Design, synthesis, biological evaluation and molecular docking of novel dabigatran derivatives as potential thrombin inhibitors

Abstract

A series of dabigatran derivatives were designed and synthesized to discover effective thrombin inhibitors. All the target compounds were characterized by ¹H NMR, ¹³C NMR and HRMS. These compounds were evaluated in vitro and showed potent anticoagulant activities against thrombin. Among these compounds, the ethyl group substitution at N-1 of benzimidazole exhibited better anticoagulant activity against thrombin. Especially, compound 10i showed an IC₅₀ of 2.04 nM. Docking simulations demonstrated that compound 10i could bind tightly with the crystal structure of the thrombin active site. Based on the results obtained, compound 10i may act as a candidate compound for further development of direct thrombin inhibitors.

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1. Introduction

Cardiovascular diseases (CVDs) are among the leading causes of death worldwide. According to the World Health Organization, CVD mortality may be expected to reach about 25 million by 2030.¹ Arterial and venous thrombotic events are major causes of CVDs. Antiplatelet drugs generally treat arterial thrombosis, whereas coagulation cascade factors primarily treat venous thrombosis.² The final mediator in the coagulation cascade is thrombin. Thrombin is a multifunctional serine protease that converts soluble fibrinogen into insoluble fibrin clots and activates platelets.^3,4 Thrombin performs an important function in blood clot formation.⁵ Thus, inhibition of thrombin will effectively treat thrombosis.

Several direct thrombin inhibitors, such as argatroban,⁶ melagatran,⁷ ximelagatran,⁸ and dabigatran etexilate,⁹ have been reported (Fig. 1.). However, upon oral administration, argatroban is inactivated and melagatran becomes unstable in humans; thus, these drugs are no longer available in the market. Oral administration of small-molecule, direct thrombin inhibitors remains a major goal for clinical development of antithrombotic therapies. The first orally available small-molecule, the direct thrombin inhibitor ximelagatran was withdrawn from the market because of uncertainties related to long-term treatment.¹⁰ Thus, dabigatran etexilate became the only orally available direct thrombin inhibitor in the market. Dabigatran etexilate is a prodrug of dabigatran. After oral administration, this prodrug is rapidly hydrolyzed to produce the direct anticoagulant activity of dabigatran in vivo.¹¹ As a potential anticoagulant agent, dabigatran etexilate is used to prevent venous thromboembolic events in patients undergoing total knee replacement surgery¹² as well as stroke and systemic embolism in patients with atrial fibrillation.¹³ Recently, Xu¹⁴ reported a novel dabigatran prodrug with excellent activity for inhibition of venous thrombosis. Thus, modified dabigatran is of great significance in developing safer and more effective thrombin inhibitors for preventing and treating thrombotic diseases.

Fig. 1 Chemical structures of direct thrombin inhibitors.

Various benzimidazole derivatives with different substitutions at the central moiety have also demonstrated diverse inhibitory activities.¹⁵ In particular, substitution at the N-1 position of benzimidazole with different linear alkyl groups enhanced the inhibitory activity of this drug. For example, when a butyl is substituted at N-1 of benzimidazole, the inhibitor activity against interleukin-2-inducible T-cell kinase becomes 17-fold better than that induced by substitution at N-1 with methyl.¹⁶ Moreover, upon isopropyl substitution at N-1 of benzimidazole, the inhibitor activity against cyclin-dependent kinase 5 exhibits more potent activity than that induced by substitution at N-1 with cyclopentyl.¹⁷ Hauel¹⁸ investigated the crystal structure of human α-thrombin binding inhibitor and reported that benzimidazole performs a crucial function in anticoagulation. The N-methyl group is highly suitable for the P-pocket, and methyl is located in the active site of the enzyme. Benzamidine of the inhibitor is also bound to the D-pocket through hydrophobic interaction. Thus, our current study aimed to optimize dabigatran by changing its N-substituents and modifying its terminal phenyl ring.

Fluorinated compounds have become widespread in medicinal chemistry.^19–21 Given the high electronegativity and small size of fluorine atoms, this element exhibits chemical properties very different from those of hydrogen. The special properties of fluorine affect the characteristics of certain drugs by enhancing receptor–ligand interactions, metabolic stability, and bioavailability and changing a number of their physical properties.^22,23 Therefore, introduction of fluorine to dabigatran is an important approach for discovering novel thrombin inhibitors. We recently designed and synthesized a series of dabigatran derivatives, and some compounds notably exhibited significant activities against thrombin in vitro. In particular, compound 10i showed maximum activity. In order to rationalize the biological results, molecular docking study was performed to analyze the probable binding models.

2. Results and discussion

2.1. Chemistry

The synthetic approach for dabigatran derivatives is presented in Scheme 1. First, the main intermediate compound 2 was prepared from 3-chloro-4-fluoro aniline (1) and ethyl acrylate with the catalyst TfOH by refluxing for 12 h under a N₂ atmosphere. Reaction of 4-chloro-3-nitrobenzoic acid (3) and the readily available amine solution yielded 4-substituted-3-nitrobenzoic acids 4a–4g in good yield. In accordance with the reported method,¹⁸ compounds 4a–4g were treated with sulfoxide chloride in the presence of N,N-dimethylformamide to obtain chlorides 5a–5g. Acylation of compound 2 with chlorides 5a–5g under alkaline conditions provided amides 6a–6g. Compounds 7a–7g were produced by nitro reduction of 6a–6g. Compounds 8a–8n were prepared by treating 7a–7g with (4-cyano-2-substituted-phenylamino)-acetic acid in the presence of 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. Cyano compounds 8a–8n were converted into the corresponding hydroxamic acids by treatment with an ethanol solution of hydroxylamine followed by Pd/C reduction under a N₂ atmosphere to obtain the amidino compounds 9a–9n. Target compounds 10a–10n were obtained from compounds 9a–9n by hydrolysis. All of the synthetic target compounds were characterized by ¹H NMR, ¹³C NMR, and ESI-HRMS.

Scheme 1 Reagents and conditions: (a) ethyl acrylate, TfOH, N₂, reflux, 12 h; (b) R₁NH₂,75 °C, reflux, 5 h; (c) SOCl₂, DMF, reflux, 2 h; (d) Et₃N, DCM, rt, 4 h; (e) Zn/NH₄Cl, THF/H₂O, reflux, 5 h; (f) ① (4-cyano-2-substituted-phenylamino)-acetic acid, HOBt, EDCI,THF, DMF, rt, 5 h; ② HOAC, 120 °C, 3 h; (g) ① Et₃N, NH₂OH·HCl, CH₃CH₂OH, reflux, 3 h; ② HCOONH₄, N₂, Pt/C (5%), HOAC, reflux 5 h; (h) NaOH, CH₃CH₂OH, rt, 2 h.

2.2. Anticoagulant assay

In order to test the biological activities of the novel dabigatran derivatives 10a–10n, compared to the potent positive control dabigatran in vitro, we evaluated their anticoagulant activities against thrombin. The IC₅₀ of each individual compound was illustrated in Table 1. Although these compounds exhibit either low or moderate activity against thrombin in vitro, the data still reflects some valuable information and structure–activity relationship. Based on the inhibitory activities of compounds 10a–10n, substitution at N-1 of benzimidazole with different alkyl side chains influences the inhibitory activities of the resultant molecule. Among compounds 10a–10g, inhibitory activities showed the order 10a, 10b > 10c > 10f > 10g > 10d > 10e; this trend demonstrates decreases in activity with increasing length or size of side chains. Similar results are obtained on compounds 10h–10n. Among them, ethyl group substitution at N-1 of benzimidazole showed better anticoagulant activity against thrombin. Moreover, fluorine was subsequently used at the C-2 position of the terminal benzene ring. The resultant compound 10i exhibited the most potent thrombin inhibitory activity with IC₅₀ of 2.04 ± 0.38 nM, which was comparable to the positive control dabigatran (IC₅₀ = 2.61 ± 0.84 nM). The result suggests that fluorine substitution may affect the inhibitory activity of this drug.

Table 1 Thrombin inhibitory activities of synthetic compounds 10a–10n

Compounds	R1	R2	IC50 (nM)
10a	–CH3	–H	8.29 ± 0.19
10b	–CH2CH3	–H	8.86 ± 0.24
10c	–CH2CH2CH3	–H	11.07 ± 0.36
10d	–CH2CH2CH2CH3	–H	40.14 ± 2.41
10e	–CH2CH2CH2CH2CH3	–H	117.53 ± 8.60
10f	–CH(CH3)2	–H	18.65 ± 2.91
10g		–H	27.53 ± 2.35
10h	–CH3	–F	11.48 ± 1.50
10i	–CH2CH3	–F	2.04 ± 0.38
10j	–CH2CH2CH3	–F	27.88 ± 2.48
10k	–CH2CH2CH2CH3	–F	36.04 ± 3.80
10l	–CH2CH2CH2CH2CH3	–F	174.18 ± 8.42
10m	–CH(CH3)2	–F	16.77 ± 1.64
10n		–F	27.08 ± 2.54
dabigatran	—	—	2.61 ± 0.84

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2.3. Molecular docking study

In order to facilitate understanding of their inhibitory mechanisms, we proceeded to detect the molecular docking of potent anticoagulant agent (10i) with the crystal structure (PDB code: 1KTS).¹⁸ As shown in Fig. 2, dabigatran was redocked to validate the docking reliability. The binding mode of redocked and co-crystallized dabigatran is almost same in the active site of thrombin receptor (PDB code: 1KTS). For example, the H atom of the acetamidine interacts with the carbonyl group of Gly219 and the O atom of Asp189 by water-mediated hydrogen bonds, respectively. The O atom of the carbonyl group interacts with the hydroxy group of Thr172. The benzimidazole ring of the two compounds are bound to thrombin by a hydrophobic interaction in the same pocket. The pyridine ring is also positioned in the same pocket, except slight rotation of bonds. After validating the docking reliability, the compound with highest activity was selected for exploring the probable binding conformation.

Fig. 2 (a) The comparison of the cognate ligand (Cyan) in the crystal structure and re-docking result of dabigatran (Violet) in the docked complex by superimposing the coordinates of protein together. (b) A molecular surface of the active site of inhibitor–thrombin complexes depicted by lipophilic potential.

Fig. 3 shows the interacting mode of compound 10i in the binding site of thrombin receptor. As shown in Fig. 3(a), compound 10i was docked into the binding cavity. It shares a similar binding mode with the compound reported by the literature:¹⁸ two hydrogen bonds were formed between the amidine group and the Asp189, Gly219 residue shown in Fig. 3(b). Except the literature reported, another two hydrogen bonds were formed between the F atom of the benzene ring with Arg173 and the N of benzimidazole ring with Gly216. The hydrogen bond distances observed are 2.11 Å (Asp189-O⋯H–N–), 1.99 Å (Gly219-C–O⋯H–N–), 2.60 Å (Arg173-N–H⋯F–C) and 2.72 Å (Gly216-N–H⋯N–C), respectively. These hydrogen bond interactions may play important roles in the interaction of dabigatran derivatives with the residues in the active site of thrombin.

Fig. 3 (a) X-ray crystal structure of compound 10i in complex with thrombin in a surface representation. The lipophilic potential is mapped on the protein surface. (b) Docking result of the compound 10i into the binding site of thrombin protease. Hydrogen bonds are shown as magenta dashed lines, with distance unit of Å. The inhibitor (Cyan) and the important residues (Atom Type) are shown as stick model.

Here, the benzimidazole ring forms π–π stacking interactions with conserved Trp60, which is crucial to the binding of dabigatran derivatives with thrombin protein, the central template is bound to thrombin by a hydrophobic interaction with the P-pocket¹⁸ and the 3-chloro-4-fluoro substituted benzene ring is positioned between Leu 99, Arg173 and Ile174. The substituents at the benzimidazole (N-1) was around gray surface, meaning the importance of the lipophilic/hydrophobic interaction in this position (His57, Tyr60A, Trp60D and Asp102). Due to steric effects, different alkyl side chains influence the inhibitory activities of the resultant molecule, which was illustrated by the potency order 10a, 10b > 10c > 10f > 10g > 10d > 10e.

