Synthesis of Either C2- or C4′-Alkylated Derivatives of Honokiol and Their Biological Evaluation for Anti-inflammatory Activity

Article abstract of DOI:10.1248/cpb.c19-00207

Honokiol, a biphenolic neolignan isolated from Magnolia officinalis, was reported to have a promising anti-inflammatory activity for the treatment of various diseases. There are many efforts on the synthesis and structure-activity relationship of honokiol derivatives. However, regioselective O-alkylation of honokiol remains a challenge and serves as a tool to provide not only some derivatives but also chemical probes for target identification and mode of action. In this study, we examined the reaction condition for regioselective O-alkylation, in which C2 and C4′-alkylated analogs of honokiol were synthesized and evaluated for inhibitory activity on nitric oxide production and cyclooxygenase-2 expression. Furthermore, we successfully synthesized a potential photoaffinity probe consisting of biotin and benzophenone based on a C4′-alkylated derivative.

Full text of DOI:10.1248/cpb.c19-00207

Chemical and Pharmaceutical Bulletin Advance Publication by J-STAGE
DOI:10.1248/cpb.c19-00207
Advance Publication
June 28, 2019
Chem. Pharm. Bull.
Regular Article
Synthesis of either C2- or C4'-alkylated derivatives of honokiol and their
biological evaluation for anti-inflammatory activity
a,#
a,#
b
b
b,*
San-ha Lee, Xiang Fei, Chaelin Lee, Hien Thi Thu Do, Inmoo Rhee, and
a,*
Seung-Yong Seo
a
College of Pharmacy, Gachon University; Incheon 21936, Republic of Korea: and
b
Department of Bioscience and Biotechnology, Sejong University; Seoul 05006, Republic
of Korea.
#
These two authors contribute equally to this study.
*
Correspondence e-mail: syseo@gachon.ac.kr (S.Y. Seo) and
nature@sejong.ac.kr. (I. Rhee); Tel: +82-32-820-4949 (S.Y. Seo)
Ⓒ
2019 The Pharmaceutical Society of Japan

Abstract
Honokiol, a biphenolic neolignan isolated from Magnolia officinalis, was reported
to have a promising anti-inflammatory activity for the treatment of various diseases. There
are many efforts on the synthesis and structure-activity relationship of honokiol derivatives.
However, regioselective O-alkylation of honokiol remains a challenge and serves as a tool
to provide not only some derivatives but also chemical probes for target identification and
mode of action. In this study, we examined the reaction condition for regioselective
O-alkylation, in which C2 and C4'-alkylated analogs of honokiol were synthesized and
evaluated for inhibitory activity on nitric oxide production and cyclooxygenase-2
expression. Furthermore, we successfully synthesized a potential photoaffinity probe
consisting of biotin and benzophenone based on a C4'-alkylated derivative.
Keywords
Magnolia officinalis, honokiol, anti-inflammatory activity, O-alkylation, photoaffinity
probe
Chemical and Pharmaceutical Bulletin Advance Publication

1
. Introduction
Macrophages and lymphocytes play an important role in the innate and adaptive
immunity of hosts. These functions are mediated by various factors, such as cytokines, that
1
have various defensive roles against pathogens. However, overproduction of inflammatory
molecules can give the host severe immunopathological symptoms, such as acute and
2
chronic inflammatory diseases. Production of inflammatory molecules is triggered by
mitogenic stimulation with various bacterial products, such as lipopolysaccharide (LPS).
Nitric oxide (NO) has been implicated in physiological and pathological processes, such as
nonspecific host defense, chronic inflammation, and vasodilation. Inducible nitric oxide
synthase (iNOS) is involved in the pathological processes related to NO overproduction
and is expressed in response to proinflammatory cytokines, such as interleukin-1β (IL-1β)
and tumor necrosis factor-α (TNF-α), in various cell types, including macrophages,
3
endothelial cells, and smooth muscle cells.
Thus, effective modulation of the overproduction state has been considered as a
4
therapeutic target in various types of inflammation. Nonsteroidal anti-inflammatory drugs
(
NSAIDs) are the most widely prescribed drugs because they exhibit anti-inflammatory
5
effects by inhibiting cyclooxygenase (COX) in arachidonic acid metabolism. There are
two human enzymes that catalyze the same biochemical reactions, namely COX-1 and
COX-2, but their amino acid sequence, tissue distribution, and physiology differ greatly.
Long-term use of NSAIDs for treating pain and inflammation is often accompanied by side
6
effects, such as gastrointestinal (GI) toxicity, hemorrhage, and inhibition of renal function.
Development of COX-2 selective inhibitors is a reasonable strategy for designing NSAIDS
to eliminate side effects because the common side effects of these NSAIDs are due to
COX-1 inhibition. Some COX-2 selective inhibitors, such as celecoxib, have an excellent
effect with minimal adverse effects on humans. Concerns regarding cardiovascular events
in thrombosis have been raised despite the initial success following introduction of
selective COX-2 inhibitors. There has been continued progress in the development of new
7
anti-inflammatory drugs with reduced risk of GI and cardiovascular diseases.
Biphenolic natural neolignans isolated from the bark of Magnolia officinalis, such as
honokiol, 4'-O-methylhonokiol, magnolol, and obovatol, have been used as traditional
medicines for treating gastrointestinal disorders, anxiety, and allergy. Recent studies
Chemical and Pharmaceutical Bulletin Advance Publication

revealed that honokiol and its analogs have various biological activities, such as
8
,9
10
11
12
antiangiogenesis,
anticancer,
anti-inflammatory,
antibacterial,
osteoclast
1
3
14
15
genesis-suppressing, hepatoprotective, and neuroprotective activities. Honokiol and its
1
6
derivatives were reported to target multiple signaling pathways, including the NF-kB,
1
7
18
19
20
p53, EGFR, GPR55, and PI3K/mTOR pathways. Previously, we have synthesized a
series of derivatives of honokiol and 4′-O-methylhonokiol for improved inhibitory
11
activities against COX-2 and PGF
production in LPS-activated macrophages.
Recently, the anti-neuroinflammatory effects of honokiol on heterocyclic bioisosteres
1
production. Some analogs were found to prevent NO
2
0
2
1
in LPS-activated BV-2 cells have been reported. However, the target of honokiol is still
unknown, and to date, only one X-ray co-crystal structure of honokiol derivative has been
2
2
suggested (PDB ID: 4OC7) to be selective for the RXR/coactivator interaction. These
promising activities have prompted us to synthesize honokiol analogs to develop
anti-inflammatory agents and photoaffinity probes to elucidate its binding targets and
mechanisms of action.
Figure 1. Structure of natural neolignans and photoaffinity probe.
2
. Results and Discussion
Honokiol and 4'-O-methylhonokiol consist of a 5,3'-diallyl-biphenyl skeleton bearing
hydroxy group in the C2 of the A ring, or hydroxyl and methoxy groups in the C4' of the B
Chemical and Pharmaceutical Bulletin Advance Publication

11
ring, respectively. The previous study showed that 4'-O-methylhonokiol exerts higher
anti-inflammatory activity than those of honokiol and various honokiol analogs, as
examined using COX-1 and COX-2 enzyme assay and LTB4 formation assay. Thus, we
contemplated efficient synthesis of mono-alkylated analogs on either the 2- or 4'-position
for elongating the regioselective chain and designing site-selective photoaffinity probes,
which would be useful to elucidate directly binding proteins.
Our first synthesis commenced with a study on regioselective O-methylation of
honokiol to determine which phenol group is more reactive under various alkylation
conditions.
a
Table 1. Regioselective O-methylation of honokiol
isolated yield (%)
entry
base
Solvent
1
a
1b
27
46
50
50
21
15
18
15
-
1c
32
30
10
15
15
15
15
7
1
LiOH
NaOH
KOH
DMSO /H
2
O
33
20
21
30
35
30
20
7
2
3
4
5
6
7
8
9
DMSO /H
DMSO /H
DMSO /H
DMSO /H
2
O
O
O
O
2
2
2
2 3
K CO
Cs
Cs
2
2
CO
3
3
CO
Acetone
THF
t-BuOK
KHMDS
NaH
DMSO
THF
-
38
1
0
Et N
THF
N/R
N/R
3
1
1
iPr
2
NEt
THF
a
Reaction conditions: a) for entry 1–6, 60 °C, overnight; b) for entry 7–9, 0 °C, 1 h; c) for
entry 10–11, reflux, overnight.
Studies on regioselective O-methylation of honokiol were performed under different
base and solvent conditions, as shown in Table 1. Briefly, 1 equivalent of commercially
available honokiol, 1.2 equivalent of iodomethane, and 3 equivalents of base were used to
Chemical and Pharmaceutical Bulletin Advance Publication

yield 4'-O-methylhonokiol (1a), 2-O-methylhonkiol (1b), and 2,4'-dimethylhonokiol (1c).
The methylation using LiOH and NaOH in aqueous dimethyl sulfoxide (DMSO) yielded
three alkylated compounds (1a–c), whereas that using KOH and K
major product. Interestingly, C4'-alkylation occurred twice as much as C2-alkylation when
Cs CO was used as a base. The result was likely to be different depending on the metal
2 3
CO afforded 1b as a
2
3
cation. The yield and selectivity with strong bases, such as t-BuOK, KHMDS, and NaH,
were not favorable, and the alkylation reaction did not work with amine bases, such as
triethylamine and N,N-diisopropylethylamine. Because the chemical environment of the
phenol group on the A ring and B ring was similar, regioselective alkylation was not
completely achieved. However, with the automatic flash column chromatography system,
both 4'-O- and 2-O-alkylated products were easily separated with more than 95% purity
8
,23,24
and fully analyzed by NMR, mass spectrometry before their biological evaluation.
1
3
Interestingly, the comparison of C-NMR spectra of honokiol, 4'-O-methylhonokiol (1a),
2
-O-methylhonkiol (1b), and 2,4'-dimethylhonokiol (1c) enable us to confirm the aspect of
their NMR spectra when other groups were alkylated at the C2 and C4'-position of
honokiol, respectively, as shown in Figure 2.
Chemical and Pharmaceutical Bulletin Advance Publication

