Rational design of mitochondria-targeted pyruvate dehydrogenase kinase 1 inhibitors with improved selectivity and antiproliferative activity

Article abstract of DOI:10.1016/j.ejmech.2018.06.012

Herein, triphenylphosphonium cation moieties were incorporated into a dichloroacetophenone derivative, leading to the discovery of novel mitochondria-targeted and tumor-specific pyruvate dehydrogenase kinase 1 (PDK1) inhibitors. Biological studies suggest

Full text of DOI:10.1016/j.ejmech.2018.06.012

Accepted Manuscript
Rational design of mitochondria-targeted pyruvate dehydrogenase kinase 1 inhibitors
with improved selectivity and antiproliferative activity
Biao Xu, Zhimei Yu, Sichuan Xiang, Yunshan Li, Shao-Lin Zhang, Yun He
PII:
S0223-5234(18)30503-8
10.1016/j.ejmech.2018.06.012
EJMECH 10482
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 April 2018
Revised Date: 31 May 2018
Accepted Date: 4 June 2018
Please cite this article as: B. Xu, Z. Yu, S. Xiang, Y. Li, S.-L. Zhang, Y. He, Rational design of
mitochondria-targeted pyruvate dehydrogenase kinase 1 inhibitors with improved selectivity
and antiproliferative activity, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.06.012.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract
With a rational design strategy, a triphenylphosphonium cation moiety and a nitro group were
incorporated into 1a, affording 1f with improved target inhibition and selectivity.
Comp.
ID
IC50 (µM)
EC (µM)
Selectivity index
5
0
NCI-H1650 (cancer)
BEAS-2B (normal)
1
a
3.8
0.1
2.25
0.21
2.78
3.28
2.78 / 2.25 = 1.2
3.28 / 0.21 = 15.6
1
f

ACCEPTED MANUSCRIPT
Title page
Title:
Rational design of mitochondria-targeted pyruvate dehydrogenase kinase 1 inhibitors with improved
selectivity and antiproliferative activity
Author names and affiliations:
Biao Xu, Zhimei Yu, Sichuan Xiang, Yunshan Li, Shao-Lin Zhang,* Yun He*
School of Pharmaceutical Sciences, Chongqing Key Laboratory of Natural Product Synthesis and Drug
Research, Chongqing University, Chongqing, 401331, P. R. China
*
Corresponding address:
Dr. Shao-Lin Zhang & Prof. Dr. Yun He, School of Pharmaceutical Sciences, Chongqing Key Laboratory of
Natural Product Synthesis and Drug Research, Chongqing University, Chongqing, 401331, P. R. China
Tel.: +86-23-65678465; E-mail: zhangsl@cqu.edu.cn; yun.he@cqu.edu.cn
Abstract:
Herein, triphenylphosphonium cation moieties were incorporated into a dichloroacetophenone derivative,
leading to the discovery of novel mitochondria-targeted and tumor-specific pyruvate dehydrogenase kinase
(PDK1) inhibitors. Biological studies suggested that all these synthesized compounds had significant in
vitro activities, in particular compound 1f, which inhibited PDK1 with an EC50 value of 0.12 µM, and
reduced the proliferation of NCI-H1650 cell with an IC50 value of 0.21 µM, but showed marginal effect
against non-cancerous BEAS-2B cell (IC50 = 3.28 µM). In addition, 1f decreased the extracellular
acidification rate and lactate formation, increased reactive oxygen species production, and depolarized the
mitochondrial membrane potential of NCI-H1650 cell, which could serve as a potential modulator to
reactive mitochondrial oxidative phosphorylation and reprogram the glucose metabolic pathways in cancer
cells. Collectively, these data demonstrated that 1f could be a promising lead for the development of
therapeutic PDK1 inhibitor in cancer treatment.
Keywords:
Cancer metabolism; Cancer proliferation; Mitochondria; Triphenylphosphonium cation; Anti-cancer drug
1

ACCEPTED MANUSCRIPT
1. Introduction
Cancer cells exhibit an increased glycolysis and suppressed oxidative phosphorylation (OXPHOS), even
under aerobic conditions [1, 2]. This metabolic alteration and adaptation is believed to provide proliferative
advantages and survival needs for cancer cells [3]. Thus, selective disruption of the metabolic aberrations
may offer therapeutic opportunities in cancer therapy [4].
Pyruvate dehydrogenase complex (PDC) is a gatekeeper enzyme, linking the glycolysis and tricarboxylic
acid cycle, which regulates the flux of the pyruvate into mitochondria for OXPHOS [5]. The activity of
PDC is regulated by the reversible phosphorylation. The phosphorylation of PDC by the four isoforms of
PDK (PDK1, 2, 3 and 4) results in its inactivation, while the dephosphorylation by two pyruvate
dehydrogenase phosphatases (PDPs) restores its activity [6, 7]. Therefore, inhibition of PDKs to activate
PDC is an attractive therapeutic strategy to reverse the abnormal metabolic pathways and inhibit cancer cell
proliferation.
Among the four PDK isoforms, PDK1 is most associated with cancer malignancy [8]. It had been
reported that PDK1 was remarkably overexpressed in various human tumor samples, such as head and neck
squamous cancer [9], gastric cancer [10] and lung cancer [11, 12]. Furthermore, overexpression of PDK1
protected cancer cells from anoikis, while depletion of the enzyme restored the susceptibility to anoikis and
reduced the metastatic potential [13]. In addition, a recent study indicated that PDK1 activity was increased
at the post-transcriptional level by diverse oncogenic tyrosine kinases [14]. This enhancement of PDK1
activity resulted in a promotion of the Warburg effect and tumor growth [15]. Collectively, these studies
suggested that PDK1 was an attractive target. Inhibition of PDK1 could provide a promising approach to
kill or, at least, greatly reduce the growth of cancer cells.
Fig. 1 Biological activities of DAP and 1a. A: Chemical structures of DAP and 1a. B: DAP and 1a at different concentrations
inhibited proteins phosphorylation in AKT, ERK and PDC signaling pathways. C: 1a bound to PDK1 protein with a K value
d
of 13.4 µM.
2

