Selective alkylation of β-anhydroicaritine and their biological evaluation on anticancer

Article abstract of DOI:10.1080/14786419.2020.1844686

A convenient and selective alkylation of icaritin has been developed. The methodology involved initial formation of β-anhydroicaritine (3) under acidic conditions followed by selective methylation at the C-3 position and then alkylation at C-5 position. Several alkylated β-anhydroicaritine derivatives were synthesised using this methodology. These newly synthesised derivatives, especially the compounds 5b, 5c and 5j, significantly suppressed cell proliferation when tested against cancer cell lines in?vitro. Compound 5j (R = Bn) exhibited a competitive inhibition against MCF7 in?vivo compared to tamoxifen.

Full text of DOI:10.1080/14786419.2020.1844686

Natural Product Research
Formerly Natural Product Letters
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Selective alkylation of β-anhydroicaritine and their
biological evaluation on anticancer
Hanqi Cui , Xianheng Wang , Changkuo Zhao , Yue Pu & Yuhe Wang
To cite this article: Hanqi Cui , Xianheng Wang , Changkuo Zhao , Yue Pu & Yuhe Wang (2020):
Selective alkylation of β-anhydroicaritine and their biological evaluation on anticancer, Natural
Product Research, DOI: 10.1080/14786419.2020.1844686
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2020.1844686
Selective alkylation of b-anhydroicaritine and
their biological evaluation on anticancer
Hanqi Cuia, Xianheng Wanga, Changkuo Zhaoa , Yue Pua and Yuhe Wangb
b
aDepartment of Medicinal Chemistry, Zunyi Medical University, Zunyi City, China; Department
of Pharmacy, Zunyi Medical University Affiliated Hospital, Zunyi, China
ABSTRACT
ARTICLE HISTORY
Received 18 July 2020
Accepted 28 October 2020
A
convenient and selective alkylation of icaritin has been
developed. The methodology involved initial formation of b-anhy-
droicaritine (3) under acidic conditions followed by selective
methylation at the C-3 position and then alkylation at C-5
position. Several alkylated b-anhydroicaritine derivatives were
synthesised using this methodology. These newly synthesised
derivatives, especially the compounds 5b, 5c and 5j, significantly
suppressed cell proliferation when tested against cancer cell lines
in vitro. Compound 5j (R ¼ Bn) exhibited a competitive inhibition
against MCF7 in vivo compared to tamoxifen.
KEYWORDS
b-anhydroicaritine;
alkylation; selectivity;
anticancer
1. Introduction
Flavones are of great interest due to their profound biological activities and
widespread occurrence in nature (Manojit et al. 2003; Bruno et al. 2005; Manojit et al.
2005; Nigel and Renee 2011; Boris et al. 2013; Christian 2020). As a natural allyl flavonoid,
icariin (1, Figure 1) has low oral absorption and low bioavailability because of its low
solubility, which blocks it enter to BBB and limits its use in clinic. Recent pharmacological
research shows that icaritin has an inhibitory effect on a variety of tumour cells,
and in metabolism, this effect is mainly hydrolysed to icaritin (2) (He et al. 2010).
After the perusal of lit, we found that the previous structural modification to
icaritin is mainly focused on the esterification of 3- and 7-hydroxy group in order to
CONTACT Changkuo Zhao
zck7103@hotmail.com; Yuhe Wang
1149076068@qq.com
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1844686.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

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H. CUI ET AL.
Figure 1. Structure of icariin, icaritin and b-anhydroicaritine.
improve their solubility or anticancer activity (Wang et al. 2000; Liu et al. 2009; Yao et al.
2010; Sun et al. 2013). Studies (Huang et al. 2007; Guo et al. 2011; Wang et al. 2015;
Vanson et al. 2017) have shown that a single substitution on the C-3 or C-7 hydroxyl
group of icaritin can improve the cytotoxicity of icaritin, and C-3 hydroxyl group may
be the preferred site for chemical modification. However, several problems arise to this
synthetic route. On the one hand, it is difficult to get mono-substituted esterification
product selectively, and always, a mixture of 3- and 7-esterified products was obtained.
On the other hand, there are few literatures were reported on the chemical modification
of C-5 hydroxyl group, and its anticancer activity is also unknown.
In view of this problem, we envisioned that a selective alkylation for icaritin can be
realised according to the difference in activity for three hydroxyl groups. And herein
we reported our research results.
2. Research and discussion
It is interesting to find that three phenolic hydroxyl groups exhibit different reaction activ-
ity during esterification or alkylation reaction (Figure 2), which makes it possible for us to
introduce alkyl group selectively. In fact, the direct alkylation to icaritin will lead to the
formation of a mixture of 3- and 3, 7-disubstituted icaritin. To overcome this problem, we
decide to protect 7-hydroxy group firstly by intra-molecular cyclisation reaction.
A
modified procedure was explored to synthesise the building block
b-anhydroicaritine (3) (Wang and Lou 2004; Wang et al. 2007; Tong et al. 2019). Upon
treatment with formic acid, icaritin underwent cyclisation reaction at reflux to give
b-anhydroicaritine in 95% yield (Dell’Agli et al. 2008). This modified procedure is more
suitable for the scale up in industrial manufacture compared to the reported micro-
wave reaction method.
In the following, in the alkaline conditions, b-anhydroicaritine was reacted with
methylation reagent in a ratio of 1:1, such as dimethyl sulfate or methyl iodide, and