3. Conclusion

In summary, a number of dabigatran derivatives was designed, synthesized and evaluated as thrombin inhibitors. During preliminary screening in vitro, all of the tested compounds showed inhibitory activities against thrombin. When the different alkyl side chains were substituted at N-1 of benzimidazole, they influenced the inhibitory activities of the resultant molecule. This may be because of the limited steric effects. Among compounds 10a–10g, the inhibitory activities showed the order 10a, 10b > 10c > 10f > 10g > 10d > 10e; this trend demonstrates decreases in activity with increasing length or size of side chains. Similar results are obtained on compounds 10h–10n. Since the ethyl group can better fill the active pocket and have the hydrophobic/lipophilic interactions with the surrounding amino acid residues, we found ethyl group substitution at N-1 of benzimidazole exhibited better anticoagulant activity against thrombin. In particular, compound 10i showed the most potent activity against thrombin with an IC₅₀ of 2.04 ± 0.38 nM; this value is considerable to that of dabigatran, with exhibits an IC₅₀ of 2.61 ± 0.84 nM. Molecular docking studies are also performed to elucidate the binding of active compounds, which will facilitate understanding of their inhibitory mechanisms. These molecular docking results revealed that compound 10i was potential thrombin inhibitor. The in vitro experimental results agree well with molecular docking results. Further evaluation of compound 10i is ongoing and will be reported in due time.

4. Experiments

4.1. Materials and measurements

All solvents were A.R. grade and purchased from Shanghai Chemical Reagent Company. Melting points were determined on a WPS-2Acapillary apparatus and were uncorrected. The ¹H NMR and ¹³C NMR spectra were recorded on a Bruker Avance III 500 NMR spectrometer using TMS as an internal standard and chemical shifts were given in ppm with tetramethylsilane (TMS). The HRMS spectra were acquired on a Solari X-70FT-MS apparatus. Elemental analyses were performed on a CHN–O–Rapid instrument. TLC was carried out on silica gel plates (GF254) with visualization of components by UV light (254 nm) or exposure to I₂. Column chromatography was carried out on silica gel (300–400 mesh). A series of dabigatron derivatives were synthesized by the method described in Scheme 1.

4.2. General procedure for the synthesis of the target compounds 10a–10n

4.2.1 General procedure for the synthesis of 3-(3-chloro-4-fluoro-phenylamino)-propionic acid ethyl ester (2). Compound 1 (8 g, 55 mmol) and ethyl acrylate (11.0 g, 10 mmol) were mixed in a 250 mL dry round bottom flask. TfOH (8.25 mmol) was added. After the addition was completed, the mixture was stirred at refluxing temperature for 12 h under a nitrogen atmosphere. After the reaction was accomplished, the unreacted ethyl acrylate was removed in vacuo. The residue was chromatographed (silica gel, petroleum ether/ethyl acetate 5 : 1) to afford the title compound 2 (10.19 g, 73.5%) as pale yellow liquid.

4.2.2 General procedure for the synthesis of 3-[(3-chloro-4-fluorophenyl)-(4-substituted-3-nitrob-benzoyl)amino]propionic acid ethylester (6a–6g). A mixture of 4-chloro-3-nitrobenzoic acid 3 (50 mmol), corresponding amine solution (865 mmol) was stirred at 75 °C for 5 h. After cooling, the resulting solution was poured into water, acidified to pH 4–5 with concentrated HCl and the solid formed was filtered and dried to obtain 4-substituted-3-nitrobenzoic acid (4a–4g).

To a solution of 4-substituted-3-nitrobenzoic acid (4a–4g) (16 mmol) in DCM (100 mL) containing N,N-dimethylformamide (DMF, 0.5 mL), thionyl chloride (18.5 mmol) was slowly added at room temperature and then the mixture was stirred at refluxing temperature for 2 h. After cooled to room temperature, the reaction mixture was concentrated to near dryness in vacuo to obtain oily residue (5a–5g). To a solution of compound 2 (16 mmol) in DCM (50 mL) containing triethylamine (TEA) (12 mmol), a DCM (40 mL) solution of the oily residue (5a–5g) was slowly added at room temperature and then the mixture was stirred for 5 h. After the completion of reaction, the reaction mixture was washed with water and extracted with DCM in the separatory funnel. The DCM layer was separated and the aqueous layer was extracted twice with DCM. The combined DCM solutions were dried by anhydrous Na₂SO₄ and then the DCM was distilled under reduced pressure. The residue was chromatographed (silica gel, petroleum ether/ethyl acetate = 2 : 1) to afford the compounds (6a–6g).

4.2.2.1 3-[(3-Chloro-4-fluoro-phenyl)-(4-methylamino-3-nitro-benzoyl)-amino]-propionic acid ethyl ester (6a). 6a was obtained in 54% yield as light yellow solid. mp 104–106 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm) 8.21 (d, J = 2.0 Hz, 1H), 7.43 (dd, J = 9.0 Hz, J = 1.9 Hz, 1H), 7.24 (dd, J = 6.3 Hz, J = 2.5 Hz, 1H), 7.09 (t, J = 8.5 Hz, 1H), 7.05–7.01 (m, 1H), 6.68 (d, J = 9.0 Hz, 1H), 4.16 (t, J = 7.1 Hz, 2H), 4.11 (q, J = 7.1 Hz, 2H), 3.02 (d, J = 5.1 Hz, 3H), 2.71 (t, J = 7.1 Hz, 2H), 1.25 (t, J = 7.1 Hz, 3H); ESI-HRMS: calcd for C₁₉H₂₀ClFN₃O₅ [M + H]⁺, 424.1070, found 424.1066.
4.2.2.2 3-[(3-Chloro-4-fluoro-phenyl)-(4-ethylamino-3-nitro-benzoyl)-amino]-propionic acid ethyl ester (6b). 6b was obtained in 40% yield as light yellow solid. mp 103–105 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm) 8.20 (d, J = 2.0 Hz, 1H), 7.41 (dd, J = 9.0 Hz, J = 1.8 Hz, 1H), 7.24 (dd, J = 6.3 Hz, J = 2.5 Hz, 1H), 7.08 (t, J = 8.5 Hz, 1H), 7.05–7.00 (m, 1H), 6.68 (d, J = 9.0 Hz, 1H), 4.16 (t, J = 7.1 Hz, 2H), 4.11 (q, J = 7.1 Hz, 2H), 3.37–3.30 (m, 2H), 2.71 (t, J = 7.1 Hz, 2H), 1.36 (t, J = 7.2 Hz, 3H), 1.25 (t, J = 7.1 Hz, 3H); ESI-HRMS: calcd for C₂₀H₂₂ClFN₃O₅ [M + H]⁺, 438.1227, found 438.1226.
4.2.2.3 3-[(3-Chloro-4-fluoro-phenyl)-(3-nitro-4-propylamino-benzoyl)-amino]-propionic acid ethyl ester (6c). 6c was obtained in 45% yield as light yellow solid. mp 93–95 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm) 8.20 (d, J = 1.7 Hz, 1H), 7.40 (dd, J = 9.0 Hz, J = 1.5 Hz, 1H), 7.24 (dd, J = 6.3 Hz, J = 2.4 Hz, 1H), 7.09 (t, J = 8.5 Hz, 1H), 7.05–7.00 (m, 1H), 6.68 (d, J = 9.1 Hz, 1H), 4.16 (t, J = 7.1 Hz, 2H), 4.11 (q, J = 7.1 Hz, 2H), 3.25 (dd, J = 12.5 Hz, J = 6.9 Hz, 2H), 2.71 (t, J = 7.1 Hz, 2H), 1.79–1.70 (m, 2H), 1.25 (t, J = 7.1 Hz, 3H), 1.05 (t, J = 7.4 Hz, 3H); ESI-HRMS: calcd for C₂₁H₂₄ClFN₃O₅ [M + H]⁺, 452.1383, found 452.1390.
4.2.2.4 3-[(4-Butylamino-3-nitro-benzoyl)-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid ethyl ester (6d). 6d was obtained in 45% yield as light yellow solid. mp 91–93 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm) 8.20 (d, J = 2.0 Hz, 1H), 7.40 (dd, J = 9.0 Hz, J = 1.8 Hz, 1H), 7.24 (dd, J = 6.3 Hz, J = 2.5 Hz, 1H), 7.09 (t, J = 8.5 Hz, 1H), 7.05–7.00 (m, 1H), 6.68 (d, J = 9.1 Hz, 1H), 4.16 (t, J = 7.1 Hz, 2H), 4.11 (q, J = 7.2 Hz, 2H), 3.28 (dd, J = 12.4 Hz, J = 7.0 Hz, 2H), 2.71 (t, J = 7.1 Hz, 2H), 1.73–1.67 (m, 2H), 1.51–1.43 (m, 2H), 1.25 (t, J = 7.2 Hz, 3H), 0.99 (t, J = 7.3 Hz, 3H); ESI-HRMS: calcd for C₂₂H₂₆ClFN₃O₅ [M + H]⁺, 466.1540, found 466.1536.
4.2.2.5 3-[(3-Chloro-4-fluoro-phenyl)-(3-nitro-4-pentylamino-benzoyl)-amino]-propionic acid ethyl ester (6e). 6e was obtained in 33% yield as light yellow solid. mp 90–91 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm) 8.20 (d, J = 2.1 Hz, 1H), 7.40 (dd, J = 9.0 Hz, J = 2.0 Hz, 1H), 7.24 (dd, J = 6.4 Hz, J = 2.5 Hz, 1H), 7.09 (t, J = 8.5 Hz, 1H), 7.05–7.01 (m, 1H), 6.67 (d, J = 9.1 Hz, 1H), 4.16 (t, J = 7.1 Hz, 2H), 4.11 (q, J = 7.1 Hz, 2H), 3.27 (dd, J = 12.4 Hz, J = 7.1 Hz, 2H), 2.71 (t, J = 7.1 Hz, 2H), 1.75–1.68 (m, 2H), 1.43–1.37 (m, 4H), 1.25 (t, J = 7.1 Hz, 3H), 0.94 (t, J = 7.0 Hz, 3H); ESI-HRMS: calcd for C₂₃H₂₈ClFN₃O₅ [M + H]⁺, 480.1696, found 480.1695.
4.2.2.6 3-[(3-Chloro-4-fluoro-phenyl)-(4-isopropylamino-3-nitro-benzoyl)-amino]-propionic acid ethyl ester (6f). 6f was obtained in 60% yield as light yellow solid. mp 104–106 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm) 8.19 (d, J = 2.0 Hz, 1H), 7.41 (dd, J = 9.0 Hz, J = 1.8 Hz, 1H), 7.25 (dd, J = 6.3 Hz, J = 2.5 Hz, 1H), 7.09 (t, J = 8.5 Hz, 1H), 7.05–7.01 (m, 1H), 6.70 (d, J = 9.1 Hz, 1H), 4.16 (t, J = 7.1 Hz, 2H), 4.11 (q, J = 7.2 Hz, 2H), 3.84–3.78 (m, 1H), 2.71 (t, J = 7.1 Hz, 2H), 1.32 (d, J = 6.4 Hz, 6H), 1.25 (t, J = 7.1 Hz, 3H); ESI-HRMS: calcd for C₂₁H₂₄ClFN₃O₅ [M + H]⁺, 452.1383, found 452.1397.
4.2.2.7 3-[(3-Chloro-4-fluoro-phenyl)-(4-cyclopropylamino-3-nitro-benzoyl)-amino]-propionic acid ethyl ester (6g). 6g was obtained in 42% yield as light yellow solid. mp 102–104 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm) 8.17 (d, J = 1.9 Hz, 1H), 7.45 (dd, J = 9.0 Hz, J = 1.9 Hz, 1H), 7.25 (dd, J = 6.3 Hz, J = 2.5 Hz, 1H), 7.15 (d, J = 9.0 Hz, 1H), 7.09 (t, J = 8.6 Hz, 1H), 7.05–7.01 (m, 1H), 4.16 (t, J = 7.1 Hz, 2H), 4.11 (q, J = 7.1 Hz, 2H), 2.71 (t, J = 7.1 Hz, 2H), 2.60–2.54 (m, 1H), 1.25 (t, J = 7.2 Hz, 3H), 0.93 (q, J = 6.6 Hz, 2H), 0.68–0.64 (m, 2H); ESI-HRMS: calcd for C₂₁H₂₂ClFN₃O₅ [M + H]⁺, 450.1227, found 450.1237.

