6-Acylamino-2-aminoquinolines as potent melanin-concentrating hormone 1 receptor antagonists. Identification, structure-activity relationship, and investigation of binding mode

Article abstract of DOI:10.1021/jm050103y

Novel 6-acylamino-2-aminoquinoline melanin-concentrating hormone 1 receptor (MCH1R) antagonists were identified by sequential in silico screening with 3D pharmacophore models derived from a series of benzamide antagonists. The structure-activity relationship exploration by synthesis of analogues found structural demands around the western part of the compounds to be quite specific, whereas much structural freedom was found in the eastern part. While these compounds in general suffered from poor solubility properties, the 4-trifluoromethoxy-phenoxyacetamide western appendage provided a favorable combination of activity and solubility properties. The amine in the eastern appendage, originally required by the pharmacophore model and believed to interact with Asp123 in transmembrane 3 of MCH1R, could be removed without diminishing affinity or functional activity of the compounds. Docking studies suggested that the Asp123 interacts preferentially with the nitrogen of the central quinoline. Synthesis and testing of specific analogues supported our revised binding mode hypothesis.

Full text of DOI:10.1021/jm050103y

5684
J. Med. Chem. 2005, 48, 5684-5697
6-Acylamino-2-aminoquinolines as Potent Melanin-Concentrating Hormone 1
Receptor Antagonists. Identification, Structure-Activity Relationship, and
Investigation of Binding Mode
Trond Ulven, Thomas M. Frimurer, Jean-Marie Receveur, Paul Brian Little, Øystein Rist,
Pia K. Nørregaard, and Thomas Ho¨gberg*
7TM Pharma A/S, Fremtidsvej 3, DK-2970 Hørsholm, Denmark
Received February 2, 2005
Novel 6-acylamino-2-aminoquinoline melanin-concentrating hormone 1 receptor (MCH1R)
antagonists were identified by sequential in silico screening with 3D pharmacophore models
derived from a series of benzamide antagonists. The structure-activity relationship exploration
by synthesis of analogues found structural demands around the western part of the compounds
to be quite specific, whereas much structural freedom was found in the eastern part. While
these compounds in general suffered from poor solubility properties, the 4-trifluoromethoxy-
phenoxyacetamide western appendage provided a favorable combination of activity and
solubility properties. The amine in the eastern appendage, originally required by the
pharmacophore model and believed to interact with Asp123 in transmembrane 3 of MCH1R,
could be removed without diminishing affinity or functional activity of the compounds. Docking
studies suggested that the Asp123 interacts preferentially with the nitrogen of the central
quinoline. Synthesis and testing of specific analogues supported our revised binding mode
hypothesis.
Introduction
Obesity has become a global epidemic with a steadily
increasing prevalence not only confined to the indus-
trialized countries.1 Thus, the condition is no longer
regarded as a cosmetic problem but a major contributor
to the development of diseases including type 2 diabetes
mellitus, coronary heart disease, certain forms of cancer,
osteoarthritis, and sleep apnoea. Recent estimates sug-
gest that 2-8% of the total healthcare costs in Western
countries are attributable to obesity.1 The treatment of
obesity may include several approaches, including (i)
reduction of food intake, (ii) prevention of fat absorption,
(iii) increase of thermogenesis, (iv) modulation of fat
metabolism or storage, and (v) modulation of central
control of body weight, and it is reasonable to expect
efficient future therapies to rely on a combination of
modalities.2 The increasing understanding of central
control mechanisms, especially hypothalamic neuropep-
tide pathways, has provided novel potential targets for
drug discovery.2,3 Thus, the orexigenic peptides agouti-
related protein (Agrp), neuropeptide Y (NPY), melanin-
concentrating hormone (MCH),4 ghrelin, and endocan-
nabinoids have been implicated in food intake and
energy homeostasis.3
MCH is a nonadecapeptide found in rat and human
brain, expressed in particular in the lateral hypothala-
mus, and the evidence for involvement of MCH in
feeding and body weight regulation is abundant.4 For
example, up-regulated MCH mRNA is observed in
fasting rats and in obese ob/ob rats, and food consump-
tion is increased upon icv injection of MCH in rats.5
Also, deletion of the MCH gene in mice results in a lean
phenotype,6 whereas overexpression of the MCH gene
Figure 1. MCH1R antagonists.10,14
in the lateral hypothalamus gives an obese and insulin-
resistant phenotype.7 Furthermore, the hormone is
reported to be involved in memory functions,8 anxiety,9
and depression.10 Two G protein-coupled seven-trans-
membrane (7TM) receptors, MCH1R (SLC-1) and
MCH2R (SLT), have been identified for MCH, although
only the former has been found in rodents.4,11,12 MCH1R-
deficient mice are reported to be lean, hyperactive, and
hyperphagic, indicating that this receptor plays a
prominent role in mediating the modulatory effect of
MCH on appetite and metabolism.13 The physiological
significance of MCH2R is still unclear.
Aminotetraline T-226296 was the first reported small-
molecule MCH1R antagonist,14 soon followed by SNAP-
794110 (Figure 1), and both these compounds were
reported to suppress food intake induced by icv-injected
MCH in rats. Furthermore, a selective peptide antago-
nist has been reported to reduce food intake and body
weight gain after chronic administration.15 Recently,
two additional compound series were reported by groups
at Schering-Plough and Abbott, both with representa-
* To whom correspondence should be addressed. Phone: +45 3925
7760. Fax: +45 3925 7776. E-mail: th@7tm.com.
10.1021/jm050103y CCC: $30.25 © 2005 American Chemical Society
Published on Web 08/13/2005

2-Aminoquinolines as Potent MCH1R Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5685
Figure 2. Representative benzamide MCH1R antagonists.17
tives exhibiting efficacy in a diet-induced obese mouse
model after oral administrations.16 Thus, MCH1R an-
tagonists are potentially interesting agents for treat-
ment of metabolic and obesity-related disorders, as well
as of disorders related to depression and anxiety.
We recently disclosed the development of a series of
substituted benzamides acting as MCH1R antagonists
(Figure 2).17 The design of these compounds relied on
the physicogenetic18 relationship of the MCH1R with
certain monoaminergic 7TM receptors with respect to
features in the putative binding site involved in recogni-
tion of small-molecule ligands. Thus, the benzamides
were derived by incorporation of structural features
from monoaminergic receptor ligands, in particular
dopamine D2 and D3 antagonists,19 combined with the
aminotetraline T-226296.14 Docking of potential ligands
in an MCH1R homology model corroborated the need
of a hydrophobic western side as present in the ami-
notetraline.20 In the present paper we describe an
efficient chemotype jump using this SAR knowledge to
construct 3D pharmacophores that are used for in silico
screening of commercial compound libraries and sub-
sequent optimization of the identified quinoline com-
pounds.21 It is notable that this compound series was
also independently discovered by Clark and co-workers
using different virtual screening approaches based on
11 compounds from the public domain.22 However, in
their case the 3D pharmacophore search did not produce
any hits, and the quinolines were identified by other
search strategies. Devita and co-workers have also
disclosed closely related quinoline derivatives.23a An-
other class of 2-aminoquinolines MCH1R antagonists
have been reported by Souers and co-workers.24
Figure 3. Four pharmacophore hypotheses on chemical
features recognized by MCH1R, based on previously disclosed
benzamide MCH1R antagonists.
representative benzamide MCH1R antagonists covering
an affinity range of 3 orders of magnitude were used as
structural input. A maximum of 255 conformers were
generated for each benzamide by Catalyst’s conforma-
tional search module, and the conformers were evalu-
ated and refined by a simulated annealing protocol (see
Experimental Section). Low-energy conformations of
representative active MCH1R benzamide ligands served
as reference structures to estimate the quality of the
alignments produced by HypoGen.
We also describe the chemical optimization and
structure-activity investigations of the appendages in
the 2 and 6 positions of the quinoline as well as the
4-substituent. We discovered that the eastern amine,
originally required by our pharmacophore models, could
be removed without loss of affinity. This prompted us
to carefully analyze the receptor interactions and revise
our original receptor binding hypothesis.
Spontaneous and induced conformational changes in
the protein can make a single static ligand-based
pharmacophore model unable to recognize the diversity
of possible binding modes induced by structurally dif-
ferent ligands.26 Taking this into account, 25 pharma-
cophore hypotheses were generated, all containing four
or five features, one of which was made optional. The
resulting pharmacophore hypotheses were clustered into
four main families, and individual models from each
family were manually evaluated. The four best hypoth-
eses, all containing five features, were chosen (Figure
3). They share a common eastern and western side, with
the western side comprising a hydrophobic and aromatic
feature and the eastern side comprising a hydrophobic
and distal positively charged group. Apart from the
spatial location of the features, the main difference
Computational Chemistry
Pharmacophore Models and in Silico Screening.
Three dimensional pharmacophore models were devel-
oped by taking advantage of binding data obtained from
the benzamide series containing more than 300 mem-
bers exhibiting various affinities to MCH1R (Figure 2).17
The models were produced using the pharmacophore
generation module HypoGen (Catalyst).25 Forty-two

