mg, 89% yield).
1
mp 213.0−215.0 °C; H NMR (CDCl
3
, 300 MHz) δ (ppm): 3.64 (s, 3 H),
(E)-N-((4-chlorophenyl)sulfonyl)-3-(4-methoxyphenyl)-2-(3,4,5-
trimethoxyphenyl)acrylamide (10b).
3
6
6
7
.67 (s, 6 H), 3.84 (s, 3 H),6.44 (s, 2H), 6.42-6.44 (d, J=12.08 Hz, 1H),
.46-6.50 (d, J=12.08 Hz, 1H), 6.60-6.63 (d, J=8.30 1H), 6.93 (s, 1H),
.96-7.00 (dd, J=8.30, 1H), 7.02-7.08 (d, J=9.06 2H), 7.40 (s, 1H), 7.65-
.70 (dd, J= 9.06 Hz 2H) ppm; mp 208.0−211.0 °C; 13C NMR (CDCl3, 75
Compound 10b was prepared according to the method described for
compound 10a, employing 4-chlorobenzenesulfonamide (50 mg, 0.261
mmol) and potassium iodide (KI) (50 mol%) (ACN) and (E)-3-(4-
methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acryloyl chloride (142 mg,
MHz) (ppm): 55.8, 56.1, 60.8, 105.8, 110.0, 110.5, 112.2, 122.2, 122.6,
1
1
24.2, 128.1, 128.5, 130.1, 132.1, 137.2, 138.1, 148.8, 150.9, 152.9,
53.5; MS(ESI): 517[M+H]+; HRMS (ESI) calculated for
S[M+H]+517.12800; found: 517.12809. HPLC: t 4.26 min,
purity 99.0%.
0
.392 mmol) in acetonitrile, obtain the pure product 10b as a white color
C H O N
24 24 9 2
R
1
solid 125mg (92%). mp 218.0−221.0 °C; H NMR (CDCl
3
, 300 MHz) δ
(
(
ppm): 3.50 (s, 3H), 3.78 (s, 6H), 3.88 (s, 3H), 6.43 (s, 2H), 6.52-6.54
d, J=8.39 Hz 1H), 6.71-6.74 (m, 1H), 6.90-6.94 (dd, J=8.39 Hz 1H),
(
Z)-4-amino-N-(2-hydroxy-3-methoxy-6-(3,4,5-
7.21-7.24 (t, 2H), 7.61 (s, 1H), 8.11-8.14 (dd, J=5.03 Hz 2H), 7.74 (s, 1H),
9.88 (s, NH 1H) ppm; 13C NMR (CDCl
, 75 MHz) (ppm): 55.4, 55.5, 60.7,
08.2, 115.7, 125.7, 128.9, 129.1, 129.8, 130.1, 131.3, 136.7, 139.5,
04.6, 141.4, 152.5, 160.4, 164.2; MS(ESI): 518 ; HRMS (ESI) calculated
N Cl S[M+H]+ 518.1000; found: 518.10260; HPLC: t 5.11
min, purity 88.0%.
trimethoxystyryl)phenyl) benzenesulfonamide (9i).
3
1
1
The compound 9a was prepared according to the general procedure by
employing (Z)-2-((tert-butyldimethylsilyl)oxy)-3-methoxy-6-(3,4,5-
trimethoxystyryl)aniline (22, 100 mg, 0.227 mmol) and 4-
methoxybenzene-1-sulfinyl chloride (23a, 55.2 mg, 0.27 mmol) to afford
for C25
H
24
O
7
R
2
6a, which on deportation with TBAF (1.0M in THF, 2.5mmol) to obtain
(E)-N-((3,4-dichlorophenyl)sulfonyl)-3-(4-methoxyphenyl)-2-(3,4,5-
trimethoxyphenyl)acrylamide (10c).
the pure product 9i as a white colour solid. (118mg, 89% yield). mp
1
222.0−225.0 °C; H NMR (CDCl
3
, 300 MHz) δ (ppm): 3.60 (s, 6 H), 3.80
(
(
s, 3 H), 3.85 (s, 3 H), 5.79-5.83 (d, J=12.08 Hz, 1H), 5.98 (s, 1H), 6.24
s, 2H), 6.28-6.32 (d, J=12.08 Hz, 1H), 6.53-6.56 (d, J=8.68, 2H), 6.63-
Compound 10c was prepared according to the method described for
compound 10a, employing 3,4-dichlorobenzenesulfonamide (50 mg,
6
8
1
1
.66 (d, J=8.876 2H), 6.70-6.73 (d, J = 8.68 Hz, 2H), 7.42-7.45 (d, J=
0
.231 mmol) and potassium iodide (KI) (50 mol%) (ACN) and (E)-3-(4-
methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acryloyl chloride (121 mg,
.334 mmol) in acetonitrile, obtain the pure product 10c as a white color
solid 115mg (90%). mp 223.0−226.0 °C; 1H NMR (CDCl
, 300 MHz) δ
ppm): 3.60 (s, 6H), 3.80 (s, 3H), 3.85 (s, 3H), 5.79-5.83 (d, J=9.39 Hz
.68 Hz 2H) ppm; 13C NMR (CDCl
3
, 75 MHz) (ppm): 152.8, 151.1, 147.8,
42.9, 137.6, 131.4, 131.3, 129.8, 128.2, 125.8, 125.4, 120.8, 120.3,
13.6, 110.6, 105.6, 60.8, 56.2, 55.7. MS(ESI): 487[M+H]+; HRMS (ESI)
0
3
calculated for C24 H27 O7 N2 S[M+H]+487.15335; found: 487.15414.
