Nitrogenated honokiol derivatives allosterically modulate GABAA receptors and act as strong partial agonists

Article abstract of DOI:10.1016/j.bmc.2015.08.034

In traditional Asian medicinal systems, preparations of the root and stem bark of Magnolia species are widely used to treat anxiety and other nervous disturbances. The biphenyl-type neolignan honokiol together with its isomer magnolol are the main constituents of Magnolia bark extracts. We have previously identified a nitrogen-containing honokiol derivative (3-acetylamino-4′-O-methylhonokiol, AMH) as a high efficient modulator of GABA_A receptors. Here we further elucidate the structure-activity relation of a series of nitrogenated biphenyl-neolignan derivatives by analysing allosteric modulation and agonistic effects on α₁β₂γ_2S GABA_A receptors. The strongest I_GABA enhancement was induced by compound 5 (3-acetamido-4′-ethoxy-3′,5-dipropylbiphenyl-2-ol, E_max: 123.4 ± 9.4% of I_GABA-max) and 6 (5′-amino-2-ethoxy-3′,5-dipropylbiphenyl-4′-ol, E_max: 117.7 ± 13.5% of I_GABA-max). Compound 5 displayed, however, a significantly higher potency (EC₅₀ = 1.8 ± 1.1 μM) than compound 6 (EC₅₀ = 20.4 ± 4.3 μM). Honokiol, AMH and four of the derivatives induced significant inward currents in the absence of GABA. Strong partial agonists were honokiol (inducing 78 ± 6% of I_GABA-max), AMH (63 ± 6%), 5′-amino-2-O-methylhonokiol (1) (59 ± 1%) and 2-methoxy-5′-nitro-3′,5-dipropylbiphenyl-4′-ol (3) (52 ± 1%). 3-N-Acetylamino-4′-ethoxy-3′,5-dipropyl-biphenyl-4′-ol (5) and 3-amino-4′-ethoxy-3′,5-dipropyl-biphenyl-4′-ol (7) were less efficacious but even more potent (5: EC₅₀ = 6.9 ± 1.0 μM; 7: EC₅₀ = 33.2 ± 5.1 μM) than the full agonist GABA.

Full text of DOI:10.1016/j.bmc.2015.08.034

Bioorganic & Medicinal Chemistry 23 (2015) 6757–6762
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Nitrogenated honokiol derivatives allosterically modulate GABAA
receptors and act as strong partial agonists
a
d
Marketa Bernaskova a, Angela Schoeffmann d, Wolfgang Schuehly b,c, , Antje Hufner , Igor Baburin ,
⇑
Steffen Hering d
a Institute of Pharmaceutical Sciences, Pharmaceutical Chemistry, University of Graz, Schubertstrasse 1, 8010 Graz, Austria
b Institute of Pharmaceutical Sciences, Pharmacognosy, University of Graz, Universitätsplatz 4, 8010 Graz, Austria
c Institute of Zoology, University of Graz, Universitätsplatz 2, 8010 Graz, Austria
d Department of Pharmacology and Toxicology, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
In traditional Asian medicinal systems, preparations of the root and stem bark of Magnolia species are
widely used to treat anxiety and other nervous disturbances. The biphenyl-type neolignan honokiol
together with its isomer magnolol are the main constituents of Magnolia bark extracts. We have previ-
ously identified a nitrogen-containing honokiol derivative (3-acetylamino-40-O-methylhonokiol, AMH)
as a high efficient modulator of GABAA receptors. Here we further elucidate the structure-activity relation
of a series of nitrogenated biphenyl-neolignan derivatives by analysing allosteric modulation and
Received 27 May 2015
Revised 6 August 2015
Accepted 25 August 2015
Available online 28 August 2015
Keywords:
agonistic effects on
a1b2c2S GABAA receptors. The strongest IGABA enhancement was induced by
Honokiol derivatives
GABAA receptor
Nitrogenation
Magnolia
compound 5 (3-acetamido-40-ethoxy-30,5-dipropylbiphenyl-2-ol, Emax: 123.4 9.4% of IGABA-max) and 6
(50-amino-2-ethoxy-30,5-dipropylbiphenyl-40-ol, Emax: 117.7 13.5% of IGABA-max). Compound 5 displayed,
however, a significantly higher potency (EC50 = 1.8 1.1 lM) than compound 6 (EC50 = 20.4 4.3 lM).
