Structure-activity relationship studies on Bax activator SMBA1 for the treatment of ER-positive and triple-negative breast cancer

Article abstract of DOI:10.1016/j.ejmech.2019.06.004

In an effort to develop novel Bax activators for breast cancer treatment, a series of diverse analogues have been designed and synthesized based on lead compound SMBA1 through several strategies, including introducing various alkylamino side chains to have a deeper access to S184 pocket, replacing carbon atoms with nitrogen, and reducing the nitro group of 9H-fluorene scaffold. Compounds 14 (CYD-2-11) and 49 (CYD-4-61) have been identified to exhibit significantly improved antiproliferative activity compared to SMBA1, with IC₅₀ values of 3.22 μM and 0.07 μM against triple-negative breast cancer MDA-MB-231 and 3.81 μM and 0.06 μM against ER-positive breast cancer MCF-7 cell lines, respectively. Mechanism of action studies of compound 49 indicated that it can activate Bax protein to induce cytochrome c release and regulate apoptotic biomarkers, leading to cancer cell apoptosis. Further in vivo efficacy studies of compounds 14 and 49 in nude mice bearing MDA-MB-231 tumor xenografts demonstrated that these drug candidates can significantly suppress tumor growth, indicating their therapeutic potential for the treatment of breast cancer.

Full text of DOI:10.1016/j.ejmech.2019.06.004

Accepted Manuscript
Structure-activity relationship studies on bax activator SMBA1 for the treatment of ER-
Positive and triple-negative breast cancer
Gang Liu, Tao Yin, Hyejin Kim, Chunyong Ding, Zhuo Yu, Hong Wang, Haiying Chen,
Ruping Yan, Eric A. Wold, Hao Zou, Xi Liu, Ye Ding, Qiang Shen, Jia Zhou
PII:
S0223-5234(19)30521-5
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.004
EJMECH 11407
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 March 2019
Revised Date: 16 May 2019
Accepted Date: 3 June 2019
Please cite this article as: G. Liu, T. Yin, H. Kim, C. Ding, Z. Yu, H. Wang, H. Chen, R. Yan, E.A. Wold,
H. Zou, X. Liu, Y. Ding, Q. Shen, J. Zhou, Structure-activity relationship studies on bax activator SMBA1
for the treatment of ER-Positive and triple-negative breast cancer, European Journal of Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.06.004.
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ACCEPTED MANUSCRIPT
Graphic Abstract

ACCEPTED MANUSCRIPT
Structure-Activity Relationship Studies on Bax Activator SMBA1 for the Treatment of ER-
Positive and Triple-Negative Breast Cancer
a,1
b,1
b
a
b
b
a
Gang Liu, Tao Yin, Hyejin Kim, Chunyong Ding, Zhuo Yu, Hong Wang, Haiying Chen,
b
a
b
b
a
b,
a,
Ruping Yan, Eric A. Wold, Hao Zou, Xi Liu, Ye Ding, Qiang Shen * and Jia Zhou *
a
Chemical Biology Program, Department of Pharmacology and Toxicology, University of Texas
Medical Branch, Galveston, Texas 77555, United States
b
Department of Clinical Cancer Prevention, Division of Cancer Prevention and Population
Sciences, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, United
States
1
These authors contribute equally to this work.
*
Corresponding authors:
Jia Zhou, PhD
Chemical Biology Program
Department of Pharmacology and Toxicology
University of Texas Medical Branch
Galveston, Texas 77555, United States
Tel.: +1-409-772-9748
Email: jizhou@utmb.edu
Qiang Shen, PhD
Department of Clinical Cancer Prevention
Division of Cancer Prevention and Population Sciences
The University of Texas MD Anderson Cancer Center
Houston, Texas 77030, United States
Tel.: +1-713-834-6357
E-mail: qshen@mdanderson.org

ACCEPTED MANUSCRIPT
Abstract
In an effort to develop novel Bax activators for breast cancer treatment, a series of diverse
analogues have been designed and synthesized based on lead compound SMBA1 through several
strategies, including introducing various alkylamino side chains to have a deeper access to S184
pocket, replacing carbon atoms with nitrogen, and reducing the nitro group of 9H-fluorene
scaffold. Compounds 14 (CYD-2-11) and 49 (CYD-4-61) have been identified to exhibit
significantly improved antiproliferative activity compared to SMBA1, with IC50 values of 3.22
µM and 0.07 µM against triple-negative breast cancer MDA-MB-231 and 3.81 µM and 0.06 µM
against ER-positive breast cancer MCF-7 cell lines, respectively. Mechanism of action studies of
compound 49 indicated that it can activate Bax protein to induce cytochrome c release and
regulate apoptotic biomarkers, leading to cancer cell apoptosis. Further in vivo efficacy studies of
compounds 14 and 49 in nude mice bearing MDA-MB-231 tumor xenografts demonstrated that
these drug candidates can significantly suppress tumor growth, indicating their therapeutic
potential for the treatment of breast cancer.
Keywords: Bax activator; SMBA1; S184; ER-Positive; Triple-Negative; Breast Cancer;
Therapeutics.
Abbreviations
BC, breast cancer; HR, hormone receptor; ER, estrogen receptor; PR, progesterone receptor;
TNBC, triple-negative breast cancer; SAR, structure-activity relationship; HTS, high-throughput
screening; TLC, thin layer chromatography; UV, ultraviolet; TMS, tetramethylsilane; HRMS,
high-resolution mass spectrometry; HPLC, high-performance liquid chromatography; DCM,

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dichloromethane; EtOAc, ethyl acetate; DMSO, dimethyl sulfoxide; EDTA,
ethylenediaminetetraacetic acid; DDT, dichlorodiphenyltrichloroethane; ADP, adenosine
diphosphate;

