506
Vol. 59, No. 4
tered, and the solvents were removed in vacuo. The residue was purified by
167.8, 167.7, 165.7, 165.6, 165.5, 144.2, 144.0, 143.9, 143.8, 143.7, 143.6,
ODS chromatography (Cosmosil, OPN; MeOH : H2Oꢂ50 : 50) to afford 142.5, 142.4, 142.3, 133.0, 132.9, 132.8, 126.8, 126.7, 126.6, 124.0, 123.9,
compound 10 (320 mg, 56%) and 4,5-di-O-caffeoylquinic acid (11, 125 mg, 123.8, 122.8, 122.7, 122.6, 110.9, 78.7, 72.1, 69.6, 67.1, 38.8, 35.6, 28.5,
1
29%): H-NMR (CD3OD, 500 MHz) d: 7.63 (1H, d, Jꢂ15.9 Hz), 7.58 (1H, 28.3, 20.7, 20.6, 20.6, 20.5, 20.5, 20.4. IR (KBr) cmꢄ1: 3448, 2925, 1774,
d, Jꢂ15.9 Hz), 7.07 (1H, d, Jꢂ8.4 Hz), 7.05 (1H, d, Jꢂ8.4 Hz), 7.00 (1H, dd, 1718, 1654, 1637, 1207, 1112, 1014. UV lmax (EtOAc) nm (e): 277 (52500).
Jꢂ8.2, 2.1 Hz), 6.89 (1H, dd, Jꢂ8.2, 2.1 Hz), 6.82 (1H, d, Jꢂ8.1 Hz), 6.75
HR-ESI-MS (positive ion) m/z: 993.2419 (MꢅNa)ꢅ (Calcd for C49H46O21Na:
(1H, d, Jꢂ8.1 Hz), 6.28 (1H, d, Jꢂ15.9 Hz), 6.27 (1H, d, Jꢂ15.9 Hz), 5.70 993.2429). [a]D25 ꢄ219° (cꢂ1.0, EtOAc).
(1H, m), 5.03 (1H, dd, Jꢂ9.8, 3.5), 4.86 (1H, m), 4.54 (1H, d, Jꢂ12 Hz),
3,4,5-tri-O-Caffeoylquinic Acid (16) 3,4,5-tri-O-Acetylcaffeoyl-1,7-
4.45 (1H, m), 2.92 (1H, dd, Jꢂ12.9, 3.2 Hz), 2.64—2.56 (2H, m), 2.07 (1H, isopropyridene-quinide (15, 104 mg, 107 mmol) was dissolved in a mixture
dd, Jꢂ13.9, 11.3 Hz). 13C-NMR (CD3OD, 125 MHz) d: 174.0, 169.4, 169.2,
154.5, 150.6, 150.4, 148.4, 148.2, 147.7, 147.6, 128.6, 128.5, 124.1, 124.0,
117.3, 117.2, 116.2, 116.0, 115.9, 115.8, 96.5, 85.1, 78.5, 77.4, 70.6, 66.0,
42.0, 33.6. IR (KBr) cmꢄ1: 3448, 2931, 1700, 1637, 1608, 1282, 1162,
of THF (5 ml) and 2 M HCl (5 ml) at room temperature. The reaction mixture
was stirred at room temperature, and progress was monitored by TLC. After
11 d, the solution was extracted with EtOAc (50 mlꢃ2) and H2O (50 ml).
The combined organic phases were washed with brine, and dried over
1116. UV lmax (MeOH) nm (e): 218 (32100), 244 (23700), 298 (30700), MgSO4, filtered, and the solvents were removed in vacuo. The residue was
320 (35400). [a]D25 ꢄ138° (cꢂ1.0, MeOH).
