Discovery of novel naphthoquinone derivatives as inhibitors of the tumor cell specific M2 isoform of pyruvate kinase

Article abstract of DOI:10.1016/j.ejmech.2017.06.064

Pyruvate kinase M2 (PKM2) is a rate-limiting enzyme of the glycolytic pathway which is highly expressed in cancer cells. Cancer cells rely heavily on PKM2 for anabolic and energy requirements, and specific targeting of PKM2 therefore has potential as stra

Full text of DOI:10.1016/j.ejmech.2017.06.064

Accepted Manuscript
Discovery of novel naphthoquinone derivatives as inhibitors of the tumor cell specific
M2 isoform of pyruvate kinase
Xianling Ning, Hailong Qi, Ridong Li, Yunqiao Li, Yan Jin, Michael A. McNutt, Junyi
Liu, Yuxin Yin
PII:
S0223-5234(17)30518-4
10.1016/j.ejmech.2017.06.064
EJMECH 9559
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 May 2017
Revised Date: 27 June 2017
Accepted Date: 28 June 2017
Please cite this article as: X. Ning, H. Qi, R. Li, Y. Li, Y. Jin, M.A. McNutt, J. Liu, Y. Yin, Discovery of
novel naphthoquinone derivatives as inhibitors of the tumor cell specific M2 isoform of pyruvate kinase,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.06.064.
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ACCEPTED MANUSCRIPT
IC50 = 0.18 µM
IC50 = 0.29 µM

ACCEPTED MANUSCRIPT
Discovery of novel naphthoquinone derivatives as inhibitors of the tumor
cell specific M2 isoform of pyruvate kinase
a, d
a, b
a, d
a, c
a, c
Xianling Ning , Hailong Qi , Ridong Li , Yunqiao Li , Yan Jin , Michael A.
a, b, c
e
a, b, c*
McNutt
, Junyi Liu and Yuxin Yin
Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center,
a
Beijing, China.
b
Peking-Tsinghua Center for Life Sciences, Peking University Health Science Center, Beijing, China.
c
Department of Pathology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing,
China.
d
Department of Pharmacology, School of Basic Medical Sciences, Peking University Health Science Center,
Beijing, China.
e
State Key Laboratory of Natural and Biomimetic Drugs, Department of Chemical Biology, School of
Pharmaceutical Sciences, Peking University Health Science Center, Beijing, China.
ABSTRACT
Pyruvate kinase M2 (PKM2) is a rate-limiting enzyme of the glycolytic pathway
which is highly expressed in cancer cells. Cancer cells rely heavily on PKM2 for
anabolic and energy requirements, and specific targeting of PKM2 therefore has
potential as strategy for cancer therapy. Here, we report the synthesis and biologic
evaluation of novel naphthoquinone derivatives as selective small molecule inhibitors
of PKM2. Some target compounds, such as compound 3k, displayed more potent
PKM2 inhibitory activity than the reported optimal PKM2 inhibitor shikonin. The
well performing compound 3k also showed nanomolar antiproliferative activity
toward a series of cancer cell lines with high expression of PKM2 including HCT116,
Hela and H1299 with IC values ranging from 0.18-1.56 µM. Moreover, compound
5
0
3
k exhibited more cytotoxicity on cancer cells than normal cells. The identification of
novel potent small molecule inhibitors of PKM2 not only offers candidate compounds
for cancer therapy, but also provides a tool with which to evaluate the function of
PKM2 in depth.
Keywords:
Pyruvate kinase M2, PKM2 inhibitor, Naphthoquinone derivatives, Antiproliferative
activity
1
. Introduction
Metabolic reprogramming is one of the hallmarks of highly proliferative cancer cells
1, 2]. Evidence suggests pyruvate kinase M2 (PKM2) makes a significant
[
contribution to cancer metabolism, especially aerobic glycolysis [3, 4]. Pyruvate
kinase converts phosphoenolpyruvate (PEP) to pyruvate and is expressed in four
isoforms in different tissues [5]. The isoforms PKL, PKR and PKM1 are mainly
*
Corresponding author
Email address: yinyuxin@hsc.pku.edu.cn
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expressed in normal tissues. However, PKM2 is preferentially expressed in various
tumors [6, 7]. Selective targeting of PKM2 therefore offers an opportunity to target
cancer cell metabolism and with reduced the side effects of therapy [8-10].
PKM2 exists in two catalytically different states and converts between high- and
low-activity state. The state with high catalytic activity contributes to catabolic
metabolism to produce energy. The state with low catalytic activity is conducive to
anabolic metabolism to support cell growth. This regulation of PKM2 activity may
provide tumor cells with the flexibility to adapt to different microenvironments [11].
Therefore, agonists or inhibitors of PKM2 may inhibit tumorigenesis by locking
PKM2 into an on or off state.
There have been a number of studies of PKM2 agonists of in recent years [12-14].
However, PKM2 inhibitors have been less well studied. Christofk et al. report that
PKM2 knockdown by RNA interference or displacement of PKM2 with PKM1
dramatically reduces the ability of human tumor cell lines to form tumor in nude mice
[
6]. Compound 3 (Fig. 1) is an effective PKM2 inhibitor that was found in a
high-throughput screen which can inhibit PKM2 activity at millimolar concentrations
in H1299 cells. However, compound 3 also has an inhibitory effect on the activity of
PKL and PKR [15]. Shikonin (Fig.1) and its its enantiomeric isomer alkannin also
inhibit PKM2 activity in cancer cells, and their inhibitory effects are much stronger
than compound 3 [16].
OH O
OH O OH
OH O OH
OH
N
OH O
OH O
Compound 3
Shikonin
Alkannin
Fig. 1. Structures of known PKM2 inhibitors
In order to further improve the PKM2 inhibitory activity and selectivity of shikonin,
we designed and synthesized a series of novel naphthoquinone derivatives (Fig. 2)
that preserve the naphthoquinone skeleton and introduce diverse dithiocarbamate
moieties. The dithiocarbamate moiety which replaces the alkyl chain of shikonin may
provide more hydrogen bond receptors or donors to enhance the interaction of
proteins and small molecules. At the same time, the dithiocarbamate moiety is well
known as a molecular scaffold which possesses diverse biologic activity, such as
anti-fungal, anti-bacterial and anti-tumor effects, which is conducive to increasing
antitumor activity of target compounds [17, 18].
O
S
R'
nS
N
R''
R
O
Fig. 2. The structure of target compounds
In this study we describe the synthesis, PKM2 inhibitory activity, PKM2 selectivity
and cytotoxicity of a series of novel naphthoquinone derivatives. Some of the
compounds we obtained, such as 3k, show higher inhibitory activity for PKM2, and
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display antiproliferative activity which is more potent than shikonin.
2
. Results and discussion
2
.1.Chemistry
To study the structure activity relationship (SAR) of target compounds, we
synthesized the compounds 3a-3w, 4a-4d and 6a-6d. The synthetic routes for the
preparation of 2-dithiocarbamate-3-methyl substituted naphthoquinones (3a-3w,
4
a-4d)
are
shown
in
Scheme
1.
Commercially
available
2
-Methyl-1,4-naphthoquinone 1 was used as a starting material and reacted with
formaldehyde in the presence of dry hydrogen chloride in a cold mixed solvent of
H O and acetic acid to provide 2-methyl-3- chloromethyl-1,4-naphthoquinone 2. The
2
target compounds 3a-3w were obtained by reaction of the intermediate 2 with carbon
disulfide (CS ) and different amines using THF as a solvent which gave good yields.
2
The target compounds 4a-4d were obtained by reaction of compound 1 with CS and
2
amines in CH CN by one step with moderate yields.
3
O
O Cl
O
O
S
R'
a
b
S
N
R''
O
O
1
2
3a-3w
c
O
R'
N
S
R'
S
O
4
a-4d
Compd.
R'
N
R''
3
3
a
N(Et)
N(n-Pr)
N(n-Bu)
N(CH CH OH)
N(allyl)
1-morpholinyl
1-thiamorpholinyl
2
b
2
3
c
2
3
d
2
2
2
3
e
2
3
f
3
3
g
h
1-(2,6-dimethyl)morpholinyl
1-thiazolidinyl
3
i
3
j
1-pyrrolidinyl
3
k
1-piperidinyl
3
l
1-(4-methyl)piperazinyl
1-(4-isopropyl)piperazinyl
1-(4-acetyl)piperazinyl
3
m
3
n
o
3
NCH cyclohexyl
3
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3
3
p
q
NHCH
3
NHCH
NHCH
NHCH
-2-Py
-3-Py
-4-Py
2
2
2
3
r
s
3
3
t
NHCH Ph
2
3
u
v
NH-1-piperidinyl
3
NH-1-(4-methyl)piperazinyl
3
w
NHCH
N(n-Pr)
2
2 2 2
CH N(Et)
4
4
a
b
1-morpholinyl
1-piperidinyl
4
c
4
d
1-(4-acetyl)piperazinyl
Scheme 1. Synthesis of 2-dithiocarbamate-3-methyl substituted naphthoquinones. Reagents and conditions: (a)
formaldehyde, HCl, HAc, H O, 0 ℃, 78.0%; (b) CS , amine, THF, rt, 75-98%. (c) CS , amine, CH CN, rt,
2
2
2
3
5
5-70%.
