Discovery of novel n-substituted prolinamido indazoles as potent rho kinase inhibitors and vasorelaxation agents

Article abstract of DOI:10.3390/molecules22101766

Inhibitors of Rho kinase (ROCK) have potential therapeutic applicability in a wide range of diseases, such as hypertension, stroke, asthma and glaucoma. In a previous article, we described the lead discovery of DL0805, a new ROCK I inhibitor, showing potent inhibitory activity (IC₅₀ 6.7 μM). Herein, we present the lead optimization of compound DL0805, resulting in the discovery of 24- and 39-fold more-active analogues 4a (IC₅₀ 0.27 μM) and 4b (IC₅₀ 0.17 μM), among other active analogues. Moreover, ex-vivo studies demonstrated that 4a and 4b exhibited comparable vasorelaxant activity to the approved drug fasudil in rat aortic rings. The research of a preliminary structure-activity relationship (SAR) indicated that the target compounds containing a β-proline moiety have improved activity against ROCK I relative to analogues bearing an β-proline moiety, and among the series of the derivatives with a β-proline-derived indazole scaffold, the inhibitory activity of the target compounds with a benzyl substituent is superior to those with a benzoyl substituent.

Full text of DOI:10.3390/molecules22101766

molecules
Article
Discovery of Novel N-Substituted Prolinamido
Indazoles as Potent Rho Kinase Inhibitors
and Vasorelaxation Agents
Yangyang Yao 1,†, Renze Li 1,†, Xiaoyu Liu 1, Feilong Yang 1, Ying Yang 1, Xiaoyu Li 1, Xiang Shi 1,
Tianyi Yuan 2, Lianhua Fang 2, Guanhua Du 2, Xiaozhen Jiao 1,* and Ping Xie 1,
*
1
State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Beijing Key Laboratory of
Active Substances Discovery and Druggability Evaluation, Institute of Materia Medica, Chinese Academy of
Medical Sciences and Peking Union Medical College, Beijing 100050, China; yao0943017@163.com (Y.Y.);
renze.li@open01.com (R.L.); lxya@imm.ac.cn (X.L.); yangfeilong@imm.ac.cn (F.Y.);
yangying@imm.ac.cn (Y.Y.); pharmli@imm.ac.cn (X.L.); xiangshi@imm.ac.cn (X.S.)
State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Beijing Key Laboratory of
Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical
Sciences and Peking Union Medical College, Beijing 100050, China; yuantianyi@imm.ac.cn (T.Y.);
fanglh@imm.ac.cn (L.F.); dugh@imm.ac.cn (G.D.)
2
*
Correspondence: jiaoxz@imm.ac.cn (X.J.); xp@imm.ac.cn (P.X.); Tel.: +86-10-63165241 (X.J.);
+86-10-63165242 (P.X.)
†
These authors contributed equally to this work.
Received: 19 September 2017; Accepted: 16 October 2017; Published: 19 October 2017
Abstract: Inhibitors of Rho kinase (ROCK) have potential therapeutic applicability in a wide range
of diseases, such as hypertension, stroke, asthma and glaucoma. In a previous article, we described
the lead discovery of DL0805, a new ROCK I inhibitor, showing potent inhibitory activity (IC50 6.7
Herein, we present the lead optimization of compound DL0805, resulting in the discovery of 24- and
39-fold more-active analogues 4a (IC50 0.27 M) and 4b (IC50 0.17 M), among other active analogues.
µM).
µ
µ
Moreover, ex-vivo studies demonstrated that 4a and 4b exhibited comparable vasorelaxant activity
to the approved drug fasudil in rat aortic rings. The research of a preliminary structure–activity
relationship (SAR) indicated that the target compounds containing a
activity against ROCK I relative to analogues bearing an -proline moiety, and among the series
of the derivatives with a -proline-derived indazole scaffold, the inhibitory activity of the target
β-proline moiety have improved
α
β
compounds with a benzyl substituent is superior to those with a benzoyl substituent.
Keywords: Rho kinase; inhibitor; N-substituted prolinamido indazoles; vasorelaxant; SAR
1. Introduction
Arterial hypertension is a long-term medical condition in which the blood pressure in the arteries
is persistently elevated. Hypertension already affects 1.1 billion people worldwide, leading to coronary
artery disease, stroke, heart failure, peripheral vascular disease, vision loss and chronic kidney disease.
Despite the development of approaches for the prevention and control of raised blood pressure, there
remains a significant need for the discovery of novel antihypertensive agents.
Rho kinase (Rho-associated coiled-coil-containing protein kinase (ROCK)) belongs to a family
of serine/threonine protein kinases and acts as a major downstream effector of Rho A [
an important role in regulating a variety of cellular functions such as smooth-muscle-cell contraction,
cell adhesion, migration and proliferation [ ]. ROCK contains two isoforms, ROCK I and ROCK II,
which share approximately 60% overall amino acid sequence identity and 92% identity within
the kinase domain [ ]. However, the expression of the two isoforms has different tissue specificity.
1]. It plays
2
3
Molecules 2017, 22, 1766; doi:10.3390/molecules22101766
www.mdpi.com/journal/molecules

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ROCK II shows high expression in brain tissue, while ROCK I is preferentially expressed in the tissues
of the pancreas, liver, lung and heart [ ]. Accumulating evidence shows that the dysregulation of
ROCK activity or expression is associated with various diseases, including hypertension [ ], stroke [ ],
asthma [ ], cancer [ ] and glaucoma [10 12], which indicates that ROCK is a potential novel target
for drug development.
3
4
5
6,
7
8
,9
–
Over the past two decades, ROCK has aroused extensive interest, and numerous ROCK
inhibitors have been developed from a variety of distinct scaffolds including isoquinoline [13],
quinazoline [14], indazole [15–20], benzimidazole [21,22], benzothiazole [23], quinazolinone [24,25],
diaminopyrazine [26], benzamide [27], chroman-3-amide [28] and urea [29]. Usually, ROCK II
inhibitors were widely applied in the central nervous system (CNS) to cure stroke, Alzheimer’s
disease and other diseases, and ROCK I inhibitors showed therapeutic potential for the treatment of
hypertension [30]. To date, only two ROCK inhibitors, fasudil (approved in 1995 for the treatment of
cerebral vasopasm in Japan) and ripasudil (approved in 2014 for treatment of glaucoma in Japan) have
been approved for clinical use. The narrow therapeutic window restricts the application of the existing
ROCK inhibitors, leading to the development of novel ROCK inhibitors. Our group has been focused
on the discovery of novel Rho kinase inhibitors for the treatment of hypertension.
5-Nitro-1H-indazole-3-carbonitrile (DL0805,
value of 6.7 M against ROCK I, which was discovered by high-throughput screening (HTS) [31].
Previous literature had disclosed the development of ROCK I inhibitors with an indazole scaffold
attached to a rigid aromatic heterocycle linking structure (I, II, III) (Figure 1) [32 34]. These studies
1) (Figure 1) is a new ROCK inhibitor with an IC50
µ
–
highlighted the importance of the indazole for ROCK I activity. Herein, we report the design,
synthesis and the structure–activity relationship (SAR) research of a series of novel N-substituted
prolinamido indazoles as ROCK I inhibitors, and their vasorelaxant activity evaluation based on
the DL0805 template.
Figure 1. The structures of screened lead compound DL0805, I, II and III.
2. Results and Discussion
2.1. Molecular Design
In order to discover potent ROCK I inhibitors, we further optimized the structure of the DL0805
template. The attractive indazole core of DL0805 was preserved, owing to the frequent presence of
this scaffold in ROCK I inhibitors. Considering the potential genotoxic hazard of the NO2 group,
we conceived of the novel N-substituted prolinamido indazole ROCK I inhibitors, wherein the NO2

