TRPV1 modulators: Structure-activity relationships using a rational combinatorial approach

Article abstract of DOI:10.1016/j.bmcl.2011.04.141

A discrete library of linear and hydantoin-containing dipeptide derivatives, based on the Lys-Trp(Nps) scaffold, was prepared by solid-phase synthesis. SAR studies indicated that potency for TRPV1 blockade and selectivity towards NMDA is mainly dictated b

Full text of DOI:10.1016/j.bmcl.2011.04.141

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3541–3545
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
TRPV1 modulators: Structure–activity relationships using a rational
combinatorial approach
Laura Zaccaro a,b, M. Teresa García-López c, Rosario González-Muñiz c, , Carolina García-Martínez d,
⇑
Antonio Ferrer-Montiel d, Fernando Albericio e,f,g, Miriam Royo a,f,
⇑
a Combinatorial Chemistry Unit, Barcelona Science Park, University of Barcelona, Barcelona 08028, Spain
b Istituto di Biostrutture e Bioimmagini CNR, Napoli 80134, Italy
c Instituto de Química Medica CSIC, Madrid 28006, Spain
d Centro de Biología Molecular y Celular, Universidad Miguel Hernandez, Elche, Alicante 03202, Spain
e Institute for Research in Biomedicine, Barcelona Science Park, University of Barcelona, Barcelona 08028, Spain
f CIBER-BBN, Networking Centre on Bioengineering Biomaterials and Nanomedicine, Barcelona 08028, Spain
g Department of Organic Chemistry, University of Barcelona, Barcelona 08028, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
A discrete library of linear and hydantoin-containing dipeptide derivatives, based on the Lys-Trp(Nps)
scaffold, was prepared by solid-phase synthesis. SAR studies indicated that potency for TRPV1 blockade
Received 20 February 2011
Revised 26 April 2011
Accepted 29 April 2011
Available online 6 May 2011
and selectivity towards NMDA is mainly dictated by the side-chain length and the basic nature of
-groups in the N-terminal residue. The 2-Nps moiety at position 2 of Trp indole ring is preferred over
the 2-pyridine one.
a,
x
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Hydantoins
Dipeptides
Combinatorial library
TRPV1
SAR studies
Capsaicin, a pungent chemical present in hot peppers, specifi-
cally activates TRPV1, a membrane-bound, nonselective, transient
receptor potential cation channel. Since its cloning in 1997,1 TRPV1
receptor has generated significant interest because of its role in
pain pathways.1–6 TRPV1 is predominantly expressed in the noci-
ceptors and is activated by a variety of endogenous stimuli that
are known to be generated as a result of tissue injury and inflam-
mation. These endogenous ligands include heat and low pH,7 endo-
cannabinoid anandamide,8 and arachidonic acid metabolites.9 The
resulting excitation of sensitive pathways is followed by a refrac-
tary state referred as desensitization during which the neurons be-
come unresponsive to capsaicin and to noxious endogenous
stimuli. In addition, activation of this receptor can be potentiated
by pro-nocicpetive mediators such as bradykinin, NGF and oth-
ers.10 These different activation pathways indicate that TRPV1
has a significant role in pathway pain and represents an intriguing
novel target for the treatment of inflammation, cancer and for
other disorders, such as urinary urge incontinence,11 chronic
cough,12 irritable bowel syndrome,13 and migraine.14 In the last
years a growing interest in developing TRPV1 agonist15–17 and
antagonist18–25 molecules as potential analgesic drugs has been
showed. Due to the side effects associated to the agonist agents,
several pharmaceutical companies have also focused their atten-
tion on the discovery of potent and selective TRPV1 antagonists
some of which are already in phase I/II clinical trials for the indica-
tions of chronic inflammatory pain and migraine.26 In this regard,
we have described a series of dipeptides containing 2-[(o-nitro-
phenyl)sulfenyl] substituted tryptophan (Trp) as a new class of
TRPV1 channel blockade, showing selective analgesic activity.27
These results prompted us to develop a methodology for the facile
preparation of a library of TRPV1 ligands based on the 2-substi-
tuted Trp moiety, to further explore the structural requirements
of TRPV1 receptor blockers.
The most convenient method for the preparation of libraries of
molecules is the solid-phase mode,28,29 which allows paralleliza-
tion, easy workups, and give typically high yields by the use of
large excess of reactants.30 The rational design of the library was
performed considering as scaffold a dipeptide composed by Trp
and a
tively (Fig. 1). Single modifications were introduced at the 2-posi-
tion of Trp, - and -N-terminus, peptide backbone, and explored
x-basic amino acid at the C- and N-termini position, respec-
a
x
⇑
combinations between them. In particular, a pyridine moiety as a
phenyl bioisostere onto the Trp indole ring and different amino
Corresponding authors.
E-mail address: mroyo@pcb.ub.cat (M. Royo).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.141

