Design, synthesis, and biological evaluation of 1,2,4-triazole bearing 5-substituted biphenyl-2-sulfonamide derivatives as potential antihypertensive candidates

Article abstract of DOI:10.1016/j.bmc.2013.10.017

A series of novel 1,2,4-triazole bearing 5-substituted biphenyl-2- sulfonamide derivatives were designed and synthesized to develop new angiotensin II subtype 2 (AT₂) receptor agonists as novel antihypertensive candidates. It was found that 14f (IC₅₀ = 0.4 nM) and 15e (IC ₅₀ = 5.0 nM) displayed potent AT₂ receptor affinity and selectivity in binding assays. Biological evaluation in vivo suggested that 14f is obviously superior to that of reference drug losartan in RHRs, and meanwhile, 14f has no significant impact on heart rate. The interesting activities of these compounds may make them promising candidates as antihypertensive agents.

Full text of DOI:10.1016/j.bmc.2013.10.017

Bioorganic & Medicinal Chemistry 21 (2013) 7742–7751
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and biological evaluation of 1,2,4-triazole bearing
5-substituted biphenyl-2-sulfonamide derivatives as potential
antihypertensive candidates
Jie Liu a,c, Qin Liu , Xue Yang a,b, Shengtao Xu , Hengyuan Zhang a,b, Renren Bai a,b, Hequan Yao a,b,
⇑
,
a,b
a,b
d
e
a,b
a,b,
⇑
Jieyun Jiang , Mingqin Shen , Xiaoming Wu , Jinyi Xu
a
State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China
Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China
Department of Organic Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China
Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, 800 Rose Street, Lexington, KY 40536, USA
Jiangsu Provincial Institute of Traditional Chinese Medicine, Nanjing 210028, PR China
b
c
d
e
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 1,2,4-triazole bearing 5-substituted biphenyl-2-sulfonamide derivatives were designed
and synthesized to develop new angiotensin II subtype 2 (AT ) receptor agonists as novel antihyperten-
sive candidates. It was found that 14f (IC50 = 0.4 nM) and 15e (IC50 = 5.0 nM) displayed potent AT recep-
tor affinity and selectivity in binding assays. Biological evaluation in vivo suggested that 14f is obviously
superior to that of reference drug losartan in RHRs, and meanwhile, 14f has no significant impact on heart
rate. The interesting activities of these compounds may make them promising candidates as antihyper-
tensive agents.
Received 6 September 2013
Revised 12 October 2013
Accepted 14 October 2013
Available online 23 October 2013
2
2
Keywords:
Antihypertensive activity
AT receptor
2
Ó 2013 Elsevier Ltd. All rights reserved.
Agonists
Biphenyl-2-sulfonamide derivatives
Structure–activity relationships
1
. Introduction
may support a significant involvement of the AT
development. In addition, most remarkably, there is substantial
evidence that the AT receptor can offset or counteract the effects
mediated by the AT receptor. Correspondingly, AT receptor
played the role of a kind of ‘natural AT
On the basis of these functions of AT
posed that the AT
2
receptor in fetal
The renin angiotensin system (RAS) plays a key role in blood
2
1
pressure regulation and electrolyte homeostasis. The octapeptide
angiotensin II (Ang II, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe), recog-
nized as the most important bioactive peptide of the RAS, is the
1
2
1
receptor antagonist.’
receptor, it has been pro-
2
receptor could be an important target in the
2
endogenous activator of the angiotensin II subtype 1 (AT
1
) and
the angiotensin II subtype 2 (AT ) receptors. The AT receptor
mediates the well-known physiological effects of Ang II, such as
2
1
therapeutic area of hypertension and cardiac remodeling. Anders
Hallberg initiated a research program aiming to identify non-
vasoconstriction, aldosterone release, stimulation of sympathetic
peptide and drug-like AT
AT receptor agonist L-162,313 was selected as the lead structure.
L-162,313 is a nonpeptidic structure that shows similar affinities to
both the AT receptor and the AT receptor. Furthermore, the com-
pound has been proven to act as an agonist at both the AT recep-
Recently, the first nonpeptidic
receptor agonist M024 was designed by introducing
2
receptor agonists. The nonselective
transmission, and cellular growth.2 AT
–4
receptor antagonists
1
1
(
the Sartans) are currently used as effective clinical antihyperten-
sive drugs. The function of the AT
2
receptor subtype, which was
1
2
cloned more recently and found to share only 32–34% sequence
identity with the AT receptor remains elusive and somewhat con-
1
1
3–15
1
tor and at the AT
selective AT
2
receptor.
5
,6
troversial. It has been suggested that it plays a role in mediating
antiproliferation, cellular differentiation, apoptosis, and vasodila-
2
a small unsubstituted imidazole in the benzylic position of the
L-162,313. The unsubstituted imidazole provided a good moiety
7
–10
tion.
One notable feature of the AT
2
receptor is the high level
of expression in most fetal tissues, including the brain. The AT
2
/
1 2
to obtain high affinity, AT /AT selectivity as well as agonism
(Fig. 1).
receptor ratio decreases dramatically after birth,1
1,12
which
16,17
AT
1
Meanwhile, the compounds where the thiophene structure has
been replaced with a phenyl maintain affinity and selectivity to-
wards the AT receptor. For example, the compound L-162,782
2
⇑
Corresponding authors. Tel.: +86 025 83271042 (H.Y.), +86 025 83271299 (J.X.).
E-mail addresses: hyao@cpu.edu.cn (H. Yao), jinyixu@china.com (J. Xu).
1
8
0
968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.10.017

J. Liu et al. / Bioorg. Med. Chem. 21 (2013) 7742–7751
7743
N
were achieved. One is by introducing 1,2,4-triazole fragment into
the benzylic position of substituted diphenyl sulfonamide scaffold,
as well as alteration of the sulfonylcarbamate part (Series I), and
the other is the replacement of the bicyclic imidazopyridine ring
with amino groups (Series II). Herein, we would like to report some
N
N
N
O
O
O
S
N
N
2
of the synthesized compounds with good AT receptor affinity and
H
O
S
selectivity including ligands proven to serve as AT
(Fig. 2).
2
agonists
O
O
O
IC50(nM)
S
M024 AT
:>10000
: 0.4
1
2
N
O
AT
H
S
agonist(AT
2
)
2. Results and discussions
2.1. Chemistry
IC50 ( nM )
L-162,313 AT
1
: 1.1
: 2.0
N
AT
2
agonist (AT
1
/AT
2
)
N
N
S
The synthetic route of the sulfonamides (3a–b, 5 and 7) is
shown in Scheme 1. The isobutyl- and methoxyphenylsulfonyl
chloride (2a–b) were synthesized from their corresponding isobu-
tyl- or methoxybenzene (1a–b) with chlorosulfonic acid, while the
methylphenylsulfonyl chloride (4) was commercially available.
These alkyl- or alkoxyphenylsulfonyl chlorides were transformed
into the corresponding t-butyl protected sulfonamides (3a–b and
O
O
O
N
H
O
IC50(nM)
L-162,782 AT
1
: 2.1
: 0.7
AT
2
2 2
5) in excellent yield by treatment with t-butylamine in CH Cl .
Meanwhile, compound 6 was achieved from compound 5 through
agonist (AT
1
/AT
2
)
a regioselective bromination with NBS in quite high yield. Subse-
1 2
Figure 1. The structures of known AT /AT agonists.
2 2
quent N-alkylation of 6 with diethylamine in CH Cl gave another
t-butyl protected sulfonamide 7.
showed the high affinities to the AT
Fig. 2).
Previously, we designed and synthesized a series of 1,2,4-
triazole derivatives with N-phenylpyrrolyl-2-tetrazole moiety.
Among them, compound ATPT was found to be an orally active
1 2
receptor and AT receptor
The benzeneboronic acids 8a–b, the key intermediates for the
synthesis of the compounds in both series, were prepared in gen-
erally good yields through two steps (Scheme 2). Treatment of
the t-butyl protected sulfonamides (3a–b, 5 and 7) with two equiv-
alents of n-BuLi formed the dianions. These anions resided at the
(
1
AT receptor antagonist, and also found to be more potent than
1
9
0
position 2 as directed there by the sulfonamide. The anions were
losartan. Furthermore, we disclosed the synthesis and biological
0
quenched with triisopropylborate and worked up with dilute acid
to afford the boronic acid products.
