Design, synthesis and evaluation of dihydro-1H-indene derivatives as novel tubulin polymerisation inhibitors with anti-angiogenic and antitumor potency

Abstract

Angiogenesis plays an important role in tumour generation and progression, which is used to supply nutrients and metastasis. Herein, a series of novel dihydro-1H-indene derivatives were designed and evaluated as tubulin polymerisation inhibitors by binding to colchicine site, exhibiting anti-angiogenic activities against new vessel forming. Through structure-activity relationships study, compound 12d was found to be the most potent derivative possessing the antiproliferative activity against four cancer lines with IC₅₀ values among 0.028−0.087 µM. Compound 12d bound to colchicine site on tubulin and inhibited tubulin polymerisation in vitro. In addition, compound 12d induced cell cycle arrest at G2/M phase, stimulated cell apoptosis, inhibited tumour metastasis and angiogenesis. Finally, the results of in vivo assay suggested that compound 12d could prevent tumour generation, inhibit tumour proliferation and angiogenesis without obvious toxicity. Collectively, all these findings suggested that compound 12d is a novel tubulin polymerisation inhibitor deserving further research.

Keywords:

• Anticancer
• angiogenesis
• tubulin
• colchicine binding site
• indene

Introduction

Tubulin plays an important role in a variety of cellular processes including cell proliferation, cell signalling and cell morphology, which is one of the attractive targets for new antitumor drugs discovery^1–3. Microtubule-targeting agents (MTAs) could inhibit the polymerisation or destabilisation of microtubules by altering its dynamics in cells⁴. Up to date, six major binding sites have been identified on tubulin, including colchicine binding site (CBS), pironetin binding site, laulimalide binding site, vinca binding site, maytansine binding site, and taxane binding site^5,6. Taxanes and vinblastine were examples as MTAs approved by FDA and used for the treatment of solid tumours^7,8. However, the application of these drugs in clinic was limited due to their multidrug resistance (MDR)^9–11.

Colchicine binding site inhibitors (CBSIs) binding to the interface of the α/β-tubulin protein¹², have been reported to overcome the MDR^13,14. Besides, some of CBSIs were also reported as anti-angiogenic agents with tumour anti-vascular activity^15–18. Combretastatin A-4 (CA-4, 2, Figure 1) was one of the most representative CBSIs, which could prevent tubulin polymerisation and inhibit cancer cell proliferation^19,20. However, only the cis-configuration of CA-4 could display potent anticancer activity, while the trans-CA-4 loses its activity completely^21,22. There are many strategies that focus on replacing linker with a nonrotatable structure to avoid the cis-trans isomerisation of CA-4²³. For example, isoCA-4 (3)²⁴ and Veru-111 (4)²⁵, which replace their cis-double bond with 1,1-double bond or five-membered heterocycle. Another common strategy is fusing the linker into ring A or ring B to afford novel compounds (e.g. compounds 5²⁶ and 6²⁷) which could maintain their potential activities against cancer cells.

Figure 1. Structures of typical CBSIs as tubulin polymerisation inhibitors.

Angiogenesis, the process of development of new capillary vessels from pre-existing ones to deliver oxygen and nutrients and remove wastes, is necessary for tumour growth, progression and metastasis²⁸. Therefore, anti-angiogenic therapies have been developed for the treatment of the tumour²⁹, and some of them have achieved success in preclinical experiments and clinical trials^30–35. It is worth noting that some of MTAs have been reported as vascular destroy agents (VDAs) and were developed as anti-cancer drugs³⁶. Thus, tubulin has become an important target for the discovery of antitumor drugs, through inhibiting the growth of solid tumour via disrupting tumour neovasculature³⁷.

Based on our previous work^38–41, in this study, we have designed a series of novel dihydro-1H-indene analogues by merging linker of CA-4 into the ring A to discover novel CBSIs, in which case the distance between ring A and B is three atoms (Figure 2). Among the analogues synthesised, compound 12d with 4-hydroxy-3-methoxyphenyl as B ring showed the most potent antiproliferative activities against four cancer lines. Compound 12d was identified as a tubulin polymerisation inhibitor by binding to colchicine site, in addition, we found that compound 12d could induce apoptosis and cell cycle arrest at the G2/M phase. Moreover, compound 12d was also found its anti-angiogenesis and anti-proliferative activity in vitro and in vivo.

Figure 2. Structure modification strategy of indene derivatives as CBSIs.

Chemistry

Target compounds 12a − 12x were synthesised as shown in Scheme 1. The key intermediate 8 was obtained by the cyclisation reaction from 3–(3,4,5-trimethoxyphenyl)propanoic acid (7). Compound 8 was then coupled with appropriate benzaldehydes 9a − 9x to generate compounds 10a − 10x. Finally, target compounds 12a − 12x were produced by two reductive reactions using LiAlH₄ and H₂-Pd/C as conditions, respectively.

Scheme 1. Synthesis of compounds 12a − 12x. Reagents and conditions: (a) PPA, 90 °C, 2 h, 71.4%; (b) substituted benzaldehydes, KOH, MeOH, rt, overnight; (c) AlCl₃, LiAlH₄, THF, 0 °C, 2–12 h; (d) H₂, Pd/C, MeOH, rt, overnight, 5.1–29.6% over three steps.

Compounds 15a − 15c were synthesised as described in Scheme 2. Compounds 13a − 13c were generated by a ring closure reaction by TfOH. Then, target compounds were afforded following the similar synthetic route as 12a − 12x. Compound 15d was obtained by reduction reaction from intermediate 10d under hydrogen atmosphere with the present of Pd/C catalyst.

Scheme 2. Synthesis of compounds 15a − 15d. Reagents and conditions: (a) TfOH, 0 °C, 0.5 h, 56.2–91.8%; (b) substituted benzaldehyde, KOH, MeOH, rt, overnight, 78.4%; (c) AlCl₃, LiAlH₄, THF, 0 °C, 2–12 h; (d) H₂, Pd/C, MeOH, rt, overnight, 23.6–71.6% over three steps.

Results and discussion

In vitro cell growth inhibition activity

The antiproliferative activities of novel target compounds were firstly evaluated against K562 cell line using CCK-8 assay, and CA-4 was used as a positive control. As the results shown in Table 1, most of synthesised compounds 12a − 12r with 4,5,6-trimethoxy-2,3-dihydro-1H-indene as core showed antiproliferative activities with the inhibition rate above 50% at the concentration of 1 µM. However, only compounds 12d, 12j and 12q exhibited great growth inhibitory effects at the lower concentration of 0.1 µM, and as presented in Table 1, compounds 12d (4-hydroxy-3-methoxyphenyl as B ring) and 12q (2,3-dihydrobenzofuran-5-yl as B ring) displayed the most promising activities with the inhibition rates of 78.82% and 83.61%, respectively. Besides, we found that compounds with electron-donating groups on B ring were more potent than that with electron-withdrawing groups (e.g. 12a vs 12p, 12d vs 12e, 12j vs 12k). In addition, compounds 12s − 12x were also designed, and these compounds obtained by introducing indole ring into 4,5,6-trimethoxy-2,3-dihydro-1H-indene could maintain the antiproliferative activities to some extent at the concentration of 0.1 µM, except for compound 12v (Table 1). And compound 12t with 5′-indole as B ring displayed inhibitory effects at 0.1 µM. Collectively, 12d, 12j, 12q and 12t showed potent anticancer effect in the preliminary toxicity assay, and was selected for further IC₅₀ evaluation.

Table 1. Preliminary evaluation of growth inhibitory effects of target compounds against K562 cell line at 1 µM and 0.1 µM.

Compound	Inhibition rate (%)^a		Compound	Inhibition rate (%)^a
	1 µM	0.1 µM		1 µM	0.1 µM
12a	72.44	5.71	12p	15.38	12.55
12b	15.22	5.80	12q	91.91	83.61
12c	9.48	6.96	12r	6.73	3.98
12d	80.57	78.82	12s	67.12	9.67
12e	66.98	10.23	12t	88.58	50.38
12f	72.00	6.65	12u	83.94	10.77
12g	7.74	4.71	12v	28.53	5.02
12h	79.78	2.25	12w	85.79	10.03
12i	66.01	3.00	12x	74.93	12.53
12j	86.61	68.65	15a	81.53	45.57
12k	13.86	4.12	15b	89.19	31.44
12l	7.29	1.03	15c	87.47	52.07
12m	16.38	2.90	15d	62.95	39.02
12n	78.84	5.81	CA-4	92.03	88.32
12o	6.97	5.65

^aThe data were expressed as the mean of at least two independent experiments.

We further evaluated the antiproliferative activities of potent compounds against four cancer lines including A549, Hela, H22 and K562, and one normal cell line HFL-1 to assess the antitumor activity and selectivity of these compounds. As shown in Table 2, compound 12d exhibited the most antiproliferative potency among these selected compounds with IC₅₀ values of 0.087, 0.078, 0.068 and 0.028 µM against A549, Hela, H22 and K562 cell lines, respectively. In addition, compound 12d showed relatively low cytotoxicity against normal cell line HFL-1 with the IC₅₀ value of 0.271 µM, suggesting that compound 12d could inhibit the growth of cancer cells with less toxicity.

Table 2. In vitro antiproliferative activities of selected compounds against four cancer cell lines and one normal cell line HFL-1.

