Development of dual inhibitors targeting pyruvate dehydrogenase kinases and human lactate dehydrogenase A: High-throughput virtual screening, synthesis and biological validation

Article abstract of DOI:10.1016/j.ejmech.2020.112579

Most cancer cells feature an altered glucose metabolism from oxidative phosphorylation to cytoplasmic glycolysis. Pyruvate dehydrogenase kinases (PDKs) and lactate dehydrogenase A (LDHA) play crucial roles in promotion of glycolysis, thus the inhibition of both enzymes is considered a promising strategy for developing of anticancer therapeutics. Herein, we describe the first discovery of series novel dual inhibitors targeting PDKs and LDHA. We identified 6 hits from a library database containing 485465 compounds through a high-throughput virtual screening assay. Hit-to-lead optimization enabled us to discover two compounds, namely 20e and 20k, which inhibited PDKs with IC₅₀ values of 0.8, and 1.6 μM, respectively, and inhibited LDHA with IC₅₀ values of 0.15 and 0.7 μM, respectively. Meanwhile, the two compounds reduced A549 cell proliferation with EC₅₀ values of 13.2, and 15.7 μM. Furthermore, 20e and 20k decreased the lactate formation, and increased oxygen consumption, suggesting the two compounds modulated the glucose metabolic pathways in cancer cells.

Full text of DOI:10.1016/j.ejmech.2020.112579

European Journal of Medicinal Chemistry 203 (2020) 112579
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Development of dual inhibitors targeting pyruvate dehydrogenase
kinases and human lactate dehydrogenase A: High-throughput virtual
screening, synthesis and biological validation
,
**
Sichuan Xiang a, Ding Huang a, Qiaolin He a, Jie Li a, Kin Yip Tam b, 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 Faculty of Health Sciences, University of Macau, Taipa, Macau, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Most cancer cells feature an altered glucose metabolism from oxidative phosphorylation to cytoplasmic
glycolysis. Pyruvate dehydrogenase kinases (PDKs) and lactate dehydrogenase A (LDHA) play crucial roles
in promotion of glycolysis, thus the inhibition of both enzymes is considered a promising strategy for
developing of anticancer therapeutics. Herein, we describe the first discovery of series novel dual in-
hibitors targeting PDKs and LDHA. We identified 6 hits from a library database containing 485465
compounds through a high-throughput virtual screening assay. Hit-to-lead optimization enabled us to
Received 15 January 2020
Received in revised form
18 May 2020
Accepted 12 June 2020
Available online 15 July 2020
discover two compounds, namely 20e and 20k, which inhibited PDKs with IC50 values of 0.8, and 1.6
respectively, and inhibited LDHA with IC50 values of 0.15 and 0.7 M, respectively. Meanwhile, the two
compounds reduced A549 cell proliferation with EC50 values of 13.2, and 15.7 M. Furthermore, 20e and
20k decreased the lactate formation, and increased oxygen consumption, suggesting the two compounds
modulated the glucose metabolic pathways in cancer cells.
mM,
Keywords:
m
Cancer metabolism
Cancer proliferation
Dual inhibitor
Pyruvate dehydrogenase kinase
Lactate dehydrogenase A
m
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
cycle (Fig. S1, see in Supporting Information) [5e7]. Many evi-
dences have shown that PDKs were closely related to the cancer
Compared to health cells, which rely on the mitochondrial
oxidative phosphorylation (OXPHOS) to generate ATP for cellular
proliferation, most cancer cells have adapted to an enhanced level
of glucose utilization and produced energy by a high rate of
glycolysis in the cytosol even under normal oxygen conditions,
which is known as Warburg effect [1,2]. Inhibition of this altered
metabolism phenotype may preferentially impede tumor growth
and proliferation [3,4].
Key factors, such as enzymes and transporters that involved in
glycolysis, are thus considered as promising targets for cancer
treatment. One of a central player in glycolysis is PDKs, which
negative regulate the activity of pyruvate dehydrogenase complex
(PDC), an important gatekeeper enzyme that catalyzes the con-
version of pyruvate to acetyl-CoA and links to the tricarboxylic acid
metabolism, and this enzyme overexpressed in variety of tumor
cells, such as lung cancer [8,9], gastric cancer [10] and head and
neck squamous cancer [11], which lead to the idea of utilizing PDKs
as a therapeutic target for the treatment of cancer. Another central
player in glycolysis is LDHA, which is responsible for the conversion
of pyruvate to lactate at the end step of the glycolytic process
[12e14]. The overexpression of LDHA has been frequently reported
in a variety of highly glycolytic human cancers, thus conferring the
growth advantages of highly glycolytic cancer cells by ensuring a
sustainable energy supply. Recent studies suggested that the inhi-
bition of LDHA reduced invasive and metastatic potential of cancer
cell by decreasing their proliferation ability [15,16]. These facts
make LDHA a potentially important therapeutic target to mitigate
the highly activated glycolysis pathway in cancer cells.
To date, various inhibitors have been reported to inhibit PDKs
and LDHA individually. As shown in Fig. 1, AZD7545 [17] is a PDKs
inhibitor with a trifluoromethylpropanamide warhead, which
binds to the lipoamide-binding site of PDKs. In contrast, Radicicol
* Corresponding author.
** Corresponding author.
E-mail addresses: zhangsl@cqu.edu.cn (S.-L. Zhang), yun.he@cqu.edu.cn (Y. He).
https://doi.org/10.1016/j.ejmech.2020.112579
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

2
S. Xiang et al. / European Journal of Medicinal Chemistry 203 (2020) 112579
Fig. 1. Reported PDKs inhibitors (a) and LDHA inhibitors (b). The inserted graphics are the binding models of PDKs with VER-246608 and LDHA with Gne140, respectively.
[18], PS10 [19], and VER-246608 [20] are ATP-competitive in-
hibitors, binding to ATP pocket of PDKs. Of note, VER-246608 was
found to inhibit PDK1 activity with an IC50 value of 35 nM, but
weakly inhibited cancer cell proliferation. Correspondingly, 1 [21]
PDK2 (PDB code: 4V26) as our binding template, which exhibited
clear electron densities and high resolution of 2.2 and 2.5 (Å),
respectively. The proteins were prepared by using Protein Prepa-
ration Workflow of the software package by hydrogens addition
and energy minimization, for LDHA, the His192 was protonated,
and Asp282 was protonated in PDK2. Then a commercially available
small library with 485465 compounds was filtered to remove
and 2 [22] are LDHA inhibitors with IC50 values of 0.5, and 0.6 mM,
respectively. 3 has been demonstrated to exhibit a low nanomolar
biochemical activity on hLDHA, but the compound displayed
limited cellular activity. In 2016, Gne140 [23] has been reported by
Genentech, which strongly inhibited LDHA activity with an IC50
value of 3 nM. The co-crystal complex of Gne140 with LDHA sug-
gested that the ligand formed direct hydrogen bond interactions
with Asn137, Arg168 and His192, which played crucial roles in the
process of LDHA catalytic function.
Considering the important biological roles of PDKs and LDHA in
glycolysis, we suppose that simultaneously inhibit both enzymes
would be more effective to reverse glucose metabolism from
cytoplasmic glycolysis to oxidative phosphorylation, thus leading
cancer cells to death. To verify this hypothesis, we herein conducted
a project to discover of dual inhibitors targeting PDKs and LDHA. To
the best of our knowledge, this is the first attempt to discover dual
inhibitors targeting PDKs and LDHA.
compounds that do not follow the rules (350
< Molecule
Weight < 500, Clogp < 5, 0 < Rotational Bond Numbers < 10,
0 < Hydrogen Bond Donor < 5, 0 < Hydrogen Bond Acceptor < 10,
30 < Polar Surface Area <120), as shown in Fig. 2. The remaining
183033 drug-like compounds were then energy minimized and
docked into the binding pocket of LDHA where Gne140 bound to.
Then 6000 top-ranked compounds with highest total binding
scores were selected for further consideration, compounds were
taken if they formed direct hydrogen bond interactions with the
amino acids of Asn137, Arg168 and His192 in LDHA. Following the
criterion, 250 potential compounds were identified, which were
then docked to PDK2. Similar to the binding model of VER-246608
in PDK2, compounds were selected if they formed hydrogen bond
interactions with amino acids of Asp282, and Thr346. Following the
workflow system, we finally identified 6 compounds (4, 5, 6, 7, 8,
and 9, see Fig. 3A), which were synthesized in multistep reactions,
as shown in Supporting Information. Meanwhile, we obtained 4-6’s
precursors 4_1, 5_1, and 6_1 for biological evaluation.
2. Results and discussion
2.1. Screening of library to identify potential compounds
We selected crystal structures of LDHA (PDB code: 4ZVV) and
Then the 9 compounds were measured anti-LDHA activity by an
enzymatic assay in the presence of pyruvate and NADH. As shown

S. Xiang et al. / European Journal of Medicinal Chemistry 203 (2020) 112579
3
line. Exposure of 4-9 at 20
mM to A549 cells for 72 h, then cell
viability was measured in a MTT assay. As shown in Fig. 3D, almost
all compounds inactive on A549, but 7 gave a moderate activity
(~50% inhibitory rate) at the tested concentration.
Then we docked 4-9 into PDK2 and LDHA to rationalize their
biochemical activities. As shown in Fig. 4A, 7 formed two hydrogen
bond interactions with Asp282, and Thr346, while one hydrogen
bond interaction is missing in 8, which was full agreement with the
biochemical activity shown in Fig. 3C. In Fig. 4B, the carbonyl group
in amide of 7 formed two hydrogen bond interactions with His192
and Asn137, the bond lengths are 1.9, and 1.7 Å, respectively, while
the lengths increase to 4.0 and 4.5 Å in 8, suggesting LDHA inhib-
itory effect for 8 should less potent than 7, which was verified in
Fig. 3B.
