Design and synthesis of thiazolylhydrazone derivatives as inhibitors of chitinolytic N-acetyl-β-D-hexosaminidase

Article abstract of DOI:10.1016/j.bmc.2018.09.014

N-acetyl-β-D-hexosaminidase (Hex) is potential target for pesticide design. Here, a series of thiazolylhydrazone derivatives were designed, synthesized and evaluated as competitive inhibitors of OfHex1, a Hex from the agricultural pest Ostrinia furnacalis. The derivative 3k, with a (benzyloxy)methyl group at the N3 atom, demonstrated greater potency with a K_i of 10.2 μM. Molecular docking analysis indicated that the (benzyloxy)methyl group of 3k was bound to a previously unexplored pocket formed by Loop478-496. Then further optimization around naphthalene ring led to find the more potency substituent phenyl. The derivative 7, with phenoxyethyl group at R₁ and a phenyl group at R₂, demonstrated an augmented potency with a K_i of 2.1 μM. Molecular docking analysis indicated that 7 was bound to the active pocket of OfHex1 more favorably than 3k. This work suggests a novel scaffold for developing specific Hex inhibitors.

Full text of DOI:10.1016/j.bmc.2018.09.014

Accepted Manuscript
Design and synthesis of thiazolylhydrazone dervatives as inhibitors of chitino-
lytic N-acetyl-β-D-hexosaminidase
Huibin Yang, Tian Liu, Huitang Qi, Zhisong Huang, Zesheng Hao, Junwu Ying,
Qing Yang, Xuhong Qian
PII:
S0968-0896(18)31174-X
https://doi.org/10.1016/j.bmc.2018.09.014
BMC 14539
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
24 July 2018
12 September 2018
15 September 2018
Please cite this article as: Yang, H., Liu, T., Qi, H., Huang, Z., Hao, Z., Ying, J., Yang, Q., Qian, X., Design and
synthesis of thiazolylhydrazone dervatives as inhibitors of chitinolytic N-acetyl-β-D-hexosaminidase, Bioorganic
&
Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.09.014
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Design and synthesis of thiazolylhydrazone dervatives as inhibitors of chitinolytic
N-acetyl-β-D-hexosaminidase
Huibin Yanga,b,#, Tian Liuc,# , Huitang Qi , Zhisong Huang , Zesheng Hao , Junwu Ying , Qing
c
a
b
b
c,
a,
Yang * , Xuhong Qian *
aShanghai Key Laboratory of Chemical Biology, Institute of Pesticides and Pharmaceuticals, School of
Pharmacy, East China University of Science and Technology, Shanghai 200237, China
bState Key Laboratory of the Discovery and Development of Novel Pesticide, Shenyang Sinochem
Agrochemicals Research and Development Co., Ltd, Shenyang 110021, China
cState Key Laboratory of Fine Chemical Engineering and School of Life Science and Biotechnology,
Dalian University of Technology, Dalian 116024, China
#
These authors contributed equally to this work.
Corresponding Author:
Xuhong Qian
xhqian@ecust.edu.cn; Shanghai Key Laboratory of Chemical Biology, Institute of Pesticides and
Pharmaceuticals, School of Pharmacy, East China University of Science and Technology, Shanghai
2
00237, China.
Qing Yang
qingyang@dlut.edu.cn; School of Life Science and Biotechnology, Dalian University of Technology,
Dalian 116024, China.

ABSTRACT
N-acetyl-β-D-hexosaminidase (Hex) is potential target for pesticide design. Here, a series of
thiazolylhydrazone derivatives were designed, synthesized and evaluated as competitive inhibitors of
OfHex1, a Hex from the agricultural pest Ostrinia furnacalis. The derivative 3k, with a
(
benzyloxy)methyl group at the N3 atom, demonstrated greater potency with a K of 10.2 µM. Molecular
i
docking analysis indicated that the (benzyloxy)methyl group of 3k was bound to a previously
unexplored pocket formed by Loop478-496. Then further optimization around naphthalene ring led to
find the more potency substituent phenyl. The derivative 7, with phenoxyethyl group at R
1
and a phenyl
group at R , demonstrated an augmented potency with a K of 2.1 µM. Molecular docking analysis
2
i
indicated that 7 was bound to the active pocket of OfHex1 more favorably than 3k. This work suggests a
novel scaffold for developing specific Hex inhibitors.

1
. Introduction
Chitin, a β-1,4-linked polymer of N-acetyl-β-D-glucosamine (GlcNAc), is one of the most abundant
1
naturally occurring polysaccharides and is a major component of the insect cuticle. During its life
cycle, an insect needs to periodically replace its old cuticle with a new one via a process called
moulting.2, 3 During insect molting, chitinolytic enzymes, such as the glycoside hydrolase (GH) family
0 N-acetyl-β-D-hexosaminidase (GH20, EC 3.2.1.52) and the GH family 18 chitinase (EC 3.2.1.14)5, 6
4
2
are needed to degrade the chitin matrix of old cuticle. In our previous work, OfHex1, a N-acetyl-β-D-
hexosaminidases (Hex) from the destructive agricultural pest, Ostrinia furnacalis, has been
7
8
demonstrated to function exclusively in chitin degradation. OfHex1 and its analogues in Tribolium
9
10
11
12
castanum, Locusta migratoria, Nilaparvata lugens and Mamestra brassicae are vital for insect
survival as RNA interference of these genes results in molting defects and insect death. Furthermore,
specifically targeting OfHex1 is considered a safer biological control strategy as chitin is absent in
higher organisms such as humans and animals.13
Several classes of compounds have been discovered as inhibitors of OfHex1, including TMG-
chitotriomycin and TMG-chitobiomycin,14-19 PUGNAc,20, 21 NAG-thiazoline (NGT) and its derivative
NMAGT,23 and Q1 and Q2.24 NGT (Figure 1) was designed to mimic the oxazolinium reaction
intermediate from the GH20 N-acetyl-β-D-hexosaminidase catalytic mechanism.22, 25 In the crystal
structure of OfHex1 complexed with NGT, the thiazoline ring of NGT formed two hydrogen bonds with
Asp367 and Tyr475 and made van der Waals or π-π stacking interactions with Glu368, Trp424, Trp448,
22
2
3
Tyr475 and Trp524. This suggests the thiazoline ring has an important function in enzyme-ligand
binding. In contrast, Q2 (Figure 1) is an unsymmetrical dyad of thiadiazole and 1,8-naphthalimide.24
The crystal structure of OfHex1 complexed with Q2 revealed that the 1,8-naphthalimide group formed
van der Waals and π-π stacking interactions with Val327 and Trp490, respectively, indicating that the
inclusion of an aromatic plane is essential in the design of effective OfHex1 inhibitors.

