Development of novel human lactate dehydrogenase A inhibitors: High-throughput screening, synthesis, and biological evaluations

Article abstract of DOI:10.1016/j.ejmech.2019.05.033

Human lactate dehydrogenase A (LDHA) plays a critical role in the glycolytic process, making the enzyme an ideal of anti-cancer drug target. Herein, we report the discovery of novel potent LDHA inhibitors by screening an in-house library. The hit-to-lead modification enabled us to identify compound 24c, which inhibited LDHA activity with an EC₅₀ value of 90 nM, and reduced MiaPaCa-2 cancer cell proliferation with an IC₅₀ value of 2.1 μM. In line with the in vitro anticancer activity, 24c suppressed the tumor growth at a dose of 10 mg/kg in a MiaPaCa-2 cells xenograft model, but with little effect to the mice weight. Moreover, 24c strongly inhibited MiaPaCa-2 cell colonies formation, induced MiaPaCa-2 cell apoptosis, and arrested MiaPaCa-2 cell cycle at G2 phase. In addition, the mitochondrial bioenergetics analysis suggested that 24c could reprogram cancer cell metabolic pathways from glycolysis to oxidation phosphorylation, which verified by decreasing the extracellular acidification rates and lactate formation, and increasing oxygen consumption rate in cancer cell. All these results indicate 24c is a promising metabolic modulator for the anticancer drug development.

Full text of DOI:10.1016/j.ejmech.2019.05.033

Accepted Manuscript
Development of novel human lactate dehydrogenase A inhibitors: High-throughput
screening, synthesis, and biological evaluations
Yuan Zhou, Pingde Tao, Meigui Wang, Peng Xu, Wei Lu, Pan Lei, Qiuyun You
PII:
S0223-5234(19)30443-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.033
EJMECH 11343
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 March 2019
Revised Date: 12 May 2019
Accepted Date: 12 May 2019
Please cite this article as: Y. Zhou, P. Tao, M. Wang, P. Xu, W. Lu, P. Lei, Q. You, Development
of novel human lactate dehydrogenase A inhibitors: High-throughput screening, synthesis, and
biological evaluations, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.05.033.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Compound 24c exhibited a potent biochemical potency, and in vitro and in vivo anticancer activity.

ACCEPTED MANUSCRIPT
Title page
Title:
Development of novel human lactate dehydrogenase A inhibitors: High-throughput screening, synthesis,
and biological evaluations
Author names and affiliations:
1
1
1
1
1
1
2, *
Yuan Zhou, Pingde Tao, Meigui Wang, Peng Xu, Wei Lu, Pan Lei, Qiuyun You
1
Department of Pharmacy, Taihe Hospital, Hubei University of Medicine, Shiyan 442000, Hubei Province,
P.R. China
2
College of Pharmacy, Hubei University of Traditional Chinese Medicine, No. 1 West Road, Huangjiahu
University Town, Hongshan District, Wuhan City 430065, Hubei Province, P.R. China
*
Corresponding address:
Prof. Qiuyun You (Q. Y. Y); Email: Qiuyun_You@hotmail.com / 1471@hbtcm.edu.cn
Abstract:
Human lactate dehydrogenase A (LDHA) plays a critical role in the glycolytic process, making the enzyme
an ideal of anti-cancer drug target. Herein, we report the discovery of novel potent LDHA inhibitors by
screening an in-house library. The hit-to-lead modification enabled us to identify compound 24c, which
inhibited LDHA activity with an EC50 value of 90 nM, and reduced MiaPaCa-2 cancer cell proliferation
with an IC value of 2.1 µM. In line with the in vitro anticancer activity, 24c suppressed the tumor growth
50
at a dose of 10 mg/kg in a MiaPaCa-2 cells xenograft model, but with little effect to the mice weight.
Moreover, 24c strongly inhibited MiaPaCa-2 cell colonies formation, induced MiaPaCa-2 cell apoptosis,
and arrested MiaPaCa-2 cell cycle at G2 phase. In addition, the mitochondrial bioenergetics analysis
suggested that 24c could reprogram cancer cell metabolic pathways from glycolysis to oxidation
phosphorylation, which verified by decreasing the extracellular acidification rates and lactate formation,
and increasing oxygen consumption rate in cancer cell. All these results indicate 24c is a promising
metabolic modulator for the anticancer drug development.
Keywords:
Anticancer drug; cancer metabolism; lactate dehydrogenase A; apoptosis
1

