Design and synthesis of indole-based peptoids as potent noncompetitive antagonists of transient receptor potential vanilloid 1

Article abstract of DOI:10.1021/jm070612v

The vanilloid receptor subunit 1, or transient receptor potential vanilloid 1 (TRPV1), integrates physical and chemical stimuli in the peripheral nervous system, playing a key role in inflammatory pain. Identification of potent TRPV1 antagonists is thus an important goal of current neuropharmacology. Herein, we describe the solid-phase synthesis of a series of indole-based peptoids (N-alkylglycines) and the biological activity of the peptoids as novel TRPV1 antagonists. The potency and selectivity of the compounds were determined by electrophysiological recordings in Xenopus oocytes. The most potent and selective noncompetitive TRPV1 antagonist of the series, compound 7, represents an interesting pharmacophoric structure for analgesic lead optimization.

Full text of DOI:10.1021/jm070612v

J. Med. Chem. 2007, 50, 6133-6143
6133
Design and Synthesis of Indole-Based Peptoids as Potent Noncompetitive Antagonists of
Transient Receptor Potential Vanilloid 1
Martina Quintanar-Audelo,† Asia Ferna´ndez-Carvajal,‡ Wim Van Den Nest,§ Cristina Carren˜o,§ Antonio Ferrer-Montiel,*,‡ and
Fernando Albericio*,†,|
IRB Barcelona, Barcelona Science Park, UniVersity of Barcelona, and Department of Organic Chemistry, 08028 Barcelona, Spain, Institute of
Molecular and Cellular Biology, UniVersity Miguel Herna´ndez, 03202 Alicante, Spain, and DiVerDrugs SL, 08850 GaVa`, Barcelona, Spain
ReceiVed May 26, 2007
The vanilloid receptor subunit 1, or transient receptor potential vanilloid 1 (TRPV1), integrates physical
and chemical stimuli in the peripheral nervous system, playing a key role in inflammatory pain. Identification
of potent TRPV1 antagonists is thus an important goal of current neuropharmacology. Herein, we describe
the solid-phase synthesis of a series of indole-based peptoids (N-alkylglycines) and the biological activity
of the peptoids as novel TRPV1 antagonists. The potency and selectivity of the compounds were determined
by electrophysiological recordings in Xenopus oocytes. The most potent and selective noncompetitive TRPV1
antagonist of the series, compound 7, represents an interesting pharmacophoric structure for analgesic lead
optimization.
Introduction
Organisms require pain to survive and to maintain structural
integrity, but sustained or chronic pain can result in side effects
and in a decreased quality of life. Inflammatory pain is a
response to tissue damage. It begins when noxious stimuli
(thermal, chemical, or mechanical) excite sensory neurons called
nociceptors.1-4 Nociceptors are sensitized by activation of PKCa
and PKA pathways, which leads to phosphorylation of sensory
receptors involved in the nociceptive function.5 The resultant
sensory signaling is conveyed to the spinal cord and, finally, to
specific brain regions, leading to the sensation of pain.6 There
is evidence that peripheral sensitization is mediated by hyper-
excitation of TRPV1 receptors. Identification of TRPV1 as a
molecular integrator of noxious stimuli that involves both
thermal nociception and inflammatory hyperalgesia has validated
it as a key therapeutic target for inflammatory pain.7-12
TRPV1 was first reported by Caterina et al.13 as a capsaicin
(1) receptor, a nonselective cation channel activated by
vanilloids and lipids, and gated at temperatures above 43 °C.
In addition, mild extracellular pH potentiates its activation by
noxious heat and vanilloids, whereas strongly acidic conditions
(pH <6) directly activate the channel.14,15 Furthermore, the
channel is notably sensitized by proalgesic substances released
Figure 1. Chemical structures of TRPV1 agonists and antagonists.
during inflammation.16 TRPV1 hyperstimulation by capsaicin
leads to long-term desensitization of the sensory neurons and
such as 1 and resiniferatoxin (RTX, 2), is limited due to side
thus produces an analgesic effect. Capsaicin is the active
effects (burning sensation, irritation, and neurotoxicity) resulting
component of various topical pain relievers and has been used
from continuous influx of Ca2+ into the cells. Structure-activity
to treat pain associated with diabetic neuropathy and arthritis.17,18
relationship (SAR) studies of vanilloids have led to other potent
However, the clinical use of TRPV1 agonists (See Figure 1),
drugs.19 However, these also exhibit irritant side effects. An
alternative approach to pain relief is to block the TRPV1-
mediated pain signaling pathways with receptor antagonists, a
promising strategy for the development of novel analgesic drugs
with potentially fewer side effects.20 In addition, some TRPV1
antagonists have shown positive effects in animal models of
neurophatic pain, which makes these kinds of compounds very
attractive as analgesic molecules.21 During the past few years,
several classes of competitive TRPV1 antagonists, either
structurally related or not to 1 or 2, have been described, and
their chemistry and pharmacology have been reviewed.22-25
Many of these antagonists are focused on structures containing
* To whom correspondence should be addressed. A.F.-M.: phone, 0034
966658727; fax, 0034 966658758; e-mail, aferrer@umh.es. F.A.: phone,
0034 934037088; fax, 0034 934037126; e-mail, albericio@pcb.ub.es.
† IRB Barcelona, Barcelona Science Park, University of Barcelona.
‡ University Miguel Herna´ndez.
§ DiverDrugs SL.
| Department of Organic Chemistry, University of Barcelona.
a Abbreviations: BAL, backbone amide linker; MTBD, 1,3,4,6,7,8-
hexahydro-1-methyl-2H-pyrimido[1,2-a]pyrimidine; NMDA, N-methyl-D-
aspartate; PKA, protein kinase A; PKC, protein kinase C; TES, 2-[(2-
hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid; TRPV1,
transient receptor potential vanilloid 1; abbreviations used for amino acids
follow the IUPAC-IUB Comission of Biochemical Nomenclature (Jones,
J. H. J. Pept. Sci. 2003, 9, 1-8).
10.1021/jm070612v CCC: $37.00 © 2007 American Chemical Society
Published on Web 11/07/2007

6134 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Scheme 1. General Synthesis of Peptoids, Method Aa
Quintanar-Audelo et al.
a Conditions: (a) ClCH2COOH, DIC, DMF; (b) R1-NH2, Et3N, DMSO/DMF; (c) ClCH2COOH, DIC, DMF; (d) R2-NH2, Et3N, DMSO/DMF; (e)
Fmoc-AA-OH; DIC/HOBt, DMF; (f) piperidine/DMF; (g) TFA cocktail cleavage.
Scheme 2. General Synthesis of Peptoids, Method Ba
a Conditions: (a) R1-NH2, NaBH3CN, AcOH/DMF; (b) ClCH2COOH, DIC, DMF; (c) R2-NH2, Et3N, DMSO/DMF; (d) Fmoc-AA-OH, DIC/HOBt,
DMF; (e) piperidine/DMF; (f) TFA cocktail cleavage.
Figure 2. Chemical structures of indole-based analogs of peptoid 4.
thiourea, urea, and amide groups.26 However, further studies
are required to assess their clinical efficacy.
Compound H-Arg-15-15C (4) (Figure 1) has been described as
a good scaffold for analgesic lead optimization, as it presents
submicromolar potency and in vivo analgesic and anti-inflam-
matory activities. However, its receptor selectivity is not
optimal.30
Herein we report the synthesis of noncompetitive antagonists
of TRPV1 by analoging of peptoid 4. A library of 20 peptoids
was constructed on solid-phase, some of which were identified
through biological assays as having higher potency than 4 and
an increase in the receptor selectivity.
