Discovery process and pharmacological characterization of 2-(S)-(4-fluoro-2-methylphenyl)piperazine-1-carboxylic acid [1-(R)-(3,5-bis- trifluoromethylphenyl)ethyl]methylamide (vestipitant) as a potent, selective, and orally active NK1 receptor

Article abstract of DOI:10.1021/jm900023b

In an effort to discover novel druglike NK₁ receptor antagonists a new series of suitably substituted C-phenylpiperazine derivatives was identified by an appropriate chemical exploration of related N-phenylpiperazine analogues, with the specifi

Full text of DOI:10.1021/jm900023b

3238
J. Med. Chem. 2009, 52, 3238–3247
Discovery Process and Pharmacological Characterization of 2-(S)-(4-Fluoro-2-
methylphenyl)piperazine-1-carboxylic Acid [1-(R)-(3,5-Bis-trifluoromethylphenyl)ethyl]methyl-
amide (Vestipitant) as a Potent, Selective, and Orally Active NK1 Receptor Antagonist
Romano Di Fabio,*,# Cristiana Griffante,# Giuseppe Alvaro,# Giorgio Pentassuglia,#,† Domenica A. Pizzi,# Daniele Donati,#,§
Tino Rossi,# Giuseppe Guercio,‡ Mario Mattioli,‡ Zadeo Cimarosti,‡ 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, Chemical DeVelopment, and Molecular DiscoVery Research, GlaxoSmithKline
Medicines Research Centre, Via A. Fleming 4, 37135 Verona, Italy
ReceiVed January 9, 2009
In an effort to discover novel druglike NK1 receptor antagonists a new series of suitably substituted
C-phenylpiperazine derivatives was identified by an appropriate chemical exploration of related N-
phenylpiperazine analogues, with the specific aim to maximize their in vitro affinity and optimize in parallel
their pharmacokinetic profile. Among the compounds synthesized, 2-(S)-(4-fluoro-2-methylphenyl)piperazine-
1-carboxylic acid [1-(R)-(3,5-bis-trifluoromethylphenyl)ethyl]methylamide (vestipitant) was identified as one
of the most in vitro potent and selective NK1 receptor antagonists ever discovered, showing appropriate
pharmacokinetic properties and in vivo activity. On the basis of its preclinical profile, this compound was
selected as a drug candidate.
Introduction
acute and delayed vomiting (CINV). Notably, all of the NK1
receptor antagonists tested in humans so far seem to be well
tolerated with a cleaner side effects profile with respect to
classicalantidepressantagents(SSRIs,SNRIs,andtricyclics).10,11,15,16
As part of a wide drug discovery program aimed toward the
identification of a novel series of NK1 receptor antagonists,
phenylpiperazine derivatives of type A, shown in Figure 2, were
designed both to maximize the recognition of the NK1 receptor
binding site and to maintain the positive physicochemical
features associated with GR205171,17 one of the most druglike
compounds identified at that time. Herein we describe the
exploration and the structure-activity relationship (SAR) of this
new class of NK1 receptor antagonists. The screening cascade
set up to select the best compounds included in vitro studies at
recombinant human NK1 receptors and an in vivo pharmaco-
dynamic model in gerbil, a species that has shown a similar
NK1 receptor pharmacology as the human NK1 receptor.18,19
Among the compounds identified, 14aa was found to be a
potent, selective, and orally active NK1 receptor antagonist.20
A large body of preclinical and clinical evidence associate
the substance P (SPa) and its preferred G-protein-coupled
receptor (GPCR) NK1 with the pathophysiology of a variety of
stress and fear-related disorders,1-5 including depression, a
pathological condition still perceived as one of the leading
disabling illnesses worldwide.6,7 In addition, since SP is widely
spread within the CNS and colocalized with relevant neurotrans-
mitters, it has been hypothesized to be critical to the control of
nociception, migraine, nausea and vomiting, inflammatory bowel
syndrome, and urinary incontinence.8,9 From the past 15 years
to date, this large therapeutic potential triggered an unprec-
edented industrial research effort aimed toward the discovery
and the clinical evaluation of NK1 receptor antagonists. In phase
II clinical trials performed by Merck and Pfizer, MK-869,10
L-759274,11 and CP-122,72112 (Figure 1) have proven to be
antidepressants. In addition, Novartis reported for NKP-60813
(Figure 1) positive result in a clinical trial in patients with social
phobia, whereas GSK showed that GR205171 (Figure 1) was
able to reduce significantly anxiety symptoms involving 36
patients suffering from social anxiety disorders (SAD).14 Finally
MK-869 (Emend) has been launched into the market as an
antiemetic agent for chemotherapy-induced nausea and both
Chemistry
The racemic derivative 7 was prepared as shown in Scheme
1. Intermediate 4 was synthesized in two steps from 2 and in
high chemical yield by sequential Suzuki-type cross coupling
reaction and followed by hydrogenation of the pyrazine ring.
Then the chemoselective protection of the less hindered nitrogen
atom as Cbz afforded key intermediate 5. The following
coupling reaction was performed with triphosgene and triethy-
lamine followed by addition of N-methyl-3,5-bis(trifluorom-
ethyl)benzylamine hydrochloride in CH2Cl2 at 0 °C in the
presence of pyridine to give intermediate compound 6, which
was then transformed into the racemate 7 by hydrogenation.
The synthesis of the pure enantiomers 7a and 7b was performed
as shown in Scheme 2. Compound 4 was transformed into the
diastereomeric mixture 8a,b in the presence of CDI and (R)-
(+)-1-phenethyl alcohol. The following elaboration of the right-
hand side of the molecule enabled us to prepare diastereoisomers
9a and 9b, which were separated by flash chromatography.
* To whom correspondence should be addressed. Phone: +39 045
8218879. Fax: +39 045 8218196. E-mail: rdf26781@gsk.com.
#
Neurosciences Centre of Excellence for Drug Discovery.
Present address: E-Pharma Trento S.p.A., Via Provina 2, 38040 Ravina
†
di Trento, Trento, Italy.
§
Present address: Nerviano Medical Sciences Srl, Via Pasteur 10, 20014
Nerviano, Milan, Italy.
‡
Chemical Development.
Molecular Discovery Research.
|
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; SSRIs,
selective serotonin reuptake inhibitors; SNRIs, selective norepinephrine
reuptake inhibitors; SAR, structure-activity relationship; CDI, carbonyl-
diimidazole; THF, tetrahydrofuran; CHO, Chinese hamster ovary; FLIPR,
fluorescence imaging plate reader; GFT, gerbil foot tapping; CRC,
concentration-response curve.
10.1021/jm900023b CCC: $40.75
2009 American Chemical Society
Published on Web 04/23/2009

A Piperazine-1-carboxylic Acid
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3239
Figure 1. Chemical structures of some NK1 receptor antagonists evaluated in clinical trials.
Figure 2. General structures of the C-phenylpiperazines and N-phenylpiperazines.
Scheme 1a
a (a) [1,2-Bis(diphenylphosphino)-ethane]dichloronickel (II), chloropyrazine, Na2CO3 1 M, toluene, EtOH 95%, 2 h, reflux; (b) 20% Pd(OH)2/C, H2 (5
atm), 95% EtOH/37% HCl, 100:1, 4 h; (d) benzylchloroformate, Et3N, CH2Cl2, T ) 0 °C, 2 h; (e) triphosgene, Et3N, CH2Cl2, T ) 0 °C, 3 h, then N-methyl-
3,5-bis(trifluoromethyl)benzylamine hydrochloride, pyridine, CH2Cl2, room temp, 12 h.; (f) (i) H2 (1 atm), 10% Pd/C, EtOH, 3 h; (ii) 1 M HCl/Et2O.
