Complanadine A, a selective agonist for the Mas-related G protein-coupled receptor X2

Article abstract of DOI:10.1016/j.bmcl.2014.05.060

The first biological target for the natural product complanadine A has been determined. The pseudosymmetric alkaloid functions as a selective agonist for the Mas-related G protein-coupled receptor X2 (MrgprX2), a G protein-coupled receptor that is highly expressed in neurons. Given the potential of MrgprX2 to function as a modulator of pain, complanadine A represents a new chemical probe to selectively interrogate the physiological function of MrgprX2 as well as a potential lead for the development of antihyperalgesics for the treatment of persistent pain. While complanadine A possess agonistic activity the related natural product lycodine, representing half of complanadine A, lacks activity providing a cursory description of the structural requirements for agonistic activity.

Full text of DOI:10.1016/j.bmcl.2014.05.060

Accepted Manuscript
Complanadine A, a selective agonist for the Mas-related G protein-coupled re-
ceptor X2
Trevor Johnson, Dionicio Siegel
PII:
S0960-894X(14)00560-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.05.060
BMCL 21674
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 February 2014
15 May 2014
16 May 2014
Please cite this article as: Johnson, T., Siegel, D., Complanadine A, a selective agonist for the Mas-related G protein-
coupled receptor X2, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.
2014.05.060
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
Leave this area blank for abstract info.
Complanadine A, a selective agonist for the
Mas-related G protein-coupled receptor X2
Trevor Johnson and Dionicio Siegel

Bioorganic & Medicinal Chemistry Letters
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m
Complanadine A, a selective agonist for the Mas-related G protein-coupled
receptor X2
Trevor Johnson and Dionicio Siegel*
Department of Chemistry, The University of Texas at Austin, Austin TX 78712
ARTICLE INFO
ABSTRACT
Article history:
Received
The first biological target for the natural product complandine A has been determined. The
pseudosymmetric alkaloid functions as a selective agonist for the Mas-related G protein-coupled
receptor X2 (MrgprX2), a G protein-coupled receptor that is highly expressed in neurons. Given
the potential of MrgprX2 to function as a modulator of pain, complandine A represents a new
chemical probe to selectively interrogate the physiological function of MrgprX2 as well as a
potential lead for the development of antihyperalgesics for the treatment of persisitent pain.
While complanadine A possess agonistic activity the related natural product lycodine,
representing half of complanadine A, lacks activity providing a cursory description of the
structural requirments for agonistic activity.
Revised
Accepted
Available online
Keywords:
Complanadine A
Mas-related G protein-coupled receptor X2
Pain management
Agonist
2014 Elsevier Ltd. All rights reserved
.
Lycodine
G protein-coupled receptors (GPCRs) expressed in human
nociceptive neurons represent a large and diverse family of
proteins that are essential for the proper detection of exogenous
stimuli. A subfamily of approximately 50 GPCRs known as Mas-
related G protein-coupled receptors (Mrgprs) are expressed in
______
Complanadine A (1), an alkaloid isolated from the club moss
Lycopodium complanatum in 2000, possesses
a
pseudosymmetric, dimeric structure.9 A subsequent reported
isolation of complanadine A (1) as well as complanadine B (3)
was guided by the compounds’ abilities to promote the secretion
of growth factors.10 The structure of complanadine A (1) is a
dimer of another natural product lycodine (2)11. As a result of
their challenging structures this class of natural products has
attracted the interest of synthetic chemists and three completed
syntheses of complandine A have been achieved,12 as well as
syntheses of lycodine (2),13 and the oxygenated derivative
complandine B (3)12c,14 (Figure 1). To our knowledge, there
have been no previous disclosures of biological targets for any
of these compounds.
sensory neurons and play
a
pivotal role in human
somatosensation.1-3 These receptors are responsible for the
detection of stimuli, the modulation of pain, and the mediation
inflammatory responses to nerve damage. While there are rodent
orthologs for a subset of Mrgprs, several receptors are unique to
∗ Corresponding author. Tel.: +1 512 471 2073; fax: +1 512 471 0397
e-mail: dsiegel@cm.utexas.edu
humans and have little homology in animals other than primates.4
Among the Mrgprs lacking murine orthologs is Mas-related G
protein-coupled receptor X2 (MrgprX2). MrgprX2 is highly
expressed in human dorsal root ganglion.5 The related MrgprX1
has been previously proposed as a target for the treatment of
acute and chronic pain.6 Agonists of Mrgprs have been shown to
be function as inhibitors of persistent pain in animal models.7
Additionally Mrgprs have also been implicated in the activation
of histamine-independent itch.8 Herein we report that the natural
product complanadine A is a highly selective agonist, functioning
solely on MrgprX2 in a screen of 165 GPCRs. Given its
selectivity for MrgprX2, complanadine A represents a new and
important chemical tool for the deconvolution of the
physiological function of MrgprX2 as well as a potential lead
compound for the therapeutic remediation of acute and persistent
pain.
Me
Me
H
H
HN
N
H
N
N
H
N
Me
H
N
complanadine A (1)
lycodine (2)
Me
H
O
HN
N
H
N
Me
H
N
complanadine B (3)

