In vitro photo-release of a TRPV1 agonist

Article abstract of DOI:10.1016/j.bmcl.2005.09.018

Intracellular photolysis of a novel 'caged' capsaicin analogue results in in vitro activation of the capsaicin receptor TRPV1.

Full text of DOI:10.1016/j.bmcl.2005.09.018

Bioorganic & Medicinal Chemistry Letters 16 (2006) 208–212
In vitro photo-release of a TRPV1 agonist
James L. Carr,a Kerrie N. Wease,c Michael P. Van Ryssen,a
Suzanne Paterson,b Ben Agate,b Katherine A. Gallagher,a C. Tom A. Brown,b
Roderick H. Scottc and Stuart J. Conwaya,*
aEaStCHEM, School of Chemistry and Centre for Biomolecular Sciences, University of St. Andrews, St. Andrews, Fife KY16 9ST, UK
bSchool of Physics and Astronomy, University of St. Andrews, St. Andrews, Fife KY16 9ST, UK
cInstitute of Medical Science, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK
Received 23 August 2005; accepted 6 September 2005
Available online 5 October 2005
Abstract—Intracellular photolysis of a novel ÔcagedÕ capsaicin analogue results in in vitro activation of the capsaicin receptor
TRPV1.
Ó 2005 Elsevier Ltd. All rights reserved.
Capsaicin (1), the main pungent component of chilli
peppers, has long been known to produce a painful irri-
tant effect if injected into the skin, applied to sensitive
structures such as the cornea, or tasted. Comprehensive
structure–activity studies have been carried out on cap-
saicin and analogues, elucidating the main requirements
for a compound to display capsaicin-like biological
activity;1–4 however, the molecular target of capsaicin
has only recently been discovered. It is now clear that
capsaicin exerts its main biological effects via activation
of a receptor.
The Ôcapsaicin receptorÕ, or transient receptor potential
channel–vanilloid subtype 1 (TRPV1), is a cation selec-
tive ligand-gated ion channel.5 Originally named the
vanilloid receptor subtype 1 (VR1), it has now been
Figure 1. Capsaicin (1) and resiniferatoxin (RTX, 2).
shown that TRPV1 is a member of the TRP family of
receptors6 and is activated by capsaicin and other nox-
rather than agonists are likely to be the therapeutically
terminal degeneration. Therefore, TRPV1 antagonists
ious compounds including resiniferatoxin (RTX, 2,
most important compounds in the treatment of
pain.12–14 Recently, many potent and selective agonists
and antagonists of TRPV1 have been reported.15,9 How-
ever, pharmacological tools required to accurately study
the function of a receptor are not limited to selective
agonists and antagonists. The use of ÔcagedÕ or photola-
bile compounds, which allow temporal and spatial con-
trol over receptor activation, has frequently proved
invaluable in the study of receptor function.16,17 Very
few chemical tools of this type to assist the study of
TRPV1 exist. Herein we report the synthesis of two nov-
el caged capsaicin analogues 8 and 9, the in vitro biolog-
ical activity of 8 in cultured dorsal root ganglia (DRG)
Fig. 1). TRPV1 is also activated by heat above ꢀ45 °C
and conditions below ꢀpH 5.5.5 This receptor repre-
sents a realistic therapeutic target for the treatment of
pain,7–10 and the use of topically applied capsaicin to
treat painful skin disorders has met with some success.11
The analgesic effect of capsaicin can be rationalised by
prolonged activation of TRPV1, causing the receptor
to desensitise. Longer-term resistance to capsaicin can
be attributed to the large influx of Ca2+ causing nerve
*
Corresponding author. Tel.: +44 1334 463478; fax: +44 1334
463808; e-mail: sjc16@st-andrews.ac.uk
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.09.018

J. L. Carr et al. / Bioorg. Med. Chem. Lett. 16 (2006) 208–212
209
neurones from neonatal rats and some studies on the
photolysis characteristics of both compounds.