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group can be replaced with a flexible N-substituted prolinamido group instead of the substituted
rigid heteroaromatic ring described in the literature (e.g., I, II, III) (Figure 1). Four related series of
N-substituted prolinamido indazoles were designed and are listed in Figure 2 (2, 3, 4, 5).
R
N
H
N
N
series I
series II
O
N
H
2
R
O
N
N
N
N
H
NC
N
H
NO2
3
R
N
H
N
H
N
DL0805
N
series III
O
N
H
4
O
R
N
H
N
N
series IV
O
N
H
5
Figure 2. Molecular design based on the template compound DL0805.
2.2. Chemistry
A concise route was developed to synthesize compounds 2a
D/L-proline as the starting material (Scheme 1). Alkylation of proline provided the intermediates 6a
–
2f and 3a
–
3c using racemic or
6f
–
.
Initially, amidation of 6a with 5-aminoindazole employing EDCI in CH2Cl2 at room temperature gave
the target compound 2a only in low yield because of 6a’s solubility. When we changed to use DMF
as reaction solvent and raised the reaction temperature to 80 ◦C, target compound 2a was obtained
in moderate yield. Thus, this reaction condition was used to synthesize the target compounds 2b
Analogues 3a 3c were similarly prepared by coupling the intermediates 6a 6c with 6-aminoindazole.
Next, we embarked on the synthesis of analogue following a similar synthetic route used
for preparing compounds and . Unfortunately, an inseparable mixture was observed during
the direct alkylation of -proline. Therefore, new synthetic strategies to access analogues and were
performed and described in Scheme 2. The target compounds 4a 4i were synthesized using racemic or
D/L- -proline as the starting material. Esterification of -proline with SOCl2 and ethyl alcohol gave
the ester . Subsequently, alkylation of ester with substituted benzyl bromides provided compounds
8a 8i, which were hydrolyzed and then reacted with 5-aminoindazole to produce compounds 4a 4i
Intermediate was also used to prepare the target compounds 5a 5f in an analogous fashion. Acylation
of ester with substituted benzoyl chloride afforded compounds 10a 10f that were saponified to
11a–11f and coupled to 5-aminoindazole to furnish the other set of target compounds 5a–5f.
–2f.
–
–
4
2
3
β
4
5
–
β
β
7
7
–
– .
7
–
7
–

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Scheme 1. Synthesis of compounds 2a–2f and 3a–3c. Reagents and conditions: (a) R-Br, KOH, isopropyl
alcohol, 40 ◦C, 12 h; (b) 5-aminoindazole, EDCI, DMF, 80 ◦C, 3 h; (c) 6-aminoindazole, EDCI, DMF,
◦
80 C, 3 h.
Scheme 2. Synthesis of compounds 4a
–
4i and 5a
–
5f. Reagents and conditions: (a) SOCl2, ethyl alcohol,
◦
0 to 40 C, 10 h; (b) R-Br, Et3N, CH2Cl2, reflux, 4 h; (c) 4 M NaOH, ethyl alcohol, room temperature, 1 h;
◦
(d) 5-aminoindazole, EDCI, DMF, 80 C, 7 h; (e) R-benzoyl chloride, Et3N, CH2Cl2, reflux, 4 h.
2.3. Bioassay Studies
2.3.1. ROCK I Inhibitory Activity Evaluation
The twenty-four target compounds were initially evaluated for their percentage inhibition
against ROCK I with a ROCK I assay kit (CY-1160, Cyclex, Nagoya, Japan) at 20 µM (Table 1).
The nine compounds with significant inhibition against ROCK I were further evaluated in full
concentration–response plots to determine their IC50 values (Table 2). The data illustrate some

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clear SAR trends. Firstly, the target compounds of series III (
4
) and IV (
5
) containing a
) and II (
-proline moiety. This implied that a near-linear molecule probably had better combination with
ROCK I than an “angular”-shaped molecule. Secondly, among the series of the derivatives with
-proline-derived indazole scaffold, the inhibitory activity of the target compounds (series III) with
a benzyl substituent ( ) was superior to those with a benzoyl substituent (series IV ). This suggested
β
-proline
moiety have improved activity against ROCK I relative to analogue series I (
an
2
3) bearing
α
a
β
4
5
that the free rotation of a single bond was probably beneficial to the ROCK I inhibitory activity. Thirdly,
the data of series III compounds showed that the substituent group had some influence on the activity
(CH3 > H > Br > OCH3 > F > NO2, CN). This implied that the activity was probably affected by multiple
factors, and the volume factor may be important for the activity. In addition, the data also revealed
the (S)-enantiomer had a higher activity than the (R)-enantiomer (4i versus 4h, respectively) with
the (1H-indazole-5-yl)-pyrrolidine-3-carboxamide scaffold. In contrast, the (R)- and (S)-enantiomers
had a similar activity (2b versus 2c; 3b versus 3c) in the (1H-indazole-5-yl)-pyrrolidine-2-carboxamide
and the (1H-indazole-6-yl)-pyrrolidine-3-carboxamide scaffolds.
Table 1. Percentage inhibition of 2a
–
2f,
3a
–3c
,
4a
–
4i and 5a
–
5f against ROCK I under the concentration
of 20µM. a
% Inhibition
of ROCK I
% Inhibition
of ROCK I
Compound
R
Configuration
Compound
R
Configuration
2a
2b
2c
2d
2e
2f
3a
3b
3c
4a
4b
4c
4d
4-H
4-H
4-H
4-CH3
4-CH3
4-CH3
4-H
4-H
4-H
4-H
(±)
(R)
(S)
(±)
(R)
(S)
(±)
(R)
(S)
(±)
(±)
(±)
(±)
a
27.2
24.3
24.6
26.6
4e
4f
4g
4h
4i
5a
5b
5c
5d
5e
5f
DL0805
fasudil
4-NO2
4-CN
4-F
4-H
4-H
(±)
(±)
(±)
(S)
(R)
(±)
(±)
(±)
(±)
(±)
(±)
44.7
24.5
63.8
54.8
73.9
42.5
54.9
19.1
27.9
45.4
55.2
72.3
71.1
21.3
16.0
15.0
23.7
73.2
75.8
53.3
62.5
4-H
4-CH3
4-OCH3
4-Br
4-NO2
4-Cl
4-CH3
4-OCH3
4-Br
These data were obtained by single determinations.
Table 2. The IC50 values of compounds against ROCK I.
Compound
ROCK I (µM)
Compound
ROCK I (µM)
Compound
ROCK I (µM)
1.41 b
0.42 b
7.32 b
11.5 b
7.26 b
6.67 b
0.36 a
4a
4b
4c
0.27 a
0.17 a
1.86 b
0.51 b
4g
4h
4i
5f
DL0805
fasudil
4d
5b
a
These data were means of multiple experiments (N = 4) with errors within 30% of the mean; b these data were
obtained by single determinations.
2.3.2. Vasorelaxant Activity Evaluation
Abnormalities in the Rho/ROCK signaling pathway are associated with various cardiovascular
diseases, especially hypertension. The inhibition of the Rho/ROCK pathway can cause the vessels to
relax [4]. The norepinephrine (NE)- or potassium chloride (KCl)-induced model of the rat aortic ring
can trigger sustained vessel contraction and is usually used to evaluate vasorelaxant activity. DL0805
has shown vasorelaxant activity [31]. Therefore, the nine compounds with significant inhibition against
ROCK I were further tested for their vasorelaxant activity in rat aortic rings in both the high-potassium
and NE models [35] (Table 3). Compounds 4a, 4b and 4c showed low micromolar EC50 values in
both vasorelaxant assays. The potent active compound 4b with excellent activity has been further
evaluated [35], and further pharmacokinetic and safety evaluations are in progress.

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Table 3. The EC50 values of compounds for vasorelaxant activity.
Vasorelexant Evaluation (µM)
Compound
In High-Potassium Model
In NE Model
4a
4b
4c
4d
4g
4h
4i
5b
5f
4.00 a
3.47 a
5.80 a
58.1 b
19.5 b
32.36 b
6.02 b
60.6 b
–
5.62 a
4.07 a
5.57 a
46.8 b
8.91 b
–
11.48 b
15.6 b
–
9.44 b
28.8 b
DL0805
fasudil
5.47 a
4.65 a
– No activity; a these data were means of multiple experiments (N = 6) with errors within 30% of the mean; b these
data were obtained by single determinations.
2.4. Molecular Docking Studies
To identify the possible binding modes of our inhibitors, molecular docking of compound 4a was
performed to elucidate key interactions within the active site of ROCK I. As shown in Figure 3, docking
of compound 4a into the binding site of Rho kinase indicates two key hydrogen-bond interactions
between the N and NH in the indazole ring and Met 156, respectively. The amide NH is predicted
to form a hydrogen bond with Ala 215. Furthermore, a pi–cation interaction between the terminal
phenyl ring and Lys 105 was also observed. In addition, molecular docking of compound 2a was
also performed. In contrast to compound 4a, two key hydrogen-bond interactions between the N and
NH in the indazole ring and Met 156 were still maintained. However, the hydrogen-bond interaction
between the amide NH with Ala 215 was not observed. The result is in agreement with the activity
result, that the activities of series III and IV were superior to those of series I and II.
Figure 3. Cont.

