Induction of enantio-selective apoptosis in human leukemia HL-60 cells by (S)-erypoegin K, an isoflavone isolated from Erythrina poeppigiana

Article abstract of DOI:10.1016/j.bmc.2020.115490

Erypoegin K, an isoflavone isolated from the stem bark of Erythrina poeppigiana, has potent apoptosis-inducing effect on human leukemia HL-60 cells. Erypoegin K has a chiral carbon at the C-2′′ position of its furan ring and naturally occurs as a racemic mixture of (S)- and (R)-isomers. In the present study, we semi-synthesized (RS)-erypoegin K from genistein and separated the optical isomers by HPLC using a chiral column to characterize its apoptosis-inducing activity. Apoptotic cell death was assessed by analyzing caspase-3 and caspase-9 activation, nuclear fragmentation, and genomic DNA ladder formation. (S)-erypoegin K showed exclusive anti-proliferative and apoptosis-inducing activity, with an IC₅₀ value of 90 nM, about 50% lower than that of its racemic mixture (175 nM). By contrast, no apoptosis-inducing activity was shown by the (R)-isomer. In addition, methylglyoxal accumulation in the culture medium was observed only in cells treated with (S)-erypoegin K. These results demonstrated that (S)-erypoegin K is a unique bioactive component that has potent apoptosis-inducing activity on HL-60 cells.

Full text of DOI:10.1016/j.bmc.2020.115490

Journal Pre-proofs
Induction of enantio-selective apoptosis in human leukemia HL-60 cells by
(S)-erypoegin K, an isoflavone isolated from Erythrina poeppigiana
Kiyomi Hikita, Satomi Saigusa, Yuto Takeuchi, Haruka Matsuyama, Rina
Nagai, Kuniki Kato, Tomiyasu Murata, Hitoshi Tanaka, Yogesh S. Wagh,
Naoki Asao, Norio Kaneda
PII:
S0968-0896(20)30311-4
https://doi.org/10.1016/j.bmc.2020.115490
BMC 115490
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
4 February 2020
2 April 2020
3 April 2020
Please cite this article as: K. Hikita, S. Saigusa, Y. Takeuchi, H. Matsuyama, R. Nagai, K. Kato, T. Murata, H.
Tanaka, Y.S. Wagh, N. Asao, N. Kaneda, Induction of enantio-selective apoptosis in human leukemia HL-60
cells by (S)-erypoegin K, an isoflavone isolated from Erythrina poeppigiana, Bioorganic & Medicinal Chemistry
(2020), doi: https://doi.org/10.1016/j.bmc.2020.115490
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Induction of enantio-selective apoptosis in human leukemia HL-60 cells by (S)-erypoegin
K, an isoflavone isolated from Erythrina poeppigiana
Kiyomi Hikitaa, Satomi Saigusaa, Yuto Takeuchia, Haruka Matsuyamaa, Rina Nagaia,
Kuniki Katob, Tomiyasu Murataa, Hitoshi Tanakab, Yogesh S. Waghc,1, Naoki Asaoc,d,
Norio Kanedaa,*
a
Laboratory of Analytical Neurobiology, Faculty of Pharmacy, Meijo University, 150
Yagotoyama, Tempaku, Nagoya 468-8503, Japan
b
Laboratory of Natural Product, Faculty of Pharmacy, Meijo University, 150
Yagotoyama, Tempaku, Nagoya 468-8503, Japan
c Advanced Institute for Materials Research, Tohoku University, 2-1-1 Katahira, Aoba-
ku, Sendai 980-8577, Japan
d
Division of Chemistry and Materials, Graduate School of Science and Technology,
Shinshu University, Ueda, Nagano 386-8567, Japan
----------------------------------------------------------------------------------------------------------
*Corresponding author at: Laboratory of Analytical Neurobiology, Faculty of Pharmacy,
Meijo University, 150 Yagotoyama, Tempaku, Nagoya 468-8503, Japan
Email address: nkaneda@meijo-u.ac.jp (N. Kaneda)
1Present address: Eternis Fine Chemicals Ltd., Rabale, Navi Mumbai, India
1

Abstract
Erypoegin K, an isoflavone isolated from the stem bark of Erythrina poeppigiana, has
potent apoptosis-inducing effect on human leukemia HL-60 cells. Erypoegin K has a
chiral carbon at the C-2′′ position of its furan ring and naturally occurs as a racemic
mixture of (S)- and (R)-isomers. In the present study, we semi-synthesized (RS)-
erypoegin K from genistein and separated the optical isomers by HPLC using a chiral
column to characterize its apoptosis-inducing activity. Apoptotic cell death was assessed
by analyzing caspase-3 and caspase-9 activation, nuclear fragmentation, and genomic
DNA ladder formation. (S)-erypoegin K showed exclusive anti-proliferative and
apoptosis-inducing activity, with an IC50 value of 90 nM, about 50% lower than that of
its racemic mixture (175 nM). By contrast, no apoptosis-inducing activity was shown by
the (R)-isomer. In addition, methylglyoxal accumulation in the culture medium was
observed only in cells treated with (S)-erypoegin K. These results demonstrated that (S)-
erypoegin K is a unique bioactive component that has potent apoptosis-inducing activity
on HL-60 cells.
Key words: isoflavone, erypoegin K, anti-proliferative activity, enantio-selective
apoptosis induction, anti-tumor agent, HL-60 cell
2

