Structural Reassignment of Hypurticin
Journal of Natural Products, 2009, Vol. 72, No. 4 707
were achieved within dihedral angle ranges of +60° to -60°. Molecular
potential energy of all structures was minimized to a rmsd gradient of
1 × 10-6 kcal/mol on the potential energy surface. An energy cutoff
of 10 kcal/mol was selected in order to have a wide window of
conformers in the Boltzmann distribution. All structures inside of the
cutoff value were geometrically optimized using the hybrid DFT method
B3LYP and basis set DGDZVP (B3LYP/DGDZVP). The optimized
structures were used to calculate the thermochemical parameters, and
the IR frequencies were estimated at 298 K and 1 atm. Magnetic
shielding tensors were calculated with the gauge invariant atomic orbital
method (GIAO), and 1H-1H vicinal coupling constants were obtained
from the B3LYP/DGDZVP optimized structures using the SpinSpin
option during the NMR job. All quantum mechanical, NMR, and optical
rotation calculations were carried out using the Gaussian 03 program42
on a Linux operating system in the KanBalam cluster from a Hewlett-
Packard HP CP 4000, which includes 1368 AMD Opteron processors
at 2.6 GHz and a RAM memory of 3 terabytes. Geometrical optimiza-
tions, frequencies, and coupling constant calculations for each stereo-
isomer required an approximate calculation time of 6450 h divided in
164 processors. Optical rotations required an additional calculation time
of 418 h divided in 38 processors.
Methyl 2,3,4,6-Tetra-O-p-toluenesulfonyl-r-D-glucose (10). This
compound was prepared from methyl R-D-glucopyranoside 9 as
described previously.34,43 Additional spectral data are as follows: ORD
[R]589 +41 (lit.43 +44.7), [R]578 +43, [R]546 +48, [R]436 +75, [R]365
+103 (c 0.80, CHCl3); IR (CHCl3) νmax 2900, 1604, 1378, 1195, 1182,
1101, 1045, 979, 817 cm-1; 1H NMR (300 MHz, CDCl3) δ 7.86-7.25
(16H, m, 4 OTs), 5.04 (1H, dd, J2,3 ) 9.8; J3,4 ) 8.9 Hz, H-3), 4.69
(1H, d, J1,2 ) 3.5 Hz, H-1), 4.51 (1H, dd, J3,4 ) 8.9; J4,5 ) 9.8 Hz,
H-4), 4.42 (1H, dd, J5,6a ) 2.1; J6a,6b ) 11.3 Hz, H-6a), 4.15 (1H, dd,
J1,2 ) 3.5; J2,3 ) 9.8 Hz, H-2), 4.03 (dd, J5,6b ) 6.6; J6a,6b ) 11.3 Hz,
H-6b), 3.93 (1H, ddd, J4,5 ) 9.8 Hz, J5,6a ) 2.1, J5,6b ) 6.6, H-5), 3.21
(3H, s, OMe), 2.46 (3H, s, OTs), 2.44 (6H, s, 2 OTs), 2.43 (3H, s,
OTs); 13C NMR (75.4 MHz, CDCl3) δ 145.7, 145.4, 145.0 (×2), 133.4,
132.8, 132.6, 132.3, 129.8 (×2), 129.8 (×2), 129.8 (×2), 129.6 (×2),
128.5 (×2), 128.4 (×2), 128.1 (×4), 96.4 (C-1), 75.2 (×2, C-2,3), 73.1
(C-4), 67.8 (C-6), 67.5 (C-5), 55.8 (OMe), 21.7 (OTs), 21.7 (×2 OTs),
21.7 (OTs); positive FAB-MS m/z 811 [M + H]+ (4), 779 [M - OMe]+
(2), 639 [M - CH3C6H4SO3]+ (2), 607 [M - CH3C6H4SO3 - OMe]+
(2), 481 (11), 281 (70), 155 (100); HRESI/APCIMS m/z 833.1029 (calcd
for C35H38O14S4+Na, 833.1042).
1236, 1216, 1206, 784, 780, 772, 754, 692 cm-1; 1H NMR (300 MHz,
CDCl3) see Table 1; 13C NMR (75.4 MHz, CDCl3) δ 170.3, 170.2,
170.2 (3CdO), 133.7 (×2), 133.5 (×2), 132.2 (×2), 129.2 (×2), 129.1
(×2), 128.3, 128.0, 72.1 (C-3′), 71.3 (C-5′), 70.2 (C-6′), 61.9 (C-2′),
30.7 (C-4′), 21.1 (OAc), 20.8 (OAc), 20.7 (OAc), 15.0 (C-7′); EIMS
m/z 476 [M]+ (4), 367 [M+ - C6H5S] (79), 307 (92), 265 (82), 205
(100), 177 (28), 147 (23), 123 (36), 95 (18); HRESI/APCIMS m/z
499.1236 (calcd for C24H28O6S2+Na, 499.1225).
Cytotoxicity Assays. Human laryngeal carcinoma (HEp-2), human
nasopharyngeal carcinoma (KB), and cervical cancer (HeLa) cells were
maintained in RMPI 1640 medium with 10% fetal bovine serum and
cultured at 37 °C in an atmosphere of 5% CO2 in air (100% humidity).
