Synthesis and Anticancer Activity Evaluation of Azepinobisindoles; the Isomeric Iheyamine A Derivatives

Introduction

Bisindole alkaloids constitute an important class of secondary metabolites with characteristic cytotoxicity and pharmacological activities.^1-5 Azepinobisindole alkaloids are a class of bisindole alkaloids containing a structural moiety of azepine (seven-membered ring) core, which demonstrated diverse biological activities especially anticancer effect. In the past years, number types of indole-fused azepines have been found with potential success in drug discovery. For example, Hyrtimomine A was reported to show anticancer activity against human epidermoid carcinoma KB cells and murine leukemia L1210 cells and showed antimicrobial activity against Candida albicans and Cryptococcus neoformans by Tanaka et al.⁶ Another example was Trigonoliimines C, which was reported with a modest anti-HIV-1 activity⁷ and their anticancer activity against human cervical cancer HeLa cells and human histiocytic lymphoma U-937 cells was also reported by Han et al.⁸ As illustrated in Fig 1, Iheyamine A (1) the azepinobisindole isolated from a colonial marine ascidian Polycitorella sp. by Sasaki et al. was reported to exhibit moderately cytotoxicity against murine leukemia P388 cells, human lung carcinoma A549 cells and human colon adenocarcinoma HT29 cells with IC₅₀ in the level of 1 µg/mL.⁹ The synthesis of Iheyamine A was firstly accomplished by Lindsay et al. using cross-Mannich reaction.¹⁰ Recently, Abe and Yamada accomplished the synthesis of Iheyamine A via dehydrative Mannich-type reaction.¹¹ Moreover, Bremner et al. synthesized the 5H-azepino[2,3-b:4,5-bʹ]-diindole system, which was the isomer of Iheyamine A.¹² However, these structure motifs have not been studied for biological activities and their core structure resemble to Hytimomine A, which could be giving promising result for anticancer activity. Therefore, a series of 5H-azepino[2,3-b:4,5-bʹ]-diindole was synthesised and evaluated for cytotoxic activity. Molecular docking study against cyclic-dependent kinase 2 (CDK2) were also investigated the inhibitory ability of the designed compounds as well as the understanding of the antitumor results.

Figure 1: Azepinobisindole alkaloids Hytimomine A, Trigonoliimine C, Iheyamine A (1) and isomeric Iheyamine A derivatives. Click here to view figure

 

Materials and Methods

General Methods

Melting points were determined with a Stuart Scientific SMP 2 melting point apparatus and were uncorrected. FT-IR spectra were recorded using a Perkin Elmer Spectrum 100. NMR spectra were recorded on a Bruker Advance 300 (300 MHz for proton NMR and 75 MHz for carbon NMR) using tetramethylsilane (TMS) as an internal reference. Mass spectra were determined on a POLARIS Q or HEWLETT PACKARD 5973.

Synthesis Method

Isatin was purchased from Fluka^TM and 5-Substituted isatins (2b-d) were prepared according to Sandmeyer isatin synthesis method.¹³

General Procedure for Synthesis of 3a–g

Isatin or 5-substituted isatin (7 mmol) and K₂CO₃ (10 mmol) were added to a dry CH₃CN solution (25 mL) and stirred for 30 min at RT. Methyl iodide or benzyl chloride (7.5 mmol) was added to the solution and then refluxed at 80°C for 3 h. The mixture was cooled to RT and the solvent was removed. The residue was dissolved in CH₂Cl₂ (30 mL) and then washed with H₂O (3×30 mL). The organic extracts were dried over anh Na₂SO₄ and concentrated to give the crude brown solid. The purification of the crude solid using flash-column chromatography (silica gel, hexane/EtOAc (2:1) gave the pure compounds 3a-g.

N–methylisatin (3a)

Orange solid; 62%; m.p. 128-129°C (lit.¹⁴129-130°C); IR (cm^–1): 1721, 1603, 1466, 1365, 1325; ¹H NMR (CDCl₃): δ 3.26 (s, 3H, CH₃), 6.90 (d, J = 8.2 Hz, 1H, ArH), 7.14 (t, J = 7.4 Hz, 1H, ArH), 7.58-7.64 (m, 2H, ArH); ¹³C NMR (CDCl₃): δ 26.2, 109.9, 117.5, 123.9, 125.3, 138.4, 151.5, 158.3, 183.4.

5–methoxy–N–methylisatin (3b)

Red solid; 82%; m.p. 171-173°C (lit.¹⁵ 175-176°C); IR (cm^–1): 1722, 1625, 1485, 1356, 1287, 1226; ¹H NMR (CDCl₃): δ 3.23 (s, 3H, CH₃), 3.81 (s, 3H, OCH₃), 6.81 (d, J = 9.3 Hz, 1H, ArH), 7.14-7.18 (m, 2H, ArH) ); ¹³C NMR (CDCl₃): δ 26.2, 56.0, 109.6, 110.9, 117.8, 124.6, 145.4, 156.6, 158.3, 183.8.

5–bromo–N–methylisatin (3c)

Orange solid; 70%; m.p. 167-169°C (lit.¹⁵  163-164°C); IR (cm^–1): 1722, 1603, 1439, 1349, 1321, 711; ¹H NMR (CDCl₃): δ 3.24 (s, 3H, CH₃), 6.80 (dd, J = 1.5, 7.4 Hz, 1H, ArH), 7.70 (s, 1H, ArH), 7.71 (dd, J = 1.8, 7.5 Hz, 1H, ArH); ¹³C NMR (CDCl₃): δ 26.4, 111.6, 116.7, 118.6, 128.1, 140.6, 150.2, 157.5, 182.2.

