Design,synthesis,and antitumor activity of novel benzoheterocycle derivatives as inhibitors of vascular endothelial growth factor receptor-2 tyrosine kinase

Abstract

The vascular endothelial growth factor receptor-2 signaling pathway promotes the formation of new blood vessels, and vascular endothelial growth factor receptor-2 tyrosine kinase exists in both active and inactive conformations. Novel indole–benzimidazole and indole–benzothiazole derivatives joined by different linkers are designed and synthesized as inhibitors of vascular endothelial growth factor receptor-2 tyrosine kinase. All the synthesized compounds were evaluated for their cytotoxicity against four human cancer cell lines (HeLa, HT29, A549, and MDA-MB-435) and human umbilical vein endothelial cell. Meanwhile, the inhibitory activities against vascular endothelial growth factor receptor-2 are estimated in vitro and the binding interactions with dual conformations of vascular endothelial growth factor receptor-2 tyrosine kinase are evaluated by molecular docking. Compounds 5a–c and 14 show inhibitory activity against vascular endothelial growth factor receptor-2 tyrosine kinase and promising cytotoxicity, specifically with IC₅₀ values ranging between 0.1 and 1 μM, which imply broad-spectrum antitumor activity. These results provide a deep insight into potential structural modifications for developing potent vascular endothelial growth factor receptor-2 tyrosine kinase inhibitors.

Introduction

Cancer, the uncontrolled, rapid, and pathological proliferation of abnormal cells, is one of the most formidable afflictions in the world, ¹ which causes about 550,000 deaths per year and represents the second leading cause of death in the world. ² Current cancer chemotherapy is severely limited by side effects and/or drug resistance, and thus it remains essential to develop new and safer anticancer drugs. At present, the development of tumor angiogenesis inhibitors has become an important field in anticancer (drug) research due to the low toxicity to the human body, the broad-spectrum of activity against tumors, and tolerance to the development of resistance.

Vascular endothelial growth factor (VEGF) shows high specificity and plays an important regulatory role in angiogenesis and vascular migration. It is frequently overexpressed in a variety of malignant tumors and is closely related to the growth, metastasis, and prognosis of tumors. Vascular endothelial growth factor receptor-2 (VEGFR-2), belonging to the protein tyrosine kinase family, ³ is a transmembrane receptor and is mainly expressed in vascular endothelial cells and hematopoietic stem cells. As a key signal sensor in the processes of physiological and pathological angiogenesis, it mainly regulates the physiological response of VEGF, including permeability, proliferation, and migration. As well as VEGF, VEGFR-2 is overexpressed in malignant tumors, including ovarian and thyroid cancer, melanoma, and medulloblastoma. Research has shown that VEGFR-2 tyrosine kinase inhibitors can interrupt VEGF/VEGFR signal pathways and control tumor cell growth. Such inhibition is a good way to develop new anticancer drugs by targeting VEGFR-2 tyrosine kinase inhibitors.

In the intracellular domain, tyrosine kinase contains ATP-binding and downstream signaling protein binding sites. One kind of VEGFR-2 kinase inhibitor occupies the ATP-binding site keeping ATP from combining with VEGFR-2 and preventing VEGFR-2 autophosphorylation. In fact, VEGFR-2 tyrosine kinase exists in both active and inactive conformations; the two conformations can be transformed by aspartate, phenylalanine, and glutamic acid (DFG) modules. ⁴ Tyrosine kinase is active when the phenyl ring on phenylalanine is directed to the N-terminal alpha helix, that is, the DFG-in conformation; however, when the benzene ring flips to the ATP binding site, it prevents the kinase from binding to ATP and tyrosine kinase is in the inactive state, that is, the DFG-out conformation.^5,6 In general, class I inhibitors bind to the ATP binding site, class II inhibitors bind to the allosteric sites of the DFG-out conformation and indirectly compete with ATP. In addition, DFG ring movement is a slow process, being a balance between the active and inactive conformational transition. Therefore, it is significant to develop inhibitors that bind to the active conformation of tyrosine kinase and/or the inactive conformation for better efficacy.

Considering that ATP binding sites are near the allosteric sites of the DFG-out conformation (DFG-out sites), dual inhibitors of the DFG-in conformation and the DFG-out conformation with double linking groups can be designed, which can bind two sites selectively. According to the research on inactive kinase inhibitors (imatinib, nilotinib, and ponatinib), a diarylamide may be one active group which can bind DFG-out sites.^7,8 Moreover, several studies also report that benzoheterocyclic compounds, such as indoles,^9-11 benzimidazoles,^12,13 benzothiazoles, ¹⁴ acridines, quinolines, and quinazolines, show tyrosine kinase inhibitory activities on different cancer cell types. Therefore, it can be speculated that the compounds will also work if diaryl moieties can be replaced by benzoheterocyclic moieties (Figure 1). Furthermore, for the DFG-in conformation, the two benzoheterocyclic moieties can bind at ATP binding sites.

Figure 1.

