Non-peptide NK1 receptor ligands based on the 4-phenylpyridine moiety

Article abstract of DOI:10.1016/j.bmc.2011.02.031

The quinoline nucleus of the previously described 4-phenylquinoline-3- carboxamides NK₁ receptor ligands 7 has been transformed into either substituted or azole - (i.e., triazole or tetrazole) fused pyridine moieties of compounds 9 and 10, resp

Full text of DOI:10.1016/j.bmc.2011.02.031

Bioorganic & Medicinal Chemistry 19 (2011) 2242–2251
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Non-peptide NK1 receptor ligands based on the 4-phenylpyridine moiety
Germano Giuliani a, Andrea Cappelli a, , Mario Matarrese b, Valeria Masiello b, Elia Anna Turolla b,
⇑
Cristina Monterisi b, Ferruccio Fazio b, Maurizio Anzini a, Gal.la Pericot Mohr a, , Daniela Riitano c,
Federica Finetti d, Lucia Morbidelli d, Marina Ziche d, Gianluca Giorgi e, Salvatore Vomero a
a Dipartimento Farmaco Chimico Tecnologico and European Research Centre for Drug Discovery and Development, Università degli Studi di Siena, Via A. Moro, 53100 Siena, Italy
b Istituto di Bioimmagini e Fisiologia Molecolare, CNR, Università di Milano/Bicocca, Istituto H. S. Raffaele, Via Olgettina 60, 20132 Milano, Italy
c Dipartimento di Farmacologia, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Roma, Italy
d Sezione di Farmacologia, Dipartimento di Biotecnologie, Università degli Studi di Siena, Via A. Moro, 53100 Siena, Italy
e Dipartimento di Chimica, Università degli Studi di Siena, Via A. Moro, 53100 Siena, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The quinoline nucleus of the previously described 4-phenylquinoline-3-carboxamides NK1 receptor
ligands 7 has been transformed into either substituted or azole—(i.e., triazole or tetrazole) fused pyridine
moieties of compounds 9 and 10, respectively, in order to obtain NK1 receptor ligands showing lower
molecular weight or higher hydrophilicity. The program of molecular manipulations produced NK1 recep-
tor ligands showing affinity in the nanomolar range. In particular, 4-methyl-1-piperazinyl derivative 9j
showed an IC50 value of 4.8 nM and was proved to behave as a NK1 antagonist blocking Sar9-SP-sulfone
induced proliferation and migration of microvascular endothelial cells. Therefore, compound 9j has been
labeled with [11C]CH3I (t1/2 = 20.4 min, b+ = 99.8%) starting from the corresponding des-methyl precursor
9i using with a radiochemical yield of about 10% (not decay corrected) and a specific radioactivity >1
Received 2 November 2010
Revised 26 January 2011
Accepted 18 February 2011
Available online 24 February 2011
Keywords:
NK1 receptor
Synthesis
Radiolabelling
Ci/
l
mol in order to be used as a radiotracer in next PET studies.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
nausea and vomiting (CINV). In 1995, the potent antagonist prop-
erties of 4-phenylisoquinolinone and naphthyridinone derivatives
3 were discovered by researchers from Takeda.6 The studies per-
formed at Takeda produced TAK-637 (4),7 which was reported to
be an orally bioavailable antagonist of NK1 receptor in the intesti-
nal smooth muscles and potentially useful in the treatment of
functional bowel diseases such as irritable bowel syndrome
(IBS).8 It is noteworthy that TAK-637 is apparently unrelated to
the best known NK1 receptor antagonists.
The structure of compound 4 was then simplified to obtain axi-
ally chiral 2,3,4,5-tetrahydro-6H-pyrido[2,3-b][1,5]oxazocin-6-one
derivative 5, which confirmed the importance of both aR axial chi-
rality and the stacked conformation of the two phenyl moieties for
receptor recognition.9
In 2006, researchers from Hoffmann-La Roche disclosed the re-
sults obtained within a large medicinal chemistry program de-
voted to the synthesis of novel NK1 receptor antagonists and
culminated in the discovery of netupitant (6a) and befetupitant
(6b).10
Early work performed in our laboratory led to the development
of a series of 4-phenylquinoline-3-carboxamide derivatives 7
(Fig. 2). Among them, secondary amide 7h (R1 = H, R2 = CH2OH)
showed affinity in the picomolar range for native human NK1
receptor expressed in astrocytoma UC11MG cells and behaved as
an agonist of NK1 receptor in endothelial cell proliferation, inositol
The neurokinin (NK) receptors belong to the family of seven-
transmembrane G-protein-coupled receptors (GPCRs) and are clas-
sified into three subtypes: NK1, NK2 and NK3. The undecapeptide
member of the tachykinin family Substance P (SP) is the endoge-
nous ligand for NK1.1 SP interacts with the NK1 receptor eliciting
a wide variety of biological effects. These include smooth muscle
contraction, the transmission of pain and stress signals, the
induction of neurogenic inflammation, endothelium-dependent
vasodilation, and angiogenesis. The first selective non-peptide
NK1 receptor antagonist CP-96,3452 (1, Fig. 1) was discovered by
Pfizer medicinal chemists and stimulated a great interest in this
area by the pharmaceutical industry. This research effort produced
a large number of potent NK1 receptor antagonists in the past dec-
ade.3 The control of both chemotherapy4 and post-surgery nausea
and vomiting5 represents the major therapeutic applications of
non-peptide NK1 receptor antagonists. In fact, aprepitant (2) was
approved by the FDA in 2003 under the brand name EmendÒ for
the prevention of acute and delayed chemotherapy-induced
⇑
Corresponding author. Tel.: +39 0577 234320; fax: +39 0577 234333.
E-mail address: cappelli@unisi.it (A. Cappelli).
Present address: Siena Biotech SpA, Strada del Petriccio e Belriguardo 35, 53100
Siena, Italy.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.02.031

G. Giuliani et al. / Bioorg. Med. Chem. 19 (2011) 2242–2251
2243
CF3
H3CO
O
i
CF3
O
NH
O
N
O
R3
O
R3
N
CH3
11 (R3 = H)
12 (R3 = CH3)
13 (R3 = H)
14 (R3 = CH3)
N
H
N
ii
F
O
F3C
N
HN
CF3
MK-869 (2)
CP-96,345 (1)
O
N
R1
CH3
O
iii, iv
OH
F3C
CH3
R
F3C
CF3
CF3
N
R3
R3
N
CH3
15 (R3 = H)
16 (R3 = CH3)
9a,b,k,l
O
O
N
N
CH3
CH3
Scheme 1. Reagents: (i) CH3–C(NH2)@CH–COOC2H5, piperidine, DMF; (ii) NaOH,
H2O, C2H5OH; (iii) SOCl2; (iv) 3,5-(CF3)2C6H3CH2NH2, CH2Cl2, TEA, or 3,5-
(CF3)2C6H3CH2N(H)CH3ÁHCl, CH2Cl2, TEA.
N
N
N
X
O
O
3
TAK-637 ( 4)
F3C
F3C
F3C
N
F3C
CF3
CF3
CF3
CF3
O
O
O
H3C
N
H3C
CH3
CH3
i
N
R1
CH3
N
R1
CH3
N
H3C
O
N
N
O
CH3
N
N
O
X
netupitant (6a, X = N-CH3)
befetupitant (6b, X = O)
9c,d
9a,b
5
ii
Figure 1. Structures of NK1 receptor antagonists 1–6 from Pfizer, Merck, Takeda,
and Hoffmann-La Roche.
F3C
F3C
CF3
+
CF3
O
O
R4
F3C
R3
CF3
N
R1
N
R1
R2
O
N
N
Cl
CH3
CH2Cl
O
X
N
N
9e,f
9g,h
R1
R1
N
R2
N
Z
iii
Y
8
7
F3C
CF3
F3C
F3C
CF3
CF3
O
O
O
N
R1
CH3
N
R1
N
R1
CH3
N
N
N
N
R3
R2
N
N
N
H3C
9i,j
X
X
9
10
Scheme 2. Reagents: (i) m-CPBA, CHCl3; (ii) POCl3; (iii) N-methylpiperazine.
