Asymmetric synthesis and receptor activity of chiral simplified resiniferatoxin (sRTX) analogues as transient receptor potential vanilloid 1 (TRPV1) ligands

Article abstract of DOI:10.1016/j.bmcl.2013.10.064

The chiral isomers of the two potent simplified RTX-based vanilloids, compounds 2 and 3, were synthesized employing highly enantioselective PTC alkylation and evaluated as hTRPV1 ligands. The analysis indicated that the R-isomer was the eutomer in binding affinity and functional activity. The agonism of compound 2R was comparable to that of RTX. Docking analysis of the chiral isomers of 3 suggested the basis for its stereospecific activity and the binding mode of 3R.

Full text of DOI:10.1016/j.bmcl.2013.10.064

Bioorganic & Medicinal Chemistry Letters 24 (2014) 382–385
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Asymmetric synthesis and receptor activity of chiral simplified
resiniferatoxin (sRTX) analogues as transient receptor potential
vanilloid 1 (TRPV1) ligands
Myeong Seop Kim a, Yooran Ki a, Song Yeon Ahn a, Suyoung Yoon a, Sung-Eun Kim a, Hyeung-Geun Park a,
Wei Sun b, Karam Son c, Minghua Cui c, Sun Choi c, Larry V. Pearce d, Timothy E. Esch d,
Ian A. DeAndrea-Lazarus d, Peter M. Blumberg d, Jeewoo Lee a,
⇑
a Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea
b Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, China
c National Leading Research Lab of Molecular Modeling & Drug Design, College of Pharmacy, Graduate School of Pharmaceutical Sciences and Global Top 5 Research Program,
Ewha Womans University, Seoul 120-750, Republic of Korea
d 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:
The chiral isomers of the two potent simplified RTX-based vanilloids, compounds 2 and 3, were synthe-
sized employing highly enantioselective PTC alkylation and evaluated as hTRPV1 ligands. The analysis
indicated that the R-isomer was the eutomer in binding affinity and functional activity. The agonism
of compound 2R was comparable to that of RTX. Docking analysis of the chiral isomers of 3 suggested
the basis for its stereospecific activity and the binding mode of 3R.
Received 25 September 2013
Revised 20 October 2013
Accepted 29 October 2013
Available online 6 November 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Vanilloid receptor 1
TRPV1 antagonist
Capsaicin
Resiniferatoxin
Molecular modeling
The transient receptor potential V1 (TRPV1) receptor1 is a
molecular integrator of nociceptive stimuli and functions as a
non-selective cation channel with high Ca2+ permeability. The
receptor is activated by protons,2 heat,3 endogenous inflammatory
mediators4,5 and natural vanilloids such as capsaicin (CAP)6 and
resiniferatoxin (RTX).7 Its activation leads to an increase in intra-
cellular Ca2+ that results in excitation of primary sensory neurons
and ultimately the central perception of pain.
RTX has proven to function pharmacologically as an ultrapotent
agonist for TRPV1, for example displaying 103- to to 104-fold great-
er potency than the prototypic agonist capsaicin.8 In order to find a
simple surrogate of RTX, we initially analyzed the pharmacophores
of RTX based on previous SAR investigations and proposed a phar-
macophoric model in which the principal pharmacophores were
the 4-hydroxy-3-methoxyphenyl (A-region), C20-ester (B-region),
orthophenyl (C1-region) and C3-keto (C2-region) groups.9 On the
basis of this model, we have extensively investigated simplified
RTX surrogates embodying the principal pharmacophores of RTX
to find potent agonists9,10 as well as antagonists11–13
These extensive efforts identified the template of N-(3-acyloxy-
2-benzylpropyl)-N0-benzyl thiourea as an optimized surrogate of
RTX. Compounds 2 and 3 represent the prototypic agonist and
antagonist with high affinity as simplified RTX-based vanilloids
in the rat TRPV1/CHO system.10,13 Their key pharmacophores are
color-coded to show their correspondence with the pharmaco-
phores of RTX (Fig. 1). Since the C-region of compounds 2 and 3,
viz. the 3-pivaloyl-2-(4-t-butylbenzyl)propyl group, has a chiral
center, its active enantiomer is expected to make a stereospecific
interaction with the receptor as previously observed in a series
of propanamides.12
Here we describe the asymmetric syntheses and receptor activ-
ities of the chiral isomers of 2 and 3 as well as modeling analysis
using our human TRPV1 homology model to explain the stereospe-
cific activity of the compounds.