ACCEPTED MANUSCRIPT
Recently, several inhibitors have been reported to inhibit the activity of PDKs and reduced cancer cells
proliferation [16-18]. Dichloroacetophenone (DAP, Fig. 1A) was identified as a PDKs inhibitor in a
screening campaign based on pyruvate dehydrogenase activity [19], which was never used in anticancer
studies. Until recently, Qin et al reported that DAP weakly inhibited the acute myeloid leukemia (AML)
cells proliferation [20]. However, DAP is not a specific PDKs inhibitor, which affects other signal pathways,
such as AKT and ERK phosphorylations (Fig. 1B), therefore inevitably leading to some off-target and / or
side effects. Continuing our endeavor to discover specific PDK1 inhibitor [21, 22], we identified a
structurally novel PDK1 inhibitor 1a, which dose-dependently inhibited PDC phosphorylation (Fig. 1B)
and bound to PDK1 protein with a K value of 13.4 µM (Fig. 1C). Unfortunately, 1a affected ERK and
d
AKT signaling pathways, similar to DAP, and therefore resulted in no selectivity. Inspired by these
preliminary results, we herein report the rational design and identification of a series of novel
mitochondria-targeted and tumor-specific PDK1 inhibitors with reduced off-target and / or side effects as
compared to their parent molecule.
2. Results and discussion
+
2
.1. Design strategy of TPP -conjugated compounds
One of the major challenges in developing anticancer drugs is to improve selectivity and reduce side
effects for normal cells. As we stated above, the PDK1 inhibitor 1a affected the ERK and AKT protein
phosphorylation, which could inevitably result in undesired side effects. As expected, comparisons of the
IC50 values between tumor cell line NCI-H1650 (IC = 2.25 µM) and non-cancerous cell BEAS-2B (IC =
50
50
2.78 µM) indicated that 1a displayed almost the same cytotoxicity between the two cell lines. To the best of
our knowledge, the ERK and AKT proteins mainly locate in cytoplasm, while PDKs exist mainly in
mitochondria. We hypothesize that if we specifically transport a PDKs inhibitor into cancer cell
mitochondria, it would avoid disrupting the ERK and AKT signal pathway.
+
Fig. 2 Design of TPP -conjugated compounds
Generally, most cancer cells exhibit an elevated mitochondrial membrane potential (∆ψm) at least 60 mV
as compared to normal cells, which could be exploited to selectively target mitochondria for cancer therapy
+
[
23]. Triphenylphosphonium cation (TPP ) is widely used as a mitochondria-targeted moiety, and the
+
utilizing of TPP as a mitochondria-targeted vehicle has been well studied [24]. With this notion in our
3

ACCEPTED MANUSCRIPT
+
mind, we tethered triphenylphosphonium cations (TPP ) to a PDK1 inhibitor, with the aim to generate
specific mitochondria-targeted compounds to avoid off-target and / or side effects. As shown in Fig. 2, the
warhead is a parent molecule that inhibits PDK1 function and cancer cell proliferation. The end of the
+
warhead is attached to a TPP moiety through alkyl linkers, which were expected to modulate the
lipophilicity and cellular uptake of the target compounds.
+
2
.2. Synthesis of TPP -conjugated compounds
+
The preparation of the TPP -conjugated compounds were shown in Schemes 1 and 2. The reaction of
arylhalides (2a and 2b) with 4-acetyl-phenylboronic acid (3) yielded compounds 4a and 4b, which were
chlorinated in the presence of copper(II) chloride dehydrate and lithium chloride to conveniently produce
dichloro-substituted compounds 1a and 1b.
Scheme 1 Synthetic routes of the parent molecules 1a and 1b. Reagents and conditions: (i) K CO , H O, (n-Bu) NBr,
2
3
2
4
o
o
Pd(OAc) , 70 C; (ii) CuCl ·2H O, LiCl, DMF, 90 C.
2
2
2
The brominated carboxylic acids (5a-d) were then stirred with triphenyl phosphines in dry acetonitrile to
afford the phosphates (6a-d) with yields ranging from 87 to 95%. Finally, 1b and phosphates (6a-d)
+
coupled efficiently and gave the TPP -conjugated compounds 1c-f. All these target compounds 1a-f were
1
13
characterized by H NMR, C NMR and HRMS spectra (Supporting information).
+
Scheme 2 Synthetic routes of the TPP -conjugated target compounds 1c-f. Reagents and conditions: (i) P(Ph) , dry CH CN,
3
3
reflux; (ii) DCC, anhydrous DCM, RT.
4

