Exploring unsymmetrical dyads as efficient inhibitors against the insect β-N-acetyl-d-hexosaminidase OfHex2

Article abstract of DOI:10.1016/j.biochi.2013.10.008

The GH20 β-N-acetyl-d-hexosaminidase OfHex2 from the insect Ostrinia furnacalis (Guene?e) is a target potential for eco-friendly pesticide development. Although carbohydrate-based inhibitors against β-N-acetyl-d- hexosaminidases are widely studied, highly

Full text of DOI:10.1016/j.biochi.2013.10.008

Biochimie 97 (2014) 152e162
Contents lists available at ScienceDirect
Biochimie
journal homepage: www.elsevier.com/locate/biochi
Research paper
Exploring unsymmetrical dyads as efficient inhibitors against
the insect
b
-N-acetyl- -hexosaminidase OfHex2
D
a
b
b
a
b
,
a,
*
Qi Chen , Peng Guo , Lin Xu , Tian Liu , Xuhong Qian **, Qing Yang
a
School of Life Science and Biotechnology, Dalian University of Technology, No.2 Linggong Road, Dalian, Liaoning 116024, China
State Key Laboratory of Bioreactor Engineering and Shanghai Key Laboratory of Chemical Biology, School of Pharmacy,
b
East China University of Science and Technology, Shanghai 200237, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The GH20
potential for eco-friendly pesticide development. Although carbohydrate-based inhibitors against
acetyl- -hexosaminidases are widely studied, highly efficient, non-carbohydrate inhibitors are more
attractive due to low cost and readily synthetic manner. Based on molecular modeling analysis of the
catalytic domain of OfHex2, a series of novel naphthalimide-scaffold conjugated with a small aromatic
moiety by an alkylamine spacer linker were designed and evaluated as efficiently competitive inhibitors
against OfHex2. The most potent one containing naphthalimide and phenyl groups spanning by an N-
alkylamine linker has a K value of 0.37 mM, which is 6 fold lower than that of M-31850, the most potent
i
non-carbohydrate inhibitor ever reported. The straightforward synthetic manners as well as the pre-
sumed binding model in this paper could be advantageous for further structural optimization for
b
-N-acetyl-
D
-hexosaminidase OfHex2 from the insect Ostrinia furnacalis (Guenée) is a target
Received 23 July 2013
Accepted 9 October 2013
Available online 17 October 2013
b
-N-
D
Keywords:
b-N-Acetyl-D-hexosaminidase
Enzyme inhibitors
Molecular docking
Naphthalimide
Tryptophan fluorescence titration
developing inhibitors against GH20
b
-N-acetyl-D-hexosaminidases.
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
diminishing or decreasing the activity of OfHex2 is perhaps an
efficient and eco-friendly path for pest control.
Glycoside hydrolase family 20 (GH20)
minidases (EC 3.2.1.52) catalyze the hydrolysis of N-glycans by
liberating N-acetyl- -glucosamine (GlcNAc) or N-acetyl- -galac-
tosamine (GalNAc) from non-reducing ends. It plays important
roles in the degradation of lysosomal or spermatozoa plasma gly-
coconjugates and the degradation of chitin [1].
b
-N-acetyl-
D
-hexosa-
A number of inhibitors against b-N-acetyl-D-hexosaminidases
have been reported during recent years [6e11]. Among them, car-
bohydrate derivatives are most potent ones. Fig. 1 lists seven
structures with high inhibitory activities. NAG-thazoline (NGT) is
D
D
the analog of the transient oxazoline intermediate with a K
0.07 M against human -N-acetyl- -hexosaminidase B (HsHexB)
and a K value of 0.06 M against a bacterial GH20 -N-acetyl-
hexosaminidase from Streptococcus gordonii [12]. Our group re-
ported that a substrate analog, TMG-(GlcNAc) with a N,N,N-tri-
methyl group substitution of the acetyl group of the GlcNAc at the
non-reducing end of (GlcNAc) , has a K value of 0.077 M against
an insect -N-acetyl- -hexosaminidase OfHex1 [13]. Hatanaka et al.
reported TMG-(GlcNAc) exhibited an inhibitory activity against a
bacterial -N-acetyl- -hexosaminidase from Streptomyces coelicolor
[14] with a K value of 0.34 M. PUGNAc, gluco-nagstain and NHAc-
i
value of
m
b
D
OfHex2 is a presumed insect lysosomal
b
-N-acetyl-
D
-hexosa-
i
m
b
D-
minidase from the destructive pest Ostrinia furnacalis (Guenée) [2].
It has been proved to play an important role in the development of
larval abdomen, pupal wing and adult appendages by means of
RNAi [3]. These properties distinguishes OfHex2 from the insect
chitinolytic enzymes, OfHex1, the homolog that functions in chitin
degradation during insect molting [2,4,5]. Besides, the comparison
between substrate spectrums of OfHex2 and its human homolog
HsHex indicates insect OfHex2 is quite different from HsHex.
Thus, OfHex2 would be an insect-specific target potential, and
2
3
i
m
b
D
2
b
D
i
m
DNJ are analogs of GlcNAc where O-5 is replaced by a nitrogen atom
in the pyranose ring and exhibit inhibitory activities against HsHex
B with K
i
values of 0.036, 0.01 and 0.003
mM, respectively [7,15,16].
An even weaker inhibitory activity (K
i
values of 7.0
m
M) of NHAc-
*
Corresponding author. Tel./fax: þ86 411 84707245.
DNJ (DNJNAc in the text) has been reported when testing against
human placenta that contained both HsHexA and HsHexB [17].
The GlcNAc-type iminocyclitiol containing iminosugar and two
** Corresponding author. Tel./fax: þ86 21 64252603.
E-mail addresses: xhqian@ecust.edu.cn (X. Qian), qingyang@dlut.edu.cn
(
Q. Yang).
0300-9084/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.biochi.2013.10.008

Q. Chen et al. / Biochimie 97 (2014) 152e162
153
Fig. 1. Reported GH20
b-N-acetyl-D-hexosaminidases inhibitors.
methoxyphenyl groups with an alkylamine chain has the lowest K
i
and had higher ligand efficiency and better inhibitory activity when
compared to M-31850. The structureeactivity relationship analysis
as well as molecular docking studies provided a binding model
between these inhibitors and OfHex2. And tryptophan fluorescent
assay indicated that the competitive inhibition benefited from the
stacking interactions between naphthalimides and tryptophan
residues in the active pocket.
value of 0.69 nM against HsHexB [18]. Comparing with non-
carbohydrate-based structure, it is more challenging to drive
these leads into large-scale application because of poor physico-
chemical properties and complex synthetic chemistry, in particular
the difficulties in further optimization by medicinal chemistry
[19,20].
Non-carbohydrate-based naphthalimides are important aromatic
heterocycles chemicals with simple synthetic chemistry and
immense pharmacologicalsignificances as theyserve as core scaffolds
formany drugs [21,22]. Naphthalimidederivativeshavebeenreported
to be efficient inhibitors of HsHex. For example, the dinaphthalimides
compound M-31850, found by high-throughput screening [23], ex-
2. Experimental section
2.1. Chemistry
2.1.1. General
hibits an IC50 value of 0.6
m
M against HsHex. The structureeactivity
All chemicals or reagents (except specially noted) were pur-
chased from standard commercial supplies and treated with stan-
dard methods before use. Solvents were dried in a routine way and
redistilled. All melting points (m.p.) were obtained with Büchi
Melting Point B540 and are uncorrected. H and C NMR spectra
were recorded on a Brucker AM-400 (400 MHz) spectrometer with
relationship analysis of both the compound M-31850 and its de-
rivatives with different linkers, suggests that one naphthalimide
group might directly bind the active site, while the second naph-
thalimide mightbindahydrophobicpatchthatisawayfromtheactive
pocket to provide additional weak interactions. Furthermore, an
appropriately-sized N-alkylamine linker is presumed but not identi-
fied to be necessary to span the two binding sites. And the amine
groupinthelinkerprobablydonateshydrogenatomtoformhydrogen
bonds with active site residues. This presumption is partially proved
by our recent work on the development of naphthalimides against
HsHex [24].
The fact that residues forming the active pocket of HsHex are
conserved in OfHex2 inspired us to deduce that the naphthalimide
derivatives might be exploited as inhibitors against OfHex2. Binding
affinityanalysis of M-31850against OfHex2 confirmed M-31850 was a
competitive inhibitor of OfHex2. Ligand efficiency (LE) value,
combining the binding free energy to the number of heavy atoms, is a
very helpful metric guiding the optimization of hits [25]. The low LE
value of M-31850 indicated there was space to increase its efficiency.
Molecular docking study suggested this low efficiency of M-31850
was caused by the oversize of the naphthalimide group binding to the
small hydrophobic cavity outside of the pocket (named out-pocket
site). Thus, the improvement of the binding efficiency in the out-
pocket site could be a way to increase inhibitors binding affinity.
To improve the ligand efficiency, a series of novel derivatives
were synthesized by replacing one of the naphthalimide in M-
1
13
CDCl
3
or DMSO-d
6
as the solvent and TMS as the internal standard.
(parts per million) values.
Chemical shifts are reported in
d
Coupling constants nJ are reported in Hz. High-resolution mass
spectra (HRMS) were recorded under electron impact (70 eV)
condition using a MicroMass GCT CA 055 instrument. Analytical
thin-layer chromatography (TLC) was carried out on precoated
plates (silica gel 60 F254), and spots were visualized with ultravi-
olet (UV) light. The mixtures were separated by gravity
chromatography.
2.1.2. Experimental details and characterization of compounds
Compounds 1 and 2 were obtained with the similar synthetic
procedure reported by Ott [26].
