Discovery of a novel class of selective non-peptide antagonists for the human neurokinin-3 receptor. 1. Identification of the 4-quinolinecarboxamide framework

Article abstract of DOI:10.1021/jm960818o

A novel class of potent and selective non-peptide neurokinin-3 (NK-3) receptor antagonists, featuring the 4-quinolinecarboxamide framework, has been designed based upon chemically diverse NK-1 receptor antagonists. The novel compounds 33-76, prompted by chemical modifications of the prototype 4, have been characterized by binding analysis using a membrane preparation of chinese hamster ovary (CHO) cells expressing the human neurokinin-3 receptors (hNK-3-CHO), and clear structure-activity relationships (SARs) have been established. From SARs, (R)-N-[α-(methoxycarbonyl)benzyl]-2- phenylquinoline-4-carboxamide (65, SB 218795, hNK-3-CHO binding K(i) = 13 nM) emerged as one of the most potent compounds of this novel class. Selectivity studies versus the other neurokinin receptors (hNK-2-CHO and hNK-1-CHO) revealed that 65 is about 90-fold selective for hNK-3 versus hNK-2 receptors (hNK-2-CHO binding K(i) = 1221 nM) and over 7000-fold selective versus hNK-1 receptors (hNK-1-CHO binding K(i) = >100 μM). In vitro functional studies in rabbit isolated iris sphincter muscle preparation demonstrated that 65 is a competitive antagonist of the contractile response induced by the potent and selective NK-3 receptor agonist senktide with a K(b) = 43 nM. Overall, the data indicate that 65 is a potent and selective hNK-3 receptor antagonist and a useful lead for further chemical optimization.

Full text of DOI:10.1021/jm960818o

1794
J . Med. Chem. 1997, 40, 1794-1807
Discover y of a Novel Cla ss of Selective Non -P ep tid e An ta gon ists for th e Hu m a n
Neu r ok in in -3 Recep tor . 1. Id en tifica tion of th e 4-Qu in olin eca r boxa m id e
F r a m ew or k
Giuseppe A. M. Giardina,*,‡ Henry M. Sarau,§ Carlo Farina,‡ Andrew D. Medhurst,† Mario Grugni,‡
Luca F. Raveglia,‡ Dulcie B. Schmidt,§ Roberto Rigolio,‡ Mark Luttmann,§ Vittorio Vecchietti,‡ and
Douglas W. P. Hay§
Department of Chemistry, SmithKline Beecham S.p.A., Via Zambeletti, 20021 Baranzate, Milano, Italy,
Department of Pulmonary Pharmacology, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road,
King of Prussia, Pennsylvania 19406-0939, and Department of Neurology Research, SmithKline Beecham Pharmaceuticals,
New Frontiers Science Park, Third Avenue, Harlow, Essex, England CM19 5AW
Received December 2, 1996X
A novel class of potent and selective non-peptide neurokinin-3 (NK-3) receptor antagonists,
featuring the 4-quinolinecarboxamide framework, has been designed based upon chemically
diverse NK-1 receptor antagonists. The novel compounds 33-76, prompted by chemical
modifications of the prototype 4, have been characterized by binding analysis using a membrane
preparation of chinese hamster ovary (CHO) cells expressing the human neurokinin-3 receptors
(hNK-3-CHO), and clear structure-activity relationships (SARs) have been established. From
SARs, (R)-N-[R-(methoxycarbonyl)benzyl]-2-phenylquinoline-4-carboxamide (65, SB 218795,
hNK-3-CHO binding Ki ) 13 nM) emerged as one of the most potent compounds of this novel
class. Selectivity studies versus the other neurokinin receptors (hNK-2-CHO and hNK-1-CHO)
revealed that 65 is about 90-fold selective for hNK-3 versus hNK-2 receptors (hNK-2-CHO
binding Ki ) 1221 nM) and over 7000-fold selective versus hNK-1 receptors (hNK-1-CHO
binding Ki ) >100 µM). In vitro functional studies in rabbit isolated iris sphincter muscle
preparation demonstrated that 65 is a competitive antagonist of the contractile response induced
by the potent and selective NK-3 receptor agonist senktide with a Kb ) 43 nM. Overall, the
data indicate that 65 is a potent and selective hNK-3 receptor antagonist and a useful lead for
further chemical optimization.
In tr od u ction
Since the discovery in 1991 of CP 96,3451 (1; Figure
1), the first potent non-peptide human neurokinin-1
(NK-1) receptor antagonist from Pfizer, and of RP
67,5802 (2), another potent NK-1 receptor antagonist
identified from random screening by Rhoˆne Poulenc in
1992, interest in the tachykinin area has dramatically
increased. Almost all the major pharmaceutical com-
panies dedicated efforts to the identification of non-
peptide antagonists selective for the three neurokinin
receptors (NK-1, NK-2, and NK-3). These receptors,
widely distributed in both the central nervous system
(CNS) and periphery,3 belong to the seven transmem-
brane G-protein-coupled receptor superfamily (7TM-
GPCR).4 Their endogenous neurotransmitters, the ta-
chykinins or neurokinins,5 constitute a group of small
peptides which are released from sensory nerves. The
tachykinins are implicated in a wide range of patho-
physiological conditions (including nociceptive, inflam-
matory, and immunoregulatory processes, airway ob-
struction and asthma, skin disorders, inflammatory
bowel disease, emesis, and various CNS disorders).4,6
The three main endogenous tachykinins identified to
date, substance P, neurokinin A (NKA), and neurokinin
B (NKB), show a preferred affinity for NK-1, NK-2, and
NK-3 receptors, respectively, although all three tachy-
kinins interact with the three neurokinin receptors.
F igu r e 1. Identification of the 4-quinolinecarboxamide frame-
work.
Initially, the focus of the research effort in the
tachykinin area was on NK-1 and NK-2 receptor an-
tagonists with much less research on NK-3 receptors;7-9
this was due to the lack of availability, until recently,
of potent and selective non-peptide NK-3 receptor
antagonists. In 1995 novel NK-3 receptor antagonists
from diverse chemical classes appeared in the litera-
ture.10-14 Our research group published in this J ournal
in 1996 a Communication to the Editor in which a novel
chemical class of potent and selective non-peptide
competitive antagonists for the human NK-3 (hNK-3)
‡ SmithKline Beecham S.p.A., Italy.
§ SmithKline Beecham Pharmaceuticals, PA.
† SmithKline Beecham Pharmaceuticals, England.
X Abstract published in Advance ACS Abstracts, May 1, 1997.
S0022-2623(96)00818-7 CCC: $14.00 © 1997 American Chemical Society

Non-Peptide Antagonists for the NK-3 Receptor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1795
receptor was disclosed.15 In this paper we report in
more detail the rational identification of the 2-phenyl-
4-quinolinecarboxamide framework and part of our work
aimed at optimizing the hNK-3 binding affinity of this
novel chemical class.
will be described. For compounds 41, 65, and 66, radio-
ligand binding affinities for the cloned hNK-2 and
hNK-1 receptors stably expressed in CHO cell lines
(hNK-2-CHO and hNK-1-CHO) and in vitro functional
activity in the rabbit isolated iris sphincter muscle
preparation (antagonism of contractions induced by the
selective NK-3 receptor agonist senktide) will also be
presented.
As most of the other research groups, we started
efforts in the tachykinin area in 1992 with focus on
NK-1 receptor antagonists, utilizing compounds 1 and
2 as prototype models from which a putative neurokinin
pharmacophore could be inferred. The identification
from Pfizer of CP 99,99416 (compound 3, described to
be much more selective than 1 in respect to Verapamil-
sensitive L-type Ca2+ binding site; Figure 1)17 suggested
that one of the two phenyl groups of the benzhydryl
moiety was not a key pharmacophore determinant for
a potent NK-1 binding affinity. Possibly, the additional
phenyl ring was responsible for the interaction with the
L-type Ca2+ channel. Another piece of important in-
formation to help the clarification of key determinants
of a putative neurokinin-1 pharmacophore was revealed
by Merck researchers in 1993. They established that
in both 1- and 3-like structures, the benzylamine group
could be replaced by the benzyloxy moiety without
affecting the binding affinity to NK-1 receptors.18 This
finding revealed that the role for the exocyclic nitrogen
in compound 1 was just to be a hydrophilic, hydrogen
bond acceptor center. Taking together all the features
described above, we focused our efforts on the pharma-
cophore atomic sequence represented in Figure 1 and
designed a number of chemical models which could offer,
in principle, realistic opportunities to be potent NK-1
receptor antagonists.
Ch em istr y
Intermediates 7a -x in Scheme 1 were obtained in
high yields (typically, greater than 80%) by the Pfitz-
inger reaction30 of isatines 5 with the appropriate
commercially available alkyl (6b-d )31 and aryl (6a ,e-
x) ketones in 95-99% EtOH and 85% KOH pellets at
80 °C for 1-3 days (occasionally, 34% KOH was
utilized). The benzyl carbamate derivative 37 was
obtained by Curtius degradation of 7a ′ in the presence
of diphenyl phosphorazidate (DPPA), triethylamine
(TEA), and benzyl alcohol, following a described proce-
dure.32 The anilide derivative 34 was prepared from 7a ′
in two steps, via preparation of the corresponding acyl
chloride and subsequent reaction with aniline in the
presence of K2CO3 in dry DMF. For the preparation of
the tertiary amide 72, the carboxylic acid 7a was treated
with (R,S)-methyl N-(methylphenyl)glycinate‚HCl,33 di-
cyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole
(HOBT), and N-methylmorpholine; for this reaction a
solvent mixture consisting of THF/MeCN in a 7:3 ratio
gave, in general, the best yields.
Secondary amides 4, 33, 38, 40-69, and 71 in Scheme
1 were synthesized from the corresponding 4-quinolin-
ecarboxylic acids 7a -x and the appropriate primary
amines of general formula R2R3CHNH2, following the
procedures described above for compound 34 or 72
(occasionally, TEA was used instead of N-methylmor-
pholine). The carboxylic acid derivative 39 was obtained
from the corresponding methyl ester 38 by treatment
with 10% HCl in refluxing dioxane for 3 h. The (p-meth-
oxyphenyl)glycine derivative 70 was obtained from the
p-hydroxy derivative 69 by treatment with MeI in the
presence of anhydrous K2CO3 in refluxing acetone
containing traces of DMF (to facilitate the solution of
the substrate).
Lithium aluminum hydride (LiAlH4) reduction of the
amide 33 to the corresponding secondary amine 35 was
unsuccessful and resulted in the formation of several,
nonidentified byproducts; thus, compound 35 was pre-
pared as described in Scheme 2. The acyl chloride 8
was treated at -70 °C in THF with lithium tri-tert-
butoxyaluminum hydride [Li(t-BuO)3AlH, 1 M solution
in THF]; attempts to stop the reaction at the aldehyde
step were unsuccessful and resulted in a 1:1 mixture of
the primary alcohol 9 and the aldehyde 10; thus, an
excess of the reducing agent was employed to obtain the
primary alcohol 9 (62%) which was oxidized to the
corresponding aldehyde (83%) by treatment with MnO2
in CH2Cl2 at room temperature for 6 days. Reduction
of the crude imine 11, obtained by refluxing 10 and
benzylamine in toluene in the presence of a catalytic
amount of p-toluenesulfonic acid, was achieved with
sodium triacetoxyborohydride [Na(AcO)3BH], in AcOH
at room temperature.
During the screening efforts it was noted that the
2-phenyl-4-quinolinecarboxamide derivative 4 had no
significant affinity for hNK-1 receptors (Ki > 10 µM, for
displacement of [3H]substance P binding from hNK-1-
CHO membranes) but possessed moderate affinity for
the hNK-3 receptor (Ki ) 870 nM, for displacement of
[
125I]MePhe7-NKB binding from hNK-3-CHO mem-
branes).
The NK-3 receptor, present in both the CNS and
periphery, is supposed to exert a neuromodulatory role,
as suggested by pharmacological studies using selective
peptide NK-3 receptor agonists. In particular, NK-3
receptor stimulation plays a key role in the modulation
of neuronal input in airways,19,20 skin,21,22 spinal cord,23,24
and nigrostriatal pathways.25-29
In contrast to the extensive investigation on the
effects of NK-3 receptor agonists, no information is
available on the activity of NK-3 receptor antagonists
in disease models. Therefore, based on available evi-
dence on NK-3 receptor distribution and agonist effects,
potential therapeutic utility for NK-3 receptor antago-
nists in pulmonary diseases, renal and bladder disor-
ders, skin disorders, neurogenic inflammation and pain,
as well as CNS disorders, including anxiety, psychosis,
movement, and convulsive disorders, can be envisiged.
Based on this information and on the moderate
affinity of compound 4 for the hNK-3 receptor, a major
effort was directed toward identifying more potent NK-3
receptor antagonists. In this paper, chemical synthesis,
radioligand binding affinity for the cloned hNK-3 recep-
tor stably expressed in chinese hamster ovary cell lines
(hNK-3-CHO), and structure-activity relationships
(SARs) of the novel hNK-3 receptor antagonists 33-76
Since alkylation of 4-amino-7-methoxy-2-phenylquin-
oline (obtained by hydrolysis of the benzyl carbamate

