Developing piperine towards TRPV1 and GABAA receptor ligands - synthesis of piperine analogs via Heck-coupling of conjugated dienes

Article abstract of DOI:10.1039/c4ob02242d

Piperine, the pungent alkaloid of black pepper, and several of its derivatives are modulators of γ-amino butyric acid type A (GABA_A) receptors. Concomitantly, this natural product has also been reported to activate transient receptor potential vanilloid type 1 (TRPV1) receptors. We have developed a Heck cross-coupling reaction of conjugated dienamides enabling the rapid assembly of piperine derivatives containing a modified aromatic core. Upon assessment of a focussed compound library, key aromatic substituents were identified selectively affecting either the GABA_A or the TRPV1 receptor.

Full text of DOI:10.1039/c4ob02242d

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Developing piperine towards TRPV1 and GABAA
receptor ligands – synthesis of piperine analogs
via Heck-coupling of conjugated dienes†
Cite this: DOI: 10.1039/c4ob02242d
Received 22nd October 2014,
Accepted 10th November 2014
Laurin Wimmer,a David Schönbauer,a Peter Pakfeifer,b Angela Schöffmann,b
DOI: 10.1039/c4ob02242d
Sophia Khom,b Steffen Heringb and Marko D. Mihovilovic*a
www.rsc.org/obc
Piperine, the pungent alkaloid of black pepper, and several of its derivatives with GABAA and TRPV1 receptors could lead to
derivatives are modulators of γ-amino butyric acid type A (GABAA) unwanted side effects.
receptors. Concomitantly, this natural product has also been
In a most recent study6 we have modified the amide func-
reported to activate transient receptor potential vanilloid type 1 tionality as well as the linker region of the natural product to
(TRPV1) receptors. We have developed a Heck cross-coupling reac- investigate the effect of such structural modifications on its
tion of conjugated dienamides enabling the rapid assembly of pharmacological activity. Analyzing the modulation of GABA-
piperine derivatives containing a modified aromatic core. Upon induced chloride currents through the GABAA activity of these
assessment of a focussed compound library, key aromatic substitu- derivatives has revealed a strong preference for di-n-butyl and
ents were identified selectively affecting either the GABAA or the di-n-propyl amide. The scaffold has proven to be highly sensi-
TRPV1 receptor.
tive to modifications of the linker region – all attempted modi-
fications led to a significant loss of efficiency.
Piperine, the pungent alkaloid of piper nigrum, was recently
identified as a positive allosteric modulator of γ-amino butyric
acid type A (GABAA) receptors.1 Pharmaceutical compounds
modulating this receptor and thus enhancing neuronal GABA-
ergic inhibition, like benzodiazepines, are widely used as
anxiolytics, sleep-inducing agents as well as for the treatment
of convulsive disorders and other disease states.2
The pungency of piperine is caused by its ability to activate
transient receptor potential vanilloid type 1 (TRPV1) recep-
tors.3 These receptors are non-selective cation channels which
serve as sensors for pain-inducing stimuli like capsaicin,
acidic conditions and heat and are also involved in tempera-
ture regulation of the human body.4 Due to the receptors’
involvement in pain processing, TRPV1 agonists and antagon-
ists are currently under investigation as agents for the treat-
ment of neuropathic pain and other diseases.5 With regard to
a further role of piperine derivatives in the formation of a
prospective pharmacological lead compound, selectivity for
either of these receptors would be highly desirable. However,
the simultaneous interaction of piperine and (potentially) its
With the goal of synthesizing a library of aryl-modified
piperine derivatives in mind, we required a robust synthetic
method which would allow us to synthesize the desired
aryldienamides in a minimum number of steps and a high
level of modularity with respect to the aryl residues.
Although at present a plethora of methods for the assembly
of 1-carbonyl-4-aryl substituted dienes exist, there is a demand
for the development of modern and efficient methods,7 includ-
ing Wittig reactions,8 metathesis,9 transition-metal catalyzed
ene–ene10 and ene–yne11 coupling reactions and C–H acti-
vation reactions.12 These methods typically assemble the 1,3-
diene from 2 + 2 or 3 + 1 carbon synthons with the require-
ment for pre-functionalization of both coupling partners. In
this context, coupling reaction of suitably substituted dienoic
acid derivatives with an aryl coupling partner is attractive.