Finally, the removal of the carbamate protecting group by
hydrogenation reaction in the presence of 10% Pd/C afforded
the title enantiomers 7a and 7b.
oisomers 13aa and 13ab were prepared from 12a by reaction
with triphosgene in CH2Cl2 at 0 °C in the presence of
triethylamine, followed by addition of racemic 1-[3,5-bis(trif-
luoromethyl)phenyl]ethyl]-N-methylamine. The final separation
by flash chromatography enabled us to get pure 13aa and 13ab,
which were then reduced in the presence of BH3 ·THF in THF
to give title compounds 14aa and 14ab. Then single diastere-
oisomers 14ba and 14bb were prepared from 12b following
the same synthetic procedure.
Single diasteroisomers 14aa, 14ab, 14ba, and 14bb were
synthesized as shown in Scheme 3. The Grignard reagent
obtained from commercially available 2-bromo-5-fluorotoluene
2 was added to LiBr and Cu2Br2 in anhydrous THF and then
reacted with methyloxalyl chloride to give intermediate 10. The
following reaction with ethylenediamine afforded the cyclized
compound 11 in good yield, which was smoothly transformed
into the key ketopiperazine 12 by hydrogenation in the presence
of 10% Pd/C. The resolution of this racemate into the pure
enantiomers 12a and 12b was achieved by crystallization of
the corresponding S-(+)-mandelic acid and R-(-)-mandelic acid
salt, respectively, in AcOEt. Then the two mixtures of diaster-
Results and Discussion
To identify an innovative series of NK1 receptor antagonists,
our chemical strategy was initially focused on the exploration
of the N-phenylpiperazines of type B, compounds discovered
by an internal screening (Figure 2). In particular, compound 1

3240 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Di Fabio et al.
Scheme 2a
a (a) CDI, (R)-(+)-1-phenylethyl alcohol, CH2Cl2, 30 min, room temp, then 4, CH3CN, reflux, 24 h; (b) triphosgene, Et3N, CH2Cl2, T ) 0 °C, 2 h, then
N-methyl-bis-3,5-(trifluoromethyl)benzylamine hydrochloride, 4 h, room temp; (c) (i) H2 (1 atm), 10% Pd/C, 95% EtOH, 4 h; (ii) 1 M HCl/Et2O.
Table 1. In Vitro Binding Affinities (pKi)/Functional Potencies (pKB) at
h-NK1 Receptors and in Vivo Activity of NK1 Receptor Antagonists in
the Gerbil Foot Tapping Model (GFT)
2. This structural modification, if tolerated, would have enabled
us to identify compounds with improved druggability, in terms
of limited molecular weight with respect to the known NK1
receptor antagonists series and appropriate basicity of the
nitrogen atom. Following this novel structural hypothesis,
compound 7 was prepared as a racemate and characterized in
terms of in vitro affinity at the NK1 receptor binding site. As
shown in Table 1, a significant improvement was observed with
respect to the corresponding N-phenylpiperazine derivative 1
(pKi of 8.89 and 8.06 for compounds 7 and 1, respectively),
confirming the possibility of getting higher in vitro affinity
depending upon a more accurate positioning of the nitrogen
atoms within the phenylpiperazine template. Notably, as shown
in Table 1, the two enantiomers 7a and 7b, prepared as reported
above, exhibited significant differences in terms of in vitro
affinity (pKi ) 9.37 and pKi ) 7.74, respectively), being that
(S)-enantiomer 7a is the most active. Further to this result, 7a
was then tested in the gerbil foot tapping (GFT) model. This
model represents a pharmacodynamic test to assess central NK1
receptor antagonism. Indeed, brain penetrant compounds when
administered systemically were able to inhibit the tapping
behavior elicited by intracerebroventricular (icv) injection of 3
pmol (5 µL) of the NK1 receptor agonist GR73632 (see
Experimental Section). As shown in Table 1, an excellent in
vivo activity was observed with 7a after oral and subcutaneous
administration (ID50 ) 0.2 mg/kg, both routes of administration).
Notably, a long lasting effect was observed because that
compound was still active 8 h after its oral administration (ID50
compd
pKi (pKB)a
n
ID50, mg/kgb
GR205171
1
7
7a
7b
14aa
14ab
14ba
14bb
10.30 ( 0.08
8.06 ( 0.07 (8.56 ( 0.02)
8.89 ( 0.05
9.37 ( 0.07
7.74 ( 0.15
9.65 ( 0.14 (9.87 ( 0.06)
9.27 ( 0.04
5
5 (3)
8
8
3
5 (3)
3
6
3
0.6
NTc
NTc
0.2
5.6
0.014
0.036
NTc
NTc
8.09 ( 0.12
8.43 ( 0.06
a pKi and pKB values have been determined as described in Experimental
Section and are the mean ( SEM of n experiments performed in duplicate.
b 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 or subcutaneous administration 1 h before
the test. c NT ) not tested.
exhibited promising in vitro potency (pKi ) 8.06; Table 1),
measured by inhibition of the binding of [3H]substance P
([3H]SP) to membranes obtained from Chinese hamster ovary
cells transfected with human NK1 receptors (h-NK1 CHO). The
antagonist profile of compound 1 was then investigated in
fluorescence imaging plate reader (FLIPR) functional studies
where it was shown to antagonize in a competitive manner SP-
induced [Ca2+]i in h-NK1 CHO cells with a pKB value of 8.56
(Table 1, Figure 4A). To maximize the in vitro affinity of this
molecule, we proposed to modify the N-phenylpiperazine core
into the C-phenylpiperazine template of type A, shown in Figure

A Piperazine-1-carboxylic Acid
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3241
Scheme 3a
a (a) (i) Mg, I2, THF, T ) 70 °C, 2 h; (ii) LiBr, Cu2Br2, THF, room temp 1 h; (iii) CH3OCOCOCl, room temp, 2 h; (b) ethylenediamine, toluene, reflux,
6 h; (c) H2 (1 atm), 10% Pd/C, MeOH, 16 h; (d) (i) S-(+)-mandelic acid or R-(-)-mandelic acid, AcOEt, T ) 3-5 °C, 2 h; (ii) filtration of the salt, then
crystallization in AcOEt; (iii) 0.73 M NaOH; (e) (i) triphosgene, Et3N, CH2Cl2, T ) 0 °C, 4 h; (ii) 1-[3,5-bis(trifluoromethyl)phenyl]ethyl]-N-methylamine,
N(i-Pr)2Et, CH3CN, T ) 70 °C, 2 h; (f) (i) 1 M BH3 ·THF, THF, reflux, 3 h; (ii) Et2O, AcOH.
) 0.3 mg/kg), suggesting that 7a was still present in the brain
after several hours from its administration.
potency of 14aa increased between 1 and 4 h (ID50 from 0.014
to 0.002 mg/kg); notably, 8 h after administration of the
compounds, the effect was still present (Table 2).
Compound 14ab was also tested in the GFT model, and in
agreement with its reduced binding potency, it was found to be
slightly less active (ID50 ) 0.036 mg/kg, po) than 14aa (Table
1).
Considering the greater in vitro and in vivo potency observed
for 14aa, an extensive pharmacological characterization of this
compound was conducted. In particular, when this compound
was preincubated for 60 min at 37 °C (3, 5, and 10 nM), it
antagonized SP-induced [Ca2+]i increase in h-NK1 CHO cells.
