New pyridin-3-ylmethyl carbamodithioic esters activate pyruvate kinase M2 and potential anticancer lead compounds

Article abstract of DOI:10.1016/j.bmc.2015.05.041

Pyruvate kinase M2 (PKM2) is a key protein responsible for cancer's Warburg effect. Activation of PKM2 may alter aberrant metabolism in cancer cells, which suggests PKM2 as a tumor selective therapeutic target. In this paper, the lead compound 8 was first discovered as a new kind of PKM2 activator from a random screening of an in-house compound library. Then, a series of lead compound 8 analogs were designed, synthesized and evaluated for their activation of PKM2 and anticancer activities. 7-Azaindole analog 32 was identified as the most potent PKM2 activator. Compounds with potent enzyme activity also exhibited selective anti-proliferation activity on cancer cell lines HCT116, Hela and H1299 compared with non-tumor cell line BEAS-2B. The structure-activity relationships of these compounds were supported by molecular docking results. Preliminary pharmacological studies also showed that compound 32 arrests the cell cycle at the G2/M phase in HCT116 cell line.

Full text of DOI:10.1016/j.bmc.2015.05.041

Accepted Manuscript
New pyridin-3-ylmethyl carbamodithioic esters activate pyruvate kinase M2
and potential anticancer lead compounds
Yu Zhang, Bin Liu, Xingyu Wu, Ridong Li, Xianling Ning, Yu Liu, Zhenming
Liu, Zemei Ge, Runtao Li, Yuxin Yin
PII:
S0968-0896(15)00451-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.05.041
BMC 12337
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
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18 May 2015
20 May 2015
Please cite this article as: Zhang, Y., Liu, B., Wu, X., Li, R., Ning, X., Liu, Y., Liu, Z., Ge, Z., Li, R., Yin, Y., New
pyridin-3-ylmethyl carbamodithioic esters activate pyruvate kinase M2 and potential anticancer lead compounds,
Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.05.041
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New pyridin-3-ylmethyl carbamodithioic
esters activate pyruvate kinase M2 and
potential anticancer lead compounds
Leave this area blank for abstract info.
Yu Zhang a, †, Bin Liu b, †, Xingyu Wu a, Ridong Li a, b, Xianling Ning a, Yu Liu a, Zhenming Liu b, Zemei Ge b,
Runtao Li b, *, Yuxin Yin a, b,
*
a Institute of Systems Biomedicine, Department of Pathology, School of Basic Medical Sciences, Beijing Key
Laboratory of Tumor Systems Biology, Peking University Health Science Center, Beijing 100191, China.
b State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China.

Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
New pyridin-3-ylmethyl carbamodithioic esters activate pyruvate kinase M2 and
potential anticancer lead compounds
Yu Zhang a, †, Bin Liu b, †, Xingyu Wu a, Ridong Li a, b, Xianling Ning a, Yu Liu a, Zhenming Liu b, Zemei
Ge b, Runtao Li b, *, Yuxin Yin a, b, *
a Institute of Systems Biomedicine, Department of Pathology, School of Basic Medical Sciences, Beijing Key Laboratory of Tumor Systems Biology,
Peking University Health Science Center, Beijing 100191, China.
b State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China.
† These authors contributed equally.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Pyruvate Kinase M2 (PKM2) is a key protein responsible for cancer’s Warburg effect.
Activation of PKM2 may alter aberrant metabolism in cancer cells, which suggests PKM2 as a
tumor selective therapeutic target. In this paper, the lead compound 8 was first discovered as a
new kind of PKM2 activator from a random screening of an in-house compound library. Then, a
series of lead compound 8 analogues were designed, synthesized and evaluated for their
activation of PKM2 and anticancer activities. 7-Azaindole analogue 32 was identified as the
most potent PKM2 activator. Compounds with potent enzyme activity also exhibited selective
anti-proliferation activity on cancer cell lines HCT116, Hela and H1299 compared with non-
tumor cell line BEAS-2B. The structure-activity relationships of these compounds were
supported by molecular docking results. Preliminary pharmacological studies also showed that
compound 32 arrests the cell cycle at the G2/M phase in HCT116 cell line.
Available online
Keywords:
Pyruvate kinase M2
Dithiocarbamic acid ester
Structure-activity relationship
Anti-proliferation
Docking
2009 Elsevier Ltd. All rights reserved.
1. Introduction
Despite their similarity in primary sequences, PKM1 and
PKM2 have different catalytic and regulatory properties. PKM1
One of the most important distinctions between healthy and
cancerous tissues was the difference in metabolism. In normal
cells, glycolysis is induced by hypoxia. Cancer cells, however,
preferentially metabolize glucose by glycolysis, even in an
aerobic environment. The phenomenon is known as Warburg
effect.1, 2 Pyruvate kinase (PK), a key enzyme that catalyzes the
last step of glycolysis is considered to be responsible for such
metabolic alteration.3
has constitutively high enzyme activity. In contrast, PKM2
requires activation by the binding of fructose-1, 6-bis-phosphate
(FBP) at an allosteric site. Additionally, PKM2 can interact with
proteins harboring phosphorylated tyrosine residues, leading to
the release of FBP, which, in turn, reduces the activity of the
enzyme.8 Thus, most of PKM2 exists as less active forms with
low catalytic rate in tumors.9 In cancer cells, independently of
their tissue of origin PKM2 gradually replaces the tissue-specific
PK isoform and becomes a predominant isoform.10 This
interesting phenomenon has raised the question of how PKM2
expression benefits tumor cells.
One hypothesis is that low PKM2 activity facilitating the flux
of glucose carbons into the anabolic pathways help meet cancer
cells’ need for biological building blocks to support the
production of new cells.11 Cancer cells’ favor for the low activity
of PKM2 isoform was further supported by the fact that
replacement of PKM2 with constitutively active PKM1 inhibits
xenograft tumor.12 Furthermore, the PKM2 form with low
activity can translocate to cell nucleus under certain
PK is a tetrameric enzyme encoded by four isozymes (PKL,
PKR, PKM1, and PKM2) that differ in their kinetic properties
and tissue distributions.4-7 PKL and PKR are encoded by the
same gene PKLR but under the control of different tissue-
specific promoters. The two isoforms are expressed in liver and
red blood cells, respectively.7 Most tissues express either PKM1
or PKM2 isoform. PKM1 is expressed in normal differentiated
tissues; whereas PKM2 is predominantly found in highly
proliferating cells including cancer cell lines and tumors.5 PKM1
and PKM2 are two different splicing forms of the same mRNA
transcribed from the PKM gene.6
———
* Corresponding author. E-mail: lirt@bjmu.edu.cn, (R. Li).
* Corresponding author. E-mail: yinyuxin@hsc.pku.edu.cn, (Y. Yin).

circumstance, which can subsequently increases the expression of
c-Myc, GLUT1, LDHA, PTB and PKM2 itself, thereby
promoting the Warburg effect and tumorigenesis.13-16 High
expression of PKM2 in cancer and its low activity form of
Compd.
8
R
Compd.