Thiazolylhydrazone derivatives have attracted continuing interest in recent years because of their
varied biological activities including anti-parasitic activity,26 anti-fungal activity,27, 28 anti-urease
activity29 and anti-viral activity. In this study, a series of thiazolylhydrazones, containing both a
thiazoline group and a naphthalimide group, were designed and synthesized (Figure 1). These
compounds were revealed as potent inhibitors of OfHex1, and their inhibition mechanisms were
predicted using structure-based molecular docking. As the thiazolylhydrazone scaffold can be readily
synthesized and modified, this work provides a convenient and novel scaffold for developing potent
OfHex1inhibitors.
30
Figure 1. Design of the thiazolylhydrazone derivatives.
2
. Results and discussion
2
.1. Chemistry
The preparation of substituted thiazolylhydrazone derivatives is summarized in Schemes 1–2.
Intermediate 1 was synthesized by reacting 1-naphthaldehyde with thiosemicarbazide at room
temperature using acetic acid as a catalyst. Intermediate 1 was subsequently reacted with ethyl 2-
bromoacetate in methanol under reflux conditions using sodium acetate as a base to yield compound 2.
Compounds 3a-l, with diverse substituents in the thiazolidinone ring, were prepared by the reaction of
intermediate 2 with commercially available halogen agents in acetonitrile under reflux conditions using

potassium carbonate as a base. Compounds 6a–d and 7, with an alternative aromatic ring substituted in
place of the naphthalene ring, were synthesized using the corresponding aromatic aldehyde as a starting
material via a condensation reaction with thiosemicarbazide, a cyclisation reaction and a substitution
1
13
reaction. The structures of compounds 3a–l, 6a–d and 7 were fully characterized by H NMR, C NMR
and HRMS spectral analysis, and the configuration of compounds were confirmed according to the
3
1
reference reported by M. Bečka, and the X-ray crystal structure of 3d was also described.
Scheme 1. Overview of synthetic methods for 3a-l. Reagents and conditions: (i) thiosemicarbazide, EtOH, acetic acid (cat.),
3 3 2 3
rt, 15h; (ii) MeOH, CH COONa, reflux, 5h; (iii) CH CN, K CO , reflux, 5h.
Scheme 2. Overview of synthetic methods for 6a-d and 7. Reagents and conditions: (i) R
rt, 15h; (ii) BrCH COOEt, MeOH, CH COONa, reflux, 5h; (iii) (chloromethoxymethyl)benzene, CH
reflux, 5h; (iv) (2-bromoethoxy)benzene, CH CN, K CO , reflux, 5h.
2
CHO, EtOH, acetic acid (cat.),
2
3
3
CN, K CO ,
2
3
3
2
3
X-ray crystal structure analysis was performed on compound 3d (CCDC 1851329) to examine
its molecular conformation. To date, there are few reports available in the literature about the
conformation of these thiazolylhydrazone derivatives. As shown in Figure 2, the configurations of
the double bonds N(2)-C(12) and N(1)-C(11) were Z and E, respectively. The torsion angles of

N(1)-N(2)-C(12)-N(3), C(11)-N(1)-N(2)-C(12) and N(2)-N(1)-C(11)-C(10) were 179.33 (15),
79.42 (16) and -178.76 (16), respectively. A dihedral angle of 2.2° was formed between the planes
1
of the naphthalene and the thiazoline rings. The proximity of the ring planes may result from the π-
π conjugation system in the thiazolylhydrazone derivatives.
Figure 2 The crystal structure of 3d.
2
.2. Inhibitory activities and mechanisms of the thiazolylhydrazone derivatives
The inhibitory activities of each of the thiazolylhydrazone derivatives against OfHex1 are shown in
Table 1. The thiazolylhydrazone derivatives, with aromatic groups at the N3 atom of the thiazolinone
ring, did exhibit potent inhibition. Conversely, derivatives with alkyl (compound 3a, 3b), alkenyl (3c),
alkynyl (3d) or cycloalkyl (3e, 3f) groups at the N3 atom of the thiazolidinone ring exhibited poor
activity. Replacement of the cycloalkyl group with 5- or 6-membered aromatic rings, as in compounds
3
g–j, increased the inhibition rates to 30%–50% at 100 µM. Surprisingly, when the chain length
between the N atom of thiazoline and benzyl was increased by the intercalation of methyleneoxyl, as in
compound 3k, the inhibitory activity was observed to dramatically increase to 86% (at 100 µM). To
characterize the inhibition mechanism, we performed a steady-state kinetic analysis of compound 3k. As
shown in Figure 3A, the compound 3k was determined to be a competitive inhibitor of OfHex1 with a
K
i
value of 10.2 µM.
To gain insights into the mechanism of inhibition of thiazolylhydrazone derivatives against OfHex1,
the binding mode of the compound 3k in OfHex1 was examined using molecular docking. As shown in
Figure 4A, compound 3k binds the entire active pocket of OfHex1 via hydrogen bonds, π-π stacking

