Triazine-based vanilloid 1 receptor open channel blockers: Design, synthesis, evaluation, and SAR analysis

Article abstract of DOI:10.1021/jm200981s

The thermosensory transient receptor potential vanilloid 1 channel (TRPV1) is a polymodal receptor activated by physical and chemical stimuli. TRPV1 activity is drastically potentiated by proinflammatory agents released upon tissue damage. Given the pivotal role of TRPV1 in human pain, there is pressing need for improved TRPV1 antagonists, the development of which will require identification of new pharmacophore scaffolds. Uncompetitive antagonists acting as open-channel blockers might serve as activity-dependent blockers that preferentially modulate the activity of overactive channels, thus displaying fewer side effects than their competitive counterparts. Herein we report the design, synthesis, biological evaluation, and SAR analysis of a family of triazine-based compounds acting as TRPV1 uncompetitive antagonists. We identified the triazine 8aA as a potent, pure antagonist that inhibits TRPV1 channel activity with nanomolar efficacy and strong voltage dependency. It represents a new class of activity-dependent TRPV1 antagonists and may serve as the basis for lead optimization in the development of new analgesics.

Full text of DOI:10.1021/jm200981s

ARTICLE
pubs.acs.org/jmc
Triazine-Based Vanilloid 1 Receptor Open Channel Blockers:
Design, Synthesis, Evaluation, and SAR Analysis†
Miquel Vidal-Mosquera,‡ Asia Fernꢀandez-Carvajal,|| Alejandra Moure,‡ Pierluigi Valente,||
Rosa Planells-Cases,^ Josꢀe M. Gonzꢀalez-Ros,|| Jordi Bujons,§ Antonio Ferrer-Montiel,|| and Angel Messeguer*,‡
‡Department of Chemical and Biomolecular Nanotechnology and §Department of Biological Chemistry and Molecular Modeling,
Institut de Química Avanc-ada de Catalunya (IQAC-CSIC), 08034 Barcelona, Spain
Instituto de Biología Molecular y Celular, Universidad Miguel Hernandez, 03202 Elche, Spain
^Centro de Investigaciꢀon Príncipe Felipe, 46012 Valencia, Spain
S Supporting Information
b
ABSTRACT: The thermosensory transient receptor potential va-
nilloid 1 channel (TRPV1) is a polymodal receptor activated by
physical and chemical stimuli. TRPV1 activity is drastically poten-
tiated by proinflammatory agents released upon tissue damage.
Given the pivotal role of TRPV1 in human pain, there is pressing
need for improved TRPV1 antagonists, the development of which
will require identification of new pharmacophore scaffolds. Uncom-
petitive antagonists acting as open-channel blockers might serve as activity-dependent blockers that preferentially modulate the
activity of overactive channels, thus displaying fewer side effects than their competitive counterparts. Herein we report the design,
synthesis, biological evaluation, and SAR analysis of a family of triazine-based compounds acting as TRPV1 uncompetitive
antagonists. We identified the triazine 8aA as a potent, pure antagonist that inhibits TRPV1 channel activity with nanomolar efficacy
and strong voltage dependency. It represents a new class of activity-dependent TRPV1 antagonists and may serve as the basis for
lead optimization in the development of new analgesics.
’ INTRODUCTION
active antagonists. Consequentially, numerous TRPV1 antago-
nists have been identified that block the receptor with high
efficacy and potency (Figure 1). However, and rather disappoint-
ingly, despite the claimed therapeutic potential of these TRPV1
antagonists, very few candidates have progressed into clinical
trials because of unpredicted side effects such as hyperthermia.
Furthermore, complete blocking of TRPV1 in some models of
chronic pain models apparently results in enhanced hypersen-
sitivity.11 These observations are consistent with its widespread
distribution in neuronal and non-neuronal tissues, which sug-
gests that it is involved in body functions other than nociception
and pain (e.g., body temperature regulation).11 Indiscriminate
pharmacological blocking of the receptor with high affinity,
quasi-irreversible, competitive vanilloid antagonists may be re-
sponsible for the observed side effects.11 Thus, high affinity
antagonists that bind to the receptor in an activity-independent
manner should show limited therapeutic indices, since these
compounds would interact with both resting and active channels.
Taken together, these data support the need for a different class
of antagonists that would either act on a specific mode of
activation or be activity-dependent, primarily targeting over-
activated receptors.
Cloning of the vanilloid receptor subunit I channel (transient
receptor potential vanilloid 1, TRPV1) has notably contributed
to our current knowledge on the molecular and cellular mecha-
nisms underlying chemical and thermal nociception and pain
transduction.1,2 TRPV1 was the first identified member of a
family of thermosensory receptors currently referred to as
thermoTRPs, which encompass cold-, warm-, and heat-activated
channels. TRPV1 is a capsaicin-operated nonselective cation
channel with high Ca2+ permeability. It is also gated by noxious
heat (g42 ꢀC), low pH, and different endogenous compounds
(endocannabinoids, phorbols, etc.) and therefore can be con-
sidered as a molecular integrator of noxious stimuli in nocicep-
tors. Genetic and pharmacological suppression of TRPV1
activity markedly reduces the thermal hyperalgesia characteristic
of inflammatory pain.3ꢀ5 Notably, enhanced expression of this
thermoTRP channel has been observed in various human
chronic pathologies, including arthritis and cancer pain. TRPV1
is essential for peripheral sensitization of nociceptors upon tissue
injury and/or inflammation produced by trauma, infection,
surgery, burns, or diseases with an inflammatory component.6,7
Thus, TRPV1 has garnered great attention as a therapeutic target
for pain management, including for neuropathic, postoperative,
chronic, or bone cancer pain.8ꢀ10
Validation of TRPV1 as a therapeutic target has prompted
intensive drug discovery programs aimed at developing orally
Received: March 15, 2011
Published: September 27, 2011
r
2011 American Chemical Society
7441
dx.doi.org/10.1021/jm200981s J. Med. Chem. 2011, 54, 7441–7452
|

Journal of Medicinal Chemistry
ARTICLE
Figure 1. Relevant TRPV1 ligands: the agonist capsaicin (1) and competitive antagonists (2ꢀ4).
electrical field at the pore entry.14 Although they are active in vivo
in animal models of pain, their development was precluded because
of unanticipated side effects arising when they were used at
submicromolar concentration, namely, release of α-CGRP from
nociceptive neurons. We have speculated that their binding site
location was also accessible in the closed-channel state and that this
could account for the observed side effects. Regardless, the chemical
diversity surrounding the glycine oligomer scaffold in 5a and 5b
constituted the basis of the present study.
The peptoids 5a and 5b, and other active TRPV1 blockers,4,15,16
share structural features that have been assumed to constitute a
basic pharmacophore for TRV1 blocking activity: two aryl
moieties and one cationic group. Herein we report the design,
synthesis, in vitro screening, and SAR analysis of a new family of
TRPV1 antagonists built around a triazine scaffold based on said
pharmacophore. Among these, triazine 8aA blocked TRPV1 with
submicromolar efficacy in a strongly voltage-dependent manner,
consistently with the receptor binding-site being located deep
within the membrane electric field.
Figure 2. N-Alkylglycine trimers that block TRPV1.14
Uncompetitive antagonists are activity-dependent inhibitors
that specifically bind to the agonistꢀreceptor complex or to the
open state of the channel.12 Because of their interaction with active
receptors, which enables them to preferentially block highly
activated receptors while only interacting minimally with phy-
siologically working or silent channels, these compounds have
attracted sizable interest as potent and safe drugs. For example,
memantine, an uncompetitive L-glutamate antagonist of the NMDA
receptor, has been approved for the treatment of Alzheimer’s
disease.13 Accordingly, identification and validation of uncom-
petitive antagonists acting as open channel blockers of TRPV1
receptors warrant exploration as a strategy to develop selective
analgesic drugs that would offer higher therapeutic indices than
do currently known channel antagonists.
Open-channel blockers are antagonists that recognize a site
within a channel’s aqueous pore; therefore, they can only access
the channel when it is open (i.e., in the conductive state).
Furthermore, the accessible time is directly proportional to the
time the channel remains in its open conformation. Therefore,
the drug-binding site is more exposed in overactivated channels
than in silent receptors or in channels that only briefly open upon
receiving a nociceptive signal.
’ RESULTS AND DISCUSSION
Design and Synthesis of New TRPV1 Blockers. To expand
upon the available SAR data obtained from our previous hit com-
pounds (the peptoids 5a and 5b), we designed compounds
representing the chemical diversity shown in Figure 3. To
modulate their TRPV1-blocking capacity, each compound was
conferred with two identical arylalkyl substituents (except entry j,
which can be compared as a cycloalkyl analogue) and one alkyl
moiety (polar cationic, polar nonionizable, or apolar). The
compounds were chiefly assembled from commercially available
primary and secondary amines (see Experimental Section).
Initially, four new peptoids were prepared (5cꢀf) using a
previously reported synthetic methodology.17 However, preli-
minary evaluation revealed that they had significantly lower
activity than did 5a and 5b (Table 1). This discrepancy was
greatest for those compounds lacking the tertiary amine group, a
trend that corroborates the proposed pharmacophore. There-
fore, a second generation of compounds was developed, which
contained a cationic guanidyl or trialkylamino group and in
which the undesired conformational flexibility of the peptoid
moiety was partially restricted by using piperazine-2,5-dione as
We have previously identified compounds with channel-block-
ing activity.14ꢀ16 These antagonists, particularly the N-alkylgly-
cine trimers (peptoids) 5a and 5b (Figure 2), block TRPV1 with
micromolar efficacy by interacting with the external vestibule of
the ionic pore, at a site apparently located outside the membrane
7442
dx.doi.org/10.1021/jm200981s |J. Med. Chem. 2011, 54, 7441–7452

Journal of Medicinal Chemistry
ARTICLE
Figure 3. Formal sources of chemical diversity incorporated in the new series of TRPV1 blockers. The wavy lines indicate the bond(s) of attachment to
the corresponding scaffold.
scaffold (6aꢀf, Table 1). This strategy has been successful to
increase the selectivity against other pharmaceutical targets
studied in our group,18,19 but in this case 6aꢀf showed poor
activity as TRPV1 antagonists (Table 1). These new heterocyclic
compounds were synthesized using a methodology developed in
our laboratory.18
We reasoned that although satisfying the requirements of the
pharmacophore, the suboptimal 3D arrangement of structural
features imposed by scaffold 6 could be responsible for the
modest or null activity observed. Furthermore, the structure of
scaffold 6 still holds a relatively high degree of flexibility and on the
other hand it contains a chiral center and three nonequivalent
positions for substitution. These features render an undesirable
number of possibilities for different combinations of R1ꢀR3. There-
fore, we turned to a more rigid and symmetric scaffold: 2,4,6-
trisubstituted-1,3,5-triazine (8, Scheme 1), which has been har-
nessed to generate compounds with antimicrobial,20 anticancer,21,22
or HIV-1 reverse transcriptase inhibitory activities.23
The initial results for some of the new compounds (e.g., 8eA;
see below) were promising, leading us to design and synthesize a
7443
dx.doi.org/10.1021/jm200981s |J. Med. Chem. 2011, 54, 7441–7452

