Novel Mcl-1/Bcl-2 dual inhibitors created by the structure-based hybridization of drug-divided building blocks and a fragment deconstructed from a known two-face BH3 mimetic

Article abstract of DOI:10.1002/ardp.201400296

We have previously reported a small-molecule two-face Bim BH3 mimetic, 2,3-dihydroxy-6-(4-isopropylphenylthio)anthracene-9,10-dione (1). Herein, we linked a polyphenol fragment, which was deconstructed from compound 1, with a drug-derived building block g

Full text of DOI:10.1002/ardp.201400296

Arch. Pharm. Chem. Life Sci. 2015, 348, 1–11
Full Paper
Novel Mcl-1/Bcl-2 Dual Inhibitors Created by the Structure-Based
Hybridization of Drug-Divided Building Blocks and a Fragment
Deconstructed from a Known Two-Face BH3 Mimetic
Zhichao Zhang1, Pengchen Su1,2, Xiangqian Li1, Ting Song1, Gaobo Chai3, Xiaoyan Yu3, and Keren Zhang1
1
State Key Laboratory of Fine Chemicals School of Chemistry, Dalian University of Technology, Dalian,
P. R. China
2
Chengde Petroleum College, Hebei, P. R. China
School of Life Science and Technology, Dalian University of Technology, Dalian, P. R. China
3
We have previously reported a small-molecule two-face Bim BH3 mimetic, 2,3-dihydroxy-6-(4-
isopropylphenylthio)anthracene-9,10-dione (1). Herein, we linked a polyphenol fragment, which was
deconstructed from compound 1, with a drug-derived building block gained from computer-aided
molecular design. 2-Phenyl-1H-benzo[d]imidazole as a new scaffold for two-face Bim mimetics was
developed; based on this, a series of Mcl-1/Bcl-2 dual inhibitors were obtained. The most potent
compound 6d binds to Mcl-1 and Bcl-2 with Ki values of 127 and 607 nM, respectively, and effectively
induces apoptosis in a dose-dependent, mechanism-based manner in multiple cancer cell lines.
Keywords: a-Helix / Drug-divided building blocks / Fragment-based / Mcl-1 / Bcl-2
Received: July 29, 2014; Revised: October 2, 2014; Accepted: October 30, 2014
DOI 10.1002/ardp.201400296
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
�
residues [3]. Additionally, the aspartyl residue (D67 on Bim a-
helix), is considered as the “hottest” residue for interaction
Introduction
with anti-apoptotic Bcl-2 proteins, and is conserved in all BH3-
only members [3, 7]. We have illustrated that D67 mimicking
plays an important role in small molecules targeting both Bcl-
2 and Mcl-1 [3, 8]. However, the potent inhibitor of Bcl-2, ABT-
737 and nonpeptide a-helix mimicry terphenyl scaffolds can
impart functionality from only one face of Bim and cannot
mimic D67 [1, 3].
Most recently, our group has reported a small molecule
two-face Bim BH3 mimetic, 2,3-dihydroxy-6-(4-isopropylphe-
nylthio)anthracene-9,10-dione (1) (Scheme 1 in Supporting
Information), which can mimic D67 to interact with R263/R146
on Mcl-1/Bcl-2 protein and then exhibits good affinities
toward the two targets, as well as specific antitumor abilities
in cells [3].
Fragment-based drug design (FBDD) has emerged in the
past decade as a practical tool for drug discovery that
capitalizes on the modular binding of low-molecular-weight,
low-affinity ligands and then modified or linked to yield
potent inhibitors [9, 10]. Besides, to fully utilize the reported
lead-like inhibitors, the inverse process – the deconstruction
of those high-affinity inhibitors – has been performed [11, 12].
Those functional groups that play the key role in binding have
Bcl-2 family proteins are a class of central arbiters of
apoptosis [1, 2]. They consist of six anti-apoptotic members
represented by Bcl-2 and Mcl-1, multi-domain pro-apoptotic
members and BH3-only proteins including Bim [1–3]. A
balance between anti-apoptotic and pro-apoptotic members
is mediated by the BH3 domain of the BH3-only proteins
inserting into a hydrophobic groove on the surface of anti-
apoptotic proteins [4–5]. Certain BH3-only proteins have
selective anti-apoptotic proteins-targets [5]. Among them,
Bim is a nonselective BH3-only protein that can neutralize
both arms of the anti-apoptotic Bcl-2 family, Bcl-2 and Mcl-1
proteins [3, 5, 6].
Five hotspot residues of Bim distribute on two faces of the
a-helix, among them D67 is the only hydrophilic residue
located on the opposite of the other four hydrophobic
Correspondence: Dr. Zhichao Zhang, State Key Laboratory of Fine
Chemicals, School of Chemistry, Dalian University of Technology,
Dalian 116012, People’s Republic of China.
E-mail: zczhang@dlut.edu.cn
Fax: þ86-411-84986032
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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–11
Z. Zhang et al.
been identified as suitable starting points for drug discovery
programs in some cases [13, 15].
consisting of a rotatable single bond. We expected that the
linked building blocks can work in synergy with polyphenol
fragment to recapitulate its binding model in 1 [21], and it can
be appropriately handled to improve the druggability.
The docking methods, which have been demonstrated to
be ideally suitable for analyzing the binding model of small,
inflexible molecules with target protein, were performed to
determine the candidates for our experiments [22]. Extensive
in vitro and cell-based evaluations were performed to identify
new scaffold for two-faced Bim BH3 mimetics.
Based on FBDD, the hybridization of a successful fragment
with the drug-derived building blocks has been put forward
by some researchers [16–19]. Recently, the first picomolar-
range Qo site inhibitor of the cytochrome bc1 complex
has been yielded by linking a drug-derived building block,
MOA pharmacophore, with
fragment [20].
a trifluoromethyl-quinoline
Herein, we linked the polyphenol fragment deconstructed
from compound 1 with drug-derived building blocks and
derived computer-aided molecular design. 2-Phenyl-1H-
benzo[d]imidazole as a new scaffold for two-face Bim BH3
mimetics was developed, based on which a dual inhibitor of
Mcl-1/Bcl-2, compound 6d, was obtained.
