2022 Volume 70 Issue 4 Pages 261-268
Habiterpenol is a G2 checkpoint inhibitor isolated from the culture broth of Phytohabitans sp. 3787_5. Here, we report the synthesis of new habiterpenol analogs through the total synthesis process of habiterpenol and evaluating the analogs for G2 checkpoint inhibitory activity. We investigated two different synthetic approaches for total synthesis, with intramolecular conjugate addition and Ti(III)-mediated radical cyclization as key reactions. Although the former was unsuccessful, the latter reaction facilitated stereoselective total synthesis and determination of the absolute configuration of habiterpenol. The extension of these chemistries to a structure–activity relationship (SAR) study gave new habiterpenol analogs, which could not be derived from natural habiterpenol and only be synthesized by applying the total synthesis. Therefore, this study provides important insights into SAR studies of habiterpenol.
According to a 2018 WHO report, cancer is the second leading cause of death worldwide, causing approximately 9.6 million deaths each year.1) Chemotherapeutic agents with various mechanisms of action are one of the primary treatments for cancer; however, these agents have many side effects. In recent years, clinicians and researchers have promoted the development of highly selective molecular-targeting therapeutic agents that are effective at low doses. Although newer therapeutic agents have relatively few side effects, their toxicity cannot be completely eliminated. Therefore, there is a need for continuous development of new anticancer drugs.
Accordingly, G2 checkpoint inhibitors have recently attracted attention as vital new molecular-targeting therapeutic agents. Cells are subjected to two checkpoints in a single cell cycle: at the G1 and G2 stages.2) Normal cells repair DNA damage at the G1 checkpoint, whereas approximately half the cancer cells repair DNA damage only at the G2 checkpoint.3,4) Therefore, inhibiting the G2 checkpoint blocks the DNA repair mechanism of cancer cells, leading to cell death; in contrast, normal cells survive as they are able to repair DNA damage at the G1 checkpoint. Therefore, combination therapy with low-dose DNA-damaging anticancer agents and G2 checkpoint inhibitors may be an effective treatment for cancer, with fewer side effects. Several G2 checkpoint inhibitors have been reported, including CBP5015) and AZD7762,6) which have already undergone clinical trials. During our ongoing screening for microbial G2 checkpoint inhibitors, we isolated a novel compound habiterpenol (1) from the culture medium of Phytohabitans sp. 3787_5, which abrogated bleomycin-induced G2 arrest in Jurkat cells without cytotoxicity7) (Fig. 1).
Habiterpenol (1) is a fused ring compound consisting of a steroid skeleton with six consecutive asymmetric carbons and a phenol unit. The CD rings in 1 are fused in a syn orientation, and the AB and BC rings are fused in an anti orientation.8) Owing to its useful biological properties, we planned a structure–activity relationship (SAR) study of 1. However, this was not an easy task because the structure of 1 has few functional groups that can be chemically converted. Therefore, we embarked on the total synthesis of 1 for synthesizing new analogs and investigated two approaches as shown in Chart 1: intramolecular Michael reaction and Ti(III)-mediated radical cyclization. Through the former reaction (3+4 → 5 → 6), total synthesis could not be achieved because the key reaction gave 6 with an undesired configuration at C7. In contrast, the latter reaction (3+7 → 8 → 9) afforded 9 with all desired configurations, leading to the completion of the total synthesis of 1.9)
Next, we moved to the SAR study of 1 and first selected a 7-hydroxymethylhabiterpenol analog (10) as shown in Fig. 2, which could be synthesized with slight modifications to the total synthesis of 1. Synthesis of analog 10 would also improve the hydrophilicity of the highly lipophilic 1.10) Moreover, we focused on a 7-epi-habiterpenol analog (11) as a second target, which could be easily derived from the previously synthesized 6. The effect of the new analog 11 on G2 checkpoint inhibitory activity is interesting because it has a conformation different from 1. Herein, we describe the synthesis of novel habiterpenol analogs and evaluate their G2 checkpoint inhibitory activity.
