Design, synthesis, and structure-affinity relationship studies in NK1 receptor ligands based on azole-fused quinolinecarboxamide moieties

Article abstract of DOI:10.1016/j.bmc.2008.05.067

The substituent in position 2 of the quinoline nucleus of NK₁ receptor ligands 5 has been constrained into different five-membered heterocyclic moieties in order to obtain information on the binding site pocket interacting with this apparently

Full text of DOI:10.1016/j.bmc.2008.05.067

Bioorganic & Medicinal Chemistry 16 (2008) 6850–6859
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and structure–affinity relationship studies in NK1
receptor ligands based on azole-fused quinolinecarboxamide moieties
a
a
b
c
Andrea Cappelli a, , Germano Giuliani , Maurizio Anzini , Daniela Riitano , Gianluca Giorgi ,
*
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 Dipartimento di Farmacologia, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Roma, Italy
c 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 substituent in position 2 of the quinoline nucleus of NK1 receptor ligands 5 has been constrained into
different five-membered heterocyclic moieties in order to obtain information on the binding site pocket
interacting with this apparently critical portion of ligands 5. This structure–affinity relationship study led
to the discovery of novel tricyclic NK1 receptor ligands 6 showing affinity in the nanomolar range to the
sub-micromolar one. The systematic structure variation suggests that electronic features of the tricyclic
moiety play a role in modulating the interaction of these amide derivatives with their receptor.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 28 December 2007
Revised 22 May 2008
Accepted 28 May 2008
Available online 12 June 2008
Keywords:
Neurokinin
NK1 receptor
Substance P
Synthesis
Amide derivatives
1. Introduction
olinone and naphthyridinone derivatives 3,6 which are appar-
ently unrelated to the best known NK1 receptor antagonists.
The neurokinin (NK) receptors are members of the seven-
transmembrane G-protein-coupled receptor family (GPCRs) and
are divided into three subtypes: NK1, NK2, and NK3. The endog-
enous ligand for NK1 is the undecapeptide member of the tach-
ykinin family, Substance P (SP).1
The studies performed at Takeda led to the development of
TAK-637 (4),7 which was reported to be an orally bioavailable
antagonist of NK1 receptor in the intestinal smooth muscles
and potentially useful in the treatment of functional bowel dis-
eases such as irritable bowel syndrome (IBS).8
By interacting with the NK1 receptor, SP elicits a wide variety
of biological responses including the transmission of pain and
stress signals, smooth muscle contraction, the induction of neu-
rogenic inflammation, endothelium-dependent vasodilation, and
angiogenesis. Since 1991, the discovery2 of the first selective
non-peptide NK1 receptor antagonist CP-96,345 (1, Fig. 1) has
catalyzed a great amount of research in this area on the part
of the pharmaceutical industry, and a large number of potent
NK1 receptor antagonists have been developed in the past dec-
ade.3 Non-peptide NK1 receptor antagonists have proved to be
highly effective in the control of both chemotherapy4 and post-
surgery nausea and vomiting.5 By way of example, aprepitant
(2, Emend) has been approved as an adjunctive therapy for che-
motherapy-induced emesis. In 1995, researchers from Takeda
discovered the potent antagonist properties of 4-phenylisoquin-
The program of structural manipulation of original Takeda
antagonists 3 led to the synthesis of 3-quinolinecarboxamide
derivatives 59 related to our quinolinecarboxamide peripheral
benzodiazepine receptor ligands.10 Several derivatives 5 showed
NK1 receptor affinity in the picomolar range, and the most active
compound (5h, R1 = H, R2 = CH2OH) behaved as an agonist of NK1
receptor in endothelial cell proliferation, inositol phosphate turn-
over, and NO-mediated cyclic GMP accumulation, thus proving
to be the first non-peptide NK1 receptor agonist showing very
high potency. Moreover, the structure–affinity relationship anal-
ysis of the series suggested a limited dimension for the pocket
accommodating the substituents in position 2.9 The outstanding
results obtained with these 3-quinolinecarboxamides stimulated
the design of tricyclic carboxamide derivatives 6, in which the
substituent in position 2 of the quinoline nucleus of compounds
5 takes part in a five-membered heterocycle (azole moiety), in
order to obtain further information on the structural prerequi-
sites for the interaction with NK1 binding site.
* Corresponding author. Tel.: +39 0577 234320; fax: +39 0577 234333.
E-mail address: cappelli@unisi.it (A. Cappelli).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.067

A. Cappelli et al. / Bioorg. Med. Chem. 16 (2008) 6850–6859
6851
CF3
H3CO
i
CF3
NH
O
N
O
NH2
N
7
8
N
ii
H
N
F
O
N
HN
O
COOH
MK-869 (2)
F3C
CP-96,345 (1)
COOC2H5
iii
CH3
R
N
N
F3C
CF3
CF3
9
10
iv
F3C
O
O
N
N
CH3
CH3
CF3
N
N
N
X
O
O
O
3
TAK-637 (4)
N
R1
R4
F3C
R3
N
CF3
6a,b
R2
O
O
Scheme 1. Reagents: (i) 2,5-Dimethoxytetrahydrofuran, CH3COOH; (ii) ClCO-
COOC2H5, C6H6; (iii) NaH, DMF; (iv) 3,5-(CF3)2C6H3CH2NH2, HOBt, DCC, CH2Cl2 or
3,5-(CF3)2C6H3CH2N(H)CH3ÁHCl, TEA, HOBt, DCC, CH2Cl2.
N
R1
N
R1
N
R2
N
X
Z
Y
6
5
Figure 1. Structures of compounds 1–6.
2. Results and discussion
2.1. Chemistry
The synthesis of pyrrolo[1,2-a]quinoline derivatives 6a,b was
accomplished starting from commercially available 2-benzylani-
line 7 as reported in Scheme 1.
Compound 7 was subjected to Clauson–Kaas reaction, with 2,5-
dimethoxytetrahydrofuran in glacial acetic acid to give intermedi-
ate 8 which reacted with ethyl oxalylchloride to give 9. This inter-
mediate was then cyclized in the presence of sodium hydride as
the base to obtain the carboxylic acid derivate 10 instead of the ex-
pected ester. Reaction between 10 and the appropriate amines in
the presence of dicyclohexylcarbodiimide (DCC) and 1-hydroxy-
benzotriazole (HOBt) afforded the target compounds 6a,b. The
structure of pyrrolo[1,2-a]quinoline derivative 6a was confirmed
by X-ray crystallography (Fig. 2).
Figure 2. Crystal structure of compound 6a. Ellipsoids enclose 50% probability.
2 M NaOH to give acid 20. The target amides 6e,f were prepared
from acid 20 by means of the same procedure described for the
corresponding pyrrolo[1,2-a]quinoline amides 6a,b.
The procedure for the preparation of imidazo[1,2-a]quinoline
amides 6c,d is shown in Scheme 2. Chloroquinoline derivate 1111
was reacted with 2-aminoethanol to obtain 12, which was cyclized
in phosphorus oxychloride to give the dihydroderivative 13. The
heating of 13 in DMSO at 120 °C gave ester 14, which was hydro-
lyzed with 2 M NaOH to give acid 15. Target amides 6c,d were pre-
pared from acid 15 by means of the same procedure described for
the corresponding pyrrolo[1,2-a]quinoline amides 6a,b (Fig. 3).
