Lead optimization of dual tubulin and Hsp27 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.04.038

Tubulin and heat shock protein 27 (Hsp27) are well-characterized molecular targets for anti-cancer drug development. We previously identified lead compounds that inhibited both Hsp27 and tubulin. These compounds exhibited extensive anti-cancer activities

Full text of DOI:10.1016/j.ejmech.2014.04.038

Accepted Manuscript
Lead optimization of dual tubulin and Hsp27 inhibitors
Bo Zhong, Rati Lama, Daniel G. Kulman, Bibo Li, Ph.D. Bin Su, Ph.D.
PII:
S0223-5234(14)00359-6
10.1016/j.ejmech.2014.04.038
EJMECH 6909
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 December 2013
Revised Date: 13 March 2014
Accepted Date: 11 April 2014
Please cite this article as: B. Zhong, R. Lama, D.G. Kulman, B. Li, B. Su , Lead optimization of
dual tubulin and Hsp27 inhibitors, European Journal of Medicinal Chemistry (2014), doi: 10.1016/
j.ejmech.2014.04.038.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical abstract
MeO
O
MeO
H
H
O
N
O
R
N
O
OMe
OMe
N
N
O S O
R
O S O
I
R= 4-MeO, 4-I
R= Et, PhCH2
Dual tubulin and Hsp27 inhibitor
Tubulin inhibitor

ACCEPTED MANUSCRIPT
Revision EJMECH-D-13-02196
Lead optimization of dual tubulin and Hsp27 inhibitors
a
a
bc
ac
Bo Zhong , Rati Lama , Daniel G. Kulman, Bibo Li *, Bin Su *
a
Department of Chemistry, College of Sciences and Health Professions, Cleveland State University, 2121
Euclid Ave., Cleveland, OH, 44115, USA
b
Department of Biological, Geo. & Env. Sciences, College of Sciences and Health Professions, Cleveland
State University, 2121 Euclid Ave., Cleveland, OH, 44115, USA
Center for Gene Regulation in Health and Disease, College of Sciences & Health Professions,
c
Cleveland State University, 2121 Euclid Ave., Cleveland, OH, 44115, USA
RECEIVED DATE:
TITLE RUNNING HEAD: Dual Tubulin and Hsp27 Inhibitors
*
To whom correspondence should be addressed:
Bin Su, Ph.D.
Bibo Li, Ph.D.
Department of Chemistry,
College of Sciences and Health Professions,
Cleveland State University,
Department of Biological, Geo. & Env. Sciences,
College of Sciences and Health Professions,
Cleveland State University,
2
121 Euclid Ave., Cleveland, OH 44115, USA
2121 Euclid Ave., Cleveland, OH 44115, USA
Phone: 216-687-2444
Fax: 216-687-6972
Phone: 216-687-9219
Fax: 216-687-9298
Email: B.su@csuohio.edu
Email: b.li37@csuohio.edu
1

ACCEPTED MANUSCRIPT
Abstract
Tubulin and heat shock protein 27 (Hsp27) are well-characterized molecular targets for anti-cancer drug
development. We previously identified lead compounds that inhibited both Hsp27 and tubulin. These
compounds exhibited extensive anti-cancer activities against the proliferation of various human cancer
cell lines. In the current study, a systematic ligand based structural optimization led to new analogs that
significantly inhibited the growth of a panel of breast cancer cell lines. Furthermore, the most potent
compounds were examined with tubulin polymerization assay and Hsp27 chaperone activity assay. The
compounds showed potent tubulin polymerization inhibition but no Hsp27 inhibitory effect. The
structural optimization dissected the dual activity and improved the selectivity of the compounds for
tubulin. The results revealed several structural moieties of the lead compounds that are critical for Hsp27
inhibition. The modification of these structural fragments eliminated Hsp27 inhibition, but did not harm
tubulin-targeting effects of the compounds. This result further defined the structure-activity relationship
between the tubulin and Hsp27 effects of these compounds.
Keyword: Tubulin, Hsp27, cancer, lead optimization.
2

ACCEPTED MANUSCRIPT
1
. Introduction
Stress factors including chemotherapies can enhance the expression of heat shock proteins (Hsp) in
mammalian cells [1-3]. Hsp27, a small Hsp with a molecular weight of 27 kDa, promotes the survival of
mammalian cells exposed to anti-cancer agents [4]. The results of several investigations demonstrate that
Hsp27 overexpression leads to the resistance of anti-cancer drugs [3,5]. The cellular protective effects of
Hsp27 rely on its molecular chaperone functions against apoptotic signals to eliminate anti-cancer agents
induced programmed cell death [6-8]. Up-regulation of Hsp27 leads to an over-active chaperone activity,
which contributes to the cancer cell survival under chemotherapeutic agents [1,2]. Increased expression
of Hsp27 has been detected in a number of cancers such as breast cancer, endometrial cancer and
leukemia [1,2,9-13], which supports the hypothesis that Hsp27 is a good target of novel cancer therapy.
Hsp27 inhibitors can decrease the protective function of the protein in cell apoptotic regulation, and
enhance the anti-cancer efficacy of chemotherapy [5,14,15]. In fact, several agents diminishing Hsp27
functions exhibited promising anti-tumor activity, and also enhanced the potency of other anti-cancer
agents in preclinical models of a variety of tumors expressing high levels of Hsp27[9,14,15].
Tubulin-containing structures are important for many cellular functions, including chromosome
segregation during cell division, intracellular transport, development and maintenance of cell shape, cell
motility, and distribution of molecules on cell membrane [16]. Tubulin inhibitors are classical
chemotherapeutic agents for cancer treatment. It has been well documented that tubulin inhibitors cause
cell cycle arrest and induce apoptosis [17-20]. Several preclinical studies demonstrated that Hsp27
inhibition could potentiate the apoptosis-inducing activity of tubulin inhibitors [21]. Compounds
targeting both Hsp27 and tubulin can fuse the two anti-cancer effects into one agent, and create a
synergistic activity by itself. These dual tubulin and Hsp27 inhibitors will generate “one stone two birds”
effect, and may represent a class of new anti-cancer agents.
Previously we developed tubulin inhibitor, Hsp27 inhibitor and dual Hsp27 and tubulin inhibitors
that exhibited potent anti-cancer activity [22]. In addition, the combination of pure tubulin inhibitor with
3

ACCEPTED MANUSCRIPT
Hsp27 inhibitor showed a synergistic effect to inhibit cancer cell proliferation [22], suggesting that the
compounds with dual molecular targets have advantages over agents with single molecular target. It is
desirable to further optimize the dual targeting compounds to generate new analogs with better potency
to inhibit both Hsp27 and tubulin, which will lead to more potent anti-cancer agents. In the current study,
systematic ligand based optimization resulted in compounds that significantly inhibited breast cancer cell
proliferation. Unexpectedly, the Hsp27 inhibitory activity of the compounds was diminished during the
lead optimization. The new derivatives became pure tubulin inhibitors. The results revealed several key
structural elements critical for Hsp27 inhibition, which provides important structural characteristics for
future optimization to improve Hsp27 inhibition.
2
2
. Results and discussions
.1. Lead optimization and summarization of the structure-activity relationship (SAR)
Previously, we have shown that structural modification of the lead compound 1 (Figure 1) by
substitution of 2,5-dimethylbenzyl moiety with 2,5-dimethoxybenzyl group dramatically increased the
inhibition of cell proliferative activity. Several new derivatives inhibited the proliferation of various
cancer cell lines with IC50 values at subnanomolar level[22]. Additionally, replacing methyl sulfonamide
group in these compounds with trifluoromethyl sulfonamide group significantly impaired their
anti-cancer activity, suggesting that the trifluoromethyl sulfonamide group is not well tolerated. Since no
other alternatives to methyl sulfonamide group were investigated in the previous study, we wonder if a
bulkier or more hydrophobic group, such as propyl, phenyl, or benzyl at this position, could be tolerated.
Therefore, in this study we kept the B moiety as 2,5-dimethoxybenzyl group, and systematically changed
the C moiety to several different sulfonamide groups including ethyl-, propyl-, phenyl-, and benzyl
sulfonamide. The acetamide group, which is smaller than the methyl sulfonamide group and shows
different electronic distribution, was also evaluated. Previous studies revealed that the N-methyl group at
the D position is important for the anti-proliferative activity of methyl sulfonamides. Removal of
4

