New chalcone dimers from Caesalpinia ferrea Mart act as potent inhibitors of DNA topoisomerase II

Article abstract of DOI:10.1016/j.tetlet.2013.07.028

New chalcone dimers, pauferrol B (2), and pauferrol C (3), were isolated from the stems of Caesalpinia ferrea Mart, and their structures were determined on the basis of 2D NMR spectroscopy. These new chalcone dimers showed potent inhibitory activities against human topoisomerase II and cell proliferation via the induction of apoptosis in human leukemia HL 60 cells. Absolute configurations of 2 and 3 were estimated by the comparison of CD spectra with a dihydrobenzofuran derivative 4, which was enantioselectively synthesized from 2-hydroxyphenyl acetic acid.

Full text of DOI:10.1016/j.tetlet.2013.07.028

Tetrahedron Letters 54 (2013) 5052–5055
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New chalcone dimers from Caesalpinia ferrea Mart act as potent
inhibitors of DNA topoisomerase II
a
a
b
a
Susumu Ohira a, , Kyouhei Takaya , Taichi Mitsui , Masahiro Kido , Kazuyuki Kakumoto ,
⇑
Ken-ichiro Hayashi a, Atsuhito Kuboki a, Hiroyuki Tani b, Shogo Ikeda a, Munekazu Iinuma c,
Yukihiro Akao c, Hiroshi Nozaki a,
⇑
a Department of Biochemistry, Faculty of Science, Okayama University of Science, 1-1 Ridai-cho, Kita-ku, Okayama 700-0005, Japan
b Integrated Center for Science (INCS), Ehime University, Bunkyou-cho, Matsuyama 790-8577, Japan
c Department of Pharmacognosy, Gifu Pharmaceutical University, 1-2-54 Daigakunishi, Gifu 501-1196, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
New chalcone dimers, pauferrol B (2), and pauferrol C (3), were isolated from the stems of Caesalpinia
ferrea Mart, and their structures were determined on the basis of 2D NMR spectroscopy. These new chal-
cone dimers showed potent inhibitory activities against human topoisomerase II and cell proliferation via
the induction of apoptosis in human leukemia HL 60 cells. Absolute configurations of 2 and 3 were esti-
mated by the comparison of CD spectra with a dihydrobenzofuran derivative 4, which was enantioselec-
tively synthesized from 2-hydroxyphenyl acetic acid.
Received 30 May 2013
Revised 2 July 2013
Accepted 4 July 2013
Available online 12 July 2013
Keywords:
Topo II inhibitor
Ó 2013 Elsevier Ltd. All rights reserved.
Caesalpinia ferrea Mart
Chalcone dimer
trans-Substituted dihydrobenzofuran
Absolute configuration
DNA topoisomerase II (topo II) is found in all prokaryotic and
eukaryotic cells; it catalyzes topological rearrangements of DNA,
such as the relaxation of supercoiled DNA and the catenation of
DNA rings.1 Topo II plays a crucial role in proliferative processes,
such as DNA replication, chromosome condensation, and chromo-
some segregation. Therefore, topo II is regarded as an important
clinical target for anti-cancer drugs.2 In the search for new classes
of inhibitors of human topo II in plant extracts,3,4 we have reported
pauferrol A (1),5 a novel chalcone trimer with a cyclobutane ring,
isolated from the stems of Caesalpinia ferrea Mart. 1 displayed
potent topo II inhibition and apoptosis-inducible activity in the
human acute myeloid leukemia HL 60 cell lines.6,7 As part of our
ongoing project to screen topo II inhibitors from extracts of C.
ferrea Mart, we succeeded in the isolation of two new chalcone
dimers (2 and 3). The chalcone dimers exhibited potent topo II
inhibitory activities and also showed significant inhibitory effects
on HL-60 cell proliferation. The structures of these chalcone dimers
were determined by spectroscopic analyses, and their absolute ste-
reochemistries were deduced on the basis of the enantioselective
synthesis of the related compound 4.
for many medicinal purposes, such as bronchitis, diabetes, and
the treatment of wounds.8 The stems of C. ferrea Mart collected
in Amazonas State, Brazil in 2000 were extracted with acetone.