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Figure 3. The 2D structure of compounds 4a and 2a, and 3D view of the key interactions between
4a 2a and the ATP binding pocket of ROCK I. Docking study was performed with the ROCK I
,
structure obtained from the Protein Data Bank (PDB code: 3NDM). Green dashed lines represent
hydrogen-bonding interactions and orange lines represent pi–cation interaction.
3. Materials and Methods
All melting points were obtained on a Yanaco melting-point apparatus and were uncorrected. ESI
mass spectra were performed on the Thermo Exactive Plus LC–MS spectrometer. Nuclear magnetic
resonance (NMR) spectra were recorded on a Bruker 300 or 400 MHz NMR spectrometer with TMS
as an internal standard. Chemical shifts (δ values) and coupling constants (J values) were given in
ppm and Hz, respectively. Column chromatography was performed with silica gel (160–200 mesh,
Qingdao Haiyang Chemical, Qingdao, China). Unless otherwise noted, reagents and solvents were
purchased from Acros Chemical Co, (Geel, Belgium) or other commercial providers and used without
further purification.
3.1. Chemistry
3.1.1. General Procedure for the Preparation of Compounds 6
To a solution of proline (2.0 g, 17 mmol) and KOH (2.85 g, 51 mmol) in i-PrOH (50 mL),
◦
4-substituted benzyl derivative (21 mmol) was added at 40 C. Afterwards, the mixture was stirred for
8 h at 40 ◦C and then the reaction mixture was cooled to room temperature. 6 M HCl was added to
adjust the pH value of the mixture to 4–5 and then CHCl3 (50 mL) was added. The mixture was stirred
for 12 h, followed by filtration and evaporation in vacuo. The residue was purified by recrystallization
in acetone at 0 ◦C to give 6 (about 90% yield).
1-Benzylpyrrolidine-2-carboxylic acid (6a): yield: 86%; white solid; 1H-NMR (400 MHz, CD3OD):
δ 7.53
(m, 2H), 7.47 (m, 3H), 4.53 (d, 1H, J = 12.0 Hz), 4.28 (d, J = 12.8 Hz, 1H), 4.14 (dd, J1 = 10.4 Hz, J2 = 7.2 Hz,
1H), 3.52 (m, 1H), 3.27 (m, 1H), 2.54 (m, 1H), 2.14 (m, 2H), 2.20 (m, 1H).
27
(R)-1-Benzylpyrrolidine-2-carboxylic acid (6b): yield: 90%; white solid; [
α
]
= +25.3 (c 0.92, MeOH);
D
1H-NMR (400 MHz, CD3OD):
δ
7.53 (m, 2H), 7.49 (m, 3H), 4.57 (d, 1H, J =10.8 Hz), 4.37 (t, J = 8.8 Hz,
1H), 4.31 (d, J = 12.8 Hz, 1H), 3.51 (m, 1H), 3.35 (m, 1H), 2.61 (m, 1H), 2.18 (m, 2H), 2.00 (m, 1H).
26
(S)-1-Benzylpyrrolidine-2-carboxylic acid (6c): yield: 92%; white solid; [
1H-NMR (400 MHz, CD3OD):
1H), 4.10 (m, 1H), 3.49 (m, 1H), 3.26 (m, 1H), 2.52 (m, 1H), 2.13 (m, 2H), 1.96 (m, 1H).
α
]
=
−
26.9 (c 0.9, MeOH);
D
δ
7.51 (m, 2H), 7.45 (m, 3H), 4.51 (d, 1H, J = 12.8 Hz), 4.26 (d, J = 12.4 Hz,

Molecules 2017, 22, 1766
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1-(4-Methylbenzyl)pyrrolidine-2-carboxylic acid (6d): yield: 84%; white solid; 1H-NMR (400 MHz,
DMSO-d6): 7.33 (d, J = 7.6 Hz, 2H), 7.19 (d, J = 7.6 Hz, 2H), 4.21 (d, J = 12.8 Hz, 1H), 4.00 (d, J = 12.8 Hz,
δ
1H), 3.76 (t, J = 7.2 Hz, 1H), 3.25 (m, 1H), 2.91 (q, J = 9.2 Hz, 1H), 2.29 (s, 4H), 2.23 (m, 1H), 1.91 (m, 2H),
1.84 (m, 1H), 1.77 (m, 1H). 13C-NMR (150 MHz, DMSO):
53.3, 28.1, 22.2, 20.8.
δ
170.6, 138.0, 130.1, 129.8, 129.1, 65.7, 56.7,
28
(R)-1-(4-Methylbenzyl)pyrrolidine-2-carboxylic acid (6e): yield: 89%; white solid; [
MeOH); H-NMR (400 MHz, DMSO-d6):
α
]
= +27.3 (c 0.92,
D
1
δ 7.31 (d, J = 7.6 Hz, 2H), 7.18 (d, J = 7.6 Hz, 2H), 4.13
(d, J = 12.8 Hz, 1H), 3.89 (d, J = 12.8 Hz, 1H), 3.52 (dd, J1 = 6.4 Hz, J2 = 8.4 Hz, 1H), 3.18 (m, 1H), 2.76
(q, J = 9.2 Hz, 1H), 2.30 (s, 4H), 2.16 (dd, J1 = 8.8 Hz, J2 = 12.4 Hz, 1H), 1.89 (m, 2H), 1.84 (m, 1H), 1.74
(m, 1H). 13C-NMR (150 MHz, DMSO):
δ
170.9, 137.8, 130.5, 130.0, 129.0, 65.8, 56.8, 53.2, 28.3, 22.4, 20.8.
26
(S)-1-(4-Methylbenzyl)pyrrolidine-2-carboxylic acid (6f): yield: 92%; white solid; [
MeOH); H-NMR (400 MHz, DMSO-d6):
α
]
=
−
23 (c 0.92,
D
1
δ 7.38 (d, J = 7.6 Hz, 2H), 7.22 (d, J = 7.6 Hz, 2H), 4.32
(d, J = 12.8 Hz, 1H), 4.13 (d, J = 12.8 Hz, 1H), 3.97 (t, J = 7.6 Hz, 1H), 3.33 (m, 1H), 3.04 (q, J = 10 Hz,
1H), 2.30 (s, 4H), 1.95 (m, 2H), 1.89 (m, 1H), 1.81 (m, 1H), 1.74 (m, 1H). 13C-NMR (150 MHz, DMSO):
δ 170.2, 138.4, 130.4, 129.1, 129.0, 65.4, 56.7, 53.6, 28.1, 22.1, 20.8.
3.1.2. General Procedure for the Preparation of Compounds 7
To a solution of ethanol (150 mL), SOCl2 (10.34 g, 86.86 mmol) was added at 0 ◦C. Afterwards,
◦
β
-proline (5.0 g, 43.43 mmol) was added in several separated portions at 0 C. The mixture was
stirred for 10 h at 40 ◦C and then evaporated in vacuo. The residue was used immediately without
further purification.
3.1.3. General Procedure for the Preparation of Compounds 8 or 10
To a solution of
7 (400 mg) in dry CH2Cl2 (8 mL), Et3N (849 mg, 8.39 mmol) and 4-substituted
benzyl or benzoyl derivative (3.36 mmol) were added at room temperature. Afterwards, the mixture was
refluxed for 4 h and then evaporated in vacuo. The residue was purified by column chromatography
on silica gel (10–20% EtOAc in CH2Cl2) to give 8 or 10 (about 80% yield).
Ethyl 1-benzylpyrrolidine-2-carboxylate (8a): yield: 79%; slightly yellow oil; 1H-NMR (CDCl3, 400 MHz):
δ
7.27–7.44 (m, 5H), 4.15 (q, J = 7.2 Hz, 2H), 3.84 (s, 2H), 3.12 (m, 2H), 2.99 (m, 1H), 2.83 (m, 1H), 2.68
(m, 1H), 2.23 (m, 1H), 2.16 (m, 1H), 1.25 (t, J = 7.2 Hz, 3H).
Ethyl 1-(4-methylbenzyl)pyrrolidine-2-carboxylate (8b): yield: 73%; slightly yellow oil; 1H-NMR (CDCl3,
400 MHz):
δ 7.21 (d, J = 7.6 Hz, 2H), 7.12 (d, J = 7.6 Hz, 2H), 4.04 (q, J = 7.2 Hz, 2H), 3.61 (s, 2H), 3.02
(p, J = 6.8 Hz, 1H), 2.94 (t, J = 8.8 Hz, 1H), 2.73 (m, 1H), 2.61 (t, J = 8.0 Hz, 1H), 2.51 (d, J = 8.0 Hz, 1H),
2.33 (s, 3H), 2.10 (p, J = 7.2 Hz, 1H), 1.24 (t, J = 7.2 Hz, 3H).
Ethyl 1-(4-methoxylbenzyl)pyrrolidine-2-carboxylate (8c): yield: 80%; slightly yellow oil; 1H-NMR (CDCl3,
400 MHz):
δ 7.43 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 8.8 Hz, 2H), 4.13 (q, J = 7.2 Hz, 2H), 3.98 (s, 2H),
3.78 (s, 3H), 3.42 (m, 1H), 3.26 (m, 2H), 3.04 (m, 1H), 2.83 (m, 1H), 2.38 (m, 1H), 2.17 (m, 1H), 1.24 (t,
J = 7.2 Hz, 3H).
Ethyl 1-(4-bromobenzyl)pyrrolidine-2-carboxylate (8d): yield: 74%; slightly yellow oil; 1H-NMR (400 MHz,
DMSO): δ 7.49 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 4.04 (q, J = 7.1 Hz, 2H), 3.54 (m, 2H), 2.98
(p, J = 6.8 Hz, 1H), 2.67 (t, J = 8.8 Hz, 1H), 2.58 (m, 1H), 2.46 (m, 2H), 1.95 (m, 2H), 1.15 (t, J = 7.1 Hz,
3H). 13C-NMR (150 MHz, DMSO):
41.31, 27.12, 14.09.
δ 174.23, 138.48, 131.02, 130.56, 119.77, 60.07, 58.10, 55.91, 53.07,
Ethyl 1-(4-nitrobenzyl)pyrrolidine-2-carboxylate (8e): yield: 83%; slightly yellow oil; 1H-NMR (500 MHz,
CDCl3): δ 8.18 (d, J = 8.2 Hz, 2H), 7.52 (m, 2H), 4.15 (q, J = 7.2 Hz, 2H), 3.74 (s, 2H), 3.27–2.36 (m, 5H),
2.13 (m, 2H), 1.26 (t, J = 7.1 Hz, 4H). 13C-NMR (100 MHz, CDCl3):
δ
129.11, 123.52, 60.68, 59.13, 56.58,
53.78, 42.12, 27.71, 14.19. HR–ESI–MS: m/z = 279.1324 [M + H]+, calcd. for C14H19N2O4: 279.1339.

