Z.-G. Li et al. / Chinese Chemical Letters 26 (2015) 267–271
269
the HCO2H-triethylamine mixture (5:2, 18
m
L). Then, the reaction
Dalian), 1
(CPT) as positive control. The reaction mixture was incubated at
37 8C for 15 min and terminated by the addition of 2
loading buffer (0.9% sodium dodecyl sulfate (SDS), 0.05% bromo-
phenol blue, and 50% glycerol). Then the DNA samples were
subjected to electrophoresis in 0.8% agarose gel in 1ꢀ TAE (Tris–
acetate–EDTA) at 8 V/cm for 1 h. Gels were stained with ethidium
mL of different compounds solution or the Top1 inhibitor
mixture was stirred at 0 8C for 8 h, diluted with water (15 mL), and
extracted with EtOAc (3 ꢀ 15 mL). The organic layer was dried over
Na2SO4 and the solvent was evaporated in vacuo. The residue was
purified by column chromatography (hexane:EtOAc = 4:1) to give
compound (S)-2 as a white solid (35 mg, yield 78%). 1H NMR
m
L of 10ꢀ
(DMSO-d6, 500 MHz):
d 2.65 (s, 3H), 2.83–2.84 (m, 2H), 3.16–3.21
(m, 1H), 4.59–4.62 (m, 1H), 6.07 (s, 1H), 7.00 (t, 1H, J = 7.7 Hz), 7.10
(t, 1H, J = 8.3 Hz), 7.18 (dd, 1H, J = 8.8 Hz, 4.4 Hz), 7.73–7.38 (m,
2H), 7.48 (d, 1H, J = 7.7 Hz), 7.52 (dd, 1H, J = 8.8 Hz, 3.3 Hz), 11.15
bromide (0.5 mg/mL) for 60 min. The DNA band was visualized
over UV light and photographed with Gel Doc Ez imager (Bio-Rad
Laboratories Ltd.).
(s, 1H). 13C NMR (DMSO-d6, 75 MHz):
d 19.6, 36.6, 69.2, 111.6,
113.2, 113.5, 118.3, 118.9, 120.4, 120.7, 122.0, 125.8, 129.6, 136.7,
2.5. Top2-mediated supercoiled pBR322 relaxation assay
146.0, 155.9, 158.7, 163.0. MS (ESI, positive) m/z calcd. for
C
19H17FN3O (M+H): 322.36; found: 322.59. HPLC (Daicel Chiralpak
AD, 0.46 cm I.D. ꢀ 25 cm L ꢀ5 m, 25 8C, i-propanol/hexane = 30/
70, flow rate = 0.8 mL/min, = 254 nm): tmajor = 8.72 min, tmi-
Relaxation assays were performed using Topoisomerase II Drug
m
l
Screening Kit (TopoGEN, Inc.) [7]. The 20
contained 50 mmol/L Tris–HCl (pH 8.0), 150 mmol/L NaCl,
10 mmol/L MgCl2, 5 mmol/L dithiothreitol, 30 g/mL bovine serum
albumin (BSA), 2 mmol/L ATP, 0.25 g pBR322 plasmid DNA,
0.75 unit of Top2 (TopoGEN, Inc.), and 1 L of different compounds
mL final reaction mixture
nor = 6.51 min, ee = 99%. a2D2 453.8 (c 1.45, MeOH).
The synthetic method for target compounds (R)-2, (S)-12, (R)-12
was similar to that of compound (S)-2.
m
m
m
(R)-3-Fluoroevodiamine ((R)-2): White solid: 38 mg (yield: 84%).
solution or the Top2 inhibitor (Eto) as positive control. The reaction
HPLC (Daicel Chiralpak AD, 0.46 cm I.D. ꢀ 25 cm L ꢀ5
m
l
m, 25 8C, i-
= 254 nm):
ꢂ 500:7 (c
mixture was incubated at 37 8C for 30 min and then terminated by
propanol/hexane = 30/70, flow rate = 0.8 mL/min,
the addition of 2
blue, 50% glycerol). The samples were analyzed on 1% agarose gel at
m
L 10ꢀ gel loading buffer (0.25% bromophenol
tmajor = 6.29 min, tminor = 8.34 min, ee = 92%.
1.45 mg/mL, MeOH).
½
a 2D2
ꢁ
8 V/cm for 1 h with 1ꢀ TAE (Tris–acetate–EDTA) as the running
(S)-10-Benzyloxyevodiamine ((S)-12): Yellow solid: 40 mg
(yield: 87%). 1H NMR (CDCl3, 500 MHz)
: 2.51 (s, 3H), 2.90–
buffer. Gels were stained with ethidium bromide (0.5 mg/mL) for
d
60 min. The DNA band was visualized over UV light and
photographed with Gel Doc Ez imager (Bio-Rad Laboratories Ltd.).
2.93 (m, 2H), 3.25–3.30 (m, 1H), 4.84–4.89 (m, 1H), 5.12 (s, 2H),
5.89 (s, 1H), 6.98 (dd, 1H, J = 9.0 Hz, 2.4 Hz), 7.10 (s, 1H), 7.14 (d,
1H, J = 8.4 Hz), 7.20 (t, 1H, J = 7.5 Hz), 7.30–7.51 (m, 7H), 8.12 (dd,
1H, J = 7.2 Hz, 1.5 Hz), 8.19 (s, 1H). MS (ESI, negative) m/z calcd. for
2.6. In vitro cytotoxicity assay
C
26H22N3O2 (M-H): 408.47; found: 408.59.
