In vitro growth inhibition of human cancer cells by novel honokiol analogs

Article abstract of DOI:10.1016/j.bmc.2012.03.062

Honokiol possesses many pharmacological activities including anti-cancer properties. Here in, we designed and synthesized honokiol analogs that block major honokiol metabolic pathway which may enhance their effectiveness. We studied their cytotoxicity in human cancer cells and evaluated possible mechanism of cell cycle arrest. Two analogs, namely 2 and 4, showed much higher growth inhibitory activity in A549 human lung cancer cells and significant increase of cell population in the G0-G1 phase. Further elucidation of the inhibition mechanism on cell cycle showed that analogs 2 and 4 inhibit both CDK1 and cyclin B1 protien levels in A549 cells.

Full text of DOI:10.1016/j.bmc.2012.03.062

Bioorganic & Medicinal Chemistry 20 (2012) 3202–3211
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
In vitro growth inhibition of human cancer cells by novel honokiol analogs
⇑
,
Jyh Ming Lin a, , A.S. Prakasha Gowda a, , Arun K. Sharma b, Shantu Amin b
a Department of Biochemistry & Molecular Biology, College of Medicine, Pennsylvania State University, Hershey, PA 17033, USA
b Department of Pharmacology, College of Medicine, Pennsylvania State University, Hershey, PA 17033, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Honokiol possesses many pharmacological activities including anti-cancer properties. Here in, we
designed and synthesized honokiol analogs that block major honokiol metabolic pathway which may
enhance their effectiveness. We studied their cytotoxicity in human cancer cells and evaluated possible
mechanism of cell cycle arrest. Two analogs, namely 2 and 4, showed much higher growth inhibitory
activity in A549 human lung cancer cells and significant increase of cell population in the G0–G1 phase.
Further elucidation of the inhibition mechanism on cell cycle showed that analogs 2 and 4 inhibit both
CDK1 and cyclin B1 protien levels in A549 cells.
Received 13 October 2011
Revised 19 March 2012
Accepted 27 March 2012
Available online 3 April 2012
Keywords:
Honokiol analogs
Cell proliferation
CDK1
Ó 2012 Elsevier Ltd. All rights reserved.
Cyclin B1
Human cancer cell
1. Introduction
last decade, there is growing evidence to challenge the classical
view of cell cycle regulation.21 Recent investigation on genetic
Honokiol is one of the biphenolic constitute isolated from the
stem bark and root of Magnolia officinalis,1 a Chinese medicine used
to treat variety of diseases. It is shown to possess many pharmaco-
logical activities, including anti-inflammatory,2 anti-thrombosis,3
antioxidant effects,4 antiviral,5,6 and induce differentiation in HL-
60 human leukemia cells.7 Studies have demonstrated that honok-
iol induces apoptosis in several cell types such as human colorec-
tal,8 rat hepatic stellate,9 human squamous lung cancer10 and B
cell chronic lymphocytic leukemia cells.11 In addition, honokiol
inhibits skin tumor promotion,12 human fibrosarcoma invasive-
ness in vitro,13 angiogenesis and tumor growth in vivo,14 and over-
comes drug resistance in multiple myeloma.15 More recently,
honokiol has also been shown to act as chemopreventive agent
against UVB induced skin cancer in a hairless mouse model.16 Sai-
bokuto, a traditional Chinese remedy containing honokiol17 as ma-
jor ingredient, is approved as effective treatment for asthma and
bronchitis by Japanese Health Ministry.
and RNA interference in mammalian cells have reported that
CDK2 and CDK4 are not necessary for cell cycle progression, and
CDK1 as the only nonredundant cell cycle driver.22,23 Several natu-
ral and synthetic small molecule inhibitors have been reported, but
cell cycle studies of these molecules are not consistent with spe-
cific CDK1 inhibition.24,25 Cyclin B1 is essential for the initiation
of mitosis. It accumulates in the S phase of the cell cycle and
reaches the maximal level at mitosis, but is absent in G1 phase
cells.26 However, recent reports indicated that cyclin B1 was ex-
pressed in the G1 phase in several synchronized and asynchro-
nously growing cancer cells.27–31 Furthermore, over expression of
cyclin B1 has been reported in tumor cells,32–35 thus targeting cy-
clin B1and its complex partner, namely CDK1 is a promising ther-
apeutic approach for controlling cancer.
There are reports of structure–activity relationship (SAR) stud-
ies on honokiol; allyl substituted biphenyl skeleton seems to be
common element in these studies.6,14,36 However, the end points
of these studies vary widely; therefore no concrete conclusion
could be drawn. We were interested in designing novel analogs
of honokiol, which could follow a similar mechanism as honokiol
but are more potent, by careful manipulation of the functional
groups. Our hypothesis for SAR study was that the hydroxyl groups
on the honokiol biphenyl skeleton may be subjected to metabolic
oxidation by phase I enzymes,37,38 and subsequently excrete out
of the system, therefore might reduce its effectiveness. Glucuroni-
dation and sulfation of the free hydroxyl groups have been shown
to be the main metabolic pathways for honokiol in rat and human
liver leading to a faster excretion and reduced half-life.39 Therefore,
Honokiol is reported to induce cell cycle arrest at G0/G1 phase
in human prostate cancer cell and vascular muscle smooth
cells.18,19 It also suppresses osteoclastogenesis and invasion
through modulation of NFj
B pathway.20 Honokiol has been shown
to interact with cell cycle regulatory proteins such as cyclin-depen-
dent kinases (CDKs) and cyclins;16,20 it down-regulates the levels
of cyclins D1, D2, E and associated CDK2, CDK4 and CDK6. In the
⇑
Corresponding author. Tel.: +1 717 531 3612; fax: +1 717 531 7072.
E-mail address: jml47@psu.edu (J.M. Lin).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.062

J. M. Lin et al. / Bioorg. Med. Chem. 20 (2012) 3202–3211
3203
we synthesized several honokiol analogs (Fig. 1), which have their
40-hydroxyl group replaced with methoxy group in A-ring, and the
substitution pattern of allyl and hydroxyl groups in B-ring altered,
in order to study their cytotoxicity and mechanism of action.