2
-Dithiocarbamate-3-hydrogen substituted naphthoquinones (6a-6d) were prepared as
shown in Scheme 2. 2-Methyl-1,4-naphthoquinone 1 was used as a starting material,
and was brominated with N-bromosuccinimide (NBS) in the presence of a catalytic
amount of 2,2-azobisisobutyronitrile (AIBN) in reflux acetic anhydride to give the
compound 2-bromomethyl-1,4-naphthoquinone 5. Compound 5 was treated with CS2
and different amines to obtain corresponding target compounds 6a-6d in moderate
yields.
O
O
Br
O
S
R'
a
b
S
N
R''
O
O
O
1
5
6a-6d
Compd.
R'
N
R''
6
6
a
N(Et)
2
b
1-morpholinyl
1-piperidinyl
6
c
6
d
1-(N-phenyl)piperazinyl
Scheme 2. Synthesis of 2-dithiocarbamate-3- hydrogen substituted naphthoquinones. Reagents and conditions: (a)
NBS, AIBN, acetic anhydride, reflux, 63%; (b) CS , amine, CH Cl , rt, 69-75%.
2
2
2
To further study the SAR of these compounds, the 1,4-dimethoxy-naphthalen
derivative 9 was also synthesized (Scheme 3). 1,4-Dimethoxy-2-hydroxymethyl
naphthalen 7 was synthesized as previously described [19], and was chloridized with
thionyl
chloride
(SOCl2)
in
refluxed
CH Cl
to
achieve
2
2
1
,4-dimethoxy-2-chloromethyl naphthalen 8. Compound 8 was treated with CS and
2
n-propylamine to obtain the corresponding target compound 9.
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O
OH
O
Cl
O
S
a
b
S
N
O
O
O
7
8
9
Scheme 3. Synthesis of dipropyl-dithiocarbamic acid 1,4-dimethoxy-naphthalen-2-ylmethyl ester
Reagents and conditions: (a) SOCl , CH Cl , reflux, 78%; (b) CS , di-n-propylamine, CH Cl , rt, 80 %.
2
2
2
2
2
2
2
2
.2.Biological evaluation
.2.1. PKM2 inhibition activity by target compounds
After the synthesis of these compounds, the cell-free PKM2 inhibitory activity of
target compounds was evaluated with a fluorescent PK-LDH coupled assay as
previously reported (Fig. 4) [15]. Shikonin was used as the positive control. The
results expressed as IC are shown in Table 1. As shown in Table 1, most of the
5
0
compounds exhibited some degree of PKM2 inhibition and some compounds, such as
k and 6d, displayed more potent activity than the positive control shikonin. The
3
amine moiety markedly influenced PKM2 inhibitory activity. Introduction of a long
chain amine slightly enhanced inhibitory activity (3a vs 3b vs 3c). When the n-propyl
amine of 3b was replaced by ethoxyl amine (3d) and allyl amine (3e), the inhibitory
activity was reduced or lost. The chain amines were further altered to various
heterocycle amines. The thiamorpholinyl 3g (IC = 5.89±1.75 µM) and thiazolidinyl
5
0
3
i (IC = 5.28±0.13 µM) substitution compounds respectively demonstrated greater
50
potency than morpholinyl 3f (IC50 = 15.24±2.72 µM) and pyrrolidinyl 3j (IC50
=
1
9.73±5.11 µM) substitution compounds. This indicated that introduction of a sulfur
atom was conductive to improving PKM2 inhibitory activity. The piperidinyl
substitution compound 3k (IC = 2.95±0.53 µM) showed slightly stronger potency
5
0
than the thiamorpholinyl substitution compound 3g (IC50 = 5.89±1.75 µM). In
addition, the 4-methylpiperazinyl substitution compound 3l (IC = 14.24±2.04 µM)
5
0
showed low activity, and increasing steric hindrance (3m and 3n) caused loss of
activity. Introduction of primary amines (3p-3s, 3u-3w) reduced their inhibitory
activity. Interestingly, compound 3t (IC = 2.21±0.60 µM) was an exception which
5
0
displayed high inhibitory activity, and this compound warrants further study. We
further investigated the contribution of the linker of naphthoquinone and
dithiocarbamate moieties and the 3-substituent in naphthoquinone. It was found that n
is 0 or 1 in target compounds rarely influenced the potency of inhibitory activity (3b
vs 4a, 3k vs 4c, 3n vs 4d). Removal of the 3-methyl substituent in the
naphthoquinone results in a 2-4-fold increase in IC (3a vs 6a, 3f vs 6b, 3k vs 6c). In
50
other words, a 3-unsubstitution at the naphthoquinone scaffold significantly improved
PKM2 inhibitory potency. The 1, 4-dimethoxy-naphthalen derivative 9 (IC50
=
1
5.66±4.47 µM) exhibited lower potency than the naphthoquinone derivative 3b (IC50
=
6.79±0.54 µM), indicating that the naphthoquinone unit is essential for PKM2
inhibitory activity.
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PK
LDH
PEP
Pyruvate
Lactate
NADH NAD+
ADP ATP
Luminescence
Fig. 4. In vitro a fluorescent PK-LDH coupled assay.
Table 1
In vitro PKM2 inhibition activity (IC ) of target compounds
50
O
S
n
S R'
R
O
Compd.
n
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
1
1
1
1
R
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
H
R’
IC50 ± SD (µM)
8.78±1.05
6.79±0.54
4.57±0.37
> 20
3
3
a
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
N(Et)
2
b
N(n-Pr)
N(n-Bu)
N(CH CH OH)
N(allyl)
1-morpholinyl
1-thiamorpholinyl
2
3
c
2
3
d
2
2
2
3
e
2
14.11±3.40
15.24±2.72
5.89±1.75
11.70±0.67
5.28±0.13
19.73±5.11
2.95±0.53
14.24±2.04
> 20
3
f
3
3
g
h
1-(2,6-dimethyl)morpholinyl
1-thiazolidinyl
3
i
3
j
1-pyrrolidinyl
3
k
1-piperidinyl
3
l
1-(4-methyl)piperazinyl
1-(4-isopropyl)piperazinyl
1-(4-acetyl)piperazinyl
3
m
3
n
o
p
q
> 20
3
NCH
3
cyclohexyl
5.08±0.34
> 20
3
3
NHCH
3
NHCH
NHCH
NHCH
-2-Py
-3-Py
-4-Py
> 20
2
2
2
3
r
s
> 20
3
> 20
3
t
NHCH
2
Ph
2.21±0.60
> 20
3
u
v
NH-1-piperidinyl
3
NH-1-(4-methyl)piperazinyl
> 20
3w
NHCH
2
CH
2
N(Et)
2
> 20
4
4
a
N(n-Pr)
2
9.98±0.14
8.50±2.06
4.93±1.78
> 20
b
1-morpholinyl
1-piperidinyl
4
c
4d
6a
6b
1-(4-acetyl)piperazinyl
N(Et)
2.58±0.76
4.23±1.02
1.74±0.71
1.40±0.41
2
H
1-morpholinyl
1-piperidinyl
6
c
H
6
d
H
1-(N-phenyl)piperazinyl
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9
-
-
-
-
-
15.66±4.47
8.82 ± 2.62
Shikonin
-
2
.2.2. PKM2 selectivity of target compounds
Because of the similarity of amino acid sequences in PKM2 and the other isoforms of
pyruvate kinase, evaluation of the selectivity for PKM2 of these target compounds
was required. Shikonin was used as the positive control. The representative
compounds 3k, 6d displayed dose-dependent inhibition of PKM2 with less inhibition
of PKM1 and PKL like shikonin. The IC50 of shikonin for PKM1 and PKL were
respectively about 1.5-fold and 4.5-fold higher than that for PKM2 according to the
assay results. However, the IC s of compounds 3k and 6d for PKM1 and PKL were
5
0
respectively about 4-5-fold and 2-3-fold higher than that for PKM2 (Table 2). Thus,
the selectivity for PKM2 of target compounds is close to that of shikonin.
Table 2
PKM2 selectivity of tested compounds on different PK isoforms
Compd.