ACCEPTED MANUSCRIPT
+
2
.3. TPP -conjugated compounds have little effect on the ERK and AKT signaling pathways
We aimed to develop PDK-selective anticancer compounds by minimizing their activities on AKT and
ERK signal pathways. Therefore, the target compounds first were chosen to assess their effect on AKT and
ERK phosphorylation through Western blotting experiments. As shown in Fig. 3, DAP, 1a and 1b at 2 µM
+
strongly reduced the AKT and ERK phosphorylation. Interestingly, the TPP -conjugated compounds 1c-f
displayed a minimal effect on AKT and ERK phosphorylation, which was in full agreement with our
hypothesis.
Fig. 3 Proteins phosphorylation in AKT and ERK signaling pathways after treated with DAP (2 µM) and 1a-1f (2 µM) in
NCI-H1650 cells for 12 h.
+
2
.4. TPP -conjugated compounds inhibit cancer cell proliferation and PDKs activity
+
With the improved selectivity of TPP -conjugated compounds, next the growth inhibitory effects of DAP,
1a−f, and 6a−d against the cancer cell lines MCF-7, A375 and NCI-H1650 were then measured. A
non-cancerous BEAS-2B cell was included to compare the selectivity of these compounds. Exposure of the
cells to the compounds for 72 h resulted in dose-dependent decreases in cell viability, except for
+
compounds 6a−d, which could rule out the possibility that the observed cytotoxicity is due to the TPP
moieties. As shown in Table 1, 1a exhibited strong antiproliferative activities against MCF-7, A375 and
NCI-H1650 cells, but its ability to inhibit PDKs (primary dehydrogenase enzymatic assay) was weak, with
an EC50 value of 3.8 µM, suggesting that 1a is not a specific PDKs inhibitor. Based on our previous study
(data are not shown here), the nitro-substitution in meta-position of benzene ring was beneficial for
inhibiting PDKs function, so a nitro group was introduced into 1a. As expected, the designed compound 1b
displayed an improved PDKs inhibitory potency with the EC50 value of 0.25 µM, however, the
+
selective-index (SI) value for 1b is only 1.6, as shown in Table 1. The introduction of TPP moieties
+
offered the TPP -conjugated compounds 1c
−f, which strongly inhibited cancer proliferation and PDKs
function. Interestingly, the SI values of 1c−f were also greatly increased, indicating that the
+
TPP -conjugated compounds selectively inhibit cancer cells, but not non-cancerous cells. Of note,
compound 1f with a linker of six carbon atoms displayed a potent growth inhibitory effect on NCI-H1650
cell (Fig. S1 in Supporting Information), the IC50 value is 0.21 µM. Compound 1f also exhibited promising
5

ACCEPTED MANUSCRIPT
PDKs inhibitory potency (EC = 0.1 µM), which warranted for further investigation.
5
0
Table 1 Biological evaluation of compounds 1a−f, 6a−d and DAP.
IC50 (µM)
PDKs inhibition
(EC , µM)
Cpds.
a
b
MCF-7
1.38 ± 0.22
0.63 ± 0.11
1.78 ± 0.30
0.97 ± 0.12
0.81 ± 0.17
0.50 ± 0.11
NI
A375
1.1 ± 0.25
0.75 ± 0.19
1.38 ± 0.21
1.27 ± 0.30
1.18 ± 0.21
0.51 ± 0.09
NI
NCI-H1650
2.25 ± 0.30
0.92 ± 0.27
0.87 ± 0.14
0.85 ± 0.14
0.88 ± 0.15
0.21 ± 0.04
NI
BEAS-2B
SI
50
1
1
a
2.78 ± 0.32
1.44 ± 0.10
6.5 ± 0.92
> 10
1.2
1.6
3.80 ± 1.41
0.25 ± 0.09
0.34 ± 0.10
0.36 ± 0.04
0.14 ± 0.02
0.10 ± 0.02
b
1
c
7.5
1
d
> 10
5.5
1
e
4.82 ± 0.10
3.28 ± 0.08
1
f
15.6
c
d
6
6
a
NI
NT
b
NI
NI
NI
NI
NI
NT
NT
NT
NI
6
c
NI
NI
NI
6
d
NI
NI
NI
NI
DAP
12.5 ± 2.1
8.5 ± 1.2
6.8 ± 0.91
7.7 ± 1.5
1.1
a
b
A normal human epithelial cells isolated from bronchial epithelium; Selective-index is calculated by comparing the IC50
c
values in human normal BEAS-2B cells against the IC50 value of the same compound in NCI-H1650 cancer cells; NI
d
represents no inhibition; NT represents not tested.
To explore the mechanism of cancer cells death, DAP and 1f were used to induce NCI-H1650 cells
apoptosis, which was then examined with the Annexin V-FITC/PI FACS assay. As shown in Fig. 4, the
percentages of apoptotic population in NCI-H1650 cells treated with DAP at 10 µM for 12 h was 10.6%,
while for 1f at 0.5, 1 and 2 µM, the percentages were 7.2, 13.8, and 17.2%, respectively, clearly
outperformed DAP in inducing cancer cell apoptosis. Moreover, the percentages of apoptosis for
NCI-H1650 cells treated with 1f at 1 µM for 6, 12 and 24 h were 6.6, 13.6, and 23.6 %, respectively,
suggesting the induction of apoptosis by 1f followed a time-dependent manner.
Fig. 4 Flow cytometer analysis of NCI-H1650 cells apoptosis. The cells were treated with DAP and 1f for 6, 12 and 24 hours,
then were stained with FITC Annexin V / PI. Cells in the upper right quadrant indicate PI positive/Annexin V positive, late
6