The cells at log phase of their growth cycle were treated in triplicate
at various concentrations of the test samples (0.16-20.0 µg/mL) and
incubated for 72 h at 37 °C in a humidified atmosphere of 5% CO2.
The cell concentrations were determined by the sulforhodamine B
method.45 Results were expressed as the dose that inhibits 50% control
growth after the incubation period (ED50).
Cell Cycle Evaluations. They were carried out according to standard
procedures.46 HeLa cells in logarithm phase were incubated for 48 h
with hyptolide (2) or spicigerolide (3) (IC50 × 10) in DMSO. Cells
were fixed with cold EtOH 70% (v/v), stained for 12 h at 4 °C with
triton X-100 (100 µL), trisodium citrate (100 mg), and RNase A (100
mg), and filtered through nylon mesh. The cell cycle analysis was
performed in a Becton Dickinson FACSCalibur flow cytometer. Data
analysis was carried out using the FlowJo program version 7.2.5.
Acknowledgment. Financial support (grant 45759-Q) was from
Consejo Nacional de Ciencia y Tecnolog´ıa. F.L.-V. received a
posdoctoral fellowship from CONACyT (grant 45861-Q). Geometry
optimizations and coupling constant calculations were performed in
the HP Cluster Platform 4000 (KanBalam) at Departamento de
Superco´mputo, Direccio´n General de Servicios de Co´mputo Acade´mico,
UNAM.
Supporting Information Available: Experimental and simulated
1H NMR spectra of 2 and tri-O-acetyl-3,6-dideoxy-D-glucose diphe-
nyldithioacetal (14). DFT B3LYP/DGDZVP free energies, population,
and comparison between DFT and experimental 1H-1H coupling
constants of stereoisomers 1b-1d. DFT B3LYP/DGDZVP optical
rotation of 1. DFT atomic Cartesian coordinates for the more relevant
conformers of 1 and 2. This material is available free of charge via the
Methyl 3,6-Dideoxy-r-D-ribo-hexapyranoside (11). Compound
1034,44 (5.0 g) was prepared as reported previously to afford 11 with
identical yields, Rf, and [R]D. The 1H NMR spectrum was in agreement
1
with the partially reported data. H NMR (300 MHz, CDCl3) δ 4.61
References and Notes
(1H, d, J1,2 ) 3.7 Hz, H-1), 3.72 (1H, ddd, J1,2 ) 3.7, J2,3e ) 4.6, J2,3a
) 11.4 Hz, H-2), 3.51 (1H, dq, J4,5 ) 9.2, J5,6 ) 6.2 Hz, H-5), 3.44
(3H, s, OMe), 3.30 (1H, ddd, J3e,4 ) 4.6, J3a,4 ) 11.6, J4,5 ) 9.2 Hz,
H-4), 2.20 (1H, dt, J2,3e ) J3e,4 ) 4.6, J3e,3a ) 11.6 Hz, H-3e), 1.64
(1H, q, J2,3a ) J3e,3a ) J3a,4 ) 11.6 Hz, H-3a), 1.26 (3H, d, J5,6 ) 6.2
Hz, Me-6); 13C NMR (75.4 MHz, CDCl3) δ 98.3 (C-1), 70.8 (C-4),
68.6 (C-5), 67.6 (C-2), 55.0 (MeO), 37.0 (C-3), 17.3 (C-6); EIMS m/z
162 [M]+ (1), 131 [M - OMe]+ (3), 118 (13), 74 (57), 58 (100), 43
(11).
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3,6-Dideoxy-D-ribo-hexose (paratose) (12). A solution of methyl
3,6-dideoxy-R-D-ribo-hexapyranoside 11 (20 mg) in 1 N HCl (5 mL)
was heated at 80 °C for 5 h. The solution was cooled, neutralized with
saturated NaHCO3 solution, evaporated to dryness, and purified by
column chromatography on silica gel. Elution with EtOAc gave a syrup
(14 mg, 76%), Rf ) 0.21 (6:1 CHCl3-MeOH), in agreement with
reported data for paratose (12).35,36
Tri-O-acetyl-3,6-dideoxy-D-glucose Diphenyldithioacetal (14). A
solution of paratose (12) (50 mg) and benzenethiol (0.15 mL) in 90%
TFA (5 mL) was heated at 55 °C for 1 h. The reaction mixture was
evaporated to dryness under an Ar flow, and the residue was purified
by column chromatography on silica gel. Elution with CH2Cl2/MeOH
(9:1) afforded diphenyldithioacetal 13 (60 mg), which was dissolved
in pyridine (1 mL) and treated with Ac2O (1 mL) at 4 °C. After 30
min, the reaction was quenched with ice and extracted with CH2Cl2.
The organic layer was washed with diluted HCl, NaHCO3-saturated
solution, and H2O, dried over NaSO4, filtered, and evaporated under
vacuum. The residue was purified by flash column chromatography
eluting with hexane/ethyl acetate (7:3) to yield 14 (33 mg, 40%) as a
colorless oil: ORD [R]589 -4, [R]578 -3, [R]546 -1, [R]436 +17, [R]365
+42 (c 0.20, CHCl3); IR (CHCl3) νmax 3032, 2928, 1736, 1440, 1372,
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