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Bioorganic & Medicinal Chemistry Letters 25 (2015) 400–403
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
m
Metal-assisted synthesis of unsymmetrical magnolol and honokiol
analogs and their biological assessment as GABAA receptor ligands
Lukas Rycek a, Roshan Puthenkalam b, Michael Schnürch a, Margot Ernst b, Marko D. Mihovilovic a,⇑
a
Vienna University of Technology, Institute of Applied Synthetic Chemistry, Getreidemarkt 9/163-OC, 1060 Vienna, Austria
Medical University of Vienna, Department of Molecular Neurosciences, Spitalgasse 4, 1090 Vienna, Austria
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Article history:
Received 20 August 2014
Revised 27 October 2014
Accepted 28 October 2014
Available online 4 November 2014
Keywords:
Magnolol
Honokiol
Cross-coupling
GABAA
Subtype selectivity
a b s t r a c t
We present the synthesis of new derivatives of natural products magnolol (1) and honokiol (2) and their
evaluation as allosteric ligands for modulation of GABAA receptor activity. New derivatives were prepared
via metal assisted cross-coupling reactions in two consecutive steps. Compounds were tested by means
of two-electrode voltage clamp electrophysiology at the a1b2c2 receptor subtype at low GABA concentrations. We have identified several compounds enhancing GABA induced current (IGABA) in the range
similar or even higher than the lead structures. At 3 lM, compound 8g enhanced IGABA by factor of
443, compared to 162 and 338 of honokiol and magnolol, respectively. Furthermore, 8g at EC10–20 features a much bigger window of separation between the a1b2c2 and the a1b1c2 subtypes compared
to honokiol, and thus improved subtype selectivity.
Ó 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/3.0/).
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Magnolol (1) and honokiol (2) (Scheme 1) are natural products
belonging to the class of neolignans isolated from Magnolia officinalis. They have been widely used in Eastern traditional medicine
for treatment of gastric and psychiatric disorders. Therapeutic
applications of Magnolia constituents were summarized comprehensively in the recent literature.1 Previously, several studies have
revealed additional pharmaceutical effects of magnolol or honokiol
as anti-angiogenetic,2 antiepileptic,3 neuroprotective,4 or antimicrobial and antiproliferative activity.5 Honokiol has been shown
to have somnogenic effects in mice.6 Furthermore, interaction with
the PPARc receptor, involved in treatment of type 2 diabetes, metabolic syndrome, and potential anti-inflammatory target, was
shown as well.7 In the central nervous system, neolignans interact
with GABAA receptors and modulate their GABA-induced activity
leading to the assumption that their antiepileptic and somnogenic
in vivo effects are mediated by these receptors.8,9
The family of GABAA receptors comprises at least 26 distinct
subtypes.10 These pentameric GABA-gated chloride channels
assemble as homo- or heteropentamers in mammals from a repertoire of 19 distinct subunits that belong to eight classes. There are
six a, three b, c and q as well as one d, p, e and h subunits. The most
abundant receptor consists of two a1, two b2 and one c2 subunits.
The GABA binding site in this type of receptor is located at the
extracellular interface between the beta and alpha subunits.11
⇑ Corresponding author. Tel.: +43 1 58801 15420; fax: +43 1 58801 15499.
E-mail address: [email protected] (M.D. Mihovilovic).
OH
OH
R1
R = 2'-OH 1
R = 4'-OH 2
Aromatic Aromatic
ring A
ring B
R2
New
derivatives
Aromatic Aromatic
ring A
ring B
Scheme 1. Structure of lead compounds and of new derivatives.
The properties and the pharmacology of the receptor subtypes
depend on the subunit composition.12 In addition to the site for
the endogenous agonist GABA, at which other agonists or antagonists such as muscimol, bicuculline or gaboxadol also bind, there
are multiple allosteric binding sites, such as the one for benzodiazepines, sites for barbiturates, neurosteroids, and several more.11
Magnolol and analogues have been shown to be allosteric modulators of GABAA receptors, thus their binding site does not overlap
with the one for GABA, muscimol, bicuculline or gaboxadol.8,9 The
antiepileptic and somnogenic effects of magnolol and honokiol,
respectively, have been connected with the benzodiazepine binding site because they can be diminished in vivo by the benzodiazepine antagonist flumazenil.4,6 However, neolignans and several
synthetic analogs have been shown to modulate both GABAA
receptors with and without a benzodiazepine binding site.8,9 The
http://dx.doi.org/10.1016/j.bmcl.2014.10.091
0960-894X/Ó 2014 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).
