Allosteric functional switch of neurokinin A-mediated signaling at the neurokinin NK2 receptor: Structural exploration

Article abstract of DOI:10.1021/jm900671k

The neurokinin NK2 receptor is known to pre-exist in equilibrium between at least three states: restinginactive, calcium-triggering, and cAMP-producing. Its endogeneous ligand, NKA, mainly induces the calcium response. Using a FRET-based assay, we have previously discovered an allosteric modulator of the NK2 receptor that has the unique ability to discriminate among the two signaling pathways: calcium-signaling is not affected while cAMP signaling is significantly decreased. A series of compounds have been prepared and studied in order to better understand the structural determinants of this allosteric functional switch of a GPCR. Most of them display the same allosteric profile, with smooth pharmacomodulation. One compound however exhibits significantly improved modulatory properties of NKA induced signaling when compared to the original modulator. 2009 American Chemical Society.

Full text of DOI:10.1021/jm900671k

J. Med. Chem. 2009, 52, 5999–6011 5999
DOI: 10.1021/jm900671k
Allosteric Functional Switch of Neurokinin A-Mediated Signaling at the Neurokinin NK2 Receptor:
Structural Exploration
†
§
†
‡
§
§
ꢀ
ꢀ
Celine Valant, Emeline Maillet, Jean-Jacques Bourguignon, Bernard Bucher, Valerie Utard, Jean-Luc Galzi, and
Marcel Hibert*,†
†UMR UdS/CNRS 7200, Laboratoire d’Innovation Theꢀrapeutique and ‡UMR UdS/CNRS 7213 Laboratoire de Biophotonique et Pharmacologie,
§
Faculteꢀ de Pharmacie, 74 Route du Rhin, BP 60024, 67401 Illkirch, France, and FRE UdS/CNRS 3211, Institut de Recherche de l’Ecole de
ꢀ
Biotechnologie, ESBS, Boulevard Seꢀbastien Brant, 67401 Illkirch, France
Received May 20, 2009
The neurokinin NK2 receptor is known to pre-exist in equilibrium between at least three states: resting-
inactive, calcium-triggering, and cAMP-producing. Its endogeneous ligand, NKA, mainly induces the
calcium response. Using a FRET-based assay, we have previously discovered an allosteric modulator of
the NK2 receptor that has the unique ability to discriminate among the two signaling pathways:
calcium-signaling is not affected while cAMP signaling is significantly decreased. A series of compounds
have been prepared and studied in order to better understand the structural determinants of this
allosteric functional switch of a GPCR. Most of them display the same allosteric profile, with smooth
pharmacomodulation. One compound however exhibits significantly improved modulatory properties
of NKA induced signaling when compared to the original modulator.
Introduction
Bodipy4,7), exists in equilibrium between predominantly three
states: inactive (R), calcium-triggering (conformation A1)
characterized by a rapid association and dissociation of the
endogeneous agonist NKA, and cAMP-producing (confor-
mation A2), characterized by slow association and slow dis-
sociation of the agonist.
We have screened 1600 drug-like molecules issued from our
laboratory in a FRET-based binding assay in order to find
antagonist ligands. Interestingly, this study led to the discov-
ery of compound 1 (Chart 1), a new allosteric ligand for the
neurokinin NK2 receptor. This ligand exhibited the particu-
larity of being a strong negative allosteric modulator for the
cAMP-producing conformation (A2) while not affecting or
slightly potentiating the calcium triggering conformation
(A1).8 In fact, in the presence of 20 nM of NKA-Bo, the
receptor was stabilized in a 30/70 ratio of A1/A2 conformat-
ions. Upon addition of 10μM of 1, stillin presence of 20nM of
NKA-Bo, the NK2 receptor was stabilized in a 50/50 ratio of
A1/A2 conformations. Finally, addition of 50 μM of 1
balances the receptor to a 95/5 maximal ratio of A1/A2
conformations (Table 1).8
Allosterism is a well-known process that has been exten-
sively studied in enzymes and in some receptors. However, the
study of this phenomenon for G protein-coupled receptors
(GPCRa) is relatively recent, probably due to the lack of
simple detection methods.1,2 Because allosteric interactions
could be quite subtle, many ways of studying allosteric
behaviors were established and can be used individually or
in tandem. Among them, the study of the effect of an allosteric
ligand on the dissociation of a labeled ligand to a receptor,
which measures the ability of an allosteric ligand to alter the
dissociation rate constant (Koff) of the orthosteric labeled
ligand in time, is one of the most sensitive, direct, and
unequivocal method to validate an allosteric interaction.
We have demonstrated that fluorescence resonance energy
transfer3 (FRET) represents an efficient and informative way
to study GPCR-ligand interaction.4 Indeed, a very specific
and sensitive FRET signal can be observed when an appro-
priate fluorescent ligand binds to a GPCR fused to a fluor-
escent protein, such as enhanced green fluorescent protein
(EGFP).5 Binding can be easily analyzed by a simple measure-
ment of EGFP emission. We have used FRET-based binding
analysis to study several GPCRs,6 including the neurokinin
A (NKA) receptor, known as NK2 receptor. In this particular
case, FRET revealed that the recombinant human neurokinin
NK2 receptor, in the presence of NKA-Bo (NKA linked to
We report here the structure-activity relationship study
around this allosteric functional switch of a GPCR, the
neurokinin NK2 receptor, and characterize our best opti-
mized hit, compound 16, in both calcium mobilization and
cAMP accumulation assays.
Methods
*To whom correspondence should be addressed. Phone: þ33390244232.
Fax: þ33390244310. E-mail: mhibert@pharma.u-strasbg.fr.
a Abbreviations: FRET, fluorescence resonance energy transfer; BRET,
bioluminescence resonance energy transfer; GPCR, G-protein-coupled re-
ceptor; EGFP, enhanced green fluorescent protein; NK2R, NK2 receptor;
NKA-Bo, NKA linked to the Bodipy fluorophore; NK2-EGFP, NK2 receptor
expressed as a fusion protein with the enhanced green fluorescent protein on
the N-terminal.
Chemistry. The synthesis of 1, [N-benzyl,N-(2-naphthyl-
methyl)-amino]-acetonitrile (Chart 1), has previously been
reported.8,9 Synthesis of 31 derivatives showing structural
similarity to 1 was undertaken, based on the substitution of
the three groups around the central tertiary amine. The 32
compounds were synthesized according to standard procedures
r
2009 American Chemical Society
Published on Web 09/11/2009
pubs.acs.org/jmc

6000 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Valant et al.
(Scheme 1). Two methods were followed to obtain the whole
set of compounds. Method A allowed the synthesis of 22
compounds (1-20, 31, 32) by two successive steps, a reduc-
tive amination between an aldehyde and an amine exhibiting
the R2 group followed by an alkylation of the secondary
amine. A further eight compounds were synthesized by
converting the N,N-(2-methylnaphthyl-benzyl)-2-amino-
ethanoic acid 18 into bulky amides 25-28 or bulky esters
21-24 in peptide coupling conditions. Method B was fol-
lowed to synthesize two additional compounds by conden-
sing first an aldehyde and an amine exhibiting the R2 group,
followed by an alkylation of the secondary amine, leading to
the compounds 29 and 30. Yields and chemical, physical and
spectroscopic characteristics of 1 and its 31 analogues are
reported in the Experimental Section and in Supporting
Information.
Biology. The FRET-based assay was a measurement
of fluorescence emission of EGFP as a function of time at
510 nm.4,6-8 It proceeds through three different phases
(Figure 1) and allows determination if a nonfluorescent
molecule behaves as a competitor of the fluorescent molecule
or if it rather modulates the interaction of the fluorescent
molecule with the receptor. The phase P1 corresponds to the
real time recording of the association of fluorescent NKA-
Bo (20 nM) to EGFP-labeled NK2 receptor. The observed
decrease of EGFP fluorescence emission at 510 nm reflects
energy transfer from excited EGFP (linked to the receptor)
to the fluorophore bodipy (linked to neurokinin A) that
develops over time. The phase P2 corresponds to the incubat-
ion period with the compound (10 μM) to be tested. If the test
compound is a competitor of NKA-Bo, an upward deflect-
ion is detected. This reflects partial-to-total dissociation of
NKA-Bo from its binding site and consequent reduction of
energy transfer. Alternatively, the compound may either not
bind to the receptor or may behave as an allosteric modul-
ator. It this case, no signal would be detected during phase
P2. The final phase, P3, corresponds to NKA-Bo dissociat-
ion from NK2-EGFP receptor following addition of 20 μM
of unlabeled NKA. During this phase, each NKA-Bo mole-
cule that dissociates from its binding site is replaced by an
unlabeled NKA molecule present in large excess. The rate of
NKA dissociation, determined from the fitting of phase P3
dissociation trace, is thus directly linked to the dissociation
constant of NKA-Bo for a given conformation of the NK2
receptor. In previous studies,6,8 fluorescent NKA was shown
to dissociate from two conformations of the NK2 receptor,
the relative abundance of which depended on the incubation
time or on the pharmacological agent added to the preparat-
ion. A rapid dissociation rate associated to a low affinity
conformation characterized as a calcium signaling state was
identified, together with a slow dissociation rate corresponding
Chart 1. Structure of 1, [N-Benzyl,N-(2-naphthylmethyl)-
amino]-acetonitrile, and Pharmacomodulation Positions (in
Circles)
Table 1. Percentages of NK2-R Active Conformations A1 and A2 with
or without Compound 1 in the Presence of 20 nM NKA-Bo
quick dissociation
(A1 conformation),
%
slow dissociation
(A2 conformation),
%
without 1
1 at 10 μM
1 at 50 μM
30
50
95
70
50
5
Scheme 1. Synthesis of Analogues of the Allosteric Modulator 1a
a Reagents and conditions: (i) MeOH, 60 ꢀC; (ii) NaBH4, RT; (iii) NEt3, DMF, 60 ꢀC; (iv) amine, BOP, NMM, DMF, RT; (v) alcohol, BOP, NMM, DMF, RT.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 6001
Figure 3. Effect of compounds 1 and 16 on NKA-evoked cAMP
production in HEK cells. cAMP dose-response relationship to
increasing concentrations of NKA (O, EC50=64.8 ( 2.1 nM) were
recorded. The effect of the presence of 10 μM compound 1 (0, EC50=
74.8 ( 3.5 nM) or compound 16 (9, EC50=64.8 ( 1.4 nM) is shown.
Each data point is the mean ( SD of six determinations. Compound 1
reduces the maximal amplitude of the cAMP response by 33 ( 5% and
compound 16 reduces it by 47 ( 4% at the same concentration,
showing improved activity over compound 1.
Figure 1. Principle of the FRET-based assay exemplified with
compound 1. Association phase P1: at time t=50 s, 20 nM NKA-
Bo are added to a suspension of cells expressing EGFP-NK2R and
association is followed as a decrease of EGFP fluorescence intensity
at 510 nm. Incubation with test compound phase P2: at 450 s, the
tested allosteric compound is added and equilibration is allowed to
proceed for 150 s. Dissociation phase P3: at 600 s, 20 μM of the
nonfluorescent ligand NKA are added and NKA-Bo dissociation is
recorded. In the absence of allosteric modulator, NKA-Bo dissoci-
ates according to the blue trace; in the presence of the allosteric
compound 1, it dissociates according to the red trace.
presented here with imposed rate constants equal to 0.03 s-1
for rapid dissociation and 0.006 s-1 for slow dissociation.
When excess of known competitive ligands such as unlabeled
NKA or the competitive NK2 receptor antagonist SR 48968
were used as competitors, dissociation of NKA-Bo followed
a biexponential time course with a rapid dissociation rate
(k1=0.03 s-1) accounting for 70% of the signal and a slow
dissociation rate (k2=0.006 s-1) accounting for 30% of the
signal amplitude, resulting in a 30/70 ratio A1/A2 conforma-
tions as described in Maillet et al.8
Results and Discussion
The FRET-based screening at 10 μM concentration of 1600
compounds from our laboratory collection on the NK2-
EGFP receptor led to the discovery of compound 1
(Chart 1). This hit was unable to fully reverse the binding of
NKA-Bo alone, but in the presence of an excess of NKA,
compound 1 significantly accelerated the dissociation kinetics
ofNKA-Bo(Table 1), showednoeffect on (orweakpotentiat-
ion of) the calcium-induced pathway (Figure 2), and drama-
tically inhibited NKA-induced cAMP response of the recep-
tor (Figure 3). Because the dissociation rate of an orthosteric
ligand can only be modified by a ligand able to bind simulta-
neously to the receptor and, therefore, on a topographically
different binding site than the orthosteric ligand, the phenom-
enon of allosteric interaction between compound 1 with
NKA-Bo to the neurokinin NK2 receptor was validated and
confirms the existence of a ÆNKA-compound 1-NK2
receptoræ ternary complex. Analysis of association kinetics
of NKA revealed that 1 does not alter the affinity of the
agonist for the different receptor conformations but rather
prevents the formation of the “cAMP-triggering conforma-
tion” while not affecting the formation of “calcium-triggering
conformation” of the NKA-NK2R complex.8 Compound 1
was thus identified as the first allosteric modulator of the
neurokinin 2 receptor that discriminates among biologically
active conformations of the receptor and exerts a selective,
allosteric functional switch of a GPCR upon endogeneous
ligand activation. Our aim here was to explore structural
elements of the structure of 1 that determine its potential to
increase the population of A1 conformation at a 10 μM
concentration of allosteric ligand.