1. Introduction
Flavones and isoflavones are present in a variety of plants and foods. Most of these
compounds have a wide range of biological activities, such as antimicrobial, antiviral,
antioxidant, anti-proliferative, apoptosis-inducing, antitumor, anti-inflammatory, and
estrogen-like activities.1-6 Numerous studies have attempted to elucidate the antitumor
effect of isoflavones using in vitro and in vivo models of various types of malignant
diseases, such as leukemia,3 lymphoma,7 prostate cancer,8 breast cancer,9 and hepatic10
cancer. The mechanism of action on cancer cells includes isoflavone-induced cell cycle
arrest and apoptosis through the regulation of relevant genes and antioxidants, DNA
repair, inhibition of angiogenesis and metastasis, potentiation of radio- and
chemotherapeutic agents, and antagonism of estrogen- and androgen-mediated signaling
pathways.8
On the basis of recent findings from molecular and cellular biological studies on the
effect of isoflavones, we focused on the anti-proliferative and cytotoxic activity of
isoflavones, aiming to develop novel compounds for use as antitumor drugs. Our first
strategy was to isolate known and unknown polyphenolic compounds from plants and to
examine their inhibitory activity against glyoxalase I (GLO-I; EC 4.4.1.5) .11 GLO-I is a
rate-limiting enzyme involved in the degradation and detoxification of a glycolytic by-
product, methylglyoxal (MG) .12,13 Inhibition of GLO-I leads to the accumulation of the
MG substrate, which is a toxic compound known to interact with proteins, lipids, and
nucleic acids, and the induction of apoptosis in various types of tumor cells.14,15
Previously, we have isolated several types of isoflavones from the stem bark of the
Leguminosae family member, Erythrina poeppigiana, which showed inhibitory activities
against purified human recombinant GLO-I and the growth of HL-60 human leukemia
cells.11,16,17 Among the isoflavones isolated, erypoegin K showed the most potent anti-
proliferative and apoptosis-inducing activity in HL-60 cells, but not in normal human
peripheral lymphocytes.17 This compound is present in the plant in a limited amount, as
a racemic mixture composed of the two optically active isomers, (S)- and (R)-erypoegin
K, based on the chiral carbon at the C-2′′ position of its furan ring.
In the present study, to assess whether one or both optical isomers possess anti-
proliferative and apoptosis-inducing activity, we semi-synthesized (RS)-erypoegin K
using genistein as the starting material. After confirming the structural and biological
equivalence between the synthesized product and the compound isolated from the natural
3

source, we separated each optical isomer by HPLC using a chiral column. Using purified
optical isomers, we found that (S)-erypoegin K enantio-selectively exhibited anti-
proliferative and apoptosis-inducing activity against HL-60 cells.
2. Materials and Methods
2.1 Synthesis of (RS)-erypoegin K
2.1.1 General
All reactions were performed in oven-dried glassware under a positive pressure of argon,
with magnetic stirring. Air-sensitive reagents and solutions were introduced to the
apparatus via a syringe through a rubber septum. All reagents, starting materials, and
solvents were obtained from commercial suppliers and were used without further
purification. Reactions were monitored by TLC with 0.25 mm pre-coated silica gel plates
(60 F254). Visualization was accomplished with either UV light, or by immersion in a
KMnO4 solution, followed by heating. All compounds were purified by column
chromatography using silica gel 60 N (spherical, neutral, 40–100 μm; KANTO Chemical
Co., Tokyo, Japan) and silica gel 60 (Merck, Darmstadt, Germany). The melting point
was measured using a model MPA100 automated melting point apparatus, with capillary
tubes closed at one end. Infrared (IR) spectra were recorded on an FT/IR-6600 apparatus
(JASCO, Tokyo, Japan). 1H NMR spectra were recorded on a model JNM AL 400 (400
1
MHz) spectrometer (JEOL, Tokyo, Japan). H NMR spectra are reported as follows:
chemical shift in ppm (δ) relative to the chemical shift of CDCl3 at 7.26 ppm and acetone-
d6 at 2.04 ppm, integration, multiplicities (s = singlet, d = doublet, t = triplet, q = quartet,
13
m = multiplet, and br = broad signal), and coupling constants (Hz). C NMR spectra
were recorded on a model JNM AL 400 (100.5 MHz) spectrometer (JEOL) with complete
proton decoupling, and the chemical shift is reported in ppm (δ) relative to the central line
for CDCl3 at 77 ppm and acetone-d6 at 29.8 ppm. High-resolution mass spectra (HRMS)
were obtained on an APEXIII spectrometer (Bruker, Billerica, MA, USA) and a JMS-700
MStation (JEOL).
2.1.2 Preparation of 4-(7-acetoxy-5-hydroxy-4-oxo-4H-chromen-3-yl) phenyl
acetate (3)
Synthesis of 3 was achieved as previously described,18 with some modifications. Ac2O
(14.8 mmol) was added dropwise to a solution of genistein (2) (2 g, 7.4 mmol) in pyridine
(40 mL) under argon at 0 °C. The resulting dark-colored solution was stirred for 24 h at
4