These extracts showed a potent inhibitory activity against topo II
and cell proliferation in HL 60 cells. Pauferrol B (2) and pauferrol
C (3) were purified as active compounds by repeated chromatogra-
phy. Stems (5 kg) of C. ferrea Mart were extracted with acetone
(18 L) at room temperature, and the solvent was removed under
reduced pressure. The extract was suspended in water and then
partitioned successively with n-hexane, EtOAc, and BuOH. The
EtOAc extracts showed a significant topo II inhibitory activity at
100 lg/ml. The EtOAc extract (50 g) was subjected to a silica gel
column chromatography elution with a gradient of CHCl3/MeOH
(10:1, 5:1, 3:1, 2:1, 1:1, MeOH) to yield six fractions. Active frac-
tions [Fr2 (500 mg) and Fr3 (580 mg)] were combined, and then
purified by silica gel column chromatography with gradient elution
(CHCl3/MeOH/water = 9:1:0.5, 6:1:0.1, 10:2:1, by vol.) to give two
active subfractions, a (280 mg) and b (120 mg), which were further
purified by octadecylsilane (ODS) medium-pressure liquid chro-
matography (MeOH/water 5:1, for each subfraction, v/v), and silica
gel flash column chromatography (n-hexane/acetone 3:1 for a and
2:1 for b, v/v) to yield pauferrol B (2: 58.6 mg) and pauferrol C (3:
20.3 mg), respectively.
C. ferrea Mart (Brazilian ironwood), commonly known as pau
ferro or juca, is a leguminous plant in Brazil and has been used
Pauferrol B (2),9 obtained as a yellow powder (mp 140 °C;
[a]D +190.3°; c 0.50, MeOH), was determined to have a molecular
⇑
Corresponding authors. Tel./fax: +81 86 256 9425.
E-mail address: sohira@dbc.ous.ac.jp (S. Ohira).
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.07.028

S. Ohira et al. / Tetrahedron Letters 54 (2013) 5052–5055
5053
Table 1
indicated that 2 is a chalcone dimer containing a dihydrofuran ring
in the structure. The HMBC [11-OH/C-11, C-12, H-70/C-20(C-60),
C-90, H-80/C-10, C-70, C-90, C-12, C-13, H-150/C-90] indicated that
the chalcone unit B fused with the unit A at C80/C-12 to form a
dihydrobenzofuran ring. Thus, the planar structure of 2 was
determined as shown in Figure 2. The 2D NMR spectral analyses,
including 1H–1H COSY, HMQC, and HMBC spectra, allowed the
assignment of all proton and carbon signals as shown in Table 1.
The relative stereostructure was elucidated by a different NOE
experiment. NOE enhancement was observed between H-20(H-60)
and H-80, indicating that the configuration between H-70 and H-80
should be in trans form. The relative structure of 2 was determined
and is shown in Figure 1.
1H and 13C NMR data of Pauferrol B(2) and Pauferrol C(3). NMR spectra were recorded
by 500 MHz NMR in acetone-d5 and TMS was used as internal standard
No.