A different approach to the synthesis of imidazo[1,5-a]quinoline
amides 6e,f is reported in Scheme 3. Chloroquinoline derivate 1611
was reacted with tert-butyl isocyanoacetate in the presence of
potassium tert-butoxide to obtain diester 17, which was cleaved
with formic acid to 18. Acid 18 was decarboxylated under nitrogen
at 200 °C to give monoester 19, which was in turn hydrolyzed with
The synthesis of [1,2,4]triazolo[4,3-a]quinoline derivates 6g,h
was carried out starting from acid 2111 as shown in Scheme 4. Acid
21 was converted into the corresponding amides 22 and 23,12
which reacted with phosphorus oxychloride to give chloroderi-
vates 24 and 25.12 The reaction of 24 and 25 with hydrazine hy-
drate gave hydrazinoderivatives 26 and 27, respectively, which
were cyclized with formic acid to obtain the target compounds
6g,h.
The synthesis of tetrazolo[1,5-a]quinoline-4-carboxamides 6i–
p is described in Scheme 5. The preparations of 2-chloroquino-
line derivative 16,11 4-(4-methylphenyl)quinoline derivative
28,13 and 4-(4-fluorophenyl)quinoline derivative 2914 were per-
formed by means of procedures described in the literature. The
reaction of 2-chloroquinoline derivates 16, 28, 29 with sodium

6852
A. Cappelli et al. / Bioorg. Med. Chem. 16 (2008) 6850–6859
COOC3H7
Cl
COOC2H5
COOC3H7
COOC2H5
COOC(CH3)3
i
i
OH
N
N
Cl
N
H
N
N
N
11
16
12
17
ii
ii
COOC3H7
COOC3H7
COOC2H5
COOH
COOC2H5
iii
iii
N
N
N
N
N
N
N
N
14
19
13
18
iv
iv
F3C
F3C
O
CF3
CF3
O
COOH
COOH
v
N
v
N
R1
R1
N
N
N
N
N
N
N
N
15
20
6c,d
6e,f
Scheme 2. Reagents: (i) HOCH2CH2NH2, C2H5OH; (ii) POCl3; (iii)
NaOH, C2H5OH; (v) 3,5-(CF3)2C6H3CH2NH2, HOBt, DCC, CH2Cl2 or 3,5-
D
, DMSO; (iv)
Scheme 3. Reagents: (i) tert-Butyl isocyanoacetate, t-BuOK, DMF; (ii) HCOOH; (iii)
, no-solvent (neat), N2; (iv) NaOH, C2H5OH; (v) 3,5-(CF3)2C6H3CH2NH2, HOBt, DCC,
D
(CF3)2C6H3CH2N(H)CH3ÁHCl, HOBt, DCC, TEA, CH2Cl2.
CH2Cl2 or 3,5-(CF3)2C6H3CH2N(H)CH3ÁHCl, HOBt, DCC, TEA, CH2Cl2.
F3C
CF3
O
COOH
i
N
R1
N
O
N
H
O
H
21
22,23
ii
F3C
F3C
CF3
CF3
O
O
N
iii
N
R1
R1
NH
N
Cl
N
H2N
26,27
iv
24,25
F3C
CF3
Figure 3. Mulliken charge distribution in the isolated tricyclic systems calculated at
B3LYP/6-31+G** level of theory using Gaussian 03 package. The code-color scheme
is also reported.
O
N
R1
N
N
N
azide gave the key intermediates 30, 31, 32 (Scheme 5) which
were hydrolyzed to obtain the required carboxylic acids 33, 34,
35. The activation of these acids with thionyl chloride followed
by the reaction with the suitable amines gave tetrazolo[1,5-
a]quinoline-4-carboxamides 6i–p.
6g,h
Scheme 4. Reagents: (i) a—SOCl2, CH2Cl2; b—3,5-(CF3)2C6H3CH2NH2, TEA, CH2Cl2 or
3,5-(CF3)2C6H3CH2N(H)CH3ÁHCl, TEA, CH2Cl2 (ii) POCl3, CH2Cl2; (iii) NH2NH2ÁH2O,
C2H5OH; (iv) HCOOH.

A. Cappelli et al. / Bioorg. Med. Chem. 16 (2008) 6850–6859
6853
cyclic amide derivatives with NK1 receptor. Indeed, the 3,5-bistri-
fluoromethylbenzyl group is confirmed to be an optimal
substituent from the point of view of the binding potency (com-
pare 6i with 6o,p) and the amide methyl group of tertiary amides
6b,d,f,h,j,l,n enhances the affinity of the secondary amides 6a,c,e,g,
i,k,m to a variable extent (from 7 to 60 times).
R4
R4
COOC2H5
COOC2H5
Cl
i
The most remarkable result of the present structure–affinity
relationship study is that the effects of the variation of the fused
azole moiety are significant (in the range of one order of magni-
tude) in both the sub-micromolar affinity secondary amides
6a,c,e,g,i,k,m, and in the series of the nanomolar affinity tertiary
amide ligands 6b,d,f,h,j,l,n. Owing to the evident similarity in the
steric features of the different tricyclic systems, this result suggests
a role of the electron distribution in the observed affinity modula-
tion. In order to evaluate this hypothesis, Mulliken charges were
calculated at B3LYP/6-31+G** level of theory in the models of the
tricyclic systems. The results obtained show a different charge dis-
tribution as an effect related to both the different position and the
number of nitrogen atoms present in the fused azole moiety. In
particular, the presence of a single (quinoline) nitrogen atom in
the simplified model of 6a,b tricyclic system leads to a uniform
electron distribution in the azole portion. The introduction of addi-
tional nitrogen atoms produces the formation of more pronounced
localized dipoles within the tricyclic system, which may find suit-
able counterparts for interaction in the binding site. In particular,
the comparison of partial charge distribution in the azole moieties
of tertiary amides 6b,d,f,h,j supports the important presence of a
nitrogen atom in position 2 of the tricyclic system (compound
6f). In fact, when the additional nitrogen is present in position 3
as in compound 6d, the affinity is about one order of magnitude
lower with respect to 6f and similar to that shown by 6b. More-
over, when two additional nitrogen atoms are present in positions
2 and 3 of 6h tricyclic system, the NK1 receptor affinity is just
slightly lower with respect to 6f.
N
N
N
N
N
16,28,29
30,31,32
ii
R4
R4
R3
R2
O
COOH
iii
N
R1
N
N
N
N
N
N
N
N
33,34,35
6i-p
Scheme 5. Reagents: (i) NaN3, DMF; (ii) NaOH, C2H5OH; (iii) a—SOCl2, CH2Cl2; b—
R2R3C6H3CH2N(H)R1, TEA, CH2Cl2.
2.2. Binding studies
Compounds 6 were assayed for their activity in inhibiting the
specific binding of [125I]BH-SP to human recombinant NK1 receptor
stably expressed in CHO cells.15 The results of the binding studies,
expressed as IC50 values in Table 1, reveal that compounds 6a–p
show NK1 receptor affinities in the nanomolar range to the micro-
molar one and the affinity modulation is affected by both the
amide substituents and the azole moiety fused at a-edge of the
quinoline nucleus.