In this study, we explore the SAR of honokiol analogs for their
cytotoxicity in human lung (A549), melanoma (UACC903) and co-
lon (HT-29) cancer cell lines and determine the underlying mech-
anism of action by evaluating the cell cycle studies of honokiol
analogs 2 and 4, the two best identified analogs based on their po-
tent inhibition of A549 human lung cancer cell growth.
nitrogen atmosphere, BBr3 (37.2 g, 148.5 mmol) was added
through double needle. The mixture was stirred for 3 h at 0 °C then
was allowed to warm up to room temperature over night. The mix-
ture was poured to ice water and organic layer separated. The
aqueous layer was extracted with CH2Cl2 (100 ml ꢁ 3). The com-
bined organic layer was dried over Na2SO4, solvent was evaporated
to yield 19.2 g black oily residue. Crude compound (7.55 g) was
purified by column chromatography eluted with hexanes/
EtOAc = 20/1 to yield 5.04 g 4-allylphenol (7) as clear oily residue
(70.9%).40 1H NMR (CDCl3) d 7.10 (2H, dt, J = 8.50 Hz, J = 2.0 Hz),
6.82 (2H, dt, J = 8.5 Hz, J = 2.0 Hz), 6.04–5.96 (1H, m), 5.19 (1H, s),
5.13–5.09 (2H, m), 3.37 (2H, d, J = 6.5 Hz). 13C NMR (CDCl3) d
153.68, 137.87, 132.41, 129.78, 115.53, 115.33, 39.35.
2. Experimental section
Reagents. All chemicals for the syntheses were obtained from
Aldrich Chemical Co. (St. Louis, MO) and used without further puri-
fication. Thin-layer chromatography (TLC) was developed on alu-
minum-supported pre-coated silica gel plates (EM industries,
Gibbstown, NJ). Column chromatography was performed on silica
gel (70–230 mesh). NMR spectra were recorded on Bruker Avance
II 500 MHz instrument and the chemical shifts are given as d values
with reference to tetramethylsilane (TMS) as internal standard. MS
spectra were recorded on Applied Biosystems API 3200. Honokiol
was supplied by Sigma–Aldrich. Stock solution of honokiol and
its analogs (final concentration, 10 mM) was prepared in DMSO,
stored at ꢀ20 °C, and diluted with fresh complete medium imme-
diately before use. DMEM was from Mediatech, Inc.; trypsin–EDTA
solution was from Invitrogen, and fetal bovine serum was from
Atlanta Biological. Protease inhibitor cocktail was from BD Biosci-
ences, propidium iodide was from Sigma. Polyclonal antibodies
to cyclin B1 and cyclin-dependent kinase 1 (CDK1) were from Mil-
lipore, and protease inhibitor from Thermo Scientific, USA.
2.1.1.2. 4-Allyl-2-bromo-phenol (8).
4-Allylphenol (7;
1.35 g; 10.06 mmol) dissolved in 300 ml of dry diethyl ether under
nitrogen atmosphere was cooled with acetone-dry ice bath. To the
reaction mixture, i-propyl magnesium chloride (2.0 M; 5.2 ml;
10.4 mmol) was added dropwise over 3 min, and mixture was stir-
red at -78 °C for 1 h before 1,3-dibromo-5,5-dimethylhydantoin41
(DBDMH; 1.44 g; 5.04 mmol) was added. The mixture was stirred
20 h at ꢀ78 °C then warmed up to room temperature over 1 h;
then 10 ml of 10% NaSO3 was added to the mixture. The aqueous
layer was extracted with ether (10 ml ꢁ 3), and the combined or-
ganic layer was dried over Na2S2O4; then concentrated in vacuo
to yield 1.40 g yellowish oil as crude product. The residue was puri-
fied by flash column chromatography on silica gel eluted with hex-
anes/EtOAc = 40/1 to give 8 as colorless oil (0.98 g, 45.7%).40 1H
NMR (CDCl3) d 7.31 (2H, d, J = 2.0 Hz), 7.07 (1H, dd, J = 8.5 Hz,
J = 2.0 Hz), 6.98 (1H, d, J = 8.50 Hz), 5.98–5.91 (1H, m), 5.45 (1H,
s), 5.13–5.08 (2H, m), 3.33 (2H, d, J = 7.0 Hz). 13C NMR (CDCl3) d
150.56, 137.03, 133.74, 131.79, 129.39, 116.17, 115.94, 110.08,
38.96. MS (m/z, Intensity): 214 (M+1+, 25%), 113 (100%), 105 (80%).
2.1. Synthesis of honokiol analogs
2.1.1.3. 4-Allyl-2,6-dibromophenol (9).
4-Allylphenol (7;
2.1.1. Syntheses of key components of Suzuki coupling reagents
1.01 g, 7.45 mmol) was dissolved in 25 ml of CHCl3 at room tem-
2.1.1.1. 4-Allylphenol (7).
4-Allylanisole (20 g, 135 mmol)
perature, 1,3-dibromo-5,5-dimethylhydantoin41 (DBDMH; 2.24 g,
was dissolved in 400 ml of CH2Cl2 and cooled by ice bath under
OMe
OMe
OMe
HO
HO
Br
HO
MeO
OH
3'-Bromo-3,5'-di-allyl-
2,6-Di-(4'-methoxy-3'-
allylphenyl)-1-phenol
3,5'-Diallyl-2'-hydroxyl-
4-methoxy-1,1'-biphenyl
2'-hydroxyl-4-methoxy-
1,1'-biphenyl
1
3
A-ring
2
HO
B-ring
OMe
OMe
HO
Honokiol
OH
OH
OH
3,3'-Diallyl-4-methoxy-
4'-hydroxy-1,1'-biphenyl
3',5-Diallyl-2,2'-di-
3,3'-Diallyl-2'-hydroxyl-4-
methoxy-1,1'-biphenyl
hydroxy-1,1'-biphenyl
4
6
5
Figure 1. Structure of honokiol analogs.

3204
J. M. Lin et al. / Bioorg. Med. Chem. 20 (2012) 3202–3211
7.83 mmol) was added in three portion over 5 min. The resulting
mixture was stirred for 2 h; the solid was removed by filtration.
The organic layer was evaporated in vacuo. The yellow residue
was purified by flash column chromatography eluted with hex-
anes/EtOAc = 20/1 to give 9 as yellowish oil residue (1.1 g, 50.6%).
1H NMR (CDCl3) d 7.29 (2H, s), 5.95–5.88 (1H, m), 5.78 (1H, s),
5.15–5.09 (2H, m), 3.36 (2H, d, J = 6.5 Hz). 13C NMR (CDCl3) d
147.66, 136.20, 134.68, 132.02, 116.88, 109.67, 38.62. MS (m/z,
Intensity): 294 (M+1+, 100%), 213 (5%).
J = 6.5 Hz). 13C NMR d (CDCl3) 152.79, 136.52, 135.81, 131.56,
129.58, 125.65, 117.54, 116.34, 99.91, 57.81, 34.73. MS (m/z,
Intensity): 258 (M+, 25%), 225 (97%), 147 (100%).
2.1.1.8.
(14).
