A novel, broad-spectrum antitumor compound containing the 1-hydroxycyclohexa-2,5-dien-4-one group: The disclosure of a new antitumor pharmacophore in protoapigenone 1

Article abstract of DOI:10.1016/j.bmcl.2011.03.108

The synthesis of a new compound 9 containing the 4-hydroxy-2,5- cyclohexadien-1-one system, a key elements toward elucidation of the protoapigenone 1 antitumor pharmacophore, was described. The compound showed potent in vitro antitumor potency with low mi

Full text of DOI:10.1016/j.bmcl.2011.03.108

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3427–3430
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A novel, broad-spectrum antitumor compound containing
the 1-hydroxycyclohexa-2,5-dien-4-one group: The disclosure of a new
antitumor pharmacophore in protoapigenone 1
⇑
Qianying Yuan, Ziwei Liu, Chaomei Xiong, Liqian Wu, Jianping Wang, Jinlan Ruan
Key Laboratory of Natural Medicinal Chemistry and Resources Evaluation of Hubei Province, College of Pharmacy, Tongji Medical Center of Huazhong
University of Science and Technology, Wuhan 430030, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of a new compound 9 containing the 4-hydroxy-2,5-cyclohexadien-1-one system, a key
elements toward elucidation of the protoapigenone 1 antitumor pharmacophore, was described. The
compound showed potent in vitro antitumor potency with low micromolar IC50’s against breast, ovarian,
prostate, liver, pancreas, and blood cancer cell lines tested and could inhibit tumor growth in vivo but no
significant impairment of hematopoiesis or immune function was observed. The minimum structural
pharmacophore of 1 has now been refined.
Received 12 February 2011
Revised 25 March 2011
Accepted 29 March 2011
Available online 5 April 2011
Keywords:
4-Hydroxy-2,5-cyclohexadien-1-one
Protoapigenone
Ó 2011 Elsevier Ltd. All rights reserved.
Synthesis
In vitro and in vivo screening
Antitumor activity
There has been considerable interest in both the chemistry and
biological activity of the ferns over the past decades. Many types of
flavonoids have been discovered from different fern species and
some of these have shown great promise in cancer therapy.1–4 Pro-
toapigenone 1, a novel flavonoid compound which has an unusual
nonaromatic B-ring, was isolated from torres’s ferns, such as
Figure 1. Chemical structures of 1 and related flavonoids.
⇑
Corresponding author. Tel.: +86 130 361 1598; fax: +86 027 83692762.
E-mail address: jinlan8152@163.com (J. Ruan).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.108

