Naphthalimide and quinoline derivatives as inhibitors for insect N-acetyl-β-D-hexosaminidase

Article abstract of DOI:10.1016/j.cclet.2019.01.023

Insect chitinolytic β-N-acetyl-D-hexosaminidase, such as OfHex1 from Ostrinia furnacalis, is a potential target for insecticide design. Among the known OfHex1 inhibitors, Q2 is of great interest because it is the first non-carbohydrate inhibitor. In this study, we designed and synthesized a series of Q2 derivatives by replacing the thiadiazole and naphthalimide groups and changing the linker length. Compound 3m showed the best inhibitory activity with a K_i value of 0.34 μmol/L against OfHex1, which is about one-quarter that of Q2 (K_i = 1.4 μmol/L). Compound 6a showed the best inhibitory activity among the quinoline-containing derivatives (K_i = 2.3 μmol/L). Molecular docking indicated that although 3m, 6a, and Q2 binding the active pocket of OfHex1 in similar mode, compound 3m engaged better than the other compounds in intermolecular interaction with OfHex1.

Full text of DOI:10.1016/j.cclet.2019.01.023

Accepted Manuscript
Title: Naphthalimide and quinoline derivatives as inhibitors
for insect N-acetyl-β-D-hexosaminidase
Authors: Huibin Yang, Huitang Qi, Tian Liu, Xusheng Shao,
Qing Yang, Xuhong Qian
PII:
DOI:
Reference:
S1001-8417(19)30051-8
https://doi.org/10.1016/j.cclet.2019.01.023
CCLET 4791
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Chinese Chemical Letters
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Accepted date:
19 December 2018
22 January 2019
22 January 2019
Please cite this article as: Yang H, Qi H, Liu T, Shao X, Yang Q, Qian X, Naphthalimide
andquinolinederivativesasinhibitorsforinsectN-acetyl-β-D-hexosaminidase, Chinese
Chemical Letters (2019), https://doi.org/10.1016/j.cclet.2019.01.023
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Communication
Naphthalimide and quinoline derivatives as inhibitors for insect N-acetyl-β-D-
hexosaminidase
c,1
c
c,*
a,*
Huibin Yanga,b,1, Huitang Qi , Tian Liu , Xusheng Shao , Qing Yang , Xuhong Qian
a
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
cSchool of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China
*
Corresponding authors:
E-mail addresses: qingyang@dlut.edu.cn (Q. Yang), xhqian@ecust.edu.cn (X. Qian).
1 These authors contributed equally to this work.
Graphical Abstract
A series of substituted naphthalimide and quinoline derivatives were designed, prepared and evaluated as potential inhibitors of
OfHex1. Compound 3m was the most potent inhibitor with a Ki value of 0.34 µmol/L. Quinoline analogs with an intramolecular
N-H hydrogen bond mimiced the naphthalimide configuration to maintain the inhibitory activity potency.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Insect chitinolytic β-N-acetyl-D-hexosaminidase, such as OfHex1 from Ostrinia furnacalis, is a
potential target for insecticide design. Among the known OfHex1 inhibitors, Q2 is of great interest
because it is the first non-carbohydrate inhibitor. In this study, we designed and synthesized a
series of Q2 derivatives by replacing the thiadiazole and naphthalimide groups and changing the
linker length. Compound 3m showed the best inhibitory activity with a Ki value of 0.34 μmol/L
against OfHex1, which is about one-quarter that of Q2 (Ki = 1.4 μmol/L). Compound 6a showed
the best inhibitory activity among the quinoline-containing derivatives (Ki = 2.3 μmol/L).
Molecular docking indicated that although 3m, 6a, and Q2 binding the active pocket of OfHex1
in similar mode, compound 3m engaged better than the other compounds in intermolecular
interaction with OfHex1.
Available online
Keywords:
OfHex1 inhibitors
Structure-activity relationship
Naphthalimide derivatives
Quinoline derivatives
Molecular docking
Chitin is the second most abundant biopolymer next to cellulose in nature, and is the major component of the extracellular matrix of
some agricultural pests such as fungi [1], nematodes and insects [2]. Notably, chitin is completely absent from vertebrates and higher
plants, and the key enzymes for chitin biosynthesis and biodegradation represent potential targets for pesticide development. For example,
insect chitinolytic β-N-acetyl-D-hexosaminidase (Hex), which is responsible for hydrolyzing chitooligosaccharides to N-acetyl-D-
glucosamine during chitin degradation, is considered to be an attractive target for pesticide development. Hex has been proved to be vital
for insect survival in different insect species, such as Tribolium castaneum [3], Ostrinia furnacalis [4], Locusta migratoria [5],
Nilaparvata lugens [6] and Mamestra brassicae [7]. Moreover, the crystal structure of OfHex1 from Ostrinia furnacalis has been resolved
and has provided a solid basis for the design of specific inhibitors [8].
Several kinds of OfHex1 inhibitors have been reported, including TMG-chitotriomycin [9-14], PUGNAc [15,16], NAG-thiazoline and
its derivative NMAGT [17,18], Q2 [19], phlegmacin B1 [20], berberine [21] and thiazolylhydrazone derivatives [22]. Among these
compounds, Q2, an unsymmetrical dyad of thiadiazole and 1,8-naphthalimide, is of great interest because it is the first non-carbohydrate
inhibitor of OfHex1 [19]. As part of our efforts toward the discovery and biological evaluation of new OfHex1 inhibitor, we report here
the synthesis and structure-activity relationships of a series of Q2 derivatives formed by replacing the thiadiazole and naphthalimide