Figure 1. Structures of complanadine A (1), lycodine (2), and complanadine
B (3)
route to this sub-target was a conjugate addition of the anion of
TMS-acetonitrile followed by protonation with ethyl salicylate to
Scheme 1. Synthesis of alkyne-nitrile 12 from pulegone (4).
afford ketone 7 with the appropriate arrangement of the three
groups on the cyclohexanone ring.15 An additional transformation
that enabled access to alkyne-nitrile 12 was a copper-catalyzed
diastereoselective displacement of the propargyl acetate of 9 to
provide the corresponding propargyl amine 10, a reaction that
Our synthesis of complanadine A focused on the subtarget
alkyne-nitrile 12, prepared in 11-steps from (+)-pulegone (4) in
14% overall yield.12a The route enabled access to alkyne-nitrile
12 on multi-gram scale (Scheme 1). A key transformation en
Me
Me
Me
Me
H
H
H
TMS
TMS
N
CpCo(CO)2
H
LiHMDS, TMSCl
TBAF
THF, 23 °C
90%
N
Bn
N
THF, 66 °C
85%
N
Bn
N
N
Bn
N
THF, 140 °C
82%
N
Bn
12
TMS
TMS
H
TMS
13
14
15
Me
N
16
H
Me
N
Me
N
CHO
H
H
CoCp(CO)2, PPh3
Pd/C, H2
TBAF
N
OHCN
N
Bn
N
OHCN
dioxane, 101 °C
98%
MeOH, 23 °C
78%
dioxane, 140 °C
43%
Bn
Me
Me
H
H
N
TMS
N
17
18
Me
H
Me
H
HCl
N
H
N
OHCN
N
H
N
HN
MeOH, 50 °C
90%
Me
Me
H
H
N
N
19
complanadine A (1)
Scheme 2. Synthesis of complanadine A (1) from alkyne-nitrile 12.

