Design and synthesis of ten biphenyl-neolignan derivatives and their in vitro inhibitory potency against cyclooxygenase-1/2 activity and 5-lipoxygenase-mediated LTB4-formation

Article abstract of DOI:10.1016/j.bmc.2009.05.018

A set of ten derivatives of methylhonokiol, an anti-inflammatory active biphenyl-type neolignan from Magnolia grandiflora, has been evaluated for their in vitro cyclooxygenase-1/2 (COX-1/2) inhibitory activity using assays with purified prostaglandin H sy

Full text of DOI:10.1016/j.bmc.2009.05.018

Bioorganic & Medicinal Chemistry 17 (2009) 4459–4465
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of ten biphenyl-neolignan derivatives and their
in vitro inhibitory potency against cyclooxygenase-1/2 activity
and 5-lipoxygenase-mediated LTB4-formation
b
a
b
a
Wolfgang Schühly a, , Antje Hüfner , Eva M. Pferschy-Wenzig , Elke Prettner , Michael Adams ,
*
Antje Bodensieck a, Olaf Kunert b, Asije Oluwemimo b, Ernst Haslinger b, Rudolf Bauer a
a Institute of Pharmaceutical Sciences, Department of Pharmacognosy, Karl-Franzens-Universität Graz, Universitätsplatz 4, 8010 Graz, Austria
b Institute of Pharmaceutical Sciences, Department of Pharmaceutical Chemistry, University of Graz, Universitätsplatz 1, 8010 Graz, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 December 2008
Revised 3 May 2009
Accepted 7 May 2009
Available online 18 May 2009
A set of ten derivatives of methylhonokiol, an anti-inflammatory active biphenyl-type neolignan from
Magnolia grandiflora, has been evaluated for their in vitro cyclooxygenase-1/2 (COX-1/2) inhibitory activ-
ity using assays with purified prostaglandin H synthase (PGHS)-1 and PGHS-2 enzymes as well as for
their 5-lipoxygenase (5-LOX) mediated LTB4 formation inhibitory activity using an assay with activated
human polymorphonuclear leukocytes. The derivatization reactions included methylation, acetylation,
hydrogenation, epoxydation and isomerization. Five of the derivatives are new to science. The most active
Key words:
compound against COX-1 and COX-2 was methylhonokiol with IC50 values of 0.1
active compound against LTB4 formation was (E)-30-propenyl-5-(2-propenyl)-biphenyl-2,40-diol with an
IC50 value of 1.0 M. Structure–activity relationship studies showed that the polarity of the derivatives
lM, whereas the most
Honokiol derivatives
Cyclooxygenase
Lipoxygenase
Anti-inflammatory
SAR
l
plays a crucial role in their activity towards COX-1/2 enzyme and 5-LOX mediated LTB4 formation.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
eral selective COX-2 inhibitors have turned out to cause side effects
such as cardiovascular problems,6 and evidence has increased that
Prostaglandins such as PGE2 are produced in the cyclooxygen-
ase pathway of the arachidonic acid cascade by the action of the
isoenzymes COX-1 and COX-2. They are not only regulating phys-
iological functions such as thromboxane production in platelets,
wound healing, kidney function, blood vessel tone and others,1
but are also involved in the development of inflammatory diseases
such as acute and chronic arthritis.2 While both isoforms COX-1
and COX-2 have a very similar protein structure and catalyze
essentially the production of the same compounds, it can be stated
in a very simplified way that COX-1 is constitutively expressed and
physiologically active in many different tissues of the body
whereas COX-2 is predominantly activated upon inflammatory
stimuli.3,4
Among the drugs known since long time and used most widely
in the treatment of inflammatory disorders are nonsteroidal anti-
inflammatory drugs (NSAIDs, e.g., aspirin). Inhibition of COX-1
has been thought to be responsible for side effects of NSAIDs such
as gastrointestinal bleeding, while inhibition of COX-2 has been
associated with their anti-inflammatory properties. This was the
rationale for the development of selective COX-2 inhibitors for
the effective treatment of inflammatory disorders.5 However, sev-
not only COX-1, but also COX-2 has physiological functions and is
constitutively expressed in certain tissues.7
Leukotrienes such as LTB4 are important mediators in inflam-
matory pathways and in allergic responses. The formation of
LTB4 from arachidonic acid in human neutrophils by 5-LOX initi-
ates a cascade of reactions which eventually causes inflammatory
symptoms.8 The biological properties of leukotrienes and their par-
ticipation in a variety of diseases suggest that 5-LOX inhibitors
should have a therapeutic potential in a range of allergic and
inflammatory disorders. However, even though several of these
compounds have turned out to be effective in the treatment of
asthma, selective 5-LOX inhibitors seem to possess an insufficient
therapeutic potential for the treatment of other inflammatory
diseases.9,10
Therefore, dual inhibitors blocking both the COX and LOX path-
way of the arachidonic acid cascade are recently regarded as a pos-
sible alternative in the treatment of inflammatory disorders.9–11
Inhibition of both pathways might offer a broader range of anti-
inflammatory effects, and several side effects known from NSAIDs
and selective COX-2 inhibitors might be reduced: the shift of ara-
chidonate metabolism towards the 5-LOX pathway observed for
NSAIDs should be avoided, resulting in a decreased production of
leukotrienes which damage the gastrointestinal mucosa and con-
tribute to allergic reactions.9,10
* Corresponding author. Tel.: +43 316 380 5527; fax: +43 316 380 9860.