The type of antagonism observed was nonsurmountable (Figure
4B), since it was characterized by a rightward shift of the SP
concentration-response curve (CRC) with a concentration-
dependent reduction of the agonist maximal response. The
apparent pKB estimated at 3 nM concentration was 9.87 (Table
1). The nonsurmountable antagonism observed in in vitro studies
Further to the identification of compound 7a, the effect of
the substitution of the benzylic position by a methyl group was
explored. To this end the single diastereoisomers 14aa, 14ab,
14ba, and 14bb were synthesized. Their in vitro affinities at
h-NK1 receptors are shown in Table 1. As expected, among
them, 14aa and 14ab were the most potent ones, suggesting
once again the importance of the correct stereochemistry of the
stereogenic center present in the phenylpiperazine core (Figure
3). Conversely, the in vitro affinities of 14aa and 14ab are only
slightly different, making the stereochemistry of the benzylic
position less critical.
Oral administration of 14aa (0.01-0.1 mg/kg), prior to icv
infusion of GR73632, caused a dose dependent and complete
inhibition of foot tapping in gerbils with an estimated ID50 of
0.014 mg/kg (Table 1), suggesting a potent central activity of
the compound. Moreover, a time course study showed that the

3242 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Di Fabio et al.
Moreover, L-733,060 partially antagonized 14aa-induced de-
pression of the SP maximal response, in agreement with the
syntopic action elicited by 14aa. Saturation binding analysis in
membranes from h-NK1 CHO cells with increasing concentra-
tions of [3H]14aa indicated the presence of a single high affinity
binding site, with a dissociation constant of 0.030 nM (pKd )
10.52) (Figure 6). Kinetic binding studies of [3H]14aa in
membranes from h-NK1 CHO cells revealed that the rate
constant for the association of 0.2 nM radioligand with the
h-NK1 receptor is 0.0988 min-1 with a t1/2 of 7.01 min and a
t99/100 of 46.6 min at 37 °C (Figure 7A). [3H]14aa dissociated
from the h-NK1 receptor with simple monophasic kinetics with
a rate of dissociation (k-1) of 0.0116 min-1 and t1/2 for receptor
occupancy of 59.7 min (Figure 7B). Calculation of the KD from
the ratio of the kinetic parameters koff/kon gave a value of 0.027
nM (pKD ) 10.57). Taking together the kinetic binding and the
functional data, it is suggested that the nonsurmountable effect
of 14aa versus SP is due to its long dissociation time from the
NK1 receptor, thus revealing a pseudoirreversible phenomenon
of the compound rather than a noncompetitive action.
Figure 3. X-ray structure of compound 14aa.
The receptogram screen (by MDS Pharma Service, Taiwan)
confirmed that 14aa is highly selective for the h-NK1 receptor,
since at 1 µM concentration it produced less than 50%
displacement of binding in the full battery of 83 receptors,
transporters, ion channels, and enzymes.
Compound 14aa also exhibited excellent pharmacokinetics
in vivo, both in rat and in dog (F ) 44% and 71%, respectively)
with a limited clearance in plasma (Clp ) 19 and 15 (mL/min)/
kg, respectively) and suitable half-life (5 and 11 h, respectively).
As far as the brain penetration in rats is concerned, a B/P ratio
of 1.3 was assessed 5 min after the intravenous administration
of the compound at 1 mg/kg dose.
Conclusion
In conclusion an appropriate optimization strategy of the
N-phenylpiperazine series available in house enabled us to
identify a new class of potent and selective NK1 receptor
antagonist exhibiting remarkable druglike properties. In par-
ticular, compound 14aa represents one of the most potent in
vitro and in vivo NK1 receptor antagonist ever identified. In
particular, when administered orally to gerbil, 14aa was found
to inhibit the animal’s foot tapping with long lasting pharma-
cological activity, exhibiting both an excellent pharmacokinetics
profile and peculiar receptor kinetic behavior, namely, a slow
dissociating rate of the ligand from the NK1 receptor. This
behavior was further supported by the nonsurmountable receptor
interaction observed in vitro. In this respect, simulation studies
demonstrated that a prolonged in vivo receptor occupancy should
take place when the antagonist receptor complex dissociates
more slowly than the antagonist gets eliminated.23 This implies
that the in vivo duration of action of an antagonist should depend
not only on its rate of elimination via excretion and/or
metabolism but also on the rate it dissociates from the receptor.
Hence, slowly dissociating NK1 receptor antagonists may exert
a substantially longer therapeutic action than the corresponding
surmountable antagonists, a characteristic that is fundamental
to drug discovery strategy to select the best possible clinical
candidates.
Figure 4. Antagonism of SP-induced increase of cytosolic calcium in
h-NK1 CHO cells by compound 1 (A) and compound 14aa (B) in h-NK1
CHO cells. Curves depicted are the mean fit of three independent
experiments performed in duplicate.
Table 2. Effect of 14aa on Gerbil Foot Tapping Test at Different Times
(h) of Oral Pretreatmenta
14aa
-1 h
-2 h
-4 h
-8 h
ID50 (mg/kg, po)
lower 95% CI
upper 95% CI
0.014
0.007
0.020
0.007
0.001
0.014
0.002
0.0009
0.009
0.027
0.020
0.035
a Data are presented as ID50 values (compound dose causing 50%
inhibition of the tapping behavior elicited by the icv injection of GR73632,
3 pmol/5 µL).
by 14aa could potentially indicate a noncompetitive behavior
of the compound interacting with nonspecific sites of the h-NK1
receptor or with the coupling systems. To rule out these
possibilities, the following in vitro studies using the combined
dose ratio analysis21 and the radioactive form of 14aa were
carried out. In particular, the competitive or noncompetitive
nature of antagonism elicited by 14aa with respect to the known
competitive antagonist L-733,06022 was investigated. As shown
in Figure 5, the cumulative effect of 10 nM 14aa and 100 nM
L-733,060 rightward-shifted the agonist CRC in an additive
model, rather than a multiplicative model, revealing a syntopic
effect of the two antagonists in inhibiting SP response.
Experimental Section
All the NMR spectra were recorded in DMSO-d6 or CDCl3 at a
constant temperature of 25 °C, and complete assignments were
made by means of several 1D and 2D techniques including 19F,
gCOSY, gHSQC, and gHMQC NMR experiments when needed.

A Piperazine-1-carboxylic Acid
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3243
Figure 5. Antagonism of SP-induced increase of cytosolic calcium in h-NK1 CHO cells. Combined dose ratio (DR) analysis between 14aa and
L-733,060 demonstrated that 14aa acts competitively with respect to L-733,060. The observed DR of the combination study was found to be closer
to the additive rather than to the multiplicative model (syntopic action).
µL). Condition B was as follows: purity of compounds 14aa, 14ab,
14ba, and 14bb was assessed using a Chiralpack AD column (4.6
mm × 250 mm) and n-hexane/ethanol, 86:14 v/v, + water, 0.2%
v/v, as mobile phase (flow 1 mL/min, UV detector at 210 nm,
injection of 4 µL). All reactions were carried out under anhydrous
nitrogen atmosphere using standard Schlenk techniques. Most
chemicals and solvents were analytical grade and used without
further purification.