5686 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18
Ulven et al.
Scheme 1a
between the four hypotheses is found in a single feature
in the central part.
Electronic catalogues were collected from the com-
mercial compound suppliers, and each compound was
transformed into a multiconformer library. The 3D
pharmacophore models (Figure 3) were used for the in
silico screening of these multiconformer libraries in a
stepwise fashion. The initial small targeted library that
was extracted produced 3% hits upon in vitro screening
and validation. Analysis of the positive and negative
information from the screening campaign was used to
modify the individual weights for the pharmacophoric
features. The 2-aminoquinolines were identified in the
subsequent in silico search of expanded commercial
libraries.
MCH1R Homology Modeling and Docking of
2-Aminoquinolines. The homology modeling study of
MCH1R utilized the crystal structure of bovine rhodop-
sin as structural template, and the primary sequence
alignment between MCH1R and bovine rhodopsin was
performed on the basis of conserved residues and
generic fingerprints in the TM segments. The 7TM
domains of the resulting MCH1R models were super-
imposed onto the 7TM domain of bovine rhodopsin
considering heavy backbone atoms only. The backbone
rms deviation was 0.08-0.58 Å. A number of residues
in the upper part of the 7TM ligand binding domain had
several possible side chain conformations that could be
mapped to different rotamer states. The most pro-
nounced side chain conformational differences were
observed for Asp123, Gln212, Tyr273, and Gln276, with
three, four, two and four rotamer states, respectively,
with less than two close contacts to other protein atoms
identified for the respective residues.
a Reagents and conditions: (a) amine (HNR2R3) (2-50 equiv),
neat or in EtOH or DMF, ∆; (b) >90% HNO3, 0 °C, 1 h; (c) Boc2O,
Et3N; (d) H2, Pd/C, THF; (e) aldehyde (R4CHO), MeONa, MeOH,
40 °C, 2 days; then NaBH4, 50 °C, 2 days; (f) acid chloride
(R5COCl), CH2Cl2, room temp, 3 h; (g) TFA, CH2Cl2.
Scheme 2a
a Reagents and conditions: (a) LDA (2 M in heptane), THF, -78
°C, 90 min, then MeI, -78 °C, 2 h f room temp over 1 h; (b)
mCPBA, CHCl3, room temp, 3 h; (c) POCl3, DIPEA, PhMe, 0 °C
f room temp, 4 h.
Scheme 3a
Finally, the MCH1R antagonists were iteratively
docked into the MCH1R model that assigned all com-
binatorial side chain conformations for the residues
specified above. The conformations used as input struc-
tures in the docking of the MCH1R antagonists were
established from a grid conformational search feature
within SYBYL to locate energy minima for the torsional
potential of the carbon-carbon bond at the central
amide.
a Reagents and conditions: (a) Me2NCH2CH2OH, NaH, DMF,
0 °C; (b) >90% HNO3, 0 °C, 1 h; (c) H2, Pd/C, THF; (d) acid chloride
(RCOCl), CH2Cl2, room temp, 12 h.
Chemistry
The majority of the compounds were synthesized by
one of the two alternative routes shown in Schemes 1
and 4, depending on which part of the molecule repre-
sented the focus for the exploration. The 2-aminoquino-
lines 2 (Scheme 1) were obtained by reacting 2-chloro-
quinolines (1) with the appropriate primary or secondary
amine. Exposure of 2 to cold fuming nitric acid gave the
6-nitro-2-aminoquinolines (3) with excellent yield and
selectivity.27 Reduction of the nitro group to afford 4 and
coupling to the appropriate acid chloride furnished the
wanted product 5. This sequence was generally per-
formed with excellent yields and without the need for
chromatography. In the cases where primary or second-
ary amines were present in the side chain of 3, these
were Boc-protected prior to reduction of the nitro group,
and the Boc group was removed in the final step.
Analogues with N-alkylated amide (R4 * H in Scheme
1) were prepared by reductive alkylation of aniline 4
prior to coupling.
The 4-ethyl analogues were synthesized from lepidine
(6) by homologation of the 4-methyl group, N-oxidation,
and treatment with phosphorus oxychloride to form
2-chloro-4-ethylquinoline 1d (Scheme 2), which served
as the substrate for the sequence shown in Scheme 1.
The 2-oxo analogues were synthesized as outlined in
Scheme 3. Reaction of 1a with dimethylaminoethoxide
afforded 8. Nitration of 8 was, as expected, less regi-
oselective than for the 2-aminoquinolines and gave 10%
of the 8-nitro isomer as a byproduct together with the
desired 9. Acylation of 9 yielded the target compounds
10.
The sequence outlined in Scheme 1 is highly efficient
for construction of libraries with diverse western parts.
However, for the exploration of the eastern part, this
protocol represents a four-step sequence for each com-
pound. Thus, an alternative approach was developed
using 2-chloro-6-nitroquinoline 11 as the starting point
(Scheme 4).28 Reduction of the nitro group by tin(II)

2-Aminoquinolines as Potent MCH1R Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5687
Scheme 4a
Out of the 411 compounds extracted in the second in
silico screening campaign, which included 1.1 million
commercially available compounds, 32 compounds ful-
filled the hit criterion of >80% displacement of [125I]-
MCH at 10 µM, corresponding to a hit rate of 8%. This
second selection contained 42 aminoquinolines, of which
22 were classified as hits. The majority of the remaining
aminoquinolines were also weak binders. The hits were
tested in full dose-response curves, where six com-
pounds, all aminoquinolines, exhibited affinities with
IC50 values below 100 nM, the most potent of which was
16 nM (Table 1). The quinolines coming out of this
campaign all shared the common core of 6-acylamino-
4-methyl-2-(1-piperazinyl)quinoline, and the compound
series displayed a number of clear structure-affinity
relationships.
a Reagents and conditions: (a) SnCl2‚2H2O, HCl (concentrated),
AcOH; (b) R1COCl, CH2Cl2, room temp, 12 h; (c) amine (R2R3NH),
∆.
In the eastern part, the piperazine was substituted
with either N-methyl (14) or N-ethyl with no significant
difference in the affinity of the compounds. The western
parts of the compounds were structurally diverse with
an aromatic ring as the common motif, preferably
separated from the 6-amide by two atom linkers such
as trans-ethenylene or methyleneoxy moieties. Out of
six compounds with a shorter methylene linker between
the amide and the aromatic ring, only one (15) displayed
affinity below 1 µM. Similarly, the majority of the
compounds had the aromatic ring connected directly to
the amide, but only 16 exhibited high affinity, presum-
ably because the p-butyl group compensates for the
aromatic ring not being positioned further westward.
The 5-chloro-2-methoxy motif that is favored in benza-
mide ligands for the related dopamine D2 receptor only
produced modest affinity (17), and only a p-methoxy (18)
is clearly insufficient. Likewise, large annelated aro-
matic moieties perpendicular to the central axis of the
molecule as in 19 did not pick up the same affinity as
the more elongated compounds. These first results
clearly suggested that separation of the aromatic ring
from the 6-amide by two atoms was the preferred motif
in the western part of the compounds.
The preference for the trans-ethenylene linker sug-
gested that the western aromatic ring is coplanar with
the quinoline system, and the 10-fold lower affinity
exhibited by the saturated two-carbon linker in 20
compared to the corresponding cinnamide derivative 21
may be ascribed, at least partly, to increased confor-
mational freedom leading to a lower population of the
preferred planar conformation. A conformational search
reinforced this assumption, since the preferred confor-
mation of saturated ethylene-linked compounds adopted
a planar (180°) or a perpendicular (80°/280°) arrange-
ment of the two-carbon linker and the western aromatic
ring relative to the amide quinoline. In contrast, the
corresponding methyleneoxy derivatives (e.g., 26) adopt
only one clearly favored coplanar conformation (0°),
where the oxygen appears to form an intramolecular
hydrogen bond with the amide (see docked structures
in Figure 4). This conformation was corroborated by a
search in the protein-ligand crystal structure database
Relibase, where seven out of eight phenoxyacetamide
hits were found to adopt this exact conformation when
bound to the proteins (see Experimental Section). The
cinnamide derivatives were found to have two sym-
metrical conformations (40°/320°) quite close to the
chloride or by platinum-catalyzed hydrogenation af-
forded 6-amino-2-chloroquinoline 12. Treatment of 12
with the appropriate acid chloride afforded the inter-
mediates 13, which were converted to target compounds
5 upon heating with the appropriate amine. The coup-
ling of 2-chloroquinolines (13) with secondary amines
was generally very efficient, whereas primary amines
required harsher reaction conditions and often gave rise
to byproducts from reaction with the 6-amide. However,
individual tuning of temperature and reaction time
frequently produced acceptable results.
Biological Evaluation
The radioligand binding assay was conducted with
stably transfected Chinese hamster ovary (CHO) cells,
expressing human MCH1R, by competition binding
using [125I]MCH. Alternatively, binding was conducted
in a scintillation proximity assay (SPA) by incubating
membranes and SPA beads with tracer in the presence
of various concentrations of test compounds. Nonspecific
binding was determined as the binding in the presence
of 1 µM MCH. Analogously, the MCH2R was expressed
by stably transfected CHO cells and screened. The SPA
MCH1R-membrane binding assay generally gives IC50
values that are up to 1 order of magnitude higher than
those from the whole-cell binding assay.
The functional activity was determined in a phos-
phatidylinositol assay. Phosphatidylinositol turnover in
stably transfected CHO cells expressing human MCH1R
was stimulated by submaximal concentrations of MCH
in the presence of increasing amounts of ligand. Com-
petitive antagonism was confirmed for selected com-
pounds by Schild analysis in a GTPγS SPA binding
assay.
Results and Discussion
The multiconformer libraries of commercial com-
pounds were screened in two rounds with iterative
refinement of the pharmacophore models. The 375
compounds selected in the first in silico screening
campaign of 0.5 million compounds resulted in 12 hits
after in vitro screening and validation. None of these
hits were pursued for optimization, but they provided
valuable information for tuning individual weights of
the four hypotheses in the query (see Supporting
Information).