purified by ODS chromatography (Cosmosil, OPN; MeOH : H2Oꢂ50 : 50) to
afford 3,4,5-tri-O-caffeoylquinic acid (16, 42 mg, 58%): H-NMR (CD3OD,
1
4,5-di-O-Caffeoylquinic Acid (11) To a solution compound 10 (234
mg, 0.33 mmol) in THF (3 ml) was added zinc powder (132 mg) and AcOH
(1.5 ml). The reaction mixture was stirred at room temperature for 26 h, and
the solution was filtered off zinc, and evaporated. The residue was extracted
with EtOAc (30 mlꢃ3) and H2O (30 ml). The combined organic phases were
dried over MgSO4, filtered and the solvents were removed in vacuo. The
residue was purified by ODS chromatography (Cosmosil, OPN; MeOH :
H2Oꢂ30 : 70) to afford 4,5-di-O-caffeoylquinic acid (11, 122 mg, 73%): 1H-
NMR (CD3OD, 500 MHz) d: 7.61 (1H, d, Jꢂ15.8 Hz), 7.58 (1H, d, Jꢂ
500 MHz) d: 7.64 (1H, d, Jꢂ15.8 Hz), 7.58 (1H, d, Jꢂ15.7 Hz), 7.56 (1H, d,
Jꢂ15.7 Hz), 7.10 (1H, m), 7.06 (1H, m), 7.03 (1H, m), 6.99 (1H, m), 6.98
(2H, m), 6.81 (1H, d, Jꢂ8.3 Hz), 6.79 (1H, d, Jꢂ8.3 Hz), 6.75 (1H, d, Jꢂ8.3
Hz), 6.36 (1H, d, Jꢂ15.8 Hz), 6.27 (1H, d, Jꢂ15.8 Hz), 6.24 (1H, d,
Jꢂ15.8 Hz), 5.71 (2H, m), 5.36 (1H, dd, Jꢂ8.2, 3.0 Hz), 2.49—2.34 (4H,
m). 13C-NMR (CD3OD, 125 MHz) d: 178.1, 169.3, 169.1, 168.9, 150.5,
150.4, 150.4, 148.7, 148.5, 148.4, 147.6, 147.5, 147.2, 128.6, 128.5, 128.4,
117.3, 117.2, 117.1, 116.6, 116.0, 115.9, 115.8, 115.4, 115.1, 73.6, 73.5,
15.9 Hz), 7.07 (1H, d, Jꢂ10.0 Hz), 7.06 (1H, d, Jꢂ10.1 Hz), 6.97 (1H, dd, 70.8, 70.0, 39.5, 37.5. IR (KBr) cmꢄ1: 3407, 2921, 1718, 1700, 1685, 1654,
Jꢂ8.1, 1.9 Hz), 6.91 (1H, dd, Jꢂ8.1, 1.9 Hz), 6.81 (1H, d, Jꢂ8.1 Hz), 6.77
(1H, d, Jꢂ8.1 Hz), 6.34 (1H, d, Jꢂ15.8 Hz), 6.30 (1H, d, Jꢂ15.9 Hz), 5.68
(1H, m), 5.03 (1H, dd, Jꢂ8.9, 3.4 Hz), 4.41 (1H, td, Jꢂ9.4, 4.3 Hz), 2.39
(1H, dd, Jꢂ14.9, 3.7 Hz), 2.26 (1H, m), 2.19—2.10 (2H, m). 13C-NMR
(CD3OD, 125 MHz) d: 178.7, 169.4, 169.3, 150.4, 150.3, 148.2, 148.1,
147.6, 147.5, 128.6, 128.5, 124.0, 123.9, 117.3, 117.2, 116.0, 115.9, 115.8,
115.7, 77.3, 76.0, 70.9, 66.6, 42.7, 37.8. IR (KBr) cmꢄ1: 3448, 2923, 1718,
1700, 1654, 1637, 1282, 1182, 1116. UV lmax (MeOH) nm (e): 217
(20000), 244 (13900), 299 (18300), 326 (22100). HR-ESI-MS (positive ion)
m/z: 539.1163 (MꢅNa)ꢅ (Calcd for C25H24O12Na: 539.1165). [a]D25 ꢄ111°
(cꢂ1.0, MeOH).
1637, 1608, 1280, 1162, 1116. UV lmax (MeOH) nm (e): 218 (36800), 236
(27500), 244 (28600), 298 (34600), 323 (36900). HR-ESI-MS (positive ion)
m/z: 701.1503 (MꢅNa)ꢅ (Calcd for C34H30O15Na: 701.1482). [a]D25 ꢄ314°
(cꢂ1.0, MeOH).