2-(4-Fluoro-2-methylphenyl)pyrazine (3). To a solution of
2-bromo-5-fluorotoluene (1.36 g, 3.28 mmol) in toluene (20 mL),
EtOH 95% (10 mL) and 1 M solution of Na2CO3, (20 mL) were
added
followed
by
[1,2-[bis(diphenylphosphino)et-
hane]dichloronickel(II) (0.21 g, 0.23 mmol) and chloropyrazine (1
mL, 11.17 mmol). The reaction mixture was refluxed for 2 h and
then poured into AcOEt (100 mL). The organic layer was extracted
with brine (4 × 25 mL), dried over Na2SO4, and evaporated in
vacuo. The crude residue was purified by flash chromatography
(cyclohexane/AcOEt, 8:2) to give the title compound as white solid
(1.4 g, 76%). Mp 66-68 °C. H NMR (400 MHz, DMSO-d6): δ
(ppm) 8.81 (d, 1H), 8.72 (m, 1H), 8.63 (d, 1H), 7.52 (m, 1H), 7.22
(m, 1H), 7.17 (m, 1H), 2.35 (s, 3H). MS: m/z 189.
Figure 6. Representative [3H]14aa saturation curve in membranes from
CHO cells stably transfected with h-NK1 receptor. Inset is the Scatchard
plot.
1
1H and gHSQC experiments were collected on a 400 Unity Varian
or 500 Inova Varian instrument, and 13C and 19F experiments were
collected using a 300 Inova Varian instrument. Chemical shifts are
reported in parts per million (ppm) downfield from the CHCl3
residual line (δ ) 7.27 ppm) or from DMSO residual line (δ )
2.49 ppm) and were assigned as singlets (s), doublets (d), triplets
(t), quartet (q), broad quartet (bq), broad singlets (bs), doublets of
doublets (dd), or multiplets (m). Coupling constants (J) are given
in Hz. IR spectra were recorded on a Nicolet Magna 760 (Thermo
Fisher Scientific) spectrometer. Mass spectra analyses were per-
formed on a VG Platform (Waters, Manchester, U.K.), with the
mass spectrometer operating in positive electrospray ion mode.
Analytical thin layer chromatography (TLC) was performed on glass
plates (Merck Kieselgel 60 F254). Visualization was accomplished
by UV light (254 nm), I2. Column chromatography was performed
on silica gel (Merck Kieselgel 70-230 mesh). Melting points (mp)
are uncorrected. Optical rotation [R]D values were obtained in
solution at the sodium D line at 20 °C. Chemical purity of
compounds has been assessed by HPLC (>95%). For chiral HPLC
analysis, condition A was as follows: purity of intermediates 12a
and 12b was assessed using a Chiralcel OJ (4.6 mm × 250 mm)
column from Daicel using n-hexane/ethanol, 80:20 v/v, as mobile
phase (flow of 1 mL/min, UV detector at 265 nm, injection of 5
2-(4-Fluoro-2-methylphenyl)piperazine (4). To a solution of
compound 3 (0.3 g, 1.59 mmol) in EtOH, 95% (20 mL), were added
HCl 37% (0.2 mL) and Pd(OH)2/C 20% (30 mg). The reaction
mixture was hydrogenated at 5 atm at room temperature for 4 h,
then filtered and concentrated in vacuo. The solid residue was
washed with a mixture of AcOEt/AcOH, 2:1 (30 mL), then with
small portions of cold AcOEt, dried overnight at 50 °C in vacuo to
give 4 (320 mg, 70%). Mp >220 °C. 1H NMR (400 MHz, DMSO-
d6): δ (ppm) 9.72 (bs, 2H), 7.90 (d, 1H), 7.21-7.17 (m, 2H), 4.85
(m, 1H), 3.57-3.20 (m, 6H), 2.40 (s, 3H). MS: m/z 195.
Phenylmethyl 3-(4-Fluoro-2-methylphenyl)-1-piperazinecarb-
oxylate (5). Compound 4 (0.25 g, 0.94 mmol) was dissolved under
a nitrogen atmosphere in dry CH2Cl2 (25 mL). Triethylamine (0.5
mL, 3.77 mmol) was added at 0 °C. Then benzylchloroformate (0.15
mL, 1.05 mmol) was dissolved in dry CH2Cl2 (10 mL) and slowly
added to the reaction mixture, then stirred at 0 °C for additional
2 h. Brine was added (10 mL), and the organic layer was separated
and washed again with brine (2 × 10 mL) and dried over Na2SO4.
The solvent was evaporated in vacuo and the crude residue was
purified by flash chromatography (cyclohexane/AcOEt, 1:1) to give
1
5 (0.208 g, 68%) as a colorless oil. H NMR (400 MHz, CDCl3):
δ (ppm) 7.52 (m, 1H), 7.40-7.30 (m, 5H), 6.90-6.80 (m, 2H),

3244 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Di Fabio et al.
Figure 7. The 0.2nM [3H]14aa association (A) and dissociation (B) on membranes from h-NK1 CHO cells. Curves depicted represent the mean
fit of three independent experiments performed in duplicate.
5.16 (dd, 2H), 4.12 (m, 2H), 3.86 (m, 1H), 3.30-2.70 (m, 4H),
2.33 (bs, 3H). MS: m/z 329.
0 °C. Then a solution of triphosgene (75 mg, 0.25 mmol) dissolved
in CH2Cl2 (2 mL) was dropped into the reaction mixture. After
2 h, a solution of N-methyl-3,5-bis(trifluoromethyl)benzylamine
hydrochloride (201 mg, 0.69 mmol) in CH2Cl2 (3 mL) was added
dropwise and the reaction mixture stirred 4 h at room temperature.
CH2Cl2 (20 mL) and HCl, 1 N (10 mL), were added. The organic
layer was separated, washed with brine (3 × 10 mL), dried over
Na2SO4, and concentrated in vacuo. The crude residue was purified
by flash chromatography (cyclohexane/AcOEt, 7:3) to give pure
separated compounds 9a and 9b (125 and 135 mg, respectively)
and some mixed fractions (45 mg) in 89% total yield.
Phenylmethyl-4-{[{[3,5-bis(trifluoromethyl)phenyl]methyl}
(methyl)amino]carbonyl}-3-(4-fluoro-2-methylphenyl)-1-
piperazinecarboxylate (6). Compound 5 (0.26 g, 0.6 mmol) was
dissolved in dry CH2Cl2 (35 mL) under a nitrogen atmosphere.
Triethylamine (0.32 mL, 2.3 mmol) was added at 0 °C, followed
by triphosgene (65 mg, 0.21 mmol) dissolved in CH2Cl2 (25 mL).
The reaction mixture was stirred at room temperature for 3 h. Then
pyridine (0.3 mL, 0.29 mmol) and N-methyl-3,5-bis(trifluorometh-
yl)benzylamine hydrochloride (0.25 g, 0.61 mmol) were added in
sequence and the solution was stirred overnight at room temperature.
HCl, 1 N (10 mL), was added and the organic layer was separated,
washed with brine (3 × 10 mL), dried over Na2SO4, and
concentrated in vacuo. The crude residue was purified by flash
chromatography (cyclohexane/AcOEt, 1:1) to give 6 (85 mg, 36%)
as a pale-yellow foam. 1H NMR (400 MHz, CDCl3): δ (ppm) 7.76
(s, 1H), 7.49 (s, 2H), 7.40-7.30 (m, 5H), 7.20 (dd, 1H), 6.86 (d,
1H), 6.79 (m, 1H) 5.17 (s, 2H), 4.66 (d, 1H), 4.64 (m, 1H), 4.36
(d, 1H), 3.97 (m, 2H), 3.4 (m, 2H), 3.16 (m, 2H), 2.93 (s, 3H),
2.38 (bs, 3H). MS: m/z 612.
9a. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 7.90 (s, 1H), 7.67
(s, 2H), 7.40-7.27 (m, 6H), 6.95 (dd, 1H), 6.80 (m, 1H), 5.74 (q,
1H), 4.60-4.40 (dd, 2H), 4.50 (m, 1H), 3.79 (m, 3H), 3.00 (m,
3H), 2.87 (s, 3H), 2.29 (s, 3H), 1.46 (d, 3H). MS: m/z 626.