2
3
dH
dC
dH
dC
1
2, 6
3, 5
4
127.6
132.0
116.8
161.2
145.7
118.3
193.3
115.9
162.6
114.3
168.1
102.6
134.5
127.4
131.6
116.9
161.1
145.3
118.0
192.6
115.0
168.6
98.8
7.77 d (8.9)
6.94 d (8.9)
7.58 d (8.6)
6.92 d (8.6)
7
8
9
7.82 d (15.6)
7.86 d (15.6)
7.78 d (15.6)
7.49 d (15.6)
10
11
12
13
14
15
Pauferrol C (3),10 a yellow powder (mp 198–200 °C: [
a]D +256.4°;
6.44 s
167.2
119.4
128.2
c 0.10, MeOH), showed a pseudo ion [M+Na]+ at m/z 533.1190 in HR-
FABMS, corresponding to a molecular formula of C30H22O8. The 1H,
13C NMR, and 1H–1H COSY spectra (Fig. 3) indicated the presence
of two phenolic protons (d 12.51 and 13.92), two sets of
ortho-coupled aromatic protons (ring A1 and B1), para-coupled aro-
matic protons on a 1,2,4,5-tetrasubstituted benzene ring, aromatic
6.57 d (8.9)
8.28 d (8.9)
13.87 s (OH)
7.98 s
13.92 s (OH)
11-OH
10
131.7
128.8
116.5
159.0
91.5
131.7
128.7
116.5
158.9
89.2
20, 60
30, 50
40
7.32 d (8.9)
6.89 d (8.9)
7.33 d (8.6)
6.88 d (8.6)
70
5.96 d (6.4)
5.45 d (6.4)
6.22 d (6.1)
5.37 d (6.1)
80
55.0
56.6
90
202.5
113.9
167.1
103.7
166.5
109.1
134.5
201.0
113.4
167.4
104.0
166.7
109.4
134.8
100
110
120
6.37 d (2.2)
6.43 d (1.8)
130
H
140
6.40 dd (8.9, 2.2)
7.78 d (8.9)
6.62 dd (8.9, 1.8)
8.01 d (8.9)
150
110-OH
12.78 s (OH)
12.51 s (OH)
formula of C30H22O8 from its HR-FABMS (m/z 511.1385 [M + H]+;
m/z 511.1393 calcd. for C30H23O8), and its IR spectra exhibited
absorption bands (
m
3337 and 1636 cmꢀ1) corresponding to a
hydroxyl and
a
, b-unsaturated ketone groups. The 1H NMR and
1H–1H COSY spectra showed the presence of two hydrogen-bonded
phenolic protons (d 13.87 and 12.78), two sets of
ortho-coupled aromatic protons (ring A1 and B1) derived from
two 4-hydroxyphenyl groups, aromatic protons (H-120, H-140,
and H-150 in ring B2) coupled in an ABX system of 2,4-dihydroxy-
phenyl group, and two ortho-coupled aromatic protons (H-14 and
H-15 in ring A2) on a 1,2,3,4-tetrasubstituted benzene ring. Further,
the 1H NMR spectra showed the presence of two mutually coupled
aliphatic methine protons (H-70 and H-80), and trans coupled
olefinic protons (H-7 and H-8, d 7.86, 7.82, each d, J = 15.6 Hz)
attributed to
a, b-unsaturated ketone groups. These evidences,
combined with the limitation imposed by twenty unsaturated
and two carbonyl carbon signals (d 193.3 and 202.5) in 13C NMR,
Figure 1.
HO
12'
11'
OH
O
B2
9'
13'
14'
O
OH
11
10'
9
15'
10
12
13
8
8'
A2
B1
OH
7' 1'
4'
7
15
O
2'
3'
14
1
A1
3
1H-1H COSY correlations
2
4
HO
HMBC correlations
Figure 2. HMBC and 1H–1H COSY of 2.

5054
S. Ohira et al. / Tetrahedron Letters 54 (2013) 5052–5055
HO
12'
11'
OH
O
B2
9'
13'
14'
O
10'
15
9
15'
10
14
13
8
8'
A2
B1
OH
11
7' 1'
4'
7
O
HO
2'
3'
1
12
A1
3
1H-1H COSY correlations
2
4
HO
HMBC correlations
Figure 3. HMBC and 1H–1H COSY of 3.
