Solid-phase synthesis of a library of amphipatic hydantoins. Discovery of new hits for TRPV1 blockade

Article abstract of DOI:10.1021/co1000986

Some heterocyclic systems, called privileged scaffolds, appear frequently in bioactive products and marketed drugs. The combination of a recognized privileged scaffold (hydantoin) and a functional group with high incidence in bioactive molecules (guanidine) guided the design of a library of amphipatic compounds, which allowed the discovery of novel TRPV1 ion channel blockers. The library was synthesized by parallel solid-phase synthesis from an orthogonally protected resin-bound Lys-Lys skeleton. Key steps of the synthetic procedure were the construction of the hydantoin ring, by reaction of the N-terminal amino group with N,N-disuccinimidyl carbonate (DSC) and subsequent base-induced cyclization, and the guanidinylation of the C-terminal Lys side-chain after removal of the Alloc protecting-group. The preliminary biological studies have allowed the identification of some of the key structural features directing the blockage of capsaicin-induced Ca²⁺ influx through TRPV1 channels, particularly, the strong preference showed for highly lipophilic acyl groups and substituted guanidine moieties. Active compounds based on this new pharmacophoric scaffold that display in vitro and in vivo inhibitory activity

Full text of DOI:10.1021/co1000986

RESEARCH ARTICLE
pubs.acs.org/acscombsci
Solid-Phase Synthesis of a Library of Amphipatic Hydantoins.
Discovery of New Hits for TRPV1 Blockade
Guillermo Gerona-Navarro,† Rosario Gonzꢀalez-Mu~niz,† Asia Fernꢀandez-Carvajal,‡ Josꢀe M. Gonzꢀalez-Ros,‡
Antonio Ferrer-Montiel,‡,§ Cristina Carre~no,§ Fernando Albericio,*,||,^,X and Miriam Royo*,O,^
†Instituto de Química Mꢀedica, CSIC, Juan de la Cierva, 3, 28006 Madrid, Spain
‡Instituto de Biología Molecular y Celular, Universidad Miguel Hernꢀandez, Av. de la Universidad, 03202 Elche, Spain
§DiverDrugs SL, Isaac Peral 17 (Pol. Ind. Camí Ral), 08850 Gavꢁa, Spain
Institute for Research in Biomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona, Spain
^CIBER-BBN, Networking Centre on Bioengineering, Biomaterials, and Nanomedicine, Barcelona Science Park,
University of Barcelona, Baldiri Reixac 10, 08028 Barcelona, Spain
XDepartment of Organic Chemistry, University of Barcelona, Martí i Franquꢀes 1-11, 08028 Barcelona, Spain
OCombinatorial Chemistry Unit, Barcelona Science Park, University of Barcelona, Baldiri Reixac 10, 08028 Barcelona, Spain
S Supporting Information
b
ABSTRACT: Some heterocyclic systems, called privileged
scaffolds, appear frequently in bioactive products and marketed
drugs. The combination of a recognized privileged scaffold
(hydantoin) and a functional group with high incidence in
bioactive molecules (guanidine) guided the design of a library of
amphipatic compounds, which allowed the discovery of novel
TRPV1 ion channel blockers. The library was synthesized by
parallel solid-phase synthesis from an orthogonally protected
resin-bound Lys-Lys skeleton. Key steps of the synthetic
procedure were the construction of the hydantoin ring, by
reaction of the N-terminal amino group with N,N-disuccinimi-
dyl carbonate (DSC) and subsequent base-induced cyclization,
and the guanidinylation of the C-terminal Lys side-chain after removal of the Alloc protecting-group. The preliminary biological
studies have allowed the identification of some of the key structural features directing the blockage of capsaicin-induced Ca2+ influx
through TRPV1 channels, particularly, the strong preference showed for highly lipophilic acyl groups and substituted guanidine
moieties. Active compounds based on this new pharmacophoric scaffold that display in vitro and in vivo inhibitory activity
KEYWORDS: hydantoins, guanidine groups, solid-phase, TRPV1 blockers, pain
’ INTRODUCTION
The impact of hydantoins in medicinal chemistry programs
has triggered an extensive development of synthetic methodol-
ogies for the generation of libraries based on this heterocyclic
skeleton.11ꢀ13 Apart from some procedures in solution, using
multicomponent reactions andfluorous synthesis,14,15 mostefforts
to prepare hydantoin-based libraries have been carried out on
solid-phase, following two main approaches: (i) cyclization of an
acyclic precursor with concomitant cleavage from the resin, and
(ii) cyclization prior to cleavage.
The vast majority of synthetic sequences, derived from the first
approach,16 comprise the attachment of an amino acid or
dipeptide to the resin through the C-terminal carboxylate, further
elaboration to an acyclic urea, and the final cyclative/cleavage
step.17ꢀ29 An alternative strategy used a urethane attachment of
5,5-Diphenylhydantoin has been used since 1938 for the treat-
ment of epilepsy because of its regulatory effect on the bioelectric
activity of the nervous system.1 Since then, many other hydantoin-
derived compounds with a wide range of therapeutic applications
have been discovered. For example, 5-arylidene derivatives have
shown antituberculosis and antiproliferative activity,2,3 whereas
Pt(II) complexes with hydantoin ligands have proven to be very
effective citotoxic agents.4 Other examples include antiviral agents,5
ligands for the glycine binding site of the NMDA receptor and for
voltage-gated sodium channels,6,7 as well as modulators of certain
proteinꢀprotein interactions related to cell adhesion.8,9 Thus,
and because of the large variety of biological applications found
for this family of compounds, the hydantoin scaffold is being
considered a “privileged structure”, a term first coined by Evans
in 1988 to define a single molecular framework able to provide
ligands for a range of different biological targets.10
Received: December 28, 2010
Revised:
June 10, 2011
Published: June 14, 2011
r
2011 American Chemical Society
458
dx.doi.org/10.1021/co1000986 ACS Comb. Sci. 2011, 13, 458–465
|

ACS Combinatorial Science
RESEARCH ARTICLE
Figure 1. General structure of the hydantoin library and selected building blocks.
amino acids to the resin (through the R-NH2 group), followed by
On the basis of the above-mentioned considerations, we
base-catalyzed cyclization of linear adducts to form and detach
hypothesized that the combination of a “privileged structure”
and the guanidine “privileged functional group” could be a
practical approach for the discovery of new compounds of
therapeutic relevance. Hence, using the hydantoin ring as central
scaffold, we have prepared a discrete library with two points of
diversity, which are decorated by an arrangement of differently
substituted guanidine groups and diverse acyl moieties, to confer
a certain amphipatic character to the library compounds. We
report herein the preparation of this collection of compounds
and the identification of TRPV1 channel blockers with in vitro
and in vivo activity. We focused on this receptor because of its
central role in pain transduction. In addition, we used in parallel
the NMDA receptor with the aim of identifying compounds that
preferentially block the TRPV1 over the glutamatergic receptor.