The synthetic plan for 10 is shown in Chart 2, in which the known epoxyaldehyde 1211) was selected over 7; 12 is a key substrate in the coupling reaction with an indene unit. Subsequent Ti(III)-mediated radical cyclization12–14) of the corresponding coupling product 13 would give rise to diol 14, which would be converted to the desired analog 10. The coupling reaction of the aldehyde 12 with the corresponding alkyl lithium species prepared by the halogen–lithium exchange of the iodide 39) with tBuLi afforded an alcohol (5S)-13 as a single diastereomer, in 97% yield (the C5 stereochemistry was determined in a later step). Dess–Martin oxidation of (5S)-13 afforded the ketone 15 in 98% yield (Table 1). The key Ti(III)-mediated radical cyclizations of (5S)-13 and 15 were examined. The treatment of (5S)-13 with 3 equivalent (equiv.) of Cp2TiCl2 and 4 equiv. of Mn as a reductant afforded the tetrahydrofuran 16 with the desired C7 stereochemistry in 25% yield but not the desired diol (5S)-14 (run 1). The use of the ketone 15 instead of (5S)-13 slightly increased the isolated yield (47%) of the tetrahydrofuran 18 with the desired C7 stereochemistry, instead of the ketone 17 (run 2). The change of reductant from Mn to Zn gave the best result, affording 18 in 74% yield. We confirmed the stereochemistry of the tetrahydrofurans 16 and 18 by rotating-frame Overhauser spectroscopy (ROESY) experiments (Fig. 3). This result revealed that the C5 stereochemistry in 13 was S. The 1,2-addition of the aldehyde 12, with the corresponding alkyl lithium species prepared from iodide 3, would have proceeded through a chelation-controlled transition state A, resulting in the stereoselective formation of the desired 5S adduct via an Si face attack on the carbonyl group by the alkyl lithium species (Chart 3).
 | ||||
|---|---|---|---|---|
| Run | SM | Cp2TiCl2 (equiv.) | Reductant (equiv.) | Result (%)a) |
| 1 | (5S)-13 | 3 | Mn (4) | 16 (25) |
| 2 | 15 | 2 | Mn (3) | 18 (47) |
| 3 | 15 | 2 | Zn (3) | 18 (74) |
a) Isolated yield.
The proposed mechanism for the formation of 16 and 18 is shown in Chart 4. The treatment of epoxides (5S)-13 and 15 with Cp2TiCl consequently resulted in the formation the α-titanoxy radical I, followed by radical cyclization with an indene moiety via a 6-exo-trig process and the expected six-membered chair-like transition state to afford the γ-titanoxy benzylic radical II with the desired C7 stereochemistry. The proximity of the Ti(IV)-bonded oxygen atom to the benzylic carbon radical in II consequently led to further stereoselective cyclization. As illustrated, radical recombination liberated Ti(III) and produced 16 or 18. This was similar to the results reported by Gansäuer et al.15) and Trost et al.16,17)
The ketone 18 was stereoselectively reduced with LiAlH4 to give the β-alcohol 16 in 92% yield (Chart 5). Barton–McCombie deoxygenation of 16 through xanthate formation (93%) and treatment with nBu3SnH and 2,2′-azobis (isobutyronitrile) (AIBN) (95%) afforded high yields of 20. Subsequent reductive C–O bond cleavage at the benzylic position was problematic to some extent. After screening the reaction conditions, the hydrogenolysis of 20 with a catalytic amount of Pd/C in the presence of 60% HClO418) afforded the primary alcohol 21 in 76% yield. Finally, 21 was quantitatively converted to the desired analog 10 by treatment with BCl3.
Next, we proceeded with the synthesis of analog 11 (Chart 6). We investigated the reduction of the C5 carbonyl group in 6; however, the reduction, performed under a variety of conditions, was unsuccessful due to steric hindrance. Therefore, the 5-oxo-7-epi-habiterpenol analog (23) was synthesized as a new habiterpenol analog instead of 11 and was synthesized in 92% yield by subjecting 6 to hydrogenolysis. The stereochemistry of 23 was confirmed by ROESY and nuclear Overhauser effect (NOE) experiments (Fig. 4).
We performed the biological evaluation of the synthetic compounds. The synthetic habiterpenol (1), synthesized by us previously, showed no cytotoxicity; however, the other analogs were cytotoxic (Table 2). When Jurkat cells were treated with bleomycin (5 µM) for 24 h, cell cycle analysis using the cell analyzer showed distribution rates of 11 and 70% in sub-G1 and G2/M phases, respectively (Table 3). On treatment with the combination of synthetic habiterpenol (1) (273 µM) with bleomycin (5 µM), cells in the G2/M phase decreased from 70 to 39%, and concomitantly, cells in the sub-G1 phase increased from 11 to 37%. These data were consistent with those obtained on treatment of natural habiterpenol, indicating that habiterpenol selectively abrogated bleomycin-induced G2 arrest in Jurkat cells.7) Cells in the G2/M phase decreased to 43% when treated with low concentrations of analog 6 (0.264 µM) along with bleomycin (5 µM), but higher concentrations of analog 6 alone increased the cells in the sub-G1 phase.19) Similarly, the other analogs on their own affected the cell cycle at concentrations below those associated with cytotoxicity.