5–nitro–N–methylisatin (3d)

Yellow solid; 76%; m.p. 195-197°C (lit.¹⁶ 197-199 °C); IR (cm^–1): 1742, 1607, 1515, 1467, 1326, 1291; ¹H NMR (DMSO-d₆): δ 7.37 (d, J = 8.8 Hz, 1H, ArH), 8.24 (d, J = 2.4 Hz, 1H, ArH), 8.55 (dd, J = 2.4, 8.7 Hz, 1H, ArH); ¹³C NMR (DMSO-d6): δ 27.0, 111.4, 118.3, 119.4, 133.5, 143.4, 156.1, 159.4, 181.7.

N–benzylisatin (3e)

Orange solid; 79%; m.p. 127-130°C (lit.¹⁷ 131°C); IR (cm^–1): 1728, 1608, 1468, 1347, 1307; ¹H NMR (CDCl₃): δ 4.94 (s, 2H, CH₂), 6.78 (d, J = 8.0 Hz, 1H, ArH), 7.09 (td, J = 0.8, 7.6 Hz, 1H, ArH), 7.30-7.37 (m, 5H, ArH), 7.48 (td, J = 1.4, 7.8 Hz, 1H, ArH), 7.62 (dd, J = 0.8, 7.5 Hz, 1H, ArH); ¹³C NMR (CDCl₃): δ 44.0, 111.0, 117.6, 123.9, 125.3, 127.4 (2C), 128.1 129.0 (2C), 134.5, 138.4, 150.7, 158.3, 183.3.

5–methoxy–N–benzylisatin (3f)

Red Solid; 52%; m.p. 123-124°C (lit.¹⁸ 127-128°C); IR (cm^–1): 1721, 1618, 1490, 1335, 1269, 1237; ¹H NMR (CDCl₃): δ 3.77 (s, 3H, OCH₃), 4.91 (s, 2H, CH₂), 6.67 (d, J = 8.6 Hz, 1H, ArH), 7.03 (dd, J = 2.7, 8.6 Hz, 1H, ArH), 7.15 (d, J = 2.7 Hz, 1H, ArH), 7.29-7.38 (m, 5H, ArH); ¹³C NMR (CDCl₃): δ 44.0, 55.9, 109.6, 112.0, 118.1, 124.6, 127.4 (2C), 128.1, 129.0 (2C), 134.6, 144.6, 156.5, 158.4, 183.6.

5–bromo–N–benzylisatin (3g)

Orange solid; 92%; m.p. 101-103°C (lit.¹⁹ 106-108°C); IR (cm^-1): 1729, 1600, 1468, 1351, 1324, 696; ¹H NMR (CDCl₃): δ 4.93 (s, 2H, CH₂), 6.67 (d, J = 8.4 Hz, 1H, ArH), 7.29-7.39 (m, 5H, ArH), 7.58 (dd, J = 2.1, 8.4 Hz, 1H, ArH), 7.72 (d, J = 2.1 Hz, 1H, ArH); ¹³C NMR (CDCl₃): δ 44.2, 112.7, 116.8, 118.8, 127.4 (2C), 128.2, 128.4, 129.2 (2C), 134.0, 140.5, 149.4, 157.5, 182.1.

General Procedure for Synthesis of 5a–g

N-substituted isatin (3a–g) (0.9 mmol) and tryptamine (4) (1 mmol) in EtOH (5mL) in the presence of (0.2 mL) glacial acetic acid and 4 Å molecular sieve was stirred at RT for 24 h. The precipitate was removed by filtration and the filtrate was concentrated and purified by column chromatography (silica gel, hexane/EtOAc (2:1)) to give compound 5a–g.

Spiro[(1–methyl–3H–indole–2(1H)-one)-3,1ʹ-(1ʹ,2ʹ,3ʹ,4ʹ–tetrahydro–9H–pyrido[3,4–b]indol)] (5a)

Cream solid; 73%; m.p. 232-233°C (lit.¹² 239-241°C); IR (cm^–1): 3139, 1714, 1609, 1469, 1347, 1296; ¹H NMR (CDCl₃): δ 2.95 (t, J = 5.7 Hz, 2H, CH₂), 3.24 (s, 3H, CH₃), 3.31 (dt, J = 5.4, 13.3 Hz, 1H, CH₂), 3.81 (dt, J = 6.1, 13.3, 1H, CH₂), 6.91 (d, J = 7.8, 1H, ArH), 7.03 (td, J = 0.9, 7.5, 1H, ArH), 7.08-7.12 (m, 3H, ArH), 7.17 (dd, J = 0.8, 7.8 Hz, 1H, ArH), 7.36 (td, J = 1.3, 7.7 Hz, 1H, ArH), 7.46 (br s, 1H, NH), 7.51-7.58 (m, 1H, ArH); ¹³C NMR (CDCl₃): δ 21.1, 25.6, 39.0, 60.5, 107.7, 110.0, 111.5, 117.5, 118.6, 121.4, 122.3, 123.7, 126.2, 128.9, 129.1, 130.5, 135.2, 142.6, 175.6.

Spiro[(5–methoxy–1–methyl–3H–indole–2(1H)-one)-3,1ʹ-(1ʹ,2ʹ,3ʹ,4ʹ–tetrahydro–9H–pyrido[3,4–b]indol)] (5b)

Beige solid; 87%; m.p. 243-245°C (lit.¹² 255-256°C); IR (cm^-1): 3292, 1708, 1600, 1496, 1341, 1296, 1231; ¹H NMR (CDCl₃): δ 2.98 (t, J = 5.7 Hz, 2H, CH₂), 3.25 (s, 3H, CH₃), 3.34 (dt, J = 5.4, 13.2 Hz, 1H, CH₂), 3.72 (s, 3H, OCH₃), 3.88 (dt, J = 6.3, 13.2 Hz, 1H, CH₂), 6.81-6.91 (m, 3H, ArH), 7.08-7.19 (m, 3H, ArH), 7.29 (br s, 1H, NH), 7.57 (d, J = 5.9 Hz, 1H, ArH); ¹³C NMR (CDCl₃): δ 22.0, 26.6, 40.0, 55.9, 62.0, 109.4, 111.2, 111.6, 111.9, 114.7, 118.4, 119.4, 122.3, 127.0, 129.9, 132.7, 136.4, 136.9, 156.6, 176.7.

