Synthesis, Optimization, and Evaluation of Glycosylated Naphthalimide Derivatives as Efficient and Selective Insect β- N-Acetylhexosaminidase OfHex1 Inhibitors

Article abstract of DOI:10.1021/acs.jafc.9b02281

Insect chitinolytic β-N-acetylhexosaminidase OfHex1, from the agricultural pest Ostrinia furnacalis (Guenée), is considered as a potential target for green pesticide design. In this study, rational molecular design and optimization led to the synthesis of compounds 15r (K_i = 5.3 μM) and 15y (K_i = 2.7 μM) that had superior activity against OfHex1 than previously reported lead compounds. Both compounds 15r and 15y had high selectivity toward OfHex1 over human β-N-acetylhexosaminidase B (HsHexB) and human O-GlcNAcase (hOGA). In addition, to investigate the basis for the potency of glycosylated naphthalimides against OfHex1, molecular docking and molecular dynamics simulations were performed to study possible binding modes. Furthermore, the in vivo biological activity of target compounds with efficient OfHex1 inhibitory potency was assayed against Myzus persicae, Plutella xylostella, and O. furnacalis. This present work indicates that glycosylated naphthalimides can be further developed as potential pest control and management agents targeting OfHex1.

Full text of DOI:10.1021/acs.jafc.9b02281

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Functional Structure/Activity Relationships
Synthesis, optimization, and evaluation of glycosylated
naphthalimide derivatives as efficient and selective
insect #-N-acetylhexosaminidase OfHex1 inhibitors
Shengqiang Shen, Lili Dong, wei chen, renjie wu, Huizhe Lu, Qing Yang, and Jianjun Zhang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02281 • Publication Date (Web): 15 May 2019
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Journal of Agricultural and Food Chemistry
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Synthesis, optimization, and evaluation of glycosylated naphthalimide
derivatives as efficient and selective insect β-N-acetylhexosaminidase
OfHex1 inhibitors
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Shengqiang Shen,†,# Lili Dong,†,# Wei Chen,‡ Renjie Wu,† Huizhe Lu,† Qing Yang,*,‡ and Jianjun Zhang*,†
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†Department of Applied Chemistry, College of Science, China Agricultural University, Beijing, China
‡Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
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ABSTRACT: Insect chitinolytic β-N-acetylhexosaminidase OfHex1, from the agricultural pest Ostrinia
furnacalis (Guenée), is considered as a potential target for green pesticide design. In this study, rational
molecular design and optimization led to the synthesis of compounds 15r (Ki =5.3 μM) and 15y (Ki =2.7 μM)
that had superior activity against OfHex1 than previously reported lead compounds. Both 15r and 15y had
high selectivity towards OfHex1 over HsHexB (human β-N-acetylhexosaminidase B) and hOGA (human
O-GlcNAcase). In addition, to investigate the basis for the potency of glycosylated naphthalimides against
OfHex1, molecular docking and MD simulations were performed to study possible binding modes.
Furthermore, the in vivo biological activity of target compounds with efficient OfHex1 inhibitory potency were
assayed against Myzus persicae, Plutella xylostella, and Ostrinia furnacalis. This present work indicates that
glycosylated naphthalimides can be further developed as potential pest control and management agents
targeting OfHex1.
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KEYWORDS: Glycosylated naphthalimids, β-N-acetylhexosaminidase, OfHex1, inhibitors, molecular
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docking, MD simulations
INTRODUCTION
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Chitin is a homopolymer of β-1,4-linked N-acetyl-β-D-glucosamine (GlcNAc), and is the second most
abundant polysaccharide in nature.1 Chitin is also a key structural component of the fungal cell wall, nematode
eggshell, and arthropod exoskeleton.2 Moreover, the cuticles of the integument and peritrophic membranes of
the midgut have found to contain chitin.3 Chitin degradation is catalyzed by two members of the glycoside
hydrolase family, GH18 chitinases (EC 3.2.1.14) and GH20 β-N-acetylhexosaminidases (EC 3.2.1.52).4,5
GH18 chitinases catalyze the cleavage of chitin into shorter chito-oligosaccharides, then GH20
β-N-acetylhexosaminidases hydrolyze these oligosaccharides into N-acetyl-D-glucosamine (GlcNAc) from
terminal non-reducing.4,5 Chitin degradation is essential to the growth and maturation of insects.6,7 Interference
with insect chitin degradation disrupts molting, pupation and eclosion processes, eventually leading to insect
death.6,8 Chitin is completely absent from higher plants and mammals,5 and thus the inhibition of chitin
degradation is a promising strategy for the development of green pesticides.5-10
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GH20 β-N-acetylhexosaminidase OfHex1 from the destructive agricultural pest, Ostrinia furnacalis
(Guenée), is an important enzyme during insect chitin degradation.11 The physiological function of OfHex1
shown via RNA interference is to degrade of old cuticles of O. furnacalis.12 Furthermore, the crystal structure
of OfHex1 (PDB: 3NSM) revealed the unique structural feature, with long substrate-binding pocket containing
two subsites (-1 and +1).11 The -1 subsite is responsible for catalysis substrates, whilst the +1 subsite is
responsible for binding related moieties of substrates to enhance affinity and specificity.11,12 The
crystallography of OfHex1 has attracted intense research attention and provides a solid foundation for the
design of specific inhibitors.10,13-15
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To date, a number of OfHex1 inhibitors have been reported, including TMG-chitotriomycin12, PUGNAc9,
NAG-thiazoline (NGT)13, naphthalimides14-15, phlegmacin B17, berberine16, thiazolylhydrazones10. Among
these inhibitors, NAG-thiazoline is the only molecule designed as an analog of the oxazolinium reaction
intermediate, but shows poor potency against OfHex1 with Ki values of 79 μM.13 Subsequently, NMAGT
bearing a methylamino substituent on the thiazoline ring was designed and synthesized by Yang, and exhibited
higher inhibitory activity (Ki value of 0.13 μM) against OfHex1.13 This suggested that the enlargement of the
functional group on the thiazoline could improve inhibitory activity against OfHex1. Naphthalimide
derivatives, including Q1 and Q2, are an important class of non-carbohydrate inhibitors of OfHex1.14 In
particular, Q1 has been shown to exhibit inhibitory activity towards OfHex1 with a Ki value of 4.28 μM.14 Q2
containing a dimethylamino group at the naphthalimide exhibited a Ki value of 0.31 μM against OfHex1.14 The
complex crystal structures of Q1-OfHex1 (PDB: 3WMB) and Q2-OfHex1 (PDB: 3WMC) show that the
thiadiazole moiety bound the -1 subsite of the active pocket, and that the naphthalimide group was sandwiched
by residues Val327 and Trp490 at the +1 subsite.14 In addition, related to the naphthalimide moiety of Q1 in
OfHex1, the 4-dimethylaminonaphthalimide of Q2 rotates approximately 180° to Trp490.14 This
conformational change ultimately results in tight binding of Q2 to OfHex1, leading to a 13-fold increase in
potency compared with Q1.14 Thus, the 4-substituent at the naphthalimide group exerts a critical effect on the
potency against OfHex1(Figure 1).
