Pierreiones A - D, solid tumor selective pyranoisoflavones and other cytotoxic constituents from antheroporum pierrei

Article abstract of DOI:10.1021/np100763p

Bioassay-guided fractionation of a solid tumor selective extract of the leaves and twigs of Antheroporum pierrei acquired from the U.S. National Cancer Institute extract repository afforded four new pyranoisoflavones, pierreiones A-D (1-4), together with rotenone (5), 12a-hydroxyrotenone (6), and tephrosin (7). The structures of all new compounds were determined on the basis of their spectroscopic data, and the absolute configuration of 1 was assigned with the help of ¹H NMR analysis of its Mosher's ester derivatives. Compounds 1 and 5-7 accounted for the majority of the biological activity in terms of either cytotoxicity and/or selective toxicity to solid tumor cell lines. Pierreiones A (1) and B (2) demonstrated solid tumor selectivity with minimal cytotoxicity, while pierreione C (3) exhibited no activity.

Full text of DOI:10.1021/np100763p

NOTE
pubs.acs.org/jnp
Pierreiones AꢀD, Solid Tumor Selective Pyranoisoflavones and Other
Cytotoxic Constituents from Antheroporum pierrei
Song Gao,† Ya-ming Xu,† Frederick A. Valeriote,*,‡ and A. A. Leslie Gunatilaka*,†
†Southwest Center for Natural Products Research and Commercialization, School of Natural Resources and the Environment, College of
Agriculture and Life Sciences, The University of Arizona, 250 E. Valencia Road, Tucson, Arizona 85706-6800, United States
‡Division of Hematology & Oncology, Department of Internal Medicine, Henry Ford Hospital, Detroit, Michigan 48202, United States
S Supporting Information
b
ABSTRACT: Bioassay-guided fractionation of a solid tumor selective
extract of the leaves and twigs of Antheroporum pierrei acquired from the
U.S. National Cancer Institute extract repository afforded four new
pyranoisoflavones, pierreiones AꢀD (1ꢀ4), together with rotenone (5),
12a-hydroxyrotenone (6), and tephrosin (7). The structures of all new
compounds were determined on the basis of their spectroscopic data, and
the absolute configuration of 1 was assigned with the help of 1H NMR
analysis of its Mosher’s ester derivatives. Compounds 1 and 5ꢀ7
accounted for the majority of the biological activity in terms of either
cytotoxicity and/or selective toxicity to solid tumor cell lines. Pierreiones A (1) and B (2) demonstrated solid tumor selectivity with
minimal cytotoxicity, while pierreione C (3) exhibited no activity.
major problem with present day cancer chemotherapy is
the serious deficiency of drugs to treat solid tumors and
A
the concurrent metastatic disease.1 The majority of cell-based
antitumor drug discovery efforts that have led to useful
discovery of standard chemotherapeutic agents have relied
frequently on their potency rather than selectivity. To address
this deficiency, we have developed a high-throughput, cost-
efficient, simple end-point disk diffusion soft agar assay based
on differential clonogenic cytotoxicity between solid tumor
cells and either normal or leukemia cells.2 Solid tumor
selectivity criteria in this approach incorporate both the
cellular and molecular targets associated with the cancer
phenotype. In continuing our efforts to uncover potential
anticancer agents from plants,3 we investigated an extract of
Antheroporum pierrei Gagnep. (Fabaceae) acquired from the
natural products repository of the U.S. National Cancer
Institute (NCI), which exhibited solid tumor selectivity in
our assay. The genus Antheroporum Gagnep. contains five
species, namely, A. banaense, A. glaucum, A. harmandii, A.
vidalli, and A. pierrei.4 None of these species have been
subjected to any detailed biological and/or chemical investi-
gations, except for a report of the occurrence of the non-
protein amino acids arginine and homoarginine in seeds of A.
pierrei,5 a perennial tree native to China, Thailand, and
Vietnam. Consequently, we investigated the solid tumor
selective extract of A. pierrei, and herein we report the isolation
and characterization of four new isoflavones, pierreiones AꢀD
(1ꢀ4), and three known compounds, rotenone (5),6 12a-
hydroxyrotenone (6),6,7 and tephrosin (7),8 and in vitro
evaluation of their potential anticancer activity.