and van der Waals interactions. The naphthalene and thiazolinone rings bind the −1 and +1 subsites and
stack with Trp524 and Trp490, respectively. Interestingly, the (benzyloxy)methyl group, which is
important for the potency of the compound, binds a subpocket formed by Trp483, Asn489 and Trp490
on Loop478-496 and Gln527 via van der Waals interactions (Figure 4A). Loop478-496 has been
demonstrated to be important for the dimerization and the substrate specificity of OfHex1, but to date
the subpocket formed by this loop has not been investigated.
Table 1. Structures and bioactivities of compounds 3a-k against OfHex1.
Inhibitory rate at 100 μM
Compds
R
1
(
%, mean±SD)
3
a
0
3
b
28.1±3.5
21.9±0.6
8.8±0.4
3
c
3
d
3
e
1.0±0.9
9.0±0.4
3
f
3
g
39.0±2.0
34.5±3.6
3
h
3
i
41.7±3.9
53.2±2.6
85.9±0.8
3
j
3
k
Q2
95.9±2.2

Table 2. Structures and bioactivities of compounds 3k, 3l, 6a-d and 7 against OfHex1.
Inhibitory rate at 10 μM
Compds
R
1
R
2
i
K （μM）
(
%, mean±SD)
3
k
35.5±2.0
56.4±4.4
43.7±1.7
10.2
7.1
3
l
6
a
8.2
6
b
59.8±3.2
62.7±3.6
63.5±2.7
79.4±3.4
81.3±3.2
6.4
3.7
4.1
2.1
1.4
6
c
6
d
7
Q2
To optimize the possible intermolecular interactions at the −1 subsite and the subpocket of OfHex1,
compounds 3l, 6a–d and 7, with modifications on the (benzyloxy)methyl and naphthalene groups of the
compound 3k, were designed and synthesized. The inhibitory activity results are shown in Table 2.
Some of these compounds exhibited good inhibitory activities at 10 µM.
Compound 3l, with an oxygen atom adjacent to the phenyl ring, exhibited an increased inhibitor
activity compared with compound 3k, which has a methylene group inserted between the oxygen atom
and the phenyl ring.

Figure 3. Inhibitory kinetics of compounds 3k (A), 3l (B), 6c (C) and 7 (D) against OfHex1.
Further studies then focused on the naphthalene ring R
2
, with a (benzyloxy)methyl group at R , as
1
shown in Table 2. The inhibitory activities of the resultant derivatives were weakly influenced by either
the substitutes or their position on the naphthalene ring (compare 3k with 6a and 6b). However,
replacement of the naphthalene ring with benzene, as in compounds 6c and 6d, resulted in a significant
inhibitory activity increase.
Based on the structure-activity relationships, we concluded that the inhibition rate of a
thiazolylhydrazone derivative against OfHex1 is increased by replacing the (benzyloxy)methyl group
with a phenoxyethyl group and the naphthalene group with a benzene group. Compound 7, which
contains both a phenoxyethyl group at R
1
and a phenyl group at R
2
, was found to be the most potent
derivative, and exhibited an inhibition rate of 79.4% at 10 µM. As shown in Figure 3D, compound 7
was also a competitive inhibitor of OfHex1 with a K
lower than that of compound 3k.
i
value of 2.1 µM, which was more than two-fold

The binding mode of compound 7 in OfHex1 was studied by molecular docking. As shown in Figure
, compound 7 binds the entire active pocket of OfHex1 in a similar manner to compound 3k. However,
4
upon closer examination, some differences in their binding modes could be found, which may explain
why compound 7 is more potent than compound 3k. First, the N2 atom of compound 7 forms a
relatively stronger hydrogen bond with Glu328 within a distance of 2.7-Å than that of compound 3k
(3.0-Å). Second, the thiazolinone ring of compound 7 had shifted approximately 1.5 Å to the inside of
the active pocket. Finally, the phenoxyethyl group of compound 7 was inserted more deeply into the
subpocket and the O2 atom forms a 2.8-Å hydrogen bond with Trp483.
Although the compound 7 (K
i
=2.1 µM) showed a similar inhibitory activity as Q2 did (K =1.4 µM)
i
2
4
against OfHex1, the predicted binding mode of 7 were different from that of Q2 (Figure 4C). First, the
five-membered heterocycle of compound 7 was located far away from the catalytic residues (Asp367
and Glu368) and formed a π-π stacking interaction with Trp490, whereas the five-membered
heterocycle of Q2 was localized near to the catalytic residues and formed a π-π stacking interaction with
Trp524. Second, the sub-pocket which was occupied by the propoxybenzene group of 7 was not
occupied by Q2. This result suggested that the efficient inhibition could be achieved through different
binding modes.
Figure 4 Inhibition mechanisms of compounds 3k and 7 against OfHex1. (A) Binding mode of 3k in the active pocket of
OfHex1. The compound 3k was shown in green. The hydrogen bonds were shown in black dashes. (B) Binding mode of 7 in
the active pocket of OfHex1. The compound 7 was shown in magenta. (C) Superimposition of compound 7 and Q2 in the
active pocket of OfHex1.