Figure 2. Structures of NK1 receptor ligands 7–10 from our laboratory.
astrocytoma UC11MG cells.11 Subsequently, the results obtained
with compounds 7 led to the design of tricyclic carboxamide deriv-
atives 8, in which the substituent in position 2 of the quinoline nu-
cleus of compounds 7 takes part in a five-membered heterocycle
(azole moiety). Within the series of compounds 8, the tertiary
amide derivatives were found to show affinity in the nanomolar
range for recombinant human NK1 receptor, whereas the
phosphate turnover, and NO-mediated cyclic GMP accumulation,
thus proving to be the first non-peptide NK1 receptor agonist
showing very high potency.11 On the other hand, the correspond-
ing tertiary amide derivative 7i (R1 = CH3, R2 = CH2OH) showed
nanomolar affinity for native human NK1 receptor expressed in

2244
G. Giuliani et al. / Bioorg. Med. Chem. 19 (2011) 2242–2251
corresponding secondary amides were about one order of magni-
tude less active.12 Furthermore, the systematic structure variation
suggested that electronic features of the tricyclic moiety play a role
in modulating the interaction of these amide derivatives with their
receptor.12 As a further step of our large program of structural
manipulations, quinoline nucleus of 7 has been transformed into
substituted or azole—(i.e., triazole or tetrazole) fused pyridine moi-
eties of compounds 9 and 10, respectively, in order to obtain NK1
receptor ligands showing lower molecular weight or higher
hydrophilicity.
F3C
F3C
CF3
CF3
O
O
i
N
H
CH3
N
11CH3
CH3
N
N
N
N
N
N
11C]9j
H3C
H3C
9i
[
Scheme 4. Reagents: (i) [11C]CH3I, TBAOH, DMF.
2. Results and discussion
2.1. Chemistry
hydrolyzed with 2 M NaOH to give acid derivatives 15 and 16.
Amidation of 15 and 16 under standard conditions provided target
amides 9a,b,k,l.
Compounds 9a and 9b were subjected to oxidation with
m-CPBA (Scheme 2) to obtain the corresponding N-oxides 9c,d,
which were in turn reacted with phosphorus oxychloride to give
chloroderivates 9e,f and 9g,h. Reaction of imidoyl chlorides 9e,f
with an excess of N-methylpiperazine at 130–140 °C gave the
target N-methylpiperazino derivatives 9i,j in good yield.
Moreover, the reaction of imidoyl chlorides 9e,f with sodium
azide gave target tetrazolo[1,5-a]pyridine derivatives 10a,b
(Scheme 3). On the other hand, the preparation of [1,2,4]triazolo-
[4,3-a]pyridine derivatives 10c,d required the two-step procedure
shown in Scheme 3, consisting in the reaction of 9e,f with hydra-
zine hydrate to obtain hydrazino derivatives 17 and 18, respec-
tively, which were cyclized with formic acid to obtain the target
compounds 10c,d.
The structure of [1,2,4]triazolo[4,3-a]pyridine derivative 10d
was confirmed by X-ray crystallography (Fig. 3). Crystallographic
conformation of 10d shows the two phenyl rings arranged in a par-
allel, stacked conformation similar to that shown by (aR)-4 in its
crystallographic structure,7 but different from the corresponding
one of secondary amide derivative 8a (R1 = R4 = H, R2 = 3-CF3,
R3 = 5-CF3, X = Y = Z = CH).12
The synthesis of 4-phenylpyridine-3-carboxamide derivatives 9
and 10 was carried out by means of the multistep procedure
reported in Schemes 1–3. Condensation between ethyl 3-amino-
crotonate and the appropriate unsaturated carbonyl derivative
(11 or 12, Scheme 1) in DMF in the presence of piperidine
gave 4-phenylnicotinate derivatives 1313 and 14,14 which were
F3C
F3C
CF3
CF3
O
O
i
N
N
R1
CH3
R1
N
N
N
Cl
CH3
N
N
10a,b
9e,f
ii
F3C
F3C
2.2. Radiosynthesis
CF3
CF3
Compound [11C]9j was synthesized by N-[11C]methylation of
the corresponding des-methyl precursor 9i with [11C]methyl iodide
in the presence of tetrabutylammonium hydroxide (TBAOH) at
80 °C in dimethylformamide (DMF) for 4 min (Scheme 4), with a
radioactivity incorporation yield of 65%.
O
O
iii
N
R1
N
R1
H
N
N
NH2
CH3
N
CH3
N
N
The overall radiosynthesis, including 11C-methylation, HPLC
purification and radiopharmaceutical formulation for intravenous
administration, was completed in an average time of 35 min from
the end of bombardment (EOB), with a radiochemical yield of
about 10% (not decay corrected) and a specific radioactivity >1
10c,d
17,18
Scheme 3. Reagents: (i) NaN3, DMF; (ii) NH2NH2ÁH2O, C2H5OH; (iii) HCOOH.
Ci/lmol at the end of synthesis (EOS). In a typical experiment,
starting from ca. 1 Ci of [11C]CO2, 80 mCi of [11C]9j were obtained
with a chemical and radiochemical purity of 95% and 100%, respec-
tively. The identity of the final 11C-radioligand was confirmed by
coinjection of the final radiopharmaceutical with its cold standard
9j on reverse-phase high-performance liquid chromatography
(HPLC) (see Section 4).
2.3. Binding studies
Compounds 9a–l and 10a–d were tested for their activity in
inhibiting the specific binding of [125I]BH-SP to human recombi-
nant NK1 receptor stably expressed in CHO cells.15 Owing to the
discrepancies observed in the structure–affinity relationships be-
tween compounds 8 and 7,11,12 some of the previously-published
3-quinolinecarboxamide derivatives 7 were tested in the above
test system. The results of the binding studies obtained with ‘old
Figure 3. X-ray crystal structure of compound 10d. Fluorine atoms bound to C21
and to C22 have site occupation factors of 0.61(3) and 0.53(3), respectively.

G. Giuliani et al. / Bioorg. Med. Chem. 19 (2011) 2242–2251
2245
Table 1
Binding affinities of compounds 7, 9 and 10 for human recombinant NK1 receptor stably expressed in CHO cells
F3C
F3C
F3C
CF3
CF3
CF3
O
O
O
N
N
N
R1
R1
CH3
R1
N
N
X
10
R2
N
X
9
R3
R2
N
N
7
Compd
R1
R2
R3
X
MW
IC50 (nM)a
7a
7b
7c
7d
7h
7i
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
H
CH3
474.4
488.4
488.4
502.4
504.4
518.5
438.4
452.6
454.4
468.4
472.8
486.8
472.8
486.8
536.5
550.5
452.4
466.4
479.4
493.4
478.4
492.4
208 13
92 5.0
257 45
19 1.2
227 27
42 5.2
653 202
98 0.20
342 47
32 2.4
270 32
91 8.0
235 47
52 8.3
53 11
4.8 1.1
434 51
41 6.7
279 26
57 18
698 130
53 15
1.5 0.10
CH3
CH2OH
CH2OH
CH3
CH3
CH3
CH3
CH3
CH3
CH2Cl
CH2Cl
CH3
CH3
CH3
H
H
H
H
Cl
Cl
H
H
O
O
N-Methylpiperazino
N-Methylpiperazino
CH3
CH3
CH3
10a
10b
10c
10d
SP
N
N
CH
CH
a
Each value was calculated from at least three independent experiments performed in duplicate and represents the concentration giving half the maximum inhibition of
125I]SP specific binding to human recombinant NK1 receptor stably expressed in CHO cells.
