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Synthetic Studies of Cortistatin A Analogue from the CD-Ring Fragment of Vitamin D2
2013 Volume 61 Issue 10 Pages 1024-1029
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2013 Volume 61 Issue 10 Pages 1024-1029
Syntheses of analogue compounds of cortistatin A (1), an anti-angiogenic steroidal alkaloid from Indonesian marine sponge, were investigated by utilizing the CD-ring fragment of vitamin D2. The incidental preparation of a new analogue having CD-cis-fused skeleton and its biological evaluation revealed the importance of the CD-trans-fused structure for the potent and selective antiproliferative activity of 1 against human umbilical vein endothelial cells (HUVECs).
Angiogenesis, the formation of new blood capillaries from preexisting blood vessels, is critical for tumor growth and metastasis. A growing tumor needs an extensive network of capillaries to provide nutrients and oxygen, etc. In addition, the new blood vessels provide a way for tumor cells to enter in the circulation and to metastasize to another organ. Therefore, substances that inhibit angiogenesis have a considerable potential to be novel therapeutic agents for the treatment of cancer.1)
In the course of our study on bioactive substances from marine organisms, we focused on a search for selective inhibitors of proliferation of human umbilical vein endothelial cells (HUVECs) as anti-angiogenic substances and isolated cortistatins,2–5) a family of novel abeo-9(10–19)-androstane-type steroidal alkaloids, from the Indonesian marine sponge of Corticium simplex. We found that cortistatin A (1, Chart 1), a major constituent, showed remarkably selective antiproliferative activity against HUVECs and also inhibited migration and tubular formation of HUVECs induced by vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF).2a Therefore, cortistatins might have considerable potential as a novel anti-angiogenic drug lead.
The unique structure and a characteristic biological property of this compound attracted many synthetic chemists, and a number of synthetic reports including five total syntheses6–13) have appeared. Nevertheless, there have been no report about in vivo anti-tumor effect of cortistatins because small quantities of the final compound could be obtained. Then we decided to engage in a synthetic study of structurally simplified and in vivo active analogues of cortistatins and achieved recently to develop analogue 2 (Chart 1). Analogue 2 exhibited not only comparable antiproliferative activity against HUVECs with high selectivity in vitro but also in vivo significant antitumor activity by oral administration.14) Based on the analogue 2 as a structural template, a development of more practical and promising anti-cancer drug candidates is highly needed. Here we report an alternative synthetic method of analogue 2 and the preparation of a new analogue having the CD-cis-fused skeleton.
In the synthesis of analogue 2, we used Hajos–Parrish ketone (3) as a CD-ring synthon. Compound 3 is commercially available as an optically active form, and some chemo-, regio-, and stereoselective transformation methods of 3 have been developed through synthetic efforts toward steroid derivatives including 1. We supposed that the degradation product (4)15) of vitamin D2 could be utilized as another useful synthon for the syntheses of cortistatin analogues. As a showcase, we intended to prepare analogue 2 from compound 4. As shown in Chart 2, the installation of an isoquinoline side chain and a methoxycarbonyl moiety should lead to compound 5, the late synthetic intermediate of analogue 2.
