Biological evaluation and SAR analysis of novel covalent inhibitors against fructose-1,6-bisphosphatase

Article abstract of DOI:10.1016/j.bmc.2020.115624

Fructose-1,6-bisphosphatase (FBPase) is an attractive target for affecting the GNG pathway. In our previous study, the C128 site of FBPase has been identified as a new allosteric site, where several nitrovinyl compounds can bind to inhibit FBPase activity. Herein, a series of nitrostyrene derivatives were further synthesized, and their inhibitory activities against FBPase were investigated in vitro. Most of the prepared nitrostyrene compounds exhibit potent FBPase inhibition (IC₅₀ < 10 μM). Specifically, when the substituents of F, Cl, OCH₃, CF₃, OH, COOH, or 2-nitrovinyl were installed at the R₂ (meta-) position of the benzene ring, the FBPase inhibitory activities of the resulting compounds increased 4.5–55 folds compared to those compounds with the same groups at the R₁ (para-) position. In addition, the preferred substituents at the R₃ position were Cl or Br, thus compound HS36 exhibited the most potent inhibitory activity (IC₅₀ = 0.15 μM). The molecular docking and site-directed mutation suggest that C128 and N125 are essential for the binding of HS36 and FBPase, which is consistent with the C128-N125-S123 allosteric inhibition mechanism. The reaction enthalpy calculations show that the order of the reactions of compounds with thiol groups at the R₃ position is Cl > H > CH₃. CoMSIA analysis is consistent with our proposed binding mode. The effect of compounds HS12 and HS36 on glucose production in primary mouse hepatocytes were further evaluated, showing that the inhibition was 71% and 41% at 100 μM, respectively.

Full text of DOI:10.1016/j.bmc.2020.115624

Journal Pre-proofs
Biological evaluation and SAR analysis of novel covalent inhibitors against
fructose-1,6-bisphosphatase
Xinya Han, Yunyuan Huang, Lin Wei, Haifeng Chen, Yanrong Guo, Zilong
Tang, Wei Hu, Qinfei Xia, Qi Wang, Jufen Yan, Yanliang Ren
PII:
DOI:
Reference:
S0968-0896(20)30454-5
https://doi.org/10.1016/j.bmc.2020.115624
BMC 115624
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Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
27 March 2020
12 June 2020
16 June 2020
Please cite this article as: Han, X., Huang, Y., Wei, L., Chen, H., Guo, Y., Tang, Z., Hu, W., Xia, Q., Wang, Q.,
Yan, J., Ren, Y., Biological evaluation and SAR analysis of novel covalent inhibitors against fructose-1,6-
bisphosphatase, Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115624
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Biological evaluation and SAR analysis of
novel covalent inhibitors against fructose-1,6-
bisphosphatase
Xinya Han1, #, Yunyuan Huang2, #, Lin Wei2, #, Haifeng Chen3, Yanrong Guo1, Zilong Tang4, Wei
Hu1, Qinfei Xia1, Qi Wang1, Jufen Yan1, *, Yanliang Ren2, *
1School of Chemistry and Chemical Engineering, Anhui University of Technology, Maanshan
243002, China
2International Cooperation Base of Pesticide and Green Synthesis (Hubei), Key Laboratory of
Pesticide & Chemical Biology (CCNU), Ministry of Education, Department of Chemistry, Central
China Normal University, Wuhan 430079, China
3Ocean College, Beibu Gulf University, Qinzhou 535011, China
4Key Laboratory of Theoretical Chemistry and Molecular Simulation of Ministry of Education,
Hunan University of Science and Technology, Xiangtan 411201, China

ABSTRACT
Fructose-1,6-bisphosphatase (FBPase) is an attractive target for affecting the GNG
pathway. In our previous study, the C128 site of FBPase has been identified as a new
allosteric site, where several nitrovinyl compounds can bind to inhibit FBPase activity.
Herein, a series of nitrostyrene derivatives were further synthesized, and their
inhibitory activities against FBPase were investigated in vitro. Most of the prepared
nitrostyrene compounds exhibit potent FBPase inhibition (IC50 < 10 μM). Specifically,
when the substituents of F, Cl, OCH3, CF3, OH, COOH, or 2-nitrovinyl were installed
at the R2 (meta-) position of the benzene ring, the FBPase inhibitory activities of the
resulting compounds increased 4.5-55 folds compared to those compounds with the
same groups at the R1 (para-) position. In addition, the preferred substituents at the R3
position were Cl or Br, thus compound HS36 exhibited the most potent inhibitory
activity (IC50 = 0.15 μM). The molecular docking and site-directed mutation suggest
that C128 and N125 are essential for the binding of HS36 and FBPase, which is
consistent with the C128-N125-S123 allosteric inhibition mechanism. The reaction
enthalpy calculations show that the order of the reactions of compounds with thiol
groups at the R3 position is Cl > H > CH3. CoMSIA analysis is consistent with our
proposed binding mode. The effect of compounds HS12 and HS36 on glucose
production in primary mouse hepatocytes were further evaluated, showing that the
inhibition was 71% and 41% at 100 μM, respectively.
KEYWORDS Fructose-1,6-bisphosphatase (FBPase), FBPase inhibitors, covalent
inhibitors

1. Introduction
T2DM (type 2 diabetes mellitus) has been increasing worldwide as a
comprehensive disease, and research interest is continuing to grow[1, 2]. The prominent
features of T2DM are insulin resistance and hyperglycemia, which mostly come from
endogenous glucose (EGP)[3]. Current antidiabetic drugs mainly focus on elevating
insulin secretion and relieving insulin resistance[2, 4-7]. Metformin is the only
available drug for the primary, albeit indirect, decrease in EGP, indicating that EGP is
an attractive target for controlling blood glucose levels, and much research remains to
be done in this field[8, 9]. Gluconeogenesis (GNG) is a major factor for EGP to generate
glucose, and in this process, the substrates pyruvate and lactate can be converted
glucose[10]. More importantly, the strategy of blocking the GNG pathway is
considered a promising strategy for developing antidiabetic drugs, that can be used to
control EGP[11, 12]. Glucose-6-phosphatase (G6Pase), fructose-1,6-bisphosphatase
(FBPase) and phosphoenolpyruvate carboxykinase (PEPCK) are important enzymes
and the primary targets for GNG. However, G6Pase and PEPCK are also involved in
other metabolic pathways, while FBPase is not, thus, it is an attractive target for only
affecting the GNG pathway[12, 13].
Human liver FBPase catalyzes the conversion of fructose-1,6-bisphosphate (FBP)
as the substrate to fructose-6-phosphate (F6P) and phosphate, and it’s one of the rate-
limiting reactions of the GNG pathway[14]. The endogenous allosteric agents
adenosine monophosphate (AMP) and fructose-2,6-bisphoshpatase (FDP) can
synergistically inhibit FBPase[15]. AMP binds to a unique allosteric site and causes a
conformational change from the active state (R) to an inactive state (T), whereas FDP

binds to the substrate site and competes with FBP[16, 17]. Recently, metformin has
been demonstrated to indirectly inhibit FBPase and then affect GNG by elevating AMP
levels[18]. On the other hand, FBPase is overexpressed in some diabetic models, and
these facts highlight the importance of FBPase as a target for regulating blood
glucose[11, 19]. Moreover, substantial efforts have been devoted to discover AMP site
inhibitors against FBPase, and a wide variety of inhibitors have been reported in the
literatures[11, 13, 20-32]. In addition, the AMP mimic MB07803, the most successful
FBPase inhibitor, has been advanced to clinical trials[11, 33].
Covalent inhibitors have higher ligand efficiencies and longer binding times
relative to noncovalent inhibitors[34, 35]. In the past decade, some covalent inhibitors,
such as ibrutinib, osimertinib and acalabrutinib, have been approved by the FDA
(United States Food and Drug Administration), initiating a new era for covalent
inhibitors[36-38]. In our previous work[39], nitrostyrene (HS1) could covalently bind
to the C128 of FBPase with an IC50 value of 3.5 μM by fragment screening. In this
work, we synthesized a series of nitrostyrene derivatives and explored their structure-
activity relationships. Furthermore, the biological activities of the prepared compounds
were evaluated in primary hepatocytes.
2. Results and discussion
2.1 Chemistry
The synthetic route to target compounds was outlined in Scheme 1 and Scheme 2
in support information. All reagents were obtained from commercial suppliers and were
used without further purification. TLC analysis were performed using precoated glass
plates. Column chromatography separations were performed using silica gel (200–300