[
compounds’ 7a–c,h (Table 1) confirm the discrepancy between the
results obtained with the two test systems. This result suggests the
existence of differences between recombinant NK1 receptors ex-
pressed in CHO and native human NK1 receptors expressed in
astrocytoma UC11MG cells. A survey of the literature showed the
existence of differences between native and recombinant proteins
in a number of different receptors. For instance, human recombi-
nant 5-HT1A receptors expressed in HEK293 cells exist in high
and low agonist affinity states, whereas rat cortex 5-HT1A receptors
are primarily in the high agonist affinity state.16 Moreover,
researchers from GlaxoSmithKline reported that NK1 receptor
affinity they observed for NKP-608 was intermediate between
the corresponding value obtained by Vassourt (pIC50 = 7.9) in ger-
bil and that obtained by Rupniak (pIC50 = 10.1) with recombinant
human NK1 receptors.17 It is noteworthy that secondary amide
7h showing the highest difference behaves as an agonist. On the
contrary, the corresponding tertiary amide 7i shows similar affini-
ties in the two different test systems. These observations suggest
that the integration between binding studies and suitable func-
tional studies is of primary importance in order to correctly char-
acterize the interaction of these amide derivatives with NK1
receptors.
affinity of the secondary amides 9a,c,e,g,i,k and 10a,c to a variable
extent (from 3 to 19 times). The structure–affinity relationships
concerning the variation of the heterocyclic moiety, suggest that
the human recombinant NK1 receptor expressed in CHO cells is
moderately sensitive to the small structure modulations shown
by compounds 9a–h,k,l and 10a–d. In particular, the presence of
the methyl group in position 2 of the quinoline moiety is beneficial
for the NK1 receptor affinity of tertiary amide 7d (compare 7d vs
7b), while the deletion of its fused benzene ring produces a signif-
icant decrease in the receptor affinity (compare 7d with 9b). The
introduction of a chlorine atom in place of the fused benzene ring
as in compound 9f does not succeed in restoring the original
NK1 receptor affinity (i.e., the one shown by 7d). Similarly, the
N-oxidation (as in compound 9d), the introduction of a methyl
group (9l) or the fusion of different azole (e.g., triazole or tetrazole)
moieties at a-edge of pyridine ring of compound 9b leads only to
small increases in the receptor affinity (compare 9d,l and 10b,d
vs 9b), whereas the introduction of a bulkier, basic N-methylpiper-
azine moiety, as in compound 9j, produces a more significant
increase in NK1 receptor affinity so that the latter compound is
the most interesting in the present small series. This result fully
agrees with those described by Hoffmann and coworkers10 and
stimulated a deeper biological characterization of 9j.
Because of its determinant role in the interaction with NK1
receptor binding site,6,7,12,18 3,5-bistrifluoromethylbenzyl was kept
unmodified in the whole series of compounds 9 and 10, while the
second amide substituent was alternatively a methyl group or a
hydrogen atom in order to evaluate the effect of the transformation
of the secondary amides in the corresponding tertiary ones. In
agreement with the results obtained with compounds 8, the amide
methyl group of tertiary amides 9b,d,f,h,j,l and 10b,d enhances the
It should be noted that the above structure–affinity relation-
ships are roughly (but not perfectly) paralleled in the secondary
amide subseries 9a,c,e,g,i,k and 10a,c. A more evident dualism in
the SAFIR trends between secondary and tertiary amides was
observed with compounds 7 using native human NK1 receptor
expressed in astrocytomas UC11MG cells as test system (see
Ref. 11).

2246
G. Giuliani et al. / Bioorg. Med. Chem. 19 (2011) 2242–2251
10b,d are able to adopt the postulated pharmacophorically rele-
vant arrangement.9 Thus, the comparison between the affinities
shown by 10d and 9j suggests for N-methylpiperazine moiety an
important role in the interaction with NK1 receptor.
2.4. Pharmacological characterization
Since N-methylpiperazino derivatives 9i,j exhibited the highest
affinity for the NK1 receptor, these compounds were selected for a
preliminary pharmacological characterization in the same test
system used in the study of parent quinoline agonist 7h.11 Post-
capillary endothelial cells isolated from bovine heart (CVEC)
proliferate and migrate when stimulated with NK1 selective ago-
nists, thus leading to angiogenesis.11 Compounds 9i and 9j were
tested on CVEC proliferation and migration and found to be lacking
of any agonist activity (Fig. 4).
On the other hand, they completely inhibited the effects induced
by NK1 receptor agonist Sar9-SP-sulfone (Fig. 4 and Table 2), sug-
gesting that the modification of 7h leading to 9i,j abolishes the ago-
nist activity. In the same assays, the negative control substance 19
(Fig. 5, IC50 >1000 nM) was devoid of any activity.
While endothelial migration was completely impaired by
100 nM and 1
were able to reduce cell proliferation only at 1
l
M drug concentrations of 9i and 9j, the compounds
M concentration
l
(Table 2), suggesting that short term biological assay (4 h migra-
tion) is more powerful. In conclusion, our results document the
activity of compounds 9i and 9j as NK1 antagonists.
3. Conclusions
Figure 4. Proliferation (top) and migration (bottom) of CVEC in response to 10 nM
Sar9-SP-sulfone (Sar9): effect of 9i,j, and 19. Top panel: Sparse and synchronized
endothelial cells were exposed to test substances for 48 h and the number of fixed
and stained cells was microscopically counted. Bottom panel: Cell migration toward
the NK1 agonist in the absence/presence of test compounds was evaluated after 4 h
of incubation. Data are means SEM of three experiments. ⁄P <0.05 versus agonist
alone (Student’s t test).
On the basis of the intriguing results obtained with compounds
7 and 8, a small series of NK1 receptor ligands based on the
4-phenylpyridine scaffold was designed, synthesized, and evalu-
ated for its potential ability to inhibit the specific binding of
[
125I]BH-SP to recombinant human NK1 receptor in order to obtain
compounds possessing lower molecular weight or higher hydro-
philicity. The structure–affinity relationship study confirms the
higher potency of tertiary amides with respect to the correspond-
ing secondary ones and suggests that the human recombinant NK1
receptor expressed in CHO cells is moderately sensitive to small
structure modulations in the heterocyclic moiety of compounds
9a–h,k,l and 10a–d. However, the introduction of a N-methylpi-
perazine moiety in position 2 of pyridine ring produced compound
9j showing a NK1 receptor affinity in the low nanomolar range. This
result stimulated a deeper biological characterization of 9j, which
was proved to behave as a NK1 receptor antagonist blocking
Sar9-SP-sulfone induced proliferation and migration of microvas-
cular endothelial cells. Thus, compound 9j was labeled with 11C
(t1/2 = 20.4 min, b+ = 99.8%) starting from the corresponding
des-methyl precursor 9i by using [11C]CH3I in the presence of
tetrabutylammonium hydroxide in DMF with a radiochemical
yield of about 10% (not decay corrected) and a specific radio-
Table 2
Inhibition of Sar9-SP-sulfone induced proliferation and migration. Data are expressed
as % inhibition of Sar9-SP-sulfone induced activity
Compound–dose
Proliferation (% inhibition)
76.3
33.5 4.5
Migration (% inhibition)
100 1.4
9i
9j
19
1
l
M
6
100 nM
1
100 nM
1 lM
100
99
100
9
8
3
2
7
lM
79.7
7.6
11
7
3
6
100 nM
0
0
O
OCH3
activity >1 Ci/
PET studies.
lmol in order to be used as a radiotracer in next
N
H
N
CH3
4. Experimental section
4.1. Chemistry
Figure 5. Structure of control substance 19 (see Section 4).
All chemicals used were of reagent grade. Yields refer to puri-
fied products and are not optimized. Melting points were deter-
mined in open capillaries on a Gallenkamp apparatus and are
uncorrected. Merck silica gel 60 (230–400 mesh) was used for col-
umn chromatography. Merck TLC plates, silica gel 60 F254 were
used for TLC. 1H NMR spectra were recorded at 200 MHz (Bruker
It is remarkable that crystallographic conformation of 10d
shows the two phenyl rings arranged in a parallel, stacked confor-
mation, which appears to be quite similar to that shown by (aR)-4
in its crystallographic structure7 and is believed to be an important
prerequisite for the interaction with NK1 receptor binding site. The
crystallographic result suggests that tertiary amides 9b,d,f,h,j,l and

G. Giuliani et al. / Bioorg. Med. Chem. 19 (2011) 2242–2251
2247
AC200 spectrometer) or at 400 MHz (Bruker DRX-400 AVANCE
spectrometer) in the indicated solvents (TMS as internal standard):
the values of the chemical shifts are expressed in ppm and the cou-
pling constants (J) in Hz. An Agilent 1100 LC/MSD operating with a
electrospray source was used in mass spectrometry experiments.