E-mail address: wolfgang.schuehly@uni-graz.at (W. Schühly).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.05.018

4460
W. Schühly et al. / Bioorg. Med. Chem. 17 (2009) 4459–4465
The impulse for the present investigation was derived from the
2. Results and discussion
fact that the active principles of Magnolia bark (e.g., from Magnolia
officinalis Rehder et Wilson) honokiol and magnolol exhibit anti-
inflammatory activities, for example, against pleurisy in mice
in vivo.12,13 Besides its strong antioxidative effect, honokiol is of
major interest because of its various pharmacological activities.14
Magnolia bark is widely known from traditional Asian medicinal
systems such as Traditional Chinese Medicine (TCM) and Kampo
medicine in Japan.15 Based on literature reports about the anti-
inflammatory activity (COX-1, COX-2 and 5-LOX) of honokiol16–18
and founded on our own finding that the anti-inflammatory activ-
ity of methylhonokiol (1b) was even higher than that of honokiol,19
we tried to fathom the effectiveness of derivatives of methylhon-
okiol, a major constituent of the seeds of the North American spe-
cies Magnolia grandiflora L. Methylhonokiol (1b) turned out to be a
promising lead structure suitable for derivatizations (Figs. 1 and 2).
The design of derivatives was undertaken by utilization of the
structural features of 1b, which allowed the alteration of the dou-
ble bond in the side chain as well as substitutions at the phenolic
OH.
A set of 10 derivatives of methylhonokiol (1b) as lead com-
pound has been synthesized and compared towards their inhibi-
tory activity in COX-1/2 in vitro test systems and against 5-LOX
mediated LTB4 formation (Fig. 1). The derivatizations encompassed
methylation and acetylation of the free phenolic OH-groups as well
as hydrogenation, isomerization and epoxidation of the side chains
(for a general scheme, see Fig. 2). The derivatizations led to yet
undescribed or only partially described compounds. With the
exception of 5 (epoxidation product) and 4c, all derivatives were
obtained in high purity and mostly high yield (67–97%). A separa-
tion of the diastereomeric mixture of 5 was not undertaken due to
its loss of activity in both COX-1/2 and 5-LOX systems (see below).
The pharmacological data for the derivatives are shown in Tables 1
and 2.
The overall best activity against COX-1 and COX-2 was exhib-
ited by methylhonokiol (1b), which showed a slight selectivity to-
wards COX-2. None of the derivatives showed a pronounced
selectivity towards COX-1 or COX-2. The highest COX-2 selectivity
was displayed by 4a and 4b with an IC50 for COX-2 about three
In a dual approach, the anti-inflammatory activity of honokiol-
derived neolignans was evaluated against COX-1 and COX-2 en-
zyme activity as well as against 5-LOX-mediated LTB4 formation
in vitro. The presented data may help to better understand the
structural requirements needed for anti-inflammatory activity of
honokiol-type neolignans.
times lower than for COX-1 (0.2 and 0.7
lM, respectively, for 4a
and 0.9 vs 2.8 M for 4b). A similar trend was observed for com-
l
pounds 2c and 3a, however, the activity displayed by these com-
pounds was much lower. Overall, for the inhibitory activity
against COX-1/2, there are two distinct trends noticeable. First,
OR1
R4
5
1'
3'
R3
1
OR2
Compound R1
R2
R3
R4
1a
1b
1c
2a
2b
2c
3a
3b
4a
4b
4c
5
-H
-H
-2-propenyl
-2-propenyl
-2-propenyl
-propyl
-2-propenyl
-2-propenyl
-2-propenyl
-propyl
-CH3
-CH3
-H
-H
-CH3
-H
-CH3
-CH3
-CH3
-H
-propyl
-propyl
-CH3
-COCH3
-COCH3
-H
-propyl
-propyl
-2-propenyl
-2-propenyl
-E-propenyl
-Z-propenyl
-E-propenyl
-oxiranylmethyl
-2-propenyl
-2-propenyl
-2-propenyl
-2-propenyl
-2-propenyl
-oxiranylmethyl
-COCH3
-CH3
-CH3
-H
-H
-H
-CH3
-H
Figure 1. Biphenyl-type neolignan derivatives prepared from methylhonokiol.

W. Schühly et al. / Bioorg. Med. Chem. 17 (2009) 4459–4465
4461
O
OH
OAc
OH
3'
d)
1'
5
1
OH
OAc
OH
1a
3b
O
5
f)
c)
OCH3
OCH3
OCH3
3'
3'
b)
a)
1'
1'
5
1
5
1
OCH3
OH
OH
1c
1b
2b
d)
e)
OCH3
OCH3
R3
OH
3'
3'
3'
1'
c) from 4a
1'
1'
5
1
5
5
1
1
OAc
OH
OH
4c
3a
4a R3 = -E-propenyl
4b R3 = -Z-propenyl
a) NaOEt / Me2SO4; 70%
b) Pd on charcoal; 97%
c) Grignard in THF; 55% (1a), 16% (4c from 4a)
d) Ac2O / pyridine; 87% (3a), 67% (3b)
e) KOtBu / THF, reflux; mixture of4a and 4b; 94%
f) MCPBA / benzene; 87%
Figure 2. Scheme of syntheses of the biphenyl-neolignan derivatives 1–5.
the very lipophilic compounds without OH group (1c, 2c, 3a and
3b), did clearly show a reduced activity. At the low concentration
used in the assay, a solubility problem could be ruled out. Second,
the variation in the side chain (hydrogenation, isomerization) did
not seem to considerably affect the overall activity against COX-
1/-2. However, the introduction of the epoxy group in 5 led to a
complete loss of activity in both COX-1/2 systems.
