Journal of Natural Products
Article
(
500.1 MHz) and 13C (125.6 MHz) NMR spectra were recorded at
raphy (Armen Spot CPC 250 mL, Armen Instrument, Saint Ave,
room temperature on a Bruker 500 Avance III NMR spectrometer
equipped with cryogenic probe head and on a Bruker Avance 500
NMR spectrometer. Amounts of approximately 0.5−5 mg of
France), in ascending mode, with a solvent system of EtOAc−
MeOH−H O (20:20:1, v/v/v), and 20 mL fractions were collected.
2
The combined CPC fractions were evaporated at 40 °C, dissolved in
20% aqueous acetonitrile, and investigated by HPLC on a gradient
system (acetonitrile−water) on a Kinetex XB-C18 column (5 μm, 4.6 ×
250 mm) at a flow rate of 1 mL/min. Further purification of the
combined CPC fractions by HPLC was scheduled 1 week later, but by
that time the chief constituents of two of the fractions were completely
converted to calonysterone (5).
compounds 1−7 were dissolved in 0.1 mL of methanol-d and
4
transferred to a 2.5 mm Bruker MATCH NMR sample tube. Chemical
shifts are given on the δ scale and are referenced to the solvent
(
methanol-d : δ = 49.1 and δ = 3.31 ppm). Pulse programs of all
4 C H
1
13
experiments ( H, C, DEPTQ, DEPT-135, sel-TOCSY (mixing time:
8
1
1
0−120 ms), sel-ROE (300 ms), gradient-selected (gs) H, H-COSY,
ROESY, edited gs-HSQC, gs-HMBC (optimized for 10 Hz)) were
taken from the Bruker software library. For 1D measurements, 64K
data points were used to yield the FID. For 2D measurements, the
sweep width in F was 4000 Hz; all data points (t × t ) were acquired
Reaction 3. A 120 mg aliquot of 20E was dissolved, and 100 mg of
NaOH was added as described above. The reaction mixture was stirred
for 8 h at room temperature, and the reaction was stopped by
neutralizing the pH as above. The reaction mixture was purified over
HPLC (35% methanol(aq), 3 mL/min) using a Zorbax XDB-C8
column (5 μm, 9.4 × 250 mm) to yield compound 6 (10.0 mg).
Following this, 120 mg of 20E was reacted with NaOH using the same
reaction setup as above, with the reaction mixture stirred for 15 h at
room temperature and the reaction stopped by neutralizing the pH as
above. The reaction mixture was fractionated by centrifugal partition
chromatography the same way as described above for reaction 2.
Fraction 4 was further purified over HPLC (30% acetonitrile(aq), 3
2
2
1
with 2K × 256. For F , linear prediction was applied to enhance the
1
1
resolution. Most H assignments were accomplished using general
knowledge of chemical shift dispersion with the aid of the proton−
1
proton coupling pattern ( H NMR spectra). The NMR signals of the
products were assigned by one- and two-dimensional NMR methods
12,13
using widely accepted strategies.
Atmospheric pressure ionization
(
APCI) MS spectra were recorded on an API 2000 triple quadrupole
tandem mass spectrometer (AB SCIEX, Foster City, CA, USA), used
in the positive-ion mode. HRMS data were recorded on a Waters-
Micromass Q-TOF Premier mass spectrometer (Waters, Milford, MA,
USA) equipped with an electrospray ion source, which was used in the
positive-ion mode. HPLC was performed on a gradient system of two
JASCO PU2080 pumps connected to a JASCO MD-2010 Plus
photodiode-array detector.
mL/min) using a Zorbax XDB-C column (5 μm, 9.4 × 250 mm) to
8
yield compound 7 (4.4 mg). It should be noted that these yields do
not correspond to the total amounts available from this reaction setup;
in order to facilitate the work, HPLC purification was performed at
once until sufficient amounts were obtained for structure elucidation
and yield optimization for subsequent reactions (see below,
longitudinal study of the reaction).
Starting Material. 20-Hydroxyecdysone isolated from the roots of
Cyanotis arachnoidea was purchased from Shaanxi KingSci Biotechnol-
ogy Co., Ltd. (Shanghai, People’s Republic of China) at 90% purity
and recrystallized from ethyl acetate−methanol (2:1, v/v) to reach a
purity of 97.8% by means of HPLC-DAD.