The A-region of compound 2 was synthesized from vanillin in 4
steps (Scheme 1). The hydroxyl of vanillin (4) was protected and
the product then reduced to give alcohol 5. The hydroxyl of 5
was converted to the azide with diphenylphosphoryl azide and
then transformed to the corresponding isothiocyanate 6 using car-
bon disulfide and triphenyphosphine.14 The A-region of compound
⇑
Corresponding author.
E-mail address: jeewoo@snu.ac.kr (J. Lee).
3
was prepared from commercially available 7 in 3 steps
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.10.064

M. S. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 382–385
383
The asymmetric synthesis of the C-region utilized the highly
enantioselective phase-transfer catalytic mono-alkylation of malo-
namic ester as a key step, previously reported by Park and Jew et al.
(Schemes 3 and 4).15 The substrate for asymmetric alkylation,
N,N-bis(p-methoxyphenyl) malonamide tert-butyl ester (11), was
prepared from malonic monoester 10. The phase-transfer catalytic
O
O
O
H
H
O
OCH3
OH
20
3
OH
a-alkylation of 11 in the presence of (R,R)-3,4,5-trifluorophenyl-
O
O
NAS bromide using 4-t-benzyl bromide afforded the highly
enantioselective 12R (99%, 92% ee). The LiAlH4 reduction of 12R
produced the 3-aminopropanol 13R, whose di-PMP protecting
group was converted into the corresponding Boc group to give
14R. The alcohol of 14R was pivaloylated and then the N-Boc group
was deprotected to yield the C-region amine 15R. Finally, the cou-
pling of 15R with isothiocyanate 6 followed by MOM-deprotection
provided the final compound 2R. The amine of 15R was converted
to the corresponding isothiocyanate, which was coupled with
amine 9 to provide the final 3R.
1 Resiniferatoxin (RTX)
R1
R2
H
N
H
N
O
O
S
R1=OH,
R2=OCH3
2
3 R1=NHSO2CH3, R2=F
To prepare the corresponding S-isomers, the phase-transfer cat-
alytic a-alkylation of 11 employing (S,S)-ligand provided 12S with
Figure 1. Resinifertoxin (RTX) and simplified RTX (sRTX).
high enantioselectivity (99%, 99% ee). With 12S, the same routes
used in Scheme 3 produced the final 2S and 3S, respectively. The
structures and optical purities of final compounds were confirmed
by spectroscopic data and chiral HPLC.16
OMOM
OMe
OMOM
OMe
OH
a, b
c, d
SCN
H
HO
OMe
O
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 human TRPV1 heterologously expressed in Chinese
hamster ovary (CHO) cells, as previously described.17,18 The results
are summarized in Table 1, together with the potencies of RTX,
I-RTX and racemates 2 and 3.
The receptor activities of compounds 2 and 3 were previously
reported for the rat TRPV1/CHO system10,13 and are reported here
for human TRPV1 compared to the activities of RTX and I-RTX. Com-
pound 2 proved to be a potent agonist for hTRPV1 with Ki = 6.1 nM
and EC50 = 1.34 nM; it was thus within ca. 5- and 1.5-fold of the
potency of RTX in binding affinity and agonism, respectively, with
human TRPV1. Compound 3 was a potent hTRPV1 antagonist with
Ki = 23 nM and Ki(ant) = 122 nM, which was ca. 2- and 20-fold less
potent than I-RTX in binding affinity and antagonism, respectively.
Analysis of the chiral isoforms indicated that the R-isomer was
the more active isomer for both compounds 2 and 3. In the case of
5
6
4
Scheme 1. Synthesis of the A-region of compound 2. Reagents and conditions:
(a) DIEA, MOMCl, CH2Cl2, rt, 2 h, 99%; (b) NaBH4, LiCl, THF, EtOH, 0 °C, 1 h , 99%;
(c) DPPA, DBU, toluene, rt, 1 h, 99%; (d) CS2, PPh3, THF, reflux, 2 h, 60%.
NH2
F
NHSO2CH3
F
NHSO2CH3
F
a, b
c
H3N
I
NC
7
8
9
Scheme 2. Synthesis of the A-region of compound 3. Reagents and conditions:
(a) MsCl, pyridine, rt, 2 h, 98%; (b) Zn(CN)2, Pd(PPh3)4, DMF, 150 °C, 15 h, 85%;
(c) 2 M BH3-SMe2 in THF, reflux, 3 h then 1 M HCl, reflux, 15 h, 92%.