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In our previous studies, we presented glycosylated naphthalimide derivatives as promising lead compounds
for β-N-acetyl-D-hexosaminidase inhibitors.15,17,18 In particular, inhibitors 15k17 (1, Ki =16.3 μM, unpublished
data), 6e15 (2, Ki =22.4 μM, unpublished data), and 6f 15 (3, Ki =21.8 μM) exhibited higher inhibitory potency
towards OfHex1. Considering the crucial roles of OfHex1 to agricultural pest control, we further modified
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these structures to improve the inhibitory efficiency. Prompted by these observations, we retained the frame
structure of glycosylated naphthalimides and focused on two aspects of optimization, namely the 2-acetamido
group at the glycosyl moiety (to increase affinity at the -1 the subsite) and the 4-substituent at the
naphthalimide (to increase affinity at the +1 subsite) (Figure 1). Accordingly, several classes of glycosylated
naphthalimide derivatives were synthesized and the inhibitory activity against OfHex1 was evaluated. To
investigate the selectivity of these naphthalimides, their inhibitory capabilities towards human
β-N-acetylhexosaminidase B (HsHexB) and human O-GlcNAcase (hOGA) were assessed. This work may
provide useful information for future design of eco-friendly pesticides.
HO
HO
HO
HO
optimization HO
HO
O
O
N
N
S
S
CH3
NGT
NHCH3
NMAGT
OfHex1: K = 0.13
OfHex1: K = 79
M

M

i
i
N
N
O
N
N
O
H
H
N
H
optimization
N
N
N
O
N
O
S
S
Q1
Q2
OfHex1: K = 4.28
M

OfHex1: K = 0.31 M

i
i
Optimization strategy for OfHex1 inhibitors NMAGT and Q2
Inspiration and molecular design
HO
HO
O
N
O
O
N
O
optimization
optimization
O
H
H
O
R
H
H
HO
HO
HO
HO
S
N
S
N
6
3
6
3
NHAc
NHAc
1
11a-11e
OfHex1: K = 16.3
M

i
HO
HO
O
O
N
N
N
N
R2
O
O
HO
HO
HO
HO
N
O
N
O
S
N
S
N
n
n
NHAc
NHR1
n=5, 6
15a-15y and 23a-23f
2: n=5; OfHex1: K = 22.4
M
M


i
3: n=6; OfHex1: K = 21.8
i
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Figure 1. Design of novel glycosylated naphthalimide derivatives for OfHex1.
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MATERIALS AND METHODS
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Materials. All commercial materials were commercially available and treated with standard methods before
use. With TMS as an internal reference, a Bruker AVANCE600 spectrometer was used to record 1H NMR-300
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MHz and 13C NMR-75 MHz in CDCl3 or DMSO-d6 at 25°C. The Bruker Daltonics Bio-TOF-Q III mass
spectrometer (Bruker Co., Karlsruhe, Germany) was used to give high-resolution mass spectra (HRMS).
Reaction progress was monitored using thin layer chromatography (TLC) on silica gel GF254 plates with
detection by charring with 15% (v/v) H2SO4 in MeOH or by UV light (254 nm).
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Synthetic Chemistry. Detailed synthetic procedures and characterization data for all of the synthesized
compounds are given in the Supporting Information.
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Enzyme Preparation. OfHex1 was overexpressed in Pichia pastoris and then purified according to
previous methods.12 HsHexB was obtained as described in the literature.17 Human O-GlcNAcase (hOGA) was
overexpressed in Escherichia coli BL21(DE3) and purified by IMAC as described previously.19
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Enzyme Inhibitory Activity Assays. The inhibitory activities of OfHex1, HsHexB, and hOGA were
assayed in end-point experiments using 4-methylumbelliferyl N-acetyl-β-D- glucosaminide (4-MU-GlcNAc)
as the substrate. OfHex1 and hOGA were assayed in 20 mM sodium phosphate buffer (pH 6.5), HsHexB was
assayed in 20 mM sodium citrate buffer (pH 4.5). In a final assay volume of 100 μL, the enzyme was
pre-incubated with inhibitors in buffer for 10 min at 30 oC, then 40 μM substrate (4-MU-GlcNAc) was added.
o
After incubation for a further 20 min at 30 C, the reaction was terminated by the addition of 100 μl 0.5 M
sodium carbonate solution. The fluorescence of the liberated MU was quantitated using a Varioskan Flash
microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at an excitation of 366 nm and emission of
445 nm. When determining the IC50 values, various inhibitor concentrations were used to detect the
corresponding inhibition rates. The inhibition constant (Ki) was obtained using Dixon plots by changing the
concentration of the 4-MU-GlcNAc at a constant concentration (40, 20 and 10 μM).
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Molecular Docking. The crystal structure of OfHex1 in complex with PUGNAc (PDB ID:3OZP)9 was
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retrieved from the Protein Data Bank and used as the starting model for molecular docking employing the
Sybyl Software (Version 7.3).20 Prior to docking calculations, the structures were optimized using MMFF94
force field. For the protein, all water molecules were removed and missing hydrogen atoms were added.
Subsequently, the ligand protomol, appropriate putative ligand pose, was generated by ligand mode based on
the Hammerhead scoring function with the molecular similarity algorithm in the active domain of the
receptor.21-23 Finally, molecular dockings were performed using the Surflex–Dock algorithm.
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Molecular Dynamics (MD) Simulations. To obtain the convincing conformations, the molecular dynamics
(MD) simulations were carried out after docking. MD simulations of three systems (OfHex1 in complex with
ligands 15r, 15y, and 23f) were performed using the Amber14 package24. AMBER03 force field25 was selected
for the protein, and GAFF force field26 was selected for the ligand. Each system was immersed in a truncated
octahedral box with TIP3P water molecules and electrostatic neutralized by adding appropriate number of
counterions (Cl− or Na+). Initially, Sander module was used to realize the energy minimization of the system.
The hydrogen atoms and water molecules were minimized with the 2500 cycles of steepest descent followed
by the conjugated-gradient methods. Then, 2500 cycles of the steepest-descent and 2500 cycles of the
conjugated gradient algorithm were carried out to minimize all atoms of the systems. After that, the system
was heated gradually from 0 to 300 K in the NVT ensemble and equilibrated in 300 K. Finally, MD
Simulations of 30 ns were performed at a constant temperature of 300 K and pressure of 1 atm employing the
PMEMD module. The SHAKE method27 was applied to constrain hydrogen atoms, and the particle mesh
Ewald (PME) method28 was used to treat the long-range electrostatic interactions under periodic boundary
conditions.