(5α)-9α,20-Dihydroxyecdysone (2): white crystals; mp 273−275
2
5
°C; [α]
D
11α-Hydroxycalonysterone (4): amorphous, transparent solid;
experimental data are limited due to the very low yield obtained and
Reaction 1. A 1.70 g aliquot of 20E was dissolved in 50 mL of 70%
MeOH(aq) (Merck), and 1.0 g of NaOH was added. The mixture was
stirred for 3 h at room temperature, and the reaction was stopped by
neutralizing the pH with concentrated acetic acid. The solvent was
evaporated under reduced pressure at 40 °C. The dry residue was
subjected to column chromatography (CC) over silica gel 60 (63−200
μm), eluting with ethyl acetate (22 fractions) and EtOAc−EtOH−
2
5
Calonysterone (5): white crystals; mp 234−235 °C; [α] +26.0 (c
D
+
+
0.2, MeOH); APCIMS m/z 477 [M + H] , 459 [M + H − H O] , 441
2
+
+
+
[M + H − 2H O] , 423 [M + H − 3H O] , 405 [M + H − 4H O] ;
2
2
H O (80:2:1, v/v/v) (77 fractions). Fractions 48−70 and 71−99 were
2
combined and evaporated to obtain dry residues of 0.24 g (A1) and
14,15-Dihydro-14α-hydroxycalonysterone (6): yellow crystals; mp
2
7
0
.13 g (A2), respectively. Sample A1 was subjected to reversed-phase
CC on Lichroprep RP-18 (40−63 μm) using a stepwise gradient of
0%, 35%, 40%, 45%, and 50% aqueous methanol (seven fractions
145−147 °C; [α] +12.0 (c 0.3, MeOH); HRMS C H O Na [M +
D
27 42
8
+
3
2
5
D
each). Fractions 8−23 were combined and repeatedly purified on the
same column with the same gradient; from fraction 10, compound 2
Isocalonysterone (7): yellow crystals; mp 140−142 °C; [α]
+
−81.0 (c 0.2, MeOH); HRMS C H O Na (M + Na ), 499.2672,
27
40
7
+
+
(
10 mg) was obtained by crystallization from EtOAc−MeOH (2:1, v/
found 499.2668; APCIMS m/z 477 [M + H] , 459 [M + H − H O] ,
2
+
+
v). The combined fractions 11−18 were purified by HPLC on a
Zorbax XDB-C (5 μm, 9.4 × 250 mm) column with an isocratic
441 [M + H − 2H O] , 423 [M + H − 3H O] , 405 [M + H −
2
2
+
8
system of 17% aqueous acetonitrile at a flow rate of 3 mL/min, to yield
compounds 1 (2.3 mg) and 4 (1.4 mg). Sample A2, obtained from the
first silica gel column, was subjected to reversed-phase CC, using a
gradient of 30%, 35%, 40%, and 50% MeOH(aq) (34, five, five, and
five fractions, respectively) as above. Fractions 21−28, eluted with 30%
MeOH, were combined and subjected to preparative TLC on Kieselgel
Compound Purity. Purity testing was performed by HPLC,
utilizing aqueous acetonitrile solvent systems on a Kinetex XB-C18
column (5 μm, 4.6 × 250 mm) at a flow rate of 1 mL/min. Integration
was performed automatically by ChromNav software 1.16.02 (slope
sensitivity: 100 μV/s, minimal area: 1000 μV·s), and purity was
this compound could neither be tested for its bioactivity nor utilized
for the quantitative determination for yield optimization. Compounds
1−3 and 5−7 possessed a purity of >98% by means of HPLC at
wavelengths of their corresponding UV absorption maxima.
6
0 F254 plates (Merck) with a CH Cl −MeOH−benzene (25:5:5, v/v/
2
2
v) solvent system, to obtain compound 3 (5 mg). When using
benzene, appropriate laboratory safety measures were taken. As also
demonstrated by the needs of the complex isolation procedure and the
very low yields, reaction 1 led to a highly complex mixture, making it
inappropriate for a larger scale preparation of any of the above
products.
Reaction 2. A 120 mg aliquot of 20E was dissolved in a mixture of
1
mL of MeOH (Merck) and 8 mL of water, and 100 mg of NaOH
Longitudinal Study of Compound Autoxidation. A 120 mg
(0.25 mmol) aliquot of 20E was dissolved, and 100 mg of NaOH was
added as above (reaction 3), in three replicates. The reaction mixture
was stirred for 2 days, and samples were taken at 0.5, 1, 2, 3, 4, 5, 6, 15,
24, and 48 h. All samples from the three independently performed
reactions were analyzed by HPLC-DAD after neutralizing the pH with
dissolved in 1 mL of water was added, so that the reaction was
performed in 10% MeOH(aq). The reaction mixture was stirred for 1
day at room temperature, and the reaction was stopped by neutralizing
the pH with a 9.6% aqueous solution of acetic acid (Merck). The
reaction mixture was fractionated by centrifugal partition chromatog-
F
J. Nat. Prod. XXXX, XXX, XXX−XXX