Synthesis of novel sulfonamide derivatives containing pyridin-3-ylmethyl 4-(benzoyl)piperazine-1-carbodithioate moiety as potent PKM2 activators

Article abstract of DOI:10.1016/j.bioorg.2021.104653

Pyruvate kinase M2 isoform (PKM2) plays a key role in cancer progression through both metabolic and non-metabolic functions, thus it is recognized as a potential target for cancer diagnosis and treatment. In this study, we discovered a sulfonamide-dithiocarbamate compound 8a as a novel PKM2 activator from a random screening of an in-house compound library. Then, a series of lead compound 8a analogs were designed and synthesized for screening as potent PKM2 activators. Among them, compound 8b (AC₅₀ = 0.136 μM) and 8k (AC₅₀ = 0.056 μM) showed higher PKM2 activation activities than positive control NZT (AC₅₀ = 0.228 μM), and they (IC₅₀ < 1 μM) exhibited more significant anti-proliferative activities against human tumor cell lines than NZT (IC₅₀ > 10 μM). Especially, compound 8k inhibited the proliferation of multiple cancer cells, but showed little toxicity on normal cells. In addition, we found that compound 8k inhibit the colony formation of MCF7 cells. Western blot analysis demonstrated that 8k could reduce PKM2 nuclear localization and block the downstream signaling pathway of PKM2, resulting in suppression of tumor cell proliferation. Overall, compound 8k may be a promising candidate for further mechanistic investigation of PKM2 and cancer therapy.

Full text of DOI:10.1016/j.bioorg.2021.104653

Bioorganic Chemistry 108 (2021) 104653
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis of novel sulfonamide derivatives containing pyridin-3-ylmethyl
4
-(benzoyl)piperazine-1-carbodithioate moiety as potent PKM2 activators
a
,
1
,
*
a,
1
e
a
a
b,*
Ridong Li
Yuxin Yin
, Xianling Ning , Jianan He , Zhiqiang Lin , Yue Su , Runtao Li
,
a,c,d,*
a
Institute of Systems Biomedicine, Beijing Key Laboratory of Tumor Systems Biology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing
00191, PR China
1
b
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, PR China
Peking-Tsinghua Center for Life Sciences, Peking University Health Science Center, Beijing 100191, PR China
Department of Pathology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, PR China
Department of Pharmacology and Therapeutics, University of Melbourne, Parkville Victoria 3010, Australia
c
d
e
A R T I C L E I N F O
A B S T R A C T
Keywords:
Pyruvate kinase M2 isoform (PKM2) plays a key role in cancer progression through both metabolic and non-
metabolic functions, thus it is recognized as a potential target for cancer diagnosis and treatment. In this
study, we discovered a sulfonamide-dithiocarbamate compound 8a as a novel PKM2 activator from a random
screening of an in-house compound library. Then, a series of lead compound 8a analogs were designed and
synthesized for screening as potent PKM2 activators. Among them, compound 8b (AC50 = 0.136 µM) and 8k
PKM2 activator
Sulfonamide
Dithiocarbamate
Nuclear translocation
Anti-tumor activity
(
AC50 = 0.056 µM) showed higher PKM2 activation activities than positive control NZT (AC50 = 0.228 µM), and
they (IC50 < 1 µM) exhibited more significant anti-proliferative activities against human tumor cell lines than
NZT (IC50 > 10 µM). Especially, compound 8k inhibited the proliferation of multiple cancer cells, but showed
little toxicity on normal cells. In addition, we found that compound 8k inhibit the colony formation of MCF7
cells. Western blot analysis demonstrated that 8k could reduce PKM2 nuclear localization and block the
downstream signaling pathway of PKM2, resulting in suppression of tumor cell proliferation. Overall, compound
8
k may be a promising candidate for further mechanistic investigation of PKM2 and cancer therapy.
1
. Introduction
The metabolic pathways of cancer cells distinguish significantly from
lead to the transformation from PKM2 tetramer to a dimer form. The
dimer form diverts the glycolytic flux to biomass production and pro-
motes oncogenesis [9,10]. Moreover, dimer form of PKM2 can trans-
locate into the nucleus to act as a transcriptional activator, even has
protein kinase activity [6,11]. For instance, PKM2 phosphorylates
STAT3 at Tyr705 resulting in the transcription of downstream genes to
promote tumorigenesis [12]. Therefore, switch PKM2 from dimer to
tetramer form by small molecule activators may become potent thera-
peutic strategy [13].
those of normal cells. Cancer cells produce energy mainly by aerobic
glycolysis [1]. Pyruvate kinase (PK) converts phosphoenolpyruvate
(
PEP) to pyruvate, catalyses the rate limiting step of glycolysis [2,3]. The
M1 isoform of pyruvate kinase (PKM1) is found in normal tissues, but
the M2 isoform of pyruvate kinase (PKM2), a splicesome variant, is
mainly found in cancer cells [4,5]. PKM1 has continuously high enzy-
matic activity as a stable tetramer. On the contrary, PKM2 exists in
balance between less active monomer or dimer and active tetramer
form, which can be regulated by some metabolic effectors or post-
translational modifications [6-8]. Some allosteric modulators in cancer
cells, such as phosphor-tyrosine and post translational modifications,
Several PKM2 activators have been reported until now. The repre-
sentative compounds of PKM2 activators include TEPP-46 (thieno-[3,2-
b]pyrrole[3,2-d]pyridazinone) [13,14], DASA-58 (substituted N,N’-
diarylsulfonamide) [13,15], NZT (quinoline sulfonamides) [16] and
MCL (Micheliolide) [17] (Fig. 1). These compounds were reported to
*
Corresponding authors at: Institute of Systems Biomedicine, Beijing Key Laboratory of Tumor Systems Biology, School of Basic Medical Sciences, Peking Uni-
versity Health Science Center, Beijing 100191, PR China (Y. Yin).
E-mail addresses: lrd@bjmu.edu.cn, lirt@bjmu.edu.cn (R. Li), yinyuxin@hsc.pku.edu.cn (Y. Yin).
1
These authors contribute equally to this work.
https://doi.org/10.1016/j.bioorg.2021.104653
Received 1 October 2020; Received in revised form 8 January 2021; Accepted 9 January 2021
Available online 19 January 2021
0
045-2068/© 2021 Elsevier Inc. All rights reserved.