5688 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18
Ulven et al.
Table 1. Representative 2-Aminoquinolines Identified in Second 3D Pharmacophore-Based and Third Scaffold Based in Silico
Screening Campaigns of Commercial Compound Collections
a SPA binding to MCH1R. b Single-point SPA binding to MCH1R minus % [125I]MCH displacement at 10 µM concentration. c Compounds
identified in the third in silico screening campaign. d Connected to the quinoline core via a thioamide group instead of an amide group.
phenoxyacetamides, only about 1 kcal/mol higher than
the fully extended conformation (180°), which repre-
sented the global minimum.
the o-chloro substituent of 22 contributes slightly to the
potency. The benzylthiourea 31 exhibited lower potency
than the corresponding amide analogue 20. Constrained
analogues, as exemplified by chromone 32, were inac-
tive. Although these compounds lack the required
western para substituent, they would be expected to
show a higher affinity if this modification represented
an improvement. Compounds 33 and 34 demonstrated
that it is possible to extend the linker to four atoms
between the 6-amide and the western phenyl ring while
preserving some of the affinity.
While compounds with highly diverse western parts
were available from commercial sources, the same was
not true for the eastern appendages of the quinolines.
Only one modification preserving the eastern basic
function was available, as well as a handful of modifica-
tions lacking this function. Whereas 22 represented one
of the most potent hits from the initial round, compound
35 showed that replacement of the eastern methyl group
with a 2-pyrimidyl led to complete loss of activity. In
contrast, and initially to our surprise, some analogues
of the most potent compounds lacking the distal ali-
phatic amine exhibited appreciable affinities (Table 1,
None of the compounds lacking the western para
substituent (e.g., 23-25) displayed impressive affinity,
whereas p-methyl (14), p-methoxy (21), and p-chloro
(26) exhibited high affinity. Increasing the size of the
para substituent to isopropoxy (27) or isopentoxy (28)
resulted in decreased affinity, suggesting that these
para substituents are larger than optimal. No clear
correlation between the electronic properties of the para
substituent and affinity was observed. Compound 29
indicated that substitution in the meta position is
forbidden, whereas compound 22 demonstrated that at
least small substituents in the ortho position are allowed
when combined with the methyleneoxy linker.
Following up on these promising initial hits, we
conducted a scaffold-based third in silico search of
commercial databases for additional compounds to
complement the SAR puzzle (Table 1, footnote c).
In general, the data from the third campaign con-
firmed our first SAR analysis. For example, compound
30, as expected, showed high affinity and indicated that

2-Aminoquinolines as Potent MCH1R Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5689
Figure 4. Binding mode of three quinolines in complex with MCH1R. The binding models differ for Asp123 on TM3, which we
propose could make interactions with two accessible rotamer states depending on the ligand considered. Proposed binding site
residues in contact with the ligand are shown. The residues that constitute the binding site are positioned on TM as follows:
(TM3) Thr120, Asp123, Gln127, Phe128; (TM5) Gln212, Phe213, Phe217, Phe221; (TM6) Trp269, Tyr272, Tyr273, Gln276; (TM7)
Tyr293, Tyr301. Ligand carbon atoms are shown in green. (A) Binding mode of 59. In this model Asp123 (ø1 ) -70°, ø2 ) -15°)
interacts with the quinoline nitrogen atom. (B) Binding mode of 65. In this model Asp123 (ø1 ) -177°, ø2 ) 65°) interacts with
the dimethylamin. (C) Binding mode of 67. In this model Asp123 (ø1 ) -70°, ø2 ) -15°) interacts with the quinoline nitrogen
atom.
bottom row). For example, piperidine analogue 36 and
morpholine analogues 37 and 48 (Table 3) all exhibited
affinities close to that of 22. The redundancy of the basic
amine was also noted by the Argenta group, although
they found a piperidine analogue to be less potent.22b
We soon realized that the compounds lacking the
eastern aliphatic amine would cause serious solubility
problems, and we tried to compensate for this by using
a morpholine combined with the less lipophilic p-
trifluoromethoxyphenoxymethylene western part as in
49 (Table 3), which led to very potent compounds with
respect to binding and functional activity. The solubility
improved for 49 (25 µM) compared to the dichlorophe-
noxypiperidine 36 (<5 µM) and the corresponding
morpholine 48 (10 µM) but not to an acceptable level.
Therefore, we decided to focus mainly on compounds in
the possession of an eastern aliphatic amine function.
The initial hits all contained a 4-methyl group on their
quinoline nuclei. Thus, to explore the potential for
optimizing this group, we synthesized analogues with
the 4-trifluoromethoxycinnamide western part and pip-
erazine or the more flexible dimethylaminoethyl(methyl)-
amine eastern side chain (Table 2). Both 4-methyl-
quinolines 38 and 39 displayed high and similar affinity.
Removal of the 4-methyl groups in 40-42 resulted in
less active compounds. An even larger drop in affinity
was observed when the methyl group was moved to the
3-position in 43 and 44. Extension of the 4-methyl to
4-ethyl in 45 led to a 10-fold less active compound. On
the basis of these results, we concluded that it would
be difficult to optimize the 4-methyl group further.
The effect of N-methylating the central amide was
also explored (46, Table 2), and this modification was
found to have a pronounced negative influence on the
affinity. A larger group in this position, like N-3,4-
difluorobenzyl (47), led to complete loss of affinity.
At this point the structural requirements of the
western and central parts of the molecule seemed
relatively clear, and the attention was shifted to the
eastern appendage. The optimization thus focused on

5690 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18
Ulven et al.
Table 2. Influence of Alkyl Substituents in the Central Part of
the Molecule
of 59 paralleled what was observed for the piperazine
analogue 22. Removal of a methyl group from either the
terminal (60) or the adjoining (61) nitrogen of the
ethylenediamine (cf. 22 and 39) had no impact on the
activity nor did introduction of a methyl group onto the
ethylene linker (62). Introduction of eastern N-ethylpyr-
rolidine groups (63) was also permitted, as was homolo-
gation to dimethylaminopropyl (64).
Exchange of the adjoining 2-amine functions of 39,
59, 61 with 2-oxy in 65 and 66 resulted in a considerable
drop in both affinity and functional activity of 1-2
orders of magnitude, suggesting that the 2-amine func-
tion is important for the affinity of the compounds. On
the other hand, exchange of the distal dimethylamine
group of compounds 39 and 59 with a hydroxyl group
(67, 68) enhanced the activity but without having the
same beneficial effect on solubility as the terminal
amine. A terminal acetoxy group (69) turned out to be
very similar to the terminal hydroxyl (67), ruling out a
hydrogen bond donor interaction in the binding of the
latter.
The majority of the compounds were counterscreened
on the receptors MCH2R and 5-HT2c, the latter as a
representative of monoamine receptors displaying cross-
affinity with MCH1R. Furthermore, 5-HT2c is also
implicated in the regulation of food intake, and 5-HT2c
agonists have been shown to suppress appetite.29 None
of the compounds displayed problematic affinities to-
ward these receptors, and a 1000-fold or higher selectiv-
ity was found for most compounds. Two compounds (39
and 65) with different eastern side chains where also
characterized in a broader panel of receptors and
transporters with no notable differences in their profile
(Table 4).
The high affinity and functional activity of the
compounds lacking an eastern amine function were not
anticipated by us because a positive charge in the
eastern distal position was a prerequisite in all phar-
macophore models. This charge was assigned to interact
with Asp123, located in TM3 of MCH1R. Representative
compounds were docked in a rhodopsin templated
MCH1R homology model. Upon analyzing the docked
quinolines in the binding site (Figure 4), we realized
that rotation of the Asp123 residue could readily ac-
commodate an interaction with the basic nitrogen in the
quinoline core. This docking mode was found to be
somewhat more favored than the originally anticipated
interaction between the terminal aliphatic amine and
the aspartate.
Figure 4A shows top and side views of the docking of
59 in MCH1R. Key interactions include a hydrogen bond
between Gln212 and the amide carbonyl and between
the western aromatic ring and a hydrophobic pocket
formed by Phe213, Phe217, Phe221, Trp269, and Tyr273.
In agreement with the SAR studies, the model accom-
modates a higher degree of freedom in the interaction
between the eastern part of the quinolines and the
upper part of the binding pocket. Two favored rotameric
states were identified for Asp123 on TM3, the first (ø1
) -70°, ø2 ) -15°) in 51% and the second (ø1 ) -177°,
ø2 ) 65°) in 21% of the structures (see Experimental
Section), both of which were invoked in the homology
models. The low-energy conformation of the phenoxy-
acetamide moiety identified in the conformational grid
a Whole-cell binding to MCH1R. b SPA binding to MCH1R. c 4-
CH3 instead of 4-CF3O. d 3,4-Difluorobenzyl. Data are given as a
range when n ) 2 and as the mean ( SEM when n ) 3-4.
compounds with the 4-trifluoromethoxyphenoxyaceta-
mide western part, which was found to combine optimal
binding properties with favorable solubility, as well as
4-trifluoromethoxycinnamide and the 4-chloro- and 2,4-
dichlorophenoxyacetamide, the last two representing
some of the most potent compounds from the com-
mercial selections (e.g., 22 and 26).
Substituting the western part of the N-methylpipera-
zine 22 with 4-trifluoromethoxyphenoxyacetamide (50)
indeed resulted in considerably improved solubility
properties as well as higher affinity (Table 3). Removal
of the N-methyl from the eastern piperazine ring (51,
52 vs 22, 26) did not appear to influence affinity or
solubility. Expansion of the piperazine ring to diazepane
in 53 also had no influence on the affinity. Substitution
of the piperazine ring with two methyl groups in the 3
and 5 positions (54) led to a considerable drop in
activity, whereas introduction of a bridging methylene
group in the piperazine to form a bicyclic system (55)
seemed to increase potency and also produced accept-
able solubility properties.
Introduction of 4-pyrrolidinylpiperidine in the place
of the piperazine (56) gave equally high affinity as well
as single-digit nanomolar functional antagonism. Re-
placement of the 4-pyrrolidine by a primary amine (57)
produced a potent compound that appeared to have
favorable solubility properties. Again in opposition to
our original pharmacophore hypotheses, removal of the
primary amine in 57 to produce piperidine 58 did not
deteriorate potency.
As seen in Table 2, affinity was preserved upon
opening of the piperazines (38, 40, 43) to form N,N,N′-
trimethylethylenediamine analogues (39, 41, 44). Cor-
respondingly, both the affinity and antagonistic activity