Molecules 2017, 22, 1766
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Ethyl 1-(4-cyanobenzyl)pyrrolidine-2-carboxylate (8f): yield: 73%; slightly yellow oil; 1H-NMR (400 MHz,
CDCl3): 7.60 (d, J = 7.9 Hz, 2H), 7.45 (d, J = 7.9 Hz, 2H), 4.14 (q, J = 7.1 Hz, 2H), 3.69 (m, 2H),
3.02 (m, 1H), 2.83 (t, J = 8.8 Hz, 1H), 2.68 (m, 2H), 2.58 (m, 1H), 2.12 (m, 2H), 1.25 (t, J = 7.1 Hz,
δ
3H).13C-NMR (100 MHz, CDCl3):
δ 174.8, 132.1, 129.1, 118.9, 110.8, 109.7, 60.7, 59.4, 56.6, 53.7, 42.1,
27.7, 14.2. HR–ESI–MS: m/z = 259.1432 [M + H]+, calcd. for C15H19N2O2: 259.1441.
Ethyl 1-benzoylpyrrolidine-2-carboxylate (10a): yield: 77%; slightly yellow oil; 1H-NMR (400 MHz, CDCl3):
δ
7.51 (m, 2H), 7.40 (m, 3H), 4.17 (q, J = 7.1 Hz, 2H), 3.71 (s, 3H), 3.09 (s, 1H), 2.20 (s, 2H), 2.04 (s, 1H),
1.26 (t, J = 7.1 Hz, 3H). 13C-NMR (150 MHz, CDCl3):
δ 169.8, 136.6, 130.0, 128.3, 127.1, 61.1, 14.2.
HR–ESI–MS: m/z = 248.1274 [M + H]+, calcd. for C14H18NO3: 248.1281.
Ethyl 1-(4-methylbenzoyl) pyrrolidine-2-carboxylate (10b): yield: 74%; slightly yellow oil; 1H-NMR
(500 MHz, CDCl3):
δ
7.42 (d, J = 7.7 Hz, 2H), 7.19 (d, J = 7.7 Hz, 2H), 4.16 (s, 2H), 3.76 (m, 3H),
173.0, 172.4,
3.07 (s, 1H), 2.37 (s, 3H), 2.19 (s, 2H), 1.26 (t, J = 7.1 Hz, 3H). 13C-NMR (150 MHz, CDCl3):
δ
169.8, 140.2, 133.6, 130.0, 129.0, 128.9, 127.2, 61.0, 51.4, 48.8, 48.5, 45.5, 43. 8, 41.9, 29.4, 27.9, 21.4, 14.1.
1
Ethyl 1-(4-methoxylbenzoyl)pyrrolidine-2-carboxylate (10c): yield: 87%; slightly yellow oil; H-NMR
(500 MHz, CDCl3):
δ 7.51 (d, J = 7.9 Hz, 2H), 6.90 (d, J = 8.1 Hz, 2H), 4.17 (q, J = 7.2 Hz, 2H), 3.83 (s, 3H),
3.66 (m, 3H), 3.08 (s, 1H), 2.19 (d, J = 7.4 Hz, 2H), 1.79 (s, 1H), 1.26 (t, J = 7.1 Hz, 3H).
Ethyl 1-(4-bromobenzoyl)pyrrolidine-2-carboxylate (10d): yield: 74%; slightly yellow oil; 1H-NMR
(400 MHz, DMSO):
δ 7.49 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 4.04 (q, J = 7.1 Hz, 2H),
3.54 (m, 2H), 2.98 (p, J = 6.8 Hz, 1H), 2.67 (t, J = 8.8 Hz, 1H), 2.58 (m, 1H), 2.46 (m, 2H), 1.95 (m, 2H),
1.15 (t, J = 7.1 Hz, 3H). 13C-NMR (150 MHz, DMSO):
δ
174.2, 138.5, 131.0, 130.6, 119.8, 60.0, 58.1, 55.9,
53.1, 41.3, 27.1, 14.1.
1
Ethyl 1-(4-nitrobenzoyl)pyrrolidine-2-carboxylate (10e): yield: 74%; slightly yellow oil; H-NMR (400
MHz, CDCl3):
2H), 3.92 (d, J = 7.1 Hz, 1H), 3.41–3.84 (m, 3H), 3.13 (dt, J = 38.4, 7.1 Hz, 1H), 2.23 (ddt, J = 32.8, 13.1,
6.7 Hz, 2H), 1.27 (m, 3H). 13C-NMR (150 MHz, CDCl3):
172.8, 172.1, 167.5, 167.3, 148.6, 142.5, 142.5,
δ 8.27 (dd, J = 8.6, 3.1 Hz, 2H), 7.68 (dd, J = 8.6, 2.5 Hz, 2H), 4.18 (dq, J = 21.6, 7.2 Hz,
δ
128.2, 128.16, 123.8, 123.708, 61.3, 61.2, 51.0, 48.7, 48.5, 45.6, 43.7, 41.8, 29.4, 27.8, 14.2, 14.1.HR–ESI–MS:
m/z = 293.1120 [M + H]+, calcd. for C14H17N2O5: 293.1132.
Ethyl 1-(4-chlorobenzoyl)pyrrolidine-2-carboxylate (10f): yield: 74%; slightly yellow oil; 1H-NMR
(600 MHz, CDCl3):
δ 7.47 (d, J = 8.0 Hz, 2H), 7.38 (dd, J = 8.4, 4.0 Hz, 2H), 4.23–4.07 (m, 2H), 3.89
(m, 1H), 3.81–3.41 (m, 3H), 3.09 (m, 1H), 2.35–2.05 (m, 2H), 1.26 (m, 3H). 13C-NMR (150 MHz, CDCl3):
δ
172.9, 172.3, 168.7, 168.5, 136.1, 134.9, 134.9, 128.7, 128.7, 128.6, 128. 6, 61.2, 61.1, 51.3, 48.7, 48.6,
45.6, 43.8, 41.8, 29.4, 27.8, 14.2, 14.1. HR–ESI–MS: m/z = 282.0877 [M + H]+, calcd. for C14H17NO3Cl:
282.0892.
3.1.4. General Procedure for the Preparation of Compounds 9 or 11
To a solution of 8 or 10 (0.775 mmol) in EtOH (3 mL), 4 M NaOH (0.7 mL, 2.8 mmol) was added
at room temperature. Afterwards, the mixture was stirred for 1 h and then evaporated in vacuo.
After being diluted with water and extracted with EtOAc, the pH of the aqueous phase was adjusted
to 1–2 with 6 M HCl. The mixture was extracted with n-BuOH and then the organic phase was washed
with saturated NaCl. The organic phase was dried over Na2SO4 and evaporated in vacuo. The residue
was used immediately without further purification.
3.1.5. General Procedure for the Preparation of Target Compounds 2a–2f; 3a–3c; 4a–4i; 5a–5f
To a solution of intermediate 9, 11 or 6 (0.39 mmol) and 5- or 6-aminoindazole (0.47 mmol) in dry
DMF (3 mL), EDCI (90 mg, 0.47 mmol) was added at r.t. Afterwards, the mixture was stirred for 7 h at
80 ◦C and then the reaction mixture was cooled to room temperature. The mixture was evaporated