| Compounds | IC50 (µM) |
|---|---|
| Habiterpenol (1) | > 273 |
| 6 | 145 |
| 10 | 14.4 |
| 23 | 1.45 |
| Compound (µM) | − Bleomycin | + Bleomycin | ||
|---|---|---|---|---|
| Sub-G1 (%) | G2/M (%) | Sub-G1 (%) | G2/M (%) | |
| None (control) | 4.1 | 26 | 11 | 70 |
| Habiterpenol (1) (273) | 12 | 21 | 37 | 39 |
| 6 (0.264) | 5.9 | 22 | 45 | 43 |
| 10 (0.262) | 4.1 | 23 | 11 | 68 |
| 23 (0.263) | 4.4 | 22 | 8.1 | 67 |
We synthesized novel habiterpenol analogs 6, 10, and 23 and conducted SAR studies of 1 and evaluated their G2 checkpoint inhibitory activity. These results revealed that the stereochemistry of 1 was crucial, and the introduction of a hydrophilic group, such as a hydroxy group, to the C7 methyl group was not necessary for abrogating bleomycin-induced G2 arrest in Jurkat cells. We will continue to use the total synthetic pathway to synthesize new analogs of 1. Additional SAR studies are currently underway and will be reported in due course.
All reactions were performed in flame-dried glassware under nitrogen by using standard techniques for handling air-sensitive materials. All the heating reactions were carried out in an oil bath. Commercial reagents were used without further purification, unless otherwise noted. Organic solvents were distilled and dried over 3 or 4 Å molecular sieves (MS). Cold baths were prepared under the following conditions: 0 °C, wet ice/water; −78 °C, dry ice/acetone. Purification by flash column chromatography was performed over silica gel 60N (spherical, neutral, particle size of 40–50 µm). TLC was performed on 0.25 mm Merck silica gel 60 F254 plates, and the effluents were visualized by UV (λ = 254 nm) and by using phosphomolybdic acid and p-anisaldehyde TLC stains. Yields corresponded to chromatographically and spectroscopically pure compounds, unless otherwise noted. 1H- and 13C-NMR spectra were recorded by using an internal deuterium lock on 400-MR, VNMRS-400, and UNITY-400 spectrometers. All NMR signals were reported in ppm relative to the internal reference standard provided by chloroform (i.e., δ = 7.26 or 77.0 ppm for 1H and 13C spectra, respectively). Multiplicity data were presented as follows: s = singlet, d = doublet, t = triplet, q = quartet, sep = septet, m = multiplet, br = broad, dd = doublet of doublets, and dt = doublet of triplets. Coupling constants (J) were reported in hertz. IR spectra were recorded on an FT/IR460-plus IR spectrometer. Absorption data were expressed in wavenumbers (cm−1). Optical rotation was recorded on a JASCO DIP-1000 polarimeter and reported as follows: [α]TD, concentration (g/100 mL), and solvent. High-resolution mass spectra were recorded on JEOL JMS 700 MStation, JEOL JMS-AX505HA, and JEOL JMS-T100LP systems equipped with FAB, electron ionization (EI), and electrospray ionization-time-of-flight (ESI-TOF) high-resolution (HR) mass spectrometers.
(S)-2-((R)-6-Isopropoxy-1-methyl-1H-inden-1-yl)-1-((1S,2R,4aS,8aS)-5,5,8a-trimethyloctahydro-1H-spiro-[naphthalene-2,2′-oxiran]-1-yl)ethan-1-ol ((5S)-13)To a solution of tBuLi (1.62 M sol. In pentane, 1.69 mL, 2.74 mmol) in Et2O (10 mL) at −78 °C was added dropwise a solution of 3 (446 mg, 1.36 mmol) in Et2O (4.2 mL). After stirring for 25 min at −78 °C, a solution of 1211) (214 mg, 0.904 mmol) in Et2O (4.2 mL) was added dropwise. The reaction mixture was kept stirring for 30 min at −78 °C and quenched with saturated aqueous NH4Cl. The aqueous layer was separated and extracted with EtOAc. The combined organic layers were washed with H2O, brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by flash silica gel column chromatography (20 : 1 hexanes/EtOAc) to afford (5S)-13 (383 mg, 97%) as a colorless amorphous solid. Rf = 0.29 (hexanes/EtOAc = 7 : 1); [α]24D −64.92 (c 1.0, CHCl3); IR (KBr) 3441, 2966, 2086, 1638, 1467, 1378, 1119, 968 cm−1; 1H-NMR (400 MHz, CDCl3) δ: 7.18 (d, J = 8.2 Hz, 1H), 6.82 (d, J = 2.4 Hz, 1H), 6.76 (dd, J = 8.2, 2.4 Hz, 1H), 6.63 (d, J = 5.4 Hz, 1H), 6.32 (d, J = 5.4 Hz, 1H), 4.56 (sep, J = 5.9 Hz, 1H), 3.53 (dd, J = 5.9, 1.8 Hz, 1H), 3.25 (m, 1H), 2.51 (dd, J = 14.6, 8.0 Hz, 1H), 2.43 (d, J = 5.9 Hz, 1H), 2.05 (dd, J = 14.6, 2.4 Hz, 1H), 1.83–1.75 (m, 1H), 1.74–1.68 (m, 1H), 1.48–0.73 (m, 10H), 1.35 (d, J = 5.9 Hz, 3H), 1.33 (d, J = 5.9 Hz, 3H), 1.26 (s, 3H), 0.94 (s, 3H), 0.80 (s, 3H), 0.75 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ: 156.7, 153.3, 143.3, 129.2, 121.8, 114.1, 110.8, 70.3, 67.8, 59.9, 59.7, 55.2, 54.2, 51.6, 49.9, 41.9, 40.6, 38.7, 38.2, 33.6, 33.3, 24.8, 22.1, 22.0, 21.4, 19.4, 18.7, 15.9; HRMS (EI) m/z: [M]+ Calcd for C29H42O3 438.3134. Found 438.3140.
