Design, synthesis and evaluation of novel potent angiotensin II receptor 1 antagonists

Article abstract of DOI:10.1016/j.ejmech.2016.07.023

A series of new angiotensin II (Ang II) receptor 1 antagonists were designed, synthesized and evaluated. All compounds showed nanomolar affinities for the angiotensin II type 1 receptor in radioligand binding assays and could reduce blood pressure signifi

Full text of DOI:10.1016/j.ejmech.2016.07.023

European Journal of Medicinal Chemistry 123 (2016) 115e127
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and evaluation of novel potent angiotensin II
receptor 1 antagonists
Xiaolu Bao a, Weibo Zhu b, Weidong Yuan b, Xingbo Zhu b, Yijia Yan c, Hesheng Tang d,
, b, *
Zhilong Chen a
a State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Materials Science and Engineering, Donghua University,
Shanghai 201620, PR China
b Department of Pharmaceutical Science and Technology, College of Chemistry and Biology, Donghua University, Shanghai 201620, PR China
c Ningbo Dongmi Pharmaceutical Co., Ltd., Ningbo 315899, Zhejiang, PR China
d Shanghai Xianhui Pharmaceutical Co., Ltd., Shanghai 200433, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new angiotensin II (Ang II) receptor 1 antagonists were designed, synthesized and evaluated.
All compounds showed nanomolar affinities for the angiotensin II type 1 receptor in radioligand binding
assays and could reduce blood pressure significantly in spontaneously hypertensive rats(SHRs). From
which, compound 2b displayed higher affinity binding to angiotensin II type 1 receptor at the same order
of magnitude to irbesartan with an IC50 value of 1.26 0.08 nM in radioligand binding assays. 2b showed
an efficient and long-lasting effect in reducing blood pressure, the maximal reducing responses were
Received 23 April 2016
Received in revised form
11 July 2016
Accepted 12 July 2016
Available online 15 July 2016
40.62
4.08 mmHg of MBP at 15 mg/kg and 28.39
2.09 mmHg at 10 mg/kg in SHRs,
Keywords:
Hypertension
ARBs
Pharmacokinetic
Tissue distribution
39.56 4.83 mmHg at 15 mg/kg and 29.05 2.20 mmHg at 10 mg/kg in RHRs, the significant antihy-
pertensive effect lasted beyond 12 h both in SHRs and in RHRs. In the single-dose pharmacokinetic
experiments, compound 2b could be absorbed efficiently and metabolized smoothly in Wistar rats after
oral administration. The values of Cmax, Tmax, AUC0e72 and MRT0e72 were 885.61
5.67 1.51 h, 6110.28 7398.33 ng/mL h and 7.87 2.30 h at 10 mg/kg, 2945.55
432.7 ng/mL,
1543.67 ng/mL,
4.33 0.82 h, 26473.62 12217.16 ng/mL h and 10.24 6.94 h at 15 mg/kg, 5759.03 1331.75 ng/mL,
1.10 h, 89488.44 18413.15 ng/mL$h and 12.89 2.0 h at 30 mg/kg respectively. The T1/2 values of the
5
three groups were similar, about 9e10 h. Compound 2b was distributed into tissues rapidly and
extensively after oral administration. The level of it was the highest in the liver, followed by in spleen,
kidney, and the lowest in brain. The acute toxicity assays of 2b proved its low acute toxicity with an LD50
value of 1551.71 mg/kg, and no toxicity reaction appeared at dose of 1200.00 mg/kg. These encouraging
results make compound 2b an effective, long-lasting and safe anti-hypertensive drug candidate and
worthy of further investigation.
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
and the prevalence of contributing factors such as obesity, seden-
tary lifestyle and smoking rise, hypertension has currently affected
Hypertension, defined as raised blood pressure greater than or
to 140 mmHg systolic or 90 mmHg diastolic, is a serious health
problem associated with an increased risk of death, stroke, and
metabolic syndromes including insulin resistance and lipid ab-
normalities [1,2]. It is recognized as one of the leading risk factors
for human morbidity and mortality [3,4]. As the population ages
approximately one billion adults globally and it may increase to
1.56 billion by the year 2025 [5]. Despite global awareness of hy-
pertension, its consequences and the availability of effective ther-
apeutics, an estimated 32% of hypertensive patients remain
untreated [6].
The rennin angiotensin system (RAS) plays a central role in the
pathophysiology of hypertension, electrolyte balance and blood
volume [7]. Angiotensin II (Ang II), an octapeptide formed within
the RAS from angiotensin I by angiotensin converting enzyme
(ACE), is the biologically active component of the rennin-
* Corresponding author. Donghua University, 2999 North Renmin Road, Shanghai
201620, PR China.
E-mail address: zlchen1967@qq.com (Z. Chen).
http://dx.doi.org/10.1016/j.ejmech.2016.07.023
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