2-Aminoquinolines as Potent MCH1R Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5691
Table 3. Binding and Antagonistic Activity on MCH1R, Binding to MCH2R and 5-HT2c, and Approximate Solubility of
Representative Quinolines
a Whole-cell binding. b SPA binding. c Inhibition of MCH-induced IP accumulation. d Solubility determined by addition of 10 mM DMSO
solution of test compound to PBS (pH 7) until opalescence. e Schild analysis from [35S]GTPγS assay: 59, KB ) 5.0 ( 1.0 nM; 65, KB ) 76
nM; 67, KB ) 3.0 nM. Data are given as a range when n ) 2 and as the mean ( SEM when n ) 3-4.
search was used in the docking of compounds 59, 65,
and 67 into the binding site of the MCH1R, shown in
Figure 4. Contrary to our original hypotheses, Asp123
appeared to preferably interact with the quinoline
nitrogen rather than the aliphatic amine, which cor-
responded to the most abundant rotamer state. The
2-amino substituent at the quinoline is expected to
increase the basicity of the quinoline nitrogen signifi-
cantly, and calculated microscopic pKa values of the
quinoline nitrogen of 59 indeed predicted it to be more
basic than the aliphatic dimethylamine (Figure 5).30
To corroborate this binding mode hypothesis, 65 was
investigated in the receptor model (Figure 4B). In this
compound the electron-donating nitrogen in the 2-posi-
tion of the quinoline of 59 is substituted with the
electron-withdrawing oxygen, resulting in a drop in
basiciy of more than 4 pKa units for the quinoline
nitrogen (Figure 5). For this compound the dimethyl-
amine was found to interact with the Asp123 in the less
abundant rotameric state.
In analogue 67 (Figure 5) the electron-donating
nitrogen is reinstated, whereas the aliphatic amine is

5692 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18
Ulven et al.
Table 4. Receptor Profiling (IC50, µM) of Compounds 39 and
65 on Select Receptors
39
65
adrenergic R2
dopamine D1
dopamine D3
1.3
0.90
2.3
0.25
7.5
1.3
histamine H1
histamine H2
>10
0.86
1.3
0.48
0.80
1.9
3.5
0.87
1.7
muscarinic M1
muscarinic M2
serotonin 5-HT1B
serotonin 5-HT2A
serotonin 5-HT2C
DA transporter
4.5
0.20
0.61
2.5
0.9
10
>10
replaced by a hydroxyl group. Docking of this compound
in the MCH1R model resulted in a binding mode
analogous to the one found for 59 (Figure 4C). It is
noteworthy that compounds 59 and 67, for which the
quinoline nitrogen was found to be the most basic site
and only differing structurally in the distal eastern end,
exhibited very similar affinities and functional activities
on MCH1R. On the other hand, 65 came out 1 order of
magnitude less potent in the binding assays and 2
orders of magnitude less potent in the functional assay.
Schild analysis confirmed the compounds to be competi-
tive antagonists, with KB values corresponding very well
to affinities obtained in the whole-cell binding assay.
These observations strengthened our confidence in the
proposed new binding mode.
Figure 5. Competitive antagonists with different potential
Asp123 interaction sites, which were docked into the MCH1R.
The presumed site of interaction with Asp123 is indicated by
circle on each compound, and calculated30 microscopic pKa
values for the basic sites are given.
The structurally related 2-aminoquinoline (Figure 6,
top) and the isoelectronic 4-aminoquinolines (Figure 6,
bottom) disclosed as MCH1R antagonists by Devita and
co-workers23 are very likely to adopt the same binding
mode in MCH1R as our quinolines. The latter compound
has the 2-amine replaced by an alkyl chain, and a
primary amine in the 4-position is instead responsible
for enhancing the basicity of the quinoline atom, cor-
roborating the idea that the electron-donating property
of the 2-amino group is more important than any specific
direct interactions with the binding site.
Figure 6. Related 2- and 4-aminoquinoline derivatives re-
ported by others to be MCH1R antagonists.23 The presumed
site of interaction with Asp123 is indicated by a circle on each
compound, and calculated30 microscopic pKa values are given.
mandatory for activity. This observation was rational-
ized by a docking study, indicating that Asp123 in its
preferred rotameric state interacts preferentially with
the quinoline nitrogen instead of the aliphatic amine.
This new binding mode found support in microscopic
pKa calculations as well as studies of analogues where
the basicity of the quinoline site was reduced or the
distal aliphatic amine was removed.
Conclusion
By using SAR knowledge in the construction of 3D
pharmacophore models and using these for in silico
screening of commercial compound collections, we ac-
complished our objective to move from our original
benzamide series to novel MCH1R antagonists of a
significantly different chemotype. Subsequent optimiza-
tion of the identified 6-acylamino-2-aminoquinoline
compounds furnished potent compounds with appre-
ciable selectivity and favorable physicochemical proper-
ties. The series displayed quite specific structural
demands around the western aromatic ring and the
linking amide moiety. A substituent in the para position
of the western phenyl ring was required, and the
trifluoromethoxyphenoxyacetamide appendage provided
a suitable combination of activity and solubility proper-
ties. We were unable to optimize the 4-methyl substitu-
ent found in the original 2-aminoquinoline hits. The
requirements in the eastern appendage were found to
be highly flexible, and this part of the molecule, when
carrying an amine function, was found to serve a more
important role in increasing the solubility of the com-
pound. The eastern distal aliphatic amine implicated
by the pharmacophore models turned out not to be
Experimental Section
General Comments. Melting points were determined on
1
an Electrothermal 9200 melting point apparatus. H and 13C
NMR spectra were recorded on a Bruker AMX 300 spectrom-
eter operating at 300.13 and 75.47 MHz, respectively. Spectra
were calibrated relative to tetramethylsilane internal standard
or residual solvent peak. High-resolution mass spectra (HRMS)
were obtained on a JEOL-SX 102 in FAB+ mode with NBA
matrix. Analytical HPLC was performed on an Agilent 1100
series instrument equipped with a UV detector and a VL mass
detector operating under electrospray ionization conditions in
positive (ESI+) or negative (ESI-) mode. HPLC method A1 had
the following parameters: column, Waters XTerra MS C18;
flow, 1.0 mL/min; gradient, 0-5 min, 15-100% acetonitrile
in water; 5-71/2 min, 100% acetonitrile; modifier, 5 mM
ammonium formate. HPLC method A2 was the same as
method A1 except for the flow, which was 0.7 mL/min. HPLC
method A3 had the following parameters: column, Waters
XTerra MS C18; flow, 1.2 mL/min; gradient, 0-4 min, 5-100%