room temperature and then poured into ice cold water. The precipitated yellow solid was
filtered, dried, and recrystallized in acetone/EtOH (3:1) to give 3 (2.25 g, 6.35 mmol), as
a slight yellowish solid with an 86% yield. mp 203–206 °C; IR (KBr) cm-1: 3077, 1751,
1
1654, 1589, 1542, 1490, 1251, 1128, 1047, 990, 897; H NMR (400 MHz, CDCl3) δ:
12.74 (s, 1H), 7.97 (s, 1H), 7.54 (d, J = 8.2 Hz, 2H), 7.19 (d, J = 8.2 Hz, 2H), 6.76 (d, J
= 2.0 Hz, 1H), 6.59 (d, J = 2.0 Hz, 1H), 2.33 (s, 3H), 2.32 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ: 180.9, 169.4, 168.3, 162.4, 156.8, 156.0, 153.4, 150.9, 130.0, 127.9, 123.6,
121.9, 109.4, 105.6, 100.9, 21.2, 21.1; HRMS calculated for C19H14O7 [M + Na]+
377.06317, found 377.06312.
2.1.3 Preparation of 4-(7-acetoxy-5-((3-methylbut-2-en-l-yl)-4-oxo-4H-chromen-3-
yl) phenyl acetate (4)
Synthesis of 4 was achieved as previously described,18 with some modifications. A
mixture of 3 (500 mg, 1.41 mmol) and triphenylphosphine (PPh3, 666 mg, 2.54 mmol)
was dissolved in CH2Cl2 (10 mL) and sonicated for 10 min at 25 °C. To this solution, 3-
methyl-2-buten-1-ol (182 mg, 2.11 mmol) and diisopropyl azodicarboxylate (DIAD, 513
mg, 2.54 mmol) in CH2Cl2 (5 mL) were added dropwise over 10 min at -10 °C. The
mixture was then stirred for 2 h at -10 °C and for 6 h at room temperature. The solvent
was removed in vacuo, and the resulting thick yellow liquid was purified by silica gel
column chromatography, with EtOAc/hexane (20:80) as an eluent, to give 4. Further
purification by recrystallization in EtOH gave 4 (464 mg, 1.09 mmol) as a white solid
with a 78% yield. mp 164–168 °C; IR (KBr) cm-1: 2913, 1750, 1611, 1542, 1477, 1304,
1250, 1173, 1112, 1045, 1012, 908; 1H NMR (400 MHz, CDCl3) δ: 7.81 (s, 1H), 7.55 (d,
J = 8.7 Hz, 2H), 7.11 (d, J = 7.8 Hz, 2H ), 6.83 (d, J = 2.0 Hz, 1H), 6.57 (d, J = 2.0 Hz,
1H), 5.60- 5.53 (m, 1H), 4.64 (d, J = 6.3 Hz, 2H), 2.33 (s, 3H), 2.30 (s, 3H), 1.76 (s, 3H),
13
1.70 (s, 3H). C NMR (100 MHz, CDCl3) δ: 174.9, 169.4, 168.3, 160.6, 158.6, 154.3,
150.9, 150.5, 138.1, 130.3, 129.4, 125.8, 121.4, 118.8, 113.4, 102.8, 102.3, 66.7, 25.7,
21.1, 21.0, 18.3; HRMS calculated for C24H22O7 [M + Na]+ 445.12577, found 445.12574.
2.1.4 Preparation of 4-(7-acetoxy-5-hydroxy-8-(3-methylbut-2-en-1-yl)-4-oxo-4H-
chromen-3-yl) phenyl acetate (5)
Synthesis of 5 was achieved as previously described,18 with some modifications. A
5

solution of 4 (1 g, 2.36 mmol) and Eu(fod)3 (246 mg, 0.236 mmol) in dry CHCl3 (2 mL)
was heated under gentle reflux for 24 h, with stirring under argon gas. The solvent was
removed in vacuo, and the resulting thick yellow liquid was purified by silica gel flash
chromatography with EtOAc/hexane (25:75) as an eluent, to give 5 (610 mg, 1.44 mmol)
as a light-yellow solid with a 61% yield. mp 143–145 °C; IR (KBr) cm-1: 3065, 2932,
1
1742, 1686, 1654, 1647, 1482, 1357, 1206, 1175, 1058, 923, 880 cm-1; H NMR (400
MHz, CDCl3) δ: 12.63 (s, 1H), 8.02 (s, 1H), 7.56 (d, J = 8.3 Hz, 2H ), 7.18 (d, J = 8.3 Hz,
2H), 6.59 (s, 1H), 5.15-5.07 (m, 1H), 3.38 (d, J = 6.8 Hz, 2H), 2.34 (s, 3H), 2.33 (s, 3H),
13
1.78 (s, 3H), 1.76 (s, 3H); C NMR (100 MHz, CDCl3) δ: 181.3, 169.4, 168.4, 159.8,
154.8, 154.6, 153.7, 150.9, 132.7, 130.0, 128.0, 123.4, 121.9, 120.9, 112.9, 109.9, 106.4,
25.6, 22.5, 21.1, 20.9, 17.8; HRMS calculated for C24H22O7 [M + Na]+ 445.12577, found
445.12575.
2.1.5 Preparation of lupiwighteone (6)
Synthesis of 6 was achieved as previously described,18 with some modifications. To a
suspension of 5 (1 g, 2.36 mmol) in a mixture of MeOH/THF (1:1, 25 mL), 10% aqueous
NaHCO3 (50 mL) was added. The mixture was stirred for 2 h at 60 °C and overnight at
room temperature. After evaporation of the solvent to half the volume under reduced
pressure, 2% aq. HCl (5 mL) was added to the mixture. The mixture was then extracted
with EtOAc, and the combined organic layers were dried over MgSO4 and filtered, after
which the solvent was removed under reduced pressure. The resulting yellow solid was
purified by silica gel flash column chromatography with CH2Cl2/MeOH (50:1) as an
eluent, to give 6 (632 mg, 1.86 mmol) as a white solid with a 79% yield. mp 134–136 °C;
1
IR (KBr) cm-1: 3527, 3173, 2913, 1660, 1614, 1514, 1365, 1254, 1178, 1064, 829; H
NMR (400 MHz, acetone-d6) δ: 12.98 (s, 1H), 9.14 (brs, 2H), 8.24 (s, 1H ), 7.47 (d, J =
8.3 Hz, 2H), 6.90 (d, J = 8.3 Hz, 2H), 6.36 (s, 1H), 5.28-5.20 (m, 1H), 3.43 (d, J = 6.8
13
Hz, 2H), 1.80 (s, 3H), 1.65 (s, 3H); C NMR (100 MHz, acetone-d6) δ: 206.3, 181.9,
162.1, 161.4, 158.3, 156.2, 154.3, 132.0, 131.1, 123.6, 123.1, 123.0, 115.9, 107.1, 106.2,
99.3, 25.8, 21.8, 17.8; HRMS calculated for C20H18O5 [M + Na]+ 361.10464, found
361.10460.
2.1.6 Preparation of (RS)-erypoegin K (1)
To a solution of 6 (169 mg, 0.5 mmol) in CH2Cl2 (15 mL), K2CO3 (207 mg, 1.5 mmol)
6