Compound	IC50 (µM)^a
	A549	Hela	H22	K562	HFL-1
12d	0.087 ± 0.009	0.078 ± 0.005	0.068 ± 0.008	0.028 ± 0.007	0.271 ± 0.031
12j	0.212 ± 0.013	0.136 ± 0.011	0.093 ± 0.005	0.068 ± 0.006	0.590 ± 0.062
12q	0.220 ± 0.015	0.183 ± 0.009	0.104 ± 0.004	0.079 ± 0.009	0.398 ± 0.023
12t	0.277 ± 0.029	0.284 ± 0.014	0.254 ± 0.020	0.176 ± 0.024	0.552 ± 0.075
15a	0.753 ± 0.040	0.317 ± 0.027	0.307 ± 0.015	0.192 ± 0.013	5.000 ± 0.306
15b	2.007 ± 0.117	3.168 ± 0.108	0.575 ± 0.041	0.425 ± 0.027	11.262 ± 1.403
15c	0.341 ± 0.064	0.309 ± 0.032	0.114 ± 0.023	0.100 ± 0.007	3.583 ± 0.548
15d	2.951 ± 0.154	3.413 ± 0.186	1.127 ± 0.058	0.774 ± 0.051	6.959 ± 0.843
CA-4	0.032 ± 0.005	0.026 ± 0.006	0.010 ± 0.002	0.004 ± 0.001	0.139 ± 0.017

^aIC₅₀ values are indicated as the mean ± SD from three independent experiments.

Besides, we investigated the structure-activity relationships (SARs) of substituted groups on the core and dihydro-1H-indene core itself, and compounds 15a − 15d were designed and screened. As is shown in Tables 1 and 2, changing trimethoxy (12d) to dimethoxy (15a) or 4-hydroxy-3-methoxyphenyl (15b) led to an about 7-fold or 15-fold decrease in antiproliferative activities of K562 cell lines. We also introduced oxygen (15c) and carbonyl group (15d) into the linker. However, compounds 15c and 15d showed a significant decrease of antiproliferative activity. Thus, both results highlighted the importance of core 4,5,6-trimethoxy-2,3-dihydro-1H-indene in the antiproliferative activities of these derivatives.

Compound 12d inhibited tubulin polymerisation in vitro

To confirm that the compound 12d was a tubulin polymerisation inhibitor, we firstly performed a confocal immunofluorescence assay to evaluate the influence of compound 12d on tubulin polymerisation of K562 cells. After treatment with or without compound 12d for 48 h, FITC and DAPI were used to mark α-tubulin and nucleus, respectively. As shown in Figure 3, the microtubule networks of blank control group displayed filamentary and fibrous structure, and mitotic spindles could be observed on some of cells in their mitotic phase. Meanwhile, 12d-treatment groups caused disorder and destruction of microtubule networks, indicating that compound 12d is a tubulin destabilizer.

Figure 3. Compound 12d disrupted tubulin distribution of K562 cells in vitro at different concentrations, and the scale bar = 10 µm.

We further evaluated whether 12d could bind to colchicine site to prevent tubulin polymerisation and the results are presented in Table 3. 12d could inhibit the potential of tubulin polymerisation with an IC₅₀ value of 3.24 µM. Besides, compound 12d could compete with [³H]-colchicine in binding to tubulin with the potency value of 71.43% at the concentration of 5 µM, confirming that compound 12d might have the similar mechanisms with these CBSIs to inhibit tubulin polymerisation and disrupt microtubule networks.

Table 3. Compound 12d inhibited tubulin polymerisation and bound to colchicine binding site on tubulin^a.

compd.	Inhibition of tubulin polymerisation IC50 (µM)	Inhibition (%) of colchicine binding^b
		1 µM	5 µM
12d	3.24 ± 0.10	59.81 ± 3.84	71.43 ± 1.03
CA-4	1.99 ± 0.15	78.55 ± 1.85	87.35 ± 1.37

^aAll data are presented as mean ± SD from three independent experiments.

^bConcentration of the tubulin was 1 µM, and the concentration of [³H]-colchicine was 5 µM.

Effect of compound 12d on the cell apoptosis in K562 cells

For further validating whether compound 12d could stimulate cell apoptosis, the Annexin V-FITC and propidium iodide (PI) staining assay were first performed to analysis the distribution of cells in the stage of early stage (Annexin V⁺/PI^-) or late stage of apoptosis (Annexin V⁺/PI⁺). As illustrated in Figure 4(A,B), compound 12d induced K562 cells apoptosis in a dose-dependent manner, with the percent of total apoptosis cells rising from 2.47% in the control group to 57.90% at the concentration of 60 nM. Besides, Hoechst 33342 staining assay demonstrated that compound 12d induced K562 cells nuclear fragmentation and cell morphology changes (Figure 4C). What’s more, we performed western blotting assay to analysis the expression level of Bcl-2 protein family related with apoptosis. As shown in Figure 4(D,E), 12d downregulated the expression of antiapoptotic proteins, such as Bcl-2 and Bcl-xl. Meanwhile, the expression level of proapoptotic proteins including Bax and Bad were upregulated by 12d in a dose-dependent manner.

Figure 4. Effect of 12d on the cell apoptosis. (A) K562 cells was stained with Annexin-V FITC/PI to analysis apoptosis. (B) Histograms showed the distribution of cell population. Error bars indicated SD of three independent experiments. (C) Apoptosis morphology and nucleus changes after treatment with compound 12d. Scale bar = 50 µm. (D) Expression of apoptosis associated proteins were evaluated by western blotting assay. (E) Histograms showed the western blotting assay.

3.4. Effect of compound 12d on inducing G2/M phase arrest

It is usually reported that tubulin polymerisation inhibitors that binding to colchicine site arrest cancer cells at G2/M phase^42,43. To investigate the cell cycle changes caused by compound 12d, we performed a flow cytometry assay and the results are shown in Figure 5. After treatment with compound 12d at the concentrations of 15, 30 and 60 nM for 48 h in K562 cells, 16.07 ± 0.83%, 21.23 ± 1.14% and 24.66 ± 0.63% of population were at the G2/M phase, respectively, compared to untreated group with the percent of 12.99 ± 0.26% of that (Figure 5(A,B)), indicating that 12d could arrest cell cycle of K562 cell in the G2/M in a dose-dependent manner. Considering that cell division cycle-associated proteins play an important role in tumour generation by regulation of cancer cell cycle, while Cyclin B1, Cdc-2 and Cdc-25c are three main proteins related to G2/M phase in the process of cell cycle⁴⁴, we further evaluated cell cycle regulatory proteins through western blotting assay. As shown in Figure 5(C,D), 12d could down-regulate expression levels of Cyclin B1, Cdc-2 and Cdc-25c in a dose-dependent manner.

Figure 5. Effect of 12d on the cell cycle. (A) K562 cells was stained with PI to analysis cell cycle. (B) Histograms showed that 12d induced cell cycle arrest at G2/M phase. Error bars indicated SD of three independent experiments. (C) Expression of G2/M phase associated proteins were evaluated by western blotting assay. (D) Histograms showed the western blotting assay.

Compound 12d downregulated mitochondrial membrane potential and induced the generation of ROS

Mitochondria plays an important role in the process of cell apoptosis, and the downregulation of mitochondrial membrane potential (MMP) is regarded as one of the hallmark events in the early stage of cell apoptosis. Besides, the generation of reactive oxygen species (ROS) could induce depolarisation of mitochondrial membrane, leading to apoptosis of cancer cells⁴⁵. We firstly investigated influences of MMP after treatment with compound 12d by JC-1 staining probe, and as illustrated in Figure 6(A,B), the percentage of green fluorescence was increasing with the treatment concentrations ascending from 0 to 60 nM, indicating that compound 12d could decrease the MMP of K562 cells. We further used DCFH-DA fluorescence probe to evaluate ROS level after treatment with compound 12d, and the results shown in Figure 6(C,D) suggested that the generation of ROS was induced by 12d in a dose-dependent manner.

Figure 6. Compound 12d influenced mitochondrial depolarisation and induced the generation of ROS. (A) MMP was measured using JC-1 staining assay after treatment with compound 12d on K562 cells. (B) Histograms showed the flow cytometry. (C) ROS was measured using DCF-DA assay after treatment with compound 12d on K562 cells. (D) Histograms showed the flow cytometry. Error bars indicated SD of three independent experiments. ***p < 0.001, ****p < 0.0001 vs control group.

Effect of compound 12d on the cell migration and invasion

Metastasis is reported as the main cause of mortality in solid tumour^46,47, while some of research found that inhibitors that bind to colchicine site on tubulin could disrupt metastasis effect of cancer cells. Therefore, we chose triple negative breast cancer cell line MDA-MB-231 for further Transwell assay considering its high migration and invasion ability. In migration assay, the number of migrated cells of the 12d-treated group (60 nM) was 124.0 ± 9.2, while number of that was 231.3 ± 12.2 in untreated group (Figure 7(A,B)). In invasion assay, the results showed the number of invaded cells was 51.3 ± 2.5 for MDA-MB-231 cells treated with 12d at the concentration of 60 nM, while the number of that was 81.3 ± 5.7 in untreated group (Figure 7(C,D)). These results in Figure 7(A–D) indicated that compound 12d could significantly suppress the migration and invasion effects of MDA-MB-231 cells in a dose-dependent manner.