Considering the array of 5-LOX product formation inhibitory
activity of the ten derivatives, the overall highest activity was
found for 4b and 4c with IC50 values of 1.0 and 1.1 lM, respec-
Table 1
Inhibition of COX-1, COX-2 enzymes and 5-LOX mediated LTB4 formation by derivatives of methylhonokiol at 8 lM
Compounds
COX-1 inhibition (% and SD)
COX-2 inhibition (% and SD)
LTB4 formation inhibition (% and SD)
Honokiol 1a
Methylhonokiol 1b
Dimethylhonokiol 1c
Tetrahydrohonokiol 2a
2b
2c
3a
3b
4a
4b
4c
5
91.0
90.8
45.1
89.6
56.5
27.3
18.0
6.8
80.5
68.6
84.8
0.5
1.5
6.5
5.8
3.1
5.6
4.0
7.0
12.7
2.4
5.4
3.0
6.5
88.3
95.3
47.8
81.6
11.6
49.4
56.3
13.8
81.5
62.7
75.1
4.2
3.8
1.6
6.0
5.7
3.8
5.4
6.3
13.6
3.1
7.2
6.0
4.8
94.0
96.4
27.8
99.9
97.1
24.4
76.3
64.9
99.3
99.9
100.0
4.0
0.9
0.3
8.5
0.2
4.0
9.4
6.1
7.9
0.5
0.1
0.0
4.6
SD: standard deviation.

4462
W. Schühly et al. / Bioorg. Med. Chem. 17 (2009) 4459–4465
Table 2
As the assay used herein is cell-based and as LTB4 concentration
is measured for the determination of inhibitory activity, no state-
ment can be made on the mechanism underlying the observed
inhibition of LTB4 formation inhibition so far. From the literature
it is known that magnolol, an isomer of honokiol, inhibits LTB4 for-
mation in neutrophils by direct inhibition of 5-LOX.21 The same
mechanism was also suggested for the LT formation inhibitory
activity of honokiol in RBL cells.18 Further pharmacological studies
will be necessary to investigate whether this is also true for the
biphenyl derivatives presented herein.
To summarize, within the set of investigated biphenyls, the
structural prerequisites for anti-inflammatory activity against
COX-1/2 seem to be a biphenylic system bearing two phenolic
OH groups or one OH and one OCH3 group together with apolar
side chains of medium length. For 5-LOX LTB4 formation inhibitory
activity, the substitution of the phenolic ring could also include
acetoxy groups. The denomination of one single clearly required
pharmacophore in both systems could not be undertaken, how-
ever, the loss of all activity in the epoxy derivative 5 indicates that
a dramatic change in the chemical character of the side chain abol-
ishes the activity.
IC50 values of derivatives of methylhonokiol and positive controls in COX-1, COX-2
enzyme and LTB4 formation assays in
l
M
Compounds
IC50 for COX-1
inhibition
IC50 for COX-2
inhibition
IC50 for LTB4
formation
inhibition (lM)
(l
M)
(
lM)
Honokiol 1a
Methylhonokiol 1b
Dimethylhonokiol 1c 11.4
2a
2b
2c
3a
3b
4a
4b
4c
5
1.8
0.1
2.1
0.06
7.7
2.1
n.d.
6.9
4.9
n.d.
0.2
0.9
2.7
n.d.
4.2
1.5
15.0
1.7
1.4
n.d.
2.2
4.5
2.1
1.1
1.0
0.8
7.7
> 20
n.d.
n.d.
0.7
2.8
0.6
n.d.
0.9
n.d.
Indomethacin
NS-398
Zileuton
2.6
5.0
n.d.: not determined.
3. Experimental
3.1. General
tively. For dimethylhonokiol (1c) and its hydrogenated sister com-
pound (2c), a significantly reduced activity was observed in com-
parison to the less highly methylated compounds (1a, 1b, 2a and
2b). However, this trend did not show its counterpart in the highly
lipophilic acetylmethylhonokiol (3a) and diacetylhonokiol (3b),
IR spectra were taken as KBr pellets on a Perkin-Elmer 281 B
spectrometer. 1H and 13C NMR spectra were recorded on a Varian
400 MHz spectrometer (400 and 100 MHz, respectively) using deu-
terated chloroform as solvent with TMS as internal standard. NMR
which showed a considerable IC50 of 2.2 and 4.5 lM, respectively.
With the exception of the epoxide 5, which lost all its LTB4 forma-
tion inhibitory activity, the influence of the chemical character of
the side chain on the activity seems to be relatively low. This could
be seen from the comparable activities of, for example, 1a, 1b, 2a,
signals marked with asterisk ( ) may be interchanged. EI-MS were
*
recorded on a Hewlett Packard HP 6890 instrument fitted with
detector HP 7890. ESI-MS were measured in ESI positive and neg-
ative mode on a Thermo Finnigan LCQ Deca XPPLUS instrument. For
TM
2b and 4a–c, where the IC50 values range from 1.0–4.5 lM.
