Synthesis and biological evaluation of n-substituted sophocarpinic acid derivatives as coxsackievirus B3 inhibitors

Article abstract of DOI:10.1002/cmdc.201300224

A series of novel N-substituted sophocarpinic acid derivatives was designed, synthesized, and evaluated for their anti-enteroviral activities against coxsackievirus typeB3 (CVB3) and coxsackievirus typeB6 (CVB6) in Vero cells. Structure-activity relationship analysis revealed that the introduction of a benzenesulfonyl moiety on the 12-nitrogen atom in (E)-β,γ-sophocarpinic acid might significantly enhance anti-CVB3 activity. Among the derivatives, (E)-12-N-(m-cyanobenzenesulfonyl)-β,γ-sophocarpinic acid (11m), possessing a meta-cyanobenzenesulfonyl group, exhibited potent activity against CVB3 with a selectivity index (SI) of 107. Furthermore, compound 11m also showed a good oral pharmacokinetic profile, with an AUC value of 7.29μMh^-1 in rats, and good safety through the oral route in mice, with an LD₅₀ value of >1000mgkg^-1; these values suggest a druggable characteristic. Therefore, compound 11m was selected for further investigation as a promising CVB3 inhibitor. We consider (E)-β,γ-N-(benzenesulfonyl)sophocarpinic acids to be a novel class of anti-CVB3 agents.

Full text of DOI:10.1002/cmdc.201300224

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DOI: 10.1002/cmdc.201300224
Synthesis and Biological Evaluation of N-Substituted
Sophocarpinic Acid Derivatives as Coxsackievirus B3
Inhibitors
Li-Mei Gao, Sheng Tang, Yan-Xiang Wang, Rong-Mei Gao, Xin Zhang, Zong-Gen Peng,
Jian-Rui Li, Jian-Dong Jiang, Yu-Huan Li,* and Dan-Qing Song*[a]
A series of novel N-substituted sophocarpinic acid derivatives
was designed, synthesized, and evaluated for their anti-entero-
viral activities against coxsackievirus type B3 (CVB3) and cox-
sackievirus type B6 (CVB6) in Vero cells. Structure–activity rela-
tionship analysis revealed that the introduction of a benzene-
sulfonyl moiety on the 12-nitrogen atom in (E)-b,g-sophocar-
pinic acid might significantly enhance anti-CVB3 activity.
Among the derivatives, (E)-12-N-(m-cyanobenzenesulfonyl)-b,g-
sophocarpinic acid (11 m), possessing a meta-cyanobenzene-
sulfonyl group, exhibited potent activity against CVB3 with
a selectivity index (SI) of 107. Furthermore, compound 11 m
also showed a good oral pharmacokinetic profile, with an AUC
value of 7.29 mmhÀ1 in rats, and good safety through the oral
route in mice, with an LD50 value of >1000 mgkgÀ1; these
values suggest a druggable characteristic. Therefore, com-
pound 11 m was selected for further investigation as a promis-
ing CVB3 inhibitor. We consider (E)-b,g-N-(benzenesulfonyl)-
sophocarpinic acids to be a novel class of anti-CVB3 agents.
Introduction
Enteroviruses are a genus of single-stranded positive
RNA viruses associated with several human diseases,
including respiratory infections, meningitis, encepha-
litis, pancreatitis, myocarditis, and neonatal sepsis.[1,2]
The genus Enterovirus comprises more than 60 sero-
types, of which Coxsackievirus type B3 (CVB3) can
be considered as the prototype for the nonpolio en-
terovirus group. CVB3 is an important human patho-
gen that induces acute and chronic viral myocarditis
in children and young adults, which eventually leads
to cardiomyopathy.[3–5] In addition to heart infec-
Figure 1. Structures of sophocarpine, matrine, and the sophocarpinic acid ana-
logues 3–6.
tions, CVB3 also causes chronic inflammatory diseas-
es of the pancreas and central nervous system.[6,7]
Numerous compounds have been reported to be se-
lective CVB3 inhibitors, some of which have entered clinical
trials.[8–10] Pleconaril had been shown to inhibit the replication
of various enteroviruses and has demonstrated potential effica-
cy against enterovirus infections.[11] Currently, there are no effi-
cient drugs approved for the infections caused by the entero-
viruses. Therefore, there is an urgent need to develop new an-
tiviral candidates for the treatment of infections caused by en-
teroviruses, especially those caused by CVB3.
demonstrated to show antiviral activity against CVB3 and used
to treat patients with viral myocarditis in China for many years
with confirmed safety,[12,13] although its mode of action against
CVB3 infection remains unknown. The unique chemical scaffold
and special anti-CVB3 activity of 1 provoked strong interest in
us to explore the structure–activity relationship (SAR) of this
kind of compound, with the aim of discovering novel potential
anti-CVB3 candidates. By considering the structural differences
between 1 and matrine (2, Figure 1), which has been used to
treat hepatitis B virus (HBV) infections in China for decades,[14]
we deduced that the a,b carbon–carbon double bond of
1 might play an important role in the anti-CVB3 activity. Based
on the primary SAR results, the SAR analysis in the present
study was initially carried out with the double bond as a re-
quired group for the anti-CVB3 effect of this kind of com-
pound.
Sophocarpine (1, Figure 1), which was extracted from the
traditional-medicine herb Sophora alopecuroides L., has been
[a] L.-M. Gao,+ Dr. S. Tang,+ Dr. Y.-X. Wang, Dr. R.-M. Gao, Dr. X. Zhang,
Dr. Z.-G. Peng, J.-R. Li, Prof. J.-D. Jiang, Prof. Y.-H. Li, Prof. D.-Q. Song
Institute of Medicinal Biotechnology
Chinese Academy of Medical Science & Peking Union Medical College
Tiantan Xili No. 1, Beijing 100050 (PR China)
E-mail: yuhuanlibj@126.com
A new chemical entity (NCE), (Z)-a,b-sophocarpinic acid or
sophocarpinic acid (3, Figure 1), could be constructed by open-
songdanqingsdq@hotmail.com
[+] These authors contributed equally to this work.
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ing ring D of 1, and a novel
class of N-substituted sophocar-
pinic acid derivatives could be
generated by introducing a vari-
ety of substituents onto the 12-
nitrogen atom in 3. On the basis
of this strategy, a series of N-
(benzoyl)-, N-(benzyl)-, and N-
(benzenesulfonyl)sophocarpinic
acid analogues were designed,
synthesized, and evaluated for
their antiviral activity against
CVB3. To further explore the in-
fluence of the configuration and
position of the a,b double bond
in 3, the cis–trans isomer (E)-
sophocarpinic acid (4) and two
structural isomers, (E)-b,g-sopho-
carpinic acid (5) and (Z)-b,g-
sophocarpinic acid (6), were cre-
ated as well. This report includes
the synthesis, SAR analysis, effi-
cacy evaluation against CVB3 in
vitro, and pharmacokinetic and
safety studies in vivo for a series
of novel N-substituted sopho-
carpinic acid derivatives.
Results and Discussion
Chemistry
Thirty new sophacarpinic acid
derivatives were prepared by
two synthetic routes, as illustrat-
ed in Scheme 1. Route one uses
commercially available com-
pound 1 as the starting material.
Compound 1 was hydrolyzed
Scheme 1. Synthetic routes for N-substituted sophocarpinic acid analogues. Reagents and conditions: a) 10% KOH/
H2O, reflux, 7 h; then 3n HCl, pH 5–6; b) 6n HCl, reflux, 9 h; c) diphenyldiazomethane, MeOH/petroleum ether
(boiling range 30–608C), overnight; d) substituted benzyl chloride/bromine or benzenesulfonyl chloride, CH2Cl2 or
MeCN, K2CO3, room temperature, overnight; e) flash column chromatography; f) m-cresol, 808C, 8–9 h; g) 2n
HCl(g)/MeOH, room temperature, overnight; h) 3n HCl, reflux, 3 h.
under either acidic or basic conditions to provide a mixture of
isomers 4 and 5 in 45–55% yields, rather than the desired Z
isomer 3. The final products in the 11 and 12 series were ac-
quired by using a three-step sequence including carboxyl pro-
tection with diphenyldiazomethane as the protective agent,
12-N-alkylation or acylation, and deprotection in m-cresol with
overall yields of 5–12%, in accordance with the methods re-
ported previously.[15–18] The isomers in the 9 and 10 series were
easily isolated because of their clear differences in polarity.