The rationale of using the NMDA receptor is that it displays a
similar Ca2+ permeability as the TRPV1 channel.
the final hydantoins.30
On the other hand, many different strategies have been
described to develop methodologies following the second ap-
proach, which allow further on-bead transformations. They differ
mainly in the nature of the reactive intermediate or in the
activating agents used to facilitate the cyclization step. Thus,
for example, base-promoted cyclization of dipeptide-derived
phenyl carbamates, thermal ring closure from dipeptide-isocya-
nate intermediates, and activation of the peptide N-terminal
amino group by means of diphosgene, triphosgene, disuccini-
mide carbonate, or carbonyldiimidazole have been successfully
employed.31ꢀ39
The guanidine functional group is also a structural motif
commonly found in natural products and in many therapeutically
active compounds, including some marketed drugs, where it
usually acts as a crucial pharmacophore entity. Thus, guanidine-
containing derivatives have been shown to have antitumor,40ꢀ42
antibiotic,43,44 and antiviral properties.45 Regarding the biological
target, the guanidine group is frequent in both enzyme
inhibitors46,47 and ligands for different receptors and ion
channels.48ꢀ51 Therefore, and by analogy with the “privileged
scaffolds”, the guanidine moiety can be considered as a “privileged
functional group” in the search for new bioactive compounds.
’ RESULTS AND DISCUSSION
A common Lys-Lys-derived hydantoin scaffold was envisaged
to incorporate the elements of diversity at the Lys side-chains.
The new chemset of 112 compounds resulted from a combina-
tion of 14 diverse acyl moieties and 8 different guanidine groups
at the N- and C-terminal side-chains, respectively (1{G1ꢀ8,
459
dx.doi.org/10.1021/co1000986 |ACS Comb. Sci. 2011, 13, 458–465

ACS Combinatorial Science
RESEARCH ARTICLE
Scheme 1. Reagents and Conditionsa
a (i) (a) Piperidine/DMF (1:4); (b) Fmoc-Lys(Alloc)ꢀOH (1.5 equiv), DIPCDI (1.5 equiv), HOBt (1.5 equiv), DMF; (c) Piperidine/DMF (1:4);
(d) Fmoc-Lys(Mtt)ꢀOH (1.5 equiv), DIPCDI (1.5 equiv), HOBt (1.5 equiv), DMF. (ii) (a) Piperidine/DMF (1:4), DSC (5 equiv), DMAP
(0.5 equiv); (b) 5% DBU/DMF. (iii) (a) Pd[PPh3]4, PhSiH3; (b) HCl.H2N(NH)CNH-X-CO2H (1.5 equiv), DIPCDI (1.5 equiv), HOBt (1.5 equiv),
DMF. (iv) (a) Pd [PPh3]4, PhSiH3; (b) Guanidylation reaction. (v) (a) TES:TFA:DCM (1:3:96); (b) RCO2H (1.5 equiv), DIPCI (1.5 equiv), HOBt
(1.5 equiv), DMF. (vi) TFA.
A1ꢀ14}, Figure 1). As diversity elements at the C-terminal Lys
side-chain, three sublibraries contain unsubstituted guanidine
groups, connected through spacers with different flexibility
(G{1ꢀ2}) or directly linked to the carbon side-chain (G3). The
other five sublibraries possess different pentasubstituted guani-
dine moieties (G{4ꢀ8}) linked to the tetramethylene side-chain.
To further incorporate diversity at the 5-(4-aminobutyl)-sub-
stituent of the hydantoin skeleton, we decided to acylate the
amino group with carboxylic acids containing short linear and
branched aliphatic chains (A{1ꢀ4}), fatty-type saturated chains
(A{5ꢀ6}), carbocyclic and aromatic moieties (A{7ꢀ11}), and
linear and cyclic substituents incorporating primary and second-
ary amino groups (A{12ꢀ14}).
’ SYNTHESIS
The orthogonally protected Fmoc-Lys(Mtt)-Lys(Alloc)-PS
(2) was prepared by conventional SPPS methods on a Fmoc-
Rink amide-MBHA-Polystyrene resin (Scheme 1). Removal of
the Fmoc group from this dipeptidyl resin, followed by treatment
with DSC in the presence of DBU allowed the cyclization to the
hydantoin scaffold,38,39 and thus the formation of the key resin-
bound intermediate 3, characterized by HPLC-MS after cleavage.
The selective removal of the Alloc protecting group, using
Pd(PPh3)4/PhSiH3, afforded a free amino at the C-terminal
Lys side-chain, used for the incorporation of the guanidine
moieties. Thus, acylation with the hydrochloride salt of 4-guani-
dinobenzoic acid and 5-guanidinovaleric acid, in the presence of
460
dx.doi.org/10.1021/co1000986 |ACS Comb. Sci. 2011, 13, 458–465

ACS Combinatorial Science
RESEARCH ARTICLE
Figure 2. Diguanidylated compounds obtained for G7 sublibrary and
cleavage product of 5{G7}.
Figure 3. In vivo activity of 1{G1ꢀ8,A1ꢀ14} library compounds with
significant TRVP1 blockade properties. (a) Black bars show latency time
to the first response of animals in a hot plate at 52 ꢀC.42 Mice were
administered intraperitoneally with saline (vehicle) or 30 mg/kg of the
compounds. (b) Gray bars show duration time of the burning sensation
evoked by intraplantar administration of capsaicin, measured as licking
and shaking the paw.59 Compound was administered ip at 30 mg/kg.
Data are given as mean ( sem, with n g 6.