groups [23-25], and altering the linker length. Moreover, to improve the pharmacological profiles and solubilities of naphthalimide
derivatives while maintaining their inhibitory activity, we replaced the naphthalimide moiety with quinoline carboxamide group via an
intramolecular N-H hydrogen bond to mimic the naphthalimide configuration. We studied the inhibition mechanisms of these compounds
using structure-based molecular docking.
The preparation of substituted naphthalimide derivatives 3a-m is summarized in Scheme 1. The dimethylamine-substituted
intermediate 1b can be obtained from the corresponding commercially available 4-bromo-1,8-naphthalic anhydride 1a. Intermediates 2
were synthesized by reacting a suspension of intermediates 1 in ethanol with excess Boc-protected amines, followed by deprotection in
acidic condition. Compounds 3 were synthesized from intermediates 2 with a halogenated compound or aryl acyl chloride under base
conditions [26].
The quinoline derivatives were prepared according the method described in Scheme 2. The quinoline acyl chloride 4, which was
prepared from quinoline carboxylic acid, was reacted with the Boc-protected amine and then deprotected under acidic condition to obtain
the intermediate 5. Compounds 6 were synthesized from intermediate 5 with the halogenated compounds or aryl acyl chloride under base
1
conditions. Details for the synthetic procedures, physical characteristics and the results of H NMR, 13C NMR and MS for all the
synthesized compounds are listed in the Supporting information.
The inhibitory activities of naphthalimide derivatives 3a-m toward OfHex1 are outlined in Table 1. For substitution of NH (R1), a
comparison of 3a with 3b showed that small rings, such as thiazole, gave better inhibitory activity than the pyridine ring. The carbonyl
group was introduced to increase the hydrogen bond between the compound and the enzyme, which could improve binding affinity.
However, a comparison of 3a with 3c–i showed that replacement of methylene with a carbonyl or sulfone group did not remarkably
improve the inhibitory activity.
Compound 3a had an acceptable level of inhibitory activity, and the raw material of 3a, 2-chloro-5-(chloromethyl)thiazole, is cheap
and easy to get. Thus, we used (2-chlorothiazol-5-yl)methyl as R1 and focused our attention on the effects of the substitution of the
naphthalimide and elongation of the alkyl chain (n). Replacement of the dimethylamino group with bromine or hydrogen at the 5-position
(R2; compound 3j and 3k) decreased the activity against OfHex1. A comparison of the inhibitory activities of compounds 3a, 3l and 3m
showed a large enhancement with elongation of the alkyl chain (n). Compound 3m stood out with comparatively higher activity with a
Ki value of 0.34 μmol/L (Fig. 1A), and exhibited about 4-fold increase in inhibitory activity when compared with compound Q2 (Ki =
1.4 μmol/L).
To improve the pharmacological profiles of naphthalimide derivatives but maintain the inhibitory activity, we replaced the
naphthalimide moiety with quinoline carboxamide group, which contains an intramolecular N-H hydrogen bond and could mimic the
naphthalimide while improving the solubility. The quinoline analogues showed obvious better inhibitory activities against OfHex1,
regardless of their content of a six-membered ring or five-membered ring (Table 2). Compounds containing a thiazole ring, including 6a,
6e and 6j, exhibited more potent inhibitory activity (>70%) than compounds containing other heterocycle ring. Compound 6a showed
the best inhibitory activity with a Ki value of 2.3 μmol/L against OfHex1 (Fig. 1B).
To elucidate the inhibition mechanism of 3m, molecular docking was performed using OfHex1 as template. We found that 3m
occupied the entire substrate binding pocket of OfHex1 and interacted via hydrogen bonds (Fig. 2A). The molecular docking study
revealed good binding of the linker and the thiazole group of 3m at the subsite −1. The linker of 3m was bent into a curved conformation
and the secondary nitrogen atom formed a hydrogen bond with the catalytic residue Glu368 and the residue Glu328. The N3 atom formed
a hydrogen bond with the phenolic hydroxyl group of Tyr475. Interestingly, the length of the linker region had a strong effect on the
inhibition mechanism. Elongation of the alkyl chain resulted in tight binding of the 4-dimethylaminonaphthalimide group of 3m. A
hydrophobic patch comprising Trp483, Trp490 and carbonyl groups from the 4-dimethylaminonaphthalimide formed hydrogen bonds
with Val327, Glu328 and Glu526.
The binding mode of compound 6a in OfHex1 was studied by molecular docking. As shown in Fig. 2B, binding of compound 6a
occurred in the entire active pocket of OfHex1 in a similar manner to compound 3m. The mechanisms of interaction of the linkers and
thiazole group with OfHex1 were similar to those of 3m, and the quinoline group bound with a hydrophobic patch comprising Trp483
and Trp490 outside of subsite −1.
Although compounds 3m (Ki = 0.34 μmol/L) and 6a (Ki = 2.3 μmol/L) showed different inhibitory activity as Q2 did (Ki = 1.4 μmol/L)
against OfHex1, the predicted binding modes of 3m and 6a were similar to that of Q2 in the crystal structure of OfHex1 in a complex
with Q2 [19] (Fig. 2C). First, binding of the thiazole group of 3m, 6a and the thiadiazole group of Q2 occurred in subsite −1 of the active
pocket in the same fashion. These groups were sandwiched by Trp524 and Trp448 and their N3 atoms formed hydrogen bonds with the
phenolic hydroxyl group of Tyr475. Second, the linkers of 3m, 6a and Q2 were bent into a curved conformation and the secondary
nitrogen atoms formed hydrogen bonds with the catalytic residue Glu368. These results suggest that the compounds discovered in this
study inhibit OfHex1 by a similar mechanism as Q2.
In summary, we designed, prepared and evaluated a series of substituted naphthalimide and quinoline derivatives as potential
inhibitors of OfHex1. Compound 3m was the most potent inhibitor with a Ki value of 0.34 µmol/L. Quinoline analogs with an
intramolecular N-H hydrogen bond mimic the naphthalimide configuration to maintain the inhibitory activity potency.

