Longeracemosones A-F, aromatase inhibitors from Dunbaria longeracemosa

Article abstract of DOI:10.1002/ejoc.201100410

Six dihydroflavonols, longeracemosones A-F (1-6), together with two known flavonoids, mundulinol (7) and sericetin (8), have been isolated from Dunbaria longeracemosa. The structures of the isolated compounds were elucidated by spectroscopic analysis and

Full text of DOI:10.1002/ejoc.201100410

FULL PAPER
DOI: 10.1002/ejoc.201100410
Longeracemosones A–F, Aromatase Inhibitors from Dunbaria longeracemosa
Vilailak Prachyawarakorn,[a] Chulabhorn Mahidol,*[a,b] and Somsak Ruchirawat*[a,b]
Keywords: Natural products / Oxygen heterocycles / Antioxidants / Biological activity / Enzyme inhibitors
Six dihydroflavonols, longeracemosones A–F (1–6), together
with two known flavonoids, mundulinol (7) and sericetin (8),
in 1 to the angular chromene in 2 was observed and the
mechanism of this unique transformation is proposed. The
have been isolated from Dunbaria longeracemosa. The struc- isolated compounds were evaluated for their cytotoxic and
tures of the isolated compounds were elucidated by spectro- antioxidant activity and compounds 3, 4, and 7 exhibited po-
scopic analysis and the absolute configurations were deter- tent aromatase inhibitory activity with IC50 values of 0.3, 0.4,
mined by analysis of the CD spectra and by the modified
Mosher’s method. A novel conversion of the linear chromene
and 1.2 μM, respectively.
7.54 (m, 2 H) and 7.35–7.43 ppm (m, 3 H), and two protons
attached to oxygenated carbon at δH = 5.15 (d, J = 12.1 Hz)
and 4.49 ppm (dd, J = 12.1, 1.4 Hz). The signals of the two
protons attached to oxygenated carbon are typical of 2-H
and 3-H in a dihydroflavonol skeleton.[3]
Introduction
Dunbaria longeracemosa (syn. D. bella) is a climber tree
belonging to the family of Leguminosae (or Fabaceae), sub-
family Papilionoideae.[1] This plant is known in Thai as
Khang-Krung. The flower of this plant is edible and its
leaves or roots mixed with the leaves of Senna hirsuta have
been used in Thai folk medicine for the treatment of fever.
To the best of our knowledge, D. longeracemosa has never
been chemically investigated. In this paper we report the
isolation and characterization of six new bioactive dihy-
droflavonols from D. longeracemosa.
The presence of two cis-coupled olefinic protons (J =
10.1 Hz) at δH = 5.55 (3ЈЈ-H) and 6.55 ppm (4ЈЈ-H) and two
methyl singlets at δH = 1.45 (5ЈЈ-H) and 1.46 ppm (6ЈЈ-H)
suggest the presence of a 2,2-dimethylchromene ring in 1;
the HMBC correlations of 4ЈЈ-H, 5ЈЈ-H, and 6ЈЈ-H to C-2ЈЈ
confirmed the presence of a 2,2-dimethylchromene unit.
The 13C NMR resonance at δC = 196.4 ppm was assigned
to a conjugated ketone carbonyl, which chelates to a hy-
droxy group showing a downfield signal at δH = 11.90 ppm
(s), and thus this hydroxy was placed at C-5. The HMBC
spectrum also showed a correlation between 5-OH and C-
5, which confirms the position of 5-OH. The HMBC corre-
lations observed between 2-H and C-3, C-4, and C-2Ј (or
C-6Ј), between 3-H and C-1Ј, and between 3-OH and C-2,
C-3, and C-4 indicate that the dihydroflavonol unit in 1 is
attached to a monosubstituted phenyl ring at C-2 and that
the hydroxy group is located at C-3. The HMBC spectrum
of 1 shows a correlation between the aldehydic proton and
C-7, C-8, and C-9, which places the aldehyde group at C-8.
The HMBC correlations between 3ЈЈ-H and C-6 and be-
tween 4ЈЈ-H and C-6 and C-7 indicate that the 2,2-dimeth-
ylpyran unit is fused to the C-6 and C-7 positions of ring
A in 1. Based upon these data, the gross structure of 1 was
established. The coupling constant of J2-H,3-H (12.1 Hz)
suggests that the two protons at C-2 and C-3 are in trans
diaxial positions. The absolute stereochemistry at C-2 in 1
is R, as revealed by the analysis of the CD spectrum in
which positive and negative Cotton effects were observed at
325 and 262–300 nm, respectively.[4] Based on the spectro-
scopic data, longeracemosone A (1) was identified as a
6,7-pyrano-fused (2R,3R)-8-formyl-3,5-dihydroxyflavanone.
Results and Discussion
A methanol extract of the air-dried whole plant of D.