Spiro[(5–nitro–1–methyl–3H–indole–2(1H)-one)-3,1ʹ-(1ʹ,2ʹ,3ʹ,4ʹ–tetrahydro–9H–pyrido[3,4–b]indol)] (5d)

Cream solid; 89%; m.p. 279-281°C; IR (cm^–1): 3259, 1702, 1606, 1509, 1489, 1324, 1293; ¹H NMR (CDCl₃): δ 3.01 (t, J = 5.6, 2H, CH₂), 3.35 (s, 3H, CH₃), 3.40-3.43 (m, 1H, CH₂), 3.79 (dt, J = 5.7, 13.5, 1H, CH₂), 7.05 (d, J = 8.7, 1H, ArH), 7.09-7.19 (m, 3H, ArH), 7.59 (d, J = 6.4, 1H, ArH), 8.08 (d, J = 2.3, 1H, ArH), 8.35 (dd, J = 2.3, 8.7, 1H, ArH); ¹³C NMR (CDCl₃): δ 21.9, 27.1, 61.1, 109.7, 111.6 (2C), 118.6, 119.1, 120.0, 122.1, 126.9, 127.0, 130.5, 133.5, 136.5, 143.1, 150.8, 177.6; HREI-MS (m/z) calculated for C₁₉H₁₇N₄O₃ (M+H)⁺ 349.1295, found 349.1308.

Spiro[(1–benzyl–3H–indole–2(1H)-one)-3,1ʹ-(1ʹ,2ʹ,3ʹ,4ʹ–tetrahydro–9H–pyrido[3,4–b]indol)] (5e)

Cream Solid; Yield 85%; m.p. 142-146°C; IR (cm^–1): 3295, 1714, 1606, 1461, 1347, 1293; ¹H NMR (CDCl₃): δ 2.98 (t, J = 5.7 Hz, 2H, CH₂), 3.37 (dt, J = 5.3, 13.2 Hz, 1H, CH₂), 3.92 (dt, J = 6.2, 13.2 Hz, 1H, CH₂), 4.86 (d, J = 15.5 Hz, 1H, CH₂), 5.00 (d, J = 15.5 Hz, 1H, CH₂), 6.85 (d, J = 7.8 Hz, 1H, ArH), 7.00 (td, J = 0.9, 7.5 Hz, 1H, ArH), 7.07-7.16 (m, 3H, ArH), 7.19 (d, J = 7.4 Hz, 1H, ArH), 7.25 (td, J = 1.3, 7.8 Hz, 1H, ArH), 7.29-7.40 (m, 5H, ArH), 7.54-7.61 (m, 1H, ArH); ¹³C NMR (CDCl₃): δ 22.1, 40.1, 44.0, 61.5, 109.6, 111.0, 112.6, 118.6, 119.7, 122.5, 123.4, 124.8, 127.3, 127.6 (2C), 127.9, 129.0 (2C), 129.7, 130.2, 131.5, 135.8, 136.3, 142.6, 176.9; HREI-MS (m/z) calculated for C₂₅H₂₂N₃O (M+H)⁺ 380.1757, found 380.1761.

Spiro[(5–bromo–1–methyl–3H–indole–2(1H)-one)-3, 1ʹ-(1ʹ,2ʹ,3ʹ,4ʹ–tetrahydro–9H–pyrido[3,4–b]indol)] (5c)

Beige solid; Yield 89%; m.p. 240-242°C; IR (cm^–1): 3267, 1708, 1607, 1459, 1348, 1229, 688; ¹H NMR (CDCl₃): δ 2.98 (t, J = 5.7 Hz, 2H, CH₂), 3.25 (s, 3H, CH₃), 3.32 (dt, J = 5.5, 13.2 Hz, 1H, CH₂), 3.83 (dt, J = 5.9, 13.2 Hz, 1H, CH₂), 6.82 (d, J = 8.3 Hz, 1H, ArH), 7.09-7.20 (m, 3H, ArH), 7.30 (br s, 1H, NH), 7.34 (d, J = 1.9 Hz, 1H, ArH), 7.50 (dd, J = 2.0, 8.3 Hz, 1H, ArH), 7.56 (d, J = 6.5 Hz, 1H, ArH); ¹³C NMR (CDCl₃): δ 19.8, 24.4, 37.8, 59.3, 108.0, 108.8, 110.6, 113.7, 116.4, 117.5, 120.5, 124.8, 125.7, 126.9, 130.4, 131.2, 134.0, 140.3, 173.9; HREI-MS (m/z) calculated for C₁₉H₁₇BrN₃O (M+H)⁺ 382.0550, found 382.0555.

Spiro[(5–methoxy–1–benzyl–3H–indole–2(1H)-one)-3,1ʹ-(1ʹ,2ʹ,3ʹ,4ʹ–tetrahydro–9H–pyrido[3,4–b]indol)] (5f)

Beige solid; Yield 84%; m.p. 180-183°C; IR (cm^–1): 3290, 1601, 1710, 1494, 1336, 1229, 1202; ¹H NMR (CDCl₃): δ 2.99 (t, J = 5.7 Hz, 2H, CH₂), 3.36 (dt, J = 5.3, 13.1 Hz, 1H, CH₂), 3.66 (s, 3H, OCH₃), 3.95 (dt, J = 6.3, 13.1 Hz, 1H, CH₂), 4.83 (d, J = 15.4 Hz, 1H CH₂), 5.0 (d, J = 15.4 Hz, 1H, CH₂), 6.75-6.80 (m, 3H, ArH), 7.09-7.18 (m, 3H, ArH), 7.30 (br s, 1H, NH), 7.31-7.36 (m, 5H, ArH), 7.58 (d, J = 6.9 Hz, 1H ArH); ¹³C NMR (CDCl₃) δ 22.0, 40.1, 44.0, 55.7, 61.9, 110.2, 111.1, 111.3, 112.5, 114.8, 118.6, 119.6, 122.5, 127.2 (2C), 127.5, 127.9, 128.9 (2C), 130.2, 132.6, 135.7, 135.9, 136.3, 156.5, 176.7; HREI-MS (m/z) calculated for C₂₆H₂₄N₃O₂ (M+H)⁺ 410.1863, found 410.1873.

