Analysis of structure-activity relationships for the 'B-region' of N-(3-acyloxy-2-benzylpropyl)-N′-[4-(methylsulfonylamino)benzyl]thiourea analogues as vanilloid receptor antagonists: Discovery of an N-hydroxythiourea analogue with potent analgesic activi

Article abstract of DOI:10.1016/j.bmcl.2004.02.002

The structural modifications on the B-region of the potent and high affinity vanilloid receptor (VR1) lead ligand N-(3-acyloxy-2-benzylpropyl)- N^′-[4-(methylsulfonylamino)benzyl]thiourea were investigated by the replacement of the thiourea with

Full text of DOI:10.1016/j.bmcl.2004.02.002

Bioorganic& Medicinal Chemistry Letters 14 (2004) 2291–2297
Analysis of structure–activity relationships for the ÔB-regionÕ
of N-(3-acyloxy-2-benzylpropyl)-N0-[4-(methylsulfonylamino)-
benzyl]thiourea analogues as vanilloid receptor antagonists:
discovery of an N-hydroxythiourea analogue with potent
analgesic activity
Jeewoo Lee,a,* Sang-Uk Kang,a Hyun-Kyung Choi,a Jiyoun Lee,a Ju-Ok Lim,a
Min-Jung Kil,a Mi-Kyung Jin,a Kang-Pil Kim,a Jong-Hyuk Sung,a Suk-Jae Chung,a
Hee-Jin Ha,b Young-Ho Kim,b Larry V. Pearce,c Richard Tran,c Daniel J. Lundberg,c
Yun Wang,c Attila Tothc and Peter M. Blumbergc
aResearch Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Shinlim-Dong,
Kwanak-Ku, Seoul 151-742, South Korea
bDigital Biotech, Ansan, Kyounggi-Do 425-839, South Korea
cLaboratory of Cellular Carcinogenesis and Tumor Promotion, Center for Cancer Research,
National Cancer Institute, NIH, Bethesda, MD 20892, USA
Received 11 December 2003; revised 30 January 2004; accepted 31 January 2004
Abstract—The structural modifications on the B-region of the potent and high affinity vanilloid receptor (VR1) lead ligand N-(3-
acyloxy-2-benzylpropyl)-N0-[4-(methylsulfonylamino)benzyl]thiourea were investigated by the replacement of the thiourea with
diverse isosteric functional groups. Structure–activity analysis indicated that the A-region in this series was the primary factor in
determining the agonistic/antagonistic activities regardless of the B-region. The NC-hydroxy thiourea analogues (12, 13) showed
excellent analgesic activities in the acetic acid writhing assay compared to the parent thiourea analogues.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
were also cloned recently from different organisms but
are not believed to be sensitive to vanilloids.1 VR1,
which is expressed predominantly on unmyelinated
pain-sensing nerve fibers (C-fibers) and small Ad fibers
in the dorsal root, trigeminal, and nodose ganglia, is
a molecular integrator of nociceptive stimuli. VR1 is
activated by protons,8 heat,9 natural ligands such as
capsaicin (CAP),10 resiniferatoxin (RTX),11 and endo-
genous substances such as anandamide12 and the
lipoxygenase product 12-HPETE.13 Since VR1 functions
as a nonselective cation channel with high Ca2þ per-
meability, its activation by these agents leads to an
increase in intracellular Ca2þ that results in excitation of
primary sensory neurons and ultimately the central
perception of pain.
The vanilloid receptor (VR1) is a member of the tran-
sient receptor potential (TRP) superfamily. Members of
this family are nonvoltage activated cation channel
proteins that play critical roles in processes ranging from
sensory physiology to vasorelaxation and male fertility,
and share structural similarities, such as six transmem-
brane segments.1;2 The vanilloid or capsaicin receptor
(VR13 or TRPV1) has been cloned from dorsal root
ganglia (DRG) of rat,4 the human,5 the chicken,6 and
the guinea pig.7 Other vanilloid receptor homologues
Keywords: Vanilloid receptor 1; TRPV1; Antagonist; Analgesic.
* Corresponding author. Tel.: +82-2-880-7846; fax: +82-2-888-0649;
e-mail: jeewoo@snu.ac.kr
The involvement of this receptor in both pathological
and physiological conditions suggests that the blocking
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.002

