Analysis of structure-activity relationships for the 'B-region' of N-(4-t-butylbenzyl)-N′-[4-(methylsulfonylamino)benzyl]-thiourea analogues as TRPV1 antagonists

Article abstract of DOI:10.1016/j.bmcl.2005.06.006

The structure-activity relationships for the 'B-region' of N-(4-t-butylbenzyl)-N′-[4-(methylsulfonylamino)benzyl]thiourea analogues have been investigated as TRPV1 receptor antagonists. A docking model of potent antagonist 2 with the sensor region of TRPV1 is proposed.

Full text of DOI:10.1016/j.bmcl.2005.06.006

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4143–4150
Analysis of structure–activity relationships for the ÔB-regionÕ of
N-(4-t-butylbenzyl)-N0-[4-(methylsulfonylamino)benzyl]-
thiourea analogues as TRPV1 antagonists
Jeewoo Lee,a,* Mi-Kyoung Jin,a Sang-Uk Kang,a Su Yeon Kim,a Jiyoun Lee,a
Myoungyoup Shin,a Jaemin Hwang,a Sookhyun Cho,a Yeon-Sil Choi,a
Hyun-Kyung Choi,a Sung-Eun Kim,a Young-Ger Suh,a Yong-Sil Lee,a Young-Ho Kim,b
Hee-Jin Ha,b Attila Toth,c Larry V. Pearce,c Richard Tran,c Tamas Szabo,c
Jacqueline D. Welter,c Daniel J. Lundberg,c Yun Wang,c Jozsef Lazar,c
Vladimir A. Pavlyukovets,c Matthew A. Morganc and Peter M. Blumbergc
aResearch Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea
bDigitalbiotech, Ansan, Kyounggi-Do 425-839, Republic of Korea
cLaboratory of Cellular Carcinogenesis and Tumor Promotion, Center for Cancer Research, National Cancer Institute, NIH,
Bethesda, MD 20892, USA
Received 9 March 2005; revised 1 June 2005; accepted 2 June 2005
Available online 29 June 2005
Abstract—The structure–activity relationships for the ÔB-regionÕ of N-(4-t-butylbenzyl)-N0-[4-(methylsulfonylamino)benzyl]thiourea
analogues have been investigated as TRPV1 receptor antagonists. A docking model of potent antagonist 2 with the sensor region of
TRPV1 is proposed.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
discovery and development of these antagonists have
been reviewed in detail.24,25
TRPV1, which is regulated by endogenous substances
and signaling pathways in concert with low pH and ele-
vated temperature, is a key nociceptor for the central
perception of pain and is expressed with enhanced den-
sity during inflammation. TRPV1 antagonists, to block
endovanilloid signaling, have thus emerged as novel
and promising analgesic and antiinflammatory agents,
particularly for chronic pain and inflammatory hyperal-
gesia. Following the identification of capsazepine as the
first competitive TRPV1 antagonist1 and of ruthenium
red2 as a noncompetitive antagonist, a growing number
of antagonists have been reported such as capsazo-
caine,3 trialkyglycines,4 hexapeptides,5 5-iodo-RTX,6,7
We previously demonstrated that a series of N-4-(meth-
ylsulfonylamino)benzyl thiourea analogues showed
potent TRPV1 antagonism with high affinity in the
rTRPV1/CHO system and, in the preceding paper, we
investigated the structure–activity relationships (SAR)
in the A-region of N-(4-t-butylbenzyl)-N0-[4-(meth-
ylsulfonylamino)benzyl]thiourea analogues. We describe
here the SAR analysis for the B-region of the high-affin-
ity prototype antagonists (1–3) (see Fig. 1).
2. Chemistry
halogenated
naphthylureas,11–13 BCTC,14 and thioureas.15–23 The
capsaicinoids,8
SB-366791/452533,9,10
The syntheses of the two types of N-hydroxy thiourea
analogues (NA and NC refer to the nitrogens next to
the A- and C-regions, respectively) are outlined in
Schemes 1 and 2. 4-tert-Butylbenzyl bromide was con-
verted to the corresponding hydroxylamine by N-alkyl-
ation of N,O-diBoc hydroxylamine followed by acid
Keywords: TRPV1 antagonist; Vanilloid Receptor
Molecular modeling; Capsaicin; Resiniferatoxin.
*
1 antagonist;
Corresponding author. Tel.: +82 2 880 7846; fax: +82 2 888 0649;
e-mail: jeewoo@snu.ac.kr
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.06.006

