Synthesis of hirtellanine B, an isoflavonoid with potent antiproliferative effects in cancer cells

Article abstract of DOI:10.1002/cjoc.201200146

We reported the first total synthesis of hirtellanine B, using oxidative coupling for the key reactions, which resulted in a high yield. The antiproliferative activity of hirtellanine B against Jurkat cells, Raji cells and K562 cells were also investigated. It was found that hirtellanine B could induce G2/M arrest and apoptosis in human lymphoid/leukemia tumor cells. Copyright

Full text of DOI:10.1002/cjoc.201200146

FULL PAPER
DOI: 10.1002/cjoc. 201200146
Synthesis of Hirtellanine B, an Isoflavonoid with Potent
Antiproliferative Effects in Cancer Cells
Zheng, Shuyana(郑书岩)
Fu, Runzhongb(符润中)
Shen, Zhengwu*,a,c(沈征武)
a School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
b Departmen of Applied Chemistry, East China University of Sciences and Technology, Shanghai 20000, China
c Basilea Pharmaceutica China Ltd., Haimen, Jiangsu 226100, China
We reported the first total synthesis of hirtellanine B, using oxidative coupling for the key reactions, which re-
sulted in a high yield. The antiproliferative activity of hirtellanine B against Jurkat cells, Raji cells and K562 cells
were also investigated. It was found that hirtellanine B could induce G2/M arrest and apoptosis in human lym-
phoid/leukemia tumor cells.
Keywords hirtellanine B, synthesis, antiproliferation, G2/M arrest, cancer cell
Introduction
Scheme 1 Retrosynthetic analysis of hirtellanine B
O
O
O
Hirtellanine B is an isoflavonoid isolated from the
roots of Campylotropis hirtella (Franch.) Schindl. A
previous study indicated that hirtellanine B showed
moderate B lymphocyte suppression activity (IC50: 3.00
O
O
O
OH
OH
O
O
O
OH
－
1
1
μmol•L ) and T lymphocyte suppression activity (IC50:
Hirtellanine B
－
1
9.55 μmol•L ).[1] However, our in vitro studies un-
veiled that hirtellanine B inhibits the proliferation of the
Jurkat, Raji and K562 cell lines. Furthermore, this
compound was found to induce G2/M arrest in these
cancer cells. Interestingly, all other flavanones isolated
from this plant, including hirtellanine A[1-4] do not show
any effects of inducing G2/M arrest of the above-men-
tioned tumor cells. Thus, it is of interest to further ex-
plore the synthesis and structure-function relationship of
hirtellanine B. In this letter, we describe the first total
synthesis of hirtellanine B and report its antiprolifera-
tive effects and induction of G2/M arrest in human lym-
phoid/leukemia tumor cells.
N
O
O
O
N
O
O
O
I
O
O
2
3
3-Iodo-5-methoxy-dimethylchromene pyran-4-one 3
was obtained from acetylphloroglucinol in five steps
with an overall yield of 71%.[4] As shown in Scheme 2,
after treated with imidazole in dimethyl formamide, 3
was smoothly converted into 2 with a 95% yield.[6] The
subsequent acid hydrolysis of 2 afforded coumarin in-
termediate 1 with a 93% yield. For the hydrolysis reac-
tion, both reaction conditions of 10% aqueous HCl in
EtOH and 10% HCl in THF were tried, however, large
amounts of by-products were observed; when AcOH
(acetic acid glacial) was used, only target compound
was obtained. The final coupling of 1 and o-dihydroxy-
benzene was achieved in the presence of K3Fe(CN)6 to
deliver hirtellanine B with a 69% yield.[6] In the oxida-
tive coupling step, catechol was oxidized by potassium
ferricyanide, and o-benzoquinone was generated in-situ
which participated into Michael addition with
4-hydroxycoumarin 1 to form hirtellanine B. The spec-
troscopic data of synthesized hirtellanine B were iden-
Results and Discussion
Our retrosynthetic analysis of hirtellanine B is de-
picted in Scheme 1. The key intermediate 3-iodo-5-
methoxy-dimethylchromene pyran-4-one 3 can be pre-
pared from commercially available acetylphlorogluci-
nol.[4] The compound 3 can readily be converted into
intermediate 2 according to the literature,[5] and the
compound 1 can be obtained from the hydrolysis of
compound 2. Finally, we anticipate the intermediate 1
can be coupled with o-dihydroxybenzene to form hirtel-
lanine B.
*
E-mail: jeff_shen_1999@yahoo.com; Tel.: ＋86-513-8219-8001; Fax: ＋86-513-8219-8003
Received February 16, 2012; accepted April 26, 2012; published online XXXX, 2012.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.201200146 or from the author.
Chin. J. Chem. 2012, XX, 1—5
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1

