Synthesis and biological evaluation of anti-cancer agents that selectively inhibit Her2 over-expressed breast cancer cell growth via down-regulation of Her2 protein

Article abstract of DOI:10.1016/j.bmcl.2018.01.016

Compound JCC76 selectively inhibited the proliferation of human epidermal growth factor 2 (Her2) over-expressed breast cancer cells. In the current study, a ligand based structural optimization was performed to generate new analogs, and we identified deri

Full text of DOI:10.1016/j.bmcl.2018.01.016

Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of anti-cancer agents that selectively
inhibit Her2 over-expressed breast cancer cell growth via down-
regulation of Her2 protein
Anran Zhao, Qiaoyun Zheng, Cody M. Orahoske, Nethrie D. Idippily, Morgan M. Ashcraft, Aicha Quamine,
⇑
Bin Su
Department of Chemistry, Center for Gene Regulation in Health and Disease, College of Sciences and Health Professions, Cleveland State University, 2121 Euclid Ave., Cleveland,
OH 44115, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Compound JCC76 selectively inhibited the proliferation of human epidermal growth factor 2 (Her2) over-
expressed breast cancer cells. In the current study, a ligand based structural optimization was performed
to generate new analogs, and we identified derivatives 16 and 17 that showed improved activity and
selectivity against Her2 positive breast cancer cells. A structure activity relationship (SAR) was summa-
rized. Compounds 16 and 17 were also examined by western blot assay to check their effect on Her2 pro-
tein. The results reveal that the compounds could decrease the Her2 protein, which explains their
selectivity to Her2 over-expressed breast cancer cells. Furthermore, the compounds inhibited the chap-
erone activity of small chaperone protein that could stabilize Her2 protein.
Received 12 September 2017
Revised 8 January 2018
Accepted 11 January 2018
Available online xxxx
Keywords:
Her2
Breast cancer
Lead optimization
SAR
Published by Elsevier Ltd.
About 25–30% of breast cancer patients have human epidermal
growth factor 2 (Her2) over-expressed tumors, and the tumor cells
depend on the Her2 pathway to proliferate.1 High expression of
Her2 protein in tumors results in constitutive activation of the
receptor and cell growth.2,3 It has been well-documented that
patients with over-expressed Her2 are associated with increased
disease recurrence, worse prognosis and lower survival.4 Targeting
the extracellular domain of Her2 receptor could result in an effi-
cient inhibition of breast cancer cell proliferation.1,2,5,6 In addition,
inhibition of intracellular signaling pathways of Her2 downstream
could lead to suppression of cancer cell growth as well.7 Currently,
there are two types of drugs that target Her2 over-expressed can-
cer. The first group is Her2 monoclonal antibody drugs such as
trastuzumab approved by FDA in 1998; the second type is intracel-
lular tyrosine kinase inhibitors such as lapatinib approved in
2007.8,9
reduce the Her2 level, and found that the cancer cells regained
the sensitivity to trastuzumab.10 Therefore, new agents that can
decrease the amount of Her2 in breast cancer cells could be used
for the Her2 over-expressed breast cancer treatment, and may also
have the potential to overcome trastuzumab resistance.11–13
Our goal is to develop new anti-cancer compounds that could
selectively inhibit the growth of Her2 over-expressed breast cancer
cells, and then investigate the molecular mechanism of the phar-
macological activity. Previously we identified a small molecule
JCC76 that showed selectivity to inhibit the growth of Her2 over-
expressed breast cancer cells.14 In this study, structural optimiza-
tion was performed to improve the biological activity and the
selectivity of the lead compound (Fig. 1).