Route two is a synthetic route with commercially available
lehmamnine (13) as the starting material. In the hydrolysis of
compound 13, the desired product 6, with the Z configuration,
was easily obtained in 6n HCl in 60% yield, whereas the two E
isomers 4 and 5 can also be obtained in 10% aqueous KOH, as
expected. Intermediate 14 was prepared by carboxyl protec-
tion, in which methanol was used as both the protective agent
and the solvent, with a good yield of 90%. Compounds 15
were gained by 12-N-alkylation or acylation of 14 in 45–75%
yields. The final products in the 16 series were easily acquired
by deprotection of compounds 15 in 3n HCl, with 60–80%
yields.
The configurations and positions of the double bonds were
1
identified by means of IR and H NMR spectra. A medium-in-
tensity absorption peak at 966–984 cmÀ1 in the IR spectrum is
the characteristic peak for the E configuration of the carbon–
carbon double bond in the 11 and 12 series. The E or Z config-
urations of the double bonds in the 12 and 16 series were fur-
ther confirmed by the coupling constant (J; trans: J3 >14 Hz,
cis: J3 <12). The J3 values for the E configuration of the 12
series range from 15.2–15.6, whereas the J3 values are ~10.4–
10.8 in the 16 series. Moreover, the chemical shifts of the a,b
hydrogen atoms in the 12 series were generally in the range
of 5.5–7.3 ppm, and those of the b,g hydrogen atoms in the 11
and 16 series were between 5.2–6.5 ppm. In addition, the a-H
signal in the 12 series was split into double peaks, which indi-
cated that the double bond was located at the a,b position.
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Biology
Table 1. SAR results for the anti-CVB3 activity of N-substituted sophocarpinic acid
analogues.[a]
SAR analysis for anti-CVB3 activity
All of the newly synthesized compounds were exam-
ined for their antiviral activities against CVB3 in Afri-
can green monkey kidney cells (Vero cells) by using
a viral cytopathogenic effect (CPE) assay with ribavir-
in (RBV) as the positive control.[19] The antiviral activi-
ty of each compound was expressed with an IC50
value and the cytotoxicity was expressed with a TC50
value. The antiviral activity of each tested compound
was evaluated by combination of its IC50 and selec-
tivity index (SI) values.
Compd
R
TC50 [mm][b]
IC50 [mm][c]
SI[d]
11 a
11 b
11 c
11 d
11 e
11 f
11 g
11 h
11 i
COC6H5
CH2C6H5
>2717.4
>2824.9
>2638.5
1113.0
1097.7
2475.2
>2392.3
>2304.1
1469.0
>2370.0
>2383.1
>2383.1
269.1
>904.9
543.5
668.3
148.4
253.7
476.4
116.6
128.8
33.7
>789.9
>761.0
326.9
2.5
–
5.2
3.9
7.5
4.3
5.2
20.5
17.9
43.6
–
CH2C6H4CN-p
CH2C6H4Br-p
CH2C6H4OCH3-p
SO2C6H5
SO2C6H4CH3-p
SO2C6H4OCH3-p
SO2C6H4CF3-p
SO2C6H4F-p
The SAR study for anti-CVB3 activity was initially
focused on the influence of the substituents on the
12-nitrogen atom in the 5 series. The different
groups, including benzoyl (11 a), substituted benzyl
(11 b–e), and substituted benzenesulfonyl (11 f–n)
groups, were attached to the nitrogen atom at the
12 position, from which 14 new (E)-b,g-sophocarpin-
ic acid analogues were created and tested (Table 1).
The results showed that compounds 11 b–e bearing
benzyl moieties showed moderate anti-CVB3 activity
with SI values ranging from 3.9–7.5, which are simi-
lar to that of RBV (SI=6.8). Most of the compounds
in the 12-benzenesulfonyl series had a good activity
against CVB3 with SI values of 5.2–107, which are
greater than or similar to that of RBV. Compounds
11 i, 11 m, and 11 n afforded potential anti-CVB3 ac-
tivity with SI values of over 43, which are much
greater than that of RBV. In particular, compound
11 m bearing an m-cyanobenzenesulfonyl group ex-
hibited the most potent anti-CVB3 effect among the
newly synthesized compounds, with an IC50 value of
2.5 mm (SI=108). The results suggested that the in-
troduction of an electron-withdrawing group such
as CF3 or CN on the sulfonylphenyl ring might be
beneficial for the increased activity against CVB3 of
this kind of compound.
11 j
11 k
11 l
SO2C6H4Cl-p
SO2C6H4Cl-o
–
7.0
106.9
46.6
–
5.2
5.1
–
3.5
3.6
–
11 m
11 n
12a
12b
12c
12d
12e
12 f
12g
12h
12i
SO2C6H4CN-m
SO2C6H4(NHCOCH3)-p
COC6H5
CH2C6H4CN-p
CH2C6H4NO2-p
SO2C6H5
SO2C6H4CH3-p
SO2C6H4OCH3-p
SO2C6H4F-p
144.1
3.1
>2717.4
>2638.5
1737.7
>2475.2
460.4
1330.3
>2369.7
1318.1
>2283.1
>2331.0
>2169.1
>520.8
331.0
>904.9
507.8
340.1
>825.1
132.8
369.2
>834.6
>761.0
>761.0
>789.1
447.1
>173.6
159.1
42.3
SO2C6H4Cl-p
SO2C6H4Cl-o
–
–
–
12j
SO2C6H4CN-p
SO2C6H4(NHCOCH3)-p
CH2C6H4OCH3-p
SO2C6H4NH2-p
CH2C6H4NO2-m
SO2C6H4CF3-m
SO2C6H4CF3-p
12k
16a
16b
16c
16d
16e
RBV
>4.85
–
2.1
11.8
6.8
10.8
6.84
501.2
423.7
293.8
8196.7
62.0
27.2
1198.6
[a] Results are given as mean values for n=3 independent experiments performed in
triplicate; standard errors are within Æ20%. [b] Cytotoxic concentration required to
inhibit Vero cell growth by 50%. [c] Concentration required to inhibit CVB3 growth by
50%. [d] Selectivity index: TC50/IC50.
Next, the SAR analysis moved on to the substitu-
ents at the 12 position in the 4 series, in which 11
new (E)-sophocarpinic acid derivatives were pro-
duced. As indicated in Table 1, most of them showed de-
creased anti-CVB3 activity compared with the corresponding
compounds in the 5 series, regardless of the electron-donating
or electron-withdrawing groups on the phenyl ring. It seems
that the trans configuration of the double bond in the a,b po-
sition does not fit well into the pocket of the speculated
target. In another variation, a series of (Z)-b,g-sophocarpinic
acid derivatives (16a–e) were prepared as well. Among these
analogues, compounds 16c and 16e afforded moderate anti-
CVB3 activity with SI values of 11.8 and 10.8, which are similar
to that of RBV. We deduced that the trans double bond in the
b,g positions might be helpful for keeping the good activity
against CVB3.
SAR analysis for anti-CVB6 activity
In parallel, the anti-CVB6 activity of each compound was also
measured in Vero cells by using a CPE assay with RBV as a posi-
tive control. Among all of them, eight compounds afforded an
antiviral effect against CVB6 (Table 2). In particular, compounds
11 i and 11 l–n showed potent anti-CVB6 activity with SI values
between 12–20, which are better than that of RBV. Out of the
newly synthesized analogues, compounds 11 i, 11 m, and 11 n
afforded the most promising activity against CVB3 and CVB6,
and the three of them were selected for further pharmaco-
kinetic (PK) behavior in vivo.