22
R
Ph
3,4,5-trimethoxy
2-naphthalene
4-ClPh
9
23
4-FPh
1-naphthalene
2-furan
10
11
12
13
14
15
16
17
18
19
20
21
24
tumorigenic function suggest that activating PKM2 is
therapeutic strategy for cancer.
a
4-OHPh
25
4-CNPh
2-thiophene
2-pyrrole
26
Several categories of PKM2 activators (Fig. 1) have been
reported including a series of N,N’-diarylsulfonamides (1),17
thieno[3,2-b]pyrrole[3,2-d]pyridazinones (2),18 2-oxo-N-aryl-
1,2,3,4-tetrahydroquinoline-6-sulfonamides (3),19 quinolone
sulfonamides (4),20 1-(sulfonyl)-5-(arylsulfonyl)indoline (5),21 2-
((1H-benzo[d]imidazol-1-yl)methyl)-4H-pyrido[1,2-a]pyrimidin-
4-ones (6)22 and 3-(trifluoromethyl)-1H-pyrazole-5-carboxamide
(7).23 Even though, the above compounds have no anti-
proliferative effects on tumor cell lines except that serine was
deprived from the medium. In this article, we describe the
discovery of new kinds of dithiocarbamatic acid esters as PKM2
selective activators with significant anti-proliferative effects on
tumor cell lines.
4-NO2Ph
4-(CH3)2NPh
4-BnOPh
4-OMePh
4-CF3Ph
2-OMePh
2-ClPh
27
2-thiazole
28
3-pyridine
29
3-coumarin
3-indole
30
31
3-azaindole
3-imidazo[1,2-a]pyridine
PhCH=CH
32
33
3,4-dichloro
3,4-difluo
34
35
3-pyrrole
Fig. 2. Lead compound 8 and strategy for its structural
modification
Scheme 1. Synthesis of compounds 8-35. Reagents and conditions: (a)
(HCHO)n, CF3COOH·(iPr)2NH, DMF, 90 oC, 24 h; (b) 3-
(aminomethyl)pyridine, CS2, TEA, DMF, rt, 12 h.
2.2. Biological evaluations in vitro
The cell-free enzymatic assay of PKM2 activity was
performed as described in Experiment Section. All the target
compounds were subjected to this assay to evaluate their affinity
toward isolated PKM2 kinase. It is clear that a slight change of
aryl group in compound 8 could induce obviously influence on
the activity (Table 1). The introduction of the para-substitutents,
including electron withdrawing or donating groups, on the
benzene ring of compound 8 led to the decrease of activity or
opposite activities (9, 10, 14, 17, 20-22). However, NO2 or CN
substituted derivatives (12, AC50 = 4.39 µM; 13, AC50 = 2.76
µM) showed 2-3 fold increase of activities compared with lead
compound 8, which could be explained by the function of
hydrogen bond receptor of them. Notably, when the benzene ring
in compound 8 was replaced with the heterocycles, we observed
the similar trend that only the heterocyle derivatives containing
hydrogen bond receptor (25, 28, 29, 32) showed remarkable
activities (AC50 = 4.30, 3.56, 3.70, 1.00 µM). More interestingly,
the activity of the azaindole derivatives 32 (AC50 = 1.00 µM) is
greatly higher than that of indole derivatives 31 (AC50 > 20 µM).
A feasible explanation for this phenomenon may be due to the
interaction of N atom in azaindole with other residues of the
PKM2 receptor, which is further discussed in docking studies
later. It was also found that ortho-substitution is more favorable
for the activity than para-substitution, such as, compounds 16 (4-
OMe, AC50 = 11.18 µM) and 18 (2-OMe AC50 = 3.93 µM);
compounds 9 (4-Cl, AC50 > 20 µM) and 19 (2-Cl, AC50 = 5.61
µM). 3-Pyrrole derivatives (35, AC50 = 4.34 µM) exhibited
higher activity than 2-pyrrole derivatives (27, AC50 > 20 µM),
which further demonstrated that the position of substituent was
crucial for the activity. Furthermore, replacing the benzene ring
in compound 8 with naphthalene ring (23 and 24), or introducing
Fig. 1. Several categories of PKM2 activators.
2. Results and discussion
2.1. Chemistry
On the basis of our established PKM2 model, the lead
compound 8 (AC50 = 7.64 µM), a new kind of PKM2 activator,
was identified from a random screening of an in-house
compound library (~ 1000 total). Our previous study has showed
that the pyridin-3-ylmethanamine moiety of compound 8 is
crucial for antitumor activity.24 Therefore, in order to further
improve the activity, we focused our attention on the
modification of phenyl ketone of compound 8, which was
replaced with various aryl or heterocyclic ketones (Fig. 2).
The synthesis of compounds 9-35 is depicted in Scheme 1.
Various aryl or hetroaryl ketones reacted respectively with
paraformaldehyde
catalyzed
by
diisopropylammonium
trifluoroacetate to form the intermediates acryloyl arenes,25 which
were followed by the addition of dithiocarbamates from CS2 and
3-(aminomethyl)pyridine to afford the corresponding target
compounds 9-35.26 Most of the starting materials, acetyl arenes,
are commercially available except that of compounds 32-34.
Their starting materials were prepared according to the
literatures.27,28

multiple substituents (20, 21, 22) led to the decrease or
completely lost of activity, which indicated that larger steric
hindrance was unfavorable to the activity.
different tumor cells ranged between 0.64 and 5.6 µM. Among
them, Hela cells were the most sensitive to these compounds with
most IC50 value in the nanomolar range. H1299 cells were the
least sensitive to these compounds among these cell lines. In
particular, compound 18 showed the greatest anti-proliferation
activity towards these cell lines, which is also potent at enzyme
level. To further explore the selectivity of these compounds
against cancer cells, they were also examined in BEAS-2B cells
derived from normal human bronchial epithelial cells. As seen in
Table 2, all tested compounds demonstrated high selectivity on
cancer cells. These results are consistent with the theory that
activating PKM2 is a tumor selective therapeutic strategy as
normal cells favor high activity of pyruvate kinase.
2.3. Growth inhibitory activity on cancer cell lines
The previous reported compounds have no anti-proliferative
effects on tumor cell lines unless serine or glutamate was
deprived from the medium.29 However, our series of PKM2
activators exhibited promising antitumor activity at cellular level.
Table 1. Activity of dithiocarbamic acid esters on PKM2.
a
R
Compd.
AC50
Max, (%)
Respb
(µM)
Table 2. In vitro cytotoxicity of selected compounds against
cancer cell lines and non-tumor cells.
8
Ph
7.64
24
9
4-ClPh
>20
NA
48
a
Compd.
IC50 (µM）
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
4-FPh
9.83
HCT116
0.01
2.00
1.86
1.81
2.80
0.64
1.53
3.66
1.90
2.39
3.15
Hela
H1299
0.05
4.90
4.25
4.51
3.89
3.91
3.08
4.04
3.35
5.56
4.24
BEAS-2B
7.38
4-OHPh
6.23
76
Taxol
8
0.0043
1.12
1.19
0.94
0.99
0.65
0.91
1.81
0.84
0.85
1.12
4-CNPh
4.39
61
19.04
24.40
22.56
28.11
12.11
13.08
14.95
15.81
19.34
21.59
4-NO2Ph
2.76
inhibitc
49
inhibitc
NA
63
11
12
13
18
19
25
28
29
32
4-(CH3)2NPh
4-BnOPh
>20
4-OMePh
4-CF3Ph
11.18
>20
23
2-OMePh
2-ClPh
3.93
88
5.61
inhibitc
63
inhibitc
3,4-dichloro
3,4-difluo
3,4,5-trimethoxy
2-naphthalene
1-naphthalene
2-furan
>20
42
10.13
inhibitc
inhibitc
4.30
131
inhibitc
inhibitc
107
29
a The anti-proliferative activities of selected compounds to tumor cells were
determined by the MTT assay.