The actual synthesis of compound 5 was depicted in Chart 3. Following the literature,15) the successive oxidative degradation of vitamin D2 provided compound 4, which was further converted to its enol triflate 6.16) Introduction of an isoquinoline moiety with desired stereochemistry was successfully achieved by a Suzuki–Miyaura cross-coupling reaction between compound 6 and isoquinolin-7-yl boronate (12)7) using standard conditions and subsequent hydrogenation using Pd–C as a catalyst. The nuclear Overhauser effect (NOE) experiment for compound 8 confirmed the desired orientation of the isoquinoline group. And the following removal of the tert-butyldimethylsilyl protecting group and Dess–Martin oxidation of the resulting secondary alcohol moiety gave a ketone 9 in good yield. The successful installation of the methoxycarbonyl moiety was achieved by the treatment of the lithium enolate of the ketone 9 with methyl cyanoformate, to give a keto-ester 10. Finally, the desired enoate 5 was obtained through conversion to an enol triflate 11 and palladium-catalyzed reduction, in 19% overall yield (7 steps) from 6 (previous method: 9 steps, 24% from known compound). No isomerization to the thermodynamically stable cis-hydrindane form was observed throughout those transformations. Compound 5 has been successfully converted to analogue 2 through three steps in 51% overall yield.14)
In an effort to install a C1 unit to the α-carbon of the ketone 9 efficiently, we tried to use Bredereck’s reagent17) (Chart 4). Thus, the ketone 9 was heated with Bredereck’s reagent in toluene to give an enaminoketone 13, and the following triflation and hydrolysis provided an aldehyde 14. Unexpectedly, the NOE experiment revealed that the complete isomerization of the hydrindane skeleton giving cis-form proceeded in these transformations. As we have not analyzed participation of the three dimensional (3-D) structure of the CD-ring part in the HUVEC-selective growth inhibitory activity of cortistatin A (1),18) we prepared a CD-cis-fused analogue 16 from the incidentally obtained aldehyde 14, through palladium-catalyzed reduction and subsequent Knoevenagel condensation–electrocyclization with 1,3-cyclohexanedione.14) The 1H-NMR and HPLC experiments revealed that analogue 16 is an equilibrated mixture (1 : 1) of 16a (8,14-cis) and 16b (8,14-trans) at the oxymethine carbon, probably caused by rapid retrocyclization–cyclization equilibrium at the B-ring.19)
Antiproliferative activities of analogue 16 against endothelial cells (HUVECs) and KB3-1 cells were evaluated (Table 1). Cortistatin A (1) and the CD-trans-fused analogue 2 have been clarified to exhibit the potent anti-proliferative activity against HUVEC (IC50: 0.0018 and 0.035 µM, respectively). On the other hand, the CD-cis-fused analogue 16 showed only weak antiproliferative activity (IC50: 1.5 µM) with poor selectivity (13-fold). This result revealed that the 3-D structure of the CD-ring of 1 would be an essential structural element for HUVEC-selective growth inhibitory activity.14) Actually, the molecular mechanics (MM) calculation analysis revealed that both isomers of 16a and 16b have a bended structures at their CD-ring part, which do not resemble with those for 1 and 2 (Chart 5).
| Cell line | 1 | 2 | 16 | |||
|---|---|---|---|---|---|---|
| IC50 | S.I. | IC50 | S.I. | IC50 | S.I. | |
| HUVEC | 0.0018 | 1 | 0.035 | 1 | 1.5 | 1 |
| KB3-1 | 7.0 | 3900 | 10.5 | 300 | 19.2 | 13 |
IC50=µM, n.d.=not determined. S.I.=selective index. IC50 against KB3-1 cells/IC50 against HUVECs.
In summary, we established the alternative synthetic method of cortistatin analogues from the degradation product (4) of vitamin D2. And the incidental preparation of a CD-cis-fused analogue 16 and its biological evaluation revealed the importance of the 3-D structure of the CD-ring part. We are further investigating to develop promising anti-cancer drug candidates, possibly through more practical method.
The following instruments were used to obtain physical data: a JASCO DIP-370 digital polarimeter (L=50 mm) for specific rotations; a JASCO FT/IR-5300 infrared spectrometer for IR spectra; a Waters Q-Tof Ultima API mass spectrometer for electrospray ionization-time-of-flight (ESI-TOF) MS; a JEOL JNM LA-500 NMR spectrometer for 1H-NMR (500 MHz) and 13C-NMR (125 MHz) using tetramethylsilane as an internal standard. HPLC was performed using a Hitachi L-6000 pump equipped with Hitachi L-4000H UV detector. Silica gel (Kanto, Silica Gel 60N) and pre-coated thin layer chromatography (TLC) plates (Merck, 60F254) were used for column chromatography and TLC. Spots on TLC plates were detected by spraying acidic p-anisaldehyde solution (p-anisaldehyde: 25 mL, c-H2SO4: 25 mL, AcOH: 5 mL, EtOH: 425 mL) with subsequent heating. Unless otherwise noted, all the reaction was performed under N2 atmosphere. After workup, the organic layer was dried over Na2SO4.