0
2.1.2.1. 2,2 -(Azanediylbis(ethane-2,1-diyl))bis(1H-benzo[de]iso-
ꢀ
1
quinoline-1,3(2H)-dione) (1). White solid. mp: 240e241 C. H NMR
(400 MHz, CDCl ):
¼ 8.38 (d, J ¼ 7.2 Hz, 4H), 8.15 (d, J ¼ 8.0 Hz, 4H),
7.64 (dd, J ¼ 8.0, 7.2 Hz, 4H), 4.32 (t, J ¼ 6.4 Hz, 4H), 3.10 (t,
3
d
1
3
J ¼ 6.4 Hz, 4H), 1.53 ppm (s, 1H); C NMR (100 MHz, CDCl
3
):
d
¼ 164.4, 133.7, 131.5, 131.1, 128.2, 126.8, 122.7, 47.4, 39.8 ppm;
þ
HRMS-ESI (m/z): calcd for C28
22
H N
3
O
4
[M þ H] , 464.1610; found,
31850 with small aromatic rings to afford new unsymmetrical
464.1604.
dyads. Optimization on the size of aromatic rings and the length of
linker was performed to improve the binding affinity of moieties
that bind outside of the active pocket.
The enzymatic testing and efficiency analysis suggested these
new unsymmetrical dyads were competitive inhibitors of OfHex2
0
2.1.2.2. 2,2 -((Ethane-1,2-diylbis(azanediyl))bis(ethane-2,1-diyl))
bis(1H-benzo[de]isoquinoline-1,3(2H)-dione) (2). White solid. mp:
decomposition (192 C). H NMR (400 MHz, CDCl
J ¼ 7.2 Hz, 4H), 8.15 (d, J ¼ 8.0 Hz, 4H), 7.70 (dd, J ¼ 8.0, 7.2 Hz, 4H),
ꢀ
1
3
):
d
¼ 8.54 (d,

154
Q. Chen et al. / Biochimie 97 (2014) 152e162
ꢀ
4
(
.28 (t, J ¼ 6.4 Hz, 4H), 2.98 (t, J ¼ 6.4 Hz, 4H), 2.82 (s, 4H), 1.91 ppm
(300 mg, 0.68 mmol, 90%) as red solid. mp 169e170 C; 1H NMR
(400 MHz, CDCl ):
s, 2H); 13C NMR (100 MHz, CDCl
):
d
¼ 164.3, 133.8, 131.5, 131.2,
d
¼ 8.52 (d, J ¼ 7.2 Hz, 2H), 8.20 (d, J ¼ 7.6 Hz,
3
3
128.1, 126.8, 122.6, 48.9, 47.5, 39.9 ppm; HRMS-ESI (m/z): calcd for
2H), 7.74 (dd, J ¼ 7.2, 7.6 Hz, 2H), 7.04e6.97 (m, 2H), 6.95 (d,
J ¼ 8.0 Hz, 1H), 4.31 (t, J ¼ 6.4 Hz, 2H), 3.79 (t, J ¼ 6.4 Hz, 2H), 3.76 (s,
þ
C
30 27
H N
4
O
4
[M þ H] , 507.2032; found, 507.2036.
1
3
3
H), 3.14e3.00 (m, 4H), 2.31 ppm (s, 1H); C NMR (100 MHz,
2
2
.1.3. The synthetic route of compound 6b
.1.3.1. 5 -Methoxy-5,5-dimethylspiro[[1,3]dioxane-2,3 -indolin]-2 -
CDCl
3
):
d
¼ 183.7, 164.4, 158.6, 156.2, 145.1, 134.0, 131.5, 131.3, 128.1,
0
0
0
126.9, 124.6, 122.4, 117.9, 111.6, 109.3, 55.9, 47.4, 46.5, 40.3,
39.4 ppm; HRMS-ESI (m/z): calcd for C25
444.1559; found,444.1560.
þ
one (3b). 5-Methoxyindoline-2,3-dione (317 mg, 1.79 mmol),
neopentyl glycol (186 mg, 1.79 mmol) and p-toluenesulfonic acid
22
H N
3
O
5
[M þ H] ,
(
31 mg, 0.18 mmol) was mixed in 15 mL toluene and stirred at
The compounds 6a and 6ced were synthesized following the
similar procedures as 6b.
reflux for 8 h. After being cooled to room temperature, the mixture
was filtrated. The filtrate was concentrated in vacuo to give a res-
idue, which was purified by silica gel column chromatography to
2.1.3.5. 2-(2-((2-(2,3-Dioxoindolin-1-yl)ethyl)amino)ethyl)-1H-
give the desired product (300 mg, 1.14 mmol) as orange solid. Yield:
benzo[de]isoquinoline-1,3(2H)-dione (6a). Yiled: 72%. Orange solid.
1
ꢀ
1
6
1
4%. H NMR (400 MHz, CDCl
H), 6.82 (dd, J ¼ 2.4, 8.0 Hz, 1H), 6.70 (d, J ¼ 8.0 Hz, 1H), 4.73 (d,
3
):
d
¼ 8.20 (s, 1H), 7.06 (d, J ¼ 2.4 Hz,
mp 82e84 C; H NMR (400 MHz, CDCl
3
):
d
¼ 8.56 (d, J ¼ 7.6 Hz,
2H), 8.23 (d, J ¼ 8.0 Hz, 2H), 7.77 (dd, J ¼ 7.6, 8.0 Hz, 2H), 7.51 (d,
J ¼ 7.6 Hz, 1H), 7.46 (dd, J ¼ 7.2, 7.6 Hz, 1H), 7.05e6.95 (m, 2H), 4.32
(t, J ¼ 6.4 Hz, 2H), 3.82 (t, J ¼ 6.4 Hz, 2H), 3.00e3.10 ppm (m, 4H);
J ¼ 10.8 Hz, 2H), 3.80 (s, 3H), 3.55 (d, J ¼ 10.8 Hz, 2H), 1.43 (s, 3H),
0
.91 ppm (s, 3H); 13C NMR (100 MHz, CDCl
3
):
d
¼ 173.7, 156.3,133.5,
1
3
128.4, 116.4, 110.9, 110.4, 93.7, 71.3, 55.9, 30.3, 23.0, 22.1 ppm.
C NMR (100 MHz, CDCl
3
):
d
¼ 183.4, 164.5, 158.5, 151.2, 138.2,
1
34.0, 131.6, 131.3, 128.2, 127.0, 125.1, 123.4, 122.5, 117.6, 110.5, 47.5,
0
0
2
.1.3.2. 1 -(2-Bromoethyl)-5 -methoxy-5,5-dimethylspiro[[1,3]
46.5, 40.5, 39.6 ppm; HRMS-ESI (m/z): calcd for C24
[M þ H] , 414.1454; found, 414.1454.
20
H N
3
O
4
0
0
þ
dioxane-2,3 -indolin]-2 -one (4b). 60% Sodium hydride (182 mg,
.60 mmol) was added portionwise to a mixture of 3b (1.0 g,
7
3
.80 mmol) dissolved in 20 mL DMF. After the reaction medium was
2.1.3.6. 6-(Dimethylamino)-2-(2-((2-(2,3-dioxoindolin-1-yl)ethyl)
ꢀ
stirred at 40 C for 1 h, 1,2-dibromoethane (1.64 mL, 18.99 mmol)
was then added. The reaction medium was stirred at 40 C until the
completion of reaction was detected by TLC. After extraction with
ethyl acetate, the organic layer was washed with water. The organic
2 4
fraction was dried over Na SO , and concentrated in vacuo to give a
amino)ethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione
(6c).
¼ 8.53 (d,
ꢀ
1
Yield: 91%. Red solid. H NMR (400 MHz, CDCl
3
):
d
J ¼ 7.2 Hz, 1H), 8.40e8.50 (m, 2H), 7.68 (dd, J ¼ 7.6, 8.0 Hz, 1H), 7.54
(d, J ¼ 7.6 Hz, 1H), 7.49 (dd, J ¼ 7.6, 8.0 Hz, 1H), 7.13 (d, J ¼ 8.0 Hz,
1H), 7.00e7.40 (m, 2H), 4.31 (t, J ¼ 6.0 Hz, 2H), 3.85 (t, J ¼ 6.0 Hz,
13
residue, which was purified by silica gel column chromatography to
give desired product (1.3 g, 3.51 mmol, 92%) as yellowish solid. H
2H), 3.14 (s, 6H), 3.07 ppm (t, J ¼ 6.0 Hz, 4H); C NMR (100 MHz,
1
CDCl
3
):
d
¼ Gp-156-125; HRMS-ESI (m/z): calcd for C26
25 4 4
H N O
þ
NMR (400 MHz, CDCl
.4 Hz, 1H), 6.74 (d, J ¼ 8.4 Hz, 1H), 4.72 (d, J ¼ 10.8 Hz, 2H), 3.99 (t,
J ¼ 7.2 Hz, 2H), 3.81 (s, 3H), 3.55e3.48 (m, 4H), 1.42 (s, 3H),
.89 ppm (s, 3H); 13C NMR (100 MHz, CDCl
):
¼ 171.1, 156.6, 135.0,
27.9, 116.0, 110.8, 109.1, 93.3, 71.4, 55.9, 41.2, 30.3, 27.0, 23.0,
3
):
d
¼ 7.07 (d, J ¼ 2.8 Hz, 1H), 6.87 (dd, J ¼ 2.8,
[M þ H] , 457.1876; found, 457.1877.
8
2.1.3.7. 2-(2-((2-(2,3-Dioxoindolin-1-yl)ethyl)amino)ethyl)-6-
methoxy-1H-benzo[de]isoquinoline-1,3(2H)-dione (6d). Yield: 56%.
0
1
3
d
1
Yellow solid. H NMR (400 MHz, CDCl
3
):
d
¼ 8.61e8.51 (m, 2H), 8.50
2
2.1 ppm.