ACCEPTED MANUSCRIPT
N-methyl group significantly decreased their cell growth inhibitory potency[22]. In this study, we further
confirmed this structure-activity relationship on this moiety with this series of compounds. Some analogs
were designed as free –NH moiety and some were designed as –Methyl NH moiety. For all the new
derivatives, moiety A was remained as 4-methoxyphenyl, 3,4-dimethoxyphenyl, 4-bromophenyl, and
4
-iodophenyl group. The previous evaluation of 39 different aryl groups at the A position revealed that
these four substituted aryl groups were superior to other ones [22].
All these new compounds were synthesized according to the previous method (Scheme 1) [22]. They
were examined for potency and selectivity on the growth inhibition of four different breast cancer cells
lines including SKBR-3, MCF-7, MDA-MB-231, and MDA-MB- 468. SKBR-3 cell is human epidermal
growth factor receptor 2 (HER2) positive and estrogen receptor (ER) negative, while MCF-7 cell is
HER2 negative and ER positive. Both MDA-MB-231 and MDA-MB- 468 cells are HER2 and ER
negative. The results are summarized in Table 1. The IC s of cell growth inhibition of the compounds
5
0
ranges from 0.001 µM to 2.44 µM for SKBR-3 cells, 0.001 µM to 186.0 µM for MCF-7cells, 0.1 µM to
8.1 µM for MDA-MB-231cells, and 0.008 µM to 1.010 µM for MDA-MB-468 cells. Most compounds
4
exhibited more potent growth inhibition to SKBR-3 and MDA-MB- 468 cells than MCF-7 and
MDA-MB-231cells. For SKBR-3 cells, SAR analysis indicated that the sulfonamide group at the C
moiety of these compounds is critical for the biological activity. The previously synthesized compounds
N-methylmethanesulfonamide 2 -5 showed potent anti-proliferative activity for SKBR-3 cells with IC50
values about 0.001-0.004 µM [22]. However N-methylethanesulfonamides (6, 14, 16),
N-methyl-propylsulfoylamides (7, 11, 15, 17), N-methyl-benzylsulfoylamide (8), and
N-methyl-phenylsulfonamides (9, 13) showed decreased activity compared to the corresponding
N-methylmethanesulfonamide
(2-5).
Only
N-methylethanesulfonamide
10
and
N-methylbenzylsulfonamide 12 maintained the potent SKBR-3 cell growth inhibition activity with IC s
50
of 0.001 µM and 0.003 µM respectively. The general order of potency of various N-methyl alkyl/aryl
5

ACCEPTED MANUSCRIPT
sulfonamides for inhibition of SKBR-3 cell growth is methanesulfonamides > ethanesulfonamides > =
propylsulfonamides > benzylsulfoylamides > phenylsulfonamides. The anti-proliferative activity of the
compounds against SKBR-3 cells seems to be inversely proportional to the size of alkyl/aryl sulfonamide
at the
C
moiety. Previous study showed that removal of the N-methyl group in
N-methylmethanesulfonamides significantly impairs their biological activity[22]. Similar phenomenon
was observed for most N-methyl alkyl/aryl sulfonamides evaluated in the current study. Removal of
N-methyl group in N-methylethanesulfonamides (10, 14, 16), N-methyl-propylsulfonamides (7, 11, 15,
1
7), N-methyl-benzylsulfonamide (8, 12), and N-methyl-phenylsulfonamides (9, 13), generated the
corresponding ethanesulfonamides (22, 26, 28), propylsulfonamides (19, 23, 27, 29), benzylsulfonamide
20, 24), and phenylsulfonamides (21, 25) with 2-43 fold loss in potency. The results confirmed that the
(
free -NH moiety of these compounds decrease the anti-cancer activity. Free-NH can act as a hydrogen
bond donor or acceptor, whereas methyl-NR can only act as hydrogen bond acceptor. The hydrogen bond
formation on this moiety may be critical for the biological activity of the corresponding compounds. A
sole hydrogen bond acceptor on this moiety benefits the anti-cancer activity based on the cell
proliferation assay results. Ethanesulfonamide 18 is exceptionally as potent as N-methyl
ethanesulfonamide 6. Four acetamide derivatives 30-33 exhibited anti-proliferative activity with IC50s in
the range of 0.110 µM and 2.44 µM. All of them are much less potent than corresponding
N-methyl-alkyl/arylsulfonamides (6-17) and alkyl/arylsulfonamides (18-23, 26-29) with the exception of
acetamide 33. This compound is as potent as the corresponding benzylsulfonamide 24 and 3-fold more
potent than phenylsulfonamide 25. The results suggest that sulfonamide group is preferred over
acetamide group for the anti-cancer activity.
A similar trend was observed in the SAR studies for MDA-MB-468, MDA-MB-231, and MCF-7
cells. For the inhibition of MDA-MB-468 cell growth, the most potent compounds were
N-methyl-benzylsulfonamide 8 and N-methyl-propylsulfonamide 11 with same IC s of 0.008 µM. Two
5
0
6

ACCEPTED MANUSCRIPT
potent compounds for growth inhibition of MCF-7 cells were N-methylethanesulfonamide 10 and
propylsulfonamide 23 with IC of 0.015 µM and 0.050 µM. Unexpectedly, N-methylethanesulfonamide
5
0
6
inhibited MCF-7 cell growth with an IC50 of 0.001µM, which is superior to all other compounds. The
result suggested that the compound may interfere with some unknown cell growth pathways to block cell
proliferation besides the identified tubulin and Hsp27 pathways. Most of the other compounds showed
activity at submicromolar levels for MCF-7 cells, and are even much less potent for inhibition of
MDA-MB-231 cell growth.
2
.2. Inhibition of tubulin polymerization
Previous studies revealed that 2, 5-dimethoxybenzyl group can enhance the tubulin polymerization
inhibitory potency of the compounds, thereby substantially improve their anti-proliferative activity. In the
current study, this critical group was remained and various alkyl/arylsulfonamides were synthesized and
evaluated by substitution of other moieties. Among these new analogs, N-methylethanesulfonamide 10
and N-methylbenzylsulfonamide 12 displayed potent cytotoxicity against SKBR-3 cells with IC s of
5
0
0
.001 µM and 0.003 µM, respectively. This potency is comparable to that of the corresponding
N-methylmethanesulfonamide 5. However, the two compounds have different sulfonamide moieties.
Therefore, it is necessary to elucidate whether the compounds act through similar anti-cancer
mechanisms compared to the lead compounds. First, their tubulin inhibitory activity was determined with
tubulin polymerization assay. Both compounds 10 and 12 showed dose-dependent inhibition of tubulin
polymerization (Figure 2). In 15 min, compound 10 inhibited tubulin polymerization by 9% at 0.1µM
and 59% at 1µM. Compound 12 displayed a similar tubulin polymerization inhibitory activity by 5% at
0
.1 µM and 56% at 1µM. These inhibitory activities of compounds 10 and 12 are comparable to that of
N-methylmethanesulfonamide 5 from the previous study[22].
7

ACCEPTED MANUSCRIPT
2
.3. Tubulin docking studies
In the previous study, lead compound 1 was found to be a colchicine site binder on tubulin[23]. We
anticipate the new derivatives 10 and 12 should also target the colchicine-binding site in tubulin, due to
their structural similarity to compound 1. To further elucidate the interaction between tubulin and
compounds 10 and 12, we performed a docking study of these two compounds into the
colchicine-binding site in tubulin. The results are shown in Figure 4. Compound 10 may form three
major interactions with tubulin. First, 2-methoxyl group in 2,5-dimethoxy benzyl moiety may form a
hydrogen bond with the side chain of Ser 178. There may be some polar contact between the sulfonamide
group and side chains such as Glu 183 and Asn 101. Additionally, the carbonyl group of benzoyl amide
moiety may form a hydrogen bond with the main chain of Ala 250. The docking investigation showed
that compound 10 has strong binding affinity to the colchicine-binding site of tubulin. Compared to
compound 10, compound 12 showed a different binding mode in the colchicine-binding site with
sulfonamide moiety and benzoyl amide moiety switched. The benzyl group in the sulfonamide moiety
can extend into a small hydrophobic pocket defined by Tyr 202, Ile 378, Leu 242, and Leu 255. A
hydrogen bond may be formed between the carbonyl group of benzoyl amide moiety and the side chain
of Asn 258. The 2,5-dimethoxybenzyl moiety in compound 12 slightly shifted away from the side chain
of Ser 178, and the possible hydrogen bond between them was forfeited. However, a hydrophobic
interaction may form between 2,5-dimethoxybenzyl moiety and Lys 352. The estimated free energies for
compound 10 and 12 for binding at the colchicine site were determined to be -7.57 and -7.86 kcal/mol,
respectively. Their similar binding energy in the colchicine-binding site may account for their similar
inhibition to tubulin polymerization.
2
.4. Hsp27 targeting effect investigation
8