1796 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Giardina et al.
Sch em e 1. Synthesis of Compounds 4, 33, 34, and 37-72 of General Formulae I-IV in Tables 1-4a
a
Reagents: (a) 34-85% aq KOH, 95-99% EtOH, 80 °C, 1-3 days; (b) DPPA, TEA, PhCH2OH, 80 °C, 20 h; (c) 1. (COCl)2, CH2Cl2, 5
°C to room temperature, 2 days, 2. PhNH2, DMF, K2CO3, room temperature, 24 h; or (R,S)-methyl N-(methylphenyl)glycinate‚HCl, DCC,
HOBT, N-methylmorpholine, THF/MeCN (7:3), 3 h, rt; (d) 1. (COCl)2, CH2Cl2, 5 °C to room temperature, 2 days, 2. R2R3CHNH2, DMF,
K2CO3, room temperature, 24 h; or R2R3CHNH2‚HCl, DCC, HOBT, N-methylmorpholine, THF/MeCN (7:3), 3 h, room temperature; (e)
10% HCl, dioxane, reflux, 3 h; (f) K2CO3, MeI, Me2CO/DMF, reflux.

Non-Peptide Antagonists for the NK-3 Receptor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1797
Sch em e 2a
Sch em e 4a
a
Reagents: (a) HCOOH (98%), HCOONH4, reflux, 2.5 h; (b)
maleic acid, 110 °C, 15 min; (c) (R,S)-methyl phenylglycinate‚HCl,
DCC, HOBT, N-methylmorpholine, THF/MeCN (7:3), 3 h, room
temperature.
Sch em e 5a
a
Reagents: (a) (COCl)2, CH2Cl2, 5 °C to room temperature, 2
days; (b) Li(t-BuO)3AlH, THF, -70 °C, 2 h, then room temperature,
16 h; (c) MnO2, CH2Cl2, room temperature, 6 days; (d) PhCH2NH2,
toluene, PTSA, reflux, 3 days; (e) Na(AcO)3BH, AcOH, room
temperature, 24 h.
Sch em e 3a
a
Reagents: (a) 180 °C, 24 h, N2, steel autoclave; (b) 85% KOH,
ethylene glycol, 200 °C, 16 h; (c) (R,S)-methyl phenylglycinate‚HCl,
DCC, HOBT, N-methylmorpholine, THF/MeCN (7:3), 3 h, room
temperature.
from the reaction mixture by adjusting to pH 8 with di-
luted NaOH, collecting the precipitate, and washing it
with hot MeOH. Treatment of the 4-quinolinol deriva-
tive 16 in refluxing POCl3 afforded the corresponding
4-chloro intermediate 17 in 68% yield. Finally, com-
pound 17 was converted to the desired derivative 36 by
refluxing with benzylamine in phenol for 3 h.
The 2-phenylpyridine-4-carboxamide derivative 73
was prepared as described in Scheme 4. Refluxing
R-bromoacetophenone with ammonium formate in 98%
HCOOH afforded 4-phenyloxazole (19).34 Compound 19
was melted with maleic acid at 110 °C for 15 min
following a reported procedure35 and afforded 2-phen-
ylpyridine-4-carboxylic acid (20) which was then coupled
with (R,S)-methyl phenylglycinate, using the same
conditions employed for compound 72, to obtain the
4-pyridinecarboxamide derivative 73.
a
Reagents: (a) 32% NH4OH, 85 °C, 2 h, steel autoclave; (b) H2O,
100 °C, 4 h; (c) m-anisidine, toluene, reflux, 8 h; (d) PPA, 135 °C,
20 min; (e) NaOH to pH 8, filtration and recrystallization of 16
from MeOH; (f) POCl3, reflux, 2 h; (g) PhCH2NH2, phenol, 150
°C, 3 h.
The 2-phenylnaphthalene-4-carboxamide derivative
74 was synthesized according to Scheme 5. In this
scheme, a mixture of 1,3-diphenylacetone (21) and N,N-
dimethylformamide dimethyl acetal (22) was heated in
a steel autoclave at 180 °C for 24 h according to the
procedure described by R. F. Abdulla et al.36 to give the
dimethylamide 23. Hydrolysis of compound 23 under
strong conditions (KOH, ethylene glycol, 200 °C) af-
forded the desired 2-phenylnaphthalene-4-carboxylic
acid 24, which was transformed in high yield into the
final secondary amide derivative 74 by the usual DCC
coupling.
37) was not possible, due to the nonbasic nature of the
4-amino group, compound 36 was prepared by a mul-
tistep procedure (Scheme 3) starting from ethyl 3-oxo-
3-phenylpropionate (12), which was converted to the
corresponding primary amide by heating in a steel
autoclave in the presence of concentrated NH4OH and
subsequent hydrolysis of the labile imine function.
Polyphosphoric acid (PPA) cyclization of compound 14,
prepared from the ketone 13 and m-anisidine under
standard conditions, was very effective and afforded a
2:3 mixture of 4-hydroxy-5-methoxy-2-phenylquinoline
(15) and 4-hydroxy-7-methoxy-2-phenylquinoline (16),
respectively. The 7-methoxy isomer 16 was obtained
1-Methyl-3-phenylisoquinoline (27; Scheme 6) was
prepared by refluxing a mixture of deoxybenzoin (25)

1798 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Giardina et al.
Sch em e 6a
P h a r m a cology
Recep tor Clon in g a n d Exp r ession . Human cD-
NAs for the NK-1, NK-2, and NK-3 tachykinin recep-
tors, with sequences identical to those in published
reports, were isolated from human placenta poly(A+)
RNA using reverse transcriptase-polymerase chain
reaction (PCR) technology and site-directed mutagen-
esis. Oligonucleotide primers for hNK-1 receptor cD-
NA,40 hNK-2 receptor cDNA,41 and hNK-3 receptor
cDNA42,43 were synthesized and used for PCR using the
human placenta cDNA as template. The individual
fragments were subcloned into the mammalian expres-
sion vector, pCDN,44 and the resulting constructs were
completely sequenced to confirm their identity and
orientation. Stable CHO cell lines for the pCDN-NK-
1, pCDN-NK-2, and pCDN-NK-3 expression vectors
were obtained by electroporation followed by clonal
selection using G418. The CHO stable cell lines were
screened for high-level receptor expression by ligand
binding assays on whole cells. From this screen, the
clonal cell line producing the highest number of recep-
tors per cell was chosen for each receptor.
a
Reagents: (a) POCl3, reflux, 29 h; (b) NBS, dibenzoyl peroxide,
CCl4, reflux, 19 h; (c) AgNO3, EtOH/H2O/THF, reflux, 1 h; (d)
AgNO3, NaOH, EtOH/H2O, 16 h; (e) (R,S)-methyl phenylgly-
cinate‚HCl, DCC, HOBT, N-methylmorpholine, THF/MeCN (7:3),
3 h, room temperature.
Sch em e 7a
Ra d ioliga n d Bin d in g Assa ys. Receptor binding
assays were performed with crude membranes from
CHO cells expressing the hNK-1, hNK-2, or hNK-3
receptors. For NK-3 receptor competition binding stud-
ies, [125I]MePhe7-NKB binding to hNK-3-CHO mem-
branes was performed using the procedure of Sadowski
and co-workers.45 Concentration-response curves for
compounds 4 and 33-76 were run using duplicate
samples; among them, compounds which gave an IC50
binding affinity lower than 1000 nM in the first experi-
ment were run in three to five independent experiments
(n ) 3-5). Specific binding was determined by sub-
tracting total binding from nonspecific binding, which
was assessed as the binding in the presence of 0.5 µM
cold MePhe7-NKB. Percent inhibition of specific binding
was determined for each concentration of the compounds
and the IC50, defined as the concentration required to
inhibit 50% of the specific binding, obtained from
concentration-response curves. Values reported in
Tables 1-4 are the apparent inhibition constants (Ki),
which were calculated from the IC50 as described by
Cheng and Prusoff.46 For selectivity studies, compounds
41, 65, and 66 were also evaluated in hNK-2-CHO and
hNK-1-CHO binding assays (Table 6). For NK-2 recep-
tor competition binding studies, [125I]NKA binding to
membranes of CHO cells stably expressing the hNK-2
receptor (hNK-2-CHO) was performed essentially as
described by Aharony et al.47 Nonspecific binding was
determined in the presence of 0.5 µM cold NKA. The
Ki was determined as described for the NK-3 assay.
Competition binding studies for the NK-1 receptor were
performed on membranes of CHO cells stably expressing
the hNK-1 receptor (hNK-1-CHO) essentially as de-
scribed by Payan et al.48 Nonspecific binding was
determined in the presence of 1 µM cold substance P
and Ki determined as for the NK-3 and NK-2 assays.
a
Reagents: (a) SOCl2, 90 °C, 3 h; (b) 1. NCCOOEt, SnCl4,
o-dichlorobenzene, 140 °C, 15 min, 2. 20% aq NaOH, dioxane
(traces), reflux, 2 h; (c) 1. (COCl)2, CH2Cl2/DMF (cat.), 5 °C to room
temperature, 2 days, 2. (R,S)-methyl phenylglycinate‚HCl, TEA,
DMF/CH2Cl2, room temperature, 24 h.
and acetonitrile with POCl3 for 29 h.37 Subsequent
oxidation of the aromatic methyl group to the corre-
sponding carboxylic acid was achieved in three steps.38
Firstly, the methyl was converted into the dibromo-
methyl group with N-bromosuccinimide (NBS); then, the
aldehyde derivative 28 was obtained by neutral silver
nitrate oxidation in refluxing EtOH/H2O/THF, and
finally, the oxidation was completed by stirring 28 at
room temperature with an excess of silver nitrate under
basic conditions. The carboxylic acid derivative 29 was
coupled under the usual conditions to obtain the desired
isoquinoline derivative 75, in which the aromatic nitro-
gen occupies the ring position between the phenyl and
the carboxamide substituents.
The 2-phenylquinazoline-4-carboxamide derivative 76
was obtained according to Scheme 7. Benzanilide
imidoyl chloride (31), easily obtained in quantitative
yield by refluxing benzanilide (30) and SOCl2, was
condensed with ethyl cyanoformate in the presence of
stannic tetrachloride at 140 °C in o-dichlorobenzene.39
The desired 2-phenylquinazoline-4-carboxylic acid (32)
was recovered after basic hydrolysis of the correspond-
ing ethyl ester. Finally, the secondary amide 76 was
prepared in two steps from 32 via its corresponding acyl
chloride.
Sen k tid e-In d u ced Con tr a ction in Ra bbit Iso-
la ted Ir is Sp h in cter (RIS) Mu scle P r ep a r a tion .
Since the rabbit isolated iris sphincter muscle prepara-
tion contains functional NK-3 receptors,49,50 the effects
of one of the most potent racemic NK-3 receptor
antagonists described in this paper (41) and of its
enantiomers (65 and 66) were investigated in this tissue