Journal of Medicinal Chemistry
ARTICLE
library of 35 triazines (Table 2, Figure S2). Preparation of triazine
derivatives using the readily available 2,4,6-trichlorotriazine as
starting material is well documented.24,25 We used the synthetic
sequence shown in Scheme 1 and various optimized workup
protocols (see Experimental Section) to efficiently obtain the
desired compounds.
Thus, for the first step the selected amine was used in excess
(4 equiv) to facilitate substitution and to further elimination of
the acid that is released via formation of the corresponding
amine hydrochloride. In most cases, the desired disubstituted
products 7 were separated from their corresponding monosubsti-
tuted derivatives by exploiting the different solubility of the two
species in alcoholic solvents. Introduction of the third substituent
required high temperature, for which microwave activation proved
invaluable. The reagent amines, already converted into their corre-
sponding hydrochlorides, were eliminated by simply washing the
crude reaction mixture with water. The workup procedures devel-
oped for the library synthesis obviated use of chromatography for
most of the products, enabling facile preparation of selected triazines
(e.g., 8aA) on gram scale.
Biological Evaluation. The inhibitory activity of each triazine
was evaluated by voltage-clamp against rat TRPV1 channels
heterologously expressed in amphibian oocytes.26 As illustrated
in Figure 4, instillation of capsaicin onto oocytes expressing
TRPV1 channels held at ꢀ60 mV generated a large inward
current that was rapidly blocked in a dose dependent-manner by
application of a single triazine compound. Note the different
blocking efficacies of the three triazines displayed (8cA, 8bA, and
8aA), of which 8aA was the most potent (at 10 μM it completely
Table 1. Structure of Peptoids 5aꢀf and Piperazine-2,5-
diones 6aꢀf and Their Corresponding Activities against
TRPV1 (Expressed as % Blocking at 10 μM)a
compd R1 R2 R3 % block compd R1 R2 R3 % block
5a
5b
5c
5d
5e
5f
e
e
e
b
e
e
e
e
e
b
e
e
B
Q
A
A
E
99
99
55
45
34
32
6a
6b
6c
6d
6e
6f
e
e
e
b
c
e
e
K
B
e
30
40
3
e
Q
b
K
e
55
28
3
B
B
C
e
a The substituents R1ꢀR3 refer to the groups shown in Figure 3.
Scheme 1. General Synthesis of the Trisubstituted Triazines 8a
a Reaction conditions: (a) R1 = arylalkyl (Figure 3), microwave irradiation at 70 ꢀC (max potency 90 W), 10 min, THF; (b) R2 = polar or apolar alkyl
(Figure 3), microwave irradiation at 100 ꢀC (max potency 110 W), 20 min, THF.
Table 2. Structures of Triazines 8 and Their Determined TRPV1 Blocking Activitya
compd
X1-R1
X2-R2
IC50 (μM) b
compd
X1-R1
X2-R2
IC50 (μM) b
8aA
8aC
8aF
8aG
8aH
8aI
NH-a
NH-a
NH-a
NH-a
NH-a
NH-a
NH-a
NH-a
NH-a
NH-a
NH-b
NH-b
NH-b
NH-b
NH-b
NH-b
NH-c
NH-d
NH-A
NH-C
NH-F
NH-G
NH-H
O-I
0.05 ( 0.007
8eA
8fA
8gA
8hA
8iA
NH-e
NH-f
NH-g
NH-h
NH-i
NH-j
NH-k
NH-l
NH-m
NH-n
NH-o
NH-p
NH-q
N-r
NH-A
NH-A
NH-A
NH-A
NH-A
NH-A
NH-A
NH-A
NH-A
NH-A
NH-A
NH-A
NH-A
NH-A
NH-A
NH-A
NH-E
1.21 ( 0.2
1.63 ( 0.37
0.45 ( 0.07
0.30 ( 0.04
2.33 ( 1.23
218 ( 104
2.62 ( 0.94
3.06 ( 0.22
0.84 ( 0.18
3.30 ( 0.61
0.66 ( 0.11
4.98 ( 0.67
69.1 ( 29.6
56.4 ( 14
1488 ( 774c
0.1 ( 0.04
2173 ( 1745c
5761 ( 6716c
6.14 ( 0.7
8jA
8aJ
NH-J
NH-K
N-O
7.05 ( 2.99
0.55 ( 0.06
70.9 ( 9.1
8kA
8lA
8aK
8aO
8aP
8bA
8bB
8bD
8bL
8bM
8bN
8cA
8dA
8mA
8nA
8oA
8pA
8qA
8rA
8sA
8tA
8tE
N-P
18.6 ( 6
NH-A
NH-B
NH-D
N-L
0.50 ( 0.15
1.2 ( 0.03
6.7 ( 2.4
1.4 ꢁ 107 ( 5.8 ꢁ 104 c
6.4 ( 2.4
NH-M
N-N
N-s
7.0 ( 2.9
0.96 ( 0.21
0.67 ( 0.06
0.97 ( 0.19
NH-t
NH-t
178 ( 47
NH-A
229 ( 23
NH-A
a Substitutions in X1-R1 and X2-R2 refer to the groups shown in Figure 3 (see also Figure S2). b Values derived from the corresponding dose response
curves. Responses were recorded at ꢀ60 mV and normalized with respect to that elicited by 10 μM capsaicin. Data are shown as the mean ( SD, with n
g 6 . c Extrapolated IC50 values.
7444
dx.doi.org/10.1021/jm200981s |J. Med. Chem. 2011, 54, 7441–7452

Journal of Medicinal Chemistry
ARTICLE
a noteworthy IC50 value of 50 ( 10 nM (nH = 0.9 ( 0.1), and most
of the triazines exhibited inhibitory micromolar IC50.
Blocking Mechanism of the Triazine 8aA. The above results
pointed to the triazine 8aA (Figure 6) as the most promising
antagonist, as it exhibited high blocking potency without sig-
nificant agonistic effects. Therefore, we next investigated the
blocking mechanism of 8aA.
Voltage dependency of inhibitory activity is a hallmark of open-
channel blockers that sense the membrane electric field, exerting
their activity within a defined range of voltages. The blocking
efficacy of positively charged channel blockers is clearly stronger at
negative membrane potentials than at positive ones (V g 0 mV).
AsdepictedinFigure6a,8aAblocksTRPV1atnegativemembrane
potentials, yet it is nearly inactive at depolarized membrane
voltages, indicating that its activity is strongly voltage-dependent.
Indeed, plotting the blocking ratio against the voltage provides a
curve that could be approximated by a Woodhull model that yields
an electric distance δ = 0.36 for the binding site within the
membrane electric field (Figure 6b). This value is consistent with
the interaction sitebeing located relatively deep within the aqueous
pore of the channel and with an uncompetitive mechanism of
channel blocking. That 8aA acts as an open-channel blocker was
further corroborated by the finding that the EC50 of capsaicin was
not altered by the presence of 8aA, indicating that this compound
is not a competitive antagonist of capsaicin (data not shown).
Regarding channel selectivity, the IC50 values found for the
triazine 8aA when screened against two related ion channels
(TRPM8 and NMDAR [NR1:NR2A]) were 7.5 ( 1.5 and 9.2
( 2.1 μM, respectively (compared to 0.05 ( 0.007 μM, against
TRPV1; see Table 2). The blocking percentages at 1 μM and at
10 μM 8aA were 37% and 55%, respectively, against TRPM8 and
were 25% and 51%, respectively, against NMDAR. Together,
these data indicate that 8aA exhibits more than 150-fold blocking
activity against TRPV1 over other ion channels that have similar
permeability.
StructureꢀActivity Relationship (SAR) Analysis. We ex-
tracted qualitative SAR information by analyzing the aforemen-
tioned results. First, and in agreement with the postulated
pharmacophore, replacing the tertiary amine with a polar non-
ionizable group or an apolar alkyl moiety either in the peptoids
(compare 5c with 5e or 5f) or the triazines (compare 8aA with
8aC, 8aG, or 8aH) caused a loss in activity (up to 40000-fold). In
contrast, changes in the length of the alkyl chain that supports the
tertiary amine in the triazine series had a relatively minor effect
(compare 8aA with 8aF or compare 8bA with 8bD). Similarly,
changing the tertiary amine group (compare 8bA with 8bB) or
replacing it with a guanidyl group (compare 8aF with 8aK) caused
only a minor decrease in activity. Regarding the atom that links the
dimethylaminopropyl moiety to the triazine, replacing nitrogen
with oxygen (compare 8aA with 8aI) resulted in a substantial
decrease in activity (∼120-fold), suggesting that the nitrogen atom
is importantfor binding. Introducing an oxygen atom into the chain
that supports the trimethylamino group (compare 8aF with 8aJ)
caused a similar decrease in activity. Conformational restriction of
the flexibility of the alkyl chain that contains the ionizable group,
induced by introducing a cycloalkyl linker, also rendered a decrease
in activity (compare 8aF with 8aP or 8bA with 8bN), which was
greater (8aO) or much greater (8bL) for the most restricted
analogues, thereby suggesting that steric interactions could be
determinant for the activity.
Figure 4. Representative ionic currents showing the blocking of capsai-
cin-evoked responses (aꢀc) and triazine-induced TRPV1 activity (dꢀf)
of compounds 8cA (a, d), 8bA (b, e), and 8aA (c, f) at the different
concentrations (μM) indicated. Ionic currents were elicited from oocytes
heterologously expressing TRPV1. The holding potential was ꢀ60 mV.
The concentration of capsaicin (Cap) was 10 μM. Ionic currents were
measured in Mg2+ Ringer buffer. The horizontal bars indicate the
experimental paradigm used for agonist stimulation and channel blocking.
abrogated the capsaicin response). The blocking was reversible,
as evidenced by the recovery of capsaicin currents after the
triazines were washed away.
The blocking activity of each triazine in the library is summar-
ized in Figure 5a, which shows that 15 compounds blocked more
than 75% of the channel activity. Because the triazines 8 can be
protonated at physiological pH (see Figure S1 for calculated pKa)
and known charged blockers have previously displayed agonistic
activity, we also sought to determine whether these compounds
actually activate the TRPV1 channel. We observed that some of
the triazines exhibited significant agonist activities, whereas
others, such as the triazine 8aA, did not (Figure 5b). Complete
screening of the library revealed that only a few of the triazines
are “pure” blockers.
To further substantiate the TRPV1-blocking activity of the
triazines 8, we obtained the corresponding dose response curves.
Figure 5c shows representative results for three triazines (8aA, 8bA,
and 8aP), and Table 2 summarizes the IC50 values for each triazine
from the library. The triazine 8aA showed the highest potency, with
Regarding the arylalkyl moieties, the >300-fold decrease in
activity of 8jA relative to 8cA indicated the importance of the
7445
dx.doi.org/10.1021/jm200981s |J. Med. Chem. 2011, 54, 7441–7452

