Synthesis of ureido thioglycosides as novel insect β?N?acetylhexosaminidase OfHex1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2020.115602

The insect β-N-acetylhexosaminidase OfHex1 from Ostrinia furnacalis (one of the most destructive agricultural pests) has been considered as a promising pesticide target. In this study, a series of novel and readily available ureido thioglycosides were designed and synthesized based on the catalytic mechanism and the co-crystal structures of OfHex1 with substrates. After evaluation via enzyme inhibition experiments, thioglycosides 11c and 15k were found to have inhibitory activities against OfHex1 with the K_i values of 25.6 μM and 53.8 μM, respectively. In addition, all these ureido thioglycosides exhibited high selectivity toward OfHex1 over hOGA and HsHexB (K_i > 100 μM). Furthermore, to investigate the inhibitory mechanism, the possible binding modes of 11c and 15k with OfHex1 were deduced based on molecular docking analysis. This work may provide useful structural starting points for further rational design of potent inhibitors of OfHex1.

Full text of DOI:10.1016/j.bmc.2020.115602

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Synthesis of ureido thioglycosides as novel insect β‑N‑acetylhexosaminidase
OfHex1 inhibitors
Shengqiang Shen, Lili Dong, Huizhe Lu, Yanhong Dong, Qing Yang, Jianjun
Zhang
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https://doi.org/10.1016/j.bmc.2020.115602
BMC 115602
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7 June 2020
9 June 2020
Please cite this article as: Shen, S., Dong, L., Lu, H., Dong, Y., Yang, Q., Zhang, J., Synthesis of ureido
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(2020), doi: https://doi.org/10.1016/j.bmc.2020.115602
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1. Synthesis of ureido thioglycosides as novel insect β‑N‑acetylhexosaminidase OfHex1 inhibitors
Shengqiang Shen,† Lili Dong,† Huizhe Lu,† Yanhong Dong,† Qing Yang,*,‡ and Jianjun Zhang*,†
†Department of Pesticide Chemistry, College of Science, China Agricultural University, Beijing,
China
‡ State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection
and Shenzhen Agricultural Genome Research Institute, Chinese Academy of Agricultural Sciences,
Beijing, China.
Corresponding Author
Jianjun Zhang; 86-10-62732219; E-mail: zhangjianjun@cau.edu.cn.
Qing Yang; 86-411-84707245; E-mail: qingyang@dlut.edu.cn.
Abstract: The insect β-N-acetylhexosaminidase OfHex1 from Ostrinia furnacalis (one of the most
destructive agricultural pests) has been considered as a promising pesticide target. In this study, a
series of novel and readily available ureido thioglycosides were designed and synthesized based on
the catalytic mechanism and the co-crystal structures of OfHex1 with substrates. After evaluation
via enzyme inhibition experiments, thioglycosides 11c and 15k were found to have inhibitory
activities against OfHex1 with the Ki values of 25.6 µM and 53.8 µM, respectively. In addition, all
these ureido thioglycosides exhibited high selectivity toward OfHex1 over hOGA and HsHexB (Ki >
100 μM). Furthermore, to investigate the inhibitory mechanism, the possible binding modes of 11c
and 15k with OfHex1 were deduced based on molecular docking analysis. This work may provide
useful structural starting points for further rational design of potent inhibitors of OfHex1.
Keywords: Ureido thioglycosides, trichloroethoxycarbonyl (Troc), β-N-acetylhexosaminidase,
OfHex1, inhibitors
1. Introduction
Chitin, the second most abundant naturally occurring polysaccharides, is the key component of
the insect exoskeleton, nematode eggshell and fungal cell wall.1 Importantly, chitin is absent from
higher mammals and plants.2 Thus, the enzymes in chitin metabolism (biosynthesis and
biodegradation) are recognized as promising targets for developing green pesticides.3-5
The insect β-N-acetylhexosaminidase OfHex1 is the important component enzyme in chitin
1