Figure 7. 12d inhibited the migration and invasion of MDA-MB-231 cells in (A) Transwell migration assay and (C) Transwell invasion assay. Scale bar = 50 µm. (B, D) Histograms showed the count of cells. Error bars indicated SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs control group.

Effect of compound 12d on anti-angiogenesis in vitro

As above mentioned, tubulin polymerisation inhibitors binding to colchicine site could serve as VDAs to disrupt blood vessels in vitro and in vivo. Human umbilical vein endothelial cells (HUVECs) are isolated from the umbilical cord tissue, and used to evaluate anti-angiogenetic potency of tested compounds. We first confirmed the IC₅₀ of 12d in inhibiting the proliferation of HUVEC to be 2.26 ± 0.38 µM, then, the inhibition of wound-healing effect of HUVECs by the treatment of compound 12d was tested. The results in Figure 8(A,B) showed that the wound healing effect was disrupted after treatment with compound 12d in a dose-dependent manner. To investigate the effect of compound 12d on the capillary-like tubules structure forming of HUVECs at different concentrations, we then performed a tube formation assay on Matrigel in vitro. As shown in Figure 8(C), the network structure formed by HUVECs was destroyed after treatment with 12d in a dose-dependent manner. And almost all of the tube-like structure was broken at the concentration of 60 nM. These results suggested that compound 12d could inhibit angiogenesis effectively.

Figure 8. Compound 12d inhibited angiogenesis of HUVECs in vitro. (A) Compound 12d prevented wound healing of HUVECs in a dose-dependence manner by comparing with wound closure percent after 24 h of culturation. Scale bar = 100 µm. (B) Histograms showed wound closure percent. Error bars indicated SD of three independent experiments. ***p < 0.001, ****p < 0.0001 vs control group. (C) Compound 12d destructed HUVECs forming normal capillary-like tubular networks. Scale bar = 100 µm.

Molecular docking study of compound 12d

We performed a molecular docking study to analyse the possible interactions between compound 12d and tubulin (PDB id:5LYJ²¹) at colchicine binding site. The docking results showed that 12d could occupy the cavity around the CA-4 formed by Thrα179, Alaα180, Valα181, Valβ238, Cysβ241, Leuβ248, Alaβ250, Leuβ255, Asnβ258, Metβ259, Alaβ317, and Lysβ352 (Figure 9A). Besides, according to the hypothesised binding pose shown in Figure 9(B), it seems that compound 12d could bind to the colchicine site in a similar manner like CA-4 by adjusting its relative flexible methylene linker and orientation of A ring to fit the cavity. Taking together, we supposed that compound 12d and positive drug CA-4 might have the similar interaction mode with tubulin.

Figure 9. Hypothesised binding interactions between compound 12d and tubulin (PDB id:5LYJ). (A) Supposed docking pose of 12d (green) in the colchicine binding site; (B) Overlap of CA-4 (orange) and compound 12d with tubulin. PyMOL was used to draw the figures.

Antitumor activity of compound 12d in vivo

Considering that compound 12d showed encouraging results in above in vitro assays and appropriate druglikeness (cLogP 3.57, tPSA57), we finally evaluated the in vivo antitumor efficacy of 12d in a mice liver cancer allograft model (Figure 10). Mice were injected with mouse liver cancer cells H22 into right flank of each mouse. Mice were then randomly divided into four groups: vehicle control (5% DMSO/5% Tween 80/90% saline, untreated control), CA-4 at a dose of 20 mg/kg (positive control) and compound 12d at 10 or 20 mg/kg, and treated via i.v. injection every once a day for three weeks. As shown in Figure 10(A-C), compared to vehicle control group, CA-4-treated group inhibited tumour growth with a tumour growth inhibition (TGI) value of 59.19%. Compound 12d-treated group could also lead to tumour growth inhibition with a TGI value of 74.94% at a dose of 20 mg/kg, while no obvious body weight loss (Figure 10D) and vital organ damage (Figure 11) were observed.

Figure 10. Antitumor activity of compound 12d in H22 allograft mice model. (A) Tumours in each of group vehicle, compound 12d (10 or 20 mg/kg/day, i.v.), and CA-4 (20 mg/kg/day, i.v.) were shown. (B) Individual tumour weights were measured after treatment for three weeks. Error bars indicated SD of three independent experiments. ****p < 0.0001 vs control group, ^###p < 0.001 vs CA-4 (20 mg/kg) group. (C) Tumour volumes and (D) mouse body weights were measured every other day for three weeks. Error bars indicated SD of three independent experiments.

Figure 11. Representative images of H&E-stained vital organ, including heart, liver, spleen, lung and kidney. Scale bar = 20 µm.

Immunohistochemistry (IHC) assay of mice tumour was further performed for quantitative analysis of the microvessel density (MVD) and Ki67-positive cells with prognostic angiogenic marker CD31 and cell proliferation marker Ki67. As shown in Figure 12, 12d-treated group demonstrated lower MVD level (Figure 12(A,B)) and decreased number of Ki-67 positive cells (Figure 12(A,C)), indicating that 12d could disrupt angiogenesis and inhibit cell proliferation of H22 tumour xenograft tumours in vivo.

Figure 12. (A) Representative images of IHC-stained markers CD31 and Ki67 in tumour tissue. Scale bar = 20 µm. Histograms showed the relative percentages of (B) microvessel density assessed by prognostic angiogenic marker CD31 and (C) expression level of cell proliferation marker Ki67 in CA-4 (20 mg/kg) or 12d (20 mg/kg) treated group compared with the control group. Error bars indicated SD of three independent experiments. ***p < 0.001, ****p < 0.0001 vs control group.

Conclusion

In summary, a series of novel indene derivatives were designed, synthesised, and evaluated as tubulin polymerisation inhibitors by binding to colchicine site. Among them, 12d was the most potent derivative possessing the antiproliferative activity against four cancer lines with IC₅₀ values among 0.028 − 0.087 µM. The following in vitro tubulin polymerisation assay revealed that compound 12d could inhibit tubulin polymerisation by binding to colchicine site in a mode similar to CA-4. Further, compound 12d induced the cell apoptosis and G2/M phase cell cycle arrest of K562, which might explain the cytotoxicity of the tested compound. As shown in mechanism assays, 12d also downregulated mitochondrial membrane potential and induced the generation of ROS. In transwell assays, wound healing and tubular networks forming assays, compound 12d displayed strong suppression on tumour metastasis and angiogenesis. The in vivo H22 allograft mice model was used to evaluate the antitumor activity of compound 12d. As the results, 12d possessed antitumor activity in a dose-dependent manner without overt toxicity. H&E and IHC analysis were further performed to find that compound 12d could inhibit tumour proliferation and angiogenesis without obvious pathological damage. Taking together, these results suggested that compound 12d is a promising tubulin polymerisation inhibitor and deserves to be developed as an anti-angiogenesis agent for further investigation.

Experimental section

Chemistry

General methods

Unless otherwise noted, all commercially available reagents were used without further purification. Solvents were dried through routine protocols. Flash column chromatography was carried out on 200–300 mesh silica gel (Qingdao Haiyang Chemical, China). Reactions were monitored by thin-layer chromatography (TLC) on 0.25 mm silicagel plates (GF254) and visualised under UV light. ¹H NMR and ¹³C NMR spectra were recorded with a Bruker AV-300 or AV-400 spectrometer (Bruker Company, Germany) in the indicated solvents (CDCl₃ or DMSO-d₆, TMS as internal standard): the values of the chemical shifts are expressed in δ values (ppm) and the coupling constants (J) in Hz. Chemical shifts (in ppm) were referenced CDCl₃ (7.26 ppm), DMSO-d₆ (2.50 ppm). ¹³C-NMR spectra were obtained by using the same NMR spectrometers and were calibrated with CDCl₃ (δ = 77.16 ppm), DMSO-d₆ (δ = 39.52 ppm). Mass spectra (MS) were measured on Agilent 1290–6125 quadrupole mass spectrometer (Agilent, US).

General procedure of synthesis of compounds 12a − 12x

To 10 ml polyphosphoric acids (PPA), 3–(3,4,5-trimethoxyphenyl)propanoic acid (7) (2 g, 8.32 mmol, 1.0 eq) was added. The mixture was allowed to stir at 90 °C for 2 h until reaction was completed as TLC monitored (petroleum ether/EtOAc, 6:1). Then the mixture was cooled to room temperature, poured into 200 ml of ice water, and extracted with EtOAc (3 × 100 ml). The organic layer was combined, washed with 5% aqueous NaHCO₃, water and saturated brine, dried over sodium sulphate, and concentrated in vacuo. The crude residue was suspended in an appropriate amount of EtOAc, heated until the mixture became clear, and cooled at 4 °C overnight to obtain deposit, and filtered to afford intermediate 8 as a gray solid (1.32 g, yield 71.4%).