R. Li et al.
Bioorganic Chemistry 108 (2021) 104653
promote tetramer formation of PKM2, affect the translocation of PKM2
into the nucleus, and inhibit the lysine433 acetylation. Although many
PKM2 activators were discovered, most of them have limited effects on
cell viability in cell culture using standard conditions [13,18]. Recently,
our group has also reported two series of PKM2 activators with different
structural types. One kind is 7-azaindole derivative containing pyridin-
intermediate 11. Without further purification, the intermediate 11
reacted with 3-(chloromethyl)pyridine and carbon disulfide using trie-
thylamine as base in DMF to gain pyridin-3-ylmethyl 4-(4-nitrobenzoyl)
piperazine-1-carbodithioate 12. The reduction of 12 using Fe and NH
4
Cl
as catalysts in H O and EtOH generated pyridin-3-ylmethyl 4-(4-ami-
2
nobenzoyl)piperazine-1-carbodithioate 13. Then a variety of commer-
cially available substituted sulfonyl chlorides reacted with compound
13 using pyridine as base in the THF and DMF mixed solvents to provide
the corresponding target compounds 8a-8m.
3
-ylmethyl dithiocarbamate moiety (5 / AC50 = 1.00 µM and 6 / AC50
=
2
.67 µM) [19,20]. Another kind is sulfonamide derivative containing 4-
hydroxythiazolidine-2-thione (7 / AC50 = 3.14 µM) [21]. To our delight,
our PKM2 activators could effectively inhibit cellular growth on tumor
cell lines, while maintaining low toxicities towards non-cancer cell lines.
Combined with literature reports [15,16,22,23] and our previous
studies [21], it was found that sulfonamides have good PKM2 activation
activity. Furthermore, our previous works have showed that dithiocar-
bamate moiety is crucial for improving PKM2 potency and selectivity
2
.2. Biological evaluation
All target compounds were evaluated for the cell-free PKM2 activa-
tion activity with a fluorescent PK-LDH coupled assay as previously
reported [19]. The reported PKM2 activator NZT was used as the posi-
tive control. The PKM2 activation rates of all target compounds at a
concentration of 5 µM were firstly examined. As shown in Table 1,
compound 8b, 8c, 8f, 8h and 8k exhibited high PKM2 activation rates
with above 100% at 5 µM. Especially, compound 8b and 8k showed the
most prominent PKM2 activation rates (275.7% and 286.4%). These
compounds with high PKM2 activation rates at 5 µM were further
evaluated for their AC50 values. Among them, compound 8b and 8k with
AC50 values of 0.136 µM and 0.056 µM respectively, displayed more
potent activities than the positive control NZT (AC50 = 0.228 µM). The
different structures at region R showed great impact on the PKM2
activation activities. Firstly, when R was a benzene ring, the PKM2
activation rate was 60.4% at 5 µM. Introduction of an ortho- or meta-
methoxyl on the benzene ring (8b, Activation rate at 5 µM = 275.7%; 8c,
Activation rate at 5 µM = 157.1%) increased the activities. On the
contrary, para-methoxyl on the benzene ring led to a decrease in potency
[
19,20]. In the past twenty years, our group has been committed to the
research of dithiocarbamates as anti-tumor agents [19,20,24-28]. As a
continuation of searching new types of PKM2 activators, we conducted a
random screening of an in-house dithiocarbamate compound library
(
~500 total) on the basis of the established PKM2 model. Fortunately,
we identified a novel compound 8a (Fig. 2) with 60.4% activation rate
on PKM2 at a concentration of 5 µM. To the best of our knowledge, this
type of sulfonamide-dithiocarbamates as PKM2 activator has not been
reported before. More interestingly, this novel sulfonamide-
dithiocarbamate showed significant anti-tumor activities in the pre-
liminary anti-tumor screening. Especially for cell lines PC3 and A375,
the IC50 values of 8a were 3.06 µM and 2.67 µM, respectively. These
results demonstrate that compound 8a is a valuable lead compound for
the development of PKM2 activators. Our previous studies [19-21] have
showed that the pyridin-3-methylene moiety of compound 8a is crucial
for antitumor activity. Therefore, in order to further improve the ac-
tivity, we focused our attention on the modification of phenyl of com-
pound 8a, which was replaced with aryl or heterocyclic rings, as well as
aliphatic chains (Fig. 2).
(
8d, Activation rate at 5 µM = 20.8%). Introduction of fluorine instead
of methoxyl on the benzene ring reduced the activation activities. The
meta-fluoro analog (8f) brought about higher activity than the ortho-
(
8e) and meta-fluoro analogs (8g). Replacing the benzene ring of com-
pound 8a with naphthalene ring (8h, Activation rate at 5 µM = 103.7%)
and quinoline ring (8k, Activation rate at 5 µM = 286.4%) resulted in
significant increase of activities. However, when the benzene ring was
replaced by thiophene (8i, Activation rate at 5 µM = 40.4%), 2,3-dihy-
drobenzo[b][1,4]dioxine (8j, Activation rate at 5 µM = 21.9%), 1H-
pyrrolo[2,3-b]pyridine (8l, Activation rate at 5 µM = 28.3%), ethenyl
2
. Results and discussion
2
.1. Chemistry
The synthetic route for designed compounds 8a-8m is shown in
Schemes 1. The 4-nitrobenzoyl chloride 9 served as a starting material
reacted with tert-butyl piperazine-1-carboxylate in THF to provide the
intermediate tert-butyl 4-(4-nitrobenzoyl)piperazine-1-carboxylate 10
under the catalysis of triethylamine. The compound 10 was deprotected
(
8m, Activation rate at 5 µM = 27.7%), the activities reduced obviously.
Above all, compound 8k not only exhibited better activity (AC50
.056 µM), but also showed the highest activation (286.4%).
To get further insight into the binding profile and reveal the struc-
ture–activity relationship of the newly synthesized compounds, a
=
0
2 2
in the present of 4 M HCl in dioxane and CH Cl and got the
Fig. 1. The structures of representative PKM2 activators.
2

R. Li et al.
Bioorganic Chemistry 108 (2021) 104653
Fig. 2. Lead compound 8a and overall strategy for its structural modification.