Bioactivity-guided fractionation of the solid tumor selective
CH2Cl2/MeOH (1:1) extract of leaves and twigs of A. pierrei
afforded compounds 1ꢀ7. Pierreione A (1) was obtained as a
colorless solid. Its molecular formula was established as
C27H30O8 from HRESIMS and 13C NMR data and indicated
13 degrees of unsaturation. The IR spectrum of 1 showed
absorption bands for OH (3433 cmꢀ1) and conjugated CO
Received: October 20, 2010
Published: March 31, 2011
r
Copyright
2011 American Chemical Society and
852
dx.doi.org/10.1021/np100763p J. Nat. Prod. 2011, 74, 852–856
American Society of Pharmacognosy
|

Journal of Natural Products
NOTE
(1639 cmꢀ1) functionalities. Its UV spectrum was typical of an
isoflavone,9,10 and this was confirmed by the presence of
characteristic NMR signals at δH 7.76 (s, H-2) and δC 150.6
substituent on ring A were at δ 6.72 (1H, d, J = 10.2 Hz), 5.70 (1H,
d, J = 10.2 Hz), and 1.45 (6H, s). Signals due to an ABX system at
δ 7.11 (1H, d, J = 1.8 Hz), 6.90 (1H, d, J = 7.8 Hz), and 6.97 (1H,
dd, J = 7.8 and 1.8 Hz) suggested that C-30 and C-40 of 3 were
oxygenated.9,14 The 1H NMR signal at δ 3.88 was assigned to an
OCH3 group (at C-30). On the basis of literature precedence, the
signals at δ 4.59 (2H, br d, J = 6.6 Hz), 5.51 (1H, t, J = 7.0 Hz), 1.75
(3H, s), and 1.72 (3H, s) were assigned to a 3000-methyl-2000-butenyl
moiety linked to C-40 through an oxygen atom.19,20 The foregoing
suggested that the structure of pierreione C should be identical to
the dimethoxy derivative of the previously known pyranoisoflavone,
isoauriculasin.20 Thus, pierreione C was identified as 30,5-di-
methoxy-40-O-(3-methyl-2-butenyl)-300,300-dimethylpyrano-(6,7)-
isoflavone (3).
Pierreione D (4), obtained as a white, amorphous solid, had
the molecular formula C26H26O5 based on its HRESIMS data.
The 1H NMR spectrum of 4 showed a very close resemblance to
that of 3, except for the presence of an aromatic proton signal at
δ 7.86 in 4 instead of the signal due to the OCH3 (δ 3.87) in 3.
The foregoing evidence identified pierreione D as 30-methoxy-40-
O-(3-methyl-2-butenyl)-300,300-dimethylpyrano-(6,7)-isoflavone
(4). Comparison of the spectroscopic and optical rotation data of
compounds 5ꢀ7 with those reported in the literature allowed
these to be identified as rotenone,6 12a-hydroxyrotenone,6,7 and
tephrosin,8 respectively.
A disk diffusion soft agar assay, using a panel of two solid
tumor cell lines [C38 (colon adenocarcinoma) and HCT-116
(colon cancer)] and one leukemia cell line [L1210 (lymphocytic
leukemia)], was used to evaluate the differential cytotoxicity
(solid tumor selectivity) of the extract, fractions, and pure com-
pounds.2 IC50 data for compounds 1ꢀ7 in the human colon
cancer cell line HCT-116 were also determined. Taken together,
these data (Table 2) suggested that pierreione A (1), rotenone
(5), 12a-hydroxyrotenone (6), and tephrosin (7) accounted for
the majority of the biological activity in terms of either cytotoxi-
city and/or selective toxicity to solid tumor cell lines, and
pierreiones A (1) and B (2) demonstrated solid tumor selectivity
with minimal cytotoxicity, while pierreione C (3) exhibited no
activity. As the only difference between the structures of pier-
reione A (1) and pierreione C (3) is the presence of a 2,
3-dihydroxyisopentane side-chain in ring C of 1 instead of an
isopentenyl moiety in 3, it is likely that the presence of the former
is required for the cytotoxic activity of 1. It is noteworthy that
cytotoxic activities for rotenone (5),21ꢀ25 12a-hydroxyrotenone
(6),23 and tephrosin (7)23,24 against several cancer cell lines have
been reported previously.
