Synthesis, biological evaluation and structure-activity relationship of novel dichloroacetophenones targeting pyruvate dehydrogenase kinases with potent anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2021.113225

Pyruvate dehydrogenase kinases (PDKs) are promising therapeutic targets that have received increasing attentions in cancer metabolism. In this paper, we report the synthesis and biological evaluation of a series of novel dichloroacetophenones as potent PDKs inhibitors. Structure-activity relationship analysis enabled us to identify a potent compound 6u, which inhibited PDKs with an EC₅₀ value of 0.09 μM, and reduced various cancer cells proliferation with IC₅₀ values ranging from 1.1 to 3.8 μM, while show weak effect against non-cancerous L02 cell (IC₅₀ > 10 μM). In the A375 xenograft model, 6u displayed an obvious antitumor activity at a dose of 5 mg/kg, but with no negative effect to the mice weight. Molecular docking suggested that 6u formed direct hydrogen bond interactions with Ser75 and Gln61 in PDK1, and meanwhile the aniline skeleton in 6u was sandwiched by the conserved hydrophobic residues Phe78 and Phe65, which contribute to the biochemical activity improvement. Moreover, 6u induced A375 cell apoptosis and cell arrest in G1 phase, and inhibited cancer cell migration. In addition, 6u altered glucose metabolic pathway in A375 cell by decreasing lactate formation and increasing ROS production and OCR consumption, which could serve as a potential modulator to reprogram the glycolysis pathway in cancer cell.

Full text of DOI:10.1016/j.ejmech.2021.113225

European Journal of Medicinal Chemistry 214 (2021) 113225
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Synthesis, biological evaluation and structure-activity relationship of
novel dichloroacetophenones targeting pyruvate dehydrogenase
kinases with potent anticancer activity
,
,
d
,
,
Biao Xu a 1, Zhi-Peng Wang b 1, Qingwang Liu e 1, Xiaohong Yang a, Xuemin Li a,
Ding Huang a, Yanfei Qiu a, Kin Yip Tam c, Shao-Lin Zhang a **, Yun He a
,
, *
a School of Pharmaceutical Sciences, Chongqing Key Laboratory of Natural Product Synthesis and Drug Research, Chongqing University, Chongqing, 401331,
PR China
b Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing, PR China
c Faculty of Health Sciences, University of Macau, Taipa, Macau, PR China
d Chongqing School, University of Chinese Academy of Sciences Chongqing, PR China
e Institute of Health and Medical Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui, 230031, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Pyruvate dehydrogenase kinases (PDKs) are promising therapeutic targets that have received increasing
attentions in cancer metabolism. In this paper, we report the synthesis and biological evaluation of a
series of novel dichloroacetophenones as potent PDKs inhibitors. Structure-activity relationship analysis
Received 4 September 2020
Received in revised form
16 January 2021
Accepted 21 January 2021
Available online 28 January 2021
enabled us to identify a potent compound 6u, which inhibited PDKs with an EC50 value of 0.09
reduced various cancer cells proliferation with IC50 values ranging from 1.1 to 3.8 M, while show weak
effect against non-cancerous L02 cell (IC50 > 10 M). In the A375 xenograft model, 6u displayed an
mM, and
m
m
obvious antitumor activity at a dose of 5 mg/kg, but with no negative effect to the mice weight. Molecular
docking suggested that 6u formed direct hydrogen bond interactions with Ser75 and Gln61 in PDK1, and
meanwhile the aniline skeleton in 6u was sandwiched by the conserved hydrophobic residues Phe78 and
Phe65, which contribute to the biochemical activity improvement. Moreover, 6u induced A375 cell
apoptosis and cell arrest in G1 phase, and inhibited cancer cell migration. In addition, 6u altered glucose
metabolic pathway in A375 cell by decreasing lactate formation and increasing ROS production and OCR
consumption, which could serve as a potential modulator to reprogram the glycolysis pathway in cancer
cell.
Keywords:
Pyruvate dehydrogenase kinase
Proliferation
Cancer metabolism
Structure-activity relationship
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
One well-researched connection between glycolysis and
oxidative phosphorylation pathway is pyruvate dehydrogenase
Proliferating cancer cells exhibit
a
metabolic alteration,
complex (PDC) [6], which directly converts pyruvate into acetyl-
CoA by the action of pyruvate dehydrogenase and is the gate-
keeping enzyme linking the glycolysis and the oxidative phos-
phorylation [7]. PDC activity is mediated primarily by reversible
phosphorylation, in which phosphorylation of any one of three
serine residues on the alpha subunit of pyruvate dehydrogenase
renders the enzyme inactive [8,9]. Phosphorylation of PDC is
catalyzed by four isoforms of PDKs in human, namely PDK1-4,
which has been widely observed in various human solid tumors
[10] and linked to the oncogenic activation of AKT and HIF path-
ways that are integrated in the aberrant cancer metabolism [11].
Moreover, the continually overexpressed PDKs protein can be
hijacked by cancer cells to inactivate PDC, which contributes to the
featuring increased glycolysis and suppressed oxidative phos-
phorylation in mitochondria, even under aerobic conditions [1,2].
These altered metabolic pathways are known to derive from
oncogene mutations and aberrant protein expressions [3]. Target-
ing these mutated oncogenes and overexpressed proteins in
metabolic pathways is thus considered promising therapeutic op-
portunities in cancer treatment [4,5].
** Corresponding author.
* Corresponding author.
E-mail addresses: zhangsl@cqu.edu.cn (S.-L. Zhang), yun.he@cqu.edu.cn (Y. He).
B. Xu, Z.-P. Wang and Q. W. Liu contributed equally to this study.
1
https://doi.org/10.1016/j.ejmech.2021.113225
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

B. Xu, Z.-P. Wang, Q. Liu et al.
European Journal of Medicinal Chemistry 214 (2021) 113225
Warburg-identified glycolytic alterations [7]. Furthermore, it has
been shown that the knockdown of PDKs restores PDC to a normal
level and reverses these glycolytic effects [12]. Therefore, down-
regulating PDKs activity by inhibitors would be an ideal strategy
to modulate the dysregulated pyruvate metabolic pathway, thus
providing a promising approach for killing or, at least, greatly
reducing the growth of cancer cells.
So far, a number of compounds have been disclosed to inhibit
PDKs activity [13e19], but none of them has successfully advanced
into clinical application. AZD7545 is a PDKs inhibitor with a tri-
fluoromethylpropanamide warhead projecting to the lipoamide-
binding site of PDKs and forms a direct hydrogen bond interac-
tion with Ser75, while the 2-chlorophenyl group of AZD7545 is
sandwiched by the conserved hydrophobic residues Phe78 and
Phe65, as shown in Fig. 1B. Another well-known PDKs inhibitor is
dichloroacetate (DCA), which was used to treat patients with
mitochondrial abnormalities [20], and has recently been studied in
a clinical trial for cancer treatment [21]. However, its high effica-
cious dosage hampered its further clinical applications [22].
Dichloroacetophenone (DAP) is a DCA derivative that was discov-
ered from a high-throughput screening campaign by GSK [23]. In
addition, DAP weakly reduced A549 cancer cell proliferation, with
3a-f. However, we failed to obtain compounds 6a-s using the same
synthetic routes. Instead, we first prepared the dichlor-
oacetophenone (5) by chlorinating 4 in acetonitrile with 2 equiv-
alents of 1,3-dichloro-5,5-dimethylhydantoin (DCDMH) at 35 ꢀC for
18 h. The fluorine group in 5 was then substituted by various pri-
mary amines or secondary amines to furnish a series of novel
dichloroacetophenones (6a-s), as shown in Scheme 2.
In our molecular simulation assay, we found that the R2 groups
in 6a-s projected into the lipoyl-binding pocket in PDK, where
adjacent to the Ser75 residue. We expected that if we introduced a
hydrogen bond donor into the end of R2 group to form a direct
hydrogen bond interaction with Ser75 in PDK, which would
contribute to biochemical activity improvement. Bearing this in
mind, we therefore designed 6t and 6u. As shown in Schemes 3 and
1-(4-aminophenyl)ethan-1-one (7) reacted with a methyl Grignard
reagent at ꢁ10 ꢀC for 3 h to form 2-(4-aminophenyl)propan-2-ol
(8), which coupled with the dichloroacetophenone (5) to afford
the target compound 6t. Meanwhile, 2-(4-nitrophenyl)acetic acid
(9) was esterified by CH3OH to give methyl 2-(4-nitrophenyl)ace-
tate (10), and reacted with iodomethane in DMF to form compound
11, which was then reduced by DIBAL-H to generate 2-methyl-2-(4-
nitrophenyl)propan-1-ol (12). Finally, 12 was reduced and coupled
with dichloroacetophenone (5) to afford the target compound 6u.
All the dichloroketones were characterized by NMR and HRMS
spectra, and their purities were confirmed by HPLC with no less
than 95% (see Supporting Information).
an IC50 value of 15.9
mM. Moreover, the compound displayed only a
36.6% inhibitory rate at the tested concentration of 80
m
M in a
biochemical assay, which was clearly far from our satisfactory [24].
We previously reported the chemical modification of DAP, leading
to the discovery of DAP-64 (Fig. 1A), which inhibited PDKs activity
(EC50 ¼ 0.33
m
M) and cancer cells proliferation (IC50 ¼ 3.87
mM
2.2. Structure-activity relationship analysis
against A549) [25]. Continuing our efforts in discovering novel
cancer metabolic modulators [19,26], herein we report the syn-
thesis, biological evaluation, and structure-activity relationship
analysis of a series of novel dichloroacetophenones targeting PDKs,
which displayed an improved enzymatic potency and cellular ac-
tivity as compared to DAP and DAP-64.
The dichloroacetophenones 3a-f and 6a-u were evaluated for
their antiproliferative activity on MCF-7 and A549 cell lines at 1 and
10
mM, respectively, and the PDKs enzymatic activity was measured
at 1
mM. The results of these biological evaluations are summarized
in Table 1. DAP displayed poor antiproliferative activities against
the two cancer cell lines at both tested concentrations, and barely
inhibited PDKs activity at 1
cellular and biochemical activities as compared with DAP, which is
in perfect agreement with our previous results [24,25].
The introduction of various aromatic substituents at 4-position
of 2,2-dichloro-1-(3-nitrophenyl)ethan-1-one yielded a series of
dichloroacetophenones (3a-f), which exhibited significant
improvement of anticancer inhibitory activities as compared with
DAP (Table 1). However, the PDKs inhibitions by these compounds
mM. DAP-64 exhibited improved
2. Results and discussion
2.1. Chemistry and design of the target compounds
The general synthetic routes for the designed dichlor-
oacetophenones 3a-f and 6a-u were shown in Schemes 1-3. As
shown in Scheme 1, 4-bromo-3-nitroacetophenone (1) reacted
with various boronic acids in water/dioxane (v/v, 1:3) to afford 2a-f
by coupling reaction catalyzed by Pd(PPh3)4 or Pd(OAc)2, which
were then chlorinated in DMF with copper(II) chloride dehydrate
and lithium chloride at 90 ꢀC for 9 h to give the target compounds
were weak, with EC50 values were more than 10 mM, suggesting
that other mechanisms, not just PDKs inhibition could be involved
in their anti-proliferative effects. While keeping the nitro and
Scheme 1. Synthetic of dichloroacetophenones 3a-f. Reaction conditions and re-
agents: (a) For 2a-d and 2f, boronic acids, K2CO3, Pd(PPh3)4, water/dioxane (v/v, 1:3),
95 ꢀC, 6 h. For 2e, cyclopropylboronic acid, tricyclohexyl phosphine, K3PO4, Pd(OAc)2,
water/dioxane (v/v, 1:3), 100 ꢀC, 4 h. (b) Copper(II) chloride dehydrate, lithium chlo-
ride, DMF, 90 ꢀC, 9 h.
Fig. 1. (A) Chemical structures of the reported PDKs inhibitors. (B) Binding model of
AZD7545 with PDK1 (PDB code: 2Q8G).
2

