Structure-activity relationships of the ultrapotent vanilloid resiniferatoxin (RTX): The homovanillyl moiety

Article abstract of DOI:10.1016/j.bmcl.2006.09.077

Starting from ROPA (2), analogues of RTX (1a) modified on the acyl side chain were prepared and evaluated for vanilloid activity in HEK-293 cells over-expressing the human recombinant TRPV1. The ROPA motif provided an enhancement of potency sufficient to

Full text of DOI:10.1016/j.bmcl.2006.09.077

Bioorganic & Medicinal Chemistry Letters 17 (2007) 132–135
Structure–activity relationships of the ultrapotent vanilloid
resiniferatoxin (RTX): The homovanillyl moiety
Giovanni Appendino,a,* Abdellah Ech-Chahad,a Alberto Minassi,a Sara Bacchiega,a
Luciano De Petrocellisb and Vincenzo Di Marzoc,*
a
`
Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Universita del Piemonte Orientale,
Viale Ferrucci 33, 28100 Novara, Italy
bEndocannabinoid Research Group, Institute of Cibernetica ‘‘Eduardo Cainiello’’, CNR, Via Campi Flegrei 34, Pozzuoli (NA), Italy
cEndocannabinoid Research Group, Institute of Biomolecular Chemistry, CNR, Via Campi Flegrei 34, 80078 Pozzuoli (NA), Italy
Received 27 August 2006; revised 23 September 2006; accepted 26 September 2006
Available online 29 September 2006
Abstract—Starting from ROPA (2), analogues of RTX (1a) modified on the acyl side chain were prepared and evaluated for vanil-
loid activity in HEK-293 cells over-expressing the human recombinant TRPV1. The ROPA motif provided an enhancement of
potency sufficient to expand the range of vanillyl surrogates to structural elements (e.g., an unsubstituted phenyl ring) that afford
inactive analogues in compounds from the capsaicin series.
Ó 2006 Elsevier Ltd. All rights reserved.
The daphnane diterpenoid resiniferatoxin (RTX, 1a)
was discovered in 1975 because of its extraordinary
activity in the mouse ear reddening assay, a surrogate
end-point for mouse skin tumor promotion.1 In this as-
say, RTX outperforms PMA (phorbol myristate ace-
tate), the most potent phorbol ester, by 2–3 orders of
magnitude.1 However, since RTX lacks tumor promot-
ing properties, its biological target remained elusive.2
An analogy with capsaicin was suggested by the
revision of the substitution pattern of the side-chain
aromatic moiety of RTX. This aryl group was original-
ly formulated as a 3-methoxy-5-hydroxyphenyl substi-
RTX as an indispensable neuropharmacological tool,
paving the way for the characterization and cloning
of the long sought capsaicin (vanilloid) receptor
(TRPV1),5 and eventually establishing the burgeon-
ing field of TRP channel research.6 RTX has also
been investigated as a drug for the treatment of a
series of conditions (lower urinary tract disfunctions,
neuropathic and cancer pain) where malfunctioning
or over-expression of TRV1 is involved.7 Very promis-
ing results were recently reported in this direction,8
while RTX was further validated as a privileged and
unique structure to investigate the biology of TRPV1
by the serendipitous discovery that its powerful agonis-
tic activity on TRPV1 (EC50 ca. 19 pM) could be re-
versed by aromatic iodination of the acyl moiety,
affording the ultrapotent vanilloid antagonist iodoresi-
niferatoxin (I-RTX, 1b).9
tuent, but was later recognized as
a
vanillyl
(3-methoxy-4-hydroxyphenyl) group,3 and in 1989 it
was indeed demonstrated that RTX behaves as an
ultrapotent analogue of capsaicin, the pungent princi-
ple of hot peppers.4 This seminal discovery established
Research on RTX has been hampered by the limit-
ed availability of the natural product. As a result,
our knowledge on its structure–activity relation-
ships is still fragmentary,10 and nothing is known
on the possibility to re-direct RTX, the most po-
tent chemical activator of a TRP channel reported
to date, to other members of this large family of
sensors, for many of which no chemical activator
is known.6
Keywords: Resiniferatoxin (RTX); TRPV1; Structure–activity rela-
tionships; Capsaicin; Vanilloids.
*
Corresponding authors. Tel.: +39 0321375744; fax: +39 0321375621
(G.A.); tel.: +39 0818675093; fax: +39 0818041770 (V.D.M.); e-mail
addresses: appendino@pharm.unipmn.it; vdimarzo@icmib.na.cnr.it
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.077

G. Appendino et al. / Bioorg. Med. Chem. Lett. 17 (2007) 132–135
133
16
ity than the other methods described in the literature for
the esterification of ROPA (Steglich esterification,10
Yamaguchi acylation,16 Mukayama coupling,3,17 and
reaction of 20-mesyl ROPA with sodium carboxy-
lates18).
15 17
12
13
O 1'
O
11
18
1
2'
4'
7'
O
3'
O14
5'
6'
O
H10
H
O
9
2
8
H
8'
H
19
7
4
5
O
3
20
6
HO
O
2''
3''
O
R2
1''
8''
HO
O
OH
20
R1 R2
4''
O
O
2
7''
R1
1a H
1b I
1c H
H
H
I
5''
H
O
OMe
6''
H
OH
O
HO
O
A
O
R4
We have recently developed an expeditious protocol to
obtain the terpenoid core of RTX (resiniferonol orth-
ophenylacetate, ROPA, 2), from Euphorbia resinifera
Berg, a household plant commercially available from
nurseries.11 Capitalizing on the availability of ‘synthetic’
amounts of ROPA, we have investigated the structure–
activity relationships of the critical homovanillyl moiety
of RTX, focusing on the aliphatic linker between the
ester carbonyl and the aryl moiety, and on the substitu-
tion pattern of the phenyl ring. While not exhaustive,
these modifications were expected to shed light on a
series of critical issues, like the optimal distance between
the vanillyl moiety and the diterpenoid core, the role
of the aryl oxygen functions, and the generality of
the switch in vanilloid activity observed upon aryl
iodination.12
R3
R1
R2
A
-
R1
R2
OH
OH
OH
H
H
H
H
OH
OH
OH
OH
R3
H
H
H
H
H
H
H
I
R4
H
H
H
H
H
H
H
H
I
3a
3b
3c
3d
3e
3f
3g
3h
3i
OMe
OMe
OMe
H
-CH2-CH2-
(E) –CH=CH-
-
-CH2
H
H
H
-CH2-CH2-
(E) –CH=CH-
-
OMe
OMe
OMe
OMe
-
H
I
H
3j
3k
-CH2-CH2-
-CH2-CH2-
H
I
The iodinated vanillic and dihydroferulic acids required
for this study were prepared from the corresponding
acids as exemplified in Scheme 1 for 5- and 6-iodovan-
illic acids. In RTX, the effect of iodination depends on
the location of the halogen atom on the homovanillyl
moiety, with the 50-iodo analogue (1b) being an ultra-
potent antagonist9 and the 60-derivative (1c) a mixed
agonist.19 The purity of the starting iodovanillic acids
was therefore of utmost relevance, and the low yields
of both iodination steps reflect the extensive purifica-
tion by chromatography and/or crystallization of the
reaction products required to secure the absence of
All RTX analogues were prepared by Mitsunobu
esterification (DIAD–TPP as the redox couple) of
ROPA with the corresponding carboxylic acids.13 Puri-
fication of the reaction products was greatly facilitated
by the crystallization of a diisopropyl hydrazodicarb-
oxylate–triphenylphosphine oxide adduct by cooling in
toluene.14 After filtration and gravity column chroma-
tography, the final products were further purified by
crystallization or, if amorphous, by preparative HPLC
on silica gel.15 Final compounds were prepared in
10–20 mg scale (30–60% overall yield) and showed
HPLC purity >96%. We found this protocol more con-
venient, higher yielding, and of more general applicabil-
1
detectable (300 MHz H NMR spectroscopy) amounts
of starting material or isomers in the final product.
The regiochemistry of the iodination step was con-
trolled by the nature of the oxygen function at C-4.
While a free hydroxyl at C-4 directed iodine to the
adjacent carbon, etherification of the phenolic hydroxyl
led to the complementary C-6 functionalization, as ob-
served for vanillin.12
COOH
COOMe
a. NIS, TFA
(25%)
I
OMe
b. LiOH, H2O
(67%)
OMe
OH
OH
Final products (3a–3k) were assayed in human embry-
onic kidney (HEK)-293 cells over-expressing the human
TRPV1, and the results are reported in Table 1.20
Mem-Cl
DIPEA
(91%)
Structure–activity data of RTX are not only scarce, but
also difficult to compare, not only because of differences
in the type of vanilloid assay employed (functional or
binding), but also because different versions of TRPV1
show different sensitivity to vanilloids, with hTRPV1
being less sensitive to RTX compared to its murine ver-
sion (rTPRV1).21 Furthermore, differences have also
been noticed between assays carried out in native
dorsal root ganglion cells and in cells transfected with
COOH
COOMe
I
a. I2, CF3COOAg
(27%)
OMe
b. LiOH, H2O
(27%)
OMe
OMem
OH
Scheme 1. Synthesis of 5- and 6-iodovanillic acids (NIS = N-iodosuc-
cinimide; TFA = trifluoroacetic acid; Mem = methoxyethoxymethyl;
DIPEA = diisopropylethylamine).

134
G. Appendino et al. / Bioorg. Med. Chem. Lett. 17 (2007) 132–135
Table 1. Vanilloid agonistic activity of aryl substituted vanillamides20
Iodination of compounds from the vanillic and the dihy-
droferulic series gave surprising results, since, in sharp
contrast with what was observed in the homovanillic
analogue (RTX), no reversal of activity was observed,
and all compounds behaved as full agonists. Interesting-
ly, iodination of vanillyl ROPA (3a) in ortho to the phe-
nolic hydroxyl (3h) caused a 5-fold increase of activity,
while introduction of an iodine atom at the C-6 meta-
position (3i) caused an almost 2-fold decrease of activi-
ty. Both iodinated analogues of dihydroferuloyl ROPA
(3k and 3l) were 2-fold less potent than their parent
compound (3b).
a
EC50 (nM)
Compound
1a (RTX)
3a
0.019
9.4
4.6
3b
3c
1.5
3d
3e
25.5
4.7
Capsaicin
40.0
51.5
69.5
1.9
3f
3g
3h
3i
15.2
8.6
3j
3k
Information on the vanilloid binding pocket of TRPV1
has been mostly obtained by experiments of oligonu-
cleotide-directed mutagenesis.23 The availability of
ROPA makes it possible to purse a complementary
and more traditional approach, based on the struc-
ture–activity relationships of the ultrapotent ligand
RTX. In this first contribution, we have shown that,
while the methylene linker and the oxygen functions
on the side chain of RTX are essential for ultrapotency,
the presence of the diterpenoid core makes it possible to
perform a surprising range of side-chain modifications,
including removal of the metabolically labile guaiacyl
oxygens. Furthermore, the lack of iodine-induced rever-
sal of activity observed in the lower- and higher-homo-
logues of RTX points to a topologically well-defined
role for this atom in the ultrapotent vanilloid antagonist
I-RTX (1b).
7.9
a EC50, concentration exerting a half-maximal effect. Data are means
of n = 3 determinations, whose SD values were never higher than
10% of the means.
cloned TRPV1.21 It was therefore important to adopt a
homogeneous end-point, and this study represents the
first attempts to explore in a systematic way the struc-
ture–activity relationships of RTX using hTRPV1.
Comparison of the activity of RTX with its vanillyl-,
coniferyl-, and dihydroconiferyl analogues showed that
the nature of the linker between the vanillyl and the ester
carbonyl is critical for ultrapotency. Thus, deletion,
homologation, or replacement with a double bond were
all detrimental for activity, with a decrease of potency in
the range of 2–2.5 orders of magnitude. Lengthening of
the linker increased activity compared to its deletion
by 3-fold (homologation) and 6-fold (replacement with
a double bond). Nevertheless, given the ultrapotency
of RTX, all these compounds (3a–3c) would qualify
as potent vanilloids. Somewhat similar results were
observed for the removal of the oxygen functions of
the vanillyl moiety of RTX. This maneuver was detri-
mental for activity, with a ca. 200-fold decrease of
potency. Nevertheless, with an EC50 in the one digit
nanomolar range (4.7 nM), ROPA phenylacetate (3d)
still showed potent vanilloid activity, one order of mag-
nitude higher than capsaicin. Remarkably, removal of
the oxygen functions in the analogues from the conife-
ryl- and dihydroconiferyl series was better tolerated,
while, compared to ROPA vanillate (3a), ROPA benzo-
ate (3e) even showed a 2-fold increase of activity. Taken
together, these observations suggest that the ROPA
motif provides an enhancement of potency sufficient to
expand the range of vanillyl surrogates to structural
elements, like an unsubstituted phenyl ring, that instead
afford inactive analogues in compounds from the
capsaicin series.22 For the same reason, maneuvers like
homologation, that are incompatible with the vanillyl
moiety of capsaicin,22 are possible with resiniferonoids.
The ultrapotency of RTX clearly requires the well-de-
fined topologic and constitutional relationship between
the side-chain aromatic moiety and the diterpenoid core
expressed by the homovanillyl group. Nevertheless, a
certain flexibility must exist in the binding pocket of
TRPV1, making it possible to accommodate a range
of acyl chains that, in the absence of ROPA, would be
incompatible with binding.
Acknowledgment
We gratefully acknowledge Indena S.p.A. for financial
support.
References and notes
1. Isolation from Euphorbia resinifera Berg: (a) Hergenhahn,
M.; Adolf, W.; Hecker, E. Tetrahedron Lett. 1975, 19,
1595; Isolation from E. poissonii Pax: (b) Schmidt, R. J.;
Evans, F. J. J. Pharm. Pharmacol. 1975, 27, 50.
2. Hergenhahn, M.; Kusumoto, S.; Hecker, E. J. Cancer Res.
Clin. Oncol. 1984, 108, 98.
3. Adolf, W.; Hergenhahn, M.; Hecker, E. J. Nat. Prod.
1982, 45, 347.
4. Szallasi, A.; Blumberg, P. M. Neuroscience 1989, 30, 515.
5. Caterina, M. J.; Schumacher, M. A.; Tominaga, M.;
Rosen, T. A.; Levine, J. D.; Julius, D. Nature 1997, 389,
816.
6. For recent reviews, see: (a) Owsianik, G.; D’hoedt, D.;
Voets, T.; Nilius, B. Rev. Physiol. Biochem. Pharmacol.
2006, 156, 6, and; Clapham, D. E. Nature 2003, 426, 517.
7. Szallasi, A. Am. J. Clin. Pathol. 2002, 118, 110.
8. Refractory detrusor overactivity: (a) Kuo, H. C.; Liu, H.
T.; Yang, W. C. J. Urol. 2006, 176, 641; Cancer pain: (b)
Hartel, M.; di Mola, F. F.; Selvaggi, F.; Mascetta, G.;
Wente, M. N.; Felix, K.; Giese, N. A.; Hinz, U.; De
Sebastiano, P.; Buchler, M. W.; Friess, H. Gut 2006, 55,
519, and; Ghilardi, J. R.; Robrich, H.; Lindsay, T. H.;
Sevick, M. A.; Sevcik, M. A.; Kubota, K.; Halvorson,
K. G.; Poblete, J.; Chaplan, S. R.; Dubin, A. E.;
Carruthers, N. I.; Swanson, D.; Kuskowski, M.; Flores,

G. Appendino et al. / Bioorg. Med. Chem. Lett. 17 (2007) 132–135
135
C. M.; Julius, D.; Manthyh, P. W. J. Neurosci. 2005,
25, 3126; RTX was found ineffective for the treatment
of interstitial cystitis (Payne, C. K.; Mosbaugh, P. G.;
Forrest, J. B.; Evans, R. J.; Withmore, K. E.; Antoci,
J. P.; Perez-Marrero, R.; Jacoby, K.; Diokno, A. C.;
O’Reilly, K. J.; Greibling, T. L.; Vasavada, S. P.; Yu,
A. S.; Frumkin, L. R. J. Urol. 2005, 173, 1590).
9. Wahl, P.; Foged, C.; Tullin, S.; Thomsen, C. Mol.
Pharmacol. 2001, 59, 9.
10. Walpole, C. S. J.; Bevan, S.; Bloomfield, G.; Breckenridge,
R.; James, I. F.; Ritchie, T.; Szallasi, A.; Winter, J.;
Wrigglesworth, R. J. Med. Chem. 1996, 39, 2939.
11. Fattorusso, E.; Lanzotti, V.; Taglialatela-Scafati, O.;
Tron, G. C.; Appendino, G. Eur. J. Org. Chem. 2002, 71.
12. Appendino, G.; Daddario, N.; Minassi, A.; Schiano
Morello, A.; De Petrocellis, L.; Di Marzo, V. J. Med.
Chem. 2005, 48, 4663.
(q, C-19), 18.9 (q, C-17), 19.9 (q, C-18), 33.1 (d, C-11),
35.8 (t, C-12), 39.2 (d, C-8), 40.3 (t, C-5), 41.1 (d, C-20),
55.5 (d, C-10), 56.2 (q, OMe), 70.9 (d, C-20), 73.4 (s, C-4),
80.7 (d, C-14), 81.2 (s, C-9), 84.5 (s, C-13), 110.8 (t, C-16),
111.9 (d, C-300), 114.1 (d, C-600), 117.9 (s, C-10), 123.0 (s, C-
30), 124.3 (d, C-700), 126.6 (d, C-60), 127.8 (d, C-7), 128.7
(d, C-50/70), 130.9 (d, C-40/80), 134.5 (s, C-200), 135.1 (s, C-
6), 136.6 (s, C-2), 146.3 (s, C-15), 150.2 (s, C-400 and C-500),
158.5 (d, C-1), 166.0 (s, C-100), 208.3 (s, C-3); CI-MS m/z
615 (M+H+) (C36H38O9+H+).
16. Wender, P. A.; Jesudason, C. D.; Nakahira, H.; Tamura,
N.; Tebbe, A. L.; Ueno, Y. J. Am. Chem. Soc. 1997, 119,
12976.
17. Gordge, P. C.; Darey, P.; Tudor-Evans, A.; Jonathan
Ryves, W.; Evans, F. J.; Hassan, N. M. Phytotherapy Res.
1994, 8, 362.
18. Shu, A. J. Y.; Heys, J. R. In Synthesis and Applications of
Isotopically Labelled Compounds; Heys, J. R., Melillo, D.
G., Eds.; Wiley, 1998; p 463.
13. Appendino, G.; Minassi, A.; Daddario, N.; Bianchi, F.;
Tron, G. C. Org. Lett. 2002, 4, 3839.
14. Anderson, N. G.; Lust, D. A.; Colapret, K. A.; Simpson,
J. H.; Malley, M. E.; Gougoutas, J. Z. J. Org. Chem. 1996,
61, 7955.
19. McDonnel, M. E.; Zhang, S.-P.; Dubin, A. E.; Dax, S. L.
Bioorg. Med. Chem. Lett. 2002, 12, 1189.
