Structural analogues of the natural products magnolol and honokiol as potent allosteric potentiators of GABAA receptors

Article abstract of DOI:10.1016/j.bmc.2014.10.027

Biphenylic compounds related to the natural products magnolol and 4′-O-methylhonokiol were synthesized, evaluated and optimized as positive allosteric modulators (PAMs) of GABA_A receptors. The most efficacious compounds were the magnolol analog 5-ethyl-5′-hexylbiphenyl-2,2′-diol (45) and the honokiol analogs 4′-methoxy-5-propylbiphenyl-2-ol (61), 5-butyl-4′-methoxybiphenyl-2-ol (62) and 5-hexyl-4′-methoxybiphenyl-2-ol (64), which showed a most powerful potentiation of GABA-induced currents (up to 20-fold at a GABA concentration of 3 μM). They were found not to interfere with the allosteric sites occupied by known allosteric modulators, such as benzodiazepines and N-arachidonoylglycerol. These new PAMs will be useful as pharmacological tools and may have therapeutic potential for mono-therapy, or in combination, for example, with GABA_A receptor agonists.

Full text of DOI:10.1016/j.bmc.2014.10.027

Bioorganic & Medicinal Chemistry 22 (2014) 6908–6917
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structural analogues of the natural products magnolol and honokiol
as potent allosteric potentiators of GABAA receptors
Alexander Fuchs a, Roland Baur b, Clara Schoeder a, Erwin Sigel b, Christa E. Müller a,b,
⇑
a PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany
b Institute of Biochemistry and Molecular Medicine, University of Bern, Bühlstrasse 28, 3012 Bern, Switzerland
a r t i c l e i n f o
a b s t r a c t
Biphenylic compounds related to the natural products magnolol and 40-O-methylhonokiol were synthe-
sized, evaluated and optimized as positive allosteric modulators (PAMs) of GABAA receptors. The most
efficacious compounds were the magnolol analog 5-ethyl-50-hexylbiphenyl-2,20-diol (45) and the honok-
iol analogs 40-methoxy-5-propylbiphenyl-2-ol (61), 5-butyl-40-methoxybiphenyl-2-ol (62) and 5-hexyl-
40-methoxybiphenyl-2-ol (64), which showed a most powerful potentiation of GABA-induced currents
Article history:
Received 25 August 2014
Revised 13 October 2014
Accepted 15 October 2014
Available online 22 October 2014
(up to 20-fold at a GABA concentration of 3 lM). They were found not to interfere with the allosteric sites
Keywords:
occupied by known allosteric modulators, such as benzodiazepines and N-arachidonoylglycerol. These
new PAMs will be useful as pharmacological tools and may have therapeutic potential for mono-therapy,
or in combination, for example, with GABAA receptor agonists.
Allosteric modulation
GABAA receptor
Honokiol
Ó 2014 Elsevier Ltd. All rights reserved.
Ion channel
Magnolol
Medicinal plant
Natural product
Positive allosteric modulator (PAM)
Synthesis
1. Introduction
expressed in the entire central nervous system (CNS), while other
isoforms have a more restricted expression pattern. The
for example, is only expressed on granule cells of the cerebellum,
and the
subunit is found on retinal cells.7 Outside of the CNS
GABAA receptors are also expressed, for example, on cells of the
immune system, in liver, on smooth muscle and in the respiratory
system.8–10
a6 subunit,
In the central nervous system of mammals, the neurotransmit-
ter
c-aminobutyric acid (GABA, 1, Fig. 1) is the main inhibitory
q
agent.1 It activates two different classes of membrane receptors:
GABAA and GABAB. The GABAA receptor is an ionotropic receptor
which controls the influx of chloride ions. It has been shown to
be modulated through different allosteric binding sites for benzo-
diazepines, barbiturates and ethanol.2,3 In contrast, the GABAB
receptor is a G protein-coupled receptor, which controls potassium
and calcium channels via G protein activation.2–4 Especially the
GABAA receptor has been an important drug target since many
years due to the anxiolytic, antidepressant, muscle relaxant and
anti-convulsive effects that it conveys.5 The GABAA receptors con-
sist of five subunits of different composition depending on the
Recent findings showed that GABAA receptors contain binding
sites for the endocannabinoid 2-arachidonoylglycerol (2-AG, 2)
and the endocannabinoid metabolite N-arachidonoylglycine
(NAGLY, 3).11,12 Moreover, a new study reported that the natural
products honokiol (4) and 40-O-methylhonokiol (5) potentiate
GABAA currents, 5 being superior to 4 (for structures see Fig. 1).13
40-O-Methylhonokiol is also known to be a ligand of the cannabi-
noid receptor (CB) 2 where it acts as a partial agonist.14
The neolignans 4 and 5 as well as magnolol (6) are constituents of
plants of the Magnolia family, and are found, for example, in
Magnolia officinalis and Magnolia grandiflora.15,16 Preparations of
Magnolia officinalis are used in traditional Chinese medicine and
Japanese Kampo medicine due to their antidepressant and anxio-
lytic effects.15,17 Furthermore, magnolol, its metabolite tetrahydro-
magnolol and their synthetic analogs have been shown to interact
with CB receptors, where they act as potent partial agonists, with
a preference for CB2.14,18
receptor subtype.6 The main subunits of GABAA
a1, b2 and c2 are
Abbreviations: iNOS, inducible NO-synthase; PAM, positive allosteric modulator;
Ro15-1788, flumazenil, ethyl 8-fluoro-5-methyl-6-oxo-5,6-dihydro-4H-benzo[f]
imidazo[1,5-a][1,4]diazepine-3-carboxylate.
Corresponding author at present address: Universität Bonn, Pharmazeutisches
Institut, An der Immenburg 4, D-53121 Bonn, Germany. Tel.: +49 228 732301; fax:
+49 228 732567.
⇑
E-mail address: christa.mueller@uni-bonn.de (C.E. Müller).
http://dx.doi.org/10.1016/j.bmc.2014.10.027
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

A. Fuchs et al. / Bioorg. Med. Chem. 22 (2014) 6908–6917
6909
O
Methylation by a phase transfer catalysis method with methyl
iodide as an alkylating agent in mixture of water and
a
R
HOOC
NH2
dichloromethane in the presence of sodium hydroxide and ben-
zyl-tri-n-butylammonium bromide led to the 2-bromo-1-meth-
oxy-4-alkylbenzenes 24–26.20 Boronic acid derivatives (28–32)
were obtained from the 2-bromo-4-alkylphenols 15–23 by treat-
ment with n-butyllithium and trimethyl borate yielding 28–32
after acid hydrolysis in moderate yields.21 The final reaction step
yielded products 37–69 via Suzuki cross-coupling (Scheme 2).22
The boronic acids 33–36 were commercially available. The final
products were purified by flash chromatography, or preparative
HPLC, respectively. The structures were confirmed by 1H and 13C
NMR spectra and by HPLC coupled to electrospray ionization mass
spectrometry (LC–ESI-MS). The purity of the compounds was
determined by (LC–ESI-MS/UV) and was in all cases greater than
>95% (for details see Section 4 and Supporting information).