Spiro[(5–bromo–1–benzyl–3H–indole–2(1H)-one)-3,1ʹ-(1ʹ,2ʹ,3ʹ,4ʹ–tetrahydro–9H–pyrido[3,4–b]indol)] (5g)

Cream solid; Yield 75%; m.p. 135-138°C; IR (ATR) 3323, 1719, 1603, 1477, 1351, 1229, 1170, 691; ¹H NMR (CDCl₃): δ 2.99 (t, J = 5.7, 2H Hz, CH₂), 3.35 (dt, J = 5.4, 13.2 Hz, 1H, CH₂), 3.88 (dt, J = 6.1 Hz, 1H, CH₂), 4.85 (d, J = 15.5 Hz, 1H, CH₂), 4.99 (d, J = 15.5 Hz, 1H, CH₂), 6.72 (d, J = 8.3 Hz, 1H, ArH), 7.08-7.20 (m, 3H, ArH), 7.22 (br s, 1H, NH), 7.30-7.45 (m, 7H, ArH), 7.58 (d, J = 6.4 Hz, 1H, ArH); ¹³C NMR (CDCl₃): δ 21.7, 39.8, 43.7, 61.2, 110.8, 110.9, 112.5, 115.8, 118.4, 119.5, 122.4, 126.8, 127.1 (2C), 127.7 (2C), 128.7 (2C), 129.0, 132.2, 133.2, 134.9, 136.0, 141.2, 176.0; HREI-MS (m/z) calculated for C₂₅H₂₁BrN₃O (M+H)⁺ 458.0863, found 458.0863.

General Procedure for Synthesis of 7a–g

A solution of spiroindolone 5a-g (0.3 mmol) in dry toluene (6 mL) was stirred and cooled to -78°C under Ar atmosphere and DIBAL-H (3 mmol) was added dropwise into the solution. After 4 h, a solution of 5% aqueous NaOH (8 mL) was quenched to the mixture and extracted with EtOAc (3×15 mL). The organic extracts were combined and washed with brine, dried over anhydrous sodium sulfate and after that concentrated under reduced pressure to give oxyindoline 6a-g. (This compound was unstable and used without further purification due to the color changed on a silica gel and in CDCl₃.)

The oxyindoline 6a-g was warmed in 5M HCl (10 mL) for 1 h at 90°C and then basified with 25% KOH until the pH was more than 12. The solid was filtered and purified by column chromatography (silica gel) to give isomeric Iheyamine derivatives 7a-g.

5–methyl–5H–azepino[2,3–b:4,5–bʹ]diindole (7a)

Following the general procedure, spiro[(1-methyl-3H-indole-2(1H)-ol)-3,1ʹ-(1ʹ,2ʹ,3ʹ,4ʹ-tetrahydro-9H-pyrido[3,4-b]indol)] (6a) was obtained as light brown viscous oil; ¹H NMR (CDCl₃): δ 2.86 (t, J = 5.6 Hz, 2H, CH₂), 2.93 (s, 3H, CH₃), 3.07 (dt, J =6.3, 12.8 Hz, 1H, CH₂), 3.42 (dt, J = 5.0, 12.9 Hz, 1H, CH₂), 4.92 (s, 1H, CH), 6.60 (d, J = 7.8 Hz, 1H, ArH), 6.69 (td, J = 0.8, 7.4 Hz, 1H, ArH), 6.92 (dd, J = 0.9, 7.4 Hz, 1H, ArH), 7.07-7.16 (m, 2H, ArH), 7.17-7.23 (m, 2H, ArH), 7.52 (dd, J = 1.7, 6.6 Hz, 1H, ArH), 7.72 (br s, 1H, NH).

The rearrangement of oxyindole 6a in 5M HCl gave compound 7a as green solid; 13 % (2 steps); m.p. 194-197°C (lit.¹² 193-195°C); IR (cm^-1): 2921, 2852, 1547, 1468, 1365; ¹H NMR (CDCl₃): δ 4.25 (s, 3H, CH₃), 7.45 (t, J = 7.8 Hz, 1H, ArH), 7.57-7.72 (m, 2H, ArH), 7.75 (d, J = 8.1 Hz, 1H, ArH), 7.83 (t, J = 8.1 Hz, 1H, ArH), 8.13 (d, J = 8.3 Hz, 1H, ArH), 8.35 (d, J = 7.8 Hz, 1H, ArH), 8.48 (d, J = 5.8 Hz, 1H, CH), 9.03 (d, J = 5.8 Hz, 1H, CH), 9.46 (d, J = 7.9 Hz, 1H, ArH); ¹³C NMR (CDCl₃): δ 28.8, 109.4, 112.3, 119.5, 121.6 (2C), 122.3, 122.8, 126.0, 126.5, 127.6, 128.7, 132.6, 139.9, 145.5, 147.2, 147.4,  156.2, 160.2; HREI-MS (m/z) calculated for C₂₀H₁₆N₃O (M+H)⁺ 284.1182, found 284.1178.