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in vacuo and the residue was purified by column chromatography on silica gel (2%–10% CH3OH in
CH2Cl2) to give the target compounds.
1-Benzyl-N-(1H-indazol-5-yl)pyrrolidine-2-carboxamide (2a): yield: 62%; white solid; m.p.: 200–201 ◦C;
1H-NMR (300 MHz, CDCl3):
δ 12.96 (s, 1H), 9.67 (s, 1H), 8.08 (s, 1H), 7.99 (s, 1H), 7.41 (m, 4H), 7.30
(t, J = 7.2 Hz, 2H), 7.22 (d, J = 6.9 Hz, 1H), 3.85 (d, J = 12.9 Hz, 1H), 3.59 (d, J = 13.2 Hz, 1H), 3.24
(dd, J1 = 4.5 Hz; J2 = 8.7 Hz, 1H), 3.03 (m, 1H), 2.39 (q, J = 8.1 Hz, 1H), 2.16 (m, 1H), 1.86 (m, 1H),
1.76 (m, 2H). 13C-NMR (150 MHz, CDCl3):
δ 172.0, 138.7, 137.0, 133.3, 131.4, 128.7, 128.2, 127.0, 122.6,
120.6, 110.0, 67.3, 58.6, 53.3, 30.0, 23.4. HR–ESI–MS: m/z = 321.1706 [M + H]+, calcd. for C19H21N4O:
321.1710.
20
(R)-1-Benzyl-N-(1H-indazol-5-yl)pyrrolidine-2-carboxamide (2b): yield: 64%; white solid; [
α
]
= +93
D
◦
(c 0.23, MeOH); m.p.: 199–201 C; 1H-NMR (400 MHz, CDCl3):
δ 12.98 (s, 1H), 9.70 (s, 1H), 8.09 (s, 1H),
8.00 (s, 1H), 7.43 (m, 4H), 7.32 (t, J = 7.5 Hz, 2H), 7.21 (t, J = 6.9 Hz, 1H), 3.85 (d, J = 12.9 Hz, 1H), 3.58
(d, J = 12.9 Hz, 1H), 3.23 (dd, J1 = 4.8 Hz; J2 = 8.7 Hz, 1H), 3.02 (m, 1H), 2.33 (q, J = 8.1 Hz, 1H), 2.14
(m, 1H), 1.86 (m, 1H), 1.79 (m, 2H). 13C-NMR (150 MHz, CDCl3):
δ 172.0, 138.7, 137.0, 133.2, 131.7,
131.4, 128.7, 128.2, 126.9, 122.6, 120.6, 109.9, 67.2, 58.6, 53.2, 29.9, 23.4. HR–ESI–MS: m/z = 321.1716
[M + H]+, calcd. for C19H21N4O: 321.1710.
20
(S)-1-Benzyl-N-(1H-indazol-5-yl)pyrrolidine-2-carboxamide (2c): yield: 59%; white solid; [
α
]
=
−
95
D
◦
(c 0.39, MeOH); m.p.: 202–203 C; 1H-NMR (300 MHz, CDCl3):
δ 12.95 (s, 1H), 9.67 (s, 1H), 8.08 (s, 1H),
7.99 (s, 1H), 7.40 (m, 4H), 7.30 (t, J = 7.2 Hz, 2H), 7.22 (d, J = 7.2 Hz, 1H), 3.85 (d, J = 12.9 Hz, 1H), 3.58
(d, J = 13.5 Hz, 1H), 3.23 (dd, J1 = 4.2 Hz; J2 = 8.7 Hz, 1H), 3.02 (m, 1H), 2.40 (q, J = 8.1 Hz, 1H), 2.16
(m, 1H), 1.86 (m, 1H), 1.77 (m, 2H). 13C-NMR (150 MHz, CDCl3):
δ 172.0, 138.7, 136.9, 136.1, 133.63,
131.4, 128.7, 128.2, 126.9, 122.6, 120.6, 109.9, 67.2, 58.6, 53.2, 29.9, 23.4. HR–ESI–MS: m/z = 321.1717
[M + H]+, calcd. for C19H21N4O: 321.1710.
N-(1H-Indazol-5-yl)-1-(4-methylbenzyl)pyrrolidine-2-carboxamide (2d): yield: 51%; white solid; m.p.:
200–202 ◦C; 1H-NMR (400 MHz, CDCl3):
δ 12.95 (s, 1H), 9.65 (s, 1H), 8.06 (s, 1H), 7.99 (s, 1H), 7.44
(q, J = 9.2 Hz, 2H), 7.27 (d, J = 7.6 Hz, 2H), 7.12 (d, J = 7.6 Hz, 2H), 3.80 (d, J = 13.2 Hz, 1H), 3.54
(d, J = 12.8 Hz, 1H), 3.21 (dd, J1 = 4.8 Hz; J2 = 8.8 Hz, 1H), 3.01 (m, 1H), 2.37 (q, J = 8.4 Hz, 1H), 2.23
(s, 3H), 2.13 (m, 1H), 1.84 (m, 1H), 1.75 (m, 2H). 13C-NMR (150 MHz, CDCl3):
δ 172.1, 137.0, 136.0,
135.6, 133.3, 131.4, 128.8, 128.7, 122.6, 120.6, 109.9, 109.3, 66.2, 58.3, 53.2, 30.0, 23.4, 20.6. HR–ESI–MS:
m/z = 335.1869 [M + H]+, calcd. for C20H23N4O: 335.1866.
(R)-N-(1H-Indazol-5-yl)-1-(4-methylbenzyl)pyrrolidine-2-carboxamide (2e): yield: 63%; white solid;
20
◦
1
[
α
]
= +66 (c 0.7, MeOH); m.p.: 203–205 C; H-NMR (400 MHz, CDCl3): δ 12.95 (s, 1H), 9.65
D
(s, 1H), 8.06 (s, 1H), 7.99 (s, 1H), 7.44 (q, J = 8.8 Hz, 2H), 7.27 (d, J = 7.6 Hz, 2H), 7.12 (d, J = 7.6 Hz, 2H),
3.80 (d, J = 12.8 Hz, 1H), 3.54 (d, J = 12.8 Hz, 1H), 3.21 (dd, J1 = 4.8 Hz; J2 = 9.2 Hz, 1H), 3.01 (m, 1H),
2.37 (q, J = 8.0 Hz, 1H), 2.23 (s, 3H), 2.14 (m, 1H), 1.83 (m, 1H), 1.75 (m, 2H). 13C-NMR (150 MHz,
CDCl3):
δ 172.0, 136.9, 136.0, 135.6, 133.3, 131.4, 128.8, 128.7, 122.6, 120.6, 109.9, 67.2, 58.3, 53.2, 29.9,
23.4, 20.6. HR–ESI–MS: m/z = 335.1869 [M + H]+, calcd. for C20H23N4O: 335.1866.
(S)-N-(1H-Indazol-5-yl)-1-(4-methylbenzyl)pyrr◦olidine-2-carboxamide (2f): yield: 56%; white solid;
20
D
1
[
α
]
=
−
69 (c 0.68, MeOH); m.p.: 204–206 C; H-NMR (400 MHz, CDCl3): δ 12.95 (s, 1H), 9.65
(s, 1H), 8.06 (s, 1H), 7.99 (s, 1H), 7.44 (q, J = 9.2 Hz, 2H), 7.27 (d, J = 7.6 Hz, 2H), 7.11 (d, J = 8.0 Hz, 2H),
3.80 (d, J = 12.8 Hz, 1H), 3.54 (d, J = 13.2 Hz, 1H), 3.21 (dd, J1 = 4.8 Hz; J2 = 8.8 Hz, 1H), 3.02 (m, 1H),
2.37 (q, J = 8.4 Hz, 1H), 2.23 (s, 3H), 2.14 (m, 1H), 1.85 (m, 1H), 1.75 (m, 2H). 13C-NMR (150 MHz,
CDCl3):
δ 172.0, 136.9, 136.0, 135.6, 133.3, 131.4, 128.8, 128.7, 122.6, 120.6, 109.3, 66.7, 58.3, 53.1, 29.9,
23.4, 20.7. HR–ESI–MS: m/z = 335.1868 [M + H]+, calcd. for C20H23N4O: 335.1866.
1-Benzyl-N-(1H-indazol-6-yl)pyrrolidine-2-carboxamide (3a): yield: 57%; white solid; m.p.: 195–196 ◦C;
1H-NMR (400 MHz, CDCl3):
δ
12.88 (s, 1H), 9.79 (s, 1H), 8.12 (s, 1H), 7.95 (s, 1H), 7.64 (d, J = 8.4 Hz,
1H), 7.39 (d, J = 7.6 Hz, 2H), 7.30 (t, J = 7.2 Hz, 2H), 7.20 (t, J = 7.2 Hz, 1H), 7.11 (d, J = 8.4 Hz, 1H), 3.84

