Novel N-benzenesulfonyl sophocarpinol derivatives as coxsackie B virus inhibitors

Article abstract of DOI:10.1021/ml500525s

Novel N-benzenesulfonyl sophocarpinic acid/ester and sophocarpinol derivatives were synthesized and evaluated for their antienteroviral activities against coxsackievirus type B3 (CVB3) from sophocarpine (1), a natural medicine isolated from Chinese herb. Structure-activity relationship (SAR) analysis revealed that the double bond and its geometrical configuration and position at the C-11 attachment did not greatly affect the potency. Among these derivatives, sophocarpinol 24d exerted the promising activities against not only CVB3 but also CVB1, CVB2, CVB5, and CVB6 with IC₅₀ ranging from 0.62 to 3.63 μM (SI from 46 to 275), indicating a broad-spectrum antienteroviral characteristic. The SAR results provided the powerful information for further strategic optimization and development of a novel scaffold of broad-spectrum antiviral candidates against enteroviruses.

Full text of DOI:10.1021/ml500525s

Letter
pubs.acs.org/acsmedchemlett
Novel N‑Benzenesulfonyl Sophocarpinol Derivatives as Coxsackie B
Virus Inhibitors
Sheng Tang,†,§ Lanying Kong,†,§ Yinghong Li,† Jiandong Jiang,†,‡ Limei Gao,† Xinyue Cheng,† Linlin Ma,†
Xin Zhang,† Yuhuan Li,*,† and Danqing Song*,†
†Institute of Medicinal Biotechnology, Chinese Academy of Medical Science & Peking Union Medical College, Beijing 100050, China
‡State Key Laboratory of Bioactive Substance and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of
Medical Sciences and Peking Union Medical College, Beijing 100050, China
S
* Supporting Information
ABSTRACT: Novel N-benzenesulfonyl sophocarpinic acid/
ester and sophocarpinol derivatives were synthesized and
evaluated for their antienteroviral activities against coxsack-
ievirus type B3 (CVB3) from sophocarpine (1), a natural
medicine isolated from Chinese herb. Structure−activity
relationship (SAR) analysis revealed that the double bond
and its geometrical configuration and position at the C-11
attachment did not greatly affect the potency. Among these
derivatives, sophocarpinol 24d exerted the promising activities
against not only CVB3 but also CVB1, CVB2, CVB5, and CVB6 with IC50 ranging from 0.62 to 3.63 μM (SI from 46 to 275),
indicating a broad-spectrum antienteroviral characteristic. The SAR results provided the powerful information for further
strategic optimization and development of a novel scaffold of broad-spectrum antiviral candidates against enteroviruses.
KEYWORDS: Sophocarpine, sophocarpinol, structure−activity relationship, enteroviruse, coxsakievirus type B3
a
Scheme 1
nteroviruses are a genus of single-stranded (+) RNA
1,2
Eviruses associated with many human diseases.
Among
Figure 1. Chemical structures of sophocarpine (1); E-β,γ-sopho-
carpinic acid (2); E-β,γ-12-N-m-cyanobenzene-sulfonyl sophocarpinic
acid (3); E-β,γ-12-N-p-trifluoromethy-benzenesulfonyl sophocarpinic
acid (4); and Z-α,β-sophocarpinic acid (14).
a
Reagents and conditions: (a) 5 N NaOH, reflux, 9 h, 2 N HCl, pH =
5−6; (b) diphenyldiazomethane, MeOH, r.t., 12 h; (c) RPhSO2Cl,
K2CO3, CH2Cl2, r.t., overnight; (d) flash column chromatography; (e)
m-cresol, reflux, 8−9 h; (f) 2 N HCl/MeOH, reflux, 2 h.
the enteroviruses, coxsackie B viruses (CVB) are important
human pathogens causing pleurodynia, myocarditis, hepatitis,
and so on.3,4 Coxsackievirus type B3 (CVB3) is an important
pathogen that induces acute and chronic viral myocarditis in
children and young adults, and eventually leads to cardiomy-
opathy.5,6 Besides heart infections, CVB3 causes chronic
inflammatory diseases of the pancreas and central nervous
system as well.7,8 In recent years, the sudden epidemic
infections of enteroviruses have caused great concerns of
society.9−11 However, there has been no special efficient drug
approved for the treatment of the infections caused by CVB3
until now.12 Therefore, there is an urgent need to develop
broad-spectrum antiviral candidates against enteroviruses to
meet an emergency of sudden infectious diseases.