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Table 2. Antiviral activity against CVB6 for some of the N-substituted
sophocarpinic acid analogues in Vero cells.[a]
Compd
TC50 [mm][b]
IC50 [mm][c]
SI[d]
11 e
11 g
11 h
11 i
11 j
11 l
11 m
11 n
RBV
>1237
>1196
>1152
>1113
>1184
>1141
>1165
251
126Æ16.3
107Æ3.7
94Æ7.5
9.8
11.2
12.3
12.6
9.8
16.5
16.6
20.9
4.85
88 Æ3.5
121Æ15.6
69Æ6.4
70Æ6.6
12Æ2.1
8190
1688Æ23.4
[a] Results are given as mean values for n=3 independent experiments
performed in triplicate; standard errors are within Æ15%. [b] Cytotoxic
concentration required to inhibit Vero cell growth by 50%. [c] Concentra-
tion required to inhibit CVB6 growth by 50%. [d] Selectivity index: TC50/
IC50.
Pharmacokinetic profiles of candidates
Compounds 11 i, 11 m, and 11 n were given individually to
Sprague–Dawley (SD) rats by the oral route (25 mgkgÀ1 intra-
gastrically). As shown in Figure 2 and Table 3, compound 11 n
had a poor area-under-curve (AUC) value of 0.13 mmh, which
indicated an unstable metabolism in vivo. However, com-
pounds 11 m and 11 i demonstrated an excellent PK profile
with AUC values of 7.29 and 43.2 mmh, respectively. In particu-
lar, the maximum concentration (Cmax) of 11 m in the plasma
was 4.54 mm, which is 1.8-fold higher than the IC50 value
(2.5 mm) for the anti-CVB3 activity in vitro. In addition, com-
Figure 2. Pharmacokinetics study of compounds 11 i, 11 m, and 11 n.
a) Mean plasma concentration–time curves for compounds 11 i and 11 m
after a single oral administration at 25 mgkgÀ1 to SD rats (n=3). b) Mean
plasma concentration–time curve of compound 11 n after a single oral ad-
ministration at 25 mgkgÀ1 to SD rats (n=3).
pound 11 m was rapidly absorbed with a maximum time (tmax
of 0.5 h, a favorable half-time (t1/2) of 1.17 h, and
a mean residence time (MRT) of 1.5 h. Therefore, it
)
was selected as a potential anti-CVB3 agent for fur-
ther investigation.
Table 3. Pharmacokinetic profiles of compounds 11 i, 11 m, and 11 n in rats.[a]
Compd
tmax [h]
Cmax [mm]
AUC0–24 h [mmh]
MRT [h]
t1/2 [h]
11 i
11 m
11 n
1.00Æ0.87
0.5Æ0.00
0.25Æ0.00
10.5Æ4.13
4.54Æ0.76
0.17Æ0.10
43.2Æ5.40
7.29Æ0.71
0.02Æ0.01
3.74Æ0.78
1.53Æ0.14
1.77Æ0.30
3.58Æ0.60
1.17Æ0.02
1.10Æ0.04
Preliminary safety evaluations
As compound 11 m had a good PK profile, we next
performed a safety evaluation of 11 m for its acute
toxicity in mice. Compound 11 m was given orally in
[a] Results are given as mean values Æ standard deviation for n=3 independent ex-
periments performed in triplicate; dose: 25 mgkgÀ1. tmax: maximum time; Cmax: maxi-
mum concentration; AUC: area under curve; MRT: mean residence time; t1/2: half-time.
a
single-dosing experiment at 250, 500, or
1000 mgkgÀ1, respectively. The mice were closely
monitored for seven days. No mouse died during
the seven-day observation period, which indicated
that the LD50 value of 11 m by the oral route was
larger than 1000 mgkgÀ1. Oral administration of 11 m at the
experimental doses did not affect the body weight of the mice
either (data not shown). Blood samples taken at the end of the
experiment were examined for liver and kidney functions. Sig-
nificant abnormality was not found in the blood aspartate ami-
notransferase, alanine aminotransferase, urea nitrogen, or crea-
tine levels in the 11 m treatment groups, even if the dose was
up to 1000 mgkgÀ1 (Figure 3). The results suggest that com-
pound 11 m was considerably safe in vivo.
Conclusions
In summary, 30 novel N-substituted sophocarpinic acid deriva-
tives were designed, synthesized, and evaluated for their anti-
enterovirus activity against CVB3 and CVB6. SAR analysis indi-
cated that the introduction of a benzenesulfonyl moiety on
the 12-nitrogen atom in the 5 series might significantly en-
hance the activity. Among these compounds, 11 m, 11 n, and
11 i were active for CVB3 and CVB6. Moreover, compound 11 m
showed a good PK profile and considerable safety in vivo,
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a solution of KOH (33.6 g, 600 mmol) in H2O (300 mL). The reaction
mixture was heated at reflux for 7 h and then stirred at room tem-
perature overnight. The reaction solution was cooled with an ice–
water bath and was acidified with HCl (3n) to pH 5–6. The solvent
was removed in vacuo, and the residue was recrystallized in meth-
anol to give a mixture of 4 and 5.
A mixture of diphenylmethanone hydrazone (14.7 g, 75 mmol,
1.5 equiv) and electrolytic manganese dioxide (13.04 g, 150 mmol,
3 equiv) in petroleum ether (boiling range 30–608C) was heated at
reflux for 0.5 h, and the mixture was filtered. The filtrate was
added to the solution of 4 and 5 in methanol, and the mixture
was then stirred overnight at room temperature. A mixture of 7
and 8 was obtained and was used in the next reaction without fur-
ther purification.
Anhydrous K2CO3 (3.5 equiv) and benzoyl chloride, benzyl chloride
(bromine), or sulfonyl chloride (1 equiv) were added a to the solu-
tion of 7 and 8 in dichloromethane or MeCN (50 mL), and the reac-
tion solution was then stirred at room temperature until TLC analy-
sis showed completion of the reaction. The reaction mixture was
filtered and the filtrate was evaporated to afford a mixture of 9
and 10.
The purified compounds 9 and 10 were obtained by splitting of
the mixture with flash column chromatography on silica gel with
ethyl acetate and hexamethylene as the eluents. Compound 9 was
then dissolved in m-cresol (10 mL), and the reaction mixture was
stirred at 808C for 8–9 h. It was then cooled, and methylisobutylke-
tone (30 mL) was added. The resulting solution was extracted with
H2O (50 mLꢁ3), and the combined extracts were evaporated to
afford the crude compound, which was purified by flash column
chromatography on silica gel with dichloromethane and methanol
as the eluents, to afford 11 a–n with yields in the range of 60–80%.
Compounds 12a–k were obtained from compounds 10 by the
same method.
Figure 3. Safety of compound 11 m in mice. a) Effect of 11 m (oral adminis-
tration) on the body weight of mice. b) Effect of 11 m on liver and kidney
functions. BUN: blood urea nitrogen [mm]; CRE: creatine [mm]; GOP: gluta-
mate-oxaloacetate transaminase [ULÀ1]; GPT: glutamate-pyruvate transami-
nase [ULÀ1]. Values are the mean Æ standard deviation of n=10 experi-
ments performed in triplicate.
which indicated a druggable nature of the structure. Thus, it
has been selected as an anti-enterovirus drug candidate for
further investigation or as a chemical probe for unraveling the
mechanism of action and studying CVB3 biology. We consider
the (E)-b,g-N-(benzenesulfonyl)sophocarpinic acids to be prom-
ising novel scaffolds for anti-CVB3 agents.
(E)-12-N-(Phenylcarbonyl)-Dbg-sophocarpinic acid (11a): White
solid (0.54 g, 6%): mp: 108–1108C; 1H NMR (400 MHz, D2O): d=
7.50 (d, J=6.8 Hz, 2H), 7.36–7.47 (m, 3H), 6.04–6.16 (m, 2H), 4.53
(s, 1H), 3.82 (s, 1H), 3.49–3.59 (m, 2H), 3.31 (d, J=10.8 Hz, 1H),
3.17 (d, J=15.6 Hz, 1H), 2.95–3.01 (m, 1H), 2.72 (s, 1H), 2.29 (d, J=
9.2 Hz, 1H), 2.14 (s, 2H), 1.95 (d, J=12.8 Hz, 2H), 1.82 (s, 2H), 1.35–
1.59 ppm (m, 5H); MS (ESI): m/z (%): 369.0 [M+H]+ (100).