ACCEPTED MANUSCRIPT
Previously, lead compound 5 inhibited both Hsp27 and tubulin [22]. The newly synthesized
derivatives 10 and 12 inhibited tubulin and showed potent anti-proliferative activity. Whether they also
target Hsp27 is still unknown. In order to further elucidate their anti-cancer mechanism, the capabilities
of both compounds to modulate the in vitro chaperone function of Hsp27 were evaluated. By monitoring
dithiothreitol (DTT)-induced insulin aggregation in the presence of Hsp27, with or without the
compounds, their Hsp27 inhibition can be examined. In this in vitro chaperone activity assays, Hsp27
exhibited potent inhibition of DTT-induced insulin aggregation. Previous study showed that the
corresponding N-methylmethanesulfonamide 5 at 10 µM inhibited Hsp27 functions by 27 % [22].
However, both compounds 10 and 12 did not show inhibitory activity against Hsp27 chaperone activity
at 10 µM, suggesting Hsp27 targeting effect decreased in the new compounds. Substitution of the
methanesulfonamide at the C moiety of compound 5 with ethanesulfonamide or benzylsulfonamide is
detrimental for its Hsp27 targeting effect. However, this modification did not affect tubulin targeting
effects. The results suggest that smaller sulfonamide moiety is preferred for Hsp27 inhibition.
3
. Conclusion
We synthesized various sulfonamide derivatives and acetamide derivatives based on the previously
reported compounds 2-5 [22]. The 2,5-dimethoxybenzyl group, which had been demonstrated to be
important for the anti-proliferative activity of these compounds, was maintained for all the new
compounds. The methanesulfonamide group at the C moiety was changed to an acetamide group or a
diversity of alkyl/aryl sulfonamide groups. The SAR study revealed that most ethyl-, propyl-, phenyl-,
benzyl-sulfonamides showed weaker cell growth inhibition, compared to the corresponding
methanesulfonamides. Only N-methylethanesulfonamide 10 and N-methylbenzylsulfonamide 12
maintained similar potency. Further mechanism investigation indicated that compounds 10 and 12 are
potent inhibitors of tubulin polymerization. Their tubulin inhibitory activities are comparable to the
9

ACCEPTED MANUSCRIPT
corresponding lead compound N-methylmethanesulfonamide 5. However, both compounds did not show
Hsp27 inhibition. The substitution of methanesulfonamide with ethanesulfonamide or benzylsulfonamide
significantly impaired the Hsp27 inhibitory effects. The molecular docking simulation suggested that
compounds 10 and 12 may adopt different binding modes to be accommodated in the colchicine binding
site of tubulin. Future study will focus on discerning the structural fragments that are important for
Hsp27 inhibition, and develop new anti-cancer agents with better potency to target both tubulin and
Hsp27.
4
4
. Experimental section
.1. Chemistry
Chemicals were commercially available and used as received without further purification. Moisture
sensitive reactions were carried out under a dry argon atmosphere in flame-dried glassware. Thin-layer
chromatography was performed on silica gel TLC plates with fluorescence indicator 254 nm (Fluka).
Flash column chromatography was performed using silica gel 60Å (BDH, 40-63 µM). Mass spectra were
obtained on the ABI QStar Electrospray mass spectrometer at Cleveland State University MS facility
1
3
Center. All the NMR spectra were recorded on a Bruker 400 MHz ( C NMR at 100 MHz) using
1
DMSO-d
6
as solvent. Chemical shifts (δ) for H NMR spectra were reported in parts per million to
residual solvent protons. The IR spectra were obtained on a Bruker ALPHA FT-IR spectrometer with
ATR module.
All the compounds were prepared according to the previously published procedures[22].
N-[3-(2,5-dimethoxy-benzyloxy)-4-(ethanesulfonyl-methyl-amino)-phenyl]-4-methoxybenzamide
1
(
6): H NMR δ 10.200 (1H, s), 7.972 (2H, d, J= 8.8 Hz), 7.732 (1H, d, J= 2.4 Hz), 7.415 (1H, dd, J= 2.4,
8
.4 Hz), 7.250 (1H, d, J= 8.8 Hz), 7.156 (1H, d, J= 2.8 Hz), 7.081 (2H, d, J= 9.2 Hz), 7.001 (1H, d, J=
1
0

ACCEPTED MANUSCRIPT
9
.2 Hz), 6.905 (1H, dd, J= 2.8, 8.8 Hz), 5.083 (2H, s), 3.848 (3H, s), 3.785 (3H, s), 3.717 (3H, s), 3.136
1
3
(
3H, s), 3.056 (2H, q, J= 7.6 Hz), 1.114 (3H, t, J= 7.6 Hz); C NMR δ 165.43, 162.52, 155.62, 153.57,
1
51.37, 140.94, 131.79, 130.10, 127.20, 125.53, 124.70, 115.45, 114.39, 114.12, 112.68, 112.28, 105.58,
+
6
5.45, 56.27, 55.93, 55.88, 46.09, 38.36, 8.34; ESI-MS calculated for C H N O S [M+H] 515.19,
2
6
31
2
7
found: 514.98
N-[3-(2,5-dimethoxy-benzyloxy)-4-(propanesulfonyl-methyl-amino)-phenyl]-4-methoxybenzamide
1
(
7): H NMR δ 10.200 (1H, s), 7.972 (2H, d, J= 9.2 Hz), 7.732 (1H, d, J= 2 Hz), 7.416 (1H, dd, J= 2,
8
9
.4 Hz), 7.246 (1H, d, J= 8.4 Hz), 7.148 (1H, d, J= 3.2 Hz), 7.081 (2H, d, J= 9.2 Hz), 7.006 (1H, d, J=
.2 Hz), 6.908 (1H, dd, J= 3.2, 9.2 Hz), 5.080 (2H, s), 3.847 (3H,s ), 3.787 (3H, s), 3.715 (3H, s), 3.134
1
3
(
3H, s), 2.984 (2H, m ), 1.624 (2H, m), 0.812 (3H,t, J= 7.2 Hz); C NMR δ 165.43, 162.52, 155.61,
53.59, 151.37, 140.91, 131.79, 130.10, 127.20, 125.55, 124.77, 115.51, 114.31, 114.12, 112.69,
12.36,105.63, 65.45, 56.32, 55.93, 55.87, 53.09, 38.15, 17.21, 13.13; ESI-MS calculated for
1
1
+
C H N O S [M+H] 529.20, found: 529.01
27
33
2
7
N-[3-(2,5-dimethoxy-benzyloxy)-4-(benzylsulfonyl-methyl-amino)-phenyl]-4-methoxybenzamide
1
(
(
(
(
8): H NMR δ 10.191 (1H, s), 7.971 (2H, d, J= 8.8 Hz), 7.722 (1H, d, J= 2.4 Hz), 7.354 (6H, m), 7.204
1H, d, J= 2.8 Hz), 7.081 (2H, d, J= 9.2 Hz), 6.991 (1H, d, J= 8.8 Hz), 6.961 (1H, d, J= 9.2 Hz), 6.867
1H, dd, J= 2.8, 8.8 Hz), 5.115 (2H, s), 4.415 (2H, s), 3.848 (3H, s), 3.741 (3H, s), 3.680 (3H, s), 3.074
1
3
3H, s); C NMR δ 165.43, 162.53, 155.70, 153.64, 151.21, 140.89, 131.40, 131.34, 130.38, 130.10,
1
5
28.74, 128.53, 127.20, 125.61, 125.03, 115.08, 114.43, 114.12, 112.63, 112.28, 105.59, 65.45, 57.21,
+
6.29, 55.93, 55.84, 38.46; ESI-MS calculated for C H N O S [M+H] 577.20, found 576.98
31
33
2
7
N-[3-(2,5-dimethoxy-benzyloxy)-4-(phenylsulfonyl-methyl-amino)-phenyl]-4-methoxybenzamide
11