4.2.3 General procedure for the synthesis of 3-[(3-amino-4-substituted-benzoyl)(3-chloro-4-fluoro-phenyl)amino]propionic acid ethyl ester (7a–7g). To a solution of compounds (6a–6g) (8.6 mmol) was added Zn (43 mmol) in 100 mL tetrahydrofuran (THF) and 50 mL H₂O, and the mixture was heated to reflux for 5 h. After cooled to room temperature, the reaction mixture was filtrated and removal of the solvent THF under reduced pressure. Then the mixture was extracted with DCM (3 × 40 mL) and the DCM layer was washed with saturated brine, dried over anhydrous Na₂SO₄, and concentrated in vacuo. The residue was chromatographed (silica gel, petroleum ether/ethyl acetate = 1 : 1) to afford compounds (7a–7g).
4.2.3.1 3-[(3-Amino-4-methylamino-benzoyl)-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid ethyl ester (7a). 7a was obtained in 89% yield as brown solid. mp 105–107 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.23 (dd, J = 6.4 Hz, J = 2.5 Hz, 1H), 7.00 (t, J = 8.6 Hz, 1H), 6.94–6.90 (m, 1H), 6.87 (s, 1H), 6.70 (d, J = 8.0 Hz, 1H), 6.33 (d, J = 8.3 Hz, 1H), 4.14 (t, J = 7.0 Hz, 2H), 4.09 (q, J = 7.1 Hz, 2H), 2.82 (s, 3H), 2.70 (t, J = 7.0 Hz, 2H), 1.23 (t, J = 7.1 Hz, 3H); ESI–HRMS: calcd for C₁₉H₂₁ClFN₃O₃Na [M + Na]⁺, 416.1148, found 416.1180.
4.2.3.2 3-[(3-Amino-4-ethylamino-benzoyl)-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid ethyl ester (7b). 7b was obtained in 86% yield as brown solid. mp 106–108 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.23 (dd, J = 6.1 Hz, J = 2.0 Hz, 1H), 7.00 (t, J = 8.6 Hz, 1H), 6.92 (dd, J = 8.0 Hz, J = 3.1 Hz, 1H), 6.87 (s, 1H), 6.68 (dd, J = 8.2 Hz, J = 1.3 Hz, 1H), 6.34 (d, J = 8.3 Hz, 1H), 4.14 (t, J = 7.1 Hz, 2H), 4.09 (q, J = 7.1 Hz, 2H), 3.11 (dd, J = 14.1 Hz, J = 7.0 Hz, 2H), 2.70 (t, J = 7.1 Hz, 2H), 1.28 (t, J = 7.1 Hz, 3H), 1.23 (t, J = 7.1 Hz, 3H); ESI-HRMS: calcd for C₂₀H₂₃ClFN₃O₃Na [M + Na]⁺, 430.1304, found 430.1338.
4.2.3.3 3-[(3-Amino-4-propylamino-benzoyl)-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid ethyl ester (7c). 7c was obtained in 85% yield as brown solid. mp 104–105 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.23 (dd, J = 6.4 Hz, J = 2.5 Hz, 1H), 7.00 (t, J = 8.6 Hz, 1H), 6.94–6.89 (m, 1H), 6.87 (d, J = 1.8 Hz, 1H), 6.68 (dd, J = 8.3 Hz, J = 1.8 Hz, 1H), 6.34 (d, J = 8.3 Hz, 1H), 4.13 (t, J = 7.1 Hz, 2H), 4.09 (q, J = 7.1 Hz, 2H), 3.03 (t, J = 7.2 Hz, 2H), 2.70 (t, J = 7.1 Hz, 2H), 1.69–1.61 (m, 2H), 1.23 (t, J = 7.1 Hz, 3H), 1.00 (t, J = 7.4 Hz, 3H); ESI-HRMS: calcd for C₂₁H₂₆ClFN₃O₃ [M + H]⁺, 422.1641, found 422.1672.
4.2.3.4 3-[(3-Amino-4-butylamino-benzoyl)-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid ethyl ester (7d). 7d was obtained in 92% yield as brown solid. mp 103–105 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.23 (dd, J = 6.4 Hz, J = 2.5 Hz, 1H), 6.99 (t, J = 8.6 Hz, 1H), 6.93–6.90 (m, 1H), 6.87 (d, J = 1.8 Hz, 1H), 6.67 (dd, J = 8.3 Hz, J = 1.7 Hz, 1H), 6.33 (d, J = 8.3 Hz, 1H), 4.13 (t, J = 7.1 Hz, 2H), 4.08 (q, J = 7.1 Hz, 2H), 3.06 (t, J = 7.2 Hz, 2H), 2.69 (t, J = 7.1 Hz, 2H), 1.64–1.58 (m, 2H), 1.47–1.38 (m, 2H), 1.23 (t, J = 7.1 Hz, 3H), 0.95 (t, J = 7.4 Hz, 3H); ESI-HRMS: calcd for C₂₂H₂₇ClFN₃O₃Na [M + Na]⁺, 458.1617, found 458.1623.
4.2.3.5 3-[(3-Amino-4-pentylamino-benzoyl)-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid ethyl ester (7e). 7e was obtained in 84% yield as brown solid. mp 105–107 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.23 (dd, J = 6.4 Hz, J = 2.6 Hz, 1H), 7.00 (t, J = 8.6 Hz, 1H), 6.93–6.90 (m, 1H), 6.87 (d, J = 1.9 Hz, 1H), 6.68 (dd, J = 8.3 Hz, J = 1.8 Hz, 1H), 6.34 (d, J = 8.3 Hz, 1H), 4.13 (t, J = 7.17 Hz, 2H), 4.09 (q, J = 7.0 Hz, 2H), 3.06 (t, J = 7.2 Hz, 2H), 2.70 (t, J = 7.1 Hz, 2H), 1.66–1.60 (m, 2H), 1.47–1.38 (m, 4H), 1.23 (t, J = 7.1 Hz, 3H), 0.92 (t, J = 7.0 Hz, 3H); ESI-HRMS: calcd for C₂₃H₃₀ClFN₃O₃ [M + H]⁺, 450.1954, found 450.1981.
4.2.3.6 3-[(3-Amino-4-isopropylamino-benzoyl)-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid ethyl ester (7f). 7f was obtained in 87% yield as brown solid. mp 101–103 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.22 (dd, J = 6.4 Hz, J = 2.6 Hz, 1H), 6.98 (t, J = 8.6 Hz, 1H), 6.92–6.89 (m, 1H), 6.86 (d, J = 1.9 Hz, 1H), 6.66 (dd, J = 8.3 Hz, J = 1.8 Hz, 1H), 6.32 (d, J = 8.3 Hz, 1H), 4.13 (t, J = 7.1 Hz, 2H), 4.09 (q, J = 7.0 Hz, 2H), 3.05–3.00 (m, 1H), 2.70 (t, J = 7.1 Hz, 2H), 1.25 (t, J = 7.1 Hz, 3H), 1.06 (d, J = 6.6 Hz, 6H); ESI-HRMS: calcd for C₂₁H₂₆ClFN₃O₃ [M + H]⁺, 422.1641, found 422.1634.
4.2.3.7 3-[(3-Amino-4-cyclopropylamino-benzoyl)-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid ethylester (7g). 7g was obtained in 86% yield as brown solid. mp 105–106 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.24 (dd, J = 6.4 Hz, J = 2.5 Hz, 1H), 7.01 (t, J = 8.6 Hz, 1H), 6.95–6.90 (m, 1H), 6.85 (d, J = 1.5 Hz, 1H), 6.75 (d, J = 8.3 Hz, 1H), 6.70 (dd, J = 8.3 Hz, J = 1.6 Hz, 1H), 4.14 (t, J = 7.1 Hz, 2H), 4.09 (q, J = 7.1 Hz, 2H), 2.70 (t, J = 7.1 Hz, 2H), 2.42–2.37 (m, 1H), 1.24 (t, J = 7.1 Hz, 3H), 0.73 (q, J = 6.5 Hz, 2H), 0.52–0.47 (m, 2H); ESI-HRMS: calcd for C₂₁H₂₄ClFN₃O₃ [M + H]⁺, 420.1485, found 420.1477.