Noncompetitive antagonists interact with additional binding
sites on the receptor structure, thereby preventing opening of
the receptor by an agonist or blocking its aqueous pore. The
first noncompetitive TRPV1 antagonist discovered was the
trinuclear polyamine complex ruthenium red (3) (Figure 1),
which binds to the pore region of the channel with high potency
(IC50 ) 0.14 µM). However, its poor selectivity seems to
underlie its proconvulsive activity in animal models, which
precludes its clinical development.25 Certain arginine-rich pep-
tides that block recombinant TRPV1 channels expressed in
Xenopus oocytes with micromolar potency have also been
described, but they are toxic.27 N-Alkylglycines, or peptoids,
were the first small molecules identified as noncompetitive
TRPV1 antagonists.28 These compounds have advantages over
arginine-rich peptides, such as higher proteolytic stability.29
Results and Discussion
Design and Synthesis of the Library. On the basis of the
previous synthesis of 4,30 we designed a series of analogs to
improve its potency. Studies of the pore region of the channel
suggest that a charged or polar side chain could be crucial to
activity, due to the presence of a cation binding site right at the

Indole-Based Peptoids as Antagonists of TRPV1
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6135
Table 1. Acidolytic Cleavage Cocktails (v/v)
mixture 1
(94:2.5:2.5:1)
mixture 2
(95:5)
mixture 3
(95:5)
mixture 4
(49:49:2)
mixture 5
(88:5:5:2)
TFA
H2O
TFA
H2O
TFA
H2O
TFA
DCM
TFA
H2O
EDT
i-Pr3SiH
anisole
phenol
i-Pr3SiH
ether washes ether washes THF washes ether washes ether washes
Table 2. HPLC Data (%) of Crude Products Obtained with Different
Cleavage Cocktailsa
peptoid mixture 1 mixture 2 mixture 3 mixture 4 mixture 5
10
11
12
13
14
15
19
25
19
20
15
22
32
43
58
26
46
40
34
45
33
40
50
46
67
61
80
70
57
64
38
60
38
21
34
59
Figure 3. Chemical structure of peptoid 8.
pore vestibule.15a Moreover, it could be complementary with
the presence of hydrophobic groups that may stabilize the
structure in the pore entrance. Several modifications were
therefore introduced on the basis of these two characteristics:
(i) substitution of 2,4-dichlorophenethyl group with a different
hydrophobic planar group such as indole; (ii) incorporation of
different amino acids at the N-terminus to evaluate the effect
of polar and charged groups of different lengths; (iii) deletion
of the acetamide group at the C-terminus; (iv) substitution of
the N-terminal amide group with a tertiary amine group; and
(v) incorporation of a cyclic group into the structure, while the
hydrophobic groups and a charged side chain are maintained.
All peptoids were prepared via the submonomer method,31
using two general routes starting on Rink amide MBHA (method
A; see Scheme 1) and BAL-MBHA (method B; see Scheme 2)
resins. In method A, the primary amine of Rink linker is acylated
with chloroacetic acid and N,N′-diisopropylcarbodiimide (DIC),
and then the chloroacetyl group undergoes nucleophilic substitu-
tion with a primary amine in basic media to yield a secondary
amine. In method B, the aldehyde group of BAL linker reacts
with a primary amine by reductive amination.32 The syntheses
then proceed similarly: acylation of the corresponding amines
with chloroacetic acid and DIC, followed by nucleophilic
substitution with tryptamine or 2,4-dichlorophenethylamine in
DMSO/DMF. A Fmoc-protected amino acid is then coupled
using DIC/HOBt, and after deprotection of the Fmoc group,
the compounds are cleaved from the resin with an optimized
TFA cocktail.
a HPLC systems: (A) H2O-TFA 99.955:0.045, (B) ACN-TFA 99.964:
0.036; gradient 10% B to 90% B in 15 min. Compounds were detected by
UV absorption at 220 nm.
cocktails was necessary to minimize side reactions and improve
the purity of the crude products. Five TFA cocktails (Table 1)
with different scavengers and washing methods were evaluated.
For all compounds, the best results were obtained with TFA-
DCM-anisole (49:49:2, v/v) (Table 2), providing purities
between 60% and 80%. Anisole was the best scavenger for the
Boc group, offering 3-4-fold higher purity of crude products.
Incorporation of a cyclic group into the peptoid was achieved
as described in Scheme 3 for the synthesis of compound 16.
Starting with BAL-MBHA resin and after reductive amination
with 2,4-dichlorophenethylamine, Alloc-Amp(Fmoc)-OH was
coupled with DIC/HOAt. Orthogonal protection of this amino
acid allowed incorporation of a 2,4-dichlorophenyl group at the
R-amine group by reductive amination, and coupling of Fmoc-
Arg(Pbf)-OH at the γ-amine group with DIC/HOAt. The
compound was cleaved from the resin with HF, as cleavage
with TFA did not yield the desired product. The presence of
the R-amino group in the cyclic moiety may explain the
increased stability of the bond between the BAL linker and the
peptoid.34
Peptoids 17-22 (Figure 5) are derivatives of 11 prepared by
different N-alkylations at the ꢀ-amine of the lysine side chain.
Peptoids 17 and 18 were obtained by exhaustive alkylation with
DIEA and an excess of MeI and EtI, respectively. Monoalky-
lation was achieved using Miller’s method:35 protecting the free
amine with o-nitrobenzenesulfonyl chloride in basic media,
followed by deprotonation and selective monoalkylation with
methyl p-nitrobenzensulfonate or EtI in DMF. Selective removal
of the sulfonamide with â-mercaptoethanol and DBU in DMF
afforded peptoids 19 and 20. The preparation of dialkylated
derivatives (21 and 22) was first evaluated using consecutive
reductive aminations, which did not proceed successfully. Thus,
the amines were first monoalkylated using Miller’s method,
obtaining 21 and 22 via reductive amination with formaldehyde
or acetaldehyde, respectively.
Incorporation of different hydrophobic groups using method
A (Scheme 1), afforded derivatives 5-7 (Figure 2), which
contain indole and 2,4-dichlorophenethyl moieties. In the
synthesis of peptoid 8 (Figure 3), the amide bond on the
N-terminus was deleted via reductive amination with Fmoc-6-
aminohexanal. Fmoc-6-aminohexanal was prepared by reduction
of the corresponding amino acid with NaBH4 to provide the
alcohol (99% yield) which, upon Swern oxidation, afforded the
desired amino aldehyde (90%).33
Removal of the acetamide group at the C-terminus from the
chemical structures of peptoids 4 and 5 results in the chemical
structures of peptoids 9 and 10 (Figure 4), respectively. This
modification was achieved by using BAL linker in method B
(Scheme 2).
Finally, to evaluate the effect of guanidinium deactivation,
we synthesized compounds 23 and 24 (Figure 6), with a nitro
and a tosyl moiety, respectively, both of which are stable to
cleavage conditions, as arginine protecting groups. The synthesis
was performed as described above for peptoid 10 but using
Fmoc-Arg(NO2)-OH and Fmoc-Arg(Tos)-OH, respectively, at
the N-terminal position.
Several amino acids (Lys, Orn, Dab, Dpr, His) were evaluated
in the N-terminal position (11-15) (Figure 4) to determine the
effects of the length and polarity of the side chain on biological
activity. Cleavage of these peptoids from the resin with TFA
yielded complex mixtures, possible due to the reactivity of the
deprotected indole group. Therefore, optimization of TFA

6136 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Quintanar-Audelo et al.
Figure 4. Analogs of peptoid 10 with different N-terminal amino acids.
Scheme 3. Synthesis of Compound 16a
a Conditions: (a) 2,4-dichlorophenethylamine, NaBH3CN, AcOH/DMF; (b) Fmoc-Amp(Alloc)-OH, DIC/HOAt, DMF; (c) (i) PhSiH3, Pd(PPh3)4, DCM,
(ii) sodium diethyldithiocarbamate, DMF; (d) 2,4-dichlorobenzaldehyde, NaBH3CN, AcOH/DMF; (e) piperidine/DMF; (f) Fmoc-Arg(Pbf)-OH, DIC/HOAt,
DMF; (g) piperidine/DMF; (h) 10% anisole/HF, -78 °C.