DIPCDI/HOBt as coupling agents, gave resins 4{1ꢀ2}. On the
other hand, direct guanidinylation with various guanidinylating
agents (G{3ꢀ8}),52 bearing different substituents, led to the
dipeptidyl chemset resins 5{3ꢀ8}. Aliquots of every member of
the chemsets 4 and 5 were subjected to cleavage and HPLC-MS
analysis, revealing a >80% conversion in all cases. Next, each resin,
4{G1ꢀ2} and 5{G3ꢀ8}, was treatedwiththe acidic cocktailTES:
TFA:DCM (1:3:96) for Mtt protecting group removal, anddivided
into 14 portions for the incorporation of the second element of
diversity (acyl groups, A{1ꢀ14}). Coupling reactions to chemsets
6 and 7 were performed with 1.5 equiv of the reagent chemset
A{1ꢀ14 } in the presence of DIPCDI/HOBt. Finally, all members
of chemsets 6 and 7 were cleaved from the resin with TFA.
The expected final compounds 1{G1ꢀ8,A1ꢀ14} were obtained
in variable yields, ranging from low to moderate for sublibraries
1{1,1ꢀ14} and 1{2,1ꢀ14}, and from moderate to good for the
other subset of compounds, 1{3ꢀ8,1ꢀ14} (see Supporting In-
formation, Table S1, for details). Ninety (80.4%) of the 112
compounds were isolated in g80% purity, and a further 14.3% in
70ꢀ80% purity. The remaining hydantoin derivatives (5.3%)
showed purities that range from 48 to 68%.
It is interesting to note that main components in library
1{7,1ꢀ14} are the diguanidinyated compounds indicated in
Figure 2 (>70%), while the expected monoguanidinylated deri-
vatives were in minor proportion within the mixtures (5ꢀ15%).
The presence of a second guanidine group was determined by
MS, and its location at position 1 of the hydantoin ring was assigned
from the 1H NMR spectra (see the chemical shifts of contiguous H5
protons, Supporting Information, Table S9). The cleavage of an
aliquot of the 5{7} resin corroborated the incorporation of the
second guanidine moiety during the guanidinylation reaction, a side-
reaction that was also observed, but to a much lower extent, during
guanidinylation of resin 3 with G4 (8%). Only compounds 1{7,12}
and 1{7,14} were obtained as monoguanidinylated products in high
purity (>80%). This result indicates that treatment of resins 7{7,12}
and 7{7,14} with piperidine for the Fmoc removal, prior to cleavage,
reverted the di- to the monoguanidylated derivative.
’ SCREENING AS TRPV1 AND NMDA CHANNEL
BLOCKERS
In the search for new bioactive chemical entities, compounds
of library 1 were assayed as blockers of the capsaicin-induced
channel activity of TRPV1, and of the glutamate-evoked activity of
NMDA, both heterogously expressed in Xenopus laevis oocytes.
TRPV1 is a neuronal receptor that integrates thermal and
chemical stimuli in the peripheral nervous system.54 From a
therapeutic point of view, TRPV1 antagonists have shown efficacy
in reducing nociception from inflammatory and neuropathic pain
in animal models.55ꢀ587 NMDA receptors are nonspecific cation
channels that directly contribute to excitatory synaptic transmis-
sion, playing a key role in a wide range of physiological and
pathological processes, such as excitotoxicity.59 The interest in
developing safe and effective NMDA channel blockers comes
from the pivotal role of glutamate and NMDA receptors in
mediating multiple neurodegenerative CNS disorders.
To identify activity-dependent TRPV1 channel blockers, we
used saturating concentrations of capsaicin, and a hyperpolarized
membrane potential. Similar conditions were used for the NMDA
receptor by using saturating concentrations of L-glutamate and
glycine. The full set of compounds blocked the capsaicin-evoked
currents from TRPV1 and the L-glutamate/glycine responses from
NMDA receptors to different extents (Supporting Information,
Figures S3 and S4). Noteworthy, some library members signifi-
cantly inhibited the TRPV1 activity induced by capsaicin, without
significantly affecting NMDA receptor function (Supporting In-
formation, Figures S3 and S4). Note that some of the compounds
produced in increase of the response probably by binding to an
allosteric site that potentiates the activity of the agonist used.
An inspection of the active components of the library provides
preliminary information on the structural requirements for TRPV1
channel blockers based on this structure. Thus, highly lipophilic
fatty-type substituents, like palmitoyl (A5) and litocholyl (A8), as
well as m-nitrocinnamyl (A10) are preferred on the 5-(4-aminobutyl)
chain. In addition, compounds with substituted guanidine groups
(G{4ꢀ8}) at the C-terminal Lys side-chain showed better TRPV1
blockade than free guanidine-derived analogues (G{1ꢀ3}). In
the last case, the distance between the guanidine and the acyl
moieties is important for the antagonist potency, with better results
for derivatives with the guanidine group directly linked to the Lys
All final compounds 1{G1ꢀ8,A1ꢀ14} were obtained as ap-
proximately 1:1 mixtures of diastereoisomers because of epimer-
ization during the hydantoin ring formation under the DBU basic
medium. The CR oftheC-terminal Lys residueismostlikely being
epimerized, since it was demonstrated that epimerization takes
place at the C-terminal residue on related hydantoin-derived Phe-
Phe model peptides.53 Although the use of the more reactive
carbonylating agent CDI in combination with the weaker base
DIEA could avoid epimerization,39 we decided to use the epimer-
izing method in our combinatorial program to initially explore a
greater diversity.
461
dx.doi.org/10.1021/co1000986 |ACS Comb. Sci. 2011, 13, 458–465

ACS Combinatorial Science
RESEARCH ARTICLE
side-chain, such as 1{3,5}, 1{3,6}, or 1{3,8}, than for the corre-
sponding analogues 1{1ꢀ2} with longer distances between the
diversity elements.