3 2 2 2
Scheme 1. Synthesis of 8a-8m. Reagents and conditions: (a) tert-butyl piperazine-1-carboxylate, Et N, THF; (b) 4 M HCl in dioxane, CH Cl ; (c) CS , 3-(chlor-
omethyl)pyridine hydrochloride, Et N, DMF; (d) Fe, NH Cl, H O, EtOH; (e) THF/DMF 1:1, RSO Cl, Pyridine.
3
4
2
2
docking study of the compound 8k with PKM2 was carried out. The
binding mode of the compound 8k in the activator pocket of PKM2 is
shown in Fig. 3. Our results demonstrate that compound 8k binds into
the pocket with an overall binding affinity of 13.3 kcal/mol, and shares a
very similar binding mode with NZT. The carbonyl group, sulfonyl
group and imino group of the compound are found to be engaged in a
hydrogen bonding with the hydrophobic side chain of Lys311B,
Tyr390A and Leu353A, respectively. Furthermore, an effective
π-π
3

R. Li et al.
Bioorganic Chemistry 108 (2021) 104653
Table 1
A375 cells ranged between 0.28 and 10 µM. Almost all target com-
pounds showed higher anti-proliferative activities than NZT. These re-
sults once again demonstrate that our PKM2 activators containing
dithiocarbamate moiety have potent antitumor activities. Those com-
pounds with excellent enzymatic activities also have significant anti-
proliferative activities. In particular, compound 8b and 8k exhibited
the optimal activities with IC50 values in the nanomolar range, which are
also potent at enzyme level. Structure-activity relationship (SAR) anal-
ysis showed that introduction of ortho-methoxyl on the benzene ring
(8b) display 3-fold increase in anti-proliferative activities against two
cell lines comparing with lead compound 8a. On the contrary, intro-
duction of meta-methoxyl (8c) or para-fluorine (8f) on the benzene ring
result in 2-fold or 3-fold decrease in anti-proliferative activities. The
different heterocyclic structures at region R also showed great impact on
the anti-proliferative activities. When the phenyl group was replaced
with thiophene ring (8i), 2,3-dihydrobenzo[b][1,4]dioxine (8j) or
quinoline (8k), the corresponding compounds showed slight or signifi-
cant improvements in comparison with compound 8a. However,
replacement with benzene ring with 7-azaindole (8l) resulted in a slight
decrease in cellular potency. In addition, replacing the benzene ring of
compound 8a with ethenyl (8m) has no significant effect on cell
viability.
PKM2
activation
activities
of
target
compounds
8a-
8
m.
a
Compd.
R
Activation rate at 5 µM (%)
AC50 (µM)
b
8
a
60.4
N.d
8
b
275.7
157.1
0.136 ± 0.040
0.508 ± 0.103
8
c
8
8
8
d
e
f
20.8
57.1
102.0
N.d
N.d
In additions, compound 8k and NZT were tested in human cervical
carcinoma cell (Hela), human breast adenocarcinoma cell (MCF7),
human carcinoma cell (CNE2) and human lung cancer cell (A549) to
further evaluate anti-proliferative effects by MTS assay. As shown in
Table 3, compound 8k exhibited remarkable inhibitory activities on all
the tested tumor cells. In contrast, NZT had little inhibitory effects on
MCF7 and CNE2 cell lines. In addition, compound 8k inhibited the
colony formation of MCF7 cells in a dose-dependent manner (Fig. 4). To
preliminary explore safety of compound 8k, we tested its cytotoxicity on
normal human primary fibroblast cells (BJ cells). The result showed that
compound 8k (IC50 > 40 µM) had little toxicity on BJ cells (Table 3),
indicating better safety windows.
0.624 ± 0.157
8
8
g
31.5
N.d
h
103.7
0.307 ± 0.081
8
8
i
j
40.4
21.9
N.d
N.d
Previous studies suggested that nuclear PKM2 can function as a
protein kinase and transcriptional coactivator to induce the expression
of genes, leading to the upregulation of glycolytic gene and promotes
tumor development [12]. For example, nuclear PKM2 was found to
induce the initiation of STAT3 signaling, which decreased the propor-
tion of apoptotic tumor cells [29]. In addition, PKM2 was also necessary
to induce sustained ERK activation and mitogen-induced cell prolifera-
tion [30]. To explore whether compound 8k can influence the nuclear
transcription of PKM2, we incubated MCF7 cells with compound 8k and
then separated cytoplasmic and nuclear protein to detect the localiza-
tion of PKM2. Western blot indicated that nuclear transcription of PKM2
decreased with compound 8k treatment in a dose-dependent manner
(Fig. 5). To demonstrate whether blocking PKM2 nuclear localization
could inhibit the expression of PKM2 downstream signaling pathway,
we treated cells with compound 8k and detected the expression of
phosphorylated Stat3 and ERK. As shown in Fig. 6, the results showed
that the expression of total Stat3 and ERK had not changed obviously,
but phosphorylated Stat3 and ERK were significantly inhibited at 5 µM
compound 8k. These results indicated that compound 8k as a potent
PKM2 activator inhibited the PKM2 nuclear translocation in tumor cells,
resulting in suppression of transcriptional regulation in tumorigenesis.
8
8
k
l
286.4
28.3
0.056 ± 0.011
N.d
8
m
27.7
N.d
NZT
——
151.9
0.228 ± 0.062
a
AC50 represents half maximal active concentration.
N.d represents not determined.
b
stacking interaction was found between the quinolone ring and Phe26B.
These interactions are identical to the pattern of binding between the
original ligand and the receptor. In addition, a pair of pi-pi stacking
interactions are present between the pyridyl group and Phe26B, and also
the benzene group and Phe26A. Overall, by analyzing the binding
pocket of PKM2 receptor, it was observed that compound 8k exhibited
stronger interaction indicating higher selectivity than PKM2 receptor
activator NZT. 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 study, the lead compound 8a was identified as a PKM2 acti-
vator from a random screening of an in-house compound library. To
explore the SAR and get more potent PKM2 activators, a series of
compound 8a analogs were designed, synthesized and evaluated for
their biological activities. Among them, compound 8b and 8k showed
higher PKM2 activation activities than positive control NZT, and also
exhibited more significant anti-proliferative activities on human tumor
cell lines than NZT. In particular, compound 8k inhibited the
To evaluate the potency of target compounds as anti-tumor agents,
we measured the effect of target compounds on cancer cells cytotoxicity.