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In Vivo Activity. Target compounds that efficiently inhibited OfHex1 were further tested for their
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insecticidal activity against Myzus persicae and Plutella xylostella according to previous studies.29-30 Test
compounds were dissolved in DMSO and diluted to a final concentration of 600 μg/mL and 200 μg/mL.
Hexaflumuron was used as a positive control and DMSO was used as a negative control. Myzus persicae and
Plutella xylostella were treated for 48h and 72h, respectively. Each bioassay was repeated in triplicate.
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Ostrinia furnacalis larvae were raised using an artificial diet with a relative humidity of 70% at 26−28 °C.
Day 1 third-instar larvae were selected for feeding experiments. Compounds 15r and 15y were dissolved in
DMSO and diluted with an artificial diet to a final concentration of 600 μg/mL. In the control group, the
artificial diet contained an equal volume of DMSO. Each group contained 30 individual larvae that were
continuously fed for 5 days.7,16
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RESULTS AND DISCUSSION
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Synthesis of Target Compounds 11a-11e and 15a-15t. The target compounds 11a-11e are outlined in
Scheme 1. Briefly, the key intermediates 6a-6e18,31 and 9b17 were obtained according to published methods
(Schemes S1-S3). Treatment of 9b with excess naphthalimides 6a-6e in the presence of K2CO3 and CH3CN
yielded the acetyl-protected compounds 10a-10e. The deacetylation of 10a-10e via methanol-ammonia
catalysis resulted in 11a-11e (Scheme S3).
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To investigate the influence of the 2-substituent group (at glycosyl moiety) and the 4-substituent group (at
naphthalimide) on the inhibitory activity and selectivity against OfHex1, triazole group-bearing
thioglycosyl-naphthalimides 15a-15t were synthesized (Scheme 2). Bromides 9a-9d were prepared according
to literature procedures15, which on further reaction with NaN3 yielded key intermediates 13a-13d (Scheme
S6). Meanwhile, naphthalimide derivatives 12a-12e were prepared as previously reported15,32 (Schemes
S4-S5). Subsequently, azides 13a-13d were reacted with 12a-12e in 2:1 THF/water mixture via Cu-catalyzed
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cycloaddition to form precursors 14a-14t. Finally, deprotection of 14a-14t was performed using
methanol-ammonia to produce the target compounds 15a-15t (Scheme S6).
141
O
N
O
O
O
O
N
O
R1'
R1'
R1'
i
ii
NH2
NHBoc
3
3
O
4a-4b
5a-5b
6a-6b
R1'= Br, Cl
O
N
O
O
N
O
N
R1''
R1''
Br
iii
ii
NH2
3
NHBoc
3
NHBoc
3
O
5c-5e
O
5a
6c-6e
R1''= OCH3, N(CH3)2, piperidyl
HO
AcO
AcO
AcO
AcO
AcO
AcO
O
O
O
iv,v,vi
vii
viii
HO
HO
S
Br
SH
OH
6a-6e
6
NHAc
NHAc
NHAc
9b
7
8a
HO
AcO
AcO
AcO
O
O
N
O
O
R1
O
R1
H
H
N
HO
HO
ix
S
N
N
3
O
S
6
6
3
NHAc
NHAc
10a-10e
11a-11e
R1= Br, Cl, OCH3, N(CH3)2, piperidyl
142
143
144
145
146
147
Scheme 1. Synthesis of glycosylated naphthalimide derivatives 11a-11e. (i) tert-butyl (3-aminopropyl) carbamate, EtOH; (ii)
DCM, CF3COOH; (iii) MeOH, KOH for 5c; dimethylamine, 2-methoxyethanol for 5d; piperidine, 2-methoxyethanol for 5e; (iv)
AcCl; (v) thiourea, acetone; (vi) Na2S2O5, DCM, H2O; (vii) 1,6-dibromohexane, K2CO3, acetone, H2O; (viii) 6a-6e, K2CO3,
CH3CN; (ix) NH3, MeOH.
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O
O
O
O
N
O
N
O
O
N
R2'
R2'
Br
R2''
ii
i
O
O
4a-4b
12a-12b
12a
12c-12e
R2'= Br, Cl
R2''= OCH3, N(CH3)2, piperidyl
AcO
AcO
AcO
AcO
AcO
O
O
O
iii
iv
v
AcO
AcO
AcO
S
N3
S
Br
SH
AcO
n
12a-12e
n
NHR3
NHR3
NHR3
13a-13d
8a-8b
9a-9d
AcO
AcO
AcO
HO
O
N
O
O
N
O
N
N
N
N
R2
R2
O
O
HO
HO
vi
S
N
S
N
n
n
NHR3
NHR3
15a-15t
14a-14t
R2= Br, Cl, OCH3, N(CH3)2, piperidyl; R3= Ac, Cbz; n= 5, 6
149
150
151
152
Scheme 2. Synthesis of glycosylated naphthalimide derivatives 15a-15t. (i) 2-propynylamine, EtOH; (ii) MeOH, KOH for 12c;
dimethylamine, 2-methoxyethanol for 12d; piperidine, 2-methoxyethanol for 12e; (iii) a, ω- dibromoalkane, K2CO3, acetone,
H2O; (iv) NaN3, K2CO3, acetone, H2O; (v) 12a-12e, CuSO4, sodium ascorbate, THF, H2O; (vi) NH3, MeOH.
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Evaluation of Enzyme Inhibitory Activity. The target compounds 11a-11e and 15a-15t were evaluated for
their inhibitory activity against OfHex1, HsHexB, and hOGA (Tables 1-3).
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158
159
160
161
162
As shown in Table 1, the substituents at the 4-position of naphthalimide moiety (11a-11e) improved the
inhibitory potency against OfHex1 compared to naphthalimide bearing no 4-substituent group (compound 1).
In particular, naphthalimides 11a-11c bearing smaller functional groups (Br, Cl, OMe) exhibited a minor
increase in activity. Naphthalimides 11d-11e bearing a basic substituent (N(CH3)2 or piperidyl) resulted in a
more effective inhibitory potency. Among these inhibitors, compound 11d bearing the dimethylamino group
showed the highest inhibitory activity against OfHex1 with an IC50 value of 13.4 μM (Table 3). Additionally,
11d showed no inhibitory activities against HsHexB and hOGA, which exhibited improved selectivity
compared to lead compound 1.
