The synthesis and evaluation of new benzophenone derivatives as tubulin polymerization inhibitors

Abstract

Inspired by the potent inhibition activity of phenstatin and millepachine against cancer cell growth, a series of new benzophenone derivatives were synthesized and evaluated as tubulin polymerization inhibitors. Among them, compound 10a exhibited 0.029–0.062 μM of IC₅₀ for five human cancer cell lines, which is much better than that of the two leading compounds. Flow cytometric analysis showed that 10a induces G2/M phase arrest and apoptosis in A549 cells. Cellular studies revealed that the induction of apoptosis by 10a was associated with the collapse of the mitochondrial membrane potential (MMP). Overall, the current study demonstrates that the benzophenone derivatives are promising anticancer agents by targeting tubulin.

---

Introduction

Discovering highly effective agents with low toxicity for the treatment of cancer is an important goal for pharmaceutical chemists. In the past decades, the development of anticancer agents that target microtubule stabilization or microtubule destabilization has attracted attention.^1–5 Tubulin plays a critical role in a variety of essential cellular processes such as the movement of organelles, intracellular transportation, and the formation and maintenance of cell shapes and so on.^6–8 Some tubulin inhibitors, such as colchicine,⁹ vinblastine^10,11 and noscapinoids,¹² have been used in clinical studies. Colchicine is a typical agent that targets the colchicine binding site. Recently, many compounds targeting the colchicine binding site have been reported. Combretastatin A-4 (CA-4) is a prominent tubulin polymerization inhibitor¹³ that strongly inhibits tubulin polymerization by binding to the colchicine binding site, and its disodium phosphate form (CA-4P) has been approved for clinical trials.^14,15 CA-4 analogues including benzophenone derivatives¹⁶ such as phenstatin and diphenylamine derivatives such as MPC-6827 (ref. 17) and its analogue^18,19 have also been developed. Recently, a tubulin inhibitor including a chalcone analogue²⁰ and others^21,22 have also been reported. To develop novel and more effective anticancer agents, many studies on the modification of CA-4 have appeared in recent years.^23–27 Among them, phenstatin (Fig. 1), in which the cis double bond structure of CA-4 is replaced by a carbonyl group, exhibited potent inhibition of cancer cell growth.^28,29 Natural products are another source of new microtubule-stabilizing agents.³⁰ Millepachine, with a chalcone structure (Fig. 1), which was first isolated from the Millettia pachycarpa, exhibits potent cytotoxicity against a variety of human cancer cells.³¹ In our study, to search for new structural compounds as anticancer agents, we have developed a new series of ortho-(3,4,5-trimethoxybenzoyl)-acetanilides as novel anti-cancer agents.³² In this work, inspired by the structure of millepachine and phenstatin, we disclose the synthesis, evaluation and mechanism of a series of new benzophenone derivatives as tubulin polymerization inhibitors.

Fig. 1 The structures of CA-4, phenstatin, MCP-6827, MCP-6827 analogue, millepachine and 10a.

Results and discussion

Chemistry

The general synthesis routes for the iso-MIL analogues are listed in Schemes 1 and 2. First, the key intermediate 3 was obtained with vanillina 1 as the starting material which reacted with 3-chloro-3-methyl-1-butyne and was then heated in pyridine at 120 °C overnight with an overall yield of 70–75% for the two-steps. Then, the reaction of 3 with a series of aryl lithium reagents generated in situ from bromobenzene or its analogues, and n-BuLi gave compounds 4a–4h at −78 °C. The oxidation of 4a–4h with pyridinium chlorochromate (PCC) provided target compounds 5a–5h (Scheme 1). On the other hand, the protection of bromobenzene analogues containing hydroxyl bromobenzene (6a–6b) with MOM was performed by treating them with chloromethyl methyl ether and sodium hydride in dichloromethane.

Scheme 1 The synthesis of target compound 5. Reagents and conditions: (a) 3-chloro-3-methylbut-1-yne, CuCl₂·2H₂O, DBU, CH₃CN, 0 °C, 5 h, 81.0%. (b) Pyridine, 120 °C, 12 h, 85.0%. (c) Substituted bromobenzene, n-BuLi, dry THF, −78 °C, 12 h, 42.9–65.3%. (d) PCC, CH₂Cl₂, 3–5 h, 43.1–76.5%.

Scheme 2 The synthesis of target compounds 10a–10b, 11, 12. (e) Chloromethyl methyl ether, DIPEA, 0 °C, 5 h, 95.1–96.1%. (f) 3, n-BuLi, dry THF, −78 °C, 12 h, 44.3–55.4%. (g) PCC, CH₂Cl₂, 3–5 h, 37.0–49.1%. (h) HCl, CH₃OH, 10 h, 32.1–41.5%. (i) Ethyl-triphenylphosphonium chloride, n-BuLi, dry THF, −78 °C, 12 h, HCl, CH₃OH, 10 h, 95.1%. (j) NaH, CH₃I, CH₂Cl₂, 48 h, 81.5%.

Using the same synthesis method for 5, compounds 9a–9b were obtained, and they were de-protected in the presence of HCl in methanol to give the target compounds 10a–10b. Finally, compound 11 was based on a Wittig reaction following a simple known method, and compound 10a was treated with CH₃I to give target compound 12.

Biological evaluation

In vitro human cancer cell lines growth inhibition

MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)) assay was first carried out for evaluation of the antiproliferative activities of the new iso-MIL analogues toward five human cancer cell lines A549 (non-small cell lung carcinoma), HeLa (human epithelial cervical cancer cell line), HCT-116 (human colon cancer cell line), A2780 (human ovarian cancer cell line) and MGC-803 (human gastric cancer cell line), and the results are listed in Table 1. It can be seen from Table 1 that most of the synthetic compounds have good anti-proliferative activity with IC₅₀ values in the sub-micromol level. It is interesting that compound 10a, which includes the replacement of the 3,4,5-trimethoxyphenyl moiety of phenstatin by the 8-methoxy-2,2-dimethyl-2H-chromen-6-yl, provided the best results with a 35 nM IC₅₀ value for A549 cell line, 29 nM for HeLa, 38 nM for HCT-116, 62 nM for A2780 and 49 nM for MGC-803, which are much better than the reference compound phenstatin (IC₅₀ values for A549: 179 nM, HeLa: 42 nM, HCT116: 123 nM, A2780: 190 nM, Mgc803: 904 nM). The preliminary structure–activity relationship analysis indicated that the 3-hydroxyl and 4-methoxy group in the B ring of the target compounds is necessary for inhibitory activity. Compound 5a, bearing a 4-methoxy group but no 3-hydroxyl group in the B ring, exhibited 0.196 to 1.02 μM IC₅₀ values, and its analogues, 5b and 5c, with a 3- or 2-methoxy group in the B ring, provided only good to moderate results (5b: 0.545 to 4.95 μM for five cancer cell lines, 5c: more than 10 μM IC₅₀ values for four cancer cell lines besides 0.985 μM for A549). Compounds 5d, 5e and 5f, with a fluorine atom or methyl instead of a hydroxyl group, all gave sub-micromol level IC₅₀ values for four cancer cell lines but was not as potent as 10a. To evaluate the effect of a sulphur atom in the synthesized compounds on the antiproliferative activities, we prepared compound 5g, which possesses a methylmercapto group in the 4-position of the B ring. 5g gave nearly the same activity level compared to 5a in most cases, but provided better results in the MGC-803 line (5a: 1.02 μM of the IC₅₀, 5g: 0.50 μM). The results also indicated that two or three methoxyl groups were unfavourable for the activities; 12 exhibited 0.717 to 3.056 IC₅₀ values and 5h gave more than 10 μM IC₅₀ values in all of the five cancer lines. Inspired by the excellent antiproliferative activity of iso-CA-4, we synthesised 11. Compound 11 showed the same level activities in all the cell lines compared with 10a, but was slightly lower than that of iso-CA-4. Considering the excellent antiproliferative activity and the briefness of the preparation, 10a was selected to further study.