Compound JCC76 has been found to selectively inhibit the
growth of SKBR-3 cells with an IC50 of 1–3 mM, and IC50s of 20–
25 mM to MCF-7 cells and MDA-MB-231 cells.14 JCC76 was also
found to inhibit a small chaperone protein heat shock protein 27
KD (HSP27).15 It has been reported that Her2 is a client protein of
HSP27. The inhibition of HSP27 by JCC76 may affect the function
of Her2, which explains the selectivity of JCC76 to Her2 over-
expressed SKBR-3 cells.10,16 To improve the potency, selectivity
and ligand efficacy of JCC76, we further optimized the structure
of this lead. Based on the structure activity relationship (SAR)
summarized before, we either maintained the 2,5-dimethylbenzyl
moiety or changed it to 2,5-dimethoxybenzyl group (Fig. 1),15,17,18
Although these drugs showed great efficiency in clinic for Her2
over-expressed breast cancer patients, resistance has been
reported in patients with long term trastuzumab treatment.9 There
are multiple reasons for the resistance, and further increased Her2
expression in the cancer cells after the treatment is one of the
resistant mechanisms.10 Researchers used different strategies to
⇑
Corresponding author.
E-mail address: B.su@csuohio.edu (B. Su).
https://doi.org/10.1016/j.bmcl.2018.01.016
0960-894X/Published by Elsevier Ltd.
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A. Zhao et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
moiety was constructed with various substituted benzamide to
explore what the best functional group could be for this moiety.
The synthesis of the new analogs is described in Schemes 1 and 2.
These new compounds were synthesized using methods
adapted from previous studies.15,17–20 Since the sulfonamide moi-
ety was flipped in the new analogs compared to JCC76, the con-
struction of the sulfonamide is different to previous synthetic
methods. Twenty-three final compounds were synthesized.
The new derivatives were then examined for the potency and
selectivity on the growth inhibition of three breast cancer cell lines
including SKBR-3, MCF-7, and MDA-MB-231. SKBR-3 cells are Her2
positive and estrogen receptor (ER) negative, while MCF-7 cells are
Her2 negative and ER positive, MDA-MB-231 cells are Her2 and ER
negative. The activity of the compounds is summarized in Table 1.
The IC50s of the cell growth inhibition of the compounds range
from 0.13 mM to 25.69 mM for SKBR-3 cells, 1.18 mM to 60.49 mM for
MCF-7 cells, and 0.27 mM to 38.99 mM for MDA-MB-231cells. The
selectivity is calculated by dividing the IC50s of the compounds
from different cell lines (Table 1). Most compounds exhibited
higher growth inhibition in SKBR-3 cells compared to MCF-7 cells
and MDA-MB-231cells, as indicated by the selective index (>1). For
SKBR-3 cells, SAR analysis suggests that the benzamide group of
Fig. 1. Lead optimization of JCC76 to improve the ligand efficiency and biological
activity.
followed by modification of the sulfonamide moiety via an orienta-
tion shift and removal of two methyl groups. These changes could
increase the ligand efficacy and solubility. In addition, the amide
Scheme 1. (a) NH3, H2O, THF; (b) CH2Cl2, BBr3; (c) 2,5-dimethoxybenzyl chloride, K2CO3, DMF; (d) 2,5-dimethylbenzyl chloride, K2CO3, DMF.
Scheme 2. (e) FeCl3, Zn, DMF/H2O; (f) RCOCl, K2CO3, 1,4-dioxane.
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A. Zhao et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
3
these compounds is critical for the biological activity. The bulky
electron-donating substitutes such as iodo group on the benza-
mide moiety overall enhance the activity, as indicated by com-
benzyl moiety, evidence shows that the methoxy contributes more
to the cell growth inhibition than the 2,5-dimethybenzyl analogs,
because compounds 19–23 are relatively less potent compared to
the corresponding di-methoxy analogs. Overall, compounds 16
with a napthyl moiety and 17 with a 4-methoxyphenyl moiety
showed the best potency and selectivity to SKBR-3 cells.
pounds
3 and 6. The electron-withdrawing groups such as
trifluoromethyl harm the activity, as indicated by compounds 7
and 15. In terms of the 2,5-dimethoxybenzyl and 2, 5-dimethyl-
Table 1
Comparison of the growth inhibitory effects of the new analogs on different breast cancer cell lines.