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L. Rycek et al. / Bioorg. Med. Chem. Lett. 25 (2015) 400–403
benzodiazepine binding site requires the presence of an a and a c2
subunit.12 Recently, it was shown that flumazenil also antagonizes
modulation by aryl pyrazoles in receptors lacking a c2 subunit and,
thus lacking the high affinity benzodiazepine binding site.13 Consequently, flumazenil sensitive effects in vivo do not necessarily indicate that the benzodiazepine binding site is eliciting them.
Certain subunits have been connected with specific pharmacological effects: a1 containing receptors are thought to mediate sedative and addictive effects of benzodiazepines, while activity of
ligands at receptors containing a b1 subunit have been proposed
to mediate ataxic effects.14 With respect to GABAA receptor subtypes, the natural neolignans have been demonstrated as relatively
unselective compounds, and safety concerns have been raised.
Subtype selective synthetic analogs have been proposed to be
potentially useful.9,13,15
Here, we present a facile synthetic strategy towards new magnolol/honokiol derivatives and their evaluation on GABAA receptors. The strength of our approach consists in a simplification of
the final molecules, allowing their easy synthetic access (compared
to the lead structures), while increasing the biological effect at the
same time.
Magnolol and honokiol are small biaryl isomeric molecules
(Scheme 1). Aromatic ring A is identical for both molecules, while
ring B differs in the position of the hydroxyl group. Our approach
was based on leaving the ring A untouched and carrying out simplification of the ring B. Instead of allyl and hydroxyl groups, single
substituents with diverse properties were introduced in all possible positions of ring B.
Very efficiently, magnolol can be accessed by metal or enzyme
mediated oxidative coupling of two 4-allylanisol units and demethylation.16–18 Such strategy, nevertheless, leads inevitably to the
symmetric product. Unsymmetrical honokiol can be prepared by
bromination of 4-allylanisole and subsequent Suzuki coupling with
4-hydroxyphenylboronic acid. Further modifications (including Oallylation, Claisen rearrangement and demethylation) lead to the
desired structure.19 4-Allyl-2-bromoanisole is the key intermediate
which could be used within the synthesis of compounds of our
interest. A drawback of this pathway is the bromination step, where
overbromination of the double bond takes place and debromination
with zinc is necessary, conflicting with aspects of atom efficiency.
Alternatively, directed ortho-lithiation and introduction of the
boronic acid into 4-allylanisole followed by subsequent coupling
with aryl bromide was reported.20,21 However, we encountered
problems with a reproduction of the protocol.
To circumvent the above outlined limitations, we have developed a simple two steps protocol, relying on palladium catalyzed
transformations. In the first step, the allyl moiety is regioselectively introduced into the more reactive 4-position of 4-bromo-2chlorophenol 3 using allylborate. In the second step, Suzuki coupling of 4-allyl-2-chlorophenol 6 with corresponding boronic acids
enabled access to the products of interest. First, we focused our
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attention on optimization of the allylation reaction of 3 and 4
(Scheme 2). Initial experiments utilizing Pd2dba3 as a catalyst, KF
as a base, dioxane/H2O (9:1) mixture as solvent at 150 °C
(30 min) under microwave irradiation led to partial success. Three
different ligands were tested (Table 1, entries 1–3) of which dppf
provided most promising results. Full consumption of the starting
material was not observed, however, formation of the desired
product was accompanied by debromination of the starting material as side reaction, leading to 2-chlorophenol. Changing the palladium source to heterogenous PdEn40Ò, which can be easily
separated by filtration, led to a decrease of the side reaction; however, full consumption of 3 could not be achieved (entry 4). Further
investigation revealed that both conversion and side reaction can
be controlled by the base used. Several bases were investigated
(entries 5–9): using triethylamine or sodium acetate led to full
consumption, however debromination was observed (entries 5
and 6). Slight improvement could be achieved by exploiting
sodium carbonate or sodium hydroxide. However, in all cases, side
product was still formed. Full conversion and complete suppression of the side reaction could be achieved finally by utilizing
potassium carbonate (entry 9). Reaction time could be successfully
shortened to 7 min and no sign of debromination was observed
(entry 10) facilitating isolation of compound 6 in 77% of yield.