Honokiol, AMH and four of the derivatives induced significant inward currents in the absence
of GABA. Strong partial agonists were honokiol (inducing 78 6% of IGABA-max), AMH (63 6%),
50-amino-2-O-methylhonokiol (1) (59 1%) and 2-methoxy-50-nitro-30,5-dipropylbiphenyl-40-ol (3)
(52 1%). 3-N-Acetylamino-40-ethoxy-30,5-dipropyl-biphenyl-40-ol (5) and 3-amino-40-ethoxy-30,5-
dipropyl-biphenyl-40-ol (7) were less efficacious but even more potent (5: EC50 = 6.9 1.0
EC50 = 33.2 5.1 M) than the full agonist GABA.
lM; 7:
l
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
receptor plays a crucial role in several disorders of the CNS such
as depression, anxiety, epilepsy. Among many other classes of
GABAA receptor modulators, two classes that are clearly identifi-
able upon their mode of action are benzodiazepines that exert their
action upon the presence of a c2 subunit within the presence of
c-Aminobutyric acid (GABA) is the most important inhibitory
neurotransmitter in the mammalian central nervous system
(CNS). The action of GABA is primarily exerted through ligand-
gated ion channels, the GABAA receptors. The GABAA receptor is a
co-assembly of five subunits, which together form a central pore
in the cell membrane for selective chloride ion transport.12 GABAA
receptors exist in different subtypes, which are characterized by
the type of subunit and the respective assemblage and they depend
on the tissue in which they occur. The different GABAA subtypes
exert different physiological effects15,17 and react differently to
GABAA receptor modulatory compounds making the search for
subtype-selective chemical entities interesting.18 The GABAA
either a1, a2,
a3 or a5 subunits21 and barbiturates, etomidate,
propofol, valerenic acid, which do not require the presence of a
c
subunit.7,19,10
The study of Asian medicinal preparations with anxiolytic and
CNS relaxing effects such as Saiboku-to from Japan led to the iden-
tification of the biphenyl neolignans honokiol and magnolol as the
major active constituents of the Asian Magnolia bark preparations
that contain, for example, Magnolia officinalis Rehd. et Wils.13
.
Besides the great multitude of pharmacological activities that are
ascribed to especially honokiol (H),14 the CNS activity of honokiol
and magnolol could be linked to their interaction with GABAA
receptors.1
⇑
Corresponding author. Tel.: +43 316 380 8754; fax: +43 316 380 9875.
E-mail address: wolfgang.schuehly@uni-graz.at (W. Schuehly).
http://dx.doi.org/10.1016/j.bmc.2015.08.034
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

6758
M. Bernaskova et al. / Bioorg. Med. Chem. 23 (2015) 6757–6762
Table 1
The modulatory effect of honokiol on chloride currents through
Structures of compounds based on nitrogenated honokiol for the evaluation of GABAA
receptor modulatory activity including the previously identified highly efficient
3-acetylamino-40-O-methylhonokiol (AMH, i.e., compd 31 in Ref. 20) and honokiol (H)
a set of GABAA receptor subtypes expressed in Xenopus oocytes was
previously investigated in our group using a series of 31 analogs of
honokiol. It led to the discovery of the very potent 3-acetylamino-
40-O-methylhonokiol (AMH) that enhanced IGABA trough
a1b2
R6
receptors by more than 2600%.20 In that communication, it was
5'
OR2
also shown that for H, the potentiation was about equal for
R3
1
a1b2c2S and
a1b2 receptor subtypes, that is, the potentiation did
1'
R4
not require the presence of a
c
2S subunit, which hints to a binding
site of H different from the benzodiazepine binding site. Accord-
ingly, Baur et al.4 could demonstrate through an indepth study
on subunit-specificity of 40-O-methylhonokiol (MH) that the cur-
rent potentiation by MH was also not depending on the presence
of a c2S subunit. The binding of benzodiazepine requires the pres-
OR1
R5
Compd R1
R2
R3
R4
R5
R6
H
AMH
–H
–H
–H
–2-Propenyl –2-Propenyl –H
–CH3 –2-Propenyl –2-Propenyl –NHCOCH3 –H
–H
ence of a c2 receptor subunit, however, benzodiazepine effects are
usually accompanied by undesired side effects23 rendering a drug
candidate interacting with a novel (non benzodiazepine) binding
site especially interesting. Recent data of Alexeev et al.2 who
analysed the effects of several point mutations on H action suggest
that its binding site may be separate from the binding site of
neurosteroids, anesthetics, ethanol and picrotoxin.
1
2
3
4
5
6
7
–CH3 –H
–C2H5 –H
–CH3 –H
–CH3 –H
–H
–C2H5 –H
–H –C2H5 Propyl
–2-Propenyl –2-Propenyl –H
–2-Propenyl –2-Propenyl –H
–NH2
–NH2
–NH2
–NHCOCH3
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
–H
–H
–C2H5 Propyl
–NHCOCH3 –H
–H
–NH2
Propyl
–NH2
–H
The structural similarity of AMH to H and MH prompted us to
further explore this lead as a candidate with potentially lacking
of benzodiazepine side-effects through the study of structure activ-
ity-relationships of nitrogenated honokiol derivatives by analysing
potential induction of chloride currents through GABAA receptors
composed of 1b2c2S subunits was analysed subsequently.
a
allosteric modulation of
a1b2c2S GABAA receptors with particular
The syntheses aimed at combining pharmacophore features
that turned out to be most promising from previous GABAA recep-
tor modulatory studies,20 that is, nitrogenation of the aromatic ring
using either an amino function or an acetylated amino function as
well as the substitution of the free hydroxy groups with either
methyl or ethyl moieties. The hydrogenation of the initial 2-prope-
nyl chain into a propyl chain was in most cases undertaken to
enhance overall chemical stability.
focus on partial agonistic effects.
