Discovery and biological characterization of (2 R,4 S)-1′-Acetyl- N -{(1 R)-1-[3,5-bis(trifluoromethyl)phenyl]ethyl}-2-(4-fluoro-2-methylphenyl)- N -methyl-4,4′-bipiperidine-1-carboxamide as a new potent and selective neurokinin 1 (NK1) recepto

Article abstract of DOI:10.1021/jm1013264

A large body of compelling preclinical evidence supports the clinical use of neurokinin (NK) receptor antagonists in a plethora of CNS and non-CNS therapeutic areas. The significant investment made in this area over the past 2 decades culminated with the

Full text of DOI:10.1021/jm1013264

J. Med. Chem. 2011, 54, 1071–1079 1071
DOI: 10.1021/jm1013264
Discovery and Biological Characterization of (2R,4S)-10-Acetyl-N-{(1R)-1-[3,5-
bis(trifluoromethyl)phenyl]ethyl}-2-(4-fluoro-2-methylphenyl)-N-methyl-4,40-bipiperidine-1-
carboxamide as a New Potent and Selective Neurokinin 1 (NK1) Receptor Antagonist Clinical
Candidate
Romano Di Fabio,*,†, Giuseppe Alvaro,†, Cristiana Griffante,†, Domenica A. Pizzi,†, Daniele Donati,†,^ Mario Mattioli,‡,
Zadeo Cimarosti,‡, Giuseppe Guercio,‡, Carla Marchioro,§, Stefano Provera,§, Laura Zonzini,†, Dino Montanari,†,
Sergio Melotto,† Philip A. Gerrard,†, David G. Trist,† Emiliangelo Ratti,† and Mauro Corsi†,
†Neurosciences Centre of Excellence for Drug Discovery, GlaxoSmithKline Medicines Research Centre, Via A. Fleming 4, 37135 Verona, Italy,
‡Chemical Development, GlaxoSmithKline Medicines Research Centre, Via A. Fleming 4, 37135 Verona, Italy, and §Molecular Discovery
Research, Analytical Chemistry, GlaxoSmithKline Medicines Research Centre, Via A. Fleming 4, 37135 Verona, Italy. Present address: Aptuit
SRL, Via A. Fleming, 4, 37135 Verona, Italy. ^ Present address: Nerviano Medical Sciences SRL, Via Pasteur 10, 20014 Nerviano (Milan), Italy.
Received October 14, 2010
A large body of compelling preclinical evidence supports the clinical use of neurokinin (NK) receptor
antagonists in a plethora of CNS and non-CNS therapeutic areas. The significant investment made in
this area over the past 2 decades culminated with the observation that NK1 receptor antagonists elicited
clinical efficacy in major depression disorders. In addition, aprepitant (Merck) was launched as a new
drug able to prevent chemotherapy-induced nausea and vomiting (CINV). After the discovery by
GlaxoSmithKline of vestipitant, a wide drug discovery program was launched aimed at identifying
additional clinical candidates. New compounds were designed to maximize affinity at the NK1 receptor
binding site while retaining suitable physicochemical characteristics to ensure excellent pharmacoki-
netic and pharmacodynamic properties in vivo. Herein we describe the discovery process of a new NK1
receptor antagonist (casopitant) selected as clinical candidate and progressed into clinical studies to
treat major depression disorders.
Introduction
aim to identify selective and potent brain penetrant NK1
receptor antagonists. Aprepitant (Merck), shown in Figure 1,
has been approved and launched into the market for
prevention of chemotherapy-induced nausea and vomiting
(CINV). In our previous communications12,13 we described
the design process and the chemical exploration of a new C-
phenylpiperazine template from which 2-(S)-(4-fluoro-2-
methylphenyl)piperazine-1-carboxylic acid [1-(R)-(3,5-bis-tri-
fluoromethylphenyl)ethyl]methylamide (vestipitant) 1 was
derived.14 Herein we report the evolution of that previous
exploration aimed at identifying additional clinical candidates
that exhibit appropriate druglike properties. The compounds
synthesized were characterized, at recombinant human NK1
receptors, in terms of in vitro receptor binding profile by
displacement of [3H]SP and in terms of functional activity,
using fluorometric imaging plate reader (FLIPR) technology.
As previously reported,12 the latter aspect is fundamental
for the selection of the most appropriate compounds to
progress: a slowly dissociating NK1 receptor antagonists
showing nonsurmountable profile exerted a potent and lon-
ger lasting pharmacodynamic action than surmountable
NK1 receptor antagonists in the gerbil foot-tapping (GFT)
model, which was chosen as the in vivo pharmacodynamic
model of election to characterize new chemical entities
(NCE) because of the similar in vitro pharmacology ob-
served in human and gerbil NK1 receptors. Conversely,
NK1 receptor antagonists were significantly less active in
mice and rats than in man.15 Moreover, over the past years,
Substance P (SPa), neurokinin (NK)A, and (NK)B1 are
all mammalian tachykinins whose biochemical effects are
mediated by the G-protein-coupled receptors (GPCR) NK1,
NK2, and NK3. These neuropeptides, acting as both neuro-
transmitters and neuromodulators, are implicated in a variety
of biological functions in the central nervous system (CNS),
namely, pain transmission, inflammation, smooth muscle
contraction, vasodilation, gland secretion, and activation of
the immune system.2-8 In particular, SP, the most abundant
tachykinin in the CNS of mammals, along with its cognate
NK1 receptor, is present in brain regions such as the amygdala,
septum, hippocampus, hypothalamus, and periaqueductal
gray9-11 cerebral areas involved in the regulation of affective
behavior and the mediation of stress responses including
anxiety and depression. In addition, SP was thought to play
a critical role in emesis. As a consequence of this large
therapeutic potential, several pharmaceutical companies have
invested in this field since the beginning of the 1990s with the
*To whom correspondence should be addressed. Phone: þ39 045
8218879. Fax: þ39 045 8218196. E-mail: romano.difabio@aptuit.com.
a Abbreviations: NK, neurokinin; SP, substance P; GPCR, G-protein-
coupled receptor; CNS, central nervous system; SAD, social anxiety
disorders; CINV, chemotherapy-induced nausea and vomiting; SAR,
structure-activity relationship; CDI, carbonyldiimidazole; THF, tetra-
hydrofuran; CHO, Chinese hamster ovary; FLIPR, fluorescence imag-
ing plate reader; GFT, gerbil foot tapping; CRC, concentration response
curve; NCE, new chemical entity; Cli, intrinsic clearance.
r
2011 American Chemical Society
Published on Web 01/13/2011
pubs.acs.org/jmc

1072 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4
Di Fabio et al.
