Synthesis and antitumor activity of novel 2, 3-didithiocarbamate substituted naphthoquinones as inhibitors of pyruvate kinase M2 isoform

Article abstract of DOI:10.1080/14756366.2017.1404591

The M2 isoform of pyruvate kinase (PKM2) is a potential antitumor therapeutic target. In this study, we designed and synthesised a series of 2, 3-didithiocarbamate substituted naphthoquinones as PKM2 inhibitors based on the lead compound 3k that we previously reported. Among them, compound 3f (IC₅₀ = 1.05 ± 0.17?μM) and 3h (IC₅₀ = 0.96 ± 0.18?μM) exhibited potent inhibition of PKM2, and their inhibitory activities are superior to compound 3k (IC₅₀ = 2.95 ± 0.53?μM) and the known PKM2 inhibitor shikonin (IC₅₀ = 8.82 ± 2.62?μM). In addition, we evaluated in vitro antiproliferative effects of target compounds using MTS assay. Most target compounds exhibited dose-dependent cytotoxicity with IC₅₀ values in nanomolar concentrations against HCT116, MCF7, Hela, H1299 and B16 cells. These small molecule PKM2 inhibitors not only provide candidate compounds for cancer therapy, but also offer a tool to probe the biological effects of PKM2 inhibition on cancer cells.

Full text of DOI:10.1080/14756366.2017.1404591

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis and antitumor activity of novel 2, 3-
didithiocarbamate substituted naphthoquinones
as inhibitors of pyruvate kinase M2 isoform
Xianling Ning, Hailong Qi, Ridong Li, Yan Jin, Michael A. McNutt & Yuxin Yin
To cite this article: Xianling Ning, Hailong Qi, Ridong Li, Yan Jin, Michael A. McNutt &
Yuxin Yin (2018) Synthesis and antitumor activity of novel 2, 3-didithiocarbamate substituted
naphthoquinones as inhibitors of pyruvate kinase M2 isoform, Journal of Enzyme Inhibition and
Medicinal Chemistry, 33:1, 126-129, DOI: 10.1080/14756366.2017.1404591
To link to this article: https://doi.org/10.1080/14756366.2017.1404591
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2017
VOL. 33, NO. 1, 126–129
https://doi.org/10.1080/14756366.2017.1404591
RESEARCH PAPER
Synthesis and antitumor activity of novel 2, 3-didithiocarbamate substituted
naphthoquinones as inhibitors of pyruvate kinase M2 isoform
Xianling Ninga,b, Hailong Qia,c, Ridong Lia,b, Yan Jina,d, Michael A. McNutta,d and Yuxin Yina,c,d
aInstitute of Systems Biomedicine, School of Basic Medical Sciences, Beijing Key Laboratory of Tumor Systems Biology, Peking University Health
b
Science Center, Beijing, China; Department of Pharmacology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing,
c
d
China; Peking-Tsinghua Center for Life Sciences, Peking University Health Science Center, Beijing, China; Department of Pathology, School of
Basic Medical Sciences, Peking University Health Science Center, Beijing, China
ABSTRACT
ARTICLE HISTORY
Received 9 September 2017
Revised 9 November 2017
Accepted 9 November 2017
The M2 isoform of pyruvate kinase (PKM2) is a potential antitumor therapeutic target. In this study, we
designed and synthesised a series of 2, 3-didithiocarbamate substituted naphthoquinones as PKM2 inhibi-
tors based on the lead compound 3k that we previously reported. Among them, compound 3f
(IC50 ¼ 1.05 0.17 mM) and 3h (IC50 ¼ 0.96 0.18 mM) exhibited potent inhibition of PKM2, and their inhibi-
tory activities are superior to compound 3k (IC50 ¼ 2.95 0.53 mM) and the known PKM2 inhibitor shikonin
(IC50 ¼ 8.82 2.62 mM). In addition, we evaluated in vitro antiproliferative effects of target compounds using
MTS assay. Most target compounds exhibited dose-dependent cytotoxicity with IC50 values in nanomolar
concentrations against HCT116, MCF7, Hela, H1299 and B16 cells. These small molecule PKM2 inhibitors
not only provide candidate compounds for cancer therapy, but also offer a tool to probe the biological
effects of PKM2 inhibition on cancer cells.