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Q. Yuan et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3427–3430
compared with 10-hydroxy analogues 1, 6, and 7, which indicate
Br
NO
O
2
O
the hydroxyl substituent at the C-10 position are essential for rec-
ognition.9 From these results we determined that the 10-hydroxy-
cyclohexa-20,50-dien-40-one B-ring is required in conjunction with
either of the C-ring 9 or the AC-ring 7. At present, compound 7 is
the structurally simplest analogue known that exhibits potent
cytotoxicity. Compound 9 may therefore be considered comple-
mentary analogues to the presently known minimum pharmaco-
phore in 7. In this Letter, we report the synthesis and biological
evaluation of 9.
NaNO2
O
Me2SO/phloroglucinol
r.t., 18 h
Et3N, PhNCO, Benzene
O
O
°
90 C, 12-18 h
11
12
O
O
N
OMe
O
Mo(CO)6
O
The synthesis of 9 is outline in Figure 2. Nitration reaction of
2-(2-bromoethyl)-1,3-dioxolane 11 with nitrite provided the
2-(2-nitroethyl)-1,3-dioxolane 1211 in 50% yield. Under Mukaiy-
ama’s dehydration conditions, the nitrile oxide intermediate was
generated with phenyl isocyanate from 12.12 1,3-dipolar cycload-
dition reaction of nitrile oxide with acetylene to give the isoxazole
13 in 45% yield.13 Reductive cleavage reaction of the isoxazole was
achieved using Mo(CO)6, with the resulting enamino ketone 14
(73% yield) being cyclized to the pyran-4-one derivative 15 in
55% yield under acidic conditions.13 Demethylation with BBr3 gave
the 2-(4-hydroxyphenyl)pyrone 16 (76% yield), which was oxi-
dized using [bis(trifluoroacetoxy)iodo]benzene (PIFA) to give the
MeCN/H2O
O
NH
O
°
2
90 C, 3-5 h
O
14
13
OMe
OH
O
O
AcOH, H2O, HCO2H
BBr3
CH2Cl2
r.t., 6-8 h
C, 0.5 h
°
60 C, 12-18 h
°
0
O
O
15
16
novel 2-(1-hydroxy-4-oxo-cyclohexa-2,5-dienyl)-pyran-4-one
9
(22% yield).16
Compound 1 and 9 were screened in vitro against eight cancer
celllines, including MCF-7 and MDA-MB-231 (breast cancer), SKOV3
(ovarian cancer), PC-3 (prostate cancer), HepG2 (liver cancer),
PANC-1 (pancreatic cancer), RPMI-8226 and U266 (multiple myelo-
ma). The results are graphically represented in Figure 3. The syn-
thetic 9 showed enhanced cytotoxic activity than natural product
O
PhI (O2CCF3)2, Me4-piperidoxyl
O
O
OH
H2O/MeCN
r.t., 3-6 h
1 with notable IC50 values of 0.38–3.43 lM against most cancer cells
9
tested (MCF-7, MDA-MB-231, SKOV3, PC-3, HepG2, and PANC-1). In
comparison with the presently structurally simplest analogue 7,
compound 9 showed about 2- to 5-fold greater cytotoxicity against
MCF-7, MDA-MB-231, and HepG2 cancer cell lines.9
The toxicity of 1 and 9 against normal cells were also investi-
gated using human peripheral blood mononuclear cell (PBMC)
and human embryonic kidney 293 cell line (HEK 293). The results
are shown in Figure 4. Compound 9 was only weakly active against
the two normal cells and exhibited selective anticancer activities
compared with 1.
The effectiveness of 1 and 9 were tested in vivo against the hep-
atoma H22 tumor bearing mice.14,15 Cyclophosphamide (CTX) was
used as reference drug. Compound 9 and CTX at the dosage of
10 mg/kg significantly inhibited the growth of implanted H22 tu-
mor in mice by 34.12% and 41.74%, respectively, while compound
1 was found to be inactive with inhibition rate less than 5%. In
addition, after intraperitoneal injection of 1 and 9, the white blood
cell (WBC), thymus index, spleen index and the body weight had
no significant difference between control and drug-treated group.
In bone marrow cells, however, the numbers of total nucleated
cells as well as the ratio of the granulocytic and erythroid cell sys-
tems were declined by 51.52% and 36.9%, respectively after treated
with 1, suggesting its damage on bone marrow hematopoiesis. By
contrast, no significant difference was observed in the 9-treated
group.
Figure 2. Synthesis of 2-(1-hydroxy-4-oxo-cyclohexa-2,5-dienyl)-pyran-4-one 9.
Thelypteris torresiana5 and Macrothelypteris torresiana.6 This kind of
flavonoid displayed potent antitumor activity against a broad spec-
trum of human cancer cell lines such as HepG2 and HepG3 (liver),
MDA-MB-231 and MCF-7 (breast), MDAH-2774 and SKOV3
(ovary), LNCap (prostate) and A549 (lung) with IC50 values of
0.94–5.59 l
M.4,5,7 Apigenin 2, however, which is the conceivable
biosynthetic precursor of 1 bearing a 40-hydroxyphenyl B-ring,
showed no significant antiproliferative activity.8 Thus, the promis-
ing compound 1 containing the novel nonaromatic B-ring system
has attracted considerable synthetic interest and several struc-
ture–activity relationship (SAR) studies related to 1 have been
reported.9
Limited structure–activity correlations of 1 as well as other nat-
ural and synthetic analogues (Fig. 1) have defined some of the
structural requirements of the cytotoxic pharmacophore. Compar-
ison of 1 to its analogues 2 and 3 showed that the latter two com-
pounds, without oxidation of 40-hydroxyphenyl, had reduced
activity compared with 1.8 In addition, compound 59, which has
only a single double bond in the B-ring exhibited in general 2–5
magnitudes less active than compound 1 and cyclohexanone 410
was devoid of biological activity. The results confirmed that a sym-
metrical double bond structure in the B-ring was crucial for potent
cytotoxicity.9 On the other hand, 7-hydrogenated 6 and 5,7-dihy-
drogenated 7 showed enhanced activity than 1, indicating that
the C-5 and C-7 hydroxyl substituents were not requirements of
the pharmacophore. Lastly, 10-methoxy 8 showed less potent
The present study help further define the minimum cytotoxic
pharmacophore shared by protoapigenone 1. The newly synthe-
sized analogue 9 showed better in vitro and in vivo activity and
less major side effects compared with 1, suggesting that the
A-ring of the bioflavonoid 1 is not the requirements of the

