Lead identification of conformationally restricted benzoxepin type combretastatin analogs: synthesis, antiproliferative activity, and tubulin effects

Abstract

We have synthesized a series of polymethoxylated rigid analogs of combretastatin A-4 which contain a benzoxepin ring in place of the usual ethylene bridge present in the natural combretastatin products. The compounds display antiproliferative activity when evaluated against the MCF-7 and MDA human breast carcinoma cell lines. 5-(3-Hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-2,3-dihydro-benzoxepine (11g) was found to be the most potent product when evaluated against the MCF-7 breast cancer cell line. A brief computational study of the structure–activity relationship for the synthesized compounds is presented. These 4,5-diarylbenzoxepins are identified as potentially useful scaffolds for the further development of antitumor agents which target tubulin.

Keywords::

• Benzoxepin
• combretastatin analogs
• antiproliferative activity

Introduction

Inhibition of microtubule function by tubulin-targeting compounds is a recognized approach to cancer chemotherapy^1,2 and there have been many known antimitotic agents derived from natural product agents^3,4. Colchicine⁵ and the vinca alkaloids⁶, which bind to distinct binding sites of tubulin, result in the destabilization of microtubules and subsequent apoptosis of the cell, while combretastatin A-4 is a powerful antimitotic agent, due to inhibition of tubulin polymerization⁷. Paclitaxel binds to an alternative site on the tubulin and produces a stabilizing effect on microtubules and leads to an accumulation of cells in metaphase arrest^8,9. The neovasculature present in solid tumors is a target for development of new agents which disrupt the microtubule complex by interacting with β-tubulin. Many synthetic, semisynthetic, and natural compounds have been investigated as tubulin inhibitors and vascular targeting agents¹⁰, and a number are in clinical trials currently, such as combretastatin A-4 (CA-4) as the phosphate ester prodrug¹¹ and the combretastatin A-4 derivative AVE8062¹². The cis configuration of the CA-4 is known to be essential for activity, together with the 3,4,5-trimethoxy groups on ring A (Figure 1). The cis-1,2-diarylethylene scaffold of CA-4 undergoes rapid cis–trans isomerization in heat, light, and protic media. Many cis-restricted analogs of CA-4 have been synthesized to improve the solubility, stability, and therapeutic index⁴ of these drugs. A number of examples where the olefinic group is replaced by a conformationally restricted ring structure have demonstrated significant antiproliferative activity¹³, including those based on coumarin (1)^14,15, 2(5H)-furanone (2)¹⁶, imidazole¹⁷, 1,3-oxazole (3)¹⁷, furazan (4)⁴ and furan (5)¹⁸, diarylindole^17,19, and arylthioindole²⁰, illustrated in Figure 1. We have previously reported the application of the benzoxepin²¹ and benzothiepin scaffolds²² for the design of antiproliferative agents as estrogen receptor (ER) antagonists. We have now investigated the development of a benzoxepin type scaffold as a conformationally restricted analog for combretastatin CA-4. From preliminary inspection, the aryl rings A and B of the target 5,6-diarylbenzoxepin structures can adopt a configuration in which they are not coplanar and now resemble the 3D orientation of the rings A and B of combretastatin CA-4. We therefore could expect activity at the colchicine binding site of tubulin for suitably substituted benzoxepins. This type of benzoxepin structure will also avoid the inactivation observed for the conventional combretastatin derivatives, which is caused by cis–trans isomerism of the olefinic bond observed in vivo²³.

Figure 1.  Combretastatin A-4, colchicines, and selected analogs of combretastatin A-4.

We have investigated the development of the benzoxepin type scaffold as a conformationally restricted analog for combretastatin, where the seven-membered oxygen containing ring forms a slightly flexible bridge on the ethylene bond linking the combretastatin rings A and B. Fifteen compounds have been synthesized and evaluated for specific antiproliferative activity in two human breast cancer cell lines, MCF-7 (estrogen receptor positive) and MDA-MB 231(estrogen receptor negative). A study of the cell cycle effects is also presented together with a docking study of the compounds in the colchicine binding site of tubulin, which can be used to rationalize the possible binding mode and activity of these novel compounds.

Materials and methods

Chemistry

All reagents were commercially available and were used without further purification unless otherwise indicated. Anhydrous tetrahydrofuran (THF) was obtained by distillation from benzophenone-sodium under nitrogen immediately before use. All reactions were performed under a nitrogen atmosphere unless specifically noted. Infrared (IR) spectra were recorded as thin films on NaCl plates or as KBr disks on a PerkinElmer Paragon 100 Fourier transform (FT)-IR spectrometer.¹H and ¹³C nuclear magnetic resonance (NMR) spectra were obtained on a Bruker Avance DPX 400 instrument at 20°C, 400.13 MHz for ¹H spectra, 100.61 MHz for ¹³C spectra, in CDCl₃, CD₃COCD₃, or CD₃OD (internal standard tetramethylsilane). Low resolution mass spectra were run on a Hewlett-Packard 5973 MSD gas chromatography-mass spectrometry (GC-MS) system in an electron impact mode, while high resolution accurate mass determinations for all final target compounds were obtained on a Micromass time-of-flight (TOF) mass spectrometer equipped with electrospray ionization (ES) interface operated in the positive ion mode at the High Resolution Mass Spectrometry Laboratory by Dr. Martin Feeney in the Department of Chemistry, Trinity College Dublin. Thin layer chromatography (TLC) was performed using Merck silica gel 60 TLC aluminum sheets with fluorescent indicator, visualizing with ultraviolet (UV) light at 254 nm. Flash chromatography was carried out using standard silica gel 60 (230–400 mesh) obtained from Merck. All products isolated were homogeneous on TLC. All samples were analyzed using reversed phase high performance liquid chromatography (Waters Alliance system). The analysis was performed at 254 nm using a Phenomonex column (4 μm, 250 × 4.60 mm) using a mobile phase of acetonitrile:water (0.1% trifluoroacetic acid (TFA)) 70:30 delivered at a flow rate of 1.0 mL/min.

4-(3,4-Dimethoxy-phenoxy)-butyric acid 6b A mixture of 3,4-dimethoxyphenol (50 mmol) and potassium carbonate (6.9 g, 55 mmol) in acetone (100 mL) was stirred for 30 min. Ethyl 4-bromobutyrate (7.9 mL, 55 mmol) was added dropwise via syringe and catalytic KI was added. The reaction mixture was refluxed for 24 h. The reaction mixture was cooled to ambient temperature and the solids removed by filtration. The solid was washed with acetone (50 mL) and the combined filtrate and washings were concentrated under reduced pressure. The residue was taken up in diethyl ether (150 mL), washed with 5% sodium hydroxide (50 mL), and the solvent removed under reduced pressure. The residue was dissolved in ethanol (20 mL) and treated with 10% sodium hydroxide in water (100 mL) and heated at 110°C for 3 h or until the solution went clear. The solution was cooled and acidified with concentrated hydrochloric acid and the product which precipitated was filtered and dried, (Yield 5g, 41%). M.P. 89–91°C²⁴. IR ν_max (film): 1682 (C=O), 1601, 1450 cm⁻¹; ¹H (400 MHz, CDCl₃): δ 6.80 (1 H, d,J = 8.52 Hz, Ar-H), 6.54 (1 H, d, J = 2.52 Hz, Ar-H), 6.41 (1 H, dd, J = 2.76 Hz, 6.04 Hz, Ar-H), 3.99 (2 H, t, J = 6.02 Hz, CH₂), 3.88 (3 H, s, OCH₃), 3.85 (3 H, s, OCH₃), 2.62 (2 H, t, J = 7.28 Hz, CH₂), 2.13 (2 H, t, J = 6.78 Hz, CH₂) ppm; ¹³C (101 MHz, CDCl₃): δ 178.4 (C), 152.9 (C), 149.4 (C), 143.1 (C), 111.3 (CH), 103.2 (CH), 100.3 (CH), 66.5 (CH₂), 55.9 (OCH₃), 55.4 (OCH₃), 30.0 (CH₂), 23.9 (CH₂) ppm.

4-(3,5-Dimethoxy-phenoxy)-butyric acid 6c Preparation as above from 3,5-dimethoxyphenol to afford a brown solid, (3.8 g, 31%). M.P. 62°C. IR ν_max (film) cm⁻¹: 1682 (C=O), 1601, 1450; ¹H (400 MHz, CDCl₃): δ 6.08 (3 H, t, J = 2.00 Hz, Ar-H), 3.98 (2 H, t, J = 6.02 Hz, OCH₂CH₂), 3.76 (6 H, s, OCH₃), 2.58 (2 H, t, J = 7.28 Hz, OCH₂CH₂), 2.09 (2 H, qn, J = 6.54 Hz, 7.00 Hz, OCH₂CH₂CH₂) ppm; ¹³C (101 MHz, CDCl₃): δ 178.5 (C), 161.0 (C), 160.2 (C), 92.9 (CH), 92.6 (CH), 66.1 (CH₂), 54.9 (OCH₃), 30.0 (CH₂), 23.8 (CH₂) ppm; Found 263.0888; C₁₂H₁₆O₅Na requires 263.0895.

3,4-Dihydro-2H-1-benzoxepin-5-one 7a 4-Phenoxybutyric acid (6a) (5 g) and polyphosphoric acid (51 g) were heated together at 80°C for 2 h. The brown syrup was poured into ice-water and the aqueous solution extracted with dichloromethane. The organic layers were washed with water (100 mL), brine (100 mL), and then dried over sodium sulfate and the solvent removed under reduced pressure. The crude product was purified by chromatography (silica, 5% diethyl ether in hexane) to give the product as a yellow oil (1.37 g, 37%)²⁵. IR ν_max (film) cm⁻¹: 1682 (C=O), 1601, 1450; ¹H NMR (400 MHz, CDCl₃) δ 7.77 (1 H, dd, J = 2.0 Hz, 8.1 Hz, Ar-H), 7.44-7.39 (1 H, td, J = 1.3 Hz, 7.0 Hz, Ar-H), 7.11–7.06 (2 H, m, Ar-H), 4.23 (2 H, t, J = 7.0 Hz, OCH₂), 2.89 (2 H, t, J = 7.0 Hz, (O=C-CH₂), 2.21 (2 H, m, C-CH_2-C); ¹³C NMR (101 MHz, CDCl₃): δ 200.6 (C), 161.9 (C), 133.6 (CH), 129.4 (CH), 128.9 (C), 122.6 (CH), 120.7 (CH), 72.6 (CH₂), 40.5 (CH₂), 26.3 (CH₂) ppm; HRMS: Found 162.1850; C₁₀H₁₀O₂ requires 162.1900.

6,7-Dimethoxy-3,4-dihydro-2H-benzo[b]oxepin-5-one 7b Preparation as above from 4-(3,4-dimethoxy-phenoxy)-butyric acid 6b (5 g) and polyphosphoric acid (51 g) or Eaton’s reagent (20 g). The crude product was purified by chromatography (silica, 5% diethyl ether in hexane) to afford the product as a brown solid, (1.36g, 29%). M.P. 77°C²⁴. IR ν_max (KBr): 2966.2, 1659.1 (C=O), 1604.3, 1502.4 cm⁻¹; ¹H (400 MHz, CDCl₃): δ 7.27 (1 H, s, Ar-H), 6.56 (1 H, s, Ar-H), 4.19 (2 H, t, J = 7.02 Hz, CH₂), 3.89 (3 H, s, OCH₃), 3.87 (3 H, s, OCH₃), 2.88 (2 H, t, J = 6.78 Hz, CH₂), 2.15 (2 H, qn, J = 6.76 Hz, 7.00 Hz, OCH₂CH₂CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 198.8 (C), 157.6 (C), 153.3 (C), 144.6 (C), 120.4 (C), 109.5 (C), 103.5 (CH), 72.6 (CH₂), 55.7 (OCH₃), 40.0 (CH₂), 25.5 (CH₂) ppm; HRMS: Found 245.0798; C₁₂H₁₄O₄Na requires 245.0790.