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J. Lee et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2291–2297
of this receptor activation, by desensitization or anta-
gonism, would have considerable therapeutic utility.
Among its therapeutictargets, pain is of partiuclar
interest. The validation of VR1 as a molecular target for
the treatment of chronic pain was confirmed using
transgenic mice lacking functional VR1 receptors. These
mice exhibited an impairment in the perception of
thermal and inflammatory pain.14
B
A
C
O
O
O
Resiniferatoxin
H
H
O
OCH3
OH
VR1 antagonists have attracted much attention as
promising drug candidates to inhibit the transmission of
painful signals from the periphery to the CNS and to
block other pathological states associated with this
receptor. The therapeutical advantage of VR1 antago-
nism over agonism is that it lacks the initial excitatory
effect preceding the desensitization. The initial acute
pain associated with capsaicin treatment has proven to
be the limiting toxicity. After the discovery of capsa-
zepine as the first VR1 antagonist,15 a number of
antagonists have been reported such as ruthenium red,16
capsazocaine,17 5-iodo-RTX,18 trialkyglycines,19 hexa-
peptides,20 SB-366791,21 amine derivatives,22 BCTC,23
halogenated capsaicin analogues,24 thioureas,25 and
thiocarbamate analogues.26 5-Iodo-RTX appears to be
the most potent among them in vitro and blocked cap-
saicin-induced current in oocytes expressing VR1 with
an IC50 ¼ 3.9 nM.18 BCTC was characterized in vivo as
a potent, orally-effective, VR1 selective antagonist.23
OH
O
O
R2
NHSO2CH3
R1
H
H
N
O
N
O
S
R2 = 3,4-Me2
1
R1 = H
R2 = 3,4-Me2
R2 = 4-t-Bu
2
3
4
R
R
1 = F
1 = F
R
2 = 3,4-Me2
R1 = OCH3
Figure 1.
structure–activity relationships, which these compounds
reveal.
We have previously reported that isostericreplacement
of the phenolichydroxyl group in potent vanilloid
receptor agonists with the alkylsulfonamido group
provided a series of compounds, which are effective
antagonists to the action of capsaicin on rat VR1 hetero-
logously expressed in Chinese hamster ovary (CHO)
cells. Among these compounds, methylsulfonamides 1,
2, and 4 showed high binding affinities with Ki values of
29.3, 54, and 49 nM, respectively, for the inhibition
of [3H]RTX binding (Fig. 1). For comparison, the
Ki ¼ 1300 nM for capsazepine in the same system.27
Whereas the 4-methylsulfonamido analogue 1 antago-
nized 45Ca2þ uptake in response to capsaicin with an
IC50 ¼ 67 nM and displayed partial agonism,28 its 3-flu-
oro analogue, 2, was a full and potent antagonist, with
a Ki ¼ 7:8 nM (compared to 520 nM for capsaze-
pine) and, conversely, its 3-methoxy analogue, 4, was
instead a full agonist with an EC50 ¼ 22 nM.27 From our
structure–activity analysis, we demonstrated that the
modifications in both the A- and C-regions of 4-meth-
ylsulfonamide ligands combined to influence the pattern
of VR1 agonism/antagonism.27
2. Chemistry
The reference thiourea 3 was prepared according to a
previous report.27 The syntheses of the two types of
N-hydroxy thiourea analogues (NC and NA refer to the
nitrogens next to the C- and A-regions, respectively)
were outlined in Schemes 1 and 2. 3-Pivaloyloxy-
2-benzylpropyl alcohols (5, 6), corresponding to the
C-region, were synthesized according to a previous
report.29 The alcohols of 5, 6 were converted into
the corresponding N,O-diBochydroxylamine by the
Mitsunobu reaction to produce 7 and 8, respectively.
After the hydrolysis of diBoc groups under acidic con-
ditions, the resulting hydroxylamines (9, 10) were con-
densed with the various isothiocyanates27 to afford the
NC-hydroxy thioureas (12–17), respectively. The alkyl-
ation of N,O-diBochydroxylamine with 18 and 1930
produced 20 and 21, whose nitro groups were converted
into the corresponding methylsulfonamides (22, 23),
respectively. The coupling of hydroxylamines (22 and
23) with the isothiocyanate (24)31 furnished the
NA-hydroxy thioureas (25, 26), respectively. The syn-
theses of thiocarbamate analogues are shown in Scheme
3. The O-alkylation of 4-nitrobenzyl alcohol with the
isothiocyanate (24) followed by reduction and mesyl-
ation provided the thiocarbamate 28. The syntheses of
its 3-fluoro and 3-methoxy analogues, 33 and 34, were
accomplished by a similar method from alcohols 30 and
32, which were prepared starting from 2932 and 31.33
The syntheses of amide and ester analogues were shown
in Scheme 4. 4-(Methylsulfonylamino)phenylacetic acid
analogues (37–39) were synthesized from 29, 35, and
As a continuation of our effort to optimize in vitro and
in vivo activities of 4-methylsulfonamide VR1 ligands,
we have investigated the structure–activity relationships
in the B-region of the high affinity lead compounds 1, 2,
and 4. We have modified the thiourea group of the lead
compounds with diverse isosteric functional groups,
such as N-hydroxy thiourea, thiocarbamate, amide, and
ester groups to provide novel VR1 ligands. Herein, we
describe the syntheses, in vitro characterization using
CHO cells transfected with rat VR1, and analgesic
activities of the B-region analogues, and we analyze the