The cytotoxicity was assessed using two PKM2 high expression human
tumor cell lines derived from human prostate cancer (PC3) and mela-
noma (A375). Paclitaxel was used as the positive control. The results are
shown in Table 2. The IC50 values of tested compounds against PC3 and
4

R. Li et al.
Bioorganic Chemistry 108 (2021) 104653
Fig. 3. The predicted docking mode of compound 8k and PKM2 receptor. a. The predicted 3D binding plot: residues of chain A and chain B of the receptor are
colored purple and white, respectively. Yellow dashes indicate hydrogen bonds or polar contacts between compound 8k and the receptor, and red dashes indicate
hydrogen bonds or polar contacts between NZT and the receptor, while the number to the next demonstrates the distance of the interaction. Compound 8k (Green
sticks) is aligned with the original ligand (Cyan sticks) from the receptor (PDB ID: 4G1N, ligand: NZT) in its crystallized conformation. These two conformations are
almost completely coincident. b. Surface plot: Surface (shown as mesh) of residues which are within 12 Å of the compound 8k. Blue, red and yellow grids represent
nitrogen, oxygen and sulfur atoms, respectively. c. The predicted 2D binding mode of compound 8k to the receptor. d. The predicted 2D binding mode of NZT to the
receptor. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
proliferation of multiple cancer cells, but showed little toxicity on
normal cells, which indicated compound 8k may have a high safety
index. Further mechanistic studies revealed that 8k could inhibit the
colony formation of MCF7 cells, as well as reduce the PKM2 nuclear
localization and block the downstream signaling pathways of PKM2,
resulting in inhibition of tumor cell proliferation. In summary, these
findings reveal that sulfonamide-dithiocarbamate derivatives represent
a new class of PKM2 activators that warrant further investigation to
generate potential anticancer agents.
2 4
anhydrous Na SO and concentrated under vacuum to afford the crude
product. The crude product was purified by column chromatography
(eluent: petroleum ether/ethyl acetate = 1:1) to provide compound 10
1
as light yellow solid (7.70 g, yield: 93%). H NMR (400 MHz, DMSO‑d
6
)
δ 8.31 – 8.28 (m, 2H), 7.72 – 7.69 (m, 2H), 3.63 (s, 1H), 3.45 (s, 1H),
3.33 (s, 1H), 3.27 (s, 1H), 1.41 (s, 9H). 13C NMR (100 MHz, DMSO‑d
6
) δ
167.26, 153.77, 147.81, 142.03, 128.32, 123.74, 79.22, 46.65, 41.39,
27.97.
4
.3. The procedure for the synthesis of (4-nitrophenyl)(piperazin-1-yl)
4
. Experimental section
methanone hydrochloride(11)
4
.1. General
To a solution of compound 10 (1.68 g, 5 mmol) in dichloromethane
(
20 mL), 4 M HCl in dioxane (5 mL, 20 mmol) was added slowly. The
All reagents and solvents were purchased from commercial sources
mixture was stirred at room temperature for 2 h. The formed precipitate
was filtrated then the precipitate was dried under vacuum to afford the
compound 11, which was used directly without further purification.
and were used without further purification. Melting points were deter-
mined on X4 microscope and are uncorrected. 1H NMR and C NMR
spectra were recorded on Bruker AVANCEIII 400 MHz and 100 MHz
spectrometer respectively. High resolution mass spectrum (HRMS) was
recorded on a Thermo Scientific Orbitrap Elite MS.
13
4
.4. The procedure for the synthesis of pyridin-3-ylmethyl 4-(4-
nitrobenzoyl)piperazine-1-carbodithioate(12)
4
.2. The procedure for the synthesis of tert-butyl 4-(4-nitrobenzoyl)
3
To a solution of compound 11 (1.09 g, 4 mmol) in DMF (20 mL), Et N
piperazine-1-carboxylate (10)
(2.8 mL, 20 mmol) was added. The reaction mixture was stirred for 5
min and CS (0.46 g, 6 mmol) was added, the reaction mixture was
2
To a solution of tert-butyl piperazine-1-carboxylate (4.66 g, 25
mmol) and triethylamine (6.9 mL, 50 mmol) in THF (40 mL), 4-nitroben-
zoyl chloride (4.64 g, 25 mmol) was added slowly. The reaction mixture
was stirred at room temperature for 12 h. Water (100 mL) was added
and the mixture was extracted with ethyl acetate (20 mL × 3), the
combined organic phase was washed with water (30 mL × 2), dried over
stirred continuously for 30 min. 3-(chloromethyl)pyridine hydrochlo-
ride (0.66 g, 4 mmol) was added and this mixture was stirred at room
temperature for 4 h. Water (50 mL) was added and the mixture was
extracted with ethyl acetate (10 mL × 3), the combined organic phase
was washed with brine (30 mL × 2), dried over anhydrous Na
2 4
SO and
concentrated. The residue was purified by column chromatography
5

R. Li et al.
Bioorganic Chemistry 108 (2021) 104653
Table 2
NMR (100 MHz, DMSO‑d
6
) δ 194.80, 167.38, 150.07, 148.38, 147.93,
The antiproliferative activities of target compounds 8a-8m against PC3 and
141.68, 136.68, 132.65, 128.49, 123.74, 123.47, 45.80, 41.11, 37.44.
4
.5. The procedure for the synthesis of pyridin-3-ylmethyl 4-(4-
A375 cells.
aminobenzoyl)piperazine-1-carbodithioate(13)
2
To a solution of compound 12 (1.21 g, 3 mmol) in H O (8 mL) and
4
EtOH (24 mL), Fe (0.84 g, 15 mmol) and NH Cl (0.23 g, 4.2 mmol) were
a
Compd.