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X. Bao et al. / European Journal of Medicinal Chemistry 123 (2016) 115e127
angiotensin system and is responsible for most of the peripheral
effects of this system and it is one of the most powerful vasocon-
strictors [8,9]. Angiotensin II receptor blockers (ARBs) are the
newest class of approved antihypertensive agents and have rapidly
become established as one of the leading therapeutic drugs in the
management of hypertension [10,11]. At present, many efficient and
safe ARBs have been widely used in the treatment of hypertension,
such as losartan, valsartan, irbesartan, candesartan, telmisartan and
so on.
2.2. Biological evaluation
2.2.1. Radioligand binding assays
Immunohistochemical staining showed that smooth muscle
actin was expressed in cytoplasm (Fig. 3), which indicated that the
obtained cells were vascular smooth muscle cells. The radioligand
binding assays showed that all the compounds have nanomolar
affinity for the AT1 receptor subtype (Table 1, Fig. 4). Generally,
compounds 1b and 2b had much stronger inhibitory ability than
compounds 1a, 1c, 2a, 2c and 2d in binding assays which indicated
that compounds 1b and 2b might have better antihypertensive
activity in vivo. And among all the compounds, compound 2b
showed higher affinity to the angiotensin AT1 receptor with an IC50
Irbesartan, a potent, long-acting angiotensin II receptor 1
antagonist, decreases blood pressure effectively in
a dose-
dependent manner in patients with mild-to-moderate hyperten-
sion [12,13]. Here a series of new compounds (compound 1aec,
2aed) (Fig. 1) were designed and synthesized taking irbesartan as
lead compound. The dominant conformations of compound 1b, 2b
and irbesartan were shown as Fig. 2 from computer software
Spartan 10. According to their overlay conformations, it is indicated
that they align well with each other. And the affinities of this series
of compounds to AT1 receptor were tested by specific radioligand
binding assay in vitro. The anti-hypertension effects of them were
evaluated in spontaneously hypertensive rats (SHRs) in vivo. The
anti-hypertension effects in renal hypertensive rats (RHRs), plasma
pharmacokinetics, tissue distribution in Wistar rats and acute
toxicity in ICR mouse of compound 2b were further investigated.
value of 1.26
0.08 nM than irbesartan whose IC50 value was
1.46 0.34 nM.
2.2.2. In vivo anti-hypertension effects of the compounds
In spontaneously hypertensive rats (SHRs), the effects of com-
pounds 1aec, 2aed (15 mg/kg) and irbesartan (15 mg/kg) on the
mean blood pressure (MBP) in vivo after oral administration were
shown in Fig. 5. The results indicated that all the new compounds,
especially compound 1b and 2b, could decrease blood pressure
significantly under the dose of 15 mg/kg compared with the
negative control group. The maximal response of compound 1b at
15 mg/kg was observed at 3 h after oral administration. It lowered
40.57 2.90 mmHg of MBP and the significant (p < 0.01) antihy-
pertensive effect sustained for at least 12 h. Among these com-
pounds, 2b has the strongest antihypertensive effect, whose
maximal response lower 40.62 4.08 mmHg MBP at 15 mg/kg and
28.39 2.26 mmHg at 10 mg/kg at 4 h after oral administration in
SHRs (Fig. 6a). The antihypertensive effects in RHRs were tested to
be coincident with which in SHRs (Fig. 6b). No influence of these
compounds to the heart rates of rats was observed. These results
suggested that the anti-hypertensive effects of compound 1b and
2b were superior to irbesartan, especially 2b, which were worthy of
further investigation.
2. Results and discussion
2.1. Chemistry
The preparation of compounds 1aec and 2aed was performed
by means of a multistep procedure described in Schemes 1 and 2.
The synthesis of 1aec was accomplished starting from the
commercially available 1-amino-cyclopentanecarboxamide (3),
which reacted with different acyl chlorides in DCM using Et3N as
base to give amide 4aed. 4aed were cyclized to produce spi-
roazacyclic compounds 5aed with 10 M KOH in MeOH. 4-
Methylindole 6a was N-protected by acylation with benzoyl chlo-
ride to give (4-methyl-1H-indol-1-yl) (phenyl) methanone 7a,
which was brominated with NBS to produce (4-bromomethyl-1H-
indol-1-yl) (phenyl) methanone 8a. 8a was substituted by 5aed
and then de-protected in the presence of K2CO3 to generate indole
compounds 9aec which were then coupled with 2-fluoro-benzo-
nitrile to give benzonitrile 10aec. The target compounds 1aec were
acquired after the formation of tetrazole using 10aec with sodium
azide and chlorotributylstannane in DMF. At each stage of the
synthetic sequence the product was isolated, purified by column
chromatography and characterized by NMR and mass spectrum
techniques.
2.2.3. Pharmacokinetic characteristics of compound 2b
The HPLC chromatograms of compound 2b in plasma and liver
tissues were showed in Fig. 7 and Fig. 8 respectively. The concen-
trations of compound 2b in rat plasma samples obtained from 6
male Wistar rats orally administered with 2b solution at 10e30 mg/
kg were quantified. The pharmacokinetic parameters were shown
in Table 2. The mean peak plasma concentration-time curve of 2b
was shown in Fig. 9. The maximum concentration (Cmax) value
ranged from 855 ng/mL at 10 mg/kg to 5759.03 ng/mL at 30 mg/kg.
The area under the concentration-time curve from 0 to 72 h
(AUC0~72) ranged from 6110.28 ng/mL$h to 89488.44 ng/mL$h. The
elimination half-lives (T1/2) ranged from 9 h to 13 h. Dose de-
pendency of AUC0~∞ and Cmax (10e30 mg/kg) was assessed and
both AUC0~∞ and Cmax met the dose proportionality criteria(Fig.10).
The compound levels in heart, liver, spleen, kidney and brain of
Wistar rats at 0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72 h post oral adminis-
tration of compound 2b at 10e30 mg/kg were shown in Fig. 11. The
pharmacokinetic parameters were shown in Table 3. It could be
found that 2b was extensively distributed in rat tissues. It was
accumulated especially in liver which exhibited the highest con-
centration ranged from 720.73 ng/g at 10 mg/kg to 1995.52 ng/g at
30 mg/kg, followed by spleen ranged from 348.02 ng/g at 10 mg/kg
to 1881.59 ng/g at 30 mg/kg. The level of compound 2b was lowest
in the brain, which ranged from 37.16 ng/g to 196.47 ng/g. As
compound 2b was detectable in brain, it was likely that 2b was able
to penetrate the blood-brain barrier (BBB). For all tissues, com-
pound 2b showed continuous increase at 0e4 h and decrease from
6 to 72 h post administration.
The preparation of 2aed was similar to 1aec.
Fig. 1. Chemical structure of irbesartan and compounds 1aec, 2aed.

X. Bao et al. / European Journal of Medicinal Chemistry 123 (2016) 115e127
117
Fig. 2. Energy-minimized conformations of 1b, 2b, irbesartan and their overlay conformations.
Scheme 1. Reagents and reaction conditions: (a) RCOCl, Et3N, DCM; (b)KOH, MeOH, reflux; (c) benzoyl chloride, Et3N, DMAP, CH2Cl2; (d) NBS, AIBN, CCl4, reflux; (e) NaH, THF, 50 ꢀC;
(f) NaOH, H2O, CH3OH, reflux; (g) 2-fluorobenzonitrile, K2CO3, DMF, reflux; (h) NaN3, Bu3SnCl, DMF, reflux.
These results showed that compound 2b could be absorbed
efficiently and metabolized gradually in plasma and in tissues of
Wistar rats.
in radioligand binding assays and could reduce blood pressure
significantly in SHRs. In the binding assay, compound 2b showed
stronger inhibitory ability against Ang II than irbesartan. In anti-
hypertensive effect tests in vivo, 2b showed an efficient and long-
lasting effect in reducing blood pressure in SHRs and RHRs at
15 mg/kg. The pharmacokinetic experiments showed that 2b could
be absorbed efficiently and metabolized smoothly in Wistar rats
after oral administration. With single-dose 2b 10, 20 and 30 mg/kg,
the mean plasma exposure (Cmax and AUC0e72) was increased in a
dose-dependent manner. It was well distributed in all tissues
including in brain tissues which suggested that 2b could cross the
BBB and play important role in the control of central nervous sys-
tem blood pressure. Besides, compound 2b has low acute toxicity.
In summary, compound 2b could be preliminary considered as
an effective candidate with high performance for novel anti-
hypertension drug and deserved for further investigation.
2.2.4. Acute toxicity test of compound 2b
The acute toxicity assay showed that compound 2b had low
acute toxicity with the LD50 value of 1551.71 mg/kg and the 95%
confidence interval was 1348.88e1745.07 mg/kg (Table 4). There
was no death and obvious untoward reaction appeared at dose of
1200 mg/kg after oral administration, and no significant change in
the weight of survival mice after 2 weeks' observation.
3. Conclusions
In this work, a series of novel angiotensin II receptor 1 antago-
nists were designed, synthesized and evaluated. All the compounds
showed nanomolar affinities for the angiotensin II type 1 receptor

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X. Bao et al. / European Journal of Medicinal Chemistry 123 (2016) 115e127
Scheme 2. Reagents and conditions: (a) benzoyl chloride, Et3N, DMAP, CH2Cl2; (b) NBS, AIBN, CCl4, reflux; (c) NaH, THF, 50 ꢀC; (d) NaOH, H2O, CH3OH, reflux; (e) 2-
fluorobenzonitrile, K2CO3, DMF, reflux; (f) NaN3, Bu3SnCl, DMF, reflux.
Fig. 3. The morphology of cultured VSMCs detected by phase-contrast microscope. (a) Untreated VSMCs, ꢁ100. (b) Untreated VSMCs, ꢁ200. (c) SM
a-actin immunocytochemical
staining of cultured VSMCs, ꢁ100. (d) SM -actin immunocytochemical staining of cultured VSMCs, ꢁ200.
a
4. Experimental section
not accurated. 1H NMR spectra were measured on a Bruker
400 MHz spectrometer using TMS (Me4Si) as internal standard. ESI-
MS spectra were recorded on a Micromass triple quadrupole mass
spectrometer. Column chromatography (CC) was performed using
silica gel H (300e400). All final compounds had a purity level
greater than 95% based upon HPLC, and 1H NMR.
4.1. Chemistry
All chemical reagents were of highest commercially available
quality and applied without further purification. Yields of the pu-
rified products were not optimized. Melting points (MP) were
measured on an electro thermal melting point apparatus and were