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1
. Introduction
Breast cancer (BC) is currently the most prevalent malignant tumor and the leading cause of
cancer-related death among women with 1.7 million cases and 521,900 deaths according to
world-wide statistics reported for 2012 [1, 2]. Based on the American Cancer Society’s estimate
in 2018, nearly 12.4% of U.S. women will develop invasive breast cancer in their lifetimes and
2
66,120 new cases are expected to be diagnosed, including 40,920 fatal BC cases. Breast cancer
can be generally classified into three subtypes according to the levels of hormone receptor (HR)
and HER2 protein: (1) estrogen receptor (ER)-/progesterone receptor (PR)- positive type (~80%),
+
(
2) triple-negative type (10-15%), and (3) HER2 positive type (~5%) [3, 4]. The vast majority
of common BC treatments include blocking hormone receptors or decreasing hormone levels
through frontline medications, such as the HR blocker tamoxifen and aromatase inhibitors.
Despite achieving a considerable overall response rate and decrease in mortality rates through
single or combination therapeutic regimens for BC, side effects from these medications, such as
bone mass loss, insomnia and others, create significant drawbacks during clinical use [5-8]. Due
to the lack of well-defined molecular targeted drugs, triple-negative breast cancer (TNBC) is
commonly treated with cytotoxic agents such as carboplatin, cisplatin and doxorubicin, which
incur off-target toxicities and lack robust response in some patients [9-12]. In recent years,
activated signaling pathways, such as ErbB, VEGF, Ras/MAPK, PI3K/Akt/mTOR, have been
identified as molecular therapeutic targets for BC and have encouraged novel drug discovery
efforts [13, 14]. However, drug resistance caused by protein mutations and failures to meet
endpoints as single agents in clinically relevant, unselected patients has, in some cases, limited
these therapeutics’ applications [12, 15]. Taken together, new agents designed to treat BC with
novel mechanisms of action are urgently needed to expand the available tools for clinical use.

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Apoptosis has long been recognized as one of the major mechanisms of programmed cell
death in response to cancer therapies [16, 17]. Certain cases of drug resistance and lack of
efficacy for marketed drugs are due to the ability of malignant cells to evade drug-induced
apoptosis [18]. Bax, an indispensable executioner protein of B-cell lymphoma 2 (Bcl-2) family,
plays a pivotal role in regulating mitochondrial dysfunction and controlling apoptosis in normal
and cancer cells [19]. Upon stimulation, it is transformed from an inactive cytosolic monomer
into a lethal mitochondrial pore protein, inducing mitochondrial outer membrane
permeabilization, the release of cytochrome c, and consequently inducing cancer cell apoptosis
[
20]. Accumulating evidence has shown that divergent levels of the active form of Bax and
inactive form of Bax (i.e. pBax) are strongly correlated with various cancers such as melanoma
21, 22], non-small cell lung cancer [23, 24], and colon cancer [25]. Specifically in BC,
[
significant downregulation of Bax has been observed and subsequent induction of Bax
expression can restore its sensitivity to apoptotic stimuli [26-28]. Thus, Bax protein activation to
induce cancer cell apoptosis represents an attractive strategy to inhibit cancer progression and
combat drug resistance to available anticancer drugs.
To date, several classes of small molecules have been identified to selectively activate Bax
to induce Bax-mediated apoptosis with micromolar to submicromolar in vitro activity and
moderate in vivo activity by binding to different sites of Bax protein, such as Zinc 14750348
binding to Bax hydrophobic groove and BTC-8 interacting with Bax at the surface used by the
BIM BH3 helix to trigger Bax (Fig. 1) [29-31]. In pursuit of novel small molecule Bax
modulators with improved drug-likeness and a distinct binding site, our team sought to target the
Ser184 phosphorylation site pocket as a potential ligand binding site and performed high
throughput screening (HTS) using the NCI compound library, discovering the lead compound

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SMBA1 as a Bax activator with 43 nM of binding affinity to Bax protein and promising in vitro
and in vivo activity against lung cancer [32]. Intriguingly, further investigation using an SMBA1
derivative CYD-2-11 (Fig. 1) revealed that it not only displayed potent antitumor activity against
KRAS-driven lung cancer in vivo, but also potentially overcame RAD001 resistance of lung
cancer through restoring Bax activation [33]. The positive performance of these compounds in
lung cancer directed our exploration into the therapeutic potential of SMBA1 and its structurally
optimized analogues for BC treatment. Since our previous in silico docking analysis revealed
that introducing an O-alkylamino side chain tethered to the hydroxyl group on the upper phenyl
ring of lead compound SMBA1 could render deeper access into a binding pocket near S184
residue and improve Bax binding affinity [33], we decided to conduct a comprehensive
medicinal chemistry campaign on the phenyl ring fragment by introducing various terminal
linkers and replacing carbon atoms with nitrogen, as well as reducing the nitro group of the 9H-
fluorene scaffold to construct a variety of SMBA1 analogues for SAR explorations (Fig. 1).
Herein, we report the structural optimization of SMBA1 with a focus on improving the
anticancer activity for BC treatment. These efforts led to the discovery of compound 14 (CYD-2-
1
1) and 49 (CYD-4-61) (Fig. 1), two promising advanced chemical leads with significantly
enhanced antiproliferative activity against breast cancer for further drug development.

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Fig. 1. Reported Bax activators and our molecular design.
2
. Results and discussion
2
.1 Chemistry
As outlined in Scheme 1, the diverse upper phenyl ring-substituted analogues were
synthesized by condensation of 9H-fluorene 1 with various aldehydes that provided compounds
as a mixture of Z/E isomer. The diastereoselectivity was determined by the appearance of two
singlets around the chemical shift of 8.50 ppm, which were assigned to the proton signals of
methylene next to nitro group on the fluorene ring. By condensation with commercially available
2
-bromobenzaldehyde in the presence of KF-Al O in MeOH [34], the 2-bromo analogue 2 was
2 3
generated with 65:35 diastereomeric ratio in 53% yield. Coupling of 2 with
diphenylmethanimine in the presence of Pd (dba) , BINAP, NaOtBu and PhMe [35], followed
2
3
by hydrolysis of the resulting intermediate 3 afforded the final product 4 in 65% total yield (dr,
1
1
6
0:40). Compound 5 was obtained through coupling of 2 with N ,N -dimethylethane-1,2-diamine
following a similar condition as that for the preparation of 3 in 29% yield (dr, 65:35).