1
3
Figure 2. C-NMR comparison of natural honokiol and its methylated analogs (1a–1c).
Chemical and Pharmaceutical Bulletin Advance Publication

Chart 1. Synthesis of O-alkylation analogs of honokiol. Reagents and conditions: a) for 1–
, iodomethane, iodoethane, 1-bromobutane, allyl bromide, 3,3-dimethylallyl bromide,
cyclopropylmethyl bromide, cyclobutylmethyl bromide, Cs CO , acetone, reflux, 24 h; b)
for 8–11, 2-bromo-4'-fluoroacetophenone, 2-bromo-2',4'-dichloroacetophenone,
-bromo-4'-nitroacetophenone, tert-butyl bromoacetate, Cs CO , THF, 1 h. Yields based on
recovered starting material in parentheses.
7
2
3
2
2
3
We turned to the alkylation using various alkylating agents and Cs CO in acetone, as
2
3
shown in Chart 1. Similar to that of methylation, the reactivity of aliphatic alkylation was
sluggish and the yield was not satisfactory. However, a slightly C4'-selectively alkylated
compound was found as a major product. Ethyl, n-butyl, cyclopropylmethyl, and
cyclobutylmethyl were not well incorporated on the phenol groups of honokiol. Allyl and
prenyl were introduced on the C4'-position slightly selectively, similar to methyl. The
O-alkylation with phenacyl bromides, such as 2-bromo-4'-fluoroacetophenone,
2
-bromo-2',4'-dichloroacetophenone, and 2-bromo-4'-nitroacetophenone, as alkylating
agents afforded C4'-alkylated analogs (8a–10a) of honokiol in moderate yield. The reaction
Chemical and Pharmaceutical Bulletin Advance Publication

of tert-butyl bromoacetate afforded a C4'-alkylated analog (11a) in moderate yield.
In a previous study, we found that modifications of 4'-O-methylhonokiol through
introduction of methyl, isopropyl, and prenyl groups at phenol slightly decrease the
11
inhibitory activity of COX-2 enzyme . Compared with other synthesized analogs, 11a
showed the most promising anti-inflammatory activity; thus, it was exploited as an
25
intermediate to obtain C4'-labeled photoaffinity probe of honokiol. By the treatment with
trifluoroacetic acid in dichloromethane, the tert-butyl ester group of 11a was removed to
afford carboxylic acid 12, as shown in Chart 2. Acid 12 was coupled with the bifunctional
2
6
molecule 13, which consists of benzophenone and biotin, under PyAOP/HOAt-mediated
reaction condition to finally afford the desired photoaffinity probe 14 in good yield.
Chart 2. Synthesis of C4′-labeled photoaffinity probe of honokiol.
The cell viability of RAW264.7 cells following treatment with honokiol and its analogs
was examined by the MTT assay. The cells were treated in the presence or absence of
honokiol analogs at different concentrations (0–20 μM). As shown in Table 2, it did not
show cell cytotoxicity at 5 μM concentration of all new synthetic compounds similar to
honokiol, suggesting that the derivatives are little toxic. The inhibitory activities of
honokiol analogs on nitric oxide (NO) production and COX-2 expression were evaluated in
RAW264.7 cells treated with 10 ng/mL or 100 ng/mL LPS in the presence or absence of
compounds at different concentrations (0–10 μM). The inhibitory effects of the synthesized
Chemical and Pharmaceutical Bulletin Advance Publication

honokiol analogs on NO production are shown in Figure 3 and Table 2. The carbon
elongation in the 2- or 4′-hydroxy position with methoxy, ethoxy, allyl, prenyl,
cyclobutylmethyl, and cyclopropylmethyl groups does not improve the inhibitory activity
on NO production regardless of the C2 or C4′ position, although ethyl, prenyl and
cyclobutyl derivatives exhibited the similar activity with honokiol (2a, 2b, 5a, 5b, 7a, and
7
b; 5.1 < IC50 < 8.3 μM). Meanwhile, the introduction of two phenacyl groups on the
hydroxyl of the C4′ position increased the NO inhibitory activity (8a and 9a; IC50 = 4.8 and
.5 μM). Next, we examined the expression level of COX-2 enzyme by western blotting.
4
All compounds showed direct inhibition of COX-2 enzyme, with a range of 65–93%
inhibition in the presence of LPS 100 nM. Contrary to the results of NO production, the
analog 3a, whose n-butoxy group was replaced with hydroxyl group, significantly
suppressed the expression of COX-2. Some analogs, such as 2a, 2b, 6a, 6b, and 7a,
moderately inhibited the expression of COX-2. Fortunately, tert-butoxyacyl derivative
(
11a) and a photoaffinity probe (15) of honokiol maintained a moderate inhibition of NO
production and COX-2 expression compared to those of honokiol without drastic loss of
activity. Based on the prediction of log P value (SLogP), most alkylated compounds were
regarded to have slightly more lipophilic properties than honokiol.
Chemical and Pharmaceutical Bulletin Advance Publication

1a
1
b
Honokiol
1
1
1
8
5
2
9
6
3
0
18
15
12
9
18
15
12
9
6
3
6
3
0
0
LPS 0
LPS 10
LPS 100
LPS 0
LPS 10
LPS 100
LPS 0
LPS 10
2a
LPS 100
3
a
1
1
1
8
5
2
9
6
3
0
18
15
12
9
2b
18
15
12
9
6
6
3
3
0
0
LPS 0
LPS 10
3b
LPS 100
LPS 0
LPS 10
4a
LPS 100
LPS 0
LPS 0
LPS 0
LPS 0
LPS 0
LPS 10
LPS 100
LPS 100
LPS 100
LPS 100
LPS 100
1
8
5
2
9
6
3
0
18
15
12
9
4b
1
8
1
1
15
12
9
6
6
3
3
0
0
LPS 0
LPS 10
LPS 100
LPS 0
LPS 10
5b
LPS 100
LPS 10
5a
1
1
1
8
5
2
9
6
3
0
18
15
12
9
18
6a
15
12
9
6
6
3
0
3
0
LPS 0
LPS 10
LPS 100
LPS 0
LPS 10
7a
LPS 100
LPS 10
6b
18
1
1
1
8
7b
1
1
8
5
1
1
5
2
9
6
3
0
5
2
9
6
3
0
12
9
6
3
0
LPS 0
LPS 10
9a
LPS 100
LPS 10
0a
LPS 0
LPS 10
8a
LPS 100
1
1
1
1
8
18
15
12
9
18
15
12
9
5
2
9
6
3
0
6
6
3
3
0
0
LPS 10
14
LPS 0
LPS 10
LPS 100
LPS 0
LPS 10
11b
LPS 100
11a
1
1
1
8
5
2
9
6
3
0
18
15
12
9
18
15
12
9
6
6
3
3
0
0
LPS 0
LPS 10
LPS 100
LPS 0
LPS 10
LPS 100
LPS 0
LPS 10
LPS 100
Figure 3. Inhibitory activity of honokiol analogs on nitric oxide production in
LPS-activated RAW264.7 cells.
Chemical and Pharmaceutical Bulletin Advance Publication

Table 2. Anti-inflammatory effect of compounds on nitric oxide production and COX-2
expression, logP value and, cell viability.
a
IC50 (μM) of NO production
COX-2
cell viability
c
compound
Honokiol
b
SLogP
c
LPS 10 ng/mL LPS 100 ng/mL expression (%)
(%)
6.2
24.9
23.7
6.1
6.7
24.2
25.9
6.3
75 ± 0.85
94 ± 9.7
86 ± 4.7
84 ± 8.1
89 ± 4.7
70 ± 7.1
93 ± 11
3.9
4.2
4.2
4.6
4.6
5.4
5.4
4.6
4.6
5.5
5.5
5.0
5.0
5.4
5.4
5.9
6.8
5.3
4.9
4.9
3.7
6.5
104.7 ± 5.8
99.9 ± 1.7
104.3 ± 2.2
107.6 ± 1.8
99.3 ± 4.3
97.4 ± 4.6
103.3 ± 0.9
97.1 ± 5.8
106.2 ± 1.2
104.0 ± 3.8
89.2 ± 3.6
96.7 ± 2.5
104.5 ± 1.8
100.5 ± 7.9
101.9 ± 4.4
91.8 ± 3.8
87.4 ± 1.6
109.9 ± 3.9
91.1 ± 4.6
102.0 ± 2.3
104.2 ± 0.8
95.6 ± 3.1
1
a
1b
2
a
2b
5.3
5.4
3
a
>100
>100
18.5
18.8
7.8
>100
>100
25.7
19.6
7.4
3b
4
a
89 ± 7.0
100 ± 1.3
93 ± 8.0
4b
5
a
e
5b
5.1
5.0
ND
6
a
39.9
>100
8.3
41.0
>100
8.7
83 ± 3.8
88 ± 6. 9
86 ± 8.2
ND
6b
7
a
7b
5.9
6.0
8
9
a
a
4.8
5.0
91 ± 13
87 ± 4.0
93 ± 12
91 ± 10
99 ± 4.5
ND
4.5
4.6
1
1
0a
1a
16.9
13.3
25.7
103.8
11.2
18.4
13.3
24.2
86.4
10.7
11b
1
2
4
1
86 ± 4.0
a
Each compound was defined as the concentration (μM) that caused 50% inhibition of NO
b
production in LPS-activated RAW 264.7 cells. The expression ratio was calculated as a
band density ratio of COX-2 to β-actin in the treatment with 5 μM of each compound.
c
Representative data from three independent experiments. SlogP was derived by applying
the rules of Wildman and Crippen and represents a good estimate of the actual logP of the
d
molecule. The effect of treatment with 5 μM of each compound was determined by MTT
assay, and the results are expressed in percentage (%)
3
. Conclusion
The regioselective O-alkylation of honokiol was aimed to synthesize either C2- or
Chemical and Pharmaceutical Bulletin Advance Publication