X. Bao et al. / European Journal of Medicinal Chemistry 123 (2016) 115e127
119
(m, 4H), 0.91 (t, 3H). MS(ESI): [MþH]þ calcd 209.2; found 209.3.
Table 1
IC50 and Ki value of the tested compounds (1aec, 2aed) and irbesartan.
4.1.1.4. 2-Hexyl-1,3-diazaspiro[4.4]non-1-en-4-one (5d). The above
general procedure was followed using heptanoyl chloride as acyl-
ation reagent to give 5d as oil. Yield: 81.1%. 1H NMR (400 MHz,
Compound
IC50 SEM (nM)
Ki (nM)
1a
1b
1c
2a
2b
2c
2d
Irbesartan
7.75 0.16
3.05 0.16
6.15 0.20
7.04 0.19
1.26 0.17
5.33 0.43
6.15 0.20
1.46 0.34
5.61 0.13
2.21 0.12
4.67 0.15
5.07 0.15
0.91 0.12
3.42 0.30
4.67 0.15
1.05 0.25
DMSO-d6, ppm) d: 10.7 (s, 1H), 2.33 (t, 2H), 1.95-1.45 (m, 12H), 1.35
(m, 4H), 0.92 (t, 3H). MS(ESI): [MþH]þ calcd 223.2; found 223.3.
4.1.2. N-benzoyl-4-methylindol (7a)
7a was prepared by the general procedure described by Dhanoa
[14].
4.1.3. N-benzoyl-5-methylindol (7b)
7b was prepared by the general procedure described by Dhanoa
[14].
4.1.4. (4-(bromomethyl)-1H-indol-1-yl)(phenyl)methanone(8a)
8a was prepared by the general procedure described by Dhanoa
[14].
4.1.5. (5-(bromomethyl)-1H-indol-1-yl)(phenyl)methanone(8b)
8b was prepared by the general procedure described by Dhanoa
[14].
4.1.6. General procedure for the synthesis of 9aec, 11aed
A solution of an appropriate spiroazacyclic compound 5aed
(5.03 mmol) and NaH (0.12 g, 5.53 mmol, 60%) in 100 mL anhydrous
THF was stirred for 30 min at 50 ꢀC. After cooled to rt, the solution
was added dropwise the mixture of an appropriate bromide 8aeb
(6.04 mmol) in anhydrous THF (50 mL). The solution was stirred for
3 h at 50 ꢀC. The resulting mixture was poured into 30 mL ice water,
and extracted with ethyl acetate (EA) (50 mL ꢁ 3). The organic layer
was dried over MgSO4. After filtration, the solvent was removed
under reduced pressure. The residue was dissolved in 10 mL
methanol and then added NaOH (0.8 g, 20 mmol) in 10 mL water
slowly. The mixture was refluxed for 3 h, and the solvent was
removed in vacuo. The residue was dissolved in 15 mL H2O and
extracted with DCM (10 mL ꢁ 4). The organic layer was dried over
MgSO4. After filtration, the solvent was removed under reduced
pressure. The residue was purified by CC to give the product as oil.
Fig. 4. Inhibitory effects of compounds 1b, 2b and irbesartan (10ꢂ5 e 10ꢂ12 M) on
specific binding of 125I-Ang II to AT1 receptors in VSMCs.
4.1.1. General procedure for the synthesis of 5aed
1-amino-cyclopentanecarboxamide (3) (8.0 g, 62.5 mmol) was
acylated with alkyl acyl chloride (93.7 mmol) and triethylamine
(17.3 mL, 125.0 mmol) in 50 mL dichloromethane(DCM) at 0 ꢀC.
After the reaction was completed, the resulting mixture was added
with 30 mL water, and extracted with DCM (25 mL ꢁ 3). The organic
layer was dried over MgSO4. After filtration, the solvent was
removed under reduced pressure. The residue was dissolved in
50 mL MeOH and then 50 mL 10 M KOH was added slowly. The
mixture was refluxed for 3 h. After cooled to room temperature, the
mixture was added 50 mL H2O and extracted with DCM
(30 mL ꢁ 4). The organic layer was dried over MgSO4, the solvent
was removed under reduced pressure. The resulting residue was
purified by CC to give 5aed.
4.1.6.1. 3-((1H-indol-4-yl)methyl)-2-propyl-1,3-diazaspiro[4.4]non-
1-en-4-one(9a). 9a was synthesized according to the above general
procedure. Yield: 79.2%. 1H NMR (400 MHz, CDCl3, ppm)
d:8.39 (brs,
1H), 7.36-7.34 (d, 1H), 7.36-6.83 (m, 3H), 6.56-6.55 (d, 1H), 4.98 (s,
2H), 2.26 (t, 2H), 2.01-1.96 (m, 8H), 1.84-1.51 (m, 2H), 0.84 (t, 3H).
MS(ESI): [MþH]þ calcd 310.2; found 310.4.
4.1.6.2. 3-((1H-indol-4-yl)methyl)-2-butyl-1,3-diazaspiro[4.4]non-1-
4.1.1.1. 2-Propyl-1,3-diazaspiro[4.4]non-1-en-4-one (5a). The above
general procedure was followed by using butyryl chloride as acyl-
ation reagent to give 5a as oil. Yield: 87.4%. 1H NMR (400 MHz,
en-4-one (9b). 9b was synthesized according to the above general
procedure. Yield: 76.8%. 1H NMR (400 MHz, CDCl3, ppm)
d: 8.35 (s,
1H), 7.37-7.34 (m, 1H), 7.26-6.84 (m, 3H), 6.56-6.55 (m, 1H), 4.94 (s,
2H), 2.28 (t, 2H), 2.01-1.96 (m, 8H), 1.52 (m, 2H), 1.28 (m, 2H), 0.84
(t, 3H). MS(ESI): [MþH]þ calcd 324.2; found 324.2.
DMSO-d6, ppm) d: 10.7 (s, 1H), 2.34 (t, 2H), 1.94-1.50 (m, 8H), 1.35
(m, 2H), 0.92 (t, 3H). MS(ESI): [MþH]þ calcd 181.1; found181.2.
4.1.1.2. 2-Butyl-1,3-diazaspiro[4.4]non-1-en-4-one (5b). The above
general procedure was followed using pentyl chloride as acylation
reagent to give 5b as oil. Yield: 89.5%. 1H NMR (400 MHz, DMSO-d6,
4.1.6.3. 3-((1H-indol-4-yl)methyl)-2-pentyl-1,3-diazaspiro[4.4]non-
1-en-4-one(9c). 9c was synthesized according to the above general
procedure. Yield: 71.3%. 1H NMR (400 MHz, CDCl3, ppm)
d: 8.39
ppm) d: 10.7 (s, 1H), 2.33 (t, 2H), 1.93-1.50 (m, 10H), 1.35 (m, 2H),
(brs, 1H), 7.36-7.34 (d, 1H), 7.26-6.84 (m, 3H), 6.56-6.55 (m, 1H),
4.98 (s, 2H), 2.27 (t, 2H), 2.01-1.96 (m, 8H),1.29-1.26 (m, 6H), 0.82 (t,
3H). MS(ESI): [MþH]þ calcd 338.2; found 338.5.
0.92 (t, 3H). MS(ESI): [MþH]þ calcd 195.1; found 195.2.
4.1.1.3. 2-Pentyl-1,3-diazaspiro[4.4]non-1-en-4-one (5c). The above
general procedure was followed using hexanoyl chloride as acyla-
tion reagent to give 5c as oil. Yield: 91.2%. 1H NMR (400 MHz,
4.1.6.4. 3-((1H-indol-5-yl)methyl)-2-propyl-1,3-diazaspiro[4.4]non-
1-en-4-one (11a). 11a was synthesized according to the above
DMSO-d6, ppm)
d: 10.7 (s, 1H), 2.32 (t, 2H), 1.92-1.49 (m, 10H), 1.34
general procedure. Yield: 77.1%. 1H NMR (400 MHz, CDCl3, ppm)
d:

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X. Bao et al. / European Journal of Medicinal Chemistry 123 (2016) 115e127
Fig. 5. Effects of compound 1aec, 2aed and irbesartan on mean blood pressure (MBP) in spontaneously hypertensive rats. *and** Significant difference from negative control,
p < 0.05 and p < 0.01, respectively.