B. Xu, Z.-P. Wang, Q. Liu et al.
European Journal of Medicinal Chemistry 214 (2021) 113225
Scheme 2. Synthetic of dichloroacetophenones 6a-s. Reaction conditions and reagents: (c) NH4Cl, acetonitrile, DCDMH, 18 h, 35 ꢀC. (d) Various primary amines or secondary
amines, K2CO3, DMF, rt, 3 h.
inhibited the growth of cancer cell lines with IC50 values ranging
from 0.33 to 1.2 mM, which suggest that 6m is not a specific PDK
inhibitor. We then modified 6m by incorporating a hydroxyl group
into the end of isopropylaniline skeleton to form 6t, though a direct
hydrogen bond interaction generated between 6t and Ser75 (Fig. 3),
the Phe78 is far away from the isopropylaniline skeleton, the
sandwiched hydrophobic interaction disappeared, so the PDK
inhibitory potency was slightly improved. While introducing a
hydroxymethyl group into 6m to afford compound 6u, which not
only retain the sandwich hydrophobic interaction, but also picked
up hydrogen bond interactions with Ser75 and Gln61. As expected,
6u displayed an improved PDK inhibitory potency, with an EC50
value of 0.09 mM, which outperform 6m and DAP-64. We finally
measured anticancer activity of 6m, 6s, 6t and 6u on various human
derived solid cancer cells, all four compounds exhibited promising
Scheme 3. Synthesis of compounds 6t and 6u. Reaction conditions and reagents: (a)
CH3MgBr, THF, ꢁ10 ꢀC, 3 h. (b) K2CO3, dichloroacetophenone 5, DMF, 12 h. (c) H2SO4,
CH3OH, reflux. (d) CH3I, NaOH, DMF, 0 ꢀC, 24 h. (e) DIBAL-H, THF, 0 ꢀC. (f) Pd/C, H2,
EtOH, rt, 12 h. (g) K2CO3, dichloroacetophenone 5, DMF, 12 h.
anticancer activities, with IC50 values ranging from 0.31 to 8.1
mM.
Meanwhile, 6m-t showed some cytotoxicity to L02 cell
(IC50 ¼ 1.5e7.8
mM), but not for 6u and DAP-64 in a high
dichloroacetophenone groups unchanged, the replacement of the
fluorine atom in compound 5 by cyclopropylamino, pyrrolidinyl,
morpholinyl, piperidinyl, and piperazinyl groups afford a series of
dichloroacetophenones (6a-6e), but they displayed a negligible
concentration.
2.3. Compound 6u bound to PDK isoforms
improvement of PDKs inhibition at 1 mM as compared to 3a-f. The
PDKs protein contains four sub-type isoforms, namely PDK1-4.
So next we have interest to explore the selective profile of 6u.
Firstly, we aligned the amino acids sequence of the lipoamide-
binding site of PDK1-4, as shown in Fig. 4A. The Ser75, Phe78,
and Phe65 residues in PDK1 is conserved in PDK2-4, suggesting 6u
might be a pan-isoform PDK inhibitor. We then docked 6u to PDK2-
4. However, the binding model for PDK2-4 is totally different from
PDK1. The 6u reaches into the deep lipoamide-binding pocket in
PDK2, missing the hydrogen bond interaction with Ser49 (Fig. 4B),
while for PDK4, 6u retains the Ser53 hydrogen bond interaction,
but missing the sandwiched hydrophobic interaction with Phe56
and Phe43 (Fig. 4D). In PDK3, 6u picked up an additional interaction
with a residue Lys173, which is a crucial amino acid in L2 domain in
PDC (Fig. 4C). Finally, we titrated 6u into PDK1-4 solution, and
measured Kd values using an isothermal titration calorimetry (ITC)
analysis. As shown in Fig. 4E and Fig S1 (see Supporting Informa-
potencies were greatly improved when some aniline substituents
were incorporated at the same position. Especially, 6m and 6s
exhibited dramatic improvements in biochemical activities, with
the PDK inhibitory rates are 80.0 and 65.8% at 1
measured a full-dose response inhibition of the compounds on
PDK, as shown in Table 2, the EC50 values are 0.34 and 0.55 M for
mM. Then we
m
6m and 6s, respectively. According our previous study [24], DAP-64
bound to the hydrophobic lipoamide-binding site in PDK. We then
docked 6m and 6s into the pocket, indicating that the substituted
anilines were sandwiched by the conserved hydrophobic residues
Phe78 and Phe65 in PDK (Fig. 2), which attributed to the
biochemical activity improvement. The replacement of the eNH in
6m by an oxygen (6q) or sulfur atom (6r) led to decrease of enzy-
matic activity, which might be explained by the restricted hydro-
phobic interaction between the benzene ring and Phe78 and Phe65
residues.
tion), the binding affinity for 6u to PDK1 is 0.21
mM, much higher
Compound 6m gave an EC50 value of 0.34 mM against PDK, and
than PDK2-4. According to our molecular docking study, 6u
3

B. Xu, Z.-P. Wang, Q. Liu et al.
European Journal of Medicinal Chemistry 214 (2021) 113225
Table 1
Biological evaluation of the prepared compounds 3a-f and 6a-u.
Comps
Antiproliferative inhibitory rate (%)
MCF-7
PDKs inhibition (%) at 1 m
M a
A549
10 (
10 (
mM)
1 (m
M)
m
M)
1 (mM)
3a
3b
3c
3d
3e
3f
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
6s
6t
6u
DAP
DAP-64
AZD7545
DOXd
56.2 1.4
89.1 1.8
91.6 1.1
97.8 0.88
87.8 3.3
43.5 2.9
96.0 0.2
94.6 2.4
90.4 1.2
95.8 0.35
57.0 2.3
90.5 2.5
11.9 4.0
21.4 0.9
97.1 0.52
95.9 0.18
88.9 1.8
93.8 2.0
94.0 0.8
90.5 2.5
93.2 1.3
97.8 1.6
85.7 5.8
79.8 7.1
96.4 7.1
92.8 3.2
95.3 4.8
42.2 5.3
73.4 3.7
NI
15.8 1.1
26.4 2.3
30.8 3.5
24.7 3.7
31.3 6.5
7.5 3.7
25.7 2.1
23.0 4.2
18.9 3.9
22.4 4.9
NH
92.6 0.6
96.0 1.8
95.3 1.0
93.6 0.95
95.9 2.1
94.4 2.1
95.7 0.54
97.3 0.34
95.6 1.6
97.1 1.7
93.1 2.2
96.6 0.30
58.9 1.6
80.9 0.81
97.4 1.2
97.7 0.36
97.3 1.1
97.4 0.70
97.0 1.7
96.6 0.30
98.6 0.8
98.9 0.2
90.7 3.4
76.3 2.5
93.2 3.8
96.3 5.8
98.6 7.5
55.2 4.8
98.2 7.8
NI
8.9 1.4
13.8 2.8
44.7 4.0
36.3 3.1
40.1 4.2
14.4 2.8
21.6 1.9
30.6 0.11
10.1 3.2
38.9 1.7
22.6 2.4
30.5 3.5
NI
25.6 0.5
5.9 1.1
12.1 0.8
21.4 3.8
17.3 2.2
26.5 4.1
17.5 5.2
32.4 2.7
36.9 3.9
40.2 10.1
48.6 2.5
20.5 3.3
17.4 2.8
8.1 1.2
60.7 7.3
55.7 4.1
64.4 4.7
68.7 3.6
80.0 7.9
66.4 7.5
88.2 5.8
50.1 2.3
39.4 1.7
19.5 5.8
65.8 9.7
48.8 3.3
98.7 10.4
24.2 5.3
NIb
NI
NI
26.3 1.2
29.2 3.5
12.1 2.2
18.8 5.1
38.6 3.3
24.2 5.3
31.6 2.5
17.9 4.2
28.7 4.0
18.9 3.2
41.2 4.7
33.9 7.8
38.9 8.8
NI
46.1 7.2
45.0 4.4
48.1 4.3
52.9 3.7
75.2 6.5
40.5 3.5
75.0 4.4
23.2 1.4
12.5 1.0
12.9 2.7
48.8 5.8
37.9 2.2
29.4 7.6
NI
c
36.6 4.4
NI
NI
18.7 1.7
NI
81.2 0.6
72.4 10.0
98.7 3.3
NTe
97.5 0.21
59.8 1.2
98.5 0.81
a
The PDKs inhibition rates were measured by the primary PDC enzymatic activity assay in the presence of the prepared compounds at 1 mM. The initial screen of cancer cells
proliferative inhibition rates (%) was run by using the MTT assay. Both bioassays were three independent measurements, and the values were reported as the average S.D; b
NI represents no inhibition; c PDKs inhibition rate was 36.6% for DAP at 80
m
M; d DOX represents Doxorubicin, a clinical anticancer drug, which was used a positive control in
e
the MTT assay; NT represents not tested.
Table 2
Effects of some selected compounds (6m, 6s, 6t, and 6u) on the growth of various cancer cell line human normal cell L02 (IC50
,
m
M) and PDKs function (EC50
, mM).
Comps.
IC50
(
mM)
PDKs (EC50, mM)
A549
MCF-7
SHSY-5Y
HCT116
A375
PC-3
L02
6m
6s
6t
6u
DAP-64
DAP
AZD7545
DCA b
0.33 0.09
1.2 0.17
3.1 0.50
3.2 0.14
3.5 0.11
18.7 1.6
NI a
1.21 0.30
1.9 0.22
2.98 0.55
2.4 0.15
7.8 1.4
21.4 4.0
NI
1.0 0.21
1.1 0.15
2.3 0.41
1.7 0.23
5.6 0.9
17.7 1.5
NI
0.7 0.11
0.5 0.08
1.5 0.22
2.1 0.31
6.2 1.1
12.9 1.7
NI
0.9 0.12
0.9 0.11
1.8 0.17
1.1 0.14
4.9 0.8
16.9 2.5
NI
0.38 0.05
0.47 0.07
2.34 0.30
3.8 0.45
8.7 1.3
23.1 2.0
NI
1.5 0.12
5.9 0.84
7.8 0.34
14.4 0.85
16.9 1.1
19.7 1.1
NI
0.34 0.07
0.55 0.11
1.49 0.20
0.09 0.01
0.33 0.07
>80
0.053
>100
19.5 2.1
13.4 0.9
17.4 2.5
9.8 0.15
20.1 3.3
15.7 1.4
21.7 3.1
a
NI indicates no inhibitory activity.
the unit of IC50 values of cell viability for DCA is mM.
b
exhibited the highest total binding scores with PDK3, but showed a
relative weak binding affinity to PDK3 in the ITC titration process,
which could be explained by the absence of the L2 domain in
protein solution in the titration process.
and 51.27%, respectively, suggesting the induction of the apoptosis
in A375 cells by 6u followed a dose-dependent manner, while for
DAP-64 at 5
mM, the percentages was 28.82%. Furthermore, we
measured the percentages of apoptotic population by 6u for 12, and
24 h, respectively, and as shown in Fig. 6, the apoptosis rates were
15.58% at 12 h, and 25.58% for 24 h, following a time-dependent
manner.
2.4. Compound 6u induced A375 cell apoptosis and cell cycle arrest
in G1 phase
We then examined the effect of 6u on cell cycle distribution
using the propidium iodide (PI) staining kit. A375 cells were treated
Since 6u exhibited the most potent enzymatic potency, it was
with 6u (1, 2.5, and 5 mM) or DAP-64 (5 mM) for 24 h, then stained
selected for further biological test. First, A375 cell was treated with
with PI and analyzed on a flow cytometer. As shown in Fig. 7, 6u led
to a significant accumulation of A375 cells at G1 phase from 52.65
6u (1, 2.5, and 5 mM) or DAP-64 (5 mM) for 36 h, then stained with
FITC Annexin V/PI dyes. As shown in Fig. 5, the percentages of
to 55.72% (1 mM), 60.75% (2.5 mM) and 66.86% (5 mM), accordingly,
apoptotic population in A375 cell treated with 6u were 14.07, 27.90
4