In agreement with the structure–affinity relationships de-
scribed in the literature,6,7,12 the amide substituents of compounds
6 appear to play a fundamental role in the interaction of these tri-
Finally, the presence of a nitrogen atom in position 3 of the tri-
cyclic nucleus is assumed to affect the conformational preferences
Table 1
Binding affinities of azole-fused quinolinecarboxamide derivatives 6 for human recombinant NK1 receptor stably expressed in CHO cells and chemical shift values of NH signal in
the 1H NMR spectra of secondary amides 6a,c,e,g,i
R4
R3
R2
O
X
N
R1
N
Z
Y
6
Compound
X–Y–Z
R1
R2
R3
R4
IC50 (nM)
NH chemical shift (ppm)
5.79
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
SP
@CH–CH@CH–
@CH–CH@CH–
@N–CH@CH–
@N–CH@CH–
@CH–N@CH–
@CH–N@CH–
@N–N@CH–
@N–N@CH–
@N–N@N–
@N–N@N–
@N–N@N–
@N–N@N–
@N–N@N–
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
2-OCH3
3-OCH3
5-CF3
5-CF3
5-CF3
5-CF3
5-CF3
5-CF3
5-CF3
5-CF3
5-CF3
5-CF3
5-CF3
5-CF3
5-CF3
5-CF3
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
F
F
H
H
390 47
36 7.5
1233 335
38 12
146 48
4.3 1.6
542 37
9.0 4.0
311 52
26 3.0
401 31
55 8.0
235 9.0
21 8.2
2749 329
780 145
1.5 0.1
10.12
5.90
9.82
9.09
@N–N@N–
@N–N@N–
@N–N@N–
H
5-OCH3

6854
A. Cappelli et al. / Bioorg. Med. Chem. 16 (2008) 6850–6859
of secondary amides 6c,g,i. This assumption appears to be sup-
ported by the chemical shift value (CDCl3 as the solvent, Table 1)
of the 1H NMR signal attributed to NH of secondary amides bearing
a nitrogen atom in position 3 of the tricyclic nucleus. Therefore, the
presence and the intensity of this intramolecular interaction may
play a role in the affinity modulation of secondary amides 6c,g,i.
On the other hand, the replacement of the hydrogen atom in
para-position of the pendent phenyl group with a methyl group
or a fluorine atom has negligible effects on the receptor affinity
(compare 6i with 6k,m and 6j with 6l,n).
matography with n-hexane/ethyl acetate (8:2) as the eluent gave
compound 8 (0.32 g) in 84% yield. 1H NMR (CDCl3): 3.93 (s, 2H),
6.35 (t, J = 1.8, 2H), 6.79 (t, J = 1.9, 2H), 7.07 (m, 2H), 7.22–7.35
(m, 7H).
4.1.2. Ethyl 2-[1-(2-Benzylphenyl)-1H-pyrrol-2-yl]-2-
oxoacetate (9)
To a solution of 8 (0.15 g, 0.65 mmol) in benzene (15 mL) was
added ethyl oxalylchloride (0.22 mL, 1.97 mmol), and the resulting
mixture was refluxed for 4 h. After concentration under reduced
pressure, the reaction mixture was diluted with ethyl acetate,
washed with saturated NaHCO3 solution, dried over sodium sul-
fate, and concentrated under reduced pressure. Purification of the
residue by flash chromatography with n-hexane/ethyl acetate
(8:2) as the eluent gave the pure product 9 (0.15 g, yield 69%). 1H
NMR (CDCl3): 1.38 (t, J = 7.0, 3H), 3.63 (d, J = 15.6, 1H), 3.80 (d,
J = 15.6, 1H), 4.36 (q, J = 7.0, 2H), 6.34 (m, 1H), 6.83 (m, 1H), 6.98
(m, 2H), 7.11–7.41 (m, 7H), 7.47 (m, 1H). MS(ESI): m/z 334 (M+H+).
3. Conclusions
A small series of 3-quinolinecarboxamides bearing different
five-membered heterocycles condensed at the a-edge of the quin-
oline nucleus were designed, synthesized, and evaluated for their
potential ability to inhibit the specific binding of [125I]BH-SP to re-
combinant human NK1 receptor in order to obtain further informa-
tion on the structural prerequisites for the interaction of NK1
binding site with this apparently central portion of ligands 5. The
structure–affinity relationship study confirms the importance of
the amide substituents in the interaction with the receptor and,
remarkably, reveals a clear modulating effect of the fused azole
moiety in both secondary and tertiary amide ligands. The results
of Mulliken charges calculations performed at B3LYP/6-31+G** le-
vel of theory with models of the different tricyclic systems support
the role of charge distribution in the interaction of tertiary amides
6b,d,f,h,j with the receptor. Moreover, the presence and the inten-
sity of an intramolecular H-bond interaction in the secondary
amides bearing a nitrogen atom in position 3 of the tricyclic system
may play an additional role in the affinity modulation within the
secondary amide derivatives.
4.1.3. 5-Phenylpyrrolo[1,2-a]quinoline-4-carboxylic acid (10)
To a solution of 9 (0.80 g, 2.4 mmol) in anhydrous DMF (8 mL)
was added NaH (0.115 g, 4.8 mmol), and the resulting mixture
was heated for 3 h at 120 °C under argon. The solvent was then re-
moved under reduced pressure, the resulting mixture was diluted
with ethyl acetate and washed with water. The organic layer was
dried over sodium sulfate and concentrated under reduced pres-
sure. Purification of the residue by flash chromatography with
ethyl acetate as the eluent gave pure acid 10 as a yellow oil which
crystallized on standing (0.50 g, yield 73%, mp 168–169 °C). 1H
NMR (CDCl3): 6.85 (m, 2H), 7.18–7.61 (m, 8H), 7.95 (m, 2H). MS(E-
SI, negative ions): m/z 286 (MÀH+).
4.1.4. N-[3,5-Bis(trifluoromethyl)benzyl]-5-phenylpyrrolo[1,2-
a]quinoline-4-carboxamide (6a)
Finally, the discrepancies observed in the structure–affinity
relationships between compounds 6 and 59 warrant further inves-
tigations (i.e., the evaluation of previously published 3-quinoline-
To an ice-cooled mixture of 10 (0.115 g, 0.40 mmol) and 3,5-
bis(trifluoromethyl)benzylamine (0.105 g, 0.43 mmol) in dry
dichloromethane (20 mL) was added HOBt (0.058 g, 0.43 mmol),
and the resulting mixture was stirred at 0–5 °C for 10 min. After-
wards, a solution of DCC (0.112 g, 0.54 mmol) in the same solvent
(10 mL) was added dropwise. The reaction mixture was stirred
overnight at room temperature, the precipitate was filtered-off,
and the filtrate was washed in sequence with water and brine,
dried, and concentrated under reduced pressure. The residue was
subjected to flash chromatography on silica gel with dichlorometh-
ane as the eluent to give 6a (0.14 g, 68%). An analytical sample
crystallized from diethyl ether melted at 202–203 °C. 1H NMR
(CDCl3): 4.40 (d, J = 6.2, 2H), 5.79 (t, J = 6.0, 1H), 6.70 (m, 1H),
6.85 (m, 1H), 7.17–7.58 (m, 10H), 7.76 (s, 1H), 7.92 (m, 2H). MS(E-
SI): m/z 513 (M+H+). Anal. (C28H18F6N2O) C,H,N.
carboxamide derivatives
5 in the human recombinant NK1
receptor expressed in CHO cells). Therefore, further studies are
now in progress in order to obtain information about the interac-
tion of these heterocyclic amide derivatives with NK1 receptor.
4. Experimental
4.1. Chemistry
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. Microanalyses were carried out by means of a Per-
kin-Elmer 240C or a Perkin-Elmer Series II CHNS/O Analyzer
2400. Merck silica gel 60 (230–400 mesh) was used for column
chromatography. Merck TLC plates, silica gel 60 F254 were used
for TLC. 1H NMR spectra were recorded with a Bruker AC 200 spec-
trometer 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. Mass spectra were recorded on either a
Varian Saturn 3 spectrometer or a ThermoFinnigan LCQ-Deca.