Such
a reaction was recently reported by Maulide and
coworkers:13 they prepared 5-halodienoic derivatives from
cyclobutene lactones and coupled these compounds in a
Suzuki–Miyaura cross-coupling reaction with arylboronic
acids.
In this project, we chose to employ a Heck cross-coupling
reaction, which is appealing for several reasons: good atom
economy, the diene coupling partner can be easily prepared in
a single step from commercial material, substituted aryl-
bromides are abundantly available and the reaction can be
expected to be E-selective.14
aInstitute of Applied Synthetic Chemistry, Vienna University of Technology,
Getreidemarkt 9, 1060 Vienna, Austria. E-mail: marko.mihovilovic@tuwien.ac.at;
Fax: +43 1 58801 154 99; Tel: +43 1 58801 163615
bDepartment of Pharmacology and Toxicology, University of Vienna, Althanstr. 14,
1090 Vienna, Austria
From the arsenal of metal assisted C–C bond formation
strategies, the Heck-cross coupling reaction, i.e. the palladium-
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4ob02242d
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catalyzed cross-coupling of olefins and aromatic or vinylic tial of this facile access to a compound library for biological
(pseudo)halides, has become an integral part of modern cross- assessment, the modulation of currents through GABAA and
coupling methods.15 The palladium-catalyzed arylation14 and TRPV1 receptors, expressed in Xenopus laevis oocytes by these
vinylation16 of conjugated dienes was first reported by Heck compounds was analyzed by means of the 2-microelectrode
and coworkers in the late 1970s and early 1980s. In the case clamp technique.
of arylation of pentadienoic acid it has been shown that
the reaction occurs at the terminal olefinic position providing
E,E-dienes as products.14
Results and discussion
Given the importance of dienes as synthetic intermediates
and final products, there are only a few precedents of direct
coupling of dienes with suitable coupling partners in the
literature. Arylation has been reported in the presence of silver
or thallium salts,17 in ionic liquids18 or under C–H activation
conditions with benzoxazole as a coupling partner19 as well as
in the total synthesis of galanthamine.20 Vinylation has been
reported under oxidative coupling conditions10 or in rhodium(I)
catalysis21 using boron compounds as coupling partners and
in a tandem hydrozirconation-coupling process.22 Trapping of
the intermediate Pd-π-allyl species by nucleophiles has been
utilized for carbo- and heteroannulation reactions.23
The conditions for the arylation of dienes initially pub-
lished by Heck were not suitable for our purpose: reactions are
conducted without a solvent, which, on a small reaction scale,
leads to impractically small volumes. In our hands, the diene
substrate was also prone to polymerization under these con-
ditions. In the present study we report the optimization of
reaction conditions and the synthesis of a focused library of
aryl-modified piperine derivatives. Demonstrating the poten-
Based on our previous findings and with the aim of further
improving activity of the hit structure towards GABAA modu-
lation,6 we focused on the preparation of piperine derivatives
bearing the non-natural dibutylamide function.
Pentadienoic acid24 was readily converted into its acid
chloride in situ by treatment with oxalylchloride/DMF, followed
by the addition of dibutylamine. Attempts to isolate the acid
chloride led to decomposition in our hands. Alternatively,
pentadienoic acid was smoothly converted into the required
amide in the presence of EDCI·HCl. When kept at −20 °C the
amide displayed a storage stability of several months without
significant degradation.