X. Bao et al. / European Journal of Medicinal Chemistry 123 (2016) 115e127
121
Fig. 6. (a) Effects of compound 2b (15 mg/kg, 10 mg/kg) and irbesartan (15 mg/kg) on mean blood pressure (MBP) in SHRs. (b) Effects of compound 2b (15 mg/kg, 10 mg/kg) and
irbesartan (15 mg/kg) on mean blood pressure (MBP) in RHRs. *and** Significant difference from negative control, p < 0.05 and p < 0.01, respectively.
Fig. 7. HPLC Chromatogram of compound 2b in plasma of Wistar rats. a, Blank plasma; b, Blank plasma spiked with 2b; c, A plasma sample from a subject after an oral of 2b at 4 h at
10 mg/kg; d, A plasma sample from a subject after an oral of 2b at 24 h at 10 mg/kg; 2- compound 2b (tR ¼ 4.2 min).
8.91 (brs, 1H), 7.50 (s, 1H), 7.40-7.37 (m, 1H), 7.28-7.26 (m, 1H), 7.07-
7.04 (m, 1H), 6.59-6.55 (m, 1H), 4.83 (s, 2H), 2.43 (t, 2H), 2.08-1.91
(m, 8H), 1.59-1.51 (m, 2H), 0.84 (t, 3H)。MS(ESI): [MþH]þ calcd
310.2; found 310.4.
(m, 1H), 6.59-6.55 (m, 1H), 4.84 (s, 2H), 2.42 (t, 2H), 2.08-1.91 (m,
8H), 1.63-1.59 (m, 2H), 1.39-1.33 (m, 2H), 0.88 (t, 3H). MS(ESI):
[MþH]þ calcd 324.2; found 324.2.
4.1.6.6. 3-((1H-indol-5-yl)methyl)-2-pentyl-1,3-diazaspiro[4.4]non-
1-en-4-one (11c). 11c was synthesized according to the above
general procedure. Yield: 76.3%. 1H NMR (400 MHz, CDCl3, ppm)
4.1.6.5. 3-((1H-indol-5-yl)methyl)-2-butyl-1,3-diazaspiro[4.4]non-1-
en-4-one(11b). 11b was synthesized according to the above general
procedure. Yield: 77.4%. 1H NMR (400 MHz, CDCl3, ppm)
d: 8.90
d:8.90 (brs, 1H), 7.50 (s, 1H), 7.40-7.37 (m, 1H), 7.28-7.26 (m, 1H),
(brs, 1H), 7.50 (s, 1H), 7.40-7.37 (m, 1H), 7.28-7.26 (m, 1H), 7.07-7.04
7.07-7.04 (m, 1H), 6.59-6.55 (m, 1H), 4.84 (s, 2H), 2.42 (t, 2H), 2.01-

122
X. Bao et al. / European Journal of Medicinal Chemistry 123 (2016) 115e127
Fig. 8. HPLC Chromatogram of compound 2b in liver tissue of Wistar rats. a, Blank liver tissue; b, Blank liver tissue spiked with 2b; c, A liver tissue sample from a subject after an
oral of 2b at 4 h at 10 mg/kg; d, A liver tissue sample from a subject after an oral of 2b at 24 h at 10 mg/kg; 1-compound 2b (tR ¼ 2.8 min).
Table 2
Pharmacokinetic parameters of compound 2b in plasma of male Wistar rats after oral administration (10 mg/kg, 15 mg/kg, 30 mg/kg) (n ¼ 6).
Dose (mg/kg)
Pharmacokinetic parameters
AUC(0e72) (ng/mL h)
AUC(0e∞) (ng/mL h)
MRT(0e72) (h)
MRT(0e∞) (h)
T1/2 (h)
Tmax(h)
Cmax(ng/mL)
10
15
30
6110.28 7398.33
23364.97 10681.56
89488.44 18413.15
6687.54 8119.11
26070.54 10505.84
88846.69 17500.03
7.87 2.30
10.24 6.94
12.89 2.0
6.61 3.97
13.66 14.83
16.06 5.24
9.93 0.51
9.03 1.20
9.93 3.40
5.67 1.51
4.33 0.86
5.00 1.10
855.61 432.7
2945.55 1543.67
5759.03 1331.75
1.96 (m, 8H), 1.64-1.26 (m, 6H), 0.82 (t, 3H). MS(ESI): [MþH]þ calcd
338.2; found 338.5.
4.1.6.7. 3-((1H-indol-5-yl)methyl)-2-hexyl-1,3-diazaspiro[4.4]non-1-
en-4-one(11d). 11d was synthesized according to the above general
procedure. Yield: 74.8%. 1H NMR (400 MHz, CDCl3, ppm)
d: 8.90
(brs, 1H), 7.51 (s, 1H), 7.40-7.36 (m, 1H), 7.28-7.25 (m, 1H), 7.06-7.04
(m, 1H), 6.59-6.54 (m, 1H), 4.83 (s, 2H), 2.41 (t, 2H), 2.01-1.96 (m,
8H), 1.65-1.26 (m, 8H), 0.80 (t, 3H). MS(ESI): [MþH]þ calcd 352.2;
found 352.2.
4.1.7. General procedure for the synthesis of 10aec, 12aed
A solution of an appropriate indole compound 9aec or 11aed
(2 mmol) in 20 mL DMF was added K2CO3 (552 mg, 4 mmol) and 2-
fluorobenzonitrile (266 mg, 2.2 mmol). The mixture was stirred-
under reflux for 5 h under nitrogen. The resulting mixture was
diluted with water 60 mL and extracted with EA (30 mL ꢁ 4). The
organic layer was washed with saturated salt water (40 mL ꢁ 4) and
dried over MgSO4. After filtration, the solvent was removed under
reduced pressure. The residue was purified by CC to give a yellow
solid.
Fig. 9. Observed plasma concentrations of 2b in rats after a signal oral administration
(10 mg/kg, 15 mg/kg, 30 mg/kg). Data are average values of 6 experiments (Mean SD).