C4'-alkylated derivatives for developing anti-inflammatory agents. To investigate
regioselectivity, we conducted O-methylation under various reaction conditions, such as
iodomethane and dimethyl sulfate as an alkylating agent, as well as potassium carbonate,
cesium carbonate, Et
all compounds, including recovered honokiol, could be separated successfully. By
treatment with Cs CO and DMSO/H O, 4'-O-methylhonokiol, which is also a natural
product, was obtained as a major product. Several analogs, namely C4'-alkyl-, C2-alkyl-,
3
N, and NaH as a base. The yield of O-methylation was moderate, and
2
3
2
2 3
and C2,C4'-dialkyl-honokiol were synthesized through O-alkylation using Cs CO as a
base.
We confirmed that almost all compounds showed low cytotoxicity in macrophages, and
several analogs showed improved inhibitory activities than that of honokiol against NO
production and/or COX-2 expression. Moreover, a photoaffinity probe in which biotin and
benzophenone were linked to the C4' position was synthesized, and it showed a moderate
inhibitory activity without drastic loss of activity. This study provided a basis for further
development of these compounds as novel therapeutics for inflammatory diseases and
cancer. Our next step is to identify the putative binding targets essential for signaling
pathways in inflammation by using the honokiol-based photoaffinity probe.
4. Experimental
4
.1 Chemistry
All starting materials and reagents were obtained from commercial suppliers and used
without further purification. Air- and moisture-sensitive reactions were performed under
nitrogen atmosphere. Flash column chromatography was performed using silica gel 60
(
230–400 mesh; Merck, Darmstadt, Germany) with the indicated solvents. Thin-layer
1
chromatography was performed using 0.25 mm silica gel plates (Merck). H-(600 MHz)
1
3
and C-NMR (150 MHz) spectra were recorded on an AVANCE III System 600 MHz
6
spectrometer (Bruker, Billerica, MA, USA) as solutions in CDCl or DMSO-d .
3
High-resolution mass spectra (HRMS) were obtained using JMS-700(JEOL) with electron
ionization or Thermo Scientific, Q Exactive™ HF-X Hybrid Quadrupole-Orbitrap™ Mass
Spectrometer (Germering, Germany) using ESI (electrospray ionization). Low-resolution
Chemical and Pharmaceutical Bulletin Advance Publication

mass spectra (LRMS) were obtained using a Waters Auto Purification instrument. Spectral
data are available in Supplementary Materials.
4
.1.1 Synthesis of honokiol analogs
',5-diallyl-4'-methoxy-[1,1'-biphenyl]-2-ol (1a) and 3,5'-diallyl-2'-methoxy-[1,1'
biphenyl]-4-ol (1b). To an acetone solution (10 mL) of honokiol (1.13 g, 4.24 mmol) was
added Cs CO (1.38 g, 4.24 mmol), by 1 M iodomethane in THF (0.5 mL, 5.08 mmol).
After stirring at reflux overnight, the reaction mixture was diluted in ethyl acetate, washed
with water and brine, dried over MgSO , and concentrated under reduced pressure. The
residue was purified by flash column chromatography on silica gel (ethyl acetate : n-hexane
3
-
2
3
4
=
1 : 19 to 1 : 2) to afford 1a (330 mg, 30%, BORSM 36%), 1b (210 mg, 15%, BORSM
1
2
3%), 1c (182 mg, 15%, BORSM 19%), and recovered honokiol (280 mg). For 1a, H
NMR (600 MHz, CDCl
3
) δ 7.28 (d, J = 8.3 Hz, 1H), 7.23 (s, 1H), 7.05 (d, J = 8.2 Hz, 1H),
.03 (s, 1H), 6.96 (d, J = 8.3 Hz, 1H), 6.90 (d, J = 8.2 Hz, 1H), 6.05–5.93 (m, 2H), 5.11–
.06 (m, 4H), 5.04 (bs, 1H), 3.88 (s, 3H), 3.43 (d, J = 6.6 Hz, 2H), 3.35 (d, J = 6.6 Hz, 2H);
7
5
1
3
3
C NMR (150 MHz, CDCl ) δ 157.2, 151, 138, 136.6, 132.3, 130.6, 130.3, 129.9, 129.1,
128.9, 128.0, 128, 116.0, 115.7, 115.7, 111.1, 55.7, 39.6, 34.4; HRMS (EI) m/z: [M] calcd
1
for C19
H
20
O
2
280.1463; found 280.1466. For 1b, H NMR (600 MHz, CDCl
3
) δ 7.31 (dd, J
=
8.2, 2.2 Hz, 1H), 7.28 (d, J = 2.2 Hz, 1H), 7.11–.09 (m, 2H), 6.90 (d, J = 8.1 Hz, 1H),
6
.85 (d, J = 8.2 Hz, 1H), 6.10–5.95 (m, 2H), 5.24–5.20 (m, 2H), 5.11–5.04 (m, 2H), 5.03
1
3
(
bs, 1H) 3.79 (s, 3H), 3.46 (d, J = 6.4 Hz, 2H), 3.37 (d, J = 6.7 Hz, 2H); C NMR (150
MHz, CDCl ) δ 154.8, 153.3, 137.8, 136.5, 132.3, 131.5, 131.2, 131, 130.3, 129.1, 128,
3
1
2
24.7, 116.6, 115.6, 115.5, 111.3, 55.7, 39.4, 35.4; HRMS (EI) m/z: [M] calcd for C H O
19 20 2
1
3
80.1463; found 280.1461. For 1c, H NMR (600 MHz, CDCl ) δ 7.41–7.39 (m, 1H), 7.34
(
s, 1H), 7.15 (s, 1H), 7.12 (d, J = 8.3 Hz, 1H), 6.92 (dd, J = 8.3, 0.9 Hz, 2H), 6.09–5.98 (m,
2
3
1
H), 5.14–5.10 (m, 2H), 5.09–5.06 (m, 2H), 3.88 (d, J = 1.0 Hz, 3H), 3.81 (d, J = 1.2 Hz,
1
3
H), 3.46 (d, J = 6.5 Hz, 2H), 3.40 (d, J = 6.4 Hz, 2H); C NMR (150 MHz, CDCl ) δ
3
56.5, 155.0, 137.9, 137.2, 132.3, 131.1, 131.1, 130.8, 130.6, 128.5, 128.2, 128.0, 115.6,
+
22 2
115.5, 111.4, 110.0, 55.8, 55.6, 39.6, 34.5; LRMS (ESI) m/z: calcd for C20H O [M+Na]
3
17.15; found 317.25.
',5-diallyl-4'-ethoxy-[1,1'-biphenyl]-2-ol (2a) and 3,5'-diallyl-2'-ethoxy-[1,1'
3
-
biphenyl]-4-ol (2b). To an acetone solution (10 mL) of honokiol (30 mg, 0.11 mmol) was
Chemical and Pharmaceutical Bulletin Advance Publication