The structures of the designed target compounds.

In our previous study, and based on speculation, a series of novel indole–benzimidazole or indole–benzothiazole VEGFR-2 inhibitors were designed by de novo drug design based on a linked-fragment approach.^15,16 The compounds were synthesized and showed certain VEGFR-2 inhibitory activity. ¹⁷ However, the activities were not high enough to be applicable to inhibit cancer cell proliferation. Herein, we have optimized the inhibitor structures for high tumor cell cytotoxicity and broad-spectrum properties.

All the target compounds were synthesized and their antitumor activity against the human lung adenocarcinoma cell line (A549), the human colon cancer cell line (HT-29), the human breast cancer high metastatic cell line (MDA-MB-435), the human cervical carcinoma cell line (HeLa), and the human umbilical vein endothelial cells (HUVECs) were evaluated in vitro, comparing with sunitinib malate as a positive control drug. Molecular docking studies were performed to understand the binding mode of the synthesized compounds with VEGFR-2 tyrosine kinase and the interactions with the kinase active site were analyzed with the aim of explaining their VEGFR-2 kinase inhibitory activity.

Results and discussion

Anti-tumor activity

The indole derivatives 5a–d, 9a,b, 13a,b, and 14 and the positive drug sunitinib malate displayed inhibitory activity on cell (HeLa, HT29, A549, MDA-MB-435, and HUVEC) proliferation in vitro. The IC₅₀ values of the tested compounds and the positive drug are shown in Table 1.

Table 1.

The IC₅₀ values of the synthesized compounds for five types of tumor cell lines in vitro.

Compound	IC50 (μM)
	HeLa	HT-29	HUVEC	A549	MDA-MB-435
5d	NA	NA	NA	77.24	NA
5a	0.2744	0.3635	0.2623	0.2185	0.546
5b	0.2698	0.3282	0.3006	0.3231	0.4535
5c	0.3317	0.3238	0.2036	0.4512	0.2736
9b	0.3945	15.44	0.2445	0.5858	43.1
9a	12.08	37.62	58.69	73.8	NA
13a	24.91	NA	8.607	67.11	5.821
13b	5.002	37.1	0.6258	26.54	0.5363
14	0.1911	0.1681	0.5601	0.5401	0.3729
SU11248	0.3036	0.3225	0.3455	0.1379	0.4274

HeLa: human cervical carcinoma cell line; HT-29: human colon cancer cell line; HUVEC: human umbilical vein endothelial cell; A549: human lung adenocarcinoma cell line; MDA-MB-435: human breast cancer high metastatic cell line; NA: no inhibitory activity.

It can be observed that three compounds 5a,b, and 14 were more potent in the inhibition of HeLa cell lines than sunitinib malate (0.3036 μM). Compounds 5c and 9b displayed significant inhibitory activity with IC₅₀ values of 0.3317 and 0.3945 μM, respectively. Compound 9a also exhibited certain cytotoxic activity against all cells (IC₅₀ = 10–50 μM). The remaining compounds showed nominal or no cytotoxic activity toward HeLa cell lines.

It is obvious that compound 14 (IC₅₀ = 0.1681) has a stronger inhibitory effect on the proliferation of HT-29 cells than the positive drug (IC₅₀ = 0.3225 μM). Compounds 5a–c displayed significant inhibitory activities with IC₅₀ values lower than 1 μM. Compound 9a also exhibited certain cytotoxic activity with IC₅₀ values of 10–50 μM. Compound 13a (with IC₅₀ >50 μM) was determined for activity against HT-29 cell lines. The compounds tested were found to be poorly active up to 50 μM, or inactive.

For the MDA-MB-435 cell line, compounds 5c (IC₅₀ = 0.2736 μM) and 14 (IC₅₀ = 0.3729 μM) showed stronger inhibitory activity than the positive control drug (with IC₅₀ values 0.4274 μM). Compounds 5a,b, and 13b (with IC₅₀ values of 0.546, 0.4535, and 0.5363 μM) were of similar activity to the positive drug. Besides, compound 13a showed obvious cytotoxic activity with IC₅₀ value of 5.821 μM, respectively. The other tested compounds displayed no toxicity toward MDA-MB-435 cell lines.

As shown in Table 1, most of the compounds can inhibit the growth of the A549 cancer cell effectively, and the relative IC₅₀ values were in the micromolar range. The positive drug and compounds 5a–c, and 14 demonstrated obvious inhibitory activity with IC₅₀ values 0.2185, 0.3231, 0.4512, and 0.5401 μM, respectively. Compound 13b exhibited certain cytotoxic activity with IC₅₀ values 10–50 μM. The remaining compounds displayed nominal or no cytotoxic activity toward A549 cell lines.

