Similarities and differences in the structure-activity relationships of capsaicin and resiniferatoxin analogues

Article abstract of DOI:10.1021/jm960139d

Structure-activity relationships in analogues of the irritant natural product capsaicin have previously been rationalized by subdivision of the molecule into three structural regions (A, B, and C). The hypothesis that resiniferatoxin (RTX), which is a hig

Full text of DOI:10.1021/jm960139d

J . Med. Chem. 1996, 39, 2939-2952
2939
Sim ila r ities a n d Differ en ces in th e Str u ctu r e-Activity Rela tion sh ip s of
Ca p sa icin a n d Resin ifer a toxin An a logu es
Christopher S. J . Walpole,* Stuart Bevan, Graham Bloomfield, Robin Breckenridge, Iain F. J ames,
Timothy Ritchie, Arpad Szallasi,† J anet Winter, and Roger Wrigglesworth
Sandoz Institute for Medical Research, 5 Gower Place, London WC1E 6BN, U.K., and Department of Physiology and
Pharmacology, Karolinska Institute, S-171 77 Stockholm, Sweden
Received February 13, 1996X
Structure-activity relationships in analogues of the irritant natural product capsaicin have
previously been rationalized by subdivision of the molecule into three structural regions (A, B,
and C). The hypothesis that resiniferatoxin (RTX), which is a high-potency ligand for the same
receptor and which has superficial structural similarities with capsaicin, could be analogously
subdivided has been investigated. The effects of making parallel changes in the two structural
series have been studied in a cellular functional assay which is predictive of analgesic activity.
Parallel structural changes in the two series lead to markedly different consequences on
biological activity; the 3- and 4-position aryl substituents (corresponding to the capsaicin ‘A-
region’) which are strictly required for activity in capsaicin analogues are not important in
RTX analogues. The homovanillyl C-20 ester group in RTX (corresponding to the capsaicin
‘B-region’) is more potent than the corresponding amide, in contrast to the capsaicin analogues.
Structural variations to the diterpene moiety suggest that the functionalized 5-membered
diterpene ring of RTX is an important structural determinant for high potency. Modeling
studies indicate that the 3D position of the R-hydroxy ketone moiety in the 5-membered ring
is markedly different in the phorbol (inactive) analogues and RTX (active) series. This difference
appears to be due to the influence of the strained ortho ester group in RTX, which acts as a
local conformational constraint. The reduced activity of an analogue substituted in this region
and the inactivity of a simplified analogue in which this unit is entirely removed support this
conclusion.
Ch a r t 1
In tr od u ction
Capsaicin and resiniferatoxin (RTX) are irritant
natural products which activate the capsaicin (or ‘va-
nilloid’) receptor in a subpopulation of primary afferent
sensory neurons. These sensory neurons are involved
in nociception, and so these agents are targets for the
design of a novel class of analgesics. Both ligands cause
a novel ion channel in the plasma membrane to become
permeable to cations, eliciting a number of biological
activities including the excitatory (algesic) as well as
the analgesic effects which both agents evoke.
In addition to these biological similarities, a number
of groups have drawn attention to structural similarities
between the two molecules.1 Both compounds possess
a 3-methoxy-4-hydroxybenzyl group, hydrogen bond
donor and acceptor species, and hydrophobic regions.
It is generally assumed that the high degree of phar-
macological similarity between these two molecules is
a consequence of receptor recognition of those structural
moieties which are identical or similar in the two
compounds. Structure-activity relationships (SAR) for
capsaicin agonists have previously been rationalized, by
ourselves and others,2-4 by dividing the capsaicin
molecule into three regions: A (aromatic ring), B (amide
bond), and C (hydrophobic side chain). We hypothesized
that it might be possible to determine if RTX could be
so simplistically subdivided and, in doing so, shed light
on the nature of the receptor-ligand interactions with
both molecules. In particular, elucidation of the struc-
tural features of the RTX diterpene unit which are
responsible for the extremely high potency of RTX would
be invaluable in attempting to increase the potency of
simple capsaicinoid analogues. For such an approach
to be successful, however, comparable SAR in both
series would need to be demonstrated. In order to
investigate this hypothesis, parallel modifications to the
structure of both ligands was undertaken, and the
consequences for biological activity were determined in
a well-characterized cellular functional assay (Ca2+
uptake assay),5 which is predictive for analgesic activity
in vivo.2
Ch em istr y
‘r-Region ’. C-20 Ester Su bstitu tion of Resin if-
er on ol 9,13,14-Or th op h en yla ceta te (ROP A): Syn -
th esis of Su bstitu ted P h en yla cetic Acid s. The
FMoc-protected 4-(aminoethoxy)-3-methoxyphenylacetic
acid, used in the synthesis of 4-(aminoethoxy)-RTX, 5d
† Karolinska Institute.
X Abstract published in Advance ACS Abstracts, J uly 1, 1996.
S0022-2623(96)00139-2 CCC: $12.00 © 1996 American Chemical Society

2940 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15
Walpole et al.
Sch em e 1
(Scheme 1), was synthesized from a commercially avail-
able acetophenone, having first introduced the 4-ph-
thalimidoethoxy group, using an oxidative thallium(III)
rearrangement by the method of McKillop.6 Saponifica-
tion followed by hydrazinolysis gave the free amino acid
which was FMoc protected using FMoc-succinimide
carbonate in the presence of triethylamine.
‘â-Region ’. The synthesis of RTX amide (7) was
achieved by the route shown in Scheme 3. The num-
bering convention for daphnane diterpenes is shown in
the structure of the starting material, resiniferonol 9,-
13,14-orthophenylacetate (ROPA). Chlorination of the
allylic alcohol ROPA, without rearrangement, was
achieved by the method of Magid,8 using hexachloroac-
etone and triphenylphosphine. The resulting allyl
chloride was treated with sodium azide in DMF to give
the allyl azide in quantitative yield. Reduction to the
allylamine, resiniferamine 9,13,14-orthophenylacetate,
was best achieved using SnCl2 in MeOH.9 Acylation of
the resulting allylamine with homovanillic acid hydrox-
ysuccinimide ester10 gave RTX amide, 7. Concurrent
with ourselves, the group of Blumberg have developed
a similar synthesis of 7.11
‘ø-Region ’. C-20 Ester Su bstitu tion of P h or bol
Ester s. In the case of the phorbol ester derivatives,
12-deoxyphorbol 13-phenylacetate 20-(3-methoxy-4-hy-
droxyphenylacetate) (8),12 12,13-diacetylphorbol 20-(3-
methoxy-4-hydroxyphenylacetate) (9a ), and 12,13-dide-
canoylphorbol 20-(3-methoxy-4-hydroxyphenylacetate)
(9b), the parent C-20 alcohol was acylated with homo-
vanillic acid using 2-(fluoromethyl)pyridinium tosylate
as the coupling reagent. The C-20 formate (presumably
The same thallium rearrangement was also used to
access the methyl ester of 3-azido-4-methoxyphenylace-
tic acid7 in the synthesis of the photoaffinity label RTX-
PAL, 5e (Scheme 2), after subjecting the commercially
available 4-hydroxy-3-aminoacetophenone to diazotiza-
tion and treatment with sodium azide followed by
methylation, all under subdued light. Saponification of
the methyl ester gave the substituted phenylacetic acid.
C-20 Ester ifica tion . For 4a ,b and 5a -d , the com-
mercially available alcohol ROPA was acylated on the
C-20 OH group with the appropriately substituted
phenylacetic (or other) acids by conversion of the acid
to the acid chloride with thionyl chloride and then
condensation with ROPA, in the presence of triethy-
lamine. The esterification yielding 5e, however, was
achieved using DCCI/DMAP. In the case of 5d , the
product of the coupling reaction was deprotected with
piperidine in dichloromethane.
Sch em e 2

SAR of Capsaicin and Resiniferatoxin Analogues
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15 2941
Sch em e 3
Sch em e 4
derived from formic acid present in DMF) was formed
as a significant side product in all reactions using this
reagent but could be separated from product chromato-
graphically.
Or th o Ester Su bstitu tion . Resiniferonol orthoac-
etate13 (Scheme 4) was kindly provided by B. Sorg. The
corresponding resiniferonol orthobenzoate was synthe-
sized from the parent diterpene polyol, resiniferonol, by
14,20-dibenzoylation with benzoic anhydride in the
presence of DMAP followed by ortho ester cyclization
with anhydrous p-toluenesulfonic acid in refluxing
dichloroethane, which gave the C-20 benzoate 9,13,14-
orthobenzoate in quantitative yield. This was C-20
deprotected with sodium methoxide in methanol to give
the orthobenzoate C-20 alcohol. Resiniferonol, prepared
from ROPA by a modification of the method of Sorg,13
was obtained in 60% yield, a substantially improved
yield compared with that reported by these authors
(16.9%) especially since 35% of unreacted ROPA was
also recovered from the reaction mixture.
C-20 esterification of resiniferonol orthoacetate and
resiniferonol orthobenzoate with acetylhomovanillic acid

2942 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15
Walpole et al.
Ta ble 1
Ta ble 2
displacement of relative stimulation of relative
[3H]RTX binding affinity
Ca2+uptake
(EC50, nM)
potency
(RTX)
compd
(Ki, nM)
(RTX)
1 (capsaicin) 2000 ( 500
300 ( 40
2 (RTX)
5a
0.12 ( 0.01
8.3 ( 1.0
1.0 ( 0.1
4.3 ( 0.2
10.6 ( 0.9
600 ( 10016
3.2 ( 1.1
0.17 ( 0.02
4.4 ( 3.4
>10 000
1
1.6 ( 0.1
1
0.015
0.120
0.028
0.011
0.0002
0.038
0.706
0.027
10.22 ( 2.71
14.89 ( 3.77
13.04 ( 2.26
122.0 ( 19.0
2450 ( 140
13.2 ( 3.8
7.6 ( 0.6
0.157
0.107
0.123
0.013
0.0007
0.122
0.210
0.028
<0.000 08
5c
5d
7
8
10a
10b
11b
ROPA
57.5 ( 21.2
<0.000 01 >20 000
Ch a r t 2
Ca2+ uptake
(EC50,2 nM)
caps
RTX
caps
RTX
A/R-region (R)
compd compd analogues
analogues
H
octyl
4a
4b
>2000
20 000
(3-H,4-H)Ar
(3-OMe,4-H)Ar
(3-OMe,4-OH)Ar
(3-OMe,4-OMe)Ar
(3-OMe,4-
3a
3b
3c
3d
3e
5a
5b
>100 000
>100 000
10.22 ( 2.71
7.89 ( 2.51
1.6 ( 0.1
2 (RTX) 550 ( 80
5c
5d
6410 ( 140 14.89 ( 3.77
410 ( 6018 13.04 ( 2.26
OCH2CH2NH2)Ar
(3-N3,4-OMe)Ar
5e
10.64 ( 1.40
was effected with DCCI/DMAP coupling, and the prod-
ucts were selectively phenol ester deprotected using
pyrrolidine14 to give orthoacetyl-RTX, 10a, and orthoben-
zoyl-RTX, 10b (Scheme 4).
It is notable, however, that the potencies in the Ca2+
uptake assay are generally about 10-fold lower than
binding potencies, presumably due to other processes,
e.g., uptake into mitochondria being necessary for
detection in this assay. Since the main aim of the
present study is to compare the SAR of parallel modi-
fications in both the RTX and capsaicin structural
series, subsequent SAR discussion will be restricted to
comparisons of Ca2+ uptake assay data, since directly
comparable data are available for a wide variety of RTX
and capsaicin analogues and since correlations between
activity in this assay and other assays, including anti-
nociceptive activity in vivo, have been established
elsewhere.2
Diter p en e Mod ifica tion s to RTX. The 3-â-OH
analogue of RTX (11b) was synthesized by direct reduc-
tion of RTX with NaBH4 in ethanol. The stereochem-
istry of the reduction product at C-3 was established
by NOE difference NMR spectroscopy. The 4-â-methoxy
analogue of RTX (11a ) was synthesized by C-20 acety-
lation of ROPA to the acetate (4a ) followed by methy-
lation with methyl iodide in the presence of silver oxide
in DMF. The crude reaction product was transesterified
with sodium methoxide in methanol to the parent C-20
alcohol. DCCI/DMAP coupling with acetylhomovanillic
acid followed by selective phenolic deacetylation, using
pyrrolidine as before, completed the synthesis.
Resu lts a n d Discu ssion
The synthesis by Bloomfield et al. of the simplified
RTX analogue based on a 2,9,10-trioxatricyclo[4.3.1.03,8]-
decane system (12) has been published elsewhere.15
Biology. The [3H]RTX binding assay was performed
using the published methods.16 The difference between
the affinity of RTX reported in this study and the value
we have previously reported17 is most probably due to
the differences in conditions under which the two assays
were carried out (37 °C16 as opposed to room tempera-
ture17 and different techniques for reducing nonspecific
binding). All compounds were tested as agonists in the
calcium uptake assay.5
Biologica l Activity. In a series of RTX analogues
which have been tested in both the Ca2+ uptake and
[3H]RTX binding assays (Table 1), the biological activity
of these compounds was similar, with comparable rank
orders of potency being found for the various ligands.
(1) C-20 Ester Su bstitu tion : A-Rin g SAR. Com-
parison of the importance of substitution of the aromatic
ring in the capsaicin A-region and RTX C-20 ester (R-
region) reveals marked differences (Table 2). The C-20
alcohol ROPA and its aliphatic esters, the acetate 4a 13
and the nonanoate 4b, are all inactive. The unsubsti-
tuted phenylacetyl ester 5a ,13 however, retains sub-
stantial activity, albeit somewhat reduced in comparison
with RTX. This contrasts markedly with the case of
capsaicin analogues, where the substituents in the 3-
and 4-positions of the A-ring are essential for potent
agonist activity, exemplified by the inactivity of the
unsubstituted analogue 3a 2. The phenolic 4-OH group
in capsaicin analogues is of particular importance and
can only effectively be substituted by the 4-aminoethoxy
group, as exemplified by 3e.18 This group, presumably,
is able to mimic the H-bond donor/acceptor properties