PKM1 (µM)
PKL (µM)
IC (PKM1)/
IC (PKL)/
50
5
0
IC (PKM2)
IC (PKM2)
50
5
0
3
6
k
d
16.71±4.66
6.26±1.75
5.7
4.5
8.20±3.43
3.80±0.25
2.8
2.7
Shikonin
12.96 ± 3.37
1.5
39.25 ± 6.53
4.5
2
.2.3. Antiproliferative effects of target compounds
To determine the effectiveness of target compounds as anti-tumor agents, we
evaluated the effect of target compounds on cancer cell viability. In vitro cytotoxicity
was assessed using three PKM2 high expression human tumor cell lines derived from
human colon cancer (HCT116), cervical cancer (Hela) and lung cancer (H1299). The
results of this evaluation are presented in Table 3. Some target compounds reduced
cancer cell viability at nanomolar concentrations with showing more pronounced
cytotoxicity than the positive control shikonin in MTS reduction assays in human
cancer cell. Among these, the most potent compounds were 3a, 3k (Fig.5) and 3r,
which exhibited IC values against HCT116 and Hela cells ranging from 0.39 to 0.41
5
0
µM, 0.18 to 0.29 µM and 0.18 to 0.38 µM, respectively. Introduction of a long chain
amine lowered the cytotoxicity of target compounds (3a vs 3b vs 3c). When the
n-propyl amine in 3b was replaced by ethoxyl amine (3d) and allyl amine (3e), the
inhibitory activity increased slightly. In addition, replacing the chain amines with
various cyclic amines, the piperidinyl (3k), morpholinyl (3f), thiamorpholinyl (3g)
substitution compounds demonstrated greater potency than pyrrolidinyl (3j),
thiazolidinyl (3i) and piperazinyl (3l-3n) substitution compounds. Compound 3r
bearing a 3-pyridylamino substitution exhibited greater cytotoxicity than 2- and
4
-pyridylamino substitution. However, replacing 3-pyridylamino (3r) with
benzylamino (3t) reduced its activity. In addition, the deletion of a 3-methyl
substituent in the naphthoquinone resulted in a decrease of cytotoxicity of the target
compounds in cell lines (3b vs 6a and 3k vs 6c). When naphthoquinone and
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dithiocarbamate moieties were linked by a methylene group (n = 1), the cytotoxicity
was higher than that by a direct connect (n = 0). The oxidization state of derivative 3b
exhibited greater potency than the reducing state of derivative 9. Thus the
naphthoquinone moiety is very important for inhibitory activity.
These results show that a weak correlation exists between antiproliferative effects and
PKM2 inhibitory activity. The discrepancies between them may be due to the different
properties of these compounds such as cell penetration which is important in the
cellular assay. It is also possible that target compounds have other mechanisms by
which tumor cells are targeted. However, some compounds which were tested showed
high PKM2 inhibitory potency with close to nanomolar activity, which supports a
ditect enzyme interaction mechanism. In addition, we found that
fructose-1,6-bisphosphate (FBP) which is a well endogenic allosteric activator of
PKM2, reduced the inhibitory activity and relieve the cytotoxicity of tested PKM2
inhibitors compounds 3a and 3k (Table 4), which also suggests that PKM2 is the
direct target of compounds which were tested.
Table 3
In vitro cytotoxicity of target compounds
a
IC50 ± SD (µM)
Compd.
HCT116
0.41±0.13
6.00±1.12
10.94±2.45
2.23±0.51
2.17±0.37
0.80±0.17
0.89±0.32
1.10±0.33
1.75±0.15
1.46±0.32
0.18±0.16
2.84±1.14
1.97±0.77
2.08±0.26
Hela
H1299
3
3
a
0.39±0.08
12.08±2.20
18.53±3.14
4.80±0.84
1.16±0.66
0.95±0.39
0.32±0.11
0.87±0.92
1.96±0.95
0.88±0.64
0.29±0.11
1.57±0.17
2.19±1.37
3.52±1.94
13.55±3.27
8.36±3.19
6.77±2.59
0.38±0.13
0.50±0.31
1.30±1.07
10.42±4.75
0.89±0.19
4.23±1.24
6.41±1.25
1.95±0.99
3.20±0.17
1.23±0.51
1.19±0.44
1.47±0.67
3.28±1.21
1.64±0.73
1.56±0.25
2.44±0.14
4.06±2.51
4.68±0.44
11.27±1.26
5.39±0.67
4.49±0.33
1.08±0.25
0.67±0.17
4.84±0.89
4.37±0.12
b
3
c
3
d
3
e
3
f
3
3
g
h
3
i
3
j
3
k
3
l
3
m
3
n
o
3
1
0.61±3.14
3
3
p
q
4
.59±1.30
.42±1.75
.18±0.05
.59±0.23
.72±0.76
.36±3.20
5
3
r
s
0
0
1
7
3
3
t
3
u
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3
v
2
3
.00±1.52
.37±0.86
3.92±1.35
5.78±2.07
> 20
> 20
3w
3.97±0.77
15.59±5.93
3.27±1.24
10.18±3.32
6.61±0.52
1.90±0.11
5.69±3.10
2.06±0.09
8.71±2.07
> 20
4
4
a
4.30±1.20
2.28±1.19
3.55±2.02
4.95±1.08
1.82±0.36
3.68±1.61
2.37±0.31
> 20
b
2.14±0.24
1.29±1.23
10.61±3.68
0.51±0.29
2.39±0.04
0.92±0.20
> 20
4
c
4d
6a
6b
6
c
6
d
9
> 20
> 20
Shikonin
1.84±0.18
2.45±0.81
1.88±0.15
HCT116
Hela
1
00
92.5
120
9
9.0
90
80
70
60
50
40
30
20
10
0
95.8
1
00
62.3
8
0
60
4
0
0
0
20.7
2
1.1
2
6
.9
4
.8
0
.078 0.156 0.313 0.625
0.078 0.156 0.313 0.625
3
k (µM)
3k (µM)
Fig. 5. Compound 3k reduces the tumor cell viability in a dose depend manner. Cells were treated with increasing
concentrations of the PKM2 inhibitor compound 3k. Cell viability was measured using MTS.
Table 4
In vitro cytotoxicity of 3a and 3k in the presence of FBP (100 µM)
a
IC50 ± SD (µM) (H1299)
FBP
+
-
3
3
a
1.58±0.33
2.01±0.16
0.89±0.19
1.56±0.25
k
2
.2.4. Cytotoxicity of some target compounds against normal cells
To further explore the selectivity of target compounds against cancer cells, they were
also tested in BEAS-2B cells derived from normal human bronchial epithelial cells.
As seen in Table 5, the tested compounds showed higher cytotoxicity in cancer cells
(H1299) than normal cells (BEAS-2B). The selectivity for tumor cells of target
compounds was superior to that of shikonin. The results indicate that target
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compounds have highly safety index.
Table 5
In vitro safety index of target compounds (BEAS-2B)
a
Compd.
IC50 ± SD
µM)
safety index
(
3
3
3
a
g
k
15.09±0.54
10.54±1.80
18.46±1.04
19.31±1.79
5.36±0.69
17.0
8.6
11.8
9.4
6
c
Shikonin
2.9
3
. Conclusions
In this study we have described the identification and characterization of a previously
undescribed chemical class of PKM2 inhibitors. These novel naphthoquinone
derivatives act as PKM2 inhibitors, and show high inhibitory responses that are more
potent than shikonin and have selectivity similar to shikonin. These compounds lock
PKM2 into a low activity conformation which forces alteration of cancer cell
metabolism that is less metabolically flexible than metabolism in the normal state.
Moreover, some of these target compounds show higher antiproliferative effects than
shikonin. However, there are unexpected results in the enzyme and cytotoxicity
experiments. The absence of correlation between PKM2 inhibitory activity and in
vitro cytotoxicity of the target compounds suggests that the cells are probably using
other mechanisms to activate these compounds, except in the case of PKM2 inhibitory
activity. However, there is no doubt that PKM2 is a target of these synthesized
naphthoquinone derivatives, through the IC50 of some compounds which inhibit
PKM2 activityis closed to nanomolar concentration range.
PKM2 has been identified as a major contributor not only of metabolic
reprogramming as for pyruvate kinases but also in direct regulation of gene
expression as a protein kinase in cancer cells [20-22]. Upon EGFR activation, PKM2
translocates from the cytoplasm into the nucleus of cancer cells where it activates
β-catenin to induce CCDN1 and c-Myc expression and upregulate GLUT1 and lactate
dehydrogenase A (LDHA) [21]. Upregulation of these glycolysis genes increases
glucose consumption and lactate production, and subsequently promotes
tumorigenesis [23].Therefore, inhibition of PKM2-coactivated tumor gene
transcription and glycolysis gene expression are also potential tumor
treatment strategies. An attempt will be made to determine whether the mechanism
works for the synthesized naphthoquinone derivatives. If so, the mechanism also can
explain the SAR discrepancy of enzyme activity and cytotoxicity of target
compounds.