To the solution of KOH (252 mg, 4.50 mmol, 5.0 eq) in 50 ml MeOH, 5,6,7-trimethoxy-2,3-dihydro-1H-inden-1-one (8) (200 mg, 0.90 mmol, 1.0 eq) was added. After stirring for 15 min, different substituted benzaldehyde (9) (1.08 mmol, 1.2 eq) in 20 ml MeOH was added dropwise, and the reaction was allowed to stir at room temperature overnight. Then the mixture was concentrated in vacuo, diluted with water, and extracted with DCM (3 × 50 ml). The organic layer was combined, washed with water and saturated brine, dried over sodium sulphate, and concentrated to afford crude product, which was purified by column chromatograph with petroleum ether/EtOAc (3:1) or DCM as eluting solvent to give the desired products 10a − 10x.

To the solution of 10a − 10x (1.0 eq) in 20 ml THF in an ice bath, AlCl₃ was added to the solution slowly. Then LiAlH₄ (3.0 eq) was added in five portions, and the mixture was stirred at the temperature of 0 °C until reaction completed monitored by TLC (petroleum ether/EtOAc, 2:1). To the mixture, saturated NH₄HCO₃ solution was added slowly. Then the mixture was filtered to remove solid, and the filtrate was extracted with EtOAc (3 × 100 ml). The combined organic layer was washed with water and saturated brine, dried over sodium sulphate, and concentrated to afford crude product, which was purified by column chromatograph with petroleum ether/EtOAc (9:1) as eluting solvent to give the desired products 11a − 11x.

To the solution of 11a − 11x (1.0 eq) in methanol (15 ml), 20 mg of 10% Pd/C (containing 55% water) was added, and the mixture was stirred overnight in a hydrogen atmosphere under atmospheric pressure. Insoluble solid was removed by filtering, and the filtrate was concentrated in vacuo to give crude product, which was purified by column chromatograph with petroleum ether/EtOAc (8:1) as eluting solvent to give the desired products 12a − 12x.

4,5,6-trimethoxy-2–(4-methoxybenzyl)-2,3-dihydro-1H-indene (12a)

Colourless liquid; three-step yield 24.0% (71 mg, 216 µmol). ¹H NMR (300 MHz, Chloroform-d) δ 7.15 (d, J = 2.0 Hz, 1H), 7.13 (d, J = 2.2 Hz, 1H), 6.87 (d, J = 2.0 Hz, 1H), 6.85 (d, J = 2.1 Hz, 1H), 6.53 (s, 1H), 3.88 (s, 3H), 3.85 (s, 3H), 3.83 (s, 3H), 3.81 (s, 3H), 3.04 − 2.86 (m, 2H), 2.72 (d, J = 2.7 Hz, 3H), 2.68 − 2.55 (m, 2H). ¹³C NMR (75 MHz, Chloroform-d) δ 157.9, 152.7, 149.9, 140.1, 139.0, 133.5, 129.8, 127.2, 113.8, 103.8, 61.2, 60.5, 56.3, 55.3, 41.9, 40.8, 39.3, 35.8. MS(ESI) m/z 329.2 [M + H]⁺.

2–(3,4-dimethoxybenzyl)-4,5,6-trimethoxy-2,3-dihydro-1H-indene (12b)

White solid; three-step yield 21.7% (70 mg, 195 µmol). ¹H NMR (300 MHz, Chloroform-d) δ 6.83 − 6.79 (m, 1H), 6.76 (d, J = 1.9 Hz, 1H), 6.74 (d, J = 1.6 Hz, 1H), 6.53 (s, 1H), 3.88 (s, 3H), 3.87 (s, 3H), 3.87 (s, 3H), 3.84 (s, 3H), 3.82 (s, 3H), 3.05 − 2.87 (m, 2H), 2.78 − 2.69 (m, 3H), 2.67 − 2.55 (m, 2H). ¹³C NMR (75 MHz, Chloroform-d) δ 152.75, 149.84, 148.81, 147.28, 140.12, 138.98, 134.01, 127.18, 120.76, 112.06, 111.13, 103.76, 61.19, 60.49, 56.26, 55.98, 55.91, 41.77, 41.30, 39.35, 35.82. MS(ESI) m/z 381.2 [M + Na]⁺.

4,5,6-trimethoxy-2–(3,4,5-trimethoxybenzyl)-2,3-dihydro-1H-indene (12c)

White solid; three-step yield 27.5% (96 mg, 247 µmol). ¹H NMR (300 MHz, Chloroform-d) δ 6.54 (s, 1H), 6.42 (s, 2H), 3.87 (s, 3H), 3.86 (s, 6H), 3.84 (d, J = 1.0 Hz, 6H), 3.82 (s, 3H), 2.98 (ddd, J = 19.5, 15.0, 6.5 Hz, 2H), 2.78 − 2.68 (m, 3H), 2.61 (dd, J = 15.1, 5.8 Hz, 2H). ¹³C NMR (75 MHz, Chloroform-d) δ 153.13, 152.79, 149.84, 140.13, 138.91, 137.21, 136.13, 127.11, 105.64, 103.75, 61.20, 61.00, 60.52, 56.27, 56.15, 42.07, 41.63, 39.37, 35.83. MS(ESI) m/z 411.2 [M + Na]⁺.

2-methoxy-4-((4,5,6-trimethoxy-2,3-dihydro-1H-inden-2-yl)methyl)phenol (12d)

White solid; three-step yield 17.8% (55 mg, 160 µmol). ¹H NMR (300 MHz, Chloroform-d) δ 6.83 − 6.75 (m, 2H), 6.68 (dd, J = 8.2, 2.1 Hz, 1H), 6.53 (s, 1H), 5.68 (s, 1H), 3.87 (s, 6H), 3.84 (s, 3H), 3.82 (s, 3H), 2.96 (ddd, J = 21.2, 14.9, 6.1 Hz, 2H), 2.77 − 2.65 (m, 3H), 2.65 − 2.54 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 152.70, 149.82, 145.46, 144.90, 140.07, 139.05, 134.71, 127.23, 120.30, 115.09, 110.60, 103.75, 61.19, 60.49, 56.25, 56.05, 41.67, 41.08, 39.32, 35.82. MS(ESI) m/z 345.2 [M + H]⁺. Purity of the title compound was estimated by a SHIMADZU HPLC system (SHIMADZU Labsolutions). UV detection was performed at λ = 254 nm, and flow rate was 1.0 ml/min. HPLC trace is in the Supporting Information. HPLC retention time 5.206 min, 95.009% pure (MeOH: H₂O = 85: 15).

2–(3-fluoro-4-methoxybenzyl)-4,5,6-trimethoxy-2,3-dihydro-1H-indene (12e)

Colourless liquid; three-step yield 14.1% (44 mg, 127 µmol). ¹H NMR (300 MHz, Chloroform-d) δ 6.98 − 6.91 (m, 1H), 6.89 (dd, J = 4.5, 1.4 Hz, 2H), 6.52 (s, 1H), 3.88 (s, 3H), 3.87 (s, 3H), 3.84 (s, 3H), 3.82 (s, 3H), 2.96 (ddd, J = 18.7, 14.8, 6.4 Hz, 2H), 2.70 (d, J = 2.6 Hz, 3H), 2.59 (dq, J = 16.7, 2.4 Hz, 2H). ¹³C NMR (75 MHz, Chloroform-d) δ 152.8, 152.3 (d, J = 245.1 Hz), 149.8, 145.8 (d, J = 10.7 Hz), 140.2, 138.8, 134.5 (d, J = 5.9 Hz), 127.1, 124.4 (d, J = 3.4 Hz), 116.5 (d, J = 17.7 Hz), 113.4 (d, J = 2.3 Hz), 103.8, 61.2, 60.5, 56.4, 56.3, 41.6, 40.7, 39.3, 35.7. MS(ESI) m/z 369.2 [M + Na]⁺.

2–(3-fluoro-4-methylbenzyl)-4,5,6-trimethoxy-2,3-dihydro-1H-indene (12f)

Colourless liquid; three-step yield 12.4% (37 mg, 112 µmol). ¹H NMR (300 MHz, Chloroform-d) δ 7.16 − 7.05 (m, 1H), 6.89 (d, J = 2.0 Hz, 1H), 6.86 (dd, J = 5.3, 1.6 Hz, 1H), 6.53 (s, 1H), 3.88 (s, 3H), 3.85 (s, 3H), 3.83 (s, 3H), 3.07 − 2.87 (m, 2H), 2.73 (q, J = 1.6 Hz, 3H), 2.66 − 2.54 (m, 2H), 2.26 (d, J = 2.0 Hz, 3H). ¹³C NMR (75 MHz, Chloroform-d) δ 161.4 (d, J = 244.2 Hz), 152.8, 149.9, 141.1 (d, J = 7.0 Hz), 140.2, 138.8, 131.3 (d, J = 5.5 Hz), 127.1, 124.3 (d, J = 3.1 Hz), 122.2 (d, J = 17.3 Hz), 115.3 (d, J = 21.6 Hz), 103.8, 61.2, 60.5, 56.3, 41.5, 41.1, 39.3, 35.8, 14.3 (d, J = 3.5 Hz). MS(ESI) m/z 353.2 [M + H]⁺.