Experimental Section
Chemistry
(E)-12-N-(Benzyl)-Dbg-sophocarpinic acid (11b): White solid
(1.02 g, 12%): mp: 103–1058C; 1H NMR (400 MHz, CD3OD): d=
7.32–7.26 (m, 5H), 6.10–6.02 (m, 1H), 5.43–5.28 (m, 1H), 4.47 (d,
J=13.6 Hz, 1H,), 3.55–3.42 (m, 2H), 3.05–2.99 (m, 4H), 2.85–2.60
(m, 3H), 2.43–2.37 (m, 2H), 1.99–1.87 (m, 3H), 1.72–1.45 ppm (m,
7H); HRMS (ESI): m/z calcd for C22H30N2O2 [M+H]+: 355.2386;
found: 355.2380.
Melting points (mp) were obtained with an MPA 100 OptiMelt au-
tomated melting point system (Stanford Research Systems, Califor-
1
nia, USA). H NMR and 13C NMR spectral analyses were performed
on a Varian Inova 400 MHz spectrometer (Varian, San Francisco, CA,
USA) or a Bruker Avance III 400 spectrometer, respectively, in CDCl3,
D2O, [D6]DMSO, or CD3OD, with Me4Si as the internal standard. In-
frared spectroscopic analysis was performed on Fourier transform
IR spectrometer (Nicolet 5700, Thermo, USA). ESI high-resolution
mass spectrometry (HRMS) was performed on an Autospec Ultima-
TOF mass spectrometer (Micromass UK Ltd, Manchester, UK). Flash
chromatography was performed on a Combiflash Rf 200 system
(Teledyne, Nebraska, USA), with a particle size of 0.038 mm.
(E)-12-N-(p-Cyanobenzyl)-Dbg-sophocarpinic acid (11c): White
solid (0.84 g, 9%): mp: 150–1528C; 1H NMR (400 MHz, D2O): d=
7.53 (d, J=8.0 Hz, 2H), 7.31 (d, J=8.0 Hz, 2H), 6.09–6.04 (m, 1H),
5.23 (dd, J=9.2, 15.6 Hz, 1H), 4.36 (d, J=14.0 Hz, 2H), 3.64 (t, J=
10.0 Hz, 1H), 3.52–3.45 (m, 2H), 3.75 (s, 1H), 3.19–3.04 (m, 3H),
2.29–2.07 (m, 3H), 2.00–1.60 (m, 5H), 1.57–1.41 ppm (m, 5H);
HRMS (ESI): m/z calcd for C23H29N3O2 [M+H]+: 380.2338; found:
380.2336.
Compound 1 and lehmamnine (13) with a purity over 98.5%, as
starting materials, were purchased from the Yanchi Dushun Biologi-
cal and Chemical Co. Ltd. (Shanxi, China) and the Ningxia Zijinghua
pharmacy Co. Ltd. (Ningxia, China).
(E)-12-N-(p-Bromobenzyl)-Dbg-sophocarpinic acid (11d): White
1
solid (1.02 g, 9%): mp: 98–1008C; H NMR (400 MHz, CD3OD): d=
General procedures for N-substituted (E)-Dbg-sophocarpinic
acids (11a–m) and N-substituted (E)-Dab-sophocarpinic acids
(12a–k): Compound 1 (12.3 g, 50 mmol, 1 equiv) was added to
7.29 (d, J=10.8 Hz, 2H), 7.25 (d, J=10.8 Hz, 2H), 6.01–5.94 (m, 1H),
5.25 (dd, J=9.2, 15.2 Hz, 1H), 4.29–4.13 (m, 1H), 3.20–2.89 (m, 5H),
2.63–2.42 (m, 4H), 2.48–1.71 (m, 5H), 1.68–1.41 ppm (m, 7H);
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HRMS (ESI): m/z calcd for C22H29BrN2O2 [M+H]+: 433.1491; found:
433.1505.
1.33 ppm (m, 3H); 13C NMR (400 MHz, CD3OD): d=176.9, 165.4,
136.5, 132.4, 132.3, 131.7, 129.8, 117.3, 117.1, 64.3, 61.2, 57.3 (2),
41.4, 40.3, 36.6, 34.7, 27.8, 26.4, 21.3, 20.5 ppm; FTIR (KBr): n˜ =
3549, 3103, 1711, 1644, 1589, 1171, 990, 839 cmÀ1; HRMS (ESI): m/z
calcd for C21H27N2O4FS [M+H]+: 423.1748; found: 423.1746.
(E)-12-N-(p-Methoxybenzyl)-Dbg-sophocarpinic acid (11e): White
1
solid (0.96 g, 9%): mp: 102–1038C; H NMR (400 MHz, CD3OD): d=
7.37 (d, J=8 Hz, 2H), 7.25 (d, J=8 Hz, 2H), 5.64–5.71 (m, 1H), 5.22
(dd, J=8.8, 15.6 Hz, 1H), 4.09–4.15 (m, 1H), 3.35–3.44 (m, 2H),
3.11–3.17 (m, 1H), 2.97–3.01 (m, 2H), 2.64–2.73 (m, 3H), 2.16–2.23
(m, 1H), 2.04 (s, 1H), 1.21–1.99 ppm (m, 11H); HRMS (ESI): m/z
calcd for C23H30F3N2O3 [M+H]+: 439.2209; found: 439.2228.
(E)-12-N-(p-Chlorobenzenesulfonyl)-Dbg-sophocarpinic acid (11k):
White solid (1.4 g, 6%): mp: 120–1228C; 1H NMR (CD3OD,
400 MHz): d=7.74 (d, J=8.0 Hz, 2H), 7.40 (d, J=8.0 Hz, 2H), 5.58–
5.36 (m, 2H), 3.79 (dd, J=4.4, 12.4 Hz, 1H), 3.72–3.67 (m, 1H),
3.25–3.05 (m, 3H), 2.96 (s, 1H), 2.85 (d, J=4.4 Hz, 2H), 2.46–2.42
(m, 2H), 2.14–2.11 (m, 1H), 1.92–1.82 (m, 2H), 1.73 (s, 3H), 1.57–
1.55 (m, 1H), 1.46–1.32 ppm (m, 3H); 13C NMR (400 MHz, CD3OD):
d=177.3, 140.0, 139.4, 131.0 (2), 130.9, 130.5, 130.3 (2), 64.2, 61.1,
57.1 (2), 47.6, 41.3, 40.8, 36.6, 27.6, 26.2, 21.1, 20.3 ppm; FTIR (KBr):
n˜ =3550, 3362, 2939, 1708, 1650, 1576, 1474, 992, 830 cmÀ1; HRMS
(ESI): m/z calcd for C21H27ClN2O4S [M+H]+: 439.1453; found:
439.1449.
(E)-12-N-(Benzenesulfonyl)-Dbg-sophocarpinic acid (11 f): White
1
solid (2.0 g, 10%): mp: 106–1088C; H NMR (400 MHz, CD3OD): d=
7.74 (d, J=5.2 Hz, 2H), 7.59–7.50 (m, 3H), 5.45–5.42 (m, 2H), 3.79
(dd, J=4.4, 12.4 Hz, 1H), 3.54–3.49 (m, 1H), 3.09 (t, J=12.4 Hz, 1H),
2.98 (t, J=12.4 Hz, 2H), 2.89–2.88 (m, 2H), 2.81 (s, 1H), 2.40–2.34
(m, 2H), 2.12–2.10 (m, 1H), 1.87–1.84 (m, 2H), 1.73–1.67 (m, 3H),
1.56–1.54 (m, 1H), 1.42–1.34 ppm (m, 3H); 13C NMR (400 MHz,
CD3OD): d=177.3, 140.9, 133.8, 130.9, 130.8, 130.1 (2), 129.1 (2),
64.3, 61.1, 57.1, 57.0, 48.4, 41.3, 40.9, 36.7, 27.6, 26.2, 21.1,
20.3 ppm; FTIR (KBr): n˜ =3599, 3429, 3064, 1715, 1571, 1446, 1372,
980, 810 cmÀ1; HRMS (ESI): m/z calcd for C21H28N2O4S [M+H]+:
405.1843; found: 405.1833.