Computer-aided molecular design
Aided by computational docking, seven building blocks
derived from commercial drugs were chosen to link with
the polyphenol fragment, yielding 9 scaffolds (Fig. 2; Figure S1
in Supporting Information). Firstly, the fused bicyclic ring
systems were selected because of the suitable length of the
obtained scaffolds that could span the required distance
between D67 and I65 on Bim BH3 (Fig. 3A, 3B). In detail, a
phenyl group was favored to occupy the hydrophobic p3
pocket, and the oxygen-, nitrogen- or carbonyl-containing
hydrophilic aromatic ring may interact with the hydrophilic,
solvent-exposed residue T266 located between R263 and
the p3 pocket in the three-dimensional structure. Secondly,
computational docking was performed to visualize the
binding model and predict the binding affinities of the 9
scaffolds. Modeling showed that all these scaffolds main-
tained the same binding model with compound 1 to span
from R263 to its opposite p3 pocket in the BH3 groove of Mcl-
1. When one or two hydrogen bonds could be formed for a1,
a2, b1, b2 and c1, compounds 6a, d1–f1 could form three
hydrogen bonds with residues N260 and R263 in Mcl-1,
suggesting benzo fused five-membered rings may display
higher binding affinities than benzo fused six-membered
rings (presented by a1 and d1, Fig. 3A, 3B). Moreover, an
additional hydrogen bond was formed between the T266 of
Mcl-1 and a nitrogen atom of each of the 6a, e1, and
f1 (presented by 6a, Fig. 3C), which had the highest level
of predicted binding affinities (Table S1 in Supporting
Information). Additionally, the binding model showed a
dihedral angle (w ¼ 15.2°) was found between the benzene
ring and the benzimidazole (Fig. 3C), suggesting that the
carbon-carbon single bond could configure polyphenol group
to the lowest free energy conformation for binding. 6a was
then selected as a starting point for further optimization
because of the chemical accessory and convenient structural
modification, and the acidic hydrogen of it could be
substituted with a hydrophobic group to mimic another
residue in Bim, L62.
Results and discussion
Rationale
The 2D 1H-15
N HSQC NMR results demonstrated that
anthraquinone scaffold of 1 spans from R263 to its opposite
p3 pocket in the BH3 groove of Mcl-1. When polyphenol
group mimics D67 residue on Bim BH3 to form hydrogen
bonds with R263, the sulfur atom occupies the opposite p3
pocket. Notably, the anthraquinone scaffold maintained its
binding model when it evolved to the compound 1 [3].
As illustrated by the crystal structure of the Bim/Mcl-1
complex, the carboxy of D67 is attached to a sp3-hybridized
carbon atom, and it can rotate freely around the central
carbon-carbon single bond in room-temperature solution
(Fig. 1). However, the D67 mimicking polyphenol group of
1 fixes on the rigid scaffold. To get a better simulation of Bim,
and to develop new scaffold for two-faced Bim BH3 mimetics,
we got a series of new scaffolds by linking the polyphenol
fragment, which was deconstructed from 1, with a small
library of building blocks extracted from known drugs
Chemistry
The synthesis of the scaffold (Scheme 1) started with the
preparation of the key intermediate 2-(3,4,5-trimethoxy-
phenyl)-1H-benzo[d]imidazole (4) from 3,4,5-trimethoxy-
benzoic acid in three steps according to the reported
methods [23–25]. 5-(1H-Benzo[d]imidazole-2-yl)benzene-
Figure 1. Positions of three hotspot residues (L62, I65 and D67)
in a ribbon diagram of the Bim/Mcl-1 complex. The p2/p3 pocket
and R263 of Mcl-1 are represented as green curve and ball
model.
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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–11
Novel Mcl-1/Bcl-2 Dual Inhibitors Created by Hybridization
Figure 2. The structures prepared for computer-aided molecular design.
Figure 3. Docking results of Mcl-1 with compounds (A) a1, (B) d1, (C) 6a and (D) 6b. Mcl-1 (PDB code: 2PQK) is shown in surface
representation where R263/N260 and T266 are colored in sky blue and purple. Hydrogen, carbon, oxygen and nitrogen atoms of a1,
d1, 6a and 6b are colored white, smudge green, orange and blue, respectively. Hydrogen bonds are depicted as black dotted lines.
OH
O
O
O
O
HO
HO
O
O
O
O
O
O
O
O
NO2
a
b
c
d
H
H
H
N
N
N
OH
Cl
N
N
O
O
O
2
4
3
6a
Scheme 1. Reagents and conditions: (a) SOCl2, reflux, 2 h; (b) o-nitroaniline, Et3N, THF, 70ºC, 6 h; (c) Fe, acetic acid, reflux, 30 min;
(d) acetic acid, 48% HBr, reflux, 3 h.
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1,2,3-triol (6a) was prepared by heating with hydrobromic
acid for 3 h under N2 [26].
another nonadjacent binding interaction is required for
efficient binding [21]. As such, the linkage of the polyphenol
fragment with benzimidazole building block is necessary for
the new scaffold, 6a, to recapitulate the binding model of
anthraquinone scaffold in compound 1.
Compounds 5b–g were synthesized by reaction of the benzyl
bromide or corresponding substituted benzyl chloride with 4 in
the presence of sodium hydride, and then 6b–g were prepared
by demethylation of the aryl methyl ethers by boron tribromide
at À 78°C in anhydrous CH2Cl2 [13–24] (Scheme 2).
By comparisons of predicted binding model of compound
6a within Mcl-1 and Bim binding within Mcl-1 evidenced by
X-ray structure, we found that 6a missed the mimicking of
L62 in Bim. Compound 6b was synthesized (Scheme 2) and
evaluated in our FPAs. An improvement of binding affinity
was found for 6b, which exhibited a 4-fold enhancement in Ki
toward Mcl-1 (0.189 mM) and a net 10-fold improvement to
Bcl-2 (0.656 mM) over 6a (Table 1). Molecular modeling studies
further demonstrated that 6b maintained the mimicking of
D67 and I65 and achieved functional mimicry of an additional
residue, L62, to occupy the p2 pocket in Mcl-1. Additionally, a
dihedral angle (w ¼ 22.3°) was also found between the two
aromatic rings (Fig. 3D).
Structure-based design, synthesis and
evaluation
Firstly, compound 6a were obtained from 3,4,5-trimethoxy-
benzoic acid in four steps (Scheme 1). The Ki values of 6a were
evaluated using fluorescence polarization assays (FPAs) that
measured its ability to competitively displace a Bid-derived
peptide from Mcl-1/Bcl-2. R-(À )-Gossypol was used as a
positive control. It was found that 6a binds to Bcl-2 and
Mcl-1 with Ki values of 7.692 mM and 0.746 mM, respectively
(Table 1). So far, we have gained a new scaffold for two-face
Bim mimetics containing a rotatable carbon-carbon single
bond.
To future optimize L62 mimicking, 5-(1-(2-methylbenzyl)-
1H-benzo[d]imidazol-2-yl)benzene-1,2,3-triol (6c), 5-(1-(3-
methylbenzyl)-1H-benzo[d]imidazol-2-yl)benzene-1,2,3-triol
Notably, 2-phenyl-1H-benzo[d]imidazole which lacks the
hydroxyl groups compared with 6a can not bind to Mcl-1/Bcl-2
efficiently, supporting the hydroxyl groups could form key
interactions with the targeting proteins. However, the
polyphenol fragment itself, such as pyrogallol, did not exhibit
obvious affinity toward the target proteins in our FPAs. It
suggested that fragments deconstructed from reported lead-
like inhibitors with the only binding interaction might be
insufficient for maintaining the original binding site and
(6d),
5-(1-(4-methylbenzyl)-1H-benzo[d]imidazol-2-yl)ben-
zene-1,2,3-triol (6e) were explored (Scheme 2). Our FPAs
showed 3/4-alkyl substituent was better accommodated than
2-alkyl substituent, conferring 6d and 6e enhanced potency
relative to 6b (Table 1). Besides, binding model of 6e to Mcl-1
protein suggested that it maintained the binding model of
6b, and the 4-methylbenzyl can interact with F270, which
located on the bottom of p2 pocket of Mcl-1 (Fig.4A, 4B).