and metachloroperoxybenzoic acid (m-CPBA, 160.24 mg, 0.65 mmol) were added. The
reaction mixture was stirred at room temperature under an argon atmosphere for 12 h.
After filtration of the mixture, the residue was washed with CH2Cl2 many times. The
filtrate was evaporated in vacuo to obtain the crude product, which was purified by silica
gel flash column chromatography with EtOAc/hexane (40:60) as an eluent to give 1 (125
mg, 0.35 mmol) with a 71% yield. mp 251–254 °C; IR (KBr) cm-1: 3487, 3403, 3281,
3078, 2978, 1900, 1831, 1671, 1615, 1575, 1479, 1252, 1170, 1068, 958, 827; 1H NMR
(400 MHz, acetone-d6) δ: 13.23 (s, 1H), 8.51 (s, 1H), 8.20 (s, 1H ), 7.40 (d, J = 8.3 Hz,
2H), 6.90 (d, J = 8.7 Hz, 2H), 6.20 (s, 1H), 4.89-4.83 (m, 1H), 3.84 (s, 1H), 3.35-3.21 (m,
2H), 1.30 (s, 3H), 1.25 (s, 3H); 13C NMR (100 MHz, acetone-d6) δ: 181.6, 167.6, 164.3,
158.4, 153.9, 153.6, 131.2, 124.0, 123.0, 115.9, 106.2, 104.7, 94.4, 92.7, 71.4, 29.2, 27.1,
25.9; HRMS calculated for C20H18O6 [M + Na]+ 377.09956, found 377.09954. 1H and 13C
NMR spectra of (RS)-erypoegin K (1) are shown in Fig. S1 and Fig. S2, respectively.
(R)-erypoegin K: mp 247-250 °C; IR (KBr) cm-1: 3488, 3403, 3078, 2978, 2696, 1832,
1774, 1671, 1618, 1572, 1479, 1291, 1203, 1252, 1171, 1025, 959, 841, 826.
(S)-erypoegin K: mp 246 - 248 °C; IR (KBr) cm-1: 3487, 3406, 3077, 2978, 2929, 2803,
2697, 1900, 1831, 1671, 1619, 1574, 1480, 1291, 1171, 1068, 959, 826.
2.2 HPLC using a chiral column
(R)- and (S)-erypoegin K were separated from the synthesized racemic mixture on a
chiral column of CHIRALPAK AD-H (10 mm × 25 cm; Daicell, Osaka, Japan), under
the following conditions: mobile phase, isoPrOH/hexane (30:70); flow rate, 4 mL/min;
and absorbance at 254 nm. Under these conditions, the retention times of (R)- and (S)-
erypoegin K were 11.4 min and 19.2 min, respectively. The solvent was removed from
each fraction by rotary evaporation.
2.3 Specific optical rotation
Samples were dissolved in acetone to a concentration of 1.9 mg/mL. Specific optical
rotation []D23 was measured using a model P-2200 polarimeter (JASCO) at 25°C.
2.4 Cell culture
Human acute promyelocytic leukemia HL-60 cells were obtained from the RIKEN cell
bank (Riken, Tokyo, Japan). Cells were cultured in RPMI-1640 medium supplemented
7

with 10% fetal bovine serum, 100 units/mL penicillin G sodium salt, and 100 μg/mL
streptomycin sulfate, at 37°C in a humidified atmosphere of 5% CO2 and 95% air.
2.5 Cell viability assay
Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTS) assay (CellTiter96 AQueous one solution cell proliferation assay kit;
Promega, Madison, WI, USA), as described previously.13 Briefly, HL-60 cells in the
logarithmic phase of growth were plated in a 96-well plate (Corning, Corning, NY, USA)
at a density of 1.2 × 104 cells/well. After 24 h of preincubation, the cells were treated with
(S)- or (R)-erypoegin K and cultured for 48 h under the conditions described above. A
stock solution of erypoegin K was prepared in DMSO, at a concentration of 2.5 mM, and
the final DMSO concentration in the cell culture medium was maintained at < 0.4%. The
water-soluble colored formozan product, tetrazolium, produced by mitochondrial
dehydrogenase, was measured at 492 nm using a Multiscan FC device (Thermo Fisher
Scientific, Waltham, MA, USA). DMSO was used as a negative control.
2.6 Caspase assay
The enzymatic activity of caspase-3 and caspase-9 in HL-60 cells was assayed using a
caspase-3/9 fluorometric assay kit (R&D Systems Inc., Minneapolis, MN, USA),
according to the manufacturer’s instructions. HL-60 cells were plated in a 24-well plate
(Corning) at a density of 5.0 × 105 cells/well. After 24 h, the cells were treated with the
test compounds for the indicated time periods (h). Subsequently, the cells were washed
twice with phosphate buffered saline (PBS (-)) and were lysed using the lysis buffer
supplied in the kit. Caspase-3 activity in the cell lysates was then fluorometrically
measured using a Wallac 1420 ARVOsx microplate counter, at an excitation
wavelength of 390 nm and an emission wavelength of 510 nm (PerkinElmer, Waltham,
MA, USA).
2.7 Detection of nuclear condensation and fragmentation
HL-60 cells were plated in a 24-well plate at a density of 8.0 × 105 cells/0.3 mL/well
and treated with 5 M erypoegin K for 6 h. The cells were then collected into a 1.5-mL
microtube by a brief centrifugation. Hoechst 33342 (final concentration: 14.3 g/mL;
Dojindo, Kumamoto, Japan) was then added, and cells were stained for 30 min in a CO2
8