Biological Results. The ability of the 20 peptoids (5-24) to
block capsaicin-mediated activation of TRPV1 channel was first
evaluated at 10 µM. The capacity of each peptoid to block the
activity of TRPV1 heterologously expressed in Xenopus oocytes
was determined using a two microelectrode voltage-clamp
amplifier at ∼20 °C. The channel activity was elicited with 10
µM capsaicin at a holding potential of -40 mV, and then the
corresponding compounds were infused.36-38 The results of this
first screening are shown in Figure 7. The receptor selectivity
of each peptoid was first evaluated by determining the extent
of blockade of the N-methyl-D-aspartate receptor (NMDA) at
10 µM (Figure 8).
Those peptoids with only one indole group in their structure
(6 and 7) showed the highest blocking activity. The introduction
of a second indole group (5) leads to a decreased activity, but
an increased selectivity, compared to the parent compound 4.

Indole-Based Peptoids as Antagonists of TRPV1
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6137
Figure 5. Nꢀ-Alkylated analogs of peptoid 11.
19 > 20 > 21 > 22). Finally, the low activity of peptoids
23 and 24 is also consistent with the fact that activity is at
least partly dependent on the presence of a free guanidinium
group.
These results agree with the reported molecular architecture
of the pore region of the TRPV1 channel.15a The location of
three acidic residues (Asp646, Glu648, and Glu651) close to
the permeation pathway ensures a strong negative electrostatic
potential, which is required to attract positive groups. This fact
is consistent with the high activity observed for peptoids
containing a positive charge, such as a guanidinium group (6,
7, 9, 10, and 16), at the opposite site of hydrophobic groups.
Essentially, the presence of these hydrophobic groups seems
required for peptoid interaction at the outer hydrophobic entrance
of the channel, whereas the positive charge fits into the internal
region of the channel. The combination of these two interactions
allows the peptoids to act as noncompetitive antagonists by
blocking the pore entrance and consequently inhibiting TRPV1
activity.
Figure 6. Chemical structure of peptoids 23 and 24.
The N-terminus (8) and C-terminus (9 and 10) modifications
retain the activity of the parent compound but result in a lower
selectivity. All of the analogs with different N-terminal amino
acids (11-15) were less active and selective than the parent
compound. This result indicates the importance of the guani-
dinium group for activity, whose replacement with a primary
amine leads to 4-fold lower activity. On the other hand, the
introduction of a rigid moiety into the peptoid skeleton (16)
does not markedly affect activity but results in a lower
selectivity. Regarding the N-alkylated peptoids (17-22), the
screening revealed a relationship between activity and the
basicity of the side chain of the N-terminal amino acid. Although
these analogs were less active than the parent compound,
comparison to peptoid 11 indicated that the more basic the
amine groups are, the higher the observed activity is (18 >
Dose-response curves were performed for the most active
compounds (5-10 and 16) and representative curves are shown
in Figure 9. The corresponding IC50 values were determined
(Table 3) and showed that five of these peptoids have similar
or higher potency than 4. For comparison, the blockade potency
of the noncompetitive TRPV1 antagonist ruthenium red has been
included. As seen, this compound is a potent TRPV1 blocker
but lacks receptor selectivity. Peptoid 7 exhibited the highest
Figure 7. Blockade activity of TRPV1 channels expressed in Xenopus oocytes. The channels were activated with 10 µM capsaicin and incubated
with peptoids at 10 µM. Oocytes were perfused at 20 °C with Ringer buffer pH 7.4 and held at -40 mV. Percentages were normalized with respect
to the value obtained in the absence of peptoids. Each point represents the mean ( SEM, n g 4.

6138 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Quintanar-Audelo et al.
Figure 8. Blockade activity of NMDA channels expressed in Xenopus oocytes. The channels were activated with 100 µM L-Glu/20 µM Gly and
exposed to 10 µM peptoids. Oocytes were perfused with Ringer buffer pH 7.4 and held at -40 mV. All measurements were performed at 20 °C.
Percentages were normalized with respect to that obtained in the absence of peptoids. Each point represents the mean ( SEM, n g 4.
Figure 9. Dose-response curves of blockade activity of capsaicin-activated TRPV1 channels expressed in Xenopus oocytes for representative
peptoids 5-7. Channels were activated with 10 µM capsaicin in Ringer buffer at pH 7.4 and held at -40 mV. Responses were normalized
with respect to that obtained in the absence of peptoids. Each point represents the mean ( SEM, n g 4; solid lines denote the best fit to the Hill
equation.
antagonistic activity (IC50 ) 0.18 µM) of our peptoids, which
is ≈4-fold stronger than 4 (IC50 ) 0.7 µM). Noteworthy, peptoid
7 also exhibited improved receptor selectivity with respect to
the parent peptoid 4, as evidenced by the 10-fold higher IC50
value for blockade of the NMDA receptor (Table 3). In addition,
this peptoid did not antagonize the ion channel activity of
voltage-gated ion channels [IC50 > 100 µM for K+ channel
subtype Kv1.1, Na+ channel subtype SKN4A, and a1H T-type
Ca2+ channel (data not shown)].28 Thus, the modification that
leads to peptoid 7 produced a noncompetitive antagonist
with higher blockade potency and selectivity than the starting
peptoid 4.
The inspection of the structure of these peptoids revealed that
deletion of the acetamide group at the C-terminus of the parent
compound leads to a higher potency, as reflected in peptoids 9
(IC50 ) 0.17 µM), 5, and 10. Interestingly, peptoid 8 has an
IC50 of 0.40 µM, despite the fact that it lacks a guanidinium
group. This result can be partially explained by considering the
absence of an amide bond in the N-terminal position of 8, which
enables a different structural conformation. However, we also
observed that a more rigid structural conformation does not
improve potency, since the introduction of a cyclic moiety into
the peptoid skeleton (16) does not greatly affect activity. Despite
the high potency of peptoids 8, 9, and 16, their poor receptor

Indole-Based Peptoids as Antagonists of TRPV1
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6139
Table 3. Blockade Efficacy and Selectivity of Most Active Peptoidsa
IC50 (µM)
chem AG (Darmstadt, Germany). HF was obtained from Air
Products and Chemicals, Inc. (Allentown, Canada), and related
equipment was obtained from Peptide Institute (Minoh, Osaka,
Japan). Other chemicals were purchased from Aldrich (Milwaukee,
WI) and the solvents were obtained from SDS (Peypin, France)
and were used as received, except THF, which was distillated over
sodium. Analytical RP-HPLC was performed using Waters (Mil-
ford, MA) chromatography systems with reverse-phase Symmetry
C18 (150 × 3.9 mm, 5 µm) columns. Semipreparative RP-HPLC
was performed on a Waters (Milford, MA) chromatography system
using a Symmetry C18 (100 × 30 mm, 5 µm) column. Compounds
were detected by UV absorption at 220 nm. Mass spectra were
obtained by RP-HPLC, UV-vis detector, and ZQ Electrospray mass
detector from Waters (Milford, MA). High-resolution mass spectra
(HRMS) were obtained in an LC/MSD TOF from Agilent Tech-
nologies (Unitat d’Espectrometria de Masses, Serveis Cient´ıfico-
peptoid
TRPV1
NMDA
RR
4
5
6
7
8
9
10
16
0.14 ( 0.09
0.7 ( 0.1
1.33 ( 0.28
0.52 ( 0.09
0.18 ( 0.03
0.4 ( 0.1
0.17 ( 0.01
1.1 ( 0.1
0.7 ( 0.2
0.5 ( 0.2
15 ( 2
207 ( 55
17 ( 3
166 ( 12
3 ( 1
0.8 ( 0.4
18 ( 4
8 ( 2
a TRPV1 ion channel activity was elicited with 10 µM capsaicin, and
NMDA receptor activity was evoked with 100 µM L-Glu/20 µM Gly.