KEYWORDS
M2 isoform of pyruvate
kinase; PKM2 inhibitors;
2,3-didithiocarbamate sub-
stituted naphthoquinones;
antiproliferative effects
Introduction
value of 10 mM (Figure 1)20. Compound A was reported to presum-
ably block the allosteric site of PKM2. In 2011, Chen et al. reported
shikonin (Figure 1) and its enantiomeric isomer alkannin to inhibit
PKM2 at low concentrations21,22. Recently, we reported a new
PKM2 inhibitor compound 3k (Figure 1) with IC50 value of
2.95 mM, which exhibits more potent PKM2 inhibitory activity than
the known optimal PKM2 inhibitor shikonin (IC50 ¼ 8.82 mM)23.
To further optimise this activity, in this study, some new ana-
logues of 3k were designed and synthesised. The PKM2 inhibitor
compound 3f and 3h have high PKM2 inhibition responses, which
are more potent than shikonin and compound 3k, and they also
exhibit high antiproliferative activity in several tumour cells.
The metabolism in cancer cells substantially differs from that in
healthy cells1–3. The tumour cells rely on glycolysis to produce
energy and have a high rate of glucose uptake but low rates of
oxidative phosphorylation (Warburg effect)4,5. Cancer cells were
found to have very strong metabolic dependencies, which are not
associated with normal cells6. The interventions on tumour gly-
colysis become
a novel strategy for selective anti-cancer
therapies7–9
.
Pyruvate kinase (PK) is the last rate-limiting enzyme in the
glycolytic pathway and catalyses the transfer of a phosphate
group from phosphoenolpyruvate (PEP) to adenosine diphosphate
(ADP) to obtain pyruvate and adenosine triphosphate (ATP)10,11
.
There are M1, M2, L and R isoforms of PK in mammalian cells: the
M1 isoform (PKM1) is expressed in many differentiated tissues
(skeletal muscle, heart and brain), PKM2 is expressed during
embryonic development or over expressed in tumour tissues, PKL
Materials and methods
Chemistry
All chemicals, reagents and solvents were purchased from com-
mercial sources. When necessary, they were purified and dried by
standard methods. Reactions were checked by thin-layer chroma-
tography (TLC) on pre-coated silica gel F254 plates. Column chro-
matography was carried out with Silica gel H (200–300 mesh or
500 mesh). Detection was by iodine vapour staining and UV
light irradiation (UV lamp, model UV-IIB). Melting points were
determined on an X4-type apparatus and are not corrected.
and PKR are expressed in liver and erythrocytes, respectively12–14
.
.
PKM2 is important for cancer metabolism and tumour growth15,16
Tumourigenesis is associated with the re-expression of PKM2
together with a downregulation of the expression of PKM1 and
other isozymes17,18. Therefore, targeting of PKM2 offers an oppor-
tunity to target cancer cell metabolism and reduce the side effects
of cancer therapy17,19. However, an exact mechanistic understand-
ing of PKM2 is still lacking. The identification of PKM2 inhibitors 1H NMR and 13C NMR spectra were recorded on a Bruker AVANCE
not only can provide candidate compounds for cancer therapy but III-400 spectrometer, Chemical shifts d in ppm with Me4Si as
internal standard, coupling constants J in Hertz. High-resolution
mass spectrum (HRMS) was recorded on a Thermo Scientific
Orbitrap Elite MS.
also is helpful to deciphering other cellular functions of PKM2.
In 2010, Vander Heiden et al. screened a library of compounds
to identify a small molecule PKM2 inhibitor compound A with IC50
CONTACT Yuxin Yin
yinyuxin@hsc.pku.edu.cn
Peking University Health Science Center, Beijing, China
ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
127
NMR (100 MHz, CDCl3) d 194.2, 183.9, 144.0, 133.8, 132.1, 126.7,
49.9, 46.8, 34.0, 12.6, 11.6. HR-MS (ESIþ) m/z: 481.1112 [M þ H]þ,
503.0931 [M þ Na]þ. Found: 481.1111 [M þ H]þ, 503.0934
[M þ Na]þ.