Changing pinacol allylboronate 4 to potassium allytrifluoroborate
5 turned out to be beneficial for purification of the product, since
pinacolester fragments often contaminated the reaction product
complicating isolation. After optimization of reaction conditions
target compound 6 was finally isolated in 80% (entry 11).
For the second step, we adopted a modified protocol by Denton
et al.21 for the coupling of phenyboronic acid with chloro compound 6 under microwave irradiation; 4-allyl-2-phenylphenol 8a
was obtained in 58% of yield (Table 2, entry 1), giving an overall
yield of 46%. For comparison, 8a was also synthesized according
to the above discussed pathway (demethylation/bromination of
4-allylanisole and Suzuki coupling), giving a 15% overall yield after
three steps in our hands.22
This facile two-step protocol was subsequently employed
within the preparation of a small library of compounds to obtain
some preliminary structure activity relationship. Indicative substitution patterns contained weakly electron donating methyl groups
O
B
OH
Cl
4
+
OH
150°C, MW,
"Pd", ligand, t
O
or
Cl
Dioxan/H 2O
BF3 K
Br
3
5
6
Scheme 2. Introduction of allyl moiety.
Table 1
Optimization of the reaction conditions for introduction of the allyl moiety
Pd source
Ligand
Borate
Base
Time (min)
Remaining SM
Debromination
Isolated yield (%)
Pd2dba3
Pd2dba3
Pd2dba3
PdEn40
PdEn40
PdEn40
PdEn40
PdEn40
PdEn40
PdEn40
PdEn40
SPhos
±BINAP
dppf
dppf
dppf
dppf
dppf
dppf
dppf
dppf
dppf
4
4
4
4
4
4
4
4
4
4
5
KF
KF
KF
KF
TEA
NaOAc
NaOH
Na2CO3
K2CO3
K2CO3
K2CO3
30
30
30
30
30
30
30
30
30
7
7
Yes
Yes
Yes
Yes
No
No
No
No
No
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Traces
Yes
No
No
No
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
65
77
80
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Entry
1
2
3
4
5
6
7
8
9
10
11
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L. Rycek et al. / Bioorg. Med. Chem. Lett. 25 (2015) 400–403
OH
OH
Cl
R
+
(HO)2 B
R
Pd 2dba 3, SPhos,
Dioxane/H 2O, KF
MW, 150°C,
10 min
7a-g,i-m
6
8a-g,i-m
O
O
(HO)2 B
7h COOMe
m
8h
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Scheme 3. Introduction of the aryl moiety.
Table 2
Synthesized derivatives and their effect at a1b2c2 GABAA receptor
Compound
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
2
1
A methoxy group in meta (8f) or para (8g) position is beneficial;
similar behavior was encountered in case of a m-methoxycarbonyl
group (8i).
Surprisingly, also high efficacy was achieved with the o-methyl
group (8b). Compounds with a nitro group did not show any effect.
Since both electron donating (methyl, methoxy) as well as electron withdrawing (methoxycarbonyl) substituents can increase the
efficacy, a hydrogen bond formation might be involved.
To investigate selectivity, the concentration dependent effect of
8g, the best performing compound at 3 lM (443 ± 60% stimulation), was characterized in a1b2c2 and a1b1c2 receptors at a
higher GABA concentration (at GABA EC10–20) for a better comparison with published literature (EC10–20),8 and compared with honokiol. In a1b2c2 the EC50 value was at approximately 7 lM,
(20 lM for honokiol) and the efficacy at 100 lM was 539 ± 98%.