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As shown in Table 2, bioassay results of 15a-15t revealed that 2-substituent of the glycosyl moiety and
4-substituent of naphthalimide significantly influenced the potency and selectivity of these compounds
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towards OfHex1. Specifically, upon increasing the size from acetyl (Ac) to benzyloxycarbonyl (Cbz), a loss in
inhibitory activity against OfHex1 was observed. These results suggest that a large substituent at the 2-position
of the sugar ring leads to steric hindrance, ultimately decreasing the binding affinity of the inhibitors and
OfHex1. The 4-substituted group on naphthalimide moiety increased the inhibitory potency against OfHex1
compared to lead compounds 2 and 3. In detail, inhibitors bearing 4-bromo (15a-15b) and 4-piperidyl
(15q-15r) groups possessed improved potency towards OfHex1 compared to inhibitors bearing Cl (15e-15f),
OCH3 (15i-15j), and N(CH3)2 (15m-15n) substituents. The length of the linker between glycosyl and triazole
also affected the activity against OfHex1, and increasing the length of the carbon atoms from five to six
slightly increased their activity (e.g. 15b>15a or 15r>15q). Further IC50 determination showed that 15r had
the highest inhibitory potency against OfHex1 (IC50 = 6.4 μM) in 15a-15t and more selective than lead
compounds 2 and 3 (Table 3).
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Table 1. Inhibition rates of 11a-11e against OfHex1, HsHexB, and hOGA at 10 μM.
HO
O
N
O
O
R1
H
HO
HO
S
N
6
3
NHAc
178
Inhibition rate at 10 μM (%)
Compd
R1
OfHex1
HsHexB
hOGA
11a
11b
11c
11d
Br
Cl
39.6 ± 3.1 13.9 ± 0.6 21.0 ± 0.5
46.0 ± 1.8 9.2 ± 0.2 18.3 ± 2.2
42.6 ± 1.6 12.5 ± 1.1 7.7 ± 1.6
54.6 ± 0.8 13.9 ± 1.7 12.7± 0.3
OMe
N(CH3)2
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1
piperidyl
H
48.3 ± 0.7 13.6 ± 0.5 14.2 ± 0.8
35.2 ± 1.7 20.1 ± 0.1 9.9 ± 0.3
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180
Table 2. Inhibition rates of 15a-15t against OfHex1, HsHexB, and hOGA at 10 μM.
HO
O
N
O
N
N
R2
O
HO
HO
S
N
n
NHR3
181
substituent group
Inhibition rate at 10 μM (%)
Compd
R2
Br
R3
Ac
n
5
6
5
6
5
6
5
6
5
6
5
6
5
OfHex1
HsHexB
hOGA
15a
15b
15c
15d
15e
15f
66.0 ± 1.3 11.2 ± 1.4 14.1 ± 0.5
68.1 ± 2.5 6.7 ± 0.8 17.6 ± 0.3
Br
Ac
Br
Cbz
Cbz
Ac
8.7 ± 1.2
7.9 ± 0.1
4.0 ± 0.4
Br
15.3 ± 3.6 7.4 ± 0.3 13.0 ± 0.5
37.6 ± 1.5 10.1 ± 1.8 11.4 ± 0.7
49.2 ± 2.8 10.3 ± 2.0 17.8 ± 0.3
Cl
Cl
Ac
15g
15h
15i
Cl
Cbz
Cbz
Ac
19.6 ± 2.6 4.8 ± 0.7
7.3 ± 1.2
Cl
11.2 ± 1.3 3.7 ± 0.8 10.9 ± 0.4
42.2 ± 2.9 10.5 ± 0.4 11.5 ± 0.6
60.1 ± 0.6 8.0 ± 1.1 13.8 ± 0.5
OMe
OMe
OMe
OMe
N(CH3)2
15j
Ac
15k
15l
Cbz
Cbz
Ac
19.6 ± 1.4 2.1 ± 1.5
7.2 ± 0.9
11.2 ± 3.0 12.4 ± 0.2 10.9 ± 0.8
56.9 ± 0.5 8.1 ± 0.3 13.1 ± 0.8
15m
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15n
15o
15p
15q
15r
15s
15t
2
N(CH3)2
Ac
6
5
6
5
6
5
6
5
6
57.9 ± 2.1
24.2 ± 2.4
19.7 ± 1.7
13.3 ± 0.2
8.2 ± 0.5
10.9 ± 0.7
9.7 ± 0.9
9.3 ± 0.7
6.1 ± 1.9
N(CH3)2 Cbz
N(CH3)2 Cbz
piperidyl Ac
piperidyl Ac
piperidyl Cbz
piperidyl Cbz
67.5 ± 1.8 5.5 ± 0.9 14.0 ± 1.1
75.1 ± 0.4 7.1 ± 1.7 12.6 ± 0.9
16.3 ± 2.9 4.3 ± 0.6
4.9 ± 0.5
6.2 ± 0.7
6.5 ± 2.3
7.9 ± 0.4
H
H
Ac
Ac
29.2 ± 0.3 16.4 ± 3.2 10.3 ± 1.8
30.6 ± 1.1 16.1 ± 0.7 14.0 ± 1.1
3
183
Table 3. IC50 values of representative compounds for OfHex1, HsHexB, and hOGA.
IC50 (μM)
Compd
OfHex1
13.4 ± 1.2
8.5 ± 0.8
8.3 ± 1.0
9.7 ± 0.3
11.4 ± 1.1
10.9 ± 0.1
7.6 ± 0.9
6.4 ± 0.3
HsHexB
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
hOGA
> 100
11d
15a
15b
15j
15m
15n
15q
15r
1
> 100
66.9 ± 2.1
> 100
> 100
> 100
> 100
> 100
18.0 ± 1.4 55.1 ± 0.5
> 100
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3
23.9 ± 0.9 97.5 ± 1.6
22.5 ± 0.2 94.2 ± 1.9
> 100
> 100
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
Optimization of Inhibitor 15r. The highest performing compound from the first stage, 15r, was selected
for further structural optimization. Based on structure−activity relationships, we fixed the 2-acetyl on the
glycosyl moiety and selected a linker with six carbon atoms (n=6, Scheme 2). Then we replaced the
4-piperidyl group with a similar sized nitrogen-containing cycloalkane. Thus, five glycosyl-naphthalimides
15u-15y were synthesized (Scheme 3). The synthetic route of 15u-15y were identical to 15a-15t and outlined
in Schemes S7 and S8. The analysis of 15u-15y against OfHex1 (Table 4) showed that the size of the
nitrogen-containing cycloalkane could affect inhibitory efficiency. Shrinking the 4-piperidyl group of 15r to
4-pyrrolyl (15u) enhanced the activity (IC50 value of 6.4 μM to 5.1μM). Enlargement of the piperidyl (15r) to
azepanyl (15y) resulted in a 1-fold increase in inhibitory efficiency. Moreover, the addition of a methyl group
at the 4-position of piperidyl (15w) led to slightly increased potency. However, changing the piperidyl group
to morpholino (15v) or methylpiperazinyl (15x) decreased the potency against OfHex1, particularly for 15x,
which resulted in a significantly weakened inhibitory effect with IC50 value of 87.1 μM. Among these
inhibitors, 15y (IC50 = 3.1 μM against OfHex1, IC50 >100 μM against HsHexB, IC50 = 90.5 μM against
hOGA) exhibited excellent potency and selectivity against OfHex1, confirming the correctness of our
optimization strategy.
