Discovery and structure-activity relationship studies of N-substituted indole derivatives as novel Mcl-1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.03.028

Myeloid cell leukemia-1 (Mcl-1) is an important antiapoptotic protein functioning through protein-protein interactions. We discovered LSL-A6 (2-((2-carbamoyl-1-(3-(4-methoxyphenoxy)propyl)-1H-indol-6-yl)oxy)acetic acid) with a novel N-substituted indole scaffold to interfere Mcl-1 binding as a novel Mcl-1 inhibitor. Molecular modeling indicated that this compound binds with Mcl-1 by interaction with P2 and R263 hot-spots. Structure modification focused on several moieties including indole core, hydrophobic tail and acidic chain were conducted and structure-activity relationship was analyzed. The most potent compound 24d which exhibited K_i value of 110?nM for interfering Mcl-1 binding was obtained after hit-to-lead modification.

Full text of DOI:10.1016/j.bmcl.2017.03.028

Accepted Manuscript
Discovery and structure-activity relationship studies of N-substituted indole de-
rivatives as novel Mcl-1 inhibitors
Shenglin Luan, Qi Ge, Yedong Chen, Mingyang Dai, Jinyu Yang, Kun Li, Dan
Liu, Linxiang Zhao
PII:
S0960-894X(17)30263-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.03.028
BMCL 24780
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 January 2017
18 February 2017
14 March 2017
Please cite this article as: Luan, S., Ge, Q., Chen, Y., Dai, M., Yang, J., Li, K., Liu, D., Zhao, L., Discovery and
structure-activity relationship studies of N-substituted indole derivatives as novel Mcl-1 inhibitors, Bioorganic &
Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.03.028
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1
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Discovery and structure-activity relationship studies of N-substituted indole
derivatives as novel Mcl-1 inhibitors
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Shenglin Luana, Qi Gea, Yedong Chena, Mingyang Daia, Jinyu Yanga, Kun Lia, Dan Liua*,
Linxiang Zhaoa*
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a Key Laboratory of Structure-Based Drugs Design & Discovery of Ministry of Education,
Shenyang Pharmaceutical University, Shenyang 110016, China
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* Corresponding authors:
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E-mail address: linxiang.zhao@vip.sina.com (Linxiang Zhao); sammyld@163.com (Dan Liu).
Tel.: +86 024 43520221;
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Abstract
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Myeloid cell leukemia-1 (Mcl-1) is an important antiapoptotic protein functioning through
protein-protein
interactions.
We
discovered
LSL-A6
(2-((2-carbamoyl-1-(3-(4-
methoxyphenoxy)propyl)-1H-indol-6-yl)oxy)acetic acid) with a novel N-substituted indole
scaffold to interfere Mcl-1 binding as a novel Mcl-1 inhibitor. Molecular modeling indicated that
this compound binds with Mcl-1 by interaction with P2 and R263 hot-spots. Structure
modification focused on several moieties including indole core, hydrophobic tail and acidic chain
were conducted and structure-activity relationship was analyzed. The most potent compound 24d
which exhibited Ki value of 110 nM for interfering Mcl-1 binding was obtained after hit-to-lead
modification.
20
Keywords: Apoptosis; Bcl-2; Mcl-1; Structure-based design
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Evasion of apoptosis is a hallmark of cancer and a contributor of resistance to current
1, 2
chemotherapies.
The B-cell lymphoma-2 (Bcl-2) family of proteins are the vital regulators of
3, 4
mitochondrial apoptosis pathway,
which include three types of proteins: the antiapoptotic
proteins (Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and Bfl-1/A1), the proapoptotic proteins (Bak, Bax and Bok)
1