2-((R)-6-Isopropoxy-1-methyl-1H-inden-1-yl)-1-((1R,2R,4aS,8aS)-5,5,8a-trimethyloctahydro-1H-spiro[naphthalene-2,2′-oxiran]-1-yl)ethan-1-one (15)To a solution of the epoxyalcohol (5S)-13 (84.1 mg, 0.192 mmol) in CH2Cl2 (3.8 mL) were added Dess–Martin periodinane (244 mg, 0.576 mmol) and NaHCO3 (96.6 mg, 1.15 mmol) at 0 °C. After stirring for 1 h at room temperature, the reaction mixture was quenched with saturated aqueous Na2S2O3 and saturated aqueous NaHCO3. The organic layer was separated, washed with H2O, brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by flash silica gel column chromatography (40 : 1 hexanes/EtOAc) to afford 15 (82.4 mg, 98%) as a colorless amorphous solid. Rf = 0.40 (hexanes/EtOAc = 8 : 1); [α]23D −116.91 (c 1.0, CHCl3); IR (film) 3444, 2932, 1710, 1631, 1377, 1195, 1120, 969 cm−1; 1H-NMR (400 MHz, CDCl3) δ: 7.15 (d, J = 8.3 Hz, 1H), 6.88 (d, J = 2.2 Hz, 1H), 6.74 (dd, J = 8.3, 2.2 Hz, 1H), 6.62 (d, J = 5.1 Hz, 1H), 6.55 (d, J = 5.1 Hz, 1H), 4.54 (sep, J = 6.1 Hz, 1H), 3.04 (d, J = 17.6 Hz, 1H), 3.04–3.02 (m, 1H), 2.71 (s, 1H), 2.59 (d, J = 4.8 Hz, 1H), 2.33 (d, J = 17.6 Hz, 1H), 1.94–1.86 (m, 1H), 1.79 (m, 1H), 1.60–0.81 (m, 9H), 1.35 (d, J = 6.1 Hz, 3H), 1.34 (d, J = 6.1 Hz, 3H), 1.29 (s, 3H), 1.14 (s, 3H), 0.85 (s, 3H), 0.81(s, 3H); 13C-NMR (100 MHz, CDCl3) δ: 209.1, 156.3, 154.2, 142.4, 135.5, 128.1, 121.6, 114.1, 110.3, 70.2, 64.2, 57.7, 55.5, 54.5, 53.1, 51.3, 41.9, 40.9, 38.8, 35.7, 33.6, 33.2, 22.2, 22.1, 21.7, 21.6, 21.5, 18.2, 14.3; HRMS (EI) m/z: [M]+ Calcd for C29H40O3 436.2977. Found 436.2974.
