Cytotoxic neolignans: An SAR study

Article abstract of DOI:10.1016/j.bmcl.2004.10.011

The cytotoxic effects of different neolignans with the structure 1 were studied. The neolignans, magnolol 1 and honokiol 2 have been reported to inhibit the growth of several tumor cell lines in vitro and in vivo. The chemical structure of magnolol and honokiol consists of biphenyl skeleton with phenolic and allylic functionalities. Analogs of 1 and 2 containing different substitution have been studies for their effect on the growth of Hep-G2 and their structure-activity relationships were reported in this work.

Full text of DOI:10.1016/j.bmcl.2004.10.011

Bioorganic & Medicinal Chemistry Letters 15 (2005) 163–166
Cytotoxic neolignans: an SAR study
Zwe-Ling Kong, Shin-Cheng Tzeng and Yeuk-Chuen Liu*
Food Science Department, National Taiwan Ocean University, Keelung, Taiwan, ROC
Received 15 September 2004; revised 1 October 2004; accepted 5 October 2004
Available online 26 October 2004
Abstract—The neolignans, magnolol 1 and honokiol 2 have been reported to inhibit the growth of several tumor cell lines in vitro
and in vivo. The chemical structure of magnolol and honokiol consists of biphenyl skeleton with phenolic and allylic functionalities.
Analogs of 1 and 2 containing different substitution have been studies for their effect on the growth of Hep-G2 and their structure–
activity relationships were reported in this work.
Ó 2004 Elsevier Ltd. All rights reserved.
Magnolol and honokiol are significant bioactive
constituents isolated from the bark of Magnoliae
officinalis, or M. obovata which have been used in
traditional Chinese medicine for the relief of flu
symptoms, treatment of anxiety and stroke. Recently,
magnolol has been demonstrated to possess potent
translocation of cytochrome c from mitochondria to
cytosol; (2) activation of caspase 3, caspase 8, and cas-
7
–9
pase 9; and (3) downregulation of bcl-2 protein.
If
the COLO cell line was incubated with magnolol in
the presence of a phospholipase C inhibitor, no apopto-
sis was observed. Honokiol was reported to possess
higher activity in the induction of apoptosis than mag-
1
–3
4
anti-oxidative activity,
5
anti-anxiety, anti-inflamma-
tory, and anti-cancer activities. Magnolol and
honokiol have cytotoxic activities against A459 cell line
6
10
nolol. Honokiol induced apoptosis by inhibition of
1
Akt and MAPK phosphorylation.
0
(
human nonsmall lung cell cancer), SK-MEL-2 (human
melanoma), SK-OV-3 (ovarian cancer), XF498 (CNS
cell line), HCT-15 (colon cancer), and Hep-G2 (liver
cancer).
The chemical structure of magnolol is an ortho,ortho-C–
C dimer of 4-allyl-phenol which consists a biphenyl skel-
eton with phenolic functionalities and two p-allyl groups
as side chains. Since the molecular target of anti-cancer
activities of magnolol was not clear, we envisioned that
further modifications of magnolol functionalities would
provide us more information concerning the basic struc-
ture–activity relationship of magnolol. Magnolol and its
analogs described in the present study were illustrated in
Schemes 1 and 2.
6
–9
Magnolol at low concentrations (3–10lM) inhibited
DNA synthesis and human cancer cell growth (COLO-
2
human untransformed cells such as keratinocytes, fibro-
05 and Hep-G2), but did not inhibit the growth of
7
blasts, and human umbilical vein endothelial cells.
Apoptosis was observed in mouse melanoma B16-BL6,
human acute monocytic leukemia THP-1, fibrosarcoma
HT-1080, COLO-205, and Hep-G2 cells when magnolol
concentration was increased to 100lM, but not in
human untransformed gingival fibroblasts and human
umbilical vein endothelial cells. The mechanism of apop-
Magnolol and the ether derivative 4 were prepared by
horseradish peroxidase-catalyzed phenolic coupling of
4-allyl-phenol 3 in the presence of H O (Scheme 1).
2
2
Briefly, the hydrogen peroxide was added slowly
(0.03equiv/min) to the mixture of 4-allyl-phenol in buffer
solution (pH6.0, 10% methanol v/v) in the presence of
horseradish peroxidase (Sigma, Type-1, 1000units/mmol
of substrate) at room temperature for 18min. Bis-euge-
nol 7 and tetrahydromagnolol 11 were also prepared
by phenolic oxidative coupling of eugenol and propyl-
phenol catalyzed by horseradish peroxidase, respectively
(Scheme 1) as previous described condition except 30%
methanol in buffer solution being employed. The
7
–9
tosis induced by magnolol has also been studied.
Several cellular phenomena related to apoptosis after
the treatment of magnolol have been observed includ-
ing: (1) intracellular calcium concentration increased,
Keywords: Neolignans; Cytotoxicities; Hep-G2; SAR.
*
Corresponding author: Tel.: +886 2 24622192x5142; fax: +886 2
4634203; e-mail: york@mail.ntou.edu.tw
2
0
960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.10.011