Oocytes were held at -40 mV and continuously perfused with Ringer’s
solution at 20 °C. IC50 values were obtained from the fit of dose-response
curves to the Hill equation. Values are given as mean (SEM, n g 4. RR
denotes ruthenium red.
1
Te`cnics, University of Barcelona). H NMR were recorded on a
Mercury 400 spectrometer (Unitat de RMN, Serveis Cientifico-
Te`cnics, University of Barcelona). Electrophysiological recordings
were performed with an electrode voltage clamp amplifier [TEC
10CD, NPI Electronic (Tamm, Germany)]. Electrodes were made
with hematocrit glass capillaries and pulled with P-97 puller of
Sutter Ins. Co. (Novato, CA).
selectivity (Table 3) led us to exclude them from further
pharmacological assays.
General Procedures. Solid-phase syntheses were performed in
polypropylene syringes equipped with a polyethylene porous disk.
Solvents and soluble reagents were removed by suction. Washes
between deprotection, coupling, and subsequent deprotection steps
were carried out with DMF (5 × 1 min) and DCM (5 × 1 min)
using 10 mL of solvent/g per wash. All final compounds were
purified to g95% purity by semipreparative RP-HPLC using
gradients of acetonitrile (0.05% TFA) and Milli-Q water (0.1%
TFA) at 20 mL/min. The purity of the compounds was determined
by analytical RP-HPLC using a gradient of acetonitrile (0.036%
TFA) and Milli-Q water (0.045% TFA) at 1 mL/min. High-
resolution mass spectrometry (HRMS) was run in ES positive
Conclusions
A series of analogs of peptoid 4 was synthesized in order to
obtain SAR information of noncompetitive TRPV1 antagonist
activity, as well as requirements for receptor selectivity. A 20-
member library was synthesized entirely on solid-phase using
Rink amide MBHA and BAL-MBHA resins via the submono-
mer method. Optimization of cleavage conditions led to the
identification of anisole as the best scavenger to minimize the
side reactions derived from an unprotected indole group. The
inhibitory activity of the analogs toward TRPV1 and NMDA
was assayed in Xenopus oocytes, and five peptoids more potent
than the original peptoid 4 were identified. These studies
revealed that the combination of two different hydrophobic
groups (indole and 2,4-dichlorophenethyl) and the presence of
the guanidinium group in the N-terminal amino acid are
favorable to activity. Deactivation of the guanidinium group or
substitution with a primary amine significantly decreased the
inhibitory activity. These results substantiate the relevance of a
positive charge to enhance the interaction with the outer pore
region of the TRPV1 channel, where acidic residues are known
to be located. The mechanism of action of these peptoids may
imply interaction of the hydrophobic moieties with the hydro-
phobic vestibule of the channel and concomitant interaction of
the positive charge with the outer pore region of the channel.
Other chemical modifications, such as elimination of the
acetamide group in the C-terminus or the introduction of a
tertiary amine at the N-terminus, increased the potency but
decreased the selectivity vs NMDA receptor.
1
ionization mode. H NMR spectra were obtained with a Varian-
Mercury 400 MHz spectrometer. Chemical shifts are reported in
part per million (ppm, δ units), and coupling constants are given
in hertz (Hz).
Coupling of Amino Acids and BAL Linker. Protected amino
acid or BAL linker (0.63 mmol) in DMF, HOBt (85 mg, 0.63 mmol)
and DIC (98 µL, 0.63 mmol) were added to the resin and allowed
to react with automatic shaking for 1 h. The solvent was removed
by filtration and the resin was washed as indicated above.
Incorporation of the BAL linker to the MBHA resin was run
overnight and confirmed by the Kaiser test.39
Removal of Protecting Groups. Fmoc: piperidine/DMF (2:8,
v/v) (2 × 10 min), washes with DMF (5 × 1 min). Alloc: Pd-
(PPh3)4 (0.1 mmol), PhSiH3 (10 mmol) in anhydrous DCM (3 ×
15 min) under an N2 atmosphere, washes with 0.02 M sodium
diethyldithiocarbamate/DMF (3 × 15 min).
Reductive Amination. The corresponding amine (2.1 mmol) was
dissolved with AcOH/DMF (1:99, v/v) and added to the resin.
NaBH3CN (132 mg, 2.1 mmol) was then added, and the mixture
was allowed to react at room temperature for 6 h. The resin was
washed with DMF and DCM.
Peptoid 7 is the most potent and selective noncompetitive
antagonist of TRPV1 based on the N-alkylglycine structure
described to date. Its ≈4-fold stronger potency and 10-fold
higher selectivity than the parent peptoid 4 converts this
noncompetitive antagonist to an interesting pharmacophoric
scaffold for analgesic lead development.
Preparation of Fmoc-6-aminohexanal. Fmoc-6-aminohexanoic
acid (1 g, 2.8 mmol) was dissolved in anhydrous THF at -78 °C
in N2 atmosphere. NMM (372 µL, 3.36 mmol) and isobutyl
chloroformate (440 µL, 3.36 mmol) were then added, and the
solution was warmed to 0 °C for 40 min. The temperature was
lowered to -78 °C, a methanolic solution of NaBH4 (214 mg,
5.6 mmol) was added, and the reaction mixture was maintained at
-78 °C for 2 h. The reaction was then gradually brought to room
temperature and quenched with aqueous ammonium chloride. The
crude mixture was extracted with EtOAc, and the organic phase
was washed with NaHCO3 and brine, dried, and concentrated in
vacuo to afford the corresponding alcohol as a white solid (955
mg, 99% yield). This alcohol was then oxidized to the target
aldehyde as follows: Oxalyl chloride (268 µL, 3 mmol) was
dissolved in anhydrous DCM, and the resulting solution was treated
Experimental Section
Materials and Equipment. Protected amino acids and HOBt
were obtained from IrisBiotech (Martdrewitz, Germany). DIC,
trifluoroacetic acid, and acetaldehyde were purchased from Fluka
Chemika (Busch, Switzerland). 2,4-Dichlorobenzaldehyde and
tryptamine were obtained from Acros (Fair Lawn, NJ). Capsaicin
was from Sigma (St Louis, MO). N-Ethyldiisopropylamine and
HPLC grade formic acid were purchased from Merck (Darmstadt,
Germany). Solid supports were supplied by Calbiochem-Novabio-

6140 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Quintanar-Audelo et al.
with DMSO (240 µL, 3.36 mmol) at -78 °C for 30 min. A DCM
solution of the alcohol was then added, and the mixture was allowed
to react at -78 °C in N2 atmosphere for 1 h. Triethylamine (1.2
mL, 8.4 mmol) was then added, and after 1 h, the reaction was
brought to room temperature and then concentrated in vacuo. The
crude was purified by flash chromatography with hexane/EtOAc
(1:1) to obtain a white solid (860 mg, 90% yield).
General Synthesis of Peptoids: Method A. Peptoids 5-7.
Starting with 0.65 mmol/g Rink Amide MBHA resin (250 mg, 0.16
mmol) the first step was deprotection of the Fmoc group, thereafter
acylation was carried out with chloroacetic acid (90 mg, 0.96 mmol)
in the presence of DIC (77 µL, 0.48 mmol) in DMF for 1 h. The
reaction was followed by the Kaiser test. After washing with DMF,
the amination was performed with tryptamine or 2,4-dichlorophen-
ethylamine (0.64 mmol), and triethylamine (89 µL, 0.64 mmol) in
DMF. The reaction was run for 4 h and followed by the De Clercq
test.40 Acylation and amination steps (tryptamine or 2,4-dichlo-
rophenethylamine) were successively repeated using the same
conditions described above. After washing with DMF and DCM,
the corresponding amino acids were coupled via the above-
mentioned procedure.