2–(3,4-difluorobenzyl)-4,5,6-trimethoxy-2,3-dihydro-1H-indene (12 g)

White solid; three-step yield 5.6% (17 mg, 51 µmol). ¹H NMR (400 MHz, Chloroform-d) δ 7.13 − 6.96 (m, 2H), 6.94 − 6.86 (m, 1H), 6.53 (s, 1H), 3.87 (s, 3H), 3.84 (s, 3H), 3.82 (s, 3H), 3.04 − 2.86 (m, 2H), 2.77 − 2.63 (m, 3H), 2.63 − 2.53 (m, 2H). ¹³C NMR (75 MHz, Chloroform-d) δ 152.9, 149.8, 140.2, 138.6, 138.3 (t, J = 4.6 Hz), 126.9, 125.3 − 124.1 (m), 117.6, 117.4, 117.2, 117.0, 103.7, 61.2, 60.5, 56.3, 41.5, 40.8, 39.2, 35.7. MS(ESI) m/z 335.1 [M + H]⁺.

2-methyl-4-((4,5,6-trimethoxy-2,3-dihydro-1H-inden-2-yl)methyl)aniline (12h)

Colourless liquid; three-step yield 8.15% (24 mg, 73 µmol). ¹H NMR (300 MHz, Chloroform-d) δ 6.89 (d, J = 9.3 Hz, 2H), 6.64 (d, J = 7.8 Hz, 1H), 6.54 (s, 1H), 3.88 (d, J = 1.3 Hz, 3H), 3.85 (d, J = 1.2 Hz, 3H), 3.83 (d, J = 1.3 Hz, 3H), 3.64 − 3.43 (m, 2H), 2.96 (ddd, J = 21.3, 14.5, 6.4 Hz, 2H), 2.82 − 2.55 (m, 5H), 2.18 (s, 3H). ¹³C NMR (75 MHz, Chloroform-d) δ 152.7, 149.8, 142.6, 140.1, 139.2, 131.6, 131.0, 127.4, 127.3, 122.5, 115.1, 103.8, 61.2, 60.5, 56.3, 41.9, 40.9, 39.4, 35.9, 17.6. MS(ESI) m/z 328.2 [M + H]⁺.

2–(3,4-dimethylbenzyl)-4,5,6-trimethoxy-2,3-dihydro-1H-indene (12i)

Colourless liquid; three-step yield 29.6% (87 mg, 267 µmol). ¹H NMR (300 MHz, Chloroform-d) δ 7.10 (d, J = 7.5 Hz, 1H), 7.02 (s, 1H), 6.98 (dd, J = 7.6, 2.0 Hz, 1H), 6.55 (s, 1H), 3.90 (s, 3H), 3.87 (s, 3H), 3.85 (s, 3H), 3.00 (ddd, J = 21.6, 15.0, 6.2 Hz, 2H), 2.83 − 2.71 (m, 3H), 2.64 (dd, J = 15.2, 5.6 Hz, 2H), 2.29 (s, 3H), 2.27 (s, 3H). ¹³C NMR (75 MHz, Chloroform-d) δ 152.7, 149.8, 140.1, 139.1, 138.8, 136.5, 134.1, 130.3, 129.7, 127.3, 126.3, 103.8, 61.2, 60.5, 56.3, 41.7, 41.3, 39.4, 35.9, 19.9, 19.4. MS(ESI) m/z 327.2 [M + H]⁺.

2-methoxy-5-((4,5,6-trimethoxy-2,3-dihydro-1H-inden-2-yl)methyl)aniline (12j)

Colourless liquid; three-step yield 12.3% (38 mg, 111 µmol). ¹H NMR (300 MHz, Chloroform-d) δ 6.72 (d, J = 8.0 Hz, 1H), 6.57 (d, J = 11.0 Hz, 2H), 6.52 (s, 1H), 3.87 (s, 3H), 3.84 (s, 6H), 3.82 (s, 3H), 3.77 (s, 2H), 2.96 (ddd, J = 21.7, 14.7, 6.5 Hz, 2H), 2.77 − 2.51 (m, 5H). ¹³C NMR (75 MHz, Chloroform-d) δ 152.7, 149.9, 145.8, 140.1, 139.2, 136.0, 134.1, 127.3, 118.8, 115.8, 110.4, 103.8, 61.2, 60.5, 56.3, 55.7, 41.7, 41.1, 39.4, 35.9. MS(ESI) m/z 344.2 [M + H]⁺.

2-methoxy-5-((4,5,6-trimethoxy-2,3-dihydro-1H-inden-2-yl)methyl)pyridine (12k)

Colourless liquid; three-step yield 27.0% (80 mg, 243 µmol). ¹H NMR (300 MHz, Chloroform-d) δ 7.98 (d, J = 2.4 Hz, 1H), 7.43 (dd, J = 8.5, 2.5 Hz, 1H), 6.74 − 6.65 (m, 1H), 6.51 (s, 1H), 3.92 (s, 3H), 3.86 (s, 3H), 3.82 (s, 3H), 3.81 (s, 3H), 2.95 (ddd, J = 21.3, 12.7, 4.4 Hz, 2H), 2.68 (d, J = 2.8 Hz, 3H), 2.64 − 2.54 (m, 2H). ¹³C NMR (75 MHz, Chloroform-d) δ 162.8, 152.9, 149.8, 146.5, 140.2, 139.3, 138.6, 129.2, 126.9, 110.6, 103.8, 61.2, 60.5, 56.3, 53.4, 41.5, 39.2, 37.8, 35.7. MS(ESI) m/z 330.2 [M + H]⁺.

2-benzyl-4,5,6-trimethoxy-2,3-dihydro-1H-indene (12 l)

Colourless liquid; three-step yield 23.1% (62 mg, 208 µmol). ¹H NMR (300 MHz, Chloroform-d) δ 7.40 − 7.32 (m, 2H), 7.27 (dt, J = 6.3, 1.6 Hz, 3H), 6.59 (s, 1H), 3.93 (s, 3H), 3.90 (s, 3H), 3.88 (s, 3H), 3.13 − 2.90 (m, 2H), 2.84 (d, J = 2.6 Hz, 3H), 2.73 − 2.63 (m, 2H). ¹³C NMR (75 MHz, Chloroform-d) δ 152.7, 149.8, 141.4, 140.1, 139.0, 129.0, 128.4, 127.2, 126.0, 103.7, 61.2, 60.5, 56.3, 41.7, 41.7, 39.4, 35.9. MS(ESI) m/z 299.2 [M + H]⁺.

4-((4,5,6-trimethoxy-2,3-dihydro-1H-inden-2-yl)methyl)phenol (12 m)

Colourless liquid; three-step yield 17.0% (48 mg, 153 µmol). ¹H NMR (300 MHz, Chloroform-d) δ 7.10 − 7.02 (m, 2H), 6.83 − 6.74 (m, 2H), 6.54 (s, 1H), 5.81 (s, 1H), 3.88 (s, 3H), 3.86 (s, 3H), 3.83 (s, 3H), 2.96 (tt, J = 16.6, 14.9, 4.9 Hz, 2H), 2.70 (d, J = 2.7 Hz, 3H), 2.61 (dd, J = 15.9, 4.9 Hz, 2H). ¹³C NMR (75 MHz, Chloroform-d) δ 154.1, 152.6, 149.7, 139.9, 139.2, 133.2 130.0, 127.3, 115.3, 103.9, 61.3, 60.5, 56.3, 41.9, 40.8, 39.3, 35.8. MS(ESI) m/z 315.2 [M + H]⁺.

4-((4,5,6-trimethoxy-2,3-dihydro-1H-inden-2-yl)methyl)aniline (12n)

Colourless liquid; three-step yield 11.0% (31 mg, 99 µmol). ¹H NMR (300 MHz, Chloroform-d) δ 7.02 (d, J = 1.9 Hz, 1H), 7.00 (d, J = 2.1 Hz, 1H), 6.66 (d, J = 1.9 Hz, 1H), 6.64 (d, J = 2.0 Hz, 1H), 6.53 (s, 1H), 3.87 (s, 3H), 3.85 (s, 3H), 3.83 (s, 3H), 3.75 − 3.31 (m, 2H), 3.04 − 2.86 (m, 2H), 2.75 − 2.55 (m, 5H). ¹³C NMR (75 MHz, Chloroform-d) δ 152.7, 149.8, 144.4, 140.1, 139.1, 131.4, 129.7, 127.3, 115.3, 103.8, 61.2, 60.4, 56.3, 41.9, 40.9, 39.3, 35.8. MS(ESI) m/z 314.2 [M + H]⁺.

N,N-dimethyl-4-((4,5,6-trimethoxy-2,3-dihydro-1H-inden-2-yl)methyl)aniline (12o)

White solid; three-step yield 25.7% (79 mg, 232 µmol). ¹H NMR (300 MHz, Chloroform-d) δ 7.12 (s, 1H), 7.09 (s, 1H), 6.74 (s, 1H), 6.72 (d, J = 1.7 Hz, 1H), 6.54 (s, 1H), 3.88 (s, 3H), 3.85 (s, 3H), 3.83 (s, 3H), 3.06 − 2.88 (m, 8H), 2.80 − 2.57 (m, 5H). ¹³C NMR (75 MHz, Chloroform-d) δ 152.7, 149.9, 149.2, 140.1, 139.2, 129.6, 127.4, 113.0, 103.8, 61.2, 60.5, 56.3, 41.9, 41.0, 40.7, 39.4, 35.9. MS(ESI) m/z 342.2 [M + H]⁺.