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Condensation of 1 with 2-methoxybenzaldhyde or 2-hydroxybenzaldhyde provided compounds 6
(dr, 60:40) or 7 (SMBA1 [32, 33], dr, 50:50) in a yield of 47% and 75%, respectively. Treatment
of 7 with various amino alcohols in mitsunobu condition [36] afforded O-alkylamino substituted
compounds 8 (dr, 50:50), 9 (dr, 50:50), 12 (dr, 50:50), 24 (dr, 55:45), 25 (dr, 50:50), 26 (dr,
5
5:45), and Boc-protected intermediates 10, 13, 16, 18, 20, 22, 27, 32, 34, 36 in 51-86% yield.
The Boc group in the latter intermediates were then deprotected with TFA to provide final
compounds 11 (dr, 50:50), 14 (CYD-2-11, dr, 65:35), 17 (dr, 60:40), 19 (dr, 50:50), 21 (dr,
6
0:40), 23 (dr, 55:45), 28 (dr, 60:40), 33 (dr, 60:40), 35 (dr, 50:50), 37 (dr, 50:50) in 48-92%
yields. Acetylation of 14 or 28 afforded amide compounds 15 (dr, 65:35) or 29 (dr, 50:50) in 83%
and 77% yield, respectively. Meanwhile, ethylpiperazine 28 was converted into the
methanesulfonamide 30 (dr, 60:40) or cyclopropanesulfonamide 31 (dr, 60:40) through the
treatment with methylsulfonyl chloride or cyclopropanesulfonyl chloride, respectively, in 75-82%
yields. It should be noted that to get chiral purity, we have successfully separated compound 14
into two single isomers by chiral HPLC with a baseline separation. However, interestingly, each
of the single isomer could dynamically tautomerize to the opposite configuration and eventually
reached an equilibrium mixture of both isomers, indicated by chiral HPLC analysis (data not
shown).

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Scheme 1. Synthesis of compounds 4-37. Reagents and conditions: (a) appropriate aldehyde,
o
KF-Al
2
O
3
, MeOH, 65 C, 16 h; (b) for 3, diphenyl-methanimine, Pd
2
(dba)
3
, BINAP, NaOtBu,
o
1
1
PhMe, 110 C, 18 h; for 5, N ,N -dimethylethane-1,2-diamine, Pd (dba) , BINAP, NaOtBu,
PhMe, 110 C, 18 h; (c) 1 N HCl, THF, rt, 2 h; (d) for 8, 2-bromoethan-1-ol, NaH, DMF, rt~80
C, 24 h; for 9-37, appropriate alcohols, Ph P, DIAD, THF, rt, 16 h; (e) TFA, CH Cl , rt,
2
3
o
o
3
2
2
3 2 2 3
overnight; (f) acetyl chloride, Et N, CH Cl , rt, 18 h; (g) appropriate sulfonyl chlorides, Et N,
CH Cl , rt, 4 h.
2
2

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The synthesis of analogues with O-alkylamino side chain substituted at different positions
or nitro reduction is described in Scheme 2. Treatment of hydroxybenzaldehydes with tert-butyl
(
2-hydroxyethyl)carbamate under Mitsunobu reaction conditions [36], followed by condensation
with 9H-fluorene and subsequent TFA-assisted de-protection generated the final compound 41a
dr, 50:50) or 41b (dr, 50:50) in moderate yields. Reduction of the nitro group of 7 or 14 by zinc
powder led to the corresponding aniline compounds 42 (dr, 65:35) or 43 (dr, 50:50) in 55% and
3% yield, respectively.
(
6
Scheme 2. Synthesis of compounds 41a-b and 42-43. Reagents and conditions: (a) tert-butyl (2-
o
hydroxyethyl)carbamate, Ph
3 2 3
P, DIAD, THF, rt, 16 h; (b) 9H-fluorene, KF-Al O , MeOH, 65 C,
1
6 h; (c) TFA, CH Cl , rt, overnight; (d) Zn, NH Cl aq, THF, rt, 4 h.
2
2
4
Pyridyl-substituted analogues were synthesized as shown in Scheme 3. Treatment of 9H-
fluorene 1 with hydroxyl-, methoxyl-pyridylaldehyde under standard condensation conditions
34] led to the corresponding compounds 44 (dr, 60:40), 45 (dr, 50:50) in 47-75% yield,
[
respectively. Compound 47 (dr, 55:45) was obtained through a condensation, coupling and
subsequent de-protection procedure following a similar method as the preparation of 4 in 23%
overall yield. Condensation of tert-butyl (2-((3-formylpyridin-2-yl)oxy)ethyl)carbamate [37]

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with 1 followed by removal of the Boc protecting group afforded compound 49 (CYD-4-61, dr,
5
0:50) in 52% overall yield. Similarly, compounds 50 (dr, 50:50) and 51 (dr, 55:45) were
obtained through condensation of 1 with 2-(piperazin-1-yl)nicotinaldehyde [38] and 2-(4-
methylpiperazin-1-yl)nicotinaldehyde [39], respectively.
Scheme 3. Synthesis of compounds 44-45, 47 and 49-51. Reagents and conditions: (a)
o
appropriate aldehyde, KF-Al
2
o
O
3
, MeOH, 65 C, 16 h; (b) diphenylmethanimine, Pd
2
(dba)
3
,
BINAP, NaOtBu, PhMe, 110 C, 18 h; (c) 1N HCl, THF, rt, 2 h; (d) TFA, CH Cl , rt, overnight.
2
2
2
2
.2 Biology
.2.1 Anti-proliferative Cellular Assay
Since lead compound SMBA1, reported to bind with an IC value of 43 nM to the S184
5
0
pocket of Bax protein [32], was previously observed to induce anti-breast cancer activity (Table
), we decided to first determine the antiproliferative effects of our newly synthesized SMBA1
derivatives against breast cancer cells to identify new compounds with enhanced cell
1