(2aR,2bS,6aS,8aR,8a1R,10aS,14bR)-13-Isopropoxy-2b,6,6,14b-tetramethyl-1,2b,3,4,5,6,6a,7,8,8a1,10a,14b-dodecahydro-9H-naphtho[2′,1′:1,2]fluoreno[9,1-bc]furan-2(2aH)-one (18)To a solution of Cp2TiCl2 (398 mg, 1.52 mmol) and Zn (152 mg, 2.28 mmol) in tetrahydrofuran (THF) (5.0 mL) at room temperature was added dropwise a solution of 15 (332 mg, 0.760 mmol) in THF (2.7 mL). After stirring for 1 h at room temperature, the reaction mixture was quenched with 2 M HCl at 0 °C. The resulting mixture was filtered through a pad of celite and the celite was washed with CH2Cl2. The filtrate was dried over Na2SO4 and evaporated in vacuo to furnish the crude product, which was purified by flash chromatography (25 : 1 hexanes/EtOAc) to afford 18 (251 mg, 74%) as a colorless amorphous solid. The stereochemistries were confirmed by ROESY experiments. Rf = 0.69 (hexanes/EtOAc = 5 : 1); [α]23D −45.52 (c 1.0, CHCl3); IR (film) 3434, 2927, 1706, 1611, 1483, 1378, 1113, 970, 816 cm−1; 1H-NMR (400 MHz, CDCl3) δ: 7.30 (d, J = 7.4 Hz, 1H), 6.77 (dd, J = 7.4, 2.4 Hz, 1H), 6.59 (d, J = 2.4 Hz, 1H), 5.43 (d, J = 6.4 Hz, 1H), 4.53 (sep, J = 5.2 Hz, 1H), 3.86 (d, J = 9.3 Hz, 1H), 3.47 (dd, J = 9.3, 2.2 Hz, 1H), 2.72–2.67 (m, 1H), 2.59 (d, J = 6.4 Hz, 1H), 2.55 (s, 2H), 2.43 (s, 1H), 2.22 (dt, J = 12.8, 3.2 Hz, 1H), 1.65–0.91 (m, 9H), 1.40 (s, 3H), 1.32 (d, J = 5.2 Hz, 6H), 0.89 (s, 3H), 0.88 (s, 3H), 0.83 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ: 209.8, 159.3, 153.6, 131.8, 126.7, 115.2, 109.5, 83.8, 75.0, 70.0, 65.7, 62.0, 56.0, 53.4, 49.0, 45.6, 41.9, 41.7, 39.2, 38.3, 33.7, 33.4, 30.9, 22.0, 22.0, 21.8, 18.8, 18.6, 14.7; HRMS (EI) m/z: [M]+ Calcd for C29H40O3 436.2977. Found 436.2969.
(2R,2aR,2bS,6aS,8aS,8a1R,10aS,14bR)-13-Isopro-poxy-2b,6,6,14b-tetramethyl-2,2a,2b,3,4,5,6,6a,7,8,8a1,9,10a,14b-tetradecahydro-1H-naphtho[2′,1′:1,2]fluoreno[9,1-bc]furan-2-ol (16)To a solution of lithium aluminum hydride (LAH) (14.3 mg, 0.369 mmol) in THF (0.5 mL) at 0 °C was added dropwise a solution of 18 (32.2 mg, 0.0737 mmol) in THF (1.0 mL). The resulting mixture was allowed to warm up to room temperature and kept stirring for 1 h. The reaction was cautiously quenched with MeOH at 0 °C, diluted with CH2Cl2, and treated with celite and Na2SO4·10H2O. The mixture was allowed to warm to room temperature and stirred for 1 h. The mixture was filtered through a pad of celite and the filtrate was concentrated in vacuo. The residue was purified by flash chromatography (10 : 1 hexanes/EtOAc) to afford 16 (29.6 mg, 92%) as a colorless amorphous solid. The stereochemistries were confirmed by ROESY experiments. Rf = 0.72 (hexanes/EtOAc = 5 : 1); [α]23D +9.65 (c 1.0, CHCl3); IR (film) 3451, 2925, 2856, 1603, 1482, 1250, 1119, 969, 858 cm−1; 1H-NMR (400 MHz, CDCl3) δ: 7.23 (d, J = 8.4 Hz, 1H), 6.78 (dd, J = 8.4, 2.4 Hz, 1H), 6.65 (d, J = 2.4 Hz, 1H), 5.58 (d, J = 7.2 Hz, 1H), 4.53 (sep, J = 6.0 Hz, 1H), 4.43 (bd, J = 4.2 Hz, 1H), 3.73 (d, J = 8.8 Hz, 1H), 3.65 (dd, J = 8.8, 2.0 Hz, 1H), 2.52 (dd, J = 15.2, 3.6 Hz, 1H), 2.18 (d, J = 7.2 Hz, 1H), 2.12 (dt, J = 12.8, 3.6 Hz, 1H), 1.82–0.88 (m, 12H), 1.31 (d, J = 6.0 Hz, 6H), 1.11 (s, 3H), 0.92 (s, 3H), 0.84 (s, 3H), 0.81 (s, 3H), 0.64 (d, J = 4.2 Hz, 1H); 13C-NMR (100 MHz, CDCl3) δ: 158.9, 153.8, 133.0, 127.0, 115.2, 108.0, 82.9, 70.2, 70.0, 67.0, 62.7, 57.4, 57.2, 46.5, 45.0, 42.8, 42.7, 42.1, 39.2, 38. 5, 36.7, 33.4, 33.3, 22.1, 22.0, 21.2, 20.0, 18.8, 16.3; HRMS (EI) m/z: [M]+ Calcd for C29H42O3 438.3134. Found 438.3128.