20. Cells were grown as monolayers in minimum essential
medium supplemented with non-essential amino acids,
10% fetal calf serum, and 2 mM glutamine, and main-
tained under 95%/5% O2/CO2 at 37 °C. The effect of the
substances on [Ca2+]i was determined by using Fluo-3, a
selective intracellular fluorescent probe for Ca2+. One day
prior to experiments cells were transferred into six-well
dishes coated with Poly-L-lysine (Sigma) and grown in the
culture medium mentioned above. On the day of the exper-
iment the cells (50–60,000 per well) were loaded for 2 h at
25 °C with 4 lM Fluo-3 methylester (Molecular Probes) in
DMSO containing 0.04% Pluoronic. After the loading,
cells were washed with Tyrode, pH 7.4, tryp-
sinized, resuspended in Tyrode, and transferred to the
cuvette of the fluorescence detector (Perkin-Elmer LS50B)
under continuous stirring. Experiments were carried out
by measuring cell fluorescence at 25 °C (kEX = 488 nm,
15. Synthesis of 3a as exemplificative: (N.B.: Resiniferonoids
are obnoxious and toxic compounds, and their handling
should be carried out only by trained personnel, wearing
gloves and face protection, and working in a well-
ventilated hood). To a cooled (0 °C) stirred solution of
ROPA (2, 50 mg, 0.11 mmol) and vanillic acid (23 mg,
0.16 mmol, 1.5 mol equiv) in dry THF (2 mL), triphenyl-
phosphine (TPP, 41 mg, 0.16 mmol, 1.5 mol equiv) and
diisopropylazodicarboxylate (DIAD, 31 lL, 32 mg,
0.16 mmol, 1.5 mol equiv) were added. The reaction
mixture was stirred at room temperature and followed
by TLC on silica gel (hexane/EtOAc 7:3 as eluant, Rf
ROPA: 0.10; Rf 3a: 0.21). After 3 h, the reaction was
worked up by evaporation, and the residue was dissolved
in toluene (ca. 5 mL) and cooled to 4 °C overnight. After
filtration of the copious white precipitate, the filtrate was
purified by gravity column chromatography on silica gel
(15 mL, petroleum ether/EtOAc 7:2 as eluant) to afford
38 mg (57%) of 3a as a white powder. Mp 89 °C (ether);
1H NMR (CDCl3, 300 MHz): d 0.96 (d, J = 7.3 Hz, 18-
Me), 1.52 (s, 17-Me), 1.55 (m, H-12a), 1.81 (br d,
J = 1.1 Hz, H-19), 2.10 (m, H-12b), 2.20 (d, J = 19.0 Hz,
H-5a), 2.56 (d, J = 19.0 Hz, H-5b), 2.60 (m, H-11), 3.16 (br
s, H-8 and H-10), 3.21 (s, H-20), 3.93 (s, OMe), 4.25 (d,
J = 2.5 Hz, H-14), 4.71 (br s, H-16a,b), 4.73 (d,
J = 12.5 Hz, H-20a), 4.76 (d, J = 12.5 Hz, H-20b), 6.00
(br s, H-7), 6.92 (d, J = 8.1 Hz, H-600), ca. 7.25 (m, 10-Ar),
ca. 7.35 (m, 10-Ar), 7.48 (s, H-1), 7.54 (s, H-300), 7.61 (d,
J = 8.1 Hz, H-700); 13C-NMR (CDCl3, 75.5 MHz): d 10.4
kEM = 540 nm) before and after the addition of the test
compounds at various concentrations. Kinetic effects
(Szallasi, A.; Blumberg, P. M.; Annicelli, L. L. K.;
Krause, J. E.; Cortright, D. N. Mol. Pharmacol. 1999,
56, 581) were not observed in the calcium fluorescence
assays as all compounds behaved exactly like RTX in
terms of onset and duration of response.
21. For a discussion, see: Appendino, G.; Szallasi, A. Prog.
Med. Chem. 2006, 44, 145.
´
´ ´
22. Szolcsanyi, J.; Jancso-Gabor, A. Arzneim. Forsch. 1975,
25, 1877.
23. Chou, M. Z.; Mtui, T.; Gao, Y.-D.; Kohler, M.; Middle-
ton, R. E. Biochemistry 2004, 43, 2501.