3-Allyl-2-methoxymethoxy-1-phenylboronic
1-Allyl-3-bromo-2-methoxymethoxybenzene
acid
(13)
(0.99 g, 3.9 mmol) was dissolved in 10 ml of dry THF and cooled
to ꢀ78 °C under nitrogen atmosphere, n-butyl lithium (2.5 M,
2.2 ml, 5.5 mmol) was added dropwise over 5 min. The mixture
was stirred for 45 min, and then tri-isopropyl borate (1.8 ml,
8.0 mmol) was added in one portion to the reaction mixture. The
mixture was warmed up to room temperature for 2 h, then 20 ml
of 0.1 N HCl was added. The organic layer was separated and aque-
ous layer was extracted with ether (25 ml ꢁ 2). The combined or-
ganic was dried over MgSO4 and concentrated in vacuo. The oily
residue was purified by chromatography on silica gel eluted with
hexane/EtOAc = 4/1, then EtOAc to give 0.93 g of 14 as colorless
residue. 1H NMR (CDCl3) d 7.73 (1H, dd, J = 7.5, 2.0 Hz), 7.35 (1H,
dd, J = 7.5, 2.0 Hz), 7.19 (1H, t, J = 2.5 Hz), 6.04–5.96 (1H, m), 5.91
(2H, s), 5.16–5.07 (2H, m), 5.06 (2H, s), 3.57 (3H, s), 3.44 (2H, d,
J = 6.0 Hz). 13C NMR (DMSO-d6) d 159.03, 137.82, 133.04, 132.04,
131.49, 123.89, 116.27, 100.13, 57.27, 34.33.
2.1.1.4. 2-Allyl-4-bromophenol (10).
4-Bromophenol was
converted to the corresponding allyl 4-bromophenyl ether. The al-
lyl ether was then undergoing thermal rearrangement according
procedure described by Sviridov et al.42 1H NMR (CDCl3) d 7.62–
7.24 (2H, m), 6.72 (1H, dd, J = 7.5 Hz, J = 1.5 Hz), 6.03–5.97 (1H,
m), 5.23–5.18 (2H, m), 4.96 (1H, s), 3.40 (2H, d, J = 6.0 Hz). 13C
NMR (CDCl3) d 153.20, 135.46, 132.98, 130.59, 127.65, 117.54,
117.21, 112.92, 34.80. MS (m/z, Intensity): 214 (M+, 100%), 199
(25%), 133 (60%), 118 (32%), 105 (28%).
2.1.1.5. 2-Allyl-4-bromoanisole (11).
2-Allyl-4-bromophenol
(10; 1.47 g; 6.89 mmol) was mixed with potassium carbonate
(6.0 g) and 30 ml of dry acetone under nitrogen atmosphere. After
stirring for 15 min, methyl iodide (3.0 ml) was added and the
resulting mixture was refluxed for 1.5 h. The reaction mixture
was filtered to remove solid, and then concentrated to give
1.55 g yellowish crude product. Further purification by column
chromatography on silica gel eluted with hexanes/EtOAc = 30/1
gave 11 as light yellowish oil (1.27 g, 81.1%). 1H NMR (CDCl3) d
7.31 (1H, dd, J = 8.5 Hz, J = 2.5 Hz), 7.27 (1H, d, J = 2.5 Hz), 6.75
(1H, d, J = 9.0 Hz), 5.98–5.93(1H, m), 5.11–5.07 (2H, m), 3.83 (3H,
s), 3.37 (2H, d, J = 3.5 Hz). 13C NMR (CDCl3) d 156.38, 136.03,
132.41, 130.99, 129.88, 116.14, 112.72, 111.97, 55.61, 33.91. MS
(m/z, Intensity): 229 (M+, 15%), 201 (100%), 171 (25%), 148 (22%).
2.1.2. General procedure for the synthesis of honokiol analogs
In
a 100 ml flask, 3-allyl-4-methoxyphenyl boronic acid
(3.9 mmol), Cs2CO3 (11.66 mmol), the corresponding bromo-allyl-
phenol (3.54 mmol), and catalyst Pd(Ph3P)4 (0.35 mmol) were
mixed in 45 ml of DME and 6 ml of water. The mixture was re-
fluxed under nitrogen overnight. Following standard workup pro-
cedure, the product was isolated by chromatography on silica
gel. The purities of all the synthetic compounds were >98% accord-
ing to the proton NMR analysis.
2.1.2.1.
(1)44,45
3,50-Diallyl-20-hydroxy-4-methoxy-1,10-biphenyl
The reaction mixture was purified by column chro-
.
2.1.1.6. 4-Methoxy-3-allyl-1-phenylboronic acid (12).
2-Al-
matography on silica gel eluted with hexanes/EtOAc = 30/1 to yield
colorless oil residue. 1H NMR (CDCl3) d 7.32 (1H, dd, J = 8.2 Hz,
2.2 Hz), 7.26 (1H, d, J = 2.2 Hz), 7.08 (1H, dd, J = 8.13, 2.2 Hz), 7.06
(1H, d, J = 2.1 Hz), 6.99 (1H, d, J = 8.2 Hz), 6.93 (1 h, d, J = 8.13 Hz),
6.08–6.97 (2H, m), 5.16 (1H,s), 5.14 (1H, q, J = 1.74 Hz), 5.11–5.06
(3H, m), 3.91 (3H, s), 3.47 (2H, d, J = 6.63 Hz), 3.38 (2H, d,
J = 6.75 Hz). 13C NMR (CDCl3) d 157.06, 150.84, 137.81, 136.51,
132.17, 130.50, 130.20, 129.80, 129.05, 128.76, 127.90, 127.85,
115.84, 115.56, 115.53, 110.00, 55.56, 39.42, 34.28. MS (m/z; Inten-
sity): 279 (Mꢀ-1, 100%), 264 (97%), 249 (32%). Total isolation yield:
52%.
lyl-4-bromoanisole (2.85 g, 12.55 mmol) was dissolved in 50 ml
of dry THF and cooled to ꢀ78 °C under nitrogen atmosphere, n-bu-
tyl lithium (2.5 M, 5.6 ml, 13.5 mmol) was added dropwise over
5 min. The mixture was stirred for 45 min, and then tri-isopropyl
borate (17.96 g, 95.7 mmol) was added in one portion to the reac-
tion mixture. The mixture was warmed up to room temperature for
2 h, then 50 ml of 0.5 N HCl was added. The organic layer was sep-
arated and aqueous layer was extracted with ether (50 ml ꢁ 3). The
combined organic layer was dried over MgSO4 and concentrated in
vacuo to give pale solid as crude product which was further puri-
fied by chromatography on silica gel and eluted with CH2Cl2/
MeOH = 30/1 to furnish 2.2 g of 12 as pale solid. 1H NMR (CDCl3)
d 8.13 (1H, dd, J = 8.0, 1.5 Hz), 8.00 (1H, d, J = 1.5 Hz), 7.01 (1H, d,
J = 8.0 Hz), 6.15–6.07 (1H, m), 5.17–5.10 (2H, m), 3.94 (3H, s),
3.51 (2H, d, J = 6.4 Hz). 13C NMR (DMSO-d6) d 159.08, 137.56,
136.25, 134.58, 126.82, 115.76, 110.13, 55.67, 34.38.
