Structure based discovery of novel hexokinase 2 inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.103609

Hexokinase 2 (HK2) is over-expressed in most of human cancers and has been proved to be a promising target for cancer therapy. In this study, based on the structure of HK2, we screened over 6 millions of compounds to obtain the lead. A total of 26 (E)-N′-(2,3,4-trihydroxybenzylidene) arylhydrazide derivatives were then designed, synthesized, and evaluated for their HK2 enzyme activity and IC₅₀ values against two cancer cell lines. Most of the 26 target compounds showed excellently in vitro activity. Among them, compound 3j showed the strongest inhibitory effects on HK2 enzyme activity with an IC₅₀ of 0.53 ± 0.13 μM and exhibited the most potent growth inhibition against SW480 cells with an IC₅₀ of 7.13 ± 1.12 μM, which deserves further studies.

Full text of DOI:10.1016/j.bioorg.2020.103609

BioorganicChemistry96(2020)103609
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Structure based discovery of novel hexokinase 2 inhibitors
Yang Liua,1, Mingxue Lia,1, Yujie Zhanga,1, Canrong Wub, Kaiyin Yangb, Suyu Gaoa,
⁎
⁎
⁎
⁎
Mengzhu Zhengb, , Xingzhou Lic, , Hua Lia,b, , Lixia Chena,
a Wuya College of Innovation, Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016,
China
b Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and
Technology, Wuhan 430030, China
c National Engineering Research Center for the Emergency Drug, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Hexokinase 2 (HK2) is over-expressed in most of human cancers and has been proved to be a promising target for
cancer therapy. In this study, based on the structure of HK2, we screened over 6 millions of compounds to obtain
the lead. A total of 26 (E)-N′-(2,3,4-trihydroxybenzylidene) arylhydrazide derivatives were then designed,
synthesized, and evaluated for their HK2 enzyme activity and IC50 values against two cancer cell lines. Most of
the 26 target compounds showed excellently in vitro activity. Among them, compound 3j showed the strongest
Cancer metabolism
Drug discovery
HK2 inhibitors
Structure-based virtual ligand screening
inhibitory effects on HK2 enzyme activity with an IC50 of 0.53
0.13 μM and exhibited the most potent growth
inhibition against SW480 cells with an IC50 of 7.13
1.12 μM, which deserves further studies.
1. Introduction
cancer cells [9–11].
Although HK2 has been a potential target for cancer treatment for
many years, several challenges still remain in the discovery and design
of efficient and selective HK2 inhibitors. These challenges are arise out
of the structural properties of HK2, including the high polarity of the
active site of the enzyme and the difficulty in specifically inhibiting the
different isoenzymes. Therefore, there is still no potent and selective
HK2 inhibitor in clinical use thus far [12]. Metformin, 2-deoxyglucose,
and 3-bromopyruvate are the most commonly reported HK2 inhibitors
[13–15]. However, there are shortcomings with each of these com-
pounds that prevent them from being developed as viable HK2 in-
hibitors. For example, metformin shows only a weak antitumor activity
in vivo and in vitro [16]. The antitumor potency of 2-deoxyglucose is
also weak, and only evident at non-physiologic concentrations [17].
There are adverse effects associated with the use of 3-bromopyruvate
because of its off-target effects. The compound also has a short half-life
[18]. Therefore, discovering more effective, selective, and less toxic
HK2 inhibitors remains a significant challenge to the development of
cancer therapies [19].
In the 1920s, Otto Warburg demonstrated that the energy metabo-
lism pattern of tumor cells was different from that of the normal tissue
cells. Normal tissues mainly produce energy through oxidative phos-
phorylation in the mitochondria, while tumor cells mainly rely on
aerobic glycolysis to meet the metabolic needs of cell proliferation [1].
The glycolytic pathway involves a variety of key enzymes, including
glucose transporter (GLUT), hexokinase (HK), phosphoric acid fructose
kinase (PFK) [2], pyruvate kinase (PK) [3] and lactate dehydrogenase
(LDH) [4]. The inhibition of these key metabolic proteins can stem the
energy supply to tumor cells, thus inhibiting cell growth [1]. Among the
4 hexokinase isomers (HK1-4), HK2 has a distinct expression pattern,
where it is not expressed in most normal tissues, but is overexpressed in
more than 70% of cancer cells [5]. This suggests that specific targeting
of HK2 may be a good treatment strategy to suppress cancer cell growth
by inhibiting glycolysis [6]. Hexokinase 2 (HK2) catalyzes the irrever-
sible first step of the glycolytic pathway, namely, converting glucose
into glucose-6-phosphate (G6P) [7]. The inhibition of HK2 can reduce
the production of ATP in cancer cells, decrease the carbon supply from
glycolysis to the carboxylic acid (TCA) cycle, and decrease the synthesis
of nucleotides and fatty acids [8], all of which culminate in the death of
In our previous study, structure-based virtual ligand screening was
adopted to screen the FDA-approved drug database, and Benserazide, a
peripherally acting DOPA decarboxylase inhibitor used to treat
⁎ Corresponding authors at: Wuya College of Innovation, Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang
Pharmaceutical University, Shenyang 110016, China (H. Li).
E-mail addresses: mengzhu_zheng@hust.edu.cn (M. Zheng), xingzhouli@aliyun.com (X. Li), li_hua@hust.edu.cn (H. Li), syzyclx@163.com (L. Chen).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2020.103609
Received 5 December 2019; Received in revised form 19 January 2020; Accepted 20 January 2020
Availableonline21January2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

Y. Liu, et al.
BioorganicChemistry96(2020)103609
Parkinson’s disease, was identified as an HK2 inhibitor [20]. En-
couraged by this finding, ZINC lead-like compound library with
6,053,287 molecules was further screened, which led to the discovery
of a new scaffold of HK2 inhibitors [21]. A novel scaffold of (E)-N′-
(2,3,4-trihydroxybenzylidene) arylhydrazide was identified as the lead
compound. A series of novel HK2 inhibitors were then designed and
synthesized based on the structure of the lead compound. Among them,
compound 3j displayed the strongest potency in inhibiting HK2 enzyme
activity and the proliferation of cancer cells.
2.1.5. (E)-4-(methylsulfonyl)-N′-(2,3,4-trihydroxybenzylidene)
benzohydrazide (3c)
Yield 79.6%. 1H NMR (400 MHz, DMSO‑d6) δ 12.20 (s, 1H), 11.36
(s, 1H), 9.59 (s, 1H), 8.56 (s, 1H), 8.50 (s, 1H), 8.16 (d, J = 8.5 Hz, 2H),
8.10 (d, J = 8.5 Hz, 2H), 6.84 (d, J = 8.5 Hz, 1H), 6.42 (d, J = 8.4 Hz,
1H), 3.30 (s, 3H); 13C NMR (100 MHz, DMSO‑d6) δ 161.7, 151.3, 149.4,
148.0, 143.8, 137.9, 133.1, 129.0, 127.6, 121.7, 111.2, 108.2, 43.7.