7-((3aS,7S,7aR)-7-((tert-Butyldimethylsilyl)oxy)-3a-methyl-3a,4,5,6,7,7a-hexahydro-1H-inden-3-yl)isoquinoline (7)Compound 12 (30.4 mg, 0.119 mmol), Pd(PPh3)4 (18.7 mg, 0.0162 mmol), and K2CO3 (44.8 mg, 0.324 mmol) were added to a solution of 616) (44.9 mg, 0.108 mmol) in N,N-dimethylformamide (DMF) (1.1 mL) and the whole mixture was stirred for 30 min at 50°C. H2O was added to the mixture and the whole mixture was extracted with AcOEt. Removal of the solvent from the AcOEt extract under reduced pressure gave a crude product, which was purified by SiO2 column (n-hexane–AcOEt=1 : 1) to give 7 (35.7 mg, 84%) as a colorless oil.
[α]D20 +3.7° (c=0.13 in CHCl3). IR (KBr): 2930, 2855, 1595, 1462, 1254, 1028 cm−1. 1H-NMR (500 MHz, CDCl3) δ: 9.20 (1H, s), 8.46 (1H, d, J=6.1 Hz), 7.91 (1H, s), 7.72 (2H, t, J=9.8 Hz), 7.58 (1H, d, J=6.1 Hz), 6.07 (1H, s), 4.19 (1H, s), 2.48 (1H, t, J=13.4 Hz), 2.17–2.09 (2H, m), 2.04–1.97 (1H, m), 1.92 (1H, dd, J=11.0, 6.0 Hz), 1.75 (1H, d, J=14.0 Hz), 1.60–1.49 (3H, m), 1.35 (3H, s), 0.91 (9H, s), 0.08 (3H, s), 0.07 (3H, s). 13C-NMR (125 MHz, CDCl3) δ: 153.8, 152.6, 142.6, 136.2, 134.6, 130.3, 128.8, 128.5, 126.0, 124.1, 120.1, 69.1, 55.1, 47.5, 36.2, 34.2, 31.4, 25.8 (3C), 19.6, 18.0, –4.8, –5.1. ESI-MS: m/z 416 (M+Na)+. High resolution (HR)-ESI-MS: m/z 416.2386, Calcd for C25H35NOSiNa. Found: 416.2390.
7-((1S,3aR,4S,7aR)-4-((tert-Butyldimethylsilyl)oxy)-7a-methyloctahydro-1H-inden-1-yl)isoquinoline (8)Pd/C (13.7 mg) was added to a solution of 7 (45.5 mg, 0.116 mmol) in AcOEt (1.2 mL) and the whole mixture was stirred for 15 h under H2 atmosphere. The whole mixture was filtered through Celite pad, eluting with AcOEt. Removal of the solvent from the filtrate under reduced pressure gave a crude product, which was purified by SiO2 column (n-hexane–AcOEt=1 : 1) to give 8 (39.9 mg, 88%) as a colorless oil.
[α]D20 −9.7° (c=0.16 in CHCl3). IR (KBr): 2930, 2855, 1591, 1471, 1252, 1022 cm−1. 1H-NMR (500 MHz, CDCl3) δ: 9.21 (1H, s), 8.46 (1H, d, J=5.5 Hz), 7.76 (1H, s), 7.71 (1H, d, J=8.5 Hz), 7.60 (1H, d, J=5.5 Hz), 7.56 (1H, d, J=7.9 Hz), 4.11 (1H, s), 2.84 (1H, t, J=9.5 Hz), 2.24 (1H, m), 2.03–2.00 (1H, m), 1.89–1.85 (1H, m), 1.72 (1H, t, J=12.5 Hz), 1.69–1.63 (1H, m), 1.59–1.55 (2H, m), 1.46–1.42 (2H, m), 1.32–1.27 (2H, m), 0.89 (9H, s), 0.74 (3H, s), 0.05 (3H, s), 0.03 (3H, s). 13C-NMR (125 MHz, CDCl3) δ: 152.3, 142.3, 140.5, 134.5, 132.5, 128.6, 126.3, 125.3, 120.0, 69.1, 57.7, 53.1, 44.1, 38.6, 34.5, 25.8 (3C), 25.3, 23.2, 18.0, 17.4, 14.8, −4.8, −5.1. ESI-MS: m/z 418 (M+Na)+. HR-ESI-MS: m/z 418.2542, Calcd for C25H37NOSiNa. Found: 418.2550.