Table 1 Antiproliferative activity of compounds against five human cancer cell lines^a

Compounds	IC50^b (μM)
	A549	HeLa	HCT116	A2780	Mgc803
a Cell lines were treated with different concentrations of the compounds for 48 h. Cell viability was measured by MTT assay as described in the Experimental section.b IC50 values are indicated as the mean ± SD (standard error) of three independent experiments.
5a	0.196 ± 0.10	0.458 ± 0.02	0.549 ± 0.12	0.476 ± 0.13	1.020 ± 0.25
5b	0.545 ± 0.011	1.23 ± 0.23	1.318 ± 0.05	4.627 ± 0.13	4.95 ± 0.34
5c	0.985 ± 0.03	>10	>10	>10	>10
5d	0.226 ± 0.11	0.279 ± 0.06	0.334 ± 0.08	0.242 ± 0.08	0.766 ± 0.13
5e	0.327 ± 0.19	1.390 ± 0.05	1.607 ± 0.12	0.358 ± 0.07	8.378 ± 0.13
5f	0.106 ± 0.01	0.125 ± 0.03	0.107 ± 0.03	0.449 ± 0.02	0.285 ± 0.08
5g	0.25 ± 0.01	0.325 ± 0.04	0.306 ± 0.04	0.636 ± 0.10	0.503 ± 0.06
5h	>10	>10	>10	>10	>10
10a	0.035 ± 0.01	0.0295 ± 0.01	0.038 ± 0.01	0.0624 ± 0.03	0.049 ± 0.01
10b	1.490 ± 0.03	1.890 ± 0.09	1.430 ± 0.23	2.680 ± 0.56	1.390 ± 0.01
11	0.059 ± 0.02	0.043 ± 0.01	0.050 ± 0.09	0.039 ± 0.06	0.079 ± 0.02
12	0.729 ± 0.23	3.056 ± 0.07	0.758 ± 0.06	0.717 ± 0.10	0.998 ± 0.12
Phenstatin	0.179 ± 0.03	0.042 ± 0.02	0.123 ± 0.05	0.190 ± 0.02	0.904 ± 0.06
Millepachine	3.750 ± 0.12	5.070 ± 0.17	5.613 ± 0.09	3.600 ± 0.14	8.900 ± 0.23
Colchicine	0.020 ± 0.04	0.046 ± 0.06	0.032 ± 0.08	0.038 ± 0.012	0.067 ± 0.04

---

Selectivity of 10a towards normal cells and cancer cells

To examine the cytotoxicity of 10a towards human normal cells (Table 2), we used BJ cells (human dermal fibroblasts) and HLF cells (human embryonic lung fibroblast) for the MTT assay comparing with the A549 cell (non-small cell lung carcinoma). The results in Table 2 showed that compound 10a has far more potent antiproliferative activity than millepachine and phenstatin. Furthermore, 10a gave 158-fold and 571-fold selectivity ratios for the BJ cell and HLF cell, respectively, which demonstrated the hypotoxicity of 10a towards the two kinds of human normal cells.

Table 2 Antiproliferative activity of compound 10a, millepachine, and phenstatin against human normal cells

Compounds	IC50^a (μM)			Selectivity ratio^b (HLF/BJ)
	A549	HLF	BJ
a Data are presented as the mean ± SE from the dose–response curves of at least three independent experiments.b Selectivity ratio = (IC50 human normal cells)/(IC50 A549).
10a	0.035 ± 0.01	5.539 ± 0.09	>20	158/>571
Millepachine	3.750 ± 0.12	>20	>20	>5/>5
Phenstatin	0.179 ± 0.03	2.421 ± 0.11	>20	14/>112

---

In vitro inhibition of tubulin polymerization

To determine whether 10a is a potent tubulin polymerization inhibitor, the tubulin polymerisation assay, which was described by Bonne, D. et al., with a moderate modification was performed, and colchicine was used as the reference compound. Fig. 2A showed that compound 10a was a potent tubulin polymerization inhibitor with an IC₅₀ value of 4.011 ± 0.14 μM, which was slightly weaker than colchicine (IC₅₀: 3.01 ± 0.12 μM, Fig. 2B).

Fig. 2 Effect of compound 10a and colchicine on microtubule dynamics. Purified tubulin protein at 10 mM in a reaction buffer was incubated at 37 °C in the absence (control) or presence of 10a (A) (10 μM, 5 μM, 3.75 μM, 2.5 μM, or 1.25 μM) and colchicine (B) (7.5 μM, 5 μM, 1 μM). Polymerizations are followed by an increase in fluorescence emission at 410 nM over a 60 min period at 37 °C (excitation wavelength is 340 nM).

Disruption of microtubule dynamics

To investigate the morphological alterations of cancer cells caused by compound 10a, A549 cells were exposed to different concentrations of 10a (25, 50 and 75 nM) for 12 h. From Fig. 3, we can see the morphological alterations such as microtubule shrinkage, membrane bleeding and chromatin condensation indeed that are normally associated with the occurrence of apoptosis occurred under the laser scanning confocal microscope.

Fig. 3 Effects of compound 10a on microtubule dynamics in vitro. A549 cells were treated with compound 10a at different concentrations (25, 50, and 75 nM) or DMSO (0.01%) for 12 h. Nucleuses were stained with Hochest 33342 (blue) and microtubules were stained with mouse anti-α-tubulin conjugated with Alex flour 488 (green). Images were taken using LSM 570 laser confocal microscope.

Apoptosis assay

To evaluate the ability of compound 10a to induce cell death, we used flow cytometry with propidium iodide (PI), which only stains DNA and enters dead cells, and fluorescent immunolabeling of the protein annexin-V (V-FITC), which selectively binds to early-apoptotic cells that expose on their surface the phospholipid phosphatidylserine. The results in Fig. 4A indicated that when A549 cells were incubated with 10a at different concentrations (25, 50, and 75 nM) or DMSO (0.01%) for 48 h, the percentages of early and late apoptosis cells were 5.94%, 38.16%, and 69.85.7%, respectively (Fig. 3A). When the incubation time was extended to 72 h, 21.13%, 69.18%, and 84.58% of early and late apoptosis cells were obtained (Fig. 4C). These data indicated that 10a induced cell apoptosis in a concentration and time dependent manner.

Fig. 4 10a induced the apoptosis of A549 cells. A549 cells were treated with 10a at the indicated concentrations for 48 h (A) and 72 h (C). Then, the cells were trypsinized, harvested and stained with Annexin V-FITC and PI solution for flow cytometry. The percentages of cells in each stage of apoptosis were quantitated by flow cytometry. ((B1) Upper left quadrant) necrotic cells; ((B2) upper right quadrant) late apoptotic cells; ((B3) bottom left quadrant) live cells; and ((B4) bottom right quadrant) early apoptotic cells. Representative images from five independent experiments were shown. Representative images from three independent experiments are shown 48 h (B) and 72 h (D).

Analysis of cell cycle arrest

To evaluate the arrest effect of compound 10a on the cell cycle, we studied the effect on cell cycle progression using propidium iodide (PI) staining by flow cytometry analysis in A549 cells. After treatment with DMSO (0.01%) or compound 10a at varied concentrations (25, 50, and 75 nM) for 24 h, the cells were harvested and analysed by flow cytometry (Fig. 5), which indicated compound 10a resulted in a significant cell-cycle arrest at the G2/M phase and showed a dose-dependent manner (38.78%, 52.11%, 68.16% G2/M cell cycle arrests were observed, respectively, compared with 18.29% of that of the control). The percentage of cells at the G2/M phase increased to 45.6%, 59.14% and 68.22% for 48 h, respectively, which showed a time-dependent manner. These results revealed that compound 10a might have the same fashion as CA-4, which arrests the cell-cycle progression at the G2/M phase due to microtubule depolymerization and cytoskeleton disruption (Fig. 5).