Figure 2. Effect of compounds 1 and 16 on NKA-evoked intracel-
lular calcium. Intracellular calcium release dose-response relation-
ship to increasing concentrations of NKA (O, EC50=0.55 ( 0.05
nM) were recorded. The effect of the presence of compound 1 (open
squares, EC50=0.50 ( 0.05 nM) or compound 16 (9, EC50=0.62 (
0.04 nM) is shown. While compound 1 does not modify the
sensitivity to NKA nor the amplitude of the response, compound
16 enhances the amplitude of the maximal response to 120 ( 5%.
to a high affinity cAMP-signaling state. In the present work,
dissociation traces are fitted using this two state model with
the equation: F(t) = λ1(k1t) þ λ2(k2t), where F(t) is the
variation of fluorescence over time, λ1 and λ2 were the
amplitudes of the rapid and slow dissociation processes
characterized by rate constants k1 and k2, respectively.
Best fits of dissociation traces were obtained according to
Maillet et al.8 with two exponential relaxations. The relative
amplitudes λ1 and λ2 for rapid and slow dissociations were
fitted using the least-squares method and assigned to the
corresponding conformations denominated A1 (for rapidly
dissociating, calcium signaling) and A2 (for slowly dissociat-
ing, cAMP-signaling) depending on the compound present.
When test compounds did not exhibit a competitive char-
acter (all but compounds 11, 30, 31, 32), determination of
relative A1 and A2 abundances did not require rapid and
slow rate constants to be fixed. For compounds 11, 30, 31,
and 32, the residual amplitude of dissociation in the prese-
nce of NKA was reduced and rate constant determinations
were less precise. For the sake of homogeneity, all data are
We attempted to modify the structure of 1 to obtain
analogues with as low as possible competitive character and

6002 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Valant et al.
Table 2. Allosteric Activity of 1 and of Its Benzyl Analogues, at 10 μM
in a Kinetic FRET-Based Assay
Table 4. Allosteric Activity of Acetonitrile Analogues of 1, at 10 μM in a
Kinetic FRET-Based Assay
% of A1
FRET
FRET
inhibitionb,
%
compd
15 propargyl
16 butyronitril
17 ethyl ethanoate
18 acetic acid
R2
conformationa inhibitionb, % nc
% of A1
conformationa
compd
R1
nc
51 ( 6
68 ( 1
51 ( 6
35 ( 2
52 ( 3
44 ( 3
38 ( 0
46 ( 2
53 ( 3
42 ( 2
54 ( 2
44
0
0
3
3
3
3
3
3
2
3
3
4
3
1
1
3
2
3
2
3
1
2
3
4
5
6
benzyl
50 ( 3
45 ( 1
43 ( 2
47 ( 3
39
0
0
10
3
0
4-bromobenzyl
2-bromobenzyl
4-iodobenzyl
phenethyl
0
0
3
19 ethyl pentoate
20 pentanoic acid
0
0
3
0
20
0
1
21 hexadecanoyl ethanoate
22 phenethyl ethanoate
23 2-hydroxyethyl ethanoate
24 morpholinoethyl ethanoate
25 butyl acetamide
26 piperidino-acetamide
27 N-methylpiperazino-acetamide
28 N-phenylpiperazino-acetamide
29 3-pyridine
0
4-methoxybenzyl
54 ( 1
2
0
a Percentage of A1 conformation, evaluated with a two exponential
equation. b FRET inhibition, expressed as a percentage of the reversal in
FRET caused by the NKA. c n, number of experiments.
0
0
0
0
Table 3. Allosteric Activity of Naphthyl Analogues of 1, at 10 μM in a
Kinetic FRET-Based Assay
58
0
43 ( 2
59 ( 2
51 ( 1
56 ( 4
61 ( 3
0
% of A1
FRET
0
compd
Ar
conformationa inhibitionb, % nc
30 2-methylpiridine
31 2-pyridine
32 4-pyrimidine
24
17
34
1
benzyl
3-chlorobenzyl
4-chlorobenzyl
50 ( 3
45
53
0
0
0
10
1
7
a Percentage of A1 conformation, evaluated with a two exponential
equation. b FRET inhibition, expressed as a percentage of the reversal in
FRET caused by the NKA. c n, number of experiments.
8
1
9
(1H)indol-3ylmethyl
quinolin-2-ylmethyl
quinolin-3-ylmethyl
cinnamyl
1
10
11
12
13
14
47 ( 4
46 ( 4
47 ( 3
49 ( 2
48 ( 3
0
21
0
3
3
The importance of the naphthalen-2-ylmethyl moiety (Ar)
of molecule 1 was then explored (Table 3). Its replacement by
halogeno-benzyl groups, such as in compounds 7 and 8, had
little influence on the allosteric activity (44-53% of A1
conformation). Replacement with an indol (analogue 9)
resulted in a cytotoxic compound, which could not be studied
further. The quinolinyl group in 10 and 11, which could be
viewed as hydrogen bond acceptor, did not lead to com-
pounds better than 1. However, it is noteworthy that among
the quinolinyl analogues, the quinolin-3-ylmethyl one (11)
promoted a strong inhibition of FRET, supporting a compe-
titive binding in addition to allosteric modulation, while the
quinolin-2-ylmethyl one (10) conserved a pure noncompeti-
tive allosteric behavior.
To probe the importance of the rigidity of the bicycle in 1,
the cinnamyl analogue 12 was synthesized. This compound
exhibited an allosteric activity quite similar to 1 (47% of A1
conformation). Interestingly, replacement of the naphthalen-
2-ylmethyl moiety with a benzyl group substituted by an
electron donating -OMe (13) or an electron withdrawing
-CF3 group (14) had no effect on the activity of the ligands.
Taken together, these results indicate that replacing the
naphthalen-2-ylmethyl group by diverse aromatic moieties
did not increase significantly the allosteric modulator activity.
This suggests that the Argrouplocated near the tertiary amine
does not make strong specific interactions with the receptor
and is most probably involved in the establishment of aroma-
tic-aromatic interactions between the allosteric ligand and
the allosteric site of the receptor.
3
4-methoxybenzyl
4-trifluoromethylbenzyl
0
3
0
3
a Percentage of A1 conformation, evaluated with a two exponential
equation. b FRET inhibition, expressed as a percentage of the reversal in
FRET caused by the NKA. c n, number of experiments.
as high as possible modulating properties. Compounds are
validated, using the FRET-based assay, by (i) a lack
of inhibition of NKA-Bo binding following the addition
of the test compound and (ii) an increase in the fraction
of receptors inducing calcium production, and further
characterized in functional assays. In this study, we report
the structure and the allosteric activity of 31 analogues of 1.
Their ability to induce an inhibition of FRET by themselves
or to affect NKA-Bo dissociation kinetics at 10 μM
was analyzed. Most compounds interact with the NK2R.
With the exception of compounds 5, 11, 30, 31, and 32,
which could not be assigned as competitive agents or as
negative allosteric effectors of NKA-Bo binding, all other
ligands behave as noncompetitive, allosteric switches favor-
ing the NKA-Bo induced calcium signaling states at the
expense of the NKA-induced cAMP signaling (Tables 2, 3,
and 4). We first modified the benzyl substituent (R1) of 1
(Chart 1 and Table 2). An introduction of a halogen atom
on the benzyl ring either in position 4 or 2, such as in
compounds 2, 3, and 4, lead to a nonsignificant reduction in
the allosteric activity of the ligands (3-5% reduction of A1
conformation). Another modification spacing the aromatic
ring from the central nitrogen atom, such as replacement of
the benzyl by a phenethyl group in analogue 5, did not
improve the efficacy of the allosteric ligand. In contrast,
introduction of an electron donor group, such as a
4-methoxy-benzyl in 6, seemed to lead to a slightly better
efficacy (54% of A1 conformation). If the substitution of
the benzyl domain by halogen atoms had a slightly negative
effect on the allosteric activity of the compounds, substitut-
ion with an electron donor group weakly reinforced the
allosteric activity, but overall, the substitution of the benzyl
group did not have a significant impact on the modulation
of allosteric activity.
The contribution of the acetonitrile substituent (R2) of the
central nitrogen was then investigated (Table 4). The carboni-
trile was first replaced by an alkyne group (compound 15).
This compound showed an allosteric activity corresponding
to 51% A1 conformation, thus equally active to compound 1,
suggesting that the nitrile was not involved in any hydrogen
bond but rather acted as an electron-rich, polarizable moiety.
The location of the nitrile was then explored in preparing
compound 16, which is the superior homologue of 1. Inter-
estingly, the allosteric activity was significantly improved up
to 68% A1 conformation, suggesting the creation or the

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 6003
Figure 4. Two putative binding domains for NK2 receptor allosteric modulators (view from the outside of the cell). The models have been built
classically by homology with bovine opsin.31 Trp1.35, Tyr1.39 on the top of TM 1, and Tyr2.64 on the top of TM2 (green) form the first aromatic
cluster, pointing toward the entrance of the orthosteric binding cleft. Phe2.56, 2.60 (TM2) and Phe3.24, 3.27, and 3.31 (top of TM3), in blue, form the
second aromatic cluster, pointing toward the outside of the receptor, in the transmembrane region. Similar domains are obtained based on the most recent
crystal structures of adrenergic and adenosine receptors.32
reinforcement of an interaction between the nitrile function
and the allosteric site. To further explore the carbonitrile
modifications, an ester or an acid function was introduced
in 17 or 18, respectively. The ethyl ester 17 retained a good
allosteric activity (51% of A1 conformation), as efficient as 1,
without promoting an inhibition of FRET. In contrast, the
corresponding acid derivative 18 lost some of its allosteric
activity with promoting only 35% of A1 conformation. The
elongation of the chain to four methylene groups (compounds
19 and 20) afforded an equi-active ester and an acid with some
rescued allosteric activity (52% and 44% of A1conformation,
respectively). The influence of the ester was then further
explored with some derivatives such as compounds 21-24.
Alkyl or aromatic hydrophobic esters 21 and 22 showed
decreased activity. A short hydrophilic hydroxyethyl chain
provided compound 23 as active as 1. The more bulky and
basic chain, as found in 24, slightly decreased the allosteric
activity to 42% of A1 conformation. Amides were then
logically synthesized and tested. Four compounds, 25-28,
were prepared. TheN-butylamide 25wasasactiveas 1, but the
cyclic piperidino analogue 26 was significantly less active than
1, with 44% of A1 conformation. The N0-methylpiperazin,
N-acetamide27 was more active than1, promoting 58% of A1
conformation, while the N0-phenyl derivative 28 was much
less active than 1, suggesting a positive contribution of the
additional N0 basic atom, possibly related to its basicity.
In a final round of chemical modifications, we replaced
the carbonitrile function with electron-rich heteroaroma-
tic groups. Compounds 29-32 were synthesized. They all
exhibited an allosteric modulator activity superior to 1, but
most of them (30-32) developed a significant inhibition of
FRET intensity, supporting the notion that some competition
with NKA may also be occurring.
oriented, and specific bonds such as H-bonds but rather a set
of diffuse hydrophobic, aromatic interactions with the allost-
eric site of the NK2 receptor. To find a possible explanation
for these results, we examined the three-dimensional model of
the NK2 receptor derived from the bovine opsin crystal
structure.10 To be consistent with our structure-activity
relationship data, we looked for aromatic domains accessible
to ligands. Only two domains could clearly be identified
(Figure 4). The first one was located at the entrance of the
binding cleft, at the top of transmembrane domains TM1 and
TM2, which corresponds to the cluster of three aromatic
amino acids, Trp37 (1.35), Tyr41 (1.39), and Tyr93 (2.64)
(in green in the left panel, Figure 4). This pocket could
be located in the direct neighborhood of the agonist binding
site,11,12 which could account for an inhibition of FRET by
exerting competitive interaction with the orthosteric ligand
and could support the hypothesis of a competitive component
of our ligands. Interestingly, a second aromatic cluster has
been found at the top of transmembrane helices TM2 and
TM3, pointing toward the outside of the helix bundle, that is,
toward the membrane (in blue in the right-hand panel,
Figure 4). It encompassed five phenylalanine residues, namely
Phe85 (2.56), 89 (2.60), 105 (3.24), 108 (3.27), and 114(3.31).