J. Lee et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2291–2297
2293
OBoc
NHBoc
R3
R3
OBoc
N
PPh3, DEAD
O
O
OH
Boc
O
O
5 R1 = 3,4-Me2
6 R1 = 4-t-Bu
7 R1 = 3,4-Me2
8 R1 = 4-t-Bu
TFA
NHSO2CH3
R1
SCN
R3
R3
R2
11
NHSO2CH3
R1
OH
N
OH
NH
H
N
O
O
O
S
R2
O
9
R1 = 3,4-Me2
12 R1 = H
R
2 = H
R3 = 3,4-Me2
10 R1 = 4-t-Bu
13
14
15
16
17
R1 = F
R1 = F
R2 = H R3 = 3,4-Me2
R2 = H R3 = 4-t-Bu
R1 = OCH3 R2 = H R3 = 3,4-Me2
R1 = H
1 = H
R
R
2 = F R3 = 3,4-Me2
2 = Cl R3 = 3,4-Me2
R
Scheme 1. Synthesis of NC-hydroxy thiourea analogues.
OBoc
NO
2
NO
2
OBoc
BocN
NHBoc
Br
R
R
NaH
18 R = H
19 R = F
20 R = H
21 R = F
i) H , Pd-C
2
ii) MsCl, pyr
iii) TFA
O
NCS
NHSO CH
2
3
NHSO CH
2
OH
3
O
OH
HN
24
H
O
N
N
R
R
O
S
22 R = H
23 R = F
25 R = H
26 R = F
Scheme 2. Synthesis of NA-hydroxy thiourea analogues.
36,33 respectively. The acids (37–39) were condensed
with 5 and 4029 under EDC coupling to provide the
corresponding amides (41–44) and the ester (45) ana-
logue, respectively.
The thiourea 3, a 4-tert-butylbenzyl surrogate of 2,
displayed 2-fold greater potency in binding affinity
(Ki ¼ 22:6 nM) but 7-fold lower potency for antagonism
(Ki ¼ 52:4 nM), as well as a shift to partial agonism,
compared to 2. The NC-hydroxy thiourea analogues
(12–15) were compared with the corresponding lead
thioureas (1–4), respectively. Their binding affinities
were attenuated by 4–35-fold. Nevertheless, their func-
tional characteristics were similar to those found in the
thioureas and depended on the effect of the 3-sub-
stituent; namely, 1/12 (3-hydrogen) were antagonists
with limited partial agonism, 2/13 (3-fluoro) were full
antagonists, and 4/15 (3-methoxy) were full agonists.
Among the 2-substituent analogues, whereas the 2-flu-
oro NC-hydroxy thiourea analogue (16) was a partial
agonist with reduced binding affinity, the 2-chloro
NC-hydroxy thiourea analogue (17) was a full antagonist
with equal binding affinity and reduced antagonistic
potency compared to 13. The NA-hydroxy thiourea
analogues (25–26) were also examined. However, they
3. Results and discussion
The binding affinities and agonistic/antagonistic poten-
cies of the synthesized VR1 ligands were assessed in
vitro by a competition assay with [3H]RTX and a 45Ca2þ
uptake assay using rat VR1 heterologously expressed in
Chinese hamster ovary (CHO) cells,34 as previously
described.28;35 The results are summarized in Table 1.
The biochemical behavior and potencies of reference
compounds (1, 2, 4, and capsazepine) were described
in detail in previous reports.27;28