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1
. Introduction
As compared to normal cells, most cancer cells exhibit an elevated of anaerobic glycolysis, converting
the glucose into lactate even in aerobic conditions, which is termed as Warburg effect [1]. This altered
metabolic pathway is recognized as a hallmark of cancer and believed to be a promising strategy for cancer
treatment [2, 3].
In the final step of aerobic glycolysis, pyruvate was catalyzed by LDHA, which used nicotinamide
adenine dinucleotide (NADH) as a co-factor, transferring a hydride to the ketone moiety of pyruvate to
generate lactate in cytoplasm [4]. This inefficient metabolic pathways enabled cancer cells for rapid energy
supply and enough precursors for cell proliferation [5]. Thus, the inhibition of LDHA protein could provide
a workable strategy for developing of anticancer therapeutics. Moreover, the LDHA protein was widely
observed to overexpress in many solid cancer cells, such as MiaPaCa-2 cell [6], breast cancer [7, 8], lung
cancer [9], gallbladder carcinoma [10], glioma cells [11], renal cell carcinoma [12], pituitary adenoma [13],
and correlate with tumor proliferation and poor prognosis [14]. While knockdown or silence of LDHA in
cancer cells induced decreases of cancer cell proliferation, migration and tumorigenesis [15, 16]. All these
evidences indicated that inhibiting of the glycolytic enzyme could offer a promising strategy for cancer
therapy.
Fig. 1 Representative of reported LDHA inhibitors
Currently, great endeavors have been made towards this target [17-19], however, no drug based on the
target has successfully advanced to clinical applications. The initial reported inhibitor is oxamate (Fig. 1),
which is the most classical LDH inhibitor and competes with pyruvate. However, oxamate is nonselective
LDHA inhibitor with very weak potencies (K > 100 ꢀM) [20]. The N-hydroxyindole NHI was reported
i
from an academic laboratory [21], which displayed a promising cellular activity, but its potency towards
2

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LDHA is far from satisfactory. Compound 33 [22] and GSK808A [23] exhibited potent biochemical
activities, but they displayed limited cellular activities and / or the inhibitors were not suitable for further
pharmacokinetics studies. Later, Genentech [24] reported the most promising LDHA inhibitor Gen140,
however, the compound does not appear to have progressed into clinical studies.
Based on these observation and continue our effort to discover metabolic modulator, herein we screened
an in-house library containing 5000 small molecules, with the aim to discover new skeleton of LDHA
inhibitors. Then we utilized the hit-to-lead optimization to identify a novel compound 24c that potent
LDHA inhibitor, and strongly inhibited cancer cells proliferation in vitro and in vivo.
2
2
. Results and discussion
.1 Screening of the library
We herein developed a biochemical assay, the inhibitory effects of the in-house library compounds were
measured by using a standard enzymatic inhibition assay. The enzyme activity was measured by checking
the decrease of absorbance at 340 nm in a microplate reader. First, the concentrations of co-factor NADH
and substrate pyruvate were adjusted to 0.06 and 0.2 mM, respectively, followed by the previous report
[18]. Then we screened different concentrations of LDHA protein in the catalysis process, as shown in Fig.
2
A, the OD values decreased with the increase of LDHA concentration, but not when the concentrations
increased to 4 nM, therefore we keep the concentration in our following screening assay.
Fig. 2 High-throughput screening (HTS) of an in-house library against LDHA activity. (A) Optimization of the LDHA
concentration for HTS assay; (B) Z-factors for each plate in the established HTS assay. The HTS assay of each plate was
repeated three times independently; (C) LDHA inhibitory activity of the 5000 compounds. The positive compound Gen140
exhibited a 100% inhibitory rate at 10 ꢀM, while oxamate gave a 46.9% inhibitory rate at 1 mM.
3

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With the established screening assay, the inhibitory activity of the 5000 compounds at 10 ꢀM were
measured. As shown in Fig. 2B, the Z factors of each round screening are greater than 0.5, indicating the
assay is reliable and reproducible. As shown in Fig. 2C, 16 compounds exhibited potent LDHA inhibitory
rates (> 90%) at 10 ꢀM, which was colored as blue, and their chemical structures were shown in Fig. 3.
Finally, the identified 16 compounds were purchased from commercial sources and used for further
biological evaluations.
Fig. 3 The chemical structures of the selected compounds from screening library
2
.2 Biological evaluations of the identified compounds
We further measured EC values of the 16 candidates against LDHA by using a primary enzymatic assay.
5
0
As shown in Table 1, all the identified compounds gave satisfactory inhibitory activities with EC values
5
0
ranging from 7.8 to 0.34 µM. For 2, 11, and 12, the three compounds gave low nanomolar activities (680, 890,
and 340 nM) against LDHA, relative weak potencies as compared to Gen140 (EC50 = 8.9 nM), but is still a
promising start for developing novel LDHA inhibitors. We then docked the most potent compounds 2, 12, and a
less potent compound 8 into the binding pocket of LDHA, as shown in Fig. 4, the three compounds picked up
direct hydrogen bond interactions with the important amino acids His192, Asp168, and Asn137. The total
binding scores for 2 and 12 were 9.98 and 10.42, while for 8, the score dropped to 8.64, much weaker as
compared to 2 and 12, which is in line with the biochemical activities.
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Fig. 4 Proposed binding model for 2 (A), 12 (B) and 8 (C) in LDHA binding pocket. The Gen140 (D) was derived from PDB
code (4ZVV). The amino acids involved in hydrogen bond interactions were shown as stick and colored as red, yellow dashes
represents the hydrogen bonds. The graphics of 3D views were drawn by PyMOL.
Table 1 Biological activities of the screened compounds
Anti-proliferation inhibition rates
c
IC50 (µM)
a
b
Compds.
EC50 (µM)
(%) at 50 µM
A549
4.5
MiaPaCa-2
NH
MCF-7
12.3
93.4
22.3
NI
MiaPaCa-2
> 50
A549
> 50
1
2
3
5
6
7
8
9
6.4 ± 1.0
0.68 ± 0.09
2.14 ± 0.3
7.5 ± 1.2
95.6
84.7
45.6
NI
12.5 ± 2.4
> 50
14.7 ± 3.9
> 50
21.4
d
NI
> 50
> 50
4.58 ± 0.12
3.3 ± 0.08
7.8 ± 1.1
NI
NI
NI
> 50
> 50
12.5
15.8
NI
25.8
36.9
NI
17.9
14.7
NI
> 50
> 50
> 50
> 50
5.7 ± 0.5
> 50
> 50
1
0
1
1.2 ± 0.1
75.6
58.7
25.8
25.8
56.8
12.4
25.7
45.8
45.6
14.7
47.8
45.7
25.8
5.9
85.7
25.7
NH
29.8 ± 4.2
> 50
22.7 ± 1.9
> 50
1
0.89 ± 0.12
0.34 ± 0.05
2.9 ± 0.11
1.8 ± 0.06
3.4 ± 0.14
4.1 ± 1.0
1
1
1
1
1
2
3
4
5
6
> 50
> 50
50.1
38.7
17.9
9.7
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
e
Oxamate
Gen140
1.2 ± 0.1 (mM)
8.9 ± 0.1 (nM)
NT
NT
NT
18.9 ± 7.8 (mM)
0.48 ± 0.1
45.8 ± 7.8 (mM)
0.36 ± 0.05
NT
NT
NT
a
b
Biochemical activity of the identified compounds 1-16 against LDHA protein. The initial screening of cancer cell
c
inhibitory rates (%) by compounds (50 µM) was run as singlet by MTT assay. The IC50 values were obtained from three
d
independent experiments by using the MTT assay. The values were expressed as the Mean ± SD. NI represents no inhibition.
e
NT represents Not tested.
5