Q. Yuan et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3427–3430
3429
Figure 3. Antitumor activity of compound 1 and 9 in vitro against (A) MCF-7 (breast), (B) MDA-MB-231 (breast), (C) SKOV3 (ovarian), (D) PC-3 (prostate), (E) HepG2 (liver),
(F) PANC-1 (pancreas), (G) RPMI-8226 (blood), and (H) U266 (blood) cell lines for 24 h. The cell proliferation rates were determined by MTT assay and they are presented as
mean SD (n = 4). Cells treated with DMSO (equivalent volume) were used as a vehicle control.
pharmacophore. We tentatively conclude that the minimum struc-
tural pharmacophore of 1 must reside in either or both of the B-
ring and C-ring. The synthesis of complementary analogues is cur-
rently under investigation in our laboratories.
Acknowledgments
This research was supported by grant (No. 30973864) from the
National Nature Science Foundation of China and grant (No.

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Q. Yuan et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3427–3430
Figure 4. Effect of compound 1 and 9 on human peripheral blood mononuclear cell (PBMC, A) and human embryonic kidney 293 cell line (HEK 293, B) for 24 h. The cell
proliferation rates were determined by MTT assay and they are presented as mean SD (n = 4). Cells treated with DMSO (equivalent volume) were used as a vehicle control.
Biological screening procedure: measurement of cell proliferation was
determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay. Briefly, 2000 cells were plated out into each well of a
2009CDA067) from the Nature Science Foundation of Hubei
Province.
96-well plate and allowed to adhere. Compound
dimethylsulfoxide (DMSO, Sigma) and diluted with tissue culture medium,
were then added at increasing concentrations (0.1–100 M, eight wells per
1 and 9 dissolved in
References and notes
l
concentration). The cells were incubated in the presence of drugs for 24 h. MTT
reagent (Sigma) dissolved in phosphate buffered saline was added to each well
1. Fang, W.; Ruan, J.; Wang, Z.; Zhao, Z.; Zou, J.; Zhou, D.; Cai, Y. J. Nat. Prod. 2006,
69, 1641.
2. Zhao, Z.; Ruan, J.; Jin, J.; Zou, J.; Zhou, D.; Fang, W.; Zeng, F. J. Nat. Prod. 2006, 69,
265.
3. Zhao, Z.; Ruan, J.; Jin, J.; Zhu, C.; Liu, Y. Fitoterapia 2010, 81, 1171.
4. Chang, H. L.; Su, J. H.; Yeh, Y. T.; Lee, Y. C.; Chen, H. M.; Wu, Y. C.; Yuan, S. S.
Cancer Lett. 2008, 267, 85.
5. Lin, A. S.; Chang, F. R.; Wu, C. C.; Liaw, C. C.; Wu, Y. C. Planta Med. 2005, 71, 867.
6. Huang, X. H.; Xiong, P. C.; Xiong, C. M.; Cai, Y. L.; Wei, A. H.; Wang, J. P.; Liang, X.
F.; Ruan, J. L. Phytomedicine 2010, 17, 930.
at
additional hours. Following this time, the medium containing the MTT reagent
was aspirated, and DMSO (150 L) was added to each well. The absorbance of
a concentration of 0.5 mg/mL, and the cells were incubated for four
l
each well was measured in a microplate reader (Power- Wave XS, Bio-Tek) at a
wavelength of 570 nm. The IC50 value for each compound tested was
determined by plotting concentration versus percent absorbance obtained in
the MTT assay.
In vivo effects of 1 and 9 were studied in hepatoma H22 tumor-bearing mice.
ICR mice (18–22 weeks) were purchased from Animal Center, Huazhong
7. Chang, H. L.; Wu, Y. C.; Su, J. H.; Yeh, Y. T.; Yuan, S. S. J. Pharmacol. Exp. Ther.
2008, 325, 841.
University of science and technology. The animals were maintained in
a
standard environmental condition and fed with rodent diet and water ad
libitum. All experiments were approved by the institutional Animal Care and
Use Committee of Huazhong University of Science and technology. Ascites was