Microanalyses were carried out by means of a Perkin–Elmer Series
II CHNS/O Analyzer 2400.
afforded 0.68 g (yield 74%) of 9a as a white solid (mp 161–
162 °C). 1H NMR (200 MHz, CDCl3): 2.65 (s, 3H), 4.43 (d, J = 5.2,
2H), 5.81 (t, J = 5.4, 1H), 7.14 (d, J = 4.5, 1H), 7.33 (m, 5H), 7.49 (s,
2H), 7.76 (s, 1H), 8.52 (d, J = 4.5, 1H). MS (ESI): m/z 439 (M+H+).
Anal. (C22H16F6N2O) C,H,N.
4.1.6. N-[3,5-Bis(trifluoromethyl)benzyl]-N,2-dimethyl-4-
phenylnicotinamide (9b)
4.1.1. Ethyl 2-methyl-4-phenylnicotinate (13)
A mixture of 15 (0.94 g, 4.4 mmol) in thionyl chloride (10 mL)
was heated to reflux for 3 h, and the excess of SOCl2 was removed
by azeotropic distillation with toluene. The chloride derivative ob-
tained was quickly dissolved in dichloromethane (25 mL) and to
the resulting solution was treated with N-[3,5-bis(trifluoro-
methyl)benzyl]methylamine hydrochloride (1.5 g, 5.1 mmol) and
TEA (1.5 mL). The reaction mixture was heated to reflux for 1 h
and concentrated under reduced pressure. The residue was parti-
tioned between dichloromethane and water and the organic layer
was dried over sodium sulfate and the solvent was removed under
reduced pressure. Purification of the residue by flash chromatogra-
phy with dichloromethane–ethyl acetate (1:1) as the eluent gave
1.4 g (yield 70%) of 9b as a colorless oil. 1H NMR (400 MHz, CDCl3;
the spectrum of this amide derivative shows the presence of two
different rotamers in the ratio of about 8:2; for the sake of simplic-
ity the integral intensities have not been given): 2.44 (s), 2.59 (s),
2.82 (s), 3.80 (d, J = 16.0), 4.20 (d, J = 16.0), 4.62 (s), 7.13–7.50
(m), 7.59 (s), 7.71 (s), 7.79 (s), 8.56 (d, J = 5.1). MS (ESI): m/z 453
(M+H+). Anal. (C23H18F6N2OÁ0.25H2O) C,H,N.
A mixture of piperidine (1.5 mL, 15.2 mmol) in DMF (130 mL)
containing trans-cinnamaldehyde 11 (50 mL, 397 mmol) and ethyl
3-aminocrotonate (38 mL, 301 mmol) was heated to reflux for 1 h.
The solvent was then evaporated under reduced pressure and the
residue was treated with 1 N HCl, neutralized with a 30% solution
of NaOH and extracted with EtOAc. The organic layer was dried
over Na2SO4 and the solvent was removed under reduced pressure.
The residue was purified by flash chromatography with petroleum
ether–ethyl acetate (2:1) as the eluent to obtain 12 g (yield 17%) of
1313 as a yellow oil. 1H NMR (200 MHz, CDCl3): 0.94 (t, J = 7.0, 3H),
2.59 (s, 3H), 4.05 (q, J = 7.0, 2H), 7.08 (d, J = 5.0, 1H), 7.37 (m, 5H),
8.49 (d, J = 5.0, 1H). MS (ESI): m/z 242 (M+H+).
4.1.2. Ethyl 2,6-dimethyl-4-phenylnicotinate (14)
A mixture of piperidine (0.8 mL, 8.1 mmol), trans-4-phenyl-3-
buten-2-one 12 (24 g, 164 mmol), ethyl 3-aminocrotonate
(13 mL, 103 mmol) in 60 mL of DMF was refluxed for 30 h. The
resulting solution was concentrated under reduced pressure and
the residue was purified by flash-chromatography with dichloro-
methane–ethyl acetate (9:1) as the eluent to obtain 4.2 g (yield
16%) of 1414 as a pale yellow oil. 1H NMR (200 MHz, CDCl3): 0.95
(t, J = 7.3, 3H), 2.54 (s, 3H), 2.58 (s, 3H), 4.05 (q, J = 7.3, 2H), 6.98
(s, 1H), 7.35 (m, 5H). MS (ESI): m/z 256 (M+H+).
4.1.7. N-[3,5-Bis(trifluoromethyl)benzyl]-2-methyl-4-
phenylnicotinamide 1-oxide (9c)
To a solution of 9a (1.5 g, 3.4 mmol) in 30 mL of chloroform was
added m-chloroperbenzoic acid (m-CPBA) (1.5 g, 8.7 mmol) and
the resulting mixture was stirred at room temperature for 4 h.
The reaction mixture was washed with a 5% aqueous solution of
K2CO3 and subsequently with water. The organic layer was dried
over sodium sulfate and the solvent was removed under reduced
pressure. Purification of the residue by flash chromatography
(ethyl acetate) afforded 1.4 g (yield 91%) of 9c as a white solid
(mp 210–212 °C). 1H NMR (200 MHz, CDCl3): 2.21 (s, 3H), 4.48
(d, J = 5.8, 2H), 7.04 (d, J = 6.7, 1H), 7.27–7.39 (m, 5H), 7.66 (s,
2H), 7.76 (s, 1H), 7.84 (d, J = 6.7, 1H), 8.91 (br t, 1H). MS (ESI, neg-
ative ions): m/z 453 (MÀH+). Anal. (C22H16F6N2O2) C,H,N.
4.1.3. 2-Methyl-4-phenylnicotinic acid (15)
A mixture of 13 (4.0 g, 16.6 mmol) in ethanol (20 mL) with a 2 N
NaOH solution (20 mL) was refluxed for 6 h. The volatile was re-
moved under reduced pressure and the residue was diluted with
water and acidified with concentrated HCl to pH 5–6. The mixture
was stirred at 0–5 °C and the solid obtained by slow precipitation
was collected by filtration, washed with n-hexane, and dried under
reduced pressure to obtain 1.6 g of 15 (yield 45%) as a white solid
(mp 233–234 °C). 1H NMR (200 MHz, DMSO-d6): 2.49 (s, 3H), 7.21
(d, J = 5.0, 1H), 7.43 (m, 5H), 8.47 (d, J = 5.3, 1H). MS (ESI, negative
ions): m/z 212 (MÀH+).
4.1.8. N-[3,5-Bis(trifluoromethyl)benzyl]-N,2-dimethyl-4-
phenylnicotinamide 1-oxide (9d)
4.1.4. 2,6-Dimethyl-4-phenylnicotinic acid (16)
To a solution of 9b (1.4 g, 3.1 mmol) in 30 mL of chloroform was
added m-chloroperbenzoic acid (m-CPBA) (1.2 g, 6.9 mmol) and
the resulting mixture was stirred at room temperature for 4 h.
The reaction mixture was washed with a 5% aqueous solution of
K2CO3 and subsequently with water. The organic layer was dried
over sodium sulfate and the solvent was removed under reduced
pressure to afford 1.4 g (yield 96%) of 9d as a pale yellow oil.
1H NMR (400 MHz, CDCl3; the spectrum of this amide derivative
shows the presence of two different rotamers in the ratio of about
8:2; for the sake of simplicity the integral intensities have not been
given): 2.49 (s), 2.55 (s), 2.58 (s), 2.81 (s), 3.84 (d, J = 16.0), 4.25 (d,
J = 16.0), 4.51 (d, J = 14.7), 4.72 (d, J = 14.7), 7.14–7.47 (m), 7.58 (s),
7.74 (s), 7.81 (s), 8.36 (d, J = 6.9), 8.38 (d, J = 6.7). MS (ESI): m/z 469
(M+H+). Anal. (C23H18F6N2O2) C,H,N.