Further investigation will also focus on structural studies of PKM2 protein and small
molecule inhibitor complex, which may lead to identification of compounds with
higher effectiveness. In any case, the findings presented here establish a foundation
for the development of PKM2-targeted anticancer therapies.
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4
4
. Experimental section
.1. General. Reactions were monitored by thin layer chromatography (TLC) on
precoated silica gel F254 plates. Detection was by iodine vapor staining and UV light
irradiation (UV lamp, model UV-IIB). Column chromatography was carried out with
Silica gel H (200-300 mesh or 500 mesh). Melting points were determined using an
1
13
X -type apparatus and left uncorrected. H NMR and C NMR spectra were recorded
4
on a Bruker AVANCE III-400 spectrometer, Chemical shifts δ in ppm with Me Si as
4
an internal standard, coupling constants J in Hertz. High resolution mass spectrum
(HRMS) was recorded on a Thermo Scientific Orbitrap Elite MS. Unless otherwise
stated, all reagents were purchased from commercial sources. When necessary,
reagents were purified and dried by standard methods. Organic solutions were dried
over anhydrous sodium sulfate.
4
.2. Procedure for preparation of 2-chloromethyl-3-methyl-[1, 4] naphthoquinone (2)
The 2-methyl-1,4-naphthaquinone (1) (2 g, 11.6 mmol) in glacial acetic acid (20 mL)
was taken in a 100 mL round-bottomed flask, and 36% aqueous formaldehyde (7 mL)
was added. The reaction solution was cooled in ice-water. Dry hydrogen chloride
passed in for 1 h. The solution turned red, and was then kept at room temperature for
2
4 h. The reaction mixture was poured on ice and extracted with ethyl acetate. The
combined organic fractions were washed with brine, dried (Na SO ), and concentrated
2
4
under reduced pressure. Purification of the crude residue by column chromatography
petroleum ether/ethyl acetate) afforded compound 2 (yellow solid). The yield of this
(
1
reaction was 78.0%. H NMR (400 MHz, CDCl ) δ 8.13-8.18 (m, 2H, ArH), 7.76-7.78
3
(
m, 2H, ArH), 4.64 (s, 2H, CH S), 2.35 (s, 3H, C=CCH ).
2 3
4
3
.3.
General
procedure
for
preparation
of
dithiocarbamic
acid
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (3)
Carbon disulfide (120 µL, 2 mmol) and amine (2 mmol) were added to THF (5 mL)
and the resulting solution was stired for 30 minutes. 2-Chloromethyl-3-methyl-[1, 4]
naphthoquinone (2) (220 mg, 1 mmol) was added in portions at frequent intervals.
The reaction mixture was then kept at room temperature until compound 2 was absent
(as evaluated by TLC). The reaction mixture was concentrated in vacuo, diluted with
H O, and extracted with CH Cl . The combined organic fractions were washed with
2
2
2
brine, dried (Na SO ), and concentrated under reduced pressure. Purification of the
2
4
crude residue by column chromatography (petroleum ether/ CH Cl ) afforded the
2
2
compound of interest.
4
3
.3.1.
Data
for
diethyl-dithiocarbamic
acid
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (3a): yellow solid
1
(89.6%); mp 155-156 °C; H NMR (400 MHz, CDCl ) δ 8.11-8.14 (m, 2H, ArH),
3
7
2
1
3
.73-7.74 (m, 2H, ArH), 4.65 (s, 2H, CH S), 4.06 (q, 2H, NCH ), 3.73 (q, 2H, NCH ),
2 2 2
1
3
.37 (s, 3H, C=CCH ), 1.30 (t, 6H, 2CH CH ). C NMR (100 MHz, CDCl ) δ 194.49,
3
2
3
3
84.81, 183.95, 146.42, 141.66, 133.67, 133.63, 132.19, 131.92, 126.49, 49.92, 46.80,
+
+
3.85, 13.70, 12.53, 11.57. HR-MS (ESI ) m/z: 334.0936 [M+H] . Found: 334.0932
+
[
M+H] .
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4
3
.3.2.
Data
for
dipropyl-dithiocarbamic
acid
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (3b): yellow solid
1
(86.0%); mp 80-81 °C; H NMR (400 MHz, CDCl ) δ 8.10-8.14 (m, 2H, ArH),
3
7
2
.72-7.74 (m, 2H, ArH), 4.63 (s, 2H, CH S), 3.93 (t, 2H, NCH ), 3.61 (t, 2H, NCH ),
2 2 2
.35 (s, 3H, C=CCH ), 1.70-1.82 (m, 4H, 2CH CH ), 0.92-0.98 (m, 6H, 2CH CH ).
3
2
3
2
3
1
3
C NMR (100 MHz, CDCl ) δ 195.02, 184.79, 183.89, 146.48, 141.64, 133.62,
3
+
1
32.19, 131.93, 126.49, 57.25, 54.43, 33.92, 20.70, 19.62, 13.66, 11.18. HR-MS (ESI )
+
+
m/z: 362.1249 [M+H] . Found: 362.1244 [M+H] .
4
3
.3.3.
Data
for
dibutyl-dithiocarbamic
acid
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (3c): yellow liquid
1
(90.5%); H NMR (400 MHz, CDCl ) δ 8.11-8.15 (m, 2H, ArH), 7.73-7.75 (m, 2H,
3
ArH), 4.63 (s, 2H, CH S), 3.98 (t, 2H, NCH ), 3.64 (t, 2H, NCH ), 2.36 (s, 3H,
2
2
2
C=CCH ), 1.67-1.75 (m, 4H, 2CH CH CH ), 1.32-1.41 (m, 4H, 2CH CH CH ),
3
2
3
2
3
2
2
3
1
0
1
2
3
4
3
.93-1.00 (m, 6H, 2CH CH CH ). C NMR (100 MHz, CDCl ) δ 194.80, 184.81,
2 2 3 3
83.88, 146.48, 141.67, 133.60, 132.21, 131.97, 126.48, 55.46, 52.60, 33.93, 29.69,
+
+
9.38, 26.36, 20.12， 13.81， 13.65. HR-MS (ESI ) m/z: 390.1562 [M+H] . Found:
+
90.1558 [M+H] .
.3.4.
Data
for
bis-(2-hydroxy-ethyl)-dithiocarbamic
acid
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (3d): yellow solid
1
(82.4%); mp 91-92 °C; H NMR (400 MHz, CDCl ) δ 8.10-8.12 (m, 2H, ArH),
3
7
2
1
5
.73-7.75 (m, 2H, ArH), 4.61 (s, 2H, CH S), 3.98-4.33 (m, 8H, 2NCH CH ), 2.78 (s,
2 2 2
13
H, 2OH), 2.35 (s, 3H, C=CCH ). C NMR (100 MHz, CDCl ) δ 197.91, 184.62,
3
3
83.85, 146.68, 141.64, 133.78, 133.72, 132.13, 131.83, 126.56, 126.50, 60.65, 59.35,
+
+
7.57, 57.52, 34.19, 13.69. HR-MS (ESI ) m/z: 366.0834 [M+H] . Found: 366.0830
+
[
M+H] .
4
3
.3.5.
Data
for
diallyl-dithiocarbamic
acid
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (3e): yellow solid
1
(75.0%); mp 95-96 °C; H NMR (400 MHz, CDCl ) δ 8.10-8.12 (m, 2H, ArH),
3
7
4
.72-7.75 (m, 2H, ArH), 5.81-5.91(m, 2H, 2CH=CH )，5.20-5.28 (m, 4H, 2CH=CH ),
2 2
.68 (m, 2H, NCH ), 4.65 (s, 2H, CH S), 4.31 (m, 2H, NCH ), 2.35 (s, 3H, C=CCH ).
2
2
2
3
1
3
C NMR (100 MHz, CDCl ) δ 196.95, 184.72, 183.83, 146.51, 141.44, 133.65,
3
1
1
4
3
33.62, 132.19, 131.92, 130.98, 130.92, 130.31, 126.48, 118.74, 56.86, 53.71, 34.25,
+
+
+
3.65. HR-MS (ESI ) m/z: 358.0936 [M+H] . Found: 358.0934 [M+H] .
.3.6. Data for morpholine-4-carbodithioic
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (3f): yellow solid
acid
1
(86.4%); mp 132-133 °C; H NMR (400 MHz, CDCl ) δ 8.11-8.14 (m, 2H, ArH),
3
7
.74-7.76 (m, 2H, ArH), 4.68 (s, 2H, CH S), 4.32 (m, 2H, NCH ), 3.99 (m, 2H,
2 2
13
NCH ), 3.78 (m, 4H,CH CH ), 2.38 (s, 3H, C=CCH ). C NMR (100 MHz, CDCl ) δ
2
2
2
3
3
1
1
96.44, 184.71, 183.93, 146.54, 141.26, 133.76, 133.71, 132.15, 131.86, 126.56,
+
+
26.50, 66.26, 66.17, 53.63, 33.70, 13.74. HR-MS (ESI ) m/z: 348.0728 [M+H] .
