Isoquinoline Alkaloids from Neolitsea sericea Var. aurata
Journal of Natural Products, 2007, Vol. 70, No. 4 641
Experimental Section
Each loaded cartridge was then washed by acetonitrile (2 mL). To the
washing was added acetonitrile to a final volume of 5 mL for UV
quantitative analysis, measured at λmax 281 nm.
General Experimental Procedures. The physical data of the
alkaloids were obtained using the following instruments: CD
(MeOH): JASCO J-710 spectropolarimeter. Off-line NMR spectra:
Bruker Avance 400. HPLC-DAD (diode array detector)-SPE-NMR (600
MHz) carried out using an Agilent 1100 liquid chromatograph
(Waldbronn, Germany) equipped with a photodiode array detector
(Bruker DAD, Bruker, Rheinstetten, Germany), followed by a Prospekt
2 automated solid-phase extraction unit (Spark Holland, Emmen,
Holland), containing 96 HySphere resin GP cartridges (10 × 2 mm,
10-12 µm), which was connected to a 30 µL inverse NMR probe
equipped in a Bruker Avance 600 NMR spectrometer. ESIMS:
Finnigan Mat TSQ 7000 and Bruker Daltonics Esquire 2000. (+)-N-
Methyllaurotetanine, (-)-pallidine, and (+)-reticuline used for partial
synthesis were previously isolated by our group.8
Plant Material. Leaves of Neolitsea sericea (Blume) Koidz. var.
aurata were collected in July 1992 on Orchid Island, Taiwan, and were
authenticated by Professor Ih-Sheng Chen, School of Pharmacy,
Kaohsiung Medical University, Kaohsiung, Taiwan. A voucher speci-
men (PHNTU920701) was stored at the herbarium library of the School
of Pharmacy, National Taiwan University.
Extraction and Isolation. Powdered, dry leaves (6.80 kg) were
percolated at room temperature with 95% EtOH (20 L × 5) to give,
upon concentration under reduced pressure, an EtOH extract (1.33 kg).
The EtOH extract was triturated with 0.1 N HCl (1.0 L × 3), and the
acidic solution was extracted with CHCl3 (0.7 L × 7) to remove neutral
constituents (13.71 g), then adjusted to pH 9 with NH4OH, and filtered
to give a residue (42 g), in which 1 was found to be a major constituent.
The filtrate was extracted with CHCl3 (1.2 L × 3) to give the total
bases (11.05 g, fraction I). The aqueous layer, after being adjusted to
pH 3.0 with concentrated HCl in an ice bath, was passed through an
Amberlite XAD-2 column, eluted with H2O and MeOH, to give the
MeOH eluate (222 g).
A small portion of the total bases (100 mg, fraction I) was reacted
with ethyl formate (0.3 mL) in DMF (2 mL) at 90 °C under nitrogen
for 1 day.16 After cooling, the reaction mixture was partitioned between
1 N HCl (20 mL) and CH2Cl2 (20 mL × 3) to give a fraction that was
soluble in CH2Cl2 (fraction Ia, 36.5 mg). The aqueous layer was adjusted
to pH 9 with aqueous NH4OH and then extracted with CH2Cl2 to give
the tertiary base major fraction (fraction Ib, 17 mg).
A portion of the total bases (1.00 g) was partitioned between ethyl
ether (50 mL × 3) and 1 N aqueous NaOH (50 mL) to give the
nonphenolic alkaloids (138 mg). The aqueous layer was adjusted to
pH 9 with NH4Cl, then extracted with CHCl3 (50 mL × 3) to give the
phenolic bases (fraction II, 610 mg). The aqueous layer was passed
through an Amberlite XAD-2 column and eluted with H2O and MeOH.
The MeOH eluate was further passed through a Sephadex LH-20
column (MeOH-CHCl3, 1:1) to give an alkaloid-containing fraction
(fraction III, 64 mg).
HPLC Conditions Used in HPLC-SPE-NMR (600 MHz) and
Separation of Alkaloids 1-14. Delivery system: Eluent A acetonitrile
containing 0.1% TFA-d, eluent B H2O containing 0.1% TFA-d, eluent
A/eluent B 5:95 to 60:40 within 30 min for fraction Ib and 10:90 to
50:50 within 40 min17 for fraction III, both linear gradient elution; flow
rate: 0.8 mL/min; HPLC column: C18 column (for fraction Ib, a Merck
LiChroCART column, 250 mm × 4.6 mm, 5 µm; for fraction III, a
Phenomenex Prodigy ODS3 100A column, 250 × 4.6 mm, 5 µm);
amount injected: fraction Ib 183.3 µg/10 µL (MeOH), fraction III 112.5
µg/10 µL (MeOH); detection at 280 nm.
Fraction Ib was separated using the HPLC conditions described
above to give alkaloids 1-5 with retention times of 13.20 (1), 13.65
(2), 14.40 (3), 17.00 (4), and 17.28 min (5) (Figure S1, Supporting
Information). Fraction III gave alkaloids 6-14 with retention times of
12.73 (6), 15.35 (7), 16.03 (8), 17.16 (9), 17.97 (10), 18.27 (11), 19.32
(12), 22.55 (13), and 23.50 min (14) (Figure 2).
Comparison of Trapping Ability of Six Different SPE Cartridges
to Boldine. The HPLC conditions, including column, eluents A and
B, and detection, used in this study were the same as those used for
analysis of fraction Ib. The delivery system was eluent A/B 1:4 and
flow rate was 0.3 mL/min, and the makeup (H2O) flow rate was 1
mL/min. Boldine (25 µg/5 µL, MeOH) was injected into the column,
and each sample eluate was trapped by a SPE cartridge including CN,
C8, C8 (EC) (8 µm), C18, C18 (HD) (7 µm), and, resin GP (10-12 µm).