Zheng, Fu & Shen
FULL PAPER
tical to those obtained from the authentic sample from
plant material. Thus, we have developed the first syn-
thetic hirtellanine B, using oxidative coupling as the key
reaction. The eight-step synthetic process converted
acetylphloroglucinol to hirtellanine B in an overall yield
of 41%.
In our preliminary studies, hirtellanine B was shown
to inhibit human lymphoid/leukemia tumor cell prolif-
eration.[7] Significant growth inhibition was observed
after 24 h of treatment with hirtellanine B in tumor cell
lines. The concentration of hirtellanine B required for
50% inhibition of cell growth (CC50) was (7.17±0.04)
μmol•L－1 in Jurkat cells, (6.58±0.40) μmol•L－1 in Raji
cells and (6.76±0.16) μmol•L－1 in K562 cells.
centage of cells (69.05%) in G2/M phase of the cell cy-
cle. As the G2/M phase population increased, the G1
phase cell population decreased, while the S phase cell
population showed slight changes within 24 h.
We also investigated the inhibitory effects of hirtel-
lanine B on Raji cells, as shown in Table 1 and Figure 1.
After 24 h of incubation, the G2/M phase cell popula-
tion of the control cells was 10.50%, while the G2/M
phase cell population of 2.5 μmol•L － hirtellanine
1
B-treated cells increased to 31.93%. In addition, when
the G2/M phase population increased, the G1 phase cell
population decreased, and the S phase cell population
remained almost unchanged.
Similar findings were obtained with K562 cells.
Compared with control cells, after 24 h incubation, the
To investigate the mechanism of hirtellanine
B-mediated cell growth inhibition, we examined cell
cycle alterations by flow cytometry.[8,9] The effects of
hirtellanine B on cell cycle progression of Jurkat, Raji
and K562 cells are shown in Figure 1 and Table 1. After
24 h of growth, the G2/M phase cell population of con-
trol Jurkat cells was 13.50%. Meanwhile, the G2/M
phase cell population of hirtellanine B-treated cells in-
creased in a dose-dependent manner. A concentration of
G2/M phase cell population of the 2.5 μmol•L－ hirtel-
1
lanine B-treated K562 cells increased to 34.26%,
whereas the cell population both at G1 phase and S
phase decreased.
This G2/M cell cycle arrest was also detected after
48 h of treatment. At the highest concentration (2.5
－
1
μmol•L ) of hirtellanine B, the proportion of cells in
2.5 μmol•L－ hirtellanine B resulted in the highest per-
1
G2/M phase increased to 79.38%, compared with the
Scheme 2 The synthesis of hirtellanine B
N
imidazole
K2CO3
5 steps
HO
OH
O
O
O
O
N
I
O
O
O
71%
DMF
80oC, 95%
I
OH
O
O
4
3
N
Catechol
O
O
O
+
O
O
O
O
O
N
K3Fe(CN)6
AcOH
95oC
93%
NaOAc, H2O
acetone, r.t.
69%
O
O
OH
2
1
O
O
O
O
O
O
O
O
O
O
O
OH
OH
O
OH
OH
O
OH
O
O
OH
Hirtellanine B
Table 1 The effects of hirtellanine B on cell cycle progressiona
Jurkat cell line/%
S/%
Raji cell line/%
S/% G2/M
K562 cell line/%
S/%
G0/G1
57.63
12.75
40.55
58.18
G2/M
13.50
69.05
48.16
14.65
G0/G1
G0/G1
42.51
32.25
40.21
43.12
G2/M
14.90
34.26
18.06
15.56
Control
28.87
56.63 32.87
41.98 26.08
57.30 23.11
69.29 27.03
10.50
31.93
19.59
12.67
42.59
Hirtellanine B, 2.5 µmol•L－
Hirtellanine B, 1.25 µmol•L－
Hirtellanine B, 0.63 µmol•L－
18.20
33.49
1
1
1
11.28
41.73
27.17
41.32
a Cells were harvested after treated with the indicated concentrations of hirtellanine B. Analysis of the cell cycle distribution was per-
formed using an equal number of cells (1×104) by flow cytometry, after staining the DNA with propidium iodide.
2
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Chin. J. Chem. 2012, XX, 1—5