HPLC: t 5.01 min, purity 92.0%.
(
R
1
1
H), 6.24 (s, 2H), 6.28-6.31 (d, J=9.39 Hz 2H), 6.53-6.56 (d, J=8.68 Hz
H), 6.66-6.72 (m, 2H), 7.42-7.45 (d, J=8.68 Hz 2H), ppm; 13C NMR
(
Z)-N-(2-hydroxy-3-methoxy-6-(3,4,5-trimethoxystyryl)phenyl)-4-
(CDCl
126.38, 129.01, 129.32, 129.46, 130.02, 131.29, 132.76, 134.44, 136.73,
38.44, 138.73, 152.59, 159.45, 165.33; MS(ESI): 552.4 ; HRMS (ESI)
3
, 75 MHz) (ppm): 54.08, 54.90, 59.52, 105.63, 112.65, 125.32,
methylbenzenesulfonamide (9k).
1
1
7
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201600643
FULL PAPER
calculated for C25
H
23
O
7
N Cl
2
S[M+H]+ 552.06450; found: 552.06432;
8.05 (t, 2H) ppm; 13C NMR (CDCl
3
+DMSO, 75 MHz) (ppm): 43.99, 55.10,
HPLC: t 3.33 min, purity 94.0%.
R
55.17, 60.0, 105.2, 111.7, 115.4, 123.3, 127.0, 127.4, 127.9, 128.6,
29.3, 131.5, 136.4, 137.4, 144.9, 150.1, 152.2, 158.0; MS(ESI): 548 ;
HRMS (ESI) calculated for C26 NClS [M+H]+ 548.11404; found:
48.11559.
1
27 8
H O
(
E)-4-(N-(3-(3,4-dichlorophenyl)-2-(3,4,5-
5
trimethoxyphenyl)acryloyl)sulfamoyl)phenyl acetate (10d)
(
E)-3-(3,4-dimethoxyphenyl)-N-((4-nitrophenyl)sulfonyl)-2-(3,4,5-
Compound 10d was prepared according to the method described for
compound 10a, employing 4-sulfamoylphenyl acetate (50 mg, 0.232
mmol) and potassium iodide (KI) (50 mol%) (ACN) and (E)-3-(3,4-
dichlorophenyl)-2-(3,4,5-trimethoxyphenyl)acrylaldehyde (128 mg, 0.348
trimethoxyphenyl) acrylamide (10h).
Compound 10h was prepared according to the method described for
compound 10a, employing 4-nitrobenzenesulfonamide (50 mg, 0.247
mmol) and potassium iodide (KI) (50 mol%) (ACN) and (E)- (E)-3-(3,4-
dimethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acryloyl chloride (140 mg,
mmol) in acetonitrile, obtain the pure product 10d as a white color solid
1
1
23mg (93%). mp 226.0−229.0 °C; H NMR (CDCl
3
, 300 MHz) δ (ppm):
2.26 (s, 3H), 3.81 (s, 6H), 3.97 (s, 3H), 6.45 (s, 2H), 6.90-6.93 (d,
J=8.49 Hz 2H), 6.99-7.02 (d, J=8.49 Hz 2H), 7.49-7.54 (m, 2H), 7.75 (s,
H), 8.19 (s, NH 1H), 8.27-8.30 (d, J= 8.68 Hz 1H), ppm; 13C NMR
CDCl , 75 MHz) (ppm): 21.0, 56.2, 61.0, 105.9, 121.8, 127.7, 128.5,
0.371 mmol) in acetonitrile, obtain the pure product 10h as a white color
1
1
(
solid 126mg (91%). mp 223.0−226.0 °C; H NMR (CDCl
3
, 300 MHz) δ
3
(ppm): 3.61 (s, 6H), 3.80 (s, 3H), 3.82 (s, 3H), 3.93 (s, 3H), 6.41 (s, 2H),
6.60-6.63 (d, J=8.49 1H), 6.72(s, 1H), 7.10 (s, 1H), 7.43 (s, 1H), 7.31-
56 (d, J=9.06 Hz 1H), 7.61-7.64 (d, J= 9.06 Hz 2H), ppm; 13C NMR
130.6, 131.0, 131.4, 132.5, 134.3, 138.9, 140.7, 141.0, 138.9, 151.8,
1
H
54.8, 163.7, 168.9; MS(ESI): 580.4 ; HRMS (ESI) calculated for C26
+
23 8
O NCl
2
S[M+H]+ 580.05969; found: 580.05965[M+Na ]calculated
(CDCl
112.76, 122.18, 123.31, 126.25, 128.80, 129.35, 131.24, 138.64, 139.84,
44.49, 148.73, 149.02, 149.79, 151.99, 165.93; MS(ESI): 558.6 ; HRMS
ESI) calculated for S[M+H]+ 559.13809; found:
3
+DMSO, 75 MHz) (ppm): 55.08, 55.51, 60.21, 107.56, 111.41,
602.04136;found: 602.04160; HPLC: t
R
3.31 min, purity 72.0%.