ACCEPTED MANUSCRIPT
1
(
9): H NMR δ 10.192 (1H, s), 7.958 (2H, d, J= 8.8 Hz), 7.624 (3H, m), 7.539 (1H, m), 7.441 (3H, m),
7
3
1
6
.074 (3H, m), 6.946 (1H, d, J= 8.8 Hz), 6.834 (2H, m), 4.802 (2H, s), 3.845 (3H, s), 3.769 (3H, s),
1
3
.714 (3H, s), 3.146 (3H, s); C NMR δ 165.46, 162.52, 155.65, 153.54, 150.67, 141.07, 139.40, 133.01,
31.05, 130.09, 129.33, 127.45, 127.20, 125.61, 124.38, 114.31, 114.11, 113.67, 112.51, 112.01, 105.66,
+
5.00, 56.21, 55.92, 55.84, 38.24; ESI-MS calculated for C H N O S [M+H] 563.19, found: 562.96
3
0
31
2
7
N-[3-(2,5-dimethoxy-benzyloxy)- 4-(ethanesulfonyl-methyl-amino)-phenyl]-4-iodobenzamide (10):
1
IR ν= 1672 (C=O); H NMR δ 10.398 (1H, s), 7.943 (2H, d, J= 8.4 Hz), 7.754 (2H, d, J= 8.4 Hz), 7.703
(
1H, d, J= 2 Hz), 7.410 (1H, dd, J= 2, 8.8 Hz), 7.267 (1H, d, J=8.8 Hz), 7.148 (1H, d, J= 3.2 Hz), 6.998
1H, d, J= 9.2 Hz), 6.904 (1H, dd, J= 3.2, 8.8 Hz), 5.083 (2H, s), 3.781 (3H, s), 3.714 (3H, s), 3.135 (3H,
(
1
3
s), 3.059 (2H, q, J= 7.2 Hz), 1.114 (3H, t, J= 7.2 Hz); C NMR δ 165.63, 155.64, 153.49, 151.40,
1
6
6
40.32, 137.83, 134.48, 131.82, 130.03, 125.39, 125.20, 115.50, 114.46, 112.96, 112.34, 105.83, 99.80,
+
5.49, 56.27, 55.86, 46.15, 38.30, 8.26; ESI-MS calculated for C H IN O S [M+H] 611.07, found:
2
5
28
2
6
10.88
N-[3-(2,5-dimethoxy-benzyloxy)- 4-(propanesulfonyl-methyl-amino)-phenyl]-4-Iodobenzamide (11):
1
H NMR δ 10.399 (1H, s), 7.944 (2H, d, J= 8.8 Hz), 7.756 (2H, d, J= 8.8 Hz), 7.704 (1H, d, J= 2.4 Hz),
7
6
1
1
5
.412 (1H, dd, J= 2. 8.4 Hz), 7.264 (1H, d, J= 8.8 Hz), 7.140 (1H, d, 3.2 Hz), 7.004 (1H, d, J= 8.8 Hz),
.907 (1H, dd, J= 2.8, 8.8 Hz), 5.080 (2H, s), 3.784 (3H, s), 3.712 (3H, s), 3.133 (3H, s), 2.985 (2H, m),
1
3
.623 (2H, m), 0.812 (3H, t, J= 7.6 Hz); C NMR δ 165.35, 155.64, 153.59, 151.36, 140.47, 137.78,
34.54, 131.86, 130.07, 125.50, 125.18, 115.50, 114.33, 112.84, 112.38, 105.78, 99.94, 65.49, 56.33,
+
5.88, 53.11, 38.14, 17.21, 13.13; ESI-MS calculated for C H IN O S [M+H] 625.09, found 624.87
2
6
30
2
6
N-[3-(2,5-dimethoxy-benzyloxy)-4-(benzylsulfonyl-methyl-amino)-phenyl]-4-iodobenzamide (12):
1
2

ACCEPTED MANUSCRIPT
1
IR ν= 1645 (C=O); H NMR δ 10.391 (1H, s), 7.944 (2H, d, J= 8.4 Hz), 7.754 (2H, d, J= 8.4 Hz),
.692 (1H, d, J= 2.4 Hz), 7.353 (6H, m), 7.196 (1H, d, J= 3.2 Hz), 7.006 (1H, d, J= 8.4 Hz), 6.959 (1H,
d, J= 9.2 Hz), 6.866 (1H, dd, J= 2.8, 8.8 Hz), 5.114 (2H, s), 4.418 (2H, s), 3.738 (3H, s), 3.677 (3H, s),
7
1
3
3
1
5
.072 (3H, s); C NMR δ 165.35, 155.72, 153.64, 151.20, 140.45, 137.79, 134.54, 131.40, 130.36,
30.07, 128.74, 128.54, 125.56, 125.43, 115.05, 114.43, 112.77, 112.29, 105.73, 99.95, 65.48, 57.22,
+
6.30, 55.84, 38.45; ESI-MS calculated for C H IN O S [M+H] 673.09, found 672.84
3
0
30
2
6
N-[3-(2,5-dimethoxy-benzyloxy)-4-(phenylsulfonyl-methyl-amino)-phenyl]-4-iodobenzamide (13):
1
H NMR δ 10.390 (1H, s), 7.937 (2H, d, J= 8.4 Hz), 7.741 (2H, d, J= 8.4 Hz), 7.619 (3H, m), 7.541 (1H,
m), 7.431 (3H, m), 7.094 (1H, d, J= 8.8 Hz), 6.945 (1H, d, J= 8.8 Hz), 6.833 (2H, m), 4.801 (2H, s),
1
3
3
1
1
6
.765 (3H, s), 3.712 (3H, s), 3.146 (3H, s); C NMR δ 165.38, 155.67, 153.53, 150.66, 140.64, 139.36,
37.77, 134.54, 133.04, 131.15, 130.07, 129.35, 127.45, 125.56, 124.77, 114.31, 113.69, 112.64, 112.03,
+
05.79, 99.95, 65.05, 56.22, 55.85, 38.22; ESI-MS calculated for C H IN O S [M+H] 659.07, found:
2
9
28
2
6
58.86
N-[3-(2,5-dimethoxy-benzyloxy)-4-(ethanesulfonyl-methyl-amino)-phenyl]-3,4-dimethoxybenzamid
1
e (14): H NMR δ 10.191 (1H, s), 7.736 (1H, d, J= 2.4 Hz), 7.643 (1H, dd, J= 2.4, 8.4 Hz), 7.534 (1H, d,
J= 2 Hz), 7.398 (1H, dd, J= 2, 8.4 Hz), 7.259 (1H, d, J= 8.4 Hz), 7.159 (1H, d, J= 3.2 Hz), 7.104 (1H, d,
J= 8.4 Hz), 7.002 (1H, d, J= 9.2 Hz), 6.907 (1H, dd, J= 2.8, 8.8 Hz), 5.086 (2H, s), 3.852 (3H, s), 3.847
(3H, s), 3.785 (3H, s), 3.719 (3H, s), 3.138 (3H, s), 3.059 (2H, q, J= 7.6 Hz), 1.115 (3H, t, J= 7.6 Hz);
1
3
C NMR δ 165.47, 155.63, 153.58, 152.32, 151.38, 148.87, 140.88, 131.79, 127.24, 125.53, 124.77,
1
8
21.54, 115.47, 114.41, 112.80, 112.29, 111.69, 111.42, 105.69, 65.47, 56.19, 56.16, 55.88, 46.10, 38.36,
+
.34; ESI-MS calculated for C H N O S [M+H] 545.20, found: 544.98
2
7
33
2
8
N-[3-(2,5-dimethoxy-benzyloxy)-4-(propanesulfonyl-methyl-amino)-phenyl]-3,4-dimethoxybenzami
1
3

ACCEPTED MANUSCRIPT
1
de (15): H NMR δ 10.192 (1H, s), 7.735 (1H, d, J= 2 Hz), 7.644 (1H, dd, J= 2, 8.4 Hz), 7.534 (1H, d,
J= 2 Hz), 7.398 (1H, dd, J= 2, 8.4 Hz), 7.256 (1H, d, J= 8.8 Hz), 7.150 (1H, d, J= 2.8 Hz), 7.104 (1H, d,
J= 8.4 Hz), 7.007 (1H, d, 8.8 Hz), 6.910 (1H, dd, J= 3.2, 8.8 Hz), 5.083 (1H, s), 3.582 (3H, s), 3.847 (3H,
s), 3.787 (3H, s), 3.717 (3H, s), 3.136 (3H, s), 2.979 (2H, m), 1.623 (2H, m), 0.811 (3H, t, J= 7.2 Hz);
1
3
C NMR δ 165.46, 155.62, 153.60, 152.31, 151.37, 148.87, 140.85, 131.77, 127.23, 125.54, 124.88,
1
3
21.53, 115.53, 114.34, 112.80, 112.39, 111.69, 111.41, 105.73, 65.47, 56.33, 56.20, 56.10, 55.88, 53.10,
+
8.15, 17.21, 13.13; ESI-MS calculated for C H N O S [M+H] 559.20, found: 559.02
2
8
35
2
8
N-[3-(2,5-dimethoxy-benzyloxy)- 4-(ethanesulfonyl-methyl-amino)-phenyl]-4-bromobenzamide (16):
1
H NMR δ 10.422 (1H, s), 7.921 (2H, d, J= 8.8 Hz), 7.773 (2H, d, J= 8.8 Hz), 7.706 (1H, d, J= 2 Hz),
7
.412 (1H, dd, J= 2.4, 8.8 Hz), 7.272 (1H, d, J= 8.8 Hz), 7.149 (1H, d, J= 3.2 Hz), 7.000 (1H, d, J= 9.2
Hz), 6.905 (1H, dd, J= 3.2, 9.2 Hz), 5.086 (2H, s), 3.782 (3H, s), 3.714 (3H, s) , 3.137 (3H, s), 3.062 (2H,
1
3
q, J= 7.6 Hz), 1.116 (3H, t, J= 7.6 Hz); C NMR δ 165.09, 155.65, 153.58, 151.37, 140.49, 134.24,
1
4
31.92, 130.27, 126.00, 125.49, 125.14, 115.43, 114.40, 112.83, 112.29, 105.74, 65.49, 56.28, 55.88,
+
6.11, 38.34, 8.34; ESI-MS calculated for C25
H28BrN
2
O
6
S [M+H] 563.09, found: 562.90
N-[3-(2,5-dimethoxy-benzyloxy)- 4-(propanesulfonyl-methyl-amino)-phenyl]-4-bromobenzamide
1
(
17): H NMR δ 10.421 (1H, s), 7.923 (2H, d, J= 8.8 Hz), 7.773 (2H, d, J= 8.8 Hz), 7.708 (1H, d, J= 1.6
Hz), 7.414 (1H, dd, J= 2, 8.8 Hz), 7.270 (1H, d, J= 8.4Hz), 7.142 (1H, d, J= 3.2 Hz), 7.005 (1H, d, J=
.8 Hz), 6.908 (1H, dd, J= 2.8, 8,8 Hz), 5.084 (2H, s), 3.785 (3H, s), 3.714 (3H, s), 3.136 (3H, s), 2.987
8
1
3
(
2H, m), 1.626 (2H, m), 0.814 (3H, t, J= 7.2 Hz); C NMR δ 165.09, 155.64, 153.59, 151.36, 140.46,
1
5
34.24, 131.92, 130.27, 126.00, 125.50, 125.21, 115.50, 114.33, 112.85, 112.38, 105.79, 65.49, 56.33,
+
5.87, 53.11, 38.13, 17.21, 13.13; ESI-MS calculated for C H BrN O S [M+H] 577.10, found 576.92
2
6
30
2
6
1
N-[3-(2,5-dimethoxy-benzyloxy)-4-(ethanesulfonyl-amino)-phenyl]-4-methoxybenzamide (18): H
1
4