NK1 receptor antagonists were found to be efficacious in
preclinical in vivo models of affective behavior.16-22 Hence,
our compounds were characterized in the gerbil social inter-
action model, one of the most sensitive in vivo models to
assess the anxiolytic-like profile of NK1 receptor ligands.23
Among the compounds synthesized, (2R,4S)-10-acetyl-N-{(1R)-
1-[3,5-bis(trifluoromethyl)phenyl]ethyl}-2-(4-fluoro-2-methyl-
phenyl)-N-methyl-4,40-bipiperidine-1-carboxamide methane-
sulfonate salt 16a (casopitant) was found to be a potent,
selective, orally active, and brain penetrant NK1 receptor
antagonist. Furthermore, as it had a favorable pharmacoki-
netic profile prediction in human, it was selected as a clinical
candidate and progressed to clinical trials to treat major
depression disorders.
Comins-type reaction with 4-methoxypyridine which was
treated with benzyl chloroformate and Grignard’s reagent
derived from commercially available 2-bromo-5-fluoro-
toluene. After the sequential reduction of the double bond with
L-selectride and the following removal of the nitrogen protec-
tive group by catalytic hydrogenation, the racemic amine
derivative 4 was purified by crystallization as camphorsulfo-
nic acidsalt. Thenthe pureenantiomer5, obtained byselective
crystallization with L-mandelic acid, was coupled with N-
methylbenzylamines 7a and 7b. The latter intermediate was
synthesized by reductive amination of the ketone 6 followed by
resolution of the racemic mixture by selective crystallization
as L-malic acid salt. The next reaction was performed in the
presence of triphosgene, to afford in good yield compounds 8a
and 8b. Finally, the target compounds 9-20 were prepared
from these common intermediates by reductive amination with
NaBH(OAc)3 in acetonitrile. In these reaction conditions a 4:1
mixture of the trans and cis diasteroisomers was obtained,
which were easily purified by flash chromatography.
Chemistry
The final compounds 9-20 were prepared as shown in
Scheme 1. The racemate intermediate 3 was obtained by
Results and Discussion
As part of a large drug discovery effort aimed at the
identification of new, druggable NK1 receptor antagonists,
the C-phenylpiperazine template, from which compound 1,
shown in Figure 1, was previously derived, was modified as
shown in Figure 2. Keeping the urea moiety present within the
right-end side chain constant (R=H or CH3), we managed to
modify the core of the template by moving the basic nitrogen
out of the piperazine ring. This chemical modification enabled
a wider exploration of the chemical space present in the left-
end side region of the molecule with respect to the correspond-
ing piperazines analogues. This was achieved by array synthesis
Figure 1. Chemical structure of aprepitant (Merck) and vestipitant
(GSK).
Scheme 1a
a (a) (i) 2-Bromo-5-fluorotoluene, Mg, THF, 60-70 ꢀC; (ii) 4-methoxypyridine, benzyl chloroformate, THF, -20 ꢀC, then Grignard’s reagent,
-20 ꢀC, 1 h; (b) (i) tris(triphenylphosphine)rhodium(I) chloride, 2-propanol, H2 (p = 5 atm), 60 ꢀC, 5 h; (ii) Pd/C 5%, H2 (p = 4 atm), 20 ꢀC, 5 h;
(iii) (R,S)-10-camphorsulfonic acid, toluene; (c) CH2Cl2, H2O, 8% NaHCO3 (aq); L-(þ)-mandelic acid, 2-propanol, heptanes; (d) MeNH2, EtOH,
NaBH4, 25 ꢀC, 1.5 h; (e) (i) ethyl acetate, NaHCO3 (aq. sat. soln), 5; (ii) triphosgene, triethylamine, ethyl acetate, then 5, 20 ꢀC, 2 h; (f) R0RNH, CH3CN,
NaBH(OAc)3, room temp, 24 h.

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4 1073
starting from ketone intermediates 8a and 8b, shown in
Scheme 1, making use of an appropriate set of commercially
available primary and secondary amines. Hence, following
this synthetic strategy, a set of compounds was rapidly pre-
pared and then characterized in terms in vitro affinity to
the NK1 receptor binding site by filtration binding assay
on membranes derived from human (h)-NK1-CHO cells,
measuring the displacement of the radiolabeled natural
agonist [3H]SP, by increasing antagonist concentrations
(1 pM to 10 μM). Then the most in vitro potent compounds were
characterized in vivo, measuring the inhibitory effect (ID50) of
the characteristic animal’s foot tapping evoked in male gerbils
by intracerebroventricular (icv) infusion of the selective NK1
receptor agonist GR73632.12 Moreover, the gerbil social
interaction model was used to assess the anxiolytic-like prop-
erties of the most interesting NK1 receptor antagonist identi-
fied. From the set of in vitro affinity data obtained, sum-
marized in Table 1, the following comments can be made: the
replacement of the C-2 substituted piperazine core, present in
compound 1, by the corresponding 2-aryl-4-aminopiperidine
resulted in the identification of an in vitro potent sub-
family of NK1 receptor antagonists. The high in vitro
affinity observed was almost independent from the specific
amine moiety introduced, accounting for the presence, within
the NK1 antagonists receptor binding site, of a sterically
tolerant region able to accept bulky lipophilic and/or polar
groups; cis diastereoisomers were consistently more potent
than the corresponding trans diastereoisomers. No significant
difference of the in vitro affinity was observed among second-
ary and tertiary amine derivatives synthesized (see pKi values
of compounds 17 and 10a, pKi = 9.70 and 9.62, respectively).
As far as the type of substitution of the secondary amine is
concerned, different kind of alkyl groups were tolerated
(see compounds 17, 18, 19, and 20) with minimal impact on
the value of the in vitro affinity at the NK1 receptor.
Finally, the in vitro affinity of the unsubtituted benzyl
derivative (R0 = H) was slightly higher than that of the
corresponding methyl substituted analogue (R0 = CH3).
However, the latter modification, as in the case of the
pyrrolidine derivative, resulted in the obtaining of more
potent NK1 receptor antagonists (pKi of 9.85 and 9.56 for
14a and 13, respectively), making the effect of this sub-
stitution subseries dependent.
The most in vitro active compounds, administered po 1 h
before the icv administration of the NK1 receptor agonist
GR73632, exhibited excellent in vivo activity in the GFT
model. In particular, the N-acetylpiperazine derivatives 15
and 16a, when given po, emerged as the most potent molecules
of the series (ID50 =0.09 and 0.08 mg/kg, respectively).
Notably, both compounds showed a long lasting inhibition
of the foot tapping behavior, with the effect still being present
Figure 2. From vestipitant to the new chemical series.
