Organic & Biomolecular Chemistry
Paper
the residue was washed with EtOAc. The combined filtrate and 2H), 3.13 (dd, J = 14.1, 6.1 Hz, 1H), 2.88 (dd, J = 14.1, 7.7 Hz,
washings was concentrated to give the crude product, which 1H), 2.59–2.51 (m, 3H), 2.36–2.28 (m, 2H), 2.27–2.20 (m, 1H),
was purified by column chromatography on Al2O3 (4 g, 2.04 (br m, 1H), 1.96 (br m, 1H), 1.90–1.80 (m, 2H), 1.75 (s,
n-hexane–Et2O 5 : 1 → 1 : 1) to give amine 18 (69 mg, 61% in 3H), 1.73 (s, 3H), 1.71 (s, 3H), 1.66 (s, 3H), 1.64 (s, 3H),
2 steps) as a yellow oil: Rf = 0.43 (CHCl3–MeOH 9 : 1); [α]2D5 +2.3 1.61–1.36 (m, 7H), 1.23–1.16 (m, 2H), 1.03 (ddd, J = 13.1, 13.1,
(c 0.50, CHCl3); IR (CHCl3) 3359, 3018, 2928, 2851, 1682, 1456, 3.3 Hz, 1H), 0.89 (s, 3H), 0.86 (s, 3H), 0.76 (s, 3H). A signal due
1418, 1366, 1252, 1167, 1137, 881 cm−1 1H NMR (600 MHz, to one proton (NH) was not observed; 13C NMR (150 MHz,
;
CDCl3) δ 5.42 (m, 1H), 5.12 (m, 1H), 3.85–3.68 (br m, 2H), CD3OD) δ 136.8, 136.0, 135.2, 123.1, 123.0 (2C), 55.0, 54.5,
3.20–3.05 (br m, 2H), 2.68 (dd, J = 12.2, 1.6 Hz, 1H), 2.61 (m, 53.1, 52.4, 51.7, 49.9, 47.6, 43.5, 40.6, 37.4, 34.0, 33.9, 28.5,
1H), 2.53 (m, 1H), 2.45 (dd, J = 12.2, 7.3 Hz, 1H), 2.00–1.36 (m, 26.1, 25.9, 25.8, 24.8, 22.9, 22.4, 19.9, 18.1, 18.0, 14.2; HRMS
11H), 1.71 (s, 3H), 1.70 (s, 3H), 1.64 (s, 3H), 1.43 (s, 9H), (ESI) m/z 429.4202, calcd for C29H53N2 [M + H]+ 429.4209.
1.20–1.14 (m, 2H), 1.06 (ddd, J = 13.1, 13.1, 3.7 Hz, 1H), 0.86
N1-(3-Methylbut-2-en-1-yl)-N4-(((1S,4aS,8aS)-2,5,5,8a-tetramethyl-
(s, 3H), 0.84 (s, 3H), 0.74 (s, 3H). A signal due to one proton 1,4,4a,5,6,7,8,8a-octahydronaphthalen-1-yl)methyl)butane-1,4-
(NH) was not observed; 13C NMR (150 MHz, CDCl3) δ 155.6, diamine (20). The amine 18 (9.4 mg, 20 μmol) was treated
134.7, 134.3, 122.9, 121.1, 79.1, 55.3, 50.0 (2C), 48.2, 46.1, 44.6, with 4.0 M HCl/MeOH (0.10 mL) at 0 °C. After being stirred at
42.2, 39.3, 36.2, 33.2, 32.9, 28.5 (3C), 27.4, 26.4, 25.7, 23.7, 21.9 room temperature for 2 h, the reaction mixture was concen-
(2C), 18.8, 17.8, 14.0; HRMS (ESI) m/z 461.4125, calcd for trated to afford diamine (20) HCl salt (8.1 mg, 92%) as a yellow
C29H53N2O2 [M + H]+ 461.4107.
solid: Rf = 0.21 (CHCl3–MeOH 9 : 1); [α]2D4 −5.5 (c 0.38, CHCl3);
tert-Butyl (3-methylbut-2-en-1-yl)(4-((3-methylbut-2-en-1-yl)- IR (CHCl3) 3378, 2964, 2849, 2771, 1588, 1457, 1386, 1241,
1
(((1S,4aS,8aS)-2,5,5,8a-tetramethyl-1,4,4a,5,6,7,8,8a-octahydro- 982 cm−1; H NMR (600 MHz, CD3OD) δ 5.59 (m, 1H), 5.33 (t,
naphthalen-1-yl)methyl)amino)butyl)carbamate (19). To
a
J = 7.2 Hz, 1H), 3.66 (d, J = 7.2 Hz, 2H), 3.24–3.02 (m, 6H),
stirred solution of amine 18 (33.0 mg, 71.6 μmol) in MeCN 2.17–1.43 (m, 11H), 1.83 (s, 3H), 1.82 (s, 3H), 1.79 (s, 3H),
(0.30 mL) were added i-Pr2NEt (12.6 μL, 72.3 μmol) and prenyl 1.32–1.16 (m, 3H), 0.92 (s, 3H), 0.89 (s, 3H), 0.83 (s, 3H).