ACCEPTED MANUSCRIPT
NMR δ 10.136 (1H, s), 8.916 (1H, s), 7.959 (2H, d, J= 8.8 Hz), 7.680 (1H, d, J= 2 Hz), 7.384 (1H, dd,
J= 2, 8.4 Hz), 7.256 (1H, d, J= 3.2 Hz), 7.215 (1H, d, J= 8.4 Hz), 7.069 (2H, d, J= 8.8 Hz), 6.984 (1H, d,
J= 8.8 Hz), 6.887 (1H, dd, J= 3.2, 8.8 Hz), 5.061 (2H, s), 3.842 (3H, s), 3.792 (3H, s), 3.724 (3H, s),
1
3
2
1
5
.956 (2H, q, J= 7.2 Hz), 1.128 (3H, t, J= 7.2 Hz); C NMR δ 165.31, 162.44, 153.65, 152.90, 151.16,
39.10, 130.04, 128.08, 127.30, 125.72, 121.26, 115.58, 114.09, 113.96, 112.84, 112.09, 105.63, 65.49,
+
6.29, 55.93, 55.91, 46.75, 8.46; ESI-MS calculated for C H N O S [M+H] : 501.17, found: 500.96
2
5
29
2
7
1
N-[3-(2,5-dimethoxy-benzyloxy)-4-(propanesulfonyl-amino)-phenyl]-4-methoxybenzamide (19): H
NMR δ10.137 (1H, s), 8.941 (1H, s), 7.960 (2H, d, J= 9.2 Hz), 7.678 (1H, d, J= 2 Hz), 7.390 (1H, dd,
J= 2, 8.8 Hz), 7.270 (1H, d, J= 3.2 Hz), 7.213 (1H, d, J= 8.8 Hz), 7.070 (2H, d, J= 9.2 Hz), 6.986 91H,
d, J= 9.2 Hz), 6.886 (1H, dd, J= 3.2, 9.2 Hz), 5.068 (2H, s), 3.843 (3H, s), 3.797 (3H, s), 3.728 (3H, s),
13
2
.918 (2H, m), 1.654 (2H, m), 0.790 (3H, t, J= 7.2 Hz); C NMR δ 165.31, 162.44, 153.70, 152.82,
1
51.07, 139.04, 130.04, 127.96, 127.30, 125.80, 121.34, 115.47, 114.09, 113.82, 112.88, 112.17, 105.70,
+
6
5.42, 56.37, 55.95, 55.92, 54.12, 17.33, 13.01; ESI-MS calculated for C H N O S [M+H] 515.19,
26
31
2
7
found: 514.99
1
N-[3-(2,5-dimethoxy-benzyloxy)-4-(benzylsulfonyl-amino)-phenyl]-4-methoxybenzamide (20): H
NMR δ 10.149 (1H, s), 8.993 (1H, s), 7.970 (2H, d, J= 8.8 Hz), 7.709 (1H, d, J= 2 Hz), 7.384 (1H, dd,
J= 2, 8.4 Hz), 7.304 (6H, m), 7.150 (1H, d, J= 8.8 Hz), 7.075 (2H, d, J= 8.8Hz), 6.949 (1H, d, J= 9.2
Hz), 6.865 (1H, dd, J= 2.8, 8.8 Hz), 5.103 (2H, s), 4.309 (2H, s), 3.846 (3H, s), 3.740 (3H, s), 3.697 (3H,
13
s); C NMR δ 165.32, 162.45, 153.67, 152.79, 151.16, 139.01, 131.29, 130.33, 130.05, 128.69, 128.46,
1
27.62, 127.31, 125.73, 121.38, 115.54, 114.10, 112.83, 112.14, 105.69, 65.55, 58.68, 56.29, 55.92;
+
ESI-MS calculated for C H N O S [M+H] 563.19, found: 562.97
3
0
31
2
7
1
N-[3-(2,5-dimethoxy-benzyloxy)-4-(phenylsulfonyl-amino)-phenyl]-4-methoxybenzamide (21): H
1
5

ACCEPTED MANUSCRIPT
NMR δ 10.082 (1H, s ), 9.551 (1H, s), 7.925 (2H, d, J= 8.8 Hz), 7.611 (2H, m), 7.473 (2H, m), 7.345
(3H, m), 7.155 (1H, d, J= 8.4 Hz), 7.099 (1H, d, J= 3.2 Hz), 7.051 (2H, d, J= 9.2 Hz), 6.943 (1H, d, J=
13
8
.8 Hz), 6.853 (1H, dd, J= 3.2, 8.8 Hz), 4.692 (2H, s), 3.833 (3H, s), 3.775 (3H, s), 3.758 (3H, s); C
NMR δ 165.30, 162.43, 153.72, 152.75, 150.48, 140.95, 139.15, 132.73, 130.02, 129.08, 127.41, 127.27,
1
27.00, 125.96, 120.82, 114.83, 114.07, 113.12, 112.78, 111.76, 105.65, 65.14, 56.25, 56.00, 55.89;
+
ESI-MS
calculated
for
C H N O S
[M+H]
549.17,
found:
548.95
2
9
29
2
7
1
N-[3-(2,5-dimethoxy-benzyloxy)-4-(ethanesulfonyl-amino)-phenyl]-4-iodobenzamide (22): H NMR
δ 10.336 (1H, s), 8.944 (1H, s), 7.931 (2H, d, J= 8.8 Hz), 7.744 (2H, d, J= 8.4 Hz), 7.657 (1H, d, J= 2.4
Hz), 7.383 (1H, dd, J= 2, 8.4 Hz), 7.245 (2H, m), 6.982 (1H, d, J= 8.8 Hz), 6.886 (1H, dd, J= 3.2, 9.2
13
Hz), 5.063 (2H, s), 3.789 (3H, s), 3.721 (3H, s), 2.962 (2H, q, J=7.2 Hz), 1.131 (3H, t, J= 7.2 Hz); C
NMR δ 165.22, 153.64, 152.85, 151.15, 138.62, 137.75, 134.63, 130.05, 128.02, 125.67, 121.69, 115.56,
113.96, 112.97, 112.10, 105.74, 99.81, 65.52, 56.30, 55.93, 46.78, 8.46; ESI-MS calculated for
+
C H IN O S [M+H] : 597.06, found: 596.85
24
26
2
6
1
N-[3-(2,5-dimethoxy-benzyloxy)-4-(propanesulfonyl-amino)-phenyl]-4-iodobenzamide (23):
H
NMR δ 10.335 (1H, s), 8.972 (1H, s), 7.931 (2H, d, J= 8.8 Hz), 7.745 (2H, d, J= 8.8 Hz), 7.655 (1H, d,
J= 2.4 Hz), 7.389 (1H, dd, J= 2, 8.4 Hz), 7.262 (1H, d, J= 3.2 Hz), 7.231 (1H, d, J= 8.4 Hz), 6.984 (1H,
d, J= 9.2 Hz), 6.885 (1H, dd, J= 3.2, 9.2 Hz), 5.070 (2H, s), 3.793 (3H, s), 3.725 (3H, s), 2.924 (2H, m),
1
3
1
1
5
.655 (2H, m), 0.792 (3H, t, J= 7.6 Hz); C NMR δ 165.22, 153.69, 152.77, 151.06, 138.57, 137.75,
34.63, 130.05, 127.89, 125.74, 121.77, 115.45, 113.83, 113.00, 112.16, 105.80, 99.81, 65.46, 56.36,
+
5.95, 54.14, 17.33, 13.01; ESI-MS calculated for C H IN O S [M+H] 611.07, found 610.88
25
28
2
6
1
N-[3-(2,5-dimethoxy-benzyloxy)-4-(benzylsulfonyl-amino)-phenyl]-4-iodobenzamide (24): H NMR
1
6