duced no significant current or voltage responses in six
neurones. The endocannabinoid, anandamide, is a more
effective activator of this receptor when applied to the
intracellular environment rather than extracellularly.19
We therefore postulated that intracellular photolysis of
compound 8 might be a more efficient way of activating
TRPV1 receptors. Intracellular photolysis of compound
8 (0.5 mM caged compound in the patch pipette solution
containing 2.5% DMSO) evoked sustained membrane
potential depolarisations of 15 2 mV in four out of
five neurones (Fig. 2A) and action potentials were
evoked in two out of these four neurones (Fig. 2B). Un-
der voltage clamp, a comparison was made between in-
ward currents activated by extracellular application of
1 lM capsaicin and intracellular photolysis of com-
pound 8. Consistent with the imaging data, capsaicin
(1 lM) failed to evoke responses in five neurones, and
subsequently intracellular photolysis of compound 8
also failed to evoke currents (Figs. 2C and D). However,
DRG neurones that responded to capsaicin also pro-
duced modest (>ꢀ10% of capsaicin-activated current)
but consistent inward currents to intracellular photolysis
of compound 8 (Figs. 2C and E). Repeated flash photol-
ysis produced a cumulative inward current but the
increases in amplitude may have been limited by desen-
sitisation. Interestingly, in one DRG neurone a transient
burst of action currents was evoked by intracellular pho-
tolysis of compound 8 (Figs. 2F and G). These events
were not preceded by a clear inward current (Fig. 2)
and are likely to be due to responses being initiated on
DRG neurone processes resulting in action potential
firing and the spread of this excitation into the voltage
clamped cell body.
Previous studies have shown that alkylation of the phe-
nolic hydroxyl group of capsaicin will remove all biolog-
ical activity.4 We have alkylated this position with a
4-methoxyphenacyl group (see 8, Scheme 1), which has
been shown to undergo in vitro photolysis to reveal
the desired biologically active product and two biologi-
cally inert by-products.18 We have opted to cage the
N-nonanoyl analogue of capsaicin (7), which has been
shown to possess pharmacology similar to that of capsa-
icin itself.3 Although capsaicin is commercially avail-
able, the use of compound 7 has allowed us to develop
a robust synthesis that can also be applied to a number
of capsaicin analogues. The synthesis commenced from
the commercially available 4-hydroxy-3-methoxybenzo-
nitrile (3, Scheme 1) and proceeded in good overall yield
to give the desired compound 8, using standard meth-
ods. All compounds displayed analytical and spectro-
scopic data consistent with the assigned structure
(please see supplementary data for spectroscopic and
analytical data for compounds 8 and 9).
Before investigating the photodeprotection of 8, it was
necessary to determine the pharmacological response
of the uncaged compound (7) in cultured dorsal root
ganglia (DRG) neurones (see supplementary data for
details). The results obtained suggest that compound 7
exerts its main biological effects through activation of
TRPV1, which was expected from literature reports.2–4
Whole cell patch clamp recording was used to examine
changes in the electrophysiological properties of cul-
tured DRG neurones as a result of intracellular flash
photolysis of compound 8 resulting in photo-release of
compound 7. Extracellular photolysis of compound 8
(0.1–1 mM 8 in saline containing 0.5–5% DMSO) pro-
Apart from the final case (Figs. 2F and G), the photol-
ysis of the phenacyl compound produced smaller
responses than those observed by Zemelman et al. when
uncaging a dimethoxynitrobenzyl caged compound.20
This observation led us to investigate the amount of
uncaging of 8 that was occurring under the experimental
conditions. In an attempt to mimic our biological exper-
iments as closely as possible, an aliquot (10 ll) of a
50 lM solution was exposed to a 300 V flash from an
identical xenon flash lamp equipped with a 360 nm filter.
The small volume of material used, however, has made it
impossible to detect whether uncaging has occurred and
1
to what degree. Attempts were made using HPLC, H
NMR and mass spectrometry (MS), but in all cases,
and even when using several combined aliquots, it was
impossible to detect uncaging and in the case of 1H
NMR it was not possible to detect even the caged
material.