(1S,3aR,7aR)-1-(Isoquinolin-7-yl)-7a-methylhexahydro-1H-inden-4(2H)-one (9)TBAF (1.0 M in tetrahydrofuran (THF), 0.20 mL, 0.204 mmol) was added to a solution of 8 (32.0 mg, 0.0818 mmol) in THF (0.45 mL) and the whole mixture was stirred for 15 h under reflux. After cooling, sat. NH4Cl aq. was added to the mixture and the whole mixture was extracted with AcOEt. Removal of the solvent from the AcOEt extract under reduced pressure gave a crude product, which was purified by SiO2 column (n-hexane–AcOEt=1 : 2) to give a deprotected alcohol (20.7 mg, 90%) as a colorless oil.
[α]D20 −4.5° (c=0.14 in CHCl3). IR (KBr): 3302, 2942, 2876, 1595, 1451, 1372, 1163 cm−1. 1H-NMR (500 MHz, CDCl3) δ: 9.21 (1H, s), 8.46 (1H, d, J=4.9 Hz), 7.75 (1H, s), 7.72 (1H, d, J=8.5 Hz), 7.61 (1H, d, J=5.5 Hz), 7.56 (1H, d, J=8.5 Hz), 4.20 (1H, s), 2.87 (1H, t, J=9.8 Hz), 2.31–2.23 (1H, m), 2.08–2.01 (1H, m), 1.94–1.86 (2H, m), 1.79–1.71 (2H, m), 1.68–1.60 (2H, m), 1.56–1.48 (2H, m), 1.34 (1H, td, J=12.7, 3.3 Hz), 0.75 (3H, s). 13C-NMR (125 MHz, CDCl3) δ: 152.3, 142.3, 140.2, 134.5, 132.4, 128.6, 126.3, 125.4, 120.1, 68.9, 57.5, 52.7, 43.8, 38.4, 33.8, 25.0, 22.7, 17.2, 14.5. ESI-MS: m/z 304 (M+Na)+. HR-ESI-MS: m/z 304.1677, Calcd for C19H23NONa. Found: 304.1673
NaHCO3 (66.0 mg, 0.786 mmol) and Dess–Martin periodinane (399 mg, 0.941 mmol) were added to a solution of the above alcohol (147 mg, 0.524 mmol) in CH2Cl2 (5.2 mL) and the whole mixture was stirred for 30 min. Sat. NaHCO3 aq. was added to the mixture, and the whole mixture was extracted with CH2Cl2. Removal of the solvent from the CH2Cl2 extract under reduced pressure gave a crude product, which was purified with SiO2 column (n-hexane–AcOEt=1 : 2) to give 9 (133 mg, 93%) as a colorless oil.
[α]D20 −114.4° (c=0.17 in CHCl3). IR (KBr): 2959, 2880, 1711, 1591, 1453, 1383, 1225 cm−1. 1H-NMR (500 MHz, CDCl3) δ: 9.21 (1H, s), 8.47 (1H, d, J=5.5 Hz), 7.78 (1H, s), 7.75 (1H, d, J=8.5 Hz), 7.61 (1H, d, J=5.5 Hz), 7.57 (1H, d, J=8.5 Hz), 3.17 (1H, t, J=9.5 Hz), 2.72 (1H, t, J=9.2 Hz), 2.30–2.23 (3H, m), 2.14–2.02 (3H, m), 1.84–1.69 (4H, m), 0.50 (3H, s). 13C-NMR (125 MHz, CDCl3) δ: 211.0, 152.3, 142.6, 139.1, 134.7, 131.8, 128.5, 126.3, 125.9, 120.0, 61.3, 57.3, 51.3, 40.7, 36.7, 25.6, 23.5, 19.3, 13.1. ESI-MS: m/z 302 (M+Na)+. HR-ESI-MS: m/z 302.1521, Calcd for C19H21NONa. Found: 302.1515.