Entry
IC50 (mM) (SKBR-3)
IC50 (mM) (MDA-231)
IC50 (mM) (MCF-7)
Selectivity MDA231/SKBR-3
Selectivity MCF-7/SKBR-3
1
2
3
4
5
6
7
8
4.03 2.47
3.44 1.35
1.09 0.97
4.38 1.25
25.07 6.64
0.52 0.15
25.69 11.03
3.14 1.03
3.14 0.46
6.84 3.18
6.25 1.28
1.97 0.4
0.71 0.34
0.67 0.15
15.81 6.98
0.13 0.06
2.09 0.11
4.17 0.78
3.01 0.49
15.74 5.35
15.05 3.89
2.94 0.75
11.22 1.16
7.95 4.34
15.92 2.56
4.58 1.7
23.01 7.67
36.74 25.02
1.63 0.61
60.49 23.83
4.08 1.1
6.91 3.42
7.44 2.25
13.5 7.83
3.7 2.02
1.54 0.94
2.22 0.85
15.77 7.39
1.18 0.86
12.8 8.98
10.79 1.36
6.63 0.62
48.88 15.8
10.29 0.72
6.5 0.77
4.1 0.22
2.0
4.6
4.2
5.3
1.5
3.1
2.4
1.3
2.2
1.1
2.2
1.9
2.2
3.3
1.0
9.1
6.1
2.6
2.2
3.1
0.7
2.2
1.9
1.0
2.3
4.8
2.4
0.6
4.4
1.5
1.6
2.9
0.9
3.5
1.3
4.0
0.9
1.6
2.1
1.6
1.2
1.5
0.8
0.6
1.3
0.9
8.02 1.65
5.25 1.53
10.52 0.69
14.7 3.25
2.26 0.36
38.99 14.65
5
1.76
9
9.01 4.48
6.44 0.4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
21.71 0.69
2.61 0.14
2.87 0.31
0.63 0.17
24.58 3.14
0.27 0.09
3.38 0.62
4.91 0.4
4.44 0.71
11.9 1.17
9.7 2.16
3.72 0.6
10.18 0.41
20.99 0.81
Fig. 2. Effect of compounds 16 and 17 on Her2 protein. SKBR-3 cells were treated with DMSO, JCC76 (1 mM), compounds 16 (0.1, 0.3, 1 mM) and 17 (0.1, 0.3, 1 mM) for 48 h.
Level of Her2 was analyzed by Western blot of cell extracts with specific antibodies. The bands of Her2 were quantified using ImageJ (NIH) and normalized to b-actin. The
results are representative of three independent experiments. *p < .05 with unpaired t test, compound 16 vs JCC76.
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A. Zhao et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Fig. 3.
a
-Crystalline lost partial activity to prevent DTT induced insulin aggregation in the presence of compounds 16 (10 mM) and compound 17 (10 mM). The kinetics of the
DTT-induced insulin aggregation was monitored in the absence of a chaperone protein, or in the presence of a chaperone protein without or with compounds. The mixture of
insulin and DTT with or without other components in the assay buffer was incubated for 45 min at 37 °C and the absorbance at 400 nm was measured. The compounds at this
concentration did not interfere with DTT and insulin interaction. The results are the representative of three independent experiments.