As a starting point for the optimization of the metal assisted
C–C bond formation reaction, the coupling of 4-bromotoluene
was conducted employing the standard Heck-reaction con-
ditions (Pd(OAc)2, (o-tolyl)3P, NEt3, MeCN, 70 °C).25
The reaction proceeded slowly, giving 52% yield after
72 hours (Table 1, entry 1). Throughout the screening process,
reactions at temperatures at or below the boiling point of the
Table 1 Optimization of coupling conditions
Entry
Solvent
Base
Ligand
T/°C
Time
GC-yield
1
2
3
3
4
5
6
7
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
PhMe
PhMe
PhMe
PhMe
THF
NEt3
NEt3
NEt3
NEt3
(o-Tolyl)3P
(o-Tolyl)3P
(o-Tolyl)3P
(o-Tolyl)3P
(o-Tolyl)3P
(o-Tolyl)3P
(o-Tolyl)3P
(o-Tolyl)3P
(o-Tolyl)3P
(o-Tolyl)3P
(o-Tolyl)3P
(o-Tolyl)3P
(o-Tolyl)3P
(o-Tolyl)3P
(o-Tolyl)3P
(o-Tolyl)3P
(o-Tolyl)3P
(o-Tolyl)3P
(o-Tolyl)3P
(o-Tolyl)3P
(o-Tolyl)3P
(o-Tolyl)3P
(2-Furyl)3P
(p-ClPh)3P
(1-Naphthyl)3P
Pd(PPh3)4
Dppf
70
72 h
3 h
3 h
1 h
1 h
1 h
1 h
1 h
1 h
1 h
1 h
1 h
1 h
1 h
1 h
1 h
13 h
1 h
1 h
1 h
1 h
1 h
1 h
1 h
1 h
1 h
1 h
1 h
1 h
1 h
52%
31%
31%
13%
4%
3%
16%
3%
1%
1%
4%
2%
140, mw
160, mw
160, mw
160, mw
160, mw
160, mw
160, mw
160, mw
160, mw
160, mw
160, mw
160, mw
160, mw
160, mw
160
160
160
160
160
160
140
140
140
NaOAc
NaHCO3
K2CO3
NEt3
NaOAc
NaHCO3
K2CO3
NEt3
NaOAc
NaHCO3
K2CO3
NEt3
NaOAc
NaHCO3
K2CO3
K2CO3 + NEt4Cl
K2CO3 + NEt4Br
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
THF
THF
THF
3%
2%
48%
16%
49%
53%
79%
76%
75%
77%
3%
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
8%
140
140
140
140
140
140
21%
42%
76%
79%
87%
89%
Cy3P
Dppp
JohnPhos
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reaction solvent were carried out in screw-cap vials heated in a
metal block. Reactions which required higher temperatures
were carried out in a microwave reactor, which facilitates auto-
mated and safe handling of pressurized vessels (an experiment
comparing these heat-sources showed that they can be used
interchangeably for this transformation). Increasing the temp-
erature to 140 °C or 160 °C both gave 31% of GC-yield after
only three hours (entries 2 and 3). An extension of the reaction
time was not attempted in these cases, since the mass balance
indicated significant decomposition of the starting materials.
First, a set of four bases (NEt3, NaHCO3, K2CO3 and NaOAc)
and four solvents (MeCN, toluene, THF and DMF) were evalu-
ated (entries 3–18). While toluene and THF did not facilitate
coupling in combination with most bases, the best results
were obtained with DMF as the solvent (entries 15–18). Out of
the set of bases tested, K2CO3 proved most effective in all sol-
vents (entries 6, 10, 14, 18), particularly in DMF (79% GC yield,
entry 18). Quaternary ammonium salts as additives, which can
be beneficial in Heck-couplings,26 did not improve the reac-
tion (entries 19 and 20).
An improvement in the side-product profile as judged by
GC was achieved by lowering the reaction temperature
to 140 °C, while the reaction yield was unaffected (entries
18 and 21).
Finally, a set of eight mono- and bidentate phosphine
ligands were tested in combination with palladium(II) acetate.
The use of (Pd(dba)2) as a palladium source was also investi-
gated, but it gave generally lower conversions (see ESI†). With
respect to the ligands the best results were obtained with John-
Phos and dppp (87–89% GC yield, Table 1, entries 28 and 29).
Compared to JohnPhos, dppp has a lower price and was there-
fore selected for the final reaction protocol.
Scheme 1 Compound library. (a)
100 °C, 16 h; (b) 44 h, 2 mol% of catalyst added after 1 h, 1 equiv. of
4-bromopyridine added after 16 h.
2
equiv. of 3-bromothiophene,
tors was studied at 100 µM. Compared to the natural product
piperine, compounds 4 (783 72%, p < 0.001), 6 (883 70%,
p < 0.001), 15 (570 113, p < 0.05), 16 (970 244%, p < 0.001)
and 18 (782 62%, p < 0.001) displayed a significantly more
pronounced IGABA enhancement, while IGABA modulation by
the other prepared compounds did not significantly differ
from that of piperine (226 26% at 100 µM; data taken from
ref. 1, see Fig. 1A).