B. Xu, Z.-P. Wang, Q. Liu et al.
European Journal of Medicinal Chemistry 214 (2021) 113225
Fig. 2. Binding model of 6m and 6s with PDK1 (PDK code: 2Q8G). (A/D) 3D diagram of the proposed 6m/6s-PDK complex; (B/E) The 2D diagram of the hydrophobic contacts
between 6m/6s and PDK. The plots were generated by LigPlotþ, the spoked arcs represent residues making non-bonded contacts with the ligand; (C/F) Stereoscopic view of 6m/6s
in the binding pocket of PDK. The graphics of 3D views were drawn by PyMOL, and the amino acids involved in hydrophobic interaction were shown as stick, while the Ser75 was
highlighted as red.
while 58.34% for DAP-64 at 5
m
M. However, the G1 phase accu-
the red fluorescent J-aggregates. Mitochondrial depolarization is
indicated by an increase of the green fluorescence intensity (J-
monomer) when the concentrations of 6u is raising, as shown in
Fig. 9 (right). All these suggested that 6u depolarized the mito-
chondrial hyperpolarization and thus revived oxidative metabolism
in cancer cell.
mulation induced by 6u and DAP-64 may result from the com-
pounds’ off-target effect. At the same time, it reduced the cells at S
phase from 34.63 to 27.43%, and G2 phase from 9.64% to 5.71%,
respectively. The cell cycle arrest at G1 phase induced by 6u was on
concentration-dependent, which might be one of the possible
mechanisms for its cytotoxicity.
2.5. Compound 6u inhibited A375 cell migration
2.7. Compound 6u suppressed the phosphorylation of PDK and
affected the glycolysis pathway in A375 cancer cell
Cell migration was widely observed in the process of cancer cell
proliferation, we therefore studied the effect of 6u on A375 cell
migration. As shown in Fig. 8A and B, the wound healing and
Transwell assays indicated that A375 cell migration was signifi-
cantly reduced after treating with 6u as compared with the control
group, suggesting that 6u strongly inhibits cancer cell migration.
To verify the PDK inhibition leading to the reduction of the PDC
phosphorylation level, A375 cells were treated with 6u (1, 2.5, and
5
m
M) or DAP-64 (5
analyzed by western blotting experiment. As shown in Fig. 10A, 6u
reduced PDC phosphorylation (p(Ser293)E1
) level at 5 or 2.5 M, as
DAP-64 did at 5 M. Inhibition of PDK activates PDC, leading to a
mM) for 24 h, then PDC phosphorylation was
a
m
m
2.6. Compound 6u depolarized mitochondria membrane potential
switch of pyruvate metabolism from lactate production to oxidative
phosphorylation. Thus, the lactate formation and oxygen con-
sumption (OCR) will change in cells. So next, A375 cells were
(
Djm)
Cancer cells exhibit hyperpolarized Djm as compared with
treated with DAP-64 (5 mM), or 6u (1, 2.5, and 5 mM) for 4 h, and the
normal cells, in which a positive voltage gradient builds across the
inner mitochondrial membrane to the outside. Depolarization of
the Djm represents an effective strategy to inhibit cancer cell
growth. When A375 cell was treated with DAP-64, or 6u for 4 h,
much reduced stain by tetramethyl rhodamine methyl ester
(TMRM) was observed, suggesting mitochondrial depolarization by
6u in the cells (Fig. 9, left). To further validate the result, we used a
cationic dye, 5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimidazo-
lylcarbocyanine iodide (JC-1), which accumulated in mitochondria
of high potential and displayed as red color due to the formation of
changes in the intracellular lactate formation and OCR were
monitored. As shown in Fig. 10B, 6u dose independently reduced
the lactate formation in A375 cells, and increased OCR in a con-
centration dependent manner (Fig. 10C). In addition, inhibition of
PDKs would lead to an increase of mitochondria activity, therefore,
reactive oxygen species (ROS) production should increase. We
therefore measured the ROS production in A375 cell after treating
with 6u (1, 2.5, and 5 mM) for 4 h. As shown in Fig. 10D, more ROS
was generated with higher concentrations of 6u, indicating
enhanced cellular respiration activity.
5

B. Xu, Z.-P. Wang, Q. Liu et al.
European Journal of Medicinal Chemistry 214 (2021) 113225
Fig. 3. Proposed binding model of 6t and 6u with PDK1 (PDK code: 2Q8G). (A/D) 3D diagram of the proposed 6t/6u-PDK complex; (B/E) The 2D diagram of hydrogen bond
interaction and hydrophobic contacts between 6t/6u and PDK; (C/F) Stereoscopic view of 6t/6u in the binding pocket of PDK.
2.8. Compound 6u suppressed tumor growth in A375 xenograft
model
increasing ROS production and OCR consumption. All these data
demonstrated that 6u could be a promising lead for the develop-
ment of potent PDKs inhibitors.
Since 6u exhibited the most potent in vitro anticancer activities,
we next established an A375 xenograft model and performed an
in vivo efficacy study for 6u. Visible tumors were developed at the
inoculation sites 7 days after A375 cells were inoculated subcuta-
neously into the animal. Tumor bearing mice were then grouped
randomly (n ¼ 4) and intramuscular injected with vehicle (5%
4. Experimental section
4.1. Chemistry
All chemicals were purchased from commercial source and used
without further purification unless otherwise stated. The reactions
were monitored by thin-layer chromatography and carried out on
commercial Merck Kieselgel 60 F254 plates, which could be visual-
ized under UV light at 254 nm. Column chromatography was per-
formed on silica gel. 1H NMR spectra were obtained on an Agilent
400 MR spectrometer, while 13C NMR spectra were obtained with
proton decoupling on an Agilent 400 MR DD2 (100 MHz) or 600 MR
DD2 spectrometer and were reported in ppm with residual solvent
DMSO þ 95% HP-
b-CD (20%)), or compound 6u (5 mg/kg) once per
three day for a continuous 21 days, respectively. As shown in the
tumor growth curve in Fig.11A, treatment of 6u at a dose of 5 mg/kg
suppressed the tumor growth, but did not affect significantly the
body weight (Fig. 11C), suggesting the compound is well tolerated
at the dose. At 21 days, the tumors were harvested and weighted,
the vehicle group is higher than the drug administrated group
(Fig. 11B).
for internal standard (d 77.16 (CDCl3)). The chemical shifts were
reported in parts per million (ppm), the coupling constants (J) were
expressed in hertz (Hz) and signals were described as singlet (s),
doublet (d), triplet (t), as well as multiplet (m). The NMR data was
analyzed by MestReNova software. High-resolution mass spectra
were obtained on a Bruker SolariX 7.0 T spectrometer. Melting
points were recorded on a WRS-2A digital melting point apparatus.
All other chemicals and solvents were commercially available, and
were used as received.
3. Conclusions
In this paper, a series of novel PDK inhibitors with improved
potencies based on the DAP or DAP-64 structure has been devel-
oped. Combining their preliminarily antiproliferative activity
against MCF-7 and A549 cells, and their ability to inhibit enzymatic
activities, we identified a promising PDK inhibitor 6u, which dis-
played good antiproliferative activities against various cancer cell
lines. The PDKs enzymatic assay revealed that 6u inhibited the
enzyme with an EC50 value of 0.09
m
M, which is a potent activity as
4.1.1. General procedures for the synthesis of compounds 2a-d, and
2f
To a round-bottom flask, 2 mmol of 1-(4-bromo-3-nitrophenyl)
ethanone (1), 3 mmol of boronic acids, 6 mmol of K2CO3 and
0.06 mmol of Pd(PPh3)4 were added. Then the flask was flushed
compared to previous reported inhibitor. We also found that 6u
induced A375 cell apoptosis and arrested the cell cycle in G1 phase,
and inhibited the cell migration. In addition, 6u altered glucose
metabolic profile in A375 cells by decreasing lactate formation and
6