2–Methoxy–5–methyl–5H–azepino[2,3–b:4,5–bʹ]diindole (7b)

Following the general procedure, Spiro[(5-methoxy-1-methyl-3H-indole-2(1H)-ol)-3,1ʹ-(1ʹ,2ʹ,3ʹ,4ʹ-tetrahydro-9H-pyrido[3,4-b]indol)] (6b) was obtained as brown viscous oil; ¹H NMR (CDCl₃): δ 2.86 (t, J = 4.9 Hz, 2H, CH₂), 2.87 (s, 3H, CH₃), 3.08 (dt, J = 6.1, 12.7 Hz, 1H, CH₂), 3.39 (dt, J = 5.2, 12.8 Hz, 1H, CH₂), 3.66 (s, 3H, OCH₃), 4.86 (s, 1H, CH), 6.53 (d, J = 8.7 Hz, 1H, ArH), 6.56 (d, J = 2.5 Hz, 1H, ArH), 6.79 (dd, J = 2.6, 8.4 Hz, 1H, ArH), 7.09-7.23 (m, 3H, ArH), 7.52 (d, J = 7.5 Hz, 1H, ArH), 7.79 (br s, 1H, NH).

The rearrangement of oxyindole 6b in 5M HCl gave compound 7b as Burgundy solid; 14% (2 steps); m.p. 148-151°C (lit.¹² 147-148°C); IR (cm^–1): 2922, 2852, 1539, 1497, 1369, 1261; ¹H NMR (CDCl₃): δ 4.15 (s, 3H, CH₃), 4.23 (s, 3H, OCH₃), 7.42 (dd, J = 2.6, 8.9 Hz, 1H, ArH), 7.43 (t, J = 7.5 Hz, 1H, ArH), 7.60 (d, J = 8.9 Hz, 1H, ArH), 7.81 (t, J = 7.7 Hz, 1H, ArH), 8.11 (d, J = 8.0 Hz, 1H, ArH), 8.32 (d, J = 7.7 Hz, 1H, ArH), 8.43 (d, J = 5.7 Hz, 1H, CH), 8.97 (d, J = 5.8 Hz, 1H, CH), 9.12 (d, J = 2.5 Hz, 1H, ArH); ¹³C NMR (CDCl₃): δ 26.7, 54.1, 105.1, 108.1, 109.5, 116.9, 117.7, 118.2, 119.2, 119.5, 120.5, 123.4, 130.2, 132.9, 142.8, 143.9, 145.3, 153.0, 153.8, 156.4; HREI-MS (m/z) calculated for C₁₉H₁₄N₃ (M+H)⁺ 314.1288, found 314.1286.

2–Bromo–5–methyl–5H–azepino[2,3–b:4,5–bʹ]diindole (7c)

Following the general procedure, spiro[(5-bromo-1-methyl-3H-indole-2(1H)-ol)-3,1ʹ-(1ʹ,2ʹ,3ʹ,4ʹ-tetrahydro-9H-pyrido[3,4-b]indol)] (6c) was obtained as brown viscous oil; ¹H NMR (CDCl₃): δ 2.86 (t, J = 5.6 Hz, 2H, CH₂), 2.90 (s, 3H, CH₃), 3.06 (dt, J = 6.3, 12.8 Hz, 1H, CH₂), 3.39 (dt, J = 5.0, 12.9 Hz, 1H, CH₂), 4.91 (s, 1H, CH), 6.46 (d, J = 8.3 Hz, 1H, ArH), 7.01 (d, J = 2.0 Hz, 1H, ArH), 7.11 (td, J = 1.4, 7.1 Hz, 1H, ArH), 7.16 (td, J = 1.5, 7.3 Hz, 1H, ArH), 7.23 (d, J = 7.0 Hz, 1H, ArH), 7.31 (dd, J = 2.0, 8.3 Hz, 1H, ArH), 7.53 (d, J = 7.2 Hz, 1H, ArH), 7.70 (br s, 1H, ArH).

The rearrangement of oxyindole 6c in 5M HCl gave compound 7c as green solid; 14% (2 steps); m.p. 213-215°C; IR (cm^–1): 2921, 2852, 1551, 1463, 1369, 589; ¹H NMR (CDCl₃): δ 4.18 (s, 3H, CH₃), 7.45 (t, J = 7.9 Hz, 1H, ArH), 7.49 (d, J = 8.8 Hz, 1H, ArH), 7.79 (dd, J = 2.0, 8.7 Hz, 1H, ArH), 7.83 (t, J = 7.7 Hz, 1H, ArH), 8.10 (d, J = 8.1 Hz, 1H, ArH), 8.30 (d, J = 7.8 Hz, 1H, ArH), 8.37 (d, J = 5.7 Hz, 1H, CH), 8.98 (d, J = 5.7 Hz, 1H, CH), 9.70 (d, J = 2.0 Hz, 1H, ArH); ¹³C NMR (CDCl₃): δ 27.0, 108.8, 113.3, 117.7, 117.8, 118.5, 119.8, 120.0, 120.9, 121.9, 123.9, 125.6, 127.4, 129.3, 130.9, 136.2, 137.8, 144.4, 145.3; HREI-MS (m/z) calculated for C₁₉H₁₃BrN₃ (M+H)⁺ 362.0287, found 362.0283.

2–nitro–5–methyl–5H–azepino[2,3–b:4,5–bʹ]diindole (7d)

Following the general procedure, spiro[(5-nitro-1-methyl-3H-indole-2(1H)-ol)-3,1ʹ-(1ʹ,2ʹ,3ʹ,4ʹ-tetrahydro-9H-pyrido[3,4-b]indol)] (6d) was obtained as brown viscous oil; ¹H NMR (CDCl₃): δ 2.85-2.91 (m, 2H, CH₂), 3.01-3.12 (m with s prominent, 4H, CH₂, CH₃), 3.45 (dt, J = 4.6, 13.0 Hz, 1H, CH2), 5.11 (s, 1H, CH), 6.49 (d, J = 8.9 Hz, 1H, ArH), 7.11-7.23 (m, 3H, ArH), 7.55 (d, J = 6.9 Hz, 1H, ArH), 7.60 (br s, 1H, NH), 7.78 (d, J = 2.3 Hz, 1H, ArH), 8.17 (dd, J = 2.3, 8.8 Hz, 1H, ArH).