degradation of insect.6 OfHex1 from the agricultural pest, Asian corn borer (Ostrinia furnacalis),
can efficiently hydrolyze β-1,4-linked chitooligosaccharides into N-acetyl-D-glucosamine
(GlcNAc).6 Interference with this physiological process can disrupt molting and metamorphosis of
Ostrinia furnacalis, eventually resulting in insect death. 6-7 Furthermore, the crystal structure of
OfHex1 (the only insect-derived β-N-acetylhexosaminidase, PDB ID: 3NSM) has been reported.8
Therefore, the development of eco-friendly pesticides with OfHex1 as the target has a good
foundation and important research significance.8-10
To date, a number of small molecule inhibitors targeting OfHex1 have been reported, including
PUGNAc, TMG-chitotriomycin8, 11, naphthalimides10, 12-14, NGT15, phlegmacin B116, berberine17,
pyrimethamine18 and thiazolylhydrazone derivatives19. Amongst these compounds, PUGNAc is a
classic and broad-spectrum β-N-acetylhexosaminidases inhibitor, with a Ki value of 0.24 μM against
OfHex19. The crystal structure of OfHex1-PUGNAc (PDB: 3OZP) showed that the sugar moiety of
PUGNAc could tightly bound to the -1 subsite of OfHex1 and the hydrophobic phenyl was
sandwiched by Val327 and Trp490 at the +1 subsite. 9 The structure-activity relationship studies
also revealed that the inhibitory potency of PUGNAc derives from the glycosyl moiety (GlcNAc),
sp2-hybridized carbon at the C-1 position, and N-phenylcarbamate group. 20 These results indicated
that glycosyl moiety and aromatic fragment were crucial in the design of potent OfHex1 inhibitors.
In addition, TMG-chitotriomycin is the most potent OfHex1 inhibitor and can only inhibit β-N-
8
acetylhexosaminidases from chitin-containing organisms. The high inhibitory potency of TMG-
chitotriomycin mainly comes from the positively charged N,N,N-triMe group, which can interacts
with catalytic Asp367 and Glu368 at the -1 subsite of OfHex1.8 This suggested that the 2-substituent
at the glycosyl moiety could exert a critical effect on the potency against OfHex1.
Prompted by these observations, we selected glycosyl moiety and hydrophobic groups
(naphthalimides or benzoyl) as the frame structures. Then, we introduced alkyl chains to connect
these two moieties and converted the glycosidic bond to thioglycosidic bond (to prevent the
compounds from being degraded by OfHex1). Considering the crucial roles of 2-substituent at the
glycosyl moiety, we further modified the acetamido group of GlcNAc to ureido group (with more
hydrogen bonding donors and hydrogen bond acceptors) at the 2-position, hoping to improve the
binding affinity toward OfHex1. Accordingly, several classes of ureido thioglycosides were
synthesized and their inhibitory activities against OfHex1, human β-N-acetylhexosaminidase B
(HsHexB), and human O-GlcNAcase (hOGA) were evaluated (Figure 1).
2

HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
O
H
O
O
O
O
O
N
O
O
O
N
OH
HO
HO
O
N
NHAc
NHAc
NHAc
NHAc
PUGNAc
TMG-chitotriomycin
design
OH
O
HO
HO
Hydrophobic
group
S
linker
linker=
n
NH
O
NH2
O
O
HN
O
O
N
H
R1
N
H
N
O
R2
N
Hydrophobic group =
N
,
,
,
,
H
et al.
Figure 1. Design of novel ureido thioglycoside derivatives for OfHex1
2. Results and discussion
2.1. Synthesis of ureido thioglycosides 7a-7d
Inspired by our previous studies12-13, we first selected naphthalimide moiety as the hydrophobic
group of the target compound. Then, we focused on studying the influences of the length of linker
on the inhibitory potency against OfHex1. The target compounds 7a-7d are synthesized and outlined
in Scheme 1. Briefly, compound 3 were obtained according to literature methods21 and reacted with
bromides 5a-5d22 to afford trichloroethyl carbamates 6a-6d. Then refer to our recently reported
synthetic method of ureido glycosides,21 precursors 6a-6d were reacted with ammonia (in MeOH,
7M) at room temperature for 60 h to yield target compounds 7a-7d in one step (Scheme S1).
HO
AcO
AcO
AcO
AcO
AcO
AcO
O
O
O
v,vi,vii
i, ii, iii, iv
OH
HO
HO
OAc
SH
NH2HCl
NHTroc
NHTroc
3
1
2
O
O
N
viii
NH
O
Br
n
ix
O
4
5a-5d
HO
O
AcO
AcO
AcO
O
N
O
O
O
HO
HO
x
N
O
S
S
n
n
NH
O
NHTroc
6a-6d
7a-7d
NH2
7a: n=3; 7b: n=4; 7c: n=5; 7d: n=6
Scheme 1. Synthesis of ureido thioglycosides 7a-7d. (i) p-anisaladehyde, NaOH, H2O; (ii) Py, Ac2O; (iii) acetone,
HCl, H2O; (iv) Et3N, CH2Cl2, TrocCl; (v) HBr, CH3COOH, CH2Cl2; (vi) thiourea, acetone; (vii) Na2S2O5, CH2Cl2,
H2O; (viii) K2CO3, CH3CN; (ix) K2CO3, acetone, H2O; (viii) NH3, MeOH
3