5
1
2
3
and the BH3-only proteins (Bim, Puma, Noxa etc.). Overexpression of antiapoptotic Bcl-2
proteins was disclosed in many cancers including leukemia, melanoma, lung, breast, prostate,
pancreatic, ovarian and cervical cancers. 6, 7
4
5
Interfering interactions of Bcl-2 antiapoptotic proteins and proapoptotic proteins to induce
apoptosis becomes a promising anticancer strategy. Potent Bcl-2 and/or Bcl-xL inhibitors have
been developed in the last decades. Navitoclax (ABT-263), binding to Bcl-2, Bcl-xL and Bcl-w,
has provided promising results in clinical trials despite of the on-target side-effect
thrombocytopenia (caused by inhibition of Bcl-xL). 8, 9 More recently, the selective Bcl-2 inhibitor
venetoclax (ABT-199) avoiding the side effect of Bcl-xL inhibition of navitoclax has been
approved by FDA for the treatment of chronic lymphocytic leukemia with 17p deletion and at
least one prior therapy.10, 11
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Neither venetoclax nor navitoclax has Mcl-1 inhibitory activity and the high Mcl-1 levels
12, 13
cause the loss of efficacy of both agents in several types of tumors.
Moreover, several
publications showed that Mcl-1 protein overexpression is a critical factor conferring resistance to
12, 14-16
navitoclax and other widely used anticancer agents.
Decreased or silenced Mcl-1
expression has shown tumor growth inhibitory effects and/or enhanced chemo-sensitivity in
17
cancer cells. Indirect inhibition of Mcl-1 through Noxa upregulation could effectively induce
18
apoptosis in acute myeloid leukemia.
target for developing therapeutics.
Mcl-1 is being considered as an attractive anticancer
20
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24
With significant effort has been devoted towards the development of Mcl-1 inhibitors,
numbers of small molecules comprising diverse chemotypes have been reported and none of
19-32
authentic Mcl-1 inhibitors enters the clinical trials.
Thus, development of novel Mcl-1
inhibitors still represents an unmet clinical need. Here we describe a hit-to-lead optimization and
related SAR studies of Mcl-1 inhibitors based on a novel N-substituted indole scaffold.
2

1
(Figure 1 should be listed here)
2
3
Figure 1. The recently reported Mcl-1 inhibitors.
4
5
A small in-house compound library comprising synthetic molecules and natural product
derivatives were screened for inhibitory activity of Mcl-1 using fluorescence polarization binding
assay (FP assay). Bid-BH3 peptide was labeled with fluorescein as the probe to monitor the
competitive binding of these compounds to Mcl-1. The traditional Mcl-1 inhibitor (−)-gossypol
was used as control. LSL-A6 (14a) bearing N-substituted indole scaffold was one of validated hits
with Ki value of 7.78 μM. To guide the further optimization of hit compound 14a, molecular
docking studies were performed using the crystal structure (PDB code: 4HW3) of Mcl-1 in
complex with a reported inhibitor (1, Figure 1). 24 A low energy docking solution was depicted in
Figure 2. The docking result reveals that compound 14a binds with Mcl-1 protein by the
occupation of P2 pocket and the formation of salt bridge with R263, the two most important hot-
spots of Mcl-1 as reported. 24, 33 In order to facilitate description, compound 14a was divided into
6
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10
11
12
13
14
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1
2
3
4
5
6
7
three moieties including indole core, hydrophobic tail and acidic chain (Figure 2). In detail, the hit
compound 14a interacts with R263 through a salt bridge mediated by carboxyl group and a
hydrogen bond by oxygen atom on the acidic chain. The 1H-indole-2-carboxamide scaffold
occupies the upper P2 pocket, while the 4-methoxyphenyl on hydrophobic tail inserts into the
bottom of P2 pocket through a saturated carbon chain. Both of these two moieties could interact
with P2 hydrophobic residues that include M250, V253 and F270. Based on the predicted binding
mode, we made further optimization of hit compound 14a to develop potent Mcl-1 inhibitors.
8
(Figure 2 should be listed here)
9
10
11
12
13
Figure 2. The structure and predicted binding mode of LSL-A6 (14a) bound to Mcl-1 (PDB:
4HW3). Pink carbons; heteroatoms colored by atom type; the amino acid side chains interacting
with ligand were colored as green. The pink dashed lines present hydrophobic interactions; the
orange dashed lines present salt bridges; the green dashed lines present hydrogen bonds.
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15
16
17
18
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20
21
22
All the side chains and indole derivatives were prepared according to methods outlined in
Schemes 1−2. Experimental details are described in the Supplementary Information. Side chains
7a-7m were prepared by substitution reaction of dibromoethane or dibromopropane with
commercial phenols or thiophenols. The synthetic route employed for preparation of the 6-
hydroxy-1H-indole-2-carboxamide (11a) was reported previously and similar routes were used to
prepare 11b-11d. 34 By nucleophilic substitution reactions of 11a-11d with ethyl bromoacetate or
methyl o-(bromomethyl)benzoate, key intermediates 12a-12e were synthesized. Intermediates
13a-13i were obtained via another nucleophilic substitution of 12a-12e with various side chains
(7a-7d and 7j) which then hydrolyzed under base condition to afford target compounds 14a-14i.
4