Such a cluster is unusual in GPCRs and could be on one hand,
the binding site of compound 1, whereby promoting a con-
formational change in the receptor results in an increase of the
calcium-induced pathway. Alternatively, it is now widely
accepted that GPCRs exist and might function as dimers
and allosteric ligands might modulate the homo- or hetero-
dimerization of GPCRs.13 A great number of GPCRs were
shown to form homodimers or multimers by Western blot-
ting, by coimmunoprecipitation in transfected/infected cells,
ormore recently, by “resonanceenergy transfer”techniquesin
transfected living cells (fluorescence and bioluminescence
resonance energy transfer: FRET or BRET).14 Therefore,
we suggest that these phenylalanine residues could represent
an attachment point between the protomers. The same cluster
can be found in homology models of NK2-R derived from the
most recently published crystallographic structures of the
adrenergic and adenosine receptors.15-17 Thus, one may
Taken all together, the results obtained in the present study
correspond to an improvement in terms of allosteric activity
supporting the calcium-induced pathway. However, the struc-
ture-activity relationship appears difficult to analyze because
the allosteric activity remained very mildly sensitive to very
diverse structural modifications. This ledus to suggest thatthe
interaction with the bindingsitemight not involve very strong,

6004 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Valant et al.
hypothesize that compound 1 may bind this aromatic bud and
thereby either reinforce or disrupt the ability of proto-
mers to dimerize, resulting in conformational and func-
tional switches.18 Though still speculative, the atypical
features and location of this aromatic cluster is worth men-
tioning to trigger experimental evaluation of its functional
relevance.
endogenous ligand. Such a pharmacological profile had been
reported in 2006 for an ionic species, gadolinium (Gd3þ),
acting at the class III metabotropic glutamate receptor
mGluR1R.28 To our knowledge, compound 1 was the first
drug-like, allosteric switch of the function triggered by the
endogenous ligand of a Class I GPCR.8 A similar profile has
been very recently considered for benzoylthiophenes modu-
lating the function of a nonendogenous agonist of the A1
adenosine receptor.29 Compound 16 represents a relatively
more potent allosteric modulator than 1 in the studied system.
Its ability to switch the function of the endogenous ligand
NKA in other cell types and in tissues relevant to human
pathologies will have to be explored. However, the present
results further illustrate the possibility to discover such mole-
cules and pave the way for not only receptor-selective but also
signaling-pathway selective therapies under the physiological
control of the endogenous ligand release.
To summarize, only the modifications of the carbonitrile
function of compound 1 were able to significantly increase the
allosteric effect of the ligands, leading to an optimized hit,
compound 16. Therefore, to further characterize the ability of
compound 16 to increase the population of A1 conformation,
intracellular calcium mobilization assays as well as cAMP
accumulation were investigated at the NK2 receptor. Figure 2
shows that 1, as previously reported,8 did not modify the
intracellular calcium response to a significant extent (EC50=
0.55 ( 0.05 nM for NKA aloneand EC50=0.50 ( 0.05 nM for
NKA in presence of 20 μM of 1). We note in this work that the
EC50 values of NKA for calcium signaling, as well as for
cAMP-signaling, are approximately 10-fold lower than those
obtained previously.8 Consequently, inhibition of cAMP-
responses by compound 1 looks less efficient. These changes
are accounted for by interbatches variations in surface ex-
pression of the EGFP-NK2 receptor as demonstrated by
other groups.19,20 The activity of all compounds studied in
the present work was evaluated in a newly selected cell line
expressing EGFP-NK2R. Interestingly, compound 16 did not
promote any drastic change in NKA potency (EC50=0.62 (
0.04 nM) but exhibited a significant increase of the agonist
efficacy (Emax=120 ( 5%), which reached a plateau approxi-
mately 20% higher than in control conditions, confirming
that the butyronitrile group was definitely able to improve the
biological activity of the modulator. Even more interestingly,
in a cAMP accumulation assay (Figure 3), compound 16
appeared more potent than compound 1, such that while
compound 1 (as previously reported8) was able to reduce the
maximal effect of NKA of 33%, compound 16 at an identical
concentration reduced the agonist efficacy of almost 50%.
These results suggest that, if compound 1 was exerting its
allosteric effect unilaterally by only inhibiting the cAMP-
induced response of NKA at the NK2 receptors while having
no significant effect on the calcium response of the endogen-
ous agonist, compound 16 is actually able to, on one hand,
potentiate the efficacy of NKA in calcium mobilization assay,
while on the other hand, inhibit the efficacy of the endogenous
agonist-induced cAMP response.
Allosteric modulation of GPCRs is now a well established
concept,1,2,13,18,21-23 originally defined by the International
Union of Pharmacology (IUPHAR: http://www.iuphar.org/)
committee as “modulators that enhance the affinity and/or
efficacy of the orthosteric ligand while having no effect on
their own”.24 Indeed many modulators that enhance or
diminish the effects of agonists or antagonists on a variety
of GPCRs are described, some of them reaching the clinical
phase.21 On another hand, molecules have been described as
being able to trigger on their own different signaling pathways
upon binding to a given GPCR.25 This mechanism has been
referred to as “agonist-” or “ligand-directed trafficking of
receptor stimulus”.25-27 Compounds 1 and 16 open new
perspectives because they combine both effects, allostery
and stimulus trafficking; they have no effect on their own
but they are able to traffic the stimulus of the endogenous
ligand, NKA, from the cAMP to the calcium signaling path-
way. They are thus pure allosteric functional switches of the
Conclusion
We have synthesized more than 30 compounds based on
compound 1, an allosteric functional switch of a class I
GPCR. Using a FRET based assay, we have characterized
most of them as allosteric modulators of the NK2 receptor.
These allosteric modulators exhibited the particularity of
stabilizing the receptor in the calcium-triggering state
(Gq coupling) to the detriment of the cAMP synthesizing
state (Gs coupling). Our structure-activity relationship study
led to the optimization of its allosteric activity, especially with
the analogue 16, which enhanced the percentage of A1 con-
formation of NK2 receptor to 68% at 10 μM and increased
the function switch amplitude. We have now a new efficient
allosteric modulator that can target specifically one among
two signaling pathways activated by the endogenous ligand at
a given receptor. This thus opens the road to a superior level of
specificity for ligands acting at GPCRs, but their detailed
mechanism of action as well as their functional and therapeu-
tic potential remain to be explored.
Experimental Section
Chemistry. General. All chemicals used were of reagent
grade. All commercialized compounds were purchased from
Accros, Aldrich, Avocado, Fluka, and Lancaster and used
without further purification. Progress of the reactions was
monitored by TLC on silica gel plates. Extracts were dried over
MgSO4, and solvents were removed under reduced pressure.
Merck silica gel (Kieselgel 60) was used for flash chromatogra-
phy (230-400 mesh) columns. Melting points were determined
using a Mettler FP62 apparatus and are uncorrected. 1H NMR
and 13C NMR spectra were recorded on Bruker DPX 200 and
300 MHz spectrometers with TMS as internal standard; the
values of the chemical shifts are given in ppm. Mass spectra were
determined with a Perkin-Elmer ESI-TOF apparatus (dilution
of the product to a concentration of 10-6 in MeOH/H2O =
90/10). LC/MS spectra were obtained on a ZQ (Z quadripole)
Waters/Micromass spectrometer equipped with an X-Terra C18
column (4.6 mm ꢀ 50 mm, 3.5 μm) using electrospray ioniza-
tion mode (ESI) with a linear gradient from water (0.1% TFA)
to CH3CN (0.1% TFA). Retention times (Rt) from analyti-
cal RP-HPLC are reported in minutes. HRMS spectra were
obtained on a MicroTof mass spectrometer from Bruker using
electrospray ionization (ESI) mode and a time-of-flight analy-
zer (TOF). Elemental analyses were performed on a Perkin-
Elmer 240C elemental analyzer, and the results are within
(0.4% of the theoretical values. Yields refer to purified pro-
ducts and are not optimized. Purity of tested compounds

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 6005
was superior to 95% as ascertained by elementary analysis or
LC-MS (see Experimental Section and Supporting In-
formation). The identity of the tested compounds was ascer-
H arom), 7.22-7.19 (1H, m, H arom), 4.02 (2H, s, -CH2-
naphth), 3.98 (2H, s, -CH2-benz).
(2-Naphthylmethyl)-4-iodobenzylamine (d). Prepared from
2-naphthaldehyde (2 g, 12.8 mmol), 4-iodobenzylamine (2.98 g,
12.8 mmol), and NaBH4 (1.5 g, 38.4 mmol) using the general
procedure for the synthesis of secondary amines (4.58 g, 96%).
Rf=0.25 (hexane/AcOEt=9/1). 1H NMR (200 MHz, CDCl3):
δ 7.85-7.81 (5H, m, H arom), 7.49-7.44 (4H, m, H arom),
7.29-7.28 (2H, m, H arom), 3.96 (2H, s, -CH2-naphth), 3.80
(2H, s, -CH2-iodobenz).
1
tained by H NMR, 13NMR, and LC/MS (see Experimental
Section and Supporting Information).
General Procedure for the Synthesis of Secondary Amines.
A mixture of aldehyde (1.0 equiv) and substituted benzylamine
(1.0 equiv) in MeOH (0.3 M) was first heated at 60 ꢀC during 4 h
and cooled at 0 ꢀC, NaBH4 (3 equiv) was added to the reaction
mixture, and the solution was stirred for 4 h at RT. After
removal of the solvent, the solid residue obtained was dissolved
in AcOEt. The organic layer was washed with a saturated
solution of NaHCO3 (ꢀ2), water, saturated solution of NaCl,
and then dried and reduced to yield an oil (40-99%). The oil
was used without further purification.
(2-Naphthylmethyl)-4-phenethylbenzylamine (e). Prepared
from 2-naphthaldehyde (73 mg, 0.46 mmol), 4-phenethylbenzyl-
amine (100 mg, 0.46 mmol), and NaBH4 (50 mg, 1.38 mmol)
using the general procedure for the synthesis of secondary
1
amines (250 mg, 99%). Rf = 0.25 (hexane/AcOEt = 8/2). H
NMR (200 MHz, CDCl3): δ 7.80-7.78 (4H, m, H arom),
7.51-7.46 (3H, m, H arom), 7.30-7.18 (9H, m, H arom), 3.98
(2H, s, -CH2-naphth), 3.83 (2H, s, -CH2-benz), 2.93 (4H, s,
-CH2-CH2-).
General Procedure for the Synthesis of Tertiary Amines. To a
mixture of secondary amine (1.0 equiv) and TEA (1.2 equiv) in
DMF (0.3 M) was added dropwise a solution of halogenated
compound (1.1 equiv) in DMF (0.3 M). The solution was stirred
overight at 60 ꢀC, and the solvent was removed in vacuo. The
resulting oil was dissolved in AcOEt; the organic layer was
washed with a saturated solution of NaHCO3 (ꢀ2), water,
saturated solution of NaCl, and then dried and reduced. The
resulting product was purified by flash chromatography to yield
an oil (32-98%).
General Procedure for the Synthesis of Amides. To a mixture of
N,N-(2-naphthylmethyl-benzyl)-2-aminoethanoic acid (1.0 equiv)
and N-methylmorpholine (3.2 equiv) in DMF (0.3 M) was added a
solution of substituted amine (1.1 equiv) in DMF (0.3 M), followed
by the coupling agent BOP. The solution was stirred for 1 night at
RT and reduced. The resulting oil was dissolved in AcOEt; the
organic layer was washed with water, with a saturated solution
of NaCl, dried, and the solvent was removed in vacuo. The result-
ing product was purified by flash chromatography to yield the
expected product (67-96%).
(2-Naphthylmethyl)-4-methoxybenzylamine (f). Prepared
from 2-naphthaldehyde (500 mg, 3.2 mmol), 4-methoxybenzyl-
amine (427 mg, 3.2 mmol), and NaBH4 (365 mg, 9.6 mmol)
using the general procedure for the synthesis of secondary
1
amines (736 mg, 83%). Rf = 0.35 (hexane/AcOEt = 9/1). H
NMR (200 MHz, CDCl3): δ 7.90-7.78 (4H, m, H arom),
7.50-7.45 (3H, m, H arom), 7.32-7.26 (2H, m, H arom),
6.93-6.88 (2H, m, H arom), 3.98 (2H, s, -CH2-naphth), 3.83
(5H, s, -CH2-methoxybenz).
(3-Chlorobenzyl)-benzylamine (g). Prepared from 3-chloro-
benzaldehyde (1 g, 7.1 mmol), benzylamine (760 mg, 7.1 mmol),
and NaBH4 (810 mg, 21.3 mmol) using the general procedure for
the synthesis of secondary amines (1.48 g, 90%). Rf = 0.25
(hexane/AcOEt=9/1). 1H NMR (300 MHz, CDCl3): δ 7.78-7.72
(4H, m, H arom), 7.49-7.43 (5H, m, H arom), 4.32 (2H, s, -CH2-
chlorobenz), 4.29 (2H, s, -CH2-benz).
General Procedure for the Synthesis of Esters. To a mixture of
N,N-(2-naphthylmethyl-benzyl)-2-aminoethanoic acid (1.0 equiv)
and N-methylmorpholine (3.2 equiv) in DMF (0.3M) was added
a solution of substituted alcohol (1.1 equiv) in DMF (0.3 M),
followed by the coupling agent BOP. The solution was stirred
overnight at RT and reduced. The resulting oil was dissolved in
AcOEt; the organic layer was washed with water, a saturated
solution of NaCl, and then dried and the solvent was removed in
vacuo. The resulting product was purified by flash chromatog-
raphy to yield the expected product (35-84%).
General Procedure for the Synthesis of Salts. HCl gas was
bubbled for 2 min into a solution of basic compound (0.3 M) in
Et2O or AcOEt. After evaporation, the resulting product was
collected as an uncolored powder.
(2-Naphthylmethyl)-benzylamine (a). Prepared from 2-napht-
haldehyde (10 g, 64 mmol), benzylamine (6.86 g, 64 mmol), and
NaBH4 (7.3 g, 196 mmol) using the general procedure for the
synthesis of secondary amines (14.4 g, 91%). Rf=0.5 (hexane/
AcOEt=2/1). 1H NMR (200 MHz, CDCl3): δ 7.89-7.82 (4H,
m, H arom), 7.55-7.28 (8H, m, H arom), 4.02 (2H, s, -CH2-
naphth), 3.89 (2H, s, -CH2-benz).