2.2. Inhibitory potency of ureido thioglycosides 7a-7d
The target compounds 7a-7d were evaluated for their inhibitory potency against OfHex1,
hOGA, and HsHexB at the concentration of 100 μM. As shown in Table 1, upon extending the
linker (carbon atoms from three to six), a gradual increase in the inhibitory activity against OfHex1
was observed. Compound 7d bearing six carbon atoms (n = 6, Scheme 1) showed the higher potency
with an inhibition rate of 59.5% at a concentration of 100 μM. In addition, the ureido group at 2-
positon of the glycosyl moiety (7d) could improve the inhibitory activity against OfHex1 compared
to that of glycosyl moiety bearing acetyl group (21e 22, unpublished data). These finding also
revealed that the 2-ureido group (of these target compounds) was helpful for increasing the potency
against OfHex1. Furthermore, 7a-7d showed lower potency against hOGA and HsHexB, suggesting
that these compounds had suitable selectivity toward OfHex1.
Table 1. Inhibition rate of compounds 7a-7d and 21e22 against OfHex1, hOGA, and HsHexB.
HO
O
N
O
O
HO
HO
S
n
NHR
Inhibition rate at 100 μM (%)
Compd
n
R
OfHex1
3.8 ± 1.0
hOGA
20.5 ± 1.3
8.8 ± 1.8
13.7 ± 2.9
7.1 ± 0.9
25.9 ± 1.8
HsHexB
34.7 ± 0.5
11.9 ± 1.9
38.9 ± 3.1
5.1 ± 2.2
7a
7b
3
4
5
6
6
CONH2
CONH2
CONH2
CONH2
COCH3
16.8 ± 2.6
43.5 ± 0.9
59.5 ± 2.1
31.9 ± 2.9
7c
7d
21ea
27.6 ± 1.3
a Structure of compound 21e were taken from ref 22.
2.3. Modification of ureido thioglycoside 7d
On the basis of structure-activity relationships from the first stage, thioglycoside 7d was selected
for structural modification. Firstly, we retained the frame structure of ureido thioglycoside and fixed
the linker with six carbon atoms (n = 6, Scheme 1). Then, we focused on structural derivatization
for naphthalimide group, including introduction of 4-substituted group on the naphthalimide or
replacement of naphthalimide with other hydrophobic groups (benzene ring, heterocyclic ring, and
alkyl group). Accordingly, several classes of ureido thioglycosides were synthesized (Scheme 2-4).
4

Firstly, we introduced nitrogen-containing cycloalkane groups (based on our previous work12) to
the 4-position of naphthalimide and synthesized ureido thioglycosides 11a-11c (Scheme 2). Briefly,
substituted naphthalic anhydrides 8a-8c were selected as the starting material and reacted with 6-
bromohexan-1-amine to afford bromide 9a-9c, and then stirred with thiol 3 in acetone/water (2:1,
v: v) to obtain acetyl-protected precursors 10a-10c. Finally, deprotected via methanol-ammonia
catalysis resulted in the target compounds 11a–11c (Scheme 2 and S2). The analysis of compounds
11a–11c against OfHex1 (Table 2) showed that the 4-substituent at the naphthalimide group could
significantly affect the inhibitory potency of these compounds. Specifically, inhibitors bearing 4-
pyrrolyl (11a) and 4-azepanyl (11c) groups increased the inhibitory activity against OfHex1
compared to lead compound 7d. However, the addition of 4-morpholino (11b) at the naphthalimide
group decreased the potency, which suggested that the oxygen atom at nitrogen-containing
cycloalkane group may be detrimental to the binding affinity with OfHex1. Further IC50
determination showed that 11c exhibited relatively good activity (OfHex1, IC50 = 28.1 μM) and
selectivity (hOGA and HsHexB, IC50 >100 μM) against OfHex1 (Table 3).
O
N
AcO
AcO
AcO
O
O
O
O
N
O
R1
R1
O
R1
i
ii
Br
6
S
6
NHTroc
O
9a-9c
10a-10c
8a-8c
HO
O
N
O
O
R1
iii
HO
HO
S
6
O
N
N
N
R1
=
NH
O
11a
11b
11c
11a-11c
NH2
Scheme 2. Synthesis of ureido thioglycosides 11a-11c. (i) 6-bromohexan-1-amine, EtOH; (ii) 3, K2CO3, acetone,
H2O; (iii) NH3, MeOH
We then synthesized a series of substituted phenyl group-bearing ureido thioglycosides 15a-
15k. As shown in Scheme 3, benzoic acid compounds 12a-12k were reacted with 6-bromohexan-
1-amine to acquire intermediates 13a-13k. Subsequently, the coupling of 13a-13k with thiol 3 in
acetone and H2O resulted in acetyl-protected precursors 14a-14k, and then stirred with ammonia
(in MeOH, 7M) at room temperature to obtain target compounds 15a-15k (Scheme S3). The
bioassay results of compounds 15a-15k against OfHex1, hOGA, and HsHexB were shown in
Tables 2-3. Most of the compounds in 15a-15k exhibited a relatively weak activity toward these
three enzymes. In detail, compounds 15a-15j displayed < 16 % inhibition rate against OfHex1, and
only one compound (15k) exhibited the higher activity with an inhibition rate of 63.7 %. These
results suggested that the size and the position of the substituent on benzene ring may be the main
5