Received: October 23, 2014
Accepted: January 7, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/ml500525s
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ACS Medicinal Chemistry Letters
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a
Scheme 2
Table 1. Structure−Activity Relationship of Newly
Synthesized Compounds against CVB3
a
b
c
compd
R1
R2
TC50 (μM)
IC50 (μM)
SI
3
m-CN
p-CF3
o-CN
o-CF3
m-CN
o-CN
o-CF3
o-CN
p-CN
o-CF3
o-CN
m-CN
p-CN
m-CF3
p-CF3
o-NO2
m-NO2
p-NO2
COOH
COOH
COOH
COOH
CO2CH3
CO2CH3
CO2CH3
COOH
COOH
COOH
CO2CH3
CO2CH3
CO2CH3
CO2CH3
CO2CH3
CO2CH3
CO2CH3
CO2CH3
CO2CH3
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
466.2
1469
466.2
393.3
72.4
13.1
33.7
22.7
11.1
7.30
8.92
11.9
>155.4
29.9
27.2
4.21
3.07
8.04
2.24
8.17
5.23
9.24
7.02
1.19
27. 6
17.4
52.4
18.6
16.2
63.6
49.8
53.1
5.47
15.0
4.17
2.31
17.03
694.6
35.5
43.6
20.5
15.6
9.9
4
11a
11b
12a
12b
12c
13a
13b
13c
17a
17b
17c
17d
17e
17f
17g
17h
17i
80.3
9.0
38.1
3.2
466.2
>466.2
423.7
289.2
542.6
292.0
198.0
54.7
>15.7
35.5
68.7
176.7
36.3
88.3
6.7
a
Reagents and conditions: (a) 5 N HCl, reflux, 9 h; (b) 2 N HCl/
MeOH, reflux, 2 h; (c) RPhSO2Cl, K2CO3, CH2Cl2, r.t., overnight; (d)
3 N HCl, reflux, 3 h; (e) Boc2O, K2CO3, CH2Cl2, r.t., overnight; (f)
LiAlH4, THF, r.t., 30 min; (g) 2 N HCl/Et2O, 30 min; (h) TBSCl,
CH2Cl2, imidazole, r.t., overnight; (i) RPhSO2Cl, TEA, CH2Cl2, r.t., 4
h; (j) 2 N HCl.
481.2
111.8
185.5
21.1
92.1
12.1
26.4
17.7
15.6
42.7
14.2
14.7
36.8
7.0
a
Scheme 3
18a
18b
18c
18d
18e
18f
18g
18h
24a
24b
24c
24d
24e
RBV
o-CN
429.6
744.8
744.8
272.4
594.6
445.4
308.8
191.2
147.7
93.5
m-CN
p-CN
p-CF3
o-NO2
m-NO2
p-NO2
6.2
3.6
m-CN
m-CF3
p-CF3
o-NO2
p-NO2
27.0
6.2
a
Reagents and conditions: (a) 5 N NaOH, reflux, 9 h, 2 N HCl, pH =
5−6; (b) 2 N MeOH/HCl, reflux, 2 h; (c) RPhSO2Cl, K2CO3,
CH2Cl2, r.t., overnight; (d) 3 N HCl, reflux, 3 h; (e) LiAlH4, THF, r.t.,
30 min; (f) Boc2O, CH2Cl2, K2CO3, r.t., overnight; (g) TBSCl,
CH2Cl2, imidazole, r.t., overnight; (h) RPhSO2Cl, TEA, 4-DMAP,
CH2Cl2, r.t.,4 h; (i) 2 N HCl, r.t., 2 h.
93.5
22.4
152.9
15.6
11.8
353.5
265.4
8197
a
Cytotoxic concentration required to inhibit Vero cell growth by 50%.
Concentration required to inhibit CVB3 growth by 50%. Selectivity
b
c
index: TC50/IC50.
Sophocarpine (1, Figure 1), a natural medicine extracted
from Chinese herb Sophora flavescens, has been used to treat
viral myocarditis caused by CVB3 for decades in China with an
unknown mechanism of action.13,14 The primary structure−
activity relationship (SAR) investigation had been carried out
with D-ring opening from compound 1, and SAR results
indicated that E-β,γ-sophocarpinic acid (2, Figure 1) with a 3-
ring core was more favorable than 1 with a 4-ring scaffold. As
described in Figure 1, the representative N-benzenesulfonyl
sophocarpinic acids 3 and 4 were identified with higher potency
than lead 1,15 and an effort to further improve the potency via
modification and optimization was carried out, in order to
develop a novel family of antienteroviral candidates against
CVB3. In the present study, using 3/4 as the leads, the SAR
study was mainly focused on the variations of the carboxyl
group and carbon−carbon double bond located at the C-11
attachment, while 12-benzenesulfonyl moiety on the N-12
atom was maintained as a required group for CVB3.15 Herein,
we reported the synthesis, SAR analysis, and in vitro anti-CVB3
evaluation of a series of novel N-benzenesulfonyl sophocarpinic
acid/ester and sophocarpinol derivatives.
Forty-one target compounds were prepared with commer-
cially available 1, lehmannine (5), or matrine (25) as the
starting material as described in Schemes 1−3, respectively.