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Then cancer cells growth inhibitory rates by the identified compounds were measured by using MTT
assay. Though all the compounds displayed good biochemical activities, which not transformed into the
cellular activity, as shown in Table 1, most of the compounds were inactive to inhibit MiaPaCa-2, A549,
and MCF-7 cells growth at 50 µM. Compound 12 gave the most potent biochemical activity, but failed to
reduce cells proliferation. The reason for the observation remains unclear, which deserved further
investigation and will be reported in our further paper. To our delight, we discovered compound 2 that
exhibited a potent biochemical activity and cellular potency (IC = 12.5, and 14.7 µM against MiaPaCa-2
5
0
and A549, respectively), which was selected for further investigation.
2
.3 Modification of compound 2 to improve its potency
Since compound 2 exhibited the most promising activities, next we would like to synthesize its
analogues with the aim to further improve its potencies. The target compounds 24a-f were prepared and the
synthetic routes were shown in Scheme 1. 1-Boc-piperazine (17) reacted with 2-furoic acid (18) in the
presence of HBTU to afford 19. Removal of the Boc group in 19 with TFA provided 20 in a 96% yield,
which was stored as one important intermediate. Subsequent the chlorination of kojic acid (21) with thionyl
chloride gave 22, which was coupled with 20 to afford compound 23. Finally, the benzylation of 23 reacted
with various benzyl bromides to offer the target compounds 24a-f.
O
O
O
OH
a
b
Boc
N
NH
HN
N
Boc
N
N
O
O
O
17
18
20
19
O
O
O
N
O
OH
Cl
O
c
d
O
N
O
O
HO
HO
HO
O
21
22
2
3
1
2
3
4
2
2
2
2
4a, R = H, R = H, R = Cl, R = H
O
O
R1
N
1
2
3
4
4b, R = H, R = H, R = F, R = H
e
O
N
1
2
3
4
4c, R = H, R = F, R = H, R = H
1
2
3
4
4d, R = H, R = Cl, R = H, R = H
O
R4
R2
1
2
3
4
24e, R = H, R = H, R = H, R = F
R3
24a-f
24f, R = F, R = Cl, R = H, R = H
1
2
3
4
Scheme 1 Synthetic routes of the targeted compounds 24a-g. Reagents and conditions: (a) HBTU, Et N, CH Cl , overnight,
3
2
2
rt; (b) TFA, CH Cl , rt; (c) SOCl , 1 h, rt; (d) Et N, CH Cl , rt; (e) Substituted benzyl bromides, Et N, CH Cl , rt.
2
2
2
3
2
2
3
2
2
With these analogues in hand, we then measured their biological roles on LDHA. As shown in Table 2,
some of the prepared compounds, such as 24b, 24d, 24e, and 24f displayed decreased activities as
compared to compound 2, while 24a and 24c exhibited improved activities, with EC values of 270 and 90
5
0
nM, respectively. We then docked 24a and 24c into LDHA, and found that the two compounds formed
6