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(d, J = 13.2 Hz, 1H), 3.59 (d, J = 12.8 Hz, 1H), 3.26 (dd, J1 = 4.8 Hz; J2 = 9.2 Hz, 1H), 3.04 (m, 1H), 2.40
(q, J = 8.4 Hz, 1H), 2.17 (m, 1H), 1.83 (m, 1H), 1.76 (m, 2H). 13C-NMR (150 MHz, CDCl3):
172.4, 140.2,
δ
138.7, 136.5, 133.2, 128.7, 128.2, 127.0, 120.4, 119.2, 114.4, 99.0, 67.3, 58.6, 53.3, 30.0, 23.4. HR–ESI–MS:
m/z = 321.1713 [M + H]+, calcd. for C19H21N4O: 321.1710.
20
(R)-1-Benzyl-N-(1H-indazol-6-yl)pyrrolidine-2-carboxamide (3b): yield: 52%; white solid; [
α
]
= +98
D
◦
(c 0.26, MeOH); m.p.: 192–193 C; 1H-NMR (400 MHz, CDCl3):
δ 12.87 (s, 1H), 9.79 (s, 1H), 8.11 (s, 1H),
7.95 (s, 1H), 7.64 (d, J = 8.4 Hz, 1H), 7.39 (d, J = 7.6 Hz, 2H), 7.30 (t, J = 7.2 Hz, 2H), 7.20 (t, J = 7.2 Hz,
1H), 7.11 (d, J = 8.8 Hz, 1H), 3.84 (d, J = 13.2 Hz, 1H), 3.60 (d, J = 12.8 Hz, 1H), 3.26 (dd, J1 = 4.8 Hz;
J2 = 9.2 Hz, 1H), 3.05 (m, 1H), 2.40 (q, J = 8.4 Hz, 1H), 2.17 (m, 1H), 1.83 (m, 1H), 1.76 (m, 2H). 13C-NMR
(150 MHz, CDCl3):
δ 172.4, 140.2, 138.7, 136.5, 133.2, 128.7, 128.2, 127.0, 120.5, 119.2, 114.4, 99.0, 67.3,
58.6, 53.3, 30.0, 23.4. HR–ESI–MS: m/z = 321.1713 [M + H]+, calcd. for C19H21N4O: 321.1710.
20
D
(S)-1-Benzyl-N-(1H-indazol-6-yl)pyrrolidine-2-carboxamide (3c): yield: 61%; white solid; [
α
]
=
−
96
◦
(c 0.49, MeOH); m.p.: 195–197 C; 1H-NMR (400 MHz, CDCl3):
δ 12.87 (s, 1H), 9.79 (s, 1H), 8.11 (s, 1H),
7.95 (s, 1H), 7.64 (d, J = 8.4 Hz, 1H), 7.39 (d, J = 7.2 Hz, 2H), 7.30 (t, J = 7.6 Hz, 2H), 7.20 (t, J = 7.2 Hz,
1H), 7.11 (d, J = 8.8 Hz, 1H), 3.84 (d, J = 12.8 Hz, 1H), 3.60 (d, J = 13.2 Hz, 1H), 3.26 (dd, J1 = 4.8 Hz;
J2 = 9.2 Hz, 1H), 3.05 (m, 1H), 2.40 (q, J = 8.4 Hz, 1H), 2.17 (m, 1H), 1.83 (m, 1H), 1.76 (m, 2H). 13C-NMR
(150 MHz, CDCl3):
δ 172.4, 140.2, 138.7, 136.5, 133.2, 128.7, 128.2, 127.0, 120.5, 119.2, 114.4, 99.0, 67.3,
58.6, 53.3, 30.0, 23.4. HR–ESI–MS: m/z = 321.1715 [M + H]+, calcd. for C19H21N4O: 321.1710.
1-Benzyl-N-(1H-indazol-5-yl)pyrrolidine-3-carboxamide (4a): yield:42%; white solid; m.p.: 203–204 ◦C;
1H-NMR (300 MHz, DMSO-d6):
δ
12.93 (s, 1H), 9.83 (s, 1H), 8.09 (s, 1H), 7.98 (s, 1H), 7.66 (d, J = 8.7 Hz,
1H), 7.38 (d, J = 9.0 Hz, 1H), 7.31 (d, J = 4.2 Hz, 4H), 7.24 (m, 1H), 3.59 (m, 2H), 3.05 (m, 1H), 2.88
(t, J = 8.7 Hz, 1H), 2.67 (m, 1H), 2.46 (m, 2H), 2.01 (m, 2H). 13C-NMR (150 MHz, DMSO-d6):
172.3, 158.1,
δ
136.7, 133.2, 132.2, 129.7, 122.6, 120.3, 113.5, 109.9, 109.5, 58.5, 56.9, 54.9, 53.4, 43.4, 27.5. HR–ESI–MS:
m/z = 321.17096 [M + H]+, calcd. for C19H21N4O: 321.17099.
N-(1H-Indazol-5-yl)-1-(4-methylbenzyl)pyrrolidine-3-carboxamide (4b): yield: 42%; white solid; 1H-NMR
(400 MHz, DMSO-d6):
δ 12.94 (s, 1H), 9.82 (s, 1H), 8.09 (s, 1H), 7.98 (s, 1H), 7.45 (d, J = 8.7 Hz, 1H),
7.38 (d, J = 8.4 Hz, 1H), 7.19 (d, J = 7.5 Hz, 2H), 7.11 (t, J = 7.8 Hz, 2H), 3.54 (s, 2H), 3.04 (m, 1H), 2.86
(t, J = 8.7 Hz, 1H), 2.66 (m, 1H), 2.42 (m, 2H), 2.28 (s, 3H), 2.01 (m, 2H). 13C-NMR (150 MHz, DMSO-d6):
δ
172.3, 136.8, 135.8, 133.3, 132.3, 128.7, 128.5, 122.7, 120.3, 110.0, 109.5, 58.9, 57.0, 53.5, 43.4, 27.6, 20.7.
N-(1H-Indazol-5-yl)-1-(4-methoxybenzyl)pyrrolidine-3-carboxamide (4c): yield: 46%; white solid; 1H-NMR
(400 MHz, DMSO-d6): 12.93 (s, 1H), 9.82 (s, 1H), 8.08 (s, 1H), 7.98 (s, 1H), 7.44 (d, J = 9.2 Hz, 1H),
δ
7.38 (d, J = 8.8 Hz, 1H), 7.22 (d, J = 8.4 Hz, 2H), 6.86 (t, J = 8.4 Hz, 2H), 3.72 (s, 3H), 3.51 (d, J = 6.8 Hz,
2H), 3.03 (m, 1H), 2.85 (t, J = 8.7 Hz, 1H), 2.65 (m, 1H), 2.43 (m, 2H), 2.00 (m, 2H). 13C-NMR (150 MHz,
DMSO-d6):
δ 170.0, 159.6, 139.7, 133.3, 132.0, 131.8, 124.3, 122.6, 120.3, 114.0, 110.2, 109.8, 106.9, 56.4,
55.2, 54.4, 52.4, 42.3, 27.7. HR–ESI–MS: m/z = 351.18106 [M + H]+, calcd. for C20H23N4O2: 351.08155.
1-(4-Bromobenzyl)-N-(1H-indazol-5-yl)pyrrolidine-3-carboxamide (4d): yield: 62%; slightly yellow solid;
◦
m.p.: 235–236 C; 1H-NMR (400 MHz, DMSO-d6):
δ 12.93 (s, 1H), 9.82 (s, 1H), 8.08 (s, 1H), 7.98 (s, 1H),
7.50 (d, J = 8.0 Hz, 2H), 7.44 (d, J = 8.9 Hz, 1H), 7.38 (d, J = 9.0 Hz, 1H), 7.28 (d, J = 7.9 Hz, 2H), 3.56
(m, 2H), 3.05 (p, J = 7.7 Hz, 1H), 2.86 (t, J = 8.5 Hz, 1H), 2.67 (q, J = 7.4 Hz, 1H), 2.43 (m, 2H), 1.99
(m, 2H). 13C-NMR (150 MHz, DMSO-d6):
δ 172.2, 138.7, 136.8, 133.3, 132.3, 131.0, 130.7, 122.7, 120.3,
119.8, 110.0, 109.6, 58.4, 57.0, 53.5, 43.5, 40.0, 27.7. HR–ESI–MS: m/z = 399.0808 [M + H]+, calcd. for
C19H20ON4Br: 399.0815.
N-(1H-Indazol-5-yl)-1-(4-methoxybenzyl)pyrrolidine-3-carboxamide (4e): yield: 64%; slightly orange solid;
m.p.: 197–199 ◦C; 1H-NMR (500 MHz, DMSO-d6):
δ 12.93 (s, 1H), 9.84 (s, 1H), 8.19 (d, J = 8.4 Hz, 2H),
8.09 (s, 1H), 7.98 (s, 1H), 7.61 (d, J = 8.4 Hz, 2H), 7.50–7.33 (m, 2H), 3.74 (s, 2H), 3.07 (q, J = 7.9 Hz, 1H),
2.90 (t, J = 8.6 Hz, 1H), 2.71 (q, J = 7.6 Hz, 1H), 2.56 (t, J = 8.2 Hz, 1H), 2.03 (m, 2H). 13C-NMR (150 MHz,