(Scheme 2). The amine of 7 was mesylated and then its iodide was
converted into the corresponding nitrile to provide nitrile 8, which
was reduced to afford the amine 9.
PMP
PMP
N
PMP
N
O
OH
O
N
a
b
c
PMP
O
HO
PMP
PMP
O
O
O
O
O
O
12R
10
11
13
R
OH
h, i
H
N
H
O
O
N
OMe
f, g
d, e
O
O
S
NH3
O
O
HO
NHBoc
2R
j, k
14R
O
H
15R
N
S
H
H
N
O
N
F
S
3
R
Scheme 3. Syntheses of (R)-sRTX isomers Reagents and conditions: (a) 4,40-dimethoxydiphenyl amine, EDC, DMAP, CH2Cl2, rt, 20 h, 98%; (b) (R,R)-3,4,5-trifluorophenyl-NAS
bromide, 4-t-butylbenzyl bromide, 50% KOH, toluene, ꢀ40 °C, 24 h, 99%; (c) LiAlH4, dibutyl ether, reflux, 1 h, 70%; (d) CAN, H2O, CH3CN, 0 °C, 30 min; (e) 8 N NaOH, Boc2O, rt,
7 h, 60% for 2 steps; (f) C(CH3)3COCl, DMAP, CH2Cl2, rt, 4 h, 95%; (g) CF3CO2H, CH2Cl2, rt, 2 h; (h) compound 6, NEt3, CH2Cl2, rt, 20 h, 80% for 2 steps; (i) CF3CO2H, CH2Cl2, 0 °C,
1 h, 60%; (j) 1,10-thiocarbonyldi-2-pyridone, NEt3, DMF, rt, 15 h, 75% for 2 steps; (k) compound 9, NEt3, CH2Cl2, rt, 15 h, 70%.

384
M. S. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 382–385
OH
H
N
H
N
O
OMe
PMP
N
Scheme 3
PMP
N
O
O
S
O
a
PMP
O
2S
PMP
O
O
O
O
12S
O
H
11
N
S
H
H
N
O
O
N
F
S
3
S
Scheme 4. Syntheses of (S)-sRTX isomers Reagents and conditions: (a) (S,S)-3,4,5-trifluorophenyl-NAS bromide, 4-t-butylbenzyl bromide, 50% KOH, toluene, ꢀ40 °C,
24 h, 99%.
isomers of 3, the eutomer 3R, with values of Ki = 17.2 nM and
Table 1
Ki(ant)=94.4 nM, showed 1.3-fold greater potency than 3 both in
binding affinity and antagonism; conversely, the distomer 3S
exhibited 3- and 1.3-fold less potency compared to 3 for binding
affinity and antagonism, respectively.
Previously, we performed docking analysis of the sRTX agonist 2
using the rat TRPV1 homology model constructed independently
and we demonstrated its binding mode compared to capsaicin
and RTX.19 In order to understand the stereospecific activities
and binding modes of 3R and 3S, we performed flexible docking
studies using our human TRPV1 model20 built based on our rat
TRPV1 model.19
Analysis of energy minimization yielded calculated binding
energies for 3R and 3S of ꢀ313.40 kcal/mol and ꢀ278.80 kcal/
mol, respectively. The more favorable binding energy of 3R than
of 3S was consistent with its stereospecific receptor activity. As
illustrated in Figure 2, 3R showed an excellent fit to the binding
site with a different binding mode compared to previous propana-
mide antagonists.20 The sulfonylaminobenzyl group (A-region)
occupied the deep bottom hole and was involved in the hydropho-
bic interactions with Val508, Tyr511, Ile564, Tyr565, and Ile569.
Furthermore, the NH of the sulfonamide group participated in
Binding affinities and functional activities to human TRPV1a
Binding affinity
Agonism
Antagonism
(Ki, nM)
Ki (nM)
(EC50, nM)
RTX
2
2R
2S
I-RTX
3
3R
3S
1.23 ( 0.22)
6.1 ( 1.7)
0.92 ( 0.19)
1.34 ( 0.27)
0.99 ( 0.10)
9.55 ( 0.41)
NE
NE
NE
NE
NE
NE
NE
NE
5.9 ( 1.3)
122 ( 29)
94 ( 19)
161 ( 38)
3.06 ( 0.57)
6.58 ( 0.40)
11.8 ( 3.1)
23.0 ( 4.6)
17.2 ( 4.3)
63 ( 17)
a
NE: not effective, mean SEM of at least three experiments.