(4-Chlorobenzyl)-benzylamine (h). Prepared from 4-chloro-
benzaldehyde (703 mg, 5 mmol), benzylamine (535 mg, 5 mmol),
and NaBH4 (570 mg, 15 mmol) using the general procedure for
the synthesis of secondary amines (1.48 g, 90%). Rf = 0.45
(hexane/AcOEt=7/3). 1H NMR (200 MHz, CDCl3): δ 7.36-7.33
(4H, m, H arom), 7.32-7.30 (5H, m, H arom), 3.81 (2H, s, -CH2-
chlorobenz), 3.79 (2H, s, -CH2-benz).
N-(1H-Indol-3-ylmethyl)-N-benzylamine (i). Prepared from
indol-3-carboxaldehyde (725 mg, 5 mmol), benzylamine (540 mg,
5 mmol), and NaBH4 (570 mg, 15 mmol) using the general
procedure for the synthesis of secondary amines (0.62 g, 52%).
1
Rf=0.85 (AcOEt). H NMR (300 MHz, CDCl3): δ 7.91-7.72
(4H, m, H arom), 7.61-7.49 (4H, m, H arom), 7.33 (1H, t, J=7.2 Hz,
Harom), 7.20(1H, t, J=7.2 Hz, H arom), 4.01 (2H, s, -CH2-indol),
3.98 (2H, s, -CH2-benz).
N-(Quinol-2-ylmethyl)-N-benzylamine (j). Prepared from qui-
nolin-2-carboxaldehyde (500 mg, 3.2 mmol), benzylamine (340 mg,
3.2 mmol), and NaBH4 (365 mg, 9.6 mmol) using the general
procedure for the synthesis of secondary amines (0.64 g, 80%).
Rf=0.25 (hexane/AcOEt=5/5). 1H NMR (300 MHz, CDCl3):
δ 7.86-7.79 (2H, m, H arom), 7.74-7.73 (1H, d, H arom),
7.65-7.60 (1H, m, H arom), 7.45-7.30 (7H, m, H arom), 4.10
(2H, s, -CH2-quinol), 3.85 (2H, s, -CH2-benz).
2-Naphthylmethyl)-4-bromobenzylamine (b). Prepared from
2-naphthaldehyde (2.34 g, 15 mmol), 4-bromobenzylamine
(2.79 g, 15 mmol), and NaBH4 (1.7 g, 45 mmol) using the general
procedure for the synthesis of secondary amines (3.42 g, 70%).
N-(Quinol-3-ylmethyl)-N-benzylamine (k). Prepared from
quinolin-3-carboxaldehyde (500 mg, 3.2 mmol), benzylamine
(340 mg, 3.2 mmol), and NaBH4 (365 mg, 9.6 mmol) using the
general procedure for the synthesis of secondary amine (0.75 g,
1
Rf=0.3 (hexane/AcOEt=9/1). H NMR (200 MHz, CDCl3):
δ 7.85-7.81 (4H, m, H arom), 7.49-7.44 (3H, m, H arom),
7.29-7.28 (4H, m, H arom), 3.96 (2H, s, -CH2-naphth), 3.80
(2H, s, -CH2-bromobenz).
(2-Naphthylmethyl)-2-bromobenzylamine (c). Prepared from
2-naphthaldehyde (1.4 g, 9 mmol), 2-bromobenzylamine (1.67 g,
9 mmol), and NaBH4 (1 g, 27 mmol) using the general procedure
for the synthesis of secondary amines (2.55 g, 87%). Rf =0.5
(hexane/AcOEt=2/1). 1H NMR (200 MHz, CDCl3): δ7.89-7.85
(4H, m, H arom), 7.53-7.34 (5H, m, H arom), 7.33-7.31 (1H, m,
1
95%). Rf =0.25 (hexane/AcOEt=5/5). H NMR (300 MHz,
CDCl3): δ 8.90 (1H, s, H arom), 8.12-8.08 (2H, m, H arom),
7.81-7.79 (1H, m, H arom), 7.79-7.77 (1H, m, H arom),
7.71-7.69 (1H, m, H arom), 7.37-7.27 (5H, m, H arom), 4.00
(2H, s, -CH2-quinol), 3.87 (2H, s, -CH2-benz).
N-(4-Trifluoromethylbenzyl)-benzylamine (l). Prepared from
4-trifluorobenzaldehyde (500 mg, 2.9 mmol), benzylamine (310 mg,
2.9 mmol), and NaBH4 (330 mg, 8.7 mmol) using the general

6006 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Valant et al.
procedure for the synthesis of secondary amines (0.65 g, 85%).
Rf=0.3 (hexane/AcOEt=9/1). H NMR (300 MHz, CDCl3):
δ7.63-7.60 (2H, d, J= 9 Hz, H arom), 7.51-7.48 (2H, d, J=8.3 Hz,
H arom), 7.36-7.30 (5H, m, H arom), 3.89 (2H, s, -CH2-
trifluorobenz), 3.84 (2H, s, -CH2-benz).
(50 MHz, DMSO-d6): δ 136.57, 134.74, 133.70, 133.49, 132.26,
131.01, 129.01, 128.37, 128.22, 127.03, 126.77, 126.56, 122.15,
58.84, 58.08, 41.27; mp=110 ꢀC. Anal. (C,H,N) C=66.01%, H=
4.67%, N=7.61%. HRMS m/z (M þ H)þ 366.0648.
1
[2-Bromobenzyl-(2-naphthylmethyl)-amino]-acetonitrile (3).
Prepared from the secondary amine c (2.55 g, 7.8 mmol),
chloroacetonitrile (648 mg, 8.6 mmol), and TEA (945 mg, 9.36 mmol)
using the general procedure for the synthesis of tertiary amines.
The resulting product was purified by flash chromatography
with hexane/AcOEt=9/1 to yield a white solid (1,7 g, 60%). Rf=
0.9 (hexane/AcOEt = 2/1). 1H NMR (200 MHz, CDCl3):
δ 7.88-7.84 (3H, m, H arom), 7.68-7.50 (4H, m, H arom),
7.41-7.18 (4H, m, H arom), 3.99 (2H, s, -CH2-naphth), 3.97
(2H, s, -CH2-bromobenz), 3.41 (2H, s, -CH2-CN). 13C NMR
(75 MHz, DMSO-d6): δ 132.91, 131.80, 129.5, 127.72, 127.0,
126.49, 126.32, 58.76, 57.97, 41.45; mp=71 ꢀC. Anal. (C,H,N)
C=63.0%, H=4.72%, N=7.22%, 1H2O. HRMS m/z (M þ H)þ
366.0648.
N-(4-Methoxybenzyl)-benzylamine (m). Prepared from 4-
methoxybenzaldehyde (500 mg, 3.7 mmol), benzylamine (393 mg,
3.7 mmol), and NaBH4 (420 mg, 11.1 mmol) using the general
procedure for the synthesis of secondary amines (0.65 g, 77%).
Rf=0.3 (hexane/AcOEt=9/1). 1H NMR (300 MHz, CDCl3):
δ 7.39-7.31 (7H, m, H arom), 6.92-6.89 (2H, m, H arom), 3.82
(3H, s, -CH3), 3.75 (2H, s, -CH2-methoxybenz), 3.70 (2H, s,
-CH2-benz).
(2E)-N-Benzyl-3-phenylprop-2-en-amine (n). Prepared from
trans-cinnamaldehyde (660 mg, 5 mmol), benzylamine (535 mg,
5 mmol), and NaBH4 (570 mg, 15 mmol) using the general
procedure for the synthesis of secondary amines (0.90 g, 81%).
Rf=0.35 (hexane/AcOEt=8/2). 1H NMR (300 MHz, CDCl3):
δ 7.42-7.25 (10H, m, H arom), 7.60-7.55 (1H, d, J=15 Hz, H
arom), 6.40-6.30 (1H, m, -CH-), 3.88 (2H, s, -CH2-benz),
3.48-3.46 (2H, d, J=6 Hz, -CH2-cinn).
N-(2-Naphthylmethyl)-3-aminopyridine (o). To a solution of
2-naphthaldehyde (1 equiv, 500 mg, 3.2 mmol) and 3-amino-
pyridine (1 equiv, 300 mg, 3.2 mmol) in ethanol (0,3 M) heated at
95 ꢀC during 48 h then cooled to 0 ꢀC, NaBH4 (3 equiv, 380 mg,
10 mmol) was added. The solution was stirred overnight at RT
and reduced. The resulting oil was dissolved in AcOEt; the
organic layer was washed with a solution of NaOH 1N (ꢀ2),
water, saturated solution of NaCl, and then dried and reduced.
The resulting product was purified by flash chromatography
with AcOEt/hexane (75/25) to yield an oil (300 mg, 40%). Rf=
0.3 (hexane/AcOEt = 1/9). 1H NMR (300 MHz, CDCl3):
δ 8.15-8.11 (1H, d, H arom), 7.97-7.94 (1H, m, H arom),
7.92-7.69 (4H, m, H arom), 7.49-7.41 (3H, m, H arom),
7.10-7.02 (1H, m, H arom), 6.94-6.88 (1H, m, H arom),
4.54-4.51 (2H, d, J=8.9 Hz, -CH2-naphth).
N-(2-Naphthylmethyl)-2-aminomethylpyridine (p). Prepared
from 2-naphthaldehyde (500 mg, 3.2 mmol), 2-aminomethyl-
pyridine (350 mg, 3.2 mmol), and NaBH4 (380 mg, 10 mmol)
using the general procedure for the synthesis of secondary
amines (550 mg, 70%). The resulting oil was purified by flash
chromatography with AcOEt/hexane/TEA (9/1/2%). Rf=0,15
(hexane/AcOEt/TEA=1/9/2%). 1H NMR (300 MHz, CDCl3):
δ 8.11-8.09 (1H, d, H arom), 7.91-7.87 (4H, m, H arom),
7.65-7.62 (1H, t, J=4.2 Hz, H arom), 7.54-7.44 (3H, m, H
arom), 7.34-7.31 (1H, d, J=7.8 Hz, H arom), 7.19-7.15 (1H,
m, H arom), 4.03 (2H, s, -CH2-pyr), 3.98 (2H, s, -CH2-
naphth).
[4-Iodobenzyl-(2-naphthylmethyl)-amino]-acetonitrile (4). Pre-
pared from the secondary amine d (4.83 g, 12.8 mmol), chloro-
acetonitrile (982 mg, 13 mmol), and TEA (1.31 g, 13 mmol)
using the general procedure for tertiary amines. The resulting
product was purified by flash chromatography with hexane/
AcOEt=95/5 to yield a white solid (3.65 g, 69%). Rf =0.25
(hexane/AcOEt = 95/5). 1H NMR (200 MHz, CDCl3):
δ 7.90-7.85 (4H, m, H arom), 7.55-7.51 (4H, m, H arom),
7.36-7.30 (3H, m, H arom), 3.92 (2H, s, -CH2-naphth), 3.78
(2H, s, -CH2-iodobenz), 3.42 (2H, s, -CH2-CN). 13C NMR
(75 MHz, DMSO-d6): δ 137.66, 135.27, 133.23, 132.88, 131.33,
128.57, 127.90, 127.63, 127.02, 126.59, 126.32, 115.78, 93.85,
57.75, 57.09, 41.50; mp = 116 ꢀC. HRMS m/z (M þ H)þ
413.0509.
[4-Phenethylbenzyl-(2-naphthylmethyl)-amino]-acetonitrile (5).
Prepared from the secondary amine e (200 mg, 0.57 mmol),
chloroacetonitrile (43 mg, 0.57 mmol), and TEA (57 mg, 0.57 mmol)
using the general procedure for the tertiary amine. The resulting
product was purified by flash chromatography with hexane/
AcOEt=95/5 to yield a white solid (195 mg, 87%). Rf=0.15
(Heptane/AcOEt = 95/5). 1H NMR (300 MHz, CDCl3):
δ 7.91-7.87 (4H, m, H arom), 7.60-7.51 (3H, m, H arom),
7.40-7.20 (9H, m, H arom), 3.94 (2H, s, -CH2-naphth), 3.81
(2H, s, -CH2-benz), 3.43 (2H,s, -CH2-CN), 2.96 (4H, s, -CH2-
CH2-). 13C NMR (75 MHz, CDCl3): δ 141.94, 140.90, 136.57,
133.02, 131.47, 130.97, 128.54, 125.01, 119.78, 113.82, 63.31,
45.83, 37.44. HRMS m/z (M þ H)þ 391.47
[4-Methoxybenzyl-(2-naphthylmethyl)-amino]-acetonitrile (6).
Prepared from the secondary amine f (720 mg, 2.6 mmol),
chloroacetonitrile (212 mg, 2.8 mmol), and TEA (323 mg, 3.2 mmol)
using the general procedure for tertiary amines. The resulting
product was purified by flash chromatography with hexane/
AcOEt=95/5 to yield a white solid (595 mg, 60%). Rf=0.45
(hexane/AcOEt=8/2). 1H NMR (200 MHz, CDCl3): δ7.86-7.82
(4H, m, H arom), 7.54-7.46 (3H, m, H arom), 7.36-7.32 (2H, m,
H arom), 3.89 (2H, s, -CH2-naphth), 3.82 (3H, s, -CH3),
3.74 (2H, s, -CH2-benz), 3.38 (2H, s, -CH2-CN). 13C NMR
(50 MHz, DMSO-d6): δ 154.44, 129.86, 128.47, 128.20, 125.40,
124.22, 123.65, 123.03, 122.97, 122.86, 121.97, 121.43, 121.20,
109.88, 109.21, 53.50, 52.95, 50.46, 35.77; mp=86ꢀC. Anal. (C,H,
N) C=79.45%, H=6.54%, N=8.74%. HRMS m/z (M þ H)þ
317.1648.