factor to affect the activity toward OfHex1 rather than the electronic properties (electron-
withdrawing and electron-donating). The presence of a larger phenyl group on benzene ring (15i-
15k) led to the increased potency. Moreover, the activity order of the substituent on benzene ring
was para > meta > ortho (15k >15i >15j, 15d >15b >15c). Further IC50 assay results showed that
15k possessed the moderate activity toward OfHex1 with the value of 55.7 μM (Table 3).
AcO
AcO
AcO
O
O
O
COOH
Br
S
ii
i
N
N
R2
H
R2
R2
H
NHTroc
12a-12k
13a-13k
14a-14k
HO
HO
HO
15a
15d
15g
15b
15c
: R2=3-Br;
: R2=H;
: R2=2-Br;
O
O
iii
15e
15f
:R2=4-Br;
:R2=4-NO2;
: R2=3-Ph;
:R2=4-Cl;
: R2=4-OMe;
: R2=2-Ph;
S
N
15h
15k
15i
:R2=3-CF3;
: R2=4-Ph
R2
H
NH
O
15j
NH2
15a-15k
Scheme 3. Synthesis of ureido thioglycosides 15a-15k. (i) 6-bromohexan-1-amine, EDCI, DMAP, Et3N, DCM; (ii)
3, K2CO3, acetone, H2O; (iii) NH3, MeOH
To further improve the structure-activity relationship of these ureido thioglycosides, we replaced
the substituted phenyl group in 15a–15k with the naphthyl, heterocyclyl, phenethyl, and alkyl group,
respectively. Thus, compounds 19a-19h were synthesized. The synthetic route of compounds 19a-
19h were identical to compounds 15a–15k and outlined in Scheme 4 and Scheme S4. Analysis of
compounds 19a-19h against OfHex1 (Table 2) showed that the naphthyl (19a), quinolinyl (19b),
furyl (19c), thienyl (19d), phenethyl (19e), and alkyl (19g, 19h) groups could not enhance the
activity. A special case was ureido thioglycoside bearing an indolyl group (19f), which exhibited
the highest inhibitory potency against OfHex1 among compounds 19a-19h, with the IC50 value of
72.3 μM (Table 3).
AcO
O
O
O
AcO
AcO
S
ii
Br
O
i
R3
N
R3COOH
R3
N
H
NHTroc
H
18a-18h
16a-16h
17a-17h
N
R3
R3
=
=
S
19d
O
HO
HO
HO
19a
19e
19b
19c
H3C
19g
O
iii
S
R3
N
H
NH
O
N
H
NH2
19a-19h
19f
19h
Scheme 4. Synthesis of ureido thioglycosides 19a-19h. (i) 6-bromohexan-1-amine, EDCI, DMAP, Et3N, DCM; (ii)
3, K2CO3, acetone, H2O; (iii) NH3, MeOH
6