Synthesis of Hydantoin Intermediate 3. Resin 2 (3.5 mmol)
was first treated with 20% piperidine in DMF (1 ꢁ 5 min, 1 ꢁ
20 min), washed with DMF (5 ꢁ 0.5 min), DCM (5 ꢁ 0.5 min),
and DMF (5 ꢁ 0.5 min), and reacted with DSC (5 equiv) and
DMAP (0.5 equiv) in DMF (minimum volume possible). The
mixture was shaken for 3 h, washed with DMF (5 ꢁ 0.5 min.) and
DCM (5 ꢁ 0.5 min.), and completion was monitored by the
ninhydrin test. Then the resin was treated with DBU in DMF
(1:4 v/v 1 ꢁ 1 min, 1 ꢁ 20 min), and washed as previously
indicated. An aliquot portion of the resin was cleaved to the
expected product, (400-amino)butyl-3-(50-allyloxycarbonylami-
no-10-carbamoyl)pentylhydantoin, which was analyzed as fol-
lows: HPLC tR = 12.50 min (>90%); ESI-MS: 406.2 (M+Na)+.
At this point the resin was divided into 8 batches for further
modifications.
Preparation of Resins 4{1ꢀ2}. Batches 1 and 2 of resin 3
(0.43 mmol each) were individually reacted with Pd(PPh3)4
(0.1 equiv) and PhSiH3 (24 equiv) in DCM under Ar, to remove
the Alloc group. The reaction mixtures were shaken under Ar for
20 min and washed with DCM. The resulting resins were swollen
in DMF and treated with G1 and G2 (1.1 mmol), repectively,
DIPCDI (1.1 mmol) and HOBt (1.1 mmol). The couplings were
accomplished for 12 h, and repeated twice with 1 equiv of each
reagent for 12 h, until negative ninhydrin test. HPLC and MS
analysis of the cleavage products indicated a >86% conversion to the
expected guanidine-derived hydantoin intermediates in both cases.
Preparation of Resins 5{3ꢀ8}. After removing the Alloc
group in batches 3ꢀ8 of resin 3 (0.43 mmol each), as indicated
above, DIEA (2.16 mmol in the case of G3 and 1.2 mmol in all
other cases) and the corresponding guanidinylating agent
(G{3ꢀ8}, 2.16 mmol for G3 and 1.2 mmol for others) were
successively added. For the preparation of resin 5{3}, the guani-
dinylation reaction was carried out for 12 h and repeated twice in
the same conditions. For the synthesis of resins 5{4ꢀ8}, the
reaction was shorter (3 h), and it needed to be repeated once with
half equivalents of each reagent. This reaction was monitored by
the Kaiser test. In all cases, after cleavage of aliquot portions, good
conversions were observed in HPLC and the (M+1)+ of the
corresponding guanidinylated intermediates were found in MS
experiments. The only exception was compound 5{7}, for which
the main product corresponded to a diguanidinylated derivative
with (M+1)+ = 601.9.
General Procedure for the Preparation of Resin-Bound
Intermediates 6 and 7. Syringes containing resins 4{1ꢀ2} and
5{3ꢀ8} were swollen in DCM and treated with TES:TFA:DCM
(1:3:96, 3 ꢁ 10 min, and 2 ꢁ 15 min) for the removal of the
Mtt group. After washing with DMF (5 ꢁ 0.5 min), DCM (5 ꢁ
0.5 min), and DMF (5 ꢁ 0.5 min), a neutralization step was
carried out with 5% DIEA in DCM (3 ꢁ 0.5 min) followed by
a final washing with DCM and drying under vacuum. Each
resulting resin was then divided into 14 sub-batches for the incor-
poration of the selected carboxylic acids. For this purpose, resins
of each sub-batch were treated with the corresponding carboxylic
acids (1.5 equiv), HOBt (1.5 equiv), and DIPCDI (1.5 equiv).
When required, the coupling reaction was repeated until the
Kaiser test was negative.
General Procedure for the Cleavage from the Resin:
Synthesis of Final Compounds 1. Cleavage from the resins
was carried out by treatment with neat TFA (1 mL/100 mg of
resin) for 1.5 h. After filtration, the acidic filtrate was evaporated
by a N2 or Ar stream, and the resultant residue was triturated with
Et2O and centrifuged. The resulting solid residue was separated,
A few of these active compounds were selected, resynthesized,
and evaluated as a mixture of enantiomers for in vivo activity.
Basically, we evaluated the effect attenuating the burning sensa-
tion evoked by intraplantar capsaicin administration,60 which is
evidenced by the duration of shaking and licking. In parallel, we
evaluated the effect on the thermal nociception by measuring the
latency to a response in a hot plate at 52 ꢀC. As illustrated in
Figure 3, intraperitoneal administration of selected compounds
did not affect the thermal nociception as evidenced by the similar
latency times of animals administered with vehicle and com-
pounds, indicating that TRPV1 blockers do not affect tempera-
ture sensitivity in physiological conditions. In contrast, compounds
significantly decreased the burning sensation of intraplantar
capsaicin application by reducing the duration of the flinching
and licking of the inflamed paw. Administration of capsaicin
induces an acute inflammatory state that results in a nocifensive
response. Therefore, these findings imply that selected hits
display anti-inflammatory activity in vivo by attenuating TRPV1
function in nociceptor terminals; although a synergistic effect on
NMDA receptors cannot be completely discarded because of
their weak interaction with this ion channel. In addition, because
these compounds act as noncompetitive channel blockers, they
may not display the hyperthermic effects observed for competi-
tive antagonists. Further optimization using medicinal chemistry
is necessary to clearly unveil the therapeutic potential of these
hits and to evolve leads for drug development.
’ CONCLUSIONS
A resin-bound Fmoc-Lys(Mtt)-Lys(Alloc) dipeptide derivative
was used as key intermediate for the generation of a hydantoin
library with two points of diversity. After the base-promoted
cyclization of the corresponding NR-DSC-activated intermediate
to the hydantoin ring, the orthogonal deprotection of Lys side-
chains allowed the parallel and successive incorporation of the
diversity elements through simple reactions. This library, joining
the privileged scaffold hydantoin and a series of differently
substituted guanidine and diverse acyl groups, allowed the
discovery of new hits for TRPV1 ion channel blockade, and to
establish the first structural requirements for activity within this
series.
