Design, synthesis and evaluation of 2,2-dimethyl-1,3-dioxolane derivatives as human rhinovirus 3C protease inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.07.049

The human rhinovirus (HRV) is the most significant cause of the common cold all over the world. The maturation and replication of this virus entirely depend on the activity of a virus-encoded 3C protease. Due to the high conservation among different serot

Full text of DOI:10.1016/j.bmcl.2017.07.049

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and evaluation of 2,2-dimethyl-1,3-dioxolane
derivatives as human rhinovirus 3C protease inhibitors
Qiyan Zhang a,b, Ruiyuan Cao , An Liu , Shihai Lei , Yuexiang Li , Jingjing Yang , Song Li
b
b
b
b
b
a,b,
, Junhai Xiao b,
⇑ ⇑
a
School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, Liaoning, China
Laboratory of Computer-Aided Drug Design and Discovery, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
The human rhinovirus (HRV) is the most significant cause of the common cold all over the world. The
maturation and replication of this virus entirely depend on the activity of a virus-encoded 3C protease.
Due to the high conservation among different serotypes and the minimal homology existing between
Received 13 May 2017
Revised 16 July 2017
Accepted 19 July 2017
Available online xxxx
3
C protease and known mammalian enzymes, 3C protease has been regarded as an attractive target
4
for the treatment of HRV infections. In this study, we identified a novel (4R,5R)-N -(2-((3-methoxyphe-
nyl)amino)ethyl)-2,2-dimethyl-N -(naphthalen-2-yl)-1,3-dioxolane-4,5-dicarboxamide (7a) to be
5
a
Keywords:
HRV
HRV 3C protease inhibitor via virtual screening. Further research has been focused on the design, synthe-
sis and in vitro biological evaluation of 7a derivatives. The studies revealed that compound 7d has an IC50
value of 2.50 ± 0.7 mM against HRV 3C protease, and it thus could serve as a promising compound for the
development of novel anti-rhinoviral medicines.
3
C protease inhibitors
Virtual screening
,2-Dimethyl-1, 3-dioxolane derivatives
2
Ó 2017 Elsevier Ltd. All rights reserved.
The human rhinovirus (HRV) is a member of the picornavirus
strate. Moreover, minimal homology exists between 3C protease
and known mammalian enzymes, making it an attractive target
for the development of anti-rhinoviral drugs.
1
8
family and a frequent cause of the common cold. Similar to other
picornaviruses, HRV has a small single-stranded, positive-sense
2
RNA genome approximately 7200 nucleotides in length, which
To date, the 3C protease inhibitors reported can be broadly
divided into two types: peptidic and non-peptidic inhibitors
can be translated into a 220 kDa polyprotein precursor in host
cells. This polyprotein precursor is proteolytically processed by
two virus-encoded proteases, 2A and 3C proteases, to produce
structural and functional viral components.3 Because the genera-
tion of structural and functional viral protease is critical for viral
replication, 3C protease has been regarded as an important target
for the treatment of HRV infections.
9
,10
11
(Fig. 1). Inhibitors such as Rupintrivir,
aldehydes,
a
-ketoamides, peptide
1
2,13
14
2-pyridone-containing peptidomimetics belong
to the peptidic inhibitor family, and typical non-peptidic inhibitors
1
5
16
17,18
are heteroaromatic esters, quinolone, benzamides,
isati-
4
19
nes and 45240. Although a large quantity of inhibitors have
been discovered, none of them have been approved by the FDA
for the treatment of rhinovirus infections.
1
5,20
Although 3C protease is a cysteine proteinase, structural analy-
sis has revealed that it belongs to the family of chymotrypsin-like
Therefore, the devel-
opment of effective antiviral therapies against HRV is important.
Recently, many studies have focused on developing non-peptidic
HRV 3C protease inhibitors owing to their smaller size and better
oral bioavailability.
Herein, we report the design and synthesis of a variety of
non-peptidic HRV 3C protease inhibitors bearing the scaffold of
4
serine proteases . However, unlike normal chymotrypsin-like ser-
5
ine proteinases, the structure of its catalytic triad is His-Glu-Cys.