4.2.4 General procedure for the synthesis of 3-((3-chloro-4-fluoro-phenyl)-{2-[(4-cyano-2-substituted-phenylamino)methyl]-1-substituted-1H-benzoimidazole-5-carbonyl}-amino)propionic acid ethyl ester (8a–8n). A mixture of (4-cyano-2-substituted-phenylamino)-acetic acid (8.64 mmol), HOBt (8.64 mmol), EDCI (8.64 mmol) and 4 mL DMF was dissolved in 30 mL tetrahydrofuran (THF), and the mixture was stirred under ice-cooling for 30 min, a tetrahydrofuran (THF) (40 mL) solution of the 7a–7g was slowly added at room temperature and then the mixture was stirred for 4 h. After the completion of reaction, removal of the solvent THF under reduced pressure. Then the mixture was extracted with DCM (3 × 40 mL) and the DCM layer was washed with saturated brine, dried over anhydrous Na₂SO₄, and concentrated in vacuo. The residue was added in HOAC (30 mL) and heated to reflux for 3 h. After cooled to room temperature, removal of the solvent HOAC under reduced pressure. After an appropriate amount of concentrated aqueous ammonia (NH₃·H₂O) was added to pH 8–9, the mixture was extracted with DCM (3 × 40 mL) and the DCM layer was washed with saturated brine, dried over anhydrous Na₂SO₄, and concentrated in vacuo. The residue was chromatographed (silica gel, dichloromethane/methanol = 100 : 1) to afford compounds (8a–8n).
4.2.4.1 3-((3-Chloro-4-fluoro-phenyl)-{2-[(4-cyano-phenylamino)-methyl]-1-methyl-1H-benzoimidazole-5-carbonyl}-amino)propionic acid ethyl ester (8a). 8a was obtained in 86% yield as white solid. mp 105–106 °C; ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) 7.62 (dd, J = 6.7 Hz, J = 2.5 Hz, 1H), 7.53 (s, 1H), 7.47 (d, J = 8.8 Hz, 2H), 7.42 (d, J = 8.4 Hz, 1H), 7.28 (dd, J = 11.7 Hz, J = 1.8 Hz, 1H), 7.20 (dd, J = 8.4 Hz, J = 1.1 Hz, 1H), 7.18–7.13 (m, 1H), 6.82 (d, J = 8.8 Hz, 2H), 4.60 (d, J = 5.5 Hz, 2H), 4.08 (t, J = 7.0 Hz, 2H), 3.99 (q, J = 7.1 Hz, 2H), 3.76 (s, 3H), 2.62 (t, J = 7.0 Hz, 2H), 1.14 (t, J = 7.1 Hz, 3H); ESI-HRMS: calcd for C₂₈H₂₆ClFN₅O₃ [M + H]⁺, 534.1703, found 534.1728.
4.2.4.2 3-((3-Chloro-4-fluoro-phenyl)-{2-[(4-cyano-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole-5-carbonyl}-amino)-propionic acid ethyl ester (8b). 8b was obtained in 83% yield as brown solid. mp 116–118 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.66 (s, 1H), 7.46 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 8.4 Hz, 1H), 7.21 (t, J = 8.3 Hz, 2H), 6.96 (d, J = 6.5 Hz, 2H), 6.73 (d, J = 8.5 Hz, 2H), 4.51 (d, J = 4.4 Hz, 2H), 4.22–4.15 (m, 4H), 4.11 (q, J = 7.1 Hz, 2H), 2.73 (t, J = 7.0 Hz, 2H), 1.41 (t, J = 7.2 Hz, 3H), 1.24 (t, J = 7.1 Hz, 3H); ESI-HRMS: calcd for C₂₉H₂₈ClFN₅O₃ [M + H]⁺, 548.1859, found 548.1838.
4.2.4.3 3-((3-Chloro-4-fluoro-phenyl)-{2-[(4-cyano-phenylamino)-methyl]-1-propyl-1H-benzoimidazole-5-carbonyl}-amino)-propionic acid ethyl ester (8c). 8c was obtained in 80% yield as brown solid. mp 110–112 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.74 (s, 1H), 7.37 (s, 2H), 7.31 (s, 1H), 7.26–7.20 (m, 2H), 6.97 (s, 2H), 6.68 (s, 2H), 4.68 (s, 2H), 4.18 (t, J = 6.9 Hz, 4H), 4.10 (q, J = 7.1 Hz, 2H), 2.71 (t, J = 7.0 Hz, 2H), 1.85 (d, J = 2.8 Hz, 2H), 1.24 (t, J = 7.3 Hz, 3H), 0.98 (s, 3H); ESI-HRMS: calcd for C₃₀H₃₀ClFN₅O₃ [M + H]⁺, 562.2016, found 562.2046.
4.2.4.4 3-[{1-Butyl-2-[(4-cyano-phenylamino)-methyl]-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid ethyl ester (8d). 8d was obtained in 95% yield as brown solid. mp 108–109 °C; ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) 7.70 (d, J = 8.5 Hz, 1H), 7.63 (dd, J = 6.9 Hz, J = 2.5 Hz, 1H), 7.50 (d, J = 8.6 Hz, 2H), 7.42 (t, J = 8.9 Hz, 1H), 7.37 (s, 1H), 7.29 (d, J = 8.5 Hz, 1H), 7.23–7.18 (m, 1H), 6.86 (d, J = 8.6 Hz, 2H), 4.64 (d, J = 5.6 Hz, 2H), 4.19 (t, J = 7.1 Hz, 2H), 4.08 (t, J = 7.0 Hz, 2H), 3.99 (q, J = 7.1 Hz, 2H), 2.68 (t, J = 7.0 Hz, 2H), 1.74–1.67 (m, 2H), 1.39–1.28 (m, 2H), 1.13 (t, J = 7.1 Hz, 3H), 0.88 (t, J = 7.4 Hz, 3H); ESI-HRMS: calcd for C₃₁H₃₂ClFN₅O₃ [M + H]⁺, 576.2172, found 576.2171.
4.2.4.5 3-((3-Chloro-4-fluoro-phenyl)-{2-[(4-cyano-phenylamino)-methyl]-1-pentyl-1H-benzoimidazole-5-carbonyl}-amino)-propionic acid ethyl ester (8e). 8e was obtained in 81% yield as brown solid. mp 112–114 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.68 (s, 1H), 7.49 (d, J = 8.6 Hz, 2H), 7.34 (dd, J = 8.4 Hz, J = 1.2 Hz, 1H), 7.24–7.20 (m, 2H), 6.97 (d, J = 6.9 Hz, 2H), 6.74 (d, J = 8.7 Hz, 2H), 4.63 (d, J = 4.3 Hz, 2H), 4.22 (t, J = 7.1 Hz, 2H), 4.14 (q, J = 7.2 Hz, 4H), 2.76 (t, J = 7.0 Hz, 2H), 1.84–1.77 (m, 2H), 1.37–1.30 (m, 4H), 1.25 (t, J = 7.2 Hz, 3H), 0.89 (t, J = 6.9 Hz, 3H); ESI-HRMS: calcd for C₃₂H₃₄ClFN₅O₃ [M + H]⁺, 590.2329, found 590.2325.
4.2.4.6 3-((3-Chloro-4-fluoro-phenyl)-{2-[(4-cyano-phenylamino)-methyl]-1-isopropyl-1H-benzoimidazole-5-carbonyl}-amino)-propionic acid ethyl ester (8f). 8f was obtained in 82% yield as brown solid. mp 113–115 °C; ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) 7.64 (dd, J = 6.9 Hz, J = 2.3 Hz, 1H), 7.57 (s, 1H), 7.49 (d, J = 8.6 Hz, 2H), 7.42 (t, J = 9.1 Hz, 1H), 7.37 (t, J = 5.1 Hz, 1H), 7.20 (d, J = 5.1 Hz, 1H), 7.16–7.11 (m, 1H), 6.86 (d, J = 8.6 Hz, 2H), 4.94–4.88 (m, 1H), 4.72 (d, J = 5.4 Hz, 2H), 4.04 (t, J = 7.0 Hz, 2H), 3.96 (q, J = 7.1 Hz, 2H), 2.61 (t, J = 7.0 Hz, 2H), 1.59 (d, J = 6.8 Hz, 6H), 1.16 (t, J = 7.1 Hz, 3H); ESI-HRMS: calcd for C₃₀H₃₀ClFN₅O₃ [M + H]⁺, 562.2016, found 562.1988.
4.2.4.7 3-((3-Chloro-4-fluoro-phenyl)-{2-[(4-cyano-phenylamino)-methyl]-1-cyclopropyl-1H-benzoimidazole-5-carbonyl}-amino)-propionic acid ethyl ester (8g). 8g was obtained in 80% yield as brown solid. mp 123–125 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.65 (s, 1H), 7.49 (d, J = 8.6 Hz, 2H), 7.40 (d, J = 8.4 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 6.6 Hz, 1H), 6.97 (d, J = 6.3 Hz, 2H), 6.74 (d, J = 8.6 Hz, 2H), 4.62 (d, J = 4.5 Hz, 2H), 4.21 (t, J = 7.1 Hz, 2H), 4.11 (q, J = 7.1 Hz, 2H), 3.29–3.23 (m, 1H), 2.74 (t, J = 7.1 Hz, 2H), 1.32–1.28 (m, 2H), 1.25 (t, J = 7.2 Hz, 3H), 1.10 (t, J = 7.9 Hz, 2H); ESI-HRMS: calcd for C₃₀H₂₈ClFN₅O₃ [M + H]⁺, 560.1859, found 560.1849.
4.2.4.8 3-((3-Chloro-4-fluoro-phenyl)-{2-[(4-cyano-2-fluoro-phenylamino)-methyl]-1-methyl-1H-benzoimidazole-5-carbonyl}-amino)-propionic acid ethyl ester (8h). 8h was obtained in 73% yield as brown solid. mp 105–107 °C; ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) 7.61 (dd, J = 6.6 Hz, J = 2.5 Hz, 1H), 7.57 (dd, J = 11.9 Hz, J = 1.7 Hz, 1H), 7.55 (s, 1H), 7.41 (d, J = 8.4 Hz, 2H), 7.27 (t, J = 9.0 Hz, 1H), 7.18 (d, J = 8.4 Hz, 1H), 7.17–7.13 (m, 1H), 7.01 (t, J = 8.7 Hz, 1H), 4.67 (d, J = 5.6 Hz, 2H), 4.08 (t, J = 7.0 Hz, 2H), 3.99 (q, J = 7.1 Hz, 2H), 3.78 (s, 3H), 2.62 (t, J = 7.0 Hz, 2H), 1.14 (t, J = 7.1 Hz, 3H); ESI-HRMS: calcd for C₂₈H₂₅ClF₂N₅O₃ [M + H]⁺, 552.1609, found 552.1609.
4.2.4.9 3-((3-Chloro-4-fluoro-phenyl)-{2-[(4-cyano-2-fluoro-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole-5-carbonyl}-amino)-propionic acid ethyl ester (8i). 8i was obtained in 83% yield as brown solid. mp 121–123 °C; ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) 7.61 (dd, J = 6.6 Hz, J = 2.5 Hz, 1H), 7.58 (dd, J = 9.8 Hz, J = 1.6 Hz, 1H), 7.54 (s, 1H), 7.41 (d, J = 8.9 Hz, 2H), 7.27 (t, J = 8.9 Hz, 1H), 7.18 (d, J = 8.5 Hz, 1H), 7.17–7.13 (m, 1H), 7.02 (t, J = 8.7 Hz, 1H), 4.67 (d, J = 5.5 Hz, 2H), 4.29 (q, J = 6.8 Hz, 2H), 4.07 (t, J = 7.0 Hz, 2H), 3.99 (q, J = 7.1 Hz, 2H), 2.61 (t, J = 7.0 Hz, 2H), 1.32 (t, J = 7.2 Hz, 3H), 1.14 (t, J = 7.1 Hz, 3H); ESI-HRMS: calcd for C₂₉H₂₇ClF₂N₅O₃ [M + H]⁺, 566.1765, found 566.1801.
4.2.4.10 3-((3-Chloro-4-fluoro-phenyl)-{2-[(4-cyano-2-fluoro-phenylamino)-methyl]-1-propyl-1H-benzoimidazole-5-carbonyl}-amino)-propionic acid ethyl ester (8j). 8j was obtained in 82% yield as brown solid. mp 117–119 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.70 (s, 1H), 7.34 (dd, J = 14.7 Hz, J = 5.8 Hz, 2H), 7.29 (dd, J = 4.1 Hz, J = 2.5 Hz, 1H), 7.22 (d, J = 8.2 Hz, 2H), 6.98 (d, J = 7.0 Hz, 2H), 6.83 (t, J = 8.4 Hz, 1H), 4.58 (d, J = 4.7 Hz, 2H), 4.21 (t, J = 7.1 Hz, 2H), 4.14–4.08 (m, 4H), 2.74 (t, J = 7.1 Hz, 2H), 1.86–1.82 (m, 2H), 1.24 (t, J = 7.3 Hz, 3H), 0.97 (t, J = 7.4 Hz, 3H); ESI-HRMS: calcd for C₃₀H₂₉ClF₂N₅O₃ [M + H]⁺, 580.1922, found 580.1925, C₃₀H₂₈ClF₂N₅O₃Na [M + Na]⁺, 602.1741, found 602.1714.
4.2.4.11 3-[{1-Butyl-2-[(4-cyano-2-fluoro-phenylamino)-methyl]-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid ethyl ester (8k). 8k was obtained in 87% yield as brown solid. mp 128–129 °C; ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) 7.70 (d, J = 8.5 Hz, 1H), 7.60 (dd, J = 6.8 Hz, J = 2.3 Hz, 1H), 7.52 (s, 1H), 7.43 (d, J = 8.5 Hz, 2H), 7.29 (d, J = 7.1 Hz, 1H), 7.20 (d, J = 7.9 Hz, 1H), 7.14–7.10 (m, 1H), 7.02 (t, J = 8.6 Hz, 1H), 4.77 (d, J = 5.4 Hz, 2H), 4.35 (t, J = 7.3 Hz, 2H), 4.13 (t, J = 6.9 Hz, 2H), 4.03 (q, J = 7.0 Hz, 2H), 2.64 (t, J = 7.1 Hz, 2H), 1.72–1.66 (m, 2H), 1.39–1.30 (m, 2H), 1.23 (t, J = 5.1 Hz, 3H), 0.89 (t, J = 7.3 Hz, 3H); ESI-HRMS: calcd for C₃₁H₃₁ClF₂N₅O₃ [M + H]⁺, 594.2078, found 594.2066.
4.2.4.12 3-((3-Chloro-4-fluoro-phenyl)-{2-[(4-cyano-2-fluoro-phenylamino)-methyl]-1-pentyl-1H-benzoimidazole-5-carbonyl}-amino)-propionic acid ethyl ester (8l). 8l was obtained in76% yield as brown solid. mp 120–122 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.68 (s, 1H), 7.32 (d, J = 8.6 Hz, 1H), 7.30 (dd, J = 9.8 Hz, J = 1.2 Hz, 1H), 7.25 (dd, J = 11.1 Hz, J = 1.5 Hz, 1H), 7.20 (d, J = 6.8 Hz, 1H) 7.18 (d, J = 8.5 Hz, 1H), 6.96 (d, J = 6.4 Hz, 2H), 6.82 (t, J = 8.4 Hz, 1H), 4.56 (d, J = 4.6 Hz, 2H), 4.20 (t, J = 7.1 Hz, 2H), 4.10 (q, J = 7.0 Hz, 4H), 2.72 (t, J = 7.1 Hz, 2H), 1.81–1.72 (m, 2H), 1.35–1.28 (m, 4H), 1.24 (t, J = 7.2 Hz, 3H), 0.87 (t, J = 6.9 Hz, 3H); ESI-HRMS: calcd for C₃₂H₃₃ClF₂N₅O₃ [M + H]⁺, 608.2235, found 608.2211.
4.2.4.13 3-((3-Chloro-4-fluoro-phenyl)-{2-[(4-cyano-2-fluoro-phenylamino)-methyl]-1-isopropyl-1H-benzoimidazole-5-carbonyl}-amino)-propionic acid ethyl ester (8m). 8m was obtained in 80% yield as brown solid. mp 119–121 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.69 (s, 1H), 7.39 (d, J = 8.5 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.31 (d, J = 8.6 Hz, 1H), 7.26 (d, J = 7.7 Hz, 1H), 7.23 (d, J = 6.0 Hz, 1H), 6.98 (d, J = 6.2 Hz, 2H), 6.83 (t, J = 8.4 Hz, 1H), 4.68–4.64 (m, 1H), 4.58 (d, J = 4.3 Hz, 2H), 4.20 (t, J = 7.0 Hz, 2H), 4.11 (q, J = 7.1 Hz, 2H), 2.73 (t, J = 7.0 Hz, 2H), 1.63 (d, J = 6.8 Hz, 6H), 1.25 (t, J = 7.2 Hz, 3H); ESI-HRMS: calcd for C₃₀H₂₉ClF₂N₅O₃ [M + H]⁺, 580.1922, found 580.1916, C₃₀H₂₈ClF₂N₅O₃Na, [M + Na]⁺, 602.1741, found 602.1724.
4.2.4.14 3-((3-Chloro-4-fluoro-phenyl)-{2-[(4-cyano-2-fluoro-phenylamino)dk-methyl]-1-cyclopropyl-1H-benzoimidazole-5-carbonyl}-amino)-propionic acid ethyl ester (8n). 8n was obtained in 79% yield as brown solid. mp 116–118 °C; ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.66 (s, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.35 (d, J = 8.7 Hz, 1H), 7.33 (dd, J = 8.5 Hz, J = 1.0 Hz, 1H), 7.26 (dd, J = 8.1 Hz, J = 1.2 Hz, 1H), 7.21 (d, J = 6.1 Hz, 1H), 6.98 (d, J = 6.9 Hz, 2H), 6.82 (t, J = 8.4 Hz, 1H), 4.68 (d, J = 4.9 Hz, 2H), 4.21 (t, J = 7.1 Hz, 2H), 4.12 (q, J = 7.1 Hz, 2H), 3.29–3.25 (m, 1H), 2.74 (t, J = 7.1 Hz, 2H), 1.30 (d, J = 5.4 Hz, 2H), 1.25 (t, J = 7.3 Hz, 3H), 1.11 (q, J = 1.9 Hz, 2H); ESI-HRMS: calcd for C₃₀H₂₇ClF₂N₅O₃ [M + H]⁺, 578.1765, found 578.1751, C₃₀H₂₆ClF₂N₅O₃Na [M + Na]⁺, 600.1585, found 600.1556.