4.1.5. N-[3,5-Bis(trifluoromethyl)benzyl]-N-methyl-5-
phenylpyrrolo[1,2-a]quinoline-4-carboxamide (6b)
This compound was prepared from 10 (0.50 g, 1.74 mmol),
15 mL of dry dichloromethane, HOBt (0.27 g, 2.0 mmol), DCC
(0.54 g, 2.6 mmol), 0.50 mL of TEA, and N-[3,5-bis(trifluoro-
methyl)benzyl]methylamine hydrochloride (0.59 g, 2.0 mmol) by
the same procedure described for the synthesis of 6a. The mixture
was purified by flash chromatography with CH2Cl2 as the eluent to
obtain 6b (0.58 g, yield 63%, melting point 161 °C). The 1H NMR
spectrum of this amide shows the presence of two different rota-
mers in equilibrium. For the sake of simplification the integral
intensities have not been given. 1H NMR (CDCl3): 2.71 (s), 2.74
(s), 3.93 (d, J = 15.7), 4.46 (d, J = 14.8), 4.65 (d, J = 15.7), 4.79 (d,
J = 14.8), 6.46 (m), 6.59 (m), 6.85 (m), 7.19–7.70 (m), 7.79 (s),
7.94 (m). MS(ESI): m/z 527 (M+H+). Anal. (C29H20F6N2O) C,H,N.
4.1.1. 1-(2-Benzylphenyl)-1H-pyrrole (8)
A mixture of 2-aminodiphenyl (7, 0.30 g, 1.64 mmol) in glacial
acetic acid (15 mL) with 2,5-dimethoxytetrahydrofuran (0.26 mL,
1.96 mmol) was stirred at 80 °C for 2 h. The solvent was then re-
moved under reduced pressure and the residue was diluted with
ethyl acetate. The mixture was washed with brine and water, the
organic layer was dried over sodium sulfate and concentrated
under reduced pressure. Purification of the residue by flash chro-

A. Cappelli et al. / Bioorg. Med. Chem. 16 (2008) 6850–6859
6855
4.1.6. Propyl 2-(2-hydroxyethylamino)-4-phenylquinoline-3-
carboxylate (12)
4.1.11. N-[3,5-Bis(trifluoromethyl)benzyl]-N-methyl-5-
phenylimidazo[1,2-a]quinoline-4-carboxamide (6d)
A
mixture of 11 (0.40 g, 1.23 mmol) in absolute ethanol
This compound was prepared from 15 (0.29 g, 1.0 mmol), 20 mL
(15 mL) with 2-aminoethanol (0.30 mL, 4.97 mmol) was refluxed
for 15 h. The solvent was then removed under reduced pressure
and the residue was diluted with ethyl acetate. The mixture was
washed with water, the organic layer was dried over sodium sul-
fate and concentrated under reduced pressure. Purification of the
residue by flash chromatography with petroleum ether/ethyl
acetate (6:4) as the eluent gave compound 12 as an orange oil,
which crystallized on standing (0.32 g, yield 74%, mp 74–
75 °C). 1H NMR (CDCl3): 0.59 (t, J = 7.5, 3H), 0.96–1.14 (m, 2H),
3.71–3.91 (m, 6H), 6.10 (br s, 1H), 7.02–7.29 (m, 4H), 7.39–
7.54 (m, 4H), 7.65 (d, J = 8.4, 1H). MS(ESI): m/z 351 (M+H+).
of dry dichloromethane, HOBt (0.20 g, 1.5 mmol), DCC (0.31 g,
1.5 mmol),
0.40 mL
of
TEA
and
N-[3,5-bis(trifluoro-
methyl)benzyl]methylamine hydrochloride (0.44 g, 1.5 mmol) by
the same procedure described for the synthesis of 6a. The crude
product was purified by flash chromatography with CHCl3/ethyl
acetate (7:3) as the eluent to obtain 6d as a white solid (0.41 g,
yield 78%). An analytical sample melted at 206 °C. The 1H NMR
spectrum of this amide shows the presence of two different rota-
mers in equilibrium. For the sake of simplification the integral
intensities have not been given. 1H NMR (CDCl3): 2.78 (s), 2.80
(s), 3.93 (d, J = 15.6), 4.49 (d, J = 15.2), 4.70 (d, J = 15.7), 5.08 (d,
J = 15.1), 7.21–7.75 (m), 7.98 (m), 8.11 (m). MS(ESI): m/z 528
(M+H+). Anal. (C28H19F6N3O) C,H,N.
4.1.7. Propyl 1,2-dihydro-5-phenylimidazo[1,2-a]quinoline-4-
carboxylate (13)
A mixture of 12 (0.18 g, 0.51 mmol) in phosphorous oxychloride
(10 mL) was refluxed for 6 h. The excess of POCl3 was then decom-
posed with ice-water, and the precipitate was extracted with
dichloromethane. The organic phase was dried over sodium sulfate
and concentrated under reduced pressure, to give compound 13 as
a yellow solid (0.12 g, yield 71%, melting point 158 °C). 1H NMR
(CDCl3): 0.66 (t, J = 7.1, 3H), 1.28 (m, 2H), 3.88–4.16 (m, 6H),
6.72–6.87 (m, 2H), 7.01 (d, J = 7.3, 1H), 7.26–7.40 (m, 6H). MS(ESI):
m/z 333 (M+H+).
4.1.12. 3-(tert-Butyl) 4-ethyl 5-phenylimidazo[1,5-a]quinoline-
3,4-dicarboxylate (17)
To an ice-cooled solution of 16 (0.60 g, 1.9 mmol) in anhydrous
DMF (15 mL) were added tert-butyl isocyanoacetate (0.76 g,
5.38 mmol) and potassium tert-butoxide (0.65 g, 5.76 mmol), and
the resulting mixture was stirred at 0–5 °C for 30 min. Afterwards,
the solution was stirred at room temperature for 30 min and then
heated at 80 °C for 10 h. The final mixture was neutralized with
glacial acetic acid, poured into crushed ice and extracted with ethyl
acetate. The combined extract was dried over sodium sulfate and
concentrated under reduced pressure. The residue was purified
by washing with diethyl ether to give 0.65 g of 17 (yield 81%). An
analytical sample melted at 159–161 °C. 1H NMR (CDCl3): 1.02 (t,
J = 7.0, 3H), 1.63 (s, 9H), 4.12 (q, J = 7.0, 2H), 7.33–7.48 (m, 7H),
7.66 (m, 1H), 8.07 (d, J = 8.3, 1H), 8.68 (s, 1H). MS(ESI): m/z 439
(M+Na+).
4.1.8. Propyl 5-phenylimidazo[1,2-a]quinoline-4-carboxylate
(14)
Compound 13 (0.30 g, 0.90 mmol) was dissolved in DMSO
(10 mL) and the resulting mixture was heated at 120 °C for
48 h. The mixture was diluted with water and extracted with
dichloromethane, the organic phase was dried over sodium sul-
fate, and concentrated under reduced pressure. Purification of
the residue by flash chromatography with CH2Cl2/ethyl acetate
(7:3) as the eluent gave 14 as an off-white solid (0.19 g, 64%,
melting point 117 °C). 1H NMR (CDCl3): 0.71 (t, J = 7.4, 3H),
1.36 (m, 2H), 4.06 (t, J = 6.8, 2H), 7.31–7.69 (m, 8H), 7.71 (s,
1H), 7.94 (d, J = 8.4, 1H), 8.08 (s, 1H). MS(ESI): m/z 331 (M+H+).