4,5,6-trimethoxy-2–(4-(trifluoromethyl)benzyl)-2,3-dihydro-1H-indene (12p)

Colourless liquid; three-step yield 25.8% (85 mg, 232 µmol). ¹H NMR (300 MHz, Chloroform-d) δ 7.58 (s, 1H), 7.55 (s, 1H), 7.34 (s, 1H), 7.31 (s, 1H), 6.53 (s, 1H), 3.88 (s, 3H), 3.84 (s, 3H), 3.83 (s, 3H), 2.97 (ddd, J = 18.3, 15.1, 6.8 Hz, 2H), 2.88 − 2.71 (m, 3H), 2.61 (ddd, J = 15.3, 6.2, 2.4 Hz, 2H). ¹³C NMR (75 MHz, Chloroform-d) δ 152.9, 149.9, 145.5, 140.2 (d, J = 6.2 Hz), 138.6, 129.2, 128.8 − 124.0 (m), 103.7, 61.2, 60.5, 56.3, 41.5, 41.4, 39.3, 35.8. MS(ESI) m/z 367.2 [M + H]⁺.

5-((4,5,6-trimethoxy-2,3-dihydro-1H-inden-2-yl)methyl)-2,3-dihydrobenzofuran (12q)

Colourless liquid; three-step yield 28.1% (86 mg, 252.6 µmol). ¹H NMR (300 MHz, Chloroform-d) δ 7.05 (d, J = 2.0 Hz, 1H), 6.94 (dd, J = 8.1, 1.9 Hz, 1H), 6.73 (d, J = 8.1 Hz, 1H), 6.54 (s, 1H), 4.56 (t, J = 8.7 Hz, 2H), 3.88 (s, 3H), 3.85 (s, 3H), 3.83 (s, 3H), 3.20 (t, J = 8.7 Hz, 2H), 3.06 − 2.86 (m, 2H), 2.71 (d, J = 2.7 Hz, 3H), 2.66 − 2.56 (m, 2H). ¹³C NMR (75 MHz, Chloroform-d) δ 158.4, 152.7, 149.8, 140.1, 139.0, 133.4, 128.3, 127.3, 127.0, 125.4, 109.0, 103.8, 71.2, 61.2, 60.5, 56.3, 42.0, 41.1, 39.3, 35.8, 29.9. MS(ESI) m/z 341.2 [M + H]⁺.

6-((4,5,6-trimethoxy-2,3-dihydro-1H-inden-2-yl)methyl)-2,3-dihydrobenzo[b]^1,4dioxine (12r)

Colourless liquid; three-step yield 24.3% (78 mg, 219 µmol). ¹H NMR (400 MHz, Chloroform-d) δ 6.80 (d, J = 8.2 Hz, 1H), 6.72 (d, J = 2.1 Hz, 1H), 6.68 (dd, J = 8.2, 2.1 Hz, 1H), 6.53 (s, 1H), 4.25 (s, 4H), 3.87 (s, 3H), 3.84 (s, 3H), 3.82 (s, 3H), 2.96 (ddd, J = 28.7, 15.2, 6.8 Hz, 2H), 2.76 − 2.65 (m, 3H), 2.64 − 2.54 (m, 2H). ¹³C NMR (75 MHz, Chloroform-d) δ 152.7, 149.8, 143.3, 141.8, 140.1, 139.0, 134.7, 127.2, 121.9, 117.5, 117.1, 103.8, 64.5, 64.4, 61.2, 60.5, 56.3, 41.6, 41.0, 39.3, 35.8. MS(ESI) m/z 379.2 [M + Na]⁺.

4-((4,5,6-trimethoxy-2,3-dihydro-1H-inden-2-yl)methyl)-1H-indole (12s)

Colourless liquid; three-step yield 10.9% (33 mg, 98 µmol). ¹H NMR (300 MHz, Chloroform-d) δ 8.18 (s, 1H), 7.23 − 7.13 (m, 1H), 7.12 − 7.01 (m, 2H), 6.86 (d, J = 7.1 Hz, 1H), 6.49 (td, J = 2.2, 1.0 Hz, 1H), 6.43 (s, 1H), 3.76 (s, 3H), 3.74 (s, 3H), 3.72 (s, 3H), 3.00 − 2.74 (m, 5H), 2.62 (td, J = 9.7, 5.0 Hz, 2H). ¹³C NMR (75 MHz, Chloroform-d) δ 152.67, 149.89, 140.56, 139.32, 135.85, 133.62, 127.63, 127.37, 123.77, 122.10, 119.79, 109.15, 103.83, 101.14, 61.24, 60.50, 56.29, 40.87, 39.67, 39.26, 36.23. MS(ESI) m/z 360.2 [M + Na]⁺.

5-((4,5,6-trimethoxy-2,3-dihydro-1H-inden-2-yl)methyl)-1H-indole (12t)

Colourless liquid; three-step yield 12.8% (39 mg, 116 µmol). ¹H NMR (300 MHz, Chloroform-d) δ 8.14 (s, 1H), 7.47 (s, 1H), 7.34 (d, J = 8.3 Hz, 1H), 7.20 (t, J = 2.8 Hz, 1H), 7.07 (dd, J = 8.3, 1.6 Hz, 1H), 6.56 − 6.48 (m, 2H), 3.86 (s, 3H), 3.84 (s, 3H), 3.82 (s, 3H), 2.97 (ddd, J = 21.9, 15.1, 6.7 Hz, 2H), 2.89 − 2.76 (m, 3H), 2.67 (dd, J = 15.0, 5.9 Hz, 2H). ¹³C NMR (101 MHz, Chloroform-d) δ 152.7, 149.9, 140.2, 139.3, 134.6, 132.8, 128.2, 127.5, 124.4, 123.5, 120.5, 110.9, 103.9, 102.4, 61.2, 60.5, 56.3, 42.3, 41.9, 39.5, 36.0. MS(ESI) m/z 360.2 [M + Na]⁺.

6-((4,5,6-trimethoxy-2,3-dihydro-1H-inden-2-yl)methyl)-1H-indole (12 u)

Colourless liquid; three-step yield 7.6% (23 mg, 68 µmol). ¹H NMR (300 MHz, Chloroform-d) δ 8.25 (s, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.23 − 7.12 (m, 2H), 7.01 (d, J = 7.9 Hz, 1H), 6.59 − 6.49 (m, 2H), 3.92 − 3.85 (m, 6H), 3.84 (s, 3H), 3.00 (dq, J = 14.2, 7.8, 7.1 Hz, 2H), 2.92 − 2.78 (m, 3H), 2.68 (dd, J = 15.0, 5.8 Hz, 2H). ¹³C NMR (75 MHz, Chloroform-d) δ 152.8, 149.9, 140.2, 139.4, 136.3, 135.4, 127.4, 126.2, 124.0, 121.4, 120.6, 111.2, 103.9, 102.4, 61.3, 60.6, 56.4, 42.2, 42.2, 39.6, 36.0. MS(ESI) m/z 360.2 [M + Na]⁺.

1-methyl-4-((4,5,6-trimethoxy-2,3-dihydro-1H-inden-2-yl)methyl)-1H-indole (12v)

Yellow liquid; three-step yield 15.8% (50 mg, 142 µmol). ¹H NMR (300 MHz, Chloroform-d) δ 7.29 − 7.19 (m, 2H), 7.07 (d, J = 3.1 Hz, 1H), 7.00 (dd, J = 6.1, 2.0 Hz, 1H), 6.55 (d, J = 3.0 Hz, 2H), 3.90 (s, 3H), 3.88 (s, 3H), 3.85 (s, 3H), 3.82 (s, 3H), 3.11 − 2.96 (m, 4H), 2.96 − 2.87 (m, 1H), 2.82 − 2.68 (m, 2H). ¹³C NMR (101 MHz, Chloroform-d) δ 152.71, 149.91, 140.14, 139.26, 136.78, 133.74, 128.34, 128.23, 127.40, 121.68, 119.38, 107.30, 103.88, 99.46, 61.18, 60.44, 56.29, 40.89, 39.68, 39.19, 36.24, 33.02. MS(ESI) m/z 352.2 [M + H]⁺.

1-methyl-5-((4,5,6-trimethoxy-2,3-dihydro-1H-inden-2-yl)methyl)-1H-indole (12w)

Brown liquid; three-step yield 21.8% (69 mg, 196 µmol). ¹H NMR (300 MHz, Chloroform-d) δ 7.48 (d, J = 1.7 Hz, 1H), 7.29 (d, J = 8.3 Hz, 1H), 7.12 (dd, J = 8.4, 1.7 Hz, 1H), 7.05 (d, J = 3.1 Hz, 1H), 6.55 (s, 1H), 6.46 (dd, J = 3.1, 0.9 Hz, 1H), 3.89 (s, 3H), 3.87 (s, 3H), 3.85 (s, 3H), 3.80 (s, 3H), 3.08 − 2.92 (m, 2H), 2.91 − 2.76 (m, 3H), 2.75 − 2.63 (m, 2H). ¹³C NMR (75 MHz, Chloroform-d) δ 152.7, 149.9, 140.1, 139.3, 135.5, 132.2, 129.0, 128.7, 127.5, 123.0, 120.6, 109.1, 103.8, 100.6, 61.2, 60.5, 56.3, 42.3, 41.9, 39.4, 36.0, 32.9. MS(ESI) m/z 374.2 [M + Na]⁺.