Despite the large number of HRV serotypes (>100), 3C protease
2,6
has a highly conservative cleavage site
located between glu-
0
1 1
tamine (P ) and glycine (P ) residues in the viral polyprotein sub-
7
2
,2-dimethyl-1,3-dioxolane, which was generated from virtual
screening. Their potencies to inhibit HRV 3C protease in vitro were
evaluated using High Performance Liquid Chromatograph (HPLC)
assay. We also explored the structure–activity relationships of
2,2-dimethyl-1,3-dioxolane derivatives with respect to inhibitory
activity against HRV 3C protease.
⇑
Corresponding authors at: School of Pharmaceutical Engineering, Shenyang
Pharmaceutical University, Shenyang 110016, Liaoning, China (S. Li); Laboratory of
Computer-Aided Drug Design and Discovery, Beijing Institute of Pharmacology and
Toxicology, Beijing 100850, China (J. Xiao).
E-mail addresses: qiyanzhang1113@163.com (Q. Zhang), 21cc@163.com (R. Cao),
anran1222@163.com (A. Liu), leishihai@126.com (S. Lei), ly19866528@126.com
Y. Li), yangjingjing@163.com (J. Yang), lis@bmi.ac.cn (S. Li), xiaojunhai@139.com
J. Xiao).
Based on the co-crystal structure of HRV 3C protease with
AG7088 [Protein Data Bank (PDB) ID: 1CQQ, 1.85 Å], we screened
(
(
http://dx.doi.org/10.1016/j.bmcl.2017.07.049
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0
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Q. Zhang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Fig. 1. HRV 3C protease inhibitors.
The HRV 3C protease inhibitory evaluation was performed
using a High Performance Liquid Chromatograph (HPLC) assay.
1
5
Compound 14 was selected as a positive control, with an IC50
value of 0.4 ± 0.06 mM in the biological evaluation system. The
structure of compound 14 is shown in Fig. 1. The HRV 3C protease
inhibitory evaluation is given in Supplementary information.
The resulting 20 compounds were tested at a concentration of
1
00 mM. Among them, the most active compound 7a (80.56% inhi-
bition at 100 mM), which is novel to inhibit HRV 3C protease, was
selected as a starting structure in this work for further optimiza-
tion. The docking models for 7a in the active sites are shown in
Fig. 3.
The docking results suggest that there were several key interac-
0
tions between 7a and HRV 3C protease in the S1 , S1 and S2 pockets
(
Fig. 3). Six H-bonds were observed in the binding mode, among
which two key H-bonds were formed between the oxygen of the
,3-dioxolane ring and the catalytic His40 (2.1 Å) and Gly145
2.1 Å) respectively. Additionally, one H-bond was formed by the
hydrogen of the amide group on the C atom with Val162 (2.7 Å)
1
(
Fig. 2. The process of virtual screening from our in-house database using DOCK 6.0.
4
in the S2 pocket, and another critical H-bond was formed by the
NH of the diamine group with Gly164 (2.3 Å) in the S1 pocket.
Moreover, the 2-naphthalene ring and the residues in the S2 pocket
can interact through hydrophobic interaction.
our in-house database using DOCK 6.0, which included nearly
1
0,000 small organic compounds. Following four rounds of rigid
and flexible virtual screens, the 200 molecules with the best shape
complementarity scores and force field scores were selected from
the DOCK 6.0 screening. The resulting 20 compounds, which occu-
The above observations indicated that increased potency might
be achieved by the introduction of proper substituents at C /C
4 5
position of the (R,R)-2,2-dimethyl-1,3-dioxolane, which was con-
siders as a core structure. Considering Gly164 in the S1 pocket
could form H-bond with the NH in diamine substituent, a series
of piperazine substituents were designed. Due to the S2 pocket
could accommodate large hydrophobic ligands, a series of aromatic
0
pied the S1, S1 and S2 pockets of HRV 3C protease and formed sev-
eral H-bonds with critical His40, Glu71, Gly145 and Cys147, were
then chosen for the biological evaluation. The process of the virtual
screening are shown in Fig. 2.