Table 1. In Vitro Binding Affinities (pKi) at h-NK1 Receptors and in Vivo Activity of NK1 Receptor Antagonists in the Gerbil Foot Tapping Model
(GFT)
stereochemistryb
pKi
n
ID50, mg/kg (t)c
a
compd
R
R0
1 (vestipitant)
9
9.65
10.1
9.62
9.03
10.09
9.75
9.19
9.56
9.85
9.70
9.93
9.48
9.36
9.70
9.49
9.77
9.68
5
2
3
2
2
2
2
3
2
2
2
2
2
2
2
2
2
0.014
0.26
0.9
N,N-dimethylamine
N,N-dimethylamine
N,N-dimethlylamine
azetidine
H
cis
cis
10a
10b
11
CH3
CH3
H
trans
cis
cis
NTd
0.13 (4 h)
NTd
NTd
0.3
12a
12b
13
azetidine
azetidine
CH3
CH3
H
trans
cis
cis
pyrrolidine
pyrrolidine
pyrrolidine
14a
14b
15
CH3
CH3
H
0.92
trans
cis
cis
>1 (4 h)
0.09
0.08
N-acetylpiperazine
N-acetylpiperazine
N-acetylpiperazine
N-methylamine
N-cyclopropylamine
N-cyclobutylamine
2-methoxy-1-ethylamine
16a
16b
17
CH3
CH3
CH3
CH3
CH3
CH3
trans
cis
cis
>0.3
NTd
18
19
0.39 (4 h)
NTd
NTd
cis
cis
20
a pKi values have been determined as described in Experimental Section, and n is the number of experiments performed in duplicate. b Relative
stereochemistry of C-2 aryl group and the C-4 R substituent. c ID50 is the compound dose causing 50% inhibition of the tapping behavior elicited by the
icv injection of GR73632, 3 pmol/5 μL. ID50 values are expressed in mg/kg after oral administration at t = 1 or 4 h (as shown in the table) before the test.
d NT = not tested.

1074 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4
Table 2. ID50 for 16a in the Gerbil Foot Tapping Modela
Di Fabio et al.
ID50, mg/kg, sc (-1 h)
ID50, mg/kg, po (-1 h)
ID50, mg/kg, po (-4 h)
ID50, mg/kg, po (-8 h)
ID50, mg/kg, po (-24 h)
0.05
(0.02-0.06)
0.08
(0.04-0.60)
0.04
(0.04-0.40)
0.19
(0.09-0.50)
1.10
(0.74-1.96)
a Data are presented as ID50 values with 95% confidence limit. Seven animals per group were utilized in the test.
Figure 4. Increase of social interaction time (s) in gerbil produced
by the ip administration of diazepam (white) and po administration
of 16a (gray) vs vehicle (black): (//) p < 0.01.
from gerbil brain cortices featuring a complete displacement
of the specific binding of 0.8 nM [3H]SP with a pKi of 9.6 ( 0.2
(n=3). In addition, [3H]GR205171 (pKi = 10.5 on h-NK1)
filtration binding experiments were carried out on homoge-
nates prepared from human cortex. As in the previous case,
16a, when tested from 1 pM to 1 μM, completely displaced
[3H]GR205171 present at 0.5 nM, with a pKi of 9.9 (n=2).
The receptor binding selectivity of compound 16a was
assessed at 10 μM in 70 different GPCRs, ion channels, and
transporter systems. This study was performed by MDS
Pharma Services according to their lead profiling screen
(MDS Pharma Sevices 2001 catalogue, adding additional
assays, namely, calcium channel type L, phenylalkylamine,
NK2, NK3). The inhibition of specific binding was greater
than 50% only for σ1, sodium channel site 2, calcium channel
type L, and dihydropyridine and benzothiazepine calcium
channel type L. For these targets a complete displacement
curve was carried out to provide Ki values. The calculated Ki
values were 2.98 μM for σ1, 4.22 μM for sodium channel site 2,
6.58 μM for dihydropyridine binding site calcium channel
type L and for calcium channel type L, 8.78 μM for ben-
zothiazepine binding site calcium channel type L. Hence, the
receptogram screen confirmed 16a as a very selective NK1
receptor antagonist. These in vitro and in vivo data high-
lighted two critical aspects for compound 16a: (a) it possesses
subnanomolar affinity and high selectivity for the NK1 re-
ceptors without sign of pharmacological species difference
between human and gerbil NK1 receptors; (b) it is active in
human native tissues and interacts at the same NK1 receptor
binding site as the chemically diverse NK1 receptor antagonist
GR205171.25 Further to its promising biological profile, 16a
was selected as a NK1 receptor antagonist satisfying the
internal criteria for the progression of compounds and was
characterized in vivo in the social interaction model in the
gerbil. As shown in Figure 4, a 0.1 mg/kg dose of diazepam
(the standard anxiolytic benzodiazepine) given ip, signifi-
cantly increased gerbil social interaction (19 ( 1.2 s with
vehicle, 29.3 ( 1.2 with diazepam, p < 0.01). No abnormal
behaviors were recorded. Then compound 16a was tested po
at the doses of 0.1, 0.3, and 1.0 mg/kg. A dose dependent
increase intime spentinactivesocial interactionwas observed.
A significant increase was recorded at the highest dose tested
Figure 3. Antagonism of SP-induced increase of cytosolic calcium
by 16a and 16b in h-NK1-CHO cells (each point is the average of
two replicates each performed in duplicate).
8 h after the oral administration of the compounds (ID50=
0.08 and 0.19 mg/kg, respectively). A time course study was
then carried out; remarkably, 16a exhibited residual activ-
ity even after 24 h also (ID50 = 1.1 mg/kg), suggesting an
appropriate exposure in the brain several hours after their
systemic administration to the animals. As shown in Table 2,
compound 16a was also active after sc administration.
Surprisingly, the corresponding trans diastereoisomer 16b,
despite the similar in vitro affinity (pKi = 9.36 vs 9.48 for
compound 16a), was found to be inactive up to 0.3 mg/kg in
the GFT model (ID50 >0.3 mg/kg) at both 1 and 4 h after its
oral administration. This unexpected result might be ex-
plained by the different receptor mode of actions (MoA)
observed for 16a and 16b as measured by the ability of these
compounds to inhibit SP-induced release of cytosolic Ca2þ in
h-NK1-CHO cells using FLIPR (fluorometric imaging plate
reader) technology, In particular, 16a showed a nonsurmount-
able mode of action, whereas 16b, as shown in Figure 3,
conversely exhibited a surmountable mode of action. Appar-
ent pA2 values of 9.7 ( 0.1 (n = 3) for 16a and 8.9 ( 0.1 (n = 2)
for 16b were calculated. Therefore, as previously high-
lighted for compound 1, the nonsurmountable MoA was an
associated feature of the receptor profile of the NK1 receptor
antagonist necessary to exert a robust and long lasting in vivo
pharmacodynamic action. To assess the in vitro affinity at
native NK1 receptors, binding experiments were performed
on homogenates derived from gerbil cerebral tissues, follow-
ing the general protocol used for membranes obtained from
h-NK1-CHO cells as previously described.24 Compound 16a
was tested in the 1 pM to 1 μM range of concentrations in the
[3H]SP filtration binding experiments on membranes prepared

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4 1075
Table 3. Pharmacokinetic Parameters of Compound 16a in Preclinical
Speciesa
introduction of an N-acetylpiperazine moiety in the left-hand
side region of the molecule resulted in the identification of
compound 16a as one of the most in vivo potent NK1 receptor
antagonist ever identified. In particular, this compound was
found to be a potent and long lasting inhibitor of the foot
tapping behavior induced in the gerbil following icv adminis-
tration of the SP agonist GR73632. This finding together with
the nonsurmountable receptor antagonism of 16a suggests
that this compound may possesses a similar NK1 receptor
kinetic profile as vestipitant, which showed a slow off-rate
from the receptor complex.12 Notably, it has been recently
reported that slowly dissociating antagonists could exert
substantially longer efficient receptor protection in vivo than
surmountable antagonists,27,28 thus making this attribute as
an important feature to optimize the pharmacodynamic
profile of GCPR antagonists. Moreover, the evaluation in
the gerbil social interaction test revealed that 16a also pos-
sesses an anxiolytic-like property, confirming the preclinical
profile of NK1 receptor antagonists on emotional behavior.