HRMS (ESI) m/z 351.0652 [M + H]+ (calcd for C15H15N2O6S,
351.0651).
2.1.6. (E)-3-(methylsulfonyl)-N′-(2,3,4-trihydroxybenzylidene)
benzohydrazide (3d)
2. Experiment
Yield 85.5%. 1H NMR (400 MHz, DMSO‑d6) δ 12.23 (s, 1H), 11.38
(s, 1H), 9.57 (s, 1H), 8.56 (brs, 1H), 8.51 (s, 1H), 8.46 (t, J = 1.5 Hz,
1H), 8.27 (d, J = 7.9 Hz, 1H), 8.16 (d, J = 9.4 Hz, 1H), 7.84 (t,
J = 7.8 Hz, 1H), 6.84 (d, J = 8.5 Hz, 1H), 6.42 (d, J = 8.5 Hz, 1H),
3.30 (s, 3H); 13C NMR (100 MHz, DMSO‑d6) δ 161.4, 151.2, 149.4,
148.0, 141.6, 134.4, 133.2, 133.0, 130.5, 130.4, 126.4, 121.6, 111.2,
2.1. Chemistry
All reactions were carried out in flame-dried glassware under an
atmosphere of argon, all reagents were procured from commercial
sources (purity > 99%) and used without further purification. Silica
gel GF254 and silica gel (200–300 mesh) were respectively used for
thin-layer chromatography and column chromatography. NMR spectra
were recorded on a Bruker AM-400 spectrometer, with 1H and 13C NMR
chemical shifts referenced to the solvent or solvent impurity peaks for
DMSO‑d6 (δH 2.50 and δC 39.52). HRESIMS data were acquired using a
Thermo Fisher LC-LTQ-Orbitrap XL spectrometer and an electrospray
ionization and a hybrid quadrupole time-of-flight (q-TOF) mass spec-
trometer (model 6540, Agilent). All other common chemicals, solvents
and reagents were of highest grade available from various commercial
sources.
108.2, 43.9. HRMS (ESI) m/z 351.0639 [M
+
H]+ (calcd for
C15H15N2O6S, 351.0651).
2.1.7. (E)-4-butyl-N′-(2,3,4-trihydroxybenzylidene)benzohydrazide (3e)
Yield 64.3%. 1H NMR (400 MHz, DMSO‑d6) δ 11.91 (s, 1H), 11.57
(s, 1H), 9.50 (s, 1H), 8.51 (brs, 1H), 8.45 (s, 1H), 7.85 (d, J = 8.2 Hz,
2H), 7.36 (d, J = 8.2 Hz, 2H), 6.78 (d, J = 8.5 Hz, 1H), 6.40 (d,
J = 8.4 Hz, 1H), 2.68 (t, J = 7.6 Hz, 2H), 1.62–1.55 (m, 2H), 1.36–1.27
(m, 2H), 0.91 (t, J = 7.3 Hz, 3H); 13C NMR (100 MHz, DMSO‑d6) δ
162.9, 150.4, 149.1, 147.9, 147.1, 133.1, 130.7, 128.9, 128.0, 121.6,
111.3, 108.0, 35.1, 33.3, 22.1, 14.2. HRMS (ESI) m/z 329.1485
[M + H]+ (calcd for C18H21N2O4, 329.1501).
2.1.1. General procedure for the synthesis of hydrazides 2
Hydrazine hydrate (5 mL, 40%) was added to a solution of required
ester (5.0 mmol) in methanol (20 mL). The solution was refluxed for
12–24 h and monitored by TLC until starting material was completely
consumed. After that, solvent was evaporated under reduced pressure
and a small amount of water (5 mL) was added to precipitate the hy-
drazide, which was filtered and dried in vacuum to give a shiny white to
yellow solid in excellent yields, without further purification.
2.1.8. (E)-3-chloro-N′-(2,3,4-trihydroxybenzylidene)benzohydrazide (3f)
Yield 88.9%. 1H NMR (400 MHz, DMSO‑d6) δ 12.06 (s, 1H), 11.40
(s, 1H), 9.56 (s, 1H), 8.55 (s, 1H), 8.47 (s, 1H), 7.97 (t, J = 1.8 Hz, 1H),
7.90–7.88 (m, 1H), 7.70–7.67 (m, 1H), 7.58 (t, J = 7.9 Hz, 1H), 6.82
(d, J = 8.5 Hz, 1H), 6.41 (d, J = 8.4 Hz, 1H); 13C NMR (100 MHz,
DMSO‑d6) δ 161.5, 151.0, 149.3, 148.0, 135.3, 133.8, 133.1, 132.1,
131.0, 127.7, 126.8, 121.6, 111.2, 108.1. HRMS (ESI) m/z 307.0494
[M + H]+ (calcd for C14H12ClN2O4, 307.0486).
2.1.2. General procedure for the synthesis of hydrazides 3a-3z
2,3,4-Trihydroxybenzaldehyde (0.55 g, 3.6 mmol) was added to a
solution of hydrazides (3.0 mmol) in methanol (20 mL). The solution
was stirred for 12 h under room temperature, which was then filtered
and finally recrystallized from ethanol, to give the target hydrazone
derivatives as white to gray powdered solid.
2.1.9. (E)-4-chloro-N′-(2,3,4-trihydroxybenzylidene)benzohydrazide (3g)
Yield 65.3%. 1H NMR (400 MHz, DMSO‑d6) δ 12.04 (s, 1H), 11.46
(s, 1H), 9.55 (s, 1H), 8.54 (s, 1H), 8.47 (s, 1H), 7.96–7.94 (m, 2H), 7.62
(d, J = 8.6 Hz, 2H), 6.81 (d, J = 8.5 Hz, 1H), 6.41 (d, J = 8.4 Hz, 1H);
13C NMR (100 MHz, DMSO‑d6) δ 161.9, 150.8, 149.3, 148.0, 137.1,
133.1, 132.1, 129.9, 129.1, 121.6, 111.2, 108.1. HRMS (ESI) m/z
307.0494 [M + H]+ (calcd for C14H12ClN2O4, 307.0486).