Fig. 5 Cell cycle arrest effect of 10a. Effects of compound 10a on DNA content/cell following the treatment of A549 cells at different concentrations (25, 50, and 75 nM) or DMSO (0.01%) for 24 h (A) and 48 h (B). The experiments were performed at least three times, and the results of the representative experiments are shown. Representative images from three independent experiments are shown 24 h (C) and 48 h (D).

10a induced mitochondrial dysfunction

As mitochondria play a vital role in the progression of apoptosis, decreased mitochondrial membrane potential (MMP, ΔΨ_m) is an early event in apoptotic cells. To detect the MMP, we used flow cytometry analysis and fluorescence microscope in this study. Fig. 6A indicated that after incubation of the cells with 10a from 25 to 75 nM, the green fluorescence intensity (JC-1 monomers) increased from 1.33% to 88.23%, while the red fluorescence intensity (JC-1 aggregates) decreased from 98.55% to 10.94%. Fig. 6B, the confocal microscopy assay, showed that the cells converted from the aggregate to the monomer form, which was consistent with the results from the flow cytometry analysis. These results implied that compound 10a induced MMP collapse and mitochondrial dysfunction and eventually triggered apoptotic cell death.

Fig. 6 Compound 10a decreased the mitochondrial membrane potential of A549 cells. The A549 cells were treated with 10a at different concentrations (25, 50, and 75 nM) or DMSO (0.01%) for 48 h, followed by incubation with the fluorescence probe JC-1 for 30 min. Then, the cells were analysed by flow cytometry (A) or a fluorescence microscope (B). The experiments were performed at least three times, and the results of the representative experiments are shown.

Docking

The X-ray crystal structure of the tubulin complex with colchicine (PDB entry 1SA0) was downloaded from RCSB PDB. The crystal structure consists of four similar chains of domains and one linear chain of helix. The colchicine binding site is between two of the domains (Fig. 7A), and the domains are named chain A and B to distinguish. The molecular docking procedure was performed with the glide protocol of Schrodinger Maestro 10.2. Compound 10a was docked into the colchicine site on the basis of the binding location of colchicine in the crystal structure. The docking included all atoms as the receptor and used extra precision with flexible ligand sampling. The ligand–protein complex with the optimum score (Fig. 7B) was selected as the initial structure for MD simulations and subsequent MM-PB(GB)SA binding free energy calculation.

Fig. 7 (A) Colchicine binding site in the crystal structure. (B) Compound 10a optimum docking pose in the colchicine binding site. (C) and (D) Representative snapshots for colchicine and compound 10a binding modes after MD simulations. The key residues that interacted with compound 10a and colchicine are shown as stick models with the corresponding colour of their ribbon. Hydrogen bonds between the key residues and compound 10a are shown as dash lines.

The MD simulations were carried out using the AMBER12 (ref. 33) molecular simulation package for both the colchicine binding crystal structure and the compound 10a docking complex. The AMBER99SB³⁴ force field was employed for the protein, and the TIP3P model³⁵ was used for the solvent water molecules. The force field parameters of ligands (colchicine and 10a) were generated from an AMBER GAFF force field. After three steps of minimizations, heating and equilibrium, 10 ns MD simulations under NVT ensemble were carried out for both systems.³⁶ The representative snapshots of the binding mode are shown in Fig. 7C and D. The trajectories of the last 2 ns were chosen for MM-PB(GB)SA binding free energy calculations. The Generalized Born (GB) method performs better in this study with −45.5878 kcal mol⁻¹ for colchicine and −39.5153 kcal mol⁻¹ for compound 10a. The binding free energy for 10a is similar to colchicine, indicating 10a is a good tubulin polymerization inhibitor in the colchicine binding site.

Conclusions

It is well known that sometimes a minute structural modification might result in great changes in bioactivity. Inspired by the structure of phenstatin and millepachine, we developed a series of new benzophenone derivatives as tubulin polymerization inhibitors. The optimal compound 10a, bearing a 8-methoxy-2,2-dimethyl-2H-chromen-6-yl moiety instead of the 3,4,5-tri-methoxyphenyl moiety of phenstatin, exhibited much better antiproliferative activity in the five tested cancer cell lines (e.g. IC₅₀ (μM) for A549 cell, 10a: 0.035; phenstatin: 0.179). The mechanism study indicated that 10a is a good tubulin polymerization inhibitor with a 4.01 μM IC₅₀ value targeting the colchicine-binding site. The 10a induced cell apoptosis, arrest cell-cycle progression, induced mitochondrial dysfunction and morphological alterations of A549 cell were also studied. The further research of 10a such as in vivo anti-tumor evaluation leading to anticancer drugs is in progress.

Experimental

Chemistry

All reagents were analytically pure and were used without further purification. All chemistry solvents (THF, CH₂Cl₂) were reagent-grade and were dried and freshly distilled before the necessary step. ¹H NMR and ¹³C NMR spectra were recorded in CDCl₃ on a Bruker Avance III spectrometer, and chemical shifts are reported as parts per million (ppm) with tetramethylsilane as the internal standard. Melting points (MP) were measured with an SRS-OptiMelt automated melting point instrument. High-resolution mass spectra (HR-MS) were recorded using an Agilent LC-MS 6120 instrument. High-performance liquid chromatography (HPLC) were run on a TC-C18 column (4.6 × 250 mm, 5 μm) with two different solvent gradients (methanol/water = 85 : 15) and a flow rate of 0.50 mL min⁻¹. All synthesized compounds were determined to be at least 95% purity by HPLC.

General procedure for the preparation of 2

3-Chloro-3-methyl-1-butyne (1.53 g, 15 mmol) was added slowly to stirred vanillina (1) (1.52 g, 10 mmol), CuCl₂·2H₂O (5 mg, 0.3 mol%), DBU (1.5 mL, 15 mmol) in CH₃CN (100 mL) at 0 °C, and the reaction was monitored by TCL. After stirring for 5 hours, the solvent was removed under reduced pressure. Water was added, and the mixture was extracted by ethyl acetate. The organic layer was dried over sodium sulfate and evaporated in vacuum to give the crude product, which was purified by flash column chromatography (petroleum ether/ethyl acetate = 5 : 1) to give a white solid (2).

3-Methoxy-4-((2-methylbut-3-yn-2-yl)oxy)benzaldehyde (2). White solid, yield: 81.0%. ¹H NMR (400 MHz, CDCl₃) δ 9.88 (s, 1H), 7.64 (d, J = 8.7 Hz, 1H), 7.43 (s, 1H), 7.42 (d, J = 6.7 Hz, 1H), 3.89 (s, 3H), 2.64 (s, 1H), 1.74 (s, 6H).

General procedure for the preparation of 3

To a solution of 2 (2.18 g, 10 mmol) in pyridine (50 mL) was stirred at 120 °C, and then the mixture was stirred for 10 h. After the solvent was removed under vacuum, the residue was subjected to column chromatography (petroleum ether/ethyl acetate = 5 : 1) to get the target compound 3.

8-Methoxy-2,2-dimethyl-2H-chromene-6-carbaldehyde (3). White solid, yield: 85.0%. ¹H NMR (400 MHz, CDCl₃) δ 9.72 (s, 1H), 7.23 (d, J = 1.6 Hz, 1H), 7.08 (d, J = 1.7 Hz, 1H), 6.28 (d, J = 9.9 Hz, 1H), 5.61 (d, J = 9.9 Hz, 1H), 3.84 (s, 3H), 1.44 (s, 6H).