Non-Peptide Antagonists for the NK-3 Receptor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1799
Ta ble 1. Physical Properties and Binding Affinity at Cloned Human Neurokinin-3 (hNK-3) Receptors Expressed in CHO Cells of
Compounds 4 and 33-43 of General Formula I
hNK-3-CHO bindinga
compd
R
R1
R2
formula
mp, °C
anal.
recryst solvent Ki, mean ( SEM (nM)b
4
33
34
35
36
37
38
39
40
41
42
43
OMe 2-Ph
OMe 2-Ph
OMe 2-Ph
OMe 2-Ph
OMe 2-Ph
OMe 2-Ph
OMe 2-Ph
OMe 2-Ph
CONHCH2Ph
CONHCH2CH2Ph
CONHPh
CH2NHCH2Ph
NHCH2Ph
NHCOOCH2Ph
CONHCH(COOMe)Ph
CONHCH(COOH)Ph
CONHCH2Ph
C24H20N2O2
C25H22N2O2
167-169 C, H, N
174-176 C, H, N
222-224 C, H, N
i-PrOH/i-Pr2O
toluene
toluene
870 ( 170
919 (2)
1671 (1)
6203 (2)
10312 (2)
8133 (1)
57 ( 10
5181 (1)
355 ( 63
31 ( 4
C
23H18N2O2
C24H22N2O‚2HCl 216-218 H, N; Cc i-PrOH/i-Pr2O
C23H20N2O
C24H20N2O3
171-174 H, N; Cd Et2O
156-158 C, H, N
187-190 C, H, N
EtOAc
C
C
26H22N2O4
25H20N2O4‚HCl 228-230 C, H, N
i-PrOH/i-Pr2O
EtOH/EtOAc
EtOAc
H
H
2-Ph
2-Ph
C23H18N2O
25H20N2O3
189-190 C, H, N
170-172 C, H, N
CONHCH(COOMe)Ph
CONHCH2Ph
C
i-PrOH/i-Pr2O
OMe 2-Me
OMe 2-CH2Ph CONHCH2Ph
C19H18N2O2‚HCl 181-183 H, N; Ce EtOAc
>33000 (1)
>33000 (1)
C25H22N2O2
149-152 C, H, N
EtOAc/i-Pr2O
a
b
Inhibition of [125I]MePhe7-NKB binding in hNK-3-CHO cell membranes. Average of three to five independent determinations (n )
3-5), unless otherwise indicated in parentheses. c C: calcd, 67.45; found, 66.80. C: calcd, 81.15; found, 80.66. e C: calcd, 66.56; found,
65.18.
d
(Table 6). Iris sphincter muscle strips were prepared
from male New Zealand White rabbits (2-3 kg, Charles
River, U.K.). Tissues were placed in 50-mL organ baths
containing Krebs-Henseleit solution for the isometric
measurement of tension as previously described.50 After
a reference contractile response to 10 µM carbachol was
obtained, experiments were conducted in the presence
of 1 µM CP 99,994 and 1 µM atropine, to block NK-1
and muscarinic receptors, respectively. Tissues were
then exposed to the NK-3 receptor antagonist under
study (10 nM) or vehicle (DMSO) for 120 min before
cumulative concentration-effect curves to senktide, the
selective NK-3 receptor agonist, were constructed. Re-
sponses to senktide are expressed as a percentage of the
carbachol-induced contraction. The dissociation con-
stant, Kb, for the antagonist-NK-3 receptor complex
was calculated from the equation: Kb ) [B]/CR - 1,
where CR is the concentration ratio of agonist used in
the presence and absence of antagonist B.51
F igu r e 2. Regions of compound 4 selected to perform signifi-
cant chemical modifications.
resulted in a 15-fold increase in affinity (cf. 38, hNK-
3-CHO binding Ki ) 57 nM, with 4). Hydrolysis to the
corresponding carboxylic acid resulted in a 6-fold de-
creased affinity (cf. 4 with 39), thus suggesting that an
acidic functionality at this position is not tolerated.
Removal of the 7-OMe substituent resulted in a 2.5-fold
increase in binding affinity (cf. 4 with 40). Replacement
of the 2-phenyl ring by an aliphatic group (e.g., Me,
compound 42) or benzylic group (compound 43) pro-
duced inactive compounds (hNK-3-CHO binding Ki >
33 µM). Combination of both the introduction of the
ester moiety and the removal of the 7-OMe substituent
gave rise to compound 41 (hNK-3-CHO binding Ki ) 31
nM), which had 28-fold higher affinity than 4 as a
hNK-3 receptor ligand. Around this molecule, a chemi-
cal program was undertaken with the aim of optimizing
hNK-3 binding affinity.
Resu lts a n d Discu ssion
Str u ctu r e-Activity Rela tion sh ip s (Ta bles 1-5).
1. Lea d Id en tifica tion . After the identification of 4
as a possible prototype to design selective hNK-3
receptor ligands, three regions of the molecule were
selected (Figure 2) to perform chemical modifications
suitable to provide expedient and significant SAR infor-
mation: (a) the carboxamide side chain, (b) the 7-OMe
substituent, and (c) the 2-phenyl ring.
Either increasing or decreasing by one carbon atom
the length of the carboxamide side chain did not affect
significantly the hNK-3-CHO binding affinity (cf. com-
pound 4 with 33 and 34, Table 1). The benzylamide
moiety (CONHBz) was therefore selected for further
modifications. Carbonyl reduction (CH2NHBz) or ex-
trusion (NHBz) or, indeed, amide inversion (NHCOBz,
data not shown) produced compounds with 7-12-fold
lower affinity for the hNK-3 receptor (cf. 4 with 35 and
36). Also the replacement of the benzylcarboxamide
with the benzyl carbamate (NHCOOBz) moiety was
detrimental (cf. 4 with 37). Simple incorporation of an
ester moiety (COOMe) at the benzylic position of 4
2. Lea d Op tim iza tion . In addition to the 2-Me and
2-Bz substitutions (compounds 42 and 43, Table 1), a
further and exhaustive examination of the optimal
steric, electronic, and lipophilic requirements of the
substituent at position 2 of the quinoline system (Table
2) was undertaken.
Incorporation of the cyclohexyl moiety decreased the
binding affinity by a factor of about 4 (cf. 41 with 44),
indicating that the aromaticity of the 2-substituent is
important for optimal hNK-3 binding affinity. Within
the (hetero)aromatic series, the sequence of potency was
3-thienyl ) Ph g 2-pyrryl g 2-thienyl ) 2-furyl >
3-pyrryl > 2-benzofuryl > 2-thiazolyl > 4-pyridyl (com-

1800 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Giardina et al.
Ta ble 2. Physical Properties and Binding Affinity at Cloned Human Neurokinin-3 (hNK-3) Receptors Expressed in CHO Cells of
Racemic Compounds 44-62 of General Formula II
hNK-3-CHO bindinga
compd
R1
formula
mp, °C
anal.
recryst solvent
Ki, mean ( SEM (nM)b
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
cyclohexyl
3-thienyl
2-pyrryl
2-thienyl
2-furyl
C25H26N2O3
151-153
169-171
181-182
188-189
178-180
202-203
240-241
209-211
177-178
146-147
175-178
180-182
124-125
212-214
161-163
170-172
172-174
182-183
226-227
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
H, N; Cc
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
EtOAc
i-PrOH
i-PrOH
EtOH
MeOH
i-Pr2O
EtOAc
i-PrOH
EtOAc
toluene
toluene/hexane
i-PrOH
toluene
132 ( 20
26 ( 3
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
23H18N2O3S
23H19N3O3
23H18N2O3S
23H18N2O4
23H19N3O3
27H20N2O4
22H17N3O3S
24H19N3O3
25H19FN2O3
25H19ClN2O3
26H22N2O3
26H22N2O4
25H20N2O4
25H19ClN2O3
25H19ClN2O3
26H22N2O3
26H22N2O4
25H20N2O4
49 ( 10
66 ( 5
77 ( 8
3-pyrryl
118 ( 11
377 ( 35
924 ( 330
2226 (1)
96 ( 12
2581 (1)
1235 (1)
786 (2)
2-benzofuryl
2-thiazolyl
4-pyridyl
Ph(2-F)
Ph(2-Cl)
Ph(2-Me)
Ph(2-OMe)
Ph(2-OH)
Ph(3-Cl)
Ph(4-Cl)
Ph(4-Me)
Ph(4-OMe)
Ph(4-OH)
i-PrOH
653 ( 274
629 ( 77
820 ( 218
142 ( 33
1853 (1)
493 ( 65
toluene/hexane
toluene/i-Pr2O
i-PrOH
i-PrOH
i-PrOH
a
b
Inhibition of [125I]MePhe7-NKB binding in hNK-3-CHO cell membranes. Average of three to five independent determinations (n )
3-5), unless otherwise indicated in parentheses. c C: calcd, 72.80; found, 71.65.
Ta ble 3. Physical Properties and Binding Affinity at Cloned Human Neurokinin-3 (hNK-3) Receptors Expressed in CHO Cells of
Racemic Compounds 63 and 64 of General Formula III
hNK-3-CHO bindinga
compd
n
formula
mp, °C
anal.
recryst solvent
Ki, mean ( SEM (nM)b
63
64
1
2
C27H22N2O3
217-219
186-188
C, H, N
C, H, N
EtOH
i-PrOH/i-Pr2O
234 ( 13
180 ( 8
C
28H24N2O3
a
b
Inhibition of [125I]MePhe7-NKB binding in hNK-3-CHO cell membranes. Average of three to five independent determinations (n )
3-5), unless otherwise indicated in parentheses.
pounds 41 and 45-52). The poor activity of the ben-
zofuran system indicates that steric hindrance of the
additional benzo-condensed ring is not tolerated (cf. 50
with 48). Furthermore, incorporation of a basic nitrogen
in the aromatic ring strongly reduced the hNK-3 binding
affinity (cf. the pyridine derivative 52 with 41 and the
thiazole 51 with the thiophenes 45 and 47 or pyrroles
46 and 48). Introduction of substituents in any position
of the 2-phenyl ring (irrespectively of their electronic
and lipophilic nature) was, in general, not tolerated (see
compounds 53-62).
mental for activity; this suggests that the 2-phenyl
substituent should be twisted out from the plane of the
quinoline ring system.
The introduction of the methyl ester moiety in the
benzylic position of the 4-amide chain (compound 41)
gave rise to a stereogenic center; preparation and testing
of the two enantiomers revealed that the binding of this
molecule to the hNK-3 receptor is enantioselective, the
R-eutomer 65 (SB 218975; Table 4, hNK-3-CHO binding
Ki ) 13 nM) having 70-fold higher affinity than the
S-distomer 66 (hNK-3-CHO binding Ki ) 926 nM) and
2-fold higher affinity than the racemate 41.
In addition, coplanarity of the 2-phenyl with the
quinoline ring, obtained by cyclizing the ortho position
of the phenyl with position 3 of the quinoline nucleus
(compounds 63 and 64, Table 3), appeared to be detri-
Within the lead optimization approach, the replace-
ment/substitution of the phenyl ring of the amide side
chain was also investigated (Table 4); the key role of