(E)-12-N-(o-Chlorobenzenesulfonyl)-Dbg-sophocarpinic acid (11l):
1
White solid (1.4 g, 6%): mp: 115–1188C; H NMR (400 MHz, CD3OD):
d=7.99 (dd, J=1.6, 8.0 Hz, 1H), 7.90 (d, J=8.4 Hz, 1H), 7.47–7.49
(m, 2H), 5.53–5.60 (m, 1H), 5.20–5.26 (m, 1H), 4.08 (dd, J=4.4,
14.0 Hz, 1H), 3.95 (t, J=10.8 Hz, 1H), 3.55 (s, 1H), 3.33–3.43 (m,
3H), 2.90–3.00 (m, 2H), 2.68–2.74 (m, 1H), 2.50–2.55 (m, 1H), 2.14–
2.26 (m, 2H), 1.72–1.90 (m, 6H), 1.53–1.67 ppm (m, 2H); HRMS
(ESI): m/z calcd for C21H28ClN2O4S [M+H]+: 439.1453; found:
439.1446.
(E)-12-N-(p-Methylbenzenesulfonyl)-Dbg-sophocarpinic
acid
1
(11g): White solid (2.0 g, 10%): mp: 104–1068C; H NMR (400 MHz,
CD3OD): d=7.62 (d, J=8.0 Hz, 2H), 7.34 (d, J=8.0 Hz, 2H), 5.49–
5.38 (m, 2H), 3.74 (dd, J=4.4, 12.0 Hz, 1H), 3.43 (t, J=9.6 Hz, 1H),
3.05–2.91 (m, 5H), 2.76 (s, 1H), 2.38 (s, 3H), 2.35–2.33 (m, 2H),
2.10–2.07 (m, 1H), 1.88–1.80 (m, 2H), 1.71–1.34 ppm (m, 8H);
13C NMR (400 MHz, CD3OD): d=177.2, 145.0, 137.5, 131.3, 130.6 (2),
130.1, 129.3 (2), 64.3, 61.1, 57.2, 57.2, 41.4, 40.7, 36.6, 34.2, 27.7,
26.3, 21.5, 21.2, 20.4 ppm; FTIR (KBr): n˜ =3395, 2935, 1715, 1596,
1574, 1445, 980, 813 cmÀ1; HRMS (ESI): m/z calcd for C22H30N2O4S
[M+H]+: 419.1999; found: 419.2006.
(E)-12-N-(m-Cyanobenzenesulfonyl)-Dbg-sophocarpinic
acid
1
(11m): White solid (1.6 g, 7%): mp: 114–1168C; H NMR (400 MHz,
CD3OD): d=8.07–8.01 (m, 2H), 7.78 (d, J=8.0 Hz, 1H), 7.61 (d, J=
8.0 Hz, 1H), 5.56–5.40 (m, 2H), 4.16–4.11 (m, 1H), 3.91–3.50 (m,
2H), 3.49–3.42 (m, 1H), 3.42–3.18 (m, 1H), 2.91–2.58 (m, 3H), 2.39–
2.19 (m, 4H), 2.06–1.42 ppm (m, 8H); 13C NMR (400 MHz, CD3OD):
d=176.2, 137.5, 135.0, 133.2, 132.2, 131.7, 130.4, 127.6, 118.3,
114.8, 64.4, 60.1, 57.1, 56.9, 40.2, 39.8, 36.6, 34.7, 27.4, 26.7, 20.5,
20.1 ppm; FTIR (KBr): n˜ =3390, 3071, 2233, 1708, 1657, 1569, 984,
(E)-12-N-(p-Methoxybenzenesulfonyl)-Dbg-sophocarpinic
acid
(11h): White solid (1.4 g, 6%): mp: 70–728C; 1H NMR (400 MHz,
CD3OD): d=7.67 (d, J=5.2 Hz, 2H), 7.04 (d, J=5.2 Hz, 2H), 5.53–
5.38 (m, 2H), 3.83 (s, 3H), 3.73 (dd, J=4.4, 12.0 Hz, 1H), 3.44–3.39
(m, 1H), 3.02–2.93 (m, 5H), 2.79 (m, 1H), 2.40–2.35 (m, 2H), 2.12–
2.08 (m, 1H), 1.91–1.80 (m, 2H), 1.72–1.65 (m, 3H), 1.55–1.31 ppm
(m, 4H); 13C NMR (400 MHz, CD3OD): d=177.3, 164.6, 132.3, 131.8,
131.4 (2), 130.2, 115.2 (2), 64.2, 61.0, 57.1, 56.3 (2), 41.3, 40.9, 36.4
(2), 27.7, 26.2, 21.1, 20.4 ppm; FTIR (KBr): n˜ =3400, 2937, 1715,
1596, 1497, 1371, 980, 836 cmÀ1; HRMS (ESI): m/z calcd for
C22H30N2O5S [M+H]+: 435.1948; found: 435.1949.
850 cmÀ1
;
HRMS (ESI): m/z calcd for C22H27N3O4S [M+H]+:
430.1795; found: 430.1782.
(E)-12-N-(p-Acetamidobenzenesulfonyl)-Dbg-sophocarpinic acid
1
(11n): White solid (2.4 g, 10%): mp: 118–1208C; H NMR (400 MHz,
CD3OD): d=7.72 (d, J=8.8 Hz, 2H), 7.66 (d, J=8.8 Hz, 2H), 5.55–
5.48 (m, 1H), 5.40–5.34 (m, 1H), 3.78 (dd, J=4.4, 12.4 Hz, 1H), 3.58
(t, J=10.0 Hz, 1H), 3.13–3.02 (m, 3H), 2.93 (s, 1H), 2.87 (d, J=
6.8 Hz, 1H), 2.53–2.42 (m, 3H), 2.12 (s, 3H), 1.87–1.82 (m, 2H), 1.72
(s, 3H), 1.69–1.55 (m, 2H), 1.48–1.34 ppm (m, 3H); 13C NMR
(400 MHz, CD3OD): d=177.6, 172.0, 144.2, 134.8, 129.8 (2), 129.5,
120.5, 120.3 (2), 64.2, 61.0, 56.9 (2), 41.3, 36.6, 27.6, 26.2, 24.1 (2),
21.1, 20.7, 20.3 ppm; FTIR (KBr): n˜ =3322, 3263, 2937, 2870, 1696,
1592, 1537, 980, 842 cmÀ1; HRMS (ESI): m/z calcd for C23H31N3O5S
[M+H]+: 462.2057; found: 462.2036.
(E)-12-N-(p-Trifluoromethybenzenesulfonyl)-Dbg-sophocarpinic
acid (11i): White solid (1.2 g, 5%): mp: 104–1078C; 1H NMR
(400 MHz, CD3OD): d=7.95 (d, J=8.4 Hz, 2H), 7.85 (d, J=8.4 Hz,
2H), 5.49–5.47 (m, 2H), 3.79 (dd, J=4.4, 12.0 Hz, 1H), 3.50–3.40 (m,
2H), 3.05 (t, J=12.0 Hz, 1H), 2.94 (s, 3H), 2.70 (s, 1H), 2.39–2.20 (m,
2H), 2.11–2.08 (m, 1H), 1.86–1.81 (m, 2H), 1.70–1.61 (m, 3H), 1.54–
1.33 ppm (m, 4H); 13C NMR (400 MHz, CD3OD): d=177.0, 143.9,
131.7, 130.2 (2), 129.7, 127.3, 121.3, 116.8, 113.3, 64.2, 61.4, 57.6 (2),
44.3, 41.7, 40.4, 36.9, 28.1, 26.7, 21.6, 20.7 ppm; FTIR (KBr): n˜ =
3578, 3414, 2869, 1715, 1569, 1446, 981, 844 cmÀ1; HRMS (ESI): m/z
calcd for C22H27F3N2O4S [M+H]+: 473.1716; found: 473.1715.