X. Bao et al. / European Journal of Medicinal Chemistry 123 (2016) 115e127
123
Fig. 10. Compound 2b AUC(0e∞) (y ¼ 4.38x ꢂ 34.67; R2 ¼ 0.85) (a) and Cmax (y ¼ 0.26x
e 1.56, R2 ¼ 0.96) (b) versus dose (mg/kg) following a signal oral administration of
male Wistar rats (n ¼ 6).
4.1.7.1. 2-(4-((4-oxo-2-propyl-1,3-diazaspiro[4.4]non-1-en-3-yl)
methyl)-1H-indol-1-yl) benzonitrile (10a). 10a was synthesized ac-
cording to the above general procedure. Yield: 85.4%. 1H NMR
(400 MHz, DMSO-d6, ppm) d: 7.98-7.92 (m, 1H), 7.83-7.78 (m, 1H),
7.72 (m, 1H), 7.66 (d, 1H), 7.33 (d, 1H), 7.01 (t, 1H), 6.87 (d, 1H), 6.73
(d, 1H), 6.66 (d, 1H), 4.94 (s, 2H), 2.27 (t, 2H), 1.93-1.80 (m, 6H), 1.76-
1.63 (m, 2H),1.54-1.40 (m, 2H), 0.80 (t, 3H)。MS(ESI): [MþH]þ calcd
411.2; found 411.5.
4.1.7.2. 2-(4-((2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)
methyl)-1H-indol-1-yl)benzonitrile (10b). 10b was synthesized ac-
cording to the above general procedure. Yield: 87.1%. 1H NMR
(400 MHz, CDCl3, ppm) d: 7.85 (d, 1H), 7.74 (t, 1H), 7.58 (d, 1H), 7.51
(t,1H), 7.43 (d,1H), 7.27 (d, 1H), 7.19 (t,1H), 6.94 (d,1H), 6.80 (d,1H),
5.01 (s, 2H), 2.33 (t, 2H), 2.07e1.83 (m, 8H), 1.52 (m, 2H), 1.28 (m,
2H), 0.82 (t, 3H). MS(ESI): [MþH]þ calcd 425.2; found 425.3.
Fig. 11. Observed tissues concentrations of 2b in Wistar rats after
administration (10 mg/kg, 15 mg/kg, 30 mg/kg). Data are average values of 6 experi-
ments (Mean SD) (a, 10 mg/kg; b, 15 mg/kg; c, 30 mg/kg).
a
signal oral
4.1.7.3. 2-(4-((4-oxo-2-pentyl-1,3-diazaspiro[4.4]non-1-en-3-yl)
methyl)-1H-indol-1-yl)benzonitrile (10c). 10c was synthesized ac-
cording to the above general procedure. Yield: 89.4%. 1H NMR
4.1.7.4. 2-(5-((4-oxo-2-propyl-1,3-diazaspiro[4.4]non-1-en-3-yl)
methyl)-1H-indol-1-yl)benzonitrile (12a). 12a was synthesized ac-
cording to the above general procedure. Yield: 87.4%. 1H NMR
(400 MHz, DMSO-d6, ppm) d: 7.94 (d, 1H), 7.77 (t, 1H), 7.69 (t, 1H),
7.62 (d, 1H), 7.32 (d, 1H), 7.01 (t, 1H), 6.87 (d, 1H), 6.72 (d, 1H), 6.63
(d,1H), 4.94 (s, 2H), 2.28 (t, 2H),1.95-1.78 (m, 6H),1.75-1.61 (m, 2H),
(400 MHz, DMSO-d6, ppm) d: 7.94 (d, 1H), 7.77 (t, 1H), 7.70 (t, 1H),
7.61 (d, 1H), 7.32 (d, 1H), 7.05-6.96 (m, 1H), 6.87 (d, 1H), 6.73 (d, 1H),
6.64 (d, 1H), 4.94 (s, 2H), 2.29 (t, 2H), 1.92-1.80 (m, 6H), 1.74e1.62
(m, 2H), 1.49-1.38 (m, 2H), 1.20-1.10 (m, 4H), 0.77 (t, 3H). MS(ESI):
[MþH]þ calcd 439.2; found 439.6.

124
X. Bao et al. / European Journal of Medicinal Chemistry 123 (2016) 115e127
Table 3
Pharmacokinetic parameters of compound 2b in tissues of male Wistar rats after oral administration (10 mg/kg, 15 mg/kg, 30 mg/kg) (n ¼ 6) .
Tissue
Heart
Liver
Dose (mg/kg)
Pharmacokinetic parameters
AUC(0e72) (ng/mL$h)
AUC(0e∞) (ng/mL h)
MRT(0e72) (h)
MRT(0e∞) (h)
T1/2 (h)
Tmax (h)
1.27
4.67 1.63
4.8 1.10
20.08
2
4
2
Cmax (ng/mL)
10
15
30
10
15
30
10
15
30
10
15
30
10
15
30
10
15
30
825.30 325.98
5835.75 189.17
7359.74 242.03
3073.19 416.59
4889.25 652.86
14473.39 1846.79
9474.46 357.51
11936.29 1625.20
18867.22 796.59
744.90 114.67
1346.97 71.89
2361.07 122.06
4335.85 131.16
5729.83 201.60
22361.07 1220.06
345.51 64.23
962.11 270.31
6699.84 170.61
8757.78 342.77
3319.04 361.25
23517.36 5041.71
15623.11 2006.10
11742.48 403.88
12046.8 1663.33
21045.84 2156.90
745.02 114.48
1346.98 71.89
2412.15 142.36
10849.31 815.64
7691.91 1045.48
29445.3 2458.1
345.52 64.23
7.68 2.01
16.93 0.13
16.18 0.79
4.93 0.98
4.77 0.56
6.30 1.13
20.25 0.32
9.71 1.84
13.27 0.67
10.63 1.90
6.64 0.31
4.85 0.29
21.03 0.13
19.37 0.25
24.85 0.28
8.10 1.90
11.60 3.20
9.03 0.50
12.00 3.80
23.85 0.13
28.07 3.07
3.58 2.86
4.77 0.56
5.49 2.83
30.42 1.92
13.08 3.88
20.38 3.15
10.63 1.90
3.64 0.31
4.10 2.02
25.30 9.31
29.40 4.03
24.10 2.0
8.10 1.90
11.60 3.20
9.02 0.51
16.92 3.07
15.85 1.91
20.18 3.13
2.33
1.80 0.71
2.12 0.26
17.85 1.36
7.57 0.8
13.36 4.58
3.04 0.86
2.46 0.08
2.35 0.03
26.95 6.95
22.58 6.01
22.35 0.03
2.73 0.06
2.59 0.09
2.99 1.53
4
104.79 14.76
282.74 16.16
400.68 35.21
720.7 61.76
1226.45 118.86
1995.52 122.77
348.02 20.19
2362.87 69.41
1881.59 326.57
62.06 6.55
369.40 39.65
395.93 31.93
179.86 6.7
210.02 10.18
395.93 31.93
37.16 9.63
Spleen
Lung
1.5 1.23
2
2
1
2
1.23
Kidney
Brain
1.17 0.41
2.33 0.81
2
1.23
6.67 1.03