5-HT1A
5-HT2A
5-HT2B
5-HT2C
5-HT6
ADRB1
ADRB2
ADRB3
APJ
CCK1
CCK2
CRF2
CX3CR1
CXCR1
CXCR2
CXCR3
CXCR4
CXCR5
CXCR6
CysLT1
CysLT2
D1
DP
EP1
Ghrelin
GIP
GPR54
GPR68
GPR91
GPR99
H1
M3
M4
NK3
NMU2
NMU1
NOP
P2Y4
PAC1
PAF
SST3
SST4
SST5
PARS†
TP
CCR1
EP2
GLP-1
M5
CCR10
CCR2B
CCR3
EP3
GLP-2
MC2
PK1
AT1
EP4
GnRH
MC5
NPBW1
NTR1
OPRD1
OPRK1
OPRM1
OT
PK2
A1
BB1
ETA
GPBA
H2
MCHR1
MCHR2
mGlu1
mGlu2
Motilin
MRGD
MRGPRX1
MRGPRX2
NK1
PRP
TRH
A2A
BB2
CCR4
ETB
GOR103
GPR109A
GPR119
GPR120
GPR14
GPR39
GPR40
GPR41
GPR43
H3
PTH1
PTH2
S1P1
S1P2
S1P3
S1P4
S1P5
Secretin
SST2
PARS‡
TSH
A2B
BB3
CCR5
FP
IP1
A3
BDKR2
BLT1
C3AR
C5AR
CAS
CCR6
FPR1
FPR2
FSH
LH
V1A
ADRA1A
ADRA1B
ADRA1D
ADRA2A
ADRA2B
ADRA2C
CCR7
LPA1
LPA2
LPA3
LPA5
M1
V1B
CCR8
OX1
V2
CCR9
D2
GABAB1b
GAL1
GAL2
GCGR
OX2
VPAC1
VPAC2
XCR1
Y2
CGRP1
ChemR23
CRF1
D3
P2Y1
CB1
D4
P2Y11
P2Y2
CB2
D5
M2
NK2
Table 1. G protein-coupled receptors tested for activity against complanadine A (1).
proved highly sensitive to the presence of oxygen.16 Lastly,
cyclization of amino-alcohol 11 generated the desired alkyne-
nitrile 12 as a crystalline solid (m.p. 138.2- 138.5 °C).
Alkyne-nitrile fragments were used twice through iterative
cobalt-mediated [2+2+2] cycloaddition reactions with substituted
alkynes to generate the 2,3’-bipyridyl core. Using this sequence,
the complanadine A core could be accessed by either a 2-pyridyl
alkyne or a 3-pyridyl alkyne.17 The higher yielding, shorter
sequence proceeding through a 2-pyridyl alkyne was used to
generate material for testing and is outlined in Scheme 2. The
first cobalt-mediated cycloaddition of alkyne-nitrile 12 and
bis(trimethylsilyl)butadiyne formed 2-pyridyl alkyne 13 in 78%
yield. The second [2+2+2] cycloaddition using pyridyl alkyne 15
with alkyne-nitrile 16, after significant optimization, provided the
correct 2,3’-bipyridyl connectivity within 17. Subsequent
replacement of the benzyl, formyl, and trimethylsilyl groups with
hydrogen afforded complanadine A (1).
Given the poly-cationic nature of complanadine A at
physiological pH, the mechanism of action studies focused on
cell surface receptors. While the compound was found to not
promote neuronal outgrowth in either primary neuronal cultures
(lacking glial cells) or in vivo through an outgrowth assay in C.
elegans,18 it was screened against a panel of GPCRs. Through the
use of stable cell lines that highly express GPCRs on the cell
surface, complanadine A’s ability to function as an agonist,
antagonist, or allosteric modulator was determined by
visualization of intracellular calcium mobilization. Out of 165
GPCRs, complanadine A was only found to significantly
modulate MrgprX2 as an agonist. Complanadine A functioned as
an agonist at a concentration of 6.3 µM, within the range of
concentrations used in the isolation study.10 This selectivity is
noteworthy as compounds can frequently bind multiple GPCRs
due to the presence of similar binding pockets.19 Importantly the
related MrgprX1 receptor was not modulated by complanadine
A, suggesting that the interaction between MrgprX2 and
complanadine A is selective.20
Synthetic material was used to determine the EC50 of
complanadine A against MrgprX2. The EC50 was 5.5 µM and
was compared to the previously known agonist PAMP21
(proadrenomedullin peptide). Other known ligands for MrgprX2
include cortistatin,5 substance P,22 compound 48/80,22 and
morphine23 however, all of these ligands are known to bind other
receptors. A small molecule dubbed “compound 12” from a study
Figure 2. Activation of Mrgprx2 by complanadine A (1) or
PAMP.
developing new agonists for MrgprX1 or MrgpX2 has been
reported to show selectivity MrgpX2, however, selectivity
towards other GPCRs has not been reported.24 Due to its unique
specificity for MrgprX2, complanadine A may be useful for
studying MrgprX2 and its biological implications.
As complanadine A is a pseudo-dimer of the natural product
lycodine, a compound that had been previously prepared by
others13 and us17, material was accessible through chemical
synthesis. Starting from the alkyne-nitrile 12, used in the

