Synthesis and in vitro antitumor evaluation of honokiol derivatives

Article abstract of DOI:10.1016/j.bmcl.2019.126849

Honokiol is a natural bioactive neolignan and has been widely researched and structural modified as an anticancer agent. In this paper, 18 honokiol derivatives were synthesized and investigated for their antitumor activity. Among these, the promising compound 5a exhibited much higher anti-proliferative activity with IC₅₀ value of 10.41 μM. Transwell assays showed that 5a could significantly inhibit the invasion and migration of I-10 cells at 2.5 μM, which was further confirmed by the western blotting experiments with down-regulation of the HIF-1α and its associated downstream proteins MMP-2 and MMP-9. Overall, these results provided useful suggestion for further structural optimization of honokiol derivatives.

Full text of DOI:10.1016/j.bmcl.2019.126849

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vitro antitumor evaluation of honokiol derivatives
Meilin Zhua,c, Bohan Lia,c, Hui Maa, Xuenan Huanga, Haotian Wanga, Yiqun Daia, Yu Lib,
⁎
⁎
Hong-Mei Lia, , Cheng-Zhu Wua,
a School of Pharmacy, Bengbu Medical College, 2600 Donghai Road, Bengbu 233030, Anhui, China
b School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Honokiol is a natural bioactive neolignan and has been widely researched and structural modified as an an-
ticancer agent. In this paper, 18 honokiol derivatives were synthesized and investigated for their antitumor
activity. Among these, the promising compound 5a exhibited much higher anti-proliferative activity with IC50
value of 10.41 μM. Transwell assays showed that 5a could significantly inhibit the invasion and migration of I-10
cells at 2.5 μM, which was further confirmed by the western blotting experiments with down-regulation of the
HIF-1α and its associated downstream proteins MMP-2 and MMP-9. Overall, these results provided useful
suggestion for further structural optimization of honokiol derivatives.
Honokiol derivatives
Cytotoxicity
Invasion
Migration
Honokiol, a biphenolic neolignan with inappreciable toxicity iso-
lated from Magnolia officinalis,1 has been reported to possess many
pharmacological activities, such as antiangiogenic,2 antitumor,3 anti-
inflammatory,4 antioxidant effects,5 antimalarial and so on.6,7 A great
number of studies have showed that honokiol could selectively inhibit
the proliferation and growth of various tumor cell lines in vitro and have
visible anticancer activity in vivo.8–10 Recently, it was suggested that the
combination of honokiol with other anticancer therapies could generate
better therapeutic effects.11,12 These research findings revealed that
honokiol could be served as a potential lead compound for anticancer
drug research and development.
promising activity with IC50 values of 10.4–11.7 μM against tested
tumor cells. Furthermore, effects of 5a on invasion and migration of I-
10 cells were evaluated in transwell assays. The ability to inhibit the
expression of relevant proteins, including HIF-1α and its associated
downstream proteins, was also evaluated by western blotting.
Based on the existing research, eighteen honokiol derivatives were
designed and synthesized using honokiol as starting material (Scheme
1). Methyl, ethyl, benzyl, 2,6-dichlorobenzyl, 2,4-dichlorobenzyl and
3,4-dichlorobenzyl were well incorporated on the phenol groups of
honokiol through Williamson alkylation of alkyl halides. Mono-
alkylated key products 1a–1b, 2a–2b, 3a–3b, 4a–b, 5a–b and 6a–b,
respectively, as well as dialkylated byproducts 1c–6c were obtained. All
reactions were accomplished by employing sodium carbonate as base,
and DMF as solvent (see in Supporting Information).
Inspired by the good anticancer effect of honokiol, a wide diversity
of honokiol derivatives have been synthesized successively during the
past 15 years. Among them, some honokiol derivatives displayed better
anti-proliferative activity than honokiol and showed good developing
potential in anticancer field.13–17 Based on these researches, the struc-
ture-activity relationship (SAR) of honokiol was studied.13,15 Although
the end points of these studies vary widely, the point that the hydro-
phobic side chain and free phenolic hydroxyl were essential for anti-
tumor activity, while the positions of phenolic hydroxyls had little in-
fluence was clear.
The anti-proliferative activities of honokiol derivatives against MCF-
7 (human breast cancer cells), CNE2Z (human nasopharyngeal carci-
noma cells) and I-10 (mouse leydig testicular cancer cells) tumor cell
lines were evaluated by MTT assay. The results were described in IC50
(concentration required for 50% inhibition) and presented in Table 1.
Effective concentration of honokiol analogs that inhibited cell
growth to 50% of control (IC50) in three different cancer cell lines was
determined (GraphPad Prism version 4 for Windows) using the MTS
assay. Cis-platinum (DDP) was used as the positive control. Values are
To find more effective antitumor honokiol derivatives, a series of
novel honokiol derivatives alkylated on either or both of the 2- or 4′-
position were synthesized (Fig. 1). All derivatives were evaluated for
their in vitro anti-proliferative activities. Among them, compound 5a
with 2, 4-dichlorobenzyl moiety located at 4′-position showed the most
given as mean
SD of three separate assays.
It is observed that O-alkylation of 4′-OH or 2-OH of honokiol
(1a–1b, 2a–2b and 3a–3b, etc.) slightly improved the anti-proliferative
⁎ Corresponding authors.
E-mail addresses: athongmei@foxmail.com (H.-M. Li), wuchengzhu0611@bbmc.edu.cn (C.-Z. Wu).
c Both authors contributed equally to this study.
https://doi.org/10.1016/j.bmcl.2019.126849
Received 10 October 2019; Received in revised form 18 November 2019; Accepted 18 November 2019
0960-894X/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:MeilinZhu,etal.,Bioorganic&MedicinalChemistryLetters,https://doi.org/10.1016/j.bmcl.2019.126849