B. Xu, Z.-P. Wang, Q. Liu et al.
European Journal of Medicinal Chemistry 214 (2021) 113225
Fig. 4. Compound 6u bound to PDK isoforms. (A) Sequence alignment of the hydrophobic lipoamide-binding pocket of PDK1-4; (BeD) The proposed binding model of 6u with PDK2
a
(PDB code: 6LIL), PDK3 (PDB code: 1Y8O), and PDK4 (PDB code: 3D2R); (4) The Kd values derived from ITC titration. Compound 6u (100
solution. not detected.
mM) was titrated into PDK1-4 (10
mM)
b
with nitrogen via a rubber septum, water (6 mL) and dioxane (18 L)
were added. The resulting suspension was stirred, degassed and
heated for 6 h at 95 ꢀC. It was then cooled to room temperature,
diluted with water, and extracted with ethyl acetate (2 x 30 mL).
The combined organic layers were dried over Na2SO4, filtered and
concentrated. The crude residue was purified by silica gel chro-
matography to afford the desired compounds.
149.81, 149.38, 139.12, 137.33, 135.09, 132.59, 131.71, 129.52, 124.24,
121.30, 26.80.
1-(3-Nitro-4-(thiophen-3-yl)phenyl)ethanone (2f) was ob-
tained as
105e106 ꢀC. 1H NMR (400 MHz, CDCl3):
J ¼ 8 Hz, 1H), 7.63 (d, J ¼ 8 Hz, 1H), 7.42 (m, 2H), 7.09e7.11 (m, 1H),
2.67 (s, 3H); 13C NMR (101 MHz, CDCl3):
(ppm) 195.42, 149.29,
a
yellow solid after flash chromatography. M.p.
d
(ppm) 8.32 (s, 1H), 8.14 (d,
d
1-(2-Nitrobiphenyl-4-yl)ethanone (2a) was obtained as a white
136.68, 136.00, 134.74, 132.16, 131.39, 127.12, 126.94, 124.71, 123.90,
26.78.
solid after flash chromatography. M.p. 85e86 ꢀC. 1H NMR
(400 MHz, CDCl3): d (ppm) 8.39 (m, 1H), 8.19e8.16 (m, 1H), 7.57 (d,
J ¼ 8 Hz, 1H), 7.45e7.44 (m, 3H), 7.32e7.34 (m, 2H), 2.68 (s, 3H); 13
C
4.1.2. General procedure for the synthesis of compound 2e
NMR (101 MHz, CDCl3):
d (ppm) 195.51, 149.56, 140.48, 136.87,
To a round-bottom flask, 2 mmol of 1-(4-bromo-3-nitrophenyl)
ethanone (1), 3 mmol of cyclopropylboronic acid, 0.4 mmol of tri-
cyclohexyl phosphine, 10 mmol of K3PO4 and 0.02 mmol of
Pd(OAc)2 were added. The flask was flushed with nitrogen via a
rubber septum, water (1 mL) and dioxane were added, the resulting
suspension was stirred, degassed and heated for 4 h at 100 ꢀC. It
was then cooled to room temperature, diluted with water, and
extracted with ethyl acetate (2 x 30 mL). The combined organic
layers were dried over Na2SO4, filtered and concentrated. The crude
residue was purified by silica gel chromatography to afford the pure
136.42, 132.59, 131.50, 129.06, 129.00, 127.83, 124.07, 26.80.
1-(40-Methyl-2-nitrobiphenyl-4-yl)ethanone (2b) was obtained
as a yellow solid after flash chromatography. M.p. 82e83 ꢀC. 1H
NMR (400 MHz, CDCl3):
d
(ppm) 8.36 (s, 1H), 8.16 (d, J ¼ 8 Hz, 1H),
7.56 (d, J ¼ 8 Hz,1H), 7.27e7.21 (m, 4H), 2.67 (s, 3H), 2.41 (s, 3H); 13
C
NMR (101 MHz, CDCl3):
d (ppm) 195.56, 149.59, 140.47, 139.22,
136.60, 133.40, 132.52, 131.44, 129.77, 127.72, 124.07, 26.80, 21.42.
1-(40-Fluoro-2-nitrobiphenyl-4-yl)ethanone (2c) was obtained
as a white solid after flash chromatography. M.p. 95e96 ꢀC. 1H NMR
compound 2e. 1H NMR (400 MHz, CDCl3):
d (ppm) 8.35 (s, 1H), 8.05
(400 MHz, CDCl3):
d
(ppm) 8.40 (s, 1H), 8.18 (d, J ¼ 8 Hz,1H), 7.55 (d,
J ¼ 8 Hz, 1H), 7.33e7.30 (m, 2H), 7.17e7.13 (m, 2H), 2.69 (s, 3H); 13
C
(d, J ¼ 8 Hz, 1H), 7.19 (d, J ¼ 8 Hz, 1H), 2.63 (s, 3H), 2.47e2.43 (m,
1H), 1.19e1.17 (m, 2H), 0.84e0.83 (m, 2H); 13C NMR (101 MHz,
NMR (101 MHz, CDCl3):
d
(ppm) 195.41, 163.27 (d, JF ¼ 251 Hz),
149.51, 139.43, 137.06, 132.59, 132.44 (d, JF ¼ 3.3 Hz), 131.57, 129.77
CDCl3): d (ppm) 195.55, 151.13, 143.52, 135.27, 131.78, 127.58, 124.18,
26.67, 12.64, 9.67.
(d, JF ¼ 8.4 Hz), 124.16, 116.11 (d, JF ¼ 22 Hz), 26.81.
1-(2-Nitro-4’-(trifluoromethoxy)biphenyl-4-yl)ethanone (2d)
was obtained as a white solid after flash chromatography. M.p.
4.1.3. General procedure for the synthesis of compound 3a-f
To a round-bottom flask, 6 mmol of copper (II) chloride dihy-
drate, 6 mmol of lithium chloride, and 1 mmol of the corresponding
ketones (2a-f) were added. Then DMF (5 mL) was added and heated
99e101 ꢀC. 1H NMR (400 MHz, CDCl3):
d
(ppm) 8.43 (s, 1H), 8.20 (d,
J ¼ 8 Hz, 1H), 7.56 (d, J ¼ 8 Hz, 1H), 7.38e7.36 (m, 2H), 7.31e7.27 (m,
2H), 2.69 (s, 3H); 13C NMR (101 MHz, CDCl3):
(ppm) 195.35,
d
7

B. Xu, Z.-P. Wang, Q. Liu et al.
European Journal of Medicinal Chemistry 214 (2021) 113225
Fig. 5. Compound 6u induced A375 cells apoptosis in a dose response manner. A375 cells were treated with 6u at 1, 2.5, and 5 mM, or DAP-64 at 5 mM for 36 h, then stained with
FITC Annexin V/PI dyes. Cells in the upper right quadrant are PI positive/Annexin V positive, indicate late apoptotic, or necrotic cells, while cells in lower right quadrant indicate
early apoptotic cells. *P < 0.05, versus control group.
to 90 ꢀC for stirring 9 h. The mixture was then cooled to room
temperature, diluted with water and extracted with ether (3 x
30 mL). The combined organic layers were wash with water (3 x
20 mL). The organic extract was dried over Na2SO4, filtered and
concentrated. The crude residue was purified by silica gel chro-
matography to afford the pure compounds (3a-f).
149.43, 140.71, 133.14, 132.73, 131.97 (d, JF ¼ 3.4 Hz), 131.05, 129.76
(d, JF ¼ 9.1 Hz), 125.74, 116.29 (d, JF ¼ 22.1 Hz), 67.83. HRMS (ESI):
calcd. for C14H7Cl2FNO3 [M-H]-: 325.9793, found: 325.9785.
2,2-Dichloro-1-(2-nitro-4’-(trifluoromethoxy)biphenyl-4-yl)
ethanone (3d) was obtained as a yellow liquid after flash chroma-
tography. 1H NMR (400 MHz, CDCl3):
d
(ppm) 8.62 (s, 1H), 8.38 (d,
J ¼ 8 Hz, 1H), 7.61 (d, J ¼ 8 Hz, 1H), 7.40e7.38 (m, 2H), 7.33e7.31 (m,
2H), 6.59 (s, 1H); 13C NMR (101 MHz, CDCl3):
(ppm) 183.93,
150.05, 149.38, 140.44, 134.61, 133.33, 132.74, 131.35, 129.52, 125.91,
2,2-Dichloro-1-(2-nitrobiphenyl-4-yl)ethanone (3a) was ob-
tained as a yellow solid after flash chromatography. M.p. 69e70 ꢀC;
d
1H NMR (400 MHz, CDCl3):
d
(ppm) 8.56 (s, 1H), 8.35 (d, J ¼ 8 Hz,
1H), 7.63 (d, J ¼ 8 Hz, 1H), 7.47e7.46 (m, 3H), 7.36e7.33 (m, 2H),
6.62 (s, 1H); 13C NMR (101 MHz, CDCl3):
(ppm) 184.00, 149.51,
121.42, 67.88. HRMS (ESI): calcd. for
392.9710, found: 391.9701.
C
15H7Cl2F3NO4 [M-H]-:
d
141.80, 135.95, 133.07, 132.76, 130.86, 129.39, 129.11, 127.78, 125.67,
67.84. HRMS (ESI): calcd. for C14H9Cl2NNaO3 [MþNa]þ: 331.9852,
found: 331.9847.
2,2-Dichloro-1-(4-cyclopropyl-3-nitrophenyl)ethanone
was obtained as a yellow solid after flash chromatography. M.p.
54e55 ꢀC; 1H NMR (400 MHz, CDCl3):
(ppm) 8.52 (s, 1H), 8.21 (d,
(3e)
d
2,2-Dichloro-1-(40-methyl-2-nitrobiphenyl-4-yl)ethanone (3b)
was obtained as a yellow solid after flash chromatography. M.p.
J ¼ 8 Hz, 1H), 7.22 (d, J ¼ 8 Hz, 1H), 6.58 (s, 1H), 2.48 (m, 1H),
1.24e1.22 (m, 2H), 0.89e0.87 (m, 2H); 13C NMR (101 MHz, CDCl3):
84e85 ꢀC; 1H NMR (400 MHz, CDCl3):
d
(ppm) 8.53 (s, 1H), 8.33 (d,
J ¼ 8 Hz, 1H), 7.62 (d, J ¼ 8 Hz, 1H), 7.29e7.25 (m, 4H), 6.60 (s, 1H),
2.41 (s, 3H); 13C NMR (101 MHz, CDCl3):
(ppm) 184.03, 149.60,
d (ppm) 183.96, 151.15, 145.40, 133.35, 129.16, 127.58, 125.72, 67.83,
12.82, 10.30. HRMS (ESI): calcd. for C11H8Cl2NO3 [M-H]-: 271.9887,
found: 271.9880.
d
141.83, 139.67, 132.98, 132.68, 130.61, 129.90, 127.71, 125.67, 67.87,
21.45. HRMS (ESI): calcd. for C15H11Cl2NNaO3 [MþNa]þ: 346.0008,
found: 346.0003.
2,2-Dichloro-1-(3-nitro-4-(thiophen-3-yl)phenyl)ethanone (3f)
was obtained as a yellow solid after flash chromatography. M.p.
92e93 ꢀC; 1H NMR (400 MHz, CDCl3):
d
(ppm) 8.50 (s, 1H), 8.32 (d,
J ¼ 8 Hz, 1H), 7.68 (d, J ¼ 8 Hz, 1H), 7.47e7.44 (m, 2H), 7.13e7.12 (m,
1H), 6.58 (s, 1H); 13C NMR (101 MHz, CDCl3):
(ppm) 183.92,
149.26, 136.07, 135.61, 132.96, 132.28, 130.64, 127.24, 127.01, 125.55,
2,2-Dichloro-1-(40-fluoro-2-nitrobiphenyl-4-yl)ethanone (3c)
was obtained as a yellow liquid after flash chromatography. 1H NMR
d
(400 MHz, CDCl3):
d
(ppm) 8.56 (s,1H), 8.35 (d, J ¼ 8 Hz,1H), 7.60 (d,
J ¼ 8 Hz, 1H), 7.34e7.30 (m, 2H), 7.17e7.13 (m, 2H), 6.61 (s, 1H); 13
C
125.27, 67.86. HRMS (ESI): calcd. for
313.9451, found: 313.9443.
C
12H6Cl2NO3S [M-H]-:
NMR (101 MHz, CDCl3):
d
(ppm) 183.94, 161.39 (d, JF ¼ 251 Hz),
8