2.1.2.2. 30-Bromo-3,50-diallyl-20-hydroxy-4-methoxy-1,10-biphe-
nyl (2).
The reaction mixture was purified by column chroma-
tography on silica gel eluted with hexanes/EtOAc = 15/1 to yield
the first compound as yellowish oil residue. 1H NMR (CDCl3) d
7.37 (1H, dd, J = 8.5, 2.0 Hz), 7.31 (1H, d, J = 2.0 Hz), 7.29 (1H, d,
J = 2.0 Hz), 7.05 (1H, d, J = 2.0 Hz), 6.96 (1H, d, J = 8.5 Hz), 6.08–
5.92 (2H, m), 5.66 (1H, s), 5.13–5.06 (4H, m), 3.90 (3H, s), 3.45
(2H, d, J = 3.5 Hz), 3.35 (2H, d, J = 3.0 Hz). MS (m/z, Intensity): 359
(Mꢀ, 100%), 344 (20%). Total isolation yield: 15%.
2.1.1.7. 1-Allyl-3-bromo-2-methoxymethoxybenzene(13). 2-
Allyl-6-bromophenol was prepared according to procedu re
reported by Palmer et al.43 In a flask, 2-allyl-6-bromo-phenol
(14.6 g) was mixed with 15 g of potassium carbonate in 250 ml of
acetone under nitrogen atmosphere, and then chloromethyl me -
thyl ether (6.0 g) was added. The mixture was refluxed overnight.
After cooling to room temperature, the solid was filtered, and
resulting solution was concentrated. The residue was purified by
column chromatography on silica gel eluted with hexanes/
EtOAc = 20/1 to yield 13 g of 1-allyl-3-bromo-2-methoxymethoxy-
benzene (13). 1H NMR (CDCl3) d 7.45 (1H, dd, J = 8.0 Hz, J = 1.5 Hz),
7.17 (1H, dd, J = 8.0 Hz, J = 1.5 Hz), 6.98 (1H, t, J = 8.0 Hz), 6.04–5.96
(1H, m), 5.14–5.09 (2H, m), 5.12 (2H, s), 3.67 (3H, s), 3.54 (2H, d,
2.1.2.3. 2,6-Di-(40-methoxy-30-allylphenyl)-phenol (3).
The
same reaction mixture was purified by column chromatography
on silica gel eluted with hexanes/ethyl acetate=15/1 to yield the
second compound as light yellowish oil residue. 1H NMR (CDCl3)
d 7.41 (2H, dd, J = 8.0, 2.0 Hz), 7.34 (2H, d, J = 2.0 Hz), 7.06(2H, s),
6.97 (2H, d, J = 8.0 Hz), 6.08–5.90 (3H, m), 5.34 (1H, s), 3.90 (6H,
s), 3.46 (4H, d, J = 6.5 Hz), 3.40 (2H, d, J = 6.5 Hz). MS (m/z, Inten-
sity): 425 (Mꢀꢀ1, 100%), 409.8 (30%), 395 (10%). Total isolation
yield: 20%.

J. M. Lin et al. / Bioorg. Med. Chem. 20 (2012) 3202–3211
3205
2.1.2.4.
(4).
3,30-Diallyl-4-methoxy-40-hydroxy-1,10-biphenyl
The reaction mixture was purified by column chromatog-
electron coupling reagent, phenazine methosulfate (PMS), were
used in the MTS assay. Viable cells reduced MTS to formazan,
which was measured by determining absorbance at 492 nm using
a spectrophotometer. Formazan production is time dependent and
proportional to the number of viable cells. After incubation for the
raphy on silica gel eluted with hexanes/EtOAc = 10/1 to yield color-
less oil residue. 1H NMR (CDCl3) d 7.38 (1H, dd, J = 7.0, 2.0 Hz),
7.35–7.31 (3H, m), 6.92 (1H, d, J = 8.4 Hz), 6.88 (1H, d, J = 7.8 Hz),
6.13–6.03 (2H, m), 5.25–5.19 (2H, m), 5.13–5.07 (2H, m), 3.89
(3H, s), 3.49 (2H,d, J = 6.6 Hz), 3.46 (2H, d, J = 6.6 Hz). 13C NMR
(CDCl3) d 156.47, 153.23, 136.94, 136.23, 134.08, 133.35, 128.87,
128.85, 128.34, 126.23, 125.46, 125.41, 116.64, 116.15, 115.49,
110.65, 55.58, 35.39, 34.41. MS (m/z; Intensity): 279 (Mꢀꢀ1,
100%), 264 (59%), 249 (11%). Total isolation yield: 12%.
indicated time in the appropriate medium, 20 ll of MTS/PMS mix-
ture was added to each well. Then the cells were incubated for 3 h
and the formazan product was quantified by measuring absor-
bance at 492 nm. The background absorbance from the control
wells was subtracted from the actual absorbance value. Three
duplicate assays were done for each experimental condition. IC50
values were calculated by GraphPad Prism version 4 for Windows
(GraphPad Software, San Diego, CA; www.graphpad.com).
2.1.2.5.
(5).
3,30-Diallyl-20-hydroxy-4-methoxy-1,10-biphenyl
The coupling mixture was purified by column chromatog-
raphy on silica gel eluted with hexanes/ethyl acetate = 20/1 to
yield colorless oil residue which was further hydrolyzed to 5 by
dissolved in 15 ml of THF and 15 ml of 6 N HCl and refluxed for
6 h. Reaction mixture was extracted with EtOAc (20 ml ꢁ 3) and
the combined organic layer was dried over MgSO4. Organic solvent
was evaporated to give a colorless residue which was further puri-
fied by column chromatography on silica gel eluted with hexanes/
EtOAc = 40/1 to give a colorless oil residue. 1H NMR (CDCl3) d 7.31
(1H, dd, J = 7.5, 2.0 Hz), 7.26 (1H, d, J = 2.0 Hz), 7.13 (2H, td, J = 7.5,
2.0 Hz), 6.98 (1H, d, J = 8.5 Hz), 6.94 (1H, t, J = 7.5 Hz), 6.13–5.90
(2H, m), 5.36 (1H, s), 5.20–5.06 (4H, m), 3.90 (3H, s), 3.49 (2H, d,
J = 6.6 Hz), 3.45 (2H, d, J = 6.7 Hz). 13C NMR (CDCl3) d 157.04,
150.49, 136.78, 136.51, 130.66, 129.77, 129.30, 129.16, 128.36,
128.03, 127.99, 126.29, 120.34, 115.86, 115.80, 110.96, 55.55,
34.79, 34.29. MS (m/z; Intensity): 279 (Mꢀꢀ1, 100%), 264 (70%),
249 (30%). Total isolation yield: 53%.