mesh). 1H NMR and 13C NMR spectra were obtained on Varian Mercury-Plus 400 and
Varian NMR System 600 spectrometers. Chemical shifts are reported in ppm (δ) with
the NMR solvent signals as an internal reference. Coupling constants (J) are reported
in Hz. The abbreviations s, d, t and m refer to singlet, doublet, triplet and multiplet,
respectively.
2.2 Nitrostyrene compounds against FBPase and structure-activity relationships.
In a previous work, phenyl groups have been shown to preferentially bind β-
nitroalkenes over alkyl chains for inhibition activity[39]. Therefore, a benzene ring was
selected as the scaffold for lead generation. Because a phenyl group linked to a β-
nitroalkene (HS1) is a suitable structure for inhibiting FBPase, the side chain of this
molecule would be the second moiety to be explored.
Table 1. Inhibitory activities of hit compounds (HS1-HS22) against FBPase.
R2
R1
NO2
Compound
R1
R2
Hu-FBPase IC50 (μM)
HS1
HS2
HS3
HS4
HS5
HS6
HS7
HS8
HS9
HS10
HS11
HS12
H
H
H
H
H
H
H
H
H
H
H
H
H
3.5 ± 0.3
4.9 ± 0.8
F
Cl
3.3 ± 0.5
CH3
0.72 ± 0.11
16.0 ± 1.8
30.0 ± 1.8
1.6 ± 0.2
OCH3
SCH3
CF3
OH
COOH
15.0 ± 1.6
25.0 ± 1.8
0.49 ± 0.07
0.36 ± 0.08
0.89 ± 0.08
NO2
CN
1,2,4-Triazole-1-yl

F
Cl
0.32 ± 0.02
0.34 ± 0.02
1.7± 0.3
HS13
HS14
HS15
HS16
HS17
HS18
HS19
HS20
HS21
HS22
H
H
H
H
H
H
H
H
H
H
CH3
OCH3
CF3
0.29 ± 0.03
0.40 ± 0.06
0.24 ± 0.03
5.5 ± 0.3
OH
COOH
NO2
1.7 ± 0.1
CN
0.38 ± 0.04
0.38 ± 0.02
2-Nitrovinyl
The SAR of substituent at the R1 (para-) position of the benzene ring was explored
using both electron-donating (e.g., CH3, OCH3, SCH3, and OH) and electron-
withdrawing (e.g., NO2, CN, Cl, CF3, and COOH) groups, and the results are
summarized in Table 1. The addition of an electron-donating group, such as OH, OCH3,
or SCH3, at the para-position of the benzene ring led to partial decrease in potency (HS8,
HS5, and HS6, Table 1). These compounds had IC50 values ranging from 15.0 to 30.0
μM, corresponding to a 4.3-8.5 folds decrease in potency relative to HS1. On the other
hand, with the introduction of an electron-withdrawing group, such as Cl, CF3, CN, and
NO2, at the para-position, the analogs showed similar or higher potencies than HS1. As
shown in Table 1, significant improvements in potency were observed when NO2, CN,
or 1,2,4-triazole-1-yl was at the para-position. The substituted analogs displayed
significantly higher potencies, the CN is most favorable (compound HS11, IC50 = 0.36
μM), followed by NO2 (compound HS10, IC50 = 0.49 μM) and 1,2,4-triazole-1-yl group
(compound HS12, IC50 = 0.89 μM). The approximately 4 to 10-fold enhancement in
potency over compound HS1 suggested that the electron-withdrawing groups (CN,
NO2, or 1,2,4-triazole-1-yl) at the para-position of compound HS1 is favorable for the

FBPase inhibitory activities. However, when a COOH was introduced, the compound
HS9 exhibited 7.1-fold decrease in potency in comparison with HS1, likely because the
carboxyl group has remarkable solvation effect.
To further elucidate the effects of the position of the substituents on the benzene
ring on inhibition, compounds HS13-HS22 were synthesized, and the relative results
were summarized in Table 1. Introducing the F, Cl, OCH3, CF3, OH, COOH or 2-
nitrovinyl at the R2 (meta-) position of the benzene ring, afforded analogs with IC50
values of 0.29-5.5 μM, corresponding to 4.5-55 folds improvements over the analogs
with substituents at the para-position. These results suggest that the F and Cl groups at
the meta-position are preferred over the same groups at the para-position and suggest
that substituents at the meta-position of the benzene ring play a key role in the binding
interactions between FBPase and compounds of this series and can enhance their
FBPase inhibitory potency.
Table 2. Inhibitory activities of hit compounds (HS23-HS36) against FBPase.
R2
R1
R3
NO2
Compound
R1
R2
R3
Hu-FBPase IC50 (μM)
HS23
HS24
HS25
HS26
HS27
HS28
HS29
HS30
H
CN
H
H
H
CH3
CH3
Br
16.0 ± 2.5
5.1 ± 0.4
H
0.21 ± 0.04
0.16 ± 0.02
0.23 ± 0.02
0.27 ± 0.02
0.23 ± 0.04
0.64 ± 0.03
H
H
Cl
H
CN
H
Br
CN
H
Br
F
Br
F
H
Br

HS31
HS32
HS33
HS34
HS35
HS36
H
Cl
Cl
H
Cl
Cl
Cl
Cl
Cl
Cl
0.34 ± 0.04
0.54 ± 0.03
0.21 ± 0.03
0.30 ± 0.04
0.37 ± 0.03
0.15 ± 0.03
H
CN
H
CN
H
NO2
H
NO2
To further optimize the 2-nitrovinylbenzene scaffold, the introduction of
substituents at the R3 position was explored and the results were summarized in Table
2. The addition of a CH3 group at the R3 position (HS23, IC50 = 16.0 μM) led to slight
decrease in activity over compound HS1, while the introduction of a CH3 group at the
R3 position of HS11 (HS24, IC50 = 5.1 μM) result in 14-fold decrease in potency
relative to that of the parent compound. These results suggest that the introduction of a
CH3 group at the R3 position is unsuitable for improving potency. However, adding Br
or Cl group at the R3 position of HS1, the corresponding compounds (HS25 and HS26)
showed 16.6-fold and 21.8-fold increases in potency, with the IC50 values of 0.21 μM
and 0.16 μM, respectively. This indicate that the chloro and bromo substituted analogs
are more potent than analogs with CH3 substituents and that the installation of a halogen
substituent at the R3 position is favorable for increasing potency. As in this case, having
determined that CN, NO2, F, Cl, and CH3 are the preferred groups on the benzene ring,
Br or Cl groups was introduced at the R3 position of the corresponding compounds, and
the FBPase inhibitory potencies of the resulting compounds were explored. As shown
in Table 2, HS27-HS36 were generally more potent than the analogs without Br or Cl
groups at the R3 position, the IC50 values of them ranging from 0.15 to 0.72 μM. Among

all the tested compounds, HS36 (IC50 = 0.15 μM) with a NO2 group at the meta-position
and a Br group at the R3 position was the most potent compound.
2.3 The binding modes of compounds HS1 and HS36 against the C128 site of FBPase.
To explore the binding model in detail, compounds HS1 and HS36 were selected
as probe molecules for studying the interactions with FBPase using the covalent
docking module of Autodock. The possible binding modes of compounds HS1 and
Figure 1. Reasonable binding modes of HS1 (A) and HS36 (C) into the C128 site of
FBPase docked by Autodock. The binding models of HS1 (B) and HS36 (D) with the
surface electrostatic potential.
HS36 are shown in Figure 1. However, compound HS1 or HS36 did not conflict with
F6P and was located at the bottom of the substrate pocket. Obviously, a covalent bond
was formed between SH of C128.A and the β carbon of compound HS1 or HS36. The
nitro group of compound HS1 could form two hydrogen bonding interactions with the