General procedure for the preparation of 4, 8a–8b

n-Butyllithium (0.8 mL, 2.5 M) was added dropwise to a solution of substituted bromobenzene (1.0 mmol) in anhydrous THF (15 mL). The mixture was stirred vigorously at −78 °C under a nitrogen atmosphere for 1 h, and then, 8-methoxy-2,2-dimethyl-2H-chromene-6-carbaldehyde (3) (218 mg, 1.0 mmol) was added. After the mixture was stirred for another 12 h, a solution of saturated NH₄Cl was added to quench the reaction. Ethyl acetate was added to the solution, and the organic layers were separated, dried over anhydrous Na₂SO₄, and evaporated in vacuum to give the crude product, which was purified by column chromatography to obtain product 4 or 8a–8b.

(8-Methoxy-2,2-dimethyl-2H-chromen-6-yl)(4-methoxyphenyl)methanol (4a). White oil, yield: 61.7%. IR (KBr, cm⁻¹) 3507, 2972, 2934, 2837, 1640, 1584, 1482, 1253, 1130, 710. ¹H NMR (400 MHz, CDCl₃) δ 7.27 (d, J = 8.6 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2H), 6.79 (d, J = 1.7 Hz, 1H), 6.58 (d, J = 1.7 Hz, 1H), 6.25 (d, J = 9.8 Hz, 1H), 5.67 (s, 1H), 5.59 (d, J = 9.8 Hz, 1H), 3.80 (s, 3H), 3.78 (s, 3H), 2.39 (s, 1H), 1.46 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 159.09 (s), 148.33 (s), 141.36 (s), 136.27 (s), 131.10 (s), 127.93 (s), 122.48 (s), 121.76 (s), 117.02 (s), 113.92 (s), 110.79 (s), 76.61 (s), 75.65 (s), 56.37 (s), 55.39 (s), 27.99 (d, J = 2.4 Hz). HRMS (ESI) (m/z) [M + Na]⁺ calcd for C₂₀H₂₂O₄, 349.1410; found, 349.1419.

(8-Methoxy-2,2-dimethyl-2H-chromen-6-yl)(3-methoxyphenyl)methanol (4b). White oil, yield: 65.3%. IR (KBr, cm⁻¹) 3506, 2976, 2934, 2828, 1640, 1585, 1506, 1246, 1133, 724. ¹H NMR (400 MHz, CDCl₃) δ 7.27–7.20 (m, 1H), 6.98–6.90 (m, 2H), 6.83–6.75 (m, 2H), 6.59 (d, J = 1.7 Hz, 1H), 6.24 (d, J = 9.8 Hz, 1H), 5.67 (s, 1H), 5.59 (d, J = 9.8 Hz, 1H), 3.80 (s, 3H), 3.77 (s, 3H), 2.50 (s, 1H), 1.45 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 159.78 (s), 148.34 (s), 145.61 (s), 141.51 (s), 135.93 (s), 131.10 (s), 129.55 (s), 122.44 (s), 121.79 (s), 118.95 (s), 117.17 (s), 112.92 (s), 112.18 (s), 110.89 (s), 76.63 (s), 75.98 (s), 56.37 (s), 55.33 (s), 27.99 (d, J = 3.0 Hz). HRMS (ESI) (m/z) [M + Na]⁺ calcd for C₂₀H₂₂O₄, 349.1410; found, 349.1409.

(8-Methoxy-2,2-dimethyl-2H-chromen-6-yl)(2-methoxyphenyl)methanol (4c). White oil, yield: 58.4%. IR (KBr, cm⁻¹) 3524, 2966, 2934, 2836, 1646, 1585, 1472, 1263, 1134, 759. ¹H NMR (400 MHz, CDCl₃) δ 7.29–7.24 (m, 1H), 7.21 (dd, J = 7.5, 1.5 Hz, 1H), 6.94 (t, J = 7.2 Hz, 1H), 6.90 (d, J = 8.2 Hz, 1H), 6.86 (d, J = 1.8 Hz, 1H), 6.60 (d, J = 1.8 Hz, 1H), 6.25 (d, J = 9.8 Hz, 1H), 5.96 (s, 1H), 5.59 (d, J = 9.8 Hz, 1H), 3.83 (s, 3H), 3.82 (s, 3H), 3.05 (s, 1H), 1.46 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 156.91 (s), 148.16 (s), 141.19 (s), 135.27 (s), 132.13 (s), 130.91 (s), 128.83 (s), 127.96 (s), 122.67 (s), 121.63 (s), 120.92 (s), 117.13 (s), 111.14 (s), 110.84 (s), 76.54 (s), 72.05 (s), 56.37 (s), 55.57 (s), 28.01 (s). HRMS (ESI) (m/z) [M + Na]⁺ calcd for C₂₀H₂₂O₄, 349.1410; found, 349.1417.

(3-Fluoro-4-methoxyphenyl)(8-methoxy-2,2-dimethyl-2H-chromen-6-yl)methanol (4d). White oil, yield: 44.4%. IR (KBr, cm⁻¹) 3506, 2970, 2935, 2842, 1628, 1585, 1499, 1272, 1124, 725. ¹H NMR (400 MHz, CDCl₃) δ 7.10 (dd, J = 12.2, 2.0 Hz, 1H), 7.05 (d, J = 9.2 Hz, 1H), 6.90 (t, J = 8.5 Hz, 1H), 6.75 (d, J = 1.8 Hz, 1H), 6.56 (d, J = 1.8 Hz, 1H), 6.25 (d, J = 9.8 Hz, 1H), 5.63 (s, 1H), 5.61 (d, J = 9.8 Hz, 1H), 3.86 (s, 3H), 3.80 (s, 3H), 2.52 (s, 1H), 1.46 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 152.28 (d, J = 245.8 Hz), 148.30 (s), 146.84 (d, J = 10.9 Hz), 141.47 (s), 137.17 (d, J = 5.5 Hz), 135.80 (s), 131.16 (s), 122.31 (s), 122.22 (d, J = 3.4 Hz), 121.77 (s), 117.00 (s), 114.42 (d, J = 19.0 Hz), 113.21 (d, J = 1.8 Hz), 110.68 (s), 76.65 (s), 75.06 (s), 56.33 (s), 56.29 (s), 27.92 (d, J = 3.7 Hz). HRMS (ESI) (m/z) [M + Na]⁺ calcd for C₂₀H₂₁O₄F, 367.1316; found, 367.1324.

(8-Methoxy-2,2-dimethyl-2H-chromen-6-yl)(4-methoxy-3-methylphenyl)methanol (4e). White oil, yield: 52.3%. IR (KBr, cm⁻¹) 3504, 2968, 2928, 2853, 1739, 1585, 1508, 1255, 1124, 725. ¹H NMR (400 MHz, CDCl₃) δ 7.13 (d, J = 9.4 Hz, 2H), 6.80 (d, J = 1.7 Hz, 1H), 6.76 (d, J = 8.1 Hz, 1H), 6.59 (d, J = 1.6 Hz, 1H), 6.25 (d, J = 9.8 Hz, 1H), 5.64 (s, 1H), 5.59 (d, J = 9.8 Hz, 1H), 3.81 (s, 3H), 3.80 (s, 3H), 2.36 (s, 1H), 2.19 (s, 3H), 1.45 (d, J = 1.3 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 157.26 (s), 148.25 (s), 141.25 (s), 136.38 (s), 135.80 (s), 131.01 (s), 129.12 (s), 126.73 (s), 125.09 (s), 122.52 (s), 121.71 (s), 116.96 (s), 110.77 (s), 109.78 (s), 76.56 (s), 75.70 (s), 56.35 (s), 55.44 (s), 27.97 (d, J = 2.4 Hz), 16.41 (s). HRMS (ESI) (m/z) [M + Na]⁺ calcd for C₂₁H₂₄O₄, 363.1567; found, 367.1566.