The compounds in series I were prepared as outlined in
Scheme 3. The 4-bromo-benzyl-1,2,4-triazole (11) was obtained
evaluation of 4 -[(benzimidazole-1-yl)-methyl]biphenyl-2-sulfon-
2
0
amides derivatives. In an effort to find novel drugs acting on
RAS, we became interested in AT receptor agonists with good
2
affinity and selectivity, and focused on the researches of deriva-
tives with diphenyl sulfonamide scaffold. On the basis of SAR infor-
2 3
by treating the 1,2,4-triazole (10) with K CO in THF and subse-
2
1
quently adding 4-bromobenzylbromide (9) to give the desired
compound in moderate yield. Compound 11 were then coupled
with the benzeneboronic acids 8a–d under Suzuki conditions with
mation of M024, especially we noticed that the replacement of
the imidazole in compound M024 with various substituted or
unsubstituted heterocycles rendered analogues with high AT
2
2
2
2 3
Pd(OAc) and PPh as catalyst and with NaOH as base to give the
receptor affinity. We decided to use compound L-162,782 as
the lead structure, meanwhile the unsubstituted 1,2,4-triazole
group was chosen as replacement for the imidazopyridine ring in
the compound L-162,782, therefore two modification strategies
tert-butyl protected compounds 12a–d in 26–87% yields. Deprotec-
tion by TFA, to give the primary sulfonamides (13a–d) followed by
reaction with the selected alkyl chloroformates, acyl chlorides and
N
N
N
N
O
O
O
N
N
S
N
H
R2
R1
Seriesൕ
PhCl
2
O
O
N
N
O
N
N
S
N
N
NH
H
O
n-Bu
O
N
R3
N
HN
ATPT
L-162,782
IC50(nM)
IC50(nM)
O
O
O
AT
AT
1
2
: 2.5
: ᧺10000
AT
AT
1
2
: 2.1
: 0.7
S
N
H
antagonist
O
agonist (AT
1
/AT
2
)
1 2
(AT /AT )
Seriesൖ
Figure 2. Strategy for the design and optimization of target AT
2
agonists.

7
744
J. Liu et al. / Bioorg. Med. Chem. 21 (2013) 7742–7751
O
benzoyl chlorides, afforded 14a–g, 15a–f, 16a–f and 17a–c
respectively.
The synthesis of series II, outlined in Scheme 4, started with a
set of different N-monoalkylated 4-bromoanilines (18a–d), which
were prepared by selective N-monoalkylation of 4-bromoaniline
O
O
O
S
S
Cl
a
b
N
H
R1
R1
R1
1
a-b
2a-b
3a-b
(
18a) with alcohols in excellent yields using triphenylphosphine
(PPh and 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) in
dichloromethane at room temperature.
R1=
,
3
)
O
2
3
O
Bromoanilines (18a–d) were then coupled with the isobutyl-
benzeneboronic acid 8a, prepared under Suzuki conditions with
O
O
O
S
24
O
O
O
S
b
S
N
c
Cl
N
H
Br
H
2 3
Pd(OAc) and PPh as catalyst and with NaOH as base to give the
tert-butyl protected compounds 19a–d in moderate yields.
5
6
4
2
5
Deprotection of compounds 19a–d by TFA, a milder deprotecting
reagent, delivered the primary sulfonamides (20a–d) that were
subsequently treated with n-butyl chloroformate, at ambient tem-
perature, in pyridine to afford the target compounds of series II
O
S
N
d
H
Et
2
N
7
(
21a–d).
Scheme 1. Synthesis of compounds 3a–b and 7. Reagents and conditions: (a)
3
chlorosulfonic acid, CHCl , rt, 1 h; (b) t-BuNH , CH Cl , rt, 8 h; (c) NBS, AIBN, CHCl ,
reflux, 4 h; (d) diethylamine, CH Cl , rt, 13 h.
2 2
3
2
2
2
2.2. Pharmacological evaluation
2
.2.1. Binding assays
All target compounds were evaluated in radioligand-binding
HO
OH
O
O
O
B
125
O
assays by displacement of [ I] Ang II from AT
1
receptors in rat
S
N
H
S
a, b
N
liver membranes and from AT receptors in pig uterus mem-
2
H
26,27
R1
branes.
The natural substrate Ang II and the selective AT
receptor antagonist losartan were used as reference substances.
The affinity results are presented in Table 1.
R1
1
8
2
3
a, 3b, 5, 7
8a-d
N
In series I all compounds exhibited a broad range (potent to
inactive) of affinity for AT receptor and none of the compounds
possessed any affinity for the AT receptor (Table 1). However,
R1=
O
2
1
Scheme 2. Synthesis of compounds 8a–d. Reagents and conditions: (a) n-BuLi, THF,
78 °C, then ꢀ20 °C, 4 h; (b) triisopropylborate, ꢀ78 °C, then rt overnight.
none of the compounds in series II encompassing amine moiety
ꢀ
N
N
N
N
Br
N
H
N
N
N
a
b
+
O
N
O
S
N
Br
9
H
Br
11
R1
10
1
2a-d
N
N
N
N
N
N
O
O
O
O
c
O
d
S
S
R2
N
NH
2
H
R1
R1
1
3a-d
Series ൕ
R1=
R1=
2
1
4a-g:
15a-f:
O
2
R = Et, n-Bu, OEt, OBu-t, OBu-n, OCH
2
Ph
3 2
R = Et, n-Bu, 4-CH Ph, OEt, OBu-t, OBu-n,OCH Ph
N
R1=
1
6a-f:
R1=
2
17a-c:
2
R = Et, n-Bu, OEt, OBu-t, OBu-n, OCH
2
Ph
R = OBu-t, OBu-n, OCH
2
Ph
Scheme 3. Synthesis of compounds 14–17. Reagents and conditions: (a) K
2 h; (d) alkyl chloroformates or benzoyl chlorides, pyridine, 0 °C, 4 h.
2
CO
3
, THF, reflux, 15 h; (b) Pd(OAc)
2
, PPh
3
, NaOH (aq), 8a–d, ethanol/toluene, reflux, 5 h; (c) TFA, rt,
1

J. Liu et al. / Bioorg. Med. Chem. 21 (2013) 7742–7751
7745
R3
NH
2
HN
a
Br
Br
18a
18b-d
R3
R3
R3
HN
HN
HN
R3
HO
OH
O
HN
B
O
S
N
O
+
H
b
c
d
O
O
O
O
O
O
S
S
S
N
O
N
NH
2
H
Br
8a-d
H
1
8a
1
9a-d
20a-d
Series II
3
2
1a-d R = H, CH
3 3 2
, Et, (CH ) CH
Scheme 4. Synthesis of compounds 21a–d. Reagents and conditions: (a) alcohols, DDQ, PPh
TFA, rt, 12 h; (d) butyl chloroformate, pyridine, 0 °C, 4 h.
3
2
, CH Cl
2
, rt, 2 h; (b) Pd(OAc)
2
, PPh , NaOH (aq), ethanol/toluene, reflux, 5 h; (c)
3
showed any AT
1
affinity nor AT
2
affinity. It is suggested that intro-
(SAP) and diastolic arterial pressure (DAP) of compound 15e was
approximately equivalent to that of losartan at the same dose.
Meanwhile, the maximum antihypertensive effect on SAP and
DAP of compound 14f was prominently more effective than that
of losartan. Furthermore, the decreases in MAP by both compounds
were significant and the reduction in MAP by 14f was even more
than that of losartan. Heart rates (HR) recorded taken from SHRs
proved that 14f and 15e, just the same as losartan, did not cause
noticeable changes of HR. (Fig. 3) Preliminary in vivo biological
evaluation showed that both compounds 14f and 15e were prom-
ising enough to warrant further investigation.
duction of unsubstituted 1,2,4-triazole group into the benzylic po-
sition of substituted diphenyl sulfonamide scaffold provided good
AT
2
selectivity and affinity.
The activities of compounds 14a–g with isobutyl substituent at
the 5-position changed obviously with modifications of the sulfon-
amide side chain. Compound 14f possessing n-butyloxy group
clearly demonstrated the most powerful AT affinity (IC50 = 0.4
2
nM). Removing the carbamate oxygen from the side chain gave a
compound (compound 14b) with an 80-fold decrease in affinity
for the AT
of the butyloxy group rendered 25 or 140 times decrease of the
affinity for the AT receptor (compounds 14d and 14e), and sur-
2
receptor as compared to 14f. Shortening or branching
2
3
. Conclusions
prisingly, introduction of the phenyl groups resulted in totally
inactive compounds (compounds 14c and 14g). Shortening of the
n-butyl group (compound 14a) also brought a twice decrease in
In summary, two series of derivatives with diphenyl sulfon-
amide scaffold were designed and synthesized to develop new
antihypertensive agents with good AT receptor affinity and selec-
tivity. One series of compounds, introducing of unsubstituted
,2,4-triazole group into the benzylic position of substituted diphe-
nyl sulfonamide scaffold, exhibited a broad range affinity for AT
receptor. However, none of the compounds in another series,
encompassing amine moiety, showed any AT affinity nor AT
affinity. Compound 14f (IC50 = 0.4 nM) and 15e (IC50 = 5.0 nM)
displayed potent AT receptor affinity and selectivity in binding
2
affinity for the AT receptor as compared to 14b. These trends were
2
also observed after replacing the isobutyl substituent with either
methoxy group as in 15a–f, or methyl group in 16a–f, or diethyl-
aminomethyl group in 17a–c. On the other hand, in the case of
constant sulfonamide side chain, change of substituents at the
1
2
5
-position of the diphenyl scaffold could also affect the activities
of these compounds. For example, among the compounds 14f,
5e, 16e and 17b, all of these compounds with the n-butyloxy
group on sulfonamide side chain, 14f showed best AT affinity, fol-
1
2
1
2
2
assays. Biological evaluation in vivo suggested that 14f is more
potent and efficacy than losartan in RHRs, and meanwhile, 14f
has no significant impact on heart rate. It is suggested that merging
together unsubstituted 1,2,4-triazole moiety with the biphenyl
sulfonamide scaffold, was a successful design strategy to obtain
lowed the 16e and 17b had the lowest affinities with IC50 value 16
and 10 nM, respectively. And the potent order of substituent was
2
as follows: i-bu > OMe > MeNEt > Me. This order was observed
after the alteration of the substituent on sulfonamide side chain.
the bioactive conformation of selective nonpeptide AT
2
receptor
agonists.