the chiral isomers of 2, eutomer 2R yielded values for binding
affinity and agonism of Ki = 3.06 nM and EC50 = 0.99 nM, which re-
flect 2- and 1.5-fold greater potency than was found for the race-
mate 2. We conclude that 2R was highly potent as an agonist for
hTRPV1 and its potency was comparable to that of the superpotent
RTX. Conversely, distomer 2S was 1.1- and 7-fold less potent than 2
in affinity and agonism, respectively. In the case of the chiral
(B)
(C)
(A)
LP
0.14
0.12
0.09
0.07
0.05
0.03
0.01
Leu547
Phe591
Met514
-0.02
-0.04
-0.06
Ile661
Tyr511
Phe587
Leu588
-0.08
-0.11
-0.13
Ala665
Ser512
-0.15
-0.17
-0.19
Ile564
Figure 2. Flexible docking of 3R in the hTRPV1 model. (A) Binding mode of 3R. The key residues are marked and displayed as capped-stick with carbon atoms in white. The
ligand is depicted as ball-and-stick with carbon atoms in cyan. The Fast Connolly surface of hTRPV1 was generated by MOLCAD and colored by the lipophilic potential
property. The surface of hTRPV1 is Z-clipped. The van der Waals surface of the ligand was presented with its carbon color for clarity. Hydrogen bonds are shown in black
dashed lines and non-polor hydrogens are undisplayed for clarity. (C) Van der Waals surface of the ligand colored by its lipophilic potential property.

M. S. Kim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 382–385
385
11. 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.
Chem. 2003, 46, 3116.
12. Ryu, H.; Jin, M.-K.; Kang, S.-U.; Kim, S. Y.; Kang, D. W.; Lee, J.; Pearce, L. V.;
Pavlyukovets, V. A.; Morgan, M. A.; Tran, R.; Toth, A.; Lundberg, D. J.; Blumberg,
P. M. J. Med. Chem. 2008, 51, 57.
13. Bhondwe, R. S.; Kang, D. W.; Kim, M. S.; Kim, H. S.; Park, S.-G.; Son, K.; Choi, S.;
Lang Kuhs, K. A.; Pavlyukovets, V. A.; Pearce, L. V.; Blumberg, P. M.; Lee, J.
Bioorg. Med. Chem. Lett. 2012, 22, 3656.
14. Isoda, T.; Hayashi, K.; Tamai, S.; Kumagai, T.; Nagao, Y. Chem. Pharm. Bull. 2006,
54, 1616.
hydrogen bonding with Ile564, and the fluorine atom of the A-re-
gion made a hydrogen bond with Ser512. The thiourea group (B re-
gion) made a hydrogen bond with Tyr511 and also contributed to
the appropriate positioning of the C-region for hydrophobic inter-
actions. The Boc group in the C-region extended toward Met514,
Leu518, and Leu547 in the hydrophobic area, and the 4-t-butylben-
zyl group made tight interactions with the adjacent monomer’s
hydrophobic region, composed of Phe587, Leu588, Phe591,
Ile661, and Ala665.
In summary, the chiral isomers of the two potent simplified
RTX-based vanilloids, agonist 2 and antagonist 3, were synthesized
with high optical purity employing highly enantioselective PTC
alkylation and evaluated as hTRPV1 ligands. The R-isomer of the
3-pivaloyloxy-2-(4-t-butylbenzyl)propyl C-region was the eutom-
er in binding affinity and functional activity, and the agonism of
compound 2 for hTRPV1 was comparable to that of RTX. The dock-
ing analysis of the chiral isomers of 3 with our hTRPV1 homology
model demonstrated the more favorable binding energy of the
preferred isomer 3R and its binding mode.
15. Kim, M.-H.; Choi, S.-H.; Lee, Y.-J.; Lee, J.; Nahm, K.; Jeong, B.-S.; Park, H.-G.; Jew,
S.-S. Chem. Commun. 2009, 782.