1
(C-2).10ꢀ12 The H NMR spectrum also showed four singlets
due to methyl groups [δ 1.24 (3H), 1.29 (3H), and 1.46 (6H)],
two OCH3 groups [δ 3.86 and 3.87], and an aromatic proton
(δ 6.59). In addition, it had signals due to an AB spin system
consisting of two doublets (J = 10.0 Hz) at δ 6.72 and 5.71. This,
together with the presence of a signal due to two methyl groups at
δ 1.46 (6H, s), suggested that 1 contained a 200,200-dimethylpyran
substituent.11 The presence of this substituent was supported
further by its 13C NMR signals at δC 130.8, 116.0, 77.7, and
28.3.11,13,14 Long-range correlations observed in the HMBC
spectrum of 1 (see Figure S17 in the Supporting Information)
allowed placement of the aromatic proton (δ 6.59) at C-8 of ring
A and one of the OCH3 groups (δ 3.86) at C-5. In the NOE
experiment, irradiation of the signal due to this OCH3 caused
enhancement of the olefinic proton at δ 6.72, suggesting that the
dimethylpyran moiety is fused to ring A in a linear manner. In the
aromatic region of the 1H NMR spectrum, the presence of signals
due an ABX spin system [δ 7.14 (1H, d, J = 2.0 Hz), 6.97 (1H, dd,
J = 8.0 and 2.0 Hz), and 6.93 (1H, d, J = 8.0 Hz)] suggested that
ring B of this isoflavone was 10,30,40-trisubstituted.10 The signal at
δH 7.14 was assigned to H-20 due to its NOE with H-2 (see
Figure S17 in the Supporting Information). The chemical shifts
of H-60 (δ 6.97) and H-50 (δ 6.93) signals were in agreement
with those reported for isoflavones.10 The NOE correlation
observed between H-20 (δ 7.14) and the OCH3 at δ 3.87
suggested that the latter group is attached to C-30. The
1
remaining signals in the H NMR spectrum indicated the
presence of an oxygenated methylene group [δ 4.29 (1H, dd,
J = 9.5 and 2.5 Hz), 4.08 (1H, dd, J = 9.5 and 6.5 Hz)], an
oxygenated methine group [δ 3.69 (1H, br s)], and two methyl
groups [δ 1.29 (s) and 1.24 (s)] attached to an oxygenated
carbon. These signals were assigned to a OCH2CH(OH)C-
(CH3)2O spin system by comparison with the data reported for
similar systems.15,16 Although no correlations were observed
between C-1000 protons (δ 4.08 and 4.29) with the signal due to
C-40 (δ 147.8) in the HMBC spectrum, this moiety was placed
at C-40 based on its NOE data (see Figure S17 in the Supporting
Information). The configuration at C-2000 was determined to be
R by application of the modified Mosher’s ester method
according to the reported procedure (see Figure S18 in the
Supporting
Information).17,18
The
structure
of pierreione A was thus established as 30,5-dimethoxy-
40-(2R,3-dihydroxy-3-methylbutoxyl)-300,300-dimethylpyrano-
(6,7)-isoflavone (1).
The molecular formula of pierreione B (2) was determined as
C26H28O7 from its HRESIMS. The 1H and 13C NMR spectra of
2 closely resembled those of 1, except that the OCH3 group at
C-5 was absent in 2; instead it showed an additional aromatic
proton (δ 7.85). The optical rotation of 2 was close to that of 1,
suggesting the R configuration at C-2000. Thus, the structure of
pierreione B was elucidated as 30-methoxy-40-(2R,3-dihydroxy-
3-methylbutoxyl)-300,300-dimethylpyrano-(6,7)-isoflavone (2).