Table 2. Inhibition rate of compounds 11a-11c, 15a-15k, and 19a-19h against OfHex1, hOGA, and
HsHexB.
Inhibition rate at 100 μM (%)
Compd
OfHex1
63.7 ± 2.5
19.1 ± 0.4
94.0 ± 0.6
4.2 ± 1.3
3.3 ± 1.9
1.0 ± 1.6
8.9 ± 2.1
7.3 ± 2.0
0.1 ± 0.5
6.3 ± 3.0
1.1 ± 0.2
15.3 ± 0.3
13.4 ± 1.7
75.3 ± 2.5
0.7 ± 1.8
1.5 ± 0.6
7.3 ± 2.6
3.2 ± 1.4
3.3 ± 0.9
76.8 ± 1.3
1.3 ± 2.7
11.5 ± 1.4
hOGA
24.6 ± 1.6
12.0 ± 1.9
43.1 ± 2.0
0.1 ± 0.1
2.3 ± 0.8
1.6 ± 2.3
13.1 ± 1.0
6.7 ± 1.4
4.0 ± 2.7
16.8 ± 0.5
48.2 ± 0.2
10.6 ± 1.7
0.2 ± 0.5
2.7 ± 2.4
16.8 ± 0.3
1.1 ± 0.6
2.8 ± 1.6
1.3 ± 1.9
6.7 ± 2.7
0.8 ± 1.1
3.5 ± 0.6
5.7 ± 1.2
HsHexB
1.3 ± 0.8
1.9 ± 1.0
1.8 ± 0.5
3.1 ± 1.6
0.4 ± 0.1
0.8 ± 0.2
0.6 ± 0.1
3.3 ± 0.9
11.6 ± 0.4
6.8 ± 1.6
12.9 ± 0.8
0.6 ± 1.7
0.3 ± 1.9
6.1 ± 2.0
1.9 ± 1.8
5.0 ± 0.7
11.8 ± 1.2
9.6 ± 0.7
4.1 ± 1.2
12.8 ± 1.3
8.7 ± 3.0
0.4 ± 0.2
11a
11b
11c
15a
15b
15c
15d
15e
15f
15g
15h
15i
15j
15k
19a
19b
19c
19d
19e
19f
19g
19h
7

Table 3. IC50 values of representative compounds for OfHex1, hOGA, and HsHexB
Inhibition rate at 20 μM (%)
IC50 values (μM)
OfHex1 hOGA HsHexB
Compd
OfHex1
hOGA
6.1 ± 2.0
12.5 ± 1.7
1.8 ± 0.6
0.2 ± 0.2
HsHexB
11a
11c
15k
19f
20.4 ± 1.5
54.2 ± 2.8
32.6 ± 0.3
20.8 ± 1.9
0.5 ± 0.3
0.1 ± 0.2
1.9 ± 1.1
3.8 ± 2.4
68.5 ± 3.1
28.1 ± 1.6
55.7 ± 2.2
72.3 ± 2.9
>100
>100
>100
>100
>100
>100
>100
>100
2.4. Inhibitory mechanism of ureido thioglycosides 11c and 15k toward OfHex1.
Two representative inhibitors, namely 11c and 15k, were selected to investigate the inhibitory
mechanism toward OfHex1. Firstly, the Dixon plots of inhibitors 11c and 15k against OfHex1 were
carried out. As shown in Figure 2, the trendlines drawn for each concentration all intersected in
quadrant two, suggesting that compounds 11c and 15k are competitive inhibitors of OfHex1.
Moreover, the Ki values of 11c and 15k were determined as 25.6 ± 0.5 μM and 53.8 ± 0.3 μM,
respectively.
Figure 2. Dixon plots for inhibitors 11c and 15k against OfHex1
To investigate the possible binding modes of compounds 11c and 15k with OfHex1, the
molecular docking studies were carried out. As shown in Figure 3, the sugar moiety of these two
inhibitors was found to be tightly bound to the -1 subsite of OfHex1 and the hydrophobic groups
extended out from the pocket. In detail, the hydroxyl groups (at glycosyl moiety) from 11c could
bind with residues Arg220, Asp367, Glu526 via H-bonding interactions. These interactions are
coherent with those found in the complex structure of PUGNAc-OfHex19. It is worth mentioning
8