B. Xu, Z.-P. Wang, Q. Liu et al.
European Journal of Medicinal Chemistry 214 (2021) 113225
Fig. 6. Compound 6u induced A375 cell apoptosis in a time-dependent manner. A375 cells were treated with 6u at 5
stained with FITC Annexin V/PI dyes. *P < 0.05, versus control group.
mM, or DAP-64 at 5 mM for 12, 24, 36 h, respectively, then
4.1.4. General procedure for the synthesis of compound 5
(101 MHz, CDCl3): d (ppm) 183.51, 149.64, 136.31, 131.44, 130.18,
A mixture of 8.0 mmol of NH4Cl, 10.0 mmol of 1-(4-fluoro-3-
nitrophenyl) ethanone, and 20 mL of acetonitrile was stirred for
5 min, then 15 mmol 1,3-dichloro-5,5-dimethylhydantoin was
added by five times within 1 h, and the mixture was stirred at 35 ꢀC
for 18 h. Afterward, solvent was distilled under reduced pressure,
100 mL of ethyl acetate was added to the residue. The ethyl acetate
layer was washed with water (2 x 30 mL), the organic layers was
dried over Na2SO4, filtered and concentrated. The crude residue
was purified by silica gel chromatography to afford a colorless oil 5.
119.03, 115.83, 67.83, 24.96, 8.17. HRMS (ESI): calcd. for
C
11H9Cl2N2O3 [M-H]-: 286.9996, found: 271.9989.
2,2-Dichloro-1-(3-nitro-4-(pyrrolidin-1-yl)phenyl)ethanone
(6b) was obtained as a yellow solid after flash chromatography.
M.p. 103e104 ꢀC; 1H NMR (400 MHz, CDCl3):
(ppm) 8.48 (s, 1H),
8.05 (d, J ¼ 9.2 Hz, 1H), 6.93 (d, J ¼ 9.2 Hz, 1H), 6.58 (s, 1H), 3.34 (m,
4H), 2.05 (m, 4H); 13C NMR (101 MHz, CDCl3):
(ppm) 183.17,
d
d
145.64, 136.15, 133.64, 129.88, 117.77, 115.94, 67.88, 50.96, 25.69.
HRMS (ESI): calcd. for C12H12Cl2N2NaO3 [MþNa]þ: 325.0117, found:
325.0113.
1H NMR (400 MHz, CDCl3):
(m, 1H), 7.51e7.47 (m, 1H), 6.57 (s, 1H); 13C NMR (101 MHz, CDCl3):
d (ppm) 8.85e8.83 (m, 1H), 8.47e8.43
2,2-Dichloro-1-(4-morpholino-3-nitrophenyl)ethanone
was obtained as a yellow solid after flash chromatography. M.p.
111e112 ꢀC; 1H NMR (400 MHz, CDCl3):
(6c)
d
(ppm) 183.11, 160.10, 157.37, 137.07 (d, JF ¼ 10.0 Hz), 128.52, 127.80
(d, JF ¼ 4.0 Hz), 119.56 (d, JF ¼ 22.0 Hz), 67.72 (d, JF ¼ 4.0 Hz).
d
(ppm) 8.56 (d, J ¼ 2.4 Hz,
1H), 8.20e8.17 (m, 1H), 7.12 (d, J ¼ 8.8 Hz, 1H), 6.55 (s, 1H), 3.86 (t,
J ¼ 4.8 Hz, 4H), 3.27 (t, J ¼ 4.8 Hz, 4H); 13C NMR (101 MHz, CDCl3):
4.1.5. General procedure for the synthesis of compound 6a-s
To a round-bottom flask, 0.2 mmol of 2,2-dichloro-1-(4-fluoro-
3-nitrophenyl)ethan-1-one (5), 0.25 mmol of various primary
amines or secondary amines, 0.25 mmol K2CO3, and DMF（0.5 mL)
were added. The reaction mixture was stirred for 3 h at room
temperature. The mixture was diluted with ethyl acetate (150 mL).
The organic layers were wash with water (3 x 20 mL), the organic
extract was dried over Na2SO4, filtered and concentrated. The crude
residue was purified by silica gel chromatography to afford the pure
compounds 6a-s.
d
(ppm) 183.32, 149.21, 139.54, 134.81, 129.63, 121.91, 119.20, 67.85,
66.35, 50.87. HRMS (ESI): calcd. for C12H11Cl2N2O4 [M-H]-: 317.0101,
found: 317.0094.
2,2-Dichloro-1-(3-nitro-4-(piperidin-1-yl)phenyl)ethanone
(6d) was obtained as a yellow solid after flash chromatography.
M.p. 64e65 ꢀC; 1H NMR (400 MHz, CDCl3):
d
(ppm) 8.52 (s, 1H), 8.10
(d, J ¼ 9.2 Hz, 1H), 7.10 (d, J ¼ 9.2 Hz, 1H), 6.57 (s, 1H), 3.25e3.24 (m,
4H), 1.72 (m, 6H); 13C NMR (101 MHz, CDCl3):
(ppm) 183.26,
d
149.74, 138.51, 134.38, 130.02, 120.11, 119.28, 67.86, 51.81, 25.63,
23.79. HRMS (ESI): calcd. for C13H13Cl2N2O3 [M-H]-: 315.0309,
found: 315.0301.
2,2-Dichloro-1-(4-(cyclopropylamino)-3-nitrophenyl)ethanone
(6a) was obtained as a yellow solid after flash chromatography. M.p.
2,2-Dichloro-1-(3-nitro-4-(piperazin-1-yl)phenyl)ethanone
104e105 ꢀC; 1H NMR (400 MHz, CDCl3):
d (ppm) 8.98e8.97 (m, 1H),
(6e) was obtained as a yellow liquid after flash chromatography. 1H
8.55 (s, 1H), 8.20 (d, J ¼ 8 Hz, 1H), 7.41 (d, J ¼ 8 Hz, 1H), 6.59 (s, 1H),
NMR (400 MHz, CDCl3): d (ppm) 8.56 (s, 1H), 8.18e8.15 (m, 1H), 7.11
2.70 (m, 1H), 1.04e1.02 (m, 2H), 0.76e0.75 (m, 2H); 13C NMR
9

B. Xu, Z.-P. Wang, Q. Liu et al.
European Journal of Medicinal Chemistry 214 (2021) 113225
Fig. 7. Compound 6u induced cell cycle arrest in A375 cells. Cells were treated with 6u at 1, 2.5 and 5 mM or DAP-64 at 5 mM for 24 h, then were harvested, washed with PBS, and
fixed in 70% cold ethanol. After incubation overnight, the cell pellets were collected by centrifugation, re-suspended in 50 mg/mL of RNase A in PBS. Then PI dye (50 mg/mL) was
added, and the mixture was incubated at 37 ꢀC for 15 min. Cell cycle analysis was performed via flow cytometer. Bar graph represents the values of cells in G1, S, and G2 phases.
(d, J ¼ 8.8 Hz, 1H), 6.53 (s, 1H), 3.29e3.28 (m, 4H), 3.09e3.08 (m,
4H); 13C NMR (101 MHz, CDCl3):
(ppm) 183.35, 149.48, 139.44,
183.26, 147.09, 137.02, 136.11, 131.97, 130.33, 130.23, 127.54, 125.55,
120.01, 116.15, 67.83. HRMS (ESI): calcd. for C14H9Cl2N2O3 [M-H]-:
322.9996, found: 322.9988.
d
134.75, 129.77, 121.57, 119.48, 67.90, 51.49, 45.48. HRMS (ESI): calcd.
for C12H14Cl2N3O3 [MþH]þ: 318.0407, found: 318.0401.
2,2-Dichloro-1-(4-(4-methoxyphenylamino)-3-nitrophenyl)
ethanone (6j) was obtained as a yellow solid after flash chroma-
1-(4-(1-Imidazol-1-yl)-3-nitrophenyl)-2,2-dichloroethanone
(6f) was obtained as a yellow liquid after flash chromatography. 1H
tography. M.p.125e126 ꢀC; 1H NMR (400 MHz, CDCl3):
d
(ppm) 9.87
(s, 1H), 9.02 (m, 1H), 8.03 (d, J ¼ 8 Hz, 1H), 7.23e7.21 (m, 2H),
7.05e6.99 (m, 3H), 6.58 (s, 1H); 13C NMR (101 MHz, CDCl3):
(ppm)
NMR (400 MHz, CDCl3):
7.71 (s, 1H), 7.66 (d, J ¼ 8 Hz, 1H), 7.27 (s, 1H), 7.12 (s, 1H), 6.58 (s,
1H); 13C NMR (101 MHz, CDCl3):
(ppm) 183.24, 144.97, 136.92,
d
(ppm) 8.73 (s, 1H), 8.50 (d, J ¼ 8 Hz, 1H),
d
d
183.29, 159.03, 148.02, 136.03, 131.57, 130.35, 129.50, 127.54, 119.56,
116.14, 115.42, 67.82, 55.73. HRMS (ESI): calcd. for C15H11Cl2N2O4
[M-H]-: 353.0101, found: 353.0092.
134.82, 131.58, 131.42, 128.65, 127.27, 119.80, 67.83. HRMS (ESI):
calcd. for C11H6Cl2N2O3 [M-H]-: 297.9792, found: 297.9785.
2,2-Dichloro-1-(3-nitro-4-(1H-1,2,4-triazol-1-yl)phenyl)etha-
none (6g) was obtained as a yellow liquid after flash chromatog-
2,2-Dichloro-1-(3-nitro-4-(p-tolylamino)phenyl)ethanone (6k)
was obtained as a yellow solid after flash chromatography. M.p.
raphy. 1H NMR (400 MHz, CDCl3):
d
(ppm) 8.73e8.72 (m, 1H),
8.54e8.51 (m, 2H), 8.18 (s, 1H), 7.82 (d, J ¼ 8.4 Hz, 1H), 6.56 (s, 1H);
13C NMR (101 MHz, CDCl3):
(ppm) 183.17, 153.83, 143.83, 134.71,
140e141 ꢀC; 1H NMR (400 MHz, CDCl3):
d
(ppm) 9.93 (s,1H), 9.02 (s,
1H), 8.04 (d, J ¼ 9.2 Hz, 1H), 7.30e7.26 (m, 2H), 7.20e7.12 (m, 3H),
6.58 (s, 1H), 2.42 (s, 3H); 13C NMR (101 MHz, CDCl3):
(ppm)
d
d
133.89, 132.06, 127.31, 127.12, 67.80. HRMS (ESI): calcd. for
C
183.30, 147.51, 137.70, 136.04, 134.27, 131.73, 130.81, 130.37, 125.63,
119.72, 116.21, 67.82, 21.24. HRMS (ESI): calcd. for C15H11Cl2N2O3
[M-H]-: 337.0152, found: 337.0146.
10H5Cl2N4O3 [M-H]-: 298.9744, found: 298.9737.
2,2-Dichloro-1-(3-nitro-4-(2-nitro-1H-imidazol-1-yl)phenyl)
ethanone (6h) was obtained as a yellow solid after flash chroma-
tography. M.p.163e164 ꢀC; 1H NMR (400 MHz, CDCl3):
(ppm) 9.05
(s, 1H), 8.61 (d, J ¼ 8 Hz, 1H), 7.69 (d, J ¼ 8 Hz, 1H), 7.41 (s, 1H), 7.20
(s, 1H), 6.62 (s, 1H); 13C NMR (101 MHz, CDCl3):
(ppm) 183.04,
141.90, 135.73, 135.03, 133.46, 130.25, 130.04, 127.82, 125.67, 67.80.
HRMS (ESI): calcd. for C11H5Cl2N4O5 [M-H]-: 342.9643, found:
342.9634.
2,2-Dichloro-1-(4-(4-ethoxyphenylamino)-3-nitrophenyl)etha-
none (6l) was obtained as a yellow solid after flash chromatog-
d
raphy. M.p. 92e93 ꢀC; 1H NMR (400 MHz, CDCl3):
d (ppm) 9.87 (s,
d
1H), 9.02 (s, 1H), 8.03 (d, J ¼ 9.2 Hz, 1H), 7.20 (d, J ¼ 8.4 Hz, 2H), 7.04
(d, J ¼ 9.2 Hz, 1H), 6.99 (d, J ¼ 8.4 Hz, 2H), 6.58 (s, 1H), 4.08 (q,
J ¼ 6.8 Hz, 2H), 1.46 (t, J ¼ 6.8 Hz, 3H); 13C NMR (101 MHz, CDCl3):
d
(ppm) 183.29, 158.42, 148.05, 136.02, 131.52, 130.36, 129.29,
2,2-Dichloro-1-(3-nitro-4-(phenylamino)phenyl)ethanone (6i)
was obtained as a yellow solid after flash chromatography. M.p.
127.51, 119.50, 116.17, 115.90, 67.82, 63.99, 14.91. HRMS (ESI): calcd.
for C16H13Cl2N2O4 [M-H]-: 367.0258, found: 367.0249.
120e121 ꢀC; 1H NMR (400 MHz, CDCl3):
d
(ppm) 9.98 (s, 1H), 9.03
(m, 1H), 8.08e8.06 (m, 1H), 7.52e7.48 (m, 2H), 7.38e7.31 (m, 3H),
7.21e7.18 (m, 1H), 6.58 (s, 1H); 13C NMR (101 MHz, CDCl3):
(ppm)
2,2-Dichloro-1-(4-(4-isopropylphenylamino)-3-nitrophenyl)
ethanone (6m) was obtained as a yellow solid after flash chroma-
d
tography. M.p.133e134 ꢀC; 1H NMR (400 MHz, CDCl3):
d (ppm) 9.95
10