The rearrangement of oxyindole 6d in 5M HCl gave compound 7d as green solid; 10% (2 steps); m.p. 328-330°C; IR (cm^–1): 2922, 2852, 1554, 1506, 1455, 1307, 1293; ¹H NMR (CDCl₃): δ 4.24 (s, 3H, CH₃), 7.49 (t, J = 7.3 Hz, 1H, ArH), 7.65 (d, J = 8.8 Hz, 1H, ArH), 7.87 (t, J = 8.2 Hz, H, ArH), 8.15 (d, J = 8.0 Hz, 1H, ArH), 8.32 (d, J = 7.4 Hz, 1H, ArH), 8.42 (d, J = 5.7 Hz, 1H, CH), 8.57 (dd, J = 2.4, 9.2 Hz, 1H, ArH), 9.04 (d, J = 5.6 Hz, 1H, CH), 10.43 (d, J = 2.1 Hz, 1H, ArH); ¹³C NMR (CDCl₃): δ 29.8, 110.7, 112.0, 117.9, 118.5, 118.6, 121.1, 122.1, 123.7, 124.4, 125.4, 126.0, 126.6, 127.7, 130.7, 136.9, 141.3, 143.8, 151.3.; HREI-MS (m/z) calculated for C₁₉H₁₂N₄O₂ (M+H)⁺ 329.1039, found 329.1041.

5–benzyl–5H–azepino[2,3–b:4,5–bʹ]diindole (7e)

Following the general procedure, spiro[(1-benzyl-3H-indole-2(1H)-ol)-3,1ʹ-(1ʹ,2ʹ,3ʹ,4ʹ-tetrahydro-9H-pyrido[3,4-b]indol)] (6e) was obtained as light brown viscous oil; ¹H NMR (CDCl₃): δ 2.81-2.88 (m, 2H, CH₂), 3.00-3.09 (m, 1H, CH₂), 3.38-3.46 (m, 1H, CH₂), 4.41 (d, J = 15.2 Hz, 1H, CH₂), 4.58 (d, J = 15.1 Hz, 1H, CH₂), 5.05 (s, 1H, CH), 6.58 (d, J = 7.9 Hz, 1H, ArH), 6.67 (td, J = 0.8, 7.5 Hz, 1H, ArH), 6.93 (dd, J = 0.9, 7.4 Hz, 1H, ArH), 7.05-7.21 (m, 4H, ArH), 7.27-7.39 (m, 3H, ArH), 7.44 (d, J = 6.7 Hz, 2H, ArH), 7.51 (d, J = 7.5 Hz, 1H, ArH), 7.64 (br s, 1H, NH).

The rearrangement of oxyindole 6e in 5M HCl gave compound 7e as Green solid; Yield 11% (2 steps); m.p. 143-146°C; IR (cm^-1): 2923, 2851, 1609, 1454, 1351; ¹H NMR (CDCl₃): δ 6.04 (s, 2H, CH₂), 7.17-7.35 (m, 5H, ArH), 7.46 (t, J = 7.5 Hz, 1H, ArH), 7.55-7.72 (m, 3H, ArH), 7.83 (t, J = 7.6 Hz, 1H, ArH), 8.14 (d, J = 8.1 Hz, 1H, ArH), 8.35 (d, J = 7.7 Hz, 1H, ArH), 8.66 (d, J = 5.7 Hz, 1H, CH), 9.02 (d, J = 5.7 Hz, 1H, CH), 9.65 (d, J = 7.7 Hz, 1H, ArH); ¹³C NMR (CDCl₃): δ 43.3, 107.7, 110.0, 117.0, 117.5, 119.3, 119.4, 120.1, 120.5, 123.6, 124.4 (2C), 124.9, 125.1, 126.2 (2C), 126.4, 130.3, 134.7, 137.0, 143.5, 144.9, 145.0, 153.9, 157.6; HREI-MS (m/z) calculated for C₂₅H₁₈N₃ (M+H)⁺ 360.1495, found 360.1485.

2–methoxy–5–benzyl–5H–azepino[2,3–b:4,5–bʹ]diindole (7f)

Following the general procedure, spiro[(5-methoxy-1-benzyl-3H-indole-2(1H)-ol)-3,1ʹ-(1ʹ,2ʹ,3ʹ,4ʹ-tetrahydro-9H-pyrido[3,4-b]indol)] (6f) was obtained as brown viscous oil; ¹H NMR (CDCl₃): δ 2.81-2.90 (m, 2H, CH₂), 2.98-3.11 (m, 1H, CH₂), 3.35-3.45 (m, 1H, CH₂), 3.64 (s, 3H, OCH₃), 4.33 (d, J = 14.9 Hz, 1H, CH₂), 4.52 (d, J = 14.8 Hz, 1H, CH₂), 5.01 (s, 1H, CH), 6.48 (d, J = 8.5 Hz, 1H, ArH), 6.56 (d, J = 2.6 Hz, 1H, ArH), 6.71 (dd, J = 2.6, 8.5 Hz, 1H, ArH), 7.06-7.22 (m, 3H, ArH), 7.27-7.45 (m, 5H, ArH), 7.51 (d, J = 7.5 Hz, 1H, ArH), 7.71 (br s, 1H, NH).

The rearrangement of oxyindole 6f in 5M HCl gave compound 7f as burgundy solid; 13% (2 steps); m.p. 209-211°C; IR (cm^–1): 2923, 2852, 1544, 1497, 1314, 1268; ¹H NMR (CDCl₃): δ 4.11 (s, 3H, OCH₃), 5.97 (s, 2H, CH₂), 7.18-7.24 (m, 5H, ArH), 7.29 (dd, J = 2.6, 8.9 Hz, 1H, ArH), 7.38-7.49 (m, 2H, ArH), 7.80 (t, J = 7.6 Hz, 1H, ArH), 8.11 (d, J = 8.0 Hz, 1H, ArH), 8.29 (d, J = 7.7 Hz, 1H, ArH), 8.38 (d, J = 5.7 Hz, 1H, ArH), 8.91 (d, J = 5.7 Hz, 1H, ArH), 9.09 (d, J = 2.5 Hz, 1H, ArH); ¹³C NMR (CDCl₃): δ 44.1, 54.4, 105.6, 109.3, 110.2, 117.4, 118.3, 118.6, 119.8, 120.1, 121.4, 124.1, 124.9 (2C), 125.7, 126.2, 126.7, 126.9 (2C), 130.8, 132.8, 135.3, 143.5, 145.0, 145.6, 154.2; HREI-MS (m/z) calculated for C₂₆H₂₀N₃O (M+H)⁺ 390.1600, found 360.1595.