The photolysis of a dimethoxynitrobenzyl caged com-
pound similar to that used by Zemelman was studied
by Katritzky et al.21 We therefore decided to investi-
gate photolysis of compound 8 using conditions similar
to those reported. Katritzky employed a 450 W immer-
sion lamp with a 363 nm filter. We used a 125 W
immersion lamp with a 375 nm filter. As we were un-
able to fully match KatritzkyÕs conditions we synthe-
sised the dimethoxynitrobenzyl caged derivative of
compound 7 (compound 9, Scheme 2) to allow direct
Scheme 1. Synthesis of the caged capsaicin analogue 8. Reagents and
conditions: (i) TIPSCl, imidazole, DMF, rt, 91%; (ii) LiAlH4, THF, rt,
90% crude yield; (iii) nonanoyl chloride, 4-DMAP, pyridine, CH2Cl2,
0 °C–rt, 78%; (iv) TBAF, THF, 81%; (v) 2-bromo-40-methoxyace-
tophenone, NaH, DMF, 0 °C–rt, 74%.

210
J. L. Carr et al. / Bioorg. Med. Chem. Lett. 16 (2006) 208–212
Figure 2. Intracellular actions of compound 8 photolysis on the excitability of cultured DRG neurones. The same extracellular solution was used for
both the electrophysiology and the Ca2+ imaging experiments. The patch pipette solution contained (in mM): KCl, 140; EGTA, 5; CaCl2, 0.1; MgCl2,
2.0; HEPES, 10.0; ATP, 2.0; compound 8 0.5–0.1% and 2.5% DMSO. This solution after correction with Tris and sucrose had a pH of 7.2 and
osmolarity of 320 mOsm Lꢁ1. An Axoclamp 2 A switching amplifier (Axon Instruments) operated at a switching frequency of 15 kHz was used. (A)
Record showing two depolarising responses to intracellular photolysis of compound 8 from the same neurone. No action potentials were evoked in
this neurone and no further depolarisation was obtained with additional photolysis (3rd flash not shown). (B) Example record showing a single action
potential and sustained depolarisation obtained in response to intracellular photolysis of compound 8. (C) Line chart showing the diversity of DRG
neurone current responses to capsaicin and intracellular photolysis of compound 8. Only 2 out of 7 DRG neurones responded to capsaicin but both
these neurones also responded to intracellular photolysis of compound 8. Five cells failed to respond to both drugs. (D) Example voltage clamp
record showing a non-responding neurone that failed to respond to both capsaicin and intracellular photolysis of compound 8. (E) Records showing
inward currents activated by capsaicin and intracellular photolysis of compound 8; note the difference in current scale. (F) Action currents evoked by
intracellular photolysis of compound 8; showing the burst firing behaviour that gradually declines as the neurone recovers to a resting state. This
neurone was voltage clamped at a holding potential of ꢁ70 mV; the excitatory action of photoreleased compound 7 appears to have occurred in an
unclamped region of the cell and a burst of action potentials has spread into the cell body to be recorded as currents. (G) The same record as (F) but
on an expanded time scale to show the high frequency action potential firing and that the first action potential was not initiated by a clear inward
current in the cell soma. Arrows mark the points at which 300 V flashes (175 mJ; lasting ꢀ1 ms) from a xenon flash lamp, equipped with a 360 nm
filter, were applied to the DRG neurones. Under voltage clamp all neurones were held at ꢁ70 mV.
comparison of the two caging groups using our photol-
ysis conditions.