TLC analysis, precoated Si60 F254 plates (Merck, Darmstadt) were
used. Detection was done by UV/254 nm and spraying with molyb-
dato-phosphoric acid and subsequent heating. Compound mix-
tures were separated by column chromatography using
cyclohexane/ethyl acetate mixtures and on a HPLC preparative col-
With the exception of 5, which was inactive in all three test sys-
tems and 3b, which selectively inhibited 5-LOX product formation,
all tested derivatives were found to inhibit COX isoenzymes as well
as 5-LOX mediated LTB4 formation. From this finding it can be as-
sumed that these compounds have a potential as dual inhibitors of
the COX and the LOX pathway of the arachidonic acid cascade.
Compounds 4a and 4b were found to be most promising so far
due to their good inhibitory activities. However, for the interpreta-
tion of the present results it has to be considered that for assessing
COX inhibitory activity, a cell-free test system with purified en-
zymes has been used, while the influence on LTB4 formation has
been investigated in a cellular system. Therefore, in order to be
able to better evaluate the potential of these compounds as dual
inhibitors, it will be necessary to perform further tests, for exam-
ple, to investigate their activity in a cell-free assay on direct 5-
LOX inhibition. This will also help to understand the mechanism
of action underlying the observed LTB4 formation inhibitory
activity.
umn (Merck, Hibar Lichrospher RP-18, 10
an acetonitrile gradient in water.
l
m, 250 ꢀ 25 mm) using
Methylhonokiol (1b) as starting material was isolated from a
dichloromethane extract of M. grandiflora seeds collected in north-
ern Mississippi. A voucher (WS-4) is deposited at the Institute of
Pharmaceutical Sciences, department of Pharmacognosy, in Graz.
The crude methylhonokiol was obtained as an oil by chromato-
graphing the extract on silica gel (40–63 lm) using n-hexane–
ethyl acetate mixtures. Honokiol (1a) was obtained by demethyla-
tion via Grignard reaction of methylhonokiol,22 its identity was
demonstrated by comparison with literature data22 and a reference
sample. A general scheme (Fig. 2) gives an overview over the prin-
cipal syntheses carried out in this investigation.
The formation of LTs can be reduced by direct inhibition of 5-
LOX or by inhibition of a number of other mechanisms playing a
role in LT formation. Indirect inhibition of LT formation can for in-
stance be caused by an inhibition of PLA2 leading to a decreased
arachidonic acid release, by an inhibition of 5-LOX activating pro-
tein (FLAP), an enzyme facilitating the access of arachidonic acid to
5-LOX, by an inhibition of the translocation of 5-LOX from the
cytosol to the nuclear membrane or by an inhibition of 5-LOX
downstream enzymes such as LTA4 hydrolase.20,21 Direct 5-LOX
inhibitors are classified according to their molecular mode of
action as redox-active 5-LOX inhibitors, iron–ligand inhibitors,
non-redox-type inhibitors and compounds acting by so far unrec-
ognized mechanisms.20,21
3.2. 2,40-Dimethoxy-5,30-di-(2-propenyl)-biphenyl (1c)
Na (25 mg, 1.1 mmol) was dissolved in abs. EtOH (16 mL) under
argon and a solution of 1a (213 mg, 0.75 mmol) in abs. EtOH (4 mL)
was added. After stirring for 5 min, the solution was heated to re-
flux. (CH3)2SO4 (195 lL, 230 mg, 1.0 mmol) was slowly added to
the hot solution and refluxing continued for 5 h. After cooling to
room temperature, the solvent was evaporated and water
(20 mL) was added. The mixture was extracted with CH2Cl2 and