2.4. Selectivity on other isoforms of pyruvate kinase
An effective antitumor agent targeting tumor specific PKM
requires a high degree of selective activation of PKM2 relative to
other PK targets with particular consideration for PKM1. Thus
the compound 32 with the highest activity on PKM2 was chosen
to test the selectivity versus other PK isoforms PKM1 and PKR.
As shown in Figure 3, compound 32 has no significant activity
on PKR and PKM1.
2-thiophene
2-pyrrole
>20
>20
126
72
2-thiazole
3-pyridine
3-coumarin
3-indole
3.56
3.70
inhibitc
106
inhibitc
NA
182
101
inhibitc
30
>20
3-azaindole
3-imidazo[1,2-a]pyridine
PhCH=CH
1.00
1.5
18.15
inhibitc
4.34
PKM1
PKM2
1.0
35
3-pyrrole
PKR
a AC50 values were determined using enzymatic PKM2 activity assay.
b %FBP indicates the extent of maximal activation, relative to FBP
(normalized to 100%).
0.5
0.0
c Compound inhibits PKM2 activity.
Ten of the compounds, which show potent PKM2 activity, were
chosen to examine the anti-proliferative effects in human colon
carcinoma cell (HCT116), human cervical carcinoma cell (Hela),
and human non-small-cell lung cancer cell (H1299). As no
reported PKM2 activators exhibit cellular activity, the antitumor
drug Taxol was selected as positive control to test the sensitivity
of the model. As shown in Table 2, the result of Taxol was
consistent with previous reports,30,31 confirming the accuracy of
our experiment. The IC50 values of tested compounds against
-2
-1
0
1
2
3
Log[32], µM
Fig. 3. Selectivity assessment for 32 versus PKM2(■), PKM1(●),
PKR(▲). Maximum activation of PKM2 was normalized to
100%.
2.5. Effect on cell cycle by Flow Cytometry

The compound 32 was also selected to be further studied
regarding to its effects on cell cycle progression. Exposure of
HCT116 cells to compound 32 resulted in an interference with
normal cell cycle distribution of this cell line. As illustrated in
Figure 4, the treatment of 32 caused blockage of the cell cycle in
the G2/M phase. Compared with control cells treated with
DMSO, when HCT116 cells were treated with increasing
concentrations of 32 (1 µM, 5 µM), the mean percentage of cells
in the G2/M phase increased from 22.74 % to 59.75 %, and the
azaindole group and Phe26C, and also the pyridine group and
Phe26D.
percentages of cells in
S and G0/G1 phase decreased
concomitantly (Fig. 4). The result is consistent with the report
that nuclear PKM2 is required for cell division by interacting
with Bub3, MCL2 in brain tumors.32,33 Our results suggest that
this series of compounds may exhibit antitumor activity by
preventing PKM2 from entering nucleus.
Fig. 5. The binding profile of compound 32 with the activator pocket
of PKM2. (A) 3D binding plot: compound 32, residues of chain C and chain
D of the receptor are colored orange, purple and green respectively. Yellow
dashes indicate hydrogen bonds or polar contacts, while the number to the
next demonstrates the distance of the interaction. (B) 2D binding plot: black
dashes, green dashes and green curves correspondingly indicate hydrogen
bonds, pi-pi stacking and hydrophobic interactions. (C) Surface plot: surface
(shown as grids) of residues which are within 12 Å of the compound. Blue,
red and yellow grids represents nitrogen, oxygen and sulfur atoms
respectively. (D) Alignment plot: compound 32 is aligned with the original
ligand (cyan sticks) from the receptor (PDB ID: 4G1N, ligand: NZT) in its
crystallized conformation within the binding site. Transparent green circles
highlighted the highly similar features of both molecules.
Fig. 4. Compound 32 blocked cell cycle. (A) Compound 32 dose-
dependent induced cell cycle arrest in G2/M phase of HCT116 cells. (B) The
structure of compound 32. (C) The profiles showed the proportions (%) in
G2/M phase of HCT116 cells treated with 32 and diluent (DMSO). The
experiments were repeated three times, and a representative experiment is
According to the docking results and the AC50 of compound
27 and 32, we hypothesized that a hydrogen bond donor (on a
five-membered ring) is expected to be present at γ-site of the
ketone so as to form a hydrogen bonding with Ala388C. If this is
the case, the low binding affinity (-7.82 kcal/mol) and activity of
2-pyrrole substitution can be attributed to its improper position of
the hydrogen bond donor. To validate it, compound 35 with a 3-
pyrrole substitution is synthesized. As expected, a significant
increase of AC50 has been observed from compound 27 to 35,
which in turn confirm the reliability of the docking results. The
introduction of other rings could always decrease the activity and
lead to an unfavorable curled conformation (see Supplementary
Information, Fig. S1.), which can be seen in compounds 23, 24,
31 and 33. But in the case of compound 32, the 7-nitrogen of the
azaindole group is elegantly positioned right at almost the same
coordinate of sulfanilamide oxygen atom of NZT. Except for
adding a necessary hydrogen bond as an acceptor, it also fixes the
azaindole plane in such a way that Phe26C could easily form a
pi-pi stacking interaction with it.
shown: ( ) p < 0.001 compared to control.
***
2.6. Docking studies and structure–activity relationship
discussion
To get further insight into the binding profile and reveal the
structure-activity relationship of the newly synthesized
compounds, a docking study of compound 32 with PKM2 was
carried out. The binding mode of compound 32 in the activator
pocket of PKM2 is shown in Figure 5.
From Figure 5 (D), our results demonstrated that compound 32
binds into the pocket with an overall binding affinity of -9.28
kcal/mol, and shares a similar binding mode with compound 4
(NZT). From Figure 5 (A) and (B), the active hydrogen on 7-
azaindole forms a strong hydrogen bond with Ala388C, while the
7-nitrogen serves as a hydrogen bond acceptor to bind with
Tyr390C. The carbonyl group of the compound is found to be
engaged in a hydrogen bonding with the positively charged side
chain of Lys311D, which highly simulates the function of the
same group of the original ligand. The active hydrogen from the
dithiocarbamic acid ester group contributes to another hydrogen
bond with Leu353D. Furthermore, a valid polar contact is found
between the nitrogen on the pyridine plane and Tyr390D. Two
pairs of pi-pi stacking interactions are present between the 7-
In this section, we found that compound 32 has a relatively
good binding profile within PKM2’s activator pocket. Predictions
from the docking results correspond to the bioassay results,
making the simulation model reliable and useful for further
compound modifications.
3. Conclusion
In this report, the lead compound 8 was identified as a PKM2
activator from a random screening of an in-house compound

library. To explore the SAR and get more potent compounds, a
series of compound 8 analogs were synthesized and biologically
evaluated. Most of them functioned as activators of PKM2 and
showed potent anti-proliferative activities against HCT116, Hela
and H1299 cell lines. The compound 32 was identified as the
most potent PKM2 selective activator. The result is supported by
molecular docking simulation, which shows that compound 32
nicely fits into the binding pocket. A flow cytometric study was
carried out to further explore the biological mechanism of this
compound. The result demonstrates that compound 32 delayed
cell cycle progression by arresting cells in G2/M phase, which
may be due to the compounds’ preventing PKM2 from entering
nucleus. This study provides a new molecular scaffold for further
development of antitumor agents that target PKM2.
133.4, 129.3, 128.4, 124.0, 47.6, 38.7, 29.3. Anal. Cald for
C16H16N2OS2: C, 60.73; H, 5.10; N 8.85; Found: C, 60.91; H,
5.16; N, 8.81.