+
Found: 348.0727 [M+H] .
4
.3.7.
Data
for
thiomorpholine-4-carbodithioic
acid
3
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (3g): yellow solid
1
(88.4%); mp 157-158 °C; H NMR (400 MHz, CDCl ) δ 8.12-8.13 (m, 2H, ArH),
3
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7
.73-7.75 (m, 2H, ArH), 4.67 (s, 2H, CH S), 4.55 (t, 2H, NCH ), 4.36 (t, 2H,
2
2
1
3
NCH ),2.77 (s, 4H, CH SCH ), 2.37 (s, 3H, C=CCH ). C NMR (100 MHz, CDCl )
2
2
2
3
3
δ 195.78, 184.68, 183.91, 146.54, 141.27, 133.74, 133.69, 132.16, 131.87, 126.54,
+
+
1
26.49, 60.19, 54.19, 33.92, 27.27, 13.71. HR-MS (ESI ) m/z: 340.0500 [M+H] .
+
Found: 364.1151 [M+H] .
4
.3.8.
Data
for
2,6-dimethyl-morpholine-4-carbodithioic
acid
3
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (3h): yellow solid
1
(76.5%); mp 141-142 °C; H NMR (400 MHz, CDCl ) δ 8.11-8.14 (m, 2H, ArH),
3
7
.73-7.76 (m, 2H, ArH), 5.48 (m, 1H, NCH CH), 4.68 (s, 2H, CH S), 4.42 (m, 1H,
2 2
NCH CH), 3.67 (t, 2H, NCH ), 2.83 (t, 2H, NCH ), 2.37 (s, 3H, C=CCH ), 1.25 (d,
2
2
2
3
13
3
1
3
H, CHCH ), 1.24 (d, 3H, CHCH ). C NMR (100 MHz, CDCl ) δ 195.87, 184.70,
3 3 3
83.97, 146.52, 141.33, 133.75, 133.69, 132.16, 131.86, 126.55, 126.49, 71.33, 71.30,
+
+
3.65, 18.56, 13.71. HR-MS (ESI ) m/z: 376.1041 [M+H] . Found: 376.1039
+
[
M+H] .
4
3
.3.9.
Data
for
thiazolidine-3-carbodithioic
acid
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (3i): yellow solid
1
(76.3%); mp 148-149 °C; H NMR (400 MHz, CDCl ) δ 8.11-8.14 (m, 2H, ArH),
3
7
.73-7.76 (m, 2H, ArH), 5.07 (s, 1H, NCH S), 4.70 (s, 1H, NCH S), 4.67 (s, 2H,
2 2
CH S), 4.35 (t, 1H, NCH CH S), 3.98 (t, 1H, NCH CH S), 3.21 (t, 1H,
2
2
2
2
2
1
3
NCH CH S),3.14 (t, 1H, NCH CH S), 2.38 (s, 3H, C=CCH ). C NMR (100 MHz,
2
2
2
2
3
CDCl ) δ 192.87, 184.80, 183.98, 146.43, 141.25, 133.78, 133.71, 132.16, 131.83,
3
+
1
[
26.56, 126.49, 56.61, 52.58, 33.82, 31.18, 13.78. HR-MS (ESI ) m/z: 350.0343
M+H] . Found: 350.0339 [M+H] .
+
+
4
3
.3.10.
Data
for
pyrrolidine-1-carbodithioic
acid
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (3j): yellow solid
1
(91.0%); mp 171-172 °C; H NMR (400 MHz, CDCl ) δ 8.11-8.12 (m, 2H, ArH),
3
7
2
.72-7.74 (m, 2H, ArH), 4.68 (s, 2H, CH S), 3.97 (t, 2H, NCH ), 3.64 (t, 2H, NCH ),
2 2 2
.38 (s, 3H, C=CCH ), 2.05-2.12 (m, 2H, CH CH CH CH ), 1.97-2.03 (m, 2H,
3
2
2
2
2
13
CH CH CH CH ). C NMR (100 MHz, CDCl ) δ 191.67, 184.80, 184.02, 146.18,
2
2
2
2
3
1
2
4
3
41.82, 133.64, 133.60, 132.20, 131.92, 126.48, 126.45, 55.37, 50.52, 33.19, 26.11,
+
+
+
4.25, 13.72. HR-MS (ESI ) m/z: 332.0779 [M+H] . Found: 332.0777 [M+H] .
.3.11. Data for piperidine-1-carbodithioic
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (3k): yellow solid
acid
1
(88.0%); mp 146-147 °C; H NMR (400 MHz, CDCl ) δ 8.10-8.12 (m, 2H, ArH),
3
7
.72-7.74 (m, 2H, ArH), 4.66 (s, 2H, CH S), 4.25 (m, 4H, CH NCH ), 2.37 (s, 3H,
2 2 2
1
3
C=CCH ), 1.72 (m, 6H, CH CH CH ). C NMR (100 MHz, CDCl ) δ 194.53, 192.77,
3
2
2
2
3
1
3
84.77,183.94, 146.38, 141.68, 133.63, 133.60, 132.20, 131.94, 126.47, 53.47, 51.41,
+
+
3.90, 25.74, 24.22, 13.66. HR-MS (ESI ) m/z: 346.0936 [M+H] . Found: 346.0932
+
[
M+H] .
4
.3.12.
Data
for
4-methyl-piperazine-1-carbodithioic
acid
3
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (3l): yellow solid
1
(97.2%); mp 124-125 °C; H NMR (400 MHz, CDCl ) δ 8.11-8.12 (m, 2H, ArH),
3
7
.72-7.75 (m, 2H, ArH), 4.66 (s, 2H, CH S), 4.37 (m, 2H, S=CNCH ), 3.96 (m, 2H,
2 2
13
S=CNCH ), 2.52 (m, 4H, CH NCH ), 2.37 (s, 3H, NCH ), 2.35 (s, 3H, C=CCH ). C
2
2
2
3
3
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NMR (100 MHz, CDCl ) δ 195.86, 184.72, 183.93, 146.47, 141.41, 133.70, 133.66,
3
+
1
3
4
3
32.17, 131.89, 126.52, 126.48, 54.34, 45.56, 33.86, 13.71. HR-MS (ESI ) m/z:
+
+
61.1045 [M+H] . Found: 361.1035 [M+H] .
.3.13. Data for 4-isopropyl-piperazine-1-carbodithioic
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester
acid
(3m): yellow solid
1
(
75.7%); mp 157-158 °C; H NMR (400 MHz, CDCl ) δ 8.11-8.12 (m, 2H, ArH),
3
7
.72-7.75 (m, 2H, ArH), 4.66 (s, 2H, CH S), 4.37 (m, 2H, S=CNCH ), 3.93 (m, 2H,
2 2
S=CNCH ), 2.76 (m, 1H, NCH), 2.61 (m, 4H, CH NCH ), 2.37 (s, 3H, NCH ), 1.07
2
2
2
3
13
(
s, 3H, CHCH ), 1.06 (s, 3H, CHCH ). C NMR (100 MHz, CDCl ) δ 195.35, 184.75,
3 3 3
1
1
4
3
83.94, 146.45, 141.48, 133.69, 133.64, 132.18, 131.90, 126.51, 54.35, 48.13, 33.78,
+
+
+
8.39, 13.71. HR-MS (ESI ) m/z: 389.1358 [M+H] . Found: 389.1348 [M+H] .