the combined organic phases were dried over Na2SO4. Compound
1c was obtained in 70% yield as a colorless oil. IR (KBr): 3446
(br), 2930, 1488, 1265, 1243, 1028, 913, 817 cmꢁ1; UV (15
lM, eth-

W. Schühly et al. / Bioorg. Med. Chem. 17 (2009) 4459–4465
4463
anol) kmax (log
e
) = 290.0 (3.87), 257.0 (4.13) 208.5 (4.60) nm; 1H
3.5. 2,40-Dimethoxy-5,30-dipropyl-biphenyl (2c)
NMR (CDCl3): d 3.43 (d, J = 7.2 Hz, 2H, H-1000), 3.47 (d, J = 7.2 Hz,
2H, H-100), 3.78 (s, 3H, 2-OCH3), 3.86 (s, 3H, 40-OCH3), 5.04 (d,
J = 10.2 Hz, 1H, H-3000), 5.05 (d, J = 10.2 Hz, 1H, H-300), 5.09 (d,
J = 18.0 Hz, 2H, H-300 and H-3000), 5.99 (m, 1H, H-200), 6.03 ( m, 1H,
H-2000), 6.90 (d, J = 8.4 Hz, 2H, H-3 and H-50), 7.09 (dd, J = 8.4 and
1.8 Hz, 1H, H-4), 7.12 (d, J = 1.8 Hz, 1H, H-6), 7.31 (d, J = 2.0 Hz,
1H, H-20), 7.37 (dd, J = 8.4 and 2.0 Hz, 1H, H-60); 13C NMR (CDCl3):
d 34.6 (C-1000), 39.7 (C-100), 55.7 (40-OCH3), 55.9 (2-OCH3), 110.1 (C-
50), 111.6 (C-3), 115.7 (C-300), 115.7 (C-3000), 128.1 (C-4), 128.5 (C-
30), 128.5 (C-60), 130.7 (C-10*), 131.0 (C-1*), 131.2 (C-20), 131.2
(C-6), 132.5 (C-5), 137.3 (C-2000), 137.9 (C-200), 155.1 (C-2), 156.7
(C-40); EI-MS 294 [M]+ (100), 238 (37), 223 (19), 165 (18). The com-
pound is yet incompletely described.22
Yield 85%, colorless oil. Compound 2c was obtained by hydroge-
nation of dimethylhonokiol (1c) with palladium on charcoal as cat-
alyst in EtOH as described for 2b. IR (KBr): 2957, 2929, 2870, 2834,
1608, 1508, 1492, 1463, 1267, 1242, 1138, 1031, 811 cmꢁ1; UV
(10 lM, ethanol) kmax (log e) = 290.0 (3.92), 257.5 (4.13), 207.0
1
(4.71) nm; H NMR (CDCl3): d 0.97 (t, J = 7.4 Hz, 3H, H-300), 0.99
(t, J = 7.4 Hz, 3H, H-3000), 1.66 (sext, J = 7.5 Hz, 4H, H-200 and H-
2000), 2.58 (dd, J = 8.4 and 6.7 Hz, 2H, H-100), 2.64 (dd, J = 8.4 and
6.7 Hz, 2H, H-1000), 3.79 (s, 3H, 2-OCH3), 3.85 (s, 3H, 40-OCH3),
6.89 (d, J = 8.4 Hz, 2H, H-3 and H-50), 7.09 (dd, J = 8.1 and 2.2 Hz,
1H, H-4), 7.13 (d, J = 2.2 Hz, 1H, H-6), 7.32 (d, J = 2.1 Hz, 1H, H-
20), 7.36 (dd, J = 8.1 and 2.2 Hz, 1H, H-60); 13C NMR (CDCl3): d
13.9 (C-300), 14.2 (C-3000), 23.0 (C-2000), 24.8 (C-200), 32.4 (C-1000),
37.2 (C-100), 55.3 (40-OCH3), 55.7 (2-OCH3), 109.8 (C-50*), 111.1
(C-3*), 127.6 (C-4), 127.8 (C-60), 130.4 (C-1**), 130.4 (C-30**),
130.6 (C-10**), 130.8 (C-6), 131.1 (C-20), 134.9 (C-5), 154.6 (C-2),
156.6 (C-40); EI-MS: 298 [M]+ (100), 269 (79), 255 (5), 211 (22),
195 (19), 165 (21), 152 (21), 1120 (40), 105 (31).
3.3. 5,30-Dipropyl-biphenyl-2,40-diol (tetrahydrohonokiol, 2a)
Compound 2a was obtained by demethylation of 2b. To a stirred
solution of 2a (100 mg, 0.352 mmol) under argon, a 10% solution of
BBr3 in CH2Cl2 (1.0 mL, 0.6 mmol) was added dropwise at rt. The
reaction mixture was poured into brine (6 mL) and stirred for
5 min. The organic layer was separated and the aqueous phase
re-extracted with CH2Cl2. The combined phases were dried over
Na2SO4 and purified by CC. 2a was obtained as crystals in 74%
yield. IR (KBr): 3357 (br, OH), 3019, 2927, 2870, 1609, 1498,
3.6. 2-Acetoxy-40-methoxy-5,30-di-(2-propenyl)-biphenyl (3a)
Compound 3a was obtained as colorless oil from 1b in pyridine
and acetic anhydride in a 87% yield.26 IR (KBr): 2908, 1763 (car-
1429, 1375, 1198, 1126, 823 cmꢁ1; UV (25
(log
) = 292.5 (4.12), 256.5 (4.30) 209.5 (4.76) nm; 1H NMR
l
M, ethanol) kmax
bonyl), 1507, 1487, 1246, 1215, 1190, 913 cmꢁ1; UV (9.9
anol) kmax (log e
l
M, eth-
e
) = 257.0 (4.40), 207.5 (4.88) nm. 1H NMR (CDCl3):