bromide (20.0 μL, 172 μmol) at 0 °C. After being stirred at Signals due to two protons (NH) were not observed; 13C NMR
room temperature for 15 h, the reaction mixture was diluted (150 MHz, CDCl3) δ 144.1, 131.7, 126.2, 115.1, 53.8, 51.0, 49.8,
with H2O (5 mL) and extracted with EtOAc (5 mL × 3). The 47.9, 47.2, 46.4, 43.0, 40.0, 37.7, 33.9, 33.5, 26.0, 24.7, 24.5,
combined extracts were washed with brine (5 mL), dried over 24.0, 22.1, 21.8, 19.6, 18.3, 14.0; HRMS (ESI) m/z 361.3574,
Na2SO4, filtered, and concentrated. The crude product was calcd for C24H45N2 [M + H]+ 361.3583.
purified by column chromatography on Al2O3 (1.0 g, n-hexane–
Cell growth analysis
Et2O 10 : 1) to give N18-Boc halichonine B (19) (37.0 mg, 97%)
as a yellow oil: Rf = 0.75 (n-hexane–Et2O 2 : 1); [α]2D5 +41.6 HL60 cells were cultured at 37 °C with 5% CO2 in RPMI
(c 0.25, CHCl3); IR (CHCl3) 2927, 2859, 1681, 1455, 1418, 1378, (Nissui) supplemented with 10% heat-inactivated FBS (FBS;
1366, 1268, 1251, 1168, 1144, 880 cm−1 1H NMR (600 MHz, Cell Culture Bioscience), 100 units per mL penicillin, 100 μg
;
CDCl3) δ 5.38 (m, 1H), 5.22 (m, 1H), 5.14 (m, 1H), 3.86–3.70 (br mL−1 streptomycin, 0.25 μg mL−1 amphotericin B, 2 mM L-glu-
m, 2H), 3.20–3.05 (br m, 3H), 2.84 (dd, J = 14.1, 7.6 Hz, 1H), tamine, and 2.25 mg mL−1 NaHCO3. HeLa cells were cultured
2.50 (m, 1H), 2.33–2.24 (m, 2H), 2.20 (m, 1H), 2.04–1.91 (br m, at 37 °C with 5% CO2 in DMEM (Nissui) supplemented with
2H), 1.89–1.31 (m, 9H), 1.75 (s, 3H), 1.71 (s, 6H), 1.65 (s, 3H), 10% heat-inactivated fetal bovine serum, 100 units per mL
1.61 (s, 3H), 1.44 (s, 9H), 1.23–1.13 (m, 2H), 0.99 (ddd, J = 13.1, penicillin, 100 μg mL−1 streptomycin, 0.25 μg mL−1 amphoteri-
13.1, 3.3 Hz, 1H), 0.87 (s, 3H), 0.85 (s, 3H), 0.73 (s, 3H); 13C cin B, 2 mM L-glutamine, and 2.25 mg mL−1 NaHCO3. HL60
NMR (150 MHz, CDCl3) δ 155.6, 136.1, 134.7, 133.8, 122.1, cells were seeded at 1 × 104 cells per well in 96-well plates
121.9, 121.2, 79.0, 53.7, 53.3, 51.5, 51.2, 50.2, 46.3, 44.6, 42.3, (Iwaki). HeLa cells were seeded at 4 × 103 cells per well in
39.2, 36.1, 33.3, 33.0, 28.5 (3C), 26.8, 25.9, 25.7, 24.3, 23.7, 96-well plates and cultured overnight. Various concentrations
22.4, 22.0, 18.8, 17.9, 17.8, 13.7; HRMS (ESI) m/z 529.4739, of compounds were then added, and cells were incubated for
calcd for C34H61N2O2 [M + H]+ 529.4733.
72 h. Cell proliferation was measured by using the MTT assay.
Halichonine B (2)
Analysis of DNA fragmentation
The N18-Boc halichonine B (19) (31.2 mg, 59.0 μmol) was HL60 cells, treated with compounds for 24 h, were washed
treated with 4.0 M HCl/MeOH (0.10 mL) at 0 °C. After being with phosphate-buffered saline (PBS; 8 g L−1 NaCl, 200 mg L−1
stirred at room temperature for 2 h, the reaction mixture was KCl, 1.15 g L−1 Na2HPO4·2H2O, 200 mg L−1 KH2PO4). The cells
concentrated to afford halichonine B (2) HCl salt. The halicho- were then resuspended in lysis buffer (10 mM Tris-HCl
nine B (2) HCl salt was purified by column chromatography on [pH 7.4], 10 mM EDTA, 0.5% Triton X-100) at 4 °C for 10 min.
Al2O3 (0.7 g, CHCl3–MeOH 10 : 1) to give halichonine B (2) free After centrifugation at 17 700g at 4 °C for 5 min, the super-
amine (20.3 mg, 74%) as a yellow oil: Rf = 0.34 (CHCl3–MeOH natant was treated with 0.2 mg mL−1 RNase A at 37 °C for 1 h.
9 : 1); [α]2D5 +68.0 (c 0.16, CHCl3); IR (CHCl3) 3367, 2964, 2851, The samples were treated with 0.2 mg mL−1 proteinase K at
1675, 1459, 1367, 1241, 1051, 983 cm−1
;
1H NMR (600 MHz, 50 °C for 30 min, and to the lysates were added 5 M NaCl
CD3OD) δ 5.36 (m, 1H), 5.28–5.20 (m, 2H), 3.18 (d, J = 7.0 Hz, (0.5 M of total) and isopropyl alcohol (50% of total). After the
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