A mixture of 14 (1.6 g, 6.3 mmol) in ethanol (20 mL) with a 2 N
NaOH solution (20 mL) was refluxed for 18 h. The reaction mixture
was concentrated under reduced pressure, diluted with water, and
acidified with concentrated HCl. The precipitate was collected by
filtration, washed with n-hexane, and dried under reduced pres-
sure to obtain 0.54 g of 16 (yield 38%) as a white solid (mp 207–
209 °C). 1H NMR (200 MHz, DMSO-d6): 2.47 (s, 6H), 7.07 (s, 1H),
7.47 (s, 5H). MS (ESI, negative ions): m/z 226 (MÀH+).
4.1.5. N-[3,5-Bis(trifluoromethyl)benzyl]-2-methyl-4-
phenylnicotinamide (9a)
A mixture of 15 (0.45 g, 2.1 mmol) in thionyl chloride (3 mL)
was heated to reflux for 3 h. The excess of SOCl2 was then removed
by azeotropic distillation with toluene and the chloride derivative
obtained was quickly dissolved in dichloromethane (15 mL). To the
resulting solution, 3,5-bis(trifluoromethyl)benzylamine (1.3 g,
5.3 mmol) and TEA (1.0 mL) were added. The reaction mixture
was heated to reflux for 1 h, and the solvent was removed under
reduced pressure. Purification of the residue by flash chromatogra-
phy with ethyl acetate–petroleum ether (2:1) as the eluent
4.1.9. N-[3,5-Bis(trifluoromethyl)benzyl]-6-chloro-2-methyl-4-
phenylnicotinamide (9e)
A mixture of 9c (0.45 g, 0.99 mmol) in 6 mL of POCl3 was re-
fluxed for 3 h. The reaction mixture was then poured into crushed
ice, stirred vigorously and extracted with dichloromethane. The
organic layer was dried over sodium sulfate and concentrated

2248
G. Giuliani et al. / Bioorg. Med. Chem. 19 (2011) 2242–2251
under reduced pressure to obtain an oil residue, which was puri-
fied by flash chromatography with petroleum ether–ethyl acetate
(2:1) as eluent to afford 9e (0.18 g, yield 38%) as a white solid
(mp 143–145 °C). 1H NMR (200 MHz, CDCl3): 2.44 (s, 3H), 4.33
(d, J = 5.9, 2H), 6.44 (t, J = 5.6, 1H), 7.09 (s, 1H), 7.14–7.35 (m,
5H), 7.44 (s, 2H), 7.70 (s, 1H). MS (ESI, negative ions): m/z 471
(MÀH+). Anal. (C22H15ClF6N2O) C,H,N.
diluted with ethyl acetate and washed with water. The organic
layer was dried over sodium sulfate and the solvent was removed
under reduced pressure. Purification of the residue by flash chro-
matography with ethyl acetate–TEA (8:2) as eluent gave 0.75 g
(yield 97%) of 9j as a yellow oil. 1H NMR (200 MHz, CDCl3; the
spectrum of this amide derivative shows the presence of two dif-
ferent rotamers in the ratio of about 75:25; for the sake of simplic-
ity the integral intensities have not been given): 2.33 (s), 2.42 (s),
2.45 (s), 2.50 (t, J = 4.9), 2.73 (s), 3.61 (t, J = 4.9), 3.71 (d, J = 16.1),
4.31 (d, J = 16.1), 4.50 (d, J = 14.9), 4.60 (d, J = 14.8), 6.39 (s),
7.19–7.45 (m), 7.57 (s), 7.69 (s), 7.75 (s). MS (ESI): m/z 551
(M+H+). Anal. (C28H28F6N4O) C,H,N.
4.1.10. N-[3,5-Bis(trifluoromethyl)benzyl]-6-chloro-N,2-
dimethyl-4-phenylnicotinamide (9f)
A mixture of 9d (1.4 g, 3.0 mmol) in 10 mL of POCl3 was re-
fluxed for 3 h. The reaction mixture was then poured into crushed
ice, stirred vigorously and extracted with dichloromethane. The or-
ganic layer was dried over sodium sulfate and concentrated under
reduced pressure to obtain an oil residue that was purified by flash
chromatography with petroleum ether–ethyl acetate (65:35) as
eluent to afford 9f (0.69 g, yield 47%) as a white foam. 1H NMR
(200 MHz, CDCl3; the spectrum of this amide derivative shows
the presence of two different rotamers in the ratio of about 8:2;
for the sake of simplicity the integral intensities have not been gi-
ven): 2.41 (s), 2.48 (s), 2.76 (s), 3.78 (d, J = 16.1), 4.17 (d, J = 15.9),
4.56 (m), 7.10–7.41 (m), 7.56 (s), 7.66 (s), 7.73 (s). MS (ESI): m/z
487 (M+H+). Anal. (C23H17ClF6N2O) C,H,N.
4.1.15. N-[3,5-Bis(trifluoromethyl)benzyl]-2,6-dimethyl-4-
phenylnicotinamide (9k)
A mixture of 16 (0.20 g, 0.88 mmol) in thionyl chloride (3 mL)
was heated to reflux for 4 h. The excess of SOCl2 was then removed
by azeotropic distillation with toluene and the chloride derivative
obtained was quickly dissolved in dichloromethane (15 mL). To the
resulting solution, 3,5-bis(trifluoromethyl)benzylamine (0.50 g,
2.1 mmol) and TEA (1.0 mL) were added. The reaction mixture
was heated to reflux for 1 h, and the solvent was removed under
reduced pressure. Purification of the residue by flash chromatogra-
phy with ethyl acetate–petroleum ether (2:1) as the eluent affor-
ded 0.30 g (yield 75%) of 9k as a white solid (mp 135–136 °C).
1H NMR (200 MHz, CDCl3): 2.54 (s, 3H), 2.60 (s, 3H), 4.39 (d,
J = 6.0, 2H), 5.64 (br t, 1H), 6.97 (s, 1H), 7.26–7.36 (m, 5H), 7.46
(s, 2H), 7.73 (s, 1H). MS (ESI): m/z 453 (M+H+). Anal. (C23H18F6N2O)
C,H,N.
4.1.11. N-[3,5-Bis(trifluoromethyl)benzyl]-2-(chloromethyl)-4-
phenylnicotinamide (9g)
Compound 9g was obtained in the synthesis of 9e and was puri-
fied by flash chromatography with petroleum ether–ethyl acetate
(2:1) as the eluent to obtain a white solid (0.14 g, yield 30%, mp
176–178 °C). 1H NMR (200 MHz, CDCl3): 4.44 (d, J = 6.0, 2H), 4.83
(s, 2H), 5.91 (br t, 1H), 7.26 (d, J = 5.4, 1H), 7.34 (m, 5H), 7.51 (s,
2H), 7.74 (s, 1H), 8.59 (d, J = 5.3, 1H). MS (ESI, negative ions): m/z
471 (MÀH+). Anal. (C22H15ClF6N2O) C,H,N.