Non-Peptide Antagonists for the NK-3 Receptor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1801
Ta ble 4. Physical Properties and Binding Affinity at Cloned Human Neurokinin-3 (hNK-3) Receptors Expressed in CHO Cells of
Compounds 41 and 65-72 of General Formula IV
recryst
solvent
[R]20
hNK-3-CHO bindinga
D
compd
*
R
R1
formula
mp, °C
anal.
(c ) 0.5, MeOH)
Ki, mean ( SEM (nM)b
41
65
66
67
68
69
70
71
72
R,S
R
S
R
R,S
R
R
R,S
R,S
Ph
Ph
Ph
CH(Me2)
CH2Ph
Ph(4-OH)
Ph(4-OMe)
2-thienyl
Ph
H
H
H
H
H
H
H
H
C25H20N2O3
170-172
180-181
180-181
115-117
152-154
140 dec
160-162
144-145
128-129
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N; Clc
C, H, N
C, H, N
C, H, N
i-PrOH
i-PrOH
i-PrOH
i-PrOH
EtOAc
Me2CO
Et2O
31 ( 4
13 ( 3
C
C
25H20N2O3
25H20N2O3
-42.0
+42.0
+28.6
926 ( 250
6208 (2)
1154 (1)
1926 (1)
666 ( 165
180 ( 49
1078 ( 388
C22H22N2O3
C26H22N2O3
C
C
C
C
25H20N2O4
26H22N2O4
23H18N2O3S
26H22N2O3
-7.0
nd
EtOAc
i-PrOH
Me
a
b
Inhibition of [125I]MePhe7-NKB binding in hNK-3-CHO cell membranes. Average of three to five independent determinations (n )
3-5), unless otherwise indicated in parentheses. c Cl: calcd, 7.90; found, 7.48.
Ta ble 5. Binding Affinity at Cloned Human Neurokinin-3
(hNK-3) Receptors Expressed in CHO Cells of Racemic
Compounds 73-76 in Figure 3
hNK-3-CHO bindinga
compd
Ki, mean ( SEM (nM)b
73
74
75
76
>10000 (1)
1202 (2)
>10000 (1)
883 (2)
a
Inhibition of [125I]MePhe7-NKB binding in hNK-3-CHO cell
membranes. b Average of three to five independent determinations
(n ) 3-5), unless otherwise indicated in parentheses.
the aromatic ring and the precise distance between its
centroid and the amidic nitrogen were assessed and
confirmed by the replacement of the phenyl with the
isopropyl moiety (cf. compound 67 with 65) and by the
incorporation of an additional methylene group (cf. 68
with 41). The latter was suggested by the similar
hNK-3 binding affinities obtained with compounds 4
and 33 (Table 1) in the unsubstituted series; in this case
a 37-fold decrease in affinity was observed with the
introduction of a methylene spacer. Also the incorpora-
tion into the phenyl ring of 4-OH (69) and 4-OMe (70)
substituents was detrimental. The hNK-3 binding
affinity decreased 6-fold when the phenyl ring was
replaced by its bioisosteric thiophene (compound 71).
The importance of the presence of an amidic hydrogen
was assessed by the synthesis and evaluation of the
corresponding NMe tertiary amide 72 (Table 4), which
F igu r e 3. Chemical structures of compounds 73-76 in Table
5.
reduced hNK-3 binding affinity with respect to the
parent quinoline 41. These results suggest that both
the benzene-condensed ring and the quinoline nitrogen
are key determinants for optimal binding affinity to
hNK-3 receptors. The inactivity of the isoquinoline
derivative 75 and the poor activity of the quinazoline
76 could be due to the presence of the isoquinoline
nitrogen, which may negatively interact with a charged
site in the receptor or negatively affect the conformation
of the 4-carboxamide side chain. Other modifications
in this regard, including imidazopyridine, pyrazinopy-
ridine, and indole derivatives, are in progress, and
biological data will be presented in a future publication.
Selectivity a n d in Vitr o F u n ction a l Activity
(Ta ble 6). The selectivity versus the other tachykinin
receptors of one of the most potent hNK-3 receptor
ligand identified in the SAR section, the R-enantiomer
65, along with that of its S-enatiomer 66 and the
racemate 41, was assessed by competitive binding
experiments using membranes prepared from CHO cells
stably expressing the hNK-2 (hNK-2-CHO) and hNK-1
had a 35-fold lower affinity than the parent 41.
A
possible explanation for this might be the capability of
the secondary amidic hydrogen to interact with a
hydrogen bond acceptor in the binding site of the NK-3
receptor, or in the case of the tertiary amide, the methyl
group might determine an unfavorable steric interaction
with the receptor.
Preliminary results of a medicinal chemistry approach
aimed at replacing the quinoline ring with various
(hetero)aromatic systems are reported in Table 5; all
the compounds prepared (i.e., the pyridine 73, the
naphthalene 74, the isoquinoline 75, and the quinazo-
line derivative 76, Figure 3) showed a dramatically

1802 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Giardina et al.
Ta ble 6. Binding Affinities at Cloned Human Neurokinin Receptors (hNK-1, hNK-2, and hNK-3) Expressed in CHO Cells and in
Vitro Functional Activities (Antagonism of senktide-induced contractions) in Rabbit Isolated Iris Sphincter (RIS) Muscle Preparation
for Compounds 41, 65, and 66
binding affinities Ki, mean ( SEM (nM)a
antagonism of senktide-induced contractions
compd
*
hNK-3b
hNK-2c
hNK-1d
in RIS muscle Kb,e mean ( SEM (nM)a
41
R,S
R
S
31 ( 4
13 ( 3
926 ( 250
2947 ( 585
1221 ( 189
8333 ( 926
>100000
>100000
>100000
42 ( 12
43 ( 6.2
3982 (2)
65 (SB 218795)
66
a
b
Average of three to five independent determinations (n ) 3-5), unless otherwise indicated in parentheses. Inhibition of [125I]MePhe7-
NKB binding in hNK-3-CHO cell membranes. c Inhibition of [125I]NKA binding in hNK-2-CHO cell membranes. Inhibition of [3H]substance
d
P binding in hNK-1-CHO cell membranes. e Equilibrium dissociation constant Kb for the antagonist-NK-3 receptor complex was calculated
from the equation: Kb ) [B]/CR - 1, where CR is the concentration ratio of agonist used in the presence and absence of antagonist B.
(hNK-1-CHO) and [125I]NKA and [3H]substance P, re-
spectively. The results, summarized in Table 6, clearly
demonstrate that the racemate 41 and the R-eutomer
65 have no affinity for the hNK-1 receptors (hNK-1-
CHO binding Ki > 100 µM) and moderate affinity for
the hNK-2 receptors (hNK-2-CHO binding Kis ) 2947
and 1221 nM, respectively), thus resulting in a 90-fold
selectivity for hNK-3 versus hNK-2 receptors.
Compounds 41 and 65 were demonstrated in vitro to
be functional antagonists of senktide-induced contrac-
tions in the rabbit isolated iris sphincter muscle, a
preparation rich in NK-3 receptors.49 Senktide was a
potent contractile agonist in this preparation with a pD2
) 9.1 ( 0.1 (n ) 4).50 Compound 65 at a concentration
of 10 nM surmountably antagonized the contractile
responses to senktide with a Kb ) 43 nM (Table 6). In
addition, the enantioselectivity for the hNK-3 receptor,
demonstrated in the binding studies for enantiomers 65
and 66, was confirmed by the in vitro functional data;
compound 65 was found to be 90-fold more potent than
its poorly active counterpart 66 (Kb ) 3982 nM).
Compounds 41, 65, and 66 were also evaluated for
their selectivity for the three tachykinin receptors (NK-
1, NK-2, and NK-3) and in vitro functional activity
(Table 6). Binding results indicated that (R)-N-[R-
(methoxycarbonyl)benzyl]-2-phenylquinoline-4-carbox-
amide (65) is about 90-fold selective for the hNK-3
receptor (hNK-3-CHO binding Ki ) 13 nM) versus the
hNK-2 receptor (hNK-2-CHO binding Ki ) 1221 nM)
and over 7000-fold selective for the hNK-3 versus the
hNK-1 receptor (hNK-1-CHO binding Ki ) >100 µM).
Functional studies in rabbit isolated iris sphincter
muscle preparation revealed that compound 65 is a
competitive antagonist of the contractile response in-
duced by the potent and selective NK-3 receptor agonist
senktide, with a Kb ) 43 nM. Overall, the data indicate
that 65 (SB 218795) is a potent and selective hNK-3
receptor antagonist and that compounds emanating
from the 2-phenylquinoline-4-carboxamide series may
represent suitable leads for further chemical optimiza-
tion. Chemical modifications aimed at (a) replacing the
methyl ester group (potentially metabolically unstable)
and (b) optimizing the hNK-3 binding affinity, in vitro
functional activity, and selectivity versus the hNK-2 re-
ceptor (based, essentially, on substitutions at the quino-
line ring) are the subject of a publication in preparation.
Con clu sion s
In this study the design and synthesis of a novel class
of potent and selective non-peptide NK-3 receptor
antagonists are described in detail. The moderately
potent hNK-3 receptor ligand 4 was identified starting
from the potent NK-1 receptor antagonists CP 96,345,
RP 67,580, and CP 99,994. From among the diverse
chemical modifications performed on the 2-phenylquino-
line-4-carboxamide skeleton of the prototype 4 (Tables
1-5), the following important features emerged as
crucial determinants for optimal hNK-3 binding affin-
ity: (i) the presence of the (benzylamino)carbonyl moiety
(CONHBz, Table 1), (ii) the absence of the 7-OMe
substituent (Table 1), (iii) the incorporation of COOMe
at the benzylic position (Table 1), (iv) the presence of
the unsubstituted phenyl ring in position 2 (Tables 1-3),
(v) the stereogenic center of the 4-amide side chain in
the R configuration (Table 4), (vi) the presence of the
unsubstituted phenyl ring in the amide side chain
(Table 4), (vii) the secondary amide at position 4 (Table
4), and (viii) the presence of the quinoline ring (Table
5).
Exp er im en ta l Section
Ra d ioliga n d Bin d in g Assa ys. The CHO cells expressing
the hNK-1, hNK-2, and hNK-3 receptors were cultured at 37
°C in a humidified incubator under 5% CO2-95% air in 1017
SO3 (in-house formulation) media containing nucleosides plus
geneticin (400 mg/L). The cells were harvested by centrifuga-
tion at 600g for 10 min. The cell pellet was resuspended in
hypotonic buffer (10 mM Tris, pH 7.4, 1.0 mM EDTA, 10 µg/
mL soybean trypsin inhibitor, 100 µg/mL bacitracin, 100 µM
benzamidine, and 10 µM phenylmethanesulfonyl fluoride) and
then rapidly frozen and thawed (three times) followed by
Dounce homogenization for preparation of crude membranes.
For NK-3 receptor competition binding studies,45 mem-
branes (∼15 µg of protein) were incubated with 0.15 nM [125I]-
MePhe7-NKB in a total of 150 µL of 50 mM Tris, pH 7.4, 4
mM MnCl2, 1 µM phosphoramidon, and 0.1% ovalbumin, with
or without various concentrations of antagonist, for 90 min at
25 °C. Incubations were stopped by rapid filtration with a
Brandell tissue harvestor (Gaithersburg, MD) through What-