Pierreione C (3) was obtained as a white, amorphous solid that
analyzed for C27H28O6 by HRESIMS. Its IR spectrum showed the
’ EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were
measured with a Jasco Dip-370 polarimeter using CHCl3 as solvent.
IR spectra using KBr disks were recorded on a Shimadzu FTIR-8300
spectrometer. 1D and 2D NMR spectra were recorded in CDCl3 with a
Bruker DRX-500 and a Bruker DRX-600 instrument at 500 or 600 MHz
for 1H NMR and 125 MHz for 13C NMR using residual CHCl3 as the
internal standard. Chemical shift values (δ) are given in parts per million
(ppm), and the coupling constants are in Hz. Low-resolution and high-
resolution MS were recorded on Shimadzu LCMS-DQ8000R and JEOL
HX110A spectrometers, respectively. HPLC was carried out on a
Hitachi instrument consisting of a DAD and an ELSD detector. The
semipreparative C-8 column (250 ꢁ 10 mm, 5 μm) used was from
Phenomenex Inc.
presence of OH (3433 cmꢀ1) and conjugated CO (1640 cmꢀ1
)
groups. Compound 3 was also suspected to be an isoflavone based
1
on its UV spectrum and the H NMR signal due to H-2 at δ
7.76.9ꢀ12 In the 1H NMR spectrum of 3, the OCH3 group at C-5
appeared at δ 3.87, while the signals of the 300,300-dimethylpyran
853
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Journal of Natural Products
NOTE
1
Table 1. H NMR Data (500 or 600 MHz, δ, Hz) for Compounds 1ꢀ4 in CDCl3
position
1a,b
2b
3c
4c
7.87 (s)
2
7.76 (s)
7.87 (s)
7.85 (s)
6.76 (s)
7.76 (s)
6.59 (s)
5
7.86 (s)
6.76 (s)
8
6.59 (s)
20
50
60
100
200
400
500
1000
7.14 (d, 2.0)
6.93 (d, 8.0)
6.97 (dd, 8.0, 2.0)
6.72 (d, 10.0)
5.71 (d, 10.0)
1.46 (s)
7.19 (d, 2.0)
6.93 (d, 8.0)
7.00 (dd, 8.0, 2.0)
6.43 (d, 10.0)
5.73 (d, 10.0)
1.47 (s)
7.11 (d, 1.8)
6.90 (d, 7.8)
6.97 (dd, 7.8, 1.8)
6.72 (d, 10.2)
5.70 (d, 10.2)
1.45 (s)
7.17 (d, 1.0)
6.90 (d, 8.0)
7.00 (br d, 8.0)
6.43 (d, 10.0)
5.72 (d, 10.0)
1.47 (s)
1.46 (s)
1.47 (s)
1.45 (s)
1.49 (s)
4.29 (dd, 9.5, 2.5)
4.08 (dd, 9.5, 6.5)
3.69 (br s)
1.29 (s)
4.30 (dd, 10.0, 2.0)
4.09 (dd, 10.0, 5.5)
3.70 (br s)
4.59 (br d, 6.6)
4.60 (br d, 6.6)
2000
4000
5.51 (t, 7.0)
1.72(s)
5.51 (t, 7.0)
1.72 (s)
1.30 (s)
5000
1.24 (s)
1.29 (s)
1.75(s)
1.75(s)
5-OCH3
30-OCH3
3.86 (s)
3.87(s)
3.87 (s)
3.88 (s)
3.88(s)
3.89 (3H, s)
a Signals were assigned by HMQC, HMBC, and 1Hꢀ H COSY experiments. b At 500 MHz. c At 600 MHz.
1
Table 2. Zone Unit Differentials in the Disk Diffusion Soft Agar Colony Formation Assaya and IC50 Values for Compounds 1ꢀ7
murine tumor selectivity
toxicity to human colon cancer cell line HCT-116
compound
conc (μg/disk)
ZC38ꢀ ZL1210
IC50 (μg/disk)
1
2
3
4
5
6
7
68
300
300
7.5
66
NAb
18
>46
30
1.9
600
500
450
550
0.23
0.26
0.24
0.045
0.055
0.040
a Measured in zone units: 200 zone units = 6 mm. Murine cell lines: C38 (colon adenocarcinoma), L1210 (lymphocytic leukemia). b NA = not active.