2.2. Synthesis of 7 analogues
Since 7 exhibited the most promising biochemical activities on
both enzymes (IC50 values are 5.9 and 9.1 mM for LDHA and PDKs,
Fig. 2. Flow chart of the multistep virtual screening strategy for the PDKs and LDHA
respectively), and moderate anti-proliferative activity on cancer
cell, we have interest to synthesize its analogues with the aim to
further improve its potency. First, we designed series of derivatives
with different substituents on benzene ring. As shown in Scheme 1,
3-hydroxy-2-methyl-4H-pyran-4-one (10) reacted with ethyl bro-
moacetate in the presence of potassium carbonate to afford 11,
which was hydrolyzed to give compound 12. Amidation of 12 with
various phenyl piperazines (13a-k) offer the targeting compounds
14a-k. In addition, pyridine-2-yl, pyrimidin-2-yl, and diphenyl
groups were incorporated to give 14l, 14m, and 14n respectively.
Removal of the carbonyl group adjacent to the piperazine ring of
14a-k to the other side of the piperazine ring, and generated series
of 20a-k. As shown in Scheme 2, 1-Boc-4-(2-hydroxyethyl)-piper-
azine (16) was brominated to offer compound 17, which was
reacted with 3-hydroxy-2-methyl-4H-pyran-4-one to a ether (18).
Removal of the Boc group in 18 with TFA provided 19 in a 98% yield,
dual inhibitors.
in Fig. 3B, 4-6 exhibited higher anti-LDHA activities as compared to
their precursors, which could be explained by the disappearance of
direct hydrogen bond interaction of the ethyl ester group with
amino acid of LDHA, while hydrogen bond interaction was picked
up in the hydroxyl group of 4-6. 7 gave the most potent inhibitory
activity with an IC50 value of 5.9
of 4-9 was measured by using an in vitro primary PDC assay. In this
assay, 4-9 were tested at an initial concentration of 10 M to disturb
mM. Then PDKs inhibitory activity
m
the PDC catalysis activity, which represents PDKs inhibitory po-
tency. As shown in Fig. 3C, most of the compounds inhibited PDKs
activity, with the inhibition rates of ~50% at 10
mM. Finally, 4-9 were
then chose to evaluate their growth inhibitory effect on cancer cell
Fig. 3. Validation of the identified compounds 4-9. Compounds 4_1, 5_1, and 6_1 were the analogues of 4, 5, and 6, respectively. (A) Chemical structures of 4-9; (B) LDHA inhibitory
effect of 4-9; (C) PDKs inhibitory effect of 4-9; (D) Cell viability of 4-9 against A549 cell lines. Compounds were used at 10 mM for the enzymatic assay, and 20 mM for cell viability
assay.

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S. Xiang et al. / European Journal of Medicinal Chemistry 203 (2020) 112579
Fig. 4. Proposed binding model of 4-9. Compounds 4-9 were docked into (A) PDK2, and (B) LDHA. The docking studies were carried out in a SYBYL-X 2.0 software, the yellow dashes
represents the hydrogen bond interactions, and the amino acids involved in hydrogen bond interactions were shown as stick and colored as red, the graphics of 3D views were
drawn by PyMOL. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
which was amidated with various benzyl carboxylic acid to afford
target compounds 20a-k.
the para-position of benzene ring of 14d greatly increase anti-LDHA
M), but the PDKs inhibitory activity dis-
appeared. However, a nitro-moiety (14g) was incorporated into the
activity (IC50 ¼ 0.56
m
same position, which contributed no anti-LDHA activity improve-
2.3. Structure-activity relationship study of the target compounds
ment, but the inhibition of PDKs activity increased to 0.8 mM. In
addition, pyridinly (14l), pyrimidinly (14m), and diphenyl (14n)
groups were introduced, but weak potencies were obtained on both
enzymes. Finally, we measure anticancer activity of compounds
14a-n. To our disappointment, all of the newly synthesized com-
pounds inactivated on A549 cancer cell.
We therefore docked 14a-n into PDK2 and LDHA, and found that
if we remove the carbonyl group in 14a-k from one side of piper-
azine to the other side, the generated compounds could pick up
hydrogen bond interaction with Lys246 in PDKs, and Arg98 in
LDHA. With this notion in mind, we designed and synthesized
chemical series of 20a-k with the hope to further improve their
biochemical activity and cellular potency. As shown in Table 2, 20a
We then measured their biochemical activities on both en-
zymes, and the results of these biological evaluations were sum-
marized in Table 1.
The hit compound 7 displayed moderate potencies on LDHA and
PDKs, the IC50 values are 5.9 and 9.1
of the chloro in 7 by fluoro group (14a, IC50 > 10
the chloro atom (14d, IC50 > 10 M) decreased anti-LDHA activity,
but a great enhancement of PDKs inhibitory effect was observed,
with the IC50 values increased to 1.0 M for 14a, and 1.8 M for 14d,
m
M, respectively. Replacement
mM) or removal of
m
m
m
respectively. Remove the fluoro group in meta- (14a) to para-
position of benzene ring, the formed compound (14b) retained
anti-LDHA activity (IC50 ¼ 3.7
mM), and increase PDKs inhibitory
potency to 1.4 M. The introduction of a methoxyl group (14e) in
displayed potent anti-LDHA activity (IC50 ¼ 2.8
mM), but inactivate
m

S. Xiang et al. / European Journal of Medicinal Chemistry 203 (2020) 112579
5
Table 1
Enzymatic inhibitory effect of 14a-n a
.
Compounds
LDHA IC50
(m
M)
PDKs IC50 (mM)
14a
14b
14c
14d
14e
14f
14g
14h
14i
14j
14k
14l
14m
14n
7
>10
3.7 0.53
>10
1.0 0.11
1.4 0.20
>10
1.8
>10
>10
0.56 0.10
4.6 0.72
6.9 1.3
>10
>10
>10
>10
>10
>10
7.3 1.3
5.9 0.8
>10
0.8 0.11
6.3 1.12
>10
8.1 1.50
>10
>10
>10
>10
9.1 1.1
a
The IC50 values were calculated from two independent experimental
measurements.
Table 2
Scheme 1. Synthetic routes for compounds 14a-n. Reactions and conditions: (a) Ethyl
bromoacetate, K2CO3, DMF, 50 ꢀC; (b) LiOH, MeOH: H2O ¼ 3:1; (c) 1-(3-chlorophenyl)
piperazines, HATU, DIPEA, DCM.
Biological activity of 20a-ka.
a
Compounds
LDHA IC50
(m
M)
PDKs IC50
(
mM)
EC50 (mM) against A549
20a
20b
20c
20d
20e
20f
20g
20h
20i
20j
20k
7
2.8 0.51
7.7 1.12
>10
>10
>10
>10
NTb
NT
NT
>20
13.2 2.24
>20
NT
>20
NT
NT
3.4 0.61
5.9 0.15
0.8 0.02
3.9 0.65
3.0
5.1 0.85
>10
>10
1.6 0.30
9.1 1.1
on PDKs, while introducing of meta-methyl (20b) or methoxyl
(20c) groups into benzene ring of 20a gave shrunk activities on
both enzymes, however, the activities recovered by incorporating
of a ortho-fluoro group into the benzene ring of 20a, with IC50
0.15 0.03
2.7 0.40
>10
4.7 0.58
3.7
>10
0.7 0.13
5.9 0.8
values of 3.4
mM for LDHA, and 5.9
mM for PDKs. This potency was
further increased to 0.15 and 0.8
mM for both enzymes by removing
15.7 3.11
18.5
the ortho-fluoro to para-position. Meanwhile, 20k was also found
to strongly inhibited PDKs and LDHA, the IC50 values are 0.7 and
1.6 mM, respectively. Then 20d-f, 20h, and 20k were selected to
measure their antiproliferative activity, as shown in Table 2, 20e
and 20k exhibited inhibitory effects with EC50 values of 13.2 and
a
The IC50 and EC50 were calculated from two independent experimental
measurements.
b
Not tested.
15.7 mM, moderate growth inhibitory activity, but still a good start
to discover cellular active PDKs and LDHA dual inhibitors.
To verify the design strategy, the most potent compounds 20e,
20k and 14b were docked to PDK2 and LDHA, respectively. As
shown in Fig. 5, the carbonyl group in 20e and 20k formed direct
hydrogen bond interaction with Lys246 of PDK2, and with Arg98 of
LDHA, while these interaction disappear in compound 14b-PDK2 or
14b-LDHA complex, indicating the improved potency of 20e and
20k was result from the extra interaction.
2.4. Compounds 20e and 20k altered mitochondrial bioenergetics
function in A549 cancer cell
PDKs and LDHA are important checkpoint enzymes that effect
pyruvate metabolism in cancer cell. Targeting the enzymes could
switch of pyruvate consumption from lactate production to
oxidative phosphorylation to form acetyl-CoA in the mitochon-
drion, therefore the oxygen consumption rate (OCR) and lactate
Scheme 2. Synthetic routes for compounds 20a-k. Reactions and conditions: (a) (Boc)2O, DCM; (b) CBr4, PPh3, THF; (c) K2CO3, DMF; (d) TFA, DCM, 0 ꢀC; (e) HATU, DIPEA, DCM.