.3.14. Data for 4-acetyl-piperazine-1-carbodithioic
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (3n): yellow solid
acid
1
(92.8%); mp 157-158 °C; H NMR (400 MHz, CDCl ) δ 8.10-8.12 (m, 2H, ArH),
3
7
2
.73-7.75 (m, 2H, ArH), 4.66 (s, 2H, CH S), 4.12-4.18 (m, 4H, CH NCH ), 3.75 (t,
2 2 2
H, S=CNCH ), 3.62 (t, 2H, S=CNCH ),2.36 (s, 3H, O=CCH ), 2.15 (s, 3H,
2
2
3
13
C=CCH ). C NMR (100 MHz, CDCl ) δ 196.75, 184.64, 183.90, 169.30, 146.59,
3
3
1
1
4
3
41.06, 133.81, 133.74, 132.12, 131.82, 126.56, 126.50, 45.20, 40.56, 33.91, 21.36,
+
+
+
3.75. HR-MS (ESI ) m/z: 389.0994 [M+H] . Found: 389.0991 [M+H] .
.3.15. Data for cyclohexyl-methyl-dithiocarbamic
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (3o): yellow solid
acid
1
(75.0%); mp 128-129 °C; H NMR (400 MHz, CDCl ) δ 8.11-8.12 (m, 2H, ArH),
3
7
.72-7.74 (m, 2H, ArH), 4.66 (s, 2H, CH S), 3.17-3.42 (d, 3H, NCH ), 2.36 (s, 3H,
2 3
13
C=CCH ), 1.11-1.87 (m, 11H, NCHCH CH CH CH CH ). C NMR (100 MHz,
3
2
2
2
2
2
CDCl ) δ 196.02, 184.75, 183.90, 146.53, 146.50, 141.60, 133.63, 133.60, 132.21,
3
1
31.96, 126.48, 62.92, 62.12, 37.75, 33.91, 33.69, 30.22, 29.42, 25.47, 25.17, 13.65.
+
+
+
HR-MS (ESI ) m/z: 374.1249 [M+H] . Found: 374.1247 [M+H] .
4
3
.3.16.
Data
for
Methyl-dithiocarbamic
acid
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (3p): yellow solid
1
(90.7%); mp 106-107°C. H NMR (400 MHz, CDCl ) δ 8.11-8.13 (m, 2H, ArH), 8.04
3
（
s, 1H，NH），7.75-7.77 (m, 2H, ArH), 4.40 (s, 2H, CH S), 3.29 (s, 3H, NCH ), 2.33
2 3
13
(s, 3H, C=CCH ). C NMR (100 MHz, CDCl ) δ 196.22, 185.22, 184.47, 146.40,
3
3
1
41.73, 134.19, 133.81, 132.10, 131.60, 126.71, 126.62, 34.03, 31.09, 13.63. HR-MS
+
+
+
(ESI ) m/z: 292.0466 [M+H] . Found: 292.0457 [M+H] .
4
3
.3.17.
Data
for
Pyridin-2-ylmethyl-dithiocarbamic
acid
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (3q): yellow solid
1
(72.5%); mp 149-150°C. H NMR (400 MHz, CDCl ) δ 8.54 (d,1H,NH), 8.09-8.12
3
(
m, 2H, ArH), 7.73-7.75 (m, 2H, ArH), 7.25-7.73(m,4H,PyH),5.02 (d, 2H, CH NH),
2
13
4
1
1
3
4
3
.62(s, 2H, CH S), 2.38 (s, 3H, C=CCH ). C NMR (100 MHz, CDCl ) δ 196.13,
2 3 3
84.75, 184.20, 153.96, 148.76, 146.00, 141.70, 137.11, 133.81, 133.69, 132.13,
+
31.80, 126.55, 126.52, 122.85, 122.22, 51.27, 31.67, 13.72. HR-MS (ESI ) m/z:
+
+
69.0731 [M+H] . Found: 369.0719 [M+H] .
.3.18. Data for Pyridin-3-ylmethyl-dithiocarbamic
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (3r): yellow solid
acid
Data
for
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1
1
(
76.1%); mp 140-141°C. H NMR (400 MHz, CDCl ) H NMR (400 MHz, CDCl ) δ
3
3
1
0.58 (t,1H,NH), 8.48-8.54 (m, 2H, PyH), 7.99-8.02 (m, 2H, ArH), 7.84-8.86 (m, 2H,
ArH), 7.71-7.73(d,1H,PyH), 7.37-7.40(m,1H,PyH),4.86 (d, 2H, CH NH), 4.49(s, 2H,
2
13
CH S), 2.21 (s, 3H, C=CCH ). C NMR (100 MHz, CDCl ) δ 196.82, 184.60, 183.79,
2
3
3
1
1
49.57, 148.95, 145.79, 141.40, 136.08, 134.64, 134.56, 133.24, 132.13, 131.77,
+
+
26.56, 126.44, 124.03, 47.94, 31.34, 13.81. HR-MS (ESI ) m/z: 369.0731 [M+H] .
+
Found: 369.0722 [M+H] .
4
.3.19.
Data
for
Pyridin-4-ylmethyl-dithiocarbamic
acid
3
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (3s): yellow solid
1
(78.5%); mp 131-132 °C. H NMR (400 MHz, CDCl ) δ 8.68 (d,1H,NH),
3
8
.55-8.57(m,
2H,
ArH),
8.11-8.13(m,2H,PyH),7.72-7.77(m,
2H,
ArH),
1
3
7
.28(s,2H,PyH),5.01(d, 2H, CH NH), 4.43(s, 2H, CH S), 2.33(s, 3H, C=CCH ). C
2
2
3
NMR (100 MHz, CDCl ) δ 196.96, 185.11, 184.37, 149.99, 146.59, 145.39, 141.42,
3
1
34.27, 133.86, 132.07, 131.49, 126.74, 126.56, 122.74, 49.34,31.22, 13.65. HR-MS
+
+
+
(ESI ) m/z: 369.0731 [M+H] . Found: 369.0722 [M+H] .
4
3
.3.20.
Data
for
Benzyl-dithiocarbamic
acid
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (3t): yellow solid
1
(74.8%); mp 113-114 °C. H NMR (400 MHz, CDCl ) δ 8.01-8.12(m, 3H, PhH),
3
7
2
1
1
.73-7.76 (m, 2H, ArH), 7.33-7.38(m, 5H, ArH,NH), 4.95 (d, 2H, CH NH), 4.44(s,
2
1
3
H, CH S), 2.34 (s, 3H, C=CCH ). C NMR (100 MHz, CDCl ) δ 195.66, 184.82,
2
3
3
84.51, 146.32, 141.60, 135.93, 134.08, 133.74, 132.09, 131.56, 128.88, 128.46,
+
+
28.14, 126.64, 126.60, 51.35, 31.19, 13.65. HR-MS (ESI ) m/z: 368.078 [M+H] .
+
Found: 368.077 [M+H] .
4
.3.21.
Data
for
Piperidin-1-yl-dithiocarbamic
acid
3
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (3u): yellow solid
1
(90.9%); mp 166-167 °C. H NMR (400 MHz, CDCl ) δ 8.1 9（ s, 1H，NH），8.10-8.12
3
(
2
m, 2H, ArH), 7.72-7.74 (m, 2H, ArH), 4.51 (s, 2H, CH S), 3.18 (m, 4H, CH NCH ),
.34 (s, 3H, C=CCH )， 1.68 (m, 6H, CH CH CH ). C NMR (100 MHz, CDCl )
3 2 2 2 3
2
2
2
13
δ201.01, 184.80, 183.88, 146.34, 142.05, 133.61, 132.17, 131.94, 126.48, 56.06,
+
+
3
0.51, 25.22, 22.87, 13.59. HR-MS (ESI ) m/z: 361.1044 [M+H] . Found: 361.1031
+
[
M+H] .
4
3
.3.22.
Data
for
(4-Methyl-piperazin-1-yl)-dithiocarbamic
acid
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (3v): yellow solid
1
(95.9%); mp 162-163 °C. H NMR (400 MHz, CDCl ) δ 11.1 1（ s, 1H，NH），8.00-8.01
3
(
2
m, 2H, ArH), 7.85-7.87 (m, 2H, ArH), 4.30 (s, 2H, CH S), 2.69-2.79（m,8H，
NCH CH ），2.20 (s, 3H, C=CCH ), 2.14 (d, 3H, NCH ). C NMR (100 MHz,
2 2 3 3
2
1
3
DMSO) δ 199.97, 183.85, 179.14, 144.78, 143.38, 136.90, 135.12, 134.92, 127.65,
+
1
3
4
3
24.81, 122.46, 122.40, 54.30, 53.41, 53.33, 45.50, 13.06. HR-MS (ESI ) m/z:
+
+
76.1153 [M+H] . Found: 374.1143 [M+H] .