1
(
26 27 10 2
C H O N
(
E)-3-(3,4-dimethoxyphenyl)-N-(phenylsulfonyl)-2-(3,4,5-
559.14020.
trimethoxyphenyl)acrylamide (10e).
(
E)-N-((4-aminophenyl)sulfonyl)-3-(3,4-dimethoxyphenyl)-2-(3,4,5-
Compound 10e was prepared according to the method described for
compound 10a, employing benzenesulfonamide (50 mg, 0.318 mmol)
and potassium iodide (KI) (50 mol%) (ACN) and (E)- (E)-3-(3,4-
dimethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acryloyl chloride (171 mg,
trimethoxyphenyl)acrylamide (10i).
Compound 10i was prepared according to the method described for
compound 10a, employing 4-aminobenzenesulfonamide (50 mg, 0.290
mmol) and potassium iodide (KI) (50 mol%) (ACN) and (E)- (E)-3-(3,4-
dimethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acryloyl chloride (164 mg,
0.436 mmol) in acetonitrile, obtain the pure product 10i as a white color
solid 132mg (86%). mp 230.0−233.0 °C; H NMR (CDCl
0
.477 mmol) in acetonitrile, obtain the pure product 10e as a white color
1
solid 147mg (90%). mp 233.0−236.0 °C; H NMR (CDCl
3
+DMSO 300
MHz) δ (ppm): 3.55 (s, 6H), 3.77 (s, 6H), 3.89 (s, 3H), 3.90 (s, 3H), 6.34
s, 2H), 6.72 -6.78(m, 2H), 6.96(s, 1H), 7.51-7.58 (m, 3H), 7.65-7.67 (d,
(
1
5
1
H), 8.0 (d, 2H), ppm; 13C NMR (CDCl
5.38, 59.87, 101.9, 102.0, 104.3, 104.6, 121.95. 122.0, 124.7, 127.6,
27.8, 127.9, 128.1, 129.6, 137.2, 137.4, 152.5, 152.7; MS(ESI): 513.6 ;
3
+DMSO 75 MHz) (ppm): 53.43,
1
3
, 300 MHz) δ
(ppm): 3.54 (s, 6H), 3.77 (s, 3H), 3.79 (s, 3H), 3.90 (s, 3H), 5.12 (s, NH
2
2H), 6.29 (s, 2H), 6.66 -6.71(m, 4H), 6.78-6.81(d, J=8.12 Hz 1H), 6.96-
6.99 (d, J=8.12 Hz 1H), 7.51-53 (d, J=11.89 Hz 1H), 7.74-7.77 (d, J=
8.68 Hz 2H), ppm; 13C NMR (CDCl
5
1
HRMS (ESI) calculated for C26
28 8
H O NS [M+H]+ 514.15301; found:
5
14.15408; HPLC: t 3.86 min, purity 82.0%.
R
3
, 75 MHz) (ppm): 54.56, 54.80, 59.56,
9.67, 105.09, 108.09, 112.9, 122.7, 126.2, 128.4, 129.1, 129.6, 131.3,
31.8, 133.53, 136.0, 137.5, 146.8, 150.8, 151.7, 153.5; MS(ESI): 528.6 ;
(
E)-3-(3,4-dimethoxyphenyl)-N-((4-fluorophenyl)sulfonyl)-2-(3,4,5-
HRMS (ESI) calculated for C26
29 8 2
H O N S[M+H]+ 529.16391; found:
trimethoxyphenyl)acrylamide (10f).
+
529.16386 [M+Na ] calculated was 551.14586;found:567.14518.
Compound 10f was prepared according to the method described for
compound 10a, employing 4-fluorobenzenesulfonamide (50 mg, 0.285
mmol) and potassium iodide (KI) (50 mol%) (ACN) and (E)- (E)-3-(3,4-
dimethoxyphenyl)-2-(3,4,5-trimethoxyphenyl) acryloyl chloride (161 mg,
(
(
E)-3-(3-fluoro-4-methoxyphenyl)-N-((4-fluorophenyl)
3,4,5-trimethoxyphenyl)acrylamide (10j).
sulfonyl)-2-
0
.427 mmol) in acetonitrile, obtain the pure product 10f as a white color
Compound 10j was prepared according to the method described for
compound 10a, employing 4-fluorobenzenesulfonamide (50 mg, 0.285
mmol) and potassium iodide (KI) (50 mol%) (ACN) and (E)- (E)-3-(3-
fluoro-4-methoxyphenyl)-2-(3,4,5-trimethoxy phenyl)acryloyl chloride
(162 mg, 0.427 mmol) in acetonitrile, obtain the pure product 10j as a
1
solid 133mg (87%). mp 225.0−228.0 °C; H NMR (CDCl
3
, 300 MHz) δ
(ppm): 3.45 (s, 3H), 3.82 (s, 6H), 3.84 (s, 3H), 3.92 (s, 3H), 6.36 (s, 1H),
6
.46 (s, 2H), 6.70-6.73 (d, J=8.30 Hz 1H), 6.78-6.80 (d, J=8.30Hz 1H),
.21-7.23 (d, J=8.49 Hz 2H), 7.74 (s, 1H), 7.99 (s, NH 1H), 8.11-8.16 (d,
7
J= 8.68 Hz 1H), ppm; 13C NMR (CDCl
, 75 MHz) (ppm): 55.14, 55.79,
6.36, 60.83, 106.44, 110.58, 111.95, 116.05, 116.24, 126.30, 126.51,
28.32, 129.55, 131.65, 134.38, 138.59, 141.52, 148.31, 150.83, 154.84,
64.01; MS(ESI): 531 ; HRMS (ESI) calculated for C26 N F
3.16 min, purity 96.0%.