longeracemosa was mixed with water and then partitioned
with CH2Cl2. The CH2Cl2 layer was sequentially subjected
to column chromatography on silica gel, HPLC, and pre-
parative TLC to obtain six new dihydroflavonols 1–6, long-
eracemosones A–F, along with two known flavonoids, mun-
dulinol (7) and sericetin (8). The spectral data of the known
flavonoids 7 and 8 were identical in all respects to those
reported in the literature.[2]
Longeracemosone A (1) was obtained as a pale-yellow
oil and its molecular formula was established by
HRFABMS as C21H18O6. The 1H NMR spectrum of 1
shows the signals of two hydroxy groups at δH = 11.90 (s)
and 3.42 ppm (d, J = 1.4 Hz), an aldehydic proton at δH
10.21 ppm (s, 1 H), a monosubstituted phenyl ring at δH
=
=
[a] Chulabhorn Research Institute,
Vibhavadi-Rangsit Road, Laksi, Bangkok 10210, Thailand
E-mail: somsak@cri.or.th
[b] Chulabhorn Graduate Institute and the Center for
Environmental Health, Toxicology and Management of
Chemicals (ETM), Chemical Biology Program,
Vibhavadi-Rangsit Road, Laksi, Bangkok 10210, Thailand
Eur. J. Org. Chem. 2011, 3803–3808
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3803

V. Prachyawarakorn, C. Mahidol, S. Ruchirawat
FULL PAPER
1
The assignments of the H and 13C NMR resonances of 1 C-7 and C-8 confirm the positions of the aldehyde and 7-
are presented in Tables 1 and 2. OH in 2. Further HMBC correlations between 3ЈЈ-H and
Longeracemosone B (2) has a molecular formula of C-6 and between 4ЈЈ-H and C-5 established the position of
C21H18O6 (by HRFABMS), the same as 1. In general, the the 2,2-dimethylpyran unit in 2 as shown. Again, the cou-
1H and 13C NMR spectroscopic data of 2 are similar to pling constant of J2-H,3-H (12.2 Hz) suggests that the two
those of 1 and analysis of the NMR data reveals that com- protons at C-2 and C-3 are in a trans diaxial position. Lon-
pound 2 is a regioisomer of 1. The downfield shifts of an geracemosones A (1) and B (2) should have a similar bio-
aldehydic carbon (from δC = 185.5 ppm for 1 to δC
=
synthetic pathway and they should also share the same ab-
191.8 ppm for 2) and a chelated hydroxy proton (from δH solute configuration. Thus, based upon these spectroscopic
= 11.90 ppm for 1 to δH = 13.04 ppm for 2) indicate that data, longeracemosone B (2) was identified as a 5,6-pyrano-
there is a hydrogen bond between the aldehydic carbonyl fused (2R,3R)-8-formyl-3,7-dihydroxyflavanone. The pro-
and 7-OH. The HMBC correlations between the 7-OH pro- tons and carbons in 2 were assigned by analysis of the spec-
ton and C-6 and C-7 and between the aldehydic proton and troscopic data shown in Tables 1 and 2.
1
Table 1. H NMR (400 MHz, CDCl3) data of compounds 1–6.[a]
Position
1
2
3
4
5
6
2
3
5.15 (d, 12.1)
4.49 (dd, 12.1, 1.4) 4.48 (d, 12.2) 4.50 (d, 11.9)
7.54 (m)
7.35–7.43 (m)
5.55 (d, 10.1)
6.55 (d, 10.1)
1.45, 1.46 (s)
10.21 (s)
5.15 (d, 12.2) 5.01 (d, 11.9)
5.05 (d, 11.9)
4.50 (d, 11.9)
7.54 (m)
7.41–7.48 (m)
5.55 (d, 10.0)
6.66 (d, 10.0)
1.47, 1.49 (s)
5.04/5.06 (d, 11.9)
4.48/4.55 (dd, 11.9, 1.3) 4.52/4.54 (d, 11.9)
7.53 (m)
7.40–7.47 (m)
5.55 (d, 10.0)
6.65/6.652 (d, 10.0)
1.46,1.47/1.47,1.48 (s)
5.04/5.05 (d, 11.9)
2Ј,6Ј
3Ј–5Ј
3ЈЈ
4ЈЈ
5ЈЈ,6ЈЈ
1ЈЈЈ
7.55 (m)
7.54 (m)
7.50 (m)
7.44–7.50 (m) 7.41–7.48 (m)
5.59 (d, 10.1) 5.54 (d, 10.0)
6.60 (d, 10.1) 6.65 (d, 10.0)
1.52, 1.61 (s) 1.48 (s)
7.41–7.46 (m)
5.57 (d, 10.1)
6.65 (d, 10.1)
1.46,1.47/1.47,1.475
10.08 (s)
2.71 (dd, 13.7, 7.7) 2.74 (dd, 13.8, 8.0) 2.60/2.64 (dd, 14.8, 8.7) 4.47 (d, 7.1)
2.87 (dd, 13.7, 5.2) 2.87 (dd, 13.8, 4.5) 2.92[b] (m)
2ЈЈЈ
4ЈЈЈ
–
–
–
–
4.18 (dd, 7.7, 5.2) 4.23 (dd, 8.0, 4.5) 2.92[b] (m)
3.57/3.61 (d, 7.1)
1.09/1.15 (s)
4.75 (br. s)
4.81 (br. s)
1.65 (s)
3.60 (br. s)
11.45 (s)
–
4.77 (br. s)
4.87 (br. s)
1.68 (s)
3.55 (br. s)
11.45 (s)
–
1.23/1.25 (s)
5ЈЈЈ
–
–
–
–
1.21/1.24 (s)
3.60/3.62 (d, 1.3)
11.48/11.49 (s)
–
–
1.15/1.27 (s)
3.49/3.50 (s)
11.62/11.622 (s)
–
3-OH
5-OH
7-OH
1ЈЈЈ-OCH3
3.42 (d, 1.4)
11.90 (s)
–
–
13.04 (s)
–
–
–
3.33/3.35 (s)
[a] Chemical shifts are given in ppm. Multiplicity and coupling constants [Hz] are presented in parentheses. [b] Overlapped signal.