Based on the selectivity and potency to Her2 over-expressed
SKBR-3 cells, compounds 16 and 17 were chosen for further inves-
tigation. JCC76 was demonstrated to be a small chaperone inhibi-
tor, and also selectively inhibited SKBR-3 cell growth.14,15 Since
Her2 is a client protein of the small chaperones and is stabilized
by the chaperone function, we hypothesize that JCC76 may be able
to increase the degradation of Her2 via inhibition of small chaper-
one protein. The hypothesis is based on the fact that chaperone
inhibitors could induce the degradation of the client proteins of
the chaperones.21–23 SKBR-3 cells were treated with JCC76, com-
pounds 16 and 17 for 48 h, and the Her2 level was determined
by western blot assay. As exhibited in Fig. 2, Her2 level was
decreased by these new derivatives, and compounds 16 and 17
showed improved activity compared to JCC76, particularly when
compound 16 was tested at 1.0 and 0.3 mM concentrations
(Fig. 2). The results demonstrate that the structural optimization
increased the potency and selectivity of JCC76, suggesting that
the targeting effect of the compound was significantly increased.
To examine if the new compounds could interfere with the
chaperone function of small chaperone proteins, an in vitro chaper-
one assay was performed. As indicated in Fig. 3, compounds 16 and
Acknowledgements
This research was supported by Center for Gene Regulation in
Health and Disease (GRHD) of Cleveland State University and the
Summer Undergraduate Research Program. Aicha Quamine was
supported by a McNair scholarship. The instruments used in the
study were supported by National Science Foundation Major
Research Instrumentation Grants (CHE-0923398 and CHE-
1126384).
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17 inhibited the protective function of small chaperone
a-crys-
talline against Dithiothreitol (DTT) induced insulin denaturing
and aggregation.24 The results demonstrate that the new analogs
retained the chaperone inhibition of the lead compound. It has
been reported that Her2 protein is a client protein of small chaper-
one protein HSP27, and inhibition of the small chaperone could
induce Her2 degradation.10 However, it is still unclear if the chap-
erone inhibition by compounds 16 and 17 is the only mechanism
for the decreased Her2 in SKBR-3 breast cancer cells. Further inves-
tigation is needed to determine if other possible mechanisms are
involved as well.
In brief, to develop new drug candidates that could selectively
target Her2 positive breast cancer cell growth, we generated 23
new derivatives based on JCC76 as a lead. Using three breast cancer
cell lines as the evaluation model, the compounds were examined
with cell growth assay. Two compounds 16 and 1725 showed good
potency and selectivity against the growth of Her2 over-expressed
SKBR-3 cells compared to two other cell lines. Compounds 16 and
17 also decreased the level of Her2 protein in SKBR-3 cells, which is
speculated to be one of the main mechanisms of the selectivity of
the compounds. In addition, the compounds inhibited the chaper-
14. Chen B, Su B, Chen S. Biochem Pharmacol. 2009;77:1787.
15. Zhong B, Chennamaneni S, Lama R, et al. J Med Chem. 2013;56:5306.
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20. Su B, Chen S. Bioorg Med Chem Lett. 2009;19:6733.
