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Molecules 2017, 22(10), 1766; doi:10.3390/molecules22101766

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
2
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
*
Correspondence: 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 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 α-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.
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 [1]. It plays an important role in regulating a variety of cellular functions such as smooth-muscle-cell contraction, cell adhesion, migration and proliferation [2]. 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 [3]. However, the expression of the two isoforms has different tissue specificity. ROCK II shows high expression in brain tissue, while ROCK I is preferentially expressed in the tissues of the pancreas, liver, lung and heart [3]. Accumulating evidence shows that the dysregulation of ROCK activity or expression is associated with various diseases, including hypertension [4], stroke [5], asthma [6,7], cancer [8,9] and glaucoma [10,11,12], which indicates that ROCK is a potential novel target for drug development.
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 [15,16,17,18,19,20], 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, 1) (Figure 1) is a new ROCK inhibitor with an IC50 value of 6.7 μM 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) [32,33,34]. These studies 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.

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 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).

2.2. Chemistry

A concise route was developed to synthesize compounds 2a2f and 3a3c using racemic or D/L-proline as the starting material (Scheme 1). Alkylation of proline provided the intermediates 6a6f. 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 2b2f. Analogues 3a3c were similarly prepared by coupling the intermediates 6a6c with 6-aminoindazole.
Next, we embarked on the synthesis of analogue 4 following a similar synthetic route used for preparing compounds 2 and 3. Unfortunately, an inseparable mixture was observed during the direct alkylation of β-proline. Therefore, new synthetic strategies to access analogues 4 and 5 were performed and described in Scheme 2. The target compounds 4a4i were synthesized using racemic or D/L-β-proline as the starting material. Esterification of β-proline with SOCl2 and ethyl alcohol gave the ester 7. Subsequently, alkylation of ester 7 with substituted benzyl bromides provided compounds 8a8i, which were hydrolyzed and then reacted with 5-aminoindazole to produce compounds 4a4i. Intermediate 7 was also used to prepare the target compounds 5a5f in an analogous fashion. Acylation of ester 7 with substituted benzoyl chloride afforded compounds 10a10f that were saponified to 11a11f and coupled to 5-aminoindazole to furnish the other set of target compounds 5a5f.

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 clear SAR trends. Firstly, the target compounds of series III (4) and IV (5) containing a β-proline moiety have improved activity against ROCK I relative to analogue series I (2) and II (3) bearing an α-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 a β-proline-derived indazole scaffold, the inhibitory activity of the target compounds (series III) with a benzyl substituent (4) was superior to those with a benzoyl substituent (series IV 5). This suggested 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.

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.

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.

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).
(R)-1-Benzylpyrrolidine-2-carboxylic acid (6b): yield: 90%; white solid; [α] D 27 = +25.3 (c 0.92, MeOH); 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).
(S)-1-Benzylpyrrolidine-2-carboxylic acid (6c): yield: 92%; white solid; [α] D 26 = −26.9 (c 0.9, MeOH); 1H-NMR (400 MHz, CD3OD): δ 7.51 (m, 2H), 7.45 (m, 3H), 4.51 (d, 1H, J = 12.8 Hz), 4.26 (d, J = 12.4 Hz, 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).
1-(4-Methylbenzyl)pyrrolidine-2-carboxylic acid (6d): yield: 84%; white solid; 1H-NMR (400 MHz, DMSO-d6): δ 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, 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): δ 170.6, 138.0, 130.1, 129.8, 129.1, 65.7, 56.7, 53.3, 28.1, 22.2, 20.8.
(R)-1-(4-Methylbenzyl)pyrrolidine-2-carboxylic acid (6e): yield: 89%; white solid; [α] D 28 = +27.3 (c 0.92, MeOH); 1H-NMR (400 MHz, DMSO-d6): 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.
(S)-1-(4-Methylbenzyl)pyrrolidine-2-carboxylic acid (6f): yield: 92%; white solid; [α] D 26 = −23 (c 0.92, MeOH); 1H-NMR (400 MHz, DMSO-d6): 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): δ 174.23, 138.48, 131.02, 130.56, 119.77, 60.07, 58.10, 55.91, 53.07, 41.31, 27.12, 14.09.
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.
Ethyl 1-(4-cyanobenzyl)pyrrolidine-2-carboxylate (8f): yield: 73%; slightly yellow oil; 1H-NMR (400 MHz, CDCl3): δ 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, 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), 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 173.0, 172.4, 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.
Ethyl 1-(4-methoxylbenzoyl)pyrrolidine-2-carboxylate (10c): yield: 87%; slightly yellow oil; 1H-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.
Ethyl 1-(4-nitrobenzoyl)pyrrolidine-2-carboxylate (10e): yield: 74%; slightly yellow oil; 1H-NMR (400 MHz, CDCl3): 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, 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): δ 172.8, 172.1, 167.5, 167.3, 148.6, 142.5, 142.5, 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 2a2f; 3a3c; 4a4i; 5a5f