OH O
OH O OH
OH
O
S
S
N
N
Data for dipropyl-dithiocarbamic acid 3-dipropylthiocarbamoylsul-
phanylmethyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (3d):
yellow solid (92.9%); mp 111–112 ꢀC. 1H NMR (400 MHz, CDCl3) d
8.12–8.14 (m, 2H, ArH), 7.73–7.76 (m, 2H, ArH), 4.80 (s, 4H, 2CH2S),
3.91 (q, 4H, 2NCH2), 3.60 (m, 4H, 2NCH2), 1.73–1.79 (m, 8H,
4CH2CH3), 0.93–0.95 (m, 12H, 4CH3). 13C NMR (100 MHz, CDCl3) d
194.7, 183.8, 144.1, 133.8, 132.1, 126.7, 57.3, 54.5, 34.1, 20.8, 19.6,
11.2. HR-MS (ESIþ) m/z: 537.1738 [M þ H]þ. Found: 537.1728
[M þ H]þ, 559.1567 [M þ Na]þ.
O
OH O
Compound A
Shikonin
Compound 3k
Figure 1. Structures of compound 3, shikonin and compound 3k.
Procedure for preparation of 2,3-bis-chloromethyl-
[1,4]naphthoquinone (2)24
Data for diallyl-dithiocarbamic acid 3-diallylthiocarbamoylsulpha-
nylmethyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (3e):
yellow liquid (83.3%); 1H NMR (400 MHz, CDCl3) d 8.11–8.14 (m,
2H, ArH), 7.74–7.76 (m, 2H, ArH), 5.80–5.91 (m, 4H, 4CH¼CH2),
5.27 (d, 2H, CH¼CH2), 5.25 (d, 2H, CH¼CH2), 5.24 (d, 2H,
CH¼CH2), 5.20 (d, 2H, CH¼CH2), 4.83 (s, 4H, 4CH2S), 4.66 (d, 4H,
2NCH2), 4.30 (d, 4H, 2NCH2). 13C NMR (100 MHz, CDCl3) d 196.6,
183.8, 143.9, 133.9, 132.0, 131.0, 130.3, 126.7, 118.9, 118.7, 56.9,
53.8, 34.4. HR-MS (ESIþ) m/z: 529.1112 [M þ H]þ. Found: 529.1113
[M þ H]þ.
The 1,4-naphthaquinone (1) (1 g, 6.3 mmol) in glacial acetic acid
(20 ml) was taken in a 100-ml round-bottomed flask, and 36%
aqueous formaldehyde (6 ml) was added. The reaction solution
was cooled in ice water. Dry hydrogen chloride passed in for 2 h.
The solution became red, then being kept at room temperature
for 48 h. The reaction mixture was poured on ice and extracted
with ethyl acetate. The combined organic fractions were washed
with brine, dried (Na2SO4) and concentrated under reduced pres-
sure. Purification of the crude residue by column chromatography
(petroleum ether/ethyl acetate) afforded the compound 2 (yellow
solid). The yield of this reaction was 68.9%. 1H NMR (400 MHz,
CDCl3) d 8.18–8.20 (m, 2H, ArH), 7.81–7.83 (m, 2H, ArH), 4.72 (s, 4H,
2CH2Cl).
Data for dithiamorpholine-dithiocarbamic acid 3-dithiamorpholi-
nethiocarbamoylsulphanylmethyl-1,4-dioxo-1,4-dihydro-naphthalen-2-
ylmethyl ester (3f): yellow solid (90.7%); mp 146–147 ꢀC. 1H NMR
(400 MHz, CDCl3) d 8.11–8.13 (m, 2H, ArH), 7.74–7.77 (m, 2H, ArH),
4.66 (s, 4H, 2CH2S), 4.57–4.58 (m, 4H, 2NCH2), 4.26–4.29 (m, 4H,
2NCH2), 2.76 (s, 8H, 4CH2S). 13C NMR (100 MHz, CDCl3) d 195.3,
183.9, 143.8, 134.0, 131.9, 126.7, 34.7, 27.3. HR-MS (ESIþ) m/z:
541.0240 [M þ H]þ, 563.0060 [M þ Na]þ. Found: 541.0343
[M þ H]þ,563.0178 [M þ Na]þ.