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Z.-L. Kong et al. / Bioorg. Med. Chem. Lett. 15 (2005) 163–166
O
OH
study. Twelve analogs (Schemes 1 and 2) were tested
for cytotoxicity against a human liver tumor cell line
Hep-G2) and the results were summarized in Table 1.
BBr
3 3 3
-CH SCH
(
refluxing, 95%
Substitution of the biphenol was required for activity
0
since the 2,2 -dihydroxybiphenyl 5 (IC : >200lM)
50
1
3
3
had no cytotoxic activity against Hep-G2. For the
sake of investigating the role of phenolic group con-
tributing to the anti-cancer activities, methoxyl analog
6 was synthesized to diminish the possibility of the
participation of phenolic ionic bonding to its biomo-
lecular target. Cytotoxicity of methylated derivative 6
OH
OH
OH
OH
O
2 2
HRP-I, H O
+
1
1
0% MeOH/buffer
(28%), 4 (5%)
(
IC : >200lM) decreased significantly which demon-
50
3
4
1
strated that free phenolic functionalities were essential
for the activities. Introducing methoxyl groups at the
0 0
3 and 3 -positions as in bis-eugenol (3,3 -dimethoxy-
OH
OH
OH
0
0
0
O
5,5 -di-2-propenyl-1,1 -biphenyl-2,2 -diol) 7 or hydr-
0
O
O
2 2
HRP-I, H O
oxyl groups at 3 and 3 -positions as in dihydroxyl
3
6
0% MeOH/buffer,
0%
magnolol 8 diminished the cytotoxicity (IC50 of 7
and 8: 117 and 104lM, respectively). It might be
due to the formation of intra-molecular hydrogen
1
4
7
BBr
refluxing
8%
3
-CH
3
SCH
3
2
OH
OH
O
O
OH
OH
OH
3 2 3
CH OCO CH
HO
OH
t-BuOK, TBAB
refluxing
5
4%
1
6
8
OH
OH
OH
OH
OH
OH
2 2
HRP-I, H O
PdCl
2
3
1
0% MeOH/buffer,
3%
MeOH, RT
3
4%
1
5
1
1
1
9
Scheme 1. Synthesis of neolignan analogs.
OH
OH
allyl bromide
CO , acetone
O
O
K
2
3
0
56%
magnolol analogs, including dimethyl 6, propenyl 9, 3,3 -
diallyl 10 derivatives and tetrahydrohonokiol were pre-
pared by the methods as illustrated in Scheme 2. Briefly,
dimethyl derivative of magnolol 6 was prepared by heat-
5
1
6
1
50 o
neat
5%
C
1
1
ing magnolol in dimethylcarbonate with base. Demeth-
ylated derivative of bis-eugenol 8 was obtained by
3
1
2
heating bis-eugenol 7 with boron tribromide. Propenyl
derivative 9 was prepared by isomerization of double
OH
OH
1
3
0
bond of magnolol catalyzed with PdCl2. 3,3 -Diallyl
derivative 10 was obtained by thermal Claisen rearrange-
0
14
1
0
ment of allyl ether of 2,2 -biphenol 5. Tetrahydro-
honokiol 12 was obtained by reduction of honokiol
OH
1
5
OH
with sodium borohydride in the presence of NiCl2.
All the chemical structures and purities of synthetic che-
micals described in this work were confirmed by NMR.
OH
OH
NaBH
4 2
-NiCl
1
6
7
0%
The chemical structure of magnolol consists of biphenyl
skeleton with phenolic and p-allylic functionalities. Since
liver cancer was the leading cause of death in Asia,
human liver tumor cell line was chosen for the present
2
1
2
Scheme 2. Synthesis of neolignan analogs.