ACCEPTED MANUSCRIPT
δ 10.349 (1H, s), 9.018 (1H, s), 7.937 (2H, d, J= 8.4 Hz), 7.755 (2H, d, J= 8.4 Hz), 7.684 (1H, d, J= 2
Hz), 7.382 (1H, dd, J= 2, 8.4 Hz), 7.304 (6H, m), 7.164 (1H, d, J= 8.4 Hz), 6.948 (1H, d, J= 9.2 Hz),
1
3
6
1
1
.864 (1H, dd, J= 2.8, 9.2 Hz), 5.104 (2H, s), 4.317 (2H, s), 3.738 (3H, s), 3.694 (3H, s); C NMR δ
65.22, 153.67, 152.71, 151.16, 138.51, 137.76, 134.65, 131.29, 130.30, 130.05, 128.70, 128.47, 127.50,
25.68, 121.82, 115.53, 114.07, 112.96, 112.14, 105.81, 99.82, 65.59, 58.70, 56.30, 55.91; ESI-MS
+
calculated for C H IN O S [M+H] 659.07, found: 658.84
2
9
28
2
6
1
N-[3-(2,5-dimethoxy-benzyloxy)-4-(phenylsulfonyl-amino)-phenyl]-4-iodobenzamide (25): H NMR
δ 10.278 (1H, s), 9.579 (1H, s), 7.911 (2H, d, J= 8.4 Hz), 7.709 (2H, d, J= 8.8 Hz), 7.614 (2H, m), 7.464
(2H, m), 7.339 (3H, m), 7.173 (1H, d, J= 8.8 Hz), 7.086 (1H, d, J= 3.2 Hz), 6.941 (1H, d, J= 9.2 Hz),
1
3
6
1
.852 (1H, dd, J= 3.2, 9.2 Hz), 4.696 (2H, s), 3.770 (3H, s), 3.753 (3H, s); C NMR δ 165.20, 153.71,
52.70, 150.48, 140.92, 138.68, 137.72, 134.61, 132.76, 130.02, 129.11, 127.35, 127.00, 125.90, 121.23,
1
14.82, 113.14, 112.89, 111.78, 105.75, 99.80, 65.17, 56.27, 56.00; ESI-MS calculated for C H IN O S
28 26 2 6
+
[
M+H] 645.06, found 644.83
N-[3-(2,5-dimethoxy-benzyloxy)- 4-(ethanesulfonyl-amino)-phenyl]-3,4-dimethoxybenzamide (26):
1
H NMR δ 10.129 (1H, s), 8.922 (1H, s), 7.682 (1H, d, J= 2.4 Hz), 7.628 (1H, dd, J= 2.4, 8.4 Hz), 7.528
(1H, d, J= 2.4 Hz), 7.366 (1H, dd, J= 2, 8.4 Hz), 7.260 (1H, d, J= 2.8 Hz), 7.227 (1H, d, J= 8.8 Hz),
7
.092 (1H, d, J= 8.4 Hz), 6.985 (1H, d, J= 9.2 Hz), 6.889 (1H, dd, J= 3.2, 8.8 Hz), 5.065 (2H, s), 3.847
(3H, s), 3.842 (3H, s), 3.792 (3H, s), 3.727 (3H, s), 2.962 (2H, q, J= 7.2 Hz), 1.131 (3H, t, J= 7.2 Hz);
1
3
C NMR δ 165.36, 153.64, 152.91, 152.22, 151.16, 148.83, 139.03, 128.08, 127.33, 125.70, 121.48,
1
8
21.32, 115.61, 113.95, 112.99, 112.08, 111.63, 111.39, 105.73, 65.49, 56.28, 56.17, 56.15, 55.92, 46.76,
+
.46; ESI-MS calculated for C H N O S [M+H] : 531.18, found: 530.97
2
6
31
2
8
N-[3-(2,5-dimethoxy-benzyloxy)-4-(propanesulfonyl-amino)-phenyl]-3,4-dimethoxybenzamide (27):
1
7

ACCEPTED MANUSCRIPT
1
H NMR δ 10.128 (1H, s), 8.950 (1H, s), 7.678 (1H, d, J= 2.4 Hz), 7.627 (1H, dd, J= 2, 8.4 Hz), 7.526
1H, d, J= 2 Hz), 7.370 (1H, dd, J= 2, 8.8 Hz), 7.273 (1H, d, J= 2.8 Hz), 7.223 (1H, d, J= 8.8 Hz), 7.092
1H, d, J= 8.8 Hz), 6.987 (1H. d, J= 9.2 Hz), 6.888 (1H, d, J= 2.8, 9.2 Hz), 5.071 (2H, s), 3.847 (3H, s),
(
(
1
3
3
.842 (3H, s), 3.797 (3H, s), 3.730 (3H, s), 2.922 (2H, m), 1.654 (2H, m), 0.790 (3H, t, J=7.2 Hz); C
NMR δ 165.35, 153.70, 152.82, 152.22, 151.07, 148.83, 138.97, 127.94, 127.33, 125.78, 121.47, 121.41,
15.50, 113.82, 113.02, 112.15, 111.63, 111.40, 105.81, 65.44, 56.35, 56.17, 56.15, 55.94, 54.13, 17.34,
1
+
1
3.00; ESI-MS calculated for C H N O S [M+H] 545.20, found: 544.98
27 33 2 8
1
N-[3-(2,5-dimethoxy-benzyloxy)-4-(ethanesulfonyl-amino)-phenyl]-4-bromobenzamide (28):
H
NMR δ 10.358 (1H, s), 8.947 (1H, s), 7.909 (2H, d, J= 8.8 Hz), 7.760 (2H, d, J= 8.8 Hz), 7.658 (1H, d,
J= 2 Hz), 7.384 (1H, dd, J= 2, 8.4 Hz), 7.248 (2H, m), 6.983 (1H, d, J= 8.8 Hz), 6.886 (1H, dd, J= 2.8,
1
3
8
.8 Hz), 5.065 (2H, s), 3.789 (3H, s), 3.721 (3H, s), 2.963 (2H, q, J= 7.2 Hz), 1.131 (3H, t, J= 7.2); C
NMR δ 164.95, 153.64, 152.85, 151.15, 138.61, 134.33, 131.88, 130.24, 128.05, 125.89, 125.66, 121.71,
15.55, 113.94, 112.97, 112.08, 105.74, 65.51, 56.28, 55.92, 46.78, 8.47; ESI-MS calculated for
1
+
C
24
H
26BrN
2
O
6
S [M+H] : 549.07, found: 548.86
N-[3-(2,5-dimethoxy-benzyloxy)-4-(propanesulfonyl-amino)-phenyl]-4-bromobenzamide (29):
NMR δ 10.356 (1H, s), 8.973 (1H, s), 7.909 (2H, d, J= 8.8 Hz), 7.760 (2H, d, J= 8.8 Hz), 7.652 (1H, s),
1
H
7
6
0
1
1
.388 (1H, d, J= 8.4 Hz), 7.262 (1H, d, J= 3.2 Hz), 7.234 (1H, d, J= 8.4 Hz), 6.985 (1H, d, J= 8.8 Hz),
.885 (1H, dd, J= 2.8, 8.8 Hz), 5.071 (2H, s), 3.794 (3H, s), 3.725 (3H, s), 2.924 (2H, m), 1.655 (2H, m),
13
.793 (3H, t, J= 7.6 Hz); C NMR δ 164.95, 153.69, 152.78, 151.04, 138.58, 134.33, 131.88, 130.24,
27.95, 125.90, 125.73, 121.78, 115.45, 113.77, 112.99, 112.11, 105.78, 65.44, 56.33, 55.93, 54.13,
+
7.35, 13.01; ESI-MS calculated for C H BrN O S [M+H] 563.09, found: 562.91
2
5
28
2
6
1
8