that the ureido group of 11c formed three hydrogen bonds with Glu328, Asp477 and Trp483. As a
comparison, the 2-acetyl on glycosyl moiety of inhibitor PUGNAc formed two hydrogen bonds
with Asp367 and Tyr475. 9 These results suggested that 2-ureido group on glycosyl moiety might
increase the binding affinity toward OfHex1. Moreover, the 4-azepanylnaphthalimide moiety of 11c
could form π-π stacking interactions with Trp448 and van der Waals interactions with Ser426,
Thr427, Asn430 (Figure 3a). The docking mode of compound 15k complex with OfHex1 (Figure
3b) showed that the glycosyl moiety was found to interact with Arg220, Asp368, Tyr475, Asp477,
Trp490, Glu526 via hydrogen bonds. The linker of 15k bound to the +1 subsite of OfHex1, and the
NH (in the linker) could form a hydrogen bond with residue Val327. In addition, the biphenyl group
was located at the loop314-355 region (near the entrance of the OfHex1 pocket) and interacted with
Lys323, Ser324, and Cys326. The binding modes of these two systems demonstrate the importance
of larger hydrophobic groups in the molecular design of OfHex1 inhibitors, which can increase the
hydrophobic interactions of compounds with the +1 subsite and loop regions of OfHex1.
(a)
(b)
、
Figure 3. Predicted binding mode of compounds 11c (a) and 15k (b) with OfHex1
3. Conclusions
In summary, we present the design, synthesis, and inhibitory potencies of various ureido
thioglycosides against β-N-acetylhexosaminidases (OfHex1, hOGA, and HsHexB). Importantly,
compounds 11c (Ki = 25.6 µM) and 15k (Ki = 53.8 µM) showed the higher efficiency and selectivity
against OfHex1. Moreover, molecular docking was carried out to allowed us to rationalize the
potency of these ureido thioglycosides toward OfHex1. The structure–activity relationship as well
as the molecular docking studies reveal that the 2-ureido group on glycosyl moiety and the larger
hydrophobic groups (such as substituted naphthalimide or biphenyl group) are important for
increasing the binding affinity of ureido thioglycosides for OfHex1. Taken in concert, the novel
ureido thioglycosides reported herein may provide useful information for the further design and
9

development of OfHex1-related green pesticides.
4. Experimental
4.1 General methods
All chemicals, reagents and solvents were commercial sources and used without further
1
purification. H NMR (300 MHz) and 13C NMR (75 MHz) spectra were recorded on a Bruker
AVANCE600 spectrometer in CDCl3 or DMSO-d6 at 25°C, and TMS was used as the internal
standard. High-resolution mass spectra (HRMS) was obtained on a Bruker Daltonics Bio-TOF-Q
III mass spectrometer (Bruker Co., Karlsruhe, Germany). Thin layer chromatography (TLC) was
performed on silica gel GF254 plates with detection by ultraviolet (UV) light (254 nm) or by
charring with 20% (v/v).
4.2 Chemical synthesis
4.2.1 Synthesis of intermediates
Detailed synthetic procedures and characterization data for all of the synthesized intermediates
(3, 5a-5d, 6a-6d, 9a-9c, 10a-10c, 13a-13k, 14a-14k, 17a-17h, 18a-18h) are given in the
Supporting Information (Schemes S1-S4).
4.2.2 Synthesis of target compounds 7a-7d, 11a-11c, 15a-15k, and 19a-19h
A solution of acetyl-protected precursors 6a-6d, 10a-10c, 14a-14k, and 18a-18h (0.5 mmol)
was suspended in anhydrous MeOH (10 mL), and solution of NH3 in MeOH (7M, 10 mL) was then
added. The reaction was stirred for 60 h at room temperature, until TLC (EtOAc/MeOH/H2O, 8:1:1
v/v/v) indicated that the reaction was complete. The mixture was concentrated in vacuo and
recrystallized from MeOH to obtain compounds 7a-7d, 11a-11c, 15a-15k, and 19a-19h.
2-[3-[(2-Ureido-β-D-glucopyranosyl) thio] propyl]-1H-benzo[de]isoquinoline-1,3(2H)-dione
(7a): white solid; (0.19 g, 79%) yield; [α]D25 -28.7(c=0.1, DMSO); 1H NMR (300 MHz, DMSO-d6)
δ 8.44 (dd, J = 9.9, 7.7 Hz, 4H, ArH), 7.92 – 7.78 (m, 2H, ArH), 5.92 (d, J = 8.7 Hz, 1H, NH), 5.46
(s, 2H, NH2), 5.05 – 4.93 (m, 2H, 2 OH), 4.52 – 4.38 (m, 2H, OH, H-1), 4.10 (t, J = 7.2 Hz, 2H,
H-3, H-4), 3.65 (dd, J = 11.5, 6.0 Hz, 1H, H-6b), 3.53 – 3.42 (m, 1H, H-6a), 3.36 – 3.19 (m, 2H,
H-2, H-5), 3.16 – 3.07 (m, 2H, NCH2), 2.72 (t, J = 7.4 Hz, 2H, SCH2), 2.02 – 1.82 (m, 2H, CH2);
13C NMR (75 MHz, DMSO-d6) δ 163.54, 158.78, 134.37, 131.36, 130.79, 127.45, 127.27, 122.14,
84.82, 81.07, 76.61, 70.89, 61.32, 55.53, 39.40, 28.01, 27.07; HRMS (ESI) calcd for C22H26N3O7S
(M+H+) 476.1491, found 476.1498.
2-[6-[(2-Ureido-β-D-glucopyranosyl)thio]hexyl]-6-(pyrrolidin-1-yl)-1H-benzo[de]isoquinoline-
1,3(2H)-dione (11a): yellow solid; (0.22 g, 76%) yield; [α]D25 -19.4 (c=0.1, DMSO); 1H NMR (300
MHz, DMSO-d6) δ 8.61 (d, J = 8.4 Hz, 1H, ArH), 8.34 (d, J = 7.4 Hz, 1H, ArH), 8.12 (d, J = 8.7
10