Despite the suboptimal in vivo pharmacokinetics observed in
different animal species, 16a was progressed based upon the
extrapolation of the in vitro clearance performed in human
liver hepatocytes, predicting half-life and oral biovailability to
be adequate for a once a day dose regime for clinical use, with
the profile fully confirmed later in clinical studies.
animal speciesb
Clp ((mL/min)/kg) Vss (l/kg) half-life (h) F (%)
mouse
rat
120
37
10
26
29
4.0
3.2
1.9
3.0
3.1
1.1
1.8
4.5
2.1
1.7
21
8
dog
cynomolgus monkey
marmoset
34
8
14
a PK parameters derived from plasma concentrations. b The PK
profile was assessed in dog, cynomolgus monkey, and marmoset at
0.5 mg/kg iv and 1 mg/kg po at 1 mg/kg iv, at 2 mg/kg po in mouse, and
at 1 mg/kg iv and po in rat.
(19.0 ( 1.2 s for vehicle to 36.6 ( 1.9 for 16a, p < 0.01) with a
maximal augmentation of 92% with respect to the vehicle
treated animals. No effect on locomotor activity was ob-
served. The data obtained indicate anxiolytic-like properties
of 16a, which parallels the profile observed for other NK1
receptor antagonists in animal models of stress and anxiety.23,26
As far as the in vivo pharmacokinetic profile is concerned,
16a was characterized in rodents (mouse and rat) and in dog
and primates (marmoset and cynomolgus monkey). As shown
in Table 3, this compound showed moderate to high plasma
clearance greater than 50% of liver blood flow in all species
and a moderate distribution volumes (Vd) again in all species
tested; the lowest value observed was in dog (Vd=1.9 L/kg).
As a result of this characteristic, the observed half-life values
were relatively short and the oral bioavailability was low in
most species (8-14%), with the only exception being dog
(34%). Subsequent in vitro and in vivo studies indicated that
the poor oral bioavailability observed was most likely driven
by an extensive first pass extraction; therefore, clearance was
the main indicator to follow for optimizing oral bioavailabil-
ity. On the basis of these results, the pharmacokinetics in
human was not expected to be favorable. However, when the
in vitro intrinsic clearance (Cli) studies were performed in
human liver hepatocytes obtained from three different do-
nors, Cli values of 0.49, 0.19, 0.07 mL/(min g), respectively,
were observed, which therefore predicted a more favorable
clinical scenario. The predicted human blood clearance along
with allometric scaling suggested in fact that the clearance in
human was potentially low, ranging from 140 to 553 mL/min
with an elimination half-life in the range from 11 to 18 h,
adequate enough for a once a day administration dose regime,
a key requirement for the progression of the compound. The
predicted low clearance in human, together with a good
correlation between oral bioavailability and hepatic extrac-
tion ratios in preclinical species, indicated that oral bioavail-
ability in human was expected to be higher than in animals
(>30%).
Experimental Section
NMR spectra were recorded in DMSO-d6 or CDCl3 at con-
stant temperature of 25 ꢀC, and complete assignment was made
bymeansofseveral1D and2D techniques including 19F, gCOSY,
gHSQC, and gHMQC NMR experiments when needed. 1H and
gHSQC experiments were collected on a 400 Unity Varian or 500
Inova Varian instrument. 13C and 19F experiments were collected
using a 300 Inova Varian instrument. Chemical shifts are re-
ported in parts per million (ppm) downfield from the CHCl3
residual line (δ = 7.27 ppm) or from the DMSO residual line
(δ =2.49ppm) andwereassignedassinglet(s), doublet (d), triplet
(t), quartet (q), broad quartet (bq), broad singlet (bs), doublet of
doublets (dd), or multiplet (m). Coupling constants (J) are given
in Hz. IR spectra were recorded on a Nicolet Magna760 (Thermo
Fisher Scientific) spectrometer. Mass spectra analyses were per-
formed on a VG Platform (Waters, Manchester, U.K.) mass
spectrometer operating in positive electrospray ion mode. Ana-
lytical thin layer chromatography (TLC) was performed on glass
plates (Merck Kieselgel 60 F254). Visualization was accom-
plished by UV light (254 nm) and I2. Column chromatography
was performed on silica gel (Merck Kieselgel 70-230 mesh).
Chemical purity of compounds was assessed by HPLC (>95%):
Zorbax Bonus RP column (dimensions 150 cm ꢀ 4.6 mm, 3.5 μm
particle size) or validated equivalent. The column temperature
was held at 20 ꢀC. Mobile phase A was a 40/60 mixture of H2O
and CH3OH þ 5 mM NH4HCO3. Mobile phase B was a 20/80
mixture of H2O and CH3OH þ 5 mM NH4HCO3. A linear
gradient from 0% to 100% of mobile phase B over 30 min was
used. Flow rate was 1.0 mL/min, and UV detector wavelength
was 210 nm. Sample concentration used was typically 0.6 mg/mL
with an injection volume of 5 μL.
On the basis of this information, 16a was progressed into
preclinical development and, upon completion of the required
toxicological package, to clinical studies. Notably, the phar-
macokinetics generated subsequently during the first time in
human phase were in good agreement with those predicted
previously, with oral bioavailability and half-lives adequate
for a once a day dose regime for clinical use.
All reactions were carried out under anhydrous N2 atmosphere
using standard Schlenk techniques. Most chemicals and solvents
were analytical grade and used without further purification.