Cells were seeded in 96-well microtiter plates at a density of
5 ꢀ 103/well in a humidified atmosphere with 5% CO2 at 37 8C for
24 h. All compounds to be tested were dissolved in DMSO at a
concentration of 10 mmol/L. Cells were treated in triplicate with
gradient concentrations of compounds at 37 8C for 72 h, with a 5%
2.3. Synthesis of (S)-10-hydroxyevodiamine ((S)-3)
A solution of (S)-12 (30 mg, 0.07 mmol) and 10% Pd/C (3 mg) in
EtOAc (10 mL) was stirred under hydrogen atmosphere at room
temperature for 12 h. The mixture was filtered, and then the
solvent was evaporated under reduced pressure. The residue was
purified by column chromatography (Hexane: EtOAc = 3:1) to give
compound (S)-3 as a yellow solid (20 mg, yield 83%). 1H NMR
CO2 environment. Then, 20
mL of MTT (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide) solution (5 mg/mL) was
added to each well. After incubated for an additional 4 h, the
formazan was dissolved in 100
mL of DMSO. The absorbance (OD)
was read on a WellscanMK-2 microplate reader (Labsystems) at
570 nm. The concentration causing 50% inhibition of cell growth
(IC50) was determined by the Logit method [11,12]. All experi-
ments were performed three times.
(DMSO-d6, 500 MHz): d 2.65–2.68 (m, 1H), 2.80–2.89 (m, 1H), 2.89
(s, 3H), 3.15–3.19 (m, 1H), 4.58–4.62 (m, 1H), 6.08 (s, 1H), 6.61–
6.62 (d, 1H, J = 8.5 Hz), 6.75 (s, 1H), 6.94 (t, 1H, J = 7.5 Hz), 7.02 (d,
1H, J = 8.2 Hz), 7.14 (d, 1H, J = 8.5 Hz), 7.46 (t, 1H, J = 7.5 Hz), 7.78
(d, 1H, J = 7.8 Hz), 8.68 (s, 1H), 10.70 (s, 1H). 13C NMR (DMSO-d6,
2.7. Molecular docking
75 MHz):
d 20.0, 31.1, 36.8, 70.4, 102.6, 111.5, 112.5, 112.5, 117.5,
119.5, 120.5, 127.1, 128.4, 131.4, 131.6, 133.9, 149.1, 151.1,
164.7. MS (ESI, positive) m/z calcd. for C19H18N3O2 (M+H): 320.37;
The X-ray crystallographic structures of CPT–DNA–Top1
ternary complex (PDB code: 1T8I [13]) and ATPase domain of
found: 320.14. HPLC (Chiralpak AD, 0.46 cm I.D. ꢀ 25 cm L ꢀ5
m
m,
human Top2a (PDB code: 1ZXM [14]) were downloaded from the
25 8C, i-propanol/hexane = 30/70, flow rate 0.6 mL/min,
protein data bank and prepared for docking using protein prepare
protocol in Discovery Studio 3.0 [15]. During this process, the
ligand was removed from the binding pocket, and polar hydrogens
were added. All the waters were deleted and each atom was
rendered with Gasteiger charges. In silico docking was carried out
using GOLD 5.1 [16] on a Linux PC as described before [6].
l
= 254 nm): tmajor = 4.399 min, ee > 99%. ½a D22
ꢁ
316.1 (c 2.5, MeOH)
The synthetic method for target compounds (R)-3 was similar to
that of compound (S)-3.
(R)-10-Hydroxyevodiamine ((R)-3): Yellow solid: 21 mg (yield:
87%). HPLC (Chiralpak AD, 0.46 cm I.D. ꢀ25 cm L ꢀ5
m
m, 25 8C,
= 254 nm):
ꢂ283.0 (c 2.5,
i-propanol/hexane = 30/70, flow rate 0.6 mL/min,
l
tmajor = 6.605 min, tminor = 4.459 min, ee = 91%. ½a D22
ꢁ
3. Results and discussion
MeOH).
3.1. In vitro antitumor activity
2.4. Top1-mediated supercoiled pBR322 relaxation assay
The growth inhibitory activities toward human cancer cell-lines
A549 (lung cancer), HCT116 (colon cancer), ZR-75-30 (human
breast cancer), CNE (human thyroid carcinoma) and KYSE-150
(human esophageal squamous cell carcinoma) were determined
using MTT assay. As shown in Table 1, all the four isomers showed
good to excellent antitumor activity against the five tested cancer
Relaxation assays were carried out as described [7]. The 20
final reaction buffer contained 35 mmol/L Tris–HCl (pH 8.0),
72 mmol/L KCl, 5 mmol/L MgCl2, 5 mmol/L dithiothreitol, 5 mmol/
mL
L spermidine, 0.1% bovine serum albumin (BSA), 0.25
mg pBR322
plasmid DNA and 1 unit of Top1 (TaKaRa Biotechnology Co., Ltd.,