6
S. Xiang et al. / European Journal of Medicinal Chemistry 203 (2020) 112579
compound 20e and 20k, which inhibited PDKs with IC50 values of
0.8, and 1.6 M, respectively, and inhibited LDHA with IC50 values of
0.15 and 0.7 M, respectively. Meanwhile, the two compounds
reduced A549 cell proliferation with EC50 values of 13.2, and
15.7 M. Furthermore, 20e and 20k significantly decreased the
m
m
m
lactate formation, and increased oxygen consumption, suggesting
the two compounds modulated the glucose metabolic pathways in
cancer cells, which could be used as promising lead for the devel-
opment of potent LDHA and PDKs dual inhibitors. Further work on
the chemical modification and SAR studies of 20e and 20k to
further improve the cellular activity are ongoing and will be the
topic of subsequent reports.
4. Experimental section
4.1. Materials and methods
All building blocks were purchased from commercial sources
and used as received. Reactions were monitored by thin-layer
chromatography (TLC) and carried out on Merck Kieselgel 60 F254
plates, which could be visualized under UV light at 254 nm. All 1H
NMR spectra were obtained on an Agilent 400 MR spectrometer at
ambient temperature and reported in ppm downfield from TMS
(0 ppm). All 13C NMR spectra were obtained with proton decou-
pling on an Agilent 400 MR DD2 (100 MHz) and reported in ppm
relative to CDCl3 (77.16 ppm). Coupling constants were reported as
Hz with multiplicity denoted as s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet), and br (broad). The NMR data was pro-
cessed by software Mest Re-Nova (Ver. 9.0.0.12821, Mestrelab
Research S.L.). Compounds exhibited purities of more than 95% as
determined by peak area integration in reverse phase
chromatography.
4.2. Chemical synthesis
4.2.1. Synthesis of compounds 11 and 12
Compounds 11 and 12 were synthesized as described by pre-
vious reported procedures [24].
4.2.2. Synthesis of 3-(2-(4-(3-chlorophenyl)piperazin-1-yl)-2-
oxoethoxy)-2-methyl-4H-pyran-4-one (7)
A mixture of 2-((2-methyl-4-oxo-4H-pyran-3-yl)oxy)acetic acid
(12, 102 mg, 0.55 mmol), HATU (232 mg, 0.61 mmol) and N,N-
Diisopropylethylamine (111 mg, 1.1 mmol) in 5 ml of DCM was
stirred for 30 min at room temperature. Then 1-(3-chlorophenyl)
piperazine (100 mg, 0.55 mmol) was added, the mixture was stirred
for another 2 h at room temperature. After removal of solvent un-
der reduced pressure. The crude product was purified by flash
chromatography (EA: PE ¼ 1:1) to give compound 7 as a colorless
oil (169 mg, yield 85%). M.p. 83.0e83.8 ꢀC. 1H NMR (400 MHz,
Fig. 5. Proposed binding model of 20e, 20k and 14b in PDK2 and LDHA.
formation in cytoplasm should be changed accordingly. So the
metabolic modulatory effect 20e and 20k on A549 cell were eval-
uated by measuring OCR on a Seahorse XFe24 extracellular flux
analyzer, and the lactate production was measured by Nova Bio-
profile Flex analyzer. As shown in Fig. 6A, lactate formation was
significantly decreased in a dose response manner with the addi-
tion of 20e and 20k, which can be explained by the reduction of
glycolysis in the A549 cell. As expected, OCR in A549 cell was
increased by treatment of 20e and 20k. Especially, the enhance-
ment of OCR for 20e and 20k is much more significant than that of
7 at same concentration, suggesting the two compounds out-
performed 7.
CDCl3)
d
7.71e7.52 (m, 1H, 1CH), 7.17 (t, J ¼ 8.1 Hz, 1H, 17CH),
6.88e6.80 (m, 2H, 13CH, 14CH), 6.79e6.72 (m, 1H, 15CH), 6.33 (d,
J ¼ 5.6 Hz, 1H, 2CH), 4.93 (s, 2H, 7CH2), 3.79e3.69 (m, 4H, 9CH2,
12CH2), 3.28e3.09 (m, 4H, 10CH2, 11CH2), 2.44 (s, 3H, 6CH3). 13C
NMR (101 MHz, CDCl3)
d 174.68 (3C), 166.79 (8C), 160.09 (5C),
153.60 (1C), 151.87 (18C), 143.86 (4C), 135.02 (16C), 130.14 (14C),
120.14 (2C), 117.15 (15C), 116.37 (17C), 114.43 (13C), 68.97 (7C),
49.17, 48.83 (9C, 12C), 44.68, 41.52 (10C, 11C), 15.14 (6C). MS (API-
ES): m/z calcd for C18H20ClN2O4 363.1 [MþH]þ; found 363.1. HRMS
3. Conclusion
(ESI): calcd. for
385.0905. HPLC purity 99.8%.
C
18H19ClN2NaO4 [MþNa]þ: 385.0925, found:
In this paper, we screened a library of 485465 molecules
through a high-throughput virtual screening, from which only
compound 7 exhibited moderate anti-LDHA activity and PDKs
inhibitory potency, but are inactive against the growth of
A549 cells. Then the hit-to-lead optimization enabled us to identify
4.2.3. Synthesis of 3-(2-(4-(3-fluorophenyl)piperazin-1-yl)-2-
oxoethoxy)-2-methyl-4H-pyran-4-one (14a)
Compound 14a was prepared in according to the procedure

S. Xiang et al. / European Journal of Medicinal Chemistry 203 (2020) 112579
7
Fig. 6. Compounds 20e and 20k altered A549 cell bioenergetics. A549 cells (20000/well) were treated with 7, 20e and 20k for 4 h. (A) Lactate formation was decreased by treatment
of 7, 20e and 20k; (B) OCR was increased with the treatment of 7, 20e and 20k.
described as compound 7. Colorless oil, yield 93%. 1H NMR
(400 MHz, CD3OD)
105.5e106.3 ꢀC. 1H NMR (400 MHz, CD3OD)
d
7.94 (d, J ¼ 5.6 Hz, 1H,
d
7.96 (d, J ¼ 5.6 Hz, 1H, 1CH), 7.21 (q, J ¼ 15.3,
1CH), 7.23 (t, J ¼ 7.9 Hz, 2H, 14CH, 16CH), 6.96 (d, J ¼ 8.1 Hz, 2H,
13CH, 17CH), 6.85 (t, J ¼ 7.3 Hz, 1H, 15CH), 6.38 (d, J ¼ 5.6 Hz, 1H,
2CH), 4.90 (s, 2H, 7CH2), 3.77e3.64 (m, 4H, 9CH2, 12CH2), 3.21e3.10
(m, 4H, 10CH2, 11CH2), 2.42 (s, 3H, 6CH3). 13C NMR (101 MHz,
8.2 Hz, 1H, 16CH), 6.76 (dd, J ¼ 8.3, 2.0 Hz, 1H, 13CH), 6.69 (dt,
J ¼ 12.4, 2.2 Hz,1H,17CH), 6.54 (td, J ¼ 8.3, 2.1 Hz,1H,15CH), 6.39 (d,
J ¼ 5.6 Hz, 1H, 2CH),
d 4.91 (s, 2H, 7CH2), 3.82e3.60 (m, 4H, 9CH2,
12CH2), 3.31e3.11 (m, 4H, 10CH2, 11CH2), 2.43 (s, 3H, 6CH3). 13C
NMR (101 MHz, CD3OD) 175.34 (3C), 167.45 (8C), 164.98, 162.57
CD3OD) d 175.35 (3C), 167.43 (8C), 160.93 (5C), 155.16 (1C), 151.08
d
(18C),143.66 (4C),128.71 (14C,16C),120.16 (2C),116.53 (15C),116.10
(13C, 17C), 68.51 (7C), 49.52, 49.12 (9C, 12C), 44.56, 41.53 (10C, 11C),
13.77 (6C). MS (API-ES): m/z calcd for C18H21N2O4 329.1 [MþH]þ;
found 329.1. HRMS (ESI): calcd. for C18H20N2NaO4 [MþNa]þ:
351.1315, found: 351.1298. HPLC purity 98.3%.
(14C), 160.94 (5C), 155.18 (1C), 152.85, 152.75 (18C), 143.64 (4C),
130.02,129.92 (16C),116.10 (2C),111.48, 111.46, (17C) 105.83,105.62,
(15C) 102.77, 102.52 (13C), 68.51 (7C), 48.65 (9C, 12C), 44.31, 41.32
(10C, 11C), 13.78 (6C). MS (API-ES): m/z calcd for C18H19FN2NaO4
369.1 [MþNa]þ; found 369.1. HRMS (ESI): calcd. for C18H19FN2NaO4
[MþNa]þ: 369.1221, found: 369.1201. HPLC purity 99.7%.
4.2.7. Synthesis of 3-(2-(4-(4-methoxyphenyl)piperazin-1-yl)-2-
oxoethoxy)-2-methyl-4H-pyran-4-one (14e)
4.2.4. Synthesis of 3-(2-(4-(4-fluorophenyl)piperazin-1-yl)-2-
oxoethoxy)-2-methyl-4H-pyran-4-one (14b)
Compound 14e was prepared in according to the procedure
described as compound 7. White solid, yield 95%. M.p.