(d, J ¼ 8.4 Hz, 1H), 7.70 (dd, J ¼ 7.6, 8.0 Hz, 1H), 7.50 (d, J ¼ 7.2 Hz,
H), 7.45 (dd, J ¼ 7.6, 8.0 Hz, 1H), 7.04 (d, J ¼ 8.0 Hz, 1H), 7.02e6.96
(m, 2H), 4.28 (t, J ¼ 6.0 Hz, 2H), 4.14 (s, 3H), 3.80 (t, J ¼ 6.0 Hz, 2H),
1
0
0
2
2
.1.3.3. 2-(2-((2-(5 -Methoxy-5,5-dimethyl-2 -oxospiro[[1,3]dioxane-
0
0
13
,3 -indolin]-1 -yl)ethyl)amino)ethyl)-1H-benzo[de]isoquinoline-
3.10e2.97 ppm (m, 4H); C NMR (100 MHz, CDCl
3
):
d
¼ 183.4,
1
,3(2H)-dione (5b). Potassium carbonate (859 mg, 6.21 mmol) was
added to a solution of 7 (1.12 g, 4.66 mmol) and 4b (1.15 g,
.11 mmol) in 30 mL acetonitrile. The mixture was stirred at reflux
164.8, 164.2, 161.0, 158.5, 151.3, 138.2, 133.6, 131.6, 129.4, 128.8,
126.0, 125.1, 123.5, 123.4, 122.2, 117.6, 114.9, 110.5, 105.2, 56.2, 47.6,
3
46.5, 40.5, 39.5 ppm; HRMS-ESI (m/z): calcd for C25
[M þ H] , 444.1559; found, 444.1564.
22
H N
3
O
5
þ
until the completion of reaction was detected by TLC and then the
undissolved substance was removed by filtration. The filtrate was
concentrated in vacuo to give a residue, which was purified by silica
2.1.3.8. 2-(2-Aminoethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione
(7). 1,2-diaminoethane (0.24 mL, 3.63 mmol) was added to a sus-
pension of 1,8-naphthalic anhydride (240 mg, 1.21 mmol) in 10 mL
ethanol. The mixture was refluxed for about 4 h until the comple-
tion was detected by TLC. Then the hot reaction mixture filtered, the
filtrate was cooled to room temperature and filtered again to get
the crude product. The crude product was purified by recrystalli-
zation in ethanol to get 7 (150 mg, 52%) as slightly yellow solid.
gel column chromatography to give desired product (900 mg,
1
1.70 mmol, 55%) as yellowish solid. H NMR (400 MHz, CDCl
3
):
d
¼ 8.55 (d, J ¼ 7.2 Hz, 2H), 8.18 (d, J ¼ 7.6 Hz, 2H), 7.72 (dd, J ¼ 7.2,
7
.6 Hz, 2H), 7.00 (d, J ¼ 1.6 Hz, 1H), 6.82-6.72 (m, 2H), 4.67 (d,
J ¼ 11.2 Hz, 2H), 4.32 (d, J ¼ 6.4 Hz, 2H), 3.77 (s, 3H), 3.70 (t,
J ¼ 6.4 Hz, 2H), 3.43 (d, J ¼ 11.2 Hz, 2H), 3.06 (d, J ¼ 6.4 Hz, 2H), 2.98
13
(
d, J ¼ 6.4 Hz, 2H), 1.95 (s, 1H), 1.38 (s, 3H), 0.83 ppm (s, 3H);
C
ꢀ
1
NMR (100 MHz, CDCl
31.2, 128.1, 127.9, 126.9, 122.6, 115.9, 110.5, 109.5, 93.4, 71.2, 56.9,
7.3, 46.5, 39.7, 39.6, 30.2, 23.0, 22.1 ppm.
3
):
d
¼ 171.4, 164.3, 156.3, 135.8, 133.9, 131.5,
R
f
¼ 0.38 (CHCl
DMSO-d ):
¼ 8.34 (d, J ¼ 7.6 Hz, 2H), 8.31 (d, J ¼ 8.0 Hz, 2H), 7.75
(dd, J ¼ 8.0, 7.6 Hz, 2H), 4.01 (t, J ¼ 6.8 Hz, 2H), 2.79 (t, J ¼ 6.8 Hz,
3
/MeOH, 10:1); mp: 141e142 C; H NMR (400 MHz,
1
4
6
d
1
3
2
H), 2.62 ppm (br, 2H); C NMR (100 MHz, DMSO-d
134.4, 131.5, 130.9, 127.7, 127.4, 122.4, 43.2, 40.2 ppm; HRMS-ESI (m/
6
):
d
¼ 163.9,
2
.1.3.4. 2-(2-((2-(5-Methoxy-2,3-dioxoindolin-1-yl)ethyl)amino)
ethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (6b). 5b (400 mg,
.76 mmol) was stirred at room temperature in a mixture of HCl-
þ
z): calcd for C14
13
H N
2
O
2
[M þ H] , 241.0977; found, 241.0978.
0
AcOH (3:1 ¼ v/v, 20 mL) for 30 min. The resulting mixture was
poured into saturated sodium bicarbonate solution and extracted
with dichloromethane. The organic layer was washed with water,
2.1.3.9. 2-(2-((2-Aminoethyl)amino)ethyl)-1H-benzo[de]isoquino-
line-1,3(2H)-dione (8). Compounds 8 were synthesized following
ꢀ
1
the same procedures as 7. White solid. mp: 54e56 C. H NMR
(400 MHz, CDCl ):
¼ 8.54 (d, J ¼ 6.8 Hz, 2H), 8.17 (d, J ¼ 8.0 Hz,
4H), 7.71 (t, J ¼ 7.6 Hz, 2H), 4.32 (t, J ¼ 6.0 Hz, 2H), 3.01 (t, J ¼ 6.4 Hz,
dried over Na
2
SO
4
, and concentrated in vacuo to give a residue,
3
d
which was purified by silica gel column chromatography to give 6b

Q. Chen et al. / Biochimie 97 (2014) 152e162
155
2
1
H), 2.77 ppm (s, 4H); 13C NMR (100 MHz, CDCl
31.2, 130.9, 127.8, 126.6, 122.2, 51.8, 47.2, 41.4, 39.7 ppm; EI MS (m/
3
):
d
¼ 164.0, 133.6,
J ¼ 6.4 Hz, 2H), 2.89 ppm (s, 6H); 13C NMR (100 MHz, DMSO-d
6
):
d
¼ 164.0, 150.7, 134.4, 131.2, 130.9, 130.7, 127.5, 127.2, 122.2, 118.1,
e): 284.6 (M þ 1, 100). HRMS-ESI (m/z): calcd for C16
H
18
N
3
O
2
111.9, 54.8, 50.2, 44.8, 36.4 ppm; HRMS-ESI (m/z): calcd for
þ
þ
[M þ H] , 284.1399; found, 284.1402.
C
23
24
H N
3
O
2
[M þ H] , 374.1869; found, 374.1873.
2
.1.3.10. 2-(2-(Benzylamino)ethyl)-1H-benzo[de]isoquinoline-
2.1.3.14. 2-(2-(Dibenzylamino)ethyl)-1H-benzo[de]isoquinoline-
1,3(2H)-dione (17). Potassium carbonate (155 mg, 1.12 mmol) was
added to a solution of 7 (162 mg, 0.68 mmol) and (bromomethyl)
benzene (231 mg, 1.35 mmol) in 10 mL acetonitrile. The mixture
was stirred at reflux until the completion of reaction was detected
by TLC and then the undissolved substance was removed by
filtration. The filtrate was concentrated in vacuo to give a residue,
which was purified by silica gel column chromatography using
1,3(2H)-dione (13). Potassium carbonate (120 mg, 0.87 mmol) was
added to a solution of 7 (316 mg, 1.32 mmol) and (bromomethyl)
benzene (74 mg, 0.43 mmol) in 10 mL acetonitrile. The mixture was
stirred at reflux until the completion of reaction was detected by
TLC and then the undissolved substance was removed by filtration.
The filtrate was concentrated in vacuo to give a residue, which was
purified by silica gel column chromatography using CH
2 2 3
Cl /CH OH
(
30:1) to give 13 (98 mg, 0.30 mmol, 68%) as white solid. mp: 110e
CH
2 2 3
Cl /CH OH (40:1) to give 17 (252 mg, 0.61 mmol, 89%) as white
ꢀ
1
ꢀ
1
1
(
2
11 C. H NMR (400 MHz, CDCl
d, J ¼ 8.0 Hz, 2H), 7.76 (dd, J ¼ 8.0, 7.6 Hz, 2H), 7.32 (d, J ¼ 7.6 Hz,
H), 7.26 (dd, J ¼ 7.6, 7.2 Hz, 2H), 7.19 (t, J ¼ 7.2 Hz, 1H), 4.39 (t,
J ¼ 6.4 Hz, 2H), 3.90 (s, 2H), 3.06 (t, J ¼ 6.4 Hz, 2H), 2.45 ppm (br,
3
):
d
¼ 8.60 (d, J ¼ 7.6 Hz, 2H), 8.22
solid. mp: 119e120 C. H NMR (400 MHz, CDCl
3
):
d
¼ 8.52 (d,
J ¼ 7.6 Hz, 2H), 8.23 (d, J ¼ 8.0 Hz, 2H), 7.77 (dd, J ¼ 8.0, 7.6 Hz, 2H),
7.24e7.17 (m, 4H), 7.05e6.95 (m, 6H), 4.38 (t, J ¼ 6.0 Hz, 2H), 3.64 (s,
13
4H), 2.82 ppm (t, J ¼ 6.0 Hz, 2H); C NMR (100 MHz, CDCl
3
):
1
3
1
H); C NMR (100 MHz, CDCl
3
):
d
¼ 164.5, 139.4, 134.0, 131.6, 131.3,
d
¼ 163.9, 139.6, 133.7, 131.5, 131.0, 128.9, 128.2, 128.0, 126.9, 126.6,
1
28.4, 128.3, 128.2, 127.0, 126.9, 122.6, 53.3, 46.9, 39.7 ppm; HRMS-
122.9, 58.3, 51.2, 37.9 ppm; HRMS-ESI (m/z): calcd for C28
H
25
N
2
O
2
þ
þ
ESI (m/z): calcd for C21
3
H
19
N
2
O
2
[M þ H] , 331.1447; found,
[M þ H] , 421.1916; found, 421.1911.