The EC50 in the b1 containing subtype is 30 lM (20 lM for honokiol), the efficacy only 206 ± 60%. The effect of 8g in a mutant
a1b2N265Sc2 receptor was investigated at EC0.5 (see Supplementary Fig. 1). This mutation in the b2 subunit is known to reduce
the efficacy of several compounds including loreclezole and etomidate. With 8g we observed a pronounced drop in response, confirming the key role of b2N265 for the beta subtype selectivity.
The stimulation in the wild type receptor at the same compound
concentration is for comparison 2462%.
Compounds not acting on b1 receptors are thought to be nonsedative—a desired property for compounds used as anxiolytic or
anticonvulsive medication (Fig. 1).14
In conclusion, we present a rapid and facile synthetic approach
to magnolol and honokiol analogs with unsymmetrical aryl substitution employing consecutive cross coupling reactions. The protocol turned out versatile towards highly diverse aryl coupling
partners.
Biological assessment of a first small product library revealed
compound 8g to possess a highly interesting combination of
increased potency compared to the natural analogue in the most
abundant a1b2c2 receptor subtype, while showing strongly
reduced responses in the b1 containing subtype that has been connected to ataxic effects.14 Such an action profile has not been
reported for synthetic neolignan derivatives or the natural products, so far, underscoring the relevance of this report.8 It has been
suggested that compounds with antiepileptic or anxiolytic properties would have fewer side effects if they are inactive in b1 containing receptor subtypes14 The activity difference we report here for
our lead compound is much more pronounced compared to the
natural products or other synthetic analogs for which this has been
studied.8,9
Based on these preliminary findings, exploitation of the synthetic strategy towards investigation of further substitution effects
R
Yield
% IGABA at 3 lM
H
o-Me
m-Me
p-Me
o-OMe
m-OMe
p-OMe
Lactone
m-COOMe
p-COOMe
o-NO2
m-NO2
p-NO2
—
—
58
34
39
46
54
48
34
31
34
36
42
86
60
—
—
Inactive
207 ± 33
Inactive
84 ± 11
Inactive
136 ± 32
443 ± 60
Inactive
406 ± 70
Inactive
Inactive
Inactive
Inactive
162 ± 31
338 ± 93
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in positions ortho, meta and para, strongly electron donating methoxy groups and methoxycarbonyl as well as nitro groups in all possible positions as representative electron withdrawing
substituents. Only in a single case we have encountered a problem
with this synthetic route: ortho-methoxycarbonyl substitution
resulted in spontaneous lactonization, since a stable six-membered
ring was formed (Scheme 3, 8h). All compounds were obtained in
moderate to good yields employing the outlined reaction sequence
(Table 2).
The ability of the synthesized honokiol derivatives to modulate
recombinantly expressed a1b2c2 GABAA receptors from rat was
determined in Xenopus laevis oocytes (Table 2). Cells were clamped
at a holding potential of 80 mV. A GABA concentration titrated to
trigger 0.5% of the respective maximum GABA-elicited current of
the individual oocyte (=GABA EC0.5) was applied together with a
final concentration of 3 lM of the compound in the measurement
buffer. Use of low GABA concentration allows detecting compounds exerting low efficacy modulation. Compounds were
applied for 20 s. None of the compounds could open the channel
without GABA, so they all are only modulators of the channel.
For details on the electrophysiological recordings see Lüscher
et al.23
Data was obtained from 5 oocytes from at least 4 different
oocyte batches for the active compounds; inactives were measured
with n = 2. The response to 3 lM honokiol (2) was 162 ± 31% of the
response to an EC0.5 GABA concentration and two of the tested
compounds (8g and 8i) possess higher efficacy than the natural
product.
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Figure 1. Dose–response curves for IGABA potentiation by 8g and honokiol in
a1b1c2 and a1b2c2 using GABA EC10–20.
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Acknowledgements
This project was in part supported by the Austrian Science Fund
(FWF): S10710 (NFN Drugs from Nature Targeting Inflammation).
Financial support by the graduate school program Molecular Drug
Targets MolTag (FWF, grant no. W1232) to L.R. and R.P. is acknowledged. The authors gratefully acknowledge Prof. Erwin Sigel (University of Bern) for hosting R.P. during an internship to collect
additional data for this publication.
Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.1
0.091.
8. Taferner, B.; Schuehly, W.; Huefner, A.; Baburin, I.; Wiesner, K.; Ecker, G. F.;
Hering, S. J. Med. Chem. 2011, 54(15), 5349.
9. Alexeev, M.; Grosenbaugh, D. K.; Mott, D. D.; Fisher, J. L. Neuropharmacology
2012, 62(8), 2507.
10. Olsen, R. W.; Sieghart, W. Neuropharmacology 2008, 56(1), 141.
11. Sieghart, W. Pharmacol. Rev. 1995, 47(2), 181.
12. Sieghart, W.; Ernst, M. Curr. Med. Chem.: Cent. Nerv. Syst. Agents 2005, 5(3), 217.
13. Mascia, M. P.; Ledda, G.; Orru, A.; Marongiu, A.; Loriga, G.; Maciocco, E.; Biggio,
G.; Ruiu, S. Eur. J. Pharmacol. 2014, 733, 1.
14. Gee, K. W.; Tran, M. B.; Hogenkamp, D. J.; Johnstone, T. B.; Bagnera, R. E.;
Yoshimura, R. F.; Huang, J.-C.; Belluzzi, J. D.; Whittemore, E. R. J. Pharmacol. Exp.
Ther. 2010, 332(3), 1040.
15. Paola Mascia, M.; Fabbri, D.; Antonietta Dettori, M.; Ledda, G.; Delogu, G.;
Biggio, G. Eur. J. Pharmacol. 2012, 693(1–3), 45.
16. Tzeng, S.-C.; Liu, Y.-C. J. Mol. Catal. B: Enzym. 2004, 32(1–2), 7.
17. Kong, Z.-L.; Tzeng, S.-C.; Liu, Y.-C. Bioorg. Med. Chem. Lett. 2005, 15(1), 163.
18. Tamura, H. Jpn. Kokai Tokkyo Koho, Chem. Abstr. 2007, 1442479.
19. Chen, C.-M.; Liu, Y.-C. Tetrahedron Lett. 2009, 50(10), 1151.
20. Denton, R. M.; Scragg, J. T. Synlett 2010, 633.
21. Denton, R. M.; Scragg, J. T.; Galofre, A. M.; Gui, X.; Lewis, W. Tetrahedron 2010,
66(40), 8029.
22. General procedures: 4-Bromo-2-chlorophenol (1.5 g, 7.25 mmol) was charged
into a microwave vial, together with potassium allyltriflouroborate (534.2 mg,
3.61 mmol), Pd(En)30TM (300 mg, 0.05 equiv), dppf (134 mg, 0.1 equiv) and
K2CO3 (667 mg, 14.5 mmol). A mixture of dioxane/water (9:1) was added
(20 mL), the vial was flushed with argon and sealed. The reaction solution was
heated to 150 °C for 7 min in a microwave oven (Biotage Initiator 60). After
complete reaction, the mixture was filtered through a pad of Celite, the solvent
was evaporated under reduced pressure and compound 6 was purified via
Kugelrohr distillation to give 80% of a yellowish oil. This material (80 mg,
0.47 mmol) was charged into a microwave vial, together with potassium
fluoride (67.2 mg, 1.17 mmol), Pd2dba3 (21.5 mg, 0.05 equiv), SPhos (19.3 mg,
0.1 equiv) and the corresponding boronic acid (0.71 mmol, 1.5 equiv). A
dioxane/water mixture (9:1) was added (5 mL), the vial was flushed with
argon and sealed. The reaction solution was heated to 150 °C for 10 min in a
microwave oven (Biotage Initiator 60). After the reaction was finished, the
mixture was filtered through a pad of Celite and the solvent was evaporated
under reduced pressure; the crude material was absorbed onto silicagel and
purified via column chromatography using PE/EtOAc mixture (0–5% gradient if
not stated differently—see electronic Supporting material).
23. Lüscher, B. P.; Baur, R.; Goeldner, M.; Sigel, E. PLoS ONE 2012, 7(7), e42101.
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in an elaborated compound library is presently underway in our
laboratories.
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