α-Methylated simplified resiniferatoxin (sRTX) thiourea analogues as potent and stereospecific TRPV1 antagonists

Article abstract of DOI:10.1016/j.bmcl.2014.04.054

A series of α-methylated analogues of the potent sRTX thiourea antagonists were investigated as rTRPV1 ligands in order to examine the effect of α-methylation on receptor activity. The SAR analysis indicated that activity was stereospecific with the (R)-configuration of the newly formed chiral center providing high binding affinity and potent antagonism while the configuration of the C-region was not significant.

Full text of DOI:10.1016/j.bmcl.2014.04.054

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2685–2688
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
a
-Methylated simplified resiniferatoxin (sRTX) thiourea analogues
as potent and stereospecific TRPV1 antagonists
Ho Shin Kim a, Mi-Kyoung Jin a, Sang-Uk Kang a, Ju-Ok Lim a, Phuong-Thao Tran a, Van-Hai Hoang a,
Jihyae Ann a, Tae-Hwan Ha a, Larry V. Pearce b, Vladimir A. Pavlyukovets b, Peter M. Blumberg b,
Jeewoo Lee a,
⇑
a Laboratory of Medicinal Chemistry, Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea
b Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 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
ligands in order to examine the effect of
that activity was stereospecific with the (R)-configuration of the newly formed chiral center providing
high binding affinity and potent antagonism while the configuration of the C-region was not significant.
Ó 2014 Elsevier Ltd. All rights reserved.
a
-methylated analogues of the potent sRTX thiourea antagonists were investigated as rTRPV1
-methylation on receptor activity. The SAR analysis indicated
Received 21 March 2014
Revised 10 April 2014
Accepted 12 April 2014
Available online 23 April 2014
a
Keywords:
Vanilloid receptor 1
TRPV1 antagonist
Capsaicin
Resiniferatoxin
The vanilloid receptor TRPV1 has emerged as an exciting
therapeutic target for a broad range on conditions, reflecting the
fundamental role of the C-fiber sensory afferent neurons in which
they are expressed.1 While TRPV1 was initially defined as the site
of action of naturally occurring agonists such as capsaicin (CAP) or
the ultrapotent resiniferatoxin (RTX), a key advance was the find-
ing that TRPV1 antagonism could be achieved with appropriate
ligands, building on the developing insights in vanilloid structure
activity relations.2,3 These initial findings have fueled vigorous
efforts in medicinal chemistry and in structural understanding of
vanilloid–TRPV1 interactions.4–12 Because of the great potency at
TRPV1 of RTX compared to CAP, one approach we have taken has
been to try to retain such enhanced potency of RTX upon structural
simplification.
O
O
H
O
H
O
OCH3
OH
N
H
OH
O
O
OCH3
OH
O
Capsaicin
Resiniferatoxin
R
OH
OH
H
N
H
N
H
N
H
N
O
OCH3
OCH3
O
S
S
2
1
Structurally, both CAP and RTX have
a vanilloid moiety
(A-region) that is connected through either ester or amide linkages
(B-region) to a long alkyl chain or diterpene moiety (C-region),
respectively (Fig. 1).
The synthetic surrogates 1 and 2 were designed to mimic the
principal pharmacophores of capsaicin and resiniferatoxin, respec-
tively, and proved to be potent agonists in the rat and human
TRPV1/CHO systems.13 The 3-pivaloyloxy-2-benzylpropyl group,
which represents the C-region of 2, was derived from the key
Figure 1. Natural and synthetic TRPV1 agonists.
pharmacophores of the diterpene in RTX in an effort to capture
the markedly higher binding affinity to TRPV1 of RTX relative to
that of capsaicin.
We have previously reported that isosteric replacement of the
phenolic hydroxyl group in the A-region of the potent agonists 1
and 2 with the methylsulfonamido group provided the potent
antagonists 3 and 5, respectively, which inhibited the activation
by capsaicin of rat and human TRPV1 expressed in CHO cells
⇑
Corresponding author. Tel.: +82 2 880 7846; fax: +82 2 888 0649.
E-mail address: jeewoo@snu.ac.kr (J. Lee).
http://dx.doi.org/10.1016/j.bmcl.2014.04.054
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