M. Zhu, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Table 1
Anti-proliferative activity of compounds (IC50, µM, 72 h).
Compound
MCF-7
CNE2Z
I-10
Honokiol
DDP
1a
41.80
16.55
24.71
37.52
> 50
26.31
19.94
> 50
23.17
22.33
> 50
11.46
19.43
> 50
14.72
27.44
> 50
15.32
17.47
> 50
1.16
1.40
2.95
1.72
31.27
1.04
0.40
31.11
4.56
0.99
0.20
0.81
25.53
30.33
> 50
24.08
20.40
> 50
30.32
24.01
> 50
16.41
19.91
> 50
11.34
24.21
> 50
17.83
14.23
> 50
0.31
0.93
26.54
37.95
> 50
21.40
24.70
> 50
29.83
21.84
> 50
16.56
17.16
> 50
10.41
21.13
> 50
12.29
12.20
> 50
1.46
0.43
1b
1c
2a
0.12
0.41
1.10
1.22
1.13
0.23
2b
Fig. 1. Design strategy for honokiol derivatives.
2c
3a
1.35
1.58
2.15
1.22
2.74
1.08
3b
activity against MCF-7, CNE2Z and I-10 cells, while both 4′-O- and 2-O-
alkylated products were inactive at 50 μM. Moreover, it is suggested
that the substituted Cl atoms on benzyl had a significant influence on
the anti-proliferative activity (4a, 5a and 6a). Among them, compound
5a showed excellent activity against I-10 cell line with IC50 value of
10.41 μM.
3c
4a
0.93
0.45
1.21
2.70
0.40
2.27
4b
4c
5a
1.99
1.07
1.67
1.51
0.30
1.67
5b
5c
Moreover, I-10 cells were treated with different concentrations of
compound 5a (2.5, 5, 10 and 20 µM). MTT assays were performed at
three time points (24 h, 48 h and 72 h). The results demonstrated that
5a treatment inhibited the proliferation of I-10 cells in a time and dose-
dependent manner. At 20 µM, cell proliferation was almost completely
inhibited. Therefore, 2.5, 5 and 10 µM 5a were used for subsequent
experiments (Fig. 2).
6a
0.30
0.51
3.15
0.93
1.54
0.81
6b
6c
Tumor invasion and migration are the main causes of treatment
failure and high mortality in all kinds of cancer patients, including
testicular cancer.18 Therefore, the effects of 5a on the migration and
invasion of I-10 cells were investigated.
Wound-healing assays were performed to investigate the effects of
5a on the migration ability of I-10 cells. As demonstrated in Fig. 3A,
10 µM 5a significantly inhibited the migration of I-10 cells compared
with the control group. To further detect the influences of 5a on the
migration and invasion of I-10 cells, transwell assays were conducted.
The results showed that 2.5 µM 5a treatments significantly reduced the
number of tumor cells that crossed the transwell chamber compared
with the control (p < 0.05). The invasion and migration of I-10 cells
were decreased to a greater extent at an increased dose of 5a, sug-
gesting that 5a inhibited the invasion and migration of I-10 cells in a
concentration-dependent manner (Fig. 3B).
Fig. 2. Cytotoxic effect of different concentrations of 5a on I-10 cells for 24 h,
48 h and 72 h by MTT assay.
Since hypoxia-inducible factor (HIF) can facilitate tumor metastasis,
Scheme 1. Synthetic route for honokiol derivatives. Reagents and conditions: iodomethane; bromoethane; benzyl chloride; 2,6-dichlorobenzylchloride; 2,4 di-
chlorobenzylchloride; 3,4-dichlorobenzylchloride; Na2CO3, DMF as solvent, oil bath, 65 °C for 3 h.
2