added Cs
mL of acetone. After stirring at reflux overnight, the reaction mixture was diluted in ethyl
acetate, washed with water and brine, dried over MgSO , and concentrated under reduced
2 3
CO (100 mg, 0.3 mmol), followed by iodoethane (21 µL, 0.11 mmol) diluted in
5
4
pressure. The residue was purified by flash column chromatography on silica gel (ethyl
acetate : n-hexane = 1 : 19 to 1 : 2) to afford 2a (16 mg, 26%, BORSM 47%), 2b (8 mg,
1
5%, BORSM 25%), 2c (5 mg, 10%, BORSM 16%), and recovered honokiol (22 mg). For
1
2
a, H NMR (600 MHz, CDCl
3
) δ 7.26–7.25 (m, 1H), 7.23 (d, J = 1.9 Hz, 1H), 7.06 (dd, J
=
8.2, 2.1 Hz, 1H), 7.03 (d, J = 1.7 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 6.90 (d, J = 8.2 Hz,
1
1
H), 6.05–5.95 (m, 2H), 5.14 (d, J = 1.1 Hz, 1H), 5.13–5.08 (m, 2H), 5.06 (d, J = 10.0 Hz,
1
3
H), 4.09 (q, J = 7.0 Hz, 2H), 3.44 (d, J = 6.7 Hz, 2H), 3.35 (d, J = 6.7 Hz, 2H); C NMR
(150 MHz, CDCl
3
) δ 156.6, 151, 138, 136.7, 132.3, 130.6, 130.3, 130.1, 128.9, 128.9,
128.0, 128, 116, 115.7, 115.6, 112.0, 63.9, 39.6, 34.6, 15.0; HRMS (EI) m/z: [M] calcd for
1
C H O 294.1620; found 294.1626. For 2b, H NMR (600 MHz, CDCl ) δ 7.34–7.33 (m,
2
0
22
2
3
2
H), 7.13 (d, J = 2.2 Hz, 1H) 7.07–7.06 (m, 1H), 6.88 (d, J = 8.3 Hz, 1H), 6.84 (d, J = 8.1
Hz, 1H), 6.09–6.03 (m, 1H), 6.02–5.95 (m, 1H), 5.23–5.16 (m, 2H), 5.11–5.04 (m, 2H),
4
.95 (s, 1H), 4.01–3.98 (q, J = 7.0 Hz, 2H), 3.45 (d, J = 6.4 Hz, 2H), 3.37 (d, J = 6.7 Hz,
13
2
H), 1.34 (t, J = 7.0 Hz, 3H); C NMR (150 MHz, CDCl ) δ 154.4, 153.2, 138, 136.7,
3
1
32.5, 131.8, 131.5, 131, 130.6, 129.1, 128.1, 124.7, 116.7, 115.7, 115.5, 113.1, 64.3, 39.6,
1
3
5.4, 15.0; HRMS (EI) m/z: [M] calcd for C H O 294.1620; found 293.1638. For 2c, H
2
0
22
2
3
NMR (600 MHz, CDCl ) δ 7.40–7.36 (m, 2H), 7.14 (d, J = 2.3 Hz, 1H), 7.06 (dd, J = 8.3,
2
2
=
.3 Hz, 1H), 6.88 (t, J = 8.2 Hz, 2H), 6.07–5.96 (m, 2H), 5.13–5.08 (m, 2H), 5.07–5.03 (m,
H), 4.08 (q, J = 7.0 Hz, 2H), 4.00 (q, J = 7.0 Hz, 2H), 3.44 (d, J = 6.7 Hz, 2H), 3.37 (d, J
1
3
6.7 Hz, 2H), 1.44 (t, J = 7.0 Hz, 3H), 1.34 (t, J = 7.0 Hz, 3H); C NMR (150 MHz,
CDCl
3
) δ 155.8, 154.4, 138.0, 137.3, 132.4, 131.2, 131.0, 130.8, 130.7, 128.3, 128.1, 127.9,
115.6, 115.5, 113.0, 111, 64.3, 63.8, 39.6, 34.6, 15.1, 15.0; LRMS (ESI) m/z: calcd for
+
C
22
H
26
2
O [M+H] 322.20; found 323.29.
3
',5-diallyl-4'-butoxy-[1,1'-biphenyl]-2-ol (3a) and 3,5'-diallyl-2'-butoxy-[1,1'
-
biphenyl]-4-ol (3b). To an acetone solution (2 mL) of honokiol (50 mg, 0.18 mmol) was
added Cs CO (183 mg, 0.5 mmol), followed by 1-bromobutane (20 µL, 0.18 mmol). After
stirring at reflux overnight, the reaction mixture was diluted in ethyl acetate, washed with
water and brine, dried over MgSO , and concentrated under reduced pressure. The residue
2
3
4
Chemical and Pharmaceutical Bulletin Advance Publication

was purified by flash column chromatography on silica gel (ethyl acetate : n-hexane = 1 :
1
9 to 1 : 2) to afford pure 3a (8 mg, 15%, BORSM 28%), 3b (9 mg, 14%, BORSM 26%),
1
3
c (5 mg, 7%, BORSM 13%), and recovered honokiol (23 mg). For 3a, H NMR (600
MHz, CDCl
3
) δ 7.26–7.23 (m, 1H), 7.22 (d, J = 2.2 Hz, 1H), 7.05 (dd, J = 8.2, 2.2 Hz, 1H),
7
2
3
.02 (d, J = 2.1 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 6.90 (d, J = 8.2 Hz, 1H), 6.04–5.94 (m,
H), 5.14 (bs, 1H), 5.12–5.03 (m, 4H), 4.02 (t, J = 6.3 Hz, 2H), 3.43 (d, J = 6.8 Hz, 2H),
.35 (d, J = 6.7 Hz, 2H), 1.84–1.79 (m, 2H), 1.57–1.50 (m, 2H), 1.00 (t, J = 7.4 Hz, 3H)；
1
3
3
C NMR (150 MHz, CDCl ) δ 156.9, 151.1, 138.1, 136.9, 132.4, 130.7, 130.5, 130.2,
129.0, 129.0, 128.2, 128.1, 116.1, 115.8, 115.8, 112.0, 68.1, 39.7, 34.8, 31.7, 19.7, 14.2;
1
HRMS (EI) m/z: [M] calcd for C22
MHz, CDCl
H
26
O
2
322.1933; found 322.1932. For 3b, H NMR (600
3
) δ 7.34 (d, J = 2.1 Hz, 1H), 7.31 (dd, J = 8.2, 2.2 Hz, 1H), 7.12 (d, J = 2.3 Hz,
1
6
2
2
3
2
2
3
1
3
1
1
H), 7.06 (dd, J = 8.3, 2.3 Hz, 1H), 6.88 (d, J = 8.3 Hz, 1H), 6.84 (d, J = 8.2 Hz, 1H), 6.10–
.02 (m, 1H), 6.02–5.88 (m, 1H), 5.22–5.15 (m, 2H), 5.12–5.02 (m, 2H), 3.92 (t, J = 6.4 Hz,
H), 3.45 (d, J = 6.4 Hz, 2H), 3.36 (d, J = 6.7 Hz, 2H), 1.72–1.64 (m, 2H), 1.47–1.38 (m,
H), 0.91 (t, J = 7.4 Hz, 3H); HRMS (EI) m/z: [M] calcd for C H O 322.1933; found
2
2
26
2
1
3
21.1926. For 3c, H NMR (600 MHz, CDCl ) δ 7.37 (d, J = 2.2 Hz, 1H), 7.33 (dd, J = 8.4,
.3 Hz, 1H), 7.12 (d, J = 2.3 Hz, 1H), 7.05 (dd, J = 8.3, 2.3 Hz, 1H), 6.87 (t, J = 8.7 Hz,
H), 6.00 (m, 2H), 5.11–5.00 (m, 4H), 4.00 (t, J = 6.4 Hz, 2H), 3.92 (t, J = 6.4 Hz, 2H),
.42 (d, J = 6.7 Hz, 2H), 3.36 (d, J = 6.7 Hz, 2H), 1.83–1.77 (m, 2H), 1.72–1.66 (m, 2H),
.53 (dd, J = 15.1, 7.5 Hz, 2H), 1.43 (m, 2H), 0.99 (t, J = 7.4 Hz, 3H), 0.91 (t, J = 7.4 Hz,
1
3
3
H); C NMR (150 MHz, CDCl ) δ 159.4, 154.4, 137.9, 137.2, 132.1, 131.1, 130.9, 130.6,
28.1, 127.9, 127.7, 115.4, 115.2, 112.5, 110.6, 68.2, 67.7, 39.5, 34.6, 31.5, 31.4, 29.7, 19.4,
+
9.4, 13.9, 13.8; LRMS (ESI) m/z: calcd for C H O [M+Na] 401.25; found 401.36.
2
6
34
2
3
,5'-diallyl-4'-(allyloxy)-[1,1'-biphenyl]-2-ol (4a) and 3,5'-diallyl-2'-(allyloxy)-[1,1'
biphenyl]-4-ol (4b). To an acetone solution (2 mL) of honokiol (60 mg, 0.22 mmol) was
added Cs CO (220 mg, 0.67 mmol), followed by allyl bromide (20 µL, 0.22 mmol). After
stirring at reflux overnight, the reaction mixture was diluted in ethyl acetate, washed with
water and brine, dried over MgSO , and concentrated under reduced pressure. The residue
was purified by flash column chromatography on silica gel (ethyl acetate : n-hexane = 1 :
-
2
3
4
1
9 to 1 : 2) to afford 4a (21 mg, 31%, BORSM 46%), 4b (11 mg, 16%, BORSM 24%), 4c
1
(
7.5 mg, 9%, BORSM 14%), and recovered honokiol (20 mg). For 4a, H NMR (600 MHz,
Chemical and Pharmaceutical Bulletin Advance Publication