General procedure for preparation of dithiocarbamic acid
3-thiocarbamoylsulphanylmethyl-1,4-dioxo-1,4-dihydro-
naphthalen-2-ylmethyl ester (3a-3h)
Carbon disulphide (180 lL, 3 mmol) and amine (3 mmol) were
added to CH3CN (5 ml) and the resulting solution was stirred for
30 min. 2, 3-Bis-chloromethyl-[1,4]naphthoquinone (2) (254 mg,
1 mmol) was added in portions at frequent intervals. Then, the
reaction mixture was kept at room temperature for 48 h. The reac-
tion mixture was concentrated in vacuo, diluted with H2O, and
extracted with CH2Cl2. The combined organic fractions were
washed with brine, dried (Na2SO4) and concentrated under
reduced pressure. Purification of the crude residue by column
chromatography (petroleum ether/CH2Cl2) afforded the title
compound.
Data for dipyrrolidine-dithiocarbamic acid 3-dipyrrolidinethiocar-
bamoylsulphanylmethyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl
1
ester (3g): yellow solid (88.2%); mp 150–151 ꢀC. H NMR (400 MHz,
CDCl3) d 8.11–8.13 (m, 2H, ArH), 7.73–7.75 (m, 2H, ArH), 4.88 (s, 4H,
2CH2S), 3.95 (q, 4H, 2NCH2), 3.64 (q, 4H, 2NCH2), 1.96–2.09 (m, 8H,
2CH2CH2). 13C NMR (100 MHz, CDCl3) d 191.3, 184.0, 143.9, 133.9,
132.0, 126.6, 55.3, 50.6, 33.5, 26.2, 24.3. HR-MS (ESIþ) m/z:
477.0799 [M þ H]þ, 499.0618[M þ Na]þ. Found: 477.0791 [M þ H]þ,
499.0626[M þ Na]þ.
Data for dithiazolidine-dithiocarbamic acid 3-dithiazolidinethio-
carbamoylsulphanylmethyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylme-
thyl ester (3h): yellow solid (88.8%); mp 158–159 ꢀC. 1H NMR
(400 MHz, CDCl3) d 8.11–8.14 (m, 2H, ArH), 7.75–7.77 (m, 2H, ArH),
4.86 (s, 4H, 2CH2S), 3.11–5.04 (m, 12H, 2NCH2CH2SCH2). 13C NMR
(100 MHz, CDCl3) d 192.5, 183.9, 143.6, 134.0, 131.9, 126.7, 56.6,
52.7, 34.2, 31.2, 29.1. HR-MS (ESIþ) m/z: 512.9927 [M þ H]þ,
534.9747 [M þ Na]þ. Found: 512.9944 [M þ H]þ, 534.9720
[M þ Na]þ.
Data for dimorpholine-dithiocarbamic acid 3-dimorpholinethiocar-
bamoylsulphanylmethyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl
ester (3a): yellow solid (78.4%); mp 153–154 ꢀC. 1H NMR (400 MHz,
CDCl3) d 8.11–8.13 (m, 2H, ArH), 7.75–7.77 (m, 2H, ArH), 4.89 (s, 4H,
2CH2S), 3.99–4.28 (m, 8H, 4OCH2), 3.76 (s, 8H, 4NCH2). 13C NMR
(100 MHz, CDCl3) d 196.1, 183.9, 143.7, 134.0, 132.0, 126.7, 66.3,
66.2, 34.0. HR-MS (ESIþ) m/z: 509.0697 [M þ H]þ. Found: 509.0686
[M þ H]þ.
Data for dimethyl-dithiocarbamic acid 3-dimethylthiocarbamoyl-
sulphanylmethyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl
Biological activity
ester
(3b): yellow solid (92.0%); mp 157–158 ꢀC. 1H NMR (400 MHz,
CDCl3) d 8.11–8.13 (m, 2H, ArH), 7.74–7.76 (m, 2H, ArH), 4.83 (s, 4H,
2CH2S), 3.56 (s, 3H, NCH3), 3.36 (s, 3H, NCH3). 13C NMR (100 MHz,
CDCl3) d 195.7, 183.9, 143.8, 133.9, 132.0, 126.7, 45.7, 41.5, 34.1.
HR-MS (ESIþ) m/z: 425.0486 [M þ H]þ,447.0305 [M þ Na]þ. Found:
425.0487 [M þ H]þ, 447.0304 [M þ Na]þ.
Purification of recombinant pyruvate kinase isoforms
Human cDNA for PKM2 was cloned into pET28aþ with a N-ter-
minal His tag and purified from Escherichia coli strain BL21
(Invitrogen) using Ni-Agarose beads (Qiagen) as described
previously25.