protons (H-120, H-140, and H-150) in a 2,4-dihydroxyphenyl group,
and a set of mutually coupled aliphatic protons (H-70 and H-80), as
well as characteristic trans coupled olefinic protons (d 7.78, 7.49,
each 1H, d, J = 15.6). These data suggested that 3 was a chalcone di-
mer with a dihydrobenzofuran ring like 2. Analysis of 1H–1H COSY,
HMQC, and HMBC spectra established full assignment of all protons
and carbons as shown in Table 1. In the HMBC spectrum, distinct
cross-peaks observed between H-7/C-2(C-6), H-8/C-1, C-9, H-15/
C-9, 11-OH/C-10,C-12, H-20(60)/C-70, H-150/C-90, and 110-OH/C-100
revealed the connections in chalcone units A and B. HMBC correla-
tions among H-80/C-14, H-15/C-80, and H-20(60)/C-70 indicated that
chalcone units A and B fused at C-80 and C-14 to form a dihydrofuran
ring. To confirm the relative stereochemistry, NOESY experiments
were performed, and NOE correlations of H-80/H-20(60) suggested
that the configuration of H-70 and H-80 was in trans. Thus, the relative
stereostructure of 3 was determined and is shown in Figure 1.
The inhibitory activities of pauferrol B (2) and C (3) against topo
II were evaluated by their inhibitory effect against the decatena-
tion activity of topo II on kinetoplast DNA.11 Chalcons 2 and 3 were
subjected to topo II assays at various concentrations. The IC50 val-
ues were determined from three individual experiments with three
replicates for each concentration. Doxorubicin (a clinically used
anti-cancer agent) was used as the positive control, and it exhib-
the aryl ketone in 2 or 3. To confirm the key transformation, we
set a simple model compound 4 as the first target, which contained
the two asymmetric centers of 2 and 3.
After some preliminary studies, we accomplished the synthesis
of 4 (Scheme 1). Fisher esterification of 2-hydroxyphenylacetic acid
(5) and p-benzyloxybenzylation of the phenol followed by hydroly-
sis of the methyl ester afforded carboxylic acid 6. Fukuyama’s
chiral auxiliary derived from methyl (R)-lactate was introduced
using water-soluble carbodiimide hydrochloride. Diazotization of
ited a potent topo II inhibition (IC50 value: 0.7
II assay conditions. Compound 2 and 3 showed significant inhibi-
tory activities (IC50: 15.3 M for 2 and 14.5 M for 3) under the
same conditions, although these activities were weak as compared
with pauferrol A (IC50: 2.1 M). Next, the effects of 2 and 3 on cell
lM) under our topo
l
l
l
proliferation in the human leukemia HL 60 cell line were exam-
ined. Chalcons 2 and 3 also exhibited significant inhibitory effects
on HL 60 cell proliferation with an IC50 value of 11.6 and 12.1
lM
(IC50: 5.2 M for pauferrol A), respectively. The HL 60 cells treated
l
with 2 and 3 displayed characteristics of apoptosis, such as nuclear
condensation and fragmentation, and DNA ladder formation. These
results suggested that 2 and 3 induced apoptosis of HL 60 cells and
consequently inhibited cell proliferation. These findings suggest
that chalcone oligomers are promising lead compounds for anti-
cancer agents.
We planned the total synthesis of 2 and 3 in order to determine
their absolute configurations and investigate structure–activity
relationship. Although enzymatic synthesis of a similar chalcone
dimer (lophirone C) was achieved in 1996,12 the product obtained
there was racemic; therefore, the new enantioselective approach to
this type of compounds had to be developed. Recently, the efficient
syntheses of optically active trans-2-aryl-2,3-dihydrobenzofuran-
3-carboxylic acid esters were reported by Fukuyama and Kan.13
The ester group of them was supposed to be a good precursor for
Scheme 1. Reagent and conditions: (a) MeOH, AcCl, 0 °C to rt (92%); (b) K2CO3, p-
benzyloxybenzyl chloride, TBAI, DMF, 60 °C (65%); (c) KOH, THF/MeOH/H2O, rt
(72%); (d) Pyrrolidinyl-(R)-lactamide, EDCIꢁHCl, DMAP, CH2Cl2, 0 °C to rt (60%); (e)
TsN3, DBU, CH3CN 0 °C to rt (94%); (f) Rh2(Oct)4, CH2Cl2, 0 °C to rt (60%, dr 8:1); (g)
Ba(OH)2ꢁ8H2O, THF/MeOH, 0 °C to rt (55%); (h) Me(MeO)NHꢁHCl, DIPEA, HBTU,
CH2Cl2, 40 °C (69%); (i) 1-Bromo-2,4-dibenzyloxybenzene, n-BuLi, THF, ꢀ78 °C
(53%); (j) 10% Pd/C, H2, THF/MeOH, rt (94%).