After establishing an optimized set of reaction parameters
for the required reaction, the robustness of the protocol was
investigated (Scheme 1). Coupling proceeded smoothly for a
variety of aryl bromides bearing electron donating (4, 6–9) or
electron withdrawing substituents (12–15). In the reactions of
bromochlorobenzenes the chloro-substituent was inert under
the reaction conditions (10 and 11). In the case of 3-bromo-
thiophene the product was obtained in a low yield of 35%.
3- and 4-bromopyridines were well accepted giving products 17
and 18 in 59% and 62% yield, respectively. However, 2-substi-
tuted heterocycles (aimed at compound 19) failed to undergo
coupling. The same was observed in the cases of 2-bromothio-
phene and 2-bromothiazole. This indicates that complexation
by the neighboring heteroatom could be responsible for the
detrimental effect on the reaction in these cases. Concerning
regio- and stereoselectivity of the reaction, all final products
were isolated as the 2E,4E-dienamides. However, GC-MS analy-
sis of the crude reaction mixture typically showed several
minor peaks with the same m/z ratio as the products, which
are likely to be stereo- and regioisomers. These side products
occurred only in trace amounts and we were therefore unable
to isolate sufficient quantities for their characterization.
The effect of aryl-modifications on the enhancement of
GABA-induced chloride currents (IGABA) through α1β2γ2S recep-
Likewise, the effect on the modulation of capsaicin-induced
currents through the TRPV1 receptors was studied at a con-
centration of 100 µM. As illustrated in Fig. 1B, compound 8
(80
22%, p < 0.001) significantly enhanced the currents
2%,
through TRPV1 channels, while compounds 4 (−90
p < 0.0015), 5 (−59 6%; p < 0.05), 7 (−63 16%; p < 0.01), 9
(−73 10%; p < 0.001), 10 (65 7%; p < 0.01) and 11 (87 2%,
p < 0.001) effectively inhibited them. Products 6, 12, 13, 14, 15,
16, 17 and 18 did not display any significant modulation of
the TRPV1 receptors (representative traces for the modulation
of GABA- and capsaicin-induced currents, respectively, by
selected compounds, see Fig. 1C).
Collectively, these data indicate that slight modifications in
the natural product piperine can lead to a high selectivity for
either the GABAA or the TRPV1 channels.
Most strikingly, compound
Icapsaicin (80 22%, p < 0.001), while it was nearly inactive on
8 significantly enhanced
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receptors (783 72%) and was thus not selective for either
receptor type. Finally, compound 6 was identified as a novel
piperine-derived effective GABAA receptor modulator (883
70%), that did not affect the TRPV1 receptors (−10 3%).
Conclusions
We have developed a facile protocol for the arylation of di-
enamides which facilitates rapid and stereoselective access to
2E,4E-products through operational simplicity and short reac-
tion times. Compared to other protocols, the use of arylbro-
mides instead of boronic acids,13 alkynes,11 alkenes10 or
aldehydes8 comprises a significant advantage in terms of price
and commercial availability. Applying this protocol we have
synthesized a library of 15 compounds. Biological testing has
revealed compounds with a high efficacy and selectivity for
either the GABAA or the TRPV1 receptors. These results are
very promising and a full pharmacological characterization of
the test compounds is currently underway in our laboratories
to be published in due course.
Experimental
The experimental procedures for compound synthesis and bio-
logical testing, as well as the compound characterization data
can be found in the ESI.†
Acknowledgements
L. W. and A. S. were supported by the Austrian Science Fund
(FWF doctoral program “Molecular drug targets” W1232).
Notes and references
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Fig. 1 (A) Modulation of GABA-induced currents through α1β2γ2s GABAA
receptors by 100 µM of the indicated compound. Dashed line represents
the IGABA enhancement by the natural product piperine 100 µM.1a Each
data point represents a mean SEM; asterisks indicate statistical signifi-
cance calculated by one-way ANOVA followed by a Dunnett mean com-
parison test. (B) Modulation of capsaicin-induced currents through
TRPV1 receptors by 100 µM of the indicated compound. Each data point
represents a mean SEM; asterisks indicate statistical significance cal-
culated by one-way ANOVA followed by a Dunnett mean comparison
test. (C) Representative currents for the modulation of GABA-induced
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