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direct hydrogen bond interactions with Asn137, Arg168, His192, and Gln99 of LDHA (Table 2), the total
binding scores are 10.2 and 10.3, respectively. Moreover, the furan ring in 24a and 24c interacted with
+
NAD , similar to the binding model of compound 2 and Gen140. Though the less potent compounds 24b,
2
4d, 24e and 24f interacted with amino acids Asn137, Arg168 and His192, but 24b, 24d and 24f not
+
interacted with NAD , while 24e failed to interacted with Gln99, accordingly, their total binding scores
dropped to 8.3, 8.4, 8.1, and 8.3, respectively. All these observations are fully in agreement with the
decreased biochemical activities. Then we evaluated anticancer activities of 24a-f on MiaPaCa-2 and A549
cancer cells. As expected, the less potent compounds 24e and 24f are inefficient to the both cell lines, while
2
4a and 24c are active, the IC values are 3.8 and 2.1 ꢀM against MiaPaCa-2 cell, respectively, 5.7 and 7.8
50
ꢀM against A549 cell. Somehow, we here made the hit-to-lead modifications that enabled us to discover
compounds 24a and 24c, which exhibited improved anticancer activities and biochemical potencies as
compared to compound 2.
Table 2 The activities of the synthesized compounds
a
Total binding
IC50 (µM)
MiaPaCa-2
Compds.
Binding model (with LDHA)
b
EC50 (µM)
0.68 ± 0.09
scores
A549
2
9.98
10.2
8.3
12.5 ± 2.4
14.7 ± 3.9
2
4a
0.27 ± 0.02
3.7 ± 0.51
0.09 ± 0.01
3.8 ± 0.5
5.7 ± 0.7
18.9 ± 1.5
7.8 ± 1.5
2
4b
24.5 ± 3.3
2
4c
10.3
2.1 ± 0.30
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2
4d
8.4
8.1
8.3
12.4 ± 0.9
> 50
> 50
> 50
> 50
> 50
> 50
2
4e
9.7 ± 1.7
2
4f
5.6 ± 1.2
a
The proposed binding models of 24a-f and 2 in active site of LDHA. The amino acids involved in hydrogen bond
+
interactions were shown as stick and colored as red, yellow dashes represents the hydrogen bonds, the substrate NAD was
b
shown as line. The total binding scores were calculated by Sybyl software.
2
.4 Anticancer activity of compound 24c
The anticancer activity of 24c was further evaluated by using the colonies formation assay. As shown in
Fig. 5, the MiaPaCa-2 cell colonies formation was totally suppressed by 24c at 1 µM, and partially
inhibited at 0.5 ꢀM, similar to the positive control drug DOX. Moreover, MiaPaCa-2 cells incubating with
2
4c formed smaller and fewer colonies in a concentration-dependent manner as compared to the untreated
group, indicating anticancer effect of 24c.
Fig. 5 Compound 24c inhibited MiaPaCa-2 cell colonies formation. The images were the colonies formed after the
compounds treatment. DOX represents doxorubicin, a positive anticancer drug.
Then 24c incubated with MiaPaCa-2 cell for 12 h, the apoptosis cells were detected by Annexin
V-FITC/PI FACS assay in a flow cytometry. As shown in Fig. 6, the percentages of apoptotic population in
MiaPaCa-2 cells treated with 24c (1, 5, and 10 ꢀM) for 12 h were 6.93, 23.33 and 31.47 %, respectively,
suggesting the induction effect in MiaPaCa-2 cells follows a dose-dependent manner.
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Fig. 6 Compound 24c induced cells apoptosis. MiaPaCa-2 cells were treated with different concentrations of 24c (1, 5, and
1
0 µM) for 24 h, then stained with annexin V / PI, the apoptosis cells were analyzed by using a flow cytometry. The insert bar
graph represents statistics of total apoptotic cell percentages from duplicate experiments. The anticancer drug DOX was
selected as positive control.
2
.5 Compound 24c arrested cell cycle at G2 phase
Compound 24c showed the most promising biochemical activity (EC = 90 nM) and anticancer potency
5
0
(
IC = 2.3 ꢀM against MiaPaCa-2), then we carried out additional studies to examine the effect of 24c on
50
cell cycle distribution. As shown in Fig. 7, compound 24c led to a significant accumulation of cells at G2
phase from 31.0% to 38.52% (1 ꢀM), 42.12% (5 ꢀM) and 59.99% (10 ꢀM), accordingly. Meanwhile, it
reduced G1 phase from 56.01 to 36.21%, respectively. In addition, we found that cell cycle arrest at G2
phase induced by 24c was concentration-dependent, which might be one of the possible mechanisms for its
cytotoxicity.
Fig. 7 Compound 24c induced cell cycle arrest in MiaPaCa-2 cells. The cells were treated with 24c for 1, 5, and 10 µM, and
DOX (5 µM) for 24ꢁh, and then fixed in ice cold 70% ethanol at 4ꢁ°C. The treated cells were then stained with propidium
iodide dye (50ꢁµg/mL). Cell cycle analysis was evaluated via flow cytometry (CytoFLEX, Beck-man, USA). The insert bar
9