Table 1. Binding affinities (by FPAs and ELISA) of compounds to Mcl-1/Bcl-2. R-(À )-Gossypol was used as a positive
control [9, 29].
FPAs [Ki (mM)]a
ELISA [IC50 (mM)]b
Compounds
Mcl-1
Bcl-2
Mcl-1
Bcl-2
NDd
NDd
Pyrogallol
2-Phenyl-1H-benzo[d]imidazole
4
5b
5c
5d
5e
5f
>50c
>50c
NDd
>50c
>50c
NDd
>50c
>50c
NDd
NDd
20c
30c
NDd
NDd
20c
30c
NDd
NDd
20c
30c
NDd
NDd
20c
30c
NDd
NDd
>50c
>50c
NDd
NDd
5g
a
6b
6c
6d
6e
6f
6g
>50c
>50c
NDd
NDd
0.746 � 0.020
0.189 � 0.009
0.171 � 0.005
0.127 � 0.005
0.125 � 0.005
0.589 � 0.016
0.687 � 0.018
0.180 � 0.010
7.692 � 0.394
0.656 � 0.018
0.627 � 0.016
0.607 � 0.014
0.591 � 0.014
0.568 � 0.014
0.587 � 0.014
0.320 � 0.020
1.1 � 0.2
0.26 � 0.06
0.20 � 0.05
0.12 � 0.03
0.14 � 0.03
0.71 � 0.12
0.79 � 0.12
0.17 � 0.02
12.1 � 1.7
0.63 � 0.15
0.56 � 0.13
0.51 � 0.13
0.60 � 0.11
0.53 � 0.10
0.51 � 0.10
0.50 � 0.10
R-(–)-Gossypol
aValues are the mean � standard deviation of three independent experiments.
bValues are the mean � standard deviation of three to seven independent experiments.
cPartial binding was observed at 100 mM, and the Ki value was an estimate.
dND ¼ Not determined.
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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–11
Novel Mcl-1/Bcl-2 Dual Inhibitors Created by Hybridization
Figure 4. (A) Detailed interactions between
compound 6e and binding site residues
(H224, F228, V253, N260, R263, T266, F270)
in Mcl-1 (PDB code: 2PQK) predicted by
modelling studies. Hydrogen, carbon, oxy-
gen and nitrogen atoms of 6e are colored
white, smudge green, orange and blue,
respectively. Hydrogen bonds are depicted
as black dotted lines. (B) The resultant
overlay of compound 6e (smudge green)
on the Bim BH3 peptide (light gray) is
displayed.
To probe p2 pocket further, we designed and synthesized
compound 6f in which the methyl group in compound 6d was
replaced by an isobutyl group (Scheme 2). Although 6f
exhibited a nanomolar Ki value (0.568 mM) to Bcl-2, which is
similar to compound 6d, a significant decrease of Ki toward
Mcl-1 (0.589 mM) was found unfortunately (Table 1).
To further illustrate the binding ability of these compounds,
we applied an enzyme-linked immunosorbent assay (ELISA).
As shown Table 1, the relative binding affinities for these
inhibitors in our ELISA are highly consistent with those
obtained from the FP-based assays. Compound 6d exhibits
the most potent affinity in that it binds to Mcl-1 and Bcl-2 with
IC50 values of 0.12 mM and 0.51 mM, respectively (Table 1).
Futhermore, we measured the Ki value of 6d and some
reported Bcl-2 inhibitors in parallel by FPAs. As shown in
Table 2, 6d exhibited similar dual binding ability to R-
(À )-Gossypol and TW-37. Although its Bcl-2 affinity is lower
than ABT-737, it could hit Mcl-1 that ABT-737 lost.
We next evaluated the apoptosis induction of compound
6a–g in Bcl-2-dependent HL60 cell line [30] and Mcl-1-
dependent K562 cell line [31]. Dual-inhibitors compound
6b, 6c, 6d and 6e can effectively induce cell death in both the
two cancer cell lines in a dose-dependent manner, as shown
by annexin-V/propidium iodide staining by flow cytometry
(Table 3). 6g, however, was found to be similar to ABT-737
that failed to kill K562 cells efficiently.
The discrepancies between the lower binding affinity of 6f
toward Mcl-1 and putative good-fitting model of the isobutyl
within the p2 pocket of Mcl-1 were perplexing. We hypothe-
sized that the loss of affinity was caused by the 2-hydroxyl
group on benzyl. To verify it, 3-methylbenzyl of 6d was
replaced by 2-hydroxy-5-methylbenzyl to yield 6g (Scheme 2).
6g binds to Mcl-1 with Ki value 0.687 mM, which is also similar
to that of 6d (Table 1). As such, we speculated that a hydrogen
bond might be formed between 2-hydroxyl group on benzyl
and 3/5-hydroxyl group in pyrogallol, and the restricted
intramolecular rotation resulted in the loss of binding energy
and affinities. Additionally, the Ki values of 6f and 6g toward
Mcl-1 decreased significantly than that of 6d, whereas no
similar influence on their affinities toward Bcl-2 was found. It
suggested that the conformation-restrained benzyl group
was not tolerated by Mcl-1, but it can still be accommodated
by Bcl-2. It is consistent with our previous finding that the p2
pocket of Mcl-1 is not as tolerant as that of Bcl-2 [8].
MCF7 cells which express high level of both Bcl-2 and Mcl-1
were then applied to understand the killing of 6d. Firstly,
treatment of 6d at 5 and 10 mM for 24 h induced 35 � 2% and
60 � 5% of cells to undergo apoptosis (Fig. 5). Consequently,
OH
R2
O
O
R1
HO
HO
O
O
O
O
a
b
H
N
N
N
N
N
N
4
5b g
6b g
Scheme 2. Reagents and conditions: (a) Benzyl bromide or substituted benzyl chloride, NaH, KI, 70ºC, 12 h; (b) BBr3, CH2Cl2, À 78ºC to
r.t., overnight.
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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–11
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Table 2. The comparisons of Ki between 6d and Mcl-1/Bcl-
2 inhibitors reported in parallel by using FPAs. The Ki
values of the reported Mcl-1/Bcl-2 inhibitors were similar
to the values reported in the literature [3, 22, 29].
FPAs [Ki (mM)]b
Compounds
Mcl-1
Bcl-2
ABT-737a
R-(À )-Gossypola
TW-37a
>100
<0.001
0.182 � 0.014
0.268 � 0.017
–
0.127 � 0.011
0.315 � 0.020
0.291 � 0.017
0.221 � 0.015
0.607 � 0.031
GX15–070a
6d
aThose reported Mcl-1/Bcl-2 inhibitors were obtained from
Selleck, China.
bValues are the mean
independent experiments.