(E)-12-N-(Phenylcarbonyl)-Dab-sophocarpinic acid (12a): White
solid (0.52 g, 6%): mp: 152–1548C; 1H NMR (400 MHz, D2O): d=
7.65–7.76 (m, 2H), 7.57–7.63 (m, 3H), 7.30 (t, J=7.6 Hz, 1H), 6.28
(d, J=15.6 Hz, 1H), 4.52 (s, 1H), 4.10 (s, 1H), 3.68–3.70 (m, 1H),
3.50–3.52 (m, 1H), 3.39–3.41 (m, 2H), 2.94–2.97 (m, 1H), 2.75 (s,
1H), 2.51 (s, 1H), 2.33–2.41 (m, 2H), 2.22–2.38 (m, 1H), 1.69–
2.07 ppm (m, 9H); HRMS (ESI): m/z calcd for C22H29N2O3 [M+H]+:
369.2178; found: 369.2196.
(E)-12-N-(p-Fluorobenzenesulfonyl)-Dbg-sophocarpinic acid (11j):
White solid (1.6 g, 8%): mp: 133–1358C; 1H NMR (400 MHz,
CD3OD): d=7.80 (dd, J=5.2, 8.8 Hz, 2H), 7.25 (t, J=8.8 Hz, 2H),
5.53–5.36 (m, 2H), 3.79 (d, J=12.0 Hz, 1H), 3.75–3.59 (m, 1H),
3.15–3.06 (m, 3H), 2.94–2.86 (m, 3H), 2.43 (s, 2H), 2.14–2.11 (m,
1H), 1.91–1.83 (m, 2H), 1.72 (s, 3H), 1.57–1.55 (m, 1H), 1.46–
(E)-12-N-(p-Cyanobenzyl)-Dab-sophocarpinic acid (12b): White
solid (0.47 g, 5%): mp: 128–1308C; 1H NMR (400 MHz, D2O): d=
7.90–7.94 (m, 2H), 7.69 (d, J=8 Hz, 2H), 6.93–7.00 (m, 1H), 6.29 (d,
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J=15.6 Hz, 1H), 4.92 (d, J=13.2 Hz, 1H), 4.36 (d, J=13.2 Hz, 1H),
4.03–4.06 (m, 1H), 3.85 (s, 1H), 3.46–3.54 (m, 2H), 3.04–3.36 (m,
6H), 2.49–2.63 (m, 2H), 2.23 (s, 1H), 1.63–2.06 ppm (m, 8H); HRMS
(ESI): m/z calcd for C23H30N3O2 [M+H]+: 380.2338; found: 380.2329.
6.60 (m, 1H), 5.68 (d, J=15.6 Hz, 1H), 3.88–3.91 (m, 1H), 3.40–3.54
(m, 2H), 3.04–3.09 (m, 2H), 2.86 (s, 1H), 2.49–2.61 (m, 2H), 2.38–
2.44 (m, 2H), 2.21 (s, 1H), 2.06–2.07 (m, 1H), 1.48–1.86 ppm (m,
9H); 13C NMR (400 MHz, CD3OD): d=172.0, 143.1, 131.0, 130.8,
130.5 (2), 130.3 (2), 128.3, 64.6, 61.1, 57.7, 57.0, 47.6, 40.3, 35.2,
34.4, 27.8, 27.7, 20.8 (2) ppm; FTIR (KBr): n˜ =3396, 3090, 2937,
2866, 1702, 1655, 1575, 1475, 1392, 1334, 1159, 1091, 982,
(E)-12-N-(p-Nitrobenzyl)-Dab-sophocarpinic acid (12c): White solid
(0.95 g, 10%): mp: 139–1418C; 1H NMR (400 MHz, CD3OD): d=
8.12–8.15 (m, 2H), 7.51–7.57 (m, 2H), 6.97–7.04 (m, 1H), 5.85 (d,
J=15.6 Hz, 1H), 4.23 (d, J=14.4 Hz, 1H), 3.27–3.47 (m, 4H), 2.90–
3.01 (m, 3H), 2.79–2.85 (m, 1H), 2.47–2.78 (m, 3H), 2.19–2.22 (m,
1H), 1.93–2.08 (m, 2H), 1.65–1.88 (m, 7H), 1.47–1.50 ppm (m, 1H);
HRMS (ESI): m/z calcd for C22H30N3O4 [M+H]+: 400.2236; found:
400.2217.
828 cmÀ1
;
HRMS (ESI): m/z calcd for C21H28ClN2O4S [M+H]+:
439.1453; found: 439.1444.
(E)-12-N-(o-Chlorobenzenesulfonyl)-Dab-sophocarpinic acid (12i):
White solid (1.4 g, 6%): mp: 129–1318C; 1H NMR (400 MHz,
CD3OD): d=7.97–8.00 (m, 2H), 7.44–7.49 (m, 2H), 6.25 (dt, J=6,
15.6 Hz, 1H), 5.44 (d, J=15.6 Hz, 1H), 4.04 (dd, J=5.6, 14.8 Hz, 1H),
3.82–3.88 (m, 1H), 3.61 (t, J=3.2 Hz, 1H), 3.39–3.51 (m, 3H), 2.97–
3.04 (m, 2H), 2.53–2.60 (m, 3H), 2.35–2.38 (m, 1H), 1.75–1.98 (m,
6H), 1.67–1.69 ppm (m, 3H); HRMS (ESI): m/z calcd for
C21H28ClN2O4S [M+H]+: 439.1453; found: 439.1441.
(E)-12-N-(Benzenesulfonyl)-Dab-sophocarpinic acid (12d): White
1
solid (2.4 g, 12%): mp: 103–1058C; H NMR (400 MHz, CD3OD): d=
7.95 (d, J=7.6 Hz, 2H), 7.67–7.80 (m, 3H), 6.52–6.59 (m, 1H), 5.57
(d, J=15.6 Hz, 1H), 3.96–3.99 (m, 1H), 3.59 (d, J=8.4 Hz, 2H), 3.54
(s, 1H), 3.44 (d, J=12.0 Hz, 2H), 3.00–3.05 (m, 2H), 2.44–2.50 (m,
2H), 2.20–2.23 (m, 2H), 1.86–1.97 ppm (m, 8H); 13C NMR (400 MHz,
CD3OD): d=172.0, 142.8, 134.3, 130.4 (2), 130.1, 129.2, 128.6 (2),
64.5, 61.2, 57.2, 57.0, 46.5, 40.0, 34.6, 34.5, 28.0, 27.9, 20.8,
20.8 ppm; FTIR (KBr): n˜ =3394, 3268, 2940, 2866, 1700, 1656, 1564,
(E)-12-N-(p-Cyanobenzenesulfonyl)-Dab-sophocarpinic acid (12j):
White solid (1.6 g, 7.4%): mp: 114–1168C; 1H NMR (400 MHz,
CD3OD): d=8.09 (d, J=8.4 Hz, 2H), 8.04 (d, J=8.4 Hz, 2H), 6.25–
6.32 (m, 1H), 5.52 (d, J=15.6 Hz, 1H), 4.02–3.97 (m, 1H), 3.64–3.62
(d, J=8.4 Hz, 2H), 3.49–3.56 (m, 1H), 3.42–3.45 (m, 2H), 3.37 (s,
1H), 3.06–3.01 (m, 2H), 2.39–2.52 (m, 2H), 2.17–2.28 (m, 2H), 1.79–
2.00 ppm (m, 8H); 13C NMR (400 MHz, CD3OD): d=176.2, 137.5,
135.0, 133.2, 132.2, 131.7, 130.4, 127.6, 118.3, 114.8, 64.4, 60.1, 57.1,
56.9, 40.2, 39.8, 36.6, 34.7, 27.4, 26.7, 20.5, 20.1 ppm; FTIR (KBr): n˜ =
3390, 3071, 2937, 2866, 2233, 1708, 1657, 1569, 1469, 1445, 1416,
1378, 1341, 1205, 1154, 984, 850, 806, 736 cmÀ1; HRMS (ESI): m/z
calcd for C22H28N3O4S [M+H]+: 430.1795; found: 430.1782.