’ EXPERIMENTAL SECTION
Preparation of Resin-Bound Dipeptide 2. The Fmoc-AM-
MBHA-PS resin (3.5 mmol) was swollen in DCM and DMF,
and treated with 20% piperidine in DMF (1 ꢁ 1 min, 3 ꢁ 5 min,
1 ꢁ 10 min) for the removal of the Fmoc group, and washed
with DMF (5 ꢁ 0.5 min), DCM (5 ꢁ 0.5 min), and DMF (5 ꢁ
0.5 min). Then, Fmoc-Lys(Alloc)ꢀOH (2.375 g, 5.25 mmol),
DIPCDI (0.82 mL, 5.25 mmol), and HOBt (0.71 g, 5.25 mmol)
were added in DMF (15 mL) and shaken for 6 h. This coupling
reaction was repeated overnight. After removing the Fmoc group
in the above indicated conditions, Fmoc-Lys(Mtt)ꢀOH (3.28 g,
5.25 mmol), DIPCDI (0.82 mL, 5.25 mmol), and HOBt (0.71 g,
5.25 mmol) were added in DMF (15 mL) and shaken for 12 h.
The second coupling was repeated twice with 1 equiv of the
amino acid derivative and coupling agents. Couplings were
monitored by the Kaiser ninhydrin test.
462
dx.doi.org/10.1021/co1000986 |ACS Comb. Sci. 2011, 13, 458–465

ACS Combinatorial Science
RESEARCH ARTICLE
dissolved in H2O or H2O/ACN mixtures, and lyophilized. All
library members were characterized by HPLC, MS, and H
Biomaterials and Nanomedicine Institute for Research in Biome-
dicine (F.A. and M.R.).
1
NMR, while 13C NMR data was recorded for some representa-
tive compounds (see Supporting Information for details).
Recombinant Rat TRPV1 and NMDAR Channels Expres-
sion in Xenopus Oocytes and Channel Blockade. All the
procedures have been described in detail elsewhere.60 Whole-
cell currents from rat TRPV1-injected oocytes were recorded in
Mg2+-Ringer’s solution (in mM: 10 Hepes pH 7.4, 115 NaCl,
2.8 KCl, 0.1 BaCl2, 2.0 MgCl2) with a two-microelectrode
voltage-clamp amplifier at 20 ꢀC. TRPV1 channels were acti-
vated by application of 10 μM capsaicin in absence or presence of
individual compounds at a holding potential (Vh) of ꢀ80 mV.
Receptor selectivity was evaluated on heterologously expressed
NMDA receptors (rat NR1:NR2A). Recombinant NMDA re-
ceptor responses were activated with 100 μM L-glutamate plus
20 μM glycine in the absence and presence of the compound
in normal Ringer solution (in mM: 10 Hepes pH 7.4, 115 NaCl,
2.8 KCl, 1.8 BaCl2), and at a holding of ꢀ80 mV.
’ DISCLOSURE
Abbreviations used for amino acids follow the IUPAC-IUB
Commission of Biochemical Nomenclature in Jones, J.H. J. Pept.
Sci., 2003, 9, 1ꢀ8.
’ ACKNOWLEDGMENT
We thank Dr. Wim Van Den Nest for kindly synthesizing
some of the active compounds.
’ ABBREVIATIONS
Ac2O, acetic anhydride; ACN, acetonitrile; Alloc, allyloxycarbo-
nyl;CDI, 1,10-carbonyldiimidazole; CNS, central nervous system;
DBU, 1,8-Diazabicyclo[5.4.0]undec-7-ene;DCM, dichlorometh-
ane; DIPCDI, N,N0-diisopropylcarbodiimide; DIEA, diisopropy-
lethylamine; DMAP, 4-Dimethylaminopyridine;DMF, dimethyl-
formamide; DSC, N,N0-Disuccinimidyl carbonate; Fmoc, Fluor-
enylmethoxycarbonyl; HOBt, hydroxybenzotriazole; NMDA, N-
methyl D-aspartate; Mtt, 4-Methyltrityl; Fmoc-Rink amide-
MBHA-PS or Fmoc-AM-MBHA-resin, 4-(20,40dimethoxyphenyl-
Fmoc-aminomethyl)-pheoxyacetamido p-methylbenhidrylamine
polystyrene resin; HPLC, high performance liquid chromatogra-
phy;HPLC-MS, high performance liquid chromatographyꢀmass
spectrometry; MS, mass spectrometry;ESI-MS, electrospray ioni-
zation mass spectrometry; SPPS, solid-phase peptide synthesis;
TES, triethyl silane; TFA, trifluoroacetic acid;TRVP1, transient
receptor potential cation channel, subfamily V, member 1
Behavioral Nociception Assays. Adult male ICR mice were
habituated to the test environment for 24 h in plexiglass chambers
prior the nociception assays. Thermal nociception was studied
using a hot plate at 52 ꢀC. The response latency for paw shaking or
licking or jumping was measured.60
Capsaicin-Induced Hyperalgesia. Capsaicin (10 μL at 0.06%
in 10% ethanol, 10% Tween 80 and 80% saline) was injected
intradermally into the heel pad with a 0.3 mm diameter needle
attached to a Hamilton syringe. The duration time of licking and
shaking the paw in response to the injection was recorded.60
’ ASSOCIATED CONTENT
’ REFERENCES
S
Supporting Information. Detailed characterization of li-
b
brary members: Yields, purities, HPLC, MS, 1H and 13C NMR data.
Full data of TRPV1 and NMDA channel blockade. This material is
available free of charge via the Internet at http://pubs.acs.org.
(1) Lꢀopez, C. A.; Trigo, G. G. In Advances in Heterocyclic Chemistry;
Brown, C., Davidson, R. M., Eds.; Academic Press: New York, 1985; Vol.
38, pp 177ꢀ228.
(2) Kiec-Kononowicz, K.; Szymanska, E. Antimycobacterial activity
of 5-arylidene derivatives of hydantoin. Il Farmaco 2002, 57, 909–916.
(3) Carmi, C.; Cavazzoni, A.; Zuliani, V.; Lodola, A.; Bordi, F.; Plazzi,
P. V.; Alfieri, R. R.; Petroni, P. G.; Mor, M. 5-Benzylidene-hydantoins as
new EGFR inhibitors with antiproliferative activity. Bioorg. Med. Chem.
Lett. 2006, 16, 4021–4025.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: albericio@irbbaracelona.org (F.A.), mroyo@pcb.ub.cat
(M.R.). Fax:+34 93-403-7126 (F.A.), +34 93-402-0496 (M.R.).
Phone: +34 93-403-7088 (F.A.), +34 93-403-7122 (M.R.).