4.1.13. 4-(Ethoxycarbonyl)-5-phenylimidazo[1,5-a]quinoline-3-
carboxylic acid (18)
A mixture of compound 17 (0.60 g, 1.44 mmol) in formic acid
(15 mL) was heated at 50 °C for 3 h. The reaction mixture was then
concentrated under reduced pressure while the remaining formic
acid was azeotropically removed with toluene. The residue was
washed with ethanol to give 0.50 g of 18 as an off-white solid
(yield 96%). An analytical sample melted at 245–247 °C. 1H NMR
(DMSO-d6): 0.87 (t, J = 6.8, 3H), 3.91 (q, J = 6.9, 2H), 7.04–7.83 (m,
8H), 8.59 (d, J = 8.3, 1H), 9.36 (s, 1H), 12.38 (s, 1H). MS(ESI, negative
ions): m/z 359 (MÀH+).
4.1.9. 5-Phenylimidazo[1,2-a]quinoline-4-carboxylic acid (15)
A mixture of 14 (0.14 g, 0.42 mmol) in ethanol (10 mL) with
2 N NaOH (4.0 mL) was refluxed for 2 h. The solvent was then
removed under reduced pressure, and the residue was diluted
with water and acidified with 2 N HCl (pH 6). The precipitate
was collected by filtration, washed with diethyl ether, and dried
under reduced pressure to give 0.10 g of 15 as a white solid
melting at 223 °C (yield 83%). 1H NMR (DMSO-d6): 7.35–7.62
(m, 7H), 7.86–7.93 (m, 2H), 8.57 (d, J = 8.4, 1H), 8.96 (s, 1H).
MS(ESI): m/z 289 (M+H+).
4.1.14. Ethyl 5-phenylimidazo[1,5-a]quinoline-4-carboxylate
(19)
Compound 18 (0.20 g, 0.55 mmol) was heated without solvent
at 190–200 °C until the end of CO2 bubbling. The cooled residue
was dissolved in ethyl acetate (30 mL), washed with water, dried
over sodium sulfate, and concentrated under reduced pressure.
Purification of the residue by flash chromatography with ethyl ace-
tate as the eluent gave 19 as a yellow solid (0.075 g, 43%, melting
point 113–114 °C). 1H NMR (CDCl3): 0.95 (t, J = 6.9, 3H), 4.09 (q,
J = 6.9, 2H), 7.27–7.48 (m, 7H), 7.59 (m, 1H), 7.72 (s, 1H), 8.01
(d, J = 8.1, 1H), 8.69 (s, 1H). MS(ESI): m/z 317 (M+H+).
4.1.10. N-[3,5-Bis(trifluoromethyl)benzyl]-5-
phenylimidazo[1,2-a]quinoline-4-carboxamide (6c)
This compound was prepared from 15 (0.10 g, 0.35 mmol),
10 mL of dry dichloromethane, HOBt (0.057 g, 0.42 mmol), DCC
(0.11 g, 0.53 mmol), and 3,5-bis(trifluoromethyl)benzylamine
(0.13 g, 0.53 mmol) by the same procedure described for the
synthesis of 6a. The mixture was purified by flash chromatogra-
phy with CHCl3/ethyl acetate (7:3) as the eluent to give 6c as a
white solid (0.11 g, 61%, melting point 208 °C). 1H NMR (CDCl3):
4.74 (d, J = 5.9, 2H), 7.28–7.51 (m, 7H), 7.68–7.74 (m, 3H), 7.82
(s, 2H), 7.98 (d, J = 8.3, 1H), 8.15 (s, 1H), 10.12 (t, J = 6.0, 1H).
MS(ESI): m/z 514 (M+H+). Anal. (C27H17F6N3O) C,H,N.
4.1.15. 5-Phenylimidazo[1,5-a]quinoline-4-carboxylic acid (20)
A mixture of 19 (0.20 g, 0.63 mmol) in ethanol (8.0 mL) with 2 N
NaOH (4.0 mL) was refluxed for 2 h. The reaction mixture was acid-
ified with 2 N HCl (pH 6), and the solvent was removed under re-
duced pressure. The residue was washed with ethanol, and the
organic solution was evaporated under reduced pressure to give
0.12 g of 20 as a brown solid (yield 66%, mp > 300 °C). 1H NMR

6856
A. Cappelli et al. / Bioorg. Med. Chem. 16 (2008) 6850–6859
(DMSO-d6): 7.18–7.54 (m, 9H), 8.34 (d, J = 8.0, 1H), 9.02 (s, 1H).
MS(ESI): m/z 289 (M+H+).
(0.60 g, yield 63%). Melting point 263–264 °C (literature12 mp 262–
264 °C). The 1H NMR spectrum of this amide shows the presence of
two different rotamers in equilibrium. For the sake of simplifica-
tion the integral intensities have not been given. 1H NMR (CDCl3):
2.73 (s), 2.87 (s), 4.08 (d, J = 16.2), 4.60 (d, J = 15.2), 4.70–4.82 (m),
7.09–7.77 (m), 12.43 (br s). MS(ESI): m/z 527 (M+Na+).
4.1.16. N-[3,5-Bis(trifluoromethyl)benzyl]-5-
phenylimidazo[1,5-a]quinoline-4-carboxamide (6e)
A solution of HOBt (0.058 g, 0.43 mmol) in dry dichloromethane
(10 mL) was added to an ice-cooled solution of 20 (0.105 g,
0.36 mmol) and 3,5-bis(trifluoromethyl)benzylamine (0.105 g,
0.43 mmol) in the same solvent (10 mL). After stirring at 0–5 °C
for 10 min, a solution of DCC (0.112 g, 0.54 mmol) in dichlorometh-
ane (10 mL) was added dropwise, and the resulting mixture was
stirred at room temperature for 20 h. The precipitate was fil-
tered-off and the filtrate was washed in sequence with water and
brine, then dried, and evaporated under reduced pressure. The res-
idue was purified by column chromatography with n-hexane/ethyl
acetate (2:8) as the eluent to give 6e as a white solid (0.12 g, yield
65%). An analytical sample melted at 191 °C. 1H NMR (CDCl3): 4.43
(d, J = 6.0, 2H), 5.90 (t, J = 5.9, 1H), 7.33 (m, 7H), 7.50 (s, 2H), 7.61
(m, 1H), 7.68 (s, 1H), 7.77 (s, 1H), 8.02 (d, J = 8.1, 1H), 8.78 (s,
1H). MS(ESI): m/z 514 (M+H+). Anal. (C27H17F6N3O) C,H,N.
4.1.20. N-[3,5-Bis(trifluoromethyl)benzyl]-2-chloro-4-
phenylquinoline-3-carboxamide (24)
A mixture of 22 (0.10 g, 0.20 mmol) in phosphorus oxychloride
(10 mL) was refluxed for 4 h. The excess of POCl3 was decomposed
with ice-water, and the precipitate was extracted with dichloro-
methane. The combined extracts were dried over sodium sulfate
and concentrated under reduced pressure to give a crude product,
which after purification by flash chromatography CH2Cl2/ethyl ace-
tate (9:1) afforded 24 as a white solid (0.085 g, yield 84%, mp
172 °C). 1H NMR (CDCl3): 4.41 (d, J = 6.0, 2H), 6.10 (t, J = 5.9, 1H),
7.26–7.53 (m, 9H), 7.73 (m, 2H), 8.01 (d, J = 8.4, 1H). MS(ESI): m/
z 509 (M+H+).
