Development of unsymmetrical dyads as potent noncarbohydrate-based inhibitors against human β-N-acetyl-D-hexosaminidase

Article abstract of DOI:10.1021/ml300475m

Human β-N-acetyl-D-hexosaminidase has gained much attention due to its roles in several pathological processes and been considered as potential targets for disease therapy. A novel and efficient skeleton, which was an unsymmetrical dyad containing naphthalimide and methoxyphenyl moieties with an alkylamine spacer linkage as a noncarbohydrate-based inhibitor, was synthesized, and the activities were valuated against human β-N-acetyl-D- hexosaminidase. The most potent inhibitor exhibits high inhibitory activity with K_i values of 0.63 μM. The straightforward synthetic manners of these unsymmetrical dyads and understanding of the binding model could be advantageous for further structure optimization and development of new therapeutic agents for Hex-related diseases.

Full text of DOI:10.1021/ml300475m

Letter
pubs.acs.org/acsmedchemlett
Development of Unsymmetrical Dyads As Potent Noncarbohydrate-
Based Inhibitors against Human β‑N‑Acetyl‑D‑hexosaminidase
Peng Guo,† Qi Chen,‡ Tian Liu,‡ Lin Xu,† Qing Yang,*,‡ and Xuhong Qian*,†
†Shanghai Key Laboratory of Chemical Biology, Institute of Pesticides and Pharmaceuticals, School of Pharmacy, East China
University of Science and Technology, Shanghai 200237, China
‡School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China
S
* Supporting Information
ABSTRACT: Human β-N-acetyl-D-hexosaminidase has gained
much attention due to its roles in several pathological processes
and been considered as potential targets for disease therapy. A
novel and efficient skeleton, which was an unsymmetrical dyad
containing naphthalimide and methoxyphenyl moieties with an
alkylamine spacer linkage as a noncarbohydrate-based inhibitor,
was synthesized, and the activities were valuated against human
β-N-acetyl-D-hexosaminidase. The most potent inhibitor
exhibits high inhibitory activity with Ki values of 0.63 μM.
The straightforward synthetic manners of these unsymmetrical
dyads and understanding of the binding model could be
advantageous for further structure optimization and develop-
ment of new therapeutic agents for Hex-related diseases.
KEYWORDS: Naphthalimide, β-N-acetyl-D-hexosaminidase, inhibitors, noncarbohydrates, GM2 gangliosides
amily 20 β-N-acetyl-D-hexosaminidase (EC 3.2.1.52) is a
Fwidely distributed enzyme that catalyzes the release of N-
acetyl-β-D-glucosamine (GlcNAc) or N-acetyl-β-D-galactos-
amine (GalNAc) residues from the nonreducing ends of
oligosaccharides and sugar components of glycoproteins and
glycolipids.1 The human β-N-acetyl-D-hexosaminidase (hHex)
has gained much attention due to its roles in several
pathological processes and been considered as potential target
for disease therapy. It is the predominant glycosaminoglycan-
degrading glycosidase in the synovial fluid from patients with
osteoarthritis.2 So the inhibition of this enzyme may prevent
cartilage matrix degradation and thus help the treatment of
osteoarthritis.3 Moreover, the human β-N-acetyl-D-hexosamini-
dase degrades GM2 gangliosides in the lysosome of neuronal
cells.4 Disfunction of this enzyme results in the accumulation of
GM2 gangliosides and leads to Tay-Sachs or Sandhoff disease.5
The adult forms of these diseases are caused by the presence of
large quantities of unstable but still active mutant enzymes,
which could not be transported to lysosome.6 So inhibitors
against β-N-acetyl-D-hexosaminidase can be used as pharmaco-
logical chaperones to stabilize these mutant enzymes and
facilitate the transport of them to increase the enzymatic
activity in the lysosome and thus help the treatment of these
diseases.7−9
Figure 1. Potency and selectivity of inhibitors against GH20 β-N-
acetyl-D-hexosaminidase.
is more challenging to convert these leads of sugars into
therapeutic agents because of poor pharmacokinetic properties
and complex synthetic chemistry, in particular, the difficulties in
further optimization by medicinal chemistry.7,15 Thus, it is of
significance to develop novel noncarbohydrate-based inhibitors
that could be readily modified or synthesized in a more
straightforward manner and is easy to improve selectivity,
solubility, and toxicity profiles.