CH3
1 GABA
OH
2 R
2-Arachidonoylglycerol
=
O
OH
OH
3 R =
N-Arachidonoylglycine
N
H
O
R
O
OH
OH
OH
4 R
5 R
Honokiol
4'-O-Methylhonokiol
= H
= CH3
6 Magnolol
2.2. Biological evaluation
Figure 1. Structures of
c-aminobutyric acid (GABA, 1) and (potential) positive
allosteric modulators of GABAA receptors (2–6).
2.2.1. Positive allosteric modulation of GABAA receptors
The compounds were initially screened at recombinant
GABAA receptors expressed in Xenopus oocytes. We determined
the potentiation of the GABA-induced current exerted by 3 M of
test compound. The agonist GABA (0.3 M; EC50 about 30 M;
the applied concentration corresponds to the EC1 at which 1% of
the maximal effect of GABA is observed) was applied several times
until a constant current response was observed. The same concen-
tration of GABA was then co-applied with the test compound.
Results are shown in Figure 2 and Table 1.
a1b2c2
In the present study we synthesized analogs of magnolol and 40-
O-methylhonokiol with the goal to study their structure–activity
relationships (SAR) as allosteric modulators of GABAA receptors
and to optimize them with regard to potency and selectivity.
l
l
l
2. Results and discussion
2.1. Chemistry
All of the tested biphenylic compounds showed a current
potentiation in the Xenopus oocytes system. In Table 1 the com-
pounds’ structures and their percent increase in GABA-induced
current are presented. In the first set of compounds — a series of
magnolol analogs — compound 45, with an ethyl and a hexyl res-
idue, was the most potent one. The weak effects of compounds
37, 38, 39, and 40 indicated that, for a high potency, an alkyl resi-
due is required on both aromatic rings of the molecule, and that a
methyl group on the one phenyl ring — in combination with a
longer alkyl residue on the second ring — was too small. However,
GABA-induced current potentiation also decreased when the mol-
ecule was too bulky. This was the case for compounds that bear a
propyl or butyl residue on one phenyl ring combined with a pentyl
or longer alkyl chain on the other phenyl residue (compare 46 to
51). Further modification of the magnolol analogs by methylation
of one of the phenolic groups resulted in a decreased effect (com-
pare 52 to 58).
In the present study 33 biphenylic compounds have been inves-
tigated, 15 of which are new compounds. The synthesis of 37–51,
55, 57 and 58 was described previously,14 while the preparation
and properties of 52–54, 56, 59–69 are described herein for the
first time.
All magnolol and 40-O-methylhonokiol analogs were synthe-
sized by a previously published procedure,14 starting from com-
mercially available 4-alkylphenols (7–14) as shown in Scheme 1.
The bromination of 4-alkylphenols via electrophilic aromatic sub-
stitution was carried out with elementary bromine in chloroform
in the presence of sodium hydrogencarbonate to provide the
2-bromo-4-alkylphenols 16–23.19 Intermediates 15 and 27 are
commercially available as well.
In the second set of compounds — the 40-O-methylhonokiol
analogs — effects of structural modifications were even much more
pronounced. Compound 64, containing a hexyl residue and a meth-
oxy group, was the most potent positive allosteric modulator
(PAM) of GABA-induced currents in the present series. Again, the
smallest compound (59), with a methyl residue and a methoxy
group, as well as the most bulky compounds (67, 68, 69), with a
hexyl residue and an ethoxy, propoxy, or isopropoxy group, respec-
tively, were the least potent ones within this set of compounds.
As a next step, concentration-dependent effects were deter-
mined for the most efficacious potentiators of GABA-induced
GABAA receptor currents, namely for the magnolol-derived
5-ethyl-50-hexylbiphenyl-2,20-diol (45), and the honokiol analogs
40-methoxy-5-propylbiphenyl-2-ol (61), 5-butyl-40-methoxybi-
phenyl-2-ol (62), and 5-hexyl-40-methoxybiphenyl-2-ol (64) (see
Fig. 3).
OH OH
27 R = H
B
28 R = methyl
OH
29 R
= ethyl
30 R = propyl
31 R = butyl
32 R = pentyl
b
c
OH
OH
R
Br
a
OMe
Br
R
R
15 R = H
7
8
9
R = methyl
R
R
16 R = methyl
= ethyl
= propyl
17 R
= ethyl
R
18 R = propyl
19 R = butyl
20 R = pentyl
21 R = hexyl
10 R = butyl
11 R = pentyl
12 R = hexyl
24 R = propyl
25 R = pentyl
26 R = hexyl
13 R
14 R
= heptyl
= octyl
22 R
= heptyl
23 R = octyl
The compounds clearly showed concentration-dependent
effects with extremely high potentiation by >1000% at the highest
Scheme 1. Reagents and conditions: Syntheses of intermediates. Reagents and
conditions: (a) Br2, NaHCO3, CHCl3, 0 °C, 80%; (b) one pot three steps, (1)
n-butyllithium, Et2O, ꢀ78 °C; (2) B(OCH3)3, ꢀ78 °C to rt; (3) HCl, 60%; (c) CH3I,
NaOH, benzyl-tri-n-butylammonium bromide, CH2Cl2/H2O (1:1), 12 h, rt, 90%.
tested concentration of 10
lM. The most potent compound, 61,
increased the GABA-induced current by 5000% at 10
lM. Higher

6910
A. Fuchs et al. / Bioorg. Med. Chem. 22 (2014) 6908–6917
R2
R3
OH OH
R3
O
O
B
Br
a
OH
OH
R2
R1
R1
15-26
27-32
37-58
for substituents
R2
see TABLE 1
R2
O
OH
O
Br
a
HO
R1
B
HO
OH
R1
7-14
33-36
59-69
Scheme 2. Reagents and conditions: Synthesis of GABAA modulators series I (magnolol analogs 37–58) and II (honokiol analogs 59–69). Reagents and conditions: (a)
Pd(PPh3)4, Na2CO3, toluene, EtOH, H2O, 100 °C, 18 h.
2400
2200
2000
1800
1600
1400
1200
1000
800
600
400
200
0
37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69
compound
Figure 2. Screening of test compounds at recombinant
a1b2
c2 GABAA receptors. The receptors were expressed in Xenopus oocytes and the current potentiation by 3 lM of
test compound was determined. The agonist GABA (0.3
l
M) was applied several times until a constant current response was obtained. The same concentration of GABA was
then co-applied with the test compound. Each bar represents a single experiment. For compounds 41 (4 independent experiments), 45 and 64 (11 independent experiments
each) mean values SD are shown).
concentrations could not be applied due to limited solubility of the
rather lipophilic compounds. Only compound 64 appeared to reach
the upper plateau of the curve with a maximal current potentiation
6000% using a GABA concentration of 0.5 l
M.13 Higher concentra-
tions led to a reduction leading to a bell-shaped concentration–
response curve.
in the range of nearly 3000% and an EC50 value of 1.8
mated. For compounds 45, 61 and 62 a concentration of 10
not sufficient to induce a maximal effect and to reach the upper
plateau of the curve. At 10 M the current potentiation for these
compounds were in the range of 1000% (45), 5000% (61) and
4000% (62). A much smaller degree of potentiation may be biolog-
ically relevant already. A potentiation of 100% was reached below
l
M was esti-
lM was
2.2.2. Direct activation of GABAA receptors
We subsequently investigated whether the most potent posi-
tive allosteric modulators additionally acted as agonists in their
own right (see Fig. 4).