Non-Peptide Antagonists for the NK-3 Receptor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1803
man GF/C filters that were presoaked for 60 min in 0.5%
bovine serum albumin (BSA). Membranes were washed with
10 mL of ice-cold 20 mM Tris, pH 7.4, containing 0.1% BSA,
then placed in vials with 10 mL of Beckman Ready Safe
cocktail, and counted in a Beckman LS 6000 (Fullerton, CA)
liquid scintillation counter.
about 90%, and compounds of formula 7a -x31 were triturated
with i-Pr2O and recrystallized from EtOH. As an example,
spectroscopic data for 7a ′ are reported: mp 226-228 °C; IR
1
(KBr) 3440 br, 1630, 1600, 1240 cm-1; H NMR (DMSO-d6) δ
8.59 (d, J ) 9.1 Hz, 1H), 8.29 (s, 1H), 8.27 (dd, J ) 7.5, 1.0
Hz, 2H), 7.61-7.50 (m, 3H), 7.55 (s, 1H), 7.36 (dd, J ) 9.1, 2.8
Hz, 1H), 3.98 (s, 3H); FAB-MS (positive, matrix NBA, gas Xe,
8 keV, source 50 °C) m/z 280 (MH+).
For NK-2 competition binding studies,47 cells were grown
and membranes were prepared as described above for the
hNK-3 binding assay. The assay buffer was the same as that
utilized for the hNK-3 binding assay with a total volume of
150 µL, ∼10 µg of membrane protein, and 0.15 nM [125I]NKA,
which was incubated for 90 min at 25 °C, with various
concentrations of antagonist. Filtration was through What-
man GF/C filters soaked for 30 min in 0.1% poly(ethylenimine)
(PEI), and membranes were washed as described above for the
hNK-3 binding assay.
Gen er a l P r oced u r es To Obta in 2-P h en ylqu in olin e-4-
ca r boxa m id es 4, 33, 34, 38, 40-69, 71, a n d 72 of Gen er a l
F or m u la e I-IV in Ta b les 1-4. Met h od A: Via Acyl
Ch lor id e. 2-Phenylquinoline-4-carboxylic acids 7a ,a ′-
c′,g,h ,j,l-n ,p ,r (7.9 mmol) were suspended in CH2Cl2 (30
mL), and oxalyl chloride (13.4 mmol) was added dropwise to
the ice-cold reaction mixture; the stirring was continued at
0-5 °C for 1 h and then at room temperature overnight.
Evaporation of the solvent afforded the crude acid chloride
which was dissolved in dry DMF (40 mL) and added dropwise,
at 0 °C, to a stirred suspension of the appropriate primary
amine or R-amino ester‚HCl (15.8 mmol) in dry DMF (15 mL),
containing anhydrous K2CO3 (15.8 mmol). After 24 h the
reaction mixture was filtered, the solvent evaporated, and the
residue, taken up in water, extracted with EtOAc, and the
organic layer was washed with H2O, 5% citric acid, 5%
NaHCO3, and brine, dried over Na2SO4, and evaporated to
dryness. The crude amide was triturated with toluene or
i-Pr2O and recrystallized from the appropriate solvent, afford-
ing the desired compounds in 68-85% yield. This method was
always used in the case of primary amines and, occasionally,
with R-amino esters‚HCl to obtain the corresponding amides
4, 33, 34, 38, 40, 42, 43, 47, 48, 50, 52, 53, 54, 56, 58, 68, and
71.
For NK-1 competition binding studies,48 the assay volume
was 300 µL, and the assay buffer (25 mM Tris, pH 7.4, 2.0
mM CaCl2, 2.0 mM MgCl2, 1 µM phosphoramidon, and 0.1%
ovalbumin) contained various concentrations of antagonist and
1.0 nM [3H]substance P. Membranes were incubated for 45
min at 25 °C; Whatman filters were presoaked with BSA and
membranes washed as described for the hNK-3 binding assay.
Ch em istr y. Melting points were determined with a Bu¨chi
530 hot stage apparatus and are uncorrected. Proton NMR
spectra were recorded on a Bruker ARX 300 spectrometer at
303 K unless otherwise indicated. Chemical shifts are re-
corded in parts per million (δ) downfield from tetramethylsi-
lane (TMS); NMR spectral data are reported as a list. IR
spectra were recorded in Nujol mull on sodium chloride disks
or in KBr with a Perkin-Elmer 1420 spectrophotometer; mass
spectra were obtained on a Finnigan MAT TSQ-700 (or TSQ-
70) spectrometer. Optical rotations were determined in MeOH
solution at the indicated concentration with a Perkin-Elmer
341 polarimeter at the sodium D-line. Silica gel used for flash
column chromatography was Kiesegel 60 (230-400 mesh) (E.
Merck AG, Darmstadt, Germany). Gas chromatographic (GC)
analyses were run on a Hewlett Packard 5890 A gas chro-
matograph (column: Supelco 2-5315 SPB-1, 30 m × 0.53 mm,
0.50 µm film). Evaporations were performed at reduced
pressure, and all oily products were dried at 0.1 mbar for 16
h. Combustion elemental analyses were performed by Redox
s.n.c., Milan, Italy, and found values were within 0.4% of the
theoretical values (unless otherwise indicated).
Syn th esis of Kn ow n In ter m ed ia tes. 6-Methoxyisatin
(5a ′) was prepared according to Mangini and Passerini;52
N-(methylphenyl)gycine was synthesized as described by
O’Donnell et al.,33 and all the R-amino acids were converted
to the corresponding R-amino methyl esters‚HCl by refluxing
in MeOH in the presence of 1.7 equiv of SOCl2. Tetrahydro-
furan (THF) was dried by distillation over LiAlH4 and stored
over 4 Å molecular sieves under nitrogen atmosphere; CH2-
Cl2 was dried over CaCl2; MeCN and DMF were stored over 4
Å molecular sieves; triethylamine (TEA) was dried by distil-
lation and stored over KOH. Isatin (5a ), ketones 6a -x,
2-phenylquinoline-4-carboxylic acid (7a ), aniline, 2-phenyl-
ethylamine, (R,S)-, (R)-, and (S)-phenylglycine, (R)-valine, (R)-
(4-hydroxyphenyl)glycine, (R,S)-2-thienylglycine, ethyl 3-oxo-
3-phenylpropionate (12), R-bromoacetophenone (18), 1,3-
diphenylacetone (21), deoxybenzoin (25), benzanilide (30), and
all reagents utilized in Schemes 1-7 are commercially avail-
able compounds and were used without further purification.
Activated manganese(IV) oxide (MnO2; <5 µm, ca. 85%) was
supplied by Aldrich Chemical Co. and used without further
activation.
Meth od B: Via DCC/HOBT. 2-Phenylquinoline-4-car-
boxylic acids 7a ,d -f,i,k ,o,q,s-x (8.0 mmol) were dissolved in
a 7:3 mixture of THF/MeCN (200 mL), and the appropriate
R-amino ester‚HCl (9.8 mmol) was added along with N-
methylmorpholine or triethylamine (TEA) (10.7 mmol) and
1-hydroxybenzotriazole (HOBT) (12.1 mmol); the reaction
mixture was cooled to -5 °C, and dicyclohexylcarbodiimide
(DCC) (12.1 mmol), dissolved in THF (10 mL), was added
dropwise; the reaction mixture was kept at 0-5 °C for 1 h and
then allowed to reach room temperature and left overnight.
The precipitated dicyclohexylurea was filtered off and the
filtrate evaporated to dryness. The residue was dissolved in
CH2Cl2 and washed with H2O, 5% citric acid, 5% NaHCO3,
and brine. The separated organic layer was dried over Na2-
SO4 and evaporated to dryness; the residue was dissolved in
CH2Cl2 (20 mL), and after overnight standing some more
dicyclohexylurea precipitated and was filtered off. The solu-
tion was evaporated to dryness, and the crude product was
triturated with i-Pr2O and recrystallized from the appropriate
solvent, affording the desired compounds in about 80% yield.
This method was used with racemic R-amino esters to obtain
the corresponding racemic amides 41, 44-46, 49, 51, 55, 57,
59-64, 67, 69, and 72; no significant difference in yields was
found between methods A and B. Method B was also employed
to prepare enantiomerically pure secondary amides 65 and 66
starting from compound 7a and (R)- and (S)-methyl phenyl-
glycinate, respectively. As an example, spectroscopic data for
compound 65 are reported: mp 180-181 °C (from i-PrOH);
[R]20 (c ) 0.5, MeOH) ) -42.0; IR (Nujol) 3300, 1750, 1640
D
cm-1
;
1H NMR (DMSO-d6) δ 9.72 (d, J ) 6.9 Hz, 1H), 8.28
(dd, J ) 8.3, 1.8 Hz, 2H), 8.20 (dd, J ) 8.3, 0.8 Hz, 1H), 8.13
(dd, J ) 8.5, 0.5 Hz, 1H), 8.11 (s, 1H), 7.83 (ddd, J ) 7, 7, 1.6
Hz, 1H), 7.66 (ddd, J ) 7, 7, 1.2 Hz, 1H), 7.60-7.50 (m, 6H),
7.47-7.37 (m, 3H), 5.78 (d, J ) 6.9 Hz, 1H), 3.72 (s, 3H); EI-
MS (source 180 °C, 70 eV, 200 mA) m/z 396 (M•+), 337, 232,
204. Anal. (C25H20N2O3) C, H, N.
Gen er a l P r oced u r e for th e P fitzin ger Rea ction 30 To
Obta in 2-P h en ylqu in olin e-4-ca r boxylic Acid s 7a -x in
Sch em e 1. Isatins 5a ,a ′ (34.0 mmol), the appropriate ketone
of formula 6a -x (40.8 mmol), and 85% KOH pellets (102
mmol) were dissolved in EtOH (40 mL), and the reaction
mixture was stirred at 80 °C for 24-72 h. Evaporation of the
solvent afforded a residue which was dissolved in H2O (50 mL),
and the solution was washed twice with Et2O (30 mL). The
ice-cold aqueous phase was acidified to pH 1 with 37% HCl,
and the precipitate was collected by suction filtration, washed
with H2O, and dried. Typically, yields of this reaction were
Enantiomeric excess of 65 and 66 was determined by chiral
HPLC eluting the compounds on Daicel Chiralcel OC column
(10 µm, 4.6 × 250 mm; 1.0 mL/min, UV 220) with a unique
mobile phase consisting of 60% hexane, 40% EtOH, and 0.1%
Et3N; both enantiomers 65 and 66 had enantiomeric excess
greater than 99%. Combustion elemental analyses of com-
pounds 4, 33, 34, 38, 40-69, 71, and 72 of general formulae
I-IV in Tables 1-4 agreed with calculated data within (0.4%