3542
L. Zaccaro et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3541–3545
followed by treatment with 1,8-diazabicyclo[5.4.0]undec-7-ene
H2N
H2N
(DBU).32 Removal of Alloc or Mtt groups from resins 2 were carried
out with tetrakis(triphenylphosphine)-palladium (Pd(PPh3)4) and
phenylsilane (PhSiH3) in DCM, or with 3% TFA-DCM, respectively.
Cleavage with TFA in the presence of triethylsilane (TES)35 afforded
final compounds 4 {1–6} (Scheme 1). Acetyl derivatives 5 {1–6}
were obtained from 2 by Fmoc-removal, followed by acetylation
with Ac2O in the presence of N,N-diisopropylethylamine (DIPEA)
and cleavage, as above indicated.
O
n
H
N
NH2
O
N
H
S
n = 4
O2N
X
X = CH
Figure 1. Starting scaffold for library generation.
Final compounds 6–9 were obtained from hydantoin-resins 3 as
indicated in Scheme 2. Thus, the sequential removal of the side-
chain protecting group, guanidilation reaction by treatment with
uromium salts, such as 1-[bis(dimethylamino)methylene]-1H-
1,2,3-triazolo-[4,5-b]pyridinium hexafluorophosphate 3-oxide
(HATU)32 and O-(benzotriazol-1-yl)-N,N,N0,N0-bis(tetramethylene)
uronium hexa-fluorophosphate (HBPyU),33 and acid cleavage affor-
ded compounds 6 and 7. A similar sequence, but changing the gua-
nidylation by a permethylation step (using CH3I), resulted in
derivatives 8. Finally, analogues 9 were prepared by simple side-
chain protecting group removal and cleavage from the resin.
Application of the above sequences of reactions to resins 2 al-
lowed the preparation of final dipeptide derivatives 11–13 and
their acetylated counterparts 15–17 (Scheme 3). In this case, spe-
cial attention is required for the guanidylation and permethylation
reactions in the presence of Fmoc groups. If these reactions are car-
ried out in the presence of DIPEA, both reactions take place at both
amino functions, due to partial removal of the Fmoc group under
reaction conditions.34 In fact, compound 18 {3} was obtained as
the main product due to double guanidinium group incorporation
on to the resin bound intermediate 10 {3} with HBPyU/DIPEA in
DMF.35
acids such as Arg, Lys, diamino butyric acid (Dba) and diamino pro-
pionic acid (Dpa) were introduced. To explore further the role of
basicity, fully pentasubstituted guanidinium groups and the
methyl quaternary amino group were also introduced at the
side-chain of the N-terminal residue. Finally, compounds with a
free or acetylated a-NH2, and cyclized backbone through a hydan-
toin structure were also considered to investigate the role of the
N-terminus as well as the rigidity of the molecule.
The library was synthesized by solid phase using Rink-p-meth-
ylbenzhydrylamine (MBHA)-polystyrene (PS) resin and fluorenyl-
methoxycarbonyl (Fmoc) based chemistry.31 Rink-MBHA-resin
was acylated with Fmoc-Trp-OH, with unprotected side-chain,
using N,N-diisopropylcarbodiimide (DIPCDI) in the presence of
aza-hydroxybenzotriazole (HOAt). Modification of Trp indole ring
at position 2 was carried with the corresponding sulfenyl deriva-
tives, 2-[(o-nitrophenyl)sulfenyl] chloride (NpsCl) or 2-[(o-nitropy-
ridinyl)sulfenyl] chloride (NpysCl). Initial trials carried out using
DMF as a solvent resulted in incomplete substitution, but the use
of HOAc/DMF under Ar atmosphere achieved total conversion to
give intermediates 1 (Scheme 1). After removal of the Fmoc group,
resin 1 was divided in aliquots and Fmoc-Arg(Pmc)-OH, Fmoc-Lys-
OH, Fmoc-Dab-OH, and Fmoc-Dap-OH, the last three Fmoc-amino
acids with the amino group of their side-chain protected either
with allyloxycarbonyl (Alloc) or methyltrityl (Mtt) groups, were
incorporated to provide resins 2 {1–6}. Most of the remaining con-
versions were carried out using standard conditions. Thus, forma-
tion of the hydantoin ring to resins 3 was performed in two steps,
first reaction with N,N0-dissucinimidyl carbonate (DSC) in the pres-
ence of N,N-dimethylamino pyridine (DMAP) in DMF as a solvent,
The activity and selectivity of the library members were tested
on recombinant TRPV1 and NMDA channels expressed in Xenopus
oocytes by electrophysiological experiments.27 The results estab-
lish some general requirements that determine the potency for
the TRPV1 blockade and selectivity towards NMDA (Tables 1 and
2).
For linear peptide derivatives (Table 1), the TRPV1 blocking
potency appears to be directly related to the increase in the ba-
sicity of
x-N side-chain functional groups, with the activity
Scheme 1. Reagents and conditions: (i) X = CH, NpsCl in HOAc/DMF; X = N, NPysCl in HOAc/DMF; (ii) piperidine/DMF; (iii) Fmoc-protected amino acid (see Table 1)-DIPCDI-
HOAt-DIPEA in DMF; (iv) (a) DSC-DMAP in DMF; (b) DBU-DMF; (v) P = Alloc, Pd(PPh3)4ꢀPhSiH3 in DCM, P = Mtt, TFA/DCM; (vi) TFA/TES/H2O; (vii) Ac2O/DIPEA in DMF.
P = side-chain protecting group. R = H for Dap, Dab, and Lys, R = C(NH)NH2 for Arg.