4.83 2.04
5.83 3.37
1181.40 295.76
1789.98 180.40
1181.40 295.76
1791.54 177.72
93.32 21.89
196.47 39.95
Table 4
The lethal dose (LD50) of compound 2b determined by acute toxicity test (n ¼ 10).
Dose(mg/kg)
Log(dose)
Mortality (%)
LD50 and 95% confidence interval (mg/kg)
1551.71 (1343.88e1745.08)
1200.00
1500.00
1875.00
2343.75
2929.69
3.08
3.18
3.27
3.37
3.47
0
50
60
80
100
1.53-1.44 (m, 2H), 0.81 (t, 3H). MS(ESI): [MþH]þ calcd 411.2; found
1.57 mmol). The solution was refluxed for 10 h under nitrogen. Then
the resulting mixture was poured into 10 mL ice water and the pH
was adjusted to 5 by addition of 1 M hydrochloric acid solution. The
mixture was extracted with DCM (30 mL ꢁ 3) and washed with
saturated salt water (50 mL ꢁ 5). The organic phase was gathered
and dried over MgSO4. After filtration, the solvent was removed
under reduced pressure. The residue was purified by CC to give
white solid.
411.5.
4.1.7.5. 2-(5-((2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)
methyl)-1H-indol-1-yl)benzonitrile (12b). 12b was synthesized ac-
cording to the above general procedure. Yield: 83.4%. 1H NMR
(400 MHz, CDCl3, ppm) d: 7.84-7.82 (m, 1H), 7.74-7.72 (m, 1H), 7.56
(d, 1H), 7.52-7.51 (m, 2H), 7.43-7.41 (m, 1H), 7.28-7.27 (m, 1H), 7.07-
7.05 (m,1H), 6.71 (d,1H), 4.79 (s, 2H), 2.32 (t, 2H), 2.06-1.83 (m, 8H),
1.56-1.51 (m, 2H), 1.30-1.26 (m, 2H), 0.85 (t, 3H). MS(ESI): [MþH]þ
calcd 425.2; found 425.3.
4.1.8.1. 3-((1-(2-(1H-tetrazol-5-yl)phenyl)-1H-indol-4-yl)methyl)-2-
propyl-1,3-diazaspiro[4.4]non-1-en-4-one (1a). 1a was synthesized
according to the above general procedure. Yield: 64.4%. 1H NMR
4.1.7.6. 2-(5-((4-oxo-2-pentyl-1,3-diazaspiro[4.4]non-1-en-3-yl)
methyl)-1H-indol-1-yl)benzonitrile (12c). 12c was synthesized ac-
cording to the above general procedure. Yield: 81.6%. 1H NMR
(400 MHz, DMSO-d6, ppm) d: 7.98-7.92 (m, 1H), 7.83-7.78 (m, 1H),
7.72 (t, J ¼ 7.3 Hz, 1H), 7.66 (d, J ¼ 7.8 Hz, 1H), 7.33 (d, J ¼ 3.3 Hz, 1H),
7.01 (t, J ¼ 7.8 Hz,1H), 6.87 (d, J ¼ 8.2 Hz, 1H), 6.73 (d, J ¼ 7.2 Hz, 1H),
6.66 (d, J ¼ 3.3 Hz, 1H), 4.94 (s, 2H), 2.27 (t, J ¼ 7.4 Hz, 2H), 1.93-1.80
(m, 6H), 1.76-1.63 (m, 2H), 1.54-1.40 (m, 2H), 0.80 (t, J ¼ 7.4 Hz, 3H).
(400 MHz, DMSO-d6, ppm) d: 7.93 (d, 1H), 7.77 (t, 1H), 7.67 (t, 1H),
7.61 (d, 1H), 7.35 (s, 1H), 7.26 (d, 1H), 7.03-6.77 (m, 2H), 6.57 (s, 1H),
4.72 (s, 2H), 2.32 (t, 2H), 1.92-1.75 (m, 6H), 1.70-1.61 (m, 2H), 1.51-
1.42 (m, 2H), 1.23-1.13 (m, 4H), 0.78 (t, 3H)。MS(ESI): [MþH]þ calcd
439.2; found 439.6.
13C NMR (101 MHz, DMSO)
d 185.97, 161.86, 154.69, 137.72, 136.76,
132.61, 131.61, 130.31, 129.15, 128.99, 128.96, 126.75, 123.28, 122.52,
117.99, 109.75, 101.57, 76.31, 41.48, 37.34, 37.34, 30.19, 25.94, 25.94,
18.40, 13.84. HRMS (ESI): m/z calculated for C26H27N7O [MþH]þ:
454.2355; found 454.2342.
4.1.7.7. 2-(5-((2-hexyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)
methyl)-1H-indol-1-yl)benzonitrile (12d). 12d was synthesized ac-
cording to the above general procedure. Yield: 79.4%. 1H NMR
4.1.8.2. 2-Butyl-3-((1-(2-(1H-tetrazol-5-yl)phenyl)-1H-indol-4-yl)
methyl)-1,3-diazaspiro[4.4]non-1-en-4-one (1b). 1b was synthe-
(400 MHz, DMSO-d6, ppm) d 7.93 (d, 1H), 7.83-7.57 (m, 3H), 7.35 (s,
1
1H), 7.26 (d, 1H), 7.04-6.78 (m, 2H), 6.57 (s, 1H), 4.71 (s, 2H), 2.32 (t,
2H),1.94-1.74 (m, 6H),1.71-1.60 (m, 2H),1.51-1.39 (m, 2H),1.25-1.06
(m, 6H), 0.79 (t, 3H). MS(ESI): [MþH]þ calcd 453.2; found 453.6.
sized according to the above general procedure. Yield: 62.1%.
NMR (400 MHz, DMSO-d6, ppm)
H
d
7.94 (d, J ¼ 7.5 Hz, 1H), 7.77 (t,
J ¼ 7.4 Hz, 1H), 7.69 (t, J ¼ 7.5 Hz, 1H), 7.62 (d, J ¼ 7.8 Hz, 1H), 7.31 (d,
J ¼ 3.2 Hz,1H), 7.00 (t, J ¼ 7.7 Hz,1H), 6.87 (d, J ¼ 8.2 Hz,1H), 6.73 (d,
J ¼ 7.2 Hz, 1H), 6.64 (d, J ¼ 3.1 Hz,1H), 4.94 (s, 2H), 2.29 (t, J ¼ 7.5 Hz,
2H), 1.94-1.78 (m, 6H), 1.74-1.61 (m, 2H), 1.47-1.37 (m, 2H), 1.28-1.16
4.1.8. General procedure for the synthesis of 1aec, 2aed
A solution of an appropriate benzonitrile compound 10aec or
12aed (0.26 mmol) in 10 mL DMF was added sodium azide
(102 mg, 1.57 mmol) and chlorotributylstannane (0.4 mL,
(m, 2H), 0.75 (t, J ¼ 7.3 Hz, 3H). 13C NMR (101 MHz, DMSO)
d 186.02,
161.98, 155.12, 137.61, 136.81, 132.20, 131.59, 130.40, 129.06, 128.94,