1
2
Compounds 17a-17p, 24a-24j were obtained by a simplified route including just two easy
substitution reactions and one hydrolysis reaction. Briefly, commercial hydroxyindoles
successively reacted with ethyl bromoacetate (or ethyl 2-bromobutyrate and methyl
bromomethylbenzoates) and side chains to afford ester analogues 16a-16p (or 23a-23j) which
were then converted to target compounds by general hydrolyzed condition. Specially, the hydroxyl
derivative 18 was achieved through a reduction of compound 16j with lithium aluminum hydride.
Compound 21 was synthesized through similar procedures with indole-6-carboxylic acid as
starting material in three-step feasible reactions including esterification, nucleophilic substitution
and hydrolysis. Compounds 20a-20c were prepared through N-alkylation reactions with different
indoles.
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5
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7
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(Scheme 1 should be listed here)
12
13
Scheme 1. Synthetic routes of side chains 7a-7m and 2-substituted indole derivatives 14a-14i
Reagents and conditions: a) substitued phenol or phenthiol, K2CO3, MeCN, reflux; (b) ethyl
azidoacetate, C2H5ONa, C2H5OH, -5-0 oC; (c) xylene, 135 oC; (d) NaOH/H2O, CH3OH, reflux; (e)
various amine, HOBt, EDCI, Et3N, CH2Cl2, r.t.; (f) BBr3, CH2Cl2, -30 oC; (g) BrCH2COOEt or
methyl o-(bromomethyl)benzoate, Cs2CO3, MeCN, r.t.; (h) side chains, Cs2CO3, MeCN, reflux; (i)
LiOH, THF/H2O, r.t..
14
15
16
17
18
5

1
(Scheme 2 should be listed here)
2
3
4
5
6
Scheme 2. Synthetic routes of N-substituted indole derivatives 17a-17p, 20a-20c, 21 and 24a-24j
Reagents and conditions: (a) Br(CH2)aCOOEt (a= 1 or 3) or methyl o,m,p-(bromomethyl)benzoate,
Cs2CO3, MeCN, r.t.; (b) side chains, Cs2CO3, MeCN, reflux; (c) LiOH, THF/H2O, r.t.; (d) LiAlH4,
THF, r.t.; (e) SOCl2, EtOH, reflux.
7
8
As shown in Figure 2, the polar 2-carbamoyl group mismatched the hydrophobic
characteristic of P2 pocket and the docking result showed no related interaction with Mcl-1. We
hypothesized that this polar group was unnecessary and might have adverse effect on potency. An
analogue without 2-carbamoyl group was prepared to verify this hypothesis. According to the data
in Table 1, compound 17a (Ki =4.42 μM) show nearly 2-fold increase than the counterpart, which
indicated that 2-carbamoyl was not an essential pharmacophore. Follow-up studies reconfirmed
the negligible contribution of that group (by comparing 14a-14d to 17a-17d). Moreover, the
deletion of 2-carbamoyl group improved the solubility of compounds which simplified the
purification process (data not show). Significantly, compounds without a carbamoyl group were
easily obtained by just three steps, which could facilitate future optimization efforts.
9
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12
13
14
15
16
6