Although HUVEC belongs to the normal cell category, it has the potential of stem cells and can be subcultured 50–60 times in theory. It is usually selected as a cell model for vascular endothelial cell experiments. The positive drug sunitinib malate demonstrated significant angiostatic effects toward HUVEC. Compounds 5a–c, 13b, and 14 displayed significant inhibitory activities with IC₅₀ values of 0.2623, 0.3006, 0.2036, 0.6258, and 0.5601 μM. 13a showed obvious cytotoxic activity with IC₅₀ values 1–10 μM. The remaining compounds do not show an inhibitory effect on neovascularization.

Enzyme inhibitory activity

According to results of enzyme inhibitory assays (Table 2), the new compounds (5a–c, 9a, 13a,b, 14) demonstrated inhibitory activity against VEGFR-2 tyrosine kinase. As shown in Table 2, amide derivative 14 showed the highest inhibition rate, with the next being ester compounds 5a–c. Though the activities of ether compounds 13a,b were low, they were also higher than that of mixture of indole and benzimidazole. It can be concluded that the linking moiety is vital to the activity of these compounds against VEGFR-2 tyrosine kinase.

Table 2.

Inhibition rates of the newly synthesized compounds against VEGFR-2 tyrosine kinases at 1 μM.

Compound	Inhibition rate (%)	Compound	Inhibition rate (%)
5a	33.9	13a	11.6
5b	36.7	13b	13.7
5c	28.8	14	34.0
9a	18.1	Indole + benzimidazole	9.4
SU11248	97.8

Molecular docking studies

To understand the possible mechanism of action, molecular docking studies of the six compounds (5a–c, 9a,b, and 14) with high antitumor activity were carried out by binding patterns with key amino acids (hot spots) at the active site of VEGFR-2. Several crystal structures of VEGFR-2 are available in the protein data bank. For this work, the cocrystallization of VEGFR-2 with sunitinib (PDB ID:4AGD) ¹⁸ was selected as the inactive DFG-out conformation, and the active DFG-in conformation (PDB ID:2IVU) ¹⁹ was the cocrystallization of VEGFR-2 with vandetanib. The results of the docking studies for the test compounds are illustrated in Table 3.

Table 3.

The docking-free-energy score of six compounds bound to the binding site of VEGFR-2 tyrosine kinase with active or inactive conformations.

Compound	Free energy score (kcal/mol)
	Inactive conformation	Active conformation
5a	–9.8	–8.4
5b	–8.8	–8.7
5c	–8.2	–8.2
9b	–10.1	–8.9
9a	–8.5	–8.5
14	–10.2	–9.0
Sunitinib	–9.4
Vandetanib		–9.7

Inactive DFG-out conformation

First of all, the confirmation of the molecular docking protocol was obtained by docking of the crystallized sunitinib ligand in the VEGFR-2 inactive site. The docking confirmation step produced the experimental binding of the crystallized ligand between the docked pose and the crystallized ligand (free energy score −9.4 kcal/mol). Meanwhile, all the key interactions between the crystallized ligand with the key amino acids in the active site (Val80, Glu149, Phe150, Cys151, Phe153; Figure 2) were produced by capability of the docking pose.

Figure 2.

2D diagram and 3D representation of compound 5a showing its interaction with the inactive conformation of VEGFR-2.

From the docking results (Figure 2), it can be observed that compound 5a was completely embedded into the active pocket of VEGFR-2, with a docking energy of −9.8 kcal/mol less than sunitinib (docking energy of −9.4 kcal/mol). For the indole ring, the benzene ring formed arene–H interactions with Val80 and the NH also had a hydrophobic interaction with phenylalanine Phe229 of DFG. For the benzimidazole ring, the N atom at position 3 forms a hydrogen bond with Lys100 of the backbone acceptor and the imidazole ring can interact with Asp228 on the conserved aspartic acid side chain.

In the inactive conformation, the NH on the indole and imidazole rings of compound 14 (energy score = −10.2 kcal/mol) forms two hydrogen bonds with two backbone receptors of Arg209 and Arg233, respectively. In addition, the pyrrole ring can form an arene–H interaction with Arg209 (Figure 3).

Figure 3.

2D diagram and 3D representation of compound 14 showing its interaction with the inactive conformation of VEGFR-2.

Active DFG-in conformation

According to the above results, compounds 5a, 9a, and 14 were able to inhibit the inactive conformation of VEGFR-2. To determine that all three compounds were also able to inhibit the active conformation of VEGFR-2, the three compounds were docked with the active conformation.

As can be seen from the docking results of compound 5a (free energy score = −8.4 kcal/mol) with the VEGFR-2 type I receptor, the indole ring forms two hydrogens bonds, one through the NH with Ser891 and the other is via the CH at the 2-position with Asp892. However, the benzene ring of benzimidazole has a polar interaction between H and Gly733, and the benzene ring forms a π–cation interaction with the skeleton receptor of Lys758 (Figure 4).

Figure 4.

2D diagram and 3D representation of compound 5a showing its interaction with the active conformation of VEGFR-2.