Reduced Chlorogenic Acid (18) Chlorogenic acid (100 mg, 0.28 mmol)
was dissolved in EtOH (5 ml). Following to addition of Pd/C (20 mg), the re-
action mixture was stirred at room temperature under a H2 atmosphere.
After 6 h, Pd/C was removed with celite and the filtrate was evaporated to af-
1
ford reduced chlorogenic acid (100 mg, quant.) as a colorless oil: H-NMR
(CD3OD, 500 MHz) d: 6.71—6.69 (2H, m), 6.55 (1H, dd, Jꢂ8.0, 2.0 Hz),
5.29 (1H, ddd, Jꢂ9.7, 4.6 Hz), 4.15 (1H, m), 3.66 (1H, dd, Jꢂ9.0, 3.1 Hz),
2.81 (2H, br t, Jꢂ7.4 Hz), 2.63—2.60 (2H, m), 2.17—2.09 (2H, m), 2.03—
1.94 (2H, m). 13C-NMR (CD3OD, 125 MHz) d: 179.1, 175.3, 147.0, 145.4,
143.4, 121.3, 117.3, 117.2, 77.5, 74.8, 73.0, 72.7, 40.1, 39.3, 38.2, 32.2. IR
(KBr) cmꢄ1: 3430, 2930, 1718, 1685, 1654, 1637, 1399, 1273, 1115. UV
lmax (MeOH) nm (e): 208 (9100), 220 (5600), 283 (2700). HR-ESI-MS
(positive ion) m/z: 379.1001 (MꢅNa)ꢅ (Calcd for C16H20O9Na: 379.1005).
[a]D25 ꢄ53° (cꢂ1.0, MeOH).
3-O-Acetylcaffeoyl-1,7-isopropyridene-quinide (14) To
a solution
compound 13 (750 mg, 1.5 mmol) in THF (5 ml) and 80% MeOH (15 ml)
was added 0.4 M HCl (30 ml) at room temperature. The reaction mixture was
stirred at room temperature for 3.5 h. The solution was extracted with EtOAc
(100, 150 ml) and H2O (150 ml). The combined organic phases were washed
with brine and dried over MgSO4, filtered and the solvent were removed in
vacuo. The residue was purified by silica gel chromatography (CHCl3 :
MeOHꢂ98 : 2) to afford compound 14 (480 mg, 69%) as a colorless oil: 1H-
Cells and Cell Culture Human dopaminergic neuroblastoma SH-SY5Y
NMR (CDCl3, 500 MHz) d: 7.65 (1H, d, Jꢂ15.9 Hz), 7.39 (1H, dd, Jꢂ8.4, cell line was obtained from American Type Culture Collection (ATCC). Cul-
2.1 Hz), 7.35 (1H, d, Jꢂ2.1 Hz), 7.22 (1H, d, Jꢂ8.4 Hz), 6.40 (1H, d, ture were maintained in DMEM (Sigma, St. Louis. MO, U.S.A.) mediujm
Jꢂ15.9 Hz), 5.34 (1H, ddd, Jꢂ11.8, 9.8, 4.6 Hz), 4.24 (1H, dd, Jꢂ7.5, supplemented with 50% F-12 (GIBCO, Carlsbad, CA, U.S.A.), 15% fetal
3.4 Hz), 3.67 (1H, m), 2.87 (1H, m), 2.34 (1H, m), 2.31 (3H, s), 2.30 (3H, s),
bovine serum (FBS) and 1% penicillin (5000 U/ml)–streptomycin (5000
2.22 (1H, dd, Jꢂ15.3, 3.3 Hz), 2.12 (1H, dd, Jꢂ15.3, 3.3 Hz), 1.68 (3H, s), mg/ml) in 100 mm fibronectin coating dish (BD, Biocoat, Franklin Lakes,
1.67 (3H, s). 13C-NMR (CDCl3, 125 MHz) d: 172.6, 168.0, 167.9, 166.6,
NJ, U.S.A.). The cells were grown at 37 °C in 5% CO2.