Table 2. 13C NMR (100 MHz, CDCl3) data of compounds 1–6.[a]
Position
1
2
3
4
5
6
2
3
4
5
6
7
8
9
10
1Ј
2Ј,6Ј
3Ј,5Ј
4Ј
83.5
72.0
84.0
72.5
83.3
72.5
83.2
72.5
83.2/83.3
72.2/72.5
196.1/196.2
156.5
103.16/103.22
160.9/161.0
105.3
83.7/83.6
72.4/72.5
196.3/196.4
157.8
103.5
161.0
106.2/106.1
159.0
196.4
161.0
103.2
164.4[b]
107.5
164.0[b]
99.7
135.3
127.2
128.7
129.3
80.2
189.3
161.7
103.6
164.2
104.3
166.2
101.4
135.6
127.4
128.8
129.5
80.5
196.2
156.4
103.2
160.7
106.0
159.9
100.3
136.2
127.2
128.7
129.2
79.0
196.1
156.4
103.1
160.7
106.0
159.9
100.3
136.2
127.2
128.6
129.2
79.1
159.8
100.28/100.34
136.1/136.2
127.3/127.5
128.6
129.2/129.3
78.9
100.2
135.6/135.8
127.1/127.2
128.7
129.3/129.4
79.2
2ЈЈ
3ЈЈ
4ЈЈ
127.0
114.4
28.4, 28.5
185.5
–
126.5
114.4
28.5, 28.7
191.8
–
126.2
115.3
28.4, 28.5
29.0
126.2
115.3
28.4, 28.5
28.9
126.3/126.4
115.3
126.58/126.61
115.0
28.3, 28.4
74.66/74.73
65.7/65.9
56.84
19.2/19.5
24.7/24.9
56.69/56.77
5ЈЈ,6ЈЈ
1ЈЈЈ
2ЈЈЈ
3ЈЈЈ
4ЈЈЈ
5ЈЈЈ
1ЈЈЈ-OCH3
28.3, 28.5/28.6
21.7/21.8
63.2/63.3
59.1/59.2
18.9/19.0
24.8
75.8
75.5
–
–
–
–
–
–
–
–
147.0
110.4
17.7
147.1
110.1
18.2
–
–
–
[a] Chemical shifts are given in ppm. [b] Interchangeable.
3804
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© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 3803–3808

Aromatase Inhibitors from Dunbaria longeracemosa
Longeracemosones A (1) and B (2) are unstable and eas- of the (R)- and (S)-MPA monoesters (Figure 1) imply that
ily decomposed during purification by silica gel TLC. Inter- the absolute configuration at C-3 is R, which confirms the
estingly, the conversion of 1 into 2 was observed at room R configuration deduced by analysis of the CD spectra of
temperature, and this can be rationalized by ring-opening compounds 1 and 2 mentioned earlier. On the basis of these
of the 2,2-dimethylchromene ring assisted by the presence data, longeracemosone C (3) was identified as a 6,7-pyrano-
of the phenolic hydroxy group in para position to the alde- fused (2R,3R,2ЈЈЈS)-3,5-dihydroxy-8-(2-hydroxy-3-methyl-
hyde group, as shown in Scheme 1. Subsequent proton but-3-enyl)flavanone.
transfer from the protonated carbonyl group to the phenox-
ide and bond rotation gives rise to the quinone methide
intermediate. The newly formed hydrogen bond between the
phenolic hydroxy group at C-7 and the aldehydic oxygen
could be the driving force for the conversion. As far as we
are aware, this novel and unique transformation of a linear
ring-fused chromene to an angularly fused chromene is un-
precedented.
Figure 1. Δδ (δR – δS) values of the (R)- and (S)-MPA diesters and
monoesters of 3.
A molecular formula of C25H26O6 was deduced from the
HRFABMS of longeracemosone D (4). The NMR spectra
of 4, as well as the UV and IR spectra, almost superim-
posed those of 3, which suggests that 4 is a stereoisomer of
3. As expected, analysis of the differences in the chemical
shifts of the (R)- and (S)-diesters of 4 indicate that C-2ЈЈЈ
in 3 has the R configuration (Figure 2), in contrast to that
of 3. Again, analysis of the chemical shift differences be-
tween the (R)- and (S)-MPA monoesters (Figure 2) suggests
the R configuration at C-2 in 4. Longeracemosone
D (4) was therefore identified as a 6,7-pyrano-fused
(2R,3R,2ЈЈЈR)-3,5-dihydroxy-8-(2-hydroxy-3-methylbut-3-
enyl)flavanone.
Scheme 1. Proposed mechanism for the conversion of 1 into 2.
Longeracemosone C (3) was isolated as a yellow
amorphous solid and its molecular formula, C25H26O6, was
established by HRFABMS. The H and 13C NMR spectro-
1
Figure 2. Δδ (δR – δS) values of the (R)- and (S)-MPA diesters and
monoesters of 4.
scopic data (Table 1 and Table 2) of 3 are similar to those
of the known flavonol mundulinol (7)[2] except for the sig-
nals of a prenyl group at C-8. Analysis of the NMR data
Longeracemosone E (5), a yellow amorphous solid, has
of 3 revealed the replacement of the prenyl group in 7 by the molecular formula C25H26O6, determined by
1
a 2-hydroxy-3-methylbut-3-ene moiety, which shows signals HRFABMS. However, the H and 13C NMR spectra of 5
typical of an exo-methylene (δH = 4.81 and 4.75 ppm), an display duplicate signals of some protons and carbons (see
oxygenated methine (δH = 4.18 ppm), and a singlet due to Tables 1 and 2), which suggests that it is a diastereomeric
methyl protons (δH = 1.65 ppm). This was further sup- mixture. The NMR data of 5 are similar to those of com-
ported by the fragment ion at m/z = 351 [M – C4H7O1]+ in pounds 3, 4, and 7, and careful analysis of the spectroscopic
the EI mass spectrum. The absolute stereochemistry of the data revealed that 5 possesses a 2,3-epoxy-3-methyl-
secondary alcohol at C-2ЈЈЈ of 3 was determined by the butane unit. Longeracemosone E (5) was therefore iden-
modified Mosher’s method.[5] Longeracemosone C (3) was tified as 6,7-(2,2-dimethylchromene)-8-(2,3-epoxy-3-meth-
treated with both (R)- and (S)-methoxyphenylacetic acid ylbutyl)-3,5-dihydroxyflavanone. As the NMR signals of
(MPAA) to afford MPA monoester (3a) and MPA diester particular positions (see Tables 1 and 2) are duplicated, it
(3b). Analysis of the differences in the chemical shifts of is possible that compound 5 contains two C-2ЈЈЈ epimers.
the (R)- and (S)-MPA diesters (Figure 1) indicates that the Previously, we isolated a flavone with a 2,3-epoxy-3-meth-
absolute stereochemistry at C-2ЈЈЈ is S. Although there is ylbutane moiety from Derris reticulata,[6] which was ob-
no proton at C-4 in 3, the differences in the chemical shifts tained as a mixture of two epimers at the C-2ЈЈЈ position.