Compound 14b was prepared in according to the procedure
143.5e144.3 ꢀC. 1H NMR (400 MHz, CD3OD)
d
7.95 (d, J ¼ 5.6 Hz, 1H,
described as compound 7. Colorless oil, yield 95%. 1H NMR
1CH), 6.95 (d, J ¼ 9.0 Hz, 2H, 14CH, 16CH), 6.83 (d, J ¼ 9.0 Hz, 2H,
13CH, 17CH), 6.39 (d, J ¼ 5.6 Hz, 1H, 2CH), 4.90 (s, 2H, 7CH2), 3.73 (s,
3H, 19OCH3), 3.72e3.62 (m, 4H, 9CH2, 12CH2), 3.12e2.97 (m, 4H,
10CH2, 11CH2), 2.42 (s, 3H, 6CH3). 13C NMR (101 MHz, CD3OD)
(400 MHz, CD3OD)
d
7.95 (d, J ¼ 5.5 Hz, 1H, 1CH), 6.97 (d, J ¼ 6.4 Hz,
4H, 13, 14, 16, 17CH), 6.38 (d, J ¼ 5.5 Hz, 1H, 2CH), 4.90 (s, 2H, 7CH2),
3.80e3.58 (m, 4H, 9CH2, 12CH2), 3.10 (s, 4H, 10CH2, 11CH2), 2.42 (s,
3H, 6CH3). 13C NMR (101 MHz, CD3OD)
d
175.35 (3C), 167.42 (8C),
d 175.37 (3C), 167.41 (8C), 160.98 (5C), 155.20 (1C), 154.63 (15C),
160.95, 158.75 (15C), 156.38 (5C), 155.18 (1C), 147.84 (18C), 143.64
(4C), 118.51, 118.44 (13C, 17C), 116.09 (2C), 115.09, 114.87 (14C, 16C),
68.50 (7C), 50.32, 49.93 (9C, 12C), 44.60, 41.56 (10C, 11C), 13.76 (6C).
MS (API-ES): m/z calcd for C18H20FN2O4 347.1 [MþH]þ; found 347.1.
HRMS (ESI): calcd. for C18H19FN2O4 [MþH]þ: 347.1401, found:
347.1385. HPLC purity 96.6%.
145.19, 143.65 (13C, 17C), 118.80 (2C), 116.08, 114.01 (14C, 16C),
68.49 (7C), 54.48 (19C), 51.00, 50.61 (9C, 12C), 44.68, 41.64 (10C,
11C), 13.75 (6C). MS (API-ES): m/z calcd for C19H23N2O5 359.1
[MþH]þ; found 359.1. HRMS (ESI): calcd. for C19H22N2NaO5
[MþNa]þ: 381.1420, found: 381.1403. HPLC purity 96.5%.
4.2.8. Synthesis of 3-(2-(4-(3-methoxyphenyl)piperazin-1-yl)-2-
oxoethoxy)-2-methyl-4H-pyran-4-one (14f)
4.2.5. Synthesis of 3-(2-(4-(2-fluorophenyl)piperazin-1-yl)-2-
oxoethoxy)-2-methyl-4H-pyran-4-one (14c)
Compound 14f was prepared in according to the procedure
Compound 14c was prepared in according to the procedure
described as compound 7. White solid, yield 92%. 1H NMR
described as compound 7. Faint yellow oil, yield 90%. 1H NMR
(400 MHz, CD3OD)
d
7.93 (d, J ¼ 5.6 Hz, 1H, 1CH), 7.12 (t, J ¼ 8.2 Hz,
(400 MHz, CD3OD)
d
7.95 (d, J ¼ 5.6 Hz, 1H, 1CH), 7.13e6.94 (m, 4H,
1H, 14CH), 6.53 (dd, J ¼ 8.2, 1.9 Hz, 1H, 13CH), 6.47 (t, J ¼ 2.0 Hz, 1H,
17CH), 6.42 (dd, J ¼ 8.1, 2.0 Hz, 1H, 15CH), 6.37 (d, J ¼ 5.6 Hz, 1H,
2CH), 4.87 (s, 2H, 7CH2), 3.73 (s, 3H, 19OCH3), 3.71e3.66 (m, 2H),
3.65e3.58 (m, 2H) (9CH2, 12CH2), 3.16e3.09 (m, 4H, 10CH2, 11CH2),
14-17CH), 6.39 (d, J ¼ 5.6 Hz, 1H, 2CH), 4.89 (s, 2H, 7CH2), 3.79e3.70
(m, 2H), 3.71e3.65 (m, 2H) (9CH2, 12CH2), 3.11e3.00 (m, 4H, 10CH2,
11CH2), 2.42 (s, 3H, 6CH3). 13C NMR (101 MHz, CD3OD)
d 175.38
(3C),167.47 (8C),161.01 (5C),156.96 (13C),155.22 (1C),154.52 (13C),
143.66 (4C), 139.57, 139.48 (18C), 124.41, 124.37 (16C), 123.02,
122.94 (15C), 119.26, 119.23 (17C), 116.09 (2C), 115.70, 115.49 (14C),
68.53 (7C), 50.54, 50.14 (9C, 12C), 44.72, 41.65 (10C, 11C), 13.77 (6C).
MS (API-ES): m/z calcd for C18H19FN2NaO4 369.1 [MþNa]þ; found
369.1. HPLC purity 97.5%.
2.40 (s, 3H, 6CH3). 13C NMR (101 MHz, CD3OD)
d 175.37 (3C), 167.40
(8C), 161.02 (16CH), 160.65 (5C), 155.24 (1C), 152.37 (18C), 143.67
(4C), 129.47 (14C), 116.10 (2C), 109.00 (13C), 105.12 (15C), 102.65
(17C), 68.54 (7C), 54.23 (19C), 49.26, 48.90 (9C, 12C), 44.42, 41.47
(10C, 11C), 13.83 (6C). MS (API-ES): m/z calcd for C19H23N2O5 359.16
[MþH]þ; found 359.2. HRMS (ESI): calcd. for C19H22N2NaO5
[MþNa]þ: 381.1420, found: 381.1403. HPLC purity 99.2%.
4.2.6. Synthesis of 2-methyl-3-(2-oxo-2-(4-phenylpiperazin-1-yl)
ethoxy)-4H-pyran-4-one (14d)
Compound 14d was prepared in according to the procedure
described as compound 7. White solid, yield 93%. M.p.
4.2.9. Synthesis of 2-methyl-3-(2-(4-(4-nitrophenyl)piperazin-1-
yl)-2-oxoethoxy)-4H-pyran-4-one (14g)
Compound 14g was prepared in according to the procedure

8
S. Xiang et al. / European Journal of Medicinal Chemistry 203 (2020) 112579
described as compound 7. Yellow solid, yield 80%. M.p.
189.1e190.8 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
J ¼ 5.6 Hz,1H, 2CH), 6.06 (s, 2H,13CH,17CH), 5.97 (s,1H,15CH), 4.87
(s, 2H, 7CH2), 3.67 (s, 6H, 19, 20OCH3), 3.58e3.47 (m, 4H, 9CH2,
12CH2), 3.16e3.02 (m, 4H, 10CH2, 11CH2), 2.34 (s, 3H, 6CH3). 13C
d
8.06 (d, J ¼ 2.3 Hz,
1H, 1CH), 8.05e8.01 (m, 2H, 14CH, 16CH), 7.00 (d, J ¼ 9.4 Hz, 2H,
13CH, 17CH), 6.33 (d, J ¼ 5.6 Hz, 1H, 2CH), 4.88 (s, 2H, 7CH2),
3.62e3.54 (m, 4H, 9CH2, 12CH2), 3.54e3.46 (m, 4H, 10CH2, 11CH2),
NMR (101 MHz, DMSO‑d6)
d 174.05 (3C), 166.75 (8C), 161.46 (14C,
16C), 159.68 (5C), 155.42 (1C), 153.05 (18C), 143.74 (4C), 116.88 (2C),
95.03 (13C, 17C), 91.99 (15C), 68.57 (7C), 55.42 (19C, 20C), 48.87,
48.57 (9C, 12C), 44.27, 41.31, (10C, 11C), 15.30 (6C). MS (API-ES): m/z
calcd for C20H25O6 389.17[MþH]þ; found 389.2. HPLC purity 97.4%.
2.35 (s, 3H, 6CH3). 13C NMR (101 MHz, DMSO‑d6)
d 174.03 (3C),
167.07 (8C), 159.67 (5C), 155.43 (1C), 154.76 (18C), 143.78 (4C),
137.41 (15C), 126.16 (14C, 16C), 116.88 (2C), 113.00 (13C, 17C), 68.62
(7C), 46.42, 46.16 (9C, 12C), 43.62, 40.91 (10C, 11C), 15.30 (6C). MS
(API-ES): m/z calcd for C18H19N3NaO6 396.1 [MþNa]þ; found 396.1.
HRMS (ESI): calcd. for C18H20N3O6 [MþH]þ: 374.1346, found
374.1327. HPLC purity 99.4%.
4.2.14. Synthesis of 2-methyl-3-(2-oxo-2-(4-(pyridin-2-yl)
piperazin-1-yl)ethoxy)-4H-pyran-4-one (14l)
Compound 14l was prepared in according to the procedure
described as compound 7. White solid, yield 90%. M.p.
4.2.10. Synthesis of 2-methyl-3-(2-(4-(2-nitrophenyl)piperazin-1-
yl)-2-oxoethoxy)-4H-pyran-4-one (14h)
101.2e102.0 ꢀC. 1H NMR (400 MHz, CD3OD)
d
8.09 (dd, J ¼ 4.9,
1.2 Hz, 1H, 13CH), 7.94 (d, J ¼ 5.6 Hz, 1H, 1CH), 7.61e7.51 (m, 1H,
15CH), 6.82 (d, J ¼ 8.6 Hz, 1H, 16CH), 6.69 (dd, J ¼ 6.8, 5.2 Hz, 1H,
14CH), 6.38 (d, J ¼ 5.6 Hz, 1H, 2CH), 4.90 (s, 2H, 7CH2), 3.72e3.61
(m, 4H, 9CH2, 12CH2), 3.59e3.48 (m, 4H, 10CH2, 11CH2), 2.41 (s, 3H,
Compound 14h was prepared in according to the procedure
described as compound 7. Yellow solid, yield 86%. M.p.