4.2.5 General procedure for the synthesis of 3-[{2-[(4-carbamimidoyl-2-substituted-phenylamino)-methyl]-1-substituted-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid ethyl ester (9a–9n). A mixture of compounds (8a–8n) (2.9 mmol), hydroxylamine hydrochloride (5.75 mmol), triethylamine (TEA) (5.75 mmol) was dissolved in 40 mL ethanol (EtOH) and heated to reflux for 3 h. The solvent was distilled off under reduced pressure. To a acetic acid (HOAC) (35 mL) solution of the residue, was added 5% Pd/C (500 mg), ammonium formate (5.75 mmol) and the mixture was heated to reflux under nitrogen atmosphere for 5 h. The reaction mixture was filtered and the filtrate was evaporated in vacuo, the residue was chromatographed (silica gel, dichloromethane/methanol = 10 : 1) to afford compounds (9a–9n).
4.2.5.1 3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid ethyl ester (9a). 9a was obtained in 42% yield as white solid. mp 182–185 °C; ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) 8.93 (s, 2H), 8.72 (s, 1H), 7.67 (d, J = 8.5 Hz, 2H), 7.62 (dd, J = 6.6 Hz, J = 2.5 Hz, 1H), 7.53 (s, 1H), 7.49 (s, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.28 (t, J = 8.9 Hz, 1H), 7.18 (d, J = 8.7 Hz, 1H), 6.87 (d, J = 8.4 Hz, 2H), 4.65 (d, J = 5.2 Hz, 2H), 4.07 (t, J = 6.8 Hz, 2H), 3.98 (q, J = 7.0 Hz, 2H), 3.78 (s, 3H), 2.62 (t, J = 6.9 Hz, 2H), 1.14 (t, J = 7.1 Hz, 3H); ESI-HRMS: calcd for C₂₈H₂₉ClFN₆O₃ [M + H]⁺, 551.1968, found 551.1947.
4.2.5.2 3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid ethyl ester (9b). 9b was obtained in 32% yield as white solid. mp 98–100 °C; ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) 8.85 (s, 3H), 7.66 (d, J = 7.5 Hz, 2H), 7.63 (d, J = 6.5 Hz, 1H), 7.53 (s, 1H), 7.46 (d, J = 8.0 Hz, 1H), 7.29 (t, J = 9.0 Hz, 1H), 7.21 (d, J = 8.5 Hz, 1H), 7.19–7.14 (m, 1H), 6.87 (d, J = 8.9 Hz, 2H), 4.65 (d, J = 5.5 Hz, 2H), 4.28 (q, J = 7.1 Hz, 2H), 4.08 (t, J = 7.0 Hz, 2H), 3.99 (q, J = 7.0 Hz, 2H), 2.62 (t, J = 7.1 Hz, 2H), 1.25 (t, J = 6.8 Hz, 3H), 1.14 (t, J = 7.1 Hz, 3H); ESI-HRMS: calcd for C₂₉H₃₁ClFN₆O₃ [M + H]⁺, 565.2125, found 565.2129.
4.2.5.3 3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-propyl-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid ethyl ester (9c). 9c was obtained in 29% yield as white solid. mp 80–81 °C; ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) 8.85 (s, 2H), 8.65 (s, 1H), 7.65 (d, J = 8.8 Hz, 2H), 7.62 (dd, J = 6.7 Hz, J = 2.5 Hz, 1H), 7.53 (s, 1H), 7.47 (d, J = 8.5 Hz, 1H), 7.43 (t, J = 5.4 Hz, 1H), 7.29 (t, J = 9.0 Hz, 1H), 7.19 (d, J = 8.0 Hz, 1H), 6.87 (d, J = 8.9 Hz, 2H), 4.65 (d, J = 5.5 Hz, 2H), 4.18 (t, J = 7.4 Hz, 2H), 4.08 (t, J = 7.1 Hz, 2H), 3.99 (q, J = 7.1 Hz, 2H), 2.62 (t, J = 7.0 Hz, 2H), 1.72–1.64 (m, 2H), 1.14 (t, J = 7.1 Hz, 3H), 0.85 (t, J = 7.3 Hz, 3H); ESI-HRMS: calcd for C₃₀H₃₃ClFN₆O₃ [M + H]⁺, 579.2281, found 579.2335.
4.2.5.4 3-[{1-Butyl-2-[(4-carbamimidoyl-phenylamino)-methyl]-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid ethyl ester (9d). 9d was obtained in 34% yield as white solid. mp 150–151 °C; ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) 9.78 (s, 1H), 8.63 (s, 1H), 7.64 (d, J = 8.3 Hz, 2H), 7.53 (s, 1H), 7.45 (d, J = 8.2 Hz, 1H), 7.33 (s, 1H), 7.29 (t, J = 8.6 Hz, 1H), 7.19 (d, J = 8.3 Hz, 2H), 6.86 (d, J = 8.1 Hz, 2H), 4.64 (d, J = 5.4 Hz, 2H), 4.21 (s, 2H), 4.08 (t, J = 6.9 Hz, 2H), 3.99 (q, J = 7.1 Hz, 2H), 2.62 (t, J = 7.0 Hz, 2H), 1.66–1.59 (m, 2H), 1.29–1.24 (m, 2H), 1.14 (t, J = 6.7 Hz, 3H), 0.84 (t, J = 6.9 Hz, 3H); ESI-HRMS: calcd for C₃₁H₃₅ClFN₆O₃ [M + H]⁺, 593.2438, found 593.2482.
4.2.5.5 3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-pentyl-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid ethyl ester (9e). 9e was obtained in 21% yield as white solid. mp 143–145 °C; ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) 8.87 (s, 2H), 8.62 (s, 1H), 7.66 (d, J = 8.7 Hz, 2H), 7.61 (dd, J = 6.5 Hz, J = 2.2 Hz, 1H), 7.53 (s, 1H), 7.44 (d, J = 8.5 Hz, 1H), 7.28 (t, J = 8.9 Hz, 1H), 7.18 (d, J = 7.6 Hz, 2H), 6.86 (d, J = 8.7 Hz, 2H), 4.65 (d, J = 5.2 Hz, 2H), 4.21 (t, J = 7.3 Hz, 2H), 4.08 (t, J = 7.1 Hz, 2H), 3.99 (q, J = 7.1 Hz, 2H), 2.62 (t, J = 7.0 Hz, 2H), 1.67–1.63 (m, 2H), 1.34–1.28 (m, 4H), 1.14 (t, J = 7.1 Hz, 3H), 0.79 (t, J = 6.6 Hz, 3H); ESI-HRMS: calcd for C₃₂H₃₇ClFN₆O₃ [M + H]⁺, 607.2594, found 607.2637.
4.2.5.6 3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-isopropyl-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid ethyl ester (9f). 9f was obtained in 49% yield as white solid. mp 103–105 °C; ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) 8.89 (s, 2H), 8.67 (s, 1H), 7.68 (d, J = 2.5 Hz, 1H), 7.66 (d, J = 9.0 Hz, 2H), 7.64 (d, J = 8.8 Hz, 1H), 7.52 (s, 1H), 7.44 (t, J = 5.3 Hz, 1H), 7.31 (t, J = 8.9 Hz, 1H), 7.20 (d, J = 7.3 Hz, 1H), 6.87 (d, J = 8.9 Hz, 2H), 4.87–4.81 (m, 1H), 4.65 (d, J = 5.3 Hz, 2H), 4.08 (t, J = 7.0 Hz, 2H), 3.99 (q, J = 7.1 Hz, 2H), 2.62 (t, J = 7.0 Hz, 2H), 1.50 (d, J = 6.8 Hz, 6H), 1.14 (t, J = 7.1 Hz, 3H); ESI-HRMS: calcd for C₃₀H₃₃ClFN₆O₃ [M + H]⁺, 579.2281, found 579.2323.
4.2.5.7 3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-cyclopropyl-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid ethyl ester (9g). 9g was obtained in 50% yield as white solid. mp 219–221 °C; ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) 8.90 (s, 2H), 8.65 (s, 1H), 7.66 (d, J = 8.9 Hz, 2H), 7.49 (s, 1H), 7.47 (d, J = 8.6 Hz, 1H), 7.35 (t, J = 5.4 Hz, 1H), 7.29 (t, J = 7.1 Hz, 1H), 7.24 (dd, J = 6.7 Hz, J = 1.0 Hz, 1H), 7.19–7.14 (m, 1H), 6.85 (d, J = 8.8 Hz, 2H), 4.69 (d, J = 5.3 Hz, 2H), 4.26–4.19 (m, 1H), 4.07 (t, J = 6.9 Hz, 2H), 3.98 (q, J = 7.1 Hz, 2H), 2.61 (t, J = 7.0 Hz, 2H), 1.19 (d, J = 6.6 Hz, 2H), 1.14 (t, J = 7.1 Hz, 3H), 1.07–1.03 (m, 2H); ESI-HRMS: calcd for C₃₀H₃₁ClFN₆O₃ [M + H]⁺, 577.2125, found 577.2185.
4.2.5.8 3-[{2-[(4-Carbamimidoyl-2-fluoro-phenylamino)-methyl]-1-methyl-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid ethyl ester (9h). 9h was obtained in 58% yield as white solid. mp 204–206 °C; ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) 8.96 (d, J = 55.9 Hz, 3H), 7.70 (d, J = 12.8 Hz, 1H), 7.61 (dd, J = 6.6 Hz, J = 2.5 Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.54 (s, 1H), 7.41 (d, J = 8.4 Hz, 1H), 7.24 (t, J = 9.0 Hz, 1H), 7.17 (d, J = 7.3 Hz, 2H), 7.06 (t, J = 8.5 Hz, 1H), 4.72 (d, J = 4.7 Hz, 2H), 4.07 (t, J = 6.5 Hz, 2H), 3.99 (q, J = 7.1 Hz, 2H), 3.79 (s, 3H), 2.61 (t, J = 6.4 Hz, 2H), 1.14 (t, J = 7.0 Hz, 3H); ESI-HRMS: calcd for C₂₈H₂₈ClF₂N₆O₃ [M + H]⁺, 569.1874, found 569.1920.
4.2.5.9 3-[{2-[(4-Carbamimidoyl-2-fluoro-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid ethyl ester (9i). 9i was obtained in 43% yield as white solid. mp 95–97 °C; ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) 9.01 (s, 3H), 7.69 (d, J = 7.5 Hz, 1H), 7.62 (dd, J = 6.5 Hz, J = 2.5 Hz, 1H), 7.57 (d, J = 8.6 Hz, 1H), 7.55 (s, 1H), 7.45 (d, J = 8.5 Hz, 1H), 7.28 (d, J = 8.5 Hz, 1H), 7.19 (d, J = 8.0 Hz, 2H), 7.07 (t, J = 8.5 Hz, 1H), 4.72 (d, J = 4.5 Hz, 2H), 4.30 (q, J = 6.5 Hz, 2H), 4.07 (t, J = 6.5 Hz, 2H), 3.99 (q, J = 7.0 Hz, 2H), 2.61 (t, J = 7.0 Hz, 2H), 1.24 (t, J = 6.0 Hz, 3H), 1.14 (t, J = 7.0 Hz, 3H); ESI-HRMS: calcd for C₂₉H₃₀ClF₂N₆O₃ [M + H]⁺, 583.2030, found 583.2081.
4.2.5.10 3-[{2-[(4-Carbamimidoyl-2-fluoro-phenylamino)-methyl]-1-propyl-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid ethyl ester (9j). 9j was obtained in 41% yield as white solid. mp 203–205 °C; ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) 9.06 (s, 2H), 8.89 (s, 1H), 7.71 (d, J = 13.1 Hz, 1H), 7.62 (dd, J = 6.6 Hz, J = 2.3 Hz, 1H), 7.59 (d, J = 8.6 Hz, 1H), 7.54 (s, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.26 (d, J = 5.6 Hz, 1H), 7.17 (d, J = 7.9 Hz, 2H), 7.07 (t, J = 8.7 Hz, 1H), 4.72 (d, J = 5.4 Hz, 2H), 4.22 (t, J = 7.1 Hz, 2H), 4.07 (t, J = 6.9 Hz, 2H), 3.99 (q, J = 7.1 Hz, 2H), 2.61 (t, J = 7.0 Hz, 2H), 1.70–1.63 (m, 2H), 1.14 (t, J = 7.1 Hz, 3H), 0.86 (t, J = 7.3 Hz, 3H); ESI-HRMS: calcd for C₃₀H₃₂ClF₂N₆O₃ [M + H]⁺, 597.2187, found 597.2228.
4.2.5.11 3-[{1-Butyl-2-[(4-carbamimidoyl-2-fluoro-phenylamino)-methyl]-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid ethyl ester (9k). 9k was obtained in 36% yield as white solid. mp 163–165 °C; ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) 8.44 (s, 1H), 7.70 (dd, J = 13.0 Hz, J = 1.8 Hz, 1H), 7.62 (dd, J = 6.7 Hz, J = 2.5 Hz, 1H), 7.57 (dd, J = 8.8 Hz, J = 2.5 Hz, 1H), 7.54 (s, 1H), 7.44 (d, J = 8.4 Hz, 1H), 7.29 (s, 1H), 7.17 (d, J = 8.0 Hz, 2H), 7.04 (t, J = 8.8 Hz, 1H), 4.71 (d, J = 5.5 Hz, 2H), 4.24 (t, J = 7.1 Hz, 2H), 4.07 (t, J = 7.0 Hz, 2H), 3.98 (q, J = 7.1 Hz, 2H), 2.61 (t, J = 7.0 Hz, 2H), 1.62–1.56 (m, 2H), 1.31–1.27 (m, 2H), 1.13 (t, J = 7.1 Hz, 3H), 0.84 (t, J = 7.3 Hz, 3H); ESI-HRMS: calcd for C₃₁H₃₄ClF₂N₆O₃ [M + H]⁺, 611.2344, found 611.2389.
4.2.5.12 3-[{2-[(4-Carbamimidoyl-2-fluoro-phenylamino)-methyl]-1-pentyl-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid ethyl ester (9l). 9l was obtained in 22% yield as white solid. mp 153–155 °C; ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) 9.01 (s, 3H), 7.70 (dd, J = 12.0 Hz, J = 7.1 Hz, 1H), 7.60 (dd, J = 6.6 Hz, J = 2.2 Hz, 1H), 7.55 (s, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.28 (t, J = 8.8 Hz, 2H), 7.17 (d, J = 8.3 Hz, 2H), 7.07 (t, J = 8.3 Hz, 1H), 4.72 (d, J = 4.9 Hz, 2H), 4.24 (t, J = 6.5 Hz, 2H), 4.07 (t, J = 6.8 Hz, 2H), 3.99 (q, J = 7.1 Hz, 2H), 2.62 (t, J = 6.9 Hz, 2H), 1.67–1.58 (m, 2H), 1.24 (d, J = 7.0 Hz, 4H), 1.14 (t, J = 7.1 Hz, 3H), 0.79 (t, J = 6.6 Hz, 3H); ESI-HRMS: calcd for C₃₂H₃₆ClF₂N₆O₃ [M + H]⁺, 625.2500, found 625.2537.
4.2.5.13 3-[{2-[(4-Carbamimidoyl-2-fluoro-phenylamino)-methyl]-1-isopropyl-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid ethyl ester (9m). 9m was obtained in 51% yield as white solid. mp 116–118 °C; ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) 9.00 (s, 2H), 8.79 (s, 1H), 7.67 (dd, J = 11.1 Hz, J = 7.3 Hz, 2H), 7.62 (d, J = 8.5 Hz, 1H), 7.55 (d, J = 9.5 Hz, 1H), 7.53 (s, 1H), 7.30 (t, J = 8.9 Hz, 1H), 7.24 (s, 1H), 7.17 (d, J = 7.8 Hz, 1H), 7.07 (t, J = 8.8 Hz, 1H), 4.96–4.91 (m, 1H), 4.72 (d, J = 5.3 Hz, 2H), 4.07 (t, J = 6.9 Hz, 2H), 3.99 (q, J = 7.1 Hz, 2H), 2.61 (t, J = 7.0 Hz, 2H), 1.49 (d, J = 6.8 Hz, 6H), 1.14 (t, J = 7.1 Hz, 3H); ESI-HRMS: calcd for C₃₀H₃₂ClF₂N₆O₃ [M + H]⁺, 597.2187, found 597.2229.
4.2.5.14 3-[{2-[(4-Carbamimidoyl-2-fluoro-phenylamino)-methyl]-1-cyclopropyl-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid ethyl ester (9n). 9n was obtained in 39% yield as white solid. mp 220–221 °C; ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) 9.02 (s, 2H), 8.77 (s, 1H), 7.70 (d, J = 12.9 Hz, 1H), 7.62 (dd, J = 6.5 Hz, J = 2.0 Hz, 1H), 7.57 (d, J = 8.6 Hz, 1H), 7.49 (s, 1H), 7.47 (d, J = 8.5 Hz, 1H), 7.29 (t, J = 8.9 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.19–7.14 (m, 1H), 6.98 (t, J = 8.7 Hz, 1H), 4.78 (d, J = 5.4 Hz, 2H), 4.06 (t, J = 6.9 Hz, 2H), 3.99 (q, J = 7.0 Hz, 2H), 3.64 (s, 1H), 2.61 (t, J = 7.0 Hz, 2H), 1.19 (d, J = 5.8 Hz, 2H), 1.14 (t, J = 7.1 Hz, 3H), 1.07 (s, 2H); ESI-HRMS: calcd for C₃₀H₃₀ClF₂N₆O₃ [M + H]^+h, 595.2030, found 595.2064.