2
1
.2.2. In vivo antihypertensive activities of compounds 14f and
5e
In vitro biological evaluation, compounds 14f and 15e showed
4. Experimental
4.1. Chemistry
potent AT
2
receptor affinity and selectivity in binding assays
among the titled compounds. Therefore, compounds 14f and 15e
were selected for further evaluation of antihypertensive effects in
SHRs (Table 2). After oral administration of losartan (20 mg/kg),
Reagents and all solvents were purchased from Shanghai Chem-
ical Reagent Company and used without further purification. All of
the experiments were monitored by analytical thin-layer chroma-
tography (TLC) performed on silica gel GF254 precoated
plates. Column chromatography was carried out using silica gel
(200–300 mesh). The purities of all of the compounds (>95%) used
1
4f (20 mg/kg) and 15e (20 mg/kg) to SHRs, the blood pressure
and heart rate were determined from 0 to 8 h. The results showed
that 14f and 15e reduced the blood pressure significantly and these
trends continued throughout the remaining time of the study. The
maximum antihypertensive effect on systolic arterial pressure

7
746
J. Liu et al. / Bioorg. Med. Chem. 21 (2013) 7742–7751
Table 1
Structures and binding assays for the AT
1
receptor and AT
2
receptor of target compounds
N
N
N
O
O
O
S
N
R2
H
R1
Compound
R1
R2
AT
2
receptor IC50 (nM)
1
AT receptor IC50 (nM)
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
4a
4b
4c
4d
4e
4f
4g
5a
5b
5c
5d
5e
5f
6a
6b
6c
6d
6e
6f
7a
7b
7c
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
i-Bu
OMe
OMe
OMe
OMe
OMe
OMe
Me
Et
n-Bu
4-MePh
OEt
OBu-t
OBu-n
OCH
Et
n-Bu
OEt
OBu-t
OBu-n
OCH
Et
n-Bu
OEt
OBu-t
OBu-n
OCH
OEt
OBu-n
OCH Ph
68 ± 5.2
31 ± 4.5
>10,000
10 ± 0.9
55 ± 3.4
0.4 ± 0.07
>10,000
184 ± 11
99 ± 10
36 ± 2.9
170 ± 12
5.0 ± 0.7
>10,000
223 ± 21
106 ± 14
64 ± 8.5
289 ± 18
16 ± 0.9
>10,000
117 ± 14
10 ± 1.1
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
2
Ph
2
Ph
Me
Me
Me
Me
Me
2
Ph
Et
2
Et
2
Et
2
NCH
NCH
NCH
2
2
2
2
R3
HN
O
O
O
S
N
H
O
Compound
R3
AT
2
receptor IC50 (nM)
1
AT receptor IC50 (nM)
2
2
2
2
1a
1b
1c
1d
H
Me
Et
>10,000
>10,000
>10,000
>10,000
>10,000
29 ± 3.0
>10,000
>10,000
>10,000
>10,000
85 ± 6.5
>10,000
Pr-n
Losartan
PD123,319
for biological screening were determined by high-performance
liquid chromatography (HPLC) (Agilent, 1260 INFINITY) using a
water (30 mL) and the resulted mixture was extracted twice with
CH Cl (20 mL). The combined organic layer was washed with
4
saturated brine (30 mL) twice and dried over anhydrous MgSO ,
and the solvent was evaporated to give the corresponding com-
pound 2a or 2b.
2
2
Gemini C18 column (4.6 ꢁ 150 mm, 5
l
m) eluted with a gradient
mixture of acetonitrile/water (1:2). H NMR spectra were recorded
on Bruker-ACF spectrometer (300 or 500 MHz) in DMSO-d . Chem-
ical shifts are reported in ppm (d) relative to tetramethylsilane
TMS) as an internal standard. Multiplicities are given as s (singlet),
d (doublet), dd (double-doublet), t (triplet), q (quadruplet), m
1
6
(
4.1.2. General procedure for synthesis of compounds 3a, 3b
and 5
ꢀ1
(
multiplet) and br s (broad signal). IR (in cm ) spectra in KBr pel-
The respective sulfonylchloride (15 mmol) was dissolved in dry
lets on a Bruker FT-IR TENSOR 27 instrument. Mass spectra were
recorded on Agilent LC/MSD TRAP SL spectrometer equipped with
an electrospray ionisation (ESI) interface; High-resolution mass
spectra were recorded using Agilent QTOF 6520. Melting points
were determined using MEL-TEMP II melting point apparatus and
are uncorrected.
2 2
CH Cl (30 mL). The mixture was cooled on an ice bath and
tert-butylamine (15 mmol) was added dropwise. After completion
of addition, the reaction mixture was left stirring at rt for 8 h. The
reaction mixture was diluted with CH
with water (30 mL ꢁ 2) and brine (30 mL ꢁ 2). The organic layer
was dried with anhydrous MgSO , filtered, and evaporated to
2 2
Cl (20 mL) and washed
4
achieve the pure compounds 3a, 3b and 5 without further
4
.1.1. General procedure for synthesis of compounds 2a–b
purification.
Chlorosulfonic acid (0.1 mol) was added slowly to a solution of
corresponding isobutyl- or methoxybenzene (1a or 1b)
25.0 mmol) in CHCl (30 mL) in an ice cold bath and the mixture
was stirred at rt for 45 min. The reaction was poured into ice-cold
4.1.3. 4-(Bromomethyl)-N-tert-butylbenzenesulfonamide (6)
(
3
Compound 5 (12.6 mmol) was dissolved in CHCl
3
(40 mL), then
NBS (10.1 mmol) and AIBN (0.288 mmol) were added respectively.

J. Liu et al. / Bioorg. Med. Chem. 21 (2013) 7742–7751
7747
Table 2
Effects on blood pressure in renal antihypertensive rats
Group
Index
Before administration
After administration
1
h
2 h
4 h
6 h
8 h
Control
SAP (mmHg)
DAP (mmHg)
MAP (mmHg)
HR (BPM)
169.01 ± 9.18
D
137.15 ± 9.31
D
147.77 ± 8.64
D
387.22 ± 15.36
D
167.68 ± 15.89
ꢀ1.33
169.99 ± 14.64
0.98
140.36 ± 13.51
3.21
150.24 ± 13.72
2.47
378.56 ± 22.98
ꢀ8.66
162.82 ± 13.38
ꢀ6.19
163.63 ± 13.04
ꢀ5.38
168.33 ± 20.27
ꢀ0.68
134.31 ± 14.11
ꢀ2.84
135.41 ± 14.66
ꢀ1.74
132.87 ± 6.06
ꢀ4.28
135.06 ± 13.23
ꢀ2.09
145.44 ± 14.33
ꢀ2.33
144.54 ± 13.79
ꢀ3.23
143.12 ± 7.26
ꢀ4.65
146.14 ± 14.74
ꢀ1.63
384.07 ± 18.09
ꢀ3.15
378.15 ± 22.25
ꢀ9.07
379.94 ± 21.56
ꢀ7.28
399.88 ± 40.05
12.66
Losartan
SAP (mmHg)
DAP (mmHg)
MAP (mmHg)
HR (BPM)
178.10 ± 10.5
D
150.15 ± 12.4
D
155.23 ± 13.46
D
389.65 ± 12.1
D
175.30 ± 4.12
ꢀ2.80
172.89 ± 10.1
ꢀ5.21
162.55 ± 11.6
157.87 ± 14.3
164.08 ± 12.1
⁄
⁄⁄
⁄
ꢀ15.55
ꢀ20.23
ꢀ14.02
142.39 ± 10.04
ꢀ7.76
141.19 ± 9.6
ꢀ8.96
135.90 ± 9.6
132.57 ± 8.1
137.06 ± 10.7
ꢀ13.09
⁄
⁄⁄
ꢀ14.25
ꢀ17.58
152.18 ± 9.85
ꢀ3.05
147.90 ± 10.87
ꢀ7.33
140.10 ± 12.19
136.11 ± 15.24
140.68 ± 10.74
⁄
⁄⁄
⁄
ꢀ15.13
ꢀ19.12
ꢀ14.55
387.65 ± 8.35
ꢀ2.00
382.55 ± 22.1
ꢀ7.10
379.08 ± 28.2
375.60 ± 12.2
376.45 ± 18.9
ꢀ10.57
ꢀ14.05
ꢀ13.20
1
4f
SAP (mmHg)
DAP (mmHg)
MAP (mmHg)
HR (BPM)
171.89 ± 13.91
D
142.45 ± 13.71
D
152.26 ± 14.96
D
383.92 ± 32.21
D
164.64 ± 11.26
ꢀ7.25
167.63 ± 17.89
ꢀ4.26
157.60 ± 15.50
150.95 ± 14.82
156.67 ± 15.44
⁄
⁄⁄
⁄⁄
ꢀ14.29
ꢀ20.94
ꢀ15.22
147.38 ± 10.22
4.93
138.90 ± 13.54
ꢀ3.55
130.13 ± 12.28
121.09 ± 15.10
128.35 ± 15.40
⁄
⁄⁄
⁄
ꢀ12.32
ꢀ21.36
ꢀ14.10
156.69 ± 7.97
4.43
391.17 ± 27.46
7.25
147.48 ± 14.19
ꢀ4.78
138.10 ± 12.19
130.38 ± 14.69
137.17 ± 14.37
⁄
⁄⁄
⁄⁄
ꢀ14.16
ꢀ21.88
ꢀ15.09
378.90 ± 27.91
ꢀ5.02
369.39 ± 27.37
367.23 ± 45.90
369.59 ± 50.47
ꢀ14.53
ꢀ16.69
ꢀ14.33
1
5e
SAP (mmHg)
DAP (mmHg)
MAP (mmHg)
HR (BPM)
175.12 ± 10.34
D
144.67 ± 11.09
D
155.34 ± 13.58
D
385.13 ± 30.11
D
171.71 ± 4.12
ꢀ3.41
170.86 ± 14.39
ꢀ4.26
164.41 ± 13.20
156.18 ± 11.60
165.02 ± 12.09
⁄
⁄⁄
⁄
ꢀ10.71
ꢀ18.94
ꢀ10.10
140.46 ± 11.55
ꢀ4.21
139.61 ± 12.02
ꢀ5.06
134.35 ± 13.70
127.31 ± 14.58
131.57 ± 12.06
⁄
⁄⁄
⁄
ꢀ10.32
ꢀ17.36
ꢀ13.1
151.25 ± 8.24
ꢀ4.09
149.72 ± 12.61
ꢀ5.62
145.19 ± 10.66
138.26 ± 15.04
143.15 ± 10.41
⁄
⁄⁄
⁄
ꢀ10.15
ꢀ17.08
ꢀ12.19
392.18 ± 24.66
7.05
377.49 ± 28.31
ꢀ7.64
371.68 ± 21.80
370.66 ± 32.38
362.23 ± 37.69
ꢀ13.45
ꢀ14.47
ꢀ22.90
Each value represents the mean ± SEM (n = 8).