B. Xu, Z.-P. Wang, Q. Liu et al.
European Journal of Medicinal Chemistry 214 (2021) 113225
Fig. 8. Compound 6u inhibited A375 cell invasion and migration. A375 cells were treated with 6u (0.5, 1.0, and 2.0 mM) or DAP-64 (2.0 mM) for 24 h in wound healing assay, while 6u
(0.6, 0.8, and 1
m
M) or DAP-64 (1
m
M) was used in Transwell assays. (A) The upper photo showed the closure of A375 cells after being scratched ( ꢂ 100) for 24 h, the ration (%) was
calculated by (start scratched area-after scratched area)/start scratched area; (B) The A375 cells were seeded onto chambers, and incubated with 6u or DAP-64 for 24 h. Cells
migrated through the chambers were stained with crystal violet, and representative images were captured. The graph indicate the changes of scratch area or number of cells vs the
control group. *P < 0.05, versus control group.
11

B. Xu, Z.-P. Wang, Q. Liu et al.
European Journal of Medicinal Chemistry 214 (2021) 113225
Fig. 9. Depolarization of Djm in A375 cell by 6u. Cell was treated with PBS, or DAP-64 (5
mM), or 6u (2, 5, and 10
m
M) at 37 ꢀC for 4 h and stained by TMRM (left) or JC-1 (right), then
analyzed with confocal microscopy. Scale bar for TMRM image is 10 mM, while 25 mM for JC-1 image.
(s, 1H), 9.02 (s, 1H), 8.07e8.04 (m, 1H), 7.35e7.33 (m, 2H), 7.22 (m,
2H), 7.17 (d, J ¼ 9.2 Hz, 1H), 6.58 (s, 1H), 3.01e2.94 (m, 1H), 1.29 (d,
2,2-Dichloro-1-(4-(4-isopropylphenoxy)-3-nitrophenyl)ethan-
1-one (6q) was obtained as a white solid after flash chromatog-
J ¼ 7.2 Hz, 6H); 13C NMR (101 MHz, CDCl3):
d
(ppm) 183.29, 148.61,
raphy. M.p. 71e72 ꢀC. 1H NMR (400 MHz, CDCl3):
d (ppm) 8.70 (s,
147.45, 136.03, 134.50, 131.73, 130.37, 128.19, 125.61, 119.72, 116.27,
67.83, 33.94, 24.08. HRMS (ESI): calcd. for C17H15Cl2N2O3 [M-H]-:
365.0465, found: 365.0456.
1H), 8.21 (d, J ¼ 4 Hz,1H), 7.31 (d, J ¼ 8 Hz, 2H), 7.06 (d, J ¼ 8 Hz, 2H),
7.01 (d, J ¼ 8 Hz, 1H), 6.52 (s, 1H), 2.99e2.92 (m, 1H), 1.28 (s, 3H),
1.27 (s, 3H); 13C NMR (101 MHz, CDCl3):
d (ppm) 183.24, 156.34,
2,2-Dichloro-1-(4-(3,4-dimethoxyphenylamino)-3-
151.36, 147.17, 139.85, 135.30, 128.40, 128.01, 124.43, 120.46, 117.98,
67.69, 33.64, 24.02. HRMS (ESI): calcd. for C17H14Cl2NOꢁ4 ([M-H]-)
366.0305, found 366.0293.
nitrophenyl)ethanone (6n) was obtained as a black solid after flash
chromatography. M.p. 143e144 ꢀC; 1H NMR (400 MHz, CDCl3):
d
(ppm) 9.74 (s, 1H), 9.04 (m, 1H), 8.09 (d, J ¼ 9.2 Hz, 1H), 7.02 (s,
1H), 6.89e6.86 (m, 2H), 6.59 (s, 1H), 3.94 (s, 3H), 3.88 (s, 3H); 13C
NMR (101 MHz, CDCl3): (ppm) 183.29, 149.29, 148.87, 147.15,
2,2-Dichloro-1-(4-((4-isopropylphenyl)thio)-3-nitrophenyl)
ethan-1-one (6r) was obtained as a pale yellow oil after flash
d
chromatography. 1H NMR (400 MHz, CDCl3):
d (ppm) 8.97 (s, 1H),
136.20, 132.01, 130.17, 126.43, 122.99, 120.22, 116.19, 112.98, 110.92,
67.80, 56.53, 29.80. HRMS (ESI): calcd. for C16H13Cl2N2O5 [M-H]-:
383.0207, found: 383.0198.
8.05 (d, J ¼ 8 Hz,1H), 7.51 (d, J ¼ 8 Hz, 2H), 7.40 (d, J ¼ 8 Hz, 2H), 7.00
(d, J ¼ 8 Hz, 1H), 6.54 (s, 1H), 3.05e2.98 (m, 1H), 1.33 (s, 3H), 1.32 (s,
3H); 13C NMR (101 MHz, CDCl3):
d (ppm) 183.76, 152.29, 148.48,
2,2-Dichloro-1-(4-(4-(dimethylamino)phenylamino)-3-
nitrophenyl)ethanone (6o) was obtained as a black solid after flash
chromatography. M.p. 165e166 ꢀC; 1H NMR (400 MHz, CDCl3):
144.27, 136.06, 133.34, 128.88, 128.53, 127.57, 127.34, 126.06, 67.82,
34.21, 23.93. HRMS (ESI): calcd. for C17H14Cl2NO3Sꢁ ([M-H]-)
382.0077, found 382.0062.
d
(ppm) 9.87 (s, 1H), 9.01 (m, 1H), 8.00 (d, J ¼ 9.2 Hz, 1H), 7.13 (d,
J ¼ 8.8 Hz, 2H), 7.05 (d, J ¼ 9.2 Hz, 1H), 6.77 (d, J ¼ 8.8 Hz, 2H), 6.58
(s, 1H), 3.02 (s, 6H); 13C NMR (101 MHz, CDCl3):
(ppm) 183.34,
1-(4-((4-(Tert-butyl)phenyl)amino)-3-nitrophenyl)-2,2-
dichloroethan-1-one (6s) was obtained as reddish brown solid after
flash chromatography. M.p. 106e107 ꢀC. 1H NMR (400 MHz, CDCl3):
d
149.94, 148.51, 135.84, 131.26, 130.45, 127.14, 125.21, 119.10, 116.43,
113.17, 67.81, 40.67. HRMS (ESI): calcd. for C16H14Cl2N3O3 [M-H]-:
366.0418, found: 366.0410.
d
(ppm) 9.95 (s, 1H), 9.04 (d, J ¼ 4 Hz, 1H), 8.06 (d, J ¼ 8 Hz, 1H), 7.50
(d, J ¼ 12 Hz, 2H), 7.25 (d, J ¼ 8 Hz, 2H), 7.22 (s, 1H), 6.57 (s, 1H), 1.36
(s, 9H); 13C NMR (101 MHz, CDCl3):
(ppm) 183.33, 150.90, 147.43,
136.07, 134.22, 131.76, 130.42, 127.14, 125.24, 119.73, 116.31, 67.84,
d
2,2-Dichloro-1-(4-((4-fluorophenyl)amino)-3-nitrophenyl)
ethan-1-one (6p) was obtained as a pure solid after flash chroma-
34.89, 31.46. HRMS (ESI): calcd. for
379.0622, found 379.0606.
C
18H17Cl2N2Oꢁ3 ([M-H]-)
tography. M.p. 145e146 ꢀC. 1H NMR (400 MHz, CDCl3):
d (ppm) 9.88
(s, 1H), 9.03 (m, 1H), 8.08 (d, J ¼ 8 Hz, 1H), 7.32e7.27 (m, 2H),
7.22e7.18 (m, 2H), 7.07 (d, J ¼ 8 Hz, 1H), 6.58 (s, 1H); 13C NMR
4.1.6. General procedure for the synthesis of compound 6t
(101 MHz, CDCl3):
d
(ppm) 183.26, 161.63 (d, JF ¼ 249 Hz), 147.40,
To a round-bottom flask, 30 mmol of 1-(4-aminophenyl)ethan-
1-one (7) was dissolved in THF (180 mL), then CH3MgBr (150 mmol,
40 mL) was added. The mixture was stirred in ꢁ10 ꢀC for 3 h. After
completion of the reaction, the mixture was quenched by NH4Cl
136.25, 132.94 (d, JF ¼ 3 Hz), 131.91, 130.31, 127.92 (d, JF ¼ 8.6 Hz),
120.06, 117.24 (d, JF ¼ 23 Hz), 115.90, 67.82. HRMS (ESI): calcd. for
C
14H8Cl2FN2O3 [M-H]-: 340.9902, found: 340.9888.
12

B. Xu, Z.-P. Wang, Q. Liu et al.
European Journal of Medicinal Chemistry 214 (2021) 113225
Fig. 10. The effect of compound 6u on the glycolysis pathway in A375 cells. (A) Compounds 6u exhibited dose response manner to inhibit p(Ser293)E1
a in A375 cells; (B) Compounds
6u affected the lactate formation in A375 cell; (C) Compound 6u increased OCR in A375 cell; (D) Compound 6u dose-independently increased ROS production in A375 cell. *P < 0.05,
versus control group.
Fig. 11. Compound 6u reduced tumor growth in A375 xenograft model. A: Average tumor volume changes during 21 days of drug exposure; B: After 21 days, the tumors were
harvested and weighted; (C) Body weigh changes of mice during 21 days of drug exposure. #P < 0.05 and * P < 0.05 between Vehicle and 6u (5 mg/kg).
solution, and the THF was removed, then was diluted with ethyl
acetate (50 mL). The organic layers were wash with water (3 x
50 mL), the organic extract was dried over Na2SO4, filtered and
concentrated. The crude residue (8) was then dissolved in DMF
(10 mL), and added compound 5 and potassium carbonate. The
mixture was stirred at room temperature for 12 h. After completion
of the reaction, the mixture was diluted with ethyl acetate (50 mL)
and water (50 mL). The organic layers were wash with water (3 x
50 mL), the organic extract was dried over Na2SO4, filtered and
concentrated. Compound 6t was purified by silica gel chromatog-
raphy to afford the reddish brown solid. M.p. 96e97 ꢀC. 1H NMR
J ¼ 12 Hz, 1H), 6.56 (s, 1H), 1.78 (s, 1H), 1.63 (s, 6H); 13C NMR
(101 MHz, CDCl3):
d (ppm) 183.30, 148.64, 147.17, 136.09, 135.41,
131.88, 130.35, 126.38, 125.25, 119.91, 116.22, 72.51, 67.81, 31.99.
HRMS (ESI): calcd. for C17H15Cl2N2Oꢁ4 ([M-H]-) 381.0414, found
381.0399.
4.1.7. General procedure for the synthesis of compound 11
To a round-bottom flask, 50 mmol of 2-(4-nitrophenyl)acetic
acid (9) and MeOH (60 mL) mixture together, then concentrated
sulfuric acid (5 mL) was added. The mixture was refluxed for 6 h.
After completion of the reaction, the mixture was quenched by
water, then MeOH was removed, and diluted with ethyl acetate
(50 mL). The organic layers were wash with water (3 x 50 mL), the
(400 MHz, CDCl3):
d (ppm) 9.96 (s, 1H), 9.03 (s, 1H), 8.06 (d,
J ¼ 12 Hz, 1H), 7.61 (d, J ¼ 8 Hz, 2H), 7.27 (d, J ¼ 8 Hz, 2H), 7.20 (d,
13