2. Results and discussion
2.1. Syntheses
Seven honokiol derivatives with nitrogen-containing moieties
(1–7; Scheme 1, Table 1) were synthesized and the enhancement
of GABA-induced chloride currents (IGABA) was studied. Aside, a
The 2-O-alkylated honokiols 2-O-methylhonokiol and
2-O-ethylhonokiol resp. were nitrated in ortho position to the free
OH
OR
OH
i)
+
R = Me
OR
R = Me
R = Et
OH
ii)
R = Et
OH
ii)
iv)
i)
iii)
v)
X
OH
OR
5'
OH
OR
+
R = Me
R = Et
R = Me
R = Et
OR
OH
OR
OH
3
NHAc
ii)
iii)
ii)
iii)
1a R = Me; X = NO2
2a R = Et; X = NO2
AMH
iii)
X
v)
v)
1 R = Me; X = NH2
2 R = Et; X = NH2
5'
OR
OH
OH
OR
X
3 R = Me; X = NH2
6 R = Et; X = NH2
7 R = Et; X = NH2
5 R = Et; X = NHAc
v)
v)
4 R = Me; X = NHAc
i) MW irradiation 1. KOH, 2. Me2SO4 or Et2SO4; ii) HNO3 (65%), EtOAc; iii) SnCl2 x 2H2O, EtOH; iv) H2, [Pd/C]; v) Ac2O, H2O
Scheme 1. Synthesis of a series of nitrogenated honokiol analogs.

M. Bernaskova et al. / Bioorg. Med. Chem. 23 (2015) 6757–6762
6759
hydroxy group according to Johnson and Corey8 resulting in 2-O-
of GABA (see Fig. 1D for representative currents evoked by 100 lM
methyl-50-nitrohonokiol (1a) and 2-O-ethyl-50-nitrohonokiol (2a),
resp., which were reduced to the corresponding amines (1) and
(2) according to literature.22 The synthesis of the five hydrogenated
honokiol derivatives 3–7 is described in Bernaskova et al.,5 the
general route to alkylated honokiols is described in Schuehly
et al.16
of the indicated compound). Figure 1C illustrates the partial ago-
nistic effects. Inward currents are expressed as fractions of IGABA-max
induced by 1 mM GABA.
H, AMH and 1 were identified as the strongest partial agonists
on
a1b2c2S GABAA receptors with maximal inward currents ranging
between 59 1% (1, n = 3) and 78 6% (H, n = 4) of Emax-dir
,
followed by the slightly less efficient compound 3 (52 1%, n = 3).
The weakest partial agonists were compounds 5 and 7, however
still inducing approximately 30% of Emax-dir (Table 3, Fig. 1C). Com-
pounds 2, 4 and 6 did not induce chloride currents in the absence
of GABA (Table 3).
2.2. Pharmacological evaluation
2.2.1. Concentration-dependent enhancement of IGABA by
honokiol derivatives
IGABA (EC3–7) modulation by derivatives 1–7 was determined
(Fig. 1, Table 2).
3. Conclusion
2.2.2. Honokiol derivatives as partial agonist on GABAA
receptors
Honokiol and its nitrogenated derivatives AMH, 1, 3, 5 and 7
induced chloride currents through GABAA receptors in the absence
In a previous study on IGABA modulation by honokiol and
derivatives, it has been found that derivatives comprising
nitrogen-containing moieties potentiate IGABA more efficiently
Figure 1. Concentration–effect curves for IGABA potentiation (
a1b2c2S) by (A) AMH (N), 2 (d), 4 (j) and 6 (s) and (B) 1 (d), 3 (j), 5 (h) and 7 (s). (C) Partial agonistic effect
induced by AMH (N), 1 (d), 3 (j), 5 (h) and 7 (s) on 1b2
a
c
2S GABAA receptors compared to IGABA induced by the full agonist GABA (4, from3). Each data point represents the
mean SE from at least three oocytes and two different frogs. (D) Typical inward currents illustrating direct activation of a1b2c2S GABAA receptors (single horizontal bar) and
IGABA modulation (double horizontal bar) by 100 lM of compounds 1, 3, 5 and 7.

6760
M. Bernaskova et al. / Bioorg. Med. Chem. 23 (2015) 6757–6762
Table 2
and modulatory effects of these compounds are mediated via sep-
arate binding sites.
Efficiency and potency of IGABA modulation (
Emax indicates maximum enhancement of chloride current through
a
1b2c
2S) by AMH and derivatives 1–7.