4.1.21. N-[3,5-Bis(trifluoromethyl)benzyl]-2-chloro-N-methyl-
4-phenylquinoline-3-carboxamide (25)
4.1.17. N-[3,5-Bis(trifluoromethyl)benzyl]-N-methyl-5-
phenylimidazo[1,5-a]quinoline-4-carboxamide (6f)
This compound was prepared from 23 (0.56 g, 1.1 mmol) and
phosphorus oxychloride (10 mL) by the same procedure described
for the synthesis of 20. It was purified by flash chromatography
with n-hexane/ethyl acetate (8:2) as the eluent to obtain 25
(0.51 g, yield 89%); melting point 148 °C (literature12 mp 147–
148 °C). The 1H NMR spectrum of this amide shows the presence
of two different rotamers in equilibrium. For the sake of simplifica-
tion the integral intensities have not been given. 1H NMR (CDCl3):
2.70 (s), 2.75 (s), 4.04 (d, J = 15.9), 4.48 (d, J = 15.6), 4.53 (d,
J = 14.9), 4.69 (d, J = 14.9), 7.21 (m), 7.34–7.65 (m), 7.77 (m), 8.09
(d, J = 8.4). MS(ESI): m/z 523 (M+H+).
This compound was prepared from 20 (0.20 g, 0.69 mmol),
15 mL of dry dichloromethane, HOBt (0.113 g, 0.83 mmol), DCC
(0.215 g, 1.04 mmol), 0.5 mL of TEA and N-[3,5-bis(trifluoro-
methyl)benzyl]methylamine hydrochloride (0.305 g, 1.04 mmol)
by means of the same procedure described for the synthesis of
6e. The crude product was purified by flash chromatography with
n-hexane/ethyl acetate (3:7) as the eluent to obtain 6f (0.22 g, yield
60%, melting point 175 °C). The 1H NMR spectrum of this amide
shows the presence of two different rotamers in equilibrium. For
the sake of simplification the integral intensities have not been gi-
ven. 1H NMR (CDCl3): 2.73 (s), 2.77 (s), 4.09 (d, J = 16.0), 4.32 (d,
J = 14.7), 4.57 (d, J = 16.0), 4.95 (d, J = 14.7), 7.20–7.71 (m), 7.79
(s), 8.03 (d, J = 8.3), 8.72 (s). MS(ESI): m/z 528 (M+H+). Anal.
(C28H19F6N3O) C,H,N.
4.1.22. N-[3,5-Bis(trifluoromethyl)benzyl]-2-hydrazino-4-
phenylquinoline-3-carboxamide (26)
To a solution of 24 (0.48 g, 0.94 mmol) in ethanol (15 mL) was
added an excess of hydrazine hydrate (200 lL, 4.1 mmol), and
4.1.18. N-[3,5-Bis(trifluoromethyl)benzyl]-1,2-dihydro-2-oxo-4-
phenylquinoline-3-carboxamide (22)
the resulting mixture was refluxed for 3 h. The solvent was then re-
moved under reduced pressure and the residue was diluted with
CH2Cl2 (20 mL); the organic phase was washed with water, dried
over sodium sulfate, and concentrated under reduced pressure to
obtain 26 as a brown solid (0.40 g, 84%, mp 163 °C). 1H NMR
(CDCl3): 4.30 (d, J = 6.0, 2H), 5.67 (t, J = 6.0, 1H), 7.11–7.61 (m,
10H), 7.77 (m, 2H). MS(ESI): m/z 505 (M+H+).
A mixture of acid 21 (0.10 g, 0.38 mmol) in thionyl chloride
(5 mL) was refluxed for 3 h. The thionyl chloride excess was azeo-
tropically removed with toluene and the residue was immediately
dissolved into dichloromethane (15 mL). To the resulting solution
3,5-bis(trifluoromethyl)benzylamine (0.14 g, 0.57 mmol) and
0.30 mL of TEA were added, and the reaction mixture was refluxed
for 4 h. The final mixture was diluted with CH2Cl2 (15 mL), washed
with water, dried over sodium sulfate, and concentrated under re-
duced pressure. The residue was purified by washing with diethyl
ether to give 0.125 g of pure 22 as a white solid (yield 67%). Melt-
ing point 251 °C (literature12 mp 251–252 °C). 1H NMR (CDCl3):
4.59 (d, J = 6.0, 2H), 7.09–7.54 (m, 10H), 7.71 (m, 2H), 8.29 (t,
J = 5.9, 1H), 11.30 (br s, 1H). MS(ESI): m/z 513 (M+Na+).
4.1.23. N-[3,5-Bis(trifluoromethyl)benzyl]-2-hydrazino-N-
methyl-4-phenylquinoline-3-carboxamide (27)
The title compound was prepared from 25 (0.48 g, 0.92 mmol),
15 mL of ethanol, and hydrazine hydrate (180 lL, 3.7 mmol) by the
same procedure described for the synthesis of 26 to obtain 27 as an
orange oil (0.40 g, yield 84%). The oil obtained was used in the next
step of the synthesis without purification. MS(ESI): m/z 519
(M+H+).
4.1.19. N-[3,5-Bis(trifluoromethyl)benzyl]-1,2-dihydro-N-
methyl-2-oxo-4-phenylquinoline-3-carboxamide (23)
4.1.24. N-[3,5-Bis(trifluoromethyl)benzyl]-5-
A mixture of acid 21 (0.50 g, 1.88 mmol) in thionyl chloride
(8 mL) was refluxed for 3 h. The thionyl chloride excess was azeo-
tropically removed with toluene and the residue was immediately
dissolved into dichloromethane (20 mL). To the resulting solution
phenyl[1,2,4]triazolo[4,3-a]quinoline-4-carboxamide (6g)
A mixture of 26 (0.36 g, 0.71 mmol) in formic acid (20 mL) was
heated at 80 °C for 4 h. The formic acid excess was removed under
reduced pressure, and the residue was dissolved into CHCl3 and
washed with water. The organic layer was dried over sodium sul-
fate and concentrated under reduced pressure. Purification of the
residue by flash chromatography with ethyl acetate/TEA (9:1) as
the eluent gave pure 6g as a white solid (0.35 g, yield 96%). An ana-
lytical sample melted at 226 °C. 1H NMR (CDCl3): 4.73 (d, J = 6.0,
2H), 7.29 (m, 2H), 7.49 (m, 6H), 7.76 (m, 3H), 8.06 (d, J = 8.3, 1H),
3,5-bis(trifluoromethyl)benzylamine
hydrochloride
(0.60 g,
2.04 mmol) and 0.5 mL of TEA were added, and the reaction mix-
ture was refluxed for 4 h. The final mixture was diluted with
CH2Cl2 (20 mL), washed with water, dried over sodium sulfate,
and concentrated under reduced pressure. Crude compound 23
was purified by washing with diethyl ether to obtain a white solid

A. Cappelli et al. / Bioorg. Med. Chem. 16 (2008) 6850–6859
6857
9.36 (s, 1H), 9.82 (t, J = 5.8, 1H). MS(ESI): m/z 515 (M+H+). Anal.
(C26H16F6N4O) C,H,N.
1H), 7.74 (m, 1H), 8.00 (m, 1H), 8.69 (d, J = 8.4, 1H), 13.80 (br s, 1H).
MS(ESI): m/z 327 (M+Na+).
4.1.25. N-[3,5-Bis(trifluoromethyl)benzyl]-N-methyl-5-
4.1.31. 5-(4-Fluorophenyl)tetrazolo[1,5-a]quinoline-4-
carboxylic acid (35)
phenyl[1,2,4]triazolo[4,3-a]quinoline-4-carboxamide (6h)
This compound was prepared from 27 (0.36 g, 0.69 mmol),
15 mL of formic acid by the same procedure described for the syn-
thesis of 6g. It was purified by flash chromatography with ethyl
acetate/TEA (9:1) as the eluent to obtain 6h as a yellow oil which
crystallized from diethyl ether (0.51 g, yield 88%). An analytical
sample melted at 224 °C. The 1H NMR spectrum of this amide
shows the presence of two different rotamers in equilibrium. For
the sake of simplification the integral intensities have not been gi-
ven. 1H NMR (CDCl3): 2.85 (s), 4.29 (d, J = 15.2), 4.63 (d, J = 16.0),
5.27 (d, J = 15.0), 7.16–7.79 (m), 8.07 (m), 9.36 (s), 9.39 (s). MS(ESI):
m/z 529 (M+H+). Anal. (C27H18F6N4O) C,H,N.