white color solid 136mg (92%). mp 233.0−236.0 °C; H NMR (CDCl3,
1
3
5
1
1
300 MHz) δ (ppm): 3.77 (s, 3H), 3.90 (s, 6H), 3.94 (s, 3H), 6.39 (s, 2H),
6.94 (d, J=8.55 Hz 1H), 7.17-7.19 (d, J=8.68 Hz 1H), 7.54-7.59 (m, 3H),
7.64-7.65 (d, J=7.70 Hz 1H), 8.08-8.09 (d, J= 5.74 Hz 2H), 10.53 (s, 1H)
H O
26 8
S[M+H]+ 532.14359; found: 532.14481; HPLC: t
R
ppm;13C NMR (CDCl
3
, 75 MHz) (ppm): 54.96, 55.09, 59.69, 105.85,
11.7, 114.76, 115.0, 116.03, 116.28, 125.96, 126.90, 128.63, 130.10,
30.22, 131.22, 134.49, 137.00, 147.45, 152.68, 165.19; MS(ESI):
19.5 ; HRMS (ESI) calculated for C25 N F S[M+H]+ 520.1236;
1
1
5
(
E)-N-((4-chlorophenyl)sulfonyl)-3-(3,4-dimethoxyphenyl)-2-(3,4,5-
H
23
O
7
2
trimethoxyphenyl) acrylamide (10g).
found: 520.12462.
Compound 10g was prepared according to the method described for
compound 10a, employing 4-chlorobenzenesulfonamide (50 mg, 0.261
mmol) and potassium iodide (KI) (50 mol%) (ACN) and (E)- (E)-3-(3,4-
dimethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acryloyl chloride (148 mg,
(
(
E)-N-((4-chlorophenyl)sulfonyl)-3-(3-fluoro-4-methoxy
3,4,5-trimethoxyphenyl)acrylamide (10k).
phenyl)-2-
0
.395 mmol) in acetonitrile, obtain the pure product 10g as a white color
solid 128mg (89%). mp 217-220.0 °C; 1H NMR (CDCl
, 300 MHz) δ
ppm): 3.45 (s, 3H), 3.78 (s, 6H), 3.85 (s, 6H), 6.42-6.46 (m, 3H), 6.76-
Compound 10k was prepared according to the method described for
compound 10a, employing 4-chlorobenzenesulfonamide (50 mg, 0.261
mmol) and potassium iodide (KI) (50 mol%) (ACN) and (E)- (E)-3-(3-
fluoro-4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)acryloyl chloride (150
3
(
6.78 (s, J=8.43 Hz 2H), 6.51-7.55 (d, J=5.30 Hz 2H), 7.61 (s, 1H), 8.02-
1
8
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201600643
FULL PAPER
mg, 0.395 mmol) in acetonitrile, obtain the pure product 10k as a white
color solid 123mg (88%). Mp231.0−234.0 °C; 1H NMR (CDCl3, 300
MHz) δ (ppm): 3.43 (s, 3H), 3.72 (s, 6H), 3.82 (s, 3H), 6.44 (s, 2H),
mmol) and potassium iodide (KI) (50 mol%) (ACN) and (E)-2-(4-
methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)acryloyl chloride (142 mg,
0.392 mmol) in acetonitrile, obtain the pure product 11b as a white color
1
6
7
.48 (s, 1H), 6.75-6.77 (d, J=2.13 Hz 1H), 7.32-7.34 (d, J=8.54 Hz, 1H),
.58-7.61 (d, J=9.15 Hz 1H), 7.66-7.69 (d, J= 9.15 Hz 2H) ppm;13C NMR
solid 127mg (94%). H NMR (CDCl
3
, 300 MHz) δ (ppm): 3.64 (s, 3H),
3.67 (s, 6H), 3.84 (s, 3H), 6.44 (s, 2H), 6.45-6.46 (d, J=6.69 Hz 2H),
6.61-6.62 (d, J=8.39 Hz 1H), 6.95 (s, 1H), 6.97-6.99 (m, 1H), 7.04-7.06 (d,
2H) 7.40 (s, NH 1H), 7.66-7.68 (d, J=8.85 Hz 2H), ppm; 13C NMR
3
(CDCl ,DMSO, 75 MHz) (ppm): 55.1, 55.9, 60.4, 106.4, 114.0, 115.5,
1
1
15.6, 115.8, 124.5, 129.1, 130.0, 130.9, 131.1, 131.8, 134.6, 137.9,
38.8, 146.7, 153.8, 165.0; MS(ESI): 536 ; HRMS (ESI) calculated for
(CDCl
121.0, 125.6, 126.1, 128.4, 130.0, 131.7, 137.8, 143.2, 148.0, 151.3,
53.1; MS(ESI): 518 HRMS (ESI) calculated for NCl
S[M+NH ]+ 536.13785; found: 536.13442; HPLC: t 8.58 min, purity
3.0%.