synthesis of complanadine A, a three-step synthesis of lycodine
was used to access material for comparison (Scheme 3). The
synthesis employed a CoCp(CO)2 mediated [2+2+2] reaction
between bis(trimethylsilyl)acetylene and 12 in chlorobenzene, in
the presence of illumination to provide pyridine 20 in 26% yield.
While the yield was low, the reaction provided sufficient material
was generated to test lycodine for its activity against MrgprX2.
Interestingly, lycodine showed no significant activity toward
MrgprX2, which suggests that the activation of MrgprX2
requires the bridge structure of complanadine A. (Figure 3).
of treatments of pain or histamine-independent itch, this study
provides a starting point for medicinal chemistry efforts to
optimize the compound’s biological performance.
Achnowledgements
Financial support from the Welch Foundation (F-1694) is
gratefully acknowledged.
References and notes
1) Dong, X.; Han, S.-k..; Zylka, M. J.; Simon, M. I.;
Anderson, D. J. Cell 2001, 106, 619.
Me
Me
2) Zylka, M. J.; Dong, X.; Southwell, A. L.; Anderson, D. J.
Proc. Natl. Acad. Sci. USA 2003, 100, 10043.
TMS
TMS
H
N
H
CoCp(CO)2
3) Lembo, P. M.; Grazzini, E.; Groblewski, T.; O’Donnell,
D.; Roy, M. O.; Zhang, J.; Hoffert, C.; Cao, J.; Schmidt,
R.; Pelletier, M.; Labarre, M.; Gosselin, M.; Fortin, Y.;
Banville, D.; Shen, S. H.; Ström, P.; Payza, K.; Dray, A.;
Walker, P.; Ahmad, S. Nat. Neurosci. 2002, 5, 201.
4) a) Zhang, L. Taylor, N.; Xie, Y.; Ford, R.; Johnson, J.;
Paulsen, J.; Bates, B. Mol. Brain. Res. 2005, 133, 187. b)
Burstein, E. S.; Ott, T. R.; Feddock, M.; Ma, J.-N.; Fuhs,
S.; Wong, S.; Schiffer, H. H.; Brann, M. R.; Nash, N. R.
Br. J. Pharmacol. 2006, 147, 73.
N
Bn
N
PhCl 131 °C, h
ν
N
Bn
26%
TMS
12
20
Me
H
Pd/C, H2
TBAF
dioxane, 101 °C
THF, 23 °C
N
Bn
N
97%
95%
5) Robas, N.; Mead, E.; Fidock, M. J. Biol. Chem. 2003,
278, 44400.
Me
H
21
6) Solinski, H. J.; Petermann, F.; Rothe, K.; Boekhoff, I.;
Gudermann, T. Breit, A. PLoS One 2013, 8, e58756.
7) Guan, Y.; Liu, Q.; Tang, Z.; Raja, S. N.; Anderson, D. J.;
Dong, X. Proc. Natl. Acad. Sci. USA 2010, 107, 15933.
8) Liu, Q.; Tang, Z.; Surdenikova, L.; Kim, S.; Patel, K. N.;
Kim, A.; Ru, F.; Guan, Y.; Weng, H.-J.; Geng, Y.;
Undem, B. J.; Kollarik, M.; Chen, Z.-F,; Anderson, D. J.;
Dong, X. Cell 2009, 139, 1353.
N
H
N
lycodine (2)
Scheme 3. Synthesis of lycodine (2) from alkyne-nitrile (12).
9) Kobayashi, J.; Hirasawa, Y.; Yoshida, N.; Morita, H.
Tetrahedron. Lett. 2000, 41, 9069.
10) Mortia, H.; Ishiuchi, K.; Haganuma, A.; Hoshino, T.;
Obara, Y.; Nakahata, N.; Kobayashi, J. Tetrahedron