Eur. J. Org. Chem. 2011, 3803–3808
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3805

V. Prachyawarakorn, C. Mahidol, S. Ruchirawat
FULL PAPER
Longeracemosone F (6) has a molecular formula of dates for the development of cancer chemoprevention
C26H28O7, determined by HRFABMS. Similarly to com- agents.[8] Unfortunately, the biological activities of com-
1
pound 5, the H and 13C NMR spectra of 6 display dupli- pounds 1 and 2 could not be assessed due to limited
cate signals of particular positions, which suggests that it is amounts of the materials.
a diastereomeric mixture. The NMR data of 6 are almost
identical to those of 5, except that one of the methylene
Table 3. Cytotoxic activity for compounds 3, 4, 7, and 8.
Compd.
Cytotoxic activity (IC50) [μm]
protons at the 1ЈЈЈ-position in 5 is replaced by a methoxy
signal (δH = 3.33/3.35 ppm and δC = 56.69/56.77 ppm) in 6.
Therefore compound 6 is a methoxy derivative of 5 and is
identified as a 6,7-pyrano-fused 8-(2,3-epoxy-1-methoxy-3-
methylbutyl)-3,5-dihydroxyflavanone. Note that compound
6 may be an artifact arising from the use of MeOH during
the extraction and purification processes. A possible path-
way proceeds via the formation of the quinone methide in-
termediate, which subsequently reacts with MeOH to give
the product 6. Such a mechanism has previously been re-
ported by our group.[7]
KB
HepG2
HuCCA-1
A549
S102
3
4
7
8
23.2
26.1
21.7
5.7
11.0
12.4
11.6
5.9
36.7
36.7
25.2
21.3
9.4
15.3
27.8
15.9
28.7
0.7
29.0
34.4
17.7
37.1
1.7
Etoposide
0.9
1.4
Conclusions
A chemical investigation of the whole plant extract of
Dunbaria longeracemosa led to the isolation of six new dihy-
droflavonols, longeracemosones A–F (1–6), and two known
flavonoids, mundulinol (7) and sericetin (8). The absolute
configurations at C-2 and C-3 of the isolated compounds
were determined by analysis of the CD spectra and the ab-
solute configuration of the secondary alcohol in the 2-hy-
droxy-3-methylbut-3-ene moiety of 3 and 4 was established
by the modified Mosher’s method. Compounds 3, 4, and 7
showed only weak cytotoxicity against five cancer cell lines,
however, they inhibited aromatase with IC50 values of 0.3,
0.4, and 1.2 μm, respectively. Sericetin (8) exhibited cyto-
toxic activity against the KB and HepG2 cancer cell lines,
but it did not inhibit the aromatase enzyme.
Experimental Section
General Methods: 1H and 13C NMR spectra were recorded with
Bruker AM 400 (400 MHz) and Varian GEMINI 2000 (200 MHz)
spectrometers in CDCl3 with Si(CH3)4 as the internal standard.
Infrared spectra (IR) were obtained with Perkin–Elmer System
2000 FT-IR and 1760 X FT-IR spectrometers. Mass spectra were
recorded with a Finnigan Mat GCQ spectrometer for EIMS and a
Finnigan MAT 90 spectrometer for HRFABMS. ESI-TOF MS
spectra were recorded with a Bruker MicroTOFLC mass spectrom-
eter. UV spectra were recorded with a Shimadzu UV/Vis 2001s
spectrophotometer. Optical rotations were measured by using the
sodium D line (590 nm) with a JASCO DIP3-70 digital polarimeter.
CD spectra were recorded in MeOH with a JASCO J-810 spectro-
polarimeter. High-performance liquid chromatography (HPLC)
was performed by using a C18 reversed-phase column as well as
an Exsil 100–10ODS HICHROM stainless-steel column (250ϫ
21.20 mm).
Compounds 3, 4, 7, and 8 were evaluated for their cyto-
toxicity against five cancer cell lines: KB (human mouth
epidermal carcinoma), HepG2 (human hepatocellular liver
carcinoma), HuCCA-1 (human cholangiocarcinoma), A549
(human lung carcinoma), and S102 (human liver cancer).
Compounds 3, 4, and 7 showed weak cytotoxic activity
with IC50 values of 11.0–36.7 μm, whereas sericetin (8) ex-
hibited cytotoxic activity against the KB (5.7 μm) and
HepG2 (5.9 μm) cell lines, the same order of magnitude as
the standard drug, etoposide (Table 3). Compounds 3, 4,
and 7 inhibited aromatase with IC50 values of 0.3, 0.4, and
1.2 μm, respectively, and were found to be more potent than
the reference compound ketoconazole (IC50 = 2.4 μm).
Compound 8 was inactive towards aromatase inhibition.
Plant Material: Whole plants of Dunbaria longeracemosa were col-
lected from Ubon Ratchathani province in May 2002 and were
identified by Dr. Wongsatit Chuakul, Department of Pharmaceuti-
cal Botany, Faculty of Pharmacy, Mahidol University. A voucher
specimen has been deposited at the Laboratory of Natural Prod-
ucts, Chulabhorn Research Institute.
Extraction and Isolation: Air-dried whole plants of D. longerace-
mosa (0.4 kg) were ground and extracted with MeOH three times
at room temperature. Evaporation of the MeOH extract gave a resi-
due, which was suspended with water and then partitioned with
Inhibitors of aromatase are known to be promising candi- CH2Cl2. The CH2Cl2 layer was evaporated to give a dark solid
3806 Eur. J. Org. Chem. 2011, 3803–3808
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Aromatase Inhibitors from Dunbaria longeracemosa
(48 g), which was subjected to column chromatography on silica
gel using hexane as eluent with increasing proportions of EtOAc,
EtOAc as eluent with increasing proportions of MeOH, and finally
with MeOH to provide 12 fractions. Fraction 4 (6.8 g), which was
eluted by 8% EtOAc in hexane, was further separated by silica gel
column chromatography using a gradient mixture of hexane and
acetone and finally with acetone to afford fractions A1–A11. Frac-
tion A2 was purified by preparative TLC using 2% acetone in hex-
ane as eluent to provide sericetin (8; 118 mg). Fraction A4 was
separated by reversed-phase HPLC and eluted with MeOH/H2O
(9:1) to give mundulinol (7; 510 mg). Fraction A6 was purified by
HPLC and eluted with MeOH/H2O (7:3) to give 2 (8 mg) and 5
(25 mg). Fraction A8 was separated by HPLC using MeOH/H2O
(7:3) as eluent to furnish compounds 1 (15 mg), 3 (40 mg), 4
(45 mg), and 6 (5 mg).