(4) Bakalova, A.; Buyukliev, R.; Tcholakova, I.; Momekov, G.;
Kostantinov, S.; Karavanova, M. Synthesis, physicochemical investiga-
tion and cytotoxic activity of new Pt(II) complexes with hydantoin
ligands. Eur. J. Med. Chem. 2003, 38, 627–632.
Author Contributions
M.R. and F.A. conceived and designed the library and supervised
its synthesis. G.G.N. performed the library synthesis and char-
acterization. R.G.M. supervised the characterization. A.F.C. and
J.M.G.R. performed the biological assays. A.F.M. designed and
supervised the biological evaluation. C.C. supervised the synthesis
of selected compounds and all biological data. R.G.M. and M.R.
cowrote the manuscript.
(5) Opacic, N.; Barbaric, M.; Zorc, B.; Cetina, M.; Nagl, A.; Frkovic,
D.; Kralj, M.; Pavelic, K.; Balzarini, J.; Andrei, G.; Snoeck, R.; De Clercq,
E.; Raic-Malic, S.; Mintas, M. Peptidyl 3-substituted 1-hydroxyureas as
isosteric analogues of succinylhydroxamate MMP inhibitors. J. Med.
Chem. 2005, 48, 475–482.
(6) Jansen, M.; Potschka, H.; Brandt, C.; L€oscher, W.; Dannhardt, G.
Hydantoin-substituted 4,6-dichloroindole-2-carboxylic acids as ligands
with high affinity for the glycine binding site of the NMDA receptor.
J. Med. Chem. 2003, 46, 64–73.
Funding Sources
(7) Zha, C.; Brown, G. B.; Brouillette, W. J. Synthesis and SAR
studies for hydantoins and analogues as voltage-gated sodium channel
ligands. J. Med. Chem. 2004, 47, 6519–6528.
(8) Last-Barney, K.; Davidson, W.; Cardozo, M.; Frye, L. L.; Grygon,
C. A.; Hopkins, J. L.; Jeanfavre, D. D.; Pav, S.; Quian, C.; Stevenson,
J. M.; Tong, L.; Zindell, R.; Kelly, T. A. Binding site elucidation of
hydantoin-based antagonists of LFA-1 using multidisciplinary technol-
ogies: evidence for the allosteric inhibition of a protein-protein interac-
tion. J. Am. Chem. Soc. 2001, 123, 5643–5650.
This research has been partially supported by grants from the
Ministry of Science and Innovation (CONSOLIDER-INGENIO
2010 (CSD2008ꢀ00005 to R.G.M. and A.F.M.), SAF2009ꢀ
09323 (to R.G.M)., BFU2009ꢀ08346 (to A.F.M.), BQU2006ꢀ
03794 and CTQ2009ꢀ20541 (to F.A.), CTQ2005ꢀ00315/
BQU and CTQ2008ꢀ00177 (to M.R.) la Generalitat Valenciana
(PROMETEO/2010/046 to A.F.M.), and la Maratꢀo de TV3 (to
A.F.M.), and CIBER-BBN, Networking Centre on Bioengineering,
463
dx.doi.org/10.1021/co1000986 |ACS Comb. Sci. 2011, 13, 458–465

ACS Combinatorial Science
RESEARCH ARTICLE
(9) Potin, D.; Launay, M.; Nicolai, E.; Fabreguette, M.; Malabre, P.;
Caussade, F.; Besse, D.; Skala, S.; Stetsko, D. K.; Todderud, G.; Reno,
B. R.; Cheney, D. L.; Chang, C. J.; Sheriff, S.; Hollenbaugh, D. L.; Barrish,
J. C.; Iwanowicz, E. J.; Suchard, S. J.; Dhar, T. G. M. De novo design,
synthesis, and in vitro activity of LFA-1 antagonists based on a bicyclic-
[5.5]hydantoin scaffold. Bioorg. Med. Chem. Lett. 2005, 15, 1161–1164.
(10) Evans, B. E.; Rittle, K. E.; Bock, M. G.; DiPardo, R. M.;
Freidinger, R. M.; Whitter, W. L.; Lundell, G. F.; Veber, D. F.; Anderson,
P. S.; Chang, R. S. L.; Lotti, V. J.; Cerino, D. J.; Chen, T. B.; Kling, P. J.;
Kunkel, K. A.; Springer, J. P.; Hirsfield, J. Methods for drug discovery:
development of potent, selective, orally effective cholecystokinin an-
tagonists. J. Med. Chem. 1988, 31, 2235–2246.
(11) Meusel, M.; G€utschow, M. Recent Developments in Hydantoin
Chemistry. A Review. Org. Prep. Proced. Int. 2004, 36, 391–443.
(12) Reyes, S.; Burgess, K. On Formation of Thiohydantoins from
Amino Acids under Acylation Conditions. J. Org. Chem. 2006, 71, 2507.
(13) Murray, R. G.; Whitehead, D.; Le Strat, F.; Conway, S. J. Facile
one-pot synthesis of 5-substituted hydantoins. Org. Biomol. Chem. 2008,
6, 988.
(14) Hulme, C.; Ma, L.; Romano, J. J.; Morton, G.; Tang, S.-Y.;
Cherrier, M.-P.; Choi, S.; Salvino, J.; Labaudiniere, R. Novel applications
of convertible isonitriles for the synthesis of mono and bicyclic γ-lactams
via a UDC strategy. Tetrahedron Lett. 2000, 41, 1889–1893.
(15) Zhang, W.; Lu, Y.; Chen, H.-T.; Zeng, L.; Kassel, D. B. Fluorous
Mixture Synthesis of Two Libraries with Hydantoin- and Benzodiazepi-
nedione-Fused Heterocyclic Scaffolds. J. Comb. Chem. 2006, 8, 687–695.
(16) DeWitt, H. S.; Kiely, J. S.; Stankovic, C. J.; Schroeder, M. C.;
Cody, D. M. R.; Pavia, M. R. ”Diversomers”: an approach to nonpeptide,
nonoligomeric chemical diversity. Proc. Natl. Acad. Sci. U.S.A. 1993, 90,
6909–6913.
(17) Mattews, J.; Rivero, R. A. Base-Promoted Solid-Phase Synthesis
of Substituted Hydantoins and Thiohydantoins. J. Org. Chem. 1997, 62,
6090–6092.