Plant Material. Leaves and twigs of Antheroporum pierrei were
collected in Thailand in August 1987 for the U.S. National Cancer
Institute (NCI) program on anticancer drug discovery and were
identified by Prof. J. S. Burley. The NCI Open Repository Sample
number N001507 was assigned to the sample.
their TLC profiles to afford 12 fractions (F1ꢀF12). Of these, fraction
F10 (78.8 mg), which was found to be active, was subjected to size-
exclusion chromatography on Sephadex LH-20 (150 g) and eluted with
CH2Cl2/MeOH (3:2) to afford three subfractions (F10aꢀc). The
subfraction F10a (34.1 mg) was further fractionated on Sephadex LH-
20 (150 g) and eluted with MeOH to obtain four subfractions, of which
only the third fraction was found to be active. Compounds 1 (5.3 mg)
and 2 (2.2 mg) were obtained from this active fraction by HPLC on a
C-8 semipreparative column. Further fractionation of the active fraction
F4 (4.3 mg) by HPLC under the same conditions afforded compounds 3
(2.3 mg) and 4 (0.9 mg). The active fraction F5 (29.2 mg) on Sephadex
LH-20 (150 g) size-exclusion chromatography and elution with
CH2Cl2/MeOH (3:2) yielded two subfractions, F5a and F5b. Further
fractionation of the active subfraction F5b by HPLC as above afforded
compounds 5 (1.4 mg), 6 (3.0 mg), and 7 (1.1 mg).
Extraction and Isolation. Dried plant material was extracted with
CH2Cl2ꢀMeOH (1:1), residual solvents were removed under vacuum,
and the extract (N001507) was stored at ꢀ20 °C in the NCI repository
at the Frederick Cancer Research and Development Center (Frederick,
MD). The crude extract (5.0 g sample from an NCI stock supply) was
partitioned between hexanes and 80% aqueous MeOH. The bioactive
MeOH(aq) fraction was diluted to 50% MeOH(aq) by the addition of
water and extracted with CHCl3. Evaporation of the CHCl3 fraction
yielded a dark green residue (474 mg), which was subjected to size-
exclusion chromatography over Sephadex LH-20 (15.0 g) made up in
hexanes/CH2Cl2 (1:4) and eluted with 250 mL each of hexanes/
CH2Cl2 (1:4), CH2Cl2/acetone (3:2), CH2Cl2/acetone (1:4), and
finally MeOH (500 mL). The bioactive hexanes/CH2Cl2 (1:4) fraction
(248 mg) was subjected to column chromatography over Lichroprep
diol (60 g) made up in hexanes/EtOAc (5:1) and eluted with hexanes/
EtOAc (5:1, 500 mL), hexanes/EtOAc (3:1, 500 mL), hexanes/EtOAc
(1:1, 500 mL), EtOAc (250 mL), EtOAc/MeOH (1:1, 250 mL), and
MeOH (500 mL). The resulting fractions were combined on the basis of
Pierreione A (1): white, amorphous solid; [R]25 ꢀ5.4 (c 0.1,
D
CHCl3); UV (MeOH) λmax (log ε) 325 (3.72), 271 (4.48), 228
(4.28), 203 (4.40) nm; IR (KBr) νmax 3433, 2974, 2931, 1639, 1604,
1
1515, 1465, 1265, 1207, 1126, 1180, 1064 cmꢀ1; H NMR data, see
Table 1; 13C NMR (125 MHz, CDCl3) δ 174.9 (C-4), 158.6 (C-9),
158.1 (C-7), 155.7 (C-5), 150.6 (C-2), 149.3 (C-30), 147.8 (C-40), 130.8
(C-200), 126.1 (C-10), 125.5 (C-3), 121.3 (C-60), 116.0 (C-100), 114.2
(C-50), 113.3 (C-6), 113.1 (C-10), 113.0 (C-20), 100.6 (C-8), 77.7
854
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Journal of Natural Products
NOTE
(C-300), 74.9 (C-2000), 71.9 (C-1000), 71.9 (C-3000), 62.8 (5-OCH3), 55.9
(30-OCH3), 28.3 (C-400), 28.3 (C-500), 26.7 (C-4000), 25.2 (C-5000);
HRESIMS m/z 483.2004 [M þ H]þ (calcd for C27H31O8, 483.2019).
evaluation of the active compounds. Both the magnitude of the zonal
difference and the potency was used for prioritization of fractions.