3
. Conclusion
This work has elucidated that thiazolylhydrazone derivatives are potent inhibitors of OfHex1, a
promising molecular target for pesticide design. Compared with previously reported OfHex1 inhibitors,
the thiazolylhydrazone scaffold is much easier to synthesize and modify, and provides a new strategy to
develop novel OfHex1 inhibitors.
4
. Experiments
4
.1. Chemistry
All starting materials and reagents were commercially available and used without further purification
except where indicated. Melting points were determined on an RY-1 melting-point apparatus (TaiKe
1
13
Co.) and were uncorrected. H NMR and C NMR spectra were recorded on a Bruker-600/400/300
(Bruker Co.) spectrometer using TMS as an internal reference. Chemical shift values (δ) are given in
ppm. High-resolution mass spectra (HRMS) were recorded under electron impact (70 eV) conditions
using a MicroMass GCT CA 055 instrument. Mass spectra were obtained with a JMA-700 mass
spectrometer (JEOL, Kyoto, Japan). Analytical thin-layer chromatography (TLC) was carried out on
precoated plates (silica gel 60 F254), and spots were visualised with ultraviolet (UV) light. Yields were
not optimised.
Schemes 1–2 summarise the methods employed to synthesise the substituted thiazolylhydrazone
derivatives 3a–l, 6a–d and 7.
4
.1.1. Synthesis of intermediates 2 and 5a–d
Intermediates 2 and 5a–d was synthesized in accordance with the literature description as shown in
Scheme 1.26
A solution of 1-naphthaldehyde (10.00 g, 64.03 mmol) in ethanol (150 mL) was reacted directly with
thiosemicarbazide (5.83 g, 64.03 mmol) in the presence of catalytic amounts of acetic acid. The mixture
was stirred at room temperature for approximately 24 h until the conversion of 1-naphthaldehyde to

intermediate 1 was complete (determined by TLC). The resulting suspension was filtered to give a crude
solid, and purified by recrystallisation from ethanol to give intermediate 1 (91% yield).
Intermediate 1 (1.00 g, 4.36 mmol) was subsequently added to a solution of ethyl-bromoacetate (0.66
g, 4.36 mmol) in methanol (50 mL) and sodium acetate (0.36 g, 4.36 mmol) and stirred at room
temperature for approximately 10 h until the conversion of intermediate 1 to compound 2 was complete
(determined by TLC). The suspension was filtered to give a crude solid, which was purified by
recrystallisation from ethanol to give intermediate 2.
4
.1.1.1. (Z)-2-(((E)-naphthalen-1-ylmethylene)hydrazono) thiazolidin-4-one (2)
1
Light yellow solid; yield 83%; m.p. 226-228 °C; H NMR (600 MHz, DMSO-d
6
): δ = 12.07 (s, 1H),
9
3
1
.05 (d, J = 8.4 Hz, 1H), 8.97 (s, 1H), 8.06-8.02 (m, 2H), 7.95 (d, J = 6.6 Hz, 1H), 7.67-7.59 (m, 3H),
1
3
.96 (s, 2H); C NMR (150 MHz, DMSO-d ): δ = 174.57, 166.89, 157.51, 133.97, 131.60, 130.61,
6
30.54, 129.89, 129.17, 127.91, 126.73, 125.89, 125.54, 33.54; HRMS: m/z calculated for C14
H
12
3
N OS
+
[
M+H] 270.0701, found 270.0700.
4
.1.1.2. (Z)-2-(((E)-(4-bromonaphthalen-1-yl)methylene) hydrazono)thiazolidin-4-one (5a)
1
Light yellow solid; yield 86%; m.p. >250 °C; H NMR (600 MHz, CDCl
3
): δ = 12.12 (br s, 1H), 9.08
(d, J = 7.8 Hz, 1H), 8.99 (s, 1H), 8.27-8.25 (m, 1H), 8.01 (d, J = 7.8 Hz, 1H),, 7.88 (d, J = 7.8 Hz, 1H),
1
3
7
1
.78-7.73 (m, 2H), 3.97 (s, 2H); C NMR (150 MHz, DMSO-d ): δ = 174.54, 166.33, 158.45, 131.81,
6
31.79, 130.34, 130.27, 130.19, 128.78, 128.56, 127.58, 126.08, 125.51, 33.55; HPLC-MS: m/z
+
calculated for C14
H11BrN
3
OS [M+H] 348.0, found 348.0.
4
.1.1.3. (Z)-2-(((E)-naphthalen-2-ylmethylene)hydrazono) thiazolidin-4-one (5b)
1
Light yellow solid; yield 88%; m.p. >250 °C; H NMR (600 MHz, DMSO-d
6
) δ = 12.00 (s, 1H), 8.56
1
3
(
s, 1H), 8.19 (s, 1H), 7.99-7.94 (m, 4H), 7.60-7.56 (m, 2H), 3.92 (s, 2H); C NMR (150 MHz, DMSO-
d
1
6
): δ = 174.79, 165.46, 156.68, 134.40, 133.15, 132.31, 130.20, 128.88, 128.83, 128.83, 127.76,
+
27.23, 123.23, 33.43; HPLC-MS: m/z calculated for C14
H
12
N
3
OS [M+H] 270.1, found 270.1.
4
.1.1.4. (Z)-2-(((E)-benzylidene)hydrazono)thiazolidin-4-one (5c)