2-Aminoquinolines as Potent MCH1R Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5693
acetonitrile in water; 4-61/2 min, 100% acetonitrile; modifier,
0.1% ammonia. HPLC method B1 had the following param-
eters: column, Agilent Zorbax Eclipse XDB-C8; flow, 0.8 mL/
min; gradient, 0-8 min, 20-100% acetonitrile in water; 8-10
min, 100% acetonitrile; modifier, 0.1% formic acid. HPLC
method B2 had the following parameters: column, Agilent
Zorbax Eclipse XDB-C8; flow, 0.8 mL/min; gradient, 0-10 min,
20-100% acetonitrile in water; 10-14 min, 100% acetonitrile;
modifier, 0.1% formic acid. Elemental analyses were performed
by Johannes Theiner at Mikroanalytisches Laboratorium,
University of Vienna, Austria, and results are within 0.4% of
the calculated values unless otherwise stated.
General Procedure I. Introduction of 2-Amine. A
stirred mixture of the 2-chloroquinoline 1 (1 equiv) and
primary or secondary amine (2-5 equiv) was heated (a) to 150
°C for 2 h, (b) to 100 °C for 16 h, or (c) in a sealed vessel in an
Emrys Optimizer (Personal Chemistry) microwave oven to 200
°C for 15 min. Excess amine was removed in vacuo if possible.
The residue was taken up in 3% aqueous HCl. The aqueous
phase was washed twice with CH2Cl2, 4 N NaOH was added
until pH 8 was reached, and the sample was extracted three
times with CH2Cl2. The extract was concentrated to give the
corresponding 2-aminoquinoline 2, which was used directly or
purified by recrystallization or chromatography, as appropri-
ate.
General Procedure II. Nitration. 2-Aminoquinoline 2
was added in small portions to cold (-10 °C), stirring, fuming
HNO3 (>90%, 1 mL/g of 2). The mixture was stirred at 0 °C
for 1 h and then poured into an ice/water mixture. To the
aqueous mixture was added 4 N NaOH until pH 12 was
reached, and the mixture was left to precipitate overnight. The
precipitate was filtered off, washed four times with water, and
dried in vacuo to give the 2-amino-6-nitroquinoline 3, which
was purified by recrystallization or used directly in the next
step.
General Procedure III. Introduction of Boc Group. To
a solution of 2-amino-6-nitroquinoline 3 (1 equiv) in CH2Cl2
(∼10 mL/g of 3) were added Boc2O (2 equiv) and Et3N (1 equiv),
and the mixture was stirred at room temperature for 12 h.
The mixture was poured into water and was extracted three
times with CH2Cl2. The extract was dried (MgSO4) and
concentrated to give to give the corresponding Boc-protected
compound.
General Procedure IV. Reduction of Nitro Group. A
mixture of 2-amino-6-nitroquinoline 3 and 5% Pd/C (∼200 mg/g
of 3) in THF or ethanol was stirred under hydrogen (1 atm)
for 12 h. The reaction mixture was filtered over a pad of Celite
and concentrated in vacuo to give the 6-aminoquinoline 4,
which was purified by chromatography or recrystallization or
used directly in the next step.
General Procedure V. Coupling of Aniline with Acid
Chloride. To the 6-aminoquinoline 4 (1 equiv) in dry CH2Cl2
was added a solution of the acid chloride (1.1 equiv) in dry
CH2Cl2. The resulting mixture was stirred for 3 h. The mixture
was diluted with CH2Cl2, washed with 1 N NaOH and brine,
dried (MgSO4), and concentrated. The crude product was
purified by recrystallization or chromatography.
General Procedure VI. Removal of Boc Group. To the
Boc-protected compound in CH2Cl2 (15 mL/g of substrate) was
added trifluoroacetic acid (1.5 mL/g of substrate), and the
mixture was stirred at room temperature for 12 h. The reaction
mixture was concentrated, and the residue was partitioned
between aqueous Na2CO3 and EtOAc. The aqueous phase was
extracted with EtOAc, and the combined organic phases were
dried (MgSO4) and concentrated. The residue was purified by
flash chromatography.
General Procedure VII. Acylation of 6-Amino-2-chlo-
rolepidine. To 6-amino-2-chloro-4-methylquinoline 12 (1
equiv) in dry CH2Cl2 (5 mL per mmol) was added dropwise or
in small portions the acyl chloride (1 equiv). The mixture was
stirred at room temperature for 2 h, then poured into MeOH
(15 mL per mmol) to give a homogeneous solution. Water (10
mL per mmol) was added in small portions, and the mixture
was left to precipitate. The crystals were collected and washed
with MeOH/water (1:1). The filtrate was concentrated, and
additional crops of crystals were collected if necessary.
N-(2-Chloro-4-methylquinolin-6-yl)-2-(4-trifluoro-meth-
oxyphenoxy)acetamide (13). The title compound was pre-
pared from 6-amino-2-chlorolepidine (12, 4.33 g, 22.4 mmol)
and 4-trifluoromethoxyphenoxyacetyl chloride (5.72 g, 22.4
mmol) according to general procedure VII to give 8.72 g (95%)
of the pure title compound as a white solid: mp 197-198 °C;
1H NMR (DMSO-d6) δ 2.62 (s, 3H), 4.84 (s, 2H), 7.13 (d, J )
9.0 Hz, 2H), 7.34 (d, J ) 9.1 Hz, 2H), 7.91 (d, J ) 9.2 Hz, 1H),
7.98 (dd, J ) 9.0, 2.3 Hz, 1H), 8.49 (d, J ) 1.9 Hz, 1H), 10.55
(s, 1H); 13C NMR (DMSO-d6) δ 18.9, 68.2, 113.1, 116.8, 123.4,
123.4, 125.1, 127.9, 129.9, 137.8, 143.1, 144.7, 148.8, 157.5,
167.7; MS ESI+ m/z 411.0 [M + H]. Anal. (C19H14ClF3N2O3)
C, H, N, Cl.
(E)-N-[4-Methyl-2-(4-methylpiperazin-1-yl)quinolin-6-
yl]-3-p-toluylacrylamide (14). The title compound was
prepared from 4-methyl-2-(4-methylpiperazin-1-yl)-quinolin-
6-ylamine (4b, 256 mg, 1.0 mmol) and 4-methylcinnamoyl
chloride (235 mg, 1.3 mmol) according to general procedure
V. The product was recrystallized from MeOH to give 305 mg
(76%) of light-yellow needles: mp 264-267 °C; 1H NMR
(CDCl3) δ 2.37 (s, 6H), 2.56 (“t”, J ) 5.1 Hz, 4H), 2.57 (s, 3H),
3.75 (“t”, J ) 5.2 Hz, 4H), 6.55 (d, J ) 15.5 Hz, 1H), 6.83 (s,
1H), 7.16 (d, J ) 8.1 Hz, 2H), 7.41 (d, J ) 7.9 Hz, 2H), 7.49
(dd, J ) 9.0, 2.3 Hz, 1H), 7.67 (d, J ) 8.9 Hz, 1H), 7.71 (br s,
1H), 7.74 (d, J ) 15.4 Hz, 1H), 8.41 (s, 1H); HRMS calcd for
C25H28N4O [M + H] 401.2341, found 401.2362. Anal. (C25H28-
N4O) C, H, N.
(E)-N-{2-[(2-Dimethylaminoethyl)methylamino]quino-
lin-6-yl}-3-p-tolylacrylamide (42). The compound was pre-
pared from N2-(2-dimethylaminoethyl)-N2-methylquinoline-
2,6-diamine (4d) and 4-methylcinnamoyl chloride according
to general procedure V: 1H NMR (CDCl3) δ 2.35 (s, 6H), 2.38
(s, 3H), 2.58 (t, J ) 7.5 Hz, 2H), 3.20 (s, 3H), 3.80 (t, J ) 7.5
Hz, 2H), 6.55 (d, J ) 15.4 Hz, 1H), 6.87 (d, J ) 9.2 Hz, 1H),
7.19 (d, J ) 7.9 Hz, 1H), 7.42-7.51 (m, 3H), 7.63 (d, J ) 9.0
Hz, 1H), 7.74 (d, J ) 15.6 Hz, 1H), 7.84 (d, J ) 9.0 Hz, 1H),
8.20 (br s, 1H); HRMS calcd for C24H29N4O [M + H] 389.2341,
found 389.2351.
(E)-N-[2-(4-Ethylpiperazin-1-yl)-3-methylquinolin-6-
yl]-3-(4-trifluoromethoxyphenyl)acrylamide (43). The title
compound was prepared from 6-amino-2-(4-ethylpiperazin-1-
yl)-3-methylquinoline (4g) and 4-trifluoromethoxycinnamoyl
chloride according to general procedure V: MS ESI+ m/z 484.9
[M + H]; HPLC (A1) >99%, (B1) 98%.
(E)-N-{2-[(2-Dimethylaminoethyl)methylamino]-4-eth-
ylquinolin-6-yl}-3-(4-trifluoromethoxyphenyl)acrylam-
ide (45). The title compound was prepared from N2-(2-di-
methylaminoethyl)-4-ethyl-N2-methylquinoline-2,6-diamine (4i,
100 mg, 0.37 mmol) and 4-trifluoromethoxycinnamoyl chloride
(93 mg, 0.37 mmol) according to general procedure V and
purified by solid-phase extraction (SCX column, CH2Cl2
f
MeOH f MeOH with 5% NH4OH) to give 96 mg (53%) of
yellow solid: 1H NMR (CDCl3) δ 1.35 (d, J ) 7.5 Hz, 3H), 2.34
(s, 6H), 2.57 (t, J ) 7.3 Hz, 2H), 2.97 (q, J ) 7.4 Hz, 2H), 3.20
(s, 3H), 3.79 (t, J ) 7.3 Hz, 2H), 6.59 (d, J ) 15.6 Hz, 1H),
6.73 (s, 1H), 7.18 (d, J ) 8.7 Hz, 2H), 7.51 (m, 3H), 7.64 (d, J
) 9.1 Hz, 1H), 7.74 (d, J ) 15.4 Hz, 1H), 7.86 (br s, 1H), 8.42
(s, 1H); HRMS calcd for C26H30F3N4O2 [M + H] 487.2321, found
487.2312; HPLC (A1) >99%.
N-(4-Methyl-2-morpholin-4-ylquinolin-6-yl)-2-(4-triflu-
oromethoxyphenoxy)acetamide (49). A solution of 6-amino-
4-methyl-2-(4-morpholinyl)quinoline (4m, 0.55 g, 2.3 mmol) in
CH2Cl2 (30 mL) was added dropwise to a solution of 4-trifluo-
romethoxyphenoxyacetyl chloride (0.64 g, 2.5 mmol) in CH2-
Cl2 (4 mL). The reaction mixture, which quickly became green
and turbid, was stirred at room temperature for 3 h, then
diluted with CH2Cl2 (150 mL). The organic phase was washed
with saturated aqueous Na2CO3 and brine, dried (MgSO4), and
concentrated. The residue was recrystallized from MeOH to
give 567 mg (55%) of woolly white crystals: mp 206-207 °C;
1H NMR (DMSO-d6) δ 2.52 (s, 3H), 3.60 (m, 4H), 3.72 (m, 4H),
4.78 (s, 2H), 7.12 (m, 2H), 7.13 (s, 1H), 7.33 (m, 2H), 7.55 (d,