incubator at 37 ℃. After fixation of the cells with an equal volume of 10%
paraformaldehyde in PBS (-) at room temperature for 30 min, cells were collected and
washed twice with PBS (-). Cells were mounted with VECTASHIELD medium (Vector
Laboratories, Burlingame, CA, USA), transferred to a slide glass, and observed with an
Axiophoto 2 fluorescence microscope, using an excitation wavelength of 352 nm and an
emission wavelength of 461 nm (Carl Zeiss, Jena, Germany).
2.8 Genomic DNA fragmentation
HL-60 cells were plated in a 3.5 cm dish (Corning) at a density of 2.0 × 105
cells/mL/dish and treated with 1 M and 3 M each of (S)- or (R)-erypoegin K for 5 h.
The cells were then collected into a 1.5-mL microtube by a brief centrifugation.
Genomic DNA was extracted from the cell pellet using an apoptosis ladder detection kit
(Wako Pure Chemical Industries, Osaka, Japan), according to the manufacturer’s
instructions. The DNA fragments were separated by 1.5% agarose gel electrophoresis,
stained with ethidium bromide, and observed with a UV transilluminator.
2.8 Measurement of MG
MG levels were measured in the culture medium as previously described,20 with some
modifications as described in detail13. Briefly, HL-60 cells were plated in a 6-cm dish at
a density of 1.5 × 106 cells/dish and preincubated for 24 h. The cells were then treated
with 1 M and 3 M each of (S)- or (R)-erypoegin K for 8 and 24 h. The culture
medium was collected and deproteinized using perchloric acid (final concentration: 0.5
M). After centrifugation, the supernatant was purified using a Sep-Pack Light tC18
cartridge (Waters, Milford, MA, USA) and subjected to derivatization with o-
phenylenediamine for 4 h at room temperature. The product, 2-methylquinoxaline (2-
MQ), was further purified on the Sep-Pack Light tC18 cartridge and analyzed by
reversed-phase HPLC with UV detection (absorbance at 315 nm), using 5-MQ as an
internal standard. The mobile phase consisted of 10 mM KH2PO4 (pH 2.5)/acetonitrile
(80:20).
2.9 Statistical analysis
Data were analyzed in Microsoft Excel 2013 using a Student’s t-test for comparison
between different groups, or in PRISM using one-way ANOVA and Dunnett’s method
9

for multiple comparison between repeated measures. p < 0.05 was considered
significant.
3. Results
3.1 Semi-synthesis of (RS)-erypoegin K
Scheme 1 shows all the steps for the semi-synthesis of (RS)-erypoegin K (1) using
commercially available genistein (2) as the starting material. Lupiwighteone (6) was
successfully prepared from genistein in 4 steps, with an overall yield of 44%.18 We
expected that the benzo-fused five-membered cyclic ether of 1 would be constructed by
the base-mediated 5-exo-tet cyclization of the epoxide intermediate, generated in situ
from 6 with the adjacent phenol group, resulting in the production of the target molecule.
Using a previously described method.18 we synthesized 6 as follows. The
chemoselective acetylation of 2 produced 3 with an 86% yield. 3 was then treated with 3-
methylbut-2-en-1-ol under modified reaction conditions using DIAD instead of diethyl
azodicarboxylate to give 4 at a 73% yield. The Eu(fod)3-catalyzed Claisen rearrangement
gave 5 at a 61% yield, which was hydrolyzed under basic conditions to give 6 at an 81%
yield. We then examined the synthesis of the cyclic ether using this procedure, including
epoxidation followed by 5-exo-tet cyclization. As expected, the reaction proceeded
smoothly with m-CPBA in the presence of K2CO3, and racemic 1 was obtained at a 71%
yield.
The reaction conditions and the yield at each step are described in detail in section 2.1.
The overall yield of 1 from 2 was 23%.
3.2 Biological activity of semi-synthesized (RS)-eryopoegin K
The biological activity of semi-synthesized (RS)-erypoegin K was assessed based on
the inhibition of HL-60 cell proliferation. The cells were treated with various
concentrations of semi-synthesized or natural (RS)-erypoegin K for 48 h. Semi-
synthesized erypoegin K exhibited potent anti-proliferative activity identical to that of
the racemic mixture of erypoegin K isolated from the plant (Fig. S3). The IC50 values of
both were 175 nM, as previously described.17
10

AcO
O
AcO
O
O
HO
O
O
b
a
O
O
OH
OH
OAc
OAc
OH
2
3
4
HO
HO
O
O
AcO
O
O
O
O
O
d
c
e
OH
OH
OH
OH
OAc
OH
5
6
1
Scheme 1. Semi-synthesis of (RS)-erypoegin K (1) from genistein (2)
Reagents and conditions: (a) Ac2O, pyridine, rt, 24 h, 86%; (b) 3-methyl-2-buten-1-ol
(1.5 equiv), DIAD (1.8 equiv.), PPh3 (1.8 equiv.), CH2Cl2, -10 °C to rt, 8 h, 78%; (c)
Eu(fod)3 (0.1 equiv.), CHCl3, 60 °C, 20 h, 61%; (d) 10% aq. NaHCO3, MeOH:THF (1:1),
60 °C to rt, 12 h, 79%; (e) m-CPBA (1.3 equiv.), K2CO3, CH2Cl2, rt, 12 h, 71%.
3.3 Separation of (R)- and (S)-erypoegin K from the racemic mixture
To examine whether either (or both) optical isomers had biological activity, we
separated semi-synthesized racemic erypoegin K into each individual isomer. Fig. 1A
shows elution profile of each isomer on HPLC using a chiral column. Their absolute
configurations were determined by measuring their specific optical rotation. Peak 1
(retention time, 11.4 min) and peak 2 (retention time, 19.2 min) showed []D23 of -89°
and +86°, and were determined to be (R)-erypoegin K and (S)-erypoegin K,
respectively, based on the previously reported []D23 value of a similar isoflavone
compound, DK-M1, which has a 2ʹ-hydroxy group on the B ring of erypoegin K. DK-
M1 is reported to be levorotatory ([D23] -76°), with an R-configuration at the C-2”
position of its furan ring.20 The chemical structures of (S)- and (R)-erypoegin K are
shown in Fig. 1B. The correctness of this assignment was further confirmed by
asymmetric semi-synthesis of each compound from 6 as the starting material
(manuscript in preparation).
11