As shown in Figure 5, the indole ring of 9a (free energy score = −8.5) interacts with Leu72 and Gly154 through arene–H bonds, meanwhile, the NH of the indole ring effects the Cys351 by H bond. For the other NH on the amide moiety, there is a H bond interaction with Leu72 as well as with the S atom on the thiazole ring. It can also be observed that the carbonyl moiety hydrogen bonds with Asn135.

Figure 5.

2D diagram and 3D representation of compound 9a showing its interaction with the active conformation of VEGFR-2 receptor active site.

Compound 14 (free energy score = −9.0 kcal/mol) extends the intermediate linkage of the ester bond, the indole heterocycle forms an arene–H interaction with Leu730. The O atom of the carbonyl group forms a hydrogen bond with Ser811, and the NH of the benzimidazole ring forms a hydrogen bond with Asp892 (Figure 6).

Figure 6.

2D diagram and 3D representation of compound 14 showing its interaction with the active conformation of VEGFR-2.

Discussion and conclusion

The target products are synthesized by condensation dehydration reaction (ester and ether) or deacidification reaction (amide). The reaction conditions are mild and easy to control. The difference is that there are many by-products in the synthesis of ether, therefore, the yields of 13a,b are low after purification by silica gel column.

Though the benzoheterocyclic compounds show good antitumor activity, the mechanism of action is complicated. In this study, the VEGFR-2 tyrosine kinase inhibitory activity was consistent with antitumor activity. For example, compounds 5a and 14, which demonstrated good VEGFR-2 tyrosine kinase inhibitory activity, also exhibited high antitumor activity. It can be concluded that, for the new indole–benzimidazole compounds, one of the pathways of antitumor action is to inhibit VEGFR-2 tyrosine kinase activity. However, the enzyme inhibitory activity of the compound was much lower than that of the positive drug with similar antitumor activity. Therefore, other key approaches exist for inhibiting tumor cell proliferation which need to be confirmed by further experiments.

Undoubtedly, the structure of the compound affects the enzyme activity, which in turn influences its antitumor activity. The data illustrated that (1H-benzimidazole-2-base-1H-3-)methyl indole acid ester compounds 5a–c with N-methyl substituents had better inhibitory activity than other indole–benzimidazole derivatives on the tested tumor cells and human umbilical vein endothelial cells (HUVEC). For compounds 5a,b, the methyl group may increase the hydrophobicity of the benzimidazole moieties, and then enhance the stability in the hydrophobic pocket of VEGFR-2 tyrosine kinase. As a result, it forms a polar interaction and a π–cation interaction with VEGFR-2 tyrosine kinase (Figure 4), which was introduced to improve the activity of the compounds. However, the activities of 5a–c were similar, and it can be concluded that the positions and types of substituents on the indole ring did not affect the results.

The different linking moieties between the benzimidazole groups and indole groups also show different activities. Comparing compounds 5a and 13a, it can be seen that the ester groups were more beneficial to improve the activities than ether groups, that is to say, the carbonyl moieties were key to enhance the cytotoxicity by contributing new binding sites. It can be further proved by the comparison between compounds 5d and 14, that the latter showed higher cytotoxicity because the carbonyl moiety engaged in an interaction with Ser811 (Figure 6). It can be speculated that the carbon chain elongation may place the functional groups (such as carbonyl moiety) into a suitable site, as a result, the cytotoxicity was improved.

Conversely, the activity of N-(benzothiazole-2-base)-1H-indole-3-carboxamide 9b was higher than N-(benzothiazole-2-base)-1H-indole-3-acetamide 9a and the carbon chain extension decreased the cytotoxic activity for amide compounds. This may be due to the benzothiazole ring having different interaction sites with the benzimidazole ring.

According to molecular docking results, the compounds bind to the different conformations of VEGFR-2 tyrosine kinase. In the DFG-out conformation, one benzoheterocyclic moiety of 5a and 14 show interactions with the DFG site, the other groups showed effects on the ATP site. It can be concluded that the design of the benzoheterocyclic groups linking with the chain moiety led to activity. This could be further proved by the free energy score, which expressed high activity based on low values. All the three compounds 5a, 14, and 9a gave low free energy scores on both the inactive and active conformations, in other words, the compounds exhibited good VEGFR-2 tyrosine kinase inhibitory activity. High cytotoxicity was based on high VEGFR-2 inhibitory activity. Therefore, compounds 5a–c, 13b, and 14 showed high cytotoxicity toward HeLa, HT29, A549, and MDA-MB-435, resulting in potential broad-spectrum anti-tumor activity.

Overall, the indole–benzoheterocycle compounds 5a and 14 showed the possibility of binding to both the inactive conformation of VEGFR-2 tyrosine kinase and the active conformation of VEGFR-2. Thus, compounds 5a–c and 14 could represent candidate drugs for a new inhibitor of VEGFR-2. Based on the inhibitory activity against VEGFR-2, compounds 5a–c and 14 possessed high inhibitory activity toward HeLa, HT29, A549, and MDA-MB-435 tumor cells in in vitro antiproliferation tests, hereby demonstrating the potential of broad-spectrum anticancer activity. Thus, indole–benzimidazole and indole–benzothiazole could be potential drug structures for anticancer agents and compounds 5a–c and 14 deserve further research.