The title compound was prepared from 32 (0.40 g, 1.19 mmol)
by the same procedure described for the synthesis of 33 to obtain
35 as a white solid (0.35 g, yield 95%). An analytical sample melted
at 267 °C. 1H NMR (DMSO-d6): 7.36–7.56 (m, 5H), 7.75 (m, 1H),
8.02 (m, 1H), 8.70 (d, J = 8.2, 1H). MS(ESI): m/z 331 (M+Na+).
4.1.32. N-[3,5-Bis(trifluoromethyl)benzyl]-5-
phenyltetrazolo[1,5-a]quinoline-4-carboxamide (6i)
The title compound was prepared from acid 33 (0.030 g,
0.10 mmol) and 3,5-bis(trifluoromethyl)benzylamine (0.050 g,
0.20 mmol) by the same procedure described for the synthesis of
compound 22, and 6i was obtained as a white solid (0.030 g, yield
58%, mp 193–195 °C). 1H NMR (CDCl3): 4.75 (d, J = 5.9, 2H), 7.31
(m, 2H), 7.51–7.69 (m, 6H), 7.75 (s, 2H), 7.96 (m, 1H), 8.79 (d,
J = 8.3, 1H), 9.09 (t, J = 6.0, 1H). MS(ESI): m/z 516 (M+H+). Anal.
(C25H15F6N5O) C,H,N.
4.1.26. Ethyl 5-phenyltetrazolo[1,5-a]quinoline-4-carboxylate
(30)
A mixture of 16 (0.80 g, 2.57 mmol) in anhydrous DMF (15 mL)
with sodium azide (0.33 g, 5.1 mmol) was heated at 80 °C for 36 h.
The solvent was then removed under reduced pressure and the res-
idue was diluted with ethyl acetate (20 mL). The organic phase was
washed with water, dried over sodium sulfate, and concentrated
under reduced pressure. The residue was purified by recrystalliza-
tion from diethyl ether to give 0.52 g of 30 as white solid (yield
64%, mp 154–156 °C). 1H NMR (CDCl3): 1.03 (t, J = 7.0, 3H), 4.21
(q, J = 7.0, 2H), 7.36–7.69 (m, 7H), 7.91 (m, 1H), 8.77 (d, J = 8.3,
1H). MS(ESI): m/z 319 (M+H+).
4.1.33. N-[3,5-Bis(trifluoromethyl)benzyl]-N-methyl-5-
phenyltetrazolo[1,5-a]quinoline-4-carboxamide (6j)
The title compound was prepared from acid 33 (0.050 g,
0.17 mmol) and N-[3,5-bis(trifluoromethyl)benzyl]methylamine
hydrochloride (0.10 g, 0.34 mmol) by the same procedure de-
scribed for the synthesis of compound 23, and 6j was obtained
as a white solid (0.071 g, yield 79%, mp 222–224 °C). The 1H
NMR spectrum of this amide shows the presence of two different
rotamers in equilibrium. For the sake of simplification the inte-
gral intensities have not been given. 1H NMR (CDCl3): 2.80 (s),
2.86 (s), 4.19 (d, J = 15.9), 4.35 (d, J = 15.0), 4.57 (d, J = 16.0),
5.19 (d, J = 15.0), 7.18–7.95 (m), 8.78 (m). MS(ESI): m/z 530
(M+H+). Anal. (C26H17F6N5O) C,H,N.
4.1.27. Ethyl 5-(4-methylphenyl)tetrazolo[1,5-a]quinoline-4-
carboxylate (31)
The title compound was prepared from 28 (0.70 g, 2.15 mmol)
by the same procedure described for the synthesis of 30 to obtain
31 (0.43 g, yield 60%) as a white solid melting at 177 °C. 1H NMR
(CDCl3): 1.08 (t, J = 6.9, 3H), 2.48 (s, 3H), 4.25 (q, J = 7.0, 2H), 7.32
(m, 4H), 7.59–7.74 (m, 2H), 7.92 (m, 1H), 8.78 (d, J = 8.4, 1H). MS(E-
SI): m/z 333 (M+H+).
4.1.34. N-[3,5-Bis(trifluoromethyl)benzyl]-5-(4-
methylphenyl)tetrazolo[1,5-a]quinoline-4-carboxamide (6k)
The title compound was prepared from acid 34 (0.090 g,
0.30 mmol) and 3,5-bis(trifluoromethyl)benzylamine (0.14 g,
0.58 mmol) by the same procedure described for the synthesis of
compound 22, and 6k was obtained as a white solid (0.12 g, yield
76%). An analytical sample of 6k melted at 227 °C. 1H NMR (CDCl3):
2.46 (s, 3H), 4.76 (d, J = 6.0, 2H), 7.18–7.35 (m, 4H), 7.62–7.77 (m,
5H), 7.94 (m, 1H), 8.75 (d, J = 8.3, 1H), 8.99 (t, J = 5.9, 1H). MS(ESI):
m/z 530 (M+H+). Anal. (C26H17F6N5O) C,H,N.
4.1.28. Ethyl 5-(4-fluorophenyl)tetrazolo[1,5-a]quinoline-4-
carboxylate (32)
The title compound was prepared from 29 (0.80 g, 2.43 mmol)
by the same procedure described for the synthesis of 30 to obtain
32 (0.70 g, yield 86%) as a white solid. An analytical sample melted
at 173 °C. 1H NMR (CDCl3): 1.09 (t, J = 7.0, 3H), 4.24 (q, J = 7.0, 2H),
7.19–7.42 (m, 4H), 7.64 (m, 2H), 7.92 (m, 1H), 8.76 (d, J = 8.2, 1H).
MS(ESI): m/z 337 (M+H+).
4.1.35. N-[3,5-Bis(trifluoromethyl)benzyl]-N-methyl-5-(4-
methylphenyl)tetrazolo[1,5-a]quinoline-4-carboxamide (6l)
The title compound was prepared from acid 34 (0.090 g,
0.30 mmol) and N-[3,5-bis(trifluoromethyl)benzyl]methylamine
hydrochloride (0.18 g, 0.61 mmol) by the same procedure de-
scribed for the synthesis of compound 23, and was obtained as
a white solid (0.13 g, yield 80%). An analytical sample of 6l
melted at 183 °C. The 1H NMR spectrum of this amide shows
the presence of two different rotamers in equilibrium. For the
sake of simplification the integral intensities have not been gi-
ven. 1H NMR (CDCl3): 2.41 (s), 2.49 (s), 2.82 (s), 2.89 (s), 4.21
(d, J = 16.0), 4.40 (d, J = 15.1), 4.57 (d, J = 15.9), 5.21 (d,
J = 15.1), 7.10 (m), 7.26 (m), 7.40–7.73 (m), 7.80 (s), 7.90 (m),
8.77 (m). MS(ESI): m/z 544 (M+H+). Anal. (C27H19F6N5O) C,H,N.
4.1.29. 5-Phenyltetrazolo[1,5-a]quinoline-4-carboxylic acid
(33)
A mixture of 30 (0.20 g, 0.63 mmol) in ethanol (10 mL) with 2 M
NaOH (4.0 mL) was refluxed for 3 h. The solvent was then removed
under reduced pressure, and the residue was diluted in water and
neutralized with 2 N HCl. The precipitate was collected by filtra-
tion, washed with n-hexane, and dried under reduced pressure to
give 0.17 g of 33 as a white solid (yield 93%). An analytical sample
melted at 256–258 °C. 1H NMR (DMSO-d6): 7.42–7.53 (m, 6H), 7.71
(m, 1H), 7.97 (m, 1H), 8.68 (d, J = 8.3, 1H), 13.81 (s, 1H). MS(ESI): m/
z 313 (M+Na+).