9b. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 7.90 (s, 1H), 7.67
(s, 2H), 7.37-7.24 (m, 6H), 6.95 (dd, 1H), 6.81 (m, 1H), 5.75 (q,
1H), 4.60-4.41 (dd, 2H), 4.52 (m, 1H), 3.83-3.00 (m, 6H), 2.88
(s, 3H), 2.33 (s, 3H), 1.48 (d, 3H). MS: m/z 626.
(2S)-N-{[3,5-Bis(trifluoromethyl)phenyl]methyl}-2-(4-fluoro-2-
methylphenyl)-N-methyl-1-piperazinecarboxamide Hydrochlo-
ride (7a). Compound 9a (120 mg, 0.2 mmol) was dissolved in
EtOH 95% (10 mL) and hydrogenated (Pd/C 5%) at 1 atm for 4 h.
The catalyst was filtered and a solution of HCl, 1 M in Et2O (0.5
mL), was added. The white precipitate was filtered, washed with
Et2O, and dried in vacuo overnight to give the title compound 7a
N-{[3,5-Bis(trifluoromethyl)phenyl]methyl}-2-(4-fluoro-2-
methylphenyl)-N-methyl-1-piperazinecarboxamide Hydrochlo-
ride (7). Compound 6 (50 mg, 0.08 mmol) was dissolved in EtOH,
95% (10 mL), and hydrogenated (Pd/C, 5%) at 1 atm for 3 h. The
catalyst was filtered, and a solution of HCl, 1 M in Et2O (0.5 mL),
was added. The white precipitate was filtered, washed with Et2O,
and dried in vacuo overnight to give the title compound 7 (20 mg).
1
(57 mg, 55%). Mp >220 °C. H NMR (400 MHz, DMSO-d6): δ
(ppm) 9.11 (bs, 1H), 8.83 (bs, 1H), 7.96 (s, 1H), 7.59 (s, 2H), 7.34
(dd, 1H), 6.94 (d, 1H), 6.86 (m, 2H), 4.66 (d, 1H), 4.53 (dd, 1H),
4.36 (m, 1H), 3.54 (m, 1H), 3.44-3.10 (m, 4H), 2.93 (s, 3H), 2.90
(m, 1H), 2.38 (s, 3H). MS: m/z 478.4. Chiral HPLC (condition A):
retention time 14.5 min (98.4% a/a). [R]2D0 +69.5 (c 0.27, CHCl3).
(2R)-N-{[3,5-Bis(trifluoromethyl)phenyl]methyl}-2-
(4-fluoro-2-methylphenyl)-N-methyl-1-piperazinecarboxamide (7b)
Hydrochloride. Compound 9b (130 mg, 0.21 mmol) was dissolved
in EtOH, 95% (10 mL), and hydrogenated (Pd/C, 5%) at 1 atm for
4 h. The catalyst was filtered, and a solution of HCl, 1 M in Et2O
(0.5 mL), was added. The white precipitate was filtered, washed
with Et2O, and dried in vacuo overnight to give the title compound
1
Mp >220 °C. H NMR (400 MHz, DMSO-d6): δ (ppm) 9.33 (bs,
1H), 9.18 (bs, 1H), 7.96 (s, 1H), 7.59 (s, 2H), 7.33 (dd, 1H), 6.99
(d, 1H), 6.85 (t, 2H), 4.66 (d, 1H), 4.53 (dd, 1H), 4.37 (d, 1H),
3.52 (d, 1H), 3.40-3.20 (m, 4H), 2.93 (s, 3H), 2.90 (m, 1H), 2.38
(s, 3H). MS: m/z 478. IR (Nujol) 3200, 1659 cm-1. MS: m/z 478.
(1R)-1-Phenylethyl 3-(4-fluoro-2-methylphenyl)-1-piperazine-
carboxylate (8a,b). To a solution of CDI (162 mg, 1 mmol) in
CH2Cl2 (5 mL) was added (R)-(+)-1-phenethyl alcohol (123 mg,
1 mmol) at room temperature, and reaction mixture was stirred for
30 min. Then compound 4 (180 mg, 0.9 mmol) dissolved in CH3CN
(15 mL) was added and the reaction mixture was refluxed for 24 h.
AcOEt (30 mL) and HCl, 1 N (10 mL), were added. The organic
layer was separated, washed with brine (3 × 10 mL), dried over
Na2SO4, and concentrated in vacuo. The crude residue was purified
by flash chromatography (cyclohexane/AcOEt, 4:1) to give a
mixture of compounds 8a and 8b (180 mg, 59%) as a colorless
1
7b (63 mg, 61%). Mp >220 °C. H NMR (400 MHz, DMSO-d6):
δ (ppm) 9.11 (bs, 1H), 8.83 (bs, 1H), 7.96 (s, 1H), 7.59 (s, 2H),
7.34 (dd, 1H), 6.94 (d, 1H), 6.86 (m, 2H), 4.66 (d, 1H), 4.65-4.35
(m, 2H), 3.54 (m, 1H), 3.44-3.10 (m, 4H), 2.93 (s, 3H), 2.90 (m,
1H), 2.38 (s, 3H). MS: m/z 478. Chiral HPLC (condition A)
retention time 15.2 min (98.4% a/a). [R]2D0 -72.6 (c 0.27, CHCl3).
(4-Fluoro-2-methylphenyl)oxoacetic Acid Methyl Ester
(10). To a suspension of magnesium turnings (6.17 g, 253.8
mmol) in dry THF (60 mL) at 20 °C under a nitrogen
atmosphere, a small amount of iodine was added, followed by
a solution of commercial 2-bromo-5-fluorotoluene 2 (40 g, 211.6
mmol) in dry THF (150 mL). The suspension was gently heated
until the brown color disappeared. The remaining bromide
solution was added dropwise, maintaining the reaction mixture
at 50-60 °C. Then the suspension was stirred at 70 °C for 2 h
until the magnesium turnings disappeared. A solution of LiBr
1
foam. H NMR (400 MHz, DMSO-d6): δ (ppm) 7.87 (m, 1H),
7.40-7.25 (m, 5H), 7.02-6.94 (m, 2H), 5.74 (m, 1H), 3.93-3.71
(m, 3H), 3.00-2.55 (m, 4H), 2.34 (s, 3H), 2.28 (s, 3H). MS: m/z
343.
(1R)-1-Phenylethyl-(3S)-4-{[{[3,5-bis(trifluoromethyl)phenyl]-
methyl}(methyl)amino]carbonyl}-3-(4-fluoro-2-methylphenyl)-1-
piperazinecarboxylate (9a) and (1R)-1-Phenylethyl-(3R)-4-{[{[3,5-bis-
(trifluoromethyl)phenyl]methyl}(methyl)amino]carbonyl}-
3-(4-fluoro-2-methylphenyl)-1-piperazinecarboxylate (9b). To
a solution of compounds 8a,b (180 mg, 0.55 mmol) dissolved in
CH2Cl2 (5 mL), triethylamine (0.35 mL, 2.5 mmol) was added at

A Piperazine-1-carboxylic Acid
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3245
(44.1 g, 507.7 mmol) in 500 mL of anhydrous THF was added
dropwise to a suspension of Cu2Br2 (36.4 g, 253.8 mmol) in
500 mL of anhydrous THF. The reaction mixture was stirred at
20 °C for 1 h (dark-green solution with a small amount of white
solid in suspension). The Grignard reagent solution previously
prepared was then added dropwise, maintaining the temperature
below 25 °C, followed by methyloxalyl chloride (19.5 mL, 212.0
mmol). The reaction mixture was stirred at 20 °C for 2 h. Then
the solvent was evaporated in vacuo and the residue was taken
up in AcOEt (1500 mL). The organic layer was washed with
saturated aqueous NH4Cl (2 × 1000 mL) and dried over
anhydrous Na2SO4, filtered, evaporated in vacuo; the crude oil
was purified by flash chromatography (cyclohexane/AcOEt, 95:
5) to give the title compound as an oil (24.4 g, 58.5%). 1H NMR
(400 MHz, CDCl3): δ (ppm) 7.74 (m, 1H), 6.98-7.04 (m, 2H),
3.96 (s, 3H), 2.61 (s, 3H). MS: m/z 197.