NATURAL PRODUCT RESEARCH
3
Figure 2. Activity difference to three phenolic hydroxyl groups for icaritin.
Table 1. Selective alkylation of b-anhydroicaritine with RX.
Entry
R
Coupling reagent
Solvent
Temp.
Time
yield
A
B
C
D
E
F
G
H
I
CH3
C2H5
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
acetone
acetone
acetone
acetone
acetone
acetone
acetone
acetone
acetone
acetone
reflux
reflux
reflux
reflux
reflux
reflux
reflux
reflux
reflux
reflux
2 d
2 d
2 d
2 d
3 d
3 d
2 d
2 d
2 d
2 d
85%
60%
60%
60%
44%
n-C3H7
n-C5H11
BrCH2CH2
BrCH2CH2CH2
BrCH2CH2CH2CH2
allyl
44%
67.3%
66.3%
44.6%
62.2%
ꢁ CH2
J
Bn
refluxed for 2 h to afford 3-methoxy b-anhydroicaritine (4) in good yield. In this step,
the molar ratio and the amount of base are essential to affect the reaction yield.
Particularly, a side product 3, 5-disubstituted b-anhydroicaritine will be formed when
the molar ratio and the amount of base are more than 1:1.
And finally, the above obtained 3-methoxy b-anhydroicaritine was further reacted
with different alkyl halides to give b-anhydroicaritine derivatives 5a–j in a moderate
yield. The detailed results are summarised in Table 1.
2.1. Proliferation inhibition assay
The cell proliferation inhibition of the four human cancer cell lines (HepG2, SW480, A549
and MCF7) by all synthetic compounds was evaluated by standard MTT methods. HepG2,
SW480, A549 and MCF7 cells were cultured in RPMI 1640 or McCoy’s 5A medium
(Invitrogen), respectively, supplemented with 10% heat-inactivated FBS and 1% penicillin/
streptomycin (Thermo Fisher Scientific, China). All cell lines were maintained at 37ꢀC with
5% CO2. Cells were seeded in 96-well plates and incubated for 24 h. A range of concen-
trations of the test compounds were added, and the plates were incubated for 72 h
before the addition of 10 lL MTT (5 mg/ml)/well. After 4 h of incubation, the medium was
removed and 100 lL DMSO was added to each well. The absorbance was measured using
a SpectraMax M5/M5e, USA microplate reader at 550 nm. The results are shown in Table
S2. The results showed that compound 5j had the best anti-cell proliferation effect.
2.1.1. Inhibition tumour cell growth in vivo
Three compounds 5b, 5c and 5j were selected as candidates for the screening test
against MCF7 in vivo. Nude mice were injected with a solution of MCF7 in Matrigel,
and inoculation for 5 days until the tumour size is up to 0.5 cm approximately. Each

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H. CUI ET AL.
mouse was fed with test compound (5 mg per day) for 20 days. The results are shown
in Table S3. The inhibition ratio was calculated as follows:
Inhibition rate ¼ ½Wðin Negative controlÞ – Wðin test compoundÞꢂ=
Wðin Negative controlÞ:
3. Experimental
3.1. Materials and methods
Melting points were determined by a Mettler Toledo FP62 melting point apparatus
and are uncorrected. 1H NMR spectra were recorded at 400 MHz on a Varian Unity
nova 400 NMR spectrometer using tetramethylsilane (TMS) as an internal standard,
and 13C NMR spectra were recorded at 100 MHz, respectively. Mass spectra were run
on TOF-SIMS instrument (PHI Nano TOF II). TLC plates (GF 254) were bought from
Branch Qingdao Haiyang Chemical Plant. Icariin (98%) was bought from Biopurify
Phytochemicals Ltd (Chengdu, China). All the solvents are bought from Sinopharm.
4. Conclusion
In summary, a convenient selective alkylation protocol for icariin was developed. By pro-
tection to form b-anhydroicaritine (3) under acidic conditions, and followed by the select-
ive alkylation to C-3 and C-5, several b-anhydroicaritine alkylated derivatives were thus
easily synthesised. These new synthesised C-5 substituted b-anhydroicaritine derivatives,
especially for compounds 5b, 5c and 5j, significantly suppress cell proliferation in vitro.
Compound 5j (R ¼ Bn) exhibits a competitive inhibition against MCF7 in vivo compared
to tamoxifen, which give us much imagination on its further development.
Acknowledgments
I am also very thankful for Dr. Yuqi He’s useful explanation on MS spectrum data.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
I
am very grateful for the financial support from Key Project of Guizhou Provincial
Administration of Traditional Chinese Medicine [QZYYZD-2019-05], Joint fund project of Science
and Technology Foundation of Zunyi City [ZunyiKeHe HZ [2020]26], Public Bidding Project
Foundation of Zunyi Medical University [[2013]F-680], Doctoral Scientific Research Foundation of
Zunyi Medical University [[2013]F-633] and [[2014]F-697] and Science and Technology
Foundation of Guizhou Province [QianKeHe ZhiCheng [2019]2829].
ORCID
Changkuo Zhao
http://orcid.org/0000-0001-7973-6333

NATURAL PRODUCT RESEARCH
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