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J. Lee et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4143–4150
The syntheses of the two types of thiocarbamate ana-
logues (OA and OC refer to the oxygens next to the A-
and C-regions, respectively) are shown in Schemes 3
and 4. The OA-thiocarbamate analogues 13–15 were
synthesized by the O-alkylation of 4-(butoxycarbonyl-
amino)benzyl alcohols26 with the 4-t-butylbenzyl isothi-
ocyanate followed by acid hydrolysis and mesylation,
respectively. The syntheses of OC-thiocarbamate ana-
logues (16, 17) were accomplished employing the above
method, starting from the 4-(butoxycarbonylamino)ben-
zyl isothiocyanates, which were prepared from 4-amin-
obenzyl amine and 3-methoxy-4-nitrobenzyl alcohol,
respectively, by straightforward functional group
interconversion.
Figure 1.
hydrolysis; the hydroxylamine was then condensed with
isothiocyanates to afford the NC-hydroxy thioureas 4–9.
The NA-hydroxy thiourea analogue (10) was obtained
by the coupling between N-[4-(methylsulfonylami-
no)benzyl]hydroxylamine26 and 4-t-butylbenzyl isothio-
cyanate. The urea (11) and NA-hydroxy urea (12)
congeners were prepared by the same protocol employed
for the syntheses of the thioureas except that the corre-
sponding isocyanates were used.
The amide, N-hydroxyamide, and its reverse analogues
(18–21) were obtained by the coupling of (4-t-butylphe-
nyl)acetic acid or 4-(methylsulfonylamino)phenylacetic
acid with the corresponding amines/hydroxylamines as
outlined in Scheme 5. The hydrazinecarbothioamide
analogues, NA-type (22) and NC-type (23), were readily
obtained by the coupling between the corresponding
isothiocyanates and hydrazines, respectively, as shown
Scheme 1. Synthesis of NC-OH thiourea analogues. Reagents: (a) BocONHBoc, NaH, DMF, 95%; (b) CF3CO2H, CH2Cl2; then NaHCO3;
(c) ArCH2NCS, DMF, 60–80% for two steps.
Scheme 2. Synthesis of NA-OH thiourea, NA-OH urea and urea analogues. Reagents: (a) (4-t-Bu)PhCH2NCO (or NCS), DMF, 70–95%.

J. Lee et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4143–4150
4145
Scheme 3. Synthesis of OA-thiocarbamate analogues. Reagents: (a) NaH, (4-t-Bu)PhCH2NCS, DMF, 60–75%; (b) CF3CO2H, CH2Cl2; (c) MsCl,
pyridine, 85–94% for two steps.
Scheme 4. Synthesis of OC-thiocarbamate analogues. Reagents: (a) CbzCl, NEt3, 92%; (b) Boc2O, THF, 95%; (c) H2, Pd–C, MeOH, 95–98%;
(d) TDI, CH2Cl2, 80–90%; (e) TBSCl, imidazole, DMF, 94%; (f) H2, Pd–C, MeOH, 98%; (g) Boc2O, THF, 92%; (h) Bu4NF, THF, 92%; (i) DPPA,
PPh3, DEAD, 86%; (l) 4-tꢀBuPhOH, NaH, DMF, 80–90%; (m) CF3CO2H, CH2Cl2; (n) MsCl, pyridine, 85–92% for two steps.
in Scheme 6. The reverse amide analogues (24–27) were
prepared by the same method employed for the synthe-
sis of 20.
NA-hydroxy thiourea analogue 10 was also examined.
It likewise exhibited reduced potency in binding affinity
and antagonism comparable to the NC-hydroxy
thiourea.
3. Results and discussion
A series of compounds with urea as the B-region have
been reported as potent TRPV1 antagonists.11–14 How-
ever, the urea analogue 11 was a weak congener of the
corresponding thiourea (1) with 40- and 4-fold less
potency in binding affinity and antagonism. The NA-hy-
droxy urea 12 showed a greater reduction in binding
affinity compared to the urea, but it was a weakly po-
tent, full agonist.
The binding affinities and agonistic/antagonistic poten-
cies of the synthesized VR1 ligands were assessed in vi-
tro by a competition binding assay with [3H]RTX and a
functional 45Ca2+ uptake assay using rat TRPV1 heter-
ologously expressed in Chinese hamster ovary (CHO)
cells (rTRPV1/CHO), as previously described.21–23 The
results are summarized in Tables 1 and 2.
The thiocarbamate analogues were also examined as
thiourea congeners and compared with the correspond-
ing thioureas. Whereas the OA-thiocarbamate analogues
13–15 were moderately weaker surrogates of the corre-
sponding thioureas with 4- to 30-fold reduced binding
affinity and antagonism, the OC-thiocarbamate ana-
logues 16 and 17 showed a dramatic decrease in binding
affinity and antagonism. The result indicated that the
NC-hydrogen of thiourea would be more significant than
the NA-hydrogen for the interaction with the receptor.
The NC-hydroxy thiourea analogues (3-hydrogen: 4, 3-
fluoro:5, and 3-methoxy:6) were compared with the cor-
responding lead thioureas (1–3), respectively. Their
binding affinities were attenuated by 15- to 20-fold. Fur-
thermore, 5 and 6 were shifted in their pattern of activity
to be weak agonists although 4 remained an antagonist
with weak agonism. Other substituents (3-chloro: 7, 3-
nitro: 8, and 2-chloro: 9) did not change the weak
potencies of the NC-hydroxy thiourea analogues. The