4.1.30. 5-(4-Methylphenyl)tetrazolo[1,5-a]quinoline-4-
carboxylic acid (34)
The title compound was prepared from 31 (0.13 g, 0.40 mmol)
by the same procedure described for the synthesis of 33 to obtain
34 as a white solid (0.10 g, yield 83%). An analytical sample melted
at 265 °C. 1H NMR (DMSO-d6): 2.40 (s, 3H), 7.34 (m, 4H), 7.57 (m,
4.1.36. N-[3,5-Bis(trifluoromethyl)benzyl]-5-(4-
fluorophenyl)tetrazolo[1,5-a]quinoline-4-carboxamide (6m)
The title compound was prepared from acid 35 (0.15 g,
0.49 mmol) and 3,5-bis(trifluoromethyl)benzylamine (0.24 g,

6858
A. Cappelli et al. / Bioorg. Med. Chem. 16 (2008) 6850–6859
0.99 mmol) by the same procedure described for the synthesis of
compound 22, and was obtained as a white solid (0.20 g, yield
77%). An analytical sample of 6m was purified by flash chroma-
tography with dichloromethane/ethyl acetate (9:1) as the eluent
(mp 232–233 °C). 1H NMR (CDCl3): 4.76 (d, J = 6.0, 2H), 7.26 (m,
4H), 7.55–7.78 (m, 5H), 7.96 (t, J = 7.4, 1H), 8.77 (d, J = 8.3, 1H),
9.21 (t, J = 6.0, 1H). MS(ESI): m/z 534 (M+H+). Anal.
(C25H14F7N5O) C,H,N.
4.3. In vitro binding assays
4.3.1. Ligands and reagents
Monoiodinated [125I]-Bolton-Hunter reagent labeled substance
P (2200 Ci/mmol) was purchased from Perkin-Elmer Life Science.
Unlabeled Substance P was purchased from Bachem. Cell culture
media, G418, and fetal calf serum were from Invitrogen.
4.3.2. Cell culture
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 Tech-
nologies, Inc.) at 37 °C in a humidified atmosphere of 5% CO2.
4.1.37. N-[3,5-Bis(trifluoromethyl)benzyl]-5-(4-fluorophenyl)-
N-methyltetrazolo[1,5-a]quinoline-4-carboxamide (6n)
The title compound was prepared from acid 35 (0.15 g,
0.49 mmol) and N-[3,5-bis(trifluoromethyl)benzyl]methylamine
hydrochloride (0.29 g, 0.99 mmol) by the same procedure de-
scribed for the synthesis of compound 23, and was obtained as
a white solid (0.19 g, yield 71%). An analytical sample of 6n
was purified by flash chromatography with dichloromethane/
ethyl acetate (9:1) as the eluent (mp 252 °C). The 1H NMR spec-
trum of this amide shows the presence of two different rotamers
in equilibrium. For the sake of simplification the integral inten-
sities have not been given. 1H NMR (CDCl3): 2.84 (s), 2.91 (s),
4.22 (d, J = 15.9), 4.42 (d, J = 15.0), 4.58 (d, J = 15.9), 5.16 (d,
J = 15.0), 7.07–7.36 (m), 7.53–7.76 (m), 7.82 (s), 7.93 (m), 8.80
(m). MS(ESI): m/z 570 (M+Na+). Anal. (C26H16F7N5O) C,H,N.
4.3.3. 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 (Geneti-
cin) after 3–6 weeks.
4.3.4. Radioreceptor binding assays
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 leu-
peptin, 0.1 mg/mL bacitracin, 0.1% (w/v) bovine serum albumin,
4.1.38. N-(2-Methoxybenzyl)-5-phenyltetrazolo[1,5-
a]quinoline-4-carboxamide (6o)
The title compound was prepared from acid 33 (0.20 g,
0.69 mmol) and 2-methoxybenzylamine (0.18 mL, 1.38 mmol) by
the same procedure described for the synthesis of compound 22,
and was obtained as a white solid (0.22 g, yield 78%). An analytical
sample of 6o melted at 222 °C. 1H NMR (CDCl3): 3.88 (s, 3H), 4.58
(d, J = 5.8, 2H), 6.86 (m, 2H), 7.18–7.32 (m, 4H), 7.45–7.59 (m, 5H),
7.89 (m, 1H), 8.47 (t, J = 5.9, 1H), 8.74 (d, J = 8.2, 1H). MS(ESI): m/z
410 (M+H+). Anal. (C24H19N5O2) C,H,N.
and 2–3 lg of membrane proteins from transfected CHO cells.
The 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-
tion (50 lL) of Microscint 20 (Packard) to each well. The data of the
4.1.39. N-(3,5-Dimethoxybenzyl)-5-phenyltetrazolo[1,5-
a]quinoline-4-carboxamide (6p)
dose–response experiments were analyzed by means of Allfit18
program to compute the IC50 values.
The title compound was prepared from acid 33 (0.20 g,
0.69 mmol) and 3,5-dimethoxybenzylamine (0.23 g, 1.38 mmol)
by the same procedure described for the synthesis of compound
22, and was obtained as a white solid (0.28 g, yield 92%). An analyt-
ical sample of 6p melted at 215 °C. 1H NMR (CDCl3): 3.78 (s, 6H),
4.54 (d, J = 5.6, 2H), 6.35 (t, J = 1.9, 1H), 6.48 (d, J = 1.8, 2H), 7.34
(m, 2H), 7.52–7.66 (m, 5H), 7.92 (m, 1H), 8.44 (t, J = 5.6, 1H), 8.76
(d, J = 8.2, 1H). MS(ESI): m/z 440 (M+H+). Anal. (C25H21N5O3) C,H,N.
4.4. Theoretical calculations
Density functional theory calculations were performed on dif-
ferent tricyclic structures by means of Gaussian 0319 implemented
on a IBM SP RS/6000 Power 5 supercomputer at Cineca in Bologna
(Italy). All geometries were fully optimized without any con-
straints at the Becke 3LYP (B3LYP)20 method with the 6-
31G+(d,p) level of theory. The final lowest energy geometries were
confirmed as a minimum on the potential energy surface by nor-
mal-mode vibrational frequency calculations that produced all real
frequencies.
4.2. X-ray crystallography
A single crystal of 6a was submitted to X-ray data collection
on
a
Siemens P4 four-circle diffractometer with
a graphite
Acknowledgments
monochromated Mo-K
a
radiation (l = 0.71069 Å) at 293 K. The
structure was solved by direct methods implemented in SHEL-
XS-97 program.16 The refinements were carried out by full-ma-
trix anisotropic least-squares on F2 for all reflections for non-H
atoms by means of SHELXL-97 program.17
Crystallographic data (excluding structure factors) for the
structure in this paper have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publication no.
CCDC 670069. 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).
The authors thank Prof. Stefania D’Agata D’Ottavi for the careful
reading of the manuscript. This work was financially supported by
MUR (Ministero dell’Università e della Ricerca)—PRIN (Programmi
di ricerca di Rilevante Interesse Nazionale).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.05.067.

A. Cappelli et al. / Bioorg. Med. Chem. 16 (2008) 6850–6859
6859
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16. Sheldrick, G. M. SHELXS-97, Rel. 97-2, A Program for Automatic Solution of
Crystal Structures, Gottingen University, 1997.
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