⁄
⁄⁄
Significance levels p < 0.1 and p < 0.05 as compared with the respective control.
Changes of BP (HR) ( ) = BP (HR) after administration—BP (HR) before administration.
D
Figure 3. (A) The acute antihypertensive activities of compound 14f, 15e and losartan in RHRs (SAP, systolic arterial pressure); (B) The acute antihypertensive activities of
compound 14f, 15e and losartan in RHRs (DAP, diastolic arterial pressure); (C) the changes of heart rate (HR) of compound 14f, 15e and losartan in RHRs. Each value
represents the mean ± SEM (n = 8).
The mixture was stirred and allowed to reflux for 4 h. The solvent
was removed leaving oil which was dissolved in ethyl acetate
removed under reduced pressure and the crude product was puri-
fied by silica gel column chromatography using CH Cl /MeOH
2 2
(
30 mL) and washed with water (30 mL). After drying over anhy-
(30:1, v/v) as eluent to afford 7.
drous MgSO and evaporating under reduced pressure, the crude
4
Yellow oil, yield 49.3%. 1H NMR (CDCl
3
, 300 MHz): d (ppm)
product was purified by silica gel column chromatography using
ethyl acetate/petroleum ether (1:5, v/v) as eluent to afford 6 as
yellow oil in 92.4% yield.
1.239 (9H, s), 1.425 (6H, t, J = 7.2 Hz), 3.132 (4H, q, J = 7.5 Hz),
4.289 (2H, s), 5.583 (1H, s, NH), 7.910 (2H, d, J = 8.4 Hz), 7.997
(2H, d, J = 8.4 Hz).
4
[
.1.4. N-tert-Butyl-4-
(diethylamino)methyl]benzenesulfonamide (7)
2
Compound 6 (20.1 mmol) was dissolved in CH Cl (30 mL) then
4.1.5. General procedure for synthesis of compounds 8a–d
To a cooled (ꢀ78 °C) solution of compounds 3a, 3b, 5 and 7 in
dry THF (30 mL) was added n-BuLi (2.5 M/L) under nitrogen and
the reaction was stirred for 1 h. The temperature was raised to
ꢀ20 °C and kept for 3 h and subsequently decreased to ꢀ78 °C.
2
diethylamine (0.101 mol, 9.7 mL) was added dropwise. The reac-
tion mixture was left stirring at rt for 13 h. Then, the solvent was

7
748
J. Liu et al. / Bioorg. Med. Chem. 21 (2013) 7742–7751
Triisopropyl borate was then added. The reaction mixture was stir-
red over night at room temperature. The reaction mixture was
cooled (0 °C) and treated with an excess of 2 M HCl solution. The
mixture was extracted with EtOAc (20 mL ꢁ 2) and the combined
organic phase was washed with water and brine. The organic layer
bath. The reaction mixture was stirred for 4 h at 0 °C under N
2
atmosphere. Evaporation and the residue taken in ethyl acetate
(15 mL), washed with HCl (1 M) followed by water, brine and dried
4
over MgSO . The residue was purified by silica gel column chroma-
3
tography using CHCl /MeOH as an eluent to give 14a–g, 15a–f,
was dried with MgSO
4
, filtered and evaporated. The crude product
16a–f and 17a–c.
was then purified by silica gel column chromatography using ethyl
acetate/petroleum ether as eluent to afford 8a–d.
0
4.1.9.1. N-{4 -[(1H-1,2,4-Triazol-1-yl)methyl]-5-isobutylbiphe-
nyl-2-ylsulfonyl}propionamide (14a).
White solid, yield.
ꢀ
1
4
(
.1.6. 2-(N-tert-Butylsulfamoyl)-5-isobutylphenylboronic acid
8a)
According to the general procedure compound 3a (14.6 mmol)
28.9% mp 144–146 °C. IR (film, cm ): 2957, 2926, 2024, 1704,
1
1467, 1333, 1141; H NMR (CDCl
3
, 300 MHz): d (ppm) 0.818 (6H,
CH), 0.905 (3H, t, J = 5.1 Hz, CH ), 2.008 (2H, q,
), 1.830–1.972 (1H, m, (CH CH), 2.542 (2H, d,
), 7.039 (1H, d, J = 1.2 Hz),
d, J = 5.1 Hz, (CH
J = 7.5, 15 Hz, CH
J = 6.9 Hz, CH
3
)
2
3
was dissolved in dry THF (30 mL) and reacted with n-BuLi (2.5 M
in hexane, 29.3 mmol) and triisopropyl borate (17.6 mmol). The
2
3 2
)
2
CH), 5.438 (s, 2H, CH
2
compound 8a was obtained as Yellow oil in 56.3% yield.
7.337 (4H, dd, J = 3.9, 2.7 Hz), 7.998 (1H, s), 8.195 (2H, d,
1
ꢀ
H NMR (CDCl
3
, 300 MHz): d (ppm) 0.863 (6H, d, J = 6.6 Hz),
J = 8.1 Hz), 8.292 (1H, s); ESI-MS, m/z: 425 [(MꢀH) ], 427
+
1
.079 (9H, s, t-Bu), 1.858 (1H, m, CH), 2.544 (2H, d, J = 6.9 Hz,
CH CH), 6.825 (1H, s, NH), 7.271 (1H, d, J = 4.2 Hz, H-4), 7.381
1H, s, H-6), 7.721 (1H, d, J = 7.8 Hz, H-3), 8.356 (2H, s, B(OH) ).
[(M+H) ]. HR-MS (ESI, M+H) m/z: calcd for
C
22
H
27
N
O
4 3
S:
2
427.1804, found 427.1806.
(
2
0
4
.1.9.2. N-{4 -[(1H-1,2,4-Triazol-1-yl)methyl]-5-isobutylbiphe-
4
.1.7. 1-(4-Bromobenzyl)-1H-1,2,4-triazole (11)
THF (30 mL) was added to K CO (7.341 mmol) and the mixture
nyl-2-ylsulfonyl}pentanemide (14b).
White solid, yield
ꢀ
1
2
3
31.5% mp 100–102 °C. IR (film, cm ): 3439, 2957, 2024, 1717,
1
was stirred for 5 min. 1,2,4-triazole 10 (29 mmol) was then added
and the mixture was stirred for 1 h. 4-Bromobenzyl bromide 9
1336, 1138; H NMR (CDCl
J = 6.9 Hz, CH ), 0.942 (6H, d, J = 6.6 Hz, (CH
(m, 2H, CH CH ), 1.879–1.961 (4H, m), 1.187–1.211 (1H, m, (CH
CH), 2.539 (2H, d, J = 6.0 Hz, CH CO), 5.435 (2H, s), 7.038 (1H, s),
7.232–7.358 (5H, m), 7.965 (1H, s), 8.199 (1H, dd, J = 1.5, 6.6 Hz),
3
, 300 MHz): d (ppm) 0.885 (3H, t,
3
3 2
) CH), 1.452–1.477
(
7.5 mmol) dissolved in DMF (5 mL) was added dropwise and the
3
2
3 2-
)
mixture was heated to reflux and stirred for 15 h before water
2
(
(
(
15 mL) was added. The mixture was extracted with ether
ꢀ
+
3 ꢁ 10 mL) and each extract was washed with water
8.248 (1H, s); ESI-MS, m/z: 453.3 [(MꢀH) ], 455 [(M+H) ].HR-MS
3 ꢁ 10 mL). The combined ether layers were dried over MgSO
4
31 4 3
(ESI, M+H) m/z: calcd for C24H N O S: 455.2117, found 455.2114.
and the solvent was evaporated. The residue was purified on silica
0
gel with CHCl
yield.