5694 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18
Ulven et al.
J ) 9.0 Hz, 1H), 7.75 (dd, J ) 8.9, 2.3 Hz, 1H), 8.20 (d, J )
2.8 Hz, 1H), 10.22 (s, 1H); 13C NMR (DMSO-d6) δ 18.6, 45.1,
66.1, 67.4, 110.4, 113.0, 116.0, 121.8, 122.5, 123.0, 127.0, 132.8,
142.1, 144.1, 144.3, 156.6, 156.7, 166.1; HRMS calcd for
C23H23F3N3O4 [M + H] 462.1641, found 462.1638. Anal.
(C23H22F3N3O4) C, H, N.
2-(2,4-Dichlorophenoxy)-N-(4-methyl-2-piperazin-1-
ylquinolin-6-yl)acetamide (51). The title compound was
prepared from tert-butyl 4-(6-amino-4-methylquinolin-2-yl)-
piperazine-1-carboxylate (4l) and 2,4-dichlorophenoxyacetyl
chloride according to general procedures V and VI: 1H NMR
(CDCl3) δ 2.61 (s, 3H), 3.02 (“t”, J ) 5.1 Hz, 4H), 3.70 (“t”, J )
5.1 Hz, 4H), 4.66 (s, 2H), 6.86 (s, 1H), 6.92 (d, J ) 8.9 Hz,
1H), 7.26 (dd, J ) 8.9, 2.6 Hz, 1H), 7.47 (d, J ) 2.5 Hz, 1H),
7.52 (dd, J ) 8.9, 2.5 Hz, 1H), 7.70 (d, J ) 8.9 Hz, 1H), 8.28
(d, J ) 2.3 Hz, 1H), 8.63 (s, 1H); HRMS calcd for C22H23Cl2N4O2
[M + H] 445.1198, found 445.1194. Anal. (C22H22Cl2N4O2‚
1/5H2O) C, H, N, Cl.
11.25 (br s, 1H), 13.10 (br s, 1H); HRMS calcd for C23H27-
Cl2N4O2 [M + H] 461.1511, found 461.1513. Anal. (C23H26-
Cl2N4O2‚2HCl‚5/2H2O) C, H, N, Cl.
(E)-N-[2-(2-Dimethylaminoethylamino)-4-methylquin-
olin-6-yl]-3-(4-trifluoromethoxyphenyl)acrylamide (61).
The title compound was prepared from tert-butyl (2-dimeth-
yl-aminoethyl){4-methyl-6-[(E)-3-(4-trifluoromethoxyphenyl)-
acryloylamino]quinolin-2-yl}carbamate (5a,1.20 g, 2.12 mmol)
according to general procedure VI. The product was purified
by flash chromatography (SiO2, CH2Cl2/MeOH/NH4OH,
80:20:1) to give 680 mg (70%) of yellow solid: mp 213-215 °C;
1H NMR (CDCl3) δ 2.30 (s, 6H), 2.52 (s, 3H), 2.59 (t, J ) 6.0
Hz, 2H), 3.56 (“q”, J ) 5.3 Hz, 2H), 5.41 (br s, 1H), 6.54 (s,
1H), 6.59 (d, J ) 15.4 Hz, 1H), 7.22 (d, J ) 8.5 Hz, 2H), 7.49-
7.59 (m, 3H), 7.60-7.71 (m, 2H), 7.75 (d, J ) 15.4 Hz, 1H),
8.34 (s, 1H); HRMS calcd for C24H26F3N4O2 [M + H] 459.2008,
found 459.1990. Anal. (C24H25F3N4O2‚1/4H2O) C, H, N.
N-{2-[(1-Ethylpyrrolidin-2-ylmethyl)amino]-4-meth-
ylquinolin-6-yl}-2-(4-trifluoromethoxyphenoxy)aceta-
mide (63). To tert-butyl (6-amino-4-methylquinolin-2-yl)-(1-
ethylpyrrolidin-2-ylmethyl)carbamate (4s, 1.34 g, 3.5 mmol)
in CH2Cl2 (30 mL) were added Et3N (0.60 mL, 4.3 mmol) and
trifluoromethoxyphenoxyacetyl chloride (0.97 g, 3.8 mmol), and
the mixture was stirred at room temperature. After 12 h, TFA
(15 mL) was added to the reaction mixture, and stirring at
room temperature was continued. After 24 h the reaction
mixture was concentrated and the residue was taken up in
CH2Cl2. The organic phase was washed with saturated K2CO3
and brine and concentrated. The residue was purified by flash
chromatography (SiO2, (MeOH with 5% NH4OH)/EtOAc, 1:5)
to give 422 mg (24%) as a yellow syrup: 1H NMR (CDCl3) δ
1.16 (t, J ) 6.8 Hz, 3H), 1.82 (m, 1H), 1.96 (m, 1H), 2.05-2.41
(m, 4H), 2.54 (s, 3H), 2.80-2.99 (m, 2H), 3.29 (m, 1H), 3.46
(m, 1H), 3.74 (m, 1H), 4.64 (s, 2H), 5.51 (br s, 1H), 6.57 (s,
1H), 7.03 (“d”, J ) 9.2 Hz, 2H), 7.22 (“d”, J ) 8.5 Hz, 2H),
7.55 (d, J ) 8.9 Hz, 1H), 7.64 (d, J ) 8.9 Hz, 1H), 8.15 (s, 1H),
8.30 (s, 1H); 13C NMR (CDCl3) δ 13.8, 19.2, 23.2, 28.7, 42.8,
48.9, 53.8, 54.0, 68.5, 113.4, 114.8, 116.3, 120.7, 123.1, 123.2,
124.2, 127.6, 130.9, 144.8, 146.2, 155.9, 157.4, 165.9.
The mesylate salt was formed by dissolving the free amine
in chloroform and adding 1.0 M methylsulfonic acid (1 equiv).
The suspension was concentrated to give the highly hygro-
scopic mesylate salt in quantitative yield: 1H NMR (DMSO-
d6) δ 1.28 (t, J ) 7.1 Hz, 3H), 1.85 (m, 2H), 1.99 (m, 1H), 2.14
(m, 1H), 2.33 (s, 3H), 2.48 (s, 3H), 3.10-3.30 (m, 4H), 3.53 (m,
2H), 3.72 (m, 3H), 4.78 (s, 2H), 6.75 (s, 1H), 7.12 (“d”, J ) 9.2
Hz, 2H), 7.34 (“d”, J ) 9.0 Hz, 2H), 7.52 (d, J ) 8.7 Hz, 1 H),
7.53 (s, 1H), 7.81 (d, J ) 9.2 Hz, 1H), 8.19 (d, J ) 1.9 Hz, 1H),
10.28 (s, 1H); HRMS calcd for C26H30F3N4O3 [M + H] 503.2270,
found 503.2287. Anal. (C26H29F3N4O3‚CH3SO3H‚7/4H2O) C, H,
N, S.
(E)-N-[2-(cis-3,5-Dimethylpiperazin-1-yl)-4-methylquin-
olin-6-yl]-2-(4-trifluoromethoxyphenoxy)acetamide (54).
The title compound was prepared from tert-butyl 4-(6-amino-
4-methylquinolin-2-yl)-cis-2,6-dimethylpiperazine-1-carboxyl-
ate (4q) and 4-trifluoromethoxycinnamoyl chloride according
to general procedures V and VI: mp 202-208 °C; 1H NMR
(DMSO-d6) δ 1.05 (d, J ) 6.0 Hz, 6H), 2.32 (t, J ) 11.7 Hz,
2H), 2.53 (s, 3H), 2.77 (m, 2H), 4.36 (d, J ) 11.1 Hz, 2H), 6.88
(d, J ) 15.8 Hz, 1H), 7.14 (s, 1H), 7.45 (d, J ) 8.7 Hz, 2H),
7.54 (d, J ) 8.9 Hz, 1H), 7.63 (d, J ) 15.6 Hz, 1H), 7.76 (s,
1H), 7.77 (d, J ) 8.3 Hz, 2H), 8.26 (d, J ) 1.3 Hz, 1H), 10.36
(s, 1H); 13C NMR (DMSO-d6) δ 18.6, 19.3, 50.2, 51.2, 110.6,
112.4, 118.3, 121.4, 121.7, 122.5, 122.8, 123.6, 126.8, 129.5,
133.3, 134.1, 138.1, 144.0, 144.2, 148.9, 156.2, 163.1; HRMS
calcd for C26H28F3N4O2 [M + H] 485.2164, found 485.2169.
Anal. (C26H27F3N4O2‚1/3H2O) C, H, N.
N-[4-Methyl-2-(4-pyrrolidin-1-ylpiperidin-1-yl)quino-
lin-6-yl]-2-(4-trifluoromethoxyphenoxy)acetamide (56).
The 2-chloroquinoline 13 (205 mg, 0.5 mmol) and 4-(1-
pyrrolidinyl)piperidine (0.80 g, 5.0 mmol) were mixed and
heated by microwaves to 180 °C for 15 min. The resulting
mixture was purified by flash chromatography (SiO2, MeOH/
CH2Cl2, 1:10 to 1:2) to give 224 mg (94%) of yellow solid: mp
1
∼180 °C (dec); H NMR (DMSO-d6) δ 1.45 (m, 2H), 1.72 (m,
4H), 1.96 (br d, J ≈ 11.3 Hz, 2H), 2.51 (s, 3H), 2.68 (m, 4H),
2.96 (t, J ) 11.3 Hz, 2H), 4.42 (d, J ) 12.4 Hz, 2H), 4.78 (s,
2H), 7.12 (m, 2H), 7.15 (s, 1H), 7.34 (d, J ) 8.3 Hz, 2H), 7.52
(d, J ) 9.1 Hz, 1H), 7.73 (dd, J ) 9.1, 2.3 Hz, 1H), 8.17 (d, J
) 2.3 Hz, 1H), 10.23 (d, 1H); 13C NMR (DMSO-d6) δ 18.6, 22.8,
30.0, 43.2, 50.7, 61.1, 67.4, 110.6, 113.0, 116.0, 120.2 (q, JCF
)
254 Hz), 122.5, 122.6, 122.9, 126.8, 132.5, 142.1, 144.2, 144.3,
156.2, 156.7, 166.0; HRMS calcd for C28H32F3N4O3 [M + H]
529.2427, found 529.2419. Anal. (C28H31F3N4O3‚3/4H2O) C, H,
N.
2-(2,4-Dichlorophenoxy)-N-[2-(2-dimethylaminoethoxy)-
4-methylquinolin-6-yl]acetamide (65). The title compound
was prepared from 2-(2-dimethylaminoethoxy)-4-methylquino-
lin-6-ylamine (4j,1.2 g, 4.9 mmol) and 2,4-dichlorophenoxy-
acetyl chloride (1.2 g, 5.0 mmol) according to general procedure
V. Purification by flash chromatography (SiO2, (MeOH with
10% NH4OH)/CH2Cl2, 1:10) followed by recrystallization from
MeOH provided 410 mg (19%) of white solid. The solid was
suspended in Et2O (10 mL), and 2 M HCl in Et2O (4 mL) was
added. The suspension was stirred at room temperature for
12 h, and the solvents were removed in vacuo to provide 517
mg of the dihydrochloride salt: 1H NMR (DMSO-d6) δ 2.57
(d, J ) 0.75 Hz, 3H), 2.85 (d, J ) 4.9 Hz, 6H), 3.55 (br q, J )
5.3 Hz, 2H), 4.72 (“t”, J ) 4.5 Hz, 2H), 4.96 (s, 2H), 6.95 (d, J
) 0.9 Hz, 1H), 7.15 (d, J ) 9.0 Hz, 1H), 7.39 (dd, J ) 8.9, 2.5
Hz, 1H), 7.62 (d, J ) 2.6 Hz, 1H), 7.76 (d, J ) 8.9 Hz, 1H),
7.85 (dd, J ) 8.9, 2.1 Hz, 1H), 8.38 (d, J ) 2.1 Hz, 1H), 10.39
(br s, 1H), 10.67 (s, 1H); HRMS calcd for C22H24Cl2N3O3 [M +
H] 448.1195, found 448.1194. Anal. (C22H23Cl2N3O3‚2HCl‚H2O)
C, H, N. HPLC (A2) >99%.
2-(2,4-Dichlorophenoxy)-N-{2-[(2-dimethylaminoeth-
yl)methylamino]-4-methylquinolin-6-yl}-acetamide (59).
The title compound was prepared from 6-amino-2-(dimethy-
laminoethyl)meth-ylamine-4-methylquinoline (4c, 1.02 g, 3.95
mmol) and 2,4-dichlorophenoxyacetyl chloride according to
general procedure V. The residue was purified by flash
chromatography (SiO2, (MeOH with 5% NH4OH)/EtOAc, 1:9
to 1:5) to give 590 mg (32%) of product: 1H NMR (DMSO-d6)
δ 2.26 (s, 3H), 2.51 (s, 6H), 3.20-3.80 (m, 4H), 3.41 (s, 3H),
4.90 (s, 2H), 6.94 (s, 1H), 7.15 (d, J ) 8.9 Hz, 1H), 7.40 (dd, J
) 9.0, 2.6 Hz, 1H), 7.49 (d, J ) 8.9 Hz, 1H), 7.60-7.70 (m,
2H), 8.16 (d, J ) 2.3 Hz, 1H), 10.29 (s, 1H); MS ESI+ m/z 461.1
[M + H]; HPLC (A1): 97%.
The product was suspended in Et2O (10 mL), and 2.0 M HCl
in Et2O was added. The turbid mixture was stirred for 30 min,
then was concentrated and dried in vacuo to produce 660 mg
(31%) of the dihydrochloride salt of the title compound as a
white powder: mp 217-221 °C; 1H NMR (DMSO-d6) δ 2.64
(s, 3H), 2.84 (s, 6H), 3.35-3.50 (m, 2H), 3.43 (s, 3H), 4.26 (m,
2H), 5.00 (s, 2H), 7.16 (d, J ) 8.9 Hz, 1H), 7.37 (dd, J ) 9.0,
2.6 Hz, 1H), 7.61 (d, J ) 2.4 Hz, 1H), 7.62 (br s, 1H), 7.93 (d,
J ) 9.2 Hz, 1H), 8.41 (s, 1H), 8.44 (br s, 1H), 11.02 (s, 1H),
(E)-N-{2-[(2-Hydroxyethyl)methylamino]-4-methylquin-
olin-6-yl}-3-(4-trifluoromethoxyphenyl)acrylamide (67).
To a suspension of 69 (2.3 g, 4.8 mmol) in THF (200 mL) was