1
Light yellow solid; yield 85%; m.p. >250 °C; H NMR (600 MHz, DMSO-d
6
): δ = 11.97 (s, 1H), 8.40
s, 1H), 7.76-7.75 (m, 2H), 7.46-7.45 (m, 3H), 3.90 (s, 2H); C NMR (150 MHz, DMSO-d ): δ =
74.57, 165.75, 156.60, 134.58, 131.00, 129.20, 128.02, 33.41; HPLC-MS: m/z calculated for
1
3
(
6
1
+
C
10
H
10
N OS [M+H] 220.0, found 220.0.
3
5
.1.1.5. (Z)-2-(((E)-4-(dimethylamino)benzylidene)hydrazono) thiazolidin-4-one (5d)
1
Light yellow solid; yield 83%; m.p. 238-240 °C; H NMR (600 MHz, DMSO-d
6
): δ = 11.82 (s, 1H),
13
8
.23 (s, 1H), 7.57-7.55 (m, 2H), 6.75-6.72 (m, 2H), 3.85 (s, 2H), 2.97 (s, 6H); C NMR (150 MHz,
DMSO-d
6
): δ = 174.51, 162.56, 156.77, 152.23, 129.45, 121.82, 112.04, 40.10, 33.28; HPLC-MS: m/z
+
calculated for C12
H
15
N
4
OS [M+H] 263.1, found 263.1.
4
.1.2. General procedure for the preparation of compounds 3a–l, 6a–d and 7
Potassium carbonate (2.28 mmol) was added to a solution of intermediate 2 or 5 (2.08 mmol) and
halogenated agents (2.08 mmol) in 20 mL acetonitrile. The mixture was heated under reflux and stirred
until the completion of reaction was detected by TLC. Any undissolved material was then removed by
filtration. The filtrate was subsequently concentrated in vacuo to give a residue, which was purified by
flash column chromatography on silica gel using ethyl acetate/petroleum ether (1:1) to yield the final
products.
4
.1.2.1. (Z)-3-methyl-2-(((E)-naphthalen-1-ylmethylene) hydrazono)thiazolidin-4-one (3a)
1
White solid; yield 20%; m.p. 179-181 °C; H NMR (600 MHz, DMSO-d
6
): δ = 9.08 (d, J =8.4 Hz, 1
H), 9.07 (s, 1H), 8.07 (d, J = 7.8 Hz, 1H), 8.03 (d, J = 8.4 Hz, 1H), 7.99 (d, J = 6.6 Hz, 1H), 7.67-7.60
13
(
m, 3H), 4.01 (s, 2H), 3.22 (s, 3H); C NMR (150 MHz, DMSO-d
6
): δ = 172.68, 165.60, 158.78,
1
33.97, 131.77, 130.78, 130.65, 129.82, 129.18, 127.96, 126.75, 125.89, 125.57, 32.67, 29.83; HRMS:
+
m/z calculated for C15
H
14
N OS [M+H] 284.0858, found 284.0834.
3
4
.1.2.2. (Z)-3-ethyl-2-(((E)-naphthalen-1-ylmethylene) hydrazono)thiazolidin-4-one (3b)
1
White solid; yield 18%; m.p. 144-146 °C; H NMR (600 MHz, DMSO-d
6
): δ = 9.08 (d, J = 8.4 Hz, 1
H), 9.06 (s, 1H), 8.07 (d, J = 8.4 Hz, 1H), 8.03 (d, J = 8.4 Hz, 1H), 7.99 (d, J = 6.6 Hz, 1H), 7.67-7.60

13
(
m, 3H), 4.02 (s, 2H), 3.82 (q, J = 7.2 Hz, 2H), 3.82 (t, J = 7.2 Hz, 3H); C NMR (150 MHz, DMSO-
d
1
2
6
): δ = 172.43, 164.86, 158.76, 133.96, 131.76, 130.85, 130.62, 129.80, 129.17, 127.95, 126.74,
+
25.88, 125.60, 38.22, 32.67, 12.78; HRMS: m/z calculated for C16
98.1018.
H
16
N
3
OS [M+H] 298.1014, found
4
.1.2.3. (Z)-3-allyl-2-(((E)-naphthalen-1-ylmethylene) hydrazono)thiazolidin-4-one (3c)
1
Yellow solid; yield 35%; m.p. 135-137 °C; H NMR (600 MHz, DMSO-d
6
): δ = 9.08 (d, J = 8.4 Hz,
1
H), 9.03 (s, 1H), 8.07 (d, J = 8.4 Hz, 1H), 8.03 (d, J = 7.8 Hz, 1H), 7.98 (d, J = 7.2 Hz, 1H), 7.67-7.60
1
3
(
m, 3H), 5.95-5.88 (m, 1H), 5.23-5.19 (m, 2H), 4.39 (d, J = 6.4 Hz, 2H), 4.07 (s, 2H); C NMR (150
MHz, DMSO-d
6
): δ = 171.96, 164.24, 158.54, 133.58, 131.41, 131.23, 130.52, 130.22, 129.34, 128.78,
OS [M+H]+
1
3
27.57, 126.34, 125.48, 125.20, 117.10, 44.64, 32.19; HRMS: m/z calculated for C17
H
16
N
3
10.1014, found 310.1008.
4
.1.2.4. (Z)-2-(((E)-naphthalen-1-ylmethylene)hydrazono)-3-(prop-2-yn-1-yl) thiazolidin-4-one (3d)
1
Yellow solid; yield 46%; m.p. 161-163 °C; H NMR (600 MHz, DMSO-d
6
): δ = 9.09-9.08 (m, 2H),
8
.07 (d, J = 7.8 Hz, 1H), 8.03 (d, J = 7.8 Hz, 1H), 8.00 (d, J = 7.2 Hz, 1H), 7.68-7.60 (m, 3H), 4.53 (d,
1
3
J = 2.4 Hz, 2H), 4.10 (s, 2H), 3.30 (t, J = 2.4 Hz, 1H); C NMR (150 MHz, DMSO-d
6
): δ = 171.85,
1
7
63.78, 159.45, 133.97, 131.94, 131.07, 130.64, 129.67, 129.02, 128.02, 126.78, 125.90, 125.60, 78.24,
+
4.57, 32.53, 32.36; HRMS: m/z calculated for C17
.1.2.5. (Z)-3-(cyclopropylmethyl)-2-(((E)-naphthalen-1-ylmethylene)hydrazono)thiazolidin-4-one
3e)
Yellow solid; yield 38%; m.p. 142-144 °C; H NMR (600 MHz, DMSO-d
H), 9.06 (s, 1H), 8.07 (d, J = 8.4 Hz, 1H), 8.03 (d, J = 7.8 Hz, 1H), 7.99(d, J = 7.2 Hz, 1H), 7.67-7.60
H
14
N
3
OS [M+H] 308.0858, found 308.0855.
4
(
1
6
) δ = 9.09 (d, J =8.4 Hz, 1
(m, 3H), 4.06 (s, 2H), 3.65 (d, J = 7.2 Hz, 2H), 1.32-1.28 (m, 1H), 0.52-0.49 (m, 2H), 0.41-0.39 (m,
1
3
2
1
H); C NMR (150 MHz, DMSO-d ): δ = 172.39, 164.89, 158.41, 133.57, 131.39, 130.48, 130.23,
6
29.40, 128.78, 127.58, 126.35, 125.50, 125.22, 47.14, 32.24, 9.41, 3.58; HRMS: m/z calculated for
+
C
18
H
18
N
3
OS [M+H] 324.1171, found 324.1172.