[Benzyl-(3-chlorobenzyl)-amino]-acetonitrile hydrochloride (7).
Prepared from the secondary amine g (1.50 g, 6.40 mmol),
chloroacetonitrile (530 mg, 7.0 mmol), and TEA (775 mg, 7.7 mmol
using the general procedure for the synthesis of tertiary amines. The
resulting product was purified by flash chromatography with
hexane/AcOEt=95/5 to yield a white solid (805 mg, 41%). Rf=
0.3 (hexane/AcOEt = 95/5). 1H NMR (300 MHz, CDCl3): δ
7.81-7.78 (4H, m, H arom), 7.51-7.46 (5H, m, H arom), 4.30
(2H, s, -CH2-chlorobenz), 4.27 (2H, s, -CH2-benz), 3.75 (2H, s,
-CH2-CN). 13C NMR (50 MHz, DMSO-d6): δ 137.54, 135.03,
[N-Benzyl,N-(2-naphthylmethyl)-amino]-acetonitrile (1). Pre-
pared from the secondary amine a (1.24 g, 5 mmol), chloroacet-
onitrile (415 mg, 5.5 mmol), and TEA (610 mg, 6 mmol) using
the general procedure for the synthesis of tertiary amines. The
resulting product was purified in isopropyl alcohol to yield a
1
white solid (1.2 g, 84%). Rf = 0.3 (hexane/AcOEt = 9/1). H
NMR (300 MHz, CDCl3): δ 7.90-7.80 (4H, m, H arom),
7.60-7.20 (8H, m, H arom), 3.92 (2H, s, -CH2-naphth), 3.81
(2H, s, -CH2-benz), 3.40 (2H, s, -CH2-CN). 13C NMR (50
MHz, DMSO-d6): δ 136.58, 134.80, 132.34, 132.20, 131.52,
130.87, 130.68, 113.81, 60.12, 59.20, 41.22; mp=92 ꢀC. Anal.
(C,H,N) C=83.75%. H=6.28%, N=9.77%. HRMS m/z (M þ
H)þ 287.1538
[4-Bromobenzyl-(2-naphthylmethyl)-amino]-acetonitrile (2).
Prepared from the secondary amine b (3.42 g, 10.5 mmol),
chloroacetonitrile (875 mg, 11.6 mmol) and TEA (1.27 g, 12.6
mmol) using the general procedure for the synthesis of tertiary
amines. The resulting product was purified by crystallization
(2.37g,65%).Rf=0,15 (hexane/AcOEt=9/1). 1H NMR (200 MHz,
CDCl3): δ 7.90-7.85 (4H, m, H arom), 7.55-7.51 (4H, m, H
arom), 7.36-7.30 (3H, m, H arom), 3.92 (2H, s, -CH2-naphth),
3.78 (2H, s, -CH2-benz), 3.42 (2H, s, -CH2-CN). 13C NMR

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 6007
133.71, 133.46, 129.41, 129.10, 128.92, 128.25, 128.10, 127.14,
126.70, 126.46, 115.08, 58.87, 58.82, 41.22. Synthesis of the salt;
mp=120 ꢀC. Anal. (C,H,N) C=62.40%, H=5.47%, N=8.14%
1HCl. HRMS m/z (M þ H)þ 271.1002.
solid (556 mg, 46%). Rf=0.3 (hexane/AcOEt=9/1). 1H NMR
(200 MHz, CDCl3): δ=7.49-7.28 (10H, m, H arom), 6.73-6.65
(1H, d, J=16 Hz, H arom), 6.29-6.14 (1H, m, -CH-), 3.75
(2H, s, -CH2-benz), 3.51 (2H, s, -CH2-cinn), 3.43-3.39 (2H, d,
J=6.8 Hz, -CH2-CN). 13C NMR (75 MHz, DMSO-d6): δ=
136.28, 136.16, 134.23, 130.26, 129.03, 128.98, 128.48, 127.00,
122.40, 114.79, 57.31, 55.68, 41.58. Synthesis of the salt; mp=
170 ꢀC. Anal. (C,H,N) C=71.96%, H=6.49%, N=9.18%,
1HCl. HRMS m/z (M þ H)þ 263.1544.
[Benzyl-(4-chlorobenzyl)-amino]-acetonitrile hydrochloride (8).
Prepared from the secondary amine h (1.05 g, 4.5 mmol), chlor-
oacetonitrile (375 mg, 4.95 mmol), and TEA (545 mg, 5.4 mmol)
using the general procedure for the synthesis of tertiary amines. The
resulting product was purified by flash chromatography with
hexane/AcOEt=95/5 to yield a white solid (580 mg, 42%). Rf=
0.3 (hexane/AcOEt = 95/5). 1H NMR (300 MHz, CDCl3):
δ 7.79-7.76 (4H, m, H arom), 7.50-7.45 (5H, m, H arom), 4.32
(2H, s, -CH2-chlorobenz), 4.28 (2H, s, -CH2-benz), 3.73 (2H, s,
-CH2-CN). 13C NMR (75 MHz, DMSO-d6): δ 136.95, 136.17,
132.58, 131.00, 129.20, 128.92, 128.89, 128.09, 115.51, 57.52, 56.76,
41.25. Mp=124 ꢀC. Anal. (C,H,N) C=62.83%, H=5.24%, N=
9.12%, 1HCl. HRMS m/z (M þ H)þ 271.0984.
[Benzyl-(4-methoxybenzyl)-amino]-acetonitrile Hydrochloride
(13). Prepared from the secondary amine
m (635 mg,
2.85 mmol), chloroacetonitrile (237 mg, 3.14 mmol), and TEA
(345 mg, 3.42 mmol) using the general procedure for the synth-
esis of tertiary amines. The resulting product was purified by
flash chromatography with hexane/AcOEt = 97/3 to yield a
white solid (485 mg, 56%). Rf=0.2 (hexane/AcOEt=97/3). 1H
NMR (300 MHz, CDCl3): δ 7.39-7.31 (7H, m, H arom),
6.92-6.89 (2H, m, H arom), 3.82 (3H, s, -CH3), 3.74 (2H, s,
-CH2-methoxybenz), 3.70 (2H, s, -CH2-benz), 3.37 (2H, s,
-CH2-CN). 13C NMR (50 MHz, DMSO-d6): δ 161.54, 133.27,
131.68, 130.86, 130.01, 128.85, 120.43, 115.30, 111.65, 58.54,
58.45, 55.81, 38.75. Synthesis of the salt; mp=114 ꢀC. HRMS
m/z (M þ H)þ 267.1485.
[Benzyl-(1H-indol-3-ylmethyl)-amino]-actonitrile Hydrochlor-
ide (9). Prepared from the secondary amine i (613 mg, 2.6 mmol),
chloroacetonitrile (216 mg, 2.85 mmol), and TEA (315 g, 3.12 mmol)
using the general procedure for the synthesis of tertiary amines.
The resulting product was purified by flash chromatography
with hexane/AcOEt=2/1 to yield a white solid (640 mg, 80%).
Rf=0.75 (hexane/AcOEt=2/1). 1H NMR (300 MHz, CDCl3): δ
7.81-7.76 (1H, d, J = 5 Hz, H arom), 7.45-7.30 (7H, m, H
arom), 7.25-7.16 (2H, m, H arom), 3.97 (2H, s, -CH2-indol),
3.82 (2H, s, -CH2-benz), 3.39 (2H, s, -CH2-CN). 13C NMR
(75 MHz, DMSO-d6): δ 139.41, 137.12, 131.94, 131.13, 129.54,
128.07, 123.60, 122.47, 120.23, 119.48, 114.75, 112.18, 102.21,
50.74, 50.06, 34.06. Synthesis of the salt; mp=150 ꢀC. Anal.
(C,H,N) C=68.71%, H=5.81%, N=13.49%, 1HCl. HRMS
m/z (M þ H)þ 276.5005.
{Benzyl-[4-(trifluoromethyl)-benzyl]-amino}-acetonitrile Hydro-
chloride (14). Prepared from the secondary amine l (660 mg,
2.5 mmol), chloroacetonitrile (210 mg, 2.75 mmol), and TEA
(300 mg, 3 mmol) using the general procedure for the synthesis
of tertiary amines. The resulting product was purified by flash
chromatography with hexane/AcOEt = 97/3 to yield a white
solid (580 mg, 68%). Rf=0.3 (hexane/AcOEt=97/3). 1H NMR
(300 MHz, CDCl3): δ 7.72-7.68 (2H, d, H arom), 7.60-7.55
(2H, m, H arom), 7.41-7.35 (5H, m, H arom), 3.83 (2H, s,
-CH2-trifluorobenz), 3.78 (2H, s, -CH2-benz), 3.41 (2H, s,
-CH2-CN). 13C NMR (75 MHz, DMSO-d6): δ 151.36, 147.16,
140.13, 133.21, 132.82, 131.76, 131.12, 129.33, 128.95, 128.52,
127.94, 127.21, 126.92, 124.76, 57.85, 54.77. Synthesis of the salt.
HRMS m/z (M þ H)þ 305.1261.
[Benzyl-(quinolin-2-ylmethyl)-amino]-acetonitrile Dihydrochloride
(10). Prepared from the secondary amine j (635 mg, 2.56 mmol),
chloroacetonitrile (212 mg, 2.81 mmol), and TEA (310 g, 3.07 mmol)
using the general procedure for the synthesis of tertiary amines.
The resulting product was purified by flash chromatography
with hexane/AcOEt=8/2 to afford a yellow solid (690 mg, 75%).
[Benzyl-(2-naphthylmethyl)-propargyl]-amine (15). Prepared
from the secondary amine a (1.24 g, 5 mmol), propargyl bromide
(655 mg, 5.5 mmol), and TEA (610 mg, 6 mmol) using the
general procedure for the synthesis of tertiary amines. The
resulting product was purified in isopropyl alcohol to yield a
1
Rf=0.2 (hexane/AcOEt=8/2). H NMR (300 MHz, CDCl3):
δ 7.85-7.83 (1H, d, H arom), 7.82-7.80 (1H, d, H arom),
7.74-7.72 (1H, d, H arom), 7.63-7.56 (1H, d, H arom),
7.44-7.37 (2H, m, H arom), 7.35-7.31 (5H, m, H arom), 4.11
(2H, s, -CH2-quinol), 3.83 (2H, s, -CH2-benz), 3.55 (2H, s,
-CH2-CN). 13C NMR (75 MHz, DMSO-d6): δ 157.79, 144.34,
140.06, 136.94, 133.69, 129.35, 128.82, 128.06, 127.93, 122.65,
122.13, 116.13, 58.35, 56.28, 42.72. Synthesis of the salt; mp=
160 ꢀC. Anal. (C,H,N) C=63.53%, H=5.35%, N=11.59%,
2HCl. HRMS m/z (M þ H)þ 288.1488.
1
white solid (1.29 g, 82%). Rf =0.3 (hexane/AcOEt=9/1). H
NMR (300 MHz, CDCl3): δ 7.90-7.80 (4H, m, H arom),
7.60-7.20 (8H, m, H arom), 3.93 (2H, s, -CH2-naphth), 3.82
(2H, s, -CH2-benz), 3.34 (2H, s, -CH2-CN). 13C NMR (50 MHz,
DMSO-d6): δ 136.58, 134.80, 132.34, 132.20, 131.52, 130.87,
130.68, 113.81, 60.12, 59.20, 41.22; mp=92 ꢀC. Anal. (C,H,N)
C = 88.40%, H=6.68%, N=4.98%. HRMS m/z (M þ H)þ
286.1538.
[Benzyl-(quinolin-3-ylmethyl)-amino]-acetonitrile Dihydrochloride
(11). Prepared from the secondary amine k (635 mg, 2.56 mmol),
chloroacetonitrile (210 mg, 2.80 mmol), and TEA (310 g,
3.07 mmol) using the general procedure for the synthesis of
tertiary amine. The resulting product was purified by flash
chromatography with hexane/AcOEt = 7/3 to yield a yellow
solid (505 mg, 55%). Rf=0.3 (hexane/AcOEt=7/3). 1H NMR
(300 MHz, CDCl3): δ 7.83-7.81 (1H, d, H arom), 7.84-7.81
(1H, d, H arom), 7.75-7.73 (1H, d, H arom), 7.63-7.55 (1H, d,
H arom), 7.44-7.37 (2H, m, H arom), 7.35-7.29 (5H, m, H
arom), 4.10 (2H, s, -CH2-quinol), 3.85 (2H, s, -CH2-benz),
3.57 (2H, s, -CH2-CN). 13C NMR (75 MHz, DMSO-d6):
δ 148.51, 140.33, 138.15, 133.92, 131.45, 130.97, 126.71,
124.61, 123.64, 123.19, 117.92, 61.54, 57.96, 44.64. Synthesis
of the salt; mp=190 ꢀC. Anal. (C,H,N) C=63.54%, H=5.36%,
N=11.79%, 2HCl. HRMS m/z (M þ H)þ 288.1509.