4.2.2. 3-(4-Chlorophenyl)-3-oxopropyl (pyridin-3-ylmethyl)c-
o
arbamodithioate (9) White solid, m.p. 113.7~113.9 C. Yield:
22%. 1H NMR (400 MHz, DMSO-d6) δ 10.52 (t, J = 5.2 Hz, 1H),
8.77-8.34 (m, 2H), 8.22-7.93 (m, 2H), 7.70 (dt, J = 7.8, 1.8 Hz,
1H), 7.68-7.52 (m, 2H), 7.36 (dd, J = 7.8, 4.8 Hz, 1H), 4.85 (d, J
= 5.3 Hz, 2H), 3.62-3.40 (m, 4H). 13C NMR (100 MHz, DMSO-
d6) δ 197.8, 197.7, 149.6, 148.9, 138.8, 136.0, 135.3, 133.4,
130.3, 129.3, 124.0, 47.6, 38.7, 29.2. HRMS Calcd. For
C16H16ClN2OS2+ [M+H]+ 351.0387, found: 351.0380.
4.2.3. 3-(4-Fluorophenyl)-3-oxopropyl (pyridin-3-ylmethyl)c-
o
4. Experimental section
arbamodithioate (10) White solid, m.p. 127.4~128.1 C. Yield:
35%. 1H NMR (400 MHz, DMSO-d6) δ 10.60 (t, J = 5.5 Hz, 1H),
8.81-8.35 (m, 2H), 8.33-7.92 (m, 2H), 7.93-7.56 (m, 1H), 7.46-
7.17 (m, 3H), 4.85 (d, J = 5.6 Hz, 2H), 3.72-3.40 (m, 4H). 13C
NMR (100 MHz, DMSO-d6) δ 197.7, 197.3, 166.8, 164.3, 149.6,
148.9, 136.0, 133.4, 131.5, 131.4, 123.9, 116.4, 116.1, 47.5, 45.8,
4.1. Materials and methods
Unless otherwise stated, starting materials were purchased
from Alfa Aesar, TCI, Energy or Arcos and used without
purification. All of the reactions were monitored by thin layer
chromatography (TLC) on GF254 silica gel plates.1H NMR
spectra and 13C NMR spectra were recorded on a Bruker
AVANCE III 400 (400 MHz) spectrometer in needful D-reagents
with tetramethylsilane (TMS) as an internal reference. NMR
chemical shifts are reported as values (ppm) relative to internal
tetramethylsilane and splitting patterns are designated as: s =
singlet, d = doublet, t = triplet, q = quartet, m = multiplet. HRMS
of additional products were carried out on Brucker Apex IV
FTMS. Melting points were measured as uncorrected values.
+
38.7. HRMS Calcd. For C16H16FN2OS2 [M+H]+ 335.0683,
found: 384.0680.
4.2.4. 3-(4-Hydroxyphenyl)-3-oxopropyl (pyridin-3-ylmethy-
l)carbamodithioate (11) White solid, m.p. 166.7~167.2 oC.
1
Yield: 11%. H NMR (400 MHz, DMSO-d6) δ 8.75-8.35 (m,
2H), 7.85 (d, J = 8.7 Hz, 2H), 7.81-7.60 (m, 1H), 7.38 (dd, J =
7.7, 4.8 Hz, 1H), 6.87 (d, J = 8.7 Hz, 2H), 4.85 (s, 2H), 3.88-3.19
(m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 197.4, 196.3, 162.0,
148.9, 148.3, 135.5, 133.0, 130.5, 127.9, 123.5, 115.2, 46.9, 37.5,
29.0. HRMS Calcd. For C16H17N2O2S2+ [M+H]+ 333.0726, found:
333.0718.
4.2. Typical procedure for the synthesis of target compounds
8-31 and 35
To a mixture of aryl ketone (5.0 mmol) and paraformaldehyde
(10.0 mmol) in DMF 10 mL was added catalyst (ammonium
salts) (5.0 mmol). The mixture was vigorously stirred at 90 oC for
24 h. After that, H2O (40 mL) was added and then extracted with
EtOAc (10 mL × 3), combined organic phase was washed with
saturated NaCl (aq) and dried over Na2SO4, and then filtered, the
solvent was removed under reduced pressure to form the
intermediates acryloyl arenes.
A mixture of 3-(aminomethyl)pyridine (5.0 mmol) and TEA
(5.0 mmol) in 20 mL DMF/H2O (10:1) was added CS2 (0.32 mL).
The solution was stirred at room temperature for 30 mins
followed by the addition of acryloyl arenes from last step. The
resulting mixture was then stirred for 12 h at room temperature.
H2O (40 mL) was added to the reaction mixture, and then it was
extracted with EtOAc (20 mL × 3), combined organic phase was
washed with saturated NaCl (aq), dried (Na2SO4), filtered, the
solvent was removed under reduced pressure. The crude product
was purified by flash column chromatography on silica gel with a
gradient eluent of EtOAc /MeOH and further recrystallized from
EtOAc/PET to give crystals of the target compounds.
4.2.5. 3-(4-Cyanophenyl)-3-oxopropyl (pyridin-3-ylmethyl)c-
arbamodithioate (12) White solid, m.p. 146.7~147. 5 oC. Yield:
17%. 1H NMR (400 MHz, DMSO-d6) δ 10.51 (t, J = 5.5 Hz, 1H),
8.64-8.41 (m, 2H), 8.11 (d, J = 8.5 Hz, 2H), 8.00 (d, J = 8.5 Hz,
2H), 7.69 (d, J = 7.8 Hz, 1H), 7.36 (dd, J = 7.7, 4.8 Hz, 1H), 4.85
(d, J = 5.6 Hz, 2H), 3.52 (s, 4H). 13C NMR (100 MHz, DMSO-
d6) δ 198.2, 197.7, 149.6, 148.9, 139.7, 136.0, 133.4, 133.3,
129.0, 124.0, 118.6, 115.8, 47.6, 39.1, 29.1. HRMS Calcd. For
C17H16N3OS2+ [M+H]+ 342.0729, found: 342.0723.
4.2.6. 3-(4-Nitrophenyl)-3-oxopropyl (pyridin-3-ylmethyl)c-
o
arbamodithioate (13) Light yellow solid, m.p. 153.0~154.1 C.
1
Yield: 27%. H NMR (400 MHz, DMSO-d6) δ 8.69-8.43 (m,
2H), 8.34 (d, J = 8.9 Hz, 2H), 8.25-8.09 (m, 2H), 7.71 (dd, J =
7.8, 1.7 Hz, 1H), 7.38 (dd, J = 7.8, 4.8 Hz, 1H), 4.86 (s, 2H), 3.54
(d, J = 15.1 Hz, 4H). 13C NMR (100 MHz, DMSO-d6) δ 197.5,
197.1, 150.0, 149.0, 148.3, 140.6, 135.5, 132.9, 129.3, 123.8,
+
123.5, 46.9, 38.8, 28.5. HRMS Calcd. For C16H16N3O3S2
[M+H]+ 362.0628, found: 362.0625.