121.0e121.8 ꢀC. 1H NMR (400 MHz, CD3OD)
d
7.95 (d, J ¼ 5.6 Hz, 1H,
1CH), 7.80e7.73 (m, 1H, 14CH), 7.58e7.52 (m, 1H, 16CH), 7.31 (d,
J ¼ 8.2 Hz, 1H, 15CH), 7.16 (t, J ¼ 7.7 Hz, 1H, 17CH), 6.38 (d, J ¼ 5.6 Hz,
1H, 2CH), 4.88 (s, 2H, 7CH2), 3.76e3.62 (m, 4H, 9CH2, 12CH2),
3.08e3.01 (m, 4H, 10CH2, 11CH2), 2.42 (s, 3H, 6CH3). 13C NMR
6CH3). 13C NMR (101 MHz, CD3OD)
d 175.34 (3C), 167.61 (8C),160.95
(17C), 159.13 (5C), 155.20 (1C), 147.04 (13C), 143.67 (4C), 137.92
(16C), 116.11 (2C), 113.63 (14C), 107.86 (16C), 68.59 (7C), 45.11, 44.76
(9C, 12C), 44.14, 41.21 (10C, 11C), 13.80 (6C). MS (API-ES): m/z calcd
for C17H19N3NaO4 352.1 [MþNa]þ; found 352.1. HPLC purity 99.4%.
(101 MHz, CD3OD)
d 175.38 (3C), 167.57 (8C), 161.06 (5C), 155.26
(1C), 145.26 (18C), 144.58, 143.67 (13C), 133.32 (16C), 124.97 (14C),
123.03 (17C), 121.91 (15C), 116.10 (2C), 68.60 (7C), 51.76, 51.37 (9C,
12C), 44.72, 41.73 (10C, 11C), 13.80 (6C). MS (API-ES): m/z calcd for
4.2.15. Synthesis of 2-methyl-3-(2-oxo-2-(4-(pyrimidin-2-yl)
piperazin-1-yl)ethoxy)-4H-pyran-4-one (14m)
C
C
18H19N3NaO6 396.1 [MþNa]þ; found 396.1. HRMS (ESI): calcd. for
Compound 14m was prepared in according to the procedure
18H19N3NaO6 [MþNa]þ: 396.1166, found 396.1148.
described as compound 7. Colorless oil, yield 91%. 1H NMR
(400 MHz, CD3OD)
d
8.33 (d, J ¼ 4.8 Hz, 2H, 13CH, 15CH), 7.95 (d,
4.2.11. Synthesis of 2-methyl-3-(2-oxo-2-(4-(o-tolyl)piperazin-1-
yl)ethoxy)-4H-pyran-4-one (14i)
J ¼ 5.6 Hz, 1H, 1CH), 6.62 (t, J ¼ 4.8 Hz, 1H, 14CH), 6.39 (d, J ¼ 5.6 Hz,
1H, 2CH), 4.90 (s, 2H, 7CH2), 3.89e3.81 (m, 4H, 9CH2, 12CH2),
3.70e3.60 (m, 4H, 10CH2, 11CH2), 2.42 (s, 3H, 6CH3). 13C NMR
Compound 14i was prepared in according to the procedure
described as compound 7. Faint yellow solid, yield 96%. M.p.
(101 MHz, CD3OD)
d 175.37 (3C), 167.74 (8C), 161.37 (16C), 160.96
108.2e109.2 ꢀC. 1H NMR (400 MHz, CD3OD)
d
7.94 (d, J ¼ 5.6 Hz, 1H,
(5C), 157.68 (13C, 15C), 155.19 (1C), 143.67 (4C), 116.09 (2C), 110.27
(14C), 68.62 (7C), 44.25, 43.44 (9C, 12C), 43.06, 41.30 (10C, 11C),
1CH), 7.18e7.09 (m, 2H, 14CH, 16CH), 7.04e6.94 (m, 2H, 15CH,
17CH), 6.38 (d, J ¼ 5.6 Hz, 1H, 2CH), 4.91 (s, 2H, 7CH2), 3.75e3.64
(m, 4H, 9CH2, 12CH2), 2.92e2.84 (m, 4H, 10CH2, 11CH2), 2.42 (s, 3H,
13.74 (6C). MS (API-ES): m/z calcd for
C16H18N4NaO4 353.1
[MþNa]þ; found 353.1. HPLC purity 99.4%.
6CH3), 2.31 (s, 3H,19CH3). 13C NMR (101 MHz, CD3OD)
d 175.35 (3C),
167.52 (8C), 160.93 (5C), 155.16 (1C), 150.62 (18C), 143.68 (4C),
132.42 (13C), 130.71 (14C)_, 126.36 (16C), 123.62 (17C),118.93 (15C),
116.12 (2C), 68.52 (7C), 51.82, 51.46 (9C,12C), 45.12, 42.07 (10C,11C),
16.56 (19C), 13.81 (6C). MS (API-ES): m/z calcd for C19H23N2O4
343.16 [MþH]þ; found 343.2.
4.2.16. Synthesis of 3-(2-(4-benzhydrylpiperazin-1-yl)-2-
oxoethoxy)-2-methyl-4H-pyran-4-one (14n)
Compound 14n was prepared in according to the procedure
described as compound 7. Colorless oil, yield 84%. 1H NMR
(400 MHz, CD3OD)
d
7.91 (d, J ¼ 5.6 Hz, 1H, 1CH), 7.42 (d, J ¼ 7.5 Hz,
4H, 14, 18, 20, 24CH), 7.26 (t, J ¼ 7.5 Hz, 4H, 15, 17, 21, 23CH), 7.16 (t,
J ¼ 7.3 Hz, 2H, 16CH, 22CH), 6.35 (d, J ¼ 5.6 Hz, 1H, 2CH), 4.80 (s, 2H,
7CH2), 4.25 (s, 1H, 13CH), 3.61e3.44 (m, 4H, 9CH2, 12CH2),
2.52e2.26 (m, 7H, 10CH2, 11CH2, 6CH3). 13C NMR (101 MHz, CD3OD)
4.2.12. Synthesis of 3-(2-(4-(3,5-dimethylphenyl)piperazin-1-yl)-2-
oxoethoxy)-2-methyl-4H-pyran-4-one (14j)
Compound 14j was prepared in according to the procedure
described as compound 7. Colorless oil, yield 96%. 1H NMR
d
175.35 (3C), 167.52 (8C), 160.93 (5C), 155.16 (1C), 150.62, 143.68
(400 MHz, CD3OD)
d
7.94 (d, J ¼ 5.6 Hz, 1H, 1CH), 6.59 (s, 2H), 6.52
(4C), 132.42 (19C, 25C), 130.71 (15, 17, 21, 23C), 126.36 (14, 18, 20,
24C), 123.62 (16C, 22C), 116.12 (2C), 68.52 (7C), 51.46 (9C, 12C),
45.12, 42.07 (10C, 11C), 16.56, 13.81 (6C). MS (API-ES): m/z calcd for
(s,1H) (13,15,17 CH), 6.38 (d, J ¼ 5.6 Hz,1H, 2CH), 4.89 (s, 2H, 7CH2),
3.73e3.63 (m, 4H, 9CH2, 12CH2), 3.14e3.08 (m, 4H, 10CH2, 17CH2),
2.42 (s, 3H, 6CH3), 2.23 (s, 6H, 19CH3, 20CH3). 13C NMR (101 MHz,
C
25H27N2O4 419.19 [MþH]þ; found 419.2. HPLC purity 98.9%.
CD3OD)
d 175.35 (3C), 167.41 (8C), 160.94 (5C), 155.17 (1C), 151.13
(18C), 143.65 (4C), 138.25 (14C, 16C), 122.03 (15C), 116.09 (2C),
114.51 (13C, 17C), 68.49 (7C), 49.72, 49.31 (9C, 12C), 44.57, 41.55
(10C, 11C), 20.26 (19C, 20C), 13.76 (6C). MS (API-ES): m/z calcd for
4.2.17. Synthesis of compounds 16 and 17
Compounds 16 and 17 were synthesized according to the pre-
vious reported procedures [25].
C
C
20H25N2O4 357.18 [MþH]þ; found 357.2. HRMS (ESI): calcd. for
20H24N2NaO4 [MþNa]þ: 379.1628, found 379.1609. HPLC purity
4.2.18. Synthesis of tert-butyl 4-(2-((2-methyl-4-oxo-4H-pyran-3-
yl)oxy)ethyl)piperazine-1-carboxylate (18)
96.8%.