IC50 Determinations. IC50 studies were carried out against human
HCT-116 colon cancer cells. These cells were grown in 5 mL of culture
medium (RPMI-1640 þ 15% FBS containing 1% penicillinꢀstrepto-
mycin and 1% glutamine) at 37 °C and 5% CO2 at a starting
concentration of 5 ꢁ 104 cells/T25 flask. On day 3, cells were exposed
to different concentrations of the drug. Flasks were incubated for 120 h
(5 d) in a 5% CO2 incubator at 37 °C, and the cells were harvested with
trypsin, washed once with HBSS, resuspended in HBSS, and then
counted using a hemocytometer. The results are normalized to an
untreated control. The IC50 values were determined using Prism 4.0
software.
Pierreione B (2): white, amorphous solid; [R]25 ꢀ5.8 (c 0.1,
D
CHCl3); UV (MeOH) λmax (log ε) 331 (3.96), 267 (4.52), 228
(4.45), 206 (4.47) nm; IR (KBr) νmax 3425, 2974, 1639, 1621, 1515,
1
1477, 1269, 1203, 1141, 1110 cmꢀ1; H NMR data, see Table 1; 13C
NMR (125 MHz, CDCl3) δ 175.6 (C-4), 158.0 (C-9), 157.4 (C-7),
152.1 (C-2), 149.4 (C-30), 147.8 (C-40), 131.7 (C-200), 126.1 (C-10),
124.5 (C-3), 123.7 (C-5), 121.2 (C-60), 121.0 (C-100), 114.20(0 C-50),
119.9 (C-6), 118.5 (C-10), 112.8 (C-20), 103.9 (C-8), 77.9 (C-3 ), 74.9
(C-2000), 71.9 (C-1000), 71.9 (C-3000), 55.8 (30-OCH3), 28.5 (C-400), 28.5
(C-500), 26.7 (C-4000), 25.2 (C-5000); HRESIMS m/z 453.1872 [M þ
H]þ (calcd for C26H29O7, 453.1913).
Pierreione C (3): white, amorphous powder; UV (MeOH) λmax (log
ε) 325 (3.84), 272 (4.60), 228 (4.42), 206 (4.54) nm; IR (KBr) νmax
3433, 2927, 1639, 1640, 1512, 1461, 1265, 1207, 1126 cmꢀ1; 1H NMR
(600 MHz, CDCl3) data, see Table 1; HRESIMS m/z 449.1968 [M þ
H]þ (calcd for C27H29O6, 449.1964).
’ ASSOCIATED CONTENT
Supporting Information. 1H NMR spectra for com-
S
b
pounds 1ꢀ7, 8a, and 8b; 13C NMR spectra for compounds
1
1
1ꢀ3 and 6; Hꢀ H COSY, HSQC, and HMBC spectra for
compound 1; key HMBC and NOE correlations for 1 (Figure
S17); Δδ values obtained for (S)- and (R)-MTPA esters (8a and
8b, respectively) of 1 (Figure S18). This material is available free
of charge via the Internet at http://pubs.acs.org.
Pierreione D (4): white, amorphous powder; UV (MeOH) λmax (log
ε) 331 (3.93), 267 (4.49), 228 (4.43), 204 (4.48) nm; IR (KBr) νmax
3461, 2931, 1647, 1612, 1517, 1473 cmꢀ1; 1H NMR (600 MHz, CDCl3)
data, see Table 1; HRESIMS m/z 419.1853 [M þ H]þ (calcd for
C26H27O5, 419.1859).