Hz, 1H, ArH), 7.52 (t, J = 7.9 Hz, 1H, ArH), 6.75 (d, J = 8.8 Hz, 1H, ArH), 5.89 (d, J = 8.4 Hz, 1H,
NH), 5.44 (s, 1H, NH), 5.15 – 4.88 (m, 2H, 2 OH), 4.58 – 4.44 (m, 1H, OH), 4.35 (d, J = 9.7 Hz,
1H, H-1), 4.07 – 3.85 (m, 2H, H-3, H-4), 3.78 – 3.57 (m, 5H, H-6b, 2 CH2), 3.56 – 3.40 (m, 1H, H-
6a), 3.34 – 3.19 (m, 2H, H-2, H-5), 3.13 – 2.98 (m, 2H, ArCH2), 2.73 – 2.51 (m, 2H, SCH2), 2.13 –
1.87 (m, 4H, 2 CH2), 1.65 – 1.40 (m, 4H, 2 CH2), 1.40 – 1.20 (m, 4H, 2 CH2); 13C NMR (75 MHz,
DMSO-d6) δ 163.82, 162.85, 158.76, 154.43, 152.17, 132.91, 132.58, 130.56, 123.17, 121.76,
121.58, 108.87, 108.44, 96.39, 84.67, 81.07, 76.63, 70.94, 61.37, 55.56, 52.92, 48.73, 29.10, 28.28,
27.61, 26.32, 25.67; HRMS (ESI) calcd for C29H39N4O7S (M+H+) 587.2539, found 587.2531.
N-[6-[(2-ureido-β-D-glucopyranosyl) thio] hexyl] benzamide (15a): white solid; (0.17 g, 77%)
1
yield; [α]D25 -22.3 (c=0.1, DMSO); H NMR (300 MHz, DMSO-d6) δ 8.42 (t, J = 5.5 Hz, 1H,
CH2NH), 7.90 – 7.74 (m, 2H, ArH), 7.59 – 7.30 (m, 3H, ArH), 5.90 (d, J = 8.6 Hz, 1H, CHNH),
5.44 (s, 2H, NH2), 5.03 – 4.91 (m, 2H, 2 OH), 4.49 (t, J = 5.8 Hz, 1H, OH), 4.34 (d, J = 9.8 Hz, 1H,
H-1), 3.67 (dd, J = 11.2, 5.8 Hz, 1H, H-6b), 3.50 – 3.39 (m, 1H, H-6a), 3.35 – 3.15 (m, 4H, H-3,
H-4, H-2, H-5), 3.14 – 3.00 (m, 2H, NCH2), 2.59 (t, J = 7.2 Hz, 2H, SCH2), 1.70 – 1.42 (m, 4H, 2
CH2), 1.41 – 1.19 (m, 4H, 2 CH2); 13C NMR (75 MHz, DMSO-d6) δ 166.26, 158.78, 134.85, 131.07,
128.33, 127.23, 84.72, 81.08, 76.59, 70.97, 61.41, 55.58, 39.29, 29.17, 29.11, 28.28, 26.21; HRMS
(ESI) calcd for C20H32N3O6S (M+H+) 442.2012, found 442.2002.
N-[6-[(2-ureido-β-D-glucopyranosyl) thio] hexyl]-1-naphthamide (19a): white solid; (0.18 g,
73%) yield; [α]D25 -15.8 (c=0.1, DMSO); 1H NMR (300 MHz, DMSO-d6) δ 8.50 (t, J = 5.6 Hz, 1H,
CH2NH), 8.24 – 8.11 (m, 1H, ArH), 8.04 – 7.91 (m, 2H, ArH), 7.63 – 7.50 (m, 4H, ArH), 5.91 (d,
J = 8.6 Hz, 1H, CHNH), 5.45 (s, 2H, NH2), 5.05 – 4.93 (m, 2H, 2 OH), 4.50 (t, J = 5.8 Hz, 1H, OH),
4.37 (d, J = 9.9 Hz, 1H, H-1), 3.69 (dd, J = 11.1, 5.9 Hz, 1H, H-6b), 3.51 – 3.40 (m, 1H, H-6a), 3.36
– 3.16 (m, 4H, H-3, H-4, H-2, H-5), 3.15 – 3.02 (m, 2H, NCH2), 2.63 (t, J = 7.2 Hz, 2H, SCH2),
1.67 – 1.48 (m, 4H, 2 CH2), 1.47 – 1.29 (m, 4H, 2 CH2); 13C NMR (75 MHz, DMSO-d6) δ 168.61,
158.78, 135.33, 133.25, 129.89, 129.65, 128.29, 126.74, 126.29, 125.50, 125.12, 125.06, 84.76,
81.11, 76.62, 70.99, 61.43, 55.61, 39.39, 29.23, 29.14, 29.09, 28.28, 26.23; HRMS (ESI) calcd for
C24H34N3O6S (M+H+) 492.2168, found 492.2171.
4.2.3 Data for target compounds 7b-7d, 11b-11c, 15b-15k, and 19b-19h
Data for compounds 7b-7d, 11b-11c, 15b-15k, and 19b-19h can be found in Supporting
Information.
4.3 Enzyme inhibitory activity assays
OfHex1 and HsHexB were overexpressed in Pichia pastoris and purified according to previous
methods.8 hOGA was overexpressed in Escherichia coli BL21(DE3) and purified as described
previously.23
The enzymatic activities of OfHex1, hOGA, and HsHexB measured at 30 °C using 4-
11