Phenylmethyl 2-(4-fluoro-2-methylphenyl)-4-oxo-3,4-dihydro-
1(2H)-pyridinecarboxylate (3). To a suspension of magnesium
turnings (55.3 kg, 2.3 kmol) in THF (467 L) at room temperature
under N2 atmosphere, a small amount of I2 (24.6 g, 97 mmol)
was added. The mixture was heated to 35-40 ꢀC. To this
suspension, a portion (62 L) of a solution of 2-bromo-5-fluoro-
toluene (357 kg, 1.89 kmol) in THF (953 L) was added. After
Conclusion
An appropriate modification of the C-phenylpiperazine
template enabled our medicinal chemistry team to expand
the scope of the exploration of the series and to identify some
in vitro potent and selective NK1 receptor antagonist exhibit-
ing remarkable druglike properties. In particular, the

1076 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4
Di Fabio et al.
the brown color disappeared, the remaining part of the solution
was added dropwise over 1 h, maintaining the temperature of
the suspension at 60-70 ꢀC. Then the reaction mixture was
refluxed for 2 h. The solution of Grignard’s reagent was then
cooled to 18-20 ꢀC. To a stirred solution of 4-methoxypyridine
1 (123 kg, 1.12 kmol) in dry THF (406 L) was added dropwise a
solution of benzyl chloroformate (263 kg, 1.54 kmol) in dry
THF (246 L) at -20 ꢀC over 30-50 min (exothermic step). The
resulting mixture was stirred at 20-30 ꢀC for 45-60 min. The
solution of the Grignard’s reagent previously prepared was
added dropwise to the suspension over 50-80 min, maintaining
the temperature below -15 ꢀC. After the addition was com-
pleted the solution was stirred for 1 h at -20 ꢀC. Then 3% HCl
(1353 L) was added slowly followed by toluene (2066 L). The
organic layer was separated and washed with a 4.5% aqueous
solution of NaHCO3 (1845 L). After separation the organic
layer was partially concentrated in vacuo. Isopropyl alcohol
(1525 L) was added, and the resulting suspension was stirred 1 h
at 20-25 ꢀC and then 2 h at 0-5 ꢀC. The solid was isolated by
centrifugation, washed with isopropyl alcohol (234 L), and dried
in a vacuum oven at 30-35 ꢀC for 10-25 h to give the title
by using a column with Chralcel OD-H as stationary phase
(25 cmꢀ4.6 mm i.d., 5 μm, flow=1 mL/min, T=30 ꢀC).
3,5-(Bistrifluoromethylbenzyl)methylamine Hydrochloride (7a).
To a stirred solution of 3,5-bis(trifluoromethyl)benzaldehyde (15 g,
58.6 mmol) in dry methanol (90 mL) a 8 M solution of MeNH2 in
methanol (8.5 mL, 68 mmol) was added. After 50 min KBH4 (3.3g,
61.2 mmol) was added. The reaction mixture was stirred for 1 h
at room temperature, then concentrated in vacuo to remove
methanol, and diluted with ethyl ether (80 mL). Finally, a 1 M
solution of HCl in diethyl ether (93 mL) was added. The solid
obtained was filtered and washed with diethyl ether (40 mL) to give
title compound 6a (16.34 g, 89.8%) as a white powder. 1H NMR
(400 MHz, DMSO-d6) δ ppm 9.30 (2H, bs), 8.32 (2H, s), 8.18 (1H,
s), 4.31 (2H, s), 2.55 (3H, s).
(1R)-1-[3,5-Bis(trifluoromethyl)phenyl]-N-methylethanamine
L-Malate (7b). To a solution of 3,5-bis-trifluoromethylaceto-
phenone (300 g, 1.17 mol) in MeOH (1120 mL), a solution of
MeNH2 8 M in EtOH (372 mL, 2.98 mol) was added dropwise in
15 min at 25 ꢀC under nitrogen. Then the mixture was stirred for
24 h. NaBH4 was added portionwise over 30 min (27.9 g, 0.74
mol) at 0 ꢀC. A second amount of NaBH4 was added over 30 min
(17.1 g, 0.45 mol) and the mixture stirred for a further 1.5 h. The
reaction mixture was concentrated in vacuo to small volume.
Then it was slowly poured into a mixture of EtOAc (1500 mL)
and a 12% aqueous solution of NH4Cl (1500 mL). The aqueous
phase was re-extracted with EtOAc (1500 mL). The organic
layers were collected, washed with 13% aqueous solution of
NaCl (300 mL), then evaporated in vacuo to obtain (1R)-1-[3,5-
bis(trifluoromethyl)phenyl]-N-methylethanamine (305 g) as a
yellow oil, which was dissolved in EtOAc (2960 mL). Then L-
malic acid was added portionwise (146.5 g). The suspension was
stirred for 2 h at 25 ꢀC and then for 3 h at 0 ꢀC. The suspension
was filtered and washed with EtOAc (300 mL). Then the solid
was dried under vacuum obtaining crude (1R)-1-[3,5-bis-
(trifluoromethyl)phenyl]-N-methylethanamine (168 g) as a
white solid (enantiomeric purity = 96.5/3.5 via capillary zone
electrophoresis). To improve chiral purity the solid (264 g) was
suspended in ethyl acetate (3430 mL) and then heated to reflux
until complete dissolution and then cooled to 3 ꢀC and stirred 90
min. The suspension was filtered, washed with ethyl acetate (264
mL), then dried to obtain title compound (250 g). 1H NMR (400
MHz, DMSO-d6) δ ppm 9.21 (3 H, bs), 8.16 (2 H, bs), 8.09 (1 H,
bs), 4.26 (1 H, q), 4.02 (1 H, dd), 2.55 (1 H, dd), 2.33 (3 H, s), 2.35
(1 H, dd), 1.45 (3 H, d). 250 g, enantiomeric purity =
100/0 a/a % via GC: Chiralsil-dex CB column (25 m ꢀ 0.25
mm i.d., 0.25 μm film thickness, T=80 ꢀC for 6 min, then 160 ꢀC
with a ramp of 10 ꢀC/min, and finally down to 80 ꢀC; flow
(helium) = 1.0 mL/min; split ratio 20:1).
(2R)-N-{[3,5-Bis(trifluoromethyl)phenyl]methyl}-2-(4-fluoro-
2-methylphenyl)-N-methyl-4-oxo-1-piperidinecarboxamide (8a).
To a solution of 5 (25.5 g, 70.9 mmol) in ethyl acetate (382.5
mL) a saturated aqueous solution of NaHCO3 (382 mL) was
added, and the resulting mixture was vigorously stirred for 15
min at room temperature. The aqueous layer was separated and
re-extracted with ethyl acetate (255 mL), and the collected
organics were dried over Na2SO4 and concentrated in vacuo
to give 5 as free base. To a solution of triphosgene (9.47 g, 31.92
mmol) in ethyl acetate (66 mL) cooled to 0 ꢀC, a solution of 5 as
free base in ethyl acetate (135 mL) and triethylamine (24.7 mL,
177 mmol) was added dropwise over 1 h, maintaining the
temperature below 10 ꢀC. After the mixture was stirred for 2 h
at 20 ꢀC, ethyl acetate (201 mL) and triethylamine (39.5 mL, 283
mmol) were added to the reaction mixture followed by 7a (33.3 g,
113 mmol) portionwise over 10 min. The mixture was stirred
for 2 h at 20 ꢀC and then quenched with a 10% aqueous solution
of NaOH (160 mL). The organics were then washed with a 1%
aqueous solution of HCl (4 ꢀ 160 mL), brine, dried over
Na2SO4, and concentrated in vacuo. The residue was purified
by flash chromatography (cyclohexane/ethyl acetate 9:1) to give
pure 8a (27.3 g, 78%). 1H NMR (500 MHz, DMSO-d6) δ (ppm):
1
compound 3 (329 kg, 86%). H NMR (600 MHz, DMSO-d6):
8.20 (dd, 1H), 7.31 (bs, 3H), 7.19 (bs, 2H), 7.11 (dd, 1H), 7.07
(dd, 1H), 6.94 (dt, 1H), 5.79 (d, 1H), 5.38 (d, 1H), 5.18 (m, 2H),
3.28 (dd, 1H), 2.35 (d, 1H), 2.27 (s, 3H). MS m/z = 340 [MH]þ.