2.1.3. (E)-N′-(2,3,4-trihydroxybenzylidene)quinoline-2-carbohydrazide
(3a)
Yield 72.1%. 1H NMR (400 MHz, DMSO‑d6) δ 12.42 (s, 1H), 11.62
(s, 1H), 9.58 (s, 1H), 8.77 (s, 1H), 8.62 (d, J = 8.5 Hz, 1H), 8.57 (brs,
1H), 8.24–8.21 (m, 2H), 8.11 (d, J = 7.8 Hz, 1H), 7.94–7.90 (m, 1H),
7.78–7.74 (m, 1H), 6.80 (d, J = 8.5 Hz, 1H), 6.44 (d, J = 8.0 Hz, 1H);
13C NMR (100 MHz, DMSO‑d6) δ 160.7, 152.3, 149.9, 149.4, 148.1,
146.5, 138.5, 133.2, 131.2, 129.6, 129.4, 128.8, 128.6, 121.9, 119.5,
111.3, 108.22. HRMS (ESI) m/z 324.0981 [M + H]+ (calcd for
2.1.10. (E)-4-methyl-N′-(2,3,4-trihydroxybenzylidene)
benzenesulfonohydrazide (3h)
Yield 76.6%. 1H NMR (400 MHz, DMSO‑d6) δ 11.28 (s, 1H), 10.06
(s, 1H), 9.57 (s, 1H), 8.50 (s, 1H), 8.02 (s, 1H), 7.73 (d, J = 8.3 Hz, 2H),
7.43 (d, J = 8.1 Hz, 2H), 6.73 (d, J = 8.6 Hz, 1H), 6.34 (d, J = 8.5 Hz,
1H), 2.37 (s, 3H); 13C NMR (100 MHz, DMSO‑d6) δ 150.3, 149.2, 147.2,
144.1, 136.1, 133.0, 130.2, 127.6, 120.4, 111.3, 108.1, 21.4. HRMS
(ESI) m/z 323.0699 [M + H]+ (calcd for C14H15N2O5S, 323.0702).
C
17H14N3O4, 324.0984).
2.1.4. (E)-4-(2-hydroxyethoxy)-N′-(2,3,4-trihydroxybenzylidene)
benzohydrazide (3b)
2.1.11. (E)-4-methoxy-N′-(2,3,4-trihydroxybenzylidene)benzohydrazide
(3i)
Yield 70.9%. 1H NMR (400 MHz, DMSO‑d6) δ 11.86 (s, 1H), 11.61
(s, 1H), 9.49 (s, 1H), 8.51 (s, 1H), 8.44 (s, 1H), 7.90 (d, J = 8.8 Hz, 2H),
7.08 (d, J = 8.9 Hz, 2H), 6.77 (d, J = 8.5 Hz, 1H), 6.40 (d, J = 8.0 Hz,
1H), 4.95 (brs, 1H), 4.08 (t, J = 4.9 Hz, 2H), 3.75 (t, J = 4.8 Hz, 2H);
13C NMR (100 MHz, DMSO‑d6) δ 162.4, 162.0, 150.1, 149.0, 147.9,
133.1, 129.9, 125.2, 121.6, 114.7, 111.3, 108.0, 70.2, 59.9. HRMS (ESI)
m/z 333.1073 [M + H]+ (calcd for C16H17N2O6, 333.1087).
Yield 74.9%. 1H NMR (400 MHz, DMSO‑d6) δ 11.86 (s, 1H), 11.62
(s, 1H), 9.49 (s, 1H), 8.52 (s, 1H), 8.45 (s, 1H), 7.92 (d, J = 8.8 Hz, 2H),
7.08 (d, J = 8.9 Hz, 2H), 6.77 (d, J = 8.5 Hz, 1H), 6.40 (d, J = 8.4 Hz,
1H), 3.84 (s, 3H); 13C NMR (100 MHz, DMSO‑d6) δ 162.5, 162.4, 150.1,
149.0, 147.9, 133.1, 129.9, 125.3, 121.6, 114.2, 111.3, 108.0, 55.9.
HRMS (ESI) m/z 303.0984 [M
+
H]+ (calcd for C15H15N2O5,
303.0981).
2

Y. Liu, et al.
BioorganicChemistry96(2020)103609
2.1.12. (E)-4-nitro-N′-(2,3,4-trihydroxybenzylidene)benzohydrazide (3j)
Yield 85.0%. 1H NMR (400 MHz, DMSO‑d6) δ 12.26 (s, 1H), 11.33
(s, 1H), 9.60 (s, 1H), 8.56 (brs, 1H), 8.50 (s, 1H), 8.38 (d, J = 12.0 Hz,
2H), 8.16 (d, J = 12.0 Hz, 2H), 6.84 (d, J = 8.5 Hz, 1H), 6.42 (d,
J = 8.5 Hz, 1H); 13C NMR (100 MHz, DMSO‑d6) δ 161.3, 151.5, 149.7,
149.5, 148.0, 139.0, 133.1, 129.5, 124.1, 121.7, 111.2, 108.2. HRMS
(ESI) m/z 318.0723 [M + H]+ (calcd for C14H12N3O6, 318.0726).
2.1.19. (E)-N-{4-[2-(2,3,4-trihydroxybenzylidene)hydrazine-1-carbonyl]
phenyl}cyclopropanecarboxamide (3q)
Yield 76.9%. 1H NMR (400 MHz, DMSO‑d6) δ 11.88 (s, 1H), 11.59
(s, 1H), 10.52 (s, 1H), 9.50 (s, 1H), 8.51 (s, 1H), 8.44 (s, 1H), 7.89 (d,
J = 8.7 Hz, 2H), 7.73 (d, J = 8.8 Hz, 2H), 6.78 (d, J = 8.5 Hz, 1H),
6.40 (d, J = 8.4 Hz, 1H), 1.85–1.79 (m, 1H), 0.85–0.83 (m, 4H); 13C
NMR (100 MHz, DMSO‑d6) δ 172.6, 162.4, 150.2, 149.1, 147.9, 142.9,
133.1, 129.0, 127.3, 121.6, 118.7, 111.3, 108.0, 15.1, 8.0. HRMS (ESI)
m/z 356.1240 [M + H]+ (calcd for C18H18N3O5, 356.1246).