Longeracemosone A (1): Pale-yellow oil. [α]2D8 = –17.8 (c = 0.74,
CHCl3). UV (MeOH): λmax [log(ε/m–1 cm–1)] = 211 [4.01], 268 nm
[4.41]. CD (MeOH): λ (Δε) = 221 (+3.03), 262 (–2.34), 276 (–1.94),
300 (–1.74), 325 nm (+0.34 m–1 cm–1). For the 1H and 13C NMR
data, see Table 1 and Table 2. MS (EI): m/z (%) = 366 (28) [M]+,
351 (100), 247 (16), 245 (13), 231 (71), 229 (25), 205 (58), 203 (26),
175 (7), 159 (12), 91 (24), 77 (17). HRMS (FAB): calcd. for
C21H19O6 [M + H]+ 367.1182; found 367.1180.
2.57 (dd, J = 13.6, 6.1 Hz, 1 H, 1aЈЈЈ-H), 2.72 (dd, J = 13.6, 8.6 Hz,
1 H, 1bЈЈЈ-H), 4.69 (m, 1 H, 4aЈЈЈ-H), 4.70 (t, J = 1.4 Hz, 1 H,
4bЈЈЈ-H), 5.06 (d, J = 12.0 Hz, 1 H, 2-H), 5.41 (dd, J = 6.1, 8.7 Hz,
1 H, 2ЈЈЈ-H), 5.44 (d, J = 10.0 Hz, 1 H, 3ЈЈ-H), 5.52 (d, J = 12.0 Hz,
1 H, 3-H), 6.51 (d, J = 10.0 Hz, 1 H, 4ЈЈ-H), 7.16–7.27 (overlapped,
B ring), 11.55 (s, 1 H, 5-OH) ppm; the α-methoxyphenylacetic acid
part exhibited δ = 7.16–7.27 (overlapped, aromatic signal), 4.61 and
4.69 (s, each 1 H), 3.25 and 3.31 (s, each 3 H, OCH3) ppm. MS
(ESI-TOF): calcd. for C43H43O10 [M + H]+ 719.2851; found
719.2827.
Preparation of the (S)-MPA Monoester (S)-3a and (S)-MPA Diester
(S)-3b: Compound 3 (5.3 mg, 0.0126 mmol) was treated with
(S)-(+)-α-methoxyphenylacetic acid (8.3 mg, 0.050 mmol) and N-
[3-(dimethylamino)propyl]-NЈ-ethylcarbodiimide
hydrochloride
(EDC; 9.55 mg, 0.050 mmol) in the presence of a catalytic amount
of 4-(dimethylamino)pyridine in CH2Cl2 at room temperature for
24 h. The reaction mixture was dried under vacuum and purified
by TLC (hexane/acetone, 8:2) to give (S)-MPA monoester (S)-3a
(6 mg) and (S)-MPA diester (S)-3b (2 mg).
(S)-MPA Monoester (S)-3a: Pale-yellow oil. 1H NMR (200 MHz,
CDCl3): δ = 1.40 (s, 6 H, 5ЈЈ-Me, 6ЈЈ-Me), 1.52 (s, 3 H, 5ЈЈЈ-Me),
2.60 (dd, J = 14.0, 8.0 Hz, 1 H, 1aЈЈЈ-H), 2.77 (dd, J = 14.0, 6.0 Hz,
1 H, 1bЈЈЈ-H), 4.08 (t, J = 6.0 Hz, 1 H, 2ЈЈЈ-H), 4.64 and 4.69 (s,
each 1 H, 4ЈЈЈ-H), 5.09 (d, J = 12.0 Hz, 1 H, 2-H), 5.47 (d, J =
10.0 Hz, 1 H, 3ЈЈ-H), 5.76 (d, J = 12.0 Hz, 1 H, 3-H), 6.57 (d, J =
10.0 Hz, 1 H, 4ЈЈ-H), 11.63 (s, 1 H, 5-OH), 7.00–7.40 (overlapped,
B ring) ppm; the α-methoxyphenylacetic acid part exhibited δ =
3.31 (3 H, OCH3), 4.74 (s, 1 H), 7.00–7.40 (overlapped, aromatic
signal) ppm. HRMS (FAB): calcd. for C34H35O8 [M + H]+
571.2332; found 571.2338.
(S)-MPA Diester (S)-3b: Pale-yellow oil. 1H NMR (400 MHz,
CDCl3): δ = 1.26 (s, 3 H, 5ЈЈЈ-Me), 1.40 and 1.44 (s, each 3 H, 5ЈЈ-
Me, 6ЈЈ-Me), 2.67 (dd, J = 13.5, 6.2 Hz, 1 H, 1aЈЈЈ-H), 2.79 (dd, J
= 13.5, 8.0 Hz, 1 H, 1bЈЈЈ-H), 4.42 and 4.53 (m, each 1 H, 4ЈЈЈ-H),
5.08 (d, J = 12.0 Hz, 1 H, 2-H), 5.37 (dd, J = 6.2, 8.0 Hz, 1 H,
2ЈЈЈ-H), 5.47 (d, J = 10.0 Hz, 1 H, 3ЈЈ-H), 5.71 (d, J = 12.0 Hz, 1
H, 3-H), 6.56 (d, J = 10.0 Hz, 1 H, 4ЈЈ-H), 11.55 (s, 1 H, 5-OH),
7.06–7.26 (overlapped, B ring) ppm; the α-methoxyphenylacetic
Longeracemosone B (2): Pale-yellow oil. [α]2D3 = –63.0 (c = 0.19,
CHCl3). UV (MeOH): λmax [log(ε/m–1 cm–1)] = 217 [4.06], 275 nm
[3.99]. CD (MeOH): λ (Δε) = 221 (+2.00), 270 (–1.50), 279 (–1.50),
304 (–1.20), 347 nm (+0.28 m–1 cm–1). For the 1H and 13C NMR
data, see Table 1 and Table 2. MS (EI): m/z (%) = 366 (23) [M]+,
351 (80), 333 (9), 309 (13), 247 (60), 231 (100), 229 (24), 218 (12),
205 (23), 203 (27). HRMS (FAB): calcd. for C21H19O6 [M + H]+
367.1182; found 367.1175.