The promising application in biology and medicine has
induced the appearance of more specific and potent inhibitors
of β-N-acetyl-D-hexosaminidases in recent years.10−14 However,
most of these inhibitors (Figure 1) possess carbohydrate
scaffolds. Comparing with noncarbohydrate-based structure, it
Received: December 23, 2012
Accepted: April 24, 2013
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
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a
High-throughput screening has been performed to find
noncarbohydrate-based inhibitors of human β-N-acetyl-D-
hexosaminidase, resulting in four structurally distinct micro-
molar competitive inhibitors.16,17 Among them, the sym-
metrical bisnapthalimide (M-31850) is the most potent one
against human β-N-acetyl-D-hexosaminidase (IC50 = 6.0 and 3.1
μM for hHex A and hHex B, respectively). Furthermore, the
bisnaphthalimide is in a framework that differs significantly
from the carbohydrate-based inhibitors of hexosaminidase and
supplies a novel scaffold that could be further optimized. In the
last two decades, many researchers have made great efforts to
promote applications of naphthalimide derivatives in the fields
of medicine and biological sensing and imaging.18−20 Most
importantly, they are in relatively simple structures whose facile
and straightforward syntheses have been established.
Scheme 1. Synthesis of Compounds 1a−k
We herein report the synthesis and the structure−function
relationship of a novel and efficient skeleton, which was an
unsymmetrical dyad containing naphthalimide and methox-
yphenyl moiety with an alkylamine spacer linkage as a
noncarbohydrate-based inhibitor against human β-N-acetyl-D-
hexosaminidases A/B.
Our strategy was first to determine the pharmacophore of M-
31850, which directly occupies the active site of the human β-
N-acetyl-D-hexosaminidase and interacts with the key residues
constituting the active pocket. Then various groups were
incorporated to this moiety to acquire additional electrostatic
and other interactions between the substrate and the enzyme.
Since these unsymmetrical dyads exhibit higher inhibitory
activity with Ki values of less than 1 μm, they show a promise
for further development of drugs.
In the structure−activity relationship of M-31850 proposed
by Tropak et al.,16 the naphthalimide group binds to the active
site of hHex and the secondary amine of the N-alkylamine
linker may form a stabilizing hydrogen bond with a residue
around. Besides, the other naphthalimide moiety binds to
another hydrophobic patch, making important contributions to
its high binding affinity. Though this binding model gives a
reasonable explanation for the inhibitory activities of
naphthalimide derivatives, there is much unknown in terms
of a specificity mechanism, which is important for further drug
development.
a
n, R1, and R2 are as defined in Table 1. Reagents and conditions: (a)
EtOH, reflux, 4 h, 50-100%; (b) MeOH, K2CO3, DMF, 60 °C, 4 h,
62% or NH(CH3)2, DMF, rt, 12 h, 82%; (c) CF3COOH, DCM, rt, 4
h, 100%; (d) AcOH, 100 °C, 1 h, 69%; (e) CF3COOH, DCM, rt, 1 h,
90%; (f) K2CO3, DMF, 50 °C, 8 h, 89%; (g) NH2NH2, MeOH, reflux,
4 h, 80%.
Table 1. Structures and Evaluation Datas of Compounds
1a−k against Human β-N-Acetyl-D-hexosaminidases A/B
a
compd
1a
n
R1
R2
NH2
Ki (μM)
2
3
4
6
2
2
2
2
2
H
H
H
H
H
H
Br
2.09
29.08
61.74
Nd
1b
NH2
1c
NH2
1d
NH2
1e
OH
30.87
2.81
Nd
1f
N(CH3)2
NH2
1g
1h
OCH3
NH2
Nd
1i
N(CH3)2
NH2
Nd
1j
Nd
1k
Nd
b
M-31850
3.22
A series of naphthalimide derivatives or analogous with
different N-alkylamine chains 1a−k has been synthesized and
assayed their inhibitory activities toward the human β-N-acetyl-
D-hexosaminidase A/B (Scheme 1).