The investigated compounds 45, 61, 62 and 64 were found to be
very weak direct agonists of GABAA receptors. All of them induced
a concentration-dependent increase in current amplitude. A con-
l
0.7 lM in each case. N-Arachidonoylglycine was previously
reported to exhibit concentration-dependent current potentiation
centration of 10 lM was not sufficient to induce a maximal effect.
showing a maximal current potentiation in the range of 400% at
At that concentration the compounds elicited currents amounting
to about 2–6% of maximal effect achieved with GABA (=100%).
Nevertheless their agonistic effect was higher than that of the par-
10
EC50 value between 1 and 10
maximal current potentiation of 138% with an EC50 value of
2.1
M was determined.23 The natural product 40-O-methylhonok-
iol was reported to strongly potentiate GABAA receptor currents. At
a concentration of 10 M the potentiation was in the range of
l
M (applying a GABA concentration of 1
lM), with an estimated
l
M.12 For 2-Arachidonoylglycerol a
ent compound 40-O-methylhonokiol (0.21% at 3 M).13
l
l
Interestingly, the rank order of efficacy for direct GABAA recep-
tor activation was similar to that observed for potentiation of
GABA effects: 61 P 62 > 64 > 45. This indicates that the identified
l

A. Fuchs et al. / Bioorg. Med. Chem. 22 (2014) 6908–6917
6911
Table 1
Activity of biphenylic compounds related to the natural products magnolol and honokiol as positive allosteric modulators of GABAA receptors
Compound
R1
R2
R3
GABAA activitya
Previously identified positive allosteric modulators
2-arachidonolyglycerol23
N-arachidonoylglycine12
Magnololb,24
+
++
++
+
Honokiol13
Methylhonokiol13
+++++
Series I: magnolol analogs
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
H
H
Pentyl
Hexyl
Butyl
Pentyl
Hexyl
Propyl
Butyl
Pentyl
Hexyl
Pentyl
Hexyl
Heptyl
Octyl
Pentyl
Hexyl
Pentyl
Hexyl
Pentyl
Hexyl
Ethyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
++
+++
++
+
+++
++
+
Methyl
Methyl
Methyl
Ethyl
Ethyl
Ethyl
Ethyl
Propyl
Propyl
Propyl
Propyl
Butyl
Butyl
Ethyl
Ethyl
Propyl
Propyl
Pentyl
Pentyl
Hexyl
+
++++
++
++
(+)
(+)
++
+
+++
++
(+)
(+)
+
Propyl
Propyl
+
(+)
Series II: 40-O-Methylhonokiol analogs
59
60
61
62
63
64
65
66
67
68
69
Methyl
Ethyl
Propyl
Butyl
Pentyl
Hexyl
Heptyl
Octyl
Hexyl
Hexyl
Hexyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Ethyl
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
(+)
+
+++++
+++++
+++
+++++
+++
(+)
+
(+)
(+)
Propyl
Isopropyl
a
allosteric potentiation at 3
Magnolol concentration 30–60 M.
l
M concentration of test compound: 0–100% (+), 100–300% +, 300–500% ++, 500–700% +++, 700–900% ++++, >1000% +++++.
l
b
positive allosteric modulators are also weak allosteric agonists and
may therefore be characterized as ago-allosteric modulators.25,26
such as loreclezole and 2-arachidonoylglycerol were found to be
no longer active at those mutated receptors, and we wanted to
investigate whether the new magnolol and honokiol analogs
shared the same binding site.11,23,27,28
2.2.3. Search for the allosteric binding site
Two of the most potent positive allosteric modulators at GABAA
receptors of the present series, the magnolol analog 45 and the
honokiol analog 64, were investigated at GABAA receptors with dif-
To evaluate the subunit specificity of test compounds 45 and 64,
first the
other -subunits. Then, the b-subunit of the
replaced by b1- and b3-subunits. Finally, the -subunit was omitted
or replaced by a d-subunit. For compound 64 a change in the
-subunit showed some alteration in its current potentiating
properties. This indicates that the -subunit has an effect on the
binding of the compound. A change of the b2-subunit to b1 showed
a-subunit of the
a
1b2c2 isoform was replaced by several
a
a1b2c2 isoform was
ferent subunit composition, including
4b2c2 5b2c2 6b2c2 1b1c2 1b3c2
Moreover, they were tested at receptors containing point muta-
tions: 1b2N265Sc2 1b2V436Tc2 (Fig. 5). The mutations were
selected because other allosteric potentiators of GABAA receptors
a1b2c2
,
a
2b2c2
,
a3b2c2
,
c
a
,
a
,
a
,
a
,
a
,
a
1b2,
a1b3d (see Fig. 5).
a
a
,
a
a

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A. Fuchs et al. / Bioorg. Med. Chem. 22 (2014) 6908–6917
(a)
(a)
7
(b)
45
61
62
64
6
5
4
3
2
1
6000
5500
5000
4500
4000
3500
3000
2500
2000
1500
1000
500
(b)
45
61
62
64
0
10-7
10-6
10-5
[compound], (M)
Figure 4. Direct activation of GABAA receptors by selected test compounds. Current
amplitudes are expressed in % of the maximal current induced by GABA (=100%) in
the corresponding cell. (a) Original current traces of an experiment with 64. (b)
Individual experiments for 45, 61, 62 and 64; data points are means SD from three
to four separate experiments.
Finally, we tested whether the magnolol and honokiol analogs
act via the allosteric benzodiazepine binding site. Thus, the current
0
was potentiated by 3
and in the presence of the benzodiazepine antagonist Ro15-1788
(1 M). Relative potentiation amounted to 81 5% (45) and
lM of 45 or 64, respectively, in the absence
-500
10-9
10-8
10-7
10-6
10-5
10-4
[compound], (M)
l
85 12% (64), respectively (n = 3) in the presence of the benzodiaz-
epine antagonist as compared to 100% in its absence. This minor
effect indicates that both PAMs do not act interact with the
benzodiazepine site (Fig. 6).
Figure 3. Concentration dependence of GABA-induced current potentiation at
recombinant 1b2 2 GABAA receptors. The GABA concentration was 0.3 M. (a)
Original current traces of an experiment with 64; numbers indicate applied
concentrations of 64 (0.003–10 M). (b) Averaged data SD for compounds 45, 61,
a
c
l
l
62 and 64 determined in four to five separate experiments.
2.2.4. Interaction with other targets
Recently several other targets have been reported to be
addressed by the Magnolia constituents magnolol, honokiol and
40-O-methylhonokiol. A study investigated the effects of 40-O-meth-
ylhonokiol (1 mg/kg) on lipopolysaccharide-induced neuroinflam-
mation, in which it was found to reduce the expression of iNOS
and COX2 and the formation of b-amyloid plaques in mouse CNS.