1804 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Giardina et al.
unless otherwise indicated in footnotes to the tables. Detailed
spectroscopic data for compounds 4, 33, 34, and 38-72 are
given in the Supporting Information.
precipitated mixture of compounds 15 and 16 was collected
by suction filtration; pure 16 (47.5 mmol, overall 34%) was
obtained by repeated washing with hot MeOH: mp >250 °C;
1H NMR (DMSO-d6) δ 8.00 (d, J ) 8.0 Hz, 1H), 7.85-7.79 (m,
2H), 7.61-7.55 (m, 3H), 7.21 (d, J ) 2.8 Hz, 1H), 6.92 (dd, J
) 8.0, 2.8 Hz, 1H), 6.28 (s, 1H), 3.88 (s, 3H); EI-MS (source
200 °C, 70 eV, 200 mA) m/z 251 (M•+), 208.
4-(Hyd r oxym eth yl)-7-m eth oxy-2-p h en ylqu in olin e (9).
The acyl chloride 8 was prepared by method A, starting from
7a ′ (50.4 mmol) and oxalyl chloride (85.7 mmol). Crude 8 was
suspended, under nitrogen atmosphere, in THF (300 mL), the
reaction mixture was cooled to -78 °C, and lithium tri-tert-
butoxyaluminum hydride (1 M solution in THF, 80 mL) was
added dropwise maintaining the temperature below -70 °C.
After 2 h at -70 °C, the reaction mixture was stirred at room
temperature overnight and ice-cooled and the reaction quenched
with H2O (150 mL). After evaporation of THF, CH2Cl2 (150
mL) was added, the mixture was filtered, and the organic layer
was separated, dried over Na2SO4, and evaporated to dryness.
Compound 9 (31.3 mmol, 62%) was obtained as a white solid:
mp 165-168 °C; IR (KBr) 3180 br, 1620, 1600, 1095 cm-1; 1H
NMR (CDCl3) δ 8.14 (dd, J ) 6.6, 1.0 Hz, 2H), 7.85 (d, J ) 9.0
Hz, 1H), 7.84 (s, 1H), 7.53 (d, J ) 2.5 Hz, 1H), 7.52-7.42 (m,
3H), 7.20 (dd, J ) 9.0, 2.5 Hz, 1H), 5.20 (s, 2H), 3.98 (s, 3H),
1.60 (s br, 1H); EI-MS (source 180 °C, 70 eV, 200 mA) m/z 265
(M•+), 264, 236.
7-Meth oxy-2-p h en ylqu in olin e-4-ca r boxa ld eh yd e (10).
MnO2 (92.0 mmol) was suspended in CH2Cl2 (300 mL); 4-(hy-
droxymethyl)-7-methoxy-2-phenylquinoline (9; 27.1 mmol),
dissolved in CH2Cl2 (1000 mL), was added dropwise, and the
reaction mixture was stirred at room temperature for 48 h.
Additional MnO2 (80.5 mmol) was added portionwise during
a period of 4 days. The reaction mixture was filtered on a
Celite pad, evaporated to dryness, and triturated with i-Pr2O
to yield 10 (22.4 mmol, 83%) as a pale yellow solid: mp 124-
126 °C; IR (KBr) 2960, 1710, 1620, 1590 cm-1; 1H NMR (CDCl3)
δ 10.52 (s, 1H), 8.87 (d, J ) 9.5 Hz, 1H), 8.21 (dd, J ) 6.1, 1.1
Hz, 2H), 8.11 (s, 1H), 7.59 (d, J ) 2.8 Hz, 1H), 7.58-7.48 (m,
3H), 7.34 (dd, J ) 9.5, 2.8 Hz, 1H), 4.00 (s, 3H); EI-MS (source
180 °C, 70 eV, 200 mA) m/z 263 (M•+), 248, 243, 235, 220.
4-[(Ben zyla m in o)m et h yl]-7-m et h oxy-2-p h en ylq u in o-
lin e Dih yd r och lor id e (35). 7-Methoxy-2-phenylquinoline-
4-carboxaldehyde (10; 3.8 mmol), benzylamine (4.1 mmol), and
toluene (30 mL) were refluxed in a Dean-Stark apparatus
with a catalytic amount of PTSA for 3 days. Formation of 11
was followed by GC. After removal of the solvent, 11 was
obtained and used without further purification. Crude 11 was
dissolved in AcOH (60 mL), and sodium triacetoxyborohydride
(9.4 mmol) was added portionwise in 15 min, maintaining the
temperature at 20 °C. The reaction mixture was maintained
at room temperature for 24 h, then the solvent was evaporated
to dryness, and the crude material was dissolved in CH2Cl2
and washed with 5% NaHCO3 and brine. The organic layer
was dried over Na2SO4, evaporated to dryness, and purified
by flash column chromatography on silica gel. The crude
product was dissolved in Et2O, acidified with HCl/Et2O; the
precipitate was collected and recrystallized from i-PrOH to
yield 35 (1.4 mmol, 37% overall yield) as a pale yellow solid:
mp 216-218 °C; IR (KBr) 3460 br, 2720 br, 1632, 1601, 1235
cm-1; 1H NMR (DMSO-d6) δ 10.00 (s br, 2H), 8.35 (s, 1H), 8.30
(dd, 2H), 8.13 (d, 1H), 7.69-7.53 (m, 6H), 7.47-7.42 (m, 4H),
7.36 (dd, 1H), 4.79 (t, 2H), 4.39 (t, 2H), 4.00 (s, 3H); EI-MS
(source 200 °C, 70 eV, 200 mA) m/z 354 (M•+), 249, 91. Anal.
(C24H22N2O‚2HCl) H, N; C: calcd, 67.45; found, 66.80.
3-Oxo-3-p h en ylp r op ion a m id e (13). Ethyl 3-oxo-3-phen-
ylpropionate (12; 0.17 mol) was heated with 32% NH4OH in a
steel autoclave at 85 °C for 2 h; the precipitated iminoamide
was recovered by suction filtration and refluxed for 4 h in
water; the precipitate was filtered and recrystallized from
water, affording the pure amide 13 (0.14 mol, 81%): mp 110-
111 °C (lit.53 mp 111-113 °C).
From the concentrated methanolic mother liquor, the re-
gioisomer 15 (32.0 mmol, 23%) crystallized on standing: mp
1
217-220 °C; H NMR (DMSO-d6) δ 7.84 (m, 2H), 7.60-7.50
(m, 5H), 7.33 (d, J ) 7.9 Hz, 1H), 6.78 (d, J ) 7.9 Hz, 1H),
6.33 (s br, 1H), 3.82 (s, 3H); EI-MS (source 200 °C, 70 eV, 200
mA) m/z 251 (M•+), 208.
4-Ch lor o-7-m eth oxy-2-ph en ylqu in olin e (17). 4-Hydroxy-
7-methoxy-2-phenylquinoline (16; 43.6 mmol) was refluxed in
POCl3 (120 mL) for 2 h; the solvent was evaporated, and the
residue was taken up with 10% NaOH and extracted with
EtOAc; the organic layer was dried over Na2SO4 and the
solvent evaporated to afford 17 (29.6 mmol, 68%): mp 99-
100 °C; IR (Nujol) 1620, 1591, 1579, 1214 cm-1 1H NMR
;
(DMSO-d6) δ 8.29 (m, 2H), 8.20 (s, 1H), 8.10 (d, J ) 9.6 Hz,
1H), 7.59-7.50 (m, 4H), 7.39 (dd, J ) 9.6, 2.5 Hz, 1H), 4.00 (s,
3H); ESI-MS (A positive, solvent methanol, spray 4.5 keV,
skimmer 60 eV, capillary 220 °C) m/z 270 (MH+); ESI-MS (B
DAU+ 270, collision gas Ar) m/z 227, 191, 165, 149, 114, 89.
4-(Ben zyla m in o)-7-m eth oxy-2-p h en ylqu in olin e (36). A
mixture of 4-chloro-7-methoxy-2-phenylquinoline (17; 3.7 mmol),
benzylamine (3.7 mmol), and phenol (4.6 g) was heated at 150
°C for 3 h; after cooling, CH2Cl2 (20 mL) and 0.1 N NaOH (20
mL) were added to the reaction mixture; the organic layer was
washed with 0.1 N NaOH until disappearance of phenol, dried
over Na2SO4, and evaporated. The residue was purified by
flash column chromatography, by eluting with CH2Cl2/MeOH/
28% NH4OH, 94:2:0.2, respectively. The resulting solid was
crystallized from Et2O, affording 36 (2.5 mmol, 67%): mp 171-
174 °C; IR (KBr) 3260 br, 1620, 1590 cm-1; 1H NMR (DMSO-
d6) δ 8.21 (d, J ) 9.0 Hz, 1H), 8.02 (dd, J ) 7.5, 0.9 Hz, 2H),
7.85 (t, J ) 6.0 Hz, 1H), 7.48-7.40 (m, 5H), 7.35 (dd, J ) 7.5,
7.5 Hz, 2H), 7.26 (d, J ) 2.8 Hz, 1H), 7.22 (dd, J ) 7.5, 7.5 Hz,
1H), 7.08 (dd, J ) 9.0, 2.8 Hz, 1H), 6.80 (s, 1H), 4.67 (d, J )
6.0 Hz, 1H), 3.90 (s, 3H); EI-MS (source 200 °C, 70 eV, 200
mA) m/z 340 (M•+), 91. Anal. (C23H20N2O) H, N; C: calcd,
81.15; found, 80.66.
N-(Ben zyloxyca r bon yl)-4-a m in o-7-m eth oxy-2-p h en yl-
qu in olin e (37). 7-Methoxy-2-phenylquinoline-4-carboxylic
acid (7a ′; 59.1 mmol), DPPA (59.1 mmol), and TEA (59.1 mmol)
were heated at 80 °C for 20 h in PhCH2OH (160 mL).32
Evaporation of the solvent afforded a residue which was taken
up in 5% NaHCO3 and extracted with CH2Cl2. The organic
layer was dried over Na2SO4 and the solvent evaporated. The
residue was chromatographed on silica gel, eluting with
EtOAc/hexane (1:1), affording 37 (27.8 mmol, 47%) which was
crystallized from EtOAc: mp 156-158 °C; IR (KBr) 3475, 1750,
1620, 1570, 1540, 1505, 1210 cm-1; 1H NMR (DMSO-d6) δ 10.20
(s, 1H), 8.39 (s, 1H), 8.29 (d, J ) 9.0 Hz, 1H), 8.13 (dd, J )
9.0, 1.7 Hz, 2H), 7.58-7.48 (m, 5H), 7.47-7.36 (m, 3H), 7.43
(s, 1H), 7.20 (dd, J ) 9.0, 2.5 Hz, 1H), 5.28 (s, 2H), 3.92 (s,
3H); EI-MS (source 180 °C, 70 eV, 200 mA) m/z 384 (M•+), 276,
91, 77. Anal. (C24H20N2O3) C, H, N.
(R,S)-N-(r-Ca r boxyben zyl)-7-m eth oxy-2-p h en ylqu in o-
lin e-4-ca r boxa m id e Hyd r och lor id e (39). (R,S)-N-[R-(Meth-
oxycarbonyl)benzyl]-7-methoxy-2-phenylquinoline-4-carbox-
amide (38; 0.40 mmol) was refluxed in 10 mL of 10% HCl and
5 mL of dioxane for 3 h; then the solvent was evaporated to
dryness. Crystallization from EtOAc (containing a few drops
of EtOH) afforded 39 (0.35 mmol, 89%): mp 228-230 °C; IR
(KBr) 3180, 1735, 1655, 1630 cm-1; 1H NMR (DMSO-d6) δ 9.6
(d, 1H), 8.26 (dd, 2H), 8.14 (d, 1H), 7.98 (s, 1H), 7.63-7.52 (m,
6H), 7.46-7.36 (m, 3H), 7.33 (dd, 1H), 5.66 (d, 1H), 3.98 (s,
3H); EI-MS (source 200 °C, 70 eV, 200 mA) m/z 412 (M•+), 368,
262, 234, 191, 77. Anal. (C25H20N2O4‚HCl) C, H, N.
(R )-N -[r-(Me t h o x y c a r b o n y l)-4-m e t h o x y b e n zy l]-2-
p h en ylqu in olin e-4-ca r boxa m id e (70). (R)-N-[R-(Methoxy-
carbonyl)-4-hydroxybenzyl]-2-phenylquinoline-4-carboxam-
ide (69; 1.5 mmol) was dissolved in 30 mL of dry acetone and
2 mL of dry DMF, and the solution was stirred for 30 min in
4-Hyd r oxy-7-m eth oxy-2-p h en ylqu in olin e (16). Com-
pound 13 (0.14 mol) was refluxed in a Dean-Stark apparatus
with m-anisidine (0.14 mol) and m-anisidine‚HCl (catalytic)
in toluene (300 mL) for 8 h. Formation of imine 14 was
followed by GC; the reaction mixture was concentrated to one-
half volume, and 14 crystallized on standing; the crude imine
intermediate was filtered, mixed with PPA (200 g), and heated
to 135 °C for 20 min. The reaction mixture was poured into
water and carefully adjusted to pH 8 with 20% NaOH; the