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AcO
AcO
AcO
AcO
O
N
O
N
N
R2
O
O
i
AcO
AcO
S
N3
S
N
6
12f-12j
6
NHAc
NHAc
13b
14u-14y
R2
HO
O
N
O
N
N
O
ii
HO
HO
S
N
6
NHAc
15u-15y
O
N
N
N
N
N
N
15u
R2
=
15v
15w
15x
15y
200
201
202
Scheme 3. Synthesis of glycosylated naphthalimide derivatives 15u-15y. (i) 12f-12j, CuSO4, sodium ascorbate, THF, H2O; (iv)
NH3, MeOH.
203
Table 4. Inhibitory activity of the optimized compounds 15u-15y against OfHex1, HsHexB, and hOGA
IC50 (μM)
Compd
R2
OfHex1
HsHexB
hOGA
N
N
15u
15v
5.1 ± 0.3
> 100
68.8 ± 2.4
O
9.4 ± 0.9
6.0 ± 0.2
> 100
> 100
> 100
N
15w
15x
71.7 ± 1.9
N
N
87.1 ± 3.5
> 100
7.8 ± 0.6
N
15y
15r
3.1 ± 0.4
6.4 ± 0.3
> 100
> 100
90.5 ± 3.2
> 100
N
204
205
Further Optimization of Inhibitor 15y. Considering that NMAGT bearing a methylamino on thiazoline
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207
208
209
210
211
212
was approximately 600-fold more potent than NGT (bearing a methyl on thiazoline) against OfHex113, we
further attempted to modify the 2-position on glycosyl moiety of 15y to improve its potency and selectivity.
Our previous experience highlighted that the large substituents at 2-position (i.e. Cbz, Table 2) would result in
activity loss, and so the methyl group of 2-acetamido was replaced with smaller substituents (i.e. NCH3, CF3,
Et, Pr). Accordingly, compounds 23a-23c (Scheme 4) bearing Et, NCH3, CF3 and compounds 23e-23f
(Scheme 5) bearing n-Pr and i-Pr were synthesized. The specific synthesis methods are shown in Schemes S9
and S10.
213
214
215
216
217
218
219
220
221
222
223
The inhibitory activity data of compounds 23a-23f are exhibited in Table 5. Unfortunately, when slightly
increasing the size of the CH3 group (at the 2-acetamido) to Et, NHCH3, CF3 substituents, the reduced activity
against OfHex1 was observed. Moreover, the larger substituents led to the lower inhibitory potency.
Specifically, replacement of the CH3 to CF3 or Et led to a 2-fold reduction in potency (from 15y to 23c or 23a).
The loss of activity from 23a to 23b suggested that NH of the the NHCH3 group was detrimental to the affinity
compared to CH2 of the Et group at this position. When the substituent was modified from CH3 to n-Pr (23e),
the inhibitory activity was reduced 10-fold. This reduction continued with i-Pr (23f) and OBn (23d) groups
leading to a loss of OfHex1 potency (IC50 >100 μM). Additionally, enlargement of the functional group at the
2-position of glycosyl moiety promoted selectivity towards OfHex1 over hOGA (compared with the IC50
values of 15y, 23a, 23b, 23c towards hOGA), and compounds 23a-23f showed no inhibitory potency towards
HsHexB.
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HO
AcO
AcO
AcO
AcO
AcO
AcO
O
O
O
v,vi,vii
i, ii, iii
iv
HO
HO
OAc
NH2HCl
17
OAc
NHCOR4
18a-18c
OH
NH2HCl
16
AcO
AcO
AcO
AcO
AcO
AcO
AcO
AcO
AcO
O
O
O
viii
ix
x
S
N3
SH
NHCOR4
19a-19c
S
Br
6
6
12j
NHCOR4
21a-21c
NHCOR4
20a-20c
AcO
AcO
AcO
HO
O
O
N
O
N
N
N
N
N
N
O
O
xi
HO
HO
N
O
S
N
S
N
6
6
NHCOR4
NHCOR4
23a-23c
22a-22c
225
226
23a: R4=CH2CH3; 23b: R4=NHCH3; 23c: R4=CF3
227
228
229
230
Scheme 4. Synthesis of glycosylated naphthalimide derivatives 23a-23e. (i) p-anisaladehyde, NaOH, H2O; (ii) Py, Ac2O; (iii)
acetone, HCl, H2O; (iv) Et3N, CH2Cl2, RCOCl for 18a-18b; Et3N, CH2Cl2, TFAA for 18c; (v) HBr, CH3COOH, CH2Cl2; (vi)
thiourea, acetone; (vii) Na2S2O5, CH2Cl2, H2O; (viii) 1,6-dibromoalkane, K2CO3, acetone, H2O; (ix) NaN3, acetone, H2O; (x) 12j,
CuSO4, sodium ascorbate, THF, H2O; (xi) NH3, MeOH.
231
AcO
AcO
AcO
AcO
AcO
AcO
AcO
AcO
AcO
O
O
O
i
ii
iii
S
N3
6
S
Br
6
SH
NHCbz
8b
12j
NHCbz
13d
NHCbz
9d
AcO
AcO
AcO
HO
O
N
O
O
N
O
N
N
N
N
N
N
N
N
O
O
iv
HO
S
N
S
N
HO
6
6
NHCbz
NHCbz
22d
23d
v
AcO
AcO
AcO
HO
O
N
O
O
N
O
N
N
N
N
O
O
vi, iv
HO
S
N
S
N
HO
6
6
NH2
NHCOR4
24
23e-23f
232
233
23e: R4=CH2CH2CH3; 23f: R4=CH(CH3)2
234
235
236
Scheme 5. Synthesis of glycosylated naphthalimide derivatives 23d-23f. (i) 1,6-dibromoalkane, K2CO3, acetone, H2O; (ii) NaN3,
acetone, H2O; (iii) 12j, CuSO4, sodium ascorbate, THF, H2O; (iv) NH3, MeOH; (v) Pd/C, H2, THF, MeOH, HCl; (vi) R4COCl,
Et3N, CH2Cl2, DMAP.
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Table 5. Inhibitory activity of the optimized compounds 23a-23f against OfHex1, HsHexB, and hOGA
IC50 (μM)
Compd
R4
OfHex1
6.8 ± 0.5
7.2 ± 0.7
6.4 ± 0.4
30.2 ± 1.8
> 100
HsHexB
> 100
> 100
> 100
> 100
> 100
> 100
> 100
hOGA
94.8 ± 0.9
97.1 ± 3.4
98.9 ± 1.4
> 100
23a
23b
23c
23e
23f
Et
NHCH3
CF3
n-Pr
i-Pr
> 100
23d
15y
OBn
CH3
> 100
> 100
3.1 ± 0.4
90.5 ± 3.2
239
240
241
242
243
244
Inhibitory Mechanism of Glycosylated Naphthalimides for OfHex1. Two representative inhibitors,
namely 15r and 15y, were selected to investigate the inhibitiory mechanism of these glycosylated
naphthalimides. Dixon plots of 15r and 15y against OfHex1 were performed. As shown in Figure 2, 15r and
15y were competitive inhibitors, with Ki values of 5.3 ± 0.2 μM and 2.7 ± 0.1 μM against OfHex1,
respectively.