4.2.6 General procedure for the synthesis of 3-[{2-[(4-carbamimidoyl-2-substituted phenylamino)-methyl]-1-substituted-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluorophenyl)amino]-propionic acid (10a–10n). Compounds 9a–9n (0.2 mmol) were dissolved in 1 mL ethanol (EtOH), was added a solution of sodium hydroxide (0.6 mmol) and kept at room temperature for 2 h. The mixture was neutralized with acetic acid to pH 6–7. The solid formed was filtered, washed with water and dried to afford the zwitterionic title compounds10a–10n.
4.2.6.1 3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid (10a). 10a was obtained in 51% yield as white solid. mp 227–229 °C; ¹H NMR (500 MHz, DMSO-d₆ + ²HCl) δ (ppm) 8.98 (s, 1H), 8.73 (s, 1H), 7.69 (d, J = 8.5 Hz, 2H), 7.67 (dd, J = 6.8 Hz, J = 2.1 Hz, 1H), 7.62 (s, 2H), 7.31 (d, J = 10.7 Hz, 1H), 7.28 (d, J = 8.8 Hz, 1H), 7.22 (s, 1H), 6.90 (d, J = 8.6 Hz, 2H), 4.80 (s, 2H), 4.04 (t, J = 6.9 Hz, 2H), 3.86 (s, 3H), 2.57 (t, J = 7.1 Hz, 2H); ¹³C NMR (125 MHz, DMSO-d₆ + ²HCl) δ (ppm) 172.57, 169.15, 164.22, 164.14, 164.06, 163.50, 160.50, 153.18, 152.93, 144.48, 135.57, 131.79, 130.62, 130.53, 129.89, 124.87, 115.32, 115.09, 113.99, 113.78, 112.89, 112.08, 111.01, 46.08, 31.45, 30.94; ESI-HRMS calcd for C₂₆H₂₅ClFN₆O₃ [M + H]⁺, 523.1655, found 523.1645. Anal. calcd for C₂₆H₂₄ClFN₆O₃: C, 59.71; H, 4.63; N, 16.07; O, 9.18%. Found: C, 59.74; H, 4.62; N, 16.10; O, 9.16%.
4.2.6.2 3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid (10b). 10b was obtained in 51% yield as white solid. mp 229–231 °C; ¹H NMR (500 MHz, D₂O + ²HCl) δ (ppm) 7.53 (d, J = 8.4 Hz, 1H), 7.45 (s, 1H), 7.41 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 7.9 Hz, 1H), 7.18 (d, J = 4.2 Hz, 1H), 6.93 (dd, J = 6.5 Hz, J = 1.8 Hz, 1H), 6.78 (t, J = 8.5 Hz, 1H), 6.58 (d, J = 8.3 Hz, 2H), 4.88 (s, 2H), 4.29 (q, J = 7.1 Hz, 2H), 4.01 (t, J = 6.2 Hz, 2H), 2.51 (t, J = 5.8 Hz, 2H), 1.27 (t, J = 6.7 Hz, 3H); ¹³C NMR (125 MHz, D₂O + ²HCl) δ (ppm) 172.35, 169.13, 165.22, 165.14, 161.69, 159.82, 151.78, 151.45, 138.27, 132.81, 132.06, 129.56, 125.95, 124.95, 117.22, 116.05, 115.83, 113.64, 113.54, 112.39, 46.34, 39.64, 38.55, 31.82, 13.36; ESI-HRMS calcd for C₂₇H₂₇ClFN₆O₃ [M + H]⁺, 537.1812, found 537.1794. Anal. calcd for C₂₇H₂₆ClFN₆O₃: C, 60.39; H, 4.88; N, 15.65; O, 8.94%. Found: C, 60.43; H, 4.86; N, 15.63; O, 8.96%.
4.2.6.3 3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-propyl-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid (10c). 10c was obtained in 35% yield as white solid. mp 220–222 °C; ¹H NMR (500 MHz, DMSO-d₆ + ²HCL) δ (ppm) 9.05 (s, 1H), 8.79 (s, 1H), 7.64 (d, J = 8.4 Hz, 2H), 7.54 (s, 1H), 7.48 (d, J = 6.2 Hz, 1H), 7.42 (d, J = 7.9 Hz, 1H), 7.26 (dd, J = 8.6 Hz, J = 3.8 Hz, 1H), 7.19 (t, J = 9.0 Hz, 1H), 7.08 (t, J = 8.5 Hz, 1H), 6.87 (d, J = 6.7 Hz, 2H), 4.64 (s, 2H), 4.15 (t, J = 7.4 Hz, 2H), 4.02 (t, J = 7.2 Hz, 2H), 2.61 (t, J = 6.5 Hz, 2H), 1.71–1.64 (m, 2H), 0.85 (t, J = 7.3 Hz, 3H); ¹³C NMR (125 MHz, DMSO-d₆ + ²HCL) δ (ppm) 173.21, 169.94, 161.31, 158.88, 154.17, 152.90, 152.74, 140.67, 136.04, 130.05, 129.57, 129.50, 129.25, 122.82, 119.39, 116.84, 115.73, 113.38, 111.63, 109.68, 46.62, 44.73, 32.74, 32.66, 22.61, 10.95; ESI-HRMS: calcd for C₂₈H₂₉ClFN₆O₃ [M + H]⁺, 551.1968, found 551.1959. Anal. calcd for C₂₈H₂₈ClFN₆O₃: C, 61.03; H, 5.12; N, 15.25; O, 8.71%. Found: C, 61.12; H, 5.10; N, 15.29; O, 8.74%.
4.2.6.4 3-[{1-Butyl-2-[(4-carbamimidoyl-phenylamino)-methyl]-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid (10d). 10d was obtained in 37% yield as white solid. mp 210–211 °C; ¹H NMR (500 MHz, D₂O + ²HCL) δ (ppm) 7.56 (d, J = 8.6 Hz, 2H), 7.53 (s, 1H), 7.46 (d, J = 5.5 Hz, 1H), 7.41 (d, J = 6.5 Hz, 1H), 7.23–7.15 (m, 3H), 6.80 (d, J = 8.6 Hz, 2H), 5.03 (s, 2H), 4.28 (t, J = 6.7 Hz, 2H), 4.06 (t, J = 6.4 Hz, 2H), 2.55 (t, J = 6.3 Hz, 2H), 1.61–1.53 (m, 2H), 1.23–1.16 (m, 2H), 0.77 (t, J = 7.3 Hz, 3H); ¹³C NMR (125 MHz, DMSO-d₆ + ²HCL) δ (ppm) 172.52, 168.90, 164.23, 164.16, 164.08, 156.86, 154.40, 152.89, 152.43, 134.09, 131.94, 130.15, 129.73, 129.28, 124.56, 119.71, 119.52, 117.21, 116.99, 114.06, 111.96, 111.49, 46.15, 44.22, 32.02, 30.85, 28.94, 19.33, 13.53; ESI-HRMS calcd for C₂₉H₃₁ClFN₆O₃ [M + H]⁺, 565.2125, found 565.2114. Anal. calcd for C₂₉H₃₀ClFN₆O₃: C, 61.64; H, 5.35; N, 14.87; O, 8.49%. Found: C, 61.73; H, 5.34; N, 14.90; O, 8.47%.
4.2.6.5 3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-pentyl-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionicacid (10e). 10e was obtained in 31% yield as white solid. mp 214–215 °C; ¹H NMR (500 MHz, DMSO-d₆ + ²HCL) δ (ppm) 8.94 (s, 1H), 8.70 (s, 1H), 7.68 (d, J = 8.4 Hz, 2H), 7.65 (d, J = 5.6 Hz, 1H), 7.61 (s, 2H), 7.28 (t, J = 8.0 Hz, 2H), 7.22 (d, J = 3.0 Hz, 1H), 6.88 (d, J = 8.5 Hz, 2H), 4.77 (s, 2H), 4.28 (t, J = 6.4 Hz, 2H), 4.03 (t, J = 6.7 Hz, 2H), 2.57 (t, J = 6.9 Hz, 2H), 1.68 (s, 2H), 1.25–1.23 (m, 4H), 0.80 (t, J = 6.6 Hz, 3H). ¹³C NMR (125 MHz, DMSO-d₆ + ²HCL) δ (ppm) 172.58, 168.10, 164.20, 159.90, 152.81, 140.46, 131.93, 130.01, 129.66, 129.18, 128.27, 127.53, 124.34, 123.03, 113.81, 113.57, 112.89, 111.68, 111.65, 111.60, 46.20, 43.49, 34.10, 32.10, 28.91, 28.24, 21.75, 13.79; ESI-HRMS calcd for C₃₀H₃₃ClFN₆O₃ [M + H]⁺, 579.2281, found 579.2285. Anal. calcd for C₃₀H₃₂ClFN₆O₃: C, 62.22; H, 5.57; N, 14.51; O, 8.29%. Found: C, 62.30; H, 5.59; N, 14.53; O, 8.27%.
4.2.6.6 3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-isopropyl-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid (10f). 10f was obtained in 29% yield as white solid. mp 230–232 °C; ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) 11.50–11.24 (brs, 1H), 8.39 (s, 2H), 7.71 (dd, J = 6.6 Hz, J = 2.3 Hz, 1H), 7.63 (d, J = 8.3 Hz, 1H), 7.59 (d, J = 8.4 Hz, 2H), 7.54 (s, 1H), 7.30 (d, J = 8.1 Hz, 1H), 7.26 (d, J = 8.9 Hz, 1H), 7.22 (d, J = 8.8 Hz, 1H), 6.85 (d, J = 8.5 Hz, 2H), 4.86–4.81 (m, 1H), 4.64 (s, 2H), 3.95 (s, 2H), 2.26 (s, 2H), 1.50 (d, J = 6.6 Hz, 6H); ¹³C NMR (125 MHz, DMSO-d₆) δ (ppm) 172.59, 172.09, 169.44, 164.27, 164.20, 156.78, 154.33, 152.70, 152.43, 140.23, 133.61, 133.44, 129.93, 129.68, 129.16, 119.74, 119.55, 117.21, 116.99, 112.26, 111.77, 48.37, 46.37, 32.06, 21.01, 20.67; ESI-HRMS: calcd for C₂₈H₂₉ClFN₆O₃ [M + H]⁺, 551.1968, found 551.1955. Anal. calcd for C₂₈H₂₈ClFN₆O₃: C, 61.03; H, 5.12; N, 15.25; O, 8.71%. Found: C, 61.10; H, 5.11; N, 15.28; O, 8.73%.
4.2.6.7 3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-cyclopropyl-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid (10g). 10g was obtained in 36% yield as white solid. mp 232–234 °C; ¹H NMR (500 MHz, D₂O + ²HCl) δ (ppm) 7.58 (d, J = 8.8 Hz, 1H), 7.38 (d, J = 7.8 Hz, 3H), 7.25 (d, J = 8.4 Hz, 1H), 7.16 (dd, J = 4.3 Hz, J = 0.9 Hz, 1H), 6.91–6.86 (m, 1H), 6.77 (t, J = 8.8 Hz, 1H), 6.55 (d, J = 8.1 Hz, 2H), 4.91 (s, 2H), 3.98 (t, J = 6.3 Hz, 2H), 3.43–3.38 (m, 1H), 2.47 (t, J = 5.5 Hz, 2H), 1.18 (d, J = 6.0 Hz, 2H), 1.01 (s, 2H); ¹³C NMR (125 MHz, D₂O + ²HCl) δ (ppm) 176.40, 175.32, 170.95, 169.49, 168.61, 164.95, 155.53, 151.33, 134.05, 132.69, 130.00, 129.52, 128.70, 125.91, 120.64, 115.61, 114.15, 113.37, 112.38, 45.81, 39.45, 31.79, 26.01, 20.27, 6.16; ESI-HRMS: calcd for C₂₈H₂₇ClFN₆O₃ [M + H]⁺, 549.1812, found 549.1823. Anal. calcd for C₂₈H₂₆ClFN₆O₃: C, 61.26; H, 4.77; N, 15.31; O, 8.74%. Found: C, 61.31; H, 4.76; N, 15.35; O, 8.76%.