A
mixture of tert-butyl 4-(2-bromoethyl)piperazine-1-
4.2.13. Synthesis of 3-(2-(4-(3,5-dimethoxyphenyl)piperazin-1-yl)-
2-oxoethoxy)-2-methyl-4H-pyran-4-one (14k)
carboxylate(17, 233 mg, 0.79 mmol), K2CO3 (219 mg, 1.59 mmol)
and 3-hydroxy-2-methyl-4H-pyran-4-one (100 mg, 0.79 mmol) in
DMF (5 mL) was stirred at room temperature until the reaction
complete. The product was collected by filtration. The solvent was
removed under reduced pressure. The crude product was purified
Compound 14k was prepared in according to the procedure
described as compound 7. Colorless oil, yield 90%. 1H NMR
(400 MHz, DMSO‑d6)
d
8.05 (d, J ¼ 5.6 Hz, 1H, 1CH), 6.33 (d,

S. Xiang et al. / European Journal of Medicinal Chemistry 203 (2020) 112579
9
by flash chromatography (DCM: MeOH ¼ 20: 1) to give a white solid
(247 mg, yield 92%). 1H NMR (400 MHz, CDCl3)
159.86 (5C), 155.21 (1C), 144.22 (4C), 136.57 (19C), 129.51 (15C),
118.50 (2C), 116.01 (14C), 115.23 (18C), 112.04 (16C), 68.44 (7C),
57.27 (8C), 54.47 (20C), 52.97, 52.49 (9C,12C), 41.65 (10C, 11C),13.55
(6C). MS (API-ES): m/z calcd for C20H25N2O5 373.17 [MþH]þ; found
373.2. HPLC purity 98.2%.
d
7.60 (d, J ¼ 5.5 Hz,
1H), 6.31 (d, J ¼ 5.5 Hz, 1H), 4.17 (t, J ¼ 4.9 Hz, 2H), 3.50e3.34 (m,
4H), 2.69 (t, J ¼ 4.9 Hz, 2H), 2.56e2.40 (m, 4H), 2.33 (s, 3H), 2.03 (s,
1H), 1.43 (s, 9H). 13C NMR (101 MHz, CDCl3)
d 174.92, 159.17, 154.73,
153.39, 144.65, 117.11, 79.59, 77.35, 77.03, 76.71, 68.58, 58.03, 53.08,
28.39, 28.22, 14.80.
4.2.23. Synthesis of 3-(2-(4-(2-fluorobenzoyl)piperazin-1-yl)
ethoxy)-2-methyl-4H-pyran-4-one (20d)
4.2.19. Synthesis of 2-methyl-3-(2-(piperazin-1-yl)ethoxy)-4H-
pyran-4-one hydrochloride (19)
Compound 20d was prepared in according to the procedure
described as compound 20a. Colorless oil, yield 86%. 1H NMR
To a solution containing tert-butyl 4-(2-((2-methyl-4-oxo-4H-
pyran-3-yl)oxy)ethyl)piperazine-1-carboxylate (18, 1.48 mmol,
500 mg) in 30 mL of THF was slowly added 2 N HCl (10 mL). After
stirring the reaction mixture for 6 h, the solvent was evaporated,
and the crude product (405 mg, 99%) was obtained, which was used
directly in the next step.
(400 MHz, CD3OD)
d
7.96 (d, J ¼ 5.6 Hz, 1H, 1CH), 7.54e7.45 (m, 1H,
14CH), 7.42e7.36 (m, 1H, 16CH), 7.28 (t, J ¼ 7.4 Hz, 1H, 15CH), 7.20 (t,
J ¼ 9.1 Hz, 1H, 17CH), 6.39 (d, J ¼ 5.6 Hz, 1H, 2CH), 4.16 (t, J ¼ 5.3 Hz,
2H, 7CH2), 3.87e3.79 (m, 2H), 3.45e3.37 (m, 2H) (10, 11CH2), 2.85
(t, J ¼ 5.3 Hz, 2H, 8CH2), 2.80e2.73 (m, 2H), 2.70e2.62 (m, 2H)
(9CH2, 12CH2), 2.39 (s, 3H, 6CH3). 13C NMR (101 MHz, CD3OD)
d
175.93 (3C), 165.77 (13C), 161.12, 159.31 (18C), 156.86 (5C), 155.36
4.2.20. Synthesis of 3-(2-(4-benzoylpiperazin-1-yl)ethoxy)-2-
methyl-4H-pyran-4-one (20a)
(1C), 144.15 (4C), 131.71, 131.63 (16C), 128.57, 128.54 (14C), 124.67,
124.64 (15C), 123.43, 123.25 (19C), 116.00 (2C), 115.59, 115.37 (17C),
68.25 (7C), 57.23 (8C), 52.85, 52.40 (9C, 12C), 46.44, 41.21 (10, 11C),
13.56 (6C). MS (API-ES): m/z calcd for C19H21FN2NaO4 383.1
[MþNa]þ; found 383.1. HRMS (ESI): calcd. for C19H22FN2O4
[MþH]þ: 361.1558, found 361.1557. HPLC purity 95.6%.
A mixture of acid (0.36 mmol), HATU (166 mg, 0.44 mmol) and
triethylamine (73.5 mg, 0.73 mmol) in 3 mL of DCM was stirred for
30 min at room temperature. Then 2-methyl-3-(2-(piperazin-1-yl)
ethoxy)-4H-pyran-4-one hydrochloride (19, 100 mg, 0.36 mmol)
was added, the mixture was stirred 2 h at room temperature. After
removal of solvent under reduced pressure. The crude product was
purified by flash chromatography to give compound 20a. Colorless
4.2.24. Synthesis of 3-(2-(4-(4-fluorobenzoyl)piperazin-1-yl)
ethoxy)-2-methyl-4H-pyran-4-one (20e)
oil, yield 77%. 1H NMR (400 MHz, CD3OD)
d
7.94 (d, J ¼ 5.6 Hz, 1H,
Compound 20e was prepared in according to the procedure
1CH), 7.52e7.42 (m, 3H, 15-17CH), 7.42e7.34 (m, 2H, 14CH, 18CH),
6.38 (d, J ¼ 5.6 Hz, 1H, 2CH), 4.15 (t, J ¼ 5.4 Hz, 2H, 7CH2), 3.77 (s,
2H), 3.45 (s, 2H) (10CH2, 11CH2), 2.76 (t, J ¼ 5.4 Hz, 2H, 8CH2), 2.66
(s, 2H), 2.55 (s, 2H) (9CH2, 12CH2), 2.39 (s, 3H, 6CH3). 13C NMR
described as compound 20a. Colorless oil, yield 85%. 1H NMR
(400 MHz, CD3OD)
d
8.23 (d, J ¼ 8.2 Hz,1H,14CH), 7.95 (d, J ¼ 5.6 Hz,
1H, 1CH), 7.86e7.78 (m, 1H, 18CH), 7.73e7.64 (m, 1H), 7.49 (d,
J ¼ 6.9 Hz, 1H) (15CH, 17CH), 6.38 (d, J ¼ 5.6 Hz, 1H, 2CH), 4.15 (t,
J ¼ 5.3 Hz, 2H, 7CH2), 3.80 (s, 2H), 3.36e3.29 (m, 2H) (10, 11CH2),
2.79 (t, J ¼ 5.3 Hz, 2H, 8CH2), 2.73 (t, J ¼ 5.1 Hz, 2H), 2.57 (t,
J ¼ 4.8 Hz, 2H) (9CH2, 12CH2), 2.39 (s, 3H, 6CH3). 13C NMR (101 MHz,
(101 MHz, CD3OD)
d 175.89 (3C), 171.00 (13C), 160.93 (5C), 155.20
(1C), 144.23 (4C), 135.32 (19C), 129.70 (16C), 128.31 (15C, 17C),
126.58 (14C, 18C), 116.01 (2C), 68.47 (7C), 57.28 (8C), 52.99, 52.53
(9C, 12C), 41.72 (10C, 11C), 13.54 (6C). MS (API-ES): m/z calcd for
CD3OD) d 175.92 (3C), 167.33 (13C), 161.00 (16C), 155.25 (1C), 145.40
C
C
19H23N2O4 343.16 [MþH]þ; found 343.2. HRMS (ESI): calcd. for
(5C), 144.22 (4C), 134.53 (19C), 131.88, 130.22 (14C, 18C), 127.90,
124.50 (15C, 17C), 116.01 (2C), 68.46 (7C), 57.30 (8C), 52.36, 52.05
(9C, 12C), 46.57, 41.37 (10, 11C), 13.57 (6C). MS (API-ES): m/z calcd
for C19H22FN2O4 361.1 [MþH]þ; found 361.1. HPLC purity 97.0%.
19H22N2NaO4 [MþNa]þ: 365.1471, found 365.1456.
4.2.21. Synthesis of 2-methyl-3-(2-(4-(3-methylbenzoyl)piperazin-
1-yl)ethoxy)-4H-pyran-4-one (20b)
Compound 20b was prepared in according to the procedure
4.2.25. Synthesis of 3-(2-(4-(4-chlorobenzoyl)piperazin-1-yl)
ethoxy)-2-methyl-4H-pyran-4-one (20f)
described as compound 20a. 1H NMR (400 MHz, CD3OD)
d 7.96 (d,
J ¼ 5.6 Hz, 1H, 1CH), 7.36e7.26 (m, 2H, 14CH, 18CH), 7.22 (s, 1H,
15CH), 7.18 (d, J ¼ 7.2 Hz, 1H, 16CH), 6.39 (d, J ¼ 5.6 Hz, 1H, 2CH),
4.15 (t, J ¼ 5.2 Hz, 2H, 7CH2), 3.79 (s, 2H), 3.48 (s, 2H) (10, 11CH2),
2.82 (t, J ¼ 5.2 Hz, 2H, 8CH2), 2.73 (s, 2H), 2.62 (s, 2H) (9CH2, 12CH2),
2.39 (s, 3H, 20CH3), 2.37 (s, 3H, 6CH3). 13C NMR (101 MHz, CD3OD)
Compound 20f was prepared in according to the procedure
described as compound 20a. Colorless oil, yield 90%. 1H NMR
(400 MHz, CD3OD)
d
7.95 (d, J ¼ 5.6 Hz, 1H, 1CH), 7.47 (d, J ¼ 8.4 Hz,
2H, 14CH, 18CH), 7.41 (d, J ¼ 8.4 Hz, 2H, 15CH, 17CH), 6.38 (d,
J ¼ 5.6 Hz, 1H, 2CH), 4.15 (t, J ¼ 5.3 Hz, 2H, 7CH2), 3.76 (s, 2H), 3.46
(s, 2H) (10, 11CH2), 2.78 (t, J ¼ 5.3 Hz, 2H, 8CH2), 2.67 (s, 2H), 2.60 (s,
2H) (9CH2, 12CH2), 2.39 (s, 3H, 6CH3). 13C NMR (101 MHz, CD3OD)
d
175.92 (3C), 171.13 (13C), 161.10 (5C), 155.35 (1C), 144.16 (4C),
138.47 (17C), 135.16 (19C), 130.40 (16C), 128.23 (15C), 127.06 (18C),
123.62 (14C), 116.01 (2C), 68.26 (7C), 57.26 (8C), 52.91, 52.48 (9C,
12C), 41.49 (10C, 11C), 19.96 (20C), 13.57 (6C). MS (API-ES): m/z
calcd for C20H25N2O4 357.18 [MþH]þ; found 357.2. HRMS (ESI):
calcd. for C20H25N2O4 [MþH]þ: 357.1808, found 357.1808. HPLC
purity 97.8%.