Molecules 2017, 22, 1766
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DMSO-d6):
δ 172.2, 147.5, 146.5, 136.8, 133.3, 132.2, 129.5, 123.4, 122.7, 120.3, 110.0, 109.6, 58.3, 57.1, 53.7,
43.5, 27.8. HR–ESI–MS: m/z = 366.1548 [M + H]+, calcd. for C19H20N5O3: 366.1561.
1-(4-Cyanobenzyl)-N-(1H-indazol-5-yl)pyrrolidine-3-carboxamide (4f): yield: 61%; slightly yellow solid;
◦
m.p.: 202–204 C; 1H-NMR (400 MHz, DMSO-d6):
δ
12.93 (s, 1H), 9.83 (s, 1H), 8.09 (s, 1H), 7.98 (s, 1H),
7.79 (d, J = 7.8 Hz, 2H), 7.53 (d, J = 7.9 Hz, 2H), 7.49–7.32 (m, 2H), 3.69 (s, 2H), 3.07 (m, 1H), 2.87
(t, J = 8.5 Hz, 1H), 2.69 (m, 1H), 2.54 (m, 1H), 2.03 (m, 3H). 13C-NMR (150 MHz, DMSO-d6):
172.2,
δ
145.3, 136.8, 133.3, 132.2, 132.2, 129.3, 122.7, 120.3, 118.9, 110.0, 109.6, 58.6, 57.1, 53.6, 43.5, 40.0, 27.8.
HR–ESI–MS: m/z = 346.1641 [M + H]+, calcd. for C20H20N5O: 346.1662.
1-(4-Fluorobenzyl)-N-(1H-indazol-5-yl)pyrrolidine-3-carboxamide (4g): yield: 50%; white solid; m.p.:
◦
1
202–203 C; H-NMR (400 MHz, CDCl3):
δ 12.93 (s, 1H), 9.82 (s, 1H), 8.09 (s, 1H), 7.98 (s, 1H),
7.44 (d, J = 8.8 Hz, 1H), 7.39 (s, 1H), 7.34 (t, J = 6.4 Hz, 2H), 7.13 (t, J = 8.8 Hz, 2H), 3.57 (d, J = 4.0 Hz,
2H), 3.04 (m, 1H), 2.86 (t, J = 8.4 Hz, 1H), 2.66 (m, 1H), 2.43 (m, 2H), 2.01 (m, 2H). 13C-NMR (150 MHz,
DMSO-d6):
δ 172.3, 162.1, 160.2, 136.8, 135.4, 133.3, 132.2, 130.3, 122.7, 120.3, 114.9, 114.7, 110.0, 109.6,
58.3, 57.0, 53.5, 27.6. HR–ESI–MS: m/z = 339.16138 [M + H]+, calcd. for C19H20N4OF: 339.16157.
(S)-1-Benzyl-N-(1H-indazol-5-yl)pyrrolidine-3-carboxamide (4h): yield: 53%; white solid; [
18 = +26 (c 1.05,
MeOH); m.p.: 203–204 ◦C; 1H-NMR (300 MHz, CDCl3):
12.93 (s, 1H), 9.83 (s, 1H), 8.09 (s, 1H), 7.98
α]
D
δ
(s, 1H), 7.66 (d, J = 8.7 Hz, 1H), 7.38 (d, J = 9.0 Hz, 1H), 7.31 (d, J = 4.2 Hz, 4H), 7.24 (m, 1H), 3.59 (s, 1H),
3.05 (m, 1H), 2.88 (t, J = 8.7 Hz, 1H), 2.67 (m, 1H), 2.46 (m, 2H), 2.01 (m, 2H).
18
(R)-1-Benzyl-N-(1H-indazol-5-yl)pyrrolidine-3-carboxamide (4i): yield: 53%; white solid; [
MeOH); m.p.: 203–204 ◦C; 1H-NMR (300 MHz, CDCl3):
α
]
=
−
25 (c 1.0,
D
δ
12.93 (s, 1H), 9.83 (s, 1H), 8.09 (s, 1H), 7.98
(s, 1H), 7.66 (d, J = 8.7 Hz, 1H), 7.38 (d, J = 9.0 Hz, 1H), 7.31 (d, J = 4.2 Hz, 4H), 7.24 (m, 1H), 3.59 (s, 1H),
3.05 (m, 1H), 2.88 (t, J = 8.7 Hz, 1H), 2.67 (m, 1H), 2.46 (m, 2H), 2.01 (m, 2H).
1-Benzoyl-N-(1H-indazol-5-yl)pyrrolidine-3-carboxamide (5a): yield: 77%; yellow solid; m.p.: 211–213 ◦C;
1H-NMR (500 MHz, DMSO-d6):
δ
12.96 (s, J = 9.5 Hz, 1H), 10.08 (s, 0.5H), 9.96 (s, 0.5H), 8.14 (s, 0.5H),
8.07 (s, 0.5H), 7.99 (d, J = 14.5 Hz, 1H), 7.52 (m, 2H), 7.48–7.35 (m, 5H), 3.83–3.45 (m, 4H), 3.28–3.09
(m, 1H), 2.13 (m, 2H). 13C-NMR (150 MHz, DMSO-d6):
170.7, 170.1, 168.2, 168.1, 136.9, 136.9, 133.3,
δ
132.0, 131.9, 129.8, 128.2, 128.2, 127.0, 122.6, 120.3, 120.3, 110.1, 109.8, 109.8, 51.4, 48.6, 45.5, 44.7, 42.9,
30.1, 27.9. HR–ESI–MS: m/z = 335.1486 [M + H]+, calcd. for C19H19N4O2: 335.1503.
N-(1H-Indazol-5-yl)-1-(4-methylbenzoyl)pyrrolidine-3-carboxamide (5b): yield: 78%; yellow solid; m.p.:
◦
224–226 C; 1H-NMR (400 MHz, DMSO-d6):
δ
12.95 (s, 1H), 10.08 (s, 0.5H), 9.97 (s, 0.5H),8.14 (s, 0.5H),
8.07 (s, 0.5H), 7.99 (d, J = 10.0 Hz, 1H), 7.55–7.32 (m, 4H), 7.24 (d, J = 7.5 Hz, 2H), 3.84–3.44 (m, 4H), 3.18
(m, 1H), 2.33 (s, 3H), 2.24–1.94 (m, 2H). 13C-NMR (150 MHz, DMSO-d6):
170.8, 170.1, 168.3, 168.1,
δ
139.5, 139.5, 136.8, 134.0, 133.9, 133.3, 132.1, 132.0, 129.6, 128.7, 127.2, 122.6, 120.3, 120.2, 110.1, 109.8,
109.7, 51.5, 48.7, 48.7, 45.5, 44.7, 42.9, 35.1, 31.3, 30.2, 29.1, 29.0, 29.0, 28.9, 28.8, 28.7, 28.7, 28.6, 27.9, 26.5,
25.1, 22.1, 20.9, 13.9. HR–ESI–MS: m/z = 349.1639 [M + H]+, calcd. for C20H21N4O2: 349.1659.
N-(1H-Indazol-5-yl)-1-(4-methoxybenzoyl)pyrrolidine-3-carboxamide (5c): yield: 79%; white solid; m.p.:
◦
242–244 C; 1H-NMR (400 MHz, DMSO-d6):
δ
12.95 (s, 1H), 10.07 (s, 0.5H), 9.95 (s, 0.5H), 8.13 (s, 0.5H),
8.07 (s, 0.5H), 7.99 (m, 1H), 7.52 (d, J = 8.3 Hz, 2H), 7.49–7.32 (m, 2H), 6.97 (d, J = 8.3 Hz, 2H), 3.79
(s, 3H), 3.73–3.45 (m, 4H), 3.17 (m, 1H), 2.14 (m, 2H). 13C-NMR (150 MHz, DMSO-d6):
170.8, 170.1,
δ
168.0, 167.8, 160.4, 136.8, 133.4, 132.0, 129.2, 128.8, 122.7, 120.3, 120.3, 113.4, 110.1, 109.8, 109.7, 55.2,
51.6, 48.8, 45.7, 44.8, 42.9, 30.2, 27.9. HR–ESI–MS: m/z = 365.1590 [M + H]+, calcd. for C20H21N4O3:
365.1608.
1-(4-Bromobenzoyl)-N-(1H-indazol-5-yl)pyrrolidine-3-carboxamide (5d): yield: 63%; slightly orange solid;
m.p.: 250–251 ◦C; 1H-NMR (500 MHz, DMSO-d6):
δ
12.95 (s, 1H), 10.08 (s, 0.5H), 9.96 (s, 0.5H), 8.13
(s, 0.5H), 8.07 (s, 0.5H), 7.99 (d, J = 11.7 Hz, 1H), 7.64 (d, J = 8.1 Hz, 2H), 7.52–7.32 (m, 4H), 3.85–3.43
(m, 4H), 3.19 (m, 1H), 2.31–1.98 (m, 2H). 13C-NMR (150 MHz, DMSO-d6):
170.7, 170.1, 167.2, 167.1,
136.9, 136.0, 135.9, 133.2, 132.0, 131.9, 131.3, 131.2, 129.3, 123.3, 123.2, 122.6, 120.3, 120.3, 110.2, 109.8,
δ