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J. Lee et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4143–4150
Scheme 5. Synthesis of amide and N-hydroxyamide analogues. Reagents: (a) NaCN, DMF, 94%; (b) NaOH, THF, 92%; (c) EDC, R-NH2 or
R-NHOH, 85–95%.
Scheme 6. Synthesis of hydrazinecarbothioamide analogues. Reagents and conditions: (a) H2, Pd–C, MeOH, 98%; (b) MsCl, pyridine, 0 °C, 80%;
(c) 4-t-BuPhCH2NCS, DMF, 52%; (d) 4-t-BuPhNHNH2, CH2Cl2, 82%.
Substitutions of the thiourea in 1 with the amide (18), N-
hydroxyamide (19), reverse amide (20), and reverse
N-hydroxyamide (21) as its isosteres also led to much
reduced potencies in binding affinity and antagonism
except for the N-hydroxy amide (19). This compound
was a weakly potent full agonist as was the NA-hydroxy
urea (12), which contains an N-hydroxy amide moiety.
We examined hydrazinecarbothioamide isosteres
containing a nitrogen next to the thiourea. Both
NA-type (22) and NC-type (23) analogues exhibited a
large decrease in binding affinity and antagonism.
4. Molecular modeling
The structural identification of the binding motif of the
TRPV1 receptor is essential to determine in detail the
mechanism of action of the TRPV1 ligands. In recent
publications, molecular determinants and models for
the vanilloid binding site were proposed based on spe-
cies-specific differences in TRPV1 sequence, site-directed
mutagenesis, homology modeling of the transmembrane
domain of TRPV1, and docking studies of capsaicin or
RTX bound to the putative binding site.28–30 The exper-
imental results indicated that Tyr511,28 Ser512,28
Met547 (only present in rTRPV1),29,30 and Thr55029
within the transmembrane domain (S1–S4) were critical
for vanilloid sensitivity. Based on these experimental
findings, several hypothetical models of RTX or capsa-
icin binding to TRPV1 were suggested. In the model
of Jordt and Julius, an aromatic residue, Tyr511, on
the intracellular S2/S3 loop interacts with the vanillyl-
moiety of capsaicin. Additional polar residues, such as
S512 or R491, could interact with capsaicin via hydro-
gen bonds, whereas lipophilic residues in TM3 might
contribute to hydrophobic interactions with the aliphat-
Since a 3-fluoro substituent on the A-region enhanced
receptor antagonism, we explored the effect of a 3-fluoro
substituent in the reverse amide surrogates as shown in
Table 2. Although 4-methylsulfonamide analogues 20
and 25 were weak antagonists with low affinities, incor-
poration of the 3-fluoro group (24, 26) improved both
binding affinity and antagonism as they did in the thio-
urea series. The increased potency was consistently 3- to
4-fold. The oleyl amide analogue (27), like the corre-
sponding thiourea analogue,27 was a poor ligand for
the receptor.