Experiment

Synthesis of the target compounds

Materials and methods

All solvents and reagents were obtained from commercial sources and were used without further purification. Thin-layer chromatography (TLC) and silica gel column chromatography used with silica gel GF₂₅₄ and 200–300 mesh, respectively (Qingdao Haiyang Chemical Co. Ltd, Shandong, China). Melting points were determined on a digital melting point apparatus (WRS-1B; Shanghai Jingke Scientific Instrument Co. Ltd, Shanghai, China) and were uncorrected. ¹H NMR and ¹³C NMR were recorded on Bruker Avance DMX 500 or 300 MHz instruments, using tetramethylsilane (TMS) as the internal standard and DMSO-d₆ as the solvent. FTIR spectra were measured on a Mattson 2020 Galaxy Series FTIR spectrometer. High-resolution mass spectrometry (HRMS) data were acquired on an Agilent Technologies LC/MSD TOF mass spectrometer, using electron ionization (EI) in either positive or negative ion mode.

Synthesis of (1H-benzoimidazol-2-yl)methyl 1H-indole-3-carboxylate derivatives (5a–c)

As shown in Scheme 1, to a mixture of 1 (10 mmol) and glycolic acid (2.28 g, 30 mmol) was added concentrated H₃PO₄ (20 mL). The reaction was refluxed at 130°C for 3 h before being quenched with 20% NaOH. 2-Hydroxymethyl-3-methyl-benzimidazole (2) was collected by filtration.^20,21

Scheme 1.

Synthesis of compounds 5a–c. Reagents and conditions: (a) HOCH₂COOH, concentrated phosphoric acid, 130°C, 3 h. (b) (CF₃CO)₂O, 0°C, DMF, then rt, 3.5 h, then 20% NaOH solution, 55 °C, 6 h. (c) DCC, DMAP, THF, rt, 7–9 h.

To produce indole-3-carboxylic acid derivatives 4a–c, compounds 3a–c (20 mmol) were dissolved in dimethyl formamide (DMF, 10 mL) and trifluoroacetic anhydride (4.2 mL, 30 mmol) was added dropwise at 0°C. After stirring for 3.5 h at room temperature (rt), water was added and the mixture was filtered. The collected solid was treated with 20% NaOH (40 mL, 0.2 mol) at 55°C for 6 h. After cooling to rt, the solution was extracted with Et₂O. The aqueous phase was acidified with concentrated HCl and the product was filtered.^22–24

Finally, compounds 4a–c (5 mmol) were dissolved in THF (25 mL), then 1,3-dicyclohexylcarbodiimide (DCC) (1.24 g, 6 mmol) and 4-dimethylaminopyridin (DMAP; 0.12 g, 1 mmol) were slowly added. After stirring for 30 min at rt, compound 2 (5 mmol) was added and the mixture was stirred for 7–9 h at rt. The by-product N,N′-dicyclohexylurea, was removed by filtration. The filtrate was concentrated in vacuo to give a solid. The solid was dissolved in CH₂Cl₂ and recrystallization. After filtration, the product was obtained.

1-Methyl-1H-benzo(d) 1H-indole-3-carboxylate (5a)

White solid; 47% yield; m.p. 207–209°C. ¹H NMR (500 MHz, DMSO-d₆): δ 3.65 (s, 3H, CH₃), 5.42 (s, 2H, CH₂), 7.02 (t, J = 7.5 Hz, 1H, ArH), 7.11 (t, J = 7.5 Hz, 1H, ArH), 7.18 (t, J = 7.5 Hz, 1H, ArH), 7.24 (t, J = 7.5 Hz, 1H, ArH), 7.35 (s, 1H, CH–N), 7.51 (d, J = 8.0 Hz, 1H, ArH), 7.55 (d, J = 8.0 Hz, 1H, ArH), 7.60 (d, J = 8.0 Hz, 2H, ArH). 11.86 (s, 1H, NH). ¹³C NMR (126 MHz, DMSO-d₆): δ 163.08, 148.96, 136.42, 134.21, 132.92, 131.51, 125.85, 125.69, 125.58, 122.64, 121.64, 120.18, 115.03, 112.64, 112.56, 104.46, 55.37, 31.38. FTIR: 3447, 3087, 2920, 1690, 1537, 1441, 1365, 1340, 1244, 1174, 778, and 748 cm^–1. HRMS (EI): m/z [M]⁺calcd for C₁₈H₁₅N₃O₂: 305.1164; found: 305.1261.