2–Bromo–5–benzyl–5H–azepino[2,3–b:4,5–bʹ]diindole (7g)

Following the general procedure, spiro[(5-bromo-1-benzyl-3H-indole-2(1H)-ol)-3,1ʹ-(1ʹ,2ʹ,3ʹ,4ʹ-tetrahydro-9H-pyrido[3,4-b]indol)] (6g) was obtained as red viscous oil; ¹H NMR (CDCl₃): δ 2.81-2.91 (m, 2H, CH₂), 3.04 (dt, J = 6.4, 13.0 Hz, 1H, CH₂), 3.40 (dt, J = 4.8, 12.9 Hz, 1H, CH₂), 4.39 (d, J = 15.1 Hz, 1H, CH₂), 4.56 (d, J = 15.1 Hz, 1H, CH₂), 5.05 (s, 1H, CH), 6.44 (d, J = 8.4 Hz, 1H, ArH), 7.02 (d, J = 2.0 Hz, 1H, ArH), 7.08-7.23 (m, 4H, ArH), 7.28-7.45 (m, 5H, ArH), 7.52 (d, J = 7.9 Hz, 1H, ArH), 7.53 (br s, 1H, NH).

The rearrangement of oxyindole 6g in 5M HCl gave compound 7g as green solid; 19% (2 steps); m.p. 213-216°C; IR (cm^–1): 2921, 2846, 1551, 1450, 1372, 688; ¹H NMR (CDCl₃): δ 5.92 (s, 2H, CH₂), 7.14-7.25 (m, 5H, ArH), 7.35 (d, J = 8.7 Hz, 1H, ArH), 7.44 (t, J = 7.7 Hz, 1H, ArH), 7.65 (dd, J = 1.5, 8.7 Hz, 1H, ArH), 7.82 (t, J = 7.6 Hz, 1H, ArH), 8.10 (d, J = 8.0 Hz, 1H, ArH), 8.26 (d, J = 7.6 Hz, 1H, ArH), 8.32 (d, J = 5.7 Hz, 1H, CH), 8.93 (d, J = 5.7 Hz, 1H, CH), 9.69 (s, 1H, ArH); ¹³C NMR (CDCl₃): δ 44.2, 109.9, 110.8, 113.9, 117.7, 118.5, 120.0, 120.5, 122.2, 123.9, 125.1 (2C), 126.0, 127.0 (3C), 127.5, 129.8, 131.3, 135.0, 135.9, 145.2, 145.5, 153.6, 157.5; HREI-MS (m/z) calculated for C₂₅H₁₇BrN₃ (M+H)⁺ 438.0600, found 438.0593.

Anticancer Activity

Cell Culture

Herein, human cervical cancer cells (HeLa), human hepatocellular carcinoma cells (HepG2) and kidney epithelial cells from African green monkey (Vero) were cultured in 10% Fetal Bovine Serum (FBS supplemented DMEM (Dulbecco’s Modified Eagle’s Medium) and antibiotics. Cells were kept in a humidified 5% CO₂ atmosphere at 37 °C with medium changes every 2 days until cells grown to 80-85% confluency. They were collected by trypsinization procedure with 0.25% trypsin and the viable cells were counted by tryphan blue exclusion.

Determination of Anticancer Activity by MTT Assay

Cells were seeded on 96-well plate at a density of 1×10⁵ cells/mL. After 24 h, the cells were administered with the test compounds at various concentrations and 20% organic solvent (acetone or DMSO) was used as a positive control. After incubation for 24 h, 100 µL of MTT (400 µg/mL) was added to each well and further incubated for 4 h. The medium was then removed and 100 µL of DMSO, which was used to dissolve the resulting formazan crystal, was added to each well. The microplate spectrometer (biochrom EZ Read 2000) was used to measure the absorbance at 540 nm. Percentage cell viability was calculated using corrected absorbance with the following equation:

% viable cells = [(Abs_sample – Abs_blank)/(Abs_control – Abs_blank)] x 100

where Abs_sample was the absorbance of the test compound, Abs_control was the absorbance of 1% organic solvent in medium and Abs_blank was the absorbance of 20% organic solvent in medium. The IC₅₀ value was determined by plotting percentage cell viability versus sample concentration.

Results and Discussion

Chemistry

The isatin (2a) was purchased from Sigma-Aldrich and the 5-substituted isatins (2b–d) were prepared by classical Sandmeyer isatin synthesis methods.¹³ N-alkylation of isatins (2a–d) with alkyl halide in the presence of K₂CO₃ were completed the N-substituted isatin derivatives (5a–g) in 52-92% yield (scheme 1).

Scheme 1: Synthesis of N-substituted isatin derivatives (3a–g). Click here to view scheme

 

The condensation reaction of N-substituted isatin derivatives (3a-g) with tryptamine (4) under acidic conditions forming the iminium ion intermediate, followed by spiro cyclization to provide racemic spiroindolone (5a-g) in good yield. Based on literature, the partial reduction of spiroindolones were achieved cleanly to the corresponding carbinolamines using sodium bis(2-methoxyethoxy)aluminium hydride.²⁰ However, the partial reduction using DIBAL-H in handed gave the carbinolamine (6a-g) in low yields. The carbinolamine (6a-g) were used without further purification by heating in 5 M HCl at 90°C to undergo rearrangement to the azepinodiindole (7a-g) and yields were reported from a 2-step process. The structure of compounds (7a-g) were compared to the previous synthesis isomeric Iheyamines¹² (scheme 2).