4.2.1. 3-Oxo-3-phenylpropyl (pyridin-3-ylmethyl)carbamodi-
thioate (8) White solid, m.p. 82.3~84.6 oC. Yield: 54%. 1H NMR
(400 MHz, DMSO-d6) δ 10.50 (s, 1H), 8.70-8.32 (m, 2H), 7.97
(d, J = 7.4 Hz, 2H), 7.66 (ddd, J = 10.8, 8.5, 4.4 Hz, 2H), 7.53 (t,
J = 7.1 Hz, 2H), 7.37 (dd, J = 7.7, 4.8 Hz, 1H), 4.86 (d, J = 5.3
Hz, 2H), 3.50 (dt, J = 11.8, 4.7 Hz, 4H). 13C NMR (100 MHz,
DMSO-d6) δ 198.7, 197.8, 149.5, 148.9, 136.7, 136.0, 133.9,
4.2.7. 3-(4-(Dimethylamino)phenyl)-3-oxopropyl (pyridin-3-
ylmethyl)carbamodithioate (14) White solid, m.p. 145.8~147.2
1
oC. Yield: 36%. H NMR (400 MHz, DMSO-d6) δ 10.46 (t, J =
5.3 Hz, 1H), 8.49 (dd, J = 11.1, 10.0 Hz, 2H), 7.75 (dd, J = 40.4,
8.4 Hz, 3H), 7.36 (dd, J = 7.7, 4.8 Hz, 1H), 6.71 (d, J = 8.9 Hz,
2H), 4.85 (d, J = 5.5 Hz, 2H), 3.49 (t, J = 6.7 Hz, 2H), 3.29 (t, J =

1
6.7 Hz, 2H), 3.01 (s, 6H). 13C NMR (100 MHz, DMSO-d6) δ
197.6, 195.3, 153.3, 149.1, 148.4, 135.5, 133.0, 129.9, 123.8,
123.5, 110.7, 47.1, 39.6, 37.1, 29.4. HRMS Calcd. For
C18H22N3OS2+ [M+H]+ 360.1199, found: 360.1197.
Yield: 56%. H NMR (400 MHz, DMSO-d6) δ 10.52 (q, J = 5.3
Hz, 1H), 8.77-8.28 (m, 2H), 8.37-8.05 (m, 1H), 7.92 (dd, J = 4.9,
3.5 Hz, 1H), 7.79 (td, J = 8.3, 4.1 Hz, 1H), 7.75-7.58 (m, 1H),
7.60-7.26 (m, 1H), 5.09-4.70 (m, 2H), 3.74-3.39 (m, 4H). 13C
NMR (100 MHz, DMSO-d6) δ 197.2, 196.5, 149.1, 148.4, 136.2,
135.5, 132.9, 131.9, 131.1, 129.8, 127.9, 123.5, 47.1, 38.4, 28.6.
4.2.8. 3-(4-(Benzyloxy)phenyl)-3-oxopropyl (pyridin-3-ylmet-
o
+
hyl)carbamodithioate (15) White solid, m.p. 148.3~148.9 C.
HRMS Calcd. For C16H15Cl2N2OS2 [M+H]+ 384.9997, found:
1
Yield: 22%. H NMR (400 MHz, DMSO-d6) δ 8.72-8.36 (m,
384.9989.
2H), 7.92 (dd, J = 9.4, 2.3 Hz, 2H), 7.69 (dt, J = 7.8, 1.9 Hz, 1H),
7.38 (dqd, J = 9.6, 8.7, 4.3 Hz, 6H), 7.11 (d, J = 8.9 Hz, 2H),
5.19 (s, 2H), 4.85 (s, 2H), 3.76-3.26 (m, 4H). 13C NMR (100
MHz, DMSO-d6) δ 197.8, 197.0, 162.8, 149.5, 148.8, 136.9,
136.0, 133.4, 130.7, 129.8, 129.0, 128.5, 128.2, 124.0, 115.2,
70.0, 47.4, 38.2, 29.5. HRMS Calcd. For C23H23N2O2S2+ [M+H]+
423.1196, found: 423.1194.
4.2.14. 3-(3,4-Difluorophenyl)-3-oxopropyl (pyridin-3-ylmet-
o
hyl)carbamodithioate (21) White solid, m.p. 128.8~129.7 C.
1
Yield: 25%. H NMR (400 MHz, DMSO-d6) δ 10.51 (s, J = 4.9
Hz, 1H), 8.62-8.35 (m, 2H), 7.99 (t, J = 8.2 Hz, 1H), 7.86 (d, J =
2.1 Hz, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.56 (d, J = 5.1 Hz, 1H),
7.42-7.28 (m, 1H), 4.86 (d, J = 5.4 Hz, 2H), 3.49 (tt, J = 8.4, 4.2
Hz, 4H). 13C NMR (100 MHz, DMSO-d6) δ 207.2, 196.6, 151.1,
149.7, 149.5, 149.0, 148.6, 136.1, 135.8, 134.1, 133.4, 124.0,
4.2.9. 3-(4-Methoxyphenyl)-3-oxopropyl (pyridin-3-ylmet-
o
+
hyl)carbamodithioate (16) White solid, m.p. 135.5~135.8 C.
117.8, 47.5, 38.8, 29.2. HRMS Calcd. For C16H15F2N2OS2
1
Yield: 54%. H NMR (400 MHz, DMSO-d6) δ 10.49 (t, J = 5.2
[M+H]+ 353.0588, found: 353.0581.
Hz, 1H), 8.50 (dd, J = 19.5, 2.4 Hz, 2H), 7.94 (d, J = 8.8 Hz, 2H),
7.70 (d, J = 7.8 Hz, 1H), 7.36 (dd, J = 7.7, 4.8 Hz, 1H), 7.03 (d, J
= 8.8 Hz, 2H), 4.86 (d, J = 5.5 Hz, 2H), 3.84 (s, 3H), 3.44 (ddd, J
= 17.6, 12.1, 5.6 Hz, 4H). 13C NMR (100 MHz, DMSO-d6) δ
197.9, 197.0, 163.7, 149.6, 148.9, 136.0, 133.4, 130.7, 129.7,
124.0, 114.4, 56.0, 47.6, 38.2, 29.5. HRMS Calcd. For
C17H19N2O2S2+ [M+H]+ 347.0883, found: 347.0876.
4.2.15. 3-Oxo-3-(3,4,5-trimethoxyphenyl)propyl (pyridin-3-
ylmethyl)carbamodi- thioate (22) White solid, m.p. 85.6~86.8
1
oC. Yield: 46%. H NMR (400 MHz, DMSO-d6) δ 10.52 (s, J =
5.0 Hz, 1H), 8.49 (dd, J = 11.1, 10.2 Hz, 2H), 7.70 (d, J = 7.8 Hz,
1H), 7.37 (dd, J = 7.6, 4.9 Hz, 1H), 7.23 (d, J = 28.6 Hz, 2H),
4.86 (d, J = 5.4 Hz, 2H), 3.85 (s, 6H), 3.74 (s, 3H), 3.61-3.40 (m,
4H). 13C NMR (100 MHz, DMSO-d6) δ 197.4, 197.2, 152.8,
149.1, 148.4, 142.0, 135.5, 132.9, 131.5, 123.5, 105.4, 60.1, 56.0,
4.2.10. 3-Oxo-3-(4-(trifluoromethyl)phenyl)propyl (pyridin-3-
+
ylmethyl)carbamodithioate (17) White solid, m.p. 135.8~136.6
47.1, 38.1, 29.1. HRMS Calcd. For C19H23N2O4S2 [M+H]+
1
oC. Yield: 11%. H NMR (400 MHz, DMSO-d6) δ 10.51 (t, J =
407.1094, found: 407.1087.