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J. Lee et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2291–2297
i) 24, NaH
NHSO2CH3
ii) Al-Hg
NO2
H
N
iii) MsCl, pyr
O
O
HO
O
S
27
29
28
i) TBSCl
ii) H2, Pd-C
iii) Boc2O
iv) Bu4NF
NO2
i) AcONEt4-4H2O
ii) K2CO3, MeOH
NHBoc
F
NHBoc
R
HO
OCH3
Br
HO
31
30 R = F
32 R = OCH3
i) 24, NaH
ii) TFA
iii) MsCl, pyr
NHSO2CH3
H
N
O
O
R
O
S
33 R = F
34 R = OCH3
Scheme 3. Synthesis of thiocarbamate analogues.
HO
O
NH
2
35
MsCl, NaOH
R
i) NaCN
i) HCl, MeOH
ii) HCl, MeOH
iii) MsCl
ii) H , Pd-C
2
iii) MsCl
iv) NaOH
F
HO
OCH
3
iv) NaOH
Br
NC
O
NHBoc
NHSO CH
NO
2
2
3
29
36
R
1
= H
= F
= OCH
37
38
39
R
2
R2
R
2
3
O
NH2
5, EDC
EDC
O
40
R
2
H
N
O
R
O
O
O
1
O
O
O
NHSO CH
NHSO CH
2 3
2
3
45
41 R = H
R
= 3,4-Me
= 3,4-Me
= 4-t-Bu
= 3,4-Me
1
2
2
2
2
2
2
42 R = F
R
R
R
1
43 R = F
1
44 R = OCH
1
3
2
Scheme 4. Syntheses of amide and ester analogues.
exhibited reduced potencies in binding affinities and
antagonisms compared to NC-hydroxy thiourea ana-
logues. The thiocarbamate analogues (28, 33, 34),
NA-oxa analogues of thiourea, showed moderate
reduction (ca. 4–10-fold) in binding affinities and
potencies in the functional assays compared to the
thioureas (1–2, 4). Like the NC-hydroxy thioureas, their
functional characteristics followed the patterns of the
A-region as determined for the thioureas. The amide
analogues (41–44) were likewise weaker congeners of the
corresponding thioureas. The ester analogue (45) was
much less potent than the amide surrogate (41).

J. Lee et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2291–2297
2295
Table 1. Potencies of vanilloid ligands for binding to rat VR1 and for inducing calcium influx in CHO/VR1 cells
R
3
NHSO CH
2
3
O
B
R
1
O
R
2
B
R1
R2
R3
Binding affinity
Ki (nM)
Agonism
EC50 (nM)
Antagonism
IC50 (nM)
Capsazepine
1300 ( 150)
NE
520 ( 12)
S
HN
NH
1
2
3
4
H
H
H
H
H
3,4-Me2
3,4-Me2
4-t-Bu
29.3 ( 7.6)
54 ( 28)
WEa
NE
67 ( 25)
7.8 ( 3.0)
52 ( 17)
NE
F
F
OCH3
22.6 ( 2.7)
49 ( 15)
WEa
3,4-Me2
22 ( 10)
S
HO
N
NH
12
13
14
15
16
17
H
H
H
H
H
F
3,4-Me2
3,4-Me2
4-t-Bu
1042 ( 73)
212 ( 40)
405 ( 15)
396 ( 62)
620 ( 150)
220 ( 54)
WEa
NE
212 ( 85)
94 ( 14)
10,100 ( 4500)
NE
F
F
WEa
OCH3
H
3,4-Me2
3,4-Me2
3,4-Me2
809 ( 126)
WEa
NE
NE
757 ( 65)
H
Cl
S
S
OH
HN
N
O
25
26
H
F
H
H
3,4-Me2
3,4-Me2
481 ( 67)
546 ( 53)
WEa
WEa
331 ( 78)
NE
HN
28
33
34
H
H
H
H
3,4-Me2
3,4-Me2
3,4-Me2
336 ( 38)
210 ( 120)
199 ( 97)
WEa
440 ( 140)b
170 ( 37)
NE
F
OCH3
WEa
373 ( 155)a
O
HN
41
42
43
44
H
H
H
H
H
3,4-Me2
3,4-Me2
4-t-Bu
500 ( 100)
157 ( 38)
74 ( 13)
WEa
460 ( 250)
150 ( 36)b
440 ( 190)b
NE
F
WEa
F
OCH3
WEa
244 ( 45)a
3,4-Me2
430 ( 120)
O
O
45
H
H
3,4-Me2
1980 ( 240)
NE
3602 (31)
NE: not effective, WE: weakly effective at 30 lM.
a Only fractional calcium uptake compared to 300 nM capsaicin (1, 15%; 3, 5%; 12, 12%; 14, 13%; 16, 20%; 25, 12%; 26, 24%; 28, 11%; 33, 7.5%; 34,
50%; 41, 5.6%; 42, 10%; 43, 7%; 44, 40%).
b Only fractional antagonism (28, 79%; 42, 65%; 43, 56%).
Previously, we have demonstrated that the A-region
makes a major contribution to the extent of agonism/
antagonism in a series of thiourea analogues;27 namely,
substitution of the 4-phenolichydroxyl of the A-region