peak 1
B
A
HO
O
O
O
OH
OH
peak 1 : (R)-erypoegin K
HO
peak 2
O
O
O
OH
OH
peak 2 : (S)-erypoegin K
0
10
20
30 min
Fig. 1 Separation of (R)- and (S)-erypoegin K optical isomers by HPLC using a
chiral column and their chemical structures
A. (R)- and (S)-erypoegin K were separated from a semi-synthesized racemic mixture of
erypoegin K by HPLC, using a chiral column of CHIRALPAK AD-H (10 mm × 25
cm). The peaks were determined to be (R)-erypoegin K (peak 1, retention time: 11.4
min) and (S)-erypoegin K (peak 2, retention time: 19.2 min).
B. Chemical structures of (R)- and (S)-erypoegin K.
3.4 Anti-proliferative and apoptosis-inducing activity of (S)-erypoegin K
The biological effect of each optical isomer on the viability of HL-60 cells was
examined. HL-60 cells were treated with racemic, (S)-, and (R)-erypoegin K at various
concentrations for 48 h. As shown in Fig. 2, (S)-erypoegin K exhibited potent anti-
proliferative activity against HL-60 cells, with an IC50 value of 90 nM, about 50% lower
than that of its racemic mixture (175 nM). By contrast, (R)-erypoegin K exhibited no
anti-proliferative activity. Though Fig. 2 shows the results up to 1 M, the (R)-isomer
showed no effect on cell proliferation at concentrations as high as 50 M (data not
shown).
12

100
100%
racemic
80
80%
racemic
(R)-eypoegin K
(R)-Erypoegin K
60
60%
(S)-eypoegin K
(S)-Erypoegin K
40
40%
20
20%
0
0%
0
0.2
0.4
0.6
0.8
1
Concentration of compounds (µM)
Fig. 2 Effects of racemic, (R)-, and (S)-erypoegin K on HL-60 cell viability
HL-60 cells (1.2 × 104 cells/ well) were treated with various concentrations of racemic,
(R)-, or (S)-erypoegin K for 48 h. The values are presented as the mean ± SE of three
independent experiments. Triplicate measurements were performed in each experiment.
To determine whether cell death induced by (S)-erypoegin K occurred via apoptosis,
nuclear condensation/fragmentation and genomic DNA ladder formation, the two
characteristic features of apoptotic cell death, was assessed in HL-60 cells. As shown in
Fig. 3A, marked nuclear condensation/fragmentation was observed in HL-60 cells
treated with (S)-erypoegin K, whereas the cells treated with (R)-erypoegin K or DMSO
(control) showed no nuclear condensation. As shown in Fig. 3B, (S)-erypoegin K
treatment resulted in genomic DNA ladder formation at concentrations of 1 M and 3
M, whereas (R)-isomer treatment did not.
To further confirm apoptotic cell death induced by (S)-erypoegin K, caspase-3 and -9
activity was measured in HL-60 cells treated with (S)- and (R)-erypoegin K. As shown
in Fig. 4A (caspase-3) and Fig. 4B (caspase-9), treatment with (S)-erypoegin K induced
marked activation of caspase-3 and -9 in HL-60 cells in a time- and dose-dependent
manner, whereas (R)-erypoegin K did not activate these enzymes. These results
13

A
B
M 1 2
3
4
5
(S)-erypoegin K
(R)-erypoegin K
Control
Fig. 3 Nuclear condensation/fragmentation and genomic DNA ladder formation
A. Representative microscopy images of nuclear condensation/fragmentation. HL-60
cells were treated with 5 M (S)-erypoegin K or (R)-erypoegin K or DMSO as a control
for 6 h, followed by staining with Hoechst 33342.
B. Genomic DNA ladder formation. Genomic DNA was extracted from the cells treated
with the test compounds for 5 h and analyzed by 1.5% agarose gel electrophoresis. Lane
1, DMSO (control); lane 2, 1 M (S)-erypoegin K; lane 3, 3 M (S)-erypoegin K; lane
4, 1 M (R)-erypoegin K; lane 5, 3 M (R)-erypoegin K. M, 100-bp DNA size marker.
suggested that (S)-erypoegin induced apoptotic cell death in HL-60 cells, at least in part,
via the mitochondrial pathway.
3.5 Production of MG in HL-60 cells treated with (S)-erypoegin K
In our previous study, MG was secreted at high levels into the culture medium of HL-
60 cells after treatment with a racemic mixture of erypoegin K obtained from a natural
product.17 Treatment with this compound resulted in higher MG levels than treatment
with a known GLO-I inhibitor, S-p-bromobenzylglutathione cyclopentyl diester
(BBGC). To examine the effect of each optical isomer on MG production, we measured
MG levels in the culture medium of HL-60 cells treated with (S)- or (R)-erypoegin K.
As shown in Fig. 5, treatment with the (S)-isomer resulted in a marked accumulation of
14