3
, 75 MHz) (ppm): 56.0, 56.4, 61.0, 105.9, 110.8, 113.8, 120.5,
25 23 7
C H O ClNF S[M+H]+ 536.09406; found: 536.09358.
1
;
25 24 7
C H O
4
R
(
(
E)-N-((4-chlorophenyl)sulfonyl)-3-(4-methoxy-3-nitrophenyl)-2-
3,4,5-trimethoxyphenyl)acrylamide (10l).
9
(
E)-2-(3,4-dimethoxyphenyl)-N-((4-fluorophenyl)sulfonyl)-3-(3,4,5-
Compound 10l was prepared according to the method described for
compound 10a, employing 4-chlorobenzenesulfonamide (50 mg, 0.261
mmol) and potassium iodide (KI) (50 mol%) (ACN) and (E)-3-(4-methoxy-
trimethoxyphenyl)acrylamide (11c).
3
-nitrophenyl)-2-(3,4,5-trimethoxyphenyl) acryloyl chloride (160 mg,
Compound 11c was prepared according to the method described for
compound 10a, employing 4-fluorobenzenesulfonamide (50 mg, 0.285
mmol) and potassium iodide (KI) (50 mol%) (ACN) and (E)-2-(3,4-
dimethoxyphenyl)-3-(3,4,5-trimethoxyphenyl)acryloyl chloride (161 mg,
0.395 mmol) in acetonitrile, obtain the pure product 10l as a white color
1
solid 131mg (89%). mp 225.0−238.0 °C; H NMR (CDCl3, 300 MHz) δ
(
(
ppm): 3.81 (s, 3H), 3.91 (s, 6H), 3.95 (s, 3H), 6.39 (s, 2H), 6.88-6.90
d, J=8.54 Hz 1H), 7.13-7.15 (d, J=8.54 Hz 2H), 7.46 (s, NH 1H), 7.53-
0.427 mmol) in acetonitrile, obtain the pure product 11c as a white color
1
7
.54 (d, J=6.71 Hz 1H), 7.69 (s,1H), 8.03-8.04 (d, J= 7.19 Hz 3H), ppm;
C NMR (CDCl3, 75 MHz) (ppm): 56.4, 56.6, 61.1, 106.06, 113.3, 126.0,
27.9, 129.2, 130.17, 131.5, 136.2, 136.5, 136.6, 138.4, 139.2, 139.3,
solid 140mg (92%). mp 221.0−224.0 °C; H NMR (CDCl
3
, 300 MHz) δ
13
(ppm): 3.45 (s, 3H), 3.82 (s, 6H), 3.84 (s, 3H), 3.92 (s, 3H), 6.36 (s, 1H),
6.46 (s, 2H), 6.70-6.73 (d, J=8.43 Hz 1H), 6.78-6.80 (d, J=8.31Hz 1H),
7.21-7.27 (dd, J=8.43 Hz 2H), 7.74 (s, 1H), 7.98 (s, NH 1H), 8.12-8.15 (d,
1
1
53.7, 153.8, 155.1, 163.4; MS(ESI): 563 ; HRMS (ESI) calculated for
+
J= 8.68 Hz 2H), ppm; 13C NMR (CDCl
C
25
H
23
O
7
Cl N
2
S[M+Na ]+ 585.07050; found: 585.07057.
3
, 75 MHz) (ppm): 55.17, 55.80,
6.39, 60.84, 106.52, 110.62, 112.03, 116.06, 116.24, 126.35, 126.50,
28.39, 129.57, 131.59, 131.66, 138.69, 141.51, 148.36, 150.88, 154.88,
64.00; MS(ESI): 531.5 HRMS (ESI) calculated for NF
3.34 min, purity 90.0%.
5
1
1
(
E)-3-(3-amino-4-methoxyphenyl)-N-(phenylsulfonyl)-2-(3,4,5-
;
26 26 8
C H O
trimethoxyphenyl)acrylamide (10m).
S[M+H]+ 532.14359; found: 532.14324; HPLC: t
R
Compound 10m was prepared according to the method described for
compound 2c, employing (E)-N-((4-chlorophenyl)sulfonyl)-3-(4-methoxy-
(
E)-N-((4-chlorophenyl)sulfonyl)-2-(3,4-dimethoxyphenyl)-3-(3,4,5-
trimethoxyphenyl)acrylamide (11d).