NH2 groups of R254.B and R243.B. In addition, the meta-substituent on HS1 is close
to the OH group of Y258.B. These results suggest that elevating the inhibitory activity
of compound HS13 compared with that of compound HS1 relies on this hydrogen
bonding interaction, and a substituent at the meta-position is better than one at the para-
position for achieving this hydrogen bonding interaction. In addition, the NO2 group of
compound HS36 could form two hydrogen bonding interactions with the NH2 group of
R254.B and R243.B. The nitro moiety at the meta-position on benzene in compound
HS36 could form a hydrogen bond with OH group of S124.A. The benzene ring of
HS36 can form π-π interaction with the phenyl ring of Y258.B.
Table 3. The IC50 of HS36 against FBPase mutations.
WT
C128S
S124A
N125A
D127A
R243A
R254A
1.9±0.1
Y258A
3.5±0.2
Specific activity
(U/mg）
4.0±0.2
2.7±0.2
5.0±0.2
2.7±0.2
1.5±0.1
2.5±0.3
HS36 IC50 (μM)
0.15±0.03 195±15
1300
1.3±0.1 26.4±3.1 0.56±0.10 1.4±0.1
8.7 176 3.7 9.3
2.2±0.2 0.91±0.12
14.7 6.1
ICM50/ICW50
1
HS36 (
)
To identify the interactions of HS36 and FBPase, the C128, S124, N125, D127,
R243, R254 and Y258 were mutated to Ser or Ala. As listed in Table 3, C128S and
N125A substitutions led to 1300-fold (195 μM) and 176-fold (26.4 μM) increases in
the IC50 values compared with those for wild-type FBPase (0.15 μM). These
experimental results suggest that C128 and N125 are of great importance for binding
of compound HS36 and FBPase, which is consistent with our previously reported
binding model and the C128-N125-S123 allosteric inhibition mechanism[39]. In
comparison, the IC50 values of S124A (1.3 μM), R243A (1.4 μM), R254A (2.2 μM) and
Y258A (0.91 μM) are 8.7-fold, 9.3-fold 14.7-fold and 6.1-fold higher, respectively,

than that of wild-type FBPase, indicating that interactions between S124, R243, R254,
Y258 and HS36 exist.
Furthermore, the compounds with a halogen at the R3 position displayed potent
inhibitory activities against FBPase, but the compounds with CH3 at R3 showed lower
activities. To elucidate the differences in the biological activity of these compounds,
the effects of different substituents at R3 on the reaction enthalpy were explored. All
local minima in water were located by geometry optimization. The level of theory was
ωB97xD/6-311++G**, and the solvation model was SMD. All quantum chemistry
calculations were performed with the Gaussian 09 package. The reaction enthalpy is -
18.65 kcal/mol for HS1, -18.24 kcal/mol for HS23 and -20.83 kcal/mol for HS26. Thus,
the order of the reactions of compounds with thiol groups at R3 is Cl (HS26) > H (HS1)
> CH3 (HS23).
2.4 CoMSIA Analysis.
Comparative Molecular Similarity Index Analysis (CoMSIA) method a is
effective tool for 3D-QSAR analysis. The PLS analysis results for the CoMSIA model
are summarized in Table 4. A predictive CoMSIA model with a leave-one-out cross-
validated coefficient (q2) of 0.489 and a correlation coefficient (R2) of 0.885 was built.
As shown in Figure 2, the predicted activity values are in good agreement with the
experimental data. The standard error (S = 0.246) and F-test value (F = 52.102) further
corroborate the predicted model. In the QSAR model, the contributions of the steric and
electrostatic fields are 9.5% and 90.5%, respectively.
Table 4. Statistical parameters of the CoMSIA Model

contribution (%)
steric electrostatic
푞2
푅2
S
F
0.489
0.885
0.246
52.102
9.5
90.5
Figure 2. Plot of predicted vs experimental values for the training and test sets based
on the CoMSIA model.
The CoMSIA contour maps shown in Figure 3 present the steric and electrostatic
fields. In the steric field map, the green contour surrounding the benzene ring suggests
that more bulky substituents in these positions would be favorable for higher activity,
while the yellow contour indicates the region of unfavorable steric effects. These results
are consistent with our binding mode proposed above. In the CoMSIA electrostatic field,
the blue region indicates that a negative charge may be favorable for inhibitory activity,
whereas a positive charge is unfavorable.

Figure 3. Steric and electrostatic maps of the CoMSIA model. HS1 is shown inside the
field. In the steric field contour plot, sterically favored areas are represented by green
polyhedra, while sterically disfavored areas are represented by yellow polyhedra. In the
electrostatic field contour plot, positive-charge-favored areas are represented by red
polyhedra, whereas negative-charge-favored areas are represented by blue polyhedra.
2.5 Inhibition of Glucose Production in Hepatocytes.
As FBPase plays a central role in GNG, the nitrostyrene compounds were further
evaluated based on glucose production in freshly isolated primary mouse hepatocytes.
As illustrated in Figure 4, the representative nitrostyrene compounds (HS12 and HS36)
at 100 μM both effectively inhibited glucose production by primary mouse hepatocytes
with lactate/pyruvate as the GNG substrate, compared with the 0.1% DMSO control
group. HS12 and HS36 at 100 μM inhibited glucose production by approximately 71%
and 41%, respectively. Metformin is as the only marketed drug recognized to acts, at

least partially, through the inhibition of GNG[12]. As a positive control, the inhibitory
ability of metformin to glucose production by mouse hepatocytes was also
evaluated[32].
Figure 4. The compounds HS12(A), HS36(B), inhibition GNG assay on the primary
mouse hepatocytes. Metformin (2 mM) was utilized as the control. *P < 0.05, **P <
0.01, ***P < 0.001 versus DMSO group.
3 Conclusion
In summary, a series of nitrostyrene derivatives were synthesized, and their
inhibitory activities against FBPase were investigated in vitro. Most of the prepared
nitrostyrene compounds could potently inhibit (<10 μM) FBPase. Specifically, when
the F, Cl, OCH3, CF3, OH, COOH, or 2-nitrovinyl substituents were installed at the
meta-position, the FBPase inhibition activities of the resulting compounds increased
4.5-55 folds compared with those of the compound with the same group at the para-
position. Moreover, the preferred substituents at the R3 position were Cl or Br over CH3
for increasing the inhibitory activity, and compound HS36 exhibited the most potent
inhibitory activity (IC50 = 0.15 μM). The structure-activity relationship (SAR) has been

analyzed by the joint use of the reaction enthalpy calculations, molecular docking, site-
directed mutagenesis and CoMSIA strategies. Subsequently, HS36 can reduce glucose
production in hepatocytes. In conclusion, this work can provide a new platform for
designing covalent inhibitors of FBPase for treating T2DM. Further structural
optimization of nitrostyrene compounds and the QSAR studies are well underway in
our group.
4 Experimental
4.1 General methods
4.1.1. General procedure for the preparation of substituted 2-nitrovinyl benzene
derivatives
An aromatic aldehyde (5 mmol) was dropped into a solution of ammonium acetate
(12 mmol) in dry nitromethane (10 mL) and acetic acid (20 mL) at 90 °C with stirring.
The mixture was then refluxed for 5 h, and the mixture was poured into water and
extracted with ethyl acetate (3 × 50 mL). The extracts were washed with saturated saline
solution, dried over Na2SO4, filtered and concentrated under reduced pressure. The
residue was recrystallized from methanol or purified by column chromatography on
silica gel (ethyl acetate / petroleum ether) to obtain the target 2-nitrovinyl benzene
derivative.
4.1.2 General procedure for the preparation of substituted 2-bromo-2-nitrovinyl
benzene derivatives
To a stirred solution of a substituted 2-nitrovinyl benzene (10.0 mmol) in sodium
acetate (12.0 mmol) in chloroform (10 mL) was dropwise added neat Br2 (12.0 mmol)
over 5 min at 0 °C. The cloudy yellow reaction mixture was then heated to reflux and
stirred for 5-8 h (monitored by TLC). The excess Br2 was removed by washing the
reaction mixture with a saturated aqueous solution of Na2S2O3. The aqueous solution
was then extracted with CH2Cl2 (3 × 20 mL). The combined CH2Cl2 layers were dried
over anhydrous Na2SO4. The solvent was removed by evaporation under reduced