1446, 1378, 1311, 1152, 1092, 982, 754, 690 cmÀ1
.
(E)-12-N-(p-Methylbenzenesulfonyl)-Dab-sophocarpinic
acid
1
(12e): White solid (2.0 g, 10%): mp: 101–1038C; H NMR (400 MHz,
CD3OD): d=7.70 (d, J=8.0 Hz, 2H), 7.35 (d, J=8.0 Hz, 2H), 6.52–
6.59 (m, 1H), 5.64 (d, J=15.6 Hz, 1H), 3.75 (s, 1H), 3.35–3.46 (m,
2H), 2.94–3.11 (m, 2H), 2.80 (s, 1H), 2.49–2.57 (m, 1H), 2.29–2.45
(m, 4H), 2.18 (s, 1H), 2.12 (s, 1H), 1.97 (s, 1H), 1.75–1.85 (m, 2H),
1.39–1.71 ppm (m, 8H); 13C NMR (400 MHz, CD3OD): d=172.1,
145.5, 142.6, 138.3, 131.0 (2), 128.7 (2), 128.5, 64.45, 57.2, 57.0 (2),
46.7, 39.9, 34.6, 34.3, 27.9 (2), 21.5, 20.7 ppm (2); FTIR (KBr): n˜ =
3399, 2935, 2867, 1702, 1654, 1597, 1557, 1447, 1329, 1155, 1091,
982, 816, 738 cmÀ1; HRMS (ESI): m/z calcd for C22H31N2O4S [M+H]+:
419.1999; found: 419.1984.
(E)-12-N-(p-Acetamidobenzenesulfonyl)-Dab-sophocarpinic acid
(12k): White solid (2.4 g, 10.4%): mp: 156–1588C; 1H NMR
(400 MHz, CD3OD): d=7.63–7.87 (m, 4H), 6.56–6.63 (m, 1H), 5.72
(d, J=15.2 Hz, 1H), 3.74–3.81 (m, 1H), 3.37–3.43 (m, 2H), 2,74–3.14
(m, 4H), 2.49–2.60 (m, 2H), 2.26–2.52 (m, 3H), 2.18 (s, 3H), 1.35–
1.83 ppm (m, 10H); 13C NMR (400 MHz, CD3OD): d=172.0, 171.7,
144.7, 142.9, 135.1, 130.0 (2), 120.5 (2), 120.2, 64.5, 57.1, 57.0, 56.9,
56.8, 46.1, 39.7, 34.3, 34.1, 27.8, 27.7, 24.1, 20.6 ppm; FTIR (KBr): n˜ =
3313, 3257, 3183, 3106, 3042, 2939, 2869, 1694, 1592, 1538, 1400,
1374, 1319, 1266, 1155, 1092, 980, 841, 751, 719 cmÀ1; HRMS (ESI):
m/z calcd for C23H32N3O5S [M+H]+: 462.2057; found: 462.2045.
(E)-12-N-(p-Methoxybenzenesulfonyl)-Dab-sophocarpinic
acid
1
(12 f): White solid (1.2 g, 6%): mp: 100–1018C; H NMR (400 MHz,
CD3OD): d=7.74–7.77 (m, 2H), 7.05–7.08 (m, 2H), 6.54–6.61 (m,
1H), 5.67 (d, J=15.2 Hz, 1H), 3.84 (s, 3H), 3.65–3.77 (m, 1H), 3.30–
3.46 (m, 2H), 3.08–3.16 (m, 2H), 2.44–2.59 (m, 3H), 2.26–2.33 (m,
1H), 2.17–2.22 (m, 2H), 1.99 (s, 1H), 1.40–1.86 ppm (m, 9H);
13C NMR (400 MHz, CD3OD): d=171.6, 165.0, 143.0, 130.9 (2), 128.7,
128.1, 115.6 (2), 64.5, 56.9, 56.8, 56.7, 56.3, 46.0, 39.6, 34.1 (2), 27.7,
27.6, 20.5, 20.4 ppm; FTIR (KBr): n˜ =3423, 2940, 2766, 1707, 1654,
1596, 1498, 1463, 1444, 1389, 1305, 1260, 1153, 1122, 1093, 1027,
981, 835, 806, 736 cmÀ1; HRMS (ESI): m/z calcd for C22H31N2O5S
[M+H]+: 435.1948; found: 435.1940.
General procedures for N-substituted (Z)-Dbg-sophocarpinic
acids (16a–e): Compound 13 (3 g, 12.2 mmol, 1 equiv) was added
to a solution of 6n HCl (30 mL). The reaction mixture was heated
at reflux for 9 h. The solvent was then removed in vacuo, and the
residue, which was crude compound 6, was dissolved in MeOH
(30 mL). Anhydrous sodium sulfate was added to the solution as
a hygroscopic agent, and the reaction mixture was stirred over-
night. Compound 14 was obtained and used in the next reaction
without further purification.
(E)-12-N-(p-Fluorobenzenesulfonyl)-Dab-sophocarpinic
acid
1
(12g): White solid (1.6 g, 8%): mp: 103–1058C; H NMR (400 MHz,
CD3OD): d=7.88 (dd, J=3.2, 4.8 Hz, 2H), 7.24 (t, J=8.8 Hz, 2H),
6.51–6.58 (m, 1H), 5.67 (d, J=15.6 Hz, 1H), 3.84–3.92 (m, 1H),
3.40–3.56 (m, 3H), 3.08–3.27 (m, 2H), 2.91 (s, 1H), 2.41–2.61 (m,
4H), 2.06–2.21 (m, 2H), 1.41–1.95 ppm (m, 8H); 13C NMR (400 MHz,
CD3OD): d=171.9, 167.9, 143.1, 132.2, 131.6, 131.6, 128.2, 117.5,
117.2, 64.5, 61.2, 57.1, 57.0, 47.1, 41.4, 40.1, 36.6, 34.9, 34.4, 27.9,
20.8 ppm; FTIR (KBr): n˜ =3069, 2936, 2866, 1703, 1654, 1591, 1493,
1398, 1333, 1152, 1090, 981, 839 cmÀ1; HRMS (ESI): m/z calcd for
C21H28FN2O4S [M+H]+: 423.1748; found: 423.1758.
Anhydrous K2CO3 (3.5 equiv) and benzoyl chloride, benzyl chloride
(bromine), or sulfonyl chloride (1.5 equiv) were added to a solution
of compound 14 in dichloromethane or MeCN (30 mL), and the re-
action solution was then stirred at room temperature until TLC
analysis showed completion of the reaction. The reaction mixture
was filtered, and the filtrate was evaporated to afford crude com-
pound 15, which was further purified by flash column chromatog-
raphy on silica gel with dichloromethane and methanol as the elu-
ents.
(E)-12-N-(p-Chlorobenzenesulfonyl)-Dab-sophocarpinic
(12h): White solid (1.6 g, 7%): mp: 116–1188C; H NMR (400 MHz,
CD3OD): d=7.79 (d, J=8.8 Hz, 2H), 7.51 (d, J=8.8 Hz, 2H), 6.53–
acid
1
Compound 15 was dissolved in 3n HCl (15 mL), and the mixture
was heated at reflux for 3 h. TLC analysis showed completion of
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the reaction. The pH value of the reaction solution was then ad-
justed to 8–9 by addition of NH4OH. The reaction solvent was re-
moved under reduced pressure, and the residue was dissolved in
MeOH and filtered to remove the organic salts. The solution was
concentrated to obtain a crude compound, which was further puri-
fied by flash column chromatography on silica gel with dichloro-
methane and methanol as the eluents to afford compounds
16a–e.
without different concentrations of test compounds. The viral cyto-
pathic effect was observed when the viral control group reached
4+, and the antiviral activity of the tested compounds was deter-
mined by the Reed and Muench analyses.