(CDCl3): d 0.95 (t, J = 7.3 Hz, 3H, H-300), 1.00 (t, J = 7.3 Hz, 3H, H-
3000), 1.65 (sext, J = 7.5 Hz, 2H, H-200), 1.68 (sext, J = 7.5 Hz, 2H, H-
2000), 2.55 (t, J = 7.5 Hz, 2H, H-100), 2.64 (t, J = 7.7 Hz, 2H, H-1000),
4.86 (br s, 2H, OH), 6.84 (d, J = 8.0 Hz, 1H, H-50), 6.90 (d,
J = 7.7 Hz, 1H, H-3), 7.03 (s, 1H, H-6), 7.05 (dd, J ꢂ 8 and 2.2 Hz,
1H, H-4), 7.17 (dd, J = 8.1 and 2.2 Hz, 1H, H-60), 7.23 (d, J = 2.2 Hz,
d 2.07 (s, 3H, acetyl-CH3), 3.40 (m, 4H, H-10 and H-1000), 3.82 (s, 3H,
40-OCH3), 5.03 (d, J = 10.2 Hz, 1H, H-3000), 5.06 (d, J = 18.0 Hz, 1H, H-
3000), 5.07 (d, J = 10.2 Hz, 1H, H-300), 5.10 (d, J = 18.0 Hz, 2H, H-300),
5.97 (m, 1H, H-2000), 6.07 (m, 1H, H-200), 6.86 (d, J = 8.4 Hz, 1H, H-
50), 7.01 (d, J = 8.4 Hz, 1H, H-3), 7.12 (d br, J = 8.1 Hz, 1H, H-4),
7.19 (s br, 1H, H-6), 7.21 (s br, 1H, H-20), 7.23 (d, J = 8.4 Hz, 1H,
13
13
1H, H-20); C NMR (CDCl3): d 13.8 (C-300), 14.0 (C-3000), 22.8 (C-
H-60); C NMR (CDCl3): d 20.9 (CH3-acetyl), 34.7 (C-1000), 40.0 (C-
2000), 24.8 (C-200), 32.0 (C-1000), 37.2 (C-100), 115.3 (C-3), 115.9 (C-
50), 127.6 (C-60), 127.7 (C-1), 128.5 (C-4), 129.5 (C-30 and C-10),
130.0 (C-6), 131.0 (C-20), 134.9 (C-5), 150.3 (C-2), 153.2 (C-40);
EI-MS: 270 [M]+ (57), 241 (100), 199 (34), 115 (12). 1H NMR values
without assignments are given in Kong et al.23
100), 55.5 (40-OCH3), 110.3 (C-50), 115.5 (C-3000), 116.2 (C-300), 122.7
(C-3), 127.7 (C-60), 128.0 (C-4), 128.4 (C-30), 129.6 (C-10), 130.6
(C-20), 130.8 (C-6), 134.4 (C-1), 136.8 (C-2000), 137.0 (C-200), 138.2
(C-5), 146.1 (C-2), 156.7 (C-40), 169.8 (CO acetyl); EI-MS: 322
[M]+ (24), 280 (100), 251 (15), 238 (18), 223 (17), 198 (20), 165
(19), 43 (70); ESI+ calcd for C21H22O3: [M+H]+ 323.16; found
323.14. Data incompletely given in El-Feraly and Li.22
3.4. 40-Methoxy-5,30-dipropyl-biphenyl-2ol (2b)
To a solution of 1b (200 mg, 0.714 mmol) in abs. EtOH (5 mL),
4 mg 10% Pd/activated charcoal was added and stirred for several
minutes. The mixture was filtered and another 8 mg of 10% Pd
on charcoal was added. The solution was stirred vigorously for
21 h at room temperature under H2 at atmospheric pressure. The
catalyst was filtered off through a Pasteur pipette filled with silica
gel and Celite. The absorbents were washed with EtOH (5 mL) and
the combined eluates were evaporated in vacuo to yield 2b in 97%
as colorless oil.24 IR (KBr): 3443 (br, OH), 2958, 2930, 2870, 1607,
3.7. 2,40-Diacetoxy-5,30-di-(2-propenyl)-biphenyl (3b)
Compound 3b was obtained as colorless oil obtained from 1a
and acetic anhydride in pyridine in a 95% yield.27 IR (KBr): 2978,
2923, 1763 (carbonyl), 1639, 1484, 1369, 1210, 1192, 1011,
914 cmꢁ1
; UV (1.6 mM, EtOH) k (log e) = 243.0 (4.93), 207.5
(5.54) nm; 1H NMR (CDCl3): d 2.08 (s, 3H, acetyl-CH3), 2.32 (s,
3H, acetyl-CH3), 3.33 (d, J = 6.6 Hz, 2H, H-1000), 3.42 (d, J = 6.6 Hz,
2H, H-100), 5.07 (m, 4H, H-300 and H-3000), 5.92 (m, 1H, H-2000), 5.95
(m, 1H, H-200), 7.04 (d, J = 8.1 Hz, 1H, H-3), 7.08 (d, J = 8.1 Hz, 1H,
H-50), 7.18 (dd, J = 8.2 and 2.2 Hz, 1H, H-4), 7.22 (d, J = 2.2 Hz, 1H,
H-6), 7.27 (dd, J ꢂ 8 and 2.2 Hz, 1H, H-60), 7.28 (s due to overlap,
1H, H-20); 13C NMR (CDCl3): d 20.9 (CH3-acetyl), 20.9 (CH3-acetyl),
34.7 (C-1000), 39.6 (C-100), 116.3 (C-3000), 116.4 (C-300), 122.3 (C-50),
122.7 (C-3), 127.9 (C-60), 128.7 (C-4), 130.9 (C-20*), 130.9 (C-6*),
131.7 (C-30), 133.8 (C-1), 135.6 (C-10), 135.7 (C-2000), 136.9 (C-200),
138.2 (C-5), 146.0 (C-2), 148.3 (C-40), 169.3 (CO acetyl), 169.6
(CO acetyl); EI-MS: 350 [M]+ (8), 308 (32), 266 (100), 224 (12).