2.1.13. (E)-3-methoxy-N′-(2,3,4-trihydroxybenzylidene)benzohydrazide
(3k)
Yield 81.8%. 1H NMR (400 MHz, DMSO‑d6) δ 11.94 (s, 1H), 11.52
(s, 1H), 9.53 (s, 1H), 8.54 (brs, 1H), 8.47 (s, 1H), 7.52–7.44 (m, 3H),
7.19–7.16 (m, 1H), 6.79 (d, J = 8.5 Hz, 1H), 6.41 (d, J = 8.4 Hz, 1H),
3.84 (s, 3H); 13C NMR (100 MHz, DMSO‑d6) δ 162.7, 159.7, 150.7,
149.2, 147.9, 134.7, 133.1, 130.2, 121.6, 120.2, 118.0, 113.2, 111.2,
2.1.20. (E)-5-hydroxy-N′-(2,3,4-trihydroxybenzylidene)-3H-indole-2-
carbohydrazide (3r)
Yield 73.0%. 1H NMR (400 MHz, DMSO‑d6) δ 11.93 (s, 1H), 11.54
(s, 1H), 11.49 (s, 1H), 9.51 (s, 1H), 8.91 (s, 1H), 8.52 (s, 1H), 8.45 (s,
1H), 7.28 (d, J = 8.8 Hz, 1H), 7.07–7.10 (m, 1H), 6.93 (d, J = 2.2 Hz,
1H), 6.82–6.77 (m, 2H), 6.41 (d, J = 8.4 Hz, 1H); 13C NMR (100 MHz,
DMSO‑d6) δ 157.6, 151.7, 149.6, 149.1, 147.8, 133.1, 132.1, 130.3,
128.1, 121.4, 115.6, 113.3, 111.4, 108.1, 104.8, 103.1. HRMS (ESI) m/
z 328.0928 [M + H]+ (calcd for C16H14N3O5, 328.0933).
108.1, 55.8. HRMS (ESI) m/z 303.0972 [M
15H15N2O5, 303.0981).
+
H]+ (calcd for
C
2.1.14. (E)-2-phenyl-N′-(2,3,4-trihydroxybenzylidene)acetohydrazide (3l)
Yield 66.3%. 1H NMR (400 MHz, DMSO‑d6) δ 11.72 (s, 1H), 11.30
(s, 1H), 9.49 (s, 1H), 8.49 (s, 1H), 8.23 (s, 1H), 7.35–7.27 (m, 5H), 6.77
(d, J = 8.5 Hz, 1H), 6.38 (d, J = 8.5 Hz, 1H), 3.54 (s, 2H); 13C NMR
(100 MHz, DMSO‑d6) δ 166.4, 149.4, 149.1, 147.8, 136.0, 133.1, 129.5,
128.8, 127.1, 121.5, 111.1, 108.0, 41.3. HRMS (ESI) m/z 287.1036
[M + H]+ (calcd for C15H15N2O4, 287.1032).
2.1.21. (E)-N′-(2,3,4-trihydroxybenzylidene)nicotinohydrazide (3s)
Yield 87.2%. 1H NMR (400 MHz, DMSO‑d6) δ 12.20 (s, 1H), 11.44
(s, 1H), 9.60 (s, 1H), 9.16 (m, 1H), 8.86 (dd, J = 4.9, 1.7 Hz, 1H), 8.60
(s, 1H), 8.55 (s, 1H), 8.35 (dt, J = 8.0, 2.0 Hz, 1H), 7.65–7.68 (m, 1H),
6.91 (d, J = 8.5 Hz, 1H), 6.49 (d, J = 8.4 Hz, 1H); 13C NMR (100 MHz,
DMSO‑d6) δ 161.5, 152.8, 151.0, 149.4, 149.0, 148.0, 135.8, 133.1,
129.2, 124.1, 121.6, 111.2, 108.2. HRMS (ESI) m/z 274.0811
[M + H]+ (calcd for C13H12N3O4, 274.0828).
2.1.15. (E)-3-nitro-N′-(2,3,4-trihydroxybenzylidene)benzohydrazide (3m)
Yield 69.2%. 1H NMR (400 MHz,DMSO‑d6) δ 12.29 (s, 1H), 11.34 (s,
1H), 9.59 (s, 1H), 8.78 (t, J = 1.9 Hz, 1H), 8.56 (brs, 1H), 8.52 (s, 1H),
8.47–8.37 (m, 2H), 7.86 (t, J = 8.0 Hz, 1H), 6.85 (d, J = 8.5 Hz, 1H),
6.42 (d, J = 8.4 Hz, 1H); 13C NMR (100 MHz, DMSO‑d6) δ 160.8,
151.3, 149.4, 148.2, 148.0, 134.8, 134.5, 133.2, 130.8, 126.8, 122.6,
121.6, 111.2, 108.2. HRMS (ESI) m/z 318.0721 [M + H]+ (calcd for
2.1.22. (E)-2-hydroxy-N′-(2,3,4-trihydroxybenzylidene)benzohydrazide
(3t)
Yield 81.0%. 1H NMR (400 MHz, DMSO‑d6) δ 11.93 (s, 1H), 11.86
(s, 1H), 11.38 (s, 1H), 9.53 (s, 1H), 8.52 (d, J = 1.6 Hz, 2H), 7.89 (dd,
J = 7.9, 1.7 Hz, 1H), 7.43–7.47 (m, 1H), 7.02–6.92 (m, 2H), 6.82 (d,
J = 8.5 Hz, 1H), 6.41 (d, J = 8.4 Hz, 1H); 13C NMR (100 MHz,
DMSO‑d6) δ 164.6, 159.5, 151.3, 149.4, 148.0, 134.3, 133.1, 128.8,
121.6, 119.4, 117.7, 115.9, 111.2, 108.2. HRMS (ESI) m/z 289.0776
[M + H]+ (calcd for C14H13N2O5, 289.0824).
C
14H12N3O6, 318.0726).
2.1.16. (E)-N′-(2,3,4-trihydroxybenzylidene)-[1,1′-biphenyl]-4-
carbohydrazide (3n)
Yield 70.5%. 1H NMR (400 MHz, DMSO‑d6) δ 12.06 (s, 1H), 11.57
(s, 1H), 9.56 (s, 1H), 8.57 (s, 1H), 8.50 (s, 1H), 8.04 (d, J = 8.3 Hz, 2H),
7.85 (d, J = 8.3 Hz, 2H), 7.76 (d, J = 7.4 Hz, 2H), 7.53–7.41 (m, 3H),
6.81 (d, J = 8.5 Hz, 1H), 6.42 (d, J = 8.4 Hz, 1H); 13C NMR (100 MHz,
DMSO‑d6) δ 162.6, 150.6, 149.2, 147.9, 143.8, 139.4, 133.1, 132.0,
129.5, 128.7, 127.4, 127.2, 121.6, 111.3, 108.1. HRMS (ESI) m/z
349.1238 [M + H]+ (calcd for C20H17N2O4, 349.1188).