SAR of Capsaicin and Resiniferatoxin Analogues
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15 2943
Ta ble 3
of the phenol which have been proposed to be important
for agonist activity.2 To compare the importance of 3-
and 4-position aromatic substitution in the two series,
a series of RTX analogues functionalized in these
positions was synthesized.
3- a n d 4-P osition Ar yl Rin g Su bstitu tion . In this
series of RTX analogues, the structure of the diterpene
and linker groups is unchanged with respect to the
natural product, whereas in the comparable capsaicin
analogues the amide B-region is retained, as in the
natural product, but the C-region has been simplified
to the bioequivalent octyl group,3 i.e., RTX analogues
have â ) CH2COO, ø ) ROPA, and capsaicin analogues
have B ) amide (CH2NHCO) C ) octyl (see Table 2).
As can be seen from the data in Table 2, the 4-OH
group in the RTX analogues is of little importance, since
activity is almost completely retained by analogues not
containing this substituent, e.g., 5b. The retention of
activity in RTX-PAL, 5e, enables its use as a photoaf-
finity label for the capsaicin/vanilloid receptor.19 In both
series the dimethyl ether compound has somewhat
lower activity than the parent catechol monomethyl
ether. In the case of the RTX analogues, however, the
aminoethoxy group in 5d apparently does not mimic the
H-bond donor/acceptor function of the 4-OH group as it
appears to in capsaicin analogues.18
Ca2+ uptake (EC50,3 nM)
caps
compd
RTX
compd
caps
RTX
B/â-region
analogues
analogues
X ) O, Y ) CO
X ) NH, Y ) CO
X ) CO, Y ) NH
6a
6b
3c
2 (RTX) 670 ( 110
1.6 ( 0.1
7
300 ( 10 122.0 ( 19.0
550 ( 80
In conclusion, the C-20 phenylacetyl ester in RTX does
appear to be required for high potency, though ring
substitution appears much less tightly proscribed than
is the case in the capsaicin A-ring. The phenolic OH
group which is critical for activity in simple capsaicin
analogues has little or no role in RTX analogues. This
divergence in SAR is particularly apparent when com-
paring the coupled pairs 3b with 5b (capsaicin ana-
logues) and 5b with 2 (RTX analogues). Previous
studies, using irritant activity as the biological read-
out,13,20 also conclude that phenylacetyl esters appear
to be most potent, although a more important role for
the phenolic OH group is suggested than is indicated
in the present study.
substituted in the para position by small hydrophobic
moieties. The nature of the diterpene group ‘ø’ has not
been thoroughly investigated in previous studies al-
though it is apparent that some derivatives in which
the daphnane ortho ester moiety is substituted or
exchanged for other diterpenes appear to be much less
active than RTX in evoking chemogenic pain12 or in [3H]-
RTX binding experiments.16 The nature of the ortho
ester side chain appears to be important, to some extent,
for irritant activity.13
Diter p en e Rep la cem en t. As well as the known 12-
deoxyphorbol 20-homovanillic acid ester (8),12 the C-20
homovanillic esters of phorbol 12,13-diacetate and phor-
bol 12,13-didecanoate (9a ,b, respectively) were pre-
pared. All three compounds showed negligible activity
in the Ca2+ flux assay (Table 4) and, as such, are even
less active than the simple octyl ester capsaicin ana-
logue 6a 3 (Table 3). Although physicochemical differ-
ences (e.g., hydrophobicity) cannot be ruled out as a
contributing factor, this result suggests that some highly
specific function of the diterpene ortho ester moiety in
RTX is responsible for its extremely high potency in this
assay.
Superficially, the tricyclic diterpene ø-regions of RTX
(2) and 8 look very similar, with the same ring junction
stereochemistry and identical functionality around the
5- and 7-membered rings, e.g., the Râ-unsaturated
R-hydroxy ketone in position C-3/C-4. The only differ-
ences occur in the substitution around the 6-membered
ring where RTX is oxygenated in a 9R,13R,14R-pattern,
while the phorbols and the 12-deoxyphorbol have a 9R,-
12â,13R-trioxygenated and 9R,13R-dioxygenated sub-
stitution pattern, respectively. In addition, the cyclo-
propane ring in the phorbols would be expected to
constrain the 6-membered ring into a rather different
conformation compared with the 13â-isopropenyl group
present in RTX. When molecular models of RTX and 8
are overlaid (Figure 1), while the majority of the
diterpene skeleton can be superimposed, the region of
(2) C-20 Lin k er : B-Region SAR. The linker region
(â) joining the substituted aromatic R-region to the C-20
position of the diterpene skeleton in RTX could be
considered comparable to a ‘reverse ester’ B-region in a
series of capsaicin analogues, i.e., B ) CH2COO. Such
a B-region is active in a series of capsaicin analogues
(e.g., 6a 3) though the ester is somewhat less potent than
the corresponding amide (Table 3). By contrast, RTX
amide, 7, is dramatically less potent than the parent
ester RTX (2). Possible explanations for this large
difference between the amide and ester include stabi-
lization of an inactive trans C-20 ester conformation by
the amide or unfavorable H-bonding or dipolar interac-
tions with the amide NH. The former explanation has
been proposed by the group of Blumberg11 as an
explanation for the much lower potency of RTX amide
(7) than RTX in [3H]RTX binding experiments and for
the induction of chemogenic pain and hypothermia in
vivo.
(3) Diter p en e Mod ifica tion : C-Region SAR. C-
Region SAR in capsaicin analogues has been previously
published4 and so is not discussed here in detail. In
summary, a hydrophobic group, e.g., an octyl chain or
substituted benzyl or phenethyl group, is required for
high potency. Optimally, such aralkyl groups are

2944 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15
Walpole et al.
Ta ble 4
F igu r e 1. Molecular modeling overlay (best fit of 6- and
7-membered rings) of RTX (2) and the 12-deoxyphorbol 13-
phenylacetyl ester 8. The common homovanillyl ester moiety
is shown in magenta, the phorbol diterpene skeleton carbon
atoms are shown in green, and the RTX diterpene carbon
atoms are shown in yellow. All diterpene oxygen atoms are
shown in red.
conformation. Another interesting feature of phorbol/
RTX analogue overlays, exemplified by Figure 1, is that
the orientation of the C-3 ketone carbonyl group with
respect to the superimposable 7-membered rings is
markedly different in the two structural series. This
effect, presumably, is a consequence of the local confor-
mational constraints imposed by the cyclic ortho ester
in the daphnane system on the 6-membered ring, being
transferred to the rings to which it is fused. Analogues
of RTX were therefore synthesized in which the nature
of the ortho ester side chain was varied in an attempt
to identify the most important feature of the ortho ester
in RTX for activity: (1) presentation of a precisely
oriented hydrophobic group or (2) a local conformational
constraint with the potential of influencing other im-
portant receptor interactions.
Or th o Ester Sid e Ch a in Mod ifica tion s. To inves-
tigate the possibility of a precisely located hydrophobic
binding site, which recognizes the ortho ester side chain,
analogues of RTX were synthesized in which the ortho-
phenylacetyl group (benzyl side chain) was replaced by
an orthoacetyl and orthobenzoyl group (methyl and
phenyl side chains, respectively). While the orthoben-
zoyl compound 10b retains high potency, comparable
to RTX (in both the Ca2+ uptake and binding assays,
Table 4; see also Table 1), there is a significant loss of
potency in the case of the orthoacetate 10a , which is
particularly apparent from the binding data (Table 1).
This compound does, however, still remain much more
potent than any of the phorbol analogues or the simple
octyl ester capsaicin analogue 6a .3 These data suggest
that the more important effect of the ortho ester is to
act as a local conformational constraint, though there
does appear to be a relatively small additive require-
ment for a hydrophobic or larger group substituting the
central ortho ester carbon atom. A further possibility,
that direct receptor recognition of the ortho ester oxygen
atoms underlies the extremely high potency of these
ligands, seems unlikely in view of the high potency of
the orthobenzoate 10b, in which these oxygen atoms
would be occluded from contact with the receptor by the
bulky phenyl ring, and the lower potency of the orthoac-
etate 10a , in which such an interaction should be more
favorable, on steric grounds.
conformational space accessible to the benzyl moiety on
the 12-phenylacetate side chain in 8 is clearly much
greater and tends to assume a rather equatorial ar-
rangement (approximately coplanar) with respect to the
6-membered ring, which exists in a chair conformation.
By contrast, in RTX, the benzyl moiety of the orthophe-
nylacetate side chain is much more constrained and held
approximately perpendicular with respect to the 6-mem-
bered ring which, in this case, is held in a boat
Da p h n a n e Sk eleton Mod ifica tion s. The above
argument suggests that the ortho ester group may exert
a remote conformational effect on the daphnane ortho