2005, 1955.
11) Anet, F. A. L.; Eves, C. R. Can. J. Chem. 1958, 36, 902.
12) a) Yuan, C.; Chang, C. T.; Axelrod, A.; Siegel, D. J. Am.
Chem. Soc. 2010, 132, 5924. b) Fischer, D. F.; Sarpong,
R. J. Am. Chem. Soc. 2010, 132, 5926. c) Zhao, L.;
Tsukano, C.; Kwon, E.; Takemoto, Y.; Hirama, M.
Angew. Chem., Int. Ed. 2013, 52, 1722.
13) a) Kleinman, E.; Heathcock, C. H. Tetrahedron Lett.
1979, 4125. b) Heathcock, C. H.; Kleinman, E. F.;
Binkley, E. S. J. Am. Chem. Soc. 1982, 104, 1054. c)
Tsukano, C.; Zhao, L.; Takemoto, Y.; Hirama, M. Eur. J.
Org. Chem. 2010, 4198.
Figure 3. Lack of activity of lycodine against MrgprX2.
14) Newton, J. N.; Fischer, D. F.; Sarpong, R. Angew. Chem.,
Complanadine A represents an important new tool for
selectively studying the activity of MrgprX2 and the subsequent
effects of this GPCR on pain. The natural product, accessible
through multiple synthetic routes, has shown a high degree of
selectivity for activating MrgprX2 while the majority of other
know agonists are known to modulate multiple targets,
complicating the determination of the down steam effects of
agonism. Structurally the dimeric structure of complanadine A
has been found to be required for activity as lycodine, which
represents half of comaplanadine A’s structure, has been found to
be inactive at similar concentrations. This provides a preliminary
understanding of the structure requirements for agonisism. As the
compound has potential for acting as a lead for the development
Int. Ed. 2013, 52, 1726.
15) a) Tomioka, K. and Koga, K. Tetrahedron Lett. 1984, 25,
1599. b) Krause, N. Angew. Chem., Int. Ed. Engl. 1994,
33, 1764.
16) Imada, Y.; Yuasa, M.; Nakamura, I.; Murahashi, S. J.
Org. Chem. 1994, 59, 2282.
17) Yuan, C.; Chang, C.; Siegel, D. J. Org. Chem. 2013, 78,
5647.
18) Zlotkowski, Z.; Pierce-Shimomura, J.; Siegel, D.
ChemBioChem 2013, 14, 307.

Iwamura, H.; Noguchi, H.; Naito, T. Biochem. Biophys.
Res. Commun. 2006, 349, 1322.
19) Kufareva, I.; Ilatovskiy, A. V.; Abagyan, R. Nucleic
Acids Research 2012, 40, D535.
23) Akuzawa, N.; Obinata, H.; Izumi, T.; Takeda, S. J. Cell
Anim. Biol. 2007, 2, 4.
24) Malik, L.; Kelly, N. M.; Ma, J.-N.; Currier, E. A.;
Burstein, E. S.; Olsson, R. Bioorg. Med. Chem. Lett.
2009, 19, 1729.
20) See supporting information.
21) Kamohara, M.; Matsuo, A.; Takasaki, J.; Kohda, M.;
Matsumoto, M.; Matsumoto, S.; Soga, T.; Hiyama, H.;
Kobori, M.; Katou, M. Biochem. Biophys. Res. Commun.
2005, 330, 1146.
22) Tatemoto, K.; Nozaki, Y.; Tsuda, R.; Konno, S.; Tomura,
K.; Furuno, M.; Ogasawara, H.; Edamura, K.; Takagi, H.;