O-((2R,2aR,2bS,6aS,8aS,8a1R,10aS,14bR)-13-Isopropoxy-2b,6,6,14b-tetramethyl-2,2a,2b,3,4,5,6,6a,7,8,8a1,9,10a,14b-tetradecahydro-1H-naphtho[2′,1′:1,2]fluoreno[9,1-bc]furan-2-yl) S-Methyl Carbonodithioate (19)To a solution of 16 (119 mg, 0.272 mmol) in THF (5.4 mL) at 0 °C was added tBuOK (108 mg, 0.816 mmol). After stirring for 20 min, the reaction mixture was allowed to warm up to room temperature and kept stirring for 40 min. To the resulting mixture was added dropwise CS2 (247 µL, 4.08 mmol). After stirring for 30 min, the mixture was treated with methyl iodide (MeI) (254 µL, 4.08 mmol) and continuously stirred for 1 h. The reaction mixture was quenched with saturated aqueous NH4Cl. The aqueous layer was separated and extracted with EtOAc. The combined organic layers were washed with H2O, brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by flash silica gel column chromatography (10 : 1 hexanes/EtOAc) to afford 19 (134 mg, 93%) as a colorless amorphous solid. Rf = 0.57 (hexanes/EtOAc = 5 : 1); [α]24D −34.21 (c 1.0, CHCl3); IR (film) 3445, 2928, 2861, 1637, 1487, 1460, 1378, 1218, 1058, 962, 943 cm−1; 1H-NMR (400 MHz, CDCl3) δ: 7.24 (d, J = 8.4 Hz, 1H), 6.76 (dd, J = 8.4, 2.4 Hz, 1H), 6.42 (d, J = 2.4 Hz, 1H), 6.19 (brs, 1H), 5.60 (d, J = 6.8 Hz, 1H), 4.44 (sep, J = 5.6 Hz, 1H), 3.74 (d, J = 8.4 Hz, 1H), 3.56 (dd, J = 8.4, 2.0 Hz, 1H), 3.01 (dd, J = 15.6, 3.6 Hz, 1H), 2.23 (d, J = 6.8 Hz, 1H), 2.19 (dt, J = 12.8, 3.6 Hz, 1H), 1.96 (s, 3H), 1,69–0.92 (m, 12H), 1.30 (d, J = 5.6 Hz, 3H), 1.27 (d, J = 5.6 Hz, 3H), 1.09 (s, 3H), 0.84 (s, 3H), 0.79 (s, 3H), 0.70 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ: 214.6, 159.2, 153.1, 131.6, 126.3, 115.8, 106.2, 83.0, 78.4, 69.7, 69.6, 62.2, 57.3, 57.1, 46.7, 43.2, 42.5, 41.9, 39.4, 39.0, 38.4, 36.8, 33.4, 33.3, 22.3, 22.0, 21.2, 19.9, 18.7, 18.1, 16.5; HRMS (EI) m/z: [M]+ Calcd for C31H44O3S2 528.2732. Found 528.2734.
(2aR,2bS,6aS,8aS,8a1R,10aS,14bR)-13-Isopropoxy-2b,6,6,14b-tetramethyl-2,2a,2b,3,4,5,6,6a,7,8,8a1,9,10a,14b-tetradecahydro-1H-naphtho[2′,1′:1,2]fluoreno[9,1-bc]furan (20)To a solution of 19 (25.2 mg, 0.0477 mmol) in benzene (950 µL) were added nBu3SnH (32 µL, 0.119 mmol) and AIBN (799 µg, 4.77 µmol). After stirring for 45 min at reflux, the reaction mixture was concentrated in vacuo. The residue was purified by flash column chromatography using a silica gel including 10% w/w K2CO3 (10 : 1 hexanes/EtOAc) to afford 20 (19.1 mg, 95%) as a colorless amorphous solid. Rf = 0.33 (hexanes/EtOAc = 8 : 1); [α]24D +39.1 (c 1.0, CHCl3); IR (film) 3446, 2928, 2862, 2090, 1637, 1480, 1258, 1213, 1120, 967 cm−1; 1H-NMR (400 MHz, CDCl3) δ: 7.26 (d, J = 8.4 Hz, 1H), 6.76 (dd, J = 8.4, 2.4 Hz, 1H), 6.58 (d, J = 2.4 Hz, 1H), 5.44 (d, J = 6.8 Hz, 1H), 4.52 (sep, J = 6.4 Hz, 1H), 3.59 (d, J = 9.2 Hz, 1H), 3.14 (dd, J = 9.2, 2.0 Hz, 1H), 2.19 (ddd, J = 13.2, 5.2, 3.6 Hz, 1H), 2.12 (d, J = 6.8 Hz, 1H), 2.06 (dt, J = 13.2, 3.6 Hz, 1H), 1.64–0.88 (m, 14H), 1.31 (d, J = 6.4 Hz, 6H), 1.11 (s, 3H), 0.84 (s, 3H), 0.78 (s, 3H), 0.58 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ: 158.8, 153.9, 133.2, 126.2, 114.9, 108.5, 83.0, 69.8, 68.9, 63.9, 56.7, 55.4, 47.5, 45.5, 42.2, 41.6, 39.2, 38.2, 37.9, 34.7, 33.4, 33.3, 22.1, 21.2, 19.6, 18.8, 17.4, 15.4; HRMS (EI) m/z: [M]+ Calcd for C29H42O2 422.3185. Found 422.3191.