3-nitrophenyl)-2-(3,4,5-trimethoxyphenyl) acrylamide (10l) (6h, 80 mg,
.15 mmol), in Methanol was added Zn (19.3 mg, 0.30 mmol) HCOONH
0
4
(
20.1 mg, 0.31 mmol) to obtain the pure product 10m as a white colour
Compound 11d was prepared according to the method described for
compound 10a, employing 4-chlorobenzenesulfonamide (50 mg, 0.261
mmol) and potassium iodide (KI) (50 mol%) (ACN) and (E)- (E)-2-(3,4-
dimethoxyphenyl)-3-(3,4,5-trimethoxyphenyl)acryloyl chloride (148 mg,
0.395 mmol) in acetonitrile, obtain the pure product 11d as a white color
solid. (72mg, 90% yield); mp 223.0−226.0 °C; 1H NMR (CDCl3, 300
MHz) δ (ppm): 3.77 (s, 3H), 3.81 (s, 6H), 3.94 (s, 3H), 6.46 (s, 2H),
6
.70-6.72 (d, J=8.80 Hz 1H), 6.95-6.97 (d, J=8.80 Hz 2H), 7.48-7.50 (d,
J=7.21 Hz 1H), 7.55 (s, 1H), 7.69 (s, 1H), 8.24-8.27(d, J= 8.55 Hz 2H),
ppm;13C NMR (CDCl3, 75 MHz) (ppm): 54.7, 55.5, 60.1, 102.4, 105.7,
1
solid 131mg (91%). mp 193.0−186.0 °C; mp 233.0−236.0 °C; H NMR
1
1
06.3, 115.9, 121.5, 126.0, 128.3, 129.2, 129.5, 135.3, 137.2, 139.1,
40.0, 147.9, 152.4, 153.1, 165.5; MS(ESI): 533.0 HRMS (ESI)
Cl S[M+H]+ 533.11438; found: 533.11617.
(CDCl3, 300 MHz) δ (ppm): 3.45 (s, 3H), 3.78 (s, 6H), 3.85 (s, 6H),
;
6.42-6.46 (m 3H), 6.76-6.84 (m, 2H), 7.51-7.55 (t, 1H), 7.61 (s, 1H),
calculated for C25
H
26
O
7
N
2
8.02-8.05 (t, 2H), 10.65 (s, NH) ppm; 13C NMR (CDCl
3
, 75 MHz) (ppm):
5
1
1
4.30, 55.01, 55.44, 59.86, 106.30, 109.90, 111.29, 125.26, 125.97,
28.22, 128.81, 129.11, 129.55, 129.76, 137.20, 38.96, 139.25, 147.37,
(
E)-N-((4-fluorophenyl)sulfonyl)-2-(4-methoxyphenyl)-3-(3,4,5-
49.62, 153.15, 165.32; MS(ESI): 548 ; HRMS (ESI) calculated for C26
trimethoxyphenyl)acrylamide (11a).
+
H
26
O
8
N Cl S[M+H]+ 548.11404; found: 548.11372[M+Na ] calculated
R
was 570.09599; found: 570.09506. HPLC: t 3.28 min, purity 83.0%.
Compound 11a was prepared according to the method described for
compound 10a, employing 4-fluorobenzenesulfonamide (50 mg, 0.285
mmol) and potassium iodide (KI) (50 mol%) (ACN) and (E)-2-(4-
methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)acryloyl chloride (142 mg,
(
E)-N-((4-fluorophenyl)sulfonyl)-2-(4-methoxyphenyl)-3-(3,4,5-
trimethoxyphenyl)acrylamide (11e).
0
.421 mmol) in acetonitrile, obtain the pure product 11a as a white color
1
solid 130mg (91%). mp 215.0−218.0 °C; H NMR (CDCl
3
, 300 MHz) δ
Compound 11e was prepared according to the method described for
compound 10a, employing 4-fluorobenzenesulfonamide (50 mg, 0.285
mmol) and potassium iodide (KI) (50 mol%) (ACN) and (E)- (E)-2-(4-
methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)acryloyl chloride (162 mg,
(
ppm): 3.75 (s, 3H), 3.80 (s, 6H), 3.96 (s, 3H), 6.41 (s, 2H), 6.68-6.70 (d,
J=9.00 Hz 2H), 6.92-6.94 (d, J=9.00 Hz 2H), 7.21-7.25 (t, 2H), 7.74 (s,
H), 7.94 (s, 1H) 8.07 (s, 1H), 8.11-8.13 (d, J=8.85 Hz 2H), ppm; 13C
NMR (CDCl , 75 MHz) (ppm): 55.47, 55.58, 60.78, 108.26, 115.77,
25.78, 128.95, 129.16, 129.82, 129.9, 130.1, 131.3, 136.8, 139.5, 140.6,
41.4, 152.5, 160.4, 164.2; MS(ESI): 501.5 ; HRMS (ESI) calculated for
N F S[M+H]+ 502.13303; found: 502.1472; HPLC: t 3.15 min,
purity 100.0%.