R
IC50 (µM)
PC3
added. The mixture was heated under reflux for 2 h. The mixture was
filtered off and the filtrate was concentrated under vacuum to afford the
crude product. The crude product was purified by column chromatog-
raphy (eluent: petroleum ether/ethyl acetate = 1:1) to provide com-
pound 13 as off- white solid (1.06 g, yield: 94%). 1H NMR (400 MHz,
A375
8
8
a
b
3.06 ± 0.28
2.67 ± 0.28
0.93 ± 0.19
0.85 ± 0.23
9.41 ± 3.48
DMSO‑d
6
) δ 8.62 (s, 1H), 8.47 (d, J = 4.0 Hz, 1H), 7.81 (d, J = 7.6 Hz,
1
H), 7.35 (dd, J = 7.6, 4.8 Hz, 1H), 7.18 (d, J = 8.8 Hz, 2H), 6.56 (d, J =
8
8
c
10.01 ± 1.38
8.4 Hz, 2H), 5.59 (s, 2H), 4.61 (s, 2H), 4.28 (s, 2H), 3.98 (s, 2H), 3.65 –
3
1
4
.63 (m, 4H). 13C NMR (100 MHz, DMSO‑d
) δ 194.50, 170.05, 150.82,
6
50.06, 148.35, 136.72, 132.71, 129.50, 123.49, 121.14, 112.62, 50.90,
9.48, 37.42.
d
6.60 ± 0.66
3.10 ± 1.83
4
.6. General procedure for the synthesis of compounds 8a-8m
8
8
e
f
6.55 ± 1.76
4.62 ± 0.58
1.98 ± 0.33
3.06 ± 0.28
To a solution of compound 13 (0.19 g, 0.5 mmol) and pyridine (0.16
mL, 2 mmol) in THF (10 mL) and DMF (5 mL), the substituted sulfonyl
chloride (0.5 mmol) was added slowly. The reaction mixture was stirred
at room temperature for 12 h. Water (50 mL) was added and the mixture
was extracted with ethyl acetate (10 mL × 3), the combined organic
phase was washed with 1 N hydrochloric acid (30 mL × 2) and water
8
8
g
6.36 ± 3.01
9.44 ± 0.59
7.61 ± 0.83
6.17 ± 0.39
(
30 mL × 2), dried over anhydrous Na
2 4
SO and concentrated under
h
vacuum to afford the crude product. The crude product was purified by
column chromatography to provide target products 8a-8m.
8
8
i
j
1.63 ± 0.09
1.86 ± 0.02
0.83 ± 0.24
0.73 ± 0.01
4.6.1. pyridin-3-ylmethyl 4-(4-(phenylsulfonamido)benzoyl)piperazine-1-
carbodithioate (8a)
Yield 74%. 1H NMR (400 MHz, DMSO‑d
) δ 10.64 (s, 1H), 8.61 (s,
6
1
3
3
1
1
H), 8.46 (dd, J = 4.8, 1.6 Hz, 1H), 7.82 – 7.79 (m, 3H), 7.62 – 7.55 (m,
H), 7.35 (d, J = 7.2 Hz, 3H), 7.16 (s, 2H), 4.60 (s, 2H), 4.27 (s, 2H),
8
8
k
l
0.97 ± 0.23
6.39 ± 1.33
0.28 ± 0.02
5.50 ± 0.35
13
.97 (s, 2H), 3.57 (s, 4H). C NMR (100 MHz, DMSO‑d ) δ 194.65,
6
68.72, 150.03, 148.34, 139.44, 139.24, 136.73, 133.10, 132.68,
30.26, 129.37, 128.67, 126.61, 123.46, 118.68, 50.98, 49.83, 37.40.
[M+H]+: 513.1089; found:
HRMS m/z: calcd for
24 25 4 3 3
C H N O S
5
13.1086.
4
.6.2. pyridin-3-ylmethyl 4-(4-(2-methoxyphenylsulfonamido)benzoyl)
8
m
2.95 ± 0.50
3.34 ± 0.64
piperazine-1- carbodithioate (8b)
NZT
——
——
> 10
> 10
1
Yield 62%. H NMR (400 MHz, DMSO‑d
6
) δ 10.36 (s, 1H), 8.60 (d, J
Paclitaxel
0.0046 ± 0.0050
0.0037 ± 0.0012
=
2.0 Hz, 1H), 8.46 (dd, J = 4.8, 1.6 Hz, 1H), 7.84 – 7.78 (m, 2H), 7.60 –
.55 (m, 1H), 7.36 – 7.29 (m, 3H), 7.18 – 7.13 (m, 3H), 7.06 (t, J = 7.6
Hz, 1H), 4.59 (s, 2H), 4.26 (s, 2H), 3.96 (s, 2H), 3.86 (s, 3H), 3.56 (s,
a
IC50 represents half inhibitory concentration.
7
1
3
4
1
1
6
H). C NMR (100 MHz, DMSO‑d ) δ 194.64, 168.79, 156.40, 150.07,
Table 3
48.38, 139.57, 136.68, 135.23, 132.65, 130.25, 129.68, 128.50,
Anti-proliferative activities of compound 8k and NZT against several tumor cell
26.32, 123.45, 120.13, 118.06, 112.98, 56.12, 50.58, 49.12, 37.41.
lines (Hela, MCF7, CNE2 and A549) and non-tumor cell line BJ.
HRMS m/z: calcd for
43.1185.
C
25
H
27
N
4
O
S
4 3
[M+H]+: 543.1194; found:
Compd.
IC50 (µM)
Hela
5
MCF7
CNE2
A549
BJ
8
k
1.23 ± 0.25
0.97 ± 0.13
>50
1.86 ± 0.10
13.4 ± 0.86
1.47 ± 0.14
>40
>40
4.6.3. pyridin-3-ylmethyl 4-(4-(3-methoxyphenylsulfonamido)benzoyl)
a
a
NZT
N.d
N.d
piperazine-1- carbodithioate (8c)
1
a
N.d represents not determined.
Yield 70%. H NMR (400 MHz, DMSO‑d
6
) δ 10.60 (d, J = 2.8 Hz, 1H),
8
.61 (s, 1H), 8.46 (dd, J = 4.8, 1.2 Hz, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.48
(
t, J = 8.0 Hz, 1H), 7.37 – 7.29 (m, 5H), 7.20 – 7.16 (m, 3H), 4.60 (s, 2H),
(
eluent: petroleum ether/ethyl acetate = 3:1) to provide compound 12
1
1
3
4.27 (s, 2H), 3.95 (s, 2H), 3.77 (s, 3H), 3.59 (s, 4H). C NMR (100 MHz,
DMSO‑d ) δ 194.67, 168.72, 159.37, 150.06, 148.37, 140.62, 139.21,
36.70, 132.66, 130.60, 130.40, 128.67, 123.45, 118.88, 118.80,
as off-white solid solid (1.45 g, yield: 90%). H NMR (400 MHz,
DMSO‑d
6
6
) δ 8.63 (s, 1H), 8.47 (d, J = 4.0 Hz, 1H), 8.32 – 8.29 (m, 2H),
.81 (d, J = 7.6 Hz, 1H), 7.75 – 7.73 (m, 2H), 7.35 (dd, J = 7.6, 4.8 Hz,
1
7
1
1
3
118.68, 111.69, 55.58, 50.69, 49.20, 37.41. HRMS m/z: calcd for
H), 4.61 (s, 2H), 4.39 – 3.97 (m, 4H), 3.78 (s, 2H), 3.47 (s, 2H).