(8-Methoxy-2,2-dimethyl-2H-chromen-6-yl)(4-methoxy-2-methylphenyl)methanol (4f). White oil, yield: 42.9%. IR (KBr, cm⁻¹) 3402, 2974, 2915, 2840, 1611, 1577, 1464, 1247, 1080, 725. ¹H NMR (400 MHz, CDCl₃) δ 7.36 (d, J = 8.5 Hz, 1H), 6.75 (dd, J = 9.3, 2.0 Hz, 2H), 6.69 (d, J = 2.5 Hz, 1H), 6.52 (d, J = 1.6 Hz, 1H), 6.23 (d, J = 9.8 Hz, 1H), 5.82 (s, 1H), 5.59 (d, J = 9.8 Hz, 1H), 3.79 (s, 3H), 3.78 (s, 3H), 2.27 (s, 1H), 2.23 (s, 3H), 1.45 (d, J = 1.7 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 158.74 (s), 148.19 (s), 141.24 (s), 137.05 (s), 135.49 (s), 134.03 (s), 130.95 (s), 127.65 (s), 122.47 (s), 121.65 (s), 117.39 (s), 116.21 (s), 111.33 (s), 110.91 (s), 76.52 (s), 72.77 (s), 56.27 (s), 55.20 (s), 27.90 (d, J = 2.9 Hz), 19.62 (s). HRMS (ESI) (m/z) [M + Na]⁺ calcd for C₂₁H₂₄O₄, 363.1567; found, 367.1572.

(8-Methoxy-2,2-dimethyl-2H-chromen-6-yl)(4-(methylthio)phenyl)methanol (4g). White oil, yield: 60.1%. IR (KBr, cm⁻¹) 3445, 2972, 2929, 2849, 1638, 1577, 1481, 1263, 1142, 742. ¹H NMR (400 MHz, CDCl₃) δ 7.30 (d, J = 8.3 Hz, 2H), 7.23 (d, J = 8.4 Hz, 2H), 6.79 (d, J = 1.7 Hz, 1H), 6.59 (d, J = 1.7 Hz, 1H), 6.26 (d, J = 9.8 Hz, 1H), 5.71 (d, J = 2.4 Hz, 1H), 5.61 (d, J = 9.8 Hz, 1H), 3.83 (s, 3H), 2.49 (d, J = 12.8 Hz, 3H), 1.46 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 148.31 (s), 141.44 (s), 140.88 (s), 137.53 (s), 135.95 (s), 131.11 (s), 127.09 (s), 126.65 (s), 122.37 (s), 121.75 (s), 117.08 (s), 110.80 (s), 76.61 (s), 75.60 (s), 56.32 (s), 27.95 (d, J = 3.4 Hz), 15.92 (s). HRMS (ESI) (m/z) [M + Na]⁺ calcd for C₂₀H₂₂O₃S, 365.1182; found, 365.1186.

(8-Methoxy-2,2-dimethyl-2H-chromen-6-yl)(3,4,5-trimethoxyphenyl)methanol (4h). White oil, yield: 55.2%. IR (KBr, cm⁻¹) 3498, 2971, 2948, 2834, 1638, 1585, 1455, 1229, 1124, 725. ¹H NMR (400 MHz, CDCl₃) δ 6.80 (d, J = 1.8 Hz, 1H), 6.63–6.57 (m, 3H), 6.26 (d, J = 9.8 Hz, 1H), 5.64 (s, 1H), 5.61 (d, J = 9.8 Hz, 1H), 3.82 (s, 3H), 3.82 (S, 9H), 2.63 (s, 1H), 1.46 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 153.04 (s), 148.11 (s), 141.29 (s), 139.67 (s), 136.96 (s), 135.88 (s), 130.98 (s), 122.31 (s), 121.61 (s), 117.09 (s), 110.87 (s), 103.50 (s), 76.51 (s), 75.77 (s), 60.76 (s), 56.22 (s), 56.03 (s), 27.80 (d, J = 2.0 Hz). HRMS (ESI) (m/z) [M + Na]⁺ calcd for C₂₂H₂₆O₆, 409.1622; found, 409.1622.

(8-Methoxy-2,2-dimethyl-2H-chromen-6-yl)(4-methoxy-3-(methoxymethoxy)phenyl)methanol (8a). White oil, yield: 55.4%. ¹H NMR (400 MHz, CDCl₃) δ 7.21 (d, J = 1.9 Hz, 1H), 6.97 (dd, J = 8.3, 1.9 Hz, 1H), 6.86 (d, J = 8.4 Hz, 1H), 6.82 (d, J = 1.8 Hz, 1H), 6.61 (d, J = 1.8 Hz, 1H), 6.26 (d, J = 9.8 Hz, 1H), 5.69 (s, 1H), 5.61 (d, J = 9.8 Hz, 1H), 5.22 (s, 2H), 3.87 (s, 3H), 3.83 (s, 3H), 3.50 (s, 3H), 2.02 (s, 1H), 1.46 (s, 6H).

(8-Methoxy-2,2-dimethyl-2H-chromen-6-yl)(3-methoxy-4-(methoxymethoxy)phenyl)methanol (8b). White oil, yield: 44.3%. ¹H NMR (400 MHz, CDCl₃) δ 7.09 (d, J = 8.3 Hz, 1H), 6.95 (d, J = 1.9 Hz, 1H), 6.86 (dd, J = 8.2, 1.9 Hz, 1H), 6.81 (d, J = 1.8 Hz, 1H), 6.60 (d, J = 1.8 Hz, 1H), 6.26 (d, J = 9.8 Hz, 1H), 5.68 (s, 1H), 5.61 (d, J = 9.8 Hz, 1H), 5.21 (s, 2H), 3.85 (s, 3H), 3.82 (s, 3H), 3.50 (s, 3H), 2.34 (s, 1H), 1.46 (s, 7H).

General procedure for the preparation of 5, 9a–9b

To a solution of 4 or 8a–8b (1.0 mmol) in 15 mL of CH₂Cl₂, PCC (258 mg 1.2 mmol) and silica gel (258 mg) were added, and the reaction was monitored by TCL. After stirring for 5 h, the mixture was filtered, and the filtrates were concentrated under vacuum to give a residue, which was purified by silica gel column chromatography to obtain the target compound as a white solid.

(8-Methoxy-2,2-dimethyl-2H-chromen-6-yl)(4-methoxyphenyl)methanone (5a). White solid, yield 75.7%. mp: 89.5–91.8 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.81 (s, 1H), 7.79 (s, 1H), 7.32 (d, J = 1.7 Hz, 1H), 7.08 (d, J = 1.7 Hz, 1H), 6.98 (s, 1H), 6.96 (s, 1H), 6.31 (d, J = 9.9 Hz, 1H), 5.67 (d, J = 9.8 Hz, 1H), 3.91 (s, 3H), 3.89 (s, 3H), 1.53 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 194.57 (s), 162.89 (s), 148.22 (s), 146.03 (s), 132.29 (s), 131.20 (s), 130.78 (s), 130.26 (s), 122.26 (s), 121.95 (s), 120.77 (s), 113.54 (s), 113.44 (s), 77.82 (s), 56.37 (s), 55.58 (s), 28.31 (s). HRMS (ESI) (m/z) [M + H]⁺ calcd for C₂₀H₂₀O₄, 325.1434; found, 325.1441. Purity: 98.6% (by HPLC).