2-Aminoquinolines as Potent MCH1R Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5695
added a solution of LiOH‚H2O (405 mg, 9.6 mmol) in water
(60 mL), and the solution was stirred at 40 °C for 60 min to
give a homogeneous solution. Then it was stirred at room
temperature for 12 h. The reaction mixture was concentrated
in vacuo to 140 mL, and water was added to form a precipitate
that was filtered off and dried in vacuo to afford 1.86 g (89%)
of white solid: 1H NMR (DMSO-d6) δ 2.49 (s, 3H), 3.14 (s, 3H),
3.63 (m, 4H), 4.79 (m, 1H), 4.89 (s, 2H), 6.96 (s, 1H), 7.14 (d,
J ) 9.1 Hz, 1H), 7.39 (dd, J ) 8.9, 2.6 Hz, 1H), 7.48 (d, J )
8.9 Hz, 1H), 7.62 (d, J ) 2.5 Hz, 1H), 7.63 (dd, J ) 9.0, 2.4 Hz,
1H), 8.15 (d, J ) 2.5 Hz, 1H), 10.28 (s, 1H); HRMS calcd for
C21H22Cl2N3O3 [M + H] 434.1038, found 434.1045. Anal.
(C21H21Cl2N3O3‚1/2H2O) C, H, N.
General Procedure for Solubility Assessment. The
procedure is based on a protocol described by Lipinski et al.31
A 10 mM DMSO solution of the compound is added in small
increments (first 1 µL, then 5 µL increments), spaced at least
5 min apart, to 1.0 mL of PBS buffer (pH 7.4) at room
temperature. The point of appearance of opalescence or
precipitate is determined visually. The highest concentration
reached, up to 200 µM, without detection of opalescence or
precipitation is reported.
Pharmacophore Evaluation and Refinement. Selected
benzamide MCH1R antagonists used as the basis for the
pharmacophore hypotheses were subjected to conformational
analysis by applying a simulated annealing protocol in Cerius2
using the MMFF force field. An initial temperature of 1000 K
was used. In the interval 1000-100 K, the temperature was
lowered in steps of 100 K. In the interval 100-0 K the
temperature was lowered in steps of 10 K. For each temper-
ature, the molecules were optimized in 1000 Monte Carlo steps
with minimization (MCM); i.e., each Monte Carlo step was
followed by 100 subsequent minimization steps. Each MCM
step was accepted or rejected on the basis of the Metropolis
criteria. In this way a total of 20 000 MCM steps were applied
in the simulated annealing protocol for each molecule. The
resulting trajectory was analyzed, and individual conformers
were ranked on the basis of their internal energies.
Conversion of Commercial Compound Libraries to
Multiconformer Libraries. Electronic catalogues of avail-
able compounds from the commercial vendors ChemBridge
Research Laboratories (CRL) (257 400 compounds), ChemDiv
(338 003 compounds), ComGenex (98 000), InterBioScreen
(192 068 compounds), Maybridge (57 046 compounds), and
SPECS (148 837 compounds) were obtained, and each com-
pound was transformed by CATALYST (Accelrys, Inc.) into a
multiconformational library with a maximum of 255 conform-
ers per molecule and an energy threshold not exceeding 10
kcal/mol of the lowest energy conformation. The first in silico
screening round included the CRL, InterBioScreen, and May-
bridge libraries, and the following rounds included all vendors.
MCH1R Homology Modeling. The homology modeling
study of MCH1R utilized the crystal structure of bovine
rhodopsin PDB code 1F88 (solved to 2.8 Å resolution), obtained
form the Brookhaven Protein Data Bank, as structural tem-
plate.32,33 The software MODELLER34 was configured to
produce 25 three-dimensional comparative homology models
of MCH1R. Individual models were evaluated on the basis of
manual visualization and MODELLER’s fitness score. Further
analyses of accessible rotamer states for these residues were
conducted within SYBYL35 and the Biopolymer module using
the Lovel rotamer library. The docking was performed using
the FlexX docking algorithm.36
Conformational Grid Search. The grid conformational
search feature within SYBYL35 was used to locate energy
minima for the torsional potential around flexible bonds in
selected linkers connecting the western aromatic ring with the
quinoline. During the grid search, designated bonds were
systematically rotated in increments of 10° through the torsion
range 0° to 360° followed by 300 conjugated minimization
steps. The resulting conformations and associated energies
were stored and analyzed to compute the potential energy
surface and subsequently to identify the global and local
energy minima, their relative energy differences, and associ-
ated energy barriers. The torsion potential around the CH2-
CONH bond of the oxyacetamide linker (-OCH2CONH-) has
a single minimum corresponding to the planar cis conformation
(0°), and the trans conformation (180°) is at the top of a ∼10
kcal/mol energy barrier. A search in Relibase (http://relibase-
.rutgers.edu) on the phenoxyacetamide substructure identified
the experimental protein-ligand complexes 1ta6, 1mui, 1idb,
1hpx, 1hiv, 1ivp, 1lee, 1lf2 containing the same linker frag-
ment. All ligands except for 1hiv have a cis conformation in
their protein-bound form, in agreement with the global
minimum of the torsion potential energy surface. The torsion
potential energy surface around the CH-CONH bond of the
acrylamide linker (-CHdCHCONH-) contains two sym-
metrical minima close to 40° and 320° and one at 180°, the
last favored with 1 kcal/mol. The torsion potential energy
surface for CH2-CONH of the propylamide linker (-CH2CH2-
CONH-) has three minima, with the global minimum located
around 180° favored by ∼1 kcal/mol over two symmetric
minima corresponding to torsion angles of 80° and 280°.
Transfections and Tissue Culture. The cDNAs encoding
the human MCH1R and MCH2R were cloned from a human
brain cDNA library and cloned into the eukaryotic expression
vectors pcDNA3.1 (Invitrogen) and pIRES (Clontech), respec-
tively. Assays were performed with stably transfected CHO-
K1 (Chinese hamster ovary; ATCC no. CCL-61) cells, express-
ing the human MCH1R or the human MCH2 receptor. Stable
MCH1R or MCH2R transfectants of CHO-K1 cells were
obtained using 5 µg of plasmid cDNA and a standard calcium
phosphate transfection method37 with subsequent selection in
1 mg/mL G418 (Life Technology) for MCH1R and 10 µg/mL
Blasticidin (Invitrogen) for MCH2R. Stably transfected CHO-
K1 cells were maintained in Ham’s F-12 culture medium
(Invitrogen) supplemented with 10% (v/v) fetal calf serum
(Invitrogen), 100 U/mL penicillin, 100 µg/mL streptomycin
(Life Technology), and either 1 mg/mL G418 or 10 µg/mL
blasticidin.
Radioligand Binding Assay. Stably transfected CHO-K1
cells, expressing either human MCH1R or human MCH2R,
were seeded in multiwell culture plates 1 day before the assay.
The number of cells per well was determined by the apparent
expression efficiency of the cell line aiming at 5-10% binding
of the added radioligand. MCH1R-expressing cells were as-
sayed by competition binding for 3 h at room temperature
using 15 pM [125I]MCH (Amersham Pharmacia Biotech) plus
variable amounts of test compound in 0.5 mL of a 25 mM
Hepes buffer, pH 7.4, supplemented with 10 mM MgCl2, 5 mM
MnCl2, 10 mM NaCl, 0.1% (w/v) bovine serum albumin (BSA),
and 100 µg/mL bacitracin. MCH2R expressing cells were
assayed by competition binding for 2 h at 37 °C using 30 pM
[
125I]MCH plus variable amounts of test compound in 0.5 mL
of a 25 mM Hepes buffer, pH 7.4, supplemented with 5 mM
MgCl2, 10 mM NaCl, 0.1% (w/v) BSA, and 100 µg/mL bacitra-
cin. Assays were performed in duplicate. Nonspecific binding
was determined as the binding in the presence of 1 µM MCH
(Bachem). Binding data were analyzed and IC50 values deter-
mined by nonlinear regression using the Prism software
(GraphPad software, San Diego, CA).
Scintillation Proximity Assay (SPA). Measurement of
[
125I]MCH binding was performed in duplicate by incubating
membranes and SPA beads with tracer in the presence of
various concentrations of test compounds at room temperature
for 2 h. Membranes and beads were preincubated for 20 min.
The MCH1R binding buffer contained 50 mM Tris (pH 7.4), 8
mM MgCl2, 12% glycerol, 0.1% (w/v) BSA, and protease
inhibitors (complete protease inhibitor cocktail tablet, Roche).
A final [125I]MCH concentration of 150 pM was applied, and
SPA beads (PVT-PEI-WGA type B, RPNQ0004 Amersham
Pharmacia Biotech) were used at a final concentration of 0.4
mg/well. Analogously, 5-HT2c competition binding was per-
formed with an SPA assay using [3H]mesulergine (Amersham
Pharmacia Biotech) as the radioligand. The 5-HT2c binding
buffer contained 50 mM Tris (pH 7.7), 50 mM MgCl2, and 0.1%
pargyline. SPA beads (YSi-WGA, RPNQ0011 Amersham Phar-
macia Biotech) were used at a final concentration of 0.4 mg/