4-[Benzyl-(2-naphthylmethyl)-amino]-butyronitrile Hydrochlor-
ide (16). Prepared from the secondary amine a (500 mg,
2.5 mmol), 4-bromobutyronitrile (410 mg, 2.75 mmol), and
TEA (303 mg, 3 mmol) using the general procedure for tertiary
amines. The resulting product was purified by flash chromatog-
raphy with hexane/AcOEt=95/5 to yield a yellow solid (550 mg,
1
63%). Rf = 0.2 (hexane/AcOEt = 9/1). H NMR (300 MHz,
CDCl3): δ 7.90-7.81 (3H, m, H arom), 7.75 (1H, s, H arom),
7.49-7.46 (3H, m, H arom), 7.37-7.35 (5H, m, H arom), 3.73
(2H, s, -CH2-naphth), 3.63 (2H, s, -CH2-benz), 2.60 (2H, t, J=
6.4 Hz, -CH2-N), 2.32 (2H, t, J=7.5 Hz, -CH2-CN), 1.85-1.81
(2H, m, -CH2-CH2-CH2-CN). 13C NMR (50 MHz, DMSO-
d6): δ 133.38, 132.88, 131.88, 131.69, 130.16, 129.83, 129.83,
129.16, 128.75, 128.57, 128.44, 128.00, 127.67, 127.44, 127.02,
119.91, 56.55, 50.13, 19.62, 14.25. Synthesis of the salt. Anal.
(C,H,N) C= 71.46%, H =6.70%, N =7.47%, 1HCl, 1H2O.
HRMS m/z (M þ H)þ 315.1850.
{Benzyl-[(2E)-3-phenylprop-2-en-1-yl]-amino}-acetonitrile Hy-
drochloride (12). Prepared from the secondary amine n (903 mg,
4.05 mmol), chloroacetonitrile (336 mg, 445 mmol), and TEA
(490 g, 4,85 mmol) using the general procedure for the synthesis
of tertiary amines. The resulting product was purified by flash
chromatography with hexane/AcOEt = 9/1 to yield a yellow
N-Ethyl-[benzyl-(2-naphthylmethyl)-amine] Acetate (17). Pre-
pared from the secondary amine a (800 mg, 4 mmol), 2-bromo-
ethylacetate (740 mg, 4.5 mmol), and TEA (505 mg, 5 mmol)

6008 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Valant et al.
using the general procedure for tertiary amines. The resulting
product was purified by flash chromatography with hexane/
AcOEt=95/5 to yield a white solid (1.12 g, 84%). Rf =0.25
(hexane/AcOEt = 95/5). 1H NMR (200 MHz, CDCl3): δ
7.90-7.78 (4H, m, H arom), 7.60-7.55 (1H, d, H arom),
7.48-7.66 (7H, m, H arom), 4.18-4.14 (2H, q, -CH2-CH3),
3.98 (2H, s, -CH2-naphth), 3.87 (2H, s, -CH2-benz), 3.32 (2H,
s, -CH2-CN), 1.27 (3H, t, J = 4.5 Hz, -CH3). 13C NMR
(50 MHz, DMSO-d6): δ 170.89, 139.13, 136.92, 133.26, 132.76,
128.98, 127.89, 127.87, 127.38, 126.39, 126.01, 60.10, 57.58,
57.36, 53.54, 14.44; mp = 69 ꢀC. Anal. (CHN) C = 71.46%,
H=6.70%, N=3.47%, HCl. HRMS m/z (M þ H)þ 334.1802.
[Benzyl-(2-naphthylmethyl)-amino]-acetic Acid (18). Prepared
from the tertiary amine 17 (400 mg, 1.7 mmol) and HCl 6N (12 mL)
heated at 100 ꢀC overnight. The solution was cooled, and the
precipitate was filtered and then washed with few mL of diethylether
to yield a white solid (510 mg, 98%). 1H NMR (300 MHz, DMSO-
d6): δ 8.05 (1H, s, H arom), 7.98-7.92 (3H, m, H arom), 7.72-7.68
(1H, d, H arom), 7.58-7.54 (4H, m, H arom), 7.43-7.40 (3H, m, H
arom), 4.49 (2H, s, -CH2-COOH), 4.38 (2H, s, -CH2-naphth),
3.76 (2H, s, -CH2-benz). 13CNMR(50MHz,DMSO-d6):δ170.21,
134.97, 133.34, 132.98, 131.91, 130.91, 129.74, 129.27, 128.55,
121.13, 60.37, 60.09, 52.91; mp = 154 ꢀC. Anal. (C,H,N) C =
79.32%, H=6.99%, N=4.19%. HRMS m/z (M þ H)þ 306.1489.
Ethyl 5-[Benzyl-(2-naphthylmethyl)-amino]-pentanoate (19).
Prepared from the secondary amine a (1 g, 4 mmol), 5-bro-
moethylvalerate (836 mg, 4 mmol), and TEA (808 mg, 8 mmol).
The resulting product was purified by flash chromatography
with hexane/AcOEt=95/5 to yield a white solid (1.07 g, 70%).
Rf = 0.15 (Heptane/AcOEt = 95/5). 1H NMR (200 MHz,
CDCl3): δ 7.89-7.77 (4H, m, H arom), 7.48-7.32 (8H, m, H
arom), 4.19-4.05 (2H, m, -CH2-naphth), 3.71 (2H, s, -CH2-
benz), 3.60 (2H, s, -CH2-CH2-CH2-CH2-COOEt), 2.51-2.45
(2H, m, -CH2-COOEt), 2.37-2.30 (2H, m, -CH2-CH3),
1.62-1.58 (4H, m, -CH2-CH2-CH2-CH2-COOEt), 1.30-1.24
(3H, m, -CH3). 13C NMR (50 MHz, DMSO-d6): δ 174.32,
133.28, 132.92, 131.78, 130.32, 129.79, 128.54, 127.97, 127.42,
127.00, 56.31, 50.98, 45.63, 31.04, 22.35, 8.74; mp=126 ꢀC. MS
m/z (M þ H)þ 376.16.
[Benzyl-(2-naphthylmethyl)-amino]-phenethyle Acetate Hydro-
chloride (22). Prepared from phenethyl alcohol (81 mg, 0.77
mmol), acid 18 (200 mg, 0.65 mmol), BOP (345 mg, 0.78 mmol),
and NMM (303 mg, 3 mmol) in a solution of acetonitrile 5 mL
and DMF 500 μL using the general procedure for the synthesis of
esters. The resulting product was purified by flash chromatogra-
phy with hexane/AcOEt = 95/5 to yield a yellow hygroscopic
1
powder (198 mg, 57%). Rf=0.25 (Heptane/AcOEt=95/5). H
NMR (300 MHz, CDCl3): δ 7.79-7.74 (3H, m, H arom), 7.62
(1H, s, H arom), 7.51-7.47 (2H, m, H arom), 7.36-7.14 (11H, m,
H arom), 4.86 (2H, t, -CH2-O-), 3.68 (2H, s, -CH2-naphth),
3.57 (2H, s, -CH2-benz), 3.21 (2H, s, -CH2-CO-), 2.97 (2H, t,
-CH2-Phen). 13C NMR (50 MHz, CDCl3): δ 166.17, 136.96,
134.20, 133.43, 132.32, 132.16, 130.69, 129.86, 129.24, 129.13,
128.85, 128.25, 127.85, 127.35, 126.65, 67.06, 58.21, 48.77, 35.20.
HRMS m/z (M þ H)þ 410.2115.
Methylsulfonyl 2-Hydroxyethyle (q). In a solution of DCM
(50 mL) with TEA (4,9 g, 48.3 mmol) and ethylene glycol (2 g,
32.3 mmol) was added mesyl chloride (5.53 mg, 48.03 mmol) at
0 ꢀC during 3 h, then stirred 2 h at RT. The resulting product was
purified by flash chromatography with hexane/AcOEt=25/75
to yield a colorless oil (905 mg, 20%). Rf=0.2 (heptane/AcOEt=
25/75). 1H NMR (300 MHz, CDCl3): δ 4.37-4.34 (2H, m,
-CH2-O-SO2), 3.92-3.89 (2H, m, -CH2-OH), 3.09 (3H, s,
-CH3), 2.20 (1H, s, -OH).
Methylsulfonyl Ethyl-[benzyl-(2-naphthylmethyl)-amino]-2-
acetate (r). To a solution of acid 18 (200 mg, 0.65 mmol) in
acetonitrile 5 mL and DMF 500 μL was added NMM (303 mg,
3 mmol), BOP (292 mg, 0.66 mmol), and ethylene glycol mono-
mesyle (100 mg, 0.7 mmol). The mixture was stirred at RT
overnight. The resulting product was purified by flash chroma-
tography with hexane/AcOEt (from 9/1 to 7/3) to yield a white
oil (98 mg, 35%). Rf=0.4 (Heptane/AcOEt=25/75). 1H NMR
(300 MHz, CDCl3): δ 7.92-7.80 (4H, m, H arom), 7.63-7.55
(1H, m, H arom), 7.44-7.28 (7H, m, H arom), 4.38-4.33 (4H,
m, -CH2-CH2-), 3.98 (2H, s, -CH2-naphth), 3.87 (2H, s,
-CH2-benz), 3.39 (2H, s, -CH2-CO), 2.93 (3H, s, -CH3).
[Benzyl-(2-naphthylmethyl)-amino]-2-hydroxyethyl Acetate (23).
The mesyl r (100 mg, 0.23 mmol) was dissolved in toluene 10 mL,
and then few mg of para-toluene sulfonic acid were added and
the mixture was stirred at reflux during 16 h. The resulting
product was purified by flash chromatography with hexane/
AcOEt=8/2 to yield a yellow powder (30 mg, 35%). 1H NMR
(300 MHz, CDCl3): δ 7.92-7.79 (4H, m, H arom), 7.63-7.57
(3H, m, H arom), 7.45-7.28 (5H, m, H arom), 4.38-4.33 (2H,
m, -CH2-OCO-), 4.15-4.10 (2H, m, -CH2-OH), 3.98 (2H, s,
-CH2-naphth), 3.93 (2H, s, -CH2-benz), 3.19 (2H, s, -CH2-
CO). 13C NMR (50 MHz, DMSO-d6): δ 170.72, 140.39, 132.94,
129.04, 128.62, 128.35, 127.96, 126.89, 124.89, 123.78, 119.78,
59.98, 58.91, 57.94, 56.87. MS m/z (M þ H)þ 350.3.
5-[Benzyl2-naphthylmethyl)-amino]-pentanoic Acid Hydro-
chloride (20). A solution of tertiary amine 18 (100 mg, 0.27 mmol)
in HCl 6N (12 mL) was heated overnight at 100 ꢀC and then
cooled at 0 ꢀC. The precipitate was filtered and suspended in
diethylether to yield a white solid (105 mg,100%). 1H NMR
(300 MHz, DMSO-d6): δ 8.16-7.84 (4H, m, H arom), 7.67-7.58
(4H, m, H arom), 7.45-7.39 (4H, m, H arom), 4.20-4.15 (2H, m,
-CH2-naphth), 3.81-3.79 (2H, m, -CH2-benz), 3.70-3.68 (2H,
m, -CH2-CH2-CH2-CH2-COOH), 2.51-2.45 (2H, m, -CH2-
COOH), 1.62-1.58 (4H, m, -CH2-CH2-CH2-CH2-COOH). 13
C
NMR (50 MHz, DMSO-d6): δ 174.30, 133.34, 132.87, 131.78,
131.59, 130.33, 129.79, 129.13, 128.69, 128.53, 128.41, 128.00,
127.80, 127.43, 127.02, 56.28, 50.98, 33.19, 31.04, 22.36, 21.97;
mp=84 ꢀC. MS m/z (M þ H)þ 348.06.
[Benzyl-(2-naphthylmethyl)-amino]-ethylmorpholine Acetate Hy-
drochloride (24). Prepared from 4-(2-hydroxyethyl)morpholine
(101 mg, 0.77 mmol), acid 18 (200 mg, 0.65 mmol), BOP
(345 mg, 0.78 mmol), and NMM (303 mg, 3 mmol) in a solution
of acetonitrile 5 mL and THF 500 μL using the general proce-
dure for the synthesis of esters. The resulting product was
purified by flash chromatography with hexane/AcOEt (from
7/3 to 5/5) to yield an oil (118 mg, 40%). Rf =0.3 (heptane/
AcOEt=7/3). 1H NMR (300 MHz, CDCl3): δ 7.89-7.79 (4H,
m, H arom), 7.65-7.60 (1H, m, H arom), 7.47-7.30 (7H, m, H
arom), 4.30-4.26 (2H, t, J=4 Hz, -O-CH2-CH2-), 4.01 (2H, s,
-CH2-naphth), 3.90 (2H, s, -CH2-benz), 3.72-3.68 (4H, t, J=4
Hz, -CH2-O-CH2-), 3.38 (2H, s, -CH2-CO), 2.68-2.62 (2H, t,
J=6 Hz, -CH2-N), 2.54-2.49 (4H, m, -CH2-N-CH2-). 13C
NMR (50 MHz, DMSO-d6): δ 167.72, 133.43, 132.85, 131.84,
131.67, 130.41, 129.92, 129.15, 128.73, 128.47, 128.01, 127.47,
127.03, 63.40, 58.52, 57.73, 55.27, 51.78, 50.92, 31.06. Synthesis
of the salt. HRMS m/z (M þ H)þ 419.2329.