pressure to give a crude solid that was purified by silica gel column chromatography
using ethyl acetate and petroleum ether as the eluent.
4.1.3 General procedure for the preparation of substituted 2-chloro-2-nitrovinyl
benzene derivatives
To a solution of substituted 2-nitrovinyl benzene (0.4 mmol) in wet DMF (2.7 mL, 0.15
M) was added iodobenzene dichloride (PhICl2) (1.5 equiv) with stirring. The resulting
mixture was maintained at room temperature, and the reaction progress was monitored
by TLC. Upon completion, the reaction mixture was poured into cold water (30 mL)
and extracted with CH2Cl2 (3 × 20 mL). The combined organic layers were washed
with saturated NaHCO3 (1 × 50 mL) and brine (1 × 50 mL) before being dried over
anhydrous Na2SO4. The solvent was removed under vacuum and the residue was
purified by silica gel chromatography, using a mixture of ethyl acetate and petroleum
ether to afford the desired product.
(E)-(2-nitrovinyl) benzene (HS1)
Yield 88%. Yellow solid. M.p.: 57 – 58 °C. 1H NMR (400 MHz, CDCl3) δ: 8.01 (d, J
= 13.7 Hz, 1H), 7.59 (d, J = 13.7 Hz, 1H), 7.55 (d, J = 7.1 Hz, 2H), 7.50 (d, J = 7.0 Hz,
1H), 7.48 – 7.42 (m, 2H). 13C NMR (101 MHz, CDCl3) δ: 133.8, 131.9, 126.9, 124.89,
124.2, 123.9. MS (EI) m/z: 149.1 [M]+.
(E)-1-fluoro-4-(2-nitrovinyl) benzene (HS2)
Yield 78%. Yellow solid. M.p.: 100 – 101 °C. 1H NMR (400 MHz, CDCl3) δ: 7.98 (d,
1H), 7.65 – 7.49 (m, 3H), 7.20 – 7.06 (m, 4H). 13C NMR (101 MHz, CDCl3) δ: 166.3,
163.8, 137.9, 137.0, 131.4, 131.4, 126.4, 117.0, 116.7. MS (EI) m/z: 167.2 [M]+.
(E)-1-chloro-4-(2-nitrovinyl) benzene (HS3)
Yield 77%. Yellow solid. M.p.: 113 – 114 °C. 1H NMR (400 MHz, CDCl3) δ: 7.96 (d,
J = 13.9 Hz, 1H), 7.57 (dd, J = 13.7 Hz, 1H), 7.53 – 7.35 (m, 4H). 13C NMR (101 MHz,
CDCl3) δ: 138.4, 137.8, 137.6, 130.4, 129.9, 128.7. MS (EI) m/z: 183.1 [M]+.
(E)-1-methyl-4-(2-nitrovinyl) benzene (HS4)
Yield 88%. Yellow solid. M.p.: 101 – 103 °C. 1H NMR (400 MHz, CDCl3) δ: 7.97 (d,
J = 13.7 Hz, 1H), 7.56 (d, J = 13.7 Hz, 1H), 7.44 (d, J = 7.7 Hz, 2H), 7.25 (d, J = 7.6
Hz, 2H), 2.40 (s, 3H).13C NMR (101 MHz, CDCl3) δ: 143.1, 139.1, 136.1, 130.1, 129.1,
127.2, 21.6. MS (EI) m/z: 163.2 [M]+.

(E)-1-methoxy-4-(2-nitrovinyl) benzene (HS5)
Yield 85%. Yellow solid. M.p.: 87 – 88 °C. 1H NMR (400 MHz, CDCl3) δ: 7.97 (d, J
= 13.6 Hz, 1H), 7.55 – 7.46 (m, 3H), 6.96 (d, J = 8.8 Hz, 2H), 3.87 (s, 3H). 13C NMR
(101 MHz, CDCl3) δ: 162.9, 139.0, 134.9, 131.2, 122.5, 114.9, 77.4, 77.1, 76.8, 55.5.
MS (EI) m/z: 179.1 [M]+.
(E)-methyl(4-(2-nitrovinyl) phenyl) sulfane (HS6)
Yield 80%. Yellow solid. M.p.: 84 – 86 °C. 1H NMR (400 MHz, CDCl3) δ: 7.94 (d, J
= 13.6 Hz, 1H), 7.56 (d, J = 13.6 Hz, 1H), 7.44 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.4 Hz,
2H), 2.51 (s, 3H). 13C NMR (101 MHz, CDCl3) δ: 145.1, 138.7, 136.0, 129.4, 126.10,
125.9, 14.8. MS (EI) m/z: 195.2 [M]+.
(E)-1-(2-nitrovinyl)-4-(trifluoromethyl) benzene (HS7)
Yield 67%. Yellow solid. M.p.: 90 – 92 °C. 1H NMR (600 MHz, CDCl3) δ: 8.03 (d, J
= 13.7 Hz, 1H), 7.73 (d, J = 8.2 Hz, 2H), 7.68 (d, J = 8.1 Hz, 2H), 7.62 (d, J = 13.7 Hz,
1H). 13C NMR (151 MHz, CDCl3) δ: 138.8, 137.1, 133.4, 129.3, 126.3. MS (EI) m/z:
217.2 [M]+.
(E)-4-(2-nitrovinyl) phenol (HS8)
Yield 69%. Yellow solid. M.p.: 163 – 165 °C. 1H NMR (600 MHz, DMSO-d6) δ: 10.45
(s, 1H), 8.07 (s, 2H), 7.73 (d, J = 8.5 Hz, 2H), 6.87 (d, J = 8.5 Hz, 2H). 13C NMR (151
MHz, DMSO-d6) δ: 162.0, 140.3, 135.2, 132.8, 121.5, 116.6. MS (EI) m/z: 165.2 [M]+.
(E)-4-(2-nitrovinyl) benzoic acid (HS9)
Yield 79%. Yellow solid. M.p.: > 250 °C. 1H NMR (600 MHz, DMSO-d6) δ: 13.38 (s,
1H), 8.32 (d, J = 13.6 Hz, 1H), 8.20 (d, J = 13.6 Hz, 1H), 8.03 (d, J = 8.1 Hz, 2H), 7.98
13
(d, J = 8.2 Hz, 2H). C NMR (151 MHz, DMSO-d6) δ: 167.1, 139.9, 138.3, 134.6,
134.3, 130.2, 130.2. MS (EI) m/z: 193.1 [M]+.
(E)-1-nitro-4-(2-nitrovinyl) benzene (HS10)
Yield 79%. Yellow solid. M.p.: 201 – 202 °C. 1H NMR (600 MHz, CDCl3) δ: 8.33 (d,
J = 8.6 Hz, 2H), 8.05 (d, J = 13.7 Hz, 1H), 7.74 (d, J = 8.6 Hz, 2H), 7.65 (d, J = 13.7
13
Hz, 1H). C NMR (151 MHz, Acetone-d6) δ: 140.6, 136.9, 136.1, 130.5, 124.1. MS
(EI) m/z: 194.1 [M]+.
(E)-4-(2-nitrovinyl) benzonitrile (HS11)
Yield 75%. Yellow solid. M.p.: 184 – 185 °C. 1H NMR (600 MHz, CDCl3) δ: 8.00 (d,
J = 13.7 Hz, 1H), 7.77 (d, J = 8.3 Hz, 2H), 7.67 (d, J = 8.3 Hz, 2H), 7.63 (d, J = 13.8
Hz, 1H). 13C NMR (151 MHz, CDCl3) δ: 139.4, 136.6, 134.3, 133.0, 129.4, 117.8, 115.2.
MS (EI) m/z: 174.0 [M]+.