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permeability and anticancer activity. Two breast cancer cell lines MDA-MB-231 (triple-negative)
and MCF-7 (ER-positive) were chosen for MTT assays using our established in vitro screening
protocol [40]. IC values were further determined for those compounds showing inhibitory rates
5
0
greater than 50% at the concentration of 10 µM. We first evaluated the antiproliferative effects
of analogues with various terminal side chains in the upper phenyl ring, as summarized in Table
1
. Replacement of terminal hydroxyl with a bromine, amino or methoxy moiety afforded
compounds 2, 4 or 6 and completely abolished the inhibitory activity. Further introducing a
N,N-dimethylamino)ethyl to 4 led to compound 5, which has no obvious activity. Then various
(
two-carbon side chains were introduced to the terminal hydroxyl of the upper ring in the parent
compound. Hydroxyl ethyl (8) and fluoroethyl (9) ether analogues did not enhance the activity.
Compound 11 with N-methylamino-ethyl as the side chain restored the antiproliferative effect
with IC values of 5.48 µM and 4.26 µM against MDA-MB-231 and MCF-7, respectively.
5
0
However, compound 12 containing (N,N-dimethylamino)ethyl fragment showed weak activity
against MDA-MB-231, indicating the terminal N-H is critical for forming a hydrogen bond with
the Bax protein in the S184 pocket [33]. Not surprisingly, compounds 13 and 15 with N-Boc and
acetyl protected side chains exhibited negligible inhibitory activity, likely due to the lack of
hydrogen bond forming capability. Notably, compound 14 (CYD-2-11) with a free amino group
at the tail significantly enhanced the anticancer potency with IC values of 3.22 µM and 3.81
5
0
µM against both MDA-MB-231 and MCF-7, respectively, which are 1.7 to 2.2-fold more potent
than the lead compound SMBA1, as well as slightly more potent than 11. Notable discrepancies
were observed in the branched alkyl amino-containing compounds 17, 19 and 21, among which
only compounds 17 and 19 retained good potency, whereas compound 21 with a chloroethyl
substituent showed no activity at our testing concentrations. In addition, piperidine-containing

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compound 23 was slightly less potent than 14. These results suggest that both the two-carbon
side chain and the basic hydrogen are important for the anticancer activity displayed by these
compounds. Furthermore, morpholine, piperidine, piperazine and 1-methylpiperazine were
incorporated into the tail of the two-carbon side chain leading to compounds 24-26 and 28,
which displayed much weaker potency overall except the piperazine analogue 28 with good
potency of 3.5 µM and 4.97 µM. Consistent with the observation described earlier, acetylation or
sulfonylation of 28 produced compounds 29-31 that displayed no activity, likely due to shielding
the hydrogen bond formed by 28. These results highlighted the critical contribution of the
terminal basic amino pharmacophore in the side chain. Further, we evaluated the length of linker
and found that all of the analogues with 3-carbon (33), 4-carbon (35) or PEG (37) linker showed
slightly weaker activity against MDA-MB-231 and MCF-7 compared to 14.
Table 1. The antiproliferative effects of analogues 2-37 against MDA-MB-231 and MCF-7
a
breast cancer cell lines.
(
IC50, µM)
MDA-MB-
31
(IC50, µM)
MDA-MB-
1
1
Entry
R
Entry
R
MCF-7
8.3 ± 1.44
>10
MCF-7
4.66 ± 0.95
>10
2
231
*
*
#
#
7
5
.6 ± 0.69
19
21
23
5.08 ± 0.19
(
SMBA1)
b
2
4
>10
>10
#
#
>10
>10
3.53 ± 0.3
5.45 ± 0.12

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c
5
6
8
9
>10
>10
>10
>10
>10
>10
ND
>10
24
25
26
28
29
30
>10
8.64 ± 3.38
>10
ND
#
>10
9.58
#
#
3.5 ± 0.23
>10
4.97 ± 0.46
>10
#
#
1
1
2
5.48 ± 0.65 4.26 ± 0.89
1
>10
>10
>10
>10
>10
ND
1
1
1
1
3
4
5
7
>10
3.22 ± 0.49
>10
31
33
35
37
ND
*
*
#
3.81 ± 0.76
5.34 ± 0.81
3.66 ± 1.71
9.35 ± 2.48
5.13
#
#
#
#
>10
5.77 ± 0.77
6.13 ± 1.48
#
#
5.04 ± 0.07
4.5 ± 0.59
a
#
Software: MasterPlex ReaderFit 2010, MiraiBio, Inc. The values are the mean ±SD, and
b
*
values are the mean ±SE of at least three independent experiments. If a specific compound is
given a value >10, it indicates that a specific IC cannot be calculated from the data points
5
0
c
collected, meaning ‘no effect’. ND – not determined.
The SAR study on the upper phenyl ring and the nitro group is summarized in Table 2. By
taking advantage of the results shown in Table 1, we established the 2-aminoethyl as a privileged
side chain and explored its location from 2- to 3- or 4-position on the phenyl ring, leading to
compounds 41a-b with slightly less potency compared to 14. Compounds 42 and 43 can be
viewed as the nitro reduction derivatives of compound SMBA1 and 14, respectively. Similarly,
both of them exhibited minor activity decreases compared to their parental compounds.
Replacement of the phenyl in SMBA1 with pyridyl afforded compound 44, which was inactive

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probably due to the tautomerism between 2-hydroxy pyridine and pyridone configuration.
Further methoxy or amino substituted analogues 45 or 47 showed no obvious activity. Excitingly,
incorporation of a 2-aminoethyl side chain into the pyridyl ring, in a similar strategy to that for
the advanced chemical lead 14, led to compound 49 (CYD-4-61), which exhibited significantly
improved antiproliferative effects against both MDA-MB-231 and MCF-7 cancer cell lines with
nanomolar IC values of 0.07 µM and 0.06 µM, respectively. Compound 49 was not only 80-
5
0
1
30 fold more potent than the initial lead SMBA1, but also 40-60 fold more potent than the lead
compound 14. We further explored the 2-aminoethyl of 49 by replacement with piperazine or
methylpiperazine to provide the terminally cyclized analogues 50 or 51, respectively.
Interestingly, compound 50 showed reduced anticancer potency (4.27 µM and 5 µM,
respectively) compared to 49, whereas compound 51 was completely inactive, indicating that the
appropriate size and shape as well as the linker length of the side chain are important to form the
key interactions within the S184 binding pocket of the Bax protein.
Table 2. The antiproliferative effects of new analogues 41-51 against MDA-MB-231 and MCF-7
a
breast cancer cell lines.
(
IC , µM)
50
1
2
Entry
R
R
X
MDA-MB-231
MCF-7
3.77
#
4
1a
4.87 ± 0.58
3
4
=
=
#
#
41b
6.4 ± 0.46
8.47 ± 0.56