X. Bao et al. / European Journal of Medicinal Chemistry 123 (2016) 115e127
125
128.89, 126.78, 124.06, 122.43, 117.99, 109.82, 101.41, 76.34, 41.49,
37.32, 37.32, 28.06, 27.04, 25.94, 25.94, 21.98,14.04. HRMS (ESI): m/z
calculated for C27H29N7O [MþH]þ: 468.2512; found 468.2505.
(s, 1H), 4.71 (s, 2H), 2.32 (t, J ¼ 7.5 Hz, 2H), 1.94-1.74 (m, 6H), 1.71-
1.60 (m, 2H), 1.51-1.39 (m, 2H), 1.25-1.06 (m, 6H), 0.79 (t, J ¼ 6.9 Hz,
3H). 13C NMR (101 MHz, DMSO)
d 186.10, 161.91, 155.61, 137.73,
136.02, 132.42, 131.66, 130.73, 129.12, 129.01, 128.90, 128.79, 128.77,
121.33, 119.02, 110.66, 103.65, 76.25, 43.29, 37.24, 37.24, 31.28,
28.46, 28.33, 25.92, 25.92, 24.91, 22.35, 14.30. HRMS (ESI): m/z
calculated for C29H33N7O [MþH]þ: 496.2825; found 496.2815.
4.1.8.3. 3-((1-(2-(1H-tetrazol-5-yl)phenyl)-1H-indol-4-yl)methyl)-2-
pentyl-1,3-diazaspiro[4.4]non-1-en-4-one (1c). 1c was synthesized
1
according to the above general procedure. Yield: 62.4%. H NMR
(400 MHz, DMSO-d6, ppm)
d
: 7.95 (d, J ¼ 6.7 Hz, 1H), 7.78 (t,
J ¼ 7.1 Hz, 1H), 7.70 (t, J ¼ 7.4 Hz, 1H), 7.62 (d, J ¼ 7.8 Hz, 1H), 7.32 (d,
J ¼ 3.3 Hz, 1H), 7.05-6.97 (m, 1H), 6.87 (d, J ¼ 8.3 Hz, 1H), 6.73 (d,
J ¼ 7.2 Hz,1H), 6.64 (d, J ¼ 3.2 Hz,1H), 4.94 (s, 2H), 2.29 (t, J ¼ 7.6 Hz,
2H), 1.92-1.80 (m, 6H), 1.74-1.62 (m, 2H), 1.49-1.38 (m, 2H),1.20-1.10
4.2. Biological evaluation
4.2.1. Radioligand binding assay
Primary vascular smooth muscle cells (VSMCs) were obtained
from thoracic aorta of SPF SD rats (Slac laboratory animal, Shanghai,
China) and cultured by the tissue explants methods. The inverted
phase contrast microscope was used to observe cell growth,
immunohistochemical staining was used to identify cells, and try-
pan blue staining was used to detect cell viability [15,16]. At first,
thoracic aorta was cut into tissues at the size of 1 mm ꢁ 1 mm after
processing, and then transferred to cell culture flasks. DMEM with
10% fetal bovine serum and 1% mixture of penicillin and strepto-
mycin were appropriately added when the tissues adhered to the
bottom surface uniformly. Culture flask was inverted and incubated
in 37 ꢀC, 5% CO2 for 3e5 h. Tissues were immersed into the culture
medium completely when attaching on the bottom. The cells were
extended when growing to covering the entire sidewall. All the
cells were incubated in 37 ꢀC, 5% CO2. And Morphological changes
was observed using an inverted phase contrast microscope and
identified by immunohistochemical methods with stained smooth
(m, 4H), 0.77 (t, J ¼ 6.8 Hz, 3H). 13C NMR (101 MHz, DMSO)
d 186.04,
162.01, 155.00, 137.65, 136.80, 132.34, 131.62, 130.39, 129.08, 128.97,
128.94, 126.79, 123.81, 122.46, 118.03, 109.80, 101.48, 76.34, 41.49,
37.32, 37.32, 31.05, 28.32, 25.95, 25.95, 24.62, 22.15, 14.26. HRMS
(ESI): m/z calculated for C28H31N7O [MþH]þ: 482.2668; found
482.2659.
4.1.8.4. 3-((1-(2-(1H-tetrazol-5-yl)phenyl)-1H-indol-5-yl)methyl)-2-
propyl-1,3-diazaspiro[4.4]non-1-en-4-one (2a). 2a was synthesized
1
according to the above general procedure. Yield: 67.4%. H NMR
(400 MHz, DMSO-d6, ppm)
d
7.95 (d, J ¼ 7.6 Hz, 1H), 7.77 (t,
J ¼ 7.5 Hz, 1H), 7.69 (t, J ¼ 7.5 Hz, 1H), 7.62 (d, J ¼ 7.8 Hz, 1H), 7.32 (d,
J ¼ 3.3 Hz,1H), 7.01 (t, J ¼ 7.7 Hz,1H), 6.87 (d, J ¼ 8.2 Hz,1H), 6.72 (d,
J ¼ 7.2 Hz,1H), 6.64 (d, J ¼ 3.2 Hz,1H), 4.94 (s, 2H), 2.28 (t, J ¼ 7.3 Hz,
2H), 1.95-1.78 (m, 6H), 1.75-1.61 (m, 2H), 1.53-1.44 (m, 2H), 0.81 (t,
J ¼ 7.3 Hz, 3H). 13C NMR (101 MHz, DMSO)
d 186.02, 161.78, 155.22,
137.57, 136.79, 132.08, 131.58, 130.42, 129.06, 128.90, 128.87, 126.73,
124.30, 122.41, 117.86, 109.83, 101.35, 76.34, 41.48, 37.34, 37.34,
30.19, 25.94, 25.94, 18.40, 13.86. HRMS (ESI): m/z calculated for
muscle-a-actin.
3e6 generations of VSMCs were used for experiments. The new
compounds and irbesartan (Shanghai Zhongkang Weiye Biological
Technology Co., Ltd. Shanghai, China) were dissolved in DMSO and
diluted to different concentrations (10ꢂ10 e 10ꢂ4 M) with PBS
before experiments. 125I-Ang II (Zhongshan Hospital, Fudan Uni-
versity, China) was dissolved with PBS and diluted to 0.2 nM.
C
26H27N7O [MþH]þ: 454.2355; found 454.2345.
4.1.8.5. 2-Butyl-3-((1-(2-(1H-tetrazol-5-yl)phenyl)-1H-indol-5-yl)
methyl)-1,3-diazaspiro[4.4]non-1-en-4-one (2b). 2b was synthe-
1
sized according to the above general procedure. Yield: 65.7%.
NMR (400 MHz, DMSO-d6, ppm)
H
VSMCs (106 cells/well, 500
37 ꢀC, 5% CO2. After the cells adhered to the wall, 500
m
m
L) were seeded into 24-well plates in
d
: 7.93 (d, J ¼ 6.3 Hz, 1H), 7.76-7.55
L 125I-Ang II
(m, 3H), 7.35 (s, 1H), 7.25 (s, 1H), 7.00-6.79 (m, 2H), 6.55 (s, 1H), 4.71
(s, 2H), 2.32 (t, 2H), 1.95-1.68 (m, 6H), 1.73-1.56 (m, 2H), 1.55-1.39
(m, 2H), 1.30-1.22 (m, 2H), 0.79 (t, J ¼ 7.2 Hz, 3H). 13C NMR
and 10 mL compound 1aec, 2aed were added respectively to the
final concentrations of 10ꢂ12 e 10ꢂ6 M and then cells were culti-
vated in 4 ꢀC for 150 min [17]. After that, cells were washed 3 times
with PBS and digested for 10 min with 0.1 M NaOH. The cells bound
(101 MHz, DMSO)
d 186.13, 161.82, 155.56, 137.52, 136.07, 131.72,
131.58, 130.89, 129.00, 129.00, 128.86, 128.65, 124.84, 121.21, 118.95,
110.72, 103.38, 76.27, 43.30, 37.25, 37.25, 28.08, 27.08, 25.93, 25.93,
22.04, 14.11. HRMS (ESI): m/z calculated for C27H29N7O [MþH]þ:
468.2512; found 468.2500.
by 125I-Ang II were counted by
g-counter (SN-682, Ri Huan Com-
pany, Shanghai). The half inhibition constant (IC50 value) of the
combination of these compounds with membrane protein were
estimated by the nonlinear portion of the competition curves. The
Ki value was calculated from the formula Ki ¼ IC50/(1 þ [L]/kd), [L]
was the concentration of radioligand present in tubes [18].
4.1.8.6. 3-((1-(2-(1H-tetrazol-5-yl)phenyl)-1H-indol-5-yl)methyl)-2-
pentyl-1,3-diazaspiro[4.4]non-1-en-4-one (2c). 2c was synthesized
according to the above general procedure. Yield: 68.1%. 1H NMR
4.2.2. In vivo study of anti-hypertensive effect
(400 MHz, DMSO-d6, ppm)
d
7.93 (d, J ¼ 7.5 Hz, 1H), 7.77 (t,
Spontaneous hypertensive rats (SHRs) (250 20 g, Beijing Vital
River Laboratory Animal Co., Ltd. Beijing, China) and renal hyper-
J ¼ 7.1 Hz, 1H), 7.68 (t, J ¼ 7.3 Hz, 1H), 7.61 (d, J ¼ 7.7 Hz, 1H), 7.35 (s,
1H), 7.26 (d, J ¼ 3.1 Hz, 1H), 7.03-6.78 (m, 2H), 6.57 (s, 1H), 4.71 (s,
2H), 2.32 (t, J ¼ 7.6 Hz, 2H), 1.92-1.75 (m, 6H), 1.70-1.61 (m, 2H),
1.51-1.42 (m, 2H), 1.23-1.13 (m, 4H), 0.78 (t, J ¼ 6.9 Hz, 3H). 13C NMR
tensive rats (RHRs) (250
20 g, Department of Pharmaceutical
Science and Technology, Donghua University, Shanghai, China)
were used to evaluate the effects on systolic blood pressure (SBP)
and diastolic blood pressure (DBP) of new compounds. 54 male
SHRs were divided into nine experimental groups randomly:
negative control group, positive control group, compound 1a, 1b,
1c, 2a, 2b, 2c and 2d groups. Each compound was suspended in a
0.5% solution of sodium carboxymethyl cellulose and administered
orally at the dose of 15 mg/kg. Irbesartan (15 mg/kg) was taken as
positive control group. The negative control group was adminis-
tered the same volume of sodium carboxymethyl cellulose solution.
Another 6 male SHRs was used to test the antihypertensive effect of
compound 2b at 10 mg/kg. Besides, 24 male RHRs were divided into
four groups randomly: negative control group, positive control
(101 MHz, DMSO)
d 186.10, 161.98, 155.56, 137.74, 136.02, 132.42,
131.68, 130.75, 129.12, 129.02, 128.90, 128.80, 128.77, 121.35, 119.02,
110.67, 103.67, 76.24, 43.30, 37.24, 37.24, 31.11, 28.33, 25.93, 25.93,
24.68, 22.22, 14.27. HRMS (ESI): m/z calculated for C28H31N7O
[MþH]þ: 482.2668; found 482.2671.
4.1.8.7. 2-Hexyl-3-((1-(2-(1H-tetrazol-5-yl)phenyl)-1H-indol-5-yl)
methyl)-1,3-diazaspiro[4.4]non-1-en-4-one (2d). 2d was synthe-
sized according to the above general procedure. Yield: 61.1%.
NMR (400 MHz, DMSO-d6, ppm)
(m, 3H), 7.35 (s, 1H), 7.26 (d, J ¼ 3.1 Hz, 1H), 7.04-6.78 (m, 2H), 6.57
1
H
d: 7.93 (d, J ¼ 7.4 Hz, 1H), 7.83-7.57