d
175.88 (3C), 169.80 (13C), 160.94 (5C), 155.23 (1C), 144.21 (4C),
135.65 (19C), 133.87 (16C), 130.68, 128.51, 128.46, 127.74 (14, 15, 17,
18C),116.02 (2C), 68.42 (7C), 57.26 (8C), 52.87, 52.52 (9C, 12C), 41.75
(10, 11C), 13.56 (6C). MS (API-ES): m/z calcd for C19H22ClN2O4 377.1
[MþH]þ; found 377.1. HRMS (ESI): calcd. for C19H22ClN2O4 [MþH]þ:
377.1262, found 377.1246. HPLC purity 97.6%.
4.2.22. Synthesis of 3-(2-(4-(3-methoxybenzoyl)piperazin-1-yl)
ethoxy)-2-methyl-4H-pyran-4-one (20c)
4.2.26. Synthesis of 2-methyl-3-(2-(4-(2-nitrobenzoyl)piperazin-1-
yl)ethoxy)-4H-pyran-4-one (20g)
Compound 20c was prepared in according to the procedure
described as compound 20a. Colorless oil, yield 80%. 1H NMR
Compound 20g was prepared in according to the procedure
(400 MHz, CD3OD)
d
7.94 (d, J ¼ 5.6 Hz, 1H, 1CH), 7.36 (t, J ¼ 8.0 Hz,
described as compound 20a. Colorless oil, yield 90%. 1H NMR
1H, 15CH), 7.04e6.99 (m, 1H, 14CH), 6.99e6.90 (m, 2H, 16CH,18CH),
6.38 (d, J ¼ 5.6 Hz,1H, 2CH), 4.15 (t, J ¼ 5.4 Hz, 2H, 7CH2), 3.81 (s, 3H,
20CH3), 3.76 (s, 2H), 3.45 (s, 2H) (10, 11CH2), 2.77 (t, J ¼ 5.4 Hz, 2H,
(400 MHz, CD3OD)
d
8.22 (d, J ¼ 8.3 Hz,1H,17CH), 7.95 (d, J ¼ 5.6 Hz,
1H, 1CH), 7.86e7.79 (m, 1H, 14CH), 7.72e7.65 (m, 1H, 16CH), 7.49
(dd, J ¼ 7.5, 1.0 Hz, 1H, 15CH), 6.38 (d, J ¼ 5.6 Hz, 1H, 2CH), 4.14 (t,
J ¼ 5.3 Hz, 2H, 7CH2), 3.85e3.73 (m, 2H), 3.31e3.27 (m, 2H) (10,
11CH2), 2.77 (t, J ¼ 5.3 Hz, 2H, 8CH2), 2.71 (t, J ¼ 5.0 Hz, 2H), 2.54 (t,
8CH2), 2.66 (s, 2H), 2.55 (s, 2H) (9CH2, 12CH2), 2.39 (s, 3H, 6CH3). 13
C
NMR (101 MHz, CD3OD)
d 175.87 (3C), 170.71 (13C), 160.92 (17C),

10
S. Xiang et al. / European Journal of Medicinal Chemistry 203 (2020) 112579
J ¼ 4.9 Hz, 2H) (9CH2, 12CH2), 2.39 (s, 3H, 6CH3). 13C NMR (101 MHz,
chromatography (EA: MeOH ¼ 20: 1) to give a canary yellow oil
(105 mg, yield 83%). 1H NMR (400 MHz, CD3OD)
CD3OD)
d
175.93 (3C), 167.32 (13C), 160.99 (5C), 155.25 (1C), 145.39
d
7.96 (d, J ¼ 5.6 Hz,
(18C), 144.24 (4C), 134.55 (15C), 131.90 (19C), 130.22 (16C), 127.91
(14C), 124.49 (17C), 116.02 (2C), 68.54 (7C), 57.31 (8C), 52.37, 52.06
(9C, 12C), 46.63, 41.43 (10, 11C), 13.58 (6C). MS (API-ES): m/z calcd
for C19H22N3O6 388.1 [MþH]þ; found 388.1.
1H, 1CH), 6.91 (s, 1H, 14CH), 6.39 (d, J ¼ 5.6 Hz, 1H, 2CH), 4.15 (t,
J ¼ 5.3 Hz, 2H, 7CH2), 3.74e3.50 (m, 4H, 10, 11CH2), 2.77 (t,
J ¼ 5.3 Hz, 2H, 8CH2), 2.72e2.54 (m, 4H, 9CH2, 12CH2), 2.40 (s, 3H,
6CH3). 13C NMR (101 MHz, CD3OD)
d 175.89 (3C), 162.86 (13C),
160.96 (5C), 155.25 (1C), 144.21 (4C), 116.02 (2C), 68.46 (7C), 64.94
(14C), 57.19 (8C), 52.55, 52.24 (9C, 12C), 45.78, 42.70 (10, 11C), 13.56
(6C). MS (API-ES): m/z calcd for C14H18Cl2N2NaO4 371.05 [MþNa]þ;
found 371.1. HPLC purity 99.8%.
4.2.27. Synthesis of 2-methyl-3-(2-(4-(4-(trifluoromethyl)benzoyl)
piperazin-1-yl)ethoxy)-4H-pyran-4-one (20h)
Compound 20h was prepared in according to the procedure
described as compound 20a. Colorless oil, yield 89%. 1H NMR
(400 MHz, CD3OD)
d
7.94 (d, J ¼ 5.6 Hz, 1H, 1CH), 7.76 (d, J ¼ 8.1 Hz,
4.3. Cell culture
2H, 14CH, 17CH), 7.59 (d, J ¼ 8.1 Hz, 2H, 15CH, 16CH), 6.37 (d,
J ¼ 5.6 Hz, 1H, 2CH), 4.13 (t, J ¼ 5.4 Hz, 2H, 7CH2), 3.78 (s, 2H), 3.40
(s, 2H) (10, 11CH2), 2.76 (t, J ¼ 5.4 Hz, 2H), 2.67 (s, 2H), 2.55 (s, 2H)
(9CH2, 12CH2), 2.38 (s, 3H, 6CH3). 13C NMR (101 MHz, CD3OD)
The cancer cell line A549 was purchased from Shanghai Cell
Bank of Chinese Academy of Sciences (Shanghai, China). The cells
were incubated in DMEM medium supplemented with 10% fetal
bovine serum, 1% penicillin and streptomycin in a 5% CO2 humidi-
fied atmosphere. Cells at exponential stage were used for all the
biological experiments.
d
175.91 (3C), 169.34 (13C), 161.00 (5C), 155.27 (1C), 144.20 (4C),
139.25 (18C), 131.48, 131.16 (19C), 127.36 (14e17C), 125.36, 125.32
(20C), 115.99 (2C), 68.43 (7C), 57.24 (8C), 52.87, 52.39 (9C, 12C),
41.69 (10, 11C), 13.54 (6C). MS (API-ES): m/z calcd for C20H22F3N2O4
411.1 [MþH]þ; found 411.1. HRMS (ESI): calcd. for C20H22F3N2O4
[MþH]þ: 411.1526, found 411.1508. HPLC purity 98.9%.
4.4. Cell viability assay
Cell viability of each synthesized was evaluated by MTT assay.
First, A549 (2000/well) were seeded in 96-well plates and cultured
4.2.28. Synthesis of 2-methyl-3-(2-(4-picolinoylpiperazin-1-yl)
ethoxy)-4H-pyran-4-one (20i)
with 20
was removed and 100
was added and incubated for 4 h. Then the supernatant was
removed and 100 L DMSO was added to each well. The absorbance
mM of compounds 4-9 for 72 h. Then the culture medium
Compound 20i was prepared in according to the procedure
mL fresh media containing 0.5 mg/mL MTT
described as compound 20a. Colorless oil, yield 95.0%. 1H NMR
(400 MHz, CD3OD)
d
8.58 (d, J ¼ 4.3 Hz, 1H, 17CH), 7.99e7.88 (m,
m
2H) (14CH, 1CH), 7.59 (d, J ¼ 7.8 Hz, 1H, 15CH), 7.49 (dd, J ¼ 6.9,
5.2 Hz, 1H, 16CH), 6.38 (d, J ¼ 5.6 Hz, 1H, 2CH), 4.16 (t, J ¼ 5.3 Hz, 2H,
7CH2), 3.85e3.73 (m, 2H), 3.54e3.42 (m, 2H) (10, 11CH2), 2.79 (t,
J ¼ 5.3 Hz, 2H, 8CH2), 2.76e2.66 (m, 2H), 2.66e2.55 (m, 2H) (9CH2,
of the each well was measured at 570 nm in a Microplate Reader,
the inhibition rate (%) could calculated from the absorbance values.