Data for diethyl-dithiocarbamic acid 3-diethylthiocarbamoylsul-
phanylmethyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (3c):
yellow solid (94.3%); mp 130–131 ꢀC. 1H NMR (400 MHz, CDCl3) d
8.12–8.14 (m, 2H, ArH), 7.74–7.76 (m, 2H, ArH), 4.83 (s, 4H, 2CH2S),
4.03 (q, 4H, 2NCH2), 3.72 (m, 4H, 2NCH2), 1.29 (m, 12H, 4CH3). 13C
PKM2 activity assay
Pyruvate kinase activity was measured with a fluorescent pyruvate
kinase-lactate dehydrogenase coupled assay as previously
described20.

128
X. NING ET AL.
Cell culture
Compound 2 was treated with CS2 and various amines to obtain
target compounds 3a-3h in moderate to high yields.
Cell lines were grown with routine culture techniques in
RPMI 1640 supplemented with 9% foetal bovine serum at 37 ꢀC in
5% CO2.
Biological activity
PKM2 inhibition activity of compounds 3a-3h
MTS cell proliferation assay
We first tested the effect of compound 3a-3h on PKM2 activity
using a fluorescent PK-LDH coupled assay according to a previ-
ously reported method20. Shikonin, which is a known inhibitor
of PKM2, was used as the positive control. Most of the
target compounds exhibited some degree of PKM2 inhibitory
Cells were plated in 96-well plates at a density of 5000 cells per
well. Twelve hours after seeding, cells were treated with various
concentrations of test compounds for 48 h. Cell viability was
assessed with the MTS assay (Promega) according to the manufac-
turer's instruction.
activity.
Compound
3f
(IC50 ¼ 1.05 0.17 mM)
and
3h
(IC50 ¼ 0.96 0.18 mM) displayed the higher inhibitory activity than
the lead compound 3k (IC50 ¼ 2.95 0.53 mM) and shikonin
(IC50 ¼ 8.82 2.62 mM) (Table 1). The preliminary SAR can be sum-
marised as follows. The amine moiety of target compounds greatly
influenced PKM2 inhibitory activity. Introduction of a short chain
N, N-dimethylamine reduced the inhibitory activity (3b vs. 3c and
3d). When the n-propyl amine in 3d was replaced by allyl amine
(3e), the inhibitory activity was lowered. The thiazolidinyl 3h
(IC50 ¼ 0.96 0.18 mM) and thiamorpholinyl 3f (IC50 ¼ 1.05
0.17 mM) substituted compounds respectively demonstrated
stronger activity than pyrrolidinyl 3g (IC50 ¼ 2.33 0.52 mM) and
morpholinyl 3a (IC50 ¼ 2.64 0.98 mM) substituted compounds.
Introduction of a sulphur atom therefore contributed to improve-
ment of PKM2 inhibitory activity.
Results and discussion
Chemistry
2, 3-Didithiocarbamate-substituted naphthoquinones (3a-3h) were
prepared as target compounds as shown in Scheme 1. 1,4-
Naphthoquinone 1 was reacted with formaldehyde in the pres-
ence of dry hydrogen chloride in a cold mixed solvent of H2O and
acetic acid to provide 2,3-dichloromethyl-1,4-naphthoquinone 2.
O
O
S
S
O
O
Cl
Cl
R'
R'
S
N
N
b
a
S
O
O
R' R'
Antiproliferative effects of target compounds 3a-3h
1
2
3a-3h
To determine the efficiency of 3a-3h as antitumour agents, we
assessed the in vitro cytotoxicity of 3a-3h using several different
tumour cell lines derived from human colon cancer (HCT116),
breast cancer (MCF7), cervical cancer (Hela) and lung cancer
(H1299) and mouse melanoma (B16). The results are presented in
Table 2. Most target compounds reduced cancer cell viability at
nanomolar concentrations in MTS reduction assays, showing
higher cytotoxicity than shikonin. Specially, compound 3b exhib-
ited an optimal dose-dependent cytotoxicity with IC50 values
against HCT116, MCF7, Hela, H1299 and B16 cells from 69 nM to
122 nM. The preliminary SAR showed that introduction of a long-
chain amine in target compounds lowered cytotoxicity (3b vs. 3c
vs 3d), which was not consistent with the enzyme activity. This
discrepancy may be due to the different properties of these com-
pounds such as cell penetration that is important in the cellular
assay. In addition, replacing the chain amines with various cyclic
amines, morpholinyl (3a), thiamorpholinyl (3f), pyrrolidinyl (3g)
and thiazolidinyl (3h) substitution compounds also demonstrated
the great potency.