Synthesis of Hirtellanine B, an Isoflavonoid with Potent Antiproliferative Effects in Cancer Cells
Figure 1 Flow cytometric analysis of the cell cycle distribution in Jurkat cells, Raji cells and K562 cells after treatment with various
concentrations of hirtellanine B.
control cells (8.93%). These results indicated that the
cytotoxicity of hirtellanine B towards human lym-
phoid/leukemia tumor cells is mainly attributable to the
induction in G2/M arrest.
Chin. J. Chem. 2012, XX, 1—5
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3

Zheng, Fu & Shen
FULL PAPER
Methods
yang Co. Ltd. All reactions were stirred with PTFE
coated magnetic stir bars and Heidolph® MR3001®
stirrers. Removal of solvents was typically accom-
plished using a rotary evaporator. Anhydrous Na2SO4
was used to dry all of the organic extracting layer for
workup. All the concentration was performed under
reduced pressure. NMR spectra were recorded at a Var-
ian Mercury Plus 400 MHz instrument with TMS as an
internal standard unless otherwise indicated. MS (ESI-
MS) were recorded on a Varian 1200L Quadrupole or
Agileant 1100 LC/MSD or Waters Acquity SQD MS
spectrometer.
Cell lines and culture
The human lymphoblast-like T-cell line Jurkat, the
human lymphoma B-cell line Raji and the human
erythroleukemia cell line K562 were obtained from
ATCC (Manassas, VA, USA) and were cultured in
RPMI 1640 medium (Gibco Invitrogen Co., Grand Is-
land, NY, USA) supplemented with 10% fetal calf se-
rum (Hyclone, UT, USA), 100 U•mL－1 of penicillin and
100 μg•mL－ of streptomycin in a humidified atmos-
1
phere at 37 ℃ in 5% CO2.
2-Imidazol-5-methoxyl-8,8-dimethyl-8H-pyrano-
[3,2-g]chromone (2) A mixture of 3 (285 mg, 0.74
mmol), imidazole (249 mg, 1.48 mmol) and K2CO3
(1.02 g, 7.40 mmol) in DMF (12 mL) was stirred at 80
℃ for 2 h. After cooling, the reaction solution was
poured into ice water (30 mL) under stirring. After stir-
ring for 5 min, the mixture was standed for 30 min. The
filtration gave the silver-gray solid product (215 mg),
and the filtrate was extract with ethyl acetate and gave
the white solid (15 mg) after concentration and purifica-
tion with prep. TLC with ethyl acetate and petroleum
ether (1∶3). The overall yield was 95.8%. m.p. 191—
MTT proliferation assay
To measure the cytotoxicity of a compound, Jurkat,
－
Raji and K562 cells were seeded (2×104 cells•mL ) in
96-well microtiter plates and incubated with different
concentrations of the compound for 48 h. Then,
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium
bromide (MTT, Sigma Chemical Co., St. Louis, MO,
USA) was added 4 h before the end of culture. After
centrifugation, the supernatants were discarded and the
cells were dissolved in dimethyl sulfoxide. Optical den-
sity values were read at 570 nm, and the percentage of
cell death was calculated.
1
1
192 ℃; H NMR (400 MHz, Acetone-d6) δ: 8.42 (s,
1H), 7.82 (s, 1H), 7.19 (s, 1H), 6.83 (s, 1H), 6.74 (d, J＝
10.0 Hz, 1H), 6.41 (s, 1H), 5.92 (d, J＝10.4 Hz, 1H),
3.86 (s, 3H), 1.47 (s, 6H); 13C NMR (100 MHz, ace-
tone-d6) δ: 176.11, 171.94, 158.76, 157.37, 155.93,
152.62, 135.90, 131.96, 131.68, 116.93, 116.12, 114.28,
101.36, 97.78, 78.52, 62.77, 28.41; IR (film) vmax: 3454,
3119, 3065, 2980, 1655, 1597, 1464, 1412, 1288, 1120,
Cell cycle analysis
To analyze the effect on the cell cycle of the com-
pound, Jurkat, Raji and K562 cells were seeded (2×
－
1
105 cells•mL ) in 24-well plates and incubated with
different concentrations of the compound for 24 h. Cells
were then harvested, washed with cold phosphate-buff-
ered saline and fixed in ice-cold 70% ethanol overnight
at 4 ℃. Cells were stained with 50 µg•mL－1 propidium
－
＋
1
864, 646 cm ; HRMS (ESI ) calcd for C18H17N2O4