1b2c2S GABAA
a
receptors induced by the indicated compound in % of the maximal IGABA induced by
1 mM GABA. Hill-coefficient (nH) and number of experiments are given
4. Experimental
4.1. General
Compound
Emax (%)
EC50
(lM)
nH
n
AMH
141.6 14.1
72.0 4.8
91.0 4.2
108.0 8.0
42.5 4.9
123.4 9.4
117.7 13.5
93.7 5.8
5.3 1.9
47.6 6.8
21.5 3.3
15.8 4.4
24.6 7.4
1.8 1.1
1.4 0.3
1.8 0.2
2.1 0.4
1.3 0.1
1.3 0.3
1.0 0.3
1.9 0.3
2.2 0.8
5
6
4
4
4
7
5
4
1
2
3
4
5
6
7
Infrared spectra were recorded on a Bruker Alpha Platinum ATR
spectrometer. 1H and 13C NMR spectra were recorded on a Varian
400 MHz spectrometer (400 and 100 MHz) using chloroform-d as
solvent and were referenced using TMS as internal standard. It is
worth to note that the carbons of the B-ring of the amines 1 and
2 give very broad signals in the 13C NMR-spectra. Therefore their
resonances are often only visible in the HMBC spectra. C-5 of 2 is
not even definitely found in HMBC.
20.4 4.3
14.4 3.2
Table 3
Direct activation of
indicates maximum chloride current through
a saturating concentration of the indicated compound in % of the maximal IGABA
induced by 1 mM GABA (see Fig. 1C). EC50 value and nH of the GABA concentration–
response curves for comparison were taken from3
EI-MS were recorded on an Agilent Technologies HP 7890A
instrument fitted with detector HP 5975C VL MSD (70 eV, ion
source 250 °C, quadrupole temperature 150 °C). Column: Agilent
HP-5MS 30 m, ID 0.25 mm, film 5% phenyl95%methylpolysiloxane
a
1b2c2S GABAA receptors by honokiol derivatives. Emax-dir
1b2c2S GABAA receptors induced by
a
9.25 lm. Oven temperature was kept at 45 °C for 2 min and pro-
grammed to 300 °C at a rate of 3 °C/min, then kept constant at
300 °C for 20 min.
ESI-MS were recorded in ESI positive and negative mode on a
Thermo Finnigan LCQ Deca XP Plus mass spectrometer with
Compound
Emax-dir (%)
EC50
(
l
M)
nH
n
GABA
H
AMH
1
2
3
4
5
6
7
100
51.0 3.0*
76.2 10.3
68.1 9.7
1.4 0.1
2.6 0.4
2.7 0.4
3.3 0.3
27
4
3
78
63
59
6
6
1
144.3 5.7**
3
autosampler. Column: Zorbax SB-C18 (3.5
lent Technologies) with guard column at a flowrate of 300
l
m; 150 ꢀ 2.1 mm; Agi-
No agonist activity
52
No agonist activity
32
No agonist activity
29
l
L/min.
1
62.2 2.4
6.9 1.0**
33.2 5.1**
2.1 0.1
1.4 0.1
1.3 0.2
3
3
3
The purity of synthesized compounds was verified using HPLC
on an Agilent 1260 series equipped with diode array detector
and by NMR spectroscopy. For analytical HPLC-DAD, an SB-C18
2
2
Zorbax column (3.5
equipped with guard column at a flow rate of 300
l
m; 150 ꢀ 2.1 mm; Agilent Technologies)
l
L/min was
Asterisks indicate statistically significant differences to IHonokiol as follows.
*
used. The gradient elution program was as follows: CH3CN
in water (0 ? 25 min/10 ? 90%, 25 ? 30 min/90 ? 100%, 30 ?
38 min/100%).
p <0.05.
p <0.01.
**
For TLC analysis, precoated Si60 F254 plates (Merck, Darmstadt)
were used. Detection was done by UV/254 nm and spraying with
molybdato-phosphoric acid and subsequent heating.
Compound mixtures were separated by PTLC (Merck; PLC silica
gel 60 F254, 1 mm), using cyclohexane/ethyl acetate mixtures.
Honokiol was purchased from APIChem Technology Co., Hangzhou,
China (purity >98%).
and also display higher potencies compared to the parent molecule
honokiol.20 Based on these findings, 7 nitrogen-containing hono-
kiol derivatives have been synthesized combining molecular fea-
tures that were recognized to be important functional groups
and subsequently studied for IGABA enhancement and direct activa-
tion of GABAA receptors composed of a1b2c2S subunits. Altogether
two compounds of the tested series and two precursors are new
4.2. Synthesis
chemical entities.
Besides their modulatory activity, H, AMH and four of the newly
synthesized derivatives activated GABAA receptors in the absence
of GABA (Fig. 1C and D, Table 3). Partial agonism was most pro-
nounced for H, AMH and 1 followed by 3. Compounds 5 and 7
are only weak partial agonists but apparently more potent on
4.2.1. Synthesis of 2-O-methyl-50-nitro-honokiol (1a)
Nitric acid (65%, 3.6 mmol, 0.25 mL) was added under intense
stirring within ca. 5 s to
a solution of 2-O-methyl-honokiol
(101 mg, 0.360 mmol; synthesis see16) in ethyl acetate (10 mL) at
room temperature. The reaction mixture was stirred for 10 min
and neutralized with NaOH (2 N). The organic phase was separated
and the water phase was extracted with ethyl acetate (3 ꢀ 15 mL).