4
.1.2.6. (Z)-3-(cyclobutylmethyl)-2-(((E)-naphthalen-1-ylmethylene)hydrazono)thiazolidin-4-one (3f)
1
Yellow solid; yield 38%; m.p. 158-160 °C; H NMR (600 MHz, DMSO-d
6
): δ = 9.09 (d, J = 8.4 Hz,
1
H), 9.05 (s, 1H), 8.07 (d, J = 8.4 Hz, 1H), 8.03 (d, J = 7.8 Hz, 1H), 7.99 (d, J = 6.6 Hz, 1H), 7.67-7.60
(m, 3H), 4.04 (s, 2H), 3.83 (d, J = 7.2 Hz, 2H), 2.83-2.79 (m, 1H), 2.01-1.98 (m, 2H),1.85-1.80 (m, 4H);
13C NMR (150 MHz, DMSO-d
): δ = 172.58, 164.94, 158.37, 133.57, 131.38, 130.54, 130.22, 129.39,
6
1
28.77, 127.55, 126.34, 125.49, 125.25, 47.52, 33.08, 32.13, 25.48, 17.72; HRMS: m/z calculated for
+
C
19
H20ClN
3
OS [M+H] 338.1327, found 338.1314.
.1.2.7. (Z)-3-((2-chlorothiazol-5-yl)methyl)-2-(((E)-naphthalen-1-ylmethylene)hydrazono)
thiazolidin-4-one (3g)
Yellow solid; yield 45%; m.p. 177-179 °C; H NMR (600 MHz, DMSO-d
J = 8.4 Hz, 1H), 8.09 (d, J = 8.4 Hz, 1H), 8.05-8.02 (m, 2H), 7.78 (s, 1H), 7.68-7.61 (m, 3H), 5.10 (s,
4
1
6
): δ = 9.14 (s, 1H), 9.09 (d,
1
3
2
1
H), 4.08 (s, 2H); C NMR (150 MHz, DMSO-d ): δ = 172.07, 164.04, 159.51, 151.51, 142.13, 134.75,
6
33.98, 132.01, 131.08, 130.65, 129.61, 129.24, 128.04, 126.80, 125.92, 125.54, 38.69, 32.85; HRMS:
+
m/z calculated for C18
H14ClN
4
OS
2
[M+H] 401.0298, found 401.0298.
4
.1.2.8. (Z)-3-((3,5-dimethylisoxazol-4-yl)methyl)-2-(((E)-naphthalen-1-ylmethylene)hydrazono)
thiazolidin-4-one (3h)
1
Yellow solid; yield 41%; m.p. 192-194 °C; H NMR (600 MHz, DMSO-d
6
): δ = 9.07 (d, J = 8.4 Hz,
1
H), 9.01 (s, 1H), 8.08 (d, J = 7.8 Hz, 1H), 8.03 (d, J = 8.4 Hz, 1H), 7.99 (d, J = 6.6 Hz, 1H), 7.68-7.60
1
3
(
m, 3H), 4.72 (s, 2H), 4.07 (s, 2H), 2.52 (s, 3H), 2.20 (s, 3H); C NMR (150 MHz, DMSO-d
6
): δ =
1
1
73.66, 168.72, 164.89, 159.84, 158.93, 133.97, 131.92, 131.18, 130.58, 129.64, 129.22, 128.03,
26.78, 125.88, 125.57, 109.61, 35.35, 32.56, 11.83, 10.69; HRMS: m/z calculated for C20
H
19
N
4
O S
2
+
[
M+H] 379.1229, found 379.1221.
4
.1.2.9. (Z)-3-(4-fluorobenzyl)-2-(((E)-naphthalen-1-yl methylene)hydrazono)thiazolidin-4-one (3i)
1
Yellow solid; yield 46%; m.p. 185-187 °C; H NMR (600 MHz, DMSO-d
6
): δ = 9.08 (d, J = 8.4 Hz,
1
H), 9.04 (s, 1H), 8.07 (d, J = 8.4 Hz, 1H), 8.03 (d, J = 8.4 Hz, 1H), 7.98 (d, J = 7.2 Hz, 1H), 7.67-7.60