(1S,3aR,7aR)-Methyl 1-(Isoquinolin-7-yl)-7a-methyl-4-oxooctahydro-1H-indene-5-carboxylate (10)Hexamethylphosphoramide (HMPA) (0.02 mL, 0.117 mmol) and lithium bis(trimethylsilyl)amide (LHMDS) (1.0 M in n-hexane, 0.47 mL, 0.47 mmol) were added to a solution of 9 (65.4 mg, 0.234 mmol) in THF (1.5 mL) at −78°C and the whole mixture was stirred for 30 min at that temperature. NCCO2CH3 (0.03 mL, 0.351 mmol) was added to the mixture and the whole mixture was stirred for 1 h. Sat. NH4Cl aq. was added to the mixture, and the whole mixture was extracted with AcOEt. Removal of the solvent from the AcOEt extract under reduced pressure gave a crude product, which was purified by SiO2 column (n-hexane–AcOEt=1 : 2) to give 10 (65.5 mg, 83%) as a mixture of two diastereomers (2 : 1).
[α]D20 −74.8° (c=0.15 in CHCl3). IR (KBr): 2955, 2882, 1742, 1717, 1591, 1437, 1385, 1275 cm−1. 1H-NMR (500 MHz, CDCl3) δ: 9.23 (1H, s), 8.50 (1H, s), 7.79 (1H, s), 7.78–7.74 (1H, m), 7.63 (1H, d, J=5.5 Hz), 7.57 (1H, d, J=8.5 Hz), 3.85–3.80 (1H, m), 3.76 (3H, d, J=2.4 Hz), 3.43 (2/3H, dd, J=12.2, 7.3 Hz), 3.36 (1/3H, d, J=6.1 Hz), 3.21 (1H, t, J=9.8 Hz), 2.92 (1/3H, dd, J=10.7, 7.6 Hz), 2.76 (2/3H, dd, J=11.0, 7.9 Hz), 2.31–2.11 (5H, m), 2.00–1.96 (1H, m), 1.83–1.79 (2H, m), 0.57 (2H, s), 0.51 (1H, s). 13C-NMR (125 MHz, CDCl3) δ: 170.5, 170.4, 152.3, 142.7, 138.6, 134.8, 131.8, 126.4, 126.1, 120.1, 61.3, 57.3, 56.8, 35.9, 31.5, 26.9, 22.6, 19.3, 14.1, 13.2. ESI-MS: m/z 360 (M+Na)+. HR-ESI-MS: m/z 360.1576, Calcd for C21H23NO3Na. Found: 360.1581.
(1S,3aR,7aR)-Methyl 1-(Isoquinolin-7-yl)-7a-methyl-4-(((trifluoromethyl)sulfonyl)oxy)-2,3,3a,6,7,7a-hexahydro-1H-indene-5-carboxylate (11)NaH (10.5 mg, 0.262 mmol) was added to a solution of 10 (44.1 mg, 0.131 mmol) in THF (0.72 mL) at −78°C and the whole mixture was stirred for 30 min at that temperature. Then PhNTf2 (93.6 mg, 0.262 mmol) was added to the mixture and the whole mixture was stirred for 30 min at −78°C and for 10 min at rt. Sat. NH4Cl aq. was added to the mixture and the whole mixture was extracted with AcOEt. Removal of the solvent from the AcOEt extract under reduced pressure gave a crude product, which was purified by SiO2 column (n-hexane–AcOEt=1 : 1) to give 11 (39.9 mg, 65%) as a colorless oil.
[α]D20 +2.3° (c=0.15 in CHCl3). IR (KBr): 2953, 2890, 1725, 1593, 1422, 1296, 1208 cm−1. 1H-NMR (500 MHz, CDCl3) δ: 9.23 (1H, s), 8.50 (1H, d, J=6.1 Hz), 7.80 (1H, s), 7.77 (1H, d, J=8.5 Hz), 7.63 (1H, d, J=5.5 Hz), 7.56 (1H, d, J=8.5 Hz), 3.79 (3H, s), 3.08 (1H, t, J=9.5 Hz), 2.87–2.80 (2H, m), 2.51–2.36 (2H, m), 2.32–2.24 (1H, m), 2.12–2.04 (1H, m), 1.95–1.88 (1H, m), 1.72–1.65 (2H, m), 0.64 (3H, s). 13C-NMR (125 MHz, CDCl3) δ: 164.9, 152.3, 142.8, 138.6, 134.8, 131.8, 128.5, 126.2, 126.1, 121.1, 120.1, 118.5 (q), 54.7, 52.1, 51.0, 47.1, 32.5, 26.3, 26.0, 21.7, 12.5. ESI-MS: m/z 492 (M+Na)+. HR-ESI-MS: m/z 492.1068, Calcd for C22H22F3NO5SNa. Found: 492.1070.