2.2.2. Synchronization and cell cycle analysis
The effect of honokiol analogs on cell cycle distribution was
determined by flow cytometry after staining the cells with DNA
intercalating dye propidium iodide staining for DNA content mea-
surement.18,19 Approximately 5 ꢁ 106 A549 cells were seeded into
6 well plate and allowed to attach by overnight incubation. Cells
were synchronized by serum deprivation, when cells reached 40–
50% cell confluency DMEM medium was added without serum
and maintained in medium containing no serum for 48 h. After
48 h restimulated by replacing with fresh medium with 10% serum
containing different (0, 0.5, 2.5 and 10 lM) concentrations of hon-
okiol analogs such as 2 and 4 in similar volume of DMSO was
added to the control (final concentration of DMSO 0.2%). Plates
were incubated for 48 h at 37 °C. After that floating and adherent
A549 cells were collected by treatment with 0.05% trypsin and pel-
leted, washed with cold PBS. Cells were fixed in 70% ethanol at
ꢀ20 °C overnight. Following overnight fixation cells were washed
twice with ice cold PBS. Afterwards, cells were resuspended 1 ml
2.1.2.6. 30,5-Diallyl-2,20-dihydroxy-1,10-biphenyl (6).
The
reaction mixture was dissolved in 15 ml of THF and refluxed 6 h
with 15 ml of 6 N HCl. After extraction with EtOAc (20 ml ꢁ 3),
the organic layer was concentrated in vacuo. The residue was puri-
fied by column chromatography on silica gel eluted with hexanes/
EtOAc = 4/1 to yield white solid. 1H NMR (CDCl3) d 7.22 (1H, dd,
J = 7.50, 2.0 Hz), 7.17 (1H, dd, J = 8.50, 2.0 Hz), 7.16 (1H, dd,
J = 7.5, 2.0 Hz), 7.09 (1H, d, J = 2.0 Hz), 7.01 (1H, t, J = 7.5 Hz), 6.99
(1H, d, J = 8.5 Hz), 6.11–6.04 (1H, m), 6.02–5.94 (1H, m), 5.21–
5.14 (2H, m), 5.13–5.07 (2H, m), 3.51 (2H, d, J = 6.50 Hz), 3.38
(2H, d, J = 6.51 Hz). 13C NMR (CDCl3) d 151.40, 151.10, 137.50,
136.47, 133.10, 131.14, 130.53, 130.11, 129.35, 127.19, 123.52,
123.46, 121.32, 116.64, 116.36, 115.85, 39.35, 34.99. MS (m/z;
Intensity): 265 (M--1, 100%), 252 (10%), 247 (8%). Total isolation
yield: 21%.
of 0.1% Triton X-100 in PBS supplemented with 50
lg/ml PI, and
100 g of DNase free RNase A. Resuspended cells were then stored
l
in the dark at room temperature for 30 min prior to analysis by
flow cytometry for the cell cycle specificity of apoptotic cells. Un-
treated cells were used as the negative controls. Stained cells were
then analyzed using FACS (FACScan; BD), and at least 100,000 cells
were counted for each sample.
2.2.3. Western blot analysis
Immunoblotting was performed as previously described.18,19
For determination of the total amount of CDK1 and cyclin B1,
A549 cells (5 ꢁ 106) were seeded onto 6 well plate and incubated
overnight to allow cells attachment and subsequently synchro-
nized by treatment with nocodazole (0.5
ter, the plates were incubated with analogs 2 and 4 (0, 0.5, 1.0, 2.0
and 5.0 M) or the DMSO for control, the final concentration of
lg/ml) for 15 h. Thereaf-
2.2. Cell culture
l
DMSO was 0.2% and the plates were incubated for 18 h at 37 °C.
The human cancer cell lines A549 (lung adenocarcinoma),
UACC903 (melanoma) and HT-29 (colon) were purchased from
American Type Culture Collection (Manassas, VA). All the cell lines
were cultured in DMEM supplemented with 10% heat-inactivated
fetal bovine serum, 5.6 mmol/l glucose, glutamine at 37 °C in a
humidified incubator with 5% CO2.
The plates were washed with ice cold PBS and then harvested in
600 ll RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Non-
idet P-40 and 0.25% sodium deoxycholate), supplemented with a
protease inhibitor cocktail (1 mM AEBSF, 800 nM Aprotatin,
50 lM Bestatin, 15 lM E64, 20 lM leupeptin, 10 lM pepstatin A
and 3 mM phenylmethylsulfonyl fluoride), and incubated on ice
for 30 min. Supernatant was recovered after removing cell debris
by centrifugation at 12–14 K rpm for 15 min at 4 °C. Protein con-
centration was determined with BCA protein assay reagents. Equal
2.2.1. Cell proliferation and IC50 determination
The culture cell lines UACC 903, A549 and HT-29 were seeded at
5 ꢁ 103 cells/well in 96-well plate and after seeding cells were
incubated at 37 °C in 5% CO2 for 24 h to allow cell attachment. Each
cell line was treated at 6 concentrations (0, 0.5, 1.0, 5, 10, 25 and
amounts of protein (40 lg) for each sample were resolved on a 12%
SDS–polyacrylamide gel and transferred onto a nitrocellulose
membrane. The membrane was blocked for 1 h with 5% skim milk
in Tris–buffered saline-Tween (TBST) buffer (100 mM Tris, 150 mM
NaCl and 0.1% Tween 20) at room temperature. After blocking
membrane were incubated with primary antibodies, a rabbit poly-
clonal CDK1 antibody against human CDK1 and a rabbit polyclonal
antibody against human cyclin B1 overnight at 4 °C. The
50 lM) of honokiol analogs with a non-treatment control for 24
and 72 h, and the final concentration of DMSO were 0.2%. The cell
viability was evaluated through the 3,4-(5-dimethylthiazol-2-yl)-
5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
salt (MTS) assay.46,47 The tetrazolium compound MTS and an

3206
J. M. Lin et al. / Bioorg. Med. Chem. 20 (2012) 3202–3211
membrane was washed with TBST buffer and incubated with sec-
ondary antibody anti-rabbit IgG (H+L) peroxidase conjugate for 2 h
at room temperature. Enhanced chemoluminescence reagents
were used to visualize the protein bands. Each experiment was
performed three times (different cell passages). The relative pro-
tein band intensities were determined by using the ImageJ 1.44
software (http://imagej.nih.gov/ij/index.html).
compound was not isolated but further reacted with methyl meth-
oxy chloride (MOMCl) to give excellent yield of 1-ally-3-bromo-2-
methoxymethoxybenzene (13). Conversion of compound 13 to the
corresponding boronic acid 14 was carried out in the similar fash-
ion as for compound 12 with n-BuLi and tri-iso-propyl borate.43
The Suzuki coupling reaction between the two moieties was
carried out by standard conditions using Pd(PPh3)4 and Cs2CO3.43
The yields of this cross-coupling varied depending on different
building blocks deployed; we obtained yields from 12% to as high
as 52% depending on the substitution pattern on ring B. Synthesis
of 66 was carried out with the coupling of 2-bromo-4-allyphenol
(8)48,51 and 3-allyl-2-methoxymethoxy-1-phenyl boronic acid
(14).43,48 The MOM group was removed by refluxing the coupled
intermediate in 6 N HCl/THF mixtures. The two steps synthesis
gave total yield of 21%. All the honokiol analogs were characterized
by proton NMR and MS.