Acknowledgments
The authors acknowledge the National Key Research and Development Program of China (Nos. 2017YFD0200500,
2017YFD0201400, 2018YFD0200100) and the National Natural Science Foundation of China (No. 31871959) for the financial
support.
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Fig. 1. Inhibitory kinetics of compounds 3m (A), 6a (B) against OfHex1.
Fig. 2. Inhibition mechanisms of compounds 3m and 6a against OfHex1. (A) Binding mode of 3m in the active pocket of OfHex1. The compound 3m was shown
in magenta. The hydrogen bonds were shown in black dashes. (B) Binding mode of 6a in the active pocket of OfHex1. The compound 6a was shown in green.
The hydrogen bonds are shown as dashed black lines. (C) Superimposition of compound 3m, 6a and Q2 in the active pocket of OfHex1. 3m, 6a and Q2 are shown
in magenta, green and yellow respectively.
Scheme 1. Synthesis of compounds 3a-m. Reagents and conditions: (a) 40% Dimethylamine aqueous solution, CuSO4·5H2O, DMF, reflux, 8 h; (b)
NH2(CH2)nNHBoc, EtOH, reflux, 3 h; (c) CF3COOH, CH2Cl2, r.t., 4 h; (d) R1Cl, K2CO3, CH3CN, reflux, 3 h; or R1Cl, Et3N, CH2Cl2, r.t., 5 h.
Scheme 2. Synthesis of compounds 6a-j. Reagents and conditions: (a) SOCl2, PhCH3, reflux, 2 h; (b) tert-butyl(2-aminoethyl)carbamate, Et3N, CH2Cl2, r.t., 5
h; (c) CF3COOH, CH2Cl2, r.t., 4 h; (d) R3Cl, K2CO3, CH3CN, reflux, 3 h; or R3Cl, Et3N, CH2Cl2, r.t., 5 h.

Table 1
Inhibitory activities of compounds 3a-m against OfHex1.
Compd. R1
R2
n
2
Inhibitory rate
(%, 10 μmol/L)
69.1 ± 4.8
3a
(CH3)2N
3b
3c
(CH3)2N
(CH3)2N
2
2
48.0 ± 0.2
54.0 ± 5.0
3d
3e
3f
(CH3)2N
(CH3)2N
(CH3)2N
2
2
2
34.0 ± 3.0
37.0 ± 1.0
23.0 ± 7.0
3g
3h
(CH3)2N
(CH3)2N
2
2
56.0 ± 1.0
35.0 ± 4.0
3i
(CH3)2N
H
2
2
2
3
4
26.0 ± 7.0
25.7 ± 1.4
31.1 ± 0.9
75.0 ± 0.0
91.0±1.0
3j
3k
3l
Br
(CH3)2N
(CH3)2N
3m
Q2
81.3 ± 3.2
Table 2
Inhibitory activities of compounds 6a-j against OfHex1.
Compd. R3
6a
Inhibitory rate (%, 10 μmol/L)
81.0 ± 0.0
6b
60.0 ± 2.0
6c
65.0 ± 3.0
66.0 ± 2.0
6d
6e
6f
70.0 ± 1.0
57.0 ± 3.0
6g
6h
66.0 ± 2.0
67.0 ± 1.0

6i
64.0 ± 1.0
6j
75.0 ± 1.0
81.3 ± 3.2
Q2