3
/MeOH (40:1) as eluent to get white solid 11 in 82%
4.1.9.3. N-{4 -[(1H-1,2,4-Triazol-1-yl)methyl]-5-isobutylbiphe-
nyl-2-ylsulfonyl}-4-methylbenzamide (14c).
White solid,
1H NMR (CDCl
, 300 MHz): d (ppm) 5.307 (2H, s, CH
yield 12.1%. mp 100–102 °C.
3
2
), 7.144
IR (film, cm 1): 3438, 2956, 2024, 1752, 1607, 1228, 1163; 1
ꢀ
H
(
2H, d, J = 8.4 Hz, Ar-H), 7.511 (2H, d, J = 8.4 Hz, Ar-H), 7.498 (1H,
s, 3H), 8.098 (1H, s, 5H);
NMR (CDCl
(1H, m), 2.142 (3H, s, CH
.114 (1H, s), 7.204–7.228 (m, 2H), 7.268–7.302 (m, 4H), 7.368–
3
, 300 MHz): d (ppm) 0.925 (6H, d, J = 6.3 Hz), 1.913
EI-MS, m/z: 237, M: [M+2] = 1:1.
3
), 2.436 (2H, d, J = 7.4 Hz), 5.164 (2H, s),
7
4
.1.8. General procedure for synthesis of compounds 12a–d
Compounds 8a–d (2.55 mmol), 11 (2.55 mmol), toluene
7.402 (m, 4H),7.908 (1H, s), 8.106 (1H, s), 8.228 (1H, d,
ꢀ
J = 7.0 Hz); ESI-MS, m/z: 487.2[(MꢀH) ]. HR-MS (ESI, M+H) m/z:
(
(
15 mL), ethanol (12 mL), NaOH (1.56 M, 7 mL), Pd(OAc)
0.05 mmol), PPh
2
calcd for C27
H
29
N
4
O
3
S: 489.6092, found 489.6095.
3
(0.204 mmol), was mixed under N
2
. The mixture
0
was warmed to reflux for 5 h. The mixture was diluted with EtOAc
20 mL), washed with water and brine, and dried over MgSO . The
solvent was removed and the residue was separated by silica gel
column chromatography using CHCl /MeOH as an eluent to give
4.1.9.4. Ethyl 4 -[(1H-1,2,4-triazol-1-yl)methyl]-5-isobutylbi-
(
4
phenyl-2-ylsulfonylcarbamate (14d).
White solid, yield
ꢀ1
55.2%. mp 96–98 °C. IR (film, cm ): 3415, 2957, 2025, 1744,
1
3
1340, 1161, 1144, 576; H NMR (CDCl
(6H, d, J = 6.6 Hz, (CH CH), 1.064 (3H, t, J = 6.9 Hz, CH
.785–1.874 (1H, m, (CH CH), 2.476 (2H, d, J = 7.2 Hz, CH
4.014 (2H, q, J = 7.2, 7.2 Hz, CH CH ), 5.300 (2H, s, CH –Ar), 6.978
3
, 300 MHz): d (ppm) 0.844
CH ),
CH),
1
2a–d.
3
)
2
3
2
1
3
)
2
2
0
4
.1.8.1.
4 -[(1H-1,2,4-Triazol-1-yl)methyl]-N-tert-butyl-5-iso-
Yellow oil, yield 86.6%.
, 300 MHz): d (ppm) 0.905 (6H, d, J = 6.6 Hz),
.987 (9H, s), 1.897 (1H, m), 2.535 (2H, d, J = 7.2 Hz), 3.515 (1H,
s, NH), 7.048 (1H, d, J = 1.5 Hz, H-4), 7.238 (1H, d, J = 0.9 Hz, H-6),
2
3
2
butylbiphenyl-2-sulfonamide (12a).
(1H, d, J = 1.5 Hz), 7.148 (2H, d, J = 8.1 Hz), 7.240–7.288 (3H, m),
7.853 (1H, s), 8.039 (s,1H), 8.103 (1H, d, J = 8.1 Hz), 8.473 (1H, s);
1
H NMR (CDCl
3
ꢀ
0
ESI-MS, m/z: 442 [(MꢀH) ]. HR-MS (ESI, M+H) m/z: calcd for
C H
22 27
N
4
O
4
S: 443.1753, found 443.1751.
7
7
. 343 (2H, d, J = 4.2 Hz, Ar-H), 7.529 (2H, d, J = 8.1 Hz, Ar-H),
.995 (1H, s, 3H), 8.051 (1H, d, J = 8.1 Hz, H-3), 8.149 (1H, s, 5H).
0
4.1.9.5.
tert-Butyl
4 -[(1H-1,2,4-triazol-1-yl)methyl]-5-iso-
butylbiphenyl-2-ylsulfonylcarbamate (14e).
White solid,
ꢀ1
4
1
.1.9. General procedure for synthesis of compounds 14a–g,
5a–f, 16a–f and 17a–c
yield 45.2%. mp 97–100 °C. IR (film, cm ): 3418, 2958, 2925,
1
3
2024, 1739, 1515, 1341, 1139; H NMR (CDCl , 300 MHz): d
TFA (4 mL) was added 12a–d (2.5 mmol) and stirred the mix-
ture under N atmosphere for 12 h at rt. The reaction mixture
2
was evaporated and most TFA was removed leaving oil which
was dissolved in ethyl acetate (15 mL) and washed with water
(ppm) 0.844 (6H, d, J = 6.6 Hz), 1.229 (9H, s, t-Bu), 1.773–1.811
(1H, m), 2.486 (2H, d, J = 7.2 Hz), 5.330 (2H, s), 6.993 (1H, s),
1.171 (1H, s), 7.199 (2H, d, J = 2.4 Hz), 7.273 (1H, d, J = 3.0 Hz),
7.315 (2H, d, J = 6.3 Hz), 7.892 (1H, s), 8.058 (1H, s), 8.084 (1H,
ꢀ
(
10 mL). After drying over anhydrous MgSO
4
and evaporating un-
s); ESI-MS, m/z: 469.3 [(MꢀH) ]. HR-MS (ESI, M+H) m/z: calcd
der reduced pressure to achieve the compounds 13a–d without
further purification. The crude 13a–d were dissolved in pyridine
4 mL), and followed by the respective alkyl chloroformates, acyl
for C24
H
31
N
4
O
4
S: 471.2066, found 471.2065.
0
(
4.1.9.6. Butyl 4 -[(1H-1,2,4-triazol-1-yl)methyl]-5-isobutylbi-
chlorides and benzoyl chlorides (2.0 mmol) were added on an ice
phenyl-2-ylsulfonylcarbamate (14f).
White solid, yield

J. Liu et al. / Bioorg. Med. Chem. 21 (2013) 7742–7751
7749
ꢀ1
4
1
0.9%. mp 78–80 °C. IR (film, cmꢀ1): 3417, 2958, 2930, 2024, 1743,
34.1%. mp 108–110 °C. IR (film, cm ): 3482, 3120, 2959, 2933,
1
1
3
510, 1342, 1160, 1144; H NMR (CDCl , 300 MHz): d (ppm) 0.767
2025, 1751, 1512, 1331; H NMR (CDCl
0.900 (3H, t, J = 9.3 Hz), 1.192–1.257 (2H, m), 1.447–1.541 (2H,
m), 3.871 (3H, s, CH O), 4.052 (2H, t, J = 6.6 Hz, CH CH O), 5.408
(2H, s), 6.760 (1H, d, J = 2.7 Hz), 7.017 (1H, dd, J = 2.4, 6.3 Hz),
7.248 (1H, s), 7.268 (1H, d, J = 3 Hz), 7.348 (2H, d, J = 8.1 Hz),
3
, 300 MHz): d (ppm)
(
3H, t, J = 7.5 Hz, CH
3
), 0.842 (6H, d, J = 6.6 Hz), 1.335–1.350 (2H, m,
), 1.809–2.022 (1H, m), 2.480 (2H, d, J = 7.2 Hz, CH CH),
O), 5.342 (2H, s), 6.993 (1H, d,
J = 1.5 Hz), 7.117 (1H, s), 7.217–7.299 (5H, m), 7.918 (1H, s),
CH
3
3
CH
2
2
3
2
2
.948 (2H, t, J = 6.6 Hz, CH
2
8
.074 (1H, s), 8.095 (1H, d, J = 3.6 Hz); ESI-MS, m/z: 469.3
7.981 (1H, s), 8.161 (1H, s), 8.203 (1H, d, J = 9.0 Hz); ESI-MS, m/z:
ꢀ
ꢀ
[
(MꢀH) ]. HR-MS (ESI, M+H) m/z: calcd for
C
H
24 31
N
4
O
4
S:
443.1 [(MꢀH) ]. HR-MS (ESI, M+H) m/z: calcd for C21
25 4 5
H N O S:
4
71.2066, found 471.2068.
445.1546, found 445.1547.
0
0
4
.1.9.7. Benzyl 4 -[(1H-1,2,4-triazol-1-yl)methyl]-5-isobutylbi-
4.1.9.13. Benzyl 4 -[(1H-1,2,4-triazol-1-yl)methyl]-5-methoxybi-
phenyl-2-ylsulfonylcarbamate (14g).