Journal of Medicinal Chemistry
ARTICLE
Figure 5. Extent of TRPV1 blocking and activation by the triazines 8. (a) Blocked response of capsaicin-evoked current at 10 μM triazine and (b) ionic
currents activated at 10 μM triazine in oocytes heterologously expressing TRPV1. (c) Representative doseꢀresponse curves displaying the blocking of
capsaicin-evoked currents at different concentrations of the triazines 8aA, 8bA, and 8aP. The solid lines depict the MichaelisꢀMenten binding isotherms
that fit the blocking activity.
aromatic groups. Substituting the aromatic moiety and/or
modifying the length of the alkyl linker seemed less important:
compounds 8bA, 8cA, 8dA, 8eA, 8fA, 8gA, 8hA, 8kA, 8lA,
8mA, 8nA, and 8oA showed activities that are within 1 order of
magnitude of each other The exceptions to these trends were
the compounds featuring a carboxylic acid (8tA) or a carboxylic
ester (8qA), which showed a g100-fold decrease in activity
relative to their unsubstituted counterpart (8cA). The com-
pounds containing either larger bicyclic aromatic systems (8iA
or 8pA) or rigid fused tricyclic systems (8sA), and even the
bulkiest compound, the quite rigid bis-aromatic substituted
triazine 8rA, did not show such a large loss in activity, which
suggests that these moieties might occupy sites on the TRPV1
receptor that are relatively open and sterically unrestricted.
Regardless, defining the role of the aromatic substituents
and of the triazine ring in binding is not trivial. This could be
expected, since most of the compounds contain the triazine
core and two aromatic rings, which, as previously stated,
show little influence on activity when their substituents are
modified. However, the low activity of 8jA, the only compound
lacking aromatic substituents, suggests that these aromatic
groups must be involved in binding. These results also support
the premise that high TRPV1-blocking activity requires a
positively charged group.
results were evaluated for SAR data. The triazines act as uncom-
petitive TRPV1 antagonists. Several compounds with activities in
the submicromolar range were identified. The triazine 8aA was the
most potent compound, exhibiting an IC50 of 50 nM. Moreover,
TRPV1 blocking by 8aA is strongly voltage dependent, which is
characteristic of open-channel blockers. Furthermore, the fast
blocking kinetics observed suggest that 8aA acts from the
extracellular side of the channel.
The triazine 8aA is among the strongest TRPV1 open channel
blockers described to date.27,28 Unlike competitive antagonists,
uncompetitive inhibitors only interact with the open channel by
recognizing a binding site located deep within the pore pathway.
This activity-dependent blocking enables preferential abrogation
of pathologically sensitized channels and implies only marginal
interaction with physiologically working receptors. Conse-
quently, uncompetitive antagonists exhibit fewer side effects
than their competitive counterparts.
Owing to its high efficacy and selectivity and to its blocking
mechanism, the triazine 8aA represents a novel class of synthetic
TRPV1 modulators and may provide a basis on which to develop
uncompetitive antagonists for other thermoTRPs (e.g., TRPA1
and TRPM8) implicated in human diseases.
’ EXPERIMENTAL SECTION
Chemistry. All solvents were obtained from VWR (Barcelona,
Spain). HPLC analyses were run on a Hewlett-Packard series 1100
(UV detector 1315A) modular system using an Xterra MS RP18
(Waters) column (5 μm, 4.6 mm ꢁ 150 mm), with CH3CNꢀH2O
mixtures containing 0.1% TFA at 1 mL/min as mobile phase and with
’ CONCLUSION
A small library of 2,4,6-trisubstitued-1,3,5-triazines designed to
satisfy the requirements of a simple pharmacophore model for
TRPV1 blocking was synthesized and screened in vitro, and the
7446
dx.doi.org/10.1021/jm200981s |J. Med. Chem. 2011, 54, 7441–7452

Journal of Medicinal Chemistry
ARTICLE
Most compounds used as diversity sources (9, Figure 3) were commer-
cially available from Sigma-Aldrich, except amine 9F, which was from Matrix
Scientific (Columbia, SC, U.S.). Amine 9J was synthesized from 5-dimethy-
lamino-3-oxapentanol in 25% overall yield using the Mitsunobu approach.29
Amine 9K was only a formal source of diversity, since technically, N-Fmoc-
1,4-butanediamine was used to prepare the corresponding triazine or
piperazinedione, which, after deprotection of the Fmoc group on the
primary amino group, was guanidinylated using the protocol described by
Feichtinger et al.30 (see Supporting Information).
N-Alkyglycine Trimers 5. These compounds were synthesized
following the general procedure reported by our laboratory for
N-alkylglycine oligomers.31
[N-(30-(N0,N0-Dimethylamino)propyl)glycyl]-[N-(20,40-dichloro-
phenethyl)glycyl]-N-(20,40-dichlorophenethyl)glycinamide
1
(5c). Yield: 100 mg, 52%, white powder. H NMR (CD3CN, 40 ꢀC)
(confs): 7.36ꢀ7.12 (m, 8H), 4.15ꢀ3.77 (m, 6H), 3.57ꢀ2.97 (m, 10H),
2.86ꢀ2.63 (m, 8H), 1.27ꢀ1.145 (m, 2H). 13C NMR (CD3CN, 40 ꢀC)
(confs): 169.43ꢀ169.09, 167.49ꢀ167.16, 139.17ꢀ138.94, 138.53ꢀ138.22,
131.95ꢀ131.61, 129.75ꢀ129.19, 55.29, 50.81ꢀ49.82, 48.80ꢀ48.48,
45.77, 43.58, 34.50ꢀ33.51, 22.25ꢀ21.83. HRMS calcd for C27H35Cl4N5O
(M + H), 618.1589; found, 618.1572.
[N-(30-(N0,N0-Dimethylamino)propyl)glycyl]-[N-(40-chloro-
phenethyl)glycyl]-N-(40-chlorophenethyl)glycinamide (5d).
Yield: 82 mg, 45%, white powder. 1H NMR (CD3CN, 40 ꢀC) (confs):
7.36ꢀ7.16 (m, 8H), 4.12ꢀ3.68 (m, 6H), 3.57ꢀ3.29 (m, 4H), 3.18ꢀ2.99
(m, 6H), 2.87ꢀ2.64 (m, 8H), 1.27ꢀ1.15 (m, 2H). 13C NMR (CD3CN,
40 ꢀC) (confs): 169.39ꢀ169.23, 168.91ꢀ168.82, 167.44ꢀ167.01,
136.70ꢀ136.42, 135.78ꢀ135.45, 133.62ꢀ133.23, 130.17ꢀ129.81,
128.66ꢀ128.22, 55.30, 50.71ꢀ50.30, 49.07ꢀ48.02, 45.74ꢀ45.20,
43.61, 32.67ꢀ32.58, 32.26ꢀ32.07, 31.57ꢀ31.37, 22.59ꢀ21.76, 20.84ꢀ19.97.
HRMS calcd for C27H37Cl2N5O3 (M + H), 550.2374; found, 550.2352.
[N-Butylglycyl]-[N-(20,40-dichlorophenethyl)glycyl]-N-(20,40-
dichlorophenethyl)glycinamide (5e). Yield: 72 mg, 37%. 1H NMR
(CD3CN) (confs): 7.49ꢀ7.20 (m, 6H), 4.15ꢀ3.36 (m, 10H), 3.06ꢀ2.79
(m, 6H), 1.67ꢀ1.55 (m, 2H), 1.40ꢀ1.14(m, 2H), 0.95ꢀ0.85 (s, 3H). 13C
NMR (CD3CN) (confs): 173.02ꢀ172.35, 169.72ꢀ169.20, 168.15ꢀ167.80,
137.02ꢀ135.87, 134.35ꢀ133.68, 130.48ꢀ130.12, 128.95ꢀ128.54,
50.25ꢀ48.62, 33.04ꢀ31.70, 28.81, 20.72, 14.14. HRMS calcd for
C26H32Cl4N4O3 (M + H), 589,1309; found, 589.1311.
Figure 6. Voltage dependency of TRPV1 blocking by the triazine 8aA
(a). Representative ionic currents were evoked by 10 μM capsaicin using
a linear ramp from ꢀ60 to +60 mV in the absence (red line) or presence
(black line) of 8aA at 10 μM. (b) Fractional blocking of TRPV1 by 8aA
as a function of the voltage. The solid line depicts the fitting to the
Woodhull model, which for the 8aA binding site yields an electric
distance (δ) value of 0.36 within the membrane electric field.
[N-(30-Hydroxypropyl)glycyl]-[N-(20,40-dichlorophenethyl)-
glycyl]-N-(20,40-dichlorophenethyl)glycinamide (5f). Yield:
monitoring at 220 nm. Compounds were purified from the crude
reaction mixture by semipreparative HPLC using an X-terra RP18
(Waters) column (15ꢀ20 μm, 47 mm ꢁ 300 mm), CH3CNꢀH2O
mixtures containing 0.1% TFA as mobile phases at a flow rate of 10 mL/
min. Unless otherwise stated, NMR spectra were recorded in CDCl3 on
a Varian Inova 400 apparatus (1H NMR, 400 MHz; 13C NMR, 100
MHz). Chemical shifts are given in ppm (δ) relative to the CDCl3 signal
(7.24 ppm for 1H NMR and 77.23 ppm for 13C NMR), and coupling
constants (J) are reported in hertz (Hz). To improve solubility, some
spectra were recorded above ambient temperature (e48 ꢀC) or in the
presence of ∼7% trifluoroacetic acid (TFA; broad signal at ∼10.5
ppm in 1H NMR and two quadruplets at 160.00 [J = 39] and 116.72
ppm [J = 290] in 13C NMR). Some of the described compounds exhibit
unexpectedly complex NMR spectra because of the presence of different
conformers in equilibrium in solution. Thus, in some cases, the spectra of
these compounds are given as absorption ranges rather than discrete
signals and are denoted with the abbreviation “confs”. High resolution
mass spectra (HRMS) were recorded at the Mass Spectrometry Service
of IQAC-CSIC (Barcelona, Spain) with an Aquity UPLC (Waters) chro-
matograph coupled to a LCT Premier Xe TOF detector (Waters), using an
Aquity UPLC BEH C18 (Waters) column (1.7 μm, 2.1 mm ꢁ 100 mm),
with CH3CNꢀH2O mixtures containing 20 mM HCOOH as mobile
phases. The purity of compounds 8 was checked by reverse phase HPLC
and confirmed to be g95%.
1
55 mg, 28%, white powder. H NMR (CD3CN) (confs): 7.50ꢀ7.20
(m, 6H), 4.14ꢀ3.82 (m, 4H), 3.67ꢀ3.56 (m, 3H), 3.13ꢀ2.83 (m, 4H),
1.87ꢀ1.83 (m, 8H), 1.28ꢀ1.24 (s, 2H). 13C NMR (CD3CN) (confs):
172.50ꢀ171.63, 169.69ꢀ169.18, 168.04ꢀ167.45, 137.16ꢀ135.95,
134.43ꢀ133.76, 130.56ꢀ130.26, 129.04ꢀ128.60, 61.54, 51.07ꢀ48.92,
33.08ꢀ32.91, 32.60- 32.45, 31.95ꢀ31.79, 30.75ꢀ30.18, 29.10. HRMS
calcd for C25H30Cl4N4O4 (M + H), 591.1107; found, 591.1099.
Piperazine-2,5-diones 6. These compounds were synthesized
following a procedure developed in our laboratory.18
3-[N-Aminocarbonylmethyl-N-(40-guanidylbutyl)amino-
carbonylmethyl]-1,4-bis(20,40-dichlorophenethyl)piperazine-
2,5-dione (6a). Yield: 10 mg, 6%. 1H NMR (DMSO-d6, 40 ꢀC)
(confs): 7.48ꢀ7.45 (m, 2H), 7.39ꢀ7.27 (m, 4H), 4.30ꢀ4.25 (m, 2H),
4.20ꢀ3.90 (m, 4H), 3.85ꢀ3.80 (m, 2H), 3.74ꢀ3.44 (m, 3H), 3.30ꢀ2.95
(m, 10 H), 1.62 (m, 2H), 1.54 (m, 2H). 13C NMR (DMSO-d6, 40 ꢀC)
(confs): 173.6, 173.0, 172.1, 171.3, 168.4, 168.2, 167.0, 166.9, 156.6,
156.3, 136.5, 136.0, 134.4, 133.5, 133.4, 130.3, 128.7, 128.6, 58.8, 58.5,
51.6, 51.4, 51.2, 50.1, 49.5, 48.8, 48.1, 47.2, 45.2, 45.0, 42.3, 42.2, 35.0,
34.7, 31.3, 31.2, 30.9, 29.8, 27.1, 27.0, 26.8, 25.7. HRMS calcd for
C29H36Cl4N7O3 (M + H), 686.1583; found, 686.1592.
3-[N-Aminocarbonylmethyl-N-(20-(100-methyl-200-pyrroli-
dinyl)ethyl)aminocarbonylmethyl]-1,4-bis(20,40-dichloro-
phenethyl)piperazine-2,5-dione (6b). Yield: 27 mg, 24%. 1H NMR
7447
dx.doi.org/10.1021/jm200981s |J. Med. Chem. 2011, 54, 7441–7452