L. Zaccaro et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3541–3545
3543
Scheme 2. Reagents and conditions: (vi) TFA/TES/H2O; (viii) R1 = CH3, HATU-DIPEA in DMF; R1 = C4H8, HBPyU-DIPEA in DMF; (ix) CH3I-DIPEA in DMF. P = side-chain
protecting group. R = H for Dap, Dab, and Lys, R = C(NH)NH2 for Arg.
Scheme 3. Reagents and conditions: (ii) Piperidine/DMF; (v) P = Alloc, Pd(PPh3)4ꢀPhSiH3 in DCM, P = Mtt, TFA/DCM; (vi) TFA/TES/H2O; (vii) Ac2O/DIPEA in DMF; (viii)
R1 = CH3, HATU-DIPEA in DMF; R1 = C4H8, HBPyU-DIPEA in DMF; (ix) CH3I-DIPEA in DMF; (x) (a) DIPEA/DMF, (b) CH3I in DMF; (xi) (a) DIPEA/DMF, (b) R1 = CH3, HATU in DMF;
R1 = C4H8, HBPyU in DMF. P = side-chain protecting group. R = H for Dap, Dab, and Lys, R = C(NH)NH2 for Arg.
following the order NH2<NðMeÞþ<(NMe2)2C@N <(pyrroli-
the enhancement of TRPV1 activity and selectivity versus NMDA.
In contrast, replacement of the Nps moiety at position 2 of the
indole ring by its pyridine bioisostere Npys generally reduces
the ability for the TRPV1 blockade as well as selectivity.
The conformational restriction of the peptide backbone through
3
dine)2C@N. Similarly, the basic character of the
a–NH2 group is
required for TRPV1 antagonist activity, since in all cases its acet-
ylation reduces considerably the percentage of blockade. In addi-
tion, these derivatives maintain or even increase the ability to
block NMDA ion channel with respect to their free amine ana-
logues, thus compromising selectivity. Remarkably, compound
a
a0
N ,N -cyclization to an hydantoin ring provided interesting re-
sults (Table 2). Thus, despite Lys –NH2 acylated, most compounds
a
18 {3} resulting from the double guanidylation of both
a
- and
increase the blocking properties on TRPV1 ion channels in compar-
ison to their acetylated linear analogues, indicating a positive role
of the backbone constraint. Again, a tendency to increase activity
and selectivity with the length and basicity of the N-terminal
side-chain is observed, and the Nps moiety is preferred over Npys,
e
-amino groups of Lys, was the most potent TRPV1-blocking
compound within this library, while maintains a nice selectivity.
With the only exception of H-Dap-, H-Dab-, and H-Lys-deriva-
tives 4 {1–3}, an increase in the side-chain length results in both