In cultured astrocytes it was shown that these effects may be
a significant decrease in current potentiation, whereas a change to
b3 resulted in a small increase in current potentiation as compared
to the
important role in compound binding. When the
omitted or replaced by a d-subunit this had almost no effect, indi-
cating that the -subunit is not important for interaction with the
a
1b2c2 isoform. This indicates that the b-subunit plays an
c
-subunit was
c
allosteric modulator. Both investigated point mutations resulted in
a decrease, but not a complete loss of current potentiation. These
findings indicate that the honokiol analog 61 may not share its
binding site with 2-arachidonoylglycerol and loreclezole, although
its activity is influenced and modulated by these sites. For the
magnolol analog 45 the subunit specificity was found to be similar
to that of honokiol analog 64. The current potentiation was
mediated via inhibitory effects on NF-
ylhonokiol was shown to be an inhibitor of cyclooxygenases (COX) 1
and 2 with an IC50 of 2.4 M for COX1 and 0.062
M for COX2.30
j
B.29 In a further study meth-
l
l
40-O-Methylhonokiol (1.5 mg/kg) as well as an ethanolic extract of
Magnolia officinalis (5 mg/kg) were shown to reduce memory
impairment effects of scopolamine via acetylcholine esterase inhi-
bition.31 Moreover, the biphenylic natural products were found to
interact with cannabinoid (CB) receptors. Magnolol acts as partial
influenced by a change of the a-subunit, but this effect was much
smaller than that observed when changing the b-subunit. The
receptor isoform in which b2 was replaced by b1 displayed the larg-
est decrease in current potentiation by both compounds, 45 as well
agonist at both CB receptor subtypes (EC50: CB1 18.3
3.28 M) whereas honokiol is a weak agonist at CB1, and an antag-
onist at CB2 receptors.18 40-O-Methylhonokiol was found to show
lM, CB2
l
as 61. The c-subunit can be considered as less important for 45,
high affinity (0.043 lM) for CB2 receptors where it behaved as a par-
and likewise for 61. A decrease in current potentiation observed
with the isoforms containing point mutations indicates that the
binding of 45 is influenced by these mutations but not abolished,
indicating that 45 does not share this binding site with 2-arachido-
noylglycerol and loreclezole.
tial agonist.14 Some synthetic analogs of magnolol were obtained by
structural optimization of compounds which showed high affinity
for CB receptors, for example, 5-hexyl-20-methoxy-50-propylbiphe-
nyl-2-ol (Ki: CB1 0.00957 lM, CB2 0.0238 l
M);14 for affinity of

A. Fuchs et al. / Bioorg. Med. Chem. 22 (2014) 6908–6917
6913
(a) 2000
1800
1600
1400
1200
1000
800
600
400
200
0
δ
2
2
2
2
2
2
2
2
2
2
2
γ
γ
γ
γ
γ
γ
γ
γ
γ
γ
β
3
2
2
2
2
2
2
1
3
β
β
S
265
1
β
β
β
β
β
β
β
α
1
36T
2V4
1
2
3
4
5
6
1
1
α
α
α
α
α
α
α
α
α
2N
β
β
1
1
α
α
Isoform
(b)
1200
1100
1000
900
800
700
600
500
400
300
200
100
0
δ
2
2
2
2
2
2
2
2
2
2
2
γ
6T
γ
γ
γ
γ
γ
γ
γ
γ
γ
β
3
2
2
2
2
2
2
1
3
β
β
S
1
β
β
β
β
β
β
β
5
α
1
3
2V4
6
1
2
3
4
5
6
1
1
α
α
α
α
α
α
α
α
α
N2
2
β
β
1
1
α
α
Isoform
Figure 5. Subunit specificity of 64 (a) and 45 (b), and effects of specific point mutations on the positive modulatory activity of the compounds. For each subunit combination
the agonistic effect (open bars; current elicited by 3 M test compound divided by that elicited by EC0.5 GABA = effect at which GABA shows 0.5% of its maximal effect) and the
l
allosteric potentiation (closed bars; current elicited by 3
alone) were determined.
lM test compound in combination with EC0.5 GABA divided by that elicited by the same concentration of GABA
red: commom features
black: different features
CH3
O
OH
OH
OH
45
61
Figure 6. Effect of the benzodiazepine antagonist Ro15-1788. GABA at a concen-
tration of 0.3 M was applied together with 3 M of compound 45 (left) or 64
M Ro15-1788. Averaged data SD
l
l
l
(right) in the absence (ꢀ) and presence (+) of 1
Figure 7. Comparison of structural features of magnolol and 40-O-methylhonokiol
analogs. Current potentiation at 10 M: 45, 1000%; 61, 5000%.
(n = 3).
l

6914
A. Fuchs et al. / Bioorg. Med. Chem. 22 (2014) 6908–6917
compounds at human CB1 and CB2 see Table S1, Supplementary
data. Another analog, 50-hexyl-20-methoxy-5-propylbiphenyl-2-ol,
was developed to inhibit the lysophosphatidylinositol-activated
Investigation of the most potent compounds of the present ser-
ies, 45, 61, 62, 64, at GABAA receptors of different subunit compo-
sition and with specific point mutations, indicated that they
interacted with a different binding site than the benzodiazepines
and the previously characterized allosteric PAM 2-arachidonoyl-
glycerol. The interaction of the biphenylic compounds with CB
receptors could be significantly reduced compared to the precursor
receptor GPR55 (IC50 3.25 l
M).14
A dual activation of CB1 and GABAA receptors could be thera-
peutically desirable, for example, for the treatment of convulsions,
spasms, and associated pain. In this respect potent GABAA receptor
PAMs that showed ancillary CB1-agonistic activity, such as 45 (Ki
40-O-methylhonokiol (Ki CB1: 8.34
lM, CB2: 0.0433 lM; see
CB1: 0.386
l
M) and 64 (Ki CB1: 0.339
lM), may be suitable candi-
Supporting information).14 This class of powerful PAMs of GABAA
receptors has the potential for further development as anxiolytic,
hypnotic, and/or muscle relaxant drugs.
dates for further evaluation, for example, in in vivo experiments.
It also appears warranted to evaluate the GABAA-selective com-
pound 61 (Ki CB1: >10 lM), which is at the same time the most
4. Experimental section
4.1. Chemistry
powerful allosteric modulator (up to 5000% current potentiation),
in extended in vitro and in vivo studies. Its potency at CB receptors
is significantly lower (>12-fold) as compared to the parent com-
pound 40-O-methylhonokiol (see Supplemenatary data).
4.1.1. Material and methods
3. Conclusions and outlook
All commercially available reagents and solvents were used
without further purification. The reactions were monitored by thin
layer chromatography (TLC) using aluminum sheets coated with
silica gel 60 F254 (Merck). Melting points were determined on a
Büchi 530 melting point apparatus and are uncorrected.