Journal of Medicinal Chemistry
ARTICLE
(DMSO-d6, 40 ꢀC) (confs): 7.51ꢀ7.49 (m, 2H), 7.40ꢀ7.27 (m, 4H),
4.12ꢀ3.60 (m, 9H), 3.50ꢀ3.30 (m, 3H), 3.19ꢀ3.02 (m, 2H), 2.98ꢀ2.70
(m, 9H), 2.30ꢀ2.10 (m, 2H), 1.98ꢀ1.79 (m, 2H), 1.70ꢀ1.50 (m, 2H).
13C NMR (DMSO-d6, 40 ꢀC) (confs): 170.6ꢀ169.3, 166.2ꢀ166.0,
164.0ꢀ164.3, 135.6ꢀ135.5, 134.2, 132.6ꢀ132.4, 132.0, 128.7, 127.5,
66.7ꢀ65.5, 66.1ꢀ66.0, 56.9ꢀ55.4, 50.4ꢀ48.5, 46.0ꢀ45.2, 44.2ꢀ41.1,
40.0ꢀ38.0, 34.8ꢀ33.7, 29.8ꢀ28.3, 21.4ꢀ21.1. HRMS calcd for
C31H39Cl4N5O4 (M + H), 684.1678; found, 684.1689.
absolute ethanol and dried to give the expected disubstituted triazine 7.
For analytical data on compounds 7, see Supporting Information.
Then a suspension of the disubstituted triazine 7 (0.5 mmol, 1 equiv)
in THF (5 mL) was allowed to react with the corresponding amine
(2 mmol, 4 equiv) for 20 min at 100 ꢀC under microwave activation
(110 W, closed system). The crude reaction mixture was diluted in
EtOAc (20 mL) and washed with H2O (20 mL), brine (20 mL) and
dried over MgSO4. Elimination of solvents yielded the expected
trisubstituted triazine 8. If higher purity than that obtained by this
procedure was required, the residue obtained after elimination of solvent
was purified by semipreparative HPLC using mixtures of CH3CN and
H2O containing 0.1% CF3COOH. The collected fractions were evapo-
rated under vacuum, redissolved in EtOAc, washed with saturated
solution aqueous NaHCO3 (20 mL) and brine (20 mL), and finally
dried over MgSO4. Elimination of solvent afforded the pure desired
trisubstituted triazine 8. Syntheses of triazines 8aF, 8aI, 8aK, 8tA, 8tE
are described in detail in the Supporting Information.
2,4-Bis(40-fluorophenethylamino)-6-(30-(N,N-dimethylamino)-
propylamino)-1,3,5-triazine (8aA). Yield: 2.28 g, 73%, colorless
oil. 1H NMR (CDCl3, 48 ꢀC): 7.11 (dd, J1 = 8, J2 = 5, 4H), 7.00 ꢀ 6.83
(m, 4H), 3.54 (d, J1 = 6, J2 = 13, 4H), 3.43ꢀ3.32 (m, 2H), 2.79 (t, J =
7, 4H), 2.32 (t, J = 7, 2H), 2.19 (s, 6H), 1.79ꢀ1.60 (m, 2H). 13C NMR
(CDCl3, 48 ꢀC): 166.39, 161.75 (d, J = 244), 135.30 (d, J = 3), 130.28
(d, J = 8,), 115.37 (d, J = 21.2), 57.91, 45.60, 42.24, 39.64, 35.53, 27.71.
HRMS calcd for C24H31N7F2 (M + H), 456.2687; found, 456.2668.
2,4-Bis(40-fluorophenethylamino)-6-(30-hydroxypropyl-
amino)-1,3,5-triazine (8aC). Yield: 100 mg, 91%, colorless oil. 1H
NMR (CDCl3, 48 ꢀC): 7.14 (dd, J1 = 8, J2 = 6, 4H), 6.96 (t, J = 9, 4H),
3.70ꢀ3.35 (m, 5H), 2.82 (t, J = 7, 4H), 1.68 (s, 2H). 13C NMR (CDCl3,
48 ꢀC): 166.67, 166.00, 161.87 (d, J = 244.3), 135.05 (d, J = 3.0), 130.33
(d, J = 7.8), 115.51 (d, J = 21.2), 58.63, 42.25, 36.87, 35.47, 33.34. HRMS
calcd for C22H26N6OF2 (M + H), 429.2214; found, 429.2207.
2,4-Bis(40-fluorophenethylamino)-6-(50-(N,N-dimethylamino)-
pentylamino)-1,3,5-triazine (8aF). Yield: 48 mg, 23%, yellow oil.
1H NMR (CDCl3, 48 ꢀC): 7.15ꢀ7.12 (m, 4H), 6.97ꢀ6.96 (m, 4H),
3.60ꢀ3.52 (m, 4H), 3.37ꢀ3.32 (m, 2H), 2.85ꢀ2.79 (m, 4H),
2.28ꢀ2.25 (m, 2H), 2.21 (s, 6H), 1.62ꢀ1.45 (m, 4H), 1.41ꢀ1.32
(m, 2H). 13C NMR (CDCl3, 48 ꢀC): 161.84 (d, J = 244), 135.21 (d, J =
2), 130.35 (d, J = 8), 115.49 (d, J = 21), 59.82, 45.48, 42.26, 35.56, 30.01,
27.41, 24.96. HRMS calcd for C26H35N7F2 (M + H), 484.3000; found,
484.2992.
3-[N-Aminocarbonylmethyl-N-(20,40-dichlorophenethyl)-
aminocarbonylmethyl]-1-[30-(N,N-diethylamino)propyl]-
4-(20,40-dichlorophenethyl)piperazine-2,5-dione (6c). Yield:
1
54 mg, 21%. H NMR (DMSO-d6, 40 ꢀC) (confs): 7.56ꢀ7.30 (m,
6H), 4.17ꢀ4.11 (m, 2H), 3.97ꢀ3.72 (m, 3H), 3.69ꢀ3.31 (m, 6H),
3.23ꢀ2.81 (m, 12H), 1.91 (m, 2H), 1.19 (t, J = 7.1 Hz, 6H). 13C NMR
(DMSO-d6, 40 ꢀC) (confs): 170.8ꢀ169.9, 167.3ꢀ167.1, 164.6, 135.5,
135.4, 135.3, 134.8, 133.9, 133.8, 132.2ꢀ131.6, 128.6ꢀ128.3,
127.3ꢀ127.1, 57.0, 50.9, 50.1, 50.0, 49.5, 49.3, 48.7, 47.3, 47.0, 43.4,
43.0, 34.5, 33.9, 31.9, 30.9, 30.4, 21.6, 21.4, 9.3. HRMS calcd for
C31H40Cl4N5O4 (M + H), 686,1834; found, 686,1849.
3-[N-Aminocarbonylmethyl-N-(40-guanidylbutyl)aminocar-
bonylmethyl]-1,4-bis(40-chlorophenethyl)piperazine-2,5-dione
(6d). Yield: 13 mg, 9%. 1H NMR (DMSO-d6, 40 ꢀC) (confs):
7.34ꢀ7.31 (m, 4H), 7.26ꢀ7.21 (m, 4H), 4.14ꢀ3.73 (m, 6H), 3.48
(m, 2H), 3.29 (m, 1H), 3.23 (m, 1H), 3.16ꢀ3.05 (m, 3H), 2.93ꢀ2.61
(m, 6H), 1.48 (m, 2H), 1.40 (m, 2H). 13C NMR (DMSO-d6, 40 ꢀC)
(confs): 169.9, 169.8, 169.6, 168.9, 165.9, 165.8, 164.0, 163.9, 156.6,
156.5, 137.7, 130.7, 130.4ꢀ130.3, 130.7, 56.5, 56.2, 49.8, 49.7, 49.6,
48.1, 47.9, 46.4, 46.3, 46.1, 44.5, 44.4, 40.4, 40.3, 34.0, 33.8, 31.7, 31.6,
31.2, 28.7, 25.6, 25.5, 24.9, 23.9. HRMS calcd for C29H38Cl2N7O4 (M +
H), 618.2362; found, 618,2367.
3-[N-Aminocarbonylmethyl-N-(20,40-dichlorophenethyl)-
aminocarbonylmethyl]-1-[20-(100-methyl-200-pyrrolidinyl)ethyl]-
4-(phenethyl)piperazine-2,5-dione (6e). Yield: 32 mg, 16%. 1H
NMR (DMSO-d6, 40 ꢀC) (confs): 7.56ꢀ7.37 (m, 2H), 7.30ꢀ7.28
(m, 3H), 7.24ꢀ7.20 (m, 3H), 4.20ꢀ4.03 (m, 2H), 3.99ꢀ3.74
(m, 5H), 3.67ꢀ3.27 (m, 4H), 3.35 (m, 2H), 3.27 (s, 3H),
3.15ꢀ2.79 (m, 6H), 2.72ꢀ2.63 (m, 1H), 2.37ꢀ2.20 (m, 2H),
2.03ꢀ1.81 (m, 2H), 1.67ꢀ1.51 (m, 2H). 13C NMR (DMSO-d6,
40 ꢀC) (confs): 173.0ꢀ169.3, 166.4ꢀ166.2, 163.7, 138.8, 138.7,
135.7, 135.0, 134.0, 132.8, 132.1, 131.7, 128.8, 128.6, 127.5ꢀ127.4,
126.3ꢀ126.2, 69.2, 66.2ꢀ62.6, 56.3ꢀ55.4, 50.5, 48.8, 48.1, 48.0, 46.5,
44.8ꢀ44.5, 41.7ꢀ41.3, 38.6ꢀ38.4, 33.7ꢀ32.4, 31.2ꢀ30.2, 29.0,
28.6ꢀ28.4, 24.4ꢀ22.0. HRMS calcd for C31H41Cl2N5O4 (M + H),
616.2457; found, 616.2445.
3-[N-Aminocarbonylmethyl-N-(20,40-dichlorophenethyl)-
aminocarbonylmethyl]-1-[20-(100-methyl-200-pyrrolidinyl)-
ethyl]-4-[20,40-dichlorophenethyl]piperazine-2,5-dione (6f).
Yield: 45 mg, 19%. 1H NMR (DMSO-d6, 40 ꢀC) (confs): 7.58ꢀ7.45
(m, 3H), 7.40ꢀ7.34 (m, 3H), 4.18ꢀ4.03 (m, 4H), 4.00ꢀ3.60 (m, 12
H), 3.52 (m, 1H), 3.19 (m, 1H), 3.07ꢀ2.93 (m, 7H), 2.82ꢀ2.63 (m,
6H), 2.35ꢀ2.25 (m, 2H), 1.95ꢀ1.86 (m, 2H), 1.68ꢀ1.51 (m, 2H). 13C
NMR (DMSO-d6, 40 ꢀC) (confs): 170.1ꢀ169.0, 166.3ꢀ166.1,
163.9ꢀ163.8, 135.6ꢀ135.0, 134.0ꢀ133.9, 132.8ꢀ131.7, 128.6ꢀ128.5,
127.5ꢀ127.3, 66.0ꢀ65.8, 65.4, 56.2ꢀ55.1, 50.2ꢀ46.4, 42.8ꢀ41.3,
38.7ꢀ38.3, 33.8ꢀ33.0, 31.1ꢀ30.1, 29.6, 29.0, 27.2ꢀ26.5, 21.1ꢀ20.8.
HRMS calcd for: C31H39Cl4N5O4 (M + H), 684.1678; found, 684.1644.
Trisubstituted Triazines 8. General Procedure. These com-
pounds were synthesized following the procedure shown in Scheme 1.
Thus, a solution of 2,4,6-trichlorotriazine (0.5 mmol, 1 equiv) in THF
(4 mL) was allowed to react with the corresponding amine (2 mmol, 4
equiv) under microwave activation for 10 min at 70 ꢀC (90 W closed
system). Then the crude reaction mixture was poured into H2O (20 mL),
heated for 10 min at 60 ꢀC, and filtered. The precipitate was subjected to
the same treatment and the insoluble material was washed with cold
2,4-Bis(40-fluorophenethylamino)-6-propylamino-1,3,5-
triazine (8aG). Yield: 84 mg, 53%, colorless oil. 1H NMR (CDCl3, 7%
TFA): 7.15ꢀ6.97 (m, 8H), 3.51ꢀ3.36 (m, 4H), 3.48ꢀ3.40 (m, 2H),
2.91 (t, J = 7, 4H), 1.66 (m, 2H), 1.00ꢀ0.95 (m, 3H). 13C NMR
(CDCl3, 7% TFA): 162.21 (d, J = 254), 154.47, 152.45, 152.11, 133.08,
130.261, 115.85 (d, J = 21), 44.31, 43.55, 34.29, 22.14, 11.15. HRMS
calcd for C22H26N6F2 (M + H), 413.2265; found, 413.2279.
2,4-Bis(40-fluorophenethylamino)-6-hexylamino-1,3,5-
triazine (8aH). Yield: 85 mg, 73%, colorless oil. 1H NMR (CDCl3, 7%
TFA) (confs): 7.15ꢀ6.97 (m, 8H), 3.76ꢀ3.62 (m, 4H), 3.47ꢀ3.39
(m, 2H), 2.91ꢀ2.88 (m, 4H), 1.64ꢀ1.58 (m, 2H), 1.33ꢀ1.28 (m,
6H), 0.92ꢀ0.84 (bb, 3H). 13C NMR (CDCl3, 7% TFA) (confs): 161.97
(d, J = 244), 154.96, 153.96, 153.46, 153.070, 133.212, 130.07 (d, J = 8),
115.70 (d, J = 21), 42.97, 42.15, 34.34, 31.30, 28.51, 26.40, 22.45, 13.84.
HRMS calcd for C25H32N6F2 (M + H), 455.2734; found, 455.2739.
2,4-Bis(40-fluorophenethylamino)-6-(30-(N,N-dimethyl-
amino)propoxy)-1,3,5-triazine (8aI). Yield: 90 mg, 38%, colorless
1
oil. H NMR (CDCl3, 48 ꢀC): 7.17ꢀ7.09 (m, 4H), 7.00ꢀ6.92 (m,
4H), 4.37ꢀ4.25 (bs, 2H), 3.67ꢀ3.55 (bs, 4H), 2.88ꢀ2.76 (bs, 4H),
2.61ꢀ2.52 (m, 2H), 2.34 s (6H), 2.25ꢀ1.92 (bb, 2H). 13C NMR
(CDCl3, 48 ꢀC): 167.38, 161.87 (d, J = 245), 134.81, 130.34 (d, J = 8),
115.56 (d, J = 21), 60.52, 56.46, 45.11, 42.28, 35.33, 25.43. HRMS calcd
for C24H30N6F2O (M + H), 457.2527; found, 457.2515.
7448
dx.doi.org/10.1021/jm200981s |J. Med. Chem. 2011, 54, 7441–7452