1-Methyl-1H-benzo(d) 5-methoxy-1H-indole-3-carboxylate (5b)

White solid; 65% yield; m.p. 227–228°C. ¹H NMR (500 MHz, DMSO-d₆), δ 3.65 (s, 3H, CH₃), 3.75 (s, 3H, OCH₃), 5.42 (s, 2H, CH₂), 7.11 (t, J = 7.5 Hz, 1H, ArH), 7.18 (t, J = 7.5 Hz, 1H, ArH), 7.24 (t, J = 7.5 Hz, 1H, ArH), 7.35 (s, 1H, CH–N), 7.51 (d, J = 8.0 Hz, 1H, ArH), 7.55 (d, J = 8.0 Hz, 1H, ArH), 7.60 (d, J = 8.0 Hz, 2H, ArH), 11.86 (s, 1H, NH). ¹³C NMR (126 MHz, DMSO-d₆): δ 163.12, 155.22, 148.96, 134.25, 132.79, 131.27, 131.16, 126.62, 125.98, 125.66, 114.87, 113.36, 112.69, 112.52, 104.05, 101.94, 55.22, 31.42, 24.39. FTIR: 3090, 2934, 1714, 1648, 1508, 1490, 1438,1270, 1250, 1181, 1150, 1070, 977, and 735 cm^–1. HRMS (EI): m/z [M]⁺calcd for C₁₉H₁₇N₃O₃: 335.1270; found: 335.1458.

1-Methyl-1H-benzo(d) 5-bromo-1H-indole-3-carboxylate (5c)

White solid; 60% yield; m.p. 213–215°C. ¹H NMR (500 MHz, DMSO-d₆): δ 3.72 (s, 3H, CH₃), 5.39 (s, 2H, CH₂), 7.14 (t, J = 7.5 Hz, 1H, ArH), 7.18 (t, J = 7.5 Hz, 1H, ArH), 7.26 (t, J = 7.5 Hz, 1H, ArH), 7.37 (s, 1H, CH–N), 7.51 (d, J = 8.0 Hz, 1H, ArH), 7.55 (d, J = 8.0 Hz, 1H, ArH), 7.63 (d, J = 8.0 Hz, 2H, ArH). 11.86 (s, 1H, NH). ¹³C NMR (126 MHz, DMSO-d₆): δ 162.82, 148.91, 135.35, 135.21, 133.09, 132.14, 127.48, 125.60, 125.40, 125.30, 122.37, 115.27, 114.70, 114.46, 112.47, 104.27, 55.70, 31.28. FTIR: 2581, 2375, 1685, 1654, 1541, 1508, 1438, 1242, 1162, and 745 cm^–1. HRMS (EI): m/z [M]⁺calcd for C₁₈H₁₄BrN₃O₂: 383.0269; found: 383.2781.

Synthesis of N-(benzothiazol-2-yl)-1H-indole-3-carboxamide derivative (9a)

As shown in Scheme 2, thionyl chloride (8 mL) was added dropwise to a solution of 1H-indole-3-carboxylic acid (6; 2.5 mmol), then the mixture was refluxed for 5 h to afford 1H-indole-3-carbonyl chloride (7). After removal of the solvent in vacuo, the solid was dissolved in CH₂Cl₂ (20 mL). Next, 6-methoxybenzo[d]thiazol-2-amine (8) (2.5 mmol)^25,26 and Et₃N were added to the mixture at 0°C. The reaction mixture was then stirred for 18 h at 30°C and filtered. The crude product was recrystallized from absolute ethanol to obtain 9a.

Scheme 2.

Synthesis of compound 9a. Reagents and conditions: (a) SOCl₂, reflux, 5 h. (b) CH₂Cl₂, Et₃N, 30 °C, 18 h.

2-(1H-indol-3-yl)-N-(6-metho[d]xybenzothiazol-2-yl)acetamide (9a)

Khaki solid; 57% yield; m.p. 254–257°C. ¹H NMR (500 MHz, DMSO-d₆): δ 2.83 (s, 3H, CH₃), 4.53 (s, 2H, CH₂), 7.06 (d, J = 6.5 Hz, 1H, ArH), 7.56 (t, J = 7.5 Hz, 2H, ArH), 7.61 (d, J = 5.5 Hz, 1H, ArH), 7.65 (d, J = 8.5 Hz, 1H, ArH), 7.69 (d, J = 8.5 Hz, 1H, ArH), 8.02 (d, 1H, J = 6.5 Hz, CH–N), 8.13 (s, 1H, ArH), 12.77 (s, 1H, NH). ¹³C NMR (126 MHz, DMSO-d₆): δ 170.41, 156.04, 142.56, 136.05, 132.69, 127.08, 124.29, 121.06, 121.01, 118.56, 118.52, 114.82, 111.43, 107.27, 104.66, 55.55, 32.32. FTIR: 3395, 3174, 3065, 2598, 2832, 1602, 1567, 1438, 1365, 1302, 1267, 1224, 1150, 1122, 1058, 1027, 750, and 817 cm^–1. HRMS (EI): m/z [M]⁺calcd for C₁₈H₁₅N₃O₂S: 337.0885; found: 337.0914.