4.1.16. N-[3,5-Bis(trifluoromethyl)benzyl]-4-phenyl-N,2,6-
trimethylnicotinamide (9l)
A mixture of 16 (0.26 g, 1.14 mmol) in thionyl chloride (3 mL)
was heated to reflux for 4 h, and the excess of SOCl2 was removed
by azeotropic distillation with toluene. The chloride derivative ob-
tained was quickly dissolved in dichloromethane (15 mL) and to
the resulting solution was treated with N-[3,5-bis(trifluoro-
methyl)benzyl]methylamine hydrochloride (0.40 g, 1.4 mmol)
and TEA (1.0 mL). The reaction mixture was heated to reflux for
1 h and concentrated under reduced pressure. The residue was par-
titioned between dichloromethane and water and the organic layer
was dried over sodium sulfate and the solvent was removed under
reduced pressure. Purification of the residue by flash chromatogra-
phy with ethyl acetate–petroleum ether (2:1) as the eluent gave
0.36 g (yield 68%) of 9l as a colorless oil. 1H NMR (200 MHz, CDCl3;
the spectrum of this amide derivative shows the presence of two
different rotamers in the ratio of about 8:2; for the sake of simplic-
ity the integral intensities have not been given): 2.42 (s), 2.53 (m),
2.78 (s), 3.77 (d, J = 16.0), 4.19 (d, J = 16.0), 4.58 (s), 6.98–7.47 (m),
7.57 (s), 7.67 (s), 7.74 (s). MS (ESI): m/z 467 (M+H+). Anal.
(C24H20F6N2O) C,H,N.
4.1.12. N-[3,5-Bis(trifluoromethyl)benzyl]-2-(chloromethyl)-N-
methyl-4-phenylnicotinamide (9h)
Compound 9h was obtained as a byproduct in the synthesis of
9f. After purification by flash chromatography with petroleum
ether–ethyl acetate (2:1) as the eluent, the title compound was ob-
tained as a yellow oil (0.42 g, yield 29%). 1H NMR (200 MHz, CDCl3;
the spectrum of this amide derivative shows the presence of two
different rotamers in the ratio of about 85:15; for the sake of sim-
plicity the integral intensities have not been given): 2.36 (s), 2.77
(s), 3.89 (d, J = 16.1), 4.22 (d, J = 16.3), 4.43 (d, J = 14.8), 4.59–4.73
(m), 4.99–5.07 (m), 7.02 (m), 7.25–7.42 (m), 7.62 (s), 7.75 (s),
8.61 (d, J = 5.5). MS (ESI): m/z 487 (M+H+). Anal. (C23H17ClF6N2O)
C,H,N.
4.1.13. N-[3,5-Bis(trifluoromethyl)benzyl]-2-methyl-6-(4-
methylpiperazin-1-yl)-4-phenylnicotinamide (9i)
A mixture of 9e (40 mg, 0.085 mmol) in 3 mL of N-methyl-
piperazine was heated at 140 °C for 20 h. The reaction mixture
was diluted with ethyl acetate and washed with water. The organic
layer was dried over sodium sulfate and the solvent was removed
under reduced pressure. Purification of the residue by flash
chromatography with ethyl acetate–TEA (8:2) as eluent gave
44 mg (yield 96%) of 9i as a white foam. 1H NMR (200 MHz,
CDCl3): 2.30 (s, 3H), 2.45 (m, 7H), 3.59 (t, J = 4.9, 4H), 4.31 (d,
J = 5.9, 2H), 5.70 (br t, 1H), 6.32 (s, 1H), 7.22–7.31 (m, 5H), 7.44
(s, 2H), 7.70 (s, 1H). MS (ESI): m/z 537 (M+H+). Anal. (C27H26F6N4O)
C,H,N.
4.1.17. N-[3,5-Bis(trifluoromethyl)benzyl]-5-methyl-7-
phenyltetrazolo[1,5-a]pyridine-6-carboxamide (10a)
A
mixture of 9e (0.36 g, 0.76 mmol) in anhydrous DMF
(15 mL) with sodium azide (0.10 g, 1.5 mmol) was heated at
130 °C for 48 h. The solvent was then removed under reduced
pressure and the residue was purified by flash-chromatography
with petroleum ether–ethyl acetate (2:1) as eluent to obtain
50 mg (yield 14%) of 10a as a white solid (mp 219–220 °C). 1H NMR
(200 MHz, CDCl3): 2.95 (s, 3H), 4.47 (d, J = 5.9, 2H), 6.59 (t, J = 5.5,
1H), 7.33–7.45 (m, 5H), 7.56 (s, 2H), 7.65 (s, 1H), 7.77 (s, 1H).
MS (ESI, negative ions): m/z 478 (MÀH+). Anal. (C22H15F6N5O)
C,H,N.
4.1.14. N-[3,5-Bis(trifluoromethyl)benzyl]-N,2-dimethyl-6-(4-
methylpiperazin-1-yl)-4-phenylnicotinamide (9j)
A mixture of 9f (0.68 g, 1.4 mmol) in 15 mL of N-methylpipera-
zine was heated at 140 °C for 20 h. The reaction mixture was

G. Giuliani et al. / Bioorg. Med. Chem. 19 (2011) 2242–2251
2249
4.1.18. N-[3,5-Bis(trifluoromethyl)benzyl]-N,5-dimethyl-7-
phenyltetrazolo[1,5-a]pyridine-6-carboxamide (10b)
resulting solution, 2-methoxybenzylamine (0.30 mL, 2.3 mmol)
and TEA (1.0 mL) were added. The reaction mixture was heated
to reflux for 1 h, and the solvent was removed under reduced pres-
sure. Purification of the residue by flash chromatography with
ethyl acetate–petroleum ether (8:2) as the eluent afforded 0.21 g
(yield 45%) of 19 as a white solid (mp 124–125 °C). 1H NMR
(200 MHz, CDCl3): 2.56 (s, 3H), 3.60 (s, 3H), 4.33 (d, J = 5.9, 2H),
6.11 (t, J = 5.6, 1H), 6.63–7.33 (m, 10H), 8.40 (d, J = 5.2, 1H). MS
(ESI): m/z 333 (M+H+). Anal. (C21H20N2O2ÁH2O) C,H,N.
A mixture of 9f (0.30 g, 0.62 mmol) in anhydrous DMF (10 mL)
with sodium azide (80 mg, 1.2 mmol) was heated at 130 °C for
48 h. The solvent was then removed under reduced pressure and
the residue was purified by flash-chromatography with petroleum
ether–ethyl acetate (2:1) as eluent to obtain 60 mg (yield 20%) of
10b as a white solid (mp 152–154 °C). 1H NMR (200 MHz, CDCl3;
the spectrum of this amide derivative shows the presence of two
different rotamers in the ratio of about 8:2; for the sake of simplic-
ity the integral intensities have not been given): 2.52 (s), 2.80 (s),
2.96 (s), 2.98 (s), 3.75 (d, J = 16.0), 4.32 (d, J = 16.0), 4.59 (m),
7.21–7.55 (m), 7.59 (s), 7.73 (s), 7.80 (s), 7.85 (s), 7.89 (s). MS
(ESI): m/z 494 (M+H+). Anal. (C23H17F6N5O) C,H,N.
4.2. X-Ray crystallography
A single crystal of 10d was submitted to X-ray data collection
on a Siemens P4 four-circle diffractometer with a graphite mono-
chromated Mo-Ka radiation (l = 0.71069 Å) at 293 K. The structure
4.1.19. N-[3,5-Bis(trifluoromethyl)benzyl]-5-methyl-7-phenyl-
[1,2,4]triazolo[4,3-a]pyridine-6-carboxamide (10c)
was solved by direct methods implemented in SHELXS-97 pro-
gram.19 The refinements were carried out by full-matrix aniso-
tropic least-squares on F2 for all reflections for non-H atoms by
means of SHELXL-97 program.20
A mixture of 9e (0.30 g, 0.635 mmol) in ethanol (15 mL) con-
taining an excess of hydrazine hydrate (10 mL) was refluxed for
8 h. The solvent was then removed under reduced pressure, and
the residue was diluted with dichloromethane. The organic layer
was washed with water and dried over sodium sulfate. After re-
moval of the solvent under reduced pressure, 17 [MS (ESI): m/z
469 (M+H+)] was promptly dissolved in formic acid (10 mL) and
the resulting solution was refluxed for 20 h. The excess of formic
acid was removed under reduced pressure, and the residue was
dissolved in chloroform. The organic layer was washed with water,
dried over sodium sulfate, and concentrated under reduced pres-
sure to get a residue that was purified by flash-chromatography
with ethyl acetate–TEA (8:2) as eluent to obtain 10c (30 mg, yield
10%) as a white solid (mp 205–206 °C). 1H NMR (200 MHz, CDCl3):
2.69 (s, 3H), 4.44 (d, J = 5.9, 2H), 6.77 (t, J = 5.8, 1H), 7.26–7.41 (m,
5H), 7.46 (s, 1H), 7.58 (s, 2H), 7.76 (s, 1H), 8.67 (s, 1H). MS (ESI,
negative ions): m/z 477 (MÀH+). Anal. (C23H16F6N4O) C,H,N.