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 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.
(R)-1-Benzyl-N-(1H-indazol-5-yl)pyrrolidine-2-carboxamide (2b): yield: 64%; white solid; [α] D 20 = +93 (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.
(S)-1-Benzyl-N-(1H-indazol-5-yl)pyrrolidine-2-carboxamide (2c): yield: 59%; white solid; [α] D 20 = −95 (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; [α] D 20 = +66 (c 0.7, MeOH); m.p.: 203–205 °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 = 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)pyrrolidine-2-carboxamide (2f): yield: 56%; white solid; [α] D 20 = −69 (c 0.68, MeOH); m.p.: 204–206 °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), 7.11 (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 (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): δ 172.4, 140.2, 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.
(R)-1-Benzyl-N-(1H-indazol-6-yl)pyrrolidine-2-carboxamide (3b): yield: 52%; white solid; [α] D 20 = +98 (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.
(S)-1-Benzyl-N-(1H-indazol-6-yl)pyrrolidine-2-carboxamide (3c): yield: 61%; white solid; [α] D 20 = −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): δ 172.3, 158.1, 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): δ 12.93 (s, 1H), 9.82 (s, 1H), 8.08 (s, 1H), 7.98 (s, 1H), 7.44 (d, J = 9.2 Hz, 1H), 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, 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): δ 172.2, 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.: 202–203 °C; 1H-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; [α] D 18 = +26 (c 1.05, MeOH); m.p.: 203–204 °C; 1H-NMR (300 MHz, CDCl3): 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).
(R)-1-Benzyl-N-(1H-indazol-5-yl)pyrrolidine-3-carboxamide (4i): yield: 53%; white solid; [α] D 18 = −25 (c 1.0, MeOH); m.p.: 203–204 °C; 1H-NMR (300 MHz, CDCl3): 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): δ 170.7, 170.1, 168.2, 168.1, 136.9, 136.9, 133.3, 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): δ 170.8, 170.1, 168.3, 168.1, 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): δ 170.8, 170.1, 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): δ 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, 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): δ 170.7, 170.0, 167.1, 167.0, 136.9, 136.8, 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 μM were pre-incubated in a system wherein ROCK I (0.02 ng/μL) phosphorylates the kinase 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 μL of horseradish 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 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 μM) to evaluate the vasorelaxant effects of the compounds. 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 μM. The tension of vessels 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.

Acknowledgment

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.