5.5 Hz, 1H), 8.69-8.43 (m, 2H), 8.16 (d, J = 8.1 Hz, 2H), 7.90 (d,
J = 8.3 Hz, 2H), 7.71 (d, J = 1.8 Hz, 1H), 7.37 (dd, J = 7.8, 4.8
Hz, 1H), 4.85 (d, J = 5.6 Hz, 2H), 3.53 (s, 4H). 13C NMR (100
MHz, DMSO-d6) δ 198.3, 197.7, 149.6, 148.9, 139.7, 136.0,
133.4, 133.0, 129.2, 126.2, 124.0, 109.9, 47.6, 39.1, 29.1. HRMS
Calcd. For C17H16F3N2OS2+ [M+H]+ 385.0651, found: 385.0644.
4.2.16. 3-(Naphthalen-2-yl)-3-oxopropyl (pyridin-3-ylmet-
o
hyl)carbamodithioate (23) White solid, m.p. 137.0~139.4 C.
1
Yield: 33%. H NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1H),
8.70 (s, 1H), 8.62-8.43 (m, 2H), 8.14 (d, J = 7.7 Hz, 1H), 8.01 (s,
3H), 7.81-7.56 (m, 3H), 7.37 (dd, J = 7.6, 4.8 Hz, 1H), 4.89 (s,
2H), 3.63 (s, 4H). 13C NMR (100 MHz, DMSO-d6) δ 198.1,
197.4, 149.1, 148.4, 135.5, 135.1, 133.4, 132.9, 132.2, 130.0,
129.6, 128.7, 128.3, 127.6, 126.9, 123.5, 123.3, 47.1, 38.3, 29.0.
4.2.11. 3-(2-Methoxyphenyl)-3-oxopropyl (pyridin-3-ylmet-
hyl)carbamodithioate (18) White solid, m.p. 96.2~97.0 oC.
1
+
Yield: 14%. H NMR (400 MHz, DMSO-d6) δ 10.47 (t, J = 5.5
HRMS Calcd. For C20H19N2OS2 [M+H]+ 367.0933, found:
Hz, 1H), 8.62-8.41 (m, 2H), 7.69 (dt, J = 7.8, 1.8 Hz, 1H), 7.64-
7.46 (m, 2H), 7.36 (dd, J = 7.6, 4.7 Hz, 1H), 7.17 (d, J = 8.3 Hz,
1H), 7.11-6.97 (m, 1H), 4.85 (d, J = 5.6 Hz, 2H), 3.86 (s, 3H),
3.58-3.32 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 199.9,
197.9, 158.9, 149.5, 148.9, 135.9, 134.5, 133.4, 130.0, 127.5,
124.0, 121.0, 113.0, 56.3, 47.5, 43.6, 29.4. HRMS Calcd. For
C17H19N2O2S2+ [M+H]+ 347.0883, found: 347.0877.
367.0928.
4.2.17. 3-(Naphthalen-1-yl)-3-oxopropyl (pyridin-3-ylmet-
o
hyl)carbamodithioate (24) White solid, m.p. 107.2~108.7 C.
1
Yield: 32%. H NMR (400 MHz, DMSO-d6) δ 10.61 (t, J = 5.5
Hz, 1H), 8.63-8.44 (m, 3H), 8.14 (d, J = 8.2 Hz, 1H), 8.08 (d, J =
7.2 Hz, 1H), 8.05-7.99 (m, 1H), 7.74 (d, J = 7.8 Hz, 1H), 7.68-
7.52 (m, 3H), 7.37 (dd, J = 7.8, 4.8 Hz, 1H), 4.92 (d, J = 5.5 Hz,
2H), 3.64 (qd, J = 7.7, 1.2 Hz, 4H). 13C NMR (100 MHz, DMSO-
d6) δ 202.2, 197.3, 149.1, 148.4, 135.5, 134.7, 133.5, 132.9,
132.7, 129.3, 128.5, 128.3, 127.9, 126.4, 125.2, 124.8, 123.5,
4.2.12. 3-(2-Chlorophenyl)-3-oxopropyl (pyridin-3-ylmet-
hyl)carbamodithioate (19) White solid, m.p. 78.0~78.6 oC.
1
Yield: 8%. H NMR (400 MHz, DMSO-d6) δ 10.53 (t, J = 5.4
+
Hz, 1H), 8.84-8.32 (m, 2H), 7.67 (dd, J = 9.9, 7.8 Hz, 2H), 7.58-
7.33 (m, 4H), 4.85 (d, J = 5.6 Hz, 2H), 3.44 (dt, J = 12.9, 6.7 Hz,
4H). 13C NMR (100 MHz, DMSO-d6) δ 201.1, 197.5, 149.5,
148.9, 138.5, 135.9, 133.4, 132.9, 131.0, 130.1, 129.7, 128.0,
47.2, 41.3, 29.3. HRMS Calcd. For C20H19N2OS2 [M+H]+
367.0933, found: 367.0933.
4.2.18. 3-(Furan-2-yl)-3-oxopropyl (pyridin-3-ylmethyl)car-
o
+
124.0, 47.6, 42.5, 29.0. HRMS Calcd. For C16H16ClN2OS2
bamodithioate (25) White solid, m.p. 122.9~123.6 C. Yield:
[M+H]+ 351.0387, found: 351.0382.
64%. 1H NMR (400 MHz, DMSO-d6) δ 10.51 (t, J = 5.5 Hz, 1H),
8.82-8.26 (m, 2H), 7.99 (dd, J = 1.6, 0.6 Hz, 1H), 7.69 (dt, J =
7.8, 1.9 Hz, 1H), 7.60-7.18 (m, 2H), 6.71 (dd, J = 3.6, 1.7 Hz,
1H), 4.85 (d, J = 5.6 Hz, 2H), 3.49 (t, J = 6.8 Hz, 2H), 3.26 (t, J =
4.2.13. 3-(3,4-Dichlorophenyl)-3-oxopropyl (pyridin-3-ylmet-
o
hyl)carbamodithioate (20) White solid, m.p. 139.3~140.2 C.

6.8 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 197.5, 186.9,
4.2.24. 3-(1H-indol-3-yl)-3-oxopropyl (pyridin-3-ylmethyl)c-
o
152.1, 149.6, 148.9, 148.4, 136.0, 133.4, 124.0, 119.1, 113.0,
arbamodithioate (31) White solid, m.p. 156.6~157.4 C. Yield:
+
47.6, 38.2, 29.0. HRMS Calcd. For C14H15N2O2S2 [M+H]+
6%. 1H NMR (400 MHz, DMSO-d6) δ 11.96 (s, 1H), 10.45 (t, J =
5.5 Hz, 1H), 8.56-8.45 (m, 2H), 8.33 (d, J = 3.1 Hz, 1H), 8.25-
8.13 (m, 1H), 7.75-7.63 (m, 1H), 7.47 (dd, J = 6.7, 1.7 Hz, 1H),
7.37 (dd, J = 7.8, 4.8 Hz, 1H), 7.26-7.12 (m, 2H), 4.86 (d, J = 5.6
Hz, 2H), 3.55 (t, J = 6.9 Hz, 2H), 3.29 (t, J = 7.0 Hz, 2H). 13C
NMR (100 MHz, DMSO-d6) δ 198.1, 193.4, 149.6, 148.9, 137.1,
135.9, 134.5, 133.5, 125.8, 124.0, 123.3, 122.3, 121.7, 116.5,
112.6, 47.5, 38.8, 30.0. HRMS Calcd. For C18H18N3OS2+ [M+H]+
356.0886, found: 356.0883.