M. Zhu, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Fig. 3. Effects of 5a under the lower con-
centration gradient on invasion and migra-
tion of I-10 cells under hypoxia conditions
by transwell assays. A: I-10 cells
(6 × 105 cells/well) were placed in 6-well
plate for 24 h and were wounded with a
scratch and rinsed to remove debris for in-
cubation with or without 5a (0, 2.5, 5 and
10 μM) for 24 h. Scale bars = 200 μm. B:
0.4 × 105 cells were seeded into the ma-
trigel coated transwell chamber and exposed
to compounds for 36 h to evaluated the in-
vasion activity and for 24 h to evaluated the
migration without matrigel. Cis-platinum
(DDP) was used as the positive control. The
cells at the lower side of the membrane were
then stained using Crystal Violet. Scale
bars = 100 μm. C and D: Quantification
analysis of
A and B presented as the
mean
standard deviation. **p < 0.01,
*p < 0.05 vs. control (n = 3).
migration, invasion, and many other important activities, as well as its
downstream proteins, matrix metalloproteinase-2 and -9 (MMP-2 and
MMP-9), inhibition of them could be a valuable strategy of modulating
the process of tumor growth.19,20 The results of the aforementioned
experiments suggest that 5a significantly inhibited the proliferation,
invasion and migration of I-10 cells. Next, the effects of 5a on the ex-
pression of invasion and migration associated proteins including HIF-
1α, MMP-2 and MMP-9 were investigated. Western blotting experi-
ments showed that treatments with 20 µM 5a significantly reduced the
expression of HIF-1α, MMP-2 and MMP-9 (Fig. 4), further confirming
that 5a plays an inhibitory role on migration and invasion in I-10 cells.
Honokiol is a small molecule with broad antitumor activity. To gain
derivatives with greater antitumor potential than honokiol, 18 honokiol
derivatives were synthesized and evaluated for their anti-proliferative
activities. It is noteworthy that benzyl group with Cl atoms substituted
at C2 or C4′ position can significantly improve the anti-proliferative
activity. In detail, compound 5a displayed the best inhibitory activity
among them. Furthermore, the anti-invasive and anti-migratory activ-
ities of 5a against I-10 cells were also researched. In conclusion, the
results of this research will promote the development of honokiol de-
rivatives in antitumor field and further mechanism, in vivo antitumor
evaluation and structural modification of 5a are in progress.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
3

M. Zhu, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Acknowledgments
This work was supported by the Education Department of Anhui
Natural Science Research Project China (Nos. KJ2018A0232,
KJ2019A0326); Anhui Provincial Natural Science Foundation (No.
1908085QH347) and Bengbu Medical College Natural Science
Foundation (BYKF1722, BYKY1768).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2019.126849.
References
1. Fujita M, Itokawa H, Sashida Y. Chem Pharm Bull. 1972;20:212.
2. Bai X, Cerimele F, Ushio-Fukai M. J Biol Chem. 2003;278:35501.
3. Fried LE, Arbiser JL. Antioxid Redox Signal. 2009;11:1139.
4. Liou KT, Shen YC, Chen CF, Tsao CM, Tsai SK. Eur J Pharmacol. 2003;475:19.
5. Lo YC, Teng CM, Chen CF, Chen CC, Hong CY. Biochem Pharmacol. 1994;47:549.
6. Sakaue Y, Domon H, Oda M, et al. Microbiol Immunol. 2016;60:10.
7. Sun L, Liao K, Wang D. PLoS One. 2015;10:e0117695.
8. Jiang QQ, Fan LY, Yang GL, et al. BMC Cancer. 2008;8:242.
9. Wang X, Duan X, Yang G, et al. PLoS One. 2011;6:18490.
10. Kumar A, Kumar-Singh U, Chaudhary A. Future Med Chem. 2013;5:809.
11. Hu J, Chen L, Liu L, et al. Exp Mol Med. 2008;40:617.
12. Liu Y, Chen L, He X, et al. Int J Gynecological Cancer. 2008;18:652.
13. Lin D, Yan Z, Chen A, et al. Bioorg Med Chem. 2019;27:3729.
14. Bernaskova M, Kretschmer N, Schuehly W, Huefner A, Weis R, Bauer R. Molecules.
2014;19:1223.
Fig. 4. Effects of 5a on the expression level of HIF-1α and its associated down-
stream proteins: MMP-2 and MMP-9. A: HIF-1α, MMP-2 and MMP-9 protein
levels were determined by western blotting following 5a treatment for 24 h. β-
actin expression was included as an internal control. B: Data are presented as
15. Ma L, Chen J, Wang X, et al. J Med Chem. 2011;54:6469.
16. Lin JM, Prakasha-Gowda AS, Sharma AK, Amin S. Bioorg Med Chem. 2012;20:3202.
17. Chou J, Chen L. Sichuan Chem Ind. 2014;17:1.
the mean
standard deviation. **p < 0.01, *p < 0.05 vs. control.
18. Liu H, Yuan M, Yao Y, Wu D, Dong S, Tong X. Biomed Pharmacother.
2019;117:109090.
19. Harris AL. Nat Rev Cancer. 2002;2:38.
20. Rapisarda A, Uranchimeg B, Scudiero DA, et al. Cancer Res. 2002;62:4316.
4