�
standard deviation of three
Figure 6. Caspase activation assays of apoptosis in MCF7 cells
induced by 6d. MCF7 cells were treated with 6d for 24 h, and
then the activation of caspase-9 or caspase-8 was evaluated by
fluorometric caspase activation assays as described in the
Experimental section. Columns, mean relative fluorescence
units (RFU) from three independent experiments; bars, SE. Data
shown are means � SE of three independent experiments in
duplicate and expressed in arbitrary fluorescence units per
milligram of protein.
Table 3. Cytotoxicity of compound 6a–g against cancer
cell lines through annexin-V/propidium iodide staining by
flow cytometry.
FCM [IC50 (mM)]a
activities of caspase-8 and caspase-9 were measured, respec-
tively to evaluate whether 6d induce apoptosis through
the mitochondria-mediated apoptotic pathway. As shown
in Fig. 6, compound 6d can induce caspase-9 activation
significantly, whereas no similar influence on caspase-8 was
found, suggesting apoptosis occurred through the mitochon-
dria pathway following 6d treatment. Furthermore, co-
immunoprecipitation (Co-IP) assays identified that the bind-
ing ability of 6d toward Bcl-2 and Mcl-1 in vitro could translate
to the disruption of Bcl-2/Bax and Mcl-1/Bak in cells (Fig. 7).
Herein, these data demonstrated that 6d can exhibit
mechanism-based apoptosis induction.
Compounds
HL60
K562
6a
6b
6c
6d
6e
6f
6g
ABT-737
>50
19.7 � 1.6
7.9 � 0.5
7.5 � 0.6
7.1 � 0.7
7.4 � 0.6
18.2 � 1.4
17.8 � 1.5
25 � 2.5
13.5 � 1.2
14.2 � 1.3
14.0 � 1.1
13.8 � 0.8
15.4 � 1.2
16.1 � 1.7
0.1 � 0.02
aValues are the mean � standard deviation of three indepen-
dent experiments.
Figure 5. Induction of apoptosis by 6d in the MCF7 cells. Cells were treated with 6d at 5 mM and 10 mM for 24 h, and apoptosis was
analyzed by annexin-V/propidium iodide staining by flow cytometry.
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Arch. Pharm. Chem. Life Sci. 2015, 348, 1–11
Novel Mcl-1/Bcl-2 Dual Inhibitors Created by Hybridization
used for multiplicity of NMR signals: s ¼ singlet, d ¼ doublet,
t ¼ triplet, q ¼ quartet, m ¼ multiplet. High-resolution mass
spectra (HRMS) were obtained on HPLC-Q-T of MS (Micro)
spectrometer. Column chromatography was performed on
silica gel 200–300 mesh. Purity of all final products was
determined by analytical HPLC to be � 95%. HPLC purity of
compounds was measured with
a reverse phase HPLC
(XBridge C18, 4.6 � 150 mm, 5 mm) with two diverse wave-
length detection systems. Compounds were eluted by
gradient elution of 40/60–0/100 H2O/CH3OH over 30 min at
a flow rate of 0.3 mL/min.
3,4,5-Trimethoxybenzoyl chloride (2) [23]
0.05 mol of 3,4,5-trimethoxybenzoic acid with 20 mL of
thionyl chloride were taken in a 100 mL round-bottomed
flask equipped with a stir bar. The mixture was stirred at room
temperature for 10 min and then heated under reflux for 2 h,
during which time the mixture turned yellow. The excess
thionyl chloride was removed by distillation.
Figure 7. Disruption of Bcl-2/Bax and Mcl-1/Bak interaction by
6d. Co-Ip method was used. MCF7 cells were treated with 6d.
Lysates equivalent to 100 mg of protein were prepared.
Harvested cells were subjected to immunoprecipitation of
Mcl-1 and Bcl-2 followed by immunoblotting for Bak and Bax.
Methyl-2-nitro-3,4,5-trimethoxybenzamide (3) [24, 25]
To o-nitroaniline (0.69 g, 5 mmol) dissolved in triethyl amine/
THF (1:4) (20 mL) was added 2 (15 mmol). The reaction mixture
was warmed at 70°C for 6 h, allowed to cool to room
temperature. And then, the mixture was poured into water
(300 mL), and extracted twice with ethyl acetate. The
combined organic phases were washed with HCl (10%),
NaHCO3 (saturated aqueous) and NaCl (saturated aqueous),
dried (Na2SO4), filtered, and concentrated. The product was
recrystallized from a mixture of water and ethanol (1:1) to
give 3 (1.08 g, 3.25 mmol, 65% yield of dark yellow crystals).
Rf ¼ 0.35 (petroleum ether/ethyl acetate 4:1). 1H NMR
(400 MHz, [D6]DMSO) d: 13.42 (s, 1H, NH), 8.54 (d, J ¼ 8.0 Hz,
1H, Ar-CH), 8.32 (d, J ¼ 8.0 Hz, 1H, Ar-CH), 7.90 (t, J ¼ 4.0 Hz,
1H, Ar-CH), 7.57 (t, J ¼ 4.0 Hz, 1H, Ar-CH), 7.30 (s, 2H, Ar-CH),
3.91 (s, 6H, 2OCH3), 3.33 (s, 3H, OCH3). 13C NMR (100 MHz, [D6]
DMSO) d: 164.7, 153.2, 142.6, 142.2, 131.3, 130.8, 125.5, 125.1,
124.1, 118.4, 106.6, 60.8, 56.1. TOF MS (EIþ): C16H16N2O6, calcd.
for 332.1008, found 332.1011.
From the data mentioned above, 2-phenyl-1H-benzo[d]-
imidazole scaffold, which get a better simulation of D67 in
Bim, was successfully applied to the design of two-face Bim
mimetics.
Conclusions
In summary, we have designed a new 2-phenyl-1H-benzo[d]-
imidazole scaffold by linking the polyphenol fragment
deconstructed from 1, with benzimidazole building block
extracted from known drugs consisting of a rotatable single
bond. Based on 1, compound 6d was obtained, which can bind
to Bcl-2 and Mcl-1 with Ki values of 0.607 mM and 0.127 mM
respectively and mimic the key binding residues located on
two faces of the Bim BH3 a-helix in the interaction with Bcl-2
and Mcl-1. Besides, compound 6d can effectively induce
apoptosis in a dose-dependent, mechanism-based manner in
MCF7 cells, which express high levels of Bcl-2 and Mcl-1
proteins. Taken together, our data indicated that compound
6d is a potent, cell-permeable, two-face Bim BH3 a-helix
mimic. We expected these results could provide some
fundamental insights into the future design and development
of Bim BH3 a-helix mimetics.