References and Notes

  1. Leung, T.; Manser, E.; Tan, L.; Lim, L. A novel serine/threonine kinase binding the Ras-related RhoA GTPase which translocates the kinase to peripheral membranes. J. Biol. Chem. 1995, 270, 29051–29504. [Google Scholar] [CrossRef] [PubMed]
  2. Riento, K.; Ridley, A.J. ROCKs: Multifunctional kinases in cell behavior. Nat. Rev. Mol. Cell Biol. 2003, 4, 446–456. [Google Scholar] [CrossRef] [PubMed]
  3. Nakagawa, O.; Fujisawa, K.; Ishizaki, T.; Saito, Y.; Nakao, K.; Narumiya, S. ROCK-I and ROCK-II, two isoforms of rho-associated coiled-coil forming protein serine/threonine kinase in mice. FEBS Lett. 1996, 392, 189–193. [Google Scholar] [CrossRef]
  4. Wirth, A. Rho kinase and hypertension. Biochim. Biophys. Acta 2010, 1802, 1276–1284. [Google Scholar] [CrossRef] [PubMed]
  5. Shin, H.; Salomone, S.; Ayata, C. Targeting cerebrovascular Rho-kinase in Stroke. Expert Opin. Ther. Targets 2008, 12, 1547–1564. [Google Scholar] [CrossRef] [PubMed]
  6. Nakahara, T.; Moriuchi, H.; Yunoki, M.; Sakamato, K. Y-27632 potentiate relaxant effects of beta 2-adrenoceptor agonists in bovine tracheal smooth muscle. Eur. J. Pharmacol. 2000, 389, 103–106. [Google Scholar] [CrossRef]
  7. Oka, M.; Fagan, K.; Jone, P.; McMutry, I. Therapeutic potential of Rho A/Rho kinase inhibitors in pulmonary hypertension. Br. J. Pharmacol. 2008, 155, 444–454. [Google Scholar] [CrossRef] [PubMed]
  8. Ying, H.; Biroc, S.L.; Li, W.W.; Alicke, B.; Xuan, J.A.; Pagila, R.; Ohashi, Y.; Okada, T.; Kamata, Y.; Dinter, H. The Rho kinase inhibitor fasudil inhibits tumor progression in human and rat tumor models. Mol. Cancer Ther. 2005, 5, 2158–2164. [Google Scholar] [CrossRef] [PubMed]
  9. Somlyo, C.; Phelps, C.; Dipiperro, C.; Eto, M.; Read, P.; Barrett, M.; Gibson, J.J.; Burnitz, M.C.; Myers, C.; Somlyo, A.P. Rho kinase and matrix metalloproteinase inhibitors cooperate to inhibit angiogenesis and growth of human prostate cancer xenotransplants. FASEB J. 2003, 17, 223–234. [Google Scholar] [CrossRef] [PubMed]
  10. Waki, M.; Yoshida, Y.; Oka, T.; Azuma, M. Reduction of intraocular pressure by topical administration of an inhibitor of the Rho-associated protein kinase. Curr. Eye Res. 2001, 22, 470–474. [Google Scholar] [CrossRef] [PubMed]
  11. Navak, G.D. Emerging drug for ophthalmic diseases. Exp. Opin. Emerg. Drugs 2003, 8, 251–266. [Google Scholar]
  12. Tanihara, H.; Inatani, M.; Honjo, M.; Tokushige, H.; Azuma, J.; Araie, M. Intraocular pressure-lowering effects and safety of topical administration of a selective ROCK inhibitor, SNJ-1656, in healthy volunteers. Arch. Ophthalmol. 2008, 3, 309–315. [Google Scholar] [CrossRef] [PubMed]
  13. Suzuki, M.; Takashima-Hirano, M.; Koyama, H.; Yamaoka, T. Efficient synthesis of [11C] H-1152, a PET probe specific for Rho-kinases, highly potential targets in diagnostic medicine and drug development. Tetrahedron 2012, 68, 2336–2341. [Google Scholar] [CrossRef]
