Synthesis, structure-activity relationship and in vitro biological evaluation of N-arylethyl isoquinoline derivatives as Coxsackievirus B3 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.08.002

Currently, there is no approved antiviral drug for the infection caused by enteroviruses. A series of novel N-arylethyl isoquinoline derivatives defined with substituents on the ring A and C were designed, synthesized and evaluated in vitro for their activities against Coxsackievirus B3 (CVB3). The primary structure-activity relationship revealed that substituents on the ring A were not beneficial for the activity. Among these analogs synthesized, compound 7f bearing a methylenedioxy at the R⁴ and R⁵ positions afforded an anti-CVB3 activity and a reasonable selectivity index (SI = 26.8); furthermore, 7f exhibited a moderate activity against enterovirus 71 (EV71) with SI value of 9.0. Thus it has been selected as an anti-enteroviral lead compound for further investigation.

Full text of DOI:10.1016/j.bmcl.2011.08.002

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5787–5790
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, structure–activity relationship and in vitro biological evaluation
of N-arylethyl isoquinoline derivatives as Coxsackievirus B3 inhibitors
Yan-Xiang Wang , Yu-Huan Li , Ying-Hong Li, Rong-Mei Gao, Hui-Qiang Wang, Yan-Xin Liu, Li-Mei Gao,
⇑
⇑
Qiao-Ni Lu, Jian-Dong Jiang , Dan-Qing Song
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Currently, there is no approved antiviral drug for the infection caused by enteroviruses. A series of novel
N-arylethyl isoquinoline derivatives defined with substituents on the ring A and C were designed, synthe-
sized and evaluated in vitro for their activities against Coxsackievirus B3 (CVB3). The primary structure–
activity relationship revealed that substituents on the ring A were not beneficial for the activity. Among
these analogs synthesized, compound 7f bearing a methylenedioxy at the R4 and R5 positions afforded an
anti-CVB3 activity and a reasonable selectivity index (SI = 26.8); furthermore, 7f exhibited a moderate
activity against enterovirus 71 (EV71) with SI value of 9.0. Thus it has been selected as an anti-enteroviral
lead compound for further investigation.
Received 7 May 2011
Revised 27 July 2011
Accepted 1 August 2011
Available online 8 August 2011
Keywords:
N-Arylethyl isoquinoline
Coxsackievirus B3
Structure–activity relationship
Enterovirus 71
Ó 2011 Elsevier Ltd. All rights reserved.
Enteroviruses
Enteroviruses are a genus of single-stranded (+) RNA viruses
associated with several human and mammalian diseases. Enterovi-
ruses infect human individuals worldwide. As children are consid-
ered to exhibit a relatively weak immune system, enteroviral
infection in neonates could be lethal or life threatening, with high
risk for morbidity and mortality. Enteroviruses are classified into
polioviruses, coxsackievirus A and B, echoviruses and enterovirus-
es 68–71 (EV68–71).1,2 Coxsackievirus B3 (CVB3) is an important
human pathogen inducing acute and chronic viral myocarditis in
children and young adults. Moreover, CVB3 is associated with the
development of aseptic meningitis, hepatitis, and meningoenceph-
alitis.3 Enterovirus 71 (EV71) might relate to severe central ner-
vous system diseases.4–8 An epidemic outbreak of EV71 infection
in Taiwan in 1998 caused many severe cases and 78 deaths.9 In
the spring of 2008, a major hand-foot-and-mouth disease (HFMD)
outbreak caused by EV71 in China resulted in a high aggregation of
fatal cases.10
urgent need for developing antiviral agents for enteroviral infec-
tion, including those against CVB3 and EV71.