2-(3,4,5-Trimethoxyphenyl)-1H-benzo[d]imidazole (4) [24,
25]
To 3 (650 mg, 1.96 mmol) suspended in acetic acid (10 mL)
was added iron powder (448 mg, 8 mmol). After 30 min at
reflux, the mixture was concentrated and the residue was
redissolved in ethyl acetate, washed with NaHCO3 (saturated
aqueous) and NaCl (saturated aqueous), dried (Na2SO4),
filtered, evaporated, and recrystallized from a mixture of
water and ethanol (2:1) to yield 340 mg (1.20 mmol, 61%
yield) of white crystals. Rf ¼ 0.4 (CH2Cl2/acetone 20:1). 1H
NMR (400 MHz, [D6]DMSO) d: 12.83 (s, 1H, NH), 7.60 (s, 2H,
Ar-CH), 7.53 (d, J ¼ 8 Hz, 2H, Ar-CH), 7.19–7.21 (m, 2H, Ar-CH),
3.91 (s, 6H, 2 OCH3), 3.33 (s, 3H, OCH3). 13C NMR (100 MHz,
[D6]DMSO) d: 153.1, 152.7, 141.7, 139.2, 124.9, 123.0, 115.2,
104.6, 60.8, 56.1. TOF MS (EIþ): C16H16N2O3, calcd. for
284.1161, found 284.1157.
Experimental
Chemistry
Materials and methods
All commercial reagents were purchased and used without
further purification or distillation unless otherwise stated.
NMR spectra were recorded with Bruker AV-400 spectrometer
with chemical shifts reported as parts per million (in DMSO,
TMS as internal standard). The following abbreviations are
� 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
7

Arch. Pharm. Chem. Life Sci. 2015, 348, 1–11
Z. Zhang et al.
5-(1H-Benzo[d]imidazole-2-yl)benzene-1,2,3-triol (6a) [26]
To 241 mg (0.85 mmol) of 4 in 5 mL of hot AcOH was added
10 mL of 48% HBr. This mixture was heated at 115–120°C
under N2 for 3 h. The volatiles were removed under reduced
pressure, and the residual solid was recrystallized from H2O to
give 151 mg (0.63 mmol, 73%) of small blue grey powder.
Rf ¼ 0.38 (H2O/acetic acid/petroleum ether/ethyl acetate
1:2:2:8). 1H NMR (400 MHz, [D6]DMSO) d: 14.98 (s, 1H, NH),
9.73 (s, 2H, 2OH), 9.60 (s, 1H, OH), 7.76 (d, J ¼ 4 Hz, 2H, Ar-CH),
7.50–7.52 (m, 2H, Ar-CH), 7.25 (s, 2H, Ar-CH). 13C NMR
(100 MHz, [D6]DMSO) d: 152.9, 141.7, 136.0, 147.5, 126.0,
115.2, 106.9, 123.0. TOF MS (EIþ): C13H10N2O3, calcd. for
242.0691, found 242.0694. HPLC (40:60 to 0:100 H2O/CH3OH)
purity ¼ 98.91%, tR ¼ 13.28 min.
d: 7.86 (d, J ¼ 8 Hz, 1H, Ar-CH), 7.69 (d, J ¼ 8 Hz, 1H, Ar-CH),
7.56–7.68 (m, 1H, Ar-CH), 7.35–7.37 (m, 2H, Ar-CH), 7.23 (d,
J ¼ 4 Hz, 1H, Ar-CH), 7.18 (s, 1H, Ar-CH), 6.98 (d, J ¼ 4 Hz, 1H,
Ar-CH), 6.84 (s, 2H, Ar-CH), 5.75 (s, 2H, CH2), 3.89 (s, 6H,
2OCH3), 3.31 (s, 3H, OCH3), 2.30 (s, 3H, CH3). 13C NMR
(100 MHz, [D6]DMSO) d: 153.3, 153.1, 142.4, 139.2, 138.3,
137.8, 136.1, 130.8, 128.5, 126.1, 124.9, 124.6, 123.1, 119.5,
119.0, 104.6, 60.8, 56.1, 52.5, 21.6. TOF MS (EIþ): C24H24N2O3,
calcd. for 388.1887, found 388.1884.
1-(4-Methylbenzyl)-2-(3,4,5-trimethoxyphenyl)-1H-benzo[d]-
imidazole (5e): Rf ¼ 0.4 (CH2Cl2/petroleum ether 3:1). Yield:
145 mg, 53%, white powder. 1H NMR (400 MHz, [D6]DMSO) d:
7.83 (d, J ¼ 8 Hz, 1H, Ar-CH), 7.66 (d, J ¼ 8 Hz, 1H, Ar-CH), 7.33–
7.39 (m, 2H, Ar-CH), 7.22 (d, J ¼ 4 Hz, 2H, Ar-CH), 7.11 (d,
J ¼ 4 Hz, 2H, Ar-CH), 6.82 (s, 2H, Ar-CH), 5.75 (s, 2H, CH2), 3.89
(s, 6H, 2OCH3), 3.31 (s, 3H, OCH3), 2.29 (s, 3H, CH3). 13C NMR
(100 MHz, [D6]DMSO) d: 153.4, 153.1, 142.3, 139.2, 137.8,
135.3, 134.3, 129.0, 127.2, 124.9, 123.1, 119.5, 119.0, 104.6,
60.8, 56.1, 52.2, 21.3. TOF MS (EIþ): C24H24N2O3, calcd. for
388.1887, found 388.1889.
General procedure for the preparation of 5b–g [24]
To 4 (200 mg, 0.7 mmol), KI (100 mg, 0.6 mmol), and benzyl
bromide or substituted benzyl chloride (1.68 mmol, 240%)
dissolved in THF (20 mL) was added NaH (0.16 g, 500 M%). The
reaction mixture was warmed at 70°C for 12 h, allowed to cool
to room temperature. And then, the mixture was quenched
with ice, and extracted with ethyl acetate about 4–5 times.
The combined organic phases were washed with NaCl
(saturated aqueous), dried (Na2SO4), filtered, and concentrat-
ed. The product was purified by flash chromatography eluting
with CH2Cl2/hexane (3:1) or recrystallization from diethyl
ether/hexane (2:1).
1-(2-Methoxy-5-isopropylbenzyl)-2-(3,4,5-trimethoxyphenyl)-
1H-benzo[d]imidazole (5f): Rf ¼ 0.4 (CH2Cl2/petroleum ether
3:1). Yield: 134 mg, 43%, orange-yellow powder. 1H NMR
(400 MHz, [D6]DMSO) d: 7.80 (d, J ¼ 8 Hz, 1H, Ar-CH), 7.70 (d,
J ¼ 8 Hz, 1H, Ar-CH), 7.57 (m, 2H, Ar-CH), 7.01 (d, J ¼ 8 Hz, 1H,
Ar-CH), 6.81–6.83 (m, 2H, Ar-CH), 6.71–6.76 (m, 2H, Ar-CH),
5.62 (s, 2H, CH2), 3.89 (s, 6H, 2OCH3), 3.35 (s, 3H, OCH3), 3.31 (s,
3H, OCH3), 2.61 (m, 1H, CH), 1.05 (s, 6H, 2CH3). 13C NMR
(100 MHz, [D6]DMSO) d: 155.8, 153.3, 153.0, 142.4, 140.6,
139.2, 137.8, 129.0, 125.4, 124.9, 124.1, 123.0, 119.5, 119.0,
111.4, 104.6, 60.8, 56.1, 46.6, 33.5, 23.3. TOF MS (EIþ):
1-Benzyl-2-(3,4,5-trimethoxyphenyl)-1H-benzo[d]imidazole
(5b): Rf ¼ 0.4 (CH2Cl2/petroleum ether 3:1). Yield: 149 mg,
64%, white powder. 1H NMR (400 MHz, [D6]DMSO) d: 7.83 (d,
J ¼ 8 Hz, 1H, Ar-CH), 7.66 (d, J ¼ 8 Hz, 1H, Ar-CH), 7.50–7.60 (m,
2H, Ar-CH), 7.33–7.39 (m, 3H, Ar-CH), 7.22 (d, J ¼ 4 Hz, 2H, Ar-
CH), 6.82 (s, 2H, Ar-CH), 5.75 (s, 2H, CH2), 3.89 (s, 6H, 2OCH3),
3.31 (s, 3H, OCH3). 13C NMR (100 MHz, [D6]DMSO) d: 153.4,
153.1, 142.4, 139.2, 137.8, 137.3, 128.6, 127.5, 125.7, 124.9,
123.1, 119.6, 119.0, 104.6, 60.7, 56.0, 52.1. TOF MS (EIþ):
C
27H30N2O4, calcd. for 446.2206, found 446.2209.