ACCEPTED MANUSCRIPT
apoptotic, or necrotic cells, and cells in lower right quadrant indicate early apoptotic cells. Bar graph represents statistics of
total apoptotic cell percentages from duplicate experiments. *P < 0.05, versus control group.
+
2
.5. TPP -conjugated compounds activate purified PDC by inhibiting PDK1
Compounds 1c-f were next evaluated for their effects on PDC activation by a primary dehydrogenase
+
enzymatic assay. As shown in Fig. 5A, the TPP -conjugated compounds 1c-f dramatically increased PDC
activity. To confirm the activation of PDC were indeed caused by direct inhibition of PDKs, we terminated
the kinase reaction with 55 mM of pyruvate and ADP before 1c-f addition. We then observed that NADH
formation was barely affected by the compounds (Fig. 5B), indicating that 1c-f had no effect on the
dehydrogenase activity. Clearly, the enhanced PDC activity was resulted from PDKs inhibition by the
compounds. The PDK1 kinase inhibitory activity was further evaluated in the presence of 1f via measuring
ATP consumption by the enzyme. As shown in Fig. 5C, 1f dose-dependently reduced PDK1 activity, with
the EC50 value of 0.12 µM. To verify that PDK1 inhibition would lead to the reduction of the PDC
phosphorylation level in cancer cells, NCI-H1650 cells were treated with DAP, 1b, and 1f for 12 h, then
PDC phosphorylation was analyzed by Western blotting experiment. As shown in Fig. 5D, 1f dramatically
reduced PDC phosphorylation level in a dose-responsive manner, while DAP and 1b had no effect at the
same concentrations.
Fig. 5 Compound 1f inhibited PDK1, and activated PDC activities. A: Compounds 1c-f effect on PDC kinetics. OD340 nm
o
values were read at 37 C. ‘w/o ATP’ represents maximal PDC activity when ATP was not included, while ‘with ATP’
represents minimal enzyme activity in the presence of 100 µM ATP. B: PDKs was inactivated, and then compounds 1c-f
effect on PDC kinetics. We run our experiments as singlet, but every dose was triplicate. The data was reported
as Mean ± SD. C: Effect of 1f on PDK1 kinase activity. D: Compounds 1f exhibited dose response manner to inhibit PDC
phosphorylation in NCI-H1650 cell, DAP and 1b were used as control groups.
7

ACCEPTED MANUSCRIPT
+
2
.6. TPP -conjugated compounds affect the mitochondrial bioenergetics function in NCI-H1650 cell
Inhibition of PDKs activates PDC, leading to a switch of pyruvate metabolism from lactate production to
OXPHOS in mitochondria. Thus, the oxygen consumption and the degree of acidification will change in
+
cancer cells. To explore whether TPP -conjugated compounds confer this switching, NCI-H1650 cells were
treated with 1b (5 µM), and 1f (1, 2, and 5 µM) for 4 h, then the changes in the extracellular acidification
rates (ECAR) and oxygen consumption rates (OCR) were monitored on a Seahorse XFe24 extracellular
flux, the lactate production was measured by a Nova Bioprofile Flex analyzer. As shown in Fig. 6A and 6B,
ECAR was significantly decreased with the addition of 1f. As expected, OCR was increased. The
enhancement of OCR for 1b at 5 µM is less significant than that of 1f at the same concentration. We
observed that 1f at 2 or 5 µM strongly inhibited the lactate formation in NCI-H1650 cell, clearly
outperforming 1b at 5 µM (Fig. 6C). In addition, we measured the reactive oxygen species (ROS)
production in NCI-H1650 cell treated with 1b and 1f. More ROS was generated with higher concentrations
of 1f, indicating an enhanced cellular respiration activity by the treatment of 1f (Fig. 6D). Based on these
results, we conclude that 1f could elicit a metabolic alteration in cancer cells to enhance oxidative
phosphorylation and ROS production, which might contribute to their anti-proliferation effect in the tumor
cells.
Fig. 6 Effect of 1b and 1f on mitochondrial bioenergetic function of NCI-H1650 cell. (A) ECAR was decreased by 1b and 1f
treatment. (B) OCR was increased with the treatment of 1b and 1f. (C) Lactate formation was decreased by treatment of 1b
and 1f. (D) ROS production was increased by treatment of 1b and 1f. * P < 0.05, versus the control group.
8

ACCEPTED MANUSCRIPT
+
2
.7. TPP -conjugated compounds depolarize mitochondrial membrane potential in NCI-H1650 cells
Cancer cells exhibit hyperpolarized mitochondrial membrane potentials (∆ψm) as compared to normal
cells. Depolarization of ∆ψm represents an effective strategy to inhibit cancer cell growth. Inhibitors
targeting PDKs enhance the OXPHOS in cancer cell, and the ∆ψm should be decreased accordingly. To
validate this hypothesis, NCI-H1650 cells were treated with 1b (5 µM) and 1f (1, 2, and 5 µM) for 4 hours,
which were then stained with 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolyl- carbocyanine iodide
(JC-1), a cationic dye, which accumulated in mitochondria of high potential and was accompanied by a
fluorescence emission shift from green to red due to the formation of the red fluorescent J-aggregates.
Mitochondrial depolarization is indicated by an increased ratio of the green / red fluorescence intensity,
which could be measured by a flow cytometer. As shown in Fig. 8, the quantitative analysis of JC-1 stained
cells revealed a significant increase in the green (low ∆ψm) / red (high ∆ψm) ratio as compared to the cells
treated with 1b at 5 µM or the control group, which suggested that 1f depolarized the mitochondrial
hyperpolarization and thus revived oxidative metabolism in cancer cells.
Fig. 7 Changes of ∆ψm in NCI-H1650 cells measured by JC-1 flow cytometry assay. Treatment of NCI-H1650 cells with 1b
(5 µM), and 1f (1, 2, and 5 µM) for 4 h led to the decrease of ∆ψm in NCI-H1650 cells. The green FL-1 represents
depolarized mitochondria (J-monomer), and the red FL-2 represents the hyperpolarized mitochondria (J-aggregates). The
depolarization of ∆ψm is indicated by the increase in the ratio of J monomer/J aggregate. * P < 0.05, versus the control
group.
9