Synthesis of 2-(((1H-indol-3-yl)methoxy)methyl)-1H-benzoimidazole derivatives (13a,b)

To a mixture of N-substituted o-phenylenediamine 11a,b (10 mmol) and chloroacetic acid (1.42 g, 15 mmol) was added 4 N HCl (30 mL). The reaction was allowed to proceed with refluxing at 100°C for 3 h prior to being quenched with 20% NaOH. The solid (12a,b) was collected by filtration. ²⁷

In the second step, indole-3-carbaldehyde derivatives 10a,b (5 mmol), anhydrous K₂CO₃ (1.38 g, 10 mmol) and acetone (25 mL) were added to a 100-mL three-neck round-bottomed flask fitted with a condenser. After heating to 100°C, compound 12a,b (5 mmol) dissolved in acetone 10 mL was added dropwise, and the mixture was stirred for 8–20 h. Anhydrous K₂CO₃ was removed by filtration and the filtrate was concentrated in vacuo. The final products 13a,b were obtained by column chromatography (EtOAC/ petroleum ether, 1:2; Scheme 3).^1,28

Scheme 3.

Synthesis of compounds 13a,b. Reagents and conditions: (a) ClCH₂COOH, 4 N HCl, reflux, 3 h and (b) (CH₃)₂CO, K₂CO₃, reflux, 8–20 h.

2-(((1H-indol-3-yl)methoxy)methyl)-1-methyl-1H-benzoimidazole (13a)

Khaki solid; 35% yield; m.p. 139–140°C. ¹ H NMR (500 MHz, DMSO-d₆), δ 3.75 (s, 3H, CH₃), 4.64 (s, 2H, CH₂), 5.72 (s, 2H, CH₂), 7.02 (t, J = 7.5 Hz, 1H, ArH), 7.11 (t, J = 7.5 Hz, 1H, ArH), 7.18 (t, J = 7.5 Hz, 1H, ArH), 7.24 (t, J = 7.5 Hz, 1H, ArH), 7.37 (s, 1H, CH–N), 7.51 (d, J = 8.0 Hz, 1H, ArH), 7.55 (d, J = 8.0 Hz, 1H, ArH), 7.60 (d, J = 8.0 Hz, 2H, ArH). ¹³C NMR (126 MHz, DMSO-d₆): δ 149.56, 141.67, 136.38, 135.89, 127.17, 122.96, 122.71, 122.01, 120.69, 119.30, 118.61, 117.98, 114.17, 111.27, 110.38, 63.02, 59.73, 29.92. FTIR: 3020, 1495, 1464, 1415, 1397, 1182, 1162, 735, and 702 cm^–1. HRMS (EI): m/z [M]⁺calcd for C₁₈H₁₇N₃O: 291.1372; found: 291.1154.

2-(((1-benzyl-1H-indol-3-yl)methoxy)methyl)-1H-benzo[d]imidazole (13b)

Orange solid; 32% yield; m.p. 149–151°C. ¹H NMR (500 MHz, DMSO-d₆): δ 5.13 (s, 2H, CH₂), 5.39 (s, 2H, CH₂), 5.73 (s, 2H, CH₂), 6.95 (t, J = 7.5 Hz, 1H, ArH), 7.08 (t, J = 7.5 Hz, 1H, ArH), 7.14 (d, J = 7.5 Hz, 2H, ArH), 7.22 (t, J = 7.5 Hz, 3H, ArH), 7.27 (d, J = 7.5 Hz, 2H, ArH), 7.40 (t, J = 8.0 Hz, 2H, ArH), 7.55 (s, 1H, CH–N), 7.58 (d, J = 6.5 Hz, 1H, ArH), 7.64 (d, J = 7.5 Hz, 1H, ArH). ¹³ C NMR (126 MHz, DMSO-d₆): δ 149.43, 141.90, 138.07, 136.16, 135.38, 128.43, 128.34, 127.28, 126.84, 126.47, 122.94, 122.00, 121.69, 119.45, 119.28, 118.54, 111.20, 110.50, 109.38, 48.94, 37.21. FTIR: 3100–3500, 3055, 2925, 2850, 1612, 1465, 1240, 1287, 1170, 1127, 1007, and 741 cm^–1. HRMS (EI): m/z [M]⁺calcd for C₂₄H₂₁N₃O: 367.1685; found: 367.0964.

Synthesis of (1H-benzoimidazol-2-yl)methyl 2-(1H-indol-3-yl)acetate (14)

1H-Indole-3-acetic acid ((6), 5 mmol) was dissolved in THF (25 mL). DCC (1.24 g, 6 mmol) and DMAP (0.12 g, 1 mmol) were slowly added to the given mixture. After stirring for 30 min at rt, benzimidazole-2-methanol (14) (5 mmol) was added and the mixture stirred for 7–9 h at rt. The by-product N,N′-dicyclohexylurea was removed by filtration. The filtrate was concentrated in vacuo to give a solid. The solid was dissolved in CH₂Cl₂ and the product 14 obtained by recrystallization (Scheme 4).