1492, 1245, 1029, 818 cmꢁ1
(log
) = 289.0 (3.85), 253.5 (4.06), 231.5 (4.06) nm; 1H NMR
;
UV (66 lM, CH2Cl2) kmax
e
(CDCl3): d 0.96 (t, J = 7.7 Hz, 3H, H-300*), 0.98 (t, J = 7.7 Hz, 3H, H-
3000*), 1.66 (sext, J = 7.7 Hz, 4H, H-2000 and H-200), 2.55 (t, J = 8.0 Hz,
2H, H-100), 2.65 (t, J = 8.0 Hz, 2H, H-1000), 3.86 (s, 3H, OCH3), 5.19
(br s, OH), 6.90 (d, J = 8.4 Hz, 1H, H-3), 6.95 (d, J = 8.1 Hz, 1H, H-
50), 7.04 (s, 1H, H-6), 7.05 (dd, J ꢂ8 and 2.1 Hz, 1H, H-4), 7.24 (d,
J = 2.1 Hz, 1H, H-20), 7.27 (dd, J = 8.2 and 2.1 Hz, 1H, H-60); 13C
NMR (CDCl3): d 13.8 (C-300*), 14.1 (C-3000*), 22.9 (C-2000), 24.8 (C-
200), 32.3 (C-1000), 37.2 (C-100), 55.4 (40-OCH3), 110.8 (C-50), 115.3
(C-3), 127.3 (C-60), 127.7 (C-1), 128.5 (C-4), 129.0 (C-10), 130.0
(C-6), 130.6 (C-20), 132.2 (C-30), 134.8 (C-5), 150.5 (C-2), 157.2
(C-40); EI-MS: 284 [M]+ (84), 255 (100), 213 (42), 181 (20), 113
(32), 98 (27). 1H NMR values without further assignments are gi-
ven in Rao and Davis.25
3.8. General procedure for isomerization of side chain in 4a and
4b
To a solution of 1b (45 mg, 0.17 mmol) in abs. THF (10 mL) un-
der argon, KOtBu (57 mg, 0.51 mmol) was added and the solution

4464
W. Schühly et al. / Bioorg. Med. Chem. 17 (2009) 4459–4465
was refluxed vigorously for 21 h. After cooling to rt, a saturated
solution of NH4Cl (10 mL) was added. The reaction mixture was re-
duced to 10 mL and extracted with CHCl3 (3 ꢀ 10 mL). The com-
bined organic layers were washed with brine (20 mL), water
(2 ꢀ 10 mL) and dried over MgSO4.27 A mixture of 4a and 4b was
obtained in 94% yield. 4a and 4b were separated by preparative
HPLC.
130.1 (C-6), 132.2 (C-5), 137.7 (C-200), 150.7 (C-2), 152.1 (C-40);
ESI+ calcd for C18H18O2: [M+H]+ 267.13; found 267.20.
3.12. 40-Methoxy-5,30-bis-oxiranylmethyl-biphenyl-2-ol (5)
Compound 5 was obtained as a dark yellow solid in a diastereo-
meric mixture from 1b by oxidation with 3-chloroperoxybenzoic
acid (MCPBA) in abs benzene in 87% yield (modified after
O’Brian).28 IR (KBr): 3423, 2920, 2850, 1733, 1608, 1492, 1245,
3.9. 40-Methoxy-30-(E)-propenyl)-5-(2-propenyl)-biphenyl-2-ol
(4a)
1027, 819 752 cmꢁ1
; UV (14 lM, EtOH) kmax (log e) = 291.5
(3.79), 256.5 (3.98), 206.5 (4.58) nm; ESI-MS: 322 [M]+ (24), 280
(100), 251 (15), 238 (18), 223 (17), 198 (20), 165 (19), 43 (70); ESIꢁ
calcd for C19H20O4: [MꢁH]ꢁ 311.12; found 311.23.
Fine crystals, mp 81 °C; IR (KBr): 3448 (br, OH), 2934, 2910,
2836, 1638, 1604, 1490, 1246, 1180, 1121, 819 cmꢁ1; UV (10
lM,
CH2Cl2) kmax (log
e) = 298.0 (3.95), 244.5 (4.38), 222.5 (4.48),
205.5 (4.43) nm; 1H NMR (CDCl3): d 1.91 (dd, J = 6.6 and 1.1 Hz,
3H, H-3000), 3.35 (d, J = 6.6 Hz, 2H, H-100), 3.88 (s, 3H, OCH3), 5.05
(d, J = 10.5 Hz, 1H, H-300), 5.08 (d, J = 16.8, 1H, H-300), 5.17 (s br,
1H, 2-OH), 5.97 (ddt, J = 16.8, 10.3 and 6.6 Hz, 1H, H-200), 6.27
(dq, J = 15.8 and 6.6 Hz, 1H, H-2000), 6.74 (d, J = 15.8 Hz, 1H, H-1000),
6.90 (d, J = 8.1 Hz, 1H, H-3), 6.94 (d, J = 8.4 Hz, 1H, H-50), 7.05 (s,
1H, H-6), 7.06 (dd, J ꢂ 8 and 1.8 Hz, 1H, H-4), 7.25 (dd, J = 8.4 and
2.2 Hz, 1H, H-60), 7.47 (s, 1H, H-20); 13C NMR (CDCl3): d 18.9 (C-
3000), 46.7 (C-100), 55.6 (OCH3), 111.4 (C-50), 115.6 (C-3 and C-300),
125.2 (C-1000), 127.1 (C-20), 127.5 (C-2000), 127.8 (C-1*), 127.9 (C-
10*), 128.5 (C-60), 128.8 (C-4), 129.2 (C-30), 130.1 (C-6), 132.1 (C-
5), 137.8 (C-200), 150.8 (C-2), 155.9(C-40); EI-MS: m/z 280 [M]+
(100), 253 (19), 224 (17), 165 (15); ESI+ calcd for C19H20O2:
[M+H]+ 281.15; found 281.14.