Fig. 3. (a) Structures of compound 7a, (b) Structure of compound 7a docked in the active site with important H-bonds shown by red lines.
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Q. Zhang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
Table 1
Inhibition and IC50 values of compounds 7a–7g tested against HRV 3C protease.
%
Compd
R1
R2
% inhibition at 100 mM
% inhibition at 10 mM
IC50 (mM)
7
a
80.56
43.99
>10
7
b
99.91
62.24
8.28 ± 1.94
7
7
c
100.00
100.00
68.98
86.05
8.57 ± 1.09
2.50 ± 0.7
d
7
7
e
f
97.80
85.51
64.41
18.78
10.62 ± 2.63
>10
7
g
78.74
20.22
>10
substituents were designed to improve potency. To explore the
relationship between compounds configuration and inhibitory
activities, a series of (S,S)-2,2-dimethyl-1,3-dioxolane derivatives
were designed.
compounds 5a or 11a only contains a single peak. These show that
the target compounds did not undergo configuration changes. The
HPLC spectrum of compound 5a and 11a are given in Supplemen-
tary information.
The structures of designed compounds are shown in Tables 1
and 2. They were synthesized following the route shown in
Schemes 1 and 2. As shown in Scheme 1, the (R,R)-2,2-dimethyl-
All synthesized target compounds 7a–7g and 13b–13g were
tested for HRV 3C protease inhibitory activity, with the data pre-
sented in Tables 1 and 2. Compared with compound 7a (inhibitory
activity <50% at 10 mM), compounds 7b (IC50 = 8.28 ± 1.94 mM)
showed significantly improved in potencies. Docking result of 7b
(Fig. 4a) suggested the benzothiazole ring could position well in
the S2 pocket and form hydrophobic interaction. Meanwhile, the
1
,3-dioxolane derivatives 7a–7g were prepared in five steps. Start-
ing from commercially available -(+)diethyl tartrate (1), 2,2-
dimethoxypropane (2) and p-toluenesulfonic acid, (R,R)-2,2-
D
21
dimethyl-1,3-dioxolane 3 was yielded. Ehrlich reagent was cho-
sen to monitor the reaction instead of TLC analysis due to the poor
UV absorption of the starting materials. After the hydrolysis of the
diethyl compound 3 with NaOH, the resulting monomethyl ester 4
was then treated with 1H-benzo[d][1,2,3]triazol-1-ol(HOBt), dicy-
clohexylcarbodiimide (DCC) and various amines to give amide 5,
which was converted to formic acid 6 by treatment with NaOH.
The condensation of 6 was performed with various amines in the
presence of HOBt and DCC to give the desired (R,R)-2,2-dimethyl-
thiazole ring was observed to form
p-stacking interaction with
His40. Therefore, compounds 7b–7g, which have benzothiazo-
lamine substitutions, were designed, synthesized and evaluated.
The inhibitory activities of compounds 7b–7e (IC50s at mM level)
revealed that a aromatic ring with various substituents seemed
to contribute less to the potency. To explore the binding modes
2
of the R substitutions, one of the most active inhibitors (7d) was
selected as a representative and docked to the crystal structure
of HRV 3C protease (PDB: 1CQQ). The docking results suggest that
there were several key interactions between 7d and HRV 3C pro-
1
,3-dioxolane derivatives 7a–7g. As shown in Scheme 2, the (S,
S)-2,2-dimethyl-1,3-dioxolane derivatives 13b–13g were prepared
0
in the same methods. All the compounds and intermediates were
tease in the S1, S1 and S2 pockets. Five H-bonds were observed
1
13
confirmed by H, C NMR and Mass spectral data. The characteri-
zation data of all the compounds are given in Supplementary
information.