  14. Chowdhury, S.; Chen, Y.T.; Fang, X.G.; Grant, W.; Pocas, J.; Cameron, M.D.; Ruiz, C.; Lin, L.; Park, H.; Schröter, T.; et al. Amino acid derived quinazolines as Rock/pka inhibitors. Bioorg. Med. Chem. Lett. 2013, 23, 1592–1599. [Google Scholar] [CrossRef] [PubMed]
  15. Feng, Y.B.; Cameron, M.D.; Frackowiak, B.; Griffin, E.; Lin, L.; Ruiz, C.; Schroter, T.; Lograsso, P. Structure-activity relationships, and drug metabolism and pharmacokinetic properties for indazolepiperazine and indazolepiperdine inhibitors of ROCK II. Bioorg. Med. Chem. Lett. 2007, 17, 2355–2360. [Google Scholar] [CrossRef] [PubMed]
  16. Chowdhury, S.; Session, E.H.; Pocas, J.R.; Grant, W.; Schroter, T.; Lin, L.; Ruiz, C.; Cameron, M.D.; Schurer, S.; Lograsso, P. Discovery and optimization of indoles and 7-azaindoles as Rho kinase (ROCK) inhibitors (Part I). Bioorg. Med. Chem. Lett. 2011, 21, 7107–7112. [Google Scholar] [CrossRef] [PubMed]
  17. Takami, A.; Iwakubo, M.; Okada, Y.; Kawata, T.; Odai, H.; Nobuaki, T.; Kazutoshi, S.; Kaname, K.; Yoshimichi, T.; Mika, M.; et al. Design and synthesis of Rho kinase inhibitors (I). Bioorg. Med. Chem. 2004, 12, 2115–2137. [Google Scholar] [CrossRef] [PubMed]
  18. Iwakubo, M.; Takami, A.; Okada, Y.; Tagami, Y.; Ohashi, H.; Sato, M.; Sugiyama, T.; Fukushima, K.; Iijima, H. Design and synthesis of Rho kinase inhibitors (II). Bioorg. Med. Chem. 2007, 15, 350–364. [Google Scholar] [CrossRef] [PubMed]
  19. Iwakubo, M.; Takami, A.; Okada, Y.; Kawata, T.; Tagami, Y.; Sato, M.; Sugiyama, T.; Fukushima, K.; Taya, S.; Amano, M.; et al. Design and synthesis of Rho kinase inhibitors (III). Bioorg. Med. Chem. 2007, 15, 1022–1033. [Google Scholar] [CrossRef] [PubMed]
  20. Oh, K.S.; Oh, B.K.; Park, C.H.; Seo, H.W. Cardiovascular effects of a noval selective Rho kinase inhibitor, 2-(1H-indazole-5-yl)amino-4-methoxy-6-piperazino triazine (DW1865). Eur. J. Pharmacol. 2013, 702, 218–226. [Google Scholar] [CrossRef] [PubMed]
  21. Session, E.H.; Yin, Y.; Bannister, T.D.; Weiser, A.; Griffin, E.; Pocas, J.; Cameron, M.D.; Ruiz, C.; Lin, L.; Schürer, S.C.; et al. Benzimidazole- and benzoxazole-based inhibitors of Rho kinase. Bioorg. Med. Chem. Lett. 2008, 18, 6390–6393. [Google Scholar] [CrossRef] [PubMed]
  22. Session, E.H.; Smolinski, M.; Wang, B.; Frackowiak, B.; Chowdhury, S.; Yin, Y.; Chen, Y.T.; Ruiz, C.; Lin, L.; Pocas, J.; et al. The development of Benzimidazole as selective rho kinase inhibitors. Bioorg. Med. Chem. Lett. 2010, 20, 1939–1943. [Google Scholar] [CrossRef] [PubMed]
  23. Yin, Y.; Lin, L.; Ruiz, C.; Cameron, M.D.; Pocas, J.R.; Grant, W.; Schröter, T.; Chen, W.; Duckett, D.; Schürer, S.; et al. Benzothiazoles as Rho-associated kinase (ROCK-II) inhibitors. Bioorg. Med. Chem. Lett. 2009, 19, 6686–6690. [Google Scholar] [CrossRef] [PubMed]
  24. Letellier, M.A.; Guillard, J.; Caignard, D.H.; Ferry, G.; Boutin, J.A.; Viaud-Massuard, M.-C. Synthesis of potential Rho-kinase inhibitors based on the chemistry of an original heterocycle: 4,4-dimethyl-3,4-dihydro-1H-quinolin-2-one. Eur. J. Med. Chem. 2008, 43, 1730–1736. [Google Scholar] [CrossRef] [PubMed]