Non-Peptide Antagonists for the NK-3 Receptor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1805
the presence of anhydrous K2CO3 (0.75 mmol); MeI (1.5 mmol)
was added and the reaction mixture heated to 40 °C for 4 h;
after cooling, anhydrous K2CO3 (0.75 mmol) and MeI (1.5
mmol) were added again, and the mixture was refluxed for
an additional 6 h. The mixture was evaporated to dryness,
dissolved in EtOAc, and washed with brine. The organic layer,
dried over Na2SO4, was evaporated to dryness. The residue
was crystallized from Et2O affording 70 (1.06 mmol, 70%): mp
160-162 °C; IR (KBr) 3210, 1750, 1635, 1625, 1590, 1530, 1515
cm-1; 1H NMR (DMSO-d6) δ 9.65 (d, 1H), 8.28 (d, 2H), 8.21 (d,
1H), 8.14 (d, 1H), 8.10 (s, 1H), 7.84 (dd, 1H), 7.67 (dd, 1H),
7.61-7.49 (m, 3H), 7.44 (d, 2H), 6.98 (d, 2H), 4.70 (d, 1H), 3.79
(s, 3H), 3.76 (s, 3H); EI-MS (source 200 °C, 70 eV, 200 mA)
m/z 426 (M•+), 367, 232, 204. Anal. (C26H22N2O4) C, H, N.
4-P h en yloxa zole (19).34 A mixture of R-bromoacetophe-
none (18; 100.5 mmol), HCOONH4 (348.8 mmol), and 98%
HCOOH (106.5 mL) was refluxed under stirring for 2.5 h. The
reaction mixture was poured into water, made alkaline with
concentrated NaOH, and extracted with Et2O. The organic
layer was washed with H2O, dried over Na2SO4, and evapo-
rated to dryness. The crude product obtained was purified by
flash column chromatography on silica gel, eluting with a
mixture of EtOAc/hexane (3:7), to afford 19 (15.8 mmol, 16%)
as a yellow oil: IR (neat) 3120, 1512 cm-1; 1H NMR (CDCl3) δ
7.94 (m, 2H), 7.75 (d, J ) 7.5 Hz, 2H), 7.42 (dd, J ) 7.5, 7.5
Hz, 2H), 7.33 (dd, J ) 7.5, 7.5 Hz, 1H); EI-MS (source 200 °C,
70 eV, 200 mA) m/z 145 (M•+), 90, 41.
2-P h en yl-4-n a p h th a len eca r boxylic Acid (24). N-(Di-
methylamino)-2-phenyl-4-naphthalenecarboxamide (23; 7.3
mmol) and 85% KOH pellets (21.4 mmol) were heated in
ethylene glycol (20 mL) at 200 °C for 16 h; the mixture was
then carefully diluted with a solution of 40% citric acid and
extracted with EtOAc; the organic layer was washed with brine
and dried over Na2SO4 and the solvent evaporated to dryness
to afford 24 (7.2 mmol, 98%) as a white solid: mp 215-218
°C; IR (KBr) 2450, 1695, 1260, 765 cm-1; 1H NMR (DMSO-d6)
δ 13.05 (s br, 1H), 8.85 (m, 1H), 8.47 (d, J ) 2.0 Hz, 1H), 8.43
(d, J ) 2.0 Hz, 1H), 8.11 (m, 1H), 7.84 (d, J ) 7.0 Hz, 2H),
7.69-7.59 (m, 2H), 7.55 (dd, J ) 7.0, 7.0 Hz, 2H), 7.45 (dd, J
) 7.0, 7.0 Hz, 1H); EI-MS (source 200 °C, 70 eV, 200 mA) m/z
248 (M•+), 231, 202.
N-[r-(Meth oxyca r bon yl)ben zyl]-2-p h en yln a p h th a len e-
4-ca r boxa m id e (74). A mixture of 24 (6.8 mmol), (R,S)-
methyl phenylglycinate‚HCl (10.3 mmol), 1-hydroxybenzotri-
azole (10.3 mmol), TEA (17.0 mmol), THF (70 mL), and CH3CN
(30 mL) was cooled, under nitrogen atmosphere, to 0 °C. DCC
(10.3 mmol), dissolved in THF (20 mL), was added dropwise,
and the reaction mixture was stirred at 0 °C for 1 h and at
room temperature overnight; 20% citric acid (20 mL) was
added, and the reaction mixture was stirred for 2 h and
filtered; the filtrate was evaporated to dryness, dissolved in
Et2O, and washed with 20% citric acid, 5% NaHCO3, and brine;
the organic layer was dried over Na2SO4 and the solvent
evaporated to dryness. The crude product was crystallized
from toluene to afford 74 (4.3 mmol, 63%) as white crystals:
mp 141-143 °C; IR (KBr) 3310, 1755, 1640, 1600, 1520, 770
cm-1; 1H NMR (DMSO-d6) δ 9.53 (d, J ) 7.0 Hz, 1H), 8.33 (d,
J ) 1.6 Hz, 1H), 8.26 (m, 1H), 8.07 (m, 1H), 7.93 (d, J ) 2.0
Hz, 1H), 7.86 (d, J ) 7.0 Hz, 2H), 7.62-7.52 (m, 6H), 7.45-
7.36 (m, 4H), 5.80 (d, J ) 7.0 Hz, 1H), 3.75 (s, 3H); EI-MS
(source 200 °C, 70 eV, 200 mA) m/z 395 (M•+), 336, 231, 202.
Anal. (C26H21NO3) C, H, N.
2-P h en ylp yr id in e-4-ca r boxylic Acid (20).35 A mixture
of 4-phenyloxazole (19; 13.8 mmol) and maleic acid (14.2 mmol)
was heated at 110 °C until melting for 15 min. The solid
obtained after cooling was triturated with Et2O, collected by
suction filtration, suspended in a mixture of H2O and MeOH,
and acidified to pH 1 with 20% HCl. The solvent was removed
by decantation, and the solid was stirred in EtOAc for 1 week
and then recrystallized with 95% EtOH to yield 20 (2.5 mmol,
18%) as an off-white solid: mp 266-268 °C; IR (KBr) 3300,
1720 cm-1; 1H NMR (DMSO-d6) δ 8.76 (dt, J ) 4 Hz, 1H), 8.20
(s, 1H), 8.35-7.45 (m, 5H), 7.71 (dt, J ) 4 Hz, 1H); FAB-MS
(negative, matrix diethanolamine, gas Xe, 8 keV, source 50
°C) m/z 198 (M - H-).
1-Meth yl-3-ph en ylisoqu in olin e (27).37 Deoxybenzoin (25;
25.5 mmol) was dissolved in CH3CN (200 mL), and POCl3 (15.6
mL) was added dropwise at room temperature. The reaction
mixture was refluxed for 29 h and then evaporated to dryness.
The crude product was dissolved in H2O, made alkaline with
2 N NaOH, and extracted with CH2Cl2; the organic layer was
washed with H2O, dried over Na2SO4, and evaporated. The
residue was purified by flash column chromatography on silica
gel eluting with a mixture of CH2Cl2/hexane (65:35) to afford
27 (5.9 mmol, 23%) as a pale yellow oil: IR (neat) 3050, 1620,
(R,S)-N-[r-(Meth oxycar bon yl)ben zyl]-2-ph en ylpyr idin e-
4-ca r boxa m id e (73). A mixture of 2-phenylpyridine-4-car-
boxylic acid (20; 2.4 mmol), (R,S)-methyl phenylglycinate‚HCl
(2.5 mmol), 1-hydroxybenzotriazole (4.8 mmol), N-methylmor-
pholine (5.4 mmol), THF (35 mL), and CH3CN (15 mL) was
cooled to 0 °C under nitrogen atmosphere; DCC (2.7 mmol),
dissolved in THF (3 mL), was added dropwise, and the reaction
mixture was stirred at 0 °C for 1 h and at room temperature
for 2 h. The precipitate was filtered off, and the solvent was
evaporated to dryness. The residue was dissolved in CH2Cl2,
washed with H2O, 20% citric acid, 5% NaHCO3, and brine,
dried over Na2SO4, and evaporated to dryness. The residue
was purified by flash column chromatography on silica gel,
eluting with a mixture of hexane/EtOAc/28% NH4OH (60:40:
0.5), respectively, and then washed with i-Pr2O and recrystal-
lized with i-PrOH to yield 73 (0.3 mmol, 12%) as a white
1590, 1570 cm-1 1H NMR (DMSO-d6) δ 8.26-8.21 (m, 4H),
;
8.01 (dd, J ) 8.3, 1.2 Hz, 1H), 7.77 (ddd, J ) 8.3, 8.3, 1.2 Hz,
1H), 7.65 (ddd, J ) 8.3, 8.3, 1.2 Hz, 1H), 7.52 (dd, J ) 7.5, 7.5
Hz, 2H), 7.41 (dd, J ) 7.5, 7.5 Hz, 1H), 2.95 (s, 3H); EI-MS
(source 200 °C, 70 eV, 200 mA) m/z 219 (M•+).
3-P h en ylisoqu in olin e-1-ca r boxylic Acid (29).38 A mix-
ture of 27 (18.7 mmol), N-bromosuccinimide (NBS; 52.4 mmol),
dibenzoyl peroxide (1.4 mmol), and CCl4 (150 mL) was refluxed
for 19 h. The insoluble material was filtered off, and the
filtrate was washed with 5% NaHCO3 and brine; the organic
layer was dried over Na2SO4 and evaporated to dryness.
1-(Dibromomethyl)-3-phenylisoquinoline was obtained as an
orange oil and utilized without further purification (TLC:
eluent CH2Cl2, Rf(27) 0.38, Rf(dibromo derivative) 0.74).
The crude 1-(dibromomethyl)-3-phenylisoquinoline was dis-
solved in a mixture of 95% EtOH (120 mL) and THF (60 mL)
and heated to reflux; AgNO3 (73.6 mmol), dissolved in H2O
(15 mL), was added; the reaction mixture was refluxed for 1 h
and filtered while still hot, washing with THF. The filtrate
was evaporated to dryness to afford 28 which was used without
further purification (TLC: eluent CH2Cl2, Rf(dibromo deriva-
tive) 0.74, Rf(28) 0.56).
solid: mp 139-141 °C; IR (KBr) 3290, 1750, 1630 cm-1 1H
;
NMR (DMSO-d6) δ 9.60 (d, J ) 6.8 Hz, 1H), 8.82 (d, J ) 4.5
Hz, 1H), 8.37 (s, 1H), 8.14 (dd, J ) 7.5, 0.8 Hz, 2H), 7.77 (dd,
J ) 4.5, 1.6 Hz, 1H), 7.57-7.37 (m, 8H), 5.73 (d, J ) 6.8 Hz,
1H), 3.70 (s, 3H); TSP-MS (DAU+ 347, -9 eV, collision gas
Ar) m/z 347 (MH+), 155. Anal. (C26H21NO3) C, H, N.
N-(Dim et h yla m in o)-2-p h en yl-4-n a p h t h a len eca r b ox-
a m id e (23).36 Diphenylacetone (21; 23.8 mmol) and N,N-
dimethylformamide dimethyl acetal (22; 0.15 mol) were heated
in a steel autoclave at 180 °C for 24 h under nitrogen
atmosphere; the reaction mixture was then diluted with a
mixture of Et2O/hexane (1:5) and stirred for 1 h. The
precipitate was filtered, washed with hexane, and dried to
afford 23 (14.9 mmol, 63%) as white needles: 137-138 °C (lit.36
The crude 28 was dissolved in EtOH and treated with
AgNO3 (45.9 mmol) dissolved in H2O (8 mL). A solution of
NaOH (158.8 mmol) in H2O (98 mL) was added dropwise, and
the reaction mixture was maintained under stirring overnight.
Insoluble material was filtered on a Celite pad and washed
with Et2O, and the filtrate was acidified with concentrated HCl
(5 mL), reduced to one-third of the volume, and ice-cooled. The
formed precipitate was collected by suction filtration, washed
with H2O, dried, and triturated with Et2O to yield 29 (8.8
mp 136-138 °C); IR (KBr) 1635, 1490, 1410, 890 cm-1 1H
;
NMR (DMSO-d6) δ 8.29 (s, 1H), 8.07 (m, 1H), 7.85 (d, J ) 7.4
Hz, 2H), 7.77 (d, J ) 1.6 Hz, 1H), 7.73 (m, 1H), 7.63-7.56 (m,
2H), 7.52 (dd, J ) 7.4, 7.4 Hz, 2H), 7.42 (dd, J ) 7.4, 7.4 Hz,
1H), 3.13 (s, 3H), 2.80 (s, 3H); EI-MS (source 200 °C, 70 eV,
200 mA) m/z 275 (M•+), 231, 202.