.3.23. Data for (2-Diethylamino-ethyl)-dithiocarbamic
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (3w): yellow solid
acid
1
(
73.3%); mp 149-150 °C. H NMR (400 MHz, DMSO) δ 10.41（s, 1H，NH），
8
.01-8.03 (m, 2H, ArH), 7.85-7.88 (m, 2H, ArH), 4.94 (s, 2H, CH S),
2
3
.99(m,2H,NHCH ), 3.31(m,2H,NHCH CH ), 3.30(q,4H,CH CH NCH CH ),2.23 (s,
2
2
2
3
2
2
3
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1
3
3
1
4
3
4
3
H, C=CCH )， 1.22 (t, 6H, 2CH CH ). C NMR (100 MHz, DMSO) δ 197.34,
3 2 2
84.60, 183.74, 145.89, 141.16, 134.68, 134.60, 132.14, 131.79, 126.58, 126.46,
8.31, 47.19, 41.62, 31.35, 13.80, 8.88. HR-MS (ESI ) m/z: 377.1357 [M+H] . Found:
+
+
+
77.1348 [M+H] .
.4.
General
procedure
for
preparation
of
dithiocarbamic
acid
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-yl ester (4)
Carbon disulfide (60 µL, 1 mmol) and amine (1 mmol) were added to CH CN (3 mL)
3
and the resulting solution was stirred for 30 minutes. 2-methyl-[1, 4] naphthoquinone
(1) (110 mg, 0.5 mmol) was added in portions at frequent intervals. The reaction
mixture was then kept at room temperature until compound 1 was absent (as
evaluated by TLC). The reaction mixture was concentrated in vacuo, diluted with H O,
2
and extracted with CH Cl . The combined organic fractions were washed with brine,
2
2
dried (Na SO ), and concentrated under reduced pressure. Purification of the crude
2
4
residue by column chromatography (petroleum ether/ CH Cl ) afforded the compound
2
2
of interest.
4
3
1
.4.1.
Data
for
dipropyl-dithiocarbamic
acid
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-yl ester (4a): yellow solid (65.1%); mp
29-130 °C; H NMR (400 MHz, CDCl ) δ 8.12-8.16 (m, 2H, ArH), 7.73-7.76 (m, 2H,
1
3
ArH), 3.80-3.89 (m, 4H, CH NCH ), 2.40 (s, 3H, C=CCH ), 1.97 (m, 2H, CH CH ),
2
2
3
2
3
1
3
1
.80 (m, 2H, CH CH ), 1.08 (m, 3H, CH CH ), 0.95 (m, 3H, CH CH ). C NMR
2 3 2 3 2 3
(
100 MHz, CDCl ) δ 191.34, 184.09, 180.30, 151.16, 142.25, 133.80, 133.50, 132.15,
3
+
1
3
4
3
1
27.20, 126.24, 57.37, 56.19, 21.28, 19.71, 15.95, 11.28, 11.19. HR-MS (ESI ) m/z:
+
+
48.1092 [M+H] . Found: 348.1088 [M+H] .
.4.2. Data for morpholine-4-carbodithioic
acid
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-yl ester (4b): yellow solid (54.2%); mp
1
40-141 °C; H NMR (400 MHz, CDCl ) δ 8.13-8.18 (m, 2H, ArH), 7.75-7.78 (m, 2H,
3
ArH), 4.15-4.27 (m, 4H, CH OCH ), 3.86-3.87 (m, 4H, CH NCH ), 2.41 (s, 3H,
2
2
2
2
13
C=CCH ). C NMR (100 MHz, CDCl ) δ 192.39, 183.85, 180.10, 152.18, 141.55,
3
3
1
33.94, 132.91, 132.11, 127.24, 126.76, 66.26, 66.22, 51.96, 51.07, 16.06. HR-MS
+
+
+
(ESI ) m/z: 334.0572 [M+H] . Found: 334.0568 [M+H] .
4
3
1
2
1
1
2
.4.3. Data for piperidine-1-carbodithioic
acid
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-yl ester (4c): yellow solid (65.4%); mp
39-140 °C; 1H NMR (400 MHz, CDCl3) δ 8.13-8.17 (m, 2H, ArH), 7.74-7.76 (m,
H, ArH), 4.21 (m, 2H, NCH2), 4.08 (m, 2H, NCH2), 2.41 (s, 3H, C=CCH3),
.77-1.87 (m, 6H, CH2CH2CH2). 13C NMR (100 MHz, CDCl3) δ 190.63, 184.07,
80.36, 151.21, 141.96, 133.83, 133.53, 133.10, 132.14, 127.22, 126.66, 53.34, 52.44,
6.37, 25.28, 24.03, 15.94. HR-MS (ESI+) m/z: 332.0779 [M+H]+. Found: 332.0775
[
M+H]+.
4
3
.4.4.
Data
for
4-acetyl-piperazine-1-carbodithioic
acid
-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-yl ester (4d): yellow solid (69.5%);
1
mp 119-120 °C; H NMR (400 MHz, CDCl ) δ 8.11-8.18 (m, 2H, ArH), 7.75-7.78 (m,
3
2
H, ArH), 4.18-4.30 (m, 4H, CH NCH ), 3.70-3.88 (m, 4H, CH NCH ), 2.41 (s, 3H,
2 2 2 2
1
3
C=CCH ), 2.19 (s, 3H, O=CCH ). C NMR (100 MHz, CDCl ) δ183.79, 180.02,
3
3
3
1
69.38, 152.39, 141.42, 133.96, 132.82, 132.09, 127.24, 126.80, 50.78, 40.69, 29.69,
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+
+
+
2
1.37, 16.10. HR-MS (ESI ) m/z: 375.0837 [M+H] . Found: 375.0833 [M+H] .
4
.5. Procedure for preparation of 2-bromomethyl-[1,4]naphthoquinone (5)
Azodiisobutyronitrile (0.05 g, 0.3 mmol) was added to a well stirred solution of
methylnaphthoquinone 1 (1 g, 5.9 mmol) in acetic anhydride (10 mL).
N-bromosuccinimide (1.05 g, 5.9 mmol) was then added in portions at frequent
intervals. The reaction mixture was refluxed for 20 min. The reaction mixture was
poured on ice and extracted with ethyl acetate. The combined organic fractions were
washed with brine, dried (Na SO ), and concentrated under reduced pressure.
2
4
Purification of the crude residue by column chromatography (petroleum ether/ ethyl
1
acetate) afforded compound 5 (yellow solid). The yield of this reaction was 62.8%. H
NMR (400 MHz, CDCl ) δ 8.10-8.18 (m, 2H, ArH), 7.70-7.81 (m, 2H, ArH), 7.13 (s,
3
1
4
1
H, C=CH), 4.42 (s, 2H, CH Br).
2
.6.
General
Procedure
for
preparation
of
dithiocarbamic
acid
,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (6)
Carbon disulfide (60 µL, 1 mmol) and amine (1 mmol) were added to CH Cl (5 mL)
2
2
and the resulting solution was stirred for 30 minutes. 2-Bromomethyl-[1, 4]
naphthoquinone (5) (250 mg, 1 mmol) was added in portions at frequent intervals.
The reaction mixture was then kept at room temperature until compound 5 was absent
(
as evaluated by TLC). The reaction mixture was diluted with H O, and extracted with
2
CH Cl . The combined organic fractions were washed with brine, dried (Na SO ), and
2
2
2
4
concentrated under reduced pressure. Purification of the crude residue by column
chromatography (petroleum ether/ ethyl acetate) afforded compound of interest.
4
1
.6.1.
Data
for
diethyl-dithiocarbamic
acid
,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (6a): yellow solid (75.3%); mp
1
118-119 °C; H NMR (400 MHz, CDCl ) δ 8.07-8.14 (m, 2H, ArH), 7.75-7.79 (m, 2H,
3
ArH), 7.23 (s, 1H, C=CH), 4.59 (s, 2H, CH S), 4.03 (q, 2H, NCH ), 3.78 (q, 2H,
2
2
13
NCH ), 1.27-1.34 (m, 6H, 2CH CH ). C NMR (100 MHz, CDCl ) δ 193.65, 185.11,
2
2
3
3
1
3
84.76, 146.42, 135.88, 133.90, 133.72, 132.21, 132.08, 126.63,126.20, 50.14, 46.84,
+
+
4.42, 12.63, 11.54. HR-MS (ESI ) m/z: 320.0779 [M+H] . Found: 320.0770
+
[
M+H] .
4
1
1
.6.2.
Data
for
morpholine-4-carbodithioic
acid
,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (6b) yellow solid (68.9%); mp
37-138 °C; H NMR (400 MHz, CDCl ) δ 8.07-8.15 (m, 2H, ArH), 7.75-7.78 (m, 2H,
1
3
ArH), 7.23 (s, 1H, C=CH), 4.61 (s, 2H, CH S), 4.27 (m, 2H, OCH ), 4.01 (m, 2H,
2
2
1
3
OCH ), 3.78 (m, 4H,CH NCH ). C NMR (100 MHz, CDCl ) δ 195.65, 184.97,
2
2
2
3
1
3
84.66, 145.92, 136.06, 133.97, 133.77, 132.19, 132.03, 126.64, 126.24, 66.25, 66.17,
+
+
4.23, 29.67, 22.68. HR-MS (ESI ) m/z: 334.0572 [M+H] . Found: 334.0567
+
[
M+H] .