31.1442.
The compounds 18e21 and 9e10 were synthesized following
the similar procedures as 13.
2
.1.3.11. 2-(2-((Pyridin-2-ylmethyl)amino)ethyl)-1H-benzo[de]iso-
quinoline-1,3(2H)-dione (14). A solution of picolinaldehyde
200 mg, 1.87 mmol) in 30 mL methanol was added to a solution of
(408 mg, 1.70 mmol) in 30 mL methanol. The mixture was stirred
at room temperature for 3 h. The white solid in the reaction mixture
was filtered and dried over infrared oven to afford the intermediate
2.1.3.15. 2-(2-(Phenethylamino)ethyl)-1H-benzo[de]isoquinoline-
ꢀ
1
(
7
1,3(2H)-dione (18). Yield: 71%. White solid. mp: 94e95 C. H NMR
(400 MHz, CDCl ):
3
d
¼ 8.66 (d, J ¼ 7.6 Hz, 2H), 8.30 (d, J ¼ 8.0 Hz,
2H), 7.84 (dd, J ¼ 8.0, 7.6 Hz, 2H), 7.34e7.27 (m, 4H), 7.24 (t,
J ¼ 6.4 Hz, 1H), 4.49 (t, J ¼ 6.4 Hz, 2H), 3.22 (t, J ¼ 6.4 Hz, 2H), 3.16 (t,
13
(
530 mg, 1.61 mmol, 95%) as white solid. Sodium triacetoxybor-
J ¼ 7.2 Hz, 2H), 3.00 ppm (t, J ¼ 7.2 Hz, 2H); C NMR (100 MHz,
ohydride (219 mg, 1.03 mmol) was added to a solution of the in-
termediate (283 mg, 0.86 mmol) and AcOH (0.05 mL, 2.08 mmol) in
CDCl
3
):
d
¼ 164.5, 139.0, 134.1, 131.6, 131.4, 128.8, 128.5, 128.2, 126.9,
126.2, 122.5, 50.4, 47.3, 39.1, 35.3 ppm; HRMS-ESI (m/z): calcd for
þ
15 mL 1,2-dichloroethane. The mixture was stirred at room tem-
C
22
H
21
N
2
O
2
[M þ H] , 345.1603; found, 345.1601.
perature overnight and then treated with water (50 mL) and
extracted with dichloromethane (50 mL*3). The organic layer was
2.1.3.16. 2-(2-((3-Phenylpropyl)amino)ethyl)-1H-benzo[de]isoquino-
ꢀ
1
dried over Na
which was purified by silica gel column chromatography using
CH Cl /CH OH (20:1) to give 14 (222 mg, 0.67 mmol, 78%) as white
solid. mp: 128e129 C. H NMR (400 MHz, CDCl
J ¼ 7.6 Hz, 2H), 8.48 (d, J ¼ 4.4 Hz, 1H), 8.21 (d, J ¼ 8.4 Hz, 2H), 7.75
2
SO
4
, and concentrated in vacuo to give a residue,
line-1,3(2H)-dione (19). Yield: 75%. White solid. mp: 47e48 C. H
NMR (400 MHz, CDCl ):
3
d
¼ 8.63 (d, J ¼ 7.6 Hz, 2H), 8.25 (d,
2
2
3
J ¼ 8.4 Hz, 2H), 7.78 (dd, J ¼ 8.4, 7.6 Hz, 2H), 7.25 (d, J ¼ 7.2 Hz, 2H),
7.21e7.13 (m, 3H), 4.40 (t, J ¼ 6.4 Hz, 2H), 3.07 (t, J ¼ 6.4 Hz, 2H),
2.79 (t, J ¼ 7.2 Hz, 2H), 2.67 (t, J ¼ 7.6 Hz, 2H), 2.20 (br, 1H), 1.88 ppm
ꢀ
1
3
):
d
¼ 8.59 (t,
1
3
(
dd, J ¼ 8.4, 7.6 Hz, 2H), 7.58 (t, J ¼ 7.6 Hz, 1H), 7.30 (d, J ¼ 8.0 Hz,
(dt, J ¼ 7.6, 7.2 Hz, 2H); C NMR (100 MHz, CDCl
3
):
d
¼ 164.2, 142.1,
1
3
d
H), 7.10 (dd, J ¼ 8.0, 6.0 Hz, 1H), 4.40 (t, J ¼ 6.4 Hz, 2H), 4.00 (s, 2H),
133.8, 131.4, 131.2, 128.4, 128.3, 128.0, 126.8, 125.7, 122.5, 49.1, 47.7,
1
3
.07 (t, J ¼ 6.4 Hz, 2H), 2.25 ppm (s, 1H); C NMR (100 MHz, CDCl
3
):
40.0, 33.5, 31.6 ppm; HRMS-ESI (m/z): calcd for C23
[M þ H] , 359.1760; found, 359.1758.
H
23
N
2
O
2
þ
¼ 164.2, 159.4, 149.2, 136.4, 133.8, 131.4, 131.1, 128.0, 126.8, 122.5,
1
C
22.3, 121.9, 54.5, 47.2, 39.8 ppm; HRMS-ESI (m/z): calcd for
þ
H
20 18
N
3
O
2
[M þ H] , 332.1399; found, 332.1401.
2.1.3.17. 2-(2-((2-Phenoxyethyl)amino)ethyl)-1H-benzo[de]isoquino-
ꢀ
1
The compounds 15 and 16 were synthesized following the same
line-1,3(2H)-dione (20). Yield: 71%. White solid. mp: 112e114 C. H
NMR (400 MHz, CDCl ):
procedures as 14.
3
d
¼ 8.59 (d, J ¼ 7.6 Hz, 2H), 8.20 (d,
J ¼ 8.0 Hz, 2H), 7.74 (dd, J ¼ 8.0, 7.6 Hz, 2H), 7.24 (t, J ¼ 7.6 Hz, 2H),
2
.1.3.12. 2-(2-((4-(Trifluoromethyl)benzyl)amino)ethyl)-1H-benzo
6.91 (t, J ¼ 7.2 Hz,1H), 6.87 (d, J ¼ 8.0 Hz, 2H), 4.37 (t, J ¼ 6.8 Hz, 2H),
13
[
1
8
de]isoquinoline-1,3(2H)-dione (15). Yield: 80%. White solid. mp:
4.05 (t, J ¼ 4.8 Hz, 2H), 3.14e3.04 (m, 4H),1.83 ppm (s,1H); C NMR
ꢀ
1
35e138 C. H NMR (400 MHz, CDCl
.19 (d, J ¼ 8.0 Hz, 2H), 7.23 (dd, J ¼ 8.0, 6.8 Hz, 2H), 7.47 (d,
3
):
d
¼ 8.56 (d, J ¼ 6.8 Hz, 2H),
(100 MHz, CDCl
3
):
d
¼ 164.2, 158.8, 133.9, 131.5, 131.2, 129.4, 128.0,
126.8, 122.5, 120.7, 114.5, 67.2, 48.5, 47.4, 39.9 ppm; HRMS-ESI (m/
þ
J ¼ 8.0 Hz, 2H), 7.39 (d, J ¼ 8.0 Hz, 2H), 4.36 (t, J ¼ 6.4 Hz, 2H), 3.90
z): calcd for C22
H
21
N
2
O
3
[M þ H] , 361.1552; found, 361.1548.
13
(
s, 2H), 3.01 (t, J ¼ 6.4 Hz, 2H), 1.56 ppm (s, 1H); C NMR (100 MHz,
CDCl ):
1
5
[
3
d
¼ 164.4, 144.6, 134.0, 131.6, 131.3, 129.0 (q, J ¼ 32.3 Hz),
2.1.3.18. 2-(2-(((5-Phenyl-1,3,4-oxadiazol-2-yl)methyl)amino)ethyl)-
1H-benzo[de]isoquinoline-1,3(2H)-dione (21). Yield: 60%. White
28.3,128.2,127.0, 125.2 (q, J ¼ 3.7 Hz),124.3 (q, J ¼ 271.9 Hz),122.6,
ꢀ
1
2.9, 47.1, 39.9 ppm; HRMS-ESI (m/z): calcd for C22
H
18
N
2
O
2
F
3
solid. mp: 166e168 C. H NMR (400 MHz, CDCl
J ¼ 7.2 Hz, 2H), 8.13 (d, J ¼ 7.6 Hz, 2H), 7.93 (dt, J ¼ 6.8, 1.6 Hz, 2H),
.67 (dd, J ¼ 7.6, 7.2 Hz, 2H), 7.47 (tt, J ¼ 7.2, 2.4 Hz, 1H), 7.41 (tt,
J ¼ 7.2, 1.6 Hz, 2H), 4.35 (t, J ¼ 6.4 Hz, 2H), 4.14 (s, 2H), 3.13 (t,
3
):
d
¼ 8.51 (d,
þ
M þ H] , 399.1320; found, 399.1324.