The natural products magnolol and 40-O-methylhonokiol were
used as lead structures to study the SARs of this new class of extre-
mely powerful GABAA receptor potentiators or PAMs. The target
compounds were accessible by straightforward procedures. Minor
modifications were found to have large effects on the compounds’
biological activity. Except for magnolol analog 45 (Fig. 7), the most
potent compounds, 61, 62, and 64, were derived from the 40-O-
methylhonokiol scaffold. Whereas 40-O-methylhonokiol has two
allyl residues, one at each of the phenyl rings, we found that only
one alkyl residue was sufficient for the compounds’ GABAA poten-
tiating effects. While methyl and ethyl residues were found to be
too small, the optimal alkyl residue was found to be propyl, as in
compound 61 (40-methoxy-5-propylbiphenyl-2-ol). The two most
powerful compounds from the two different scaffolds share several
features, which appear to be a required for high GABAA potentiat-
ing activity. There are the directly connected phenyl rings and a
free phenolic group para to a C2- or C3-alkyl residue (ethyl in 45,
propyl in 61, see Table 1). The substitution pattern of the second
ring, which differentiates magnolol from 40-O-methylhonokiol ana-
logs, was found to be responsible for high GABAA potentiating
activity and selectivity over CB receptors. A single methoxy group
in the para-position was found to be superior to an ortho-hydroxyl
group in combination with an alkyl residue (see Fig. 7).
Methylation of one of the hydroxyl groups in magnolol analogs
resulted in a loss of activity, no matter whether the methoxy group
was in the para-position to the short or the long alkyl residue
(compare compounds 52–58). Due to the different SARs it appears
likely that both, magnolol and honokiol analogs exhibit—at least
somewhat—different binding modes.
Preparative HPLC was performed on
20 mm, particle size 10 m, Eurospher 100) using a mixture of
methanol and H2O as eluent at flow rate of 20 mL/min.
a
C18 column (250 ꢁ
l
a
Microwave reactions were performed in a CEM Discover micro-
wave reactor. 1H NMR and 13C NMR data were recorded on a
Bruker Avance spectrometer at 500 MHz for proton and 125 MHz
for carbon. Shifts are given in ppm relative to the remaining pro-
tons of the deuterated solvents. Mass spectra were recorded on
an API 2000 mass spectrometer (electron spray ion source, Applied
Biosystems, Darmstadt, Germany) coupled with an Agilent 1100
HPLC system using
(50 ꢁ 2.00 mm, particle size 3
a
Phenomenex Luna HPLC C18 column
m). The purity of the tested com-
l
pounds was determined by HPLC-UV obtained on an LC-MS instru-
ment (Applied Biosystems API 2000 LC–MS/MS, HPLC Agilent
1100) using the procedure as follows: dissolving of the compounds
at a concentration of 1.0 mg/mL in methanol and if necessary
sonication to complete dissolution. Then, 10
was injected into Phenomenex Luna C18 HPLC column
(50 ꢁ 2.00 mm, particle size 3 m) and elution was performed for
30 min at a flow rate of 250 L/min with a gradient of water/meth-
lL of the solution
a
l
l
anol either containing 2 mM ammonium acetate from 90:10 up to
0:100, starting the gradient after 10 min (system A), or containing
2 mM ammonium acetate and 0.1% formic acid from 90:10 up to
0:100, starting the gradient after 10 min (system B), or containing
2 mM ammonium acetate from 60:40 up to 0:100 for 30 min, start-
ing the gradient after 0 min and ending after 20 min (system C). UV
absorption was detected from 220 to 400 nm using a diode array
detector.
All compounds were synthesized according to or in analogy to
previously prescribed procedures.14 Compounds 37–51, 55, 57
and 58 were prescribed previously.14 Compounds 52–54, 56,
59–69 represent new compounds.
In case of the more efficacious 40-O-methylhonokiol analogs
(61, 62, 64) it could be interesting to evaluate the effects of further
substituents on the methoxy-substituted phenyl ring in the future
with the goal to potentially increase the PAM effect.
Alexeev et al., had reported on a current potentiation by honok-
iol of 351.2 79.0% at 30–60
lM (hippocampal dentate granule
neurons) which was in a similar range as in our study (192 63%
Analytical data for new products are described below. For
detailed synthetic procedures, analytical data of compounds
15–32, 37–51, 55, 57 and 58 as well as affinity of all compounds
for human CB1/2 receptors see Supplementary data.
at 3 lM, Xenopus oocytes), considering that cell types and applied
concentrations were different.13,24
The most potent compound of the present series, 40-methoxy-5-
propylbiphenyl-2-ol (61) potentiated the current amplitude of
GABAA receptors (
by up to 5000% at 10
as weak direct agonists at the GABAA receptor with a current poten-
tiation at a concentration of 10 M of up to 6% of the maximal effect
of GABA; they can therefore be characterized as ago-allosteric mod-
ulators, although their direct effect was only moderate, in contrast
to the extremely powerful indirect GABA-activating effect.
a
1b2c2 isoform) expressed in Xenopus oocytes
4.1.2. Analytical data
4.1.2.1. 5-Ethyl-20-methoxy-50-pentylbiphenyl-2-ol, C20H26O2,
Mr: 298.42, (52). 1H NMR (500 MHz, CDCl3) d 7.19 (dd, J = 8.3,
2.3 Hz, Har, 1H), 7.16 (d, J = 2.2 Hz, Har, 1H), 7.13 (dd, J = 8.2,
2.3 Hz, Har, 1H), 7.09 (d, J = 2.2 Hz, Har, 1H), 6.96 (d, J = 8.2 Hz,
Har, 2H), 6.25 (s, OH, 1H), 3.88 (s, O-CH3, 3H), 2.68–2.59
(m, ar-CH2, 4H), 1.66–1.59 (m, CH2, 2H), 1.38–1.31 (m, CH2, 4H),
lM concentration. The compounds also acted
l

A. Fuchs et al. / Bioorg. Med. Chem. 22 (2014) 6908–6917
6915
1.26 (t, J = 7.6 Hz, CH3, 3H), 0.90 (t, J = 7.0 Hz, CH3, 3H). 13C NMR
(126 MHz, CDCl3) d 153.46 (Car-OCH3), 151.68 (Car-O), 136.71
(Car), 136.60 (Car), 132.42 (Car), 130.49 (Car), 128.86 (Car), 128.52
(Car), 127.09 (Car), 126.24 (Car), 117.37 (Car), 111.45 (Car), 56.31
(O-CH3), 35.05 (CH2), 31.48 (CH2), 31.34 (CH2), 28.04 (CH2), 22.52
(CH2), 15.78 (CH3), 14.03 (CH3). LC/ESI-MS (positive mode) m/z
299 (M+H)+ (negative mode) m/z 297 (MꢀH)ꢀ 96.3%, colorless
liquid.
7.09–7.00 (m, CHar, 2H), 6.90 (d, J = 8.1 Hz, CHar, 2H), 5.04 (s, OH
1H), 3.86 (s, CH3, 3H), 2.62 (q, J = 7.6 Hz, ar-CH2, 2H), 1.24
(t, J = 7.6 Hz, CH3, 3H). 13C NMR (126 MHz, CDCl3) d 159.23
(Car-OCH3), 150.42 (Car-O), 136.45 (Car), 130.23 (Car, 2C), 129.50
(Car, 2C), 128.04 (Car), 127.52 (Car), 115.47 (Car), 114.63 (Car, 2C),
55.34 (O-CH3), 27.99 (CH2), 15.82 (CH3). LC/ESI-MS (positive mode)
m/z 229 (M+H)+ (negative mode) m/z 227 (MꢀH)ꢀ 98.1%. mp 52 °C,
off-white powder.