In search for small molecule anti-CVB3 agents, the compound
library established in our laboratory was screened against CVB3
using viral cytopathogenic effect (CPE) assay.14 In the screening test,
ribavirin (RBV, Fig. 1) was selected as the positive control. Although
antiviral effect of ribarivin is not considered potent, the agent is
relatively effective against CVB312 with respect to other antiviral
agents. As the reference drug is currently used for CVB3 in hospitals
and has a broad spectrum antiviral effect in cell culture we select this
agent as a control in the cell-based test. Through the screening hit 1
(Fig. 1) was identified, because it showed inhibitory activity with
IC50 value of 209 lM, stronger than that of RBV (IC50 = 910 lM).
The anti-CVB3 activity and unique chemical structure of 1 provoked
O
+ Cl-
A
N
Numerous compounds have been reported to be selective inhib-
itors for enteroviruses, some of which have entered into clinical tri-
als.11–13 Unfortunately, there is no approved antiviral drug for the
treatment of acute enteroviral infections. Therefore, there is an
NH2
N
N
N
B
HO
O
OCH3
OCH3
C
OH
OH
⇑
Corresponding authors. Tel.: +86 10 6318 8423; fax: +86 10 6301 7302 (J.-D.J.);
tel./fax: +86 10 6316 5268 (D.-Q.S.).
1
E-mail addresses: jiang.jdong@163.com (J.-D. Jiang), songdanqingsdq@hotmail.
com (D.-Q. Song).
RBV
These authors made equal contributions to the work.
Figure 1. Chemical structures of compound 1 and RBV.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.002

5788
Y.-X. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5787–5790
HCl
CHO
R
R
N
HN
NH2
b
a
+
R
R'
85 93%
>95%
R'
R'
5
2
4
3
R1
R2
R3
+ Cl-
N
+ COOH-
R
N
c
d / e
78 92%
43 65%
R4
R5
R'
R7
7a
u
6
R6
R1, R3 = −OCH3, −CH3, −Cl, −F, −H, etc;
R2 = −Cl, −H, etc;
R4, R5, R6, R7 = −OCH3, −OH, −OCH2O, etc.
Scheme 1. Synthesis of the aimed compounds (7a–u). Reagents and conditions: (a) 100 °C, 5 h; (b) NaBH4, methanol, 4 h; (c) CHO–CHO, anhydrous formic acid, CuSO4,
concentrated HCl, 100 °C, 6 h; (d) methanol/H2O, CaO, 1.5 h; (e) concentrated HCl.
Table 1
our interest to explore the structure–activity relationship (SAR) of
Structures of the synthetic N-arylethyl isoquinoline derivatives (7a–u)
this group of compounds, aiming for discovery of anti-CVB3 agents.
Working through this strategy, a series of novel N-arylethyl isoquin-
oline derivatives were designed, synthesized and evaluated for their
activities against CVB3 in vitro with 1 as the lead in the present
R1
R2
+ Cl-
study.
After selection of the hit compound, a novel and convenient
A
N
B
R3
synthetic route was developed for the desired N-arylethyl isoquin-
oline derivatives, which allows an extensive SAR analysis. Twenty-
one analogs (7a–u) were de novo synthesized as described in
Scheme 1, which demonstrates a four-step process. The method
used commercially available derivatives of phenylethylamine (2)
and benzaldehyde (3) as the starting materials, as described in
our previous reports.15–18 Twenty-one analogs were selectively
prepared through varying substituents on the aromatic ring A
and C. The activating group (–OCH3, –OH) at the R5 position is of
assistance to the cyclization of the ring B, and the hydrogen atom
or halogen such as –Cl at position R2 on the ring A might be bene-
ficial for the formation of the product 6 with yields of 43–65%. In
the other hand, berberine analogs were obtained while an activat-
ing group exists at the R2 position.15–18 Then, the target com-
pounds were purified with silica gel column chromatography
using methanol/chloroform (1:20) as the eluent.
All of the study compounds were evaluated for their antiviral
activity against CVB3 in African green monkey kidney cells (Vero
cells), which were infected with CVB3 and measured with CPE assay.
Anti-CVB3 activity was expressed with IC50 value and cytotoxicity
with TC50 value. As animportanttherapeuticindication, theselectiv-
ity index (SI) was calculated as the ratio of TC50 to IC50. Antiviral
activity of each compound was evaluated by combining its IC50 with
SI. Structures of the 21 N-arylethylisoquinoline derivatives and their
anti-CVB3 effect are shown in Tables 1 and 2, respectively.
R4
R5
C
R7
R6
Compd
R1
R2
R3
R4
R5
R6
R7
1
H
H
H
H
H
CH3
H
H
H
H
F
H
CH3
CH3
F
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
Cl
H
H
H
H
H
H
F
OCH3
H
OCH3
H
H
F
H
F
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OH
OH
OH
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
7a
7b
7c
7d
7e
7f
7g
7h
7i
OCH2O
OCH2O
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
OCH3
OCH3
OCH3
OCH3
OCH3
OH
OH
H
H
H
H
H
H
H
H
H
7j
7k
7l
7m
7n
7o
7p
7q
7r
7s
7t
OH
H
H
H
F
H
H
H
OH
H
H
CH3
OCH3
H
OH
H
H
OH
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
The SAR study was mainly concentrated on the variations of
substituents on the aromatic ring A and C. First, substituting –H
7u
H
OCH3

Y.-X. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5787–5790
5789
Table 2
Anti-CVB3 activity and cytotoxicity for target compounds in Vero cells
Table 3
Antiviral activity against EV71 and cytotoxocity of the key compounds
a
b
a
b
Compd
TC50
(
lM)
IC50
(lM)
SIc
Compd
TC50
(lM)
IC50
(
lM)
SIc
1
584 0.00
1383 59.4
49.5 4.55
352 9.04
464 19.8
584 0.00
815 37.4
581 0.00
1012 42.2
665 30.0
206 20.5
131 6.23
970 42.2
234 12.5
333 24.7
389 24.7
200 7.34
1098 42.4
772 30.0
1057 42.2
2782 0.00
46 2.20
209 13.3
>320
>11.4
91.1 5.74
>107
>337
30.4 0.00
>335
>337
>320
>53.3
>33.9
>323
11.2 0.00
>111
25.0 2.07
>38.6
>336
371 0.00
>352
>927
>10.6
2.8
—
—
3.9
—
—
26.8
—
—
—
—
—
—
2.1
—
15.6
—
—
2.1
—
—
—
7f
7g
7i
7o
RBV
820 28.7
420 25.9
864 33.0
534 16.5
8190 0.00
91.1 5.02
45.8 3.13
107 5.35
152 6.64
2119 44.1
9.0
9.2
8.1
3.5
3.9
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
a
Cytotoxic concentration required to inhibit Vero cell growth by 50%. Vero cells
(2.5 Â 104/well) were plated into a 96-well plate. A total of 24 h later, the mono-
layer cells were incubated in the present of various concentrations of test com-
pounds. After 48 h of culture at 37 °C, the cells were monitored by CPE. TC50 value
was calculated by Reed and Muench analyzes. Each experiment was repeated three
times.
b
Concentration required to inhibit CVB3 replication by 50%. Vero cells were
infected with 100 median tissue culture infective dose (100TCID50) Cox B3. Then,
confluent Vero cells were cultured in the maintenance medium (MEM plus 2% FBS)
and incubated, respectively with or without different concentration of test com-
pounds. Viral CPE was observed when the viral control reached 4+ and IC50 was
determined by the Reed and Muench analyzes. Each experiment was repeated three
times.
c
Selectivity index value equaled to TC50/IC50
.
7s
7t
7u
RBV
pounds 7f and 7o showed potential activity against either CVB3 or
EV71, suggesting a broad-spectrum activity against enteroviruses
by the two compounds.
8190 0.00
910 46.5
9.0
a
Cytotoxic concentration required to inhibit Vero cell growth by 50%. Vero cells
(2.5 Â 104/well) were plated into a 96-well plate. A total of 24 h later, the mono-
layer cells were incubated in the present of various concentrations of test com-
pounds. After 48 h of culture at 37 °C, the cells were monitored by CPE. TC50 value
was calculated by Reed and Muench analyzes. Each experiment was repeated three
times.