Longeracemosone C (3): Yellow amorphous solid. [α]2D6 = +25.7 (c
= 0.61, CHCl3). UV (MeOH): λmax [log(ε/m–1 cm–1)] = 204 [4.21],
267 [4.37], 275 [4.41], 314 nm [3.92]. CD (MeOH): λ (Δε) = 233
(+4.59), 272 (+6.25), 302 (–6.93), 325 (+1.75), 353 nm
(+2.34 m–1 cm–1). FTIR (KBr): ν = 3436, 2967, 1627, 1457, 1287,
˜
1193, 1127, 1025, 900 cm–1. For the 1H and 13C NMR data, see
Tables 1 and 2. MS (EI): m/z (%) = 422 (14) [M]+, 352 (29), 351
(100), 261 (8), 231 (55), 205 (34), 189 (33), 163 (12), 135 (9). HRMS acid part exhibited δ = 3.24 and 3.32 (s, each 3 H, OCH3), 4.57
(FAB): calcd. for C25H27O6 [M + H]+ 423.1808; found 423.1804.
and 4.75 (s, each 1 H), 7.06–7.26 (overlapped, aromatic sig-
nal) ppm. MS (ESI-TOF): calcd. for C43H43O10 [M
719.2851; found 719.2862.
Longeracemosone D (4): Yellow amorphous solid. [α]2D6 = +18.2 (c
= 0.63, CHCl3). UV (MeOH): λmax [log(ε/m–1 cm–1)] = 203 [4.31],
267 [4.44], 275 [4.47], 314 nm [3.98]. CD (MeOH): λ (Δε) = 233
+
H]+
Preparation of the (R)-MPA Monoester (R)-3a and (R)-MPA Di-
ester (R)-3b: Compound 3 (5.3 mg, 0.0126 mmol) was treated with
(R)-(–)-α-methoxyphenylacetic acid (8.3 mg, 0.050 mmol) and N-
[3-(dimethylamino)propyl]-NЈ-ethylcarbodiimide
hydrochloride
(EDC; 9.55 mg, 0.050 mmol) in the presence of a catalytic amount
of 4-(dimethylamino)pyridine in CH2Cl2 at room temperature for (+7.88), 299 (–4.85), 325 (+1.79), 351 nm (+2.43 m–1 cm–1). FTIR
24 h. The reaction mixture was dried under vacuum and purified
by preparative TLC (hexane/acetone, 8:2) to give (R)-MPA mono-
ester (R)-3a (5 mg) and (R)-MPA diester (R)-3b (2 mg).
(KBr): ν = 3428, 2978, 1626, 1457, 1284, 1193, 1126, 1025, 901,
˜
768, 698 cm–1. For the 1H and 13C NMR data, see Table 1 and
Table 2. MS (EI): m/z (%) = 423 (10) [M + H]+, 422 (13) [M]+, 352
(27), 351 (100), 231 (32), 205 (27), 189 (14), 163 (7), 135 (6). HRMS
(FAB): calcd. for C25H27O6 [M + H]+ 423.1808; found 423.1807.
1
(R)-MPA Monoester (R)-3a: Pale-yellow oil. H NMR (200 MHz,
CDCl3): δ = 1.40 (s, 6 H, 5ЈЈ-Me, 6ЈЈ-Me), 1.54 (s, 3 H, 5ЈЈЈ-Me),
2.61 (dd, J = 14.0, 8.0 Hz, 1 H, 1aЈЈЈ-H), 2.78 (dd, J = 14.0, 6.0 Hz,
Preparation of the (R)-MPA Monoester (R)-4a and (R)-MPA
1 H, 1bЈЈЈ-H), 3.31 (3 H, OCH3), 4.09 (t, J = 6.0 Hz, 1 H, 2ЈЈЈ-H), Diester (R)-4b: Compound 4 (6.3 mg, 0.015 mmol) was treated with
4.66 and 4.69 (s, each 1 H, 4ЈЈЈ-H), 5.25 (d, J = 12.0 Hz, 1 H, 2-
H), 5.46 (d, J = 10.0 Hz, 1 H, 3ЈЈ-H), 5.75 (d, J = 12.0 Hz, 1 H, 3-
H), 6.56 (d, J = 10.0 Hz, 1 H, 4ЈЈ-H), 7.10–7.40 (overlapped, B
ring), 11.61 (s, 1 H, 5-OH) ppm; the α-methoxyphenylacetic acid
part exhibited δH = 3.37 (OCH3, 3 H), 4.69 (s, 1 H), 7.10–7.40
(overlapped, aromatic signal) ppm. HRMS (FAB): calcd. for
C34H35O8 [M + H]+ 571.2332; found 571.2333.
(R)-(–)-α-methoxyphenylacetic acid (10 mg, 0.06 mmol) and
N-[3-(dimethylamino)propyl]-NЈ-ethylcarbodiimide hydrochloride
(EDC; 10 mg, 0.052 mmol) in the presence of a catalytic amount
of 4-(dimethylamino)pyridine in CH2Cl2 at room temperature for
24 h. The reaction mixture was dried under vacuum and purified
by TLC (hexane/acetone, 8:2) to give (R)-MPA monoester (R)-4a
(6 mg) and (R)-MPA diester (R)-4b (3 mg).