Journal of Medicinal Chemistry
ARTICLE
2,4-Bis(40-fluorophenethylamino)-6-(20-(200-(N,N-dimethyl-
amino)ethoxy)ethylamino)-1,3,5-triazine (8aJ). Yield: 50 mg,
23%, yellow oil. 1H NMR (CDCl3, 7% TFA) (confs): 7.15ꢀ7.09 (m,
4H), 7.00ꢀ6.94 (m, 4H), 3.82ꢀ3.61 (m, 10H), 3.36 (m, 2H), 3.00ꢀ2.82
(m, 10H). 13C NMR (CDCl3, 7% TFA) (confs): 161.95 (d, J = 243),
155.18, 153.89, 133.32, 130.08 (d, J = 8), 115.65 (d, J = 21), 68.82, 63.80,
58.11, 43.88, 42.99, 40.99, 34.33. HRMS calcd for C25H33N7F2O
(M + H), 486.2793; found, 486.2796.
(CDCl3, 7% TFA) (confs): 162.01, 155.28, 136.56, 132.70, 130.10,
128.84, 53.99, 44.08, 42.51, 40.73, 34.94. HRMS calcd for C24H29N7Cl2
(M + H), 486.1940; found, 486.1986.
2,4-Bis(40-chlorophenethylamino)-6-(40-methylpipera-
zin-10-ylamino)-1,3,5-triazine (8bM). Yield: 81 mg, 55%, white
1
powder. H NMR (CDCl3, 7% TFA) (confs): 7.29ꢀ7.04 (m, 8H),
3.77ꢀ3.43 (m, 10H), 3.16ꢀ3.01 (m, 2H), 2.93ꢀ2.82 (m, 7H). 13C
NMR (CDCl3, 7% TFA) (confs): 157.54, 154.29, 135.50, 133.43,
129.87, 129.20, 53.50, 51.60, 43.61, 43.24, 34.26. HRMS calcd
C24H30N8Cl2 (M + H), 501.2049; found, 501.2058.
2,4-Bis(40-fluorophenethylamino)-6-(40-guanidylbutyl-
1
amino)-1,3,5-triazine (8aK). Yield: 60 mg, 23%, colorless oil. H
2,4-Bis(40-chlorophenethylamino)-6-(40-(200-aminoethyl)-
piperazin-10-yl)-1,3,5-triazine (8bN). Yield: 116 mg, 64%, white
powder. 1H NMR (CDCl3, 48 ꢀC): 7.52ꢀ7.50 (m, 4H), 7.38ꢀ7.24 (m,
4H), 5.18ꢀ5.04 (bs, 2H), 4.10ꢀ3.95 (bsb, 4H), 3.91ꢀ3.74 (m, 4H),
3.19ꢀ3.04 (m, 6H), 2.80ꢀ2ꢀ66 (m, 6H). 13C NMR (CDCl3, 48 ꢀC):
166.43, 165.27, 138.10, 132.30, 130.24, 128.77, 77.48, 77.16, 76.84,
53.37, 43.28, 42.05, 38.87, 35.71, 30.54. HRMS calcd for C25H32N8Cl2
(M + H), 515.2205; found, 515.2204.
2,4-Bis(phenethylamino)-6-(30-(N,N-dimethylamino)pro-
pylamino)-1,3,5-triazine (8cA). Yield: 105 mg, 74%, colorless oil.
1H NMR (CDCl3, 48 ꢀC): 7.28ꢀ7.252 (m, 4), 7.20ꢀ7,17 (m, 6H),
3.61 (dd, J1 = 13, J2 = 6, 4H), 3.4ꢀ3.40 (m, 2H), 2.86 (t, J = 7, 4H),
2.41 (t, J = 7, 2H), 2.27 (s, 6H), 1.72ꢀ1.79 (m, 2H). 13C NMR (CDCl3,
48 ꢀC): 166.20, 139.65, 129.00, 128.74, 126.50, 57.86, 45.43, 42.28,
39.61, 36.40, 27.51. HRMS calcd for C24H33N7 (M + H), 432.1703;
found, 432.1703.
NMR (CDCl3, 7% TFA) (confs): 7.17ꢀ7.09 (m, 4H, 7.03ꢀ6.95 (m,
4H), 3.74ꢀ3.60 (m, 4H), 3.54ꢀ3.15 (m, 4H), 2.94ꢀ2.83 (m, 4H),
1.76ꢀ1.59 (m, 4H). 13C NMR (CD3CN, 7% TFA) (confs): 162.84 (d,
J = 240), 158.30, 156.54, 136.23, 131.75 (d, J = 8), 116.20 (d, J = 21),
43.30, 42.47, 41.27, 35.376, 30,16, 26.83, 26.79. HRMS calcd for
C24H31N9F2 (M + H), 484.2749; found, 484.2729.
2,4-Bis(40-fluorophenethylamino)-6-(40-(100-methylpiperi-
din-400-yl)piperazin-10-yl)-1,3,5-triazine (8aO). Yield: 250 mg,
95%, yellow oil. 1H NMR (CDCl3, 48 ꢀC): 7.17ꢀ7.14 (m, 4H),
6.99ꢀ6.96 (m, 4H), 3.82ꢀ3.77 (m, 4H), 3.62ꢀ3.56 (m, 4H),
3.36ꢀ3.29 (m, 2H), 2.85 (t, J = 14, 4H), 2.65ꢀ2.45 (m, 10 H),
2.06ꢀ1.94 (m, 4H). 13C NMR (CDCl3, 48 ꢀC): 161.87 (d, J = 244 Hz),
134.70, 130.32 (d, J = 8 Hz), 115.52 (d, J = 21 Hz), 59.25, 54.06, 49.12,
44.35, 43.86, 42.33, 35.16, 26.22. HRMS calcd for C29H38N8F2 (M + H),
537.3265; found, 537.3209.
2,4-Bis(40-fluorophenethylamino)-6-(40-(200-(N,N-dimeth-
ylamino)ethyl)piperazin-10-yl)-1,3,5-triazine (8aP). Yield: 192
2,4-Bis(40-methoxyphenethylamino)-6-(30-(N,N-dimethyl-
amino)propylamino)-1,3,5-triazine (8dA). Yield: 87 mg, 57%,
colorless oil. 1H NMR (CDCl3, 7% TFA) (confs): 7.12ꢀ7.04 (m, 4H),
6.89ꢀ6.83 (m, 4H), 3.82 (s, 6H), 3.76ꢀ3.65 (m, 4H), 3.62ꢀ3.52 (m,
2H), 3.25ꢀ3.18 (m, 2H), 2.94ꢀ2.80 (m, 10H), 2.16ꢀ2.07 (m, 2H).
13C NMR (CDCl3, 7% TFA) (confs): 158.35, 154.55, 130.07, 129.66,
114.44, 56.16, 55.94, 43.59, 42.94, 37.64, 34.25, 24.18. HRMS calcd for
C26H37N7O2 (M + H), 480.3087; found, 480.3090.
1
mg, 73%, yellow oil. H NMR (CDCl3, 48 ꢀC): 7.15ꢀ7.12 (m, 4H),
6.97ꢀ6.94 (m, 4H), 3.79ꢀ3.72 (bs, 4H), 3.60ꢀ3.53 (m, 4H),
2.85ꢀ2.79 (m, 4H), 2.53ꢀ2.43 (m, 8H), 2.27 (s, 6H). 13C NMR
(CDCl3, 48 ꢀC): 166.15, 164.99, 161.62 (d, J = 244), 135.12 (d, J = 3),
130.25 (d, J = 8), 115.38 (d, J = 21), 56.88, 56.83, 53.67, 45.98, 42.96,
42.20, 35.38. HRMS calcd for C27H36N8F2 (M + H), 511.3109; found,
511.3092.
2,4-Bis(40-chlorophenethylamino)-6-(30-(N,N-dimethyl-
amino)propylamino)-1,3,5-triazine (8bA). Yield: 64 mg, 71%,
white powder. 1H NMR (CD3CN3, 7% TFA) (confs): 7.33ꢀ7.20
(m, 8H), 3.65ꢀ3.52 (m, 4H), 3.48ꢀ3.38 (m, 2H), 3.12ꢀ3.04
(m, 2H), 2.89ꢀ2.83 (m, 4H), 2.79ꢀ2.75 (m, 6H), 1.98ꢀ1.90
(m, 2H). 13C NMR (CDCl3, 7% TFA) (confs): 163.32,
155.57ꢀ154.78, 136.50, 132.71, 129.97, 128.852, 55.93, 43.57, 42.34,
37.39, 34.63, 24.33. HRMS calcd for C24H31N7Cl2 (M + H), 488.2096;
found, 488.2103.
2,4-Bis(40-chlorophenethylamino)-6-(20-(100-methylpyroli-
din-200-yl)ethylamino)-1,3,5-triazine (8bB). Yield: 70 mg, 60%,
colorless oil. 1H NMR (CDCl3, 7% TFA) (confs): 7.30ꢀ7.10 (m, 8H),
3.91ꢀ3.08 (m, 8H), 2.97ꢀ2.70 (8H), 2.36ꢀ1.76 (m, 6H). 13C NMR
(CDCl3, 7% TFA) (confs): 162.35, 155.43, 154.80, 136.30, 132.86,
129.94, 128.93, 68.23, 56.78, 42.50, 40.66, 38.00, 34.59, 29.60, 29.32,
21.55. HRMS calcd for C26H33N7Cl2 (M + H), 514.2253; found,
514.2230.
2,4-Bis(20,40-dichlorophenethylamino)-6-(30-(N,N-dimeth-
ylamino)propylamino)-1,3,5-triazine (8eA). Yield: 100 mg,
88%, yellow oil. 1H NMR (CDCl3, 7% TFA) (confs): 7.39ꢀ7.36 (m,
2H), 7.20ꢀ7.12 (m, 4H), 3.73ꢀ3.60 (m, 4H), 3.57ꢀ3.48 (m, 2H),
3.22ꢀ3.14 (m, 2H), 3.02ꢀ2.96 (m, 4H), 2.9 (s, 6H), 2.11ꢀ2.05 (m,
2H). 13C NMR (CDCl3, 7% TFA) (confs): 162.78, 155.60, 154.74,
134.77, 134.30, 133.50, 131.62, 129.43, 127.36, 55.73, 43.24, 40.39,
37.36, 32.64, 24.03. HRMS calcd for C24H29N7Cl4 (M + H), 556.1317;
found, 556.1320.
2,4-Bis(40-nitrophenethylamino)-6-(30-(N,N-dimethylamino)-
propylamino)-1,3,5-triazine (8fA). Yield: 224 mg, 97%, yellowish
oil. 1H NMR (CDCl3, 48 ꢀC): 8.10 (d, J = 8.7, 4H), 7.34 (d, J = 8.7, 4H),
3.65ꢀ3.54 (m, 4H), 3.43ꢀ3.35 (m, 2H), 2.96 (t, J = 7, 4H), 2.47 (t, J =
7, 2H), 2.31 (s, 6H), 1.77 (m, 2H). 13C NMR(CDCl3, 48 ꢀC): 166.26,
147.53, 147.03, 129.82, 123.85, 57.59, 45.23, 41.65, 39.41, 36.31, 27.20.
HRMS calcd for C24H31N9O4 (M + H), 510.2577; found, 510.2594.
2,4-Bis(20-fluorophenethylamino)-6-(30-(N,N-dimethyl-
amino)propylamino)-1,3,5-triazine (8gA). Yield: 143 mg, 57%,
white solid. 1H NMR (CDCl3, 48 ꢀC): 7.25ꢀ7.15 (m, 4H), 7.11ꢀ6.99
(m, 4H), 3.76ꢀ3.65 (m, 4H), 3.63ꢀ3.50 (m, 2H), 3.28ꢀ3.18 (m,
2H), 2.97ꢀ2.92 (m, 10H), 2.16ꢀ2.07 (m, 2H). 13C NMR (CDCl3,
48 ꢀC): 163.87, 161.26 (d, J = 243), 155.89, 155.06, 153.91, 131.05,
128.85, 124.54, 124.33, 115.38 (d, J =22), 56.00, 43.58, 41.48, 37.76,
28.51, 24.11. HRMS calcd for C24H31N7F2 (M + H), 456.2687; found,
456.2701.
2,4-Bis(40-chlorophenethylamino)-6-(20-(N,N-dimethyl-
amino)ethylamino)-1,3,5-triazine (8bD). Yield: 102 mg, 71%,
white powder. 1H NMR (CDCl3, 7% TFA) (confs): 7.30ꢀ7.21
(m, 4H), 7.14ꢀ7.04 (m, 4H), 3.80ꢀ3.93 (m, 2H), 3.67ꢀ3.54 (m,
4H), 3.45ꢀ3.34 (m, 2H), 3.05ꢀ2.94 (m, 6H), 2.88ꢀ2.79 (m, 4H). 13C
NMR (CDCl3, 7% TFA): 154.86, 153.64, 135.96, 132.80, 129.89,
128.90, 57.02, 44.15, 42.59, 35.88, 34.42. HRMS calcd for C23H29N7Cl2
(M + H), 474.1940; found, 474.1946.
2,4-Bis(40-chlorophenethylamino)-6-(40-methylpiperazin-
2,4-Bis(30-fluorophenethylamino)-6-(30-(N,N-dimethylamino)-
propylamino)-1,3,5-triazine (8hA). Yield: 133 mg, 53%, white
solid. 1H NMR (CDCl3, 7% TFA) (confs): 7.27ꢀ7.23 (m, 2H),
6.97ꢀ6.88 (m, 6H), 3.70ꢀ3.61 (m, 4H), 3.57ꢀ3.49 (m, 2H),
3.23ꢀ3.12 (m, 2H), 2.92ꢀ2.86 (m, 10H), 2.14ꢀ2.03 (m, 2H).
1
10-yl)-1,3,5-triazine (8bL). Yield: 52 mg, 36%, white powder. H
NMR (CDCl3, 7% TFA) (confs): 7.26ꢀ7.24 (m, 4H), 7.10ꢀ7.08 (m,
4H), 4.74 (d, J =15, 2H), 3.82 (d, J =6, 2H), 3.64 (q, J =6, 4H), 3.37 (t,
J =13, 2H), 3.02 (d, J =3.5, 3H), 2.88ꢀ2.76 (d, J =3.5, 6H). 13C NMR
7449
dx.doi.org/10.1021/jm200981s |J. Med. Chem. 2011, 54, 7441–7452