4.2.6.8 3-[{2-[(4-Carbamimidoyl-2-fluoro-phenylamino)-methyl]-1-methyl-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid (10h). 10h was obtained in 20% yield as white solid. mp 225–227 °C; ¹H NMR (500 MHz, DMSO-d₆ + ²HCL) δ (ppm) 9.16 (s, 1H), 8.91 (s, 1H), 7.84 (s, 1H), 7.78 (dd, J = 12.2 Hz, J = 3.8 Hz, 1H), 7.71 (d, J = 7.1 Hz, 2H), 7.60 (d, J = 6.0 Hz, 1H), 7.47 (d, J = 7.8 Hz, 1H), 7.31 (d, J = 8.2 Hz, 1H), 7.28–7.25 (m, 1H), 7.06 (t, J = 9.7 Hz, 1H), 5.03 (s, 2H), 4.04 (t, J = 6.9 Hz, 2H), 3.97 (s, 3H), 2.58 (t, J = 7.0 Hz, 2H); ¹³C NMR (125 MHz, DMSO-d₆ + ²HCl) δ (ppm) 172.54, 169.29, 164.24, 154.33, 153.40, 152.68, 140.21, 135.39, 134.91, 130.11, 129.66, 129.30, 129.21, 123.91, 119.65, 119.46, 117.17, 116.95, 111.92, 110.61, 46.15, 32.05, 30.54, 21.02; ESI-HRMS: calcd for C₂₆H₂₄ClF₂N₆O₃ [M + H]⁺, 541.1561, found 541.1549. Anal. calcd for C₂₆H₂₃ClF₂N₆O₃: C, 57.73; H, 4.29; N, 15.54; O, 8.87%. Found: C, 57.75; H, 4.28; N, 15.56; O, 8.89%.
4.2.6.9 3-[{2-[(4-Carbamimidoyl-2-fluoro-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid (10i). 10i was obtained in 24% yield as white solid. mp 227–229 °C; ¹H NMR (500 MHz, DMSO-d₆ + ²HCL) δ (ppm) 9.13 (s, 1H), 8.91 (s, 1H), 7.75 (d, J = 12.7 Hz, 1H), 7.72 (s, 1H), 7.69 (dd, J = 6.8 Hz, J = 2.3 Hz, 1H), 7.66 (s, 1H), 7.61 (d, J = 8.5 Hz, 1H), 7.37 (d, J = 8.3 Hz, 1H), 7.30 (t, J = 8.9 Hz, 1H), 7.26–7.22 (m, 1H), 7.09 (t, J = 8.8 Hz, 1H), 4.92 (s, 2H), 4.42 (q, J = 7.1 Hz, 2H), 4.04 (t, J = 7.2 Hz, 2H), 2.57 (t, J = 7.2 Hz, 2H), 1.32 (t, J = 7.2 Hz, 3H); ¹³C NMR (125 MHz, DMSO-d₆ + ²HCL) δ (ppm) 172.53, 168.99, 163.51, 156.86, 154.40, 152.36, 150.83, 133.86, 130.14, 129.34, 129.27, 126.03, 124.45, 119.71, 119.53, 117.21, 117.00, 114.52, 111.61, 111.21, 99.49, 46.18, 32.03, 14.32; ESI-HRMS calcd for C₂₇H₂₆ClF₂N₆O₃ [M + H]⁺, 555.1717, found 555.1685. Anal. calcd for C₂₇H₂₅ClF₂N₆O₃: C, 58.43; H, 4.54; N, 15.14; O, 8.65%. Found: C, 58.49; H, 4.53; N, 15.10; O, 8.62%.
4.2.6.10 3-[{2-[(4-Carbamimidoyl-2-fluoro-phenylamino)-methyl]-1-propyl-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid (10j). 10j was obtained in 42% yield as white solid. mp 232–234 °C; ¹H NMR (500 MHz, DMSO-d₆ + ²HCL) δ (ppm) 9.10 (s, 1H), 8.86 (s, 1H), 7.74 (d, J = 12.8 Hz, 1H), 7.71 (s, 1H), 7.68 (dd, J = 6.6 Hz, J = 2.3 Hz, 1H), 7.65 (s, 1H), 7.60 (d, J = 8.7 Hz, 1H), 7.35 (d, J = 8.5 Hz, 1H), 7.29 (t, J = 8.9 Hz, 1H), 7.26–7.21 (m, 1H), 7.07 (t, J = 8.8 Hz, 1H), 4.91 (s, 2H), 4.32 (t, J = 7.4 Hz, 2H), 4.03 (t, J = 7.0 Hz, 2H), 2.57 (t, J = 7.2 Hz, 2H), 1.77–1.70 (m, 2H), 0.90 (t, J = 7.3 Hz), 3H; ¹³C NMR (125 MHz, DMSO-d₆ + ²HCL) δ (ppm) 172.49, 171.99, 168.50, 163.40, 156.95, 154.48, 152.59, 133.34, 133.08, 130.24, 129.39, 129.31, 126.05, 125.23, 119.75, 119.57, 117.25, 117.03, 115.31, 114.43, 112.21, 111.66, 46.13, 46.06, 31.99, 22.05, 21.0, 10.74; ESI-HRMS calcd for C₂₈H₂₈ClF₂N₆O₃ [M + H]⁺, 569.1874, found 569.1859. Anal. calcd for C₂₈H₂₇ClF₂N₆O₃: C, 59.10; H, 4.78; N, 14.77; O, 8.44%. Found: C, 59.14; H, 4.79; N, 14.74; O, 8.42%.
4.2.6.11 3-[{1-Butyl-2-[(4-carbamimidoyl-2-fluoro-phenylamino)-methyl]-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid (10k). 10k was obtained in 17% yield as white solid. mp 221–223 °C; ¹H NMR (500 MHz, D₂O + ²HCL) δ (ppm) 7.57 (d, J = 8.7 Hz, 1H), 7.49 (s, 1H), 7.40 (dd, J = 12.5 Hz, J = 0.9 Hz, 1H), 7.34 (d, J = 8.5 Hz, 1H), 7.25 (d, J = 8.4 Hz, 1H), 7.20 (d, J = 6.4 Hz, 1H), 7.00–6.95 (m, 1H), 6.83 (t, J = 8.7 Hz, 1H), 6.49 (t, J = 7.1 Hz, 1H), 4.96 (s, 2H), 4.28 (t, J = 7.1 Hz, 2H), 4.06 (t, J = 6.4 Hz, 2H), 2.55 (t, J = 6.4 Hz, 2H), 1.73–1.64 (m, 2H), 1.23–1.15 (m, 2H), 0.77 (t, J = 7.2 Hz, 3H); ¹³C NMR (125 MHz, DMSO-d₆ + ²HCL) δ (ppm) 172.52, 166.15, 163.49, 156.87, 154.41, 152.52, 140.67, 135.40, 134.13, 134.01, 132.10, 131.90, 130.17, 129.31, 129.24, 126.04, 119.70, 119.52, 117.09, 116.98, 114.31, 111.60, 48.11, 46.15, 44.22, 32.02, 30.88, 19.30, 13.53; ESI-HRMS calcd for C₂₉H₃₀ClF₂N₆O₃ [M + H]⁺, 583.2030, found 583.2009. Anal. calcd for C₂₉H₂₉ClF₂N₆O₃: C, 59.74; H, 5.01; N, 14.41; O, 8.23%. Found: C, 59.79; H, 5.02; N, 14.43; O, 8.21%.
4.2.6.12 3-[{2-[(4-Carbamimidoyl-2-fluoro-phenylamino)-methyl]-1-pentyl-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid (10l). 10l was obtained in 27% yield as white solid. mp 214–216 °C; ¹H NMR (500 MHz, DMSO-d₆ + ²HCL) δ (ppm) 9.14 (s, 1H), 8.93 (s, 1H), 7.76 (d, J = 12.7 Hz, 1H), 7.68 (d, J = 9.8 Hz, 1H), 7.62 (d, J = 6.0 Hz, 2H), 7.34 (d, J = 8.4 Hz, 1H), 7.30 (dd, J = 8.3 Hz, J = 5.2 Hz, 2H), 7.09 (dd, J = 16.6 Hz, J = 8.4 Hz, 2H), 4.92 (s, 2H), 4.35 (t, J = 6.4 Hz, 2H), 4.04 (t, J = 7.1 Hz, 2H), 2.56 (t, J = 7.3 Hz, 2H), 1.69 (s, 2H), 1.27–1.23 (m, 4H), 0.81 (t, J = 6.4 Hz, 3H); ¹³C NMR (125 MHz, DMSO-d₆ + ²HCL) δ (ppm) 172.62, 169.95, 163.49, 161.35, 158.93, 152.41, 150.66, 148.27, 141.20, 139.56, 135.88, 130.09, 130.00, 129.66, 125.97, 122.97, 119.31, 115.98, 115.75, 114.23, 114.02, 111.41, 109.75, 46.27, 43.33, 32.09, 29.00, 28.20, 21.76, 13.73; ESI-HRMS calcd for C₃₀H₃₂ClF₂N₆O₃ [M + H]⁺, 597.2187, found 597.2167. Anal. calcd for C₃₀H₃₁ClF₂N₆O₃: C, 60.35; H, 5.23; N, 14.08; O, 8.04%. Found: C, 60.28; H, 5.21; N, 14.10; O, 8.06%.
4.2.6.13 3-[{2-[(4-Carbamimidoyl-2-fluoro-phenylamino)-methyl]-1-isopropyl-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid (10m). 10m was obtained in 31% yield as white solid. mp 237–238 °C; ¹H NMR (500 MHz, DMSO + ²HCl) δ (ppm) 7.66 (dd, J = 12.3 Hz, J = 1.5 Hz, 1H), 7.61 (d, J = 8.7 Hz, 1H), 7.56 (s, 1H), 7.52 (d, J = 8.3 Hz, 1H), 7.27 (t, J = 8.9 Hz, 1H), 7.19 (d, J = 7.8 Hz, 2H), 7.13 (dd, J = 11.8 Hz, J = 5.2 Hz, 1H), 7.06 (t, J = 8.6 Hz, 1H), 4.99–4.91 (m, 1H), 4.70 (s, 2H), 3.83 (t, J = 7.1 Hz, 2H), 2.62 (t, J = 7.2 Hz, 2H), 1.49 (d, J = 6.6 Hz, 6H); ¹³C NMR (125 MHz, DMSO-d₆ + ²HCL) δ (ppm) 172.66, 172.26, 169.89, 163.64, 162.85, 161.21, 156.72, 154.26, 152.14, 141.02, 140.31, 134.18, 133.21, 129.90, 129.11, 129.03, 125.95, 122.70, 119.57, 116.94 (s), 111.85, 99.49, 94.40, 47.75, 46.36, 32.08, 20.99, 20.68; ESI-HRMS calcd for C₂₈H₂₈ClF₂N₆O₃ [M + H]⁺, 569.1874, found 569.1863. Anal. calcd for C₂₈H₂₇ClF₂N₆O₃: C, 59.10; H, 4.78; N, 14.77; O, 8.44%. Found: C, 59.16; H, 4.77; N, 14.80; O, 8.41%.
4.2.6.14 3-[{2-[(4-Carbamimidoyl-2-fluoro-phenylamino)-methyl]-1-cyclopropyl-1H-benzoimidazole-5-carbonyl}-(3-chloro-4-fluoro-phenyl)-amino]-propionic acid (10n). 10n was obtained in 45% yield as white solid. mp 258–259 °C; ¹H NMR (500 MHz, DMSO-d₆ + ²HCl) δ (ppm) 7.84 (d, J = 11.8 Hz, 2H), 7.72 (d, J = 4.9 Hz, 1H), 7.68 (s, 1H), 7.65 (d, J = 8.5 Hz, 1H), 7.54–7.49 (m, 1H), 7.31 (d, J = 9.1 Hz, 1H), 7.27 (dd, J = 10.6 Hz, J = 2.6 Hz, 1H), 7.04 (t, J = 8.8 Hz, 1H), 5.07 (s, 2H), 4.01 (s, 2H), 3.64 (s, 1H), 2.55 (s, 2H), 1.22 (d, J = 17.1 Hz, 4H); ¹³C NMR (125 MHz, DMSO-d₆ + ²HCL) δ (ppm) 172.52, 168.31, 163.41, 157.02, 155.46, 154.56, 151.09, 148.69, 140.57, 133.64, 130.15, 129.34, 128.97, 125.99, 119.89, 117.34, 117.13, 114.58, 114.39, 113.05, 111.91, 55.97, 46.23, 31.92, 26.19, 20.97, 18.24, 6.20; ESI-HRMS calcd for C₂₈H₂₆ClF₂N₆O₃ [M + H]⁺, 567.1717, found 567.1732. Anal. calcd for C₂₈H₂₅ClF₂N₆O₃: C, 59.31; H, 4.44; N, 14.82; O, 8.47%. Found: C, 59.39; H, 4.43; N, 14.85; O, 8.45%.