(E)-1-(4-(2-nitrovinyl) phenyl)-1H-1,2,4-triazole (HS12)
Yield 65%. Yellow solid. M.p.: > 200 °C. 1H NMR (600 MHz, DMSO-d6) δ: 9.45 (s,
1H), 8.33 (d, J = 14.0 Hz, 2H), 8.20 (d, J = 13.6 Hz, 1H), 8.09 (d, J = 8.7 Hz, 2H), 8.01
13
(d, J = 8.7 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ: 157.7, 147.7, 143.8, 143.3,
143.0, 136.4, 134.5, 124.5. MS (EI) m/z: 216.1 [M]+.
(E)-1-fluoro-3-(2-nitrovinyl) benzene (HS13)
Yield 72%. Yellow solid. M.p.: 45 – 47 °C. 1H NMR (600 MHz, CDCl3) δ: 7.97 (d, J
= 13.6 Hz, 1H), 7.57 (d, J = 13.7 Hz, 1H), 7.45 (q, J = 7.5 Hz, 1H), 7.35 (d, J = 7.7 Hz,
1H), 7.25 (d, J = 9.2 Hz, 1H), 7.21 (t, J = 8.2 Hz, 1H). 13C NMR (151 MHz, CDCl3) δ:
163.8, 162.1, 138.1, 137.7, 132.1, 132.1, 131.1, 131.1, 125.2, 119.1, 119.0, 115.5, 115.3.
MS (EI) m/z: 167.0 [M]+.
(E)-1-chloro-3-(2-nitrovinyl) benzene (HS14)
Yield 77%. Yellow solid. M.p.: 47 – 48 °C. 1H NMR (600 MHz, CDCl3) δ: 7.98 – 7.90
(m, 1H), 7.59 (d, J = 13.8 Hz, 1H), 7.55 (s, 1H), 7.51 – 7.44 (m, 2H), 7.44 – 7.39 (m,
1H). 13C NMR (151 MHz, CDCl3) δ: 138.0, 137.5, 135.3, 132.0, 131.8, 130.6, 128.7,
127.3. MS (EI) m/z: 183.3 [M]+.
(E)-1-methyl-3-(2-nitrovinyl) benzene (HS15)
1
Yield 81%. Yellow liquid. H NMR (600 MHz, CDCl3) δ 7.97 (d, J = 13.7 Hz, 1H),
13
7.58 (d, J = 13.7 Hz, 1H), 7.37 – 7.29 (m, 4H), 2.40 (s, 3H). C NMR (151 MHz,
CDCl3) δ: 134.1, 134.1, 131.7, 127.9, 124.8, 124.6, 124.1, 121.2, 16.1. MS (EI) m/z:
163.1 [M]+.
(E)-1-methoxy-3-(2-nitrovinyl) benzene (HS16)
Yield 77%. Yellow solid. M.p.: 91 – 93 °C. 1H NMR (600 MHz, CDCl3) δ: 7.97 (d, J
= 13.6 Hz, 1H), 7.57 (d, J = 13.6 Hz, 1H), 7.37 (t, J = 8.2 Hz, 1H), 7.14 (d, J = 7.6 Hz,
13
1H), 7.04 (d, J = 6.5 Hz, 2H), 3.85 (s, 3H). C NMR (151 MHz, CDCl3) δ: 154.9,
133.9, 132.1, 126.1, 125.3, 116.6, 112.8, 108.8, 50.3. MS (EI) m/z: 179.1 [M]+.
(E)-1-(2-nitrovinyl)-3-(trifluoromethyl) benzene (HS17)
Yield 69%. Yellow solid. M.p.: 75 – 76 °C. 1H NMR (600 MHz, CDCl3) δ: 8.04 (d, J
= 13.7 Hz, 1H), 7.81 (s, 1H), 7.76 (d, J = 7.9 Hz, 2H), 7.65 (d, J = 13.6 Hz, 1H), 7.62
(d, J = 7.8 Hz, 1H). 13C NMR (151 MHz, CDCl3) δ: 138.4, 137.3, 132.0, 131.8, 130.8,
130.0, 128.4, 125.7, 124.3, 122.5. MS (EI) m/z: 216.9 [M]+.

(E)-3-(2-nitrovinyl) phenol (HS18)
Yield 85%. Yellow solid. M.p.: 135 – 136 °C. 1H NMR (600 MHz, CDCl3) δ: 7.95 (d,
J = 13.7 Hz, 1H), 7.56 (d, J = 13.6 Hz, 1H), 7.33 (t, J = 7.9 Hz, 1H), 7.13 (d, J = 7.6
Hz, 1H), 7.02 (s, 1H), 6.98 (d, J = 8.1 Hz, 1H). 13C NMR (151 MHz, DMSO-d6) δ:
158.1, 139.8, 138.2, 131.8, 130.5, 121.0, 119.6, 116.5. MS (EI) m/z: 115.1 [M]+.
(E)-3-(2-nitrovinyl) benzoic acid (HS19)
Yield 85%. Yellow solid. M.p.: 194 – 195 °C. 1H NMR (600 MHz, CDCl3) δ: 8.30 (s,
1H), 8.23 (d, J = 7.8 Hz, 1H), 8.07 (d, J = 13.7 Hz, 1H), 7.80 (d, J = 7.7 Hz, 1H), 7.68
(d, J = 13.7 Hz, 1H), 7.61 (t, J = 7.7 Hz, 1H). 13C NMR (151 MHz, DMSO-d6) δ: 166.8,
139.1, 138.5, 133.4, 132.6, 132.1, 131.0, 129.7. MS (EI) m/z: 193.2 [M]+.
(E)-1-nitro-3-(2-nitrovinyl) benzene (HS20)
Yield 75%. Yellow solid. M.p.: 122 – 123 °C. 1H NMR (600 MHz, CDCl3) δ: 8.43 (s,
1H), 8.36 (d, J = 8.2 Hz, 1H), 8.07 (d, J = 13.8 Hz, 1H), 7.89 (d, J = 7.6 Hz, 1H), 7.73
– 7.65 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ: 153.2, 145.1, 141.8, 140.4, 137.2,
135.5, 130.9, 129.4. MS (EI) m/z: 194.1 [M]+.
(E)-3-(2-nitrovinyl) benzonitrile (HS21)
1
Yield 82%. Yellow solid. M.p.: 201 – 202 °C. H NMR (600 MHz, Chloroform-d) δ
7.99 (d, J = 13.7 Hz, 1H), 7.85 (s, 1H), 7.79 (d, J = 7.8 Hz, 2H), 7.64 – 7.60 (m, 2H).
13C NMR (151 MHz, cdcl3) δ: 138.9, 136.4, 134.9, 132.7, 132.3, 131.4, 130.4, 117.5,
114.0. MS (EI) m/z: 174.1 [M]+.
1,3-bis((E)-2-nitrovinyl) benzene (HS22)
Yield 78%. Yellow solid. M.p.: 203 – 204 °C. 1H NMR (400 MHz, CDCl3) δ: 8.02 (d,
J = 13.7 Hz, 2H), 7.69 (d, J = 8.0 Hz, 3H), 7.63 (d, J = 13.7 Hz, 2H), 7.60 – 7.55 (m,
1H). 13C NMR (101 MHz, DMSO-d6) δ: 143.9, 143.0, 138.4, 136.2, 135.0, 134.2.
(E)-4-(2-nitroprop-1-en-1-yl) benzonitrile (HS24)
Yield 70%. Yellow solid. M.p.: 108 – 110 °C. 1H NMR (600 MHz, CDCl3) δ: 8.07 (s,
13
1H), 7.78 (d, J = 8.2 Hz, 2H), 7.56 (d, J = 8.1 Hz, 2H), 2.46 (s, 3H). C NMR (151
MHz, CDCl3) δ: 149.6, 136.7, 132.3, 131.0, 130.0, 117.9, 112.9, 13.8. MS (EI) m/z:
188.1 [M]+.
(Z)-(2-bromo-2-nitrovinyl) benzene (HS25)