The combined organic layers were washed with brine (15 mL),
dried over Na2SO4 and concentrated under reduced pressure yield-
ing 115 mg (98%) of methyl-50-nitro-honokiol (1a) as orange oil.
a
1b2
ity was previously reported for H and magnolol (at concentrations
>10
M) by Alexeev et al.,2 though in a different cell system. Our
c2S receptors than the full agonist GABA. Partial agonist activ-
l
data confirm and extend this finding to nitrogenated derivatives
such as AMH, 1, 3, 5 and 7 (Table 3). Remarkably, small structural
changes completely diminish partial agonism while preserving
positive allosteric modulation of GABAA receptors (2, 4, 6 in Tables
2 and 3). First studies with H on mutated GABAA receptors (includ-
1a: IR (ATR, m
max, cmꢁ1): 3209, 3079, 2909, 2835, 1638, 1621,
1536, 1498, 1464, 1431, 1323, 1239, 1179, 1129, 1027, 912, 810,
768, 676, 606; 1H NMR (CDCl3) d 10.97 (s, 1H, OH), 8.15 (d,
J = 1.8 Hz, 1H, H-60), 7.65 (d, J = 1.8 Hz, 1H, H-20), 7.16 (dd, J = 8.4,
2.2 Hz, 1H, H-4), 7.10 (d, J = 2.2 Hz, 1H, H-6), 6.92 (d, J = 8.4 Hz,
1H, H-3), 6.01 (ddt, J ꢂ17, 10.3, 6.8 Hz, 1H, H-2000), 5.97 (ddt, J
ꢂ17, 10.2, 6.6 Hz, 1H, H-200), 5.13 (m, 2H, H-3000), 5.09 (m, 2H,
H-300), 3.80 (s, 3H, OCH3), 3.53 (d, J = 6.6 Hz, 2H, H-1000), 3.38 (d,
ing
a
1(Q240W), essential for the action of neurosteroids; b3(M286W)
,
preventing the action of general anesthetics; b3(T256F) or
a1(T260F)
essential for the interaction with picrotoxine) did not affect allo-
steric modulation of GABAA receptors by either H or magnolol sug-
gesting that these molecules interact with an yet unidentified
binding site.2 We show here that the agonistic activity of H and
the studied nitrogenated derivatives does not correlate with allo-
steric modulation. Future studies will show whether agonistic
13
J = 6.6 Hz, 2H, H-100); C NMR (CDCl3) d 154.8 (C-2), 152.3 (C-40),
139.1 (C-20), 137.5 (C-200), 135.3 (C-2000), 133.3 (C-50), 132.6 (C-5),

M. Bernaskova et al. / Bioorg. Med. Chem. 23 (2015) 6757–6762
6761
130.6 (C-30), 130.5 (C-6), 130.2 (C-10), 129.2 (C-4), 127.8 (C-1),
123.4 (C-60), 116.7 (C-3000), 115.8ꢁ(C-300), 111.4 (C-3), 55.7 (OCH3),
39.3 (C-100), 33.8 (C-1000); MS (ESI ) m/z (%): 324.22 ([MꢁH]ꢁ, 100).
solution was extracted with dichloromethane (3 ꢀ 10 mL). The
organic layer was concentrated to a final volume of 15 mL, washed
with brine, dried over Na2SO4, concentrated under reduced pres-
sure and purified by PTLC (silica, cyclohexane/ethyl acetate 5:3)
4.2.2. Synthesis of 50-amino-2-O-methylhonokiol (1)
to yield 2 (22 mg, 34%) as a brown oil yield 2: IR (ATR, mmax,
SnCl2 ꢀ 2H2O (70 mg, 0.310 mmol) was added to a solution of
2-O-methyl-50-nitro-honokiol (1a) (98 mg, 0.301 mmol) in MeOH
(10 mL) and was stirred for 72 h at room temperature, an addi-
tional amount of SnCl2 ꢀ 2H2O (100 mg, 0.443 mmol) was added
and stirring was continued for 24 h. The foamy precipitate result-
ing from the addition of NaHCO3 (1 N, 20 mL) was filtered off with
CeliteÒ and rinsed with EtOH (30 mL). After evaporation of the
alcohols the resulting mixture was extracted with dichloro-
methane (3 ꢀ 10 mL). The combined extracts were dried over
Na2SO4, concentrated under reduced pressure and purified by PTLC
(silica, cyclohexane/ethyl acetate 5:3) to yield 1 (25 mg, 39%) as a
cmꢁ1): 3374, 3313, 3075, 2976, 2922, 1638, 1607, 1489, 1437,
1472; 1410, 1392, 1236, 1142, 993, 909, 805, 732; 1H NMR (CDCl3)
d 7.13 (s, 1H, H-6), 7.06 (d, J = 8.3 Hz, 1H, H-4), 6.91 (s, 1H, H-60),
6.88 (d, J = 8.3 Hz, 1H, H-3), 6.82 (s, 1H, H-20), 6.06 (ddt, J = 16.9,