Method B: Peptoids 9-24 (Except for 16). The BAL handle
(0.63 mmol) was coupled to 300 mg of MBHA resin (0.7 mmol/
g). Reductive amination of the handle was the performed with a
primary amine (tryptamine or 2,4-dichlorophenethylamine, 2.1
mmol) as described above, and the reaction was followed by the
aldehydes test.41 The resulting amine was then acylated with
chloroacetic acid (119 mg, 1.26 mmol) in the presence of DIC (99
µL, 0.63 mmol) in DMF for 1 h. After washing with DMF, the
amination was performed with tryptamine or 2,4-dichlorophen-
ethylamine (0.84 mmol) and triethylamine (117 µL, 0.84 mmol) in
DMF, and the reaction was left for 4 h. After washing, the
corresponding amino acids were incorporated via the above-
mentioned procedure.
Synthesis of Peptoid 8. Peptoid 8 was synthesized via method
A with 2,4-dichlorophenethylamine for amination. In the last step,
instead of coupling an amino acid, a reductive amination was
performed with Fmoc-aminohexanal (542 mg, 1.6 mmol) and
NaBH3CN (100 mg, 1.6 mmol) dissolved in AcOH/DMF (1:99 v/v)
under agitation overnight. After Fmoc deprotection, the peptoid was
cleaved from the resin with TFA/H2O (95:5 v/v).
µL, 1 mmol) in DCM for 2 h. The reaction was followed by the
Kaiser test. After several washes, N-alkylation was carried out with
MeI (62 µL, 0.84 mmol) or EtI (68 µL, 0.84 mmol) in the presence
of MTBD (91 µL, 0.63 mmol) in DMF for 30 min. After washing,
the protecting group was eliminated with â-mercaptoethanol (147
µL, 2.1 mmol) and DBU (140 µL, 1 mmol) in DMF (2 × 20 min).
Dialkylation to obtain compounds 21 and 22 was performed in
two steps: a first alkylation achieved via Miller’s method as
describe above and then a second alkylation via reductive amination.
The latter comprised acetaldehyde (118 µL, 2.1 mmol) or formal-
dehyde (90 µL, 2.1 mmol) and NaBH3CN (132 mg, 2.1 mmol) in
AcOH/DMF overnight and was followed by the De Clercq test.
Cleavage of Peptoids from the Resin. Removal of the side chain
protecting groups and cleavage of the peptoids from the resin were
accomplished by treatment with a mixture of cold TFA (10 mL/g
of resin) for 1 h, allowing the slurry to achieve room temperature.
Compounds 8 and 17-24 were treated with TFA-H2O (95:5, v/v)
for 1 h; compounds 5-7 and 9 were treated with TFA-phenol-
H2O-iPr3SiH (88:5:5:2, v/v) for 2 h; and compounds 10-15 were
treated with TFA-DCM-anisole (49:49:2, v/v) for 1 h. The crude
peptoids were concentrated in vacuo, washed with ether, dissolved
in water, and then lyophilized to yield white powders.
Characterization of Peptoids. [L-Arginyl]-[N-[2-(indol-3-yl)-
ethyl]glycyl]-N-[(2-(indol-3-yl)ethyl)] Glycinamide (5). Yield: 4
mg, 5%. HRMS: m/z found (M + H)+ 574.3248, calcd (M + H)+
1
574.3247. H NMR (DMSO-d6): δ 1.45 (m, 2H), 1.59 (m, 2H),
2.89 (m, 2H), 3.03 (m, 4H), 3.47 (m, 2H), 3.6 (m, 2H), 4.05 (m,
4H), 4.12 (m, 1H), 6.98 (m, 2H), 7.04 (m, 2H), 7.13 (m, 1H), 7.17
(m, 1H), 7.23 (m, 1H), 7.32 (m, 2H), 7.53 (m, 1H), 7.60 (m, 1H),
8.15 (m, 3H), 10.81-10.93 (m, 2H). HPLC [gradient 0% to 100%
MeCN (+0.036% TFA) in H2O (+0.045% TFA) in 15 min]: tR )
6.9 min.
[L-Arginyl]-[N-[2-(indol-3-yl)ethyl]glycyl]-N-(2,4-dichlorophen-
ethyl) Glycinamide (6). Yield: 4 mg, 5%. HRMS: m/z found (M
1
+ H)+ 603.2355, calcd (M + H)+ 603.2382. H NMR (DMSO-
d6): δ 1.31 (m, 2H), 1.43 (m, 2H), 2.86 (m, 2H), 2.9 (m, 2H), 3.11
(m, 2H), 3.48 (m, 2H), 3.56 (m, 2H), 4.07 (m, 4H), 4.21 (m, 1H),
7.14 (m, 4H), 7.3 (m, 2H), 7.36 (m, 2H), 7.52 (m, 2H), 8.08 (m,
2H), 8.18 (m, 3H), 10.82 (br s, 1H). HPLC [gradient 20% to 100%
MeCN (+0.036% TFA) in H2O (+0.045% TFA) in 15 min]: tR )
7.4 min.
Synthesis of Peptoid 16. Peptoid 16 was synthesized starting
with 1.1 mmol/g BAL-aminomethyl resin (380 mg, 0.41 mmol)
followed by reductive amination with 2,4-dichlorophenethylamine
(630 µL, 4.1 mmol) and NaBH3CN (263 mg, 4.1 mmol) as describe
above. Next, Alloc-Amp(Fmoc)-OH (265 mg, 0.83 mmol) was then
coupled with HOAt (114 mg, 0.83 mmol) and DIC (130 µL, 0.82
mmol) in DMF for 6 h. After washing with DMF and DCM, the
Alloc group was eliminated as describe above. Reductive amination
with 2,4-dichlorobenzaldehyde (731 mg, 4.1 mmol) and NaB3HCN
(263 mg, 4.1 mmol) was carried out in AcOH/DMF (1:99 v/v)
overnight. After washing and deprotection of Fmoc group, Fmoc-
Arg(Pbf)-OH (343 mg, 1.23 mmol) was incorporated in DMF using
the general procedure. After Fmoc deprotection, cleavage from the
resin was performed with 10% anisole in HF for 1 h at -78 °C.
The HF was evaporated, and the resin was washed with methyl
tert-butyl ether (3 × 5 min). The product was extracted with AcOH
and acetonitrile and then lyophilized.
General Procedure for N-Alkylation of Lysine Side Chains
(17-22). The basic structure was synthesized as peptoid 11, in
which an N-terminus Boc-Lys(Fmoc)-OH (295 mg, 0.63 mmol)
was incorporated with HOBt (85 mg, 0.63 mmol) and DIC (98
µL, 0.63 mmol) as coupling reagents. N-Alkylations were performed
after Fmoc deprotection and washing with DMF and DCM.
Exhaustive alkylation was carried out with MeI (156 µL, 2.1
mmol) and DIEA (277 µL, 2.1 mmol) in DMF for 4 h to obtain
peptoid 17. Peptoid 18 was synthesized with the same procedure
using EtI (327 mg, 2.1 mmol) as alkylating reagent.
[L-Arginyl]-[N-(2,4-dichlorophenethyl)glycyl]-N-[2-(indol-3-
yl)ethyl]glycinamide (7). Yield: 5 mg, 6%. HRMS: m/z found
(M + H)+ 603.2344, calcd (M + H)+ 603.2397. 1H NMR (DMSO-
d6): δ 1.3 (m, 2H), 1.45 (m, 2H), 2.89 (m, 2H), 2.91 (m, 2H), 3.1
(m, 2H), 3.5 (m, 2H), 3.59 (m, 2H), 4.08 (m, 4H), 4.21 (m, 1H),
7.14 (m, 4H), 7.3 (m, 4H), 7.32 (m, 2H), 7.53 (m, 2H), 8.16 (m,
3H), 10.85 (br s, 1H). HPLC [gradient 20% to 100% MeCN
(+0.036% TFA) in H2O (+0.045% TFA) in 15 min]: tR ) 5.0
min.