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4
4
4
4
4
4
2
3
4
5
7
9
OH
NH
NH
NO
NO
NO
NO
2
2
2
2
2
2
CH
CH
N
>10
>10
#
6.03 ± 2.35
6.31
b
c
OH
>10
ND
OMe
N
>10
>10
>10
>10
NH
N
2
*
#
*
N
0.07 ± 0.03
0.06 ± 0.03
#
5
5
0
1
NO
2
2
N
N
4.27 ± 0.36
>10
5.0 ± 1.73
>10
NO
a
#
Software: MasterPlex ReaderFit 2010, MiraiBio, Inc. The values are the mean ±SD, and
b
*
values are the mean ±SE of at least three independent experiments. If a specific compound is
given a value >10, it indicates that a specific IC cannot be calculated from the data points
5
0
c
collected, meaning ‘no effect’. ND – not determined.
2
.2.2 Docking Analysis of Compound 49 (CYD-4-61) to Bax Protein.
To investigate the possible binding mode of compound 49 to Bax protein and illustrate the
significantly improved potency of 49, we performed a virtual molecular docking study using the
published solution structure of Bax (PDB ID: 1F16) and software Small Molecule Drug
Discovery Suite 2019-1 (Schrödinger, LLC, New York, NY, 2019). As shown in Fig. 2A-B, the
molecular docking analysis revealed that the introduced O-alkylamino side chain on the upper
ring could form two hydrogen bonds with residue Asp102 around binding pocket near S184 site,
which is consistent with our design. In addition, the nitrogen atom of the pyridine ring formed a
hydrogen bond with residue Arg109, which was accounted for the better antiproliferative activity
of compound 49 than 14.

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Fig. 2. (A) Predicted binding mode of compound 49 (magenta) docking into S184 site binding
pocket of Bax protein (blue ribbon). Key residues of binding site are shown in yellow sticks.
Hydrogen bonds are depicted as dashed purple lines, and salt bridge is shown in dashed blue line.
(B) Interaction diagram of binding site between compound 49 and the Bax protein.
2
.2.3 The Inhibitory Activities of Compound 49 (CYD-4-61) against a Variety of BC Cells.
To investigate whether our Bax activators retain efficacy in several breast cancer cell lines,
+
the most potent compound 49 was evaluated for its growth inhibitory activity against two ER
BC cell lines (T47D and MCF-7), two TNBC cell lines (MDA-MB-231 and MDA-MB-468),
Adriamycin (ADR)-resistant BC cell line MCF-7 as well as immortal mammary epithelial cell
line (MCF-10A) at the indicated concentrations using MTT assays [41]. As shown in Fig. 3 and
+
Table 3, compound 49 was found to significantly inhibit the growth of ER and TNBC cell lines,
and the growth-inhibitory rate was greater than 50% at 1 µM with submicromolar to nanomolar
IC50 values, which were more potent than those of SMBA1 and 14 (CYD-2-11). Notably,
compound 49 inhibited the growth of drug-resistant MCF-7 cells in a dose-dependent manner

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(Fig. 3, bottom right panel), indicating Bax activation is a promising approach of overcoming
breast cancer chemoresistance.
MDA-MB-231
1.5
1.0
0.5
0.0
DMSO
1
0.1
3.3
0.33
10
SMBA1
CYD461
Fig. 3. Growth-inhibitory effect of 49 against various breast cancer cells. Breast cell lines were
treated with 49 at indicated concentrations for 72 h. The values are the mean ± SE of at least
three independent experiments.
Table 3. The antiproliferative effects of SMBA1, 14 (CYD-2-11) and 49 (CYD-4-61) against
a, b
various cell lines.
(
IC , µM)
50
Compound
SMBA1
MCF-10A
T47D
MCF-7
MDA-MB-231 MDA-MB-468
7.58 ± 0.20 10.44 ± 0.38 8.30 ± 1.44
5.60 ± 0.69
3.22 ± 0.49
0.07 ± 0.03
16.12 ± 11.09
8.73 ± 2.89
1.61 ± 1.04
1
4
4
9
4.37 ± 0.74
0.11 ± 0.02
9.47 ± 0.42 3.81 ± 0.76
1.20 ± 0.35 0.06 ± 0.03
a
b
Software: MasterPlex ReaderFit 2010, MiraiBio, Inc. The values are the mean ±SE of at least
three independent experiments.

ACCEPTED MANUSCRIPT
2
.2.4 Effects of Compound 49 (CYD-4-61) on Colony Formation and Apoptosis Induction in BC
Cells.
Considering the potent antiproliferative activities against MCF-7 and MDA-MB-231 BC
cell lines, we further evaluated the effect of compound 49 on the clonogenicity of cancer cells
42]. The colony formation assay was performed with 49 at a 1 M concentration, as shown in
[
µ
Figs. 4A-B, and resulted in significantly blocking the clonogenic ability of both BC cells and
almost no colony formation was observed following the addition of 49. Hoechst nuclear staining
was subsequently employed to verify if compound 49 was able to induce cancer cell apoptosis.
As shown in Fig. 4C, compound 49 generated an increase of apoptotic cells as detected by the
concentrated dense granular fluorescence compared to untreated cells, whereas no relevant
apoptosis was detectable in control cells.

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Fig. 4. Effects of compound 49 on colony formation and apoptosis in breast cancer cells. (A) 49
was used to treat MDA-MB-231 and MCF-7 cancer cells at 1 µM as indicated. Culture areas
were scanned and digitally quantified with GelCount instrument. (B) Density data from scanning
the stained culture areas were graphed as shown. Error bars represent standard deviations (p <
0
.001). (C) Hoechst nuclear staining of MDA-MB-231 and MCF-7 to show the level of cells
with apoptotic bodies by treated with 49 at 5 M and 10 M.
µ
µ
2
.2.5 Effects of Compound 49 (CYD-4-61) on Apoptosis-related Biomarkers.
For illuminating the effects of our compounds on the expression of Bax protein in cancer
cell, we directly measured the levels of the Bax active form (6A7) and the p-Bax induced by

ACCEPTED MANUSCRIPT
compound 49. As shown in Fig. 5A, levels of p-Bax were reduced in a time-dependent fashion in
MCF-7 cells and the active Bax (6A7) isoform could be continuously observed through the 48 h
treatment, while the total Bax protein level was increased after the treatment of 49. Furthermore,
dose-dependent increases for cytochrome c and decreasing levels of Bax protein were detected in
cytosolic fractions for both MCF-7 and MDA-MB-231 after the addition of 49 at 5
µM
concentration (Fig. 5B). This result suggests that Bax activation by 49 results in mitochondrial
outer membrane permeabilization, thereby leading to cytochrome c release from mitochondria
into the cytoplasm. To elucidate the potential mechanisms contributing to apoptosis, the
expression levels of several protein markers related to apoptosis were determined by Western
blotting analysis [43, 44]. As shown in Fig. 5C, treatment of MDA-MB-231 and MCF-7 with 49
dose-dependently led to the upregulation of cleaved PARP-1, cleaved caspase 3, and
downregulation of cyclin D1. This data clearly demonstrates that the antiproliferative effect of
compound 49 may be attributed to its induction of active Bax protein to trigger the intrinsic
apoptotic signaling pathway, the mode of action which is consistent with our previously reported
Bax activators [32, 33].