B. Xu, Z.-P. Wang, Q. Liu et al.
European Journal of Medicinal Chemistry 214 (2021) 113225
organic extract was dried over Na2SO4, filtered and concentrated.
The crude residue (10) was then dissolved in DMF (10 mL), then
CH3I and sodium hydroxide was added. The mixture was stirred at
4.2.2. Cell viability assay
Cell viability after treated with the prepared compounds and
DAP, DAP-64 were tested by using the MTT assay. A suspension of
cells (3000e6000/well for cancer cell lines, and 7000/well for L02)
were seeded in 96-well plates and cultured overnight. Then two
concentrations of all synthesized compounds (100, and 10
buffer containing 1% DMSO) were added into the 96-well plates, in
which cells were incubated for 72 h. Then,10 L of MTT (0.5 mg/mL)
solution was added to each well of the plates. After 4 h incubation,
the supernatant was removed and 100 L of DMSO was added. The
absorbance of each well was measured at 570 nm in a microplate
reader. After identified the potential compounds, the full dose
concentration of desired compounds (6m, 6s, 6t, and 6u) was
measured, resultant OD570 nm values were expressed as IC50 values,
which were the mean values derived from three independent
experiments.
0
ꢀC for 24 h. After completion of the reaction, the mixture was
diluted with ethyl acetate (50 mL) and water (50 mL). The organic
layers were wash with water (3 x 50 mL), the organic extract was
dried over Na2SO4, filtered and concentrated. Compound 11 was
purified by silica gel chromatography to afford a white solid. M.p.
mM, PBS
m
40e41 ꢀC. 1H NMR (400 MHz, CDCl3):
d
(ppm) 8.15 (d, J ¼ 8.0 Hz,
2H), 7.48 (d, J ¼ 8.0 Hz, 2H), 3.65 (s, 3H), 1.60 (s, 6H); 13C NMR
m
(101 MHz, CDCl3):
52.60, 47.01, 26.46.
d
(ppm) 176.07, 152.00, 146.75, 126.90, 123.65,
4.1.8. General procedure for the synthesis of compound 12
To a round-bottom flask, compound 11 (5.0 g, 22.4 mmol) and
THF (40 mL) were added, then DIBAL-H (67.0 mL, 67.2 mmol) was
added dropwise. The mixture was stirred at 0 ꢀC for 24 h. After
completion of the reaction, the mixture was quenched by NH4Cl
solution, then diluted with ethyl acetate (50 mL). The organic layers
were collected and washed with water (3 x 50 mL). The organic
extract was dried over Na2SO4, filtered and concentrated. Com-
pound 12 was purified by silica gel chromatography to afford a pale
4.2.3. Lactate measurement
Suspension of A375 cells (2.5 ꢂ 105/well) were seeded in 24-
well plates and cultured overnight. Then DAP-64 (5 mM) and 6u
(1, 2.5, and 5 mM) were added to the corresponding wells, the plates
were incubated at 37 ꢀC for 4 h. Then the medium was transferred
into EP tubes, which were centrifuged for 4 min (12000 rpm/min).
At last, 1 mL of medium was collected, which was used to test the
lactate production on a Nova Bioprofile Flex analyzer.
yellow solid. M.p. 60e61 ꢀC. 1H NMR (400 MHz, CDCl3):
d (ppm)
8.16 (d, J ¼ 8.0 Hz, 2H), 7.55 (d, J ¼ 8.0 Hz, 2H), 3.67 (s, 3H), 1.61 (s,
1H),1.37 (s, 6H); 13C NMR (101 MHz, CDCl3):
127.39, 123.44, 72.42, 40.78, 25.32.
d (ppm) 154.92,146.23,
4.2.4. OCR measurement
4.1.9. General procedure for the synthesis of compound 13
The OCR was measured as previous report [17]. A375 cells (5 x
A mixture of compound 12 (2.0 g, 10.2 mmol) and 10% Pd/C
(200 mg, 0.2 mmol) in EtOH (20 mL) was evacuated and back-filled
with H2. The mixture was stirred at room temperature for 12 h.
After completion of the reaction, the mixture was filtered and the
solvent was evaporated in vacuo. The crude product was purified by
flash chromatography to provide a colorless oil 13. 1H NMR
104/well) were cultured in XF24-well cell culture microplates
(Seahorse Bioscience) in 100
1 h at 37 ꢀC in 5% CO2 atmosphere. After the cells were attached, an
additional 100 L growth medium was added and the cells were
incubated for another 24 h, which was then treated with DAP-64
(5 M), 6u (1, 2.5, and 5 M) for 4 h. The culture medium was then
removed and the cells were rinsed two times with 100 L of pre-
warmed XFe base medium. Finally 170 L of XFe cell Mito stress
mL growth medium and incubated for
m
m
m
(400 MHz, CDCl3):
d
(ppm) 7.16 (d, J ¼ 8.0 Hz, 2H), 6.65 (d,
m
J ¼ 12.0 Hz, 2H), 3.51 (s, 2H), 2.99 (br, 3H), 1.28 (s, 6H); 13C NMR
m
(101 MHz, CDCl3):
39.30, 25.43.
d
(ppm) 144.40, 136.32, 127.16, 115.24, 73.11,
test assay medium was added to each well and the plate was placed
at 37 ꢀC without CO2 for 1 h prior to measurement.
4.1.10. General procedure for the synthesis of compound 6u
4.2.5. ITC analysis
A mixture of compound 13 (236 mg, 1.43 mmol), compound 5
(300 mg, 1.2 mmol) and potassium carbonate in DMF (10 mL) was
stirred at room temperature for 12 h. After completion of the re-
action, the mixture was diluted with ethyl acetate (20 mL). The
organic layers were collected and washed with water (3 x 20 mL).
The organic extract was dried over Na2SO4, filtered and concen-
trated. Compound 6u was purified by silica gel chromatography to
afford a reddish brown solid. M.p. 90e91 ꢀC. 1H NMR (400 MHz,
Proteins was prepared to 10
KH2PO4/K2HPO4, 100 mM KCl, and 1 mM MgCl2). Compound 6u
was dissolved in pure DMSO and diluted to 100 M by using the
buffer solution. Then 250 L of proteins solution were transferred to
reaction cell and the compound solution was transferred to the
titration syringe. Titrations were conducted with the injection of
mM in the buffer solution (20 mM
m
m
2.5 mL titrant in every increment into the reaction cell, which was
maintained at 25 ꢀC.
CDCl3):
d
(ppm) 9.95 (s, 1H), 9.02 (s, 1H), 8.06 (d, J ¼ 12 Hz, 1H), 7.50
(d, J ¼ 8 Hz, 2H), 7.27 (d, J ¼ 8 Hz, 2H), 7.21 (d, J ¼ 8 Hz, 1H), 6.57 (s,
4.2.6. ROS measurement
1H), 3.67 (s, 2H), 1.53 (s, 1H), 1.37 (s, 6H); 13C NMR (101 MHz,
A375 cells were seeded at a density of 4 ꢂ 104 cells/mL on a dark,
clear bottom 96-well plates and were allowed to grow overnight.
Then culture medium was removed and a diluted DCFH-DA solu-
CDCl3):
d (ppm) 183.13, 146.98, 146.09, 135.92, 134.79, 131.70,
130.21, 127.96, 125.17, 119.73, 116.09, 72.77, 67.66, 40.04, 25.37.
HRMS (ESI): calcd. for C18H17Cl2N2Oꢁ4 ([M-H]-) 395.0571, found
395.0553.
tion was added (100 mL/well) to incubate the cells for 45 min at
37 ꢀC. Remove the medium and washed with 1 ꢂ PBS for three
times. Then the cells were treated with 6u (1, 2.5, and 5 mM), and
4.2. Biological evaluation
DAP-64 (5 m
M) at 37 ꢀC for 4 h. At last, the plate was measured on a
microplate reader at Ex/Em ¼ 485/535 nm.
4.2.1. Cell culture
A549, MCF-7, SHSY-5Y, HCT-116, A375, PC-3 and L02 cell lines
were purchased from Shanghai Cell Bank of Chinese Academy of
Sciences (Shanghai, China), which were cultured in DMEM medium
and supplemented with 10% FBS, 1% penicillin and streptomycin at
37 ꢀC in a 5% CO2 humidified atmosphere. Cells grown at expo-
nential stage were used for all the biological experiments.
4.2.7. PDKs inhibitory potency assay
The PDKs inhibitory potency assay was measured as previous
report [24].
4.2.8. Cell cycle assay
A375 cells were seeded in 6-well plates at density of
14