CDCl
Hz, 1H), 6.13–5.99 (m, 1H), 6.03–5.93 (m, 1H), 5.46 (dd, J = 17.3, 1.6 Hz, 1H), 5.31 (dd, J
10.6, 1.5 Hz, 1H), 5.13–5.04 (m, 5H), 4.60 (d, J = 5.0 Hz, 1H), 3.47 (d, J = 6.7 Hz, 3H),
3
) δ 7.29–7.22 (m, 2H), 7.08–7.00 (m, 3H), 6.94 (d, J = 8.2 Hz, 1H), 6.90 (d, J = 8.2
=
1
3
3
1
6
4
7
.35 (d, J = 6.7 Hz, 3H); C NMR (150 MHz, CDCl
3
) δ 156.1, 150.9, 137.9, 136.6, 133.4,
32.3, 130.7, 130.3, 130.2, 129.3, 128.9, 127.9, 127.9, 117.3, 116.0, 115.6, 115.6, 112.3,
22 2
9.0, 39.5, 34.6; HRMS (EI) m/z: [M] calcd for C21H O 306.1620; found 306.1614. For
1
b, H NMR (600 MHz, CDCl
3
) δ 7.37 (dd, J = 6.9, 2.0 Hz, 2H), 7.13 (d, J = 2.2 Hz, 1H),
.07 (dd, J = 8.3, 2.3 Hz, 1H), 6.89 (d, J = 8.3 Hz, 1H), 6.85 (d, J = 8.9 Hz, 1H), 6.09–5.95
m, 3H), 5.33 (dd, J = 17.3, 1.7 Hz, 1H), 5.24–5.15 (m, 3H), 5.12–5.04 (m, 2H), 4.96 (bs,
(
1
3
1
H), 4.50 (d, J = 4.8 Hz, 2H), 3.46 (d, J = 6.4 Hz, 2H), 3.37 (d, J = 6.7 Hz, 2H); C NMR
(150 MHz, CDCl
3
) δ 153.9, 153.3, 137.9, 136.6, 133.6, 132.7, 131.8, 131.3, 131.0, 130.8,
1
29.2, 128.0, 124.7, 116.8, 116.7, 115.7, 115.5, 113.3, 69.4, 39.5, 35.4; HRMS (EI) m/z:
1
[
M] calcd for C H O 306.1620; found 306.1614. For 4c, H NMR (600 MHz, CDCl ) δ
2
1
22
2
3
7
8
1
4
.44–7.35 (m, 2H), 7.16 (d, J = 2.3 Hz, 1H), 7.09 (dd, J = 8.3, 2.3 Hz, 1H), 6.91 (dd, J =
.3, 6.2 Hz, 2H), 6.14–5.97 (m, 4H), 5.49–5.46 (m, 1H), 5.38–5.37 (m, 1H), 5.32–5.29 (m,
H), 5.23–5.21 (m, 1H), 5.14–5.10 (m, 2H), 5.08–5.05 (m, 2H), 4.61–4.60 (m, 2H), 4.53–
1
3
.52 (m, 2H), 3.48 (d, J = 6.6 Hz, 2H), 3.39 (d, J = 6.7 Hz, 2H); C NMR (150 MHz,
) δ 155.5, 154.0, 137.9, 137.2, 133.8, 133.6, 132.7, 131.3, 131.1, 131.0, 130.9, 128.4,
28, 117.0, 116.9, 115.7, 115.6, 113.3, 111.3, 69.5, 69, 39.6, 34.7; LRMS (ESI) m/z: calcd
CDCl
3
1
+
for C24
H
26
O
2
[M+Na] 401.24; found 401.36.
3
',5-diallyl-4'-((3-methylbut-2-en-1-yl)oxy)-[1,1'-biphenyl]-2-ol (5a) and 3,5'
-
diallyl-2'-((3-methylbut-2-en-1-yl)oxy)-[1,1'-biphenyl]-4-ol (5b). To an acetone solution
(
2 mL) of honokiol (40 mg, 0.15 mmol) was added Cs CO (146 mg, 0.45 mmol), followed
2
3
by 3,3-dimethylallyl bromide (22 mg, 0.15 mmol). After stirring at reflux overnight, the
reaction mixture was diluted in ethyl acetate, washed with water and brine, dried over
MgSO , and concentrated under reduced pressure. The residue was purified by flash
4
column chromatography on silica gel (ethyl acetate : n-hexane = 1 : 19 to 1 : 2) to afford 5a
(25 mg, 50%, BORSM 73%), 5b (1.6 mg, 3%, BORSM 5%), and recovered honokiol (13
1
mg). For 5a, H NMR (600 MHz, CDCl
3
) δ 7.26–7.21 (m, 2H), 7.05–7.00 (m, 2H), 6.94 (d,
1H, J = 8.3 Hz), 6.90 (d, 1H, J = 8.2 Hz), 6.04–5.93 (m, 2H), 5.52–5.48 (m, 1H), 5.19 (s,
1
H), 5.11–5.03 (m, 4H), 4.57 (d, 2H, J = 6.5 Hz), 3.43 (d, 2H, J = 6.8 Hz), 3.34 (d, 2H, J =
Chemical and Pharmaceutical Bulletin Advance Publication

1
3
6
1
6
3
6
5
3
1
.7 Hz), 1.80 (s, 3H), 1.75 (s, 3H); C NMR (150 MHz, CDCl
3
) δ 156.3, 150.8, 137.8,
37.5, 136.5, 132.1, 130.4, 130.1, 128.7, 127.8, 127.7, 119.9, 115.8, 115.5, 115.4, 112.2,
26 2
5.2, 39.4, 34.4, 25.7, 18.3; HRMS (EI) m/z: [M] calcd for C23H O 334.1933; found
1
34.1935. For 5b, H NMR (600 MHz, CDCl
3
) δ 7.25–7.20 (m, 2H), 6.92–6.91 (m, 1H),
.90–6.88 (m, 1H), 6.09–5.91 (m, 3H), 5.37–5.33 (m, 1H), 5.25 (s, 1H), 5.23–5.17 (m, 2H),
.11–5.06 (m, 2H), 5.05–5.02 (m, 1H), 3.46 (d, 2H, J = 6.4 Hz), 3.37 (d, 2H, J = 7.2 Hz),
1
3
.32 (d, 2H, J = 6.8 Hz), 1.75 (s, 6H); C NMR (150 MHz, CDCl
36.2, 133.6, 131.8, 131.4, 130.2, 129.2, 128.9, 128.1, 127.9, 127.7, 126.1, 122.4, 117.0,
16.5, 115.5, 39.6, 35.4, 29.6, 25.9, 18.0; HRMS (EI) m/z: [M] calcd for C23
3
) δ 153.9, 149.0, 138.0,
1
3
26 2
H O
34.1933; found 334.1927.
',5-diallyl-4'-(cyclopropylmethoxy)-[1,1'-biphenyl]-2-ol
-(2-(cyclopropylmethoxy)-5-(prop-2-en-1-yl)phenyl)-2-(prop-2-en-1-yl)phenol (6b).
CO (0.15
3
(6a)
and
4
To an acetone solution (2 mL) of honokiol (50 mg, 0.18 mmol) was added Cs
2
3
mg, 0.45 mmol), followed by cyclopropylmethyl bromide (25 mg, 0.18 mmol). After
stirring at reflux overnight, the reaction mixture was diluted in ethyl acetate, washed with
water and brine, dried over MgSO , and concentrated under reduced pressure. The residue
4
was purified by flash column chromatography on silica gel (ethyl acetate : n-hexane = 1 :
1
9 to 1 : 2) to afford 6a (10 mg, 16%, BORSM 40%), 6b (2 mg, 3%, BORSM 8%), 6c (5
1
mg, 7%, BORSM 18%), and recovered honokiol (30 mg). For 6a, H NMR (600 MHz,
CDCl
.92–6.89 (m, 2H), 6.06–5.93 (m, 2H), 5.13–5.0.4 (m, 5H), 3.88 (d, 2H, J = 6.7 Hz), 3.46
d, 2H, J = 6.8 Hz), 3.34 (d, 2H, J = 6.8 Hz), 1.32–1.27 (m, 1H), 0.64–0.63 (m, 2H), 0.38–
3
) δ 7.25 (d, J = 2.4 Hz, 2H), 7.04 (dd, J = 8.2, 2.2 Hz, 1H), 7.02(d, , J = 2.1 Hz, 1H)
6
(
1
3
0
1
.37 (m, 2H); C NMR (150 MHz, CDCl ) δ 156.4, 150.8, 137.8, 136.6, 132.1, 130.4,
3
30.1, 128.8, 128.7, 127.8, 127.8, 115.8, 115.5, 115.4, 112.1, 72.7, 39.4, 34.5, 10.3, 3.0;
1
HRMS (EI) m/z: [M] calcd for C22
MHz, CDCl
H
24
O
2
320.1776; found 320.1774. For 6b, H NMR (600
3
) δ 7.46–7.32 (m, 2H), 7.13 (d, J = 2.3 Hz, 1H), 7.05 (dd, J = 8.3, 2.3 Hz, 1H),
6
5
2
2
1
.86 (dd, J = 17.2, 8.3 Hz, 2H), 6.1–6.03 (m, 1H), 6.01–5.94 (m, 1H), 5.23–5.22 (m, 1H),
.17–5.15 (m, 1H), 5.10–5.05 (m, 1H), 5.04 (m, 1H), 4.96 (bs, 1H), 3.77 (d, J = 6.7 Hz,
H), 3.46 (d, J = 6.4 Hz, 2H), 3.36 (d, J = 6.7 Hz, 2H), 1.20–1.16 (m, 1H), 0.55–0.52 (m,
1
3
3
H), 0.28–0.25 (m, 2H); C NMR (150 MHz, CDCl ) δ 154.4, 153.2, 137.9, 136.6, 132.6,
31.9, 131.4, 130.9, 130.8, 129.1, 128.0, 124.6, 116.6, 115.6, 115.5, 113.8, 73.5, 39.6, 35.4,
Chemical and Pharmaceutical Bulletin Advance Publication