in the binding mode, as shown in Fig. 4b, among which two key
H-bonds were formed between the oxygen of the 1,3-dioxolane
ring and the catalytic His40 (2.3 Å) and Gly145 (2.9 Å), respec-
tively. Additionally, one H-bond was formed by the NH of the
In order to determine the configuration of the target com-
pounds, amide 11a were synthesized according to Scheme 2. The
compounds 5a and 11a could be completely separated by
reverse-phase HPLC (Agilent, Chiralpak (4.6 ꢀ 250 mm, 5 mm) with
amide group on the C
and another critical H-bond was formed by the O of the amide
group on the C with Gly164 (2.7 Å) in the S1 pocket. Moreover,
the thiazole ring was observed to form -stacking interaction with
His40, which might be resulted in the dramatic inhibitory activity
4
atom with Val162 (1.8 Å) in the S2 pocket,
5
a isocratic of 50 mM KH
2
PO
4
(40%, v/v) and methanol (60%, v/v) for
p
8
0 min. The peaks were monitored at 220 nm. HPLC spectrum of
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Q. Zhang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Table 2
%
Inhibition and IC50 values of compounds 13b–13g tested against HRV 3C protease.
Compd
R1
R2
% inhibition at 100 mM
% inhibition at 10 mM
IC50 (mM)
1
3b
85.22
43.61
>10
1
1
3c
67.80
84.20
17.51
48.11
>10
>10
3d
1
1
3e
3f
82.47
56.36
10.77
20.75
>10
>10
1
3g
85.35
34.30
>10
3 3 2
Scheme 1. Reagents and conditions: a) CHCl , p-toluenesulfonic acid, reflux 17 h, 42.8%, b) KOH, CH OH, rt, 24 h, 95.0%, c) HOBt, DCC, THF, rt, 12 h, 62.5–95%, d) H O/dioxane
(
1:1), NaOH dropped, rt, 2 h, 84.4–95.0%, e) HOBt, DCC, THF, rt, 2 h, 8.6–66.7%.
Scheme 2. Reagents and conditions: a) CHCl
3 3
, p-toluenesulfonic acid, reflux 17 h, 42.8%, b) KOH, CH OH, rt, 24 h, 95.0%, c) HOBt, DCC, THF, rt, 12 h, 70.0–95.5%, d)
H
2
O/dioxane (1:1), NaOH dropped, rt, 2 h, 91.2%, e) HOBt, DCC, THF, rt, 2 h, 8.6–66.7%.
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Q. Zhang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
5
Fig. 4. (a) Structure of compound 7b docked in the active site with important H-bonds shown by red lines. (b) Structure of compound 7d docked in the active site with
important H-bonds shown by red lines. (c) Structure of compound 7f docked in the active site with important H-bonds shown by red lines.
1
changes depending on the substituents R . Just as expected, the
benzothiazole ring and the residues in the S2 pocket can interact
through hydrophobic interaction.
National Science Foundation for Distinguished Young Scholars of
China to Dr. Cao (81302702) and State Key Laboratory of
Toxicology and Medical Countermeasures (Academy of Military
Medical Sciences).
Compared with compound 7b–7e (IC50s at mM level), com-
pounds 7f and 7g (inhibitory activity <50% at 10 mM) exhibited
poor inhibitory activities. To explain the poor activities, compound
A. Supplementary data
(
7f) was selected as a representative and docked to the crystal
structure of HRV 3C protease (PDB: 1CQQ). Docking result of com-
pound 7f (Fig. 4c) suggested the 1-benzylpiperazine substitution
could not position well in the S1 pocket and fewer H-bonds were
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.07.
0
49.
2
observed. Thus, increasing the number of C atoms at the R sub-
stituents will reduce the inhibitory activities.
References
Compared with compound 7b–7g, compounds 13b–13g (inhibi-
tory activity <50% at 10 mM) showed lower inhibitory activities.
This result indicates that the activity varies significantly depending
on the compound configuration. Moreover, (R,R)-2,2-dimethyl-1,3-
dioxolane derivatives exhibited better inhibitory activities.
In conclusion, a computer-based database screening strategy
was employed to identify a novel series of 2,2-dimethyl-1,3-diox-
olane derivatives as ligands for the HRV 3C protease pocket. More-
over, thirteen 2,2-dimethyl-1,3-dioxolane derivatives were
designed, synthesized and evaluated as HRV 3C protease inhibitors
in an enzymatic assay. Four compounds (7b, 7c, 7d,7e) were found
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This work was supported by the National Science and
Technology Major Project of China (2014zx09304-001), the
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