245
246
247
248
To investigate the basis for the potency of these glycosylated naphthalimides towards OfHex1, the binding
modes of 15r, 15y and 23f with OfHex1 were carried out via molecular docking. As shown in Figure 3a, the
glycosyl moiety from all three inhibitors was found to bind to -1 subsite of OfHex1 and the naphthalimide
group extended out from the active pocket. Moreover, the conformation of inhibitor 15r was similar to that of
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250
15y but buried shallower. For the binding mode of 23f, the naphthalimide group had a great degree of rotation
compared to 15r, which moved away from residues Gly485 and Ala486.
251
252
253
254
255
In an effort to further illustrate the appropriate binding modes of compounds 15r, 15y and 23f in complex
with OfHex1, 30 ns MD simulations were performed (Figures 3b-3c). The root-mean-square deviations
(RMSD) values were maintained at around 1.5-3.1 Å for these three systems, suggesting the complexes
underwent reasonable conformational changes (Figure 3b). The conformations of 15r, 15y and 23f combined
with OfHex1 at 30 ns of the MD simulations were superimposed (Figure 3c).
256
257
258
259
260
261
262
263
264
265
266
267
268
269
In comparison to the docking conformations (Figure 3a), the alkyl chain of 15r and 15y showed a high
degree of folding after MD simulations (Figure 3c). Thus, naphthalimide moiety of 15r and 15y could bind to
+1 subsite of the OfHex1 pocket with high affinity, forming strong π-π stacking interactions with Trp490 and
van der Waals interactions with Val327 (Figures 3c-3e). The glycosyl moiety of 15r was found to bind to the
-1 subsite via H-bonding interactions with residues His246, Glu297, Asp299, Glu367, and the triazole ring
bound with residue Tyr475 at a distance of 2.1 Å (Figure 3d). These interactions in -1 subsite are much less
than that of PUGNAc (Ki = 0.1 μM against OfHex1)9, which may suggest that the efficiency of these
glycosylated naphthalimids is in micromolar range. As a comparison, the glycosyl moiety of 15y buried
shallower than 15r, which might be due to the increased size of cycloalkane (from piperidyl to azepanyl)
leading to the steric hindrance in -1 subsite (Figure 3c). Accordingly, the glycosyl moiety of 15y formed four
fewer hydrogen bonds than 15r (five hydrogen bonds) with residues Arg220, Glu297, Asp299, Tyr475
(Figures 3d-3e). However, the triazole ring of 15y could form three hydrogen bonds (more than 15r) with
residues Trp483, val484, and Trp490 (Figures 3d-3e). On the basis of these observations, 15y (Ki = 2.7 μM)
ultimately displayed a higher inhibitory potency against OfHex1 than 15r (Ki = 5.3 μM).
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272
273
274
It was noteworthy that the conformation of 23f significantly differed to 15r and 15y (Figures 3c and 3f). In
detail, the glycosyl moiety of 23f was buried at the entrance of the OfHex1 pocket and only interacted with
Arg 220, whilst the triazole bearing naphthalimide moiety extended out from the active pocket (Figure 3f).
These results revealed that the branched and large i-Pr group led to the glycosyl moiety of 23f being unable to
move deeply into the -1 subsite of OfHex1, explaining the basis for the loss of activity.
275
276
277
Figure 2. Dixon plots for inhibitors 15r and 15y against OfHex1
278
(b)
(a)
279
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(d)
(c)
280
(f)
(e)
281
282
283
284
285
286
Figure 3. Binding mechanism of 15r, 15y and 23f with OfHex1 revealed by molecular docking and MD simulations. (a)
Molecular docking and (c) 30 ns MD simulations of OfHex1 in complex with 15r, 15y and 23f. (b) RMSD changes of 15r, 15y
and 23f in the active pocket of OfHex1. Specific binding modes of (d) OfHex1-15r, (e) OfHex1-15y, and (f) OfHex1-23f
systems at 30 ns after MD simulations. The protein is shown as a cartoon style. Compound 15r is shown in green, 15y is shown
in yellow, 23f is shown in cyan (colored according to the element).
287
288
289
290
291
292
Insecticidal Activity. The glycosylated naphthalimides that showed higher inhibitory activity against
OfHex1 were further selected to evaluate their insecticidal activity. As shown in Table 6, the activity of
compounds 15b and 15y were higher than the control drug hexaflumuron towards Myzus persicae with the
mortalities of 41.5% and 47.2% at a concentration of 200 μg/mL. Moreover, Compounds 15m, 15n and 23a
displayed > 50% mortality against Myzus persicae at 600 μg/mL, and the remaining compounds exhibited
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295
296
297
298
299
relatively weak activity. For Plutella xylostella, compounds 15m, 15n, 15r, 15v, 15w and 15y exhibited high
activity with mortality rates over 50% at 600 μg/mL, which were lower than those of hexaflumuron (93.3%).
Although many of the compounds displayed moderate or a lack of insecticidal activity against Plutella
xylostella, a smaller body size of the treated insects was noted after 72 hours feeding, compared to the negative
control groups. The structure–activity relationships of these glycosylated naphthalimides against Myzus
persicae and Plutella xylostella showed that the N(CH3)2 substituent at the 4-position of the naphthalimides
was perhaps the active functional group for insecticidal activity.
300
301
Table 6. Insecticidal activity of glycosylated naphthalimides (mortality (%) ± SD).
Plutella
Myzus persicae
xylostella
Compd
600 μg/mL 200 μg/mL 600 μg/mL
11d
15a
15b
15j
46.2 ± 2.8
7.9 ± 0.5
16.7 ± 0.9
33.3 ± 1.2
23.3 ± 0.8
10.0 ± 0.8
53.3 ± 1.6
66.7 ± 2.4
33.3 ± 2.0
56.7 ± 1.2
13.3 ± 1.2
76.7 ± 3.7
19.6 ± 3.5
74.1 ± 1.1
68.0 ± 1.7
31.9 ± 2.5
48.9 ± 3.6
39.2 ± 0.5
41.5 ± 0.9
15m
15n
15q
15r
15u
38.8 ± 2.5
29.4 ± 1.2
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15v
9.5 ± 0.9
43.1 ± 1.3
82.7 ± 2.4
55.1 ± 4.1
39.2 ± 3.0
26.2 ± 0.8
75.6 ± 2.2
50.0 ± 0.0
53.3 ± 0.9
63.3 ± 3.1
43.3 ± 1.2
16.7 ± 0.4
20.0 ± 1.6
93.3 ± 0.5
15w
15y
47.2 ± 1.9
17.1 ± 1.7
23a
23b
23c
Hexaflumuron
40.7 ± 3.1
302
303
304
305
306
307
308
Effects of 15r and 15y on Ostrinia furnacalis Larvae. Compounds 15r and 15y were the most effective
OfHex1 inhibitors observed in this study. Both compounds thus fed to 3th-instar day 1 Ostrinia furnacalis
larvaes for 5 days. As shown in Figure 4, the larvaes of inhibitor-containing feeding groups (Figures 4c and
4d) exhibited slower growth than the control group (Figure 4b). Especially inhibitor 15y led to the death of >
76 % of larvaes after 5 days of feeding. The Ostrinia furnacalis larvaes that survived had significantly reduced
body sizes (Figure 4d). These results suggested that OfHex1 inhibitors are promising pest control reagents.