1
1
0.4, 3.2; HRMS (EI) m/z: [M] calcd for C22
H
24
O
2
320.1776; found 320.1775. For 6c, H
NMR (600 MHz, CDCl
3
) δ 7.44 (s, 1H), 7.39–7.35 (m, 1H), 7.14 (s, 1H), 7.06 (dd, J = 8.2,
1
5
2
2
.7 Hz, 1H), 6.90–6.85 (m, 2H), 6.11–6.03 (m, 1H), 6.03–5.95 (m, 1H), 5.16–5.08 (m, 2H),
.08–5.02 (m, 2H), 3.88 (d, J = 6.6 Hz, 2H), 3.77 (d, J = 6.7 Hz, 2H), 3.48 (d, J = 5.8 Hz,
H), 3.37 (d, J = 6.5 Hz, 2H), 1.33–1.28 (m, 1H), 1.23–1.16 (m, 1H), 0.63 (d, J = 7.0 Hz,
13
H), 0.55 (d, J = 8.0 Hz, 2H), 0.38 (d, J = 4.8 Hz, 2H), 0.28 (d, J = 4.8 Hz, 2H); C NMR
) δ 155.9, 154.5, 138, 137.4, 132.6, 131.3, 131.1, 131.0, 130.9, 128.3,
(150 MHz, CDCl
3
128.3, 127.9, 115.6, 115.5, 113.7, 111.3, 73.5, 72.7, 39.6, 34.8, 10.6, 10.5, 3.2, 3.2; LRMS
+
(
ESI) m/z: calcd for C26
H
30
O
2
[M+Na] 397.21; found 397.38.
3
',5-diallyl-4'-(cyclobutylmethoxy)-[1,1'-biphenyl]-2-ol (7a) and 3,5'-diallyl-2'
(cyclo butylmethoxy)-[1,1'-biphenyl]-4-ol (7b). To an acetone solution (4 mL) of
CO (0.51 g, 1.6 mmol), followed by
-
honokiol (140 mg, 0.52 mmol) was added Cs
2
3
cyclobutylmethyl bromide (72 µl, 0.63 mmol). After stirring at reflux overnight, the
reaction mixture was diluted with ethyl acetate, washed with water and brine, dried over
MgSO , and concentrated under reduced pressure. The residue was purified by flash
4
column chromatography on silica gel (ethyl acetate : n-hexane = 1/19 : 1/1) to afford 7a (10
mg, 6%, BORSM 36%), 7b (4.5 mg, 3%, BORSM 16%), 7c (1 mg, 0.5%, BORSM 3%),
1
and recovered honokiol (112 mg). For 7a, H NMR (600 MHz, CDCl
3
) δ 7.27–7.23 (m,
2
H), 7.05 (dd, J = 8.2, 2.2 Hz, 1H), 7.03 (d, J = 2.1 Hz, 1H), 6.93 (d, J = 8.3 Hz, 1H), 6.90
(
d, J = 8.2 Hz, 1H), 6.04–5.94 (m, 2H), 5.12–5.04 (m, 5H), 3.97 (d, J = 6.3 Hz, 2H), 3.44
d, J = 6.8 Hz, 2H), 3.34 (d, J = 6.7 Hz, 2H), 2.86–2.79 (m, 2H), 2.17–2.13 (m, 2H), 2.01–
(
1
3
1
1
3
3
.92 (m, 4H); C NMR (150 MHz, CDCl ) δ 156.1, 150.9, 137.9, 136.6, 133.4, 132.3,
30.7, 130.3, 130.2, 129.3, 128.9, 127.9, 127.9, 117.3, 116.0, 115.6, 115.6, 112.3, 69.0,
1
9.5, 34.6; HRMS (EI) m/z: [M] calcd for C23
H
26
O
2
334.1933; found 334.1935.. For 7b, H
NMR (600 MHz, CDCl
3
) δ 7.35 (d, J = 2.1 Hz, 1H), 7.32 (dd, J = 8.2, 2.2 Hz, 1H), 7.13 (d,
J = 2.2 Hz, 1H), 7.06 (dd, J = 8.3, 2.3 Hz, 1H), 6.88 (d, J = 8.3 Hz, 1H), 6.84 (d, J = 8.2 Hz,
1
5
H), 6.09–6.01 (m, 1H), 6.00–5.95 (m, 1H), 5.22–5.18 (m, 1H), 5.18–5.16 (m, 1H), 5.10–
.07 (m, 1H), 5.06–5.04 (m, 1H), 4.94 (bs, 1H), 3.88 (d, J = 6.3 Hz, 2H), 3.45 (d, J = 6.3
Hz, 2H), 3.36 (d, J = 6.7 Hz, 2H), 2.74–2.66 (m, 1H), 2.06–2.00 (m, 2H), 1.94–1.78 (m,
1
3
4
1
3
H); C NMR (150 MHz, CDCl ) δ 154.6, 153.3, 138.0, 136.7, 132.4, 132, 131.5, 130.9,
30.6, 129.2, 128.0, 124.5, 116.7, 115.6, 115.5, 113, 72.7, 39.6, 35.6, 34.9, 25, 18.6.;
Chemical and Pharmaceutical Bulletin Advance Publication

HRMS (EI) m/z: [M] calcd for C23
26 2
H O 334.1933; found 334.1940.
2
-((3,5'-diallyl-2'-hydroxy-[1,1'-biphenyl]-4-yl)oxy)-1-(4-fluorophenyl)ethan-1-on
e (8a) and 2-((3',5-diallyl-4'-hydroxy-[1,1'-biphenyl]-2-yl)oxy)-1-(4-fluorophenyl)
ethan-1-one (8b). To a Tetrahydrofuran solution (4 mL) of honokiol (140 mg, 0.52 mmol)
was added Cs
mmol). After stirring 1 h at ambient temperature, the reaction mixture was diluted in ethyl
acetate, washed with water and brine, dried over MgSO , and concentrated under reduced
2 3
CO (0.51 g, 1.6 mmol), followed by 4-fluorophenacyl bromide (0.14 g, 0.62
4
pressure. The residue was purified by flash column chromatography on silica gel (ethyl
acetate : n-hexane = 1 : 19 to 1 : 2) to afford 8a (67 mg, 32%, BORSM 40%), 8b (21 mg,
1
0%, BORSM 12%), 8c (50 mg, 18%, BORSM 22%), and recovered honokiol (27 mg).
1
For 8a, H NMR (600 MHz, CDCl
3
) δ 8.09–8.06 (m, 2H), 7.27 (d, J = 2.2 Hz, 1H), 7.24
(
dd, J = 8.3, 2.3 Hz, 1H), 7.21–7.16 (m, 2H), 7.05 (dd, J = 8.2, 2.1 Hz, 1H), 7.00 (d, J = 2.1
Hz, 1H), 6.89 (d, J = 8.2 Hz, 1H), 6.85 (d, J = 8.3 Hz, 1H), 6.04–5.93 (m, 2H), 5.27 (s, 2H),
1
3
5
.10–5.04 (m, 5H), 3.50 (d, J = 6.6 Hz, 2H), 3.34 (d, J = 6.7 Hz, 2H); C NMR (150 MHz,
CDCl ) δ 193.4, 155.5, 150.9, 137.9, 136.5, 132.4, 131.3, 131.2, 131.2, 130.3, 129.1, 128.1,
3
1
27.7, 116.3, 116.3, 116.2, 115.8, 115.7, 112.2, 71.3, 39.5, 34.5.; HRMS (EI) m/z: [M]
1
calcd for C26
H23FO
3
402.1631; found 402.1635. For 8b, H NMR (600 MHz, CDCl
3
) δ
7
.95–7.92 (m, 2H), 7.33–7.30 (m, 2H), 7.09–7.01 (m, 4H), 6.83 (dd, J = 8.3, 5.4 Hz, 2H),
.04–5.97 (m, 2H), 5.10–5.06 (m, 5H), 3.41 (d, J = 6.4 Hz , 2H), 3.36 (d, J = 6.8 Hz, 2H);
6
1
3
3
C NMR (150 MHz, CDCl ) δ 193.8, 166.9, 165.2, 153.4, 153.3, 137.5, 136.4, 131.7,
1
31.3, 131.2, 131.1, 129.1, 128.0, 116.6, 115.9, 115.8, 115.8, 115.5, 113.4, 71.9, 39.4, 35.3.
1
For 8c, H NMR (600 MHz, CDCl ) δ 8.11–8.05 (m, 2H), 7.95–7.90 (m, 2H), 7.36–7.34 (m,
3
2
1
4
1
1
H), 7.20–7.15 (m, 2H), 7.12 (d, J = 2.2 Hz, 1H), 7.10–7.03 (m, 3H), 6.83 (d, J = 8.4 Hz,
H), 6.78 (d, J = 9.0 Hz, 1H), 6.01–5.92 (m, 2H), 5.22 (s, 2H), 5.09 (s, 2H), 5.07–5.00 (m,
1
3
3
H), 3.44 (d, J = 6.6 Hz, 2H), 3.35 (d, J = 6.7 Hz, 2H); C NMR (150 MHz, CDCl ) δ
93.8, 193.7, 155.0, 153.4, 137.6, 136.9, 133.9, 131.7, 131.4, 131.3, 131.2, 131.0, 128.7,
28.3, 116.2, 116.0, 115.9, 115.9, 115.8, 113.4, 111.2, 72.0, 71.5, 39.5, 34.5; LRMS (ESI)
+
m/z: calcd for C H F O [M+Na] 561.18; found 561.32.
3
4
28
2
2
2
-((3,5'-diallyl-2'-hydroxy-[1,1'-biphenyl]-4-yl)oxy)-1-(2,4-dichlorophenyl)ethan-1
-one (9a) and 2-((3',5-diallyl-4'-hydroxy-[1,1'-biphenyl]-2-yl)oxy)-1
-(2,4-dichlorophenyl)ethan-1-one (9b). To an acetone solution (4 mL) of honokiol (140
Chemical and Pharmaceutical Bulletin Advance Publication