A
B
14000
12000
10000
8000
6000
4000
2000
0
3000
2500
2000
1500
1000
500
control
control
Control
Control
**
(S)-EpK 0.1 µM
（S)-EryK 0.1µM
(S)-EpK 0.1 µM
（S)-EryK 0.1µM
**
(S)-EpK 0.5 µM
（S)-EryK 0.5µM
(S)-EpK 0.5 µM
（S)-EryK 0.5µM
(R)-EpK 0.1 µM
(R)-EpK 0.1 µM
(R)-EpK 0.1 µM
(R)-EpK 0.1 µM
((RR))--EEppKK 00..55µµMM
(R)-EpK 0.5 µM
(R)-EpK 0.5 µM
**
**
*
0
0
3
6
9
12 15 18 21 24
Time (h)
0
3
6
9
12 15 18 21 24
Time (h)
Fig. 4 Effect of (S)-, and (R)-erypoegin K on caspase-3 and caspase-9 activity in
HL-60 cells
HL-60 cells were treated with (S)- or (R)-erypoegin K at concentrations of 0.1 and 0.5
M. After incubation for the indicated time periods, enzyme activities of caspase-3 (A)
and caspase-9 (B) in cell extract were assayed using each specific fluorescent substrate.
Enzyme activity is expressed as relative fluorescence intensity in an arbitrary unit. The
values are presented as the mean ± SE of four independent experiments. Triplicate
measurements were performed in each experiment. The significance of differences was
evaluated using one-way ANOVA and Dunnett’s method. *p < 0.05, **p < 0.01 vs.
control. S-EpK, (S)-erypoegin K; R-EpK, (R)-erypoegin K.
MG principally in a time- and dose-dependent manner, whereas treatment with the (R)-
isomer showed no such effect. A lower level of MG accumulation than that expected in
the treatment with 3 M (S)-erypoegin K for 24 h may be attributed to the enhanced cell
death induced by the compound.
3.6 Effect of (S)- and (R)-erypoegin K on purified GLO-I enzyme activity
Considering that MG was only detected in the culture medium of cells treated with (S)-
erypoegin K, it was plausible that the production of MG was due to the selective
inhibition of GLO-I by (S)-erypoegin K. To confirm this, we examined the effect of (S)-
erypoegin K on the enzyme activity of purified human GLO-I produced using an
Escherichia coli expression system, as previously described.11 Unexpectedly, there was
no significant difference in GLO-I-inhibitory activity between the two isomers (Fig.
15

S4). Their IC50 values for enzyme inhibition were both approximately 100 M, which
coincided with the IC50 value of the racemic mixture (109.4 M), as previously
described.11 These data suggested that the selective anti-proliferative and apoptosis-
Fig. 5 Effect of (S)- and (R)-erypoegin K on the production of MG in HL-60 cells.
0.6
*
**
8 h
**
0.5
24 h
0.4
0.3
0.2
0.1
0
DMSO
1 µM
3 µM
1 µM
3 µM
(S)-erypoegin K
(R)-erypoegin K
HL-60 cells were treated with (S)- and (R)-erypoegin K (1 or 3 M), and cultured for 8
or 24 h. Subsequently, MG level in the culture medium was determined by the o-
phenylenediamine derivatization and HPLC/UV method. The values are presented as
the mean ± SE of four independent experiments. Triplicate measurements were
performed in each experiment. The significance of differences was evaluated using one-
way ANOVA and Dunnett’s method. *p < 0.05, **p < 0.01 vs. control. MG:
methylglyoxal.
inducing activity of (S)-erypoegin K may be mediated by a mechanism other than the
inhibition of GLO-I.
4. Discussion
In our previous study, we isolated an isoflavone, designated erypoegin K, from the stem
bark of Erythrina poeppigiana. Erypoegin K showed potent anti-proliferative and
16

apoptosis-inducing activities against human leukemia HL-60 cells, but not against normal
human peripheral lymphocytes.17 In the present study, we semi-synthesized erypoegin K
(1) as a racemic mixture, from genistein (2) as the starting material, with a final yield of
23%. The product, (RS)-erypoegin K, showed bioequivalence with the natural compound,
as indicated by the identical IC50 values (175 nM) against HL-60 cell proliferation.
Chemical synthesis and chiral separation of (S)- and (R)-erypoegin K made it possible
to examine their activity against tumor cells. Using the purified isomers, anti-proliferative
and apoptosis-inducing activities in HL-60 cells were found to be exclusive to (S)-
erypoegin K. Cell death was induced by apoptosis, as demonstrated by (S)-erypoegin K-
selective activation of caspase-3 and -9, nuclear condensation/fragmentation, and
genomic DNA ladder formation. By contrast, the (R)-isomer showed no anti-proliferative
or apoptosis-inducing activity. These data also suggested that apoptosis induced by (S)-
erypoegin K was mediated at least partly by the mitochondrial signaling pathway.
MG is a carbonyl compound produced as a by-product of glycolysis and other pathways.
MG is highly toxic to cells, because it disturbs cellular function and metabolism through
the formation of covalent adducts with proteins, lipids, and nucleic acids.13 Since GLO-I
is a rate-limiting enzyme for the detoxification of MG, inhibition of GLO-I is expected
to induce apoptosis, via accumulation of toxic MG substrate. We have previously reported
that treatment of HL-60 cells with racemic erypoegin K induces accumulation of MG in
the culture medium of the treated cells, with MG levels being comparable to or even
higher than after treatment with BBGC, a known GLO-I inhibitor.17 In the present study,
we observed that HL-60 cell death and the accumulation of MG both occurred exclusively
with (S)-erypoegin K treatment (Fig. 2 and 5). However, a marked discrepancy occurred
between the two IC50 values; i.e., 109.4 M, for inhibition of the purified GLO-I enzyme11
and 175 nM, for cytotoxicity against HL-60 cells,17 the latter being 625-fold lower. These
results suggested that the potent cytotoxic activity of (RS)-erypoegin K cannot be
explained by a simple mechanism involving GLO-I inhibition and the subsequent
accumulation of MG in the culture medium. In agreement with this notion, (S)- and (R)-
isomers exhibited no significant difference in the inhibition of purified human GLO-I
enzyme activity (Fig. S4). This strongly suggested that although the production and
accumulation of MG clearly correlated with enantio-selective anti-proliferative and
apoptosis-inducing activity of (S)-erypoegin K, it may be attributable to some
intracellular mechanism(s) other than the inhibition of the glyoxalase system, which
17