Measurement of Intracellular ATP Content Intracellular ATP level
143.8, 143.6, 142.4, 133.0, 126.5, 124.0, 122.8, 118.7, 111.3, 79.8, 73.3,
70.1, 70.0, 38.0, 37.6, 28.6, 28.4, 20.6, 20.5. IR (KBr) cmꢄ1: 3448, 2923, was assessed by firefly bioluminescence using the luminescence luciferase
1774, 1718, 1706, 1654, 1637, 1209, 1182, 1112, 1016. UV lmax (MeOH)
assay kit (TOYO Ink, Tokyo, Japan). SH-SY5Y cells were cultured in 100 ml
nm (e): 218 (18100), 278 (22300). HR-ESI-MS (positive ion) m/z: 501.1424 test medium at a density of 2.0ꢃ104 cells/well in fibronectin coated 96 well
(MꢅNa)ꢅ (Calcd for C23H26O11Na: 501.1373). [a]D25 ꢄ7° (cꢂ1.0, MeOH).
3,4,5-tri-O-Acetylcaffeoyl-1,7-isopropyridene-quinide (15) To a solu-
tion of compound 14 (170 mg, 0.36 mmol) and DMAP (5 mg, 40 mmol) in (GIBCO). After 48 h, the medium was changed to 100 ml of OPTI-MEM
micro-plate (BD, Biocoat). After 24 h incubation at 37 °C, 5% CO2, cells
were treated with each compound dissolved 100 ml of OPTI-MEM
dry benzene (20 ml) was added dry pyridine (1 ml) at room temperature
under N2 atmosphere. Acid chloride (b, 410 mg, 1.4 mmol) was added and
the reaction mixture was refluxed for 24 h. The solution was cooled to room
temperature and quenched by the addition of 1 M HCl. The aqueous phase
was extracted with EtOAc (30, 50 ml). The organic layer was washed with
brine and dried over MgSO4, filtered, and the solvent were removed in
vacuo. The residue was purified by silica gel chromatography (hexane :
without test compounds. ATP content was measured according to the manu-
facturer’s protocol.
Statistical Analysis Statistical differences between means were as-
sessed for significance using the Student’s t-test. The value of pꢀ0.05 was
considered significant.
Acknowledgement The work described in this paper was partially
supported by Sasakawa Scientific Research Grant from The Japan Science
1
EtOAcꢂ2 : 3) to afford compound 15 (140 mg, 40%) as a colorless oil: H-
NMR (CDCl3, 500 MHz) d: 7.69 (1H, d, Jꢂ15.9 Hz), 7.60 (1H, d, Society.
Jꢂ15.9 Hz), 7.59 (1H, d, Jꢂ15.9 Hz), 7.46 (1H, dd, Jꢂ8.4, 2.0 Hz), 7.43
(1H, m), 7.37 (1H, dd, Jꢂ8.4, 2.0 Hz), 7.34 (1H, d, Jꢂ2.0 Hz), 7.31 (1H, m),
7.31 (1H, m), 7.25 (1H, m), 7.20 (1H, d, Jꢂ8.3 Hz), 7.17 (1H, d, Jꢂ8.3 Hz),
6.49 (1H, d, Jꢂ15.9 Hz), 6.33 (1H, d, Jꢂ15.9 Hz), 6.32 (1H, d, Jꢂ15.9 Hz),
5.80 (1H, m), 5.56 (1H, m), 5.28 (1H, dd, Jꢂ10.1, 3.2 Hz), 2.48—2.40 (2H,
m), 2.32 (3H, s), 2.31 (3H, s), 2.29 (3H, s), 2.28 (3H, s), 2.27 (3H, s), 2.26
(3H, s), 2.23—2.14 (2H, m), 1.64 (3H, s), 1.57 (3H, s), 1.56 (3H, s), 1.48
(3H, s). 13C-NMR (CDCl3, 125 MHz) d: 173.0, 168.1, 168.1, 168.0, 167.9,
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