2.1.23. (E)-4-fluoro-N′-(2,3,4-trihydroxybenzylidene)benzohydrazide
(3u)
Yield 62.5%. 1H NMR (400 MHz, DMSO‑d6) δ 11.96 (s, 1H), 11.48
(s, 1H), 9.46 (s, 1H), 8.47 (s, 1H), 8.05–7.96 (m, 2H), 7.38 (t,
J = 8.8 Hz, 2H), 6.80 (d, J = 8.4 Hz, 1H), 6.41 (d, J = 8.4 Hz, 1H); 13
C
NMR (100 MHz, DMSO‑d6) δ 163.4, 161.9, 150.6, 149.2, 147.9, 133.1,
130.7, 130.6, 129.8, 129.8, 121.6, 116.1, 115.8, 111.2, 108.1. HRMS
(ESI) m/z 291.0739 [M + H]+ (calcd for C14H12FN2O4, 291.0781).
2.1.17. (E)-N′-(2,3,4-trihydroxybenzylidene)-2,3-dihydrobenzo[b][1,4]
dioxine-2-carbohydrazide (3o)
Yield 82.2%. 1H NMR (400 MHz, DMSO‑d6) δ 11.74 (s, 1H), 11.18
(s, 1H), 9.56 (s, 1H), 8.54 (s, 1H), 8.41 (s, 1H), 7.05–7.02 (m, 1H),
6.92–6.85 (m, 3H), 6.77 (d, J = 8.5 Hz, 1H), 6.39 (d, J = 8.5 Hz, 1H),
5.00–4.99 (m, 1H), 4.43–4.33 (m, 2H); 13C NMR (100 MHz, DMSO‑d6) δ
163.4, 151.5, 149.4, 147.9, 143.4, 142.5, 133.1, 122.2, 122.1, 121.6,
117.8, 117.6, 111.0, 108.2, 72.5, 65.0. HRMS (ESI) m/z 331.0913
[M + H]+ (calcd for C16H15N2O6, 331.0930).
2.1.24. (E)-4-chloro-3-nitro-N′-(2,3,4-trihydroxybenzylidene)
benzohydrazide (3v)
Yield 73.6%. 1H NMR (400 MHz, DMSO‑d6) δ 12.21 (s, 1H), 11.24
(s, 1H), 9.53 (s, 1H), 8.60 (d, J = 2.1 Hz, 1H), 8.50 (d, J = 7.9 Hz, 2H),
8.23 (dd, J = 8.4, 2.2 Hz, 1H), 7.99 (d, J = 8.4 Hz, 1H), 6.85 (d,
J = 8.5 Hz, 1H), 6.42 (d, J = 8.5 Hz, 1H); 13C NMR (100 MHz,
DMSO‑d6) δ 160.0, 151.3, 149.5, 148.0, 147.8, 133.5, 133.1, 133.1,
132.6, 128.8, 125.2, 121.5, 111.2, 108.2. HRMS (ESI) m/z 352.0306
[M + H]+ (calcd for C14H11ClN3O6, 352.0336).
2.1.18. (E)-6-hydroxy-N′-(2,3,4-trihydroxybenzylidene)-2-
naphthohydrazide (3p)
Yield 63.8%. 1H NMR (400 MHz, DMSO‑d6) δ 12.04 (s, 1H), 11.64
(s, 1H), 10.16 (s, 1H), 9.51 (s, 1H), 8.54 (brs, 1H), 8.50 (s, 1H), 8.43 (s,
1H), 7.95–7.89 (m, 2H), 7.81 (d, J = 8.7 Hz, 1H), 7.21 (s, 1H), 7.19 (d,
2.1.25. (E)-N′-(2,3,4-trihydroxybenzylidene)thiophene-2-carbohydrazide
(3w)
Yield 60.4%. 1H NMR (400 MHz, DMSO‑d6) δ 11.99 (s, 1H), 11.34
(s, 1H), 9.49 (s, 1H), 8.52 (s, 1H), 8.44 (s, 2H), 7.89 (dd, J = 4.3,
2.8 Hz, 2H), 7.26–7.22 (m, 1H), 6.81 (d, J = 8.5 Hz, 1H), 6.40 (d,
J = 8.4 Hz, 1H); 13C NMR (100 MHz, DMSO‑d6) δ 157.6, 150.2, 149.2,
147.8, 138.2, 133.1, 132.3, 129.3, 128.6, 121.4, 111.3, 108.1. HRMS
(ESI) m/z 279.0465 [M + H]+ (calcd for C12H11N2O4S, 279.0440).
J = 8.7 Hz, 2H), 6.80 (d, J = 8.5 Hz, 1H), 6.42 (d, J = 8.4 Hz, 1H); 13
C
NMR (100 MHz, DMSO‑d6) δ 163.1, 157.6, 150.2, 149.1, 147.9, 136.8,
133.1, 131.2, 128.5, 127.3, 127.0, 126.7, 124.8, 121.6, 120.0, 111.3,
109.1, 108.1. HRMS (ESI) m/z 339.0973 [M + H]+ (calcd for
C
18H15N2O5, 339.0981).
3

Y. Liu, et al.
BioorganicChemistry96(2020)103609
Fig. 1. Discovery of leads as HK2 inhibitor. (A) Procedure for screening active lead compounds. (B) Chemical structure of 22 compounds by virtual screening from
ZINC database.
Table 1
Predicted binding free energies and inhibitory activities of screening hits.
ZINC number
ICM scoresa
IC50 for enzyme activities (μM)
Kd valuesb (μM)
Cell IC50 (μM)d
> 200
02944755
03232404
02337606
00361702
15671389
03233449
18168839
00050584
05379912
00355832
84343815
17951523
13153763
17135347
13144212
40268091
32455482
02241008
39948337
19938708
16513910
15777479
−38.28
−37.82
−37.14
−37.02
−34.49
−33.77
−40.66
−32.99
−34.30
−33.87
−32.29
−32.20
−32.05
−34.72
−32.47
−32.39
−32.84
−38.14
−32.64
−35.38
−44.62
−35.91
> 50
69.80
69.30
431.20
6920.00
286.20
41.70
242.00
n.b.c
5.90
3.20
8.17
0.12
0.11
102.30
> 200
> 200
> 200
87.30
11.20
> 50
> 50
> 50
9.39
65.20
99.80
34.80
3.60
6.90
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
10.10
> 50
> 50
> 50
38.60
17.30
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
94.70
206.20
n.b
n.b.
n.b.
n.b.
313.20
n.b.
16.20
1530.00
n.b.
194.00
n.b.
0.21
233.00
n.b.
35.10
6.50
7.20
> 200
> 200
> 200
296.00
n.b.
a
b
c
Docking score/interaction potential of compounds with HK2 (kcal/mol).
The Kd value is automatic calculated by the curve fitting, and presents as means
n.b is no clear binding detected in the MST measurement.
SW480 cell was used to determine IC50.