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Fig. 5. Bax activation-mediated intrinsic apoptosis mechanism studies on compound 49. (a)
Effects of compound 49 on Bax active form (6A7) and inactive p-Bax. MCF-7 was treated with
4
9 (5
by Western blot analysis. (B) Western-blot analysis of apoptotic biomarkers in cytosolic fraction
after the treatment with 49 (5 M) through 0-48 h. (C) Western blot analysis of apoptotic
biomarkers induced by 49 at various concentrations (48 h).
µM) and the p-Bax, active Bax (6A7) and total Bax levels were determined through 0-48 h
µ
2
.2.6 In Vivo Anticancer Efficacy of Compounds 14 (CYD-2-11) and 49 (CYD-4-61).
To evaluate whether the promising in vitro anticancer activities of our compounds could be
translated into in vivo antitumor efficacy for the proof-of-concept studies, we preliminarily
examined the effects of compounds 14 and 49 in suppression of tumor growth in an aggressive
triple-negative breast cancer MDA-MB-231 xenograft model [42]. SMBA1 was chosen as the
positive control. As shown in Fig. 6A, compared to the vehicle control, compound SMBA1 was
found to significantly suppress the growth of MDA-MB-231 tumors following intraperitoneal
injection (i.p.) administration at a dose and regimen of 40 mg/kg once daily for 7 consecutive
days, with an inhibition rate of 51%, and compound 14 at a half-dose level of SMBA1 exhibited
a comparable inhibition rate (57%). Importantly, compound 49 at a very low dose of 2.5
mg/kg/day once daily exhibited significant in vivo efficacy with an inhibition rate of 55%. As
depicted in Fig. 6B, slight body weight loss was observed for compound 49 in the treatment
groups with potentially higher toxicity than SMBA1 and compound 14 (CYD-2-11), but within
the tolerable range at the testing dose. The notable in vivo efficacy suggests that compounds 14
and 49 are improved lead compounds for further structural optimization and preclinical
development. In addition, appropriate formulation and/or drug delivery strategies applicable to

ACCEPTED MANUSCRIPT
compound 49 (e.g. nanoparticle encapsulation) are expected to further enhance the therapeutic
and safety window.
A
B
Fig. 6. In vivo efficacy of compounds 14 and 49 in comparison with the lead SMBA1 in
inhibiting growth of MDA-MB-231 xenograft tumors in mice (ip). (A) Data are presented as the
mean ± SD of tumor volume at each time point. Significant differences between compounds 14,
4
9, SMBA1 treatment group, and control were determined using one way ANOVA. p
≤ 0.05.
(B) Data showed mean ± SD of mouse body weight at each time point.
3
. Conclusion
To identify potent Bax activators for breast cancer treatment, we have conducted a
comprehensive structural optimization campaign based on our lead compound SMBA1 through
several strategies, including the introduction of various alkylamino side chains to render deeper
access to the S184 pocket, replacement of carbon atoms with nitrogen, and the reduction of the
nitro group of 9H-fluorene scaffold. Diverse analogues were synthesized and evaluated for their
antiproliferative effects against both triple-negative BC (e.g. MDA-MB-231 and MDA-MB-468)

ACCEPTED MANUSCRIPT
and ER-positive BC (e.g. MCF-7 and T47D) cell lines. Among them, compounds 14 and 49
displayed improved potency against the these BC cell lines, especially 49, which not only
exhibited 80 to 130-fold enhancement of antiproliferative effects in both MDA-MB-231 and
MCF-7 cancer cell lines with nanomolar IC values of 0.07 µM and 0.06 µM, respectively, but
5
0
also displays the potential to overcome Adriamycin (ADR)-resistant MCF-7. Mechanism of
action studies based on compound 49 suggest that it activates Bax protein by inhibiting its
phosphorylation, resulting in mitochondrial outer membrane permeabilization, induction of
cytochrome c release, and promotion of breast cancer cell apoptosis. Furthermore, compound 14
at 20 mg/kg and compound 49 at 2.5 mg/kg, respectively, showed significant tumor growth
inhibition efficacy in a MDA-MB-231 tumor xenograft model compared to SMBA1 at a dose of
4
0 mg/kg. Further structural optimization based on compounds 14 and 49 to discover novel Bax
activators with improved potency and safer toxicity profiles, as well as enhanced druggability for
cancer treatment are ongoing.
4
. Experimental protocols
4
.1 Chemistry
All reactions were performed in glassware containing a stir bar and the commercially
available starting materials and all the commercial available chemical reagents and solvents were
reagent grade and used without further purification. The reactions were performed under an air
atmosphere unless otherwise stated. Preparative column chromatography was performed using
silica gel 60 and a particle size of 0.063−0.200 mm (70−230 mesh, flash). Analytical TLC was
carried out employing silica gel 60 F254 plates (Merck, Darmstadt, Germany). Visualization of
1
13
the developed chromatograms was performed with detection by UV (254 nm). H and
C
1
13
spectra were recorded on a Bruker-600 ( H, 300 MHz or 600 MHz; C, 75 MHz or 150 MHz;