2-(4-Fluoro-2-methylphenyl)-4-piperidinone Camphor-10-
sulfonate (4). A solution of compound 3 (200 kg, 59 kmol) in
isopropyl alcohol (2200 L) was heated to 50 ( 5 ꢀC until the solid
was dissolved. A solution of tris(triphenylphosphine)rhodium-
(I) chloride (2 kg, 2.2 mol) in isopropyl alcohol (70 L) was added
to the reaction mixture. The solution was then hydrogenated at
P = 5 bar of H2 for 5 h at 60 ( 5 ꢀC. The reaction mixture was
cooled to 20 ( 5 ꢀC, and a suspension of 5% Pd/C (48 kg) in
isopropyl alcohol (200 L) was added. The mixture was hydro-
genated at P = 4 ( 1 bar of H2 for 5 h at 20 ( 5 ꢀC. Then the
catalyst was filtered and washed with isopropyl alcohol (500 L).
10-Camphorsulfonic acid (140 kg) was added, and the solution
was concentrated to 700 L. Toluene (2200 L) was added and the
solution concentrated again to 700 L. Further toluene (1400 L)
was added. The solution was seeded, and the obtained slurry was
stirred for 10-12 h at 20-21 ꢀC and then for 6-7 h at 2 ( 2 ꢀC.
The solid was isolated by centrifugation, washed with toluene
(200 L), and dried in a vacuum oven at 30-35 ꢀC for 11-18 h to
1
give title compound 4 (166 kg, 64.3%). H NMR (600 MHz,
DMSO-d6): 9.51 (bs, 1H), 9.32 (bs, 1H), 7.70 (dd, 1H), 7.19 (m,
1H), 7.16 (dd, 1H), 4.98 (d, 1H), 3.62 (m, 2H), 2.95 (dd, 1H), 2.90
(m, 1H), 2.89 (d, 1H), 2.63 (m, 1H), 2.55 (m, 1H), 2.53 (m, 1H),
2.40 (d, 1H), 2.37 (s, 3H), 2.24 (m, 1H), 1.94 (t, 1H), 1.85 (m, 1H),
1.80 (d, 1H), 1.28 (m, 2H), 1.03 (s, 3H), 0.74 (s, 3H). MS m/z =
208 [MH]þ.
(2R)-2-(4-Fluoro-2-methylphenyl)-4-piperidinone L-(þ)-
Mandelate (5). To a suspension of 4 (310 kg, 0.7 kmol) in
CH2Cl2 (2170 L), H2O (700 L) was added. The suspension was
stirred until a clear solution was obtained. Then an 8% aqueous
solution of NaHCO3 (1364 L) was added and the system stirred
for about 30 min. The organic layer was separated, washed with
H2O (700 L), and the solvent was partially evaporated in vacuo
followed by addition of isopropyl alcohol (1240 L) and complete
evaporation of the solvent. A solution of L-(þ)-mandelic acid
(99 kg, 0.65 kmol) in isopropyl alcohol (310 L) was added to the
organic phase and the resulting suspension stirred for at least 5 h
at 20 ( 3 ꢀC. The solid was isolated by centrifugation, washed
with isopropanol (208 L), heptane (93 L), and dried in a vacuum
oven at 20 ( 3 ꢀC for 14 h to give 5 (117 kg, 46%). 1H NMR (600
MHz, DMSO-d6): 7.52 (dd, 1H), 7.40 (d, 2H), 7.33 (t, 2H), 7.27
(t, 1H), 7.02 (m, 2H), 4.96 (s, 1H), 4.06 (dd, 1H), 3.32 (ddd, 1H),
2.90 (td, 1H), 2.52 (m, 1H), 2.46 (m, 1H), 2.32 (s, 3H), 2.29 (m,
1H), 2.22 (m, 1H). MS: m/z 208 [MH]þ. The enantiomeric purity
of compound 5 was determined by chiral HPLC (99.5:0.5 a/a %)

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4 1077
7.97 (1H, s), 7.77 (2H, s), 7.26 (1H, dd), 6.97 (1H, dd), 6.92 (1H,
td), 5.24 (1H, t), 4.60 (1H, d), 4.44 (1H, d), 3.58 (2H, m), 2.80
(3H, s), 2.70 (2H, m), 2.50 (2H, m), 2.28 (3H, s).
The reaction mixture was stirred at room temperature under
N2 atmosphere for 24 h and quenched with saturated NaHCO3
(23.1 mL) and water (61.6 mL).
(2R)-N-{(1R)-1-[3,5-Bis(trifluoromethyl)phenyl]ethyl}-2-(4-
fluoro-2-methylphenyl)-N-methyl-4-oxo-1-piperidinecarbox-
amide (8b). To a solution of 7b (100 g, 280 mmol) in ethyl acetate
(400 mL) a 15% aqueous solution of Na2CO3 (400 mL) was
added. The mixture was stirred at room temperature for 15 min.
The organic layer was separated and washed with a 10%
aqueous solution of NaCl (400 mL), dried over Na2SO4, and
concentrated in vacuo to 300 mL to give 7b as free base. To a
solution of 5 (130 g, 320 mmol) in ethyl acetate (500 mL) a 13%
aqueous solution of NH4OH (325 mL) was added, and the
resulting mixture was stirred for 15 min. The organic layer was
washed with H2O (4 ꢀ 650 mL) and brine (650 mL), dried over
Na2SO4, and concentrated in vacuo to 300 mL to give 5 as free
base. Then to triphosgene (31.4 g, 104 mmol) dissolved in ethyl
acetate (250 mL) the previously prepared solution of intermedi-
ate 5 was added at 10 ꢀC under N2 atmosphere, along with
triethylamine (89 mL, 638 mmol). After the mixture was stirred
for 15 min at room temperature, 7b as free base and triethyl-
amine (77.6 mL, 640 mmol) were added and the reaction mixture
was heated to reflux for 5 h.
Saturated NH4OH aqueous solution (210 mL) was added.