C
+
C
25
H
27
N
4
O
S
4 3
[M+H] : 543.1194; found: 543.1191.
6

R. Li et al.
Bioorganic Chemistry 108 (2021) 104653
Fig. 4. Compound 8k inhibited the clone formation of MCF7 cells. MCF7 cells were exposed to 0, 2.5 and 5
μ
M compound 8k for 10 days.
4
.6.5. pyridin-3-ylmethyl 4-(4-(2-fluorophenylsulfonamido)benzoyl)
piperazine-1- carbodithioate (8e)
1
Yield 60%. H NMR (400 MHz, DMSO‑d
6
) δ 10.97 (s, 1H), 8.60 (d, J
=
2.0 Hz, 1H), 8.46 (dd, J = 4.8, 1.6 Hz, 1H), 7.92 – 7.88 (m, 1H), 7.81 –
7
.78 (m, 1H), 7.74 – 7.68 (m, 1H), 7.46 – 7.33 (m, 5H), 7.15 (d, J = 8.4
1
3
Hz, 1H), 4.59 (s, 2H), 4.26 (s, 2H), 3.96 (s, 2H), 3.57 (s, 4H). C NMR
100 MHz, DMSO‑d
(
6
) δ 194.66, 168.67, 158.13 (d, J = 253.0 Hz),
1
1
1
50.07, 148.37, 138.75, 136.67, 136.13 (d, J = 9.0 Hz), 132.64, 130.40,
30.31, 128.68, 126.97 (d, J = 14.0 Hz), 125.06 (d, J = 2.0 Hz), 123.43,
18.27, 117.37 (d, J = 20.0 Hz), 50.85, 49.24, 37.41. HRMS m/z: calcd
+
for C24
H24FN
4
O
3
S
3
[M+H] : 531.0995; found: 531.0991.
4
.6.6. pyridin-3-ylmethyl 4-(4-(3-fluorophenylsulfonamido)benzoyl)
piperazine-1- carbodithioate (8f)
Fig. 5. Compound 8k reduced the nuclear localization of PKM2. Cells were
Yield 67%. 1H NMR (400 MHz, DMSO‑d
) δ 10.73 (s, 1H), 8.61 (s,
treated with 0, 5 and 20 M compound 8k for 24 h, and the cytoplasmic and
μ
6
nuclear fractions were separated. Tubulin and HDAC1 were used as cytosolic
and nuclear loading controls, respectively.
1H), 8.46 (d, J = 4.8 Hz, 1H), 7.80 (d, J = 7.2 Hz, 1H), 7.65 – 7.62 (m,
3H), 7.51 (d, J = 4.4 Hz, 1H), 7.39 – 7.33 (m, 3H), 7.18 (d, J = 3.2 Hz,
2
H), 4.60 (s, 2H), 4.28 (s, 2H), 3.97 (s, 2H), 3.59 (s, 4H). 13C NMR (100
MHz, DMSO‑d
6
) δ 194.68, 168.68, 161.66 (d, J = 247.0 Hz), 150.08,
1
1
1
48.38, 141.41 (d, J = 6.0 Hz), 138.82, 136.70, 132.66, 131.82, 130.70,
28.74, 123.45, 122.95 (d, J = 3.0 Hz), 120.38 (d, J = 21.0 Hz), 119.12,
13.69 (d, J = 24.0 Hz), 50.68, 49.20, 37.43. HRMS m/z: calcd for
+
C
24
H24FN
4
O
S
3 3
[M+H] : 531.0995; found: 531.0989.
4
.6.7. pyridin-3-ylmethyl 4-(4-(4-fluorophenylsulfonamido)benzoyl)
piperazine-1- carbodithioate (8g)
1
Yield 64%. H NMR (400 MHz, DMSO‑d
6
) δ 10.63 (s, 1H), 8.60 (d, J
=
1.6 Hz, 1H), 8.46 (dd, J = 4.8, 1.6 Hz, 1H), 7.86 (dd, J = 8.8, 5.2 Hz,
2
3
3
1
1
3
H), 7.80 (d, J = 8.0 Hz, 1H), 7.41 (t, J = 8.8 Hz, 2H), 7.37 – 7.33 (m,
H), 7.15 (d, J = 8.0 Hz, 2H), 4.60 (s, 2H), 4.27 (s, 2H), 3.97 (s, 2H),
.58 (s, 4H). 13C NMR (100 MHz, DMSO‑d
) δ 194.66, 168.68, 163.14,
6
50.06, 148.37, 139.02, 136.69, 135.74, 132.66, 130.51, 129.72 (d, J =
0.0 Hz), 128.69, 123.45, 118.93, 116.61 (d, J = 22.0 Hz), 50.56, 49.48,
Fig. 6. Compound 8k down-regulated the downstream pathway of PKM2. Cells
+
7.40 (s). HRMS m/z: calcd for C24
H24FN
4
O
3
S
3
[M+H] : 531.0995;
were treated with 0, 1.25 and 5 M compound 8k for 24 h.
μ
found: 531.0992.