4.1.2.2. 5-Ethyl-50-hexyl-20-methoxybiphenyl-2-ol, C21H28O2, Mr:
312.45, (53). 1H NMR (500 MHz, CDCl3) d 7.19 (dd, J = 8.3, 2.3 Hz,
CHar, 1H), 7.16 (d, J = 2.2 Hz, CHar, 1H), 7.13 (dd, J = 8.2, 2.3 Hz, CHar,
1H), 7.09 (d, J = 2.2 Hz, CHar, 1H), 6.96 (d, J = 8.3 Hz, CHar, 2H), 6.25
(s, OH, 1H), 3.88 (s, CH3, 3H), 2.65 (q, J = 7.5 Hz, ar-CH2, 2H), 2.61 (t,
J = 7.8 Hz, ar-CH2, 2H), 1.62 (dt, J = 15.4, 7.5 Hz, CH2, 2H), 1.37–1.28
(m, CH2 6H), 1.26 (t, J = 7.6 Hz, CH3, 3H), 0.89 (t, J = 7.0 Hz, CH3, 3H).
13C NMR (126 MHz, CDCl3) d 153.46 (Car-OCH3), 151.68 (Car-O),
136.71 (Car), 136.60 (Car), 132.42 (Car), 130.49 (Car), 128.86 (Car),
128.52 (Car), 127.08 (Car), 126.24 (Car), 117.37 (Car), 111.45 (Car),
56.31 (O-CH3), 35.08 (CH2), 31.70 (CH2), 31.63 (CH2), 28.97 (CH2),
28.04 (CH2), 22.61 (CH2), 15.78 (CH3), 14.08 (CH3). LC/ESI-MS
(positive mode) m/z 313 (M+H)+ (negative mode) m/z 311 (MꢀH)ꢀ
97.3%, colorless liquid.
4.1.2.7. 40-Methoxy-5-propylbiphenyl-2-ol, C16H18O2, Mr: 242.31,
(61). 1H NMR (500 MHz, CDCl3) d 7.42–7.39 (m, CHar, 1H), 7.06–
7.00 (m, CHar, 1H), 6.90–6.88 (m, CHar, 1H), 5.04 (s, OH, 1H), 3.86
(s, CH3, 3H), 2.55 (t, J = 7.8 Hz, ar-CH2, 2H), 1.68–1.60 (m, CH2,
2H), 0.96 (t, J = 7.3 Hz, CH3, 3H). 13C NMR (126 MHz, CDCl3) d
159.22 (Car-OCH3), 150.42 (Car-O), 134.92 (Car), 130.23 (Car, 2C),
130.10 (Car), 129.50 (Car), 128.64 (Car), 127.44 (Car), 115.39 (Car),
114.63 (Car, 2C), 55.34 (O-CH3), 37.19 (CH2), 24.76 (CH2), 13.82
(CH3). LC/ESI-MS (positive mode) m/z 243 (M+H)+ (negative mode)
m/z 241 (MꢀH)ꢀ 96.7%. mp 63 °C, off-white powder.
4.1.2.8. 5-Butyl-40-methoxybiphenyl-2-ol, C17H20O2, Mr: 256.34,
(62). 1H NMR (500 MHz, Chloroform-d) OH signal is missing d
7.41–7.38 (m, Har, 2H), 7.06–7.00 (m, Har, 4H), 6.88 (d, J = 8.0 Hz,
Har, 1H), 3.86 (s, O-CH3, 3H), 2.57 (t, J = 7.8 Hz, ar-CH2, 2H), 1.63–
1.55 (m, CH2, 2H), 1.41–1.32 (m, CH2, 2H), 0.93 (t, J = 7.3 Hz, CH3,
3H). 13C NMR (126 MHz, CDCl3) d 159.19 (Car-OCH3), 150.39
(Car-OH), 135.09 (Car), 130.22 (Car, 2C), 130.04 (Car), 129.53 (Car),
128.55 (Car), 127.44 (Car), 115.39 (Car, 2C), 114.59 (Car), 55.34
(O-CH3), 34.76 (ar-CH2), 33.88 (CH2), 22.33 (CH2), 13.95 (CH3).
LC/ESI-MS (positive mode) m/z 257 (M+H)+ 97.9%, colorless liquid.
4.1.2.3. 20-Methoxy-50-pentyl-5-propylbiphenyl-2-ol, C21H28O2,
Mr: 312.45, (54). 1H NMR (500 MHz, CDCl3) d 7.19 (dd, J = 8.3,
2.3 Hz, CHar, 1H), 7.16 (d, J = 2.2 Hz, CHar, 1H), 7.11 (dd, J = 8.2,
2.2 Hz, CHar, 1H), 7.08 (d, J = 2.2 Hz, CHar, 1H), 6.97 (d, J = 3.9 Hz,
CHar, 1H), 6.95 (d, J = 3.7 Hz, CHar, 1H), 6.28 (s, OH, 1H), 3.88 (s,
CH3, 3H), 2.64–2.56 (m, 1H), 1.70–1.62 (m, 1H), 1.37–1.32 (m,
1H), 0.97 (t, J = 7.3 Hz, CH3, 3H), 0.91 (t, J = 7.0 Hz, CH3, 3H). 13C
NMR (126 MHz, CDCl3)
d 153.46 (Car-OCH3), 151.69 (Car-O),
4.1.2.9. 40-Methoxy-5-pentylbiphenyl-2-ol, C18H22O2, Mr: 270.37,
(63). 1H NMR (500 MHz, Chloroform-d) d 7.42–7.38 (m, 2H),
7.06–7.00 (m, 4H), 6.88 (d, J = 8.1 Hz, 1H), 3.86 (s, 3H), 2.56 (t,
J = 7.8 Hz, 2H), 1.63–1.58 (m, 2H), 1.36–1.31 (m, 4H), 0.90 (t,
J = 7.0 Hz, 3H). 13C NMR (126 MHz, CDCl3) d 159.19 (Car-OCH3),
150.37 (Car-OH), 135.14 (Car), 130.22 (Car, 2C), 130.01 (Car),
129.51 (Car), 128.55 (Car), 127.43 (Car), 115.38 (Car, 2C), 114.60
(Car), 55.33 (O-CH3), 35.05 (ar-CH2), 31.49 (CH2), 31.40 (CH2),
22.52 (CH2), 14.02 (CH3). LC/ESI-MS (positive mode) m/z 271
(M+H)+ 97.8%, colorless liquid.