307.0570, found: 307.0563.
4.2.19. 3-Oxo-3-(thiophen-2-yl)propyl (pyridin-3-ylmethyl)c-
o
arbamodithioate (26) White solid, m.p. 135.3~136.2 C. Yield:
53%. 1H NMR (400 MHz, DMSO-d6) δ 10.49 (t, J = 5.3 Hz, 1H),
8.56-8.45 (m, 2H), 8.02 (dd, J = 4.9, 1.1 Hz, 1H), 7.96 (dd, J =
3.8, 1.1 Hz, 1H), 7.69 (dt, J = 7.8, 1.8 Hz, 1H), 7.37 (ddd, J =
7.8, 4.8, 0.7 Hz, 1H), 7.24 (dd, J = 4.9, 3.8 Hz, 1H), 4.85 (d, J =
5.6 Hz, 2H), 3.51 (t, J = 6.5 Hz, 2H), 3.39 (dd, J = 10.3, 3.6 Hz,
2H). 13C NMR (100 MHz, DMSO-d6) δ 197.6, 191.7, 149.6,
148.9, 143.7, 136.0, 135.5, 134.0, 133.4, 129.3, 124.0, 47.6, 38.9,
29.4. HRMS Calcd. For C14H15N2OS3+ [M+H]+ 323.0341, found:
323.0339.
4.2.25. The preparation of compound 32
1
1-(1H-pyrrolo[2,3-b]pyridin-3-yl)ethan-1-one (36)27, H NMR
(400 MHz, CDCl3) δ 13.11-12.78 (m, 1H), 8.79-8.69 (m, 1H),
8.47-8.37 (m, 1H), 8.15-7.98 (m, 1H), 7.36-7.28 (m, 1H), 2.64-
2.52 (m, 3H).
4.2.20. 3-Oxo-3-(1H-pyrrol-2-yl)propyl (pyridin-3-ylmethyl)c-
o
3-Oxo-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propyl
(pyridin-3-
arbamodithioate (27) White solid, m.p. 128.7~129.3 C. Yield:
ylmethyl)carbamodithioate (32) White solid, m.p. 147.8~148.2
oC. Yield: 12%. 1H NMR (400 MHz, DMSO-d6) δ 12.50 (s, 1H),
10.47 (t, J = 5.5 Hz, 1H), 8.56-8.43 (m, 4H), 8.33 (dd, J = 4.7,
1.6 Hz, 1H), 7.74-7.65 (m, 1H), 7.37 (dd, J = 7.7, 4.8 Hz, 1H),
7.26 (dd, J = 7.9, 4.7 Hz, 1H), 4.86 (d, J = 5.6 Hz, 2H), 3.55 (t, J
= 6.8 Hz, 2H), 3.31 (d, J = 7.0 Hz, 2H). 13C NMR (100 MHz,
DMSO-d6) δ 198.0, 193.6, 149.6, 149.4, 148.9, 144.7, 135.9,
134.9, 133.4, 130.0, 124.0, 118.6, 118.1, 115.1, 47.6, 38.7, 29.8.
19%. 1H NMR (400 MHz, DMSO-d6) δ 11.85 (s, 1H), 10.47 (t, J
= 5.3 Hz, 1H), 8.57-8.39 (m, 2H), 7.69 (d, J = 7.8 Hz, 1H), 7.36
(dd, J = 7.7, 4.8 Hz, 1H), 7.09 (s, 1H), 6.97 (s, 1H), 6.22-6.13 (m,
1H), 4.86 (d, J = 5.5 Hz, 2H), 3.50 (t, J = 6.8 Hz, 2H), 3.18 (t, J =
6.9 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 197.8, 187.8,
149.6, 148.9, 136.0, 133.4, 131.8, 126.1, 124.0, 117.2, 110.3,
+
47.6, 37.6, 29.9. HRMS Calcd. For C14H16N3OS2 [M+H]+
+
306.0729, found: 306.0724.
HRMS Calcd. For C17H17N4OS2 [M+H]+ 357.0838, found:
357.0832.
4.2.21. 3-Oxo-3-(thiazol-2-yl)propyl (pyridin-3-ylmethyl)c-
arbamodithioate (28) Yellow solid, m.p. 114.0~114.9 oC. Yield:
10%. 1H NMR (400 MHz, CDCl3) δ 9.12-8.96 (m, 1H), 8.51-8.36
(m, 2H), 8.02-7.93 (m, 1H), 7.70 (dd, J = 7.1, 4.2 Hz, 2H), 7.28-
7.20 (m, 1H), 5.03-4.89 (m, 2H), 3.77-3.62 (m, 4H). 13C NMR
(100 MHz, CDCl3) δ 198.2, 191.9, 166.2, 149.2, 148.8, 144.9,
136.3, 132.6, 126.6, 123.8, 48.1, 38.9, 29.0. HRMS Calcd. For
C13H14N3OS3+ [M+H]+ 324.0294, found: 324.0290.
4.2.26. The preparation of compound 33
1-(Imidazo[1,2-a]pyridin-3-yl)ethan-1-one (37)28, 1H NMR
(400 MHz, CDCl3) δ 9.73-9.47 (m, 1H), 8.46-8.27 (m, 1H), 7.88-
7.69 (m, 1H), 7.56-7.42 (m, 1H), 7.25-6.94 (m, 1H), 2.86-2.35
(m, 3H).
3-(Imidazo[1,2-a]pyridin-3-yl)-3-oxopropyl (pyridin-3-ylme-
o
thyl)carbamodithioate (33) White solid, m.p. 164.8~165.6 C.
1
Yield: 14%. H NMR (400 MHz, DMSO-d6) δ 10.51 (t, J = 5.1
4.2.22. 3-Oxo-3-(pyridin-3-yl)propyl (pyridin-3-ylmethyl)car-
o
Hz, 1H), 9.54 (d, J = 6.8 Hz, 1H), 8.64 (s, 1H), 8.50 (dd, J =
10.4, 8.8 Hz, 3H), 7.85 (d, J = 8.9 Hz, 1H), 7.70 (d, J = 7.7 Hz,
1H), 7.38 (d, J = 5.0 Hz, 1H), 7.29 (t, J = 6.7 Hz, 1H), 4.86 (d, J
= 5.0 Hz, 2H), 3.59 (t, J = 6.8 Hz, 2H), 3.40 (t, J = 6.8 Hz, 2H).
13C NMR (100 MHz, DMSO-d6) δ 197.1, 187.6, 149.1, 148.4,
143.8, 135.5, 132.9, 132.6, 129.6, 128.0, 123.5, 123.0, 117.4,
115.6, 47.1, 38.3, 29.3. HRMS Calcd. For C17H17N4OS2+ [M+H]+
357.0838, found: 357.0832.
bamodithioate (29) Yellow solid, m.p. 95.1~195.9 C. Yield:
11%. 1H NMR (400 MHz, DMSO-d6) δ 10.52 (t, J = 5.3 Hz, 1H),
9.13 (t, J = 4.3 Hz, 1H), 8.80 (dd, J = 4.7, 1.4 Hz, 1H), 8.50 (dd,
J = 20.1, 2.4 Hz, 2H), 8.30 (dd, J = 6.1, 1.8 Hz, 1H), 7.70 (d, J =
7.8 Hz, 1H), 7.56 (dd, J = 7.8, 4.8 Hz, 1H), 7.36 (dd, J = 7.7, 4.8
Hz, 1H), 4.86 (d, J = 5.5 Hz, 2H), 3.53 (t, J = 4.3 Hz, 4H). 13C
NMR (100 MHz, DMSO-d6) δ 198.3, 197.7, 154.1, 149.6, 149.6,
148.9, 136.0, 135.9, 133.4, 131.9, 124.4, 124.0, 47.6, 39.0, 29.1.