ACCEPTED MANUSCRIPT
3. Conclusion
+
We have developed novel TPP -conjugated PDK1 inhibitors with improved potency and selectivity. The
+
TPP -conjugated compounds have abilities to enhance PDC enzymatic activity and PDK1 kinase inhibition.
+
The MTT assay suggested that TPP -conjugated compounds possessed prominent antiproliferative
activities against NCI-H1650 cells with IC50 values of 0.87, 0.85, 0.88 and 0.21 µM, respectively, while
exhibiting little effect on the non-cancerous BEAS-2B cells. Meanwhile, the most potent 1f among these
compounds could alter the glucose metabolic profile in NCI-H1650 cells by decreasing ECRA and lactate
formation, and increasing OCR values as well as ROS production. At last, 1f was found to depolarize ∆ψm
in NCI-H1650 cancer cells, which could trigger apoptotic stimuli and lead to cancer cell death. Collectively,
+
our data demonstrated that TPP -conjugated compounds, especially 1f could be a promising lead for the
developing therapeutic PDK1 inhibitor for cancer therapy.
4. Experimental protocols
4.1. General methods and target compounds synthesis
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 visualized under UV light at 254ꢀnm. Column
1
chromatography was performed on silica gel. H NMR spectra were obtained on an Agilent 400ꢀMR
1
3
spectrometer, while C NMR spectra were obtained with proton decoupling on an Agilent 400ꢀMR DD2
(100ꢀMHz) or 600ꢀMR DD2 spectrometer and were reported in ppm with residual solvent for internal
standard [δ 77.16 (CDCl )]. The chemical shifts were reported in parts per million (ppm), the coupling
3
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 7.0ꢀT 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.
4
.1.1 Synthesis of compounds 4a
To a round-bottom flask, 4.0 mmol of arylhalides (2a or 2b), 4.4 mmol of 1-(4-acetyl-phenyl)-boronic
acid (3), 0.2 mol % of Pd(OAc) , 10.0 mmol of powdered K CO and 4.0 mmol of (n-Bu) NBr were added.
−b
2
2
3
4
The flask was flushed with nitrogen, water (4.4 mL) was added and the resulting suspension was stirred and
o
heated for 4 h at 70 C. After completion of the reaction, the mixture was diluted with water, and extracted
with EtOAc (2 x 30 mL). The combined organic layers were dried over Na SO , filtered and concentrated.
2
4
The crude residues were purified by silica gel chromatography to afford the pure compound (4a in 89 %
yield, and 4b in 83 %) [25].
1
0

ACCEPTED MANUSCRIPT
1
-(4'-Hydroxy-3'-nitro-[1,1'-biphenyl]-4-yl)ethan-1-one (4b). Compound 4b was obtained as a yellow
o
1
solid after the flash chromatography. M.p. 117-119 C; H NMR (400 MHz, CDCl ): δ (ppm) 10.62 (s, 1H),
3
8
1
1
.36-8.37 (m, 1H), 8.04-8.06 (m, 2H), 7.86-7.88 (m, 1H), 7.65-7.67 (m, 2H), 7.27-7.29 (m, 1H), 2.65 (s,
1
3
H); C NMR (101 MHz, CDCl ): δ (ppm) 197.35, 154.89, 142.54, 136.35, 136.10, 133.81, 132.39,
3
-
29.15, 126.73, 123.15, 120.73, 26.63; MS (ESI): m/z, 256.0 [M-H] .
4.1.2 Synthesis of compounds 1a
−b
To a round-bottom flask 9.0 mmol of copper(II) chloride dihydrate, 9.0 mmol of lithium chloride and 1.5
mmol of the ketones (4a or 4b) were added. Then DMF (7.0 mL) was added, the mixture was heated to 90
ºC, and stirred for 6 h. Then the mixture was cooled to room temperature, diluted with water and extracted
with ether (3 x 30 mL). The combined organic layers were washed with water (3 x 20 mL) and dried over
Na SO , filtered and concentrated. The crude residues were purified by silica gel chromatography to afford
2
4
the pure compounds.
,2-Dichloro-1-(4'-hydroxy-[1,1'-biphenyl]-4-yl)ethan-1-one (1a). Compound 1a was obtained as a
2
o
1
yellow solid after flash chromatography. Yield: 48%; M.p. 114-117 C; H NMR (400 MHz, CDCl ): δ
3
1
3
(
ppm) 8.13 (m, 2H), 7.67 (m, 2H), 7.54 (m, 2H), 6.93 (m, 2H), 6.68 (s, 1H); C NMR (101 MHz, CDCl3):
δ (ppm) 185.67, 156.71, 146.93, 131.55, 130.39, 129.17, 128.66, 126.76, 116.03, 67.82; MS (ESI): m/z,
-
-
2
79.0 [M-H] ; HRMS (ESI): calcd. for C H Cl O [M-H] : 278.9985, found: 278.9986.
14 9 2 2
2
,2-Dichloro-1-(4'-hydroxy-3'-nitro-[1,1'-biphenyl]-4-yl)ethan-1-one (1b). Compound 1b was obtained
o
1
as a yellow solid after flash chromatography. Yield: 64%; M.p. 90-93 C; H NMR (400 MHz, CDCl ): δ
3
(ppm) 10.66 (s, 1H), 8.41 (s, 1H), 8.20-8.22 (m, 2H), 7.88-7.90 (m, 1H), 7.71-7.73 (m, 2H), 7.30-7.32 (m,
1
3
1
1
H), 6.67 (s, 1H); C NMR (101 MHz, CDCl ): δ (ppm) 185.29, 155.17, 143.96, 136.05, 133.85, 131.83,
3
-
30.67, 130.42, 126.96, 123.35, 120.93, 67.87; MS (ESI): m/z, 324.0 [M-H] ; HRMS (ESI): calcd. for
-
C H Cl NO [M-H] : 323.9836, found: 323.9834.
14
8
2
4
4
.1.3 Synthesis of compounds 1c
−f
To a round-bottom flask was added 5a (5.0 mmol), P(Ph) (20.0 mmol) and dry MeCN (10.0 mL). The
3
mixture was stirred vigorously and heated to reflux. After the refluxing was ceased (15 h), the solution was
concentrated. The residue was rinsed consecutively with benzene (3 x 10 mL), hexanes (10 mL), and ether
(2 x 10 mL). The crystalline white solid was dried to give 6a (1.8 g, 87%) [26]. 6b-d were prepared in
according to the procedure described as 6a.
To a round-bottom flask was added 1b (0.25 mmol), 6a (0.2 mmol) and DCC (0.246 mmol). The flask
was flushed with nitrogen, and then anhydrous DCM (6 mL) was added. The reaction was stirred at room
temperature for one day. Then was filtered and concentrated, and purified by silica gel chromatography to
afford compound 1c in 70% yield [27]. Compounds 1d-f were prepared in according to the procedure
11