1-methyl-6-((4,5,6-trimethoxy-2,3-dihydro-1H-inden-2-yl)methyl)-1H-indole (12x)

Brown liquid; three-step yield 5.1% (16 mg, 45 µmol). ¹H NMR (400 MHz, Chloroform-d) δ 7.57 (d, J = 7.9 Hz, 1H), 7.16 (s, 1H), 7.04 − 6.99 (m, 2H), 6.55 (d, J = 1.7 Hz, 1H), 6.47 (dd, J = 3.1, 0.9 Hz, 1H), 3.88 (s, 3H), 3.86 (s, 3H), 3.84 (s, 3H), 3.79 (s, 3H), 3.08 − 2.96 (m, 2H), 2.96 − 2.83 (m, 3H), 2.82 − 2.60 (m, 2H). ¹³C NMR (100 MHz, Chloroform-d) δ 152.8, 149.9, 140.2, 139.2, 137.1, 135.0, 128.6, 127.4, 126.8, 120.9, 120.7, 109.2, 103.9, 100.8, 61.2, 60.5, 56.3, 42.2, 39.5, 39.5, 36.0, 32.9. MS(ESI) m/z 352.2 [M + H]⁺.

General procedure of synthesis of compounds 15a − 15c

To 50 ml of trifluoromethanesulfonic acid (TfOH) in an ice bath, substituted phenylpropanoic acid (13a − 13b) or 2–(3,4,5-trimethoxyphenoxy)acetic acid (13c) was added. The mixture was allowed to stir at 0 °C for 0.5 h until reaction was completed as TLC monitored (petroleum ether/EtOAc, 6:1). Then the mixture was poured into appropriate amount of ice water, and the red solid was separated out. The precipitate was filtered and dried under reduced pressure to afford intermediates 14a − 14c. Compound 15a − 15c were synthesised from 14a − 14c followed the similar procedure of compound 12a − 12x.

5-((5,6-dimethoxy-2,3-dihydro-1H-inden-2-yl)methyl)-2-methoxyphenol (15a)

White solid; three-step yield 23.5% (77 mg, 245 µmol). ¹H NMR (300 MHz, Chloroform-d) δ 6.83 − 6.76 (m, 2H), 6.73 (s, 2H), 6.69 (dd, J = 8.2, 2.1 Hz, 1H), 5.64 (s, 1H), 3.88 (s, 3H), 3.85 (s, 6H), 2.93 (dd, J = 14.9, 6.7 Hz, 2H), 2.76 − 2.65 (m, 3H), 2.61 (dd, J = 14.9, 5.9 Hz, 2H). ¹³C NMR (75 MHz, Chloroform-d) δ 147.8, 145.4, 144.8, 134.9, 134.8, 120.3, 115.1, 110.6, 107.9, 56.1, 56.1, 41.9, 41.1, 38.9. MS(ESI) m/z 337.1 [M + Na]⁺.

2–(3-hydroxy-4-methoxybenzyl)-6-methoxy-2,3-dihydro-1H-inden-5-ol (15b)

Colourless liquid; three-step yield 40.7% (91 mg, 303 µmol). ¹H NMR (300 MHz, Chloroform-d) δ 6.86 − 6.74 (m, 3H), 6.74 − 6.64 (m, 2H), 5.70 (s, 1H), 5.58 (s, 1H), 3.88 (s, 3H), 3.86 (s, 3H), 2.92 (ddd, J = 15.2, 6.6, 4.5 Hz, 2H), 2.83 − 2.65 (m, 3H), 2.59 (dt, J = 15.5, 5.4 Hz, 2H). ¹³C NMR (75 MHz, Chloroform-d) δ 145.5, 145.3, 144.9, 144.3, 135.6, 135.0, 134.2, 120.3, 115.2, 110.7, 110.6, 107.4, 56.2, 56.1, 42.0, 41.1, 38.9, 38.7. MS(ESI) m/z 323.1 [M + Na]⁺.

2-methoxy-5-((4,5,6-trimethoxy-2,3-dihydrobenzofuran-2-yl)methyl)phenol (15c)

Colourless liquid; three-step yield 20.7% (64 mg, 185 µmol). ¹H NMR (300 MHz, Chloroform-d) δ 6.85 (d, J = 2.0 Hz, 1H), 6.83 − 6.77 (m, 1H), 6.73 (dd, J = 8.2, 2.0 Hz, 1H), 6.19 (s, 1H), 5.72 (s, 1H), 4.94 (dq, J = 8.5, 6.9 Hz, 1H), 3.90 (s, 3H), 3.86 (s, 3H), 3.79 (s, 3H), 3.77 (s, 3H), 3.22 (dd, J = 15.0, 8.8 Hz, 1H), 3.06 (dd, J = 13.8, 6.9 Hz, 1H), 2.94 (dd, J = 15.1, 7.3 Hz, 1H), 2.82 (dd, J = 13.8, 6.7 Hz, 1H). ¹³C NMR (75 MHz, Chloroform-d) δ 156.1, 153.8, 150.1, 145.6, 145.5, 134.9, 130.7, 120.9, 115.6, 110.8, 108.6, 90.1, 84.5, 61.3, 60.0, 56.2, 56.0, 41.4, 33.4. MS(ESI) m/z 369.1 [M + Na]⁺.

Synthesis of compound 15d

To the solution of 10d (100 mg, 280 µmol, 1.0 eq) in methanol (20 ml), 10 mg of 10% Pd/C (containing 55% water) was added, and the mixture was stirred overnight in a hydrogen atmosphere under atmospheric pressure. Insoluble solid was removed by filtering, and the filtrate was concentrated in vacuo to give crude product, which was purified by column chromatograph with petroleum ether/EtOAc (6:1) as eluting solvent to give 2–(3-hydroxy-4-methoxybenzyl)-5,6,7-trimethoxy-2,3-dihydro-1H-inden-1-one (15d) as a white solid (72 mg, 201 µmol, yield 71.6%). ¹H NMR (300 MHz, Chloroform-d) δ 6.80 (d, J = 2.0 Hz, 1H), 6.75 (d, J = 8.2 Hz, 1H), 6.69 (dd, J = 8.1, 2.0 Hz, 1H), 6.57 (s, 1H), 5.68 (s, 1H), 4.05 (s, 3H), 3.89 (s, 3H), 3.84 (s, 3H), 3.83 (s, 3H), 3.24 (dd, J = 13.9, 4.2 Hz, 1H), 3.02 (dd, J = 16.8, 7.7 Hz, 1H), 2.95 − 2.84 (m, 1H), 2.73 (dd, J = 16.8, 3.7 Hz, 1H), 2.54 (dd, J = 13.9, 10.1 Hz, 1H). ¹³C NMR (100 MHz, Chloroform-d) δ 204.1, 159.9, 151.8, 151.7, 145.6, 145.2, 140.8, 133.0, 122.3, 120.5, 115.2, 110.8, 103.8, 62.0, 61.5, 56.3, 56.0, 49.7, 36.8, 32.0. MS(ESI) m/z 359.1 [M + H]⁺.

In vitro biological experiments

Antiproliferative assay

The four cancer cell lines (K562, A549, Hela and H22) and one normal cell line HFL-1 were used in the CCK-8 antiproliferative activity assay. All of cell lines were grown at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air in RPMI-1640 medium with 10% foetal bovine serum. Cells were seed into 96-well plates with a density of about 3.5 × 10³ cells/well and cultured for 24 h. Then the cells were treated with 100 µL of medium with or without tested compounds at different concentrations and cultured for 72 h in three replicates, and CA-4 was used as a positive control. Subsequently, 10 µL of CCK-8 was added to each well and incubated for another 2–3 h. Finally, the optical density at a wavelength of 450 nm was detected with BioTek EL-x800 microplate reader (BioTek Instruments, US). The inhibition rate or the IC₅₀ value were calculated using GraphPad Prism.

Immunofluorescence assay

The exponentially growing K562 cells were seeded into 6-well cell plates (5 × 10⁵ cells/well). After treatment with different concentrations of tested compound (15, 30, and 60 nM) for 48 h, the cells were fixed with 4% paraformaldehyde and washed with PBS for three times, and blank control was set. After blocking by added 50–100 µL of goat serum albumin at room temperature for 20 min, the cells were incubated with a monoclonal antibody (anti-α-tubulin, Abcam, UK) at 37 °C for 2 h. Then the cells were washed with PBS three times, and then stained by the FITC fluorescence secondary antibody at 37 °C for another 1 h. After washed with PBS, the nucleus was stained by 100 µL of 4,6-diamidino-2-phenylindole (DAPI) in the dark for 5 min. Finally, cells were visualised using Confocal microscope (Olympus, Japan).