5696 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18
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well. Nonspecific binding was determined as the binding in
the presence of 1 µM cold mesulergine.
Membranes prepared from CHO-K1 cells expressing either
the hMCH1R or the h5-HT2c receptor were purchased from
Euroscreen (ES-370-M or ES-315-M, respectively), and a final
concentration of 2 µg/well (hMCH1R) or 4 µg/well (h5-HT2c)
was used. Binding data were analyzed and IC50 values
determined by nonlinear regression using the Prism software
(GraphPad software, San Diego, CA).
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Bergsma, D.; Muir, A.; Fitzgerald, L. R.; Hervieu, G.; Dytko, G.
M.; Foley, J. J.; Martins, J.; Liu, W.-S.; Park, J.; Ellis, C.;
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ligand for the orphan G protein-coupled receptor SLC-1. Nature
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Lin, S. H. S.; Leslie, F.; Civelli, O. Molecular characterization
of the melanin-concentrating-hormone receptor. Nature 1999,
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McDonald, T. P.; Pan, J.; Pong, S.-S.; Feighner, S. D.; Tan, C.
P.; Fukami, T.; Iwaasa, H.; Hreniuk, D. L.; Morin, N. R.;
Sadowski, S. J.; Ito, M.; Ito, M.; Bansal, A.; Ky, B.; Figueroa, D.
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Q. Identification and characterization of a second melanin-
concentrating hormone receptor, MCH-2R. Proc. Natl. Acad. Sci.
U.S.A. 2001, 98, 7564-7569. (b) An, S.; Cutler, G.; Zhao, J. J.;
Huang, S.-G.; Tian, H.; Li, W.; Liang, L.; Rich, M.; Bakleh, A.;
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Trumbauer, M. E.; Chen, A. S.; Guan, X.-M.; Jiang, M. M.; Feng,
Y.; Camacho, R. E.; Shen, Z.; Frazier, E. G.; Yu, H.; Metzger, J.
M.; Kuca, S. J.; Shearman, L. P.; Gopal-Truter, S.; MacNeil, D.
J.; Strack, A. M.; MacIntyre, D. E.; Van der Ploeg, L. H. T.; Qian,
S. Melanin-concentrating hormone 1 receptor-deficient mice are
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selective melanin-concentrating hormone receptor antagonist.
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MacNeil, D. J.; Van der Ploeg, L. H. Y.; Howard, A. D.; Feighner,
S. D. Synthesis and biological evaluation in vitro of selective,
high affinity peptide antagonists of human melanin-concentrat-
ing hormone action at human melanin-concentrating hormone
receptor 1. Biochemistry 2002, 41, 6383-6390. (b) Shearman,
Phosphatidylinositol Assay. Stably transfected CHO-K1
cells, expressing human MCH1R (2 × 105 cells/well), were
incubated overnight with 5 µCi of [3H]myoinositol (Amersham
Pharmacia Biotech). Phosphatidylinositol turnover was stimu-
lated by submaximal concentrations of MCH, i.e., 10 nM, in
the presence of increasing amounts of test compound. The test
compound was added 5 min before addition of the agonist
(MCH). Incubation was in HBSS (Hanks’ balanced salt solu-
tion) supplemented with 10 mM LiCl at 37 °C for 45 min. Cells
were lysed with 10 mM ice-cold formic acid, and the generated
[3H]inositol phosphates were purified on Bio-Rad AG 1-X8
anion-exchange resin. Determinations were made in duplicate.
Data were analyzed and IC50 values determined by nonlinear
regression using the Prism software (GraphPad software, San
Diego, CA).
[
35S]GTPγS SPA Binding Assay. Schild analysis was
conducted with a [35S]GTPγS SPA binding assay. The assay
was performed by incubating 5 µg/well hMCH1R membranes
(Euroscreen) and 0.4 mg/well SPA beads (PVT-WGA, RP-
NQ0001 Amersham Pharmacia Biotech) with 1 nM [35S]GTPγS
(Perkin-Elmer, NEG 030H) in the presence of various concen-
trations of MCH and test compounds at room temperature for
1 h. The assay buffer contained 50 mM HEPES (pH 7.5), 100
mM NaCl, 5 mM MgCl2, 0.1% BSA, 3 µM GDP, and 10 µg/mL
saponin. Dose ratios calculated from EC50 values from MCH
concentration response curves were utilized to prepare Schild
plots and predict KB values: compound 59, KB ) 5.0 ( 1.0 nM;
compound 65, KB ) 76 nM; compound 67, KB ) 3.0 nM.
Acknowledgment. The authors thank Bo Aastrup,
Rokhsana Andersen, Ann Christensen, Joan Gredal,
Stina Hansen, and Helle Iversen for valuable discus-
sions and excellent technical assistance.
Supporting Information Available: Purity of central
target compounds determined by elemental analysis and/or
HPLC, synthetic details, yields, and characterization (mp, 1H
NMR, 13C NMR, and/or ESI-MS) for intermediates 1b, 2a-k,
3a-q, 4a-v, and 5a,b and target compounds 22, 26, 38-41,
44, 46-48, 50, 52, 53, 55, 57, 58, 60, 62, 64, 66, 68, and 69,
and coordinates and weights for pharmacophore hypotheses.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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2-Aminoquinolines as Potent MCH1R Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 18 5697
L. P.; Camacho, R. E.; Stribling, D. S.; Zhoua, D.; Bednarek, M.
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