ACCEPTED MANUSCRIPT
1
N-[3-(2,5-dimethoxy-benzyloxy)-4-acetamido-phenyl]-4-methoxybenzamide (30): H NMR δ 10.063
1H, s), 9.171 (1H, s), 7.950 (2H, d, J = 8.8 Hz), 7.677 (1H, d, J= 8.4 Hz), 7.623 (1H, s), 7.355 (1H, d,
J= 8.8 Hz), 7.141 (1H, d, J= 2.8 Hz), 7.058 (2H, d, J= 8.8 Hz), 6.981 (1H, d, J= 9.2 Hz), 6.859 (1H, dd,
(
1
3
J= 2.8, 8.8 Hz), 5.097 (2H, s), 3.838 (3H, s), 3.805 (3H, s), 3.718 (3H, s), 2.070 (3H, s); C NMR δ
68.63, 165.15, 162.36, 153.68, 150.90, 149.91, 136.92, 129.98, 127.40, 126.16, 123.99, 123.61, 114.50,
14.05, 113.66, 112.74, 112.24, 105.88, 65.44, 56.39, 55.90, 55.82, 24.06; ESI-MS calculated for
1
1
+
C H N O [M+H] 451.19, found: 450.99
25
27
2
6
1
N-[3-(2,5-dimethoxy-benzyloxy)-4-acetamido-phenyl]-3,4-dimethoxybenzamide (31): H NMR δ
1
0.056 (1H, s), 9.172 (1H, s), 7.690 (1H, d, J= 8.8 Hz), 7.606 (2H, m), 7.522 (1H, d, J= 1.6 Hz), 7.327
(
(
(
1H, d, J= 9.2 Hz), 7.142 (1H, d, J= 3.2 Hz), 7.080 (1H, d, J= 8.4 z), 6.982 (1H, d, J= 8.8 Hz), 6.861
1H, dd, J= 2.8, 8.8 Hz), 5.099 (2H, s), 3.842 (3H, s), 3.837 (3H, s), 3.804 (3H, s), 3.720 (3H, s), 2.072
1
3
3H, s); C NMR δ 168.65, 165.21, 153.68, 152.13, 150.91, 149.90, 148.81, 136.85, 127.45, 126.16,
1
5
23.96, 123.70, 121.42, 114.55, 113.65, 112.92, 112.21, 111.61, 111.38, 106.03, 65.48, 56.36, 56.14,
+
5.80, 24.07; ESI-MS calculated for C26
H
29
N
2
O
7
[M+H] 481.20, found 481.01
N-[3-(2,5-dimethoxy-benzyloxy)-4-acetamido-phenyl]-4-bromobenzamide (32):
1
H NMR δ 10.286 (1H, s), 9.192 (1H, s), 7.901 (2H, d, J= 8.4 Hz), 7.748 (2H, d, J= 8.4 Hz), 7.710 (1H,
d, J= 8.8 Hz), 7.600 (1H, d, J= 2 Hz), 7.357 (1H, d, J= 8.4 Hz), 7.133 ( 1H, d, J= 2.8 Hz), 6.981 (1H, d,
1
3
J= 9.2 Hz), 6.860 (1H, dd, J= 3.2, 8.8 Hz), 5.102 (2H, s), 3.803 (3H, s), 3.715 (3H, s), 2.075 (3H, s); C
NMR δ 168.65, 164.77, 153.67, 150.91, 149.85, 136.45, 134.43, 131.84, 130.20, 126.10, 125.76, 123.95,
114.51, 113.67, 112.86, 112.25, 105.96, 99.99, 65.47, 56.39, 55.82, 24.08; ESI-MS calculated for
+
C H BrN O [M+H] 499.09, found: 498.89
24
24
2
5
N-[3-(2,5-dimethoxy-benzyloxy)-4-acetamido-phenyl]-4-iodobenzamide (33):
1
9

ACCEPTED MANUSCRIPT
1
H NMR δ 10.263 (1H, s), 9.188 (1H, s), 7.918 (2H, d, J= 8.8 Hz), 7.730 (3H, m), 7.599 (1H, d, J= 2
Hz), 7.253 (1H, dd, J= 2, 8.8 Hz), 7.131 (1H, d, J= 3.2 Hz), 6.979 (1H, d, J= 8.8 Hz), 6.858 (1H, dd, J=
1
3
3
1
.2, 9.2 Hz), 5.099 (2H, s), 3.801 (3H, s), 3.714 (3H, s), 2.073 (3H, s); C NMR δ 168.66, 165.04,
53.67, 150.91, 149.86, 137.71, 136.45, 134.73, 130.02, 126.11, 123.99, 123.95, 114.51, 113.68, 112.86,
+
1
12.25, 105.95, 99.66, 65.47, 56.40, 55.82, 24.08; ESI-MS calculated for C H IN O [M+H] 547.07,
2
4
24
2
5
found 546.86
4
4
.2. Biological studies
.2.1. Cell culture. Four breast cancer cell lines including SKBR-3, MCF-7, MDA-MB-231, and
MDA-MB- 468 were obtained from ATCC (Rockville, MD). The cells were maintained in RPMI1640
medium supplemented with 10% fetal bovine serum (FBS), 2 mmol/L L-Glutamine, 1 mmol/L sodium
pyruvate, 100 U/mL penicillin-streptomycin. FBS was heat inactivated for 30 min in a 56 ºC water bath
before use. Cell cultures were grown at 37 ºC, in a humidified atmosphere of 5% CO in a VWR CO
2
2
incubator (Bridgeport NJ).
.2.2. Cell viability analysis. The effects of the new derivatives on the viability of four breast cancer cell
4
lines were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H- tetrazolium bromide assay in
six replicates. Cells were grown in RPMI1640 medium in 96-well, flat-bottomed plates for 24 h, and were
exposed to various concentrations of the compounds dissolved in DMSO (final concentration 0.1%) in
media for 48 h. Controls received DMSO vehicle at a concentration equal to that in drug-treated cells. The
medium was removed, replaced by 200 µL of 0.5 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-
tetrazolium bromide in fresh media, and cells were incubated in the CO incubator at 37°C for 2 h.
2
Supernatants were removed from the wells, and the reduced 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
2
H-tetrazolium bromide dye was solubilized in 200 µL/well DMSO. Absorbance at 570 nm was
2
0

ACCEPTED MANUSCRIPT
determined on a plate reader. Statistical and graphical information was determined using GraphPad Prism
software (GraphPad Software Incorporated) and Microsoft Excel (Microsoft Corporation). IC50 values
were determined using nonlinear regression analysis.
4
.2.3. Tubulin polymerization assay. 100 µL microtubule-associated protein-rich tubulin (2 mg/mL,
bovine brain, Cytoskeleton) in buffer containing 80 mM PIPES (pH 6.9), 2 mM MgCl , 0.5 mM EGTA,
2
and 5% glycerol was mixed with DMSO (as control) or various concentrations of 10 and 12 in DMSO
and incubated at 37 ºC. 1 µL of 100 mM GTP was added to the mixture to initiate the tubulin
polymerization. The absorbance was monitored at 340 nm over 20 min using a Varian Cary 50 series
spectrophotometer.
4
.2.4. Hsp27 chaperone activity assay. A mixture of 0.24 mg/ml human recombinant insulin (Life
Technologies), 0.02 mg/ml Hsp27 (Cell Sciences), and compounds 10 and 12 in 98 µl sodium phosphate
buffer, pH 7.4, was incubated at 37 ºC for 5min, whereupon 2 µL of 1 M DTT in the same assay buffer
was added to initiate the insulin aggregation. The absorbance was monitored at 400 nm over 30 min
using a Varian Cary 50 series spectrophotometer. A mixture of 0.24 mg/ml insulin in the absence or
presence of 0.02mg/ml Hsp27 with DMSO was used as control.
5
. Molecular docking simulation. Molecular docking of compounds 10 and 12 into the crystal structure
of tubulin was performed using software AUTODOCK 4. The X-ray crystal structures of tubulin with
PDB code 1SA0 from Protein Data Bank was used for docking simulation of compounds into the
colchicine binding site. To prepare tubulin for autodock, all hydrogens were added and the ligand with
identifier CN2700 in 1SA0 was deleted. Compound 10 and 12 were drawn and energy minimized with
MM2 force field using Chem3D Ultra 10.0 (Cambridge Soft Corp., US). Autogrid was used to
pre-calculate the grid maps of the binding energy between tubulin and compounds. A grid box size of 44
2
1

ACCEPTED MANUSCRIPT
×
46 ×42 points in x, y and z directions was built and the grid center was located in x=116.909, y= 89.688,
z=7.094. For running autodock, Lamarckian genetic algorithm was chosen as the search method. Genetic
algorithm parameters were set as default. The top docked conformation for each compound was retained
for analysis.
6
. Acknowledgements
This research was supported by a startup fund from Cleveland State University, and also partially
supported by grant R15AI 103889 (B. Su), Center for Gene Regulation in Health and Disease (GRHD)
of Cleveland State University and Ohio Department of Development (ODOD) and National Science
Foundation Major Research Instrumentation Grants (CHE-0923398 and CHE-1126384).
7
. Supporting Information
HPLC analysis for compounds 6–33; IR spectra for compounds 10 and 12; H NMR and C NMR for
1
13
compounds 6-33
Abbreviations: heat shock protein 27 (Hsp27), structure-activity relationship (SAR), human epidermal
growth factor receptor 2 (Her2), fetal bovine serum (FBS), dithiothreitol (DTT).
2
2