(8-Methoxy-2,2-dimethyl-2H-chromen-6-yl)(3-methoxyphenyl)methanone (5b). White solid, yield 63.7%. mp: 83.8–85.4 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.43–7.34 (m, 2H), 7.33–7.28 (m, 2H), 7.15–7.05 (m, 2H), 6.30 (d, J = 9.9 Hz, 1H), 5.67 (d, J = 9.9 Hz, 1H), 3.90 (s, 3H), 3.86 (s, 3H), 1.58 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 195.36 (s), 159.54 (s), 148.30 (s), 146.55 (s), 139.69 (s), 131.16 (s), 129.61 (s), 129.22 (s), 122.79 (s), 122.42 (s), 121.88 (s), 120.76 (s), 118.26 (s), 114.22 (s), 113.38 (s), 77.97 (s), 56.38 (s), 55.56 (s), 28.36 (s). HRMS (ESI) (m/z) [M + H]⁺ calcd for C₂₀H₂₀O₄, 325.1434; found, 325.1442. Purity: 96.8% (by HPLC).

(8-Methoxy-2,2-dimethyl-2H-chromen-6-yl)(2-methoxyphenyl)methanone (5c). White solid, yield 63.7%. mp: 131.7–133.1 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.50–7.39 (m, 2H), 7.30 (d, J = 8.6 Hz, 1H), 7.04 (d, J = 7.4 Hz, 1H), 7.00 (d, J = 6.9 Hz, 2H), 6.25 (d, J = 9.9 Hz, 1H), 5.63 (d, J = 9.9 Hz, 1H), 3.89 (s, 3H), 3.77 (s, 3H), 1.51 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 195.08 (s), 157.08 (s), 148.32 (s), 146.98 (s), 131.40 (s), 130.86 (s), 130.09 (s), 129.18 (s), 123.06 (s), 121.90 (s), 120.74 (s), 120.39 (s), 112.48 (s), 111.48 (s), 78.00 (s), 56.32 (s), 55.77 (s), 28.39 (s). HRMS (ESI) (m/z) [M + H]⁺ calcd for C₂₀H₂₀O₄, 325.1434; found, 325.1445. Purity: 98.4% (by HPLC).

(3-Fluoro-4-methoxyphenyl)(8-methoxy-2,2-dimethyl-2H-chromen-6-yl)methanone (5d). White solid, yield 43.1%. mp: 121.2–122.1 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.63–7.55 (m, 2H), 7.31 (s, 1H), 7.06 (s, 1H), 7.03 (t, J = 8.2 Hz, 1H), 6.31 (d, J = 9.9 Hz, 1H), 5.68 (d, J = 9.8 Hz, 1H), 3.98 (s, 3H), 3.91 (s, 3H), 1.53 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 193.45 (s), 153.03 (s), 151.21 (d, J = 10.8 Hz), 149.47 (d, J = 219.5 Hz), 146.38 (s), 131.33 (s), 131.07 (d, J = 5.1 Hz), 129.67 (s), 127.30 (d, J = 3.2 Hz), 122.29 (s), 121.87 (s), 120.84 (s), 117.80 (d, J = 19.2 Hz), 113.37 (s), 112.24 (d, J = 1.0 Hz), 77.97 (s), 56.43 (s), 28.37 (s). HRMS (ESI) (m/z) [M + H]⁺ calcd for C₂₀H₁₉O₄F, 343.1340; found, 343.1338. Purity: 95.0% (by HPLC).

(8-Methoxy-2,2-dimethyl-2H-chromen-6-yl)(4-methoxy-3-methylphenyl)methanone (5e). White solid, yield 76.5%. mp: 145.6–147.1 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.65 (d, J = 6.5 Hz, 2H), 7.32 (s, 1H), 7.08 (s, 1H), 6.87 (d, J = 9.0 Hz, 1H), 6.31 (d, J = 9.9 Hz, 1H), 5.67 (d, J = 9.8 Hz, 1H), 3.91 (s, 3H), 3.91 (s, 3H), 2.27 (s, 3H), 1.53 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 194.87 (s), 161.19 (s), 148.18 (s), 145.93 (s), 132.58 (s), 131.16 (s), 130.48 (s), 130.30 (s), 130.22 (s), 126.75 (s), 122.24 (s), 122.02 (s), 120.76 (s), 113.50 (s), 108.96 (s), 77.79 (s), 56.40 (s), 55.64 (s), 28.34 (s), 16.41 (s). HRMS (ESI) (m/z) [M + H]⁺ calcd for C₂₁H₂₂O₄, 339.1591; found, 339.1592. Purity: 98.4% (by HPLC).

(8-Methoxy-2,2-dimethyl-2H-chromen-6-yl)(4-methoxy-2-methylphenyl)methanone (5f). White solid, yield 76.5%. mp: 116.2–117.9 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.38 (s, 1H), 7.31 (d, J = 8.4 Hz, 1H), 6.98 (s, 1H), 6.81 (s, 1H), 6.75 (d, J = 8.4 Hz, 1H), 6.26 (d, J = 9.9 Hz, 1H), 5.65 (d, J = 9.9 Hz, 1H), 3.90 (s, 3H), 3.86 (s, 3H), 2.37 (s, 3H), 1.51 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 196.64 (s), 160.94 (s), 148.35 (s), 146.61 (s), 139.87 (s), 133.31–133.24 (m), 131.31 (s), 131.10 (d, J = 11.7 Hz), 130.82 (s), 122.88 (s), 121.88 (s), 120.74 (s), 116.62 (s), 112.85 (s), 110.17 (s), 77.96 (s), 56.33 (s), 55.39 (s), 28.36 (s), 20.60 (s). HRMS (ESI) (m/z) [M + H]⁺ calcd for C₂₁H₂₂O₄, 339.1591; found, 339.1596. Purity: 96.0% (by HPLC).

(8-Methoxy-2,2-dimethyl-2H-chromen-6-yl)(4-(methylthio)phenyl)methanone (5g). White solid, yield 44.5%. mp: 117.1–118.2 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.72 (d, J = 7.3 Hz, 2H), 7.34 (s, 1H), 7.30 (d, J = 7.5 Hz, 2H), 7.08 (s, 1H), 6.30 (d, J = 9.5 Hz, 1H), 5.67 (d, J = 9.5 Hz, 1H), 3.91 (s, 3H), 2.54 (s, 3H), 1.53 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 194.72 (s), 148.30 (s), 146.31 (s), 144.52 (s), 134.39 (s), 131.22 (s), 130.46 (s), 129.86 (s), 124.88 (s), 122.42 (s), 121.88 (s), 120.78 (s), 113.36 (s), 77.90 (s), 56.37 (s), 28.33 (s), 14.98 (s). HRMS (ESI) (m/z) [M + H]⁺ calcd for C₂₀H₂₀O₃S, 341.1206; found, 341.1211. Purity: 97.6% (by HPLC).

(8-Methoxy-2,2-dimethyl-2H-chromen-6-yl)(3,4,5-trimethoxyphenyl)methanone (5h). White solid, yield 70.5%. mp: 151.8–152.9 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.35 (s, 1H), 7.14 (s, 1H), 7.03 (s, 2H), 6.33 (d, J = 9.9 Hz, 1H), 5.69 (d, J = 9.8 Hz, 1H), 3.94 (s, 3H), 3.92 (s, 3H), 3.89 (s, 6H), 1.54 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 194.71 (s), 152.89 (s), 148.19 (s), 146.41 (s), 141.52 (s), 133.44 (s), 131.29 (s), 129.73 (s), 122.42 (s), 121.80 (s), 120.88 (s), 113.55 (s), 107.39 (s), 78.00 (s), 61.09 (s), 56.42 (s), 28.42 (s). HRMS (ESI) (m/z) [M + H]⁺ calcd for C₂₂H₂₄O₆, 385.1646 found, 385.1638. Purity: 100.0% (by HPLC).