(1S,3aS,7aS)-Methyl 1-(Isoquinolin-7-yl)-7a-methyl-2,3,3a,6,7,7a-hexahydro-1H-indene-5-carboxylate (5)Pd(OAc)2 (0.7 mg, 3.3 µmol), PPh3 (1.5 mg, 5.6 µmol), Et3N (0.023 mL, 0.162 mmol) and HCOOH (4 µL, 0.108 mmol) were successively added to a solution of 11 (25.4 mg, 0.054 mmol) in THF (1.1 mL) and the whole mixture was stirred for 15 min at 70°C. After cooling to rt, H2O was added to the mixture and the whole mixture was extracted with AcOEt. Removal of the solvent from the AcOEt extract under reduced pressure gave a crude product, which was purified by SiO2 column (n-hexane–AcOEt=1 : 1) to give 5 (10.1 mg, 58%) as a colorless oil.
[α]D20 +15.7° (c=0.13 in CHCl3). IR (KBr): 2949, 2926, 2878, 2857, 1711, 1632, 1591, 1435, 1254 cm−1. 1H-NMR (500 MHz, CDCl3) δ: 9.22 (1H, s), 8.48 (1H, d, J=5.5 Hz), 7.80 (1H, s), 7.76 (1H, d, J=8.5 Hz), 7.62 (1H, d, J=6.1 Hz), 7.59 (1H, d, J=8.5 Hz), 7.04 (1H, d, J=1.8 Hz), 3.72 (3H, s), 2.99 (1H, t, J=9.8 Hz), 2.53–2.46 (2H, m), 2.40–2.34 (1H, m), 2.27–2.18 (2H, m), 2.05–2.00 (1H, m), 1.75 (1H, q, J=6.7 Hz), 1.71–1.64 (2H, m), 0.49 (3H, s). 13C-NMR (125 MHz, CDCl3) δ: 167.8, 152.3, 142.5, 140.6, 140.0, 134.6, 132.1, 129.9, 128.6, 126.1, 125.8, 120.1, 55.1, 51.5, 49.4, 44.0, 33.8, 26.5, 24.3, 23.4, 12.3. ESI-MS: m/z 344 (M+Na)+. HR-ESI-MS: m/z 344.1626, Calcd for C21H23NO2Na. Found: 344.1632.
(1S,3aS,7aR)-5-Formyl-1-(isoquinolin-7-yl)-7a-methyl-2,3,3a,6,7,7a-hexahydro-1H-inden-4-yl Trifluoromethanesulfonate (14)tert-Butoxy bis(dimethylamino)methane (Bredereck’s reagent, 0.17 mL, 0.820 mmol) was added to a solution of 9 (22.9 mg, 0.0820 mmol) in toluene (0.8 mL) and the whole mixture was stirred for 15 h at 55°C. Removal of the solvent from the reaction mixture under reduced pressure gave a crude enamine 13, which was then dissolved in CH2Cl2 (0.8 mL). 2,6-Lutidine (0.021 mL, 0.180 mmol) and Tf2O (0.015 mL, 0.0902 mmol) were added to the solution at −78°C and the whole mixture was stirred for 15 min at that temperature. H2O was added to the mixture and the whole mixture was extracted with CH2Cl2. Removal of the solvent from the CH2Cl2 extract under reduced pressure gave a crude product, which was purified with SiO2 column (n-hexane–AcOEt=2 : 1) to give 14 (11.2 mg, 31%) as a colorless oil.