10.2, 5.9 Hz, 1H, H-2000), 5.99 (ddt, J = 16.8, 9.9, 6.6 Hz, 1H, H-200),
5.26 (d, J = 17.2 Hz, 1H, H-3000), 5.20 (d, J = 10.1 Hz, 1H, H-3000), 5.09
(d, J = 17.0 Hz, 1H, H-300), 5.08 (d, J = 10.4 Hz, 1H, H-300), 4.00 (q,
J = 6.8 Hz, 2H, OCH2), 3.44 (d, J = 5.9 Hz, 2H, H-1000), 3.36 (d,
J = 6.6 Hz, 2H, H-100), 1.34 (t, J = 6.8 Hz, 3H, OCH3CH3); 13C NMR
(CDCl3) d 154.2 (C-2), 142.3 (C-4), 137.8 (C-200), 136.7 (C-2000),
132.3 (C-5), 131.4 (C-10), 130.9 (C-6), 130.8 (C-1), 127.8 (C-4),
125.2 (C-30), 122.1 (C-20), 117.0 (C-60), 116.4 (C-3000), 115.4 (C-300),
111.3 (C-3), 55.6 (OCH3), 39.3 (C-100), 35.6 (C-1000); MS (ESI) m/z
(%): 310.14 [M+H]+ (100).
brown oil. 1: IR spectra (ATR,
m
max, cmꢁ1): 3373, 3313, 3074,
3000, 2974, 2903, 2832, 1637, 1606, 1488, 1240, 1141, 907, 809;
1H NMR (CDCl3) d 7.12 (s, 1H, H-6), 7.10 (d, J = 8.8 Hz, 1H, H-4),
6.90 (d, J ꢂ8 Hz, 1H, H-3), 6.89 (s, 1H, H-60), 6.75 (s, 1H, H-20),
6.00 (ddt, J = 16.9, 10.2, 6.4 Hz, 1H, H-2000), 6.04 (ddt, J = 16.9, 10.9,
6.6 Hz, 1H, H-200), 5.28 (d, J = 17.6 Hz, 1H, H-3000), 5.21 (d,
J = 9.9 Hz, 1H, H-3000), 5.11 (dq, J = 16.9, 1.2 Hz, 1H, H-300), 5.07 (d,
J ꢂ8 Hz, 1H, H-300), 3.79 (s, 3H, OCH3), 3.45 (d, J = 6.0 Hz, 2H,
H-1000), 3.37 (d, J = 6.5 Hz, 2H, H-100); 13C NMR (CDCl3) d 154.8
(C-2), 142.3 (C-4), 137.8 (C-200), 136.7 (C-2000), 134.4 (C-50), 132.1
(C-5), 131.3 (C-10), 131.0 (C-6), 130.5 (C-1), 127.8 (C-4), 124.7
(C-30),122.1 (C-20), 117.0 (C-60), 116.7 (C-3000), 115.5 (C-300),
111.2 (C-3), 55.7 (OCH3), 39.4 (C-100), 36.0 (C-1000); MS (ESI) m/z
(%): 296.17 [M+H]+ (100).
4.3. Pharmacological experiments
4.3.1. Expression of GABAA receptors in Xenopus laevis oocytes
and two-microelectrode voltage-clamp experiments
Preparation of stage V–VI oocytes from Xenopus laevis and syn-
thesis of capped runoff poly(A) cRNA transcripts from linearized
cDNA templates (pCMV vector) was performed as previously
described.9 Female Xenopus laevis frogs (NASCO, USA) were anes-
thetized by 15 min incubation in a 0.2% MS-222 (methane sul-
fonate salt of 3-aminobenzoic acid ethyl ester; Sigma Aldrich,
Vienna, Austria) solution before removal of parts of the ovaries.
Follicle membranes from isolated oocytes were enzymatically
digested with 2 mg/mL collagenase (Type 1A, Sigma–Aldrich,
Vienna, Austria).
Selected oocytes were injected with 10–50 nL of DEPC-treated
water (diethyl pyrocarbonate, Sigma, Vienna, Austria) containing
the different GABAA cRNAs at a concentration of approximately
300–3000 pg/nL/subunit. To ensure expression of the c2S subunit
4.2.3. Synthesis of 2-O-ethyl-50-nitro-honokiol (2a)
Nitric acid (65%, 0.182 mL, 2.62 mmol) was added under intense
stirring within ca. 5 s to a solution of 2-O-ethyl-honokiol (77 mg,
0.262 mmol; synthesis see16) in ethyl acetate (10 mL) at room tem-
perature. The reaction mixture was stirred for 60 s and carefully
neutralized with NaOH (2 N). The organic phase was separated,
and the aqueous phase was extracted with ethyl acetate
(3 ꢀ 15 mL). The combined organic phases were washed with brine
(3 ꢀ 15 mL), dried over Na2SO4, and concentrated under reduced
pressure. Because of incomplete reaction the residue was solved
again in ethyl acetate (10 mL) nitration and workup were repeated
but with a reaction time of 10 min resulting in 88 mg of 2-O-ethyl-
50-nitro-honokiol (2a) as an orange oil, yield 98%.
in the case of
a1b2c2S receptors, cRNAs were mixed in a ratio of
1:1:10. The amount of cRNAs was determined by means of a Nano-
Drop ND-1000 (Kisker-Biotech, Steinfurt, Germany).