SAR of Capsaicin and Resiniferatoxin Analogues
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15 2945
College London Chemistry Department) and Bruker AM500
500 MHz (Sandoz, Basel) instruments. All spectra were
recorded using tetramethylsilane (TMS) as an internal stan-
dard, and chemical shifts are reported in parts per million (δ)
downfield from TMS. Coupling constants are reported in
hertz. Mass spectra were recorded by the Mass Spectrometry
Department of University College London, using a VG 7070F/H
spectrometer, and FAB spectra were recorded in Sandoz,
Basel, using a VG 70-SE spectrometer. Accurate mass deter-
minations were made by M. Cocksedge and Dr. D. Carter,
London School of Pharmacy, using a VG ZAB SE mass
spectrometer and FAB ionization.
TLC was performed using Merck Kiesel gel 60 F254 silica
plates, and components were visualized using UV light and
iodine vapor. HPLC was performed using a Waters 600
system (µ-Bondapak C-18 column (RP18), using gradients or
isocratic solvent systems of compositions stated in the text).
Compounds were purified by flash column chromatography21
using Merck Kiesel gel 60 (230-400 mesh) or preparative
HPLC using a Waters Delta Prep 3000 preparative chroma-
tography system equipped with a Dynamax 300A C18 12 µm
particle size column (83-243-C), dimensions 41.4 × 250 mm.
Solvents were HLPC grade and used without further purifica-
tion. Solvents were dried according to the standard proce-
dures.22 Test compounds were homogeneous by TLC or HPLC
unless otherwise stated. Chemical yields were not optimized.
Resiniferatoxin (RTX) and resiniferonol 9,13,14-orthophe-
nylacetate (ROPA) were obtained from CCR Inc., KS, and were
pure by HPLC. In both cases, NMR and MS spectra, including
HRMS data, confirmed the identity of the compound. Resin-
iferonol 9,13,14-orthoacetate13 was kindly provided by B. Sorg.
12-Deoxyphorbol 13-phenylacetate, phorbol 12,13-diacetate,
and phorbol 12,13-didecanoate were obtained from LC Ser-
vices.
Sa fety In for m a tion . All the diterpene final compounds
described in this article should be assumed to be extremely
irritant compounds. All diterpene intermediates and final
products should, in addition, be treated as potential tumor
promotors, especially esters of phorbol. Great care should be
taken to avoid exposure. When transferring a known weight
of compound is necessary, solutions in a known volume of dry
dichloromethane or acetone should be made and the desired
amount of solution aliquoted into the reaction vessel or vial
for testing, as required. Solvent should then be removed by
rotory evaporation in vacuo or by passing a stream of dry
nitrogen over the sample until a constant weight of diterpene,
as a glassy resin, remains as a film inside the vessel.
R TX An a logu es: 3-Azid o-4-h yd r oxya cet op h en on e.
3-Amino-4-hydroxyacetophenone (3.1 g, 20 mmol) was dis-
solved in water (8 mL), and concentrated HCl solution (4.5
mL) was added. The cream suspension was stirred at 0 °C
while a solution of sodium nitrite (1.45 g, 21 mmol) in water
(5 mL) was added, dropwise. After stirring for 15 min, the
orange solution was filtered and stirred at 0 °C during the
addition of a solution of sodium azide (1.3 g, 20 mmol) in water
(5 mL). The frothing solution was stirred until N2 evolution
ceased (30 min), and the yellow precipitate was then collected
by filtration under subdued light and dried in vacuo to give a
yellow solid, yield 2.3 g (64%): TLC (silica gel, CH2Cl2/MeOH,
20:1) Rf ) 0.44; 1H NMR (CDCl3, 200 MHz) δ 2.58 (3H, s,
ArCH3), 6.25 (1H, br s, ArOH), 6.98 (1H, d, J ) 8 Hz, ArH5),
7.69 (1H, d of d, J ) 8 Hz, J ′ ) 2 Hz, ArH6), 7.76 (1H, d, J ′ )
2 Hz, ArH2).
F igu r e 2. Molecular modeling overlay (best fit of 6-membered
ring and ortho ester) of RTX (2; shown in magenta) and the
simplified RTX analogue 12 (shown in blue).
ester skeleton which places key structural features in
the correct orientation for receptor recognition. From
the modeling work, a likely candidate for such an
important structural feature is the Râ-unsaturated
R-hydroxy ketone moiety in positions C-3 and C-4 of the
diterpene. In order to test this hypothesis, analogues
of RTX were prepared in which both the carbonyl and
hydroxy groups were modified. While methylation of
the 4-â-OH group, in compound 11a , has a negligible
effect on activity, reduction of the 3-keto carbonyl group
to the 3-â,4-â-diol (11b) results in a substantial loss of
potency (Table 4).
A simplified analogue (12), described by Bloomfield
et al.,15 which possesses a cyclohexane phenylacetyl
ortho ester linked to a homovanillic ester via an allylic
alcohol, following the backbone of RTX, has very low
potency, being less active than the simple octyl ester
capsaicin analogue 6a 3. Since 12 and RTX can easily
be overlaid, as shown in Figure 2, the correct orientation
of the substituted phenylacetate ortho ester per se does
not seem to be sufficient for high potency. It is doubtful
that the increased conformational freedom which results
from the formal removal, in 12, of the fused 7-membered
diterpene ring present in RTX could alone result in such
a dramatic loss in activity.
Over a ll Con clu sion s. The loss of potency on reduc-
tion of the 3-keto group, taken together with the
different orientation of this group in RTX (active) and
phorbol (inactive) analogues with otherwise comparable
diterpene functionality, suggests an important role for
the functional groups substituting the 5-membered ring
in RTX, especially the 3-keto group. The inactivity of
the simplified RTX analogue described by Bloomfield
et al.,18 which contains the phenylacetyl ortho ester
moiety but not the fused 7- or 5-membered rings, is
consistent with this suggestion. Since the 5-membered
ring substituents in RTX could make potential hydrogen-
bonding interactions with the receptor, perhaps these
groups, and not the linker group â, should be viewed
as a counterpart of the capsaicin B-region, especially
since the B-region/â-region SAR is so different between
the two series.
3-Azid o-4-m eth oxya cetop h en on e. A solution of 3-azido-
4-hydroxyacetophenone (2.2 g, 12.4 mmol) in acetone (60 mL)
and solid K2CO3 (1.71 g, 12.4 mmol) was stirred under a N2
atmosphere. Methyl iodide (7.1 g, 50 mmol) was added and
the mixture refluxed, under subdued light, for 2 h. The crude
product was purified by flash column chromatography (silica
gel, cyclohexane/EtOAc, 2:1) to give a beige solid, yield 1.8 g
(76%): TLC (silica gel, cyclohexane/EtOAc, 1:1) Rf ) 0.40; 1H
NMR (CDCl3, 200 MHz) δ 2.53 (3H, s, ArCH3), 3.92 (3H, s,
ArOCH3), 5.90 (1H, d, J ) 8 Hz, ArH5), 7.60 (1H, d, J ′ ) 2 Hz,
ArH2), 7.72 (1H, d of d, J ) 8 Hz, J ′ ) 2 Hz, ArH6).
Attempts to mimic the 3D position and orientation of
the 3-keto group in the RTX diterpene moiety in a
simple capsaicin analogue would be an important step
in testing this hypothesis and could lead to the design
of simple capsaicin analogues with higher potencies
than have so far been achieved.
Exp er im en ta l Section
Gen er a l In for m a tion . Routine NMR spectra were re-
corded using a Varian Gemini 200 machine. High-field spectra
were recorded using Varian VX400 400 MHz (University
Meth yl 3-Azid o-4-m eth oxyp h en yla ceta te.7 Thallium-
(III) nitrate (2.75 g, 6.3 mmol) was dissolved in a 17% solution

2946 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15
Walpole et al.
of perchloric acid (70% aqueous) in methanol (18.6 mL) and
stirred at room temperature. A solution of 3-azido-4-meth-
oxyacetophenone (1.2 g, 6.3 mmol) in the same methanolic
perchloric acid solution (10 mL) was added and the reaction
mixture stirred, under subdued light, for 18 h. After this time
significant starting material (∼30%) remained by TLC, and
so additional thallium nitrate (1 g, 2.3 mmol) was added and
the reaction mixture stirred for a further 1 h, after which time
no starting material remained by TLC. The reaction mixture
was poured into water (1 L) and extracted with EtOAc, and
the extract was dried over Na2SO4. The crude product was
purified by flash column chromatography (silica gel, cyclohex-
ane/EtOAc, 2:1) to give a pale yellow oil, yield 0.7 g (50%):
3.15 (2H, br s, H-10, H-8), 3.21 (2H, s, ortho ester CH2Ph),
4.12 (2H, AB, CH2Cl H2-20), 4.24 (1H, d, H-14), 4.71 (2H, s,
H2-16), 6.01 (1H, m, H-7), 7.20-7.40 (5H, m, phenylacetyl ortho
ester ArH), 7.46 (1H, m, H-1); FAB-MS (M + 1)+ 483 (100).
9,13,14-Or th op h en yla cetylr esin ifer on yl 20-Azid e.11 9,-
13,14-Orthophenylacetylresiniferonyl 20-chloride (70 mg, 0.15
mmol) was dissolved in DMF and stirred at room temperature.
A solution of sodium azide (10.7 mg, 0.17 mmol) in 10:2 DMF/
H2O was added and the mixture stirred for 90 min, after which
time reaction was complete by TLC. The solvent was removed
in vacuo and 50/50% diethyl ether/CH2Cl2 (10 mL) added to
the resulting resin. The insoluble salts were removed by
filtration, and the organic solution was concentrated in vacuo
to leave a colorless glass (70 mg, 100%): pure by TLC (silica
1
TLC (silica gel, cyclohexane/EtOAc, 1:1) Rf ) 0.51; H NMR
1
(CDCl3, 200 MHz) δ 3.51 (2H, s, ArCH2CO), 3.66 (3H, s, ArCH2-
COOCH3), 3.82 (3H, s, ArOCH3), 6.81 (1H, d, J ) 8 Hz, ArH5),
6.90 (1H, d, J ′ ) 2 Hz, ArH2), 6.96 (1H, d of d, J ) 8 Hz, J ′ )
2 Hz, ArH6).
gel, cyclohexane/EtOAc, 1:1) Rf ) 0.52; H NMR (CDCl3, 200
MHz) δ 0.95 (3H, d, CH3 H3-18), 1.52 (3H, s, CH3 H3-17), 1.65
(1H, d, H-12R), 1.82 (3H, br d, CH3 H3-19), 2.12 (1H, t, H-12â),
2.18 (1H, AB, H-5R), 2.50 (1H, m, H-11), 2.65 (1H, AB, H-5â),
3-Azid o-4-m eth oxyp h en yla cetic Acid . Methyl 3-azido-
4-methoxyphenylacetate (0.65 g, 2.2 mmol) was dissolved in
dioxane (25 mL), 5 N NaOH (6 mL, 29 mmol) was added, and
the reaction mixture was stirred for 4 h at room temperature
under subdued light. The dioxane was then removed in vacuo,
and the remaining aqueous solution was carefully acidified
with HCl (concentrated) until a beige solid precipitated. The
mixture was extracted with EtOAc, and dried over Na2SO4,
and evaporated to give a yellow gum. The crude product was
purified by flash column chromatography (silica gel, cyclohex-
ane/EtOAc, 1:1) to give a pale yellow glass, yield 0.42 g (69%):
1H NMR (CD3OD, 200 MHz) δ 3.39 (2H, s, ArCH2CO), 3.83
(3H, s, ArOCH3), 6.92 (1H, d, J ) 8 Hz, ArH5), 6.96 (1H, d, J ′
) 2 Hz, ArH2), 7.06 (1H, d of d, J ) 8 Hz, J ′ ) 2 Hz, ArH6).
9,13,14-Or th op h en yla cetylr esin ifer on yl 20-(3-Azid o-4-
m eth oxyp h en yla ceta te) (5e). Resiniferonol 9,13,14-ortho-
phenylacetate (20 mg, 0.043 mmol) was dissolved in dry
CH2Cl2 (2 mL), (dimethylamino)pyridine (0.57 mg, 0.0047
mmol) was added, and the solution was stirred at room
temperature under a N2 atmosphere in the dark. A solution
of dicyclohexylcarbodiimide (DCCI; 9.7 mg, 0.047 mmol) in
CH2Cl2 (0.5 mL) and then a solution of 3-azido-4-methoxyphe-
nylacetic acid (9.8 mg, 0.047 mmol) in CH2Cl2 (0.5 mL) was
added, and the reaction mixture was stirred for 1 h. The
solution was washed with 1 M NaHCO3 and then NaCl
(saturated) and dried over MgSO4. After evaporation in vacuo,
the crude product was purified by preparative reversed-phase
HPLC (isocratic MeOH (83%)/H2O (17%), no UV monitor). The
pure fractions identified by analytical HPLC were pooled and
evaporated under subdued light to give a colorless glass, yield
27.6 mg (98%): TLC (silica gel, cyclohexane/EtOAc, 1:1) Rf )
0.48 (colorless, darkens in light); HPLC (isocratic MeOH (83%)/
H2O (17%)) tR ) 5.8 min, 100% pure; 1H NMR (CDCl3, 200
MHz) δ 0.96 (3H, d, CH3 H3-18), 1.53 (3H, s, CH3 H3-17), 1.83
(3H, br d, CH3 H3-19), 2.02-2.24 (3H, m, H-5R, H-12R,â), 2.47
(1H, AB, H-5â), 2.56 (1H, m, H-11), 3.09 (2H, br s, H-10, H-8),
3.22 (2H, s, ortho ester CH2Ph), 3.57 (2H, s, phenylacetyl ester
ArCH2CO), 3.84 (3H, s, ArOCH3), 4.20 (1H, d, H-14), 4.56 (2H,
AB, H2-20), 4.71 (2H, s, H2-16), 5.87 (1H, m, H-7), 6.83 (1H, d,
J ) 8 Hz, ArH5), 6.94 (1H, d, J ′ ) 2 Hz, ArH2), 7.03 (1H, d of
d, J ) 8 Hz, J ′ ) 2 Hz, ArH6), 7.25-7.50 (6H, m, phenylacetyl
ortho ester 5ArH, H-1); FAB-MS (M + 1)+ 654 (92). Accurate
mass (FAB MH+): calcd for C37H40N3O8, 654.2315; found,
654.2310.
3.15 (2H, br s, H-10, H-8), 3.22 (2H, s, ortho ester CH2Ph),
3.79 (2H, br s, CH2N3 H2-20), 4.26 (1H, d, H-14), 4.63 (2H, s,
H2-16), 5.89 (1H, br m, H-7), 7.20-7.40 (5H, m, phenylacetyl
ortho ester ArH), 7.46 (1H, br s, H-1); FAB-MS (M + 1)+ 490
(100).
9,13,14-Or th op h en yla cetylr esin ifer on yla m in e.11 Tin-
(II) chloride (67 mg, 0.36 mmol) was dissolved in anhydrous
methanol (1 mL) and stirred at room temperature under a N2
atmosphere. 9,13,14-Orthophenylacetylresiniferonyl 20-azide
(42 mg, 0.086 mmol), in solution in anhydrous methanol (0.5
mL), was slowly added. The reaction mixture was stirred for
8 h, after which time no starting material remained by TLC.
The solvent was removed in vacuo, and the residue was
redissolved in 1 M NaOH (2 mL) and then extracted with CH2-
Cl2 (10 mL). The organic solution was dried over MgSO4 and
then evaporated to give a colorless glass, yield 30 mg (75%)
which was >90% pure by TLC and used in the next step
without purification: TLC (silica gel, CH2Cl2/MeOH/HOAc, 90:
9:1) Rf ) 0.08 (stains strongly with ninhydrin and fluorescam-
ine); 1H NMR (CDCl3, 200 MHz) δ 0.95 (3H, d, CH3 H3-18),
1.52 (3H, s, CH3 H3-17), 1.55 (1H, d, H-12R), 1.86 (3H, br d,
CH3 H3-19), 2.10-2.25 (2H, m, H-12b, H-5R), 2.55-2.65 (2H,
m, H-11, H-5â), 3.11 (2H, br s, H-10, H-8), 3.21 (2H, s, ortho
ester CH2Ph), 3.28 (2H, d, CH2NH2 H2-20) 4.25 (1H, d, H-14),
4.72 (2H, s, H2-16), 5.76 (1H, br s, H-7), 7.25-7.40 (5H, m,
phenylacetyl ortho ester ArH), 7.46 (1H, br s, H-1); FAB-MS
(M + 1)+ 490 (100).
N-(9,13,14-Or th oph en ylacetylr esin ifer on yl)-4-h ydr oxy-
3-m et h oxyp h en yla cet a m id e11 (7). 9,13,14-Orthopheny-
lacetylresiniferonylamine (22 mg, 0.048 mmol) was dissolved
in dry CH2Cl2 (2 mL) and stirred on ice under a N2 atmosphere.
Homovanillic acid N-hydroxysuccinimide ester8 (12.7 mg, 0.048
mmol), as a solution in CH2Cl2 (0.5 mL), was added and the
reaction mixture stirred for 18 h. The reaction mixture was
washed with water and then saturated NaCl and dried over
MgSO4
glass which was purified by preparative reversed-phase HPLC
O (30%)). The pure fractions identi-
. The solvent was removed in vacuo leaving a colorless
(isocratic MeOH (70%)/H2
fied by analytical HPLC were pooled and evaporated to give a
colorless glass, yield 8 mg (27%): TLC (silica gel, CH2Cl2/
MeOH, 25:1) Rf ) 0.19; HPLC (MeOH/H2O gradient 60-90%
MeOH) tR ) 10.6 min, 100% pure; 1H NMR (CDCl3, 200 MHz)
δ 0.94 (3H, d, CH3 H3-18), 1.20 (1H, d, H-12 R), 1.54 (3H, s,
CH3 H3-17), 1.81 (3H, br d, CH3 H3-19), 2.02 (1H, AB, H-5R),
2.12 (1H, d, H-12â), 2.40 (1H, AB, H-5â), 2.54 (1H, m, H-11),
3.01 (2H, br s, H-10, H-8), 3.21 (2H, s, ortho ester CH2Ph),
3.56 (2H, s, ArCH2CONH), 3.66 (2H, m, CONHCH2 H2-20),
3.82 (1H, br s, ArOH), 3.86 (3H, s, ArOCH3), 4.16 (1H, d, H-14),
4.71 (2H, s, H2-16), 5.52-5.60 (2H, br m, ArOH, CH2NHCO),
5.62 (1H, s, H-7), 6.74-6.90 (3H, m, vanillyl ArH), 7.25-7.50
(6H, m, phenylacetyl ortho ester 5ArH, H-1). FAB-MS (M +
9,13,14-Or th op h en yla cetylr esin ifer on yl 20-Ch lor id e.11
Resiniferonol 9,13,14-orthophenylacetate (100 mg, 0.22 mmol)
was dissolved in hexachloroacetone (700 µL, 4.8 mmol) and
stirred on an ice bath. Triphenylphosphine (62.1 mg, 0.24
mmol) in CH2Cl2 (200 µL) was slowly added. After stirring
for 15 min, no starting material remained by TLC. The solvent
was evaporated in vacuo, and the crude product was purified
by preparative reversed-phase HPLC (isocratic MeOH (83%)/
H2O (17%)). The pure fractions were pooled and evaporated
to give a colorless glass, yield 90 mg (87%): TLC (silica gel,
cyclohexane/EtOAc, 1:1) Rf ) 0.59; 1H NMR (CDCl3, 200 MHz)
δ 0.96 (3H, d, CH3 H3-18), 1.52 (3H, s, CH3 H3-17), 1.58 (1H,
d, H-12 R), 1.83 (3H, br d, CH3 H3-19), 2.15 (1H, m, H-12â),
2.32 (1H, AB, H-5R), 2.56 (1H, m, H-11), 2.70 (1H, AB, H-5â),
1)+ 628 (100). Accurate mass (FAB MH+): calcd for C37H42
NO8, 628.2910; found, 628.2914.
-
4-(Br om oeth oxy)-3-m eth oxya cetop h en on e. Acetovanil-
lone (5 g, 30 mmol), 1,2-dibromoethane (89 mL, 194 g, 1.04
mol), 40% KOH (21 mL, 150 mmol), and 40% tetrabutylam-
monium hydroxide (20 mmol) were combined and heated to
55 °C with stirring. No acetovanillone remained by TLC after

SAR of Capsaicin and Resiniferatoxin Analogues
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15 2947
3 h. The cooled reaction mixture was diluted with CH2Cl2 (200
mL), extracted with water (2 × 100 mL), washed with
saturated NaCl, and then dried over Na2SO4. After removal
of the solvents in vacuo, a yellow crystalline solid remained,
reaction mixture stirred at room temperature for 2 h. The
solvent was removed in vacuo, and the residue was redissolved
in EtOAc, washed with an aqueous solution of KHSO4 (1%)
and NaCl (saturated), and then dried over MgSO4. The solvent
was removed in vacuo, and the crude product was purified by
flash column chromatography (silica gel, CH2Cl2/MeOH, 10:
1). The pure fractions were evaporated to give a cream solid,
yield 320 mg (40%): TLC (silica gel, CH2Cl2/MeOH, 10:1) Rf )
1
yield 7.89 g (95%): H NMR (CDCl3, 200 MHz) δ 2.60 (3H, s,
ArCOCH3), 3.66 (2H, m, CH2CH2Br), 3.96 (3H, s, ArOCH3),
4.42 (2H, m, ArOCH2CH2Br), 6.93 (1H, d, J ) 8 Hz, ArH5),
7.50-7.70 (2H, m, ArH2, ArH6).
0.23; 1H NMR (CD3OD, 200 MHz)
δ 3.35 (2H, m,
4-(P h t h a lim id o e t h o x y )-3-m e t h o x y a c e t o p h e n o n e .
4-(Bromoethoxy)-3-methoxyacetophenone (7.5 g, 28 mmol) was
added to dry DMF (50 mL) and stirred at 55 °C until
solubilized. Potassium phthalimide (6.5 g, 35 mmol) was
added and the mixture stirred at 55 °C for 2 h, after which
time no starting material remained by TLC. The solvent was
removed in vacuo, redissolved in EtOAc, washed with water
and saturated NaCl, and dried over MgSO4. The product, on
evaporation of the solvent, was recrystallized from ethanol to
give cream needles, yield 6.7 g (72%): TLC (silica gel, cyclo-
fluorenylCH2O), 3.50 (2H, s, ArCH2CO), 3.75 (3H, s, ArOCH3),
3.90 (1H, m, fluorenyl 5-ring CH), 4.10-4.50 (4H, m,
ArOCH2CH2NH), 6.82-6.89 (3H, m, vanillyl ArH), 7.19-7.79
(8H, m, fluorenyl ArH).
9,13,14-Or th op h en yla cetylr esin ifer on yl 20-[4-[[(F lu o-
r en ylm eth yloxyca r bon yl)a m in o]eth oxy]-3-m eth oxyp h e-
n yla ceta te]. 4-[[(Fluorenylmethyloxycarbonyl)amino]ethoxy]-
3-methoxyphenylacetic acid (50 mg, 0.11 mmol) was dissolved
in dry CH2Cl2 (1 mL) and stirred at room temperature during
the addition of freshly distilled thionyl chloride (130 mg, 1.1
mmol). The mixture was then refluxed for 15 min, after which
time the solvent was removed in vacuo to leave an orange oil.
The oil was redissolved in dry CH2Cl2 (1 mL) and diluted with
dry hexane (10 mL), causing an oil to separate. The solution
was decanted from the oil which was washed with dry hexane,
and the solvent residues were removed in vacuo, leaving the
acid chloride as an orange glass which was used without
further purification, yield 42 mg (81%).
A solution of resiniferonol 9,13,14-orthophenylacetate (24
mg, 0.052 mmol) in dry CH2Cl2 (0.5 mL) was stirred on ice,
under a N2 atmosphere, and triethylamine (14.6 µL, 0.1 mmol)
was added. A solution of the acid chloride (36.4 mg, 0.08
mmol) in dry CH2Cl2 (0.5 mL) was slowly added and the
reaction mixture allowed to come to room temperature with
stirring over 18 h. The solvent was removed in vacuo, and
the crude product was purified by flash column chromatogra-
phy (silica gel, cyclohexane/EtOAc, 3:2). The solvent was
removed from the pure fractions to leave a colorless glass, yield
31.3 mg (65%): TLC (silica gel, CH2Cl2/MeOH, 10:1) Rf ) 0.73;
1H NMR (CDCl3, 200 MHz) δ 0.95 (3H, d, CH3 H3-18), 1.52
(3H, s, CH3 H3-17), 1.83 (3H, br d, CH3 H3-19), 2.05 (1H, AB,
H-5R), 2.14 (2H, t, 2H-12), 2.43 (1H, AB, H-5â), 2.54 (1H, m,
H-11), 3.07 (2H, br m, H-10, H-8), 3.20 (2H, s, ortho ester CH2-
Ph), 3.57-3.61 (4H, br m, phenylacetyl ester ArCH2CO,
fluorenylCH2O), 3.82 (3H, s, ArOCH3), 4.07 (2H, br t,
ArOCH2CH2NH), 4.21 (1H, d, H-14), 4.23 (1H, t, fluorenyl
5-ring CH), 4.40 (2H, m, ArOCH2CH2NH), 4.56 (2H, AB, H2-
20), 4.70 (2H, s, H2-16), 5.29 (1H, s, 4-OH), 5.53 (1H, br t,
carbamate NH), 5.87 (1H, m, H-7), 6.80-6.90 (3H, m, vanillyl
ArH), 7.25-7.75 (14H, m, phenylacetyl ortho ester 5ArH,
fluorenyl ArH, H-1).
9,13,14-Or th op h en yla cetylr esin ifer on yl 20-[4-(Am in o-
eth oxy)-3-m eth oxyp h en yla ceta te] (5d ). 9,13,14-Orthophe-
nylacetylresiniferonyl 20-[4-[[(fluorenylmethyloxycarbonyl) ami-
no]ethoxy]-3-methoxyphenylacetate] (25 mg, 0.028 mmol) was
dissolved in dry CH2Cl2 (3 mL) and stirred at room tempera-
ture. Distilled piperidine (3 mL, 30.3 mmol) was added and
the reaction mixture stirred for 15 min, after which time no
starting material remained by TLC. The solvents were
removed in vacuo to leave a white solid which was purified by
preparative reversed-phase HPLC (gradient 10-60% CH3CN/
H2O). The pure fractions were evaporated, and the residue
was redissolved in CH2Cl2, washed with water and NaCl
(saturated), and then dried over Na2SO4 to give a colorless
glass, yield 13.2 mg (69%): TLC (silica gel, CH2Cl2/MeOH/
HOAc, 80:18:2) Rf ) 0.33 (stains strongly with ninhydrin and
fluorescamine); analytical reversed-phase HPLC (gradient 10-
100% CH3CN/H2O) tR ) 12.4 min, 100% pure; 1H NMR (CDCl3,
200 MHz) δ 0.95 (3H, d, CH3 H3-18), 1.52 (3H, s, CH3 H3-17),
1.80 (3H, br d, CH3 H3-19), 1.90-2.22 (5H, br m, H-5R, 2H-
12, ArOCH2CH2NH2), 2.30 (2H, AB, H-5â), 2.56 (1H, m, H-11),
3.04-3.10 (2H, br m, H-10, H-8), 3.20 (2H, s, ortho ester CH2-
Ph), 3.40 (2H, br m, ArOCH2CH2NH2), 3.56 (2H, s, ArCH2-
CO), 3.85 (3H, s, ArOCH3), 4.15 (2H, br t, ArOCH2CH2NH2),
4.22 (1H, d, H-14), 4.55 (2H, m, H2-20), 4.70 (2H, s, H2-16),
5.88 (1H, m, H-7), 6.80-6.90 (3H, m, vanillyl ArH), 7.25-7.40
(5H, m, phenylacetyl ortho ester ArH), 7.45 (1H, m, H-1); FAB-
1
hexane/EtOAc, 1:2) Rf ) 0.50; H NMR (CDCl3, 200 MHz) δ
2.50 (3H, s, ArCOCH3), 3.70 (3H, s, ArOCH3), 4.02 (2H, t,
ArOCH2CH2N[phthal]), 4.35 (2H, t, ArOCH2CH2N[phthal]),
7.10 (1H, d, J ) 8 Hz, ArH5), 7.45-7.70 (2H, m, ArH2, ArH6).
Meth yl 4-(P h th a lim id oeth oxy)-3-m eth oxyp h en yla ce-
ta te. Thallium(III) nitrate (2.62 g, 6.0 mmol) was dissolved
in a 17% solution of perchloric acid (70% aqueous) in methanol
(17.7 mL) and stirred at room temperature. A solution of
4-(phthalimidoethoxy)-3-methoxyacetophenone (2 g, 6.0 mmol)
in the same methanolic perchloric acid solution (10 mL) was
added and the reaction mixture stirred for 18 h, after which
time no starting material remained by TLC. The reaction
mixture was poured into water (1 L) and extracted with EtOAc,
and the extract was dried over Na2SO4. The crude product
was purified by flash column chromatography (silica gel,
cyclohexane/EtOAc, 2:1) to give a white solid, yield 1.5 g (69%):
1
TLC (silica gel, cyclohexane/EtOAc, 1:1) Rf ) 0.26; H NMR
(CDCl3, 200 MHz) δ 3.52 (2H, s, ArCH2COCH3), 3.70 (3H, s,
ArCH2COCH3), 3.80 (3H, s, ArOCH3), 4.10-4.30 (4H, m,
ArOCH2CH2N[phthal], ArOCH2CH2N[phthal]), 6.70-6.90 (3H,
m, vanillyl ArH), 7.60-7.90 (4H, m, phthalimide ArH).
4-(P h th a lim id oeth oxy)-3-m eth oxyp h en yla cetic Acid .
Methyl 4-(phthalimidoethoxy)-3-methoxyphenylacetate (3.0 g,
8.2 mmol) was dissolved in dioxane (65 mL), and 5 M NaOH
(16.3 mL, 82 mmol) was added. The reaction mixture was
stirred at room temperature for 18 h, after which time no
starting material reamained by TLC. The dioxane was
removed in vacuo leaving an aqueous solution which was
acidified with HCl (concentrated) causing precipitation of a
white solid which was extracted with EtOAc. The extract was
washed with NaCl (saturated) and dried over Na2SO4. Evapo-
ration of the solvent gave a white crystalline solid, yield 2.75
g (100%): TLC (silica gel, cyclohexane/EtOAc, 1:1) Rf ) 0.02;
1H NMR (CD3OD, 200 MHz) δ 3.55 (2H, s, ArCH2CO2H), 3.72
(2H, m, ArOCH2CH2N[phthal]), 3.78 (3H, s, ArOCH3), 4.15
(2H, t, ArOCH2CH2N[phthal]), 6.80-6.95 (3H, m, vanillyl
ArH), 7.30-8.00 (4H, m, phthalimide ArH).
4-(Am in oeth oxy)-3-m eth oxyp h en yla cetic Acid . 4-(Ph-
thalimidoethoxy)-3-methoxyphenylacetic acid (1.5 g, 4.4 mmol)
was dissolved in ethanol (7.5 mL), hydrazine monohydrate
(1.74 mL, 35.8 mmol) was added, and the mixture was refluxed
for 2 h. The cooled reaction mixture was filtered and the
filtrate evaporated in vacuo to give a gum which was redis-
solved in MeOH (20 mL). The solution was diluted with EtOAc
(100 mL) which caused an oil to separate. The solvent was
decanted from the oil, the residue was washed with hexane,
and solvent residues were evaporated in vacuo, leaving a
colorless gum, yield 0.8 g (80%): TLC (silica gel, CH2Cl2/MeOH/
1
HOAc, 120:90:5) Rf ) 0.43 (purple stain with ninhydrin); H
NMR (CD3OD, 200 MHz) δ 3.15 (2H, t, ArOCH2CH2NH2), 3.42
(2H, s, ArCH2CO), 3.84 (3H, s, ArOCH3), 4.10 (2H, br t,
ArOCH2CH2NH2), 6.80-7.00 (3H, m, vanillyl ArH).
4-[[(F lu or en ylm et h yloxyca r b on yl)a m in o]et h oxy]-3-
m et h oxyp h en yla cet ic Acid . 4-(Aminoethoxy)-3-methox-
yphenylacetic acid (0.4 g, 1.78 mmol) was dissolved in water
(3 mL), and triethylamine (0.27 mL, 1.96 mmol) was added
(final pH ∼ 10). A solution of FMoc-succinimide carbonate
(0.65 g, 1.93 mmol) in CH3CN (3 mL) was added and the

2948 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15
Walpole et al.
MS (M + 1)+ 672 (100). Accurate mass (FAB MH+): calcd for
C39H46NO9, 672.3173; found, 672.3170.
a N2 atmosphere, and triethylamine (5.5 µL, 0.041 mmol) was
added. A solution of acetyl chloride (2 µM, 0.027 mmol) in
dry CH2Cl2 (0.5 mL) was slowly added and the reaction
mixture allowed to come to room temperature with stirring
over 3 h. The solvent was removed in vacuo, and the crude
product was purified by flash column chromatography (silica
gel, cyclohexane/EtOAc, 3:1). The pure fractions were evapo-
rated to give a colorless glass, yield 11.2 mg (83%): TLC (silica
gel, cyclohexane/EtOAc, 1:1) Rf ) 0.50; analytical reversed-
phase HPLC (gradient 10-70% MeOH/water) tR ) 14.92 min,
100% pure; 1H NMR (CDCl3, 200 MHz) δ 0.96 (3H, d, CH3 H3-
18), 1.54 (3H, s, CH3 H3-17), 1.83 (3H, br d, CH3 H3-19), 2.09
(3H, OCOCH3), 2.10-2.23 (1H, m, H-5R, 2H-12), 2.50-2.62
(2H, m, H-11, H-5â), 3.14 (2H, br m, H-10, H-8), 3.21 (2H, s,
ortho ester CH2Ph), 4.26 (1H, d, H-14), 4.54 (2H, AB, H2-20),
4.70 (2H, s, H2-16), 5.92 (1H, m, H-7), 7.20-7.40 (5H, m,
phenylacetyl ortho ester ArH), 7.46 (1H, m, H-1); FAB-MS (M
+ 1)+ 507 (100). Accurate mass (FAB MH+): calcd for
C30H35O7, 507.2383; found, 507.2380.
9,13,14-Or th op h en yla cetylr esin ifer on yl 20-P h en yla c-
eta te (5a ). A solution of resiniferonol 9,13,14-orthopheny-
lacetate (12 mg, 0.026 mmol) in dry CH2Cl2 (0.5 mL) was
stirred on ice, under a N2 atmosphere, and triethylamine (7.5
µL, 0.052 mmol) was added. A solution of phenylacetyl
chloride (6.1 mg, 0.039 mmol) in dry CH2Cl2 (0.5 mL) was
slowly added and the reaction mixture allowed to come to room
temperature with stirring over 18 h. The solvent was removed
in vacuo, and the crude product was purified by flash column
chromatography (silica gel, cyclohexane/EtOAc, 3:1). The pure
fractions were evaporated to give a colorless glass, yield 10.5
mg (70%): TLC (silica gel, CH2Cl2/MeOH, 25:1) Rf ) 0.90;
analytical reversed-phase HPLC (isocratic 80% MeOH/water)
tR ) 7.37 min, >98% pure; 1H NMR (CDCl3, 200 MHz) δ 0.95
(3H, d, CH3 H3-18), 1.62 (3H, s, CH3 H3-17), 1.83 (3H, br d,
CH3 H3-19), 2.05 (1H, AB, H-5R), 2.15 (2H, m, 2H-12), 2.43
(2H, AB, H-5â), 2.56 (1H, m, H-11), 3.06 (2H, br m, H-10, H-8),
3.21 (2H, s, ortho ester CH2Ph), 3.66 (2H, s, ArCH2CO), 4.19
(1H, d, H-14), 4.56 (2H, AB, H2-20), 4.69 (2H, s, H2-16), 5.85
(1H, m, H-7), 7.15-7.45 (11H, m, phenylacetyl ortho ester ArH,
C-20 ester ArH, H-1); FAB-MS (M + 1)+ 583 (55). Accurate
mass (FAB MH+): calcd for C36H39O7, 583.2695; found,
583.2692.
9,13,14-Or th op h en yla cetylr esin ifer on yl 20-(3-Meth ox-
yp h en yla ceta te) (5b). A solution of resiniferonol 9,13,14-
orthophenylacetate (10 mg, 0.022 mmol) in dry CH2Cl2 (0.5
mL) was stirred on ice, under a N2 atmosphere, and triethy-
lamine (6.2 µL, 0.044 mmol) was added. A solution of the
3-methoxyphenylacetyl chloride (6.1 mg, 0.033 mmol) in dry
CH2Cl2 (0.5 mL) was slowly added and the reaction mixture
allowed to come to room temperature with stirring over 18 h.
The solvent was removed in vacuo, and the crude product was
purified by flash column chromatography (silica gel, cyclohex-
ane/EtOAc, 3:1). The pure fractions were evaporated to give
a colorless glass, yield 6.2 mg (47%): TLC (silica gel, CH2Cl2/
MeOH, 25:1) Rf ) 0.82; analytical reversed-phase HPLC
(isocratic 80% MeOH/water) tR ) 5.15 min, 100% pure; 1H
NMR (CDCl3, 200 MHz) δ 0.95 (3H, d, CH3 H3-18), 1.55 (3H,
s, CH3 H3-17), 1.82 (3H, br d, CH3 H3-19), 2.05 (1H, AB, H-5R),
2.13 (1H, m, H-12), 2.42 (2H, AB, H-5â), 2.55 (1H, m, H-11),
3.05 (2H, br m, H-10, H-8), 3.21 (2H, s, ortho ester CH2Ph),
3.62 (2H, s, ArCH2CO), 3.78 (3H, s, ArOCH3), 4.20 (1H, d,
H-14), 4.56 (2H, AB, H2-20), 4.70 (2H, s, H2-16), 5.86 (1H, m,
H-7), 6.80-6.90 (3H, m, C-20 ester ArH2,4,6), 7.20-7.40 (6H,
m, phenylacetyl ortho ester ArH, C-20 ester ArH5), 7.42 (1H,
m, H-1); FAB-MS (M + 1)+ 613 (60). Accurate mass (FAB
MH+): calcd for C37H41O8, 613.2801; found, 613.2805.
9,13,14-Or th op h en yla cetylr esin ifer on yl 20-Non a n oa te
(4b). A solution of resiniferonol 9,13,14-orthophenylacetate
(9 mg, 0.020 mmol) in dry CH2Cl2 (0.5 mL) was stirred on ice,
under a N2 atmosphere, and triethylamine (3.1 µL, 0.022
mmol) was added. A solution of the nonanoyl chloride (3.9
mg, 0.022 mmol) in dry CH2Cl2 (0.5 mL) was slowly added and
the reaction mixture allowed to come to room temperature with
stirring over 18 h. The solvent was removed in vacuo, and
the crude product was purified by flash column chromatogra-
phy (silica gel, cyclohexane/EtOAc, 4:1). The pure fractions
were evaporated to give a colorless glass, yield 5 mg (36%):
TLC (silica gel, CH2Cl2/MeOH, 10:1) Rf ) 0.85; analytical
reversed-phase HPLC (gradient 10-70% MeOH/water) tR
)
17.8 min, >98% pure; 1H NMR (CDCl3, 200 MHz) δ 0.89 (3H,
br t, alkyl CH3), 0.96 (3H, d, CH3 H3-18), 1.21-1.35 (10H, env,
alkyl CH2), 1.54 (3H, s, CH3 H3-17), 1.60-1.70 (2H, br m,
COCH2CH2), 1.83 (3H, br d, CH3 H3-19), 2.11-2.18 (3H, m,
H-5a, 2H-12), 2.32 (2H, t, COCH2CH2), 2.50-2.62 (2H, m,
H-11, H-5â), 3.13 (2H, br m, H-10, H-8), 3.22 (2H, s, ortho ester
CH2Ph), 4.26 (1H, d, H-14), 4.55 (2H, AB, H2-20), 4.70 (2H, s,
H2-16), 5.90 (1H, m, H-7), 7.20-7.40 (5H, m, phenylacetyl ortho
ester ArH), 7.46 (1H, m, H-1). FAB-MS (M + 1)+ 605 (100).
Accurate mass (FAB MH+): calcd for C37H49O7, 605.3478;
found, 605.3474.
9,13,14-Or th oacetylr esin ifer on yl 20-(4-Acetoxy-3-m eth -
oxyp h en yla ceta te). A solution of resiniferonol 9,13,14-
orthoacetate13 (4.4 mg, 0.011 mmol) and (dimethylamino)py-
ridine (0.14 g, 0.0013 mmol) in dry CH2Cl2 (0.5 mL) was stirred
at room temperature, and a solution of acetylhomovanillic acid
(2.72 mg, 0.013 mmol) in dry CH2Cl2 (0.5 mL) and a solution
of DCCI (2.48 mg, 0.013 mmol) were added. The reaction
mixture was stirred for 1 h, and then the solvent was removed
in vacuo. Diethyl ether (2 mL) was added to the residue, the
suspension was filtered, and the filtrate was evaporated to
leave a colorless glass. The crude product was purified by
9,13,14-Or t h op h en yla cet ylr esin ifer on yl
20-(3,4-Di-
m eth oxyp h en yla ceta te) (5c). A solution of resiniferonol 9,-
13,14-orthophenylacetate (10 mg, 0.022 mmol) in dry CH2Cl2
(0.5 mL) was stirred on ice, under a N2 atmosphere, and
triethylamine (6.2 µL, 0.044 mmol) was added. A solution of
the 3,4-dimethoxyphenylacetyl chloride (7.1 mg, 0.033 mmol)
in dry CH2Cl2 (0.5 mL) was slowly added and the reaction
mixture allowed to come to room temperature with stirring
over 18 h. The solvent was removed in vacuo, and the crude
product was purified by flash column chromatography (silica
gel, cyclohexane/EtOAc, 3:1). The pure fractions were evapo-
rated to give a colorless glass, yield 5 mg (36%): TLC (silica
gel, CH2Cl2/MeOH, 25:1) Rf ) 0.60; analytical reversed-phase
HPLC (isocratic 80% MeOH/water) tR ) 4.34 min, 100% pure;
1H NMR (CDCl3, 200 MHz) δ 0.95 (3H, d, CH3 H3-18), 1.55
(3H, s, CH3 H3-17), 1.80 (3H, br d, CH3 H3-19), 2.08 (1H, AB,
H-5R), 2.15 (1H, m, H-12), 2.45 (2H, AB, H-5â), 2.55 (1H, m,
H-11), 3.08 (2H, br m, H-10, H-8), 3.20 (2H, s, ortho ester CH2-
Ph), 3.58 (2H, s, ArCH2CO), 3.86 (3H, s, ArOCH3), 3.88 (3H,
s, ArOCH3), 4.21 (1H, d, H-14), 4.58 (2H, AB, H2-20), 4.70 (2H,
s, H2-16), 5.87 (1H, m, H-7), 6.80 (3H, m, C-20 ester ArH),
7.20-7.40 (5H, m, phenylacetyl ortho ester ArH), 7.43 (1H,
m, H-1); FAB-MS (M + 1)+ 643 (50). Accurate mass (FAB
MH+): calcd for C38H43O9, 643.2907; found, 643.2902.
preparative HPLC (isocratic 70% MeOH/H2
O), yield 7.5 mg
(89%): TLC (silica gel, cyclohexane/EtOAc, 1:1) Rf ) 0.36; FAB-
MS (M + 1)+ 595 (100).
9,13,14-Or th oacetylr esin ifer on yl 20-(4-Hydr oxy-3-m eth -
oxyp h en yla ceta te) (10a ). 9,13,14-Orthoacetylresiniferonyl
4-acetoxy-3-methoxyphenylacetate (7 mg, 0.011 mmol) was
dissolved in dry CH2Cl2 (1 mL) and stirred at room tempera-
ture, under a N2 atmosphere. Pyrrolidine (28.4 mg, 0.37 mmol)
in CH2Cl2 (0.1 mL) was added. After 70 min, no starting
material remained by TLC. The solvent was evaporated in
vacuo, and the crude product was purified by preparative
HPLC (isocratic 70% MeOH/H2O). The pure fractions were
evaporated to give a colorless glass, yield 5 mg (86%): TLC
(silica gel, CH2Cl2/MeOH, 50:1) Rf ) 0.41; analytical reversed-
phase HPLC (gradient 10-100% MeCN/0.1% aqueous TFA)
tR ) 12.0 min, >97% pure; 1H NMR (CDCl3, 200 MHz) δ 1.15
(3H, d, CH3 H3-18), 1.65 (3H, s, orthoacetyl CH3), 1.74 (3H, br
d, CH3 H3-19), 1.82 (3H, s, CH3 H3-17), 2.03 (1H, AB, H-5R),
2.18 (2H, m, H-12), 2.41 (2H, AB, H-5â), 2.59 (1H, m, H-11),
3.01 (1H, br m, H-10), 3.08 (1H, br m, H-8), 3.55 (2H, s, ArCH2-
CO), 3.89 (3H, s, ArOCH3), 4.22 (1H, d, H-14), 4.54 (2H, AB,
H2-20), 4.88 (1H, s, H-16), 4.98 (1H, s, H-16), 5.84 (1H, m, H-7),
9,13,14-Or th oph en ylacetylr esin ifer on yl 20-Acetate (4a).
A solution of resiniferonol 9,13,14-orthophenylacetate (12.3 mg,
0.027 mmol) in dry CH2Cl2 (0.5 mL) was stirred on ice, under

SAR of Capsaicin and Resiniferatoxin Analogues
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15 2949
6.80 (3H, m, vanillyl ArH), 7.51 (1H, m, H-1). FAB-MS (M +
1)+ 533 (40). Accurate mass (FAB MH+): calcd for C31H37O9,
553.2438; found, 553.2434.
H3-18), 1.72 (1H, d, H-12R), 1.84 (3H, s, CH3 H3-17), 1.86 (3H,
br m, CH3 H3-19), 2.20-2.38 (2H, m, H-5R, H-12â), 2.59 (1H,
AB, H-5â), 2.76 (1H, m, H-11), 3.26 (2H, br m, H-8, H-10), 4.08
(2H, s, H2-20), 4.49 (1H, d, H-14), 4.91 (1H, m, H-16), 5.06 (1H,
s, H-16), 5.92 (1H, br m, H-7), 7.40 (3H, m, orthobenzoyl
ArH3,4,5), 7.62 (1H, m, H-1), 7.75 (2H, m, orthobenzoyl ArH2,6).
9,13,14-Or t h ob en zoylr esin ifer on yl 20-(4-Acet oxy-3-
m eth oxyp h en yla ceta te). 9,13,14-Orthobenzoylresiniferonol
(14 mg, 0.031 mmol) was dissolved in dry CH2Cl2 (3 mL) and
Resin ifer on ol. Resiniferonol 9,13,14-orthophenylacetate
(105 mg, 0.23 mmol) was dissolved in MeOH (70 mL), and 1
N HCl (24 mL, 24 mmol) was added. The reaction mixture
was stirred for 4 h at room temperature before the addition of
1 M NaOMe solution in MeOH (30 mL, final pH ) 10) and
stirring for a further 30 min. After this time the reaction
mixture contained only starting material and resiniferonol by
HPLC. The solvent was evaporated, and the crude product
was purified by preparative HPLC (MeOH/H2O gradient, 10-
70%). The pure fractions were evaporated to give a colorless
glass, yield 53.7 mg (60%), as well as 37 mg (35%) of recovered
starting material: TLC (silica gel, CH2Cl2/MeOH, 10:1) Rf )
0.16; analytical reversed-phase HPLC (gradient 10-70%
MeOH/H2O) tR ) 11.2 min, >99% pure; 1H NMR (CD3OD, 200
MHz) δ 0.94 (3H, d, CH3 H3-18), 1.74 (3H, br d, CH3 H3-19),
1.77 (3H, s, CH3 H3-17), 1.90-2.02, (2H, m, H2-12), 2.33-2.35
(2H, m, AB, H-5R, H-11), 2.50 (1H, AB, H-5â), 3.16 (1H, br
m, H-10), 3.42 (1H, br m, H-8), 3.96 (2H, m, H2-20), 4.01 (1H,
d, H-14), 5.05 (2H, m, H2-16), 5.90 (1H, m, H-7), 7.52 (1H, m,
H-1); FAB-MS (M + 1)+ 391 (100).
Resin ifer on ol 14,20-Diben zoa te. Resiniferonol (54 mg,
0.14 mmol) was dissolved in dry EtOAc (6 mL), DMAP (38 mg,
0.31 mmol) was added, and the mixture was stirred at room
temperature under a N2 atmosphere. A solution of benzoic
anhydride (70 mg, 0.31 mmol) in EtOAc (3 mL) was slowly
added, and the reaction mixture was stirred for 18 h. TLC
indicated the absence of resiniferonol but the presence of
significant monobenzoylated material. Further benzoic an-
hydride (14 mg, 0.062 mmol) and DMAP (7.6 mg, 0.062 mmol)
were added, and the solution was stirred for a further 18 h.
After this time TLC indicated that the reaction mixture was
mainly dibenzoylated material. The solution was washed with
water and NaCl (saturated) and dried over MgSO4. The crude
product was purified by flash column chromatography (silica
gel, EtOAc/cyclohexane, 1:4) to give a colorless glass, yield 40.7
mg (51.5%): TLC (silica gel, EtOAc/cyclohexane, 1:1) Rf ) 0.35;
1H NMR (CD3OD, 200 MHz) δ 1.05 (3H, d, CH3 H3-18), 1.70
(1H, d, H-12R), 1.75 (3H, s, CH3 H3-17), 1.86 (3H, br d, CH3
H3-19), 2.25-2.46 (3H, m, H-5R, H-11, H-12â), 2.64 (1H, AB,
H-5â), 3.11 (1H, br m, H-8), 4.02 (1H, br m, H-10), 4.68 (2H,
m, H2-20), 5.12 (1H, s, H-16), 5.18 (1H, 2, H-16), 5.77 (1H, br
stirred at room temperature under a N2 atmosphere.
A
solution of DCCI (7.2 mg, 0.034 mmol) and DMAP (0.42 mg,
0.0034 mmol) in CH2Cl2 (0.5 mL) was added followed by a
solution of acetylhomovanillic acid (7.8 mg, 0.034 mmol) in
CH2Cl2 (0.5 mL). The reaction mixture was stirred for 1 h at
room temperature, after which time no starting material
remained by TLC. The solvent was evaporated in vacuo, and
the residue was suspended in diethyl ether, the solid removed
by filtration, and the filtrate evaporated in vacuo to leave the
crude product which was purified by preparative HPLC
(isocratic 73% MeOH/H2
O); the pure fractions were evaporated
in vacuo to give a colorless glass, yield 14 mg (66%): TLC (silica
1
gel, EtOAc/cyclohexane, 1:1) Rf ) 0.42; H NMR (CDCl3, 200
MHz) δ 1.23 (3H, d, CH3 H3-18), 1.70 (1H, d, H-12a), 1.82 (6H,
br s, CH3 H3-17, H3-19), 2.05 (1H, d, H-5R), 2.20-2.38 (2H, s,
m, ArOCOCH3, H-5â, H-12â), 2.76 (1H, m, H-11), 3.15 (1H,
br m, H-8), 3.22 (1H, br m, H-10), 3.58 (2H, s, ArCH2CO), 3.81
(3H, s, ArOCH3), 4.44 (1H, d, H-14), 4.54 (2H, s, H2-20), 4.89
(1H, m, H-16), 5.05 (1H, s, H-16), 5.94 (1H, br m, H-7), 6.83-
7.02 (3H, m, vanillyl ArH), 7.40 (3H, m, orthobenzoyl ArH3,4,5),
7.55 (1H, m, H-1), 7.75 (2H, m, orthobenzoyl ArH2,6).
9,13,14-Or t h ob en zoylr esin ifer on yl 20-(4-H yd r oxy-3-
m eth oxyp h en yla ceta te) (10b). 9,13,14-Orthobenzoylresin-
iferonyl 20-(4-acetoxy-3-methoxyphenylacetate) (8 mg, 0.012
mmol) was dissolved in dry CH2Cl2 (1 mL) and stirred at room
temperature under N2. A solution of pyrrolidine (50 µL, 0.60
mmol) in CH2Cl2 (0.5 mL) was added and the reaction mixture
was stirred for 90 min, after which time no starting material
remained by TLC. The solvent was removed in vacuo, the
crude product was purified by preparative HPLC (isocratic 70%
MeOH/H2O), and the pure fractions were evaporated in vacuo
to give a colorless glass, yield 7 mg (93%): TLC (silica gel,
EtOAc/cyclohexane, 1:1) Rf ) 0.32; HPLC (isocratic 70%
1
MeOH/H2O) tR ) 10.5 min, 100% pure; H NMR (CDCl3, 200
MHz) δ 1.25 (3H, d, CH3 H3-18), 1.70 (1H, d, H-12R), 1.82 (6H,
br s, CH3 H3-17, H3-19), 2.05 (1H, d, H-5R), 2.30 (1H, m,
H-12â), 2.44 (1H, AB, H-5â), 2.72 (1H, m, H-11), 3.15 (1H, br
m, H-8), 3.24 (1H, br m, H-10), 3.52 (2H, s, ArCH2CO), 3.81
(3H, s, ArOCH3), 4.43 (1H, d, H-14), 4.53 (2H, AB, H2-20), 4.91
(1H, m, H-16), 5.06 (1H, s, H-16), 5.92 (1H, br m, H-7), 6.70-
6.85 (3H, m, vanillyl ArH), 7.35-7.44 (3H, m, orthobenzoyl
d, H-7), 5.87 (1H, s, H-14), 7.34-7.66 (7H, m, benzoyl ArH3,4,5
,
H-1), 7.92-8.08 (4H, m, benzoyl ArH2,6); FAB-MS (M + 1)+
573 (30).
9,13,14-Or th oben zoylr esin ifer on ol 20-Ben zoa te. A so-
lution of resiniferonol 14,20-dibenzoate (20.6 mg, 0.036 mmol)
in dry dichloroethane (20 mL) was added by syringe to a dry
flask containing anhydrous CaCl2 (206 mg, 1.86 mmol) and
anhydrous toluenesulfonic acid (6 mg, 0.036 mmol). The
reaction mixture was heated to 80 °C for 1 h after which no
starting material remained. The precipitate was removed by
filtration, the solvent was evaporated, and the crude product
was purified by preparative HPLC (isocratic 75% MeOH/H2O).
The pure fractions were evaporated in vacuo to give a colorless
glass, yield 19.6 mg (98%): TLC (silica gel, EtOAc/cyclohexane,
1:1) Rf ) 0.62; 1H NMR (CDCl3, 200 MHz) δ 1.28 (3H, d, CH3
H3-18), 1.75 (1H, d, H-12R), 1.83 (3H, s, CH3 H3-17), 1.86 (3H,
br m, CH3 H3-19), 2.20-2.38 (2H, m, H-5R, H-12â), 2.68 (1H,
AB, H-5â), 2.77 (1H, m, H-11), 3.31 (2H, br m, H-8, H-10), 4.53
(1H, d, H-14), 4.78 (2H, AB, H2-20), 4.92 (1H, s, H-16), 5.07
(1H, s, H-16), 6.10 (1H, br m, H-7), 7.38-8.04 (11H, m, benzoyl,
orthobenzoyl ArH, H-1).
9,13,14-Or th oben zoylr esin ifer on ol. 9,13,14-Orthoben-
zoylresiniferonol 20-benzoate (19.6 mg, 0.035 mmol) was
dissolved in dry MeOH (10 mL) and stirred at room temper-
ature under a N2 atmosphere. A solution of NaOMe (390 µL
of 1 M solution, 0.39 mmol) in dry methanol was added, and
the reaction mixture was stirred for 1 h, after which time no
starting material remained by TLC. The crude product was
purified by preparative HPLC (isocratic 65% MeOH/H2O), and
the pure fractions were evaporated in vacuo to give a colorless
glass, yield 14.5 mg (91%): TLC (silica gel, EtOAc/cyclohexane,
1:1) Rf ) 0.19; 1H NMR (CDCl3, 200 MHz) δ 1.25 (3H, d, CH3
ArH3,4,5
FAB-MS (M + 1)
for C37H39O9, 615.2594; found, 615.2590.
), 7.60 (1H, m, H-1), 7.75 (2H, m, orthobenzoyl ArH2,6);
+ 615 (20). Accurate mass (FAB MH+): calcd
9,13,14-Or th oph en ylacetyl-3â-h ydr oxyr esin ifer on yl 20-
(4-Hyd r oxy-3-m eth oxyp h en yla ceta te) (11b). Resinifera-
toxin (11.3 mg, 0.018 mmol) was dissolved in absolute ethanol
(1 mL) and stirred at room temperature. NaBH4 (3.4 mg, 0.089
mmol) was added and the reaction mixture stirred for 2 h.
After this time no starting material remained by TLC, and so
AcOH (15 µM) was added and the solvent removed in vacuo.
The residue was redissolved in CH2Cl2, washed with water
and saturated NaCl, and dried over Na2SO4. The solvent was
removed in vacuo to leave a glass which was purified by
preparative HPLC (isocratic 75% MeOH/H2O). The pure
fractions were evaporated to give a colorless glass, yield 6.8
mg (60%): TLC (silica gel, EtOAc/cyclohexane, 1:1) Rf ) 0.24;
analytical reversed-phase HPLC (gradient 10-100% MeCN/
0.1% aqueous TFA) tR ) 17.2 min, 100% pure; 1H NMR (CD3-
OD, 200 MHz) δ 0.98 (3H, d, CH3 H3-18), 1.42 (1H, d, H-12R),
1.51 (3H, s, CH3 H3-17), 1.72 (3H, br d, CH3 H3-19), 2.10 (1H,
AB, H-5R), 2.16 (1H, m, H-12â), 2.40 (1H, AB, H-5â), 2.65 (1H,
m, H-11), 2.69 (1H, br m, H-10), 3.02 (1H, br m, H-8), 3.12
(2H, s, ortho ester CH2Ph), 3.54 (2H, AB, ArCH2CO), 3.80 (3H,
s, ArOCH3), 3.89 (1H, br, H-3), 4.15 (1H, d, H-14), 4.55 (2H,
AB, H2-20), 4.66 (1H, s, H-16), 4.71 (1H, s, H-16), 5.48 (1H, br
m, H-1), 5.83 (1H, m, H-7), 6.70-6.83 (3H, m, vanillyl ArH),

2950 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15
Walpole et al.
7.15-7.36 (5H, m, phenylacetyl ortho ester ArH); FAB-MS (M
+ 1)+ 631 (32). Accurate mass (FAB MH+): calcd for C37H43O9,
631.2907; found, 631.2903.
3â-Epimer was assigned by NOE difference NMR
spectroscopy: H-10 irradiated (δ 2.70); NOE-H-5R, H-3; H-3
irradiated (δ 3.89); NOE-H-10, H-5R, 3H-19; H-8 identified by
NOE from irradiation of H-14 (δ 4.18)-NOE to H-8, H-7, 3H-
17.
and purified by flash column chromatography (silica gel,
EtOAc/cyclohexane, 1:2). The pure fractions were evaporated
in vacuo to give a colorless glass, yield 4 mg (12%): TLC (silica
gel, CH2Cl2/MeOH, 25:1) Rf ) 0.21; analytical reversed-phase
HPLC (gradient 10-100% CH3CN/0.1% aqueous TFA) tR
)
12.4 min, >98% pure; 1H NMR (CDCl3, 200 MHz) δ 0.66 (1H,
d, H-14), 0.85 (3H, d, CH3 H3-18), 1.02 (3H, s, CH3 H3-16), 1.04
(3H, s, CH3 H3-17), 1.75 (3H, d, CH3 H3-19), 2.05 (2H, m, H-12),
2.26 (1H, AB, H-5R), 2.40 (1H, AB, H-5â), 2.90 (1H, m, H-8),
3.18 (1H, m, H-10), 3.52 (2H, s, ArCH2CO), 3.61 (2H, s, ArCH2-
CO), 3.78 (3H, s, ArOCH3), 4.46 (2H, m, H2-20), 5.34 (1H, br s,
ArOH), 5.60 (1H, br m, H-7), 6.73-6.81 (3H, m, vanillyl ArH),
7.25-7.35 (5H, m, phenylacetyl ester ArH), 7.56 (1H, s, H-1);
FAB-MS (M + 1)+ 631 (7). Accurate mass (FAB MH+): calcd
for C37H43O9, 631.2907; found, 631.2903.
9,13,14-Or th oph en ylacetyl-4â-m eth oxyr esin ifer on ol. 9,-
13,14-Orthophenylacetylresiniferonyl 20-acetate, 4a (50 mg,
0.099 mmol), in dry DMF (0.3 mL) was stirred under a N2
atmosphere with Ag2O (35 mg, 0.15 mmol). A solution of
methyl iodide (100 µL, 1.62 mmol) in DMF (50 µL) was added,
and the reaction mixture was stirred for 18 h at room
temperature. The solvent was evaporated, and methanol (0.5
mL) was added to the residue. The insoluble material was
removed by filtration and the acetyl protecting group removed
from the crude product in situ by transesterification by the
addition of a methanolic solution of NaOMe (1.1 mmol). After
stirring for 1 h at room temperature, the solvent was removed
in vacuo and the product was purified by preparative HPLC
(isocratic 72% MeOH/H2O); the pure fractions were evaporated
to give a colorless glass, yield 32 mg (67%): TLC (silica gel,
EtOAc/cyclohexane, 1:1) Rf ) 0.27; analytical reversed-phase
HPLC (isocratic 70% MeOH/H2O) tR ) 11.2 min, 100% pure;
1H NMR (CDCl3, 200 MHz) δ 0.99 (3H, d, CH3 H3-18), 1.53
(3H, s, CH3 H3-17), 1.56 (1H, d, H-12R), 1.80 (3H, br d, CH3
H3-19), 2.00 (1H, AB, H-5R), 2.12 (1H, m, H-12â), 2.62 (1H,
m, H-11), 2.93 (1H, br m, H-8), 3.02 (1H, AB, H-5â), 3.12 (1H,
m, H-10), 3.20 (2H, s, ortho ester CH2Ph), 3.32 (3H, s, 4-OCH3),
4.10 (2H, m, H2-20), 4.24 (1H, d, H-14), 4.70 (2H, s, H2-16),
5.86 (1H, m, H-7), 7.20-7.40 (6H, m, phenylacetyl ortho ester
ArH, H-1); FAB-MS (M + 1)+ 479 (100).
P h or bol 12,13-Dia ceta te 20-(4-Hyd r oxy-3-m eth oxyp h e-
n yla ceta te) (9a ). Phorbol 12,13-diacetate (90 mg, 0.2 mmol)
was dissolved in dry DMF (1 mL), and to this were added
triethylamine (46 µL, 0.36 mmol) and 2-(fluoromethyl)pyri-
dinium tosylate (109 mg, 0.39 mmol) in DMF (0.2 mL). The
reaction mixture was stirred at room temperature, under N2,
for 30 min before the addition of further triethylamine (71 µL,
0.54 mmol) and homovanillic acid (99 mg, 0.54 mmol) in DMF
(0.2 mL). The reaction mixture was heated to 60 °C for 2 h,
after which time the solvent was removed in vacuo and
purified by flash column chromatography (silica gel, EtOAc/
cyclohexane, 1:2). The pure fractions were evaporated in vacuo
to give a colorless glass, yield 29 mg (24%): TLC (silica gel,
CH2Cl2/MeOH, 25:1) Rf ) 0.41; analytical reversed-phase
HPLC (gradient 10-100% CH3CN/0.1% aqueous TFA) tR
)
1
12.8 min, 100% pure; H NMR (CDCl3, 200 MHz) δ 0.76 (3H,
d, CH3 H3-18), 1.00 (1H, d, H-14), 1.12 (3H, s, CH3 H3-16), 1.14
(3H, s, CH3 H3-17), 1.68 (3H, d, CH3 H3-19), 2.02 (6H, s, 2 ×
OCOCH3), 2.22 (1H, AB, H-5R), 2.42 (1H, AB, H-5â), 2.94 (1H,
m, H-8), 3.08 (1H, m, H-10), 3.52 (2H, s, ArCH2CO), 3.74 (3H,
s, ArOCH3), 4.44 (2H, AB, H2-20), 5.07 (1H, br s, OH), 5.32
(1H, d, H-12), 5.54 (1H, br m, H-7), 5.90 (1H, br s, OH), 6.62-
6.81 (3H, m, vanillyl ArH), 7.50 (1H, s, H-1), 8.88 (1H, br s,
ArOH). FAB-MS (M + 1)+ 613 (12). Accurate mass (FAB
MH+): calcd for C33H41O11, 613.2649; found, 613.2645.
9,13,14-Or th oph en ylacetyl-4â-m eth oxyr esin ifer on yl 20-
(4-Hyd r oxy-3-m eth oxyp h en yla ceta te) (11a ). 9,13,14-Or-
thophenylacetyl-4â-methoxyresiniferonol (27 mg, 0.056 mmol)
was dissolved in CH2Cl2 (2.5 mL), and to this were added DCCI
(12.7 mg, 0.062 mmol) and DMAP (0.75 mg, 0.0062 mmol).
The solution was stirred at room temperature, and acetylho-
movanillic acid (13.9 mg, 0.062 mmol) in CH2Cl2 (0.5 mL) was
added. Stirring was continued for 2 h, after which time the
solution was washed with 1 N HCl, water, and then NaCl
(saturated) and dried over Na2SO4. The crude product was
evaporated in vacuo to give a colorless glass which was >95%
pure by TLC (silica gel, EtOAc/cyclohexane, 1:1; Rf ) 0.43).
This material, in CH2Cl2 (2 mL), was deprotected without
further purification by addition of pyrrolidine (200 µL, 2.2
mmol) and stirring at room temperature for 30 min. The
solution was washed with 1 N HCl, water, and then NaCl
(saturated) and dried over Na2SO4. The crude product was
purified by preparative HPLC (isocratic 73% MeOH/H2O), and
the pure fractions were evaporated to give a colorless glass,
yield 20.7 mg (57%): TLC (silica gel, EtOAc/cyclohexane, 1:1)
Rf ) 0.44; analytical reversed-phase HPLC (isocratic 75%
P h or bol 12,13-Did eca n oa te 20-(4-Hyd r oxy-3-m eth ox-
yp h en yla ceta te) (9b). This compound was synthesized by
an analogous method to that described for phorbol-12,13-
diacetate 20-(4-hydroxy-3-methoxyphenylacetate) from phorbol
12,13-didecanoate and purified by flash column chromatogra-
phy (silica gel, EtOAc/cyclohexane, 1:2). The pure fractions
were evaporated in vacuo to give a colorless glass, yield 5.5
mg (17.7%): TLC (silica gel, EtOAc/cyclohexane, 1:1) Rf
) 0.49;
analytical reversed-phase HPLC (gradient 10-100% CH3CN/
0.1% aqueous TFA) tR ) 13.6 min, >98% pure; 1H NMR
(CDCl3
, 200 MHz) δ 0.88 (9H, m, CH H3-18, 2 × decanoate
3
CH3
), 0.94 (1H, d, H-14), 1.19 (3H, s, CH3 H3-16), 1.21 (3H, s,
CH3 H3-17), 1.77 (3H, d, CH3 H3-19), 2.32 (5H, m, 2 × OCOCH2-
CH2, H-5R), 2.45 (1H, AB, H-5â), 3.15-3.19 (2H, m, H-8, H-10),
1
MeOH/H2O) tR ) 8.6 min, >98% pure; H NMR (CDCl3, 200
3.55 (2H, s, ArCH2CO), 3.88 (3H, s, ArOCH3), 4.46 (2H, AB,
MHz) δ 0.97 (3H, d, CH3 H3-18), 1.52 (3H, s, CH3 H3-17), 1.58
(1H, d, H-12R), 1.75-1.82 (4H, br m, CH3 H3-19, H-5R), 2.09
(1H, m, H-12â), 2.57 (1H, m, H-11), 2.87-2.94 (1H, br m, d,
H-8, H-5â), 3.04 (1H, br m, H-10), 3.21 (2H, s, ortho ester CH2-
Ph), 3.24 (3H, s, 4-OCH3), 3.57 (2H, AB, ArCH2CO), 3.90 (3H,
s, ArOCH3), 4.19 (1H, d, H-14), 4.58 (2H, AB, H2-20), 4.71 (2H,
d, H2-16), 5.86 (1H, br m, H-7), 6.75-6.87 (3H, m, vanillyl
ArH), 7.20-7.40 (6H, m, phenylacetyl ortho ester ArH, H-1);
FAB-MS (M + 1)+ 643 (50). Accurate mass (FAB MH+): calcd
for C38H43O9, 643.2907; found, 643.2903.
H2-20), 5.39 (1H, d, H-12), 5.54 (1H, br s, OH), 5.62-5.65 (2H,
br m, s, H-7, OH), 6.73-6.82 (3H, m, vanillyl ArH), 7.56
(1H, s, H-1); FAB-MS (M + 1)+ 838 (10). Accurate mass (FAB
MH
+): calcd for C49H73O11, 837.5153; found, 837.5150.
Molecu la r Mod elin g. Studies on compounds 2, 8, and 12
were performed on an Alliant Fx2800 computer using a Silicon
Graphics workstation as the graphics display unit. The
molecules were constructed using a proprietary modeling
package, Draw, and the structures optimized using the
algorithms of the molecular mechanics program Minimax.23
The conformational space available to these compounds was
explored using molecular dynamic (MD) simulations, ac-
complished using the Insight/Discover suite of programs.24
Thus each compound was submitted to the following protocol:
A starting structure was energy minimized using the CVFF
force field and the conjugate gradient method until the rms
derivative of the energy was below 0.01 kcal/mol/Å. The
system was then brought to equilibrium, over 1 ps at a
temperature of 300 K, before a molecular simulation study
spanning a further 250 ps (at 300 K) was undertaken. Snap
shots taken at 1 ps intervals were minimized using the
P h or bol An a logu es: 12-Deoxyp h or bol 13-P h en yla c-
eta te 20-(4-Hyd r oxy-3-m eth oxyp h en yla ceta te) (8).12 12-
Deoxyphorbol 13-phenylacetate (25 mg, 0.054 mmol) was
dissolved in dry DMF (0.5 mL), and to this were added
triethylamine (12 µL, 0.096 mmol) and 2-(fluoromethyl)-
pyridinium tosylate (29.4 mg, 0.105 mmol) in DMF (0.2 mL).
The reaction mixture was stirred at room temperature, under
N2, for 30 min before the addition of further triethylamine (19.2
µL, 0.146 mmol) and homovanillic acid (28 mg, 0.146 mmol)
in DMF (0.2 mL). The reaction mixture was heated to 60 °C
for 2 h, after which time the solvent was removed in vacuo