3
2
White solid, yield
1.1%. mp 100–102 °C. IR (film, cm ): 3419, 3033, 2957, 2926,
025, 1745, 1510, 1342, 1160, 1144; 1H NMR (CDCl
, 300 MHz):
phenyl-2-ylsulfonylcarbamate (15f).
White solid, yield
ꢀ1
ꢀ1
37.2%. mp 164–167 °C. IR (film, cm ): 3437, 3032, 2941, 2024,
1
3
1748, 1593; H NMR (CDCl
3
, 300 MHz): d (ppm) 2.429 (3H, s,
d (ppm) 0.895 (6H, d, J = 8.4 Hz), 1.854–1.944 (m, 1H), 2.546 (2H,
d, J = 7.2 Hz), 5.022 (2H, s, CH O), 5.327 (2H, s, CH ), 6.998 (1H,
s), 7.111–7.198 (5H, m), 7.274 (1H, s),7.307–7.328 (3H, m), 7.730
CH O), 5.017 (2H, s, CH O), 5.265 (2H, s), 7.014 (1H, s), 7.158–
3
2
2
2
7.183 (m, 4H), 7.260–7.320 (m, 6H), 7.867 (1H, s), 8.050 (1H, s),
ꢀ
8.128 (1H, d, J = 8.1 Hz); ESI-MS, m/z: 477.2 [(MꢀH) ].HR-MS
(
[
1H, s), 8.126 (1H, s), 8.166 (2H, d, J = 7.5 Hz); ESI-MS, m/z: 503
23 4 5
(ESI, M+H) m/z: calcd for C24H N O S: 479.1389, found 479.1391.
ꢀ
(MꢀH) ]. HR-MS (ESI, M+H) m/z: calcd for
27 29 4 4
C H N O S:
0
5
05.1910, found 505.1912.
4.1.9.14. N-{4 -[(1H-1,2,4-Triazol-1-yl)methyl]-5-methylbiphe-
nyl-2-ylsulfonyl}propionamide (16a).
White solid, yield
, 300 MHz): d (ppm) 0.981
), 1.982 (2H, q, J = 7.5 Hz, J = 7.5 Hz,
), 3.445 (s, 3H, CH ), 5.439 (2H, s), 7.079 (1H, d,
0
1
4
.1.9.8. N-{4 -[(1H-1,2,4-Triazol-1-yl)methyl]- 5-methoxybi-
31.3%. mp 88–90 °C. H NMR (CDCl
(3H, t, J = 7.5 Hz, CH CH
CH CH
3
phenyl-2-ylsulfonyl}propionamide (15a).
White solid, yield
0.2%. mp 181–185 °C. IR (film, cm ): 3440, 3113, 3033, 2353,
024, 1706, 1599, 1477, 1324, 1146; 1H NMR (CDCl
, 300 MHz):
CH ), 1.969 (2H, q, J = 7.5,
O), 5.425 (2H, s), 6.730 (1H, d,
J = 2.1 Hz), 7.023 (1H, dd, J = 2.7, 6.3 Hz), 7.209–7.304 (4H, m),
3
2
ꢀ
1
3
2
3
2
3
3
J = 1.2 Hz), 7.023 (1H, dd, J = 2.7, 6.3 Hz), 7.231–7.259 (2H, m),
7.322–7.373 (2H, m), 7.400 (1H, dd, J = 1.8, 7.8 Hz), 8.200 (1H,
d d (ppm) 0.972 (3H, t, J = 7.5 Hz, CH
.5 Hz, CH CH ), 3.862 (s, 3H, CH
3
2
7
3
2
3
d, J = 8.4 Hz), 8.252 (1H, s), 8.661 (1H, s). ESI-MS, m/z: 383.2
ꢀ
[(MꢀH) ]. HR-MS (ESI, M+H) m/z: calcd for
19 21 4 3
C H N O S:
7
.950 (1H, s), 8.225 (1H, s), 8.248 (1H, d, J = 3.9 Hz), 8.669 (1H,
385.1334, found 385.1331.
ꢀ
s); ESI-MS, m/z: 399.1 [(MꢀH) ]. HR-MS (ESI, M+H) m/z: calcd
0
for C19
H
21
N
4
O
4
S: 401.1284, found 401.1285.
4.1.9.15. N-{4 -[(1H-1,2,4-Triazol-1-yl)methyl]-5-methylbiphe-
nyl-2-ylsulfonyl}pentanamide (16b).
White solid, yield
0
ꢀ1
4
.1.9.9.
N-{4 -[(1H-1,2,4-Triazol-1-yl)methyl]-5-methoxybi-
38.0%. mp 91–93 °C. IR (film, cm ): 3391, 2957, 2024, 1702,
1
phenyl-2-ylsulfonyl}pentanamide (15b).
White solid, yield
5.1%. mp 94–98 °C. IR (film, cm ): 3417, 3122, 2959, 2932,
871, 2025, 1715, 1595, 1130,1138; 1H NMR (CDCl
, 300 MHz): d
CH ), 1.195–1.219 (m, 2H,
), 1.414–1.463 (m, 2H, CH CH CH ), 1.941 (3H, t,
CH CO), 3.864 (3H, s, CH O), 5.429 (2H, s), 6.732
1H, d, J = 2.4 Hz), 7.023 (1H, dd, J = 2.4, 6.3 Hz), 7.238–7.311 (3H,
3
1512, 1468, 1334, 1142; H NMR (CDCl , 300 MHz): d (ppm)
ꢀ1
4
2
(
0.832 (3H, t, J = 6.6 Hz), 1.235–1.431 (m, 4H), 1.937 (t, 2H), 2.429
(s, 3H), 5.429 (s, 2H), 7.069 (s, 1H), 7.262–7.376 (m, 4H), 7.968
(s, 1H), 8.179 (1H, d, J = 7.8 Hz), 8.266 (1H, s), 8.418 (1H, s);
3
ppm) 0.864 (3H, t, J = 4.5 Hz, CH
3
2
ꢀ
CH
3
CH
2
2
2
2
ESI-MS, m/z: 411.1 [(MꢀH) ]. HR-MS (ESI, M+H) m/z: calcd for
J = 7.2 Hz, CH
(
2
2
3
C
21
25
H N
4
O
3
S: 413.1647, found 413.1651.
0
m), 7.956 (1H, s), 7.227 (1H, s), 7.256 (1H, s), 8.758 (1H, s); ESI-
4.1.9.16. Ethyl 4 -[(1H-1,2,4-triazol-1-yl)methyl]-5-methylbi-
ꢀ
MS, m/z: 427 [(MꢀH) ]. HR-MS (ESI, M+H) m/z: calcd for C21
H
25
N
4-
phenyl-2-ylsulfonylcarbamate (16c).
White solid, yield
ꢀ1
O
4
S: 429.1597, found 429.1599.
26.3%. mp 89–90 °C. IR (film, cm ): 3452, 3004, 2784, 2669,
1
2
3
024, 1736, 1507, 1333, 1236, 1145; H NMR (CDCl , 300 MHz):
0
4
.1.9.10. Ethyl 4 -[(1H-1,2,4-triazol-1-yl)methyl]-5-methoxybi-
d (ppm) 1.143 (t, 3H, J = 7.2 Hz), 2.438 (3H, s), 4.066 (2H, q,
J = 6.9, 7.2 Hz), 5.397 (2H, s), 7.093 (1H, s), 7.242–7.262 (m, 2H),
7.242–7.332 (m, 3H), 7.967 (1H, s), 8.136 (1H, d, J = 1.5 Hz), 8.167
phenyl-2-ylsulfonylcarbamate (15c).
White solid, yield
, 300 MHz): d (ppm) 1.160
1
2
(
7.3%. mp 155–158 °C. H NMR (CDCl
3H, t, J = 6.9 Hz, CH CH
CO), 5.433 (2H, s), 6.744 (1H, d, J = 1.5 Hz),
3
ꢀ
3
2
), 3.869 (s, 3H, CH
3
O), 4.068 (2H, q,
(1H, s); ESI-MS, m/z: 399.1 [(MꢀH) ]. HR-MS (ESI, M+H) m/z: calcd
J = 7.2, 7.2 Hz, CH
3
CH
2
for C19
H
21
N
4
O
4
S: 401.1284, found 401.1286.
6
.965–7.023 (1H, q, J = 2.4, 5.1 Hz), 7.337–7.397 (4H, m), 8.029
0
(
[
4
1H, s), 8.182 (1H, d, J = 9.0 Hz), 8.370 (1H, s); ESI-MS, m/z: 415.1
(MꢀH) ]. HR-MS (ESI, M+H) m/z: calcd for
4.1.9.17. tert-Butyl 4 -[(1H-1,2,4-triazol-1-yl)methyl]-5-meth-
ꢀ
C
H
19 21
N
4
O
5
S:
ylbiphenyl-2-ylsulfonylcarbamate (16d).
Colorless oil, yield
, 300 MHz): d (ppm) 1.256 (9H, s, t-Bu),
.405 (3H, s), 5.406 (2H, s), 6.708 (1H, s), 7.011 (1H, d, J = 8.5 Hz),
1
17.1233, found 417.1231.