3-(4-Fluoro-2-methylphenyl)-5,6-dihydro-1H-pyrazin-2-one
(11). To a solution of intermediate 10 (20.1 g, 101.8 mmol) and
ethylenediamine (6.8 mL, 101.8 mmol) in 400 mL of toluene,
anhydrous Na2SO4 (20 g) was added at 20 °C under nitrogen
atmosphere. The reaction mixture was heated at reflux for 6 h. Then
the reaction mixture was cooled and filtered. The solvent was
evaporated in vacuo and the crude oil was purified by flash
chromatography (AcOEt), affording the title compound as a white
solid (12.9 g, 61.4%). 1H NMR (400 MHz, CDCl3): δ (ppm) 7.33
(m, 1H), 6.95-6.90 (m, 2H), 6.56 (m, 1H), 3.97 (m, 2H), 3.58 (m,
2H), 2.31 (s, 3H). MS: m/z 207.
solid. This material was suspended in AcOEt (45 mL), and an
amount of 22 mL of a 0.73 M solution of NaOH, saturated with
NaCl, was added. The organic layer was separated and washed
with H2O (15 mL). The aqueous layer was extracted with AcOEt
(4 × 15 mL). The combined organic phases were dried over
Na2SO4 and concentrated in vacuo to give the title compound
1
(2.17 g, 41.3%) as a white foam. H NMR (400 MHz, DMSO-
d6): δ (ppm) 7.79 (bm, 1H), 7.23 (dd, 1H), 6.97 (dd, 1H), 6.91
(td, 1H), 4.45 (s, 1H), 3.31 (m, 1H), 3.14 (m, 1H), 2.93 (m,
1H), 2.84 (m, 2H), 2.32 (s, 3H). Chiral HPLC (condition A):
retention time 7.2 min (98.1% a/a). [R]2D0 -18 (c 1.17, CHCl3).
MS: m/z 209.
2-(S)-(4-Fluoro-2-methylphenyl)-3-oxopiperazine-1-carboxylic
Acid [1-(R)-(3,5-Bis-trifluoromethylphenyl)ethyl]methylamide
(13aa) and 2(S)-(4-Fluoro-2-methylphenyl)-3-oxopiperazine-1-carb-
oxylic Acid [1-(S)-(3,5-Bis-trifluoromethylphenyl)ethyl]methyl-
amide (13ab). To a solution of intermediate 12a (1.21 g, 5.8 mmol)
in 27 mL of dry CH2Cl2, Et3N (1.64 mL, 11.8 mmol) was added.
The solution was cooled to 0 °C, and a solution of triphosgene
(0.73 g, 2.46 mmol) in dry CH2Cl2 (6 mL) was dropped into the
reaction mixture in 40 min. The solution was stirred 4 h at 0 °C
and then brought back to room temperature. Diisopropylethylamine
(2 mL, 11.5 mmol) was added, followed by a solution of 1-[3,5-
bis(trifluoromethyl)phenyl]ethyl]-N-methylamine (2.36 g, 8.7 mmol)
dissolved in 60 mL of CH3CN in acetonitrile. After evaporation of
the CH2Cl2, the internal temperature was brought to 70 °C and the
reaction flask was equipped with a water condenser, and the mixture
was stirred for 2 h. Then the solvent was evaporated in vacuo and
the residue was partitioned between CH2Cl2 and HCl, 1 M, and
the separated organic layer was dried over anhydrous Na2SO4,
filtered, and evaporated in vacuo to give a crude residue which
was purified by flash chromatography (AcOEt/cyclohexane, 8:2)
to afford the title compounds 13aa (0.88 g, 1.74mmol, 30.0%) and
13ab (0.89 g, 30.3%) as white foams.
3-(4-Fluoro-2-methylphenyl)piperazin-2-one (12). To a solu-
tion of intermediate 11 (11.76 g, 57.0 mmol) in CH3OH (170 mL),
Pd/C, 10% (3 g), was added, and the reaction mixture was
hydrogenated at 1 atm at room temperature for 16 h. Then the
catalyst was filtered and the solvent was evaporated to low volume
(30 mL). CH3OH (140 mL) and AcOEt (700 mL) were added, and
the solution was rapidly filtered by a silica pad (60 g). The eluted
solution was evaporated in vacuo to obtain the title compound
1
13aa. H NMR (500 MHz, DMSO-d6): δ 8.16 (s. 1H), 7.98 (s,
1
2H), 7.71 (bs, 2H), 7.19 (dd, 1H), 6.97 (dd, 1H), 6.87 (td, 1H),
5.34 (s, 1H), 5.14 (q, 1H), 3.45-3.2 (m, 4H), 2.53 (s, 3H), 2.27 (s,
3H), 1.56 (d, 3H). MS: m/z 506.
(11.76 g, 99.0%). H NMR (400 MHz, DMSO-d6): δ (ppm) 7.77
(bm, 1H), 7.24 (dd, 1H), 6.96 (dd, 1H), 6.92 (td, 1H), 4.43 (s, 1H),
3.30 (m, 1H), 3.14 (m, 1H), 2.92 (m, 1H), 2.82 (m, 2H), 2.33 (s,
3H). MS: m/z 209.
1
13ab. H NMR (500 MHz, DMSO-d6): δ 8.16 (s, 1H), 7.95 (s,
2H), 7.70 (bs, 2H), 7.19 (dd, 1H), 6.98 (dd, 1H), 6.90 (td, 1H),
5.29 (q, 1H), 5.28 (s, 1H), 3.45-3.15 (m, 4H), 2.66 (s, 3H), 2.27
(s, 3H), 1.52 (d, 3H). MS: m/z 506.
(+)-(S)-3-(4-Fluoro-2-methylphenyl)piperazin-2-one (12a). To
a suspension of racemic intermediate 12 (5.25 g, 25.2 mmol) in
135 mL of AcOEt, S-(+)-mandelic acid (4.095 g, 26.9 mmol)
was added. The suspension was stirred at room temperature for
1 h and then at 3-5 °C for an additional 2 h, filtered, and
evaporated in vacuo to give crude L-(+)mandelate 3-(4-fluoro-
2-methylphenyl)piperaz- in-2-one 12a, which was suspended in
AcOEt (55 mL). The solution was refluxed until complete
dissolution occurred, then cooled to room temperature and stirred
for 2 h, filtered, and dried in vacuo to obtain a white solid. This
material was suspended in AcOEt (45 mL), and an amount of
22 mL of a 0.73 M solution of NaOH, saturated with NaCl, was
added. The organic layer was separated and washed with H2O
(15 mL). Then the aqueous layer was extracted with AcOEt (4
× 15 mL). The combined organic layers were dried over
anhydrous Na2SO4 and concentrated in vacuo to give the title
compound (2.17 g, 41.3%) as a white foam. 1H NMR (400 MHz,
DMSO-d6): δ (ppm) 7.77 (bm, 1H), 7.24 (dd, 1H), 6.96 (dd,
1H), 6.92 (td, 1H), 4.43 (s, 1H), 3.30 (m, 1H), 3.14 (m, 1H),
2.92 (m, 1H), 2.82 (m, 2H), 2.33 (s, 3H). Chiral HPLC (condition
A) retention time 7.9 min (98.2% a/a). [R]2D0 +17.9 (c 1.17,
CHCl3). MS: m/z 209.