4.2.27. 3-Oxo-5-phenylpent-4-en-1-yl (pyridin-3-ylmethyl)ca-
o
+
HRMS Calcd. For C15H16N3OS2 [M+H]+ 318.0729, found:
rbamodithioate (34) White solid, m.p. 119.9~120.6 C. Yield:
318.0725.
1
28 %. H NMR (400 MHz, DMSO-d6) δ 10.50 (t, J = 5.4 Hz,
1H), 8.64-8.36 (m, 2H), 7.77-7.61 (m, 4H), 7.47-7.34 (m, 4H),
6.91 (d, J = 16.3 Hz, 1H), 4.86 (d, J = 5.6 Hz, 2H), 3.47 (t, J =
6.7 Hz, 2H), 3.16 (t, J = 6.8 Hz, 2H). 13C NMR (100 MHz,
DMSO-d6) δ 198.1, 197.3, 149.1, 148.4, 142.6, 135.5, 134.3,
132.9, 130.5, 128.9, 128.5, 126.1, 123.5, 47.1, 39.9, 28.7. HRMS
Calcd. For C18H19N2OS2+ [M+H]+ 343.0933, found: 367.0930.
4.2.23. 3-Oxo-3-(2-oxo-2H-chromen-3-yl)propyl (pyridin-3-
ylmethyl)carbamodithioate (30) White solid, m.p. 127.8~128.7
oC. Yield: 6%. 1H NMR (400 MHz, DMSO-d6) δ 10.49 (t, J = 5.5
Hz, 1H), 8.75-8.68 (m, 1H), 8.54-8.42 (m, 2H), 7.98 (dd, J = 7.8,
1.2 Hz, 1H), 7.74 (ddd, J = 22.0, 12.9, 4.6 Hz, 2H), 7.50-7.34 (m,
3H), 4.85 (d, J = 5.6 Hz, 2H), 3.58-3.37 (m, 4H). 13C NMR (100
MHz, DMSO-d6) δ 197.7, 196.1, 158.8, 155.1, 149.6, 148.9,
147.9, 136.0, 135.1, 133.4, 131.3, 125.4, 124.3, 124.0, 118.6,
4.2.28 3-Oxo-3-(1H-pyrrol-3-yl)propyl (pyridin-3-ylmethyl)c-
o
arbamodithioate (35) White solid, m.p. 145.5~146.8 C. Yield:
+
116.6, 47.6, 42.4, 29.0. HRMS Calcd. For C19H17N2O3S2
24 %. 1H NMR (400 MHz, DMSO-d6) δ 11.47 (s, 1H), 10.45 (t, J
= 5.5 Hz, 1H), 8.65-8.32 (m, 2H), 7.84-7.62 (m, 1H), 7.59 (dt, J
= 3.1, 1.6 Hz, 1H), 7.37 (dd, J = 7.8, 4.8 Hz, 1H), 6.83 (dd, J =
[M+H]+ 385.0675, found: 385.0672.

4.6, 2.4 Hz, 1H), 6.45 (dd, J = 4.1, 2.5 Hz, 1H), 4.85 (d, J = 5.6
Hz, 2H), 3.46 (t, J = 6.9 Hz, 2H), 3.14 (t, J = 6.9 Hz, 2H). 13C
NMR (100 MHz, DMSO-d6) δ 198.1, 193.1, 149.6, 148.9, 135.9,
133.5, 125.1, 124.8, 124.0, 120.4, 107.9, 47.5, 38.8, 29.8. HRMS
Calcd. For C14H16N3OS2+ [M+H]+ 305.0708, found: 367.0728.
washed twice with 1 × PBS, and resuspended in 200 µL of 1 ×
PBS. The cells were fixed in 4 mL of ice-cold 75 % ethanol at
−20 °C overnight and stained with 500 µL of propidium iodide
(50 µg/mL, Sigma) containing 0.1 % RNase (1 mg/mL, Sigma)
for 15 min in dark conditions at room temperature. The cells
were then analyzed by flow cytometry (FACSVerseTM, BD). The
results were indicated as mean values from three independent
determinations.
4.3. Purification of recombinant pyruvate kinase isoforms
The human cDNA for PKM2, PKM1 and PKR were cloned
into pET28a with a N-terminal His tag and purified from E.coli
strain BL21 (Invitrogen) using Ni-Agarose beads (Qiagen) as
described previously.12 Briefly, E.coli grown to an OD (600 nm)
of 0.6 were induced with 0.5 mM IPTG at 21 ℃ for 4 h. Cells
were collected and lysed by freeze/thaw cycles and sonication.
Lysate was passed over a Ni-NTA agarose column and eluted
with 250 mM imidazole in 1 ml fractions. Fractions with high
concentration of pyruvate kinase were determined using SDS-
PAGE and coomassie staining according to standard protocol.
4.7. Procedures for molecular docking:
Molecular docking was carried out by using AutoDock 4.2
(Scripps Research Institute),37 assessed by OpenSource PyMOL
1.3.x (Schrödinger, LLC) and PoseView web service (Universität
Hamburg) afterwards.38 The X-ray crystal structure of PKM2 and
its reported activator compound 4 (NZT) taken from PDB
(4G1N) was used as the input structure. Receptor preparation: All
HETATM and hydrogen atoms are deleted and crystal cell is
removed. Alternative conformers of residues are also deleted to
retain one set. Then the receptor is further processed via
AutoDock Tools 1.5.6 rc3. Polar hydrogen and Kollman charge
is added to finally give the prepared protein. Ligand preparation:
Polar hydrogen and Gasteiger charge is given and there is no
further modifications on the ligand torsion tree. Molecular
docking: Parameter selection is done via AutoDock Tools 1.5.6
rc3. Whilst other parameters for AutoDock were set as default,
the Number of GA Run was modified to 100 for reliability and
accuracy reasons.
4.4. Measurement of enzyme activity
Fluorescent Pyruvate Kinase-Lactate Dehydrogenase Coupled
Assay. 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.34 For PKM2, 45 µL of
substrate mix (final concentration, 50 mM Tris-Cl pH 8.0, 200
mM KCl, 15 mM MgCl2, 0.1 mM PEP, 4.0 mM ADP and 0.2
mM NADH) was dispensed into Corning black solid 96-well
plates and 1 uL of compounds were delivered using a Eppendorf
pin tool and then 5 µL of enzyme mix (final concentrations, 10
nM hPKM2 and 1 µM of LDH) was added. Plates were
immediately placed in FlexStation 3 (Molecular Devices), and
NADH fluorescence was determined at 30 s exposure intervals
for between 3 and 6 min. Data were normalized to the untreated
and AC100 activation using known activators such as FBP. Data
was fit in GraphPad prism. Conversion of fluorescent units to
pmols of NADH was performed using a standard curve of known
NADH concentrations. Data was collected on the FlexStation 3.
Acknowledgments
We gratefully acknowledge the generous support provided by
The National Natural Science Funds of China (No. 21172011，
81372491, 81430056, 31420103905 & 81321003) and the 111
Project (B07001).
References and notes
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Supplementary Material
1H NMR and 13C NMR spectra for all the compounds and
additional data and software were available in supporting
information.