2686
H. S. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2685–2688
chirality of the C-region. Modeling analysis suggested that the
-methyl in the propanamide B-region constituted an additional
O
S
O
O
S
O
H
N
H
N
R
a
H
N
H
N
pharmacophore group which interacted with a small hydrophobic
H
N
H
N
O
pocket on the receptor.18
*
R
F
*
S
O
S
As part of our continuing effort to examine the effect of
a-meth-
3
5a R=H
R=H
ylation on potent TRPV1 ligands, we investigated the -methylated
a
5b
4 R=Me
R=3,4-Me2
analogues of simplified RTX thiourea antagonist 5a–c and its phen-
5c R=4-t-Bu
ylalanol analogues. In this paper, we described the efficient synthe-
ses of the racemic/chiral
receptor activities and SAR analysis.
The synthesis of the chiral C-region of the target compounds
was accomplished by Seebach’s diastereoselective alkylation of
a-methylated thiourea derivatives, their
R
R
R
H
H
N
F
O
N
*
F
*
*
O
S
O
O
S
O
O
O
O
N
H
N
H
the
a
-alkoxide enolate as a key reaction (Scheme 1). Anti-selective
-malate 10 with substituted benzyl bro-
6 R=H
R=Me
8 R=H
alkylations of dibenzyl
L
7
9
R=Me
mides afforded 3R-benzyl-2S-hydroxysuccinates, which were
reduced and then protected to provide the diverging intermediates
11. For the synthesis of the (S)-isomeric isothiocyanate 13, the
hydroxyl group of 11 was converted into the corresponding azide
12 and then its acetonide was transformed into the 3-pivaloyloxy
group in three steps. For the synthesis of the chiral (R)-isomeric
isothiocyanate 15, the hydroxyl group of 11 was pivaloylated to
afford 14 and then its acetonide was transformed into the corre-
sponding isothiocyanate in four steps. The racemic C-region was
prepared as described in the previous report.19
Figure 2. Lead TRPV1 antagonists.
(Fig. 2).14,15 The SAR investigation of the B-region of 3 indicated
that compound 4, the -methyl substituted analogue of 3, showed
a
similar potency to that of 3 but displayed enhanced binding to the
receptor with stereospecificity for the (R)-configuration.16
Impressively, in the amide B-region surrogates 6 and 8, a-meth-
ylation led to greater improvement in receptor potency and speci-
ficity (Fig. 2). Although compound 6 displayed moderate binding
affinity and antagonism, its
a-methylated analogue 7 showed an
The syntheses of the stereo isomeric compounds 19–30 were
generally accomplished by the coupling of the corresponding
C-region isothiocyanates with A-region amines (Scheme 2). For the
approximately 10-fold increase in receptor potency.17 Of its two
chiral analogues, the (S)-configuration showed approximately a
further two-fold enhancement in receptor activity compared to
the corresponding racemate, whereas the (R)-configuration pro-
syntheses of the racemic
a-methyl A-region analogues, commer-
cially available 4-aminoacetophenone 16 was mesylated and then
its methylketone was converted into the corresponding amine via
the oxime to afford 17, which was condensed with the C-region
vided weak antagonism. In addition, the a-methylation of the sim-
plified RTX (sRTX) amide antagonist 8 provided compound 9 as a
diastereomeric mixture, which also showed a dramatic increase
in receptor potency compared to 8.18 The receptor activities of
the four different isomers of 9 indicated that the (S)-configuration
isothiocyanates to afford the
28. For the syntheses of the chiral
commercially available optical (R or S)-
a
-methyl analogues 23, 24, 27 and
-methyl A-region analogues,
-methyl-4-nitrobenzyl-
a
a
of the
a
-methyl group displayed high potency irrespective of the
amine 18 were condensed with the C-region isothiocyanates,
respectively, and then the 4-nitro group was converted to the cor-
R
responding 4-methylsulfonamide to afford the chiral
a-methyl
R
O
g
a-c
analogues 19–22, 25, 26, 29 and 30. As references, the chiral iso-
mers of 5a was prepared from (4-methylsulfonylamino)benzyl-
amine by the same method shown in Scheme 2.
OBn
BnO
O
OH
O
O
OH
10
O
O
O
O
14
11
For the syntheses of phenylalanol-type chiral C-region ana-
logues, commercially available optical (R or S)-phenylalanol 31
was transformed into the corresponding isothiocyanate 32 in four
steps, respectively, which was converted to the thioureas 33–36 by
following the same methods described in Scheme 2 (Scheme 3).
The binding affinities and potencies as agonists/antagonists of
the synthesized TRPV1 ligands were assessed in vitro by a binding
competition assay with [3H]RTX and by a functional 45Ca2+ uptake
assay using rat TRPV1 heterologously expressed in Chinese ham-
ster ovary (CHO) cells, as previously described.3 The results are
summarized in Tables 1 and 2, together with the potencies of
5a–c as references.
d
e,f,d,h
R
R
R
e-h
N3
O
NCS
O
NCS
O
(R)
15
(S)
O
O
O
13
12
Scheme 1. Synthesis of chiral C-region. Reagents and conditions: (a) LHMDS,
ArCH2Br, HMPA, THF, 55–68%; (b) LiAlH4, THF, 55–60%; (c) pTsOH, acetone, 76–82%;
(d) PPh3, DPPA, DEAD, THF, 88–90%; (e) H5IO6, ether, 98%; (f) NaBH4, MeOH,
72–89%; (g) Me3CCOCl, pyridine, 80–82%; (h) CS2, PPh3, THF, reflux, 80–90%.
O
S
H
N
NH2
a-c
O
O
H2N
d
O
S
H
N
R
16
17
NO2
H
N
H
N
O
O
*
*
O
S
+H3N
e-g
*
19-30
18
Scheme 2. Synthesis of 3-pivaloyl-2-benzyl-propyl C-region analogues. Reagents and conditions: (a) CH3SO2Cl, pyridine, 95%; (b) NH2OH–HCl, pyridine, 85–88%; (c) H2,
Pd–C, HCl, MeOH, 96–98%; (d) RNCS, CH2Cl2 or DMF, 82–93%; (e) RNCS, NEt3, CH2Cl2, 82–84%; (f) H2, Pd–C, MeOH, 96–98%; (g) CH3SO2Cl, pyridine, 92–94%.