[α]D20 +26.3° (c=0.14 in CHCl3). IR (KBr): 2961, 2928, 2884, 1688, 1657, 1419, 1217, 1140 cm−1. 1H-NMR (500 MHz, CDCl3) δ: 10.07 (1H, s), 9.23 (1H, s), 8.50 (1H, d, J=5.5 Hz), 7.78 (1H, d, J=9.5 Hz), 7.73 (1H, s), 7.64 (1H, d, J=6.1 Hz), 7.52 (1H, d, J=8.5 Hz), 3.13 (1H, dd, J=11.0, 7.3 Hz), 2.85 (1H, t, J=7.9 Hz), 2.62–2.55 (2H, m), 2.49 (1H, td, J=11.0, 7.5 Hz), 2.40–2.32 (1H, m), 2.22 (1H, dt, J=14.0, 6.0 Hz), 1.98–1.91 (1H, m), 1.67–1.62 (1H, m), 1.57–1.52 (1H, m), 0.77 (3H, s). 13C-NMR (125 MHz, CDCl3) δ: 187.6, 163.2, 152.3, 142.8, 139.3, 134.8, 131.6, 128.5, 128.2, 126.7, 126.3, 120.1, 117.0, 51.3, 49.7, 46.6, 30.2, 29.0, 27.8, 23.4, 19.4. ESI-MS: m/z 462 (M+Na)+. HR-ESI-MS: m/z 462.0963, Calcd for C21H20F3NO4SNa. Found: 462.0969.
(1S,3aR,7aS)-1-(Isoquinolin-7-yl)-7a-methyl-2,3,3a,6,7,7a-hexahydro-1H-indene-5-carbaldehyde (15)Pd(OAc)2 (2.6 mg, 0.012 mmol), PPh3 (5.3 mg, 0.02 mmol), Et3N (0.081 mL, 0.579 mmol) and HCOOH (0.015 mL, 0.386 mmol) were successively added to a solution of 14 (84.9 mg, 0.193 mmol) in THF (5.7 mL) and the whole mixture was stirred for 15 min at 70°C. After cooling to rt, H2O was added to the mixture and the whole mixture was extracted with AcOEt. Removal of the solvent from the AcOEt extract under reduced pressure gave a crude product, which was purified by SiO2 column (n-hexane–AcOEt=1 : 1) to give 15 (45.5 mg, 81%) as a colorless oil.
[α]D20 +68.7° (c=0.15 in CHCl3). IR (KBr): 2955, 2930, 2874, 1682, 1640, 1591, 1449, 1379 cm−1. 1H-NMR (500 MHz, CDCl3) δ: 9.50 (1H, s), 9.22 (1H, s), 8.49 (1H, d, J=5.5 Hz), 7.77 (1H, s), 7.75 (1H, d, J=5.0 Hz), 7.62 (1H, d, J=5.5 Hz), 7.55 (1H, d, J=8.5 Hz), 6.75 (1H, s), 3.09 (1H, dd, J=10.7, 7.6 Hz), 2.60 (1H, s), 2.45–2.25 (4H, m), 2.16–2.11 (1H, m), 1.64–1.49 (3H, m), 0.68 (3H, s). 13C-NMR (125 MHz, CDCl3) δ: 194.1, 154.9, 152.3, 142.6, 140.7, 138.8, 134.6, 131.9, 128.6, 126.4, 125.9, 120.1, 51.1, 47.8, 43.8, 31.1, 29.3, 28.9, 23.5, 18.3. ESI-MS: m/z 314 (M+Na)+. HR-ESI-MS: m/z 314.1521, Calcd for C20H21NONa. Found: 314.1518.
(3S,3aR,11bS)-3-(Isoquinolin-7-yl)-3a-methyl-1,3,3a,4,5,8,9,10,11a,11b-decahydrocyclopenta[c]xanthen-7(2H)-one (16)1,3-Cyclohexanedione (11.3 mg, 0.101 mmol) and ethylenediamine (0.004 mL, 0.061 mmol) were added to a solution of 15 (14.7 mg, 0.0505 mmol) in AcOEt (2.5 mL) and the whole mixture was stirred for 28 h. Removal of the solvent from the reaction mixture under reduced pressure gave a crude product, which was purified with SiO2 column (CHCl3–MeOH–H2O=30 : 3 : 1 (lower phase), then n-hexane–AcOEt=1 : 1) to give 16 (12.6 mg, 65%) as a mixture of two diastereomers (1 : 1).