3. Result
3.1. Synthesis of honokiol analogs
We prepared honokiol analogs with the phenol group replaced
by methoxy group in A-ring and/or by changing the substitution
pattern on B-ring of the biphenyl structure. A total of six honokiol
analogs were synthesized and their SAR evaluated (Fig. 1). Our syn-
thetic approach for honokiol analogs was based on the Pd-cata-
lyzed Myaura–Suzuki coupling reaction36 which provides the
flexibility of changing substitution on both rings of biphenyl moi-
ety and also makes synthetic scheme extremely converge.
3.2. Inhibition of human cancer cells growth by honokiol
analogs
Literature reports have shown that honokiol inhibit prolifera-
tion, suppress tumor growth and induce apoptosis18,19,46 in human
cancer cells. We first tested for growth inhibitory activity of the
newly synthesized analogs in three different human cancer cell
lines UACC903, A549 and HT-29, by MTS assay. The cells were
maintained in the presence or absence of different concentration
The syntheses of analogs 1–6 are illustrated in Scheme 1. The
important building block for the synthesis are substituted isomers
of 4-allyl-2-bromophenol (8) and 4-allyl-2,6-dibromophenol (9)
which were prepared independently from same reagent 1,3dibro-
mo-5,5-dimethylhydantoin (DBDMH)48,49 under different reaction
condition (Scheme 2). The common reagent for honokiol analogs
1–5 is 3-allyl-4-methoxyphenyl boronic acid (12). Compound 11
was prepared from Claisen Rearrangement of 1-allyloxy-4-bromo-
benzene50 according to standard procedure43 to give 2-allyl-4-bro-
mophenol 10. Standard methylation with methyl iodide and
potassium carbonate yielded 2-allyl-4-bromo-anisole 11. Further
treatment of 11 with n-BuLi followed by tri-isopropyl borate43 gave
the desire Suzuki coupling reagent 12.
(0–50 lM) of honokiol analogs for 24 and 72 h. Cells were treated
with increasing concentrations of honokiol analogs. Honokiol ana-
logs significantly inhibited cell viability, as evidenced by a MTS as-
say, in a time and concentration-dependent manner. The IC50 of
honokiol analogs in different cancer cells were examined and the
results are shown in Table 1. All new analogs gave relatively low
IC50 values as compared to honokiol in all three cancer cell lines,
except that analogs 1, 5, and 6 were less effective in UACC903 cells.
Analog 2 and 4 were consistently the most cytotoxic than honokiol
and other analogs in all three cell lines tested. There were no sig-
Claisen rearrangement of 1-allyloxy-2-bromobenzene to the
corresponding 1-ally-3-bromo-2-phenol was carried out according
to procedure reported by Palmer et al.43 under mild condition, the
OMe
OMe
R2
R3
R4
R1
Pd(Ph3P)4
Cs2CO3
+
R3
R4
R1
B
R5
HO
OH
8: R1=H,R2=Br,R3=OH
R4=H,R5=H
12
R5
9: R1=H,R2=Br,R3=OH
R4=Br,R5=H
1: R1=H,R3=OH,R4,R5=H.
2: R1,R5=H,R3=OH,R4=Br.
3: R1,R5=H,R3=OH,R4=4-MeO-3-allylphenyl
4: R1,R3,R4=H,R5=OH
10: R1=H,R2=H,R3=Br
R4=H,R5=OH
11: R1=OH,R2=Br,
R3,R4,R5=H
5: R1=OH,R3,R4,R5=H
HO
OH
B
1).Pd(Ph3P)4/Cs2CO3
2). 6N HCl/THF
O
O
HO
+
OH
HO
Br
14
8
6
Scheme 1. Synthetic of honokiol analogs.

J. M. Lin et al. / Bioorg. Med. Chem. 20 (2012) 3202–3211
3207
OMe
Br
OH
OMe
1. i-PrMgCl
2. DBDMH
BBr3/CH2Cl2
8
DBDMH
OMe
Br
Br
7
9
OMe
OH
Reflux
1). K2CO3/MeI
2). n-BuLi/(i-PrO)3B
O
R1
Br
Br
11: R1=Br
10
OMOM
13
12: R1=B(OH)2
OH
OMOM
O
B
Br
Br
HO
1). Et2AlCl
2). MOMCl
n-BuLi/(i-PrO)3B
14
Scheme 2. Syntheses of Suzuki reagents.
nificant differences in growth inhibition between analog 2 and 4
(Table 1).
cycle distribution of A549 cells was determined. Flow cytometry
was employed to examine the changes in DNA content. Synchro-
nized A549 cells were treated with analog 2 and 4 at various con-
centrations. We observed that both analog 2 and 4 treated A549
cells induced G0–G1 cell cycle arrest as observed in the case of
honokiol (Fig. 2A–D). Further analysis indicated that 2 and 4
3.3. Effects on cell cycle distribution
Honokiol arrests cells in the G1 phase.18,19 We examined
whether the new honokiol analogs can regulate the cell cycle sim-
ilar to honokiol. Therefore, the effect of these analogs on the cell
(2.5
lM and 10 lM) induced the accumulation of significant num-
ber of cells in the G0–G1 phase of the cell cycle (Table 2). These
Table 1
IC50 (l
M) values of honokiol analogs in human cancer cell lines (24 and 72 h exposure)a
Analog
UACC903
A549
HT-29
24 h
72 h
24 h
72 h
24 h
72 h
1
2
3
4
5
6
37.68 27.86
2.98 1.78
3.94 4.28
3.05 1.83
29.04 22.37
22.28 15.92
7.45 5.28
24.13 19.75
1.29 0.80
1.56 0.88
1.73 0.88
1.74 1.10
2.47 1.32
11.29 7.78
2.04 1.15
12.51 7.94
1.24 0.77
1.04 0.60
1.25 0.78
1.99 1.09
6.28 4.42
1.08 0.66
7.75 4.96
22.45 17.20
3.99 2.62
16.09 12.57
3.19 1.78
16.24 13.30
17.38 14.02
23.05 18.04
14.13 10.84
1.87 1.23
11.39 9.14
2.00 1.27
11.50 8.92
12.37 10.31
13.24 10.66
2.93 1.73
1.39 0.84
24.66 18.26
17.39 12.60
5.10 3.60
Honokiol
a
Cells were incubated in the presence or absence of honokiol analogs at different concentrations (lM). 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. Values are given as
mean SD of three separate assays.