1
3
(
m, 3H), 7.48-7.47 (m, 2H), 7.21-7.18 (m, 2H), 4.95 (s, 2H), 4.11 (s, 2H); C NMR (150 MHz, DMSO-
d
1
6
): δ = 172.35, 164.44, 161.57, 158.73, 133.56, 132.12, 131.46, 130.54, 130.22, 129.99, 129.93,
29.27, 128.79, 127.57, 126.36, 125.49, 125.18, 115.34, 115.19, 45.09, 32.24; HRMS: m/z calculated
+
for C21
H17FN
3
OS [M+H] 378.1076, found 378.1073.
4
.1.2.10. (Z)-3-(4-chlorobenzyl)-2-(((E)-naphthalen-1-yl methylene)hydrazono)thiazolidin-4-one (3j)
1
Yellow solid; yield 40%; m.p. 175-177 °C; H NMR (600 MHz, DMSO-d
6
): δ = 9.08 (d, J = 8.4 Hz,
1
H), 9.02 (s, 1H), 8.07 (d, J = 7.8 Hz, 1H), 8.03 (d, J = 8.4 Hz, 1H), 7.98 (d, J = 7.2 Hz, 1H), 7.67-7.60
1
3
(
m, 3H), 7.45-7.42 (m, 4H), 4.96 (s, 2H), 4.11 (s, 2H); C NMR (150 MHz, DMSO-d
6
): δ =172.36,
1
1
3
64.41, 158.77, 134.87, 133.56, 132.20, 131.48, 130.57, 130.21, 129.61, 129.26, 128.80, 128.46,
27.58, 126.36, 125.49, 125.18, 45.14, 32.27. HRMS: m/z calculated for C21
94.0781, found 394.0776.
H
17ClN
OS [M+H]+
3
4
.1.2.11. (Z)-3-((benzyloxy)methyl)-2-(((E)-naphthalen-1-ylmethylene)hydrazono) thiazolidin-4-one
3k)
Yellow solid; yield 47%; m.p. 152-154 °C; H NMR (600 MHz, DMSO-d
.08-7.99 (m, 3H), 7.68-7.60 (m, 3H), 7.37-7.35 (m, 4H), 7.31-7.29 (m, 1H), 5.26 (s, 2H), 4.68 (s, 2H),
(
1
6
): δ = 9.09-9.07 (m, 2H),
8
4
1
1
3
.06 (s, 2H); C NMR (150 MHz, DMSO-d ): δ = 173.18, 164.77, 159.26, 138.12, 133.98, 131.92,
6
31.01, 130.65, 129.71, 129.21, 128.65, 128.03, 127.96, 126.79, 125.92, 125.58, 71.77, 71.54, 32.78;
+
HRMS: m/z calculated for C22
H
20
N
3
O
2
S [M+H] 390.1276, found 390.1273.
4
.1.2.12. (Z)-2-(((E)-naphthalen-1-ylmethylene)hydrazono)-3-(2-phenoxyethyl) thiazolidin-4-one (3l)
1
Yellow solid; yield 46%; m.p. 182-184 °C; H NMR (600 MHz, DMSO-d
6
): δ = 9.07 (d, J = 8.4 Hz,
1
H), 9.03 (s, 1H), 8.07-7.98 (m, 3H), 7.67-7.60 (m, 3H), 7.31-7.28 (m, 2H), 7.97-6.94 (m, 3H), 4.30 (t, J
1
3
=
6.0 Hz, 2H), 4.16 (t, J = 6.0 Hz, 2H), 4.06 (s, 2H); C NMR (150 MHz, DMSO-d ): δ = 172.60,
6
1
1
3
64.84, 159.04, 158.41, 133.97, 131.85, 130.85, 130.64, 129.93, 129.82, 129.73, 127.98, 126.77,
25.91, 125.55, 121.29, 114.94, 63.60, 42.13, 32.64; HRMS: m/z calculated for C22
90.1276, found 390.1280.
H
20
N
OS [M+H]+
3

4
.1.2.13. (Z)-3-((benzyloxy)methyl)-2-(((E)-(4-bromo naphthalen-1-yl)methylene)hydrazono)
thiazolidin-4-one (6a)
Light yellow solid; yield 38%; m.p. 102-103 °C; H NMR (600 MHz, DMSO-d
.08 (dd, J = 1.6, 4.2 Hz, 1H), 8.60 (dd, J = 1.6, 8.4 Hz, 1H), 8.29 (d, J = 7.8 Hz, 1H), 8.13 (d, J = 8.4
Hz, 1H), 7.81 (dd, J = 4.2, 8.4 Hz, 1H), 7.36-7.36 (m, 4H), 7.31-7.27 (m, 2H), 5.26 (s, 2H), 4.67 (s, 2H),
1
6
): δ = 9.61 (s, 1H),
9
1
3
4
1
.05 (s, 2H); C NMR (100 MHz, CDCl ): δ = 183.06, 167.63, 153.39, 140.94, 132.68, 131.85, 127.49,
3
27.06, 124.99, 123.81, 123.72, 123.68, 123.49, 123.29, 123.06, 122.39, 119.49, 71.41, 67.17, 34.60;
+
HRMS: m/z calculated for C22
H19BrN
3
O
2
S [M+H] 468.0381, found 468.0391.
4
.1.2.14. (Z)-3-((benzyloxy)methyl)-2-(((E)-naphthalen-2-ylmethylene)hydrazono)thiazolidin-4-one
(6b)
1
Light yellow solid; yield 37%; m.p. 174-175 °C; H NMR (600 MHz, DMSO-d
6
): δ = 8.62 (s, 1H),
8
4
1
6
.21 (s, 1H), 7.98-7.94 (m, 4H), 7.58-7.57 (m, 2H), 7.38-7.34 (m, 4H), 7.30-7.29 (m, 1H), 4.94 (s, 2H),
1
3
.10 (s, 2H), 3.33 (s, 2H); C NMR (100 MHz, CDCl ): δ = 167.69, 163.03, 158.47, 132.74, 130.01,
3
28.39, 127.71, 127.17, 126.16, 125.58, 124.07,123.88, 123.83, 123.68, 122.62, 121.88, 118.79, 67.69,
+
6.91, 33.99; HRMS: m/z calculated for C22
H
20
N O
3 2
S [M+H] 390.1276, found 390.1279.
4
.1.2.15. (Z)-2-(((E)-benzylidene)hydrazono)-3-((benzyloxy) methyl)thiazolidin-4-one (6c)
1
Light yellow solid; yield 37%; m.p. 131-132 °C; H NMR (600 MHz, CDCl
3
): δ = 8.47 (s, 1H), 7.79-
7
.77 (m, 2H), 7.43-7.41 (m, 3H), 7.37-7.32 (m, 4H), 7.29-7.27 (m, 1H), 5.34 (s, 2H), 4.72 (s, 2H), 3.70
1
3
(
s, 2H); C NMR (100 MHz, CDCl
3
): δ = 171.89, 162.62, 158.50, 136.99, 133.68, 130.41, 128.22,
NaO S
1
27.88, 127.72, 127.37, 127.25, 71.93, 71.16, 31.99; HRMS: m/z calculated for C18
H
17
N
3
2
+
[
M+Na] 362.0939, found 362.0938.
.1.2.16. (Z)-3-((benzyloxy)methyl)-2-(((E)-4-(dimethylamino) benzylidene)hydrazono)thiazolidin-4-
one (6d)
Yellow solid; yield 42%; m.p. 152-153 °C; H NMR (600 MHz, CDCl
.0 Hz, 2H), 7.35-7.33 (m, 5H), 6.76 (d, J = 9.0 Hz, 2H), 5.19 (s, 2H), 4.63 (s, 2H), 3.97 (s, 2H), 2.98 (s,
4
1
3
): δ = 8.33 (s, 1H), 7.60 (d, J =
9