3544
L. Zaccaro et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3541–3545
Table 1
Table 2
Blockade of TRPV1 and NMDA receptors by Xaa-Trp(Nps) and Xaa-Trp(Npys) linear
derivatives
Blockade of TRPV1 and NMDA receptors by Xaa-Trp(Nps)- and Xaa-Trp(Npys)-derived
hydantoins
R3
R3
R2
R3
X
O
n
H
N
H
N
B
N
NH2
NH2
A
NH2
N
N
H
O2N
S
O
A
B
O
N
N
N
E
N
N
N
R3 ( )n
HN
NH2
N
H
N
S
H2N NH2
C
N
N
N
N
H2N NH2
N
N
N
N
O
O2N
X
O
D
C
D
E
Compd R2
R3
X
n
Activity (% blockade at
10 M)
Compd
R3
X
n
Activity (% blockade at 10 lM)
l
TRPV1 (1
lM)
NMDA
TRPV1 (1
l
M)
NMDA
6 {1}
6 {2}
6 {3}
6 {5}
7 {3}
7 {5}
8 {3}
8 {5}
9 {1}
9 {2}
9 {3}
9 {4}
9 {5}
9 {6}
E
E
E
E
D
D
B
B
A
A
A
C
A
C
CH
CH
CH
N
CH
N
1
2
4
4
4
4
4
4
1
2
4
3
4
3
51.3
56.3
60.4
52.5
13.0
40.9
66.5 (17.4)
19.9
29.9
11.0
15.4
50.2
24.6
43.7
12.0
10.7
12.9
9.3
4 {1}
4 {2}
4 {3}
4 {4}
4 {5}
4 {6}
5 {1}
5 {2}
5 {3}
5 {4}
5 {5}
5 {6}
11 {3}
12 {2}
12 {3}
13 {3}
15 {3}
16 {1}
16 {2}
16 {3}
16 {5}
17 {3}
17 {5}
H
H
H
H
H
H
Ac
Ac
Ac
Ac
Ac
Ac
H
H
H
H
Ac
Ac
Ac
Ac
Ac
Ac
Ac
A
A
A
C
A
C
A
A
A
C
A
C
B
E
E
D
B
E
E
E
E
D
D
CH
CH
CH
CH
N
1
2
4
3
4
3
1
2
4
3
4
3
4
2
4
4
4
1
2
4
4
4
4
39.3
20.6
26.3
62.8 (18.7)
17.0
44.8
14.6
27.6
14.3
29.2
12.7
23.9
36.7
24.0
29.7
13.5
7.7
23.6
20.6
27.0
27.2
15.4
25.8
13.6
26.3
5.5
10.8
9.2
CH
N
N
20.6
18.4
16.0
10.5
20.2
26.8
24.2
CH
CH
CH
CH
N
CH
CH
CH
CH
N
N
N
54.3
CH
CH
CH
CH
CH
CH
CH
CH
N
76.1 (23.5)
55.8
13.9
8.3
49.7 (21.1)
29.0
49.9
69.7
32.2
30.3
9.6
Acknowledgments
42.4
This search have been partially supported by grants from the
Ministry of Science and Innovation (CONSOLIDER-INGENIO
44.0
16.6
19.9
12.0
6.4
30.2
6.1
CH
N
2010
CSD2008-00005,
SAF2009-09323,
BFU2009-08346,
20.6
BQU2006-03794 and CTQ2009-20541, CTQ2005-00315/BQU and
CTQ2008-00177, la Generalitat Valenciana (PROMETEO 2010/
046), la Marató de TV3, and CIBER-BBN, Networking Centre on Bio-
engineering, Biomaterials and Nanomedicine Institute for Research
in Biomedicine.
N
18 {3}
E
CH
4
97.2 (27.3)
6.8
N
N
Supplementary data
although the differences are smaller than for linear analogues.
Derivative with a quaternary trimethylamino group at the Lys
side-chain, 8 {3}, proved to be the most potent compound within
the hydantoin series, with a blockade potency similar to that of
the bispyrrolidine-containing guanidine derivative 6 {3}.
In conclusion, the solid-phase synthesis of a combinatorial li-
brary based on a Lys-Trp(Nps) peptide scaffold has contributed to
a quick establishment of the minimal requirements for efficient
TRPV1 ion channel blockade and selectivity over ionotropic NMDA
receptor. Preferred structural issues are a side-chain length of 3-4-
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.04.141.
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