1-(2-Methoxy-5-methylbenzyl)-2-(3,4,5-trimethoxyphenyl)-
1H-benzo[d]imidazole (5g): Rf ¼ 0.4 (CH2Cl2/petroleum ether
3:1). Yield: 139 mg, 47.5%, orange-yellow powder. 1H NMR
(400 MHz, [D6]DMSO) d: 7.80 (d, J ¼ 8 Hz, 1H, Ar-CH), 7.70 (d,
J ¼ 8 Hz, 1H, Ar-CH), 7.57 (m, 2H, Ar-CH), 7.01 (d, J ¼ 8 Hz, 1H,
Ar-CH), 6.81–6.83 (m, 2H, Ar-CH), 6.71–6.76 (m, 2H, Ar-CH),
5.62 (s, 2H, CH2), 3.89 (s, 6H, 2OCH3), 3.34 (s, 3H, OCH3), 3.29 (s,
3H, OCH3), 2.25 (s, 3H, CH3). 13C NMR (100 MHz, [D6]DMSO) d:
155.6, 153.4, 153.0, 142.4, 139.2, 137.7, 130.6, 129.7, 127.0,
125.6, 124.9, 123.0, 119.5, 119.0, 111.6, 104.4, 60.8, 56.1, 46.5,
21.7. TOF MS (EIþ): C25H26N2O4, calcd. for 418.1993, found
418.1989.
C
23H22N2O3, calcd. for 374.1630, found 374.1627.
The step to obtain 5c–g is the same with the process of
compound 5b. The starting material is 200 mg separately.
1-(2-Methylbenzyl)-2-(3,4,5-trimethoxyphenyl)-1H-benzo[d]-
imidazole (5c): Rf ¼ 0.4 (CH2Cl2/petroleum ether 3:1). Yield:
150 mg, 55%, white powder. 1H NMR (400 MHz, [D6]DMSO) d:
7.84 (d, J ¼ 8 Hz, 1H, Ar-CH), 7.67 (d, J ¼ 8 Hz, 1H, Ar-CH), 7.34–
7.40 (m, 3H, Ar-CH), 7.27 (m, 1H, Ar-CH), 7.24 (d, J ¼ 8 Hz, 1H,
Ar-CH), 7.18 (m, 1H, Ar-CH), 6.83 (s, 2H, Ar-CH), 5.75 (s, 2H,
CH2), 3.89 (s, 6H, 2OCH3), 3.32 (s, 3H, OCH3), 2.29 (s, 3H, CH3).
13C NMR (100 MHz, [D6]DMSO) d: 153.4, 153.0, 142.4, 139.2,
137.8, 137.4, 136.7, 130.3, 128.9, 128.4, 125.6, 124.9, 123.1,
119.5, 119.0, 104.6, 60.7, 56.1, 49.6, 18.8. TOF MS (EIþ):
General procedure for the preparation of 6b–g [13]
5-(1-Benzyl-1H-benzo[d]imidazol-2-yl)benzene-1,2,3-triol
(6b): In this step, all reagents and reactors are required to be
anhydrous. 1-Benzyl-2-(3,4,5-trimethoxyphenyl)-1H-benzo[d]-
imidazole (5b) (150 mg, 0.4 mmol) in 5 mL of anhydrous CH2Cl2
was placed in a flask under liquid nitrogen condition. Then
boron tribromide (0.378 mL, 4.0 mmol) in CH2Cl2 (5 mL) was
slowly added at ultralow temperature and the solution was
C
24H24N2O3, calcd. for 388.1887, found 388.1885.
1-(3-Methylbenzyl)-2-(3,4,5-trimethoxyphenyl)-1H-benzo[d]-
imidazole (5d): Rf ¼ 0.4 (CH2Cl2/petroleum ether 3:1). Yield:
159 mg, 58.5%, white powder. 1H NMR (400 MHz, [D6]DMSO)
� 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
8

Arch. Pharm. Chem. Life Sci. 2015, 348, 1–11
Novel Mcl-1/Bcl-2 Dual Inhibitors Created by Hybridization
stirred for the time and temperature slowly returned to room
temperature. After reacting overnight, methanol (1 mL) was
added. The mixture was stirred for another 15 min after
adding additional H2O (1 mL). The solid was filtered, washed
with water, recrystallized from ethanol/H2O (1:20) as white
rosettes, yield 49.5%. Rf ¼ 0.55 (H2O/acetic acid/petroleum
C21H18N2O3, calcd. for 346.1317, found 346.1315. HPLC
(40:60 to 0:100 H2O/CH3OH) purity ¼ 98.56%, tR ¼ 11.39 min.
5-(1-(2-Hydroxy-5-isopropylbenzyl)-1H-benzo[d]imidazol-2-
yl)benzene-1,2,3-triol (6f): Rf ¼ 0.45 (H2O/acetic acid/petro-
leum ether/ethyl acetate 1:2:2:8). Yield: 41 mg, 46.9%,
orange-yellow powder. 1H NMR (400 MHz, [D6]DMSO) d:
10.19 (s, 1H, OH), 9.65 (s, 2H, 2OH), 9.30 (s, 1H, OH), 7.80 (d,
J ¼ 8 Hz, 1H, Ar-CH), 7.70 (d, J ¼ 8 Hz, 1H, Ar-CH), 7.57 (m, 2H,
Ar-CH), 7.01 (d, J ¼ 8 Hz, 1H, Ar-CH), 6.81–6.83 (m, 2H, Ar-CH),
6.71–6.76 (m, 2H, Ar-CH), 5.62 (d, J ¼ 8 Hz, 2H, CH2), 2.61 (m, H,
CH), 1.05 (s, 6H, 2CH3). 13C NMR (100 MHz, [D6]DMSO) d: 153.3,
147.5, 142.4, 140.9, 137.8, 136.0, 129.4, 127.0, 126.0, 124.5,
123.0, 119.5, 119.0, 115.5, 106.9, 46.3, 33.5, 23.3. TOF MS (EIþ):
1
ether/ethyl acetate 1:2:2:8). H NMR (400 MHz, [D6]DMSO) d:
9.71 (s, 2H, 2OH), 9.37 (s, 1H, OH), 7.84 (d, J ¼ 8 Hz, 1H, Ar-CH),
7.67 (d, J ¼ 8 Hz, 1H, Ar-CH), 7.51–7.61 (m, 2H, Ar-CH), 7.34–
7.39 (m, 3H, Ar-CH), 7.22 (d, J ¼ 4 Hz, 2H, Ar-CH), 6.82 (s, 2H,
Ar-CH), 5.76 (s, 2H, CH2). 13C NMR (100 MHz, [D6]DMSO) d:
153.3, 147.5, 142.4, 137.8, 137.3, 136.0, 128.6, 127.6, 126.0,
125.7, 123.0, 119.5, 119.0, 106.9, 52.2. TOF MS (EIþ):
C
20H16N2O3, calcd. for 332.1161, found 332.1165. HPLC
(40:60 to 0:100 H2O/CH3OH) purity ¼ 99.12%, tR ¼ 11.02 min.