4
.6.4. pyridin-3-ylmethyl 4-(4-(4-methoxyphenylsulfonamido)benzoyl)
4
.6.8. pyridin-3-ylmethyl 4-(4-(naphthalene-1-sulfonamido)benzoyl)
piperazine-1- carbodithioate (8d)
1
piperazine-1- carbodithioate (8h)
Yield 66%. H NMR (400 MHz, DMSO‑d
2.0 Hz, 1H), 8.46 (dd, J = 4.8, 1.6 Hz, 1H), 7.81 – 7.72 (m, 3H), 7.36 –
.33 (m, 3H), 7.14 (d, J = 8.8 Hz, 2H), 7.07 (d, J = 8.8 Hz, 2H), 4.60 (s,
6
) δ 10.48 (s, 1H), 8.60 (d, J
1
Yield 71%. H NMR (400 MHz, DMSO‑d
6
) δ 11.04 (s, 1H), 8.71 (d, J
=
=
8.4 Hz, 1H), 8.59 (d, J = 2.0 Hz, 1H), 8.45 (dd, J = 4.8, 1.6 Hz, 1H),
7
2
H), 4.27 (s, 2H), 3.97 (s, 2H), 3.79 (s, 3H), 3.58 (s, 4H). 1 C NMR (100
) δ 194.66, 168.78, 162.56, 150.08, 148.38, 139.50,
36.69, 132.66, 131.01, 130.02, 128.90, 128.66, 123.45, 118.45,
3
8.29 (dd, J = 7.4, 1.0 Hz, 1H), 8.24 (d, J = 8.4 Hz, 1H), 8.08 (d, J = 7.6
Hz, 1H), 7.80 – 7.73 (m, 2H), 7.69 – 7.63 (m, 2H), 7.33 (dd, J = 7.8, 4.2
Hz, 1H), 7.27 (d, J = 8.4 Hz, 2H), 7.08 (d, J = 8.8 Hz, 2H), 4.58 (s, 2H),
MHz, DMSO‑d
6
1
1
4
.22 (s, 2H), 3.92 (s, 2H), 3.51 (s, 4H). 13C NMR (100 MHz, DMSO‑d
) δ
6
14.49, 55.63, 50.79, 49.44, 37.42. HRMS m/z: calcd for C25
H
27
N
4
O
4
S
3
+
194.75, 168.88, 150.06, 148.37, 139.13, 136.67, 134.63, 134.21,
[
M+H] : 543.1194; found: 543.1191.
1
33.77, 132.64, 129.94, 129.76, 129.15, 128.65, 128.23, 127.34,
7

R. Li et al.
Bioorganic Chemistry 108 (2021) 104653
1
27.03, 124.51, 124.10, 123.44, 117.65, 50.23, 49.49, 37.38. HRMS m/
[19].
.8. Anti-proliferation activity assay
+
z: calcd for C28
H
27
4
N O
3
S
3
[M+H] : 563.1245; found: 563.1240.
4
4
.6.9. pyridin-3-ylmethyl 4-(4-(thiophene-2-sulfonamido)benzoyl)
piperazine-1- carbodithioate (8i)
Cell lines (PC3, A375, Hela, MCF7, CNE2, A549 and BJ) were
1
◦
Yield 65%. H NMR (400 MHz, DMSO‑d
6
) δ 10.75 (s, 1H), 8.61 (d, J
cultured in DMEM containing 9% fetal bovine serum (FBS) at 37 C in
=
2.0 Hz, 1H), 8.46 (dd, J = 4.8, 1.6 Hz, 1H), 7.92 (d, J = 4.8 Hz, 1H),
2
5% CO . Cell viability was detected with the MTS assay (Promega) ac-
7
.80 (d, J = 8.0 Hz, 1H), 7.62 (dd, J = 2.6, 1.0 Hz, 1H), 7.39 (d, J = 8.4
cording to the manufacturer’s instructions. Briefly, 3000–5000 cells in
per well were plated in 96-well plates. After incubated for 12 h, the cells
were treated with different concentrations of tested compounds or
DMSO (as negative control) for 48 h. Then 20 µL MTS was added in per
Hz, 2H), 7.34 (dd, J = 7.8, 5.0 Hz, 1H), 7.21 (d, J = 8.0 Hz, 2H), 7.13
(
dd, J = 4.8, 4.0 Hz, 1H), 4.60 (s, 2H), 4.28 (s, 2H), 3.98 (s, 2H), 3.60 (s,
13
4
1
1
6
H). C NMR (100 MHz, DMSO‑d ) δ 194.66, 168.72, 150.06, 148.37,
◦
39.82, 138.96, 136.71, 134.32, 133.63, 132.68, 130.63, 128.65,
well and incubated at 37 C for 3 h. Absorbance of each well was
27.70, 123.46, 119.02, 50.51, 49.07, 37.41. HRMS m/z: calcd for
determined by a microplate reader (Flexstation 3) at a 490 nm wave
length. The IC50 values were calculated using Prism Graphpad software
of the triplicate experiment.
+
C
22
H
23
4
N O
3
S
4
[M+H] : 519.0653; found: 519.0642.
4
.6.10. pyridin-3-ylmethyl 4-(4-(2,3-dihydrobenzo[b][1,4]dioxine-6-
sulfonamido) benzoyl)piperazine-1-carbodithioate (8j)
Yield 63%. 1H NMR (400 MHz, DMSO‑d
) δ 10.71 (s, 1H), 8.61 (s,
4.9. Procedures for molecular docking
6
1
H), 8.46 (d, J = 4.0 Hz, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.36 – 7.28 (m,
Molecular docking was carried out by using Open-Source Auto-
dockvina (Scripps Research Institute), pre-processed by Schrodinger
Maestro (Peking University) and AutoDock Tools 1.5.6 rc3, assessed by
Schrodinger Maestro and Open-Source PyMOL 1.3.x (Schrodinger, LLC)
afterwards. The X-ray crystal structure of PKM2 and its reported acti-
vator NZT taken from PDB (4G1N) was obtained from protein data bank
(https://www.rcsb.org) and used as the input structure. Receptor
preparation: All solvent molecules or any other heteromolecules are
removed. All HETATM and hydrogen atoms are deleted and crystal cell
is removed. Alternative conformers of residues are also deleted to retain
one set. 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 modification on the ligand torsion tree.
Molecular docking: Parameter selection is done via AutoDock Tools
1.5.6 rc3. All the remaining parameters were kept as default.
5
H), 7.20 (d, J = 8.8 Hz, 2H), 7.00 (d, J = 8.4 Hz, 1H), 4.60 (s, 2H), 4.28
1
3
(
dd, J = 8.6, 4.6 Hz, 6H), 3.98 (s, 2H), 3.59 (s, 4H). C NMR (100 MHz,
DMSO‑d
6
) δ 194.65, 168.80, 150.05, 148.36, 147.29, 143.26, 139.52,
1
1
36.70, 132.68, 131.85, 130.02, 128.60, 123.47, 120.33, 118.50,
17.61, 115.65, 64.33, 64.02, 50.82, 49.43, 37.40. HRMS m/z: calcd for
+
C
26
H
27
4
N O
5
S
3
[M+H] : 571.1144; found: 571.1130.