(8-Methoxy-2,2-dimethyl-2H-chromen-6-yl)(4-methoxy-3-(methoxymethoxy)phenyl)methanone (9a). White oil, yield 49.1%. ¹H NMR (400 MHz, CDCl₃) δ 7.65 (d, J = 1.7 Hz, 1H), 7.49 (dd, J = 8.4, 1.7 Hz, 1H), 7.32 (d, J = 1.4 Hz, 1H), 7.11 (d, J = 1.5 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H), 6.31 (d, J = 9.9 Hz, 1H), 5.67 (d, J = 9.8 Hz, 1H), 5.27 (s, 2H), 3.97 (s, 3H), 3.90 (s, 3H), 3.52 (s, 3H), 1.53 (s, 6H).

(8-Methoxy-2,2-dimethyl-2H-chromen-6-yl)(3-methoxy-4-(methoxymethoxy)phenyl)methanone (9b). White oil, yield 37.0%.¹H NMR (400 MHz, CDCl₃) δ 7.44 (d, J = 1.9 Hz, 1H), 7.34 (dt, J = 3.8, 1.9 Hz, 2H), 7.19 (d, J = 8.4 Hz, 1H), 7.11 (d, J = 1.8 Hz, 1H), 6.32 (d, J = 9.9 Hz, 1H), 5.68 (d, J = 9.8 Hz, 1H), 5.33 (s, 2H), 3.95 (s, 3H), 3.91 (s, 3H), 3.55 (s, 3H), 1.53 (s, 6H).

General procedure for the preparation of 7a–7b

DIPEA (258 mg, 2 mmol) and chloromethyl methyl ether (121 mg, 1.5 mmol) were added dropwise to a 6a–6b (201 mg, 1 mmol) solution in anhydrous CH₂Cl₂ (20 mL) over 5 minutes at 0 °C. After stirring for 3 hours, the mixture was quenched with saturated NaHCO₃ solution and extracted by EtOAc. The combined organic extracts were dried over MgSO₄ and evaporated in a vacuum to give a 7a–7b.

4-Bromo-1-methoxy-2-(methoxymethoxy)benzene (7a). Yellow oil, yield 95.1%. ¹H NMR (400 MHz, CDCl₃) δ 7.29 (d, J = 2.3 Hz, 1H), 7.09 (dd, J = 8.6, 2.3 Hz, 1H), 6.75 (d, J = 8.7 Hz, 1H), 5.20 (s, 2H), 3.85 (s, 3H), 3.51 (s, 3H).

4-Bromo-2-methoxy-1-(methoxymethoxy)benzene (7b). Yellow oil, yield 96.1%. ¹H NMR (400 MHz, CDCl₃) δ 6.99 (s, 3H), 5.16 (s, 2H), 3.83 (s, 3H), 3.48 (s, 3H).

General procedure for the preparation of 10a–10b

Compound 9a–9b (384 mg, 1 mmol) was dissolved in methanol (10 mL), and then HCl (12 N, 1 mL) was added. After stirring for 3 h, the reaction mixture was neutralized with NaHCO₃, extracted with ethyl acetate and purified by flash column chromatography (petroleum ether/ethyl acetate = 5 : 1) to provide the target compound 10a–10b.

(3-Hydroxy-4-methoxyphenyl)(8-methoxy-2,2-dimethyl-2H-chromen-6-yl)methanone (10a). White solid, yield 41.5%. mp: 116.9–118.4 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.41 (s, 1H), 7.38 (d, J = 8.3 Hz, 1H), 7.33 (s, 1H), 7.09 (d, J = 1.6 Hz, 1H), 6.92 (d, J = 8.3 Hz, 1H), 6.31 (d, J = 9.9 Hz, 1H), 5.71 (s, 1H), 5.67 (d, J = 9.9 Hz, 1H), 3.98 (s, 3H), 3.91 (s, 3H), 1.52 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 194.56 (s), 150.03 (s), 148.22 (s), 146.11 (s), 145.26 (s), 131.55 (s), 131.16 (s), 130.08 (s), 123.49 (s), 122.40 (s), 121.98 (s), 120.73 (s), 116.26 (s), 113.45 (s), 109.80 (s), 77.83 (s), 56.38 (s), 56.18 (s), 28.30 (s). HRMS (ESI) (m/z) [M + H]⁺ calcd for C₂₀H₂₀O₅, 341.1384 found, 341.1381. Purity: 98.2% (by HPLC).

(4-Hydroxy-3-methoxyphenyl)(8-methoxy-2,2-dimethyl-2H-chromen-6-yl)methanone (10b). White solid, yield 32.1%. mp: 168.7–170.5 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.44 (s, 1H), 7.35 (d, J = 8.2 Hz, 1H), 7.31 (s, 1H), 7.10 (s, 1H), 6.96 (d, J = 8.2 Hz, 1H), 6.31 (d, J = 7.2 Hz, 2H), 5.68 (d, J = 9.8 Hz, 1H), 3.94 (s, 3H), 3.90 (s, 3H), 1.53 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 194.56 (s), 149.81 (s), 148.14 (s), 146.69 (s), 146.00 (s), 131.21 (s), 130.43 (s), 130.23 (s), 125.59 (s), 122.18 (s), 121.91 (s), 120.78 (s), 113.57 (s), 111.99 (s), 77.81 (s), 56.36 (s), 56.20 (s), 28.30 (s). HRMS (ESI) (m/z) [M + H]⁺ calcd for C₂₀H₂₀O₅, 341.1384 found, 341.1380. Purity: 99.0% (by HPLC).

General procedure for the preparation of 11

To a solution of ethyl-triphenylphosphonium chloride (392 mg 1.2 mmol) in anhydrous THF (15 mL) at −78 °C under a nitrogen atmosphere, n-butyllithium (0.8 mL, 2.5 M) was added dropwise. The resulting yellow solution was stirred for 1 h and then warmed to room temperature and stirred for 1 h. A solution of 9a (384 mg 1 mmol) in dry THF was added at −78 °C and warmed to room temperature. Once completed, the reaction mixtures were stopped with a saturated NH₄Cl solution and extracted with ethyl acetate. The organic layer was washed with brine, dried and filtered. Removal of the solvent gave a residue that was purified by flash column chromatography (petroleum ether/ethyl acetate = 8 : 1) to furnish yellow oil, which was dissolved in methanol (10 mL). HCl (12 N, 1 mL) was added to remove the MOM and the product 2-methoxy-5-(1-(8-methoxy-2,2-dimethyl-2H-chromen-6-yl)vinyl)phenol (11) was obtained.

2-Methoxy-5-(1-(8-methoxy-2,2-dimethyl-2H-chromen-6-yl)vinyl)phenol (11). White solid, yield 95.1%. mp: 137.3–139.0 °C. ¹H NMR (400 MHz, CDCl₃) δ 6.98 (s, 1H), 6.85 (d, J = 8.3 Hz, 1H), 6.81 (d, J = 8.3 Hz, 1H), 6.77 (s, 1H), 6.62 (s, 1H), 6.26 (d, J = 9.8 Hz, 1H), 5.62 (d, J = 9.4 Hz, 2H), 5.29 (d, J = 1.8 Hz, 2H), 3.91 (s, 3H), 3.82 (s, 3H), 1.49 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 149.31 (s), 147.85 (s), 146.42 (s), 145.21 (s), 141.79 (s), 135.13 (s), 133.97 (s), 131.00 (s), 122.48 (s), 121.42 (s), 120.36 (s), 119.08 (s), 114.67 (s), 112.53 (s), 112.15 (s), 110.21 (s), 76.75 (s), 56.38 (s), 56.07 (s), 28.07 (s). HRMS (ESI) (m/z) [M + H]⁺ calcd for C₂₁H₂₂O₄, 339.1591 found, 339.1587. Purity: 99.8% (by HPLC).