Z.-L. Kong et al. / Bioorg. Med. Chem. Lett. 15 (2005) 163–166
Table 1. Cytotoxicities of neolignan analogs against Hep-G2
165
References and notes
Compounds
IC50 (lM)
1
2
. Chan, P.; Cheng, J. T.; Tsao, C. W.; Niu, C. S.; Hong, C.
Y. Life Sci. 1996, 59, 2067–2073.
. Lee, Y. M.; Hsiao, G.; Chen, H. R.; Chen, Y. C.; Sheu, J.
R.; Yen, M. H. Eur. J. Pharmacol. 2001, 422, 159–
1
2
3
4
5
6
7
8
9
32.0
16.5
>200
51.8
1
67.
>200
>200
116.8
103.5
38.0
3
4
. Haraguchi, H.; Ishikawa, H.; Shirataki, N.; Fukuda, A. J.
Pharm. Pharmacol. 1997, 49, 209–212.
. Maruyama, Y.; Kuribara, H.; Morita, M.; Yuzurihara,
M.; Weintraub, S. T. J. Nat. Prod. 1998, 61, 135–
1
38.
1
1
1
0
1
2
42.1
23.4
5
. Wang, J. P.; Ho, T. F.; Chang, L. C.; Chen, C. C. J.
Pharm. Pharmacol. 1995, 47, 857–860.
13.1
6
7
. Kim, Y. K.; Ryu, S. Y. Planta Med. 1999, 65, 291–292.
. Lin, S. Y.; Chang, Y. T.; Liu, J. D.; Yu, C. H.; Hp, Y. S.;
Lee, Y.-H.; Lee, W.-S. Mol. Carcinog. 2001, 32, 73–83.
. Ikeda, K.; Nagase, H. Biol. Pharm. Bull. 2002, 25, 1546–
1549.
8
9
bonding between the hydroxyl groups and methoxyl
groups in the bis-eugenol molecule 7 and the catechol
groups in the dihydroxyl-magnolol moiety 8. The intra-
molecular hydrogen bonding could prohibit the
formation of intermolecular ionic or hydrogen bond-
ing between the phenoxyl groups of bis-eugenol ana-
logs and the biomolecular target. Since the
. Yang, S. E.; Hsieh, M. T.; Tsai, T. H.; Hsu, S. L. Br. J.
Pharmacol. 2003, 138, 193–201.
1
0. Bai, X. H.; Cerimele, F.; Ushio-Fukai, M.; Waqas, M.;
Campbell, P. M.; Govindarajan, B.; Der, C. J.; Battle, T.;
Frank, D. A.; Ye, K.; Murad, E.; Dubiel, W.; Soff, G.;
Arbiser, J. L. J. Biol. Chem. 2003, 278, 35501–
3
5507.
cytotoxicity of ortho-O-ether 4 (IC : 52lM) was com-
5
0
11. Ouk, S.; Thiebaud, S.; Borredon, E.; Legars, P.; Lecomte,
L. Tetrahedron Lett. 2002, 43, 2661–2663.
12. Agharahimi, M. R.; LeBel, N. A. J. Org. Chem. 1995, 60,
1856–1863.
parable to magnolol or honokiol, the significant activ-
ity of the derivative 4 demonstrated that at least one
free hydroxyl group was essential to the induction of
cytotoxicity. Although the highly conjugated deriva-
tive was known to be pro-oxidants and prone to form
DNA-adduct, oxidative stress might not play an
important role of highly conjugated derivative of mag-
nolol for the cytotoxic activity since the cytotoxicity
1
1
1
3. Jing, X. B.; Gu, W. X.; Ren, X. F.; Bie, P. Y.; Pan, X. F. J.
Chin. Chem. Soc. 2001, 48, 59–63.
4. Li, C. Y.; Wang, Y.; Hu, M. K. Bioorg. Med. Chem. 2003,
1
1, 3665–3671.
5. Narisada, M.; Horibe, I.; Watanabe, F.; Takeda, K. J.
Org. Chem. 1989, 54, 5808.
16. Structures were confirmed with a 500MHz NMR
of highly conjugated magnolol analog
8lM) was close to the activity of its parent com-
pound 1 (IC : 32lM). No significant differences of
9 (IC :
50
3
spectrometer (Bruker, Rheinstetten, Germany) spectro-
meter with TMS as an internal reference. The purities of
all the synthetic compounds are >98% according to the
5
0
cytotoxicities of saturated allyl analogs (11 and 12)
comparing to the corresponding parent compounds
might imply that the double bonds did not contribute
p–p-interaction with the biomolecular target. The
position and nature of substituents on the benzene
rings also modulated anti-tumor activity of magnolol
derivatives. Honokiol is also one of a significant bio-
active neolignans other than magnolol isolated from
Magnolia bark. The structure of honokiol consists of
para-allyl-phenol and an ortho-allyl-phenol which link
together through ortho,para- C–C-coupling. According
NMR analysis.
1
H 6
H NMR of magnolol 1 d (acetone-d ): 7.09 (2H, d,
J = 2.04Hz), 7.04 (2H, dd, J = 8.2, 2.1Hz), 6.90 (2H, d,
J = 8.2Hz), 5.97 (2H, m), 5.07 (2H, dd, J = 17.0, 1.8Hz),
4
.99 (2H, dd, J = 10.1, 1.8Hz), 3.33 (4H, d, J = 6.7Hz) 4-
allyl-phenol 3 dH (acetone-d ): 6.97 (2H, d, J = 8.35Hz),
6
6
.73 (2H, d, J = 8.45Hz), 5.92 (1H, m), 5.02 (1H, dd,
J = 18.7, 1.8Hz), 4.97 (1H, dd, J = 9.8, 1.8Hz), 3.26 (2H,
d, J = 6.15Hz).
1
H NMR of isomagnolol 4 d (acetone-d ): 8.1 (1H), 7.14
H
6
0
0
(2H, d, J = 8.5Hz, H-3 /5 ), 6.93 (1H, d, J = 8.2Hz), 6.88
(1H, dd, J = 8.2, 1.6Hz), 6.83 (2H, d, J = 8.5Hz), 6.77
6
to the previous report, the cytotoxicity of honokiol
(
dd, J = 6.7Hz), 3.27 (2H, d, J = 6.6Hz).
1H, d, J = 1.6Hz), 5.95 (2H, m), 5.05 (4H, m), 3.32 (2H,
was comparable to magnolol against different cell
lines. The cytotoxic activity of honokiol was also close
to magnolol against Hep-G2 in the present study. The
1
3
H NMR of dimethoxymagnolol 6 (CDCl ): 6.90 (2H,
d, J = 8.3Hz), 7.06 (2H, d, J = 2.15Hz), 7.13 (2H, dd,
J = 8.3, 2.15Hz), 5.90 (2H, m), 5.08 (2H, dd, J =
0
3
-allyl group of honokiol (both allyl groups of magno-
lol are para to the phenolic-OH groups) might play
1
3
7.0, 1.65Hz), 5.05 (2H, d, J = 9.9Hz), 3.75 (6H, s),
.36 (4H, d, J = 6.7Hz).
an important role to the cytotoxicity since the ether
0
1
derivative and 3,3 -diallyl derivative 10 (IC : 42lM)
5
0
H NMR of bis-eugenol 7 dH (CDCl ): 6.79 (2H, d,
3
also have significant activities against Hep-G2.
J = 1.85Hz), 6.68 (2H, J = 1.9Hz), 5.99 (2H, m), 5.11 (2H,
dd, J = 17.0, 1.75Hz), 5.00 (2H, dd, J = 10.05, 0.8Hz),
3
.85 (6H, s), 3.33 (4H, d, J = 6.70Hz).
1
H NMR of demethylated bis-eugenol 8 d
2H, d, J = 1.80Hz), 6.71 (2H, J = 1.7Hz), 6.11 (2H, s),
.96 (2H, m), 5.57 (2H, s), 5.11 (2H, dd, J = 18.28, 1.55
H 3
(CDCl ): 6.80
Acknowledgements
(
5
This work was partly supported by the National Science
Council of the Republic of China under Grant NSC 91-
Hz), 5.07 (2H, d, J = 10.55Hz), 3.33 (4H, d, J = 6.70Hz).
1
H NMR of highly conjugated magnolol derivative 9 dH
2
113-M-019-001.
(CDCl ): 7.29 (2H, dd, J = 8.35, 2.05Hz), 7.23 (2H, d,
3