The organic layer was separated and washed with a 5% aqueous
solution of H2SO4 (4 ꢀ 400 mL) and finally brine (400 mL). The
organic phase was concentrated in vacuo to 500 mL, isooctane
(500 mL) was added, and the solution was concentrated to
500 mL. The resulting slurry was filtered, the solvent was
evaporated in vacuo, and the residue was purified by chroma-
tography (cyclohexane/ethyl acetate 8/2) to give title compound
8b (90.4 g, 65%). 1H NMR (600 MHz, DMSO-d6): 7.98 (s, 1H),
7.78 (s, 2H), 7.25 (dd, 1H), 6.98 (dd, 1H), 6.89 (dt, 1H), 5.25 (dd,
1H), 5.16 (q, 1H), 3.63 (m, 1H), 3.56 (m, 1H), 2.75 (dd, 1H), 2.68
(dd, 1H), 2.57 (s, 3H), 2.53 (m, 1H), 2.46 (dt, 1H), 2.27 (s, 3H),
1.58 (d, 3H). MS: m/z 505 [MH]þ.
General Procedure of the Synthesis of Compounds 9-22:
Synthesis of 15 and 16a Presented as Representative Examples.
(2R,4S)-4-(4-Acetyl-1-piperazinyl)-N-{[3,5-bis(trifluoromethyl)-
phenyl]methyl}-2-(4-fluoro-2-methylphenyl)-N-methyl-1-piper-
idinecarboxamide (15). 1-Acetylpiperazine (6.27 g, 48.89 mmol)
was dissolved in acetonitrile (20 mL). Then the intermediate 8a
(12 g, 24.4 mmol) in acetonitrile (270 mL) was added followed by
NaBH(OAc)3 (21.5 g, 34.2 mmol). The reaction mixture was
stirred at room temperature for 24 h. The reaction was quenched
with aqueous saturated NaHCO3 (40 mL) and water (100 mL),
and the resulting solution was concentrated in vacuo to about
140 mL. EtOAc (300 mL) was added, and the organic layer was
separated. The aqueous layer was extracted with EtOAc (2ꢀ
125 mL). The collected organic layers were washed with brine
(200 mL), dried over Na2SO4, and concentrated in vacuo to
obtain the crude 15 which was purified by flash chromatography
(methanol/ethyl acetate 25/75) to give pure 15 (4.1 g). A portion
of the pure free base 15 (3.6 g, 6 mmol) was dissolved in ethyl
ether (36 mL) cooled to 0 ꢀC. HCl 1 M in ethyl ether is added (6
mL, 6 mol) and the resulting suspension was stirred for 4.5 h at
3 ꢀC, filtered, and dried in vacuo to give 15 (3.24 g, 23.7%). 1H
NMR (400 MHz, DMSO-d6) δ (ppm): 11.13 (1H, bs), 7.95 (1H,
s), 7.60 (2H, s), 7.26 (1H, m), 6.94 (1H, dd), 6.83 (1H, m), 4.63
(1H, d), 4.38 (2H, m), 4.20 (1H, d), 3.95 (1H, m), 3.60-3.30 (6H,
m), 3.08 (2H, m), 2.93 (3H, m), 2.73 (1H, t), 2.36(3H, s), 2.20 (2H,
m), 2.01 (3H, s), 1.93 (1H, m), 1.69 (1H, q).
The resulting solution was concentrated in vacuo. EtOAc
(208 mL) was added. The organic layer was separated, and the
aqueous layer was re-extracted with ethyl acetate (2 ꢀ 77 mL).
The collected organics layers were washed with brine (2 ꢀ 118
mL), dried over Na2SO4, and concentrated in vacuo to give
crude 16a which was dissolved in THF (85.4 mL) and mixed with
a solution of methanesulfonic acid (0.89 mL, 13.73 mmol) in
THF (6.1 mL). The slurry was stirred for 3 h at 0 ꢀC and the solid
filtered, washed with THF (15.4 mL), then dried in vacuo for 48
h to give title compound 16a as a white solid (4.44 g, 41%). 1H
NMR (600 MHz, DMSO-d6): 9.57 (bs, 1H), 7.99 (bs, 1H), 7.68
(bs, 2H), 7.23 (m, 1H), 6.95 (dd, 1H), 6.82 (m, 1H), 5.31 (q, 1H),
4.45 (m, 1H), 4.20 (dd, 1H), 3.99 (m, 1H), 3.56 (m, 1H), 3.47 (m,
3H), 3.37 (m, 1H), 3.15 (m, 1H), 2.96 (m, 1H), 2.87 (m, 1H), 2.80
(t, 1H), 2.74 (s, 3H), 2.36 (s, 3H), 2.30 (s, 3H), 2.13 (m, 1H), 2.08
(m, 1H), 2.10 (s, 3H), 1.87 (m, 1H), 1.73 (m, 1H), 1.46 (d, 3H).
MS: m/z 617 [MH]þ, as free base.
Receptor Binding Studies. Materials. [3H]SP (1.26 TBq/mmol)
was purchased from Amersham Life Science. SP and GR73632
(δ-aminovaleryl,6 Pro,9 N-Me-Leu10)-substance P (6-11) were
obtained from Bachem. GR205171 was sensitized in the medicinal
chemistry laboratories of GSK. All drugs were prepared as
10 mM solution. In binding experiments vs [3H]SP, all competing
drugs were serially diluted in the assay buffer. In binding experi-
ments and FLIPR assays, drugs were serially diluted in DMSO to
have a final concentration of the solvent equal to 1%. Fluo-4 was
purchased from Molecular Probes. Probenecid was purchased
from Aldrich.
Binding Experiments. Membranes from Chinese hamster
ovary cells stably transfected with human NK1 receptor (h
NK1-CHO) were prepared essentially as previously described.29
[3H]SP binding assay was carried out in 96-well plates in a final
volume of 400 μL of 50 mM HEPES, pH 7.4, 3 mM MnCl2, and
0.02% BSA. Incubations proceeded at 22 ꢀC for 60 min and
displacement experiments were performed by using 0.5 nM
[3H]SP. Nonspecific binding was defined by the addition of
1 μM GR205171. Reactions were stopped by filtration through
GF/C filtermats using a cell harvester.
Brain cortical samples from one donor were obtained under
approved ethical guidelines. [3H]SP and [3H]GR205171 binding
experiments in cerebral tissues were carried out in 96-well deep
well polypropylene plates. The assay volume of 400 μL consisted
of 100 μL of incubation buffer (containing 50 mM HEPES, pH
7.4, 3 mM MnCl2, and 0.02% BSA), 4 μL of DMSO, or
increasing concentrations of 16a dissolved in DMSO (1 pM to
1 μM final concentration). When [3H]SP binding was per-
formed, an amount of 100 μL of [3H]SP (0.5-0.8 nM final
concentration) was used in incubation buffer and 200 μL of
membrane suspension (0.6 mg of protein) in incubation buffer
containing 2 μg/mL leupeptin, 20 μg/mL bacitracin, and 0.5 μM
phosphoramidon. When [3H]GR205171 binding was carried
out, an amount of 100 μL of [3H]GR205171 (0.8 nM final
concentration) was used in incubation buffer and 200 μL of
membrane suspension containing 0.14 mg of protein dissolved
in incubation buffer with the addition of 2 μg/mL leupeptin, 20
μg/mL bacitracin, and 0.5 μM phosphoramidon. For both
radioligands the incubation proceeded at room temperature
for 60 min. Nonspecific binding was defined by the addition
of cold SP (1 μM) or GR205171 (1 μM) for the gerbil and human
cerebral tissues, respectively. The reaction was stopped by rapid
filtration through GF/C filtermats presoaked in 0.5% poly-
etylenimmine (PEI) using a Brandel M/96R cell harvester.