methylumbelliferyl N-acetyl-β-D-glucosaminide (4-MU-GlcNAc) as the substrate. HsHexB was
assayed in 20 mM sodium citrate buffer (pH 4.5), OfHex1 and hOGA were assayed in 20 mM
sodium phosphate buffer (pH 6.5). Firstly, the enzyme was pre-incubated with inhibitors in buffer
for 10 min, then 4-MU-GlcNAc was added. After incubation for a further 20 min at 30 °C, the
reaction mixture was terminated by the addition of 100 μL of 0.5 M sodium carbonate solution. The
fluorescence was quantified (excitation at 366 nm, emission at 445 nm) on a Varioskan Flash
microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The inhibition constant (Ki) was
acquired using Dixon plots by linear fitting of data in Dixon plots.
4.4 Molecular docking
The Sybyl Software (Version7.3) was used for molecular docking studies. The crystal structure
8
of OfHex1-PUGNAc (PDB code: 3OZP) was used as the docking model. Prior to molecular
docking, the structures of inhibitors were optimized using the MMFF94 force field. Then, the ligand
protomol was created based on the Hammerhead scoring function with the molecular similarity
algorithm in the active domain of receptor.24-25 Finally, molecular docking between the ligands and
the receptors were performed using the Surflex-Dock algorithm in Sybyl 7.3.
Conflicts of interest
None.
Acknowledgments
We acknowledge the financial support by the National Natural Science Foundation (No.
21772230, No. 31425021), and Chinese Universities Scientific Fund (No. 2019TC135).
Supporting Information
Experimental procedures, 1H NMR and 13C NMR spectrum.
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HO
O
N
O
HO
HO
HO
HO
HO
HO
HO
HO
HO
HO
O
N
O
H
O
O
O
HO
HO
O
O
N
S
O
O
O
N
6
OH
HO
HO
NH
O
NHAc
N
NHAc
NHAc
NHAc
O
NH2
PUGNAc
TMG-chitotriomycin
11c
OfHex1: Ki=25.6M; HsHexB: Ki>100M
design
OH
O
HO
HO
Hydrophobic
group
S
linker
linker=
n
NH
To provide more binding
affinity at -1 subsite
O
NH2
O
O
HN
O
O
N
H
R1
N
H
N
O
R2
N
Hydrophobic group =
N
,
,
,
,
H
et al.
Docking result of OfHex1 in complex with 11c
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which
may be considered as potential competing interests:
14

15