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graph showed the percentages (%) of cells in G1, S and G2 phases, respectively.
2
.6 Compound 24c altered mitochondrial bioenergetics function in cancer cell
LDHA is an important checkpoint enzyme that catalyzes the conversion of pyruvate to lactate production.
Therefore, targeting this enzyme could alter the metabolic profile of cancer cell, the oxygen consumption
rate (OCR) and the extracellular acidification rates (ECAR) should be changed accordingly. To prove the
hypothesis, MiaPaCa-2 cells were treated with 24c (1, 5, and 10 µM) for 4 h, then the changes of the lactate
formation, OCR and ECAR were evaluated. As shown in Fig. 8, the lactate formation was reduced in the
medium of 24c treated group. Similar to the lactate formation, ECAR was significantly decreased with the
addition of 24c, which can be explained by the reduction of glycolysis in the cancer cells after treating of
2
4c. OCR was gradually increased by the treatment of 24 at 1, 5, and 10 µM. Here we proved that 24c
could cause a metabolic alteration by enhancing oxidative phosphorylation and reducing lactate formation
in cancer cell, which might contribute to their anti-proliferation effect.
Fig. 8 Compound 24c altered bioenergetics function of mitochondria for MiaPaCa-2 cells.
(A) Compound 24c (1, 5, and 10 µM) reduced the lactate formation in MiaPaCa-2 cells. (B / C) MiaPaCa-2 cells were treated
with 24c (1, 5, and 10 µM) for 4 h, then ECAR / OCR were assessed under basal condition. * P < 0.05, versus the untreated
control group.
2
.7 Compound 24c suppressed tumor growth in a MiaPaCa-2 cells xenograft model
To further verify the anticancer activity of 24c, MiaPaCa-2 cells xenograft model was prepared, and was
treated with 24c at a single dose of 10 mg/kg. As shown in Fig. 9A, compound 24c significantly suppressed
the tumor growth over the time. At end of the administration, the tumors were harvested and weighted. In
Fig. 9B, the tumor weight is much less in 24c treatment group than the vehicle group. From the observation,
we could conclude that 24c inhibited the tumor growth in vivo. To prove the compound is safe, the body
weight of each mice was recorded. As shown in Fig. 9C, the dose administration of 24c did not affect the
body weight of the mice, indicating low toxicity of the compound.
1
0

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Fig. 9 Compound 24c attenuated tumor growth in vivo. MiaPaCa-2 cell xenograft mouse model was not treated (vehicle,
control group), or treated with compound 24c at a dose of 10 mg/kg daily for 21 days. A, tumor volume changes were
recorded twice a week. B, tumor weight of each samples was recorded. C, body weight of each mouse was recorded.
3
. Results and discussion
In summary, we screened an in-house library containing 5000 small molecules, from which 16
compounds exhibited potent anti-LDHA activity, but most of the candidates are inactive against the growth
of cancer cells. Then the hit-to-lead optimization enabled us to identify compound 24c, which strongly
inhibited LDHA with an EC value of 90 nM, and reduced cancer cell growth with IC values of 2.1 and
5
0
50
7
.8 ꢀM against MiaPaCa-2 and A549 cells, respectively. In line with the in vitro anticancer activity, the
compound suppressed the tumor growth at a dose of 10 mg/kg in MiaPaCa-2 cells xenograft model, but
with no negative effect to the mice weight. Meanwhile, the compound strongly inhibited MiaPaCa-2 cells
colonies formation at 0.5 µM, induced the cancer cell apoptosis and cell cycle arrest in dose response
manners. As a potent LDHA inhibitor, 24c significantly modulated pyruvate metabolic profile in
MiaPaCa-2 cells, reflecting of the decreasing ECRA and lactate production, and increasing OCR values.
These observations suggested that 24c could be used as a promising lead for the development of potent
LDHA inhibitor.
4
. Experimental section
4
.1 Compound library and chemistry
The screening library contains 5000 small molecules that are commercially available, part of them are
not disclosed in literature. The library is stored in 384 plates at a concentration of 10 mM in DMSO, the
purities are more than 95%, which could be used in any biological experiments. The potential compounds
1
-16 were bought from local agent directly. Control drug oxamate sodium (CAS: 565-73-1) was bought
from Sigma-Aldrich, the Gen140 was synthesized according to the literature [24]. The chemicals and
solvents used in the experiments were bought from commercial sources and used without any treatments.
Column chromatography was carried out on silica gel (100−200 mesh). The NMR spectra was recorded on
a Bruker AV-400 instrument and the signals were reported in ppm downfield from TMS (0 ppm). High
1
1