Journal of Medicinal Chemistry
ARTICLE
13C NMR (CDCl3, 7% TFA) (confs): 163.88, 161.28 (d, J = 236),
155.88, 155.60, 131.00 (d, J = 5), 128.52, 125.00, 124.16, 115.27 (d,
J = 22), 55.60, 43.26, 41.18, 37.11, 28.97, 24.32. HRMS calcd for
C24H31N7F2 (M + H), 456.2687; found, 456.2705.
(m, 4H), 4.48ꢀ4.40 (m, 4H), 3.48ꢀ3.34 (bs, 2H), 2.9ꢀ2.36 (m, 8 H),
1.83ꢀ1.73 (m, 2H). 13C NMR (CDCl3, 48 ꢀC): 165.24, 148.06, 147.00,
133.41, 120.98, 108.48, 101.18, 77.55, 77.23, 76.91, 57.52, 44.89, 44.73,
39.40, 26.72. HRMS calcd for C24H29N7O4 (M + H), 480.2359; found,
480.2365.
2,4-Bis(20-(300-indolyl)ethylamino)-6-(30-(N,N-dimethylamino)-
propylamino)-1,3,5-triazine (8iA). Yield: 20 mg, 34%, brown
2,4-Bis(40-methoxycarbonylbenzylamino)-6-(30-(N,N-dimeth-
ylamino)propylamino)-1,3,5-triazine (8qA). Yield: 97 mg, 42%,
colorless oil. 1H NMR (CDCl3, 48 ꢀC): 7.93 (d, J = 8, 4H), 7.31 (d, J = 6,
4H), 4.57 (d, J = 5, 4H), 3.88 (s, 6H), 3.35 (dd, J1 = 12, J2 = 6, 2H), 2.31
(t, J = 6, 2H), 2.19 (s, 6H), 1.66 (m, 2H). 13C NMR (CDCl3, 48 ꢀC):
167.11 C1, 166.58, 166.53, 145.20, 130.01, 129.24, 127.40, 57.96, 52.20,
45.61, 44.62, 39.81, 27.51. HRMS calcd for C28H33N7O4 (M + H),
508.2630; found, 508.2633.
1
solid. H NMR (CD3CN, 48 ꢀC): 9.03 (s, 2H), 7.62 (d, J = 8, 2H),
7.38 (d, J = 8, 2H), 7.12 (t, J = 7, 2H), 7.06 (s, 2H), 7.02 (t, J = 7, 2H),
3.69ꢀ3.572 (m, 4H), 3.40ꢀ3.30 (s, 2H), 2.99 (t, J = 7, 4H), 2.30 (t, J =
7, 2H), 2.16 (s, 6H), 1.73ꢀ1.63 (m, 2H). 13C NMR (CD3CN, 48 ꢀC):
167.86, 138.03, 129.04, 123.77, 122.71, 120.05, 119.88, 114.38, 112.55,
58.76, 45.95, 42.38, 40.45, 28.76, 26.82. HRMS calcd for C28H35N9
(M + H), 498.3094; found, 498.3084.
2,4-Bis(40-(bis(400-fluorophenyl)methyl)piperazin-10-yl)-
6-(30-(N,N-dimethylamino)propylamino-1,3,5-triazine (8rA).
Yield: 120 mg, 80%, white powder. 1H NMR (CDCl3, 48 ꢀC): 7.33 (dd,
J1 = 8.7, J2 = 5.4, 8H), 6.96 (dd, J1 = 11.3, J2 = 6.1, 8H), 4.23 (s, 2H),
3.75ꢀ3.65 (m, 8H), 3.41 (dd, J = 13, 2H), 2.69 (t, J = 9, 2H), 2.48 (s,
6H), 2.38ꢀ2.28 (m, 8H), 1.91ꢀ1.85 (m, 2H). 13C NMR (CDCl3,
48 ꢀC): 166.10ꢀ166.84, 162.09 (d, J = 247), 138.11 (d, J = 3.0), 129.51
(d, J = 7.8), 115.60 (d, J = 21), 74.66, 56.87, 51.85, 44.21, 43.51, 38.60,
26.39. HRMS calcd for C42H47N9F4 (M + H), 754.3969; found,
754.3958.
2,4-Bis(20-(100-cyc1ohexenyl)ethylamino)-6-(30-(N,N-dimeth-
ylamino)propylamino)-1,3,5-triazine (8jA). Yield: 220 mg, 95%,
white solid. 1H NMR (CDCl3, 48 ꢀC): 5.46 (s, 2H), 3.35ꢀ3.47 (m, 6H),
2.34 (t, J = 7.0, 2H), 2.21 (s, 6H), 2.14 (t, J = 8, 2H), 2.00ꢀ1.86 (m, 8H),
1.70 (m, 2H), 1.62ꢀ1.50 (m, 8H). 13C NMR (CDCl3, 48 ꢀC): 166.24,
135.08, 123.50, 57.95, 45.65, 39.66, 38.84, 38.26, 28.29, 27.79, 25.48,
23.13, 22.64. HRMS calcd for C24H41N7 (M + H), 428.3502; found,
428.3505.
2,4-Bis(40-chlorobenzylamino)-6-(30-(N,N-dimethylamino)-
propylamino)-1,3,5-triazine (8kA). Yield: 110 mg, 68%, white oil.
1H NMR (CDCl3, 7% TFA) (confs): 7.31ꢀ7.10 m (8H), 4.56ꢀ4.58
(m, 4H), 3.56ꢀ3.53 (m, 2H), 3.23ꢀ3.04 (m, 2H), 2.93ꢀ2.86
(m, 6H), 2.24ꢀ2.01 (2H). 13C NMR (CDCl3, 7% TFA) (confs):
162.50, 155.10, 134.26, 129.17, 129.15, 56.01, 44.89, 43.74, 38.15, 24.09.
HRMS calcd for C22H27N7Cl2 (M + H), 460.1783; found, 460.1779.
2,4-Bis(40-fluorobenzylamino)-6-(30-(N,N-dimethylamino)-
propylamino)-1,3,5-triazine (8lA). Yield: 105 mg, 72%, white solid.
1H NMR (CDCl3, 7% TFA) (confs): 7.24ꢀ6.98 (m, 8H), 4.63ꢀ4.53 (m,
4H), 3.57ꢀ3.53 (m, 2H), 3.25ꢀ3.15 (m, 2H), 2.93ꢀ2.89 (m, 6H),
2.16ꢀ2.03 (m, 2H). 13C NMR (CDCl3, 7% TFA) (confs): 162.80 (d, J =
251), 160.05, 154.73, 153.80, 131.49, 129.49, 115.93 (d, J = 22), 56.01,
45.05, 43.82, 38.29, 24.10. HRMS calcd for C22H27N7F2 (M + H),
428.2374; found, 428.2394.
2,4-Bis(2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-2(9H)-yl)-
6-(30-(N,N-dimethylamino)propylamino-1,3,5-triazine (8sA).
1
Yield: 80 mg, 63%, brownish oil. H NMR (CD3CN, 48 ꢀC): 9.00
(s, 2H), 7.38 (dt, J1 = 22, J2 = 7, 4H), 7.06 (ddd, J1 = 15, J2 = 14, J3 =
7, 4H), 4.98ꢀ4.89 (bs, 4H), 4.17ꢀ4.10 (bs, 4H), 3.544ꢀ3.34 (m, 2H),
2.81ꢀ2.75 (bs, 4H), 2.33 (t, J = 7, 2H), 2.21ꢀ2.15 (m, 6H), 1.74ꢀ1.67
(m, 2H). 13C NMR (CD3CN, 48 ꢀC): 167.70, 167.00, 137.60, 133.27,
128.38, 122.18, 120.07, 118.71, 112.06, 109.45, 58.68, 45.90, 42.48, 42.35,
40.36, 28.64, 21.97. HRMS calcd for C30H35N9 (M + H), 522.3094;
found, 522.3079.
2,4-Bis(40-hydroxycarbonylbenzylamino)-6-(30-(N,N-dimethyl-
amino)propylamino)-1,3,5-triazine (8tA). Yield: 160 mg, 61%,
colorless oil. 1H NMR (C5D5N, 48 ꢀC): 8.38 (d, J = 8, 4H), 7.63 (d, J = 8,
4H), 4.87 (s, 4H), 3.61 (t, J = 6, 2H), 3.09 (t, J = 7, 2H), 2.70 (s, 6H),
2.15ꢀ2.05 (m, 2H). 13C NMR (C5D5N, 7% TFA, 48 ꢀC): 169.28,
164.91, 145.54, 131.85, 131.12, 130.49, 128.38, 128.05, 56.31, 45.15,
43.19, 38.74, 25.81. HRMS calcd for C24H29N7O4 (M + H), 480.2395;
found, 480.2384.
2,4-Bis(20,40-dichlorobenzylamino)-6-(30-(N,N-dimethyl-
amino)propylamino)-1,3,5-triazine (8 mA). Yield: 38 mg, 20%,
1
white powder. H NMR (CDCl3, 7% TFA) (confs): 7.41ꢀ7.05 (m,
6H), 4.67ꢀ4.61 (m, 4H), 3.55ꢀ3.46 (m, 2H), 3.17ꢀ3.10 (m, 2H),
2.90ꢀ2.86 (m, 6H), 2.12ꢀ2.05 (m, 2H). 13C NMR (CDCl3, 7% TFA)
(confs): 163.64, 155.95, 155.39, 134.40, 133.88, 132.59, 129.85, 129.48,
127.28, 55.92, 43.54, 42.18, 37.95, 24.23. HRMS calcd for C22H25N7Cl4
(M + H), 528.1004; found, 528.0992.
2,4-Bis(40-hydroxycarbonylbenzylamino)-6-(propylamino)-
1,3,5-triazine (8tE). Yield: 25 mg, 27%, colorless oil. 1H NMR
(C5D5N, 48 ꢀC): 8.37 (d, J = 8, 4H), 7.62 (d, J = 8, 4H), 4.88
(s, 4H), 3.56 (t, J = 7, 2H), 1.66ꢀ1.61 (m, 2H), 1.44ꢀ1.35 (m, 2H),
0.90 (t, J = 7, 3H). 13C NMR (C5D5N, 48 ꢀC): 169.32, 166.61, 146.20,
131.73, 131.12, 130.47, 128.40, 128.05, 45.21, 41.30, 32.76, 20.83, 14.42.
HRMS calcd for C23H26N6O4 (M + H), 451.2094; found, 451.2087.
Biological Assays. Recombinant Rat TRPV1 Channels Ex-
pression in Xenopus oocytes and Channel Blocking. All the
procedures have been described in detail elsewhere.4,5 Whole-cell
currents from rat TRPV1-injected oocytes were recorded in Mg2+
Ringer’s solution (10 mM Hepes, pH 7.4, 115 mM NaCl, 2.8 mM
KCl, 0.1 mM BaCl2, 2.0 mM MgCl2) with a two-microelectrode voltage-
clamp amplifier at 20 ꢀC. The TRPV1 channels were activated by
applying 10 μM capsaicin in the absence or the presence of individual
compounds at a holding potential (Vh) of ꢀ60 mV. Doseꢀresponse
curves for individual peptoids were fitted to the Hill equation:
2,4-Bis(20,40-difluorobenzylamino)-6-(30-(N,N-dimethyl-
amino)propylamino)-1,3,5-triazine (8nA). Yield: 52 mg, 40%,
1
white powder. H NMR (CDCl3, 7% TFA) (confs): 7.29ꢀ7.18 (m,
2H), 6.88ꢀ6.79 (m, 4H), 4.67ꢀ4.59 (m, 4H), 3.65ꢀ3.47 (m, 2H),
3.22ꢀ3.17 (m, 2H), 2.91 (m, 6H), 2.14ꢀ2.05 (m, 2H). 13C NMR
(CDCl3, 7% TFA) (confs): 163 (dd, J1 = 197, J2 = 12), 163.02, 160.88
(dd, J1 = 197, J2 = 12), 155.39, 154.79, 131.08ꢀ130.66, 112.09ꢀ 111.60,
104.56 (t, J = 25), 56.17, 43.80, 39.11, 37.94, 24.26. HRMS calcd for
C22H25N7F4 (M + H), 464.2186; found, 464.2199.
2,4-Bis(40-trifluoromethylbenzylamino)-6-(30-(N,N-dimethyl-
amino)propylamino)-1,3,5-triazine (8oA). Yield: 220 mg, 96%,
colorless oil. 1H NMR (CDCl3, 48 ꢀC): 7.54ꢀ7.39 (m, 8H), 4.58
(bs, 4H), 3.37 (dd, J1 = 12 J2 = 6, 2H), 2.39ꢀ2.122 (m, 8H),
1.67ꢀ1.60 (m, 2H). 13C NMR (CDCl3, 48 ꢀC): 166.47, 166.38, 144.07,
129.57 (q, J=32), 127.67, 125.54 (q, J=4), 124.39 (q, J=270), 57.71, 45.41,
44.32, 38.57, 27.29. HRMS calcd for C24H27N7F6 (M + H), 528.2310;
found, 528.2292.
2,4-Bis(30-40-methylenedioxybenzylamino)-6-(30(N,N-dimeth-
ylamino)propylamino)-1,3,5-triazine (8pA). Yield: 226 mg, 94%,
colorless oil. 1H NMR (CDCl3, 48 ꢀC): 6.84ꢀ6.70 (m, 6H), 5.92ꢀ5.88
I
1
ꢀ
ꢁ
¼
nH
½blockerꢂ
IC50
Imax
1 þ
where IC50 denotes the channel blocker concentration that inhibits half
of the response obtained in its absence (Imax) and nH denotes the Hill
7450
dx.doi.org/10.1021/jm200981s |J. Med. Chem. 2011, 54, 7441–7452