B. Xu, Z.-P. Wang, Q. Liu et al.
European Journal of Medicinal Chemistry 214 (2021) 113225
1e2 ꢂ 105 cells/well and treated with 6u (1, 2.5 and 5
m
M) for 24 h.
residues making non-bonded contacts with the ligand.
Then, cells were harvested and fixed in 70% pre-cooled ethanol.
Finally, cells were stained by PI/RNase A and measured by a flow
cytometer (Accuri C6, BD Biosciences).
4.2.14. Statistical analysis
Data was reported as Mean SD. Statistical analysis was per-
formed using GraphPad Prism version 6.0 for Windows. *P < 0.05
was considered as statistically significant.
4.2.9. Cell apoptosis assay
A375 cells were seeded at a density of 1e2 ꢂ 105 cells/well on 6-
well plates and allowed to grow overnight. The cells were treated
Declaration of competing interest
with 6u (1, 2.5 and 5 mM) or DAP-64 (5 mM) for 24 h. Then the cells
were trypsinized, repeatedly washed with cold PBS for three times,
centrifuged at 1200 rpm/min for 5 min, and the supernatants were
discarded. Then cells were stained by a FITC Annexin V/PI kit in the
binding buffer for 15 min at room temperature. Subsequently, cells
were measured by a flow cytometer (Accuri C6, BD Biosciences).
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
4.2.10. Western blotting
A375 cells were seeded in 6-well plates and treated with 6u (1,
2.5, and 5 mM), and DAP-64 (5 mM) for 24 h. Then the treated cells
This work was supported by the National Natural Science
Foundation of China (No. 21807008), Chongqing Science and
Technology Bureau (csts2019jcyj-zdxmX0021), the Science and
Technology Development Fund, Macau SAR （File no. 0057/2018/
A2) and University of Macau (File no. MYRG2019-00034-FHS).
were incubated with cell lysate buffer for 15 min, and centrifuged
with the speed of 12000 rpm/min at 4 ꢀC for 15 min. Protein con-
centrations were assessed by Pierces BCA Protein Assay Kit and
balanced to the same level, followed by a 8 min protein denatur-
ation with SDS loading buffer at 100 ꢀC. Proteins in the samples
were separated by SDS-PAGE electrophoresis, transferred onto
nitrocellulose filter membrane, blocked in 5% fat free milk for 2 h,
breezed with desired primary antibodies overnight, followed with
secondary HRP-conjugated anti-rabbit IgG for 2 h. Membranes
were finally scanned in a ChemiDoc MP Imaging System (Bio-Rad)
after 2 min incubation in Clarity Western ECLSubstrate (Bio-Rad).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113225.
References
[1] J.L. Counihan, E.A. Grossman, D.K. Nomura, Cancer metabolism: current un-
derstanding and therapies, Chem. Rev. 118 (2018) 6893e6923.
[2] M.G. Vander Heiden, L.C. Cantley, C.B. Thompson, Understanding the Warburg
effect: the metabolic requirements of cell proliferation, Science 324 (2009)
1029e1033.
4.2.11. JC-1 and TMRM assay for mitochondria membrane potential
A375 cells were seeded as 4 x 104 cells/mL and allowed to grow
overnight at 37 ꢀC. Cells were then treated with DAP-64 (5
mM) or
[3] M.V. Liberti, J.W. Locasale, The Warburg effect: how does it benefit cancer
cells? Trends Biochem. Sci. 41 (2016) 211e218.
6u (1, 2.5, and 5 m mM JC-1
M) for 4 h at 37 ꢀC. Then a solution of 0.5
[4] S.L. Zhang, X.H. Hu, W. Zhang, H.K. Yao, K.Y. Tam, Development of pyruvate
dehydrogenase kinase inhibitors in medicinal chemistry with particular
emphasis as anticancer agents, Drug Discov. Today 20 (2015) 1112e1119.
[5] S.L. Zhang, Y. He, K.Y. Tam, Targeting cancer metabolism to develop human
lactate dehydrogenase (hLDH) 5 inhibitors, Drug Discov, Today Off. 23 (2018)
1407e1415.
[6] W. Zhang, S.L. Zhang, X.H. Hu, K.Y. Tam, Targeting tumor metabolism for
cancer treatment: is pyruvate dehydrogenase kinases (PDKs) a viable anti-
cancer target? Int. J. Biol. Sci. 11 (2015) 1390e1400.
[7] T. McFate, A. Mohyeldin, H. Lu, J. Thakar, J. Henriques, N.D. Halim, H. Wu,
M.J. Schell, T.M. Tsang, O. Teahan, S. Zhou, J.A. Califano, N.H. Jeoung,
R.A. Harris, A. Verma, Pyruvate dehydrogenase complex activity controls
metabolic and malignant phenotype in cancer cells, J. Biol. Chem. 283 (2008)
22700e22708.
[8] G. Chen, L. Wang, S. Liu, C. Chuang, T.E. Roche, Activated function of the py-
ruvate dehydrogenase phosphatase through Ca2þ-facilitated binding to the
inner lipoyl domain of the dihydrolipoyl acetyltransferase, J. Biol. Chem. 271
(1996) 28064e28070.
[9] C.A. Brautigam, R.M. Wynn, J.L. Chuang, D.T. Chuang, Subunit and catalytic
component stoichiometries of an in vitro reconstituted human pyruvate de-
hydrogenase complex, J. Biol. Chem. 284 (2009) 13086e13098.
[10] H. Hur, Y. Xuan, Y.B. Kim, G. Lee, W. Shim, J. Yun, I.H. Ham, S.U. Han,
Expression of pyruvate dehydrogenase kinase-1 in gastric cancer as a po-
tential therapeutic target, Int. J. Oncol. 42 (2013) 44e54.
[11] M.I. Koukourakis, A. Giatromanolaki, G. Bougioukas, E. Sivridis, Lung cancer: a
comparative study of metabolism related protein expression in cancer cells
and tumor associated stroma, Canc. Biol. Ther. 6 (2007) 1476e1479.
[12] (a) S.M. Wigfield, S.C. Winter, A. Giatromanolaki, J. Taylor, M.L. Koukourakis,
A.L. Harris, PDK-1 regulates lactate production in hypoxia and is associated
with poor prognosis in head and neck squamous cancer, Br. J. Canc. 98 (2008)
1975e1984;
or TMRM reagents in fresh medium was added and incubated at
37 ꢀC for 15 min. Then the culture medium was removed. The cells
were washed 3 times with cool PBS, the depolarized mitochondria
membrane potential was recorded in a Laser scanning confocal
microscope (Leica TCS SP8).
4.2.12. Animal study
The animal study was approved by the Laboratory Animal
Welfare and Ethics Committee of the Third Military Medical Uni-
versity. Each animal was housed under standard conditions and
had free access to food and water. A375 cells at 80% confluence
were harvested with trypsin/EDTA, and resuspended in serum-free
DMEM medium. Cells were then inoculated in the right flank of
each mouse subcutaneously. Within days after inoculation, the
tumor bearing mice were randomly assigned into 2 treatment
groups (4 mice/group) of vehicle, 6u (5 mg/kg) and treated with
compound by intramuscular injection once per three days. Tumor
volume and body weight of each mouse were recorded once per
three days. Then, tumor samples were harvested and weighted.
4.2.13. Molecular docking
Molecular docking was performed using a Sybyl-X 2.0 software.
The crystal structure of PDKs protein was downloaded from the
Protein Data Bank (PDB ID: 2Q8G). The crystal structure of PDKs
was optimized with H added and charge added by AMBER7 FF99
method. The structures of small molecular database were subjected
to the polar H adding and being energy optimized with a tripos
force field and charged optimized with Gasteiger-Huckel method.
The graphics of 3D views were drawn by PyMOL, and the amino
acids involved in hydrophobic interaction were shown as stick.
Plots were generated by LigPlotþ, the spoked arcs represent
(b) M.I. Koukourakis, A. Giatromanolaki, E. Sivridis, K.C. Gatter, A.L. Harris,
Pyruvate dehydrogenase and pyruvate dehydrogenase kinase expression in
non-small cell lung cancer and tumor-associated stroma, Neoplasia 7 (2005)
1e6.
[13] T. Meng, D.D. Zhang, Z.Q. Xie, T. Yu, S.C. Wu, L. Wyder, U. Regenass, K. Hilpert,
M. Huang, M.Y. Geng, J.K. Shen, Discovery and optimization of 4,5-
diarylisoxazoles as potent dual inhibitors of pyruvate dehydrogenase kinase
and heat shock protein 90, J. Med. Chem. 57 (2014) 9832e9843.
[14] P.A. Brough, L. Baker, S. Bedford, K. Brown, S. Chavda, V. Chell, J. D’Alessandro,
N.G.M. Davies, B. Davis, L.L. Strat, A.T. Macias, D. Maddox, P.C. Mahon,
15

B. Xu, Z.-P. Wang, Q. Liu et al.
European Journal of Medicinal Chemistry 214 (2021) 113225
A.J. Massey, N. Matassova, S. McKenna, J.W.G. Meissner, J.D. Moore,
J.B. Murray, C.J. Northfield, C. Parry, R. Parsons, S.D. Roughley, T. Shaw,
H. Simmonite, S. Stokes, A. Surgenor, E. Stefaniak, A. Robertson, Y.K. Wang,
P. Webb, N. Whitehead, M. Wood, Application of off-rate screening in the
identification of novel pan-isoform inhibitors of pyruvate dehydrogenase ki-
nase, J. Med. Chem. 60 (2017) 2271e2286.
(2018) 275e284.
[20] I. Papandreou, T. Goliasova, N.C. Denko, Anticancer drugs that target meta-
bolism: is dichloroacetatethe new paradigm? Int. J. Canc. 128 (2011)
1001e1008.
[21] E.D. Michelakis, G. Sutendra, P. Dromparis, L. Webster, A. Haromy, E. Niven,
C. Maguire, T.L. Gammer, J.R. Mackey, D. Fulton, B. Abdulkarim,
M.S. McMurtry, K.C. Petruk, Metabolic modulation of glioblastoma with
dichloroacetate, Sci. Transl. Med. 2 (2010) 31ra34.
[22] P.W. Stacpoole, T.L. kurtz, Z. Han, T. Langaee, Role of dichloroacetate in the
treatment of genetic mitochondrial diseases, Adv. Drug Deliv. Rev. 60 (2008)
1478e1487.
[23] J. Espinal, T. Leesnitzer, A. Hassman, M. Beggs, J. Cobb, Inhibition of pyruvate
dehydrogenase kinase by halogenated acetophenones, Drug Dev. Res. 35
(1995) 130e136.
[15] W.Y. Sun, Z.Q. Xie, Y.F. Liu, D. Zhao, Z.X. Wu, D.D. Zhang, H. Lv, S. Tang, N. Jin,
H.L. Jiang, M.J. Tan, J. Ding, C. Luo, J. Li, M. Huang, M.Y. Geng, JX06 selectively
inhibits pyruvate dehydrogenase kinase PDK1 by a covalent cysteine modi-
fication, Canc. Res. 75 (2015) 4923e4936.
[16] Y.F. Liu, Z.Q. Xie, D. Zhao, J. Zhu, F. Mao, S. Tang, H. Xu, C. Luo, M.Y. Geng,
M. Huang, J. Li, Development of the first generation of disulfide-based sub-
type-selective and potent covalent pyruvate dehydrogenase kinase 1 (PDK1)
inhibitors, J. Med. Chem. 60 (2017) 2227e2244.
[17] S.L. Zhang, X.H. Hu, W. Zhang, K.Y. Tam, Unexpected discovery of dichlor-
oacetate derived adenosine triphosphate competitors targeting pyruvate de-
hydrogenase kinase to inhibit cancer proliferation, J. Med. Chem. 59 (2016)
3562e3568.
[24] S.L. Zhang, Z. Yang, X.H. Hu, H. Chakravarty, K.Y. Tam, Anticancer effects of
some novel dichloroacetophenones through the inhibition of pyruvate de-
hydrogenase kinase, Eur. J. Pharmaceut. Sci. 123 (2018) 43e55.
[25] S.L. Zhang, Z. Yang, X.H. Hu, K.Y. Tam, Dichloroacetophenones targeting at
[18] S.C. Tso, M.L. Lou, C.Y. Wu, W.J. Gui, J.L. Chuang, L.K. Morlock, N.S. Williams,
R.M. Wynn, X.B. Qi, D.T. Chuang, Development of dihydroxyphenyl sulfony-
lisoindoline derivatives as liver-targeting pyruvate dehydrogenase kinase
inhibitors, J. Med. Chem. 60 (2017) 1142e1150.
pyruvate dehydrogenase kinase 1 with improved selectivity and anti-
proliferative activity: synthesis and structure activity relationships, Bioorg.
Med. Chem. Lett 28 (2018) 3441e3445.
[26] S.C. Xiang, D. Huang, Q.L. He, J. Li, K.P. Tam, S.L. Zhang, Y. He, Development of
dual inhibitors targeting pyruvate dehydrogenase kinases and human lactate
dehydrogenase A: high-throughput virtual screening, synthesis and biological
validation, Eur. J. Med. Chem. 203 (2020), 155 112579.
[19] B. Xu, Z.M. Yu, S.C. Xiang, Y.S. Li, S.L. Zhang, Y. He, Rational design of
mitochondria-targeted pyruvate dehydrogenase kinase
1 inhibitors with
improved selectivity and antiproliferative activity, Eur. J. Med. Chem. 155
16