1
2
The predicted binding mode represented that the hydrophobic P2 pocket of Mcl-1 was
partially occupied by p-methoxyphenyl of hit compound 14a, we assumed that sufficient
exploiting of this sub-pocket by introducing steric and hydrophobic groups could be beneficial to
potency. Then, the SAR studies of hydrophobic tail were conducted. Initially, removal of para-
methoxy group (17e) totally deprived the Mcl-1 inhibitory activity, indicating the significant role
of substituent groups of distal phenyl. The SAR analysis was done based on a series of analogues
with various substituted phenyl on the hydrophobic tail. Installation of simple small groups such
as methyl and chlorine (17f-17i) exhibited slight decreased potency (Ki varied from 9.89 μM to
12.87 μM) except for the incorporation of 3’,5’-di-Me-4’-Cl-phenyl which produced a 5-fold more
potent inhibitor 17j than 14a (Ki =1.50 μM). Interestingly, this substitution pattern was preferred
3
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16
17
18
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20
21
22
23
24
25
21-24
in several recent publications and considered as anchoring unit of some Mcl-1 inhibitors.
Several kinds of steric and hydrophobic groups were also introduced into P2 binding moiety and
produced 2-fold to 4-fold improvement on binding affinity such as t-butyl (14b, Ki=1.87 μM),
phenoxyl (14c, Ki=3.24 μM) and naphthyl (14d, Ki=2.57 μM). The tolerance of larger and more
hydrophobic groups manifested that the hydrophobic tail was probably positioned at the Mcl-1
deepest hydrophobic P2 pocket as our prediction. The linker length and heteroatom of
hydrophobic tail also influenced the binding ability. Based on the molecular modeling, only 3-
atoms or 4-atoms linker could properly place the substituted phenyl into the bottom of P2 pocket.
Compounds 17k and 17l bearded shorten linkers and produced 8-fold and 5-fold reduction
compared to counterparts 17b and 17j respectively, indicating that the 3-atoms linker was
inappropriate. Moreover, replacement of the oxygen on the linker with a more hydrophobic sulfur
atom (17m) resulted in marginal decrease on potency. In summary, the substitutions on
hydrophobic tail and linker length have crucial roles on binding affinity, which should be
optimized precisely. In our SAR research, the preferable substitution patterns were 4’-t-butyl and
3’, 5’-di-Me-4’-Cl and the original linker should be retained.
26
27
(Table 1 should be listed here)
Table 1. The structures and in vitro Mcl-1 inhibitory activity of 14a-14d and 17a-17m
7