7
2
[
.1.3.13. 2-(2-((4-(Dimethylamino)benzyl)amino)ethyl)-1H-benzo
de]isoquinoline-1,3(2H)-dione (16). Orange solid. Yield: 70& 1
H
J ¼ 6.4 Hz, 2H), 1.97 ppm (s, 1H); C NMR (100 MHz, CDCl
13
3
):
NMR (400 MHz, DMSO-d
6
):
d
¼ 8.59 (s, 1H), 8.52 (d, J ¼ 7.6 Hz, 2H),
d
¼ 165.0, 164.8, 164.2, 133.8, 131.4, 131.3, 131.0, 128.7, 127.9, 126.7,
8
6
.50 (d, J ¼ 7.6 Hz, 2H), 7.90 (t, J ¼ 7.6 Hz, 2H), 7.27 (d, J ¼ 7.6 Hz, 2H),
126.6, 123.6, 122.2, 46.9, 43.2, 39.3 ppm; HRMS-ESI (m/z): calcd for
þ
.72 (d, J ¼ 7.6 Hz, 2H), 4.39 (t, J ¼ 6.4 Hz, 2H), 4.08 (s, 2H), 3.29 (t,
C
23
H
19
N
4
O
3
[M þ H] , 399.1457; found, 399.1457.

156
Q. Chen et al. / Biochimie 97 (2014) 152e162
2
.1.3.19. 2-(2-((2-(1,3-Dioxoisoindolin-2-yl)ethyl)amino)ethyl)-1H-
55.3, 47.9, 47.4, 39.7 ppm; HRMS-ESI (m/z): calcd for C24
[M þ H] , 418.2; found, 418.2.
H
24
N
3
O
4
þ
benzo[de]isoquinoline-1,3(2H)-dione (9). Yield: 70%. White solid.
mp 172e174 C; H NMR (400 MHz, CDCl
2
4
3
d
ꢀ
1
3
):
H), 8.21 (d, J ¼ 8.0 Hz, 2H), 7.72 (dd, J ¼ 7.2, 8.0 Hz, 2H), 7.63 (s, 4H),
.32 (t, J ¼ 6.0 Hz, 2H), 3.80 (t, J ¼ 6.0 Hz, 2H), 3.07 (t, J ¼ 6.0 Hz, 2H),
d
¼ 8.49 (d, J ¼ 7.2 Hz,
2.2. Inhibitory activity and ligand efficiency analysis
1
3
.02 (t, J ¼ 6.0 Hz, 2H), 1.59 ppm (s, 1H); C NMR (100 MHz, CDCl
3
):
The insect GH20 enzyme OfHex2 was prepared as described by
Liu et al. [2] the bacterial hexosaminidase from Serratia marcescens
and another insect hexosaminidase OfHex1 was prepared as
describled by Liu et al. [27]. GH20 hexosaminidases from Tricho-
derma viride, Canavalia ensiformis and Homo sapiens were pur-
chased from SigmaeAldrich. All of the synthesized compounds
were evaluated for their inhibitory activities against hexosamini-
¼ 168.4, 164.4, 133.8, 133.6, 132.1, 126.9, 123.0, 122.7, 47.4, 47.2,
þ
3
9.7, 37.6 ppm; HRMS-ESI (m/z): calcd for C24
19
H N
3
O
4
[M þ H] ,
414.1454; found, 414.1456.
2
.1.3.20. 2-(2-((2-(2-Oxobenzo[cd]indol-1(2H)-yl)ethyl)amino)
ethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (10). Yellow solid.
ꢀ
1
mp: 147e149 C. H NMR (400 MHz, CDCl
H), 8.14 (d, J ¼ 8.0 Hz, 2H), 7.92 (d, J ¼ 8.0 Hz, 1H), 7.87 (d,
J ¼ 6.8 Hz, 1H), 7.67 (dd, J ¼ 8.0, 7.2 Hz, 2H), 7.61 (dd, J ¼ 7.6, 7.2 Hz,
H), 7.42 (d, J ¼ 8.4 Hz, 1H), 7.33 (dd, J ¼ 8.0, 7.6 Hz, 1H), 6.94 (d,
J ¼ 6.8 Hz, 1H), 4.29 (t, J ¼ 6.4 Hz, 2H), 3.99 (t, J ¼ 6.4 Hz, 2H), 3.09 (t,
3
):
d
¼ 8.44 (d, J ¼ 7.2 Hz,
dases by using the artificial substrate, pNP-GlcNAc. The assay
ꢀ
2
components were incubated in a final volume of 60
ml at 25 C for
30 min in the presence of Britton-Robinson buffer(OfHex2, pH 6.0;
OfHex1, TvHex, and SmHex, pH 7.0; HsHex and CeHex, pH 4.5),
ethanol at the final concentration of 20%, enzyme, compounds and
pNP-GlcNAc. Then enzyme reaction was stopped by the addition of
1
13
J ¼ 6.4 Hz, 2H), 3.05 (t, J ¼ 6.4 Hz, 2H), 1.76 ppm (s, 1H); C NMR
100 MHz, CDCl ):
¼ 168.2, 164.3, 139.6, 133.8, 131.5, 131.1, 130.7,
29.0, 128.5, 128.5, 128.1, 126.8, 126.5, 125.1, 124.1, 122.5, 120.1,
(
1
3
d
2 3 i
60 ml 0.5 M Na CO solution. The inhibition constant (K [mM]) was
obtained by Dixon plots by changing the concentration of the
compound at a constant concentration (0.2 and 0.5 mM) of the
substrate. Ligand efficiency values were calculated as follows [28]:
105.3, 47.9, 47.4, 40.4, 39.7 ppm; HRMS-ESI (m/z): calcd for
þ
C
27
22
H N
3
O
3
[M þ H] , 436.1661; found, 436.1668.
i
LE ¼ ꢁ1.35 log(K )/N, where N is the number of heavy atoms.
2
.1.3.21. 2-((2-(1,3-Dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)ethyl)
amino)-N-(quinolin-8-yl)acetamide (11).
-Chloro-N-(quinol-8-yl)acetamide (107 mg, 0.49 mmol),
117 mg, 0.49 mmol), N,N-diisopropylethylamine (0.86 mL,
.9 mmol) and potassium iodide (10 mg) were added to acetonitrile
30 mL), after stirred and refluxed for 10 h under nitrogen atmo-
sphere, the mixture was cooled to rt and concentrated in vacuo to
get crude product which was purified by silica gel column chro-
matography to afford 11 (140 mg, 0.33 mmol, 68%) as white solid.
2.3. Molecular modeling
2
(
4
(
7
Homology modeling of the catalytic domain of OfHex2 (Gen-
Bank: ABO65045.1, residues 192e503), was carried out by using
SWISS-MODEL Workspace [29]. The crystal structure of HsHex (PDB
code: 1O7A, chain B) with amino acid sequence identity of 48.8% of
the catalytic domain of OfHex2 was used as template. The quality of
the homology model was evaluated by QMEAN global score and
QMEAN Z-score and estimation of the local quality was performed
using graphical plots of Anolea mean force potential, GROMOS
empirical force field energy and QMEAN, indicated the modeled
catalytic domain of OfHex2 was reliable according to Z-score of
ꢁ0.18 and a high QMEAN global score of 0.759 [30]. The structure of
modeled catalytic domain was depicted using the software PyMol.
ꢀ
1
mp: 176e178 C. H NMR (400 MHz, CDCl
J ¼ 7.6 Hz, 1H), 8.51 (d, J ¼ 7.2 Hz, 2H), 8.16 (d, J ¼ 8.0 Hz, 2H), 8.01
d, J ¼ 8.0 Hz, 1H), 7.99 (d, J ¼ 4.4 Hz, 1H), 7.67 (dd, J ¼ 8.0, 7.2 Hz,
H), 7.48 (dd, J ¼ 8.0, 7.6 Hz, 1H), 7.42 (d, J ¼ 8.0 Hz, 1H), 7.16 (dd,
J ¼ 8.0, 4.4 Hz, 1H), 4.47 (t, J ¼ 6.4 Hz, 2H), 3.64 (s, 2H), 3.21 (t,
3
):
d
¼ 11.1 (s,1H), 8.75 (d,
(
2
13
J ¼ 6.4 Hz, 2H), 1.97 ppm (s, 1H); C NMR (100 MHz, CDCl
3
):
d
¼ 170.4, 164.4, 147.9, 138.7, 135.8, 134.2, 133.8, 131.5, 131.3, 128.2,
1
27.8, 127.1, 126.8, 122.5, 121.5, 121.1, 116.5, 53.4, 47.9, 39.7 ppm;
2.4. Molecular docking
þ
HRMS-ESI (m/z): calcd for C25
H
21
N
4
O
3
[M þ H] , 425.1614; found,
4
25.1617.
The PRODRG2 server [31,32] has been used to generate and
optimize the initial structure of our compounds before docking. The
molecular docking methodology, by running autodock4.2 software
[33,34], consists of two steps: the protein-ligand complex was
obtained firstly by rigid docking and then by flexible docking via
setting the active pocket-outside-ligand binding residues flexible.
Polar hydrogen atoms and Gasteiger charges were added using
AutoDockTools. All maps were calculated with 0.375 A spacing
between grid points. The centre of the grid box was placed at the
centre of the active pocket of Modeled OfHex2. The dimensions of
2
.1.3.22. N-(2-((2-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)
ethyl)amino)ethyl)-4-methoxybenzamide (12). To a mixture of 4-
methoxybenzoic acid (2.0 g, 13.2 mmol) in 20 mL benzene was
ꢀ
dropwised thionyl dichloride (4.8 mL, 65.7 mmol) at 0 C. And then
the mixture was heated and refluxed for 8 h. After completion, the
reaction mixture was cooled to room temperature and concen-
trated under vacuum to give intermediate (2.2 g, 98%) as colorless
oil. To a solution of 8 (150 mg, 0.53 mmol) and pyridine (2 mL) in
ꢀ
ꢀ
10 mL dichloromethane was dropwised a solution of intermediate
the active site box were set at 52 ꢂ 50 ꢂ 58 A. Docking calculations
obtained above (60 mg, 0.35 mmol) in 10 mL dichloromethane. The
mixture was stirred at room temperature and monitored by TCL.
After completion, the reaction mixture was treated with water
were carried out using the Lamarckian genetic algorithm (LGA) and
all parameters were the same for each docking. A population of
random individuals (population size: 150), a maximum number of
25,000,000 energy evaluations, a maximum number of generations
of 27,000 was used.