The D-ring in compound 1 was opened in alkaline conditions
to produce an isomer mixture of E-sophocarpinic acid (6), and
diphenyldiazomethane was then chosen as the protective agent
to facilitate the separation of mixture 8 into its two isomers 9
and 10 with good yields. The desired acid products in 11 and
13 series were acquired by deprotection of 9 or 10 in m-cresol
with overall yields of 5−12% as reported previously.15,16 The
sophocarpinic esters 12a−c were obtained via methyl
esterification of 11 at refluxing temperature in 2 N HCl/
CH3OH in 85−90% yields.
As shown in Scheme 2, methanol was used as the protective
agent in compound 16 so as to conveniently obtain the target
B
DOI: 10.1021/ml500525s
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ACS Medicinal Chemistry Letters
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The influence of the carboxyl group at the C-11 attachment
was first explored. As shown in Table 1, E-β,γ-sophocarpinic
esters (12a−c) exerted potent anti-CVB3 activities with IC50
Table 2. Structure−Activity Relationship for CVB3 of Some
Aimed Compounds
between 7.3 and 11.9 μM and high cytotoxic activities (TC50
=
38−80 μM) to give SI values 3.2−9.9. Their corresponding
sophocarpinic acid (3 and 11a−b) with weaker activities and
toxicities gave SI values ranging from 15.6 to 35.5. Then, E-α,β-
sophocarpinic acids (13a−c) were generated to investigate
whether the position of double bond would affect the activity.
Compounds 13b−c afforded activities comparable to their
geometric isomers; while compound 13a lost its activity,
suggesting the double bond located in β,γ-position is a little
more beneficial than that in α,β-position.
Next, SAR was moved on the impact of Z-configuration of
double bond on the anti-CVB3 effect. Among Z-sophacarpinic
ester analogues (17a−i), all of them displayed excellent
activities with IC50 between 1.19 and 9.24 μM, much better
than that of their corresponding sophacarpinic acids 18a−h.
Especially, compound 17b had a promising potency with an
IC50 of 3.07 μM and SI value of 176. Sophocarpinols 24a−e
showed good potency with IC50 below 17.0 μM, particularly,
compound 24d exhibited an excellent potency with an IC50 of
2.31 (SI = 153), suggesting that the o-NO2 substituent might be
helpful for the improved activity. The SAR results indicated
that E- or Z-configuration of the double-bond was not an
important factor for potency. Then, the double-bond was
removed, and a couple of matrinic acids/esters (28a−e and
29a−c) and matrinols (34a−c) were constructed. As shown in
Table 2, all of them displayed a favorable activity against CVB3
with SI ranging from 5.2 to 92, indicating that the double bond
might not play the key role for keeping good potency.
Out of the 41 new derivatives, compounds 17b, 24d, and 28e
demonstrated the promising anti-CVB3 effects with SI over
92.4, and then all of them were chosen for next investigation.
Their antienteroviral activities against another four coxsack-
ievirus B subtypes, including CVB1, CVB2, CVB5, and CVB6,
were carried out using RBV as the positive control. As
described in Table 3, compounds 17b and 24d afforded
potencies against the four tested CVBs with IC50 ranging from
0.62 to 12.6 μM (SI from 39 and 275), indicating broad-
spectrum antienteroviral activities against different types of
CVB. It was noteworthy that compound 24d exhibited a
promising broad-spectrum antienteroviral effect against five
coxsakievirus B subtypes with average IC50 of 1.4 μM (average
SI = 142), much better than that of RBV with average SI of 6.7,
and was thus selected for further investigation.
a
b
c
compd
R1
R2
TC50 (μM)
IC50 (μM)
SI
28a
28b
28c
28d
28e
29a
29b
29c
34a
34b
34c
RBV
m-CN
p-CF3
o-NO2
m-NO2
m-CF3
m-CN
p-CF3
o-NO2
m-CN
p-CF3
o-NO2
CO2CH3
CO2CH3
CO2CH3
CO2CH3
CO2CH3
COOH
288.0
73.4
11.7
2.54
23.9
3.51
1.98
82.3
75.4
78.9
11.3
6.55
35.7
694.6
24.6
28.9
16.7
19.7
92.4
5.2
398.8
68.9
193.3
427.8
391.7
410.3
147.0
232.6
243.9
8197
COOH
5.2
COOH
5.2
CH2OH
CH2OH
CH2OH
13.0
35.5
6.8
11.8
a
Cytotoxic concentration required to inhibit Vero cell growth by 50%.