In conclusion, 21 novel N-arylethyl isoquinoline derivatives de-
fined with modifications on the ring A or C were designed, synthe-
sized and evaluated for anti-CVB3 and anti-EV71 activities. The
primary SAR for anti-CVB3 indicated that substituents on the ring
A might reduce the antiviral activity. Among these analogs, com-
pounds 7f and 7o exhibited reasonable activity against CVB3 with
SI value of 26.8 and 15.6, respectively, stronger than that of RBV. In
addition, compound 7f showed moderate activity against EV71
with SI value of 9.0, higher than that of RBV. Thus, 7f has been se-
lected in our laboratory as a promising anti-enteroviral candidate
for further investigation. The antiviral results and SAR of the N-
arylethyl isoquinoline derivatives are of interest for further chem-
ical modifications.
b
Concentration required to inhibit CVB3 replication by 50%. Vero cells were
infected with 100 median tissue culture infective dose (100TCID50) Cox B3. Then,
confluent Vero cells were cultured in the maintenance medium (MEM plus 2% FBS)
and incubated, respectively with or without different concentration of test com-
pounds. Viral CPE was observed when the viral control reached 4+ and IC50 was
determined by the Reed and Muench analyzes. Each experiment was repeated three
times.
c
Selectivity index value equaled to TC50/IC50
.
at the R3 position with –F or –OCH3 (7a–b) decreased the SI value,
owing to the low activity or high cytotoxicity. Next, compounds
(7c–e) possessing –OH at R4 and –OCH3 at the R5 position were
synthesized and analyzed. Compound 7c with no substituent on
the ring A exhibited a moderate activity with IC50 value of
Acknowledgements
This work was supported by the Research Funds of Peking
Union Medical College for Young Scientists and the National S&T
Major Special Project on Major New Drug Innovation
(2009ZX09301-003).
91 lM, stronger than that of 1. With the replacement by a methyl-
enedioxy at the R4 and R5 positions (7f–g), compound 7f19 with the
absence of substituent on the ring A exhibited a reasonable anti-
CVB3 activity with SI value of 26.8, nearly threefold of that of the
lead 1 (SI = 9.0). It appeared that side groups on the ring A might
be a negative factor for the activity against CVB3.
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much stronger than that of 1. In addition, tri-methoxy was added
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19. 8-(2-Phenylethyl)[1,3]dioxolo[4,5-h]isoquinolin-8-ium chloride (7f): Yield:
52%. Melt point: 276–278 °C (dec); White solid; IR (KBr): 3446, 3021, 2959,
1612, 1501, 1459, 1293, 1265, 1198, 1176, 1017, 915; 1H NMR (DMSO-d6,
ppm): d 9.57 (s, 1H, 8-CH), 8.59 (d, 1H, J = 6.8 Hz, Ph), 8.25 (d, 1H, J = 6.8 Hz,
Ph), 7.69 (s, 1H, Ph), 7.68 (s, 1H, Ph), 7.26–7.20 (m, 5H, Ph), 6.42 (s, 2H, CH2),
4.87 (t, 2H, J = 7.2 Hz, CH2), 3.31 (t, 2H, J = 7.6 Hz, CH2); 13C NMR (DMSO-d6,
ppm): 156.2, 151.2, 145.5, 137.4, 136.3, 134.1, 128.8, 128.6, 127.0, 125.2, 123.7,
104.11, 104.07, 103.0, 60.8, 36.3; HRMS-ESI calcd for C18H16NO2Cl: 278.11810
[MÀCl]+, found 278.11898.
18. Wang, Y. X.; Wang, L.; Xu, Y. N.; Li, Y. H.; Jiang, J. D.; Si, S. Y.; Li, Y. B.; Ren, G.;
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