ACCEPTED MANUSCRIPT
1
9
3 3 3
F, 300 MHz) spectrometer with CDCl , CDCl + CD OD or DMSO-d6 as the solvent and TMS
as an internal reference. Chemical shifts down-field from TMS were expressed in parts per
million, and J values were given in hertz. Purity of final compounds was determined by
analytical HPLC, which was carried out on a Shimadzu HPLC system (model: CBM-20A LC-
2
0AD SPD-20AUV−vis). All biologically evaluated compounds are >95% pure.
.1.1 (E/Z)-9-(2-Bromobenzylidene)-2-nitro-9H-fluorene (2).
4
To a solution of 2-nitrofluorene (422 mg, 2.0 mmol) and 2-bromobenzalhyde (370 mg,
2
2 3
.0 mmol) in 10 mL of methanol was added KF-Al O (320 mg, 2.0 mmol). The reaction mixture
o
was stirred at 65 C for 16 h, and then evaporated in vacuo. The residue was purified by silica gel
column chromatography (ethyl acetate/CH Cl , 1:4) to give compound 2 as a yellow foam. Yield
2
2
1
4
01 mg, 53%. H NMR (600 MHz, DMSO-d )
δ
8.97 (d, J = 2.1 Hz, 1H), 8.35 (dd, J = 8.4, 2.1
6
Hz, 0.65H), 8.29 (dd, J = 8.4, 2.1 Hz, 0.35H), 8.19 – 8.16 (m, 1.65H), 8.10 (d, J = 7.6 Hz, 1H),
8
7
.03 (s, 0.35H), 7.91 – 7.85 (m, 1.35H), 7.72 (d, J = 7.5 Hz, 0.35H), 7.66 (d, J = 7.5 Hz, 0.65H),
1
3
.60 – 7.46 (m, 3.35H), 7.26 (t, J = 7.6 Hz, 0.65H), 7.10 (d, J = 7.9 Hz, 0.65H). C NMR (150
147.61, 146.88, 146.76, 144.74, 140.25, 139.57, 138.99, 137.64, 137.06,
36.94, 136.52, 136.46, 135.54, 135.45, 133.48, 133.35, 131.68, 131.40, 131.18, 130.91, 130.37,
6
MHz, DMSO-d ) δ
1
1
30.04, 129.94, 129.89, 129.54, 128.55, 125.01, 124.66, 124.35, 123.36, 123.13, 122.53, 122.24,
22.06, 121.47, 121.26, 119.10, 117.14. HRMS (ESI) calcd for C H BrNO , 378.0130 [M + H]
1
2
0
12
2
＋
;
found, 378.0122.
.1.2 (E/Z)-2-((2-Nitro-9H-fluoren-9-ylidene)methyl)aniline (4).
To a solution of 2 (189 mg, 0.5 mmol) in 5 mL of toluene was sequentially added
diphenylmethanimine (99 mg, 0.55 mmol), Pd (dba) (46 mg, 0.05 mmol), BINAP (31 mg, 0.05
4
2
3
mmol), and NaOtBu (67 mg, 0.6 mmol) under N atmosphere. The reaction mixture was stirred
2

ACCEPTED MANUSCRIPT
o
at 110 C for 18 h, and then evaporated in vacuo. The resulting residue was purified by silica gel
column chromatography (MeOH/CH Cl , 1:40) to give compound 3 as a yellow solid. Yield 170
2
2
mg, 71%.
To a solution of 3 (143 mg, 0.3 mmol) in 8 mL of THF was added 2 mL of 1N HCl. The
mixture was stirred at rt for 2 h and then adjusted to pH 8 by adding saturated NaHCO solution
3
and extracted with CH Cl (3 × 10 mL). The combined organic layer was washed with water,
2
2
2 4
dried over Na SO , filtered, and concentrated in vacuo. The residue was purified by silica
column chromatography (MeOH/CH
2
Cl
2
, 1:10) to give the compound 4 as a yellow solid. Yield
+ CD OD) 8.59 (d, J = 2.1 Hz, 0.4H), 8.29 (d, J = 2.1
1
8
7 mg, 92%. H NMR (600 MHz, CDCl
3
3
δ
Hz, 0.6H), 8.18 (dd, J = 8.3, 2.1 Hz, 0.4H), 8.11 (dd, J = 8.4, 2.1 Hz, 0.6H), 7.84 – 7.82 (m,
0
7
.6H), 7.76 – 7.69 (m, 2H), 7.66 (s, 0.6H), 7.64 (s, 0.4H), 7.57 (dd, J = 7.9, 1.0 Hz, 0.4H), 7.42 –
1
3
.30 (m, 2.6H), 7.25 – 7.15 (m, 1.4H), 6.84 – 6.77 (m, 2H). C NMR (150 MHz, CDCl +
3
CD OD)
δ
146.97, 146.69, 146.33, 144.66, 144.55, 140.53, 139.82, 138.56, 137.87, 136.84,
3
1
1
1
36.78, 135.25, 135.16, 130.47, 130.21, 130.09, 130.06, 128.97, 128.93, 128.81, 128.61, 126.57,
26.48, 124.85, 123.80, 123.45, 120.93, 120.89, 120.65, 120.61, 120.03, 119.62, 119.43, 118.58,
＋
18.34, 116.07, 115.90, 115.87. HRMS (ESI) calcd for C H N O , 315.1134 [M + H] ; found,
2
0
15
2
2
3
15.1125.
1
1
2
4
.1.3
(E/Z)-N ,N -Dimethyl-N -(2-((2-nitro-9H-fluoren-9-ylidene)methyl)phenyl)ethane-1,2-
diamine (5).
1
1
To a solution of 2 (189 mg, 0.5 mmol) in 5 mL of toluene was sequentially added N ,N -
dimethylethane-1,2-diamine (53 mg, 0.6 mmol), Pd (dba) (46 mg, 0.05 mmol), BINAP (31 mg,
2
3
0
.05 mmol), and NaOtBu (67 mg, 0.6 mmol) under N atmosphere. The reaction mixture was
2
o
stirred at 110 C for 18 h, and then evaporated in vacuo. The resulting residue was purified by