S. Ohira et al. / Tetrahedron Letters 54 (2013) 5052–5055
5055
Acknowledgments
We thank Professor H. Akashi (Okayama University of Science)
and Dr. H. Sato (Rigaku Corporation, Japan) for X-ray diffraction
analyses.
Scheme 2. Reagent and conditions: (a) Ba(OH)2ꢁ8H2O, THF/MeOH, 0 °C to rt (55%);
(b) 10% Pd/C, H2, THF/MeOH, rt (94%); (c) MeOH, AcCl, 0 °C to rt (71%); (d) p-BrBzCl,
DMAP, CH2Cl2, rt (71%).
References and notes
1. Wang, J. C. Annu. Rev. Biochem. 1985, 54, 665–697.
2. D’Arpa, P.; Liu, L. F. Biochem. Biophys. Acta 1989, 989, 163–177.
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K.; Iimuma, M.; Nozaki, H. Phytochemistry 2006, 67, 307–313.
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Hoshino, T.; Tsutsui, K.; Tsutsui, K.; Iimuma, M.; Nozaki, H. Chem. Pharm. Bull.
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5. Nozaki, H.; Hayashi, K.; Kido, M.; Kakumoto, K.; Ikeda, S.; Matsuura, N.; Tani,
H.; Takaoka, D.; Iinuma, M.; Akao, Y. Tetrahedron Lett. 2007, 48, 8290–8292.
6. Jarvinen, T. A.; Tanner, M.; Rantanen, V.; Barlund, M.; Borg, A.; Grenman, S.;
Isola, J. Am. J. Pathol. 2000, 156, 839–847.
the active methylene of 7 with p-toluenesulfonyl azide proceeded
smoothly to give diazo compound 8. C–H insertion of rhodium
carbenoid was achieved using rhodium (II) octanoate dimer14 as
the catalyst to afford an 8:1 inseparable diastereomeric mixture
of trans-2-aryl-2,3-dihydrobenzofuran-3-carboxylic acid ester (9
and the isomer) in 60% yield.15 The relative stereochemistry on
the dihydrobenzofuran ring of 9 was confirmed by the NOE and
the cis isomers of 9 were not observed. Hydrolysis of 9 followed
by condensation with N,O-dimethylhydroxylamine afforded the
Weinreb amide 10. Reaction of 10 with the aryl lithium which
was generated from 1-bromo-2,4-dibenzyloxybenzene proceeded
smoothly to give aryl ketone 11 in 53% yield. Hydrogenolysis of
11 produced 4 in high yield.
7. Chen, G.; Shu, J.; Stacey, D. W. Oncogene 1997, 15, 1643–1651.
8. Hashimoto, G. Illustrated Cyclopedia of Brazilian Medical Plant; Aboc-sha Co. Ltd:
Japan, 1996. pp 1–646.
9. Pauferrol B (2), yellow powder, mp 140 °C; [
a
]
D +190.3°(c 0.5, MeOH); UV
); 214.2 (4.43), 228.0 (4.30), 229.2 (4.38), 285.2 (4.13),
mmax; 3337, 2933, 1701, 1636, 1514, 1442, 1362, 1281,
(MeOH) kmax nm (log
e
370.8 (4.39); IR (KBr)
1229, 1102, 1057, 1011, 972 cmꢀ1; HR-FABMS m/z: 511.1385 [M+H]+ (calcd for
C
30H23O8,
10. Pauferrol C (3), yellow amorphous powder, mp 198–200 °C; [
MeOH); UV (MeOH) kmax nm (log ); 215.0 (4.49), 229.8 (4.44), 283.0 (4.29),
324.6 (4.22), 375.8 (4.31); IR (KBr) max; 3400, 2923, 1699, 1634 1514, 1443,
1362, 1231, 1122, 975 cmꢀ1; HR-FABMS m/z: 533.1190 [M+Na]+ (calcd for
30H22O8Na, 2.2 mmu).