Oocytes were stored at +18 °C in modified ND96 solution
(90 mM NaCl, 1 mM CaCl2, 1 mM KCl, 1 mM MgCl2 ꢀ 6H2O, and
5 mM HEPES (4-(2-hydroxyethyl)-1-piperazine-ethane-sulfonic
acid); pH 7.4, all from Sigma–Aldrich, Vienna, Austria).
IR (ATR, m
max, cmꢁ1): 3204, 3079, 2978, 1638, 1621, 1536, 1499,
1466, 1323, 1238, 1129, 1042, 912, 674, 551; 1H NMR (CDCl3) d
10.97 (s, 1H, OH), 8.20 (d, J = 2.2 Hz, 1H, H-60), 7.73 (d, J = 1.5 Hz,
1H, H-20), 7.13 (d, J ꢂ7, 1H, H-4), 7.12 (s, 1H, H-6), 6.90 (d,
J = 8.8 Hz, 1H, H-3), 6.02 (ddt, J ꢂ17, 10.3, 6.7 Hz, 1H, H-2000), 5.98
(ddt, J ꢂ17, 9.9, 6.6 Hz, 1H, H-200), 5.15 (m, 2H, H-3000), 5.09 (m, 2H,
H-300), 4.03 (q, J = 6.9 Hz, 2H, OCH2CH3), 3.53 (d, J = 6.6 Hz, 2H,
H-1000), 3.38 (d, J = 6.6 Hz, 2H, H-100), 1.35 (t, J = 6.9 Hz, 3H, OCH2CH3);
13C NMR (CDCl3) d 154.1 (C-2), 152.2 (C-40), 139.3 (C-20), 137.5 (C-
200), 135.3 (C-2000), 133.3 (C-50), 132.5 (C-5), 130.4, 2 x 130.3 (C-6,
C-10, C-30), 129.2 (C-4), 127.7 (C-1), 123.3 (C-60), 116.8 (C-3000),
115.8 (C-300), 112.5 (C-3), 64.1 (OCH2CH3), 39.3 (C-100), 33.7 (C-1000),
14.8 (OCH2CH3); MS (ESI) m/z (%): 340.24 ([M+H]+, 100).
Chloride currents through GABAA receptors (IGABA) were mea-
sured at room temperature (+21 1 °C) by means of the two-mi-
croelectrode voltage clamp technique making use of a TURBO
TEC-05X amplifier (npi electronic, Tamm, Germany). IGABA were eli-
cited at a holding potential of ꢁ70 mV. Data acquisition was car-
ried out by means of an Axon Digidata 1322A interface using
pCLAMP v.10 (Molecular Devices, Sunnyvale, CA, USA). The modi-
fied ND96 solution was used as bath solution. Microelectrodes
were filled with 2 M KCl and had resistances between 1 and 3 MX.
4.3.2. Perfusion system
GABA and the studied derivatives were applied by means of the
ScreeningTool (npi electronic, Tamm, Germany) perfusion system
as described previously.6,9 To elicit IGABA, the chamber was per-
4.2.4. Synthesis of 50-amino-2-O-ethylhonokiol (2)
SnCl2 ꢀ 2H2O (426 mg, 1.89 mmol) was added to a solution of
2-O-ethyl-50-nitro-honokiol (2a) (71 mg, 0.21 mmol) in EtOH
(10 mL). After stirring for 72 h at room temperature NaHCO3
(1 N, 30 mL) was added. The foamy precipitate was filtered off with
CeliteÒ and rinsed with EtOH (5 ꢀ 10 mL). The solutions were
concentrated under reduced pressure and the resulting aqueous
fused with 120
respectively, at a volume rate of 300
account for possible slow recovery from increasing levels of desen-
sitization in the presence of high drug concentrations. The duration
of washout periods was therefore extended from 1.5 min (<10
compounds) to 30 min (P10 compounds), respectively.
l
L of GABA- or compound-containing solutions,
l
L/s.11 Care was taken to
lM
lM

6762
M. Bernaskova et al. / Bioorg. Med. Chem. 23 (2015) 6757–6762
Oocytes with maximal current amplitudes >3
exclude voltage clamp errors.
lA were discarded to
References and notes
1. Ai, J.; Wang, X.; Nielsen, M. Pharmacology 2001, 63, 34.
2. Alexeev, M.; Grosenbaugh, D. K.; Mott, D. D.; Fisher, J. L. Neuropharmacology
2012, 62, 2507.