4.3 Anticoagulant assay

The in vitro anticoagulant activities of the target compounds 10a–10n were evaluated against thrombin. Dabigatran was served as the reference. Human thrombin (national standard) (5.4 μg mL⁻¹) was pre-incubated for 10 min at 37 °C with the test compounds. The specificity substrate, Ac-FVR-AMC (5 μM) was added to the preincubation mixture, which was measured in a microtiter plate detector Envision (PerkinElmer) at room temperature. By the changing of dynamic detection 10 minutes RFU (relative fluorescence unit), got the initial velocity of enzymatic reaction. Then the inhibition rate and concentration that induced a 50% inhibition of thrombin (IC₅₀) were calculated.

4.4 Docking simulations

The molecular docking was performed using Surflex-Dock module in SYBYL-X 2.0. package (Tripos Inc., St. Louis, USA) running on windows 7 workstation. Each molecule was optimized using Tripos force field and Gasteiger–Huckel charges. Structural energy minimization was performed using Powell gradient algorithm with a convergence criterion of 0.005 kcal (mol⁻¹ Å) and a maximum of 10 000 iterations. For the protein preparation, all water existing in the protein, as well as the ligand were removed from the crystal structure (PDB entry code: 1KTS).¹⁸ The side-chain was repaired, the polar hydrogen atoms were added and Gasteiger–Huckel charges were assigned to protein atoms.

Acknowledgements

This work was supported by the Science and Technology Commission of Shanghai Municipality (no. 13142201001, 13DZ1930402, 13DZ1930403).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra01828e

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