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resolution mass spectra (HRMS) were obtained on a Xevo G2-XS QTof spectrometer (Waters, USA).
Melting points (M.p.) were determined using a SMP20 instrument from Stuart Company.
4
.2 Compound synthesis
4
.2.1 Synthesis of tert-butyl 4-(furan-2-carbonyl)piperazine-1-carboxylate (19)
To triethylamine (1.65 mmol) solution in dichloromethane (2.0 mL) was added carboxylic acid 18 (0.55
mmol) and HBTU (0.55 mmol) at room temperature. Then N-boc-piperazine 17 (0.5 mmol) in
dichloromethane (5 mL) was added dropwise and stirred at room temperature overnight. After the reaction
completed, the mixture was washed with saturated sodium carbonate solution. The mixture was extracted
with dichloromethane, the organic phase was washed twice with water, and dried over anhydrous sodium
sulfate. Subsequently the residues were purified by silica gel column chromatography to give 19 as white
o
+
solid. M.p. > 300 C. HRMS (ESI) calcd for C H N O [M + H] , 281.1501; found, 281.1498.
1
4
21
2
4
4
.2.2 Synthesis of furan-2-yl(piperazin-1-yl)methanone (20)
N-Boc protected piperazine 19 (10 mM) was dissolved in a mixed solution of dichloromethane (20 mL)
and TFA (0.5 mL), which was stirred at room temperature for 1 h. After completion of the reaction, the
mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography to give
o
2
0 as white solid. M.p. 139−140 C, which is full agreement with the literature [25].
4
.2.3 Synthesis of 2-(chloromethyl)-5-hydroxy-4H-pyran-4-one (22)
To a rapidly stirring suspension of kojic acid 21 (7.0 mmol) in dichloromethane (20 mL) was added
thionyl chloride (8.0 mmol) dropwise. After 6 h of stirring, the suspension was filtered, and the solids were
o
recrystallized from ethanol. Kojic chloride as white needles was obtained. M.p. 169−170 C. HRMS (ESI)
+
calcd for C H N O [M + H] , 281.1501; found, 281.1498.
14
21
2
4
4
.2.4 Synthesis of 2-((4-(furan-2-carbonyl)piperazin-1-yl)methyl)-5-hydroxy-4H-pyran-4-one (23)
To triethylamine (1.2 mM) solution in dichloromethane (10 mL) were added 20 (1.0 mM), then kojic
chloride (1.0 mM) in dichloromethane (10 mL) was added dropwise. The reaction was stirred at room
temperature overnight. After completion of the reaction, the mixture was washed with water, and exacted
with dichloromethane. Then the organic phase was washed twice with water and dried over anhydrous
sodium sulfate. Subsequently the residues were purified by silica gel column chromatography to give 23 as
o
+
white solid. M.p. 107−108 C. HRMS (ESI) calcd for C H N O [M + H] , 305.1137; found, 305.1132.
15
17
2
5
4
.2.5 Synthesis of 5-((3-chlorobenzyl)oxy)-2-((4-(furan-2-carbonyl)piperazin-1-yl)methyl)-4H-pyran-4-one
24a)
Dichloromethane (10 mL) and triethylamine (1.1 mmol) were added to 100 mL round-bottom flask.
(
Then 23 (1.0 mmol) in dichloromethane (20 mL) was added dropwise. The mixture was stirred for 15 min
at room temperature, then 1-(bromomethyl)-3-chlorobenzene (1.0 mM) was added. The reaction was stirred
1
2

ACCEPTED MANUSCRIPT
at room temperature overnight. Then the mixture was poured into 50 mL of brine and was extracted with
dichloromethane. The organic layer was dried over anhydrous sodium sulfate, and the crude compound was
o
1
purified by column chromatography to give the desired compound 24a. M.p. 122−123 C. H NMR (400
MHz, CDCl ): δ 7.59 (s, 1H), 7.48 (s, 1H), 7.40 (s, H), 7.32-7.29 (m, 3H), 7.01 (d, J = 2.4 Hz, 1H), 6.48 (t,
3
1
3
J = 2 Hz, 2H), 5.05 (s, 2H), 3.84 (s, 4H), 3.40 (s, 2H), 2.58 (t, J = 4 Hz, 4H); C NMR (100 MHz, CDCl ):
3
1
70.42, 164.01, 158.93, 147.64, 146.85, 143.69, 141.95, 137.71, 134.50, 129.93, 128.50, 127.73, 125.75,
+
1
16.60, 114.95, 111.27, 71.05, 59.01, 53.39, 53.11. HRMS (ESI) calcd for C H ClN O [M + H] ,
2
2
22
2
5
4
29.1217; found, 429.1210.
.2.6 Synthesis of 5-((3-fluorobenzyl)oxy)-2-((4-(furan-2-carbonyl)piperazin-1-yl)methyl)-4H-pyran-4-one
4
(
24b)
According to the procedure for 24a, a white solid 24b was obtained. Yield: 72 %. M.p. 130−131 C. H
NMR (400 MHz, CDCl ): δ 7.56 (s, 1H), 7.46 (s, 1H), 7.34-7.29 (m, H), 7.16-7.09 (m, 2H), 7.02-6.98 (m,
o
1
3
1
3
2
H), 6.46 (t, J = 4 Hz, 2H), 5.04 (s, 2H), 3.81 (s, 4H), 3.38 (s, 2H), 2.55 (t, J = 6.0 Hz, 4H); C NMR (100
MHz, CDCl ): 174.43, 164.03 (d, J = 20 Hz), 161.63, 158.94, 147.64, 146.88, 143.69, 141.86, 138.20 (d, J
3
=
=
14 Hz), 130.22 (d, J = 16 Hz), 123.14 (d, J = 16 Hz), 116.61, 115.26 (d, J = 100 Hz), 114.93, 114.53 (d, J
+
100 Hz), 111.28. HRMS (ESI) calcd for C H FN O [M + H] , 413.1513; found, 413.1513.
2
2
22
2
5
4
.2.7 Synthesis of 5-((2-fluorobenzyl)oxy)-2-((4-(furan-2-carbonyl)piperazin-1-yl)methyl)-4H-pyran-4-one
24c)
According to the procedure for 24a, a white solid 24c was obtained. Yield: 75 %. M.p. 135−136 C. H
NMR (400 MHz, CDCl ): δ 7.61 (s, 1H), 7.49-7.46 (m, 2H), 7.33-7.28 (m, 1H), 7.14 (t, J = 8 Hz, 1H), 7.05
(
o
1
3
(t, J = 8 Hz, 1H), 6.98 (d, J = 4 Hz, 1H), 6.46 (s, 2H), 5.11 (s, 2H), 3.82 (s, 4H), 3.38 (s, 2H), 2.56 (t, J =
13
4
.0 Hz, 4H); C NMR (100 MHz, CDCl ): 174.43, 163.94, 161.76, 159.30, 158.94, 147.67, 146.96, 143.68,
3
1
41.57, 130.36 (t, J = 6.5 Hz), 124.38 (d, J = 1.5 Hz), 122.73 (d, J = 7 Hz), 116.59, 115.35 (d, J = 10 Hz),
1
14.90, 111.27, 65.44, 65.40, 59.04, 53.08. HRMS (ESI) calcd for C H FN O [M + H]+, 413.1513;
2
2
22
2
5
found, 413.1510.
4
.2.8 Synthesis of 5-((2-chlorobenzyl)oxy)-2-((4-(furan-2-carbonyl)piperazin-1-yl)methyl)-4H-pyran-4-one
24d)
According to the procedure for 24a, a white solid 24d was obtained. Yield: 64 %. M.p. 122−123 C. H
NMR (400 MHz, CDCl ): δ 7.61 (s, 1H), 7.56 (t, J = 4 Hz, 1H), 7.48 (s, 1H), 7.40-7.38 (m, 1H), 7.31-7.28
(
o
1
3
(m, 2H), 7.02 (d, J = 2 Hz, 1H), 6.50-6.48 (m, 2H), 5.17 (s, 2H), 3.84 (s, 4H), 3.41 (s, 2H), 2.58 (t, J = 6 Hz,
1
3
4
H); C NMR (100 MHz, CDCl ): 174.41, 163.97, 158.98, 147.73, 147.09, 143.71, 141.51, 133.31, 132.89,
3
1
29.56, 129.48, 129.44, 127.13, 116.66, 114.93, 111.32, 68.82, 59.10, 53.11. HRMS (ESI) calcd for
+
C H ClN O [M + H] , 429.1217; found, 429.1214.
2
2
22
2
5
1
3