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J. Lee et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2291–2297
analysis indicated that the functional characteristics of
40
the 4-methylsulfonamide series depended primarily on
the A-region in which 3-fluoro favored antagonism and
3-methoxy favored agonism. Despite its relatively weak
in vitro potency, compound 13, an NC-hydroxy thiourea
analogue, exhibited high analgesicpotenyc in the
writhing assay. Compound 13 has been chosen for fur-
ther investigation as an analgesicacndidate and for
further mechanistic study.
vehicle
1
30
20
10
12
2
13
0
Acknowledgement
0
0.001 0.01
0.1
1
10
100
Dose (mg/kg)
This work was supported by a grant (02-PJ1-PG11-
VN01-SV01-0009) from the Ministry of Health & Wel-
fare, R.O.K.
Figure 2. Analgesic activities of compounds 1, 2, 12, and 13 by acetic
acid-induced writhing test.
in agonists with a 4-methylsulfonamido group shifted
the ligands from agonism toward antagonism, and a
3-fluoro substitution further favored antagonism,
whereas a 3-methoxy group favored agonism. This
observation was also examined in this structural series
with different B-regions. Although their potencies in the
binding and functional assays were less than those of
thiourea analogues, their extent of agonism/antagonism
depended primarily on the A-region as was the case for
the thiourea analogues described previously, regardless
of B-region. In this structural series, 4-methylsulfon-
amido compounds showed antagonism with a low level
of agonism. The incorporation of the 3-fluoro further
enhanced the antagonism; the 3-methoxy group shifted
the ligands to full agonists.
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potencies.
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ability of the compounds to compete with specific
[3H]RTX binding in the CHO/VR1 system and were
expressed as Ki SEM. Potencies as agonists were
expressed as EC50 SEM, and absolute levels of 45Ca2þ
uptake were compared with that induced by a maximally
effective concentration of capsaicin in this system
(300 nM). The in vitro antagonisticpotenices of the
compounds were evaluated by measuring antagonism of
the 45Ca2þ uptake induced by 50 nM capsaicin and
expressed as the IC50 SEM, respectively, correcting for
competition by capsaicin. All Ki and EC50 values represent
the mean of at least three experiments. Levels of partial
agonism are from one to four experiments.
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36. Experimental protocols involving animals in this study
were reviewed by the Animal Care and Use Committee of
Digital Biotech Co., according to the NIH guidelines
(NIH publication number 85-23, revised 1985) of ÔPrinci-
ples of Laboratory Animal CareÕ.
37. Since 1 and 12 are structurally related, we have studied
whether 1 may be generated after the systemicadminis-
tration of 12. Thus, 5 mg/kg of 12 was administered
intravenously to male Sprague–Dawley rats (250–280 g)
and 0.25 mL of blood was collected at various times up to
120 min. Plasma was collected and an aliquot (0.15 mL)
was deproteinated by the addition of 0.3 mL acetonitrile.
An aliquot of the supernatant (0.35 mL) was evaporated to
dryness under a stream of nitrogen and the residue was
reconstituted by the addition of 0.1 mL of mobile phase
(water/methanol/acetonitrile ¼ 20:40:40). An aliquot of the
mixture was injected directly onto the LC–MS system.
Although both 1 and 12 could be readily quantified by the
LC–MS system, the concentration of 12, but not 1, was
detected in the sample. Since we could not have detected
any conversion of 12 into 1, we conclude that the action of
12 cannot be explained by its conversion to 1 in vivo.