((2aS,6aS,6bR,8aR,13aR,13bS)-10-Isopropoxy-3,3,6a,8a-tetramethyl-2,2a,3,4,5,6,6a,6b,7,8,8a,13,13a,13b-tetradecahydro-1H-indeno[2,1-a]henanthrene-13b-yl) Methanol (21)A suspension of 20 (53.3 mg, 0.126 mmol), 10% Pd/C (5.3 mg), and 60% HClO4 (420 µL, 0.300 M) in a 1 : 1 mixture of THF and EtOH (7.6 mL) was vigorously stirred under a H2 atmosphere at room temperature for 20 min. The reaction mixture was quenched saturated aqueous NaHCO3 and filtered through a pad of celite. The filtrate was concentrated in vacuo. The residue was purified by flash silica gel column chromatography (10 : 1 hexanes/EtOAc) to afford 21 (40.7 mg, 76%) as a colorless amorphous solid. Rf = 0.61 (hexanes/EtOAc = 5 : 1); [α]24D −52.45 (c 1.0, CHCl3); IR (film) 3454, 2929, 2857, 1634, 1472, 1377, 1120, 1031, 969 cm−1; 1H-NMR (400 MHz, CDCl3) δ: 7.10 (d, J = 8.4 Hz, 1H), 6.68–6.65 (m, 2H), 4.51 (sep, J = 5.8 Hz, 1H), 3.44 (dd, J = 13.0, 3.9 Hz), 3.18 (dd, J = 13.0, 6.5 Hz, 1H), 2.95 (d, J = 3.8 Hz, 2H), 2.32 (dt, J = 13.4, 4.0 Hz, 1H), 2.23 (dd, J = 13.4, 3.3 Hz, 1H), 1.79 (t, J = 3.8 Hz, 1H), 1.75–0.86 (m, 14H), 1.32 (d, J = 5.8 Hz, 6H), 1.10 (s, 3H), 0.86 (s, 3H), 0.82 (s, 3H), 0.79 (s, 3H), 0.41 (brt, J = 6.5 Hz, 1H); 13C-NMR (100 MHz, CDCl3) δ: 157.3, 153.7, 134.4, 124.9, 113.3, 109.5, 70.0, 63.6, 62.7, 58.4, 57.0, 46.4, 42.1, 40.6, 40.2, 37.5, 37.2, 35.0, 33.4, 33.3, 33.0, 30.9, 22.1, 21.5, 18.8, 18.7, 17.7, 16.4; HRMS (EI) m/z: [M]+ Calcd for C29H44O2 424.3341. Found 424.3324.
(2aS,6aS,6bR,8aR,13aR,13bS)-13b-(hydroxymethyl)-3,3,6a,8a-tetramethyl-2,2a,3,4,5,6,6a,6b,7,8,8a,13,13a,13b-tetradecahydro-1H-indeno[2,1-a]henanthrene-10-ol (10)To a solution of 21 (15.0 mg, 0.0354 mmol) in CH2Cl2 (354 µL) at −78 °C was added dropwise BCl3 (1.0 M sol. In CH2Cl2, 177 µL, 0.177 mmol). The reaction mixture was warmed to room temperature over 30 min and quenched with saturated aqueous NaHCO3 at 0 °C. The aqueous layer was separated and extracted with CH2Cl2. The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by flash silica gel column chromatography (100 : 1 hexanes/EtOAc) to afford 10 (13.4 mg, 99%) as a colorless amorphous solid. Rf = 0.47 (hexanes/EtOAc = 5 : 1); [α]24D −49.09 (c 1.0, CHCl3); IR (film) 3429, 2928, 2855, 1622, 1462, 1363, 1218, 1036, 937, 859, 810 cm−1; 1H-NMR (400 MHz, CDCl3) δ: 7.07 (d, J = 7.9 Hz, 1H), 6.60 (d, J = 2.4 Hz, 1H), 6.53 (dd, J = 7.9, 2.4 Hz, 1H), 5.66 (s, 1H), 3.45 (d, J = 12.8 Hz, 1H), 3.28 (d, J = 12.8 Hz, 1H), 2.94 (d, J = 4.4 Hz, 2H), 2.31 (dt, J = 12.5, 3.4 Hz, 1H), 2.15 (dt, J = 12.5, 3.4 Hz, 1H), 1.80 (d, J = 4.4 Hz, 1H), 1.74 (d, J = 12.4 Hz, 1H), 1.68–0.78 (m, 13H), 1.10 (s, 3H), 0.86 (s, 6H), 0.78 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ: 155.1, 153.8, 134.3, 125.2, 113.6, 108.5, 63.6, 63.1, 58.4, 57.0, 46.4, 42.1, 40.6, 40.2, 37.7, 37.4, 35.0, 33.3, 33.0, 30.9, 21.5, 18.8, 18.6, 17.8, 16.4; HRMS (EI) m/z: [M]+ Calcd for C26H38O2 382.2872. Found 382.2872.