taken from hepatoma H22 mice passing on from generation to generation for
6–7 days under aseptic condition, which was diluted with normal saline into a
suspended solution in a concentration of 1 ꢁ 107 cells/ml. A volume of 0.2 ml
of H22 tumor cell suspension was hypodermically injected into the right flank
of each mouse. Twenty-four hours after injection of the H22 cells, the mice
were randomly divided into four groups, that is, a model group, a compound 1
group, a compound 9 group, a Cyclophosphamide (CTX) group, 10 mice in each
group, and with a normal group set as the controls (0.2 ml saline was injected
into the right axillary fossa for each mouse of this group). From the second day
after inoculation, 1 and 9 dissolved in 10% ethanol solution were administered
for seven successive days by intraperitoneal injection (10 ml/kg for each
mouse) at daily dose of 10 mg/kg. CTX (10 mg/kg) was used as reference drug.
Normal animals and untreated tumor bearing animals were treated with the
same volume of 10% ethanol solution. The mice were killed at day nine. The
tumors, thymus, and spleen were separated and weighed, the blood sample
was taken for count of WBC and the femurs were dissected and marrow cells
flushed from the bone. The tumor inhibition rate was calculated: (the tumor
weight of the model group—the tumor weight of the treatment group)/the
tumor weight of the model group ꢁ 100% and the thymus index and the spleen
index were calculated: The thymus index = the thymus weight (mg)/body
weight (g); while the spleen index = the spleen weight (mg)/body weight (g).
Numbers of nucleated cells of the bone marrow were determined using a
Coulter Counter. The counts of granulocytic or erythroid cells per femur were
performed on marrow smears stained with the May-Grünwald-Giemsa
method.
8. Manthey, J. A.; Guthrie, N. J. Agric. Food Chem. 2002, 50, 5837.
9. Lin, A. S.; Nakagawa-Goto, K.; Chang, F. R.; Yu, D.; Morris-Natschke, S. L.; Wu, C.
C.; Chen, S. L.; Wu, Y. C.; Lee, K. H. J. Med. Chem. 2007, 50, 3921.
10. Jerz, G.; Waibel, R.; Achenbach, H. Phytochemistry 2005, 66, 1698.
11. Robyn, L. C.; Jonathan, S. N.; Harry, S. M. J. Org. Chem. 1982, 47, 4040.
12. Mukaiyama, T.; Hoshino, T. J. Am. Chem. Soc. 1960, 82, 5339.
13. Li, C. S.; Lacasse, E. Tetrahedron Lett. 2002, 43, 3565.
14. Dai, Z.; Liu, X.; Ji, Z.; Liu, L.; Kang, H.; Wang, X.; Diao, Y. J. Tradit. Chin. Med.
2008, 28, 205.
15. Zhang, W.; Li, J.; Qiu, S.; Chen, J.; Zheng, Y. Fitoterapia 2008, 79, 168.
16. Experimental: physico-chemical properties of the synthetic compounds are as
follows:
2-(4-Methoxyphenyl)-4H-pyran-4-one (15): 1H NMR (400 MHz, in DMSO): d
8.15 (1H, d, J = 5.6 Hz, H-6), d 7.85 (2H, d, J = 8.8 Hz, H-2’,6’), d 7.08 (2H, d,
J = 8.8 Hz, H-3’,5’), d 6.81 (1H, d, J = 2.4 Hz, H-3), d 6.41 (1H, dd, J = 5.6, 2.4 Hz,
H-5), d 3.88 (3H, s, methoxy-CH3).
2-(4-Hydroxyphenyl)-4H-pyran-4-one (16): 1H NMR (400 MHz, in DMSO): d
8.17 (1H, d, J = 5.6 Hz, H-6), d 7.74 (2H, d, J = 8.8 Hz, H-2’,6’), d 6.89 (2H, d,
J = 8.8 Hz, H-3’,5’), d 6.75 (1H, d, J = 2.4 Hz, H-3), d 6.28 (1H, dd, J = 5.6, 2.4 Hz,
H-5).
2-(1-Hydroxy-4-oxo-cyclohexa-2,5-dienyl)-pyran-4-one (9): yellow solid; UV
kmax(MeOH) 252.5 nm; 1H NMR (400 MHz, in DMSO):
d 8.10 (1H, d,
J = 6.0 Hz, H-6), d 6.96 (2H, d, J = 10.0 Hz, H-2’,6’), d 6.30 (2H, d, J = 10.0 Hz,
H-3’,5’), d 6.54 (1H, d, J = 2.4 Hz, H-3), d 6.29 (1H, dd, J = 6.0, 2.4 Hz, H-5); 13C
NMR (100 MHz, in DMSO): d 185.3 (C-4’), d 178.2 (C-4), d 167.1 (C-2), d 156.9
(C-6), d 148.4 (C-2’,6’), d 129.1 (C-3’,5’), d 116.9 (C-3), d 113.5 (C-5), d 69.0 (C-
1’); ESI–MS (m/z): 203 [Mꢀ-H, 100]; HRMS (FAB) calcd for C11H8O4: m/z
204.0422; found: 204.0425.