ACCEPTED MANUSCRIPT
silica gel column chromatography (MeOH/CH
2
Cl
2
, 1:10) to give compound 5 as a yellow solid.
1
Yield 56 mg, 29%. H NMR (600 MHz, CDCl )
δ 8.71 (d, J = 2.1 Hz, 0.35H), 8.46 (d, J = 2.1
3
Hz, 0.65H), 8.31 (dd, J = 8.3, 2.1 Hz, 0.35H), 8.24 (dd, J = 8.4, 2.1 Hz, 0.65H), 7.95 – 7.93 (m,
0
7
3
.65H), 7.86 – 7.82 (m, 2H), 7.75 – 7.71 (m, 1.35H), 7.52 – 7.47 (m, 1.7H), 7.45 – 7.41(m, 1.3H),
.40 – 7.36 (m, 0.65H), 7.26 (td, J = 7.6, 1.1 Hz, 0.35H), 6.89 – 6.61 (m, 2H), 4.53 (brs, 1H),
1
3
.29 – 3.27 (m, 2H), 2.56 – 2.52 (m, 2H), 2.17 (s, 2.1H), 2.15 (s, 3.9H). C NMR (150 MHz,
147.15, 146.88, 146.52, 146.36, 146.17, 144.46, 140.90, 140.10, 138.57, 138.19,
37.02, 136.87, 135.11, 135.03, 130.92, 130.59, 130.43, 130.33, 128.90, 128.76, 128.47, 126.93,
3
CDCl ) δ
1
1
1
3
26.73, 124.99, 123.73, 123.41, 120.96, 120.91, 120.76, 120.54, 120.30, 119.62, 119.41, 117.09,
16.85, 115.99, 111.29, 111.18, 57.92, 45.12, 41.35, 41.18. HRMS (ESI) calcd for C H N O ,
2
4
24
3
2
＋
86.1869 [M + H] ; found, 386.1859.
4
.1.4 (E/Z)-9-(2-Methoxybenzylidene)-2-nitro-9H-fluorene (6).
To a solution of 2-nitrofluorene (211 mg, 1.0 mmol) and o-anisaldehyde (272 mg, 2.0
mmol) in 5 mL of methanol was added KF-Al O (160 mg, 1.0 mmol). The reaction mixture was
2
3
o
stirred at 65 C for 16 h, and then evaporated in vacuo. The residue was purified by silica gel
column chromatography (ethyl acetate/CH Cl , 1:2) to provide compound 6 as a yellow foam.
2
2
1
3
Yield 155 mg, 47%. H NMR (300 MHz, CDCl ) δ 8.70 (d, J = 2.1 Hz, 0.4H), 8.49 (d, J = 2.1
Hz, 0.6H), 8.30 – 8.21 (m, 1H), 7.94 – 7.77 (m, 4H), 7.69 (d, J = 7.5 Hz, 0.4H), 7.62 (dd, J = 7.5
Hz, 0.6H), 7.53 – 7.38 (m, 2.6H), 7.23 (t, J = 7.8 Hz, 0.4H), 7.15 – 7.04 (m, 2H), 3.93 and 3.91
1
3
(
s, 3H). C NMR (75 MHz, CDCl )
δ
157.82, 157.69, 147.11, 146.73, 146.46, 144.38, 141.13,
40.37, 138.83, 138.25, 137.16, 136.81, 134.38, 134.18, 131.00, 130.91, 130.71, 128.83, 128.79,
28.55, 128.41, 127.15, 124.50, 124.20, 123.66, 123.33, 121.00, 120.82, 120.61, 120.34, 119.79,
3
1
1

ACCEPTED MANUSCRIPT
1
3
4
3
19.52, 119.47, 116.09, 111.12, 110.92, 55.55, 55.51. HRMS (ESI) calcd for C21H16NO ,
＋
30.1130 [M + H] ; found, 330.1121.
.1.5 (E/Z)-2-(2-((2-Nitro-9H-fluoren-9-ylidene)methyl)phenoxy)ethan-1-ol (8).
To a solution of compound 7 (158 mg, 0.5 mmol) in 5 mL of DMF was added NaH (40
mg, 1.0 mmol). After stirred at rt for 20 min, 2-bromoethan-1-ol (125 mg, 1.0 mmol) was added
o
and the reaction was stirred at 80 C for 24 h. Water (20 mL) was added and the reaction was
extracted with CH Cl (3 × 10 mL). The combined organic layer was washed with water, dried
2
2
over Na SO , filtered, and concentrated in vacuo. The residue was purified by silica column
2
4
chromatography (MeOH/CH
2
Cl
2
, 1:20) to give compound 8 as a yellow oil. Yield 136 mg, 68%.
1
H NMR (600 MHz, DMSO-d ) δ 8.85 (d, J = 2.1 Hz, 0.5H), 8.34 (d, J = 2.1 Hz, 0.5H), 8.32 (d,
6
J = 2.0 Hz, 0.5H), 8.27 (dd, J = 8.4, 2.1 Hz, 0.5H), 8.17 – 8.15 (m, 1.5H), 8.11 – 8.08 (m, 2H),
δ
7
7
4
1
1
1
6
.64 – 7.61 (m, 1.5H), 7.54 – 7.51 (m, 1.5H), 7.47 (q, J = 7.4 Hz, 1H), 7.29 (t, J = 7.6 Hz, 0.5H),
.25 – 7.21 (m, 1H), 7.12– 7.07 (m, 1H), 4.86 (t, J = 5.7 Hz, 0.5H), 4.82 (t, J = 5.6 Hz, 0.5H),
1
3
.12 – 4.10 (m, 2H), 3.69 – 3.64 (m, 2H). C NMR (150 MHz, CDCl ) δ 156.68, 156.63, 147.11,
3
46.72, 146.45, 144.42, 140.99, 140.19, 138.80, 138.19, 137.05, 136.84, 134.65, 131.19, 131.01,
30.69, 128.96, 128.66, 128.59, 126.68, 124.86, 124.55, 124.52, 123.83, 123.47, 121.22, 121.10,
21.01, 120.93, 120.81, 119.82, 119.63, 119.58, 116.12, 112.46, 112.26, 77.25, 77.04, 76.83,
＋
9.87, 69.78, 61.37, 61.33. HRMS (ESI) calcd for C H NO , 360.1236 [M + H] ; found,
2
2
18
4
3
60.1226.
.1.6 (E/Z)-9-(2-(2-Fluoroethoxy)benzylidene)-2-nitro-9H-fluorene (9).
4
o
Compound 7 (158 mg, 0.5 mmol) was dissolved in 5 mL of THF and colded to 0 C
o
under ice bath, then DIAD (182 mg, 0.9 mmol) was added. After stirred at 0 C for 20 min, the
2
-fluoroethan-1-ol (58 mg, 0.9 mmol) and Ph P (236 mg, 0.9 mmol) was added and the reaction
3