6,8-Dimethoxy-3,4-dihydro-2H-benzo[b]oxepin-5-one 7c Preparation as above from 4-(3,5-dimethoxy-phenoxy)-butyric acid 6c (2 g, 8.32 mmol) and polyphosphoric acid (20 g) or Eaton’s reagent (20 g). The product was purified by flash column chromatography (SiO₂, 5% diethyl ether/hexane) to afford the product as a yellow gel, (1.25 g, 67.6%). IR ν_max (film):1573.58, 1604, 1682 (C=O), 2941 cm⁻¹; ¹H (400 MHz, CDCl₃): δ 6.19 (2 H, s, Ar-H), 4.16 (2 H, t, J = 6.26 Hz, CH₂), 3.82 (3 H, s, OMe), 3.81 (3H, s, OMe), 2.78 (2H, t, J = 6.76 Hz, CH₂), 2.14 (2H, qn, J = 6.65 Hz, CH₂); ¹³C NMR (101 MHz, CDCl₃): δ 199.9 (C), 163.1 (C), 163.0 (C), 115.8 (C), 113.6 (C), 97.0 (CH), 94.1 (CH), 71.9 (CH₂), 56.0 (OCH₃), 53.0 (OCH₃), 41.5 (CH₂), 26.2 (CH₂) ppm; HRMS: Found 245.0793, C₁₂H₁₄O₄Na requires 245.0790.

5-(4-Methoxyphenyl)-2,3-dihydro-1-benzoxepin 9a To a mixture of 3,4-dihydro-2H-1-benzoxepin-5-one 7a (0.40 g, 2.50 mmol) and sodium carbonate (0.79 g, 7.2 mmol) in dichloromethane (30 mL) at 0°C under nitrogen was added trifluoromethanesulfonic anhydride (1.20 mL, 7.2 mmol). The mixture was stirred overnight at room temperature and water (30 mL) was added. The aqueous layer was extracted with dichloromethane (2 × 50 mL), washed with brine (30 mL), dried over sodium sulfate, and the solvent was removed under reduced pressure. The triflate (8a) was dissolved in THF (30 mL), and 4-methoxyphenylboronic acid (493 mg, 2.86 mmol) and 2 M Na₂CO₃ (4.8 mL) were added and the mixture stirred under nitrogen for 10 min. Tetrakis(triphenylphosphine)palladium(0) Pd(PPh₃)₄ (149 mg) was added and the reaction refluxed for 6 h at 85°C. The solution was cooled to room temperature and acidified with 2 M hydrochloric acid. The aqueous layer was extracted with dichloromethane (3 × 30 mL) and the combined organic layers were washed with water (30 mL), brine (30 mL), dried over sodium sulfate, and the solvent removed under reduced pressure. The product was purified by column chromatography (silica, 5% diethyl ether/hexane) to give the product as a yellow solid, (198 mg, 31%). M.P. 71°C²¹. IR ν_max (KBr):1657.7, 1602.3, 1510.3, 1510.3, 1484.6, 1247.8, 1177.5 cm⁻¹; ¹H (400 MHz, CDCl₃): δ 7.25 (3H, m, Ar-H), 7.13 (1H, d, J = 2.0 Hz, Ar-H), 7.03 (2H, m, Ar-H), 6.91 (2H, d, J = 8.80 Hz, Ar-H), 6.29 (1H, t, J = 6.36 Hz, CH), 4.53 (2 H, t, J = 6.36 Hz, CH₂), 3.85 (3 H, s, OCH₃), 2.51 (2 H, t, J = 6.20 Hz, CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 158.4 (C), 157.4 (C), 140.5 (C), 134.8 (C), 132.60 (C), 130.7 (CH), 129.2 (CH), 129.1 (CH), 128.9 (CH), 125.4 (CH), 122.7 (CH), 114.1 (CH), 114.0 (CH), 113.1 (CH), 77.5 (CH₂), 54.8 (CH₃), 29.4 (CH₂) ppm; HRMS: Found 253.1241; C₁₇H₁₇O₂ requires 253.1229.

5-(4-Hydroxy-phenyl)-2,3-dihydro-1-benzoxepin 9b A mixture of 3,4-dihydro-2H-benzoxepin-5-one 7a (0.33 g, 2.04 mmol) and phosphorus tribromide (1.00 mL, 10.4 mmol) was heated at 90°C for 24 h. The reaction was cooled to room temperature, added slowly dropwise to ice-water (20 mL), and the aqueous layer extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with water (20 mL), dried over sodium sulfate, and the solvent removed under reduced pressure. The product was immediately purified by chromatography (silica, 5% diethyl ether in hexane) to give a yellow oil, 5-bromo-2,3-dihydro-benzo[b]oxepin (0.26 g, 56%), which was used immediately in the following reaction. Pd(PPh₃)₄ (40 mg) was added to a solution, 4-hydroxyphenylboronic acid (0.76 g, 5.5 mmol) and 2 M Na₂CO₃ (12.8 mL, 25 mmol) were added to this product, and the solution heated to 80°C for 24 h. The solution was cooled, water was added (20 mL), and the aqueous layer extracted with dichloromethane (4 × 50 mL). The combined organic layers were washed with brine (20 mL), dried over sodium sulfate, and the solvent removed under reduced pressure. The crude product was purified by column chromatography (silica, 5% diethyl ether in hexane) to give 4-(2,3-dihydro-benzo[b]oxepin-5-yl)-phenol as a white solid, (0.62 g, 52%), which was used in subsequent reactions without further purification. M.P. 110°C. IR ν_max (KBr): 3384 (OH), 1606, 1513, 1485, 1263, 1201, 758 cm⁻¹; ¹H (400 MHz, CDCl₃): δ 7.23 (1 H, m, Ar-H), 7.14 (3 H, m, Ar-H), 7.00 (2 H, m, Ar-H), 6.77 (2 H, d, J = 6.0Hz, Ar-H), 6.27 (1 H, t, J = 6.0 Hz, CH), 2.51 (2 H, t, J = 6.0 Hz, OCH₂), 2.46 (2 H, m, CH₂);¹³C NMR (101 MHz, CDCl₃): δ 157.6 (C), 154.8 (C), 140.9 (C), 135.3 (C), 133.0 (CH), 131.0 (CH), 129.8 (CH), 128.4 (CH), 126.9 (CH), 123.3 (CH), 121.9 (CH), 114.9 (CH), 78.0 (CH₂), 29.7 (CH₂) ppm; HRMS: Found: 261.0902; C₁₆H₁₄O₂Na requires 261.0891.

5-(3,4-Dimethoxyphenyl)-2,3-dihydro-1-benzoxepin 9c Preparation as described above for 9a from 3,4-dihydro-2H-benzo[b]oxepin-5-one 7a (1.36 g, 8.88 mmol) and 3,4-dimethoxyphenylboronic acid (1.29 g, 8.88 mmol) via the triflate 8c. The crude product was purified by column chromatography (silica, 5% diethyl ether/hexane) to afford the product as a yellow oil which was used in subsequent reactions without further purification, (1.12 g, 45%). IR ν_max (KBr): 2935, 1606, 1508, 1246 (C=C) cm⁻¹; ¹H (400 MHz, CDCl₃): δ 7.23–7.21 (1 H, m, Ar-H), 7.13 (1 H, d, J = 7.52 Hz, Ar-H), 7.04–7.02 (2 H, m, Ar-H), 6.86 (1 H, d, 1.52 Hz, Ar-H), 6.85 (1 H, s, Ar-H), 6.82 (1 H, d, J = 2.04 Hz, Ar-H), 6.29 (1H, t, J = 6.28 Hz, CH), 4.50 (2 H, t, J = 6.02 Hz, OCH₂), 3.90 (3 H, s, OCH₃), 3.83 (3 H, s, OCH₃), 2.47 (2 H, q, J = 6.03 Hz, CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 157.4 (C), 148.1 (C), 147.9 (C), 140.7 (C), 135.2 (C), 132.4 (C), 130.7 (CH), 128.0 (CH), 126.8 (CH), 122.7 (CH), 121.4 (CH), 120.5 (CH), 111.4 (CH), 110.3 (CH), 77.3 (CH₂), 55.5 (OCH₃), 55.4 (OCH₃), 29.4 (CH₂) ppm.

5-(3-Fluoro-4-methoxyphenyl)-2,3-dihydro-1-benzoxepin 9d Preparation as described above for 9a from 3,4-dihydro-2H-benzo[b]oxepin-5-one 7a (1.36 g, 8.88 mmol) and 3-fluoro-4-methoxyphenylboronic acid (0.76 g, 3.82 mmol) via the triflate 8a. The crude product was purified by column chromatography (silica, 5% diethyl ether/hexane) to give the product as a white solid, (0.60 g, 58%). M.P. 88°C. IR ν_max (KBr): 2932.9, 1596.7, 1574.9, 1483.3, 1209.4 cm⁻¹; ¹H (400 MHz, CDCl₃): δ 7.27–7.24 (1 H, m, Ar-H), 7.14 (1 H, dd, J = 1.00 Hz, 7.04 Hz, Ar-H), 7.09 (1 H, d, J = 3.48 Hz, Ar-H), 7.04–7.01 (3 H, m, Ar-H), 6.94 (1 H, t, J = 8.54 Hz, Ar-H), 6.31 (1H, t, J = 6.02 Hz, CH), 4.52 (2 H, t, J = 6.02 Hz, OCH₂), 3.93 (3 H, s, OCH₃), 2.50 (2 H, q, J = 6.19 Hz, CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 157.4 (C), 152.8 (C), 150.4 (C), 146.5 (C), 146.4 (C), 139.7 (C), 135.4 (C), 132.0 (CH), 130.5 (C), 129.1 (CH), 128.9 (CH), 128.2 (CH), 127.4 (CH), 123.8 (CH), 122.9 (CH), 121.1 (CH), 115.9 (CH), 115.7 (CH), 114.1 (CH), 112.5 (CH), 112.4 (CH), 77.4 (CH₂), 58.8 (OCH₃), 29.3 (CH₂) ppm; HRMS: Found 271.1169; C₁₇H₁₆O₂F requires 271.1134.

5-(3-Formyl -4-methoxyphenyl)-2,3-dihydro-1-benzoxepin 9e Preparation as described above for 9a from 3,4-dihydro-2H-benzo[b]oxepin-5-one 7a (1.36 g, 8.88 mmol) and 3-formyl-4-methoxyphenylboronic acid (0.61 g, 3.37 mmol) via the triflate 8a. The product was purified by column chromatography (silica, 2.5% methanol/dichloromethane) to give the product as a yellow oil, (0.57 g, 26%). IR ν_max (film): 1759 (C=O), 3384 (OH), 1603, 1494, 1269, 1180 cm⁻¹. ¹H (400 MHz, CDCl₃): δ 10.5 (1 H, s, CHO), 7.81 (1 H, d, J = 2.52 Hz, Ar-H), 7.42 (1 H, dd, J = 2.52 Hz, 6.00 Hz, Ar-H), 7.25 (1 H, d, J = 7.56 Hz, Ar-H), 7.09 (1 H, d, J = 3.00 Hz, Ar-H), 6.89–7.01 (3H, m, Ar-H), 6.29 (1H, t, J = 6.04 Hz, CH), 4.47 (2 H, t, J = 6.02 Hz, OCH₂), 3.93 (3 H, s, OCH₃), 2.47 (2 H, q, J = 6.01 Hz, CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 189.7 (CHO), 161.1 (C), 157.9 (C), 139.8 (CH), 136.1 (C), 135.3 (C), 132.2 (C), 130.7 (CH), 129.4 (C), 129.3 (C), 128.8 (CH), 128.6 (CH), 128.3 (CH), 126.6 (C), 124.4 (CH), 123.3 (CH), 121.9 (CH), 121.5 (CH), 114.4 (CH), 111.4 (CH), 76.7 (CH₂), 55.7 (CH₃), 29.9 (CH₂) ppm; HRMS: Found 281.1185; C₁₈H₁₇O₃ requires 281.1178.