  25. Fang, X.G.; Chen, Y.T.; Session, E.H.; Chowdhury, S.; Vojkovsky, T.; Yin, Y.; Pocas, J.R.; Grant, W.; Schröter, T.; Lin, L.; et al. Synthesis and biological evalution of 4-quinazolinones as Rho kinase inhibitors. Bioorg. Med. Chem. Lett. 2011, 21, 1844–1848. [Google Scholar] [CrossRef] [PubMed]
  26. Herderson, A.J.; Hadden, M.; Guo, C.; Douglas, N.; Decornez, H.; Hellberg, M.R.; Rusinko, A.; McLaughlin, M.; Sharif, N.; Drace, C.; et al. 2,3-Diaminopyrazines as rho kinase inhibitors. Bioorg. Med. Chem. Lett. 2010, 20, 1137–1140. [Google Scholar] [CrossRef] [PubMed]
  27. Rajagopalan, L.E.; Davies, M.S.; Kahn, L.E.; Kornmeier, C.M.; Shimada, H.; Steiner, T.A.; Zweifel, B.S.; Wendling, J.M.; Payne, M.A.; Loeffler, R.F.; et al. Biochemical, cellular, and anti-inflammatory properties of a potent, selective, orally bioavailable benzamide inhibitor of Rho kinase activity. J. Pharmacol. Exp. Ther. 2010, 333, 707–716. [Google Scholar] [CrossRef] [PubMed]
  28. Chen, Y.T.; Bannister, T.D.; Weiser, A.; Griffin, E.; Lin, L.; Ruiz, C.; Cameron, M.D.; Schürer, S.; Duckett, D.; Schröter, T.; et al. Chroman-3-amides as potent Rho kinase inhibitors. Bioorg. Med. Chem. Lett. 2008, 18, 6406–6409. [Google Scholar] [CrossRef] [PubMed]
  29. Yin, Y.; Lin, L.; Ruiz, C.; Khan, S.; Cameron, M.D.; Grant, W.; Pocas, J.; Eid, N.; Park, H.; Schroter, T.; et al. Synthesis and biological evalution of urea derivatives as highly potent and selective Rho kinase inhibitors. J. Med. Chem. 2013, 56, 3568–3581. [Google Scholar] [CrossRef] [PubMed]
  30. Feng, Y.B.; LoGrasso, P.V.; Defert, O.; Li, R. Rho kinase (ROCK) inhibitors and their therapeutic potential. J. Med. Chem. 2016, 59, 2269–2300. [Google Scholar] [CrossRef] [PubMed]
  31. Gong, L.L.; Peng, J.H.; Fang, L.L.; Ping, X.; Jiao, X.-Z.; Xie, P.; Fang, L.-H.; Du, G.-H. The vasorelaxant mechanisms of a Rho kinase inhibitor in rat thoracic aorta. Molecules 2012, 17, 5935–5944. [Google Scholar] [CrossRef] [PubMed]
  32. Goodman, K.B.; Cui, H.; Dowdell, S.E.; Gaitanopoulos, D.E.; Ivy, R.L.; Sehon, C.A.; Stavenger, R.A.; Wang, G.Z.; Viet, A.Q.; Xu, W.; et al. Development of dihydropyridoneindazole amides as selective rho-kinase inhibitors. J. Med. Chem. 2007, 50, 6–9. [Google Scholar] [CrossRef] [PubMed]
  33. Sehon, C.A.; Wang, G.Z.; Viet, A.Q.; Goodman, K.B.; Dowdell, S.E.; Elkins, P.A.; Semus, S.F.; Evans, C.; Jolivette, L.J.; Kirkpatrick, R.B.; et al. Potent, selective and orally bioavailable dihydropyrimidine inhibitors of Rho kinase (ROCK i) as potential therapeutic agents for cardiovascular diseases. J. Med. Chem. 2008, 51, 6631–6634. [Google Scholar] [CrossRef] [PubMed]
  34. Feurer, A.; Bennabi, S.; Heckroth, H.; Schirok, H.; Mittendorf, J. Heteroaryl-Substituted Phenylaminopyridines as Rho Kinase Inhibitors. International Patent WO2004039796, 13 May 2004. [Google Scholar]
  35. Yuan, T.Y.; Chen, Y.C.; Zhang, H.F.; Li, L.; Jiao, X.-Z.; Xie, P.; Fang, L.-H.; Du, G.-H. A novel indazole derivative, DL0805–2, relaxes the angiotensin II-inducedvascular smooth muscle contration by inhibiting Rho kinase and calcium fluxes. Acta Pharmacol. Sin. 2016, 37, 1–13. [Google Scholar] [CrossRef] [PubMed]