(c)
(a)
(b)
(d)
309
310
Figure 4. Bioactivity of OfHex1 inhibitors 15r and 15y for Ostrinia furnacalis larvaes. (a) 3th-instar day 1 larvaes prior to
311
inhibitor treatment; larvaes of (b) DMSO, (c) 15r, (d) 15y containing diet-fed group after 5 days of feeding.
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313
314
315
316
317
318
319
320
321
322
323
324
325
326
In conclusion, we present the molecular design, synthesis, and inhibitory activity against OfHex1, HsHexB,
and hOGA of glycosylated naphthalimides 11a-11e and 15a-15t. As a result, compound 15r exhibited the
higher efficiency against OfHex1 with a Ki value of 5.3 μM and excellent selectivity (IC50 >100 μM against
HsHexB and hOGA). After in-depth SAR studies, an azepanyl naphthalimide derivative, compound 15y (Ki
=2.7 μM against OfHex1, IC50 >100 μM against HsHexB, IC50 = 90.5 μM against hOGA), was identified as a
promising OfHex1 inhibitor. Moreover, molecular docking and MD simulations studies of 15r, 15y and 23f
were performed to allow us to rationalize the basis for the potency of these glycosylated naphthalimides
towards OfHex1. In addition, the efficient OfHex1 inhibitors were further selected to evaluate their insecticidal
activity against Myzus persicae and Plutella xylostella. The results showed that compounds 15b and 15y had
higher insecticidal activity against Myzus persicae than the commercial pesticide hexaflumuron. Furthermore,
feeding experiments demonstrated that the OfHex1 inhibitors 15r and 15y could effectively inhibit the growth
of Ostrinia furnacalis larvae. Thus, the structure−activity relationships combined with docking and MD
simulations studies provide direction for the further structural optimization for OfHex1, and the development
of green pest control and management agents.
327
328
ASSOCIATED CONTENT
329
330
331
■ Supporting Information
Experimental procedures, Molecular docking and MD simulations results, 1H NMR and 13C NMR spectrum.
■ AUTHOR INFORMATION
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Corresponding Author
*E-mail: zhangjianjun@cau.edu.cn.
*E-mail: qingyang@dlut.edu.cn.
Author Contributions
336
337
#These authors contributed equally to this work. All authors have given approval to the final version of the
manuscript.
338
339
340
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
341
342
We acknowledge the financial support by the National Natural Science Foundation (21772230, 31425021),
and Chinese Universities Scientific Fund (No.2019TC135).
343
344
■ ABBREVIATIONS
GH18, glycoside hydrolase family 18; GH20, glycoside hydrolase family 20; MD, molecular dynamics.
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346
REFERENCES
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1. Sutherland, T. E.; Andersen, O. A.; Betou, M.; Eggleston, I. M.; Maizels, R. M.; van Aalten, D.; Allen, J. E., Analyzing airway
inflammation with chemical biology: dissection of acidic mammalian chitinase function with a selective drug-like Inhibitor.
Chem. Biol. 2011, 18 (5), 569-579.
350
351
2. Merzendorfer, H.; Zimoch, L., Chitin metabolism in insects: structure, function and regulation of chitin synthases and
chitinases. J. Exp. Biol. 2003, 206 (24), 4393-4412.
352
3. Liu, J.; Liu, M.; Yao, Y.; Wang, J.; Li, Y.; Li, G.; Wang, Y., Identification of novel potential β-N-acetyl-D-hexosaminidase
24
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Page 25 of 27
Journal of Agricultural and Food Chemistry
353
354
inhibitors by virtual screening, molecular dynamics simulation and MM-PBSA calculations. Int. J. Mol. Sci. 2012, 13,
4545-4563.
355
356
4. Dahiya, N.; Tewari, R.; Hoondal, G. S., Biotechnological aspects of chitinolytic enzymes: a review. Appl. Microbiol.
Biotechnol. 2006, 71 (6), 773-782.
357
358
5. Liu, T.; Chen, L.; Ma, Q.; Shen, X.; Yang, Q., Structural insights into chitinolytic enzymes and inhibition mechanisms of
selective inhibitors. Current Pharmaceutical Design 2014, 20 (5), 754-770.
359
360
6. Zhu, K. Y.; Merzendorfer, H.; Zhang, W.; Zhang, J.; Muthukrishnan, S., Biosynthesis, turnover, and functions of chitin in
insects. Annu. Rev. Entomol. 2016, 61, 177-196.
361
362
7. Chen, L.; Liu, T.; Duan, Y.; Lu, X.; Yang, Q., Microbial secondary metabolite, Phlegmacin B-1, as a novel inhibitor of insect
chitinolytic enzymes. J. Agric. Food Chem. 2017, 65 (19), 3851-3857.
363
364
8. Dong, Y.; Jiang, X.; Liu, T.; Ling, Y.; Yang, Q.; Zhang, L.; He, X., Structure-based virtual screening, compound synthesis,
and bioassay for the design of chitinase inhibitors. J. Agric. Food Chem. 2018, 66 (13), 3351-3357.
365
366
9. Liu, T.; Zhang, H.; Liu, F.; Chen, L.; Shen, X.; Yang, Q., Active-pocket size differentiating insectile from bacterial chitinolytic
β-N-acetyl-D-hexosaminidases. Biochem. J. 2011, 438 (3), 467-474.
367
368
10. Yang, H.; Liu, T.; Qi, H.; Huang, Z.; Hao, Z.; Ying, J.; Yang, Q.; Qian, X., Design and synthesis of thiazolylhydrazone
derivatives as inhibitors of chitinolytic N-acetyl-β-D-hexosaminidase. Bioorg Med Chem 2018, 26 (20), 5420-5426.
369
370
11. Yang, Q.; Liu, T.; Liu, F.; Qu, M.; Qian, X., A novel β-N-acetyl-D-hexosaminidase from the insect Ostrinia furnacalis
(Guenee). FEBS J 2008, 275 (22), 5690-702.
371
372
12. Liu, T.; Zhang, H.-T.; Liu, F.-Y.; Wu, Q.-Y.; Shen, X.; Yang, Q., Structural determinants of an insect
β-N-acetyl-D-hexosaminidase specialized as a chitinolytic enzyme. J. Biol. Chem. 2011, 286 (6), 4049-4058.