1
(R)-MPA Diester (R)-3b: Pale-yellow oil. 1H NMR (400 MHz,
CDCl3): δ = 1.39 (s, 6 H, 5ЈЈ-Me, 6ЈЈ-Me), 1.52 (s, 3 H, 5ЈЈЈ-Me),
(R)-MPA Monoester (R)-4a: Pale-yellow oil. H NMR (200 MHz,
CDCl3): δ = 1.47 and 1.48 (s, each 3 H, 5ЈЈ-Me, 6ЈЈ-Me), 1.63 (s, 3
Eur. J. Org. Chem. 2011, 3803–3808
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3807

V. Prachyawarakorn, C. Mahidol, S. Ruchirawat
FULL PAPER
H, 5ЈЈЈ-Me), 2.71 (dd, J = 13.9, 7.3 Hz, 1 H, 1aЈЈЈ-H), 2.86 (dd, J (6) [M + H]+, 422 (13) [M]+, 408 (25), 407 (100), 335 (10), 287 (12),
= 13.9, 5.1 Hz, 1 H, 1bЈЈЈ-H), 4.21 (t, J = 6.0 Hz, 1 H, 2ЈЈЈ-H), 4.75 261 (18), 243 (11), 229 (10), 215 (27), 189 (20). HRMS (FAB):
and 4.82 (s, each 1 H, 4ЈЈЈ-H), 5.34 (d, J = 12.5 Hz, 1 H, 2-H), 5.53 calcd. for C25H27O6 [M + H]+ 423.1808; found 423.1808.
(d, J = 10.3 Hz, 1 H, 3ЈЈ-H), 5.81 (d, J = 12.5 Hz, 1 H, 3-H), 6.64
Longeracemosone F (6): Yellow amorphous solid. [α]2D7 = + 23.57
(d, J = 10.3 Hz, 1 H, 4ЈЈ-H), 7.22–7.32 (overlapped, B ring), 11.67
(c = 0.52, CHCl3). CD (MeOH): λ (Δε) = 235 (+5.35), 297 (–2.73),
(s, 1 H, 5-OH) ppm; the α-methoxyphenylacetic acid part exhibited
353 nm (+1.70 m–1 cm–1). For the 1H and 13C NMR data, see
δ = 3.37 (3 H, OCH3), 4.75 (s, 1 H), 7.22–7.32 (overlapped, aro-
Tables 1 and 2. MS (EI): m/z (%) = 452 (7) [M]+, 438 (8), 437 (30),
matic signal) ppm. HRMS (FAB): calcd. for C34H35O8 [M + H]+
382 (23), 381 (100), 261 (25), 235 (15), 201 (13). HRMS (FAB):
571.2332; found 571.2333.
calcd. for C26H29O7 [M + H]+ 453.1913; found 453.1916.
(R)-MPA Diester (R)-4b: Pale-yellow oil. 1H NMR (400 MHz,
Bioassays: The cytotoxic assays were performed following the
CDCl3): δ = 1.51 (s, 3 H, 5ЈЈЈ-Me), 1.43 and 1.46 (s, each 3 H, 5ЈЈ-
method described previously.[9] In brief, cell lines suspended in
Me, 6ЈЈ-Me), 2.75 (dd, J = 13.6, 5.8 Hz, 1 H, 1aЈЈЈ-H), 2.87 (dd, J
RPMI 1640 containing 10% FBS were seeded at 1ϫ 104 cells
= 13.6, 8.3 Hz, 1 H, 1bЈЈЈ-H), 4.47 (br. s, 1 H, 4aЈЈЈ-H), 4.62 (t, J
(100 μL) per well in a 96-well plate and incubated in a humidified
= 1.4 Hz, 1 H, 4bЈЈЈ-H), 5.28 (d, J = 11.9 Hz, 1 H, 2-H), 5.44 (dd,
atmosphere of 95% air and 5% CO2 at 37 °C. After 24 h, ad-
J = 8.3, 5.8 Hz, 1 H, 2ЈЈЈ-H), 5.54 (d, J = 10.0 Hz, 1 H, 3ЈЈ-H),
ditional medium (100 μL) was added containing the test compound
5.76 (d, J = 11.9 Hz, 1 H, 3-H), 6.63 (d, J = 10.0 Hz, 1 H, 4ЈЈ-
and vehicle, giving a final concentration of 50 μg/mL, and 0.2%
H), 11.67 (s, 1 H, 5-OH) ppm; the α-methoxyphenylacetic acid part
DMSO and the solutions were further incubated for 3 d. Cells were
exhibited δ = 4.60 and 4.76 (s, each 1 H), 3.30 and 3.39 (s, each 3
subsequently fixed with 95% EtOH, stained with crystal violet
H, OCH3) ppm; the signals of the aromatic groups of the B ring
solution, and lysed with a solution of 0.1 n HCl in MeOH, after
and α-methoxyphenylacetic acid were observed at δ = 6.98 (m),
which the absorbance was measured at 550 nm. The number of
7.20–7.38 (m), 7.75 (m) ppm. MS (ESI-TOF): calcd. for C43H43O10
surviving cells was determined from the absorbance. The results
[M + H]+ 719.2851; found 719.2837.
are expressed as percentage survival compared with the control.
Preparation of the (S)-MPA Monoester (S)-4a and (S)-MPA Diester
(S)-4b: Compound 4 (6.3 mg, 0.015 mmol) was treated with (S)-
(+)-α-methoxyphenylacetic acid (10 mg, 0.06 mmol) and N-[3-(di-
methylamino)propyl]-NЈ-ethylcarbodiimide hydrochloride (EDC;
10 mg, 0.052 mmol) in the presence of a catalytic amount of 4-
(dimethylamino)pyridine in CH2Cl2 at room temperature for 24 h.
The reaction mixture was dried under vacuum and purified by TLC
(hexane/acetone, 8:2) to give (S)-MPA monoester (S)-4a (6 mg) and
(S)-MPA diester (S)-4b (3 mg).