166
Z.-L. Kong et al. / Bioorg. Med. Chem. Lett. 15 (2005) 163–166
J = 1.95Hz), 6.96 (2H, d, J = 8.35Hz), 6.35 (2H, dd,
J = 15.7, 1.05 HZ), 6.13 (2H, m), 1.87 (6H, dd, J = 6.6,
.35Hz).
dd, J = 8.25, 2.05Hz), 7.07 (2H, dd, J = 2.0Hz), 6.94 (2H,
d, J = 8.25Hz), 5.48 (2H, s), 2.56 (4H, t, J = 7.4Hz), 1.64
(4H, m), 0.95 (6H, d, J = 7.3Hz).
1
1
0
1
H NMR of 3,3 -diallyl-biphenol 10 d
H
(CDCl
d, J = 6.7, 0.7Hz), 7.14 (2H, J = 7.5, 1.4Hz), 7.00 (2H, t,
3
): 7.20 (2H,
H 3
H NMR of tetrahydronokiol 12 d (CDCl ): 7.21 (1H, d,
J = 2.05Hz), 7.04 (1H, d, J = 8.15, 2.05Hz), 7.01 (1H,
J = 2.10Hz), 6.88 (1H, d, J = 7.95Hz), 6.87 (1H,
J = 7.95Hz), 5.09 (1H, s), 4.90 (1H, s), 2.63 (2H, t,
7.55Hz), 2.54 (1H, s, 7.50Hz), 1.67 (4, m), 1.0 (3H, t,
J = 7.35Hz), 0.95 (3H, t, J = 7.35Hz).
J = 7.55Hz), 6.06 (2H, m), 5.48 (2H, s), 5.17 (2H, dd, J =
17.2, 0.55Hz), 5.12 (2H, dd, J = 11.5, 1.3Hz), 3.48 (4H, d,
J = 6.55Hz).
1
H 3
H NMR of tetrahydromagnolol 11 d (CDCl ): 7.13 (2H,