was irradiated for 60 min showed a trace of the uncaged
material (see supplementary data). 1H NMR analysis of
a sample that had been irradiated for 16 h 40 min
showed that the caged material had degraded (data
not shown). However, the expected peaks for compound
7 were not clearly present, indicating that this
compound may be further broken down on extended
irradiation. Irradiation of compound 8 using a 125 W
Aliquots (1 ml) of a 7.3 mM solution of compound 8
were irradiated using a 125 W mercury arc lamp through
1
a 375 nm filter. H NMR analysis of samples irradiated
for 1, 15 and 30 min showed no detectable uncaging
occurring. The H NMR spectrum of the sample that
1

J. L. Carr et al. / Bioorg. Med. Chem. Lett. 16 (2006) 208–212
211
of compound 9 releases a greater amount of compound
7 and this is reflected in the more prominent biological
response observed by Zemelman et al.20 The different
amounts of photolysis observed on irradiation of these
compounds will render them useful and complementary
biological tools for the investigation of TRPV1. The fact
that the two caging groups behave in different manners
on irradiation at the same wavelength suggests that
compounds 8 and 9 may be of use in investigating the
concept of wavelength orthogonality as applied to bio-
logically active compounds. This is currently under
investigation and our findings will be published in due
course.
Scheme 2. Synthesis of the caged capsaicin analogue 9. Reagents and
conditions: (i) 4,5-dimethoxy-2-nitrobenzyl bromide, tBuOK, THF, rt,
66%.
mercury arc lamp with no filter showed a trace of com-
pound 7 after 15 min.
Acknowledgments
We thank Dr. Ruth Ross (University of Aberdeen) and
Dr. Douglas Philp (University of St Andrews) for help-
ful discussions and critical reading of the manuscript.
We acknowledge the School of Chemistry, University
of St. Andrews and Pfizer Research UK (Sandwich)
for financial support.
Irradiation of aliquots (1 ml) of a 7.3 mM solution of
compound 9 using a 125 W mercury arc lamp through
a 375 nm filter showed more rapid uncaging (Fig. 3).
After 30 min 35% uncaging had occurred. Examination
of the UV/vis spectra of these compounds explains these
results clearly as the absorption maximum for the car-
bonyl of the compound 8 is at 278 nm, whereas the
absorption maximum for the nitro group of compound
9 is at 345 nm, much closer to the wavelength of irradi-
ation (see supplementary data).
Supplementary data
The biological characterisation of compound 1, selected
partial NMR spectra for the photolysis of compound 8
and full analytical and spectroscropic data for
compounds 8 and 9 are available in the supplementary
material, in the online version, at doi:10.1016/
j.bmcl.2005.09.018.
Zemelman observed a 1–2 s interval between flash pho-
tolysis and TRPV1 activation.20 We did not observe this
with our studies on compound 8. It is not clear to us
why Zemelman and co-workers observed the interval
that they did; we will employ compound 9 to investigate
this further.
References and notes
Although it is not possible to draw any quantitative con-
clusions from these studies, our investigations into the
photolysis of compounds 8 and 9 correlate with biolog-
ical activity observed for these compounds. It can be as-
sumed that during in vitro flash photolysis of compound
8, using the conditions described above, only a very
small amount of compound 7 is being released. The fact
that a biological response is observed reflects the high
potency of compound 7 for TRPV1. Flash photolysis
1. Janusz, J. M.; Buckwalter, B. L.; Young, P. A.; Lahann,
T. R.; Farmer, R. W.; Kasting, G. B.; Loomans, M. E.;
Kerckaert, G. A.; Maddin, C. S.; Berman, E. F.; Bohne,
R. L.; Cupps, T. L.; Milstein, J. R. J. Med. Chem. 1993,
36, 2595.
2. Walpole, C. S. J.; Wrigglesworth, R.; Bevan, S.; Campbell,
E. A.; Dray, A.; James, I. F.; Masdin, K. J.; Perkins, M.
N.; Winter, J. J. Med. Chem. 1993, 36, 2373.
3. Walpole, C. S. J.; Wrigglesworth, R.; Bevan, S.; Campbell,
E. A.; Dray, A.; James, I. F.; Masdin, K. J.; Perkins, M.
N.; Winter, J. J. Med. Chem. 1993, 36, 2381.
4. Walpole, C. S. J.; Wrigglesworth, R.; Bevan, S.; Campbell,
E. A.; Dray, A.; James, I. F.; Perkins, M. N.; Reid, D. J.;
Winter, J. J. Med. Chem. 1993, 36, 2362.