1
Mcl-1 a
(Ki±SD, μM)
7.78±1.21
5.38±1.13
4.13±0.44
10.26±0.71
4.42±0.83
1.87±0.28
3.24±0.57
2.57±0.79
>30
Compd.
R1
Ar
A
n
CONH2
CONH2
CONH2
CONH2
4-OCH3-phenyl
4-t-Bu-phenyl
4-phenoxyphenyl
1-Naphthyl
4-OCH3-phenyl
4-t-Bu-phenyl
4-phenoxyphenyl
1-Naphthyl
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
S
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
2
3
14a
14b
14c
14d
17a
17b
17c
17d
17e
17f
17g
17h
17i
17j
17k
17l
H
H
H
H
H
H
H
H
H
H
H
H
H
phenyl
3-Me-phenyl
3,5-di-Me-phenyl
4-Cl-phenyl
3-Me-4-Cl-phenyl
3,5-di-Me-4-Cl-phenyl
4-t-Bu-phenyl
>30
12.53±1.80
12.87±1.33
9.89±1.13
1.50±0.19
8.19±1.01
11.61±2.05
2.38±0.35
0.20±0.05
3,5-di-Me-4-Cl-phenyl
4-t-Bu-phenyl
17m
Gossypol
a
2
3
Values were measured by FP assay for inhibition constant (Ki). The values are the mean ± SD of three
independent experiments.
4
5
The acidic chain which mimics the conserved Asp of BH3-only peptides was considered as a
crucial pharmacophore and further optimization was focused on that. Firstly, adjustment of the
attached site (17j, 17n and 17o) and length of acidic chain (17j, 17p and 21) produced no obvious
discrepancy among these analogs, which showed that this moiety was rather tolerated of
modification. Several literatures have reported the extreme importance of acidic group without
which the binding affinity for Mcl-1 was totally deprived. 24 Interestingly, our compounds whose
carboxyl was masked by ethyl ester (16j) or reduced to alcohol (18) just exhibited marginal
decreased potency. By contrast, compounds 20a-20c showed no inhibitory activity against Mcl-1.
Docking experiments were employed to clarify the different potency of 16j, 18 and 20a-20c
(Supplementary information, Figure S1). Their different capacity of forming H-bond with R263
might be the main factor which has impact on binding affinity. Based on our docking results,
compounds 16j and 18 still could form three H-bonds with R263 mediated by oxygen atoms at
acidic chain despite the carboxyl group has been masked. By comparison, compound 20a just
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12
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14
15
16
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1
2
form one H-bond with R263 while 20b and 20c form no interactions with R263, which probably
result in their deprivation of potency.
3
4
(Table 2 should be listed here)
Table 2. The structures and in vitro Mcl-1 inhibitory activity of 16j, 17n-17p, 18-21
5
Mcl-1 a
Compd.
a
R1
(Ki±SD, μM)
2.0±0.34
1.49±0.17
2.68±0.72
3.37±0.36
2.10±0.33
>30
4
5
6
6
6
6
6
6
6
OCH2COOH
OCH2COOH
O(CH2)3COOH
OCH2COOEt
OCH2CH2OH
COOEt
17n
17o
17p
16j
18
20a
21
20b
20c
COOH
CH3
3.0±0.84
>30
H
>30
0.20±0.05
Gossypol
a
6
7
Values were measured by FP assay for inhibition constant (Ki). The values are the mean ± SD of three
independent experiments.
8
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The substantial increase on potency was still not obtained after the first round optimization,
although we indeed achieved a little progress such as 5-fold improved potency. We speculated that
the volume of our compounds were too small to occupy the entire Mcl-1 groove effectively, when
compared with the natural binding partners (e.g. BH3 peptides) or other recently reported
inhibitors exemplified by A-1210477 (5). 27 Based on the structural data of Mcl-1, the residue of
R263 is rather flexible and solvent-exposed, leaving large room for further optimization on acidic
chain. In addition, the previous SAR results indicated that modification on this moiety seems to be
more tolerated. Consequently, we made more optimizations on the acidic chain. To match the
hydrophobic characteristic of Mcl-1 groove, the aliphatic carboxylic acid was replaced by benzoic
acid. Rationally, the extra benzene ring is expected to form additional interactions with
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11
12
13
14
15
16
17
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1
2
surrounding hydrophobic residues while the carboxyl maintains the favorable interactions with
R263. Besides, the enlarged size of molecules might restrict the flexible conformations of our
molecules, which might be beneficial to potency. Novel indole analogue incorporating with the
benzoic acid (24a) confirmed this assumption. A sub-micromole level binding affinity (Ki= 0.80
μM) was observed by FP assay, which showed almost 10-fold improvement by comparison with
compound 14a. The attached positions of carboxyl group on phenyl might have influence on
potency. As shown in Table 3, both the ortho (24a, 24d, 24e, 24i with Ki= 0.11~0.80 μM) and
meta (24b, 24f, 24g, 24j with Ki= 0.15~0.74 μM) carboxyl group on phenyl have more favorable
effect on binding affinity than the para-substituted derivatives (24c, 24h with Ki= 1.19, 1.63 μM),
which probably due to the different distance and spatial accessibility to R263. Besides, the
attached position of acidic chain also had influence on potency. Generally, attaching the acidic
chain on C4 (24i, 24j) and C5 (24d-24g) were more preferable than C6 (24a-24b). The most
potent compound 24d (Ki= 0.11 μM) was obtained with 70-fold increased binding affinity than hit
compound 14a, exhibiting higher potency for Mcl-1 than (−)-gossypol (Ki= 0.20 μM) in FP assay.
Molecular modeling experiments were employed to explain the improvement of binding affinity.
The docking results of 24a, 24d and 24i were depicted in Figure 3. The hydrophobic tail inserts
into the deep P2 pocket while the indole core occupy the upper P2 pocket, which is consistent with
the binding mode of hit compound 14a (Figure 1). Besides, the benzoic acid moiety could form
not only charge-charge interactions but also hydrophobic interactions with R263. Specially, 24d
and 24i could form additional H-bonds with N260 which may make extra contributions on binding
affinity. The overlay of 24d with bound conformation of Bim peptide (Figure 3D) showed that
compound 24d could mimic the hotspots h2 and D67 of Bim peptide.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
(Figure 3 should be listed here)
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1
2
3
4
5
6
7
Figure 3. The binding modes of typical compounds 24a (A), 24d (B) and 24i (C) and the overlay
of 24d with bound conformation of Bim peptide (D). Pink carbons; heteroatoms colored by atom
type; the amino acid side chains interacting with ligand were colored as green. The pink dashed
lines present hydrophobic interactions; the orange dashed lines present salt bridges; the green
dashed lines present hydrogen bonds. The Bim peptide was colored as offwhite and the hot spots
was colored as green and labeled as h1-h4 and D67.
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As we can see from Figure 3A-3C, the C2 position of indole core is in proximity to the
hydrophobic amino acid side chains of M231, V253 and F270. We assumed that introducing
additional hydrophobic groups at this position could form more interactions and improve the
binding affinity. Consequently, compounds 14e-14i were prepared to expand our inhibitors (Table
3). The results were preliminary yet promising which indicated that C2 is a modifiable position to
improve the potency. For instance, with the increase of the volume of R1, the binding affinity
showed rising trend and compound 14h (Ki= 0.20 μM) obtained 4-fold improvement compared
with parental compound 24a (Ki= 0.80 μM). The docking results showed that compound 14h
could form extra hydrophobic interactions with M231, L235 and V253 mediated by aliphatic ring
on C2 as anticipated (Supplementary information, Figure S2).
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11
12
13
14
15
16
17
18
19
20
21
22
23
Finally, we evaluated the selectivity profile of typical compounds for Bcl-2 protein. The data
in Table 3 indicated that our inhibitors could bind to Bcl-2 as similar potency as Mcl-1. Despite of
the structural similarity with Fesik’s inhibitor (1) and A-1210477 (5), our compounds showed no
obvious selectivity for Mcl-1. Compared with the Mcl-1 selective inhibitors 1 and 5, the acidic
groups of our inhibitors exhibit more flexibility which probably could adapt to the subtle
positional difference of conserved arginine between Mcl-1 and Bcl-2.
11