1
3
0.427 mmol) in acetonitrile, obtain the pure product 11e as a white color
1
1
1
C
solid 133mg (90%). mp 228.0−231.0 °C; H NMR (CDCl
3
, 300 MHz) δ
(ppm): 3.50 (s, 3H), 3.82 (s, 6H), 3.93 (s, 3H), 6.43 (s, 2H), 6.47-6.49
(d, J=8.12 Hz 1H), 6.68-6.72 (m, 1H), 6.89-6.95 (t, 1H), 7.21-7.24 (d,
J=8.49 Hz 2H), 7.71 (s,1H), 8.00 (s, NH 1H), 8.11-8.16 (dd, J= 8.68 Hz
25
H
24
O
7
R
3
1
1
H), ppm; 13C NMR (CDCl
, 75 MHz) (ppm): 54.3, 54.7, 59.2, 106.7,
3
10.7, 110.7, 112.2, 121.4, 125.9, 126.8, 127.5, 128.3, 131.3, 132.1,
37.5, 137.7, 138.3, 147.8, 148.1, 151.2, 165.2; MS(ESI): 519.5 ; HRMS
(
E)-N-((4-chlorophenyl)sulfonyl)-2-(4-methoxyphenyl)-3-(3,4,5-
trimethoxyphenyl)acrylamide (11b).
(ESI) calculated for
C
25
H
23
O
7
N
F
2
S[M+H]+ 520.12361; found:
5
20.12287; HPLC: t 3.30 min, purity 93.0%.
R
Compound 11b was prepared according to the method described for
compound 10a, employing 4-chlorobenzenesulfonamide (50 mg, 0.261
1
9
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ChemMedChem
10.1002/cmdc.201600643
FULL PAPER
º
(
E)-N-(4-chlorophenylsulfonyl)-2-(4-methoxyphenyl)-3-(3,4,5-
at 37 C. CA-4 was used as a positive control whereas taxol was used as
negative control which binds at the taxane site. After incubation the
fluorescence of tubulin–colchicine complex was determined by using
Tecan multimode reader with excitation wavelength of 350 nm and
emission wavelength of 435 nm. 30 mM Tris buffer was used as blank
which was subtracted from all the samples and the fluorescence values
are normalized to DMSO (control).[50]
trimethoxyphenyl)acrylamide (11f).
Compound 11f was prepared according to the method described for
compound 10a, employing 4-chlorobenzenesulfonamide (50 mg, 0.261
mmol) and potassium iodide (KI) (50 mol%) (ACN) and (E)- (E)-2-(4-
methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)acryloyl chloride (150 mg,
0
.395 mmol) in acetonitrile, obtain the pure product 11f as a white color
1
solid 127mg (91%). mp 233.0−237.0 °C; H NMR (CDCl
3
, 300 MHz) δ
Immunohistochemistry
(
(
6
ppm): 3.62 (s, 6H), 3.68 (s, 6H), 3.83 (s, 3H), 6.41 (s, 2H), 6.43-6.46
d, J=9.12 Hz 1H), 6.48-6.52 (d, J=8.28 2H), 6.61-6.63 (d, J=8.65 1H),
.92-6.95 (m, 2H), 7.39 (s,1H), 7.44-7.46 (d, J= 8.28 Hz 2H), ppm; 13C
,DMSO, 75 MHz) (ppm): 54.8, 55.8, 60.5, 106.0, 113.5,
25.8, 127.2, 128.6, 129.2, 130.9, 132.1, 133.7, 134.5, 137.9, 140.1,
40.5, 154.0, 160.6, 164.6; MS(ESI): 536; HRMS (ESI) calculated for C25
N Cl F S[M+H]+ 536.09406; found: 536.09342; HPLC: t 3.23 min,
HeLa and MCF-7cells were seeded on glass cover slips, incubated for
48 h in the presence or absence of test compounds 7a, 9a and CA-4 at
NMR (CDCl
1
1
3
25 nM concentrations. Following the termination of incubation, cells were
fixed with 3% paraformaldehyde, 0.02% glutaraldehyde in PBS and
permeabilized by dipping the cells in 100% methanol (20 °C). Later,
cover slips were blocked with 1% BSA in phosphate buffered saline for 1
h followed by incubation with a primary anti tubulin (mouse monoclonal)
antibody and FITC conjugated secondary mouse anti IgG antibody.
Photographs were taken using the confocal microscope, equipped with
FITC settings and the pictures were analyzed for the integrity of
microtubule network.[51]
H
23
O
7
R
purity 94.0%.
MTT assay.
The anticancer activity of the compounds was determined using MTT (3-
(
4, 5-dimethylthiazol-2 yl)-2,5-diphenyl tetrazolium bromide) reduction
[
47]
1×106 cells/well were seeded in 100 µl DMEM, supplemented
assay.
Hoechst staining for morphological analysis of apoptosis
with 10% FBS in each well of 96-well micro culture plates and incubated
for 24 h at 37 °C in a CO2 incubator. Compounds, diluted to the desired
concentrations in culture medium, were added to the wells with
respective vehicle control. After 48 h of incubation, MTT (10 µl, 5 mg/mL)
was added to each well and the plates were further incubated for 4 h.
The supernatant from each well was carefully removed, formazon
crystals were dissolved in DMSO (100 µl) and absorbance at 540 nm
wavelength was recorded.