[α]D20 +1.7° (c=0.18 in CHCl3). IR (KBr): 2957, 2880, 1725, 1645, 1605, 1454, 1402, 1167 cm−1. 1H-NMR (500 MHz, CDCl3) δ: 9.22 (1H, d, J=3.7 Hz), 8.49 (1H, d, J=6.1 Hz), 7.76 (1H, d, J=2.4 Hz), 7.74 (1H, s), 7.66 (1H, s), 7.62 (1H, d, J=6.1 Hz), 7.52 (1H, dt, J=16.7, 7.0 Hz), 6.29 (1/2H, s), 6.20 (1/2H, s), 5.24 (1/2H, d, J=5.5 Hz, 8-H for 16a), 4.93 (1/2H, d, J=10.4 Hz, 8-H for 16b), 3.36 (1/2H, dd, J=11.0, 8.5 Hz), 3.04 (1/2H, q, J=3.9 Hz), 2.45–2.32 (7H, m), 2.24–1.93 (6H, m), 1.68 (1/2H, dd, J=13.1, 8.2 Hz), 1.61–1.59 (1/2H, m), 1.49–1.44 (1/2H, m), 1.40–1.35 (1/2H, m), 0.74 (3/2H, s), 0.65 (3/2H, s). 13C-NMR (125 MHz, CDCl3) δ: 152.3, 142.6, 142.5, 139.7, 134.7, 134.4, 132.0, 127.1, 126.8, 126.0, 125.8, 120.0, 111.2, 109.7, 81.6, 79.7, 56.8, 54.2, 50.0, 47.4, 47.2, 36.4, 36.3, 35.4, 33.3, 31.6, 29.1, 28.8, 28.2, 28.1, 27.7, 27.5, 26.0, 25.1, 24.2, 22.6, 22.3, 20.6, 14.1. ESI-MS: m/z 408 (M+Na)+. HR-ESI-MS: m/z 408.1939, Calcd for C26H27NO2Na. Found: 408.1935.
Cell CultureHUVECs (5×105 cells/vial) was obtained from Kurabo Inc. and grown in the HuMedia-EG2 medium with growth supplements (Kurabo Inc.). Human KB epidermoid carcinoma cells (KB3-1) was cultured in the RPMI 1640 medium supplemented with heat-inactivated 10% fetal bovine serum (FBS) and kanamycin (50 µg/mL) in a humidified atmosphere of 5% CO2 at 37°C.
Growth Inhibition AssayA suspension of HUVECs in the proliferation medium (HuMedia-EG2) with growth supplements was plated into well of 96-well plate (2×103 cells/well/100 µL). After 24 h, the culture medium was removed and replaced with fresh essential minimal medium (HuMedia-EB2) with growth factor [bFGF (30 ng/mL) or VEGF (30 ng/mL)] of endothelial cells and various concentrations of testing compound. Plates were incubated for an additional 72 h in a humidified atmosphere of 5% CO2 at 37°C, and cell proliferation was detected by WST-8 colorimetric reagent. KB 3-1 cells in RPMI 1640 medium (2×103 cells/well/100 µL) were also inoculated into well of 96-well plate and treated as same as in the case of HUVECs for the evaluation of anti-proliferative effect. The IC50 value was determined by linear interpolation from the growth inhibition curve. We assessed selectivity of anti-proliferative activity [selective index (S.I.)] from the differences of IC50 values against HUVECs and KB 3-1 cells.
Conformational AnalysisMolecular modeling experiments were executed using MacroModel 9.1 equipped with Maestro 6.5 graphical interface installed on a Linux RedHat ver. 3 system. Initial structures were built with standard bond lengths and angles, and energy minimization was carried out using MMFFs force field and Polak–Ribiere conjugate gradient (PRCG). Optimization was performed with a gradient RMSD less than 0.05 kJ/mol-Å or 1000 iterations. Aqueous solution conditions were simulated using the continuum dielectric water solvent model (GB/SA) installed in MacroModel. A total of 5000 search steps were performed, and the conformations with energy difference of 15 kJ/mol from the global minimum were saved. Extended cut-off distances were defined at 8 Å for van der Waals, 20 Å for electrostatics, and 4 Å for H-bonds.
This study was financially supported by Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, The Uehara Memorial Foundation, and Adaptable and Seamless Technology Transfer Program through Target-Driven R&D (A-STEP, Exploratory Research) from Japan Science and Technology Agency.