[[N-(2,4-Dichlorophenethyl)-N-(6-aminohexyl)]glycyl]-N-(2,4-
dichlorophenethyl) Glycinamide (8). Yield: 24 mg, 19%.
HRMS: m/z found (M + H)+ 577.1485, calcd (M + H)+ 577.1488.
1H NMR (DMSO-d6): δ 1.21-1.33 (m, 4H), 1.5-1.6 (m, 4H),
2.75 (m, 2H), 2.87 (m, 2H), 3.0 (m, 4H), 3.2 (m, 2H), 3.49 (m,
2H), 4.0 (m, 2H), 7.13 (br s, 1H), 7.27 (dd, J ) 2.12, 8.24 Hz,
1H), 7.36 (m, 1H), 7.43 (m, 2H), 7.5 (m, 1H), 7.62 (m, 1H). HPLC
[gradient 5% to 70% MeCN (+0.036% TFA) in H2O (+0.045%
TFA) in 15 min]: tR ) 10.0 min.
[L-Arginyl]-N-(2,4-dichlorophenethyl)glycine 2,4-(Dichlo-
rophenethylamide) (9). Yield: 5 mg, 5%. HRMS: m/z found (M
1
+ H)+ 577.1230, calcd (M + H)+ 577.1236. H NMR (DMSO-
d6): δ 1.49 (m, 2H), 1.66 (m, 2H), 2.86 (m, 6H), 3.39 (m, 2H),
3.51 (m, 2H), 3.91(d, J ) 9.2 Hz, 2H), 4.17 (m, 1H), 7.25 (d, J )
2.0 Hz, 1H), 7.32 (s, 2H), 7.37 (dd, J ) 8.2, 2.0 HZ, 1H), 7.43
(dd, J ) 8.2, 2.0 Hz, 1H), 7.54 (m, 1H), 7.57 (d, J ) 2.0 Hz, 1H),
7.62 (d, J ) 2.0 Hz, 1H), 8.15 (m, 5H). HPLC retention [gradient
5% to 100% MeCN (+0.036% TFA) in H2O (+0.045% TFA) in
15 min]: tR ) 8.2 min.
Monoalkylation to obtain 19 and 20 was performed via Miller’s
method. First, the amine group was protected by treatment with
o-nitrobenzensulfonyl chloride (139 mg, 0.63 mmol) and DIEA (138
[L-Arginyl]-N-[2-(indol-3-yl)ethyl]glycine 2-(Indol-3-yl)ethy-
lamide (10). Yield: 2 mg, 3%. HRMS: m/z found (M + H)+

Indole-Based Peptoids as Antagonists of TRPV1
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24 6141
517.3032, calcd (M + H)+ 517.3039. 1H NMR (DMSO-d6): δ 1.21
(m, 2H), 1.38 (m. 2H), 2.84 (m, 2H), 2.96 (m, 2H), 3.04 (m, 2H),
4.33 (m, 2H), 4.41 (m, 2H), 4.43 (m, 2H), 4.91 (m, 2H), 3.68 (br
s, 1H), 4.48 (m, 3H), 4.91 (m, 1H), 6.94 (m, 5H), 7.06 (m, 2H),
7.09 (s, 1H), 7.51-7.58 (m, 2H), 9.21 (m, 2H), 10.82 (m, 2H).
HPLC [gradient 25% to 55% MeCN (+0.036% TFA) in H2O
(+0.045% TFA) in 15 min]: tR ) 15.5 min.
[NE-Triethyl-L-lysyl]-N-[2-(indol-3-yl)ethyl]glycine 2-(Indol-
3-yl)ethylamide (18). Yield: 12 mg, 11%. HRMS: m/z found (M)+
573.3904, calcd (M)+ 573.3917. 1H NMR (DMSO-d6): δ 1.16 (m,
9H), 1.31 (m, 2H), 1.49 (m, 2H), 1.69 (m, 2H), 2.8 (m, 2H), 2.9
(m, 4H), 3.1 (m, 6H), 3.33 (m, 2H), 3.52 (m, 2H), 4.1 (m, 1H), 4.2
(m, 2H), 6.9 (m, 2H), 7.0 (m, 2H), 7.1 (m, 1H), 7.2 (m, 1H), 7.3
(m, 2H), 7.51 (dd, J ) 3.65, 7.75 Hz, 1H), 7.58 (dd, J ) 7.87,
14.15 Hz, 1H), 8.2 (br s, 1H), 10.85 (m, 2H). HPLC [gradient 10%
to 80% MeCN (+0.036% TFA) in H2O (+0.045% TFA) in 15 min]:
tR ) 7.1 min.
[L-Lysyl]-N-[2-(indol-3-yl)ethyl]glycine 2-(Indol-3-yl)ethyla-
mide (11). Yield: 5 mg, 6%. HRMS: m/z found (M + H)+
489.2871, calcd (M + H)+ 489.2879. 1H NMR (DMSO-d6): δ 1.2
(m, 2H), 1.4 (m, 4H), 2.67 (m, 2H), 2.82 (m, 2H), 2.95 (m, 2H),
3.4 (m, 2H), 3.52 (m, 2H), 4.34 (m, 1H), 4.44 (m, 2H), 5.0 (br s,
1H), 6.96 (m, 2H), 7.08 (m, 4H), 7.18 (br s, 1H), 7.23 (m, 1H),
7.32 (d, J ) 8.13 Hz, 1H), 7.5 (d, J ) 8.26 Hz, 1H), 7.56 (m, 1H),
10.84 (m, 2H). HPLC [gradient 25% to 55% MeCN (+0.036%
TFA) in H2O (+0.045% TFA) in 15 min]: tR ) 14.7 min.
[L-Ornityl]-N-[2-(indol-3-yl)ethyl]glycine 2-(Indol-3-yl)ethyl-
amide (12). Yield: 5 mg, 7%. HRMS: m/z found (M + H)+
475.2814, calcd (M + H)+ 475.2821. 1H NMR (DMSO-d6): δ 1.58
(m, 2H), 1.69 (m, 2H), 2.86 (m, 4H), 2.73 (m, 2H), 3.41 (m, 2H),
3.50 (m, 2H), 3.9 (s, 2H), 4.12 (m, 1H), 6.97 (m, 2H), 7.05 (m,
2H), 7.12 (m, 2H), 7.22 (br s, 1H), 7.32 (m, 2H), 7.5 (d, J ) 7.81
Hz, 1H), 7.57 (dd, J ) 7.81, 19.9 Hz, 1H), 7.72 (br s, 2H), 8.23
(br s, 2H), 10.87 (m, 2H). HPLC [gradient 5% to 70% MeCN
(+0.036% TFA) in H2O (+0.045% TFA) in 15 min]: tR ) 8.2
min.
[NE-Methyl-L-lysyl]-N-[2-(indol-3-yl)ethyl]glycine 2-(Indol-3-
yl)ethylamide (19). Yield: 15 mg, 16%. HRMS: m/z found (M
1
+ H)+ 503.3130, calcd (M + H)+ 503.3134. H NMR (DMSO-
d6): δ 1.21 (s, 3H), 1.28 (m, 2H), 1.47 (m, 2H), 1.63 (m, 2H), 2.8
(m, 6H), 3.39 (m, 2H), 3.59 (m, 2H), 4.09 (m, 2H), 4.16 (m, 1H),
6.96 (m, 2H), 7.05 (m, 2H), 7.14 (m, 2H), 7.33 (m, 2H), 7.5 (d, J
) 7.93 Hz, 1H), 7.59 (dd, J ) 7.87, 17,23 Hz, 1H), 8.17 (br s,
2H), 8.44 (br s, 2H), 10.88 (m, 2H). HPLC [gradient 10% to 80%
MeCN (+0.036% TFA) in H2O (+0.045% TFA) in 15 min]: tR )
6.8 min.