3208
J. M. Lin et al. / Bioorg. Med. Chem. 20 (2012) 3202–3211
Table 2
results indicated that the observed growth inhibitory effects of 2
and 4 in A549 cells are due to cell cycle arrest, and is accompanied
by a decrease in both S phase and G2-M phase cells in a concentra-
tion-dependent manner (Table 2).
Cell cycle distribution (%) with honokiol analogs in A549 cells after 48 h treatmenta
Compound
Concentration
Control
G0/G1 (%)
S (%)
G2/M (%)
DMSO
Honokiol
40.6 4.6
50.5 5.3
61.1 3.1
72.9 5.2
55.8 2.9
63.1 3.6
76.4 2.6
69.8 1.1
75.7 3.5
82.8 2.4
41.2 3.1
37.9 2.3
28.1 1.5
21.7 4.2
32.8 2.8
30.6 1.5
20.7 14.3
23.0 5.2
20.2 2.7
14.3 3.3
18.2 2.1
11.6 1.7
10.8 4.3
5.4 1.8
11.4 1.8
6.3 2.0
3.0 0.7
7.2 0.9
4.2 1.0
2.8 0.6
0.5
2.5
10
l
l
M
M
3.4. Effects of honokiol analogs on inhibition of CDK1 and cyclin
B1 level in A549 cells
lM
2
4
0.5
2.5
10
l
l
M
M
The profound effect of 2 and 4 on cell cycle distribution led to
the question of how they interact with cell cycle regulatory
machinery, namely CDKs and cyclins. In order to address this ques-
tion, we investigated CDK1 and cyclin B1, the two important regu-
latory proteins, as initial targets.21,32,52,53 We examined the ability
of analogs 2 and 4 to inhibit the total cellular content of human
CDK1 and cyclin B1 proteins in human lung cancer cells (A549).
The cells were arrested and released, and then incubated for 18 h
with four different concentrations of 2 and 4. The total cellular con-
tent of human CDK1 and cyclin B1 proteins was determined by
Western blot analysis using polyclonal antibodies. A459 cells trea-
ted with analogs 2 and 4 caused a rapid decrease in CDK1and cy-
lM
0.5
2.5
10
l
l
M
M
lM
a
Approximately 5 ꢁ 106 A549 cells were starved by serum deprivation, and then
incubated in the presence or absence of analog 2 and 4 for 48 h. After incubation,
cells were fixed, treated with RNAse A and stained for DNA with propidium iodide.
Cell cycle distribution was determined by FACS analysis. Results are presented as a
percentage of the total cell population. The whole experiment was repeated twice.
Values are given as mean SD.
treated with 2 and 5 lM of analogs 2 and 4 (Fig. 3B and C). The con-
centration dependent change of CDK1 and cyclin B1 protein level is
comparable to the results for honokiol.
clin B1 protein level in
(Fig. 3B and C). In comparison, honokiol was less effective
(Fig. 3A). A significant reduction was observed in the groups
a concentration-dependent manner
A
10 µM
2.5 µM
0.5 µM
● G0/G1
● S
● G0/G1
● S
● G0/G1
● S
● G2/M
О Debris
● G2/M
О Debris
● G2/M
О Debris
0
0
0
50 100 150 200
0
50 100 150 200
0
0
0
50 100 150 200
Propidiumiodide
Propidiumiodide
Propidiumiodide
B
C
● G0/G1
● S
● G0/G1
● S
● G0/G1
● S
● G2/M
О Debris
● G2/M
О Debris
● G2/M
О Debris
50 100
150 200
50 100 150 200
0
0
50 100 150 200
Propidiumiodide
Propidiumiodide
Propidiumiodide
● G0/G1
● S
● G0/G1
● S
● G0/G1
● S
● G2/M
О Debris
● G2/M
О Debris
● G2/M
О Debris
50 100 150 200
50 100 150 200
50 100 150 200
Propidium iodide
Propidiumiodide
Propidiumiodide
DMSO
D
● G0/G1
● S
● G2/M
О Debris
0
50 100 150 200
Propidiumiodide
Figure 2. Percentage of cell cycle distribution in A549 cells treated for 48 h. (A) Percentage of cells distribution by honokiol treatment, (B) Percentage of cell distribution by
analog 2 treatment and (C) Percentage of cell distribution by analog 4 treatment. (D) Percentage of cell distribution by DMSO treatment. The results are presented as means
( SD) and similar results were observed in two independent experiments.

J. M. Lin et al. / Bioorg. Med. Chem. 20 (2012) 3202–3211
3209
A μM
C μM
B μM
5.0
5.0
0
0.5 1.0 2.0
5.0
0.5 1.0 2.0
0
0.5 1.0 2.0
0
CDK1
CDK1
CDK1
β-Actin
β-Actin
β-Actin
Cyclin B1
β-Actin
Cyclin B1
β-Actin
Cyclin B1
β-Actin
Figure 3. Effects of honokiol analogs on CDK1 and cyclin B1 proteins. A549 cells were treated with honokiol analogs with 0.0, 0.5, 1.0, 2.0 and 5.0
l
M for 18 h. After that cell
lysates were prepared and the level of CDK1 and cyclin B1 proteins were determined by western blot analysis. A. Cells treated with honokiol, B. Cells treated with 2 and (C)
Cells treated with 4. The experiments were repeated more than twice using independently prepared lysates with similar results. b-Actin is shown as a loading control. The
blots were stripped and reported with anti-actin antibody to normalize for differences in protein loading.
A
Cyclin B1
Cdk1
100
80
60
40
20
0
0
0.5
1.0
2.0
5.0
100
0
0.5
80
60
40
20
0
1.0
2.0
5.0
0
0.5
1.0
2.0
5.0
0.5
1.0
2.0
5.0
0
Honokiol (µM)
Honokiol (µM)
Cyclin B1
B
Cdk1
100
80
60
40
20
0
100
0
0
0.5
0.5
80
60
40
20
0
1.0
2.0
5.0
1.0
2.0
5.0
0
0.5
1.0
2.0
5.0
0
0.5
1.0
2.0
5.0
Analog 2 (µM)
Analog 2 (µM)
C
Cyclin B1
Cdk1
100
80
60
40
20
0
100
80
60
40
20
0
0
0
0.5
0.5
1.0
2.0
5.0
1.0
2.0
5.0
0
0.5
1.0
2.0
5.0
0
0.5
1.0
2.0
5.0
Analog 4 (µM)
Analog 4 (µM)
Figure 4. Percentage of CDK1 and cyclin B1 inhibition in A549 cells treated with honokiol analogs. Western blot analysis for CDK1 and cyclin B1 using whole cell lysates from
A549 cells treated with honokiol analogs for the indicated concentrations for 18 h. Western blotting for CDK1 and cyclin B1 proteins was done more than two times and the
results were consistent. (A) Percentage of CDK1 and cyclin B1 inhibition cells treated with honokiol, (B) Percentage of CDK1 and cyclin B1 inhibition cells treated with 2 and
(C) Percentage of CDK1 and cyclin B1 inhibition cells treated with 4.