Scheme 2: Synthesis of isomeric Iheyamine 7a-g. Click here to view figure

 

Anticancer Activity

The in vitro anticancer activity of the synthesized compounds (7a-g) were evaluated for cytotoxicity against HeLa, HepG2, and Vero cell lines by MTT assay.²¹ All of the tested compounds exhibited good activity against both cancer cell lines and the IC₅₀ values were shown in Table 1. The N-methyl derivative of azepinodiindoles (7a-c) were more potent than the N-benzyl derivatives (7e-g). The methoxy substituent on the aromatic ring of compound (7b) and bromine substituent (7c) had similar inhibitory effect against HepG2 and HeLa cell lines with the same IC₅₀ value of 15.91 µg/mL. However, compound (7b) had lower activity on normal cell lines (Vero cell lines). The unsubstituted aromatic ring of azepinodiindoles (7a) and (7e) showed the selective inhibitory effect against HepG2 cell lines with no effect from the R² substitution. On the aromatic ring, the electron withdrawing nitro group, (7d) was the least active compound compared with other compounds containing methyl groups on R².

Table 1: Cytotoxicity of test compounds against cancer cell lines (HeLa and HepG2) and normal cell (Vero).

Compounds	R1	R2	IC50 (µg/mL)
			HeLa	HepG2	Vero
7a	H	CH3	71.30 ± 1.11	27.27 ± 0.38	101.01 ± 0.15
7b	OCH3	CH3	15.91 ± 0.36	22.62 ± 0.22	117.87 ± 0.37
7c	Br	CH3	15.91 ± 0.30	30.46 ± 0.83	43.30 ± 0.52
7d	NO2	CH3	66.63 ± 0.61	95.07 ± 0.36	205.48 ± 0.26
7e	H	Bn	61.01 ± 0.99	28.57 ± 0.48	75.99 ± 0.76
7f	OCH3	Bn	82.61 ± 0.47	63.00 ± 0.42	330.50 ± 0.72
7g	Br	Bn	149.56 ± 0.29	122.97 ± 0.14	234.38 ± 0.29
Acridine orange	–	–	4.11	6.36	5.82
Actinomycin D	–	–	NT	2.70	NT

 

NT: not tested

Molecular Docking

Using the iGEMDOCK v2.1 software²², molecular docking was performed in order to explore binding positions along with intermolecular interactions between compounds (7a–g) and binding site of cyclin-dependent kinase 2 (CDK2) which was key protein for cell cycle progression.^23,24 Compounds (7a–g) were docked into the active site of CDK2 co-crystallized with LZ8 (PDB ID: 2VTO). LZ8 was also redocked into the CDK2 and its binding energy and hydrogen bond was then compared to those of azepinodiindoles (7a–g). The docking results revealed that the binding position of redocked LZ8 (shown in orange in Fig 2) was more or less the same to that of co-crystallized LZ8 in CPX2. Table 2 showed that the binding energy of all synthesized compounds (7a–g) were -103.98 to -84.35 kcal/mol, which were higher than that of LZ8. Among these, compound 7b fit well in the binding cavity of CPK2 and had the most similar orientation and binding position to those of LZ8, as illustrated in Fig. 2. Additionally, this compound had three hydrogen bonding interaction with LEU83, LYS89 and GLN131. For 7a, 7c and 7d, it was found that they fit in the binding site of CPK2, however their orientation and binding pose were rather different to those of LZ8 (Fig. 3a). In contrast, although the azepinodiindoles (7e–g) containing Bn group for R² substituent had lower binding energy than 7b, they could not fit in the binding site of CDK2, especially 7g.

Table 2: Summary of binding energy, amino acid interactions and hydrogen bond length of azepenodiindoles (7a–g) in binding site of CDK2.

Compounds	Binding energy (kcal/mol)	Amino acid residue	Hydrogen bond length (Å)
7a	-84.35	ILE10, GLY11	2.48, 2.47
7b	-89.74	LEU83, LYS89, GLN131	2.28, 2.39, 2.42
7c	-86.07	–	–
7d	-103.98	ILE10, GLU12, LYS129	2.45, 2.30, 1.63
7e	-100.44	ILE10, GLY11	2.84, 2.73
7f	-99.11	GLU12, THR14, GLN131	2.98, 2.62, 2.89
7g	-101.47	ILE10	2.69
LZ8	-114.60	PHE82, LEU83	2.61, 2.21

 

Figure 2: Comparison of the binding of 7b (blue), redocked LZ8 (orange) and LZ8 (pink) in the cavity of CPK2(PDB ID: 2VTO). Click here to view figure

Figure 3: The binding of 7a (green), 7c (yellow), 7d (deep orange), 7e (red), 7f (light blue), 7g (purple) and LZ8 (pink) in the cavity of CPK2 (PDB ID: 2VTO).Click here to view figure

 

Conclusion

In summary, syntheses of isomeric Iheyamine A derivatives 9a–g can be prepared from the synthetic isatin derivatives and commercially available tryptamine. There have been achieved in 3-11% overall yield with 4-6 steps. The key reactions including Sandmeyer isatin synthesis method, N-alkylation, condensation, reduction and acid-catalyzed rearrangement. Out of the synthesized compounds 9a–g, compound 9b and 9c exhibited higher cytotoxicity against HeLa cell lines more than other compounds and exhibited cytotoxicity against HepG2 cell lines in similar to compound 9a and 9e but compound 9c is toxic to normal cell lines (Vero). Thus, the substitutions on the isomeric Iheaymine A core structure have to further investigation on their biological activities.

Acknowledgements

We thanks Faculty of Science, Silpakorn University; the Department of Chemistry for the financial support for Rattanopas, S. and the Department of microbiology for the bioactivity assay facilitation and we also thanks the Chulabhorn Research Institute for HREI-MS spectroscopy.

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