(-)-(R)-3-(4-Fluoro-2-methylphenyl)piperazin-2-one (12b). To
a suspension of racemic intermediate 12 (5.25 g, 25.2 mmol) in
AcOEt (135 mL), R-(-)-mandelic acid (4.095 g, 26.9 mmol)
was added. The suspension was stirred at room temperature for
1 h and then at 3-5 °C for 2 h, filtered, and dried in vacuo to
obtain crude R-(-)mandelate 3-(4-fluoro-2-methylphenyl)pip-
erazin-2-one (5.55 g), which was suspended in AcOEt (55.5 mL)
and heated to reflux until complete dissolution. Then the solution
was cooled to room temperature and stirred for 2 h, filtered,
washed with AcOEt (22 mL), and dried in vacuo to give a white
2-(R)-(4-Fluoro-2-methylphenyl)-3-oxopiperazine-1-
carboxylic Acid [1-(R)-(3,5-Bis-trifluoromethylphenyl)ethyl]-
methylamide (13ba) and 2(R)-(4-Fluoro-2-methylphenyl)-3-
oxopiperazine-1-carboxylic Acid [1-(S)-(3,5-Bis-trifluoromethyl-
phenyl)ethyl]methylamide (13bb). To a solution of intermediate
12b (1.2 g, 5.76 mmol) in dry CH2Cl2 (27 mL), Et3N (1.64 mL,
11.8 mmol) was added. The solution was cooled to 0 °C, and a
solution of triphosgene (0.72 g, 2.43 mmol) in dry CH2Cl2 (6 mL)
was added dropwise over 40 min. The reaction mixture was stirred
at 0 °C for 4 h, then was brought back to room temperature.
Diisopropyltriethylamine (2 mL, 11.5 mmol) was then added,
followed by a solution of 1-[3,5-bis(trifluoromethyl)phenyl]ethyl]-
N-methylamine (2.35 g, 8.66 mmol) in CH3CN (60 mL). After
evaporation of the CH2Cl2, the mixture was heated at 70 °C for an
additional 2 h. The solvent was evaporated in vacuo, and the residue
was partitioned between CH2Cl2 and HCl, 1M. The aqueous layer
was extracted with CH2Cl2, and the combined organic extracts were
dried over anhydrous Na2SO4, filtered, and evaporated in vacuo to
give a crude residue which was purified by flash chromatography
(AcOEt/cyclohexanes, 8:2) to obtain the title compounds 13ba (0.88
g, 1.74 mmol, 30%) and 13bb (0.9 g, 30.7%) as white foams.
1
13ba. H NMR (500 MHz, DMSO-d6): δ 8.15 (s, 1H), 7.97 (s,
2H), 7.71 (bs, 2H), 7.18 (dd, 1H), 6.97 (dd, 1H), 6.85 (td, 1H),
5.33 (s, 1H), 5.14 (q, 1H), 3.45-3.2 (m, 4H), 2.52 (s, 3H), 2.26 (s,
3H), 1.55 (d, 3H). MS: m/z 506.
1
13bb. H NMR (500 MHz, DMSO-d6): δ 8.15 (s, 1H), 7.95 (s,
2H), 7.70 (bs, 2H), 7.19 (dd, 1H), 6.99 (dd, 1H), 6.89 (td, 1H),
5.29 (q, 1H), 5.28 (s, 1H), 3.45-3.15 (m, 4H), 2.66 (s, 3H), 2.27
(s, 3H), 1.52 (d, 3H). MS: m/z 506.

3246 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Di Fabio et al.
2-(S)-(4-Fluoro-2-methylphenyl)piperazine-1-carboxylic Acid
[1-(R)-(3,5-Bis-trifluoromethylphenyl)ethyl]methylamide
Acetate Salt (14aa), 2-(S)-4-Fluoro-2-methylphenyl)piperazine-
1-carboxylic Acid [1-(S)-(3,5-Bis-trifluoromethylphenyl)ethyl]-
methylamide Acetate Salt (14ab), 2-(R)-(4-Fluoro-2-methyl-
phenyl)piperazine-1-carboxylic Acid [1-(S)-(3,5-Bis-trifluoromethyl-
phenyl)ethyl]methyl-amide Acetate Salt (14bb), and 2-(R)-4-
Fluoro-2-methylphenyl)piperazine-1-carboxylic Acid [1-(R)-(3,5-Bis-tri-
fluoromethylphenyl)ethyl]methylamide Acetate Salt (14ba).
To a solution of pure intermediate 13aa, 13ab, 13ba, or 13bb (0.76
g 1.5 mmol) in dry THF (10 mL) was added under nitrogen
atmosphere 1 M BH3 ·THF solution in THF (8 mL, 8 mmol), and
the reaction mixture was refluxed for 3 h. Then an amount of 2.5
mL of HCl, 12 N, was added at 0 °C and the solution was stirred
at room temperature for 45 min. Then 6.5 mL of H2O was added
along with 3.2 g of NaHCO3 until neutral pH was attained. The
solvent was evaporated, and the aqueous phase was extracted with
Et2O (3 × 10 mL). The combined organic layers were washed with
saturated NaHCO3 (10 mL), brine, dried over anhydrous Na2SO4,
and evaporated in vacuo to give 14aa, 14ab, 14ba, or 14bb,
respectively (0.660, 0.42, 0.18, and 0.19 g), as colorless oils
Theses materials were dissolved in Et2O (8 mL), and acetic acid
(0.07 mL) was added dropwise. The mixture was stirred at 20 °C
for 2 h and then at 0 °C for 2 h. The suspensions were filtered and
dried in vacuo to give the title compounds (0.26 g and 31.4%, 0.17 g
and 31.1%, 0.128 g and 34.5%, 0.069 g and 21.8%, respectively)
as a white solids.
mM HEPES, pH 7.4, 3 mM MnCl2, and 0.02% BSA. In [3H]14aa
binding experiments, 1% DMSO was included in the assay.
Incubations proceeded at 22 °C ([3H]SP) or 37 °C ([3H]14aa) for
60 min. 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. Association of [3H]14aa to h-NK1
receptors was studied by incubating the membranes with 0.2 nM
radioligand for increasing times (from 2 to 120 min). In dissociation
experiments, membranes were incubated with 0.2 nM [3H]14aa for
60 min; separation of [3H]14aa from the receptor was then obtained
by blocking the reassociation of the radioligand at different times
(from 2 to 180 min) through the addition of 100 nM cold 14aa.
In Vitro NK1 Receptor Functional Studies. In functional
experiments, the ability of 14aa to inhibit SP-induced release of
[Ca2+]i in h-NK1 CHO cells was evaluated. After 30 min of labeling
with the cytoplasmic calcium indicator Fluo-4 AM (2 µM), h-NK1
CHO cells were washed and incubated in the absence or presence
of antagonists for 60 min at 37 °C in Hank’s balanced salts with
20mMHEPESand2.5mMprobenecid,andthenconcentration-response
curves of SP (nM) were performed. Fluorescence was monitored
using a FLIPR (λEX ) 488 nm, λEM ) 510-570 nm).