5-(3,4,5-Trimethoxyphenyl)-2,3-dihydro-1-benzoxepin 9f Preparation as described above for 9a from 3,4-dihydro-2H-benzo[b]oxepin-5-one 7a (1.36 g, 8.88 mmol) and 3,4,5-trimethoxyphenylboronic acid (0.93 g, 4.38 mmol) via the triflate 8a. The crude product was purified by column chromatography (silica, 5% diethyl ether/hexane) to give the product as a yellow oil, (0.311 g, 27%). IR ν_max (film): 2936.9, 1579.9, 1124.3, 1007.4 cm⁻¹; ¹H (400 MHz, CDCl₃): δ 7.25–7.21 (1 H, m, Ar-H), 7.12 (1 H, d, J = 8.00 Hz, Ar-H), 7.04 (2 H, d, J = 4.00 Hz, Ar-H), 6.52 (2 H, s, Ar-H), 4.50 (2 H, t, J = 6.04 Hz, OCH₂), 3.89 (3 H, s, OCH₃), 3.83 (3 H, s, OCH₃), 2.53 (2 H, q, J = 6.19 Hz, CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 157.5 (C), 152.4 (C), 140.9 (C), 138.2 (C), 136.8 (C), 131.9 (CH), 130.8 (C), 128.1 (CH), 127.5 (CH), 122.7 (CH), 121.4 (CH), 105.4 (CH), 76.9 (CH₂), 60.5 (OCH₃), 55.6 (OCH₃), 29.6 (CH₂) ppm; HRMS: Found 335.1272; C₁₉H₂₀O₄Na requires 335.1259.

6,8-Dimethoxy-5-(4-methoxyphenyl)-2,3-dihydro-benzoxepin 9g Preparation as described above for 9a from 6,7-dimethoxy-3,4-dihydro-2H-benzo[b]oxepin-5-one 7b (1.36 g, 8.88 mmol) and 4-methoxyphenylboronic acid (0.532 g, 3.5mmol) via the triflate 8b. The crude product was purified by column chromatography using silica and 5% diethylether in hexane to afford the product as an oil which was used in subsequent reactions without further purification, (0.232 g, 27.5%). IR ν_max (film): 2967, 1604, 1573, 1251 cm⁻¹; ¹H (400 MHz, CDCl₃): δ 7.16 (2 H, d, J = 9.0 Hz, Ar-H), 6.79 (2 H, d, J = 8.5 Hz, Ar-H), 6.39 (2 H, m, Ar-H), 6.29 (1 H, d, J = 2.5 Hz, Ar-H), 4.48 (2 H, t, J = 6.0 Hz, CH₂), 3.82 (3 H, s, OCH₃), 3.79 (3 H, s, OCH₃), 3.46 (3 H, s, OCH₃), 2.31 (2 H, t, J = 6.5 Hz, CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 160.7 (C), 158.4 (C), 158.0 (C), 139.3 (C), 134.4 (C), 126.9 (CH), 124.9 (CH), 115.0 (C), 113.1 (CH), 99.4 (CH), 95.8 (CH), 80.4 (CH₂), 55.6 (OCH₃), 55.3 (OCH₃), 55.2 (OCH₃), 27.3 (CH₂) ppm.

7,8-Dimethoxy-5-(4-methoxy-phenyl)-2,3-dihydro-benzo[b]oxepin 9h Preparation as described above for 9a from 6,8-dimethoxy-3,4-dihydro-2H-benzo[b]oxepin-5-one 7c (0.5 g, 2.25 mmol) and 4-methoxyphenylboronic acid (0.445 g, 2.93 mmol) via the triflate 8b. The product was purified by column chromatography (silica, 5% diethyl ether/hexane) to give the product as a white solid, (0.393g, 55.9%). M.P. 81–90°C. IR ν_max (KBr): 2967, 1604, 1573, 1251 cm⁻¹; ¹H (400 MHz, CDCl₃): δ 7.23 (2 H, m, Ar-H), 6.86 (2 H, m, Ar-H), 6.69 (1 H, s, Ar-H), 6.48 (1 H, s, Ar-H), 6.21 (1 H, t, OCH₂CH₂CH), 4.48 (2 H, t, CH₂), 3.88 (3 H, s, OCH₃), 3.81 (3 H, s, OCH₃), 3.65 (3 H, s, 3 × OCH₃), 2.44 (2H, t, CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 158.8 (C), 151.8 (C), 148.7 (C), 144.5 (C), 140.8 (C), 134.8 (C), 129.4 (CH), 125.7 (CH), 124.0 (CH), 113.4 (CH), 105.4 (CH), 78.5(CH₂), 56.0 (OCH₃), 55.8 (OCH₃), 55.1 (OCH₃), 29.6 (CH₂) ppm; HRMS: Found 335.1260; C₁₉H₂₀O₄Na requires 335.1259.

4-Bromo-5-(4-methoxyphenyl)-2,3-dihydro-1-benzoxepin 10a To a solution of 5-(4-methoxyphenyl)-2,3-dihydro-1-benzoxepin 9a (1.62 g, 5.74 mmol) in dichloromethane (30 mL) at 0°C was added pyridinium tribromide (1.08 g, 5.74 mmol, technical grade 90%) and stirred for 1 h with careful monitoring by TLC. Water (30 mL) was added and the solution extracted with dichloromethane (2 × 30 mL). The combined organic layers were washed with a saturated solution of sodium hydrogen carbonate (30 mL), brine (30 mL), dried over sodium sulfate, and the solvent removed under reduced pressure. The residue was purified by column chromatography (silica, 5% diethyl ether/hexane) to give the product as a brown solid which was used in the following reaction without further purification, (1.64 g, 86%). M.P. 69°C²¹. IR ν_max (KBr): 1604.7, 1509.3, 1248.5, 1172.9, 1036.9, 753.9 cm⁻¹; ¹H (400 MHz, CDCl₃): δ 7.21 (2H, m, Ar-H), 7.13 (1H, dd J = 0.98 Hz, 6.84 Hz, Ar-H), 6.95 (1H, m, Ar-H), 6.91 (2H, d, J = 8.80 Hz, Ar-H), 6.82 (1H, dd, J = 1.46 Hz, 6.36 Hz, Ar-H), 4.62 (2 H, t, J = 6.86 Hz, CH₂), 3.86 (3 H, s, OCH₃), 3.04 (2 H, t, J = 5.86 Hz, CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 158.4 (C), 156.0 (C), 139.0 (C), 134.3 (C), 133.2 (C), 130.9 (CH), 130.8 (CH), 129.1 (CH), 129.0 (CH), 128.5 (CH), 123.0 (CH), 121.6 (C), 121.4 (CH), 115.3 (CH), 114.1 (CH), 113.0 (CH), 77.1 (CH₂), 54.7 (CH₃), 40.7 (CH₂) ppm.

4-Bromo-5-(4-hydroxyphenyl)-2,3-dihydro-1-benzoxepin 10b Preparation as described above for 10a from 4-(2,3-dihydro-benzoxepin-5-yl)phenol 9b (0.67 g, 2.8 mmol). The impure product was purified by column chromatography (silica, dichloromethane:hexane 1:1) to give the product as a white solid, (0.59 g, 67%)²¹. M.P. 58°C. IR ν_max (KBr): 3384 (OH), 1606, 1513, 1485, 1263, 1201, 758cm⁻¹; ¹H (400 MHz, CDCl₃): δ 7.20 (1 H, t, J = 6.0 Hz, m, Ar-H), 7.14 (2 H, d, J = 8.5 Hz, Ar-H), 7.07 (1 H, dd, J = 8.0 Hz, 1.0 Hz, Ar-H), 6.95 (1 H, t, J = 6.5Hz, Ar-H), 6.85–6.77 (3 H, m, Ar-H), 4.59 (2 H, t, J = 6.0 Hz, OCH₂), 3.00 (2 H, t, J = 6.0 Hz, CH₂);¹³C NMR (101 MHz, CDCl₃): δ 156.4 (C), 154.8 (C), 131.4 (CH), 128.9 (CH), 123.4 (CH), 122.0 (CH), 114.9 (CH), 77.4 (CH₂), 41.1 (CH₂) ppm; HRMS: Found 317.1842; C₁₆H₁₄BrO₂ requires 317.1848.

4-Bromo-5-(3,4-dimethoxyphenyl)-2,3-dihydro-1-benzoxepin 10c Preparation as described above for 10a from 5-(3,4-dimethoxyphenyl)-2,3-dihydro-1-benzoxepin 9c (1.62 g, 5.74 mmol). The crude residue was purified by column chromatography (silica, 5% diethyl ether/hexane) to give the product, (1.64 g, 79%) as a white solid. M.P. 88°C. IR ν_max (KBr): 2934.1, 1582.4, 1512.4, 1440.1, 1247.5, 1223.7 cm⁻¹; ¹H (400 MHz, CDCl₃): δ 7.18 (1 H, m, Ar-H), 7.06 (1 H, m, Ar-H), 6.92 (1 H, m, Ar-H), 6.80 (1 H, dd, J = 8.00 Hz, 1.50 Hz, Ar-H), 4.55 (2 H, t, J = 5.80 Hz, CH₂), 3.87 (3 H, s, OCH₃), 3.80 (3 H, s, OCH₃), 2.99 (2 H, t, J = 6.00 Hz, CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 156.0 (C), 148.0 (C), 147.8 (C), 139.1 (C), 134.5 (C), 132.9 (C), 130.9 (CH), 128.5 (CH), 123.0 (CH), 122.0 (CH), 121.6 (CH), 121.5 (CH), 112.7 (CH), 110.1 (CH), 76.8 (CH₂), 55.5 (OCH₃), 55.4 (OCH₃), 40.8 (CH₂) ppm; HRMS: Found 359.0300; C₁₈H₁₇BrO₃ requires 359.0283.

4-Bromo-5-(3-fluoro-4-methoxyphenyl)-2,3-dihydro-1-benzoxepin 10d Preparation as described above for 10a from 5-(3-fluoro-4-methoxyphenyl)-2,3-dihydro-1-benzoxepin 9d (0.60 g, 2.22 mmol). The crude residue was purified by column chromatography (silica, 5% diethyl ether/hexane) to give the product as a brown solid which was used in subsequent reactions without further purification, (0.72 g, 93%). M.P. 47°C. IR ν_max (KBr): 2924.9, 1511.2, 1483.9, 1267.4, 1126.8 cm⁻¹; ¹H (400 MHz, CDCl₃): δ 7.28–7.22 (1 H, m, Ar-H), 7.09 (1 H, d, J = 7.36 Hz, Ar-H), 7.02 (1 H, d, J = 11.24 Hz, Ar-H), 6.99–6.96 (3 H, m, Ar-H), 6.81 (1 H, dd, J = 1.48 Hz, 6.32 Hz, Ar-H), 4.60 (2 H, t, J = 5.86 Hz, CH₂), 3.94 (3 H, s, OCH₃), 3.03 (2 H, t, J = 5.86 Hz, CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 156.2 (C), 152.8 (C), 150.3 (C), 147.8 (C), 146.7 (C), 138.2 (C), 134.9 (C), 134.8 (C), 132.7 (C), 131.0 (CH), 128.8 (CH), 125.8 (CH), 123.3 (CH), 122.3 (C), 121.9 (CH), 117.7 (CH), 117.5 (CH), 112.5 (CH), 76.5 (CH₂), 55.9 (CH₃), 40.9 (CH₂) ppm; ¹⁹F NMR (376 MHz, CDCl₃, Me₄Si): δ–135.94 ppm; HRMS: Found: 371.0060; C₁₇H₁₄O₂FBrNa requires 371.0059.

4-Bromo-5-(3-formyl-4-methoxyphenyl)-2,3-dihydro-1-benzoxepin 10e Preparation as described above for 10a from 5-(3-formyl-4-methoxyphenyl)-2,3-dihydro-1-benzoxepin 9e (217 mg, 0.77 mmol). The crude residue was purified by chromatography (silica, 5% diethyl ether/hexane) to give the product as a white solid which was used in subsequent reactions without further purification, (184 mg, 60%). M.P. 145°C. IR ν_max (KBr): 2917.5, 1677.2, 1599.8, 1484.9, 1257.0 cm⁻¹; ¹H (400 MHz, CDCl₃): δ 10.50 (1 H, s, CHO), 7.76 (1 H, d, J = 2.48 Hz, Ar-H), 7.43 (1 H, dd, J = 2.52 Hz, 6.00 Hz, Ar-H), 7.23 (1 H, dt, J = 1.52 Hz, 6.76 Hz, Ar-H), 7.10 (1 H, d, J = 8.04 Hz, Ar-H), 7.03 (1 H, d, J = 8.56 Hz, Ar-H), 6.95 (1 H, dt, J = 1.00 Hz, 7.52 Hz, Ar-H), 6.74 (1H, dd, J = 1.52 Hz, 6.52 Hz, Ar-H), 4.58 (2 H, t, J = 6.20 Hz, OCH₂), 3.96 (3 H, s, OCH₃), 3.01 (2 H, t, J = 6.02 Hz, CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 189.5 (CHO), 160.9 (C), 156.5 (C), 138.3 (C), 137.4 (CH), 134.8 (CH), 132.8 (CH), 131.1 (CH), 130.2 (C), 129.4 (CH), 129.3 (CH), 129.0 (CH), 126.6 (C), 124.3 (CH), 123.5 (CH), 123.3 (CH), 122.8 (CH), 122.1 (CH), 121.5 (CH), 116.2 (CH), 111.4 (CH), 77.2 (CH₂), 55.7 (CH₃), 53.1 (CH₃), 41.1 (CH₂) ppm; HRMS: Found: 359.0292; C₁₈H₁₆BrO₃ requires 359.0283.

4-Bromo-5-(3,4,5-trimethoxyphenyl)-2,3-dihydro-1-benzoxepin 10f Preparation as described above for 10a from 5-(3,4,5-trimethoxyphenyl)-2,3-dihydro-1-benzoxepin 9f (0.95 g, 3.02mmol). The crude residue was purified by column chromatography (silica, 5% diethyl ether/hexane) to give the product as a white solid, (0.80 g, 67%). M.P. 111°C. IR ν_max (KBr): 2929.6, 1581.9, 1482.6, 1459.3, 1410.0, 1235.3, 1130.2 cm⁻¹; ¹H (400 MHz, CDCl₃): δ 7.23 (1 H, dt, J = 1.32 Hz, 6.54 Hz, Ar-H), 7.09 (1 H, dd, J = 1.00 Hz, 7.00 Hz, Ar-H), 6.98 (1 H, dt, J = 1.35 Hz, 6.26 Hz, Ar-H), 6.87 (1 H, dd, J = 1.50 Hz, 6.52 Hz, Ar-H), 6.47 (2 H, s, Ar-H), 4.61 (2 H, t, J = 5.78 Hz, OCH₂), 3.92 (3 H, s, OCH₃), 3.83 (6 H, s, OCH₃), 3.05 (2 H, t, J = 6.02 Hz, CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 156.0 (C), 152.4 (C), 139.3 (C), 137.4 (C), 136.8 (C), 132.4 (CH), 130.9 (C), 128.5 (CH), 123.1 (CH), 121.9 (CH), 121.5 (C), 106.6 (CH), 77.1 (CH₂), 60.5 (OCH₃), 55.7 (OCH₃), 40.9 (CH₂) ppm; HRMS: Found 413.0381; C₁₉H₁₉O₄NaBr requires 413.0364.

6,8-Dimethoxy-4-bromo-5-(4-methoxyphenyl)-2,3-dihydrobenzoxepin 10g Preparation as described above for 10a from 6,8-dimethoxy-5-(4-methoxyphenyl)-2,3-dihydrobenzoxepin 9g (0.232 g, 0.74 mmol). The crude product was then purified by column chromatography over silica gel (eluent: hexane 95%, diethylether 5%). The product was isolated as a brown solid which was used in subsequent reactions without further purification, (0.083 g, 28.5%). M.P. 96–102°C. IR ν_max (KBr): 2927.1, 1610.3, 1573.7, 1241.8 (C=C), 736.58 (C-Br) cm⁻¹; ¹H (400 MHz, CDCl₃): δ 7.20 (2 H, d, J = 8.52 Hz, Ar-H), 6.84 (2 H, d, J = 8.52 Hz, Ar-H), 6.33 (1 H, d, J = 2.52 Hz, Ar-H), 6.22 (1 H, d, J = 2.52 Hz, Ar-H), 4.59 (2 H, t, J = 6.16 Hz, CH₂), 3.80 (3 H, s, OCH₃), 3.79 (3 H, s, OCH₃), 3.35 (3 H, s, OCH₃), 2.86 (2 H, t, J = 5.78 Hz, CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 161.1 (C), 158.3 (C), 158.1 (C), 156.7 (C), 136.9 (C), 134.0 (C), 129.9 (CH), 119.1 (C), 116.6 (C), 112.5 (C), 99.3 (CH), 96.2 (CH), 78.8 (CH₂), 77.3 (C), 77.0 (C), 76.7 (C), 55.7 (OCH₃), 55.3 (OCH₃), 55.1 (OCH₃), 39.2 (CH₂) ppm; HRMS: Found 413.0376; C₁₉H₁₉O₄NaBr requires 413.0364.

4-Bromo-7,8-dimethoxy-5-(4-methoxy-phenyl)-2,3-dihydro-benzo[b]oxepin 10h Preparation as described for 10a from 7,8-dimethoxy-5-(4-methoxy-phenyl)-2,3-dihydro-benzo[b]oxepin 9h (262 mg, 0.96 mmol). The residue was purified by flash column chromatography (silica, 5% diethyl ether/hexane) to give the product as off-white crystals, (284 mg, 75.6 %). M.P. 79°C. IR ν_max (KBr): 2916.7, 1607,9, 1510.7, 1465.5, 1208.9, 1026.6, 854.0 cm⁻¹; ¹H (400 MHz, CDCl₃, Me₄Si): δ 7.22 (2 H, d, J = 9.04 Hz, Ar-H), 6.93 (2 H, d, J = 8.52 Hz, Ar-H), 6.65 (1 H, s, Ar-H), 6.24 (1 H, s, Ar-H), 4.59 (2 H, t, J = 5.76 Hz, CH₂), 3.88 (3 H, s, OCH₃), 3.86 (3 H, s, OCH₃), 3.59 (3 H, s, OCH₃), 3.03 (2H, t, J = 5.78 Hz, CH₂) ppm; ¹³C (101 MHz, CDCl₃, Me₄Si): δ 158.3 (C), 150.3 (C), 148.8 (C), 144.3 (C), 138.7 (C), 133.9 (C), 130.8 (CH), 124.2 (C), 119.9 (C), 113.1 (CH), 104.9 (CH), 78.1 (CH₂), 55.7 (OCH₃), 55.5 (OCH₃), 54.8 (OCH₃), 40.7 (CH₂) ppm; HRMS: Found 413.0344; C₁₉H₁₉O₄NaBr requires 413.0364.

5-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-2,3-dihydro-1-benzoxepin 11a To a solution of 4-bromo-5-(4-methoxyphenyl)-2,3-dihydro-1-benzoxepin 10a (208 mg, 0.63 mmol) in THF (20 mL) under nitrogen was added tetrakis(triphenylphosphine)palladium(0), Pd(PPh₃)₄ (96 mg) and the reaction stirred for 10 min. 3,4,5-Trimethoxyphenylboronic acid (200 mg, 0.94 mmol) and 2 M sodium carbonate (1.55 mL, 3.14 mmol) were added and the solution heated to 80°C and refluxed overnight. The solution was cooled and 2 M hydrochloric acid was added. The solution was extracted with dichloromethane (3 × 50 mL) and the combined organic layers were washed with water (50 mL), brine (50 mL), dried over sodium sulfate, and the solvent removed under reduced pressure. The residue was purified by column chromatography (silica, 10% diethyl ether/hexane) to give the product as a white solid, (193mg, 73%). IR ν_max (KBr): 1685 (C=O) cm⁻¹; ¹H (400 MHz, CDCl₃): δ 7.27 (1H, m, Ar-H), 7.16 (1H, dd J = 1.02 Hz, 7.00 Hz, Ar-H), 7.05 (1H, dt, J = 1.49 Hz, 6.04 Hz, Ar-H), 6.91 (3H, d, J = 5.00 Hz, Ar-H), 6.70 (2H, d, J = 9.04 Hz, Ar-H), 6.39 (2H, s, Ar-H), 4.66 (2 H, t, J = 6.26 Hz, CH₂), 3.84 (3 H, s, OCH₃), 3.76 (3 H, s, OCH₃), 3.65 (6 H, s, 2 × OCH₃), 2.73 (2 H, t, J = 6.02 Hz, CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 158.1 (C), 154.9 (C), 152.5 (C), 137.7 (C), 137.2 (C), 137.1 (C), 136.5 (C), 133.9 (CH), 132.2 (CH), 130.9 (CH), 128.4 (CH), 123.6 (CH), 122.0 (CH), 113.1 (CH), 106.8 (CH), 80.7 (CH₂), 60.8 (CH₃), 55.9 (CH₃), 55.1 (CH₃), 35.3 (CH₂) ppm.

4-[4-(3,4,5-Trimethoxyphenyl)-2,3-dihydro-1-benzoxepin-5-yl-phenol 11b Preparation as described above for 11a from 4-bromo-5-(4-hydroxyphenyl)-2,3-dihydro-1-benzoxepin 10b (158 mg, 0.5 mmol) and 3,4,5-trimethoxyphenylboronic acid (138 mg, 0.65 mmol). The crude product residue was purified by column chromatography (silica, 10% diethyl ether/hexane) to give the product as a beige solid, (133 mg, 67%). M.P. 151°C. IR ν_max (KBr): 3365.9 (OH), 1610.9, 1578.2, 1127.3 cm⁻¹; ¹H (400 MHz, CDCl₃): δ 7.22 (1 H, t, J = 7.00 Hz, Ar-H), 7.13 (1 H, dd, J = 1.00 Hz, 8.00 Hz, Ar-H), 7.03 (1 H, dt, J = 1.50 Hz, 7.50 Hz, Ar-H), 6.90 (1 H, dt, J = 1.50 Hz, 7.50 Hz, Ar-H), 6.82 (2 H, d, J = 5.00 Hz, Ar-H), 6.57 (2 H, d, J = 8.50 Hz, Ar-H), 6.37 (2 H, s, Ar-H), 4.64 (2 H, t, J = 6.00 Hz, CH₂), 3.82 (3 H, s, OCH₃), 3.62 (6 H, s, OCH₃), 2.70 (2 H, t, J = 6.00 Hz, CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 155.7 (C), 154.4 (C), 152.5 (C), 137.8 (C), 137.6 (C), 137.2 (C), 137.1 (C), 133.7 (C), 132.4 (CH), 130.9 (CH), 128.4 (CH), 123.7 (CH), 122.0 (CH), 114.6 (CH), 106.8 (CH), 80.7 (CH₂), 60.9 (OCH₃), 55.9 (OCH₃), 35.2 (CH₂) ppm; HRMS: Found 427.1519; C₂₅H₂₄O₅Na requires 427.1521.

5-(3,4-Dimethoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-2, 3-dihydro-1-benzoxepin 11c Preparation as described above for 11a from 4-bromo-5-(3,4-dimethoxyphenyl)-2,3-dihydro-1-benzoxepin 10c (240 mg, 851 μmol) and 3,4,5-trimethoxyphenylboronic acid (225 mg, 1.06 mmol). The crude residue was purified by column chromatography (silica, 10% diethyl ether/hexane) to give the product as a white solid, (200 mg, 52%). M.P. 152°C. IR ν_max (KBr): 1577.9, 1515.9, 1407.5, 1232.9, 1125.2, 1024.4 cm⁻¹; ¹H (400 MHz, CDCl₃): δ 7.22 (1 H, dt, J = 1.51 Hz, 6.28 Hz, Ar-H), 7.09 (1 H, dt, J = 1.50 Hz, 6.02 Hz, Ar-H), 7.02 (1 H, d, J = 8.56 Hz, Ar-H), 6.94 (1 H, dd, J = 1.48 Hz, 7.04 Hz, Ar-H), 6.84–6.81 (1 H, m, Ar-H), 6.78 (1 H, d, J = 2.00 Hz, Ar-H), 6.60 (2 H, s, Ar-H), 4.60 (2 H, t, J = 5.76 Hz, CH₂), 3.92 (3 H, s, OCH₃), 3.87 (9 H, s, OCH₃), 3.84 (3 H, s, OCH₃), 3.02 (2 H, t, J = 6.02 Hz, CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 156.0 (C), 153.1 (C), 148.0 (C), 147.8 (C), 139.1 (C), 137.6 (C), 134.5 (C), 132.8 (C), 130.1 (CH), 128.5 (CH), 123.2 (CH), 123.0 (CH), 122.0 (CH), 121.6 (CH), 121.5 (CH), 112.7 (CH), 110.1 (CH), 104.7 (CH), 76.8 (CH₂), 60.4 (OCH₃), 55.7 (OCH₃), 55.6 (OCH₃), 54.5 (OCH₃), 55.3 (OCH₃), 40.8 (CH₂) ppm; HRMS: Found 471.1786; C₂₇H₂₈O₆Na requires 471.1784.

5-(3,4-Dimethoxyphenyl)-4-(2,3,4-trimethoxyphenyl)-2,3-dihydro-benzoxepin 11d Preparation as described above for 11a from 4-bromo-5-(3,4-dimethoxyphenyl)-2,3-dihydro-1-benzoxepin 10c (1.26 g, 3.49 mmol) and 2,3,4-trimethoxyphenylboronic acid (0.92 g, 4.36 mmol). The crude residue was purified by column chromatography (silica, 10% diethyl ether/hexane) to give the product as a white solid, (1.42 g, 90%). M.P. 130°C. IR ν_max (KBr): 2932.1, 1587.4, 1514.2, 1488.9, 1289.3, 1091.5, 1025.7 cm⁻¹; ¹H (400 MHz, CDCl₃): δ 7.26–7.23 (1 H, m, Ar-H), 7.15 (1 H, dd, J = 1.00 Hz, 7.04 Hz, Ar-H), 7.05 (1 H, dt, J = 1.17 Hz, 6.28 Hz, Ar-H), 6.97 (1 H, dd, J = 1.52 Hz, 6.00 Hz, Ar-H), 6.66 (2 H, t, J = 8.28 Hz, Ar-H), 6.57 (2H, d, J = 11.52 Hz, Ar-H), 6.45 (1H, d, J = 8.52 Hz, Ar-H), 4.64 (2 H, t, J = 6.26 Hz, OCH₂), 3.96 (3 H, s, OCH₃), 3.86 (3 H, s, OCH₃), 3.83 (3 H, s, OCH₃), 3.82 (3 H, s, OCH₃), 3.58 (3 H, s, OCH₃), 2.69 (2 H, t, J = 6.26 Hz, CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 155.7 (C), 152.3 (C), 150.9 (C), 147.2 (C), 146.9 (C), 141.6 (C), 137.4 (C), 136.3 (C), 135.4 (C), 133.7 (C), 130.7 (CH), 128.8 (C), 127.9 (CH), 125.6 (CH), 122.9 (CH), 121.6 (CH), 113.8 (CH), 109.7 (CH), 106.3 (CH), 79.9 (CH₂), 60.4 (CH₃), 60.2 (CH₃), 55.4 (CH₃), 55.2 (CH₃), 55.1 (CH₃), 34.6 (CH₂) ppm; HRMS: Found 471.1784; C₂₇H₂₈O₆Na requires 471.1784.

5-(3–Fluoro-4-methoxyphenyl)-4-(2,3,4-trimethoxyphenyl)-2,3-dihydro-benzoxepin 11e Preparation as described above for 11a from 4-bromo-5-(3-fluoro-4-methoxyphenyl)-2,3-dihydro-1-benzoxepin 10d (146 mg, 420 μmol) and 3,4,5-trimethoxyphenylboronic acid (111 mg, 515 μmol). The product was purified by column chromatography (silica, 5% diethyl ether/hexane) to give an orange solid, (170 mg, 94%). M.P. 69°C. IR ν_max (KBr): 2919.1, 1578.3, 1507.8, 1407.2, 1125.7 cm⁻¹; ¹H (400 MHz, CDCl₃): δ 7.33–7.30 (1 H, m, Ar-H), 7.18 (1 H, dd, J = 1.00 Hz, 7.00 Hz, Ar-H), 7.08 (1 H, dt, J = 1.02 Hz, 6.52 Hz, Ar-H), 6.91 (1 H, dd, J = 1.52 Hz, 6.52 Hz, Ar-H), 6.79–6.74 (3 H, m, Ar-H), 6.40 (1H, s, Ar-H), 4.68 (2 H, t, J = 6.02 Hz, OCH₂), 3.87 (3 H, s, OCH₃), 3.86 (3 H, s, OCH₃), 3.69 (6 H, s, OCH₃), 2.74 (2 H, t, J = 6.02 Hz, CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 155.6 (C), 152.3 (C), 150.1 (C), 145.8 (C), 145.7 (C), 138.3 (C), 136.9 (C), 136.4 (CH), 136.1 (C), 135.7 (C), 134.2 (C), 134.1 (C), 130.4 (CH), 130.2 (CH), 130.1 (CH), 128.3 (CH), 126.7 (CH), 126.6 (CH), 125.9 (CH), 123.3 (CH), 121.8 (CH), 118.3 (CH), 118.1 (CH), 112.0 (CH), 106.3 (CH), 80.1 (CH₂), 60.5 (OCH₃), 55.7 (OCH₃), 55.5 (OCH₃), 35.0 (CH₂) ppm; HRMS: Found 459.1584; C₂₆H₂₅O₅FNa requires 459.1584.

5-(3–Formyl-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-2,3-dihydro-benzoxepin 11f Preparation as described above for 11a from 4-bromo-5-(3-formyl-4-methoxyphenyl)-2,3-dihydro-1-benzoxepin 10e (146 mg, 407 μmol) and 3,4,5-trimethoxyphenylboronic acid (147 mg, 682 μmol). The product was purified by column chromatography (silica, 5% diethyl ether/hexane) to give a yellow solid, (175 mg, 96%). M.P. 98°C. IR ν_max (KBr): 2839, 1682 (C=O), 1603, 1579, 1505cm⁻¹; ¹H (400 MHz, CDCl₃): δ 10.3 (1 H, s, CHO), 7.50 (1 H, d, J = 2.04 Hz, Ar-H), 7.26 (1 H, dt, J = 1.68 Hz, 6.26 Hz, Ar-H), 7.17 (2 H, dt, J = 2.01 Hz, 6.01 Hz, Ar-H), 7.01 (1 H, t, J = 6.52 Hz, Ar-H), 6.83 (1 H, dd J = 1.50 Hz, 6.04 Hz, Ar-H), 6.77 (1 H, d, J = 8.52 Hz, Ar-H), 6.37 (2H, s, Ar-H), 4.67 (2 H, t, J = 6.02 Hz, OCH₂), 3.94 (6 H, s, OCH₃), 3.90 (3 H, s, OCH₃), 3.88 (3 H, s, OCH₃), 2.71 (2 H, t, J = 6.02 Hz, CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 189.5 (CHO), 160.3 (C), 156.0 (C), 153.3 (C), 152.7 (C), 139.1 (C), 138.6 (CH), 137.5 (C), 137.4 (C), 136.2 (C), 136.0 (C), 134.1 (C), 131.1 (CH), 130.6 (CH), 128.7 (CH), 124.1 (C), 123.8 (C), 122.2 (CH), 110.9 (CH), 106.7 (CH), 104.5 (CH), 92.7 (CH), 80.4 (CH₂), 60.9 (OCH₃), 60.8 (OCH₃), 56.2 (OCH₃), 55.8 (OCH₃), 35.5 (CH₂) ppm; HRMS: Found 469.1615; C₂₇H₂₆O₆Na requires 469.1627.

5-(3–Hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-2,3-dihydro-benzoxepin 11g Hydrogen peroxide (30%, 10 drops) and sulfuric acid (2.0 mL) were added to a solution of 5-(3-formyl-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-2,3-dihydrobenzoxepin 11f (132 mg, 300 μmol) in methanol (10 mL) and the solution stirred at room temperature for 6 h. Water (20 mL) was added, the solution neutralized to pH 7, the aqueous layer extracted with ethyl acetate (2 × 25 mL), dried over sodium sulfate, and the solvent removed under reduced pressure. The product was purified by column chromatography (silica, 5% diethyl ether/hexane) to give the product as a yellow solid, (75 mg, 58%). IR ν_max (film): 3436.9 (OH), 1580.5, 1509.4, 1242.4, 1125.6 cm⁻¹. ¹H (400 MHz, CDCl₃): δ 7.22 (1 H, dt, J = 1.76 Hz, 6.78 Hz, Ar-H), 7.12 (1 H, dd, J = 1.00 Hz, 7.04 Hz, Ar-H), 7.03 (1 H, dt, J = 1.00 Hz, 7.02 Hz, Ar-H), 6.91 (1 H, dd J = 1.76 Hz, 6.00 Hz, Ar-H), 6.60 (1H, d, J = 8.04 Hz, Ar-H), 6.58 (1 H, d, J = 2.00 Hz, Ar-H), 6.48 (1 H, dd J = 2.00 Hz, 6.04 Hz, Ar-H), 6.21 (2 H, s, Ar-H), 4.63 (2 H, t, J = 6.02 Hz, OCH₂), 3.81 (6 H, s, 2 × OCH₃), 3.65 (6 H, s, 2 × OCH₃), 2.71 (2 H, t, J = 6.26 Hz, CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 155.4 (C), 152.1 (C), 144.8 (C), 144.4 (C), 137.5 (C), 137.3 (C), 136.8 (C), 136.5 (C), 136.1 (C), 134.5 (C), 130.6 (CH), 127.9 (CH), 123.2 (CH), 122.7 (CH), 121.6 (CH), 116.9 (CH), 109.4 (CH), 106.3 (CH), 80.2 (CH₂), 60.4 (OCH₃), 55.8 (OCH₃), 55.5 (OCH₃), 55.4 (OCH₃), 34.9 (CH₂) ppm; HRMS: Found 457.1640; C₂₆H₂₆O₆Na requires 457.1627.

4-(3,4-Dimethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-2, 3-dihydro-1-benzoxepin 11h Preparation as described above for 11a from 4-bromo-5-(3,4,5-trimethoxyphenyl)-2,3-dihydro-1-benzoxepin 10f (196 mg, 0.5 mmol) and 3,4-dimethoxyphenylboronic acid (114 mg, 0.625 mmol). The crude residue was purified by column chromatography (silica, gradient ethyl acetate/hexane) to give the product as a white solid, (185 mg, 83%). M.P. 131°C. IR ν_max (KBr): 1579.6, 1465.6, 1127.3 cm⁻¹; ¹H (400 MHz, CDCl₃): δ 7.28–7.24 (1 H, m, Ar-H), 7.14 (1 H, dd, J = 1.00 Hz, 7.00 Hz, Ar-H), 7.04 (1 H, dt, J = 1.50 Hz, 6.78 Hz, Ar-H), 6.96 (1 H, dd, J = 1.74 Hz, 6.04 Hz, Ar-H), 6.66 (1 H, d, J = 8.00 Hz, Ar-H), 6.55 (1 H, dd, J = 2.02 Hz, 6.00 Hz, Ar-H), 6.52 (1 H, d, J = 2.00 Hz, Ar-H), 6.41 (2 H, s, Ar-H), 4.67 (2 H, t, J = 6.26 Hz, OCH₂), 3.84 (3 H, s, OCH₃), 3.83 (3 H, s, OCH₃), 3.66 (6 H, s, OCH₃), 3.57 (3 H, s, OCH₃), 2.78 (2 H, t, J = 6.02 Hz, CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 155.9 (C), 152.7 (C), 147.9 (C), 147.6 (C), 137.9 (C), 137.8 (C), 137.3 (C), 136.8 (C), 136.6 (C), 133.9 (C), 130.9 (CH), 128.5 (CH), 123.7 (CH), 123.6 (CH), 122.1 (CH), 114.5 (CH), 110.3 (CH), 106.8 (CH), 104.5 (CH), 80.7 (CH₂), 60.9 (OCH₃), 60.8 (OCH₃), 56.2 (OCH₃), 55.9 (OCH₃), 55.6 (OCH₃), 35.2 (CH₂) ppm; HRMS: Found 471.1791; C₂₇H₂₈O₆Na requires 471.1784.

2-Methoxy-5-[5-(3,4,5-trimethoxyphenyl)-2,3-dihydro-1-benzoxepin-4-yl]-benzaldehyde 11i Preparation as described above for 11a from 4-bromo-5-(3,4,5-trimethoxyphenyl)-2,3-dihydro-1-benzoxepin 10f (389 mg, 1 mmol) and 3-formyl-4-methoxyphenylboronic acid (243 mg, 1.3 mmol). The product was purified by column chromatography (silica, 5% diethyl ether/hexane) to give the product as a yellow solid, (307 mg, 69%). M.P. 141°C. IR ν_max (KBr): 1681.0 (C=O), 1496.3, 1237.9, 1121.6 cm⁻¹; ¹H (400 MHz, CDCl₃): δ 10.41 (1 H, s), 7.76 (1 H, d, J = 2.00 Hz, Ar-H), 7.30 (1 H, t, J = 1.50 Hz, Ar-H), 7.25 (1 H, d, J = 2.00 Hz, Ar-H), 7.13 (1 H, dd, J = 1.00 Hz, 8.00 Hz, Ar-H), 7.04 (1 H, dt, J = 1.00 Hz, 7.50 Hz, Ar-H), 6.95 (1 H, dd, J = 1.50 Hz, 8.00 Hz, Ar-H), 6.76 (1 H, d, J = 8.50 Hz, Ar-H), 6.19 (2 H, s, Ar-H), 4.61 (1 H, t, J = 6.00 Hz, O-CH₂), 3.87 (3 H, s, OCH₃), 3.80 (3 H, s, OCH₃), 3.56 (6 H, s, OCH₃), 2.72 (2 H, t, J = 6.00 Hz, CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 189.2 (CH), 160.3 (C), 155.8 (C), 152.3 (C), 137.9 (CH), 137.6 (C), 136.7 (C), 136.5 (C), 136.3 (C), 136.1 (C), 134.7 (C), 131.9 (CH), 130.8 (CH), 128.5 (CH), 127.9 (CH), 124.0 (C), 123.5 (CH), 121.9 (CH), 111.1 (CH), 108.5 (CH), 80.2 (CH₂), 60.6 (CH₃), 55.7 (CH₃), 55.5 (CH₃), 35.1 (CH₂) ppm; HRMS: Found 469.1626; C₂₇H₂₆O₆Na requires 469.1627.

2-Methoxy-5-[5-(3,4,5-trimethoxyphenyl)-2,3-dihydro-1-benzoxepin-4-yl]-phenol 11j Hydrogen peroxide (30%, 233fL) and sulfuric acid (50 μL) were added to a solution of 2-methoxy-5-[5-(3,4,5-trimethoxyphenyl)-2,3-dihydro-1-benzoxepin-4-yl]-benzaldehyde 11i (45 mg, 0.1 mmol) in methanol (10 mL) and the solution stirred at room temperature for 6 h. Water (20 mL) was added, the solution neutralized to pH 7, the aqueous layer extracted with ethyl acetate (2 × 25 mL), dried over sodium sulfate, and the solvent removed under reduced pressure and the product obtained as an orange solid, (21 mg, 50%). IR ν_max (KBr): 3410.4 (OH), 1579.9, 1115.3 cm⁻¹; ¹H (400 MHz, CDCl₃): δ 7.25–7.24 (1 H, m, Ar-H), 7.13 (1 H, d, J = 8.04 Hz, Ar-H), 7.06 (1 H, t, J = 6.78 Hz, Ar-H), 6.96 (1 H, dd, J = 1.50 Hz, 6.04 Hz, Ar-H), 6.82 (1 H, d, J = 6.04 Hz, Ar-H), 6.68–6.62 (2 H, m, Ar-H), 6.22 (2 H, s, Ar-H), 4.65 (3 H, t, J = 6.28 Hz, O-CH₂), 3.86 (3 H, s, OCH₃), 3.84 (3 H, s, OCH₃), 3.58 (6 H, s, 2 × OCH₃), 2.69 (2 H, t, J = 6.02 Hz, CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 156.0 (C), 152.3 (C), 145.2 (C), 138.2 (C), 137.1 (C), 136.9 (C), 136.7 (C), 136.5 (C), 135.9 (C), 131.0 (CH), 128.4 (CH), 123.5 (CH), 122.1 (CH), 121.6 (CH), 114.9 (CH), 110.2 (CH), 108.7 (CH), 80.6 (CH₂), 60.9 (CH₃), 55.9 (CH₃), 35.7 (CH₂) ppm; HRMS: Found 457.1645; C₂₆H₂₆O₆Na requires 457.1627.

6,8-Dimethoxy-5-(4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-2,3-dihydrobenzoxepin 11k Preparation as described above for 11a from 4-bromo-6,8-dimethoxy-5-(4-methoxyphenyl)-2,3-dihydro-benzoxepin 10j (0.069 g, 0.14 mmol) and. 3,4,5-trimethoxyphenylboronic acid (1.3 eq., 0.19 mmol, 0.039 g). The crude product was purified using column chromatography (silica and 5% diethylether in hexane) to afford the product as a white solid, (13 mg, 18.9%). ¹H (400 MHz, CDCl₃): δ 6.91 (2 H, d, J = 9.05 Hz, Ar-H), 6.71 (1 H, s, Ar-H), 6.67 (2 H, d, J = 8.56 Hz, Ar-H), 6.37 (3 H, d, J = 3.00 Hz, Ar-H), 4.64 (2 H, t, J = 6.02 Hz, CH₂), 3.84 (6 H, s, 2 × OCH₃), 3.76 (3 H, s, OCH₃), 3.62 (9 H, s, 3 × OCH₃), 2.72 (2 H, t, J = 6.02 Hz, CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 160.8 (C), 158.8 (C), 157.4 (C), 157.2 (C), 152.5 (C), 137.4 (C), 136.9 (C), 134.4 (C), 130.0 (C), 118.0 (CH), 112.5 (C), 107.1 (CH), 104.5 (CH), 99.2 (CH), 96.3 (CH), 79.8(CH₂), 60.9 (OCH₃), 56.3(OCH₃), 55.4(OCH₃), 55.1(OCH₃), 35.6 (CH₂) ppm; HRMS: Found 501.1897; C₂₈H₃₀O₇Na requires 501.1889.

7,8-Dimethoxy-5-(4-methoxy-phenyl)-4-(3,4,5-trimethoxy-phenyl)-2,3-dihydro-benzo[b]oxepin 11l Preparation as described above for 11a from 4-bromo-7,8-dimethoxy-5-(4-methoxy-phenyl)-2,3-dihydro-benzo[b]oxepin 10k (100 mg, 0.256 mmol) and 3,4,5-trimethoxyphenyl boronic acid (70 mg, 0.332 mmol). The crude product was purified using column chromatography over silica gel (eluent 5% diethylether in hexane) to give the product as a pale yellow solid, (0.141 g, 100%). M.P. 81–90°C. IR ν_max (KBr): 2933, 1607, 1577, 1244 cm⁻¹; ¹H (400 MHz, CDCl₃): δ 6.91 (2 H, d, J = 8.56 Hz, Ar-H), 6.71 (1 H, s, Ar-H), 6.67 (2 H, d, J = 6.76 Hz, Ar-H), 6.38 (3 H, m, Ar-H), 4.62 (2 H, t, J = 6.76 Hz, CH₂), 3.91 (3 H, s, OCH₃), 3.82 (3 H, s, OCH₃), 3.76 (3 H, s, OCH₃), 3.64 (9 H, s, OCH₃), 2.75 (2H, t, J = 6.76 Hz, CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 158.1 (C), 152.6 (C), 150.2 (C), 148.9 (C), 145.0 (C), 138.0 (C), 137.1 (C), 137.0 (C), 133.5 (C), 132.3 (CH), 128.0 (C), 113.0 (CH), 113.0 (CH), 106.8 (CH), 105.5 (CH), 81.1 (CH₂), 60.9 (OCH₃), 56.1 (OCH₃), 55.9 (OCH₃), 55.1 (OCH₃), 35.5 (CH₂) ppm; HRMS: Found 501.1877; C₂₈H₃₀O₇Na requires 501.1889.

5-Naphthalen-1-yl-2,3-dihydrobenzoxepin 12 Preparation as described above for 9a from 3,4-dihydro-2H-1-benzoxepin-5-one 7a (0.40 g, 2.50 mmol) and 1-naphthylboronic acid (493 mg, 2.86 mmol) via the triflate 8a. The product was purified by column chromatography (silica, 5% diethyl ether/hexane) to give a yellow solid which was used in subsequent reactions without further purification, (497 mg, 73%). M.P. 68°C. IR ν_max (KBr): 2961.4, 1587.8, 1479.3, 1429.5, 1261.3, 799.7 cm⁻¹; ¹H (400 MHz, CDCl₃): δ 7.92 (2 H, t, J = 7.54 Hz, Ar-H), 7.84 (1 H, d, J = 8.56 Hz, Ar-H), 7.57 (1 H, t, J = 7.54 Hz, Ar-H), 7.51–7.48 (1 H, m, Ar-H) 7.43-7.39 (2 H, m, Ar-H), 7.19 (2 H, J = 7.54 Hz, Ar-H), 6.84–6.79 (1 H, m, Ar-H), 6.74 (1 H, d, J = 8.04 Hz, Ar-H), 6.25 (1 H, t, J = 5.02 Hz, CH), 4.57 (2 H, t, J = 5.20 Hz, OCH₂), 2.86 (2 H, q, J = 5.52 Hz, CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 158.4 (C), 141.7 (C), 138.2 (C), 133.6 (C), 132.2 (C), 131.7 (CH), 131.6 (CH), 130.6 (C), 128.1 (CH), 128.0 (CH), 127.8 (C), 127.5 (C), 127.5 (CH), 127.1 (CH), 126.3 (CH), 125.9 (CH), 125.8 (CH), 125.6 (CH), 122.8 (CH), 120.9 (CH), 73.1 (CH₂), 32.8 (CH₂) ppm.

4-Bromo-5-naphthalen-1-yl-2,3-dihydrobenzoxepin 13 Pyridinium tribromide (339 mg, 0.96 mmol) was added to a solution of 5-naphthalen-1-y-2,3-dihydrobenzoxepin 12 (262 mg, 0.96 mmol) in dichloromethane (20 mL) at 0°C and stirred for 5 min. Water (20 mL) was added and the aqueous layer extracted with dichloromethane (2 × 20 mL). The organic layer was washed with a saturated solution of sodium hydrogen carbonate (20 mL), brine (20 mL), dried over sodium sulfate, and the solvent removed under reduced pressure. The residue was purified by chromatography (silica, 5% diethyl ether/hexane) to give the product as an orange solid which was used in subsequent reactions without further purification, (284 mg, 84 %). M.P. 107°C. IR ν_max (KBr): 2962.1, 1429.3, 1260.4, 1125.9, 1027.9, 801.3 cm⁻¹; ¹H (400 MHz, CDCl₃): δ 7.92 (2 H, d, J = 8.00 Hz, Ar-H), 7.88 (1 H, d, J = 8.00 Hz, Ar-H), 7.60 (1 H, t, J = 7.78 Hz, Ar-H), 7.54–7.48 (3 H, m, Ar-H), 7.24–7.17 (2 H, m, Ar-H), 6.87–6.79 (2 H, m, Ar-H), 4.79–4.75 (1 H, m, OCH₂), 4.74–4.62 (1 H, m, OCH₂), 3.37–3.32 (1 H, m, CH₂), 3.30–3.18 (1 H, m, CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 156.2 (C), 140.2 (C), 137.7 (C), 133.4 (C), 132.9 (C), 131.5 (C), 130.7 (CH), 130.5 (C), 128.5 (CH), 127.9 (CH), 127.6 (C), 127.5 (C), 127.2 (CH), 126.8 (CH), 126.1 (CH), 125.6 (CH), 125.0 (CH), 124.9 (CH), 123.0 (CH), 121.5 (CH), 75.7 (CH₂), 41.5 (CH₂) ppm.

5-Naphthalen-1-yl-4-(3,4,5-trimethoxyphenyl)-2,3-dihydro-benzoxepin 14 4-Bromo-5-naphthalen-1-yl-2,3-dihydrobenzoxepin 13 (139 mg, 396 μmol) was dissolved in THF (30 mL). 3,4,5-Trimethoxyphenylboronic acid (109 mg, 515 μmol) and 2 M sodium carbonate (0.99 mL) were added and the mixture stirred under nitrogen for 10 min. Pd(PPh₃)₄ (30 mg, 0.025 m mol) was added and the reaction refluxed for 6 h at 85°C. The solution was cooled to room temperature and acidified with 2 M hydrochloric acid. The aqueous layer was extracted with dichloromethane (3 × 30 mL) and the combined organic layers were washed with water (30 mL), brine (30 mL), dried over sodium sulfate, and the solvent removed under reduced pressure. The product was purified by column chromatography (silica, 5% diethyl ether/hexane) to give the product as a yellow solid, (134 mg, 77%). M.P. 124°C. IR ν_max (KBr): 2930.3, 1579.3, 1505.7, 1462.2, 1246.5, 1123.7, 1019.5 cm⁻¹; ¹H (400 MHz, CDCl₃): δ 7.87 (1 H, m, Ar-H), 7.74 (1 H, m, Ar-H), 7.67 (1 H, m, Ar-H), 7.33 (4 H, m, Ar-H), 7.16 (3 H, m, Ar-H), 6.85 (2 H, m, Ar-H), 6.24 (2 H, s, Ar-H), 4.83 (1 H, m, OCH₂), 4.73 (1 H, m, OCH₂), 3.68 (3 H, s, OCH₃), 3.29 (6 H, s, 2 × OCH₃,), 2.98 (1 H, m, CH₂), 2.77 (1 H, m, CH₂) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 155.1 (C), 152.2 (C), 140.5 (C), 137.3 (C), 136.5 (C), 133.5 (C), 129.6 (CH), 128.4 (CH), 128.2 (CH), 128.0 (CH), 127.2 (CH), 126.3 (CH), 126.2 (CH), 125.5 (CH), 125.4 (CH), 123.6 (CH), 122.1 (CH), 105.7 (CH), 80.4 (CH₂), 60.7 (CH₃), 55.5 (CH₃), 34.8 (CH₂) ppm; HRMS: Found 461.1725; C₂₉H₂₆O₄Na requires 461.1729.

Biochemical evaluation of activity

Antiproliferation studies

All assays were performed in triplicate for the determination of mean values reported. Compounds were assayed as the free bases isolated from reaction. The human breast tumor cell line MCF-7 was cultured in Eagle’s minimum essential medium in a 95% O₂/5% CO₂ atmosphere with 10% fetal calf serum. The medium was supplemented with 1% non-essential amino acids. MDA-MB 231 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% (v/v) fetal bovine serum (FBS), 2mM l-glutamine and 100 μg/mL penicillin/streptomycin (complete medium). Cells were trypsinized and seeded at a density of 2.5 × 10⁴ cells/mL (or 1.0 × 10⁴ cells/well) into a 96-well plate and incubated at 37°C, 95%O₂/5% CO₂ atmosphere, for 24 h. After this time they were treated with 2 μL volumes of test compound which had been pre-prepared as stock solutions in ethanol to furnish the concentration range of study, 1 nM–100 μM, and re-incubated for a further 72 h. Control wells contained the equivalent volume of the vehicle ethanol (1%, v/v). The culture medium was then removed and the cells washed with 100 μL phosphate buffered saline (PBS) and 50 μL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) added, to reach a final concentration of 1 mg/mL MTT. Cells were incubated for 2 h in darkness at 37°C. At this point, solubilization was begun through the addition of 200 μL dimethylsulfoxide (DMSO) and the cells maintained at room temperature in darkness for 20 min to ensure thorough color diffusion before reading the absorbance. The absorbance value of control cells (no added compound) was set to 100% cell viability, and, from this, graphs of absorbance versus cell density per well were prepared to assess cell viability, and, from these, graphs of percentage cell viability versus concentration of subject compound added were drawn.

Analysis of pro-apoptotic effects

Flow cytometry The MCF-7 cells were seeded at a density of 18 × 10⁴ cells/mL in 5 mL of medium (900,000 cells per flask). After 24 h, cells were treated with either vehicle (50 μL of ethanol; 1%, v/v) or selected compound (1 μM) and incubated for 72 h. Following incubation, the cells were removed from the bottom of the flask by scraping and the medium placed in a 20 mL Sterilin tube. They were centrifuged for 10 min at 600 × g. The supernatant was decanted and the pellet resuspended in 1 mL of ice-cold PBS; cells were again centrifuged for 10 min at 600 × g. The supernatant was decanted and the pellet resuspended in 200 μL of ice-cold phosphate buffered saline (PBS). Subsequently ice-cold 70% ethanol (2 mL) was slowly added to the tube as it was gently vortexed. The cells were kept at–20°C for at least 1 h (could be left overnight). After the fixation, 5 μL of FBS was added to the samples. The cells were harvested by centrifugation at 600 × g for 10 min. The ethanol was carefully removed and the pellet resuspended in 400 μL of PBS and transferred to FACS (fluorescence-activated cell sorting) microtubes. A 25 μL aliquot of RNase A (1 mg/mL) and 75 μL of propidium iodide (PI) 1 mg/mL, a DNA binding fluorescent dye, were added to each tube. The samples were wrapped in aluminum foil and incubated for a minimum of 30 min at 37°C. The samples were read at 488 nM using a FACscalibur flow cytometer from Becton Dickinson. The FACS data for 10,000 cells were analyzed using the Macintosh-based application Cellquest and the data were stored as frequency histograms. An increase in the percentage of cells in the pre-G1 peak is indicative of apoptosis.

Computational procedure

Docking was carried out using FREDv2.2.3 software in conjunction with Chemgauss3.

Ligand preparation

All compounds were drawn using ACD/Chemsketch v10²⁶ and SMILES strings generated. A single conformer was generated using Corina v3.4 and ensuring Omega v2.2.1 was subsequently employed to generate a maximum of 1000 conformations of each compound.

Receptor preparation

PDB entries 1SA0 and 1SA1 were downloaded from the Protein Data Bank (PDB). All waters were retained in both isoforms. Addition and optimization of hydrogen positions for these waters was carried out using MOE 2007.09²⁷ ensuring that all other atom positions remained fixed.

Results and discussion

Chemistry

The synthesis of the benzoxepin analogs of combretastatin CA-4 is illustrated in Scheme 1 and is achieved by ligand coupling reactions of polymethoxylated 5-arylated benzoxepins with arylboronic acids in the presence of tetrakis(triphenylphosphine)palladium(0) catalyst. The target benzoxepin compounds chosen for synthesis were designed to have comparable structures to the reported combretastatin analogs in ring A and ring B substitution pattern¹¹. The trimethoxy substitution pattern was retained in all compounds, which is a common structural feature of colchicines, CA-4, and many other tubulin inhibitors^11,28. The required 2,3,4,5-tetrahydro-1-benzoxepin-2-ones 7a–c were obtained by acid-promoted cyclization of the appropriate 4-phenoxybutyric acids 6a–c (obtained by alkylation of the appropriate phenols with ethyl bromobutyrate). Polyphosphoric acid was found to be the most efficient reagent for the preparation of the unsubstitued benzoxepin 7a, R = H, while Eaton’s reagent was found to be superior for 7b and 7c, with yields of up to 67% achieved for 7c. To facilitate the subsequent arylation reaction, the 2,3,4,5-tetrahydro-1-benzoxepin-2-ones 7a–c were first converted to the triflates 8a–c by treatment with triflic anhydride and then subsequently coupled in situ with a number of substituted arylboronic acids in a Suzuki reaction²⁹ promoted by Pd(PPh₃)₄ to afford the 5-arylbenzoxepins 9a–h in moderate yield. These products were converted to the vinylic bromides 10a–h on treatment with pyridine hydrobromide perbromide. This reaction was completed rapidly and required careful monitoring to prevent the subsequent aryl bromination reaction. A second Suzuki reaction promoted by Pd(PPh₃)₄ was then completed on compounds 10a–h to afford the required products 11a–l in relatively good yield for most of the products (Table 1). For synthesis of the phenolic products 11g and 11j, the direct coupling of the vinyl triflate 8a or the vinyl bromide 10f with the protected 3-benzyloxy-4-methoxyboronic acid was first attempted, but was not found to be successful. An alternative approach was necessary to obtain these products in which the aldehyde compounds 11f and 11i were treated with hydrogen peroxide in a Baeyer–Villiger oxidation reaction to afford the phenols 11g and 11j (Scheme 2). The 5-naphthylbenzoxepin product 14 was synthesized in a similar synthetic sequence to compounds 9a–h, by first arylation of the benzoxepin 7a with 1-naphthylboronic acid to afford the product 12 in 73% yield. Subsequent bromination of 12 proceeded in high yield (84%) to afford the vinyl bromide 13. Suzuki reaction of the bromide 13 promoted by Pd(PPh₃)₄ afforded the required product 14, again in high yield (Scheme 3). The ¹H NMR spectra of the products 11a–l and 14 revealed the characteristic methylene protons at C-2 and C-3 of the benzoxepin ring as coupled triplet signals, e.g. for compound 11g these signals were observed at δ 4.63 and δ 2.71, respectively, with J = 6.02 Hz.

Scheme 1.  Synthesis of benzoxepins 11a–l. Scheme reagents and conditions: (i) PPA, 110°C, 4 h, or Eaton’s reagent, 80°C, 2 h; (ii) (TfO)₂O, Na₂CO₃, 18 h, rt; (iii) Pd(PPh₃)₄, ArB(OH)₂, Na₂CO₃(aq), THF; (iv) PyHBr₃, CHCl₂, 20°C, 18 h.

Scheme 2.  Synthesis of benzoxepins 11g and 11j. Scheme reagents and conditions: (i) H₂O₂, H₂SO₄, CH₃OH, 20°C, 6 h.

Scheme 3.  Synthesis of benzoxepin 14. Scheme reagents and conditions: (i) (TfO)₂O, Na₂CO₃, 18 h, rt; (ii) Pd(PPh₃)₄, ArB(OH)₂, Na₂CO₃(aq), THF; (iii) PyHBr₃, CHCl₂, 20°C, 18 h.

Table 1.  Yield and antiproliferative activity for benzoxepins 11a–l and 14.