Concentration required to inhibit CVB3 growth by 50%. Selectivity
b
c
index: TC50/IC50.
products 17 with high yields as previously reported.17
Sophocarpinols 24b−c bearing a CF3 substitution were directly
gained through selective reduction of 17d−e using LiAlH4 as
the reducing agent in THF in 82−85% yields.17 In another
synthetic route, the key intermediate 21 was acquired via N-
tert-butoxycarbonyl (Boc) protection, ester reduction with
LiAlH4, and Boc removal in 2 N HCl from 16 with good yields.
The sophocarpinols 24a and 24d−e possessing CN or NO2
substitution were successfully achieved through hydroxyl
protection of 21 with tert-butyldimethylsilyl (TBS), 12-N-
benzenesulfonyl substitution, and deprotection of 23 in 2 N
HCl with overall yields of 26−32%. Similarly, as depicted in
Scheme 3, matrinic acids/esters (28a−e and 29a−c) were
semisynthesized from matrine with good yields as reported
previously.16,17 As mentioned above, matrinol 34b possessing
CF3 substitution was directly acquired from 28b; while
compounds 34a and 34c with CN or NO2 were obtained via
a six-step sequence from compound 27 in high yields.
All the new target compounds were measured for their in
vitro anti-CVB3 activities in African green monkey kidney
(Vero) cells using viral cytopathogenic effect (CPE) assay with
ribavirin (RBV) as the positive control.18 The potency against
CVB3 of each tested compound was evaluated by the
combination of its IC50 and selectivity index (SI) value as the
important therapeutic indication.
Taken together, 41 new N-benzenesulfonyl sophocarpinic
acid/ester and sophocarpinol derivatives were designed,
synthesized, and evaluated for their antienterovirus activities
against CVB3. SAR analysis revealed that (i) the double-bond
and its configuration and position at the 11-attachment could
Table 3. Antienteroviral Activities against Four CVB Subtypes of Representative Compounds
17b
compd TC50 (μM) IC50 (μM)
24d
TC50 (μM) IC50 (μM)
28e
TC50 (μM) IC50 (μM)
RBV
a
b
c
a
b
c
a
b
c
a
b
c
SI
SI
SI
TC50 (μM) IC50 (μM)
SI
CVB1
CVB2
CVB5
CVB6
489.8
489.8
489.8
489.8
2.60
12.6
10.5
2.81
188.4
38.9
169.8
169.8
169.8
169.8
1.46
1.44
3.63
0.62
116.0
117.2
46.8
183.3
127.1
73.4
1.28
7.61
143.5
16.7
8196
8196
8196
8196
1951
2101
910.8
1708
4.2
3.9
9.0
4.8
46.7
174.3
275.5
61.1
0.44
139.4
a
b
c
Cytotoxic concentration required to inhibit Vero cell growth by 50%. Concentration required to inhibit CVB growth by 50%. Selectivity index:
TC50/IC50.
C
DOI: 10.1021/ml500525s
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ACS Medicinal Chemistry Letters
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not greatly affect the potency; (ii) the replacement of carboxyl
with ester or alcohol might significantly improve the activity
against CVB3. Among them, sophocarpinol 24d exhibited the
highest potency against CVB3 as well as CVB1, CVB2, CVB5,
and CVB6, indicating a broad-spectrum antienteroviral feature.
The SAR results provided the powerful information on further
strategic modification and optimization. Overall, N-benzene-
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optimization and development of a novel scaffold of broad-
spectrum antiviral agents against enteroviruses.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic procedure, analytical data, antiviral assays, and
cytotoxicity assay. This material is available free of charge via
the Internet at http://pubs.acs.org.
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Z. G.; Gao, L. M.; Jiang, J. D.; Song, D. Q. Synthesis, structure-activity
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AUTHOR INFORMATION
Corresponding Authors
*(D.S.) Tel: +86 10 63165268. Fax: +86 10 63165268. E-mail:
songdanqingsdq@hotmail.com.
*(Y.L.) Tel: +86 10 63010984. Fax: +86 10 63010984. E-mail:
yuhuanlibj@126.com.
■
Author Contributions
§S.T. and L.K contributed equally to this work.
Funding
This work was supported by the National Natural Science
Foundation of China (81321004 and 81402799), the Beijing
Natural Science Foundation (7142107), and 863 Youth Project
(SS2015AA020910).
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
CVB, coxsackievirus B; SAR, structure−activity relationship;
THF, tetrahydrofuran; TLC, thin layer chromatography; TBS,
tert-butyldimethylsilyl; Boc2O, di-tert-butyl pyrocarbonate;
TEA, triethylamine; RBV, ribavirin; SI, selective index; Vero
cells, African green monkey kidney cells; CPE, viral
cytopathogenic effect
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Cercenado, E.; Tenorio, A.; Bouza, E. Cytopathic effect inhibition
assay for determining the in-vitro susceptibility of herpes simplex virus
to antiviral agents. J. Antimicrob. Chemother. 1999, 44, 705−708.
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D
DOI: 10.1021/ml500525s
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX