Non-vanillyl resiniferatoxin analogues as potent and metabolically stable transient receptor potential vanilloid 1 agonists

Article abstract of DOI:10.1016/j.bmc.2008.11.085

A series of non-vanillyl resiniferatoxin analogues, having 4-methylsulfonylaminophenyl and fluorophenyl moieties as vanillyl surrogates, have been investigated as ligands for rat TRPV1 heterologously expressed in Chinese hamster ovary cells. Although lack

Full text of DOI:10.1016/j.bmc.2008.11.085

Bioorganic & Medicinal Chemistry 17 (2009) 690–698
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Non-vanillyl resiniferatoxin analogues as potent and metabolically stable
transient receptor potential vanilloid 1 agonists
Hyun-Kyung Choi a, Sun Choi b, Yoonji Lee b, Dong Wook Kang a, HyungChul Ryu a, Han-Joo Maeng a,
Suk-Jae Chung a, Vladimir A. Pavlyukovets c, Larry V. Pearce c, Attila Toth c, Richard Tran c, Yun Wang c,
Matthew A. Morgan c, Peter M. Blumberg c, Jeewoo Lee a,
*
a Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Shinlim-Dong, Kwanak-Ku, Seoul 151-742, South Korea
b College of Pharmacy, Division of Life and Pharmaceutical Sciences, and National Core Research Center for Cell Signaling and Drug Discovery, Ewha Womans University,
Seoul 120-750, South Korea
c Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MA 20892, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of non-vanillyl resiniferatoxin analogues, having 4-methylsulfonylaminophenyl and fluoro-
phenyl moieties as vanillyl surrogates, have been investigated as ligands for rat TRPV1 heterologously
expressed in Chinese hamster ovary cells. Although lacking the metabolically problematic 4-hydroxy
substituent on the A-region phenyl ring, the compounds retained substantial agonist potency. Indeed,
the 3-methoxy-4-methylsulfonylaminophenyl analog (1) was modestly (2.5-fold) more potent than
RTX, with an EC50 = 0.106 nM. Further, it resembled RTX in its kinetics and pattern of stimulation of
the levels of intracellular calcium in individual cells, as revealed by imaging. Compound 1 displayed mod-
estly enhanced in vitro stability in rat liver microsomes and in plasma, suggesting that it might be a phar-
macokinetically more favorable surrogate of resiniferatoxin. Molecular modeling analyses with selected
analogues provide evidence that the conformational differences could affect their binding affinities, espe-
cially for the ester versus amide at the B-region.
Received 18 October 2008
Revised 20 November 2008
Accepted 20 November 2008
Available online 25 December 2008
Keywords:
Resiniferatoxin
TRPV1 agonist
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
pain.10,11 An enantiocontrolled total synthesis12 and a conforma-
tional analysis13 of RTX have been reported.
Resiniferatoxin (RTX),1,2 isolated from Euphorbia resinifera, is an
extremely potent irritant tricyclic diterpene which is structurally
related to phorbol-related diterpenes except for its homovanillyl
ester group at C-20.3,4 RTX has proven to function pharmacologi-
cally as an ultrapotent agonist for the transient receptor potential
vanilloid 1 (TRPV1) channel, displaying 103- to 104-fold greater po-
tency than the prototypic agonist capsaicin.5
RTX was found to evoke large inward currents in dorsal root
ganglion (DRG) neurons and TRPV1 transfected cell lines.6 The ac-
tions of RTX are mediated by binding, with picomolar affinity, di-
rectly to the capsaicin-binding site on the TRPV1 receptor.7
Whereas capsaicin under normal conditions produces only short
term desensitization of the TRPV1 mediated currents, the apparent
desensitization to RTX can be of very long duration, lasting for
weeks.8,9 RTX is being developed as a potent desensitizing agent
for neurons in the treatment of urinary urge incontinence and
the pain associated with diabetic neuropathy, as well as for cancer
Structure–activity relations for RTX derivatives have been inves-
tigated employing partial modifications starting from RTX or ROPA
(resiniferonol orthophenylacetate) based on the three structural re-
gions including the A-region (4-hydroxy-3-methoxyphenyl), B-re-
gion (C20 ester), and C-region (diterpene).14–20 The analysis
indicated that the 4-hydroxy-3-methoxyphenyl, C20-ester, C3-keto,
and orthophenyl groups represent principal pharmacophores. In-
deed, simplified RTX analogues containing these pharmacophores
showed potent TRPV1 agonism with high binding affinity.21
In the SAR of the A-region of RTX (4-hydroxy-3-methoxy-
phenyl), any modifications on the phenolic hydroxyl, such as meth-
ylation and 2-aminoethylation, led to the reduction in binding
affinity and agonism in rat DRG. Although its phenyl surrogate
showed dramatic reduction in binding affinity (70-fold), its agonist
potency was reduced just by 6-fold compared to RTX. This diver-
gence in potencies between ligand binding and agonism for TRPV1
ligands is not unusual20 and may reflect the distinction between
the properties of the small fraction of the receptor at the cell
surface and the predominant proportion in internal membranes.22
5-Iodo RTX, prepared semisynthetically from RTX by iodination,
displayed good potency in rat and human TRPV1 and shifted the
* Corresponding author. Tel.: +82 2 880 7846; fax: +82 2 888 0649.
E-mail address: jeewoo@snu.ac.kr (J. Lee).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.085

H.-K. Choi et al. / Bioorg. Med. Chem. 17 (2009) 690–698
691
activity from agonism to antagonism.17 The SAR of the B-region
(ester) has been investigated through the replacement of the ester
by the isosteric amide or thiourea and by insertion or deletion of a
carbon between the A and B-regions.18,23 However, all caused the
reduction in binding affinity and agonism. The C-region (diterpene)
of RTX was partly modified or replaced by the simplified skeleton.
The SAR analysis of the C-region indicated that the C3-keto group,
as well as the orthophenylacetate, appeared to be critical for activ-
ity in the diterpene.18
Although resiniferatoxin represents a promising therapeutic
candidate as a potent TRPV1 agonist, its structure includes a vanil-
lyl group (4-hydroxy-3-methoxybenzyl) which was regarded as
essential for its activity but which is was reported in the case of
capsaicin to be subject to rapid metabolism, leading to reduced
activity in biological systems.24,25
Recently, we reported that the phenolic hydroxyl group in po-
tent vanilloid receptor agonists could be been replaced with an
isosteric methanesulfonamide, resulting in a series of capsaicin
antagonists that retained potent binding affinity for rat TRPV1 het-
erologously expressed in Chinese hamster ovary (CHO) cells.26 It
was also demonstrated that, in contrast with capsaicin, the C-20
phenylacetyl ester of RTX retained substantial activity, albeit
somewhat reduced in comparison with RTX, and ring substitutions
appeared not to be essential for agonism.4
On the basis of this SAR analysis, we have investigated non-van-
illyl RTX analogues in which the 4-hydroxy-3-methoxyphenyl
group was replaced with non-phenolic phenyl groups with the
objective that this might reduce in vivo instability of RTX but retain
its high potency for TRPV1. We describe that replacement of the 4-
hydroxy-3-methoxyphenyl group with fluorophenyl derivatives or
methanesulfonamide generated compounds which still retain
much activity in cellular assays. Here, we report the syntheses,
receptor activities and in vitro metabolic stabilities of 4-methane-
sulfonamidephenyl and fluorophenyl analogues of RTX.
20-amine equivalent23 by the condensation with the correspond-
ing acids using EDC (1-[3-(dimethylamino)propyl]-3-ethylcarbo-
diimide hydrochloride) coupling reagent as shown in Schemes
1 and 2.
2.2. Receptor assay
The synthesized resiniferatoxin analogues were evaluated for
their binding affinities (expressed as Ki values) using a competitive
binding assay with [3H]RTX and for their agonism/antagonism
using a functional 45Ca2+ uptake assay (expressed as EC50 values)
for rat TRPV1 heterologously expressed in Chinese hamster ovary
(CHO) cells.26 Capsaicin and resiniferatoxin were used as reference
compounds for comparison; the potencies of capsaicin and resinif-
eratoxin were Ki = 1.81 and 0.043 nM, respectively, and EC50 = 45
and 0.27 nM, respectively.
The 4-phenolic hydroxyl group of RTX was substituted with an
isosteric methanesulfonylamido group to give compound 1. The
methanesulfonylamido group was an attractive choice because it
has a pKa value similar to that of the hydroxyl group but appears
to be more stable metabolically. Whereas the binding affinity of
1 was reduced by 5-fold compared to RTX, its functional activity
(for agonism) was in fact enhanced 2.5-fold, yielding an
EC50 = 0.106 nM (Table 1). Compound 1 thus appeared to be a
superior agonist as a surrogate of RTX. It is noteworthy that, for a
series of simplified RTX analogues that we had investigated, the
substitution of the phenolic hydroxyl with the methylsulfonylami-
no group had switched the activity of the ligand from agonism to
antagonism.26 However, here in the case of the actual ROPA struc-
ture constituting the C-region, the methylsulfonylamino group
serves as an isostere conferring agonism.
Next, the 3-methoxy group of compound 1 was replaced by 3-flu-
oro or removed to provide compounds 2 and 3, respectively. The
modifications led to dramatic reductions in binding affinity by 20-
fold in 2 and by 120-fold in 3, and it caused a moderate decrease in
agonism by 9-fold in 2 and 7-fold in 3 (Table 1). The results indicated
that, in the presence of the 4-methylsulfonylamino group, the 3-
methoxy group is of importance for binding to the receptor. This re-
sult is in contrast to that with RTX, where removal of the 4-methoxy
group to yield tinyatoxin caused only a 3-fold decrease in binding
potency27 and a 6-fold loss of biological potency.28 The results are
2. Results and discussion
2.1. Chemistry
The RTX analogues were prepared starting from commercially
available ROPA (resiniferonol-9,13,14-orthophenylacetate) or its
R
5
R
R
R
6
4
O
O
O
O
HO
3
O
H
O
H
O
R5
R2
R
2
H
H
R6
R4
R3
O
EDC, CH2Cl2
OH
OH
O
O
O
OH
1-13
Scheme 1. Synthesis of RTX analogues.
1. MsCl, pyridine, DMF
2. NaN3, DMF
3. H2, Lindler's cat, EtOH
4. RCO2H, EDC, CH2Cl2
O
O
O
O
O
H
H
O
H
H
NHSO2CH3
O
OH
OH
O
OH
O
N
H
14
Scheme 2. Synthesis of RTX-amide analogue.

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H.-K. Choi et al. / Bioorg. Med. Chem. 17 (2009) 690–698
Table 1
Potencies of RTX analogues for binding to rat VR1 and for inducing calcium influx in CHO/VR1 cells
O
O
H
O
R5
R2
H
R6
R4
R3
O
OH
O
X
X
R2
R3
R4
R5
R6
RTX bindinga (Ki = nM)
Agob (EC50 = nM)
Antc
CAP
RTX
1
2
3
4
5
6
7
1808
0.0426
44.8
0.27
O
O
O
O
O
O
O
O
O
O
O
O
O
O
NH
H
H
H
H
F
H
H
F
F
F
F
H
H
F
OCH3
OCH3
F
H
H
F
H
F
H
H
H
F
F
F
OH
NHSO2CH3
NHSO2CH3
NHSO2CH3
H
H
F
H
F
H
H
F
H
F
H
H
H
H
H
H
H
H
H
F
H
H
F
F
H
H
H
H
H
H
H
H
H
H
H
F
NEd
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
WEf
NE
0.228 ( 0.051)
0.84 ( 0.22)
6.81 ( 0.91)
0.73 ( 0.23)
2.4 ( 0.31)
3.1 ( 1.7)
3.53 ( 0.41)
0.89 ( 0.31)
0.4 ( 0.14)
2.69 ( 0.66)
0.98 ( 0.28)
8.3 ( 2.0)
0.106 ( 0.031)
2.45 ( 0.2)
1.78 ( 0.53)
4.65 ( 0.62)
1.92 ( 0.6)
3.88 ( 0.67)
9.1 ( 1.6)
1.80 ( 0.59)
4.21 ( 1.5)
11.3 ( 2.5)
5.5 ( 1.0)
59( 10)
8
9
10
11
12
13
14
H
H
F
135 ( 25)
320 ( 41)
WEe
375 ( 90)
H
H
NHSO2CH3
H
a
Receptor binding affinities were assessed in terms of the ability of the compounds to compete for specific binding of [3H]RTX in the CHO/VR1 system and were expressed
as the Ki SEM.
b
Potencies as agonists were expressed as EC50 SEM, and absolute levels of 45Ca2+ uptake were compared with that induced by a maximally effective concentration of
capsaicin in this system, namely 300 nM.
c
The in vitro antagonistic potencies of the compounds were evaluated by measuring antagonism of the 45Ca2+ uptake induced by 50 nM capsaicin and expressed as the Ki
SEM, respectively, correcting for competition by capsaicin.
d
NE: not effective.
Only 23% fractional calcium uptake compared to 300 nM capsaicin.
WE: weakly effective.
e
f
similarly in contrast to those with a simplified RTX C region.26 When
the ester group of compound 3 was changed to amide 14, the recep-
tor activity dropped by 50-fold for binding and by 210-fold for agon-
ism. This result mirrors that for the RTX amide, which was 450-fold
less potent for binding than was RTX.23 The result confirms that the
ester group provides an optimal B-region for receptor activity of RTX
analogs, and stands in contrast to the findings with capsaicin, where
the ester and amide are very similar.29
Previous structure–activity relationship of RTX analogues dem-
onstrated that the unsubstituted phenylacetyl ester retained sub-
stantial activity, albeit reduced potency in comparison with RTX
(relative potency: Ki: 0.015, EC50: 0.157).18 This contrasts markedly
with the case of capsaicin analogues, where 4-hydroxy-3-methoxy
substituents are essential for potent agonism.30
As a complementary strategy to that of substituting the 4-hy-
droxy group with a methylsulfonylamino group to generate ligands
less subject to metabolism, we synthesized the fluorophenyl ana-
logues (4–13), which might reduce the electron density of the phe-
nyl group and protect against electrophilic hydroxylation mediated
by cytochrome enzymes.
The three possible monofluorophenyl analogues (4–6) were
synthesized. They exhibited moderately reduced activities in
both binding affinity (17–74-fold) and agonism (7–17-fold) com-
pared to RTX, which are comparable to those of unsubstituted
phenyl analogues (70-fold, 6.5-fold, respectively) (Table 1). The
position appears not to be critical for potency. As a next step,
the six possible difluorophenyl analogues (7–12) were investi-
gated. Their activities showed moderate to marked reductions
in both binding (9.4–195-fold) and agonism (6.7–217-fold), with
the more potent ligands similar to those of the monosubstituted
analogues. In particular, compounds 8 (2,5-F2) and 9 (2,6-F2)
showed significant potency without the 4-phenolic OH. Com-
pared to RTX, the relative affinity of compound 9 was 0.1 and
the relative agonism of compound 8 was 0.15.
The pentafluorophenyl analogue (13) was also prepared. It
showed relatively very low affinity and a shift in functional activity
to partial agonism/antagonism, with the antagonism being
predominant.
2.3. Calcium imaging
Changes in intracellular calcium levels in Chinese hamster
ovary cells over-expressing TRPV1 show marked differences in re-
sponse to different vanilloids.31 In particular, the response to cap-
saicin (at a capsaicin dose approximating its EC50) is rapid and
uniform within the population of individual cells and diminishes
after reaching an initial maximum. In contrast, the response to res-
iniferatoxin shows a gradual onset, reflecting marked heterogene-
ity in the time to response of individual cells. This pattern of
response may contribute to the partial dissociation between pun-
gency and desensitization/defunctionalization observed for RTX,
where the more gradual activation of TRPV1 in C fiber neurons
might reduce the perception of the activation as pain. We wished
to determine if the introduction of the methylsulfonylamino group
into RTX was able to maintain this pattern of response characteris-
tic of RTX. Compound 1 induced a similar pattern of response in
intracellular calcium to that observed for RTX ( Fig. 1). As with
RTX, the rate of response to compound 1 was gradual and this

H.-K. Choi et al. / Bioorg. Med. Chem. 17 (2009) 690–698
693
Capsaicin
Resiniferatoxin
Compound 1
A
B
1.50
1.25
1.00
0.75
0.50
0.25
0.00
1.50
1.50
1.25
1.00
0.75
0.50
0.25
0.00
1.25
1.00
0.75
0.50
0.25
0.00
0
100 200 300 400 500 600 700
Time (s)
0
100 200 300 400 500 600 700
Time (s)
0
100 200 300 400 500 600 700
Time (s)
Capsaicin
Resiniferatoxin
Compound 1
100
75
50
25
0
100
75
50
25
0
100
75
50
25
0
0
1
2
3
4
5
6
7
8
9
10 11 12
0
1
2
3
4
5
6
7
8
9
10 11 12
0
1
2
3
4
5
6
7
8
9 10 11 12
Time (min)
Time (min)
Time (min)
Figure 1. Modulation of intracellular calcium levels in response to vanilloid treatment. Chinese hamster ovary cells heterologously expressing rat TRPV1 were preloaded with
Fura-2 and treated with capsaicin, RTX, or compound 1 as indicated. The cells were imaged and the 340/380 fluorescence ratio of the Fura-2 was monitored to detect changes
in intracellular calcium levels as described.31 Ligand concentrations, chosen to be approximately at their EC50’s for TRPV1, were: capsaicin, 30 nM; RTX, 75 pM; compound 1,
100 pM. (a) In each panel, 20 cells responding to the stimuli are shown; each line represents the response of an individual cell. Data are from a single experiment
representative of three independent experiments for capsaicin and RTX and of four experiments for compound 1. (b) The average percent of responding cells as a function of
time. Values represent the mean data for 3 (capsaicin and RTX) or 4 (compound 1) experiments. Bars, SEM. The data for capsaicin and RTX are from experiments reported
previously;31 the experiments with compound 1 were done in parallel.
gradual response again reflected heterogeneity in the time to re-
sponse of individual cells, with individual cells when responding
tending to respond fully.
2 kcal/mol (Fig. 3a), so that the low energy conformers can be inter-
changeable among them and adjust well to the active conforma-
tion at the binding site of TRPV1. These results coincide with the
previous report, confirmed by NMR and modeling, that the A-re-
gion phenyl ring of RTX was found extended away from the diter-
pene core in non polar solution but in polar solution it showed bent
and intermediate conformers along with the extended
conformer.13
In addition, the sulfonamide analogues of RTX were found to ex-
ist in two major conformers as shown in Figure 3b. One conformer,
which is shown in purple, is similar to that of RTX. But surprisingly,
the other one, which is the lowest energy conformer displayed in
magenta, indicates that the sulfonamide group forms very distinc-
tive internal H-bonding with the hydroxyl group of the diterpene
core. The energy difference between the extended conformer and
the bent conformer was about 4–7 kcal/mol. Therefore, it appears
to require more energy to break that internal H-bond to establish
the active conformation at the binding site. That might be why
the sulfonamide series have lower binding affinities than RTX.
On the other hand, the absence of methoxy group in the sulfon-
amide analogue 3 lowers the binding affinity about one order of
magnitude compared with the analogue 1. It seems that the meth-
oxy group might play an important role for binding, and according
to Chou et al.,32 the methoxy group of RTX interacts with Tyr555 at
the binding site of TRPV1.
2.4. In vitro metabolic stability
The two potent RTX analogues 1 and 8 were selected for fur-
ther examination of in vitro metabolic elimination and plasma
stability compared to RTX. The three compounds were incubated
with a rat liver microsomal fraction or with plasma and their
concentrations before and after the incubation were compared.
The in vitro metabolic stability at 60 min of incubation was ex-
pressed as the percent of the initial concentration. Compared to
57.6 ( 6.4)% of RTX remaining after 60 min, 81.4 ( 5.8)% of com-
pound 1 and 57.9 ( 8.6)% of compound 8 remained (Fig. 2). Like-
wise, relative to 71.7 ( 4.0)% of RTX remaining after 240 min
incubation with rat plasma, 90.8 ( 5.3)% of compound 1 and
88.3 ( 4.1)% of compound 8 remained (Fig. 2). Collectively, this
observation indicates that compound 1 is more stable than
RTX and compound 8 in liver microsomes and plasma, suggest-
ing that compound 1 is a pharmacokinetically more favorable
RTX surrogate.
2.5. Molecular modeling
In order to investigate the structural basis for the binding affin-
ity differences of the synthesized ligands, we performed conforma-
tional analyses of 1, 3, 14 and RTX using Grid Search in Tripos
SYBYL molecular modeling program. The resulting low energy con-
formers in the gas phase were solvated with water, and then the
solvated molecules were further energy minimized to find the low-
est energy conformations in aqueous solution.
In the sulfonamide series, the replacement of ester to amide at
the B-region decreases the binding affinity and that can be explained
by the conformational differences between them. As shown in
Figure 4, the resulting conformers of the compound 14, which has
an amide group at the B-region, show distinctive conformational dif-
ferences compared with compound 3. The A-region phenyl ring of
compound 14 has a quite different orientation than that of RTX,
Our conformational study showed that RTX can exist in three
major conformers in water with small energy differences less than
making it difficult to maintain the
p–p stacking interaction with
Phe543 at the binding site.32 In addition, the amide analogue might

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H.-K. Choi et al. / Bioorg. Med. Chem. 17 (2009) 690–698
a
b
**
100
80
60
40
20
0
**
120
100
80
60
40
20
0
RTX
compound 1
compound 8
RTX
compound 1 compound 8
Figure 2. (a) The comparison of in vitro metabolic stability for RTX derivatives in the presence of rat liver microsomes. The initial concentration was fixed at 10
lg/mL for the
three derivatives. The vertical axis indicates the percent of the initial concentration remaining after incubation for 60 min with the microsomes. RTX, resiniferatoxin. Values
**
represent the mean SEM of three replicates. p < 0.01, from RTX value by the unpaired t-test. (b) Comparative metabolic stability of RTX derivatives in the presence of rat
plasma. The initial concentration was fixed at 10
240 min of incubation. RTX, resiniferatoxin. Values represent the mean SEM of three replicates. p < 0.01, from RTX value by the unpaired t-test.
l
g/mL for the three derivatives. The vertical axis indicates the percent of the initial concentration remaining after the
**
Figure 3. The representative low energy conformers of RTX and its analogue 1. (a) The low energy conformers of RTX. Carbon atoms are shown in green colors. (b) Two major
low energy conformers of compound 1, a sulfonamide analogue of RTX. The lowest energy conformer shows a bent conformation with a distinctive internal H-bonding.
Carbon atoms are shown in magenta or purple. Non-polar hydrogens are undisplayed for clarity.
not be able to fit well at the binding site because the amide confor-
mation is much more restricted than that of the ester group.
For more direct comparison of the effect of the amide and ester,
we performed another conformational study with an RTX analogue
which has an amide at the B-region.18 Consistent with the mark-
edly reduced biological potency of the RTX-amide analogue, the
lowest energy conformer of the RTX-amide analogue was quite dif-
ferent from that of RTX. In the RTX-amide analogue, the B-region is
more bent toward the diterpene core (Fig. 5). Even though it does
not form an internal H-bond as occurs with compounds in the sul-
fonamide series, the planar amide bond seems to make it possible
to form this bent conformation. As mentioned above, it was re-
ported that the low energy conformers of RTX can be clustered into
several groups in a polar environment13 and that coincides with
our computational results. In the case of RTX-amide analogue,
our study showed that its several possible conformers converged
on one lowest energy conformer after further minimization, result-
ing in the global minimum.
group at the terminus of the C region or an amide at the B-region
give somewhat distinctive conformational restrictions to the abil-
ity of the RTX analogues to adjust their bioactive conformation at
the binding site and this may underlie their decreased binding
affinities. Although the lowest energy conformer in solution might
not be the precise active conformation at the binding site, it pro-
vides an insight into structural factors that should contribute to
their binding affinity differences. For more precise simulation,
homology modeling of TRPV1 and docking studies of the ligands
are under investigation.
3. Conclusion
We have investigated the structure activity relationships and the
metabolic stability of RTX analogues by replacing the 4-hydroxyl-3-
methoxyphenyl group in the A-region with 4-methylsulfonylamid-
ophenyl or fluorophenyl groups. Among the derivatives, compound
1, having
a 4-methylsulfonylamino-3-methoxyphenyl group,
Based on our conformational analyses of a series of RTX ana-
logues along with RTX, we could conclude that the sulfonamide
showed excellent agonism with a value of EC50 = 0.106 nM for rat
TRPV1 in CHO cells, which was 2.5-fold more potent than RTX. Com-

H.-K. Choi et al. / Bioorg. Med. Chem. 17 (2009) 690–698
695
Figure 4. Low energy conformers of sulfonamide analogues of RTX, 3, and 14. (a) Lowest energy conformers, showing bent conformation due to internal H-bonding, (b)
Extended conformers as alternative low energy conformations. Carbon atoms are shown in sky-blue and white for compounds 3 and 14, respectively. Non-polar hydrogens
are undisplayed for clarity.
antagonist character of TRPV1 ligands is dependent on the entire
structure and cannot be isolated to the A-region of the molecule.
4. Experimental
4.1. General
All chemical reagents were commercially available. ROPA was
purchased from LC Laboratories, Woburn, MA. Melting points were
determined on a Büchi Melting Point B-540 apparatus and are
uncorrected. Silica gel column chromatography was performed
on silica gel 60, 230–400 mesh, Merck. Proton NMR spectra were
recorded on a JEOL JNM-LA 300 at 300 MHz. Chemical shifts are re-
ported in ppm units with Me4Si as a reference standard. Infrared
spectra were recorded on a Perkin–Elmer 1710 Series FTIR. Mass
spectra were recorded on a VG Trio-2 GC–MS. Elemental analyses
were performed with an EA 1110 Automatic Elemental Analyzer,
CE Instruments.
4.2. General procedure for coupling
Figure 5. Low energy conformers of RTX and RTX-amide. The representative low
energy conformers of RTX-amide analogue are converged on one bent conformer,
To
a solution of resiniferonol-9,13,14-orthophenylacetate
resulting in the global minimum. Carbon atoms are displayed in green and brown
for RTX and RTX-amide, respectively. Non-polar hydrogens are undisplayed for
clarity.
(14 mg, 0.03 mmol) and acid (0.06 mmol) in CH2Cl2 (1 mL) was
added 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydro-
chloride (15 mg, 0.08 mmol) and 4-dimethylaminopyridine
(1.3 mg). After being stirred for 2 h at room temperature, the reac-
tionmixturewasdilutedwithH2OandextractedwithCH2Cl2 several
times. The combined organic layers were dried over anhydrous
MgSO4, filtered, and concentrated in vacuo. The residue was purified
by flash column chromatography over silica gel with EtOAc/hexanes
(1:2) as eluent to afford the product.
pound 1 likewise induced a similar kinetic pattern of the cellular re-
sponse as evidenced by intracellular calcium levels as did RTX,
whereas capsaicin induced a very different pattern of response. Fi-
nally, the stability studies with rat liver microsomes and plasma
incubation demonstrated that compound 1 was a metabolically
more stable surrogate of RTX. Our conformational study indicated
that the sulfonamide group in the lowest energy conformer of com-
pound 1 could form an internal H-bond with the diterpene core,
resulting in a more bent conformation than that of RTX. Also, com-
pared with the ester, the amide bond at the B-region might confer
conformational restriction, which could affect the binding affinity.
The results not only suggest that compound 1 is a promising
RTX-like agonist but further emphasize that the relative agonist/
4.2.1. 3-Methoxy-4-(methylsulfonylamino) analogue (1)
82% Yield, white solid, mp = 203–205 °C, 1H NMR (CDCl3,
500 MHz) d 7.47–7.49 (m, 2H, H-1 and ArH-5), 7.37–7.4 (m, 2H,
Ph), 7.2–7.3 (m, 3H, Ph), 6.85–6.92 (m, 2H, ArH-2,6), 6.74 (s, 1H,
NHSO2), 5.92 (s, 1H, H-7), 4.74 (s, 2H, H-16), 4.60 (dd of AB, 2H,
J = 12.1, 19.3 Hz, H-20), 4.24 (s, 1H, H-14), 3.91 (s, 3H, OCH3),

696
H.-K. Choi et al. / Bioorg. Med. Chem. 17 (2009) 690–698
3.64 (s, 2H, H-200), 3.23 (s, 2H, H-20), 3.12 (s, 1H, H-8), 3.10 (s, 1H, H-
10), 2.98 (s, 3H, SO2CH3), 2.59 (m, 1H, H-11), 2.45 (d, 1H,
J = 18.7 Hz, H-5a), 2.20 (br s, 1H, OH), 2.16 (dd, 1H, J = 8.8 and
14.2 Hz, H-12a), 2.09 (d, 1H, J = 18.7 Hz, H-5b), 1.85 (s, 3H, H-19),
1.56 (d, 1H, J = 14.2 Hz, H-12b), 1.55 (s, 3H, H-17), 0.98 (d, 3H,
J = 7.1 Hz, H-18); MS (FAB) 706 (MH+); Anal. Calcd for
C38H43NO10S: C, 64.66; H, 6.14; N, 1.98; S, 4.54. Found: C, 64.89;
H, 6.17; N, 1.94; S, 4.50.
3H, Ph), 6.98–7.1 (m, 4H, Ar), 5.89 (s, 1H, H-7), 4.73 (d, 2H,
J = 4 Hz, H-16), 4.58 (dd of AB, 2H, J = 12.1, 25.7 Hz, H-20), 4.22
(d, 1H, J = 3.3 Hz, H-14), 3.64 (s, 2H, H-200), 3.23 (t of AB, 2H, H-
20), 3.10 (s, 1H, H-8), 3.06 (s, 1H, H-10), 2.57 (m, 1H, H-11), 2.46
(d, 1H, J = 23.5 Hz, H-5a), 2.12–2.2 (m, 2H, H-12a and OH), 2.06
(d, 1H, J = 23.5 Hz, H-5b), 1.85 (m, 3H, H-19), 1.57 (d, 1H,
J = 17.9 Hz, H-12b), 1.54 (s, 3H, H-17), 0.99 (d, 3H, J = 7.1 Hz, H-
18); MS (FAB) 601 (MH+); Anal. Calcd for C36H37FO7: C, 71.98; H,
6.21. Found: C, 72.27; H, 6.25.
4.2.2. 3-Fluoro-4-(methylsulfonylamino) analogue (2)
84% Yield, white solid, mp = 76–78 °C, 1H NMR (CDCl3, 500 MHz)
d 7.53 (t, 1H, J = 8.05 Hz, ArH-5), 7.48 (s, 1H, H-1), 7.37–7.4 (m, 2H,
Ph), 7.2–7.3 (m, 3H, Ph), 7.14 (d, 1H, J = 10.9 Hz, ArH-2), 7.10 (d, 1H,
J = 7.93 Hz, ArH-6), 6.59 (s, 1H, NHSO2), 5.90 (s, 1H, H-7), 4.74 (s,
2H, H-16), 4.60 (t of AB, 2H, H-20), 4.23 (s, 1H, H-14), 3.65 (s, 2H,
H-200), 3.23 (s, 2H, H-20), 3.11 (d, 2H, H-8 and H-10), 3.07 (s, 3H,
SO2CH3), 2.59 (m, 1H, H-11), 2.45 (d, 1H, J = 18.7 Hz, H-5a), 2.33
(br s, 1H, OH), 2.16 (dd, 1H, J = 8.8 and 14.2 Hz, H-12a), 2.07 (d, 1H,
J = 18.7 Hz, H-5b), 1.85 (s, 3H, H-19), 1.57 (d, 1H, J = 14 Hz, H-12b),
1.55 (s, 3H, H-17), 0.98 (d, 3H, J = 7 Hz, H-18); MS (FAB) 694
(MH+); Anal. Calcd for C37H40FNO9S: C, 64.05; H, 5.81; N, 2.02; S,
4.62. Found: C, 64.29; H, 5.86; N, 1.98; S, 4.57.
4.2.7. 2,3-Difluoro analogue (7)
92% Yield, white solid, mp = 62–64 °C, 1H NMR (CDCl3,
500 MHz) d 7.46 (s, 1H, H-1), 7.35–7.4 (m, 2H, Ph), 7.2–7.3 (m,
3H, Ph), 7.0–7.1 (m, 3H, Ar), 5.89 (s, 1H, H-7), 4.71 (s, 2H, H-16),
4.58 (dd of AB, 2H, J = 12.1, 25.3 Hz, H-20), 4.22 (d, 1H, J = 2.6 Hz,
H-14), 3.73 (s, 2H, H-200), 3.24 (t of AB, 2H, H-20), 3.09 (br s, 1H,
H-8 and H-10), 2.56 (m, 1H, H-11), 2.47 (d, 1H, J = 18.8 Hz, H-5a),
2.06–2.18 (m, 3H, H-12a, H-5b and OH), 1.83 (s, 3H, H-19), 1.55
(d, 1H, J = 14.6 Hz, H-12b), 1.52 (s, 3H, H-17), 0.96 (d, 3H,
J = 7.1 Hz, H-18); MS (FAB) 619 (MH+); Anal. Calcd for
C36H36F2O7: C, 69.89; H, 5.87. Found: C, 70.18; H, 5.84.
4.2.8. 2,4-Difluoro analogue (8)
4.2.3. 4-(Methylsulfonylamino) analogue (3)
94% Yield, white solid, mp = 54–56 °C, 1H NMR (CDCl3,
500 MHz) d 7.45 (s, 1H, H-1), 7.35–7.4 (m, 2H, Ph), 7.2–7.3 (m,
4H, Ph, 1 H of Ar), 6.79–6.86 (m, 2H, Ar), 5.89 (s, 1H, H-7), 4.71
(s, 2H, H-16), 4.58 (dd of AB, 2H, J = 12.1, 22.1 Hz, H-20), 4.21 (d,
1H, J = 2.7 Hz, H-14), 3.66 (s, 2H, H-200), 3.21 (dd of AB, 2H, H-20),
3.10 (s, 1H, H-8), 3.07 (s, 1H, H-10), 2.56 (m, 1H, H-11), 2.47 (d,
1H, J = 18.8 Hz, H-5a), 2.06–2.17 (m, 3H, H-12a, H-5b and OH),
1.83 (m, 3H, H-19), 1.56 (d, 1H, J = 14.3 Hz, H-12b), 1.52 (s, 3H,
H-17), 0.97 (d, 3H, J = 7.1 Hz, H-18); MS (FAB) 619 (MH+); Anal.
Calcd for C36H36F2O7: C, 69.89; H, 5.87. Found: C, 70.20; H, 5.83.
84% Yield, white solid, mp = 95–96 °C, 1H NMR (CDCl3,
500 MHz) d 7.45 (s, 1H, H-1), 7.35–7.38 (m, 2H, Ph), 7.2–7.3 (m,
5H, 3 H of Ph and 2 H of Ar), 7.18 (d, 2H, J = 8.3 Hz, Ar), 6.38 (s,
1H, NHSO2), 5.85 (s, 1H, H-7), 4.72 (s, 2H, H-16), 4.56 (s, 2H, H-
20), 4.18 (d, 1H, J = 2.6 Hz, H-14), 3.63 (s, 2H, H-200), 3.21 (s, 2H,
H-20), 3.07 (m, 2H, H-8 and H-10), 3.02 (s, 3H, SO2CH3), 2.56 (m,
1H, H-11), 2.35 (d, 1H, J = 18.7 Hz, H-5a), 2.15 (br s, 1H, OH), 2.13
(dd, 1H, J = 8.7, 14.2 Hz, H-12a), 2.03 (d, 1H, J = 18.7 Hz, H-5b),
1.83 (s, 3H, H-19), 1.55 (d, 1H, J = 14.2 Hz, H-12b), 1.54 (s, 3H, H-
17), 0.96 (d, 3H, J = 7.2 Hz, H-18); MS (FAB) 676 (MH+); Anal. Calcd
for C37H41NO9S: C, 65.76; H, 6.12; N, 2.07; S, 4.74. Found: C, 65.98;
H, 6.17; N, 2.04; S, 4.70.
4.2.9. 2,5-Difluoro analogue (9)
94% Yield, pale yellow solid, mp = 50–52 °C, 1H NMR (CDCl3,
500 MHz) d 7.45 (s, 1H, H-1), 7.35–7.4 (m, 2H, Ph), 7.2–7.3 (m,
3H, Ph), 6.88–7.05 (m, 3H, Ar), 5.89 (s, 1H, H-7), 4.73 (m, 2H, H-
16), 4.59 (dd of AB, 2H, J = 12.1, 23.6 Hz, H-20), 4.22 (d, 1H,
J = 2.7 Hz, H-14), 3.67 (s, 2H, H-200), 3.21 (t of AB, 2H, H-20), 3.10
(s, 1H, H-8), 3.08 (s, 1H, H-10), 2.57 (m, 1H, H-11), 2.47 (d, 1H,
J = 18.9 Hz, H-5a), 2.14 (dd, 1H, J = 8.7, 14.3 Hz, H-12a), 2.10 (d,
1H, J = 18.9 Hz, H-5b), 2.03 (br s, 1H, OH), 1.83 (dd, 3H, J = 1.3,
2.7 Hz, H-19), 1.56 (d, 1H, J = 14.3 Hz, H-12b), 1.51 (s, 3H, H-17),
0.97 (d, 3H, J = 7.1 Hz, H-18); MS (FAB) 619 (MH+); Anal. Calcd
for C36H36F2O7: C, 69.89; H, 5.87. Found: C, 70.18; H, 5.83.
4.2.4. 2-Fluoro analogue (4)
83% Yield, white solid, mp = 54–56 °C, 1H NMR (CDCl3,
500 MHz) d 7.48 (s, 1H, H-1), 7.37–7.4 (m, 2H, Ph), 7.24–7.3 (m,
4H, Ph, 1H of Ar), 7.05–7.2 (m, 3H, Ar), 5.90 (s, 1H, H-7), 4.73 (s,
2H, H-16), 4.60 (dd of AB, 2H, J = 12.1, 24 Hz, H-20), 4.23 (d, 1H,
J = 2.6 Hz, H-14), 3.72 (s, 2H, H-200), 3.23 (dd of AB, 2H, J = 13.9,
16 Hz, H-20), 3.11 (br s, 1H, H-8 and H-10), 2.59 (m, 1H, H-11),
2.48 (d, 1H, J = 19 Hz, H-5a), 2.09–2.2 (m, 3H, H-12a, H-5b and
OH), 1.85 (d, 3H, J = 1.3 Hz, H-19), 1.58 (d, 1H, J = 14.3 Hz, H-
12b), 1.54 (s, 3H, H-17), 0.99 (d, 3H, J = 7.1 Hz, H-18); MS (FAB)
601 (MH+); Anal. Calcd for C36H37FO7: C, 71.98; H, 6.21. Found: C,
72.28; H, 6.23.
4.2.10. 2,6-Difluoro analogue (10)
93% Yield, pale yellow solid, mp = 52–54 °C, 1H NMR (CDCl3,
500 MHz) d 7.45 (br s, 1H, H-1), 7.35–7.4 (m, 2H, Ph), 7.2–7.3 (m,
4H, Ph and 1 H of Ar), 6.90 (m, 2H, Ar), 5.89 (s, 1H, H-7), 4.71 (d,
2H, J = 1 Hz, H-16), 4.59 (dd of AB, 2H, J = 12.2, 23.5 Hz, H-20),
4.22 (d, 1H, J = 2.7 Hz, H-14), 3.74 (s, 2H, H-200), 3.21 (dd of AB,
2H, J = 14.2, 15.8 Hz, H-20), 3.10 (br s, 2H, H-8 and H-10), 2.57
(m, 1H, H-11), 2.48 (d, 1H, J = 18.9 Hz, H-5a), 2.06–2.17 (m, 3H,
H-12a, H-5b and OH), 1.83 (dd, 3H, J = 1.4, 2.8 Hz, H-19), 1.55 (d,
1H, J = 14.2 Hz, H-12b), 1.52 (s, 3H, H-17), 0.96 (d, 3H, J = 7.1 Hz,
H-18); MS (FAB) 619 (MH+); Anal. Calcd for C36H36F2O7: C, 69.89;
H, 5.87. Found: C, 70.22; H, 5.82.
4.2.5. 3-Fluoro analogue (5)
80% Yield, white solid, mp = 54–56 °C, 1H NMR (CDCl3,
500 MHz) d 7.48 (s, 1H, H-1), 7.37–7.4 (m, 2H, Ph), 7.24–7.3 (m,
3H, Ph), 6.95–7.1 (m, 4H, Ar), 5.90 (s, 1H, H-7), 4.73 (d, 2H,
J = 3.5 Hz, H-16), 4.60 (dd of AB, 2H, J = 12.1, 25.9 Hz, H-20), 4.23
(d, 1H, J = 2.3 Hz, H-14), 3.67 (s, 2H, H-200), 3.24 (t of AB, 2H, H-
20), 3.11 (s, 1H, H-8), 3.09 (s, 1H, H-10), 2.58 (m, 1H, H-11), 2.47
(d, 1H, J = 18.4 Hz, H-5a), 2.17 (dd, 1H, , J = 8.7, 14.3 Hz, H-12a),
2.06–2.1 (m, 2H, H-5b and OH), 1.86 (s, 3H, H-19), 1.55 (d, 1H,
J = 14.3 Hz, H-12b), 1.54 (s, 3H, H-17), 0.99 (d, 3H, J = 7.1 Hz, H-
18); MS (FAB) 601 (MH+); Anal. Calcd for C36H37FO7: C, 71.98; H,
6.21. Found: C, 72.30; H, 6.24.
4.2.11. 3,4-Difluoro analogue (11)
94% Yield, white solid, mp = 70–72 °C, 1H NMR (CDCl3,
500 MHz) d 7.46 (s, 1H, H-1), 7.35–7.4 (m, 2H, Ph), 7.2–7.3 (m,
3H, Ph), 7.05–7.15 (m, 2H, Ar), 6.98 (m, 1H, Ar), 5.88 (s, 1H, H-7),
4.71 (d, 2H, J = 5 Hz, H-16), 4.57 (dd of AB, 2H, J = 12.1, 24.8 Hz,
H-20), 4.21 (d, 1H, J = 2.6 Hz, H-14), 3.60 (s, 2H, H-200), 3.21 (t of
4.2.6. 4-Fluoro analogue (6)
82% Yield, white solid, mp = 56–58 °C, 1H NMR (CDCl3,
500 MHz) d 7.47 (s, 1H, H-1), 7.37–7.4 (m, 2H, Ph), 7.24–7.3 (m,

H.-K. Choi et al. / Bioorg. Med. Chem. 17 (2009) 690–698
697
AB, 2H, H-20), 3.07 (s, 1H, H-8), 3.05 (s, 1H, H-10), 2.56 (m, 1H, H-
11), 2.45 (d, 1H, J = 18.8 Hz, H-5a), 2.14 (dd, 1H, J = 8.7, 14.3 Hz, H-
12a), 2.07 (br s, 1H, OH), 2.05 (d, 1H, J = 18.8 Hz, H-5b), 1.83 (d, 3H,
J = 1.2 Hz, H-19), 1.53 (d, 1H, J = 14.3 Hz, H-12b), 1.52 (s, 3H, H-17),
0.96 (d, 3H, J = 7.1 Hz, H-18); MS (FAB) 619 (MH+); Anal. Calcd for
C36H36F2O7: C, 69.89; H, 5.87. Found: C, 70.13; H, 5.85.
60 min for microsomal stability and 240 min for the plasma stabil-
ity. The concentration of the analogues in the incubation media
was measured by LC–MS system. HPLC separation was carried
out in a Waters 2695 HPLC system with an analytical column (Phe-
nomenex C18, 4.6 ꢀ 150 m, i.d. of 5
l
m). The mobile phase, 0.1%
HCOOH:acetonitrile (10:90, v/v), was run at
a
flow rate of
0.2 mL/min. MS analysis was performed by a Waters micromass
ZQ in the positive ESI mode. In this study, the selective ion moni-
toring at m/z 629, m/z 706 and m/z 619 for RTX, compounds 1
and 8 was selected for the quantification of the derivatives.
4.2.12. 3,5-Difluoro analogue (12)
93% Yield, white solid, mp = 50–52 °C, 1H NMR (CDCl3, 500 MHz)
d 7.46 (s, 1H, H-1), 7.35–7.4 (m, 2H, Ph), 7.2–7.3 (m, 3H, Ph), 6.82 (m,
1H, Ar), 6.68–6.78 (m, 2H, Ar), 5.89 (s, 1H, H-7), 4.71 (s, 2H, H-16),
4.58 (dd of AB, 2H, J = 12.1, 27.3 Hz, H-20), 4.21 (d, 1H, J = 2.6 Hz,
H-14), 3.62 (s, 2H, H-200), 3.21 (t of AB, 2H, H-20), 3.10 (s, 1H, H-8),
3.07 (s, 1H, H-10), 2.56 (m, 1H, H-11), 2.47 (d, 1H, J = 18.8 Hz, H-
5a), 2.06–2.17 (m, 3H, H-12a, H-5b and OH), 1.83 (s, 3H, H-19),
1.55 (dd, 1H, J = 4.1, 14.3 Hz, H-12b), 1.52 (s, 3H, H-17), 0.96 (d,
3H, J = 7.1 Hz, H-18); MS (FAB) 619 (MH+); Anal. Calcd for
C36H36F2O7: C, 69.89; H, 5.87. Found: C, 70.15; H, 5.84.
4.4. Molecular modeling
The structures of tested compounds were built with Concord for
3D and energy minimized using the MMFF94s force field (method:
powell; termination: gradient 0.05 kcal/mol Å; and max iterations:
1,000,000) implemented in the SYBYL molecular modeling pro-
gram (Tripos, Inc.).
The conformational analyses of each compound were per-
formed using the SYBYL Grid Search method (force field:
MMFF94s; charges: MMFF94; minimization method: powell; ter-
mination: gradient 0.05 kcal/mol Å; and max iterations: 10,000).
The torsion angles which were defined for the grid search included
C1–C2–X3–C4, C2–X3–C4–C5, and C3–C4–C5–C6.
In the analyses, 1728 unique conformers were found for com-
pounds 1, 3 and RTX, respectively, and 288 conformers were found
for compound 14. Among them, the lowest energy conformer and
highly probable conformers were chosen and solvated with water
(shape of the cluster: box; radii type: Tripos; and with the default
number of solvent layers). The solvated conformers were energy
minimized using the Tripos force field and the Gasteiger–Hückel
charges. Then, the resulting conformers were superimposed using
the fit atoms function in SYBYL. All computational studies were
performed with Tripos SYBYL molecular modeling program pack-
age version 8.0 on a Linux (RHEL 4.0 Intel Xeon processor 5050)
workstation.
4.2.13. Pentafluoro analogue (13)
92% Yield, white solid, mp = 73–75 °C, 1H NMR (CDCl3,
500 MHz) d 7.46 (s, 1H, H-1), 7.35–7.38 (m, 2H, Ph), 7.2–7.3 (m,
3H, Ph), 5.91 (s, 1H, H-7), 4.72 (s, 2H, H-16), 4.60 (dd of AB, 2H,
J = 12.1, 21.9 Hz, H-20), 4.23 (d, 1H, J = 2.7 Hz, H-14), 3.76 (s, 2H,
H-200), 3.21 (t of AB, 2H, H-20), 3.12 (s, 1H, H-8), 3.10 (s, 1H, H-
10), 2.57 (m, 1H, H-11), 2.51 (d, 1H, J = 18.8 Hz, H-5a), 2.1–2.18
(m, 2H, H-12a and H-5b), 2.07 (s, 1H, OH), 1.84 (s, 3H, H-19),
1.56 (d, 1H, J = 14.3 Hz, H-12b); MS (FAB) 673 (MH+); Anal. Calcd
for C36H33F5O7: C, 64.28; H, 4.95. Found: C, 64.49; H, 4.91.
4.2.14. Amide analogue (14)
82% Yield, white solid, mp = 143–147 °C, 1H NMR (CDCl3,
500 MHz) d 7.44 (s, 1H, H-1), 7.35–7.38 (m, 2H, Ph), 7.2–7.3 (m,
5H, 3 H of Ph and 2 H of Ar), 7.18 (d, 2H, J = 8.3 Hz, Ar), 6.48 (br
s, 1H, NHSO2), 5.58 (br s, 1H, H-7), 5.51 (t, 1H, J = 5.9 Hz, NHCO),
4.72 (m, 2H, H-16), 4.12 (d, 1H, J = 2.98 Hz, H-14), 3.92 (dd, 1H,
J = 6.3 and 15.2 Hz, H-2a00), 3.79 (dd, 1H, J = 5.6 and 15.2 Hz, H-
2b00), 3.56 (s, 2H, H-20), 3.20 (br s, 2H, H-20), 3.01 (m, 5H, H-8, H-
10 and CH3), 2.54 (m, 1H, H-11), 2.36 (d, 1H, J = 18.8 Hz, H-5a),
2.26 (br s, 1H, OH), 2.11 (dd, 1H, J = 8.7 and 14.2 Hz, H-12a), 2.00
(d, 1H, J = 18.8 Hz, H-5b), 1.81 (m, 3H, H-19), 1.54 (m, 4H, H-12b
and H-17), 0.94 (d, 3H, J = 7.2 Hz, H-18); MS (FAB) 675 (MH+); Anal.
Calcd for C37H42N2O8S: C, 65.86; H, 6.27; N, 4.15; S, 4.75. Found: C,
66.09; H, 6.30; N, 4.13; S, 4.71.
O
O
H
O
R5
R2
H
R6
R4
R3
O
1
OH
O
X
6
4
5
2
3
4.3. Comparison of in vitro metabolic stability for
resinferatoxin (RTX), compounds 1 and 8 in rat liver microsome
and plasma
Acknowledgments
The stability of RTX analogues was compared in the presence of
a Sprague-Dawley rat liver microsomal fraction (BD Bioscience,
Bedford, USA) or in freshly prepared rat plasma. For the micro-
This research was supported by Grants R11-2007-107-02001-0
and R01-2007-000-20052-0 from the Korea Science and Engineer-
ing Foundation (KOSEF), the National Core Research Center pro-
gram (No. R15-2006-020) of the Ministry of Education, Science
and Technology and KOSEF through the Center for Cell Signaling
& Drug Discovery Research at Ewha Womans University, and by
the Intramural Research Program of the National Institutes of
Health, Center for Cancer Research, National Cancer Institute.
somal stability study, 1 mL of reaction mixture contained a 10 lL
aliquot of the aqueous solutions of 1 mM NADPH and 5 mM MgCl2
in 0.5 mL of phosphate buffer (100 mM, pH adjusted to 7.4), and
0.5 mg microsomal protein. The mixture was pre-warmed in a
37 °C water bath under constant agitation (i.e., 50 oscillations per
min). The temperature and agitation were maintained throughout
the experiment until the termination of the reaction. The reaction
was initiated by the addition of a 10
(final concentration of the derivatives in the mixture was 10
mL). For the rat plasma stability study, a 10 L aliquot of the drug
solution (final concentration of the analogues in the mixture was
10 g/mL) was added to pre-warmed rat plasma (0.5 mL). The
l
L aliquot of the drug solution
References and notes
lg/
l
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