ACCEPTED MANUSCRIPT
described as 1c.
3-((4'-(2,2-Dichloroacetyl)-3-nitro-[1,1'-biphenyl]-4-yl)oxy)-3-oxopropyl)triphenylphosphonium
(
o
1
bromide (1c). Compound 1c was obtained as a white solid after flash chromatography. M.p. 89-93 C; H
NMR (400 MHz, CDCl ): (ppm) 8.27-8.28 (m, 1H), 8.21-8.23 (m, 2H), 7.82-7.93 (m, 10H), 7.69-7.76 (m,
3
1
3
9
1
1
H), 6.67 (s, 1H), 4.41-4.47 (m, 2H), 3.31-3.37 (m, 2H); C NMR (101 MHz, CDCl ): δ (ppm) 185.37,
3
61.04, 143.94, 143.58, 141.39, 138.47, 135.33, 133.73 (d, J = 10.1 Hz), 133.55, 130.67 (d, J = 12.1 Hz),
30.55, 130.42, 127.51, 127.26, 124.01, 117.61 (d, J = 86.9 Hz), 67.91, 27.62, 18.44 (d, J = 58.6 Hz); MS
+
+
(
ESI): m/z, 642.1 [M-Br] ; HRMS (ESI): calcd. for C H Cl NO P [M-Br] : 642.0998, found: 642.1027.
35 27 2 5
(4-((4'-(2,2-Dichloroacetyl)-3-nitro-[1,1'-biphenyl]-4-yl)oxy)-4-oxobutyl)triphenylphosphonium
(1d).
o
1
Compound 1d was obtained as a white solid after the flash chromatography. M.p. 117-120 C; H NMR
(
400 MHz, CDCl ): (ppm) 8.32 (m, 1H), 8.21-8.24 (m, 2H), 7.86-7.92 (m, 7H), 7.78-7.80 (m, 3H),
3
1
3
7
.65-7.75 (m, 9H), 6.68 (s, 1H), 4.15-4.23 (m, 2H), 3.37-3.40 (m, 2H), 2.09 (m, 2H); C NMR (101 MHz,
CDCl ): δ (ppm) 185.36, 171.18, 144.02, 143.62, 141.74, 138.30, 135.06, 133.68 (d, J = 10.1 Hz), 133.66,
3
130.90, 130.74, 130.51 (d, J = 13.1 Hz), 127.51, 126.76, 124.17, 118.01 (d, J = 86.9 Hz), 67.91, 33.31 (d, J
+
=
19.2 Hz), 21.47 (d, J = 51.5 Hz), 17.80; MS (ESI): m/z, 656.1 [M-Br] ; HRMS (ESI): calcd. for
+
C H Cl NO P [M-Br] : 656.1155, found: 656.1155.
36
29
2
5
(5-((4'-(2,2-Dichloroacetyl)-3-nitro-[1,1'-biphenyl]-4-yl)oxy)-5-oxopentyl)triphenylphosphonium (1e).
o
1
Compound 1e was obtained as a white solid after the flash chromatography. M.p. 100-104 C; H NMR
400 MHz, CDCl ): (ppm) 8.31 (m, 1H), 8.22-8.24 (m, 2H), 7.86-7.92 (m, 7H), 7.69-7.80 (m, 11H),
(
3
7
2
1
.43-7.45 (m, 1H), 6.68 (s, 1H), 3.99-4.02 (m, 2H), 2.86-2.89 (m, 2H), 2.22-2.26 (m, 2H), 1.86-1.88 (m,
13
H); C NMR (101 MHz, CDCl ): δ (ppm) 185.42, 170.65, 143.78, 143.21, 141.64, 138.03, 134.96, 134.93,
3
33.50 (d, J = 10.1 Hz), 133.45, 130.95, 130.60, 130.39 (d, J = 13.1 Hz), 127.32, 126.15, 123.95, 117.92 (d,
J = 85.9 Hz), 68.03, 32.87, 24.78 (d, J = 18.2 Hz), 22.36 (d, J = 69.7 Hz), 21.50; MS (ESI): m/z, 670.1
+
+
[
M-Br] ; HRMS (ESI): calcd. for C H Cl NO P [M-Br] : 670.1311, found: 670.1311.
37 31 2 5
(6-((4'-(2,2-Dichloroacetyl)-3-nitro-[1,1'-biphenyl]-4-yl)oxy)-6-oxohexyl)triphenylphosphonium
(1f).
o
1
Compound 1f was obtained as a white solid after the flash chromatography. M.p. 94-97 C; H NMR (400
MHz, CDCl ): (ppm) 8.31 (m, 1H), 8.21-8.23 (m, 2H), 7.86-7.95 (m, 7H), 7.68-7.81 (m, 11H), 7.49-7.51
3
1
3
(
m, 1H), 6.69 (s, 1H), 3.91-3.98 (m, 2H), 2.67-2.71 (m, 2H), 1.80-1.89 (m, 4H), 1.69-1.75 (m, 2H); C
NMR (101 MHz, CDCl ): δ (ppm) 185.41, 170.98, 144.05, 143.53, 141.91, 138.10, 135.00, 133.63 (d, J =
3
1
6
0.1 Hz), 133.45, 130.98, 130.69, 130.49 (d, J = 13.1 Hz), 127.46, 126.48, 124.10, 118.24 (d, J = 85.9 Hz),
+
7.98, 33.50, 29.50 (d, J = 17.2 Hz), 23.89, 22.86, 22.36; MS (ESI): m/z, 684.1 [M-Br] ; HRMS (ESI):
+
calcd. for C H Cl NO P [M-Br] : 684.1468, found: 684.1463.
3
8
33
2
5
1
2