[Benzyl-(2-naphthylmethyl)-amino]-hexadecanoyl Acetate Hydro-
chloride (21). Prepared from 1-hexadecanol (187 mg, 0.77 mmol),
acid 18 (200 mg, 0.65 mmol), BOP (345 mg, 0.78 mmol), and
NMM (303 mg, 3 mmol) in a solution of acetonitrile 5 mL and
DMF 500 μL using the general procedure for the synthesis of
esters. The resulting product was purified by flash chromatog-
raphy with hexane/AcOEt=98/2 to yield an oil (221 mg, 60%).
Rf=0.5 (Heptane/AcOEt: 95/5). 1H NMR (300 MHz, CDCl3):
δ 7.89-7.75 (4H, m, H arom), 7.51-7.32 (8H, m, H arom),
4.17-4.11 (2H, t, J = 6 Hz, -CH2-O-), 4.02 (2H, s, -CH2-
naphth), 3.91 (2H, s, -CH2-benz), 3.37 (2H, s, -CH2-CO), 1.30
(31H, m, -(CH2)14-CH3). 13C NMR (50 MHz, CDCl3):
δ 166.39, 134.18, 133.42, 132.12, 130.65, 129.95, 129.41,
128.75, 128.25, 127.82, 127.25, 126.86, 66.89, 58.07, 48.05,
32.32, 30.09, 29.84, 29.77, 29.49, 28.65, 26.06, 23.03, 14.53.
Synthesis of the salt; mp = 140 ꢀC. HRMS m/z (M þ H)þ
530.3993.
[Benzyl-(2-naphthylmethyl)-amino]-N-butyl-acetamide Hydro-
chloride (25). Prepared from n-butylamine (45 mg, 0.62 mmol),

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 6009
acid 18 (200 mg, 0.59 mmol), BOP (273 mg, 0.62 mmol), and
NMM (130 mg, 1.29 mmol) using the general procedure for the
synthesis of amides. The resulting product was purified by flash
chromatography with hexane/AcOEt = 7/3 to yield a white
powder (200 mg, 85%). Rf = 0.2 (hexane/AcOEt = 7/3). 1H
NMR (300 MHz, CDCl3): δ 7.91-7.88 (3H, m, H arom), 7.75
(1H, s, H arom), 7.38-7.36 (3H, m, H arom), 7.35-7.30 (5H, m,
H arom), 3.79 (2H, s, -CH2-naphth), 3.69 (2H, s, -CH2-benz),
3.18 (2H, s, -CH2-CO), 1.48-1.40 (2H, m, -NH-CH2-),
1.35-1.25 (2H, m, -CH2-CH2-CH2-CH3), 0.97-0.88 (5H, m,
-CH2-CH3). 13C NMR (50 MHz, DMSO-d6): δ 165.76, 135.72,
134.91, 133.37, 132.95, 131.85, 130.85, 130.63, 129.77, 129.27,
129.08, 128.48, 128.04, 60.91, 60.75, 53.29, 40.59, 32.45, 21.27,
14.31. Synthesis of the salt; mp=160 ꢀC. HRMS m/z (M þ H)þ
361.2274.
60 ꢀC and reduced. The resulting oil was dissolved in AcOEt; the
organic layer was washed with a saturated solution of NaHCO3
(ꢀ2), water, saturated solution of NaCl, and then dried and
reduced. The resulting product was purified by flash chromato-
graphy with hexane/AcOEt=7/3 to yield a beige powder (123 mg,
1
34%). Rf =0.25 (hexane/AcOEt=7/3). H NMR (300 MHz,
CDCl3): δ 8.32-8.29 (1H, d, H arom), 8.05-8.01 (1H, m, H
arom), 7.92-7.75 (4H, m, H arom), 7.62 (1H, s, H arom),
7.50-7.45 (3H, m, H arom), 7.40-7.20 (3H, m, H arom),
7.07-7.03 (3H, m, H arom), 4.86 (2H, s, -CH2-naphth), 4.78
(2H, s, -CH2-benz). 13C NMR (50 MHz, DMSO-d6): δ 147.50,
134.74, 133.77, 133.43, 132.07, 130.01, 129.87, 128.72, 128.23,
128.12, 127.25, 127.18, 126.90, 126.72, 126.61, 125.23, 124.73,
124.38, 55.39, 55.16. Synthesis of the salt; mp=160 ꢀC. HRMS
m/z (M þ H)þ 325.1699.
[Benzyl-(2-naphthylmethyl)-amino]-piperidino-acetamide Hydro-
chloride (26). Prepared from piperidine (52 mg, 0.62 mmol),
acid 18 (200 mg, 0.59 mmol), BOP (273 mg, 0.62 mmol), and
NMM (130 mg, 1.29 mmol) using the general procedure for
the synthesis of amides. The resulting product was purified by
flash chromatography with hexane/AcOEt = 8/2 to yield a
[Benzyl-(2-naphthylmethyl)-amino]-2-methylpyridine Hydro-
chloride (30). Prepared from the secondary amine o (250 mg, 1
equiv), benzylbromide (200 mg, 1.1 equiv) and TEA (1.2 equiv)
using the general procedure for tertiary amines. The resulting
product was purified by flash chromatography with hexane/
AcOEt=9/1, 8/2, and then 7/3 to yield a yellow solid (77%). Rf=
1
1
white powder (220 mg, 91%). H NMR (300 MHz, CDCl3): δ
0.15 (hexane/AcOEt = 9/1). H NMR (300 MHz, CDCl3): δ
7.85-7.75 (4H, m, H arom), 7.57-7.48 (3H, m, H arom),
7.41-7.29 (5H, m, H arom), 3.85 (2H, s, -CH2-naphth), 3.74
(2H, s, -CH2-benz), 3.51-3.47 (2H, t, J=5.28 Hz), 3.33 (2H, s,
-CH2-CO), 3.24-3.21 (2H, t, J=5.28 Hz, -N-CH2), 1.61-1.51
(4H, m, -N-CH2-CH2-), 1.46-1.42 (2H, m, -CH2-piper). 13C
NMR(50 MHz, DMSO-d6): δ 180.02, 145.90, 132.79, 131.97,
129.10, 128.63, 128.43, 127.97, 127.00, 58.93, 45.51, 42.66,
25.43, 25.00, 23.77. Synthesis of the salt. HRMS m/z (M þ H)þ
373.2274.
[Benzyl-(2-naphthylmethyl)-amino]-(N0-methyl)-piperazido-aceta-
mide Hydrochloride (27). Prepared from N-methylpiperazine
(62 mg, 0.62 mmol), acid 18 (200 mg, 0.59 mmol), BOP (273 mg,
0.62 mmol), and NMM (130 mg, 1.29 mmol) using the general
procedure for the synthesis of amides. The resulting product was
purified by flash chromatography with AcOEt/MeOH/TEA (9/1/
2%) to yield a white powder (168 mg, 67%). Rf =0.3 (AcOEt/
MeOH/TEA=9/1/2%). 1H NMR (300 MHz, CDCl3):δ7.91-7.88
(3H, m, H arom), 7.77 (1H, s, H arom), 7.54-7.48 (3H, m, H arom),
7.37-7.32 (5H, m, H arom), 3.87 (2H, s, -CH2-naphth), 3.76 (2H,
s, -CH2-benz), 3.61-3.55 (2H, t, J = 4.9 Hz, -CH2-NCO),
3.36-3.32 (4H, m, -CH2-CO-N-CH2-), 2.38-2.29 (7H, m,
-CH2-NCH3-CH2-). 13C NMR (50 MHz, DMSO-d6): δ 164.99,
135.64, 134.82, 133.52, 133.05, 131.88, 130.53, 129.87, 129.36,
129.21, 128.64, 128.29, 61.11, 54.10, 52.98, 44.03, 43.21, 40.40.
Synthesis of the salt. HRMS m/z (M þ H)þ 388.2383.
8.06-8.04 (1H, d, H arom), 7.93-7.89 (4H, m, H arom),
7.65-7.57 (3H, m, H arom), 7.50-7.42 (4H, m, H arom),
7.37-7.30 (2H, m, H arom), 7.29-7.25 (1H, m, H arom),
7.11-7.09 (1H, m, H arom), 3.81 (2H, s, -CH2-pyr), 3.79
(2H, s, -CH2-naphth), 3.68 (2H, s, -CH2-benz). 13C NMR
(50 MHz, DMSO-d6): δ 161.29, 142.82, 137.52, 129.59, 128.93,
127.98, 127.32, 125.79, 115.77, 57.66, 57.04. Synthesis of the salt.
HRMS m/z (M þ H)þ 339.1856.
[Benzyl-(2-naphthylmethyl)-amino]-2-pyridine (31). A solution
of secondary amine a (500 mg,1 equiv) with BuLi (1.5 equiv) in
DMF (0.3 M) was stirred at 0 ꢀC then cooled at RT during 1 h.
2-Chloropyridine (253 mg, 1.1 equiv) was added, and the solution
was stirred at RT during 1 h and then at 60 ꢀC overnight and
reduced. The resulting oil was dissolved in AcOEt; the organic
layer was washed with a saturated solution of NaHCO3 (ꢀ2),
water, saturated solution of NaCl, and then dried and reduced.
The resulting product was purified by flash chromatography with
hexane/AcOEt = 7/3 to yield an oil (32%). Rf = 0.2 (hexane/
AcOEt = 7/3). 1H NMR (300 MHz, CDCl3): δ 8.36-8.33(1H, d,
J=6 Hz, H arom), 8.33-7.84 (3H, m, H arom), 7.72 (1H, s, H
arom), 7.53-7.35 (9H, m, H arom), 6.70-6.67(1H, dd, J1=5 Hz,
J2=1 Hz, H arom), 6.60-6.55 (1H, d, J=8 Hz, H arom), 5.04
(2H,s,-CH2-naphth), 4.93 (2H, s, -CH2-benz). 13CNMR(50MHz,
DMSO-d6): δ 159.04, 148.43, 138.78, 137.95, 136.32, 133.87,
133.11, 112.78, 106.44, 78.03, 77.45, 76.82, 51.49, 51.26; mp=
76 ꢀC. HRMS m/z (M þ H)þ 325.1540.
[Benzyl-(2-naphthylmethyl)-amino]-(N0-phenyl)-piperazido-acet-
amide Hydrochloride (28). Prepared from N-phenylpiperazine
(100 mg, 0.65 mmol), acid 18 (200 mg, 0.59 mmol), BOP (273 mg,
0.62 mmol), and NMM (130 mg, 1.29 mmol) using the general
procedure for the synthesis of amides. The resulting product was
purified by flash chromatography with hexane/AcOEt=7/3 to yield
a white powder (240 mg, 84%). Rf=0.25 (hexane/AcOEt=7/3). 1H
NMR (300 MHz, CDCl3): δ 7.92-7.87 (3H, m, H arom), 7.77 (1H,
s, H arom), 7.54-7.47 (3H, m, H arom), 7.37-7.32 (7H, m, H
arom), 6.95-6.89 (3H, m, H arom), 3.87 (2H, s, -CH2-naphth),
3.76 (2H, s, -CH2-benz), 3.74-3.72 (2H, m, -CH2-N-phenyl),
3.47-3.45 (2H, m, -CH2-N-phenyl), 3.35 (2H, s, -CH2-CO),
3.13-3.10 (2H, t, J=5.3 Hz, -CO-NH-CH2-), 3.03-3.00 (2H,
t, J=5.0 Hz, -CO-NH-CH2-). 13C NMR (50 MHz, DMSO-d6):
δ 163.27, 149.35, 133.48, 132.78, 132.09, 131.96, 130.04, 129.48,
129.14, 128.73, 128.66, 128.48, 127.97, 127.55, 127.03, 121.55,
117.20, 58.82, 50.47, 49.06, 43.99, 43.99, 41.07. Synthesis of the salt;
mp=100 ꢀC. HRMS m/z (M þ H)þ 450.2540.
[Benzyl-(2-naphthylmethyl)-amino]-4-pyrimidine Hydrochlor-
ide (32). Prepared from the secondary amine a (350 mg, 1.3 mmol),
4-chloropyrimidine (150 mg, 1.3 mmol) and TEA (150 mg, 1.5 mmol)
using the general procedure for tertiary amines. The resulting
product was purified by flash chromatography with hexane/
AcOEt = 5/5 to yield a yellow solid (250 mg, 53%). Rf = 0.2
(hexane/AcOEt=7/3). 1H NMR (300 MHz, CDCl3): δ8.72 (1H, s,
H arom), 8.28-8.17 (1H, d, J=6 Hz, H arom), 7.86-7.75 (4H, m,
H arom), 7.60 (2H, m, H arom), 7.50-7.46 (3H, m, H arom),
7.40-7.25 (3H, m, H arom), 6.48-6.45 (1H, d, J=6 Hz, H arom),
4.95 (2H, s, -CH2-naphth), 4.83 (2H, s, -CH2-benz). 13C NMR
(50 MHz, CDCl3): δ 162.34, 158.79, 155.96, 137.10, 134.56, 133.79,
133.24, 129.19, 128.13, 127.96, 127.52, 126.78, 126.37, 126.09,
125.64, 103.55, 50.77, 50.57, 30.11. Synthesis of the salt. HRMS
m/z (M þ H)þ 326.1652.