J. Lee et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4143–4150
4147
Table 1. Potencies of TRPV1 ligands for binding to rat VR1 and for inducing calcium influx in CHO/VR1 cells
B
R1
R2
Ki (nM)c binding affinity
EC50 (nM)c agonism
NE
Ki (nM)c antagonism
Capsazepine
1300 ( 150)
520 ( 12)
1
2
3
H
H
H
H
63 ( 10)
53.5 ( 6.5)
50.4 ( 16.5)
NE
NE
WEa
54 ( 8.7)
9.16 ( 1.6)
3.4 ( 0.5)b
F
OCH3
4
5
6
7
8
9
H
H
H
H
H
H
Cl
1090 ( 150)
800 ( 190)
926 ( 74)
WEa
470 ( 200)b
NE
NE
F
>7060
2000 ( 200)a
NE
OCH3
Cl
1310 ( 210)
1330 ( 310)
2270 ( 730)
579 ( 43)
635 ( 52)
NE
NO2
H
NE
NE
10
11
12
H
H
H
H
H
H
4260 ( 370)
2560 ( 370)
3500 ( 620)
NE
470 ( 100)b
225 ( 65)
NE
NE
1055 ( 36)
13
14
15
H
H
H
H
772 ( 21)
450 ( 170)
461 ( 41)
NE
222 ( 71)
120 ( 21)
110 ( 9.6)
F
OCH3
WEa
NE
16
17
H
OCH3
H
H
4600 ( 1400)
2170 ( 150)
WEa
NE
6250 ( 2300)
8400 ( 2700)
18
19
20
H
H
H
H
H
H
5220 ( 1500)
5300 ( 730)
2100 ( 440)
WEa
6760 ( 760)
1960 ( 400)
NE
NE
398 ( 78)
(continued on next page)

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J. Lee et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4143–4150
Table 1 (continued)
B
R1
H
R2
H
Ki (nM)c binding affinity
EC50 (nM)c agonism
Ki (nM)c antagonism
21
22
6400 ( 3100)
WEa
6600 ( 2000)b
H
H
H
H
12100 ( 4100)
6400 ( 3200)
NE
NE
1460 ( 420)
2010 ( 580)
23
NE: not effective, WE: weakly effective.
a Only fractional calcium uptake compared with that induced by 300 nM capsaicin (4, 16%; 6, 72%; 14, 7%; 16, 13%; 18, 20%; 21, 32%).
b Fractional antagonism at 10 lM capsaicin (4, 61%; 10, 78%; 21, 87%).
c Values represent the means SEM of three or more experiments. Values for extent of partial agonism or partial antagonism are from one to three
experiments. The extent of agonism was compared to the effect of a maximal concentration of capsaicin (300 nM) determined in the same assay.
Antagonist values were determined from inhibition of the response evoked by 50 nM capsaicin. WE and NE values were determined at ligand
concentrations of 10–30 lM.
Table 2. Potencies of TRPV1 ligands for binding to rat VR1 and for inducing calcium influx in CHO/VR1 cells
R1
R2
Ki (nM) binding affinity
EC50 (nM) agonism
Ki (nM) antagonism
20
24
25
26
27
H
F
4-t-BuPhCH2
4-t-BuPhCH2
3,4-Me2Ph(CH2)3
3,4-Me2Ph(CH2)3
Oleyl
2100 ( 440)
472 ( 134)
7180 ( 770)
2710 ( 700)
>25,000
NE
NE
NE
NE
NE
398 ( 78)
118 ( 35)
13,555 ( 6905)
4656 ( 1638)
NE
H
F
H
ic moiety of capsaicin within the plane of the mem-
brane.28 In the model of Gavva and co-workers,
Tyr511, Met547, and Thr550 are suggested to be present
in the binding pocket, since all three residues are impor-
tant molecular determinants for vanilloid sensitivity.
This model has the interactions of the vanillyl-moiety
with Thr550 and the Ôtail endÕ hydrophobic group of
capsaicin or RTX interacting with Tyr511.29 In the
model of Middleton and co-workers, Met547 and
Tyr555 interact with the vanillyl-moiety and Tyr511
contacts the orthophenyl group of RTX. The model also
shows the additional interactions of the C3-carbonyl and
the C2-methyl of RTX with Asn551 and Leu515, respec-
tively.30 The most distinctive difference among the three
models is that whereas Tyr 511 interacts with the vanillyl
group (A-region) in the model of Jordt and Julius, it
contacts the hydrophobic ends of RTX or capsaicin
(C-region) in the models of Gavva or Middleton and
co-workers.
constructed through homology modeling by Gavva
and colleagues.31 Two alternative binding modes are
possible in the molecular docking simulations. However,
the molecular surface mapping of ligand 2 and of the
receptor is able to distinguish their regions of hydropho-
bic and hydrophilic surface. The calculations indicated
that, in ligand 2, the 4-tert-butylbenzyl group represents
the area of highest lipophilicity (brown), and the sulf-
onylaminobenzyl segment represents the area of highest
hydrophilicity (blue), as shown in Fig. 2a. In the recep-
tor, the hydrophobic binding pocket is surrounded by
Trp549, Met552, and Leu553, and the hydrophilic pock-
et is formed by Ser510, Try511, and Ser512. On the basis
of this mapping, the docking study was conducted such
that the nonpolar part of the ligand was fitted to the
hydrophobic region of the pocket, and the polar subunit
was bound to the hydrophilic region. The resulting bind-
ing model is shown in Fig. 2b. In this model, the NH of
the sulfonamide acts as a hydrogen bond donor for the
˚
phenolic hydroxyl group of Tyr511 (2.01 A) and the 3-
fluoro atom engages in a hydrogen bond to the amide
˚
proton of Gly563 (1.77 A). The sulfur atom of the thio-
urea group makes a hydrogen bond to the side chain of
To investigate the binding mode of the thiourea TRPV1
antagonists, the potent antagonist 2 was docked into the
transmembrane helices TM3/4 of TRPV1, which was