136.69 (Car), 135.07 (Car), 132.41 (Car), 131.09 (Car), 129.09 (Car),
128.83 (Car), 127.11 (Car), 126.16 (Car), 117.28 (Car), 111.48 (Car),
56.30 (O-CH3), 37.28 (CH2), 35.04 (CH2), 31.48 (CH2), 31.34 (CH2),
24.75 (CH2), 22.52 (CH2), 14.02 (CH3), 13.89 (CH3). LC/ESI-MS
(positive mode) m/z 313 (M+H)+ (negative mode) m/z 311 (MꢀH)ꢀ
98.3%, colorless liquid.
4.1.2.4. 50-Ethyl-20-methoxy-5-pentylbiphenyl-2-ol, C20H26O2,
Mr: 298.42, (56). 1H NMR (500 MHz, CDCl3) d 7.24–7.20 (m, Har,
1H), 7.19 (d, J = 2.3 Hz, Har, 1H), 7.12 (dd, J = 8.2, 2.1 Hz, Har, 1H),
7.09 (d, J = 2.2 Hz, Har, 1H), 6.96 (dd, J = 14.1, 5.7 Hz, Har, 2H),
6.27 (s, OH, 1H), 3.89 (s, O-CH3, 3H), 2.68 (q, J = 7.6 Hz, ar-CH2,
2H), 2.60 (t, J = 7,5 Hz, ar-CH2, 2H), 1.68–1.61 (m, CH2, 2H), 1.39–
1.34 (m, CH2-CH2, 4H), 1.27 (t, J = 7.6 Hz, CH3, 3H), 0.92 (t,
J = 6.9 Hz, CH3, 3H). 13C NMR (126 MHz, CDCl3) d 153.49 (Car-
OCH3), 151.65 (Car-O), 137.98 (Car), 135.32 (Car), 131.92 (Car),
131.01 (Car), 129.05 (Car), 128.29 (Car), 127.19 (Car), 126.16 (Car),
117.29 (Car), 111.58 (Car), 56.32 (O-CH3), 35.12 (CH2), 31.56
(CH2), 31.38 (CH2), 28.01 (CH2), 22.55 (CH2), 15.79 (CH3), 14.04
(CH3). LC/ESI-MS (positive mode) m/z 299 (M+H)+ (negative mode)
m/z 297 (MꢀH)ꢀ 97.8%. mp 41 °C, off-white powder.
4.1.2.10. 5-Hexyl-40-methoxybiphenyl-2-ol, C19H24O2, Mr:
284.39, (64). 1H NMR (500 MHz, CDCl3) d 7.42–7.38 (m, CHar,
2H), 7.06–7.00 (m, CHar, 2H), 6.88 (d, J = 8.1 Hz, CHar, 2H), 5.02 (s,
OH 1H), 3.86 (s, CH3, 3H), 2.56 (t, J = 7.8 Hz, ar-CH2, 2H), 1.65–
1.57 (m, CH2, 2H), 1.37–1.26 (m, CH2-CH2-CH2, 6H), 0.89 (t,
J = 6.9 Hz, CH3, 3H). 13C NMR (126 MHz, CDCl3)
d 159.21
(Car-OCH3), 150.36 (Car-O), 135.17 (Car), 130.22 (Car), 130.01 (Car),
129.48 (Car), 128.56 (Car), 127.43 (Car), 115.38 (Car), 114.62 (Car),
55.33 (O-CH3), 35.09 (CH2), 31.71 (CH2), 31.68 (CH2), 28.97 (CH2),
22.58 (CH2), 14.06 (CH3). LC/ESI-MS (positive mode) m/z 285
(M+H)+ (negative mode) m/z 283 (MꢀH)ꢀ 97.7%. mp 61 °C, off-
white powder.
4.1.2.5. 40-Methoxy-5-methylbiphenyl-2-ol, C14H14O2, Mr:
214.26, (59). 1H NMR (500 MHz, Chloroform-d) d 7.41–7.37 (m,
Har, 2H), 7.07–6.99 (m, Har, 4H), 6.87 (d, J = 8.0 Hz, Har, 1H), 5.02
(s, OH, 1H), 3.86 (s, O-CH3, 3H), 2.31 (s, CH3, 3H). 13C NMR
(126 MHz, CDCl3) d 159.22 (Car-OCH3), 150.23 (Car-OH), 130.65
(Car), 130.19 (Car, 2C), 129.87 (Car), 129.35 (Car), 129.20 (Car),
127.49 (Car), 115.43 (Car, 2C), 114.62 (Car), 55.33 (O-CH3), 20.46
(CH3). LC/ESI-MS (positive mode) m/z 299 (M+H)+ 98.9%, colorless
liquid.
4.1.2.11. 5-Heptyl-40-methoxybiphenyl-2-ol, C20H26O2, Mr:
298.42, (65). 1H NMR (600 MHz, Chloroform-d) d 7.41–7.38 (m,
2H), 7.06–7.00 (m, 4H), 6.88 (d, J = 8.1 Hz, Har, 1H), 5.02 (s, OH),
3.86 (s, O-CH3, 3H), 2.56 (t, J = 7.8 Hz, ar-CH2, 2H), 1.63–1.58 (m,
2H), 1.36–1.24 (m, 8H), 0.88 (t, J = 6.9 Hz, CH3, 3H). 13C NMR
(151 MHz, CDCl3) d 159.21 (Car-OCH3), 150.35 (Car-OH), 135.18
(Car), 130.23 (Car, 2C), 130.02 (Car), 129.47 (Car), 128.57 (Car),
127.42 (Car), 115.38 (Car), 114.62 (Car, 2C), 55.35 (O-CH3), 35.11
(CH2), 31.82 (CH2), 31.76 (CH2), 29.28 (CH2), 29.18 (CH2), 22.66
4.1.2.6. 5-Ethyl-40-methoxybiphenyl-2-ol, C15H16O2, Mr: 228.29,
(60). 1H NMR (500 MHz, CDCl3) d 7.42–7.38 (m, CHar, 2H),

6916
A. Fuchs et al. / Bioorg. Med. Chem. 22 (2014) 6908–6917
(CH2), 14.10 (CH3). LC/ESI-MS (positive mode) m/z 299 (M+H)+
97.7%, colorless liquid.
precipitated in ethanol/isoamylalcohol 19:1, the dried pellet
dissolved in water and stored at ꢀ80 °C. cRNA mixtures were pre-
pared from these stock solutions and stored at ꢀ80 °C. Xenopus
laevis oocytes were prepared, injected and defolliculated as
described previously.32,33 They were injected with 50 nL of the
4.1.2.12. 40-Methoxy-5-octylbiphenyl-2-ol, C21H28O2, Mr: 312.45,
(66). 1H NMR (600 MHz, Chloroform-d) d 7.44–7.36 (m, Har, 2H),
7.08–6.99 (m, Har, 4H), 6.88 (d, J = 8.1 Hz, Har, 1H), 5.05 (s, OH,
1H), 3.86 (s, O-CH3, 3H), 2.56 (t, J = 7.8 Hz, ar-CH2, 2H), 1.60 (m,
CH2, 2H), 1.33–1.25 (m, CH2, 10H), 0.88 (t, J = 7.0 Hz, CH3, 3H).