The fluorine atoms show a statistical disorder and two sites ro-
tated by 60° from one another have been refined for each trifluoro-
methyl group. The two sets of fluorine atoms bound to C21 have
site occupation factors (s.o.f.) of 0.61(3) and 0.39(3) while those
bound to C22 have s.o.f. of 0.53(3) and 0.47(3), respectively.
Crystallographic data (excluding structure factors) for the struc-
ture in this paper have been deposited with the Cambridge Crystal-
lographic Data Centre as supplementary publication no. CCDC
708808. Copies of the data can be obtained, free of charge, on
application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK;
(fax: +44 (0) 1223 336 033; or e-mail: deposit@ccdc.cam.ac.uk).
4.3. Radiochemistry
4.3.1. Radiosynthesis
Reagents and solvents were obtained from Sigma–Aldrich Italia
S.p.A. (Milano, Italy) and from ABX (Radeberg, Germany) and were
HPLC or ACS grade.
4.1.20. N-[3,5-Bis(trifluoromethyl)benzyl]-N,5-dimethyl-7-
phenyl-[1,2,4]triazolo[4,3-a]pyridine-6-carboxamide (10d)
A mixture of 9f (0.20 g, 0.41 mmol) in ethanol (10 mL) contain-
ing an excess of hydrazine hydrate (8 mL) was refluxed for 8 h. The
solvent was then removed under reduced pressure, and the residue
was diluted with dichloromethane. The organic layer was washed
with water and dried over sodium sulfate. After removal of the sol-
vent under reduced pressure, 18 [MS (ESI): m/z 483 (M+H+)] was
promptly dissolved in formic acid (8 mL) and the resulting solution
was refluxed for 18 h. The excess of formic acid was removed un-
der reduced pressure, and the residue was dissolved in chloroform.
The organic layer was washed with water, dried over sodium sul-
fate, and concentrated under reduced pressure to obtain a residue
that was purified by flash-chromatography with ethyl acetate–TEA
(8:2) as eluent to afford 10d (61 mg, yield 30%) as a white solid. An
analytical sample was obtained by recrystallization from ethyl ace-
tate by slow evaporation (colorless prisms, mp 217–218 °C).
1H NMR (200 MHz, CDCl3; the spectrum of this amide derivative
shows the presence of two different rotamers in the ratio of about
9:1; for the sake of simplicity the integral intensities have not been
given): 2.48 (s), 2.70 (s), 2.75 (s), 3.64 (d, J = 16.0), 4.32 (d, J = 16.0),
4.44 (d, J = 14.7), 4.63 (d, J = 14.7), 7.22–7.52 (m), 7.58 (s), 7.65 (s),
7.70 (s), 7.78 (s), 8.78 (s), 8.82 (s). MS (ESI): m/z 493 (M+H+). Anal.
(C24H18F6N4O) C,H,N.
[
11C]Carbon dioxide was produced by the 14N(p, 11C reaction
a)
on a IBA Cyclone 18/9 cyclotron, using 18 MeV proton beam at cur-
rents 15–30 A, and trapped in a hollow stainless steel loop, cooled
with liquid nitrogen.
l
[
11C]Methyl iodide was substantially synthesized as described
by Crouzel21 involving the reduction of [11C]CO2 with 0.07 M
LiAlH4 to lithium aluminum-[11C]methylate, hydrolysis of this
intermediate organometallic complex, iodination of the formed
[
11C]methanol with hydriodic acid and distillation through an
Ascarite–Sicapent purification column. Radiochemical synthesis
was performed on the partially modified fully automated synthesis
module (PET Tracer Synthesizer, Nuclear Interface Datentechnik
GmbH, Münster, Germany) for [11C]-methylation described in de-
tail elsewhere.22
For the methylation reaction, [11C]Methyl iodide was trans-
ported by a stream of argon (6 mL/min) into the reaction vessel
containing 1.0 mg (0.0019 mmol) of des-methyl precursor 9i. The
precursor was dissolved in 100
lL of dimethylformamide (ABX)
containing 1.0 L of tetrabutylammonium hydroxide (60% aqueous
l
solution, Fluka) at 80 °C. After 4 min, the reaction mixture was
diluted with 1.2 mL of mobile phase (CH3CN/sodium dihydrogen
orthophosphate monohydrate 0.05 M, 60/40 (v/v); pH 7.2) and
injected in semipreparative HPLC for the purification of the com-
pound of interest. After this step, the fraction corresponding to
4.1.21. N-(2-Metoxybenzyl)- 2-methyl-4-phenylnicotinamide
(19)
[
11C]9j was collected in 30 mL of sterile water and the product
A mixture of 15 (0.30 g, 1.4 mmol) in thionyl chloride (5 mL)
was heated to reflux for 3 h. The excess of SOCl2 was then removed
by azeotropic distillation with toluene and the chloride derivative
obtained was quickly dissolved in dichloromethane (15 mL). To the
was recovered by solid-phase extraction (SPE) on pre-activated
Sep-Pak tC18 plus cartridge (Waters) with 0.7 mL of ethanol.
The purification by semipreparative HPLC was performed with
an ACE C18 column (5
lm, 250 Â 10 mm, CPS) using a Sykam HPLC

2250
G. Giuliani et al. / Bioorg. Med. Chem. 19 (2011) 2242–2251
Pump and monitoring with an UV detector K-2001 (Knauer) set at
254 nm, in series with a radiochemical detector, with a flow rate of
5 mL/min. Retention times were 8.5 min for 9i and 12 min and
14 min for the first and the second peak of the [11C]9j rotamers,
respectively. The pH of the final solution was neutral.
concentration of the radiotracer was kept constant at 10 pM in
the presence of increasing concentrations of the compounds to
be tested. Reactions lasted for 90 min at room temperature and
were terminated by rapid filtration into GF/B glass fiber filtering
microplates (Filtermate 196; Packard Instruments, Meriden, CT).
Filters were washed three times with 1 mL of ice-cold 50 mM
Tris–HCl pH 7.4, and allowed to dry for a few hours. The plates
were counted in a Top Count (Packard Instruments) after the addi-
For the quality control of the final radiopharmaceutical, an
X-Terra RP18 analytical column (5
lm, 250 Â 4.6 mm, Waters)
was used and the chromatographic process was performed with
a Waters 1515 isocratic pump and monitored with both UV and
Radiochemical Detectors (a Waters 2487 variable-wavelength UV
detector in series with a b+-Bioscan Flow Count detector); for the
analysis, a mobile phase of CH3CN/Sodium dihydrogen orthophos-
phate 1-hydrate 0.05 M, 60/40 (v:v), pH 7.2 was used, with a wave-
length of 254 nm and 1.0 mL/min flow rate. The retention times of
the two rotamers of [11C]9j were 6.6 min and 7.4 min for the first
peak and the second one, respectively. Data collection and HPLC
control were performed with the use of the chromatography soft-
ware package Empower. The identification of the radiolabelled
compound was also confirmed by the coinjection of [11C]9j with
its cold standard (9j).
tion (50 lL) of Microscint 20 (Packard) to each well. The data of the
dose–response experiments were analyzed by means of Allfit23
program to compute the IC50 values.