16. Compound 2R: white solid. mp 60–73 °C, a2D0 ꢀ3.53 (c 0.8, CHCl3), 1H NMR
(400 MHz, CDCl3) d 7.27 (d, J = 8.24 Hz, 2H), 7.07 (d, J = 8.12 Hz, 2H), 6.83 (t,
2H), 6.77 (d, 1H), 6.22 (br t, 1H), 6.02 (br s, 1H), 5.60 (br s, 1H), 4.37 (br s, 2H),
4.12 (dd, J = 11.52, 3.84 Hz, 1H), 3.84 (s, 3H), 3.78 (dd, J = 11.48, 4.64 Hz, 1H),
3.72 (br s, 1H), 3.24 (pentet, 1H), 2.56 (qd, 2H), 2.28 (m ,1H), 1.26 (s, 9H), 1.19
(s, 9H). MS (FAB) m/z 501 (MH+), HRMS calcd for C28H40N2O4S (M+H),
501.2786, found 501.2774. Optical purity: ee 96% (Daicel Chiralcel OD-H,
Retention time = 30 min, Eluent: n-Hep:IPA = 9:1.
Compound 2S: white solid, mp 64–74 °C, a2D0 + 5.85 (c 0.8, CHCl3), 1H NMR
(400 MHz, CDCl3) d 7.27 (d, J = 8.24 Hz, 2H), 7.07 (d, J = 8.12 Hz, 2H), 6.84 (t,
2H), 6.77 (d, 1H), 6.20 (br t, 1H), 5.97 (br s, 1H), 5.58 (br s, 1H), 4.37 (br s, 2H),
4.13 (dd, J = 11.52, 3.88 Hz, 1H), 3.85 (s, 3H), 3.78 (dd, J = 11.48, 4.64 Hz, 1H),
3.72 (br s, 1H), 3.24 (pentet, 1H), 2.55 (qd, 2H), 2.28 (m ,1H), 1.26 (s, 9H), 1.20
(s, 9H). MS (FAB) m/z 501 (MH+), HRMS calcd for C28H40N2O4S (M+H),
501.2787, found 501.2774. Optical purity: ee 97 % (Daicel Chiralcel OD-H,
Retention time = 34 min, Eluent: n-Hep:IPA=9:1.
Acknowledgments
This research was supported by the National Research Founda-
tion of Korea (NRF) Grant (2007-0056817) and NLRL program
Grant (2011-0028885) funded by the Korea government MSIP,
and was supported in part by the Intramural Research Program
of the National Institutes of Health, Center for Cancer Research,
National Cancer Institute (Project Z1A BC 005270).
Compound 3R: white solid, mp 79-88 °C, a2D0 ꢀ9.4 (c 1.0, CHCl3). 1H NMR
(500 MHz, CDCl3) d 7.47 (t, J = 8.2 Hz, 1H), 7.28 (d, J = 8.2 Hz, 2H), 7.12 (dd,
J = 11 Hz, 1H), 7.08 (t, 3H), 6.62 (s, 1H), 6.41 (br t, 1H), 6.17 (br s, 1H), 4.57 (s,
2H), 4.15 (dd, J = 11.5, 3.75 Hz, 1H), 3.81 (d, J = 7.7 Hz, 1H), 3.72 (s, 1H), 3.18 (m,
1H), 2.98 (s, 3H), 2.58 (qd, 2H), 2.30 (m, 1H), 1.26 (s, 9H), 1.20 (s, 9H). MS (FAB)
m/z 566 (MH+), HRMS calcd for C28H40FN3O4S2 (M+H), 566.2523, found
566.2512. Optical purity: ee 95% (Daicel Chiralpak IA, Retention
time = 18.1 min, Eluent: n-Hex:EtOH=9:1).
Compound 3S: white solid, mp 77–88 °C, a2D0 7.524 (c 1.0, CHCl3). 1H NMR
(500 MHz, CDCl3) d 7.48 (t, J = 8.15 Hz, 1H), 7.28 (d, J = 8.15 Hz, 2H), 7.12 (dd,
J = 11, 1.3 Hz, 1H), 7.08 (t, 3H), 6.59 (s, 1H), 6.39 (br t, 1H), 6.13 (br s, 1H), 4.57
(s, 2H), 4.16 (dd, J = 11.5, 3.75 Hz, 1H), 3.80 (d, J = 7.7 Hz, 1H), 3.72 (s, 1H), 3.18
(m, 1H), 2.98 (s, 3H), 2.58 (qd, 2H), 2.30 (m, 1H), 1.26 (s, 9H), 1.20 (s, 9H).
MS (FAB) m/z 566 (MH+), HRMS calcd for C28H40FN3O4S2 (M+H), 566.2522,
found 566.2512. Optical purity: ee 97% (Daicel Chiralpak IA, Retention
time = 19.9 min, Eluent: n-Hex:EtOH = 9:1).
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