(
100 mL) and then extracted with dichloromethane (3 * 50 mL). The
combined organic layers were washed with brine, dried over
Na SO , and concentrated in vacuo. The residue was purified by
silica gel column chromatography to give 12 (100 mg, 68%) as
2
4
2.5. Fluorescence spectroscopy
1
yellow solid. H NMR (400 MHz, CDCl
.10 (d, J ¼ 7.2 Hz, 2H), 7.65e7.56 (m, 4H), 7.04 (s, 1H), 6.70 (d,
J ¼ 6.4 Hz, 2H), 4.26 (t, J ¼ 6.0 Hz, 2H), 3.75 (s, 3H), 3.80 (t, J ¼ 6.0 Hz,
H), 3.07 (t, J ¼ 6.0 Hz, 2H), 3.02 (t, J ¼ 6.0 Hz, 2H), 1.59 ppm (s, 1H);
3
):
d
¼ 8.39 (d, J ¼ 6.8 Hz, 2H),
8
In the tryptophan fluorescence titration experiment, compound
was titrated into 100ul titration buffer (0.5 M NaCl, 20 mM
Na HPO , pH 6.0) containing 0.32 mg OfHex2 from 0 to 185 mM. The
2 4
samples were incubated at 298 K for various durations. The tryp-
2
1
3
C NMR (100 MHz, CDCl
3
):
d
¼ 167.2, 164.4, 161.8, 133.9, 131.4,
1
31.2, 128.8, 127.9, 127.0, 126.8, 122.3, 113.4, 77.5, 77.4, 77.2, 76.9,
tophan fluorescence titration was performed in the 96 well solid

Q. Chen et al. / Biochimie 97 (2014) 152e162
157
black polystyrene microplate using a Varioskan Flash (Thermo
3.2. Synthesis of designed naphthalimides
Scientific Co.). The excitation wavelength was 280 nm, and the
emission data were collected between 300 and 480 nm. To deter-
1 and 2 were obtained by reacting a suspension of 1,8-
naphthalic anhydride in ethanol with 0.5 equivalence of amines
(Fig. 3).
mine the dissociation constant, the fluorescence changes (
22 nm after addition of test compounds were fit with the specific
binding equation: maxX/(K max is the
DF) at
3
D
F ¼
D
F
d
þ X), where
D
F
Compound 6aed were synthesized by following steps (Fig. 4).
The carbonyl-protection of isatins were generated by neopentyl
glycol and tosylic acid as catalyst in toluene. After treated with
sodium hydride in dry DMF, 3a or 3b reacted with 1,2-
dibromoethane to yield bromides 4aeb. The preparations of
carbonyl-protected 5aed were finished by the reactions of corre-
sponding naphthalimide derivatives with 4a or 4b under the con-
dition of potassium carbonate and acetonitrile. 20aed were finally
obtained by removal of the protecting group in the system of hy-
drochloric acid and acetic acid.
maximum fluorescence changes and X is the concentration of
added inhibitor [35]. This nonlinear fitting method was performed
with the GraphPad Prism software program (San Diego, CA).
3
. Results and discussion
3.1. Mechanism of symmetrical dyads of naphthalimide M-31850
binding to OfHex2
Compounds 7 and 8 were obtained by reacting a suspension of
,8-naphthalic anhydride in ethanol with excessive amines fol-
lowed by recrystallization. Compound 7 was refluxed with different
bromides or chlorides and potassium carbonate in acetonitrile for
Inhibitory activity testing suggested M-31850 is a competitive
inhibitor against OfHex2 with a K value of 2.5 mM. However, the
i
low LE value (0.21) indicated the binding between M-31850 and
OfHex2 was not efficient enough. To study the mechanism of M-
1850 interacting with OfHex2, molecular docking was performed
1
1
2 h to afford compounds 9, 10, 13 and 17-21. Compound 7 was
3
easily transformed into 14e16 by performing a Schiff condensation
in methanol in the presence of equivalent of different benzalde-
hydes followed reducing reaction with sodium triacetoxyborohy-
dride. Compound 11 was synthesized by conjugating 7 and 2-
chloro-N-(quinol-8-yl)-acetamide in acetonitrile with diisopropy-
using the homology modeled structure of OfHex2 using HsHex as
template. The molecular docking study revealed that one of the
naphthalimide groups of M-31850 localized in the active pocket
and the secondary amine in the linker formed a hydrogen bond
with the side chain of the residue E345. And the second naph-
thalimide group stacked poorly in the narrow out-pocket site. As
shown in Fig. 2, only part of the imide ring and one aromatic ring of
naphthaline of the second naphthalimide group stacked with the
lethylamine and potassium iodide.
8
reacted with 4-
methoxybenzoyl chloride in basic condition to afford 12 (Fig. 5).
ꢀ
3.3. Bioevaluation of inhibitory activity
imidazole group of the residue H285 at a dihedral of about 35 in
the out-pocket site, while the other aromatic ring of naphthaline
group was away from the out-pocket site. The inefficiency binding
in the out-pocket site might be caused by the oversize of naph-
thalimide group or the lack of flexibility of the linker between
secondary amine and the second naphthalimide group. Thus, the
second naphthalimide group is better to be replaced by a smaller
group and to be conjugated by an appropriately-lengthed N-
alkylamine linker.
All of the synthesized compounds were evaluated for their in-
hibition activities against OfHex2 by using the artificial pNP-GlcNAc
as substrate.
To improve the binding efficiency to the out-pocket site, com-
pounds 1e12 containing less than three conjugate aromatic rings
were synthesized (Table 1). The slightly increase in inhibitory ac-
tivity of 2 suggested that the increase of linker flexibility could
improve binding affinity. Compounds 6a, and 9e12 possess similar
structures as 1 (M-31850). All of them have amide and phenyl
group, but are smaller than 1. Reserving two carbonyl groups and
reducing naphthyl to phenyl (6a and 9) improved about 1-fold in
inhibitory activity, though the ligand efficiency were little
increased from 0.21 to 0.26 and 0.25, respectively. It was suggested
the reduced phenyl group of 1 favored its binding. The activity loss
of 10 revealed that the fragment containing three aromatic rings
could not improve the affinity even if minished its size comparing
with naphthalimide. The decreased inhibitory activity of 6b,
substituted with methoxy group on the 4-position of isatin group of
6a, suggested additional substitution on this position would lead to
steric hindrance. However, the change on the similar position of
monocycle compound 12 remarkably improved inhibitory activity
if compared with 1. This suggested that aromatic monocycle frag-
ment could improve the potency and the steric position of the
monoaromatic ring would influence the affinity. Besides, the
addition of hydrophilic group at 4-position of naphthalimide (6c
and 6d) significantly interfered the inhibitory activity. It indicated
naphthalimide group might play an irreplaceable role for inhibitory
activity.
Then, a series of aromatic monocycles compounds 13e21 with
different linker were synthesized (Table 2). The compound 20 was
identified to be the best that exhibited about 6-fold increase in
inhibitory activity when compared with compound 1. The com-
parison of the inhibitory activity of compounds 13 and 18e20,
indicated the activity improvement was linked to length of the
linker between phenyl group and secondary amine. Flexible linker
Fig. 2. Molecular docking of M-31850 into OfHex2. The active pocket and out-pocket
site were shown in light orange and pale cyan surface, respectively. The catalytic
residue E345 was shown in blue stick. The compound M-31850 was shown in magenta
stick.

158
Q. Chen et al. / Biochimie 97 (2014) 152e162
Fig. 3. Synthesis of compounds 1 and 2. a) EtOH, reflux, 7 h.
ꢀ
Fig. 4. Synthesis of compounds 6aed. a) NPG, TsOH, toluene, reflux, 8 h; b) NaH, BrCH
2
CH
2
Br, DMF, 40 C, 3 h; c) K
2
CO
3
, MeCN, reflux, 12 h; d) AcOH, HCl, rt, 30 min.
Fig. 5. Synthesis of compounds 7e21. a) amines, EtOH, reflux, 4 h; b) 9, 10, 13 and 17e21: 7, bromides or chlorides, K
2 3
CO , MeCN, reflux, 12 h; 14e16: 1, benzaldehydes, MeOH, rt,
1
2 h, then NaBH(CH COO) , AcOH, DCM, rt, 12 h; 11: 7, chloride, diisopropylethylamine, KI, MeCN, reflux, 10 h; 12: 8, acyl chloride, pyridine, rt, 12 h.
3
3

Q. Chen et al. / Biochimie 97 (2014) 152e162
159
Table 1
Table 2
Inhibitory activities of compounds 1e12 against OfHex2.
Inhibitory activities of compounds 13e21 against OfHex2.
No.
R1
R2
K
i
[m
M]
LE
No.
R
K
i
[
m
M]
LE
Parenta
Parenta
1
1
1
3
4
5
1.57
1.74
0.31
0.31
e
1
2
H
H
2.50
2.16
0.21
0.20
N.D.b
2.47
6
6
a
H
H
1.21
2.09
0.26
0.23
1
6
0.27
b
17
N.D.
e
1
8
9
1.31
0.55
0.37
0.30
0.31
0.31
6
6
c
OCH
3
N.D.b
N.D.
e
1
d
N(CH
3
)
2
20
21
N.D.
e
9
1
H
H
1.40
N.D.
0.25
a
General structure naphthalimide derivatives evaluated.
Not determined because of the low activity (less than 50% inhibition at 10 mm).
b
0
e
hexosaminidases from either bacterium or fungi. In comparison
with other -N-acetyl- -hexosaminidases, the compound 20
against OfHex2 exhibited a K of 0.37 M, while against the plant
CeHex with K of 4.88 M, the human HsHex with K of 1.29 M and
the insect chitinolytic enzyme OfHex1 with K of 224.51 M.