1
Yield 87%. Yellow solid. M.p.: 63 – 64 °C. H NMR (600 MHz, CDCl3) δ: 8.65 (s,
13
1H), 7.90 (d, J = 7.1 Hz, 2H), 7.56 – 7.48 (m, 3H). C NMR (151 MHz, CDCl3) δ:
136.4, 131.9, 130.9, 130.1, 128.9, 127.9. MS (EI) m/z: 226.9 [M−1]+ 228.9 [M+1]+.
(Z)-(2-chloro-2-nitrovinyl) benzene (HS26)
1
Yield 78%. Yellow solid. M.p.: 43 – 44 °C. H NMR (600 MHz, CDCl3) δ: 8.38 (s,
13
1H), 7.88 – 7.83 (m, 2H), 7.55 – 7.48 (m, 3H). C NMR (151 MHz, DMSO-d6) δ:
137.1, 132.5, 132.4, 131.6, 129.9, 129.4. MS (EI) m/z: 183.0 [M]+.
(Z)-3-(2-bromo-2-nitrovinyl) benzonitrile (HS27)
1
Yield 65%. Yellow solid. M.p.: 92 – 93 °C. H NMR (600 MHz, CDCl3) δ: 8.60 (s,
1H), 8.18 (s, 1H), 8.05 (d, J = 8.2 Hz, 1H), 7.80 (d, J = 7.8 Hz, 1H), 7.65 (t, J = 7.9 Hz,
1H). 13C NMR (151 MHz, CDCl3) δ: 134.6, 134.5, 133.8, 133.5, 131.6, 130.6, 129.9,
117.7, 113.5. MS (EI) m/z: 251.9 [M−1]+, 253.9 [M+1]+.
(Z)-4-(2-bromo-2-nitrovinyl) benzonitrile (HS28)
Yield 67%. Yellow solid. M.p.: 148 – 150 °C. 1H NMR (600 MHz, CDCl3) δ: 8.62 (s,
1H), 7.96 (d, J = 8.2 Hz, 2H), 7.79 (d, J = 8.3 Hz, 2H). 13C NMR (151 MHz, CDCl3) δ:
134.6, 134.2, 132.5, 130.9, 117.9, 114.8. MS (EI) m/z: 251.9 [M−1]+, 253.9 [M+1]+.
(Z)-1-(2-bromo-2-nitrovinyl)-3-fluorobenzene (HS29)
Yield 64%. Yellow solid. M.p.: 41 – 42 °C. 1H NMR (600 MHz, CDCl3) δ 8.58 (s, 1H),
7.67 (d, J = 9.6 Hz, 1H), 7.60 (d, J = 7.8 Hz, 1H), 7.48 (q, J = 7.8 Hz, 1H), 7.23 (t, J =
8.2 Hz, 1H). 13C NMR (151 MHz, CDCl3) δ: 163.3, 161.7, 135.1, 132.1, 130.6, 129.2,
127.2, 118.8, 116.8, 94.8. MS (EI) m/z: 244.9 [M-1]+, 246.9 [M+1]+.
(Z)-1-(2-bromo-2-nitrovinyl)-4-fluorobenzene (HS30)
Yield 68%. Yellow solid. M.p.: 52 – 53 °C. 1H NMR (600 MHz, CDCl3) δ 8.63 (s, 1H),
7.94 (dd, J = 8.7, 5.4 Hz, 2H), 7.20 (t, J = 8.6 Hz, 2H). 13C NMR (151 MHz, CDCl3) δ:
165.4, 163.7, 135.3, 133.4, 127.8, 126.3, 116.4. MS (EI) m/z: 244.8 [M-1]+, 246.9
[M+1]+. HRMS (ESI) m/z calcd C8H5NO2FBr [M + CH3O]+ 275.9677, found 275.9676.
(Z)-1-chloro-3-(2-chloro-2-nitrovinyl) benzene (HS31)
1
Yield 79%. Yellow solid. M.p.: 45 – 46 °C. H NMR (600 MHz, CDCl3) δ: 8.28 (s,
1H), 7.83 (s, 1H), 7.70 (d, J = 7.6 Hz, 1H), 7.48 (d, J = 8.1 Hz, 1H), 7.44 (t, J = 7.8 Hz,
1H). 13C NMR (151 MHz, CDCl3) δ: 133.4, 129.9, 126.6, 126.1, 125.3, 125.1, 124.9,
124.2. MS (EI) m/z: 216.9 [M]+.

(Z)-1-chloro-4-(2-chloro-2-nitrovinyl) benzene (HS32)
1
Yield 80%. Yellow solid. M.p.: 58 – 60 °C. H NMR (600 MHz, CDCl3) δ: 8.38 (s,
1H), 7.85 (d, J = 8.1 Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H). 13C NMR (151 MHz, CDCl3) δ:
137.7, 137.5, 131.9, 129.9, 129.1, 127.6. MS (EI) m/z: 216.9 [M]+.
(Z)-3-(2-chloro-2-nitrovinyl) benzonitrile (HS33)
Yield 72%. Yellow solid. M.p.: 106 – 108 °C. 1H NMR (600 MHz, CDCl3) δ: 8.36 (s,
1H), 8.16 (s, 1H), 8.05 (d, J = 7.9 Hz, 1H), 7.81 (d, J = 7.7 Hz, 1H), 7.66 (t, J = 7.9 Hz,
1H). 13C NMR (151 MHz, CDCl3) δ: 134.4, 129.7, 129.4, 128.6, 125.8, 124.9, 123.8,
112.5, 108.5. MS (EI) m/z: 208.1 [M]+. MS (EI) m/z: 208.0 [M]+. HRMS (ESI) m/z
calcd C9H5N2O2Cl [M + CH3O]+ 239.0229, found 239.0236.
(Z)-4-(2-chloro-2-nitrovinyl) benzonitrile (HS34)
Yield 73%. Yellow solid. M.p.: 138 – 140 °C. 1H NMR (600 MHz, CDCl3) δ: 8.37 (s,
1H), 7.95 (d, J = 8.2 Hz, 2H), 7.80 (d, J = 8.2 Hz, 2H). 13C NMR (151 MHz, CDCl3) δ:
134.8, 128.8, 127.5, 126.0, 124.1, 112.7, 109.7. MS (EI) m/z: 208.0 [M]+. HRMS (ESI)
m/z calcd C9H5N2O2Cl [M + CH3O]+ 239.0229, found 239.0236.
(Z)-1-(2-chloro-2-nitrovinyl)-3-nitrobenzene (HS35)
Yield 67%. Yellow solid. M.p.: 111 – 112 °C. 1H NMR (600 MHz, CDCl3) δ: 8.76 (s,
1H), 8.43 (s, 1H), 8.38 (d, J = 8.2 Hz, 1H), 8.15 (d, J = 7.6 Hz, 1H), 7.78 – 7.70 (m,
1H). 13C NMR (151 MHz, CDCl3) δ: 136.0, 135.8, 133.3, 130.9, 129.8, 128.4, 125.6,
124.8. MS (EI) m/z: 217.1 [M]+. HRMS (ESI) m/z calcd C8H5N2O4Cl [M + CH3O]+
259.0127, found 259.0127.
(Z)-1-(2-chloro-2-nitrovinyl)-4-nitrobenzene (HS36)
Yield 68%. Yellow solid. M.p.: 98 – 100 °C. 1H NMR (600 MHz, CDCl3) δ: 8.42 (s,
1H), 8.36 (d, J = 8.7 Hz, 2H), 8.02 (d, J = 8.7 Hz, 2H). 13C NMR (151 MHz, CDCl3) δ:
148.5, 135.3, 133.4, 131.2, 128.4, 123.7. MS (EI) m/z: 217.1 [M]+.
4.2 FBPase inhibition assays
The protein expression and purity, and FBPase inhibition assays were performed
for detection compounds inhibitory activities as previously described[39].
4.3 Gluconeogenesis inhibition in mouse hepatocytes.