4.3.3. Data analysis
Stimulation of chloride currents by modulators of the GABAA
receptor was measured at a GABA concentration eliciting between
3% and 7% of the maximal current amplitude (EC3–7). The GABA
EC3–7 was determined for each oocyte individually. Enhancement
3. Baburin, I.; Khom, S.; Timin, E.; Hohaus, A.; Sieghart, W.; Hering, S. Brit. J.
Pharmacol. 2008, 424.
4. Baur, R.; Schuehly, W.; Sigel, E. Biochim. Biophys. Acta 2014, 1840, 3017.
5. Bernaskova, M.; Kretschmer, N.; Schuehly, W.; Huefner, A.; Weis, R.; Bauer, R.
Molecules 2014, 19, 1223.
6. Baburin, I.; Beyl, S.; Hering, S. Pflugers Arch.—Eur. J. Physiol. 2006, 453, 117.
7. Hevers, W.; Luddens, H. Mol. Neurobiol. 1998, 18, 35.
8. Johnson, T. W.; Corey, E. J. J. Am. Chem. Soc. 2001, 123, 4475.
9. Khom, S.; Baburin, I.; Timin, E. N.; Hohaus, A.; Sieghart, W.; Hering, S. Mol.
Pharm. 2006, 69, 640.
10. Khom, S.; Strommer, B.; Ramharter, J.; Schwarz, T.; Schwarzer, C.; Erker, T.;
Ecker, G. F.; Mulzer, J.; Hering, S. Br. J. Pharmacol. 2010, 161, 65.
11. Khom, S.; Strommer, B.; Schöffmann, A.; Hintersteiner, J.; Baburin, I.; Erker, T.;
Schwarz, T.; Schwarzer, C.; Zaugg, J.; Hamburger, M.; Hering, S. Biochem. Pharm.
2013, 85, 1827.
12. Macdonald, R. L.; Olsen, R. W. Annu. Rev. Neurosci. 1994, 17, 569.
13. Maruyama, Y.; Kuribara, H.; Morita, M.; Yuzurihara, M.; Weintraub, S. T. J. Nat.
Prod. 1998, 61, 135.
14. Maruyama, Y.; Kuribara, H. CNS Drug Rev. 2000, 6, 35.
15. Rudolph, U.; Crestani, F.; Mohler, H. Trends Pharmacol. Sci. 2001, 22, 188.
16. Schuehly, W.; Viveros Paredes, J. M.; Kleyer, J.; Huefner, A.; Raduner, S.;
Altmann, K.-H.; Gertsch, J. Chem. Biol. 2011, 18, 1053.
of the chloride current was defined as I(GABA+compound)
/
IGABA-max * 100%, where I(GABA+compound) is the current response in
the presence of a given compound and IGABA-max is the current
response induced by 1 mM GABA. Concentration–response curves
were generated and the data were fitted by nonlinear regression
analysis using Origin Software (OriginLab Corporation, USA). Data
were fitted to the equation 1/(1 + (EC50/[compound])nH), where
nH is the Hill coefficient. Each data point represents the mean SE
from at least 3 oocytes and P2 oocyte batches. Statistical signifi-
cance was calculated using paired Student t-test with a confidence
interval of <0.05.
Acknowledgements
17. Sieghart, W.; Sperk, G. Curr. Top. Med. Chem. 2002, 2, 795.
18. Sieghart, W.; Ernst, M. Curr. Med. Chem. 2005, 5, 217.
19. Sieghart, W. Adv. Pharmacol. 2015, 72, 53.
20. Taferner, B.; Schuehly, W.; Huefner, A.; Baburin, I.; Wiesner, K.; Ecker, G. F.;
Hering, S. J. Med. Chem. 2011, 54, 5349.
21. Wafford, K. A.; Bain, C. J.; Whiting, P. J.; Kemp, J. A. Mol. Pharmacol. 1993, 44,
437.
22. Widdowson, K. L.; Elliott, J. D.; Veber, D. F.; Nie, H.; Rutledge, M. C.; McCleland,
B. W.; Xiang, J.-N.; Jurewicz, A. J.; Hertzberg, R. P.; Foley, J. J.; Griswold, D. E.;
Martin, G. L.; Lee, J. M.; White, J. R.; Sarau, H. M. J. Med. Chem. 2004, 47, 1319.
23. Wieland, H. A.; Luddens, H.; Seeburg, P. H. J. Biol. Chem. 1992, 267, 1426.
Parts of this work were supported by the Austrian Science Fund
(FWF) under project P21241. This work was supported by the Aus-
trian Science Fund (FWF doctoral program ‘‘Molecular Drug Tar-
gets” W1232 to S.H). The authors wish to express their gratitude
to R. Bauer (Graz) for allowing access to analytical HPLC equipment
as well as to the ESI-MS facility.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.08.034.