Filters were washed 3 times with 1 mL of ice cold wash buffer
(containing 50 mM HEPES, pH 7.4 and 3 mM MnCl2), and
radioactivity was counted in a liquid scintillation counter (β
counter, Packard). In each experiment, every concentration of
displacer was tested in duplicate.
(2R,4S)-10-Acetyl-N-{(1R)-1-[3,5-bis(trifluoromethyl)phenyl]-
ethyl}-2-(4-fluoro-2-methylphenyl)-N-methyl-4,40-bipiperidine-
1-carboxamide Methanesulfonate Salt (16a). 1-Acetylpiperazine
(3.9 g, 30.5 mmol) was dissolved in acetonitrile (17.7 mL). Then
the intermediate 8b (7.7 g, 15.7 mmol) dissolved in acetonitrile
(177 mL) was added followed by NaBH(OAc)3 (13.6 g,
22 mmol).

1078 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4
Di Fabio et al.
Measurement of [Ca2þ]i Using FLIPR in h-NK1-CHO Cells. h-
NK1-CHO cells were seeded into black walled clear-bottom 96-
well plates (Costar, U.K.) at a density of 60 000 cells per well and
cultured overnight. The cells were then incubated for the label-
ing in the culture medium containing the fluorescent calcium
indicator Fluo-4 AM (2 μM), the organic anion transport
blocker probenecid (5 mM), and HEPES (20 mM) for 30 min
in a humidified atmosphere of 5% CO2. After being washed with
Hanks’ balanced salts solution (HBSS) containing 20 mM
HEPES and 2.5 mM probenecid (wash buffer), the cells were
incubated for 60 min at 37 ꢀC in wash buffer containing 0.02%
BSA (assay buffer) either in the absence (control) or in the
presence of 16a and 16b (1-10 nM). The plates were then placed
into a FLIPR (Molecular Devices, Sunnyvale, CA) to monitor
cell fluorescence (λex = 488 nm, λem = 510-570 nm) before and
after the addition of different concentrations of SP (2 pM to 300
nM) in assay buffer. FLIPR experiments were carried out by
using a laser setting of 1.0 W and a 0.4 s CCD (charge coupled
device) camera shutter speed.
In Vivo Studies. Pharmacological Studies. All experiments
were prereviewed and approved by a local animal care commit-
tee in accordance with the guidelines of the “Principles of
Laboratory Animal Care” (NIH Publication No. 86-23, revised
1985) and with a project license that was obtained according to
Italian law (Art. 7, Legislative Decree No. 116, January 27,
1992), which acknowledges European Directive 86/609/EEC on
the care and welfare of laboratory animals.
doses of 1 mg/kg, iv, and 2 mg/kg, po, using a composite design
with three animals per time point (n = 21 for the iv arm; n = 21
for the po arm). In all studies, blood samples were collected at
nominal times after dosing, centrifuged to obtain plasma, and
then assayed for test compound concentration using a method
based on protein precipitation followed by HPLC-MS/MS
analysis.
Data Analysis. Data of competition binding experiments on
membranes from hNK1-CHO cells were analyzed by using the
nonlinear curve-fitting program MicroSAT 5.2.1. For [3H]SP
displacement binding experiments, IC50 values were obtained
constraining the slope factor to unity. IC50 values were con-
verted to Ki using the Cheng-Prusoff equation.30 Data from
competition binding experiments on brain homogenates were
analyzed using the nonlinear curve-fitting program LIGAND.31
KD of radioligands was assessed by elaborating satura-
tion experiments with LIGAND. In FLIPR experiments on
h-NK1-CHO cells, functional responses were measured as
fluorescence intensity (FI) produced after receptor stimulation
minus basal FI. Curve fitting was determined by nonlinear
regression analysis using GraFit, version 4.0.32 In functional
experiments, the potency of compounds 16a/b was determined
by using either the Schild’s analysis33 (competitive antagonism)
or the Gaddum equation [nonsurmountable antagonism; pA2 =
log(concn ratio-1) - log (antag concn)], where concn ratio
represents the ratio between the EC50 values of the agonist in
the presence and in the absence of the antagonist.
A characteristic intense foot tapping behavior is invoked by
icv administration of selective NK1 receptor agonists in Mongolian
gerbils. This behavior can be selectively antagonized by CNS
penetrating NK1 antagonists. The model is robust and is
particularly useful, as standard NK1 receptor ligands recognize
the gerbil NK1 receptor with the same affinity as the human
NK1 receptor. Direct icv infusion of a highly selective tachykinin
NK1 receptor agonist, GR73632, in gerbils evokes a vigorous
species-specific behavior. This rhythmic hind limb thumping
is not induced by agonists for the other tachykinin receptors.
This particular action was used to detect the central or peri-
pheral activity of the tested compound. The skull is exposed, and
an amount of 5 μL (3 pmol/5 μL of GR73632) is injected by the
insertion of a cuffed 25 gauge needle to a depth of 4 mm below
bregma, directly into the lateral ventricle. Immediately the
animals were individually placed in a clear Perspex observation
box. Following 1 min of recovery, the duration of repetitive hind
foot tapping is recorded for 3 min, with a maximum possible
duration of 180 s. 1, 4, 8, and 24 h before GR73632 administra-
tion, at least seven animals each received treatment of vehicle or
test compound (10 mL/kg). All treatments were randomly
distributed between the five animals of each cage.
In the social interaction test it has been hypothesized that the
time spent in active social interaction reflects the level of stress
induced in the animals by the environment and the presence of
an unfamiliar partner.20 Clinically effective anxiolytic drugs
(e.g., diazepam) increase the interaction exhibited by the two
unfamiliar animals, and these anxiolytic-like effects can be
distinguished from potential sedative effects (assessed by the
locomotor activity as line crossing). On the day of the experi-
ment pairs of male Mongolian gerbils (50-70 g) matched for the
treatment condition but unfamiliar to each other were placed in
the center of the arena and their social interaction (sniffing,
following, grooming the partner, wrestling) was recorded for
5 min. One hour prior to the test animals received vehicle or 16a
(ip 2 mL/kg or po 10 mL/kg; 10 animals for groups) according to
a randomized experimental design.
Behavioral data were subjected to one way analysis of var-
iance (ANOVA) followed by Dunnett’s test or a paired t test
comparing each compound dose with the related vehicle treat-
ment, using GB-stat software (version 6.5, Dynamic Microsys-
tems Inc.). The dose of 16a inhibiting foot tapping by 50%
(ID50) was determined by a linear regression analysis using RS-1
software and is presented with 95% confidence limits.
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