Primary hepatocytes were isolated from C57BLKS mice that were fasted
overnight as described previously[40, 41], and then seeded in 24-well plates (2×105
cells per cell) in DMEM (InvitrogenTM) containing 1 g/L glucose and 10% fetal bovine
serum (FBS). After a 4-h attachment period, the cells were incubated overnight after
being added fresh medium. Then the medium was replaced with 500 μL of DMEM
without glucose and phenol red, but supplemented with 20 mM sodium lactate and 2
mM sodium pyruvate as well as the test compounds at the desired concentrations in
three parallel wells. After 4 h of incubation, 50 μL of culture supernatant was collected,
and the glucose concentration was determined using a colorimetric glucose assay kit
(Fudan-Zhang-jiangTM, Shanghai, China).
4.4 Molecular docking
The crystal structure of FBPase (PDB: 4H46) complexed with F6P and AMP was
downloaded from the RSCB Protein Data Bank. Compound HS13 and HS36 were built
and optimized in SYBYL 1.3. Then, HS13 and HS36 were docked into the C128 site
of FBPase by using the covalent module of Autodock[42]. The docking process used
the default parameters except the “generate diverse solution” option was chosen to
enhance the variety of docked conformations.
Author information
Corresponding author
To whom correspondence should be addressed. Phone: +86-27-67862022. Fax: +86-
27-67862022. E-mails : yanjvfen@163.com; E-mails: renyl@mail.ccnu.edu.cn.

Author Contributions
The manuscript was written through contributions of all authors. All authors have given
approval to the final version of the manuscript. # These authors contributed equally.
Notes
The authors declare no competing financial interest.
Acknowledgments
This work was supported by the National Key R&D Program of China (No.
2017YFE0118200), the Anhui Key Research and Development Program (No.
201904b11020025), the Natural Science Foundation of Anhui Province, China (No.
1908085MB35), the Natural Science Foundation for the Higher Education Institutions
of Anhui Province of China (No. KJ2018A0040), the China-Hungary Cooperation
Committee Regular Meeting Exchange Project (No. 2018-II-2), the China-Ukraine
Cooperation Committee Regular Meeting Exchange Project (No. CU03-04), the Open
Project Program of Key Laboratory of Pesticide & Chemical Biology (CCNU),
Ministry of Education (No. 201801A02). We would like to thank professor Wenjing
Xiao from central China normal university for providing us with compound HS23.
ABBREVIATIONS
FBPase, fructose-1,6-bisphosphatase; FBP, fructose-1,6-biphosphate; F6P, fructose-6-
biphosphate; IC50, half maximal inhibitory concentration; T2DM, Type 2 diabetes
mellitus; SAR, structure−activity relationship; GNG, gluconeogenesis; AMP,
adenosine monophosphate; DMEM, Dulbecco's modified Eagle's medium.

REFERENCES
[1] W. Yang, J. Lu, J. Weng, W. Jia, L. Ji, J. Xiao, Z. Shan, J. Liu, H. Tian, Q. Ji, D.
Zhu, J. Ge, L. Lin, L. Chen, X. Guo, Z. Zhao, Q. Li, Z. Zhou, G. Shan, J. He, D. China
National, G. Metabolic Disorders Study, Prevalence of diabetes among men and women
in China, N. Engl. J. Med., 362 (2010) 1090-1101.
[2] S.E. Kahn, M.E. Cooper, S. Del Prato, Pathophysiology and treatment of type 2
diabetes: perspectives on the past, present, and future, The Lancet, 383 (2014) 1068-
1083.
[3] S. Chatterjee, K. Khunti, M.J. Davies, Type 2 diabetes, The Lancet, 389 (2017)
2239-2251.
[4] A. Wagman, J. Nuss, Current Therapies and Emerging Targets for the Treatment of
Diabetes, Curr. Pharm. Des., 7 (2001) 417-450.
[5] D.E. Moller, New drug targets for type 2 diabetes and the metabolic syndrome,
Nature, 414 (2001) 821-827.
[6] N. Kerru, A. Singh-Pillay, P. Awolade, P. Singh, Current anti-diabetic agents and
their molecular targets: A review, Eur. J. Med. Chem., 152 (2018) 436-488.
[7] L.K.J. Stadler, I.S. Farooqi, A New Drug Target for Type 2 Diabetes, Cell, 170
(2017) 12-14.
[8] A.K. Madiraju, D.M. Erion, Y. Rahimi, X.M. Zhang, D.T. Braddock, R.A. Albright,
B.J. Prigaro, J.L. Wood, S. Bhanot, M.J. MacDonald, M.J. Jurczak, J.P. Camporez, H.Y.
Lee, G.W. Cline, V.T. Samuel, R.G. Kibbey, G.I. Shulman, Metformin suppresses
gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase, Nature,
510 (2014) 542-546.
[9] R.A. Miller, Q. Chu, J. Xie, M. Foretz, B. Viollet, M.J. Birnbaum, Biguanides
suppress hepatic glucagon signalling by decreasing production of cyclic AMP, Nature,
494 (2013) 256-260.
[10] B.R. Landau, J. Wahren, V. Chandramouli, W.C. Schumann, K. Ekberg, S.C.
Kalhan, Contributions of gluconeogenesis to glucose production in the fasted state, J.
Clin. Invest., 98 (1996) 378-385.

[11] P.D. van Poelje, S.C. Potter, V.C. Chandramouli, B.R. Landau, Q. Dang, M.D.
Erion, Inhibition of fructose 1,6-bisphosphatase reduces excessive endogenous glucose
production and attenuates hyperglycemia in Zucker diabetic fatty rats, Diabetes, 55
(2006) 1747-1754.
[12] A.K. Rines, K. Sharabi, C.D.J. Tavares, P. Puigserver, Targeting hepatic glucose
metabolism in the treatment of type 2 diabetes, Nat. Rev. Drug Discov. 15 (2016) 786-
804.
[13] Q. Dang, S.R. Kasibhatla, K.R. Reddy, T. Jiang, M.R. Reddy, S.C. Potter, J.M.
Fujitaki, P.D. van Poelje, J. Huang, W.N. Lipscomb, M.D. Erion, Discovery of potent
and specific fructose-1,6-bisphosphatase inhibitors and a series of orally-bioavailable
phosphoramidase-sensitive prodrugs for the treatment of type 2 diabetes, J. Am. Chem.
Soc., 129 (2007) 15491-15502.
[14] H. Ke, Thorpe, C. M., Seaton, B. A., Lipscomb, W. N., & Marcus, F., Structure
refinement of fructose-1,6-bisphosphatase and its fructose 2,6-bisphosphate complex at
2.8 Å resolution. , J. Mol. Biol., 212 (1990) 513-539.
[15] Y. Gao, L. Shen, R.B. Honzatko, Central cavity of fructose-1,6-bisphosphatase
and the evolution of AMP/fructose 2,6-bisphosphate synergism in eukaryotic
organisms, J. Biol. Chem., 289 (2014) 8450-8461.
[16] J.K. Hines, X. Chen, J.C. Nix, H.J. Fromm, R.B. Honzatko, Structures of
mammalian and bacterial fructose-1,6-bisphosphatase reveal the basis for synergism in
AMP/fructose 2,6-bisphosphate inhibition, J. Biol. Chem., 282 (2007) 36121-36131.
[17] M. Gidh-Jain, Y. Zhang, P. van Poelje, J. Liang, S. Huang, J. Kim, J. Elliott, M.
Erion, The allosteric site of human liver fructose-1,6-bisphosphatase. Analysis of six
AMP site mutants based on the crystal structure, J. Biol. Chem., 269 (1994) 27732-
27738.
[18] R.W. Hunter, C.C. Hughey, L. Lantier, E.I. Sundelin, M. Peggie, E. Zeqiraj, F.
Sicheri, N. Jessen, D.H. Wasserman, K. Sakamoto, Metformin reduces liver glucose
production by inhibition of fructose-1-6-bisphosphatase, Nat. Med., 24 (2018) 1395-
1406.
[19] M. Kebede, J. Favaloro, J.E. Gunton, D.R. Laybutt, M. Shaw, N. Wong, B.C. Fam,