ACCEPTED MANUSCRIPT
4
.2. Biological evaluations
.2.1 Cell cultures
4
Cancer cell lines NCI-H1650, A375, and MCF-7, and the non-cancerous BEAS-2B cell line were purchased
from Shanghai cell bank of Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM
medium supplemented with 10% FBS, 1% penicillin and streptomycin. Cells grown at exponential stage were
used for all the biological experiments.
4
.2.2 Cell viability assay
Cell viability upon the treatment of the prepared compounds and DAP was tested by using the MTT
assay. A suspension of cells (5000/well for NCI-H1650, 3500/well for MCF-7 and A375, and 4000/well for
BEAS-2B) were seeded in 96-well plates and cultured for 24 h. Then seven different concentrations of 1a-f
ranging from 10-0.16 µM (PBS buffer containing 1% DMSO) and DAP (40-0.63 µM) were added into the
96-well plates, in which cells were incubated for 72 h. Then, 10 µL of MTT (0.5 mg/mL) solution was
added to each well of the plates. After 4 h incubation at 37 °C, the supernatant was removed and 100 µL
DMSO was added, with the aid of gentle shaking to dissolve the formazan crystals in the plates. The
absorbance of the each well was measured at 570 nm in a microplate reader (Bio-Rad Laboratories,
Shanghai, USA). At last, resultant OD570 nm values were expressed as IC50 values, which were the mean
values derived from three independent experiments.
4
.2.3 Cell apoptosis detection
NCI-H1650 cells were seeded at a density of 4 × 10 cells/mL on each well of 6-well plates and were
5
allowed to grow overnight. Then cells were treated with DAP (10 µM) and 1f (0.5, 1, and 2 µM) for 12 h,
and 1f at 1 µM for 6, 12, 24 h. 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 resuspended
5
in 1× Annexin binding buffer to ∼5 × 10 cells/mL. To the 100 µL of cell suspension, 5 µL of Annexin V
and 10 µL of PI were added and incubated for 15 min at room temperature. After the incubation, 400 µL of
binding buffer was added to each sample and was gently mixed and analyzed immediately on a flow
cytometer (Accuri C6, BD Biosciences).
4.2.4 JC-1 assay for flow cytometry analysis
5
NCI-H1650 cells were cultured on 6-well plates at a density of 4 x 10 cells/mL and allowed to grow
overnight at 37 °C. Cells were treated with 1b (5 µM) and 1f (1, 2, and 5 µM) for 4 h at 37 °C. A solution
of 1 µM JC-1 reagent 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 and trypsinized for 5 min at 37 °C.
The cells were isolated and washed 3 times in PBS and centrifuged (1000 rpm / min for 5 min). The
1
3

ACCEPTED MANUSCRIPT
resulting cell pellet was resuspended in 100 µL PBS and analyzed by BD FACSCalibur flow cytometry.
4.2.5 ROS measurement
4
NCI-H1650 cells were seeded at a density of 4 × 10 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 (Beyotime,
Jiangsu, China) solution was added (100 µL/well) to incubate the cells for 45 min at 37 °C. Remove the
medium and washed with 1 × PBS for three times. Then the cells were treated with 1b (5 µM), and 1f (1, 2,
and 5 µM) at 37 °C for 4 h. At last, the plate was measured on a microplate reader at Ex / Em = 485 / 535
nm.
4.2.6 Western blotting assay
NCI-H1650 cells were seeded in 6-well plates and treated with compounds at desired concentrations for
2 h. Then the treated cells were incubated with cell lysate buffer for 15 min, and centrifuged at 12000 rpm
min at 4 °C for 15 min. Protein concentration for each sample was assessed by Pierces BCA Protein Assay
1
/
Kit and balanced to the same level, followed by a 8 min protein denaturation with SDS loading buffer at
00 °C. Proteins in the samples were separated by SDS-PAGE electrophoresis, transferred onto
1
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).
4.2.7 Statistical analysis
Data was reported as Mean ± SD. Statistical analysis was performed using GraphPad Prism version 6.0
for Windows. *p < 0.05 was considered as statistically significant.
Acknowledgments
We thank the financial support from Chongqing University of the Start-up Research Grant (No:
0247001104409), and the National Natural Science Foundation of China (No. 21572027).
References
[
1]
2]
D. Hanahan, R.A. Weinberg, Hallmarks of cancer: the next generation, Cell 144 (2011) 646−674.
S.L. Zhang, Y. He, K.Y. Tam, Targeting cancer metabolism to develop human lactate
dehydrogenase (hLDH)5 inhibitors, Drug Discov. Today (2018) https://doi.org/
[
1
0.1016/j.drudis.2018.05.014.
[3]
M.G.V. Heiden, L.C. Cantley, C.B. Thompson, Understanding the Warburg effect: the metabolic
1
4