([M＋1]＋) 325.1183, found 325.1187.
iodide (PI) and 100 U•mL－ RNase, and analyzed
4-Hydroxycoumarin (1) The solution of 2 (80 mg,
0.25 mmol) in AcOH (acetic acid glacial) (5 mL) was
heated at 95 ℃ for 30 h. The reaction solution was
cooled to r.t., the ice water (15 mL) was added under
stirring and large amount of solid was formed. Further
stirring for 20 min and then standing for 30 min, the
solid was filtrated and the solid cake was washed with
cold water to give the off-white solid product (55 mg).
The filtrate was treated with ethyl acetate to afford addi-
tional white solid (8 mg) after purification by perp. TLC
with ethyl acetate and petroleum ether (1∶2). The
1
(10000 events were collected per sample) in a FACS-
Calibur flow cytometer (BD Biosciences, San Jose, CA,
USA) using the CellQuest software.
Conclusions
In summary, we report the first practical synthesis of
hirtellanine B, involving an eight-step reaction and re-
sulting in an overall yield of 41%. We have also dem-
onstrated the inhibitory effect of hirtellanine B on Jurkat,
Raji and K562 cell proliferation. Its cytotoxicity to-
wards these tumor cells is mainly attributable to the in-
duction of G2/M arrest.
1
overall yield was 93.1%. m.p. 124—125 ℃; H NMR
(400 MHz, Acetone-d6) δ: 6.66 (d, J＝10.0 Hz, 1H),
6.51 (s, 1H), 5.92 (d, J＝10.0 Hz, 1H), 5.41 (s, 1H),
3.98 (s, 3H), 1.47 (s, 6H); 13C NMR (100 MHz, acetone-
d6) δ: 166.13, 161.51, 157.77, 155.94, 153.51, 131.21,
115.52, 111.49, 102.44, 100.87, 90.11, 77.74, 63.94,
27.35; IR (film) vmax: 3524, 3346, 2978, 2854, 2613,
1678, 1616, 1319, 1165, 1083, 939, 825; ESI-MS m/z:
275.3 ([M＋1]＋, 100%). Spectral data were identical to
the literature data.[10]
Experimental
General methods
Reagents were purchased from commercial vendors
and used as received unless otherwise stated. Acetone
and DMF were dried by 4 Å molecular sieves. Reac-
tions were monitored by analytical thin-layer chroma-
tography (TLC) with silica gel GF254 with a layer
thickness of 0.25 mm purchased from Tsingdao Hai-
Hirtellanine B Compound 1 (50 mg, 0.18 mmol)
and o-diphenol (35 mg, 0.32 mmol) were dissolved into
acetone (7 mL). The solution of NaOAc (177 mg, 2.15
4
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Synthesis of Hirtellanine B, an Isoflavonoid with Potent Antiproliferative Effects in Cancer Cells
mmol) in H2O (2 mL) was added, and subsequently, the
solution of K3Fe(CN)6 (214 mg, 0.65 mmol) in 10 mL
H2O was added dropwise in 30 min. After stirring for 2
h, the mixture was filtered and washed with acetone.
The filtrate was concentrated under reduced pressure.
Ethyl acetate (50 mL) was added, and washed with wa-
ter and brine, respectively. After dryness and concentra-
tion, the residue was suspended into DCM (6 mL). After
filtration, the light yellow solid (48 mg, 69%) was ob-
tained. m.p. 283—285 ℃; 1H NMR (400 MHz,
DMSO-d6) δ: 9.30—9.70 (br, s, 2H), 7.24 (s, 1H), 7.17
(s, 1H), 6.77 (s, 1H), 6.67 (d, J＝10.4 Hz, 1H), 5.94 (d,
J＝10.0 Hz, 1H), 3.92 (s, 3H), 1.43 (s, 6H); 13C NMR
(100 MHz, DMSO-d6) δ: 158.03, 157.84, 156.50,
154.44, 151.29, 149.89, 146.64, 145.23, 132.23, 115.65,
114.23, 112.76, 105.29, 103.97, 102.29, 101.51, 99.52,
78.39, 63.80, 28.45; IR (film) vmax: 3502, 3414, 3126,
1707, 1624, 1371, 1280, 1122, 862; HRMS (ESI＋) calcd
for C21H17O7 ([M＋1]＋) 381.0969, found 381.0976.
Shanghai Institute of Material Medica, Chinese Acad-
emy of Sciences, Shanghai, China.
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Acknowledgement
The authors would like to thank Basilea Pharmaceu-
tica China for supporting these studies. The authors also
appreciate the help provided by Prof. Peilan He from
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