3.13. In vitro-assays
COX-1 and COX-2 inhibition assays were performed in a 96-
well-plate format with purified prostaglandin H synthase (PGHS-
1) from ram seminal vesicles for COX-1 and purified PGHS-2 from
sheep placental cotyledons for COX-2 (both Cayman Chemical
Company, Ann Arbor, USA) as previously described.29,30 The con-
centration of PGE2, the main arachidonic acid metabolite in the
reaction, was determined by a competitive PGE2 EIA kit (Assay De-
signs Inc., Ann Arbor, MI, USA). Indomethacin (ICN, Aurora, USA;
IC50 COX-1 0.9
lM), a dual COX-1/2 inhibitor was used as positive
control in COX-1 experiments and NS-398, a selective COX-2 inhib-
itor, (Cayman Chemical Company, IC50 COX-2 2.6
positive control.
lM) was used as
The bioassay for inhibition of 5-LOX mediated LTB4 formation
was carried out in a 96-well-plate format with stimulated human
polymorphonuclear leukocytes as described by Adams et al.31 with
3.10. 40-Methoxy-30-(Z)-propenyl)-5-(2-propenyl)-biphenyl-2-
ol (4b)
slight modifications.20 Zileuton (Sequoia, Oxford, UK; IC50 5.0
was used as positive control.
lM)
Fine crystals, IR (KBr): 3529 (br, OH), 3449 (br), 3021, 2975,
2910, 2836, 1603, 1489, 1249, 1181, 1117, 819 cmꢁ1; UV (10
lM,
Test samples were dissolved in absolute ethanol. Pure com-
pounds were tested at a final concentration of 8 M. Samples were
EtOH) kmax (log
e
) = 297.0 (4.05), 221.5 (4.59) nm; 1H NMR (CDCl3):
l
d 1.84 (dd, J = 7.1 and 1.6 Hz, 3H, H-3000), 3.35 (d, J = 6.7 Hz, 2H, H-
100), 3.88 (s, 3H, OCH3), 5.06 (d, J = 10 Hz, 1H, H-300), 5.09 (dq, J = 17
and 1.5 Hz, 1H, H-300), 5.14 (s br, 2-OH), 5.89 (dq, J = 11.5 and
7.0 Hz, 1H, H-2000), 5.96 (dq, J = 16.8, 10.1 and 6.6 Hz, 1H, H-200),
6.56 (d, J = 11.6 Hz, 1H, H-1000), 6.91 (d, J = 7.9 Hz, 1H, H-3), 6.98
(d J = 8.4 Hz, 1H, H-50), 7.05 (s, 1H, H-6), 7.06 (dd, J ꢂ 8 and
2.0 Hz, 1H, H-4), 7.32 (dd, J = 8.3 and 2.2 Hz, 1H, H-60), 7.35 (d,
tested in at least 3 independent experiments run in duplicate. Re-
sults are given as means S.D. The samples possessing inhibition
values higher than 50% at this 8 lM were then selected for the
determination of IC50. For IC50 determination, active samples were
tested in at least 3 concentrations in at least 3 independent exper-
iments, each time in duplicate. Calculation of IC50 values was per-
formed by semilogarithmic presentation of dose vs. activity and
logarithmic regression analysis.
13
J = 2.0 Hz, 1H, H-20); C NMR (CDCl3): d 14.7 (C-3000), 39.4 (C-100),
55.6 (OCH3), 111.0 (C-50), 115.6 (C-3* and C-300*), 124.7 (C-1000),
127.1 (C-30), 127.6 (C-2000), 127.8 (C-1), 128.5 (C-60), 128.5 (C-10),
128.8 (C-4), 130.2 (C-6), 130.8 (C-20), 132.2 (C-5), 137.7 (C-200),
150.8 (C-2), 156.7 (C-40); ESI+ calcd for C19H20O2: [M+H]+ 281.15;
found 281.38.
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Compound 4c was obtained as crystals from 4a by demethyla-
tion using Grignard reagent22 in 16% yield. IR (KBr): 3300 (OH),
2912, 1637, 1609, 1491, 1433, 1219, 825 cmꢁ1; UV (10
lM, EtOH)
kmax (log
e) = 299.0 (4.00), 244.5 (4.41), 222 (4.52), 204.0 (4.51)
1
nm; H NMR (CDCl3): d 1.92 (dd, J = 6.6 and 1.5 Hz, 3H, H-3000),
3.34 (d, J = 6.6 Hz, 2H, H-100), 5.05 (d, J = 10.3 Hz, 1H, H-300), 5.08
(dq, J = 16.8 and 1.5 Hz, 1H, H-300), 5.12 (s br, 1H, 2-OH), 5.19 (s
br, 1H, 40-OH), 5.96 (ddt, J = 16.9, 10.3 and 6.6 Hz, 1H, H-200), 6.25
(dq, J = 15.8 and 6.6 Hz, 1H, H-2000), 6.59 (dq, J = 15.8 and 1.5 Hz,
1H, H-1000), 6.87 (d, J = 8.4 Hz, 1H, H-50), 6.90 (d, J = 8.1 Hz, 1H, H-
3), 7.02 (d, J = 1.8 Hz, 1H, H-6), 7.05 (dd, J = 8.1 and 2.2 Hz, 1H, H-
4), 7.16 (dd, J = 8.0 and 2.2 Hz, 1H, H-60), 7.37 (d, J = 2.2 Hz, 1H,
13
H-20); C NMR (CDCl3): d 18.9 (C-3000), 39.4 (C-100), 115.6 (C-300*
and C-3*), 116.5 (C-50), 124.8 (C-1000), 125.9 (C-30), 127.7 (C-1),
128.0 (C-20), 128.6 (C-60), 128.9 (C-4), 129.2 (C-2000), 129.5 (C-10),
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