ACCEPTED MANUSCRIPT
requirements of cell proliferation, Science 324 (2009) 1029–1033.
[4]
[5]
[6]
M.G.V. Heiden, Targeting cancer metabolism: a therapeutic window opens, Nat. Rev. Drug
Discovery 10 (2012) 671−684.
M.S. Patel, L.G. Korotchkina, Regulation of the pyruvate dehydrogenase complex, Biochem. Soc.
Trans. 34 (2006) 217−222.
X.K. Yu, Y. Hiromasa, H. Tsen, J.K. Stoops, T.E. Roche, Z.H. Zhou, Structures of the human
pyruvate dehydrogenase complex cores: a highly conserved catalytic center with flexible N-terminal
domains, Structure 16 (2008) 104–114.
[
[
[
[
[
[
7]
8]
9]
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) 1112−1119.
W. Zhang, S.L. Zhang, X.H. Hu, K.Y. Tam, Targeting tumor metabolism for cancer treatment: is
pyruvate dehydrogenase kinases (PDKs) a viable anticancer target? Int. J. Biol. Sci. 11 (2015)
1
390−1400.
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. Cancer 98 (2008) 1975−1984.
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 potential therapeutic target, Int. J. Oncol. 42 (2013)
4
4–54.
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, Cancer Biol.
Ther. 6 (2007) 1476−1479.
12] 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) 1−6.
[
13] S. Kamarajugadda, L. Stemboroski, Q. Cai, N.E. Simpson, S. Nayak, M. Tan, J. Lu, Glucose
oxidation modulates anoikis and tumor metastasis, Mol. Cell. Biol. 32 (2012) 1893–1907.
14] T. Hitosugi, J. Fan, T.W. Chung, K. Lythgoe, X. Wang, J. Xie, Q. Ge, T.L. Gu, R.D. Polakiewicz,
J.L. Roesel, G.Z. Chen, T.J. Boggon, S. Lonial, H. Fu, F.R. Khuri, S. Kang, J. Chen, Tyrosine
phosphorylation of mitochondrial pyruvate dehydrogenase kinase 1 is important for cancer
metabolism, Mol. Cell 44 (2011) 864–877.
[
[15] E. Saunier, C. Benelli, S. Bortoli, The pyruvate dehydrogenase complex in cancer: an old metabolic
1
5

ACCEPTED MANUSCRIPT
gatekeeper regulated by new pathways and pharmacological agents, Int. J. Cancer 138 (2016)
8
09–817.
[
16] 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) 9832−9843.
17] 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 modification, Cancer Res. 75 (2015)
[
4
923−4936.
[
18] 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 subtype-selective and potent covalent
pyruvate dehydrogenase kinase 1 (PDK1) inhibitors, J. Med. Chem. 60 (2017) 2227–2244.
19] J. Espinal, T. Leesnitzer, A. Hassman, M. Beggs, J. Cobb, Inhibition of pyruvate dehydrogenase
kinase by halogenated acetophenones, Drug Develop. Res. 35 (1995) 130–135.
[
[
20] L.J. Qin, Y. Tian, Z.L. Yu, D.B. Shi, J.S. Wang, C.L. Zhang, R.Y. Peng, X.Z. Chen, C.C. Liu, Y.M.
Chen, W.L. Huang, W.G. Deng, Targeting PDK1 with dichloroacetophenone to inhibit acute
myeloid leukemia (AML) cell growth, Oncotarget 7 (2016) 1395–1407.
[
21] S.L. Zhang, X.H. Hu, W. Zhang, K.Y. Tam, Unexpected discovery of dichloroacetate derived
adenosine triphosphate competitors targeting pyruvate dehydrogenase kinase to inhibit cancer
proliferation, J. Med. Chem. 59 (2016) 3562–3568.
[
22] S.L. Zhang, W. Zhang, Q.P. Xiao, Z. Yang, X.H. Hu, Z.Y. Wei, K.Y. Tam, Development of
dichloroacetamide pyrimidines as pyruvate dehydrogenase kinase inhibitors to reduce cancer cell
growth: synthesis and biological evaluation, RSC Adv. 6 (2016) 78762–78767.
[
23] D.C. Wallace, Mitochondria and cancer, Nat. Rev. Cancer 12 (2012) 685−698.
24] J.A. Jara, V. Castro-Castillo, J. Saavedra-Olavarria, L. Peredo, M. Pavanni, F. Jana, M.E. Letelier, E.
Parra, M.I. Becker, A. Morello, U. Kemmerling, J.D. Maya, J. Ferreira, Antiproliferative and
uncoupling effects of delocalized, lipophilic, cationic gallic acid derivatives on cancer cell lines.
Validation in vivo in singenic mice, J. Med. Chem. 57 (2014) 2440−2454.
[
[
25] M. Eleni, Ladopoulou, A.N. Matralis, A. Nikitakis, A.P. Kourounakis, Antihyperlipidemic
morpholine derivatives with antioxidant activity: an investigation of the aromatic substitution,
Bioorg. Med. Chem. 23 (2015) 7015–7023.
[
26] E. Momán, D. Nicoletti, A. Mouriňo, Synthesis of novel analogues of 1α,25-dihydroxyvitamin D3
with side chains at C-18, J. Org. Chem. 69 (2004) 4615–4625.
1
6

ACCEPTED MANUSCRIPT
[27] V. Cuchelkar, P. Kopečková, J. Kopeček, Novel HPMA copolymer-bound constructs for combined
tumor and mitochondrial targeting, Mol. Pharmaceutics 5 (2008) 776–786.
1
7

ACCEPTED MANUSCRIPT
Highlights
►
Target compounds have less side effect and more specific as compared with their parent
molecule
►
►
1f displayed a very strong PDKs inhibitory activity with an EC value of 0.1 µM
50
1f showed very promising cytotoxicity to cancer cells but little effect to normal cells