Scheme 4.

Synthesis of compound 14. Reagents and conditions: (a) DCC, DMAP, THF, rt, 7–9 h.

(1H-benzoimidazol-2-yl)methyl 2-(1H-indol-3-yl)acetate (14)

White solid; 79% yield; m.p. 133–135°C. ¹ H NMR (500 MHz, DMSO-d₆: δ 12.77 (d, J = 25.2 Hz, 1H), 10.97 (s, 1H), 7.69–7.60 (m, 1H), 7.55 (d, J = 15.0 Hz, 1H), 7.51 (d, J = 7.9 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.29 (s, 1H), 7.23 (dd, J = 21.0, 8.5 Hz,1H), 7.07 (t, J = 7.5 Hz, 1H), 6.96 (t, J = 7.4 Hz, 1H), 5.31 (d, J = 3.0 Hz, 2H), 3.8 (s, 2H). ¹³C NMR (126 MHz, DMSO-d₆): δ 171.20,149.16, 141.35, 136.05, 127.04, 124.19, 121.07, 118.49, 111.38, 106.59, 59.54, 30.45. FTIR: 3310, 3055, 2930, 1715, 1622, 1544, 1441, 1251, 1128, 1108, 995, and 738 cm^–1. HRMS (EI): m/z (M)⁺calcd for C₁₈H₁₅N₃O₂: 305.1164; found: 305.1257.

VEGFR-2 kinase assay in vitro

The inhibitory activities of seven compounds (5a–c, 9a, 13a,b, 14) and a mixture of indole and benzimidazole (1:1) against VEGFR-2 were measured according to the effect on the phosphorylation level of the substrate using the enzyme-linked immunosorbent assay (ELISA) method. Briefly, samples and positive control (SU11248) in phosphate buffer saline (PBS) were added to ELISA plates, which were coated with the substrate poly(Glu, Tyr)_4:1. Next, ATP (0.5 mol) and enzyme solution were added to give a final volume of 100 μL. After reaction for 1 h in the dark, the plate was washed with T-PBS solution. A total of 100 μL of the antibody PY99 was placed into every well and reacted for 30 min at 37 ℃. Next, horseradish peroxidase (HRP) labeled goat anti-mouse IgG (100 μL/well) was added after washing, and the reaction allowed to continue for 30 min at 37 ℃. The next step was coloration for 1–10 min by o-phenylenediamine (OPD) solution in the dark at rt. Finally, the reaction was stopped by addition of sulfuric acid (2 M, 50 μL), and the absorbance was measured using a microplate reader (SPECTRAMAX190, Molecular Devices Corporation, America).

The inhibition rate against VEGFR-2 (IRE) was calculated by the following formula

$$\mathrm{IRE}=\left(1-\frac{\begin{array}{l}\mathrm{OD}\mathrm{value}\mathrm{of}\mathrm{sample}\\ -\mathrm{OD}\mathrm{value}\mathrm{of}\mathrm{control}\mathrm{with}\mathrm{no}\mathrm{ATP}\end{array}}{\begin{array}{l}\mathrm{OD}\mathrm{value}\mathrm{of}\mathrm{neagtive}\mathrm{control}\\ -\mathrm{OD}\mathrm{value}\mathrm{of}\mathrm{control}\mathrm{with}\mathrm{no}\mathrm{ATP}\end{array}}\right)\times 100\%$$

Anti-tumor assay in vitro

In this study, the cytotoxicities of seven new compounds and sunitinib malate (a positive drug) against four cancer cells (A549, HT-29, MDA-MB-435, and HeLa) and HUVEC were estimated in vitro. To explain the structure–activity relationship, the activities of (1H-benzoimidazol-2-yl)methyl-1H-indole-3-carboxylate (5d) and N-(6-methoxy-benzothiazol-2-yl)-1H-indole-3-carboxamide (9b) were also tested. The method used was the cell proliferation inhibition test using the EnoGeneCell^™ Counting Kit-8 (CCK-8) cell viability assay kit. The cells were digested and counted to prepare a cell suspension with a concentration of 1 × 10⁵cells/mL. About 100 μL of the cell suspension (1 × 10⁴ cells per well) was added to each well of the 96-well plate and incubated at 37°C in a 5% CO₂ incubator for 24 h. Next, 100 μL of the corresponding drug-containing medium was added to each well, while establishing a negative control group, a vehicle control group, and a positive control group (sunitinib malate) with five wells, respectively. After incubating at 37°C in a 5% CO₂ incubator for 72 h, each well was supplemented with 10 μL of CCK-8 solution and incubated for 4 h. Finally, the OD value at 450 nm was measured with a microplate reader.

Molecular docking

The molecular docking study was performed using AutoDock Vina 1.1.2. The crystal structure of VEGFR-2 tyrosine kinase (PDB ID: 4AGD, 2IVU) was obtained from the RCSB Protein Data Bank. The ligand and receptor were prepared and the binding model was analyzed using Molecular Operating Environment.