4
1
1
.6.3.
Data
for
piperidine-1-carbodithioic
acid
,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (6c) yellow solid (70.9%); mp
22-123 °C; H NMR (400 MHz, CDCl ) δ 8.07-8.14 (m, 2H, ArH), 7.74-7.77 (m, 2H,
1
3
ArH), 7.23 (s, 1H, C=CH), 4.59 (s, 2H, CH S), 4.03 (m, 2H, CH NCH ), 3.78 (m, 2H,
2
2
2
1
3
CH NCH ), 1.27-1.34 (m, 6H, CH CH CH ). C NMR (100 MHz, CDCl ) δ 193.65,
2
2
2
2
2
3
1
85.60, 184.78, 146.41, 135.92, 133.90, 133.71, 132.20, 132.08, 126.63, 126.20，
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+
+
5
4.62, 53.58, 34.44, 25.97, 25.48, 24.21. HR-MS (ESI ) m/z: 332.0779 [M+H] .
+
Found: 332.0770 [M+H] .
4
1
1
.6.4.
Data
for
4-phenyl-piperazine-1-carbodithioic
acid
,4-dioxo-1,4-dihydro-naphthalen-2-ylmethyl ester (6d): yellow solid (72.7%); mp
44-145 °C; H NMR (400 MHz, CDCl ) δ 8.08-8.16 (m, 2H, ArH), 7.75-7.78 (m, 2H,
1
3
ArH), 6.93-7.33 (m, 6H, PhH, C=CH), 4.63 (s, 2H, CH S), 4.49 (m, 2H, NCH ), 4.15
2
2
13
(
m, 2H, NCH ), 3.33 (m, 4H,CH NCH ). C NMR (100 MHz, CDCl ) δ 196.09,
2 2 2 3
1
1
84.71, 183.95, 146.52, 141.34, 133.74, 133.68, 132.18, 131.89, 129.36, 126.55,
+
+
26.50, 120.82, 116.44, 48.87, 33.86, 13.74. HR-MS (ESI ) m/z: 409.1045 [M+H] .
+
Found: 409.1037 [M+H] .
.7. Procedure for preparation of 2-chloromethyl-1,4-dimethoxy-naphthalene (8)
4
The compound (1,4-dimethoxy-naphthalen-2-yl)-methanol (7) (436 mg, 2 mmol) in
CH Cl (10 mL) was placed in a 50 mL round-bottomed flask, and thionyl chloride
2
2
(
0.22 mL, 3 mmol) in CH Cl (5 mL) was added in portions at frequent intervals,
2 2
keeping the temperature below 0℃. The reaction mixture was then refluxed for 2 h,
and diluted with H O, followed by extracted with CH Cl . The combined organic
2
2
2
fractions were washed with brine, dried (Na SO ), and concentrated under reduced
2
4
pressure. Purification of the crude residue by column chromatography (petroleum
ether/ ethyl acetate) afforded compound 8 (yellow solid). The yield of this reaction
1
was 78.2%. H NMR (400 MHz, CDCl ) δ 8.06-8.28 (m, 2H, ArH), 7.52-7.61 (m, 2H,
3
ArH), 6.79 (s, 1H, C=CH), 4.88 (s, 2H, CH Cl), 4.03 (s, 3H, OCH ), 4.02 (s, 3H,
2
3
OCH3).
4
1
.8.
Procedure
for
preparation
of
dipropyl-dithiocarbamic
acid
,4-dimethoxy-naphthalen-2-ylmethyl ester (9)
Carbon disulfide (60 µL, 1 mmol) and dipropyl amine (136µL, 1 mmol) were added
to CH Cl (5 mL) and the resulting solution was stirred for 30 minutes.
2
2
2
-chloromethyl-1,4-dimethoxy-naphthalene (8) (377mg, 1 mmol) was added in
portions at frequent intervals. The reaction mixture was then kept at room temperature
until absence of compound 8 (checked by TLC). The reaction mixture was diluted
with H O, and extracted with CH Cl . The combined organic fractions were washed
2
2
2
with brine, dried (Na SO ), and concentrated under reduced pressure. Purification of
2
4
the crude residue by column chromatography (petroleum ether/ ethyl acetate) afforded
1
the compound 9 (white solid). The yield of this reaction was 76.0%; mp 70-71 °C; H
NMR (400 MHz, CDCl ) δ 7.47-8.24 (m, 4H, ArH), 6.88 (s, 1H, C=CH), 4.80 (s, 2H,
3
CH S), 3.97-3.98 (m, 8H, 2OCH , NCH ), 3.62-3.68 (m, 2H, NCH ), 1.73-1.85 (m,
2
3
2
2
13
4
1
1
H, 2CH CH CH ), 0.91-1.01(m, 6H, 2CH CH CH ). C NMR (100 MHz, CDCl ) δ
2 2 3 2 2 3 3
96.25, 151.94, 148.11, 128.45, 126.70, 126.40, 125.56, 123.95, 122.43，121.98,
+
05.54, 62.78, 56.89, 55.72, 54.37, 37.32, 20.70, 19.70, 11.25, 11.19. HR-MS (ESI )
+
+
m/z: 378.1561 [M+H] . Found: 378.1553 [M+H] .
.9. Purification of recombinant pyruvate kinase isoforms
4
Human cDNA for PKM2 or PKM1 was cloned into pET28a with a N-terminal His tag
and purified from Escherichia coli strain BL21 (Invitrogen) using Ni-Agarose beads
(Qiagen) as described previously.[6, 24] The PKL was purchased from Sino
Biological Inc.
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.10. PKM2 activity assay
Pyruvate kinase activity was measured with a fluorescent pyruvate kinase-lactate
dehydrogenase coupled assay as described previously [15].All compounds were tested
in a kinetic mode by coupling the generation of pyruvate by pyruvate kinase to the
depletion of NADH through lactate dehydrogenase. For PKM2, 40 µL of buffer (50
mM Tris–HCl, pH 7.5, 10 mM KCl, 5 mM MgCl ), 1 µL of compound and 5 µL of
2
enzyme solution were dispensed into Corning black solid 96-well plates and
incubated for 15 min. 55 µL of substrate mix (final concentration, 0.5 mM PEP, 4.0
mM ADP, 0.12 mM NADH, 0.25 mM FBP and 1 unit LDH) was then added, and the
plates were placed in a FlexStation 3 (Molecular Devices), followed by determination
of NADH fluorescence at 30 s exposure intervals for 3 to 6 min. Data was collected
on the FlexStation 3.
4
.11. Cell culture
Cell lines (HCT116, Hela, H1299, BEAS-2B) were cultured in RPMI 1640 containing
9
% fetal bovine serum (FBS) at 37°C in 5% CO2.
4
.12. Cell viability experiments
Cell viability was detected with the MTS assay (Promega) according to the
manufacturer’s instructions. Briefly, 5000 cells in per well were plated in 96-well
plates. After incubated for 12 h, the cells were treated with different concentration of
tested compound or DMSO (as negative control) for 48 h. Then 20 µL MTS was
added in per well and incubated at 37°C for 3h. Absorbance of each well was
determined by a microplate reader (Flexstation 3) at a 490 nm wavelength. The IC50
values were calculated using Prism Graphpad software of the triplicate experiment.
Acknowledgments
This study was supported by the National Key Research and Development Program of
China (2016YFA0500302), National Natural Science Foundation of China (81402777)
and the China Postdoctoral Science Foundation (2014M560026 and 2015T80028).
References
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1] G. Kroemer, J. Pouyssegur, Tumor cell metabolism: cancer's Achilles' heel, Cancer cell, 13 (2008)
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[
Fleming, S.L. Schreiber, L.C. Cantley, The M2 splice isoform of pyruvate kinase is important for
cancer metabolism and tumour growth, Nature, 452 (2008) 230-233.
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[
7] Z.X. Liu, L. Hong, S.Q. Fang, G.H. Tan, P.G. Huang, Z. Zeng, X. Xia, X.X. Wang, Overexpression
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8] W. Yang, Z. Lu, Pyruvate kinase M2 at a glance, Journal of cell science, 128 (2015) 1655-1660.
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Highlights
ò
The novel dithiocarbamate substituted naphthoquinone derivatives were
designed and synthesized.
ò All compounds were evaluated for in vitro PKM2 inhibitory activity and
antiproliferative activity.
ò
ò
Compound 3k showed more potent PKM2 inhibitory activity than the positive
control shikonin.
Compound 3k exhibited nanomolar antitumor activity toward PKM2 high
expression cancer cell lines.