1806 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
mmol, 47% overall yield) as a yellow solid: mp 132-135 °C;
Giardina et al.
Hz, 1H), 7.64-7.53 (m, 5H), 7.47-7.37 (m, 3H), 5.85 (d, J )
7.5 Hz, 1H), 3.79 (s, 3H); EI-MS (source 200 °C, 70 eV, 200
mA) m/z 338, 205. Anal. (C24H19N3O3) C, H, N.
1
IR (KBr) 1760 cm-1; H NMR (DMSO-d6) δ 13.70 (s br, 1H),
8.63 (s, 1H), 8.51 (dd, J ) 9.0, 1.2 Hz, 1H), 8.26 (d, J ) 7.5
Hz, 2H), 8.14 (dd, J ) 8.8, 1.0 Hz, 1H), 7.85 (ddd, J ) 8.8, 8.8,
1.0 Hz, 1H), 7.74 (ddd, J ) 9.0, 8.8, 1.0 Hz, 1H), 7.55 (dd, J )
7.5, 7.5 Hz, 2H), 7.46 (dd, J ) 7.5, 7.5 Hz, 1H); EI-MS (source
200 °C, 70 eV, 200 mA) m/z 249 (M•+), 205, 204.
Ack n ow led gm en t. We are grateful to J oseph Wein-
stock,| Geoffrey D. Clarke, and Paola Zaratin for
helpful discussions; we wish to thank Karl F. Erhard|
for chiral HPLC determinations; we acknowledge Da-
vide Graziani,# Stefano Cavagnera,# and Roseanna
Muccitelli∇ for their chemical and biological technical
expertise; we also thank Renzo Mena# and Alberto
Cerri# for mass and NMR spectroscopic determinations
(R,S)-N-[r-(Meth oxycar bon yl)ben zyl]-3-ph en ylisoqu in -
olin e-1-ca r boxa m id e (75). A mixture of 29 (6.0 mmol),
(R,S)-methyl phenylglycinate‚HCl (6.4 mmol), 1-hydroxyben-
zotriazole (11.8 mmol), N-methylmorpholine (7.3 mmol), THF
(50 mL), and CH3CN (20 mL) was cooled to 0 °C under nitrogen
atmosphere. DCC (6.8 mmol), dissolved in THF (7 mL), was
added dropwise, and the reaction mixture was stirred at 0 °C
for 1 h and at room temperature for 2 h. The insoluble
material was filtered off, and the solvent was evaporated to
dryness. The crude product was dissolved in CH2Cl2, washed
with H2O, 20% citric acid, 5% NaHCO3, and brine, and dried
over Na2SO4 and the solvent evaporated. The residue was
triturated with warm i-Pr2O and recrystallized from 95%
EtOH to yield 75 (4.1 mmol, 68%) as a white solid: mp 182-
(|Medicinal Chemistry and Pulmonary Pharmacology,
SmithKline Beecham Pharmaceuticals, PA; Depart-
ment of Biology and Department of Chemistry, Smith-
∇
#
Kline Beecham S.p.A., Italy).
Su p p or tin g In for m a tion Ava ila ble: Detailed spectro-
scopic data (IR, MS, and 1H NMR) for compounds 4, 33, 34,
and 38-72 (9 pages). Ordering information is given on any
current masthead page.
183 °C; IR (KBr) 3380, 1745, 1680, 1620 cm-1 1H NMR
;
(DMSO-d6) δ 9.65 (d, J ) 7.2 Hz, 1H), 8.80 (d, J ) 9.0 Hz,
1H), 8.61 (s, 1H), 8.28 (d, J ) 7.2 Hz, 2H), 8.12 (d, J ) 9.0 Hz,
1H), 7.82 (dd, J ) 9.0, 9.0 Hz, 1H), 7.71 (dd, J ) 9.0, 9.0 Hz,
1H), 7.60-7.54 (m, 4H), 7.50-7.35 (m, 4H), 5.80 (d, J ) 7.2
Hz, 1H), 3.75 (s, 3H); EI-MS (source 200 °C, 70 eV, 200 mA)
m/z 396 (M•+), 337, 232, 204. Anal. (C25H20N2O3) C, H, N.
2-P h en ylqu in a zolin e-4-ca r boxylic Acid (32).39 Benzani-
lide (30; 39.0 mmol) and SOCl2 (10 mL) were refluxed for 3 h,
and the solvent was evaporated to dryness to afford compound
31 as a yellow oil. SnCl4 (39.0 mmol) was added dropwise to
a solution of crude 31 and ethyl cyanoformate (39.0 mmol) in
o-dichlorobenzene (30 mL); the reaction mixture was heated
at 140 °C for 15 min, poured into water, made alkaline with
20% NaOH, and extracted with CH2Cl2; the organic layer was
washed with brine and dried over Na2SO4. The dark oily
residue was treated with warm i-Pr2O and the insoluble
material discarded by filtration; the filtrate was evaporated
to dryness and the residue flash chromatographed on silica
gel, eluting with hexane/EtOAc (98:2) to afford ethyl 2-phen-
ylquinazoline-4-carboxylate as a yellow oil: IR (KBr) 1740,
1550, 1210, 770 cm-1; 1H NMR (DMSO-d6) δ 8.55 (m, 2H), 8.35
(d, 1H), 8.15 (m, 2H), 7.80 (m, 1H), 7.60 (m, 3H), 4.60 (q, 2H),
1.40 (t, 3H).
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The corresponding carboxylic acid 32 was obtained by
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and 95% EtOH (15 mL) for 2 h; the solvent was evaporated
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1
°C; IR (KBr) 3420, 1715, 1565, 1550 cm-1; H NMR (DMSO-
d6) δ 14.40 (s br, 1H), 8.61-8.55 (m, 2H), 8.41 (dd, J ) 9.0,
1.5 Hz, 1H), 8.17 (dd, J ) 9.0, 1.6 Hz, 1H), 8.10 (ddd, J ) 9.0,
9.0, 1.5 Hz, 1H), 7.81 (ddd, J ) 9.0, 9.0, 1.6 Hz, 1H), 7.63-
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