includes GLO-I. To further study the detailed mechanisms underlying the bioactivity of
(S)-erypoegin K, screening for possible (S)-erypoegin K target molecules by affinity-bead
technology and tumor cell-panel profiling analysis is currently underway. The availability
of active (S)- and inactive (R)-isomers of erypoegin K provides us with a specific tool to
investigate the action of these compounds in tumor cells.
In conclusion, the isoflavone (S)-erypoegin K isolated from Erythrina poeppigiana
exhibited potent and enantio-selective anti-proliferative and apoptosis-inducing activities
against HL-60 cells. (S)-erypoegin K may be a useful compound to use for the
development of tumor-selective antitumor agents.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was partly supported by JSPS KAKENHI from the Ministry of Education,
Culture, Sports, Science, and Technology (MEXT), Japan [grant number
JP16K08311(C)]; the “Nanotechnology Platform” of the MEXT of Japan, at the Center
for Integrated Nanotechnology Support, Tohoku University [S-14-TU-0003, S-15-TU-
0001, S-16-TU-0001] and at Shinshu University [S-17-SH-0017]; and the Research
Institute of Meijo University (2017).
We would like to thank Editage (www.editage.com) for English language editing.
Appendix A. Supplementary Data
Supplementary data associated with this article can be found in the online version.
18

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11. Hikita K, Yamada S, Shibata R, Katoh M, Murata T, Kato K, Tanaka H, Kaneda N.
Phenolic constituents from stem bark of Erythrina poeppigiana and their inhibitory
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21

Supplementary Data
Induction of enantio-selective apoptosis in human leukemia HL-60 cells by (S)-erypoegin
K, an isoflavone isolated from Erythrina poeppigiana
Kiyomi Hikitaa, Satomi Saigusaa, Yuto Takeuchia, Haruka Matsuyamaa, Rina Nagaia,
Kuniki Katob, Tomiyasu Murataa, Hitoshi Tanakab, Yogesh S. Waghc,1, Naoki Asaoc,d,
Norio Kanedaa,*
a
Laboratory of Analytical Neurobiology, Faculty of Pharmacy, Meijo University, 150
Yagotoyama, Tempaku, Nagoya 468-8503, Japan
b
Laboratory of Natural Product, Faculty of Pharmacy, Meijo University, 150
Yagotoyama, Tempaku, Nagoya 468-8503, Japan
c Advanced Institute for Materials Research, Tohoku University, 2-1-1 Katahira, Aoba-
ku, Sendai 980-8577, Japan
d
Division of Chemistry and Materials, Graduate School of Science and Technology,
Shinshu University, Ueda, Nagano 386-8567, Japan
----------------------------------------------------------------------------------------------------------
*Corresponding author: Laboratory of Analytical Neurobiology, Faculty of Pharmacy,
Meijo University, 150 Yagotoyama, Tempaku, Nagoya 468-8503, Japan
Email address: nkaneda@meijo-u.ac.jp (N. Kaneda)
1Present address: Eternis Fine Chemicals Ltd., Rabale, Navi Mumbai, India
22

Fig. S1 1H NMR spectrum of semi-synthesized (RS)-erypoegin K
23

Fig. S2 13C NMR spectrum of semi-synthesized (RS)-erypoegin K
24

100
100%
Semi-synthesized
Natural product
80
80%
60
60%
40
40%
20
20%
0
0%
0
1
2
3
4
5
Concentration of compounds (µM)
Fig. S3 Comparison of anti-proliferative activity of natural and semi-synthesized
(RS)-erypoegin K
Human leukemia HL-60 cells were treated for 48 h with various concentrations of
(RS)-erypoegin K obtained from the natural or semi-synthetic sources. Cell viability
was determined using the MTS-based assay, as previously described.1 The data are
presented as the mean ± SE of three independent experiments. Triplicate measurements
were performed in each experiment.
Results
As shown in Fig. S3, semi-synthesized (RS)-erypoegin K exhibited potent anti-
proliferative activity against HL-60 cells, identical to the activity of the natural
product.1 The IC50 values of the products from the two origins were both 175 nM.
25

100
80
60
40
20
0
50μM
100μM
n.s.
n.s.
Fig. S4 Inhibitory effect of (S)- and (R)-erypoegin K on the enzyme activity of
purified human GLO-I
Recombinant human GLO-I was purified to homogeneity and its activity was
measured in the presence or absence of (S)- and (R)-erypoegin K, as previously
described.2 The enzyme reaction was monitored by measuring the increase in optical
density at 240 nm because of the formation of S-D-lactoylglutathione. Enzyme activity
was expressed relative to the activity of the control treated with vehicle (DMSO), which
was taken as 100%. The data are presented as the mean ± SE of three independent
experiments. Triplicate measurements were performed in each experiment. BBG: S-p-
bromobenzylglutathione. n.s.: not significant by Student’s t-test.
Results
The effect of each optical isomer of erypoegin K on the activity of purified human GLO-
I enzyme was examined. As shown in Fig. S4, (S)- and (R)-erypoegin K exhibited almost
the same inhibitory activity at concentrations of 50 M and 100 M. BBG, a known
inhibitor of GLO-I, was used as a positive control.
26

References
1. Hikita K, Hattori N, Takeda A, Yamakage Y, Shibata R, Yamada S, Kato K, Murata
T, Tanaka H, Kaneda N. Potent apoptosis-inducing activity of erypoegin K, an isoflavone
isolated from Erythrina poeppigiana, against human leukemia HL-60 cells. J Nat
Med.2018;72:260-266. doi:10.1007/s11418-017-1147-9.
2. Hikita K, Yamada S, Shibata R, Katoh M, Murata T, Kato K, Tanaka H, Kaneda N.
Phenolic constituents from stem bark of Erythrina poeppigiana and their inhibitory
activity on human glyoxalase I. J Nat Med.2014;68:636-42. doi:10.1007/s11418-014-
0834-z.
27

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
28

29