(18) Kim, S. W.; Ahn, S. Y.; Koh, J. S.; Lee, J. H.; Ro, S.; Cho, H. Y.
Solid phase synthesis of hydantoin library using a novel cyclization and
traceless cleavage step. Tetrahedron Lett. 1997, 38, 4603–4606.
(19) Kim, S. W.; Koh, J. S.; Lee, E. J.; Ro, S. Solid phase synthesis of
benzamidine and butylamine-derived hydantoin libraries. Mol. Diversity
1998, 3, 129–132.
(20) Boeijen, A.; Kruijtzer, J. A. W.; Liskamp, R. M. J. Combinatorial
chemistry of hydantoins. Bioorg. Med. Chem. Lett. 1998, 8, 2375–2380.
(21) Karnbrock, W.; Deeg, M.; Gerhardt, J.; Rapp, W. Solid phase
synthesis of hydantoins by thermal cyclization and screening of reaction
conditions using APOS 1200. Mol. Diversity 1998, 3, 165–171.
(22) Gong, Y.-D.; Najdi, S.; Olmstead, M. M.; Kurth, M. J.
Solid-Phase Synthesis: Intramolecular Azomethine Ylide Cycloaddition
(fProline) and Carbanilide Cyclization (fHydantoin) Reactions.
J. Org. Chem. 1998, 63, 3081–3086.
(23) Park, K.-H.; Kurth, M. J. An uncatalyzed cyclo-elimination
process for the release ofN3-alkylated hydantoins from solid-phase:
synthesis of novel isoxazoloimidazolidinediones. Tetrahedron Lett. 1999,
40, 5841–5844.
Spiro-2,5-diketopiperazines via Resin-Bound Cyclic R,R-Disubstituted
R-Amino Esters. J. Comb. Chem. 2006, 8, 85–94.
(29) Yeh, W.-P.; Chang, W.-J.; Sun, M.-L.; Sun, C.-M. Microwave-
assisted traceless synthesis of hydantion-fused β-carboline scaffold.
Tetrahedron 2007, 63, 11809–11816.
(30) Dressman, B. A.; Spangle, L. A.; Kaldor, S. W. Solid phase
synthesis of hydantoins using a carbamate linker and a novel cyclization/
cleavage step. Tetrahedron Lett. 1996, 37, 937–940.
(31) Xiao, X.-Y.; Ngu, K.; Chao, C.; Patel, D. V. Selective Solid Phase
Synthesis of Ureas and Hydantoins from Common Phenyl Carbamate
Intermediates. J. Org. Chem. 1997, 62, 6968–6973.
(32) Chong, P. Y.; Petillo, P. A. Solid phase hydantoin synthesis: An
efficient and direct conversion of Fmoc-protected dipeptides to hydan-
toins. Tetrahedron Lett. 1999, 40, 2493–2496.
(33) Bhalay, G.; Cowell, D.; Hone, N. D.; Scobie, M.; Baxter, A. D.
Multiple solid-phase synthesis of hydantoins and thiohydantoins. Mol.
Diversity 1997, 3, 195–198.
(34) Nefzi, A.; Dooley, C.; Ostresh, J. M.; Houghten, R. A. Combi-
natorial chemistry: From peptides and peptidomimetics to small organic
and heterocyclic compounds. Bioorg. Med. Chem. Lett. 1998, 8, 2273–2278.
(35) Nefzi, A.; Ostresh, J. M.; Giulianotti, M.; Houghten, R. A.
Efficient solid phase synthesis of 3,5-disubstituted hydantoins. Tetra-
hedron Lett. 1998, 39, 8199–8202.
(36) Nefzi, A.; Giulianotti, M.; Truong, L.; Rattan, S.; Ostresh, J. M.;
Houghten, R. A. Solid-Phase Synthesis of Linear Ureas Tethered to
Hydantoins and Thiohydantoins. J. Comb. Chem. 2002, 4, 175–178.
(37) Severinsen, R.; Lau, J. F.; Bondensgaard, K.; Hansen, B. S.;
Begtrup, M.; Ankersen, M. Parallel solid-phase synthesis of disubstituted
(5-biphenyltetrazolyl) hydantoins and thiohydantoins targeting the
growth hormone secretagogue receptor. Bioorg. Med. Chem. Lett. 2004,
14, 317–320.
(38) Royo, M.; Van der Nest, W.; Del Fresno, M.; Frieden, A.;
Yahalom, D.; Rosenblatt, M.; Chorev, M.; Albericio, F. Solid-phase
syntheses of constrained RGD scaffolds and their binding to the Rvβ3
integrin receptor. Tetrahedron Lett. 2001, 42, 7387–7391.
(39) Vazquez, J.; García-Jare~no, A.; Mondragꢀon, L.; Rubio-Martínez,
J.; Pꢀerez-Payꢀa, E.; Albericio, F. Conformationally Restricted Hydantoin-
Based Peptidomimetics as Inhibitors of Caspase-3 with Basic Groups
Allowed at the S3 Enzyme Subsite. ChemMedChem 2008, 3, 979–985.
(40) Ekelund, S.; Nygren, P.; Larsson, R. Guanidino-containing
drugs in cancer chemotherapy: biochemical and clinical pharmacology.
Biochem. Pharmacol. 2001, 61, 1183–1193.
(41) Ravaud, A.; Cerny, T.; Terret, C.; Wanders, J.; Bui, B. N.; Hess,
D.; Droz, J.-P.; Fumoleau, P.; Twelves, C. Phase I study and pharma-
cokinetic of CHS-828, a guanidino-containing compound, administered
orally as a single dose every 3 weeks in solid tumours: An ECSG/
EORTC study. Eur. J. Cancer 2005, 41, 702–707.
(42) Sienczyk, M.; Oleksyszyn, J. Inhibition of trypsin and urokinase
by Cbz-amino(4-guanidinophenyl)methanephosphonate aromatic ester
derivatives: The influence of the ester group on their biological activity.
Bioorg. Med. Chem. Lett. 2006, 16, 2886–2890.
(43) Katsura, Y.; Nishino, Y.; Inoue, Y.; Sakane, K.; Matsumoto, Y.;
Morinaga, C.; Ishikawa, H.; Takasugi, H. Anti-Helicobacter pylori agents.