1
3
6
1
H); C NMR (100 MHz, CDCl ): δ = 169.48, 163.03, 154.77, 153.41, 147.56, 142.71, 132.10, 124.73,
3
23.73, 123.28, 116.09, 106.98, 71.39, 66.88, 35.52, 34.42; HRMS: m/z calculated for C20
H
23
N
4
O S
2
+
[
M+H] 383.1542, found 383.1545.
4
.1.2.17. (Z)-2-(((E)-benzylidene)hydrazono)-3-(2-phenoxyethyl)thiazolidin-4-one (7)
1
Yellow solid; yield 38%; m.p. 100-102 °C; H NMR (600 MHz, CDCl
3
): δ = 8.40 (s, 1H), 7.78-7.77
(m, 2H), 7.42-7.41 (m, 3H), 7.28-7.24 (m, 2H), 6.96-6.91 (m, 3H), 4.29 (t, J = 5.4 Hz, 2H), 4.25 (t, J =
1
3
5
1
.4 Hz, 2H), 3.77 (s, 2H); C NMR (150 MHz, CDCl ): δ = 171.90, 163.35, 156.56, 158.28, 134.13,
3
30.82, 129.42, 128.67, 128.11, 121.06, 114.60, 63.42, 42.25, 32.36; HRMS: m/z calculated for
+
C
18
H
18
N
3
O
2
S [M+H] 340.1120, found 340.1115.
4
.2. X-ray structure determination and refinement
Single X-ray quality crystals of compound 3d were obtained by slow evaporation from a
dichloromethane solution under ambient conditions. The X-ray crystallographic data were collected at
2
0
96 K on a Bruker SMART Apex II CCD diffractometer using graphite-monochromated Mo-Kα (λ =
.71073 Å) radiation. Data integration and reduction were performed using the SAINT program, and
empirical absorption corrections were performed by the multi-scan method implemented in the
SADABS program. The phase problem was solved using SHELXS-97 (Agilent Technologies, Inc.,
3
2
Santa Clara, CA), and the model was refined by full-matrix least-squares on F2 using SHELXL-97.
All non-hydrogen atoms were refined anisotropically.
Crystallographic data for the structures in this paper have been deposited with the Cambridge
Crystallographic Data Center as supplementary publication nos. CCDC 1851329 (compound 3d).
Figure 2 shows the asymmetric units of compound 3d obtained using SHELXL-97.
4
.3. Inhibitory activity assay
OfHex1 was expressed in Pichia pastoris GS115 and purified by metal-chelating chromatography as
3
3
described previously. The enzymatic activity of OfHex1 was measured using 4-methylumbelliferyl-β-
N-acetyl-D-glucosamine (MU-β-GlcNAc, kindly provided by Prof. Jianjun Zhang at China Agricultural

University) as a substrate. The reaction mixtures consisted of 100 µl of 0.4 nM enzyme, 50 µM MU-β-
GlcNAc, 100 µM or 10 µM inhibitors and 2% DMSO in 20 mM sodium phosphate buffer (pH 6.5).
Reaction mixtures without inhibitors were used as positive controls. After incubating at 30 °C for
specific periods, 0.5 M sodium carbonate was added to the reaction mixture, and the fluorescence
produced was quantified using a Varioskan Flash microplate reader (Thermo Fisher Scientific) using
excitation and emission wavelengths of 360 and 450 nm, respectively. Experiments were performed in
triplicate. Three substrate concentrations (12.5, 25 and 50 µM) were used to determine K
i
values. The K
i
values and types of inhibition were determined by linear fitting of data in Dixon plots.
4
.4. Molecular docking
3
4
The program database (PDB) files of compounds 3k and 7 were generated by PRODRG. The PDB
file of the ligand-free OfHex1 was prepared by PyMOL (DeLano Scientific LLC, San Carlos, CA, USA)
from the TMG-chitotriomycin-complexed structure of OfHex1 (PDB code: 3NSN). The PDBQT files of
3
4
the proteins and compounds were prepared by MGLTools. Affinity grids of 50 × 50 × 50 Å were
calculated using AutoGrid4.35
3
4
Molecular dockings were performed by AutoDock4 using the Lamarckian genetic algorithm with a
population size of 100 individuals, 25 000 000 energy evaluations and 27 000 generations. Plausible
docking models were selected from the abundant clusters [root-mean-square deviation (RMSD) = 2 Å]
with the lowest binding energies. All structural figures were prepared using PyMOL.
Acknowledgments
The authors acknowledge financial support from the National Key R&D Program of China
(2016YFD0300708, 2017YFD0200500, 2017YFD0201400, 2018YFD0200100) and the National
Natural Science Foundation of China (31871959). We thank Dr. Bin Li, Haibo Yu and Pengfei Liu for
assistance with the manuscript preparation.

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Graphical Abstract