1
1
2
H
H
5.77
2.31
0.22
0.24
b
D
i
m
i
m
i
m
1
i
m
a
General structure naphthalimide derivatives evaluated.
Not determined because of the low activity (less than 50% inhibition at 10 mm).
b
3.5. Mechanism analysis of unsymmetrical dyads of naphthalimide
derivatives inhibiting OfHex2
with proper length facilitated the phenyl group staking well with
the hydrophobic patch outside of the active pocket. The highest
inhibitory activity of 20 indicated that the oxygen in the linker
might contribute an interaction. The activity loss of 21 which has an
additional heterocycle on the linker also supported that the binding
required a flexible linker. The approximate inhibitory activity of 13
and 14 indicated that substitution on the 2-position of the phenyl
group of 13 with nitrogen atom would not influence the inhibitory
activity obviously. The lower activity of 15 and 16, the analogs of 13,
suggested that a hydrophobic group on the 4-position of the phenyl
group of 13 would weaken the binding of phenyl group to the out-
pocket site.
To further understand the molecular mechanism of naph-
thalimide derivatives, we investigated the binding mode of the
most potent compound 20. As shown in Fig. 6, dixon plot of 20
against OfHex2 indicated 20 is a competitive inhibitor of OfHex2,
because of the trendlines drawn for each concentration of substrate
meeting in quadrant two. Thus, 20 could specific bind to the active
pocket of OfHex2.
Molecular docking was also performed using the structure of
OfHex2 constructed by the homology modeling using HsHex as
template. The interactions between 20 and OfHex2 were then
analyzed.
As shown in Fig. 7A, the naphthalimide group of 20 was stacking
with the W478 and/or the W411 in the active pocket. The secondary
3.4. Effects of the compound 20 on b-N-acetyl-D-hexosaminidases
Table 3
Inhibition constants (K
The inhibitory activity analysis of the compound 20, the most
potential compound in Table 2, was further performed against
GH20 -N-acetyl- -hexosaminidases from bacteria (SmChb), fungi
TvHex), plant (CeHex) and human (HsHex) and the insect chiti-
nolytic enzyme, OfHex1, as shown in Table 3. The data indicated
that the compound 20 did not inhibit -N-acetyl-D-
i
,
m
M) of 20 against GH20 b-N-acetyl-D-hexosaminidases.
SmChb
TvHex
N.D. (11.8)
OfHex1
OfHex2
0.37
CeHex
4.88
HsHex
1.29
b
D
N.D.a (5.6)b
224.51
(
a
Not determined because of the low activity (less than 20% inhibition at 100 mm).
Inhibition (%) at 100 mm are given in parentheses.
b
b

160
Q. Chen et al. / Biochimie 97 (2014) 152e162
amine in the linker of 20 formed hydrogen bonds with the side
ꢀ
chain of the catalytic amino acid E345 within the distance of 2.8 A.
It was worth to note the phenyl group of 20 might stack with two
different hydrophobic patch consisted of (I) the residues W411 and
Y436, or (II) H285.
The stacking with hydrophobic patch (I) was observed both in
rigid-body docking and flexible docking by setting the residues
W411, Y436 and H439 flexible. But the stacking with the patch (II)
was only observed in flexible docking. This might be due to the
steric hindrance in active pocket caused by the side chain of Y436.
Interestingly, Y436 (Y450 in HsHex B) and H439 (L454 in HsHex B)
were located in a loop. Like in the b-N-acetyl-D-hexosaminidase
from Streptococcus pneumonia (PDB code: 3RPM) [36], these two
residues were close enough to form a stacking interaction. And the
shift of Y436 was also observed in the comparison of the crystal
complexes of HsHexB:NGT and HsHexB:PYR [37]. In comparison
with OfHex2, inhibitory activities of 20 against OfHex1 and SmChb
dramatically decreased. This might be due to the residue H285 was
not solvent exposed in OfHex1 (H303) and SmChb (H452) [27,38].
As a result the naphthalimide group would insert more deeply in
the pocket, forming a sandwiched stacking interaction with both
Fig. 6. Dixon plots for inhibition of OfHex2 by 20. The trendlines represent two sub-
strate concentrations (þ, 0.2 mM; ,, 0.5 mM). The insert graph is the partial enlarged
view around the intersection.
Fig. 7. Molecular docking of 20 against OfHex2. Hydrophobic patch (I) and (II) were colored pale cyan and light pink, respectively. Residues were shown in slate sticks. A) Two
conformations of 20 were shown in cyan and magenta sticks. B) The interaction between 20 of conformation (I) and OfHex2.
Fig. 8. Fluorescence spectra of OfHex2 (0.32 mg) in the presence of different concentrations (0e120
22 nm are plotted against the inhibitor concentration.
mM) of 20 (A) and 17 (B). In the inserted figure, the fluorescence changes at
3

Q. Chen et al. / Biochimie 97 (2014) 152e162
161
the W441 and W478 and hydrogen bonds with H285, D344 and
4. Conclusion
E345 by oxygen atom on the imide ring (Fig. 7B). And the oxygen in
the linker improved the affinity of 20 by forming a hydrogen bond
with the side chain of residue E345 in a distance of 2.8 A.
In conclusion, by analyzing the structure of the catalytic domain
of the insect enzyme OfHex2, we designed and synthesized a series
of novel naphthalimide-scaffolded compounds. The efficiency
evaluation by enzymatic testing suggested most of these unsym-
metrical dyads exhibited high binding efficiency and competitive
inhibitory activities. The structureeactivity relationship and dock-
ing studies provided a binding model between inhibitors and
OfHex2. And tryptophan fluorescent assay supported that the
competitive inhibition was introduced by stacking interactions
between naphthalimides and tryptophans. This work provides an
alternative for developing insecticides as well as inhibitors against
ꢀ
Tryptophan fluorescence titration data also supported that the
competitive activity of 20 benefited from the stacking interactions
between naphthalimide and tryptophans. OfHex2 had a strong
fluorescence emission band at around 330 nm by fixing the exci-
tation wavelength at 280 nm (Fig. 8A). However, under the same
condition, 20 had no fluorescence band but had an absorbance
spectra ranging from 300 to 370 nm instead, making it a suitable
potential quencher for tryptophan fluorescence. Since tryptophan
residues comprised the hydrophobic wall of the active pocket [2],
the interaction between 20 and the active pocket would result in
the fluorescence quench of tryptophans.
GH20
b-N-acetyl-D-hexosaminidases.
As expected, by adding 20 to OfHex2, the fluorescence intensity
of OfHex2 decreased in a compound-concentration dependent
manner (Fig. 8A). The degrees of fluorescence quenching of OfHex2
were plotted versus the concentration of 20 (Fig. 8A, the inserted
Acknowledgments
This work was supported by the National High Technology
Research and Development Program of China (2011AA10A204), the
National Key Project for Basic Research (2010CB126100), the Na-
tional Key Technology R&D Program (2011BAE06B05), the National
Natural Science Foundation of China (21236002, 31070715,
figure), and a K
d
value (dissociation constant) was calculated to be
3
.31 ꢃ 0.169 M. This confirmed the interactions between 20 and
m
tryptophan residues at the active pocket. The same conclusion was
true for 17 (Fig. 8B), which exhibited much lower inhibitory activity
and weaker fluorescent quenching ability than 20.
31101671), the Fundamental Research Funds for the Central Uni-
versities (DUT11RC(3)73).
As shown in Fig. 9, the hydrophobic interaction between the
phenyl group and the out-pocket residue H285 was strengthened,
when the number of atoms between the secondary amine and
phenyl fragment increased from one to three. This improvement
benefited from both the length and flexibility of the linker and was
consistent with inhibitory activity data. The hydrophobic group on
the 4-position of the phenyl group of 13 will hurt the binding,
resulting in the inhibitory activity loss of 15 and 16.
Thus, a model of naphthalimide derivatives binding to OfHex2
was established: naphthalimide group bound tothe active pocket by
stacking with the tryptophan residues, the secondary amine in the
flexible linker formed hydrogen bonds with the side chain of residue
E345, and the small aromatic group was stacked with the H285 that
located at a hydrophobic patch outside of the active pocket.
References
[
1] J. Intra, G. Pavesi, D.S. Horner, Phylogenetic analyses suggest multiple
changes of substrate specificity within the glycosyl hydrolase 20 family,
BMC Evol. Biol.
F1471-2148-8-214.
2] F. Liu, T. Liu, M. Qu, Q. Yang, Molecular and biochemical characterization of a
novel -N-acetyl- -hexosaminidase with broad substrate-spectrum from the
Asian corn borer, Ostrinia furnacalis, Int. J. Biol. Sci. 8 (2012) 1085e1096.
3] F. Liu, Q. Yang, Investigation of -N-acetyl- -hexosaminidase as a potential
target with RNA interference in Ostrinia furnacalis, Chin. J. Pestic. Sci. 15
2013) 145e152.
[4] T. Liu, F. Liu, Q. Yang, Jun Yang, Expression, purification and characterization of
the chitinolytic -N-acetyl- -hexosaminidase from the insect Ostrinia furna-
8 (2008) 214. http://link.springer.com/article/10.1186%
2
[
[
b
D
b
D
(
b
D
calis, Protein Expr. Purif. 68 (2009) 99e103.
[
[
[
[
[
5] T. Liu, H. Zhang, F. Liu, Q. Wu, X. Shen, Q. Yang, Structural determinants of an
insect beta-N-Acetyl-D-hexosaminidase specialized as a chitinolytic enzyme,
J. Biol. Chem. 286 (2011) 4049e4058.
6] T. Liu, J. Yan, Q. Yang, Comparative biochemistry of GH3, GH20 and GH84 b-N-
acetyl-
Curr. Drug Targets 13 (2012) 512e525.