a
Measured with 4MU-β-GlcNAc as substrate and calculated by linear
regression of data in Dixon plots. Values with SD were shown in Table
S1, Supporting Information. Nd: Not determined (less than 50%
inhibition at 50 μM). For comparison, the Ki of M-31850 was also
determined with the same condition.
b
Compounds 1a−g were obtained by reacting a suspension of
the commercially available 1,8-naphthalic anhydrides in ethanol
with excess amines followed by recrystallization. 4-Nitro-1,8-
naphthalic anhydride was refluxed with tert-butyl (2-
aminoethyl)carbamate in ethanol to afford intermediate in an
almost quantitative yield. The preparations of 1h−i were
finished by the reactions of the intermediate with dimethyl-
amine or methanol followed by deprotection in acidic
conditions. Compound 1j was prepared by reaction of phthalic
anhydride in acetic acid with Boc-protected ethylenediamine
followed by deprotection in the presence of trifluoroacetic acid.
The preparation of 1k started with the substitution of 1,8-
naphtholactam with 1,2-dibromoethane in basic conditions.
Then, the bromine of the intermediate was converted to
primary amine by Gabriel synthesis to afford 1m.
the N-alkylamine chain make important contributions, and
perhaps, the second naphthalimide group of M-31850 may be
not necessary or suitable for binding the hydrophobic aglycon
pocket outside the active site.
With the extension of chain length between the terminal N
atom and naphthalimide group, the inhibitory activities of 1a−d
decrease dramatically. This may indicate that the terminal
amine of 1a should be in an appropriate distance away from the
naphthalimide group so as to form a hydrogen bond with a key
residue in charge of catalysis. The change of terminal amine to
alcohol (1e) also affects the activity tremendously. However,
the change of the primary amine to tertiary amine as 1f
maintains the activity. Thus, we deduce that it may be a good
strategy to modify and optimize this scaffold from the terminal
amine.
To our surprise, the single naphthalimide moiety with the N-
alkylamine chain, 1a, is a bit more potent than the symmetrical
dyad M-31850. This suggests that the naphthalimide group and
B
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ACS Medicinal Chemistry Letters
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The affinity difference between 1g−k and 1a suggests that
the naphthalimide group plays an irreplaceable role for
inhibitory activity. Any modification to the naphthalimide
group would lead to a decrease or loss in activity, further
confirming its importance.
Computation-based molecular modeling of β chain of hHex
in complex with 1a explains the above observations (Figure 2).
other interactions in our case, different methoxybenzyl groups
with different linkers were then introduced to obtain
compounds 3a−m, 6a−c, and 7a−c, which were also evaluated
under the same condition as 1a−m.
Compounds 3a−m were first prepared by corresponding
phenols or phenthiols reacting with 1,2-dibromoethane or 1,3-
dibromopropane in a solution of sodium hydroxide and
tetrabutylammonium bromide in ethanol or a solution of
sodium hydroxide in water (Scheme 2). Then 1a was refluxed
a
Scheme 2. Synthesis of Compounds 3a−m, 6a−c, and 7a−c
a
Reagents and conditions: (a) NaOH, H2O, 90 °C, 48 h, 68−73% or
NaOH, TBAB, EtOH, reflux, 24 h, 32−61%; (b) 1a, K2CO3, MeCN,
reflux, 12 h, 44−72%; (c) HO(CH2)2NH2, K2CO3, CuI, L-proline,
DMSO, 60 °C, 12 h, 78−90%; (d) TBAB, PPh3, DDQ, DCM, rt, 30
min, 44−82%; (e) 1a, K2CO3, MeCN, reflux, 12 h, 55−70%; (f) 1a,
MeOH, rt; (g) NaBH(CH3COO)3, AcOH, DCM, rt, 12 h, 50−65%
(for steps f and g). X, n, and R are as defined in Table 2.