4
.6.11. pyridin-3-ylmethyl 4-(4-(quinoline-8-sulfonamido)benzoyl)
piperazine-1- carbodithioate (8k)
1
Yield 58%. H NMR (400 MHz, DMSO‑d
6
) δ 10.49 (s, 1H), 9.13 (dd, J
=
4.4, 1.6 Hz, 1H), 8.59 (d, J = 2.0 Hz, 1H), 8.51 (dd, J = 8.0, 1.2 Hz,
1
7
H), 8.46 – 8.43 (m, 2H), 8.29 (d, J = 7.6 Hz, 1H), 7.80 – 7.70 (m, 3H),
.33 (dd, J = 7.6, 4.8 Hz, 1H), 7.22 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 8.4
1
3
Hz, 2H), 4.58 (s, 2H), 4.22 (s, 2H), 3.92 (s, 2H), 3.51 (s, 4H). C NMR
(
100 MHz, DMSO‑d
6
) δ 194.63, 168.72, 151.47, 150.06, 148.36, 142.70,
1
1
39.47, 136.97, 136.65, 135.17, 134.41, 132.63, 132.23, 129.76,
28.40, 125.62, 123.42, 122.63, 118.33, 50.62, 49.08, 37.42. HRMS m/
4.10. Colony formation assay
+
z: calcd for C27
H
26
5
N O
3
S
3
[M+H] : 564.1198; found: 564.1185.
To test the clonogenic capacity of cells in vitro, 600 cells (MCF7)
were plated in six-well culture plates. After 24 h incubation, cells were
4
.6.12. pyridin-3-ylmethyl 4-(4-(1H-pyrrolo[2,3-b]pyridine-3-
sulfonamido)benzoyl) piperazine-1-carbodithioate (8l)
treated with 0, 2.5 and 5 M compound 8k for 24 h. Then the medium
μ
1
Yield 57%. H NMR (400 MHz, DMSO‑d
H), 8.60 (d, J = 2.0 Hz, 1H), 8.45 (dd, J = 4.6, 1.4 Hz, 1H), 8.34 (dd, J
4.8, 1.6 Hz, 1H), 8.21 (s, 1H), 8.18 (d, J = 8.0 Hz, 1H), 7.79 (d, J = 8.0
Hz, 1H), 7.35 – 7.24 (m, 4H), 7.17 – 7.15 (m, 2H), 4.59 (s, 2H), 4.24 (s,
6
) δ 12.65 (s, 1H), 10.55 (s,
was changed into fresh medium without compound 8k and the cells
continued to grow for 10 days to form colonies, and then fixed with
methanol and stained with crystal violet.
1
=
2
1
1
1
H), 3.94 (s, 2H), 3.56 (s, 4H). 13C NMR (100 MHz, DMSO‑d
6
) δ 194.63,
4.11. The separation of cytoplasmic and nuclear protein
68.79, 150.06, 148.37, 147.96, 144.67, 139.69, 136.69, 132.66,
31.80, 129.56, 128.55, 127.47, 123.45, 117.88, 117.61, 115.48,
MCF7 cells were treated with 0, 5 and 20 M compound 8k for 24 h,
μ
11.84, 50.52, 49.99, 37.39. HRMS m/z: calcd for C25
H
25
N
6
O
S
3 3
and cells were harvested and rinsed with PBS. Cells were suspended in 5
volumes of cold Buffer A (10 mM HEPES pH = 7.9, 0.1 mM EDTA, 0.1
mM EGTA, 10 mM KCl, protease inhibitor cocktail, 1 mM DTT, 0.15%
NP-40), and allowed to swell on ice for 15 min, the homogenate was
centrifuged for 1 min at 12,000g. The supernatant containing the cyto-
plasm fraction was transferred to a fresh tube. The crude nuclear fraction
+
[
M+H] : 553.1150; found: 553.1146.
4
.6.13. pyridin-3-ylmethyl 4-(4-(vinylsulfonamido)benzoyl)piperazine-1-
carbodithioate (8m)
1
Yield 42%. H NMR (400 MHz, DMSO‑d
6
) δ 10.35 (s, 1H), 8.61 (d, J
=
2.0 Hz, 1H), 8.46 (dd, J = 4.8, 1.6 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H),
was suspended in buffer B (10 mM Tris pH = 7.9, 1.5 mM MgCl , 10 mM
2
7
.41 (d, J = 8.4 Hz, 2H), 7.35 (dd, J = 7.6, 4.8 Hz, 1H), 7.18 (d, J = 8.8
KCl, 400 mM NaCl, 0.4% Triton X-100 and protease inhibitor cocktail),
◦
Hz, 2H), 6.87 – 6.81 (m, 1H), 6.18 (d, J = 16.4 Hz, 1H), 6.07 (d, J = 10.0
and vortexed at 4 C for 30 min. The homogenate was centrifuged for 15
Hz, 1H), 4.60 (s, 2H), 4.29 (s, 2H), 3.99 (s, 2H), 3.63 (s, 4H). 13C NMR
min at 12,000g, and the nuclear extract was transferred to a fresh tube.
(
100 MHz, DMSO‑d
6
) δ 194.69, 168.89, 150.09, 148.40, 139.61, 136.71,
1
3
4
36.26, 132.68, 129.86, 128.70, 127.96, 123.47, 118.34, 50.79, 49.15,
4.12. Western blot
+
7.45. HRMS m/z: calcd for C20
H
23
4
N O
3
S
3
[M+H] : 463.0932; found:
63.0929.
After cells were treated with compound 8k, western blotting was
performed according to the standard protocol. The Stat3, phospho-Stat3
(Tyr 705), Erk, phospho-Erkand PKM2 antibodies were purchased from
Cell Signaling Technology. β-Actin antibody was purchased from MBL.
HDAC1 was purchased from Santa Cruz biotechnology. Tubulin was
purchased from TransGen Biotech.
4
.7. PKM2 activity assay
Pyruvate kinase activity was detected with a fluorescent pyruvate
kinase-lactate dehydrogenase coupled assay as previously described
8

R. Li et al.
Bioorganic Chemistry 108 (2021) 104653
Declaration of Competing Interest
S. Auld, C.J. Thomas, Evaluation of thieno[3,2-b]pyrrole[3,2-d]pyridazinones as
activators of the tumor cell specific M2 isoform of pyruvate kinase, Bioorg. Med.
Chem. Lett. 20 (2010) 3387–3393.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104653.
4
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