enhance honokiol anti-tumor activity. Our investigation is to gain
insights into the interaction of newly prepared honokiol analogs
and the mechanism of cell cycle regulation using A549 human lung
cancer cells as a model. To validate our hypothesis, we prepared six
modified honokiol analogs and their cytotoxicity tested in vitro in
human cancer cell lines A549, UACC903 and HT-29 for different
time and concentration dependent manner. Our results show that
4. Discussion
The SAR studies on honokiol analogs have been reported in the
literature with varying success.6,36,54–56 Our goal of using SAR to
enhance the activity of honokiol as antitumor agents is based on
observed pharmacological properties of honokiol and our hypoth-
esis is that the non-availability of phenol group in the A-ring would

3210
J. M. Lin et al. / Bioorg. Med. Chem. 20 (2012) 3202–3211
treatment of honokiol analogs for 24 and 72 h induces a rapid
growth arrest in A549, UACC903 and HT-29 cell lines. This result
is comparable with previous studies with honokiol alone.8,9,11,54
The blocking of hydroxyl group by methylation on A-ring analogs,
such as analogs 2 and 4 showed higher cytotoxicity for all three cell
lines. These results support our hypothesis that making phenol
group in A-ring unavailable would enhance activity of honokiol.
Specifically, both 2 and 4 showed lower IC50 in A549 cell line com-
pared with other analogs and honokiol. For further investigation
we selected 2 and 4 to elucidate underlying mechanisms of growth
inhibition, cell cycle arrest and apoptosis. We studied the cell cycle
distribution and observed significantly induced a G1 phase arrest
of cells after treatment of 2 and 4 in the synchronized A549 cell
line compared to the untreated control cells.
The intracellular events governing the cell division cycle are
conserved in many eukaryotic cell types. The kinase activities of
CDKs are the driving force for the progression of the cell cycle
through the transition check points, due to their ability to activate
cyclins, essential components of cyclin and CDK complexes.57 Stud-
ies have documented a G1 phase cell cycle arrest by honokiol in hu-
man prostate cancer18 and VSMC cells19 treatment at
concentrations within the range of analogs 2 and 4 used in the
present study. The present study reveals that exposure of A549
cells to pharmacologically achievable concentrations of honokiol
analogs 2 and 4 showed growth suppression and G0–G1 phase ar-
rest (Fig. 2A–D). The G0–G1 phase cell cycle arrest is modest as evi-
denced by 10–50% increase in percentage of G0–G1 phase over
DMSO treated control (Table 2). The observation is consistent with
that reported for honokiol and may suggest that the mechanism of
G0–G1 phase arrest for honokiol, 2, and 4 is similar.
To further understand interaction between honokiol analogs
and cell cycle regulatory proteins, we determined total protein lev-
els of CDK1 and cyclin B1 after treating A549 cells with honokiol
analogs. Our results indicated that analogs 2 and 4 significantly
lowered the protein levels of CDK1 and cyclin B1 in a concentration
dependent manner (Fig. 4). Taken together, the cell cycle distribu-
tion studies and the lowering of CDK1 and cyclin B1 may imply
that other CDKs are also involved in the cell cycle arrest. The pro-
tein kinase CDK1 and cyclin B1 play a crucial role in the onset of
mitosis in human cells. The CDK1 and cyclin B1 complex accumu-
lates in the cytoplasm during the G2 phase, being translocated into
the nucleus at the beginning of mitosis.58 The decrease in cyclin B1
levels leads to inhibition of cell division owing to low CDK1 activ-
ity. Therefore, a sub-cytotoxic concentration of 2 and 4 are likely to
inhibit CDK1 activity through a decrease in the levels of cyclin B1.
Suppression of CDK1 activity not only inhibits cell division, but
also allows the assembly of pre-replication complexes for licensing
the DNA for another round of replication.59
The introduction of methoxy group on the A-ring of honokiol in-
creases the inhibitory activity in human cancer cell lines. The cell
cycle passage in mammalian cells is regulated by a complex system
of activating and inhibiting factors. The different cyclin-dependent
kinases act in distinct phases of the cell cycle. Thus, potent and
selective inhibitors of CDKs might induce different effects on the
cell cycle distribution, depending on the respective phase in which
the cells are targeted. The results of cell cycle distribution study
indicated that analogs 2 and 4 can interfere with cell cycle regula-
tion at G0–G1 phase. In addition, the protein levels of CDK1 and cy-
clin B1 were reduced significantly upon treatment of 2 and 4 which
indicated both analogs can interfere with the regulation of cell cy-
cle at M phase. Taken together, these results suggest that honokiol
analogs could exert control at two critical stages of cell cycle,
namely, G0-–G1 and M phase. It is also interesting to note that re-
cently CDK1 was discovered to be able to drive the mammalian cell
cycle.21 Therefore, results of the present study are a significant step
forward in understanding the activity of 2 and 4; they also shed
new light on the molecular mechanisms underlying the potent ef-
fects of the 2 and 4. Honokiol was shown to cause apoptosis in can-
cer cell lines that have defective p53 gene and activated ras gene.60
Honokiol can also induced cyclophilin D and cause death in cells
with wild-type p53.60 Further studies on other human cancer cell
lines will be tested in the future.
This investigation confirmed that blocking the possible oxida-
tion of the phenolic hydroxyl group in the biphenyl group skeleton
will enhance the anti-tumor activity of honokiol. The newly syn-
thesized analogs are capable of arresting the progress of cell cycle
at G0–G1 phase. These studies provide experimental evidence that
newly synthesized honokiol analogs are potent tumor cell growth
inhibitors and they are also capable of inhibiting CDK1 and cyclin
B1, important cell cycle regulatory proteins. In conclusion, honok-
iol analogs 2 and 4 are potent anti-cancer agents that exhibit
strong inhibitory property toward cell growth at lower concentra-
tion than the honokiol, and induce cell cycle arrest at G0–G1 phase.
Most importantly, both analogs effectively lower CDK1 and cyclin
B1 protein levels in the treated cells, which suggest that they inter-
fered with key kinases that promote mitosis. The mechanistic
bases for these apparent synergies need further investigation.
Acknowledgments
The author thanks the financial support from Penn State Her-
shey Cancer Institute (J.M.L). This study was supported in part by
the NCI Grant R03-CA143999 (A.K.S.). The authors like to thank
the Core Research Facilities at College of Medicine, Pennsylvania
State University for recording of flow cytometry and NMR facility
for NMR spectra. In addition, we appreciate Dr. Kun-Ming Chen
for obtaining mass spectra for our compounds.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.03.062.
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