4.5. Pharmacological studies
Bovine post-capillary venular endothelial cells (CVEC) were
obtained and cultured as previously described.24 Cellular prolifera-
tion was quantified in subconfluent and synchronized CVEC by
total cell number counted per well after 48 h of incubation.11 Cell
migration was evaluated by means of the Neuroprobe micro-
chemotaxis chamber.25
4.3.2. Mass spectrometry characterization of [11C]9j
Acknowledgment
After the transfer of the collected fraction on the tC18 plus
Sep-Pak cartridge, [11C]9j (prepared by carrier added radiosynthe-
sis) was recovered by elution with 5.0 mL of ethanol. The peak was
collected in the final sterile vial and its identity was confirmed
(after decay of radioactivity) by mass spectrometry. An API QStar
Pulsar instrument (Applied Biosystems) was used, with an off-line
nanospray ionization source and a QqTOF analyzer.
The mass spectrum shows a peak at m/z 551.22 corresponding
to the protonated molecule [M+H]+, whose structure has been
further confirmed by collision-induced decomposition experi-
ments showing the main elimination of 57 u attributed to a
methylaziridine.
The authors thank Dr. Francesco Berrettini (CIADS, Università di
Siena) for the X-ray data collection. This work was financially sup-
ported by MIUR (Ministero dell’Istruzione, dell’Università e della
Ricerca)—PRIN (Programmi di ricerca di Rilevante Interesse
Nazionale).
Supplementary data
Supplementary data (analytical data for compounds 9a–l, 10a–
d, and 19) associated with this article can be found, in the online
version, at doi:10.1016/j.bmc.2011.02.031. These data include
MOL files and InChiKeys of the most important compounds de-
scribed in this article.
4.4. In vitro binding assays
4.4.1. Ligands and reagents
References and notes
Monoiodinated
[
125I]-Bolton-Hunter reagent labeled SP
1. Hokfelt, T.; Pernow, B.; Wahren, J. J. Int. Med. 2001, 249, 27.
2. Snider, R. M.; Constantine, J. W.; Lowe, J. A., III; Longo, K. P.; Lebel, W. S.;
Woody, H. A.; Drozda, S. E.; Desai, M. C.; Vinick, F. J.; Spencer, R. W.; Hess, H.-J.
Science 1991, 251, 437.
3. Sorbera, L. A.; Silvestre, J.; Castaner, J. Drugs Future 1999, 24, 254.
4. Bleiberg, H. Curr. Opin. Oncol. 2000, 12, 284.
(2200 Ci/mmol) was purchased from Perkin–Elmer Life Science.
Unlabeled SP was purchased from Bachem. Cell culture media,
G418, and fetal calf serum were from Invitrogen.
4.4.2. Cell culture
5. Diemunsch, P.; Grelot, L. Drugs 2000, 60, 533.
6. Natsugari, H.; Ikeura, Y.; Kiyota, Y.; Ishichi, Y.; Ishimaru, T.; Saga, O.;
Shirafuji, H.; Tanaka, T.; Kamo, I.; Doi, T.; Otsuka, M. J. Med. Chem. 1995, 38,
3106.
Permanently transfected CHO cells were grown in a mixture of
Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium (1:1)
supplemented with 10% fetal calf serum, 100 U/mL penicillin G,
100 mg/mL streptomycin sulfate, and 100 mg/mL G418 (Life
Technologies, Inc.) at 37 °C in a humidified atmosphere of 5% CO2.
7. Natsugari, H.; Ikeura, Y.; Kamo, I.; Ishimaru, T.; Ishichi, Y.; Fujishima, A.;
Tanaka, T.; Kasahara, F.; Kawada, M.; Doi, T. J. Med. Chem. 1999, 42, 3982.
8. (a) Venkova, K.; Sutkowski-Markmann, D. M.; Greenwood-Van Meerveld, B. J.
Pharmacol. Exp. Ther. 2002, 300, 1046; (b) Okano, S.; Nagaya, H.; Ikeura, Y.;
Natsugari, H.; Inatomi, N. J. Pharmacol. Exp. Ther. 2001, 298, 559.
9. Ishichi, Y.; Ikeura, Y.; Natsugari, H. Tetrahedron 2004, 60, 4481.
10. Hoffman, T.; Bös, M.; Stadler, H.; Schnider, P.; Hunkeler, W.; Godel, T.; Galley,
G.; Ballard, T. M.; Higgins, G. A.; Poli, S. M.; Sleight, A. J. Bioorg. Med. Chem. Lett.
2006, 16, 1362.
4.4.3. Transfection of cells
Wild-type h-NK1 receptor were transfected in CHO cells using
Lipofectin (Life Technologies, Inc.), and stably expressing clones
were isolated following selection with 400 mg/mL G418
(Geneticin) after 3–6 weeks.
11. Cappelli, A.; Giuliani, G.; Pericot Mohr, G.; Gallelli, A.; Anzini, M.; Vomero, S.;
Cupello, A.; Scarrone, S.; Matarrese, M.; Moresco, R. M.; Fazio, F.; Finetti, F.;
Morbidelli, L.; Ziche, M. J. Med. Chem. 2004, 47, 1315.
12. Cappelli, A.; Giuliani, G.; Anzini, M.; Riitano, D.; Giorgi, G.; Vomero Bioorg. Med.
Chem. 2008, 16, 6850.
4.4.4. Radioreceptor binding assays
13. Weller, D. D.; Rapoport, H. J. Am. Chem. Soc. 1976, 98, 6650.
14. Bagley, M. C.; Brace, C.; Dale, J. W.; Ohnesorge, M.; Phillips, N. G.; Xiong, X.;
Bower, J. J. Chem. Soc., Perkin Trans. 1 2002, 1663.
15. Riitano, D.; Werge, T. M.; Costa, T. J. Biol. Chem. 1997, 272, 7646.
16. Watson, J.; Collin, L.; Ho, M.; Riley, G.; Scott, C.; Selkirk, J. V.; Price, G. W. Br. J.
Pharmacol. 2000, 130, 1108.
17. Griffante, C.; Carletti, R.; Andreetta, F.; Corsi, M. Br. J. Pharmacol. 2006, 148, 39.
18. Natsugari, H.; Ikeda, H.; Ishimaru, T.; Doi, T. (Takeda Chemical Industries, Ltd)
Condensed Heterocyclic Compounds, their Production and Use. Eur. Pat. Appl.
Enriched plasma membranes from transfected stable cells were
prepared as described15 and stored (2 mg/mL) at À80 °C. The com-
pounds were dissolved into 1 mM DMSO and stored at À20 °C.
Radioreceptor binding assays were made in 1 mL reaction mixture
containing 50 mM Hepes–Tris pH 7.4, 5 mM MgCl2, 10 mM leupep-
tin, 0.1 mg/mL bacitracin, 0.1% (w/v) bovine serum albumin, and
2–3 lg of membrane proteins from transfected CHO cells. The

G. Giuliani et al. / Bioorg. Med. Chem. 19 (2011) 2242–2251
2251
EP 585,913, 9 Mar 1994, JP Appl. 92/237,481, 4 Sep 1992; Chem. Abstr. 1995,
122, 56051x.
19. Sheldrick, G. M. SHELXS-97, Rel. 97-2, A Program for Automatic Solution of
Crystal Structures, Gottingen University, 1997.
22. Matarrese, M.; Soloviev, D.; Todde, S.; Neutro, F.; Petta, P.; Carpinelli, A.;
Brüssermann, M.; Galli Kienle, M.; Fazio, F. Nucl. Med. Biol. 2003, 30, 79.
23. De Lean, K. W.; Munson, P. J.; Rodbard, D. Am. J. Physiol. 1978, 235,
E97.
20. Sheldrick, G. M. SHELXL-97, Rel. 97-2,
Refinement, Gottingen University, 1997.
A
Program for Crystal Structure
24. Schelling, M. E.; Meininger, C. J.; Hawker, J. R., Jr.; Granger, H. J. Am. J. Physiol.
1988, 254, H1211.
21. Crouzel, C.; Långström, B.; Pike, V. W.; Coenen, H. H. Appl. Radiat. Isot. 1987, 38,
25. Ziche, M.; Morbidelli, L.; Masini, E.; Amerini, S.; Granger, H. J.; Maggi, C. A.;
Geppetti, P.; Ledda, F. J. Clin. Invest. 1994, 94, 2036.
601.