Then EC50 values were determined according to the procedure
mentioned above.
12CH2), 2.40 (s, 3H, 6CH3). 13C NMR (101 MHz, CD3OD)
d 175.91
(3C), 167.86 (13C), 160.97 (5C), 155.24 (1C), 153.39 (17C), 148.32
(18C), 144.22 (4C), 137.70 (15C), 124.82 (16C), 123.02 (14C), 115.99
(2C), 68.41 (7C), 57.29 (8C), 52.88, 52.41 (9C, 12C), 46.66, 41.66 (10,
11C), 13.54 (6C). MS (API-ES): m/z calcd for C18H22N3O4 344.16
[MþH]þ; found 344.2. HRMS (ESI): calcd. for C18H22N3O4 [MþH]þ:
344.1604, found 344.1593. HPLC purity 99.7%.
4.5. Lactate production assay
A549 (2 ꢁ 105/well) were incubated in 6-well plates for over-
night. Then compound 24e and 20k (40, 20, and 10 mM) were added
to the corresponding wells, which were incubated at 37 ꢀC for 4 h.
Finally, the medium of every well was collected and centrifuged for
10 min (10000 rpm) to remove any cell fragments. The lactate
production in medium medium was evaluated on a Nova Bioprofile
Flex analyzer (Nova Biomedical).
4.2.29. Synthesis of 3-(2-(4-(furan-2-carbonyl)piperazin-1-yl)
ethoxy)-2-methyl-4H-pyran-4-one (20k)
Compound 20k was prepared in according to the procedure
described as compound 20a. Colorless oil, yield 89%. 1H NMR
4.6. LDHA biochemical assay
(400 MHz, CD3OD)
d
7.97 (d, J ¼ 5.6 Hz, 1H, 1CH), 7.67 (d, J ¼ 0.9 Hz,
1H, 16CH), 7.04 (d, J ¼ 3.4 Hz, 1H, 14CH), 6.57 (dd, J ¼ 3.4, 1.7 Hz, 1H,
15CH), 6.41 (d, J ¼ 5.6 Hz, 1H, 2CH), 4.18 (t, J ¼ 5.3 Hz, 2H, 7CH2),
3.87 (s, 4H, 10, 11CH2), 2.88 (t, J ¼ 5.3 Hz, 2H, 8CH2), 2.83e2.75 (m,
4H, 9CH2, 12CH2), 2.40 (s, 3H, 6CH3). 13C NMR (101 MHz, CD3OD)
LDHA biochemical assay was conducted according to the pre-
vious literature [26]. Briefly, 5
protein buffer (150 mM Tris HCl, pH ¼ 7.4, 80
Tween-20). Then 10 L of LDHA stocking solution was added to a
384-well assay plate, then 10 L of DMSO-solubilized compounds
(1% DMSO in protein buffer) were transformed to each 96-well
assay plate. Following compound transfer, 80 L of substrate solu-
tion containing NADH and sodium pyruvate in protein assay buffer
was dispensed to initiate the reaction. In this assay, the final con-
centrations of LDHA should fix to 5 nM, while NADH and sodium
pyruvate should fix to 0.06 and 0.2 mM, respectively. Following
10 min of incubation at room temperature, the plates were recor-
ded on the Microplate Reader with excitation at 340 nm.
m
L of LDHA was added into 95
mL of
mM EDTA, and 0.01%
m
d
175.96 (3C), 161.17 (13C), 159.49 (5C), 155.40 (1C), 146.81 (17C),
m
144.61 (16C), 144.15 (4C), 116.39 (2C), 116.00 (14C), 111.04 (15C),
68.15 (7C), 57.27 (8C), 52.82 (9C, 12C), 13.55 (6C). MS (API-ES): m/z
calcd for C17H21N2O5 333.1 [MþH]þ; found 333.1. HRMS (ESI): calcd.
for C17H21N2O5 [MþH]þ: 333.1445, found 333.1439.
m
4.2.30. Synthesis of 3-(2-(4-(2,2-dichloroacetyl)piperazin-1-yl)
ethoxy)-2-methyl-4H-pyran-4-one (20j)
2-methyl-3-(2-(piperazin-1-yl)ethoxy)-4H-pyran-4-one hydro-
chloride (100 mg, 0.36 mmol) was dissolved in DCM (2 mL), TEA
(73.5 mg, 0.73 mmol) and cat. DMAP (4 mg) were added. The
mixture was cooled to 0 ꢀC, 2,2-dichloroacetyl chloride (54 mg,
0.36 mmol) was added very slowly at 0 ꢀC. Finally the reaction was
stirred at room temperature for overnight. The reaction mixture
was washed with 1 N NaOH solution. The solvent was removed
under reduced pressure. The crude product was purified by flash
4.7. PDKs biochemical assay
Initially, Pig PDC was incubated for 30 min at 37 ꢀC in buffer A
(40 mM Mops (pH ¼ 7.20), 0.5 mM EDTA, 30 mM KCl,1.5 mM MgCl2,
0.25 mM acetyl-CoA, 0.05 mM NADH, 2 mM dithiothreitol, 10 mM
NaF). Then, ATP-containing buffer A was added to the enzyme to

S. Xiang et al. / European Journal of Medicinal Chemistry 203 (2020) 112579
11
[7] 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.
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trigger the kinase reaction, in which PDKs catalyzed the phos-
phorylation of dehydrogenase in the presence or absence of the
compounds. The reaction was performed in a total volume of
100
fold in 1.8 x buffer A containing 55
m
L at 37 ꢀC, and was initiated by diluting the PDC mixture 2.2-
mM ADP, 100 mM ATP (ATP was
omitted in control reactions representing the absence of PDK ac-
tivity). After 3 min the kinase reaction was terminated by the
addition of 10
last the remaining PDC activity was tested in a total volume of
200 L of buffer B (120 mM Tris
L at 37 ꢀC by addition of 90
mL of stopping buffer (55 mM ADP and pyruvate). At
m
m
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(pH ¼ 7.8), 0.61 mM EDTA, 0.73 mM MgCl2, 2.2 mM thiamine py-
rophosphate, 11 mM 2-mercaptoethanol, 2.2 mM NADþ, 2.2 mM
pyruvate, 1.1 mM CoA). The absorbance of each well was measured
at 340 nm in a SpectraMax M Series Multi-mode Microplate Reader.
The results were expressed as IC50 values, which were the mean
values derived from three independent experiments.
4.8. Molecular docking
The PDB codes 4ZVV and 4V26 were selected as our binding
model, compounds were docked to the binding pocket of LDHA,
and analyzed their binding behaviors. Firstly, the crystal structure
LDHA (4ZVV) was prepared by using the software preparation
procedure, such as H atoms were added to the amino acids, and
charged by AMBER7 FF99 method. The small molecules was pre-
pared by H adding and energy optimizing with a tripos force field.
The total binding scores were obtained from the docking of the
prepared molecules to the protein.
Declaration of competing interest
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.
[20] J.D. Moore, A. Staniszewska, T. Shaw, J. D’Alessandro, B. Davis, A. Surgenor,
L. Baker, N. Matassova, J. Murray, A. Macias, P. Brough, M. Wood, P.C. Mahon,
VER-246608, a novel pan-isoform ATP competitive inhibitor of pyruvate de-
hydrogenase kinase, disrupts Warburg metabolism and induces context-
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[21] P.S. Dragovich, B.P. Fauber, L.B. Corson, C.Z. Ding, C. Eigenbrot, H. Ge,
A.M. Giannetti, T. Hunsaker, S. Labadie, Y. Liu, S. Malek, B. Pan, D. Peterson,
K. Pitts, H.E. Purkey, S. Sideris, M. Ultsch, E. VanderPorten, B. Wei, Q. Xu, I. Yen,
Q. Yue, H. Zhang, X. Zhang, Identification of substituted 2-thio-6-oxo-1,6-
dihydropyrimidines as inhibitors of human lactate dehydrogenase, Bioorg.
Med. Chem. Lett 23 (2013) 3186e3194.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China ((No. 21807008), the Fundamental Research
Funds for the Central Universities (2019CDXYYX0008).
Appendix A. Supplementary data
[22] B.P. Fauber, P.S. Dragovich, J. Chen, L.B. Corson, C.Z. Ding, C. Eigenbrot,
A.M. Giannetti, T. Hunsaker, S. Labadie, Y. Liu, S. Malek, D. Peterson, K. Pitts,
S. Sideris, M. Ultsch, E. VanderPorten, J. Wang, B.-Q. Wei, I. Yen, Q. Yue,
Identification of 2-amino-5-aryl-pyrazines as inhibitors of human lactate
dehydrogenase, Bioorg. Med. Chem. Lett 23 (2013) 5533e5539.
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112579.
[23] A. Boudreau, H.E. Purkey, A. Hitz, K. Robarge, D. Peterson, S. Labadie,
M. Kwong, R. Hong, M. Gao, C. Del Nagro, R. Pusapati, S. Ma, L. Salphati,
P. Pang, A. Zhou, T. Lai, Y. Li, Z. Chen, B. Wei, I. Yen, S. Sideris, M. McCleland,
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C. Eigenbrot, P.K. Jackson, S. Malek, G. Hatzivassiliou, D. Sampath,
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