SPE-NMR Procedure. A makeup flow of pure water with a flow
rate of 2.4 mL/min and 2.0 mL/min for fractions I and II, respectively,
was added to the postcolumn eluent, and the mixed sample volume of
each peak was passed through a HySphere resin GP cartridge (10 × 2
mm). After the cartridges were dried by flushing with dry nitrogen,
the compound trapped in each cartridge was eluted with deuterated
acetonitrile into a 30 µL inverse NMR probe. The 1D 1H NMR spectrum
of each separated compound was recorded using a multiple solvent
suppression pulse program for residual protons and water signals in
the d-solvent. Shaped low-power rf pulse and CW decoupling on the
F2 channel for the decoupling of the 13C satellites were utilized. All
spectra were measured at 300 K, and the 1H chemical shift was
referenced to a residual signal of CD2HCN at δ 1.93. For each
measurement 256-1024 scans were accumulated into 16k data points
with a sweep width of 12 000 Hz. 2D NMR spectra were recorded by
using standard pulse programs (COSY and NOESY), and the correlation
maps consisted of 2048 × 256 data points per spectrum.
ESIMS and CD Procedure. Alkaloids 6-14, corresponding to
peaks 6-14 in Figure 2, were obtained by another HPLC run using
the same conditions as in HPLC-SPE-NMR. These alkaloids were
analyzed by ESIMS (Finnigan MAT TSQ 7000) and CD.
Effect of TFA on the Production of Artifacts. Fraction III at a
concentration of 1 mg/10 mL (MeOH) was analyzed directly by ESIMS
(Bruker Daltonics Esquire 2000) without HPLC separation, and the
result is shown in Figure S3, Supporting Information.
9S,17S-Pallidine NR-oxide (6): tR 14.83 min (Figure 2); CD (MeOH)
1
(Cotton effect, CE) 279 (-), 264 (-), 252 (-), 235 (-), 219 (+); H
NMR data, see Table 1; ESIMS m/z 344 [M + H]+ (calcd for C19H21-
NO5 + H).
1S,2S-Reticuline NR-oxide (8): tR 18.51 min (Figure 2); CD
(MeOH) (CE) 300 (+), 291 (+), 236 (+), 227 (+); 1H NMR data, see
Table 1; ESIMS m/z 346 [M + H]+ (calcd for C19H23NO5 + H).
1
6R,6aS-Boldine Nâ-oxide (9): tR 19.55 min (Figure 2); H NMR
data, see Table 1; ESIMS m/z 344 [M + H]+ (calcd for C19H21NO5 +
H).
1S,2R-Reticuline Nâ-oxide (10): tR 20.35 min (Figure 2); CD
(MeOH) (CE) 291 (+), 237 (+), 226 (+); 1H NMR data, see Table 1;
ESIMS m/z 346 [M + H]+ (calcd for C19H23NO5 + H).
1R,2R-Juziphine Nâ-oxide (12): tR 22.24 min (Figure 2); CD
1
(MeOH) (CE) 279 (-), 234 (-), 224 (-); H NMR data, see Table
S1, Supporting Information; ESIMS m/z 316 [M + H]+ (calcd for
C18H21NO4 + H).
6S,6aS-N-Methyllaurotetanine NR-oxide (13): tR 24.67 min (Figure
2); CD (MeOH) (CE) 317 (-), 300 (-), 281 (-), 246 (+), 221 (-);
1H NMR data, see Table 1; ESIMS m/z 358 [M + H]+ (calcd for C20H23-
NO5 + H).
6R,6aS-N-Methyllaurotetanine Nâ-oxide (14): tR 25.55 min (Figure
2); CD (MeOH) (CE) 316 (-), 291 (-), 280 (-1.35), 244 (+15.17),
1
220 (-8.07); H NMR data, see Table 1; ESIMS m/z 358 [M + H]+
(calcd for C20H23NO5 + H).
Preparation of 6S,6aS- and 6R,6aS-N-Methyllaurotetanine N-
Oxides (13 and 14), 1S,2S- and 1S,2R-Reticuline N-Oxide (8 and
10), and 9S,17S-Pallidine N-Oxide (6). (+)-N-Methyllaurotetanine (7.0
mg, 20.5 µmoL) was reacted with Ac2O-py (2:1, 0.3 mL) in a sealed
tube for 21 h at room temperature. Absolute EtOH (5 mL) was then
added, and the solution was stirred for 2 h. Evaporation of the reaction
mixture gave a residue that was partitioned between 2.5% aqueous NH4-
OH (5 mL) and CH2Cl2 (5 mL × 2). The combined CH2Cl2 layers
were dried (Na2SO4) and evaporated. To the residue dissolved in CH2-
Cl2 (2 mL) was added m-CPBA (2.7 mg, 16 µmoL), and the solution
was stirred for 1 h.11 NaHCO3 powder (ca. 10 mg) was then added,
and the mixture was filtered through cotton wool. The filtrate was
evaporated to dryness. The residue was dissolved in MeOH (3 mL)
and O-deacetylated by reaction with 25% aqueous NH4OH (1 mL) at
room temperature for 3 h. Evaporation of the reaction mixture gave a
residue that was further purified via a Sephadex LH-20 column (MeOH)
to give a mixture of 13 and 14 (4.1 mg, overall yield 55.9%) in a ratio
of 10:1, based on HPLC analysis using the conditions for fraction III.
Alkaloid 6 was obtained as the major product from pallidine (6a)8 by
the same procedure, in a yield of 40%. Alkaloids 8 and 10 were obtained
in an approximately 1:1 ratio (1.6 mg and 1.3 mg, 67%) using reticuline