23.4%. H NMR (CDCl
3
2
0
4
.1.9.11.
tert-Butyl
4 -[(1H-1,2,4-triazol-1-yl)methyl]-5-
White so-
lid, yield 23.4%. mp 94–96 °C. IR (film, cm ): 3368, 3129, 2973,
024, 1744, 1592, 1317, 1156; 1H NMR (CDCl
, 300 MHz): d
ppm) 1.235 (9H, s, t-Bu), 3.862 (3H, s), 5.425 (2H, s), 6.788 (1H,
7.332–7.420 (4H, m), 8.008 (1H, s), 8.098 (1H, d, J = 8.9 Hz), 8.159
ꢀ
methoxybiphenyl-2-ylsulfonylcarbamate (15d).
(1H, s). ESI-MS, m/z: 427.1 [(MꢀH) ]. HR-MS (ESI, M+H) m/z: calcd
ꢀ
1
for C21
H
25
N
4
O
4
S: 429.1597, found 429.1595.
2
(
3
0
4.1.9.18. Butyl 4 -[(1H-1,2,4-triazol-1-yl)methyl]-5-methylbi-
s), 6.961 (1H, d, J = 8.7 Hz), 7.325 (2H, d, J = 7.8 Hz), 7.486 (2H, d,
J = 7.2 Hz), 7.997 (1H, s), 8.070 (1H, d, J = 9.0 Hz), 8.198 (1H, s);
phenyl-2-ylsulfonylcarbamate (16e).
White solid, yield
ꢀ1
32.4%. mp 92–94 °C. IR (film, cm ): 3441, 2960, 2872, 2024,
ꢀ
1
ESI-MS, m/z: 443.2 [(MꢀH) ]. HR-MS (ESI, M+H) m/z: calcd for
1743, 1510, 1341, 1160, 1143, 889; H NMR (CDCl
(ppm) 0.861 (3H, t, J = 7.5 Hz), 1.143–1.256 (2H, m), 1.429–1.608
2H, m), 2.435 (3H, s), 4.015 (2H, t, J = 6.6 Hz), 5.377 (2H, s),
3
, 300 MHz): d
C
21
H
25
N
4
O
5
S: 445.1546, found 445.1548.
(
0
4
.1.9.12. Butyl 4 -[(1H-1,2,4-triazol-1-yl)methyl]-5-methoxybi-
7.079 (1H, s), 7.214–7.241 (2H, d, J = 6.6 Hz), 7.265–7.364 (3H,
m), 7.938 (1H, s), 8.138 (s, 1H), 8.166 (s, 1H) ESI-MS, m/z: 427.1
phenyl-2-ylsulfonylcarbamate (15e).
White solid, yield

7
750
J. Liu et al. / Bioorg. Med. Chem. 21 (2013) 7742–7751
ꢀ
[
4
(MꢀH) ]. HR-MS (ESI, M+H) m/z: calcd for
C
H
21 25
N
4
O
4
S:
was evaporated and most TFA was removed leaving oil which
was dissolved in ethyl acetate (15 mL) and washed with water
29.1597, found 429.1599.
(
4
10 mL). After drying over anhydrous MgSO and evaporating un-
0
4
.1.9.19. Benzyl 4 -[(1H-1,2,4-triazol-1-yl)methyl]-5-methylbi-
der reduced pressure to achieve the compounds 20a–d without
further purification. The crude 20a–d was dissolved in pyridine
(4 mL), and followed by n-butyl chloroformate (2.0 mmol) were
added on an ice bath. The reaction mixture was stirred for 4 h at
0 °C under N
ethyl acetate (15 mL), washed with HCl (1 M) followed by water,
brine and dried over MgSO . The residue was purified by silica
phenyl-2-ylsulfonylcarbamate (16f).
4.2%. mp 145–147 °C. IR (film, cm ): 3464, 3035, 2627, 2024,
744, 1508, 1337, 1229, 1154, 885, 786;
00 MHz): d (ppm) 0.855 (3H, s, CH ), 5.026 (2H, s, CH
.289 (2H, s), 7.019 (1H, s), 7.158–7.183 (m, 2H), 7.248–7.253
White solid, yield
ꢀ1
3
1
3
5
1
H
NMR (CDCl
3
,
3
2
O),
2
atmosphere. Evaporation and the residue taken in
(
m, 2H), 7.271–7.305 (m, 4H), 7.315–7.330 (m, 3H), 7.868 (1H,
4
s), 8.019 (1H, s), 8.156 (1H, d, J = 7.9 Hz); ESI-MS, m/z: 461.1
gel column chromatography using CHCl
give 21a–d.
3
/MeOH as an eluent to
ꢀ
[
4
(MꢀH) ]. HR-MS (ESI, M+H) m/z: calcd for
C
H
24 23
N
4
O
4
S:
63.1440, found 463.1442.
0
4
.1.12.1. Butyl 4 -amino-5-isobutylbiphenyl-2-ylsulfonylcarba-
0
1
4
.1.9.20. Ethyl 4 -[(1H-1,2,4-triazol-1-yl)methyl]-5-[(diethyl-
mate (21a).
Yellow oil, yield 22.4%.
3
H NMR (CDCl ,
amino)methyl]biphenyl-2-ylsulfonylcarbamate (17a).
Yel-
, 300 MHz): d (ppm) 1.082
N), 1.468 (3H, s), 2.560 (d, 4H,
N), 3.508 (2H, d, J = 7.2 Hz), 4.096 (2H, q,
J = 7.0, 7.2 Hz), 5.520 (2H, s), 7.738–7.764 (m, 4H), 7.805–7.829
300 MHz): d (ppm) 0.868 (3H, t, J = 4.5 Hz), 0.920 (6H, d,
1
low oil, yield 14.3%. H NMR (CDCl
6H, t, J = 6.4 Hz, (CH CH
J = 6.1 Hz, (CH CH
3
J = 3.0 Hz), 1.191–1.221 (2H, m), 1.416–1.472 (2H, m), 1.882–
1.923 (1H, m), 2.539 (2H, d, J = 4.2 Hz), 3.988 (3H, t, J = 3.9 Hz),
6.730 (2H, d, J = 5.1 Hz), 7.088 (1H, d, J = 0.9 Hz), 7.128–7.150 (m,
(
3
2 2
)
3
2 2
)
2H), 7.250–7.269 (m, 1H), 8.116 (1H, d, J = 4.8 Hz). ESI-MS, m/z:
ꢀ
(
m, 3H), 8.011 (1H, s), 8.102 (1H, d, J = 8.1 Hz), 8.201 (1H, s).
403.1 [(MꢀH) ]. HR-MS (ESI, M+H) m/z: calcd for C21
29 2 4
H N O S:
ꢀ
ESI-MS, m/z: 470.2 [(MꢀH) ]. HR-MS (ESI, M+H) m/z: calcd for
405.1848, found 405.1851.
C
23
30
H N
5
O
4
S: 472.2019, found 472.2016.
0
4
.1.12.2. Butyl 5-isobutyl-4 -(methylamino)biphenyl-2-ylsulfo-
0
1
4
.1.9.21. Butyl 4 -[(1H-1,2,4-triazol-1-yl)methyl]-5-[(diethyl-
nylcarbamate (21b).
3
Yellow oil, yield 12.1%. H NMR (CDCl ,
amino)methyl]biphenyl-2-ylsulfonylcarbamate (17b).
Yel-
300 MHz): d (ppm) 0.821–1.864 (5H, m), 0.879–0.962 (10H, m),
1.890–1.934 (1H, m), 2.548 (2H, d, J = 4.2 Hz), 3.353 (3H, s), 7.107
(1H, t, J = 1.8, 1.5 Hz), 7.237–7.367 (m, 5H), 8.148 (1H, d,
low oil, yield 14.3%.
1
H NMR (CDCl
3
, 300 MHz): d (ppm) 0.902 (3H, t, J = 7.0 Hz),
CH N), 1.166–1.287 (2H, m), 1.321–
.514 (2H, m), 2.450 (d, 4H, J = 6.2 Hz, (CH CH N), 3.798 (2H, d,
ꢀ
1
1
.078 (6H, t, J = 6.0 Hz, (CH
3
2
)
2
J = 8.4 Hz). ESI-MS, m/z: 417.4 [(MꢀH) ]. HR-MS (ESI, M+H) m/z:
3
2
)
2
calcd for C22
H
31
N
2
O
4
S: 419.2005, found 419.2002.
J = 7.0 Hz), 4.115 (2H, t, J = 6.8 Hz), 5.680 (2H, s), 7.633–7.678 (m,
0
4
8
H), 7.705–7.729 (m, 3H), 7.908 (1H, s), 8.002 (1H, d, J = 8.0 Hz),
.101 (1H, s). ESI-MS, m/z: 498.5 [(MꢀH) ]. HR-MS (ESI, M+H)
4.1.12.3. Butyl 5-isobutyl-4 -(ethylamino)biphenyl-2-ylsulfo-
nylcarbamate (21c).
ꢀ
1
3
Yellow oil, yield 10.3%. H NMR (CDCl ,
300 MHz): (ppm) 0.832 (3H, t, J = 4.5 Hz), 0.923 (6H, d,
m/z: calcd for C25
H
34
N
O
5 4
S: 500.2332, found 500.2334.
d
J = 3.1 Hz), 1.174–1.198 (2H, m), 1.467–1.489 (2H, m), 1.869–
1.937 (1H, m), 2.563 (2H, d, J = 2.1 Hz), 7.269–7.298 (m, 1H),
0
4
.1.9.22. Benzyl 4 -[(1H-1,2,4-triazol-1-yl)methyl]-5-[(diethyl-
amino)methyl]- biphenyl-2-ylsulfonylcarbamate (17c).