SD for three experiments.
d
2.1.26. (E)-N′-(2,3,4-trihydroxybenzylidene)isonicotinohydrazide (3x)
Yield 54.8%. 1H NMR (400 MHz, DMSO‑d6) δ 12.18 (s, 1H), 11.29
(s, 1H), 9.56 (s, 1H), 8.79–8.81 (m, 2H), 8.55 (s, 1H), 8.50 (s, 1H),
7.83–7.85 (m, 2H), 6.84 (d, J = 8.5 Hz, 1H), 6.42 (d, J = 8.5 Hz, 1H);
13C NMR (100 MHz, DMSO‑d6) δ 161.4, 151.5, 150.8, 149.5, 148.0,
140.5, 133.1, 133.1, 121.9, 121.6, 111.2, 108.2. HRMS (ESI) m/z
274.0822 [M + H]+ (calcd for C13H12N3O4, 274.0828).
J = 3.0 Hz, 1H), 6.90 (dd, J = 8.8, 3.0 Hz, 1H), 6.85–6.76 (m, 2H),
6.40 (d, J = 8.5 Hz, 1H); 13C NMR (100 MHz, DMSO‑d6) δ 164.2,
151.8, 151.2, 150.0, 149.3, 148.0, 133.1, 121.9, 121.7, 118.3, 116.1,
114.2, 111.2, 108.1. HRMS (ESI) m/z 305.0779 [M + H]+ (calcd for
C14H13N2O6, 305.0774).
2.1.28. (E)-N′-(2,3,4-trihydroxybenzylidene)benzohydrazide (3z)
Yield 76.6%. 1H NMR (400 MHz, DMSO‑d6) δ 11.94 (s, 1H), 11.52
(s, 1H), 9.46 (s, 1H), 8.49 (s, 1H), 8.44 (s, 1H), 7.91–7.88 (m, 2H),
7.59–7.55 (m, 1H), 7.53–7.49 (m, 2H), 6.76 (d, J = 8.5 Hz,1H), 6.37
(d, J = 8.5 Hz, 1H); 13C NMR (100 MHz, DMSO‑d6) δ 162.9, 150.5,
149.1, 147.9, 133.3, 133.1, 132.2, 128.9, 127.9, 121.6, 111.2, 108.0.
2.1.27. (E)-2,5-dihydroxy-N′-(2,3,4-trihydroxybenzylidene)
benzohydrazide (3y)
Yield 68.8%. 1H NMR (400 MHz, DMSO‑d6) δ 11.84 (s, 1H), 11.44
(s, 1H), 11.10 (s, 1H), 9.51 (s, 1H), 9.13 (s, 1H), 8.49 (s, 1H), 7.28 (d,
4

Y. Liu, et al.
BioorganicChemistry96(2020)103609
Fig. 2. The analysis of the lead compound
binding mode in the HK2 pocket. (A) Ligand
interaction diagram of the lead compound zinc
03233449 with HK2 (PDB code: 2NZT) (B) Low-
energy binding conformations of zinc03233449
bound to HK2 generated by virtual ligand
docking. Zinc03233449 formed a series of H-
bond
interactions
with
HK2
residues.
Zinc03233449 (yellow), substrate glucose
(purple) and product glucose-6-phosphate
(brown) were depicted as ball-and-stick model.
The trihydroxybenzene ring fragment of
zinc03233449 occupied the binding site of the
substrate glucose and they are almost completely
overlapped. The trifluoromethylpyrazole part of
zinc03233449 is inserted into the binding pocket
of product glucose-6-phosphate.
HRMS (ESI) m/z 273.0877 [M
+
H]+ (calcd for C14H13N2O4,
at 340 nm every minute for 15 min on a SpectraMax M5e plate read.
Briefly, 30 μL recombinant HK2 (0.3 μM) was incubated with 10 μL
compounds at 37 °C for 10 min. Then 70 μL assay mix containing
273.0875).
100 mM Tris-HCl pH 8.0, 200 mM Glucose, 5 mM MgCl2, 1 mM NAD+
,
2.2. Biology
0.8 mM ATP, 0.25 Units of G6P-DH, was added. The enzyme inhibition
IC50 values for compounds were calculated based on the changes in
absorbance. No Compounds interference with G6P-DH activity was
observed.
2.2.1. Clone, expression and purification of HK2
Human HK2 aa17-916 with an N-terminal His tag and thrombin
cleavage site was cloned into pET28-LIC. All proteins were expressed in
E. coli BL21 (DE3) Codon Plus (Invitrogen). Cultures were grown in LB
media containing 50 mg/L kanamycin and 50 mg/L chloramphenicol at
37 °C to OD 0.6–0.8, then induced at 20 °C with 0.5 mM IPTG for 20 h.
Cultures were harvested by centrifugation at 4000 g for 10 min. HK2
was purified with Ni-agarose affinity chromatography, followed by
anion exchange chromatography and size exclusion chromatography.
The purified proteins were stored in 25 mM phosphate pH 8 containing
50 mM Tris pH 8 containing 150 mM NaCl.
2.2.3. Microscale thermophoresis (MST) assay
Specific details were referred to our previous work and with a little
modification. Briefly, Labelled HK2 was kept constant at 20 nM, and all
tested samples were 1:1 diluted in a 20 mM HEPES (pH 7.4), 1% (v/v)
DMSO and 0.05 (v/v) % Tween-20. After 10 min incubation at room
temperature, samples were loaded into Monolith™ standard-treated
capillaries. And the thermophoresis was measured at room temperature
after 30 min incubation on a Monolith NT.115 instrument (Germany).
Furthermore, laser power was set to 40% using 30 sec on-time. The LED
power was set to 50%. The dissociation constant Kd values were fitted
by using the NT-Analysis software.
2.2.2. In vitro enzyme inhibition assay
Specific details were referred to our previous work and with a little
modification. To assay the inhibitory effects of compounds on HK2,
NADH which was generated through a coupled reaction with glucose-6-
phosphate dehydrogenase (G6P-DH) was monitored by optical density
5

Y. Liu, et al.
BioorganicChemistry96(2020)103609
Scheme 1. Rational design and synthetic strategy for the HK2 inhibitors. (A) Reagents and conditions: (i) 40% Hydrazine hydrate (5.0 equiv.), in MeOH, reflux. (ii)
2,3,4-Trihydroxybenzaldehyde (1.1 equiv), in MeOH, rt. (B) Synthesized compounds 3a-3z and theirenzymatic inhibition IC50
.
2.2.4. Molecular docking
(CCK-8).