Rotenone (5): white solid; [R]25 ꢀ173.3 (c 0.03, CHCl3); LR-
D
APCIMS and 1H NMR data were consistent with literature values.6
’ AUTHOR INFORMATION
12a-Hydroxyrotenone (6): white solid; [R]25 ꢀ90.7 (c 0.07,
D
Corresponding Author
*Tel: (520) 621-9932. Fax: (520) 621-8378. E-mail (A.A.L.G.):
leslieg@cals.arizona.edu. E-mail (F.A.V.): fvaleri1@hfhs.org.
1
CHCl3); LR-APCIMS, H NMR, and 13C NMR data were consistent
with those reported.6,7
Tephrosin (7): white solid; [R]25 ꢀ100.0 (c 0.09, CHCl3); LR-
D
APCIMS and 1H NMR data were consistent with literature values.8
Preparation of the (R)- and (S)-MTPA Ester Derivatives (8a
’ ACKNOWLEDGMENT
and 8b) of 1 by a Convenient Mosher Ester Procedure.18,19
.
Financial support for this work was provided by Grant R01
CA092143 funded by the National Cancer Institute (NCI). We
thank Dr. D. Newman of the NCI for providing the crude extract
and Dr. X.-N. Wang for reading the manuscript and suggesting
some modifications.
Compound 1 (1.0 mg) was transferred into a clean NMR tube and was
dried under vacuum. Pyridine-d5 (0.5 mL) and (S)-(ꢀ)-R-methoxy-
R-(trifluoromethyl)phenylacetyl (MTPA) chloride (5 μL) were added
to the NMR tube immediately under a stream of N2, and the tube was
shaken carefully to mix the sample and the MTPA chloride. The NMR
tube was allowed to stand at room temperature for 2 h to afford the (S)-
1
MTPA ester derivative (8a) of 1. H NMR data of 8a (600 MHz,
’ REFERENCES
pyridine-d5): δ 8.08 (1H, s, H-2), 7.50 (1H, br s, H-20), 7.27 (1H, d, J =
8.4 Hz, H-50), 6.83 (1H, d, J = 10.2 Hz, H-100), 6.03 (1H, d, J = 9.0 Hz,
H-2000), 5.76 (1H, d, J = 9.6 Hz, H-200), 4.89 (1H, d, J = 11.4 Hz, H-1000a),
4.56 (1H, dd, J = 11.4, 9.0 Hz, H-1000b), 4.01 (3H, s, OCH3, OCH3-5 or
OCH3-30), 3.80 (3H, s), 3.77 (3H, s, OCH3-5 or OCH3-30), 1.43 (3H, s,
H-4000 or H-5000), 1.42 (6H, s, H-400 and 500), 1.37 (3H, s, H-4000 or
H-5000). In the manner described for 8a, another portion of 1 (0.9 mg)
was reacted in a second NMR tube with (R)-(þ)-R-methoxy-
R-(trifluoromethyl)phenylacetyl chloride (5 μL) at room temperature
for 2 h in pyridine-d5 (0.5 mL) to afford the (R)-MTPA ester derivative
(8b). 1H NMR data of 8b (600 MHz, pyridine-d5): δ 8.07 (1H, s, H-2),
7.50 (1H, d, J = 1.8 Hz, H-20), 7.25 (1H, dd, J = 8.4, 1.8 Hz, H-60), 7.12
(1H, d, J = 8.4 Hz, H-50), 6.83 (1H, d, J = 10.2 Hz, H-100), 6.77 (1H, s,
H-8), 6.07 (1H, dd, J = 9.0, 1.8 Hz, H-2000), 5.77 (1H, d, J = 10.2 Hz,
H-200), 4.76 (1H, dd, J = 10.8, 1.8 Hz, H-1000a), 4.42 (1H, dd, J = 10.8, 9.0
Hz, H-1000b), 4.00 (3H, s, OCH3-5 or OCH3-30), 3.79 (3H, s), 3.77 (3H,
s), 1.53 (3H, s, H-4000 or H-5000), 1.52 (3H, s, H-4000 or H-5000), 1.43 (6H,
s, H-400 and H-500).
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