21. Rocchi P, Beraldi E, Ettinger S, et al. Cancer Res. 2005;65:11083.
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25. N-(3-((2,5-dimethoxybenzyl)oxy)-4-sulfamoylphenyl)-1-naphthamide(16). 1H
NMR (400 MHz, DMSO-d6) d 8.606 (1H, s), 8.110 (1H, d, J = 9.0 Hz), 8.073 (1H,
s), 8.040 (2H, s), 7.845 (1H, s), 7.768 (1H, d, J = 8.6 Hz), 7.663 (2H, t, J = 6.1 Hz),
7.606 (1H, d, J = 8.6 Hz), 7.280 (1H, s), 7.007 (1H, d, J = 8.8 Hz), 6.877 (1H, d, J =
8.3 Hz), 5.2720 (2H, s), 3.825 (3H, s), 3.714 (3H, s); 13C NMR (100 MHz, DMSO-
d6) d 166.466, 155.557, 153.721, 150.835 144.635, 134.893, 132.490, 132.208,
129.499, 128.802, 128.734, 128.596, 128.520, 128.180, 127.428, 126.778,
125.479, 124.869, 114.713, 113.988, 112.169, 111.562, 105.108, 65.578,
56.282, 55.806; DUIS-MS calculated for C26H24N2O6S, [MÀH]À: 491.15, found
491.1; Purity: 98.7% N-(3-((2,5-dimethoxybenzyl)oxy)-4-sulfamoylphenyl)-4-
methoxybenzamide(17). 1H NMR (400 MHz, DMSO-d6) 10.468 (1H, s), 7.807
(1H, s), 7.742 (1H, d, J = 8.4 Hz), 7.520 (4H, m), 7.264(1H, s), 7.200 (1H, d, J = 7.6
Hz), 7.003 (1H, d, J = 9.0 Hz), 6.932 (2H, s), 6.870 (1H, d, J = 6.8 Hz), 5.248 (2H,
s), 3.856 (3H, s), 3.817 (3H, s), 3.711 (3H, s); 13C NMR (100 MHz, DMSO-d6) d
166.116, 159.700, 155.520, 153.721, 150.849, 144.431, 136.287, 130.136,
128.739, 126.831, 125.453, 120.401, 118.111, 114.726, 114.041, 113.555,
112.199, 111.603, 105.145, 65.553, 56.290, 55.867, 55.807; DUIS-MS
calculated for C23H24N2O7S, [MÀH]À: 471.14, found 471.0; Purity: 98.1%.
one activity of
a-crystalline, suggesting that they are potential
small chaperone inhibitors. The biological assays in the current
study are included in the Refs. 26–28.
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A. Zhao et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
5
26. Cell growth assay: The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-
tetrazolium bromide (MTT) cell viability assay was used to examine the
growth inhibitory effect of the analogs on the three breast cancer cell lines in
four replications. 5000 cells per well were seeded with RPMI1640 medium in
96-well flat-bottomed plates for 24 h and were then exposed to various
concentrations of test compounds dissolved in DMSO (final concentration 0.1%)
in medium for 48 h. Controls received DMSO at a same concentration as that in
drug-treated cells. Cells were incubated in 200 mL of 0.5 mg/ml of MTT reagent
diluted in fresh media at 37 °C for 2 h. Supernatants were removed from the
wells, and the reduced MTT dye was solubilized with 200 mL/well DMSO.
separated on 12% SDS-polyacrylmide gel and transferred onto a polyvinylidene
diflouride (PVDF) membrane (Pall Cooperation, FL.). After blocking for 1 h, the
membrane was incubated in PBST containing 5% BSA and primary antibody
specific to Her2 or b-actin (Cell signaling, MA) overnight at 4 °C. HRP-
conjugated anti-rabbit IgG or anti-mouse IgG (Cell signaling, MA) were used
as secondary antibody and incubated at room temperature for 1 h. The
membrane was incubated in ECL plus reagent (GE health) and then exposed to
hyper film. The results are based on three independent experiments, and only
one of the representative bands is presented.
28. Alpha-crystalline chaperone activity assay: 24 mL 1 mg/ml insulin stock
solution was added to the single well of 384 well plate, 3 mL 5 mg/ml alpha-
crystalline, 71 mL PBS buffer with appropriate concentration of compound
dissolved inside were added as well. The mixture was thoroughly mixed and
incubated at 37 °C for 5 min, whereupon 2 mL of 1 M DTT in water was added to
initiate the insulin aggregation. The absorbance (A) at 400 nm was monitored
over 45 min using a plate reader. A mixture of insulin in the absence or
presence of alpha-crystalline with 0.1% DMSO was used as control.
Absorbance at 570 nm was determined on
a
SpectraMax Plus384
spectrophotometer (Molecular Devices). Data obtained with quadreplication
were normalized and fitted to a dose–response curve using GraphPad Prism v.5
(GraphPad).
27. Western blot assay: SKBR-3 cells were treated with different compounds at
indicated concentrations respectively for 48 h. Total cell lysates were extracted
using M-PER reagent (Pierce). Equal amounts of proteins (50 lg) samples were
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