In vitro tubulin polymerization inhibitory assay

The tubulin was resuspended in PEM buffer (containing 80 mM PIPES(pH 6.9), 0.5 mM EGTA, 2 mM MgCl₂, and 15% glycerol). Then, the mixture was incubated with compound 12d or CA-4 at different concentrations (0.1, 0.5, 1, 5, 10 µM) for 5 min. After PEG (containing 3 mg/mL of GTP) was added for 30 min, the absorbance at a wavelength of 340 nm was detected with Berthold LB941 microplate reader (Berthold, Germany). Competition scintillation proximity assay (SPA) was performed to evaluate the competitive binding activity of test compounds against radiolabeled [³H]-colchicine. Compound 12d or CA-4 was added to a 100 µL of buffer (containing 80 mM PIPES (pH 6.8), 1 mM EGTA, 1 mM MgCl₂, 10% glycerol, and 1 mM GTP), then [³H]-colchicine (5 µM) was added subsequently. After the mixture was incubated for 2 h, 80 µg of streptavidin-labeled SPA beads were added, and the radioactive counts were measured with a scintillation counter⁴⁸.

Analysis of cell apoptosis

The exponentially growing K562 cells were seeded into 6-well cell plates. After treating with different concentrations of test compound for 48 h, 5 × 10⁵ cells of each group were harvested and resuspended with 500 µL of binding buffer. Then, 5 µL of Annexin V-FITC and 5 µL of PI were added respectively. Finally, after incubating for 15–30 min in the dark at room temperature for 5–15 min, the samples were analysed by a CytoFLEX flow cytometer (Beckman Coulter, US).

Hoechst 33342 staining

After K562 cells were treated with or without different concentrations of tested compound for 48 h, the medium was removed and air dried. Then the samples were fixed with 4% paraformaldehyde overnight and washed in PBS for three times, then resuspended in 500 µL PBS. Thereafter, cells were stained by Hoechst 33342 solution for 15 min in the dark at room temperature, the images of cells were captured (magnification 200×) by a IX51 biological inverted microscope (Olympus, Japan)⁴⁹.

Western blot analysis

After K562 cells were treated with or without different concentrations of tested compound for 48 h, cells were harvested and washed with PBS for two times. The collected cells were lysed by the cold Lysis Buffer containing 5 µL of 100 mM phenylmethylsulfonyl fluoride (PMSF), following by centrifugation with 14,000 rpm for 15 min. Protein concentrations of supernatant were determined by using BCA assay, and then equal amounts of protein were separated by SDS-PAGE and transferred to poly(vinylidene fluoride) (PDVF) membranes. After blocked with 5% skim milk for 2 h, the membranes were incubated with primary antibodies against Cdc-2, Cyclin B1, Cdc25c, Bad, Bax, Bcl-2, Bcl-xl (Abcam, UK) and GAPDH (KeyGEN BioTECH, China) overnight at 4 °C. The membranes were then washed with TBST three times for 10 min, incubated with secondary antibody for 1–2 h, and washed with TBST again. Finally, the proteins were stained with ECL chemiluminescence immunoassay kit, and the images were captured by bio-rad ChemiDoc Touch (Bio-rad, US). The data was analysed by software Gel-Pro32.

Cell cycle analysis

The exponentially growing K562 cells were seeded into 6-well cell plates, and the cells were treated with or without different concentrations of tested compound for 48 h. After washed with PBS for two times, 5 × 10⁵ cells were harvested by centrifugal method with 1000 rpm for 5 min, and then fixed in 70% cold ethanol for 2 h. The fixed cells were incubated with 100 µL RNase A solution for 30 min at 37 °C and 400 µL PI solution for 30 min at 4 °C in dark. Finally, samples were analysed at an excitation wavelength of 488 nm by a CytoFLEX flow cytometer (Beckman Coulter, US).

Mitochondrial membrane potential analysis

The exponentially growing K562 cells were seeded into 6-well cell plates, and the cells were treated with or without different concentrations of tested compound for 48 h. After washed with PBS for two times, K562 cells were resuspended in 500 µL of JC-1 incubation buffer at 37 °C for 15 min. Finally, the samples were detected by a CytoFLEX flow cytometer (Beckman Coulter, US).

Measurement of intracellular ROS generation

The exponentially growing K562 cells were seeded into 6-well cell plates, and the cells were treated with or without different concentrations of tested compound for 48 h. K562 cells were washed with PBS for two times and adjusted to the concentration of 1 × 10⁶ cells/mL. Then, the medium was removed and the cells were washed with PBS, stained by DCFH-DA according to ROS assay kit operating instruction, and analysed at an excitation wavelength of 488 nm by an IX51 biological inverted microscope (Olympus, Japan).

Transwell assay

In migration assay, MDA-MB-231 cells were seeded into 6-well plates at a density of 1 × 10⁵ cells/well and cultured for 24 h at 37 °C. After treatment with or without tested compound for 48 h, cells were harvested and 100 µL of cell suspension was inoculated on Transwell upper plates at a concentration of 1 × 10⁵ cells/mL. The cells in the bottom chamber were cultured in the medium containing 10% FBS for another 24 h, and the cells that had migrated to the bottom from the top chambers. Then, the Transwell was removed, and the cells were washed with PBS for two times, fixed with 4% paraformaldehyde and then stained with 0.1% crystal violet. The images of migrated cells were captured (magnification 200×) from at least three different fields of vision by an IX51 biological inverted microscope (Olympus, Japan). The difference of the migration and invasion assay is that in invasion assay, 30 µL Matrigel was added to the upper chamber to form gel, whereas in migration assay, the chamber was not coated with Matrigel⁵⁰.

Wound healing assays

After seeded in six-well plates at a concentration of 5 × 10⁵ cells/well overnight, the HUVEC cells were incubated with or without different concentrations of tested compound for 24 h. Then, the surface of cells was scratch-wounded by a 200 µL sterilised pipette, washed with PBS, and the images of wound (magnification 100×) were obtained by an IX51 biological inverted microscope (Olympus, Japan). After culturation for 24 h, images of wound healing were captured again.

. Tube formation assay

The Matrigel matrix was thawed at 4 °C overnight, and the matrix was diluted and incubated for 2 h at 37 °C to form gel in 12-well cell plates. Then HUVEC cells were seeded in 12-well cell plates and after treatment with or without tested compound for 24 h, cells were harvested and 1 ml of cell suspension was inoculated on 12-well cell plates at a concentration of 2 × 10⁵ cells/mL. After incubating for 12 h, the tube formation and morphological changes were captured by an IX51 biological inverted microscope (Olympus, Japan)⁵¹.

Molecular modelling

The structure of α- and β-tubulin complex with CA-4 (PDB id: 5LYJ) was downloaded from the RCSB PDB protein data bank (https://www.rcsb.org/). The protein was prepared by Protein Preparation Wizard module of Schrodinger. Then molecular docking was performed on Maestro 11.5, and PyMOL was used to draw the figures.

Evaluation of in vivo antitumor effect

H22 allograft mice model was used to evaluate in vivo antitumor effect of compound 12d. H22 cells at logarithmic growth phase were collected and adjusted to the concentration of 1 × 10⁷ cells/mL, then the cells were subcutaneously injected 0.1 ml into the right flank of each mouse. Next day, the mice were randomly divided into four groups (n = 6), and mice in control group were administrated with 5% DMSO/5% Tween 80/90% saline once a day by intravenous injection, while mice of each treated groups were administrated with CA-4 (20 mg/kg) dissolved in 5% DMSO/5% Tween 80/90% saline, or 12d (10 or 20 mg/kg) dissolved in 5% DMSO/5% Tween 80/90% saline once a day by intravenous injection, respectively. The tumour size and mice body weight were recorded once two days. About 21 days after the initiation of treatment, the mice were sacrificed. Then the tumours and major organs (heart, liver, spleen, lung, and kidney) were isolated for further experiments. Tumours were weighted to calculate the tumour growth inhibition (TGI) value by the formula: TGI (%) = (1-W_t/W_c) × 100%, where W_t and W_c are the mean tumour weight of the treatment group and vehicle control group, respectively.

Immunohistochemistry analysis

Immunohistochemistry (IHC) analysis was used to evaluate the expression of prognostic angiogenic marker CD31 and cell proliferation marker Ki67. The tumours were isolated from mice (control group, CA-4-treated group at 20 mg/kg, and compound 12d-treated group at 20 mg/kg), and then fixed with 4% paraformaldehyde overnight, sliced into sections, and paraffin-embedded. Then, all of these slices were blocked with 1% BSA for 20 min followed by incubation with CD31 primary antibody or Ki67 primary antibody overnight at 4 °C. Next, the slides were treated with secondary antibody with horseradish peroxidase goat anti-rabbit for 1 h and developed with 3,3′-diaminobenzidine tetrahydrochloride (DAB). After washed with water for 15 min, the slices were counterstained with haematoxylin, and finally photographed by a microscope.

Evaluation of in vivo safety profile

1.2 H&E staining was used to evaluate in vivo safety of compounds. The major organs were isolated from sacrificed mice in in vivo antitumor effect experiment (control group, CA-4-treated group at 20 mg/kg, and compound 12d-treated group at 20 mg/kg), and then fixed with 4% paraformaldehyde overnight, sliced into sections, paraffin-embedded, stained with haematoxylin and eosin (H&E). Finally, the tissue morphology was observed using a microscope.

Authors’ contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Disclosure statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability statement

Data will be made available on request.

Additional information

Funding

This study was financially supported by the National Natural Science Foundation of China (no. 81973167 and 82273472), Kunshan High-level Medical Talent Post Training Project (2018), Suzhou Health Key Medical Talent Training Project (GSWS2020112) and Suzhou Key Laboratory of Neuro-Oncology and Nano-Bionics.