(2aS,6aS,6bR,8aR,13aS,13bR)-10-hydroxy-3,3,6a,8a,13b-pentamethyl-1,2,2a,3,4,5,6,6a,6b,8,8a,13,13a,13b-tetradecahydro-7H-indeno[2,1-a]henanthrene-7-one (23)A suspension of 6 (20.6 mg, 0.054 mmol) and 10% Pd/C (4.1 mg) in EtOH (544 µL) was vigorously stirred for 1 h under an H2 atmosphere at room temperature. The resulting mixture was filtered through a pad of Celite and the filtrate was concentrated in vacuo. The residue was purified by flash silica gel column chromatography (30 : 1 hexanes/EtOAc) to afford 23 (19.1 mg, 92%) as a colorless oil. The stereochemistries were confirmed by ROESY and NOE experiments. Rf = 0.23 (hexanes/EtOAc = 3 : 1); [α]25D −200.04 (c 0.1, CHCl3); IR (film) 3374, 2953, 2926, 1684, 1612, 1498, 1460, 1217, 756 cm−1; 1H-NMR (400 MHz, CD3OD) δ: 6.93 (d, J = 8.0 Hz, 1H), 6.57 (dd, J = 8.0, 2.3 Hz, 1H), 6.51 (d, J = 2.3 Hz, 1H), 2.98 (dd, J = 16.5, 8.6 Hz, 1H), 2.75 (dd, J = 16.5, 5.1 Hz, 1H), 2.70 (d, J = 12.0 Hz, 1H), 2.62 (dd, J = 12.0, 1.6 Hz, 1H), 2.03–1.94 (m, 2H), 1.76–0.87 (m, 9H), 1.38 (s, 3H), 1.32 (s, 3H), 1.00 (s, 3H), 0.92 (s, 3H), 0.85 (s, 3H) ; 13C-NMR (100 MHz, CD3OD) δ: 216.5, 157.9, 154.3, 132.9, 125.3, 115.2, 109.2, 70.3, 58.8, 53.0, 50.6, 47.9, 43.6, 43.4, 42.7, 39.0, 34.8, 34.3, 33.6, 33.2, 31.4, 28.4, 22.0, 19.7, 18.9, 18.6; HRMS (EI) m/z: [M]+ Calcd for C26H36O2 380.2715. Found 380.2718.
BiologyEvaluation Methods of the Biological ActivitiesCell Culture and Cell Cycle Analysis of Jurkat CellsJurkat cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% Fetal Bovine Serum (FBS), 100 units/mL−1 penicillin, and 100 µg/mL−1 streptomycin at 37 °C in a humidified atmosphere of 5% CO2. Jurkat cells (1.0 × 105 cells in 200 µL) prepared in a 96-well microplate were treated with samples at 37 °C for 24 h. Cells were then suspended in 200 µL of the cell cycle reagent kit. The cell cycle status was assessed by analyzing the DNA content using the MUSE Cell Analyzer.
Cell Viability AssayWe evaluated the effect that compounds had on the cell viability of Jurkat cells using WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulfonate) reagent. Jurkat cells (1.0 × 105 cells in 200 µL) in a 96-well microplate were treated with samples in the presence or absence of bleomycin (5.0 µM) at 37 °C for 20 h. After incubation, cells were treated with 20 µL of the Water Soluble Tetrazolium Salts-1 (WST-1) reagent and then incubated at 37 °C for 30 min. The absorbance at 480 nm of each well was measured using a microplate reader.
The inhibition of cell growth was defined as (absorbance-sample/absorbance-control) × 100. The IC50 value was defined as the sample concentration that inhibited cell growth by 50%.
This work was supported by a Grant-in-Aid for Scientific Research (C) and a Kitasato University Research Grant for Young Researchers (T.N.). We also thank Ms. N. Sato and Dr. K. Nagai (Kitasato University) for kindly measuring the NMR and MS spectra.
The authors declare no conflict of interest.
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