Figure 2. Computational molecular model of 1a against β chain of
hHex (PDB code, 3LMY) in the active site as shown in cartoon
(hHexB) and stick (1a). Key residues around the active site are shown
as lines including carbon (yellow), nitrogen (blue), and oxygen (red)
atoms. The carbon atoms of 1a are colored in green. The hydrogen
bonds are labeled as a black dash. The unit of hydrogen bonds is Å.
The molecular model was created using the software PyMOL.
with the bromide 2a−m and potassium carbonate in
acetonitrile for 12 h to afford compounds 3a−m. Compounds
6a−c were synthesized by following steps. Compounds 4a−c
were obtained by CuI-catalyzed Ullmann-type coupling
reactions of bromobenzene and ethanolamine.22 These alcohols
were converted into the corresponding bromides 5a−c using a
system of Ph3P, DDQ, and TBAB in high yield. Compounds
5a−c were substituted and refluxed with 1a and potassium
carbonate in acetonitrile, giving the compounds 6a−c.
Compound 1a was easily transformed into 7a−c by performing
a Schiff condensation in methanol in the presence of equivalent
different benzaldehydes following the reducing reaction with
sodium triacetoxyborohydride.
The introduction of anisole groups results in an increase in
the inhibitory activity when comparing these dyads 3a−e, 3h,
and 7a with 1a, suggesting the importance of the methox-
yphenoxy group attached to the terminal nitrogen of N-
alkylamine naphthalimide by an appropriate linker. In contrast,
the absence of methoxyl group dramatically decreases the
inhibitory activities of 3i−j and 3l−m. By the activity
comparison of 3a−h, 6a−c, and 7a−c, it can be drawn that
the molecules with a para-methoxyl group of the benzene ring
are more potent. This may suggest that the methoxyl group and
the NH of the alkylamine linker must be spaced appropriately
in order to effectively bind a corresponding residue. The
connecting type of the methoxyphenoxy group and the
alkylamine linker also affects the activity. The dyads 3a−d
The naphthalimide group binds the hydrophobic pocket
composed of residues, W405, W424, and W489, and interacts
with the indole ring of the W489 by a π−π stacking force, and
the hydrogen atom of terminal amine interacts through a
hydrogen bond with the catalytic residue E355, which is for
protonation of the glycosidic oxygen atom during catalysis.21
Computational studies also support that, if there is increase in
the length of the alkyl chain, the NH group would be within a
more appropriate distance to interact with the D452 instead of
the catalytic E355 (Figure S2, Supporting Information). The
hydrophilic groups at 4-position of naphthalimide leads to a
restriction for the interaction between the compound and the
active site.
So compound 1a was selected as an excellent scaffold for
developing noncarbohydrate-based inhibitors. To further
improve the activity, we proposed to attach other groups to
the terminal nitrogen of naphthalimide 1a, which afford
unsymmetrical dyads. A series of planar conjugate structures
was first introduced to acquire other contact with the
hydrophobic aglycon pockets near the active site. After failure
trials, we found the anisole analogues, instead of largely
hydrophobic structures, could enhance binding affinity. On the
basis of studies by Lin et al.,14 the p-methoxybenzyl substituents
of GlcNAc-type iminocyclitiol (Figure 1) could be surrounded
by a hydrophobic pocket, and thus, the potency and selectivity
of inhibitors are improved. To acquire similar electrostatic or
C
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ACS Medicinal Chemistry Letters
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Molecular docking has also been performed to analyze the
binding model of these unsymmetrical dyads. However, the
results can not give perfect explanations to the inhibitory
activity difference (Figure S3, Supporting Information).
In summary, we have described the molecular design,
synthesis, and inhibitory activities against human β-N-acetyl-
D-hexosaminidase of a series of unsymmetrical dyads containing
naphthalimide and methoxyphenyl moiety with an alkylamine
spacer linkage. The enzymatic activity testing indicated that the
potent unsymmetrical dyads exhibit better competitive
inhibition activity than the HTS hit symmetrical dyad M-
31850 when the human β-N-acetyl-D-hexosaminidase is the
target. Especially, dyads 3h and 7a have the highest activities
with Ki values of 0.69 and 0.63 μM, respectively. Furthermore,
these structures are much different from the reported
carbohydrate-based inhibitors and supply a novel and efficient
skeleton, which is easier and more straightforward to be
synthesized and modified. The understanding of the binding
model of these inhibitor potentors against the human β-N-
acetyl-D-hexosaminidase may be advantageous for further
structure optimization and thereby provides some insight into
the rational design of new therapeutic agents for Hex-related
diseases.