7] T. Sumida, K.A. Stubbs, M. Ito, S. Yokoyama, Gaining insight into the inhibition
of glycoside hydrolase family 20 exo- -N-acetylhexosaminidases using a
D-hexosaminidases and recent progress in selective inhibitor discovery,
b
structural approach, Org. Biomol. Chem. 10 (2012) 2607e2612.
8] M. Terinek, A. Vasella, Synthesis of N-acetylglucosamine-derived nagstatin
analogues and their evaluation as glycosidase inhibitors, Helv. Chim. Acta 88
(2005) 10e22.
9] H.C. Dorfmueller, V.S. Borodkin, M. Schimpl, S.M. Shepherd, N.A. Shpiro,
D.M. van Aalten, GlcNAcstatin: a picomolar, selective O-GlcNAcase inhibitor
that modulates intracellular O-GlcNAcylation levels, J. Am. Chem. Soc. 128
(2005) 16484e16485.
[
10] A.F. Glawar, D. Best, B.J. Ayers, S. Miyauchi, S. Nakagawa, M. Aguilar-Moncayo,
J.M. García Fernández, C. Ortiz Mellet, E.V. Crabtree, T.D. Butters, F.X. Wilson,
A. Kato, G.W. Fleet, Scalable syntheses of both enantiomers of DNJNAc and
DGJNAc from glucuronolactone: the effect of N-alkylation on hexosaminidase
inhibition, Chemistry 18 (2012) 9341e9359.
[
11] M.B. Tropak, S.P. Reid, M. Guiral, S.G. Withers, D. Mahuran, Pharmacological
enhancement of beta-hexosaminidase activity in fibroblasts from adult Tay-
Sachs and Sandhoff Patients, J. Biol. Chem. 279 (2004) 13478e13487.
12] D.B. Langley, D.W. Harty, N.A. Jacques, N. Hunter, J.M. Guss, C.A. Collyer,
[
Structure of N-acetyl-beta-D-glucosaminidase (GcnA) from the endocarditis
pathogen Streptococcus gordonii and its complex with the mechanism-based
inhibitor NAG-thiazoline, J. Mol. Biol. 377 (2008) 104e116.
[
[
13] Y. You, T. Liu, Y. Yang, Q. Wu, Q. Yang, B. Yu, Synthesis, evaluation, and
mechanism of N, N, N-trimethyl-
saccharides as selective inhibitors of glycosyl hydrolase family 20
-hexosaminidases, ChemBioChem 12 (2011) 457e467.
D
-glucosamine-(1 / 4)-chitooligo-
b
-N-acetyl-
D
14] H. Usuki, Y. Yamamoto, Y. Kumagai, T. Nitoda, H. Kanzaki, T. Hatanaka, MS/MS
fragmentation-guided search of TMG-chitooligomycins and their structure-
Fig. 9. Comparison of the binding model of 20 and its analogs 13 and 18. Residues
H285, E345 and V346 were shown in slate sticks. 13, 18 and 20 were shown in
magenta, salmon and cyan stick, respectively.
activity relationship in specific
b-N-acetylglucosaminidase inhibition, Org.
Biomol. Chem. 9 (2011) 2943e2951.

162
Q. Chen et al. / Biochimie 97 (2014) 152e162
[
[
[
15] M. Horsch, L. Hoesch, A. Vasella, D.M. Rast, N-acetylglucosaminono-1,5-lactone
[28] M.G.J. Baud, P. Haus, T. Leiser, F.J. Meyer-Almes, M.J. Fuchter, Highly ligand
efficient and selective N-2-(Thioethyl)picolinamide histone deacetylase in-
hibitors inspired by the natural product psammaplin A, ChemMedChem 8
(2013) 149e156.
[29] K. Arnold, L. Bordoli, J. Kopp, T. Schwede, The SWISS-MODEL workspace: a
web-based environment for protein structure homology modelling, Bioin-
formatics 22 (2006) 195e201.
[30] P. Benkert, M. Biasini, T. Schwede, Toward the estimation of the absolute
quality of individual protein structure models, Bioinformatics 27 (2011)
343e350.
oxime and the corresponding (phenylcarbamoyl)oxime. Novel and potent in-
hibitors of beta-N-acetylglucosaminidase, Eur. J. Biochem. 197 (1991) 815e818.
16] B. Shanmugasundaram, A.W. Debowski, R.J. Dennis, G.J. Davies, D.J. Vocadlo,
A. Vasella, Inhibition of O-GlcNAcase by a gluco-conFig.d nagstatin and a
PUGNAc-imidazole hybrid inhibitor, Chem. Commun. 42 (2006) 4372e4374.
17] A. de la Fuente, R. Martin, T. Mena-Barragán, X. Verdaguer, J.M. García Fer-
nández, C. Ortiz Mellet, A. Riera, Stereoselective synthesis of 2-Acetamido-1,2-
dideoxyallonojirimycin (DAJNAc), a new potent hexosaminidase inhibitor,
Org. Lett. 15 (2013) 3638e3641.
[
18] C.W. Ho, S.D. Popat, T.W. Liu, K.C. Tsai, M.J. Ho, W.H. Chen, A.S. Yang, C.H. Lin,
Development of GlcNAc-inspired iminocyclitiols as potent and selective N-
acetyl-beta-hexosaminidase inhibitors, ACS Chem. Biol. 5 (2010) 489e497.
19] M.B. Tropak, D. Mahuran, Lending a helping hand, screening chemical libraries
for compounds that enhance beta-hexosaminidase A activity in GM2 gan-
gliosidosis cells, FEBS J. 274 (2007) 4951e4961.
[31] D.M. van Aalten, R. Bywater, J.B. Findlay, M. Hendlich, R.W. Hooft, G. Vriend,
PRODRG, a program for generating molecular topologies and unique molec-
ular descriptors from coordinates of small molecules, J. Comput. Aided Mol.
Des. 10 (1996) 255e262.
[32] A.W. Schüttelkopf, D.M. van Aalten, Acta. PRODRG: a tool for high-throughput
crystallography of protein-ligand complexes, Crystallogr. D Biol. Crystallogr.
60 (2004) 1355e1363.
[33] G.M. Morris, D.S. Goodsell, R.S. Halliday, R. Huey, W.E. Hart, R.K. Belew,
A.J. Olson, Automated docking using a lamarckian genetic algorithm and
empirical binding free energy function, J. Comput. Chem. 19 (1998) 1639e
1662.
[
[20] H.C. Dorfmueller, D.M.F. van Aalten, Screening-based discovery of drug-like O-
GlcNAcase inhibitor scaffolds, FEBS Lett. 584 (2010) 694e700.
[21] A. Kamal, N.R. Bolla, P.S. Srikanth, A.K. Srivastava, Naphthalimide derivatives
with therapeutic characteristics: a patent review, Expert. Opin. Ther. Pat. 23
(
2013) 299e317.
[
22] S. Banerjee, E.B. Veale, C.M. Phelan, S.A. Murphy, G.M. Tocci, L.J. Gillespie,
D.O. Frimannsson, J.M. Kelly, T. Gunnlaugsson, Recent advances in the devel-
opment of 1,8-naphthalimide based DNA targeting binders, anticancer and
fluorescent cellular imaging agents, Chem. Soc. Rev. 42 (2013) 1601e1618.
23] M.B. Tropak, J.E. Blanchard, S.G. Withers, E.D. Brown, D. Mahuran, High-
throughput screening for human lysosomal beta-N-Acetyl hexosaminidase in-
hibitors acting as pharmacological chaperones, Chem. Biol. 14 (2007) 153e164.
24] P. Guo, Q. Chen, T. Liu, L. Xu, Q. Yang, X.H. Qian, Development of unsym-
metrical dyads as potent noncarbohydrate-based inhibitors against human
[34] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell,
A.J. Olson, AutoDock4 and AutoDockTools4: automated docking with selective
receptor flexibility, J. Comput. Chem. 30 (2009) 2785e2791.
[35] W.C. Chao, J.F. Lu, J.S. Wang, H.C. Yang, T.A. Pan, S.C. Chou, L.H. Wang,
P.T. Chou, Probing ligand binding to thromboxane synthase, Biochemistry 52
(2013) 1113e1121.
[
[
[36] Y. Jiang, W. Yu, J. Zhang, C. Frolet, A. Di Guilmi, C. Zhou, T. Vernet, Y. Chen,
Structural basis for the substrate specificity of
a novel b-N-acetylhex-
osaminidase StrH protein from Streptococcus pneumoniae R6, J. Biol. Chem.
286 (2011) 43004e43012.
beta-N-Acetyl-D-hexosaminidase, ACS Med. Chem. Lett. 4 (2013) 527e531.
[25] A.L. Hopkins, C.R. Groom, A. Alex, Ligand efficiency: a useful metric for lead
selection, Drug Discov. Today 9 (2004) 430e431.
[37] K.S. Bateman, M.M. Cherney, D.J. Mahuran, M. Tropak, M.N. James, Crystal
structure of b-hexosaminidase B in complex with pyrimethamine, a potential
[
26] I. Ott, Y. Xu, X. Qian, Fluorescence properties and antiproliferative effects of
mono-, bis-, and tris- thiophenylnaphthalimides: results of a comparative pilot
study, J. Photochem. Photobiol. B 105 (2011) 75e80.
pharmacological chaperone, J. Med. Chem. 54 (2011) 1421e1429.
[38] Y. Wang, T. Liu, Q. Yang, Z. Li, X.H. Qian, A modeling study for structure fea-
tures of b-N-acetyl-D-hexosaminidase from Ostrinia furnacalis and its novel
[27] T. Liu, H. Zhang, F. Liu, L. Chen, X. Shen, Q. Yang, Active-pocket size differ-
inhibitor allosamidin: species selectivity and multi-target characteristics,
entiating insectile from bacterial chitinolytic
b
-N-acetyl-
D
-hexosaminidases,
Chem. Biol. Drug Des. 79 (2012) 572e582.
Biochem. J. 438 (2011) 467e474.