[NE-Ethyl-L-lysyl]-N-[2-(indol-3-yl)ethyl]glycine 2-(Indol-3-yl)-
ethylamide (20). Yield: 13 mg, 15%. HRMS: m/z found (M +
1
H)+ 517.3293, calcd (M + H)+ 517.3291. H NMR (DMSO-d6):
δ 1.13 (m, 3H), 1.31 (m, 2H), 1.47 (m, 2H), 1.64 (m, 2H), 2.79
(m, 2H), 2.87 (m, 4H), 2.98 (m, 2H), 3.4 (m, 2H), 3.54 (m, 2H),
4.09 (m, 1H), 4.11 (br s, 2H), 6.9 (m, 2H), 7.05 (m, 2H), 7.1 (d, J
) 2.1 Hz, 1H), 7.14 (br s, 1H), 7.32 (d, J ) 8.09 Hz, 2H), 7.5 (d,
J ) 7.87 Hz, 1H), 7.57 (dd, J ) 7.87, 17.41 Hz, 1H), 8.18 (m,
2H), 8.49 (m, 2H), 10.85 (m, 2H). HPLC [gradient 10% to 80%
MeCN (+0.036% TFA) in H2O (+0.045% TFA) in 15 min]: tR )
8.8 min.
[L-Diaminobutiryl]-N-[2-(indol-3-yl)ethyl]glycine 2-(Indol-3-
yl)ethylamide (13). Yield: 10 mg, 13%. HRMS: m/z found (M
1
+ H)+ 461.2661, calcd (M + H)+ 461.2665. H NMR (DMSO-
d6): δ 2.49 (m, 4H), 2.88 (m, 6H), 3.28 (m, 2H), 3.72 (m, 2H),
4.13 (br s, 1H), 4.24 (m, 2H), 6.96 (m, 2H), 7.05 (m, 2H), 7.13
(m, 2H), 7.22 (br s, 1H), 7.33 m, 2H), 7.49 (m, 1H), 7.57 (m, 1H),
8.12 (br s, 1H), 8.33 (br s, 1H), 8.47 (br s, 1H), 10.88 (m, 2H).
HPLC [gradient 25% to 55% MeCN (+0.036% TFA) in H2O
(+0.045% TFA) in 15 min]: tR ) 14.6 min.
[NE-Dimethyl-L-lysyl]-N-[2-(indol-3-yl)ethyl]glycine 2-(Indol-
3-yl)ethylamide (21). Yield: 9 mg, 10%. HRMS: m/z found (M
1
+ H)+ 517.3287, calcd (M + H)+ 517.3243. H NMR (DMSO-
d6): δ 1.21 (s, 6H), 1.26 (m, 2H), 1.5 (m, 2H), 1.62 (m, 2H), 2.82
(m, 2H), 2.86 (m, 2H), 2.98 (m, 2H), 3.4 (m, 2H), 3.59 (m, 2H),
4.04 (m, 1H), 4.11 (s, 2H), 6.97 (m, 2H), 7.05 (m, 2H), 7.14 (m,
2H), 7.34 (m, 2H), 7.5 (d, J ) 7.76 Hz, 1H), 7.57 (dd, J ) 8.17,
16.64 Hz, 1H), 8.14 (m, 3H), 10.87 (m, 2H). HPLC [gradient 10%
to 80% MeCN (+0.036% TFA) in H2O (+0.045% TFA) in 15 min]:
tR ) 8.7 min.
[L-Diaminopropionyl]-N-[2-(indol-3-yl)ethyl]glycine 2-(Indol-
3-yl)ethylamide (14). Yield: 8 mg, 11%. HRMS: m/z found (M
1
+ H)+ 447.2505, calcd (M + H)+ 447.2508. H NMR (DMSO-
d6): δ 0.83 (m, 2H), 2.97 (m, 2H), 3.06 (m, 2H), 3.14 (m, 2H),
3.46 (m, 2H), 3.95 (m, 2H), 4.24 (m, 2H), 6.96 (m, 2H), 7.05 (m,
2H), 7.13 (m, 1H), 7.2 (m, 1H), 7.32 (m, 2H), 7.53 (m, 2H), 8.04
(br s, 1H), 8.22 (br s, 1H), 8.33 (br s, 1H), 10.88 (m, 2H). HPLC
[gradient 25% to 55% MeCN (+0.036% TFA) in H2O (+0.045%
TFA) in 15 min]: tR ) 15.2 min.
[NE-Diethyl-L-lysyl]-N-[2-(indol-3-yl)ethyl]glycine 2-(Indol-3-
yl)ethylamide (22). Yield: 2 mg, 6%. HRMS: m/z found (M +
1
H)+ 545.3598, calcd (M + H)+ 545.3604. H NMR (DMSO-d6):
[L-Histidyl]-N-[2-(indol-3-yl)ethyl]glycine 2-(Indol-3-yl)ethy-
lamide (15). Yield: 1 mg, 2%. HRMS: m/z found (M + H)+
498.2610, calcd (M + H)+ 498.2617. 1H NMR (DMSO-d6): δ 2.85
(m, 2H), 2.99 (m, 2H), 3.1 (m, 1H), 3.22 (m, 1H), 3.41 (m, 2H),
3.49 (m, 2H), 3.85 (m, 2H), 4.2 (m, 1H), 6.96 (m, 2H), 7.05 (m,
2H), 7.11 (m, 1H), 7.14 (m, 1H), 7.2 (m, 1H), 7.32 (m, 2H), 7.37
(m, 1H), 7.49 (m, 2H), 8.4 (m, 1H), 8.59 (m, 1H), 8.91 (m, 1H),
10.88 (m, 2H). HPLC [gradient 5% to 70% MeCN (+0.036% TFA)
in H2O (+0.045% TFA) in 15 min]: tR ) 8.3 min.
cis-(L-Arginyl)amino-N-(2,4-dichlorophenylmethyl)-L-pro-
line 2,4-Dichlorophenethylamide (16). Yield: 10 mg, 4%.
HRMS: m/z found (M + H)+ 618.2335, calcd (M + H)+ 618.2339.
1H NMR (DMSO-d6): δ 1.21 (m, 2H), 1.42 (m, 2H), 1.63 (m, 2H),
2.75 (m, 4H), 3.07 (m, 2H), 3.3 (m, 2H), 3.72 (m, 3H), 4.2 (m,
2H), 7.22 (m, 2H), 7.37 (m, 1H), 7.47 (br s, 1H), 7.49 (m,1H),
7.59 (m, 1H) 7.63 (m, 1H), 8.0 (br s, 1H), 8.12 (m, 2H), 8.64 (m,
1H). HPLC [gradient 20% to 100% MeCN (+0.036% TFA) in H2O
(+0.045% TFA) in 15 min]: tR ) 5.4 min.
δ 1.14 (m, 6H), 1.31 (m, 2H), 1.50 (m, 2H), 1.65 (m, 2H), 2.81
(m, 2H), 2.87 (m, 4H), 3.0 (m, 4H), 3.4 (m, 2H), 3.5 (m, 2H), 4.1
(m, 1H), 4.2 (m, 2H), 6.9 (m, 2H), 7.0 (m, 2H), 7.11 (d, J ) 12.1
Hz, 1H), 7.14 (m, 1H), 7.34 (m, 2H), 7.51 (d, J ) 8.2 Hz, 1H),
7.55 (dd, J ) 8.2, 16.78 Hz, 1H), 8.15 (br s, 3H), 10.81 (m, 2H).
HPLC [gradient 5% to 95% MeCN (+0.036% TFA) in H2O
(+0.045% TFA) in 15 min]: tR ) 7.0 min.