General procedure for the preparation of 12

To a solution of 10a (340 mg, 1 mmol) in anhydrous THF (15 mL), NaH (60%, 60 mg, 1.5 mmol) and CH₃I (213 mg, 1.5 mmol) were added at 0 °C. After stirring for 48 h at room temperature, the reaction mixture was extracted with ethyl acetate. The solvents were removed under reduced pressure, and the solution was purified by flash column chromatography (petroleum ether/ethyl acetate = 5 : 1) to give (3,4-dimethoxyphenyl)(8-methoxy-2,2-dimethyl-2H-chromen-6-yl)methanone (12).

(3,4-Dimethoxyphenyl)(8-methoxy-2,2-dimethyl-2H-chromen-6-yl)methanone (12). Yellow oil, yield 81.5%. ¹H NMR (400 MHz, CDCl₃) δ 7.44 (s, 1H), 7.39 (d, J = 8.3 Hz, 1H), 7.32 (s, 1H), 7.10 (d, J = 1.4 Hz, 1H), 6.91 (d, J = 8.3 Hz, 1H), 6.32 (d, J = 9.9 Hz, 1H), 5.68 (d, J = 9.8 Hz, 1H), 3.97 (s, 3H), 3.95 (s, 3H), 3.91 (s, 3H), 1.53 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 194.50 (s), 152.57 (s), 148.91 (s), 148.13 (s), 146.00 (s), 131.20 (s), 130.80 (s), 130.20 (s), 124.80 (s), 122.17 (s), 121.88 (s), 120.76 (s), 113.50 (s), 112.21 (s), 109.73 (s), 77.80 (s), 56.35 (s), 56.12 (s), 28.30 (s). HRMS (ESI) (m/z) [M + H]⁺ calcd for C₂₁H₂₂O₅, 355.1540 found, 355.1536. Purity: 99.8% (by HPLC).

Biological

The cell lines (A549, HeLa, A2780, HCT116, Mgc803, BJ, HLF) were purchased from the Laboratory Animal Service Center at Sun Yat-sen University (Guangzhou, China). We performed the tubulin polymerisation assay using a commercial kit (cytoskeleton, cat. #B011P) that was purchased from Cytoskeleton (Danvers, MA, USA). FITC-conjugated mouse anti-tubulin antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). Goat anti-mouse IgG/Alexa-Fluor 488 antibody was obtained from Invitrogen (Camarillo, California, USA). A purified brain tubulin polymerisation kit was purchased from Cytoskeleton (Danvers, MA, USA). Annexin-V/FITC and Cell cycle were purchased from Keygen Biotech, China. MTT was purchased from Sigma, USA. A lipophilic cationic dye, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolcarbocyanine (JC-1) was obtained from Beyotime, China.

Biological assays such as cell culture, MTT assay, in vitro tubulin polymerisation assay can be seen in the ESI.†

Acknowledgements

We thank the National Natural Science Foundation of China (No. 21302235, 20972198), Guangdong Natural Science Foundation (2014A030313124) and PhD Programs Foundation of Ministry of Education of China (20120171120045) for financial support of this study.

Notes and references

1. Y. Zhao, X. Mu and G. Du, Pharmacol. Ther., 2016, 162, 134–143.
2. V. Nitika and K. Kapil, Curr. Drug Ther., 2013, 8, 189–196.
3. C. F. Brogdon, F. Y. Lee and R. M. Canetta, Anti-Cancer Drugs, 2014, 25, 599–609.
4. M. Joshi, X. Liu and C. P. Belani, Anti-Cancer Drugs, 2014, 25, 571–583.
5. C. C. Rohena and S. L. Mooberry, Nat. Prod. Rep., 2014, 31, 335–355.
6. A. E. Prota, K. Bargsten and D. Zurwerra, Science, 2013, 339, 587–590.
7. R. A. Stanton, K. M. Gernert and J. H. Nettles, Med. Res. Rev., 2011, 31, 443–481.
8. F. Pellegrini and D. R. Budman, Cancer Invest., 2005, 23, 264–273.
9. T. Graening and H. G. Schmalz, Angew. Chem., Int. Ed., 2004, 43, 3230–3256.
10. M. Gorman, N. Neuss and K. Biemann, J. Am. Chem. Soc., 1962, 84, 1058–1059.
11. J. W. Moncrief and W. N. Lipscomb, J. Am. Chem. Soc., 1965, 87, 4963–4964.
12. A. DeBono, B. Capuano and P. J. Scammells, J. Med. Chem., 2015, 58, 5699–5727.
13. G. R. Pettit, G. M. Cragg and D. L. Herald, Can. J. Chem., 1982, 60, 1374–1376.
14. G. J. Rustin, G. Shreeves and P. D. Nathan, Br. J. Cancer, 2010, 102, 1355–1360.
15. D. W. Siemann, D. J. Chaplin and P. A. Walicke, Expert Opin. Invest. Drugs, 2009, 18, 189–197.
16. H.-P. Hsieh, J.-P. Liou and Y.-T. Lin, Bioorg. Med. Chem., 2003, 13, 101–105.
17. K. F. Grossmann, H. Colman and W. A. Akerley, J. Neuro-Oncol., 2012, 110, 257–264.
18. D. Cao, Y. Liu and W. Yan, J. Med. Chem., 2016, 59, 5721–5739.
19. R. K. V. Devambatla, O. A. Namjoshi and S. Choudhary, J. Med. Chem., 2016, 59, 5752–5765.
20. J. Yan, J. Chen and S. Zhang, J. Med. Chem., 2016, 59, 5264–5283.
21. V. Chaudhary, J. B. Venghateri and H. P. S. Dhaked, J. Med. Chem., 2016, 59, 3439–3451.
22. Y.-N. Liu, J.-J. Wang and Y.-T. Ji, J. Med. Chem., 2016, 59, 5341–5355.
23. A. Brancale and R. Silvestri, Med. Res. Rev., 2007, 27, 209–238.
24. R. Kaur, G. Kaur and R. K. Gill, Eur. J. Med. Chem., 2014, 87, 89–124.
25. H. Chen, Y. Li and C. Sheng, J. Med. Chem., 2013, 56, 685–699.
26. B. L. Flynn, G. S. Gill and D. W. Grobelny, J. Med. Chem., 2011, 54, 6014–6027.
27. Y. S. Shan, J. Zhang and Z. Liu, Curr. Med. Chem., 2011, 18, 523–538.
28. G. R. Pettit, M. P. Grealish and D. L. Herald, J. Med. Chem., 2000, 43, 2731.
29. G. R. Pettit, B. Toki and D. L. Herald, J. Med. Chem., 1998, 41, 1688.
30. K. von Schwarzenberg and A. M. Vollmar, Cancer Lett., 2013, 332, 295–303.
31. W. Wu, H. Ye and L. Wan, Carcinogenesis, 2013, 34, 1636–1643.
32. J. Sheng, F. Mao and J. Yan, RSC Adv., 2014, 4, 41510–41520.
33. D. A. Case, T. Darden and T. E. Cheatham III, Amber12 2012, University of California, San Francisco, CA, 2012.
34. Y. Duan, C. Wu and S. Chowdhury, J. Comput. Chem., 2003, 24, 1999–2012.
35. W. L. Jorgensen, J. Chandrasekhar and J. D. Madura, J. Chem. Phys., 1983, 79, 926–935.
36. J. Wang, R. M. Wolf, J. W. Caldwell and P. A. Kollman, J. Comput. Chem., 2004, 25, 1157–1174.

---

Footnote

† Electronic supplementary information (ESI) available: NMR spectrums, HPLC and HR-MS of target compounds. See DOI: 10.1039/c6ra16948a

---