SAR of Capsaicin and Resiniferatoxin Analogues
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15 2951
optimization criteria outlined above. For each compound, the
resulting minimized structures were analyzed by superimposi-
tion of the diterpene 7-membered ring moiety.
in a Beckman 12 microfuge; a 200 µL aliquot of the superna-
tant was removed to determine free [3H]RTX concentration,
the remainder of the supernatant was removed by aspiration,
the tip of the microfuge tube containing the pelleted mem-
branes was cut off with a razor blade after the pellet had been
carefully dried with the tip of a rolled kimwipe, and the bound
radioactivity was determined by scintillation counting. Spe-
cific dpm ranged from approximately 50 dpm at 6 pM [3H]-
RTX to 250 dpm at 50 pM [3H]RTX.
Biology. In Vitr o Assa ys: Stim u la tion of 45Ca 2+ Up -
ta k e in to Dor sa l Root Ga n glion (DRG) Neu r on s. The
uptake and accumulation of 45Ca2+ by capsaicin analogues
were studied in neonatal rat cultured spinal sensory neurons
by the method described in detail by Wood et al.5 In brief,
spinal (dorsal root) ganglia were dissected aseptically from
newborn rats and incubated sequentially at 37 °C for 30 min
with collagenase (Boeringer Mannheim) followed by 30 min
in 2.5 mg/mL trypsin (Worthington), both enzymes made up
in Ham’s F-14 medium. The ganglia were then washed in
medium supplemented with 10% horse serum and the cells
dissociated by trituration through a Pasteur pipet. The cells
were collected by centrifugation and resuspended in Ham’s
F-14 medium with 10% horse serum plus 1 µg/mL nerve
growth factor. The neuronal preparation was plated onto poly-
D-ornithine Terasaki plates (Flow Laboratories) at a density
of 1000 neurons/well. Cultures were incubated at 37 °C in a
humidified incubator gassed with 3% CO2 in air. After the
cells had adhered, 10-4 M cytosine arabinoside, a mitotic
inhibitor, was added to the culture for 48 h to kill the dividing
non-neuronal cells.
Binding data from saturation experiments using increasing
concentrations of radioactive ligand were analyzed by com-
puter fit to the Hill equation:
n
n
B ) (BmaxLHn)/(Kd + LH
)
(1)
where B is the concentration of the receptor-ligand complex,
Bmax is the maximum binding capacity, LH is the concentration
of the radioactive ligand, Kd is the concentration of ligand
required to occupy one-half of the receptors, and n is the
cooperativity index, also known as the Hill coefficient. Equa-
tion 1 predicts that the plot of the specific binding vs ligand
concentration will be a hyperbola if the binding sites are
independent of each other (n ) 1) but will be sigmoidal if there
is positive cooperativity among receptors (n >1).
45Ca2+ uptake assays were made on 3-7 day old cultures.
The Terasaki plates were washed four times with calcium-
free Hank’s balanced salt solution (BSS) buffered with 10 mM
HEPES (pH ) 7.4). Excess medium was drained from the
plate and then 10 µL of remaining medium removed from the
individual wells; 10 µL of medium containing the test concen-
tration of compound plus 10 µCi/mL 45Ca2+ (Amersham) was
added to each well. All media contained 1% dimethyl sulfoxide
(DMSO) to keep the compounds in solution. The neurons were
incubated at room temperature for 10 min and then the
Terasaki plates washed six times in BSS and dried in an oven;
10 µL of 0.3% sodium dodecyl sulfate was added to each well
to dissolve the cells and extract the 45Ca2+. The contents of
each well were transferred to scintillation vials and counted
in 1 mL of Beckman CP scintillation fluid. In all experiments
one group of replicates was treated with medium alone to
estimate the background uptake.
Binding data from experiments in which [3H]RTX was
displaced by increasing concentrations of nonradioactive ligands
were analyzed by the modified Hill equation:25
n
B ) ((Bmax(LH + Lc)n)/(Kd + (LH + Lc)n)) ×
((LH/(LH + Lc)))
in which Lc is the concentration of the nonradioactive ligand.
This equation describes a sigmoidal competition curve if n )
1. In contrast, if there is positive cooperativity among binding
sites and LH < Lc (low receptor occupancy), the equation
predicts that low concentrations of nonradioactive ligand will
enhance rather than inhibit binding; the resulting competition
curve will be distorted accordingly. Computer analysis was
performed on an IBM PC using the program Fit P. Results
are given as mean ( SEM from three independent experi-
ments.
EC50 values (the concentration of drug necessary to produce
50% of the maximal response) were estimated with at least
six replicates at each concentration. Each compound was
tested in two or more independent experiments. Data were
fitted with a sigmoidal function of the form:
Su p p or t in g In for m a t ion Ava ila b le: Tables for HPLC
gradient programs (2 pages). Ordering information is given
on any current masthead page.
total uptake ) a/(1 + (EC50/conc)b) + c
Refer en ces
(1) Szallasi, A.; Blumberg, P. M. Resiniferatoxin, a phorbol-related
diterpene, acts as an ultrapotent analogue of capsaicin, the
irritant contituent in red pepper. Neuroscience 1989, 30 (2), 515-
520.
(2) Walpole, C. S. J .; Wrigglesworth, R.; Bevan, S. J .; Campbell, E.
A.; Dray, A.; J ames, I. F.; Perkins, M. N.; Reid, D. J .; Winter, J .
Analogues of Capsaicin as Novel Analgesic Agents: Structure-
Activity Studies. Part 1. The Aromatic ‘A’ Region. J . Med. Chem.
1993, 36, 2362-2372.
(3) Walpole, C. S. J .; Wrigglesworth, R.; Bevan, S. J .; Campbell, E.
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J . Analogues of Capsaicin as Novel Analgesic Agents: Structure-
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(4) Walpole, C. S. J .; Wrigglesworth, R.; Bevan, S. J .; Campbell, E.
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J . Analogues of Capsaicin as Novel Analgesic Agents: Structure-
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where a ) the maximum evoked uptake, b ) the slope factor,
and c ) the background uptake in the absence of compound.
Results are given as mean ( SEM.
Disp la cem en t of [3H]RTX Bin d in g fr om DRG Mem -
br a n es. Binding assays were carried out as described in detail
by Szallasi et al.16 Briefly, female Sprague-Dawley rats (250-
300 g) were sacrificed by decapitation under CO2 anesthesia;
the cervical and upper thoracic DRG were removed and
disrupted using a Polytron tissue homogenizer in ice-cold
buffer (pH ) 7.4) containing (in mM) KCl, 5; NaCl, 5.8; MgCl2,
2; CaCl2, 0.75; sucrose, 137 and 4-(2-hydroxyethyl)-1-pipera-
zineethanesulfonic acid, 10. Tissue homogenates were washed
twice in the same buffer, and the particulate fraction was
stored at -70 °C; 25-30 µg aliquots of the DRG particulate
fraction in 0.5 mL of the above buffer containing 0.25 mg/mL
bovine serum albumin (Cohn fraction V; Sigma Chemical Co.,
St. Louis, MO), a carrier protein included to stabilize RTX in
aqueous solution, were incubated in triplicate with [3H]RTX
and nonradioactive ligands at 37 °C for 30 min. Nonspecific
binding was determined in the presence of 100 nM nonradio-
active RTX. Tubes were kept on ice while the additions were
made. After the binding reaction had been terminated by
chilling the assay mixture on ice, 100 µg of bovine R1-acid
glycoprotein (Sigma) in 50 µL of Dulbecco’s phosphate-buffered
saline was added to reduce nonspecific binding. Bound and
free [3H]RTX were then separated by pelleting the membranes

2952 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 15
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(9) Maiti, S. N.; Singh, M. P.; Michetich, R. G. Facile Conversion of
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