mg, 0.52 mmol) was added Cs
bromide (77 µl, 0.62 mmol). After stirring for 1 h at ambient temperature, the reaction
mixture was diluted in ethyl acetate, washed with water and brine, dried over MgSO , and
2 3
CO (0.51 g, 1.6 mmol), followed by 4-nitrophenacyl
4
concentrated under reduced pressure. The residue was purified by flash column
chromatography on silica gel (ethyl acetate : n-hexane = 1 : 19 to 1 : 2) to afford 9a (14 mg,
6
%, BORSM 17%), 9b (14 mg, 6%, BORSM 17%), 9c (23 mg, 7%, BORSM 19%), and
1
recovered honokiol (64 mg). For 9a, H NMR (600 MHz, CDCl
3
) δ 7.56 (d, J = 8.3 Hz,
1
1
5
1
1
H), 7.48 (s, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.24 (s, 1H), 7.05 (d, J = 7.9 Hz, 1H), 7.00 (s,
H), 6.89 (d, J = 8.2 Hz, 1H), 6.83 (d, J = 8.2 Hz, 1H), 5.96–5.85 (m, 3H), 5.18 (s, 2H),
13
3
.09–4.99 (m, 4H), 3.35 (t, J = 7.3 Hz, 4H); C NMR (150 MHz, CDCl ) δ 197.5, 155.2,
50.9, 138.6, 137.9, 136.3, 134.9. 132.9, 132.4, 131.2, 130.5, 130.4, 130.3, 130.2, 129.1,
28.1, 127.7, 127.6, 116.1, 115.8, 115.7, 112.0, 73.1, 39.5, 34.4; HRMS (EI) m/z: [M]
1
calcd for C26
H22Cl
2
O
3
452.0946; found 452.0947. For 9c, H NMR (600 MHz, CDCl
3
) δ
7
1
1
4
.54 (d, J = 8.3 Hz, 1H), 7.48 (d, J = 1.9 Hz, 1H), 7.38–7.33 (m, 2H), 7.29 (d, J = 1.9 Hz,
H), 7.22–7.16 (m, 3H), 7.08–7.04 (m, 2H), 6.79 (d, J = 8.2 Hz, 1H), 6.64 (d, J = 8.8 Hz,
H), 5.99–5.92 (m, 1H), 5.89–5.82 (m, 1H), 5.13 (s, 2H), 5.10–5.04 (m, 2H), 4.99 (s, 2H),
1
3
.97–4.91 (m, 2H), 3.35 (d, J = 6.7 Hz, 2H), 3.27 (d, J = 6.6 Hz, 2H); C NMR (150 MHz,
) δ 198.25, 197.92, 154.56, 153.13, 138.38, 138.02, 137.60, 136.73, 135.10, 135.04,
33.83, 132.85, 132.74, 131.57, 131.46, 131.38, 131.12, 130.98, 130.90, 130.43, 130.16,
CDCl
3
1
1
28.46, 128.22, 127.57, 127.32, 115.94, 115.60, 112.95, 110.57, 73.68, 73.00, 39.49, 34.48;
+
LRMS (ESI) m/z: calcd for C34
H26Cl
4
O
4
[M+Na] 661.04; found 661.25.
2
-((3,5'-diallyl-2'-hydroxy-[1,1'-biphenyl]-4-yl)oxy)-1-(4-nitrophenyl)ethan-1-one
10a). To a tetrahydrofuran solution (2 mL) of honokiol (50 mg, 0.18 mmol) was added
Cs CO (183 mg, 0.56 mmol), followed by 2-bromo-4′-nitroacetophenone (46 mg, 0.18).
(
2
3
After stirring 1 h at room temperature, the reaction mixture was diluted in ethyl acetate,
washed with water and brine, dried over MgSO , and concentrated under reduced pressure.
4
The residue was purified by flash column chromatography on silica gel (ethyl acetate :
n-hexane = 1 : 19 to 1 : 2) to afford 10a (35 mg, 43%, BORSM 54%), 10c (10 mg, 9%,
1
BORSM 11%), and recovered honokiol (10 mg). H NMR (600 MHz, CDCl
3
) δ 8.37–8.34
(
m, 2H), 8.21–8.18 (m, 2H), 7.28 (dd, 2H, J = 5.1, 2.1 Hz), 7.05 (dd, 1H, J = 8.2, 2.2 Hz),
7
.01 (d, 1H, J = 2.1 Hz), 6.88 (dd, 2H, J = 14.2, 8.2 Hz), 6.00–5.94 (m, 2H), 5.30 (s, 2H),
Chemical and Pharmaceutical Bulletin Advance Publication

13
5
.10–5.02 (m, 5H), 3.47 (d, 2H, J = 6.6 Hz), 3.34 (d, 2H, J = 6.7 Hz); C NMR (150 MHz,
CDCl ) δ 194.0, 155.1, 150.8, 150.8, 139.1, 137.8, 136.3, 132.4, 131.4, 130.9, 130.3, 130.2,
29.7, 129.1, 128.1, 127.5, 124.1, 116.3, 115.8, 115.7, 112.0, 71.6, 39.5, 34.4; HRMS (EI)
3
1
1
m/z: [M] calcd for C26
CDCl ) δ 8.24 (dd, J = 8.9, 1.7 Hz, 2H), 7.98 (dd, J = 9.0, 1.0 Hz, 2H), 7.80 (dd, J = 9.0,
.1 Hz, 2H), 7.42 (dd, J = 9.0, 2.1 Hz, 1H), 7.20 (d, J = 2.0 Hz, 1H), 7.16–7.11 (m, 1H),
5
H23NO 429.1576; found 429.1574. For 10c, H NMR (600 MHz,
3
1
7
1
3
1
1
.08–7.00 (m, 1H), 6.86–6.76 (m, 1H), 5.99–5.88 (m, 1H), 5.09–4.98 (m, 2H), 4.82 (d, J =
5.0 Hz, 1H), 4.57 (d, J = 10.8 Hz, 1H), 4.23–4.18 (m, 1H), 3.90 (dd, J = 9.2, 6.9 Hz, 1H),
1
3
3
.34 (dd, J = 19.1, 7.0 Hz, 2H); C NMR (150 MHz, CDCl ) δ 209.8, 155.2, 153.5, 151.1,
47.5, 147.1, 137.6, 136.9, 133.6, 131.9, 131.7, 131.2, 130.9, 128.8, 128.6, 126.6, 126.5,
23.7, 123.3, 116, 113.2, 111.0, 75.0, 74.6, 49.3, 39.5; LRMS (ESI) m/z: calcd for
+
C
34
H
28
O
2
[M+H] 593.19; found 593.35.
tert-butyl 2-((3,5'-diallyl-2'-hydroxy-[1,1'-biphenyl]-4-yl)oxy)acetate (11a) and
tert-butyl 2-((3',5-diallyl-4'-hydroxy-[1,1'-biphenyl]-2-yl)oxy)acetate (11b). To a
tetrahydrofuran solution (2 mL) of honokiol (200 mg, 0.75 mmol) was added Cs CO (245
2
3
mg, 0.75 mmol), followed by tert-butyl bromoacetate (220 mg, 1.13). After stirring 1 h at
ambient temperature, the reaction mixture was diluted in ethyl acetate, washed with water
and brine, dried over MgSO
4
, and concentrated under reduced pressure. The residue was
purified by flash column chromatography on silica gel (ethyl acetate : n-hexane = 1 : 19 to
1
: 2) to afford 11a (107 mg, 37%, BORSM 48%), 11b (36 mg, 13%, BORSM 16%), 11c
1
(
97 mg, 26%, BORSM 33%), and recovered honokiol (43 mg). For 11a, H NMR (600
MHz, CDCl ) δ 7.24 (d, 2H, J = 2.3 Hz), 7.06–7.04 (m, 1H), 7.02 (d, 1H, J = 2.2 Hz), 6.90
3
(d, 1H, J = 8.2 Hz), 6.80 (d, 1H, J = 8.0 Hz), 6.01 (dd, 2H, J = 44.3, 6.9 Hz), 5.14–5.04 (m,
1
3
5
H), 4.58 (s, 2H), 3.51 (d, 2H, J = 6.7 Hz), 3.35 (d, 2H, J = 6.7 Hz), 1.51 (s, 9H); C NMR
(150 MHz, CDCl
3
) δ 167.9, 155.4, 150.8, 137.7, 136.3, 132.1, 130.8, 130.2, 130.1, 129.9,
1
28.8, 127.7, 127.6, 116.0, 115.5, 115.5, 111.8, 82.4, 65.9, 39.4, 34.3, 28.1; HRMS (EI)
1
m/z: [M] calcd for C H O 380.1988; found 380.1987. For 11b, H NMR (600 MHz,
2
4
28
4
CDCl
.06 (dd, J = 8.3, 2.2 Hz, 1H), 6.84 (d, J = 8.3 Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H), 6.10–6.03
m, 1H), 6.00–5.93 (m, 1H), 5.23–5.20 (m, 1H), 5.18–5.16 (m, 1H), 5.10–5.04 (m, 3H),
3
) δ 7.40 (dd, J = 8.2, 2.2 Hz, 1H), 7.36 (d, J = 2.1 Hz, 1H), 7.14 (d, J = 2.2 Hz, 1H),
7
(
5.01 (bs, 1H), 4.46 (s, 2H), 3.46 (d, J = 6.4 Hz, 2H), 3.37 (d, J = 6.7 Hz, 2H), 1.48 (s, 9H);
Chemical and Pharmaceutical Bulletin Advance Publication