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4
.2.9 Synthesis of 5-((4-fluorobenzyl)oxy)-2-((4-(furan-2-carbonyl)piperazin-1-yl)methyl)-4H-pyran-4-one
(
24e)
According to the procedure for 24a, a white solid 24e was obtained. Yield: 85 %. M.p. 126−127 C. H
NMR (400 MHz, CDCl ): δ 7.55 (s, 1H), 7.46 (d, J = 2.0 Hz, 1H), 7.38-7.36 (m, 2H), 7.06-6.98 (m, 3H),
o
1
3
1
3
6
.46 (s, 2H), 5.01 (s, 2H), 3.81 (s, 4H), 3.38 (s, 2H), 2.56 (t, J = 6.0 Hz, 4H); C NMR (100 MHz, CDCl ):
3
1
74.53, 163.95, 161.42, 158.95, 147.63, 146.90, 143.70, 141.87, 131.47, 129.79 (d, J = 4.5 Hz), 116.64,
+
1
15.55 (d, J = 11 Hz). HRMS (ESI) calcd for C H FN O [M + H] , 413.1513; found, 413.1507.
2
2
22
2
5
4
.2.10
Synthesis
of
5
-((2-chloro-6-fluorobenzyl)oxy)-2-((4-(furan-2-carbonyl)piperazin-1-yl)methyl)-4H-pyran-4-one (24f)
o
1
According to the procedure for 24a, a white solid 24f was obtained. Yield: 74 %. M.p. 109−110 C. H
NMR (400 MHz, CDCl ): δ 7.67 (s, 1H), 7.49 (s, 1H), 7.33-7.22 (m, 2H), 7.05-7.01 (m, 2H), 6.49 (m, 2H),
3
1
3
5
1
1
5
.21 (s, 2H), 3.85 (s, 4H), 3.42 (s, 2H), 2.59 (t, J = 4.0 Hz, 4H); C NMR (100 MHz, CDCl ): 174.46,
3
63.89, 160.80, 158.94, 147.66, 147.12, 143.68, 142.33, 136.72 (d, J = 12.5 Hz), 131.03 (d, J = 5 Hz),
25.49 (d, J = 2 Hz), 121.56 (d, J = 8.5 Hz), 116.57, 115.13, 114.25 (d, J = 11.5 Hz), 111.26, 62.99, 62.96,
+
9.03, 53.05. HRMS (ESI) calcd for C H ClFN O [M + H] , 447.1123; found, 447.1120.
2
2
21
2
5
4
.3 Cell culture
The cancer cell lines MCF-7, A549 and MiaPaCa-2 were purchased from Shanghai Cell Bank of Chinese
Academy of Sciences (Shanghai, China). The cells were incubated in RPMI 1640 medium supplemented
with 10% fetal bovine serum, 1% penicillin and streptomycin in a 5% CO humidified atmosphere. Cells at
2
exponential stage were used for all the biological experiments.
4
.4 Cell viability assay
Cell viability was evaluated by MTT assay according to the manufacturer’s instructions. A549 and
MiaPaCa-2 cells (5000/well) were seeded in 96-well plates and cultured with 50 ꢀM of compounds 1-16
for 72 h. Then the culture medium was removed and 100 ꢀL fresh media containing 0.5 mg/mL MTT was
added and incubated for 4 h. Then the supernatant was removed and 100 ꢀL DMSO was added to each well.
The absorbance of the each well was measured at 570 nm in a Microplate Reader. The IC50 values were
determined following the procedure mentioned above.
4
.5 Cell colony formation
MiaPaCa-2 cell was seeded in 6-well plates, and each well contained 300 cells in 2 mL medium. Then
compound 24c (0.1, 0.5, and 1 µM) and positive control drug DOX (0.5 µM) were added into the
corresponding well, and treated the cancer cells for 3 days, then replaced by medium without drug for every
3
days. 15 days after, cell colonies were fixed in 95% ethanol for 15 min, and stained by 0.1% crystal violet,
then imaged.
1
4