H. S. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2685–2688
2687
Table 2
In vitro rTRPV1 activities for 2-pivaloyloxy-1-benzylethyl C-region analogues
e-g
NCS
a-d
NH2
S
O
O
HO
N
H
N
H
*
*
*
O
S
O
*
31
S
O
O
N
H
O
32
33-36
*
*
N
H
N
H
1
2
O
S
O
O
N
H
Scheme 3. Synthesis of 2-pivaloyl-1-benzyl-ethyl C-region analogues. Reagents
and conditions: (a) Boc2O, CH2Cl2, rt, 94%; (b) Me3CCOCl, NEt3, CH2Cl2, 0 °C, 88%; (c)
CF3CO2H, CH2Cl2, 0 °C; (d) thiocarbonyldiimidazole, NEt3, DMF, 50 °C, 82%; (e)
RNCS, NEt3, CH2Cl2, 82–84%; (f) H2, Pd–C, MeOH, 96–98%; (g) CH3SO2Cl, pyridine,
92–94%.
C1
C2
Ki (nM)
EC50 (nM)
Ki (nM)
33
34
35
36
R
R
S
S
R
S
R
S
420 ( 32)
272 ( 28)
NE
NE
NE
NE
NE
193 ( 95)
290 ( 66)
WEa
NE
WEa
First, we examined the effect of a-methylation on 5a with R = H.
In the two chiral isomers of 5a, its (R)-configuration showed
a
Only fractional antagonism: 35, 47%; 36, 4%.
slightly better antagonism than that of (S)-configuration. Incorpo-
ration of
stereo-isomers 19–22 (C1:
respectively. The result indicated that the
a
-methyl group into 5a provided the four different
-methyl, C2: C-region chiral centers),
-methylation produced
a
a
demonstrated a similar SAR pattern in the amide B-region surro-
gate of 27–30 in which the receptor activity resided only in the
(S)-configuration of C1 while the C2-configuration was not critical
for receptor activity.18 Nevertheless, among this series, the (R,S)-
configuration of C1 and C2 seemed to be the optimal configuration
in which compounds 26 and 30 showed high binding affinities
with Ki = 2.12 nM and 6.75 nM, respectively.
the stereospecific antagonism and the C1-configuration in the two
chiral centers was critical for the receptor activity in which (R)-con-
figuration of C1 (19, 20) showed much better binding affinity and
antagonismthanthose of(S)-configurationofC1 (21, 22)irrespective
of C2 chirality. Compounds 19 and 20 showed approximately a 10-
fold increase in binding affinity and slight or no enhancement in
antagonism compared to (R)-5a and (S)-5a, respectively. The prefer-
ence for the R-configuration of C1 in receptor activity was also exam-
ined in the thiourea antagonist 4 with a 4-t-butylbenzyl C-region.16
In the presence of the same C1 chirality, the receptor activity upon
changing the C2-configuration gave mixed results.
We also investigated the
a-methylated thiourea analogues with
a
2-pivaloyloxy-1-benzylethyl C-region (Table 2). Similar to
the above, the receptor activity in this series resided only in the
(R)-isomers of C1, 33 and 34, while the C2-configuration did not
have a significant effect on activity.
We next investigated the a-methylated analogues of 5b and 5c
In summary, we have investigated a series of a-methylated ana-
logues of the potent sRTX thiourea antagonists 5a–c. The SAR anal-
ysis indicated that they showed stereospecific antagonism
with R = 3,4-Me2 and 4-t-Bu, respectively, which were found to be
the most potent antagonists in this series.14 Since the R-configura-
tion of C1 in the series of the
active one, we examined only the (R)-configuration of C1 of the
-methylated 5b and 5c. As examined above, -methylation of
5b and 5c also provided stereospecific antagonism in which the
(R)-isomers 24 and 28 in the -methyl analogues of 5b and 5c
a-methylated 5a proved to be the
dependent on the a-methyl configuration, with the (R)-configura-
tion proving to be the active one as had been found for the previous
thiourea antagonists 3 and 4. In addition, the C2-configuration in
the C-region appeared not to be significant for activity. Overall,
a
a
a
we concluded that
a-methylation at the benzylic position of
showed 5- and 6-fold increases in binding affinity and 10- and
4-fold increases in antagonism, compared to 5b and 5c, respec-
tively. The C2-configuration in 24 and 28 also appeared not to be
significant for receptor activity (25 vs 26, 29 vs 30). We previously
4-methylsulfonamidophenyl antagonists provided stereospecific
and potent antagonists. Whereas the amide B-region analogues
preferred the (S)-configuration for receptor activity, the thiourea
B-region ones favored the (R)-configuration.
Table 1
In vitro rTRPV1 activities for 3-pivaloyloxy-2-benzylpropyl C-region analogues
O
H
R
N
S
H
H
N
O
O
N
*
*
2
1
O
S
R
Chirality (C1) (C2)
Ki (nM)
EC50 (nM)
Ki(CAP) (nM)
(R)-5a
(S)-5a
19
20
21
22
5b
23
24
25
26
5c
27
28
29
30
H
H
H
H
H
H
R
252 ( 77)
236 ( 4.0)
22.7 ( 4.4)
19.4 ( 1.9)
1140 ( 160)
710 ( 240)
29.3
37.2 ( 5.9)
6.1 ( 2.3)
15.2 ( 3.4)
2.12 ( 0.73)
64
NE
NE
NE
NE
NE
NE
WE
NE
NE
NE
NE
WE
NE
NE
NE
NE
64.3 ( 9.8)
80.6 ( 6.4)
44.5 ( 9.9)
89 ( 20)
3500 ( 300)
133 ( 24)
67
25.9 ( 9.1)
6.9 ( 1.4)
7.1 ( 1.3)
13.2 ( 5.6)
86
28.7 ( 8.7)
23.9 ( 6.1)
53 ( 11)
30.6 ( 7.3)
S
R
S
R
S
R
R
S
S
3,4-Me2
3,4-Me2
3,4-Me2
3,4-Me2
R
R
R
R
S
4-t-Bu
4-t-Bu
4-t-Bu
4-t-Bu
42.7 ( 5.6)
10.0 ( 1.3)
17.9 ( 1.2)
6.75 ( 0.99)
R
R
R
R
S