5. Caterina, M. J.; Schumacher, M. A.; Tominaga, M.;
Rosen, T. A.; Levine, J. D.; Julius, D. Nature 1997, 389,
816.
6. Montell, C.; Birnbaumer, L.; Flockerzi, V.; Bindels, R. J.;
Brudorf, E. A.; Caterina, M. J.; Clapham, D. E.; Harte-
neck, C.; Heller, S.; Julius, D.; Kojima, I.; Mori, Y.;
Penner, R.; Prawitt, D.; Scharenberg, A. M.; Schultz, G.;
Shimizu, N.; Zhu, M. X. Mol. Cell 2002, 9, 229.
7. Szallasi, A.; Blumberg, P. M. Pharmacol. Rev. 1999, 51,
159.
8. Julius, D.; Basbaum, A. I. Nature 2001, 413, 203.
9. Szallasi, A.; Appendino, G. J. Med. Chem. 2004, 47, 2717.
10. Ghilardi, J. R.; Rohrich, H.; Lindsay, T. H.; Sevcik, M.
A.; Schwei, M. J.; Kubota, K.; Halvorson, K. G.; Poblete,
Figure 3. Partial 300 MHz 1H NMR spectra of compound 9 irradi-
ated, for the time shown, with a 125 W mercury arc lamp through a
375 nm filter. Thirty-five percentage uncaging (as adjudged by
appearance of compound 7 vs total material) was observed after
30 min. The doublet at 4.19 ppm is from compound 9; the doublet at
4.14 ppm is from compound 7.

212
J. L. Carr et al. / Bioorg. Med. Chem. Lett. 16 (2006) 208–212
J.; Chaplan, S. R.; Dubin, A. E.; Carruthers, N. I.;
Swanson, D.; Kuskowski, M.; Flores, C. M.; Julius, D.;
Wang, X. H.; Zhu, J. W.; Norman, N. H.; Treanor, J. J. S.
J. Med. Chem. 2005, 48, 71.
Mantyh, P. W. J. Neurosci. 2005, 25, 3126.
11. Mason, L.; Moore, R. A.; Derry, S.; Edwards, J. E.;
McQuay, H. J. Br. Med. J. 2004, 328, 991.
12. Gunthorpe, M. J.; Rami, H. K.; Jerman, J. C.; Smart, D.;
Gill, C. H.; Soffin, E. M.; Hannan, S. L.; Lappin, S. C.;
Egerton, J.; Smith, G. D.; Worby, A.; Howett, L.; Owen,
D.; Nasir, S.; Davies, C. H.; Thompson, M.; Wyman, P.
A.; Randall, A. D.; Davis, J. B. Neuropharmacology 2004,
46, 133.
13. Valenzano, K. J.; Sun, Q. Curr. Med. Chem. 2004, 11,
3185.
14. Doherty, E. M.; Fotsch, C.; Bo, Y. X.; Chakrabarti, P. P.;
Chen, N.; Gavva, N.; Han, N. H.; Kelly, M. G.; Kincaid,
J.; Klionsky, L.; Liu, Q. Y.; Ognyanov, V. I.; Tamir, R.;
15. Planells-Cases, R.; Garcia-Martinez, C.; Royo, M.; Perez-
Paya, E.; Carreno, C.; Albericio, F.; Messeguer, A.;
Ferrer-Montiel, A. Drug Future 2003, 28, 787.
16. Dorman, G.; Prestwich, G. D. Trends Biotechnol. 2000, 18,
64.
17. Bochet, C. G. J. Chem. Soc., Perkin Trans. 1 2002, 125.
18. Park, C. H.; Givens, R. S. J. Am. Chem. Soc. 1997, 119,
2453.
19. Evans, R. M.; Scott, R. H.; Ross, R. A. Br. J. Pharmacol.
2004, 141, 1223.
20. Zemelman, B. V.; Nesnas, N.; Lee, G. A.; Miesenbock, G.
Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 1352.
21. Katritzky, A. R.; Xu, Y. J.; Vakulenko, A. V.; Wilcox, A.
L.; Bley, K. R. J. Org. Chem. 2003, 68, 9100.