J. Lee et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4143–4150
4149
Figure 2. (a) Surface maps color-coded by lipophilic potential (LP) of antagonist 2 and TRPV1. The color for LP ranges from brown (highest
lipophilic area of the molecule) to blue (highest hydrophilic area); (b) proposed model of 2 bound to TRPV1 binding site.
˚
Gln560 (2.26 A). The hydrophobic 4-tert-butylbenzyl
group contacts closely the hydrophobic parts of
Phe517, Trp549, Leu553, and Arg557. The 4-(meth-
ylsulfonylamino)phenyl ring (A-region) forms a parallel
stack with the 4-hydroxyphenyl ring of Tyr511 with an
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ton, R. E. Biochemistry 2004, 43, 2501.
18. Yoon, J.; Choi, H.; Lee, H. J.; Ryu, C. H.; Park, H.-G.;
Suh, Y.-G.; Oh, U.; Jeong, Y. S.; Choi, J. K.; Park, Y.-H.;
Kim, H.-D. Bioorg. Med. Chem. Lett. 2003, 13, 1549.
19. Ryu, C. H.; Jang, M. J.; Jung, J. W.; Park, J.-H.; Choi, H.
Y.; Suh, Y.-G.; Oh, U.; Park, H.-G.; Lee, J.; Koh, H.-J.;
Mo, J.-H.; Joo, Y. H.; Park, Y.-H.; Kim, H.-D. Bioorg.
Med. Chem. Lett. 2004, 14, 1751.
31. The structure of compound 2 was built using the Sybyl
molecular modeling program (Tripos, Inc.), and then the
geometry was fully optimized using the Tripos force
field with the following nondefault options: (method)
conjugate gradient; (termination) gradient 0.01
ꢀ1
˚
A ; (max iterations) 10,000. The partial
kcal molꢀ1
atomic charges were calculated by the Gasteiger–Huckel
¨
20. Suh, Y.-G.; Lee, Y.-S.; Min, K.-H.; Park, O.-H.; Seung,
H.-S.; Kim, H.-D.; Park, H.-G.; Choi, J.-Y.; Lee, J.;
Kang, S.-W.; Oh, U.-T.; Koo, J.-Y.; Joo, Y.-H.; Kim,
S.-Y.; Kim, J. K.; Park, Y.-H. Bioorg. Med. Chem. Lett.
2003, 13, 4389.
21. Wang, Y.; Szabo, T.; Welter, J. D.; Toth, A.; Tran, R.;
Lee, Ji.; Kang, S. U.; Suh, Y-G.; Blumberg, P. M.; Lee, J.
[published erratum appears in Mol. Pharm. 2003, 63, 958]
Mol. Pharm. 2002, 62, 947.
method in the Sybyl program. The molecular surface
maps color-coded by lipophilic potential of the binding
site of TRPV1 and compound 2 were calculated using
the MOLCAD program implemented in Sybyl 6.9, prior
to the docking study. The docking study was conducted
using the program DOCK implemented in Sybyl 6.9.
Distance constraint docking was applied to keep the
closest atom–atom pairs from hydrogen-bonding inter-
˚
actions with 2.3 A constraint and the force field constant
22. Wang, Y.; Toth, A.; Tran, R.; Szabo, T.; Welter, J. D.;
Blumberg, P. M.; Lee, J.; Kang, S.-U.; Lim, J.-O.; Lee, J.
Mol. Pharm. 2003, 64, 325.
23. 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.;
set to 200, which is used in calculating penalty for
deviation from the equilibrium distance, for the NH of
the sulfonamide to the sp3 oxygen of Tyr511 and the S
atom of the thiourea group to the NH of Gln560. All
computational work was done on a Silicon Graphics O2
R10000 workstation.