13C NMR (151 MHz, CDCl3) d 159.20 (Car-OCH3), 150.36 (Car-OH),
135.18 (Car), 130.23 (Car, 2C), 130.02 (Car), 129.49 (Car), 128.57
(Car), 127.43 (Car), 115.38 (Car), 114.61 (Car, 2C), 55.35 (O-CH3),
35.11 (CH2), 31.88 (CH2), 31.75 (CH2), 29.48 (CH2), 29.32 (CH2),
29.26 (CH2), 22.66 (CH2), 14.10 (CH3). LC/ESI-MS (positive mode)
m/z 313 (M+H)+ 97.1%, colorless liquid.
cRNA solution containing, in the case of
units at a concentration of 75 nM:75 nM, in the case of
tors 1 or a2 6 and b2 and 2 subunits at a concentration of
10 nM:10 nM:50 nM and in the case of bd receptors 1, b3 and d
subunits at
concentration of 10 nM:10 nM:50 nM.34
Subsequently, the oocytes were incubated in modified Barth’s solu-
tion at +18 °C for at least 24 h before the measurements for b and
receptors and 48 h for bd receptors.
ab receptors
a1 and b2 sub-
abc
recep-
a
,
a3,
a5
,
a
c
a
a
a
a
abc
a
4.2.2. Functional characterization of the GABAA receptors
4.1.2.13. 40-Ethoxy-5-hexylbiphenyl-2-ol, C20H26O2, Mr: 298.42,
(67). 1H NMR (500 MHz, Chloroform-d) d 7.40–7.36 (m, Har, 2H),
7.07–6.98 (m, Har, 4H), 6.88 (d, J = 8.1 Hz, Har, 1H), 5.04 (s, OH,
1H), 4.09 (q, J = 7.0 Hz, O-CH2, 2H), 2.56 (t, J = 7.8 Hz, ar-CH2, 2H),
1.65–1.57 (m, CH2, 2H), 1.45 (t, J = 7.0 Hz, CH3, 3H), 1.37–1.28
(m, CH2, 6H), 0.89 (t, J = 6.8 Hz, CH3, 3H). 13C NMR (126 MHz,
Currents were measured using a modified two-electrode volt-
age clamp amplifier Oocyte clamp OC-725 (Warner Instruments)
in combination with a XY-recorder (90% response time 0.1 s) or
digitized at 100 Hz using a PowerLab 2/20 (AD Instruments) using
the computer programs Chart (ADInstruments GmbH, Spechbach,
Germany). Tests with a model oocyte were performed to ensure
linearity in the larger current range. The response was linear up
CDCl3)
d 158.59 (Car-OCH2), 150.36 (Car-OH), 135.14 (Car),
130.19 (Car, 2C), 129.99 (Car), 129.29 (Car), 128.53 (Car), 127.48
(Car), 115.35 (Car), 115.17 (Car, 2C), 63.53 (O-CH2), 35.10 (ar-CH2),
31.72 (CH2), 31.70 (CH2), 28.98 (CH2), 22.59 (CH2), 14.82 (CH3),
14.08 (CH3). LC/ESI-MS (positive mode) m/z 299 (M+H)+ 97.7%, col-
orless liquid.
to 15 lA.
Electrophysiological experiments were performed by using the
two-electrode voltage clamp method at a holding potential of
ꢀ80 mV. The perfusion medium contained 90 mM NaCl, 1 mM
KCl, 1 mM MgCl2, 1 mM CaCl2, and 5 mM Na-HEPES (pH 7.4) and
was applied by gravity flow 6 ml/min. The perfusion medium
was applied through a glass capillary with an inner diameter of
1.35 mm, the mouth of which was placed about 0.4 mm from the
surface of the oocyte.
Allosteric modulation was measured at a GABA concentration
eliciting 0.5–1.0% of the maximal GABA current amplitude in the
corresponding receptor. GABA was applied for 20 s alone or in
combination with allosteric compound. Potentiation of GABA
currents was expressed as (I(modulator+GABA)/IGABA ꢀ 1) ⁄ 100%. The
perfusion system was cleaned between drug applications by wash-
ing with DMSO to avoid contamination. In each case DMSO was
washed out before placing the next oocyte.
4.1.2.14. 5-Hexyl-40-propoxybiphenyl-2-ol, C21H28O2, Mr: 312.45,
(68). 1H NMR (500 MHz, Chloroform-d) d 7.40–7.36 (m, Har, 2H),
7.05–6.99 (m, Har, 4H), 6.88 (d, J = 8.1 Hz, Har, 1H), 5.03 (s, OH,
1H), 3.97 (t, J = 6.6 Hz, O-CH2, 2H), 2.56 (t, J = 7.8 Hz, ar-CH2, 2H),
1.90–1.79 (m, CH2, 2H), 1.63–1.57 (m, CH2, 2H), 1.37–1.27 (m,
CH2, 6H), 1.06 (t, J = 7.4 Hz, CH3, 3H), 0.88 (t, J = 7.0 Hz, CH3, 3H).
13C NMR (126 MHz, CDCl3) d 158.81 (Car-OCH2), 150.37 (Car-OH),
135.14 (Car), 130.17 (Car, 2C), 129.99 (Car), 129.22 (Car), 128.52
(Car), 127.50 (Car), 115.34 (Car), 115.21 (Car, 2C), 69.61 (O-CH2),
35.10 (ar-CH2), 31.72 (CH2), 31.70 (CH2), 28.98 (CH2), 22.60
(CH2), 22.57 (CH2), 14.08 (CH2), 10.51 (CH3). LC/ESI-MS (positive
mode) m/z 313 (M+H)+ 98.5%, colorless liquid.
Acknowledgements
4.1.2.15. 5-Hexyl-40-isopropoxybiphenyl-2-ol, C21H28O2, Mr:
312.45, (69). 1H NMR (600 MHz, Chloroform-d) d 7.39–7.35 (m,
Har, 2H), 7.05–6.98 (m, Har, 4H), 6.88 (d, J = 8.0 Hz, Har, 1H), 5.05
(s, OH, 1H), 4.60 (hept, J = 6.1 Hz, CH, 1H), 2.56 (t, J = 7.8 Hz, ar-
CH2, 2H), 1.63–1.57 (m, CH2, 2H), 1.38 (d, J = 6.1 Hz, CH3, 6H),
1.36–1.28 (m, 6H), 0.88 (t, J = 7.2 Hz, CH3, 3H). 13C NMR
(151 MHz, CDCl3) d 157.57 (Car-OCH), 150.37 (Car-OH), 135.14
(Car), 130.22 (Car, 2C), 130.00 (Car), 129.14 (Car), 128.52 (Car),
127.49 (Car), 116.41 (Car, 2C), 115.34 (Car), 69.94 (O-CH), 35.12
(ar-CH2), 31.73 (CH2), 31.72 (CH2), 29.00 (CH2), 22.61 (CH), 22.06
(2CH3), 14.10 (CH3). LC/ESI-MS (positive mode) m/z 313 (M+H)+
99.2%, colorless liquid.
E.S. was supported by the Swiss National Foundation grant
31003A_132806/1. We thank Dr. Viktor Rempel for experimental
advice and support and for valuable discussions.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.10.027.
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