ACCEPTED MANUSCRIPT
References
[
1] H.J. Arts, H. Hollema, W. Lemstra, P.H. Willemse, E.G. De Vries, H.H. Kampinga, A.G. Van der Zee,
Heat-shock-protein-27 (hsp27) expression in ovarian carcinoma: relation in response to
chemotherapy and prognosis, Int. J. Cancer. 84 (1999) 234-238.
[
2] C. Garrido, P. Mehlen, A. Fromentin, A. Hammann, M. Assem, A.P. Arrigo, B. Chauffert, Inconstant
association between 27-kDa heat-shock protein (Hsp27) content and doxorubicin resistance in human
colon cancer cells. The doxorubicin-protecting effect of Hsp27, Eur. J. Biochem. 237 (1996) 653-659.
3] H.S. Hsu, J.H. Lin, W.C. Huang, T.W. Hsu, K. Su, S.H. Chiou, Y.T. Tsai, S.C. Hung, Chemoresistance
of lung cancer stemlike cells depends on activation of Hsp27, Cancer. 117 (2011) 1516-1528.
4] C. Garrido, M. Brunet, C. Didelot, Y. Zermati, E. Schmitt, G. Kroemer, Heat shock proteins 27 and 70:
anti-apoptotic proteins with tumorigenic properties, Cell. Cycle. 5 (2006) 2592-2601.
5] M. Kamada, A. So, M. Muramaki, P. Rocchi, E. Beraldi, M. Gleave, Hsp27 knockdown using
nucleotide-based therapies inhibit tumor growth and enhance chemotherapy in human bladder cancer
cells, Mol. Cancer. Ther. 6 (2007) 299-308.
[
[
[
[
[
[
[
6] C. Garrido, E. Solary, A role of HSPs in apoptosis through "protein triage"? Cell Death Differ. 10
(2003) 619-620.
7] C. Garrido, J.M. Bruey, A. Fromentin, A. Hammann, A.P. Arrigo, E. Solary, HSP27 inhibits
cytochrome c-dependent activation of procaspase-9, FASEB J. 13 (1999) 2061-2070.
8] C.G. Concannon, A.M. Gorman, A. Samali, On the role of Hsp27 in regulating apoptosis, Apoptosis.
8
(2003) 61-70.
9] V. Baylot, C. Andrieu, M. Katsogiannou, D. Taieb, S. Garcia, S. Giusiano, J. Acunzo, J. Iovanna, M.
Gleave, C. Garrido, P. Rocchi, OGX-427 inhibits tumor progression and enhances gemcitabine
chemotherapy in pancreatic cancer, Cell. Death Dis. 2 (2011) e221.
[10] G.O. Elpek, S. Karaveli, T. Simsek, N. Keles, N.H. Aksoy, Expression of heat-shock proteins hsp27,
hsp70 and hsp90 in malignant epithelial tumour of the ovaries, APMIS. 111 (2003) 523-530.
2
3

ACCEPTED MANUSCRIPT
[
11] S.H. Kang, K.W. Kang, K.H. Kim, B. Kwon, S.K. Kim, H.Y. Lee, S.Y. Kong, E.S. Lee, S.G. Jang,
B.C. Yoo, Upregulated HSP27 in human breast cancer cells reduces Herceptin susceptibility by
increasing Her2 protein stability, BMC Cancer. 8 (2008) 286-2407-8-286.
[
12] R. Langer, K. Ott, K. Specht, K. Becker, F. Lordick, M. Burian, K. Herrmann, A. Schrattenholz,
M.A. Cahill, M. Schwaiger, H. Hofler, H.J. Wester, Protein expression profiling in esophageal
adenocarcinoma patients indicates association of heat-shock protein 27 expression and chemotherapy
response, Clin. Cancer Res. 14 (2008) 8279-8287.
[13] M. Sherman, G. Multhoff, Heat shock proteins in cancer, Ann. N. Y. Acad. Sci. 1113 (2007)
1
92-201.
[
14] J.C. Heinrich, A. Tuukkanen, M. Schroeder, T. Fahrig, R. Fahrig, RP101 (brivudine) binds to heat
shock protein HSP27 (HSPB1) and enhances survival in animals and pancreatic cancer patients, J.
Cancer Res. Clin. Oncol. 137 (2011) 1349-1361.
[
15] D. Chauhan, G. Li, R. Shringarpure, K. Podar, Y. Ohtake, T. Hideshima, K.C. Anderson, Blockade of
Hsp27 overcomes Bortezomib/proteasome inhibitor PS-341 resistance in lymphoma cells, Cancer
Res. 63 (2003) 6174-6177.
[
16] R. Heald, E. Nogales, Microtubule dynamics, J. Cell. Sci. 115 (2002) 3-4.
17] H.J. Ahn, Y.S. Kim, J.U. Kim, S.M. Han, J.W. Shin, H.O. Yang, Mechanism of taxol-induced
apoptosis in human SKOV3 ovarian carcinoma cells, J. Cell. Biochem. 91 (2004) 1043-1052.
18] G.C. Das, D. Holiday, R. Gallardo, C. Haas, Taxol-induced cell cycle arrest and apoptosis:
dose-response relationship in lung cancer cells of different wild-type p53 status and under isogenic
condition, Cancer Lett. 165 (2001) 147-153.
[
[
[
[
19] V.M. Pushkarev, D.V. Starenki, V.A. Saenko, H. Namba, J. Kurebayashi, M.D. Tronko, S. Yamashita,
Molecular mechanisms of the effects of low concentrations of taxol in anaplastic thyroid cancer cells,
Endocrinology. 145 (2004) 3143-3152.
20] V.M. Pushkarev, D.V. Starenki, V.A. Saenko, S. Yamashita, O.I. Kovzun, I.D. Popadiuk, V.V.
2
4

ACCEPTED MANUSCRIPT
Pushkarev, M.D. Tronko, Effects of low and high concentrations of antitumour drug taxol in
anaplastic thyroid cancer cells, Exp. Oncol. 31 (2009) 16-21.
[
[
[
21] Y. Matsui, B.A. Hadaschik, L. Fazli, R.J. Andersen, M.E. Gleave, A.I. So, Intravesical combination
treatment with antisense oligonucleotides targeting heat shock protein-27 and HTI-286 as a novel
strategy for high-grade bladder cancer, Mol. Cancer. Ther. 8 (2009) 2402-2411.
22] B. Zhong, S. Chennamaneni, R. Lama, X. Yi, W.J. Geldenhuys, J.J. Pink, A. Dowlati, Y. Xu, A.
Zhou, B. Su, Synthesis and Anticancer Mechanism Investigation of Dual Hsp27 and Tubulin
Inhibitors, J. Med. Chem. 56 (2013) 5306-5320.
23] X. Yi, B. Zhong, K.M. Smith, W.J. Geldenhuys, Y. Feng, J.J. Pink, A. Dowlati, Y. Xu, A. Zhou, B.
Su, Identification of a class of novel tubulin inhibitors, J. Med. Chem. 55 (2012) 3425-3435.
2
5

ACCEPTED MANUSCRIPT
Figure 1. Ligand based optimization of compound 1
MeO
O
H
B
O
N
MeO
O
OMe
H
H
R1
R3
O
N
O
O
R
N
N
O S O
R2
OMe
N
D
N
O S O
O S O
O
MeO
O
O
A
C
H
O
N
2
, 3, 4, 5
OMe
1
R= 4-MeO, 3,4-diMeO, 4-Br, 4-I
R1
NH
O
2
6

ACCEPTED MANUSCRIPT
Figure 2. Tubulin polymerization in the presence of different concentrations of compounds 10 and
1
2.
B
A
0
0
0
0
0
0
.25
0.25
.20
.15
.10
.05
.00
0.20
0.15
0.10
0.05
0.00
0
5
10
15
1
20
0
5
10
15
M 12
20
Time (min)
Time (min)
DMSO
100 nM 12
1
µ
DMSO
100 nM 10
µM 10
Figure 3. Hsp27 chaperone activity for the protection of DTT induced insulin aggregation in the
presence of different concentrations of compounds 10 and 12. The kinetic of the DTT
reduction-induced insulin aggregation was monitored in the absence of Hsp27 or in the presence of
Hsp27, with or without compounds.
Insulin + DTT
A
B
Insulin + 10
Insulin + HSP27 + DTT
Insulin + HSP27 + 10 M 10 + DTT
µM 10 + DTT
Insulin + DTT
Insulin + 10 M 12 + DTT
Insulin + HSP27 + DTT
Insulin + HSP27 + 10 M 12 + DTT
0
0
0
0
0
0
0
0
.35
0.35
µ
µ
.30
.25
.20
.15
.10
.05
.00
0.30
0.25
0.20
0.15
0.10
0.05
0.00
µ
0
5
10
15
20
25
30
0
5
10
15
20
25
30
Time (min)
Time (min)
2
7

ACCEPTED MANUSCRIPT
Figure 4. The molecular docking simulation of the interaction of tubulin with compounds 10 and
1
2. Proposed binding mode of compound 10 (A) and 12 (B) at the colchicine binding site .The tubulin
polypeptide backbones are shown as ribbons. Only important active site amino acids around compounds
are shown for clarity.
A.
B.
2
8