Crystal structures of human HK2 (PDB code: 2NZT) was obtained
from the Protein Data Bank. The docking was operated by using ICM
3.8.2 modeling software on an Intel i7 4960 processor (MolSoft LLC,
San Diego, CA). Ligand binding pocket residues were selected by using
graphical tools in the ICM software, to create the boundaries of the
docking search. In the docking calculation, potential energy maps of the
receptor were calculated using default parameters. Compounds were
inputted into ICM and an index file was created. Conformational sam-
pling was based on the Monte Carlo procedure30, and finally the
lowest-energy and the most favorable orientation of the ligand were
selected.
2.2.7. Western blot analysis
Lysate protein (20–40 μg) was subjected to 10% SDS-PAGE and
electrophoretically transferred to polyvinylidenedifluoride membranes
(PVDF) (Millipore, USA). The membranes were sequentially blocked
with 5% non-fat milk, and incubated overnight with the following
primary antibodies: β-actin (A2228) (1:1000), HK2 (#2867) (1:1000.
Protein bands were visualized using an enhanced chemiluminescence
reagent (ECL Plus) (GE Healthcare, USA) after hybridization with a HRP
conjugated secondary antibody.
2.3. Statistical analysis
2.2.5. Cell culture
Human colon cancer cells SW480 and human pancreatic cancer cells
SW1990 were obtained from the American Type Culture Collection
(ATCC, USA) and grown in high Glucose Dulbecco’s modified Eagle’s
medium supplemented with 2 mM glutamine, 10% (v/v) fetal bovine
serum (FBS), 100 U/mL penicillin and 100 mg/mL streptomycin. Cell
cultures were grown and maintained in culture at 37 °C in a humidified
tissue culture incubator with 5% CO2 using standard tissue procedures.
Cell lines, HCT116(CCL-247), SW620(CCL-227), PANC-1(CRL-1469),
Aspc-1(CRL-1682) were purchased from ATCC.
All values are expressed as the mean
SD. Comparisons between
multiple-group means were performed using one-way analysis of var-
iance (one-way ANOVA). P values < 0.05 were statistically significant.
All statistical analyses were performed using GraphPad Prism software
version 5.0.
3. Results and discussion
3.1. Screening, rational design of HK2 inhibitors
2.2.6. Cytotoxicity test
Structure-based virtual ligand screening was performed using the
ICM-Pro 3.8.2 modeling software (MolSoft LLC, San Diego, CA) [22].
From 6,053,287 molecules, 130 screen hits with significant ICM scores
SW480 or SW1990 cells were incubated with tested compounds for
48 h and its cytotoxicity was measured using a Cell Counting Kit-8
6

Y. Liu, et al.
BioorganicChemistry96(2020)103609
Fig. 3. Compound 3j binds to HK2 and inhibits HK2 enzyme activity in vitro. (A) The equilibrium dissociation constants (Kd) of compounds (3a-3z). (B) MST assay
identifying the binding of compound 3j to HK2. (C) The enzymatic activity inhibition assay of compounds (3a-3z). (D) The binding pockets analysis of compound 3j
on HK2 (docking model from PDB code 2NZT).
and high occupancy of the active binding pocket were obtained. After
further screening to eliminate highly similar structures, 22 hits (Mol-
Table 2
Port, Latvia) were manually selected for further evaluation (Fig. 1A and
The equilibrium dissociation constants (Kd) and inhibitory activity on HK2
B, Table 1). Three lead compounds with ZINC codes of 03232404,
enzyme activity in vitro.
Compounds
03233449, and 39948337, sharing the same fragment as (E)-N′-(2,3,4-
Kd valuesa (μM)
Enzyme IC50 (μM)
trihydroxybenzylidene) arylhydrazide, exhibited significant enzyme
inhibitory effects. A simple structure–activity relationship (SAR) ana-
lysis showed that the trihydroxybenzene ring was essential for enzyme
inhibitory activity.
3a
149.00
198.00
n.b.b
19.30
0.69
1.51
3.32
4.19
1.15
1.08
0.98
> 12.5
5.21
0.53
5.89
> 12.50
0.70
> 12.5
0.74
1.26
2.02
2.30
1.72
2.07
5.16
1.17
4.80
6.43
1.02
1.08
0.02
0.08
0.15
0.23
0.12
0.11
0.05
3b
43.80
3c
3d
78.40
73.30
75.90
40.60
n.b.
11.40
22.90
11.90
7.42
Further modifications were made to these compounds based on the
analysis of their binding mode in the HK2 pocket (Fig. 2A). From a
generated docking model, the compound adopted an extended con-
formation, in which the pyrogallol part of the compound was inserted
into the binding pocket of the substrate glucose and appeared as an
overlapped conformation (Fig. 2B). As stated above, the trihydrox-
ybenzene ring was vital for the activity, and it could form strong hy-
drogen bonds with Asn656, Asp657 and Thr620, thus competing with
glucose for the binding site. The hydrazide fragment predicted to form
hydrogen bonds with Asp657, Gly681 and Thr680 in the polar pocket,
was also preserved in our modifications. Since restriction of rotatable
bonds is an effective means of reducing the number of possible spatial
conformations and improving target selectivity for small-molecule
drugs, the double bond of hydrazine was still retained after further
modification. In order to enhance the binding affinity of the lead
compounds for HK2, and their pharmacokinetic properties, some ap-
propriate groups were substituted onto the acyl group to improve oc-
cupancy of the active binding pocket and their hydrophobicity (Scheme
1).
3e
3f
3g
3h
3i
47.10
4.99
5.80
0.23
0.13
0.31
3j
0.41
3k
55.40
248.00
96.60
n.b.
14.60
22.50
19.60
3l
3m
0.12
3n
3o
107.00
349.00
67.50
331.00
562.00
n.b.
18.50
23.80
9.32
0.21
0.41
0.23
0.52
0.32
0.25
0.34
0.11
0.35
0.67
0.09
0.04
3p
3q
3r
23.10
83.10
3s
3t
3u
669.00
306.00
495.00
n.b.
179.00
38.60
33.90
3v
3w
3x
3y
268.00
7.63
28.40
3z
0.77
Metformin
2-Deoxyglucose
3-Bromopyruvate
31400.00
n.b.
1300.00
3.13
> 10,000
> 10,000
26.10
0.60
0.05
3.2. Chemistry
a
The Kd value is automatically calculated by the curve fitting and presents as
The structures and preparation of target compounds (3a-3z) are
described in Scheme 1. The esters derived from commercially available
acids were selected as starting materials, which were converted into the
corresponding hydrazides by reacting with hydrazine hydrate in
means
SD for three experiments.
b
n.b. is no clear binding detected in the MST measurement.