Col-
orless oil, yield 18.2%. H NMR (DMSO, 300 MHz): d (ppm) 0.980
6H, t, J = 7.2 Hz), 3.603 (4H, t, J = 18.9, 24 Hz), 4.997 (2H, s),
.516 (2H, s), 6.832–6.841 (2H, d, J = 2.7 Hz), 7.035–7.080 (4H, d,
7.312–7.367 (m, 5H), 8.142 (1H, d, J = 8.4 Hz). ESI-MS, m/z: 431.1
1
ꢀ
[(MꢀH) ].HR-MS (ESI, M+H) m/z: calcd for
23 33 2 4
C H N O S:
(
5
433.2161, found 433.2165.
0
J = 2.7 Hz), 7.229–7.305 (5H, m), 7.379–7.406 (3H, m), 7.956 (1H,
4.1.12.4. Butyl 5-isobutyl-4 -(isopropylamino)biphenyl-2-yls-
s), 8.009 (1H, d, J = 8.4 Hz), 8.666 (1H, s). ESI-MS, m/z: 532.3
ulfonylcarbamate (21d).
(CDCl , 300 MHz): d (ppm) 0.856 (3H, t, J = 4.5 Hz), 0.939 (6H, d,
J = 3.0 Hz), 0.948 (6H, d, J = 3.2 Hz),1.256–1.296 (2H, m), 1.467–
.537 (2H, m), 1.879–1.969 (1H, m), 2.550 (d, 2H, J = 4.2 Hz),
Yellow oil, yield 20.2%. 1H NMR
ꢀ
[
5
(MꢀH) ]. HR-MS (ESI, M+H) m/z: calcd for
C
28
H
32
N
5
O
4
S:
3
34.2175, found 534.2177.
1
4
.1.10. General procedure for synthesis of compounds 18b–d
The respective alcohol (10.0 mmol) was added to a mixture of
PPh (12.0 mmol), DDQ (12.0 mmol), and 4-bromoanilines 18a
10.0 mmol) in CH Cl (30 mL). The reaction was stirred at room
7.154 (2H, d, J = 5.1 Hz), 7.266 (1H, d, J = 0.9 Hz), 7.433–7.502 (m,
ꢀ
3H), 8.038 (1H, d, J = 2.1 Hz). ESI-MS, m/z: 445.2 [(MꢀH) ]. HR-
3
33 2 4
MS (ESI, M+H) m/z: calcd for C23H N O S: 447.2318, found
(
2
2
447.2315.
temperature for 2 h. Then, the solvent was evaporated under re-
duced pressure and the crude product was purified by silica gel
column chromatography using ethyl acetate/petroleum ether as
eluent to afford 18b–d.
4.2. Pharmacological evaluation
4.2.1. Rat liver membrane AT
AT receptor affinity assay was determined using rat liver mem-
brane as described previously by Wallinder, et al. and Dudley
1
receptor-binding assay
1
2
1
4
.1.11. General procedure for synthesis of compounds 19a–d
2
6
Compound 18a–d (2.55 mmol), 8a (2.55 mmol), toluene (15 mL),
et al.
ethanol (12 mL), NaOH (1.56 M, 7 mL), Pd(OAc)
0.204 mmol), was mixed under N . The mixture was warmed to re-
flux for 5 h. The mixture was diluted with EtOAc (20 mL), washed
with water and brine, and dried over MgSO . The solvent was re-
moved and the residue was separated by silica gel column chroma-
tography using CHCl /MeOH as an eluent to give 19a–d.
2 3
(0.05 mmol), PPh
(
2
4.2.2. Porcine myometrial membrane AT
assay
2
receptor-binding
4
Porcine myometrial membranes were prepared from porcine
2
7
uteri according to the method by Nielsen et al.
A presumable interference by binding to AT
blocked by addition of 1 M losartan. Binding of [ I]Ang II to
membranes was conducted in a final volume of 0.5 mL containing
50 mM Tris–HCl (pH 7.4), 100 mM NaCl, 10 mM MgCl , 1 mM
EDTA, 0.025% bacitracin, 0.2% BSA, homogenate corresponding to
3
1
receptors was
1
25
l
4
.1.12. General procedure for synthesis of compounds 21a–d
TFA (4 mL) was added 19a–d (2.5 mmol) and stirred the mix-
ture under N atmosphere for 12 h at rt. The reaction mixture
2
2

J. Liu et al. / Bioorg. Med. Chem. 21 (2013) 7742–7751
7751
1
8
0 mg of the original tissue weight, [125I]Ang II (80,000–
5,000 cpm, 0.03 nM) and variable concentrations of test sub-
4. Kaschina, E.; Unger, T. Blood Press. 2003, 12, 70.
5.
Mukoyama, M.; Nakajima, M.; Horiuchi, M.; Sasamura, H.; Pratt, R. E.; Dzau, V.
J. J. Biol. Chem. 1993, 268, 24539.
stance. Samples were incubated at 25 °C for 1.5 h. At the end of
incubation, bound complex was trapped on filters (GF/C) and
washed with cold Tris buffer (pH 7.4; 3 ꢁ 250
to tubes. The radioactivity was measured in a 1470 Wizard r-coun-
ter. The characteristics of the Ang II-binding AT receptor were
determined by using six different concentrations (0.03–5 nmol/L)
6.
Kambayashi, Y.; Bardhan, S.; Takahashi, K.; Tsuzuki, S.; Inui, H.; Hamakubo, T.;
Inagami, T. J. Biol. Chem. 1993, 268, 24543.
7
8
9
.
.
.
Horiuchi, M. Adv. Exp. Med. Biol. 1996, 396, 217.
lL), and transferred
Csikos, T.; Chung, O.; Unger, T. J. Hum. Hypertens. 1998, 12, 311.
Laflamme, L.; de Gasparo, M.; Gallo, J.-M.; Payet, M. D.; Gallo-Payet, N. J. Biol.
Chem. 1996, 271, 22729.
2
1
0. Gendron, L.; Oligny, J.-F.; Payet, M. D.; Gallo-Payet, N. J. Biol. Chem. 2003, 278,
606.
1. Tsutsumi, K.; Saavedra, J. M. Am. J. Physiol. 1991, 261, 209.
3
1
25
of the labeled [ I]Ang II. Nonspecific binding was determined in
the presence of 1 M Ang II. The specific binding was determined
by subtracting the nonspecific binding from the total bound
1
l
12. Grady, E. F.; Sechi, L. A.; Griffin, C. A.; Schambelan, M.; Kalinyak, J. E. J. Clin.
Invest. 1991, 88, 921.
13. Perlman, S.; Costa-Neto, C. M.; Miyakawa, A. A.; Schambye, H. T.; Hjort, S. A.;
Paiva, A. C. M.; Rivero, R. A.; Greenlee, W. J.; Schwartz, T. W. Mol. Pharmacol.
1997, 51, 301.
1
25
[
I]Ang II. The IC50 of an inhibitor was determined as the concen-
125
tration that displaced the specifically bound [ I]Ang II by 50%. All
determinations were performed in triplicate.
1
4. Kivlighn, S. D.; Huckle, W. R.; Zingaro, G. J.; Rivero, R. A.; Lotti, V. J.; Chang, R. S.
L.; Schorn, T. W.; Kevin, N.; Johnson, R. G. Am. J. Physiol. 1995, 268, 820.
5. Perlman, S.; Schambye, H. T.; Rivero, R. A.; Greenlee, W. J.; Hjorth, S. A.;
Schwartz, T. W. J. Biol. Chem. 1995, 270, 1493.
1
4
.2.3. Antihypertensive effects in the spontaneously
hypertensive rats (RHRs)
Male SHRs were purchased from Vital River Laboratory Animal
Technology Co. Ltd, (Beijing, China). After one week of acclimation,
16. Wan, Y.; Wallinder, C.; Johansson, B.; Holm, H.; Mahalingam, A. K.; Wu, X.;
Botros, M.; Karlén, A.; Pettersson, A.; Nyberg, F.; Fändriks, L.; Hallberg, A.;
Alterman, M. J. Med. Chem. 2004, 47, 1536.
1
7. Wan, Y.; Wallinder, C.; Plouffe, B.; Beaudry, H.; Mahalingam, A. K.; Wu, X.;
Johansson, B.; Holm, M.; Botoros, M.; Karlen, A.; Pettersson, A.; Nyberg, F.;
Fändriks, L.; Gallo-Payet, N.; Hallberg, A.; Alterman, M. J. Med. Chem. 2004, 47,
3
2 SHRs (10-weeks-old, 180–200 g body weight) were randomly
divided into four groups, namely the SHR model group, the losar-
tan control group, the compounds 14f and 15e control groups.
After oral administration with saline water, losartan (20 mg/kg),
5995.
1
1
8. Wu, X.; Wan, Y.; Mahalingam, A. K.; Murugaiah, A. M. S.; Plouffe, B.; Botros, M.;
Karlen, A.; Hallberg, A.; Gallo-Payet, N.; Alterman, M. J. Med. Chem. 2006, 49,
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1
4f (20 mg/kg) and 15e (20 mg/kg) to SHRs respectively, the SAP,
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2
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Acknowledgments
This study was financially supported by a grant from National
Natural Science Fund (No. 81302635) and Project Program of State
Key Laboratory of Natural Medicines, China Pharmaceutical
University (JKGQ201115).
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