Scheme 1. Synthesis of 2,3-didithiocarbamate-substituted naphthoquinones.
Reagents and conditions: (a) formaldehyde, HCl, HAc, H2O, 0 ꢀC, 68.9%; (b) CS2,
amine, CH3CN, rt, 78–94%.
Table 1. PKM2 inhibitory activity of compounds 3a-3h.
O
S
S
R
R
O
S
S
Compound
R
IC50 SD (lM)
3a
3b
3c
3d
morpholinyl
dimethylamino
diethylamino
di-n-propylamino
diallylamino
2.64 0.98
>10
2.78 0.97
3.08 1.23
>10
3e
3f
3g
3h
3k
thiamorpholinyl
pyrrolidinyl
thiazolidinyl
1.05 0.17
2.33 0.52
0.96 0.18
2.95 0.53
8.82 2.62
To further explore the selectivity of target compounds against
cancer cells, we tested the cytotoxicity of representative com-
pound 3f in BEAS-2B cells derived from normal human bronchial
Shikonin
Table 2. In vitro cytotoxicity of target compounds
IC50 SDa (lM)
compd
HCT116
MCF7
Hela
H1299
B16
3a
3b
3c
3d
3e
3f
3g
3h
0.214 0.003
0.088 0.004
0.093 0.002
0.597 0.014
0.742 0.045
0.189 0.035
0.164 0.003
0.374 0.015
1.060 0.182
0.340 0.003
0.069 0.009
0.084 0.003
>10
1.092 0.421
0.194 0.055
0.638 0.020
0.234 0.045
2.405 0.346
0.337 0.054
0.122 0.017
0.251 0.059
>10
3.456 3.188
0.412 0.018
0.298 0.053
1.126 0.080
0.813 0.081
0.331 0.091
0.109 0.002
0.144 0.013
0.830 0.162
1.019 0.111
0.206 0.018
0.247 0.015
0.306 0.008
0.954 0.186
0.272 0.010
0.104 0.011
0.108 0.010
0.787 0.203
0.709 0.027
0.189 0.006
0.159 0.002
0.317 0.022
1.220 0.155
Shikonin

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
129
epithelial cells. The result showed that IC50 value of compound 3f
was 11.49 0.62 mM, which indicated the target compound had
higher selectivity for cancer cells than normal cells.
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We have described the identification and characterisation of a pre-
viously undescribed chemical class of PKM2 inhibitors. Most target
compounds as PKM2 inhibitors, especially compound 3f and 3h,
have high inhibition responses and are more potent than shikonin
and compound 3k. These compounds are therefore optimal PKM2
inhibitors with the best characteristics reported to date. They may
lock PKM2 into a low activity conformation and forces disruption
of cancer cell metabolism in a manner that is less metabolically
flexible than the normal state.
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In addition, we have investigated in vitro cytotoxicity of these
PKM2 inhibitors. Most target compounds show higher antitumour
effects than shikonin in MTS assay. The compound 3b and 3c
exhibited optimal dose-dependent cytotoxicity with IC50 values
against HCT116, MCF7, Hela, H1299 and B16 cells, respectively,
from 69 nM to 122 nM and from 84 nM to 251 nM.
However, there is absence of correlation between the PKM2
inhibitory activity and in vitro antitumor activity of the target com-
pounds. This suggests that these compounds may have other
mechanisms to influence the tumour cells. In future studies, we
will focus on evaluation as yet unidentified mechanisms of these
compounds.
12. Gupta V, Bamezai RN. Human pyruvate kinase M2: a multi-
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Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This study was supported by the National Natural Science 19. Dayton TL, Jacks T, Vander Heiden MG. PKM2, cancer metab-
olism, and the road ahead. EMBO Rep 2016;17:1721–30.
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Identification of small molecule inhibitors of pyruvate kinase
M2. Biochem Pharmacol 2010;79:1118–24.
Foundation of China (Key grants #81430056, #81372491 and
#81402777) and the China Postdoctoral Science Foundation
(#2014M560026 and #2015T80028).
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