373
374
13. Liu, T.; Xia, M.; Wang, J.; Zhang, H.; Shen, X.; Zhou, H.; Yang, Q., Exploring NAG-thiazoline and its derivatives as
inhibitors of chitinolytic β-acetylglucosaminidases. FEBS Lett 2015, 589 (1), 110-6.
375
376
14. Liu, T.; Guo, P.; Zhou, Y.; Wang, J.; Chen, L.; Yang, H.; Qian, X.; Yang, Q., A crystal structure-guided rational design
switching non-carbohydrate inhibitors' specificity between two β-GlcNAcase homologs. Sci. Rep. 2014, 4, 6188.
377
378
15. Chen, W.; Shen, S.; Dong, L.; Zhang, J.; Yang, Q., Selective inhibition of β-N-acetylhexosaminidases by
thioglycosyl-naphthalimide hybrid molecules. Bioorg. Med. Chem. 2018, 26 (2), 394-400.
379
380
16. Duan, Y.; Liu, T.; Zhou, Y.; Dou, T.; Yang, Q., Glycoside hydrolase family 18 and 20 enzymes are novel targets of the
traditional medicine berberine. J. Biol. Chem. 2018, 293 (40), 15429-15438.
381
382
17. Shen, S.; Chen, W.; Dong, L.; Yang, Q.; Lu, H.; Zhang, J., Design and synthesis of naphthalimide group-bearing
thioglycosides as novel β-N-acetylhexosaminidases inhibitors. J. Enzyme Inhib. Med. Chem. 2018, 33 (1), 445-452.
383
384
18. Shen, S.; Dong, L.; Chen, W.; Zeng, X.; Lu, H.; Yang, Q.; Zhang, J., Modification of the thioglycosyl-naphthalimides as
potent and selective human O-GlcNAcase inhibitors. Acs Med. Chem. Lett. 2018, 9 (12), 1241-1246.
385
19. Kong, H.; Lu, H.; Dong, Y.; Liang, X.; Jin, S.; Chen, W.; Liu, T.; Yang, Q.; Zhang, J., Synthesis of NAM-thiazoline
25
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 26 of 27
386
387
derivatives as novel O-GlcNAcase inhibitors. Carbohydr Res 2016, 429, 54-61.
20. SYBYL Molecular Modeling Software, 7.3, 2006, Tripos, St. Louis, MO.
388
389
21. Jain, A. N.; Scoring noncovalent protein–ligand interactions: a continuous differentiable function tuned to compute binding
affinities. J. Comput. Aided. Mol. Des. 1996, 10, 427–440.
390
391
22. Welch, W.; Ruppert J.; Jain, A. N.; Hammerhead: fast, fully automated docking of flexible ligands to protein binding sites.
Chem. Biol. 1996, 3, 449–462.
392
393
23. Clark, R. D.; Strizhev, A.; Leonard, J. M.; Blake, J. F.; Matthew, J. B.; Consensus scoring for ligand/protein interactions. J.
Mol. Graph. Model. 2002, 20, 281–295.
394
395
396
397
24. Case DA, Babin V, Berryman JT, Betz RM, Cai Q, Cerutti DS, Cheatham III TE, Darden TA, Duke RE, Gohlke H, Goetz
AW, Gusarov S, Homeyer N, Janowski P, Kaus J, Kolossváry I, Kovalenko A, Lee TS, LeGrand S, Luchko T, Luo R, Madej B,
Merz KM, Paesani F, Roe DR, Roitberg A, Sagui C, Salomon-Ferrer R, Seabra G, Simmerling CL, Smith W, Swails J, Walker
RC, Wang J, Wolf RM, Wu X, Kollman PA. 2014, AMBER 14. University of California, San rancisco
398
399
400
25. Duan, Y.; Wu, C.; Chowdhury, S.; Lee, M. C.; Xiong, G.; Zhang, W.; Yang, R.; Cieplak, P.; Luo, R.; Lee, T.; Caldwell, J.;
Wang, J.; Kollman, P., A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase
quantum mechanical calculations. J. Comput. Chem. 2003, 24, 1999-2012.
401
402
26. Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D. A., Development and testing of a general amber force field.
J. Comput. Chem. 2004, 26, 114.
403
404
27. Darden, T.; York, D.; Pedersen, L., Particle mesh Ewald: An N·log(N) method for Ewald sums in large systems. J. Chem.
Phys. 1993, 98, 10089-92.
405
406
28. Ryckaert, J. P.; Ciccotti, G.; Berendsen, H. J. C., Numerical integration of the Cartesian equations of motion of a system with
constraints: molecular dynamics of n-alkanes. J. Comput. Phys. 1977, 23, 327-41.
407
408
29. Sun, G.; Zhang, J.; Jin, S.; Zhang, J., Synthesis and insecticidal activities of 5-deoxyavermectin B2a oxime ester derivatives.
RSC Adv. 2018, 8 (7), 3774-3781.
409
410
30. Sun, G.-S.; Xu, X.; Jin, S.-H.; Lin, L.; Zhang, J.-J., Ovicidal and insecticidal activities of pyriproxyfen derivatives with an
oxime ester group. Molecules 2017, 22 (6), 958/1-958/11.
411
412
31. Guo, P.; Chen, Q.; Liu, T.; Xu, L.; Yang, Q.; Qian, X., Development of unsymmetrical dyads as potent
noncarbohydrate-based inhibitors against human β-N-Acetyl-D-hexosaminidase. ACS Med. Chem. Lett. 2013, 4 (6), 527-531.
413
414
32. Berchel, M.; Haelters, J.-P.; Couthon-Gourves, H.; Deschamps, L.; Midoux, P.; Lehn, P.; Jaffres, P.-A., Modular construction
of fluorescent lipophosphoramidates by Click Chemistry. Eur. J. Org. Chem. 2011, (31), 6294-6303.
415
416
33. Berchel, M.; Haelters, J.-P.; Couthon-Gourves, H.; Deschamps, L.; Midoux, P.; Lehn, P.; Jaffres, P.-A., Modular construction
of fluorescent lipophosphoramidates by Click Chemistry. Eur. J. Org. Chem. 2011, 2011 (31), 6294-6303.
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Graphic for table of contents
HO
HO
O
N
O
O
N
O
HO
O
N
O
N
N
N
N
N
N
N
N
N
N
H
N
O
O
O
HO
HO
HO
HO
HO
HO
S
S
S
6
6
6
NHAc
NHAc
NHAc
Lead compound
Compound 15r
Compound 15y
OfHex1: K = 21.8
M
M
M
OfHex1: K = 5.3
HsHexB: Ki > 100
M



OfHex1: K = 2.7
HsHexB: Ki > 100
M
M
M



i
i
i
HsHexB: K = 92.4
M

i
hOGA: K > 100
hOGA: K > 100
M


hOGA: K = 90.1
i
i
i
419
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