Table 2. Structures and Evaluation Datas of Dyads 3a−m,
6a−c and 7a−c against Human β-N-Acetyl-D-
hexosaminidases A/B
a
compd
n
X
R
Ki (μM)
3a
3b
3c
3d
3e
3f
2
2
2
2
3
3
3
2
2
2
2
2
2
O
O
O
S
p-OMe
1.04
0.93
1.35
1.13
1.23
4.39
Nd
m-OMe
o-OMe
p-OMe
O
O
S
p-OMe
m-OMe
p-OMe
3g
3h
3i
O
O
O
O
O
O
m,p-(OMe)2
[1,3]dioxole
p-OCF3
p-COOMe
p-Cl
0.69
3.00
Nd
3j
3k
3l
4.20
Nd
3m
6a
6b
6c
7a
7b
7c
p-NO2
Nd
p-OMe
1.08
Nd
m-OMe
o, p-(OMe)2
p-OMe
4.63
0.63
6.22
4.99
0.27, 0.29
m-OMe
m, p-(OMe)2
b
NGT
a
ASSOCIATED CONTENT
Measured with 4MU-β-GlcNAc as substrate and calculated by linear
■
regression of data in Dixon plots. Values with SD were shown in Table
S1, Supporting Information. Nd: not determined (less than 50%
inhibition at 50 μM). Ki against hHex A and hHex B, respectively;
S
* Supporting Information
Experimental procedures for the synthesis, characterization, and
enzymatic assay protocols of compounds 1a−k, 3a−m, 6a−c,
and 7a−c as well as molecular docking of selected compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
b
Values from ref 16.
containing the oxygen or sulfur atom between benzene ring and
alkane chain are more potent than dyads 6a−c containing a
nitrogen atom.
AUTHOR INFORMATION
Besides determining the Ki value, Dixon plots (Figure 3) also
revealed that these active dyads are competitive inhibitors
■
Corresponding Author
*(Q.Y.) Fax: (86)-411-84707245. E-mail: qingyang@dlut.edu.
cn. (X.Q.) Fax: (86)-21-64252603. E-mail: xhqian@ecust.edu.
cn.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
This work is supported by National Natural Science
Foundation of China (21236002, 31070715, and 31101671),
National Special Fund for State Key Laboratory of Bioreactor
Engineering (ECUST) (Shanghai, China), National Key
Project for Basic Research (2010CB126100), the National
High Technology Research and Development Program of
China (2011AA10A204), the National Key Technology R&D
Program (2011BAE06B05), and the Fundamental Research
Funds for the Central Universities (DUT11ZD113 and
DUT11RC(3)73).
Figure 3. Representative Dixon plots (A) for inhibition of human β-N-
acetyl-D-hexosaminidases A/B by 7a. The trendlines represent two
substrate concentrations ( , 0.1 mM; , 0.2 mM). The inset B is the
partial enlarged view of A.
■
●
Notes
The authors declare no competing financial interest.
against human β-N-acetyl-D-hexosaminidase. The trendlines
drawn for each concentration of substrate intersect in a single
point above the x-axis, indicating competitive inhibition. These
results supported that these inhibitors are able to compete with
the substrate for binding directly to the catalytic pocket and
interact with critical residues locating in the active site.
ACKNOWLEDGMENTS
■
The authors thank the technical guidance for molecular
docking provided by Dr. Yongliang Yang at Dalian University
of Technology.
D
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(17) Maegawa, G. H. B.; Tropak, M.; Buttner, J.; Stockley, T.; Kok,
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ABBREVIATIONS
■
Hex, β-N-acetyl-D-hexosaminidase; hHex, human β-N-acetyl-D-
hexosaminidase; HTS, high-throughput screening; 4MU-β-
GlcNAc, 4-methylumbelliferyl N-acetyl-β-D-glucosaminide;
PDB, protein data bank
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