1
2
In conclusion, a series of novel N-substituted indole derivatives as Mcl-1 inhibitors based on
an in-house hit LSL-A6 (14a) have been designed and synthesized. The related structure-activity
relationship was conducted and the most potent compound 24d which exhibited Ki value of 110
nM for Mcl-1 was obtained. Although the sub-micromole level binding affinity of 24d might not
be expected to confer on-target cellular effects and the selectivity of 24d is not very specific, 35 this
molecule is still a promising lead as the several potential modifiable sites it has. Specially,
preliminary data indicated that C2 position of indole core was a promising modifiable position to
enhance the potency. The SAR studies and molecular modeling results indicated that all these
compounds probably bind with P2 pocket and R263 of Mcl-1 while barely with other hot-spots
such as P1, P3 and P4 pockets. Hence, further work will be focused on effectively occupying more
hot-spots within BH3 groove and enhance the selectivity for Mcl-1.
3
4
5
6
7
8
9
10
11
12
13
(Table 3 should be listed here)
Table 3. The structures and in vitro Mcl-1 inhibitory activity of 24a-24j and 14e-14h
14
Mcl-1 a
(Ki±SD, μM)
0.80±0.15
0.74±0.09
1.19±0.21
0.11±0.0058
0.31±0.04
0.20±0.04
0.25±0.02
1.63±0.20
0.14±0.01
0.15±0.01
0.95±0.04
0.89±0.09
0.57±0.07
Bcl-2 a
(Ki±SD, μM)
1.05±0.14
1.19±0.21
1.24±0.28
0.76±0.05
0.17±0.02
0.41±0.06
0.47±0.03
1.41±0.23
0.23±0.03
0.14±0.02
0.85±0.04
0.81±0.01
0.73±0.10
Compd.
a
b
R1
R2
2’
3’
4’
2’
2’
3’
3’
4’
2’
3’
2’
2’
2’
6
6
6
5
5
5
5
5
4
4
6
6
6
H
H
H
H
H
H
H
H
H
3,5-di-Me-4-Cl
3,5-di-Me-4-Cl
3,5-di-Me-4-Cl
3,5-di-Me-4-Cl
4-t-Bu
24a
24b
24c
24d
24e
24f
24g
24h
24i
24j
14e
14f
14g
3,5-di-Me-4-Cl
4-t-Bu
3,5-di-Me-4-Cl
3,5-di-Me-4-Cl
3,5-di-Me-4-Cl
3,5-di-Me-4-Cl
3,5-di-Me-4-Cl
3,5-di-Me-4-Cl
H
CONH2
CONHCH3
CONH(CH3)2
2’
6
3,5-di-Me-4-Cl
0.20±0.01
0.27±0.03
14h
2’
6
4-t-Bu
0.36±0.06
0.15±0.02
14i
12

0.20±0.05
0.42±0.11
Gossypol
a
1
2
Values were measured by FP assay for inhibition constant (Ki). The values are the mean ± SD of three
independent experiments.
3
Supplementary information
4
5
All the experimental protocols (chemistry, biological, computational protocols) and
spectrums of target compounds are detailed in Supplementary information.
6
Acknowledgment
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We thank Dr. Ting song (Dalian University of Technology) for the binding assay for Mcl-1
protein and Jian Wang (Shenyang Pharmaceutical University) for the insightful discussions on
molecular docking research. The work was supported by National Natural Science Foundation of
China(Grant No. 8173293).
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*Graphical Abstract