The step to obtain 6c–g is the same with the process of
compound 6b. The starting material is 100 mg separately.
C23H22N2O4, calcd. for 390.1580, found 390.1584. HPLC (40:60
to 0:100 H2O/CH3OH) purity ¼ 99.35%, tR ¼ 12.78 min.
5-(1-(2-Hydroxy-5-methylbenzyl)-1H-benzo[d]imidazol-2-yl)-
benzene-1,2,3-triol (6g): Rf ¼ 0.45 (H2O/acetic acid/petroleum
ether/ethyl acetate 1:2:2:8). Yield: 38 mg, 43.9%, orange-
yellow powder. 1H NMR (400 MHz, [D6]DMSO) d: 10.28 (s, 1H,
OH), 9.76 (s, 2H, 2OH), 9.39 (s, 1H, OH), 7.80 (d, J ¼ 8 Hz, 1H, Ar-
CH), 7.70 (d, J ¼ 8 Hz, 1H, Ar-CH), 7.57 (m, 2H, Ar-CH), 7.01 (d,
J ¼ 8 Hz, 1H, Ar-CH), 6.81–6.83 (m, 2H, Ar-CH), 6.71–6.76 (m,
2H, Ar-CH), 5.61 (d, J ¼ 8 Hz, 2H, CH2), 2.25 (s, 3H, CH3). 13C
NMR (100 MHz, [D6]DMSO) d: 153.4, 153.1, 147.5, 142.4, 137.8,
136.0, 130.9, 130.1, 127.4, 127.1, 126.0, 123.0, 119.5, 119.0,
115.7, 106.9, 46.3, 21.3. TOF MS (EIþ): C21H18N2O4, calcd. for
362.1267, found 362.1269. HPLC (40:60 to 0:100 H2O/CH3OH)
purity ¼ 98.68%, tR ¼ 12.79 min.
5-(1-(2-Methylbenzyl)-1H-benzo[d]imidazol-2-yl)benzene-
1,2,3-triol (6c): Rf ¼ 0.55 (H2O/acetic acid/petroleum ether/
ethyl acetate 1:2:2:8). Yield: 48.7 mg, 54.7%, orange-yellow
powder. 1H NMR (400 MHz, [D6]DMSO) d: 9.89 (s, 2H, 2OH),
9.39 (s, 1H, OH), 7.84 (d, J ¼ 8 Hz, 1H, Ar-CH), 7.67 (d, J ¼ 8 Hz,
1H, Ar-CH), 7.34–7.40 (m, 3H, Ar-CH), 7.27 (m, 1H, Ar-CH), 7.24
(d, J ¼ 8 Hz, 1H, Ar-CH), 7.18 (m, 1H, Ar-CH), 6.83 (s, 2H, Ar-CH),
5.75 (s, 2H, CH2), 2.29 (s, 3H, CH3). 13C NMR (100 MHz, [D6]
DMSO) d: 153.3, 147.5, 142.4, 137.8, 137.4, 136.8, 136.0, 130.3,
128.9, 128.4, 126.0, 125.6, 123.0, 119.5, 119.0, 106.9, 49.7, 18.8.
TOF MS (EIþ): C21H18N2O3, calcd. for 346.1317, found
346.1314. HPLC (40:60 to 0:100 H2O/CH3OH) purity ¼ 98.87%,
tR ¼ 11.19 min.
Bioevaluation
5-(1-(3-Methylbenzyl)-1H-benzo[d]imidazol-2-yl)benzene-
1,2,3-triol (6d): Rf ¼ 0.55 (H2O/acetic acid/petroleum ether/
ethyl acetate 1:2:2:8). Yield: 46 mg, 53.9%, orange-yellow
powder. 1H NMR (400 MHz, [D6]DMSO) d: 9.89 (s, 2H, 2OH),
9.39 (s, 1H, OH), 7.86 (d, J ¼ 8 Hz, 1H, Ar-CH), 7.69 (d, J ¼ 8 Hz,
1H, Ar-CH), 7.56–7.68 (m, 1H, Ar-CH), 7.35–7.37 (m, 2H, Ar-CH),
7.23 (d, J ¼ 4 Hz, 1H, Ar-CH), 7.18 (s, 1H, Ar-CH), 6.98 (d,
J ¼ 4 Hz, 1H, Ar-CH), 6.84 (s, 2H, Ar-CH), 5.75 (s, 2H, CH2), 2.31
(s, 3H, CH3). 13C NMR (100 MHz, [D6]DMSO) d: 153.3, 147.5,
142.4, 138.3, 137.8, 136.3, 136.0, 130.9, 128.5, 126.0, 124.6,
123.0, 119.5, 119.0, 106.9, 52.5, 21.6. TOF MS (EIþ):
Fluorescence polarization-based binding assay (FPAs)
A 21-residue Bid BH3 peptide (residues 79–99) [28] bearing a
6-carboxy-fluorescein succinimidyl ester fluorescence tag
(FAM-Bid) was synthesized at HD Biosciences (Shanghai,
China) [32]. Recombinant protein of Bcl-2 and Mcl-1 was
synthesized and purified from bacteria of Escherichia coli
BL21 as described in our previous study [33].
For the competitive binding assay for Bcl-2 protein, FAM-
Bid peptide (5 nM) and Bcl-2 protein (40 nM) were preincu-
bated in the assay buffer (100 mM potassium phosphate, pH
7.5; 100 mg/mL bovine gamma globulin; 0.02% sodium azide).
Each inhibitor was first dissolved in pure DMSO to obtain a
4 mM stock solution. Then the stock solution was diluted
successively to get the solution with different concentration
gradients (400 mM, 200 mM, 100 mM, 50 mM, 25 mM,
12.5 mM, 6.25 mM, 3.125 mM, 1.5625 mM and 0.78125 mM).
Next, serial dilutions of compounds were added. After 30 min
incubation, the polarization values were measured using the
Spectra Max M5 Detection System in a black 96-well plate.