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4
.6 Annexin V-FITC/PI double-staining assay
Apoptotic cell death was determined by Annexin V fluorescein isothiocyanate (FITC). MiaPaCa-2 cell
5
was seeded in 6-well plates at 2.0 × 10 cells per well and cultured for 24 h, then treated with compound
2
4c at 1, 5, and 10 ꢀM for 12 h. Then the cells were collected and washed with cold PBS twice, which were
then stained by the kit according to the manufacturer’s instructions. Finally, the prepared cells were
analyzed by BD FACSCanto II flow cytometer (BD Biosciences, CA).
4
.7 Cell cycle analysis
Cell cycle was analyzed by cycle test kit after the instructions. MiaPaCa-2 cell was seeded in 6-well
5
plates at 1.0 × 10 cells per well and cultured for 24 h, then was treated with compound 24c (1, 5, and 10
ꢀM) for 24 h. Then the treated cells were collected and fixed by 70% precooled ethanol, which were then
treated with RNase A, and stained by PI (Beyotime, Jiangsu, China). At last, the stained cells were analyzed
by BD FACSCanto II flow cytometer (BD Biosciences, CA).
4
.8 Lactate production assay
5
MiaPaCa-2 cells (2 × 10 / well) were incubated in 6-well plates for overnight. Then compound 24c (1, 5,
and 10 ꢀM) 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 5 min (12000 rpm) to remove any cell fragments.
The lactate production in medium medium was evaluated on a Nova Bioprofile Flex analyzer (Nova
Biomedical).
4
.9 LDHA biochemical assay
Recombinant human LDHA protein (His tag) was bought from abcam (ab93699). Briefly, 5 ꢀL of LDHA
was added into 95 ꢀL of protein buffer (200 mM Tris HCl, pH = 7.4, 100 ꢀM EDTA, and 0.01% Tween-20),
which was prepared for stocking solution. Then 3 ꢀL of LDHA stocking solution was added to a 384-well
assay plate, then 3 ꢀL of DMSO-solubilized compounds (1% DMSO in protein buffer) were transformed to
to each 386-well assay plate. Following compound transfer, 24 ꢀL of substrate solution containing NADH
and sodium pyruvate in protein assay buffer was dispensed to initiate the reaction. In this assay, the final
concentrations of LDHA should fix to 4 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 recorded on the
Microplate Reader with excitation at 340 nm.
4
.10 MiaPaCa-2 xenograft assay
The animal studies were approved by the Ethics Committee of the Hubei University of Medicine, and
carried out strictly under their supervision. Six to eight weeks nude mice were inoculated subcutaneously
6
with 3.0 × 10 MiaPaCa-2 cells suspended in serum-free RPMI 1640 in the right flank. One week after the
inoculation, the mice were randomly divided into two groups, the vehicle control treat with 0.5% CMC-Na,
1
5

ACCEPTED MANUSCRIPT
while the experimental group was treated with compound 24c at one single dose at 10 mg/kg daily for 21
days. Tumor volume and body weight of the mice were recorded two times in a week. At the end of the
experiment, mice were humanely killed, the tumor samples were weighted.
4
.11 Molecular docking
The selected the PDB code 4ZVV 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 prepared 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.
4
.12 Statistical analysis
Data were analyzed using GraphPad Prism 5.0. Dose–response curves were generated using log(inhibitor)
vs. response equation in nonlinear regression analysis. The data was expressed as Mean±SD of three
independent experiments. Two-way analysis of variance (ANOVA test) was used for statistical analysis of
differences in values among multiple groups. P < 0.05 was considered to be statistically significant.
Notes
The authors declare no competing financial interest for this manuscript.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (81573865).
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Highlights
►
►
►
Compound 24c inhibited LDHA with an EC value of 90 nM
50
Compound 24c reduced MiaPaCa-2 cell proliferation with an IC value of 2.1 µM
5
0
Compound 24c suppressed tumor growth in a xenograft model