7

Y. Liu, et al.
BioorganicChemistry96(2020)103609
Fig. 4. Compound 3j inhibits cell proliferation. (A) Western blot analysis of HK2 expression in colon cancer cells and pancreatic cancer cells. (B, C) IC50 values of
compounds (3a-3z) in SW1990 and SW480 cell lines. (D) The colony formation assay of compound 3j on SW1990 cells.
Table 3
methanol. Treatment of the latter with 2,3,4-trihydroxybenzaldehyde
in anhydrous methanol produced sufficient yields of the desired hy-
drazone derivatives. Their structures were confirmed by NMR and mass
spectrum analyses. The trans(E) configuration of the imine double
bonds was determined based on the observation that the hydroxyl
protons at the meta- and para-position of imine moiety appeared at δH
8.4–9.6, whereas those at the ortho-position shifted down-field to δH
11.3–12.4 [23,24]. This difference in chemical shift could be explained
by the formation of intramolecular hydrogen bonds between the ortho-
OH group and the N atom of imine when the double bonds were in the
trans(E) configuration.
IC50 values of designed compounds against two cell lines.
Compounds
Cell IC50 (μM)
SW1990
SW480
3a
139.00
200.00
174.80
117.00
67.30
13.60
72.00
187.10
174.80
115.00
34.50
23.80
40.60
> 200
169.30
7.13
11.40
9.86
8.93
11.90
4.33
6.76
7.23
3b
23.10
10.80
16.80
3c
3d
3e
5.79
3f
47.50
5.51
7.37
3g
58.40
3h
> 200
165.50
24.00
3i
14.90
8.45
23.30
20.50
11.70
3.3. Biological evaluation
3j
1.12
3k
175.00
196.00
58.80
93.80
> 200
29.30
123.80
186.10
190.20
> 200
> 200
107.50
18.76
70.65
33.29
60.39
193.90
115.60
83.25
n.p.
17.80
3.3.1. Binding affinity and selective inhibition of compound 3j for the HK2
enzyme
3l
3m
5.65
3.11
3n
126.50
191.60
196.30
193.60
195.30
> 200
38.60
10.20
14.10
12.90
7.86
6.26
The microscale thermophoresis (MST) technique has been used to
assess protein–protein, small molecule–protein, nucleic acid–protein,
and antibody–antigen interactions [25–27]. MST was used to assay the
binding affinity of the lead compounds (3a–3z) for HK2 (Fig. 3A).
Among the synthesized compounds, 3j with an equilibrium dissociation
3o
14.50
13.20
3p
3q
3r
8.74
3s
23.10
3.52
3t
8.26
constant (Kd) of 4.99
0.41 μM displayed the strongest binding af-
3u
155.90
101.60
104.70
183.00
> 200
118.50
n.p.a
39.70
30.08
33.20
23.30
8.82
finity (Fig. 3B, Table 2). The inhibitory activity of the compounds (3a-
3z) on HK2 enzyme in vitro was evaluated by spectrophotometry [28].
Compound 3j showed the strongest inhibitory effects against HK2 with
3v
4.21
3w
19.15
30.02
24.10
17.11
3x
3y
an IC50 value of 0.53
0.13 μM (Fig. 3C, Table 2). The molecular
3z
19.30
docking result of compound 3j further confirm its same binding mode
Metformin
2-Deoxyglucose
3-Bromopyruvate
as the lead zinc03233449 (Fig. 3D).
n.p.
n.p.
n.p.
n.p.
3.3.2. Compound 3j inhibits cell proliferation
a
n.p. is not performed.
The protein expression of HK2 by colon cancer and pancreatic
cancer cell lines was evaluated via western blot analysis. HK2 was
8

Y. Liu, et al.
BioorganicChemistry96(2020)103609
overexpressed in SW480 and SW1990 cell lines (Fig. 4A). The in-
hibitory effect of compound 3j on cell proliferation was further ex-
amined on SW480 and SW1990 cells. After treatment with compound
3j for 48 h, the IC50 values of compound 3j for SW1990 and SW480
24 cancer classes, Genomics 84 (2004) 1014–1020.
[6] K.C. Patra, Q. Wang, P.T. Bhaskar, L. Miller, Z. Wang, W. Wheaton, N. Chandel,
M. Laakso, W.J. Muller, E.L. Allen, A.K. Jha, G.A. Smolen, M.F. Clasquin,
R.B. Robey, N. Hay, Hexokinase 2 is required for tumor initiation and maintenance
and its systemic deletion is therapeutic in mouse models of cancer, Cancer Cell 24
(2013) 213–228.
cells were 24.0
8.45 μM and 7.13
1.12 μM, respectively
[7] J.E. Wilson, Isozymes of mammalian hexokinase: structure, subcellular localization
and metabolic function, J. Exp. Biol. 206 (2003) 2049–2057.
(Fig. 4B–C, Table 3). The antiproliferative effect of compound 3j on
SW1990 was also confirmed by colony formation assay (Fig. 4D).
[8] D.A. Tennant, R.V. Duran, E. Gottlieb, Targeting metabolic transformation for
cancer therapy, Nat. Rev. Cancer 10 (2010) 267–277.
[9] W. Guo, Z. Qiu, Z. Wang, Q. Wang, N. Tan, T. Chen, Z. Chen, S. Huang, J. Gu, J. Li,
M. Yao, Y. Zhao, X. He, MiR-199a-5p is negatively associated with malignancies and
regulates glycolysis and lactate production by targeting hexokinase 2 in liver
cancer, Hepatology 62 (2015) 1132–1144.
4. Conclusion
In summary, with the aid of structure-based virtual ligand
screening, 3 lead compounds with the same active moiety were iden-
tified from the ZINC lead-like database. After a careful analysis of their
ligand–protein binding mode, we designed a series of novel HK2 in-
hibitors. These hydrazone derivatives with a trans(E) configuration of
their imine double bonds were synthesized directly from the corre-
sponding hydrazides and 2,3,4-trihydroxybenzaldehyde. The trans(E)
configuration is vital to help compounds bind to the pocket in an ex-
tended conformation. Substitution by a nitro group at the para-position
of the benzoyl hydrazine dramatically improved the binding affinity to
HK2, which led to the synthesis of compound 3j. Compound 3j sig-
nificantly inhibited cancer cell proliferation by directly targeting HK2.
Therefore, compound 3j is a novel inhibitor of HK2 due to its high
activity and deserve further study for future cancer therapeutics.
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Acknowledgements
We acknowledge support from National Natural Science Foundation
of China (NSFC) (grant number Nos. U1803122, 81773637, 81773594,
U1703111), National Mega-project for Innovative Drugs (grant number
2019ZX09721001-004-007), and the Fundamental Research Fund for
the Central Universities (grant number 2017KFYXJJ151).
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