126
X. Bao et al. / European Journal of Medicinal Chemistry 123 (2016) 115e127
group and compound 2b (15 mg/kg and 10 mg/kg) groups. Then
blood pressure and heart rates were monitored before and after the
administration by a biological signal analysis system (MPA - 2000,
Alcott Biotech, China). Six determinations were made in every
session of blood pressure measurements and the means of the six
values were taken as the systolic blood pressure level and diastolic
blood pressure level, respectively. The mean arterial pressure
(MBP) was calculated by the formula: MBP ¼ (SBP ꢂ DBP)/3 þ DBP,
and all the results were expressed as mean SD. A probability level
of less than 0.05 was considered significant.
4.2.4. Acute toxicity of compound 2b
To detect the safety of compound 2b, the acute toxicity assay
was carried out with 50 normal ICRs (20 2 g, Academia Sinica,
China). The LD50 value was delivered via intragastric administration
(ig) at doses of 2929.69, 2343.75, 1875, 1500, 1200 mg/kg. Survival
was assessed daily for two weeks. The lethal dose (LD50) and 95%
confidence limits were determined from logistic regression analysis
(GLM) curve fitting of the 14 days' mortality data. Survival rats were
observed continuously and recorded systematically for the physical
signs of toxicity including weight, mobility, aggressiveness and
respiratory movements. At the 15th day, the survivals were
dissected to examine the pathological changes of organs [23].
4.2.3. Pharmacokinetic characteristics of compound 2b
4.2.5. Statistics
High performance liquid chromatography (HPLC) method was
used to analyze the drug concentration of compound 2b in plasma
and in tissues. The method was developed, validated and operated
on two separate but similar Waters-brand systems. The flow rate
Results were expressed as means standard error of the means.
Data were analyzed by one-way analysis of variance. When overall
statistical significance was achieved (P < 0.05), anova one way and
t-test statistical were used to compare each of the doses to the
vehicle control. Probability values less than 0.05 were considered to
be significant. Binding isotherms from competition studies were
obtained using the nonlinear regression program GraphPad Prism 5
software (Network of Science Software of China) and pharmaco-
kinetic parameters were acquired by DAS.2.0 software. And the
completed animal research here adhered to the “Principles of
Laboratory Animal Care” and was approved by IACUC.
was 1.0 mL/min and the injection volume was 20
were performed at ambient temperature on a reverse phase
Wondasilicon C18-WR column (4.6 ꢁ 150 mm, 5 m). For the
mL. Separations
m
analysis of drug concentration in plasma, Mobile Phases A was
0.02 mol/L potassium dihydrogen phosphate solution (55%) and
Mobile Phases B was acetonitrile solution (45%). For the analysis of
drug concentration in tissues, Mobile Phases A was acetonitrile
(60%) and Mobile Phases B was pure water (40%). Microsoft Excel
program, Origin 8.0 and DAS 2.0 software were used to calculate the
pharmacokinetic parameters.
Notes
18 male Wistar rats (180 20 g, Shanghai Slac Laboratory Ani-
mal Company, Shanghai, China) were randomly divided into three
groups and administrated with 2b at dose of 10 mg/kg, 5 mg/kg and
2 mg/kg, respectively. Blood samples were collected from each rat
by retro-orbital puncture at a predetermined time interval of pre-
dose, 0.5, 1, 2, 4, 6, 8, 12, 24, 48 and 72 h into the polypropylene
microfuge tubes containing heparin. Plasma was separated by
centrifuging the blood samples at 4000 ꢁ g at 4 ꢀC for 10 min. Then
The authors declare no competing financial interest.
Acknowledgement
This work was supported by Science and Technology Commis-
sion of Shanghai Municipality (Grants No. 13431900700,
14431906200), Science and Technology Bureau of Ningbo (Grant
No. 2015A610274), and the Fundamental Research Funds for the
Central Universities (Grant No. CUSF-DH-D-2016041).
200
mL plasma samples for analysis were placed into a tube fol-
lowed by 100
m
L of internal standard. Acetonitrile (200 L) was
m
added to precipitate proteins and the tube was vortexed for 1 min.
Precipitated proteins were separated by centrifugation for 5 min at
12,000 ꢁ g at 4 ꢀC. The supernatant was filtered by needle filter of
Abbreviations
RAS
AII
renin angiotensin system
angiotensin II
0.45 mm and the resulting 20 mL filtrate was taken to analyze in
HPLC. Linearity for 2b was tested by extracting plasma standards
spiked at nominal concentrations of 0.1, 0.5, 1, 5, 10 g/mL. The
ARBs
ACE
SHR
RHR
ICR
angiotensin II receptor blockers
angiotensin converting enzyme
spontaneously hypertensive rat
renal hypertensive rats
m
calibration line was generated by least squares linear regression of
the peak height ratio (PHR) of analyte/internal standard against
nominal concentration with a weighting of concentrationꢂ2 [19,20].
198 Wistar rats (180 20 g, Shanghai Slac Laboratory Animal
Company, Shanghai, China) were used to investigate the tissue
distribution of compound 2b. Animals were orally administrated
with 2b at 10 mg/kg, 15 mg/kg and 30 mg/kg respectively. Heart,
liver, spleen, lung, kidney and brain samples were collected at
0 (prior to dosing), 0.5, 1, 2, 4, 6, 8, 12, 24, 48 and 72 h post-dosing
(n ¼ 6). 0.25 g samples from different tissues were obtained after
being rinsed with normal saline solution and blotted with filter
paper. The samples were grinded into tissue fluid (0.25 g/1 mL
normal saline solution) with High-speed Homogenizer (PRO200,
Bio-gen series, USA). The tissue fluid was centrifuged at 10,000 ꢁ g
for 10 min. 500 mL supernate was collected and added with 2 mL
methanol, and then it was vortexed for 60 s and centrifuged at
12,000 ꢁ g for 5 min. Supernatant was obtained and dried in gentle
stream of nitrogen flow at 50 ꢀC. The residue was spun for 2 min
Institute for Cancer Research
VSMCs Vascular Smooth Muscle Cells
MBP
BBB
LD50
IC50
mean blood pressure
blood-brain barrier
median lethal dose
half maximal inhibitory concentration
maximum concentration
Cmax
AUC0~72 area under the concentration-time curve
T1/2
DCM
DMF
NMR
MP
TMS
CC
DCM
ESI
elimination half-lives
methylene chloride
N,N-dimethylformamide
nuclear magnetic resonance
melting points
Me4Si
column chromatography
dichloromethane
electrospray ionization
mass spectrometry
after being reconstituted with 100
sample. 20 L supernatant samples were taken to analyze through
HPLC [21,22].
mL methanol to get supernatant
m
MS
DMSO dimethylsulfoxide

X. Bao et al. / European Journal of Medicinal Chemistry 123 (2016) 115e127
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