Inhibition of GR73632-Induced Foot Tapping in Gerbils. A
well validated procedure to explore the central inhibitory activity
of selective NK1 receptor antagonists was applied to characterize
the in vivo activity of the tested compounds.25 A characteristic
intense foot tapping behavior was invoked in male mongolian
gerbils (45-70 g, Charles River Italy) by intracerebroventricular
(icv) infusion of a highly selective tachykinin NK1 receptor agonist,
GR73632, in gerbils evoking a vigorous species-specific behavior.
This rhythmic hind limb thumping is not induced by agonist for
the other tachykinin receptors. The skull was exposed, and an
amount of 5 µL [3 pmol/5 µL of GR73632] was injected by the
insertion of a cuffed 25 gauge needle to a depth of 4 mm below
bregma, directly into the lateral ventricle. The scalp incision was
closed. The animal was allowed to recover from anesthesia and
was individually placed in a clear Perspex observation box (25 cm
× 20 cm × 20 cm). Following a minute of recovery, the duration
of repetitive hind foot tapping was recorded for 3 min, giving a
maximum possible duration of 180 s.
1
14aa. H NMR (500 MHz, DMSO-d6): δ (ppm) 7.99 (s, 1H),
7.71 (s, 2H), 7.28 (m, 1H), 6.92 (m, 1H), 6.77 (m, 1H), 5.28 (q, J
) 6.5 Hz, 1H), 4.22 (dd, J ) 11.7, 3.4 Hz, 1H), 3.3-2.7 (m, 6H),
2.69 (s, 3H), 2.32 (s, 3H), 1.90 (s, 3H), 1.48 (d, J ) 6.9 Hz, 3H);
19F-NMR (300 MHz, DMSO-d6): δ (ppm) -61.6 (m), -116.3 (s).
MS: m/z 492 [M - CH3COO]+, 341 [C14H15F6N2O]+, 221
[C12H14FN2O]+. Chiral HPLC (condition B): retention time 4.56
min (98.5% a/a). [R]2D0 -120.4 (c 1, CHCl3).
1
14ab. H NMR (400 MHz, DMSO-d6): δ (ppm) 7.93 (s, 1H),
7.59 (s, 1H), 7.29 (m, 1H), 6.90 (m, 1H), 6.77 (m, 1H), 5.33 (q,
1H), 4.19 (m, 1H), 3.2-2.6 (m, 6H), 2.80 (s, 3H), 2.32 (s, 3H),
1.89 (s, 3H), 1.49 (d, 3H). MS (m/z): 492 [M - CH3COO]+. Chiral
HPLC (condition B): retention time 14.28 min (97% a/a). [R]D20
-164.9 (c 1, CHCl3)
The ability of different compounds to inhibit GR73632-induced
foot tapping following oral administration is included here for the
purpose of comparison. At 1, 4, 8, or 24 h before the GR73632
administration, at least seven animals each received treatment of
vehicle or test compound (10 mL/kg).
1
14ba. H NMR (400 MHz, DMSO-d6): δ (ppm) 7.92 (bs, 1H),
7.58 (bs, 1H), 7.28 (m, 1H), 6.89 (m, 1H), 6.76 (m, 1H), 5.32 (q,
1H), 4.18 (dd, 1H), 3.23 (m, 1H), 2.90-2.65 (m, 5H), 2.79 (s, 3H),
2.31 (s, 3H), 1.88 (s, 3H), 1.47 (d, 3H). MS: m/z 492 [M -
CH3COO]+. Chiral HPLC (condition B): retention time 5.20 min
(97.8% a/a). [R]2D0 +2.2 (c 1.17, CHCl3).
Data Analysiss. Receptor Binding and Functional Studies.
Radioligand binding data were analyzed by nonlinear regression
analysis using GraphPad Prism 4.0 (GraphPad Software, CA). Deter-
mination of KD and Bmax of radioligands was assessed by elaborating
saturation experiments using one site binding (hyperbola) equation.
Curve fitting from competition binding experiments was determined
by using one site competition equation after checking, with the F test
(P < 0.05), that the Hill slope in the four-parameter logistic equation
was not statistically different from 1.0. In this condition, IC50 values
were converted to Ki using the Cheng-Prusoff equation.26 Results
are expressed as the mean pKi ( SEM. [3H]14aa kinetic association
and dissociation expriments were elaborated with GraphPad Prism by
using one phase exponential association and one phase exponential
decay, respectively. The ratio of kinetic parameters Koff/Kon was used
to estimate the pKD value of [3H]14aa.
In functional studies, SP concentration-response curves in the
presence or absence of fixed concentrations of test drugs where
fitted using a four-parameter logistic equation in GraphPad Prism.
The potency (pKB) of compound 1 was determined by using Schild
analysis.27 The apparent pKB value of 14aa was estimated by using
the Gaddum equation: pKB ) log(DR - 1) - log[A], where DR is
the ratio of equiactive agonist concentrations in the presence and
absence of a fixed antagonist concentration [A].
1
14bb. H NMR (500 MHz, DMSO-d6): δ (ppm) 7.98 (s, 1H),
7.71 (s, 2H), 7.26 (m, 1H), 6.90 (m, 1H), 6.76 (m, 1H), 5.27 (q,
1H), 4.21 (dd, 1H), 3.2-2.6 (m, 6H), 2.68 (s. 3H), 2.3 (s, 3H),
1.88 (s, 3H), 1.47 (d, 3H). MS: m/z 492 [M - CH3COO]+. Chiral
HPLC (condition B): retention time 4.15 min (98.1% a/a). [R]D20
+207 (c 1, CHCl3).
In Vitro and in Vivo Biological Characterization.
Materials. [3H]SP (1.26 TBq/mmol) was purchased from Amer-
sham Life Science. 14aa acetate salt, labeled with [3H] by
Amersham Life Science, has been provided as a 3.11 TBq/mmol,
ethanol solution. GR205171 was synthesized in house. L-733,060,
hydrochloride, was purchased from RBI. SP and GR73632 (δ-
aminovaleryl6, Pro9, N-Me-Leu10)substance P (6-11) were obtained
from Bachem. 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 [3H]14aa binding experiments 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.
In Vitro NK1 Receptor Binding Experiments. Membranes from
Chinese hamster ovary cells stably transfected with human NK1
receptor (h-NK1 CHO) were prepared essentially as described by
Beattie et al. (1995).23 [3H]SP and [3H]14aa binding assays were
carried out in 96-well plates in a final volume of 400 µL of 50
Combined dose ratio analysis to investigate the competitive or
noncompetitive nature of 14aa antagonism was performed according
to the procedure developed by Shankley et al.21 In brief, the theory

A Piperazine-1-carboxylic Acid
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3247
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predicts that when two antagonists (A and C) act through a syntopic
interaction at the receptor (additive model), then the measured dose
ratio in the presence of both the antagonists should be DRA+C
)
DRA + DRC - 1, where DRA and DRC are the dose ratios obtained
independently in the presence of the antagonists A and C. On the
contrary, when the two antagonists act independently through an
allotropic action (multiplicative model), the measured dose ratio
should be DRAC ) DRADRC.
Data from gerbil foot tapping studies were subjected to one-way
analysis of variance (ANOVA) followed by Dunnet’s t test, using GB-
stat software (version 6.5, Dynamic Microsystems Inc.). The doses of
compounds inhibiting foot tapping by 50% (ID50) were determined
by a linear regression analysis using RS-1 software ((95% CI).
Data of number of postures or jumps reduction were subjected to a
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Supporting Information Available: HPLC chromatograms of
compounds 7a, 7b, 14aa, 14ab, 14ba, 14bb. This material is
available free of charge via the Internet at http://pubs.acs.org.
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