Cells (Hela and MCF-7) were seeded at a density of 10,000 cells over
18-mm cover slips and incubated for 24 h. Then, the medium was
replaced, and cells were treated with 7a and 9a at 25 nM concentration
for 48 h. Cells treated with vehicle (0.001% DMSO) were included as
controls for all experiments. After 48 h treatment, Hoechst 33258
(SigmaeAldrich) staining was added to the medium at a concentration of
0.5 mg/ml. After incubation for 30 min at 37 °C, cells from each dish were
captured from randomly selected fields under fluorescent microscope
(Olympus microscope) to qualitatively determine the proportion of viable
and apoptotic cells based on their relative fluorescence and nuclear
fragmentation.[52]
Cell cycle analysis
Cell cycle analysis Flow cytometric analysis (FACS) was performed to
evaluate the distribution of the cells through the cell-cycle phases. HeLa
and MCF-7cells were incubated for 48 h with compounds 7a and 9a at
concentrations 25 and 50 nM. Untreated and treated cells were
harvested, washed with phosphate-buffered saline (PBS), fixed in ice-
cold 70% ethanol, and stained with propidium iodide (Sigma–Aldrich).
Cell-cycle analysis was performed by flow cytometry (Becton Dickinson
FACS Caliber instrument) as described earlier.[48]
Mitochondrial membrane potential
6
HeLa and MCF-7(1×10 cells/well) cells were cultured in six-well plates
after treatment with compounds 7a and 9a at 25 and 50 nM
concentrations for 48 h. After 48 h of treatment, cells were collected by
trypsinization and washed with PBS followed by resuspending in JC-1 (5
µg/ml) and incubated at 37 0C for 15 min. Cells were rinsed three times
with medium and suspended in pre warmed medium. The cells were then
subjected to flow cytometric analysis on a flow cytometer (Becton
Dickinson) in the FL1, FL2 channel to detect mitochondrial potential.[53]
Tubulin polymerization assay
A
fluorescence based in vitro tubulin polymerization assay was
performed according to the manufacturer’s protocol (BK011,
Cytoskeleton, Inc.). Briefly, the reaction mixture in a total volume of 10 lL
contained PEM buffer, GTP (l µM) in the presence or absence of test
compounds (final concentration of 3 µM). Tubulin polymerization was
followed by a time dependent increase in fluorescence due to the
Annexin staining assay for apoptosis
6
HeLa and MCF-7(1×10 ) cells were seeded in six-well plates and
allowed to grow overnight. The medium was then replaced with complete
medium containing compounds 7a and 9a at 25 and 50 nM
concentrations. After 48 h of drug treatment, cells from the supernatant
and adherent monolayer cells were harvested by trypsinization, washed
with PBS at 5000 rpm. Then the cells were stained with Annexin VFITC
and propidium iodide using the Annexin-V-FITC apoptosis detection kit
incorporation of
a
fluorescence reporter into microtubules as
polymerization proceeds. Fluorescence emission at 420 nM (excitation
wavelength is 360 nM) was measured by using a Varioscan multimode
plate reader (Thermo scientific Inc.). CA-4 was used as positive control in
each assay. The IC50 value was defined as the drug concentration
required inhibiting 50% of tubulin assembly compared to control. The
reaction mixture for these experiments include: tubulin (3 mg/ml) in PEM
buffer, GTP (1 l µM), in the presence or absence of test compounds at
(
Sigma aldrich). Flow cytometry was performed for this study as
[54]
described earlier.
0
.1, 0.5, 1, 2 and 4 µM concentrations. Polymerization was monitored by
Protein extraction and Western blot analysis of caspase 3
º
[49]
increase in the Fluorescence as mentioned above at 37 C.
After treatment with test compounds 7a and 9a at 25 nM concentrations
for 48 h. Protein was isolated with RIPA (radioimmunoprecipitation
assay) buffer. Protein (50 µg per lane) was applied in 10 % SDS-PAGE
Colchicine competition assay
The test compounds (7a and 9a) of various concentrations 0.1 µM, 0.5
µM, 1 µM, 5 µM and 10 µM were incubated with 3 µM tubulin in the
presence and absence of 3 µM colchicine in 30 µM Tris buffer for 60 min
(
Sodium dodecyl sulfate Polyacrylamide gel electrophoresis). After
electrophoresis, the protein was transferred to a polyvinylidine difluoride
PVDF) membrane (Thermo Scientific Inc.) and blocked with BSA
(
2
0
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201600643
FULL PAPER
(
bovine serum albumin). The membrane was washed with TBST for 5
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incubated with the corresponding horseradish peroxidase labeled
secondary antibody and incubated for another 1 h. Membranes were
washed with TBST three times for 15 min and the protein blots were
visualized with chemiluminescence reagent (Thermo Scientific Inc.).
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A series of (Z)-3,4,5-Trimethoxy styryl Benzene sulfonamides /Sulfonates have been designed, synthesized and
evaluated for their cytotoxicity against the NCI panel of sixty human cancer cell lines, majority of these compounds
exhibited promising cytotoxicity with GI50 values ranging between 18-50 nM. Further studies elucidate the
mechanism of action of these new analogues inhibited the in vitro tubulin polymerization and disorganized the
microtubule assembly in MCF-7 and HeLa cancer cells. The lead compounds 7a and 9a displayed notable in vivo
antitumor activity in HeLa tumor xenograft model. Our studies resulted in the identification of a scaffold that can
target tubulin polymerization having significant potential towards the development of new antitumor drugs.
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