[NG-Nitro-L-arginyl]-N-[2-(indol-3-yl)ethyl]glycine 2-(Indol-
3-yl)ethylamide (23). Yield: 7 mg, 7%. HRMS: m/z found (M +
1
H)+ 562.2881, calcd (M + H)+ 562.2890. H NMR (DMSO-d6):
δ 1.5 (m, 2H), 1.64 (m, 2H), 2.85 (m, 2H), 3.0 (m, 2H), 3.12 (m,
2H), 3.45 (m, 2H), 3.6 (m, 2H), 4.11 (br s, 2H), 4.18 (m, 1H), 6.97
(m, 2H), 7.04 (m, 2H), 7.12 (m, 2H), 7.23 (d, J ) 8.1 Hz, 1H),
7.33 (m, 2H), 7.5 (d, J ) 7.92 Hz, 1H), 7.55 (dd, J ) 8.12, 15.06
Hz, 1H), 8.17 (m, 4H), 8.43 (m, 1H), 10.81-10.92 (m, 2H). HPLC
[gradient 10% to 80% MeCN (+0.036% TFA) in H2O (+0.045%
TFA) in 15 min]: tR ) 8.1 min.
[NG-Tosyl-L-arginyl]-N-[2-(indol-3-yl)ethyl]glycine 2-(Indol-
3-yl)ethylamide (24). Yield: 9 mg, 8%. HRMS: m/z found (M +
[NE-Trimethyl-L-lysyl]-N-[2-(indol-3-yl)ethyl]glycine 2-(Indol-
3-yl)ethylamide (17). Yield: 7 mg, 5%. HRMS: m/z found (M)+
531.3443, calcd (M)+ 531.3448. 1H NMR (DMSO-d6): δ 1.21 (s,
9H), 1.28 (m, 2H), 1.47 (m, 2H), 1.63 (m, 2H), 2.82 (m, 2H), 2.86
(m, 2H), 3.15 (m, 2H), 3.43 (m, 2H), 3.53 (m, 2H), 4.19 (s, 1H),
4.23 (m, 2H), 6.9 (m, 2H), 7.06 (m, 2H), 7.14 (m, 2H), 7.32 (m,
2H), 7.54 (m, 2H), 8.16 (m, 3H), 10.86 (m, 2H). HPLC [gradient
0% to 90% MeCN (+0.036% TFA) in H2O (+0.045% TFA) in 15
min]: tR ) 7.7 min.
1
H)+ 671.3120, calcd (M + H)+ 671.3128. H NMR (DMSO-d6):
δ 1.21 (s, 3H), 1.38 (m, 2H), 1.61 (m, 2H), 2.27 (m, 3H), 2.85 (m,
2H), 2.97 (m, 2H), 3.35 (m, 2H), 3.6 (m, 4H), 4.0 (m, 2H), 4.19
(m, 1H), 6.59 (m, 1H), 6.78 (m, 1H), 6.96 (m, 2H), 7.07 (m, 2H),
7.24 (m, 3H), 7.33 (m, 2H), 7.49 (m, 1H), 7.61 (m, 3H), 8.2 (br s,
2H), 10.8-10.86 (m, 2H). HPLC [gradient 0% to 90% MeCN
(+0.036% TFA) in H2O (+0.045% TFA) in 15 min]: tR ) 9.7
min.

6142 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 24
Quintanar-Audelo et al.
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Biological Assays. Electrophysiological Recordings. Xenopus
oocytes were defolliculated using calcium-free Barth’s solution,
collagenase (2 mg/mL), and slow agitation (50-60 rpm) for 1-2
h. The oocytes were then stored at 18 °C for 12 h before injection.
In vitro transcribed RNA encoding the rat TRPV1 or rat NR1
and NR2A subunits of the NMDA receptor (injected at 1:1 w/w)
was injected into Xenopus oocytes (5-10 ng/oocyte). Electro-
physiological recordings were made 2-4 days after injection.
Electrophysiological recordings were performed using an electrode
voltage clamp amplifier. Throughout the experiments, the oocytes
were continuously perfused with an external Ringer’s solution
containing (in mM) 115 NaCl, 3 KCl, 0.1 BaCl2, 2 MgCl26H2O,
and 10 HEPES-Na+, pH 7.4 for TRPV1 experiments and 115 NaCl,
3 KCl, 2 BaCl2 and 10 HEPES-Na+, pH 7.4 for NMDA experi-
ments. Electrodes were filled with 1 M KCl buffered with 10 mM
TES and typically had resistance of 300-500 kΩ. The currents
were sampled at 4-5 Hz after filtering at 1 kHz. Leak subtraction
was accomplished with two inverted quarter-amplitude prepulses
that were scaled and subtracted from the test pulse (P/4). TRPV1
channels were activated by application of 10 µM of capsaicin in
the absence or presence of the inhibitors of the channel at a holding
potential (Vh) of -40 mV. For the selectivity assays, NMDA
channels were activated with a solution of 100 µM L-glutamate
and 20 µM glycine. Whole currents were recorded at ca. 20 °C.
Responses were normalized with respect to that evoked in the
absence of peptoids. Dose-response curves for blockade activity
were fitted to the Hill equation
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I
1
1 + [peptoid]
IC50
)
ηH
Imax
(21) Culshaw, A. J.; Bevan, S.; Christiansen, M.; Copp, P.; Davis, A.;
Davis, C.; Dyson, A.; Dziadulewicz, E. K.; Edwards, L.; Eggelte,
H.; Fox, A.; Gentry, C.; Groarke, A.; Hallett, A.; Hart, T. W.; Hughes,
G. A.; Knights, S.; Kotsonis, P.; Lee, W.; Lyothier, I.; McBryde,
A.; McIntyre, P.; Paloumbis, G.; Panesar, M.; Patel, S.; Seiler, M-P.;
Yaqoob, M.; Zimmermann, K. Identification and Biological Char-
acterization of 6-Aryl-7-isopropylquinazolinones as Novel TRPV1
Antagonists that are Effective in Models of Chronic Pain. J. Med.
Chem. 2006, 49, 471-474.
(
)
where IC50 denotes the concentration of channel peptoid that inhibits
half of the channel response obtained in its absence (Imax) and nH
denotes the Hill coefficient. Experimental data were processed using
ORIGIN version 7.0 SMO (OriginLab Corp.).
Acknowledgment. The authors thank Laura Garc´ıa de
Diego, for her work on some of the electrophysiological
experiments, and Reme Torres, for mRNA preparation and
oocytes injection and maintenance. This work was partially
supported by funds from The Ministry of Education and Science
(SAF2006-02580 to A.F.-M.) and Fundacio´n Ramo´n Areces (to
A.F.-M.). M.Q.-A. thanks DURSI, the Generalitat of Catalunya,
and European Social Founds for a predoctoral (FI-IQUC)
fellowship.
(22) Rami, H. K.; Gunthorpe, M. J. The Therapeutic Potential of TRPV1
(VR1) Antagonists; Clinical Answers Await. Drug DiscoVery To-
day: Ther. Strateg. 2004, 1, 97-104.
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as the Next Generation of Painkillers. Are We Putting the Cart before
the Horse? J. Med. Chem. 2004, 47, 2717-2723.
(24) Planells-Cases, R.; Garc´ıa-Mart´ınez, C.; Royo, M.; Pe´rez-Paya´, E.;
Carren˜o, C.; Albericio, F.; Messeguer, A.; Ferrer-Montiel, A. Small
Molecules Targeting the Vanilloid Receptor Complex as Drugs for
Inflammatory Pain. Drugs Future 2003, 28, 787-795.
(25) Messeguer, A.; Planells-Cases, R.; Ferrer-Montiel, A. Physiology and
Pharmacology of the Vanilloid Receptor. Curr. Neuropharmacol.
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Vanilloid Type 1 Channel Modulators for the Treatment of Pain. J.
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E.; Belmonte, C.; Gonza´lez-Ros, J. M.; Ferrer-Montiel, A. Arginine-
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Supporting Information Available: A table containing HPLC
data of all purified peptoids and dose-response curves of selected
peptoids assayed in TRPV1 and NMDA receptors. This material
is available free of charge via the Internet at http://www.pubs.
acs.org.
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JM070612V