Saturation experiments determined that FAM-Bid binds to the
Bcl-2 protein with a Kd value of 8 nM. For Mcl-1, assays were
performed in the same manner as that for Bcl-2 with the
following exceptions: 50 nM Mcl-1 and 10 nM FAM-Bid
peptide were used in the assay buffer (25 mM Tris, pH 8.0;
C
21H18N2O3, calcd. for 346.1317, found 346.1319. HPLC
(40:60 to 0:100 H2O/CH3OH) purity ¼ 98.36%, tR ¼ 11.14 min.
5-(1-(4-Methylbenzyl)-1H-benzo[d]imidazol-2-yl)benzene-
1,2,3-triol (6e): Rf ¼ 0.55 (H2O/acetic acid/petroleum ether/
ethyl acetate 1:2:2:8). Yield: 48 mg, 53%, orange-yellow
powder. 1H NMR (400 MHz, [D6]DMSO) d: 9.98 (s, 2H, 2OH),
9.45 (s, 1H, OH), 7.83 (d, J ¼ 8 Hz, 1H, Ar-CH), 7.66 (d, J ¼ 8 Hz,
1H, Ar-CH), 7.33–7.39 (m, 2H, Ar-CH), 7.22 (d, J ¼ 4 Hz, 2H, Ar-
CH), 7.11 (d, J ¼ 4 Hz, 2H, Ar-CH), 6.82 (s, 2H, Ar-CH), 5.75 (s,
2H, CH2), 2.16 (s, 3H, CH3). 13C NMR (100 MHz, [D6]DMSO) d:
153.3, 147.5, 142.4, 137.8, 136.0, 134.3, 128.9, 127.2, 126.0,
123.0, 119.5, 119.0, 106.9, 52.2, 21.3. TOF MS (EIþ):
� 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
9

Arch. Pharm. Chem. Life Sci. 2015, 348, 1–11
Z. Zhang et al.
150 mM NaCl). FAM-Bid peptide binds to the Mcl-1 protein
with a Kd value of 1.9 nM. R-(À )-Gossypol was used as
comparison in our assay. The Ki value for each inhibitor was
calculated using the equation Wang et al. have developed for
FP-based assays. The computer program for calculating Ki
values for FP-based assays is available free of charge at: http://
sw16.im.med.umich.edu/software/calc ki/ [33].
Caspase activation assays [8]
The activity of caspase-8 and caspase-9 was determined using
ApoAlert Profiling Plate (Clontech, #630225) according to the
manufacturer’s protocol. The release of fluorochrome 7-
amino-4-methyl coumarin was analyzed at 380 nm excitation
and 460 nm emission using a multiplate fluorescence spec-
trophotometer. Data shown were mean � SE of three
independent experiments in duplicate and expressed in
arbitrary fluorescence units per milligram of protein.
Annexin-V/propidium iodide assay [34]
Apoptosis analysis was performed using an annexin-V/
propidium iodide (PI) apoptosis detection kit (Roche, Indian-
apolis, IN) according to manufacturer’s instructions. Briefly,
cells were treated with inhibitors at different concentrations
and time-points. Cells were harvested, washed with ice-cold
PBS and then stained with annexin-V-FITC and PI for 15 min at
room temperature in the dark. Stained cells were analyzed in
a FACS calibur flow cytometer. Annexin-V (þ) and PI (À ) cells
wereconsideredearly-stageapoptoticcells, whileannexinV(þ)
and PI (þ) cells were measured as late-stage apoptotic cells.
Immunoprecipitation [8]
Cells (25 � 106) were lysed in 1% CHAPS buffer. Whole-cell
lysates were obtained, 500 mg total proteins were precleared
with protein A-Sepharose and incubated overnight with 5 mg
of the specific antibody. Immunocomplexes were captured
with either protein A-Sepharose or protein G-Agarose (Sigma
Chemical Co.). The presence of immunocomplexes was
determined by immunoblot analysis.
Molecular docking
Enzyme-linked immunosorbent assay (ELISA) [28]
The 3D structure of Mcl-1 (PDB ID: 2PQK) was obtained from
the protein bank in the RCSB [35]. The mol2 structures of
the inhibitors were generated using Chembio3D Ultra 11.0
followed by energy minimization. AutoDock 4.2 program
equipped with ADT was used to perform the automated
molecular docking [36]. Grid maps covering residues that
were perturbed more than the threshold value of 0.1 ppm in
the binding groove of the proteins were defined for all
inhibitors in the AutoDock calculations using a grid spacing
about of 0.375 Å. For each docking job, 1000 hybrid GALS runs
were carried-out. A total of 1000 possible binding conforma-
tions were generated and grouped into clusters based on a
1.0 Å cluster tolerance. The docking models were analyzed
and represented using ADT.
An enzyme-linked immunosorbent assay was reported as
previous [24]. R-(À )-Gossypol was used as a positive con-
trol [28, 31]. For this assay, biotinylated Bim peptide (residues
81–106, biotin-(b)A-(b)A-D-M-R-P-E-I-W-I-A-Q-E-L-R-R-I-G-D-
EF-N-A-Y-Y-A-R-R-amide, hereafter called biotin-Bim) was
diluted to 0.09 mg/mL in SuperBlock blocking buffer in PBS
(Pierce Biotechnology, Inc, Rockford, IL, catalog #37515) and
incubated for 1.5 h in 96-well microtiter plates already coated
with streptavidin (Qiagen, catalog #15500) to allow the
formation of the complex between Biotin-Bim and strepta-
vidin. All incubations were performed at room temperature
unless otherwise noted. Each inhibitor was first dissolved in
pure DMSO to obtain a 10 mM stock solution. Then the stock
solution was diluted successively to get the solution with
different concentration gradients (200 mM, 100 mM, 10 mM,
5 mM, 1 mM, 0.5 mM, 0.1 mM, 0.05 mM, 0.01 mM, 0.005 mM, and
0.001 mM). For each tested inhibitor, different concentrations
of the inhibitor were incubated with 20 nM His-tagged Mcl-1
protein in PBS for 1 h with a final DMSO concentration of 4%.
The plates were washed three times with PBS containing
0.05% Tween-20. The inhibitor and protein mixture (100 mL)
was transferred to the plate containing the biotin-Bim/
streptavidin complex and incubated for 2 h. The plate was
then washed as before and mouse anti-His antibody that was
conjugated with horseradish peroxidase (Qiagen, catalog
#34460) was added into the wells and incubated for 1 h. The
plate was then washed with PBS containing 0.05% Tween-20.
Finally, TMB (100 mL, Beyotime, catalog #P0209) was added to
each well; the enzymatic reaction was stopped after 30 min by
addition of H2SO4 (100 mL, 2 M). Absorbances were measured
with a TECAN GENios (Swiss, TECAN) microplate reader using
a wavelength of 450 nm. Three independent experiments
were performed with each inhibitor to calculate average IC50
value and standard deviation (SD). (À )-Gossypol was used as
comparison in our assay.
This work was supported by the National Natural Science
Foundation of China (21372036, 81272876, 21402022).
The authors have declared no conflict of interest.
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