Compound number	Yield (%)	Antiproliferative activity, IC50 (µM)
		MCF-7 cells^a	MDA-MB 231 cells^a
11a	73	4.86 ± 0.559	12.60 ± 0.141
11b	67	8.83 ± 2.79	13.04 ± 4.06
11c	52	5.26 ± 1.08	7.51 ± 1.14
11d	90	9.84 ± 1.80	10.50 ± 1.12
11e	94	10.70 ± 5.92	12.40 ± 1.27
11f	96	6.24 ± 1.33	17.99 ± 6.40
11g	58	0.85 ± 0.09	11.56 ± 2.51
11h	83	2.55 ± 3.15	20.40 ± 14.4
11i	69	37.19 ± 27.23	23.42 ± 9.95
11j	50	1.635 ± 1.393	4.57 ± 0.363
11k	19	8.25 ± 3.05	5.33 ± 2.38
11l	100	12.79 ± 0.50	46.43 ± 3.26
14	77	25.9 ± 20.7	96.43 ± 9.66

^aExperimental values represent the average for experiments performed in triplicate along with the standard deviation (SD) between the assay values. IC₅₀ values are half maximal inhibitory concentrations required to block the growth stimulation of MCF-7 cells. The IC₅₀ values obtained for combretastatin CA-4 are 0.0031 µM for MCF-7 and 0.043 µM for MDA-MB 231, which are in good agreement with the reported values for combretastatin CA-4 using the MTT assay on human MCF-7 and MDA-MB 231 breast cancer cell lines, respectively.

Biochemistry

The benzoxepin compounds prepared above were evaluated in a series of in vitro assays which determined their antiproliferative activity in MCF-7 and MDA-MB 231 breast cancer cell lines and also their pro-apoptotic effects in MCF-7 cells by flow cytometry.

Antiproliferative activity in MCF-7 and MDA-MB 231 breast cancer cells

Compounds 11a–l and 14 were initially screened for their antiproliferative activity using the ER expressing (ER dependent) MCF-7 human breast cancer cell line by means of the MTT (tetrazolium) based assay. The drug concentration required to inhibit the cell growth by 50% (IC₅₀) following incubation of the cells in the culture medium for 72 h was determined and the results are displayed in Table 1. The IC₅₀ values obtained for combretastatin CA-4 were 0.0031 μM for MCF-7 and 0.043 μM for MDA-MB 231, which are in good agreement with the reported values for combretastatin CA-4 using the MTT assay on human MCF-7 and MDA-MB 231 breast cancer cell lines^11,20,30.

Compounds 11a–e and 11k, which display polymethoxylated aryl substitution, were found to exert modest antiproliferative effects on MCF-7 cells, with IC₅₀ in the range 4.86–10.70 μM, when compared with our determined values for combretastatin CA-4 (IC₅₀ = 0.0031 μM). A common substitution pattern of 3,4,5-trimethoxy or 2,3,4-trimethoxyphenyl for the aryl ring located on position 4 of the benzoxepin scaffold was present in these compounds, together with 4-methoxy, 4-hydroxy, 4,5-dimethoxy, and 4-fluoro-5-methoxyphenyl substitution at C-5. Compound 11e was designed to mimic a fluorinated combretastatin CA-4 analog in which the hydroxy group on ring B was replaced with a fluorine without substantial loss of activity^31,32. However, compound 11e was found to exhibit only moderate antiproliferative activity, with IC₅₀ = 10.7 μM. Compound 14, containing the 1-naphthyl substituent at C-4 of the benzoxepin scaffold, was found to have poor antiproliferative activity, with IC₅₀ = 25.9 μM. This compound could be considered as an analog of the known naphthylcombretastatins, in which the 1-naphthyl ring mimics the 3-hydroxy-4-methoxy ring B of combretastatin A4³³. A possible explanation for the low activity of compound 14 is the steric hindrance caused by the presence of the 1-naphthyl substituent in the conformationally restricted benzoxepin analog, which hinders the required favorable aryl alignment predicted for ring B in the binding site. The most active compound in the series was identified as 11g (IC₅₀ = 850 nM), in which the aromatic rings located at C-5 of the benzoxepin ring contain a similar substitution pattern to that found for ring A (3,4,5-trimethoxyphenyl) of the combretastatin CA-4, and the aromatic rings located at C-4 of the benzoxepin ring contain a similar substitution pattern to that found for ring B (3-hydroxy-4-methoxyphenyl) of the combretastatin CA-4 molecule. Compound 11h, also containing the C-5 trimethoxyphenyl ring substituent together with 3,4-dimethoxyphenyl substitution at C-4, was active (IC₅₀ = 2.55 μM). Compound 11j (IC₅₀ = 1.635 μM), in which the aryl substitution pattern for the aromatic rings located at C-4 and C-5 is also similar to combretastatin CA-4, reversed in orientation from compound 11g, is slightly less active than 11g, indicating that the most favorable orientation of aryl substitution on the benzoxepin scaffold is for the 3,4,5-trimethoxyaryl ring A to be positioned at C-4 and the 3-hydroxy-4-methoxyaryl ring B to be located at C-5. The aldehyde compounds 11f and 11i, which are synthetic precursors of the active compounds 11g and 11j, were found to be considerably less active than their phenolic products 11g and 11j, with IC₅₀ values of 6.24 μM and 37.19 μM, respectively. Compounds 11k and 11l, which contain the 6,8- and 6,7-dimethoxy substitution pattern, respectively, on the benzoxepin aryl ring, are less active (IC₅₀ = 8.25, 12.79 μM) than the unsubstituted analog 11a (IC₅₀ = 4.86 μM).

The compounds were also evaluated for antiproliferative activity in the MDA-MB 231 human breast cancer cell line and the results are displayed in Table 1. The compounds were shown to have moderate activity when compared with combretastatin CA-4, IC₅₀ = 0.043 μM, with 11k, 11j being the most active, displaying IC₅₀ values of 5.33 μM, 4.57 μM respectively.

Preliminary apoptosis experiments were performed by flow cytometry analysis on propidium iodide labeled MCF-7 cells, and showed that compound 11g caused an increase in the pre-G1 peak from 11.1% (control) to 19.8%, indicating a possible pro-apoptotic effect for the compound.

To assess the similarity in structure of combretastatin CA-4 and the most potent benzoxepin synthesized 11g, an overlay of the two structures was first examined, as illustrated in Figure 2. The MOE flexible alignment tool²⁷ was used for the above illustration, retaining default settings. It can be confirmed that the aryl rings A and B of the benzoxepin 11g structure are aligned in an identical orientation to that of the combretastatin CA-4, in which they are not coplanar, thus indicating that the presence of the seven-membered ring on the alkene bridge in compound 11g facilitates the constraint of the two aryl rings in the required Z configuration.

Figure 2.  Overlay of combretastatin CA-4 (blue) with benzoxepin 11g (gray).

Molecular modeling studies of novel benzoxepin compounds

To investigate the possible tubulin binding mode of these benzoxepin compounds, a docking study was carried out to examine the docked orientations of the most potent benzoxepins 11g and 11j in the colchicine binding site of tubulin, using the reported X-ray structures of tubulin co-crystallized with a colchicine derivative, DAMA-colchicine (PDB entry 1SA0) and also podophyllotoxin (PDB entry 1SA1)³⁴. Figure 3 illustrates the docked positions for each benzoxepin 11g and 11j in tubulin, together with DAMA-colchicine and combretastatin CA-4, and importantly shows that 11g and 11j should exhibit similar tubulin binding modes when compared with both CA-4 and colchicine. To dock the benzoxepins, PDB entry 1SA0 (tubulin–colchicine: RB3-SLD) was selected, as it can be seen from Figure 3 that steric hindrance from Thr179 and movement of Asn249 would have rendered the benzoxepins in a docking pose unattainable in reality for PDB entry 1SA1 (tubulin–podophyllotoxin: RB3-SLD³⁴). For the most active benzoxepin 11g, it can be seen that the trimethoxy ring A is located in the colchicine binding site in the region of Cys241, and with a very similar orientation to the trimethoxy ring of the DAMA-colchicine in the reported co-crystallized structure. There is also clear interaction between the benzoxepin aryl ring and the Thr179, and also between the benzoxepin heterocyclic and Asn249. Additional hydrophobic contacts are also observed, which stabilize the binding of compounds 11g and 11j to the protein. Computational analysis by ter Haar et al. has shown that the colchicine binding site of tubulin can accommodate structurally diverse ligands⁵, suggesting a high plasticity of the tubulin protein at this site³⁵.

Figure 3.  Docked poses of benzoxepins 11g (red) and 11j (pink) in tubulin overlayed by backbone with docked poses of CA-4 (blue) and colchicine (yellow). 1SA0, yellow; 1SA1, gray.

To assess the propensity of each ligand to dock in different manners using FREDv2.2.3 in combination with Chemgauss3, an analysis of the docked poses was carried out using PostDOCK. A new visualization tool—PostDOCK³⁶—was utilized to examine the resulting docked poses for 11g and 11j generated by FREDv2.2.3. The program was developed using SVL (Scientific Vector Language) to integrate within MOE and analyzes a set of docked poses for a given compound whereby a pseudo-3D snapshot is generated, representing the conformations and energies of the docked set. Docking energies are represented by a transparency scale whereas the poses themselves are represented by color. Briefly, the Boltzmann population of each pose is calculated whereby a high Boltzmann is equivalent to an opaque surface and a low Boltzmann is related to a transparent surface. A color scale is produced, and the more yellow the structural representation, the closer the total population is in conformation to the lowest energy binding pose. Importantly we have added confidence that our docking protocol is working effectively, as a significant population of poses exhibited a low rmsd (root mean square deviation) with the lowest energy pose for each. The Boltzmann distributions were as follows: 55.22% of docked structures have an rmsd of 0 with the lowest energy pose of combretastatin docked in tubulin, 95.57% for 11j and 97.61% for 11g, indicating that the conformations depicted for 11g and 11j in Figure 4 are accurate representations of the lowest energy conformations for the docked molecules 11g and 11j.

Figure 4.  PostDOCK view of docked compounds combretstatin A-4, 11j, and 11g using FREDv2.2.1 combined with Chemgauss3 as a scoring function.

Conclusion

A series of polymethoxylated rigid analogs of combretastatin which contain a benzoxepin ring in place of the usual ethylene bridge present in the natural combretastatin products have been synthesized. The compounds displayed moderate antiproliferative activity when evaluated against the MCF-7 and MDA-MB 231 human breast carcinoma cell lines. 5-(3-Hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-2,3-dihydro-benzoxepin (11g) was found to be the most potent derivative and showed an IC₅₀ value of 850 nM when evaluated against the MCF-7 breast cancer cell line. From molecular modeling studies, the structures of the active compounds 11g and 11j were shown to adopt a conformation in which the two aromatic rings are not coplanar, and can align within the colchicine binding site of tubulin. The conformationally restricted 4,5-diarylbenzoxepins 11a–l (Table 2), structurally similar to colchicines and combretastatin, are shown to be potentially useful scaffolds for the further development of antitumor agents which are designed to target tubulin polymerization.

Table 2.  Structures of benzoxepins 11a–l.

	R1	R2	R3	R4	R5	R6	R7	R8	R9	R10	R11
11a	H	H	H	H	OCH3	H	H	OCH3	OCH3	OCH3	H
11b	H	H	H	H	OH	H	H	OCH3	OCH3	OCH3	H
11c	H	H	H	OCH3	OCH3	H	H	OCH3	OCH3	OCH3	H
11d	H	H	H	OCH3	OCH3	H	H	H	OCH3	OCH3	OCH3
11e	H	H	H	F	OCH3	H	H	OCH3	OCH3	OCH3	H
11f	H	H	H	CHO	OCH3	H	H	OCH3	OCH3	OCH3	H
11g	H	H	H	OH	OCH3	H	H	OCH3	OCH3	OCH3	H
11h	H	H	H	OCH3	OCH3	H	H	OCH3	OCH3	OCH3	H
11i	H	H	H	OCH3	OCH3	OCH3	H	H	OCH3	CHO	H
11j	H	H	H	OCH3	OCH3	OCH3	H	H	OCH3	OH	H
11k	OCH3	H	OCH3	H	OCH3	H	H	OCH3	OCH3	OCH3	H
11l	OCH3	OCH3	H	H	OCH3	H	H	OCH3	OCH3	OCH3	H

Declaration of interest

This work was supported through funding from the Trinity College IITAC research initiative (HEA PRTLI), Enterprise Ireland (EI), Science Foundation Ireland (SFI), and the Health Research Board (HRB), with additional support for computational facilities from the Wellcome Trust. A postgraduate research award from Trinity College is gratefully acknowledged.