Cytotoxicity assay: The cytotoxicity of the compounds in the pres-
ence of Vero cells was monitored by a CPE assay. Vero cells (3ꢁ104
in each well) were plated into a 96-well plate. After 24 h, the mon-
olayer cells were incubated in the presence of various concentra-
tions of test compounds. After 48 h of culture at 378C and 5% CO2
in a carbon dioxide incubator, the cells were monitored with the
CPE assay. The median TC50 value was calculated by Reed and
Muench analyses.
(Z)-12-N-(p-Methoxybenzyl)-Dbg-sophocarpinic acid (16a): White
1
solid (1.00 g, 21%): mp: 141–1428C; H NMR ([D6]DMSO, 400 MHz):
d=7.21 (d, J=8.4 Hz, 2H), 6.87 (d, J=8.4 Hz, 2H), 5.87–5.81 (m,
1H), 5.38 (t, J=10.4 Hz, 1H), 4.03 (d, J=12.8 Hz,1H), 3.73 (s, 3H),
3.39–3.15 (m, 3H), 2.89–2.85 (d, J=13.2 Hz, 1H), 2.72–2.75 (t, J=
13.2 Hz, 2H), 2.51–2.44 (m, 1H), 2.21–2.18 (d, J=4.0 Hz, 1H), 1.85
(s, 1H), 1.84–1.75 (dd, J=11.2 Hz, 2H), 1.65–1.32 ppm (m, 10H);
HRMS (ESI): m/z calcd for C23H32N2O3 [M+H]+: 385.2505; found:
385.2486.
Pharmacokinetic evaluation of compounds 11 l, 11 m, and 11 n in
vivo: Animals were purchased from the Institute of Laboratory
Animal Sciences, Chinese Academy of Medical Sciences and Peking
Union Medical College, Beijing, China. All experimental procedures
were approved by the Biomedical Ethics Committee of the Chinese
Academy of Medical Sciences for Animal Use and Protection. The
rats were cared for according to the institutional guidelines of the
Chinese Academy of Medical Sciences. Three male SD rats with
weights in the range of 180–220 g were used in each study. Each
of these rats was administered orally with compounds 11 l, 11 m,
or 11 n at a concentration of 25 mgkgÀ1. Blood samples (0.3 mL)
were collected at 0, 0.25, 0.5, 1.0, 1.5, 2.0, 4.0, 6.0, and 24 h and
were immediately centrifuged to separate the plasma fraction. The
plasma samples obtained were stored at À208C until analysis. Con-
centration–time profiles were obtained for each analyte, and stan-
dard noncompartmental analysis was performed on the data by
using WinNonlin, Version 5.3,[17] to determine the AUC and other
noncompartmental parameters. Data for each pharmacokinetic
parameter were averaged and reported as the mean Æ standard
deviation.
(Z)-12-N-p-(Aminobenzenesulfonyl)-Dbg-sophocarpinic
acid
(16b): White solid (1.05 g, 21%): mp: 165–1668C; 1H NMR
([D6]DMSO, 400 MHz): d=7.29 (d, J=8.4 Hz, 2H), 6.61 (d, J=8.8 Hz,
2H), 6.06 (s, 2H), 5.61–5.54 (m,1H), 5.46 (t, J=10.4 Hz, 1H), 3.62–
3.24 (m, 5H), 3.17 (s, 1H), 3.00–2.65 (m, 4H), 2.07–1.79 (m, 3H),
1.67–1.24 ppm (m, 7H); HRMS (ESI): m/z calcd for C21H29N3O4S
[M+H]+: 420.1971; found: 420.1952.
(Z)-12-N-(m-Nitrobenzyl)-Dbg-sophocarpinic acid (16c): White
1
solid (1.37 g, 28%): mp: 206–2088C; H NMR ([D6]DMSO, 400 MHz):
d=8.44 (s, 1H), 8.30 (d, J=8.4 Hz, 1H), 8.05 (d, J=7.6 Hz, 1H), 7.75
(t, J=8.0 Hz, 1H), 6.29–6.22 (m, 1H), 5.83 (t, J=10.4 Hz, 1H), 5.31
(d, J=9.2 Hz, 1H), 4.76 (d, J=12.4 Hz, 1H), 4.08–4.03 (m, 2H), 3.66–
3.17 (m, 6H), 3.00–2.91 (m, 3H), 2.62–2.50 (m, 2H), 1.91–1.56 ppm
(m, 7H); 13C NMR (CD3OD, 400 MHz): d=171.8, 147.8, 138.0, 134.29,
131.6, 130.4, 126.3, 124.4, 124.3, 59.6, 58.7, 56.5, 54.3, 54.1, 47.7,
35.3, 33.7, 30.3, 24.0, 23.9, 18.1, 17.9 ppm; HRMS (ESI): m/z calcd
for C22H29N3O4 [M+H]+: 400.2222; found: 400.2231.
Safety evaluation of compound 11 m in vivo: Male and female
Kunming mice with weights between 18–20 g were purchased
from the Laboratory Animal Center of the Academy of Military
Medical Sciences (Beijing, China). The mice were randomly divided
into four groups with ten mice each (five male and five female).
After fasting for 5 h before drug administration, the four groups of
mice orally received compound 11 m at concentrations of 0 (1%
polysorbate-80 in saline as a control), 250, 500, or 1000 mgkgÀ1, re-
spectively. Compound 11 m was given in a single dose. The body
weight and survival were monitored. Seven days later, blood sam-
ples were taken for liver- and kidney-function examinations.
(Z)-12-N-(m-Trifluoromethylbenzenesulfonyl)-Dbg-sophocarpinic
acid (16d): White solid (1.01 g, 18%): mp: 137–1388C; 1H NMR
([D6]DMSO, 400 MHz): d=8.05 (d, J=8.0 Hz, 2H), 7.91 (s, 1H), 7.82
(t, J=8.0 Hz, 1H), 5.62–5.55 (m, 1H), 5.43 (t, J=10.4 Hz, 1H), 3.91–
3.83 (m, 3H), 3.54–3.36 (m, 2H), 3.17 (s, 1H), 3.03–2.73 (m, 3H),
2.37 (d, J=16.4 Hz, 1H), 2.03–1.24 ppm (m, 10H); HRMS (ESI): m/z
calcd for C22H27F3N2O4S [M+H]+: 473.1738; found: 473.1716.
(Z)-12-N-(p-Trifluoromethylbenzenesulfonyl)-Dbg-sophocarpinic
acid (16e): White solid (1.59 g, 27.6%): mp: 134–1368C; 1H NMR
([D6]DMSO, 400 MHz): d=7.96–7.90 (m, 4H), 5.64–5.59 (m, 1H),
5.48 (t, J=10.8 Hz, 1H), 4.51 (d, J=9.2 Hz, 1H), 3.93–3.82 (m, 2H),
3.59–3.17 (m, 2H), 2.95–2.86 (m, 3H), 2.54–2.34 (m, 1H), 2.25 (d,
J=8.4 Hz, 1H), 1.91–1.43 ppm (m, 10H); HRMS (ESI): m/z calcd for
C22H27F3N2O4S [M+H]+: 473.1735; found: 473.1716.
Acknowledgements
This work was supported by the Chinese National Mega-Project
for Innovation Drugs (2012ZX09301002-001-017).
Biological methods
Keywords: antiviral agents · druggability · enteroviruses ·
sophocarpinic acids · structure–activity relationships
Anti-CVB3 and anti-CVB6 assays: Vero cells (African green monkey
kidney cells, ATCC accession no. CCL-81) were obtained from the
American Type Culture Collection. CVB3 and CVB6 (Nancy strain) vi-
ruses were all from the Institute of Virology, Chinese Academy of
Preventive Medicine. Confluent Vero cells grown in 96-well micro-
plates were infected with 100 median tissue culture infective doses
of CVB3 or CVB6, respectively. After 1 h of adsorption at 378C, the
monolayers were washed with phosphate-buffered saline (PBS)
and incubated at 378C in the maintenance medium (minimum es-
sential medium (MEM) plus 2% fetal bovine serum (FBS)) with or
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ChemMedChem 2013, 8, 1545 – 1553 1553