5. 2-(substituted guanidino)-4-arylthiazoles and Aryloxazole Analogues.
J. Med. Chem. 2002, 45, 143–150.
(24) Huang, W.; Cheng, S.; Sun, W. 2-Polystyrylsulfonyl ethanol
supports for the solid-phase syntheses of hydantoins and ureas. Tetra-
hedron Lett. 2001, 42, 1973–1974.
(25) Park, K.-H.; Ehrler, J.; Spoerri, H.; Kurth, M. J. Preparation of a
990-Member Chemical Compound Library of Hydantoin- and Isoxazo-
line-Containing Heterocycles Using Multipin Technology. J. Comb.
Chem. 2001, 3, 171–176.
(26) Lamothe, M.; Lannuzel, M.; Perez, M. Solid-Phase Preparation
of Hydantoins through a New Cyclization/Cleavage Step. J. Comb.
Chem. 2002, 4, 73–78.
(44) Oh, C.-H.; Cho, J.-H. Synthesis and biological evaluation of 1β-
methylcarbapenems having guanidino moieties. Eur. J. Med. Chem. 2006,
41, 50–55.
(45) Masuda, T.; Yoshida, S.; Arai, M.; Kaneko, S.; Yamashita, M.;
Honda, T. Synthesis and anti-influenza evaluation of polyvalent pialidase
inhibitors bearing 4-guanidino-Neu5Ac2en derivatives. Chem. Pharm.
Bull. 2003, 51, 1386–1398.
(27) Alsina, J.; Scott, W. L.; O’Donnell, M. J. Solid-phase synthesis of
R-substituted proline hydantoins and analogs. Tetrahedron Lett. 2005,
46, 3131–3135.
(28) Kuster, G. J. T.; van Berkom, L. W. A.; Kalmoua, M.; van
Loevezijn, A.; Sliedregt, L. A. J. M.; van Steen, B. J.; Kruse, C. G.;
Rutjes, F. P. J. T.; Scheeren, H. W. Synthesis of Spirohydantoins and
(46) Huang, H.; Martasek, P.; Roma, L. J.; Silverman, R. B. Synthesis
and evaluation of peptidomimetics as selective inhibitors and active site
probes of Nitric Oxide Synthases. J. Med. Chem. 2000, 43, 2938–2945.
(47) Lam, P. Y. S.; Clark, C. G.; Li, R.; Pinto, D. J. P.; Orwat, M. J.;
Galemmo, R. A.; Fevig, J. M.; Teleha, C. A.; Alexander, R. S.; Smallwood,
A. M.; Rossi, K. A.; Wright, M. R.; Bai, S. A.; He, K.; Luettgen, J. M.;
464
dx.doi.org/10.1021/co1000986 |ACS Comb. Sci. 2011, 13, 458–465

ACS Combinatorial Science
RESEARCH ARTICLE
Wong, P. C.; Knabb, R. M.; Wexler, R. R. Structure-based design of novel
guanidine/benzamidine mimics: potent and orally bioavailable factor Xa
inhibitors as novel anticoagulants. J. Med. Chem. 2003, 46, 4405–4418.
(48) Soyka, R.; Guth, B. D.; Weisenberger, H. M.; Luger, P.; M€uller,
T. H. Guanidine derivatives as combined thromboxane A2 receptor
antagonists and synthase Inhibitors. J. Med. Chem. 1999, 42, 1235–1249.
(49) Lack, S. L.; Chaovignac, C.; Grundt, P.; Miller, C. N.; Wood, S.;
Traynor, J. R.; Lewis, J. W.; Husbands, S. M. Guanidino N-substituted
and N,N-disubstituted derivatives of the k-opioid antagonist GNTI.
J. Med. Chem. 2003, 46, 5505–5511.
(50) Suagse, K.; Horikawa, M.; Sugiyama, M.; Ishiguro, M. Restric-
tion of a peptide turn conformation and Conformational analysis of
guanidino group using arginine-proline fused amino acids: application to
mini Atrial Natriuretic peptide on binding to the receptor. J. Med. Chem.
2004, 47, 489–492.
(51) Lee, S.; Yi, K. Y.; Hwang, S. K.; Lee, B. H.; Yoo, S.-E.; Lee, K.
(5-Arylfuran-2-ylcarbonyl)guanidines as cardioprotectives through the
inhibition of Na+/H+ exchanger isoform-1. J. Med. Chem. 2005, 48,
2882–2891.
(52) DelFresno, M.; El-Faham, A.; Carpino, L. A.; Royo, M.;Albericio,
F. Substituted guanidines: introducing diversity in combinatorial chem-
istry. Org. Lett. 2000, 2, 3539–3542.
(53) Vꢀazquez, J.; Royo, M.; Albericio, F. Re-evaluation of a solid-
phase hydantoin synthesis. Lett. Org. Chem. 2004, 1, 224–226.
(54) Caterina, M. J.; Julius, D. The Vanilloid receptor: a molecular
gateway to the pain pathway. Annu. Rev. Neurosci. 2001, 24, 487–517.
(55) Jara-Oseguera, A.; Simon, S. A.; Rosenbaum, T. TRPV1: on the
road to pain relief. Curr. Mol. Pharmacol. 2008, 1, 255–269.
(56) 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.
(57) Messeguer, A.; Planells-Cases, R.; Ferrer-Montiel, A. Physiol-
ogy and pharmacology of the vanilloid receptor. Curr. Neuropharmacol.
2006, 4, 1–5.
(58) Garcia-Martinez, 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 non competitive
antagonist of the TRVP1channel with in vivo analgesic and anti-
inflammatory activity. J. Pain 2006, 7, 735–746.
(59) Albensi, B. C. The NMDA receptor/ion channel complex: a
drug target for modulating synaptic plasticity and excitotoxicity. Curr.
Pharm. Des. 2007, 13, 3185–3194.
(60) García-Martínez, C.; Humet, M.; Planells-Cases, R.; Gomis, A.;
Caprini, M.; Viana, F.; De la Pe~na, E.; Sanchez-Baeza, F.; Carbonell, T.;
De Felipe, C.; 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.
465
dx.doi.org/10.1021/co1000986 |ACS Comb. Sci. 2011, 13, 458–465