D0.8 mmu).
a]D +256.4°(c 0.1,
After completion of the synthesis of 4,16 we decided to confirm
the absolute configuration of 9, as the opposite stereoselectivity to
Fukuyama’s results in the C–H insertion reaction was reported
with a similar substrate.17 Benzyl ether 9 was transformed to
p-bromobenzyl ether 12 which yielded a good crystal (Scheme 2).
X-ray diffraction analysis of 12 confirmed the absolute configura-
tion of intermediate 9.18
Optical rotation of 4 (ꢀ253°) was opposite to the natural prod-
ucts 2 and 3. Since the dihydrobenzofuran ring with two stereo-
genic centers may largely contribute to the rotation of these
compounds, absolute configurations of both the natural products
would be opposite to that of 4. Comparison of CD spectra of 4 (neg-
ative Cotton effect) and natural products (both positive Cotton ef-
fects) also supported this inspection. Thus, absolute configurations
of Pauferrol B and C were estimated as 2 and 3 as shown in
Figure 1. No biological activity was observed for 4, under similar
conditions to 2 and 3.
e
m
C
D
11. Miller, K. G.; Liu, L. F.; England, P. T. J. Biol. Chem. 1981, 256, 9334–9339.
12. Shimamura, T.; Arakawa, Y.; Hikita, K.; Niwa, M. Heterocycles 1996, 43, 2223–
2227.
13. (a) Kurosawa, W.; Kan, T.; Fukuyama, T. Synlett 2003, 1028–1030; (b)
Kurosawa, W.; Kan, T.; Fukuyama, T. J. Am. Chem. Soc. 2003, 125, 8112–8113;
(c) Kurosawa, W.; Kobayashi, H.; Kan, T.; Fukuyama, T. Tetrahedron 2004, 60,
9615–9628; (d) Koizumi, Y.; Kobayashi, H.; Wakimoto, T.; Furuta, T.;
Fukuyama, T.; Kan, T. J. Am. Chem. Soc. 2008, 130, 16854–16855; (e)
Matsumoto, S.; Asakawa, T.; Hamashima, Y.; Kan, T. Synlett 2012, 1082–1084.
14. (a) Padwa, A.; Austin, D. J. Angew. Chem., Int. Ed. Engl. 1994, 33, 1797–1815; (b)
Taber, D. F.; You, K. K.; Rheingold, A. L. J. Am. Chem. Soc. 1996, 118, 547–556.
15. Use of tetrakis[(S)-(-)-N-(p-dodecylphenylsulfonyl)prolinato]dirhodium (II)
produced a 13:1 diastereomeric mixture in 41% yield. On the other hand,
rhodium (II) acetate dimer gave only a complex mixture.
16. 4, brown solid, mp 82 °C; [
281.8 (5.48), 316.1 (4.24)); IR (MeOH)
1457, 1362, 1277, 1200, 1138, 970 cmꢀ1; HR-FABMS m/z: 349.1077 [M+H]+
(calcd for C21H17O5, 0.1 mmu).
a
]
D
ꢀ253° (c 5.3, MeOH); UV (MeOH) kmax (log
e
);
m
max; 3320, 1627, 1615, 1594, 1516,
D
17. Wang, D.-H.; Yu, J.-Q. J. Am. Chem. Soc. 2011, 133, 5767–5769.
18. Crystallographic data for 12 have been deposited at the Cambridge
Crystallographic Data Center under the reference number CCDC 934518.
Copies of the data can be obtained, free of charge, on application to the
Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK.
Efforts for the total synthesis of 2 or 3 and the derivatives are in
progress, which are based on a similar strategy. Final confirmation
of structures and the structure–activity relationships of these
chalcone oligomers will be reported in due course.