(S)-MPA Monoester (S)-4a: Pale-yellow oil. 1H NMR (200 MHz,
CDCl3): δ = 1.46 and 1.48 (s, each 3 H, 5ЈЈ-Me, 6ЈЈ-Me), 1.62 (s, 3
H, 5ЈЈЈ-Me), 2.69 (dd, J = 13.9, 7.3 Hz, 1 H, 1aЈЈЈ-H), 2.84 (dd, J
= 13.9, 5.1 Hz, 1 H, 1bЈЈЈ-H), 4.20 (t, J = 6.0 Hz, 1 H, 2ЈЈЈ-H), 4.74
and 4.81 (s, each 1 H, 4ЈЈЈ-H), 5.18 (d, J = 12.5 Hz, 1 H, 2-H), 5.53
(d, J = 10.3 Hz, 1 H, 3ЈЈ-H), 5.82 (d, J = 12.5 Hz, 1 H, 3-H),
6.65 (d, J = 10.3 Hz, 1 H, 4ЈЈ-H), 11.70 (s, 1 H, 5-OH), 7.05–7.35
(overlapped, B ring) ppm; the α-methoxyphenylacetic acid part ex-
hibited δ = 3.38 (3 H, OCH3), 4.81 (s, 1 H), 7.05–7.35 (overlapped,
aromatic signal) ppm. HRMS (FAB): calcd. for C34H35O8
[M + H]+ 571.2332; found 571.2333.
Etoposide was used as the reference compound (Table 3). The aro-
matase inhibitory assay was performed using the method reported
by Stresser et al.[10] The reference compound, ketoconazole, exhib-
ited an IC50 value of 2.4 μm.
Acknowledgments
We thank S. Sitthimonchai for inhibitory assay of aromatase,
P. Intachote and K. Chiablaem for cytotoxicity testing, P. Kit-
takoop for helpful discussions and preparation of the manuscript,
and the Institute of Molecular Biosciences, Mahidol University, for
partial financial support.
[1] T. Smitinand, Thai plant names (botanical name-vernacular
names), Royal Forest Department, The Forest Herbarium,
Bangkok, Thailand, 2001.
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Gómez-Garibay, I. Chavez-Uribe, M. Sousa-Sánchez, Phyto-
chemistry 2000, 55, 953–957; b) A. C. Jain, M. K. Zutshi, Tetra-
hedron 1973, 29, 3347–3350; c) J. J. Van Zyl, G. J. H. Rall, D. G.
Roux, J. Chem. Res. (M) 1979, 1301–1320.
[3] S. Cao, J. K. Schilling, J. S. Miller, R. Andriantsiferana, V. E.
Rasamison, D. G. I. Kingston, J. Nat. Prod. 2004, 67, 454–456.
[4] T. M. Smalberger, R. Vleggaar, J. C. Weber, Tetrahedron 1974,
30, 3927–3931.
[5] Sh. K. Latypov, J. M. Seco, E. Quiñoá, R. Riguera, J. Org.
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[6] C. Mahidol, H. Prawat, S. Ruchirawat, K. Lihkitwitayawuid,
L.-Z. Lin, G. A. Cordell, Phytochemistry 1997, 45, 825–829.
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[8] a) L. W. Rice, Gynecol. Oncol. 2010, 118, 202–207; b) A. B.
Castrellon, S. Gluck, Expert Rev. Anticancer Ther. 2008, 8,
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[9] T. Tengchaisri, R. Chawengkirttikul, N. Rachaphaew, V. Re-
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[10] D. M. Stresser, S. D. Turner, J. McNamara, P. Stocker, V. P.
Miller, C. L. Crespi, C. J. Patten, Anal. Biochem. 2000, 284,
427–430.
(S)-MPA Diester (S)-4b: Pale-yellow oil. 1H NMR (400 MHz,
CDCl3) δ = 1.45 and 1.49 (s, each 3 H, 5ЈЈ-Me, 6ЈЈ-Me), 1.63 (s, 3
H, 5ЈЈЈ-Me), 2.63 (dd, J = 13.6, 5.6 Hz, 1 H, 1aЈЈЈ-H), 2.82 (dd, J
= 13.6, 9.2 Hz, 1 H, 1bЈЈЈ-H), 4.75 and 4.78 (m, each 1 H, 4ЈЈЈ-H),
4.79 (d, J = 12.0 Hz, 1 H, 2-H), 5.50 (dd, J = 5.6, 9.2 Hz, 1 H,
2ЈЈЈ-H), 5.52 (d, J = 10.0 Hz, 1 H, 3ЈЈ-H), 5.66 (d, J = 12.0 Hz, 1
H, 3-H), 6.61 (d, J = 10.0 Hz, 1 H, 4ЈЈ-H), 11.67 (s, 1 H, 5-OH),
7.00–7.32 (overlapped, B ring) ppm; the α-methoxyphenylacetic
acid part exhibited δ = 3.28 and 3.38 (s, each 3 H, OCH3), 4.61
and 4.82 (s, each 1 H), 7.00–7.32 (overlapped, aromatic sig-
nal) ppm. MS (ESI-TOF): calcd. for C43H43O10 [M
719.2851; found 719.2855.
+
H]+
Longeracemosone E (5): Yellow amorphous solid. [α]2D7 = +16.86 (c
= 0.49, CHCl3). UV (MeOH): λmax [log(ε/m–1 cm–1)] = 267 [4.59],
274 [4.64], 313 nm [4.13]. CD (MeOH): λ (Δε) = 233 (+6.22), 300
(–3.30), 330 (+1.20), 362 nm (+1.79 m–1 cm–1). FTIR (KBr): ν =
˜
3433, 2971, 1629, 1587, 1463, 1380, 1286, 1128 cm–1. For the 1H
Received: March 25, 2011
Published Online: June 1, 2011
and 13C NMR data, see Tables 1 and 2. MS (EI): m/z (%) = 423
3808
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© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 3803–3808