2688
H. S. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2685–2688
11. Lee, J. H.; Lee, Y.; Ryu, H.; Kang, D. W.; Lee, J.; Lazar, J.; Pearce, L. V.;
Acknowledgments
Pavlyukovets, V. A.; Blumberg, P. M.; Choi, S. J. Comput. Aided Mol. Des. 2011, 25,
317.
This research was supported by Grants from the National
Research Foundation of Korea (NRF) (R11-2007-107-02001-0),
and by the Intramural Research Program of NIH, Center for Cancer
Research, NCI (Project Z1A BC 005270).
12. Liao, M.; Cao, E.; Julius, D.; Cheng, Y. Nature 2013, 504, 107.
13. Kim, M. S.; Ki, Y.; Ahn, S. Y.; Yoon, S.; Kim, S.-E.; Park, H.-G.; Sun, W.; Son, K.;
Cui, M.; Choi, S.; Pearce, L. V.; Esch, T. E.; DeAndrea-Lazarus, I. A.; Blumberg, P.
M.; Lee, J. Bioorg. Med. Chem. Lett. 2014, 24, 382. and references therein.
14. Lee, J.; Lee, J.; Kang, M.; Shin, M.-Y.; Kim, J.-M.; Kang, S.-U.; Lim, J.-O.; Choi, H.-
K.; Suh, Y.-G.; Park, H.-G.; Oh, U.; Kim, H.-D.; Park, Y.-H.; Ha, H.-J.; Kim, Y.-H.;
Toth, A.; Wang, Y.; Tran, R.; Pearce, L. V.; Lundberg, D. J.; Blumberg, P. M. J. Med.
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