Journal of Medicinal Chemistry
ARTICLE
coefficient, which is an estimate of the number of drug binding sites. IꢀV
characteristics were recorded using a ramp protocol.5,32 Oocytes were
depolarized from ꢀ60 to 60 mV in 5 s (25 mV/s). Leak currents were
measured in the absence of agonist in the external bath medium and
subtracted from the ionic current recorded in the presence of the ligand.
Voltage dependence of channel blocking was studied as described in
Ferrer-Montiel et al.32 Experimental data were fitted to either the Hill or
Woodhull equation32 with a nonlinear least-squares regression algo-
rithm using GraphPad Prism 5 software.
(7) Szallasi, A. G.; Martin, J. Peripheral TRPV1 receptors as targets
for drug development: new molecules and mechanisms. Curr. Pharm.
Des. 2008, 14, 32–41.
(8) Kim, H. Y.; Park, C.-K.; Cho, I.-H.; Jung, S. J.; Kim, J. S.; Oh, S. B.
Differential changes in TRPV1 expression after trigeminal sensory nerve
injury. J. Pain 2008, 9, 280–288.
(9) 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 characterization of 6-aryl-
7-isopropylquinazolinones as novel TRPV1 antagonists that are effective
in models of chronic pain. J. Med. Chem. 2006, 49, 471–474.
(10) Ghilardi, J. R.; Rohrich, H.; Lindsay, T. H.; Sevcik, M. A.; Schwei,
M. J.; Kubota, K.; Halvorson, K. G.; Poblete, J.; Chaplan, S. R.; Dubin,
A. E.; Carruthers, N. I.; Swanson, D.; Kuskowski, M.; Flores, C. M.; Julius,
D.; Mantyh, P. W. Selective blockade of the capsaicin receptor TRPV1
attenuates bone cancer pain. J. Neurosci. 2005, 25, 3126–3131.
(11) Gavva, N. R.; Treanor, J. J. S.; Garami, A.; Fang, L.; Surapaneni,
S.; Akrami, A.; Alvarez, F.; Bak, A.; Darling, M.; Gore, A.; Jang, G. R.;
Kesslak, J. P.; Ni, L.; Norman, M. H.; Palluconi, G.; Rose, M. J.; Salfi, M.;
Tan, E.; Romanovsky, A. A.; Banfield, C.; Davar, G. Pharmacological
blockade of the vanilloid receptor TRPV1 elicits marked hyperthermia
in humans. Pain 2008, 136, 202–210.
’ ASSOCIATED CONTENT
S
Supporting Information. Full experimental details con-
b
cerning concise product synthesis, NMR spectra of synthesized
compounds, and full structures of triazines 8. This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: +34-934006121. E-mail: angel.messeguer@iqac.csic.es.
’ ACKNOWLEDGMENT
(12) Planells-Cases, R.; Garcia-Martinez, C.; Royo, M.; Perez-Paya,
E.; Carreno, C.; Albericio, F.; Messeguer, A.; Ferrer-Montiel, A. Small
molecules targeting the vanilloid receptor complex as drugs for inflam-
matory pain. Drugs Future 2003, 28, 787–797.
(13) Johnson, J. W.; Kotermanski, S. E. Mechanism of action of
memantine. Curr. Opin. Pharmacol. 2006, 6, 61–67.
This work was supported by grants from Spanish Ministry of
Science and Innovation (Grant SAF2008-00048 to A.M., Grant
BFU2009-08346 to A.F.-M., CONSOLIDER-INGENIO 2010
(Grant CSD2008-00005) to A.F.-M., J.M.G.-R., and A.M.), from
Fundaciꢀo La Maratꢀo de TV3 (to A.F.-M. and A.M.), and from
PROMETEO/ 2010/046 from the GVA to A.F.-M.
(14) García-Martínez, C.; Humet, M.; Planells-Cases, R.; Gomis, A.;
Caprini, M.; Viana, F.; Pe~na, E. D. l.; Sanchez-Baeza, F.; Carbonell, T.;
Felipe, C. D.; Pꢀerez-Payꢀa, E.; Belmonte, C.; Messeguer, A.; Ferrer-
Montiel, A. Attenuation of thermal nociception and hyperalgesia by VR1
blockers. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 2374–2379.
(15) Quintanar-Audelo, M.; Fernꢀandez-Carvajal, A.; Van Den Nest,
W.; Carre~no, C.; Ferrer-Montiel, A.; Albericio, F. Design and synthesis
of indole-based peptoids as potent noncompetitive antagonists of
transient receptor potential vanilloid 1. J. Med. Chem. 2007, 50,
6133–6143.
(16) Bonache, M.; García-Martinez, C.; García de Diego, L.;
Carre~no, C.; Pꢀerez de Vega, M.; García-Lꢀopez, M.; Ferrer-Montiel, A.;
Gonzꢀalez-Mun~iz, R. Old molecules for new receptors: Trp(Nps)
dipeptide derivatives as vanilloid TRPV1 channel blockers. ChemMed-
Chem 2006, 1, 429–438.
’ DEDICATION
†Dedicated to the memory of Prof. Rafael Suau.
’ ABBREVIATIONS USED
DMEM, Dulbecco’s modified Eagle’s medium; DRG, dorsal root
ganglion; FBS, fetal bovine serum; NGF, nerve growth factor;
TRPV1, transient receptor potential vanilloid 1
’ REFERENCES
(1) Szallasi, A.; Blumberg, P. M. Vanilloid (capsaicin) receptors and
mechanisms. Pharmacol. Rev. 1999, 51, 159–212.
(2) Szallasi, A.; Fowler, C. J. After a decade of intravesical vanilloid
therapy: still more questions than answers. Lancet Neurol. 2002,
1, 167–172.
(17) Masip, I.; Cortꢀes, N.; Abad, M.-J.; Guardiola, M.; Pꢀerez-Payꢀa,
E.; Ferragut, J.; Ferrer-Montiel, A.; Messeguer, A. Design and synthesis
of an optimized positional scanning library of peptoids: identification of
novel multidrug resistance reversal agents. Bioorg. Med. Chem. 2005,
13, 1923–1929.
(18) Moure, A.; Sanclimens, G.; Bujons, J.; Masip, I.; Alvarez-Larena,
A.; Pꢀerez-Payꢀa, E.; Alfonso, I.; Messeguer, A. Chemical modulation of
peptoids: synthesis and conformational studies on partially constrained
derivatives. Chem.—Eur. J. 2011, 17, 7927–7939.
(3) Messeguer, A.; Planells-Cases, R.; Ferrer-Montiel, A. Physiology
and pharmacology of the vanilloid receptor. Curr Neuropharmacol. 2006,
4, 1–15.
(4) García-Martínez, C.; Fernꢀandez-Carvajal, A.; Valenzuela, B.;
Gomis, A.; Van Den Nest, W.; Ferroni, S.; Carre~no, C.; Belmonte, C.;
Ferrer-Montiel, A. Design and characterization of a noncompetitive
antagonist of the transient receptor potential vanilloid subunit 1 channel
with in vivo analgesic and anti-inflammatory activity. J. Pain 2006,
7, 735–746.
(5) Valente, P.; Garcia-Sanz, N.; Gomis, A.; Fernandez-Carvajal, A.;
Fernandez-Ballester, G.; Viana, F.; Belmonte, C.; Ferrer-Montiel, A.
Identification of molecular determinants of channel gating in the
transient receptor potential box of vanilloid receptor I. FASEB J. 2008,
22, 3298–3309.
(6) Szallasi, A.; Cortright, D. N.; Blum, C. A.; Eid, S. R. The vanilloid
receptor TRPV1: 10 years from channel cloning to antagonist proof-of-
concept. Nat. Rev. Drug Discovery 2007, 6, 357–372.
(19) Scheper, J.; Guerra-Rebollo, M.; Sanclimens, G.; Moure, A.;
ꢀ
Masip, I.; Gonzꢀalez-Ruiz, D.; Rubio, N.; Crosas, B.; Meca-Cortꢀes, O.;
Loukili, N.; Plans, V.; Morreale, A.; Blanco, J.; Ortiz, A. R.; Messeguer,
ꢀ
A.; Thomson, T. M. Proteinꢀprotein interaction antagonists as novel
inhibitors of non-canonical polyubiquitylation. PLoS One 2010,
5, e11403.
(20) Saleh, M.; Abbott, S.; Perron, V.; Lauzon, C.; Penney, C.; Zacharie,
B. Synthesis and antimicrobial activity of 2-fluorophenyl-4,6-disubstituted
[1,3,5]triazines. Bioorg. Med. Chem. Lett. 2010, 20, 945–949.
(21) Menicagli, R.; Samaritani, S.; Signore, G.; Vaglini, F.; Dalla Via,
L. In vitro cytotoxic activities of 2-alkyl-4,6-diheteroalkyl-1,3,5-triazines:
new molecules in anticancer research. J. Med. Chem. 2004, 47,
4649–4652.
7451
dx.doi.org/10.1021/jm200981s |J. Med. Chem. 2011, 54, 7441–7452