Biology. Cloning. NK2R cDNA: Rat NK2R cDNA in 50
frame fusion with EGFP cDNA was cloned in pCEP4 expres-
sion vector (Invitrogen) as described.6
[Benzyl-(2-naphthylmethyl)-amino]-3-pyridine Hydrochloride
(29). A solution of secondary amine p (234 mg, 1 mmol) and
NaH (48 mg, 2 mmol) in DMF (0.3 M) was stirred at 0 ꢀC few
minutes then at RT during one hour. The benzylbromide (190 mg,
1.1 mmol) was added and the reaction was stirred during 1 h at
Cell Culture and Buffer. HEK293 cells were stably transfected
by calcium phosphate precipitation4 and selected with 500 μg/
mL hygromycin B. Cells were assayed in Hepes-BSA buffer
(137.5 mM NaCl, 1.25 mM MgCl2, 1.25 mM CaCl2, 6 mM KCl,

6010 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19
Valant et al.
5.6 mM glucose, 10 mM Hepes, 0.4 mM NaH2PO4, 1% bovine
serum albumin (w/v), pH 7.4) supplemented with protease
inhibitors.6
FRET Measurements. Fluorescence binding: Interaction of
fluorescent peptides with chimeric EGFP-rNK2 was monitored
on a Fluorolog 2 spectrofluorometer (SPEx) with excitation set
at 470 nm (bandwidth=5 nm) as a decrease of EGFP emission at
510 nm (bandwidth=10 nm) reflecting fluorescence resonance
energy transfer toward the acceptor group Bodipy as described.4
(2) May, L. T.; Leach, K.; Sexton, P. M.; Christopoulos, A. Allosteric
modulation of G protein-coupled receptors. Annu. Rev. Pharmacol.
Toxicol. 2007, 47, 1–51.
(3) (a) Forster, T. Energiewanderung und fluoreszenz. Naturwis-
senschaften 1946, 6, 166–175. (b) Clegg, R. M. Fluorescence energy
transfer. Curr. Opin. Biotechnol. 1995, 6, 103–110.
(4) Vollmer, J. Y.; Alix, P.; Chollet, A.; Takeda, K.; Galzi, J. L.
Subcellular compartmentalization of activation and desensitization
of responses mediated by NK2 neurokinin receptors. J. Biol. Chem.
1999, 274, 37915–37922.
(5) Zimmer, M. Green fluorescent protein (GFP): applications, structure
and related photophysical behaviour. Chem. Rev. 2002, 102, 759–781.
(6) Palanche, T.; Ilien, B.; Zoffmann, S.; Reck, M. P.; Bucher, B.;
Edelstein, S. J.; Galzi, J. L. The neurokinin A receptor activates
calcium and cAMP responses through distinct conformational
states. J. Biol. Chem. 2001, 276, 34853–34861.
(7) Ilien, B.; Franchet, C.; Bernard, P.; Morisset, S.; Weill, C. O.;
Bourguignon, J. J.; Hibert, M.; Galzi, J. L. Fluorescence resonance
energy transfer to probe human M1 muscarinic receptor structure
and drug binding properties. J. Neurochem. 2003, 85, 768–778.
(8) Maillet, E. L.; Pellegrini, N.; Valant, C.; Bucher, B.; Hibert, M.;
Bourguignon, J. J.; Galzi, J. L. A novel conformation specific
allosteric inhibitor of the tachykinin NK2 receptor (NK2R) with
functionally selective properties. FASEB J. 2007, 21, 2124–2134.
(9) Galzi, J. L.; Hibert, M.; Bourguignon, J. J.; Maillet, E. Method for
isolating an allosteric effector of a receptor. WO Patent 03107004,
December, 2003.
(10) Palczewski, K.; Kumasaka, T.; Hori, T.; Behnke, C. A.; Motoshima,
H.; Fox, B. A.; Le Trong, I.; Teller, D. C.; Okada, T.; Stenkamp,
R. E.; Yamamoto, M.; Miyano, M. Crystal structure of rhodopsin:
a G protein coupled receptor. Science 2000, 289, 739–745.
(11) Bhogal, N.; Donnelly, D.; Findlay, J. B. The ligand binding site of
the neurokinin 2 receptor. Site-directed mutagenesis and identification
of neurokinin A binding residues in the human neurokinin 2
receptor. J. Biol. Chem. 1994, 269, 27269–27274.
(12) Labrou, N. E.; Bhogal, N.; Hurrell, C. R.; Findlay, J. B. Interaction
of Met297 in the seventh transmembrane segment of the tachykin-
in NK2 receptor with neurokinin A. J. Biol. Chem. 2001, 276,
37944–9.
(13) Milligan, G.; Smith, N. J. Allosteric modulation of heterodime-
ric G-protein coupled receptors. Trends Pharmacol. Sci. 2007, 28,
615–20.
(14) Milligan, G.; Bouvier, M. Methods to monitor the quaternary structure
of G protein-coupled receptors. FEBS J. 2005, 272, 2914–2925.
(15) Cherezov, V.; Rosenbaum, D. M.; Hanson, M. A.; Rasmussen, S.;
Thian, F.; Kobilka, T. S.; Choi, H.-J.; Kuhn, P.; Weis, W.;
Kobilka, B.; Stevens, R. High-Resolution Crystal Structure of an
Engineered Human β2-Adrenergic G Protein-Coupled Receptor.
Science 2007, 318, 1258–1265.
(16) Warne, T.; Maria, J.; Serrano-Vega, M.; Baker, J.; Moukhametzianov,
R; Edwards, P.; Henderson, R.; Leslie, A.; Tate, C.; Schertler, G.
Structure of a β1-adrenergic G-protein-coupled receptor. Nature 2008,
454, 486–491.
(17) Veli-Pekka Jaakola, V.; Griffith, M.; Hanson, M.; Cherezov, V.;
Chien, E.; Lane, R.; IJzerman, A.; Stevens, R. The 2.6 Angstrom
Crystal Structure of a Human A2A Adenosine Receptor Bound to
an Antagonist. Science 2008, 322, 1211–1217.
(18) Springael, J.-Y.; Urizar, E.; Costagliola, S.; Vassart, G.; Parmentier,
M. Allosteric properties of G protein coupled receptor oligomers.
Pharmacol. Ther. 2007, 115, 410–418.
(19) Adham, N.; Ellerbrock, B.; Hartig, P.; Weinshank, R. L.; Branchek,
T. Receptor reserve masks partial agonist activity of drugs in a
cloned rat 5-hydroxytryptamine1B receptor expression system. Mol.
Pharmacol. 1993, 43, 427–433.
Cell (106 cells/mL) or membrane suspensions (100 μg protein/
3
mL) in Hepes-BSA buffer were placed at 20 ꢀC in a 1 mL quartz
cuvette under constant stirring. Addition of fluorescent agonist
to the suspension results in a decrease of EGFP fluorescence at
510 nm. The intensity of the fluorescence variation is propor-
tional to receptor sites occupancy. Typically, saturation of
receptor sites results in 40% decrease of total preparation
fluorescence. Dissociation experiments are initiated by addition
of an excess of unlabeled NKA (1 μM) and recorded as a
recovery of EGFP fluorescence intensity as a function of time.
Data Analysis. The KaleidaGraphTM software was used for
fitting NKAbo binding traces. Biexponential dissociation was
defined by equation: [F]=f(t)=A1 exp(-V1 ꢀ t) þ A2 exp(-V2
ꢀ t). Where, t is the time in s, [F] is the FRET signal accounting
for ligand bound to the receptor, V1 and V2 the off rate
constants koff of respectively rapid and slow dissociation events
in s-1, and A1 and A2 are the amplitudes of respectively rapid
and slow dissociation.
Intracellular Calcium Determinations. Determinations were
carried out as described.6,30 Briefly, adherent cells were loaded
with 5 μM Indo-1 for 45 min at 37 ꢀC, dissociated in PBS and
suspended in Hepes buffer (in mM: 137.5 NaCl, 1.25 MgCl2,
1.25 CaCl2, 6 KCl, 5.6 glucose, 10 Hepes, 0.4 NaH2PO4, 1%
BSA (w/v), pH 7.4) in Hepes buffer (in mM: 137.5 NaCl, 1.25
MgCl2, 1.25 CaCl2, 6 KCl, 5.6 glucose, 10 Hepes, 0.4 NaH2PO4,
1% BSA (w/v), pH 7.4). Cell suspension measurements were
made at 21 ꢀC in a 1 mL cuvette on the SPEX Fluorolog 2
spectrofluorometer with excitation set at 355 nm and time-based
emission detected at 405 and 475 nm. Recordings were normal-
ized according to the maximal intensity of fluorescence detected
upon addition of the detergent digitonin and plotted as 475/405
ratios. Data points reported in the figure correspond to the area
under the response curve in arbitrary units.
cAMP Measurements. Functional NK2R-mediated coupling
to adenylyl cyclase was assessed by measuring the dose-depen-
dent stimulatory effects of neurokinin A on cAMP accumulat-
ion. HEK293 cultures expressing NK2 receptors were harvested
and seeded in 24-well plates coated with collagen (60 mg/mL) at
an initial density of 50000. After 3 days of culture (∼80%
confluency), the cells were treated as previously described.6,30
The inhibitory effect of LPI805 and LPI827 on NKA-stimulated
cAMP accumulation was determined in the presence of 10 μM of
each compound. The reaction was stopped by addition of one
volume ice-cold 0.2 M HCl and cAMP was determined by
radioimmunoassay.
(20) Whaley, B. S.; Yuan, N.; Birnbaumer, L.; Clark, R. B.; Barber, R.
Differential expression of the beta-adrenergic receptor modifies
agonist stimulation of adenylyl cyclase: a quantitative evaluation.
Mol. Pharmacol. 1994, 45, 481–489.
(21) Soudijn, W.; van Wijngaarden, I.; IJzerman, A. Allosteric modula-
tion of G protein coupled receptors: perspectives and recent
developments. Drug Discovery Today 2004, 9, 752–758.
(22) Leach, K.; Sexton, P. M.; Christopoulos, A. Allosteric GPCR
modulators: taking advantage of permissive receptor pharmacol-
ogy. Trends Pharmacol. Sci. 2007, 28, 382–389.
Acknowledgment. This work was supported by the Centre
ꢀ
National de la Recherche Scientifique, the Universite de
Strasbourg, and the European Community’s Sixth Frame-
work Program (grant LSHB-CT-2003-503337).
Supporting Information Available: Extended analytical data,
mass spectrometry, and LC-MS purity on the most potent
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
(23) Schwartz, T.; Holst, B. Allosteric enhancers, allosteric agonists and
ago-allosteric modulators:where do they bind and how do they act?
Trends Pharmacol. Sci. 2007, 28, 367–373.
(24) Neubig, R. R.; Spedding, M.; Kenakin, T.; Christopoulos, A.
International Union of Pharmacology Committee on Receptor
Nomenclature and Drug Classification. XXXVIII. Update on
terms and symbols in quantitative pharmacology. Pharmacol.
Rev. 2003, 55, 597–606.
References
(1) Christopoulos, A. Allosteric binding sites on cell-surface receptors:
novel targets for drug discovery. Nat. Rev. Drug Discovery 2002, 1,
198–210.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 19 6011
(25) Urban, J. D.; Clarke, W. P.; von Zastrow, M.; Nichols, D. E.;
Kobilka, B.; Weinstein, H.; Javitch, J. A.; Roth, B. L.; Christopoulos,
A.; Sexton, P. M.; Miller, K. J.; Spedding, M.; Mailman, R. B.
Functional selectivity and classical concepts of quantitative pharma-
cology. J. Pharmacol. Exp. Ther. 2007, 320, 1–13.
(26) Kenakin, T. Agonist-receptor efficacy. II. Agonist trafficking of
receptor signals. Trends Pharmacol. Sci. 1995, 16, 232–238.
(27) Hermans, E. Biochemical and pharmacological control of the
multiplicity of coupling of G protein coupled receptors. Pharmacol.
Ther. 2003, 99, 25–44.
(29) Aurelio, L.; Valant, C.; Flynn, B.; Sexton, P.; Christopoulos, A.;
Scammells, P. Allosteric modulators of the adenosine A1 receptor:
synthesis and pharmacological evaluation of 4 substituted 2-ami-
no-3-bezoylthiophenes. J. Med. Chem. 2009, 52, 4543–4547.
(30) Lecat, S.; Bucher, B.; Mely, Y.; Galzi, J. L. Mutations in the
extracellular amino-terminal domain of the NK2 neurokinin re-
ceptor abolish cAMP signaling but preserve intracellular calcium
responses. J. Biol. Chem. 2002, 277, 42034–42048.
(31) Palczewski, K.; Kumasaka, T.; Hori, T.; Behnke, C. A.; Motoshi-
ma, H.; Fox, B. A.; et al. Crystal structure of rhodopsin: a G
protein-coupled receptor. Science 2000, 289, 739–745.
(28) Tateyama, M.; Kubo, Y. Dual signalling is differentially affected
by different active states of the metabotropic glutamate receptor
1r. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 1124–1128.
(32) Topiol, S.; Sabioa, M. X-ray structure breakthroughs in the GPCR
transmembrane region. Biochem. Pharmacol. 2009, 38, 11–20.