1
3
3
C NMR (150 MHz, CDCl ) δ 168.5, 153.5, 153.3, 137.7, 136.7, 133.4, 131.6, 131.3,
1
30.8, 130.8, 129.2, 127.8, 125.0, 116.4, 115.7, 115.5, 112.8, 82.3, 66.5, 39.5, 35.3, 28.1;
1
HRMS (EI) m/z: [M] calcd for C24
600 MHz, CDCl
.3 Hz, 1H), 7.05 (dd, J = 8.3, 2.3 Hz, 1H), 6.77 (dd, J = 10.7, 8.4 Hz, 2H), 6.11–6.04 (m,
H), 6.00–5.94 (m, 1H), 5.14–5.03 (m, 4H), 4.55 (s, 2H), 4.44 (s, 2H), 3.51 (d, J = 6.7 Hz,
H
28
O
4
380.1988; found 380.1986. For 11c, H NMR
(
3
) δ 7.44 (dd, J = 8.4, 2.3 Hz, 1H), 7.40 (d, J = 2.2 Hz, 1H), 7.13 (d, J =
2
1
2
1
1
1
3
3
H), 3.36 (d, J = 6.7 Hz, 2H), 1.51 (s, 9H), 1.46 (s, 9H); C NMR (150 MHz, CDCl ) δ
68.3, 168.3, 155.0, 153.4, 137.8, 137.1, 133.4, 131.5, 131.4, 131.4, 130.8, 128.8, 128.5,
28, 115.8, 115.6, 112.9, 111.1, 82.3, 82.2, 66.5, 66.4, 39.5, 34.6, 28.2, 28.2; LRMS (ESI)
+
m/z: calcd for C30
H
38
O
6
[M+Na] 517.25; found 517.37.
2
2 2
-((3,5'-diallyl-2'-hydroxy-[1,1'-biphenyl]-4-yl)oxy)acetic acid (12). To a CH Cl
solution (3.0 mL) of tert-butyl acetate (11a) (50 mg, 0.13 mmol) was added trifluoroacetic
acid (1 mL). After stirring for 2 h at ambient temperature, the reaction mixture was
concentrated under reduced pressure to afford crude the carboxylic acid (12) (45 mg, 95%).
1
H NMR (600 MHz, DMSO-d
6
) δ 9.25 (s, 1H), 7.30 (d, J = 8.5 Hz, 1H), 6.99 (s, 1H), 6.91
(
d, J = 8.1 Hz, 1H), 6.86 (d, J = 8.4 Hz, 1H), 6.82 (d, J = 8.2 Hz, 1H), 6.05–5.97 (m, 1H),
5
3
1
3
.97–5.90 (m, 1H), 5.12–4.98 (m, 6H), 4.69 (s, 2H), 3.39 (d, J = 6.6 Hz, 2H), 3.33 (s, 1H),
1
3
.28 (d, J = 6.6 Hz, 2H). C NMR (150 MHz, DMSO-d
31.2, 130.4, 130.2, 130.1, 127.8, 127.8, 127.4, 127.3, 115.9, 115.6, 115.3, 111.1, 65.0,
8.8, 34.0; HRMS (EI) m/z: [M] calcd for C20 324.1362; found 324.1362.
N-(2-(2-(2-(2-(4-((4-(4-(3-(2-((3,5'-diallyl-2'-hydroxy-[1,1'-biphenyl]-4-yl)oxy)acet
6
) δ 170.4, 154.3, 152.4, 138.3, 137,
20 4
H O
amido)propoxy)benzoyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)
ethyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide
(
14). To a CH Cl solution (3.0 mL) of tert-butyl acetate (12b) (23 mg, 0.05 mmol) was
2 2
added trifluoroacetic acid (0.3 mL). After stirring for 2 h at ambient temperature, the
reaction mixture was concentrated under reduced pressure to afford crude the carboxylic
acid (13) (20 mg, 98%). To a DMF solution (3 mL) of carboxylic acid (13) (50 mg, 0.15
mmol) were added HOAt (63 mg, 0.46 mmol), PyAOP (161 mg, 0.308 mmol), and DIPEA
(
0.22 mL, 1.26 mmol). After stirring for 24 h, the reaction mixture was diluted in ethyl
acetate, dried over MgSO , and concentrated under reduced pressure. The residue was
purified by flash column chromatography on silica gel (MeOH : CH Cl = 1 : 10) to afford
4
2
2
Chemical and Pharmaceutical Bulletin Advance Publication

biotin probe (14) (25 mg, 20%, BORSM 31%) and recovered starting material 13 (18
1
mg). H NMR (600 MHz, CDCl
3
) δ 9.16 (s, 1H), 7.84 (s, 1H), 7.72–7.67 (m, 4H), 7.41 (dd,
1
6
2
4
3
H, J = 8.4, 2.1 Hz), 7.37 (d, 1H, J = 1.7 Hz), 7.01 (dd, 2H, J = 10.5, 2.9 Hz), 6.98 (s, 1H),
.91–6.85 (m, 4H), 6.80–6.77 (m, 1H), 6.72 (d, 1H, J = 4.6 Hz), 6.28 (s, 1H), 5.95–5.86 (m,
H), 5.57 (s, 1H), 5.23 (s, 2H), 5.03–4.94 (m, 4H), 4.52 (t, 2H, J = 4.8 Hz), 4.49 (s, 1H),
.42–4.38 (m, 1H), 4.23–4.19 (m, 1H), 4.00 (t, 2H, J = 5.6 Hz), 3.84 (t, 2H, J = 4.8 Hz),
.56–3.54 (m, 2H), 3.52 (d, 8H, J = 1.2 Hz), 3.47 (t, 2H, J = 4.9 Hz), 3.37 (d, 2H, J = 5.9
Hz), 3.35–3.33 (m, 2H), 3.26 (d, 2H, J = 6.6 Hz), 3.07–2.97 (m, 2H), 2.82–2.78 (m, 1H),
2
6
1
1
6
2
1
.66 (d, 1H, J = 12.7 Hz), 2.13 (dd, 2H, J = 9.7, 4.9 Hz), 2.05–2.00 (m, 2H), 1.69–1.53 (m,
1
3
3
H); C NMR (150 MHz, CDCl ) δ 194.3, 173.3, 168.8, 163.8, 162.0, 161.4, 153.9, 152.9,
43.2, 138.1, 137.5, 132.9, 132.2, 132.2, 131.8, 131.1, 130.6, 130.5, 130.2, 128.7, 128.2,
27.6, 127.3, 124.3, 116.2, 115.3, 115.2, 114.3, 114.0, 111.3, 70.5, 70.3, 70.3, 70.0, 69.8,
9.4, 67.5, 65.6, 62.0, 61.7, 60.2, 55.5, 50.3, 40.5, 39.4, 39.1, 36.2, 35.8, 35.1, 28.9, 28.2,
8.1, 25.5; HRMS (ESI/QTOF) m/z: [M+Na] calcd for C57
082.4673.
69 7
H N NaO11S 1082.4668; found
4
4
.2 Biological assays
.2.1 Cell viability assay
The cell viability was measured using soluble 3-(4, 5-dimethyl- thiazol-2-yl)-2,
-diphenyl-tetrazolium bromide (MTT; Sigma, Cat. No. M5655) that converts into
5
insoluble purple formazan by metabolic enzymes in live cells. 5×104 RAW264.7 cells were
seeded in 96 well plates and treated with various concentration of honokiol derivatives.
Following 24 h incubation, the MTT solution were added to each well and additionally
incubated at 37 °C for 2 h. Then, the supernatant was removed and the formazan was
dissolved with Dimethyl sulfoxide (DMSO, Sigma, D-2650). The quantitative absorbance
was measured at 540nm with ELISA reader (Molecular Devices, SpectraMax M2).
4
.2.2 Nitric oxide production assay
To observe the production of nitric oxide (NO), 4×105 RAW264.7 cells were seeded
and activated by LPS (InVivogen, Cat. No. tlrl-eklps) in 24 well plates. Consistent with
LPS activation, the cells also treated with honokiol derivatives and incubated for 24 h at
3
7°C. After 24 h incubation, the supernatant was transferred to 96-well plate and Griess
Chemical and Pharmaceutical Bulletin Advance Publication

reagent A (1% Sulfanilamide, Sigma, Cat. No. S9251; 2% Phosphoric acid, Sigma, Cat. No.
45245) and Gries reagent B (N-(1-Naphthyl) ethylenediamine dihydrochloride (NED),
3
TCI, Cat. No. N0063) were sequentially incubated for 20 min each at room temperature.
The NO concentration was determined by absorbance at 540 nm using ELISA reader
(
Molecular Devices, SpectraMax M2).
.2.3 Western blotting
.5 × 106 RAW 264.7 cells were stimulated with LPS (10 ng/ml or 100 ng/ml)
InvivoGen, Cat. No. 13106MM) with or without honokiol derivatives (5 μM) respectively
4
2
(
and lysed with a cold lysis buffer containing 50 mM Tris-HCl (Amresco, Cat No.0826), 2
mM EDTA (Biosesang, Cat No.E2002), 1% NP-40 (Millipore, Cat. No.492016), protease
inhibitors (Roche, Cat No. 11836170001), and PhoSTOP (Roche, Cat. No. 04906837001).
Lysates were clearly collected and electrophoresed by SDS-PAGE and transferred to PVDF
blotting membrane (GE Healthcare, Cat. No. 10600023). The membrane was incubated
with rabbit anti-COX-2 antibody (Cell Signaling Technology, Cat. No. 4842), mouse
anti-iNOS antibody (BD Biosciences, Cat. No 610329), mouse anti-β-actin antibody (Santa
Cruz Biotechnology, Cat. No sc-47778) respectively, followed by detection using the
corresponding horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Jackson
ImmunoResearch Laboratories, Cat. No. 115-036-003), goat anti-rabbit IgG (Bio-Rad, Cat.
No. 170-6515) respectively, and enhanced chemiluminescence (ECL) (Bio-Rad, Cat. No.
1
70-0561). The membranes were scanned with a Fusion Solo chemiluminescence imaging
system (Vilber).
Acknowledgments
This work was supported by the Gachon University research fund of
017(GCU-2018-0666) and grant from the National Research Foundation of Korea
grant funded by the Korean government (grant number:
NRF-2016R1D1A1A09917300).
2
Conflict of Interest (COI)
The authors declare no conflict of interest.
Chemical and Pharmaceutical Bulletin Advance Publication

Supplementary Materials
The online version of this article contains supplementary materials.
Chemical and Pharmaceutical Bulletin Advance Publication

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