Journal of Medicinal Chemistry
ARTICLE
(22) Zheng, M.; Xu, C.; Ma, J.; Sun, Y.; Du, F.; Liu, H.; Lin, L.; Li, C.;
Ding, J.; Chen, K.; Jiang, H. Synthesis and antitumor evaluation of a
novel series of triaminotriazine derivatives. Bioorg. Med. Chem. 2007,
15, 1815–1827.
(23) Xiong, Y.-Z.;Chen, F.-E.;Balzarini, J.; DeClercq, E.;Pannecouque,
C. Non-nucleoside HIV-1 reverse transcriptase inhibitors. Part 11: Structural
modulations of diaryltriazines with potent anti-HIV activity. Eur. J. Med.
Chem. 2008, 43, 1230–1236.
(24) Uccello-Barretta, G.; Samaritani, S.; Menicagli, R.; Salvadori, P.
2,4,6-Tri[(S)-10-methylbenzylamino]-1,3,5-triazine: a new NMR chiral
solvating agent for 3,5-dinitrophenyl derivatives; an attempt at a chiral
discrimination rationale. Tetrahedron: Asymmetry 2000, 11, 3901–3912.
(25) Díaz-Ortiz, A.; Elguero, J.; DelaHoz, A.; Jimꢀenez, A.; Moreno,
A.; Moreno, S.; Sꢀanchez-Migallꢀon, A. Microwave-Assisted Synthesis and
Dynamic Behaviour of Tris(1pyrazolyl)-1,3,5-triazine-2,4,6-triamines.
QSAR Comb. Sci. 2005, 24, 649–659.
(26) Baumgartner, W.; Islas, L.; Sigworth, F. J. Two-microelectrode
voltage clamp of Xenopus oocytes: voltage errors and compensation for
local current flow. Biophys. J. 1999, 77, 1980–1991.
(27) Mellor, I. R.; Ogilvie, J.; Pluteanu, F.; Clothier, R. H.; Parker,
T. L.; Rosini, M.; Minarini, A.; Tumiatti, V.; Melchiorre, C. Methoc-
tramine analogues inhibit responses to capsaicin and protons in rat
dorsal root ganglion neurons. Eur. J. Pharmacol. 2004, 505, 37–50.
(28) Kitaguchi, T.; Swartz, K. J. An inhibitor of TRPV1 channels
isolated from funnel web spider venom. Biochemistry 2005, 44,
15544–15549.
(29) Swamy, K. C. K.; Kumar, N. N. B.; Balaraman, E.; Kumar, K. V.
P. P. Mitsunobu and related reactions: advances and applications. Chem.
Rev. 2009, 109, 2551–2651.
(30) Feichtinger, K.; Zapf, C.; Sings, H. L.; Goodman, M. Dipro-
tected triflylguanidines: a new class of guanidinylation reagents. J. Org.
Chem. 1998, 63, 3804–3805.
(31) Messeguer, J.; Cortꢀes, N.; García-Sanz, N.; Navarro-Vendrell,
G.; Ferrer-Montiel, A.; Messeguer, A. Synthesis of a positional scanning
library of pentamers of N-alkylglycines assisted by microwave activation
and validation via the identification of trypsin inhibitors. J. Comb. Chem.
2008, 10, 974–980.
(32) Ferrer-Montiel, A. V.; Merino, J. M.; Planells-Cases, R.; Sun,
W.; Montal, M. Structural determinants of the blocker binding site in
glutamate and NMDA receptor channels. Neuropharmacology 1998,
37, 139–147.
7452
dx.doi.org/10.1021/jm200981s |J. Med. Chem. 2011, 54, 7441–7452