  36. Bosanac, T.; Hickey, E.R.; Ginn, J.; Kashem, M.; Kerr, S.; Kugler, S.; Li, X.; Olague, A.; Schlyer, S.; Young, E.R. Substituted 2H-isoquinolin-1-ones as potent Rho-kinase inhibitors: part 3, aryl substituted pyrrolidines. Bioorg. Med. Chem. Lett. 2010, 20, 3746–3749. [Google Scholar] [CrossRef] [PubMed]
  • Sample Availability: Samples of the compounds 2a-2f; 3a-3c; 4a-4i; 5a-5f are available from the authors.
Figure 1. The structures of screened lead compound DL0805, I, II and III.
Figure 1. The structures of screened lead compound DL0805, I, II and III.
Molecules 22 01766 g001
Figure 2. Molecular design based on the template compound DL0805.
Figure 2. Molecular design based on the template compound DL0805.
Molecules 22 01766 g002
Scheme 1. Synthesis of compounds 2a2f and 3a3c. 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 1. Synthesis of compounds 2a2f and 3a3c. 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.
Molecules 22 01766 sch001
Scheme 2. Synthesis of compounds 4a4i and 5a5f. 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.
Scheme 2. Synthesis of compounds 4a4i and 5a5f. 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.
Molecules 22 01766 sch002
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.
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.
Molecules 22 01766 g003aMolecules 22 01766 g003b
Table 1. Percentage inhibition of 2a2f, 3a3c, 4a4i and 5a5f against ROCK I under the concentration of 20μM. a
Table 1. Percentage inhibition of 2a2f, 3a3c, 4a4i and 5a5f against ROCK I under the concentration of 20μM. a
CompoundRConfiguration% Inhibition of ROCK ICompoundRConfiguration% Inhibition of ROCK I
2a4-H(±)27.24e4-NO2(±)44.7
2b4-H(R)24.34f4-CN(±)24.5
2c4-H(S)24.64g4-F(±)63.8
2d4-CH3(±)26.64h4-H(S)54.8
2e4-CH3(R) 4i4-H(R)73.9
2f4-CH3(S)21.35a4-H(±)42.5
3a4-H(±)16.05b4-CH3(±)54.9
3b4-H(R)15.05c4-OCH3(±)19.1
3c4-H(S)23.75d4-Br(±)27.9
4a4-H(±)73.25e4-NO2(±)45.4
4b4-CH3(±)75.85f4-Cl(±)55.2
4c4-OCH3(±)53.3DL0805 72.3
4d4-Br(±)62.5fasudil 71.1
a These data were obtained by single determinations.
Table 2. The IC50 values of compounds against ROCK I.
Table 2. The IC50 values of compounds against ROCK I.
CompoundROCK I (μM)CompoundROCK I (μM)CompoundROCK I (μM)
4a0.27 a4g1.41 b5f7.26 b
4b0.17 a4h0.42 bDL08056.67 b
4c1.86 b4i7.32 bfasudil0.36 a
4d0.51 b5b11.5 b
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.
Table 3. The EC50 values of compounds for vasorelaxant activity.
Table 3. The EC50 values of compounds for vasorelaxant activity.
CompoundVasorelexant Evaluation (μM)
In High-Potassium ModelIn NE Model
4a4.00 a5.62 a
4b3.47 a4.07 a
4c5.80 a5.57 a
4d58.1 b46.8 b
4g19.5 b8.91 b
4h32.36 b--
4i6.02 b11.48 b
5b60.6 b15.6 b
5f----
DL08059.44 b28.8 b
fasudil5.47 a4.65 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.
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