Molecules 2017, 22, 1766
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109.8, 51.3, 48.7, 48.6, 45.6, 44.7, 42.9, 30.2, 27.9. HR–ESI–MS: m/z = 413.0591 [M + H]+, calcd. for
C19H18N4O2Br: 413.0608.
N-(1H-Indazol-5-yl)-1-(4-nitrobenzoyl)pyrrolidine-3-carboxamide (5e): yield: 78%; yellow solid; m.p.:
177–178 ◦C; 1H-NMR (500 MHz, DMSO-d6):
δ 13.00 (d, J = 9.8 Hz, 1H), 10.29 (d, J = 6.0 Hz, 1H), 10.18
(d, J = 6.5 Hz, 1H), 8.27 (d, J = 8.3 Hz, 2H), 8.16 (s, 0.5H), 8.09 (s, 0.5H), 7.99 (d, J = 14.3 Hz, 1H), 7.79
(dd, J = 8.3, 6.1 Hz, 2H), 7.48 (s, 1H), 7.43 (t, J = 8.7 Hz, 1H), 3.87–3.40 (m, 4H), 3.32–3.19 (m, 1H),
2.34–2.01 (m, 2H). 13C-NMR (150 MHz, DMSO-d6):
δ 170.7, 170.1, 166.4, 166.3, 147.9, 147.9, 142.9, 142.9,
136.9, 136.8, 133.3, 133.3, 132.2, 132.1, 128.5, 123.6, 123.6, 122.6, 122.6, 120.3, 120.2, 110.1, 110.0, 109.7,
109.7, 51.2, 48.7, 48.4, 45.6, 44.6, 42.8, 30.1, 28.0. HR–ESI–MS: m/z = 380.1338 [M + H]+, calcd. for
C19H18N5O4: 380.1353.
1-(4-Chlorobenzoyl)-N-(1H-indazol-5-yl)pyrrolidine-3-carboxamide (5f): yield: 77%; white solid; m.p.:
◦
225–227 C; 1H-NMR (400 MHz, DMSO-d6):
δ
12.95 (s, 1H), 10.09 (s, 0.5H), 9.97 (s, 0.5H), 8.14 (s, 0.5H),
8.07 (s, 0.5H), 8.00 (d, J = 9.4 Hz, 1H), 7.68–7.28 (m, 6H), 3.88–3.55 (m, 3H), 3.55–3.46 (m, 1H), 3.19
(m, 1H), 2.26–2.00 (m, 2H). 13C-NMR (150 MHz, DMSO-d6):
170.7, 170.0, 167.1, 167.0, 136.9, 136.8,
δ
135.6, 135.6, 134.5, 134.5, 133.4, 133.3, 132.0, 131.9, 129.1, 128.3, 128.3, 122.7, 122.6, 120.3, 120.3, 110.1,
110.1, 109.8, 109.8, 51.3, 48.7, 48.6, 45.6, 44.7, 42.9, 30.2, 27.9. HR–ESI–MS: m/z = 369.1101 [M + H]+,
calcd. for C19H18N4O2Cl: 369.1113.
3.2. Bioassay Studies
3.2.1. Rho Kinase Activity Assay
Rho kinase assay kit (CY-1160, Cyclex, Nagoya, Japan) was employed to detect the inhibitory
effect of the compounds on ROCK I following the manufacturer’s instructions. Briefly, the compounds
at 20
substrate’s myosin-binding subunit (MBS) pre-absorbed onto the microplate in the presence of Mg2+
and ATP. Then, the system was washed after incubation at 30 ◦C for 30 min. 100
L of horseradish
µM were pre-incubated in a system wherein ROCK I (0.02 ng/µL) phosphorylates the kinase
µ
peroxidase-conjugated anti-AF20 antibody was added to the wells and then incubated for 30 min at
room temperature. The reaction system was then incubated with the substrate tetra-methylbenzidine
(TMB), which can be catalyzed from a colorless solution to a blue solution. The absorbance was
measured at a wavelength of 450 nm on a microplate reader. The color reflects the relative level of
ROCK I activity. The inhibitory rates of the compounds on ROCK I were obtained from OD 450 nm
and the IC50 values were calculated by nonlinear regression.
3.2.2. Vasorelaxant Activity Test
Male Sprague-Dawley rats weighing 240–280 g (Vital River Laboratories, Beijing, China) were
maintained in a 12-h light/dark cycle at 24 ◦C in a relative humidity of 60% room, and received food
and water ad libitum. The animal care and handling were performed in accordance with the Guide
for the Care and Use of Laboratory Animals published by the US National Institutes of Health and
the Laboratories Institutional Animal Care and Use Committee of the Chinese Academy of Medical
Science and Peking Union Medical College (ethic approval number: 00001012).
The vasorelaxant activity assay was performed as described previously [35]. Briefly, the descending
thoracic aorta was isolated after rats were euthanized by cervical dislocation. Then, the aorta was
cut into ring segments (3–4 mm in length). The endothelial layer of the aorta was destroyed by
gently rubbing the luminal surface with a moist cotton swab when necessary. Two stainless-steel
triangles were inserted into the lumen of each ring. One triangle was fixed, and the other attached to
a force transducer. Changes in isometric force were recorded on a BIOPAC polygraph (MP100WSW,
Biopac Systems, Inc, Goleta, CA, USA). The rings were mounted in organ baths containing 10 mL
Krebs-Henseleit (K-H) buffer with the following composition (mM): NaCl 120, KCl 4.8, MgSO4 1.4,
KH2PO4 1.2, glucose 11.0, CaCl2 2.5, NaHCO3 25.0 and EDTA 0.01. The K-H buffer was continuously

Molecules 2017, 22, 1766
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◦
bubbled with 95% O2/5% CO2 at 37 C. Rings were allowed to equilibrate for 60 min at a resting
tension of 1.2 g with changes of buffer every 20 min.
After the equilibration period, the aortic rings were constricted with a high K+ (60 mM) K-H
solution to stimulate the tissue and to test its availability. Then, the rings were washed with normal
K-H buffer to restore the basic tension of 1.2 g. The aortic rings were then stimulated with a high K+
(60 mmol/L) K-H buffer ornorepinephrine (1
When the contraction reached the platform, compounds were added to the bath in batches at 5-min
intervals to reach the accumulative concentrations of 1, 2, 5, 10, 25 and 50 M. The tension of vessels
µM) to evaluate the vasorelaxant effects of the compounds.
µ
was recorded and nonlinear regression was used to calculate the EC50 values.
3.3. Molecular Docking
Molecular docking was performed with Discovery Studio 2016 software package (version 2016,
BIOVIA, San Diego: Dassault Systèmes, CA, USA, 2016). The crystal structure of ROCK I, with co-crystal
ligand obtained from the Protein Data Bank (PDB code: 3NDM) [36], was used to simulate the binding
mode between our compounds and ROCK I protein. The original water molecules were removed
from the coordinates set. The co-crystal ligand was used to determine the binding site and was then
removed prior to docking. The docking calculation was carried out by using the LibDock protocol.
Smart Minimizer algorithm was used to minimize docked poses with CHARMm force field. The default
parameter settings were used. The obtained docking poses were ranked by LibDock score and the best-
scored pose was chosen.
4. Conclusions
In summary, a series of potent ROCK I inhibitors based on a hybrid prolinamido indazole scaffold
were designed and synthesized. Six compounds 4a, 4b, 4c, 4d, 4g and 4h showed activity profiles
superior to DL0805, validating our design strategy. These results have established preliminary SAR
trends, and molecular docking studies on the active compound 4a provide a foundation for future
molecular optimization of this promising scaffold.
Acknowledgments: This investigation was supported by the National Natural Scientific & Technological Major
Special Project “significant creation of new drugs” (No. 2013ZX09103001-008), CAMS Innovation Fund for Medical
Sciences (CIFMS, 2016-I2M-3-009).
Author Contributions: Xiaozhen jiao and Ping Xie designed the compounds; Yangyang Yao, Renze Li and
Feilong Yang synthesized the compounds; Ying Yang performed the molecular docking; Xiaoyu Li and Xiang Shi
collected the data; Biological Evaluation was performed by Tianyi Yuan, Lianhua Fang and Guanhua Du;
Xiaozhen Jiao, xiaoyu Liu and Ping Xie wrote or contributed to the writing of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 2a-2f; 3a-3c; 4a-4i; 5a-5f are available from the authors.
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