K. Aston-Mourney, C. Rantzau, A. Zulli, J. Proietto, S. Andrikopoulos, Fructose-1,6-
Bisphosphatase Overexpression in Pancreatic -Cells Results in Reduced Insulin
Secretion: A New Mechanism for Fat-Induced Impairment of -Cell Function, Diabetes,
57 (2008) 1887-1895.
[20] S.W. Wright, D.L. Hageman, L.D. McClure, A.A. Carlo, J.L. Treadway, A.M.
Mathiowetz, J.M. Withka, P.H. Bauer, Allosteric inhibition of fructose-1,6-
bisphosphatase by anilinoquinazolines, Bioorg. Med. Chem. Lett., 11 (2001) 17-21.
[21] C. Lai, R.J. Gum, M. Daly, E.H. Fry, C. Hutchins, C. Abad-Zapatero, T.W. von
Geldern, Benzoxazole benzenesulfonamides as allosteric inhibitors of fructose-1,6-
bisphosphatase, Bioorg. Med. Chem. Lett., 16 (2006) 1807-1810.
[22] M.D. Erion, Q. Dang, M.R. Reddy, S.R. Kasibhatla, J. Huang, W.N. Lipscomb,
P.D. van Poelje, Structure-guided design of AMP mimics that inhibit fructose-1,6-
bisphosphatase with high affinity and specificity, J. Am. Chem. Soc., 129 (2007)
15480-15490.
[23] Q. Dang, S.R. Kasibhatla, T. Jiang, K. Fan, Y. Liu, F. Taplin, W. Schulz, D.K.
Cashion, K.R. Reddy, P.D. van Poelje, J.M. Fujitaki, S.C. Potter, M.D. Erion,
Discovery of phosphonic diamide prodrugs and their use for the oral delivery of a series
of fructose 1,6-bisphosphatase inhibitors, J. Med. Chem., 51 (2008) 4331-4339.
[24] P. Hebeisen, B. Kuhn, P. Kohler, M. Gubler, W. Huber, E. Kitas, B. Schott, J.
Benz, C. Joseph, A. Ruf, Allosteric FBPase inhibitors gain 10(5) times in potency when
simultaneously binding two neighboring AMP sites, Bioorg. Med. Chem. Lett., 18
(2008) 4708-4712.
[25] S. Heng, K.R. Gryncel, E.R. Kantrowitz, A library of novel allosteric inhibitors
against fructose 1,6-bisphosphatase, Bioorg. Med. Chem., 17 (2009) 3916-3922.
[26] A. Rudnitskaya, K. Huynh, B. Torok, K. Stieglitz, Novel heteroaromatic
organofluorine inhibitors of fructose-1,6-bisphosphatase, J. Med. Chem., 52 (2009)
878-882.
[27] T. Tsukada, M. Takahashi, T. Takemoto, O. Kanno, T. Yamane, S. Kawamura, T.
Nishi, Synthesis, SAR, and X-ray structure of tricyclic compounds as potent FBPase
inhibitors, Bioorg. Med. Chem. Lett., 19 (2009) 5909-5912.

[28] E. Kitas, P. Mohr, B. Kuhn, P. Hebeisen, H.P. Wessel, W. Haap, A. Ruf, J. Benz,
C. Joseph, W. Huber, R.A. Sanchez, A. Paehler, A. Benardeau, M. Gubler, B. Schott,
E. Tozzo, Sulfonylureido thiazoles as fructose-1,6-bisphosphatase inhibitors for the
treatment of type-2 diabetes, Bioorg. Med. Chem. Lett., 20 (2010) 594-599.
[29] P. Yi, Y.T. Di, W. Liu, X.J. Hao, Y. Ming, D.S. Huang, J. Yang, Z.Z. Yi, Z.J. Li,
R.D. Yang, J.C. Zhang, Protein-based alignment in 3D-QSAR of FBPase inhibitors,
Eur. J. Med. Chem., 46 (2011) 885-892.
[30] R.F. Tayyem, H.M. Zalloum, M.R. Elmaghrabi, A.M. Yousef, M.S. Mubarak,
Ligand-based designing, in silico screening, and biological evaluation of new potent
fructose-1,6-bisphosphatase (FBPase) inhibitors, Eur. J. Med. Chem., 56 (2012) 70-95.
[31] J. Bie, S. Liu, Z. Li, Y. Mu, B. Xu, Z. Shen, Discovery of novel indole derivatives
as allosteric inhibitors of fructose-1,6-bisphosphatase, Eur. J. Med. Chem., 90 (2015)
394-405.
[32] B.R. Liao, H.B. He, L.L. Yang, L.X. Gao, L. Chang, J. Tang, J.Y. Li, J. Li, F.
Yang, Synthesis and structure-activity relationship of non-phosphorus-based fructose-
1,6-bisphosphatase inhibitors: 2,5-Diphenyl-1,3,4-oxadiazoles, Eur. J. Med. Chem., 83
(2014) 15-25.
[33] M.D. Erion, P.D. van Poelje, Q. Dang, S.R. Kasibhatla, S.C. Potter, M.R. Reddy,
K.R. Reddy, T. Jiang, W.N. Lipscomb, MB06322 (CS-917): A potent and selective
inhibitor of fructose 1,6-bisphosphatase for controlling gluconeogenesis in type 2
diabetes, Proc. Natl. Acad. Sci. U S A, 102 (2005) 7970-7975.
[34] J. Singh, R.C. Petter, T.A. Baillie, A. Whitty, The resurgence of covalent drugs,
Nat. Rev. Drug Discov., 10 (2011) 307-317.
[35] T.A. Baillie, Targeted Covalent Inhibitors for Drug Design, Angew. Chem. Int.
Ed. Engl., 55 (2016) 13408-13421.
[36] R. Lonsdale, R.A. Ward, Structure-based design of targeted covalent inhibitors,
Chem. Soc. Rev., 47 (2018) 3816-3830.
[37] P. Chatterjee, W.M. Botello-Smith, H. Zhang, L. Qian, A. Alsamarah, D. Kent,
J.J. Lacroix, M. Baudry, Y. Luo, Can Relative Binding Free Energy Predict Selectivity
of Reversible Covalent Inhibitors?, J. Am. Chem. Soc., (2017).

[38] S. De Cesco, J. Kurian, C. Dufresne, A.K. Mittermaier, N. Moitessier, Covalent
inhibitors design and discovery, Eur. J. Med. Chem., 138 (2017) 96-114.
[39] Y. Huang, L. Wei, X. Han, H. Chen, Y. Ren, Y. Xu, R. Song, L. Rao, C. Su, C.
Peng, L. Feng, J. Wan, Discovery of novel allosteric site and covalent inhibitors of
FBPase with potent hypoglycemic effects, Eur. J. Med. Chem., 184 (2019) 111749.
[40] J.C. Yoon, P. Puigserver, G. Chen, J. Donovan, Z. Wu, J. Rhee, G. Adelmant, J.
Stafford, C.R. Kahn, D.K. Granner, Control of hepatic gluconeogenesis through the
transcriptional coactivator PGC-1, Nature, 413 (2001) 131-138.
[41] S.-H. Koo, H.C. Towle, Glucose Regulation of Mouse S14 Gene Expression in
Hepatocytes INVOLVEMENT OF A NOVEL TRANSCRIPTION FACTOR
COMPLEX, J. Biol. Chem., 275 (2000) 5200-5207.
[42] G.M. Morris, H. Ruth, L. William, M.F. Sanner, R.K. Belew, D.S. Goodsell, A.J.
Olson, AutoDock4 and AutoDockTools4: Automated docking with selective receptor
flexibility, J. Comput. Chem., 30 (2010) 2785-2791.
Conflict of interest
The authors declared that they have no conflicts of interest to this work.
We declare that we do not have any commercial or associative interest that represents
a conflict of interest in connection with the work submitted.